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Sample records for high-sensitivity fluorescence hybridization

  1. Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

    PubMed

    Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

    2016-07-15

    Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis.

  2. Enzyme-free and label-free fluorescence aptasensing strategy for highly sensitive detection of protein based on target-triggered hybridization chain reaction amplification.

    PubMed

    Wang, Xiuzhong; Jiang, Aiwen; Hou, Ting; Li, Haiyin; Li, Feng

    2015-08-15

    Proteins are of great importance in medical and biological fields. In this paper, a novel fluorescent aptasensing strategy for protein assay has been developed based on target-triggered hybridization chain reaction (HCR) and graphene oxide (GO)-based selective fluorescence quenching. Three DNA probes, a helper DNA probe (HP), hairpin probe 1 (H1) and hairpin probe 2 (H2) are ingeniously designed. In the presence of the target, the aptamer sequences in HP recognize the target to form a target-aptamer complex, which causes the HP conformation change, and then triggers the chain-like assembly of H1 and H2 through the hybridization chain reaction, generating a long chain of HP leading complex of H1 and H2. At last the fluorescence indicator SYBR Green I (SG) binds with the long double strands of the HCR product through both intercalation and minor groove binding. When GO was added into the solutions after HCR, the free H1, H2 and SG would be closely adsorbed onto GO surface via π-π stacking. However, the HCR product cannot be adsorbed on GO surface, thereby the SG bound to HCR product gives a strong fluorescence signal dependent on the concentration of the target. With the use of platelet-derived growth factor BB (PDGF-BB) as the model analyte, this newly designed protocol provides a highly sensitive fluorescence detection of PDGF-BB with a limit of detection down to 1.25 pM, and also exhibit good selectivity and applicability in complex matrixes. Therefore, the proposed aptasensing strategy based on target-triggered hybridization chain reaction amplification should have wide applications in the diagnosis of genetic diseases due to its simplicity, low cost, and high sensitivity at extremely low target concentrations.

  3. A highly sensitive target-primed rolling circle amplification (TPRCA) method for fluorescent in situ hybridization detection of microRNA in tumor cells.

    PubMed

    Ge, Jia; Zhang, Liang-Liang; Liu, Si-Jia; Yu, Ru-Qin; Chu, Xia

    2014-02-04

    The ability to detect spatial and temporal microRNA (miRNA) distribution at the single-cell level is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. We report for the first time the development of a target-primed RCA (TPRCA) strategy for highly sensitive and selective in situ visualization of miRNA expression patterns at the single-cell level. This strategy uses a circular DNA as the probe for in situ hybridization (ISH) with the target miRNA molecules, and the free 3' terminus of miRNA then initiates an in situ RCA reaction to generate a long tandem repeated sequence with thousands of complementary segments. After hybridization with fluorescent detection probes, target miRNA molecules can be visualized with ultrahigh sensitivity. Because the RCA reaction can only be initiated by the free 3' end of target miRNA, the developed strategy offers the advantage over existing ISH methods in eliminating the interference from precursor miRNA or mRNA. This strategy is demonstrated to show high sensitivity and selectivity for the detection of miR-222 expression levels in human hepatoma SMMC-7721 cells and hepatocyte L02 cells. Moreover, the developed TPRCA-based ISH strategy is successfully applied to multiplexed detection using two-color fluorescent probes for two miRNAs that are differentially expressed in the two cell lines. The results reveal that the developed strategy may have great potential for in situ miRNA expression analysis for basic research and clinical diagnostics.

  4. A CMOS In-Pixel CTIA High Sensitivity Fluorescence Imager

    PubMed Central

    Murari, Kartikeya; Etienne-Cummings, Ralph; Thakor, Nitish; Cauwenberghs, Gert

    2012-01-01

    Traditionally, charge coupled device (CCD) based image sensors have held sway over the field of biomedical imaging. Complementary metal oxide semiconductor (CMOS) based imagers so far lack sensitivity leading to poor low-light imaging. Certain applications including our work on animal-mountable systems for imaging in awake and unrestrained rodents require the high sensitivity and image quality of CCDs and the low power consumption, flexibility and compactness of CMOS imagers. We present a 132×124 high sensitivity imager array with a 20.1 μm pixel pitch fabricated in a standard 0.5 μ CMOS process. The chip incorporates n-well/p-sub photodiodes, capacitive transimpedance amplifier (CTIA) based in-pixel amplification, pixel scanners and delta differencing circuits. The 5-transistor all-nMOS pixel interfaces with peripheral pMOS transistors for column-parallel CTIA. At 70 fps, the array has a minimum detectable signal of 4 nW/cm2 at a wavelength of 450 nm while consuming 718 μA from a 3.3 V supply. Peak signal to noise ratio (SNR) was 44 dB at an incident intensity of 1 μW/cm2. Implementing 4×4 binning allowed the frame rate to be increased to 675 fps. Alternately, sensitivity could be increased to detect about 0.8 nW/cm2 while maintaining 70 fps. The chip was used to image single cell fluorescence at 28 fps with an average SNR of 32 dB. For comparison, a cooled CCD camera imaged the same cell at 20 fps with an average SNR of 33.2 dB under the same illumination while consuming over a watt. PMID:23136624

  5. Highly sensitive turn-on fluorescence detection of thrombomodulin based on fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Kong, Liyan; Zhu, Jiaming; Wang, Wen; Jin, Lehe; Fu, Yanjiao; Duan, Bohui; Tan, Liang

    2017-02-01

    As an integral glycoprotein on the surface of endothelial cells, thrombomodulin (TM) has very high affinity for thrombin. TM has been regarded to be a marker of endothelial damage since it can be released during endothelial cell injury. In this work, a highly sensitive fluorescence method for the quantitative detection of TM was developed. TM antibody (Ab) and bovine serum albumin (BSA) were bound on gold nanoparticles (AuNPs) to construct BSA-AuNPs-Ab nanocomposites and they were characterized by transmission electron microscope and UV-vis spectrophotometry. The fluorescence of acridine orange (AO) was quenched by the prepared gold nanocomposites based on fluorescence resonance energy transfer (FRET). In the presence of TM, the fluorescence was turned on due to the effective separation of AO from the surface of gold nanocomposites. Under optimum conditions, the enhanced fluorescence intensity displayed a linear relationship with the logarithm of the TM concentration from 0.1 pg mL- 1 to 5 ng mL- 1 with a low detection limit of 12 fg mL- 1. The release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells in the presence of H2O2 was investigated using the proposed method. The released sTM content in the growth medium was found to be increased with the enhancement of contact time of the cells with H2O2.

  6. Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

    NASA Astrophysics Data System (ADS)

    Wang, Xue F.; Periasamy, Ammasi; Wodnicki, Pawel; Siadat-Pajouh, M.; Herman, Brian

    1995-04-01

    We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using

  7. High Sensitivity Stress Sensor Based on Hybrid Materials

    NASA Technical Reports Server (NTRS)

    Cao, Xian-An (Inventor)

    2014-01-01

    A sensing device is used to detect the spatial distributions of stresses applied by physical contact with the surface of the sensor or induced by pressure, temperature gradients, and surface absorption. The sensor comprises a hybrid active layer that includes luminophores doped in a polymeric or organic host, altogether embedded in a matrix. Under an electrical bias, the sensor simultaneously converts stresses into electrical and optical signals. Among many applications, the device may be used for tactile sensing and biometric imaging.

  8. Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells

    NASA Astrophysics Data System (ADS)

    Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

    2016-06-01

    A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells.

  9. Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

    PubMed

    Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

    2014-11-01

    A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity.

  10. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    NASA Astrophysics Data System (ADS)

    Zhang, Yi; Keegan, Gemma L.; Stranik, Ondrej; Brennan-Fournet, Margaret E.; McDonagh, Colette

    2015-07-01

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of 19-fold compared to a control assay without AgNPs.

  11. Mesoporous structured MIPs@CDs fluorescence sensor for highly sensitive detection of TNT.

    PubMed

    Xu, Shoufang; Lu, Hongzhi

    2016-11-15

    A facile strategy was developed to prepare mesoporous structured molecularly imprinted polymers capped carbon dots (M-MIPs@CDs) fluorescence sensor for highly sensitive and selective determination of TNT. The strategy using amino-CDs directly as "functional monomer" for imprinting simplify the imprinting process and provide well recognition sites accessibility. The as-prepared M-MIPs@CDs sensor, using periodic mesoporous silica as imprinting matrix, and amino-CDs directly as "functional monomer", exhibited excellent selectivity and sensitivity toward TNT with detection limit of 17nM. The recycling process was sustainable for 10 times without obvious efficiency decrease. The feasibility of the developed method in real samples was successfully evaluated through the analysis of TNT in soil and water samples with satisfactory recoveries of 88.6-95.7%. The method proposed in this work was proved to be a convenient and practical way to prepare high sensitive and selective fluorescence MIPs@CDs sensors.

  12. Highly sensitive ratiometric fluorescent chemosensor for silver ion and silver nanoparticles in aqueous solution.

    PubMed

    Jang, Sujung; Thirupathi, Ponnaboina; Neupane, Lok Nath; Seong, Junho; Lee, Hyunsook; Lee, Wan In; Lee, Keun-Hyeung

    2012-09-21

    A pyrene derivative chemosensor (Pyr-WH) based on a dipeptide shows a highly sensitive ratiometric response to Ag(I) as well as silver nanoparticles in aqueous solution at physiological pH. Pyr-WH penetrated live HeLa cells and exhibits a ratiometric response to intracellular Ag(I). The binding mode of Pyr-WH with Ag(I) was characterized based on fluorescence changes in different pH, NMR, and ESI mass spectrometer experiments.

  13. Highly sensitive detection of superoxide dismutase based on an immunoassay with surface-enhanced fluorescence.

    PubMed

    Yang, Xiaoming; Dou, Yao; Zhu, Shanshan

    2013-06-07

    Herein, a novel highly sensitive enhanced-fluorescence immunoassay for detection of superoxide dismutase (SOD) is established by combining surface-enhanced fluorescence (SEF) with immuno-magnetic separation. Based on a sandwich-type immunoassay, analytes in samples are first captured by magnetic beads coated with a monoclonal antibody and then "sandwiched" by another monoclonal antibody on silver nanoparticles labeled with fluorescein-labeled oligonucleotides in the presence of a magnet. Subsequently, the immune complex is enriched by exposure to a magnetic field. Lastly, the fluorescence intensity is measured according to the number of dissociated fluoresceins. The increased fluorescence intensity permits highly sensitive detection of SOD in a linear range of 10-8 × 10(5) pg mL(-1), with a detection limit of 4 pg mL(-1) at a signal-to-noise ratio of 3. Significantly, this method was validated for detection of SOD in human serum, human urine, and cosmetic samples. Moreover, the reliability and accuracy of results obtained by the enhanced-fluorescence method was confirmed by the analysis of high performance liquid chromatography (HPLC).

  14. A virus-MIPs fluorescent sensor based on FRET for highly sensitive detection of JEV.

    PubMed

    Liang, Caishuang; Wang, Huan; He, Kui; Chen, Chunyan; Chen, Xiaoming; Gong, Hang; Cai, Changqun

    2016-11-01

    Major stumbling blocks in the recognition and detection of virus are the unstable biological recognition element or the complex detection means. Here a fluorescent sensor based on virus-molecular imprinted polymers (virus-MIPs) was designed for specific recognition and highly sensitive detection of Japanese encephalitis virus (JEV). The virus-MIPs were anchored on the surface of silica microspheres modified by fluorescent dye, pyrene-1-carboxaldehyde (PC). The fluorescence intensity of PC can be enhanced by the principle of fluorescence resonance energy transfer (FRET), where virus acted as energy donor and PC acted as energy acceptor. The enhanced fluorescence intensity was proportional to the concentration of virus in the range of 24-960pM, with a limit of detection (LOD, 3σ) of 9.6pM, and the relative standard deviation was 1.99%. In additional, the specificity study confirmed the resultant MIPs has high-selectivity for JEV. This sensor would become a new key for the detection of virus because of its high sensitive, simple operation, high stability and low cost.

  15. Highly sensitive fluorescence detection of avidin/streptavidin with an optical interference mirror slide.

    PubMed

    Yasuda, Mitsuru; Akimoto, Takuo

    2012-01-01

    This paper presents highly sensitive fluorescence detections of avidin and streptavidin using an optical interference mirror (OIM) slide consisting of a plane mirror covered with an optical interference layer. Compared with a common glass slide, the OIM slide can enhance the fluorescence from a dye by more than 100-fold. We fabricated an OIM slide by depositing an optical interference layer of Al(2)O(3) on an Ag mirror. To enhance the fluorescence maximally, the optimal thickness of the Al(2)O(3) layer was estimated from optical interference theory. For detections of protein, avidin/streptavidin labeled with fluorescein, Cy3, and Cy5 were detected with biotin immobilized on an OIM slide with the optimal Al(2)O(3) thickness. We achieved a sensitivity improvement of more than 50-fold, comparing with a glass slide. Such a high degree of improvement would be a significant contribution to further progress in biomedical research and medical diagnostics.

  16. A novel high-sensitive miniaturized optical system for fluorescence detection

    NASA Astrophysics Data System (ADS)

    Yao, Mingjin; Fang, Ji

    2011-03-01

    This paper presents a novel, high sensitive and miniaturized fluorescence detection system which integrated a LED light source, all necessary optical components and a photodiode with preamplifier into one package about 2 cm x 2 cm x 2 cm especially for the applications of lab-on-a-chip, portable bio-detection system and point-of-care diagnostic system. The prototype has been tested using the fluorescence dye 5-Carboxyfluorescein (5-FAM) dissolved into solvent DMSO (Dimethyl Sulfoxide) and diluted with DI water as the testing solution samples. Resolution approximation method is accepted to evaluate the sensitivity. The testing results prove a remarkable sensitivity at pico-scale molar, around 1.08 pM/L, which should meet the most of bio-detection requirements. This cost-effective detection system can be widely integrated to the portable device and system for fluorescent detection in biological, chemical, medical, point-of-care applications.

  17. Fluorescent hybridization probes for nucleic acid detection.

    PubMed

    Guo, Jia; Ju, Jingyue; Turro, Nicholas J

    2012-04-01

    Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

  18. Highly sensitive and simple SERS substrate based on photochemically generated carbon nanotubes-gold nanorods hybrids.

    PubMed

    Caires, A J; Vaz, R P; Fantini, C; Ladeira, L O

    2015-10-01

    We report a simple and easy formation of hybrids between multi-wall carbon nanotubes and gold nanorods by one-pot in situ photochemical synthesis. Measurements of surface-enhanced Raman scattering (SERS) through the effect "coffee ring" in visible and near infrared (NIR) show high sensitivity with detection of nanomolar concentrations of aromatic dyes. The formation of nanocomposites between carbon nanotubes and gold nanorods without chemical binders simplifies the preparation. Photochemical synthesis is an advance over the techniques previously published.

  19. Magnetic-fluorescent-targeting multifunctional aptasensorfor highly sensitive and one-step rapid detection of ochratoxin A.

    PubMed

    Wang, Chengquan; Qian, Jing; Wang, Kan; Wang, Kun; Liu, Qian; Dong, Xiaoya; Wang, Chengke; Huang, Xingyi

    2015-06-15

    A multifunctional aptasensor for highly sensitive and one-step rapid detection of ochratoxin A (OTA), has been developed using aptamer-conjugated magnetic beads (MBs) as the recognition and concentration element and a heavy CdTe quantum dots (QDs) as the label. Initially, the thiolated aptamer was conjugated on the Fe3O4@Au MBs through Au-S covalent binding. Subsequently, multiple CdTe QDs were loaded both in and on a versatile SiO2 nanocarrier to produce a large amplification factor of hybrid fluorescent nanoparticles (HFNPs) labeled complementary DNA (cDNA). The magnetic-fluorescent-targeting multifunctional aptasensor was thus fabricated by immobilizing the HFNPs onto MBs' surface through the hybrid reaction between the aptamer and cDNA. This aptasensor can be produced at large scale in a single run, and then can be conveniently used for rapid detection of OTA through a one-step incubation procedure. The presence of OTA would trigger aptamer-OTA binding, resulting in the partial release of the HFNPs into bulk solution. After a simple magnetic separation, the supernatant liquid of the above solution contained a great number of CdTe QDs produced an intense fluorescence emission. Under the optimal conditions, the fluorescence intensity of the released HFNPs was proportional to the concentration of OTA in a wide range of 15 pg mL(-1) -100 ng mL(-1) with a detection limit of 5.4 pg mL(-1) (S/N=3). This multifunctional aptasensor represents a promising path toward routine quality control of food safety, and also creates the opportunity to develop aptasensors for other targets using this strategy.

  20. A highly sensitive ratiometric fluorescent probe for the detection of cytoplasmic and nuclear hydrogen peroxide.

    PubMed

    Wen, Ying; Liu, Keyin; Yang, Huiran; Li, Yi; Lan, Haichuang; Liu, Yi; Zhang, Xinyu; Yi, Tao

    2014-10-07

    As a marker for oxidative stress and a second messenger in signal transduction, hydrogen peroxide (H2O2) plays an important role in living systems. It is thus critical to monitor the changes in H2O2 in cells and tissues. Here, we developed a highly sensitive and versatile ratiometric H2O2 fluorescent probe (NP1) based on 1,8-naphthalimide and boric acid ester. In response to H2O2, the ratio of its fluorescent intensities at 555 and 403 nm changed 1020-fold within 200 min. The detecting limit of NP1 toward H2O2 is estimated as 0.17 μM. It was capable of imaging endogenous H2O2 generated in live RAW 264.7 macrophages as a cellular inflammation response, and especially, it was able to detect H2O2 produced as a signaling molecule in A431 human epidermoid carcinoma cells through stimulation by epidermal growth factor. This probe contains an azide group and thus has the potential to be linked to various molecules via the click reaction. After binding to a Nuclear Localization Signal peptide, the peptide-based combination probe (pep-NP1) was successfully targeted to nuclei and was capable of ratiometrically detecting nuclear H2O2 in living cells. These results indicated that NP1 was a highly sensitive ratiometric H2O2 dye with promising biological applications.

  1. Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.

    PubMed

    Mehta, Pramod Kumar; Lee, Hyeri; Lee, Keun-Hyeung

    2017-05-15

    The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R(2)=0.99) in aqueous solutions, 2.98nM (R(2)=0.98) in 1% serum samples, and 3.43nM (R(2)=0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response.

  2. Tuning the Aggregation/Disaggregation Behavior of Graphene Quantum Dots by Structure-Switching Aptamer for High-Sensitivity Fluorescent Ochratoxin A Sensor.

    PubMed

    Wang, Song; Zhang, Yajun; Pang, Guangsheng; Zhang, Yingwei; Guo, Shaojun

    2017-02-07

    The design of graphene quantum dots (GQDs)-aptamer bioconjugates as the new sensing platform is very important for developing high-sensitivity fluorescent biosensors; however, achieving new bioconjugates is still a great challenge. Herein, we report the development of a new high-sensitivity fluorescent aptasensor for the detection of ochratoxin A (OTA) based on tuning aggregation/disaggregation behavior of GQDs by structure-switching aptamers. The fluorescence sensing process for OTA detection involved two key steps: (1) cDNA-aptamer (cDNA, complementary to part of the OTA aptamer) hybridization induced the aggregation of GQD (fluorescence quenching) after cDNA was added into the GQDs-aptamer bioconjugate solution, and (2) the target of OTA triggered disaggregation of GQD aggregates (fluorescence recovery). Such new fluorescent sensing platform can be used to monitor OTA with a linear range of 0 to 1 ng/mL and very low detection limit of 13 pg/mL, which is among the best in all the developed fluorescent nanoparticles-based sensors. Such sensing strategy is also successful in analyzing OTA in practical red wine sample with 94.4-102.7% of recoveries and relative standard deviation in the range of 2.9-5.8%. The present works open a new way for signaling the target-aptamer binding event by tuning aggregation/disaggregation behavior of GQDs-bioconjugates.

  3. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    NASA Astrophysics Data System (ADS)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  4. Fluorescent trimethyl-substituted naphthyridine as a label-free signal reporter for one-step and highly sensitive fluorescent detection of DNA in serum samples.

    PubMed

    Wang, Jiamian; Wang, Xiuyun; Wu, Shuo; Che, Ruping; Luo, Pinchen; Meng, Changgong

    2017-01-15

    A facile label-free sensing method is developed for the one-step and highly sensitive fluorescent detection of DNA, which couples the specific C-C mismatch bonding and fluorescent quenching property of a trimethyl-substituted naphthyridine dye (ATMND) with the exonuclease III (Exo III) assisted cascade target recycling amplification strategy. In the absence of target DNA, the DNA hairpin probe with a C-C mismatch in the stem and more than 4 bases overhung at the 3' terminus could entrap and quench the fluorescence of ATMND and resist the digestion of Exo III, thus showing a low fluorescence background. In the presence of the target, however, the hybridization event between the two protruding segments and the target triggers the digestion reaction of Exo III, recycles the initial target, and simultaneously releases both the secondary target analogue and the ATMND caged in the stem. The released initial and secondary targets take part in another cycle of digestion, thus leading to the release of a huge amount of free ATMND for signal transducing. Based on the fluorescence recovery, the as-proposed label-free fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 10pM to 1μM, a low limit of detection of 6pM, good selectivity, and a facile one-step operation at room temperature. Practical sample analysis in serum samples indicates the method has good precision and accuracy, which may thus have application potentials for point-of-care screening of DNA in complex clinical and environmental samples.

  5. Highly Sensitive Detection of S-Nitrosylated Proteins by Capillary Gel Electrophoresis with Laser Induced Fluorescence

    PubMed Central

    Wang, Siyang; Circu, Magdalena L.; Zhou, Hu; Figeys, Daniel; Aw, Tak Y.; Feng, June

    2011-01-01

    S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer’s disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol(S-NO) to SH in cysteine using the “fluorescence switch” assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione(GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM)concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions. PMID:21820121

  6. Isotachophoresis with ionic spacer and two-stage separation for high sensitivity DNA hybridization assay.

    PubMed

    Eid, Charbel; Garcia-Schwarz, Giancarlo; Santiago, Juan G

    2013-06-07

    We present an on-chip electrophoretic assay for rapid and high sensitivity nucleic acid (NA) detection. The assay uses isotachophoresis (ITP) to enhance NA hybridization and an ionic spacer molecule to subsequently separate reaction products. In the first stage, the probe and target focus and mix rapidly in free solution under ITP. The reaction mixture then enters a region containing a sieving matrix, which allows the spacer ion to overtake and separate the slower probe-target complex from free, unhybridized probes. This results in the formation of two focused ITP peaks corresponding to probe and probe-target complex signals. For a 149 nt DNA target, we achieve a 220 fM limit of detection (LOD) within 10 min, with a 3.5 decade dynamic range. This LOD constitutes a 12× improvement over previous ITP-based hybridization assays. The technique offers an alternative to traditional DNA hybridization assays, and can be multiplexed and extended to detect other biomolecules.

  7. Highly Sensitive Ultraviolet Photodetectors Fabricated from ZnO Quantum Dots/Carbon Nanodots Hybrid Films

    PubMed Central

    Guo, Deng-Yang; Shan, Chong-Xin; Qu, Song-Nan; Shen, De-Zhen

    2014-01-01

    Ultraviolet photodetectors have been fabricated from ZnO quantum dots/carbon nanodots hybrid films, and the introduction of carbon nanodots improves the performance of the photodetectors greatly. The photodetectors can be used to detect very weak ultraviolet signals (as low as 12 nW/cm2). The detectivity and noise equivalent power of the photodetector can reach 3.1 × 1017 cmHz1/2/W and 7.8 × 10−20 W, respectively, both of which are the best values ever reported for ZnO-based photodetectors. The mechanism for the high sensitivity of the photodetectors has been attributed to the enhanced carrier-separation at the ZnO/C interface. PMID:25502422

  8. Self-assembled dipeptide-gold nanoparticle hybrid spheres for highly sensitive amperometric hydrogen peroxide biosensors.

    PubMed

    Gong, Yufei; Chen, Xu; Lu, Yanluo; Yang, Wensheng

    2015-04-15

    Novel self-assembled dipeptide-gold nanoparticle (DP-AuNP) hybrid microspheres with a hollow structure have been prepared in aqueous solution by a simple one-step method. Diphenylalanine (FF) dipeptide was used as a precursor to form simultaneously peptide spheres and a reducing agent to reduce gold ions to gold nanoparticles in water at 60°C. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that formed AuNPs were localized both inside and on the surface of the dipeptide spheres. Horseradish peroxidase (HRP) as a model enzyme was further immobilized on the dipeptide-AuNP hybrid spheres to construct a mediate H2O2 amperometric biosensor. UV-vis spectroscopy showed that the immobilized HRP retained its original structure. Cyclic voltammetry characterization demonstrated that the HRP/dipeptide-AuNP hybrid spheres modified glassy carbon electrode showed high electrocatalytic activity to H2O2. The proposed biosensor exhibited a wide linear response in the range from 5.0×10(-7) to 9.7×10(-4)M with a high sensitivity of 28.3µAmM(-1). A low detection limit of 1.0×10(-7)M was estimated at S/N=3. In addition, the biosensor possessed satisfactory reproducibility and long-term stability. These results indicated that the dipeptide-AuNP hybrid sphere is a promising matrix for application in the fabrication of electrochemical biosensors due to its excellent biocompatibility and good charge-transfer ability.

  9. Construction of optical glucose nanobiosensor with high sensitivity and selectivity at physiological pH on the basis of organic-inorganic hybrid microgels.

    PubMed

    Wu, Weitai; Zhou, Ting; Aiello, Michael; Zhou, Shuiqin

    2010-08-15

    A new class of optical glucose nanobiosensors with high sensitivity and selectivity at physiological pH is described. To construct these glucose nanobiosensors, the fluorescent CdS quantum dots (QDs), serving as the optical code, were incorporated into the glucose-sensitive poly(N-isopropylacrylamide-acrylamide-2-acrylamidomethyl-5-fluorophenylboronic acid) copolymer microgels, via both in situ growth method and "breathing in" method, respectively. The polymeric gel can adapt to surrounding glucose concentrations, and regulate the fluorescence of the embedded QDs, converting biochemical signals into optical signals. The gradual swelling of the gel would lead to the quenching of the fluorescence at the elevated glucose concentrations. The hybrid microgels displayed high selectivity to glucose over the potential primary interferents of lactate and human serum albumin in the physiologically important glucose concentration range. The stability, reversibility, and sensitivity of the organic-inorganic hybrid microgel-based biosensors were also systematically studied. These general properties of our nanobiosensors are well tunable under appropriate tailor on the hybrid microgels, in particular, simply through the change in the crosslinking degree of the microgels. The optical glucose nanobiosensors based on the organic-inorganic hybrid microgels have shown the potential for a third generation fluorescent biosensor.

  10. [Development of novel fluorescence-derivatization-HPLC methods enabling highly sensitive and selective analysis of biological compounds].

    PubMed

    Todoroki, Kenichiro

    2011-01-01

    Fluorescence-derivatization-HPLC methods are powerful tools for performing the analysis of bioactive compounds with high sensitivity and selectivity. In this paper, the author reviews the development of the following four types of novel fluorescence-derivatization-HPLC analytical systems: (1) simultaneous HPLC analysis of melatonin and its related compounds through post-column electrochemical demethylation and fluorescence derivatization, (2) HPLC analysis of 5-hydroxyindoles based on fluorescence derivatization by online pre-column photocatalytic oxidation with benzylamine, (3) reagent peak-free HPLC analysis for aliphatic amines and amino acids using F-trap pyrene as a fluorous tag-bound fluorescence derivatization reagent, and (4) reagent peak-free HPLC analysis for carboxylic acids using a fluorous scavenging-derivatization method. The authors have also successfully applied these systems to biological and pharmaceutical analyses.

  11. Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn-On DNA Probes.

    PubMed

    Uno, Kakishi; Sasaki, Taeko; Sugimoto, Nagisa; Ito, Hideto; Nishihara, Taishi; Hagihara, Shinya; Higashiyama, Tetsuya; Sasaki, Narie; Sato, Yoshikatsu; Itami, Kenichiro

    2017-01-17

    Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure-property relationship study has revealed that the dialkylamino group at the 2-position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double-stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2-position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser-scanning microscope.

  12. A Highly Sensitive Fluorescent Sensor for Palladium and Direct Imaging of Its Ecotoxicity in Living Model Organisms.

    PubMed

    Liu, Fei; Du, Juan; Xu, Meiying; Sun, Guoping

    2016-01-01

    Rhodamine is an ideal platform for fluorescence probes owing to its spiro-lactam framework and excellent photochemical properties. Herein, a novel rhodamine-based palladium fluorescent chemosensor, Rd-Eb, showing a fast response time (3 min), high sensitivity for palladium species over other ions, and a low detection limit (1.91×10(-7)  m), was synthesized. It can act as an obvious colorimetric as well as a fluorescent "off/on" sensor for Pd(2+) . In addition, it is also an excellent sensor for in vivo imaging of Pd(2+) in zebra fish and Daphnia magna, illuminating the impact of palladium on organisms at different growth stages with respect to biological toxicology.

  13. High sensitivity fluorescent single particle and single molecule detection apparatus and method

    DOEpatents

    Mathies, Richard A.; Peck, Konan; Stryer, Lubert

    1990-01-01

    Apparatus is described for ultrasensitive detection of single fluorescent particles down to the single fluorescent molecule limit in a fluid or on a substrate comprising means for illuminating a predetermined volume of the fluid or area of the substrate whereby to emit light including background light from the fluid and burst of photons from particles residing in the area. The photon burst is detected in real time to generate output representative signal. The signal is received and the burst of energy from the fluorescent particles is distinguished from the background energy to provide an indication of the number, location or concentration of the particles or molecules.

  14. Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases

    NASA Astrophysics Data System (ADS)

    Kumaraswamy, Sriram; Bergstedt, Troy; Shi, Xiaobo; Rininsland, Frauke; Kushon, Stuart; Xia, Wensheng; Ley, Kevin; Achyuthan, Komandoor; McBranch, Duncan; Whitten, David

    2004-05-01

    Sensor formats have been developed for detecting the activity of proteolytic enzymes based on fluorescent conjugated polymer superquenching. These sensors employ a reactive peptide sequence within a tether linking a quencher to a biotin. The peptide binds to sensors containing colocated biotin-binding protein and fluorescent polymer by means of biotin-biotin binding protein interactions, resulting in a strong quenching of polymer fluorescence. Enzyme-mediated cleavage of the peptide results in a reversal of the fluorescence quenching. These assays for protease activity are simple, sensitive, fast, and have the specificity required for screening chemical libraries for novel protease inhibitors in a high-throughput screening assay environment. These assays have been demonstrated for enterokinase, caspase-3/7, and -secretase.

  15. A fluorescent graphitic carbon nitride nanosheet biosensor for highly sensitive, label-free detection of alkaline phosphatase

    NASA Astrophysics Data System (ADS)

    Xiang, Mei-Hao; Liu, Jin-Wen; Li, Na; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

    2016-02-01

    Graphitic C3N4 (g-C3N4) nanosheets provide an attractive option for bioprobes and bioimaging applications. Utilizing highly fluorescent and water-dispersible ultrathin g-C3N4 nanosheets, a highly sensitive, selective and label-free biosensor has been developed for ALP detection for the first time. The developed approach utilizes a natural substrate of ALP in biological systems and thus affords very high catalytic efficiency. This novel biosensor is demonstrated to enable quantitative analysis of ALP in a wide range from 0.1 to 1000 U L-1 with a low detection limit of 0.08 U L-1, which is among the most sensitive assays for ALP. It is expected that the developed method may provide a low-cost, convenient, rapid and highly sensitive platform for ALP-based clinical diagnostics and biomedical applications.Graphitic C3N4 (g-C3N4) nanosheets provide an attractive option for bioprobes and bioimaging applications. Utilizing highly fluorescent and water-dispersible ultrathin g-C3N4 nanosheets, a highly sensitive, selective and label-free biosensor has been developed for ALP detection for the first time. The developed approach utilizes a natural substrate of ALP in biological systems and thus affords very high catalytic efficiency. This novel biosensor is demonstrated to enable quantitative analysis of ALP in a wide range from 0.1 to 1000 U L-1 with a low detection limit of 0.08 U L-1, which is among the most sensitive assays for ALP. It is expected that the developed method may provide a low-cost, convenient, rapid and highly sensitive platform for ALP-based clinical diagnostics and biomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08278a

  16. A highly sensitive fluorescence probe for metallothioneins based on tiron-copper complex

    NASA Astrophysics Data System (ADS)

    Xiao, Xilin; Xue, Jinhua; Liao, Lifu; Huang, Mingyang; Zhou, Bin; He, Bo

    2015-06-01

    The fabrication of tiron-copper complex as a novel fluorescence probe for the sensitive directly detection of metallothioneins at nanomolar levels was demonstrated. In Britton-Robinson (B-R) buffer (pH 7.50), the interaction of bis(tiron)copper(II) complex cation [Cu(tiron)2]2+ and metallothioneins enhanced the fluorescence intensity of the system. The fluorescence enhancement at 347 nm was proportional to the concentration of metallothioneins. The mechanism was studied and discussed in terms of the fluorescence spectra. Under the optimal experimental conditions, at 347 nm, there was a linear relationship between the fluorescence intensity and the concentration of the metallothioneins in the range of 8.80 × 10-9-7.70 × 10-7 mol L-1, with a correlation coefficient of r = 0.995 and detection limit 2.60 × 10-9 mol L-1. The relative standard deviation was 0.77% (n = 11), and the average recovery 94.4%. The method proposed was successfully reliable, selective and sensitive in determining of trace metallothioneins in fish visceral organ samples with the results in good agreement with those obtained by HPLC.

  17. Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method.

    PubMed

    Minami, Akira; Shimizu, Hirotaka; Meguro, Yuko; Shibata, Naoki; Kanazawa, Hiroaki; Ikeda, Kiyoshi; Suzuki, Takashi

    2011-09-01

    Sialidase (EC 3.2.1.18) removes sialic acid from sialoglycoconjugates. Since sialidase extracellularly applied to the rat hippocampus influences many neural functions, including synaptic plasticity and innervations of glutamatergic neurons, endogenous sialidase activities on the extracellular membrane surface could also affect neural functions. However, the distribution of sialidase activity in the brain remains unknown. To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB (FRV LB) at pH 7.3. After 1h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. These fluorescence intensities were attenuated with a sialidase-specific inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA, 1mM). Additionally, the fluorescence intensities caused by X-Neu5Ac and FRV LB were correlated with the sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac), a classical substrate for quantitative measurement of sialidase activity, in each brain region. Therefore, staining with X-Neu5Ac and FRV LB is specific for sialidase and useful for quantitative analysis of sialidase activities. The results suggest that white matter of the rat brain has intense sialidase activity.

  18. Radiochemical Analysis by High Sensitivity Micro X-Ray Fluorescence Detection

    SciTech Connect

    Ning Gao

    2006-05-12

    The primary objective of the project was to develop a novel dual-optic x-ray fluorescence instrument capable of doing radiochemical analysis of high-level radioactive wastes at DOE sites such as Savannah River Site and Hanford Site.

  19. Ag Nanoparticles-enhanced Fluorescence of Terbium-Deferasirox Complexes for the Highly Sensitive Determination of Deferasirox.

    PubMed

    Abolhasani, Jafar; Naderali, Roza; Hassanzadeh, Javad

    2016-01-01

    We describe the effect of different sized gold and silver nanoparticles on the terbium sensitized fluorescence of deferasirox. It is indicated that silver nanostructures, especially 18 nm Ag nanoparticles (AgNPs), have a remarkable amplifying effect compared to Au nanoparticles. Based on this observation, a highly sensitive and selective method was developed for the determination of deferasirox. Effects of various parameters like AgNPs and Tb(3+) concentration and pH of media were investigated. Under the optimal conditions, a calibration curve was plotted as the fluorescence intensities versus the concentration of deferasirox in the range of 0.1 to 200 nmol L(-1), and detection limit of 0.03 nmol L(-1) was obtained. The method has good linearity, recovery, reproducibility and sensitivity, and was satisfactorily applied for the determination of deferasirox in urine and pharmaceutical samples.

  20. High sensitivity automated multiplexed immunoassays using photonic crystal enhanced fluorescence microfluidic system.

    PubMed

    Tan, Yafang; Tang, Tiantian; Xu, Haisheng; Zhu, Chenqi; Cunningham, Brian T

    2015-11-15

    We demonstrate a platform that integrates photonic crystal enhanced fluorescence (PCEF) detection of a surface-based microspot fluorescent assay with a microfluidic cartridge to achieve simultaneous goals of high analytic sensitivity (single digit pg/mL), high selectivity, low sample volume, and assay automation. The PC surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cyanines 5 (Cy5), was used to amplify the fluorescence signal intensity measured from a multiplexed biomarker microarray. The assay system is comprised of a plastic microfluidic cartridge for holding the PC and an assay automation system that provides a leak-free fluid interface during introduction of a sequence of fluids under computer control. Through the use of the assay automation system and the PC embedded within the microfluidic cartridge, we demonstrate pg/mL-level limits of detection by performing representative biomarker assays for interleukin 3 (IL3) and Tumor Necrosis Factor (TNF-α). The results are consistent with limits of detection achieved without the use of the microfluidic device with the exception that coefficients of variability from spot-to-spot are substantially lower than those obtained by performing assays with manual manipulation of assay liquids. The system's capabilities are compatible with the goal of diagnostic instruments for point-of-care settings.

  1. High Sensitivity Automated Multiplexed Immunoassays Using Photonic Crystal Enhanced Fluorescence Microfluidic System

    PubMed Central

    Tan, Yafang; Tang, Tiantian; Xu, Haisheng; Zhu, Chenqi; Cunningham, Brian T.

    2015-01-01

    We demonstrate a platform that integrates photonic crystal enhanced fluorescence (PCEF) detection of a surface-based microspot fluorescent assay with a microfluidic cartridge to achieve simultaneous goals of high analytic sensitivity (single digit pg/mL), high selectivity, low sample volume, and assay automation. The PC surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cyanines 5 (Cy5), was used to amplify the fluorescence signal intensity measured from a multiplexed biomarker microarray. The assay system is comprised of a plastic microfluidic cartridge for holding the PC and an assay automation system that provides a leak-free fluid interface during introduction of a sequence of fluids under computer control. Through the use of the assay automation system and the PC embedded within the microfluidic cartridge, we demonstrate pg/mL-level limits of detection by performing representative biomarker assays for interleukin 3 (IL3) and Tumor Necrosis Factor (TNF-α). The results are consistent with limits of detection achieved without the use of the microfluidic device with the exception that coefficients of variability from spot-to-spot are substantially lower than those obtained by performing assays with manual manipulation of assay liquids. The system’s capabilities are compatible with the goal of diagnostic instruments for point-of-care settings. PMID:26043313

  2. Graphitic Carbon Nitride Nanosheets-Based Ratiometric Fluorescent Probe for Highly Sensitive Detection of H2O2 and Glucose.

    PubMed

    Liu, Jin-Wen; Luo, Ying; Wang, Yu-Min; Duan, Lu-Ying; Jiang, Jian-Hui; Yu, Ru-Qin

    2016-12-14

    Graphitic carbon nitride (g-C3N4) nanosheets, an emerging graphene-like carbon-based nanomaterial with high fluorescence and large specific surface areas, hold great potential for biosensor applications. Current g-C3N4 nanosheets based fluorescent biosensors majorly rely on single fluorescent intensity reading through fluorescence quenching interactions between the nanosheets and metal ions. Here we report for the first time the development of a novel g-C3N4 nanosheets-based ratiometric fluorescence sensing strategy for highly sensitive detection of H2O2 and glucose. With o-phenylenediamine (OPD) oxidized by H2O2 in the presence of horseradish peroxidase (HRP), the oxidization product can assemble on the g-C3N4 nanosheets through hydrogen bonding and π-π stacking, which effectively quenches the fluorescence of g-C3N4 while delivering a new emission peak. The ratiometric signal variations enable robust and sensitive detection of H2O2. On the basis of the glucose converting into H2O2 through the catalysis of glucose oxidase, the g-C3N4-based ratiometric fluorescence sensing platform is also exploited for glucose assay. The developed strategy is demonstrated to give a detection limit of 50 nM for H2O2 and 0.4 μM for glucose, at the same time, it has been successfully used for glucose levels detection in human serum. This strategy may provide a cost-efficient, robust, and high-throughput platform for detecting various species involving H2O2-generation reactions for biomedical applications.

  3. Improved Diffuse Fluorescence Flow Cytometer Prototype for High Sensitivity Detection of Rare Circulating Cells In Vivo

    NASA Astrophysics Data System (ADS)

    Pestana, Noah Benjamin

    Accurate quantification of circulating cell populations is important in many areas of pre-clinical and clinical biomedical research, for example, in the study of cancer metastasis or the immune response following tissue and organ transplants. Normally this is done "ex-vivo" by drawing and purifying a small volume of blood and then analyzing it with flow cytometry, hemocytometry or microfludic devices, but the sensitivity of these techniques are poor and the process of handling samples has been shown to affect cell viability and behavior. More recently "in vivo flow cytometry" (IVFC) techniques have been developed where fluorescently-labeled cells flowing in a small blood vessel in the ear or retina are analyzed, but the sensitivity is generally poor due to the small sampling volume. To address this, our group recently developed a method known as "Diffuse Fluorescence Flow Cytometry" (DFFC) that allows detection and counting of rare circulating cells with diffuse photons, offering extremely high single cell counting sensitivity. In this thesis, an improved DFFC prototype was designed and validated. The chief improvements were three-fold, i) improved optical collection efficiency, ii) improved detection electronics, and iii) development of a method to mitigate motion artifacts during in vivo measurements. In combination, these improvements yielded an overall instrument detection sensitivity better than 1 cell/mL in vivo, which is the most sensitive IVFC system reported to date. Second, development and validation of a low-cost microfluidic device reader for analysis of ocular fluids is described. We demonstrate that this device has equivalent or better sensitivity and accuracy compared a fluorescence microscope, but at an order-of-magnitude reduced cost with simplified operation. Future improvements to both instruments are also discussed.

  4. Highly sensitive rapid fluorescence detection of protein residues on surgical instruments

    NASA Astrophysics Data System (ADS)

    Kovalev, Valeri I.; Bartona, James S.; Richardson, Patricia R.; Jones, Anita C.

    2006-07-01

    There is a risk of contamination of surgical instruments by infectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect ~10 attomole/cm2 with a scan speed of ~3-10 cm2/s of the test instrument's surface. A theoretical analysis and experimental measurements will be discussed.

  5. Peptide-Induced AIEgen Self-Assembly: A New Strategy to Realize Highly Sensitive Fluorescent Light-Up Probes.

    PubMed

    Han, Aitian; Wang, Huaimin; Kwok, Ryan T K; Ji, Shenglu; Li, Jun; Kong, Deling; Tang, Ben Zhong; Liu, Bin; Yang, Zhimou; Ding, Dan

    2016-04-05

    Fluorescent light-up probes with aggregation-induced emission (AIE) characteristics have recently attracted great research interest due to their intelligent fluorescence activation mechanism and excellent photobleaching resistance. In this work, we report a new, simple, and generic strategy to design and prepare highly sensitive AIE fluorescent light-up bioprobe through facile incorporation of a self-assembling peptide sequence GFFY between the recognition element and the AIE luminogen (AIEgen). After the bioprobes respond to the targets, the peptide GFFY is capable of inducing the ordered self-assembly of AIEgens, yielding close and tight intermolecular steric interactions to restrict the intramolecular motions of AIEgens for excellent signal output. Using two proof-of-concepts, we have demonstrated that self-assembling peptide-incorporating AIE light-up probes show much higher sensitivity in sensing the corresponding targets in both solutions and cancer cells as compared to those without GFFY induced self-assembly. Taking the probe TPE-GFFYK(DVEDEE-Ac), for example, a detection limit as low as 0.54 pM can be achieved for TPE-GFFYK(DVEDEE-Ac) in caspase-3 detection, which is much lower than that of TPE-K(DVED-Ac) (3.50 pM). This study may inspire new insights into the design of advanced fluorescent molecular probes.

  6. A convenient and label-free fluorescence "turn off-on" nanosensor with high sensitivity and selectivity for acid phosphatase.

    PubMed

    Liu, Ziping; Lin, Zihan; Liu, Linlin; Su, Xingguang

    2015-05-30

    In this study, we reported a convenient label-free fluorescence nanosensor for rapid detection of acid phosphatase on the basis of aggregation-caused quenching (ACQ) and enzymolysis approach. The selectivity nanosensor was based on the fluorescence "turn off-on" mode, which possessed high sensitivity features. The original strong fluorescence intensity of CuInS2 QDs was quenched by sodium hexametaphosphate (NaPO3)6. The high efficiency of the quenching was caused by the non-covalent binding of positively charged CuInS2 QDs to the negatively charged (NaPO3)6 through electrostatic interactions, aggregating to form a CuInS2 QDs/(NaPO3)6 complex. Adding acid phosphatase caused intense fluorescence of CuInS2 QDs/(NaPO3)6 to be recovered, and this was because of enzymolysis. (NaPO3)6 was hydrolyzed into small fragments and the high negative charge density decreased, which would weaken the strong electrostatic interactions. As a result, the quenched fluorescence "turned on". Under the optimum conditions, there was a good linear relationship between I/I0 (I and I0 were the fluorescence intensity of CuInS2 QDs/(NaPO3)6 system in the presence and absence of acid phosphatase, respectively) and acid phosphatase concentration in the range of 75-1500 nU mL(-1) with the detection limit of 9.02 nU mL(-1). The proposed nanosensor had been utilized to detect and accurately quantify acid phosphatase in human serum samples with satisfactory results.

  7. Highly sensitive rapid fluorescence detection of protein residues on surgical instruments

    NASA Astrophysics Data System (ADS)

    Kovalev, Valeri I.; Barton, James S.; Richardson, Patricia R.; Jones, Anita C.

    2006-02-01

    There is a risk of contamination of surgical instruments by nfectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect ~100 zeptomoles/mm2 with an area scan speed of ~20 cm2/s and for using the system to detect other agents of biomedical interest. A theoretical analysis and experimental measurements will be discussed.

  8. Highly sensitive and selective detection of Al(III) ions in aqueous buffered solution with fluorescent peptide-based sensor.

    PubMed

    In, Byunggyu; Hwang, Gi Won; Lee, Keun-Hyeung

    2016-09-15

    A fluorescent sensor based on a tripeptide (SerGluGlu) with a dansyl fluorophore detected selectively Al(III) among 16 metal ions in aqueous buffered solutions without any organic cosolvent. The peptide-based sensor showed a highly sensitive turn on response to aluminium ion with high binding affinity (1.84×10(4)M(-1)) in aqueous buffered solutions. The detection limit (230nM, 5.98ppb) of the peptide-based sensor was much lower than the maximum allowable level (7.41μM) of aluminium ions in drinking water demanded by EPA. The binding mode of the peptide sensor with aluminium ions was characterized using ESI mass spectrometry, NMR titration, and pH titration experiments.

  9. Highly sensitive colorimetric and fluorescent sensor for cyanazine based on the inner filter effect of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Dong, Liang; Hou, Changjun; Yang, Mei; Fa, Huanbao; Wu, Huixiang; Shen, Caihong; Huo, Danqun

    2016-06-01

    Cyanazine residue poses a great threat to human health and its derivatives would remain in soils, natural waters, and other environmental domains for a long time. Herein, a simple, rapid, and ultra-sensitive analytical method for the determination of cyanazine (CZ) based on inner filter effect (IFE) of Au nanoparticles (AuNPs) on the fluorescence of CdTe quantum dots (QDs) is first described in this study. With the presence of citrate-stabilized AuNPs, the fluorescence of GSH-capped CdTe QDs was remarkably quenched by AuNPs via IFE. The fluorescence of the AuNP-CdTe QD system was recovered upon addition of CZ. CZ can adsorb on to the surface of AuNPs due to its cyano group that has good affinity with gold, which could induce the aggregation of AuNPs accompanying color change from red to blue. Thus, the IFE of AuNPs on CdTe QDs was weakened, and the fluorescence intensity of CdTe QDs was recovered accordingly. A good linear correlation for detection of CZ was exhibited from 0.05 to 9 μM, and the detection limit reached 0.1568 μM, which was much lower than the safety limit required by the USA, the UK, and China. In order to probe into the selectivity of AuNPs towards CZ over other pesticides, various frequently used pesticides were mixed with AuNPs. AuNP composite solution shows good selectivity towards CZ among other pesticides. This method was successfully carried out for the assessment of CZ in real samples with satisfactory results, which revealed many advantages such as high sensitivity, low cost, and non-time-consuming compared with traditional methods.

  10. Highly sensitive and rapid bacteria detection using molecular beacon-Au nanoparticles hybrid nanoprobes.

    PubMed

    Cao, Jing; Feng, Chao; Liu, Yan; Wang, Shouyu; Liu, Fei

    2014-07-15

    Since many diseases are caused by pathogenic bacterial infections, accurate and rapid detection of pathogenic bacteria is in urgent need to timely apply appropriate treatments and to reduce economic costs. To end this, we designed molecular beacon-Au nanoparticle hybrid nanoprobes to improve the bacterial detection efficiency and sensitivity. Here, we show that the designed molecular beacon modified Au nanoparticles could specifically recognize synthetic DNAs targets and can readily detect targets in clinical samples. Moreover, the hybrid nanoprobes can recognize Escherichia coli within an hour at a concentration of 10(2) cfu/ml, which is 1000-folds sensitive than using molecular beacon directly. Our results show that the molecular beacon-Au nanoparticle hybrid nanoprobes have great potential in medical and biological applications.

  11. M-DNA/Transition Metal Dichalcogenide Hybrid Structure-based Bio-FET sensor with Ultra-high Sensitivity

    NASA Astrophysics Data System (ADS)

    Park, Hyung-Youl; Dugasani, Sreekantha Reddy; Kang, Dong-Ho; Yoo, Gwangwe; Kim, Jinok; Gnapareddy, Bramaramba; Jeon, Jaeho; Kim, Minwoo; Song, Young Jae; Lee, Sungjoo; Heo, Jonggon; Jeon, Young Jin; Park, Sung Ha; Park, Jin-Hong

    2016-10-01

    Here, we report a high performance biosensor based on (i) a Cu2+-DNA/MoS2 hybrid structure and (ii) a field effect transistor, which we refer to as a bio-FET, presenting a high sensitivity of 1.7 × 103 A/A. This high sensitivity was achieved by using a DNA nanostructure with copper ions (Cu2+) that induced a positive polarity in the DNA (receptor). This strategy improved the detecting ability for doxorubicin-like molecules (target) that have a negative polarity. Very short distance between the biomolecules and the sensor surface was obtained without using a dielectric layer, contributing to the high sensitivity. We first investigated the effect of doxorubicin on DNA/MoS2 and Cu2+-DNA/MoS2 nanostructures using Raman spectroscopy and Kelvin force probe microscopy. Then, we analyzed the sensing mechanism and performance in DNA/MoS2- and Cu2+-DNA/MoS2-based bio-FETs by electrical measurements (ID-VG at various VD) for various concentrations of doxorubicin. Finally, successful operation of the Cu2+-DNA/MoS2 bio-FET was demonstrated for six cycles (each cycle consisted of four steps: 2 preparation steps, a sensing step, and an erasing step) with different doxorubicin concentrations. The bio-FET showed excellent reusability, which has not been achieved previously in 2D biosensors.

  12. M-DNA/Transition Metal Dichalcogenide Hybrid Structure-based Bio-FET sensor with Ultra-high Sensitivity

    PubMed Central

    Park, Hyung-Youl; Dugasani, Sreekantha Reddy; Kang, Dong-Ho; Yoo, Gwangwe; Kim, Jinok; Gnapareddy, Bramaramba; Jeon, Jaeho; Kim, Minwoo; Song, Young Jae; Lee, Sungjoo; Heo, Jonggon; Jeon, Young Jin; Park, Sung Ha; Park, Jin-Hong

    2016-01-01

    Here, we report a high performance biosensor based on (i) a Cu2+-DNA/MoS2 hybrid structure and (ii) a field effect transistor, which we refer to as a bio-FET, presenting a high sensitivity of 1.7 × 103 A/A. This high sensitivity was achieved by using a DNA nanostructure with copper ions (Cu2+) that induced a positive polarity in the DNA (receptor). This strategy improved the detecting ability for doxorubicin-like molecules (target) that have a negative polarity. Very short distance between the biomolecules and the sensor surface was obtained without using a dielectric layer, contributing to the high sensitivity. We first investigated the effect of doxorubicin on DNA/MoS2 and Cu2+-DNA/MoS2 nanostructures using Raman spectroscopy and Kelvin force probe microscopy. Then, we analyzed the sensing mechanism and performance in DNA/MoS2- and Cu2+-DNA/MoS2-based bio-FETs by electrical measurements (ID-VG at various VD) for various concentrations of doxorubicin. Finally, successful operation of the Cu2+-DNA/MoS2 bio-FET was demonstrated for six cycles (each cycle consisted of four steps: 2 preparation steps, a sensing step, and an erasing step) with different doxorubicin concentrations. The bio-FET showed excellent reusability, which has not been achieved previously in 2D biosensors. PMID:27775004

  13. HistoFlex--a microfluidic device providing uniform flow conditions enabling highly sensitive, reproducible and quantitative in situ hybridizations.

    PubMed

    Søe, Martin Jensen; Okkels, Fridolin; Sabourin, David; Alberti, Massimo; Holmstrøm, Kim; Dufva, Martin

    2011-11-21

    A microfluidic device (the HistoFlex) designed to perform and monitor molecular biological assays under dynamic flow conditions on microscope slide-substrates, with special emphasis on analyzing histological tissue sections, is presented. Microscope slides were reversibly sealed onto a cast polydimethylsiloxane (PDMS) insert, patterned with distribution channels and reaction chambers. Topology optimization was used to design reaction chambers with uniform flow conditions. The HistoFlex provided uniform hybridization conditions, across the reaction chamber, as determined by hybridization to microscope slides of spotted DNA microarrays when applying probe concentrations generally used in in situ hybridization (ISH) assays. The HistoFlex's novel ability in online monitoring of an in situ hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Tissue sections were not visually damaged during assaying, which enabled adapting a complete ISH assay for detection of microRNAs (miRNA). The effects of flow based incubations on hybridization, antibody incubation and Tyramide Signal Amplification (TSA) steps were investigated upon adapting the ISH assay for performing in the HistoFlex. The hybridization step was significantly enhanced using flow based incubations due to improved hybridization efficiency. The HistoFlex device enabled a fast miRNA ISH assay (3 hours) which provided higher hybridization signal intensity compared to using conventional techniques (5 h 40 min). We further demonstrate that the improved hybridization efficiency using the HistoFlex permits more complex assays e.g. those comprising sequential hybridization and detection of two miRNAs to be performed with significantly increased sensitivity. The HistoFlex provides a new histological analysis platform that will allow multiple and sequential assays to be performed under their individual optimum assay conditions. Images can subsequently be recorded either in

  14. High-sensitivity determination of Zn(II) and Cu(II) in vitro by fluorescence polarization

    NASA Astrophysics Data System (ADS)

    Thompson, Richard B.; Maliwal, Badri P.; Feliccia, Vincent; Fierke, Carol A.

    1998-04-01

    Recent work has suggested that free Cu(II) may play a role in syndromes such as Crohn's and Wilson's diseases, as well as being a pollutant toxic at low levels to shellfish and sheep. Similarly, Zn(II) has been implicated in some neural damage in the brain resulting from epilepsy and ischemia. Several high sensitivity methods exist for determining these ions in solution, including GFAAS, ICP-MS, ICP-ES, and electrochemical techniques. However, these techniques are generally slow and costly, require pretreatment of the sample, require complex instruments and skilled personnel, and are incapable of imaging at the cellular and subcellular level. To address these shortcomings we developed fluorescence polarization (anisotropy) biosensing methods for these ions which are very sensitivity, highly selective, require simple instrumentation and little pretreatment, and are inexpensive. Thus free Cu(II) or Zn(II) can be determined at picomolar levels by changes in fluorescence polarization, lifetime, or wavelength ratio using these methods; these techniques may be adapted to microscopy.

