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Sample records for labeling probes enables

  1. Enabling interstellar probe

    NASA Astrophysics Data System (ADS)

    McNutt, Ralph L.; Wimmer-Schweingruber, Robert F.; International Interstellar Probe Team

    2011-04-01

    The scientific community has advocated a scientific probe to the interstellar medium for over 30 years. While the Voyager spacecraft have passed through the termination shock of the solar wind, they have limited lifetimes as their radioisotope power supplies decay. It remains unclear whether they can reach the heliopause, the boundary between shocked solar wind and interstellar plasmas, and, in any case, they will not reach the undisturbed interstellar medium. As with most exploratory space missions, their ongoing observations continue to raise even more questions about the nature of the interaction of our heliosphere and the interstellar medium. Scientific questions including: What is the nature of the nearby interstellar medium? How do the Sun and galaxy affect the dynamics of the heliosphere? What is the structure of the heliosphere? How did matter in the solar system and interstellar medium originate and evolve? can only be answered by an "interstellar precursor" probe. Such a mission is required to make in situ measurements in the interaction region and interstellar medium itself at distances far from the Sun, but in a finite mission lifetime. By launching a probe toward the incoming "interstellar wind," whose direction is known, the distance to be traveled can be minimized but is still large. The current consensus is that a scientifically compelling mission must function to at least a distance of 200 astronomical units (AU) from the Sun and return a reasonable stream of data during the voyage. The central problem is that of providing a means of propulsion to accelerate a probe from the Solar System. Even with a low-mass payload and spacecraft, achieving the high speeds needed, even with gravity assists, have remained problematic. Voyager 1, the fastest object ever to leave the system is now traveling ˜3.6 AU/yr, and a credible probe must reach at least 2-3 times this speed. The use of an Ares V is an approach for enabling a fast interstellar precursor

  2. Nanoelectronic Coating Enabled Versatile Multifunctional Neural Probes.

    PubMed

    Zhao, Zhengtuo; Luan, Lan; Wei, Xiaoling; Zhu, Hanlin; Li, Xue; Lin, Shengqing; Siegel, Jennifer J; Chitwood, Raymond A; Xie, Chong

    2017-08-09

    Brain function can be best studied by simultaneous measurements and modulation of the multifaceted signaling at the cellular scale. Extensive efforts have been made to develop multifunctional neural probes, typically involving highly specialized fabrication processes. Here, we report a novel multifunctional neural probe platform realized by applying ultrathin nanoelectronic coating (NEC) on the surfaces of conventional microscale devices such as optical fibers and micropipettes. We fabricated the NECs by planar photolithography techniques using a substrate-less and multilayer design, which host arrays of individually addressed electrodes with an overall thickness below 1 μm. Guided by an analytic model and taking advantage of the surface tension, we precisely aligned and coated the NEC devices on the surfaces of these conventional microprobes and enabled electrical recording capabilities on par with the state-of-the-art neural electrodes. We further demonstrated optogenetic stimulation and controlled drug infusion with simultaneous, spatially resolved neural recording in a rodent model. This study provides a low-cost, versatile approach to construct multifunctional neural probes that can be applied to both fundamental and translational neuroscience.

  3. Labeled nucleotide phosphate (NP) probes

    DOEpatents

    Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  4. The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method.

    PubMed

    do Nascimento, Cássio; Santos Barbosa, Rodrigo Edson; Mardegan Issa, João Paulo; Watanabe, Evandro; Yoko Ito, Izabel; Monesi, Nadia; Albuquerque Junior, Rubens Ferreira de

    2008-01-01

    Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.

  5. Labeled line drawing of Galileo spacecraft's atmospheric probe

    NASA Image and Video Library

    1989-09-11

    Labeled line drawing entitled GALILEO PROBE identifies the deceleration module aft cover, descent module, and deceleration module aeroshell configurations and dimensions prior to and during entry into Jupiter's atmosphere.

  6. Probes labelled with energy transfer coupled dyes

    DOEpatents

    Mathies, R.A.; Glazer, A.; Ju, J.

    1997-11-18

    Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids. 7 figs.

  7. Probes labelled with energy transfer coupled dyes

    DOEpatents

    Mathies, Richard A.; Glazer, Alexander; Ju, Jingyue

    1997-01-01

    Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids.

  8. 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.

  9. In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

    PubMed

    Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

    2017-08-01

    Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

  10. Hybridization probe pairs and single-labeled probes: an alternative approach for genotyping and quantification.

    PubMed

    Froehlich, Thomas; Geulen, Oliver

    2008-01-01

    Real-time polymerase chain reaction (PCR) has become a standard tool in both quantitative gene expression and genetic variation analysis. Data collection is performed throughout the PCR process, thus combining amplification and detection into a single step. This can be achieved by combining a variety of different fluorescent chemistries that correlate the concentration of an amplified PCR product to changes in fluorescence intensity. Hybridization probe pairs and single-labeled probes are sequence-specific, dye-labeled oligonucleotides, used in real-time PCR approaches, in particular for genotyping of single nucleotide polymorphisms (SNPs). In that case, a detector probe is designed to cover the polymorphism. Allelic variants are identified and differentiated via post-PCR melting curve analysis. A single melting curve can distinguish different T (m)s, and differently labeled probes may be used, theoretically allowing multiplexed genotyping of several SNPs.

  11. Comparative examination of probe labeling methods for microarray hybridization

    NASA Astrophysics Data System (ADS)

    Burke, David I.; Woodward, Karen; Setterquist, Robert A.; Kawasaki, Ernest S.

    2001-06-01

    For detection of differential gene expression, confocal laser based scanners are now capable of analyzing microarrays using one to five wavelengths. This allows investigators to choose among several labeling methods. Here we compare direct incorporation and indirect methods (amino-allyl and dendrimers) for labeling cDNA probes. We assessed reproducible sensitivity of each probe preparation method in two ways. First, by comparing hybridization intensities for limit of signal detection and second by measuring the lowest detectable concentration of a known ratio of mixed DNA (spikes). Limit of detection assay was done using arrays of mixed targets consisting of a serially diluted human specific gene fragment (HU1) and an undiluted DNA of chloramphenicol acetyl tranferase (CAT) gene. Then, individual single target arrays of CAT and HU1 DNA were used to determine the lowest detectable spike ratio of each labeling method. The results of this study will be presented and their significance for the analysis of microarrays will be discussed.

  12. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    NASA Astrophysics Data System (ADS)

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-06-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

  13. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    PubMed Central

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-01-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging. PMID:27339882

  14. Photolytic Labeling to Probe Molecular Interactions in Lyophilized Powders

    PubMed Central

    Iyer, Lavanya K.; Moorthy, Balakrishnan S.; Topp, Elizabeth M.

    2014-01-01

    Local side-chain interactions in lyophilized protein formulations were mapped using solid-state photolytic labeling-mass spectrometry (ssPL-MS). Photoactive amino acid analogs (PAAs) were used as probes and either added to the lyophilized matrix or incorporated within the amino acid sequence of a peptide. In the first approach, apomyoglobin was lyophilized with sucrose and varying concentrations of photo-leucine (L-2-amino-4, 4′-azipentanoic acid; pLeu). The lyophilized solid was irradiated at 365 nm to initiate photolabeling. The rate and extent of labeling were measured using ESI-HPLC-MS, with labeling reaching a plateau at ∼ 30 min, forming up to 6 labeled populations. Bottom-up MS/MS analysis was able to provide peptidelevel resolution of the location of pLeu. ssPL-MS was also able to detect differences in side-chain environment between sucrose and guanidine hydrochloride formulations. In the second approach, peptide GCG (1-8)* containing p-benzoyl-L-phenylalanine (pBpA) in the amino acid sequence was lyophilized with various excipients and irradiated. Peptide-peptide and peptide-excipient adducts were detected using MS. Top-down MS/MS on the peptide dimer provided amino acidlevel resolution regarding interactions and the cross-linking partner for pBpA in the solid state. The results show that ssPL-MS can provide high-resolution information about protein interactions in the lyophilized environment. PMID:24125175

  15. Method for producing labeled single-stranded nucleic acid probes

    DOEpatents

    Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

    1999-10-19

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

  16. Photolytic labeling to probe molecular interactions in lyophilized powders.

    PubMed

    Iyer, Lavanya K; Moorthy, Balakrishnan S; Topp, Elizabeth M

    2013-12-02

    Local side-chain interactions in lyophilized protein formulations were mapped using solid-state photolytic labeling-mass spectrometry (ssPL-MS). Photoactive amino acid analogues (PAAs) were used as probes and either added to the lyophilized matrix or incorporated within the amino acid sequence of a peptide. In the first approach, apomyoglobin was lyophilized with sucrose and varying concentrations of photoleucine (L-2-amino-4,4'-azipentanoic acid; pLeu). The lyophilized solid was irradiated at 365 nm to initiate photolabeling. The rate and extent of labeling were measured using electrospray ionization/high-performance liquid chromatography/mass spectrometry (ESI-HPLC-MS), with labeling reaching a plateau at ~30 min, forming up to six labeled populations. Bottom-up MS/MS analysis was able to provide peptide-level resolution of the location of pLeu. ssPL-MS was also able to detect differences in side-chain environment between sucrose and guanidine hydrochloride formulations. In the second approach, peptide GCG (1-8)* containing p-benzoyl-L-phenylalanine (pBpA) in the amino acid sequence was lyophilized with various excipients and irradiated. Peptide-peptide and peptide-excipient adducts were detected using MS. Top-down MS/MS on the peptide dimer provided amino acid-level resolution regarding interactions and the cross-linking partner for pBpA in the solid state. The results show that ssPL-MS can provide high-resolution information about protein interactions in the lyophilized environment.

  17. A set of external reference controls/probes that enable quality assurance between different microarray platforms.

    PubMed

    Akiyama, Hideo; Ueda, Yoji; Nobumasa, Hitoshi; Ooshima, Hiroyuki; Ishizawa, Yohei; Kitahiro, Koji; Miyagawa, Isao; Watanabe, Kazufumi; Nakamura, Takazumi; Tanaka, Ritsuka; Yamamoto, Nobuko; Nakae, Hiroki; Kawase, Mitsuo; Gemma, Nobuhiro; Sekiguchi, Yuji; Fujibuchi, Wataru; Matoba, Ryo

    2015-03-01

    RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration-response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT-PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT-PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.

  18. Directly labeled DNA probes using fluorescent nucleotides with different length linkers.

    PubMed Central

    Zhu, Z; Chao, J; Yu, H; Waggoner, A S

    1994-01-01

    Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR. Images PMID:8078779

  19. Protein labeling with fluorogenic probes for no-wash live-cell imaging of proteins.

    PubMed

    Hori, Yuichiro; Kikuchi, Kazuya

    2013-08-01

    Protein labeling by using a protein tag and its specific fluorescent probe is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. Recently, fluorogenic probes for protein labeling were developed. When using these probes, a washing step is not required for the removal of free probes from the cells, thus, allowing rapid detection of proteins in living cells with high signal-to-noise ratio. Various chemical principles have been applied in the designing of probes to include a turn-on fluorescence switch that is activated by the protein labeling reaction. In this review, we describe about the design strategy of the probes and the advances in fluorogenic protein labeling systems. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

    PubMed

    Chen, Ye; Gu, Min; Gunning, Peter W; Russell, Sarah M

    2016-09-01

    Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.

  1. Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.

    PubMed

    Fortin, Dale A; Tillo, Shane E; Yang, Guang; Rah, Jong-Cheol; Melander, Joshua B; Bai, Suxia; Soler-Cedeño, Omar; Qin, Maozhen; Zemelman, Boris V; Guo, Caiying; Mao, Tianyi; Zhong, Haining

    2014-12-10

    Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo. Copyright © 2014 the authors 0270-6474/14/3416698-15$15.00/0.

  2. Live Imaging of Endogenous PSD-95 Using ENABLED: A Conditional Strategy to Fluorescently Label Endogenous Proteins

    PubMed Central

    Fortin, Dale A.; Tillo, Shane E.; Yang, Guang; Rah, Jong-Cheol; Melander, Joshua B.; Bai, Suxia; Soler-Cedeño, Omar; Qin, Maozhen; Zemelman, Boris V.; Guo, Caiying

    2014-01-01

    Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo. PMID:25505322

  3. Label-free DNA hybridization detection by various spectroscopy methods using triphenylmethane dyes as a probe

    NASA Astrophysics Data System (ADS)

    Tu, Jiaojiao; Cai, Changqun; Ma, Ying; Luo, Lin; Weng, Chao; Chen, Xiaoming

    2012-12-01

    A new assay is developed for direct detection of DNA hybridization using triphenylmethane dye as a probe. It is based on various spectroscopic methods including resonance light scattering (RLS), circular dichroism (CD), ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy (AFM), six triphenylmethane dyes interact with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated, respectively. The interaction results in amplified resonance light scattering signals and enables the detection of hybridization without the need for labeling DNA. Mechanism investigations have shown that groove binding occurs between dsDNA and these triphenylmethane dyes, which depends on G-C sequences of dsDNA and the molecular volumes of triphenylmethane dyes. Our present approaches display the advantages of simple and fast, accurate and reliable, and the artificial samples were determined with satisfactory results.

  4. Label-free DNA hybridization detection by various spectroscopy methods using triphenylmethane dyes as a probe.

    PubMed

    Tu, Jiaojiao; Cai, Changqun; Ma, Ying; Luo, Lin; Weng, Chao; Chen, Xiaoming

    2012-12-01

    A new assay is developed for direct detection of DNA hybridization using triphenylmethane dye as a probe. It is based on various spectroscopic methods including resonance light scattering (RLS), circular dichroism (CD), ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy (AFM), six triphenylmethane dyes interact with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated, respectively. The interaction results in amplified resonance light scattering signals and enables the detection of hybridization without the need for labeling DNA. Mechanism investigations have shown that groove binding occurs between dsDNA and these triphenylmethane dyes, which depends on G-C sequences of dsDNA and the molecular volumes of triphenylmethane dyes. Our present approaches display the advantages of simple and fast, accurate and reliable, and the artificial samples were determined with satisfactory results. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Cleavable Biotin Probes for Labeling of Biomolecules via the Azide – Alkyne Cycloaddition

    PubMed Central

    Szychowski, Janek; Mahdavi, Alborz; Hodas, Jennifer J. L.; Bagert, John D.; Ngo, John T.; Landgraf, Peter; Dieterich, Daniela C.; Schuman, Erin M.; Tirrell, David A.

    2010-01-01

    The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via the azide – alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na2S2O4, 2% HOCH2CH2SH, 10% HCO2H, 95% CF3CO2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO2H for 0.5 h. A model GFP protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies. PMID:21141861

  6. AZIDE-SPECIFIC LABELLING OF BIOMOLECULES BY STAUDINGER-BERTOZZI LIGATION: PHOSPHINE DERIVATIVES OF FLUORESCENT PROBES SUITABLE FOR SINGLE-MOLECULE FLUORESCENCE SPECTROSCOPY

    PubMed Central

    Chakraborty, Anirban; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2010-01-01

    We describe the synthesis of phosphine derivatives of three fluorescent probes that have brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labelling through use of the Staudinger-Bertozzi ligation and procedures for quantitation of labelling specificity and labelling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labelling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy. PMID:20580957

  7. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    PubMed

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  8. Mobile, Multi-modal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia

    DTIC Science & Technology

    2015-10-01

    eyes and image choroidal vessels/capillaries using CARS intravital microscopy Subtask 3: Measure oxy-hemoglobin levels in PBI test and control eyes...AWARD NUMBER: W81XWH-14-1-0537 TITLE: Mobile, Multi-modal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia...4. TITLE AND SUBTITLE Mobile, Multimodal, Label-Free Imaging Probe Analysis of Choroidal Oximetry and Retinal Hypoxia 5a. CONTRACT NUMBER W81XWH

  9. Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements.

    PubMed

    Rachel, Natalie M; Quaglia, Daniela; Lévesque, Éric; Charette, André B; Pelletier, Joelle N

    2017-08-31

    Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG towards its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, β-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity towards the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling. This article is protected by copyright. All rights reserved. © 2017 The Protein Society.

  10. Using phylogenetic probes for quantification of stable isotope labeling and microbial community analysis

    SciTech Connect

    Brodie, Eoin L; DeSantis, Todd Z; Karaoz, Ulas; Andersen, Gary L

    2014-12-09

    Herein is described methods for a high-sensitivity means to measure the incorporation of stable isotope labeled substrates into RNA following stable isotope probing experiments (SIP). RNA is hybridized to a set of probes such as phylogenetic microarrays and isotope incorporation is quantified such as by secondary ion mass spectrometer imaging (NanoSIMS).

  11. Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

    PubMed Central

    Medon, P P; Lanser, J A; Monckton, P R; Li, P; Symons, R H

    1988-01-01

    Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods. PMID:3053766

  12. Cu(2+)-labeled dansyl compounds as fluorescent and PET probes for imaging apoptosis.

    PubMed

    Han, Junyan; Wang, Xukui; Yu, MeiXiang

    2016-11-15

    Compound DNSTT-Cu(2+), a novel chelate of Cu(2+) with DOTA conjugated to a fluorescent dansyl fragment, is developed for imaging cell apoptosis. Apoptotic U-87MG cells could be selectively visualized by the fluorescence of DNSTT-Cu(2+) from cytoplasm of cells, confirmed by the fluorescence of apoptosis cells co-labeled with Alexa Fluor 568-labeled annexin V, a conventional probe for selectively labeling membranes of apoptosis cells. A radioactive (64)Cu(2)(+) analog, DNSTT-(64)Cu(2+), was easily synthesized, providing a potential PET probe for imaging apoptosis in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Synthesis and evaluation of new thiodigalactoside-based chemical probes to label galectin-3.

    PubMed

    van Scherpenzeel, Monique; Moret, Ed E; Ballell, Lluis; Liskamp, Rob M J; Nilsson, Ulf J; Leffler, Hakon; Pieters, Roland J

    2009-07-06

    New chemical probes were synthesized to label galectin-3. They are based on the high affinity thiodigalactoside ligand. The probes were synthesized with benzophenone or acetophenone moieties as the photolabel for covalent attachment to the protein. Besides labeling the protein, these aromatic photolabels also greatly enhance the affinity of the probes towards galectin-3, due to the interaction of the photolabel with two arginine guanidinium groups of the protein. The linkage between the sugar and the photolabel was varied as an ester, an amide, and a triazole. For the amide and triazole derivatives, a versatile synthetic route towards a symmetrical 3-azido-3-deoxy-thiodigalactoside was developed. The new probes were evaluated for their binding affinity of human galectin-3. They were subsequently tested for their labeling efficiency, as well as specificity in the presence of a protein mixture and a human cancer cell lysate.

  14. Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

    PubMed

    Pan'kov, S V; Chechetkin, V R; Somova, O G; Antonova, O V; Moiseeva, O V; Prokopenko, D V; Yurasov, R A; Gryadunov, D A; Chudinov, A V

    2009-10-01

    Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

  15. Label-free detection of polynucleotide single-base mismatch via pyrene probe excimer emission

    NASA Astrophysics Data System (ADS)

    Tang, Dan; Lu, Ping; Liao, Dongli; Yang, Xiangyu; Zhang, Yujing; Yu, Cong

    2011-02-01

    The pyrene probe and pyrene-labeled oligonucleotides (ODNs) probe are expected to be candidates as fluorescent probe for DNA assay. In particular, label-free detection is a very hot because of its simpleness, speediness and cheapness. Herein, we have investigated the use of a pyrenylakylammonium salt, a novel fluorescent probe for the detection of one single nucleotide polymorphism (SNP) in double stranded DNA. After S1 nuclease digestion, the pyrene probes bind electrostatically to the perfect complement DNA and emit a strong excimer emission. However, treatment of the non-complementary DNA with S1 nuclease caused nucleotide fragments of less than 5 bases, which could not induce excimer emission. By comparing ratio of excimer to monomer fluorescence between normal and mutant DNA after S1 nuclease digestion, One-base mutation in DNA was detected easily. This new method may be applied to the detection of SNP.

  16. FRET-labeled siRNA probes for tracking assembly and disassembly of siRNA nanocomplexes.

    PubMed

    Alabi, Christopher A; Love, Kevin T; Sahay, Gaurav; Stutzman, Tina; Young, Whitney T; Langer, Robert; Anderson, Daniel G

    2012-07-24

    The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability, and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which may be difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (K(D)) and intracellular disassembly half-life for several in-house and proprietary commercial DVs. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing.

  17. Label Transfer Reagents to Probe p38 MAPK Binding Partners

    PubMed Central

    Andrews, Simeon S.; Hill, Zachary B.; Perera, B. Gayani K.; Maly, Dustin J.

    2013-01-01

    Protein kinases are essential enzymes for cellular signalling, and are often regulated by participation in protein complexes. The mitogen-activated protein kinase (MAPK) p38 is involved in multiple pathways, and its regulation depends on its interactions with other signalling proteins. However, the identification of p38 interacting proteins is challenging. For this reason, we have developed label transfer reagents (LTRs) which allow labelling of p38 signalling complexes. These LTRs leverage the potency and selectivity of known p38 inhibitors to place a photo-crosslinker and tag in the vicinity of p38 and its binding partners. Upon UV irradiation, proteins that are in close proximity to p38 are covalently crosslinked, and labelled proteins are detected and/or purified through an orthogonal chemical handle. Here we demonstrate that p38-selective LTRs selectively label a diversity of p38 binding partners, including substrates, activators, and inactivators. Furthermore, these LTRs can be used in immunoprecipitations to provide low-resolution structural information on p38-containing complexes. PMID:23319368

  18. Label transfer reagents to probe p38 MAPK binding partners.

    PubMed

    Andrews, Simeon S; Hill, Zachary B; Perera, B Gayani K; Maly, Dustin J

    2013-01-21

    Protein kinases are essential enzymes for cellular signaling, and are often regulated by participation in protein complexes. The mitogen-activated protein kinase (MAPK) p38 is involved in multiple pathways, and its regulation depends on its interactions with other signaling proteins. However, the identification of p38-interacting proteins is challenging. For this reason, we have developed label transfer reagents (LTRs) that allow labeling of p38 signaling complexes. These LTRs leverage the potency and selectivity of known p38 inhibitors to place a photo-crosslinker and tag in the vicinity of p38 and its binding partners. Upon UV irradiation, proteins that are in close proximity to p38 are covalently crosslinked, and labeled proteins are detected and/or purified with an orthogonal chemical handle. Here we demonstrate that p38-selective LTRs selectively label a diversity of p38 binding partners, including substrates, activators, and inactivators. Furthermore, these LTRs can be used in immunoprecipitations to provide low-resolution structural information on p38-containing complexes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Mediator probe PCR: detection of real-time PCR by label-free probes and a universal fluorogenic reporter.

    PubMed

    Wadle, Simon; Rubenwolf, Stefanie; Lehnert, Michael; Faltin, Bernd; Weidmann, Manfred; Hufert, Frank; Zengerle, Roland; von Stetten, Felix

    2014-01-01

    Mediator probe PCR (MP PCR) is a novel detection format for real-time nucleic acid analysis. Label-free mediator probes (MP) and fluorogenic universal reporter (UR) oligonucleotides are combined to accomplish signal generation. Compared to conventional hydrolysis probe PCRs costs can thus be saved by using the same fluorogenic UR for signal generation in different assays. This tutorial provides a practical guideline to MP and UR design. MP design rules are very similar to those of hydrolysis probes. The major difference is in the replacement of the fluorophore and quencher by one UR-specific sequence tag, the mediator. Further protocols for the setup of reactions, to detect either DNA or RNA targets with clinical diagnostic target detection as models, are explained. Ready to use designs for URs are suggested and guidelines for their de novo design are provided as well, including a protocol for UR signal generation characterization.

  20. Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

    PubMed Central

    Yu, H; Chao, J; Patek, D; Mujumdar, R; Mujumdar, S; Waggoner, A S

    1994-01-01

    Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps. Images PMID:8065939

  1. Thiol- and Biotin-Labeled Probes for Oligonucleotide Quartz Crystal Microbalance Biosensors of Microalga Alexandrium Minutum

    PubMed Central

    Lazerges, Mathieu; Perrot, Hubert; Rabehagasoa, Niriniony; Compère, Chantal

    2012-01-01

    Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency. PMID:25585927

  2. Thiol- and biotin-labeled probes for oligonucleotide quartz crystal microbalance biosensors of microalga alexandrium minutum.

    PubMed

    Lazerges, Mathieu; Perrot, Hubert; Rabehagasoa, Niriniony; Compère, Chantal

    2012-07-04

    Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency.

  3. Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.

    PubMed

    Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin

    2009-06-01

    Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that

  4. Labeled Putrescine as a Probe in Brain Tumors

    NASA Astrophysics Data System (ADS)

    Volkow, Nora; Goldman, Stephen S.; Flamm, Eugene S.; Cravioto, Humberto; Wolf, Alfred P.; Brodie, Jonathan D.

    1983-08-01

    The polyamine metabolism of transplanted N-nitrosomethylurea-derived rat glioma was determined with radiolabeled putrescine used as a marker for malignancy. The uptake of putrescine in vivo was complete within 5 minutes and was specific for tumor tissue. The conversion of putrescine to spermine and other metabolites by the tumor was rapid, in contrast to the case for adjacent normal brain. These results suggest that putrescine labeled with carbon-11 may be used as a positron-emission tomographic tracer for the selective metabolic imaging of brain tumor and may be used in an appropriate model as a marker for tumor growth rate.

  5. Detection of Potato spindle tuber viroid and Other Related Viroids by a DIG Labelled RNA Probe.

    PubMed

    Monger, Wendy A; Jeffries, Colin

    2015-01-01

    Viroids can cause diseases of considerable economic importance; in Europe the main concern is with pospiviroids that may affect the tomato and potato industries. Methods for detection are required that are both sensitive and robust. The detection method described here is a probe hybridization method with a commercially available digoxigenin (DIG) labelled full-length Potato spindle tuber viroid (PSTVd) RNA probe. This method detects PSTVd and all other known pospiviroids.

  6. Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes.

    PubMed

    Withana, Nimali P; Garland, Megan; Verdoes, Martijn; Ofori, Leslie O; Segal, Ehud; Bogyo, Matthew

    2016-01-01

    Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

  7. Biotin-labeled synthetic oligodeoxyribonucleotides: chemical synthesis and uses as hybridization probes.

    PubMed Central

    Chollet, A; Kawashima, E H

    1985-01-01

    Oligodeoxynucleotides have been selectively labeled with biotin at their 5'-termini through an aminoalkylphosphoramide linker arm by an efficient chemical method. The reactions were performed in aqueous solution on unprotected oligonucleotides and were insensitive of the sequence and length of the oligonucleotide. 5'-biotin-labeled oligonucleotides were hybridized to dot, Southern and genomic blots of target plasmid DNA immobilized on nitrocellulose filters. Detection level is about 2 fmole. There is no noticeable disturbance of the strength and selectivity of hybridization of the 5'-biotin-labeled probes in comparison with non-modified DNA. Images PMID:4000941

  8. DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes.

    PubMed

    Rasche, Frank; Lueders, Tillmann; Schloter, Michael; Schaefer, Sabine; Buegger, Franz; Gattinger, Andreas; Hood-Nowotny, Rebecca C; Sessitsch, Angela

    2009-03-01

    A (13)CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of (13)CO2 into (13)C-labeled metabolites by the plant, the most directly active, cultivar specific heterotrophic endophytic bacteria that consume these labeled metabolite scan be identified by DNA stable isotope probing (DNA-SIP).Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries.Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the 'light' fractions of (13)CO2-incubated potato cultivars and of potato cultivars not incubated with (13)CO2. In the 'heavy' fractions of the (13)CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée,respectively, indicating cultivar-dependent distinctions in (13)C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities.The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant-microbe interactions and plant-dependent microbial metabolisms within the endosphere.

  9. Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications.

    PubMed

    Mizukami, Shin; Hori, Yuichiro; Kikuchi, Kazuya

    2014-01-21

    The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In

  10. [Application of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe in cellular magnetic resonance imaging].

    PubMed

    Wen, Ming; Li, Bi-Bo; Ouyang, Yu; Jiang, Ming-Dong; Luo, Yi; Li, Shao-Lin

    2008-10-01

    To prepare the superparamagnetic iron oxide (SPIO)-labeled antisense oligodeoxynucleotide (ASODN) probe and evaluate the application of this probe in cellular magnetic resonance imaging (MRI). We prepared the SPIO-labeled ASODN probe using chemical cross linking method to conjugate SPIO to ASODN, detected its configuration by atomic force microscopy, determined the conjugating rate and biology activation by high performance liquid chromatography, and detected the stability by polyacrylamide gel electrophoresis. After that, we transfected the SK-Br3 oncocytes which had over-expression of the c-erbB2 oncogene by this probes, observed the intracellular iron distribution by optical microscope, measured iron content by atomic absorption spectroscopy, and observed the signal change by MRI. Atomic force microscope showed that the SPIO-labeled ASODN probe was mostly spherical and well-distributed, with a diameter of 25-40 nm and a conjugating rate of 100%. This probe had inhered biological activity and stability. In addition, light microscopy revealed an intracellular uptake of iron oxides in the transfected SK-Br3 oncocyte, and the iron content of the group of transfected SK-Br3 oncocytes was significantly higher than those of other contrast groups (all P < 0.01). MRI showed that transfected SK-Br3 oncocyte had the lowest signal among all other cells (all P < 0.05). We prepared the SPIO-labeled ASODN probe successfully. It can effectively transfect SK-Br3 oncocyte and enter SK-Br3 oncocyte, and thus reduce the signal intension in MRI.

  11. Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

    PubMed

    Uttamapinant, Chayasith; Howe, Jonathan D; Lang, Kathrin; Beránek, Václav; Davis, Lloyd; Mahesh, Mohan; Barry, Nicholas P; Chin, Jason W

    2015-04-15

    Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

  12. Resonant waveguide grating biosensor-enabled label-free and fluorescence detection of cell adhesion

    PubMed Central

    Zaytseva, Natalya; Lynn, Jeffery G.; Wu, Qi; Mudaliar, Deepti J.; Sun, Haiyan; Kuang, Patty Q.; Fang, Ye

    2013-01-01

    Cell adhesion to extracellular matrix (ECM) is fundamental to many distinct aspects of cell biology, and has been an active topic for label-free biosensors. However, little attention has been paid to study the impact of receptor signaling on the cell adhesion process. We here report the development of resonant waveguide grating biosensor-enabled label-free and fluorescent approaches, and their use for investigating the adhesion of an engineered HEK-293 cell line stably expressing green fluorescent protein (GFP) tagged β2-adrenergic receptor (β2-AR) onto distinct surfaces under both ambient and physiological conditions. Results showed that cell adhesion is sensitive to both temperature and ECM coating, and distinct mechanisms govern the cell adhesion process under different conditions. The β2-AR agonists, but not its antagonists or partial agonists, were found to be capable of triggering signaling during the adhesion process, leading to an increase in the adhesion of the engineered cells onto fibronectin-coated biosensor surfaces. These results suggest that the dual approach presented is useful to investigate the mechanism of cell adhesion, and to identify drug molecules and receptor signaling that interfere with cell adhesion. PMID:24319319

  13. Preparation and chromatographic use of 5'-fluorescent-labelled DNA probes.

    PubMed

    Tous, G; Fausnaugh, J; Vieira, P; Stein, S

    1988-07-01

    A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'-terminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversed-phase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

  14. Real-time, haptics-enabled simulator for probing ex vivo liver tissue.

    PubMed

    Lister, Kevin; Gao, Zhan; Desai, Jaydev P

    2009-01-01

    The advent of complex surgical procedures has driven the need for realistic surgical training simulators. Comprehensive simulators that provide realistic visual and haptic feedback during surgical tasks are required to familiarize surgeons with the procedures they are to perform. Complex organ geometry inherent to biological tissues and intricate material properties drive the need for finite element methods to assure accurate tissue displacement and force calculations. Advances in real-time finite element methods have not reached the state where they are applicable to soft tissue surgical simulation. Therefore a real-time, haptics-enabled simulator for probing of soft tissue has been developed which utilizes preprocessed finite element data (derived from accurate constitutive model of the soft-tissue obtained from carefully collected experimental data) to accurately replicate the probing task in real-time.

  15. Convergent synthesis and evaluation of (18)F-labeled azulenic COX2 probes for cancer imaging.

    PubMed

    Nolting, Donald D; Nickels, Michael; Tantawy, Mohammed N; Yu, James Y H; Xie, Jingping; Peterson, Todd E; Crews, Brenda C; Marnett, Larry; Gore, John C; Pham, Wellington

    2012-01-01

    The overall objectives of this research are to (i) develop azulene-based positron emission tomography (PET) probes and (ii) image COX2 as a potential biomarker of breast cancer. Several lines of research have demonstrated that COX2 is overexpressed in breast cancer and that its presence correlates with poor prognoses. While other studies have reported that COX2 inhibition can be modulated and used beneficially as a chemopreventive strategy in cancer, no viable mechanism for achieving that approach has yet been developed. This shortfall could be circumvented through in vivo imaging of COX2 activity, particularly using sensitive imaging techniques such as PET. Toward that goal, our laboratory focuses on the development of novel (18)F-labled COX2 probes. We began the synthesis of the probes by transforming tropolone into a lactone, which was subjected to an [8 + 2] cycloaddition reaction to yield 2-methylazulene as the core ring of the probe. After exploring numerous synthetic routes, the final target molecule and precursor PET compounds were prepared successfully using convergent synthesis. Conventional (18)F labeling methods caused precursor decomposition, which prompted us to hypothesize that the acidic protons of the methylene moiety between the azulene and thiazole rings were readily abstracted by a strong base such as potassium carbonate. Ultimately, this caused the precursors to disintegrate. This observation was supported after successfully using an (18)F labeling strategy that employed a much milder phosphate buffer. The (18)F-labeled COX2 probe was tested in a breast cancer xenograft mouse model. The data obtained via successive whole-body PET/CT scans indicated probe accumulation and retention in the tumor. Overall, the probe was stable in vivo and no defluorination was observed. A biodistribution study and Western blot analysis corroborate with the imaging data. In conclusion, this novel COX2 PET probe was shown to be a promising agent for cancer imaging

  16. Convergent synthesis and evaluation of 18F-labeled azulenic COX2 probes for cancer imaging

    PubMed Central

    Nolting, Donald D.; Nickels, Michael; Tantawy, Mohammed N.; Yu, James Y. H.; Xie, Jingping; Peterson, Todd E.; Crews, Brenda C.; Marnett, Larry; Gore, John C.; Pham, Wellington

    2013-01-01

    The overall objectives of this research are to (i) develop azulene-based positron emission tomography (PET) probes and (ii) image COX2 as a potential biomarker of breast cancer. Several lines of research have demonstrated that COX2 is overexpressed in breast cancer and that its presence correlates with poor prognoses. While other studies have reported that COX2 inhibition can be modulated and used beneficially as a chemopreventive strategy in cancer, no viable mechanism for achieving that approach has yet been developed. This shortfall could be circumvented through in vivo imaging of COX2 activity, particularly using sensitive imaging techniques such as PET. Toward that goal, our laboratory focuses on the development of novel 18F-labled COX2 probes. We began the synthesis of the probes by transforming tropolone into a lactone, which was subjected to an [8 + 2] cycloaddition reaction to yield 2-methylazulene as the core ring of the probe. After exploring numerous synthetic routes, the final target molecule and precursor PET compounds were prepared successfully using convergent synthesis. Conventional 18F labeling methods caused precursor decomposition, which prompted us to hypothesize that the acidic protons of the methylene moiety between the azulene and thiazole rings were readily abstracted by a strong base such as potassium carbonate. Ultimately, this caused the precursors to disintegrate. This observation was supported after successfully using an 18F labeling strategy that employed a much milder phosphate buffer. The 18F-labeled COX2 probe was tested in a breast cancer xenograft mouse model. The data obtained via successive whole-body PET/CT scans indicated probe accumulation and retention in the tumor. Overall, the probe was stable in vivo and no defluorination was observed. A biodistribution study and Western blot analysis corroborate with the imaging data. In conclusion, this novel COX2 PET probe was shown to be a promising agent for cancer imaging and

  17. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  18. Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy.

    PubMed Central

    Murakami, A; Nakaura, M; Nakatsuji, Y; Nagahara, S; Tran-Cong, Q; Makino, K

    1991-01-01

    Fluorescein-labeled oligonucleotides as DNA-probes were synthesized and used to monitor hybrid formation, namely to detect DNA or oligonucleotide sequence in solution. The introduction of fluorescein to oligonucleotides was carried out by oxidation of a hydrogen phosphonate linkage with ethylenediamine or hexamethylenediamine as a tether and by a subsequent labeling of the primary amine moiety by FITC. Fluorescence anisotropy, r, was adopted as an index to monitor the behavior of F-probe in solution. An increase in the anisotropy was observed upon an increase in the chain-length of F-probe. When F-Probe formed a hybrid with its complementary oligonucleotide in solution, the r value increased compared to that of F-Probe itself. These observations clearly indicate that measurements of r in solution will readily lead to the monitoring of the presence of a hybrid in solution. Consequently, it is promising to detect a certain nucleic acid sequence in solution using fluorescent-labeled oligonucleotides. PMID:1870966

  19. In situ hybridization with labeled probes: assessment of african Swine Fever virus in formalin-fixed paraffin-embedded tissues.

    PubMed

    Ballester, Maria; Rodríguez, Fernando

    2015-01-01

    In situ hybridization (ISH) has become a very valuable molecular diagnostic tool to detect specific DNA or RNA sequences in biological samples through the use of complementary DNA- or RNA-labeled probes. Here, we describe an optimized in situ hybridization protocol to detect African swine fever virus (ASFV) DNA in formalin-fixed, paraffin-embedded tissues using digoxigenin-labeled probes.

  20. (19)F labelled glycosaminoglycan probes for solution NMR and non-linear (CARS) microscopy.

    PubMed

    Lima, Marcelo A; Cavalheiro, Renan P; M Viana, Gustavo; Meneghetti, Maria C Z; Rudd, Timothy R; Skidmore, Mark A; Powell, Andrew K; Yates, Edwin A

    2016-08-15

    Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of (19)F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing (19)F NMR spectroscopy is followed. Furthermore, the ability of (19)F labelled GAGs to be imaged using CARS microscopy is demonstrated. (19)F labelled GAGs enable both (19)F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.

  1. Combined Labelled and Label-free SERS Probes for Triplex Three-dimensional Cellular Imaging

    NASA Astrophysics Data System (ADS)

    Chen, Yong; Bai, Xiangru; Su, Le; Du, Zhanwei; Shen, Aiguo; Materny, Arnulf; Hu, Jiming

    2016-01-01

    Cells are complex chemical systems, where the molecular composition at different cellular locations and specific intracellular chemical interactions determine the biological function. An in-situ nondestructive characterization of the complicated chemical processes (like e.g. apoptosis) is the goal of our study. Here, we present the results of simultaneous and three-dimensional imaging of double organelles (nucleus and membrane) in single HeLa cells by means of either labelled or label-free surface-enhanced Raman spectroscopy (SERS). This combination of imaging with and without labels is not possible when using fluorescence microscopy. The SERS technique is used for a stereoscopic description of the intrinsic chemical nature of nuclei and the precise localization of folate (FA) and luteinizing hormone-releasing hormone (LHRH) on the membrane under highly confocal conditions. We also report on the time-dependent changes of cell nuclei as well as membrane receptor proteins during apoptosis analyzed by statistical multivariate methods. The multiplex three-dimensional SERS imaging technique allows for both temporal (real time) and spatial (multiple organelles and molecules in three-dimensional space) live-cell imaging and therefore provides a new and attractive 2D/3D tracing method in biomedicine on subcellular level.

  2. Wireless Displacement Sensing Enabled by Metamaterial Probes for Remote Structural Health Monitoring

    PubMed Central

    Ozbey, Burak; Unal, Emre; Ertugrul, Hatice; Kurc, Ozgur; Puttlitz, Christian M.; Erturk, Vakur B.; Altintas, Ayhan; Demir, Hilmi Volkan

    2014-01-01

    We propose and demonstrate a wireless, passive, metamaterial-based sensor that allows for remotely monitoring submicron displacements over millimeter ranges. The sensor comprises a probe made of multiple nested split ring resonators (NSRRs) in a double-comb architecture coupled to an external antenna in its near-field. In operation, the sensor detects displacement of a structure onto which the NSRR probe is attached by telemetrically tracking the shift in its local frequency peaks. Owing to the NSRR's near-field excitation response, which is highly sensitive to the displaced comb-teeth over a wide separation, the wireless sensing system exhibits a relatively high resolution (<1 μm) and a large dynamic range (over 7 mm), along with high levels of linearity (R2 > 0.99 over 5 mm) and sensitivity (>12.7 MHz/mm in the 1–3 mm range). The sensor is also shown to be working in the linear region in a scenario where it is attached to a standard structural reinforcing bar. Because of its wireless and passive nature, together with its low cost, the proposed system enabled by the metamaterial probes holds a great promise for applications in remote structural health monitoring. PMID:24445416

  3. DNA quantification via ICP-MS using lanthanide-labeled probes and ligation-mediated amplification.

    PubMed

    Brückner, Kathrin; Schwarz, Kathleen; Beck, Sebastian; Linscheid, Michael W

    2014-01-07

    The combination of lanthanide-tagged oligonucleotide probes with inductively coupled plasma mass spectrometry (ICP-MS) as the detection technique is a novel labeling and analysis strategy for heterogeneous nucleic acid quantification assays. We describe a hybridization assay based on biotin-streptavidin affinity using lanthanide-labeled reporter probes and biotinylated capture probes. For the basic sandwich type assay, performed in streptavidin-coated microtitration wells, the limit of detection (LOD) was 7.2 fmol of DNA target, corresponding to a final concentration of 6 pM terbium-labeled probes detectable by ICP-MS after elution from the solid support. To improve the sensitivity and sequence specificity of the approach, it was combined with established molecular biological techniques, i.e., elution with a restriction endonuclease and signal and target amplification by the ligase detection reaction (LDR) and ligase chain reaction (LCR), respectively. Initial experiments showed that the enzymes facilitated the discrimination of single-base mismatches within the recognition or ligation site. Furthermore, LCR as a target amplification step resulted in a 6000-fold increase of sensitivity, and finally an LOD of 2.6 amol was achieved with an artificial double-stranded DNA target.

  4. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

    PubMed

    Bohl, Cornelius; Pomorski, Adam; Seemann, Susanne; Knospe, Anne-Marie; Zheng, Chaonan; Krężel, Artur; Rolfs, Arndt; Lukas, Jan

    2017-08-15

    Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC-tag) detection. We tested the efficiency of halogen substituted biarsenical probes to interact with C-terminally TC-tagged GALA peptide at pH 4.5 in vitro and identified F2FlAsH-EDT2 as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS-7 cells transiently expressing TC-tagged GALA. The investigated fluorescence-based LCI technology of TC-tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.-Bohl, C., Pomorski, A., Seemann, S., Knospe, A.-M., Zheng, C., Krężel, A., Rolfs, A., Lukas, J. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A. © FASEB.

  5. Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

    NASA Astrophysics Data System (ADS)

    Koh, Eunjung; Song, Ha Jeong; Kwon, Na Young; Kim, Gi Won; Lee, Kwang Ho; Jo, Soyeon; Park, Sujin; Park, Jihyun; Park, Eun Kyeong; Hwang, Seung Yong

    2017-06-01

    Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).

  6. 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.

  7. Microfluidic technology platforms for synthesizing, labeling and measuring the kinetics of transport and biochemical reactions for developing molecular imaging probes

    SciTech Connect

    Phelps, Michael E.

    2009-09-01

    Radiotracer techniques are used in environmental sciences, geology, biology and medicine. Radiotracers with Positron Emission Tomography (PET) provided biological examinations of ~3 million patients 2008. Despite the success of positron labeled tracers in many sciences, there is limited access in an affordable and convenient manner to develop and use new tracers. Integrated microfluidic chips are a new technology well matched to the concentrations of tracers. Our goal is to develop microfluidic chips and new synthesis approaches to enable wide dissemination of diverse types of tracers at low cost, and to produce new generations of radiochemists for which there are many unfilled jobs. The program objectives are to: 1. Develop an integrated microfluidic platform technology for synthesizing and 18F-labeling diverse arrays of different classes of molecules. 2. Incorporate microfluidic chips into small PC controlled devices (“Synthesizer”) with a platform interfaced to PC for electronic and fluid input/out control. 3. Establish a de-centralized model with Synthesizers for discovering and producing molecular imaging probes, only requiring delivery of inexpensive [18F]fluoride ion from commercial PET radiopharmacies vs the centralized approach of cyclotron facilities synthesizing and shipping a few different types of 18F-probes. 4. Develop a position sensitive avalanche photo diode (PSAPD) camera for beta particles embedded in a microfluidic chip for imaging and measuring transport and biochemical reaction rates to valid new 18F-labeled probes in an array of cell cultures. These objectives are met within a research and educational program integrating radio-chemistry, synthetic chemistry, biochemistry, engineering and biology in the Crump Institute for Molecular Imaging. The Radiochemistry Training Program exposes PhD and post doctoral students to molecular imaging in vitro in cells and microorganisms in microfluidic chips and in vivo with PET, from new technologies

  8. Microbial food web mapping: linking carbon cycling and community structure in soils through pyrosequencing enabled stable isotope probing

    SciTech Connect

    Buckley, Daniel H.