  15. A practical and highly sensitive C3N4-TYR fluorescent probe for convenient detection of dopamine

    NASA Astrophysics Data System (ADS)

    Li, Hao; Yang, Manman; Liu, Juan; Zhang, Yalin; Yang, Yanmei; Huang, Hui; Liu, Yang; Kang, Zhenhui

    2015-07-01

    The C3N4-tyrosinase (TYR) hybrid is a highly accurate, sensitive and simple fluorescent probe for the detection of dopamine (DOPA). Under optimized conditions, the relative fluorescence intensity of C3N4-TYR is proportional to the DOPA concentration in the range from 1 × 10-3 to 3 × 10-8 mol L-1 with a correlation coefficient of 0.995. In the present system, the detection limit achieved is as low as 3 × 10-8 mol L-1. Notably, these quantitative detection results for clinical samples are comparable to those of high performance liquid chromatography. Moreover, the enzyme-encapsulated C3N4 sensing arrays on both glass slide and test paper were evaluated, which revealed sensitive detection and excellent stability. The results reported here provide a new approach for the design of a multifunctional nanosensor for the detection of bio-molecules.The C3N4-tyrosinase (TYR) hybrid is a highly accurate, sensitive and simple fluorescent probe for the detection of dopamine (DOPA). Under optimized conditions, the relative fluorescence intensity of C3N4-TYR is proportional to the DOPA concentration in the range from 1 × 10-3 to 3 × 10-8 mol L-1 with a correlation coefficient of 0.995. In the present system, the detection limit achieved is as low as 3 × 10-8 mol L-1. Notably, these quantitative detection results for clinical samples are comparable to those of high performance liquid chromatography. Moreover, the enzyme-encapsulated C3N4 sensing arrays on both glass slide and test paper were evaluated, which revealed sensitive detection and excellent stability. The results reported here provide a new approach for the design of a multifunctional nanosensor for the detection of bio-molecules. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr03316k

  16. Highly sensitive DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization.

    PubMed

    Yu, Xu; Zhang, Zhi-Ling; Zheng, Si-Yang

    2015-04-15

    A novel highly sensitive colorimetric assay for DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization was established. The DNA modified superparamagnetic beads were demonstrated to capture and enrich the target DNA in the hybridization buffer or human plasma. The hybridization chain reaction and enzyme-induced silver metallization on the gold nanoparticles were used as cascade signal amplification for the detection of target DNA. The metalization of silver on the gold nanoparticles induced a significant color change from red to yellow until black depending on the concentration of the target DNA, which could be recognized by naked eyes. This method showed a good specificity for the target DNA detection, with the capabilty to discriminate single-base-pair mismatched DNA mutation (single nucleotide polymorphism). Meanwhile, this approach exhibited an excellent anti-interference capability with the convenience of the magentic seperation and washing, which enabled its usage in complex biological systems such as human blood plasma. As an added benefit, the utilization of hybridization chain reaction and enzyme-induced metallization improved detection sensitivity down to 10pM, which is about 100-fold lower than that of traditional unamplified homogeneous assays.

  17. Tungsten disulfide nanosheet and exonuclease III co-assisted amplification strategy for highly sensitive fluorescence polarization detection of DNA glycosylase activity.

    PubMed

    Zhao, Jingjin; Ma, Yefei; Kong, Rongmei; Zhang, Liangliang; Yang, Wen; Zhao, Shulin

    2015-08-05

    Herein, we introduced a tungsten disulfide (WS2) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity. Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as the substrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3'-terminal of DP. This design facilitated the Exo III-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS2 nanosheet. Thus, the final system exhibited a small FP signal. However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS2 nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, this method was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics.

  18. Synergistic Effect of Hybrid Multilayer In2Se3 and Nanodiamonds for Highly Sensitive Photodetectors.

    PubMed

    Zheng, Zhaoqiang; Yao, Jiandong; Xiao, Jun; Yang, Guowei

    2016-08-10

    Layered materials have rapidly established themselves as intriguing building blocks for next-generation photodetection platforms in view of their exotic electronic and optical attributes. However, both relatively low mobility and heavier electron effective mass limit layered materials for high-performance applications. Herein, we employed nanodiamonds (NDs) to promote the performance of multilayer In2Se3 photodetectors for the first time. This hybrid NDs-In2Se3 photodetector showed a tremendous promotion of photodetection performance in comparison to pristine In2Se3 ones. This hybrid devices exhibited remarkable detectivity (5.12 × 10(12) jones), fast response speed (less than 16.6 ms), and decent current on/off ratio (∼2285) simultaneously. These parameters are superior to most reported layered materials based photodetectors and even comparable to the state-of-the-art commercial photodetectors. Meanwhile, we attributed this excellent performance to the synergistic effect between NDs and the In2Se3. They can greatly enhance the broad spectrum absorption and promote the injection of photoexcited carrier in NDs to In2Se3. These results actually open up a new scenario for designing and fabricating innovative optoelectronic systems.

  19. An aptamer-based effective method for highly sensitive detection of chloramphenicol residues in animal-sourced food using real-time fluorescent quantitative PCR.

    PubMed

    Duan, Ye; Wang, Lihui; Gao, Zhiqiang; Wang, Huishan; Zhang, Hexiao; Li, Hao

    2017-04-01

    Chloramphenicol (CAP) residues can not only harm human health through entering food chain, but also cause the spreading of drug-resistant bacteria, thereby leading to secondary environmental pollution. Therefore, it is in urgent need of establishing an efficient technology to detect CAP residues in animal-sourced food. In this study, a novel sensitive approach for detection of CAP was designed based on a CAP specific aptamer and real-time fluorescent quantitative PCR (qRT-PCR). The CAP specific aptamer was firstly hybridized with a biotin modified complementary probe, and then was immobilized on streptavidin conjugated magnetic beads through biotin. When CAP was added, the aptamer would specifically bind with CAP by forming a hairpin structure and be released from the magnetic beads for CAP detection by qRT-PCR. Factors (i.e., probe strand length, aptamer concentration, NaCl concentration and incubation time) that would influence the determination accuracy of this aptamer-based detection system were optimized. Under the optimized conditions, the present detection system exhibited a high sensitivity toward CAP with a limit of detection of 0.1ng/mL (linear range from 0.1 to 20ng/mL). Moreover, this detection system also showed high selectivity against thiamphenicol (TAP) and florfenicol (FF), which are CAP's structure analogs. Eventually, this detection system was applied for detecting CAP in real spiked milk. The recovery rate of CAP from spiked milk samples ranged from 94.0-102.0%. These results indicated this developed detection system a promising high sensitive and specific method of CAP residues detection in animal-sourced food.

  20. Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families.

    PubMed

    Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M; Kaczmarek, Leszek; Salvesen, Guy S; Drag, Marcin

    2017-02-23

    Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid.

  1. Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

    PubMed Central

    Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J.; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M.; Kaczmarek, Leszek; Salvesen, Guy S.; Drag, Marcin

    2017-01-01

    Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. PMID:28230157

  2. Bioelectrochemistry of heme peptide at seamless three-dimensional carbon nanotubes/graphene hybrid films for highly sensitive electrochemical biosensing.

    PubMed

    Komori, Kikuo; Terse-Thakoor, Trupti; Mulchandani, Ashok

    2015-02-18

    A seamless three-dimensional hybrid film consisting of carbon nanotubes grown at the graphene surface (CNTs/G) is a promising material for the application to highly sensitive enzyme-based electrochemical biosensors. The CNTs/G film was used as a conductive nanoscaffold for enzymes. The heme peptide (HP) was immobilized on the surface of the CNTs/G film for amperometric sensing of H2O2. Compared with flat graphene electrodes modified with HP, the catalytic current for H2O2 reduction at the HP-modified CNTs/G electrode increased due to the increase in the surface coverage of HP. In addition, microvoids in the CNTs/G film contributed to diffusion of H2O2 to modified HP, resulting in the enhancement of the catalytic cathodic currents. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HP was also analyzed.

  3. A method for conducting highly sensitive microRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer.

    PubMed

    Sempere, Lorenzo F; Korc, Murray

    2013-01-01

    Profiling experiments in whole tissue biopsies have linked altered expression of microRNAs (miRNAs) to different types of cancer, including pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that altered miRNA expression can occur in different cellular compartments (cancer and non-cancer cells) in tumor lesions, and thus it is important to ascertain which specific cell type expresses a particulars miRNA in PDAC tissues. Here, we describe a highly sensitive fluorescence-based ISH method to visualize miRNA accumulation within individual cells in formalin-fixed paraffin-embedded (FFPE) tissue sections using 5' and 3' terminally fluorescein-labeled locked nucleic acid (LNA)-modified probes. We describe a multicolor ISH/IHC method based on sequential rounds of horseradish peroxidase (HRP)-mediated tyramide signal amplification (TSA) reactions with different in-house synthesized fluorochrome-conjugated substrates that enable co-detection of miRNAs, abundant noncoding RNAs and protein markers for signal quantification, and cell type co-localization studies in FFPE pancreatic tissue sections from clinical specimens and mouse models of PDAC.

  4. Fluorescence-guided tumor visualization using a custom designed NIR attachment to a surgical microscope for high sensitivity imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kittle, David S.; Patil, Chirag G.; Mamelak, Adam; Hansen, Stacey; Perry, Jeff; Ishak, Laura; Black, Keith L.; Butte, Pramod V.

    2016-03-01

    Current surgical microscopes are limited in sensitivity for NIR fluorescence. Recent developments in tumor markers attached with NIR dyes require newer, more sensitive imaging systems with high resolution to guide surgical resection. We report on a small, single camera solution enabling advanced image processing opportunities previously unavailable for ultra-high sensitivity imaging of these agents. The system captures both visible reflectance and NIR fluorescence at 300 fps while displaying full HD resolution video at 60 fps. The camera head has been designed to easily mount onto the Zeiss Pentero microscope head for seamless integration into surgical procedures.

  5. One-pot synthesis of mesoporous structured ratiometric fluorescence molecularly imprinted sensor for highly sensitive detection of melamine from milk samples.

    PubMed

    Xu, Shoufang; Lu, Hongzhi

    2015-11-15

    A facile strategy was developed to prepare mesoporous structured ratiometric fluorescence molecularly imprinted sensor for highly sensitive and selective determination of melamine using CdTe QDs as target sensitive dye and hematoporphyrin as reference dyes. One-pot synthesis method was employed because it could simplify the imprinting process and shorten the experimental period. The as-prepared fluorescence MIPs sensor, which combined ratiometric fluorescence technique with mesoporous silica materials into one system, exhibited excellent selectivity and sensitivity. Under optimum conditions, these mesoporous structured ratiometric fluorescence MIP@QDs sensors showed detection limit as low as 38 nM, which was much lower than those non-mesoporous one. The recycling process was sustainable at least 10 times without obvious efficiency decrease. The feasibility of the developed method in real samples was successfully evaluated through the analysis of melamine in raw milk and milk powder samples with satisfactory recoveries of 92-101%. The developed method proposed in this work proved to be a convenient, rapid, reliable and practical way to prepared high sensitive and selective fluorescence sensors with potentially applicable for trace pollutants analysis in complicated samples.

  6. Amplified electrochemiluminescence of luminol based on hybridization chain reaction and in situ generate co-reactant for highly sensitive immunoassay.

    PubMed

    Xiao, Lijuan; Chai, Yaqin; Yuan, Ruo; Cao, Yaling; Wang, Haijun; Bai, Lijuan

    2013-10-15

    In this work, we described a simple and highly sensitive electrochemiluminescence (ECL) strategy for IgG detection. Firstly, L-cysteine functionalized reduced graphene oxide composite (L-cys-rGO) was decorated on the glassy carbon electrode (GCE) surface. Then anti-IgG was immobilized on the modified electrode surface through the interaction between the carboxylic groups of the L-cys-rGO and the amine groups in anti-IgG. And then biotinylated anti-IgG (bio-anti-IgG) was assembled onto the electrode surface based on the sandwich-type immunoreactions. By the conjunction of biotin and streptavidin (SA), SA was immobilized, which in turn, combined with the biotin labeled initiator strand (S1). In the presence of two single DNA strands of glucose oxidase labeled S2 (GOD-S2) and complementary strand (S3), S1 could trigger the hybridization chain reaction (HCR) among S1, GOD-S2 and S3. Herein, due to HCR, numerous GOD was efficiently immobilizated on the sensing surface and exhibited excellent catalysis towards glucose to in situ generate amounts of hydrogen peroxide (H2O2), which acted as luminol's co-reactant to significantly enhance the ECL signal. The proposed ECL immunosensor presented predominate stability and high sensibility for determination of IgG in the range from 0.1 pg mL(-1) to 100 ng mL(-1) with a detection limit of 33 fg mL(-1) (S/N=3). Additionally, the designed ECL immunosensor exhibited a promising application for other protein detection.

  7. Molecular cytogenetics using fluorescence in situ hybridization

    SciTech Connect

    Gray, J.W.; Kuo, Wen-Lin; Lucas, J.; Pinkel, D.; Weier, H-U.; Yu, Loh-Chung.

    1990-12-07

    Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.

  8. Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.

    PubMed

    Wellinghausen, Nele; Wirths, Beate; Poppert, Sven

    2006-09-01

    Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients.

  9. "Turn-off" fluorescent sensor for highly sensitive and specific simultaneous recognition of 29 famous green teas based on quantum dots combined with chemometrics.

    PubMed

    Liu, Li; Fan, Yao; Fu, Haiyan; Chen, Feng; Ni, Chuang; Wang, Jinxing; Yin, Qiaobo; Mu, Qingling; Yang, Tianming; She, Yuanbin

    2017-04-22

    Fluorescent "turn-off" sensors based on water-soluble quantum dots (QDs) have drawn increasing attention owing to their unique properties such as high fluorescence quantum yields, chemical stability and low toxicity. In this work, a novel method based on the fluorescence "turn-off" model with water-soluble CdTe QDs as the fluorescent probes for differentiation of 29 different famous green teas is established. The fluorescence of the QDs can be quenched in different degrees in light of positions and intensities of the fluorescent peaks for the green teas. Subsequently, with aid of classic partial least square discriminant analysis (PLSDA), all the green teas can be discriminated with high sensitivity, specificity and a satisfactory recognition rate of 100% for training set and 98.3% for prediction set, respectively. Especially, the "turn-off" fluorescence PLSDA model based on second-order derivatives (2nd der) with reduced least complexity (LVs = 3) was the most effective one for modeling. Most importantly, we further demonstrated the established "turn-off" fluorescent sensor mode has several significant advantages and appealing properties over the conventional fluorescent method for large-class-number classification (LCNC) of green teas. This work is, to the best of our knowledge, the first report on the rapid and effective identification of so many kinds of famous green teas based on the "turn-off" model of QDs combined with chemometrics, which also implies other potential applications on complex LCNC classification system with weak fluorescence or even without fluorescence to achieve higher detective response and specificity.

  10. A ratiometric fluorescent nanoprobe based on terbium functionalized carbon dots for highly sensitive detection of an anthrax biomarker.

    PubMed

    Chen, Hao; Xie, Yujie; Kirillov, Alexander M; Liu, Liangliang; Yu, Minghui; Liu, Weisheng; Tang, Yu

    2015-03-25

    A ratiometric fluorescent nanoprobe based on terbium functionalized carbon dots (CDs) was designed to detect dipicolinic acid (DPA) as an anthrax biomarker with high selectivity and sensitivity. CDs were generated by one-step synthesis using an ethylenediaminetetraacetic acid precursor, and served as a scaffold for coordination with Tb(3+) and a fluorescence reference.

  11. Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

    PubMed

    Marras, Salvatore A E

    2008-03-01

    The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

  12. A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2.

    PubMed

    Xi, Gaina; Wang, Xiaoping; Chen, Tongsheng

    2016-01-01

    A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02-0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe.

  13. A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2

    PubMed Central

    Xi, Gaina; Wang, Xiaoping; Chen, Tongsheng

    2016-01-01

    A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02–0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe. PMID:27143876

  14. Molecular switching fluorescence based high sensitive detection of label-free C-reactive protein on biochip.

    PubMed

    Islam, Md Shahinul; Yu, Hyunung; Lee, Hee Gu; Kang, Seong Ho

    2010-11-15

    A novel detection technique on biochip for the quantification of label-free C-reactive protein (CRP) based on molecular switching of fluorescence (MSF) is demonstrated by total internal reflection fluorescence microscopy. It alters fluorescence intensity of fluoreseinamine isomer 1 (FAI) upon binding with its specific ligand, O-phosphorylethanolamine (PEA). In the MSF-based detection, FAI was used as an ink, printed on a 3-glycidoxypropyl-trimethoxysilane (GPTS)-coated glass coverslip. With the addition of GPTS conjugated PEA solution to the FAI-printed coverslip, the fluorescence intensity was remarkably decreased. Addition of CRP increased fluorescence intensity linearly in the range of 800 aM to 500 fM (R=0.997). The MSF-based biochip assay for the estimation of CRP in human sera showed ∼200 times increased detection sensitivity in less than a third of the time to obtain results using a conventional enzyme-linked immunosorbent assay. This biochip detection is a promising new technique for the quantification of CRP molecules from trace amounts of clinical samples.

  15. Magic sized ZnS quantum dots as a highly sensitive and selective fluorescence sensor probe for Ag+ ions.

    PubMed

    Mandal, Abhijit; Dandapat, Anirban; De, Goutam

    2012-02-07

    A green and simple chemical synthesis of magic sized water soluble blue-emitting ZnS quantum dots (QDs) has been accomplished by reacting anhydrous Zn acetate, sodium sulfide and thiolactic acid (TLA) at room temperature in aqueous solution. Refluxing of this mixture in open air yielded ZnS clusters of about 3.5 nm in diameter showing very strong and narrow photoluminescence properties with long stability. Refluxing did not cause any noticeable size increment of the clusters. As a result, the QDs obtained after different refluxing conditions showed similar absorption and photoluminescence (PL) features. Use of TLA as a capping agent effectively yielded such stable and magic sized QDs. The as-synthesized and 0.5 h refluxed ZnS QDs were used as a fluorescence sensor for Ag(+) ions. It has been observed that after addition of Ag(+) ions of concentration 0.5-1 μM the strong fluorescence of ZnS QDs was almost quenched. The quenched fluorescence can be recovered by adding ethylenediamine to form a complex with Ag(+) ions. The other metal ions (K(+), Ca(2+), Au(3+), Cu(2+), Fe(3+), Mn(2+), Mg(2+), Co(2+)) showed little or no effect on the fluorescence of ZnS QDs when tested individually or as a mixture. In the presence of all these ions, Ag(+) responded well and therefore ZnS QDs reported in this work can be used as a Ag(+) ion fluorescence sensor.

  16. Rhodamine 6G hydrazone bearing thiophene unit: A highly sensitive and selective off-on fluorescent chemosensor for Al3+

    NASA Astrophysics Data System (ADS)

    Wu, Wei-Na; Mao, Pan-Dong; Wang, Yuan; Zhao, Xiao-Lei; Jia, Lei; Xu, Zhou-Qing

    2016-10-01

    A rhodamine derivative (R1) has been synthesized by a hydrazone formation of rhodamine 6G hydrazide with 3-methylthiophene-2-carbaldehyde, which exhibits high selectivity and sensitivity as an "off-on" fluorescent sensor toward Al3+ in water containing media. The binding process was confirmed by UV-vis absorption, fluorescence measurements, mass spectroscopy and DFT calculation. The probe functions by Al3+ induced hydrolytic cleavage of the imine-bond to produce an intense rhodamine-based emission. To test the practical use of the probe, the determination of Al3+ in real water samples was also evaluated.

  17. Supramolecular spectrally encoded microgels with double strand probes for absolute and direct miRNA fluorescence detection at high sensitivity.

    PubMed

    Causa, Filippo; Aliberti, Anna; Cusano, Angela M; Battista, Edmondo; Netti, Paolo A

    2015-02-11

    We present novel microgels as a particle-based suspension array for direct and absolute microRNA (miRNA) detection. The microgels feature a flexible molecular architecture, antifouling properties, and enhanced sensitivity with a large dynamic range of detection. Specifically, they possess a core-shell molecular architecture with two different fluorescent dyes for multiplex spectral analyses and are endowed with a fluorescent probe for miRNA detection. Encoding and detection fluorescence signals are distinguishable by nonoverlapping emission spectra. Tunable fluorescence probe conjugation and emission confinement on single microgels allow for ultrasensitive miRNA detection. Indeed, the suspension array has high selectivity and sensitivity with absolute quantification, a detection limit of 10(-15) M, a dynamic range from 10(-9) to 10(-15) M, and higher accuracy than qRT-PCR. The antifouling properties of the microgels also permit the direct measurement of miRNAs in serum, without sample pretreatment or target amplification. A multiplexed assay has been tested for a set of miRNAs chosen as cancer biomarkers.

  18. Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Nallathamby, Prakash D.; Foster, Carmen M.; Morrell-Falvey, Jennifer L.; Mortensen, Ninell P.; Doktycz, Mitchel J.; Gu, Baohua; Retterer, Scott T.

    2013-10-01

    A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2

  19. Volume Labeling with Alexa-Fluor Dyes and Surface Functionalization of Highly Sensitive Fluorescent SiO2 Nanoparticles

    SciTech Connect

    Wang, Wei; Foster, Carmen M; Morrell-Falvey, Jennifer L; Nallathamby, Prakash D; Mortensen, Ninell P; Doktycz, Mitchel John; Gu, Baohua; Retterer, Scott T; Gu, Baohua

    2013-01-01

    A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or free surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.

  20. Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles.

    PubMed

    Wang, Wei; Nallathamby, Prakash D; Foster, Carmen M; Morrell-Falvey, Jennifer L; Mortensen, Ninell P; Doktycz, Mitchel J; Gu, Baohua; Retterer, Scott T

    2013-11-07

    A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or "free" surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.

  1. A comparison of planar, laser-induced fluorescence, and high-sensitivity interferometry techniques for gas-puff nozzle density measurements

    SciTech Connect

    Jackson, S. L.; Weber, B. V.; Mosher, D.; Phipps, D. G.; Stephanakis, S. J.; Commisso, R. J.; Qi, N.; Failor, B. H.; Coleman, P. L.

    2008-10-15

    The distribution of argon gas injected by a 12-cm-diameter triple-shell nozzle was characterized using both planar, laser-induced fluorescence (PLIF) and high-sensitivity interferometry. PLIF is used to measure the density distribution at a given time by detecting fluorescence from an acetone tracer added to the gas. Interferometry involves making time-dependent, line-integrated gas density measurements at a series of chordal locations that are then Abel inverted to obtain the gas density distribution. Measurements were made on nominally identical nozzles later used for gas-puff Z-pinch experiments on the Saturn pulsed-power generator. Significant differences in the mass distributions obtained by the two techniques are presented and discussed, along with the strengths and weaknesses of each method.

  2. Highly sensitive dual-mode fluorescence detection of lead ion in water using aggregation-induced emissive polymers.

    PubMed

    Saha, Sukanta Kumar; Ghosh, Khama Rani; Gao, Jian Ping; Wang, Zhi Yuan

    2014-09-01

    A series of fluorene-based conjugated polymers containing the aggregation-induced emissive (AIE)-active tetraphenylethene and dicarboxylate pseudocrown as a receptor exhibits a unique dual-mode sensing ability for selective detection of lead ion in water. Fluorescence turn-off and turn-on detections are realized in 80%-90% and 20% water in tetrahydrofuran (THF), respectively, for lead ion with a concentration as low as 10(-8) m.

  3. A highly sensitive and selective fluorescent sensor for detection of Al(3+) using a europium(III) quinolinecarboxylate.

    PubMed

    Xu, Wentao; Zhou, Youfu; Huang, Decai; Su, Mingyi; Wang, Kun; Hong, Maochun

    2014-07-07

    Eu2PQC6 has been developed to detect Al(3+) by monitoring the quenching of the europium-based emission, with the lowest detection limit of ∼32 pM and the quantitative detection range to 150 μM. Eu2PQC6 is the first ever example that the europium(III) complex serves as an Al(3+) fluorescent sensor based on "competition-displacement" mode.

  4. A highly sensitive fluorescent chemosensor for selective detection of zinc (II) ion based on the oxadiazole derivative

    NASA Astrophysics Data System (ADS)

    Lin, Lu; Wang, Dan; Chen, Si-Hong; Wang, Dun-Jia; Yin, Guo-Dong

    2017-03-01

    A novel fluorescent chemosensor based on the oxadiazole, 2-(2-hydroxyphenyl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole, was designed and synthesized. The interaction of the oxadiazole with different metal ions had been investigated through UV-vis absorption and fluorescence spectra in 9:1 (v/v) ethanol-water (pH = 7.0) solution. The oxadiazole showed a pronounced fluorescence enhancement at 430 nm upon addition of Zn2 + in aqueous solution, whereas it had no apparent interference from other metal ions. The results indicated that the oxadiazole possessed high selectivity and sensitivity to Zn2 + ion. The stoichiometric ratio between the oxadiazole and Zn2 + ion was calculated to be 2:1 by Job plot experiment, meanwhile their binding modes was confirmed by 1H NMR and mass spectrometry. Their association constant was determined to be 1.95 × 105 M- 1 and the detection limit for Zn2 + ion was 6.14 × 10- 7 mol/L.

  5. TU-G-207-03: High Spatial Resolution and High Sensitivity X-Ray Fluorescence Imaging

    SciTech Connect

    Xing, L.

    2015-06-15

    Last few years has witnessed the development of novel of X-ray imaging modalities, such as spectral CT, phase contrast CT, and X-ray acoustic/fluorescence/luminescence imaging. This symposium will present the recent advances of these emerging X-ray imaging modalities and update the attendees with knowledge in various related topics, including X-ray photon-counting detectors, X-ray physics underlying the emerging applications beyond the traditional X-ray imaging, image reconstruction for the novel modalities, characterization and evaluation of the systems, and their practical implications. In addition, the concept and practical aspects of X-ray activatable targeted nanoparticles for molecular X-ray imaging will be discussed in the context of X-ray fluorescence and luminescence CT. Learning Objectives: Present background knowledge of various emerging X-ray imaging techniques, such as spectral CT, phase contrast CT and X-ray fluorescence/luminescence CT. Discuss the practical need, technical aspects and current status of the emerging X-ray imaging modalities. Describe utility and future impact of the new generation of X-ray imaging applications.

  6. A novel lab-on-chip platform with integrated solid phase PCR and Supercritical Angle Fluorescence (SAF) microlens array for highly sensitive and multiplexed pathogen detection.

    PubMed

    Hung, Tran Quang; Chin, Wai Hoe; Sun, Yi; Wolff, Anders; Bang, Dang Duong

    2017-04-15

    Solid-phase PCR (SP-PCR) has become increasingly popular for molecular diagnosis and there have been a few attempts to incorporate SP-PCR into lab-on-a-chip (LOC) devices. However, their applicability for on-line diagnosis is hindered by the lack of sensitive and portable on-chip optical detection technology. In this paper, we addressed this challenge by combining the SP-PCR with super critical angle fluorescence (SAF) microlens array embedded in a microchip. We fabricated miniaturized SAF microlens array as part of a microfluidic chamber in thermoplastic material and performed multiplexed SP-PCR directly on top of the SAF microlens array. Attribute to the high fluorescence collection efficiency of the SAF microlens array, the SP-PCR assay on the LOC platform demonstrated a high sensitivity of 1.6 copies/µL, comparable to off-chip detection using conventional laser scanner. The combination of SP-PCR and SAF microlens array allows for on-chip highly sensitive and multiplexed pathogen detection with low-cost and compact optical components. The LOC platform would be widely used as a high-throughput biosensor to analyze food, clinical and environmental samples.

  7. Rhodamine 6G conjugated-quantum dots used for highly sensitive and selective ratiometric fluorescence sensor of glutathione.

    PubMed

    Gui, Rijun; An, Xueqin; Su, Hongjuan; Shen, Weiguo; Zhu, Linyong; Ma, Xingyuan; Chen, Zhiyun; Wang, Xiaoyong

    2012-05-30

    Rhodamine 6G (R6G) and 3-mercaptopropionic acid (MPA) capped-CdTe quantum dots (QDs) were conjugated by electrostatic interactions in aqueous solution. The R6G-QDs conjugate was utilized as a photoluminescence (PL) ratiometric sensor for the detection of glutathione (GSH). In this method, intentional introduction of GSH destroyed the conjugation of R6G and QDs, and induced regular PL change of R6G-QDs conjugates due to the competitive chelation between GSH and MPA ligand on the surface of QDs. The ratio of PL intensity of R6G (I(R6G)) to that of QDs (I(QDs)) in this conjugate was near linear toward the concentration of GSH in the range from 0.05 to 80 μM, and corresponding regression equation showed a good linear coefficient of 0.9954. The limit of detection of 15 nM in this proposed method was about 40-fold lower than that of other QDs-based PL sensors. Interferential experiments testified that R6G-QDs conjugates-based ratiometric PL sensor of GSH showed high selectivity over other related thiols and amino acids. Real sample assays further verified perfect analysis performance of the PL sensor of GSH. In comparison with conventional analytical techniques for the measurement of GSH, this ratiometric PL sensor was facile, economic, highly sensitive and selective.

  8. A high-sensitive and fast-fabricated glucose biosensor based on Prussian blue/topological insulator Bi2Se3 hybrid film.

    PubMed

    Wu, Shouguo; Liu, Gang; Li, Ping; Liu, Hao; Xu, Haihong

    2012-01-01

    A novel and fast-fabricated Prussian blue (PB)/topological insulator Bi(2)Se(3) hybrid film has been prepared by coelectrodeposition technique. Taking advantages of topological insulator in possessing exotic metallic surface states with bulk insulating gap, Prussian blue nanoparticles in the hybrid film have smaller size as well as more compact structure, showing excellent pH stability even in the alkalescent solution of pH 8.0. Based on the Laviron theory, the electron transfer rate constant of PB/Bi(2)Se(3) hybrid film modified electrode was calculated to be 4.05 ± 0.49 s(-1), a relatively big value which may be in favor of establishing a high-sensitive biosensor. An amperometric glucose biosensor was then fabricated by immobilizing glucose oxidase (GOD) on the hybrid film. Under the optimal conditions, a wide linear range extending over 3 orders of magnitude of glucose concentrations (1.0 × 10(-5)-1.1 × 10(-2)M) was obtained with a high sensitivity of 24.55 μA mM(-1) cm(-2). The detection limit was estimated for 3.8 μM defined from a signal/noise of 3. Furthermore, the resulting biosensor was applied to detect the blood sugar in human serum samples without any pretreatment, and the results were comparatively in agreement with the clinical assay.

  9. Fluorescent-dye-doped sol-gel sensor for highly sensitive carbon dioxide gas detection below atmospheric concentrations.

    PubMed

    Dansby-Sparks, Royce N; Jin, Jun; Mechery, Shelly J; Sampathkumaran, Uma; Owen, Thomas William; Yu, Bi Dan; Goswami, Kisholoy; Hong, Kunlun; Grant, Joseph; Xue, Zi-Ling

    2010-01-15

    Optical fluorescence sol-gel sensors have been developed for the detection of carbon dioxide gas in the 0.03-30% range with a detection limit of 0.008% (or 80 ppm) and a quantitation limit of 0.02% (or 200 ppm) CO(2). Sol-gels were spin-coated on glass slides to create an organically modified silica-doped matrix with the 1-hydroxypyrene-3,6,8-trisulfonate (HPTS) fluorescent indicator. The luminescence intensity of the HPTS indicator (513 nm) is quenched by CO(2), which protonates the anionic form of HPTS. An ion pair technique was used to incorporate the lipophilic dye into the hydrophilic sol-gel matrix. TiO(2) particles (<5 microm diameter) were added to induce Mie scattering and increase the incident light interaction with the sensing film, thus increasing the signal-to-noise ratio. Moisture-proof overcoatings have been used to maintain a constant level of water inside the sensor films. The optical sensors are inexpensive to prepare and can be easily coupled to fiber optics for remote sensing capabilities. A fiber-optic bundle was used for the gas detection and shown to work as part of a multianalyte platform for simultaneous detection of multiple analytes. The studies reported here resulted in the development of sol-gel optical fluorescent sensors for CO(2) gas with sensitivity below that in the atmosphere (ca. 387 ppm). These sensors are a complementary approach to current FT-IR measurements for real-time carbon dioxide detection in environmental applications.

  10. Fluorescent SSCP of overlapping fragments (FSSCP-OF): a highly sensitive method for the screening of mitochondrial DNA variation.

    PubMed

    Salas, A; Rasmussen, E M; Lareu, M V; Morling, N; Carracedo, A

    2001-12-27

    The mtDNA analysis (mtDNA) is increasingly being demanded for forensic purposes due to the fact that many times the use of standard nuclear marker fails to analyze degraded samples (such as bones) and specially for the analysis of hair shafts (a common sample in the crime scene). However, analysis of mtDNA sequencing implies a great lab effort when a high number of samples must be analyzed. The present work introduces a novel and reliable method for the screening of mtDNA variation in the first and second hypervariables (HV1 and HV2) regions which we have denominated fluorescent single strand conformation polymorphism (SSCP) of overlapping fragments (FSSCP-OF). FSSCP-OF is based on the basic theory of SSCP analysis and combines two complementary strategies: the use of PCR amplified overlapping fragments and fluorescent detection technology. The overlap region contains a high percentage (50%) of the d-loop mtDNA variation and for this reason, the probability to detect a polymorphic position by SSCP analysis is clearly increased in comparison to conventional SSCP methods due to the fact that the same polymorphic position is usually placed in a different "relative" position in the two overlapped fragments. The use of multicolor fluorescent technology allows also the multiplex amplification of overlapping fragment and its subsequent analysis in an automatic sequencer. We have analyzed 50 samples of unrelated individuals through the FSSCP-OF technique and we have found that using this methodology the probability to distinguish two samples with different sequences is close to 100%. FSSCP-OF has other important advantages with respect to previous screening methods, such as the automation and standardization of the protocols, which is of special interest for the forensic routine.

  11. Copper nanoclusters as a highly sensitive and selective fluorescence sensor for ferric ions in serum and living cells by imaging.

    PubMed

    Cao, Haiyan; Chen, Zhaohui; Zheng, Huzhi; Huang, Yuming

    2014-12-15

    A simple, one-step facile route for preparation of water soluble and fluorescent Cu nanoclusters (NCs) stabilized by tannic acid (TA) is described. The as-prepared TA capped Cu NCs (TA-Cu NCs) are characterized by UV-vis spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, luminescence, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The TA-Cu NCs show luminescence properties having excitation and emission maxima at 360 nm and 430 nm, respectively, with a quantum yield of about 14%. The TA-Cu NCs are very stable even in 0.3 M NaCl, and their luminescent properties show pH independent. The fluorescence (FL) of Cu NCs is strongly quenched by Fe(3+) through an electron transfer mechanism, but not by other metal ions. Furthermore, the FL of the TA-Cu NCs shows no changes with the addition of Fe(2+) or H2O2 individually. On this basis, a facile chemosensor was developed for rapid, reliable, sensitive, and selective sensing of Fe(3+) ions with detection limit as low as 10 nM and a dynamic range from 10 nM to 10 μM. The proposed sensor was successfully used for the determination of iron contents in serum samples. Importantly, the Cu NCs-based FL probe showed long-term stability, good biocompatibility and very low cytotoxicity. It was successfully used for imaging ferric ions in living cells, suggesting the potential application of Cu NCs fluorescent probe in clinical analysis and cell imaging.

  12. Highly sensitive fluorescence and SERS detection of azide through a simple click reaction of 8-chloroquinoline and phenylacetylene.

    PubMed

    Zeng, Qing; Ye, Lingling; Ma, Lu; Yin, Wenqing; Li, Tingsheng; Liang, Aihui; Jiang, Zhiliang

    2015-05-01

    In 0.19 mol/L acetic acid (HAc), a click reaction of 8-chloroquinoline/azide/phenylacetylene take places in aqueous solution without Cu(I) as a catalyst. 8-Chloroquinoline (CQN) exhibited a strong fluorescence peak at 430 nm that was quenched linearly as the concentration of azide increased from 20 to 1000 ng/mL. This quenching was due to consumption of CQN in the click reaction and a decrease in the number of efficiently excited photons due to the presence of triazole-quinoline ramification molecules with strong hydrophobicity. Using blue nanosilver sol as the substrate, CQN absorbed onto the surface of nanosilver particles, showing a strong surface-enhanced Raman scattering (SERS) peak at 1585 cm(-1) that decreased linearly as the azide concentration increased from 8 to 500 ng/mL; the detection limit was 4 ng/mL. Thus, two new, simple and sensitive fluorescence and SERS methods have been developed for the determination of azide via the click reaction.

  13. High-sensitivity capillary electrophoresis of double-stranded DNA fragments using monomeric and dimeric fluorescent intercalating dyes

    SciTech Connect

    Zhu, H.; Clark, S.M.; Benson, S.C.; Rye, H.S.; Glazer, A.N.; Mathies, R.A. )

    1994-07-01

    Fluorescence-detected capillary electrophoresis separations of [phi]X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from [phi]X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. 43 refs., 10 figs., 1 tab.

  14. Peroxyoxalate chemiluminescence detection for the highly sensitive determination of fluorescence-labeled chlorpheniramine with Suzuki coupling reaction.

    PubMed

    Adutwum, Lawrence Asamoah; Kishikawa, Naoya; Ohyama, Kaname; Harada, Shiro; Nakashima, Kenichiro; Kuroda, Naotaka

    2010-09-01

    A sensitive and selective high performance liquid chromatography-peroxyoxalate chemiluminescence (PO-CL) method has been developed for the simultaneous determination of chlorpheniramine (CPA) and monodesmethyl chlorpheniramine (MDCPA) in human serum. The method combines fluorescent labeling with 4-(4,5-diphenyl-1H-imidazole-2-yl)phenyl boronic acid using Suzuki coupling reaction with PO-CL detection. CPA and MDCPA were extracted from human serum by liquid-liquid extraction with n-hexane. Excess labeling reagent, which interfered with trace level determination of analytes, was removed by solid-phase extraction using a C18 cartridge. Separation of derivatives of both analytes was achieved isocratically on a silica column with a mixture of acetonitrile and 60 mM imidazole-HNO(3) buffer (pH 7.2; 85:15, v/v) containing 0.015% triethylamine. The proposed method exhibited a good linearity with a correlation coefficient of 0.999 for CPA and MDCPA within the concentration range of 0.5-100 ng/mL. The limits of detection (S/N = 3) were 0.14 and 0.16 ng/mL for CPA and MDCPA, respectively. Using the proposed method, CPA could be selectively determined in human serum after oral administration.

  15. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.

    PubMed

    Tada, Mayumi; Takeuchi, Atsuya; Hashizume, Miki; Kitamura, Kazuo; Kano, Masanobu

    2014-06-01

    Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

  16. Highly Sensitive Fluorescence Methods for the Determination of Alfuzosin, Doxazosin, Terazosin and Prazosin in Pharmaceutical Formulations, Plasma and Urine.

    PubMed

    Guo, Xiaozhen; Wu, Hao; Guo, Shiwen; Shi, Yating; DU, Juanli; Zhu, Panpan; DU, Liming

    2016-01-01

    Polymeric ionic liquid-coated magnetic nanoparticles have been successfully prepared as adsorbents for the magnetic solid-phase extraction of four drugs, namely alfuzosin, doxazosin, terazosin and prazosin, from pharmaceutical preparations, urine samples and plasma samples. The four drugs were detected by fluorescence spectrophotometer. Several extraction parameters, including the pH of the solution; the type, ratio and volume of the desorbing reagent; the amount of adsorbent; the time of the extraction and desorption processes; and the addition of NaCl, were investigated and optimized. Linear responses were determined for the four drugs in the concentration range of 0.5 - 45 ng mL(-1). The limit of detection values for alfuzosin, doxazosin, terazosin and prazosin, which were defined as three times the standard deviation of a blank sample, were determined to be 0.035, 0.034, 0.027 and 0.028 ng mL(-1) (n = 11), respectively. Furthermore, this new method gave preconcentration factors of 114.5, 111.3, 111.1 and 108.5 for these four drugs.

  17. High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents

    PubMed Central

    Cheng, Bingbing; Bandi, Venugopal; Wei, Ming-Yuan; Pei, Yanbo; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Yuan, Baohong

    2016-01-01

    For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena—such as the presence of immune system cells, tumor angiogenesis, and metastasis—may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 μm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging. PMID:27829050

  18. Focused upon hybridization: rapid and high sensitivity detection of DNA using isotachophoresis and peptide nucleic acid probes.

    PubMed

    Ostromohov, Nadya; Schwartz, Ortal; Bercovici, Moran

    2015-09-15

    We present a novel assay for rapid and high sensitivity detection of nucleic acids without amplification. Utilizing the neutral backbone of peptide nucleic acids (PNA), our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis (ITP) unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, significantly improving the signal-to-noise ratio while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model and experimentally demonstrate the ability to detect targets as short as 17 nucleotides (nt) and a limit of detection of 100 fM with a dynamic range of 5 decades. We also demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal. The assay requires 15 min to complete, and it could potentially be used in applications where rapid and highly sensitive amplification-free detection of nucleic acids is desired.

  19. Exonuclease III-Assisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification: One-Step and Dual-Amplification Strategy for Highly Sensitive Fluorescence Detection of DNA.

    PubMed

    Zhou, Fulin; Li, Baoxin

    2015-07-21

    Detection of ultralow concentration of specific DNA sequence is a central challenge in the early diagnosis of gene-related disease and biodefense application. Herein, we report a dual-amplification strategy for highly sensitive fluorescence detection of DNA. In this proposed strategy, a dumbbell-shaped DNA probe is designed to integrate target binding, magnetic separation, and signal response. In the presence of specific DNA target, the multifunctional dumbbell probe can initiate exonuclease III (Exo III)-aided target recycling amplification, and, in the meantime, generate a large number of fluorescein (FAM)-encapsulated liposomes. The developed method offers very high sensitivity due to primary amplification via numerous FAM from a liposome and secondary amplification via target recycling amplification. The detection limit of the proposed method can reach 4 aM, which is much lower than that of the Exo III-aided target recycling technique applied for DNA quantification without FAM-encapsulated liposomes amplification. Moreover, the dual-signal amplification process can be completed one-step in this system. Therefore, this method provides a simple, isothermal, and low-cost approach for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases.

  20. A turn-on fluorescent probe based on coumarin-anhydride for highly sensitive detection of hydrazine in the aqueous solution and gas states

    NASA Astrophysics Data System (ADS)

    Wu, Guangfu; Tang, Xin; Ji, Wengang; Chiu Lai, King Wai; Tong, Qingxiao

    2017-03-01

    In this paper, a new coumarin-based fluorescent probe for hydrazine was rationally designed and successfully synthesized based on the Gabriel reaction. This novel probe enabled highly sensitive and selective detection of hydrazine. The detection limit was 43.6 nM (1.49 ppb). It displayed distinct changes in the intensity of both absorption and emission spectra upon the addition of hydrazine and remarkable color changes can be visually observed. The sensing mechanism of this probe toward hydrazine was characterized by 1H NMR and mass spectroscopy. Moreover, with the help of theoretical calculations, the sensing capability of this probe for hydrazine detection was described: the electron structure was modulated by a three-substituted group of coumarin once upon the addition of hydrazine. In addition, a test paper experiment indicated its great potential in environment monitoring.

  1. Fluorescence biosensing strategy based on mercury ion-mediated DNA conformational switch and nicking enzyme-assisted cycling amplification for highly sensitive detection of carbamate pesticide.

    PubMed

    Wang, Xiuzhong; Hou, Ting; Dong, Shanshan; Liu, Xiaojuan; Li, Feng

    2016-03-15

    Pesticides are of great importance in agricultural and biological fields, but pesticide residues may harm the environment and human health. A highly sensitive fluorescent biosensor for the detection of carbamate pesticide has been developed based on acetylcholinesterase (AChE)-catalyzed hydrolysis product triggered Hg(2+) release coupled with subsequent nicking enzyme-induced cleavage of a duplex DNA for cycling amplification. In this protocol, two DNA probes, an unmodified single-stranded helper DNA probe 1 (HP1) and a quencher-fluorophore probe (QFP) are ingeniously designed. HP1 can be folded into hairpin configuration through T-Hg(2+)-T base pair formation. QFP, labeled with FAM and BHQ1 at its two terminals, contains the recognition sequence and the cleavage site of the nicking enzyme. In the presence of carbamate pesticide, the activity of AChE is inhibited, and the amount of the product containing the thiol group generated by the hydrolysis reaction of acetylthiocholine chloride (ACh) decreases, resulting in the release of a low concentration of Hg(2+). The number of HP1 that can be selectively unfolded would be reduced and the subsequent nicking enzyme-assisted cleavage processes would be affected, resulting in decreased fluorescence signals. The fluorescence intensity further decreases with the increase of the pesticide concentration. Therefore, the pesticide content can be easily obtained by monitoring the fluorescence signal change, which is inversely proportional to the logarithm of the pesticide concentration. The detection limit of aldicarb, the model analyte, is 3.3 μgL(-1), which is much lower than the Chinese National Standards or those previously reported. The as-proposed method has also been applied to detect carbamate pesticide residues in fresh ginger and artificial lake water samples with satisfactory results, which demonstrates that the method has great potential for practical application in biological or food safety field.

  2. G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection.

    PubMed

    Jiang, Hong-Xin; Liang, Zhen-Zhen; Ma, Yan-Hong; Kong, De-Ming; Hong, Zhang-Yong

    2016-11-02

    Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.

  3. Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Au(nano)-CNT/PAN(nano) films.

    PubMed

    Zhou, Na; Yang, Tao; Jiang, Chen; Du, Meng; Jiao, Kui

    2009-01-15

    A polyaniline nanofibers (PAN(nano))/carbon paste electrode (CPE) was prepared via dopping PAN(nano) in the carbon paste. The nanogold (Au(nano)) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN(nano)/CPE. The immobilization and hybridization of the DNA probe on the Au(nano)-CNT/PAN(nano) films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)(6)](3-/4-) as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au(nano)-CNT/PAN(nano) films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R(et)) of the electrode surface increased after the immobilization of the probe DNA on the Au(nano)-CNT/PAN(nano) films and rose further after the hybridization of the probe DNA. The remarkable difference between the R(et) value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au(nano)-CNT/PAN(nano) films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 x 10(-12)mol/L to 1.0 x 10(-6)mol/L with a detection limit of 5.6 x 10(-13)mol/L.

  4. Highly sensitive and selective high-performance liquid chromatography method for bioequivalence study of cefpodoxime proxetil in rabbit plasma via fluorescence labeling of its active metabolite.

    PubMed

    Ahmed, Sameh; Abdel-Wadood, Hanaa M; Mohamed, Niveen A

    2013-09-01

    Cefpodoxime proxetil (CFP), a broad-spectrum third-generation cephalosporin, has been used most widely in the treatment of respiratory and urinary tract infections. For bioequivalence study of CFP in rabbit plasma, it was necessary to develop a highly sensitive and selective high-performance liquid chromatographic (HPLC) method with fluorescence (FL) detection. The pre-column labeling of cefpodoxime acid (CFA) (active metabolite) with an efficient benzofurazan type fluorogenic reagent, 4-N,N-dimethyl aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was carried out in the present study in 100mM borate buffer (pH=8.5) at 50°C for 15min. The obtained fluorescent products were separated on C18 column with an isocratic elution of the mobile phase, which consists of 10mM phosphate buffer (pH=3.5)/CH3CN (70:30, v/v). The fluorescent product (DBD-CFA) was detected fluorimetrically at 556nm with an excitation wavelength of 430nm. Cefotaxime sodium was used as internal standard. The method was validated according to the requirements of US-FDA guidelines. The correlation coefficient of 0.999 was obtained in the concentration ranges of 10-1000ngmL(-1). The limits of detection and quantification (S/N=3) were 3 and 10ngmL(-1), respectively. Plasma CFA levels were successfully determined in rabbit with satisfactory precision and accuracy. The proposed HPLC-FL method was successfully applied to study bioequivalence in rabbits for two formulations of different brands contained CFP (prodrug) in a randomized, two-way, single-dose, crossover study and all pharmacokinetic parameters for the two formulations were assessed.

  5. Carbon nanotube-polyamidoamine dendrimer hybrid-modified electrodes for highly sensitive electrochemical detection of microRNA24.

    PubMed

    Li, Fengye; Peng, Jing; Zheng, Qiong; Guo, Xiang; Tang, Hao; Yao, Shouzhuo

    2015-01-01

    A simple and ultrasensitive microRNA (miRNA) electrochemical biosensor employing multiwalled carbon nanotube (MWCNT)-polyamidoamine (PAMAM) dendrimer and methylene blue (MB) redox indicator is reported in this work. The assay utilizes a glass carbon (GC) electrode modified with MWCNT-PAMAM, on which the oligonucleotide capture probes are immobilized. The electrochemical detection of miRNAs is completed by measuring the reduction signal change of MB before and after the probe hybridization with target miRNA (miRNA24 is used as a model case). The MWCNT-PAMAM/GC electrode shows greatly enhanced signal to MB reduction in contrast to bare GC electrode. The functionalization of MWCNT with PAMAM maintains the electrochemical property of MWCNT to MB reduction but minimizes the undesired adsorption of MB on the MWCNT surface. The effect of experimental variables on the miRNA detection is investigated and optimized. A detection limit of 0.5 fM and a linear peak current density-concentration relationship up to 100 nM are obtained following 60 min hybridization. The proposed assay is successfully used to detect miRNA24 in total RNA sample extracted from HeLa cells.

  6. Highly sensitive photodetectors based on hybrid 2D-0D SnS{sub 2}-copper indium sulfide quantum dots

    SciTech Connect

    Huang, Yun; Zhan, Xueying; Xu, Kai; Yin, Lei; Cheng, Zhongzhou; Jiang, Chao; Wang, Zhenxing E-mail: hej@nanoctr.cn; He, Jun E-mail: hej@nanoctr.cn

    2016-01-04

    Both high speed and efficiency of photoelectric conversion are essential for photodetectors. As an emerging layered metal dichalcogenide (LMD), tin disulfide owns intrinsic faster photodetection ability than most other LMDs but poor light absorption and low photoelectric conversion efficiency. We develop an efficient method to enhance its performance by constructing a SnS{sub 2}-copper indium sulfide hybrid structure. As a result, the responsivity reaches 630 A/W, six times stronger than pristine SnS{sub 2} and much higher than most other LMDs photodetectors. Additionally, the photocurrents are enhanced by more than 1 order of magnitude. Our work may open up a pathway to improve the performance of photodetectors based on LMDs.

  7. High-resolution and high sensitivity mesoscopic fluorescence tomography based on de-scanning EMCCD: System design and thick tissue imaging applications

    NASA Astrophysics Data System (ADS)

    Ozturk, Mehmet Saadeddin

    Optical microscopy has been one of the essential tools for biological studies for decades, however, its application areas was limited to superficial investigation due to strong scattering in live tissues. Even though advanced techniques such as confocal or multiphoton methods have been recently developed to penetrate beyond a few hundreds of microns deep in tissues, they still cannot perform in the mesoscopic regime (millimeter scale) without using destructive sample preparation protocols such as clearing techniques. They provide rich cellular information; however, they cannot be readily employed to investigate the biological processes at larger scales. Herein, we will present our effort to establish a novel imaging approach that can quantify molecular expression in intact tissues, well beyond the current microscopy depth limits. Mesoscopic Fluorescence Molecular Tomography (MFMT) is an emerging imaging modality that offers unique potential for the non-invasive molecular assessment of thick in-vitro and in-vivo live tissues. This novel imaging modality is based on an optical inverse problem that allows for retrieval of the quantitative spatial distribution of fluorescent tagged bio-markers at millimeter depth. MFMT is well-suited for in-vivo subsurface tissue imaging and thick bio-printed specimens due to its high sensitivity and fast acquisition times, as well as relatively large fields of view. Herein, we will first demonstrate the potential of this technique using our first generation MFMT system applied to multiplexed reporter gene imaging (in-vitro) and determination of Photodynamic Therapy (PDT) agent bio-distribution in a mouse model (in-vivo). Second, we will present the design rationale, in silico benchmarking, and experimental validation of a second generation MFMT (2GMFMT) system. We will demonstrate the gain in resolution and sensitivity achieved due to the de-scanned dense detector configuration implemented. The potential of this novel platform will be

  8. Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

    PubMed

    Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

    2015-04-21

    Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

  9. Green Fluorescent Protein as a Visual Marker in Somatic Hybridization

    PubMed Central

    OLIVARES‐FUSTER, O.; PEÑA, L.; DURAN‐VILA, N.; NAVARRO, L.

    2002-01-01

    Using a transgenic citrus plant expressing Green Fluorescent Protein (GFP) as a parent in somatic fusion experiments, we investigated the suitability of GFP as an in vivo marker to follow the processes of protoplast fusion, regeneration and selection of hybrid plants. A high level of GFP expression was detected in transgenic citrus protoplasts, hybrid callus, embryos and plants. It is demonstrated that GFP can be used for the continuous monitoring of the fusion process, localization of hybrid colonies and callus, and selection of somatic hybrid embryos and plants. PMID:12096810

  10. Hybrid silver nanoparticle/conjugated polyelectrolyte nanocomposites exhibiting controllable metal-enhanced fluorescence

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoyu; He, Fang; Zhu, Xi; Tang, Fu; Li, Lidong

    2014-03-01

    Metal-enhanced fluorescence of conjugated polyelectrolytes (CPs) is realized using a simple, green hybrid Ag nanocomposite film. Ag nanoparticles (Ag NPs) are pre-prepared by sodium citrate reduction and incorporated into agarose by mixing to form an Ag-containing agarose film (Ag@agarose). Through variation of the amount of Ag NPs in the Ag@agarose film as well as the thickness of the interlayer between CPs and the Ag@agarose film prepared of layer-by-layer assembly of chitosan and sodium alginate, a maximum 8.5-fold increase in the fluorescence of CPs is obtained. After introducing tyrosinase, this system also can be used to detect phenolic compounds with high sensitivity and good visualization under ultraviolet light.

  11. Highly sensitive quantitation of pesticides in fruit juice samples by modeling four-way data gathered with high-performance liquid chromatography with fluorescence excitation-emission detection.

    PubMed

    Montemurro, Milagros; Pinto, Licarion; Véras, Germano; de Araújo Gomes, Adriano; Culzoni, María J; Ugulino de Araújo, Mário C; Goicoechea, Héctor C

    2016-07-01

    A study regarding the acquisition and analytical utilization of four-way data acquired by monitoring excitation-emission fluorescence matrices at different elution time points in a fast HPLC procedure is presented. The data were modeled with three well-known algorithms: PARAFAC, U-PLS/RTL and MCR-ALS, the latter conveniently adapted to model third-order data. The second-order advantage was exploited when analyzing samples containing uncalibrated components. The best results were furnished with the algorithm U-PLS/RTL. This fact is indicative of both no peak time shifts occurrence among samples and high colinearity among spectra. Besides, this latent-variable structured algorithm is capable of better handle the need of achieving high sensitivity for the analysis of one of the analytes. In addition, a significant enhancement in both predictions and analytical figures of merit was observed for carbendazim, thiabendazole, fuberidazole, carbofuran, carbaryl and 1-naphtol, when going from second- to third-order data. LODs obtained were ranged between 0.02 and 2.4μgL(-1).

  12. A novel fluorescent probe: europium complex hybridized T7 phage.

    PubMed

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  13. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

    1995-01-01

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

  14. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

    1995-11-28

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

  15. Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.

    PubMed

    Lécuyer, Eric; Parthasarathy, Neela; Krause, Henry M

    2008-01-01

    Fluorescent in situ hybridization is the standard method for visualizing the spatial distribution of RNA. Although traditional histochemical RNA detection methods suffered from limitations in resolution or sensitivity, the recent development of peroxidase-mediated tyramide signal amplification provides strikingly enhanced sensitivity and subcellular resolution. In this chapter, we describe optimized fluorescent in situ hybridization protocols for Drosophila embryos and tissues utilizing tyramide signal amplification, either for single genes or in a high-throughput format, which greatly increases the sensitivity, consistency, economy, and throughput of the procedure. We also describe variations of the method for RNA-RNA and RNA-protein codetection.

  16. Highly sensitive routine method for urinary 3-hydroxybenzo[a]pyrene quantitation using liquid chromatography-fluorescence detection and automated off-line solid phase extraction.

    PubMed

    Barbeau, Damien; Maître, Anne; Marques, Marie

    2011-03-21

    Many workers and also the general population are exposed to polycyclic aromatic hydrocarbons (PAHs), and benzo[a]pyrene (BaP) was recently classified as carcinogenic for humans (group 1) by the International Agency for Research on Cancer. Biomonitoring of PAHs exposure is usually performed by urinary 1-hydroxypyrene (1-OHP) analysis. 1-OHP is a metabolite of pyrene, a non-carcinogenic PAH. In this work, we developed a very simple but highly sensitive analytical method of quantifying one urinary metabolite of BaP, 3-hydroxybenzo[a]pyrene (3-OHBaP), to evaluate carcinogenic PAHs exposure. After hydrolysis of 10 mL urine for two hours and concentration by automated off-line solid phase extraction, the sample was injected in a column-switching high-performance liquid chromatography fluorescence detection system. The limit of quantification was 0.2 pmol L(-1) (0.05 ng L(-1)) and the limit of detection was estimated at 0.07 pmol L(-1) (0.02 ng L(-1)). Linearity was established for 3-OHBaP concentrations ranging from 0.4 to 74.5 pmol L(-1) (0.1 to 20 ng L(-1)). Relative within-day standard deviation was less than 3% and relative between-day standard deviation was less than 4%. In non-occupationally exposed subjects, median concentrations for smokers compared with non-smokers were 3.5 times higher for 1-OHP (p<0.001) and 2 times higher for 3-OHBaP (p<0.05). The two urinary biomarkers were correlated in smokers (ρ=0.636; p<0.05; n=10) but not in non-smokers (ρ=0.09; p>0.05; n=21).