    2015-03-15

    Soil represents a massive reservoir of active carbon and climate models vary dramatically in predicting how this carbon will respond to climate change over the coming century. A major cause of uncertainty is that we still have a very limited understand the microorganisms that dominate the soil carbon cycle. The vast majority of soil microbes cannot be cultivated in the laboratory and the diversity of organisms and enzymes that participate in the carbon cycle is staggeringly complex. We have developed a new toolbox for exploring the carbon cycle and the metabolic and ecological characteristics of uncultivated microorganisms. The high-resolution nucleic acid stable isotope probing approach that we have developed makes it possible to characterize microbial carbon cycling dynamics in soil. The approach allows us to track multiple 13C-labeled substrates into thousands of microbial taxa over time. Using this approach we have discovered several major lineages of uncultivated microorganisms that participate in cellulose metabolism and are found widely in soils (including Verrucomicrobia and Chloroflexi, which have not previously been implicated as major players in the soil carbon cycle). Furthermore, isotopic labelling of nucleic acids enables community genomics and permits genome fragment binning for a majority of these cellulolytic microorganisms allowing us to explore the metabolic underpinnings of cellulose degradation. This approach has allowed us to describe unexpected dynamics of carbon metabolism with different microbial taxa exhibiting characteristic patterns of carbon substrate incorporation, indicative of distinct ecological strategies. The data we describe allows us to characterize the activity of novel microorganisms as they occur in the environment and these data provide a basis for understanding how the physiological traits of discrete microorganisms sum to govern the complex responses of the soil carbon cycle.

  9. Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

    PubMed Central

    Wu, Jun; Shao, Fangwei; Zhang, Li-Feng

    2014-01-01

    Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts. PMID:25226542

  10. Labeling of target mRNAs using a photo-reactive microRNA probe.

    PubMed

    Nakamoto, Kosuke; Minami, Koichiro; Akao, Yukihiro; Ueno, Yoshihito

    2016-05-10

    To identify target mRNAs of an miRNA, we synthesized photo-reactive miRNA probes, which contained a photo-reactive nucleoside analog, 1-O-[4-(3-trifluoromethyl-3H-diazirine-3-yl)]benzyl-β-d-ribofuranose, in the middle of the strand. The photo-reactive miRNA-145 probe was found to specifically label the target mRNAs, FSCN1 and KLF4, by UV-A irradiation in human colon cancer DLD-1 cells.

  11. Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

    PubMed Central

    Sotoma, Shingo; Iimura, Jun; Igarashi, Ryuji; Hirosawa, Koichiro M.; Ohnishi, Hidenori; Mizukami, Shin; Kikuchi, Kazuya; Fujiwara, Takahiro K.; Shirakawa, Masahiro; Tochio, Hidehito

    2016-01-01

    The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface. PMID:28335184

  12. Ultrasensitive and label-free molecular level detection enabled by light phase control in magnetoplasmonic nanoantennas

    PubMed Central

    Maccaferri, Nicolò; Gregorczyk, Keith; de Oliveira, Thales V. A. G.; Kataja, Mikko; van Dijken, Sebastiaan; Pirzadeh, Zhaleh; Dmitriev, Alexandre; Åkerman, Johan; Knez, Mato; Vavassori, Paolo

    2015-01-01

    Systems allowing label-free molecular detection are expected to have enormous impact on biochemical sciences. Research focuses on materials and technologies based on exploiting localized surface plasmon resonances in metallic nanostructures. The reason for this focused attention is their suitability for single molecule sensing, arising from intrinsically nanoscopic sensing volume, and the high sensitivity to the local environment. Here we propose an alternative route, which enables radically improved sensitivity compared torecently reported plasmon-based sensors. Such high sensitivity is achieved by exploiting the control of the phase of light in magnetoplasmonic nanoantennas. We demonstrate a manifold improvement of refractometric sensing figure-of-merit. Most remarkably, we show a raw surface sensitivity (i.e., without applying fitting procedures) of two orders of magnitude higher than the current values reported for nanoplasmonic sensors. Such sensitivity corresponds to a mass of ~0.8 ag per nanoantenna of polyamide-6.6 (n=1.51), which is representative for a large variety of polymers, peptides and proteins. PMID:25639190

  13. Isoindoline nitroxide-labeled porphyrins as potential fluorescence-suppressed spin probes.

    PubMed

    Liu, F; Zou, T J; Tan, Z L; Chen, S; Wu, Z H; Yan, G P; Zhang, Q; Liang, S C; Yang, J

    2017-02-07

    A series of isoindoline nitroxide-labeled porphyrins were synthesized by the reaction of 5-phenyldipyrromethane and 5-(4'-carboethoxy-methyleneoxyphenyl)dipyrromethane with 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO) using the Lindsey method. The corresponding water-soluble spin-labeled porphyrins were also prepared. Subsequently, these compounds were characterized and their in vitro properties were evaluated. The electrochemical assay demonstrated that these isoindoline nitroxide-labeled porphyrins had similar electrochemical and redox properties to 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). The electron paramagnetic resonance test showed that these porphyrins exhibited hyperfine splittings and characteristic spectra of CTMIO with typical nitroxide g-values and nitrogen isotropic hyperfine coupling constants. The in vitro cytotoxicity assay indicated that these porphyrins possessed low cytotoxicity to human renal tubular epithelial 293T cells (normal cells) and human hepatoma HepG2 cells (tumor cells). Fluorescence spectroscopy revealed that free base isoindoline nitroxide-labeled porphyrins exhibited fluorescence suppression characteristic of nitroxide-fluorophore systems. In vitro fluorescene imaging demonstrated that the reduced isoindoline nitroxide-labeled porphyrins eliminated fluorescence suppression and displayed strong red fluorescence imaging in HepG2 cells. Thus these isoindoline nitroxide-labeled porphyrins may be considered potentially as biological spin probes for fluorescence imaging and EPR spectroscopy.

  14. Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns

    PubMed Central

    Spencer, J.; Schwarzacher, W.

    2016-01-01

    ABSTRACT In order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, with S. aureus being immobilized to a greater extent than E. coli. Finally, low numbers of magnetically labeled E. coli bacteria (<100 CFU per ml) were immobilized with 100% efficiency and concentrated 7-fold within 15 min. Therefore, our study provides a novel protocol for rapid and highly efficient magnetic labeling, capture, and concentration of both Gram-positive and Gram-negative bacteria. IMPORTANCE Antimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved

  15. Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns.

    PubMed

    Correia Carreira, S; Spencer, J; Schwarzacher, W; Seddon, A M

    2016-06-15

    In order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, with S. aureus being immobilized to a greater extent than E. coli Finally, low numbers of magnetically labeled E. coli bacteria (<100 CFU per ml) were immobilized with 100% efficiency and concentrated 7-fold within 15 min. Therefore, our study provides a novel protocol for rapid and highly efficient magnetic labeling, capture, and concentration of both Gram-positive and Gram-negative bacteria. Antimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved within 15 min. This

  16. Could Nano-Structured Materials Enable the Improved Pressure Vessels for Deep Atmospheric Probes?

    NASA Technical Reports Server (NTRS)

    Srivastava, D.; Fuentes, A.; Bienstock, B.; Arnold, J. O.

    2005-01-01

    A viewgraph presentation on the use of Nano-Structured Materials to enable pressure vessel structures for deep atmospheric probes is shown. The topics include: 1) High Temperature/Pressure in Key X-Environments; 2) The Case for Use of Nano-Structured Materials Pressure Vessel Design; 3) Carbon based Nanomaterials; 4) Nanotube production & purification; 5) Nanomechanics of Carbon Nanotubes; 6) CNT-composites: Example (Polymer); 7) Effect of Loading sequence on Composite with 8% by volume; 8) Models for Particulate Reinforced Composites; 9) Fullerene/Ti Composite for High Strength-Insulating Layer; 10) Fullerene/Epoxy Composite for High Strength-Insulating Layer; 11) Models for Continuous Fiber Reinforced Composites; 12) Tensile Strength for Discontinuous Fiber Composite; 13) Ti + SWNT Composites: Thermal/Mechanical; 14) Ti + SWNT Composites: Tensile Strength; and 15) Nano-structured Shell for Pressure Vessels.

  17. Combining Metabolic ¹⁵N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

    PubMed

    Thomas, Martin; Huck, Nicola; Hoehenwarter, Wolfgang; Conrath, Uwe; Beckers, Gerold J M

    2015-01-01

    that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope ((15)N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while (15)N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.

  18. A Nuclear Receptor Ligand-based Probe Enables Temporal Control of C. elegans Development

    PubMed Central

    Judkins, Joshua C.; Mahanti, Parag; Hoffman, Jacob; Yim, Isaiah; Antebi, Adam; Schroeder, Frank C.

    2014-01-01

    C. elegans development and lifespan are controlled by the nuclear hormone receptor DAF-12, an important model for vertebrate vitamin D and liver-X receptors. Similar to its mammalian homologs, DAF-12 function is regulated by bile acid-like steroidal ligands, the dafachronic acids; however, tools for investigating their biosynthesis and function in vivo are lacking. We report a flexible synthesis for DAF-12 ligands and masked ligand derivatives that enable precise temporal control of DAF-12 function. For ligand masking, we introduce photocleavable amides of 5-methoxy-N-methyl-2-nitroaniline (MMNA). MMNA-masked ligands are bioavailable and after incorporation into the worm can be used to trigger expression of DAF-12 target genes and initiate development from dauer larvae to adults by brief, innocuous UV-irradiation. In-vivo release of DAF-12 ligands and other small-molecule signals using MMNA-based probes will enable functional studies with precise spatial and temporal resolution. PMID:24453122

  19. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. [Visualization and Functional Regulation of Live Cell Proteins Based on Labeling Probe Design].

    PubMed

    Mizukami, Shin; Kikuchi, Kazuya

    2016-01-01

      There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant β-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various β-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the β-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.

  1. Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells.

    PubMed

    Nakanishi, J; Nakajima, T; Sato, M; Ozawa, T; Tohda, K; Umezawa, Y

    2001-07-01

    We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

  2. Fluorescent labelling of in situ hybridisation probes through the copper-catalysed azide-alkyne cycloaddition reaction.

    PubMed

    Hesse, Susann; Manetto, Antonio; Cassinelli, Valentina; Fuchs, Jörg; Ma, Lu; Raddaoui, Nada; Houben, Andreas

    2016-09-01

    In situ hybridisation is a powerful tool to investigate the genome and chromosome architecture. Nick translation (NT) is widely used to label DNA probes for fluorescence in situ hybridisation (FISH). However, NT is limited to the use of long double-stranded DNA and does not allow the labelling of single-stranded and short DNA, e.g. oligonucleotides. An alternative technique is the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC), at which azide and alkyne functional groups react in a multistep process catalysed by copper(I) ions to give 1,4-distributed 1,2,3-triazoles at a high yield (also called 'click reaction'). We successfully applied this technique to label short single-stranded DNA probes as well as long PCR-derived double-stranded probes and tested them by FISH on plant chromosomes and nuclei. The hybridisation efficiency of differently labelled probes was compared to those obtained by conventional labelling techniques. We show that copper(I)-catalysed azide-alkyne cycloaddition-labelled probes are reliable tools to detect different types of repetitive sequences on chromosomes opening new promising routes for the detection of single copy gene. Moreover, a combination of FISH using such probes with other techniques, e.g. immunohistochemistry (IHC) and cell proliferation assays using 5-ethynyl-deoxyuridine, is herein shown to be easily feasible.

  3. Spin-labeled psoralen probes for the study of DNA dynamics

    SciTech Connect

    Spielmann, H.P.; Chi, D.Y.; Hunt, N.G.

    1995-11-14

    Six nitroxide spin-labeled psoralen derivatives have been synthesized and evaluated as probes for structural and dynamic studies. Sequence specific photoaddition of these derivatives to DNA oligonucleotides resulted in site-specifically cross-linked and spin-labeled oligomers. Comparison of the general line shape features of the observed electron paramagnetic resonance (EPR) spectra of several duplexes ranging in size from 8 to 46 base pairs with simulated EPR spectra indicate that the nitroxide spin-labeled probe reports the global tumbling motion of the oligomers. While there is no apparent large amplitude motion of the psoralen other than the overall tumbling of DNA on the time scales investigated, there are no indications of bending and other residual motions. The (A)BC excinuclease DNA repair system detects structural or dynamic features of the DNA that distinguish between damaged and undamaged DNA and are independent of the intrinsic structure of the lesion. NMR studies have shown that psoralen-cross-linked DNA has altered backbone dynamics and conformational populations in the immediate vicinity of the adduct. We suggested that the signal for recognition of a lesion to be repaired is in the sugar-phosphate backbone and not in the damaged base(s). 71 refs., 11 figs., 1 tab.

  4. 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.

  5. Carbon-13 Labeling Used to Probe Cure and Degradation Reactions of High- Temperature Polymers

    NASA Technical Reports Server (NTRS)

    Meador, Mary Ann B.; Johnston, J. Christopher

    1998-01-01

    High-temperature, crosslinked polyimides are typically insoluble, intractible materials. Consequently, in these systems it has been difficult to follow high-temperature curing or long-term degradation reactions on a molecular level. Selective labeling of the polymers with carbon-13, coupled with solid nuclear magnetic resonance spectrometry (NMR), enables these reactions to be followed. We successfully employed this technique to provide insight into both curing and degradation reactions of PMR-15, a polymer matrix resin used extensively in aircraft engine applications.

  6. Probing metabolic processes of intact soil microbial communities using position-specific 13C-labeled glucose

    NASA Astrophysics Data System (ADS)

    Fairbanks, D. E.; Hungate, B. A.; KOCH, G. W.; Schwartz, E.; Dijkstra, P.

    2012-12-01

    Soils represent one of the largest carbon pools in the terrestrial biosphere and fluxes into or out of this pool may feedback to current climate change. Understanding the mechanisms behind microbial processes regulating C cycling, microbial turnover, and soil organic matter stabilization is hindered by our lack of understanding of the details of microbial physiology in soils. Position-specific 13C labeled metabolic tracers are proposed as a new way to probe microbial community energy production, biosynthesis, C use efficiency (the proportion of substrate incorporated into microbial biomass), and enables the determination of C fluxes through the various C metabolic pathways. We determined the 13CO2 production from microbial communities within a one hour time frame by adding six isotopomers (1-13C, 2-13C, 3-13C, 4-13C, 5-13C, 6-13C) of glucose in parallel incubations using a young volcanic soil (Pinyon-juniper wood, near Sunset Crater, Flagstaff, Arizona). We compared the measured rates of position-specific 13CO2 production with modeled results based on glucose (1-13C and U-13C) and pyruvate (1-13C and 2,3-13C) incubations. These labeling and modeling techniques may improve our ability to analyze the biochemistry and ecophysiology of intact soil microbial communities.

  7. Synthesis of amino-group functionalized superparamagnetic iron oxide nanoparticles and applications as biomedical labeling probes

    NASA Astrophysics Data System (ADS)

    Ma, Ming; Zhan, Yanqiang; Shen, Yaqi; Xia, Xing; Zhang, Suming; Liu, Zuli

    2011-08-01

    Superparamagnetic iron oxide (SPIO) nanoparticles were synthesized by coprecipitation technique and further functionalized with amino-group to obtain amino-group functionalized (amino-SPIO) nanoparticles. The X-ray diffraction results reveal the structure of amino-SPIO nanoparticles, from which the average iron core diameter is approximately 10 nm by calculation; while Zetasizer reveals their hydrodynamic diameter are mainly distributed in the range of 40-60 nm. These nanoparticles can be taken up by liver tissue, resulting in dramatically darkening of liver tissue under T2-magnetic resonance imaging (MRI). The spin-spin relaxivity coefficient of these nanoparticles is 179.20 mM-1 s-1 in a 1.5 T magnetic resonance system. In addition, amino-SPIO nanoparticles were conjugated to Tat (FITC) peptide and incubated with neural stem cells in vitro, the authors can detect the positive-labeling (labeled) neural stem cells showing green fluorescence, which indicates Tat (FITC) peptide-derivated amino-SPIO nanoparticles are able to enter cells. Furthermore, it was also find significant negative T2 contrast enhancement when compared with the non-nanoparticles-labeled neural stem cells in T2-weighted MRI. The amino-SPIO nanoparticles show promising potential as a new type of labeling probes, which can be used in magnetic resonance-enhanced imaging and fluorescence diagnosis.

  8. Development of Label-Free Bioaffinity Sensor Using a Lumped-Constant Microwave Resonator Probe

    NASA Astrophysics Data System (ADS)

    Okazaki, Noriaki; Nishino, Taito; Chikyow, Toyohiro; Cho, Yasuo

    2011-01-01

    A novel label-free bioaffinity sensor using a lumped-constant microwave resonator was developed. A gold probe with a spherical tip immobilizing anti-chicken egg albumin antibody or anti-human albumin antibody on its surface was connected to the resonator and inserted into a flow cell to monitor the target antigen binding. The sensor showed specific sensitivity against its target antigen evidenced by the change of resonance frequency after the antigen injection. The large frequency shift observed during the injection of antigen solution was ascribed to the change of solution dielectric constant which showed a systematic increase with antigen concentration.

  9. Intra-albumin migration of bound fatty acid probed by spin label ESR

    SciTech Connect

    Gurachevsky, Andrey . E-mail: a.gurachevsky@medinnovation.de; Shimanovitch, Ekaterina; Gurachevskaya, Tatjana; Muravsky, Vladimir

    2007-09-07

    Conventional ESR spectra of 16-doxyl-stearic acid bound to bovine and human serum albumin were recorded at different temperatures in order to investigate the status of spin-labeled fatty acid in the interior of the protein globule. A computer spectrum simulation of measured spectra, performed by non-linear least-squares fits, clearly showed two components corresponding to strongly and weakly immobilized fatty acid molecules. The two-component model was verified on spectra measured at different pH. Thermodynamic parameters of the spin probe exchange between two spin probe states were analyzed. It was concluded that at physiological conditions, fatty acid molecules permanently migrate in the globule interior between the specific binding sites and a space among albumin domains.

  10. Synthesis, spectroscopic properties, and biological applications of eight novel chlorinated fluorescent proteins-labeling probes.

    PubMed

    Wu, Xianglong; Tian, Min; Fan, Wutu; Pan, Yalei; Zhai, Yuankun; Niu, Yinbo; Li, Chenrui; Lu, Tingli; Mei, Qibing

    2014-05-01

    Eight novel chlorinated fluorescent proteins-labeling probes with a linker and reactive group were prepared in 7 steps by the reaction of chlorinated resorcinols with 3, 6-dichloro-4-carboxyphthalic anhydride in the presence of methanesulfonic acid. Structures of target compounds and intermediates were determined via IR, MS, (1)H NMR and element analysis. The spectral properties of the chlorinated fluoresceins were studied. These fluorescent probes showed absorbance peaks at 508-536 nm and fluorescence peaks at 524-550 nm. It was found that they have absorption and emission maxima at long wavelengths and high fluorescence quantum yields. Emission spectra of chlorinated fluoresceins shifted towards long wavelength with increase in chlorine. The probes were used for fluorescence imaging of cells in order to investigate whether they can conjugate to cells. The fluorescence imaging of living cells showed that they were localized in cell nucleus. However, they were localized in cytosol of chemically fixed cells. These probes will be useful reagents for the preparation of stable fluorescent conjugates.

  11. Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

    NASA Astrophysics Data System (ADS)

    He, Hui; Niu, Cheng-Gang; Zeng, Guang-Ming; Ruan, Min; Qin, Pin-Zhu; Liu, Jing

    2011-11-01

    The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA) 3(5-NH 2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA) 3(5-NH 2-phen) on the 5'-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10 -10 mol L -1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.

  12. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    PubMed Central

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-01-01

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

  13. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    PubMed

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  14. 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.

  15. A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

    PubMed

    Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

    2014-03-26

    Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

  16. Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis.

    PubMed

    Hur, Deokhwe; Kim, Myoung Sug; Song, Minsik; Jung, Jinwook; Park, Heekyung

    2015-01-01

    Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization. Here, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe. The PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.

  17. Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe.

    PubMed

    Carpy, Alejandro; Patel, Avinash; Tay, Ye Dee; Hagan, Iain M; Macek, Boris

    2015-01-01

    Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, (13)C(6) (15)N(4)-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Global analysis of fluorescence decays to probe the internal dynamics of fluorescently labeled macromolecules.

    PubMed

    Duhamel, Jean

    2014-03-11

    The aim of this review is to introduce the reader first to the mathematical complexity associated with the analysis of fluorescence decays acquired with solutions of macromolecules labeled with a fluorophore and its quencher that are capable of interacting with each other via photophysical processes within the macromolecular volume, second to the experimental and mathematical approaches that have been proposed over the years to handle this mathematical complexity, and third to the information that one can expect to retrieve with respect to the internal dynamics of such fluorescently labeled macromolecules. In my view, the ideal fluorophore-quencher pair to use in studying the internal dynamics of fluorescently labeled macromolecules would involve a long-lived fluorophore, a fluorophore and a quencher that do not undergo energy migration, and a photophysical process that results in a change in fluorophore emission upon contact between the excited fluorophore and quencher. Pyrene, with its ability to form an excimer on contact between excited-state and ground-state species, happens to possess all of these properties. Although the concepts described in this review apply to any fluorophore and quencher pair sharing pyrene's exceptional photophysical properties, this review focuses on the study of pyrene-labeled macromolecules that have been characterized in great detail over the past 40 years and presents the main models that are being used today to analyze the fluorescence decays of pyrene-labeled macromolecules reliably. These models are based on Birks' scheme, the DMD model, the fluorescence blob model, and the model free analysis. The review also provides a step-by-step protocol that should enable the noneducated user to achieve a successful decay analysis exempt of artifacts. Finally, some examples of studies of pyrene-labeled macromolecules are also presented to illustrate the different types of information that can be retrieved from these fluorescence decay

  19. Bioorthogonal Labeling of 5-Hydroxymethylcytosine in Genomic DNA and Diazirine-Based DNA Photo-Cross-Linking Probes

    PubMed Central

    SONG, CHUN-XIAO; HE, CHUAN

    2013-01-01

    CONSPECTUS DNA is not merely a combination of four genetic codes, namely A, T, C, and G. It also contains minor modifications that play crucial roles throughout biology. For example, the fifth DNA base, 5-methylcytosine (5-mC), which accounts for ~1% of all the nucleotides in mammalian genomic DNA, is a vital epigenetic mark. It impacts a broad range of biological functions, from development to cancer. Recently, an oxidized form of 5-methylcytosine, 5-hydroxymethylcytosine (5-hmC), was found to constitute the sixth base in the mammalian genome; it was believed to be another crucial epigenetic mark. Unfortunately, further study of this newly discovered DNA base modification has been hampered by inadequate detection and sequencing methods, because current techniques fail to differentiate 5-hmC from 5-mC. The immediate challenge, therefore, is to develop robust methods for ascertaining the positions of 5-hmC within the mammalian genome. In this Account, we describe our development of the first bioorthogonal, selective labeling of 5-hmC to specifically address this challenge. We utilize β-glucosyltransferase (βGT) to transfer an azide-modified glucose onto 5-hmC in genomic DNA. The azide moiety enables further bioorthogonal click chemistry to install a biotin group, which allows for detection, affinity enrichment, and, most importantly, deep sequencing of the 5-hmC-containing DNA. With this highly effective and selective method, we revealed the first genome-wide distribution of 5-hmC in the mouse genome and began to shed further light on the biology of 5-hmC. The strategy lays the foundation for developing high-throughput, single-base-resolution sequencing methods for 5-hmC in mammalian genomes in the future. DNA and RNA are not static inside cells. They interact with protein and other DNA and RNA in fundamental biological processes such as replication, transcription, translation, and DNA and RNA modification and repair. The ability to investigate these interactions

  20. Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

    PubMed

    Paradh, A D; Hill, A E; Mitchell, W J

    2014-01-01

    DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

  1. Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes and standardized secondary universal fluorogenic reporters.

    PubMed

    Faltin, Bernd; Wadle, Simon; Roth, Günter; Zengerle, Roland; von Stetten, Felix

    2012-11-01

    The majority of established techniques for monitoring real-time PCR amplification involve individual target-specific fluorogenic probes. For analysis of numerous different targets the synthesis of these probes contributes to the overall cost during assay development. Sequence-dependent universal detection techniques overcome this drawback but are prone to detection of unspecific amplification products. We developed the mediator probe PCR as a solution to these problems. A set of label-free sequence-specific primary probes (mediator probes), each comprising a target-specific region and a standardized mediator tag, is cleaved upon annealing to its target sequence by the polymerases' 5' nuclease activity. Release of a mediator triggers signal generation by cleavage of a complementary fluorogenic reporter probe. Real-time PCR amplification of human papillomavirus 18 (HPV18), Staphylococcus aureus, Escherichia coli, and Homo sapiens DNA dilution series showed exceptional linearity when detected either by novel mediator probes (r(2) = 0.991-0.999) or state-of-the-art hydrolysis probes (TaqMan probes) (r(2) = 0.975-0.993). For amplification of HPV18 DNA the limits of detection were 78.3 and 85.1 copies per 10-μL reaction when analyzed with the mediator probe and hydrolysis probe, respectively. Duplex amplification of HPV18 target DNA and internal standard had no effects on back calculation of target copy numbers when quantified with either the mediator probe PCR (r(2) = 0.998) or the hydrolysis probe PCR (r(2) = 0.988). The mediator probe PCR has equal performance to hydrolysis probe PCR and has reduced costs because of the use of universal fluorogenic reporters. © 2012 American Association for Clinical Chemistry

  2. Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay.

    PubMed

    Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

    2017-01-25

    Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.

  3. Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

    PubMed Central

    Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

    2017-01-01

    Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates. PMID:28120891

  4. Imaging viral RNA using multiply labeled tetravalent RNA imaging probes in live cells.

    PubMed

    Alonas, Eric; Vanover, Daryll; Blanchard, Emmeline; Zurla, Chiara; Santangelo, Philip J

    2016-04-01

    Viruses represent an important class of pathogens that have had an enormous impact on the health of the human race. They are extraordinarily diverse; viral particles can range in size from ∼80nm to ∼10μm in length, and contain genomes with RNA or DNA strands. Regardless of their genome type, RNA species are frequently generated as a part of their replication process, and for viruses with RNA genomes, their loading into the virion represents a critical step in the creation of infectious particles. RNA imaging tools represent a powerful approach to gain insight into fundamental viral processes, including virus entry, replication, and virion assembly. Imaging viral processes in live cells is critical due to both the heterogeneity of these processes on a per cell basis, and the inherent dynamics of these processes. There are a number of methods for labeling RNA in live cells; we'll introduce the myriad of methods and then focus on one approach for labeling viral RNA, using multiply-labeled tetravalent RNA imaging probes (MTRIPs), which do not require engineering of the target RNAs. We feel this approach is advantageous given many viral genomes may not tolerate large nucleotide insertions into their sequences. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe.

    PubMed Central

    Wright, A C; Miceli, G A; Landry, W L; Christy, J B; Watkins, W D; Morris, J G

    1993-01-01

    An oligonucleotide DNA probe (VVAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hylA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with VVAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100% specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by VVAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 10(2) and 2 x 10(1) bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by VVAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The VVAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests. Images PMID:8434919

  6. Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology.

    PubMed

    Laiho, Jutta E; Oikarinen, Sami; Oikarinen, Maarit; Larsson, Pär G; Stone, Virginia M; Hober, Didier; Oberste, Steven; Flodström-Tullberg, Malin; Isola, Jorma; Hyöty, Heikki

    2015-08-01

    Enteroviral infections are common, affecting humans across all age groups. RT-PCR is widely used to detect these viruses in clinical samples. However, there is a need for sensitive and specific in situ detection methods for formalin-fixed tissues, allowing for the anatomical localization of the virus and identification of its serotype. The aim was to design novel enterovirus probes, assess the impact of probe design for the detection and optimize the new single molecule in situ hybridization technology for the detection of enteroviruses in formalin-fixed paraffin-embedded samples. Four enterovirus RNA-targeted oligonucleotide RNA probes - two probes for wide range enterovirus detection and two for serotype-targeted detection of Coxsackievirus B1 (CVB1) - were designed and validated for the commercially available QuantiGene ViewRNA in situ hybridization method. The probe specificities were tested using a panel of cell lines infected with different enterovirus serotypes and CVB infected mouse pancreata. The two widely reactive probe sets recognized 19 and 20 of the 20 enterovirus serotypes tested, as well as 27 and 31 of the 31 CVB1 strains tested. The two CVB1 specific probe sets detected 30 and 14 of the 31 CVB1 strains, with only minor cross-reactivity to other serotypes. Similar results were observed in stained tissues from CVB -infected mice. These novel in-house designed probe sets enable the detection of enteroviruses from formalin-fixed tissue samples. Optimization of probe sequences makes it possible to tailor the assay for the detection of enteroviruses on the serotype or species level. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling

    PubMed Central

    2017-01-01

    Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling of solvent accessible residues followed by their MS-based identification yields modeling restraints that allow mapping the location and orientation of subunits within protein assemblies. Together with complementary restraints derived from cross-linking and native MS, we built native-like models of four heterocomplexes with known subunit structures and compared them with available X-ray crystal structures. The results demonstrated that covalent labeling followed by MS markedly increased the predictive power of the integrative modeling strategy enabling more accurate protein assembly models. We applied this strategy to the F-type ATP synthase from spinach chloroplasts (cATPase) providing a structural basis for its function as a nanomotor. By subjecting the models generated by our restraint-based strategy to molecular dynamics (MD) simulations, we revealed the conformational states of the peripheral stalk and assigned flexible regions in the enzyme. Our strategy can readily incorporate complementary chemical labeling strategies and we anticipate that it will be applicable to many other systems providing new insights into the structure and function of protein complexes. PMID:28208298

  8. Probing meaningfulness of oscillatory EEG components with bootstrapping, label noise and reduced training sets.

    PubMed

    Castaño-Candamil, Sebastián; Meinel, Andreas; Dähne, Sven; Tangermann, Michael

    2015-01-01

    As oscillatory components of the Electroencephalogram (EEG) and other electrophysiological signals may co-modulate in power with a target variable of interest (e.g. reaction time), data-driven supervised methods have been developed to automatically identify such components based on labeled example trials. Under conditions of challenging signal-to-noise ratio, high-dimensional data and small training sets, however, these methods may overfit to meaningless solutions. Examples are spatial filtering methods like Common Spatial Patterns (CSP) and Source Power Comodulation (SPoC). It is difficult for the practitioner to tell apart meaningful from arbitrary, random components. We propose three approaches to probe the robustness of extracted oscillatory components and show their application to both, simulated and EEG data recorded during a visually cued hand motor reaction time task.

  9. Biconically tapered fiber optic dip probe for rapid label-free immunoassays

    NASA Astrophysics Data System (ADS)

    Miller, John; Castaneda, Angelica; Lee, Kun Ho; Sanchez, Martin; Murinda, Shelton; Lin, Wei-Jen; Salik, Ertan

    2014-02-01

    We report U-shaped biconically tapered optical fibers (BTOF) as dip probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 0.5, 5.0, and 50 μg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G) -antigen (rabbit anti-mouse IgG) reactions was studied. The limit of detection for the sensor was estimated to be less than 0.5 μg/mL with low temperature sensitivity. Utilization of the rate of the sensor peak shift within the first few minutes of antibody-antigen reaction is proposed as a rapid detection method.

  10. New cross-linking quinoline- and quinolone-based luminescent lanthanide probes for sensitive labeling

    NASA Astrophysics Data System (ADS)

    Pillai, Shyamala; Wirpsza, Laura; Kozlov, Maxim; Marras, Salvatore A. E.; Krasnoperov, Lev N.; Mustaev, Arkady

    2012-03-01

    New luminescent lanthanide chelates containing thiol-, amine-, and click-reactive groups in antenna-fluorophore moieties were synthesized. The chelates include diethylenetriaminepentaacetic acid (DTPA) coupled to two types of chromophores: 7-amino-4-trifluoromethyl-2(1H) quinolinone, and 7-amino-4-trifluoromethyl-2-alkoxyquinoline. The synthesized compounds were characterized using NMR, light absorption, steady-state and time-resolved fluorescent spectroscopy. Some of the compounds displayed high brightness with Tb3+, Eu3+, and Dy3+. Obtained reactive lanthanide chelates can be easily attached to biological molecules. The probes demonstrated high performance in molecular beaconbased DNA hybridization assays (sub-pico molar detection limit), in bacterial proteome labeling, and in live cell imaging.

  11. Dual-beam histotripsy: a low-frequency pump enabling a high-frequency probe for precise lesion formation.

    PubMed

    Lin, Kuang-Wei; Duryea, Alexander P; Kim, Yohan; Hall, Timothy L; Xu, Zhen; Cain, Charles A

    2014-02-01

    Histotripsy produces tissue fractionation through dense energetic bubble clouds generated by short, high-pressure, ultrasound pulses. When using pulses shorter than 2 cycles, the generation of these energetic bubble clouds only depends on where the peak negative pressure (P-) exceeds the intrinsic threshold of the medium (26 to 30 MPa in soft tissue with high water content). This paper investigates a strategic method for precise lesion generation in which a low-frequency pump pulse is applied to enable a sub-threshold high-frequency probe pulse to exceed the intrinsic threshold. This pump-probe method of controlling a supra-threshold volume can be called dual-beam histotripsy. A 20-element dual-frequency (500-kHz and 3-MHz elements confocally aligned) array transducer was used to generate dual-beam histotripsy pulses in red blood cell phantoms and porcine hepatic tissue specimens. The results showed that when sub-intrinsic-threshold pump (500-kHz) and probe (3-MHz) pulses were applied together, dense bubble clouds (and resulting lesions) were only generated when their peak negative pressures combined constructively to exceed the intrinsic threshold. The smallest reproducible lesion varied with the relative amplitude between the pump and probe pulses, and, with a higher proportion of the probe pulse, smaller lesions could be generated. When the propagation direction of the probe pulse relative to the pump pulse was altered, the shape of the produced lesion changed based on the region that exceeded intrinsic threshold. Because the low-frequency pump pulse is more immune to attenuation and aberrations, and the high-frequency probe pulse can provide precision in lesion formation, this dual-beam histotripsy approach would be very useful in situations in which precise lesion formation is required through a highly attenuative and aberrative medium, such as transcranial therapy. This is particularly true if a small low-attenuation acoustic window is available for the high

  12. Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections

    SciTech Connect

    Park, J.S.; Kurman, R.J.; Kessis, T.D.; Shah, K.V. )

    1991-01-01

    A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

  13. Effects of oxygen on EPR spectra of nitroxide spin-label probes of model membranes

    NASA Astrophysics Data System (ADS)

    Popp, Carol A.; Hyde, James S.

    The use of a methylpentene polymer, TPX, for construction of sample containers that allow easy equilibration of electron paramagnetic resonance samples with nitrogen is described. The effects of oxygen-dependent shortening of the electron spin relaxation times of nitroxide spin labels were studied in dispersions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylchohne (DPPC). First-harmonic, in-phase, absorption spectra of deoxygenated samples of 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16SASL) in DMPC display decreased linewidths and increased peak-to-peak heigths and resolution of 13C splittings. Continuous-wave (cw) saturation studies of 16SASL/DMPC and both lipid- and aqueous-phase components of 2,2,6,6-tetramethyl-piperidinooxyl (Tempo) partitioned into DPPC show that the rf field at which the signal intensity is maximized decreases when aerated samples are equilibrated with nitrogen. Second-harmonic, out-of-phase, absorption (saturation transfer) spectra of deoxygenated samples of 16SASUDMPC at -22°C and 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (5SASL) in DPPC at 35°C display increased signal intensity and lineshape changes. Electron-electron double resonance (ELDOR) spectra display much greater ELDOR reduction in signal intensity when a deoxygenated sample of 16SASL/DMPC is used. Our results indicate that the routine use of deoxygenated samples in biologically relevant studies using spin-label probes should be considered.

  14. Simultaneous electrochemical immunoassay using graphene-Au grafted recombinant apoferritin-encoded metallic labels as signal tags and dual-template magnetic molecular imprinted polymer as capture probes.

    PubMed

    Wang, De; Gan, Ning; Zhang, Huairong; Li, Tianhua; Qiao, Li; Cao, Yuting; Su, Xiurong; Jiang, Shan

    2015-03-15

    A novel electrochemical multiplexed immunoassay was designed for simultaneous determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using recombinant apoferritin-encoded metallic nanoparticles (rApo-M) as labels and dual-template magnetic molecularly imprinted polymers (MMIPs) as capture probes. The labels were prepared by loading recombinant apoferritin (r-Apo) and separately immobilize primary antibodies (anti-AFP and anti-CEA) via Au nanoparticles of in site growth on graphene (G). The capture probes were synthesized by self-polymerization of dopamine (DA) on the Fe3O4 nanoparticles (Fe3O4 NPs) and using AFP and CEA as the template proteins, which were used to enrich the targets simultaneously. After a sandwich-type immunoreaction, the labels were captured to the surface of MMIPs. The subsequent electrochemical stripping analysis of the metal components from the immunocomplex provide a means for quantification of targets based on the peak currents of Cd and Pb. Experimental results showed the immunoassay enabled the simultaneous determination of AFP and CEA in a single run with wide dynamic ranges of 0.001-5ngmL(-1). And the detection limits of AFP and CEA were 0.3 and 0.35pgmL(-1) (S/N=3), respectively. These results suggested that the proposed multiplexed immunoassay would be applied for clinical screening of other biomarkers.

  15. Node-pore sensing enables label-free surface-marker profiling of single cells.

    PubMed

    Balakrishnan, Karthik R; Whang, Jeremy C; Hwang, Richard; Hack, James H; Godley, Lucy A; Sohn, Lydia L

    2015-03-03

    Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside.

  16. A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes.

    PubMed

    Henry, Olivier; Lopez-Gallego, Fernando; Agger, Sean A; Schmidt-Dannert, Claudia; Sen, Stephanie; Shintani, David; Cornish, Katrina; Distefano, Mark D

    2009-07-01

    Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC(50) values of 5.8 (meta isomer) and 3.0 microM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K(I) of 0.46 microM. Radiolabeled forms of both analogues selectively labeled the beta-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.

  17. Construction of magnetic-carbon-quantum-dots-probe-labeled apoferritin nanocages for bioimaging and targeted therapy.

    PubMed

    Yao, Hanchun; Su, Li; Zeng, Man; Cao, Li; Zhao, Weiwei; Chen, Chengqun; Du, Bin; Zhou, Jie

    Carbon dots (CDs) are one of the most highlighted carbon-based materials for biological applications, such as optical imaging nanoprobes, which are used for labeling cells in cancer treatment mainly due to their biocompatibility and unique optical properties. In this study, gadolinium (Gd)-complex-containing CDs were obtained through a one-step microwave method to develop multimodal nanoprobes integrating the advantages of optical and magnetic imaging. The obtained Gd-CDs exhibited highly fluorescent properties with excellent water solubility and biological compatibility. Natural apoferritin (AFn) nanocages, an excellent drug delivery carrier, are hollow in structure, with their pH-dependent, unfolding-refolding process at pH 2.0 and 7.4. The chemotherapeutic drug doxorubicin (DOX) can be highly effective and encapsulated into AFn cavity. A widely used tumor-targeting molecule, folic acid (FA), functionalized the surface of AFn to obtain an active tumor targeting effect on MCF-7 cells and malignant tumors in mice models. In this study, an AFn nanocarrier encapsulating high concentration of DOX labeled with magnetic and fluorescent Gd-CDs probe was developed. Gd-CDs exhibited a unique green photoluminescence and almost no toxicity compared with free GdCl3. Furthermore, Gd-doped CDs significantly increased the circulation time and decreased the toxicity of Gd(3+) in in vitro and in vivo magnetic resonance imaging, which demonstrated that the AFn nanocages labeled with Gd-CD compounds could serve as an excellent T1 contrast agent for magnetic resonance imaging. The self-assembling multifunctional Gd-CDs/AFn (DOX)/FA nanoparticles have a great potential for cancer theranostic applications.

  18. Construction of magnetic-carbon-quantum-dots-probe-labeled apoferritin nanocages for bioimaging and targeted therapy

    PubMed Central

    Yao, Hanchun; Su, Li; Zeng, Man; Cao, Li; Zhao, Weiwei; Chen, Chengqun; Du, Bin; Zhou, Jie

    2016-01-01

    Carbon dots (CDs) are one of the most highlighted carbon-based materials for biological applications, such as optical imaging nanoprobes, which are used for labeling cells in cancer treatment mainly due to their biocompatibility and unique optical properties. In this study, gadolinium (Gd)-complex-containing CDs were obtained through a one-step microwave method to develop multimodal nanoprobes integrating the advantages of optical and magnetic imaging. The obtained Gd-CDs exhibited highly fluorescent properties with excellent water solubility and biological compatibility. Natural apoferritin (AFn) nanocages, an excellent drug delivery carrier, are hollow in structure, with their pH-dependent, unfolding–refolding process at pH 2.0 and 7.4. The chemotherapeutic drug doxorubicin (DOX) can be highly effective and encapsulated into AFn cavity. A widely used tumor-targeting molecule, folic acid (FA), functionalized the surface of AFn to obtain an active tumor targeting effect on MCF-7 cells and malignant tumors in mice models. In this study, an AFn nanocarrier encapsulating high concentration of DOX labeled with magnetic and fluorescent Gd-CDs probe was developed. Gd-CDs exhibited a unique green photoluminescence and almost no toxicity compared with free GdCl3. Furthermore, Gd-doped CDs significantly increased the circulation time and decreased the toxicity of Gd3+ in in vitro and in vivo magnetic resonance imaging, which demonstrated that the AFn nanocages labeled with Gd-CD compounds could serve as an excellent T1 contrast agent for magnetic resonance imaging. The self-assembling multifunctional Gd-CDs/AFn (DOX)/FA nanoparticles have a great potential for cancer theranostic applications. PMID:27660437

  19. A Versatile Photoactivatable Probe Designed to Label the Diphosphate Binding Site of Farnesyl Diphosphate Utilizing Enzymes

    PubMed Central

    Henry, Olivier; Lopez-Gallego, Fernando; Agger, Sean A.; Schmidt-Dannert, Claudia; Sen, Stephanie; Shintani, David; Cornish, Katrina; Distefano, Mark D.

    2009-01-01

    Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit S. cerevisiae protein farnesyltransferase (ScPFTase) with IC50 values of 5.8 (meta isomer) and 3.0 µM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a KI of 0.46 µM. Radiolabeled forms of both analogues selectively labelled the β-subunit of ScPFTase. The para isomer was also shown to label E. coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes. PMID:19447628

  20. A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

    PubMed Central

    Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg

    2015-01-01

    Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways. PMID:26496085

  1. Method for sequential staining of GTL-banded metaphases with fluorescent-labeled chromosome-specific paint probes.

    PubMed

    Jalal, S M; Law, M E; Christensen, E R; Spurbeck, J L; Dewald, G W

    1993-04-01

    We describe a method for use of fluorescent-labeled whole chromosome-specific paint probes on GTL-banded metaphases to utilize the combined potential of these techniques for defining chromosome abnormalities. The efficacy of this method was tested on 6 cases involving different chromosome abnormalities and various tissues, including blood, amniotic fluid, skin fibroblasts, and bone marrow.

  2. Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

    PubMed Central

    Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

    2000-01-01

    A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

  3. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA.

    PubMed Central

    Leone, G; van Schijndel, H; van Gemen, B; Kramer, F R; Schoen, C D

    1998-01-01

    Molecular beacon probes can be employed in a NASBA amplicon detection system to generate a specific fluorescent signal concomitantly with amplification. A molecular beacon, designed to hybridize within the target sequence, was introduced into NASBA reactions that amplify the genomic RNA of potato leafroll virus (PLRV). During amplification, the probe anneals to the antisense RNA amplicon generated by NASBA, producing a specific fluorescent signal that can be monitored in real-time. The assay is rapid, sensitive and specific. As RNA amplification and detection can be carried out in unopened vessels, it minimizes the risk of carry-over contaminations. Robustness has been verified on real-world samples. This homogeneous assay, called AmpliDet RNA, is a significant improvement over current detection methods for NASBA amplicons and is suitable for one-tube applications ranging from high-throughput diagnostics to in vivo studies of biological activities. PMID:9547273

  4. Supramolecular Probes for Assessing Glutamine Uptake Enable Semi-Quantitative Metabolic Models in Single Cells

    PubMed Central

    Xue, Min; Wei, Wei; Su, Yapeng; Johnson, Dazy; Heath, James R.