  17. Coherent fluorescence emission by using hybrid photonic-plasmonic crystals.

    PubMed

    Shi, Lei; Yuan, Xiaowen; Zhang, Yafeng; Hakala, Tommi; Yin, Shaoyu; Han, Dezhuan; Zhu, Xiaolong; Zhang, Bo; Liu, Xiaohan; Törmä, Päivi; Lu, Wei; Zi, Jian

    2014-09-01

    The spatial and temporal coherence of the fluorescence emission controlled by a quasi-two-dimensional hybrid photonic-plasmonic crystal structure covered with a thin fluorescent-molecular-doped dielectric film is investigated experimentally. A simple theoretical model to describe how a confined quasi-two-dimensional optical mode may induce coherent fluorescence emission is also presented. Concerning the spatial coherence, it is experimentally observed that the coherence area in the plane of the light source is in excess of 49 μm(2), which results in enhanced directional fluorescence emission. Concerning temporal coherence, the obtained coherence time is 4 times longer than that of the normal fluorescence emission in vacuum. Moreover, a Young's double-slit interference experiment is performed to directly confirm the spatially coherent emission. This smoking gun proof of spatial coherence is reported here for the first time for the optical-mode-modified emission.

  18. FISH-ing for Genes: Modeling Fluorescence "in situ" Hybridization

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck

    2006-01-01

    Teaching methods of genetic analysis such as fluorescence in situ hybridization (FISH) can be an important part of instructional units in biology, microbiology, and biotechnology. Experience, however, indicates that these topics are difficult for many students. The authors of this article describe how they created an activity that effectively…

  19. Highly sensitive detection of cancer-related genes based on complete fluorescence restoration of a molecular beacon with a functional overhang.

    PubMed

    Li, Feng; Zhou, Ying-Ying; Peng, Ting; Xu, Huo; Zhang, Rong-Bo; Zhao, Hui; Wang, Zheng-Yong; Lv, Jian-Xin; Wu, Zai-Sheng; Shen, Zhi-Fa

    2016-07-21

    The accurate detection of cancer-related genes is of great significance for early diagnosis and targeted therapy of cancer. In this contribution, an automatically cycling operation of a functional overhang-containing molecular beacon (OMB)-based sensing system was proposed to perform amplification detection of the p53 gene. Contrary to the common molecular beacon (MB), a target DNA is designated to hybridize with a label-free recognition probe (RP) with a hairpin structure rather than OMB. In the presence of a target DNA of interest, the locked primer in RP opens and triggers the subsequent amplification procedures. The newly-developed OMB is not only capable of accomplishing cyclical nucleic acid strand-displacement polymerization (CNDP) with the help of polymerase and nicking endonuclease, but is also cleaved by restriction endonucleases, removing the quencher away from the fluorophore. Thus, the target DNA at an extremely low concentration is expected to generate a considerable amount of double-stranded and cleaved OMBs, and the quenched fluorescence is completely restored, leading to a dramatic increase in fluorescence intensity. Utilizing this sensing platform, the target gene can be detected down to 8.2 pM in a homogeneous way, and a linear response range of 0.01 to 150 nM could be obtained. More strikingly, the mutant genes can be easily distinguished from the wild-type ones. The proof-of-concept demonstrations reported herein are expected to promote the development of DNA biosensing systems, showing great potential in basic research and clinical diagnosis.

  20. Fluorescence labeling of carbon nanotubes and visualization of a nanotube-protein hybrid under fluorescence microscope.

    PubMed

    Yoshimura, Shige H; Khan, Shahbaz; Maruyama, Hiroyuki; Nakayama, Yoshikazu; Takeyasu, Kunio

    2011-04-11

    Biological applications of carbon nanotubes have been hampered by the inability to visualize them using conventional optical microscope, which is the most common tool for the observation and measurement of biological processes. Recently, a number of fluorescence labeling methods for biomolecules and various fluorescence probes have been developed and widely utilized in biological fields. Therefore, labeling carbon nanotubes with such fluorophores under physiological conditions will be highly useful in their biological applications. In this Article, we present a method to fluorescently label nanotubes by combining a detergent and a fluorophore commonly used in biological experiments. Fluorophores carrying an amino group (Texas Red hydrazide or BODIPY FL-hydrazide) were covalently attached to the hydroxyl groups of Tween 20 using carbonyldiimidazole. Fluorescence microscopy demonstrated that nanotubes were efficiently solubilized and labeled by this fluorescently labeled detergent. By using this technique, we also demonstrated multicolor fluorescence imaging of a nanotube-protein hybrid.

  1. Hybridization assay based on evanescent fluorescence excitation and collection

    NASA Astrophysics Data System (ADS)

    Sumner, James J.; Mmerole, Robert U.; Stratis-Cullum, Dimitra N.; Yi, Hyunmin; Bentley, William E.; Gillespie, James B.

    2003-08-01

    There is a great need for high throughput and sensitive sensors for genetic analysis. These sensors can be used for varied purposes from monitoring gene expression in organims to speciation of possible pathogens. Consequently, an instrument capable of these tasks would be a great benefit for food and water safety, medical diagnostics and defense of military and civilian populations from biological threats. This work examines the development of a hybridization-based biosensor using a novel tapered fiber optic rpobe. The immobilization of single-stranded, synthetic ologinucleotides utilizing aminoproplytriethoxysilane and glutaraldehyde was implemented on the fiber optic sensor. Hybridization takes place with a complementary analyte sequence followed by a fluorescent, labeled signaling probe to form a sandwich assay. Following hybridization, the fiber is interrogated with a diode laser source and the resulting fluorescence signal is detected using a miniature spectrometer.

  2. Hybrid native phosphorescence and fluorescence spectroscopy for cancer detection

    NASA Astrophysics Data System (ADS)

    Alimova, Alexandra; Katz, A.; Sriramoju, Vidyasagar; Budansky, Yuri; Bykov, Alexei A.; Zeylikovich, Roman; Alfano, R. R.

    2006-02-01

    Native fluorescence of tissues in the UV and visible spectral regions has been investigated for over two decades. Native fluorescence has been demonstrated to be an accurate tools for distinguish normal tissue from malignant and pre-malignant. Prior investigations have demonstrated that there are several ratio-based algorithms, which can distinguish malignant tissue from normal with high sensitivity and specificity.1 The wavelength combinations used in these ratios isolate the contributions from pairs of tissue fluorophors, one of which is frequently tryptophan (trp), the predominant tissue fluorophore with excitation in the UV (250-300 nm). In this work, algorithms using a combination of native fluorescence and trp phosphorescence were developed which show promise for providing enhanced detection accuracy. Using optical fibers to collect the emission from the specimen allowed interrogation of small regions of tissue, providing precise spatial information. Using a specially designed setup, specimens were excited in the UV and spectra were collected in the range of 300 to 700 nm. Three main emission bands were selected for analysis: 340 nm (trp fluorescence); 420 - 460 nm band (fluorescence from the extra cellular matrix); and 500 - 520 nm (trp phosphorescence). Normal specimens consistently exhibited a low ratio (<10) of 345 to 500 nm emission intensity while this same ratio was consistently high (>15) for cancer specimens. Creating intensities ratio maps from the tissue allows one to localize the malignant regions with high spatial precision. The study was performed on ex vivo human breast tissues. The ratio analysis correlated well with histopathology.

  3. Largely Enhanced Single-molecule Fluorescence in Plasmonic Nanogaps formed by Hybrid Silver Nanostructures

    PubMed Central

    Zhang, Jian; Lakowicz, Joseph R.

    2013-01-01

    It has been suggested that narrow gaps between metallic nanostructures can be practical for producing large field enhancement. We design a hybrid silver nanostructure geometry in which fluorescent emitters are sandwiched between silver nanoparticles and silver island film (SIF). A desired number of polyelectrolyte layers are deposited on the SIF surface before the self-assembly of a second silver nanoparticle layer. Layer-by-layer configuration provides a well-defined dye position. It allows us to study the photophyical behaviors of fluorophores in the resulting gap at the single molecule level. The enhancement factor of a fluorophore located in the gap is much higher than those on silver surfaces alone and on glass. These effects may be used for increased detectability of single molecules bound to surfaces which contain metallic structures for either biophysical studies or high sensitivity assays. PMID:23373787

  4. Highly sensitive detection of neodymium ion in small amount of spent nuclear fuel samples using novel fluorescent macrocyclic hexadentate polyaminocarboxylate probe in capillary electrophoresis-laser-induced fluorescence detection.

    PubMed

    Saito, Shingo; Sato, Yoshiyuki; Haraga, Tomoko; Nakano, Yuta; Asai, Shiho; Kameo, Yutaka; Takahashi, Kuniaki; Shibukawa, Masami

    2012-04-06

    A rapid and high-sensitive detection method for the total concentration of Nd ion (total Nd) in a small amount of a spent nuclear fuel sample is urgently required since the precise quantification of total Nd ion makes it possible for burnup (degree of fuel consumption) to be determined. In this work, a capillary electrophoresis-laser-induced fluorescent detection method (CE-LIF) is proposed for the analysis of total Nd in a spent fuel sample solution, with the use of a newly synthesized metal fluorescent probe which has a fluorescein and a macrocylic hexadentate chelating group, FTC-ABNOTA, for lanthanide (Ln) ions. Ln ions were derivatized to form a strongly fluorescent complex with the probe to suppress the quenching of the ligand-centered emission. The detection of Ln complexes in the CE-LIF indicated that the interaction between Ln ions and the FTC-ABNOTA was strong enough not to dissociate during migration. The mutual separation among the Ln-FTC-ABNOTA complexes in CE-LIF was achieved by pH control providing a dynamic ternary complexation (DTC) with hydroxide ions. Using the DTC separation mode, a high resolution of Nd from other Ln ions with high resolution of 1.3-1.9 and a theoretical plate number of 68,000, and a very low detection limit of 22 pM (3.2 ppt, 0.11 attomole amount basis) were successfully obtained. A simulated spent fuel sample containing various metal ions was examined in this method with a good quantification result of 102.1% recovery obtained even with a large excess of U.

  5. Fluorescence In Situ Hybridization Probe Preparation.

    PubMed

    Tolomeo, Doron; Stanyon, Roscoe R; Rocchi, Mariano

    2017-01-01

    The public human genome sequencing project utilized a hierarchical approach. A large number of BAC/PAC clones, with an insert size approximate from 50 kb to 300 kb, were identified and finely mapped with respect to the Sequence Tagged Site (STS) physical map and with respect to each other. A "golden path" of BACs, covering the entire human genome, was then selected and each clone was fully sequenced. The large number of remaining BACs was not fully sequenced, but the availability of the end sequence (~800-1000 bp) at each end allowed them to be very precisely mapped on the human genome.The search for copy number variations of the human genome used several strategies. One of these approaches took advantage of the fact that fosmid clones, contrary to BAC/PAC clones, have a fixed insert size (~40 kb) (Kidd et al., Nature 453: 56-64, 2008). In this context, the ends of ~7 million fosmid clones were sequenced, and therefore it was possible to precisely map these clones on the human genome.In summary, a large number of genomic clones (GC) are available for FISH experiments. They usually yield bright FISH signals and are extremely precious for molecular cytogenetics, and in particular cancer cytogenetics. The already-labeled probes available commercially are usually based on a combination of such GCs. The present chapter summarizes the protocols for extracting, labeling, and hybridization onto slides of DNA obtained from GC.

  6. Matching base-pair number dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy.

    PubMed

    Tawa, Keiko; Yao, Danfeng; Knoll, Wolfgang

    2005-08-15

    Two single-stranded DNA oligonucleotides consisting of complementary base-pairs can form double strands. This phenomenon is well studied in solutions, however, in order to clarify the physical mechanism of the hybridization occurring at a solid/solution interface, we studied the kinetics by surface plasmon fluorescence spectroscopy (SPFS): one single-stranded oligo-DNA (probe-DNA) was immobilized on the substrate, the other one (target-DNA) labelled with a fluorescent probe was added to the flow cell. After hybridization, the chromophores could be excited by the surface plasmon mode and their fluorescence detected with high sensitivity. The dependence of the k(on) and k(off) rate constants on the length of the hybridizing oligonucleotides was investigated by using a MM0 series (no mismatch) and the kinetics was found to be well described by a Langmuir adsorption model. From these measurements we found that also in the case of surface hybridization the affinity of the duplexes decreases as the number of matching base-pairs decreases from 15 to 10. In order to show that SPFS is the powerful technique with high sensitivity, the hybridization process for mixed target-oligos was measured by SPFS and analyzed by an expanded Langmuir model in which two components of target-oligo can bind to probe-DNA at the sensor surface competitively. Two sets of the k(on) and k(off) obtained from the experiment are successfully consistent with the k(on) and k(off) obtained from experiments for single (pure) target-DNA.

  7. Fluorescence Image Analyzer - FLIMA: software for quantitative analysis of fluorescence in situ hybridization.

    PubMed

    Silva, H C M; Martins-Júnior, M M C; Ribeiro, L B; Matoso, D A

    2017-03-30

    The Fluorescence Image Analyzer (FLIMA) software was developed for the quantitative analysis of images generated by fluorescence in situ hybridization (FISH). Currently, the images of FISH are examined without a coefficient that enables a comparison between them. Through GD Graphics Library, the FLIMA software calculates the amount of pixels on image and recognizes each present color. The coefficient generated by the algorithm shows the percentage of marks (probes) hybridized on the chromosomes. This software can be used for any type of image generated by a fluorescence microscope and is able to quantify digoxigenin probes exhibiting a red color, biotin probes exhibiting a green color, and double-FISH probes (digoxigenin and biotin used together), where the white color is displayed.

  8. Ratiometric fluorescent paper sensor utilizing hybrid carbon dots-quantum dots for the visual determination of copper ions

    NASA Astrophysics Data System (ADS)

    Wang, Yahui; Zhang, Cheng; Chen, Xiaochun; Yang, Bo; Yang, Liang; Jiang, Changlong; Zhang, Zhongping

    2016-03-01

    A simple and effective ratiometric fluorescence nanosensor for the selective detection of Cu2+ has been developed by covalently connecting the carboxyl-modified red fluorescent cadmium telluride (CdTe) quantum dots (QDs) to the amino-functionalized blue fluorescent carbon nanodots (CDs). The sensor exhibits the dual-emissions peaked at 437 and 654 nm, under a single excitation wavelength of 340 nm. The red fluorescence can be selectively quenched by Cu2+, while the blue fluorescence is a internal reference, resulting in a distinguishable fluorescence color change from pink to blue under a UV lamp. The detection limit of this highly sensitive ratiometric probe is as low as 0.36 nM, which is lower than the U.S. Environmental Protection Agency (EPA) defined limit (20 μM). Moreover, a paper-based sensor has been prepared by printing the hybrid carbon dots-quantum dots probe on a microporous membrane, which provides a convenient and simple approach for the visual detection of Cu2+. Therefore, the as-synthesized probe shows great potential application for the determination of Cu2+ in real samples.A simple and effective ratiometric fluorescence nanosensor for the selective detection of Cu2+ has been developed by covalently connecting the carboxyl-modified red fluorescent cadmium telluride (CdTe) quantum dots (QDs) to the amino-functionalized blue fluorescent carbon nanodots (CDs). The sensor exhibits the dual-emissions peaked at 437 and 654 nm, under a single excitation wavelength of 340 nm. The red fluorescence can be selectively quenched by Cu2+, while the blue fluorescence is a internal reference, resulting in a distinguishable fluorescence color change from pink to blue under a UV lamp. The detection limit of this highly sensitive ratiometric probe is as low as 0.36 nM, which is lower than the U.S. Environmental Protection Agency (EPA) defined limit (20 μM). Moreover, a paper-based sensor has been prepared by printing the hybrid carbon dots-quantum dots probe on a

  9. Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

    PubMed

    Cano, R J; Torres, M J; Klem, R E; Palomares, J C; Casadesus, J

    1992-05-01

    This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.

  10. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    PubMed

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  11. Fluorescent probes for "off-on" highly sensitive detection of Hg²⁺ and L-cysteine based on nitrogen-doped carbon dots.

    PubMed

    Zhang, Yi; Cui, Peipei; Zhang, Feng; Feng, Xiaoting; Wang, Yaling; Yang, Yongzhen; Liu, Xuguang

    2016-05-15

    Fluorescent nitrogen-doped carbon dots (NCDs) were synthesized by a facile, and low-cost one-step hydrothermal strategy using citric acid as carbon source and ammonia solution as nitrogen source for the first time. The obtained NCDs show stable blue fluorescence with a high quantum yield of 35.4%, along with the fluorescence lifetime of ca. 6.75 ns. Most importantly, Hg(2+) can completely quench the fluorescence of NCDs as a result of the formation of a non-fluorescent stable NCDs-Hg(2+) complex. Static fluorescence quenching towards Hg(2+) is proved by the Stern-Volmer equation, ultraviolet-visible absorption spectra, temperature dependent quenching and fluorescence lifetime measurements. Subsequently, the fluorescence of the NCDs-Hg(2+) system is completely recovered with the addition L-cysteine (L-Cys) owing to the dissociation of NCDs-Hg(2+) complex to form a more stable Hg(2+)-L-Cys complex by Hg(2+)-S bonding. Therefore, such NCDs can be used as an effective fluorescent "turn-off" probe for rapid, rather highly selective and sensitive detection of Hg(2+), with a limit of detection (LOD) as low as 1.48 nM and a linear detection range of 0-10 μM. Interestingly, NCDs-Hg(2+) system can be conveniently employed as a fluorescent "turn-on" sensor for highly selective and sensitive detection of L-Cys with a low LOD of 0.79 nM and a wide linear detection range of 0-50 μM. Further, the sensitivity of NCDs to Hg(2+) is preserved in tap water with a LOD of 1.65 nM and a linear detection range of 0-10 μM.

  12. A simple protocol for attenuating the auto-fluorescence of cyanobacteria for optimized fluorescence in situ hybridization (FISH) imaging.

    PubMed

    Zeller, Perrine; Ploux, Olivier; Méjean, Annick

    2016-03-01

    Cyanobacteria contain pigments, which generate auto-fluorescence that interferes with fluorescence in situ hybridization (FISH) imaging of cyanobacteria. We describe simple chemical treatments using CuSO4 or H2O2 that significantly reduce the auto-fluorescence of Microcystis strains. These protocols were successfully applied in FISH experiments using 16S rRNA specific probes and filamentous cyanobacteria.

  13. Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications

    PubMed Central

    Cui, Chenghua; Shu, Wei; Li, Peining

    2016-01-01

    Fluorescence in situ hybridization (FISH) is a macromolecule recognition technology based on the complementary nature of DNA or DNA/RNA double strands. Selected DNA strands incorporated with fluorophore-coupled nucleotides can be used as probes to hybridize onto the complementary sequences in tested cells and tissues and then visualized through a fluorescence microscope or an imaging system. This technology was initially developed as a physical mapping tool to delineate genes within chromosomes. Its high analytical resolution to a single gene level and high sensitivity and specificity enabled an immediate application for genetic diagnosis of constitutional common aneuploidies, microdeletion/microduplication syndromes, and subtelomeric rearrangements. FISH tests using panels of gene-specific probes for somatic recurrent losses, gains, and translocations have been routinely applied for hematologic and solid tumors and are one of the fastest-growing areas in cancer diagnosis. FISH has also been used to detect infectious microbias and parasites like malaria in human blood cells. Recent advances in FISH technology involve various methods for improving probe labeling efficiency and the use of super resolution imaging systems for direct visualization of intra-nuclear chromosomal organization and profiling of RNA transcription in single cells. Cas9-mediated FISH (CASFISH) allowed in situ labeling of repetitive sequences and single-copy sequences without the disruption of nuclear genomic organization in fixed or living cells. Using oligopaint-FISH and super-resolution imaging enabled in situ visualization of chromosome haplotypes from differentially specified single-nucleotide polymorphism loci. Single molecule RNA FISH (smRNA-FISH) using combinatorial labeling or sequential barcoding by multiple round of hybridization were applied to measure mRNA expression of multiple genes within single cells. Research applications of these single molecule single cells DNA and RNA FISH

  14. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

    PubMed Central

    Moffitt, Jeffrey R.; Zhuang, Xiaowei

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

  15. Detection of prokaryotic cells with fluorescence in situ hybridization.

    PubMed

    Zwirglmaier, Katrin

    2010-01-01

    Fluorescence in situ hybridization with rRNA targeted oligonucleotide probes is nowadays one of the core techniques in microbial ecology, allowing the identification and quantification of microbial cells in environmental samples in situ. Next to the classic FISH protocol, which uses fluorescently monolabelled probes, the more sensitive CARD-FISH (also known as TSA-FISH), which involves an enzyme catalyzed signal amplification step, is becoming increasingly popular. This chapter describes protocols for both methods. While classic FISH has the advantage of being relatively cheap and easy to do on morphologically diverse samples, CARD-FISH offers a significantly higher sensitivity, allowing the detection of slow growing or metabolically inactive cells, which are below the detection limit of classic FISH. The drawback here is the considerably higher price for the probes and advanced cell fixation and permeabilization requirements that have to be optimized for different target cells.

  16. Genetic analysis of human milk by fluorescence in situ hybridization.

    PubMed

    Densmore, Lezlie; Pflueger, Solveig

    2006-01-15

    Ductal lavage is a technique for early breast cancer detection in high-risk women. During this procedure, exfoliated epithelial cells are flushed out of the milk ducts of nonlactating women and the collected cells are analyzed for cellular changes associated with breast cancer. A recently developed protocol uses interphase fluorescence in situ hybridization (I-FISH) to detect specific chromosomal aneusomies known to be associated with breast cancer. The ability to perform I-FISH on breast milk will prove to be valuable to lactating women who are at high risk for breast cancer or who develop symptoms while breastfeeding. Using established protocols for analyzing peripheral blood lymphocytes, amniocytes, and epithelial cells in urine and breast duct lavage samples as a guide, a protocol for I-FISH of human breast milk has been developed. Cell isolation, fixation, pretreatment, denaturation, hybridization, and washings were optimized to produce slides of high quality, sensitivity, and specificity.

  17. 10p Duplication characterized by fluorescence in situ hybridization

    SciTech Connect

    Wiktor, A.; Feldman, G.L.; Van Dyke, D.L.; Kratkoczki, P.; Ditmars, D.M. Jr.

    1994-09-01

    We describe a patient with severe failure to thrive, mild-moderate developmental delay, cleft lip and palate, and other anomalies. Routine cytogenetic analysis documented a de novo chromosome rearrangement involving chromosome 4, but the origin of the derived material was unknown. Using chromosome specific painting probes, the karyotype was defined as 46,XY,der(4)t(4;10)(q35;p11.23). Characterization of the dup(10p) by fluorescence in situ hybridization (FISH) analysis provides another example of the usefulness of this technology in identifying small deletions, duplications, or supernumerary marker chromosomes. 19 refs., 4 figs.

  18. "Turn-off" fluorescent data array sensor based on double quantum dots coupled with chemometrics for highly sensitive and selective detection of multicomponent pesticides.

    PubMed

    Fan, Yao; Liu, Li; Sun, Donglei; Lan, Hanyue; Fu, Haiyan; Yang, Tianming; She, Yuanbin; Ni, Chuang

    2016-04-15

    As a popular detection model, the fluorescence "turn-off" sensor based on quantum dots (QDs) has already been successfully employed in the detections of many materials, especially in the researches on the interactions between pesticides. However, the previous studies are mainly focused on simple single track or the comparison based on similar concentration of drugs. In this work, a new detection method based on the fluorescence "turn-off" model with water-soluble ZnCdSe and CdSe QDs simultaneously as the fluorescent probes is established to detect various pesticides. The fluorescence of the two QDs can be quenched by different pesticides with varying degrees, which leads to the differences in positions and intensities of two peaks. By combining with chemometrics methods, all the pesticides can be qualitative and quantitative respectively even in real samples with the limit of detection was 2 × 10(-8) mol L(-1) and a recognition rate of 100%. This work is, to the best of our knowledge, the first report on the detection of pesticides based on the fluorescence quenching phenomenon of double quantum dots combined with chemometrics methods. What's more, the excellent selectivity of the system has been verified in different mediums such as mixed ion disruption, waste water, tea and water extraction liquid drugs.

  19. A highly sensitive label-free sensor for Mercury ion (Hg²⁺) by inhibiting thioflavin T as DNA G-quadruplexes fluorescent inducer.

    PubMed

    Ge, Jia; Li, Xi-Ping; Jiang, Jian-Hui; Yu, Ru-Qin

    2014-05-01

    DNA sequences with guanine repeats can be induced to form G-quartets that adopt G-quadruplex structures in the presence of thioflavin T (ThT). ThT plays a dual role of inducing DNA sequences to fold into quadruplex structures and of sensing the change by its remarkable fluorescence enhancement. ThT binding to the DNA sequences with guanine repeats showed highly specific fluorescence enhancement compared with single/double-stranded DNA. In this work, we have utilized the conformational switch from G-quadruplex complex induced by fluorogenic dye ThT to Hg(2+) mediated T-Hg-T double-stranded DNA formation, thereby pioneering a facile approach to detect Hg(2+) with fluorescence spectrometry. Through this approach, Hg(2+) in aqueous solutions can be detected at 5 nM with fluorescence spectrometry in a facile way, with high selectivity against other metal ions. These results indicate the introduced label-free method for fluorescence spectrometric Hg(2+) detection is simple, quantitative, sensitive, and highly selective.

  20. High sensitivity detection of cancer in vivo using a dual-controlled activation fluorescent imaging probe based on H-dimer formation and pH activation.

    PubMed

    Ogawa, Mikako; Kosaka, Nobuyuki; Regino, Celeste A S; Mitsunaga, Makoto; Choyke, Peter L; Kobayashi, Hisataka

    2010-05-01

    The key to improving the sensitivity of in vivo molecular imaging is to increase the target-to-background signal ratio (TBR). Optical imaging has a distinct advantage over other molecular imaging methods in that the fluorescent signal can be activated at the target thus reducing background signal. Previously, we found that H-dimer formation quenches fluorescence of xanthene fluorophores, and among these, TAMRA had the highest quenching ratio. Another approach to lowering background signal is to employ pH activation based on the photon-induced electron transfer (PeT) theory. We hypothesized that combining these two strategies could lead to greater quenching capacity than was possible with either probe alone. A pH-sensitive fluorophore, pHrodo or TAMRA was conjugated to the cancer targeting molecules, avidin (Av) and trastuzumab (Tra). As expected, both pHrodo and TAMRA formed H-dimers when conjugated to avidin or antibody and the dimerization resulted in efficient fluorescence quenching. In addition, pHrodo conjugated probes showed pH-dependent fluorescence activation. When the probes were used in an in vivo animal model, fluorescence endoscopy with Av-pHrodo depicted tumors with high TBR 1 h and 2 h after injection. Av-TAMRA also visualized tumors 1 h and 2 h after the injection, however, TBR was lower due to the background signal from non-specific binding 1 h after the injection as well as background fluorescence from the unbound agent. Thus, we demonstrate that a dual-controlled activatable optical probe based on the combination of H-dimer formation and pH activation can achieve high TBR at early time points during in vivo molecular imaging.

  1. High-sensitivity detection of breast tumors in vivo by use of a pH-sensitive near-infrared fluorescence probe

    NASA Astrophysics Data System (ADS)

    Mathejczyk, Julia Eva; Pauli, Jutta; Dullin, Christian; Resch-Genger, Ute; Alves, Frauke; Napp, Joanna

    2012-07-01

    We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

  2. A label-free fluorescence DNA probe based on ligation reaction with quadruplex formation for highly sensitive and selective detection of nicotinamide adenine dinucleotide.

    PubMed

    Zhao, Jingjin; Zhang, Liangliang; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

    2012-05-11

    A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs.

  3. Microwave assisted one-pot synthesis of graphene quantum dots as highly sensitive fluorescent probes for detection of iron ions and pH value.

    PubMed

    Zhang, Chunfang; Cui, Yanyan; Song, Li; Liu, Xiangfeng; Hu, Zhongbo

    2016-04-01

    Recently, carbon nanomaterials have received considerable attention as fluorescent probes owing to their low toxicity, water solubility and stable photochemical properties. However, the development of graphene quantum dots (GQDs) is still on its early stage. In this work, GQDs were successfully synthesized by one-step microwave assisted pyrolysis of aspartic acid (Asp) and NH4HCO3 mixture. The as-prepared GQDs exhibited strongly blue fluorescence with high quantum yield up to 14%. Strong fluorescence quenching effect of Fe(3+) on GQDs can be used for its high selectivity detection among of general metal ions. The probe exhibited a wide linear response concentration range (0-50 μM) to Fe(3+) and the limit of detection (LOD) was calculated to be 0.26 μM. In addition, GQDs are also sensitive to the pH value in the range from 2 to 12 indicating a great potential as optical pH sensors. More importantly, the GQDs possess lower cellular toxicity and high photostability and can be directly used as fluorescent probes for cell imaging.

  4. Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

    PubMed Central

    Poppert, Sven; Essig, Andreas; Marre, Reinhard; Wagner, Michael; Horn, Matthias

    2002-01-01

    Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step. PMID:12147510

  5. Engineering cell-fluorescent ion track hybrid detectors

    PubMed Central

    2013-01-01

    Background The lack of sensitive biocompatible particle track detectors has so far limited parallel detection of physical energy deposition and biological response. Fluorescent nuclear track detectors (FNTDs) based on Al2O3:C,Mg single crystals combined with confocal laser scanning microscopy (CLSM) provide 3D information on ion tracks with a resolution limited by light diffraction. Here we report the development of next generation cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). Methods The biocompatibility of FNTDs was tested using six different cell lines, i.e. human non-small cell lung carcinoma (A549), glioblastoma (U87), androgen independent prostate cancer (PC3), epidermoid cancer (A431) and murine (VmDk) glioma SMA-560. To evaluate cell adherence, viability and conformal coverage of the crystals different seeding densities and alternative coating with extracellular matrix (fibronectin) was tested. Carbon irradiation was performed in Bragg peak (initial 270.55 MeV u−1). A series of cell compartment specific fluorescence stains including nuclear (HOECHST), membrane (Glut-1), cytoplasm (Calcein AM, CM-DiI) were tested on Cell-Fit-HDs and a single CLSM was employed to co-detect the physical (crystal) as well as the biological (cell layer) information. Results The FNTD provides a biocompatible surface. Among the cells tested, A549 cells formed the most uniform, viable, tightly packed epithelial like monolayer. The ion track information was not compromised in Cell-Fit-HD as compared to the FNTD alone. Neither cell coating and culturing, nor additional staining procedures affected the properties of the FNTD surface to detect ion tracks. Standard immunofluorescence and live staining procedures could be employed to co-register cell biology and ion track information. Conclusions The Cell-Fit-Hybrid Detector system is a promising platform for a multitude of studies linking biological response to energy deposition at high level of optical microscopy

  6. Paper-based fluorescent sensor for rapid naked-eye detection of acetylcholinesterase activity and organophosphorus pesticides with high sensitivity and selectivity.

    PubMed

    Chang, Jiafu; Li, Haiyin; Hou, Ting; Li, Feng

    2016-12-15

    Various strategies have been proposed for the sensing of acetylcholinesterase (AChE) activity and organophosphorus pesticides (OPs). However, the practical application of most methods is restricted by their intrinsic drawbacks such as complexity, long analysis time, and high cost. Thus, it is highly desirable to develop simple, fast and sensitive approaches for AChE activity and OPs detection. Herein, we reported a simple paper-based fluorescent sensor (PFS) based on the aggregation induced emission (AIE) effect of tetraphenylethylene (TPE) and the addition reaction capability of maleimide, which has been used as a powerful tool for rapid naked-eye detection of AChE activity and OPs. The introduction of TPE provides the probe with unique fluorescence property in solid state and is of great importance for improving the sensitivity of PFS. The hydrolysis product of acetylthiocholine catalyzed by AChE induced the maleimide ring destruction and activated the fluorescence performance of TPE. Given that AChE activity can be specifically inhibited by OPs, the as-proposed PFS can also be utilized for sensitive detection of OPs. Meanwhile, the variation of fluorescence signal can be readily detected by naked eyes, and low detection limits of 2.5mUmL(-1) and 0.5ngmL(-1) for AChE activity and OPs are obtained, respectively. Moreover, it has been successfully applied for AChE activity and OPs detection in diluted human serum samples, showing its great potential to be applied in real samples. Thus, this strategy possesses considerable advantages of simplicity, rapid detection, portability, cost efficiency and visualization.

  7. Efficient ensemble system based on the copper binding motif for highly sensitive and selective detection of cyanide ions in 100% aqueous solutions by fluorescent and colorimetric changes.

    PubMed

    Jung, Kwan Ho; Lee, Keun-Hyeung

    2015-09-15

    A peptide-based ensemble for the detection of cyanide ions in 100% aqueous solutions was designed on the basis of the copper binding motif. 7-Nitro-2,1,3-benzoxadiazole-labeled tripeptide (NBD-SSH, NBD-SerSerHis) formed the ensemble with Cu(2+), leading to a change in the color of the solution from yellow to orange and a complete decrease of fluorescence emission. The ensemble (NBD-SSH-Cu(2+)) sensitively and selectively detected a low concentration of cyanide ions in 100% aqueous solutions by a colorimetric change as well as a fluorescent change. The addition of cyanide ions instantly removed Cu(2+) from the ensemble (NBD-SSH-Cu(2+)) in 100% aqueous solutions, resulting in a color change of the solution from orange to yellow and a "turn-on" fluorescent response. The detection limits for cyanide ions were lower than the maximum allowable level of cyanide ions in drinking water set by the World Health Organization. The peptide-based ensemble system is expected to be a potential and practical way for the detection of submicromolar concentrations of cyanide ions in 100% aqueous solutions.

  8. High-Sensitivity Spectrophotometry.

    ERIC Educational Resources Information Center

    Harris, T. D.

    1982-01-01

    Selected high-sensitivity spectrophotometric methods are examined, and comparisons are made of their relative strengths and weaknesses and the circumstances for which each can best be applied. Methods include long path cells, noise reduction, laser intracavity absorption, thermocouple calorimetry, photoacoustic methods, and thermo-optical methods.…

  9. Highly sensitive determination of nitric oxide in biologic samples by a near-infrared BODIPY-based fluorescent probe coupled with high-performance liquid chromatography.

    PubMed

    Zhang, Hui-Xian; Chen, Jian-Bo; Guo, Xiao-Feng; Wang, Hong; Zhang, Hua-Shan

    2013-11-15

    Nitric oxide (NO) acts as an important regulator and mediator in numerous processes of biological systems. In this work, the analytical potential of a novel near-infrared (NIR, >600 nm) BODIPY-based fluorescent probe for NO, 8-(3,4-diaminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-di(1,2-dihydro) naphtho[b, g]s-indacene (DANPBO-H) has been evaluated in high performance liquid chromatography (HPLC). In 25 mM pH 6.50 borate buffer, DANPBO-H reacted with NO to give the corresponding triazole, DANPBO-H-T, at 35 °C for 20 min. DANPBO-H-T was eluted using a mobile phase of methanol/tetrahydrofuran/50mM pH 7.00 H3Cit-NaOH buffer (81:7:12, v/v/v) in 4 min on a C8 column and detected with fluorescence detection at excitation and emission wavelengths of 621 and 631 nm, respectively. The limit of detection (LOD) (signal-to-noise=3) reached to 5.50×10(-10) M. Excellent selectivity was observed against other reactive oxygen/nitrogen species. Various representative biological matrixes including the whole blood and organs of mice, the pangen and radical of rice, human vascular endothelial (ECV-304) cells and mouse macrophage (RAW 264.7) cells were used to verify the feasibility and resistance to interfering effects from complex biological sample matrixes of the developed DANPBO-H-based HPLC method. Compared to the existing derivatization-based HPLC methods for NO, the proposed method eliminates interfering effects from complex biological sample matrixes efficiently owing to the fluorescence detection in the NIR region, and is more advantageous and robust for the sensitive and selective determination of NO in complex biological samples.

  10. Nitrogen and Phosphorus Co-Doped Carbon Nanodots as a Novel Fluorescent Probe for Highly Sensitive Detection of Fe(3+) in Human Serum and Living Cells.

    PubMed

    Shi, Bingfang; Su, Yubin; Zhang, Liangliang; Huang, Mengjiao; Liu, Rongjun; Zhao, Shulin

    2016-05-04

    Chemical doping with heteroatoms can effectively modulate physicochemical and photochemical properties of carbon dots (CDs). However, the development of multi heteroatoms codoped carbon nanodots is still in its early stage. In this work, a facile hydrothermal synthesis strategy was applied to synthesize multi heteroatoms (nitrogen and phosphorus) codoped carbon nanodots (N,P-CDs) using glucose as carbon source, and ammonia, phosphoric acid as dopant, respectively. Compared with CDs, the multi heteroatoms doped CDs resulted in dramatic improvement in the electronic characteristics and surface chemical activities. Therefore, the N,P-CDs prepared as described above exhibited a strong blue emission and a sensitive response to Fe(3+). The N,P-CDs based fluorescent sensor was then applied to sensitively determine Fe(3+) with a detection limit of 1.8 nM. Notably, the prepared N,P-CDs possessed negligible cytotoxicity, excellent biocompatibility, and high photostability. It was also applied for label-free detection of Fe(3+) in complex biological samples and the fluorescence imaging of intracellular Fe(3+), which indicated its potential applications in clinical diagnosis and other biologically related study.

  11. The application of fluorescence in situ hybridization in different ploidy levels cross-breeding of lily.

    PubMed

    Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

    2015-01-01

    21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid 'Freya' had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true.

  12. Labeling-free fluorescent detection of DNA hybridization through FRET from pyrene excimer to DNA intercalator SYBR green I.

    PubMed

    Zhou, Ruyi; Xu, Chen; Dong, Jie; Wang, Guojie

    2015-03-15

    A novel labeling-free fluorescence complex probe has been developed for DNA hybridization detection based on fluorescence resonance energy transfer (FRET) mechanism from pyrene excimer of pyrene-functionalized poly [2-(N, N-dimethylamino) ethyl methacrylate] (PFP) to SYBR Green I (SG, a specific intercalator of double-stranded DNA) in a cost-effective, rapid and simple manner. The complex probe consists of the positively charged PFP, SG and negatively charged single-stranded DNA (ssDNA). Upon adding a complementary strand to the complex probe solution, double-stranded DNA (dsDNA) was formed, followed by the intercalation of SG into dsDNA. The pyrene excimer emission was overlapped with the absorption of SG very well and the electrostatic interactions between PFP and dsDNA kept them in close proximity, enabling efficient FRET from pyrene excimer to SG. The fluorescence of SG in the duplex DNA resulting from FRET can be successfully applied to detect DNA hybridization with high sensitivity for a very low detection limit of 10nM and excellent selectivity for detection of single base pair mismatch.

  13. Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay.

    PubMed

    Hu, Pan; Yang, Bin

    2016-01-15

    Due to its unique features such as high sensitivity, homogeneous format, and independence on fluorescent intensity, fluorescence anisotropy (FA) assay has become a hotspot of study in oligonucleotide-based bioassays. However, until now most FA probes require carefully customized structure designs, and thus are neither generalizable for different sensing systems nor effective to obtain sufficient signal response. To address this issue, a cleavable DNA-protein hybrid molecular beacon was successfully engineered for signal amplified FA bioassay, via combining the unique stable structure of molecular beacon and the large molecular mass of streptavidin. Compared with single DNA strand probe or conventional molecular beacon, the DNA-protein hybrid molecular beacon exhibited a much higher FA value, which was potential to obtain high signal-background ratio in sensing process. As proof-of-principle, this novel DNA-protein hybrid molecular beacon was further applied for FA bioassay using DNAzyme-Pb(2+) as a model sensing system. This FA assay approach could selectively detect as low as 0.5nM Pb(2+) in buffer solution, and also be successful for real samples analysis with good recovery values. Compatible with most of oligonucleotide probes' designs and enzyme-based signal amplification strategies, the molecular beacon can serve as a novel signal translator to expand the application prospect of FA technology in various bioassays.

  14. eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM).

    PubMed

    Schmitt, Franz-Josef; Thaa, Bastian; Junghans, Cornelia; Vitali, Marco; Veit, Michael; Friedrich, Thomas

    2014-09-01

    The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO

  15. Human cDNA mapping using fluorescence in situ hybridization

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  16. Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes

    NASA Astrophysics Data System (ADS)

    Harrington, Walter N.; Haji, Mwafaq R.; Galanzha, Ekaterina I.; Nedosekin, Dmitry A.; Nima, Zeid A.; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S.; Zharov, Vladimir P.

    2016-11-01

    Photoswitchable fluorescent proteins with controllable light–dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light–dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

  17. Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes

    PubMed Central

    Harrington, Walter N.; Haji, Mwafaq R.; Galanzha, Ekaterina I.; Nedosekin, Dmitry A.; Nima, Zeid A.; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S.; Zharov, Vladimir P.

    2016-01-01

    Photoswitchable fluorescent proteins with controllable light–dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light–dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy. PMID:27824110

  18. Hybrid Integrated Silicon Microfluidic Platform for Fluorescence Based Biodetection

    PubMed Central

    Chandrasekaran, Arvind; Acharya, Ashwin; You, Jian Liang; Soo, Kim Young; Packirisamy, Muthukumaran; Stiharu, Ion; Darveau, Andre

    2007-01-01

    The desideratum to develop a fully integrated Lab-on-a-chip device capable of rapid specimen detection for high throughput in-situ biomedical diagnoses and Point-of-Care testing applications has called for the integration of some of the novel technologies such as the microfluidics, microphotonics, immunoproteomics and Micro Electro Mechanical Systems (MEMS). In the present work, a silicon based microfluidic device has been developed for carrying out fluorescence based immunoassay. By hybrid attachment of the microfluidic device with a Spectrometer-on-chip, the feasibility of synthesizing an integrated Lab-on-a-chip type device for fluorescence based biosensing has been demonstrated. Biodetection using the microfluidic device has been carried out using antigen sheep IgG and Alexafluor-647 tagged antibody particles and the experimental results prove that silicon is a compatible material for the present application given the various advantages it offers such as cost-effectiveness, ease of bulk microfabrication, superior surface affinity to biomolecules, ease of disposability of the device etc., and is thus suitable for fabricating Lab-on-a-chip type devices.

  19. Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes.

    PubMed

    Harrington, Walter N; Haji, Mwafaq R; Galanzha, Ekaterina I; Nedosekin, Dmitry A; Nima, Zeid A; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S; Zharov, Vladimir P

    2016-11-08

    Photoswitchable fluorescent proteins with controllable light-dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light-dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

  20. Hybridization-triggered fluorescence detection of DNA with minor groove binder-conjugated probes

    NASA Astrophysics Data System (ADS)

    Afonina, Irina A.; Lokhov, Sergey G.; Belousov, Yevheniy S.; Reed, Michael W.; Lukhtanov, Eugeny A.; Shishkina, Irina G.; Gorn, Vladimir V.; Sanders, Silvia M.; Walburger, David K.; Hoekstra, Merl F.; Vermeulen, Nicolaas M. J.

    2002-06-01

    Fluorogenic 2'-deoxynucleotide probes containing a minor groove binding-quencher compound at the 5'-end and a fluorophore at the 3'-end, were recently described. These probes fluoresce upon hybridization to the complementary target. The 5'-MGB-quencher group prevents 5'-nuclease digestion by Taq polymerase during homogeneous amplification. The 5'-MGB-quencher-oligonucleotide-fluor (MGB-Q-ODN-Fl) probes displayed a dynamic range of 7 order of magnitude, with an ultimate sensitivity of better than 5 copies per sample. The high sensitivity and specificity is illustrated by the application of the probes in single nucleotide polymorphism detection, final load determination and gene expression analyses. This paper summarizes new developments in sequence detection, gene expression and SNP analysis using new Tm prediction software to design robust 5'-MGB-Q-ODN-Fl probes. Furthermore, the software is capable of estimating the Tm of probes containing a modified base. Due to G:G self-association, many G-rich probes and primers are poor performers in amplification reactions. The software recognizes such sequences and substitution of G with 6-Amino-1,5-dihydro-pyrazolo(3,4- d)pyrimidin-4-one (PPG) is indicated, when necessary to eliminate G:G self-association. Examples of improved performance of PPG containing primers and probes is demonstrated.

  1. Staining with highly sensitive Coomassie brilliant blue SeePico™ Stain after Flamingo™ fluorescent gel stain is useful for cancer proteomic analysis by means of two-dimensional gel electrophoresis.

    PubMed

    Kuramitsu, Yasuhiro; Hayashi, Eiko; Okada, Futoshi; Zhang, Xiulian; Tanaka, Toshiyuki; Ueyama, Yoshiya; Nakamura, Kazuyuki

    2010-10-01

    Highly sensitive Coomassie brilliant blue SeePico™ Stain was applied for proteomic analysis using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). After staining with Flamingo™ Fluorescent Gel Stain, the images of the protein spots were analyzed, and 424 protein spots were detected. After washing with Milli-Q water three times, the gels were re-stained with SeePico™ Stain and the images of the protein spots were analyzed; 272 spots were detected. To assess whether SeePico™ Stain alters MS analysis, a spot was picked up and was analyzed by LC-MS/MS. The MS analysis showed good protein identification. These results show a possible role for SeePico™ Stain in cancer proteomics using 2-DE and MS.

  2. Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics.

    PubMed

    Kondo, Tadashi; Hirohashi, Setsuo

    2006-01-01

    Proteome data combined with histopathological information provides important, novel clues for understanding cancer biology and reveals candidates for tumor markers and therapeutic targets. We have established an application of a highly sensitive fluorescent dye (CyDye DIGE Fluor saturation dye), developed for two-dimensional difference gel electrophoresis (2D-DIGE), to the labeling of proteins extracted from laser microdissected tissues. The use of the dye dramatically decreases the protein amount and, in turn, the number of cells required for 2D-DIGE; the cells obtained from a 1 mm2 area of an 8-12 microm thick tissue section generate up to 5,000 protein spots in a large-format 2D gel. This protocol allows the execution of large-scale proteomics in a more efficient, accurate and reproducible way. The protocol can be used to examine a single sample in 5 d or to examine hundreds of samples in large-scale proteomics.

  3. Low Cost, Low Power, High Sensitivity Magnetometer

    DTIC Science & Technology

    2008-12-01

    Guedes , A.; et al., 2008: Hybrid - LOW COST, LOW POWER, HIGH SENSITIVITY MAGNETOMETER A.S. Edelstein*, James E. Burnette, Greg A. Fischer, M.G...Edelstein, 2004; Burnette, 2008), we suggested a method for mitigating the problem of 1/f noise. We and others ( Guedes , 2008) have been utilizing...6. Guedes , A.; et al., 2008: Hybrid - 3magnetoresistive/microelectromechanical devices for static field modulation and sensor 1/f noise

  4. Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice.

    PubMed

    Pathak, Rupak; Koturbash, Igor; Hauer-Jensen, Martin

    2017-01-11

    Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

  5. Mapping neurofibromatosis 1 homologous loci by fluorescence in situ hybridization

    SciTech Connect

    Viskochil, D.; Breidenbach, H.H.; Cawthon, R.

    1994-09-01

    Neurofibromatosis 1 maps to chromosome band 17q11.2 and the NF1 gene is comprised of 59 exons that span approximately 335 kb of genomic DNA. In order to further analyze the structure of NF1 from exons 2 through 27b, we isolated a number of cosmid and bacteriophage P-1 genomic clones using NF1-exon probes under high-stringency hybridization conditions. Using tagged, intron-based primers and DNA from various clones as a template, we PCR-amplified and sequenced individual NF1 exons. The exon sequences in PCR products from several genomic clones differed from the exon sequence derived from cloned NF1 cDNAs. Clones with variant sequences were mapped by fluorescence in situ hybridization under high-stringency conditions. Three clones mapped to chromosome band 15q11.2, one mapped to 14q11.2, one mapped to both 2q14.1-14.3 and 14q11.2, one mapped to 2q33-34, and one mapped to both 18q11.2 and 21q21. Even though some PCR-product sequences retained proper splice junctions and open reading frames, we have yet to identify cDNAs that correspond to the variant exon sequences. We are now sequencing clones that map to NF1-homologous loci in order to develop discriminating primer pairs for the exclusive amplification of NF1-specific sequences in our efforts to develop a comprehensive NF1 mutation screen using genomic DNA as template. The role of NF1-homologous sequences may play in neurofibromatosis 1 is not clear.

  6. A highly sensitive NADH sensor based on a mycelium-like nanocomposite using graphene oxide and multi-walled carbon nanotubes to co-immobilize poly(luminol) and poly(neutral red) hybrid films.

    PubMed

    Chiang Lin, Kuo; Yu Lai, Szu; Ming Chen, Shen

    2014-08-21

    Hybridization of poly(luminol) (PLM) and poly(neutral red) (PNR) has been successfully performed and further enhanced by a conductive and steric hybrid nanotemplate using graphene oxide (GO) and multi-walled carbon nanotubes (MWCNTs). The morphology of the PLM-PNR-MWCNT-GO mycelium-like nanocomposite is studied by SEM and AFM and it is found to be electroactive, pH-dependent, and stable in the electrochemical system. It shows electrocatalytic activity towards NADH with a high current response and low overpotential. Using amperometry, it has been shown to have a high sensitivity of 288.9 μA mM(-1) cm(-2) to NADH (Eapp. = +0.1 V). Linearity is estimated in a concentration range of 1.33 × 10(-8) to 1.95 × 10(-4) M with a detection limit of 1.33 × 10(-8) M (S/N = 3). Particularly, it also shows another linear range of 2.08 × 10(-4) to 5.81 × 10(-4) M with a sensitivity of 151.3 μA mM(-1) cm(-2). The hybridization and activity of PLM and PNR can be effectively enhanced by MWCNTs and GO, resulting in an active hybrid nanocomposite for determination of NADH.

  7. FMT-PCCT: hybrid fluorescence molecular tomography-x-ray phase-contrast CT imaging of mouse models.

    PubMed

    Mohajerani, Pouyan; Hipp, Alexander; Willner, Marian; Marschner, Mathias; Trajkovic-Arsic, Marija; Ma, Xiaopeng; Burton, Neal C; Klemm, Uwe; Radrich, Karin; Ermolayev, Vladimir; Tzoumas, Stratis; Siveke, Jens T; Bech, Martin; Pfeiffer, Franz; Ntziachristos, Vasilis

    2014-07-01

    The implementation of hybrid fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) has been shown to be a necessary development, not only for combining anatomical with functional and molecular contrast, but also for generating optical images of high accuracy. FMT affords highly sensitive 3-D imaging of fluorescence bio-distribution, but in stand-alone form it offers images of low resolution. It was shown that FMT accuracy significantly improves by considering anatomical priors from CT. Conversely, CT generally suffers from low soft tissue contrast. Therefore utilization of CT data as prior information in FMT inversion is challenging when different internal organs are not clearly differentiated. Instead, we combined herein FMT with emerging X-ray phase-contrast CT (PCCT). PCCT relies on phase shift differences in tissue to achieve soft tissue contrast superior to conventional CT. We demonstrate for the first time FMT-PCCT imaging of different animal models, where FMT and PCCT scans were performed in vivo and ex vivo, respectively. The results show that FMT-PCCT expands the potential of FMT in imaging lesions with otherwise low or no CT contrast, while retaining the cost benefits of CT and simplicity of hybrid device realizations. The results point to the most accurate FMT performance to date.

  8. Fluorescence from a quantum dot and metallic nanosphere hybrid system

    NASA Astrophysics Data System (ADS)

    Schindel, Daniel G.; Singh, Mahi R.

    2014-03-01

    We present energy absorption and interference in a quantum dot-metallic nanosphere system embedded on a dielectric substrate. A control field is applied to induce dipole moments in the nanosphere and the quantum dot, and a probe field is applied to monitor absorption. Dipole moments in the quantum dot or the metal nanosphere are induced, both by the external fields and by each other's dipole fields. Thus, in addition to direct polarization, the metal nanosphere and the quantum dot will sense one another via the dipole-dipole interaction. The density matrix method was used to show that the absorption spectrum can be split from one peak to two peaks by the control field, and this can also be done by placing the metal sphere close to the quantum dot. When the two are extremely close together, a self-interaction in the quantum dot produces an asymmetry in the absorption peaks. In addition, the fluorescence efficiency can be quenched by the addition of a metal nanosphere. This hybrid system could be used to create ultra-fast switching and sensing nanodevices.

  9. Pallister-Killian syndrome detected by fluorescence in situ hybridization

    SciTech Connect

    Butler, M.G.; Dev, V.G.

    1995-07-03

    The Pallister-Killian syndrome is a rare cytogenetic condition first described in 1977 by Pallister et al. in 3 adults; the first affected child was reported in 1981. This syndrome (also known as Pallister mosaic aneuploidy syndrome or isochromosome 12p mosaicism) is characterized by postnatal growth retardation, seizures, hypotonia, deafness, profound mental retardation, minimal speech development, and a distinctive facial appearance (high prominent forehead, ocular hypertelorism, sparse anterior scalp hair, prominent lower lip, large ears with thick protruding lobules, cupid-bow shaped upper lip, and a long philtrum). A chromosome 12 abnormality (tetrasomy 12p) has been reported in skin biopsies from these patients but this chromosome anomaly is usually not found (or in only a small proportion, e.g., <0.5%, of blood cells) in peripheral blood. We report on an additional patient with Pallister-Killian syndrome confirmed with fluorescence in situ hybridization (FISH) using an alpha satellite DNA probe for chromosome 12. This report further illustrates the application of FISH in identifying the source of chromosomal markers of unknown origin in infants with multiple congenital anomalies specifically before the natural history of a condition allows for definitive diagnosis based on clinical findings. 9 refs., 2 figs.

  10. DNA fluorescence shift sensor: a rapid method for the detection of DNA hybridization using silver nanoclusters.

    PubMed

    Lee, Shin Yong; Hairul Bahara, Nur Hidayah; Choong, Yee Siew; Lim, Theam Soon; Tye, Gee Jun

    2014-11-01

    DNA-templated silver nanoclusters (AgNC) are a class of subnanometer sized fluorophores with good photostability and brightness. It has been applied as a diagnostic tool mainly for deoxyribonucleic acid (DNA) detection. Integration of DNA oligomers to generate AgNCs is interesting as varying DNA sequences can result in different fluorescence spectra. This allows a simple fluorescence shifting effect to occur upon DNA hybridization with the hybridization efficiency being a pronominal factor for successful shifting. The ability to shift the fluorescence spectra as a result of hybridization overcomes the issue of background intensities in most fluorescent based assays. Here we describe an optimized method for the detection of single-stranded and double-stranded synthetic forkhead box P3 (FOXP3) target by hybridization with the DNA fluorescence shift sensor. The system forms a three-way junction by successful hybridization of AgNC, G-rich strand (G-rich) to the target DNA, which generated a shift in fluorescence spectra with a marked increase in fluorescence intensity. The DNA fluorescence shift sensor presents a rapid and specific alternative to conventional DNA detection.

  11. Enzyme-triggered tyramine-enzyme repeats on prussian blue-gold hybrid nanostructures for highly sensitive electrochemical immunoassay of tissue polypeptide antigen.

    PubMed

    Xu, Tisen; Zhang, Haiying; Li, Xuegui; Xie, Zhaohui; Li, Xiangyong

    2015-11-15

    A novel sandwich-type electrochemical immunoassay with sensitivity enhancement was developed for quantitative detection of tissue polypeptide antigen (TPA) by coupling with target-induced tyramine signal amplification on prussian blue-gold hybrid nanostructures. The immunosensor was prepared through immobilizing anti-TPA capture antibody on a cleaned screen-printed carbon electrode (SPCE). Prussian blue-gold hybrid nanostructures (PBGNS) labeled with horseradish peroxidase (HRP) and detection antibody were utilized as the signal-transduction tags. Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the electrode. The carried HRP could trigger the formation of tyramine-HRP repeats on the PBGNS in the presence of H2O2. Using the doped prussian blue as the electron mediator, the conjugated HRP could catalyze the reduction of H2O2. Under the optimal conditions, the catalytic currents increased with the increasing target TPA in the dynamic range from 1.0 pg mL(-1) to 100 ng mL(-1) with a detection limit of 0.3 pg mL(-1). The reproducibility and specificity of the electrochemical immunoassay were acceptable. In addition, the contents of target TPA in nine human serum specimens were evaluated by using the developed electrochemical immunosensor, and the obtained results correlated well with those from commercially enzyme-linked immunosorbent assay (ELISA) method with a correlation coefficient of 0.9975.

  12. Competitive RNA-RNA hybridization-based integrated nanostructured-disposable electrode for highly sensitive determination of miRNAs in cancer cells.

    PubMed

    Zouari, M; Campuzano, S; Pingarrón, J M; Raouafi, N

    2017-05-15

    A new method for the detection of miRNAs making use of a competitive RNA/RNA hybridization configuration is described in this work. A biotinylated miRNA (biotin-miRNA) of identical sequence to that of the target miRNA is mixed with the samples to be analyzed allowing competition to be accomplished with the target miRNA for a thiolated RNA probe assembled onto a gold nanoparticles (AuNPs) modified screen-printed electrode. After labeling the hybridized biotin-miRNA with streptavidin-HRP conjugates, amperometric detection at -0.20V was carried out using the H2O2/hydroquinone (HQ) system. The decrease in the amperometric response was proportional to the concentration of model target miRNA-21 in the 100 fM to 25.0 pM range. The integrated sensor provided a very low detection limit (25 fM, 0.25 attomol in 10μL sample) for miRNA-21 without any amplification step, a complete discrimination against single nucleotide mismatched sequences under practical conditions and high storage stability. The usefulness of the developed method was demonstrated by determining the endogenous levels of the mature target miRNA in total RNA (RNAt) extracted from cancerous and non-cancerous cells.

  13. The Application of Fluorescence In Situ Hybridization in Different Ploidy Levels Cross-Breeding of Lily

    PubMed Central

    Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

    2015-01-01

    21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid ‘Freya’ had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true. PMID:26010356

  14. New multifunctional porous materials based on inorganic-organic hybrid single-walled carbon nanotubes: gas storage and high-sensitive detection of pesticides.

    PubMed

    Wang, Feng; Zhao, Jinbo; Gong, Jingming; Wen, Lili; Zhou, Li; Li, Dongfeng

    2012-09-10

    Single-walled carbon nanotubes (SWNTs) that are covalently functionalized with benzoic acid (SWNT-PhCOOH) can be integrated with transition-metal ions to form 3D porous inorganic-organic hybrid frameworks (SWNT-Zn). In particular, N(2)-adsorption analysis shows that the BET surface area increases notably from 645.3 to 1209.9 m(2)  g(-1) for SWNTs and SWNT-Zn, respectively. This remarkable enhancement in the surface area of SWNT-Zn is presumably due to the microporous motifs from benzoates coordinated to intercalated zinc ions between the functionalized SWNTs; this assignment was also corroborated by NLDFT pore-size distributions. In addition, the excess-H(2)-uptake maximum of SWNT-Zn reaches about 3.1 wt. % (12 bar, 77 K), which is almost three times that of the original SWNTs (1.2 wt. % at 12 bar, 77 K). Owing to its inherent conductivity and pore structure, as well as good dispersibility, SWNT-Zn is an effective candidate as a sensitive electrochemical stripping voltammetric sensor for organophosphate pesticides (OPs): By using solid-phase extraction (SPE) with SWNT-Zn-modified glassy carbon electrode, the detection limit of methyl parathion (MP) is 2.3 ng mL(-1).

  15. Synergetic signal amplification of multi-walled carbon nanotubes-Fe3O4 hybrid and trimethyloctadecylammonium bromide as a highly sensitive detection platform for tetrabromobisphenol A

    NASA Astrophysics Data System (ADS)

    Zhou, Feng; Wang, Yue; Wu, Wei; Jing, Tao; Mei, Surong; Zhou, Yikai

    2016-11-01

    In this work, we fabricated an electrochemical sensor based on trimethyloctadecylammonium bromide and multi-walled carbon nanotubes-Fe3O4 hybrid (TOAB/MWCNTs-Fe3O4) for sensitive detection of tetrabromobisphenol A (TBBPA). The nanocomposite was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) techniques. The electrochemical behaviors of TBBPA on TOAB/MWCNTs-Fe3O4 composite film modified glassy carbon electrode (GCE) were investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) method. The experimental results indicated that the incorporation of MWCNTs-Fe3O4 with TOAB greatly enhanced the electrochemical response of TBBPA. This fabricated sensor displayed excellent analytical performance for TBBPA detection over a range from 3.0 nM to 1000.0 nM with a detection limit of 0.73 nM (S/N = 3). Moreover, the proposed electrochemical sensor exhibited good reproducibility and stability, and could be successfully applied to detect TBBPA in water samples with satisfactory results.

  16. Synergetic signal amplification of multi-walled carbon nanotubes-Fe3O4 hybrid and trimethyloctadecylammonium bromide as a highly sensitive detection platform for tetrabromobisphenol A

    PubMed Central

    Zhou, Feng; Wang, Yue; Wu, Wei; Jing, Tao; Mei, Surong; Zhou, Yikai

    2016-01-01

    In this work, we fabricated an electrochemical sensor based on trimethyloctadecylammonium bromide and multi-walled carbon nanotubes-Fe3O4 hybrid (TOAB/MWCNTs-Fe3O4) for sensitive detection of tetrabromobisphenol A (TBBPA). The nanocomposite was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) techniques. The electrochemical behaviors of TBBPA on TOAB/MWCNTs-Fe3O4 composite film modified glassy carbon electrode (GCE) were investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) method. The experimental results indicated that the incorporation of MWCNTs-Fe3O4 with TOAB greatly enhanced the electrochemical response of TBBPA. This fabricated sensor displayed excellent analytical performance for TBBPA detection over a range from 3.0 nM to 1000.0 nM with a detection limit of 0.73 nM (S/N = 3). Moreover, the proposed electrochemical sensor exhibited good reproducibility and stability, and could be successfully applied to detect TBBPA in water samples with satisfactory results. PMID:27897238

  17. Role of fluorescence in situ hybridization (FISH) in sequencing the tomato genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosomes at various stages of the cell cycle can be used for localization of DNA probes via Fluorescence in situ hybridization (FISH). While mitotic metaphase chromosomes are demonstrably too short and compact for this purpose, long pachytene chromosomes are ideal. BACs that hybridize to euchrom...

  18. Fluorescent In Situ Hybridization: A New Tool for the Direct Identification and Detection of F. psychrophilum

    PubMed Central

    Strepparava, Nicole; Wahli, Thomas; Segner, Helmut; Polli, Bruno; Petrini, Orlando

    2012-01-01

    F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium (“Pan-Flavo”) and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum. PMID:23152887

  19. Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

    PubMed Central

    Smith, Leslie; Thayer, Mathew

    2012-01-01

    Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of >3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5. Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within

  20. Control of fluorescence in quantum emitter and metallic nanoshell hybrids for medical applications

    NASA Astrophysics Data System (ADS)

    Singh, Mahi R.; Guo, Jiaohan; J. Cid, José M.; De Hoyos Martinez, Jesús E.

    2017-03-01

    We study the light emission from a quantum emitter and double metallic nanoshell hybrid systems. Quantum emitters act as local sources which transmit their light efficiently due to a double nanoshell near field. The double nanoshell consists of a dielectric core and two outer nanoshells. The first nanoshell is made of a metal, and the second spacer nanoshell is made of a dielectric material or human serum albumin. We have calculated the fluorescence emission for a quantum emitter-double nanoshell hybrid when it is injected in an animal or a human body. Surface plasmon polariton resonances in the double nanoshell are calculated using Maxwell's equations in the quasi-static approximation, and the fluorescence emission is evaluated using the density matrix method in the presence of dipole-dipole interactions. We have compared our theory with two fluorescence experiments in hybrid systems in which the quantum emitter is Indocyanine Green or infrared fluorescent molecules. The outer spacer nanoshell of double metallic nanoshells consists of silica and human serum albumin with variable thicknesses. Our theory explains the enhancement of fluorescence spectra in both experiments. We find that the thickness of the spacer nanoshell layer increases the enhancement when the fluorescence decreases. The enhancement of the fluorescence depends on the type of quantum emitter, spacer layer, and double nanoshell. We also found that the peak of the fluorescence spectrum can be shifted by changing the shape and the size of the nanoshell. The fluorescence spectra can be switched from one peak to two peaks by removing the degeneracy of excitonic states in the quantum emitter. Hence, using these properties, one can use these hybrids as sensing and switching devices for applications in medicine.

  1. A PDMS-Based Cylindrical Hybrid Lens for Enhanced Fluorescence Detection in Microfluidic Systems

    PubMed Central

    Lin, Bor-Shyh; Yang, Yu-Ching; Ho, Chong-Yi; Yang, Han-Yu; Wang, Hsiang-Yu

    2014-01-01

    Microfluidic systems based on fluorescence detection have been developed and applied for many biological and chemical applications. Because of the tiny amount of sample in the system; the induced fluorescence can be weak. Therefore, most microfluidic systems deploy multiple optical components or sophisticated equipment to enhance the efficiency of fluorescence detection. However, these strategies encounter common issues of complex manufacturing processes and high costs. In this study; a miniature, cylindrical and hybrid lens made of polydimethylsiloxane (PDMS) to improve the fluorescence detection in microfluidic systems is proposed. The hybrid lens integrates a laser focusing lens and a fluorescence collecting lens to achieve dual functions and simplify optical setup. Moreover, PDMS has advantages of low-cost and straightforward fabrication compared with conventional optical components. The performance of the proposed lens is first examined with two fluorescent dyes and the results show that the lens provides satisfactory enhancement for fluorescence detection of Rhodamine 6G and Nile Red. The overall increments in collected fluorescence signal and detection sensitivity are more than 220% of those without lens, and the detection limits of Rhodamine 6G and Nile red are lowered to 0.01 μg/mL and 0.05 μg/mL, respectively. The hybrid lens is further applied to the detection of Nile red-labeled Chlorella vulgaris cells and it increases both signal intensity and detection sensitivity by more than 520%. The proposed hybrid lens also dramatically reduces the variation in detected signal caused by the deviation in incident angle of excitation light. PMID:24531300

  2. Mapping pachytene chromosomes of coffee using a modified protocol for fluorescence in situ hybridization

    PubMed Central

    Iacia, Ana Amélia Sanchez; Pinto-Maglio, Cecília A. F.

    2013-01-01

    Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes. PMID:24244840

  3. Magnetic-activated cell sorting (MACS) significantly decreases the hybridization efficiency of fluorescence in situ hybridization (FISH).

    PubMed

    Kuo, P L; Guo, H R

    2001-05-01

    Fetal cells were enriched from maternal blood using density gradient centrifugation of Histopaque followed by magnetic-activated cell sorting (MACS) to select CD71-positive cells. For each specimen, cells partially purified by Histopaque were split into equal portions, and each portion was subjected to purification by MACS in parallel. Cells before and after MACS were subjected to dual-color fluorescence in situ hybridization (FISH) analysis with X- and Y-chromosome-specific probes. We found that the hybridization rates were decreased by approximately 10% after MACS based on duplicated analysis for each sample.

  4. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  5. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  6. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  7. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  8. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 Section 866.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES...

  9. De Novo nonreciprocal translocation 1;8 confirmed by fluorescent in situ hybridization

    SciTech Connect

    Wiley, J.E.; Stout, C.; Palmer, S.M.

    1995-07-17

    Constitutional nonreciprocal translocations are extremely rare, and even their existence is controversial. We report on a newborn infant with a de novo nonreciprocal translocation between chromosomes 1 and 8 resulting in 1q42.3 deletion syndrome. Fluorescent in situ hybridization with whole chromosome paints confirmed the conventional cytogenetic diagnosis. 3 refs., 2 figs., 1 tab.

  10. Fluorescent Properties of Manganese Halide Benzothiazole Inorganic-Organic Hybrids.

    PubMed

    Yu, Hui; Mei, YingXuan; Wei, ZhenHong; Mei, GuangQuan; Cai, Hu

    2016-11-01

    The reaction of manganese (II) halides MnX2 and benzothiazole (btz) in the concentrated acids HX (X = Cl, Br) at 80 °C resulted in the formation of two inorganic-organic hybrid complexes: [(btz)2(MnX4)]·2H2O (X = Cl, 1; X = Br, 2). Both compounds showed green luminescence and exhibited moderate quantum yields of 43.17 % for 1 and 26.18 % for 2, which were directly originated from the tetrahedral coordination of Mn(2+) ion. Two organic - inorganic hybrids [(btz)2(MnX4)]·2H2O based on MnCl2, benzothiazole and halide acids emitted green light with the moderate quantum efficiencies when excited by 365 nm light. Graphical abstract Two organic-inorganic hybrids [(btz)2(MnX4)]·2H2O based on MnCl2, benzothiazole and halide acids emitted green light with the moderate quantum efficiencies when excited by 365 nm light.

  11. RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH).

    PubMed

    Moffitt, J R; Zhuang, X

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule fluorescence in situ hybridization (smFISH)-an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context-provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here, we describe multiplexed error-robust fluorescence in situ hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves.

  12. Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae.

    PubMed

    Cano, R J; Palomares, J C; Torres, M J; Klem, R E

    1992-07-01

    This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.

  13. Hybrid magnetic nanoparticle/nanogold clusters and their distance-dependent metal-enhanced fluorescence effect via DNA hybridization

    NASA Astrophysics Data System (ADS)

    GuThese Authors Contributed Equally To This Study., Xuefan; Wu, Youshen; Zhang, Lingze; Liu, Yongchun; Li, Yan; Yan, Yongli; Wu, Daocheng

    2014-07-01

    To improve the metal-enhanced fluorescence (MEF) effect of nanogolds (AuNPs) and accurately detect specific DNA sequences via DNA hybridization, novel hybrid magnetic nanoparticles/nanogold clusters (HMNCs) were designed based on finite-difference time-domain simulation results and prepared by using Fe3O4 and nanogolds. The nanogolds outside the HMNC were then conjugated with thiol-terminated DNA molecules, thus DNA modified-HMNCs (DNA-HMNCs) were obtained. The size distributions of these nanostructures were measured by a Malvern size analyzer, and their morphology was observed via transmission electron microscopy (TEM). The ultraviolet (UV)-visible (vis) absorption spectra of the samples were recorded with a UV-2600 spectrophotometer. Fluorescence spectra and the MEF effect were recorded using a spectrophotofluorometer, and lifetimes were determined using a time-correlated single photon counting apparatus. The prepared HMNCs were stable in aqueous solutions and had an average diameter of 87 +/- 3.2 nm, with six to eight AuNPs around a single Fe3O4 nanoparticle. Fluorescein isothiocyanate (FITC) tagged DNA-HMNC conjugates exhibited a significant MEF effect and could accurately detect specific DNA sequences after DNA hybridization. This result indicates their various potential applications in sensors and biomedical fields.To improve the metal-enhanced fluorescence (MEF) effect of nanogolds (AuNPs) and accurately detect specific DNA sequences via DNA hybridization, novel hybrid magnetic nanoparticles/nanogold clusters (HMNCs) were designed based on finite-difference time-domain simulation results and prepared by using Fe3O4 and nanogolds. The nanogolds outside the HMNC were then conjugated with thiol-terminated DNA molecules, thus DNA modified-HMNCs (DNA-HMNCs) were obtained. The size distributions of these nanostructures were measured by a Malvern size analyzer, and their morphology was observed via transmission electron microscopy (TEM). The ultraviolet (UV

  14. Fluorescent In Situ Hybridization to Detect Transgene Integration into Plant Genomes

    NASA Astrophysics Data System (ADS)

    Schwarzacher, Trude

    Fluorescent chromosome analysis technologies have advanced our understanding of genome organization during the last 30 years and have enabled the investigation of DNA organization and structure as well as the evolution of chromosomes. Fluorescent chromosome staining allows even small chromosomes to be visualized, characterized by their composition and morphology, and counted. Aneuploidies and polyploidies can be established for species, breeding lines, and individuals, including changes occurring during hybridization or tissue culture and transformation protocols. Fluorescent in situ hybridization correlates molecular information of a DNA sequence with its physical location on chromosomes and genomes. It thus allows determination of the physical position of sequences and often is the only means to determine the abundance and distribution of DNA sequences that are difficult to map with any other molecular method or would require segregation analysis, in particular multicopy or repetitive DNA. Equally, it is often the best way to establish the incorporation of transgenes, their numbers, and physical organization along chromosomes. This chapter presents protocols for probe and chromosome preparation, fluorescent in situ hybridization, chromosome staining, and the analysis of results.

  15. Absorption Reconstruction Improves Biodistribution Assessment of Fluorescent Nanoprobes Using Hybrid Fluorescence-mediated Tomography

    PubMed Central

    Gremse, Felix; Theek, Benjamin; Kunjachan, Sijumon; Lederle, Wiltrud; Pardo, Alessa; Barth, Stefan; Lammers, Twan; Naumann, Uwe; Kiessling, Fabian

    2014-01-01

    Aim: Fluorescence-mediated tomography (FMT) holds potential for accelerating diagnostic and theranostic drug development. However, for proper quantitative fluorescence reconstruction, knowledge on optical scattering and absorption, which are highly heterogeneous in different (mouse) tissues, is required. We here describe methods to assess these parameters using co-registered micro Computed Tomography (µCT) data and nonlinear whole-animal absorption reconstruction, and evaluate their importance for assessment of the biodistribution and target site accumulation of fluorophore-labeled drug delivery systems. Methods: Besides phantoms with varying degrees of absorption, mice bearing A431 tumors were imaged 15 min and 48 h after i.v. injection of a fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) using µCT-FMT. The outer shape of mice and a scattering map were derived using automated segmentation of the µCT data. Furthermore, a 3D absorption map was reconstructed from the trans-illumination data. We determined the absorption of five interactively segmented regions (heart, liver, kidney, muscle, tumor). Since blood is the main near-infrared absorber in vivo, the absorption was also estimated from the relative blood volume (rBV), determined by contrast-enhanced µCT. We compared the reconstructed absorption with the rBV-based values and analyzed the effect of using the absorption map on the fluorescence reconstruction. Results: Phantom experiments demonstrated that absorption reconstruction is possible and necessary for quantitative fluorescence reconstruction. In vivo, the reconstructed absorption showed high values in strongly blood-perfused organs such as the heart, liver and kidney. The absorption values correlated strongly with the rBV-based absorption values, confirming the accuracy of the absorption reconstruction. Usage of homogenous absorption instead of the reconstructed absorption map resulted in reduced values in the heart, liver and kidney, by

  16. Assessment of asthmatic inflammation using hybrid fluorescence molecular tomography-x-ray computed tomography

    NASA Astrophysics Data System (ADS)

    Ma, Xiaopeng; Prakash, Jaya; Ruscitti, Francesca; Glasl, Sarah; Stellari, Fabio Franco; Villetti, Gino; Ntziachristos, Vasilis

    2016-01-01

    Nuclear imaging plays a critical role in asthma research but is limited in its readings of biology due to the short-lived signals of radio-isotopes. We employed hybrid fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) for the assessment of asthmatic inflammation based on resolving cathepsin activity and matrix metalloproteinase activity in dust mite, ragweed, and Aspergillus species-challenged mice. The reconstructed multimodal fluorescence distribution showed good correspondence with ex vivo cryosection images and histological images, confirming FMT-XCT as an interesting alternative for asthma research.

  17. Evaluation of three-dimensional microchannel glass biochips for multiplexed nucleic acid fluorescence hybridization assays.

    PubMed

    Benoit, V; Steel, A; Torres, M; Yu, Y Y; Yang, H; Cooper, J

    2001-06-01

    Three-dimensional, flow-through microchannel glass substrates have a potential for enhanced performance, including increased sensitivity and dynamic range, over traditional planar substrates used in medium-density microarray platforms. This paper presents a methodology for the implementation of multiplexed nucleic acid hybridization fluorescence assays on microchannel glass substrates. Fluorescence detection was achieved, in a first instance, using conventional low-magnification microscope objective lenses, as imaging optics whose depth-of-field characteristics match the thickness of the microchannel glass chip. The optical properties of microchannel glass were shown, through experimental results and simulations, to be compatible with the quantitative detection of heterogeneous hybridization events taking place along the microchannel sidewalls, with detection limits for oligonucleotide targets in the low-attomole range.

  18. Identification of mosaicism in Prader-Willi syndrome using fluorescent in situ hybridization

    SciTech Connect

    Mowery-Rushton, P.A.; Surti, U.; Hanchett, J.M.

    1996-12-30

    We report on our findings of 4 patients with mosaicism for a deletion of chromosome 15, most commonly associated with Prader-Willi syndrome (PWS). We examined a series of typical and atypical PWS patients in order to identify cytogenetically undetected deletions, using fluorescence in situ hybridization. In 4 of the patients analyzed we detected a deletion in 14-60% of peripheral blood leukocytes, using four commercially available probes. Our results indicate that mosaicism may play a role in the etiology of some PWS cases. These findings may be especially useful in patients who display discrepancies between clinical phenotype and established diagnostic criteria. Methylation and microsatellite polymorphism analyses of 2 patients with low-level mosaicism failed to identify the deletion. We propose that fluorescence in situ hybridization is the most effective method for detecting somatic mosaicism, since a large number of cells can be individually examined for the presence or absence of a specific deletion. 47 refs., 5 figs., 3 tabs.

  19. Cytodiagnosis of Extraskeletal Ewing’s Sarcoma and its Confirmation by Fluorescence in situ Hybridization

    PubMed Central

    Singh, Ashish Ranjan; Barwad, Adarsh; Dange, Prasad; Siddaraju, Neelaiah

    2016-01-01

    Extraskeletal Ewing’s sarcoma is an aggressive malignant small round cell tumour usually occuring in children and adolescents. It needs to be differentiated from other malignant small round cell tumours and immunohistochemistry plays a pivotal role in establishing the diagnosis. Fluorescence in situ hybridization or real time-polymerase chain reaction helps in confirming the diagnosis by demonstration of EWS-FLI1 translocation, which is found in approximately 85% of the cases. We report a case of extraskeletal Ewing’s sarcoma in a10-year-old male, who presented with a right gluteal region mass. Fine needle aspiration and cell block preparation followed by a panel of immunohistochemical markers were performed. Immunohistochemistry for CD99 and FLI1 was positive. EWS-FLI1 translocation was confirmed by fluorescence in situ hybridization. PMID:27656453

  20. Toward robust high resolution fluorescence tomography: a hybrid row-action edge preserving regularization

    NASA Astrophysics Data System (ADS)

    Behrooz, Ali; Zhou, Hao-Min; Eftekhar, Ali A.; Adibi, Ali

    2011-02-01

    Depth-resolved localization and quantification of fluorescence distribution in tissue, called Fluorescence Molecular Tomography (FMT), is highly ill-conditioned as depth information should be extracted from limited number of surface measurements. Inverse solvers resort to regularization algorithms that penalize Euclidean norm of the solution to overcome ill-posedness. While these regularization algorithms offer good accuracy, their smoothing effects result in continuous distributions which lack high-frequency edge-type features of the actual fluorescence distribution and hence limit the resolution offered by FMT. We propose an algorithm that penalizes the total variation (TV) norm of the solution to preserve sharp transitions and high-frequency components in the reconstructed fluorescence map while overcoming ill-posedness. The hybrid algorithm is composed of two levels: 1) An Algebraic Reconstruction Technique (ART), performed on FMT data for fast recovery of a smooth solution that serves as an initial guess for the iterative TV regularization, 2) A time marching TV regularization algorithm, inspired by the Rudin-Osher-Fatemi TV image restoration, performed on the initial guess to further enhance the resolution and accuracy of the reconstruction. The performance of the proposed method in resolving fluorescent tubes inserted in a liquid tissue phantom imaged by a non-contact CW trans-illumination FMT system is studied and compared to conventional regularization schemes. It is observed that the proposed method performs better in resolving fluorescence inclusions at higher depths.

  1. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions.

    PubMed

    Hua, Mengjuan; Wang, Chengquan; Qian, Jing; Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping; Wang, Kun

    2015-08-12

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg(2+). The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg(2+), the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg(2+) in a broad linear range of 10 nM-22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg(2+) contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg(2+) quantification related biological systems.

  2. Magneto-fluorescent hybrid of dye and SPION with ordered and radially distributed porous structures

    NASA Astrophysics Data System (ADS)

    Gogoi, Madhulekha; Deb, Pritam

    2014-04-01

    We have reported the development of a silica based magneto-fluorescent hybrid of a newly synthesized dye and superparamagnetic iron oxide nanoparticles with ordered and radially distributed porous structure. The dye is synthesized by a novel yet simple synthetic approach based on Michael addition between dimer of glutaraldehyde and oleylamine molecule. The surfactant used for phase transformation of the dye from organic to aqueous phase, also acts as a structure directing agent for the porous structure evolution of the hybrid with radial distribution. The evolution of the radially distributed pores in the hybrids can be attributed to the formation of rod-like micelles containing nanoparticles, for concentration of micelles greater than critical micelle concentration. A novel water extraction method is applied to remove the surfactants resulting in the characteristic porous structure of the hybrid. Adsorption isotherm analysis confirms the porous nature of the hybrids with pore diameter ∼2.4 nm. A distinct modification in optical and magnetic property is observed due to interaction of the dye and SPION within the silica matrix. The integration of multiple structural components in the so developed hybrid nanosystem results into a potential agent for multifunctional biomedical application.

  3. Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

    1998-01-01

    A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

  4. Bioinspired synthesis of fluorescent calcium carbonate/carbon dot hybrid composites.

    PubMed

    Guo, Shanshan; Yang, Miao; Chen, Min; Zhang, Juan; Liu, Kang; Ye, Ling; Gu, Wei

    2015-05-07

    Herein, we report a novel method to synthesise fluorescent calcium carbonate/carbon dots (CaCO3/CDs) by simply mixing CaCl2 and Na2CO3 solutions in the presence of CDs. There are two roles of CDs in this easy and cost-effective biomimetic strategy, that is as the template to direct the formation and assembly of calcite nanocrystals into hierarchical spheres with diameters in the range of 200-300 nm and simultaneously as the phosphor to enable the CaCO3 to emit blue fluorescence under UV (365 nm) irradiation with a quantum yield of 56.2%. The CaCO3/CD hybrid composites possessing unique fluorescence properties are potentially useful in various applications.

  5. Hybrid lanthanide nanoparticles with paramagnetic shell coated on upconversion fluorescent nanocrystals.

    PubMed

    Li, Zhengquan; Zhang, Yong; Shuter, Borys; Muhammad Idris, Niagara

    2009-10-20

    Nanoparticles comprising of fluorescent probes and MRI contrast agents are highly desirable for biomedical applications due to their ability to be detected at different modes, optically and magnetically. However, most fluorescent probes in such nanoparticles synthesized so far are down-conversion phosphors such as organic dyes and quantum dots, which are known to display many intrinsic limitations. Here, we report a core-shell hybrid lanthanide nanoparticle consisting of an upconverting lanthanide nanocrystal core and a paramagnetic lanthanide complex shell. These nanoparticles are uniform in size, stable in water, and show both high MR relaxivities and upconversion fluorescence, which may have the potential to serve as a versatile imaging tool for smart detection or diagnosis in future biomedical engineering.

  6. MCM-enzyme-supramolecular hydrogel hybrid as a fluorescence sensing material for polyanions of biological significance.

    PubMed

    Wada, Atsuhiko; Tamaru, Shun-ichi; Ikeda, Masato; Hamachi, Itaru

    2009-04-15

    Polyanions are important sensing targets because of their wide variety of biological activities. We report a novel polyanion-selective fluorescence sensing system composed of a hybrid material of supramolecular hydrogel, enzymes, and aminoethyl-modified MCM41-type mesoporous silica particles (NH(2)-MCM41) encapsulating anionic fluorescent dyes. The rational combination of the polyanion-exchange ability of NH(2)-MCM41 and semi-wet supramolecular hydrogel matrix successfully produced three distinct domains; namely, cationic nanopores, hydrophobic nano/microfibers, and aqueous bulk gel phase, which are orthogonal to each other. The coupling of anion-selective probe release from NH(2)-MCM41 with translocation of the probe facilitated by enzymatic reaction enabled fluorescence resonance energy transfer-type sensing in the hybrid materials for polyanions such as heparin, chondroitin sulfate, sucrose octasulfate, and so forth. The enzymatic dephosphorylation catalyzed by phosphatase (alkaline phosphatase or acid phosphatase) that is embedded in gel matrix with retention of activity also contributed to improving the sensing selectivity toward polysulfates relative to polyphosphates. It is clear that the orthogonal domain formation of these materials and maintaining the mobility of the fluorescent dyes between the three domains are crucial for the rapid and convenient sensing provided by this system.

  7. Synthesis and fluorescence properties of six fluorescein-nitroxide radical hybrid-compounds.

    PubMed

    Sato, Shingo; Endo, Susumu; Kurokawa, Yusuke; Yamaguchi, Masaki; Nagai, Akio; Ito, Tomohiro; Ogata, Tateaki

    2016-12-05

    Six fluorescein-nitroxide radical hybrid-compounds (2ab, 3ab, 4, and 5) were synthesized by the condensation of 5- or 6-carboxy-fluorescein and 4-amino-TEMPO (2ab), 5- or 6-aminofluorescein and 4-carboxy-TEMPO (3ab), and fluorescein and 4-carboxy-TEMPO (4), or by reaction of the 3-hydroxyl group of fluorescein with DPROXYL-3-ylmethyl methanesulfonate (5). Fluorescence intensities (around 520nm) after reduction of the radical increased to 1.43-, 1.38-, and 1.61-folds for 2a, 2b and 3b respectively; 3a alone exhibited a decrease in intensity on reduction. Since 4 was readily solvolyzed in PBS or even methanol to afford fluorescein and 4-carboxy-TEMPO, its fluorescence change could not be measured. Hybrid compound 5 containing an ether-linkage between the fluorescein phenol and 3-hydroxymethyl-DPROXYL hydroxyl centers, was stable and on reduction, showed a maximum increase (3.21-fold) in relative fluorescence intensity in PBS (pH5.0), despite its remarkably low absolute fluorescence intensity.

  8. Fluorescent and cross-linked organic-inorganic hybrid nanoshells for monitoring drug delivery.

    PubMed

    Sun, Lijuan; Liu, Tianhui; Li, Hua; Yang, Liang; Meng, Lingjie; Lu, Qinghua; Long, Jiangang

    2015-03-04

    Functionalized and monodisperse nanoshells have attracted significant attention owing to their well-defined structure, unique properties, and wide range of potential applications. Here, the synthesis of cross-linked organic-inorganic hybrid nanoshells with strong fluorescence properties was reported via a facile precipitation polymerization of hexachlorocyclotriphosphazene (HCCP) and fluorescein on silica particles used as templates. The resulting poly(cyclotriphosphazene-co-fluorescein) (PCTPF) nanoshells were firm cross-linked shells with ∼2.2 nm mesopores that facilitated the transport of drug molecules. The fluorescent nanoshells also exhibited excellent water dispersibility and biocompatibility; thus, they can be considered as ideal drug vehicles with high doxorubicin storage capacity (26.2 wt %) and excellent sustained release (up to 14 days). Compared to doxorubicin (DOX) alone, the PCTPF nanoshells more efficiently delivered DOX into and killed cancer cells. Moreover, the PCTPF nanoshells also exhibited remarkable fluorescent emission properties and improved photobleaching stability in both suspension and solid state owing to the covalent immobilization of fluorescein in the highly cross-linked organic-inorganic hybrids. The exceptional fluorescent properties enabled the release of DOX as well as the distribution of nanoshells and DOX to be monitored.

  9. Synthesis and fluorescence properties of six fluorescein-nitroxide radical hybrid-compounds

    NASA Astrophysics Data System (ADS)

    Sato, Shingo; Endo, Susumu; Kurokawa, Yusuke; Yamaguchi, Masaki; Nagai, Akio; Ito, Tomohiro; Ogata, Tateaki

    2016-12-01

    Six fluorescein-nitroxide radical hybrid-compounds (2ab, 3ab, 4, and 5) were synthesized by the condensation of 5- or 6-carboxy-fluorescein and 4-amino-TEMPO (2ab), 5- or 6-aminofluorescein and 4-carboxy-TEMPO (3ab), and fluorescein and 4-carboxy-TEMPO (4), or by reaction of the 3-hydroxyl group of fluorescein with DPROXYL-3-ylmethyl methanesulfonate (5). Fluorescence intensities (around 520 nm) after reduction of the radical increased to 1.43-, 1.38-, and 1.61-folds for 2a, 2b and 3b respectively; 3a alone exhibited a decrease in intensity on reduction. Since 4 was readily solvolyzed in PBS or even methanol to afford fluorescein and 4-carboxy-TEMPO, its fluorescence change could not be measured. Hybrid compound 5 containing an ether-linkage between the fluorescein phenol and 3-hydroxymethyl-DPROXYL hydroxyl centers, was stable and on reduction, showed a maximum increase (3.21-fold) in relative fluorescence intensity in PBS (pH 5.0), despite its remarkably low absolute fluorescence intensity.

  10. Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.

    PubMed

    Nowicka, Anna; Grzebelus, Ewa; Grzebelus, Dariusz

    2012-03-01

    Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot.

  11. Homogeneous competitive hybridization assay based on two-photon excitation fluorescence resonance energy transfer.

    PubMed

    Liu, Lingzhi; Dong, Xiaohu; Lian, Wenlong; Peng, Xiaoniu; Liu, Zhihong; He, Zhike; Wang, Ququan

    2010-02-15

    Recently, we have successfully developed a two-photon excitation fluorescence resonance energy transfer (TPE-FRET)-based homogeneous immunoassay using two-photon excitable small organic molecule as the energy donor. In the present work, the newly emerging TPE-FRET technique was extended to the determination of oligonucleotide. A new TPE molecule with favorable two-photon action cross section was synthesized [2-(2,5-bis(4-(dimethylamino)styryl)-1H-pyrrol-1-yl)acetic acid, abbreviated as TP-COOH], with the tagged reactive carboxyl group allowing facile conjugation with streptavidin (SA). Employing the TP-COOH molecule as energy donor and black hole quencher 1 (BHQ-1) as acceptor, a TPE-FRET-based homogeneous competitive hybridization model was constructed via a biotin-streptavidin bridge. Through the hybridization between a biotinylated single-stranded DNA (ssDNA) and a BHQ-1-linked ssDNA, and the subsequent capture of the as-formed hybrid by TP-COOH labeled SA, the donor fluorescence was quenched due to the FRET between TP-COOH and BHQ-1. Upon the competition between a target ssDNA and the quencher-linked ssDNA toward the biotinylated oligonucleotide, the donor fluorescence was recovered in a target-dependent manner. Good linearity was obtained with the target oligonucleotide ranging from 0.08 to 1.52 microM. The method was applied to spiked serum and urine samples with satisfying recoveries obtained. The results of this work verified the applicability of TPE-FRET technique in hybridization assay and confirmed the advantages of TPE-FRET in complicated matrix.

  12. Fluorescence background subtraction technique for hybrid fluorescence molecular tomography/x-ray computed tomography imaging of a mouse model of early stage lung cancer

    NASA Astrophysics Data System (ADS)

    Ale, Angelique; Ermolayev, Vladimir; Deliolanis, Nikolaos C.; Ntziachristos, Vasilis

    2013-05-01

    The ability to visualize early stage lung cancer is important in the study of biomarkers and targeting agents that could lead to earlier diagnosis. The recent development of hybrid free-space 360-deg fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) imaging yields a superior optical imaging modality for three-dimensional small animal fluorescence imaging over stand-alone optical systems. Imaging accuracy was improved by using XCT information in the fluorescence reconstruction method. Despite this progress, the detection sensitivity of targeted fluorescence agents remains limited by nonspecific background accumulation of the fluorochrome employed, which complicates early detection of murine cancers. Therefore we examine whether x-ray CT information and bulk fluorescence detection can be combined to increase detection sensitivity. Correspondingly, we research the performance of a data-driven fluorescence background estimator employed for subtraction of background fluorescence from acquisition data. Using mice containing known fluorochromes ex vivo, we demonstrate the reduction of background signals from reconstructed images and sensitivity improvements. Finally, by applying the method to in vivo data from K-ras transgenic mice developing lung cancer, we find small tumors at an early stage compared with reconstructions performed using raw data. We conclude with the benefits of employing fluorescence subtraction in hybrid FMT-XCT for early detection studies.

  13. Comparison of culture, polymerase chain reaction, and fluorescent in situ hybridization for detection of Brachyspira hyodysenteriae and "Brachyspira hampsonii" in pig feces.

    PubMed

    Wilberts, Bailey L; Warneke, Hallie L; Bower, Leslie P; Kinyon, Joann M; Burrough, Eric R

    2015-01-01

    Swine dysentery is characterized by mucohemorrhagic diarrhea and can occur following infection by Brachyspira hyodysenteriae or "Brachyspira hampsonii ". A definitive diagnosis is often based on the isolation of strongly beta-hemolytic spirochetes from selective culture or by the application of species-specific polymerase chain reaction (PCR) assays directly to feces. While culture is highly sensitive, it typically requires 6 or more days to complete, and PCR, although rapid, can be limited by fecal inhibition. Fluorescent in situ hybridization (FISH) has been described in formalin-fixed tissues; however, completion requires approximately 2 days. Because of the time constraints of available assays, a same-day FISH assay was developed to detect B. hyodysenteriae and "B. hampsonii " in pig feces using previously described oligonucleotide probes Hyo1210 and Hamp1210 for B. hyodysenteriae and "B. hampsonii", respectively. In situ hybridization was simultaneously compared with culture and PCR on feces spiked with progressive dilutions of spirochetes to determine the threshold of detection for each assay at 0 and 48 hr. The PCR assay on fresh feces and FISH on formalin-fixed feces had similar levels of detection. Culture was the most sensitive method, detecting the target spirochetes at least 2 log-dilutions less when compared to other assays 48 hr after sample preparation. Fluorescent in situ hybridization also effectively detected both target species in formalin-fixed feces from inoculated pigs as part of a previous experiment. Accordingly, FISH on formalin-fixed feces from clinically affected pigs can provide same-day identification and preliminary speciation of spirochetes associated with swine dysentery in North America.

  14. A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes.

    PubMed

    Wang, Dan Ohtan; Matsuno, Hitomi; Ikeda, Shuji; Nakamura, Akiko; Yanagisawa, Hiroyuki; Hayashi, Yasunori; Okamoto, Akimitsu

    2012-01-01

    Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.

  15. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

    PubMed

    Fontenete, Silvia; Silvia, Fontenete; Barros, Joana; Joana, Barros; Madureira, Pedro; Pedro, Madureira; Figueiredo, Céu; Céu, Figueiredo; Wengel, Jesper; Jesper, Wengel; Azevedo, Nuno Filipe; Filipe, Azevedo Nuno

    2015-05-01

    In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

  16. Self-Assembly of Fluorescent Hybrid Core-Shell Nanoparticles and Their Application.

    PubMed

    Wang, Chun; Tang, Fu; Wang, Xiaoyu; Li, Lidong

    2015-06-24

    In this work, a fluorescent hybrid core-shell nanoparticle was prepared by coating a functional polymer shell onto silver nanoparticles via a facile one-pot method. The biomolecule poly-L-lysine (PLL) was chosen as the polymer shell and assembled onto the silver core via the amine-reactive cross-linker, 3,3'-dithiobis(sulfosuccinimidylpropionate). The fluorescent anticancer drug, doxorubicin, was incorporated into the PLL shell through the same linkage. As the cross-linker possesses a thiol-cleavable disulfide bond, disassembly of the PLL shell was observed in the presence of glutathione, leading to controllable doxorubicin release. The silver core there provided an easily modified surface to facilitate the shell coating and ensures the efficient separation of as-prepared nanoparticles from their reaction mixture through centrifugation. Cell assays show that the prepared hybrid fluorescent nanoparticles can internalize into cells possessing excellent biocompatibility prior to the release of doxorubicin, terminating cancer cells efficiently as the doxorubicin is released at the intracellular glutathione level. Such properties are important for designing smart containers for target drug delivery and cellular imaging.

  17. PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples.

    PubMed

    Fazli, Mustafa; Bjarnsholt, Thomas; Høiby, Niels; Givskov, Michael; Tolker-Nielsen, Tim

    2014-01-01

    Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA-FISH can be performed with high specificity due to the extraordinary thermal stability of RNA-PNA hybrid complexes. We describe a PNA-FISH procedure and provide examples of the application of PNA-FISH for the identification of bacteria in chronic wounds, cystic fibrosis lungs, and soft tissue fillers. In all these cases, bacteria can be identified in biofilm aggregates, which may explain their recalcitrance to antibiotic treatment.

  18. Exploring the origin of the D genome of oat by fluorescence in situ hybridization.

    PubMed

    Luo, Xiaomei; Zhang, Haiqin; Kang, Houyang; Fan, Xing; Wang, Yi; Sha, Lina; Zhou, Yonghong

    2014-09-01

    Further understanding of the origin of cultivated oat would accelerate its genetic improvement. In particular, it would be useful to clarify which diploid progenitor contributed the D genome of this allohexaploid species. In this study, we demonstrate that the landmarks produced by fluorescence in situ hybridization (FISH) of species of Avena using probes derived from Avena sativa can be used to explore the origin of the D genome. Selected sets of probes were hybridized in several sequential experiments performed on exactly the same chromosome spreads, with multiple probes of cytological preparations. Probes pITS and A3-19 showed there might be a similar distribution of pITS between the Ac and D genomes. These results indicated that the Ac genome is closely related to the D genome, and that Avena canariensis (AcAc) could be the D-genome donor of cultivated oat.

  19. Fluorescent properties of a hybrid cadmium sulfide-dendrimer nanocomposite and its quenching with nitromethane.

    PubMed

    Campos, Bruno B; Algarra, Manuel; Esteves da Silva, Joaquim C G

    2010-01-01

    A fluorescent hybrid cadmium sulphide quantum dots (QDs) dendrimer nanocomposite (DAB-CdS) synthesised in water and stable in aqueous solution is described. The dendrimer, DAB-G5 dendrimer (polypropylenimine tetrahexacontaamine) generation 5, a diaminobutene core with 64 amine terminal primary groups. The maximum of the excitation and emission spectra, Stokes' shift and the emission full width of half maximum of this nanocomposite are, respectively: 351, 535, 204 and 212 nm. The fluorescence time decay was complex and a four component decay time model originated a good fit (chi = 1.20) with the following lifetimes: tau (1) = 657 ps; tau (2) = 10.0 ns; tau (3) = 59.42 ns; and tau (4) = 265 ns. The fluorescence intensity of the nanocomposite is markedly quenched by the presence of nitromethane with a dynamic Stern-Volmer constant of 25 M(-1). The quenching profiles show that about 81% of the CdS QDs are located in the external layer of the dendrimer accessible to the quencher. PARAFAC analysis of the excitation emission matrices (EEM) acquired as function of the nitromethane concentration showed a trilinear data structure with only one linearly independent component describing the quenching which allows robust estimation of the excitation and emission spectra and of the quenching profiles. This water soluble and fluorescent nanocomposite shows a set of favourable properties to its use in sensor applications.

  20. Multiplexed miRNA fluorescence in situ hybridization for formalin-fixed paraffin-embedded tissues.

    PubMed

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2014-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections.

  1. [Atypical cat eye syndrome. Fluorescence in situ hybridization of metaphase chromosomes].

    PubMed

    Bartsch, O; Aksu, F; Fenner, A; Schwinger, E

    1992-08-01

    In 1983, a chromosome analysis was carried out in a newborn preterm infant with minor anomalies (preauricular skin tag, maldescensus testis). All analysed metaphases showed a small extra chromosome, which was symmetric, dicentric and bi-satellited. In spite of in depth analysis, its origin remained obscure. Recent re-evaluation using fluorescence in situ hybridization (FISH) led to the diagnosis of a dicentric chromosome 22. The FISH technique is an important new tool in chromosome diagnostics. The phenotype of this infant only vaguely resembles the cat eye syndrome. The syndrome should be diagnosed clinically and not only based on the results of chromosome analysis.

  2. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  3. Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

    PubMed

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

  4. Fluorescent in situ hybridization analyses of human oocytes in trisomy 18 and 21

    SciTech Connect

    Cheng, E.Y.; Chen, Y.J.; Gartler, S.M.

    1994-09-01

    The commonly accepted view of synapsis is that only 2 homologues can synapse at any one site and that this restriction applies to polyploids as well. However, triple synapsis has been observed is some triploid plants and in triploid chicken. In humans, triple synapsis of the long arm of chromosome 21 was detected in sperm of a trisomic 21 individual. More recently, studies of oocytes from trisomic 21 and 18 fetuses also indicated extensive triple synapsis along the entire length of the chromosomes. To further investigate this question, we undertook an evaluation of trivalent synapsis in fetal oocytes from 2 trisomic 21 and 2 trisomic 18 fetuses using fluorescent in situ hybridization (FISH) with whole chromosome probes. Oocytes were hybridized with whole chromosome probes obtained from ONCOR, Inc. after fixation with methanol and acetic acid. Slides were scored for the distribution of prophase stages, hybridization efficiency, and hybridization characteristics of chromosomes 18 and 21 in the trisomic 18 and 21 fetuses respectively. Fifty-eight per cent (379/650) of pachytenes analyzed for chromosome 18 contained a conspicous trivalent and 319 (48%) of these nuclei contained a single, thick, continuous fluorescent signal consistent with complete triple synapsis along the entire length of all 3 chromosomes. Sixteen per cent (104/650) of pachytene contained 2 signals consistent with a bivalent and a univalent, and 9 cells contained 3 thin signals consistent with asynapsis of all 3 chromosomes. The remaining 158 pachytenes had unusual pairing configurations that we could not classify, but they most likely represent trivalents with partial pairing between different homologues. In the 2 trisomic 21 fetuses, the majority (143/232) of pachytenes also contained one signal while only 52 cells contained a bivalent and univalent. Five cells contained 3 separate signals. These results confirm the existence of triple synapsis in human meiosis.

  5. 1p36 deletion syndrome confirmed by fluorescence in situ hybridization and array-comparative genomic hybridization analysis

    PubMed Central

    Kang, Dong Soo; Shin, Eunsim

    2016-01-01

    Pediatric epilepsy can be caused by various conditions, including specific syndromes. 1p36 deletion syndrome is reported in 1 in 5,000–10,000 newborns, and its characteristic clinical features include developmental delay, mental retardation, hypotonia, congenital heart defects, seizure, and facial dysmorphism. However, detection of the terminal deletion in chromosome 1p by conventional G-banded karyotyping is difficult. Here we present a case of epilepsy with profound developmental delay and characteristic phenotypes. A 7-year- and 6-month-old boy experienced afebrile generalized seizure at the age of 5 years and 3 months. He had recurrent febrile seizures since 12 months of age and showed severe global developmental delay, remarkable hypotonia, short stature, and dysmorphic features such as microcephaly; small, low-set ears; dark, straight eyebrows; deep-set eyes; flat nasal bridge; midface hypoplasia; and a small, pointed chin. Previous diagnostic work-up, including conventional chromosomal analysis, revealed no definite causes. However, array-comparative genomic hybridization analysis revealed 1p36 deletion syndrome with a 9.15-Mb copy loss of the 1p36.33-1p36.22 region, and fluorescence in situ hybridization analysis (FISH) confirmed this diagnosis. This case highlights the need to consider detailed chromosomal study for patients with delayed development and epilepsy. Furthermore, 1p36 deletion syndrome should be considered for patients presenting seizure and moderate-to-severe developmental delay, particularly if the patient exhibits dysmorphic features, short stature, and hypotonia. PMID:28018437

  6. 1p36 deletion syndrome confirmed by fluorescence in situ hybridization and array-comparative genomic hybridization analysis.

    PubMed

    Kang, Dong Soo; Shin, Eunsim; Yu, Jeesuk

    2016-11-01

    Pediatric epilepsy can be caused by various conditions, including specific syndromes. 1p36 deletion syndrome is reported in 1 in 5,000-10,000 newborns, and its characteristic clinical features include developmental delay, mental retardation, hypotonia, congenital heart defects, seizure, and facial dysmorphism. However, detection of the terminal deletion in chromosome 1p by conventional G-banded karyotyping is difficult. Here we present a case of epilepsy with profound developmental delay and characteristic phenotypes. A 7-year- and 6-month-old boy experienced afebrile generalized seizure at the age of 5 years and 3 months. He had recurrent febrile seizures since 12 months of age and showed severe global developmental delay, remarkable hypotonia, short stature, and dysmorphic features such as microcephaly; small, low-set ears; dark, straight eyebrows; deep-set eyes; flat nasal bridge; midface hypoplasia; and a small, pointed chin. Previous diagnostic work-up, including conventional chromosomal analysis, revealed no definite causes. However, array-comparative genomic hybridization analysis revealed 1p36 deletion syndrome with a 9.15-Mb copy loss of the 1p36.33-1p36.22 region, and fluorescence in situ hybridization analysis (FISH) confirmed this diagnosis. This case highlights the need to consider detailed chromosomal study for patients with delayed development and epilepsy. Furthermore, 1p36 deletion syndrome should be considered for patients presenting seizure and moderate-to-severe developmental delay, particularly if the patient exhibits dysmorphic features, short stature, and hypotonia.

  7. Label-free fluorescent catalytic biosensor for highly sensitive and selective detection of the ferrous ion in water samples using a layered molybdenum disulfide nanozyme coupled with an advanced chemometric model.

    PubMed

    Hu, Jie; Zhuang, Qianfen; Wang, Yong; Ni, Yongnian

    2016-03-07

    In this work, we developed a novel layered molybdenum disulfide (MoS2) nanosheet peroxidase mimetic-based fluorescent catalytic biosensor for the sensitive and selective detection of Fe(2+). It was found that Fe(2+) remarkably enhanced the catalytic activity of the MoS2 nanosheet for oxidation of OPD to form a highly fluorescent substance, 2,3-diaminophenazine (DAPN), and the MoS2/OPD/H2O2 biosensor displayed substantial fluorescence enhancement after addition of Fe(2+) in a concentration-dependent manner. The fluorescence intensity was proportional to the concentration of Fe(2+) over a range of 0.005-0.20 μM with a limit of detection of 3.5 nM (signal/noise = 3). When compared with the OPD/H2O2 biosensor, the MoS2/OPD/H2O2 biosensor provided a higher sensitivity and selectivity for Fe(2+), suggesting the validity of the use of the MoS2 nanosheets. To further demonstrate the feasibility of the MoS2/OPD/H2O2 biosensor for Fe(2+) detection in real water samples, we measured the three-dimensional excitation-emission spectra of the real system, and submitted the excitation-emission matrix (EEM) data to an advanced chemometrics model based on parallel factor analysis (PARAFAC). The results showed that the use of the PARAFAC model could further enhance the selectivity of the biosensor and determine Fe(2+) concentration in the presence of unexpected interferents from real water samples. This work opens up new opportunities for the use of the catalytic properties of the MoS2 nanosheets and advanced chemometrics models in the field of biosensors.

  8. Apoferritin fibers: a new template for 1D fluorescent hybrid nanostructures

    NASA Astrophysics Data System (ADS)

    Jurado, Rocío; Castello, Fabio; Bondia, Patricia; Casado, Santiago; Flors, Cristina; Cuesta, Rafael; Domínguez-Vera, José M.; Orte, Angel; Gálvez, Natividad

    2016-05-01

    Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials.Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a

  9. High-sensitivity magnetic profiling

    SciTech Connect

    Unterberger, R.R.

    1983-05-01

    A high sensitivity rubidium 87 magnetometer, designed and built by the author, is used at sea to make magnetic profiles over subsurface structures of interest. The Texas AandM University Research Vessel GYRE was used to launch, tow and recover a nonmagnetic fiberglass skiff that carried the magnetometer. To avoid magnetic field disturbances of the GYRE, the skiff with the magnetometer was towed 600 ft behind the ship. Loran C, and sometimes SATNAV, position data were used to determine the ship location. Two recording depth finders using 3.5 kHz and 12 kHz respectively were used to profile the bottom. Time marks were plotted on the magnetic and sonar data in accordance with WWV time signals received on 10 MHz. (15 MHz and 5 MHz were also available if there happened to be poor radio transmission at 10 MHz). Magnetic data were recorded in digital form on a strip chart recorder, using the last two digits of the six digit resonance frequency of the Rb 87 atoms.

  10. Detection of Helicobacter pylori in raw bovine milk by fluorescence in situ hybridization (FISH).

    PubMed

    Angelidis, Apostolos S; Tirodimos, Ilias; Bobos, Mattheos; Kalamaki, Mary S; Papageorgiou, Demetrios K; Arvanitidou, Malamatenia

    2011-12-02

    The transmission pathways of Helicobacter pylori in humans have not been fully elucidated. Research in the last decade has proposed that foodborne transmission, among others, may be a plausible route of human infection. Owing to the organism's fastidious growth characteristics and its ability to convert to viable, yet unculturable states upon exposure to stress conditions, the detection of H. pylori in foods via culture-dependent methods has been proven to be laborious, difficult and in most cases unsuccessful. Hence, nucleic acid-based methods have been proposed as alternative methods but, to date, only PCR-based methods have been reported in the literature. In the current study, fluorescence in situ hybridization (FISH) was used for the detection of H. pylori in raw, bulk-tank bovine milk. After repeated milk centrifugation and washing steps, the bacterial flora of raw milk was subjected to fixation and permeabilization and H. pylori detection was conducted by FISH after hybridization with an H. pylori-specific 16S rRNA-directed fluorescent oligonucleotide probe. Using this protocol, H. pylori was detected in four out of the twenty (20%) raw milk samples examined. The data presented in this manuscript indicate that FISH can serve as an alternative molecular method for screening raw bovine milk for the presence of H. pylori.

  11. Same-day prenatal diagnosis of common chromosomal aneuploidies using microfluidics-fluorescence in situ hybridization.

    PubMed

    Ho, Sherry S Y; Chua, Cuiwen; Gole, Leena; Biswas, Arijit; Koay, Evelyn; Choolani, Mahesh

    2012-04-01

    Rapid molecular prenatal diagnostic methods, such as fluorescence in situ hybridization (FISH), quantitative fluorescence-PCR, and multiplex ligation-dependent probe amplification, can detect common fetal aneuploidies within 24 to 48 h. However, specific diagnosis or aneuploidy exclusion should be ideally available within the same day as fetal sampling to alleviate parental anxiety. Microfluidic technologies integrate different steps into a microchip, saving time and costs. We have developed a cost-effective, same-day prenatal diagnostic FISH assay using microfluidics. Amniotic fluids (1-4 mL from 40 pregnant women at 15-22 weeks of gestation) were fixed with Carnoy's before loading into the microchannels of a microfluidic FISH-integrated nanostructured device. The glass slides were coated with nanostructured titanium dioxide to facilitate cell adhesion. Pretreatment and hybridization were performed within the microchannels. Fifty nuclei were counted by two independent analysts, and all results were validated with their respective karyotypes. Of the 40 samples, we found three cases of fetal aneuploidies (trisomies 13, 18, and 21), whereas the remaining 37 cases were normal. Results were concordant with their karyotypes and ready to be released within 3 h of sample receipt. Microfluidic FISH, using 20-fold less than the recommended amount of probe, is a cost-effective method to diagnose common fetal aneuploidies within the same day of fetal sampling.

  12. Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.

    PubMed

    Markaki, Yolanda; Smeets, Daniel; Cremer, Marion; Schermelleh, Lothar

    2013-01-01

    Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry.

  13. Application of locked nucleic acid-based probes in fluorescence in situ hybridization.

    PubMed

    Fontenete, Sílvia; Carvalho, Daniel; Guimarães, Nuno; Madureira, Pedro; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-07-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.

  14. Detection of a complex translocation using fluorescent in situ hybridization (FISH)

    SciTech Connect

    Rosen, B.A.; Abuelo, D.N.; Mark, H.F.

    1994-09-01

    The use of fluorescent in situ hybridization (FISH) allowed the detection of a complex 3-way translocation in a patient with multiple congenital malformations and mental retardation. The patient was a 10-year-old girl with mental retardation, seizures, repaired cleft palate, esotropia, epicanthal folds, broad nasal bridge, upward slanting palpebral fissures, single transverse palmar crease, brachydactyly, hypoplastic nails, ectrodactyly between the third and fourth right toes, and hypoplasia of the left third toe. Chromosome analysis performed at birth was reported as normal. We performed high resolution banding analysis which revealed an apparently balanced translocation between chromosomes 2 and 9. However, because of her multiple abnormalities, further studies were ordered. Fluorescent in situ hybridization (FISH) using chromosome painting probes revealed a karyotype of 46,XX,t(2;8;9) (2pter{yields}q31::8q21.2{yields}8qter; 8pter{yields}q21.2::2q31{yields}q34::9q34{yields}qter; 9pter{yields}q34::2q34{yields}qter). The 3-way translocation appears to be de novo, as neither parent is a translocation carrier. This case illustrates the importance of using FISH to further investigate cases of apparently balanced translocations in the presence of phenotypic abnormalities and/or mental retardation.

  15. Highly Sensitive DNA Sensor Based on Upconversion Nanoparticles and Graphene Oxide.

    PubMed

    Alonso-Cristobal, P; Vilela, P; El-Sagheer, A; Lopez-Cabarcos, E; Brown, T; Muskens, O L; Rubio-Retama, J; Kanaras, A G

    2015-06-17

    In this work we demonstrate a DNA biosensor based on fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er nanoparticles and graphene oxide (GO). Monodisperse NaYF4:Yb,Er nanoparticles with a mean diameter of 29.1 ± 2.2 nm were synthesized and coated with a SiO2 shell of 11 nm, which allowed the attachment of single strands of DNA. When these DNA-functionalized NaYF4:Yb,Er@SiO2 nanoparticles were in the proximity of the GO surface, the π-π stacking interaction between the nucleobases of the DNA and the sp(2) carbons of the GO induced a FRET fluorescence quenching due to the overlap of the fluorescence emission of the NaYF4:Yb,Er@SiO2 and the absorption spectrum of GO. By contrast, in the presence of the complementary DNA strands, the hybridization leads to double-stranded DNA that does not interact with the GO surface, and thus the NaYF4:Yb,Er@SiO2 nanoparticles remain unquenched and fluorescent. The high sensitivity and specificity of this sensor introduces a new method for the detection of DNA with a detection limit of 5 pM.