    2016-01-01

    We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip (SCBC) with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady state model that describes energy potential in single cells, and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors. PMID:26916347

  5. Feedback and control in integrated optics enabled by contactLess integrated photonic probe

    NASA Astrophysics Data System (ADS)

    Annoni, Andrea; Morichetti, Francesco; Grillanda, Stefano; Peserico, Nicola; Carminati, Marco; Ciccarella, Pietro; Ferrari, Giorgio; Sampietro, Marco; Melloni, Andrea

    2015-02-01

    We demonstrate non-invasive light observation in silicon photonics with a ContactLess Integrated Photonics Probe (CLIPP), neither introducing appreciable perturbations of the optical field nor requiring photon tapping from the waveguide. Light monitoring with sensitivity down to -30 dBm, across 40 dB dynamic range, in few tens of microseconds, on TE and TM polarizations, and on monomode and multimode waveguides is achieved. Moreover, we show wavelength tuning, locking and swapping of high-Q resonators assisted by the CLIPP that is integrated inside the microring. CLIPP readout and feedback control is managed by a CMOS microelectronic circuit bridged to the silicon photonic chip.

  6. Concise synthesis of a probe molecule enabling analysis and imaging of vizantin.

    PubMed

    Yamamoto, Hirofumi; Oda, Masataka; Nakano, Mayo; Yabiku, Kenta; Shibutani, Masahiro; Nakanishi, Toshiyuki; Suenaga, Midori; Inoue, Masahisa; Imagawa, Hiroshi; Nagahama, Masahiro; Matsunaga, Yoichi; Himeno, Seiichiro; Setsu, Kojun; Sakurai, Jun; Nishizawa, Mugio

    2013-01-01

    Trehalose 6,6'-dicorynomycolate (TDCM) was first characterized in 1963 as a cell surface glycolipid of Corynebacterium spp. by Ioneda and co-workers. TDCM shows potent anti-tumor activity due to its immunoadjuvant properties. Furthermore, the toxicity of TDCM in mice is much weaker than the related trehalose diester of mycolic acid; trehalose 6,6'-dimycolate (TDM, formerly known as cord factor). We have investigated the chemical modification of this class of compound to generate novel agents that display increased immunoadjuvant activity with minimal associated toxicity. During the course of this work we recently developed 6,6'-bis-O-(3-nonyldodecanoyl)-α,α'-trehalose (designated as vizantin). Our results show that vizantin exhibited a potent prophylactic effect on experimental lung metastasis of B16-F0 melanoma cells without a loss of body weight and death in mice. Furthermore, vizantin effectively stimulated human macrophages in an in vitro model, making it a promising candidate for a safe adjuvant in clinical applications. In order to elucidate the pharmacokinetics of vizantin, a probe molecule with similar activity was developed on the basis of a structure-activity relationship (SAR) study with vizantin. The distribution of the probe molecule after intravenous administration into a mouse was assessed by macro confocal microscopy, where it was found to accumulate in the lungs and liver.

  7. Characterization of alkaline phosphatase labeled UidA(Gus) probe and its application in testing of transgenic tritordeum.

    PubMed

    Tu, Zhiming; Zhang, Jiangzhou; Yang, Guangxiao; He, Guangyuan

    2011-08-01

    Hybridization is a very important molecular biology technique to measure the degree of genetic similarity between DNA sequences, and detect the foreign genes in transgenic organisms. To label a DNA or RNA probe plays a key role in hybridization. A method using nonradioactive material alkaline phosphatase to label UidA(Gus) DNA as probe has been studied. On that basis of Renz and our previous work, alkaline phosphatase-labeled DNA was used as a probe to examine the transformation of the foreign UidA(Gus) gene in transgenic tritordeum. Such DNA-enzyme complexes were characterized and examined carefully, the results showed that it was a sensitive, specific, safe and economical probe. For dot hybridization and Southern blot under full-stringency conditions with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-Nitro Blue Tetrazolium (NBT) as the substrate, dot hybridization showed that the UidA(Gus) gene was transformed into the target plants and inherited stable, Southern blot showed that at least two copies of UidA(Gus) gene were inserted into one line of our transgenic tritordeum. Histochemical staining with X-Gluc of transgenic tritordeum also certified that the foreign UidA(Gus) DNA were transformed into the transgenic tritordeum.

  8. Mechanisms of catalytic dehydrogenation of alkanes by rhodium clusters Rhn+ probed by isotope labelling

    NASA Astrophysics Data System (ADS)

    Adlhart, Christian; Uggerud, Einar

    2006-03-01

    The regioselectivity for the dehydrogenation of alkanes by rhodium clusters was investigated by reacting Rhn+, n = 1-20, with the isotopically labelled alkanes ethane-1,1,1-d3 and propane-1,1,1,3,3,3-d6. For Rh+ reacting with propane a clear preference for a 1,2- over a 1,1- and 1,3-mechanism was observed. For larger clusters, hydrogen scrambling is faster than hydrogen elimination, which essentially leads to statistical formation of the neutrals H2, HD, and D2. Isotope scrambling with D2 was also used as a structural probe for the reaction products of rhodium clusters with ethane. The intactness of the CH bonds was demonstrated for (n > 6). The studies are completed with a detailed kinetic analysis for the reaction of Rh7+ with ethane and ethane/hydrogen and ethane/helium mixtures. An over-all picture with efficient CH bond activation and fast and reversible hydrogen rearrangements emerges on the basis of these experiments. Some of the dehydrogenation reactions appear to be reversible.

  9. Positron-labeled dopamine agonists for probing the high affinity states of dopamine subtype 2 receptors.

    PubMed

    Hwang, Dah-Ren; Narendran, Raj; Laruelle, Marc

    2005-01-01

    It is well documented that guanidine nucleotide-coupled dopamine subtype 2 receptors (D2) are configured in high and low affinity states for the dopamine agonist in vitro. However, it is still unclear whether these functional states exist in vivo. We hypothesized that positron-labeled D2 agonist and Positron Emission Tomography can be used to probe these functional states noninvasively. Recently, we demonstrated in nonhuman primates that N-[11C]propyl-norapomorphine (NPA), a full D2 agonist, is a suitable tracer for imaging the high affinity states of D2 receptors in vivo. We also developed kinetic modeling method to derive receptor parameters, such as binding potential (BP) and specific uptake ratios (V3''). When coupled with a dopamine releasing drug, amphetamine, NPA was found to be more sensitive than antagonist tracers, such as [11C]raclopride (RAC), to endogenous dopamine concentration changes (by about 42%). This finding suggests that NPA is a superior tracer for reporting endogenous DA concentration. In addition, the difference of the BP or V3'' of NPA and RAC under control and amphetamine challenge conditions could be used to estimate the functional states of D2 receptors in vivo. On the basis of our findings and the assumptions that NPA binds only to the high affinity states and RAC binds equally to both affinity states, we proposed that about 70% of the D2 receptors are configured in the high affinity states in vivo.

  10. Biconically Tapered Fiber Optic Probes for Rapid Label-Free Immunoassays ǂ

    PubMed Central

    Miller, John; Castaneda, Angelica; Lee, Kun Ho; Sanchez, Martin; Ortiz, Adrian; Almaz, Ekrem; Turkoglu Almaz, Zuleyha; Murinda, Shelton; Lin, Wei-Jen; Salik, Ertan

    2015-01-01

    We report use of U-shaped biconically tapered optical fibers (BTOF) as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G)-antigen (rabbit anti-mouse IgG) reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method. PMID:25836359

  11. Enabling freehand lateral scanning of optical coherence tomography needle probes with a magnetic tracking system

    PubMed Central

    Yeo, Boon Y.; McLaughlin, Robert A.; Kirk, Rodney W.; Sampson, David D.

    2012-01-01

    We present a high-resolution three-dimensional position tracking method that allows an optical coherence tomography (OCT) needle probe to be scanned laterally by hand, providing the high degree of flexibility and freedom required in clinical usage. The method is based on a magnetic tracking system, which is augmented by cross-correlation-based resampling and a two-stage moving window average algorithm to improve upon the tracker's limited intrinsic spatial resolution, achieving 18 µm RMS position accuracy. A proof-of-principle system was developed, with successful image reconstruction demonstrated on phantoms and on ex vivo human breast tissue validated against histology. This freehand scanning method could contribute toward clinical implementation of OCT needle imaging. PMID:22808429

  12. Method to detect the end-point for PCR DNA amplification using an ionically labeled probe and measuring impedance change

    DOEpatents

    Miles, Robin R.; Belgrader, Phillip; Fuller, Christopher D.

    2007-01-02

    Impedance measurements are used to detect the end-point for PCR DNA amplification. A pair of spaced electrodes are located on a surface of a microfluidic channel and an AC or DC voltage is applied across the electrodes to produce an electric field. An ionically labeled probe will attach to a complementary DNA segment, and a polymerase enzyme will release the ionic label. This causes the conductivity of the solution in the area of the electrode to change. This change in conductivity is measured as a change in the impedance been the two electrodes.

  13. Hard real-time beam scheduler enables adaptive images in multi-probe systems

    NASA Astrophysics Data System (ADS)

    Tobias, Richard J.

    2014-03-01

    Real-time embedded-system concepts were adapted to allow an imaging system to responsively control the firing of multiple probes. Large-volume, operator-independent (LVOI) imaging would increase the diagnostic utility of ultrasound. An obstacle to this innovation is the inability of current systems to drive multiple transducers dynamically. Commercial systems schedule scanning with static lists of beams to be fired and processed; here we allow an imager to adapt to changing beam schedule demands, as an intelligent response to incoming image data. An example of scheduling changes is demonstrated with a flexible duplex mode two-transducer application mimicking LVOI imaging. Embedded-system concepts allow an imager to responsively control the firing of multiple probes. Operating systems use powerful dynamic scheduling algorithms, such as fixed priority preemptive scheduling. Even real-time operating systems lack the timing constraints required for ultrasound. Particularly for Doppler modes, events must be scheduled with sub-nanosecond precision, and acquired data is useless without this requirement. A successful scheduler needs unique characteristics. To get close to what would be needed in LVOI imaging, we show two transducers scanning different parts of a subjects leg. When one transducer notices flow in a region where their scans overlap, the system reschedules the other transducer to start flow mode and alter its beams to get a view of the observed vessel and produce a flow measurement. The second transducer does this in a focused region only. This demonstrates key attributes of a successful LVOI system, such as robustness against obstructions and adaptive self-correction.

  14. Miniature environmental chamber enabling in situ scanning probe microscopy within reactive environments.

    PubMed

    Nonnenmann, Stephen S; Bonnell, Dawn A

    2013-07-01

    Developments in solid oxide fuel cells (SOFCs) continue to be one of the most intensely studied areas involving energy-producing systems, in an attempt to partially alleviate rapidly growing energy concerns. Direct, experimental observation of the governing electrochemical processes have remained largely elusive, due to high operating temperatures in the range of 400 °C-1000 °C. Here we outline the design and development of a miniature environmental chamber that enables a standard atomic force microscopes access to realistic SOFC operating conditions (T = 600 °C) for direct interrogation of electrochemical phenomena within SOFC cross-sections.

  15. Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe.

    PubMed

    Yang, Haiying; Li, Zhejian; Shan, Meng; Li, Congcong; Qi, Honglan; Gao, Qiang; Wang, Jinyi; Zhang, Chengxiao

    2015-03-10

    A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0×10(2) to 3.0×10(4) cells mL(-1), with a detection limit of 2.6×10(2) cells mL(-1). The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL(-1). The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.

  16. Confined Activation and Subdiffractive Localization Enables Whole-Cell PALM with Genetically Expressed Probes

    PubMed Central

    York, Andrew G.; Ghitani, Alireza; Vaziri, Alipasha; Davidson, Michael W.; Shroff, Hari

    2011-01-01

    We demonstrate 3D superresolution microscopy in whole fixed cells using photoactivated localization microscopy (PALM). The use of the bright, genetically expressed fluorescent marker photoactivatable mCherry (PA-mCherry1) in combination with near diffraction-limited confinement of photoactivation using two-photon illumination and 3D localization methods allowed us to investigate a variety of cellular structures at <50 nm lateral and <100 nm axial resolution. Compared to existing methods, we substantially reduce excitation and bleaching of unlocalized markers, enabling 3D PALM imaging with high localization density in thick structures. Our 3D localization algorithms based on cross-correlation do not rely on idealized noise models or specific optical configurations, allowing flexible instrument design. Generation of appropriate fusion constructs and expression in Cos7 cells allowed us to image invaginations of the nuclear membrane, vimentin fibrils, the mitochondrial network, and the endoplasmic reticulum at depths greater than 8 μm. PMID:21317909

  17. Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels.

    PubMed

    Oztug Durer, Zeynep A; Kamal, J K Amisha; Benchaar, Sabrina; Chance, Mark R; Reisler, Emil

    2011-11-25

    Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex-rigor state-is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function.

  18. A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid.

    PubMed

    Yang, Wen; Tian, Jianniao; Ma, Yefei; Wang, Lijun; Zhao, Yanchun; Zhao, Shulin

    2015-11-05

    A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3'-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Creating and virtually screening databases of fluorescently-labelled compounds for the discovery of target-specific molecular probes

    NASA Astrophysics Data System (ADS)

    Kamstra, Rhiannon L.; Dadgar, Saedeh; Wigg, John; Chowdhury, Morshed A.; Phenix, Christopher P.; Floriano, Wely B.

    2014-11-01

    Our group has recently demonstrated that virtual screening is a useful technique for the identification of target-specific molecular probes. In this paper, we discuss some of our proof-of-concept results involving two biologically relevant target proteins, and report the development of a computational script to generate large databases of fluorescence-labelled compounds for computer-assisted molecular design. The virtual screening of a small library of 1,153 fluorescently-labelled compounds against two targets, and the experimental testing of selected hits reveal that this approach is efficient at identifying molecular probes, and that the screening of a labelled library is preferred over the screening of base compounds followed by conjugation of confirmed hits. The automated script for library generation explores the known reactivity of commercially available dyes, such as NHS-esters, to create large virtual databases of fluorescence-tagged small molecules that can be easily synthesized in a laboratory. A database of 14,862 compounds, each tagged with the ATTO680 fluorophore was generated with the automated script reported here. This library is available for downloading and it is suitable for virtual ligand screening aiming at the identification of target-specific fluorescent molecular probes.

  20. Generation of an intramolecular three-color fluorescence resonance energy transfer probe by site-specific protein labeling.

    PubMed

    Voss, Stephanie; Zhao, Lei; Chen, Xi; Gerhard, Frank; Wu, Yao-Wen

    2014-02-01

    Fluorescence resonance energy transfer (FRET) is a valuable tool for studying protein structure, folding and interactions. The steep distance dependence of the FRET efficiency requires the donor and acceptor to be in close proximity (1-7.5 nm) to exhibit sufficient energy transfer. One possibility to overcome this limitation is the usage of a FRET cascade that utilizes more than one FRET pair. Essential for realizing this FRET cascade is the site-specific introduction of multiple fluorophores to a given protein, which remains a great challenge. In this study, orthogonal labeling techniques, including fluorescent protein tagging, oxime ligation and kinetically controlled cysteine conjugation, are employed to introduce three fluorophores at specific sites of Rab1b GTPase, yielding a triple-labeled FRET probe. The generated protein probe exhibits efficient energy transfer from the primary donor enhanced green fluorescent protein over the intermediate acceptor rhodamine to the final acceptor Dy630. The labeling strategy opens up a new avenue for multi-color labeling of proteins, facilitating long-distance FRET studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  1. Characterization of the Cricket Hindgut Microbiota with Fluorescently Labeled rRNA-Targeted Oligonucleotide Probes

    PubMed Central

    Santo Domingo, Jorge W.; Kaufman, Michael G.; Klug, Michael J.; Tiedje, James M.

    1998-01-01

    Most cricket hindgut microorganisms (60 to 80%) were detected with a universal fluorescent rRNA-targeted probe and found to be eubacteria. Group-specific probes showed that the hindguts of five different cricket species harbor similar bacterial groups, although in different proportions, and that different diets shifted the structure of the hindgut microbial community. The Bacteroides-Prevotella probe, of the eight eubacterial probes tested, stained the largest percentage of cells in all crickets. PMID:16349506

  2. 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.

  3. Detection of novel biomarkers for ovarian cancer with an optical nanotechnology detection system enabling label-free diagnostics

    NASA Astrophysics Data System (ADS)

    Kaja, Simon; Hilgenberg, Jill D.; Collins, Julie L.; Shah, Anna A.; Wawro, Debra; Zimmerman, Shelby; Magnusson, Robert; Koulen, Peter

    2012-08-01

    Ovarian carcinoma has the highest lethality rate of gynecologic tumors, largely attributed to the late-stage diagnosis of the disease. Reliable tools for both accurate diagnosis and early detection of disease onset are lacking, and presently less than 20% of ovarian cancers are detected at an early stage. Protein biomarkers that allow the discrimination of early and late stages of ovarian serous carcinomas are urgently needed as they would enable monitoring pre-symptomatic aspects of the disease, disease progression, and the efficacy of intervention therapies. We compare the absolute and relative protein levels of six protein biomarkers for ovarian cancer in five different established ovarian cancer cell lines, utilizing both quantitative immunoblot analysis and a guided-mode resonance (GMR) bioassay detection system that utilizes a label-free optical biosensor readout. The GMR sensor approach provided highly accurate, consistent, and reproducible quantification of protein biomarkers as validated by quantitative immunoblotting, as well as enhanced sensitivity, and is therefore suitable for quantification and detection of novel biomarkers for ovarian cancer. We identified fibronectin, apolipoprotein A1, and TIMP3 as potential protein biomarkers for the differential diagnosis of primary versus metastatic ovarian carcinoma. Future studies are needed to confirm the suitability of protein biomarkers tested herein in patient samples.

  4. Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR.

    PubMed

    Klein, Jennifer C; Burr, Adam R; Svensson, Bengt; Kennedy, Daniel J; Allingham, John; Titus, Margaret A; Rayment, Ivan; Thomas, David D

    2008-09-02

    We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.

  5. Imidazolium-tagged glycan probes for non-covalent labeling of live cells.

    PubMed

    Benito-Alifonso, David; Tremell, Shirley; Sadler, Joanna C; Berry, Monica; Galan, M Carmen

    2016-04-07

    Selective, bioorthogonal and fast labeling of glycoconjugates in living cells is a major challenge for synthetic and cellular biology. Here we report the use imidazolium tagged-mannosamine derivative (ITag-Man) for the non-covalent, rapid and site-specific labeling of sialic acid containing glycoproteins using commercial N-nitrilotriacetate fluorescent reagents in a range of cell lines.

  6. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

    PubMed Central

    2013-01-01

    The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses. PMID:24351207

  7. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  8. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling.

    PubMed

    Zhang, Haizhen; Brown, Roslyn N; Qian, Wei-Jun; Monroe, Matthew E; Purvine, Samuel O; Moore, Ronald J; Gritsenko, Marina A; Shi, Liang; Romine, Margaret F; Fredrickson, James K; Pasa-Tolić, Ljiljana; Smith, Richard D; Lipton, Mary S

    2010-05-07

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope (18)O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a Gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level (16)O and (18)O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in Delta gspD mutant cells of many outer membrane proteins including the outer membrane c-type cytochromes OmcA and MtrC, in agreement with a previous report that these proteins are substrates of the type II secretion system.

  9. A novel single-labeled fluorescent oligonucleotide probe for silver(I) ion detection in water, drugs, and food.

    PubMed

    Bian, Liujiao; Ji, Xu; Hu, Wei

    2014-05-28

    Due to the high toxicity of silver(I) ions, a method for the rapid, sensitive, and selective detection for silver(I) ions in water, pharmaceutical products, and food is of great importance. Herein, a novel single-labeled fluorescent oligonucleotide (OND) probe based on cytosine-Ag(I)-cytosine coordination and the inherent fluorescence quenching ability of the G-quadruplex is designed to detect silver(I) ions. The formation of a hairpin structure in the OND-Ag(I) complex brings the hexachloro fluorescein (HEX) labeled at the 5'-end of the OND probe close to the G-quadruplex located at the 3'-end of the OND probe, leading to a fluorescence quenching due to photoinduced electron transfer between HEX and the G-quadruplex. Through this method, silver(I) ions can be detected quantitatively, the linear response range is from 1 to 100 nmol/L with a detection limit of 50 pmol/L, and no obvious interference occurs with other metal ions with a 10-fold concentration. This assay is simple, sensitive, and selective, and it can be used to detect silver(I) ions in actual water, drug, and food samples.

  10. Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

    NASA Astrophysics Data System (ADS)

    Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

    2016-05-01

    A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

  11. Detection of human cytomegalovirus by slot-blot hybridization assay employing oligo-primed /sup 32/P-labelled probe

    SciTech Connect

    Agha, S.A.; Coleman, J.C.; Selwyn, S.; Mahmound, L.A.; Abd-Elaal, A.M.; Archard, L.C.

    1988-12-01

    A /sup 32/P-labelled Hind III-0 DNA fragment (nine Kilobases; Kb) from human cytomegalovirus AD-169 (HCMV) was used in slot-blot hybridization assay for the detection of HCMV in clinical samples. The results obtained with DNA hybridization assay (DNA HA) were compared with virus isolation using conventional tube cell culture (CTC) and centrifugation vial culture (CVC), immunofluorescence (IF), and complement fixation test (CFT). Of 15 CTC-positive samples, 13 were positive with DNA HA (sensitivity 86.7%). Also, 14 additional samples were DNA HA-positive but CTC-negative. CVC and/or IF confirmed the diagnosis in nine of 14; the remaining five samples were from three patients who showed fourfold rising antibody titre by CFT. Although DNA HA using /sup 32/P-labelled probes is relatively cumbersome and expensive, it is a valuable test for quantitation of viral shedding in patients with HCMV infections who may benefit from antiviral therapy.

  12. Frequency-domain flow cytometry: fluorescence-lifetime-based sensing technology for analyzing cells and chromosomes labeled with fluorescent probes

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Crissman, Harry A.; Lehnert, Bruce E.; Lehnert, Nancy M.; Deka, Chiranjit

    1997-05-01

    A flow cytometer has been developed that combines flow cytometry (FCM) and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making frequency-domain, excited-state lifetime measurements on cells/chromosomes labeled with fluorescent probes, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine-wave) laser excitation beam. Fluorescence signals are processed by (1) low-pass filtering to obtain conventional FCM dc-excited signals and (2) phase-sensitive detection electronics to resolve heterogeneous fluorescence based on differences in lifetimes expressed as phase-shifts and to quantify fluorescence lifetimes in real time. Processed signals are displayed as frequency distribution histograms and bivariate contour diagrams. Recent examples of biological applications include: (1) lifetime histograms recorded on autofluorescent human lung fibroblasts, murine thymus cells labeled with antibodies conjugated to fluorophores for studying fluorescence quenching as a function of antibody dilution and F/P ratio, and on cultured cells, nuclei, and chromosomes stained with DNA-binding fluorochromes and (2) phase-resolved, fluorescence signal- intensity histograms recorded on autofluorescent HLFs labeled with immunofluorescence markers and on murine thymus cells labeled with Red 613-antiThy 1.2 and propidium iodide (PI positive `dead' cells) to demonstrate the resolution of signals from highly overlapping emission spectra. This technology will increase the number of fluorescent markers usable in multilabeling studies and lifetimes can be used as spectroscopic probes to study the interaction of markers with their targets, each other, and the surrounding microenvironment.

  13. Redox-labelled poly(ethylene glycol) used as a diffusion probe in poly(ethylene glycol) melts

    SciTech Connect

    Haas, O.; Velasquez, C.; Porat, Z.

    1995-12-01

    Ferrocene labelled monomethyl poly(ethylene glycol) MPEG with molecular weights of 1900 and 750 was prepared and used as an electrochemical diffusion probe in poly(ethylene glycol) melts. Cyclic voltammetry and chronoamperometry were used in connection with microdisk electrodes to measure the diffusion coefficient of redox tagged molecules using melted poly(ethylene glycol) as a solvent. The molecular weight of the solvent polymer was 750, 2000 and 20000. Results from the temperature dependency of the diffusion process and of the viscosity and conductivity of the polymer electrolyte are presented and discussed.

  14. A H+/Ag+ dual-target responsive label-free light-up probe based on a DNA triplex.

    PubMed

    Xu, Lijun; Guo, Yahui; Wang, Jine; Zhou, Lu; Zhang, Yuanyuan; Hong, Shanni; Wang, Zhili; Zhang, Jianye; Pei, Renjun

    2015-05-01

    We developed a dual-target responsive sensor for label-free light-up fluorescent detection of protons (H(+)) and silver ions (Ag(+)) using an "OR'' logic gate. Berberine, a cost-effective and non-toxic indicator, partially intercalates the formed triplex DNA in the presence of H(+) or Ag(+), generating enhanced fluorescence. The designed Ag(+) probe has high selectivity and desirable sensitivity, which is necessary for practical use. The robust "OR" logic gate is capable of a rapid and reversible response to the H(+) and/or Ag(+) inputs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Pump-probe microscopy of respiratory chain pigments: towards non-fluorescent label-free metabolic imaging

    NASA Astrophysics Data System (ADS)

    Domingue, Scott R.; Chicco, Adam J.; Bartels, Randy A.; Wilson, Jesse W.

    2017-02-01

    Current label-free metabolic microscopy techniques are limited to obtaining contrast from fluorescent molecules NAD(P)H and FAD+, and are unable to determine redox state along the mitochondrial respiratory chain itself. The respiratory chain electron carriers do not fluoresce, but some are heme proteins that have redox-dependent absorption spectra. The most prominent of these, cytochrome c, has been extensively characterized by transient absorption spectroscopy, which suggests that pump-probe measurements in the vicinity of 450 - 600 nm can provide strong contrast between its redox states. Motivated by the success of pump-probe microscopy targeting another heme protein, hemoglobin, we seek to extend the technique to the cytochromes, with the ultimate goal of dissecting respiratory chain function of individual cells in live tissue. To that end, we have developed a new optical system producing ultrafast, visible, independently-tunable pulse pairs via sum-frequency generation of nonlinearly broadened pulses in periodically-poled lithium niobate. The system is pumped by a homebuilt fiber-based oscillator/amplifier emitting 1060 nm pulses at 1.3 W (63 MHz repetition rate), and produces tunable pulses in the vicinity of 488 and 532 nm. Pump-probe spectroscopy of cytochrome c with this source reveals differences in excited-state absorption relaxation times between redox states. Though redox contrast is weak with this setup, we argue that this can be improved with a resonant galvo-scanning microscope. Moreover, pump-probe images were acquired of brown adipose tissue (which contains dense mitochondria), demonstrating label-free contrast from excited-state absorption in respiratory chain hemes.

  16. Monitoring methanol-induced protein unfolding by fluorescence anisotropy measurements of covalently labelled rhodamine probe*

    NASA Astrophysics Data System (ADS)

    Soleilhac, Antonin; Bertorelle, Franck; Dugourd, Philippe; Girod, Marion; Antoine, Rodolphe

    2017-06-01

    We describe the use of an extrinsic fluorophore (rhodamine B isothiocyanate) as a versatile probe to measure rotational motions of proteins. To illustrate the usefulness of this probe, we describe the fluorescence anisotropy values of this fluorophore covalently linked to myoglobin protein measured in aqueous solutions of increased methanol content. Methanol-induced unfolding is revealed by the transition from constrained to free rotation of the covalently attached rhodamine B fluorophore.

  17. Multimodal nonlinear endo-microscopy probe design for high resolution, label-free intraoperative imaging

    PubMed Central

    Chen, Xu; Xu, Xiaoyun; McCormick, Daniel T.; Wong, Kelvin; Wong, Stephen T.C.

    2015-01-01

    We present a portable, multimodal, nonlinear endo-microscopy probe designed for intraoperative oncological imaging. Application of a four-wave mixing noise suppression scheme using dual wavelength wave plates (DWW) and a polarization-maintaining fiber improves tissue signal collection efficiency, allowing for miniaturization. The probe, with a small 14 mm transversal diameter, includes a customized miniaturized two-axis MEMS (micro-electromechanical system) raster scanning mirror and micro-optics with an illumination laser delivered by a polarization-maintaining fiber. The probe can potentially be integrated into the arms of a surgical robot, such as da Vinci robotic surgery system, due to its minimal cross sectional area. It has the ability to incorporate multiple imaging modalities including CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation), and TPEF (two-photon excited fluorescence) in order to allow the surgeon to locate tumor cells within the context of normal stromal tissue. The resolution of the endo-microscope is experimentally determined to be 0.78 µm, a high level of accuracy for such a compact probe setup. The expected resolution of the as-built multimodal, nonlinear, endo-microscopy probe is 1 µm based on the calculation tolerance allocation using Monte-Carlo simulation. The reported probe is intended for use in laparoscopic or radical prostatectomy, including detection of tumor margins and avoidance of nerve impairment during surgery. PMID:26203361

  18. Monoclonal antibody-targeted fluorescein-5-isothiocyanate-labeled biomimetic nanoapatites: a promising fluorescent probe for imaging applications.

    PubMed

    Oltolina, Francesca; Gregoletto, Luca; Colangelo, Donato; Gómez-Morales, Jaime; Delgado-López, José Manuel; Prat, Maria

    2015-02-10

    Multifunctional biomimetic nanoparticles (NPs) are acquiring increasing interest as carriers in medicine and basic research since they can efficiently combine labels for subsequent tracking, moieties for specific cell targeting, and bioactive molecules, e.g., drugs. In particular, because of their easy synthesis, low cost, good biocompatibility, high resorbability, easy surface functionalization, and pH-dependent solubility, nanocrystalline apatites are promising candidates as nanocarriers. This work describes the synthesis and characterization of bioinspired apatite nanoparticles to be used as fluorescent nanocarriers targeted against the Met/hepatocyte growth factor receptor, which is considered a tumor associated cell surface marker of many cancers. To this aim the nanoparticles have been labeled with Fluorescein-5-isothiocyanate (FITC) by simple isothermal adsorption, in the absence of organic, possibly toxic, molecules, and then functionalized with a monoclonal antibody (mAb) directed against such a receptor. Direct labeling of the nanoparticles allowed tracking the moieties with spatiotemporal resolution and thus following their interaction with cells, expressing or not the targeted receptor, as well as their fate in vitro. Cytofluorometry and confocal microscopy experiments showed that the functionalized nanocarriers, which emitted a strong fluorescent signal, were rapidly and specifically internalized in cells expressing the receptor. Indeed, we found that, once inside the cells expressing the receptor, mAb-functionalized FITC nanoparticles partially dissociated in their two components, with some mAbs being recycled to the cell surface and the FITC-labeled nanoparticles remaining in the cytosol. This work thus shows that FITC-labeled nanoapatites are very promising probes for targeted cell imaging applications.

  19. Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes.

    PubMed

    Herrera Moro Chao, Daniela; Kallemeijn, Wouter W; Marques, Andre R A; Orre, Marie; Ottenhoff, Roelof; van Roomen, Cindy; Foppen, Ewout; Renner, Maria C; Moeton, Martina; van Eijk, Marco; Boot, Rolf G; Kamphuis, Willem; Hol, Elly M; Aten, Jan; Overkleeft, Hermen S; Kalsbeek, Andries; Aerts, Johannes M F G

    2015-01-01

    Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.

  20. Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples.

    PubMed

    Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Liu, J Q; Zhang, Y; Wang, N N; Hu, P; Lu, S Y; Ren, H L; Liu, Z S

    2014-11-15

    A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample.

  1. Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled "universal" reference sample.

    PubMed

    Qian, Wei-Jun; Liu, Tao; Petyuk, Vladislav A; Gritsenko, Marina A; Petritis, Brianne O; Polpitiya, Ashoka D; Kaushal, Amit; Xiao, Wenzhong; Finnerty, Celeste C; Jeschke, Marc G; Jaitly, Navdeep; Monroe, Matthew E; Moore, Ronald J; Moldawer, Lyle L; Davis, Ronald W; Tompkins, Ronald G; Herndon, David N; Camp, David G; Smith, Richard D

    2009-01-01

    The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope (18)O-labeled "universal" reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the (18)O-labeled "universal" reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on (16)O/(18)O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label-free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dual-quantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinity depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free (16)O-MS intensity data extracted from the same data sets. The results showed that the (18)O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the (18)O-reference for accurate quantitation makes this

  2. Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry.

    PubMed

    Zheng, Qiuling; Zhang, Hao; Wu, Shiyong; Chen, Hao

    2016-05-01

    This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d0 and DSP-d8 in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d0 and DSP-d8 separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca(2+) on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology. Graphical Abstract ᅟ.

  3. Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zheng, Qiuling; Zhang, Hao; Wu, Shiyong; Chen, Hao

    2016-05-01

    This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d0 and DSP-d8 in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d0 and DSP-d8 separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca2+ on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology.

  4. Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe.

    PubMed

    Xu, Min; Li, Baoxin

    2015-01-01

    A label-free and sensitive fluorescence assay for exonuclease activity is developed using commercially available SYBR Green I (SG) dye as signal probe. A proof-of-concept of this assay has been demonstrated by using exonuclease III (Exo III) as a model enzyme. In this assay, double-stranded DNA (dsDNA) can bind SG, resulting in a strong fluorescence signal of SG. Upon the addition of Exo III, dsDNA would be digested, and SG emits very weak fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. This method has a linear detection range from 1 U/mL to 200 U/mL with a detection limit of 0.7 U/mL. This label-free approach is selective, simple, convenient and cost-efficient without any complex DNA sequence design or fluorescence dye label. The method not only provides a platform for monitoring activity and inhibition of exonuclease but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.

  5. Probing the Metabolic Network in Bloodstream-Form Trypanosoma brucei Using Untargeted Metabolomics with Stable Isotope Labelled Glucose

    PubMed Central

    Creek, Darren J.; Mazet, Muriel; Achcar, Fiona; Anderson, Jana; Kim, Dong-Hyun; Kamour, Ruwida; Morand, Pauline; Millerioux, Yoann; Biran, Marc; Kerkhoven, Eduard J.; Chokkathukalam, Achuthanunni; Weidt, Stefan K.; Burgess, Karl E. V.; Breitling, Rainer; Watson, David G.; Bringaud, Frédéric; Barrett, Michael P.

    2015-01-01

    Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate. PMID:25775470

  6. Fluorescently Labeled Virus Probes Show that Natural Virus Populations Can Control the Structure of Marine Microbial Communities

    PubMed Central

    Hennes, K. P.; Suttle, C. A.; Chan, A. M.

    1995-01-01

    Fluorescently stained viruses were used as probes to label, identify, and enumerate specific strains of bacteria and cyanobacteria in mixed microbial assemblages. Several marine virus isolates were fluorescently stained with YOYO-1 or POPO-1 (Molecular Probes, Inc.) and added to seawater samples that contained natural microbial communities. Cells to which the stained viruses adsorbed were easily distinguished from nonhost cells; typically, there was undetectable binding of stained viruses to natural microbial assemblages containing >10(sup6) bacteria ml(sup-1) but to which host cells were not added. Host cells that were added to natural seawater were quantified with 99% (plusmn) 2% (mean (plusmn) range) efficiency with fluorescently labeled virus probes (FLVPs). A marine bacterial isolate (strain PWH3a), tentatively identified as Vibrio natriegens, was introduced into natural microbial communities that were either supplemented with nutrients or untreated, and changes in the abundance of the isolate were monitored with FLVPs. Simultaneously, the concentrations of viruses that infected strain PWH3a were monitored by plaque assay. Following the addition of PWH3a, the concentration of viruses infecting this strain increased from undetectable levels (<1 ml(sup-1)) to 2.9 x 10(sup7) and 8.3 x 10(sup8) ml(sup-1) for the untreated and nutrient-enriched samples, respectively. The increase in viruses was associated with a collapse in populations of strain PWH3a from ca. 30 to 2% and 43 to 0.01% of the microbial communities in untreated and nutrient-enriched samples, respectively. These results clearly demonstrate that FLVPs can be used to identify and quantify specific groups of bacteria in mixed microbial communities. The data show as well that viruses which are present at low abundances in natural aquatic viral communities can control microbial community structure. PMID:16535146

  7. Fluorescently Labeled Virus Probes Show that Natural Virus Populations Can Control the Structure of Marine Microbial Communities.

    PubMed

    Hennes, K P; Suttle, C A; Chan, A M

    1995-10-01

    Fluorescently stained viruses were used as probes to label, identify, and enumerate specific strains of bacteria and cyanobacteria in mixed microbial assemblages. Several marine virus isolates were fluorescently stained with YOYO-1 or POPO-1 (Molecular Probes, Inc.) and added to seawater samples that contained natural microbial communities. Cells to which the stained viruses adsorbed were easily distinguished from nonhost cells; typically, there was undetectable binding of stained viruses to natural microbial assemblages containing >10(sup6) bacteria ml(sup-1) but to which host cells were not added. Host cells that were added to natural seawater were quantified with 99% (plusmn) 2% (mean (plusmn) range) efficiency with fluorescently labeled virus probes (FLVPs). A marine bacterial isolate (strain PWH3a), tentatively identified as Vibrio natriegens, was introduced into natural microbial communities that were either supplemented with nutrients or untreated, and changes in the abundance of the isolate were monitored with FLVPs. Simultaneously, the concentrations of viruses that infected strain PWH3a were monitored by plaque assay. Following the addition of PWH3a, the concentration of viruses infecting this strain increased from undetectable levels (<1 ml(sup-1)) to 2.9 x 10(sup7) and 8.3 x 10(sup8) ml(sup-1) for the untreated and nutrient-enriched samples, respectively. The increase in viruses was associated with a collapse in populations of strain PWH3a from ca. 30 to 2% and 43 to 0.01% of the microbial communities in untreated and nutrient-enriched samples, respectively. These results clearly demonstrate that FLVPs can be used to identify and quantify specific groups of bacteria in mixed microbial communities. The data show as well that viruses which are present at low abundances in natural aquatic viral communities can control microbial community structure.

  8. Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

    NASA Astrophysics Data System (ADS)

    Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

    2011-10-01

    Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

  9. Novel Benzodiazepine Photoaffinity Probe Stereoselectively Labels a Site Deep Within the Membrane-spanning Domain of the Cholecystokinin Receptor

    PubMed Central

    Hadac, Elizabeth M.; Dawson, Eric S.; Darrow, James W.; Sugg, Elizabeth E.; Lybrand, Terry P.; Miller, Laurence J.

    2008-01-01

    An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties and for development of more potent and selective molecules that act at the same biological target. In this work, we have identified the active enantiomers in racemic mixtures of structurally related benzophenone derivatives of 1,5-benzodiazepines, representing both antagonist and agonist ligands of the type A cholecystokinin receptor. The parent compounds of the 1,5-benzodiazepine CCK receptor photoaffinity ligands were originally prepared in an effort to develop orally active drugs. The enantiomeric compounds reported in this study selectively photoaffinity-labeled the CCK receptor, resulting in the identification of a site of attachment for the photolabile moiety of the antagonist probe deep within the receptor’s membrane-spanning region at Leu88, a residue within transmembrane segment two. In contrast, the agonist probe labeled a region including extracellular loop one and a portion of transmembrane segment three. The antagonist covalent attachment site to the receptor served as a guide in the construction of theoretical three-dimensional molecular models for the antagonist-receptor complex. These models provided a means for visualization of physically plausible ligand-receptor interactions in the context of all currently available biological data that address small molecule interactions with the CCK receptor. Our approach, featuring the use of novel photolabile compounds targeting the membrane-spanning receptor domain to probe the binding site region, introduces powerful tools and a strategy for direct and selective investigation of non-peptidyl ligand binding to peptide receptors. PMID:16451051

  10. A label-free and universal platform for antibiotics detection based on microchip electrophoresis using aptamer probes.

    PubMed

    Zhou, Lingying; Gan, Ning; Zhou, You; Li, Tianhua; Cao, Yuting; Chen, Yinji

    2017-05-15

    A novel label-free, universal, and high throughput aptasensor was developed based on a microchip electrophoresis (MCE) platform for automatic detection of antibiotic residues in food. Firstly, chloramphenicol (CAP) was employed as a model to be captured by its aptamer probe (Apt). Then, the partial complementary oligonucleotide of CAP's aptamer (C-DNA) was introduced into the reaction system. Because the Apt-CAP complex can't further hybrid with free C-DNA, the amount of hybrid Apt-C-DNA double strand DNA (dsDNA) was less than that without adding the target. Finally, the above mixture was introduced into the microchip electrophoresis (MCE) platform for detection, both dsDNA and Apt-CAP can be separated and produce different fluorescence signals in the MCE. In a certain concentration range, the ratio of signal between dsDNA and Apt-CAP (IdsDNA/I Apt-CAP) was proportional to the concentration of targets. Under the optimum conditions, the ratio showed a satisfactory linearity range from 0.008 to 1ng/mL of CAP with a detection limit of 0.003ng/mL. Thus, a universal MCE-based assay was developed for quantifying CAP automatically. The method was also successfully applied in the different food samples for CAP detection, which showed a good recovery (Milk: 91.1-108%, Fish: 86.1-114%) and the results were consistent with that of ELISA. This method owned many merits as follows: firstly, MCE was a high throughput screening platform and the detection time is limited to 3min for each sample. Secondly, the aptamer probes can be directly used for detection without labeling any signal tag which can facilitate the preparation procedures of probes. Thirdly, the operation was easy just by the following steps: firstly, the mixture of aptamer probes were incubated followed adding C-DNA; then measurement was performed. Moreover, the assay with MCE platform can be used to detect other targets just by changing the corresponding aptamer probe; it can even realize simultaneous detection

  11. Design Strategies for Bioorthogonal Smart Probes

    PubMed Central

    Shieh, Peyton; Bertozzi, Carolyn R.

    2014-01-01

    Bioorthogonal chemistry has enabled the selective labeling and detection of biomolecules in living systems. Bioorthogonal smart probes, which become fluorescent or deliver imaging or therapeutic agents upon reaction, allow for the visualization of biomolecules or targeted delivery even in the presence of excess unreacted probe. This review discusses the strategies used in the development of bioorthogonal smart probes and highlights the potential of these probes to further our understanding of biology. PMID:25315039

  12. Survival of free-living Acholeplasma in aerated pig manure slurry revealed by 13C-labeled bacterial biomass probing

    PubMed Central

    Hanajima, Dai; Aoyagi, Tomo; Hori, Tomoyuki

    2015-01-01

    Many studies have been performed on microbial community succession and/or predominant taxa during the composting process; however, the ecophysiological roles of microorganisms are not well understood because microbial community structures are highly diverse and dynamic. Bacteria are the most important contributors to the organic-waste decomposition process, while decayed bacterial cells can serve as readily digested substrates for other microbial populations. In this study, we investigated the active bacterial species responsible for the assimilation of dead bacterial cells and their components in aerated pig manure slurry by using 13C-labeled bacterial biomass probing. After 3 days of forced aeration, 13C-labeled and unlabeled dead Escherichia coli cell suspensions were added to the slurry. The suspensions contained 13C-labeled and unlabeled bacterial cell components, possibly including the cell wall and membrane, as well as intracellular materials. RNA extracted from each slurry sample 2 h after addition of E. coli suspension was density-resolved by isopycnic centrifugation and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of bacterial 16S rRNA genes. In the heavy isotopically labeled RNA fraction, the predominant 13C-assimilating population was identified as belonging to the genus Acholeplasma, which was not detected in control heavy RNA. Acholeplasma spp. have limited biosynthetic capabilities and possess a wide variety of transporters, resulting in their metabolic dependence on external carbon and energy sources. The prevalence of Acholeplasma spp. was further confirmed in aerated pig manure slurry from four different pig farms by pyrosequencing of 16S rRNA genes; their relative abundance was ∼4.4%. Free-living Acholeplasma spp. had a competitive advantage for utilizing dead bacterial cells and their components more rapidly relative to other microbial populations, thus allowing the survival and prevalence

  13. Detection of atomic spin labels in a lipid bilayer using a single-spin nanodiamond probe.

    PubMed

    Kaufmann, Stefan; Simpson, David A; Hall, Liam T; Perunicic, Viktor; Senn, Philipp; Steinert, Steffen; McGuinness, Liam P; Johnson, Brett C; Ohshima, Takeshi; Caruso, Frank; Wrachtrup, Jörg; Scholten, Robert E; Mulvaney, Paul; Hollenberg, Lloyd

    2013-07-02

    Magnetic field fluctuations arising from fundamental spins are ubiquitous in nanoscale biology, and are a rich source of information about the processes that generate them. However, the ability to detect the few spins involved without averaging over large ensembles has remained elusive. Here, we demonstrate the detection of gadolinium spin labels in an artificial cell membrane under ambient conditions using a single-spin nanodiamond sensor. Changes in the spin relaxation time of the sensor located in the lipid bilayer were optically detected and found to be sensitive to near-individual (4 ± 2) proximal gadolinium atomic labels. The detection of such small numbers of spins in a model biological setting, with projected detection times of 1 s [corresponding to a sensitivity of ∼5 Gd spins per Hz(1/2)], opens a pathway for in situ nanoscale detection of dynamical processes in biology.