  16. Estimation of protein concentration at high sensitivity using SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling.

    PubMed

    Arrell, Miriam S; Kálmán, Franka

    2016-11-01

    3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS-CGE with LIF detection (SDS-CGE-LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N-termini, which vary in number and accessibility for different proteins. This work investigates the accuracy of estimation of protein concentration with SDS-CGE-LIF in real biological samples, where a different protein must be used as a standard. Sixteen purified proteins varying in molecular weight, structure, and sequence were labeled with FQ at constant mass concentration applying a commonly used procedure for SDS-CGE-LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a RSD of 36%. This compares favorably with other less sensitive methods for estimation of protein concentration such as SDS-CGE-UV and SDS-PAGE-Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the number of labels bound with UHPLC-ESI-QTOF-MS revealed large variations in the labeling efficiency (percentage of labels to the number of labeling sites given by the sequence) for different proteins (from 3 to 30%). This explains the observation that fluorescence per mole of protein was not proportional to the number of lysines in the sequence.

  17. Chromosome orientation fluorescence in situ hybridization (CO-FISH) to study sister chromatid segregation in vivo

    PubMed Central

    Falconer, Ester; Chavez, Elizabeth; Henderson, Alexander; Lansdorp, Peter M.

    2013-01-01

    Previously, assays for sister chromatid segregation patterns relied on incorporation of BrdU and indirect methods to infer segregation patterns after two cell divisions. Here we describe a method to differentially label sister chromatids of murine cells and directly assay sister chromatid segregation patterns following one cell division in vitro and in vivo by adaptation of the well-established CO-FISH (chromosome orientation fluorescent in situ hybridization) technique. 5-bromo-2′-deoxyuridine (BrdU) is incorporated into newly-formed DNA strands, followed by photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially-labeled sister chromatids in post-mitotic cells are visualized using fluorescence microscopy and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires four days for in vivo mouse tissues, and two days for in vitro cultured cells. PMID:20595964

  18. Fluorescent and Magnetic Mesoporous Hybrid Material: A Chemical and Biological Nanosensor for Hg2+ Ions

    PubMed Central

    Suresh, Moorthy; Anand, Chokkalingam; Frith, Jessica E.; Dhawale, Dattatray S.; Subramaniam, Vishnu P.; Strounina, Ekaterina; Sathish, Clastinrusselraj I.; Yamaura, Kazunari; Cooper-White, Justin J.; Vinu, Ajayan

    2016-01-01

    We introduce “sense, track and separate” approach for the removal of Hg2+ ion from aqueous media using highly ordered and magnetic mesoporous ferrosilicate nanocages functionalised with rhodamine fluorophore derivative. These functionalised materials offer both fluorescent and magnetic properties in a single system which help not only to selectively sense the Hg2+ ions with a high precision but also adsorb and separate a significant amount of Hg2+ ion in aqueous media. We demonstrate that the magnetic affinity of these materials, generated from the ultrafine γ-Fe2O3 nanoparticles present inside the nanochannels of the support, can efficiently be used as a fluorescent tag to sense the Hg2+ ions present in NIH3T3 fibroblasts live cells and to track the movement of the cells by external magnetic field monitored using confocal fluorescence microscopy. This simple approach of introducing multiple functions in the magnetic mesoporous materials raise the prospect of creating new advanced functional materials by fusing organic, inorganic and biomolecules to create advanced hybrid nanoporous materials which have a potential use not only for sensing and the separation of toxic metal ions but also for cell tracking in bio-separation and the drug delivery. PMID:26911660

  19. The design of a microscopic system for typical fluorescent in-situ hybridization applications

    NASA Astrophysics Data System (ADS)

    Yi, Dingrong; Xie, Shaochuan

    2013-12-01

    Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

  20. Fluorescent and Magnetic Mesoporous Hybrid Material: A Chemical and Biological Nanosensor for Hg2+ Ions

    NASA Astrophysics Data System (ADS)

    Suresh, Moorthy; Anand, Chokkalingam; Frith, Jessica E.; Dhawale, Dattatray S.; Subramaniam, Vishnu P.; Strounina, Ekaterina; Sathish, Clastinrusselraj I.; Yamaura, Kazunari; Cooper-White, Justin J.; Vinu, Ajayan

    2016-02-01

    We introduce “sense, track and separate” approach for the removal of Hg2+ ion from aqueous media using highly ordered and magnetic mesoporous ferrosilicate nanocages functionalised with rhodamine fluorophore derivative. These functionalised materials offer both fluorescent and magnetic properties in a single system which help not only to selectively sense the Hg2+ ions with a high precision but also adsorb and separate a significant amount of Hg2+ ion in aqueous media. We demonstrate that the magnetic affinity of these materials, generated from the ultrafine γ-Fe2O3 nanoparticles present inside the nanochannels of the support, can efficiently be used as a fluorescent tag to sense the Hg2+ ions present in NIH3T3 fibroblasts live cells and to track the movement of the cells by external magnetic field monitored using confocal fluorescence microscopy. This simple approach of introducing multiple functions in the magnetic mesoporous materials raise the prospect of creating new advanced functional materials by fusing organic, inorganic and biomolecules to create advanced hybrid nanoporous materials which have a potential use not only for sensing and the separation of toxic metal ions but also for cell tracking in bio-separation and the drug delivery.

  1. Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

    PubMed Central

    Larracuente, Amanda M.; Ferree, Patrick M.

    2015-01-01

    DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH. PMID:25591075

  2. Toward a hybridization assay using fluorescence resonance energy transfer and quantum dots immobilized in microfluidic channels

    NASA Astrophysics Data System (ADS)

    Tavares, Anthony J.; Petryayeva, Eleonora; Algar, W. Russ; Chen, Lu; Krull, Ulrich J.

    2010-06-01

    Quantum dots (QDs) have been widely adopted as integrated components of bioassays and biosensors. In particular, solid phase nucleic acid hybridization assays have been demonstrated to have several advantages and permit the detection of up to four DNA targets simultaneously using fluorescence resonance energy transfer (FRET). This work explores the potential for miniaturization of a solid-phase nucleic acid hybridization assay using QDs and FRET on a microfluidics platform. A method was developed for the immobilization of Streptavidin coated QDs and the preparation of QD-probe oligonucleotide conjugates within microfluidic channels using electrokinetic delivery. Proof-of-concept was demonstrated for the selective detection of target DNA using FRET-sensitized emission from a Cy3 acceptor paired with a green emitting QD donor. The microfluidic platform offered the advantages of smaller sample volumes, nearly undetectable non-specific adsorption, and hybridization within minutes. This work is an important first step toward the development of biochips that enable the multiplexed detection of nucleic acid targets.

  3. [Fluorescence in situ hybridization with DNA probes derived from individual chromosomes and chromosome regions].

    PubMed

    Bogomolov, A G; Karamysheva, T V; Rubtsov, N B

    2014-01-01

    A significant part of the eukaryotic genomes consists of repetitive DNA, which can form large clusters or distributed along euchromatic chromosome regions. Repeats located in chromosomal regions make a problem in analysis and identification of the chromosomal material with fluorescence in situ hybridization (FISH). In most cases, the identification of chromosome regions using FISH requires detection of the signal produced with unique sequences. The feasibility, advantages and disadvantages of traditional methods of suppression of repetitive DNA hybridization, methods of repeats-free probe construction and methods of chromosome-specific DNA sequences visualization using image processing of multicolor FISH results are considered in the paper. The efficiency of different techniques for DNA probe generation, different FISH protocols, and image processing of obtained microscopic images depends on the genomic size and structure of analyzing species. This problem was discussed and different approaches were considered for the analysis of the species with very large genome, rare species and species which specimens are too small in size to obtain the amount of genomic and Cot-1 DNA required for suppression of repetitive DNA hybridization.

  4. A simple and rapid fluorescence in situ hybridization microwave protocol for reliable dicentric chromosome analysis.

    PubMed

    Cartwright, Ian M; Genet, Matthew D; Kato, Takamitsu A

    2013-03-01

    Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80-90°C) step followed by 1-3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well.

  5. Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models.

    PubMed

    Fontenete, Sílvia; Guimarães, Nuno; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-01-01

    The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.

  6. Fast and sensitive DNA hybridization assays using microwave-accelerated metal-enhanced fluorescence.

    PubMed

    Aslan, Kadir; Malyn, Stuart N; Geddes, Chris D

    2006-09-22

    A new, fast, and sensitive DNA hybridization assay platform based on microwave-accelerated metal-enhanced fluorescence (MAMEF) is presented. Thiolated oligonucleotide anchors were immobilized onto silver nanoparticles on a glass substrate. The hybridization of the complementary fluorescein-labeled DNA target with the surface-bound oligonucleotides was completed within 20 s upon heating with low-power microwaves. In addition, the signal is optically amplified, a consequence of close proximity of the fluorophore to the silvered substrate. In this proof-of-principle methodology, as low as 50 nM of a target DNA was detected, although we envisage far-lower detection limits. Control experiments, where the surface-bound oligonucleotide was omitted, were also performed to determine the extent of non-specific binding. In these studies a significantly reduced non-specific adsorption was found when using microwave heating near to silvered structures as compared to room temperature incubation. These findings suggest that MAMEF could be a most useful alternative to the DNA hybridization assays used today, especially with regard to substantially increasing both the assay rapidity and sensitivity.

  7. Quantum-dot-labeled DNA probes for fluorescence in situ hybridization (FISH) in the microorganism Escherichia coli.

    PubMed

    Wu, Sheng-Mei; Zhao, Xiang; Zhang, Zhi-Ling; Xie, Hai-Yan; Tian, Zhi-Quan; Peng, Jun; Lu, Zhe-Xue; Pang, Dai-Wen; Xie, Zhi-Xiong

    2006-05-12

    Semiconductor quantum dots (QDs) as a kind of nonisotopic biological labeling material have many unique fluorescent properties relative to conventional organic dyes and fluorescent proteins, such as composition- and size-dependent absorption and emission, a broad absorption spectrum, photostability, and single-dot sensitivity. These properties make them a promising stable and sensitive label, which can be used for long-term fluorescent tracking and subcellular location of genes and proteins. Here, a simple approach for the construction of QD-labeled DNA probes was developed by attaching thiol-ssDNA to QDs via a metal-thiol bond. The as-prepared QD-labeled DNA probes had high dispersivity, bioactivity, and specificity for hybridization. Based on such a kind of probe with a sequence complementary to multiple clone sites in plasmid pUC18, fluorescence in situ hybridization of the tiny bacterium Escherichia coli has been realized for the first time.

  8. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification.

    PubMed

    Wright, Erik S; Yilmaz, L Safak; Corcoran, Andrew M; Ökten, Hatice E; Noguera, Daniel R

    2014-08-01

    Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).

  9. Novel ultra-high sensitive 'metal resist' for EUV lithography

    NASA Astrophysics Data System (ADS)

    Fujimori, Toru; Tsuchihashi, Toru; Minegishi, Shinya; Kamizono, Takashi; Itani, Toshiro

    2016-03-01

    This study describes the use of a novel ultra-high sensitive `metal resist' for use in extreme ultraviolet (EUV) lithography. Herein, the development of a metal resist has been studied for improving the sensitivity when using metal-containing non-chemically amplified resist materials; such materials are metal-containing organic-inorganic hybrid compounds and are referred to as EUVL Infrastructure Development Center, Inc. (EIDEC) standard metal EUV resist (ESMR). The novel metal resist's ultra-high sensitivity has previously been investigated for use with electron beam (EB) lithography. The first demonstration of ESMR performance was presented in SPIE2015, where it was shown to achieve 17-nm lines with 1.5 mJ/cm2: equivalent in EUV lithography tool. The sensitivity of ESMR using EUV open-flame exposure was also observed to have the same high sensitivity as that when using EB lithography tool. Therefore, ESMR has been confirmed to have the potential of being used as an ultra-high sensitive EUV resist material. The metal-containing organic-inorganic hybrid compounds and the resist formulations were investigated by measuring their sensitivity and line-width roughness (LWR) improvement. Furthermore, new processing conditions, such as new development and rinse procedures, are an extremely effective way of improving lithographic performance. In addition, the optimal dry-etching selective conditions between the metal resist and spin-on carbon (SOC) were obtained. The etched SOC pattern was successfully constructed from a stacked film of metal resist and SOC.

  10. A dumbell probe-mediated rolling circle amplification strategy for highly sensitive transcription factor detection.

    PubMed

    Li, Chunxiang; Qiu, Xiyang; Hou, Zhaohui; Deng, Keqin

    2015-02-15

    Highly sensitive detection of transcription factors (TF) is essential to proteome and genomics research as well as clinical diagnosis. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, quantitative, and inexpensive detection of TF. The strategy consists of a hairpin DNA probe containing a TF binding sequence for target TF, a dumbbell-shaped probe, a primer DNA probe designed partly complementary to hairpin DNA probe, and a dumbbell probe. In the presence of target TF, the binding of the TF with hairpin DNA probe will prohibit the hybridization of the primer DNA probe with the "stem" and "loop" region of the hairpin DNA probe, then the unhybridized region of the primer DNA will hybridize with dumbbell probe, subsequently promote the ligation reaction and the rolling circle amplification (RCA), finally, the RCA products are quantified via the fluorescent intensity of SYBR Green I (SG). Using TATA-binding protein (TBP) as a model transcription factor, the proposed assay system can specifically detect TBP with a detection limit as low as 40.7 fM, and with a linear range from 100 fM to 1 nM. Moreover, this assay related DNA probe does not involve any modification and the whole assay proceeds in one tube, which makes the assay simple and low cost. It is expected to become a powerful tool for bioanalysis and clinic diagnostic application.

  11. Multiplex fluorophore systems on DNA with new diverse fluorescence properties and ability to sense the hybridization dynamics.

    PubMed

    Lee, Dong Gyu; Kim, In Sun; Park, Jung Woo; Seo, Young Jun

    2014-07-14

    We developed a multiplexed fluorophore system on a DNA scaffold (MFD) that produced new and diverse fluorescence properties depending on the mixing pattern and sequence that could not be obtained from each monomer fluorophore. Our approach for producing new fluorescence properties is relatively facile: simply mixing fluorophores on a DNA scaffold provides large variations in the color and intensity using only one excitation wavelength with high "Stokes shifts" (~190 nm). Furthermore these special fluorescence properties could be controlled by the hybridization pattern and were therefore dependent on the structural changes in DNA.

  12. High sensitivity detection of HPV-16 in SiHa and CaSki cells utilizing FISH enhanced by TSA.

    PubMed

    Adler, K; Erickson, T; Bobrow, M

    1997-01-01

    Detection of integrated human papillomavirus type 16 (HPV-16) DNA in SiHa and CaSki cells was used as a model system to demonstrate sensitivity and resolution of a well defined target. Using 293- to 1987-base polymerase chain reaction (PCR)-synthesized probes to the E6 and E7 open reading frames of HPV-16, several fluorescent in situ hybridization (FISH) detection methods, enhanced with tyramide signal amplification (TSA), were compared. The synthetic probes were biotin labeled by a nick translation method and the hybridized probes were detected by various fluorescent TSA methods using cyanine 3 tyramide, biotinyl tyramide and a biotin TSA Plus reagent. High sensitivity detection in SiHa cells was demonstrated using a 619-base probe to detect two single copies of integrated HPV-16 DNA. In CaSki cells, which contain up to 600 copies of HPV-16 DNA, a 293-base probe was used for detection. The results of these comparisons show that with refinement of TSA methods and reagents, increasing levels of high sensitivity detection can be achieved and that these methods allow subnuclear localization as well.

  13. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    PubMed

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay.

  14. Triggering hairpin-free chain-branching growth of fluorescent DNA dendrimers for nonlinear hybridization chain reaction.

    PubMed

    Xuan, Feng; Hsing, I-Ming

    2014-07-16

    We present a nonlinear hybridization chain reaction (HCR) system in which a trigger DNA initiates self-sustained assembly of quenched double-stranded substrates into fluorescent dendritic nanostructures. During the process, an increasing number of originally sequestered trigger sequences labeled with fluorescent reporters are freed up from quenched substrates, leading to chain-branching growth of the assembled DNA dendrimers and an exponential increase in the fluorescence intensity. The triggered assembly behavior was examined by PAGE analysis, and the morphologies of the grown dendrimers were verified by AFM imaging. The exponential kinetics of the fluorescence accumulation was also confirmed by time-dependent fluorescence spectroscopy. This method adopts a simple sequence design strategy, the concept of which could be adapted to program assembly systems with higher-order growth kinetics.

  15. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    PubMed Central

    Hsu, Joanne H.; Zeng, Hui; Lemke, Kalistyn H.; Polyzos, Aris A.; Weier, Jingly F.; Wang, Mei; Lawin-O’Brien, Anna R.; Weier, Heinz-Ulrich G.; O’Brien, Benjamin

    2013-01-01

    Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols. PMID:23344021

  16. Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH)

    PubMed Central

    Lecrenier, M. C.; Ledoux, Q.; Berben, G.; Fumière, O.; Saegerman, C.; Baeten, V.; Veys, P.

    2014-01-01

    Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as “mad cow disease”), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application. PMID:25034259

  17. Chromosome translocations measured by fluorescence in-situ hybridization: A promising biomarker

    SciTech Connect

    Lucas, J.N.; Straume, T.

    1995-10-01

    A biomarker for exposure and risk assessment would be most useful if it employs an endpoint that is highly quantitative, is stable with time, and is relevant to human risk. Recent advances in chromosome staining using fluorescence in situ hybridization (FISH) facilitate fast and reliable measurement of reciprocal translocations, a kind of DNA damage linked to both prior exposure and risk. In contrast to other biomarkers available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time post exposure, has a rather small inter-individual variability, and can be measured accurately at the low levels. Here, the authors discuss results from their studies demonstrating that chromosome painting can be used to reconstruct radiation dose for workers exposed within the dose limits, for individuals exposed a long time ago, and even for those who have been diagnosed with leukemia but not yet undergone therapy.

  18. A hybrid fluorescence tomography and x-ray CT system for quantitative molecular imaging

    NASA Astrophysics Data System (ADS)

    Lin, Yuting; Barber, William C.; Iwanczk, Jan S.; Roeck, Werner W.; Nalcioglu, Orhan; Gulsen, Gultekin

    2010-02-01

    A gantry-based hybrid fluorescence and x-ray computed tomography (FT/CT) system is developed for quantitative molecular imaging. The performance of the dual modality FT/CT system is evaluated using an irregular shaped phantom with an inclusion containing Indocyanine-Green (ICG). The anatomical data from CT provides structural a priori information for the FT inverse problem. Although a 4.2 mm diameter inclusion can be resolved in the reconstructed concentration image without any a priori information, ICG concentration in the inclusion is recovered with 75% error. On the other hand, the error in the recovered ICG concentration reduces to 15% when a priori information from CT is utilized. The results demonstrate that accurate fluorophore concentration can only be obtained when x-ray CT structural a priori information is available.

  19. Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization

    SciTech Connect

    Chen, H.; Tuck-Muller, C.M.; Wertelecki, W.

    1995-03-27

    We report on a 15-year-old black boy with severe mental retardation, multiple congenital anomalies, and a supernumerary ring chromosome mosaicism. Fluorescence in situ hybridization with a chromosome 1 painting probe (pBS1) identified the ring as derived from chromosome 1. The karyotype was 46,XY/47,XY,+r(1)(p13q23). A review showed 8 reports of ring chromosome 1. In 5 cases, the patients had a non-supernumerary ring chromosome 1 resulting in partial monosomies of the short and/or long arm of chromosome 1. In 3 cases, the presence of a supernumerary ring resulted in partial trisomy of different segments of chromosome 1. In one of these cases of the supernumerary ring was composed primarily of the centromere and the heterochromatic region of chromosome 1, resulting in normal phenotype. Our patient represents the third report of a supernumerary ring chromosome 1 resulting in abnormal phenotype. 28 refs., 5 figs., 1 tab.

  20. Partial trisomy 13q identified by sequential fluorescence in situ hybridization

    SciTech Connect

    Gopal Rao, V.V.N.; Carpenter, N.J.; Gucsavas, M.

    1995-07-31

    We report on a 19-month-old boy with partial trisomy 13q resulting from a probable balanced translocation involving chromosomes 1 and 13. The infant presented with omphalocele, malrotation, microcephaly with overriding skull bones, micrognathia, apparently low-set ears, rocker-bottom feet, and congenital heart disease, findings suggestive of trisomy 13. Karyotypic studies from peripheral blood lymphocytes documented an unbalanced karyotype 46,XY,-1,+der. The mother`s chromosomes were normal, and the father was not available. Conventional cytogenetic techniques were unable to identify the extra material on the terminal 1q. Using fluorescence in situ hybridization (FISH) on the GTL-banded metaphases, the extra material on 1q was identified as the terminal long arm of 13, thus resulting in partial trisomy 13 (q32-qter). 8 refs., 2 figs., 1 tab.

  1. Comparison of immunohistochemical and fluorescence in situ hybridization assessment of HER-2 status in routine practice.

    PubMed

    Dolan, Michelle; Snover, Dale

    2005-05-01

    Because HER-2 expression in invasive carcinoma of the breast has well-documented ramifications for treatment and prognosis, accurate assessment of HER-2 status is critical. Comparative studies have shown high concordance rates between immunohistochemical analysis and fluorescence in situ hybridization (FISH) in cases with immunohistochemical scores of 0 or 1+ (negative) and 3+ (strongly positive) and low concordance rates among cases with immunohistochemical scores of 2+. The present study was performed to determine concordance rates in a setting more representative of routine clinical practice, in which multiple pathologists submit specimens to a single cytogenetics referral laboratory. We found a higher rate of discordance between immunohistochemical analysis and FISH (approximately 92%) in the groups with immunohistochemical scores of 2+ than reported in other studies. These results strongly support the practice of performing FISH in all cases with immunohistochemical scores of 2+, particularly in routine practice, in which interobserver variability in immunohistochemical scoring among multiple pathologists is likely to be high.

  2. DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) in buccal cells.

    PubMed

    Cortés-Gutiérrez, E I; Dávila-Rodríguez, M I; Fernández, J L; López-Fernández, C; Gosálvez, J

    2012-12-28

    DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91). In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

  3. High-sensitivity immunoassay with surface plasmon field-enhanced fluorescence spectroscopy using a plastic sensor chip: application to quantitative analysis of total prostate-specific antigen and GalNAcβ1-4GlcNAc-linked prostate-specific antigen for prostate cancer diagnosis.

    PubMed

    Kaya, Takatoshi; Kaneko, Tomonori; Kojima, Shun; Nakamura, Yukito; Ide, Youichi; Ishida, Kenji; Suda, Yoshihiko; Yamashita, Katsuko

    2015-02-03

    A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcβ1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

  4. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

    PubMed Central

    Ronander, Elena; Bengtsson, Dominique C.; Joergensen, Louise; Jensen, Anja T. R.; Arnot, David E.

    2012-01-01

    Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1. Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types. PMID:23070076

  5. Analysis of single-cell gene transcription by RNA fluorescent in situ hybridization (FISH).

    PubMed

    Ronander, Elena; Bengtsson, Dominique C; Joergensen, Louise; Jensen, Anja T R; Arnot, David E

    2012-10-07

    Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System(2) (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

  6. FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

    PubMed Central

    Giorgi, Debora; Farina, Anna; Grosso, Valentina; Gennaro, Andrea; Ceoloni, Carla; Lucretti, Sergio

    2013-01-01

    The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic

  7. Fluorescence in-situ hybridization (FISH) as a tool for visualization and enumeration of Campylobacter in broiler ceca

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food-borne human pathogens are typically detected and enumerated by either cultural methods or PCR-based approaches. Fluorescence in-situ hybridization (FISH) is a standard microscopy tool for microbial ecology but has not been widely used for food safety applications despite important advantages o...

  8. Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures

    PubMed Central

    Kempf, Volkhard A. J.; Trebesius, Karlheinz; Autenrieth, Ingo B.

    2000-01-01

    Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients. PMID:10655393

  9. Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

    PubMed

    Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

    2015-01-01

    Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

  10. Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector

    PubMed Central

    Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

    2015-01-01

    Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD. PMID:26697410

  11. Micro fluorescence in situ hybridization (μFISH) for spatially multiplexed analysis of a cell monolayer.

    PubMed

    Huber, D; Autebert, J; Kaigala, G V

    2016-04-01

    We here present a micrometer-scale implementation of fluorescence in situ hybridization that we term μFISH. This μFISH implementation makes use of a non-contact scanning probe technology, namely, a microfluidic probe (MFP) that hydrodynamically shapes nanoliter volumes of liquid on a surface with micrometer resolution. By confining FISH probes at the tip of this microfabricated scanning probe, we locally exposed approximately 1000 selected MCF-7 cells of a monolayer to perform incubation of probes - the rate-limiting step in conventional FISH. This method is compatible with the standard workflow of conventional FISH, allows re-budgeting of the sample for various tests, and results in a ~ 15-fold reduction in probe consumption. The continuous flow of probes and shaping liquid on these selected cells resulted in a 120-fold reduction of the hybridization time compared with the standard protocol (3 min vs. 6 h) and efficient rinsing, thereby shortening the total FISH assay time for centromeric probes. We further demonstrated spatially multiplexed μFISH, enabling the use of spectrally equivalent probes for detailed and real-time analysis of a cell monolayer, which paves the way towards rapid and automated multiplexed FISH on standard cytological supports.

  12. The role of fluorescence in situ hybridization technologies in molecular diagnostics and disease management.

    PubMed

    King, W; Proffitt, J; Morrison, L; Piper, J; Lane, D; Seelig, S

    2000-12-01

    Large genomic changes, such as aneuploidy, deletions, and other chromosomal rearrangements, have long been associated with pregnancy loss, congenital abnormalities, and malignancy. These genomic changes are quantitative, unambiguous, and fundamental in the transition of normal cells to abnormal ones. Detection of these large genetic changes has an increasingly important role in determining patient diagnosis and care, including therapeutic selection. We have developed two major product platforms that assess genomic changes at various levels of resolution. Fluorescence in situ hybridization (FISH) techniques and the related technology of array-based comparative genomic hybridization (CGH) allow detection of genesized or larger alterations in the genome. FISH is a robust DNA probe technology that can measure both balanced and unbalanced genomic changes on a cell-by-cell basis. In most instances, it is not dependent on metaphase chromosomes, and it is widely used in clinical diagnostics. Array-based CGH has much greater multiplexing capabilities than FISH. This technology has the potential to examine many regions of the genome simultaneously for changes in DNA copy number and identify complex patterns of gains and losses within the genome. In this article, we review several of the current medical applications of FISH and discuss such advanced techniques as CGH and array-based CGH.

  13. Combined RNA/DNA fluorescence in situ hybridization on whole-mount Drosophila ovaries.

    PubMed

    Shpiz, Sergey; Lavrov, Sergey; Kalmykova, Alla

    2014-01-01

    DNA FISH (fluorescent in situ hybridization) analysis reveals the chromosomal location of the gene of interest. RNA in situ hybridization is used to examine the amounts and cell location of transcripts. This method is commonly used to describe the localization of processed transcripts in different tissues or cell lines. Gene activation studies are often aimed at determining the mechanism of this activation (transcriptional or posttranscriptional). Elucidation of the mechanism of piRNA-mediated silencing of genomic repeats is at the cutting edge of small RNA research. The RNA/DNA FISH technique is a powerful method for assessing transcriptional changes at any particular genomic locus. Colocalization of the RNA and DNA FISH signals allows a determination of the accumulation of nascent transcripts at the transcribed genomic locus. This would be suggest that this gene is activated at the transcriptional (or co-transcriptional) level. Moreover, this method allows for the identification of transcriptional derepression of a distinct copy (copies) among a genomic repeat family. Here, a RNA/DNA FISH protocol is presented for the simultaneous detection of RNA and DNA in situ on whole-mount Drosophila ovaries using tyramide signal amplification. With subsequent immunostaining of chromatin components, this protocol can be easily extended for studying the interdependence between chromatin changes at genomic loci and their transcriptional activity.

  14. Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH).

    PubMed

    Deloge-Abarkan, Magali; Ha, Thi-Lan; Robine, Enric; Zmirou-Navier, Denis; Mathieu, Laurence

    2007-01-01

    Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

  15. Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes.

    PubMed

    Macnaughton, S J; O'Donnell, A G; Embley, T M

    1994-10-01

    The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coli and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coli and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl.

  16. X chromosome aneuploidy in infertile women: Analysis by interphase fluorescent in situ hybridization

    SciTech Connect

    Morris, M.A.; Moix, I.; Mermillod, B.

    1994-09-01

    Up to 1 in 3 couples have a problem of infertility at some time in their lives. Sex chromosome anomalies are found in 5-10% of couples, with mosaic aneuploidy being a common finding in primary infertility. Recurrent spontaneous abortion (RSA), in contrast, is frequently associated with autosomal structural anomalies. We hypothesized that low-level mosaic X chromosome aneuploidy was associated with primary infertility but not with RSA. Three groups were studied: women from couples with primary infertillity (n=26); women with three or more spontaneous abortions (n=22); and age-matched normally fertile women (at least two pregnancies; n=28). Interphase fluorescent in situ hybridization (FISH) was used to determine X chromosome ploidy in 100 nuclei per patient, using a contig of three cosmids from MAO locus (kindly donated by W. Berger, Nijmegen). A control probe (chr. 15 centromere) was simultaneously hybridized, and only nuclei containing two control signals were scored for the X chromosome. The mean numbers of nuclei with two X chromosome signals were the same in all groups (Welch equality of means test: p>0.97). However, there is a significant difference between the variances of the primary infertile and RSA groups (Levene`s test: p=0.025 after Bonferrone correction for multiple testing). This provides preliminary support for the hypothesis of an association between primary infertility and low-level mosaic X chromosome aneuploidy.

  17. Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis.

    PubMed

    Milinovich, Gabriel J; Trott, Darren J; Burrell, Paul C; Croser, Emma L; Al Jassim, Rafat A M; Morton, John M; van Eps, Andrew W; Pollitt, Christopher C

    2007-08-01

    Carbohydrate-induced laminitis in horses is characterized by marked changes in the composition of the hindgut microbiota, from a predominantly Gram-negative population to one dominated by Gram-positive bacteria. The objective of this study was to monitor changes in the relative abundance of selected hindgut bacteria that have previously been implicated in the pathophysiology of equine laminitis using fluorescence in situ hybridization (FISH). Caecal cannulae were surgically implanted in five Standardbred horses and laminitis induced by oral administration of a bolus dose of oligofructose. Caecal fluid and faecal specimens were collected over a 48 h period at 2 to 4 h intervals post-oligofructose administration and subjected to FISH using probes specific for nine bacterial groups to determine changes in their relative abundance compared with total bacteria hybridizing to the generic EUBMIX probe. Additionally, hoof biopsies were taken over the course of the experiment at 6 h intervals and evaluated for histopathological changes consistent with laminitis, allowing changes in hindgut microbiota to be correlated with the onset of lesions in the foot. Of the microorganisms specifically targeted, streptococci of the Streptococcus bovis/equinus complex were the only bacteria that consistently proliferated in both caecal fluid and faeces immediately before the onset of histological signs of laminitis. Furthermore, lactobacilli, Enterobacteriaceae, Allisonella histaminiformans, enterococci, Bacteroides fragilis, Mitsuokella jalaludinii and Clostridium difficile did not establish significant populations in the hindgut before the onset of equine laminitis.

  18. PNA-DNA hybridization study using labeled streptavidin by voltammetry and surface plasmon fluorescence spectroscopy.

    PubMed

    Liu, Jianyun; Tiefenauer, Louis; Tian, Shengjun; Nielsen, Peter Eigil; Knoll, Wolfgang

    2006-01-15

    Using ferrocene-streptavidin conjugates as amplifiers, we recently have demonstrated the simultaneous detection of DNA hybridization to peptide nucleic acid (PNA)-modified gold surfaces at the femtomole level by electrochemical and surface plasmon resonance techniques (Liu, J.; Tian, S.; Tiefenauer, L.; Nielsen, P. E.; Knoll, W. Anal. Chem. 2005, 77, 2756-2761). In this paper, a detailed study of the binding behavior of PNA-DNA is presented by square wave voltammetry and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The different binding constants for fully matched and single-mismatched DNA were obtained. The effect of the buffer concentration on the PNA-DNA hybrids was investigated using labeled streptavidin by cyclic voltammetry (CV) and SPFS. At high ionic strength, both the CV and SPFS signals were restrained dramatically, which is most probably due to a conformational change of the short-strand PNA-DNA helices on the surface. We conclude that the combination of electrochemical techniques with SPFS is very useful for the study of short DNA structure transformation.

  19. Effect of Triplet Harvesting on the Lifetime Based on Fluorescence and Phosphorescence in Hybrid White Organic Light Emitting Diodes.

    PubMed

    Lee, Eun; Lee, Ho Won; Yang, Hyung Jin; Sun, Yong; Lee, Jae Woo; Hwang, Kyo Min; Kim, Woo Young; Kim, Young Kwan

    2016-03-01

    We investigated efficient hybrid white organic light emitting diodes (WOLEDs) apply to triplet harvesting (TH) concept based on three complementary colors by mixing containing blue fluorescent emitter with phosphorescent emitters. The TH is to transfer these triplet excitons from a fluorescence to a phosphorescence, where they can decay radiatively. We fabricated several hybrid WOLEDs, having various emitting layer structures with blue fluorescent emitter and red, green phosphorescent emitter. The WOLED exhibited maximum luminous efficiency of 9.02 cd/A, and a maximum external quantum efficiency of 4.17%. The WOLED showed a highly color-stable white emission with the Commission International de L'Éclairage chromaticity of (0.38, 0.36) at 1,000 cd/m2.

  20. Highly bright broadband red light produced by fluorescence polymer/InGaN hybrid light-emitting diodes.

    PubMed

    Lai, Chun-Feng; Chang, Chi-Jung; Hsieh, Cheng-Liang; Chen, Yung-Lin; Tuan, Chi-Shen

    2013-10-15

    The fabrication of fluorescence polymer/InGaN hybrid light-emitting diodes (LEDs) that emit highly bright broadband red light is presented in this Letter. The absorption peak of the fluorescence polymer was 455 nm, and the emission peak was 640 nm. The light output power and external quantum efficiency of hybrid LEDs at a driving current of 100 mA were 46.6 mW and 24.1%, respectively. The emission spectrum of hybrid LEDs was located at a wavelength of 641 nm, with a broadband FWHM of 106 nm. Thus this study offers potential methods for enhancing the output power of commercial white-light-emitting devices.

  1. Enzyme-free fluorescent biosensor for the detection of DNA based on core-shell Fe3O4 polydopamine nanoparticles and hybridization chain reaction amplification.

    PubMed

    Li, Na; Hao, Xia; Kang, Bei Hua; Xu, Zhen; Shi, Yan; Li, Nian Bing; Luo, Hong Qun

    2016-03-15

    A novel, highly sensitive assay for quantitative determination of DNA is developed based on hybridization chain reaction (HCR) amplification and the separation via core-shell Fe3O4 polydopamine nanoparticles (Fe3O4@PDA NPs). In this assay, two hairpin probes are designed, one of which is labeled with a 6-carboxyfluorescein (FAM). Without target DNA, auxiliary hairpin probes are stable in solution. However, when target DNA is present, the HCR between the two hairpins is triggered. The HCR products have sticky ends of 24 nt, which are much longer than the length of sticky ends of auxiliary hairpins (6 nt) and make the adsorption much easier by Fe3O4@PDA NPs. With the addition of Fe3O4@PDA NPs, HCR products could be adsorbed because of the strong interaction between their sticky ends and Fe3O4@PDA NPs. As a result, supernatant of the solution with target DNA emits weak fluorescence after separation by magnet, which is much lower than that of the blank solution. The detection limit of the proposed method is as low as 0.05 nM. And the sensing method exhibits high selectivity for the determination between perfectly complementary sequence and target with single base-pair mismatch. Importantly, the application of the sensor for DNA detection in human serum shows that the proposed method works well for biological samples.

  2. Distribution of 45S rDNA in Modern Rose Cultivars (Rosa hybrida), Rosa rugosa, and Their Interspecific Hybrids Revealed by Fluorescence in situ Hybridization.

    PubMed

    Ding, Xiao-Liu; Xu, Ting-Liang; Wang, Jing; Luo, Le; Yu, Chao; Dong, Gui-Min; Pan, Hui-Tang; Zhang, Qi-Xiang

    2016-01-01

    To elucidate the evolutionary dynamics of the location and number of rDNA loci in the process of polyploidization in the genus Rosa, we examined 45S rDNA sites in the chromosomes of 6 modern rose cultivars (R. hybrida), 5 R. rugosa cultivars, and 20 hybrid progenies by fluorescence in situ hybridization. Variation in the number of rDNA sites in parents and their interspecific hybrids was detected. As expected, 4 rDNA sites were observed in the genomes of 4 modern rose cultivars, while 3 hybridization sites were observed in the 2 others. Two expected rDNA sites were found in 2 R. rugosa cultivars, while in the other 3 R. rugosa cultivars 4 sites were present. Among the 20 R. hybrida × R. rugosa offspring, 13 carried the expected number of rDNA sites, and 1 had 6 hybridization sites, which exceeded the expected number by far. The other 6 offspring had either 2 or 3 hybridization sites, which was less than expected. Differences in the number of rDNA loci were observed in interspecific offspring, indicating that rDNA loci exhibit instability after distant hybridization events. Abnormal chromosome pairing may be the main factor explaining the variation in rDNA sites during polyploidization.

  3. Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

    PubMed

    Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

    2017-03-01

    This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace.

  4. Label-free fluorescence dual-amplified detection of adenosine based on exonuclease III-assisted DNA cycling and hybridization chain reaction.

    PubMed

    Sun, Jiewei; Jiang, Wei; Zhu, Jing; Li, Wei; Wang, Lei

    2015-08-15

    In this work, we constructed a label-free and dual-amplified fluorescence aptasensor for sensitive analysis of adenosine based on exonuclease III (Exo III)-assisted DNA cycling and hybridization chain reaction (HCR). Firstly, we fabricated a trifunctional probe that consisting of the catalytic strand, the aptamer sequence and a streptavidin-magnetic nanobead (streptavidin-MNB). The streptavidin-MNB played a role of enrichment and separation to achieve a low background. The aptamer sequence was employed as a recognition element to bind the target adenosine, leading to the releasing of the catalytic stand. Then, the catalytic strand induced the Exo III-assisted DNA cycling reaction and produced a large amount of DNA fragments, which got a primary amplification. Subsequently, the DNA fragments acted as trigger strands to initiate HCR, forming nicked double helices with multiple G-quadruplex structures, which achieved a secondary amplification. Finally, the G-quadruplex structures bonded with the N-nethyl mesopor-phyrin IX (NMM) and yielded an enhanced fluorescence signal, realizing the label-free detection. In the proposed strategy, a small amount of adenosine can be converted to a large amount of DNA triggers, leading to a significant amplification for the target. This method exhibited a high sensitivity toward adenosine with a detection limit of 4.2×10(-7) mol L(-1), which was about 10 times lower than that of the reported label-free strategies. Moreover, this assay can significantly distinguish the content of adenosine in urine samples of cancer patients and normal human, indicating that our method will offer a new strategy for reliable quantification of adenosine in medical research and early clinical diagnosis.

  5. Magnified fluorescence detection of silver(I) ion in aqueous solutions by using nano-graphite-DNA hybrid and DNase I.

    PubMed

    Wei, Yin; Li, Bianmiao; Wang, Xu; Duan, Yixiang

    2014-08-15

    This paper describes a novel approach utilizing nano-graphite-DNA hybrid and DNase I for the amplified detection of silver(I) ion in aqueous solutions for the first time. Nano-graphite can effectively quench the fluorescence of dye-labeled cytosine-rich single-stranded DNA due to its strong π-π stacking interactions; however, in the presence of Ag(+), C-Ag(+)-C coordination induces the probe to fold into a hairpin structure, which does not adsorb on the surface of nano-graphite and thus retains the dye fluorescence. Meanwhile, the hairpin structure can be cleaved by DNase I, and in such case Ag(+) is delivered from the complex. The released Ag(+) then binds other dye-labeled single-stranded DNA on the nano-graphite surface, and touches off another target recycling, resulting in the successive release of dye-labeled single-stranded DNA from the nano-graphite, which leads to significant amplification of the signal. The present magnification sensing system exhibits high sensitivity toward Ag(+) with a limit of detection of 0.3nM (S/N=3), which is much lower than the standard for Ag(+) in drinking water recommended by the Environmental Protection Agency (EPA). The selectivity of the sensor for Ag(+) against other biologically and environmentally related metal ions is outstanding due to the high specificity of C-Ag(+)-C formation. Moreover, the sensing system is used for the determination of Ag(+) in river water samples with satisfying results. The proposed assay is simple, cost-effective, and might open the door for the development of new assays for other metal ions or biomolecules.

  6. Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions

    PubMed Central

    Masuko, Masayuki; Ohuchi, Shohkichi; Sode, Koji; Ohtani, Hiroyuki; Shimadzu, Akira

    2000-01-01

    We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32mer and its complementary two sequential 16mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of ~100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 Å (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleotides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 Å. Comparison between the fluorescent properties of a pyrene–perylene pair with those of a widely used fluorescein–rhodamine pair showed that the pyrene–perylene FRET is suitable for hybridization assays. PMID:10734211

  7. Dynamics of hybrid amoeba proteus containing zoochlorellae studied using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, C.-H.; Fong, B. A.; Alfano, S. A., Jr.; Rakhlin, I.; Wang, W. B.; Ni, X. H.; Yang, Y. L.; Zhou, F.; Zuzolo, R. C.; Alfano, R. R.

    2011-03-01

    The microinjection of organelles, plants, particles or chemical solutions into Amoeba proteus coupled with spectroscopic analysis and observed for a period of time provides a unique new model for cancer treatment and studies. The amoeba is a eukaryote having many similar features of mammalian cells. The amoeba biochemical functions monitored spectroscopically can provide time sequence in vivo information about many metabolic transitions and metabolic exchanges between cellar organelles and substances microinjected into the amoeba. It is possible to microinject algae, plant mitochondria, drugs or carcinogenic solutions followed by recording the native fluorescence spectra of these composites. This model can be used to spectroscopically monitor the pre-metabolic transitions in developing diseased cells such as a cancer. Knowing specific metabolic transitions could offer solutions to inhibit cancer or reverse it as well as many other diseases. In the present study a simple experiment was designed to test the feasibility of this unique new model by injecting algae and chloroplasts into amoeba. The nonradiative dynamics found from these composites are evidence in terms of the emission ratios between the intensities at 337nm and 419nm; and 684nm bands. There were reductions in the metabolic and photosynthetic processes in amoebae that were microinjected with chloroplasts and zoochlorellae as well of those amoebae that ingested the algae and chloroplasts. The changes in the intensity of the emissions of the peaks indicate that the zoochlorellae lived in the amoebae for ten days. Spectral changes in intensity under the UV and 633nm wavelength excitation are from the energy transfer of DNA and RNA, protein-bound chromophores and chlorophylls present in zoochlorellae undergoing photosynthesis. The fluorescence spectroscopic probes established the biochemical interplay between the cell organelles and the algae present in the cell cytoplasm. This hybrid state is indicative

  8. Fluorescent Nanodiamond-Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging.

    PubMed

    Liu, Weina; Naydenov, Boris; Chakrabortty, Sabyasachi; Wuensch, Bettina; Hübner, Kristina; Ritz, Sandra; Cölfen, Helmut; Barth, Holger; Koynov, Kaloian; Qi, Haoyuan; Leiter, Robert; Reuter, Rolf; Wrachtrup, Jörg; Boldt, Felix; Scheuer, Jonas; Kaiser, Ute; Sison, Miguel; Lasser, Theo; Tinnefeld, Philip; Jelezko, Fedor; Walther, Paul; Wu, Yuzhou; Weil, Tanja

    2016-10-12

    There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analysis of FND-Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-dimensional cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of FND-Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in subcellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual FND-Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND-Au unique candidates for multimodal optical-electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

  9. Fluorescent in situ hybridization (FISH) assessment of chromosome copy number in sperm

    SciTech Connect

    Sheu, M.; Sigman, M.; Mark, H.F.L.

    1994-09-01

    Approximately 15% of all recognized pregnancies end in spontaneous abortions. The overall frequency of chromosome abnormalities in spontaneous abortions is approximately 50%. Thus aneuploidy is a significant cause of fetal wastage. In addition, structural and numerical abnormalities of chromosomes can also lead to birth defects, developmental delay, mental retardation and infertility. Conventional cytogenetic analysis via GTG- and other banding techniques is a powerful tool in the elucidation of the nature of chromosomal abnormalities. Fluorescent in situ hybridization (FISH) enables detection of numerical chromosomal abnormalities, especially trisomies, in intact cells. Using FISH and commercially available biotin-labeled probes, we have initiated a prospective study to assess specific chromosome copy number of preparations of unstained smears from men referred for a male infertility evaluation as well as smears from normal control males chosen randomly from the sample of sperm donors. A total of approximately 19,000 sperm nuclei have been examined thus far. Of those suitable for analysis, 7382 (38.75%) were normal possessing one copy of chromosome 8, 155 (0.81%) were disomic, and 15 (0.079%) had more than two copies of chromosome 8. Comparisons with data available in the literature will be discussed. Work is ongoing to increase the efficiency of hybridization using both reported and previously untried pretreatment and fixation protocols. We have also initiated studies using multicolor FISH with various chromosome enumeration probes. The assay described here is a potentially powerful tool for detecting rare events such as spontaneous germ cell aneuploidy, aneuploidy detected in semen from men with carcinoma in situ of the testis and aneuploidy induced by potential environmental genotoxicants. It can also be utilized for segregation analysis and for correlating chromosome copy number with germ cell morphology.

  10. Faster identification of pathogens in positive blood cultures by fluorescence in situ hybridization in routine practice.

    PubMed

    Peters, Remco P H; Savelkoul, Paul H M; Simoons-Smit, Alberdina M; Danner, Sven A; Vandenbroucke-Grauls, Christina M J E; van Agtmael, Michiel A

    2006-01-01

    Rapid identification of microorganisms in blood cultures is required to optimize empirical treatment at an early stage. Fluorescence in situ hybridization (FISH) can reduce the time to identification of microorganisms in growth-positive blood cultures. In this study, we evaluated the performance, time to identification, and potential clinical benefits of FISH compared to those of conventional culture methods in routine practice. After Gram staining, blood culture fluids were simultaneously further identified with FISH and with conventional culture methods. Results and points in time of FISH and culture identification (provisional and final identifications) were collected and compared. For 91% of microorganisms, the genus or family name was identified, and for 79%, the species name could be attributed. The sensitivity and specificity of the individual probes exceeded 95%, except for the Enterobacteriaceae probe (sensitivity, 89%). Cross-hybridization was obtained with the Klebsiella pneumoniae probe for Klebsiella oxytoca. The time gains of FISH and final culture identification were more than 18 h for bacteria and 42 h for yeasts. With FISH, Staphylococcus aureus was differentiated from coagulase-negative staphylococci 1.4 h faster than by provisional identification (P < 0.001). In conclusion, FISH allows rapid and reliable identification of the majority of microorganisms in growth-positive blood cultures. The substantial time gain of identification with FISH may allow same-day adjustment of antimicrobial therapy, and FISH is especially useful if no provisional identification is obtained. With further extension of the number of probes and a reduction in turnaround time, FISH will become a very useful diagnostic tool in the diagnosis of bloodstream infections.

  11. Camera-based ratiometric fluorescence transduction of nucleic acid hybridization with reagentless signal amplification on a paper-based platform using immobilized quantum dots as donors.

    PubMed

    Noor, M Omair; Krull, Ulrich J

    2014-10-21

    Paper-based diagnostic assays are gaining increasing popularity for their potential application in resource-limited settings and for point-of-care screening. Achievement of high sensitivity with precision and accuracy can be challenging when using paper substrates. Herein, we implement the red-green-blue color palette of a digital camera for quantitative ratiometric transduction of nucleic acid hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). A nonenzymatic and reagentless means of signal enhancement for QD-FRET assays on paper substrates is based on the use of dry paper substrates for data acquisition. This approach offered at least a 10-fold higher assay sensitivity and at least a 10-fold lower limit of detection (LOD) as compared to hydrated paper substrates. The surface of paper was modified with imidazole groups to assemble a transduction interface that consisted of immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs was responsible for a FRET-sensitized emission from the acceptor dye, which served as an analytical signal. A hand-held UV lamp was used as an excitation source and ratiometric analysis using an iPad camera was possible by a relative intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an

  12. Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

    PubMed Central

    Stender, Henrik; Kurtzman, Cletus; Hyldig-Nielsen, Jens J.; Sørensen, Ditte; Broomer, Adam; Oliveira, Kenneth; Perry-O'Keefe, Heather; Sage, Andrew; Young, Barbara; Coull, James

    2001-01-01

    A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity. PMID:11157265

  13. Use of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Definitive, Rapid Identification of Five Common Candida Species▿

    PubMed Central

    Reller, Megan E.; Mallonee, Amanda B.; Kwiatkowski, Nicole P.; Merz, William G.

    2007-01-01

    We investigated a 2.5-h peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay with five Candida species-specific probes to identify Candida colonies and compared it to standard 2-h to 5-day phenotypic identification methods. Suspensions were made and slides were prepared and read for fluorescence per the manufacturer's instructions. Sensitivity was 99% (109/110), and specificity was 99% (129/130). PNA-FISH can rapidly identify those Candida species isolated most frequently. PMID:17804657

  14. Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization

    SciTech Connect

    Giraldez, R.A.; Harris, C.

    1994-09-01

    Hybridization of dual-labeled DNA probes for the repetitive sequences on the X and Y chromosomes allows a fast, non-invasive, more reliable method for sex determination that current cytogenetic Barr body and Y chromatin assays. Scrapes of squamous epithelial cells were collected from the oral cavity of 14 subjects (5{male}, 9{female}) and smeared onto silanized slides. The smears were allowed to air dry. Samples were blinded and then fixed in 50% methanol/50% glacial acetic acid for 10 minutes, and allowed to dry. The slides were incubated in a pretreatment solution containing 30% sodium bisulfite at 45{degrees}C for 10 minutes. They were rinsed in 2XSSC pH 7.0 and then dehydrated through a series of 70%, 85%, and 100% ethanols at room temperature and allowed to air dry. A probe mixture (30 {mu}L containing 10 ng/{mu}L biotin-labeled DXZ1 and digoxigenin-labeled DYZ1/DYZ3 in 70% Formamide/2XSSC) was aliquoted onto each slide, coverslipped, and sealed with rubber cement. Probe and target DNA were simultaneously denatured at 72{degrees}C on a slide warmer for 6 minutes. Probe was allowed to hybridize overnight in a humidified chamber at 37{degrees}C. Slides were postwashed at 72{degrees}C in 0.5xSSC pH 7.0 for 5 minutes, then soaked at room temperature 1XPBD for 2 minutes, and detected with rhodamine/anti-digoxigenin-FITC/avidin for 15 minutes at 37{degrees}C. Slides were soaked 3X in 1XPBD and then counterstained with 15 {mu}L 0.05 {mu}g/mL DAP1/Antifade. 200 nuclei were scored for the presence of one green (X), two green (XX), one green and one red (XY), or a single red (Y) signal, using a fluorescent microscope equipped with a triple band pass filter. Greater than 90% of the hybridized nuclei from each of the 14 cases studied conformed to the sex chromosome pattern. The modal number in 9 cases showed two green signals (XX), and a green and a red signal (XY) in the other 5 cases; this was in complete agreement with the cytogenetic results.

  15. Correlations between arsenic in Maine groundwater and microbial populations as determined by fluorescence in situ hybridization.

    PubMed

    Weldon, Jennifer M; MacRae, Jean D

    2006-04-01

    Arsenic is known to cause serious health effects when consumed in drinking water. In the state of Maine, approximately half of the population relies on private groundwater wells for their drinking water. Of those wells, as many as 13% may contain arsenic levels above the current EPA maximum contaminant level of 10 microgl(-1). Microorganisms can potentially contribute to arsenic release into groundwater through several mechanisms. Some can reduce arsenate to arsenite, which is more toxic and may be more mobile. Sulfurospirillum species NP4, which was isolated from well water, respires arsenate and could act in this way. Microorganisms can also act indirectly by reducing bedrock surface coatings, such as iron oxyhydroxides, that adsorb arsenic in the groundwater environment. The genus Geobacter contains many species that are capable of iron reduction that could play a role in the indirect release of arsenic into groundwater. Water samples from Northport, ME and the Branch Lake region of Ellsworth, ME, which both have elevated groundwater arsenic levels, have been probed using fluorescence in situ hybridization (FISH), to determine the percentage of the population that is NP4 and the percentage that are Geobacter species. Geobacter abundance correlates well with the total arsenic concentration indicating that indirect mechanisms could be important in releasing arsenic. NP4 appears to be reducing arsenate since its prevalence correlates well with arsenite, the end product of arsenate respiration.

  16. Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).

    PubMed

    Ferreira, André M; Cruz-Moreira, Daniela; Cerqueira, Laura; Miranda, João M; Azevedo, Nuno F

    2017-03-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is a highly specific molecular method widely used for microbial identification. Nonetheless, and due to the detection limit of this technique, a time-consuming pre-enrichment step is typically required before identification. In here we have developed a lab-on-a-chip device to concentrate cell suspensions and speed up the identification process in yeasts. The PNA-FISH protocol was optimized to target Saccharomyces cerevisiae, a common yeast that is very relevant for several types of food industries. Then, several coin-sized microfluidic devices with different geometries were developed. Using Computational fluid dynamics (CFD), we modeled the hydrodynamics inside the microchannels and selected the most promising options. SU-8 structures were fabricated based on the selected designs and used to produce polydimethylsiloxane-based microchips by soft lithography. As a result, an integrated approach combining microfluidics and PNA-FISH for the rapid identification of S. cerevisiae was achieved. To improve fluid flow inside microchannels and the PNA-FISH labeling, oxygen plasma treatment was applied to the microfluidic devices and a new methodology to introduce the cell suspension and solutions into the microchannels was devised. A strong PNA-FISH signal was observed in cells trapped inside the microchannels, proving that the proposed methodology works as intended. The microfluidic designs and PNA-FISH procedure described in here should be easily adaptable for detection of other microorganisms of similar size.

  17. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  18. Fluorescent in situ hybridization of human sperm – diagnostics, indications, and therapeutic implications

    PubMed Central

    Ramasamy, Ranjith; Besada, Stefan; Lamb, Dolores J.

    2015-01-01

    Male factor infertility is a relatively common condition, affecting at least 6% of men of reproductive age. Typically men with unknown genetic abnormalities resort to using assisted reproductive technologies (ART) to achieve their reproductive goals. Infertile men who father biological children using ART could have a higher incidence of aneuploidy, which is a deviation from the normal haploid or diploid chromosomal state. Aneuploidy can be evaluated using fluorescent in situ hybridization (FISH), a cytogenetic assay that gives an estimate of the frequencies of chromosomal abnormalities. The chromosomes that are generally analyzed in FISH are associated with aneuploidies that are compatible with life, that is, chr.13, 18, 21, X, and Y. The technique is indicated for a variety of reasons, but most importantly in the following: 1) men who despite normal semen parameters suffer recurrent pregnancy loss and 2) men with normal semen parameters, undergoing IVF, but still experiencing recurrent implantation failure. It may be used as a screening tool to help in reproductive and genetic counseling of affected couples, or those who have previously experienced failure of ART. A qualitative analysis of FISH study results allows them to make informed reproductive choices. Given its increasing clinical use in various infertility diagnoses and the development of novel adjunct technologies, one can expect much progress in the areas of preimplantation genetic screening, diagnostics, and therapeutics. PMID:25439797

  19. Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues.

    PubMed

    Shargil, D; Zemach, H; Belausov, E; Lachman, O; Kamenetsky, R; Dombrovsky, A

    2015-10-01

    Cucumber green mottle mosaic virus (CGMMV), which belongs to the genus Tobamovirus, is a major pathogen of cucurbit crops grown indoors and in open fields. Currently, immunology (e.g., ELISA) and molecular amplification techniques (e.g., RT-PCR) are employed extensively for virus detection in plant tissues and commercial seed lots diagnostics. In this study, a fluorescent in situ hybridization (FISH) technique, using oligonucleotides whose 5'-terminals were labeled with red cyanine 3 (Cy3) or green fluorescein isothiocyanate (FITC), was developed for the visualization of the pathogen in situ. This simple and reliable method allows detection and localization of CGMMV in the vegetative and reproductive tissues of cucumber and melon. When this technique was applied in male flowers, anther tissues were found to be infected; whereas the pollen grains were found to be virus-free. These results have meaningful epidemiological implications for the management of CGMMV, particularly with regard to virus transfer via seed and the role of insects as CGMMV vectors.