  14. Near-infrared dyes and upconverting phosphors as biomolecule labels and probes

    NASA Astrophysics Data System (ADS)

    Patonay, Gabor; Strekowski, Lucjan; Nguyen, Diem-Ngoc; Seok, Kim Jun

    2007-02-01

    Near-Infrared (NIR) absorbing chromophores have been used in analytical and bioanalytical chemistry extensively, including for determination of properties of biomolecules, DNA sequencing, immunoassays, capillary electrophoresis (CE) separations, etc. The major analytical advantages of these dyes are low background interference and high molar absorptivities. NIR dyes have additional advantages due to their sensitivity to microenvironmental changes. Spectral changes induced by the microenvironment are not desirable if the labels are used as a simple reporting group, e.g., during a biorecognition reaction. For these applications upconverting phosphors seem to be a better choice. There are several difficulties in utilizing upconverting phosphors as reporting labels. These are: large physical size, no reactive groups and insolubility in aqueous systems. This presentation will discuss how these difficulties can be overcome for bioanalytical and forensic applications. During these studies we also have investigated how to reduce physical size of the phosphor by simple grinding without losing activity and how to attach reactive moiety to the phosphor to covalently bind to the biomolecule of interest. It has to be emphasized that the described approach is not suitable for medical applications and the results of this research are not applicable in medical applications. For bioanalytical and forensic applications upconverting phosphors used as reporting labels have several advantages. They are excited with lasers that are red shifted respective to phosphorescence, resulting in no light scatter issues during detection. Also some phosphors are excited using eye safe lasers. In addition energy transfer to NIR dyes is possible, allowing detection schemes using donor-acceptor pairs. Data is presented to illustrate the feasibility of this phenomenon. If microenvironmental sensitivity is required, then specially designed NIR dyes can be used as acceptor labels. Several novel dyes

  15. Probing Xylan-Specific Raman Bands for Label-Free Imaging Xylan in Plant Cell Wall

    SciTech Connect

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; Tucker, Melvin P.; Vinzant, Todd; Himmel, Michael E.

    2015-06-15

    Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike lignin and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.

  16. Membrane-Sugar Interactions Probed by Pulsed Electron Paramagnetic Resonance of Spin Labels.

    PubMed

    Konov, Konstantin B; Leonov, Dmitry V; Isaev, Nikolay P; Fedotov, Kirill Yu; Voronkova, Violeta K; Dzuba, Sergei A

    2015-08-13

    Sugars can stabilize biological systems under extreme desiccation and freezing conditions. Hypothetical molecular mechanisms suggest that the stabilization effect may be determined either by specific interactions of sugars with biological molecules or by the influence of sugars on the solvating shell of the biomolecule. To explore membrane-sugar interactions, we applied electron spin echo envelope modulation (ESEEM) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), to phospholipid bilayers with spin-labeled lipids added and solvated by aqueous deuterated sucrose and trehalose solutions. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The spin-labeled lipids were 1,2-dipalmitoyl-sn-glycero-3-phospho(TEMPO)choline (T-PCSL), with spin-label TEMPO at the lipid polar headgroup. The deuterium ESEEM amplitude was calibrated using known concentrations of glassy deuterated sugar solvents. The data obtained indicated that the sugar concentration near the membrane surface obeyed a simple Langmuir model of monolayer adsorption, which assumes direct sugar-molecule bonding to the bilayer surface.

  17. Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells.

    PubMed

    Ramil, Carlo P; Dong, Maoqing; An, Peng; Lewandowski, Tracey M; Yu, Zhipeng; Miller, Laurence J; Lin, Qing

    2017-09-15

    A new bioorthogonal reactant pair, spiro[2.3]hex-1-ene (Sph) and 3,6-di(2-pyridyl)-s-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels-Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as trans-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells.

  18. Subclass-specific labeling of protein-reactive natural products with customized nucleophilic probes.

    PubMed

    Rudolf, Georg C; Koch, Maximilian F; Mandl, Franziska A M; Sieber, Stephan A

    2015-02-23

    Natural products represent a rich source of bioactive compounds that constitute a large fraction of approved drugs. Among those are molecules with electrophilic scaffolds, such as Michael acceptors, β-lactams, and epoxides that irreversibly inhibit essential enzymes based on their catalytic mechanism. In the search for novel bioactive molecules, current methods are challenged by the frequent rediscovery of known chemical entities. Herein small nucleophilic probes that attack electrophilic natural products and enhance their detection by HPLC-UV and HPLC-MS are introduced. A screen of diverse probe designs revealed one compound with a desired selectivity for epoxide- and maleimide-based antibiotics. Correspondingly, the natural products showdomycin and phosphomycin could be selectively targeted in extracts of their natural producing organism, in which the probe-modified molecules exhibited superior retention and MS detection relative to their unmodified counterparts. This method may thus help to discover small, electrophilic molecules that might otherwise easily elude detection in complex samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Label-free detection of the aptamer binding on protein patterns using Kelvin probe force microscopy (KPFM).

    PubMed

    Gao, Pei; Cai, Yuguang

    2009-05-01

    Anti-lysozyme aptamers are found to preferentially bind to the edge of a tightly packed lysozyme pattern. Such edge-binding is due to the better accessibility and flexibility of the edge lysozyme molecules. Kelvin probe force microscopy (KPFM) was used to study the aptamer-lysozyme binding. Our results show that KPFM is capable of detecting the aptamer-protein binding down to the 30 nm scale. The surface potential of the aptamer-lysozyme complex is approximately 12 mV lower than that of the lysozyme. The surface potential images of the aptamer-bound lysozyme patterns have the characteristic shoulder steps around the pattern edge, which is much wider than that of a clean lysozyme pattern. These results demonstrate the potentials of KPFM as a label-free method for the detection of protein-DNA interactions.

  20. A constellation of deuterium-labeled silanes as a simple mechanistic probe not requiring absolute configuration determination.

    PubMed

    Fallon, Thomas; Oestreich, Martin

    2015-10-12

    A new stereochemical probe for mechanisms at the silicon atom that is based on a deuterium-labeled silolane is synthesized and evaluated. The key synthetic step involves the hydrogenation of a 2,5-dihydrosilole with deuterium gas, giving a complex mixture of isochronic stereoisotopologues. The overall stereochemical imbalance of this mixture is evident in its (2) H NMR spectrum, which provides a good qualitative measure of changes in the configuration at the silicon atom. The technique is rapid, easy to use, and overcomes limitations and biases of traditional methods. The utility of this new procedure is demonstrated by tracking the stereochemical course of several classical reactions as well as contemporary catalytic transformations involving bond formation at the silicon atom.

  1. Magnetic Particle Spectroscopy Reveals Dynamic Changes in the Magnetic Behavior of Very Small Superparamagnetic Iron Oxide Nanoparticles During Cellular Uptake and Enables Determination of Cell-Labeling Efficacy.

    PubMed

    Poller, Wolfram C; Löwa, Norbert; Wiekhorst, Frank; Taupitz, Matthias; Wagner, Susanne; Möller, Konstantin; Baumann, Gert; Stangl, Verena; Trahms, Lutz; Ludwig, Antje

    2016-02-01

    In vivo tracking of nanoparticle-labeled cells by magnetic resonance imaging (MRI) crucially depends on accurate determination of cell-labeling efficacy prior to transplantation. Here, we analyzed the feasibility and accuracy of magnetic particle spectroscopy (MPS) for estimation of cell-labeling efficacy in living THP-1 cells incubated with very small superparamagnetic iron oxide nanoparticles (VSOP). Cell viability and proliferation capacity were not affected by the MPS measurement procedure. In VSOP samples without cell contact, MPS enabled highly accurate quantification. In contrast, MPS constantly overestimated the amount of cell associated and internalized VSOP. Analyses of the MPS spectrum shape expressed as harmonic ratio A₅/A₃ revealed distinct changes in the magnetic behavior of VSOP in response to cellular uptake. These changes were proportional to the deviation between MPS and actual iron amount, therefore allowing for adjusted iron quantification. Transmission electron microscopy provided visual evidence that changes in the magnetic properties correlated with cell surface interaction of VSOP as well as with alterations of particle structure and arrangement during the phagocytic process. Altogether, A₅/A₃-adjusted MPS enables highly accurate, cell-preserving VSOP quantification and furthermore provides information on the magnetic characteristics of internalized VSOP.

  2. Dopamine transport sites selectively labeled by a novel photoaffinity probe: 125I-DEEP

    SciTech Connect

    Grigoriadis, D.E.; Wilson, A.A.; Lew, R.; Sharkey, J.S.; Kuhar, M.J. )

    1989-08-01

    The dopamine transporter was labeled using a photosensitive compound related to GBR-12909, {sup 125}I-1-(2-(diphenylmethoxy)ethyl)-4-(2- (4-azido-3-iodophenyl)ethyl)piperazine ({sup 125}I-DEEP). {sup 125}I-DEEP bound reversibly and with high affinity to the dopamine transport protein in the absence of light and could be covalently attached to the protein following exposure to UV light. In rat striatal homogenates, {sup 125}I-DEEP was found to incorporate covalently into a protein with apparent molecular weight of 58,000 Da. The properties of this binding protein were characteristic of the dopamine transporter since covalent attachment could be inhibited by dopamine-uptake blockers with the proper pharmacological rank order of potencies. Covalent binding was also inhibited in a stereospecific manner by (+) and (-) cocaine, as well as other cocaine analogs. The protein was not found in the cerebellum. The dopamine transporter appears to exist in a glycosylated form since photoaffinity-labeled transport sites could adsorb to wheat germ-agglutinin and could be specifically eluted from the column by beta-N-acetylglucosamine.

  3. Functional molecular lumino-materials to probe serum albumins: solid phase selective staining through noncovalent fluorescent labeling.

    PubMed

    Dey, Gourab; Gupta, Abhishek; Mukherjee, Trinetra; Gaur, Pankaj; Chaudhary, Abhishek; Mukhopadhyay, Subhra Kanti; Nandi, Chayan K; Ghosh, Subrata

    2014-07-09

    Selective staining of human serum albumin protein in gel electrophoresis over wide range of other protein(s) is extremely important because it contains more than 60% volume of serum fluid in human body. Given the nonexistence of suitable dye materials for selective staining of serum albumins in gel electrophoresis, we report a new class of easy synthesizable and low molecular weight staining agents based on 3-amino-N-alkyl-carbazole scaffold for selective staining of serum albumins in solid phase. A detailed structure-efficiency relationship (SER) study enabled us to develop two such potent functional molecular probes which stain both human and bovine serum albumin selectively in gel electrophoresis in the presence of other proteins and enzymes. The present gel staining process was found to be very simple and less time-consuming as compared to the conventional coomassie blue staining which in turn makes these probes a new class of serum albumin-specific staining materials in proteome research. Moreover, these molecular lumino-materials can detect serum albumins at subnanomolar level in the presence of broad spectrum of other proteins/enzymes in aqueous buffer (99.9% water, pH = 7.3) keeping the protein secondary structure intact. Our experimental and the docking simulation results show that these probes bind preferentially at 'binding site I' of both the serum proteins.

  4. Designed diblock hairpin probes for the nonenzymatic and label-free detection of nucleic acid.

    PubMed

    Wen, Junlin; Chen, Junhua; Zhuang, Li; Zhou, Shungui

    2016-05-15

    The detection of nucleic acid sequences is of great importance in a variety of fields. An ultrasensitive DNA sensing platform is constructed using elaborately designed diblock hairpin probes (DHPs) that are composed of hairpin and poly-adenine blocks. The introduction of an initiator DNA target triggers the catalytic assembly of probes DHP1, DHP2 and DHP3 to fabricate numerous poly-adenine-tailed branched DNA junctions, which significantly amplify the signal of the target-DNA-recognizing event without any enzyme. Coupled to a gold nanoparticle-based colorimetric assay, the amplified recognition signal can be quantitatively detected or visually read with the naked eye. The combination of the high-efficiency target-catalyzed DHP assembly and sensitive gold-based colorimetric assay offers an ultrasensitive detection of DNA with a detection limit of 0.1 pM and a dynamic range from 0.01 to 5 pM. The proposed sensing platform can discriminate even single-base mutations. Moreover, the sensing platform can be expanded to detect pollutant-degrading-bacteria-specific DNA sequences. The proposed sensing system should offer an alternative approach for the detection of nucleic acids in the fields of microbiology, biogeochemistry, and environmental sciences. Copyright © 2016. Published by Elsevier B.V.

  5. Label-free silicon quantum dots as fluorescent probe for selective and sensitive detection of copper ions.

    PubMed

    Zhao, Jiangna; Deng, Jianhui; Yi, Yinhui; Li, Haitai; Zhang, Youyu; Yao, Shouzhuo

    2014-07-01

    In this work, label-free silicon quantum dots (SiQDs) were used as a novel fluorescence probe for the sensitive and selective detection of Cu(2+). The fluorescence of the SiQDs was effectively quenched by H2O2 from the reaction of ascorbic acid with O2, and hydroxyl radicals from Fenton reaction between H2O2 and Cu(+). The fluorescence intensity of SiQDs was quenched about 25% in 15 min after the addition of H2O2 (1mM). While the SiQDs was incubated with AA (1mM) and Cu(2+) (1 µM) under the same conditions, the fluorescence intensity of SiQDs decreased about 55%. Obviously, the recycling of Cu(2+) in the test system may lead to a dramatical decrease in the fluorescence of SiQDs. Under the optimized experimental conditions, the rate of fluorescence quenching of SiQDs was linearly dependent on the Cu(2+) concentration ranging from 25 to 600 nM with the limit of detection as low as 8 nM, which was much lower than that of existing methods. Moreover, the probe was successfully applied to the determination of Cu(2+) in different environmental water samples and human hair.

  6. Proteome-wide Discovery and Characterizations of Nucleotide-binding Proteins with Affinity-labeled Chemical Probes

    PubMed Central

    Xiao, Yongsheng; Guo, Lei; Jiang, Xinning; Wang, Yinsheng

    2013-01-01

    Nucleotide-binding proteins play pivotal roles in many cellular processes including cell signaling. However, targeted study of sub-proteome of nucleotide-binding proteins, especially protein kinases and GTP-binding proteins, remained challenging. Here, we reported a general strategy in using affinity-labeled chemical probes to enrich, identify, and quantify ATP- and GTP-binding proteins in the entire human proteome. Our results revealed that the ATP/GTP affinity probes facilitated the identification of 100 GTP-binding proteins and 206 kinases with the use of low mg quantities of lysate of HL-60 cells. In combination with the use of SILAC-based quantitative proteomics method, we assessed the ATP/GTP binding selectivities of nucleotide-binding proteins at the global proteome scale. Our results confirmed known and, more importantly, unveiled new ATP/GTP-binding preferences of hundreds of nucleotide-binding proteins. Additionally, our strategy led to the identification of three and one unique nucleotide-binding motifs for kinases and GTP-binding proteins, respectively, and the characterizations of the nucleotide binding selectivities of individual motifs. Our strategy for capturing and characterizing ATP/GTP-binding proteins should be generally applicable for those proteins that can interact with other nucleotides. PMID:23413923

  7. Dynamical transition in molecular glasses and proteins observed by spin relaxation of nitroxide spin probes and labels

    NASA Astrophysics Data System (ADS)

    Golysheva, Elena A.; Shevelev, Georgiy Yu.; Dzuba, Sergei A.

    2017-08-01

    In glassy substances and biological media, dynamical transitions are observed in neutron scattering that manifests itself as deviations of the translational mean-squared displacement, , of hydrogen atoms from harmonic dynamics. In biological media, the deviation occurs at two temperature intervals, at ˜100-150 K and at ˜170-230 K, and it is attributed to the motion of methyl groups in the former case and to the transition from harmonic to anharmonic or diffusive motions in the latter case. In this work, electron spin echo (ESE) spectroscopy—a pulsed version of electron paramagnetic resonance—is applied to study the spin relaxation of nitroxide spin probes and labels introduced in molecular glass former o-terphenyl and in protein lysozyme. The anisotropic contribution to the rate of the two-pulse ESE decay, ΔW, is induced by spin relaxation appearing because of restricted orientational stochastic molecular motion; it is proportional to <α2>τc, where <α2> is the mean-squared angle of reorientation of the nitroxide molecule around the equilibrium position and τc is the correlation time of reorientation. The ESE time window allows us to study motions with τc < 10-7 s. For glassy o-terphenyl, the <α2>τc temperature dependence shows a transition near 240 K, which is in agreement with the literature data on . For spin probes of essentially different size, the obtained data were found to be close, which evidences that motion is cooperative, involving a nanocluster of several neighboring molecules. For the dry lysozyme, the <α2>τc values below 260 K were found to linearly depend on the temperature in the same way as it was observed in neutron scattering for . As spin relaxation is influenced only by stochastic motion, the harmonic motions seen in ESE must be overdamped. In the hydrated lysozyme, ESE data show transitions near 130 K for all nitroxides, near 160 K for the probe located in the hydration layer, and near 180 K for the label in the

  8. Correction: NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro.

    PubMed

    Lee, Ronald F S; Escrig, Stéphane; Croisier, Marie; Clerc-Rosset, Stéphanie; Knott, Graham W; Meibom, Anders; Davey, Curt A; Johnsson, Kai; Dyson, Paul J

    2015-11-28

    Correction for 'NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro' by Ronald F. S. Lee et al., Chem. Commun., 2015, DOI: 10.1039/c5cc06983a.

  9. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.

    PubMed

    Němcová, Kateřina; Sebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-09-01

    In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT20 tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.

  10. Hydrophobic Treatment Enabling Analysis of Wettable Surfaces using a Liquid Microjunction Surface Sampling Probe/Electrospray Ionization-Mass Spectrometry System

    SciTech Connect

    Walworth, Matthew J; Stankovich, Joseph J; Van Berkel, Gary J; Schulz, Michael; Minarik, susanne; Nichols, Judy; Reich, Eike

    2011-01-01

    An aerosol application procedure involving one or more commercially available silicone based products was developed to create hydrophobic surfaces that enable analysis of otherwise wettable, absorbent surfaces using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system. The treatment process resulted in a hydrophobic surface that enabled formation of the requisite probe - to - surface liquid microjunction for sampling and allowed efficient extraction of the analytes from the surface, but did not contribute significant chemical background in the mass spectra. The utility of this treatment process was demonstrated with the treatment of wettable high performance thin layer chromatography plates, post plate development, and their subsequent analysis with the sampling probe. The surface treatment process for different surface types was described and explained and the effectiveness of the treatment and subsequent analysis was illustrated using alkaloids from Goldenseal (Hydrastis canadensis) root separated on a normal phase silica gel 60 F254S plate and peptides from protein tryptic digests separated on a Protochrom HPTLC Silica gel 60 F254S plate and a Protochrom HPTLC cellulose sheet. This simple surface treatment process significantly expands the analytical surfaces that can be analyzed with the liquid microjunction surface sampling probe, and therefore, also expands the analytical utility of this liquid extraction based surface sampling approach.

  11. Hydrophobic treatment enabling analysis of wettable surfaces using a liquid microjunction surface sampling probe/electrospray ionization-mass spectrometry system.

    PubMed

    Walworth, Matthew J; Stankovich, Joseph J; Van Berkel, Gary J; Schulz, Michael; Minarik, Susanne; Nichols, Judy; Reich, Eike

    2011-01-15

    An aerosol application procedure involving one or more commercially available silicone-based products was developed to create hydrophobic surfaces that enable analysis of otherwise wettable, absorbent surfaces using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system. The treatment process resulted in a hydrophobic surface that enabled formation of the requisite probe-to-surface liquid microjunction for sampling and allowed efficient extraction of the analytes from the surface, but did not contribute significant chemical background in the mass spectra. The utility of this treatment process was demonstrated with the treatment of wettable high-performance thin layer chromatography plates, post-plate development, and their subsequent analysis with the sampling probe. The surface treatment process for different surface types was described and explained and the effectiveness of the treatment and subsequent analysis was illustrated using alkaloids from goldenseal (Hydrastis canadensis) root separated on a normal phase silica gel 60 F(254S) plate and peptides from protein tryptic digests separated on a ProteoChrom HPTLC Silica gel 60 F(254S) plate and a ProteoChrom HPTLC Cellulose sheet. This simple surface treatment process significantly expands the analytical surfaces that can be analyzed with the liquid microjunction surface sampling probe, and therefore, also expands the analytical utility of this liquid extraction based surface sampling approach.

  12. F-18 Labeled RGD Probes Based on Bioorthogonal Strain-Promoted Click Reaction for PET Imaging.

    PubMed

    Kim, Hye Lan; Sachin, Kalme; Jeong, Hyeon Jin; Choi, Wonsil; Lee, Hyun Soo; Kim, Dong Wook

    2015-04-09

    A series of fluorine-substituted monomeric and dimeric cRGD peptide derivatives, such as cRGD-ADIBOT-F (ADIBOT = azadibenzocyclooctatriazole), di-cRGD-ADIBOT-F, cRGD-PEG5-ADIBOT-F, and di-cRGD-PEG5-ADIBOT-F, were prepared by strain-promoted alkyne azide cycloaddition (SPAAC) reaction of the corresponding aza-dibenzocyclooctyne (ADIBO) substituted peptides with a fluorinated azide 3. Among these cRGD derivatives, di-cRGD-PEG5-ADIBOT-F had the highest binding affinity in a competitive binding assay compared to other derivatives and even the original cRGDyk. On the basis of the in vitro study results, di-cRGD-PEG5-ADIBOT-(18)F was prepared from a SPAAC reaction with (18)F-labeled azide and subsequent chemo-orthogonal scavenger-assisted separation without high performance liquid chromatography (HPLC) purification in 92% decay-corrected radiochemical yield (dcRCY) with high specific activity for further in vivo positron emission tomography (PET) imaging study. In vivo PET imaging study and biodistribution data showed that this radiotracer allowed successful visualization of tumors with good tumor-to-background contrast and significantly higher tumor uptake compared to other major organs.

  13. Fluorescent probes as a tool for labelling and tracking the amphibian chytrid fungus Batrachochytrium dendrobatidis.

    PubMed

    Herbert, Sarah M; Leung, Tommy L F; Bishop, Phillip J

    2011-09-09

    The dissemination of the virulent pathogen Batrachochytrium dendrobatidis (Bd) has contributed to the decline and extinction of many amphibian species worldwide. Several different strains have been identified, some of which are sympatric. Interactions between co-infecting strains of a pathogen can have significant influences on disease epidemiology and evolution; therefore the dynamics of multi-strain infections is an important area of research. We stained Bd cells with 2 fluorescent BODIPY fatty acid probes to determine whether these can potentially be used to distinguish and track Bd cell lines in multi-strain experiments. Bd cells in broth culture were stained with 5 concentrations of green-fluorescent BODIPY FL and red-fluorescent BODIPY 558/568 and visualised under an epifluorescent microscope for up to 16 d post-dye. Dyed strains were also assessed for growth inhibition. The most effective concentration for both dyes was 10 pM. This concentration of dye produced strong fluorescence for 12 to 16 d in Bd cultures held at 23 degrees C (3 to 4 generations), and did not inhibit Bd growth. Cells dyed with BODIPY FL and BODIPY 558/568 can be distinguished from each other on the basis of their fluorescence characteristics. Therefore, it is likely that this technique will be useful for research into multi-strain dynamics of Bd infections.

  14. The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.

    PubMed

    Liu, Yu; Miao, Kun; Dunham, Noah P; Liu, Hongbin; Fares, Matthew; Boal, Amie K; Li, Xiaosong; Zhang, Xin

    2017-03-21

    The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a "channel dye" into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation-π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation-π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation-π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors.

  15. The Cation−π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification

    PubMed Central

    2017-01-01

    The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a “channel dye” into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation−π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation−π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation−π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors. PMID:28221782

  16. Motility imaging via optical coherence phase microscopy enables label-free monitoring of tissue growth and viability in 3D tissue-engineering scaffolds.

    PubMed

    Holmes, Christina; Tabrizian, Maryam; Bagnaninchi, Pierre O

    2015-05-01

    As the field of tissue engineering continues to progress, there is a deep need for non-invasive, label-free imaging technologies that can monitor tissue growth and health within thick three-dimensional (3D) constructs. Amongst the many imaging modalities under investigation, optical coherence tomography (OCT) has emerged as a promising tool, enabling non-destructive in situ characterization of scaffolds and engineered tissues. However, the lack of optical contrast between cells and scaffold materials using this technique remains a challenge. In this communication, we show that mapping the optical phase fluctuations resulting from cellular viability and motility allows for the distinction of live cells from their surrounding scaffold environment. Motility imaging was performed via a common-path optical coherence phase microscope (OCPM), an OCT modality that has been shown to be sensitive to nanometer-level fluctuations. More specifically, we examined the development of human adipose-derived stem cells and/or murine pre-osteoblasts within two distinct scaffold systems, commercially available alginate sponges and custom-microfabricated poly(d, l-lactic-co-glycolic acid) fibrous scaffolds. Cellular motility is demonstrated as an endogenous source of contrast for OCPM, enabling real-time, label-free monitoring of 3D engineered tissue development.

  17. The Internal Dynamics of Mini c TAR DNA Probed by EPR of Nitroxide Spin Labels at the Lower Stem, the Loop, and the Bulge †

    PubMed Central

    Sun, Yan; Zhang, Ziwei; Grigoryants, Vladimir M.; Myers, William K.; Liu, Fei; Earle, Keith A.; Freed, Jack H.; Scholes, Charles P.

    2012-01-01

    Electron paramagnetic resonance (EPR) at 236.6 GHz and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, the upper loop, and near the bulge of mini c TAR DNA. High frequency 236.6 GHz EPR, not previously applied to spin labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2′-amino cytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously suggested to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ3 RNA, but at 5 °C the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from zero to two. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA all labels reported tumbling times > 5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bi-labels attached near the 3′ and 5′ terminals showed evidence for an NCp7-induced increase in the 3′ - 5 ′end-to-end distance distribution and a partially melted stem. PMID:23009298

  18. Deuteration of Hyperpolarized (13) C-Labeled Zymonic Acid Enables Sensitivity-Enhanced Dynamic MRI of pH.

    PubMed

    Hundshammer, Christian; Düwel, Stephan; Köcher, Simone S; Gersch, Malte; Feuerecker, Benedikt; Scheurer, Christoph; Haase, Axel; Glaser, Steffen J; Schwaiger, Markus; Schilling, Franz

    2017-09-20

    Aberrant pH is characteristic of many pathologies such as ischemia, inflammation or cancer. Therefore, a non-invasive and spatially resolved pH determination is valuable for disease diagnosis, characterization of response to treatment and the design of pH-sensitive drug-delivery systems. We recently introduced hyperpolarized [1,5-(13) C2 ]zymonic acid (ZA) as a novel MRI probe of extracellular pH utilizing dissolution dynamic polarization (DNP) for a more than 10000-fold signal enhancement of the MRI signal. Here we present a strategy to enhance the sensitivity of this approach by deuteration of ZA yielding [1,5-(13) C2 , 3,6,6,6-D4 ]zymonic acid (ZAd ), which prolongs the liquid state spin lattice relaxation time (T1 ) by up to 39 % in vitro. Measurements with ZA and ZAd on subcutaneous MAT B III adenocarcinoma in rats show that deuteration increases the signal-to-noise ratio (SNR) by up to 46 % in vivo. Furthermore, we demonstrate a proof of concept for real-time imaging of dynamic pH changes in vitro using ZAd , potentially allowing for the characterization of rapid acidification/basification processes in vivo. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Protein tethering enables rapid and label-free SERS platform for screening drugs of abuse (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Siddhanta, Soumik; Wróbel, Maciej S.; Barman, Ishan

    2017-02-01

    A quick, cost-effective method for detection of drugs of abuse in biological fluids would be of great value in healthcare, law enforcement, and home testing applications. The alarming rise in narcotics abuse has led to considerable focus on developing potent and versatile analytical tools that can address this societal problem. While laboratory testing plays a key role in the current detection of drug misuse and the evaluation of patients with drug induced intoxication, these typically require expensive reagents and trained personnel, and may take hours to complete. Thus, a significant unmet need is to engineer a facile method that can rapidly detect drugs with little sample preparation, especially the bound fraction that is typically dominant in the blood stream. Here we report an approach that combines the exquisite sensitivity of surface enhanced Raman spectroscopy (SERS) and a facile protein tethering mechanism to reliably detect four different classes of drugs, barbiturate, benzodiazepine, amphetamine and benzoylecgonine. The proposed approach harnesses the reliable and specific attachment of proteins to both drugs and nanoparticle to facilitate the enhancement of spectral markers that are sensitive to the presence of the drugs. In conjunction with chemometric tools, we have shown the ability to quantify these drugs lower than levels achievable by existing clinical immunoassays. Through molecular docking simulations, we also probe the mechanistic underpinnings of the protein tethering approach, opening the door to detection of a broad class of narcotics in biological fluids within a few minutes as well as for groundwater analysis and toxin detection.

  20. Two-photon excited fluorescence of intrinsic fluorophores enables label-free assessment of adipose tissue function

    NASA Astrophysics Data System (ADS)

    Alonzo, Carlo Amadeo; Karaliota, Sevasti; Pouli, Dimitra; Liu, Zhiyi; Karalis, Katia P.; Georgakoudi, Irene

    2016-08-01

    Current methods for evaluating adipose tissue function are destructive or have low spatial resolution. These limit our ability to assess dynamic changes and heterogeneous responses that occur in healthy or diseased subjects, or during treatment. Here, we demonstrate that intrinsic two-photon excited fluorescence enables functional imaging of adipocyte metabolism with subcellular resolution. Steady-state and time-resolved fluorescence from intracellular metabolic co-factors and lipid droplets can distinguish the functional states of excised white, brown, and cold-induced beige fat. Similar optical changes are identified when white and brown fat are assessed in vivo. Therefore, these studies establish the potential of non-invasive, high resolution, endogenous contrast, two-photon imaging to identify distinct adipose tissue types, monitor their functional state, and characterize heterogeneity of induced responses.

  1. Fiber optic probe enabled by surface-enhanced Raman scattering for early diagnosis of potential acute rejection of kidney transplant

    NASA Astrophysics Data System (ADS)

    Chi, Jingmao; Chen, Hui; Tolias, Peter; Du, Henry

    2014-06-01

    We have explored the use of a fiber-optic probe with surface-enhanced Raman scattering (SERS) sensing modality for early, noninvasive and, rapid diagnosis of potential renal acute rejection (AR) and other renal graft dysfunction of kidney transplant patients. Multimode silica optical fiber immobilized with colloidal Ag nanoparticles at the distal end was used for SERS measurements of as-collected urine samples at 632.8 nm excitation wavelength. All patients with abnormal renal graft function (3 AR episodes and 2 graft failure episodes) who were clinically diagnosed independently show common unique SERS spectral features in the urines collected just one day after transplant. SERS-based fiber-optic probe has excellent potential to be a bedside tool for early diagnosis of kidney transplant patients for timely medical intervention of patients at high risk of transplant dysfunction.

  2. Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics

    PubMed Central

    Lo, Camden Yeung-Wah; Chen, Sijie; Creed, Sarah Jayne; Kang, Miaomiao; Zhao, Na; Tang, Ben Zhong; Elgass, Kirstin Diana

    2016-01-01

    Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology. PMID:27492961

  3. Luminol functionalized gold nanoparticles as colorimetric and chemiluminescent probes for visual, label free, highly sensitive and selective detection of minocycline

    NASA Astrophysics Data System (ADS)

    He, Yi; Peng, Rufang

    2014-11-01

    In this work, luminol functionalized gold nanoparticles (LuAuNPs) were used as colorimetric and chemiluminescent probes for visual, label free, sensitive and selective detection of minocycline (MC). The LuAuNPs were prepared by simple one-pot reduction of HAuCl4 with luminol, which exhibited a good chemiluminescence (CL) activity owing to the presence of luminol molecules on their surface and surface plasmon resonance absorption. In the absence of MC, the color of LuAuNPs was wine red and their size was relatively small (˜25 nm), which could react with silver nitrate, producing a strong CL emission. Upon the addition of MC at acidic buffer solutions, the electrostatic interaction between positively charged MC and negatively charged LuAuNPs caused the aggregation of LuAuNPs, generating a purple or blue color. Simultaneously, the aggregated LuAuNPs did not effectively react with silver nitrate, producing a weak CL emission. The signal change was linearly dependent on the logarithm of MC concentration in the range from 30 ng to 1.0 μg for colorimetric detection and from 10 ng to 1.0 μg for CL detection. With colorimetry, a detection limit of 22 ng was achieved, while the detection limit for CL detection modality was 9.7 ng.

  4. Selective and sensitive detection of acetylcholinesterase activity using denatured protein-protected gold nanoclusters as a label-free probe.

    PubMed

    Li, Hongchang; Guo, Yuxin; Xiao, Lehui; Chen, Bo

    2014-01-07

    Based on the fluorescence quenching of novel denatured protein-protected gold nanoclusters, a label-free detection method of acetylcholinesterase (AChE) activity has been developed. Using denatured bovine serum albumin (dBSA), in which 35 cysteine residues can interact polyvalently with Au nanoclusters (AuNCs) as a stabilizing agent, water-soluble and stable fluorescent gold nanoclusters were synthesized. The fluorescence of the AuNCs was quenched by thiocholine that was produced from the AChE hydrolysis of S-acetylthiocholine iodide (ACTI) to detect the AChE activity. The linear range of the method was 0.005-0.15 U mL(-1). The limit of detection (LOD) was 0.02 mU mL(-1). Other enzymes and metal ions, i.e., GPT, γ-GT, GOx, K(+), Ca(2+) and Na(+), showed minimal interference. Using the fluorescence probe, satisfactory results for the detection of the AChE activity in human serum were obtained.

  5. Luminol functionalized gold nanoparticles as colorimetric and chemiluminescent probes for visual, label free, highly sensitive and selective detection of minocycline.

    PubMed

    He, Yi; Peng, Rufang

    2014-11-14

    In this work, luminol functionalized gold nanoparticles (LuAuNPs) were used as colorimetric and chemiluminescent probes for visual, label free, sensitive and selective detection of minocycline (MC). The LuAuNPs were prepared by simple one-pot reduction of HAuCl₄ with luminol, which exhibited a good chemiluminescence (CL) activity owing to the presence of luminol molecules on their surface and surface plasmon resonance absorption. In the absence of MC, the color of LuAuNPs was wine red and their size was relatively small (∼25 nm), which could react with silver nitrate, producing a strong CL emission. Upon the addition of MC at acidic buffer solutions, the electrostatic interaction between positively charged MC and negatively charged LuAuNPs caused the aggregation of LuAuNPs, generating a purple or blue color. Simultaneously, the aggregated LuAuNPs did not effectively react with silver nitrate, producing a weak CL emission. The signal change was linearly dependent on the logarithm of MC concentration in the range from 30 ng to 1.0 μg for colorimetric detection and from 10 ng to 1.0 μg for CL detection. With colorimetry, a detection limit of 22 ng was achieved, while the detection limit for CL detection modality was 9.7 ng.

  6. Apoferritin Protein Nanoparticles Dually labeled with Aptamer and Horseradish Peroxidase as a Sensing Probe for Thrombin Detection

    SciTech Connect

    Zhao, Jie; Liu, Meiling; Zhang, Youyu; Li, Haitao; Lin, Yuehe; Yao, Shouzhuo

    2013-01-08

    A sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Aptamer1 (Apt1) loaded on core/shell Fe3O4/Au magnetic nanoparticle (AuMNP) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5 pM to 100 pM with a detection limit of 0.07 pM (S/N = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in biomarker detection and disease diagnosis. --------------------------------------------------------------------------------

  7. Probing the Secondary Structure of Membrane Peptides Using (2)H-Labeled d(10)-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy.

    PubMed

    Liu, Lishan; Sahu, Indra D; McCarrick, Robert M; Lorigan, Gary A

    2016-02-04

    Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing (2)H isotopic labeling and site-directed spin-labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was (2)H-labeled, such as (2)H-labeled d10-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as i + 1 to i + 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with (2)H-labeled d10-Leu, four Leu residues within the AChR M2δ peptide were fully mapped out using this ESEEM method. Unique (2)H-ESEEM patterns were observed with the (2)H-labeled d10-Leu for the AChR M2δ α-helical model peptide. For proteins and peptides with an α-helical secondary structure, deuterium modulation can be clearly observed for i ± 3 and i ± 4 samples, but not for i ± 2 samples. Also, a deuterium peak centered at the (2)H Larmor frequency of each i ± 4 sample always had a significantly higher intensity than the corresponding i + 3 sample. This unique feature can be potentially used to distinguish an α-helix from a π-helix or 310-helix. Moreover, (2)H modulation depth for ESEEM samples on Leu10 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2δ peptide as suggested by previous MD simulations and NMR experiments.

  8. The internal dynamics of mini c TAR DNA probed by electron paramagnetic resonance of nitroxide spin-labels at the lower stem, the loop, and the bulge.

    PubMed

    Sun, Yan; Zhang, Ziwei; Grigoryants, Vladimir M; Myers, William K; Liu, Fei; Earle, Keith A; Freed, Jack H; Scholes, Charles P

    2012-10-30

    Electron paramagnetic resonance (EPR) at 236.6 and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, in the upper loop, and near the bulge of mini c TAR DNA. High-frequency 236.6 GHz EPR, not previously applied to spin-labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2'-aminocytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously thought to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ(3) RNA, but at 5 °C, the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from 0 to 2. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA, all labels reported tumbling times of >5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bilabels attached near the 3' and 5' termini showed evidence of an NCp7-induced increase in the 3'-5' end-to-end distance distribution and a partially melted stem.

  9. Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences.

    PubMed

    Ahn, Jeong Jin; Kim, Youngjoo; Lee, Seung Yong; Hong, Ji Young; Kim, Gi Won; Hwang, Seung Yong

    2015-09-01

    Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (Tm) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Bias assisted scanning probe microscopy direct write lithography enables local oxygen enrichment of lanthanum cuprates thin films

    SciTech Connect

    Lavini, Francesco; Yang, Nan; Vasudevan, Rama K.; Strelcov, Evgheni; Jesse, Stephen; Okatan, Mahmut Baris; Kravchenko, Ivan I.; Di Castro, Daniele; Kalinin, Sergei V.; Balestrino, Giuseppe; Foglietti, Vittorio; Aruta, Carmela

    2015-01-01

    Here, scanning probe bias techniques have been used as a method to locally dope thin epitaxial films of La2CuO4 (LCO) fabricated by pulsed laser deposition. The local electrochemical oxidation of LCO very efficiently introduces interstitial oxygen defects in the thin film. Details on the influence of the tip voltage bias and environmental conditions on the surface morphology have been investigated. The results show that a local uptake of oxygen occurs in the oxidized films.

  11. Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

    PubMed

    Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

    2014-09-01

    A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Study of tropomyosin labelled with a fluorescent probe by pulse fluorimetry in polarized light. Interaction of that protein with troponin and actin.

    PubMed

    Wahl, P; Tawada, K; Auchet, J C

    1978-08-01

    Tropomyosin has been labelled with a fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine which is presumed to bind preferentially to the unique reactive cysteine residue of the alpha chain. Anisotropy decay measurements show that tropomyosin monomer and polymer are flexible molecules. This flexibility decreases when troponin interacts with tropomyosin, and is partially restored by a micromolar concentration of Ca2+.

  13. Evaluation of 68Ga-Labeled MG7 Antibody: A Targeted Probe for PET/CT Imaging of Gastric Cancer

    PubMed Central

    Xu, Bing; Li, Xiaowei; Yin, Jipeng; Liang, Cong; Liu, Lijuan; Qiu, Zhaoyan; Yao, Liping; Nie, Yongzhan; Wang, Jing; Wu, Kaichun

    2015-01-01

    MG7-Ag, a specific gastric cancer-associated antigen, can be used to non-invasively monitor gastric cancer by molecular imaging with positron emission tomography/computed tomography (PET/CT). In this study, we prepared and evaluated a 68Ga-labeled MG7 antibody as a molecular probe for nanoPET/CT imaging of gastric cancer in a BGC-823 tumor xenografted mouse model. Macrocyclic chelator 1,4,7-triazacyclononane-N,N0,N00-triacetic acid (NOTA)-conjugated MG7 antibody was synthesized and radiolabeled with 68Ga (t1/2 = 67.71 min). Then, 68Ga-NOTA-MG7 was tested using in vitro cytological studies, in vivo nanoPET/CT and Cerenkov imaging studies as well as ex vivo biodistribution and histology studies. The in vitro experiments demonstrated that 68Ga-NOTA-MG7 has an excellent radiolabeling efficiency of approximately 99% without purification, and it is stable in serum after 120 min of incubation. Cell uptake and retention studies confirmed that 68Ga-NOTA-MG7 has good binding affinity and tumor cell retention. For the nanoPET imaging study, the predominant uptake of 68Ga-NOTA-MG7 was visualized in tumor, liver and kidneys. The tumor uptake reached at its peak (2.53 ± 0.28%ID/g) at 60 min pi. Cherenkov imaging also confirmed the specificity of tumor uptake. Moreover, the biodistribution results were consistent with the quantification data of nanoPET/CT imaging. Histologic analysis also demonstrated specific staining of BGC-823 tumor cell lines. PMID:25733152

  14. Ionic liquid-capped graphene quantum dots as label-free fluorescent probe for direct detection of ferricyanide.

    PubMed

    Sun, Xue; Qian, Yuting; Jiao, Yajie; Liu, Jiyang; Xi, Fengna; Dong, Xiaoping

    2017-04-01

    Despite complex molecular and atomic doping, efficient post-functionalization strategies for graphene quantum dots (GQDs) are of key importance to control the physicochemical properties and broaden the practical applications. With ionic liquid as specific modification agents, herein, the preparation of ionic liquid-capped GQDs (IL-GQDs) and its application as label-free fluorescent probe for direct detection of anion were reported. Hydroxyl-functionalized GQDs that could be easily gram-scale synthesized and possessed single-crystalline were chosen as the model GQDs. Also, the most commonly used ionic liquids, water-soluble 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIMBF4) was chosen as the model IL. Under the ultrasonic treatment, BMIMBF4 easily composited with GQDs to form IL-GQDs. The synthesized IL-GQDs were characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and fluorescence (FL) spectrum. After successful combination with IL, the excitation-independent photoluminescence behavior of GQDs presented almost no change, whereas, the anion responsiveness of IL-GQDs drastically improved, which afforded the IL-GQDs a sensitive response to Fe(CN)6(3-). Based on the strong fluorescence quench, a facile and sensitive detection of Fe(CN)6(3-) was achieved. A wide linear range of 1.0×10(-7) to 2.5×10(-3)moll(-1) with a low detection limit of 40 nmol l(-1) was obtained. As the composition and properties of IL and GQDs could be easily tuned by varying the structure, ionic liquids-capped GQDs might present promising potential for their applications in sensing and catalysis.

  15. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

    PubMed

    Ren, Xiaomei; El-Sagheer, Afaf H; Brown, Tom

    2016-05-05

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.

  16. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    PubMed Central

    Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

  17. [Depth-dependent investigation of the apolar zone of lipid membranes using a series of fluorescent probes, Me4-BODIPY-8-labeled phosphatidylcholines].

    PubMed

    Omel'kov, A V; Pavlova, Iu B; Boldyrev, I A; Molotkovskiĭ, Iu G

    2007-01-01

    A series of lipid probes, phosphatidylcholines labeled with Me4-BODIPY-8 (4,4-difluoro-1,3,5,7- tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl) fluorophore attached to the end of an acyl residue at different distances from the polar head, were used as depth-dependent probes for the apolar zone of model membrane systems, large unilamellar vesicles (LUVs). Data on the anisotropy of probe fluorescence demonstrated different mobility profiles for the fluorophore microenvironment in LUVs of different composition at various temperatures, which indicates a high sensitivity of these probes as tools for studying membrane systems. An interesting anomaly was observed for LUVs from dimiristoylphosphatidylcholine (DMPC) or from a DMPC-cholesterol mixture: the anisotropy of the fluorophore located near the bilayer center is larger than that of the fluorophore located further from the center; i.e., the mobility of the microenvironment is lower in the first case. This anomaly is supposed to result from the penetration of the unlabeled long chain of the probes into the opposite bilayer leaflet. Such a possibility should be taken into account in constructing fluorescent probes and interpreting the results.