  20. The importance of using fluorescence in situ hybridization for the diagnosis of Smith-Magenis syndrome

    SciTech Connect

    Juyal, R.C.; Greenberg, F.; Lupski, J.R.

    1994-09-01

    Smith-Magenis syndrome (SMS) is a clinically recognizable multiple congenital anomaly/mental retardation syndrome associated with deletion of chromosome 17p11.2. Quality metaphase preparations are required for unambiguous detection of the deletion. We and others have reported cases of SMS due to mosaicism for del(17)(p11.2). Examination of peripheral blood lymphocyte cultures of a patient with the SMS phenotype at 850 band level of resolution revealed a low level mosaicism (11%) for the deletion. Examination of fibroblasts at relatively low resolution revealed the deletion in all cells. In a second study, we reported molecular evidence for mosaicism in the unaffected mother of an SMS patient who demonstrated mosaicism (55%) for the deletion at a resolution level of < 500 bands. We now report a different SMS patient who was initially diagnosed as mosaic del(17)(p11.2) in two different cytogenetic laboratories. A third blinded cytogenetic study yielded a questionable diagnosis. Fluorescence in situ hybridization (FISH) conducted in two different laboratories with two different markers shown to be within the deletion region and a control marker from chromosome 17 demonstrated a deletion in 20/20 and 25/25 metaphases scored, respectively. It appears the latter patient may harbor a very small deletion and that FISH is a more reliable test for the Smith-Magenis deletion. Furthermore, FISH should be used to confirm or refute mosaicism seen in routine cytogenetics studies.

  1. Rapid and accurate identification of Xanthomonas citri subspecies citri by fluorescence in situ hybridization.

    PubMed

    Waite, D W; Griffin, R; Taylor, R; George, S

    2016-11-01

    Citrus canker is an economically important disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). This organism targets a wide range of citrus plants, including sweet orange, grapefruit, lemon and lime. As Xcc is spread by environmental factors such as wind and rain, it is difficult to control its movement once the disease has established. In order to facilitate monitoring of citrus canker we sought to design a novel diagnostic protocol based on fluorescence in situ hybridization (FISH) for identification of bacterial cells directly from canker pustules without cultivation or DNA extraction. This method was validated for specificity against a range of Xanthomonas species and strains. We show that our assay is extremely rapid (typically requiring between 2 and 3 h), and possesses a similar specificity to existing PCR diagnostic tools. The sensitivity of the assay is comparable to that of an existing PCR-based technique and sufficient for identifying Xcc in symptomatic plant material. The method is easily transferable to diagnosticians without prior experience using FISH.

  2. Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes

    NASA Technical Reports Server (NTRS)

    Wu, H.; George, K.; Yang, T. C.

    1998-01-01

    PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

  3. Directly incorporating fluorochromes into DNA probes by PCR increases the efficience of fluorescence in situ hybridization

    SciTech Connect

    Dittmer, Joy

    1996-05-01

    The object of this study was to produce a directly labeled whole chromosome probe in a Degenerative Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) that will identify chromosome breaks, deletions, inversions and translocations caused by radiation damage. In this study we amplified flow sorted chromosome 19 using DOP-PCR. The product was then subjected to a secondary DOP PCR amplification, After the secondary amplification the DOP-PCR product was directly labeled in a tertiary PCR reaction with rhodamine conjugated with dUTP (FluoroRed) to produce a DNA fluorescent probe. The probe was then hybridized to human metaphase lymphocytes on slides, washed and counterstained with 4{prime},6-diamino-2-phenylindole (DAPI). The signal of the FluoroRed probe was then compared to a signal of a probe labeled with biotin and stained with avidin fluorescein isothio cynate (FITC) and anti-avidin FITC. The results show that the probe labeled with FluoroRed gave signals as bright as the probe with biotin labeling. The FluoroRed probe had less noise than the biotin labeled probe. Therefore, a directly labeled probe has been successfully produced in a DOP-PCR reaction. In future a probe labeled with FluoroRed will be produced instead of a probe labeled with biotin to increase efficiency.

  4. Does polyomavirus infection interfere with bladder cancer fluorescence in situ hybridization?

    PubMed

    Hossain, Deloar; Hull, David; Kalantarpour, Fatemeh; Maitlen, Rebecca; Qian, Junqi; Bostwick, David G

    2014-03-01

    Urine cytology is a proven and widely used screening tool for the detection of urothelial carcinoma. However, morphologic features of polyomavirus infected cells, characterized by nuclear inclusions (decoy cells) are a known source of diagnostic confusion with malignancy. Fluorescence in situ hybridization (FISH) is now routinely used to support the cytological diagnosis of urothelial carcinoma and monitor for recurrence. We sought to determine whether polyomavirus infection could result in positive FISH results (aneuploidy). This study deals with retrospective study of 100 polyomavirus-infected urine samples from patients with no history of urothelial carcinoma or organ transplantation. All cases were stained with Papanicolaou and acid hematoxylin stain. One slide from each sample was de-stained and FISH was performed using chromosome enumeration probes 3, 7, 17, and locus-specific probe 9p21. Adequate cells for FISH analysis (25 cells) were present in 81 cases; 19 cases were insufficient due to loss of cells during de-staining and FISH preparation process. All polyomavirus-infected cells (decoy cells) exhibited a normal chromosome pattern. Four cases were FISH positive, but there were no positive decoy cells. Decoy cells did not exhibit aneuploidy by FISH. The presence of decoy cells does not exclude the possibility of concurrent urothelial carcinoma. Acid hematoxylin stain appeared to supplement the Papanicolou stain in identifying and confirming the presence of polyomavirus infection.

  5. Potential clinical impact of three-dimensional visualization for fluorescent in situ hybridization image analysis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Li, Shibo; Bin, Zheng; Zhang, Roy; Li, Yuhua; Tian, Huimin; Chen, Wei; Liu, Hong

    2012-05-01

    Chromosomal translocation is strong indication of cancers. Fluorescent in situ hybridization (FISH) can effectively detect this translocation and achieve high accuracy in disease diagnosis and prognosis assessment. For this purpose, whole chromosome paint probes are utilized to image the configuration of DNA fragments. Although two-dimensional (2-D) microscopic images are typically used in FISH signal analysis, we present a case where the translocation occurs in the depth direction where two probed FISH signals are overlapped in the projected image plane. Thus, the translocation cannot be identified. However, when imaging the whole specimen with a confocal microscope at 27 focal planes with 0.5-μm step interval, the translocation can be clearly identified due to the free rotation capability by the three-dimensional (3-D) visualization. Such a translocation detection error of using 2-D images might be critical in detecting and diagnosing early or subtle disease cases where detecting a small number of abnormal cells can make diagnostic difference. Hence, the underlying implication of this report suggests that utilizing 3-D visualization may improve the overall accuracy of FISH analysis for some clinical cases. However, the clinical efficiency and cost of using 3-D versus 2-D imaging methods are also to be assessed carefully.

  6. Fifty probands with extra structurally abnormal chromosomes characterized by fluorescence in situ hybridization

    SciTech Connect

    Blennow, E.; Telenius, H.; Nordenskjoeld, M.

    1995-01-02

    Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 (inv dup(15)), including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality. 42 refs., 2 figs., 2 tabs.

  7. Molecular cytogenetic characterization of the DiGeorge syndrome region using fluorescence in situ hybridization

    SciTech Connect

    Lindsay, E.A. Imperial Cancer Research Fund, London ); Halford, S.; Wadey, R.; Scambler, P.J. ); Baldini, A. )

    1993-08-01

    DiGeorge syndrome (DGS) is a developmental defect characterized by cardiac defects, facial dysmorphism, and mental retardation. Several studies have described a critical region for DGS at 22q11, within which the majority of DGS patients have deletions. The authors have isolated nine cosmid and three YAC clones using previously described and newly isolated probes that have been shown to be deleted in many DGS patients. Using fluorescence in situ hybridization and digital imaging, they have mapped and ordered these clones relative to the breakpoints of two balanced translocations at 22q11 (one in a DGS patient and one in the unaffected parent of a DGS child). The data indicate that the breakpoint in the unaffected individual distally limits the DGS critical region (defined as the smallest region of overlap), while proximally the region is limited by repeat-rich DNA. The critical region includes the balanced translocation breakpoint of the DGS patient that presumably disrupts the gene causing this syndrome.

  8. Fluorescence in situ hybridization with Bacterial Artificial Chromosomes (BACs) to mitotic heterochromatin of Drosophila.

    PubMed

    Accardo, Maria Carmela; Dimitri, Patrizio

    2010-01-01

    The organization of eukaryotic chromosomes into euchromatin and heterochromatin represents an enigmatic aspect of genome evolution. Constitutive heterochromatin is a basic, yet still poorly understood component of eukaryotic genomes and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Drosophila melanogaster polytene chromosomes do not seem to be particularly useful to map heterochromatin sequences because the typical features of heterochromatin, organized as it is into a chromocenter, limit cytogenetic analysis. In contrast, constitutive heterochromatin has been well-defined at the cytological level in mitotic chromosomes of neuroblasts and has been subdivided into several bands with differential staining properties. Fluorescence in situ hybridization (FISH) using Bacterial Artificial Chromosomes (BAC) probes that carry large genomic portions defined by sequence annotation has yielded a "revolution" in the field of cytogenetics because it has allowed the mapping of multiple genes at once, thus rendering constitutive heterochromatin amenable to easy and fast cytogenetics analyses. Indeed, BAC-based FISH approaches on Drosophila mitotic chromosomes have made it possible to correlate genomic sequences to their cytogenetic location, aiming to build an integrated map of the pericentric heterochromatin. This chapter presents our standard protocols for BAC-based FISH, aimed at mapping large chromosomal regions of mitotic heterochromatin in Drosophila melanogaster.

  9. A new approach to screen transgenic offspring using fluorescence in situ hybridization

    SciTech Connect

    Swiger, R.R.; Tucker, J.D.; Heddle, J.A.

    1995-11-01

    In order to identify the presence of vector, specifically Lacl or LacZ, in putative transgenic mice rapidly and reliably, we developed a new method which utilizes fluorescence in situ hybridization (FISH). Transgenic mouse models are being used with increasing frequency for mutational and toxicological studies. By applying our method, an investigator can reliably determine the presence and the number of integration sites of a transgenic vector in numerous samples with less effort compared to conventional methods. This approach involves gazing a tail with a scalpel to obtain a blood smear on a microscope slide. After fixing the slide in 3:1 methanol: acetic acid, typical FISH analysis using biotinylated lambda DNA as the probe in performed. Our method eliminates the need for DNA extraction, blotting, and PCR, and yields results from a large number of individually identifiable cells from each animal. This assay is more accurate, reliable and easier to perform than conventional schemes presently used for screening transgenic animals. A particular advantage of this assay is the ability to discriminate between animals that are heterozygous and homozygous, something that eludes the PCR-based methods and Southern blotting. We have successfully analyzed over 95 samples with this method. Based on these results, we believe our system is more sensitive and accurate than conventional means of screening.

  10. Recurrent gene amplifications in human type I endometrial adenocarcinoma detected by fluorescence in situ hybridization.

    PubMed

    Samuelson, Emma; Levan, Kristina; Adamovic, Tatjana; Levan, Göran; Horvath, György

    2008-02-01

    Determining what genes are actively involved in tumor development is important, because they may provide targets for directed therapy. Human tumors are greatly heterogeneous with respect to etiology and genetic background, which complicates the identification of common genetic aberrations. In contrast, genetic and environmental variation can be in part controlled in experimental animals, which facilitates identification of the important changes. In inbred BDII rats, which are genetically predisposed to endometrial adenocarcinomas (EAC), certain chromosome regions exhibit recurrent amplification in the tumors. Previous CGH analysis had shown that a subset of human EAC tumors exhibited increased copy numbers in the homologous chromosomal regions, located in human 2p21 approximately p25 and 7q21 approximately q31. Using fluorescence in situ hybridization analysis on imprints from 13 human EAC tumors, we determined the average copy numbers of each of 15 probes derived from cancer-related genes situated in these chromosome regions. Among the genes analyzed, those most often targeted by amplification were SDC1 and CYP1B1 in 2p21 approximately p25 and CDK6 and MET in 7q21 approximately q31, but all of the 15 genes tested were found to be amplified in at least two tumors.

  11. Gonadoblastomas in 45,X/46,XY mosaicism: analysis of Y chromosome distribution by fluorescence in situ hybridization.

    PubMed

    Iezzoni, J C; Von Kap-Herr, C; Golden, W L; Gaffey, M J

    1997-08-01

    Gonadoblastomas are composed of nests of neoplastic germ cells and sex cord derivatives surrounded by ovarian-type stroma. These tumors are found almost exclusively in persons with gonadal dysgenesis associated with a Y chromosome or Y chromosome fragment, and accordingly, the Y chromosome has been implicated in gonadoblastoma oncogenesis. To evaluate this association, we used two-color fluorescence in situ hybridization with chromosome-specific probes to determine the distribution of the X and Y chromosomes in the tumor nests and surrounding stromal cells in paraffin tissue sections of three gonadoblastomas in two patients with gonadal dysgenesis and 45,X/46,XY mosaicism. Statistical analysis of the data from the fluorescence in situ hybridization demonstrated that in all three gonadoblastomas, the proportion of nuclei with a Y chromosome signal was significantly higher in the tumor cells than in the nontumoral cells of the surrounding stroma (P<.001). These results suggest that Y chromosome material participates in gonadoblastoma tumorigenesis.

  12. Ribosomal DNA location in the scarab beetle Thorectes intermedius (Costa) (Coleoptera: Geotrupidae) using banding and fluorescent in-situ hybridization.

    PubMed

    Vitturi, R; Colomba, M S; Barbieri, R; Zunino, M

    1999-01-01

    Mitotic metaphase chromosomes of the scarab beetle Thorectes intermedius (Costa) (Coleoptera Scarabaeoidea: Geotrupidae) were analyzed using various banding methods and fluorescent in-situ hybridization (FISH) with a ribosomal probe. The results obtained indicate that silver and CMA3 staining are unable to localize the chromosome sites of nucleolar organizer regions (NORs). Such an inadequacy is a consequence of the extensive silver and CMA3 stainability of both constitutive heterochromatin and heterochromatin associated to the NORs.

  13. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    PubMed

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH.

  14. Localization of the human OB gene (OBS) to chromosome 7q32 by fluorescence in situ hybridization

    SciTech Connect

    Geffroy, S.; Duban, B.; Martinville, B. de

    1995-08-10

    An important gene involved in the pathogenesis of obesity is the product of the human homologue of the murine obese gene (gene symbol OBS). Using fluorescence in situ hybridization (FISH), we have localized the human OB gene to human chromosome 7, specifically to region 7q32.1. The FISH data of human OBS provide a gene-associated marker for genetic mapping. 8 refs., 1 fig.

  15. Ecophysiological Analysis of Microorganisms in Complex Microbial Systems by Combination of Fluorescence In Situ Hybridization with Extracellular Staining Techniques

    NASA Astrophysics Data System (ADS)

    Nielsen, Jeppe Lund; Kragelund, Caroline; Nielsen, Per Halkjær

    Ecophysiological analysis and functions of single cells in complex microbial systems can be examined by simple combinations of Fluorescence in situ hybridization (FISH) for identification with various staining techniques targeting functional phenotypes. In this chapter, we describe methods and protocols optimized for the study of extracellular enzymes, surface hydrophobicity and specific surface structures. Although primarily applied to the study of microbes in wastewater treatment (activated sludge and biofilms), the methods may also be used with minor modifications in several other ecosystems.

  16. A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

    PubMed

    Speel, E J; Schutte, B; Wiegant, J; Ramaekers, F C; Hopman, A H

    1992-09-01

    We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.

  17. Multiplexed hybridization detection of quantum dot-conjugated DNA sequences using surface plasmon enhanced fluorescence microscopy and spectrometry.

    PubMed

    Robelek, Rudolf; Niu, Lifang; Schmid, Evelyne L; Knoll, Wolfgang

    2004-10-15

    In this study, the general suitability of quantum dot (QD)-DNA conjugates for the surface plasmon enhanced fluorescence spectroscopy technique is demonstrated. Furthermore, the QD-DNA system is transferred to the platform of surface plasmon enhanced fluorescence microscopy. Using this technique together with a microarray format, in which the sensor-bound single-stranded catcher probes are organized in individual surface spots, results in a simultaneous qualitative analysis of QD-conjugated analyte DNA strands as multicolor images. A clear decomposition of different QD(x)()-DNA(y)() mixtures can be achieved for sequential, as well as mixture injections. Besides this, the study describes the successful approach of measuring spectrally resolved surface plasmon enhanced fluorescence signals derived from catcher probe hybridized QD-DNA conjugates.

  18. Super-resolution Localization and Defocused Fluorescence Microscopy on Resonantly Coupled Single-Molecule, Single-Nanorod Hybrids.

    PubMed

    Su, Liang; Yuan, Haifeng; Lu, Gang; Rocha, Susana; Orrit, Michel; Hofkens, Johan; Uji-i, Hiroshi

    2016-02-23

    Optical antennas made of metallic nanostructures dramatically enhance single-molecule fluorescence to boost the detection sensitivity. Moreover, emission properties detected at the optical far field are dictated by the antenna. Here we study the emission from molecule-antenna hybrids by means of super-resolution localization and defocused imaging. Whereas gold nanorods make single-crystal violet molecules in the tip's vicinity visible in fluorescence, super-resolution localization on the enhanced molecular fluorescence reveals geometrical centers of the nanorod antenna instead. Furthermore, emission angular distributions of dyes linked to the nanorod surface resemble that of nanorods in defocused imaging. The experimental observations are consistent with numerical calculations using the finite-difference time-domain method.

  19. Super-resolution Localization and Defocused Fluorescence Microscopy on Resonantly Coupled Single-Molecule, Single-Nanorod Hybrids

    PubMed Central

    2016-01-01

    Optical antennas made of metallic nanostructures dramatically enhance single-molecule fluorescence to boost the detection sensitivity. Moreover, emission properties detected at the optical far field are dictated by the antenna. Here we study the emission from molecule–antenna hybrids by means of super-resolution localization and defocused imaging. Whereas gold nanorods make single-crystal violet molecules in the tip’s vicinity visible in fluorescence, super-resolution localization on the enhanced molecular fluorescence reveals geometrical centers of the nanorod antenna instead. Furthermore, emission angular distributions of dyes linked to the nanorod surface resemble that of nanorods in defocused imaging. The experimental observations are consistent with numerical calculations using the finite-difference time-domain method. PMID:26815168

  20. A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

    PubMed

    Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroyuki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

    2013-01-14

    A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

  1. Efficient assembly of multi-walled carbon nanotube-CdSe/ZnS quantum dot hybrids with high biocompatibility and fluorescence property.

    PubMed

    Zhang, Yingying; Qin, Weiling; Tang, Hao; Yan, Feng; Tan, Liang; Xie, Qingji; Ma, Ming; Zhang, Youyu; Yao, Shouzhuo

    2011-10-15

    CdSe/ZnS core-shell quantum dots (QDs) were efficiently tethered onto polyamidoamine dendrimer-modified multi-walled carbon nanotubes (MWCNTs) by covalent linkage and mercapto-mediated assembly. The obtained MWCNT-QD hybrids were both photophysically and morphologically characterized. The QDs are well-distributed on single nanotube surface in high density and the assembly of QDs onto MWCNTs does not change the fluorescence emission wavelength of QDs but significantly decreases the emission density. Cytotoxicity of MWCNT-QD hybrids to HeLa cells and their fluorescence property in living cell system were evaluated in detail. The hybrids show a little effect on cell viability even at very high concentration (100 μg mL(-1)). Moreover, they possess intense red fluorescence signal under optical fluorescence microscopy and good fluorescence stability over 72-h exposure in living cell system.

  2. Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes

    PubMed Central

    Oliveira, Kenneth; Haase, Gerhard; Kurtzman, Cletus; Hyldig-Nielsen, Jens Jo/rgen; Stender, Henrik

    2001-01-01

    The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified as Candida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs of C. albicans and C. dubliniensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79 C. dubliniensis and 70 C. albicans strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation of C. albicans and C. dubliniensis. PMID:11682542

  3. Chromogenic in situ hybridization is a reliable alternative to fluorescence in situ hybridization for diagnostic testing of 1p and 19q loss in paraffin-embedded gliomas.

    PubMed

    Lass, Ulrike; Hartmann, Christian; Capper, David; Herold-Mende, Christel; von Deimling, Andreas; Meiboom, Maren; Mueller, Wolf

    2013-05-01

    Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual-color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side-by-side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n = 39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)-based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin-embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis.

  4. Interfacial transduction of nucleic acid hybridization using immobilized quantum dots as donors in fluorescence resonance energy transfer.

    PubMed

    Algar, W Russ; Krull, Ulrich J

    2009-01-06

    Fluorescence resonance energy transfer (FRET) using immobilized quantum dots (QDs) as energy donors was explored as a transduction method for the detection of nucleic acid hybridization at an interface. This research was motivated by the success of the QD-FRET-based transduction of nucleic acid hybridization in solution-phase assays. This new work represents a fundamental step toward the assembly of a biosensor, where immobilization of the selective chemistry on a surface is desired. After immobilizing QD-probe oligonucleotide conjugates on optical fibers, a demonstration of the retention of selectivity was achieved by the introduction of acceptor (Cy3)-labeled single-stranded target oligonucleotides. Hybridization generated the proximity required for FRET, and the resulting fluorescence spectra provided an analytical signal proportional to the amount of target. This research provides an important framework for the future development of nucleic acid biosensors based on QDs and FRET. The most important findings of this work are that (1) a QD-FRET solid-phase hybridization assay is viable and (2) a passivating layer of denatured bovine serum albumin alleviates nonspecific adsorption, ultimately resulting in (3) the potential for a reusable assay format and mismatch discrimination. In this, the first incarnation of a solid-phase QD-FRET hybridization assay, the limit of detection was found to be 5 nM, and the dynamic range was almost 2 orders of magnitude. Selective discrimination of the target was shown using a three-base-pairs mismatch from a fully complementary sequence. Despite a gradual loss of signal, reuse of the optical fibers over multiple cycles of hybridization and dehybridization was possible. Directions for further improvement of the analytical performance by optimizing the design of the QD-probe oligonucleotide interface are identified.

  5. Fluorescence in situ hybridization investigation of potentially pathogenic bacteria involved in neonatal porcine diarrhea

    PubMed Central

    2014-01-01

    Background Neonatal diarrhea is a multifactorial condition commonly present on pig farms and leads to economic losses due to increased morbidity and mortality of piglets. Immature immune system and lack of fully established microbiota at birth predispose neonatal piglets to infection with enteric pathogens. The microorganisms that for decades have been associated with enteritis and diarrhea in suckling piglets are: rotavirus A, coronavirus, enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens type C, Cryptosporidium spp., Giardia spp., Cystoisospora suis and Strongyloides ransomi. However, in recent years, the pig industry has experienced an increased number of neonatal diarrhea cases in which the above mentioned pathogens are no longer detected. Potentially pathogenic bacteria have recently received focus in the research on the possible etiology of neonatal diarrhea not caused by common pathogens. The primary aim of this study was to investigate the role of E. coli, Enterococcus spp., C. perfringens and C. difficile in the pathogenesis of neonatal porcine diarrhea with no established casual agents. Fluorescence in situ hybridization with oligonucleotide probes was applied on the fixed intestinal tissue samples from 51 diarrheic and 50 non-diarrheic piglets collected from four Danish farms during outbreaks of neonatal diarrhea not caused by well-known enteric pathogens. Furthermore, an association between the presence of these bacteria and histological lesions was evaluated. Results The prevalence of fluorescence signals specific for E. coli, C. perfringens and C. difficile was similar in both groups of piglets. However, Enterococcus spp. was primarily detected in the diarrheic piglets. Furthermore, adherent bacteria were detected in 37 % diarrheic and 14 % non-diarrheic piglets. These bacteria were identified as E. coli and Enterococcus spp. and their presence in the intestinal mucosa was associated with histopathological changes. Conclusions The

  6. High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres.

    PubMed

    Fransz, P F; Alonso-Blanco, C; Liharska, T B; Peeters, A J; Zabel, P; de Jong, J H

    1996-03-01

    A technique to detect DNA sequences on extended DNA fibres (EDF) prepared from interphase nuclei from tomato (Lycopersicon esculentum) and Arabidopsis thaliana leaves by fluorescence in situ hybridization (FISH) is described. Three nuclear lysis procedures have been tested for their ability to decondense chromatin and to generate highly extended intact DNA fibres on microscopic slides. DNA probes of various sizes have been used in FISH experiments to EDFs to establish the resolution and sensitivity of the technique. The fluorescent signals of a 5S rDNA probe hybridized to tomato EDFs revealed continuous strings of about 200 microns, that corresponded to a molecular size of about 660 kb. In A. thaliana, a contig of three cosmids spanning a genomic region with a total length of about 89 kb was analysed. By means of multicolour hybridization the physical positions of the cosmids were visualized as red and green fluorescence strings with overlapping regions in yellow. Comparison of the length of the fluorescent signals with the molecular data revealed a stretching degree of the DNA fibres at 3.27 kb microns-1, which is close to the Watson-Crick DNA length estimate of 2.9 kb microns-1. Other experiments on small size molecular probes with both lambda clones (13.5-17 kb insert sizes) and plasmids (4.2 and 5 kb) in a contig of A. thaliana, and the 5S rDNA region in tomato showed close agreement with molecular data. The lower limit of the detection, which was established in a hybridization experiment with two DNA probes from the 45S ribosomal gene on extended fibres of tomato, was about 0.7 kb. Consistent patterns of alternating fluorescent red and green spots were obtained reflecting the tandemly repeated arrangement of the 18S and 25S ribosomal sequences. On the basis of the microscopic distance between these hybridization spots the size of the ribosomal unit was estimated at 8.2 kb. This implies a drastic improvement of high-resolution physical mapping of DNA sequences

  7. Fluorescence In-Situ Hybridization Detects Increased Sperm Aneuploidy in Men with Recurrent Pregnancy Loss

    PubMed Central

    Ramasamy, Ranjith; Scovell, Jason M.; Kovac, Jason R.; Cook, Peter J.; Lamb, Dolores J.; Lipshultz, Larry I.

    2015-01-01

    Objective To investigate, in men presenting with recurrent pregnancy loss (RPL), the prevalence of sperm autosome and sex chromosome aneuploidy. Design Retrospective Study. Setting Male infertility clinic at a tertiary referral center. Patients 140 men with recurrent pregnancy loss provided semen samples and five normozoospermic controls provided 140 semen samples for comparison. RPL, documented in the female partners, was defined as a prior miscarriage and/or recurrent IVF/ICSI failure. Interventions Fluorescent In situ hybridization (FISH) was used to detect numerical abnormalities in sex chromosomes (X,Y) and autosomes (13, 18, 21) in ejaculated sperm. Main Outcome Measures Sperm aneuploidy in men with RPL and normozoospermic controls. Results Men with RPL had a greater percentage of sperm aneuploidy within the sex chromosomes, chromosomes 18 and 13/21 (1.04% vs. 0.38%; 0.18% vs. 0.03%; 0.26% vs. 0.08%). In total, 40% of men with normal sperm density and motility had abnormal sperm aneuploidy in the all the chromosomes analyzed. Men with abnormal sperm density and motility had a higher proportion of sperm sex chromosome aneuploidy than men with normal density/motility (62% vs. 45%). Men with normal strict morphology (>4%) had lower rates of sex chromosome and sperm aneuploidy than men with abnormal strict morphology (28% vs. 57%). There was no association between sperm DNA fragmentation and sperm aneuploidy. Conclusions Men with RPL have increased sperm aneuploidy compared to controls. A total of 40% of men with RPL and normal sperm density/motility had abnormal sperm aneuploidy. Men with oligoasthenozoospermia and abnormal strict morphology had greater percentage of sperm aneuploidy compared to men with normal semen parameters. PMID:25707335

  8. Quantification of Enterococcus italicus in traditional Italian cheeses by fluorescence whole-cell hybridization.

    PubMed

    Fornasari, Maria Emanuela; Rossetti, Lia; Remagni, Chiara; Giraffa, Giorgio

    2008-08-01

    The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91+/-0.17 to 7.34+/-0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening.

  9. Melanocytic nevi with an atypical epithelioid cell component: clinical, histopathologic, and fluorescence in situ hybridization findings.

    PubMed

    Pouryazdanparast, Pedram; Haghighat, Zahra; Beilfuss, Beth Ann; Guitart, Joan; Gerami, Pedram

    2011-09-01

    Combined melanocytic nevi can contain a phenotypically distinct population of large atypical epithelioid cells in a background of smaller banal-appearing melanocytes. On the basis of the pattern of proliferation and degree of pigmentation, nevi with this pattern have been referred to as nevi with an atypical epithelioid cell component (N-AECC). When N-AECC display sheet-like or an expansile nodular growth pattern, notable cytologic atypia, and any level of mitotic activity, they can be difficult to distinguish from melanoma. The clinical history and appearance of these lesions may similarly raise concern for melanoma. In view of this diagnostic problem, we present 28 cases of N-AECC from our dermatopathology consultation and in-house practice. All 28 cases were found to be negative on the basis of fluorescence in situ hybridization (FISH) for imbalanced chromosomal aberrations commonly found in melanoma. The clinical outcomes showed a benign clinical course for all cases for which the outcome information was available. FISH analysis also revealed that, in 4 of 28 cases (14%), the AECC of the lesion demonstrated polyploidy localized to the large epithelioid cell component. This is likely more common among cases of N-AECC that have an atypical spitzoid epithelioid cell component, particularly those with obvious senescent nuclear changes. Care must be taken to avoid the pitfall of misinterpreting these FISH findings as changes consistent with melanoma. The use of ancillary testing methods including FISH may be beneficial in improving the diagnostic accuracy involved in making the distinction of N-AECC from melanoma. Further, we report a novel finding of polyploidy seen in certain cases of benign N-AECC.

  10. Fluorescence In Situ Hybridization and Optical Mapping to Correct Scaffold Arrangement in the Tomato Genome

    PubMed Central

    Shearer, Lindsay A.; Anderson, Lorinda K.; de Jong, Hans; Smit, Sandra; Goicoechea, José Luis; Roe, Bruce A.; Hua, Axin; Giovannoni, James J.; Stack, Stephen M.

    2014-01-01

    The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome–fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps. PMID:24879607

  11. LINE-1 elements: analysis by fluorescence in-situ hybridization and nucleotide sequences.

    PubMed

    Waters, Paul D; Dobigny, Gauthier; Waddell, Peter J; Robinson, Terence J

    2008-01-01

    Long-interspersed nuclear element-1 (LINE-1) is a non-terminal repeat transposon that constitutes a major component of the mammalian genome. LINE-1 has a dynamic evolutionary history characterized by the rise, fall, and replacement of subfamilies. The distribution of LINE-1 elements can be viewed from a chromosomal perspective using fluorescence in-situ hybridization (FISH), as well as at the sequence level. We have designed LINE-1 primers from regions conserved among mouse, rat, rabbit, and human L1, which were able to amplify part of ORF2 from all eutherian (placental) mammals tested thus far. The product generated can be used as a FISH painting probe to examine the genomic distribution of L1 in different species. It can also be cloned and sequenced for phylogenetic analysis. Although FISH patterns resulting from LINE-1 chromosome painting and bioinformatic analyses have shown that this element accumulates in AT-rich regions of the genomes of mouse and human, our PCR amplified LINE-1 probe suggests that this is not a universal phenomenon, and that the patterns displayed in laurasiatherian, afrotherian and xenarthran species are less prominent. The "banding" like distribution of LINE-1 observed in human and mouse, therefore, appears to reflect aspects of genome architecture unique to Euarchontoglires (Supraprimates), the superordinal clade to which they belong. By sequencing the cloned amplicons used for FISH experiments and supplementing these with L1 sequences obtained from public databases, analysis by parsimony, distance-based, maximum likelihood, and "hierarchical Bayesian" or "marginal likelihood" methods provides a powerful adjunct to the FISH data. Using this approach, relatively intact LINE-1 from most placental orders tend to reflect accepted eutherian evolutionary relationships. This suggests that there were often only closely related copies active near branch points in the tree, that inactive copies tended to become extinct quite readily, and that for

  12. Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome.

    PubMed

    Shearer, Lindsay A; Anderson, Lorinda K; de Jong, Hans; Smit, Sandra; Goicoechea, José Luis; Roe, Bruce A; Hua, Axin; Giovannoni, James J; Stack, Stephen M

    2014-05-30

    The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.

  13. Identification of a centromeric exchange of acrocentric chromosomes by fluorescence in situ hybridization

    SciTech Connect

    Yu, C.W.; Immken, L.; Curry, C.J.R.

    1994-09-01

    Exchanges of the peri-centromeric area of acrocentric chromosomes are difficult to identify using the conventional cytogenetic techniques. Fluorescence in situ hybridization (FISH) provides a new way for precisely identifying such rearrangements. Here we report a case of centromeric rearrangement in an amniotic fluid specimen with an extra marker chromosome. M.G., a 41-year-old G1, was referred for advanced maternal age. Chromosome studies revealed a 47,XX +mar karyotype. The marker appeared to be bi-satallited with a single C band. Chromosome studies from the parents were normal. The parents elected to terminate the pregnancy. Anatomical examination of the abortus revealed a very short neck, posteriorly rotated ears, high set cecum, absent hepatic lobation and low abdominal kidneys with short ureters. FISH studies with alpha-satellite probes of 13/21, 14/22, and 15, and the DiGeorge probe, indicated that there is a translocation of 21 alpha-satellite to the 22, and that the marker chromosome probably consists of 14/22 alpha-satellite material. FISH analysis of the parents chromosome revealed that father had the translocation of 21 alpha-satellite to the 22 as well. Exchanges of centromeric material among the acrocentric chromosomes may not be an uncommon event in humans. Although it probably has no clinical significance, it may result in non-disjunction or marker chromosome formation from an uncommon satellite association. With the use of FISH techniques, exchanges involving the centromeric regions of acrocentric chromosomes can be identified.

  14. ESHRE PGD consortium best practice guidelines for fluorescence in situ hybridization-based PGD.

    PubMed

    Harton, G L; Harper, J C; Coonen, E; Pehlivan, T; Vesela, K; Wilton, L

    2011-01-01

    In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of new technologies as well as evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. organization of a PGD centre, fluorescence in situ hybridization (FISH)-based testing, amplification-based testing and polar body and embryo biopsy for PGD/preimplantation genetic screening (PGS). Here, we have updated the sections that pertain to FISH-based PGD. PGS has become a highly controversial technique. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Whereas some believe that PGS does not have a place in clinical medicine, others disagree; therefore, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible. Topics covered in this guideline include inclusion/exclusion criteria for FISH-based PGD testing, referrals and genetic counselling, preclinical validation of tests, FISH-based testing methods, spreading of cells for analysis, set-up of local IVF centre and transport PGD centres, quality control/ quality assurance and diagnostic confirmation of untransferred embryos.

  15. A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay.

    PubMed

    Bou, Gerelchimeg; Sun, Mingju; Lv, Ming; Zhu, Jiang; Li, Hui; Wang, Juan; Li, Lu; Liu, Zhongfeng; Zheng, Zhong; He, Wenteng; Kong, Qingran; Liu, Zhonghua

    2014-08-01

    For efficient transgenic herd expansion, only the transgenic animals that possess the ability to transmit transgene into next generation are considered for breeding. However, for transgenic pig, practically lacking a pre-breeding screening program, time, labor and money is always wasted to maintain non-transgenic pigs, low or null transgenic transmission pigs and the related fruitless gestations. Developing a pre-breeding screening program would make the transgenic herd expansion more economical and efficient. In this technical report, we proposed a three-step pre-breeding screening program for transgenic boars simply through combining the fluorescence in situ hybridization (FISH) assay with the common pre-breeding screening workflow. In the first step of screening, combined with general transgenic phenotype analysis, FISH is used to identify transgenic boars. In the second step of screening, combined with conventional semen test, FISH is used to detect transgenic sperm, thus to identify the individuals producing high quality semen and transgenic sperm. In the third step of screening, FISH is used to assess the in vitro fertilization embryos, thus finally to identify the individuals with the ability to produce transgenic embryos. By this three-step screening, the non-transgenic boars and boars with no ability to produce transgenic sperm or transgenic embryos would be eliminated; therefore only those boars could produce transgenic offspring are maintained and used for breeding and herd expansion. It is the first time a systematic pre-breeding screening program is proposed for transgenic pigs. This program might also be applied in other transgenic large animals, and provide an economical and efficient strategy for herd expansion.

  16. High Sensitivity deflection detection of nanowires

    SciTech Connect

    Sanii, Babak; Ashby, Paul

    2009-10-28

    A critical limitation of nanoelectromechanical systems (NEMS) is the lack of a high-sensitivity position detection mechanism. We introduce a noninterferometric optical approach to determine the position of nanowires with a high sensitivity and bandwidth. Its physical origins and limitations are determined by Mie scattering analysis. This enables a dramatic miniaturization of detectable cantilevers, with attendant reductions to the fundamental minimum force noise in highly damping environments. We measure the force noise of an 81{+-}9??nm radius Ag{sub 2}Ga nanowire cantilever in water at 6{+-}3??fN/{radical}Hz.

  17. Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis

    PubMed Central

    Nikolakakis, K.; Lehnert, E.

    2015-01-01

    The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization. PMID:25956763

  18. Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.

    PubMed

    Tanner, M; Gancberg, D; Di Leo, A; Larsimont, D; Rouas, G; Piccart, M J; Isola, J

    2000-11-01

    Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.

  19. RNA containing pyrrolocytidine base analogs: good binding affinity and fluorescence that responds to hybridization.

    PubMed

    Wahba, Alexander S; Damha, Masad J; Hudson, Robert H E

    2008-01-01

    6-Phenylpyrrolocytidine and 6-methoxymethylene-pyrrolocytidine are base-modified nucleosides with remarkable fluorescence properties. When incorporated into RNA, these analogs enhance binding affinity towards RNA and DNA targets with a concomitant change in their fluorescence upon duplex formation. The fluorescence response depends on the nature of the 6-substituent and the sequence position of the modified nucleoside. The fluorescence response of these structurally conservative, well-tolerated fluorescent nucleosides may be exploited as probes in the study of nucleic acid processing enzymes.

  20. In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading.

    PubMed

    Picanço-Albuquerque, C G; Morais, C L; Carvalho, F L F; Peskoe, S B; Hicks, J L; Ludkovski, O; Vidotto, T; Fedor, H; Humphreys, E; Han, M; Platz, E A; De Marzo, A M; Berman, D M; Lotan, T L; Squire, J A

    2016-05-01

    The prognostic value of phosphatase and tensin homolog (PTEN) loss in prostate cancer has primarily been evaluated by either fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). Previously, we found that PTEN loss by IHC was associated with increased risk of upgrading from biopsy (Gleason 3 + 3) to prostatectomy (Gleason 7+). Now, using an evaluable subset of 111 patients with adjacent biopsy sections, we analyzed the association between PTEN deletion in cancer and the odds of upgrading by a highly sensitive and specific four-color FISH assay. We also compared the concordance of PTEN loss by IHC and PTEN deletion by FISH. PTEN deletion was found in 27 % (12/45) of upgraded cases compared with 11 % (7/66) of controls (P = 0.03). Cancers with PTEN deletions were more likely to be upgraded than those without deletions (adjusting for age odds ratio = 3.40, 95 % confidence interval 1.14-10.11). With respect to concordance, of 93 biopsies with PTEN protein detected by IHC, 89 (96 %) had no PTEN deletion by FISH, and of 18 biopsies without PTEN protein by IHC, 15 had homozygous or hemizygous PTEN deletion by FISH. Only 4 biopsies of the 93 (4 %) with PTEN protein intact had PTEN deletion by FISH. When the regions of uncertainty in these biopsies were systematically studied by FISH, intra-tumoral variation of PTEN deletion was found, which could account for variation in immunoreactivity. Thus, FISH provides a different approach to determining PTEN loss when IHC is uncertain. Both FISH and IHC are concordant, showing consistent positive associations between PTEN loss and upgrading.

  1. BAP1 immunohistochemistry has limited prognostic utility as a complement of CDKN2A (p16) fluorescence in situ hybridization in malignant pleural mesothelioma.

    PubMed

    M McGregor, Stephanie; McElherne, James; Minor, Agata; Keller-Ramey, Jennifer; Dunning, Ryan; Husain, Aliya N; Vigneswaran, Wickii; Fitzpatrick, Carrie; Krausz, Thomas

    2017-02-01

    BRCA-associated protein 1 (BAP1) immunohistochemistry (IHC) and CDKN2A (p16) fluorescence in situ hybridization (FISH) have shown clinical utility in confirming the diagnosis of malignant pleural mesothelioma (MPM), but the role for using these 2 markers to guide clinical management is not yet clear. Although p16 loss is predictive of poor prognosis, there is controversy as to whether BAP1 loss is predictive of a more favorable prognosis; how these results interact with one another has not been explored. We performed CDKN2A FISH on a previously published tissue microarray on which we had performed BAP1 IHC, revealing combined BAP1/p16 status for 93 MPM cases. As expected, BAP1 IHC in combination with CDKN2A FISH resulted in high sensitivity (84%) and specificity (100%) for MPM, and p16 loss was an independent predictor of poor survival (hazard ratio, 2.2553; P = .0135). There was no association between BAP1 loss and p16 loss, as 26%, 28%, 30%, and 16% of overall cases demonstrated loss of BAP1 alone, loss of p16 alone, loss of both BAP1 and p16, or neither abnormality, respectively. Although multivariate analysis demonstrated that BAP1 IHC is not an independent predictor of prognosis, when viewed in combination with homozygous CDKN2A deletion, risk stratification was evident. More specifically, patients with CDKN2A disomy and loss of BAP1 expression had improved outcomes compared with those with CDKN2A disomy and retained BAP1 expression (hazard ratio, 0.2286; P = .0017), and this finding was notably evident among epithelioid cases. We conclude that BAP1 IHC provides prognostic information within the context of CDKN2A FISH that may have clinical utility beyond diagnosis.

  2. Toward a multiplexed solid-phase nucleic acid hybridization assay using quantum dots as donors in fluorescence resonance energy transfer.

    PubMed

    Algar, W Russ; Krull, Ulrich J

    2009-05-15

    Solid-phase assays using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) have been developed for the selective detection of nucleic acids. QDs were immobilized on optical fibers and conjugated with probe oligonucleotides. Hybridization with acceptor labeled target oligonucleotides generated FRET-sensitized acceptor fluorescence that was used as the analytical signal. A sandwich assay was also introduced and avoided the need for target labeling. Green and red emitting CdSe/ZnS QDs were used as donors with Cy3 and Alexa Fluor 647 acceptors, respectively. Quantitative measurements were made via spectrofluorimetry or fluorescence microscopy. Detection limits as low as 1 nM were obtained, and the discrimination of single nucleotide polymorphisms (SNPs) with contrast ratios as high as 31:1 was possible. The assays retained their selectivity and at least 50% of their signal when tested in bovine serum and against a large background of noncomplementary genomic DNA. Mixed films of the two colors of QD and two probe oligonucleotide sequences were prepared for multiplexed solid-phase hybridization assays. It was possible to simultaneously detect two target sequences with retention of selectivity, including SNP discrimination. This research provides an important precedent and framework for the future development of QD-based bioassays and biosensors.

  3. High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization.

    PubMed

    Moffitt, Jeffrey R; Hao, Junjie; Wang, Guiping; Chen, Kok Hao; Babcock, Hazen P; Zhuang, Xiaowei

    2016-09-27

    Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.

  4. High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization

    PubMed Central

    Moffitt, Jeffrey R.; Hao, Junjie; Wang, Guiping; Chen, Kok Hao

    2016-01-01

    Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH. PMID:27625426

  5. Efficient ratiometric fluorescence probe based on dual-emission quantum dots hybrid for on-site determination of copper ions.

    PubMed

    Yao, Jianlei; Zhang, Kui; Zhu, Houjuan; Ma, Fang; Sun, Mingtai; Yu, Huan; Sun, Jian; Wang, Suhua

    2013-07-02

    Of various chemosensory protocols, the color change observed by the naked eye is considered to be a conceivable and on-site way to indicate the presence of an analyte. We herein designed a ratiometric fluorescence probe by hybridizing dual-emission quantum dots (QDs) and demonstrated its efficiency for on-site visual determination of copper ions. The hybrid probe comprises two sizes of cadmium telluride QDs emitting red and green fluorescence, respectively, in which the red-emitting ones are embedded in silica nanoparticles and the green-emitting ones are covalently linked onto the surface. The fluorescence of the embedded QDs is insensitive to the analyte, whereas the green emissive QDs are functionalized to be selectively quenched by the analyte. Upon exposure to different amounts of copper ions, the variations of the dual emission intensity ratios display continuous color changes from green to red, which can be clearly observed by the naked eye. The limit of detection for copper is estimated to be 1.1 nM, much lower than the allowable level of copper (~20 μM) in drinking water set by U.S. Environmental Protection Agency. The probe is demonstrated for the determination of copper ions in lake water and mineral water samples, especially for visually monitoring copper residues on herb leaves. This prototype ratiometric probe is simple, fully self-contained, and thus potentially attractive for visual identification without the need for elaborate equipment.

  6. Unlocked nucleic acids with a pyrene-modified uracil: synthesis, hybridization studies, fluorescent properties and i-motif stability.

    PubMed

    Perlíková, Pavla; Karlsen, Kasper K; Pedersen, Erik B; Wengel, Jesper

    2014-01-03

    The synthesis of two new phosphoramidite building blocks for the incorporation of 5-(pyren-1-yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene-modified nucleobase component were found to destabilize an i-motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene-modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene-modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady-state fluorescence emission studies of oligonucleotides containing pyrene-modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene-modified UNA monomers was observed after formation of i-motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene-modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.

  7. Mesoporous cerium phosphonate nanostructured hybrid spheres as label-free Hg²⁺ fluorescent probes.

    PubMed

    Zhu, Yun-Pei; Ma, Tian-Yi; Ren, Tie-Zhen; Yuan, Zhong-Yong

    2014-09-24

    Porous phosphonate-based organic-inorganic hybrid materials have been shown to have novel and amazing physicochemical properties due to the integration of superiorities from both inorganic components and organic moieties. Herein, mesoporous cerium phosphonate nanostructured hybrid spheres are prepared with the assistance of cationic surfactant cetyltrimethylammonium bromide while using ethylene diamine tetra(methylene phosphonic acid) as the coupling molecule. The resulting hybrid is constructed from the cerium phosphonate nanoparticles, accompanied by high specific surface area of 455 m(2) g(-1). The uniform incorporation of rare-earth element cerium and organophosphonic functionalities endows mesoporous cerium phosphonate with excellent fluorescence properties for the development of an optical sensor for selective Hg(2+) detection on the basis of the fluorescence-quenching mechanism. The signal response of mesoporous cerium phosphonate against the Hg(2+) concentration is linear over the range from 0.05 to 1.5 μmol L(-1), giving a limit of detection of 16 nmol L(-1) (at a signal-to-noise ratio of 3). Most of the common physiologically relevant cations and anions did not interfere with the detection of Hg(2+). This label-free system provides a promising platform for further use in bioimaging and biomedical fields.

  8. Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization.

    PubMed

    Ding, Z F; Xia, S Y; Xue, H; Tang, J Q; Ren, Q; Gu, W; Meng, Q G; Wang, W

    2015-09-01

    Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.

  9. Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization

    SciTech Connect

    Lawrence, J.B.; Marselle, L.M.; Byron, K.S.; Johnson, C.V.; Sullivan, J.L.; Singer, R.H. )

    1990-07-01

    Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

  10. Synthesis and spectral properties of polymethine-cyanine dye-nitroxide radical hybrid compounds for use as fluorescence probes to monitor reducing species and radicals

    NASA Astrophysics Data System (ADS)

    Sato, Shingo; Tsunoda, Minoru; Suzuki, Minoru; Kutsuna, Masahiro; Takido-uchi, Kiyomi; Shindo, Mitsuru; Mizuguchi, Hitoshi; Obara, Heitaro; Ohya, Hiroaki

    2009-01-01

    Various hybrid compounds comprised of two types of nitroxide radicals and either a pentamethine (Cy5) or trimethine cyanine (Cy3) were synthesized. The nitroxide radicals were linked either via an ester-bond to one or two N-alkyl carboxyl-terminated groups of Cy5, or via two amido-bonds (aminocarbonyl or carbonylamino group) to the 5-position of the indolenine moieties of Cy5 and Cy3. Changes in fluorescence and ESR intensities of the hybrid compounds were measured before and after addition of Na ascorbate in PBS (pH 7.0) to reduce the radicals. Among the hybrid compounds synthesized, those that linked the nitroxide radicals via an aminocarbonyl residue at the 5-position of the indolenine moieties on Cy5 and Cy3 exhibited a 1.8- and 5.1-fold increase in fluorescence intensity with the reduction of the nitroxide segment by the addition of Na ascorbate, respectively. In contrast, fluorescence intensity was not enhanced in the other hybrid compounds. Thus, the hybrid compounds which exhibited an increase in fluorescent intensity with radical reduction can be used in the quantitative measurement of reducing species such as Fe 2+ and ascorbic acid, and hydroxyl radicals. Because these hybrid compounds have the advantage of fluorescing at longer wavelengths—661 (Cy5) or 568 (Cy3) nm, respectively, they can be used to measure radical-reducing species or radicals either in solution or in vivo.

  11. A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Wu, Changfeng; Barnhill, Hannah; Liang, Xiaoping; Wang, Qian; Jiang, Huabei

    2005-11-01

    A fluorescent probe based on bionanoparticle cowpea mosaic virus has been developed for near-infrared fluorescence tomography. A unique advantage of this probe is that over 30 dye molecules can be loaded onto each viral nanoparticle with an average diameter of 30 nm, making high local dye concentration (∼1.8 mM) possible without significant fluorescence quenching. This ability of high loading of local dye concentration would increase the signal-to-noise ratio considerably, thus sensitivity for detection. We demonstrate successful tomographic fluorescence imaging of a target containing the virus-dye nanoparticles embedded in a tissue-like phantom. Tomographic fluorescence data were obtained through a multi-channel frequency-domain system and the spatial maps of fluorescence quantum yield were recovered with a finite-element-based reconstruction algorithm.

  12. High concordance between immunohistochemistry and fluorescence in situ hybridization testing for HER2 status in breast cancer requires a normalized IHC scoring system.

    PubMed

    Gown, Allen M; Goldstein, Lynn C; Barry, Todd S; Kussick, Steven J; Kandalaft, Patricia L; Kim, Patricia M; Tse, Christopher C

    2008-10-01

    The American Society of Clinical Oncologists and College of American Pathologists have recently released new guidelines for laboratory testing of HER2 status in breast cancer, which require high levels (95%) of concordance between immunohistochemistry positive (3+) and fluorescence in situ hybridization-amplified cases, and between immunohistochemistry negative (0/1+) and fluorescence in situ hybridization-nonamplified cases; these required levels of concordance are significantly higher than those found in most published studies. We tested the hypothesis that a modification of the HER2 immunohistochemistry scoring system could significantly improve immunohistochemistry and fluorescence in situ hybridization concordance. A total of 6604 breast cancer specimens were evaluated for HER2 status by both immunohistochemistry and fluorescence in situ hybridization using standard methodologies. Results were compared when the standard immunohistochemistry scoring system was replaced by a normalized scoring system in which the HER2 score was derived by subtracting the score on the non-neoplastic breast epithelium from that on the tumor cells. Among the 6604 tumors, using a non-normalized immunohistochemistry scoring system, 267/872 (30.6%) of the immunohistochemistry 3+ cases proved to be fluorescence in situ hybridization nonamplified, whereas using the normalized scoring system only 30/562 (5.3%) of immunohistochemistry 3+ cases proved to be 'false positive'. The concordance rate between immunohistochemistry 3+ and fluorescence in situ hybridization-amplified cases using the normalized scoring method was 94.7%, whereas the concordance using the non-normalized method was only 69.4%. Extremely high concordance between immunohistochemistry and fluorescence in situ hybridization assessment of HER2 status in breast cancer is achievable, but to attain this high level of concordance, modification of the FDA-approved immunohistochemistry scoring system is required.

  13. RNA containing pyrrolocytidine base analogs: increased binding affinity and fluorescence that responds to hybridization.

    PubMed

    Wahba, Alexander S; Damha, Masad J; Hudson, Robert H E

    2008-01-01

    6-Phenylpyrrolocytidine and 6-methoxymethylene-pyrrolocytidine are base-modified nucleosides with remarkable fluorescence properties. These analogs produce increased binding affinity to both RNA and DNA targets when incorporated into oligoribonucleotides. The fluorescence observed for the single-stranded oligomers is quenched upon duplex formation with either RNA or DNA targets. The fluorescence response depends on the nature of the 6-substituent and the sequence position of the modified nucleoside.

  14. Potential actionable targets in appendiceal cancer detected by immunohistochemistry, fluorescent in situ hybridization, and mutational analysis

    PubMed Central

    Millis, Sherri Z.; Kimbrough, Jeffery; Doll, Nancy; Von Hoff, Daniel; Ramanathan, Ramesh K.

    2017-01-01

    Background Appendiceal cancers are rare and consist of carcinoid, mucocele, pseudomyxoma peritonei (PMP), goblet cell carcinoma, lymphoma, and adenocarcinoma histologies. Current treatment involves surgical resection or debulking, but no standard exists for adjuvant chemotherapy or treatment for metastatic disease. Methods Samples were identified from approximately 60,000 global tumors analyzed at a referral molecular profiling CLIA-certified laboratory. A total of 588 samples with appendix primary tumor sites were identified (male/female ratio of 2:3; mean age =55). Sixty-two percent of samples were adenocarcinomas (used for analysis); the rest consisted of 9% goblet cell, 15% mucinous; 6% pseudomyxoma, and less than 5% carcinoids and 2% neuroendocrine. Tests included sequencing [Sanger, next generation sequencing (NGS)], protein expression/immunohistochemistry (IHC), and gene amplification [fluorescent in situ hybridization (FISH) or CISH]. Results Profiling across all appendiceal cancer histological subtypes for IHC revealed: 97% BRCP, 81% MRP1, 81% COX-2, 71% MGMT, 56% TOPO1, 5% PTEN, 52% EGFR, 40% ERCC1, 38% SPARC, 35% PDGFR, 35% TOPO2A, 25% RRM1, 21% TS, 16% cKIT, and 12% for TLE3. NGS revealed mutations in the following genes: 50.4% KRAS, 21.9% P53, 17.6% GNAS, 16.5% SMAD4, 10% APC, 7.5% ATM, 5.5% PIK3CA, 5.0% FBXW7, and 1.8% BRAF. Conclusions Appendiceal cancers show considerable heterogeneity with high levels of drug resistance proteins (BCRP and MRP1), which highlight the difficulty in treating these tumors and suggest an individualized approach to treatment. The incidence of low TS (79%) could be used as a backbone of therapy (using inhibitors such as 5FU/capecitabine or newer agents). Therapeutic options includeTOPO1 inhibitors (irinotecan/topotecan), EGFR inhibitors (erlotinib, cetuximab), PDGFR antagonists (regorafenib, axitinib), MGMT (temozolomide). Clinical trials targeting pathways involving KRAS, p53, GNAS, SMAD4, APC, ATM, PIK3CA, FBXW7, and

  15. Segmental duplications within the Glycine max genome revealed by fluorescence in situ hybridization of bacterial artificial chromosomes.

    PubMed

    Pagel, Janice; Walling, Jason G; Young, Nevin D; Shoemaker, Randy C; Jackson, Scott A

    2004-08-01

    Soybean (Glycine max L. Merr.) is presumed to be an ancient polyploid based on chromosome number and multiple RFLP fragments in genetic mapping. Direct cytogenetic observation of duplicated regions within the soybean genome has not heretofore been reported. Employing fluorescence in situ hybridization (FISH) of genetically anchored bacterial artificial chromosomes (BACs) in soybean, we were able to observe that the distal ends of molecular linkage group E had duplicated regions on linkage groups A2 and B2. Further, using fiber-FISH, it was possible to measure the molecular size and organization of one of the duplicated regions. As FISH did not require repetitive DNA for blocking fluorescence signals, we assume that the 200-kb genome region is relatively low in repetitive sequences. This observation, along with the observation that the BACs are located in distal euchromatin regions, has implications for genome structure/evolution and the approach used to sequence the soybean genome.

  16. Karyotype analysis, banding, and fluorescent in situ hybridization in the scarab beetle Gymnopleurus sturmi McLeay (Coleoptera Scarabaeoidea : Scarabaeidae).