  18. An interference-free and label-free sandwich-type magnetic silicon microsphere -rGO-based probe for fluorescence detection of microRNA.

    PubMed

    Li, Shiyu; He, Kui; Liao, Rong; Chen, Chunyan; Chen, Xiaoming; Cai, Changqun

    2017-11-01

    An interference-free and label-free sensing platform was developed for the highly sensitive detection of microRNA-21 (miRNA-21) in vitro by magnetic silicon microsphere (MNP)-reduced graphene oxide (rGO)-based sandwich probe. In this method, DNA capture probes (P1) were connected with MNPs at the 5' end and hybridized with completely complementary target miRNA. Subsequently, rGO was retained and induced the fluorescence quenching in the supernatant. Through the magnetic separation, the supernatant environment was simplified and the interference to analytical signal was eliminated. When DNA capture probe-modified magnetic silicon microspheres (MNP-P1) were adsorbed through rGO in the absence of a target and formed a sandwich structure, the formed nanostructure was easily removed from the solution by a magnetic field and the fluorescence intensity was maximally recovered. This proposed strategy, which both overcame the expensive and cumbersome fluorescent labeling, and eliminated interference to analytical signal for guaranteeing high signal-to-background ratio, exhibited high sensitivity with a detection limit as low as 0.098nM and special selectivity toward miRNA-21. The method was potentially applicable for not only detection of miRNA-21 but also various biomarker analyses just by changing capture probes. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Evaluation of the biotinylated (Blugene) vs sup 32 P-labeled cDNA probes of beta-glucocerebrosidase: Relative sensitivities in genomic and other systems

    SciTech Connect

    Strasberg, P. )

    1989-07-01

    The sensitivity, rapidity, and ease of use of biotinylated (Blugene) and {sup 32}P cDNA probes have been compared, the probe being the cDNA for beta-glucocerebrosidase. With the Blugene kit I could detect 2 pg of biotinylated DNA on dot blots. However, under conditions of hybridization, the lower limit of detection for unlabeled cDNA (transblotted onto nitrocellulose) by its labeled counterpart was 5000-fold smaller (10 pg vs 50 ng) for the isotopically labeled probe. {sup 32}P- and Blugene-probes hybridized detectably with 0.5 and 10 micrograms, respectively, of transblotted EcoR 1-digested genomic DNA, making the radioactive method 20 times as sensitive. However, color development was complete within 30 min to 3 h, whereas radioautoradiography required 12 h to one week. Blugene was also safer, easy to use, and effective under appropriate conditions. The {sup 32}P method is expensive, hazardous, time-consuming, and technically difficult. This nonisotopic procedure represents a desirable improvement in biotechnology.

  20. A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe.

    PubMed

    Gao, Zhong Feng; Chen, Dong Mei; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing

    2015-10-15

    Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications.

  1. In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

    PubMed Central

    Assmus, B.; Hutzler, P.; Kirchhof, G.; Amann, R.; Lawrence, J. R.; Hartmann, A.

    1995-01-01

    The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology. PMID:16534951

  2. Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2

    PubMed Central

    Vaidya, Aditya Sudheer; Karumudi, Bhargava; Mendonca, Emma; Madriaga, Antonett; Abdelkarim, Hazem; van Breemen, Richard B.; Petukhov, Pavel A.

    2012-01-01

    The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs. PMID:22771007

  3. Taking label-free optical spectroscopy techniques into the operating theatre: biopsy needles and surgical guidance probes (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Leblond, Frédéric

    2017-02-01

    Recent advances will be described relating to the development and clinical translation of optical spectroscopy techniques designed to guide surgical interventions in brain and prostate oncology applications. The use of molecular imaging guidance systems can enable true intra-operative tissue identification, increasing the effectiveness of cancer surgery and potentially positively impacting patient survival. Surgical resection is a fundamental cancer treatment, but its effectiveness is reduced by the inability to rapidly and accurately identify cancer margins. We will introduce a portable intraoperative label-free multimodal optical spectroscopy system combining intrinsic fluorescence, diffuse reflectance, and Raman spectroscopy that can identify cancer in situ during surgery. We will show that this on-line guidance system can detect primary cancer such as glioma as well as metastatic melanoma and cancer of the lung and colon with an accuracy, sensitivity, and specificity of 97%, 100%, and 93% respectively. Moreover, a method will be presented, along with preliminary tissue classification results, based on the interrogation of whole human prostates from prostatectomies. The development and in vivo validation of an optical brain needle biopsy instrument will be presented demonstrating its ability to detect bulk tumor using Raman spectroscopy with the goal of reducing the number of non-diagnostic samples during a procedure. The extraction of tissue can cause life-threatening hemorrhage because of significant blood vessel injury during the procedure. We will demonstrate that a sub-diffuse optical tomography technique integrated with a commercial biopsy needle can detect the presence of blood vessels to limit the hemorrhage risk.

  4. A highly sensitive and facile graphene oxide-based nucleic acid probe: Label-free detection of telomerase activity in cancer patient's urine using AIEgens.

    PubMed

    Ou, Xiaowen; Hong, Fan; Zhang, Zhenyu; Cheng, Yong; Zhao, Zujin; Gao, Pengcheng; Lou, Xiaoding; Xia, Fan; Wang, Shutao

    2017-03-15

    Molecular beacon (MB)-based sensing platforms that consist of a fluorogen-quencher pair play an important role in medical and biological researches. However, the synthesis of both fluorogen and quencher in the nucleic acid probes will increase the burden of organic synthesis works and induce the difficulties for precisely controlling the relative distance between fluorogen and quencher, which may lead to false-positive and false-negative results. In this work, initially we report a single labeled MB (FAM-MB, with carboxyfluorescein as fluorogen and without quencher) thus simplifies MBs with the aid of graphene oxide (GO) to detect telomerase activity. To further simplify this structure, namely label-free strategy, we design a facile, sensitive and selective platform using a label-free beacon (AIE-MB, without fluorogen and quencher), based on aggregation-induced emission fluorogen (silole-R). Upon the addition of telomerase, AIE-MB induced comb-like DNA structure leads to high aggregation of silole-R and thus exhibits strong fluorescence emission. By exploitation of this, we can detect telomerase with superior sensitivity and demonstrate their applications in bladder cancer diagnosis. Compared to single-labeled FAM-MB based telomerase activity assay, the label-free AIE-MB induced method could perform the sensitive detection with high signal-to-background ratio. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. A Novel 99mTc-Labeled Molecular Probe for Tumor Angiogenesis Imaging in Hepatoma Xenografts Model: A Pilot Study

    PubMed Central

    Zhao, Qian; Yan, Ping; Wang, Rong Fu; Zhang, Chun Li; Li, Ling; Yin, Lei

    2013-01-01

    Introduction Visualization of tumor angiogenesis using radionuclide targeting provides important diagnostic information. In previous study, we proved that an arginine-arginine-leucine (RRL) peptide should be a tumor endothelial cell specific binding sequence. The overall aim of this study was to evaluate whether 99mTc-radiolabeled RRL could be noninvasively used for imaging of malignant tumors in vivo, and act as a new molecular probe targeting tumor angiogenesis. Methods The RRL peptide was designed and radiosynthesized with 99mTc by a one-step method. The radiolabeling efficiency and radiochemical purity were then characterized in vitro. 99mTc-RRL was injected intravenously in HepG2 xenograft-bearing BALB/c nude mice. Biodistribution and in vivo imaging were performed periodically. The relationship between tumor size and %ID uptake of 99mTc-RRL was also explored. Results The labeling efficiencies of 99mTc-RRL reached 76.9%±4.5% (n = 6) within 30–60 min at room temperature, and the radiochemical purity exceeded 96% after purification. In vitro stability experiment revealed the radiolabeled peptide was stable. Biodistribution data showed that 99mTc-RRL rapidly cleared from the blood and predominantly accumulated in the kidneys and tumor. The specific uptake of 99mTc-RRL in tumor was significantly higher than that of unlabeled RRL blocking and free pertechnetate control test after injection (p<0.05). The ratio of the tumor-to-muscle exceeded 6.5, tumor-to-liver reached 1.98 and tumor-to-blood reached 1.95. In planar gamma imaging study, the tumors were imaged clearly at 2–6 h after injection of 99mTc-RRL, whereas the tumor was not imaged clearly in blocking group. The tumor-to-muscle ratio of images with 99mTc-RRL was comparable with that of 18F-FDG PET images. Immunohistochemical analysis verified the excessive vasculature of tumor. There was a linear relationship between the tumor size and uptake of 99mTc-RRL with R2 = 0.821. Conclusion 99mTc-RRL can

  6. Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.

    PubMed

    Cole, C G; Patel, K; Shipley, J; Sheer, D; Bobrow, M; Bentley, D R; Dunham, I

    1992-12-01

    The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.

  7. On-Chip Bioorthogonal Chemistry Enables Immobilization of In Situ Modified Nanoparticles and Small Molecules for Label-Free Monitoring of Protein Binding and Reaction Kinetics

    PubMed Central

    Tassa, Carlos; Liong, Monty; Hilderbrand, Scott; Sandler, Jason E.; Reiner, Thomas; Keliher, Edmund J.; Weissleder, Ralph; Shaw, Stanley Y.

    2012-01-01

    Efficient methods to immobilize small molecules under continuous-flow microfluidic conditions would greatly improve label-free molecular interaction studies using biosensor technology. At present, small-molecule immobilization chemistries require special conditions and in many cases must be performed outside the detector and microfluidic system where real-time monitoring is not possible. Here, we have developed and optimized a method for on-chip bioorthogonal chemistry that enables rapid, reversible immobilization of small molecules with control over orientation and immobilization density, and apply this technique to surface plasmon resonance (SPR) studies. Immobilized small molecules reverse the orientation of canonical SPR interaction studies, and also enable a variety of new SPR applications including on-chip assembly and interaction studies of multicomponent structures such as functionalized nanoparticles, and measurement of bioorthogonal reaction rates. We use this approach to demonstrate that on-chip assembled functionalized nanoparticles show a preserved ability to interact with their target protein, and to measure rapid bioorthogonal reaction rates with k2 > 103 M−1 s−1. This method offers multiple benefits for microfluidic biological applications, including rapid screening of targeted nanoparticles with vastly decreased nanoparticle synthetic requirements, robust immobilization chemistry in the presence of serum, and a continuous flow technique that mimics biologic contexts better than current methods used to measure bioorthogonal reaction kinetics such as NMR or UV-vis spectroscopy (e.g., stopped flow kinetics). Taken together, this approach constitutes a flexible and powerful technique for evaluating a wide variety of reactions and intermolecular interactions for in vitro or in vivo applications. PMID:22760641

  8. On-chip bioorthogonal chemistry enables immobilization of in situ modified nanoparticles and small molecules for label-free monitoring of protein binding and reaction kinetics.

    PubMed

    Tassa, C; Liong, M; Hilderbrand, S; Sandler, J E; Reiner, T; Keliher, E J; Weissleder, R; Shaw, S Y

    2012-09-07

    Efficient methods to immobilize small molecules under continuous-flow microfluidic conditions would greatly improve label-free molecular interaction studies using biosensor technology. At present, small-molecule immobilization chemistries require special conditions and in many cases must be performed outside the detector and microfluidic system where real-time monitoring is not possible. Here, we have developed and optimized a method for on-chip bioorthogonal chemistry that enables rapid, reversible immobilization of small molecules with control over orientation and immobilization density, and apply this technique to surface plasmon resonance (SPR) studies. Immobilized small molecules reverse the orientation of canonical SPR interaction studies, and also enable a variety of new SPR applications including on-chip assembly and interaction studies of multicomponent structures, such as functionalized nanoparticles, and measurement of bioorthogonal reaction rates. We use this approach to demonstrate that on-chip assembled functionalized nanoparticles show a preserved ability to interact with their target protein, and to measure rapid bioorthogonal reaction rates with k(2) > 10(3) M(-1) s(-1). This method offers multiple benefits for microfluidic biological applications, including rapid screening of targeted nanoparticles with vastly decreased nanoparticle synthetic requirements, robust immobilization chemistry in the presence of serum, and a continuous flow technique that mimics biologic contexts better than current methods used to measure bioorthogonal reaction kinetics such as NMR or UV-vis spectroscopy (e.g., stopped flow kinetics). Taken together, this approach constitutes a flexible and powerful technique for evaluating a wide variety of reactions and intermolecular interactions for in vitro or in vivo applications.

  9. H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes.

    PubMed

    Chutinimitkul, Salin; Suwannakarn, Kamol; Chieochansin, Thaweesak; Mai, Le Quynh; Damrongwatanapokin, Sudarat; Chaisingh, Arunee; Amonsin, Alongkorn; Landt, Olfert; Songserm, Thaweesak; Theamboonlers, Apiradee; Poovorawan, Yong

    2007-01-01

    A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.

  10. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

    SciTech Connect

    Hwang, Gyoyeon; Lee, Hansol; Lee, Jiyeon

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  11. Conformationally Strained trans-Cyclooctene (sTCO) Enables the Rapid Construction of 18F-PET Probes via Tetrazine Ligation

    PubMed Central

    Wang, Mengzhe; Svatunek, Dennis; Rohlfing, Katarina; Liu, Yu; Wang, Hui; Giglio, Ben; Yuan, Hong; Wu, Zhanghong; Li, Zibo; Fox, Joseph

    2016-01-01

    The bioorthogonal reaction between tetrazines and trans-cyclooctenes is a method for the rapid construction of F-18 probes for PET imaging. Described here is a second generation 18F-labeling system based on a conformationally strained trans-cyclooctene (sTCO)—a dienophile that is approximately 2 orders of magnitude more reactive than conventional TCO dienophiles. Starting from a readily prepared tosylate precursor, an 18F labeled sTCO derivative (18F-sTCO) could be synthesized in 29.3 +/- 5.1% isolated yield and with high specific activity. Tetrazine ligation was carried out with a cyclic RGD-conjugate of a diphenyl-s-tetrazine analogue (RGD-Tz) chosen from a diene class with an excellent combination of fast reactivity and stability both for the diene as well as the Diels-Alder adduct. For both the tetrazine and the sTCO, mini-PEG spacers were included to enhance solubility and improve the in vivo distribution profile of the resulting probe. Extremely fast reactivity (up to 2.86 x 105 M-1s-1 at 25 °C in water) has been observed in kinetic studies in the reaction of sTCO with diphenyl-s-tetrazine derivatives. A kinetic study on sTCO diastereomers in 55:45 MeOH:water showed that the syn-diastereomer displayed slightly faster reactivity than the anti-diastereomer. An 18F-sTCO conjugate with RGD-Tz demonstrated prominent and persistent tumor uptake in vivo with good tumor-to-background contrast. Unlike most radiolabeled RGD peptides, the tumor uptake of this PET agent increased from 5.3 +/- 0.2% ID/g at 1 h post injection (p.i.), to 8.9 +/- 0.5% ID/g at 4 h p.i., providing evidence for prolonged blood circulation. These findings suggest that tetrazine ligations employing 18F-sTCO should serve as a powerful and general platform for the rapid construction of peptide or protein derived PET agents. PMID:27162558

  12. A single fluorescent probe enables clearly discriminating and simultaneously imaging liquid-ordered and liquid-disordered microdomains in plasma membrane of living cells.

    PubMed

    Tian, Minggang; Liu, Yong; Sun, Yuming; Zhang, Ruoyao; Feng, Ruiqing; Zhang, Ge; Guo, Lifang; Li, Xuechen; Yu, Xiaoqiang; Sun, Jing Zhi; He, Xiuquan

    2017-03-01

    Liquid-ordered (Lo) and liquid-disordered (Ld) microdomains in plasma membrane play different yet essential roles in various bioactivities. However, discrimination of the two microdomains in living cells is difficult, due to the similarity in their constituents and structures. Up to now, polarity sensitive probes are the only tool for imaging the two microdomains, but their small difference between emission spectra in the two microdomains (less than 50 nm) limited their application in living cells. In this work, we first presented an aggregation/monomer type of fluorescent probe (2,7-9E-BHVC12) with much larger separation in emission wavelength (up to 100 nm), for dual-color visualizing the two membrane microdomains in living cells. The probe can form red-emissive aggregates and yellow-emissive monomers when induced by Lo and Ld microdomains, respectively, and thus enables clear visualization of the two membrane microdomains in living cells with dual colors, and thus high-fidelity images of substructures of plasma membrane have been obtained. According to the images of three kinds of normal cells and three kinds of cancer cells stained with 2,7-9E-BHVC12, significant difference in plasma membrane microstructure of cancer cells was found. In terms of 2,7-9E-BHVC12, normal cells were mainly consisted of either Lo or Ld microdomains all over their membranes, while cancer cells all clearly display coexistence of Lo and Ld membrane microdomains. Therefore, 2,7-9E-BHVC12 can serve as a powerful tool for studies of membrane microdomains, and the different results of normal and cancer cells would also deepen our understanding in cancer science. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Use of stable isotope labeled probes to facilitate liquid chromatography/mass spectrometry based high-throughput screening of time-dependent CYP inhibitors.

    PubMed

    Dasgupta, Malini; Tang, Weimin; Caldwell, Gary W; Yan, Zhengyin

    2010-08-15

    Inhibition curve shift is a commonly used approach for screening of time-dependent CYP inhibitors which requires parallel paired incubations to obtain two inhibition curves for comparison. For the control incubation, a test compound is co-incubated with a probe substrate in human liver microsomes (HLM) fortified with NADPH; for the time-dependent incubation (TDI), the test compound is pre-incubated with NADPH-fortified HLM followed by a secondary incubation with a probe substrate. For both incubations, enzyme activity is measured respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of the CYP-specific metabolite, and a TDI inhibitor can be readily identified by inhibition curve shifting as a result of CYP inactivation by the test compound during the pre-incubation. In the present study, we describe an alternative approach to facilitate TDI screening in which stable isotope labeled CYP-specific probes are used for the TDI, and non-labeled substrates are included in the control incubation. Because CYP-specific metabolites produced in the TDI are stable isotope labeled, two sets of incubation samples can be combined and then simultaneously analyzed by LC/MS/MS in the same batch run to reduce the run time. This new method has been extensively validated using both a number of known competitive and TDI inhibitors specific to five most common CYPs such as 1A2, 2C9, 2C19, 2D6, and 3A4. The assay is performed in a 96-well format and can be fully automated. Compared to the traditional method, this approach in combination with sample pooling and a short LC/MS/MS gradient significantly enhances the throughput of TDI screening and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resource.

  14. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

  15. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

  16. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RIBOSOMAL RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluroescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonu...

  17. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

    SciTech Connect

    Price, E.M.; Freisheim, J.H.

    1987-07-28

    A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.

  18. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

    PubMed Central

    Peterkin, P I; Idziak, E S; Sharpe, A N

    1991-01-01

    A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera. Images PMID:1901711

  19. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

    PubMed

    Peterkin, P I; Idziak, E S; Sharpe, A N

    1991-02-01

    A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.

  20. Isotope-labeled aspartate sidechain as a non-perturbing infrared probe: Application to investigate the dynamics of a carboxylate buried inside a protein

    NASA Astrophysics Data System (ADS)

    Abaskharon, Rachel M.; Brown, Stephen P.; Zhang, Wenkai; Chen, Jianxin; Smith, Amos B.; Gai, Feng

    2017-09-01

    Because of their negatively charged carboxylates, aspartate and glutamate are frequently found at the active or binding site of proteins. However, studying a specific carboxylate in proteins that contain multiple aspartates and/or glutamates via infrared spectroscopy is difficult due to spectral overlap. We show, herein, that isotopic-labeling of the aspartate sidechain can overcome this limitation as the resultant 13COO- asymmetric stretching vibration resides in a transparent region of the protein IR spectrum. Applicability of this site-specific vibrational probe is demonstrated by using it to assess the dynamics of an aspartate ion buried inside a small protein via two-dimensional infrared spectroscopy.

  1. Sensitive and high resolution in situ hybridization to human chromosomes using biotin labelled probes: assignment of the human thymocyte CD1 antigen genes to chromosome 1.

    PubMed Central

    Albertson, D G; Fishpool, R; Sherrington, P; Nacheva, E; Milstein, C

    1988-01-01

    A method for in situ hybridization originally developed for mapping genes in the nematode, Caenorhabditis elegans has been adapted for high resolution cytological mapping of genes in the human. The probe DNAs are labelled by incorporation of biotin dUTP and the site of hybridization detected by immunofluorescence. For the accurate assignment of the hybridization signal to chromosome bands, visualized by staining with Hoechst 33258, a heterologous ribosomal DNA probe is also included in the hybridization reaction. These rDNA signals are used as fiducial markers when aligning the two fluorescent images. We demonstrate the method by assignment of the human thymocyte CD1 antigen genes to human chromosome 1q22-23. Images PMID:3053166

  2. Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS).

    PubMed

    Dean, Lee; Kwon, Ye Jin; Philpott, M Katherine; Stanciu, Cristina E; Seashols-Williams, Sarah J; Dawson Cruz, Tracey; Sturgill, Jamie; Ehrhardt, Christopher J

    2015-07-01

    Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of

  3. Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.

    PubMed

    Röder, Christoph; König, Helmut; Fröhlich, Jürgen

    2007-09-01

    Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the construction of partially complementary 'side'-labeled probes, based on secondary structure models of the rRNA sequences. The specificity and routine applicability of the FISH-based method for yeast identification were tested by analyzing different wine isolates. Investigation of the prevalence of Dekkera/Brettanomyces yeasts in the German viticultural regions Wonnegau, Nierstein and Bingen (Rhinehesse, Rhineland-Palatinate) resulted in the isolation of 37 D. bruxellensis strains from 291 wine samples.

  4. A label-free fluorescence strategy for selective detection of nicotinamide adenine dinucleotide based on a dumbbell-like probe with low background noise.

    PubMed

    Chen, Xuexu; Lin, Chunshui; Chen, Yiying; Wang, Yiru; Chen, Xi

    2016-03-15

    In this work we developed a novel label-free fluorescence sensing approach for the detection of nicotinamide adenine dinucleotide (NAD(+)) based on a dumbbell-like DNA probe designed for both ligation reaction and digestion reaction with low background noise. SYBR Green I (SG I), a double-helix dye, was chosen as the readout fluorescence signal. In the absence of NAD(+), the ligation reaction did not occur, but the probe was digested to mononucleotides after the addition of exonuclease I (Exo I) and exonuclease I (Exo III), resulting in a weak fluorescence intensity due to the weak interaction between SG I and mononucleotides. In the presence of NAD(+), the DNA probe was ligated by Escherichia coli DNA ligase, blocking the digestion by Exo I and Exo III. As a result, SG I was intercalated into the stem part of the DNA dumbbell probe and fluorescence enhancement was achieved. This method was simple in design, fast to operate, with good sensitivity and selectivity which could discriminate NAD(+) from its analogs.

  5. Monitoring triplex DNA formation with fluorescence resonance energy transfer between a fluorophore-labeled probe and intercalating dyes.

    PubMed

    Chiou, Chiuan-Chian; Chen, Shiau-Wen; Luo, Ji-Dung; Chien, Yu-Tzu

    2011-09-01

    Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Design and synthesis of a FlAsH-type Mg2+ fluorescent probe for specific protein labeling.

    PubMed

    Fujii, Tomohiko; Shindo, Yutaka; Hotta, Kohji; Citterio, Daniel; Nishiyama, Shigeru; Suzuki, Koji; Oka, Kotaro

    2014-02-12

    Although the magnesium ion (Mg(2+)) is one of the most abundant divalent cations in cells and is known to play critical roles in many physiological processes, its mobilization and underlying mechanisms are still unknown. Here, we describe a novel fluorescent Mg(2+) probe, "KMG-104-AsH", composed of a highly selective fluorescent Mg(2+) probe, "KMG-104", and a fluorescence-recoverable probe, "FlAsH", bound specifically to a tetracysteine peptide tag (TCtag), which can be genetically incorporated into any protein. This probe was developed for molecular imaging of local changes in intracellular Mg(2+) concentration. KMG-104-AsH was synthesized, and its optical properties were investigated in solution. The fluorescence intensity of KMG-104-AsH (at λ(em/max) = 540 nm) increases by more than 10-fold by binding to both the TCtag peptide and Mg(2+), and the probe is highly selective for Mg(2+) (K(d/Mg) = 1.7 mM, K(d/Ca) ≫ 100 mM). Application of the probe for imaging of Mg(2+) in HeLa cells showed that this FlAsH-type Mg(2+) sensing probe is membrane-permeable and binds specifically to tagged proteins, such as TCtag-actin and mKeima-TCtag targeted to the cytoplasm and the mitochondrial intermembrane space. KMG-104-AsH bound to TCtag responded to an increase in intracellular Mg(2+) concentration caused by the release of Mg(2+) from mitochondria induced by FCCP, a protonophore that eliminates the inner membrane potential of mitochondria. This probe is expected to be a strong tool for elucidating the dynamics and mechanisms of intracellular localization of Mg(2+).

  7. In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA.

    PubMed

    Choi, Yun Sik; Kim, Yong Cheol; Baek, Keum Jin; Choi, Youngnim

    2015-05-21

    The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

  8. Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napus Embryos1

    PubMed Central

    Schwender, Jörg; Ohlrogge, John B.

    2002-01-01

    Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mm carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mm amino acids as well as 6 to 15 mm malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing 13C-labeled carbohydrates. The 13C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of 13C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of 13C label into plastid-formed fatty acids, but substantially diluted 13C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. 13C label in the terminal acetate units of C20 and C22 fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute 13C label incorporation into fatty acids or

  9. Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification

    NASA Astrophysics Data System (ADS)

    Tian, Qianqian; Wang, Ying; Deng, Ruijie; Lin, Lei; Liu, Yang; Li, Jinghong

    2014-12-01

    The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development.The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification

  10. Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry.

    PubMed

    Zhu, Quan-Fei; Hao, Yan-Hong; Liu, Ming-Zhou; Yue, Jiang; Ni, Jian; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-09-04

    Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3-104 folds in standard solution and 5-138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes

  11. Dual-Labeled Near-Infrared/(99m)Tc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells.

    PubMed

    Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

    2016-07-07

    We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m ((99m)Tc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with (99m)Tc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner.

  12. Dual-Labeled Near-Infrared/99mTc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells

    PubMed Central

    Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

    2016-01-01

    We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m (99mTc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with 99mTc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner. PMID:27399687

  13. An ultrasensitive method for the detection of gene fragment from transgenics using label-free gold nanoparticle probe and dynamic light scattering.

    PubMed

    Gao, Duyang; Sheng, Zonghai; Han, Heyou

    2011-06-24

    The detection of transgenic products is of great significance for the development of transgenic technique. In this paper, we developed a simple, rapid and ultrasensitive method for the detection of sequence-specific Nopaline synthase (NOS) gene from the transgenic plants using label-free gold nanoparticle (NP) probe and dynamic light scattering (DLS) technology. Gold NPs were stable in NaCl solution with the presence of NOS gene probe. On the contrary, they were aggregated in NaCl solution when the probe sequence was hybridized with target sequence. The change in the size of gold NPs can be detected by DLS technology with high sensitivity. Under the optimal conditions, the average hydrodynamic diameter of gold NPs was linear with the concentration of the target sequence ranging from 1.0×10(-13)mol L(-1) to 5.0×10(-9)mol L(-1), with a detection limit of 3.0×10(-14)mol L(-1) (S/N=3). The relative standard deviation (at 1.0×10(-9)mol L(-1) of target sequence) was 4.8% (n=11). The result shows that gold NPs-based DLS method has great potential in the analysis of transgenic products. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. A novel homogenous detection method based on the self-assembled DNAzyme labeled DNA probes with SWNT conjugates and its application in detecting pathogen.

    PubMed

    Ding, Xinghua; Li, Hua; Deng, Le; Peng, Zhihui; Chen, Hui; Wang, Dan

    2011-07-15

    In this paper, a novel and cost-effective homogeneous detection method was constructed for the detection of genomic DNA and Staphylococcus aureus (S. aureus), based on the noncovalent assembly of DNAzyme-labeled detection probe and single-walled carbon nanotubes (SWNTs). When the target genomic DNA and hemin was existed in the detection solution, the detection probe wrapped on the SWNTs by π-stacking interactions would keep away from SWNTs and form a DNAzyme-self-assembly construction. This DNAzyme construction could catalyze 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS²⁻) and generate a colored product which could lead to the absorbance changes. Hence, according to its catalyzed capacity, the DNAzyme construction could amplify the detection signal. The concentration of target DNA could be quantified by exploiting their optical absorption changes at 414 nm and the concentration limit of detection of the method was 30 nM. And this detection method detected S. aureus quantitatively. In addition, this work proved that the method obtain higher detection sensitivity compared with the method without SWNTs because of the protection profile of SWNTs towards the detection probe.

  15. A simple and label-free aptasensor based on nickel hexacyanoferrate nanoparticles as signal probe for highly sensitive detection of 17β-estradiol.

    PubMed

    Fan, Lifang; Zhao, Guohua; Shi, Huijie; Liu, Meichun

    2015-06-15

    A simple and label-free electrochemical aptasensor was developed for detecting 17β-estradiol (E2). To translate the binding events between aptamer and E2 into the measurable electrochemical signal, the nickel hexacyanoferrate nanoparticles (NiHCF NPs) as signal probe was in situ introduced on the electrode by a simple two-step deposition method, exhibiting well-defined peaks with good stability and reproducibility. Subsequently, Au nanoparticles (Au NPs) was covered on the NiHCF NPs, which not only provided a platform for immobilizing the aptamer by S-Au interaction, but further enhanced the conductivity and stability of the signal probe. With the addition of E2, the formation of E2-aptamer complexes on the sensing interface retarded the interfacial electron transfer reaction of the probe, resulting in the decrease of the electrochemical signal. E2 could be readily examined by measuring the signal change. A linear range of 1×10(-12)-6×10(-10) M was obtained with a low detection limit of 0.8×10(-12) M. The aptasensor also exhibited high specificity to E2 in control experiments employing seven endocrine disrupting compounds as the interferents that had similar structure or coexisted with E2 in the environment. Besides, the applicability of the aptasensor was successfully evaluated by determining E2 in the real samples.

  16. Monodisperse REPO4 (RE = Yb, Gd, Y) hollow microspheres covered with nanothorns as affinity probes for selectively capturing and labeling phosphopeptides.

    PubMed

    Cheng, Gong; Zhang, Ji-Lin; Liu, Yan-Lin; Sun, De-Hui; Ni, Jia-Zuan

    2012-02-13

    Rare-earth phosphate microspheres with unique structures were developed as affinity probes for the selective capture and tagging of phosphopeptides. Prickly REPO(4) (RE = Yb, Gd, Y) monodisperse microspheres, that have hollow structures, low densities, high specific surface areas, and large adsorptive capacities were prepared by an ion-exchange method. The elemental compositions and crystal structures of these affinity probes were confirmed by energy-dispersive spectroscopy (EDS), powder X-ray diffraction (XRD), and Fourier-transform infrared (FTIR) spectroscopy. The morphologies of these compounds were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and nitrogen-adsorption isotherms. The potential ability of these microspheres for selectively capturing and labeling target biological molecules was evaluated by using protein-digestion analysis and a real sample as well as by comparison with the widely used TiO(2) affinity microspheres. These results show that these porous rare-earth phosphate microspheres are highly promising probes for the rapid purification and recognition of phosphopeptides. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Chemiluminescence-imaging detection of DNA on a solid-phase membrane by using a peroxidase-labeled macromolecular probe.

    PubMed

    Azam, Md Golam; Yamasuji, Mutsumi; Krawczyk, Tomasz; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki

    2015-07-01

    We have developed a novel method for sensitive chemiluminescence (CL)-imaging detection of DNA by using a macromolecular probe synthesized by attaching multiple molecules of horseradish peroxidase (HRP) and biotin in dextran backbone. The probe formed a macromolecular assembly by binding to streptavidin which specifically recognized biotinylated complementary DNA, which was hybridized to a target DNA on a solid-phase membrane. This methodology was applied to CL-imaging detection of a synthetic telomere DNA (TTAGGG)10 and human telomere DNA by using the CL probe comprising of dextranT2000 (MW=ca. 2000kDa) bonded to approximately 42 molecules of HRP and 210 molecules of biotin. The human telomere DNA in a small number of buccal mucous cells (ca. 70 cell numbers) of cheek tissue was quantitatively determined by the proposed CL detection method that afforded approximately 10 times higher sensitivity than that of the conventional CL method using commercially available HRP-avidin probe. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane.

    PubMed

    Azam, Md Golam; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki

    2011-09-01

    In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran backbone after the formation of aldehyde groups into the macromolecule by periodate oxidation. The synthesized probes contained 27-31 molecules of ALP in their macromolecules when 50-fold molar ratio of ALP to the dextran was used for the synthesis. These probes provided 14-20 times stronger chemiluminescence (CL) than that of the equimolar free ALP adsorbed on a nylon membrane. The velocity of the CL reaction of ALP-catalyzed adamantlyl-1,2-dioxetane substrate was improved from a slower emission (glow type) of CL to a faster one (flash type). The CL signal integrated for 2 min under strongly alkaline conditions (pH 13.0) was about ten times greater than that obtained by the conventional conditions (pH 9.5). Therefore, the synthesized macromolecular probe could be successfully utilized for the high-throughput CL detection of biotin-conjugated anti-rabbit IgG antibody with a lower detection limit of 880 amol per spot on the nylon membrane. This study provides analytical strategy for the rapid, convenient, and sensitive detection of target proteins in immunoassays.

  19. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

    PubMed

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer.

  20. Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

    NASA Astrophysics Data System (ADS)

    Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

    2009-07-01

    Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55+/-0.01 s-1 and 1.12+/-0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

  1. Label-Free Imaging of Female Genital Tract Melanocytic Lesions With Pump-Probe Microscopy: A Promising Diagnostic Tool

    PubMed Central

    Robles, Francisco E.; Deb, Sanghamitra; Fischer, Martin C.; Warren, Warren S.; Selim, Maria Angelica

    2017-01-01

    Objectives Melanomas of the female genital tract present a unique clinical challenge. Not only are these lesions in an anatomically sensitive area, but also they tend to be multifocal and have high recurrence rates. Furthermore, several benign melanocytic proliferations resemble early-stage melanoma clinically and/or histopathologically. Thus, there is a significant need for additional tools that can help correctly diagnose and stage these lesions. Here, we quantitatively and nondestructively analyze the chemical composition of melanin in excised pigmented lesions of the female genital tract using pump-probe microscopy, a high-resolution optical imaging technique that is sensitive to many biochemical properties of melanin. Materials and Methods Thirty-one thin (~5 μm) tissue sections previously excised from female genital tract melanocytic lesions were imaged with pump-probe microscopy and analyzed. Results We find significant quantitative differences in melanin type and structure between melanoma and nonmalignant melanocytic proliferations. Our analysis also suggests a link between the molecular signatures of melanins and lesion-specific genetic mutations. Finally, significant differences are found between metastatic and nonmetastatic melanomas. The limitations of this work include the fact that molecular information is restricted to melanin pigment and the sample size is relatively small. Conclusions Pump-probe microscopy provides unique information regarding the biochemical composition of genital tract melanocytic lesions, which can be used to improve the diagnosis and staging of vulvar melanomas. PMID:28157824

  2. Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes.

    PubMed

    Marras, Salvatore A E

    2006-01-01

    With the introduction of simple and relatively inexpensive methods for labeling nucleic acids with nonradioactive labels, doors have been opened that enable nucleic acid hybridization probes to be used for research and development, as well as for clinical diagnostic applications. The use of fluorescent hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. The use of hybridization probes that bind to the amplification products in real-time markedly improves the ability to obtain quantitative results. Furthermore, real-time nucleic acid amplification assays can be carried out in sealed tubes, eliminating carryover contamination. Because fluorescent hybridization probes are available in a wide range of colors, 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. It is therefore important to carefully select the labels of hybridization probes, 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 chapter outlines different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers.

  3. Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain.

    PubMed

    Agafonov, Roman V; Nesmelov, Yuri E; Titus, Margaret A; Thomas, David D

    2008-09-09

    We have engineered a mutant of Dictyostelium discoideum (Dicty) myosin II that contains the same fast-reacting "SH1" thiol as in muscle myosin, spin-labeled it, and performed electron paramagnetic resonance (EPR) to compare the structure of the force-generating region of the two myosins. Dicty myosin serves as a model system for muscle myosin because of greater ease of mutagenesis, expression, and crystallization. The catalytic domains of these myosins have nearly identical crystal structures in the apo state, but there are significant differences in ATPase kinetics, and there are no crystal structures of skeletal muscle myosin with bound nucleotides, so another structural technique is needed. Previous EPR studies, with a spin label attached to SH1 in muscle myosin, have resolved the key structural states of this region. Therefore, we have performed identical experiments on both myosins spin-labeled at equivalent sites. Spectra were identical for the two myosins in the apo and ADP-bound states. With bound ADP and phosphate analogs, (i) both proteins exhibit two resolved structural states (prepowerstroke, postpowerstroke) in a single biochemical state (defined by the bound nucleotide), and (ii) these structural states are essentially identical in the two myosins but (iii) are occupied to different extents as a function of the biochemical state. We conclude that (i) myosin structural and biochemical states do not have a one-to-one correspondence, and (ii) Dicty myosin can serve as a good analog for structural studies of muscle myosin only if differences in the coupling between biochemical and structural states are taken into account.

  4. A DNA-templated silver nanocluster probe for label-free, turn-on fluorescence-based screening of homo-adenine binding molecules.

    PubMed

    Park, Ki Soo; Park, Hyun Gyu

    2015-02-15

    A novel, label-free, turn-on fluorescence strategy to detect molecules that bind to adenine-rich DNA sequences has been developed. The probe employs DNA-templated silver nanoclusters (DNA-AgNCs) as the key detection component. The new strategy relies on the formation of non-Watson-Crick homo-adenine DNA duplex, triggered by strong interactions with homo-adenine binding molecules, which brings a guanine-rich sequence in one strand close to DNA-AgNCs located on the opposite strand. This phenomenon transforms weakly fluorescent AgNCs into highly emissive species that display bright red fluorescence. Finally, we have shown that the new fluorescence turn-on strategy can be employed to detect coralyne, the most representative homo-adenine binding molecule that triggers formation of a non-Watson-Crick homo-adenine DNA duplex.

  5. Terminal protection of a small molecule-linked loop DNA probe for turn-on label-free fluorescence detection of proteins.

    PubMed

    Chen, Xuexu; Lin, Chunshui; Chen, Yiying; Luo, Feng; Wang, Yiru; Chen, Xi

    2016-09-15

    A novel label-free turn-on fluorescence biosensor for the determination of streptavidin (SA) was proposed. Using terminal protection of small molecule-linked DNA chimeras, which can protect DNA from degradation by various exonucleases when the small molecule moieties are bound to their protein target, we designed a loop probe, where the 3'-end was modified with biotin to resist digestion by exonucleases in the presence of target SA. Coupled with an intercalating dye, SYBR Green I, strong enhancement of the fluorescence signals was obtained compared with that in the absence of SA. A linear correlation equation was obtained for SA from 0 to 200nM with a limit detection of 0.4nM. This strategy holds great promise for practical applications with good specificity and sensitivity.

  6. Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes

    NASA Astrophysics Data System (ADS)

    Qi, Yingying; Li, Li; Li, Baoxin

    2009-09-01

    A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV-vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 × 10 -13 to 4.5 × 10 -6 mol/L. The detection limit (3 σ) of this method has decreased to pico-molar level.

  7. Probing the origin of acetyl-CoA and oxaloacetate entering the citric acid cycle from the 13C labeling of citrate released by perfused rat hearts.

    PubMed

    Comte, B; Vincent, G; Bouchard, B; Des Rosiers, C

    1997-10-17

    We present a strategy for simultaneous assessment of the relative contributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylation, and fatty acid oxidation to citrate formation in the perfused rat heart. This requires perfusing with a mix of 13C-substrates and determining the 13C labeling pattern of a single metabolite, citrate, by gas chromatography-mass spectrometry. The mass isotopomer distributions of the oxaloacetate and acetyl moieties of citrate allow calculation of the flux ratios: (pyruvate carboxylation)/(pyruvate decarboxylation), (pyruvate carboxylation)/(citrate synthesis), (pyruvate decarboxylation)/(citrate synthesis) (pyruvate carboxylation)/(fatty acid oxidation), and (pyruvate decarboxylation)/(fatty acid oxidation). Calculations, based on precursor-product relationship, are independent of pool size. The utility of our method was demonstrated for hearts perfused under normoxia with [U-13C3](lactate + pyruvate) and [1-13C]octanoate under steady-state conditions. Under these conditions, effluent and tissue citrate were similarly enriched in all 13C mass isotopomers. The use of effluent citrate instead of tissue citrate allows probing substrate fluxes through the various reactions non-invasively in the intact heart. The methodology should also be applicable to hearts perfused with other 13C-substrates, such as 1-13C-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.

  8. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

    SciTech Connect

    Price, E.M.; Smith, P.L.; Klein, T.E.; Freisheim, J.H.

    1987-07-28

    N/sup ..cap alpha../-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-(/sup 125/I)iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the /sup 125/I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.

  9. Synthesis of a Targeted Biarsenical Cy3-Cy5 Affinity Probe for Superresolution Fluorescence Imaging

    SciTech Connect

    Fu, Na; Xiong, Yijia; Squier, Thomas C.

    2012-11-01

    Photoswitchable fluorescent probes capable of the targeted labeling of tagged proteins are of significant interest due to their ability to enable in situ imaging of protein complexes within native biomolecular assemblies. Here we describe the synthesis of a fluorescent probe (AsCy3Cy5), and demonstrate the targeted labeling and super-resolution imaging of a tagged protein within a supramolecular protein complex.

  10. In vivo quantifying molecular specificity of Cy5.5-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging

    PubMed Central

    Dai, Yunpeng; Yin, Jipeng; Huang, Yu; Chen, Xueli; Wang, Guodong; Liu, Yajun; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

    2016-01-01

    We quantified molecular specificity of Cy5.5-GX1 in vivo with dynamic fluorescence imaging to better understand its kinetic properties. According to whether or not free GX1 was injected and when it was injected, twelve of BGC-823 xenografted mice were randomly divided into three groups and underwent a 60 minute dynamic fluorescence scanning. Combined with a principal-component analysis, the binding potential (Bp) of the probe was determined by both Logan graphical analysis with reference tissue model (GARTM) and Lammertsma simplified reference tissue model (SRTM). The sum of the pharmacokinetic rate constants (SKRC) was quantified by the Gurfinkel exponential model (GEXPM). Cy5.5-GX1 specifically targeted tumor both in vitro and in vivo. We obtained similar quantification results of Bp (GARTM Bp = 0.582 ± 0.2655, SRTM Bp = 0.618 ± 0.2923), and obtained a good linear relation between the Bp value and the SKRC value. Our results indicate that the SKRC value is more suitable for an early-stage kinetic data analysis, and the Bp value depicts kinetic characteristics under the equilibrium state. Dynamic fluorescence imaging in conjunction with various kinetic models are optimal tools to quantify molecular specificity of the Cy5.5-GX1 probe in vivo. PMID:27446643

  11. Label-free selective sensing of mercury(II) via reduced aggregation of the perylene fluorescent probe.

    PubMed

    Wang, Bin; Wang, Fangyuan; Jiao, Huping; Yang, Xiangyu; Yu, Cong

    2010-08-01

    In the present work, we report a fluorescence turn-on approach for the sensitive and selective detection of Hg(2+). A cationic perylene derivative (compound 1) was used as the fluorescence probe, and a thymine-rich oligonucleotide (oligo-M) was employed for the specific interaction with Hg(2+). Compound 1 shows strong tendency to self-aggregate into linear chain structures in aqueous media because of the pi-pi stacking interactions of its planar aromatic ring structure. The compound 1 free monomer is strongly fluorescent, whereas its aggregates are not fluorescent. When oligo-M and compound 1 were mixed, oligo-M induced strong compound 1 aggregation and resulted in significant fluorescence quenching. In the presence of Hg(2+), the specific interactions between oligo-M and Hg(2+) induced hairpin structure formation of oligo-M and thus weakened its binding to compound 1 aggregates. As a result, free probe monomers were released, and increased fluorescence was observed. The fluorescence intensity increase was in direct proportion to the concentration of Hg(2+) added. Our method provides a simple, fast, and efficient means for Hg(2+) quantification, it is highly sensitive with a limit of detection of 1 nM, and is also highly selective against other common metal ions.