    PubMed

    Colomba, M S; Vitturi, R; Zunino, M

    2000-01-01

    Conventional staining, differential banding, and in situ hybridization with both ribosomal and telomeric probes to mitotic chromosomes of Gymnopleurus sturmi (Scarabaeoidea : Scarabaeidae) are described. The karyotype is distinguished by a pericentric inversion polymorphism in chromosome 3, which is either acrocentric or subtelocentric. Silver staining (Ag-NOR) and chromomycin A3 (CMA3), failed to study the detection of nucleolar organizer regions (NORs), due to the extensive silver and CMA3 stainability of all GC-rich heterochromatin. Fluorescent in situ hybridization (FISH) using a Paracentrotus lividus (Echinodermata) rDNA probe mapped the ribosomal RNA genes (rDNA). FISH with the all-human telomeric sequences (TTAGGG)n revealed a lack of homology between the telomeric probe and the telomeres of G. sturmi. This suggests that the telomeric hesanucleotide (TTAGGG)n is not so conserved within eukaryotes as it has been hypothesized.

  17. Rapid Ovary Mass-Isolation (ROMi) to Obtain Large Quantities of Drosophila Egg Chambers for Fluorescent In Situ Hybridization.

    PubMed

    Jambor, Helena; Mejstrik, Pavel; Tomancak, Pavel

    2016-01-01

    Isolation of large quantities of tissue from organisms is essential for many techniques such as genome-wide screens and biochemistry. However, obtaining large quantities of tissues or cells is often the rate-limiting step when working in vivo. Here, we present a rapid method that allows the isolation of intact, single egg chambers at various developmental stages from ovaries of adult female Drosophila flies. The isolated egg chambers are amenable for a variety of procedures such as fluorescent in situ hybridization, RNA isolation, extract preparation, or immunostaining. Isolation of egg chambers from adult flies can be completed in 5 min and results, depending on the input amount of flies, in several milliliters of material. The isolated egg chambers are then further processed depending on the exact requirements of the subsequent application. We describe high-throughput in situ hybridization in 96-well plates as example application for the mass-isolated egg chambers.

  18. Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: Clinical experience with 4,500 specimens

    SciTech Connect

    Ward, B.E.; Gersen, S.L.; Carelli, M.P.; McGuire, N.M.; Dackowski, W.R.; Klinger, K.W. ); Weinstein, M. ); Sandlin, C. ); Klinger, K.W. )

    1993-05-01

    Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). The authors herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies. 40 refs., 1 fig., 5 tabs.,

  19. Statistical treatment of fluorescence in situ hybridization validation data to generate normal reference ranges using Excel functions.

    PubMed

    Ciolino, Allison L; Tang, Mary E; Bryant, Ron

    2009-07-01

    Fluorescent in situ hybridization has become an essential tool for diagnosing and monitoring hematological disease. Testing for minimal residual disease requires precise and accurate normal cut-offs. There is no consensus in the field on the correct method of establishing a normal reference range. We discuss and compare several proposed statistical methods to calculate normal reference ranges, including Gaussian statistics, the beta inverse function, and a binomial treatment of the data. We demonstrate that a binomial treatment of the data is an accurate and simple method to calculate a normal reference range.

  20. Enumeration of respiring Pseudomonas spp. in milk within 6 hours by fluorescence in situ hybridization following formazan reduction.

    PubMed

    Kitaguchi, Akiko; Yamaguchi, Nobuyasu; Nasu, Masao

    2005-05-01

    Respiring Pseudomonas spp. in milk were quantified within 6 h by fluorescence in situ hybridization (FISH) with vital staining. FISH with an oligonucleotide probe based on 16S rRNA sequences was used for the specific detection of Pseudomonas spp. at the single cell level. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to estimate bacterial respiratory activity. The numbers of respiring Pseudomonas cells as determined by FISH with CTC staining (CTC-FISH) were almost the same or higher than the numbers of CFU as determined by the conventional culture method.

  1. A nano-graphite-DNA hybrid sensor for magnified fluorescent detection of mercury(II) ions in aqueous solution.

    PubMed

    Wei, Yin; Li, Bianmiao; Wang, Xu; Duan, Yixiang

    2014-04-07

    In this communication, we present a nano-graphite-DNA hybrid sensor for fluorescent detection of mercury(II) ions in aqueous solution for the first time. Furthermore, an amplification strategy based on nano-graphite for Hg(2+) detection by using DNase I was demonstrated. The proposed amplified assay was simple and cost-effective with a limit of detection (LOD) for Hg(2+) of 0.5 nM, which was about 20-fold lower than that of traditional unamplified homogeneous assays. We further demonstrated its practical application to detect Hg(2+) in a real sample.

  2. Prenatal diagnosis of the derivative chromosome 22 associated with cat eye syndrome by fluorescence in situ hybridization.

    PubMed

    Reeser, S L; Donnenfeld, A E; Miller, R C; Sellinger, B S; Emanuel, B S; Driscoll, D A

    1994-11-01

    Cytogenetic studies of cultured amniocytes demonstrated a karyotype of 46,XX/47,XX, +mar. A bisatellited, dicentric, distamycin-DAPI negative, NOR-positive marker was present in 76 per cent of the metaphases examined. Similar markers have been associated with cat eye syndrome (CES). We report on the utilization of fluorescence in situ hybridization (FISH) with a 14/22 alpha-satellite probe and a chromosome 22-specific cosmid for locus D22S9 to determine the origin of the prenatally detected supernumerary marker chromosome. FISH studies demonstrated that the marker is a derivative of chromosome 22 and enabled us to provide the family with additional prognostic information.

  3. Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.

    PubMed

    van de Corput, M P; Dirks, R W; van Gijlswijk, R P; van Binnendijk, E; Hattinger, C M; de Paus, R A; Landegent, J E; Raap, A K

    1998-11-01

    With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH.

  4. High sensitivity knitted fabric strain sensors

    NASA Astrophysics Data System (ADS)

    Xie, Juan; Long, Hairu; Miao, Menghe

    2016-10-01

    Wearable sensors are increasingly used in smart garments for detecting and transferring vital signals and body posture, movement and respiration. Existing fabric strain sensors made from metallized yarns have low sensitivity, poor comfort and low durability to washing. Here we report a knitted fabric strain sensor made from a cotton/stainless steel (SS) fibre blended yarn which shows much higher sensitivity than sensors knitted from metallized yarns. The fabric feels softer than pure cotton textiles owing to the ultrafine stainless steel fibres and does not lose its electrical property after washing. The reason for the high sensitivity of the cotton/SS knitted fabric sensor was explored by comparing its sensing mechanism with the knitted fabric sensor made from metallized yarns. The results show that the cotton/SS yarn-to-yarn contact resistance is highly sensitive to strain applied to hooked yarn loops.

  5. Identification of peanut (Arachis hypogaea) chromosomes using a fluorescence in situ hybridization system reveals multiple hybridization events during tetraploid peanut formation.

    PubMed

    Zhang, Laining; Yang, Xiaoyu; Tian, Li; Chen, Lei; Yu, Weichang

    2016-09-01

    The cultivated peanut Arachis hypogaea (AABB) is thought to have originated from the hybridization of Arachis duranensis (AA) and Arachis ipaënsis (BB) followed by spontaneous chromosome doubling. In this study, we cloned and analyzed chromosome markers from cultivated peanut and its wild relatives. A fluorescence in situ hybridization (FISH)-based karyotyping cocktail was developed with which to study the karyotypes and chromosome evolution of peanut and its wild relatives. Karyotypes were constructed in cultivated peanut and its two putative progenitors using our FISH-based karyotyping system. Comparative karyotyping analysis revealed that chromosome organization was highly conserved in cultivated peanut and its two putative progenitors, especially in the B genome chromosomes. However, variations existed between A. duranensis and the A genome chromosomes in cultivated peanut, especially for the distribution of the interstitial telomere repeats (ITRs). A search of additional A. duranensis varieties from different geographic regions revealed both numeric and positional variations of ITRs, which were similar to the variations in tetraploid peanut varieties. The results provide evidence for the origin of cultivated peanut from the two diploid ancestors, and also suggest that multiple hybridization events of A. ipaënsis with different varieties of A. duranensis may have occurred during the origination of peanut.

  6. Developments of highly sensitive DNA sensors

    NASA Astrophysics Data System (ADS)

    Ogata, Naoya

    2011-09-01

    The large enhancements of optical properties of the dye-intercalated DNA lead us to apply the dye-intercalated DNA as various sensors with a high sensitivity to detect environmentally toxic gases such as dioxine, NOx or carbon monoxide. This paper retorts on DNA sensors for the further applications of DNA as materials. Also, bio-medical applications of DNA sensors such as a glucose sensor are reported.

  7. [Clinical interpretation of high sensitivity troponin T].

    PubMed

    Alquézar Arbé, Aitor; Santaló Bel, Miguel; Sionis, Alessandro

    2015-09-21

    Determination of cardiac troponin (cTn) is necessary for the diagnosis of acute myocardial infarction without ST segment elevation. However Tnc can be released in other clinical situations. The development of high-sensitive cTn T assays (hs-cTnT) improves the management of patients with suspected acute coronary syndrome. Here, we provide an overview of the diverse causes of hs-cTnT elevation and recommend strategies for the clinical interpretation of the test result.

  8. High sensitivity neutron detector for Z

    SciTech Connect

    Ruggles, L.E.; Porter, J.L. Jr.; Simpson, W.W.; Vargas, M.F.; Zagar, D.M.; Hartke, R.; Buersgens, F.; Symes, D.R.; Ditmire, T.

    2004-10-01

    We have developed, calibrated, and tested a high sensitivity neutron detector that can be operated in the harsh x-ray bremsstrahlung environment that exists in experiments conducted on the 20 MA Z z-pinch facility located at Sandia National Laboratories in Albuquerque, New Mexico. The detector uses a scintillator coupled to a microchannel-plate photomultiplier tube detector and extensive x-ray shielding.

  9. Above 20% external quantum efficiency in novel hybrid white organic light-emitting diodes having green thermally activated delayed fluorescent emitter

    PubMed Central

    Kim, Bo Seong; Yook, Kyoung Soo; Lee, Jun Yeob

    2014-01-01

    High efficiency hybrid type white organic light-emitting diodes (WOLEDs) combining a green thermally activated delayed fluorescent (TADF) emitting material with red/blue phosphorescent emitting materials were developed by manipulating the device architecture of WOLEDs. Energy transfer between a blue phosphorescent emitting material and a green TADF emitter was efficient and could be managed by controlling the doping concentration of emitters. A high quantum efficiency above 20% was achieved in the hybrid WOLEDs by optimizing the device structure of the hybrid type WOLEDs for the first time and the device performances of the hybrid WOLEDs were comparable to those of all phosphorescent WOLEDs. PMID:25317855

  10. Above 20% external quantum efficiency in novel hybrid white organic light-emitting diodes having green thermally activated delayed fluorescent emitter

    NASA Astrophysics Data System (ADS)

    Kim, Bo Seong; Yook, Kyoung Soo; Lee, Jun Yeob

    2014-08-01

    High efficiency hybrid type white organic light-emitting diodes (WOLEDs) combining a green thermally activated delayed fluorescent (TADF) emitting material with red/blue phosphorescent emitting materials were developed by manipulating the device architecture of WOLEDs. Energy transfer between a blue phosphorescent emitting material and a green TADF emitter was efficient and could be managed by controlling the doping concentration of emitters. A high quantum efficiency above 20% was achieved in the hybrid WOLEDs by optimizing the device structure of the hybrid type WOLEDs for the first time and the device performances of the hybrid WOLEDs were comparable to those of all phosphorescent WOLEDs.

  11. Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water.

    PubMed

    Savichtcheva, Olga; Okayama, Noriko; Ito, Tsukasa; Okabe, Satoshi

    2005-11-05

    To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.

  12. Portable evanescent wave fiber biosensor for highly sensitive detection of Shigella

    NASA Astrophysics Data System (ADS)

    Xiao, Rui; Rong, Zhen; Long, Feng; Liu, Qiqi

    2014-11-01

    A portable evanescent wave fiber biosensor was developed to achieve the rapid and highly sensitive detection of Shigella. In this study, a DNA probe was covalently immobilized onto fiber-optic biosensors that can hybridize with a fluorescently labeled complementary DNA. The sensitivity of detection for synthesized oligonucleotides can reach 10-10 M. The surface of the sensor can be regenerated with 0.5% sodium dodecyl sulfate solution (pH 1.9) for over 30 times without significant deterioration of performance. The total analysis time for a single sample, including the time for measurement and surface regeneration, was less than 6 min. We employed real-time polymerase chain reaction (PCR) and compared the results of both methods to investigate the actual Shigella DNA detection capability of the fiber-optic biosensor. The fiber-optic biosensor could detect as low as 102 colony-forming unit/mL Shigella. This finding was comparable with that by real-time PCR, which suggests that this method is a potential alternative to existing detection methods.

  13. Luminescent Lanthanide Reporters for High-Sensitivity Novel Bioassays.

    SciTech Connect

    Anstey, Mitchell R.; Fruetel, Julia A.; Foster, Michael E.; Hayden, Carl C.; Buckley, Heather L.; Arnold, John

    2013-09-01

    Biological imaging and assay technologies rely on fluorescent organic dyes as reporters for a number of interesting targets and processes. However, limitations of organic dyes such as small Stokes shifts, spectral overlap of emission signals with native biological fluorescence background, and photobleaching have all inhibited the development of highly sensitive assays. To overcome the limitations of organic dyes for bioassays, we propose to develop lanthanide-based luminescent dyes and demonstrate them for molecular reporting applications. This relatively new family of dyes was selected for their attractive spectral and chemical properties. Luminescence is imparted by the lanthanide atom and allows for relatively simple chemical structures that can be tailored to the application. The photophysical properties offer unique features such as narrow and non-overlapping emission bands, long luminescent lifetimes, and long wavelength emission, which enable significant sensitivity improvements over organic dyes through spectral and temporal gating of the luminescent signal.Growth in this field has been hindered due to the necessary advanced synthetic chemistry techniques and access to experts in biological assay development. Our strategy for the development of a new lanthanide-based fluorescent reporter system is based on chelation of the lanthanide metal center using absorbing chromophores. Our first strategy involves "Click" chemistry to develop 3-fold symmetric chelators and the other involves use of a new class of tetrapyrrole ligands called corroles. This two-pronged approach is geared towards the optimization of chromophores to enhance light output.

  14. Localized surface plasmon resonance-mediated fluorescence signals in plasmonic nanoparticle-quantum dot hybrids for ultrasensitive Zika virus RNA detection via hairpin hybridization assays.

    PubMed

    Adegoke, Oluwasesan; Morita, Masahiro; Kato, Tatsuya; Ito, Masahito; Suzuki, Tetsuro; Park, Enoch Y

    2017-03-22

    The current epidemic caused by the Zika virus (ZIKV) and the devastating effects of this virus on fetal development, which result in an increased incidence of congenital microcephaly symptoms, have prompted the World Health Organization (WHO) to declare the ZIKV a public health issue of global concern. Efficient probes that offer high detection sensitivity and specificity are urgently required to aid in the point-of-care treatment of the virus. In this study, we show that localized surface plasmon resonance (LSPR) signals from plasmonic nanoparticles (NPs) can be used to mediate the fluorescence signal from semiconductor quantum dot (Qdot) nanocrystals in a molecular beacon (MB) biosensor probe for ZIKV RNA detection. Four different plasmonic NPs functionalized with 3-mercaptopropionic acid (MPA), namely MPA-AgNPs, MPA-AuNPs, core/shell (CS) Au/AgNPs, and alloyed AuAgNPs, were synthesized and conjugated to L-glutathione-capped CdSeS alloyed Qdots to form the respective LSPR-mediated fluorescence nanohybrid. The concept of the plasmonic NP-Qdot-MB biosensor involves using LSPR from the plasmonic NPs to mediate a fluorescence signal to the Qdots, triggered by the hybridization of the target ZIKV RNA with the DNA loop sequence of the MB. The extent of the fluorescence enhancement based on ZIKV RNA detection was proportional to the LSPR-mediated fluorescence signal. The limits of detection (LODs) of the nanohybrids were as follows: alloyed AuAgNP-Qdot646-MB (1.7 copies/mL)) > CS Au/AgNP-Qdot646-MB (LOD =2.4 copies/mL) > AuNP-Qdot646-MB (LOD =2.9 copies/mL) > AgNP-Qdot646-MB (LOD =7.6 copies/mL). The LSPR-mediated fluorescence signal was stronger for the bimetallic plasmonic NP-Qdots than the single metallic plasmonic NP-Qdots. The plasmonic NP-Qdot-MB biosensor probes exhibited excellent selectivity toward ZIKV RNA and could serve as potential diagnostic probes for the point-of care detection of the virus.

  15. Novel hybrid structure silica/CdTe/molecularly imprinted polymer: synthesis, specific recognition, and quantitative fluorescence detection of bovine hemoglobin.

    PubMed

    Li, Dong-Yan; He, Xi-Wen; Chen, Yang; Li, Wen-You; Zhang, Yu-Kui

    2013-12-11

    This work presented a novel strategy for the synthesis of the hybrid structure silica/CdTe/molecularly imprinted polymer (Si-NP/CdTe/MIP) to recognize and detect the template bovine hemoglobin (BHb). First, amino-functionalized silica nanoparticles (Si-NP) and carboxyl-terminated CdTe quantum dots (QDs) were assembled into composite nanoparticles (Si-NP/CdTe) using the EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) chemistry. Next, Si-NP/CdTe/MIP was synthesized by anchoring molecularly imprinted polymer (MIP) layer on the surface of Si-NP/CdTe through the sol-gel technique and surface imprinting technique. The hybrid structure possessed the selectivity of molecular imprinting technique and the sensitivity of CdTe QDs as well as well-defined morphology. The binding experiment and fluorescence method demonstrated its special recognition performance toward the template BHb. Under the optimized conditions, the fluorescence intensity of the Si-NP/CdTe/MIP decreased linearly with the increase of BHb in the concentration range 0.02-2.1 μM, and the detection limit was 9.4 nM. Moreover, the reusability and reproducibility and the successful applications in practical samples indicated the synthesis of Si-NP/CdTe/MIP provided an alternative solution for special recognition and determination of protein from real samples.

  16. An integrated hybrid interference and absorption filter for fluorescence detection in lab-on-a-chip devices.

    PubMed

    Richard, Charles; Renaudin, Alan; Aimez, Vincent; Charette, Paul G

    2009-05-21

    We present a hybrid optical filter design that combines interference and absorbing components for enhanced fluorescence detection in miniaturized highly-integrated lab-on-a-chip devices. The filter is designed in such a way that the advantages of each technology are used to offset the disadvantages of the other. The filter is fabricated with microfabrication compatible processes and materials for monolithic integration with microelectronics and microfluidics devices. The particular embodiment of the filter described herein is designed to discriminate fluorescence emission at 650 nm from excitation at 532 nm. The 9-layer interference filter component is fabricated with alternating TiO(2) and SiO(2) thin-film layers and has an attenuation of -12.6 dB at 532 nm and -0.76 dB at 650 nm. The absorbing filter component is fabricated using a dyed photopolymer (KMPR + Orasol Red) having an attenuation of -32.6 dB at 532 nm and -1.28 dB at 650 nm. The total rejection ratio of the hybrid filter is 43 dB. The filter exhibits very low autofluorescence and performs equally well at off-axis incidence angles.

  17. Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH).

    PubMed

    Ueno, Ryohei

    2009-04-01

    Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

  18. Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

    PubMed Central

    Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

  19. Detection of aneuploidy in sperm of an ataxia telangiectasia patient using three-chromosome fluorescence in situ hybridization

    SciTech Connect

    Lowe, X.R.; Baulch, J.E.; Arnheim, N.

    1994-09-01

    Ataxia telangiectasia (A-T) is an inherited, recessive, cancer-prone disorder. Fluorescence in situ hybridization (FISH) with DNA probes specific for three chromosomes was applied to sperm of an A-T patient to determine if there may be an increased germinal risk for aneuploidy. Air-dried sperm smears were treated with proteinase K and were decondensed with DTT and LIS. The slides were then hybridized with fluorescently labeled repetitive DNA probes specific for chromosomes X, Y and 8, and a total of 11,825 sperm cells were scored. The ratio of sperm bearing X-8 and Y-8 was 1:1, as predicted. The frequencies of hyperhaploidy were 3.9, 1.0, 17.6 and 7.8 per 10,000 cells for categories X-X-8, Y-Y-8, X-Y-8 and 8-8-(X or Y), respectively, In addition, the frequency of diploidy (X-Y-8-8) was 18.6 and auto-diploidies (X-X-8-8 and Y-Y-8-8) were 1.0 and 2.0, respectively. These frequencies were not significantly different when compared with levels in healthy men (p > 0.1). Our finding suggests that chromosome X, Y and 8 aneuploidies are not elevated in the sperm of A-T patients, but studies with additional patients and chromosomes are needed.

  20. Nucleic acid in-situ hybridization detection of infectious agents

    NASA Astrophysics Data System (ADS)

    Thompson, Curtis T.

    2000-04-01

    Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

  1. Quantum dots as FRET acceptors for highly sensitive multiplexing immunoassays

    NASA Astrophysics Data System (ADS)

    Geissler, Daniel; Hildebrandt, Niko; Charbonnière, Loïc J.; Ziessel, Raymond F.; Löhmannsröben, Hans-Gerd

    2009-02-01

    Homogeneous immunoassays have the benefit that they do not require any time-consuming separation steps. FRET is one of the most sensitive homogeneous methods used for immunoassays. Due to their extremely strong absorption over a broad wavelength range the use of quantum dots as FRET acceptors allows for large Foerster radii, an important advantage for assays in the 5 to 10 nm distance range. Moreover, because of their size-tunable emission, quantum dots of different sizes can be used with a single donor for the detection of different analytes (multiplexing). As the use of organic dyes with short fluorescence decay times as donors is known to be inefficient with quantum dot acceptors, lanthanide complexes with long luminescence decays are very efficient alternatives. In this contribution we present the application of commercially available biocompatible CdSe/ZnS core/shell quantum dots as multiplexing FRET acceptors together with a single terbium complex as donor in a homogeneous immunoassay system. Foerster radii of 10 nm and FRET efficiencies of 75 % are demonstrated. The high sensitivity of the terbium-toquantum dot FRET assay is shown by sub-100-femtomolar detection limits for two different quantum dots (emitting at 605 and 655 nm) within the same biotin-streptavidin assay. Direct comparison to the FRET immunoassay "gold standard" (FRET from Eu-TBP to APC) yields a three orders of magnitude sensitivity improvement, demonstrating the big advantages of quantum dots not only for multiplexing but also for highly sensitive nanoscale analysis.

  2. Cooled membrane for high sensitivity gas sampling.

    PubMed

    Jiang, Ruifen; Pawliszyn, Janusz

    2014-04-18

    A novel sample preparation method that combines the advantages of high surface area geometry and cold surface effect was proposed to achieve high sensitivity gas sampling. To accomplish this goal, a device that enables the membrane to be cooled down was developed for sampling, and a gas chromatograph-mass spectrometer was used for separation and quantification analysis. Method development included investigation of the effect of membrane temperature, membrane size, gas flow rate and humidity. Results showed that high sensitivity for equilibrium sampling, such as limonene sampling in the current study could be achieved by either cooling down the membrane and/or using a large volume extraction phase. On the other hand, for pre-equilibrium extraction, in which the extracted amount was mainly determined by membrane surface area and diffusion coefficient, high sensitivity could be obtained by using thinner membranes with a larger surface and/or a higher sampling flow rate. In addition, humidity showed no significant influence on extraction efficiency, due to the absorption property of the liquid extraction phase. Next, the limit of detection (LOD) was found, and the reproducibility of the developed cooled membrane gas sampling method was evaluated. Results showed that LODs with a membrane diameter of 19mm at room temperature sampling were 9.2ng/L, 0.12ng/L, 0.10ng/L for limonene, cinnamaldehyde and 2-pentadecanone, respectively. Intra- and inter-membrane sampling reproducibility revealed RSD% lower than 8% and 13%, respectively. Results uniformly demonstrated that the proposed cooled membrane device could serve as an alternative powerful tool for future gas sampling.

  3. Highly sensitive terahertz sensor for glucose detection

    NASA Astrophysics Data System (ADS)

    Kim, Hyo-Suk; Lee, Dong-Kyu; Lee, Seok; Chung, Youngchul; Seo, Minah

    2015-07-01

    In this report, we present a new type of non-contact detection method for glucose molecule using nano antenna array based bio sensing chip that operates at terahertz frequency range (0.5 - 2.5 THz). Localized and hugely enhanced transmitted terahertz field by nano antenna array in the sensing chip induced enhancement of absorption coefficient of glucose molecule that enables us to detect even very small volume of molecules. Nano antenna based terahertz sensing chip can be expected to offer accurate identification of glucose level as a non-invasive and painless sensing tool with high sensitivity.

  4. Sets of RNA Repeated Tags and Hybridization-Sensitive Fluorescent Probes for Distinct Images of RNA in a Living Cell

    PubMed Central

    Kubota, Takeshi; Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

    2010-01-01

    Background Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. Methodology/Principal Findings Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs. Conclusions/Significance A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging. PMID:20885944

  5. Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

    SciTech Connect

    Park, June-Woo; Tompsett, Amber; Zhang, Xiaowei; Newsted, John L.; Jones, Paul D.; Au, Doris; Kong, Richard; Wu, Rudolf S.S.; Giesy, John P. Hecker, Markus

    2008-10-15

    The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 {mu}g/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells.

  6. A compact frequency-domain photon migration system for integration into commercial hybrid small animal imaging scanners for fluorescence tomography

    PubMed Central

    Darne, Chinmay D.; Lu, Yujie; Tan, I-Chih; Zhu, Banghe; Rasmussen, John C.; Smith, Anne M.; Yan, Shikui; Sevick-Muraca, Eva M

    2012-01-01

    The work presented herein describes system design and performance evaluation of a miniaturized near-infrared fluorescence (NIRF) frequency-domain photon migration (FDPM) system with non-contact excitation and homodyne detection capability for small animal fluorescence tomography. The FDPM system was developed specifically for incorporation into a Siemens microPET/CT commercial scanner for hybrid small animal imaging, but could be adapted to other systems. Operating at 100 MHz, the system noise was minimized and the associated amplitude and phase errors were characterized to be ±0.7% and ±0.3°, respectively. To demonstrate the tomographic ability, a commercial mouse-shaped phantom with 50 μM IRDye800CW and 68Ga containing inclusion was used to associate PET and NIRF tomography. 3-D mesh generation and anatomical referencing was accomplished through CT. A simplified spherical harmonics approximation (SP3) algorithm, for efficient prediction of light propagation in small animals, was tailored to incorporate FDPM approach. Finally, PET-NIRF target co-localization accuracy was analyzed in vivo with a dual-labeled imaging agent targeting orthotopic growth of human prostate cancer. The results obtained validate the integration of time-dependent fluorescence tomography system within a commercial microPET/CT scanner for multimodality small animal imaging. PMID:23171509

  7. A compact frequency-domain photon migration system for integration into commercial hybrid small animal imaging scanners for fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Darne, Chinmay D.; Lu, Yujie; Tan, I.-Chih; Zhu, Banghe; Rasmussen, John C.; Smith, Anne M.; Yan, Shikui; Sevick-Muraca, Eva M.

    2012-12-01

    The work presented herein describes the system design and performance evaluation of a miniaturized near-infrared fluorescence (NIRF) frequency-domain photon migration (FDPM) system with non-contact excitation and homodyne detection capability for small animal fluorescence tomography. The FDPM system was developed specifically for incorporation into a Siemens micro positron emission tomography/computed tomography (microPET/CT) commercial scanner for hybrid small animal imaging, but could be adapted to other systems. Operating at 100 MHz, the system noise was minimized and the associated amplitude and phase errors were characterized to be ±0.7% and ±0.3°, respectively. To demonstrate the tomographic ability, a commercial mouse-shaped phantom with 50 µM IRDye800CW and 68Ga containing inclusion was used to associate PET and NIRF tomography. Three-dimensional mesh generation and anatomical referencing was accomplished through CT. A third-order simplified spherical harmonics approximation (SP3) algorithm, for efficient prediction of light propagation in small animals, was tailored to incorporate the FDPM approach. Finally, the PET-NIRF target co-localization accuracy was analyzed in vivo with a dual-labeled imaging agent targeting orthotopic growth of human prostate cancer. The obtained results validate the integration of time-dependent fluorescence tomography system within a commercial microPET/CT scanner for multimodality small animal imaging.

  8. A compact frequency-domain photon migration system for integration into commercial hybrid small animal imaging scanners for fluorescence tomography.

    PubMed

    Darne, Chinmay D; Lu, Yujie; Tan, I-Chih; Zhu, Banghe; Rasmussen, John C; Smith, Anne M; Yan, Shikui; Sevick-Muraca, Eva M

    2012-12-21

    The work presented herein describes the system design and performance evaluation of a miniaturized near-infrared fluorescence (NIRF) frequency-domain photon migration (FDPM) system with non-contact excitation and homodyne detection capability for small animal fluorescence tomography. The FDPM system was developed specifically for incorporation into a Siemens micro positron emission tomography/computed tomography (microPET/CT) commercial scanner for hybrid small animal imaging, but could be adapted to other systems. Operating at 100 MHz, the system noise was minimized and the associated amplitude and phase errors were characterized to be ±0.7% and ±0.3°, respectively. To demonstrate the tomographic ability, a commercial mouse-shaped phantom with 50 µM IRDye800CW and ⁶⁸Ga containing inclusion was used to associate PET and NIRF tomography. Three-dimensional mesh generation and anatomical referencing was accomplished through CT. A third-order simplified spherical harmonics approximation (SP₃) algorithm, for efficient prediction of light propagation in small animals, was tailored to incorporate the FDPM approach. Finally, the PET-NIRF target co-localization accuracy was analyzed in vivo with a dual-labeled imaging agent targeting orthotopic growth of human prostate cancer. The obtained results validate the integration of time-dependent fluorescence tomography system within a commercial microPET/CT scanner for multimodality small animal imaging.

  9. Identification and characterization of marker chromosomes, de novo rearrangements and microdeletions in 100 cases with fluorescence in situ hybridization (FISH)

    SciTech Connect

    Anderson, S.M.; Liu, Y.; Papenhausen, P.R.

    1994-09-01

    Results of molecular cytogenetic analysis are presented for 100 cases in which fluorescence in situ hybridization (FISH) was used as an adjunct to standard cytogenetics. Commercially available centromeric, telomeric, chromosome painting and unique sequence probes were used. Cases were from a 12-month period (June 1993-May 1994) and included examples of sex chromosome abnormalities (8), duplications (5), de novo translocations (6), satellited (12) and non-satellited (7) markers, and microdeletion syndromes (62). Satellited marker chromosomes were evaluated using a combination of DAPI/Distamycin A staining, hybridization with a classical satellite probe for chromosome 15 and hybridization with alpha-satellite probes for chromosomes 13, 14, 21 and 22. Markers positive for the chromosome 15 probe were further evaluated using unique sequence probes for the Prader-Willi/Angelman region. Microdeletion analysis was performed for Prader-Willi/Angelman (49) and DiGeorge/VCF (13) syndromes. Seven cases evaluated for Prader-Willi/Angelman syndrome demonstrated evidence of a deletion within this region. Uniparental disomy analysis was available in cases where a deletion was not detected by FISH, yet follow-up was clinically indicated. Two cases evaluated for DiGeorge/VCF syndrome demonstrated molecular evidence of a deletion. Included in our analysis is an example of familial DiGeorge syndrome.

  10. Nucleic acid sandwich hybridization assay with quantum dot-induced fluorescence resonance energy transfer for pathogen detection.

    PubMed

    Chou, Cheng-Chung; Huang, Yi-Han

    2012-12-04

    This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection.

  11. High sensitivity troponin and valvular heart disease.

    PubMed

    McCarthy, Cian P; Donnellan, Eoin; Phelan, Dermot; Griffin, Brian P; Sarano, Maurice Enriquez-; McEvoy, John W

    2017-01-16

    Blood-based biomarkers have been extensively studied in a range of cardiovascular diseases and have established utility in routine clinical care, most notably in the diagnosis of acute coronary syndrome (e.g., troponin) and the management of heart failure (e.g., brain-natriuretic peptide). The role of biomarkers is less well established in the management of valvular heart disease (VHD), in which the optimal timing of surgical intervention is often challenging. One promising biomarker that has been the subject of a number of recent VHD research studies is high sensitivity troponin (hs-cTn). Novel high-sensitivity assays can detect subclinical myocardial damage in asymptomatic individuals. Thus, hs-cTn may have utility in the assessment of asymptomatic patients with severe VHD who do not have a clear traditional indication for surgical intervention. In this state-of-the-art review, we examine the current evidence for hs-cTn as a potential biomarker in the most commonly encountered VHD conditions, aortic stenosis and mitral regurgitation. This review provides a synopsis of early evidence indicating that hs-cTn has promise as a biomarker in VHD. However, the impact of its measurement on clinical practice and VHD outcomes needs to be further assessed in prospective studies before routine clinical use becomes a reality.

  12. Pallister-Killian syndrome: A mild case diagnosed by fluorescence in situ hybridization. Review of the literature and expansion of the phenotype

    SciTech Connect

    Bielanska, M.M.; Khalifa, M.M.; Duncan, A.M.V.

    1996-10-16

    Pallister-Killian syndrome (PKS) is a rare disorder characterized by a specific combination of anomalies, mental retardation and mosaic presence of a supernumerary isochromosome 12p which is tissue-limited. We report an atypical case of PKS with a mild phenotype. Fluorescence in situ hybridization (FISH) was used to demonstrate that the supernumerary marker chromosome identified in the patient`s fibroblasts was an isochromosome 12p. This study broadens the spectrum of PKS phenotype. It also illustrates the usefulness of fluorescence in situ hybridization in diagnosis of patients with chromosomal abnormalities and mild or atypical clinical findings. 40 refs., 2 figs., 1 tab.

  13. Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J.; Robbins, W.A. |; Pinkel, D.; Weier, H.U.; Mehraein, Y. |

    1994-10-15

    Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of {approximately} 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of {approximately} 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring. 44 refs., 1 fig., 3 tabs.

  14. Photochemical properties in flag leaves of a super-high-yielding hybrid rice and a traditional hybrid rice (Oryza sativa L.) probed by chlorophyll a fluorescence transient.

    PubMed

    Zhang, Meiping; Shan, YongJie; Kochian, Leon; Strasser, Reto J; Chen, GuoXiang

    2015-12-01

    Chlorophyll a fluorescence of flag leaves in a super-high-yielding hybrid rice (Oryza sativa L.) LYPJ, and a traditional hybrid rice SY63 cultivar with lower grain yield, which were grown in the field, were investigated from emergence through senescence of flag leaves. As the flag leaf matured, there was an increasing trend in photosynthetic parameters such as quantum efficiency of primary photochemistry ([Formula: see text] Po) and efficiency of electron transport from PS II to PS I (Ψ Eo). The overall photosynthetic performance index (PIABS) was significantly higher in the high-yielding LYPJ compared to SY63 during the entire reproductive stage of the plant, the same to MDA content. However, [Formula: see text] Po(=F V/F M), an indicator of the primary photochemistry of the flag leaf, did not display significant changes with leaf age and was not significantly different between the two cultivars, suggesting that PIABS is a more sensitive parameter than [Formula: see text] Po (=F V/F M) during leaf age for distinguishing between cultivars differing in yield.

  15. Identification of neurofibromatosis 1 (NF1) homologous loci by direct sequencing, fluorescence in situ hybridization, and PCR amplification of somatic cell hybrids

    SciTech Connect

    Purandare, S.M.; Neil, S.M.; Brothman, A. |

    1995-12-10

    Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1-homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5{prime} end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template. 41 refs., 3 figs., 3 tabs.

  16. Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

    PubMed

    Kiyose, Shinichiro; Igarashi, Hisaki; Nagura, Kiyoko; Kamo, Takaharu; Kawane, Kazunori; Mori, Hiroki; Ozawa, Takachika; Maeda, Matsuyoshi; Konno, Keisuke; Hoshino, Hideaki; Konno, Hiroyuki; Ogura, Hiroyuki; Shinmura, Kazuya; Hattori, Naohiko; Sugimura, Haruhiko

    2012-11-01

    The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.

  17. Fluorescent-magnetic hybrid nanostructures: preparation, properties, and applications in biology.

    PubMed

    Quarta, Alessandra; Di Corato, Riccardo; Manna, Liberato; Ragusa, Andrea; Pellegrino, Teresa

    2007-12-01

    Research on nanocomposite materials aims at developing nanoscale composites with innovative optical, chemical, and magnetic properties, all combined in one single nanostructure. In this scenario, nanostructures which show simultaneously fluorescent and magnetic features are of particular interest for pharmaceutical and biomedical applications. In this review, we will focus our attention on magnetic-fluorescent nanocomposite based on colloidal iron oxide nanocrystals combined with different classes of fluorophores which can be either organic dyes, such as fluoresceins, cyanines, porphyrins, or colloidal quantum dots. We will give an overview of the preparation methods of the magnetic-fluorescent nanocomposites that are now available and we will outline the most significant in vitro studies of such nanocomposites on living cells. Some examples of their applications in biology and medicine will also be discussed.

  18. Capture antibody targeted fluorescence in situ hybridization (CAT-FISH): dual labeling allows for increased specificity in complex samples.

    PubMed

    Stroot, Joyce M; Leach, Kelly M; Stroot, Peter G; Lim, Daniel V

    2012-02-01

    Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~10(3)cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence.

  19. Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect.

    PubMed

    Santos, Rita S; Guimarães, Nuno; Madureira, Pedro; Azevedo, Nuno F

    2014-10-10

    Despite the fact that fluorescence in situ hybridization (FISH) is a well-established technique to identify microorganisms, there is a lack of understanding concerning the interaction of the different factors affecting the obtained fluorescence. In here, we used flow cytometry to study the influence of three essential factors in hybridization - temperature, time and formamide concentration - in an effort to optimize the performance of a Peptide Nucleic Acid (PNA) probe targeting bacteria (EUB338). The PNA-FISH optimization was performed with bacteria representing different families employing response surface methodology. Surprisingly, the optimum concentration of formamide varied according to the bacterium tested. While hybridization on the bacteria possessing the thickest peptidoglycan was more successful at nearly 50% (v/v) formamide, hybridization on all other microorganisms appeared to improve with much lower formamide concentrations. Gram staining and transmission electron microscopy allowed us to confirm that the overall effect of formamide concentration on the fluorescence intensity is a balance between a harmful effect on the bacterial cell envelope, affecting cellular integrity, and the beneficial denaturant effect in the hybridization process. We also conclude that microorganisms belonging to different families will require different hybridization parameters for the same FISH probe, meaning that an optimum universal PNA-FISH procedure is non-existent for these situations.

  20. A green method for the preparation of fluorescent hybrid structures of gold and corrole

    NASA Astrophysics Data System (ADS)

    Pereira, Ângela S.; Barata, Joana F. B.; Vaz Serra, Vanda I. R. C.; Pereira, Sérgio; Trindade, Tito

    2015-10-01

    Gold/soap nanostructures were prepared by a green methodology using saponified household sunflower oil, as reducing and organic dispersing agent of auric acid. The incorporation of hydrophobic molecules on the novel water-soluble gold nanoparticles was followed by fluorescence lifetime imaging microscopy, using as model hydrophobic compound 5,10,15-tris-(pentafluorophenyl)corrolatogallium(III)(pyridine) (GaPFC), a highly fluorescent corrole. The results showed the hydrophobic GaPFC located between the organic bilayer surrounding several Au nanoparticles, which in turn were coated with fatty acids salts anchored by the double bond at the gold's surface.

  1. Detection of the AML translocation (8;21) by two-color fluorescent in situ hybridization

    SciTech Connect

    Sacchi, N.; Magnani, I.; Kearney, L.

    1994-09-01

    In the translocation (8;21)(q22;q22) associated with acute myelogenous leukemia (AML), part of the long arm of chromosome 8 is reciprocally translocated onto chromosome 21. At the molecular level the translocation results in the fusion of the 5{prime} region of the AML1 gene on chromosome 21 and almost the entire CDR gene (also ETO or MTG8) on chromosome 8. To detection the translocation at the single cell level, we used two probes, a cosmid clone containing the first five exons of AML1 and a P1 clone containing the entire CDR gene. Hybridization of the two probes to the distal and proximal sides of the translocation breakpoint was expected to highlight the derivative 8q-chromosome in an interphase cell. To demonstrate the ability to identify the translocation in interphase cells using two-color FISH, these two probes were hybridized simultaneously to a cell line containing the 8;21 translocation, Kasumi-1. Each probe was detected with a different color so that their relationship in the sample could be determined within the same interphase cell. Simultaneous hybridization of the CDR and AML1 probes to interphase Kasumi-1 cells resulted in one orange and one green hybridization signal randomly located in the cell, from the hybridization to the normal 8 and 21 chromosomes, and one orange-green pair of signals from the close hybridization of the two probes to the fusion gene on the derivative 8q-chromosome, indicating the translocation. The translocation was identified by an abnormal pairing of the two differently colored signals in the same interphase cell. This technique allows for the detection of the translocation in all cells, not just those arrested in metaphase, and also permits the analysis of a small number of cells. Therefore, useful information can still be obtained from samples not suited for RT-PCR analysis and conventional cytogenetic techniques.

  2. Visualization and quantification of archaeal and bacterial metabolically active cells in soil using fluorescence in situ hybridization method

    NASA Astrophysics Data System (ADS)

    Semenov, Mikhail; Manucharova, Natalia; Stepanov, Alexey

    2015-04-01

    The method of in situ hybridization using fluorescent labeled 16S rRNA-targeted oligonucleotide probes (FISH - fluorescence in situ hybridization) combines identification and quantification of groups of microorganisms at different phylogenetic levels, from domain to species. The FISH method enables to study the soil microbial community in situ, avoiding plating on nutrient media, and allows to identify and quantify living, metabolically active cells of Bacteria and Archaea. The full procedure consists of the following steps: desorption of the cells from the soil particles, fixation of cells, coating a fixed sample on the glass slide, hybridization with the specific probes and, finally, microscopic observation and cell counting. For the FISH analysis of Bacteria and Archaea, the paraformaldehyde-fixed samples were hybridized with Cy3-labeled Archaea-specific probe(Arc915) and 6-carboxyfluorescein (FAM)-labeled Bacteria-specific probe(EUB338). When a molecular probe is incorporated into a cell, it can hybridize solely with a complementary rRNA sequence. The hybridization can be visualized under the fluorescent microscope and counted. The application of FISH will be demonstrated by the abundance of metabolically active cells of Archaea and Bacteria depending on soil properties, depth and land use. The research was carried out at field and natural ecosystems of European part of Russia. Samples were collected within the soil profiles (3-6 horizons) of Chernozem and Kastanozem with distinct land use. Quantification of metabolically active cells in virgin and arable Chernozem revealed that the abundance of Archaea in topsoil of virgin Chernozem was doubled as compared with arable soil, but it leveled off in the deeper horizons. Plowing of Chernozem decreased an amount of archaeal and bacterial active cells simultaneously, however, Bacteria were more resistant to agrogenic impact than Archaea. In Kastanozem, a significant change in the abundance of metabolically active

  3. High-sensitivity detection of TNT.

    PubMed

    Pushkarsky, Michael B; Dunayevskiy, Ilya G; Prasanna, Manu; Tsekoun, Alexei G; Go, Rowel; Patel, C Kumar N

    2006-12-26

    We report high-sensitivity detection of 2,4,6-trinitrotoluene (TNT) by using laser photoacoustic spectroscopy where the laser radiation is obtained from a continuous-wave room temperature high-power quantum cascade laser in an external grating cavity geometry. The external grating cavity quantum cascade laser is continuously tunable over approximately 400 nm around 7.3 microm and produces a maximum continuous-wave power of approximately 200 mW. The IR spectroscopic signature of TNT is sufficiently different from that of nitroglycerine so that unambiguous detection of TNT without false positives from traces of nitroglycerine is possible. We also report the results of spectroscopy of acetylene in the 7.3-microm region to demonstrate continuous tunability of the IR source.

  4. High-sensitivity fiber optic acoustic sensors

    NASA Astrophysics Data System (ADS)

    Lu, Ping; Liu, Deming; Liao, Hao

    2016-11-01

    Due to the overwhelming advantages compared with traditional electronicsensors, fiber-optic acoustic sensors have arisen enormous interest in multiple disciplines. In this paper we present the recent research achievements of our group on fiber-optic acoustic sensors. The main point of our research is high sensitivity interferometric acoustic sensors, including Michelson, Sagnac, and Fabry-Pérot interferometers. In addition, some advanced technologies have been proposed for acoustic or acoustic pressure sensing such as single-mode/multimode fiber coupler, dual FBGs and multi-longitudinal mode fiber laser based acoustic sensors. Moreover, our attention we have also been paid on signal demodulation schemes. The intensity-based quadrature point (Q-point) demodulation, two-wavelength quadrature demodulation and symmetric 3×3 coupler methodare discussed and compared in this paper.

  5. Highly sensitive silicon microreactor for catalyst testing

    SciTech Connect

    Henriksen, Toke R.; Hansen, Ole; Olsen, Jakob L.; Vesborg, Peter; Chorkendorff, Ib

    2009-12-15

    A novel microfabricated chemical reactor for highly sensitive measurements of catalytic activity and surface kinetics is presented. The reactor is fabricated in a silicon chip and is intended for gas-phase reactions at pressures ranging from 0.1 to 5.0 bar. A high sensitivity is obtained by directing the entire gas flow through the catalyst bed to a mass spectrometer, thus ensuring that nearly all reaction products are present in the analyzed gas flow. Although the device can be employed for testing a wide range of catalysts, the primary aim of the design is to allow characterization of model catalysts which can only be obtained in small quantities. Such measurements are of significant fundamental interest but are challenging because of the low surface areas involved. The relationship between the reaction zone gas flow and the pressure in the reaction zone is investigated experimentally. A corresponding theoretical model is presented, and the gas flow through an on-chip flow-limiting capillary is predicted to be in the intermediate regime. The experimental data for the gas flow are found to be in good agreement with the theoretical model. At typical experimental conditions, the total gas flow through the reaction zone is around 3x10{sup 14} molecules s{sup -1}, corresponding to a gas residence time in the reaction zone of about 11 s. To demonstrate the operation of the microreactor, CO oxidation on low-area platinum thin film circles is employed as a test reaction. Using temperature ramping, it is found that platinum catalysts with areas as small as 15 {mu}m{sup 2} are conveniently characterized with the device.

  6. Highly sensitive silicon microreactor for catalyst testing

    NASA Astrophysics Data System (ADS)

    Henriksen, Toke R.; Olsen, Jakob L.; Vesborg, Peter; Chorkendorff, Ib; Hansen, Ole

    2009-12-01

    A novel microfabricated chemical reactor for highly sensitive measurements of catalytic activity and surface kinetics is presented. The reactor is fabricated in a silicon chip and is intended for gas-phase reactions at pressures ranging from 0.1 to 5.0 bar. A high sensitivity is obtained by directing the entire gas flow through the catalyst bed to a mass spectrometer, thus ensuring that nearly all reaction products are present in the analyzed gas flow. Although the device can be employed for testing a wide range of catalysts, the primary aim of the design is to allow characterization of model catalysts which can only be obtained in small quantities. Such measurements are of significant fundamental interest but are challenging because of the low surface areas involved. The relationship between the reaction zone gas flow and the pressure in the reaction zone is investigated experimentally. A corresponding theoretical model is presented, and the gas flow through an on-chip flow-limiting capillary is predicted to be in the intermediate regime. The experimental data for the gas flow are found to be in good agreement with the theoretical model. At typical experimental conditions, the total gas flow through the reaction zone is around 3×1014 molecules s-1, corresponding to a gas residence time in the reaction zone of about 11 s. To demonstrate the operation of the microreactor, CO oxidation on low-area platinum thin film circles is employed as a test reaction. Using temperature ramping, it is found that platinum catalysts with areas as small as 15 μm2 are conveniently characterized with the device.

  7. Quantifying Substrate Uptake by Individual Cells of Marine Bacterioplankton by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Microautoradiography▿

    PubMed Central

    Sintes, Eva; Herndl, Gerhard J.

    2006-01-01

    Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up 3H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions. PMID:16950912

  8. Molecular cytogenetics: unraveling of the genetic composition of individual cells by fluorescence in situ hybridization and digital imaging microscopy.

    PubMed

    Tanke, H J; Florijn, R J; Vrolijk, J; Raap, A K

    1995-01-01

    Molecular biology techniques allow the unraveling of the genetic alterations that cause or accompany malignant disease. Since tumors are often heterogeneous, biochemical analysis of tissue homogenates is of limited diagnostic value. This paper gives examples of methods that are presently operational to analyze the genetic composition of individual cells. They are based on fluorescence in situ hybridization (FISH) and digital imaging microscopy. First, the current status of indirect and direct FISH staining methods with respect to probe labeling, detection sensitivity, multiplicity, and DNA resolution is summarized. Microscope hardware as well as charge-coupled device (CCD) cameras required for FISH analysis are then described. Applications potentially important for the analysis of urological malignancies, such as the automated enumeration of chromosomal abnormalities (counting of dots in interphase cells) and high-resolution DNA mapping on highly extended chromatin, are described in detail. Finally, the limitations of the present methodology and its future prospects are discussed.

  9. An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines

    PubMed Central

    Perroud, Pierre-François; Cove, David J.; Quatrano, Ralph S.; McDaniel, Stuart F.

    2011-01-01

    Summary The sequencing of the Physcomitrella patens genome, combined with the high frequency of gene targeting in this species, makes it ideal for reverse genetic studies. For forward genetic studies, experimental crosses and genetic analysis of progeny are essential.Since P. patens is monoicous, producing both male and female gametes on the same gametophore, and undergoing self-fertilization at a high frequency, the identification of crossed sporophytes is difficult. Usually spores from many sporophytes from a mixed culture must be testsed for the production of recombinant progeny.Here, we describe the use of transgenic lines that express a fluorescent transgene constitutively, to provide a direct visual screen for hybrid sporophytes.We show that segregations in crosses obtained with this technique are as expected, and demonstrate its utility for the study of the rate of outcrossing between three isolates of P. patens. PMID:21366596

  10. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1, 1992--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  11. Peptide nucleic acid fluorescence in-situ hybridization for identification of Vibrio spp. in aquatic products and environments.

    PubMed

    Zhang, Xiaofeng; Li, Ke; Wu, Shan; Shuai, Jiangbing; Fang, Weihuan

    2015-08-03

    A peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for specific detection of the Vibrio genus. In silico analysis by BLAST and ProbeCheck showed that the designed PNA probe targeting the 16S rRNAs was suitable for specific identification of Vibrio. Specificity and sensitivity of the probe Vib-16S-1 were experimentally verified by its reactivity against 18 strains of 9 Vibrio species and 14 non-Vibrio strains of 14 representative species. The PNA-FISH assay was able to identify 47 Vibrio positive samples from selectively enriched cultures of 510 samples of aquatic products and environments, comparable with the results obtained by biochemical identification and real-time PCR. We conclude that PNA-FISH can be an alternative method for rapid identification of Vibrio species in a broad spectrum of seafood or related samples.

  12. Diagnostics of common microdeletion syndromes using fluorescence in situ hybridization: Single center experience in a developing country

    PubMed Central

    Kurtovic-Kozaric, Amina; Mehinovic, Lejla; Stomornjak-Vukadin, Meliha; Kurtovic-Basic, Ilvana; Catibusic, Feriha; Kozaric, Mirza; Dinarevic, Senka Mesihovic; Hasanhodzic, Mensuda; Sumanovic-Glamuzina, Darinka

    2016-01-01

    Microdeletion syndromes are caused by chromosomal deletions of less than 5 megabases which can be detected by fluorescence in situ hybridization (FISH). We evaluated the most commonly detected microdeletions for the period from June 01, 2008 to June 01, 2015 in the Federation of Bosnia and Herzegovina, including DiGeorge, Prader-Willi/Angelman, Wolf-Hirschhorn, and Williams syndromes. We report 4 patients with DiGeorge syndromes, 4 patients with Prader-Willi/Angelman, 4 patients with Wolf-Hirschhorn syndrome, and 3 patients with Williams syndrome in the analyzed 7 year period. Based on the positive FISH results for each syndrome, the incidence was calculated for the Federation of Bosnia and Herzegovina. These are the first reported frequencies of the microdeletion syndromes in the Federation of Bosnia and Herzegovina. PMID:26937776

  13. Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization.

    PubMed

    Wang, Xiaozhu; Takebayashi, Shin-Ichiro; Bernardin, Evans; Gilbert, David M; Chella, Ravindran; Guan, Jingjiao

    2012-06-01

    We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells.

  14. Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

    PubMed Central

    Moreno, Yolanda; Botella, Salut; Alonso, José Luis; Ferrús, María A.; Hernández, Manuel; Hernández, Javier

    2003-01-01

    The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 103 cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples. PMID:12571045

  15. A highly sensitive and multiplexed method for focused transcript analysis.

    PubMed

    Kataja, Kari; Satokari, Reetta M; Arvas, Mikko; Takkinen, Kristiina; Söderlund, Hans

    2006-10-01

    We describe a novel, multiplexed method for focused transcript analysis of tens to hundreds of genes. In this method TRAC (transcript analysis with aid of affinity capture) mRNA targets, a set of amplifiable detection probes of distinct sizes and biotinylated oligo(dT) capture probe are hybridized in solution. The formed sandwich hybrids are collected on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted, optionally amplified by a PCR using a universal primer pair and detected with laser-induced fluorescence and capillary electrophoresis. The probes were designed by using a computer program developed for the purpose. The TRAC method was adapted to 96-well format by utilizing an automated magnetic particle processor. Here we demonstrate a simultaneous analysis of 18 Saccharomyces cerevisiae transcripts from two experimental conditions and show a comparison with a qPCR system. The sensitivity of the method is significantly increased by the PCR amplification of the hybridized and eluted probes. Our data demonstrate a bias-free use of at least 16 cycles of PCR amplification to increase probe signal, allowing transcript analysis from 2.5 ng of the total mRNA sample. The method is fast and simple and avoids cDNA conversion. These qualifications make it a potential, new means for routine analysis and a complementing method for microarrays and high density chips.

  16. Using respirometric techniques and fluorescent in situ hybridization to evaluate the heterotrophic active biomass in activated sludge.

    PubMed

    Ismail, A; Wentzel, M C; Bux, F

    2007-10-15

    The separation and accurate quantification of active biomass components in activated sludge is of paramount importance in models, used for the management and design of waste water (WW) treatment plants. Accurate estimates of microbial population concentrations and the direct, in situ determination of kinetic parameters could improve the calibration and validation of existing models of biological nutrient removal activated sludge systems. The aim of this study was to obtain correlations between heterotrophic active biomass (Z(BH)) concentrations predicted by mathematical models and quantitative information obtained by Fluorescent in situ hybridizations (FISH). Respirometric batch test were applied to mixed liquors drawn from a well-defined parent anoxic/aerobic activated sludge system to quantify the Z(BH) concentrations. Similarly fluorescent labeled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidizers were used in combination with DAPI staining to validate the Z(BH) active biomass component in activate sludge respirometric batch tests. For the direct enumeration and simultaneous in situ analysis of the distribution of nitrifying bacteria, in situ hybridization with oligonucleotide probes were used. Probes (NSO 1225, NSR 1156, and NIT3) were used to target the nitrifiers and the universal probe (EUB MIX) was used to target all Eubacteria. Deducting the lithoautotrophic population from the total bacteria population revealed the Z(BH) population. A conversion factor of 8.49 x 10(-11) mg VSS/cell was applied to express the Z(BH) in terms of COD concentration. Z(BH) values obtained by molecular probing correlated closely with values obtained from the modified batch test. However, the trend of consistently poor correspondence of measured and theoretical concentrations were evident. Therefore, the focus of this study was to investigate alternative technology, such as FISH to validate or replace kinetic parameters which are invariably

  17. Assignment of the human aggrecan gene (AGC1) to 15q26 using fluorescence in situ hybridization analysis

    SciTech Connect

    Korenberg, J.R.; Chen, X.N.; Doege, K.; Grover, J.; Roughley, P.J.

    1993-05-01

    The large aggregating proteoglycan aggrecan is a major structural component of the extracellular matrix of articular cartilage. Recent cDNA cloning of the human aggrecan gene (AGC1) reveals a core protein of at least 2316 amino acids characterized by several distinct structural domains. Two globular domains, termed G1 and G2, are present at the amino terminus of the molecule and a third, termed G3, is present at the carboxy terminus. The G1 domain is homologous in structure to the cartilage link protein and accounts for the aggregating potential of aggrecan through its ability to interact with hyaluronic acid. The aggrecan gene is known to consist of 15 exons, with each exon encoding a distinct functional region of the mature protein. However, while the link protein gene is known to reside on chromosome 5 in the human, the location of the aggrecan gene is currently undetermined in any species. The probe (pAGG2) for the aggrecan gene was mapped on chromosome band 15q26, most likely in the subregion of 15q26.1, using fluorescence in situ hybridization. Clear signals were noted on both chromatids of chromosome band 15q26 in over 80% of the 300 metaphase cells examined in three independent experiments using pAGG2. No other sites of hybridization were noted on both chromatids of any other chromosome band. The precise band location was identified by using chromsomes of about 650 bands and employing fluorescence reverse banding with chromomycin A3 and distamycin. 14 refs., 1 fig.