  12. In vivo quantifying molecular specificity of Cy5.5-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging.

    PubMed

    Dai, Yunpeng; Yin, Jipeng; Huang, Yu; Chen, Xueli; Wang, Guodong; Liu, Yajun; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

    2016-04-01

    We quantified molecular specificity of Cy5.5-GX1 in vivo with dynamic fluorescence imaging to better understand its kinetic properties. According to whether or not free GX1 was injected and when it was injected, twelve of BGC-823 xenografted mice were randomly divided into three groups and underwent a 60 minute dynamic fluorescence scanning. Combined with a principal-component analysis, the binding potential (Bp) of the probe was determined by both Logan graphical analysis with reference tissue model (GARTM) and Lammertsma simplified reference tissue model (SRTM). The sum of the pharmacokinetic rate constants (SKRC) was quantified by the Gurfinkel exponential model (GEXPM). Cy5.5-GX1 specifically targeted tumor both in vitro and in vivo. We obtained similar quantification results of Bp (GARTM Bp = 0.582 ± 0.2655, SRTM Bp = 0.618 ± 0.2923), and obtained a good linear relation between the Bp value and the SKRC value. Our results indicate that the SKRC value is more suitable for an early-stage kinetic data analysis, and the Bp value depicts kinetic characteristics under the equilibrium state. Dynamic fluorescence imaging in conjunction with various kinetic models are optimal tools to quantify molecular specificity of the Cy5.5-GX1 probe in vivo.

  13. Early-Stage Aggregation of Islet Amyloid Polypeptide on Membrane Surfaces Probed by Label-Free Chiral Sum Frequency Generation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Wang, Zhuguang; Fu, Li; Yan, Elsa

    2013-03-01

    The aggregation of human islet amyloid polypeptide (hIAPP) into fibrils is associated with type II diabetes. It can be catalyzed by interactions with membranes. Recent studies have shown that cytotoxicity arises from the intermediates of aggregation instead of mature fibrils. However, the pathogenic mechanism is still unknown and it remains challenging to probe structures of the intermediates on membrane surfaces due to a lack of biophysical methods that are sensitive to both protein secondary structures and interfaces. Here, we used label-free chiral sum frequency generation spectroscopy (cSFG) to probe the intermediates. Recently, we have discovered cSFG provides highly specific peptide vibrational signatures that can distinguish protein secondary structures at interfaces. Using cSFG, we observed in situ and in real time the aggregation of hIAPP from disordered structures to α-helices and then β-sheets on membrane surfaces. We also obtained the orientation of the β-sheet aggregates inserted into the membranes. We further studied the S20G mutant, which is linked to the early onset of type II diabetes among Asian populations. We compared the mutant with the wild-type hIAPP to evaluate the effect of S20G in the early-stage aggregation on membrane surfaces.

  14. A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA

    PubMed Central

    Hosoda, Kazufumi; Matsuura, Tomoaki; Kita, Hiroshi; Ichihashi, Norikazu; Tsukada, Koji; Urabe, Itaru; Yomo, Tetsuya

    2008-01-01

    We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (L-assay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformation-interchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.1 nM. Moreover, this assay allows quantification of target RNA even in the presence of a several thousandfold excess by weight of cellular RNA. In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). The L-assay requires only incubation under isothermal conditions, is inexpensive, and is expected to be useful for basic research requiring high-accuracy, easy-to-use RNA quantification, and real-time quantification. PMID:18230763

  15. Carbon Nanotubes Labeled with Aptamer and Horseradish Peroxidase as a Probe for Highly Sensitive Protein Biosensing by Postelectropolymerization of Insoluble Precipitates on Electrodes.

    PubMed

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

    2015-08-04

    Carbon nanotubes (CNTs) labeled with aptamer and horseradish peroxidase (HRP) were used as a probe to amplify the impedimetric sensing of the aptamer-protein (with thrombin as the model) interaction. The HRP-biocatalyzed oxidation of 3,3-diaminobenzidine (DAB) in the presence of H2O2 and the postelectropolymerization of insoluble precipitates produced on the electrode supports were used as a signal amplification route for the sensing process. Thrombin was sensed by aptamer 1 immobilized on a glassy carbon electrode. The multiwalled CNT-aptamer 2-HRP probe was linked to the aptamer 1-thrombin complex through the thrombin-aptamer 2 interaction. The postelectropolymerization of biocatalyzed precipitates of DAB on the electrode greatly increased the electron-transfer resistance at the electrode-solution interface. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to follow the stepwise fabrication of the aptasensor and impedimetric detection of thrombin. Thrombin concentration as low as 0.05 pM could be detected by this method. In addition, the proposed impedimetric aptasensor exhibits good sensitivity (5195 Ω decade(-1)), selectivity, and reproducibility. The aptasensor also has acceptable recovery for thrombin detection in complex protein sample.

  16. Sensitivity of a digoxigenin-labelled DNA probe in detecting Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), in oysters.

    PubMed

    Meyer, Gary R; Bower, Susan M; Carnegie, Ryan B

    2005-02-01

    The protistan parasite Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), induces mortality and reduces marketability in the Pacific oyster, Crassostrea gigas, in British Columbia, Canada. This parasite is a pathogen of international concern because it infects a range of oyster species, and because its life cycle and mode of transmission are unknown. A digoxigenin-labelled DNA probe in situ hybridisation technique (DIG-ISH) was developed, and its detection sensitivity was compared to standard histological sections stained with haematoxylin and eosin stain (H&E-histo). In H&E-histo preparations, the detection of M. mackini was certain only when the parasite occurred within the vesicular connective tissue of adult oysters. However, the DIG-ISH technique clearly demonstrated the presence of infection in all other host tissues as well as in juvenile oysters with poorly developed vesicular connective tissue. The probe hybridised strongly to M. mackini, did not hybridise to oyster tissues or with the other shellfish parasites tested, and was more sensitive for detecting infections when compared to H&E-histo.

  17. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe.

    PubMed

    Dinhopl, N; Mostegl, M M; Richter, B; Nedorost, N; Maderner, A; Fragner, K; Weissenböck, H

    2011-11-12

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.

  18. In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

    PubMed Central

    Dinhopl, N.; Mostegl, M. M.; Richter, B.; Nedorost, N.; Maderner, A.; Fragner, K.; Weissenböck, H.

    2011-01-01

    The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues. PMID:21921059

  19. Custom 4-Plex DiLeu Isobaric Labels Enable Relative Quantification of Urinary Proteins in Men with Lower Urinary Tract Symptoms (LUTS).

    PubMed

    Greer, Tyler; Hao, Ling; Nechyporenko, Anatoliy; Lee, Sanghee; Vezina, Chad M; Ricke, Will A; Marker, Paul C; Bjorling, Dale E; Bushman, Wade; Li, Lingjun

    2015-01-01

    The relative quantification of proteins using liquid chromatography mass spectrometry (LC-MS) has allowed researchers to compile lists of potential disease markers. These complex quantitative workflows often include isobaric labeling of enzymatically-produced peptides to analyze their relative abundances across multiple samples in a single LC-MS run. Recent efforts by our lab have provided scientists with cost-effective alternatives to expensive commercial labels. Although the quantitative performance of these dimethyl leucine (DiLeu) labels has been reported using known ratios of complex protein and peptide standards, their potential in large-scale proteomics studies using a clinically relevant system has never been investigated. Our work rectifies this oversight by implementing 4-plex DiLeu to quantify proteins in the urine of aging human males who suffer from lower urinary tract symptoms (LUTS). Protein abundances in 25 LUTS and 15 control patients were compared, revealing that of the 836 proteins quantified, 50 were found to be differentially expressed (>20% change) and statistically significant (p-value <0.05). Gene ontology (GO) analysis of the differentiated proteins showed that many were involved in inflammatory responses and implicated in fibrosis. While confirmation of individual protein abundance changes would be required to verify protein expression, this study represents the first report using the custom isobaric label, 4-plex DiLeu, to quantify protein abundances in a clinically relevant system.

  20. Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label.

    PubMed

    Kloiber, Karin; Spitzer, Romana; Tollinger, Martin; Konrat, Robert; Kreutz, Christoph

    2011-05-01

    The refolding kinetics of bistable RNA sequences were studied in unperturbed equilibrium via (13)C exchange NMR spectroscopy. For this purpose a straightforward labeling technique was elaborated using a 2'-(13)C-methoxy uridine modification, which was prepared by a two-step synthesis and introduced into RNA using standard protocols. Using (13)C longitudinal exchange NMR spectroscopy the refolding kinetics of a 20 nt bistable RNA were characterized at temperatures between 298 and 310K, yielding the enthalpy and entropy differences between the conformers at equilibrium and the activation energy of the refolding process. The kinetics of a more stable 32 nt bistable RNA could be analyzed by the same approach at elevated temperatures, i.e. at 314 and 316 K. Finally, the dynamics of a multi-stable RNA able to fold into two hairpin- and a pseudo-knotted conformation was studied by (13)C relaxation dispersion NMR spectroscopy.

  1. Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR

    PubMed Central

    Nußbaumer, Felix; Juen, Michael Andreas; Gasser, Catherina; Kremser, Johannes; Müller, Thomas; Tollinger, Martin

    2017-01-01

    Abstract We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min−1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s−1 and with a backward rate constant of 10.6 s−1.

  2. Synthesis of iodine-125 labeled (+/-)-15-(4-azidobenzyl)carazolol: a potent beta-adrenergic photoaffinity probe

    SciTech Connect

    Heald, S.L.; Jeffs, P.W.; Lavin, T.N.; Nambi, P.; Lefkowitz, R.J.; Caron, M.G.

    1983-06-01

    (+/-)-15-(4-Azidobenzyl)carazolol (2), a potent beta-adrenergic photoaffinity ligand has been radioiodinated to theoretical specific activity (2175 Ci/mmol) and shown to label covalently beta-adrenergic receptor peptides in avian and amphibian erythrocyte membrane preparations. The radioiodinated analogues of the desired compound (2) were optimally prepared by two synthetic steps from (+/-)-15-(4-aminobenzyl)carazolol (8). The latter was iodinated with carrier-free Na/sup 125/I and chloramine T to yield two major isotopomers (the monoiodinated derivatives 9 and 10), which were separated by thin-layer chromatography and converted via diazonium salt formation to their respective 4-azides, 12 and 6. These azides can be used interchangeably in ligand binding or photoaffinity labeling experiments. Compound 8 was obtained by catalytic reduction of the nitro derivative (7), which was arrived at by direct reaction of 1,1-dimethyl-2-(4-nitrophenyl)ethylamine (3) with 4-(2,3-epoxypropoxy)carbazole (5). Of the desired isomers, (+/-)-15-(4-azido-3-iodobenzyl)carazolol (6) could be synthesized from 1,1-dimethyl-2-(4-azido-3-iodophenyl)ethylamine (4) by direct reaction with 5. This and the preceding sequence of reactions were carried out by using nonradioactive materials, and separation and purification of products were accomplished by high-performance liquid chromatography. The compounds described have been shown to be potent beta-adrenergic antagonistsec The photoactive azide derivatives of these compounds (6 and 12) have been shown to covalently incorporate into the beta-adrenergic receptor binding subunit of frog and turkey erythrocyte membrane preparations. Incorporation of the ligands into these polypeptides can be blocked specifically by both beta-adrenergic agonists and antagonists.

  3. Pre-embedding immunogold labeling of TUNEL stain enables evaluation of DNA strand breaks and ultrastructural alterations in individual cells of neuronal tissue.

    PubMed

    Barth, Martin; Oulmi, Yasmina; Ehrenreich, Hannelore; Schilling, Lothar

    2002-12-01

    In the brain apoptosis may occur as a physiological phenomenon during periods of programmed cell death as well as under pathological conditions such as ischemia, trauma, tumor, and degenerative diseases. While the definition of apoptotic cell death was originally based on ultrastructural alterations, the detection of DNA double-strand breaks has become an important feature in studies of apoptosis. Currently, the terminal transferase-mediated dUTP nick-end labeling (TUNEL) procedure is widely used for detection of apoptotic cell death. However, there is a growing body of evidence to suggest that the TUNEL staining does not label apoptotic alterations exclusively. Therefore, a new staining procedure was developed combining TUNEL methodology with pre-embedding nanogold labeling to detect DNA double-strand breaks in individual cells by electron microscopy and assess the accompanying ultrastructural alterations. In vitro DNAse-treated vibratome sections (thickness, 20 micro m) from normal adult rat brains were used to develop the staining procedure consisting of the following steps: (i) TUNEL staining of free-floating vibratome sections using fluorescein isothiocyanate (FITC)-labeled UTP, (ii) conversion of the fluorescence signal into an electron-dense signal using an anti-FITC antibody coupled with ultrasmall (diameter, 0.8 nm) gold particles followed by silver enhancement, and (iii) osmification, embedding in Spurr resin and cutting of ultrathin sections. Early postnatal brain tissue was used to study physiologically occurring apoptotic cell death. Under these conditions different patterns of gold staining were observed probably representing different states of cellular decay along the apoptotic avenue. Severe focal brain ischemia was studied as a pathological situation in which intense TUNEL staining occurs. Under these conditions TUNEL labeling of cells was regularly observed in conjunction with ultrastructural alterations indicative of necrosis. These results

  4. Probing biochemical mechanisms of action of muscarinic M3 receptor antagonists with label-free whole cell assays.

    PubMed

    Deng, Huayun; Wang, Chaoming; Su, Ming; Fang, Ye

    2012-10-02

    Binding kinetics of drugs is increasingly recognized to be important for their in vivo efficacy and safety profiles. However, little is known about the effect of drug binding kinetics on receptor signaling in native cells. Here we used label-free whole cell dynamic mass redistribution (DMR) assays under persistent and duration-controlled stimulation conditions to investigate the influence of the binding kinetics of four antagonists on the signaling of endogenous muscarinic M3 receptor in native HT-29 cells. Results showed that DMR assays under different conditions differentiated the biochemical mechanisms of action of distinct M3 antagonists. When co-stimulated with acetylcholine, tiotropium, a relatively slow binding antagonist, was found to selectively block the late signaling of the receptor, suggesting that acetylcholine attains its binding equilibrium faster than tiotropium does, thereby still being able to initiate its rapid response until the antagonist draws up and fully blocks the signaling. Furthermore, DMR assays under microfluidics allowed estimation of the residence times of these antagonists acting at the receptor in native cells, which were found to be the determining factor for the blockage efficiency of M3 receptor signaling under duration-controlled conditions. This study demonstrates that DMR assays can be used to elucidate the functional consequence of kinetics-driven antagonist occupancy in native cells.

  5. Tritium-labelled isovaleryl-RYYRIK-NH2 as potential antagonist probe for ORL1 nociceptin receptor.

    PubMed

    Inamine, Shogo; Nishimura, Hirokazu; Li, Jinglan; Isozaki, Kaname; Matsushima, Ayami; Costa, Tommaso; Shimohigashi, Yasuyuki

    2014-11-01

    IsoVa-RYYRIK-NH2 is a highly specific antagonist ligand of the opioid receptor-like 1 (ORL1) receptor, an endogenous ligand of which is 17-mer peptide nociceptin. ORL1 antagonists have potential for clinical use as analgesic and antineuropathic drugs, and thus information on the receptor-binding characteristics of antagonists is very important for rational drug design. In the present study, we prepared tritium-labelled isova-RYYRIK-NH2 from its precursor with the 3-methylcrotonyl (CH3)2CCHCO group by a catalytic reduction using tritium gas. The resulting [(3)H]isoVa-RYYRIK-NH2 was evaluated in a saturation binding assay using the COS-7 cell membrane preparations of transiently expressed ORL1. It exhibited more than 90% specific binding with a dissociation constant of 1.21±0.03nM. From the mutual heterologous binding assays using [(3)H]isoVa-RYYRIK-NH2 and [(3)H]nociceptin, isoVa-RYYRIK-NH2 and nociceptin were found to share the receptor-binding site, but each also had a separate specific binding site of its own. They differentiated the two different binding states or conformations of ORL1, which might represent the agonist-active and antagonist-inactive conformations of ORL1. [(3)H]isoVa-RYYRIK-NH2 is thus a key tracer to uncover the amino acid residues important for receptor inactivation.

  6. Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label

    PubMed Central

    Kloiber, Karin; Spitzer, Romana; Tollinger, Martin; Konrat, Robert; Kreutz, Christoph

    2011-01-01

    The refolding kinetics of bistable RNA sequences were studied in unperturbed equilibrium via 13C exchange NMR spectroscopy. For this purpose a straightforward labeling technique was elaborated using a 2′-13C-methoxy uridine modification, which was prepared by a two-step synthesis and introduced into RNA using standard protocols. Using 13C longitudinal exchange NMR spectroscopy the refolding kinetics of a 20 nt bistable RNA were characterized at temperatures between 298 and 310 K, yielding the enthalpy and entropy differences between the conformers at equilibrium and the activation energy of the refolding process. The kinetics of a more stable 32 nt bistable RNA could be analyzed by the same approach at elevated temperatures, i.e. at 314 and 316 K. Finally, the dynamics of a multi-stable RNA able to fold into two hairpin- and a pseudo-knotted conformation was studied by 13C relaxation dispersion NMR spectroscopy. PMID:21252295

  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. Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

    PubMed

    Niu, Mingming; Cho, Ji-Hoon; Kodali, Kiran; Pagala, Vishwajeeth; High, Anthony A; Wang, Hong; Wu, Zhiping; Li, Yuxin; Bi, Wenjian; Zhang, Hui; Wang, Xusheng; Zou, Wei; Peng, Junmin

    2017-02-22

    Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

  9. Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.

    PubMed

    Fan, Ying; Shi, Lichi; Ladizhansky, Vladimir; Brown, Leonid S

    2011-02-01

    Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly (13)C,(15)N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution (13)C and (15)N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

  10. Label-free electronic probing of nucleic acids and proteins at the nanoscale using the nanoneedle biosensor

    PubMed Central

    Esfandyarpour, Rahim; Javanmard, Mehdi; Koochak, Zahra; Esfandyarpour, Hesaam; Harris, James S.; Davis, Ronald W.

    2013-01-01

    Detection of proteins and nucleic acids is dominantly performed using optical fluorescence based techniques, which are more costly and timely than electrical detection due to the need for expensive and bulky optical equipment and the process of fluorescent tagging. In this paper, we discuss our study of the electrical properties of nucleic acids and proteins at the nanoscale using a nanoelectronic probe we have developed, which we refer to as the Nanoneedle biosensor. The nanoneedle consists of four thin film layers: a conductive layer at the bottom acting as an electrode, an oxide layer on top, and another conductive layer on top of that, with a protective oxide above. The presence of proteins and nucleic acids near the tip results in a decrease in impedance across the sensing electrodes. There are three basic mechanisms behind the electrical response of DNA and protein molecules in solution under an applied alternating electrical field. The first change stems from modulation of the relative permittivity at the interface. The second mechanism is the formation and relaxation of the induced dipole moment. The third mechanism is the tunneling of electrons through the biomolecules. The results presented in this paper can be extended to develop low cost point-of-care diagnostic assays for the clinical setting. PMID:24404047

  11. Selective Luminescent Labeling of DNA and RNA Quadruplexes by π-Extended Ruthenium Light-Up Probes.

    PubMed

    Saadallah, Dounia; Bellakhal, Mehdi; Amor, Souheila; Lefebvre, Jean-François; Chavarot-Kerlidou, Murielle; Baussanne, Isabelle; Moucheron, Cécile; Demeunynck, Martine; Monchaud, David

    2017-01-26

    A series of Ru(II) complexes exhibiting π-extended, acridine-based ancillary chelating heterocycles display high affinity and selectivity for DNA and RNA quadruplexes. The most promising candidates (3, 4) possess remarkable light-up luminophore properties (up to 330-fold luminescence enhancement upon interaction with quadruplexes), enabling them to discriminate quadruplexes from genomic DNA owing to a photochemical mechanism involving DNA protection against non-radiative decay (DAND), thus deviating from the other complexes of this series of ligands that exhibit an excited-state intramolecular proton transfer (ESIPT) that quenches their luminescence. The in vitro and preliminary in cellulo results shown here confirm the interest of this new family of fluorophores as invaluable molecular tools to detect G-quadruplexes in cells.

  12. Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts.

    PubMed Central

    Millard, P J; Roth, B L; Thi, H P; Yue, S T; Haugland, R P

    1997-01-01

    A new family of fluorescent probes has been developed for assessing the viability and metabolic activity of yeasts. This class of halogenated unsymmetric cyanine dyes is exemplified by the FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide] stain, a membrane-permeant nucleic acid-binding dye that has been found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae. Biochemical processing of the dye by active yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye. The formation of CIVS occurred under both aerobic and anaerobic conditions and was highly temperature dependent. Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels approximately 6-fold and completely inhibited CIVS formation. Under aerobic conditions, the formation of CIVS was abrogated by the cytochrome oxidase inhibitors azide and cyanide; however, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited CIVS formation under both aerobic and anaerobic conditions. Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited CIVS production. Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione S-transferase, suggested that dye processing can involve enzyme-mediated formation of glutathione conjugates. The conversion of FUN-1 by S. cerevisiae was studied quantitatively by using several techniques, including fluorometry, flow cytometry, and wide-field and confocal laser scanning fluorescence microscopy. PMID:9212436

  13. Probing pyruvate metabolism in normal and mutant fibroblast cell lines using 13C-labeled mass isotopomer analysis and mass spectrometry.

    PubMed

    Riazi, Roya; Khairallah, Maya; Cameron, Jessie M; Pencharz, Paul B; Des Rosiers, Christine; Robinson, Brian H

    2009-12-01

    Fibroblast cell lines are frequently used to diagnose genetic mitochondrial defects in children. The effect of enzyme deficiency on overall flux rate through metabolic pathways is, however, not generally considered. We have transposed an experimental paradigm that was developed for isolated perfused organs using (13)C-labeled substrates and (13)C-isotopomer analysis to probe pyruvate mitochondrial metabolism in cultured human fibroblast cell lines with normal or genetically mutant pyruvate decarboxylation (PDC) or carboxylation (PC) activity. Cells were incubated with 1mM [U-(13)C]pyruvate, and the (13)C-molar percent enrichment (MPE) of intracellular pyruvate, citrate, malate (as a surrogate of oxaloacetate) and aspartate was assessed by mass spectrometry. We estimated various flux ratios relevant to metabolic pathways involved in energy production, namely pyruvate formation, PDC, PC, and citrate recycling in the citric acid cycle (CAC). In all cell lines, exogenous pyruvate was predominately decarboxylated (PC/PDC ratios 0.01-0.3). PC-deficient cell lines displayed an expected negligible contribution of PC flux to oxaloacetate formation for citrate synthesis (PC/CS), which was associated with a greater contribution of PDC to acetyl-CoA formation (PDC/CS), and greater recycling of (13)C-labeled citrate into the CAC. In PDH-deficient cell lines, metabolic flux alterations were most apparent in cells with more than 50% reduction in enzyme activity. This led to an unexpected lower PC/CS flux ratio, while the PDC/CS flux ratio was unchanged. These data illustrate the usefulness of this approach in identifying unexpected metabolic consequences of genetic defects related to pyruvate metabolism.

  14. Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities.

    PubMed

    Xie, Ran; Hong, Senlian; Chen, Xing

    2013-10-01

    Metabolic labeling of biomolecules with bioorthogonal functionalities enables visualization, enrichment, and analysis of the biomolecules of interest in their physiological environments. This versatile strategy has found utility in probing various classes of biomolecules in a broad range of biological processes. On the other hand, metabolic labeling is nonselective with respect to cell type, which imposes limitations for studies performed in complex biological systems. Herein, we review the recent methodological developments aiming to endow metabolic labeling strategies with cell-type selectivity. The cell-selective metabolic labeling strategies have emerged from protein and glycan labeling. We envision that these strategies can be readily extended to labeling of other classes of biomolecules. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Two-photon excitation in chip electrophoresis enabling label-free fluorescence detection in non-UV transparent full-body polymer chips.

    PubMed

    Geissler, David; Belder, Detlev

    2015-12-01

    One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep-ultraviolet (UV) excitation (<300 nm) allows label-free detection of a broader range of analytes but also mandates the use of expensive fused silica glass, which is transparent to UV light. Herein, we report the first application of label-free deep UV fluorescence detection in non-UV transparent full-body polymer microfluidic devices. This was achieved by means of two-photon excitation in the visible range (λex = 532 nm). Issues associated with the low optical transmittance of plastics in the UV range were successfully circumvented in this way. The technique was investigated by application to microchip electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. 203Pb-Labeled Alpha-Melanocyte-Stimulating Hormone Peptide as an Imaging Probe for Melanoma Detection

    SciTech Connect

    Yubin, Miao; Figueroa, Said D.; Fisher, Darrell R.; Moore, Herbert A.; Testa, Richard F.; Hoffman, Timothy J.; Quinn, Thomas P.

    2008-05-01

    Abbreviations: a-MSH; alpha melanocyte stimulating hormone, DOTA; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Re(Arg11)CCMSH; DOTA-[Cys3,4,10, D-Phe7, Arg11]a-MSH3-13, NDP; [Nle4,d-Phe7] a-MSH3-13. Abstract Peptide-targeted alpha therapy with 200 mCi of 212Pb-DOTA-Re(Arg11)CCMSH cured 45% of B16/F1 murine melanoma-bearing C57 mice in a 120-day study, highlighting its melanoma treatment potential. However, there is a need to develop an imaging surrogate for patient specific dosimetry and to monitor the tumor response to 212Pb-DOTA-Re(Arg11)CCMSH therapy. The purpose of this study was to evaluate the potential of 203Pb-DOTA-Re(Arg11)CCMSH as a matched-pair SPECT imaging agent for 212Pb-DOTA-Re(Arg11)CCMSH. Method: DOTA-Re(Arg11)CCMSH was labeled with 203Pb in 0.5 M NH4OAc buffer at pH 5.4. The internalization and efflux of 203Pb-DOTA-Re(Arg11)CCMSH were determined in B16/F1 melanoma cells. The pharmacokinetics of 203Pb-DOTA-Re(Arg11)CCMSH were examined in B16/F1 melanoma-bearing C57 mice. A micro-SPECT/CT imaging study was performed with 203Pb-DOTA-Re(Arg11)CCMSH in a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection. Results: 203Pb-DOTA-Re(Arg11)CCMSH was easily prepared in NH4OAc buffer and completely separated from the excess non-radiolabeled peptide by RP-HPLC. 203Pb-DOTA-Re(Arg11)CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of 203Pb-DOTA-Re(Arg11)CCMSH activity internalized after a 20-min incubation at 25C. After incubating the cells in culture media for 20 min, 78% of internalized activity remained in the cells. 203Pb-DOTA-Re(Arg11)CCMSH exhibited similar biodistribution pattern with 212Pb-DOTA-Re(Arg11)CCMSH in B16/F1 melanoma-bearing mice. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor uptake of 12.00 +/- 3.20 %ID/g at 1 h post-injection. The tumor uptake gradually decreased to 3.43 +/- 1.12 %ID/g at 48 h post-injection. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor to kidney

  17. Tritium-labeled (E,E)-2,5-bis(4'-hydroxy-3'-carboxystyryl)benzene as a probe for β-amyloid fibrils.

    PubMed

    Matveev, Sergey V; Kwiatkowski, Stefan; Sviripa, Vitaliy M; Fazio, Robert C; Watt, David S; LeVine, Harry

    2014-12-01

    Accumulation of Aβ in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aβ production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aβ40 and Aβ42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[(3)H]-2,5-bis(4'-hydroxy-3'-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aβ plaque and tau pathology in brain tissue and in vitro studies with synthetic Aβ and tau fibrils.

  18. Simultaneous electrochemical detection of cervical cancer markers using reduced graphene oxide-tetraethylene pentamine as electrode materials and distinguishable redox probes as labels.

    PubMed

    Wu, Dan; Guo, Aiping; Guo, Zhankui; Xie, Lili; Wei, Qin; Du, Bin

    2014-04-15

    A novel, highly sensitive electrochemical immunoassay was proposed for the simultaneous determination of carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCCA) for the diagnosis of cervical cancer. Using an electrochemical analysis technique, two well-separated peaks were generated by neutral red and thionine, making the simultaneous detection of the two analytes on the electrode possible. Reduced graphene oxide-tetraethylene pentamine (rGO-TEPA), containing more amino groups, was of benefit to immobilize the primary antibody (Ab1) through an amidation reaction. Au@mesoporous carbon CMK-3 was synthesized and incubated with two secondary antibodies (Ab2) and different redox probes (neutral red and thionine) to fabricate the electrochemical immunosensor label intending to improve the analytical performance of the immunosensor. The immunosensor was prepared with a sandwich structure based on the peak current change of neutral red and thionine before and after the antigen-antibody reaction. The results showed that the immunosensor had a wide linear range, low detection limit, good reproducibility and stability. The method has been applied to the analysis of serum samples with satisfactory results.

  19. Tritium-labeled (E,E)-2,5-Bis(4’-hydroxy-3’-carboxystyryl)benzene as a Probe for β-Amyloid Fibrils

    PubMed Central

    Matveev, Sergey V.; Kwiatkowski, Stefan; Sviripa, Vitaliy M.; Fazio, Robert C.; Watt, David S.; LeVine, Harry

    2014-01-01

    Accumulation of Aβ in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aβ production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aβ40 and Aβ42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[3H]-2,5-bis(4’-hydroxy-3’-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aβ plaque and tau pathology in brain tissue and in vitro studies with synthetic Aβ and tau fibrils. PMID:25452000

  20. /sup 125/I-FK 33-824: a selective probe for radioautographic labeling of mu opioid receptors in the brain

    SciTech Connect

    Moyse, E.; Pasquini, F.; Quirion, R.; Beaudet, A.

    1986-03-01

    The selectivity of the Met-enkephalin analog FK 33-824 (FK) for mu opioid receptors has been, over the years, a matter of controversy. We report here pharmacological and radioautographic data demonstrating that at nanomolar concentrations. /sup 125/I-FK interacts exclusively with mu sites. (1) Specific binding of /sup 125/I-FK to rat striatal membranes is totally inhibited by mu- and/or delta-preferring ligands according to monovalent, Michaelian kinetics, with a potency proportional to the affinity of competing drugs for mu receptors. (2) Unlabeled FK competes only at high concentration with the delta-selective ligand 3H-DPLPE and according to the same kinetics as the mu-selective agonist DAGO. (3) /sup 125/I-FK generates the same regional radioautographic labeling pattern as 3H-DAGO. We conclude that when used at nanomolar concentrations /sup 125/I-FK constitutes a selective probe for the radioautographic detection of mu opioid receptors at both light and electron microscopic levels.

  1. Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

    PubMed Central

    Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2015-01-01

    Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

  2. A signal-on electrochemical DNA biosensor based on potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition mediated labeling of hairpin-like oligonucleotide with electroactive probe.

    PubMed

    Hu, Qiong; Kong, Jinming; Li, Yajie; Zhang, Xueji

    2016-01-15

    A novel electrochemical biosensor was developed for the signal-on detection of sequence-specific DNA by exploiting potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (φCuAAC) as an efficient approach for the labeling of hairpin-like oligonucleotide (hairpin) with electroactive probe. The hairpins, dually labeled with thiol and azide at either terminal, were firstly self-assembled on gold electrode and served as the capture probes for the specific recognition of target DNA. Upon hybridization with target DNA, the surface-confined hairpins were unfolded, liberating the azide-containing terminals away from electrode surface. Subsequently, the unfolded hairpins were conveniently and efficiently labeled with ethynylferrocene (EFC) via the φCuAAC. The quantitatively labeled EFC was finally measured via differential pulse voltammetry (DPV) for the signal-on electrochemical detection of sequence-specific DNA. The biosensor presented a good linear response over the range from 1pM to 1nM with a detection limit of 0.62pM. Results also revealed that it was highly specific and held a good detection capability in serum samples. Furthermore, the ability to chemoselectively label hairpin-like oligonucleotide with signal reporter by electrical addressing, together with the simplicity and efficiency of the φCuAAC, makes it compatible with microfluidic devices and microelectrode arrays to achieve the miniaturized and multiplexed detections. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Enabling low-noise null-point scanning thermal microscopy by the optimization of scanning thermal microscope probe through a rigorous theory of quantitative measurement

    NASA Astrophysics Data System (ADS)

    Hwang, Gwangseok; Chung, Jaehun; Kwon, Ohmyoung

    2014-11-01

    The application of conventional scanning thermal microscopy (SThM) is severely limited by three major problems: (i) distortion of the measured signal due to heat transfer through the air, (ii) the unknown and variable value of the tip-sample thermal contact resistance, and (iii) perturbation of the sample temperature due to the heat flux through the tip-sample thermal contact. Recently, we proposed null-point scanning thermal microscopy (NP SThM) as a way of overcoming these problems in principle by tracking the thermal equilibrium between the end of the SThM tip and the sample surface. However, in order to obtain high spatial resolution, which is the primary motivation for SThM, NP SThM requires an extremely sensitive SThM probe that can trace the vanishingly small heat flux through the tip-sample nano-thermal contact. Herein, we derive a relation between the spatial resolution and the design parameters of a SThM probe, optimize the thermal and electrical design, and develop a batch-fabrication process. We also quantitatively demonstrate significantly improved sensitivity, lower measurement noise, and higher spatial resolution of the fabricated SThM probes. By utilizing the exceptional performance of these fabricated probes, we show that NP SThM can be used to obtain a quantitative temperature profile with nanoscale resolution independent of the changing tip-sample thermal contact resistance and without perturbation of the sample temperature or distortion due to the heat transfer through the air.

  4. Analysis of M1G-dR in DNA by aldehyde reactive probe labeling and liquid chromatography tandem mass spectrometry.

    PubMed

    Jeong, Yo-Chan; Sangaiah, Ramiah; Nakamura, Jun; Pachkowski, Brian F; Ranasinghe, Asoka; Gold, Avram; Ball, Louise M; Swenberg, James A

    2005-01-01

    A novel method for the measurement of pyrimido[1,2-a]purin-10(3H)one (M1G) has been developed. Previous methods for analysis of M1G have been confounded by the fact that this lesion exists in equilibrium between a ring-closed form and a ring-opened aldehyde form. Poor detection sensitivity of the aldehydic form can result from loss of the adduct during analysis by its reaction with amines or proteins. We utilized the aldehyde reactive probe (ARP) to produce a stable ARP-M1G-deoxyribose (ARP-M1G-dR) conjugate to minimize adduct loss. This conjugate has increased the hydrophobicity that enhances separation of ARP-M1G-dR from unmodified DNA nucleosides by using solid phase extraction. In addition, measuring ARP-M1G-dR by selective reaction monitoring (SRM) of the [ARP-M1G-dR + H]+ (635) --> [M1G + H]+ (188) transition increases the detection sensitivity by nearly an order of magnitude relative to the measurement of M1G-dR by SRM. For accurate measurement, analytical standard (AS) DNA and internal standard (IS) DNA were used. High purity 15N-labeled DNA was isolated from Escherichia coli that had been grown in minimum salt medium containing (15NH4)2SO4. The 15N-DNA and calf thymus DNA were treated with malondialdehyde to induce a high number of M1G adducts to prepare the IS and AS DNA, respectively. A consistent calibration curve was established from the analysis of 200 microg of blank DNA, 23 ng of IS DNA (400 fmol of 15N5-M1G-dR), and AS DNA containing 0-810 fmol of M1G-dR. With the use of this novel IS DNA and selective labeling, this assay is a useful tool for monitoring oxidative stress-induced DNA damage from small amounts of DNA without the need of a specific antibody or laborious procedures. By this assay, two M1G adducts/10(8) guanines can readily be detected. Furthermore, this approach should be applicable to the analysis of other aldehydic DNA adducts as well as the measurement of an array of DNA lesions.

  5. Targeting CD146 with a 64Cu-labeled antibody enables in vivo immunoPET imaging of high-grade gliomas

    PubMed Central

    Yang, Yunan; Hernandez, Reinier; Rao, Jun; Yin, Li; Qu, Yazhuo; Wu, Jinrong; England, Christopher G.; Graves, Stephen A.; Lewis, Christina M.; Wang, Pu; Meyerand, Mary E.; Nickles, Robert J.; Bian, Xiu-wu; Cai, Weibo

    2015-01-01

    Given the highly heterogeneous character of brain malignancies and the associated implication for its proper diagnosis and treatment, finding biomarkers that better characterize this disease from a molecular standpoint is imperative. In this study, we evaluated CD146 as a potential molecular target for diagnosis and targeted therapy of glioblastoma multiforme (GBM), the most common and lethal brain malignancy. YY146, an anti-CD146 monoclonal antibody, was generated and radiolabeled for noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. 64Cu-labeled YY146 preferentially accumulated in the tumors of mice bearing U87MG xenografts, which allowed the acquisition of high-contrast PET images of small tumor nodules (∼2 mm). Additionally, we found that tumor uptake correlated with the levels of CD146 expression in a highly specific manner. We also explored the potential therapeutic effects of YY146 on the cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) properties of U87MG cells, demonstrating that YY146 can mitigate those aggressive phenotypes. Using YY146 as the primary antibody, we performed histological studies of World Health Organization (WHO) grades I through IV primary gliomas. The positive correlation found between CD146-positive staining and high tumor grade (χ2 = 9.028; P = 0.029) concurred with the GBM data available in The Cancer Genome Atlas (TCGA) and validated the clinical value of YY146. In addition, we demonstrate that YY146 can be used to detect CD146 in various cancer cell lines and human resected tumor tissues of multiple other tumor types (gastric, ovarian, liver, and lung), indicating a broad applicability of YY146 in solid tumors. PMID:26553993

  6. Trace fluorescent labeling for protein crystallization

    PubMed Central

    Pusey, Marc; Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-01-01

    Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent. PMID:26144224

  7. Label-free and enzyme-free detection of transcription factors with graphene oxide fluorescence switch-based multifunctional G-quadruplex-hairpin probe.

    PubMed

    Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2016-01-15

    Transcription factors (TFs) play pivotal roles in the regulation of a variety of essential cellular processes and some of them have been recognized as potential diagnostic markers and therapeutic targets of some diseases. Sensitive and accurate detection of TFs is of great importance to better understanding their roles in gene regulation and evaluation of disease state. Here, we developed a simple, label-free and enzyme-free new fluorescent strategy for the detection of TFs by graphene oxide (GO) fluorescence switch-based multifunctional G-quadruplex-hairpin probe (MGHP). The MGHP possessed of three functions simultaneously, adsorbing onto GO with the loop part, binding to target with the stem part and serving as signal carrier with the terminal G-quadruplex. First, the MGHP was adsorbed quickly to GO. Next, the TF bound to the stem part of MGHP to form a huge target-MGHP complex, which led to desorption of the complex from GO. Finally, NMM was inserted into G-quadruplex in the complex to yield an enhanced fluorescence response. The GO used here, as a fluorescence switch, could quickly and efficiently quench the fluorescence of NMM inserted into the MGHP absorbed on the GO, guaranteeing a high signal-to-noise ratio. Sensitive detection of purified NF-κB p50 and HeLa cell nuclear extracts were achieved with detection limits of 0.2nM and 7.8ng/µL, respectively. Moreover, this proposed strategy could be used to screen inhibitors of NF-κB p50 activity. The strategy proposed here might offer a new potential approach for reliable quantification of TFs in clinical diagnostics and treatment research of some diseases.

  8. Europium-decorated graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection of Cu(2+) and L-cysteine.

    PubMed

    Lin, Liping; Song, Xinhong; Chen, Yiying; Rong, Mingcong; Wang, Yiru; Zhao, Li; Zhao, Tingting; Chen, Xi

    2015-09-03

    In this work, europium-decorated graphene quantum dots (Eu-GQDs) were prepared by treating three-dimensional Eu-decorated graphene (3D Eu-graphene) via a strong acid treatment. Various characterizations revealed that Eu atoms were successfully complexed with the oxygen functional groups on the surface of graphene quantum dots (GQDs) with the atomic ratio of 2.54%. Compared with Eu free GQDs, the introduction of Eu atoms enhanced the electron density and improved the surface chemical activities of Eu-GQDs. Therefore, the obtained Eu-GQDs were used as a novel "off-on" fluorescent probe for the label-free determination of Cu(2+) and l-cysteine (L-Cys) with high sensitivity and selectivity. The fluorescence intensity of Eu-GQDs was quenched in the presence of Cu(2+) owing to the coordination reaction between Cu(2+) and carboxyl groups on the surface of the Eu-GQDs. The fluorescence intensity of Eu-GQDs recovered with the subsequent addition of L-Cys because of the strong affinity of Cu(2+) to L-Cys via the Cu-S bond. The experimental results showed that the fluorescence variation of the proposed approach had a good linear relationship in the range of 0.1-10 μM for Cu(2+) and 0.5-50 μM for L-Cys with corresponding detection limits of 0.056 μM for Cu(2+) and 0.31 μM for L-Cys. The current approach also displayed a special response to Cu(2+) and L-Cys over the other co-existing metal ions and amino acids, and the results obtained from buffer-diluted serum samples suggested its applicability in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using ( sup 125 I)iodinated probes

    SciTech Connect

    Ruat, M.; Traiffort, E.; Bouthenet, M.L.; Schwartz, J.C.; Hirschfeld, J.; Buschauer, A.; Schunack, W. )

    1990-03-01

    Iodoaminopotentidine (I-APT)--i.e., N-(2-(4-amino-3-iodobenzamido)ethyl)-N'-cyano-N''-(3-(3- (1-piperidinylmethyl)phenoxy)propyl)guanidine--represents one of the most potent H2-receptor antagonists known so far. In membranes of guinea pig brain 125I-APT bound reversibly, selectively, and with high affinity (Kd = 0.3 nM) to a homogeneous population of sites unambiguously identified as H2 receptors by inhibition studies conducted with a large panel of antagonists. 125I-APT binding was also inhibited by histamine, and the effect was modulated by a guanyl nucleotide, which is consistent with the association of the H2 receptor with a guanine nucleotide binding regulatory protein. The low nonspecific binding of 125I-APT generated high contrast autoradiographic pictures in brain sections and established the precise distribution of H2 receptors. Their highly heterogeneous distribution and laminated pattern in some areas suggest their major association with neuronal elements. These localizations were more consistent than those of H1 receptors with the distribution of histaminergic projections, indicating that H2 receptors mediate a larger number of postsynaptic actions of histamine--e.g., in striatum. Colocalizations of H1 and H2 receptors in some areas account for their known synergistic interactions in cAMP formation induced by histamine. The distribution of 125I-APT binding sites did not strictly parallel that of the H2-receptor-linked adenylate cyclase activity, which may reflect heterogeneity among H2 receptors. After UV irradiation and SDS/PAGE analysis, (125I)iodoazidopotentidine (125I-AZPT), a photoaffinity probe derived from 125I-APT, was covalently incorporated in several peptides, among which the labeling of two peptides of 59 and 32 kDa was prevented by H2 antagonists, suggesting that they correspond to H2-receptor binding peptides or proteolysis products of the latter.

  10. Synthesis and evaluation of 18F-labeled ATP competitive inhibitors of topoisomerase II as probes for imaging topoisomerase II expression

    PubMed Central

    Daumar, Pierre; Zeglis, Brian M.; Ramos, Nicholas; Divilov, Vadim; Sevak, Kuntal Kumar; Pillarsetty, NagaVaraKishore; Lewis, Jason S.

    2015-01-01

    Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription, replication, and chromosome segregation processes and, as such, represents an attractive target for cancer therapy. Numerous studies indicate that the response to treatment with Topo-II inhibitors is highly dependent on both the levels and the activity of the enzyme. Consequently, a non-invasive assay to measure tumoral Topo-II levels has the potential to differentiate responders from non-responders. With the ultimate goal of developing a radiofluorinated tracer for positron emission tomography (PET) imaging, we have designed, synthesized, and evaluated a set of fluorinated compounds based on the structure of the ATP-competitive Topo-II inhibitor QAP1. Compounds 18 and 19b showed inhibition of Topo-II in in vitro assays and exhibited moderate, Topo-II level dependent cytotoxicity in SK-BR-3 and MCF-7 cell lines. Based on these results, 18F-labeled analogs of these two compounds were synthesized and evaluated as PET probes for imaging Topo-II overexpression in mice bearing SK-BR-3 xenografts. [18F]-18 and [18F]-19b were synthesized from their corresponding protected tosylated derivatives by fluorination and subsequent deprotection. Small animal PET imaging studies indicated that both compounds do not accumulate in tumors and exhibit poor pharmacokinetics, clearing from the blood pool very rapidly and getting metabolized over. The insights gained from the current study will surely aid in the design and construction of future generations of PET agents for the non-invasive delineation of Topo-II expression. PMID:25240701

  11. Design principles and theory of paramagnetic fluorine-labelled lanthanide complexes as probes for (19)F magnetic resonance: a proof-of-concept study.