  18. Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

    SciTech Connect

    Martin, R.H. |; Rademaker, A.

    1994-09-01

    Aneuploidy estimates for individual chromosomes in human sperm have varied more than 10-fold in different laboratories using fluorescence in situ hybridization (FISH). These laboratories use different scoring criteria in the assessment of a disomic sperm. In order to determine reliable estimates of aneuploidy, we have investigated whether scoring criteria affect the aneuploidy frequency in human sperm. Aneuploidy estimates for chromosomes 1(pUC1.77), 12(pBR12), X(XC) and Y(DYZ3Z) were obtained in human sperm from five donors using multicolor FISH analysis to provide an internal control to differentiate between nullisomy and lack of hybridization and between disomy and diploidy. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one scoring criterion used one-half a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other scoring criterion set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half domain as the scoring criterion and 113,478 were scored using one domain as the criterion. The mean percent disomy for chromosomes 1, 12, X, Y and XY was .18, .16, .15, .19, .25 respectively using the one-half domain criterion and .08, .17, .07, .12, .16 respectively using the one domain criterion. The percent disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X and Y split into more than one domain in decondensed interphase sperm and use of the one-half domain criterion leads to an overestimate of aneuploidy frequencies.

  19. Detection of aneuploid human sperm by fluorescence in situ hybridization: Evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y

    SciTech Connect

    Robbins, W.A. Lawrence Livermore National Lab., CA ); Segraves, R.; Pinkel, D. ); Wyrobek, A.J. )

    1993-04-01

    Fluorescence in situ hybridization with repetitive-sequence DNA probes was used to detect human sperm disomic for chromosomes 1 and Y in three healthy men. Data on these same men had been obtained previously, using the human-sperm/hamster-egg cytogenetic technique, providing a cytogenetic reference for validating sperm hybridization measurements. Air-dried smears were prepared from semen samples and treated with DTT and lithium diiodosalicylate to expand sperm chromatin. Hybridization with fluorescently tagged DNA probes for chromosomes 1 (pUC177) or Y (pY3.4) yielded average frequencies of sperm with two fluorescent domains of 14.2[+-]2.4/10,000 and 5.6[+-]1.6/10,000 sperm, respectively. These frequencies did not differ statistically from frequencies of hyperploidy observed for these chromosomes with the hamster technique. In addition, frequencies of disomic sperm from one donor were elevated [approximately]2.5-fold above those of other donors, for both chromosomes 1 (P = .045) and Y (P = .01), consistent with a trend found with the hamster technique. The authors conclude that fluorescence in situ hybridization to sperm chromosomes provides a valid and promising measure of the frequency of disomic human sperm. 43 refs., 1 fig., 4 tabs.

  20. Cytogenetic follow-up by karyotyping and fluorescence in situ hybridization: implications for monitoring patients with myelodysplastic syndrome and deletion 5q treated with lenalidomide

    PubMed Central

    Göhring, Gudrun; Giagounidis, Aristoteles; Büsche, Guntram; Hofmann, Winfried; Kreipe, Hans Heinrich; Fenaux, Pierre; Hellström-Lindberg, Eva; Schlegelberger, Brigitte

    2011-01-01

    In patients with low and intermediate risk myelodysplastic syndrome and deletion 5q (del(5q)) treated with lenalidomide, monitoring of cytogenetic response is mandatory, since patients without cytogenetic response have a significantly increased risk of progression. Therefore, we have reviewed cytogenetic data of 302 patients. Patients were analyzed by karyotyping and fluorescence in situ hybridization. In 85 patients, del(5q) was only detected by karyotyping. In 8 patients undergoing karyotypic evolution, the del(5q) and additional chromosomal aberrations were only detected by karyotyping. In 3 patients, del(5q) was only detected by fluorescence in situ hybridization, but not by karyotyping due to a low number of metaphases. Karyotyping was significantly more sensitive than fluorescence in situ hybridization in detecting the del(5q) clone. In conclusion, to optimize therapy control of myelodysplastic syndrome patients with del(5q) treated with lenalidomide and to identify cytogenetic non-response or progression as early as possible, fluorescence in situ hybridization alone is inadequate for evaluation. Karyotyping must be performed to optimally evaluate response. (clinicaltrials.gov identifier: NCT01099267 and NCT00179621) PMID:21109690

  1. Sky-Blue Organic Light Emitting Diode with 37% External Quantum Efficiency Using Thermally Activated Delayed Fluorescence from Spiroacridine-Triazine Hybrid.

    PubMed

    Lin, Ting-An; Chatterjee, Tanmay; Tsai, Wei-Lung; Lee, Wei-Kai; Wu, Meng-Jung; Jiao, Min; Pan, Kuan-Chung; Yi, Chih-Lung; Chung, Chin-Lung; Wong, Ken-Tsung; Wu, Chung-Chih

    2016-08-01

    Extremely efficient sky-blue organic electroluminescence with external quantum efficiency of ≈37% is achieved in a conventional planar device structure, using a highly efficient thermally activated delayed fluorescence emitter based on the spiroacridine-triazine hybrid and simultaneously possessing nearly unitary (100%) photoluminescence quantum yield, excellent thermal stability, and strongly horizontally oriented emitting dipoles (with a horizontal dipole ratio of 83%).

  2. Assignment of the gastric inhibitory polypeptide receptor gene (GIPR) to chromosome bands 19q13.2-q13.3 by fluorescence in situ hybridization

    SciTech Connect

    Stoffel, M.; Fernald, A.A.; Bell, G.I.; Le Beau, M.M.

    1995-08-10

    The gastric inhibitory polypeptide receptor gene (GIPR) was localized, using fluorescence in situ hybridization (FISH), to human chromosome bands 19q13.2-q13.3. Gastric inhibitory polypeptide (GIP) is a potent stimulator of insulin secretion and mutations in the GIPR gene may be related to non-insulin-dependent diabetes mellitus (NIDDM). 13 refs., 1 fig.

  3. Localization of the DCTN1 gene encoding p150{sup Glued} to human chromosome 2p13 by fluorescence in situ hybridization

    SciTech Connect

    Holzbaur, E.L.F.; Tokito, M.K.

    1996-02-01

    This report discusses the genetic mapping of the DCTN1 gene to human chromosome 2p13 using fluorescence in situ hybridization. This gene encodes the largest polypeptide of the dynactin complex, which is one of two microtubule-based biological motor protein complexes. 12 refs., 1 fig.

  4. Midkine gene (MDK), a gene for prenatal differentiation and neuroregulation, maps to band 11p11. 2 by fluorescence in situ hybridization

    SciTech Connect

    Kaname, Tadashi; Uehara, Kazuyoshi; Muramatsu, Taskashi ); Kuwano, Akira; Murano, Ichiro; Kajii, Tadashi )

    1993-08-01

    Midkine (MDK) is a retinoic acid-responsive gene concerned with prenatal development and neurite growth. The authors mapped the gene to band p11.2 of chromosome 11 through fluorescence in situ hybridization analysis and using a 4.5-kb fragment of its human genomic DNA. 11 refs., 1 fig.

  5. Chromosomal localization of the human natural killer cell class I receptor family genes to 19q13.4 by fluorescence in situ hybridization

    SciTech Connect

    Suto, Yumiko; Maenaka, Katsumi; yabe, Toshio

    1996-07-01

    This report describes the localization of the human natural killer cell I receptor family genes to human chromosome 19q13.4 using fluorescence in situ hybridization. These genes mediate the inhibition of the cytotoxicity of subsets of natural killer cells. 8 refs., 1 fig.

  6. An environmentally sensitive fluorescent purine nucleoside that changes emission wavelength upon hybridization.

    PubMed

    Saito, Yoshio; Suzuki, Azusa; Okada, Yuji; Yamasaka, Yuki; Nemoto, Nobukatsu; Saito, Isao

    2013-06-25

    C7-naphthylethynylated 8-aza-7-deaza-2'-deoxyguanosine (na)G was synthesized and its photophysical properties were examined. The fluorescent nucleoside exhibited solvatofluorochromic properties (Δλ(fl)(max) = 67 nm). An ODN probe containing (na)G forms a stable base pair only with C and discriminates structural changes such as mismatches and deletions by a distinct change in its emission wavelength.

  7. In vivo volumetric fluorescence sectioning microscopy with mechanical-scan-free hybrid illumination imaging

    PubMed Central

    Lin, Chen-Yen; Lin, Wei-Hsin; Chien, Ju-Hsuan; Tsai, Jui-Chang; Luo, Yuan

    2016-01-01

    Optical sectioning microscopy in wide-field fashion has been widely used to obtain three-dimensional images of biological samples; however, it requires scanning in depth and considerable time to acquire multiple depth information of a volumetric sample. In this paper, in vivo optical sectioning microscopy with volumetric hybrid illumination, with no mechanical moving parts, is presented. The proposed system is configured such that the optical sectioning is provided by hybrid illumination using a digital micro-mirror device (DMD) for uniform and non-uniform pattern projection, while the depth of imaging planes is varied by using an electrically tunable-focus lens with invariant magnification and resolution. We present and characterize the design, implementation, and experimentally demonstrate the proposed system’s ability through 3D imaging of in vivo Canenorhabditis elegans’ growth cones. PMID:27867708

  8. Automated detection and analysis of fluorescent in situ hybridization spots depicted in digital microscopic images of Pap-smear specimens

    NASA Astrophysics Data System (ADS)

    Wang, Xingwei; Zheng, Bin; Li, Shibo; Zhang, Roy; Mulvihill, John J.; Chen, Wei R.; Liu, Hong

    2009-03-01

    Fluorescence in situ hybridization (FISH) technology has been widely recognized as a promising molecular and biomedical optical imaging tool to screen and diagnose cervical cancer. However, manual FISH analysis is time-consuming and may introduce large inter-reader variability. In this study, a computerized scheme is developed and tested. It automatically detects and analyzes FISH spots depicted on microscopic fluorescence images. The scheme includes two stages: (1) a feature-based classification rule to detect useful interphase cells, and (2) a knowledge-based expert classifier to identify splitting FISH spots and improve the accuracy of counting independent FISH spots. The scheme then classifies detected analyzable cells as normal or abnormal. In this study, 150 FISH images were acquired from Pap-smear specimens and examined by both an experienced cytogeneticist and the scheme. The results showed that (1) the agreement between the cytogeneticist and the scheme was 96.9% in classifying between analyzable and unanalyzable cells (Kappa=0.917), and (2) agreements in detecting normal and abnormal cells based on FISH spots were 90.5% and 95.8% with Kappa=0.867. This study demonstrated the feasibility of automated FISH analysis, which may potentially improve detection efficiency and produce more accurate and consistent results than manual FISH analysis.

  9. Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry.

    PubMed Central

    Simon, N; LeBot, N; Marie, D; Partensky, F; Vaulot, D

    1995-01-01

    Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features. PMID:7618862

  10. Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.

    PubMed

    Gifford, Lida K; Opalinska, Joanna B; Jordan, David; Pattanayak, Vikram; Greenham, Paul; Kalota, Anna; Robbins, Michelle; Vernovsky, Kathy; Rodriguez, Lesbeth C; Do, Bao T; Lu, Ponzy; Gewirtz, Alan M

    2005-02-17

    We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

  11. Rapid detection of viable Bacillus pumilus SAFR-032 encapsulated spores using novel propidium monoazide-linked fluorescence in situ hybridization.

    PubMed

    Mohapatra, Bidyut R; La Duc, Myron T

    2012-07-01

    The survival of Bacillus pumilus SAFR-032 spores to standard industrial clean room sterilization practices necessitates the development of rapid molecular diagnostic tool(s) for detection and enumeration of viable bacterial spores in industrial clean room environments. This is of importance to maintaining the sterility of clean room processing products. This paper describes the effect of propidium monoazide (PMA) on fluorescence in situ hybridization (FISH) for detecting and enumerating B. pumilus SAFR-032 viable spores having been artificially encapsulated within poly(methylmethacrylate) (Lucite, Plexiglas) and released via an organic solvent (PolyGone-500). The results of the PMA-FISH experiments discussed herein indicate that PMA was able to permeate only the compromised coat layers of non-viable spores, identifying PMA treatment of bacterial spores prior to FISH analysis as a novel method for selecting out the fraction of the spore population that is non-viable from fluorescence detection. The ability of novel PMA-FISH to selectively distinguish and enumerate only the living spores present in a sample is of potential significance for development of improved strategies to minimize spore-specific microbial burden in a given environment.

  12. The human sorbitol dehydrogenase gene: cDNA cloning, sequence determination, and mapping by fluorescence in situ hybridization

    SciTech Connect

    Lee, F.K.; Chung, S. ); Cheung, M.C. )

    1994-05-15

    The cDNA for human sorbitol dehydrogenase (SORD) has been cloned and sequenced. It translates into a peptide of 356 amino acid residues, one more than the sequence previously reported from peptide analysis. An extra alanine was found at the acetyl-blocked N-terminal, between positions 1 and 4. This matches the rat cDNA, which also has 356 amino acids, with an extra proline at position 3. Four other mismatches were also observed, but these are all amino acid substitutions that occur outside proposed functionally important regions. Further work must be performed to determine whether these discrepancies represent polymorphic forms of the enzyme. The SORD gene was mapped by fluorescence in situ hybridization and found to occupy a single site on chromosome 15q15, indicating that it is a single-copy gene. This was confirmed by Southern blot hybridization. SORD is thought to be involved in the etiology of diabetic complications, and its deficiency has been linked to congenital cataracts. The cloned gene could be used as a probe to study the role of this enzyme in the pathogenesis of these diseases. 24 refs., 4 figs.

  13. Retrieval of four adaptive lineages in duiker antelope: evidence from mitochondrial DNA sequences and fluorescence in situ hybridization.

    PubMed

    van Vuuren, B J; Robinson, T J

    2001-09-01

    Independent molecular markers (mitochondrial DNA sequences from two genes and fluorescence in situ hybridization with satellite DNA sequences as hybridization probes) were employed to investigate phylogenetic relationships among duiker antelope. When analyzed singly or taken together, the molecular and cytogenetic data allowed for the delimitation of four adaptive groups: the conservative dwarfs which are basal, a savanna specialist which groups apart from the forest duikers, the giant duikers, and the red duikers. Within the latter, a further subdivision comprising an east African and a west African red duiker clade is evident. The placement of the endangered zebra duiker and Aders' duiker remains problematic. Several of the nomenclatural divisions in current use are questioned by our results. These include the recognition of Philantomba as genus name for the blue and Maxwell's duiker and that Harvey's duiker be relegated to a subspecies of the Natal red duiker. We place our results in a biogeographic context and argue that duiker speciation has been driven predominantly by habitat fragmentation which probably led to the disruption of gene flow between geographic populations.

  14. Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii.

    PubMed

    Zhang, Xiaofeng; Wu, Shan; Li, Ke; Shuai, Jiangbing; Dong, Qiang; Fang, Weihuan

    2012-07-02

    A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.

  15. Breakpoints in Robertsonian translocations are localized to satellite III DNA by fluorescence in situ hybridization

    SciTech Connect

    Gravholt, C.H.; Friedrich, U.; Caprani, M.; Jorgensen, A.L. )

    1992-12-01

    The authors characterized 21 t(13;14) and 3 t(14;21) Robertsonian translocations for the presence of DNA derived from the short arms of the translocated acrocentric chromosomes and identified their centromeres. Nineteen of these 24 translocation carriers were unrelated. Using centromeric [alpha]-repeat DNA as chromosome-specific probe, they found by in situ hybridization that all 24 translocation chromosomes were dicentric. The chromatin between the two centromeres did not stain with silver, and no hybridization signal was detected with probes for rDNA or [beta]-satellite DNA that flank the distal and proximal ends of the rDNA region on the short arm of the acrocentrics. By contrast, all 24 translocation chromosomes gave a distinct hybridization signal when satellite III DNA was used as probe. This result strongly suggests that the chromosomal rearrangements leading to Robertsonian translocations occur preferentially in satellite III DNA. The authors hypothesize that guanine-rich satellite III repeats may promote chromosomal recombination by formation of tetraplex structures. The findings localize satellite III DNA to the short arm of the acrocentric chromosomes distal to centromeric [alpha]-repeat DNA and proximal to [beta]-satellite DNA. 32 refs., 4 figs., 2 tabs.

  16. Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor

    PubMed Central

    2013-01-01

    Background The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable. Results Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively. Conclusions The approach described in this study enables for the first time the

  17. Highly sensitive beam steering with plasmonic antenna.

    PubMed

    Rui, Guanghao; Zhan, Qiwen

    2014-08-05

    In this work, we design and study a highly sensitive beam steering device that integrates a spiral plasmonic antenna with a subwavelength metallic waveguide. The short effective wavelength of the surface plasmon polaritons (SPPs) mode supported by the metallic waveguide is exploited to dramatically miniaturize the device and improve the sensitivity of the beam steering. Through introducing a tiny displacement of feed point with respect to the geometrical center of the spiral plasmonic antenna, the direction of the radiation can be steered at considerably high angles. Simulation results show that steering angles of 8°, 17° and 34° are obtainable for a displacement of 50 nm, 100 nm and 200 nm, respectively. Benefiting from the reduced device size and the shorter SPP wavelength, the beam steering sensitivity of the beam steering is improved by 10-fold compared with the case reported previously. This miniature plasmonic beam steering device may find many potential applications in quantum optical information processing and integrated photonic circuits.

  18. Face Transplantation in a Highly Sensitized Recipient.

    PubMed

    Chandraker, Anil; Arscott, Ramon; Murphy, George; Lian, Christine; Bueno, Ericka; Marty, Francisco; Rennke, Helmut; Milford, Edgar; Tullius, Stefan; Pomahac, Bodhan

    2016-05-01

    Face transplantation was performed in a highly sensitized recipient with positive preoperative crossmatch and subsequent antibody-mediated rejection. The recipient was a 45-year-old female with extensive conventional reconstructions after chemical burns over the majority of the body. Residual quality of life and facial functions were poor. Levels of circulating anti-human leukocyte antigen (HLA) antibodies were high, and panel reactive antibody score was 98%. A potential donor was identified; however, with positive T and B cell flow crossmatches. The transplant team proceeded with face transplantation from this donor, under tailored immune suppression and with available salvage options. The operation was successful. Plasmapheresis and induction immune suppression (i.e., thymoglobulin followed by mycophenolate mofetil, tacrolimus, and steroids) were provided. Five days later, there was significant facial swelling, rising anti-HLA antibody titers, and unprecedented evidence of C4d deposits on skin. High doses of steroids and thymoglobulin were provided; however, rejection increased such that by day 19 it was diagnosed grade III in the BANFF scale. After stopping thymoglobulin because of serum sickness, combination therapy of plasmapheresis, eculizumab, bortezomib, and alemtuzumab was provided. HLA antibody levels decreased while swelling and redness improved. At 3 months, there were no longer signs of rejection on biopsy.

  19. Demonstration of high sensitivity laser ranging system

    NASA Technical Reports Server (NTRS)

    Millar, Pamela S.; Christian, Kent D.; Field, Christopher T.

    1994-01-01

    We report on a high sensitivity semiconductor laser ranging system developed for the Gravity and Magnetic Earth Surveyor (GAMES) for measuring variations in the planet's gravity field. The GAMES laser ranging instrument (LRI) consists of a pair of co-orbiting satellites, one which contains the laser transmitter and receiver and one with a passive retro-reflector mounted in an drag-stabilized housing. The LRI will range up to 200 km in space to the retro-reflector satellite. As the spacecraft pair pass over the spatial variations in the gravity field, they experience along-track accelerations which change their relative velocity. These time displaced velocity changes are sensed by the LRI with a resolution of 20-50 microns/sec. In addition, the pair may at any given time be drifting together or apart at a rate of up to 1 m/sec, introducing a Doppler shift into the ranging signals. An AlGaAs laser transmitter intensity modulated at 2 GHz and 10 MHz is used as fine and medium ranging channels. Range is measured by comparing phase difference between the transmit and received signals at each frequency. A separate laser modulated with a digital code, not reported in this paper, will be used for coarse ranging to unambiguously determine the distance up to 200 km.

  20. Transportable high sensitivity small sample radiometric calorimeter

    SciTech Connect

    Wetzel, J.R.; Biddle, R.S.; Cordova, B.S.; Sampson, T.E.; Dye, H.R.; McDow, J.G.

    1998-12-31

    A new small-sample, high-sensitivity transportable radiometric calorimeter, which can be operated in different modes, contains an electrical calibration method, and can be used to develop secondary standards, will be described in this presentation. The data taken from preliminary tests will be presented to indicate the precision and accuracy of the instrument. The calorimeter and temperature-controlled bath, at present, require only a 30-in. by 20-in. tabletop area. The calorimeter is operated from a laptop computer system using unique measurement module capable of monitoring all necessary calorimeter signals. The calorimeter can be operated in the normal calorimeter equilibration mode, as a comparison instrument, using twin chambers and an external electrical calibration method. The sample chamber is 0.75 in (1.9 cm) in diameter by 2.5 in. (6.35 cm) long. This size will accommodate most {sup 238}Pu heat standards manufactured in the past. The power range runs from 0.001 W to <20 W. The high end is only limited by sample size.

  1. Highly sensitive direct conversion ultrasound interferometer

    NASA Astrophysics Data System (ADS)

    Svitelskiy, Oleksiy; Grossmann, John; Suslov, Alexey

    2015-03-01

    Being invented more than fifty years ago, the ultrasonic pulse-echo technique has proven itself as a valuable and indispensable non-destructive tool to explore elastic properties of materials in engineering and scientific tasks. We propose a new design for the instrument based on mass-produced integral microchips. In our design the radiofrequency echo-pulse signal is processed by AD8302 RF gain and phase detector (www.analog.com).Its phase output is linearly proportional to the phase difference between the exciting and response signals. The gain output is proportional to the log of the ratio of amplitudes of the received to the exciting signals. To exclude the non-linear fragments and to enable exploring large phase changes, we employ parallel connection of two detectors, fed by in-phase and quadrature signals respectively. The instrument allowed us exploring phase transitions with precision of ΔV / V ~10-7 (V is the ultrasound speed). The high sensitivity of the logarithmic amplifiers embedded into AD8302 requires good grounding and screening of the receiving circuitry.

  2. Photofunctional hybrids of lanthanide functionalized bio-MOF-1 for fluorescence tuning and sensing.

    PubMed

    Shen, Xiang; Yan, Bing

    2015-08-01

    A series of luminescent Ln(3+)@bio-MOF-1 (Ln=Eu, Tb, bio-MOF-1=Zn8(ad)4(BPDC)6O⋅2Me2NH2 (ad=adeninate, BPDC=biphenyldicarboxylate)) are synthesized via postsynthetic cation exchange by encapsulating lanthanide ions into an anionic metal-organic framework (MOF), and their photophysical properties are studied. After loading 2-thenoyltrifluroacetone (TTA) as sensitized ligand by a gas diffusion ("ship-in-bottle") method, it is found that the luminescent intensity of Eu(3+) is enhanced. Especially, when loading two different lanthanide cations into bio-MOF-1, the luminescent color can be tuned to close white (light pink) light output. Additionally, bio-MOF-1 and Eu(3+)@bio-MOF-1 are selected as representative samples for sensing metal ions. When bio-MOF-1 is immersed in the aqueous solutions of different metal ions, it shows highly sensitive sensing for Fe(3+) as well as Eu(3+)@bio-MOF-1 immersed in the DMF solutions of different metal ion. The results are benefit for the further application of functionalized bio-MOFs in practical fields.

  3. Aneuploidy detection for chromosomes 1, X and Y by fluorescence in situ hybridization in human sperm from oligoasthenoteratozoospermic patients

    SciTech Connect

    Pang, M.G.; Zackowski, J.L.; Acosta, A.A.

    1994-09-01

    Oligoasthenoteratozoospermic males (n=15) were investigated for infertility as compared with proven fertile donors. The oligoasthenoteratozoospermic population showed a mean sperm concentration of 9.7 x 10{sup 6}/ml (Range 4.2-19.7), mean motility of 38.5% (Range 10.6-76.8) and morphology (measured by the percentage of normal forms evaluated by strict criteria) with a mean of 3.49% (Range 1.5-5.0). Fluorescence in situ hybridization (FISH) using satellite DNA probes specific for chromosomes 1 (puc 1.77), X (alpha satellite), and Y (satellite-III at Yqh) was performed on human interphase sperm nuclei. DNA probes were either directly labelled with rhodamine-dUTP, FITC-dUTP, or biotinylated by nick translation. Hybridization and signal detection were done by routine laboratory protocols. Microscopic analysis was performed using a cooled CCD camera attached to an epi-fluorescent microscope. After hybridization, fertile donors yielded a frequency of 0.96% (n=12) nullisomic, 98.5% (n=1231) monosomic and 0.96% (n=12) disomic for chromosome 1, whereas oligoasthenoteratozoospermic males yielded a frequency of 16% (n=600) nullisomic, 74.5% (n=2792) monosomic and 9.9% (n=370) disomic. In addition, fertile donors yielded a frequency of 45.7% (n=633) monosomic and 0.7% (n=11) disomic for chromosome X, whereas oligoasthenoteratozoospermic males yielded a frequency of 38.7% (n=760) monosomic and 0.8% (n=13) disomic. Chromosome Y frequencies for fertile donors showed 44.6% (n=614) monosomic and 0.6% (n=2) disomic, whereas oligoasthenoteratozoospermic males yielded a frequency of 33.2% (n=701) monosomic and 0.8% (n=15) disomic. This suggests that the frequency of nullisomy for chromosome 1 is significantly higher (p<0.001) in sperm from oligoasthenoteratozoospermic makes versus sperm from our fertile donors. We conclude that FISH is a powerful tool to determine the frequency of aneuploidy in sperm from oligoasthenoteratozoospermic patients.

  4. Multicolour hybrid nanoprobes of molecular beacon conjugated quantum dots: FRET and gel electrophoresis assisted target DNA detection

    NASA Astrophysics Data System (ADS)

    Kim, Joong Hyun; Chaudhary, Sumit; Ozkan, Mihrimah

    2007-05-01

    We have developed multicolour hybrid DNA probes employing green, yellow and orange colour quantum dot conjugated molecular beacons with black hole quencher 2. Optical and electrophoretic characterization revealed fluorescent energy transfer that follows the FRET mechanism with single nucleotide discrimination. Target DNA identification was observed to be highly sensitive up to 8 ng in gel electrophoresis. Comparison with the conventional organic dye SYBR Gold™ showed that our hybrid nanoprobes exhibit more stable performance with less background signal.

  5. Fluorescent Whole-Cell Hybridization with 16S rRNA-Targeted Oligonucleotide Probes To Identify Brucella spp. by Flow Cytometry

    PubMed Central

    Fernández-Lago, Luis; Vallejo, F. Javier; Trujillano, Ignacio; Vizcaíno, Nieves

    2000-01-01

    A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of Brucella abortus in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of Brucella and with a total of nine different Brucella clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate between Brucella suis biovars 2, 3, 4, and 5 and almost all the other Brucella spp. On the basis of differences in fluorescence intensities, no discrimination was established between Brucella spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions with Brucella spp. and with a total of 17 clinical isolates not belonging to the genus Brucella. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification of Brucella spp. PMID:10878084

  6. Photocatalytic oxidation removal of Hg0 using ternary Ag/AgI-Ag2CO3 hybrids in wet scrubbing process under fluorescent light

    NASA Astrophysics Data System (ADS)

    Zhang, Anchao; Zhang, Lixiang; Chen, Xiaozhuan; Zhu, Qifeng; Liu, Zhichao; Xiang, Jun

    2017-01-01

    A series of ternary Ag/AgI-Ag2CO3 photocatalysts synthesized using a facile coprecipitation method were employed to investigate their performances of Hg0 removal in a wet scrubbing reactor. The hybrids were characterized by N2 adsorption-desorption, XRD, SEM-EDS, HRTEM, XPS, DRS and ESR. The photocatalytic activities of Hg0 removal were evaluated under fluorescent light. The results showed that AgI content, fluorescent light irradiation, reaction temperature all showed significant influences on Hg0 removal. NO exhibited significant effect on Hg0 removal in comparison to SO2. Among these ternary Ag/AgI-Ag2CO3 hybrids, Ag/AgI(0.1)-Ag2CO3 showed the highest Hg0 removal efficiency, which could be ascribed to the effective separation of photogenerated electron-hole pairs between AgI and Ag2CO3 and the surface plasmon resonance (SPR) effect in the visible region by metallic silver nanoparticles (Ag0 NPs). The trapping studies of reactive radicals showed that the superoxide radicals (rad O2-) may play a key role in Hg0 removal under fluorescent light. According to the experimental and characterization results, a possible photocatalytic oxidation mechanism for enhanced Hg0 removal over Ag/AgI(0.1)-Ag2CO3 hybrid under fluorescent light was proposed.

  7. Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.

    PubMed

    Langenbacher, Adam D; Rodriguez, Delany; Di Maio, Alessandro; De Tomaso, Anthony W

    2015-01-01

    Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments.

  8. Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure.

    PubMed

    Frischer, M E; Floriani, P J; Nierzwicki-Bauer, S A

    1996-10-01

    The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.

  9. Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment.

    PubMed

    Klaus, Joseph P; Botten, Jason

    2016-03-02

    Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

  10. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    NASA Astrophysics Data System (ADS)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  11. Fully automated fluorescent in situ hybridization (FISH) staining and digital analysis of HER2 in breast cancer: a validation study.

    PubMed

    van der Logt, Elise M J; Kuperus, Deborah A J; van Setten, Jan W; van den Heuvel, Marius C; Boers, James E; Schuuring, Ed; Kibbelaar, Robby E

    2015-01-01

    HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA) and 100 (50 selected IHC 2+ and 50 random IHC scores) full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted) with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (κ = 0.94) and 93.8% (κ = 0.88), respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer.

  12. Contribution of fluorescence in situ hybridization to immunohistochemistry for the evaluation of HER-2 in breast cancer.

    PubMed

    Cianciulli, Anna M; Botti, Claudio; Coletta, Angela M; Buglioni, Simonetta; Marzano, Raffaella; Benevolo, Maria; Cione, Antonio; Mottolese, Marcella

    2002-02-01

    The main focus of the present study was to assess the efficacy of interphase cytogenetics using fluorescence in situ hybridization (FISH) as a valid alternative to immunohistochemistry (IHC) in paraffin-embedded tissue sections and/or the efficacy of the combination of the two methods, while, at the same time, aiming to provide additional information on the use of the two methods. For this study, selected breast cancer patients (n=66) were tested for HER-2 gene amplification by FISH. The probe contains DNA sequences specific for the HER-2 human gene locus and hybridizes to the 17q11.2 through q12 region of human chromosome 17. The same samples were tested previously for HER-2 overexpression by two monoclonal antibodies (300G9 and CB11), recognizing an extracellular and an internal domain of gp185(Her-2), respectively. HER-2 overexpression also was evaluated using the HerceptTest Kit (Dako, Milan, Italy). The HerceptTest was performed according to the manufacturer's standard procedures, and results were scored on a 0 to 3+ scale. A total of 34 (51%) of 66 breast tumors enrolled in this study were positive by FISH. Of the 34 cases amplified by FISH, 9 were negative by IHC using both monoclonal antibody (MoAb) 300G9 and MoAb CB11, with a concordance rate from 80.3% to 83.3%. A higher concordance was verified (92.4%) when we used the HerceptTest Kit. Of the 32 cases found negative with the HerceptTest, FISH analysis identified HER-2 gene amplification in more than 10%. Our results indicate that with the combined use of both methods, several amplified samples classified negative by IHC can be used thus improving therapeutic planning for specific therapy with the monoclonal antibody trastuzumab.

  13. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage.

    PubMed

    Nicomrat, Duongruitai; Dick, Warren A; Tuovinen, Olli H

    2006-01-01

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at < 0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant. Heterotrophs in the Acidiphilium genus totaled 20% of the bacterial population. Leptospirillum ferrooxidans was below the level of detection in the bacterial community. The results from the FISH technique from this field study are consistent with results from other experiments involving enumeration by most probable number, dot-blot hybridization, and denaturing gradient gel electrophoresis analyses and with the geochemistry of the site.

  14. Ultrasensitive fluorescence detection of DNA sequencing gels

    SciTech Connect

    Mathies, R.A.

    1991-01-01

    During the three years of this grant we have: (1) Developed and applied a new theory for optimizing high-sensitivity fluorescence detection. (2) Developed and patented a new high-sensitivity confocal-fluorescence laser-excited gel-scanner. (3) Applied this scanner to the development of a new class of versatile and sensitive fluorescent dyes for DNA detection. (4) Developed methods for the detection of single fluorescent molecules by fluorescence burst detection. 11 refs., 10 figs.

  15. Evaluation of a radiative transfer equation and diffusion approximation hybrid forward solver for fluorescence molecular imaging.

    PubMed

    Gorpas, Dimitris; Andersson-Engels, Stefan

    2012-12-01

    The solution of the forward problem in fluorescence molecular imaging strongly influences the successful convergence of the fluorophore reconstruction. The most common approach to meeting this problem has been to apply the diffusion approximation. However, this model is a first-order angular approximation of the radiative transfer equation, and thus is subject to some well-known limitations. This manuscript proposes a methodology that confronts these limitations by applying the radiative transfer equation in spatial regions in which the diffusion approximation gives decreased accuracy. The explicit integro differential equations that formulate this model were solved by applying the Galerkin finite element approximation. The required spatial discretization of the investigated domain was implemented through the Delaunay triangulation, while the azimuthal discretization scheme was used for the angular space. This model has been evaluated on two simulation geometries and the results were compared with results from an independent Monte Carlo method and the radiative transfer equation by calculating the absolute values of the relative errors between these models. The results show that the proposed forward solver can approximate the radiative transfer equation and the Monte Carlo method with better than 95% accuracy, while the accuracy of the diffusion approximation is approximately 10% lower.

  16. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    PubMed

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  17. microFIND(®) approach to fluorescent in situ hybridization (FISH).

    PubMed

    Zanardi, Andrea; Barborini, Emanuele; Carbone, Roberta

    2013-01-01

    FISH technology has gained increasing attention in the management of cancer disease, either for predictive or prognostic indications. Molecular cytogenetics has greatly improved diagnostic capability of classical cytogenetics analysis of metaphase-based chromosome for the identification of genetic aberrations. The availability of a large number of fluorescent probes, each specific for different genetic lesions, together with a robust protocol for interphase FISH, provide the pathologist with the essential tools for an accurate evaluation of patient's disease. Hemato-oncological and many of the solid tumors have been comprehensively characterized by peculiar genetic defects and are now routinely evaluated by interphase FISH. Despite the reliability of the method, which has undergone only minor changes since the 1970s, FISH assay is still hampered by reagents cost, preventing its adoption in large-scale oncological screening. In this chapter we describe a major improvement of interphase FISH assay for cytological samples through the description of the miniaturized device microFIND(®) that offers, besides reduction of cost per assay, a completely novel vision to the FISH technology, thanks to the perspective of full automation of FISH assay using a dedicated robotic platform for microFIND(®) handling, (not presently described in the chapter).

  18. Fluorescence in-situ hybridization analysis of chromosomal constitution in spermatozoa from a mosaic 47,XYY/46,XY male.

    PubMed

    Wang, J Y; Samura, O; Zhen, D K; Cowan, J M; Cardone, V; Summers, M; Bianchi, D W

    2000-07-01

    Sex-chromosome mosaicism in spermatozoa from a mosaic 47,XYY[20%]/46, XY[80%] male with fertility problems was assessed using triple-probe fluorescence in-situ hybridization (FISH) studies. Chromosome-specific probes for X, Y and 18 were used, and the possible outcomes were deduced. In normal haploid spermatozoa of the patient and a normal 46,XY male control, the X:Y ratio was close to 1:1. There was a significant difference in the total incidence of karyotypically abnormal spermatozoa between the patient and the 46, XY male control (2.31% versus 1.46%, P < 0.0001). The incidence of some types of disomic spermatozoa X+Y+18 (24,XY) and X+18+18 (24,X, +18), or diploid X+Y+18+18 (46,XY) spermatozoa was significantly increased in the patient's semen sample. There was, however, no significant difference in the incidence of disomic Y+Y+18 (24,YY) spermatozoa. Because the majority of the patient's spermatozoa was karyotypically normal, the aetiology of his fertility problems was unclear. These results add to the growing body of information regarding chromosome abnormalities in spermatozoa from men who are mosaic for sex chromosome abnormalities. In these men, FISH analysis of spermatozoa may be warranted to determine the relative percentages of abnormal cells, and to determine if in-vitro fertilization with preimplantation genetic diagnosis may increase the likelihood of a successful pregnancy.

  19. Fluorescence in-situ hybridization for the identification of bacterial species in archival heart valve sections of canine bacterial endocarditis.

    PubMed

    Kornreich, B G; Craven, M; McDonough, S P; Nydam, D V; Scorza, V; Assarasakorn, S; Lappin, M; Simpson, K W

    2012-05-01

    Bacterial endocarditis (BE) is defined as inflammation of cardiac valve structures and/or the endocardium secondary to bacterial infection. Canine valvular BE is associated with significant morbidity and mortality and ante-mortem diagnosis and post-mortem identification of causative organisms is problematic. Identification of bacteria in canine BE has traditionally relied on visualization of organisms on histological sections stained with haematoxylin and eosin (HE), Gram and modified Steiner's stains. Each of these staining techniques has limitations with respect to identification of bacterial species in cases of BE. Fluorescence in-situ hybridization (FISH) has been introduced recently as a technique to identify bacteria in biological specimens. To our knowledge, FISH has not been used previously to identify bacteria in archival samples of heart valves from dogs with naturally occurring BE. We sought to determine whether FISH could detect the presence and species of bacteria in archival heart valve sections from dogs with BE, and to compare FISH to histochemical stains in the identification of bacteria. FISH detected bacteria in seven of 17 cases of canine BE and showed near perfect agreement with modified Steiner's stain for the detection of bacteria. FISH identified Streptococcus spp. and/or Staphylococcus spp. in all of these cases, but Bartonella spp. were not identified.

  20. Validation of break-apart and fusion MYC probes using a digital fluorescence in situ hybridization capture and imaging system

    PubMed Central

    Liew, Michael; Rowe, Leslie; Clement, Parker W.; Miles, Rodney R.; Salama, Mohamed E.

    2016-01-01

    Introduction: Detection of MYC translocations using fluorescence in situ hybridization (FISH) is important in the evaluation of lymphomas, in particular, Burkitt lymphoma and diffuse large B-cell lymphoma. Our aim was to validate a digital FISH capture and imaging system for the detection of MYC 8q24 translocations using LSI-MYC (a break-apart probe) and MYC 8;14 translocation using IGH-MYC (a fusion probe). Materials and Methods: LSI-MYC probe was evaluated using tissue sections from 35 patients. IGH-MYC probe was evaluated using tissue sections from forty patients. Sections were processed for FISH and analyzed using traditional methods. FISH slides were then analyzed using the GenASIs capture and analysis system. Results: Results for LSI-MYC had a high degree of correlation between traditional method of FISH analysis and digital FISH analysis. Results for IGH-MYC had a 100% concordance between traditional method of FISH analysis and digital FISH analysis. Conclusion: Annotated whole slide images of H and E and FISH sections can be digitally aligned, so that areas of tumor within a section can be matched and evaluated with a greater degree of accuracy. Images can be archived permanently, providing a means for examining the results retrospectively. Digital FISH imaging of the MYC translocations provides a better diagnostic tool compared to traditional methods for evaluating lymphomas. PMID:27217970

  1. Interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction as a diagnostic aid for synovial sarcoma.

    PubMed Central

    Shipley, J.; Crew, J.; Birdsall, S.; Gill, S.; Clark, J.; Fisher, C.; Kelsey, A.; Nojima, T.; Sonobe, H.; Cooper, C.; Gusterson, B.

    1996-01-01

    Identification of the t(X;18)(p11.2;q11.2) that is associated with a high proportion of synovial sarcoma can be a useful diagnostic aid. The translocation results in fusion of the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene, two homologous genes within Xp11.2. Two-color interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction were assessed as approaches to identify the rearrangement in well characterized cases. The presence of the translocation, and the specific chromosome X gene disrupted, were inferred from the configuration of signals from chromosome-specific centromere probes, paints, and markers flanking each gene in preparations of interphase nuclei. Rearrangement was found in two cell lines and eight of nine tumor samples, including analysis of five touch imprints. This was consistent with cytogenetic data in four cases and reverse transcription polymerase chain reaction analysis using primers known to amplify both SYT-SSX1 and SYT-SSX2 transcripts. The transcripts were distinguished by restriction with LspI and SmaI. Contrary to previous suggestions, there was no obvious correlation between histological subtype and involvement of the SSX1 or SSX2 gene. These approaches could also be applied to the identification of tumor-free margins and metastatic disease. Images Figure 1 Figure 3 PMID:8579118

  2. Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

    PubMed Central

    Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

    2014-01-01

    Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation. PMID:25489864

  3. Analysis of methanogenic activity in a thermophilic-dry anaerobic reactor: use of fluorescent in situ hybridization.

    PubMed

    Montero, B; García-Morales, J L; Sales, D; Solera, R

    2009-03-01

    Methanogenic activity in a thermophilic-dry anaerobic reactor was determined by comparing the amount of methane generated for each of the organic loading rates with the size of the total and specific methanogenic population, as determined by fluorescent in situ hybridization. A high correlation was evident between the total methanogenic activity and retention time [-0.6988Ln(x)+2.667] (R(2) 0.8866). The total methanogenic activity increased from 0.04x10(-8) mLCH(4) cell(-1)day(-1) to 0.38x10(-8) mLCH(4) cell(-1)day(-1) while the retention time decreased, augmenting the organic loading rates. The specific methanogenic activities of H(2)-utilizing methanogens and acetate-utilizing methanogens increased until they stabilised at 0.64x10(-8) mLCH(4) cell(-1)day(-1) and 0.33x10(-8) mLCH(4) cell(-1)day(-1), respectively. The methanogenic activity of H(2)-utilizing methanogens was higher than acetate-utilizing methanogens, indicating that maintaining a low partial pressure of hydrogen does not inhibit the acetoclastic methanogenesis or the anaerobic process.

  4. HER-2 and cancer antigen 125 evaluation in ovarian borderline tumors by immunohistochemistry and fluorescence in situ hybridization.

    PubMed

    Heinrich, J K R; Böttcher-Luiz, F; Andrade, L L A; Davidson, S; Bonds, L; Stephens, J; Varella-Garcia, M

    2004-01-01

    The study determined the expression of cancer antigen (CA) 125 and HER-2 in 45 borderline ovarian tumors (BOTs) and investigated the correlation of these biologic markers with histologic type, clinical stage, and outcome. The level of CA 125 protein was assessed using DAKO's M-11 clone antibody in immunohistochemistry (IHC) assays (Carpinteria, CA). The HER-2 protein expression was assessed in IHC assays using the HercepTest (DAKO), and the HER-2 gene copy number per cell was investigated through fluorescence in situ hybridization (FISH) assays using VYSIS' PathVysion DNA Probe (Downers Grove, IL). Expression of the CA 125 protein was detected in 49% of the samples (22 out of 45 tumors) and significantly associated with the serous histologic type. However, CA 125 expression did not associate with clinical stage or outcome. Protein overexpression or gene amplification of HER-2 was not found. However, abnormal FISH results were detected in 16% (seven out of 45 patients) of specimens comprising extranumerary copies of HER-2 and/or chromosome 17 per cell. Abnormal FISH results were found to be independent of CA 125 expression and histologic type whereas they positively associate with advanced clinical stage. Our data show that HER-2 is not altered in BOTs, and the presence of aneusomy for chromosome 17 and HER-2 may predict tumor progression.

  5. Smith-Magenis syndrome deletion: A case with equivocal cytogenetic findings resolved by fluorescence in situ hybridization

    SciTech Connect

    Juyal, R.C.; Patel, P.I.; Greenberg, F.

    1995-09-11

    The availability of markers for the 17p11.2 region has enabled the diagnosis of Smith-Magenis syndrome (SMS) by fluorescence in situ hybridization (FISH). SMS is typically associated with a discernible deletion of band 17p11.2 upon cytogenetic analysis at a resolution of 400-550 bands. We present a case that illustrates the importance of using FISH to confirm a cytogenetic diagnosis of del(17)(p11.2). Four independent cytogenetic analyses were performed with different conclusions. Results of low resolution analyses of amniocytes and peripheral blood lymphocytes were apparently normal, while high resolution analyses of peripheral blood samples in two laboratories indicated mosaicism for del(17)(p11.2). FISH clearly demonstrated a 17p deletion on one chromosome of all peripheral blood cells analyzed and ruled out mosaicism unambiguously. The deletion was undetectable by flow cytometric quantitation of chromosomal DNA content, suggesting that it is less than 2 Mb. We conclude that FISH should be used to detect the SMS deletion when routine chromosome analysis fails to detect it and to verify mosaicism. 23 refs., 3 figs., 1 tab.

  6. Locked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs

    PubMed Central

    Robertson, Kelly L.

    2012-01-01

    We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population. PMID:22057868

  7. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage

    SciTech Connect

    Nicomrat, D.; Dick, W.A.; Tuovinen, O.H.

    2006-07-15

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at < 0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant.

  8. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-12-31

    The ultimate goal of this proposal is to create a cDNA map of the human genome. Mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach will generate 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  9. Fluorescent in situ hybridization (FISH) and high resolution karyotype analysis reveal a novel inversion duplication of 10q

    SciTech Connect

    Czarnecki, P.; Dyke, D.L. Van; Dowling, P.K.

    1994-09-01

    A white male born with dysmorphic features, including upslanting palpebral fissures, bilateral simian creases, posteriorly rotated ears, bitemporal narrowing, frontal bossing, camptodactyly and head circumference and weight less than the 5th percentile was found to have a de novo add(10)(q26.1). High resolution karyotype analysis revealed a novel chromosomal abnormality: 46,XY,inv dup(10)(q26.3-q25.1). Fluorescent in situ hybridization using a chromosome 10-specific painting probe (Oncor, Inc.) confirmed that the extra material was derived from chromosome 10. Duplication of 10q24 or 10q25 is associated with characteristic craniofacial malformations, minor malformations of the hands and feet, major malformations of the heart, skeleton, and kidneys and severe mental retardation. Our patient, currently 7 months old, has many of the skeletal and craniofacial manifestations of other patients, but is developmentally normal at this early age. This is the first FISH confirmation of a 10q duplication and demonstrates the utility of this technology in addition to karyotype analysis. Molecular studies to determine the parental origin and extent of the duplication are in progress, since the apparent lack of developmental delay was unexpected. Identification of the origin of duplicated material will help assist in genetic counseling by further delineating new genetic syndromes.

  10. Physical mapping of immune-related genes in Yesso scallop (Patinopecten yessoensis) using fluorescent in situ hybridization

    PubMed Central

    Yang, Zujing; Li, Xuan; Liao, Huan; Hu, Liping; Zhang, Zhengrui; Zhao, Bosong; Huang, Xiaoting; Bao, Zhenmin

    2016-01-01

    Abstract The innate immune system plays a pivotal role in defending invasion of microorganisms for scallops. Previous studies on immune-related genes in the Yesso scallop, Patinopecten yessoensis (Jay, 1857) have mainly focused on characterization and expression pattern in response to bacterial challenge, no research has been carried out on the cytogenetic level yet. In the present study, eight fosmid clones containing the sequences of key immune-related genes (PyNFkB, PyTRAF2, PyTRAF4, PyTRAF7, PyMyd88-1, PyMyd88-3, PyMKK-7 and PyTNFR) were isolated and seven of them were successfully mapped on chromosomes of Patinopecten yessoensis utilizing fluorescence in situ hybridization. Wherein, PyMyd88-1, PyMyd88-3 and PyMKK-7 located on the same chromosome pair with adjacent positions and the other genes were mapped on four non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of such genes of the Yesso scallop will lay a foundation for studies such as assignment of interested genes to chromosomes, construction cytogenetic maps and so on. PMID:28123676

  11. Spatial organization of bacterial flora in normal and inflamed intestine: A fluorescence in situ hybridization study in mice

    PubMed Central

    Swidsinski, Alexander; Loening-Baucke, Vera; Lochs, Herbert; Hale, Laura P.

    2005-01-01

    AIM: To study the role of intestinal flora in inflammatory bowel disease (IBD). METHODS: The spatial organization of intestinal flora was investigated in normal mice and in two models of murine colitis using fluorescence in situ hybridization. RESULTS: The murine small intestine was nearly bacteria-free. The normal colonic flora was organized in three distinct compartments (crypt, interlaced, and fecal), each with different bacterial compositions. Crypt bacteria were present in the cecum and proximal colon. The fecal compartment was composed of homogeneously mixed bacterial groups that directly contacted the colonic wall in the cecum but were separated from the proximal colonic wall by a dense interlaced layer. Beginning in the middle colon, a mucus gap of growing thickness physically separated all intestinal bacteria from contact with the epithelium. Colonic inflammation was accompanied with a depletion of bacteria within the fecal compartment, a reduced surface area in which feces had direct contact with the colonic wall, increased thickness and spread of the mucus gap, and massive increases of bacterial concentrations in the crypt and interlaced compartments. Adhesive and infiltrative bacteria were observed in inflamed colon only, with dominant Bacteroides species. CONCLUSION: The proximal and distal colons are functionally different organs with respect to the intestinal flora, representing a bioreactor and a segregation device. The highly organized structure of the colonic flora, its specific arrangement in different colonic segments, and its specialized response to inflammatory stimuli indicate that the intestinal flora is an innate part of host immunity that is under complex control. PMID:15754393

  12. Paternal-age effects on sperm aneuploidy investigated in mice and humans by three-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J.; Lowe, X.; Holland, N.T.

    1994-09-01

    We conducted a cross-species comparison of the effects of paternal age on sperm aneuploidy in mice and humans. A new murine assay was developed to detect sperm hyperhaploidy and polyploidy for chromosomes X, Y, and 8 using fluorescence in situ hybridization with chromosome-specific DNA probes, to serve as a direct corollate to the three-chromosome method developed early for human sperm. Sperm aneuploidy was evaluated in eight male B6C3F1 male mice (aged 22.5-30.5 mo) and compared to young controls (2.4 mo). The aged group showed significant ({approximately}2.0-fold) increases in hyperhaploidies involving chromosomes X, Y and 8, with the greatest effects seen in the oldest animals. Sperm aneuploidy was also evaluated in two groups of healthy men who differed in mean age [46.8{plus_minus}3.1 (n=4) vs. 28.5{plus_minus}5.0 (n=10) yrs], using the three-chromosome method. The older group showed a statistically significant increase in hyperhaploid sperm for both sex chromosomes. Additional controlled human studies are planned. Taken together, the murine and human data are consistent with a positive effect of paternal age on sperm aneuploidy. In both species, the strongest age effect was observed for hyperhaploidies of chromosome Y. Future studies are needed to investigate the shape of the age-effect curve and to evaluate chromosomal differences, especially for humans in their late reproductive years.

  13. Feasibility of using fluorescence in situ hybridization (FISH) to detect early gene changes in sputum cells from uranium miners

    SciTech Connect

    Neft, R.E.; Rogers, J.L.; Belinsky, S.A.

    1995-12-01

    Epidemiological studies have shown that combined exposure to radon progeny and tobacco smoke produce a greater than additive or synergistic increase in lung cancer risk. Lung cancer results from multiple genetic changes over a long period of time. An early change that occurs in lung cancer is trisomy 7 which is found in 50% of non-small cell lung cancer and in the far margins of resected lung tumors. The 80% mortality associated with lung cancer is in part related to the high proportion of patients who present with an advanced, unresectable tumor. Therefore, early detection of patients at risk for tumor development is critical to improve treatment of this disease. Currently, it is difficult to detect lung cancer early while it is still amendable by surgery. Saccomanno, G. has shown that premalignant cytologic changes in sputum cells collected from uranium miners can be detected by a skilled, highly trained cytopathologist. A more objective alternative for identifying premalignant cells in sputum may be to determine whether an early genetic change such as trisomy 7 is present in these cells. Fluorescence in situ hybridization (FISH) can be used to identify cells with trisomy 7. The results of this investigation indicate that FISH may prove to be an accurate, efficient method to test at-risk individuals for genetic alterations in bronchial epithelial cells from sputum.

  14. Monitoring of chimerism using fluorescence in situ hybridization in a child with severe combined immune deficiency following bone marrow transplant

    SciTech Connect

    Wenger, S.L.; Chen, X.O.; Katz, A.J. |

    1994-09-01

    A boy with severe combined immunodeficiency received a bone marrow transplant from his sister when he was approximately 3 years of age. His peripheral blood karyotype at age 3 and 4 years was 46,XX (20 cells analyzed). Because of a decline in antibody production at 19 years of age, the patient`s peripheral blood was analyzed again for suspected chimerism. His karyotype in phytohemagglutinin (PHA)-stimulated culture was 46,XX in 49 cells and 46,XY in one cell. Both metaphase and interphase cells were examined for sex chromosome constitution using X and Y dual-color alpha-satellite probes for fluorescence in situ hybridization (FISH). FISH results for metaphase cells showed 1/50 XY cells, but 38% of interphase cells showed the presence of both X and Y centromere. Pokeweed mitogen (PWM)-stimulated cultures grew poorly and were therefore analyzed using FISH only: 81% of interphase cells were 46,XX. The discrepancy between metaphase and interphase in the PHA-stimulated cultures most likely represents a failure of this boy`s own XY T-cells to be stimulated.

  15. Cytogenetic characterization of complex karyotypes in seven established melanoma cell lines by multiplex fluorescence in situ hybridization and DAPI banding.

    PubMed

    Schulten, Hans Jürgen; Gunawan, Bastian; Otto, Friedrich; Hassmann, René; Hallermann, Christian; Noebel, Albrecht; Füzesi, László

    2002-03-01

    We report the use of multiplex fluorescence in situ hybridization (M-FISH) to resolve chromosomal aberrations in seven established melanoma cell lines with hypotriploid to hypertetraploid complex karyotypes. By simultaneous identification of all human chromosomes in single FISH experiments using a set of 52 directly labeled, whole chromosome painting probes, cryptic chromosomal translocations and the origin of unclear chromosomal material in structural rearranged and marker chromosomes could be identified, refining the tumor karyotypes in all seven cell lines. The number of structural aberrations in each cell line assigned with combined M-FISH and DAPI banding analysis ranged from 15 to 45. Altogether, 275 breakpoints could be assigned to defined chromosomal regions or bands. The chromosome arms 1p, 6q, 7p, 9p, and 11q which are known to be nonrandomly associated with melanoma tumorigenesis, were frequently involved in chromosomal breaks and/or copy number changes. This study also demonstrated the practical usefulness of combining M-FISH with conventional cytogenetic banding techniques for the characterization of complex tumor karyotypes with massive genomic alterations.

  16. Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

    SciTech Connect

    Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B.

    1995-07-17

    Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

  17. Effects of fixative and fixation protocols on assessment of Her-2/neu oncogene amplification status by fluorescence in situ hybridization.

    PubMed

    Willmore-Payne, Carlynn; Metzger, Ken; Layfield, Lester J

    2007-03-01

    Fluorescence in situ hybridization (FISH) is used to determine amplification status of the Her-2/neu gene in specimens of newly diagnosed breast carcinoma. The Vysis kit for FISH analysis stipulates that the tissue be formalin-fixed and paraffin-embedded. Concerns regarding carcinogenicity of formalin and environmental effects of formalin waste have led to the development of formalin replacement products. An increasing number of breast biopsy specimens are being fixed in these substitutes. We tested 6 non-formalin-based fixatives to determine their impact on FISH testing for Her-2/neu gene amplification status by comparison with formalin-fixed control specimens from the same neoplasm. Specimens fixed in Pen-Fix, Prefer, Histochoice, UniFix, and GTF were associated with absent or technically compromised staining in at least one of the 3 neoplasms tested for each fixative when compared to the formalin-fixed control. O-Fix did not seem to compromise staining quality in 3 paired specimens tested.

  18. The utility of fluorescence in situ hybridization for detection of bladder urothelial carcinoma in routine clinical practice.

    PubMed

    Kwak, Kyung Won; Kim, Sun Hee; Lee, Hyun Moo

    2009-12-01

    To evaluate the ability of fluorescence in situ hybridization (FISH) in detecting bladder urothelial carcinoma (BUC), FISH and cytology were compared for the evaluation of 308 consecutive urine samples from patients suspected of having BUC. All patients underwent cystoscopy for identification of bladder lesions. The FISH results were compared with the cytology assessment. In all, 122 patients had confirmed BUC. Among them, 68 (55.7%) were FISH-positive, while only 33 (27%) were positive on cytology. According to disease st