    PubMed

    Chalmers, Kirsten H; De Luca, Elena; Hogg, Naomi H M; Kenwright, Alan M; Kuprov, Ilya; Parker, David; Botta, Mauro; Wilson, J Ian; Blamire, Andrew M

    2010-01-04

    The synthesis and spectroscopic properties of a series of CF(3)-labelled lanthanide(III) complexes (Ln=Gd, Tb, Dy, Ho, Er, Tm) with amide-substituted ligands based on 1,4,7,10-tetraazacyclododecane are described. The theoretical contributions of the (19)F magnetic relaxation processes in these systems are critically assessed and selected volumetric plots are presented. These plots allow an accurate estimation of the increase in the rates of longitudinal and transverse relaxation as a function of the distance between the Ln(III) ion and the fluorine nucleus, the applied magnetic field, and the re-rotational correlation time of the complex, for a given Ln(III) ion. Selected complexes exhibit pH-dependent chemical shift behaviour, and a pK(a) of 7.0 was determined in one example based on the holmium complex of an ortho-cyano DO3A-monoamide ligand, which allowed the pH to be assessed by measuring the difference in chemical shift (varying by over 14 ppm) between two (19)F resonances. Relaxation analyses of variable-temperature and variable-field (19)F, (17)O and (1)H NMR spectroscopy experiments are reported, aided by identification of salient low-energy conformers by using density functional theory. The study of fluorine relaxation rates, over a field range of 4.7 to 16.5 T allowed precise computation of the distance between the Ln(III) ion and the CF(3) reporter group by using global fitting methods. The sensitivity benefits of using such paramagnetic fluorinated probes in (19)F NMR spectroscopic studies are quantified in preliminary spectroscopic and imaging experiments with respect to a diamagnetic yttrium(III) analogue.

  12. Isolation of reducing oligosaccharide chains from the chondroitin/dermatan sulfate-protein linkage region and preparation of analytical probes by fluorescent labeling with 2-aminobenzamide.

    PubMed

    Sakaguchi, H; Watanabe, M; Ueoka, C; Sugiyama, E; Taketomi, T; Yamada, S; Sugahara, K

    2001-01-01

    The glycosaminoglycan (GAG)-protein linkage regions of various proteoglycans share the common tetrasaccharide GlcA-Gal-Gal-Xyl-attached to Ser residues in the core proteins. In previous analysis we demonstrated unique modifications by epimerization, sulfation and phosphorylation of the component sugars. Here we developed a sensitive analytical method for the linkage region oligosaccharides to detect or monitor structural variations and changes. This will be useful for investigation of their biological roles, which are largely unknown, but they have been implicated in biosynthesis. A variety of linkage region-derived hexasaccharides was first prepared as reducing sugar chains from peptide chondroitin/dermatan sulfate of whale cartilage, shark cartilage, and bovine aorta by means of chondroitinase digestion in conjunction with beta-elimination in the absence of reducing reagents, but involving a mild alkali, 0.5 M LiOH, at 4 degrees C to prevent peeling reactions. The structures of these oligosaccharides were determined by the combination of HPLC, enzymatic digestion, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and (1)H NMR spectroscopy, which revealed eleven different hexasaccharides including a novel structure, DeltaHexAalpha1-3GalNAcbeta1-4IdoAalpha1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl (DeltaHexA and IdoA represent unsaturated hexuronic acid and L-iduronic acid, respectively). These oligosaccharides were labeled with a fluorophore, 2-aminobenzamide, to prepare analytical probes using the recently developed procedure [Kinoshita and Sugahara (1999) Anal. Biochem. 269, 367-378]. The fluorophore-tagged hexasacharides of low picomoles were well separated by HPLC and successfully analyzed by MALDI-TOF mass spectrometry. The principle of the method should be applicable to the analysis of the linkage region oligosaccharides derived from heparin and heparan sulfate as well.

  13. Enabling user-guided segmentation and tracking of surface-labeled cells in time-lapse image sets of living tissues.

    PubMed

    Mashburn, David N; Lynch, Holley E; Ma, Xiaoyan; Hutson, M Shane

    2012-05-01

    To study the process of morphogenesis, one often needs to collect and segment time-lapse images of living tissues to accurately track changing cellular morphology. This task typically involves segmenting and tracking tens to hundreds of individual cells over hundreds of image frames, a scale that would certainly benefit from automated routines; however, any automated routine would need to reliably handle a large number of sporadic, and yet typical problems (e.g., illumination inconsistency, photobleaching, rapid cell motions, and drift of focus or of cells moving through the imaging plane). Here, we present a segmentation and cell tracking approach based on the premise that users know their data best-interpreting and using image features that are not accounted for in any a priori algorithm design. We have developed a program, SeedWater Segmenter, that combines a parameter-less and fast automated watershed algorithm with a suite of manual intervention tools that enables users with little to no specialized knowledge of image processing to efficiently segment images with near-perfect accuracy based on simple user interactions.

  14. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy

    PubMed Central

    2015-01-01

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  15. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

    PubMed

    Hammers, Matthew D; Taormina, Michael J; Cerda, Matthew M; Montoya, Leticia A; Seidenkranz, Daniel T; Parthasarathy, Raghuveer; Pluth, Michael D

    2015-08-19

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.

  16. Zip nucleic acids are potent hydrolysis probes for quantitative PCR

    PubMed Central

    Paris, Clément; Moreau, Valérie; Deglane, Gaëlle; Voirin, Emilie; Erbacher, Patrick; Lenne-Samuel, Nathalie

    2010-01-01

    Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3′ end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes. PMID:20071749

  17. Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy.

    PubMed

    Flaender, M; Sicoli, G; Fontecave, Th; Mathis, G; Saint-Pierre, C; Boulard, Y; Gambarelli, S; Gasparutto, D

    2008-01-01

    A new approach is described for the insertion of nitroxide spin-labels at specific positions within DNA oligomers. The latter bioconjugaison strategy is based on a click chemistry 1,3-dipolar cycloaddition between a spin-labeling reagent, namely the 4-azido-TEMPO, and alkyne modified uridine-containing oligonucleotides. This highly efficient labeling method was applied for site-specific incorporation of two TEMPO units within a set of double-stranded DNA constructs. Then the determination of the inter-nitroxide distances was achieved by using a four-pulses DEER technique that successfully validates the new site-directed spin labeling strategy.

  18. Hydrophobic pocket targeting probes for enteroviruses

    NASA Astrophysics Data System (ADS)

    Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

    2015-10-01

    Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content

  19. Sensitive detection of Fusarium circinatum in pine seed by combining an enrichment procedure with a real-time polymerase chain reaction using dual-labeled probe chemistry.

    PubMed

    Ioos, Renaud; Fourrier, Céline; Iancu, Gabriela; Gordon, Thomas R

    2009-05-01

    Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

  20. Probing the micellization kinetics of pyrene end-labeled diblock copolymer via a combination of stopped-flow light-scattering and fluorescence techniques.

    PubMed

    Zhang, Jingyan; Li, Yuting; Armes, Steven P; Liu, Shiyong

    2007-10-25

    A pyrene end-labeled double hydrophilic diblock copolymer, poly(2-(diethylamino)ethyl methacrylate)-b-poly(2-(dimethylamino)ethyl methacrylate) (Py-PDEA-b-PDMA), was synthesized by sequential monomer addition via oxyanionic polymerization using a 1-pyrenemethanol-based initiator. This diblock copolymer exhibits reversible pH-responsive micellization behavior in aqueous solution, forming PDEA-core micelles stabilized by the soluble PDMA block at neutral or alkaline pH. Taking advantage of the pyrene probe covalently attached to the end of the PDEA block, the pH-induced micellization kinetics of Py-PDEA-b-PDMA was monitored by stopped-flow light scattering using a fluorescence detector. Upon a pH jump from 4.0 to 9.0, both the scattered light intensity and excimer/monomer fluorescence intensity ratios (IE/IM) increase abruptly initially, followed by a more gradual increase to reach plateau values. Interestingly, the IE/IM ratio increases abruptly within the first 10 ms: a triple exponential function is needed to fit the corresponding dynamic trace, leading to three characteristic relaxation time constants (tau(1,fluo) < tau(2,fluo) < tau(3,fluo)). On the other hand, dynamic traces for the scattered light intensity can be well-fitted by double exponential functions: the resulting time constants tau(1,scat) and tau(2,scat) can be ascribed to formation of the quasi-equilibrium micelles and relaxation into their final equilibrium state, respectively. Most importantly, tau(1,scat) obtained from stopped-flow light scattering is in general agreement with tau(2,fluo) obtained from stopped-flow fluorescence. The fastest process (tau(1,fluo) approximately 4 ms) detected by stopped-flow fluorescence is ascribed to the burst formation of small transient micelles comprising only a few chains, which are too small to be detected by conventional light scattering. These nascent micelles undergo rapid fusion and grow into quasi-equilibrium micelles and then slowly approach their final

  1. Development of a (11)C-labeled tetrazine for rapid tetrazine-trans-cyclooctene ligation.

    PubMed

    Herth, Matthias M; Andersen, Valdemar L; Lehel, Szabolcs; Madsen, Jacob; Knudsen, Gitte M; Kristensen, Jesper L

    2013-05-08

    Tetrazine-trans-cyclooctene ligations are remarkably fast and selective reactions even at low micro-molar concentrations. In bioorthogonal radiochemistry, tools that enable conjugation of radioactive probes to pre-targeted vectors are of great interest. Herein, we describe the successful development of the first (11)C-labelled tetrazine and its reaction with trans-cyclooctenol.

  2. The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

    SciTech Connect

    Cooper, L.; Cooper, R.D.; Burkot, T.R. )

    1991-07-01

    Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.

  3. Benzimidazole covalent probes and the gastric H+/K+-ATPase as a model system for protein labeling in a copper-free setting

    PubMed Central

    Paresi, Chelsea J.; Liu, Qi; Li, Yue-Ming

    2016-01-01

    Affinity probes are useful tools for determining molecular targets and elucidating mechanism of action for novel, bioactive compounds. In the case of covalent inhibitors, activity based probes are particularly valuable for ensuring acceptable selectivity margins. However, there is a variety of bioorthogonalchemisty reactions available for modifying compounds of interest with clickable tags. Here, we describe a direct comparison of tetrazine ligation and strain promoted azide-alkyne cycloaddition using benzimidazole based probes to bind their known target, the gastric proton pump, ATP4A. This study validates the use of chemical probes for target identification and illustrates the superior efficiency of tetrazine ligation for copper-free click systems. In addition, we have identified several novel binding partners of benzimidazole probes: Isoform 2 of deleted in malignant brain tumors 1 protein (DMBT1) and three uncharacterized proteins. PMID:26952080

  4. Use of oligodeoxynucleotide signature probes for identification of physiological groups of methylotrophic bacteria

    SciTech Connect

    Tsien, H.C.; Bratina, B.J.; Tsuji, K.; Hanson, R.S. )

    1990-09-01

    Oligodeoxynucleotide sequences that uniquely complemented 16S rRNAs of each group of methylotrophs were synthesized and used as hybridization probes for the identification of methylotrophic bacteria possessing the serine and ribulose monophosphate (RuMP) pathways for formaldehyde fixation. The specificity of the probes was determined by hybridizing radiolabeled probes with slot-blotted RNAs of methylotrophs and other eubacteria followed by autoradiography. The washing temperature was determined experimentally to be 50 and 52{degrees}C for 9-{alpha} (serine pathway) and 10-{gamma} (RuMP pathway) probes, respectively. RNAs isolated from serine pathway methylotrophs bound to probe 9-{alpha}, and RNAs from RuMP pathway methylotrophs bound to probe 10-{gamma}. Nonmethylotrophic eubacterial RNAs did not bind to either probe. The probes were also labeled with fluorescent dyes. Cells fixed to microscope slides were hybridized with these probes, washed, and examined in a fluorescence microscope equipped with appropriate filter sets. Cells of methylotrophic bacteria possessing the serine or RuMP pathway specifically bind probes designed for each group. Samples with a mixture of cells of type I and II methanotrophs were detected and differentiated with single probes or mixed probes labeled with different fluorescent dyes, which enabled the detection of both types of cells in the same microscopic field.

  5. DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05420j Click here for additional data file.

    PubMed Central

    Agasti, Sarit S.; Wang, Yu; Schueder, Florian; Sukumar, Aishwarya

    2017-01-01

    Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed cells. PMID:28451377

  6. Spin-Labeled Analogs of CMP-NeuAc as NMR Probes of the α-2,6-Sialyltransferase ST6Gal I

    PubMed Central

    Liu, Shan; Venot, Andre; Meng, Lu; Tian, Fang; Moremen, Kelley W.; Boons, Geert-Jan; Prestegard, James H.

    2014-01-01

    SUMMARY Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I. PMID:17462576

  7. Trace fluorescent labeling for protein crystallization

    SciTech Connect

    Pusey, Marc Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-06-27

    The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent.

  8. Two-step labeling of Staphylococcus aureus with Lysostaphin-Azide and DIBO-Alexa using click chemistry.

    PubMed

    Potapova, Inga; Eglin, David; Laschke, Matthias W; Bischoff, Markus; Richards, R Geoff; Moriarty, T Fintan

    2013-01-01

    Specific bacteria imaging is highly desirable in clinical diagnostics. Probes enabling rapid and specific diagnostics of bacteria are limited. Current clinical infection diagnostics is time consuming and invasive, relies on microbiological cultures. We investigated the potential of Lysostaphin as a specific probe to label staphylococci in a new labeling protocol. We used azido (N(3))-modified Lysostaphin-N(3) and DIBO-dye in a two-step bacteria-labeling protocol. N(3) and DIBO (di-benzocyclooctyne) are the counterparts of the "click" chemistry. In the first step, Lysostaphin-N(3) binds specifically to Staphylococcus aureus. In the second step, N(3) clicks to DIBO thus achieving the selective for S. aureus labeling. Such a two-step approach effectively distinguishes S. aureus from Escherichia coli; non-toxic and was proven to work in vivo. The two-step labeling protocol is a promising approach for diagnostic imaging of staphylococcal infections in clinical settings.

  9. Using infrared spectroscopy of a nitrile labeled phenylalanine and tryptophan fluorescence to probe the α-MSH peptide's side-chain interactions with a micelle model membrane

    NASA Astrophysics Data System (ADS)

    Gonzalez, Javier D.; Levonyak, Nicholas S.; Schneider, Sydney C.; Smith, Matthew J.; Cremeens, Matthew E.

    2014-01-01

    The interactions of α-MSH (Ac-SYSMEHFRWGKPV-NH2) side-chains were biophysically characterized with a micelle model membrane and in model intracellular bacterial conditions using infrared (IR) spectroscopy of a nitrile labeled α-MSH analogue, circular dichroism (CD), and tryptophan fluorescence. Local changes detected by the tryptophan and a nitrile-labeled phenylalanine using fluorescence and infrared spectroscopies, respectively, suggest that the Trp9 side-chain in the conserved core (HisPheArgTrp) of α-MSH is buried in an SDS micellar environment, while Phe(CN)7 does not appear to be buried.

  10. Photoacoustic lifetime contrast between methylene blue monomers and self-quenched dimers as a model for dual-labeled activatable probes

    PubMed Central

    Shao, Qi; Hackel, Benjamin J.; Thomas, David D.; Ashkenazi, Shai

    2013-01-01

    Abstract. Activatable photoacoustic probes efficiently combine the high spatial resolution and penetration depth of ultrasound with the high optical contrast and versatility of molecular imaging agents. Our approach is based on photoacoustic probing of the excited-state lifetime of methylene blue (MB), a fluorophore widely used in clinical therapeutic and diagnostic applications. Upon aggregation, static quenching between the bound molecules dramatically shortens their lifetime by three orders of magnitude. We present preliminary results demonstrating the ability of photoacoustic imaging to probe the lifetime contrast between monomers and dimers with high sensitivity in cylindrical phantoms. Gradual dimerization enhancement, driven by the addition of increasing concentrations of sodium sulfate to a MB solution, showed that lifetime-based photoacoustic probing decreases linearly with monomer concentration. Similarly, the addition of 4 mM sodium dodecyl sulfate, a concentration that amplifies MB aggregation and reduces the monomer concentration by more than 20-fold, led to a signal decrease of more than 20 dB compared to a solution free of surfactant. These results suggest that photoacoustic imaging can be used to selectively detect the presence of monomers. We conclude by discussing the implementation of the monomer–dimer contrast mechanism for the development of an enzyme-specific activatable probe. PMID:23640075

  11. 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.

  12. Acidic pH-induced membrane insertion of colicin A into E. coli natural lipids probed by site-directed spin labeling.

    PubMed

    Pulagam, Lakshmi Padmavathi; Steinhoff, Heinz-Jürgen

    2013-05-27

    Colicin A is a pore-forming toxin that forms a voltage-gated channel in the inner membrane of the target bacteria. The structures of the closed and open channel states of membrane-bound colicin A are not resolved. In the present site-directed spin-labeling study, the insertion-competent state of colicin A is provoked by an acidic pH jump prior to the insertion into liposomes prepared from Escherichia coli natural lipids. The membrane-bound colicin A is able to open a voltage-dependent channel as demonstrated by the efflux of tempophosphate spin label from the lumen of liposomes. The EPR spectra of spin-labeled colicin A variants in the membrane-bound closed channel state reveal a conformational equilibrium with resolved interhelical tertiary contacts. The spin label accessibility and polarity profiles suggest the amphipathic helices (H1-H7 and H10) to be located in the membrane close to the membrane-water interface and the hydrophobic hairpin (H8 and H9) to be immersed more deeply in the membrane.

  13. Thin filament activation probed by fluorescence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle.

    PubMed

    Brenner, B; Kraft, T; Yu, L C; Chalovich, J M

    1999-11-01

    A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole, IANBD) on Cys(133) of the troponin I subunit. IANBD-labeled troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (. Proc. Natl. Acad. Sci. USA. 77:7209-7213). In the skinned fibers, the fluorescence of this probe is unaffected when cross-bridges in their weak binding states attach to actin filaments but decreases either upon the addition of Ca(2+) or when cross-bridges in their strong binding states attach to actin. Maximum reduction is observed when Ca(2+) is raised to saturating concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-bridges in strong binding states alone (low Ca(2+) concentration) gives only about half of the maximum reduction seen with the addition of calcium. This illustrates that fluorescence of IANBD-labeled troponin I can be used to evaluate thin filament activation, as previously introduced for solution studies. In addition, at nonsaturating Ca(2+) concentrations IANBD fluorescence can be used for straightforward classification of states of the myosin head as weak binding (nonactivating) and strong binding (activating), irrespective of ionic strength or other experimental conditions. Furthermore, the approach presented here not only can be used as a means of exchanging native skeletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal troponin for cardiac troponin.

  14. CdTe/CdS-MPA quantum dots as fluorescent probes to label yeast cells: synthesis, characterization and conjugation with Concanavalin A

    NASA Astrophysics Data System (ADS)

    Kato, Ilka T.; Santos, Camila C.; Benetti, Endi; Tenório, Denise P. L. A.; Cabral Filho, Paulo E.; Sabino, Caetano P.; Fontes, Adriana; Santos, Beate S.; Prates, Renato A.; Ribeiro, Martha S.

    2012-03-01

    Candida albicans is the most frequent human opportunistic pathogenic fungus and one of the most important causes of nosocomial infections. In fact, diagnosis of invasive candidiasis presents unique problems. The aim of this work was to evaluate, by fluorescence image analysis, cellular labeling of C. albicans with CdTe/CdS quantum dots conjugated or not to concanavalin A (ConA). Yeast cells were incubated with CdTe/CdS quantum dots (QD) stabilized with mercaptopropionic acid (MPA) (emission peak at 530 nm) for 1 hour. In the overall study we observed no morphological alterations. The fluorescence microscopic analysis of the yeast cells showed that the non-functionalized QDs do not label C. albicans cells, while for the QD conjugated to ConA the cells showed a fluorescence profile indicating that the membrane was preferentially marked. This profile was expected since Concanavalin A is a protein that binds specifically to terminal carbohydrate residues at the membrane cell surface. The results suggest that the QD-labeled Candida cells represent a promising tool to open new possibilities for a precise evaluation of fungal infections in pathological conditions.

  15. The sandwich-type electrochemiluminescence immunosensor for α-fetoprotein based on enrichment by Fe3O4-Au magnetic nano probes and signal amplification by CdS-Au composite nanoparticles labeled anti-AFP.

    PubMed

    Zhou, Hankun; Gan, Ning; Li, Tianhua; Cao, Yuting; Zeng, Saolin; Zheng, Lei; Guo, Zhiyong

    2012-10-09

    A novel and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor was fabricated on a glassy carbon electrode (GCE) for ultra trace levels of α-fetoprotein (AFP) based on sandwich immunoreaction strategy by enrichment using magnetic capture probes and quantum dots coated with Au shell (CdS-Au) as the signal tag. The capture probe was prepared by immobilizing the primary antibody of AFP (Ab1) on the core/shell Fe(3)O(4)-Au nanoparticles, which was first employed to capture AFP antigens to form Fe(3)O(4)-Au/Ab1/AFP complex from the serum after incubation. The product can be separated from the background solution through the magnetic separation. Then the CdS-Au labeled secondary antibody (Ab2) as signal tag (CdS-Au/Ab2) was conjugated successfully with Fe(3)O(4)-Au/Ab1/AFP complex to form a sandwich-type immunocomplex (Fe(3)O(4)-Au/Ab1/AFP/Ab2/CdS-Au), which can be further separated by an external magnetic field and produce ECL signals at a fixed voltage. The signal was proportional to a certain concentration range of AFP for quantification. Thus, an easy-to-use immunosensor with magnetic probes and a quantum dots signal tag was obtained. The immunosensor performed at a level of high sensitivity and a broad concentration range for AFP between 0.0005 and 5.0 ng mL(-1) with a detection limit of 0.2 pg mL(-1). The use of magnetic probes was combined with pre-concentration and separation for trace levels of tumor markers in the serum. Due to the amplification of the signal tag, the immunosensor is highly sensitive, which can offer great promise for rapid, simple, selective and cost-effective detection of effective biomonitoring for clinical application.

  16. Validating Transcripts with Probes and Imaging Technology

    PubMed Central

    Itzkovitz, Shalev; van Oudenaarden, Alexander

    2011-01-01

    High throughput gene expression screens provide a quantitative picture of the average expression signature of biological samples. However, the analysis of spatial gene expression patterns with single cell resolution requires quantitative in-situ measurement techniques. Here we describe recent technological advances in RNA fluorescent in-situ hybridization (FISH) techniques that facilitate detection of individual fluorescently labeled mRNA molecules of practically any endogenous gene. These methods, which are based on advances in probe design, imaging technology, and image processing, enable the absolute measurement of transcript abundance in individual cells with single-molecule resolution. PMID:21451512

  17. (13)C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging.

    PubMed

    Salamanca-Cardona, Lucia; Keshari, Kayvan R

    2015-01-01

    In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-(13)C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.

  18. 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.

  19. Lab in a Tube: Sensitive Detection of MicroRNAs in Urine Samples from Bladder Cancer Patients Using a Single-Label DNA Probe with AIEgens.

    PubMed

    Min, Xuehong; Zhuang, Yuan; Zhang, Zhenyu; Jia, Yongmei; Hakeem, Abdul; Zheng, Fuxin; Cheng, Yong; Tang, Ben Zhong; Lou, Xiaoding; Xia, Fan

    2015-08-05

    We demonstrate an ultrasensitive microRNA detection method based on an extremely simple probe with only fluorogens but without quencher groups. It avoids complex and difficult steps to accurately design the relative distance between the fluorogens and quencher groups in the probes. Furthermore, the assay could accomplish various detection limits by tuning the reaction temperature due to the different activity of exonuclease III corresponding to the diverse temperature. Specifically, 1 pM miR-21 can be detected in 40 min at 37 °C, and 10 aM (about 300 molecules in 50 μL) miR-21 could be discriminated in 7 days at 4 °C. The great specificity of the assay guarantees that the real 21 urine samples from the bladder cancer patients are successfully detected by our method.

  20. SERS quantitative detection of trace human chorionic gonadotropin using a label-free Victoria blue B as probe in the aggregated immunonanogold sol substrate.

    PubMed

    Ma, Lu; Wen, Guiqing; Ye, Lingling; Lu, Zujun; Luo, Yanghe; Liang, Aihui; Jiang, Zhiliang

    2015-09-01

    Nanogold particles (NG) were modified by anti-rabbit antibody (RAb) against human chorionic gonadotropin to obtain an immunonanogold probe (ING). In pH 7.0 Na2HPO4-citrate buffer solution containing KCl, ING probes formed large aggregates in which Victoria blue B (VBB) molecules were adsorbed on the surface and which exhibited strong surface-enhanced Raman scattering (SERS) at a peak of 1612 cm(-1). After addition of human chorionic gonadotropin (hCG) an immune reaction with the ING probe occurred to form dispersive ING-hCG complexes with non-SERS activity that led to a decreased SERS peak at 1612 cm(-1). The decreased SERS intensity was linear to the concentration of hCG over 2.4-73.2 ng/mL. The ING reaction was studied in detail by SERS, scanning electron microscope (SEM), resonance Rayleigh scattering (RRS), surface plasmon resonance (SPR) absorption and laser scattering techniques. SERS quenching was observed and discussed. Copyright © 2014 John Wiley & Sons, Ltd.

  1. A label-free aptasensor based on polyethyleneimine wrapped carbon nanotubes in situ formed gold nanoparticles as signal probe for highly sensitive detection of dopamine.

    PubMed

    Azadbakht, Azadeh; Roushani, Mahmoud; Abbasi, Amir Reza; Menati, Saeid; Derikvand, Zohreh

    2016-11-01

    Herein, a highly sensitive and selective aptamer biosensor for quantitative detection of a model target, dopamine (DA), was developed by using a gold (Au) electrode modified with highly dispersed gold nanoparticles (AuNPs) and acid-oxidized carbon nanotubes (CNTs-COOH) functionalized with polyethyleneimine (PEI). Amine-terminated12-mercaptureprobe (ssDNA1) as a capture probe and specific DA-aptamer (ssDNA2) as a detection probe was immobilized on the surface of a modified electrode via the formation of covalent amide bond and hybridization, respectively. Methylene blue (MB) was used as the redox probe, which was intercalated into the aptamer through the specific interaction with its guanine bases. In the presence of DA, the interaction between aptamer and DA displaced the MB from the electrode surface, rendering a lowered electrochemical signal attributed to decreased amount of adsorbed MB. The developed electrochemical DA aptasensor showed a good linear response to DA from 5 to 300nM with detection limit of 2.1nM. The biosensor also exhibited satisfactory selectivity and could be successfully used to detect DA in blood serum sample.

  2. Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression.

    PubMed

    Nguyen, Minh Hong; Ojima, Yoshihiro; Sakka, Makiko; Sakka, Kazuo; Taya, Masahito

    2014-10-01

    Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing.

  3. Food Labels

    MedlinePlus

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Food Labels KidsHealth > For Teens > Food Labels Print A ... have at least 95% organic ingredients. continue Making Food Labels Work for You The first step in ...

  4. Photoaffinity-labeled Cytokinins

    PubMed Central

    Theiler, Jane B.; Leonard, Nelson J.; Schmitz, Ruth Y.; Skoog, Folke

    1976-01-01

    Two new azidopurine derivatives, 2-azido-N6-(Δ2-isopentenyl)adenine and 2-azido-N6-benzyladenine, have been synthesized as potential photoaffinity labels for probing cytokinin-binding sites. The preparation and the biological activity of these compounds are described. PMID:16659772

  5. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, B.F.; Chen, B.X.

    1997-07-22

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest. 8 figs.

  6. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, Bernard F.; Chen, Bi-Xing

    1997-01-01

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest.

  7. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, Bernard F.; Chen, Bi-Xing

    1999-01-01

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example, detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest, detecting a polypeptide such as those expressed by infectious agents, fungi or parasites.

  8. NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro.

    PubMed

    Lee, Ronald F S; Escrig, Stéphane; Croisier, Marie; Clerc-Rosset, Stéphanie; Knott, Graham W; Meibom, Anders; Davey, Curt A; Johnsson, Kai; Dyson, Paul J

    2015-11-28

    The in vitro inter- and intra-cellular distribution of an isotopically labelled ruthenium(II)-arene (RAPTA) anti-metastatic compound in human ovarian cancer cells was imaged using nano-scale secondary ion mass spectrometry (NanoSIMS). Ultra-high resolution isotopic images of (13)C, (15)N, and Ru indicate that the phosphine ligand remains coordinated to the ruthenium(II) ion whereas the arene detaches. The complex localizes mainly on the membrane or at the interface between cells which correlates with its anti-metastatic effects.

  9. Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

    DOEpatents

    Erlanger, B.F.; Chen, B.

    1999-07-20

    The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example, detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest, detecting a polypeptide such as those expressed by infectious agents, fungi or parasites. 25 figs.

  10. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    PubMed

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  11. A fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip.

    PubMed

    Li, Xuepu; Lu, Donglian; Sheng, Zonghai; Chen, Kun; Guo, Xuebo; Jin, Meilin; Han, Heyou

    2012-10-15

    A novel fluorescence immunoassay method for fast and ultrasensitive detection of avian influenza virus (AIV) was developed. The immunoassay method which integrated lateral flow test strip technique with fluorescence immunoassay used the label-free and high luminescent quantum dots (QDs) as signal output. By the sandwich immunoreaction performed on lateral flow test strip, the gold nanoparticle (NP) labels were captured in the test zone and further dissolved to release a large number of gold ions as a signal transduction bridge that was detected by the QDs-based fluorescence quenching method. Under the optimal conditions, the relative fluorescence intensity of QDs was linear over the range of 0.27-12 ng mL(-1) AIV, and the limit of detection was estimated to be 0.09 ng mL(-1) which was 100-fold greater than enzyme-linked immunosorbent assay (ELISA). The sensitive and specific response was also coupled with high reproducibility in the proposed method. A series of six parallel measurements produced reproducible fluorescent signals with a relative standard deviation of 4.7%. The proposed method can be used to directly detect clinical sample without any pretreatment, and showed high efficiency (90.0%), sensitivity (100.0%) and specificity (88.2%) compared with virus isolation (gold method). The new method shows great promise for rapid, sensitive, and quantitative detection of AIV in-field or point-of-care diagnosis.

  12. 99mTc- and Re-labeled 6-dialkylamino-2-naphthylethylidene derivatives as imaging probes for β-amyloid plaques.

    PubMed

    Cui, Mengchao; Tang, Ruikun; Li, Zijing; Ren, Huiying; Liu, Boli

    2011-02-01

    Based on the conjugate strategy, two neutral (99m)Tc labeled 2-(1-(6-(dialkylamino)naphthalen-2-yl)ethylidene)malononitrile (DDNP) and 1-(6-(dialkylamino)naphthalen-2-yl)ethanone (ENE) derivatives, and their corresponding rhenium complexes were synthesized. In vitro fluorescent staining indicated that the corresponding rhenium derivatives selectively stained the β-amyloid (Aβ) plaques in the brain sections of AD model mice with low background. Compared with FDDNP and FENE, the affinities of the corresponding rhenium derivatives to Aβ aggregates decreased about 10-14-fold. In vivo biodistribution experiments in normal mice showed that (99m)Tc-MAMA-ENE displayed medium initial brain uptake (0.65%ID/g at 2min) with a reasonable washout from the brain (0.19%ID/g at 2h) while (99m)Tc-MAMA-DDNP showed a low brain uptake (0.28%ID/g at 2 min). Further optimize these (99m)Tc-labeled tracers in order to improve their binding affinities to Aβ plaques and diffusion through the blood brain barrier may generate useful imaging agents for SPECT. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. A novel nanocatalytic SERS detection of trace human chorionic gonadotropin using labeled-free Vitoria blue 4R as molecular probe.

    PubMed

    Wen, Guiqing; Liang, Xiaojing; Liu, Qingye; Liang, Aihui; Jiang, Zhiliang

    2016-11-15

    In pH 7.4 Na2HPO4-NaH2PO4 buffer solution containing the peptide probes for human chorionic gonadotropin (hCG), silver nanoparticles (AgNPs) were aggregated to big AgNPs clusters that exhibited very weak catalytic effect on the gold nanoparticle reaction of H2O2-HAuCl4. When hCG was present in the peptide probe solution, the AgNPs did not aggregate and it had strong catalytic effect on the gold nanoparticle reaction with a strong resonance Rayleigh scattering (RRS) peak at 370nm and a strong surface enhanced Raman scattering (SERS) peak at 1615cm(-1) in the presence of molecular probe of Victoria blue 4R (VB4R). With the increase of the hCG concentration, the catalysis enhanced due to the nanocatalyst of AgNPs increasing, and the RRS intensity increased at 370nm. The increased RRS intensity was linear to the hCG concentration in 0.05-10ng/mL, with a linear regression equation of ΔI370nm=409.8C +294. And the SERS intensity at 1615cm(-1) increased linearly with the hCG concentration in the range of 0.05-20ng/mL, with a linear regression equation of ΔI1615cm-1=142C+134. Based on this, two new methods of nanocatalytic SERS and RRS were proposed for the determination of trace hCG.

  14. Thiazole orange as a fluorescent probe: Label-free and selective detection of silver ions based on the structural change of i-motif DNA at neutral pH.

    PubMed

    Kang, Bei Hua; Gao, Zhong Feng; Li, Na; Shi, Yan; Li, Nian Bing; Luo, Hong Qun

    2016-08-15

    Silver ions have been widely applied to many fields and have harmful effects on environments and human health. Herein, a label-free optical sensor for Ag(+) detection is constructed based on thiazole orange (TO) as a fluorescent probe for the recognition of i-motif DNA structure change at neutral pH. Ag(+) can fold a C-rich single stranded DNA sequence into i-motif DNA structure at neutral pH and that folding is reversible by chelation with cysteine (Cys). The DNA folding process can be indicated by the fluorescence change of TO, which is non-fluorescent in free molecule state and emits strong fluorescence after the incorporation with i-motif DNA. Thus, a rapid, sensitive, and selective method for the detection of Ag(+) and Cys is developed with a detection limit of 17 and 280nM, respectively. It is worth noting that the mechanism underlying the increase of the fluorescence of thiazole orange in the presence of i-motif structure is explained. Moreover, a fluorescent DNA logic gate is successfully designed based on the Ag(+)/Cys-mediated reversible fluorescence changes. The proposed detection strategy is label-free and economical. In addition, this system shows a great promise for i-motif/TO complex to analyze Ag(+) in the real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Biopatterning for label-free detection.

    PubMed

    Goddard, Julie M; Mandal, Sudeep; Nugen, Sam R; Baeumner, Antje J; Erickson, David

    2010-03-01

    We present a biopatterning technique suitable for applications which demand a high degree of surface cleanliness, such as immobilization of biological recognition elements onto label-free biosensors. In the case of label-free biosensing, the mechanism of signal transduction is based on surface bound matter, making them highly sensitive to surface contamination including residues left during the biopatterning process. In this communication we introduce a simple, rapid processing step that removes 98% of the residues that often remain after standard parylene lift-off patterning. Residue-free parylene biopatterning is combined with microfluidics to localize biomolecule immobilization onto the sensing region and to enable multiplexed biopatterning. We demonstrate the applicability of this method to multiplexed label-free detection platforms by patterning nucleic acid capture probes corresponding to the four different serotypes of Dengue virus onto parallel 1D photonic crystal resonator sensors. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to quantify surface cleanliness and uniformity. In addition to label-free biosensors, this technique is well suited to other nanobiotechnology patterning applications which demand a pristine, residue-free surface, such as immobilization of enzymes, antibodies, growth factors, or cell cultures.

  16. Biopatterning for label-free detection

    PubMed Central

    Goddard, Julie M.; Mandal, Sudeep; Nugen, Sam R.; Baeumner, Antje J.; Erickson, David

    2009-01-01

    We present a biopatterning technique suitable for applications which demand a high degree of surface cleanliness, such as immobilization of biological recognition elements onto label-free biosensors. In the case of label-free biosensing, the mechanism of signal transduction is based on surface bound matter, making them highly sensitive to surface contamination including residues left during the biopatterning process. In this communication we introduce a simple, rapid processing step that removes 98% of the residues that often remain after standard parylene lift-off patterning. Residue-free parylene biopatterning is combined with microfluidics to localize biomolecule immobilization onto the sensing region and to enable multiplexed biopatterning. We demonstrate the applicability of this method to multiplexed label-free detection platforms by patterning nucleic acid capture probes corresponding to the four different serotypes of Dengue virus onto parallel 1D photonic crystal resonator sensors. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to quantify surface cleanliness and uniformity. In addition to label-free biosensors, this technique is well suited to other nanobiotechnology patterning applications which demand a pristine, residue-free surface, such as immobilization of enzymes, antibodies, growth factors, or cell cultures. PMID:19939644

  17. Crystal violet as an i-motif structure probe for reversible and label-free pH-driven electrochemical switch.

    PubMed

    Zhang, Xi Yuan; Luo, Hong Qun; Li, Nian Bing

    2014-06-15

    A simple pH-induced electrochemical switch based on an i-motif structure is developed by using crystal violet as a selective electrochemical probe for the i-motif structure. Thiol-modified cytosine-rich single-strand oligonucleotide (C-rich ssDNA) can be self-assembled on the gold electrode surface via gold-sulfur interaction. Crystal violet is employed as an electrochemical probe for the i-motif structure because of its capability of binding with the i-motif structure through an end-stacking mode. In acidic aqueous solution, crystal violet may approach the electrode surface owing to the formation of the i-motif structure, resulting in an obvious signal, so-called "ON" state. Whereas in neutral or basic aqueous solution, the i-motif structure unfolds to dissociative single strand, which causes crystal violet to leave from the electrode surface, and a weak signal is obtained, so-called "OFF" state. In addition, in the range of pH 4.6-7.3, the increase in current has a good linear relationship (R=0.989) with pH value in the testing solutions. This pH-driven electrochemical switch has the advantages of simplicity, sensitivity, high selectivity, and good reversibility. Furthermore, it provides a possible platform for pH measurement.

  18. A potential fluorescent probe: Maillard reaction product from glutathione and ascorbic acid for rapid and label-free dual detection of Hg(2+) and biothiols.

    PubMed

    Dong, Jiang Xue; Song, Xiao Fang; Shi, Yan; Gao, Zhong Feng; Li, Bang Lin; Li, Nian Bing; Luo, Hong Qun

    2016-07-15

    Maillard reactions and their fluorescent products have drawn much attention in the fields of food and life science, however, the application of fluorescent products separated from the reaction as an indicator for detection of certain substances in sensor field has not been mentioned. In this article, we report on an easy-to-synthesize and water-soluble fluorescent probe separated from the typical Maillard reaction products of glutathione and ascorbic acid, with excellent stability and high quantum yield (18.2%). The further application of the probe has been explored for dual detection of Hg(2+) and biothiols including cysteine, homocysteine, and glutathione, which is based on Hg(2+)-induced fluorescence quenching of the Maillard reaction fluorescent products (MRFPs) and the fluorescence recovery as the introduction of biothiols. This sensing system exhibits a good selectivity and sensitivity, and the linear ranges for Hg(2+), cysteine, homocysteine, and glutathione are 0.05-12, 0.5-10, 0.3-20, and 0.3-20μM, respectively. The detection limits for Hg(2+), cysteine, homocysteine, and glutathione are 22, 47, 96, and 30nM at a signal-to-noise ratio of 3, respectively. Furthermore, the practical applications of this sensor for Hg(2+) and biothiols determination in water samples and human plasma sample have been demonstrated with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Studies of inactivation, retardation and accumulation of viruses in porous media by a combination of dye labeled and native bacteriophage probes

    NASA Astrophysics Data System (ADS)

    Gitis, Vitaly; Dlugy, Christina; Gun, Jenny; Lev, Ovadia

    2011-06-01

    Penetration of viruses through soils is governed by the processes of transport, reversible adsorption, accumulation and inactivation. Until now, it was difficult to decouple the latter two processes and accurately predict viral fate. The present work describes a novel method—tracer studies with a mixture of native and fluorescent-dyed bacteriophages—that facilitates parallel quantification of the two processes. When the native phages are experiencing both accumulation and inactivation, the labeled ones are inactivated already and therefore can only be accumulated. Thus the effect of inactivation is applicable to native bacteriophages only and depletion of phage concentration due to inactivation can be elucidated from a total phage balance. The novel approach is exemplified by batch and column studies of the effects of temperature, pH, and saturation, on inactivation of MS2 bacteriophage. A three-parameter model accounting for inactivation, reversible adsorption (i.e., retardation), and accumulation is implemented.

  20. Noninvasive positron emission tomography imaging of cell death using a novel small-molecule probe, (18)F labeled bis(zinc(II)-dipicolylamine) complex.

    PubMed

    Wang, Hongliang; Tang, Xiaolan; Tang, Ganghua; Huang, Tingting; Liang, Xiang; Hu, Kongzhen; Deng, Huaifu; Yi, Chang; Shi, Xinchong; Wu, Kening

    2013-08-01

    The synthetic bis(zinc(II)-dipicolylamine) (DPAZn2) coordination complexes are known to have a high specific and selective affinity to target the exposed phosphatidylserine (PS) on the surface of dead and dying cells. An (18)F-labeled DPAZn2 complex (4-(18)F-Fluoro-benzoyl-bis(zinc(II)-dipicolylamine), (18)F-FB-DPAZn2) as positron emission tomography (PET) tracer was developed and evaluated for in vivo imaging of tumor treated with a chemical agent. The in vitro cell stain studies revealed that fluorescent DPAZn2 complexes (Dansyl-DPAZn2) stained the same cells (apoptotic and necrotic cells) as fluorescein isothiocyanate (FITC) labeled Annexin V (FITC-Annexin V). The radiosynthesis of (18)F-FB-DPAZn2 was achieved through the amidation the precursor bis(2,2'-dipicolylamine) derivative (DPA2) with the prosthetic group N-succinimidyl-4-[(18)F]-fluorobenzoate ((18)F-SFB) and chelation with zinc nitrate. In the biodistribution study, the fast clearance of (18)F-FB-DPAZn2 from blood and kidney was observed and high uptake in liver and intestine within 90 min postinjection was also found. For the PET imaging, significantly higher tumor uptake of (18)F-FB-DPAZn2 was observed in the adriamycin (ADM)-treated Hepa1-6 hepatocellular carcinoma-bearing mice than that in the untreated tumor-model mice, while a slightly decreased tumor uptake of (18)F-FDG was found in the ADM-treated tumor-bearing mice. The results indicate that (18)F-FB-DPAZn2 has the similar capability of apoptosis detection as FITC-Annexin V and seems to be a potential PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy. The high uptake of (18)F-FB-DPAZn2 in the abdomen needs to optimize the structure for improving its pharmacokinetics characteristics in the future work.

  1. Multicolor combinatorial probe coding for real-time PCR.

    PubMed

    Huang, Qiuying; Zheng, Linlin; Zhu, Yumei; Zhang, Jiafeng; Wen, Huixin; Huang, Jianwei; Niu, Jianjun; Zhao, Xilin; Li, Qingge

    2011-01-14

    The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed "Multicolor Combinatorial Probe Coding" (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2(n)-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

  2. Cardiovascular clinical trials in Japan and controversies regarding prospective randomized open-label blinded end-point design.

    PubMed

    Kohro, Takahide; Yamazaki, Tsutomu

    2009-02-01

    Recently, results of several cardiovascular clinical trials conducted in Japan were published. Most of them were designed as prospective randomized open-label blinded end-point (PROBE)-type trials, in which patients were randomly allocated to different regimens and both the patients and doctors are aware of the regimen being administered. Although the PROBE design enables performing trials resembling real-world practices, entails low costs and renders patient recruitment easier, it presents several conditions that have to be satisfied to acquire accurate results, due to its open-label nature. Principally, the so-called hard end points, which are judged by objective criteria, should be used as primary end points in order to prevent biases. In this article, a general description of various designs of clinical studies is provided, followed by a description of the PROBE design, and the precautions to be taken while conducting PROBE-designed trials by comparing trials conducted in Japan and the West.

  3. Autonomous folding of the recombinant large cytoplasmic loop of sarcoplasmic reticulum Ca2+-ATPase probed by affinity labeling and trypsin digestion.

    PubMed

    Moutin, M J; Rapin, C; Miras, R; Vinçon, M; Dupont, Y; McIntosh, D B

    1998-02-01

    Recombinant large cytoplasmic loop (LCL, residues 329-740) of sarcoplasmic reticulum Ca2+-ATPase, expressed in and purified from Escherichia coli, comprises most of the active site and binds ATP [Moutin, M.-J., Cuillel, M., Rapin, C., Miras, R., Anger, M., Lompré, A.-M. & Dupont, Y. (1994) J. Biol. Chem. 269, 11147-11154]. In this study, we show that fluorescein-5' isothiocyanate (FITC) specifically labels the same lysine residue as in the native Ca2+-ATPase (Lys515), with similar kinetics and pH dependence. ATP blocks the reaction with the lysine residue, but at higher concentrations compared with those for the native pump, in agreement with the lower ATP-binding affinity found previously. Graded tryptic digestion of LCL shows that favored cleavage is at the T1 site and that the N-terminal 75% of LCL are resistant to trypsin, as is native Ca2+-ATPase. Other experiments reveal differences to the native pump. (a) FITC derivatizes some -SH groups of LCL. (b) The C-terminal 25% of the polypeptide is susceptible to end-clipping by trypsin. (c) 2',3'-O-(2,4,6-trinitrophenyl)-ATP fails to specifically label the LCL (on the equivalent of Lys492), although it binds tightly (KD = 1.3 microM) and (d) Glutaraldehyde does not specifically cross-link LCL (between the equivalent of Lys492 and Arg678). These results could be explained by a flexible and loose structure of the hinge region of LCL (C-terminal 25%). Anchoring this region in the membrane and/or interaction with the missing beta-strand domain may be required for its compact folding and proper interaction with the rest of LCL. The results suggest that the N-terminal 75% of LCL expressed in E. coli folds autonomously to a fairly stable unit and native-like structure, encompassing the phosphorylation and central ATP binding sections. The hinge region does not appear to be part of the FITC-binding site but constitutes portions of the 2',3'-O-(2,4,6-trinitrophenyl)-ATP and, probably, ATP-binding site.

  4. Probing of Ehrlich ascites carcinoma cell using in situ aggregates of Au-NPs as SERS label created by plasmon exciting hybrid- TEM*11 laser mode

    NASA Astrophysics Data System (ADS)

    Kumar, R.; Mehta, D. S.; Saraswati, S.; Shakher, C.

    2012-02-01

    Apart from commonly employed target-specific labeling/adsorption of antibodies over Au-NPs surface for the creation of localized aggregates, an alternative approach using optical tweezers (OT) driven by hybrid-TEM*11 mode has been devised and exploited for in vitro detection of Ehrlich ascites carcinoma cells (EAC) relying on enhanced scattering. Intra-cavity generated spatially featured asymmetric (SFA) laser beam (λ = 532 nm) has effected simultaneous trapping of mice-EAC cells and in-situ crowd/assembly of incubated Au-NPs/small gold nano-aggregates (created from two or more individual Au-NPs). Relatively larger focus spot created by tightly focused SFA beam than frequently employed Gaussian-mode in OT has offered an extended working area and hence dilute heating has taken care of EAC cells. GNA improves significantly the sensitivity of diagnostics relying on scattered light and the safety and efficacy of therapeutic nanotechnologies for the diseases of cancer and vascular system in medicine.

  5. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain

    PubMed Central

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23–230) as detected by [1H, 15N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn2+-binding to the octarepeat motif. PMID:27341298

  6. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

    PubMed

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-06-24

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

  7. Facile synthesis of boron- and nitride-doped MoS2 nanosheets as fluorescent probes for the ultrafast, sensitive, and label-free detection of Hg(2+).

    PubMed

    Liu, Xiaojia; Li, Liping; Wei, Yuanjie; Zheng, Yizhi; Xiao, Qian; Feng, Bo

    2015-07-07

    Bulk MoS2, a prototypical transition metal chalcogenide material, is an indirect band gap semiconductor with negligible photoluminescence. In this study, we have developed, for the first time, a simple and low-cost synthetic strategy to prepare boron- and nitrogen-doped MoS2 (B,N-MoS2) nanosheets. Through boron and nitrogen doping, the band gap of MoS2 increases from 1.20 eV to 1.61 eV, and the obtained B,N-MoS2 nanosheets exhibit an enhanced fluorescence. The B,N-MoS2 nanosheets can be used as a green and facile sensing platform for label-free detection of Hg(2+) because of their high sensitivity and selectivity toward Hg(2+). In addition, detection can be easily accomplished through one-step rapid (within 2 min) operation, with a limit as low as 1 nM. This study demonstrates that the introduction of boron and nitrogen elements into ultrathin MoS2 nanosheets for enhanced fluorescence properties is feasible through a facile and general preparation strategy and may also offer a unique idea as a potential way to design more efficient MoS2-based sensors and fluorescent materials.

  8. Studies of inactivation, retardation and accumulation of viruses in porous media by a combination of dye labeled and native bacteriophage probes.

    PubMed

    Gitis, Vitaly; Dlugy, Christina; Gun, Jenny; Lev, Ovadia

    2011-06-01

    Penetration of viruses through soils is governed by the processes of transport, reversible adsorption, accumulation and inactivation. Until now, it was difficult to decouple the latter two processes and accurately predict viral fate. The present work describes a novel method-tracer studies with a mixture of native and fluorescent-dyed bacteriophages-that facilitates parallel quantification of the two processes. When the native phages are experiencing both accumulation and inactivation, the labeled ones are inactivated already and therefore can only be accumulated. Thus the effect of inactivation is applicable to native bacteriophages only and depletion of phage concentration due to inactivation can be elucidated from a total phage balance. The novel approach is exemplified by batch and column studies of the effects of temperature, pH, and saturation, on inactivation of MS2 bacteriophage. A three-parameter model accounting for inactivation, reversible adsorption (i.e., retardation), and accumulation is implemented. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Fluorinated vitamin b(12) analogs are cofactors of corrinoid-dependent enzymes: a f-labeled nuclear magnetic resonance probe for identifying corrinoid-protein interactions.

    PubMed

    Stupperich, E; Eisinger, H J; Kerssebaum, R; Nexø, E

    1993-02-01

    The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B(12) analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B(12). Hence, it is possible to use F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.

  10. Fluorinated Vitamin B12 Analogs Are Cofactors of Corrinoid-Dependent Enzymes: a 19F-Labeled Nuclear Magnetic Resonance Probe for Identifying Corrinoid-Protein Interactions

    PubMed Central

    Stupperich, Erhard; Eisinger, Hans-Jürgen; Kerssebaum, Rainer; Nexø, Ebba

    1993-01-01

    The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B12 analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B12. Hence, it is possible to use 19F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides. PMID:16348877

  11. Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2.

    PubMed

    Thompson, Andrew R; Naber, Nariman; Wilson, Clyde; Cooke, Roger; Thomas, David D

    2008-12-01

    We have used a bifunctional spin label (BSL) to cross-link Cys(707) (SH1) and Cys(697) (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A.M.ADP.P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A.M.ADP. In contrast, helical order is observed for the strongly bound state A.M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A.M.ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A.M.ADP, slow rotational mobility comparable to that of A.M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.

  12. CLAVATA3 Dodecapeptide Modified CdTe Nanoparticles: A Biocompatible Quantum Dot Probe for In Vivo Labeling of Plant Stem Cells

    PubMed Central

    Yu, Guanghui; Tan, Yanping; He, Xiangzhu; Qin, Yonghua; Liang, Jiangong

    2014-01-01

    CLAVATA3 (CLV3) dodecapeptides function in plant stem cell maintenance, but CLV3 function in cell-cell communication remains less clear. Here, we coupled CLV3 dodecapeptides to synthesized CdTe nanoparticles to track their bioactivity on stem cells in the root apical meristem. To achieve this, we first synthesized CdTe quantum dots (QDs) using a one-pot method, and then evaluated the cytotoxicity of the QDs in BY-2 cells. The results showed that QDs in plant cells must be used at low concentrations and for short treatment time. To make biocompatible probes to track stem cell fate, we conjugated CLV3 dodecapeptides to the QDs by the zero-coupling method; this modification greatly reduced the cytotoxicity of the QDs. Furthermore, we detected CLV3-QDs localized on the cell membrane, consistent with the known localization of CLV3. Our results indicate that using surface-modified QDs at low concentrations and for short time treatment can improve their utility for plant cell imaging. PMID:24586624

  13. CLAVATA3 dodecapeptide modified CdTe nanoparticles: a biocompatible quantum dot probe for in vivo labeling of plant stem cells.

    PubMed

    Yu, Guanghui; Tan, Yanping; He, Xiangzhu; Qin, Yonghua; Liang, Jiangong

    2014-01-01

    CLAVATA3 (CLV3) dodecapeptides function in plant stem cell maintenance, but CLV3 function in cell-cell communication remains less clear. Here, we coupled CLV3 dodecapeptides to synthesized CdTe nanoparticles to track their bioactivity on stem cells in the root apical meristem. To achieve this, we first synthesized CdTe quantum dots (QDs) using a one-pot method, and then evaluated the cytotoxicity of the QDs in BY-2 cells. The results showed that QDs in plant cells must be used at low concentrations and for short treatment time. To make biocompatible probes to track stem cell fate, we conjugated CLV3 dodecapeptides to the QDs by the zero-coupling method; this modification greatly reduced the cytotoxicity of the QDs. Furthermore, we detected CLV3-QDs localized on the cell membrane, consistent with the known localization of CLV3. Our results indicate that using surface-modified QDs at low concentrations and for short time treatment can improve their utility for plant cell imaging.

  14. Biologically green synthesized silver nanoparticles as a facile and rapid label-free colorimetric probe for determination of Cu2 + in water samples

    NASA Astrophysics Data System (ADS)

    Basiri, Sedigheh; Mehdinia, Ali; Jabbari, Ali

    2017-01-01

    A highly sensitive and cost-effective colorimetric sensing platform for the selective trace analysis of Cu2 + ions was developed based on the accelerated etching of Riboflavin stabilized silver nanoparticles (R/AgNPs). The R/AgNPs were prepared from the Cucumis melo juice by a green chemistry approach. The bio-synthesized AgNPs were studied by UV-Vis spectroscopy and showed an intense absorption band at 404 nm that were further confirmed by FTIR and EDS analysis. Simultaneous presence of Cu2 + and thiosulfate decreased the absorption intensity of green synthesized AgNPs which resulted in sensitive and selective determination of Cu2 +. The selectivity of R/AgNPs detection system for Cu2 + was excellent. Furthermore, the method offered a wide linear detection range from 5 nM to 100 nM with a detection limit of 1.12 nM. Surprisingly, it was a quick approach and the decolorization of the R/AgNPs solutions occurred only within 5 min. Our results clearly indicate these R/AgNPs could be used as an efficient probe for the colorimetric sensing of Cu2 + in environmental water samples.

  15. Stable-isotope labeling and probing of recent photosynthates into respired CO2, soil microbes and soil mesofauna using a xylem and phloem stem-injection technique on Sitka spruce (Picea sitchensis).

    PubMed

    Churchland, Carolyn; Weatherall, Andrew; Briones, Maria J I; Grayston, Sue J

    2012-11-15

    Here we report on the successful application of a novel stem-injection stable-isotope-labeling and probing technique in mature trees to trace the spatial and temporal distribution of rhizosphere carbon belowground. Three 22-year-old Sitka spruce trees were injected with 6.66 g of (13)C-labeled aspartic acid. Over the succeeding 30 days, soil CO(2) efflux, phospholipid fatty-acid (PLFA) microbial biomarkers and soil invertebrates (mites, collembolans and enchytraeids) were analyzed along a 50 m transect from each tree to determine the temporal and spatial patterns in the translocation of recently fixed photosynthates belowground. Soil δ(13)CO(2) values peaked 13-23 days after injection, up to 5 m from the base of the injected tree and was, on average, 3.5‰ enriched in (13)C relative to the baseline. Fungal PLFA biomarkers peaked 2-4 days after stem-injection, up to 20 m from the base of the injected tree and were (13)C-enriched by up to 50‰. Significant (13)C enrichment in mites and enchytraeids occurred 4-6 days after injection (by, on average, 1.5‰). Stem injection of large trees with (13)C-enriched compounds is a successful tool to trace C-translocation belowground. In particular, the significant (13)C enrichment of CO(2) and enchytraeids near the base of the tree and the significant (13)C enrichment of PLFAs up to 20 m away indicate that mature Sitka spruce (Picea sitchensis) have the capacity to support soil communities over large distances. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Radioiodinated Exendin-4 Is Superior to the Radiometal-Labelled Glucagon-Like Peptide-1 Receptor Probes Overcoming Their High Kidney Uptake

    PubMed Central

    Läppchen, Tilman; Tönnesmann, Roswitha; Eersels, Jos; Meyer, Philipp T.; Maecke, Helmut R.; Rylova, Svetlana N.

    2017-01-01

    GLP-1 receptors are ideal targets for preoperative imaging of benign insulinoma and for quantifying the beta cell mass. The existing clinical tracers targeting GLP-1R are all agonists with low specific activity and very high kidney uptake. In order to solve those issues we evaluated GLP-1R agonist Ex-4 and antagonist Ex(9–39) radioiodinated at Tyr40 side by side with [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 (68Ga-Ex-4) used in the clinic. The Kd, Bmax, internalization and binding kinetics of [Nle14,125I-Tyr40-NH2]Ex-4 and [Nle14,125I-Tyr40-NH2]Ex(9–39) were studied in vitro using Ins-1E cells. Biodistribution and imaging studies were performed in nude mice bearing Ins-1E xenografts. In vitro evaluation demonstrated high affinity binding of the [Nle14,125I-Tyr40-NH2]Ex-4 agonist to the Ins-1E cells with fast internalization kinetics reaching a plateau after 30 min. The antagonist [Nle14,125I-Tyr40-NH2]Ex(9–39) did not internalize and had a 4–fold higher Kd value compared to the agonist. In contrast to [Nle14,125I-Tyr40-NH2]Ex(9–39), which showed low and transient tumor uptake, [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated excellent in vivo binding properties with tumor uptake identical to that of 68Ga-Ex-4, but substantially lower kidney uptake resulting in a tumor-to-kidney ratio of 9.7 at 1 h compared to 0.3 with 68Ga-Ex-4. Accumulation of activity in thyroid and stomach for both peptides, which was effectively blocked by irenat, confirms that in vivo deiodination is the mechanism behind the low kidney retention of iodinated peptides. The 124I congener of [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated a similar favourable biodistribution profile in the PET imaging studies in contrast to the typical biodistribution pattern of [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4. Our results demonstrate that iodinated Ex-4 is a very promising tracer for imaging of benign insulinomas. It solves the problem of high kidney uptake of the radiometal-labelled tracers by improving the tumor

  17. Advanced Molecular Probes for Sequence-Specific DNA Recognition

    NASA Astrophysics Data System (ADS)

    Bertucci, Alessandro; Manicardi, Alex; Corradini, Roberto

    DNA detection can be achieved using the Watson-Crick base pairing with oligonucleotides or oligonucleotide analogs, followed by generation of a physical or chemical signal coupled with a transducer device. The nature of the probe is an essential feature which determines the performances of the sensing device. Many synthetic processes are presently available for "molecular engineering" of DNA probes, enabling label-free and PCR-free detection to be performed. Furthermore, many DNA analogs with improved performances are available and are under development; locked nucleic acids (LNA), peptide nucleic acids (PNA) and their analogs, morpholino oligonucleotides (MO) and other modified probes have shown improved properties of affinity and selectivity in target recognition compared to those of simple DNA probes. The performances of these probes in sensing devices, and the requirements for detection of unamplified DNA will be discussed in this chapter. Chemistry and architectures for conjugation of probes to reporter units, surfaces and nanostructures will also be discussed. Examples of probes used in ultrasensitive detection of unamplified DNA are listed.

  18. A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA

    PubMed Central

    Sawant, Anupam A.; Tanpure, Arun A.; Mukherjee, Progya P.; Athavale, Soumitra; Kelkar, Ashwin; Galande, Sanjeev; Srivatsan, Seergazhi G.

    2016-01-01

    Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells. PMID:26384420

  19. Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

    PubMed Central

    Yilmaz, L. Safak; Loy, Alexander; Wright, Erik S.; Wagner, Michael; Noguera, Daniel R.

    2012-01-01

    Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu. PMID:22952791

  20. A surface enhanced Raman scattering quantitative analytical platform for detection of trace Cu coupled the catalytic reaction and gold nanoparticle aggregation with label-free Victoria blue B molecular probe.

    PubMed

    Li, Chongning; Ouyang, Huixiang; Tang, Xueping; Wen, Guiqing; Liang, Aihui; Jiang, Zhiliang

    2017-01-15

    With development of economy and society, there is an urgent need to develop convenient and sensitive methods for detection of Cu(2+) pollution in water. In this article, a simple and sensitive SERS sensor was proposed to quantitative analysis of trace Cu(2+) in water. The SERS sensor platform was prepared a common gold nanoparticle (AuNP)-SiO2 sol substrate platform by adsorbing HSA, coupling with the catalytic reaction of Cu(2+)-ascorbic acid (H2A)-dissolved oxygen, and using label-free Victoria blue B (VBB) as SERS molecular probes. The SERS sensor platform response to the AuNP aggregations by hydroxyl radicals (•OH) oxidizing from the Cu(2+) catalytic reaction, which caused the SERS signal enhancement. Therefore, by monitoring the increase of SERS signal, Cu(2+) in water can be determined accurately. The results show that the SERS sensor platforms owns a linear response with a range from 0.025 to 25μmol/L Cu(2+), and with a detection limit of 0.008μmol/L. In addition, the SERS method demonstrated good specificity for Cu(2+), which can determined accurately trace Cu(2+) in water samples, and good recovery and accuracy are obtained for the water samples. With its high selectivity and good accuracy, the sensitive SERS quantitative analysis method is expected to be a promising candidate for determining copper ions in environmental monitoring and food safety.

  1. Enhanced Fluorescence ELISA Based on HAT Triggering Fluorescence "Turn-on" with Enzyme-Antibody Dual Labeled AuNP Probes for Ultrasensitive Detection of AFP and HBsAg.

    PubMed

    Wu, Yudong; Guo, Weisheng; Peng, Weipan; Zhao, Qian; Piao, Jiafang; Zhang, Bo; Wu, Xiaoli; Wang, Hanjie; Gong, Xiaoqun; Chang, Jin

    2017-03-22

    At present, enzyme-linked immunosorbent assay (ELISA) is considered to be the most appropriate approach in clinical biomarker detection, with good specificity, low cost, and straightforward readout. However, unsatisfactory sensitivity severely hampers its wide application in clinical diagnosis. Herein, we designed a new kind of enhanced fluorescence enzyme-linked immunosorbent assay (FELISA) based on the human alpha-thrombin (HAT) triggering fluorescence "turn-on" signals. In this system, detection antibodies (Ab2) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine110 with fluorescence quenched served as the substrate of HAT. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, leading to a strong fluorescence signal for sensing ultralow levels of alpha fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg). Under the optimized reaction conditions, AFP and HBsAg were detected at the ultralow concentrations of 10(-8) ng mL(-1) and 5 × 10(-4) IU mL(-1), respectively, which were at least 10(4) times lower than those of the conventional fluorescence assay and 10(6) times lower than those of the conventional ELISA. In addition, we further discussed the efficiency of the sensitive FELISA in clinical serum samples, showing great potential in practical applications.

  2. Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near-infrared laser-induced fluorescence detection using a new BODIPY-based probe as labeling reagent.

    PubMed

    Zhang, Li-Yun; Tu, Feng-Qin; Guo, Xiao-Feng; Wang, Hong; Wang, Peng; Zhang, Hua-Shan

    2014-10-01

    A CZE with near-infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ-glutamylcysteine, cysteinylglycine, and N-acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N-acetylcysteine to 0.31 nmol/L for γ-glutamylcysteine, which are better than or comparable to those reported with other derivatization-based CE-LIF methods. As the first trial of NIR CE-LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5-104.3%.

  3. Fluorescent biosensors enabled by graphene and graphene oxide.

    PubMed

    Zhang, Huan; Zhang, Honglu; Aldalbahi, Ali; Zuo, Xiaolei; Fan, Chunhai; Mi, Xianqiang

    2017-03-15

    During the past few years, graphene and graphene oxide (GO) have attracted numerous attentions for the potential applications in various fields from energy technology, biosensing to biomedical diagnosis and therapy due to their various functionalization, high volume surface ratio, unique physical and electrical properties. Among which, graphene and graphene oxide based fluorescent biosensors enabled by their fluorescence-quenching properties have attracted great interests. The fluorescence of fluorophore or dye labeled on probes (such as molecular beacon, aptamer, DNAzymes and so on) was quenched after adsorbed on to the surface of graphene. While in the present of the targets, due to the strong interactions between probes and targets, the probes were detached from the surface of graphene, generating dramatic fluorescence, which could be used as signals for detection of the targets. This strategy was simple and economy, together with great programmable abilities of probes; we could realize detection of different kinds of species. In this review, we first briefly introduced the history of graphene and graphene oxide, and then summarized the fluorescent biosensors enabled by graphene and GO, with a detailed account of the design mechanism and comparison with other nanomaterials (e.g. carbon nanotubes and gold nanoparticles). Following that, different sensing platforms for detection of DNAs, ions, biomolecules and pathogens or cells as well as the cytotoxicity issue of graphene and GO based in vivo biosensing were further discussed. We hope that this review would do some help to researchers who are interested in graphene related biosening research work. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Label-Free Receptor Assays

    PubMed Central

    Fang, Ye

    2010-01-01

    Label-free biosensors offer integrated, kinetic and multi-parametric measures of receptor biology and ligand pharmacology in whole cells. Being highly sensitive and pathway-unbiased, label-free receptor assays can be used to probe the systems cell biology including pleiotropic signaling of receptors, and to characterize the functional selectivity and phenotypic pharmacology of ligand molecules. These assays provide a new dimension for elucidating receptor biology and for facilitating drug discovery. PMID:21221420

  5. Label-Free Receptor Assays.

    PubMed

    Fang, Ye

    2011-01-01

    Label-free biosensors offer integrated, kinetic and multi-parametric measures of receptor biology and ligand pharmacology in whole cells. Being highly sensitive and pathway-unbiased, label-free receptor assays can be used to probe the systems cell biology including pleiotropic signaling of receptors, and to characterize the functional selectivity and phenotypic pharmacology of ligand molecules. These assays provide a new dimension for elucidating receptor biology and for facilitating drug discovery.

  6. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  7. Aerobic, Palladium-Catalyzed Dioxygenation of Alkenes Enabled by Catalytic Nitrite**

    PubMed Central

    Wickens, Zachary K.; Guzmán, Pablo E.; Grubbs, Robert H.

    2014-01-01

    Catalytic nitrite was found to enable carbon-oxygen bond-forming reductive elimination from unstable alkyl palladium intermediates, providing dioxygenated products from alkenes. A variety of functional groups are tolerated and high yields (up to 94%) are observed with many substrates, including a multi-gram scale reaction. Nitrogen dioxide, which could form from nitrite under the reaction conditions, was shown to be kinetically competent in the dioxygenation of alkenes. Furthermore, the reductive elimination event was probed with 18 O-labeling experiments, which demonstrated that both oxygen atoms in the difunctionalized products are derived from one molecule of acetic acid. PMID:25376666

  8. SIMPLE: a sequential immunoperoxidase labeling and erasing method.

    PubMed

    Glass, George; Papin, Jason A; Mandell, James W

    2009-10-01

    The ability to simultaneously visualize expression of multiple antigens in cells and tissues can provide powerful insights into cellular and organismal biology. However, standard methods are limited to the use of just two or three simultaneous probes and have not been widely adopted for routine use in paraffin-embedded tissue. We have developed a novel approach called sequential immunoperoxidase labeling and erasing (SIMPLE) that enables the simultaneous visualization of at least five markers within a single tissue section. Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole, combined with a rapid non-destructive method for antibody-antigen dissociation, we demonstrate the ability to erase the results of a single immunohistochemical stain while preserving tissue antigenicity for repeated rounds of labeling. SIMPLE is greatly facilitated by the use of a whole-slide scanner, which can capture the results of each sequential stain without any information loss.

  9. NMR studies of active-site properties of human carbonic anhydrase II by using (15) N-labeled 4-methylimidazole as a local probe and histidine hydrogen-bond correlations.

    PubMed

    Shenderovich, Ilya G; Lesnichin, Stepan B; Tu, Chingkuang; Silverman, David N; Tolstoy, Peter M; Denisov, Gleb S; Limbach, Hans-Heinrich

    2015-02-09

    By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Probing the conformation and 2D-distribution of pyrene-terminated redox-labeled poly(ethylene glycol) chains end-adsorbed on HOPG using cyclic voltammetry and atomic force electrochemical microscopy.

    PubMed

    Anne, Agnès; Bahri, Mohamed Ali; Chovin, Arnaud; Demaille, Christophe; Taofifenua, Cécilia

    2014-03-14

    The present paper aims at illustrating how end-attachment of water-soluble flexible chains bearing a terminal functional group onto graphene-like surfaces has to be carefully tuned to ensure the proper positioning of the functional moiety with respect to the anchoring surface. The model experimental system considered here consists of a layer of poly(ethylene glycol) (PEG) chains, bearing an adsorbing pyrene foot and a ferrocene (Fc) redox functional head, self-assembled onto highly oriented pyrolytic graphite (HOPG). Cyclic voltammetry is used to accurately measure the chain coverage and gain insights into the microenvironment experienced by the Fc heads. Molecule-touching atomic force electrochemical microscopy (Mt/AFM-SECM) is used to simultaneously probe the chain conformation and the position of the Fc heads within the layer, and also to map the 2D-distribution of the chains over the surface. This multiscale electrochemical approach allows us to show that whereas Fc-PEG-pyrene readily self-assembles to form extremely homogeneous layers, the strongly hydrophobic nature of graphite planes results in a complex coverage-dependent structure of the PEG layer due to the interaction of the ferrocene label with the HOPG surface. It is shown that, even though pyrene is known to adsorb particularly strongly onto HOPG, the more weakly adsorbing terminal ferrocene can also act as the chain anchoring moiety especially at low coverage. However we show that beyond a critical coverage value the Fc-PEG-pyrene chains adopt an ideal "foot-on" end-attached conformation allowing the Fc head to explore a volume away from the surface solely limited by the PEG chain elasticity.

  11. Formative Assessment Probes: Labeling versus Explaining

    ERIC Educational Resources Information Center

    Keeley, Page

    2013-01-01

    In the elementary grades, the butterfly is a commonly used curricular context for children to learn about growth and development of organisms as they progress through their life cycle. "A Framework for K-12 Science Education's" life science core idea LS1.B, Growth and Development of Organisms, states that by the end of grade 5,…

  12. Formative Assessment Probes: Labeling versus Explaining

    ERIC Educational Resources Information Center

    Keeley, Page

    2013-01-01

    In the elementary grades, the butterfly is a commonly used curricular context for children to learn about growth and development of organisms as they progress through their life cycle. "A Framework for K-12 Science Education's" life science core idea LS1.B, Growth and Development of Organisms, states that by the end of grade 5,…

  13. Karyotyping human and mouse cells using probes from single-sorted chromosomes and open source software.

    PubMed

    Potapova, Tamara A; Unruh, Jay R; Box, Andrew C; Bradford, William D; Seidel, Christopher W; Slaughter, Brian D; Sivagnanam, Shamilene; Wu, Yuping; Li, Rong

    2015-12-01

    Multispectral karyotyping analyzes all chromosomes in a single cell by labeling them with chromosome-specific probes conjugated to unique combinations of fluorophores. Currently available multispectral karyotyping systems require the purchase of specialized equipment and reagents. However, conventional laser scanning confocal microscopes that are capable of separating multiple overlapping emission spectra through spectral imaging and linear unmixing can be utilized for classifying chromosomes painted with multicolor probes. Here, we generated multicolor chromosome paints from single-sorted human and mouse chromosomes and developed the Karyotype Identification via Spectral Separation (KISS) analysis package, a set of freely available open source ImageJ tools for spectral unmixing and karyotyping. Chromosome spreads painted with our multispectral probe sets can be imaged on widely available spectral laser scanning confocal microscopes and analyzed using our ImageJ tools. Together, our probes and software enable academic labs with access to a laser-scanning spectral microscope to perform multicolor karyotyping in a cost-effective manner.

  14. Structure of the Ribosomal RNA Decoding Site Containing a Selenium-Modified Responsive Fluorescent Ribonucleoside Probe.

    PubMed

    Nuthanakanti, Ashok; Boerneke, Mark A; Hermann, Thomas; Srivatsan, Seergazhi G

    2017-03-01

    Comprehensive understanding of the structure-function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation-sensitive fluorophore and an anomalous X-ray diffraction label (5-selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand-induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

  15. Use of Sloppy Molecular Beacon Probes for Identification of Mycobacterial Species ▿ †

    PubMed Central

    El-Hajj, Hiyam H.; Marras, Salvatore A. E.; Tyagi, Sanjay; Shashkina, Elena; Kamboj, Mini; Kiehn, Timothy E.; Glickman, Michael S.; Kramer, Fred Russell; Alland, David

    2009-01-01

    We report here the use of novel “sloppy” molecular beacon probes in homogeneous PCR screening assays in which thermal denaturation of the resulting probe-amplicon hybrids provides a characteristic set of amplicon melting temperature (Tm) values that identify which species is present in a sample. Sloppy molecular beacons possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different species despite the presence of mismatched base pairs. By using four sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore, four different Tm values can be determined simultaneously. We tested this technique with 27 different species of mycobacteria and found that each species generates a unique, highly reproducible signature that is unaffected by the initial bacterial DNA concentration. Utilizing this general paradigm, screening assays can be designed for the identification of a wide range of species. PMID:19171684

  16. Enhanced vibrational spectroscopy, intracellular refractive indexing for label-free biosensing and bioimaging by multiband plasmonic-antenna array.

    PubMed

    Chen, Cheng-Kuang; Chang, Ming-Hsuan; Wu, Hsieh-Ting; Lee, Yao-Chang; Yen, Ta-Jen

    2014-10-15

    In this study, we report a multiband plasmonic-antenna array that bridges optical biosensing and intracellular bioimaging without requiring a labeling process or coupler. First, a compact plasmonic-antenna array is designed exhibiting a bandwidth of several octaves for use in both multi-band plasmonic resonance-enhanced vibrational spectroscopy and refractive index probing. Second, a single-element plasmonic antenna can be used as a multifunctional sensing pixel that enables mapping the distribution of targets in thin films and biological specimens by enhancing the signals of vibrational signatures and sensing the refractive index contrast. Finally, using the fabricated plasmonic-antenna array yielded reliable intracellular observation was demonstrated from the vibrational signatures and intracellular refractive index contrast requiring neither labeling nor a coupler. These unique features enable the plasmonic-antenna array to function in a label-free manner, facilitating bio-sensing and imaging development.

  17. Integrated microfluidic probe station

    NASA Astrophysics Data System (ADS)

    Perrault, C. M.; Qasaimeh, M. A.; Brastaviceanu, T.; Anderson, K.; Kabakibo, Y.; Juncker, D.

    2010-11-01

    The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution—thus hydrodynamically confining the microjet—and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

  18. Integrated microfluidic probe station.

    PubMed

    Perrault, C M; Qasaimeh, M A; Brastaviceanu, T; Anderson, K; Kabakibo, Y; Juncker, D

    2010-11-01

    The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution--thus hydrodynamically confining the microjet--and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

  19. F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

    SciTech Connect

    Wu, Anna M

    2013-01-18

    The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2. Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.

  20. Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction

    PubMed Central

    Kutyavin, Igor V.

    2013-01-01

    Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations. PMID:24013564

  1. Emerging applications of label-free optical biosensors

    NASA Astrophysics Data System (ADS)

    Zanchetta, Giuliano; Lanfranco, Roberta; Giavazzi, Fabio; Bellini, Tommaso; Buscaglia, Marco

    2017-01-01

    Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

  2. Peptide nucleic acid probes with charged photocleavable mass markers

    PubMed Central

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  3. Quantitative Microbial Ecology through Stable Isotope Probing

    PubMed Central

    Mau, Rebecca L.; Schwartz, Egbert; Caporaso, J. Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J.; Liu, Cindy M.; McHugh, Theresa A.; Marks, Jane C.; Morrissey, Ember M.; Price, Lance B.

    2015-01-01

    Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in 18O and 13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of 18O into DNA from [18O]water. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing. PMID:26296731

  4. Functional investigations on embryonic stem cells labeled with clinically translatable iron oxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Liu, Jing; Wang, Liqin; Cao, Jianbo; Huang, Yue; Lin, Yu; Wu, Xiaoyun; Wang, Zhiyong; Zhang, Fan; Xu, Xiuqin; Liu, Gang

    2014-07-01

    Stem cell based therapies offer significant potential in the field of regenerative medicine. The development of superparamagnetic iron oxide (SPIO) nanoparticle labeling and magnetic resonance imaging (MRI) have been increasingly used to track the transplanted cells, enabling in vivo determination of cell fate. However, the impact of SPIO-labeling on the cell phenotype and differentiation capacity of embryonic stem cells (ESCs) remains unclear. In this study, we wrapped SPIO nanoparticles with stearic acid grafted PEI600, termed as Stearic-LWPEI-SPIO, to generate efficient and non-toxic ESC labeling tools. Our results showed that efficient labeling of ESCs at an optimized low dosage of Stearic-LWPEI-SPIO nanoparticles did not alter the differentiation and self-renewal properties of ESCs. The localization of the transplanted ESCs observed by MRI correlated well with histological studies. These findings demonstrate that Stearic-LWPEI-SPIO nanoparticles have potential to be clinically translatable MRI probes and may enable non-invasive in vivo tracking of ESCs in experimental and clinical settings during cell-based therapies.Stem cell based therapies offer significant potential in the field of regenerative medicine. The development of superparamagnetic iron oxide (SPIO) nanoparticle labeling and magnetic resonance imaging (MRI) have been increasingly used to track the transplanted cells, enabling in vivo determination of cell fate. However, the impact of SPIO-labeling on the cell phenotype and differentiation capacity of embryonic stem cells (ESCs) remains unclear. In this study, we wrapped SPIO nanoparticles with stearic acid grafted PEI600, termed as Stearic-LWPEI-SPIO, to generate efficient and non-toxic ESC labeling tools. Our results showed that efficient labeling of ESCs at an optimized low dosage of Stearic-LWPEI-SPIO nanoparticles did not alter the differentiation and self-renewal properties of ESCs. The localization of the transplanted ESCs observed by MRI

  5. Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

    PubMed Central

    Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.

    2015-01-01

    Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379

  6. Electrostatic nucleic acid nanoassembly enables hybridization chain reaction in living cells for ultrasensitive mRNA imaging.

    PubMed

    Wu, Zhan; Liu, Gao-Qin; Yang, Xiao-Li; Jiang, Jian-Hui

    2015-06-03

    Efficient approaches for intracellular delivery of nucleic acid reagents to achieve sensitive detection and regulation of gene and protein expressions are essential for chemistry and biology. We develop a novel electrostatic DNA nanoassembly that, for the first time, realizes hybridization chain reaction (HCR), a target-initiated alternating hybridization reaction between two hairpin probes, for signal amplification in living cells. The DNA nanoassembly has a designed structure with a core gold nanoparticle, a cationic peptide interlayer, and an electrostatically assembled outer layer of fluorophore-labeled hairpin DNA probes. It is shown to have high efficiency for cellular delivery of DNA probes via a unique endocytosis-independent mechanism that confers a significant advantage of overcoming endosomal entrapment. Moreover, electrostatic assembly of DNA probes enables target-initialized release of the probes from the nanoassembly via HCR. This intracellular HCR offers efficient signal amplification and enables ultrasensitive fluorescence activation imaging of mRNA expression with a picomolar detection limit. The results imply that the developed nanoassembly may provide an invaluable platform in low-abundance biomarker discovery and regulation for cell biology and theranostics.

  7. Fluorescent carbohydrate probes for cell lectins

    NASA Astrophysics Data System (ADS)

    Galanina, Oxana; Feofanov, Alexei; Tuzikov, Alexander B.; Rapoport, Evgenia; Crocker, Paul R.; Grichine, Alexei; Egret-Charlier, Marguerite; Vigny, Paul; Le Pendu, Jacques; Bovin, Nicolai V.

    2001-09-01

    Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe x-PAA-flu, Sia 2-PAA-flu, GlcNAc 2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe x-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia 2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe x-PAA- 3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe x-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

  8. Various routes of administration of (99m)Tc-labeled synthetic lactoferrin antimicrobial peptide hLF 1-11 enables monitoring and effective killing of multidrug-resistant Staphylococcus aureus infections in mice.

    PubMed

    Brouwer, Carlo P J M; Welling, Mick M

    2008-07-01

    The synthetic antimicrobial peptide representative of the first 11 N-terminal amino acids of human lactoferrin (hLF 1-11) kills multidrug-resistant Staphylococcus aureus (MRSA). This study displays antimicrobial activity of hLF 1-11, via various routes of administration, against MRSA infections in mice. Radiolabeling hLF 1-11 with technetium-99m ((99m)Tc-hLF 1-11) enables scintigraphic monitoring directly after administration. (99m)Tc-hLF 1-11 was taken up by the gall bladder, intestines, and kidneys. Most of the radioactivity was captured in the urinary bladder and about 1% of the injected dose accumulated into infected thigh muscles. At 2 or 24h after either intravenously, subcutaneously, intraperitoneally, or orally injected a single dose of 0.04 mg/kg hLF 1-11 in mice significantly reduced (20-60 times) the number of viable MRSA. In a dose-response setting in immunocompetent mice maximum bactericidal effects (10,000 times reduction) of intravenously injected (99m)Tc-hLF 1-11 was seen with 40 mg/kg whereas the same dose of orally administered (99m)Tc-hLF 1-11 induced about approximately 100 times reduction. In conclusion, intravenously and orally administrated (99m)Tc-hLF 1-11 accumulates in infected tissues and is highly effective against experimental infections with MRSA. Moreover, scintigraphy is an excellent tool to study the pharmacology of experimental compounds and to determine the uptake in infected tissues.

  9. DNA probe for lactobacillus delbrueckii

    SciTech Connect

    Delley, M.; Mollet, B.; Hottinger, H. )

    1990-06-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

  10. Spectrophotometric probe

    DOEpatents

    Prather, William S.; O'Rourke, Patrick E.

    1994-01-01

    A support structure bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe.

  11. Spectrophotometric probe

    DOEpatents

    Prather, W.S.; O'Rourke, P.E.

    1994-08-02

    A support structure is described bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe. 3 figs.

  12. A signal amplification probe enhances sensitivity of antibodies and aptamers based Immuno-diagnostic assays.

    PubMed

    Nussbaum, Ofer; Bar Oz, Michal; Tilayov, Tal; Atiya, Helly; Dagan, Shlomo

    2017-09-01

    One major unmet need is improving the sensitivity of immune-diagnostic assays. This is particularly important in the field of biomarker discoveries and monitoring. We have established a novel signal amplification probe system enabling a highly sensitive target detection platform to be used in immuno-assays. The probe consists of a double stranded DNA that can carry a large number of signaling elements such as biotin or fluorescent molecules. The DNA probe anchors to the recognition unit, whether an antibody or an aptamer, by covalent conjugation or by a simple and rapid molecular association process. Binding curves obtained by using the DNA amplification probe are dose dependent and linear over a wide range of antigen concentration. The optimal slopes are characterized by high signals and low background increasing the assay sensitivity and reducing the limit of detection by up to 10-fold compared to biotinylated antibodies commonly used in ELISA systems. When using aptamers in combination with the amplification probe for antigen recognition, the limit of detection is comparable to that obtained by biotinylated antibodies. Biotin labeled aptamers practically cannot be used for detection of low target levels. The DNA amplification probe system enables to expand the range of diagnostic assays including clinical samples and meet research needs. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. High-Resolution and Specific Detection of Bacteria on Complex Surfaces Using Nanoparticle Probes and Electron Microscopy

    PubMed Central

    Ye, Jun; Nielsen, Shaun; Joseph, Stephen; Thomas, Torsten

    2015-01-01

    The study of the interaction of bacteria with surfaces requires the detection of specific bacterial groups with high spatial resolution. Here, we describe a method to rapidly and efficiently add nanogold particles to oligonucleotide probes, which target bacterial ribosomal RNA. These nanogold-labeled probes are then used in an in situ hybridization procedure that ensures both cellular integrity and high specificity. Electron microscopy subsequently enables the visualization of specific cells with high local precision on complex surface structures. This method will contribute to an increased understanding of how bacteria interact with surface structures on a sub-micron scale. PMID:26018431

  14. High-resolution and specific detection of bacteria on complex surfaces using nanoparticle probes and electron microscopy.

    PubMed

    Ye, Jun; Nielsen, Shaun; Joseph, Stephen; Thomas, Torsten

    2015-01-01

    The study of the interaction of bacteria with surfaces requires the detection of specific bacterial groups with high spatial resolution. Here, we describe a method to rapidly and efficiently add nanogold particles to oligonucleotide probes, which target bacterial ribosomal RNA. These nanogold-labeled probes are then used in an in situ hybridization procedure that ensures both cellular integrity and high specificity. Electron microscopy subsequently enables the visualization of specific cells with high local precision on complex surface structures. This method will contribute to an increased understanding of how bacteria interact with surface structures on a sub-micron scale.

  15. Monitoring membrane binding and insertion of peptides by two-color fluorescent label.

    PubMed

    Postupalenko, V Y; Shvadchak, V V; Duportail, G; Pivovarenko, V G; Klymchenko, A S; Mély, Y

    2011-01-01

    Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

  16. Food labeling

    MedlinePlus

    ... States Food and Drug Administration (FDA) has proposed making changes to the food labels that may correct these problems. AMOUNTS PER SERVING The total calories and the calories from fat are listed. These numbers help consumers make decisions about fat intake. The list of nutrients includes ...

  17. Review of methods to probe single cell metabolism and bioenergetics

    PubMed Central

    Vasdekis, Andreas E.; Stephanopoulos, Gregory

    2015-01-01

    Single cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed. PMID:25448400

  18. Synthesis and properties of acridone-labeled base-discriminating fluorescent (BDF) nucleosides.

    PubMed

    Saito, Yoshio; Hanawa, Kazuo; Bag, Subhendu Sekhar; Motegi, Kaori; Saito, Isao

    2006-01-01

    We have developed novel acridone-labelled BDF probe which showed its potential in recognizing opposite matched base from its target sequence via enhancement of fiuorescence intensity. This probe emit at a longer wavelength than previously reported pyrene-labelled BDF probe and thus can be used in DNA chip.

  19. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, Tuan

    1998-01-01

    The subject invention disclosed herein is a new gene probe biosensor and methods thereof based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays.

  20. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-07-21

    The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  1. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-09-29

    The subject invention disclosed herein is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  2. Surface enhanced Raman gene probe and methods thereof

    DOEpatents

    Vo-Dinh, T.

    1998-02-24

    The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

  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. Labeling proteins inside living cells using external fluorophores for microscopy

    PubMed Central

    Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

    2016-01-01

    Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial toxin which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG’s to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20–30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes. DOI: http://dx.doi.org/10.7554/eLife.20378.001 PMID:27935478

  5. Fixture For Calibrating Pressure Probe

    NASA Technical Reports Server (NTRS)

    Ashby, George C., Jr.; Vasquez, Peter; Horsley, Lewis A.; Bowman, John T.; Zumbrun, Henry N.; Eves, John W.

    1994-01-01

    Fixture in form of specially designed clamshell housing enables in situ calibration of pressure transducer mounted in body of pressure probe in wind tunnel. Includes two metal half shells machined with necks and matching cavities, when put together, define larger neck and cavity accommodating probe. Probe secured to bottom half shell by use of clamp before installing top half shell: necessary to follow sequence to protect probe during assembly. Clamshell calibration fixture attached to pressure probe in few minutes, making it possible to calibrate pressure transducer at convenient times. Calibrations performed before and after wind-tunnel runs each day, between runs in event of delays or suspected malfunctions, and essentially any other time, without having to remove probe from wind tunnel.

  6. Fixture For Calibrating Pressure Probe

    NASA Technical Reports Server (NTRS)

    Ashby, George C., Jr.; Vasquez, Peter; Horsley, Lewis A.; Bowman, John T.; Zumbrun, Henry N.; Eves, John W.

    1994-01-01

    Fixture in form of specially designed clamshell housing enables in situ calibration of pressure transducer mounted in body of pressure probe in wind tunnel. Includes two metal half shells machined with necks and matching cavities, when put together, define larger neck and cavity accommodating probe. Probe secured to bottom half shell by use of clamp before installing top half shell: necessary to follow sequence to protect probe during assembly. Clamshell calibration fixture attached to pressure probe in few minutes, making it possible to calibrate pressure transducer at convenient times. Calibrations performed before and after wind-tunnel runs each day, between runs in event of delays or suspected malfunctions, and essentially any other time, without having to remove probe from wind tunnel.

  7. Utilization of physiological and taxonomic fluorescent probes to study Lactobacilli cells and response to pH challenge.

    PubMed

    Olszewska, Magdalena A; Kocot, Aleksandra M; Nynca, Anna; Łaniewska-Trokenheim, Łucja

    2016-11-01

    pH stress is recognized as an important feature for Lactobacillus in relation to lifestyle and commercial utility. Hence, this study aims to investigate the cell function of Lactobacilli cells subjected to pHs between 7.0 and 2.0. For this purpose, the Lactobacilli isolates of vegetable origin were first hybridized with fluorescent oligonucleotide rRNA probes for detecting Lactobacillus species. Then, cells were exposed to pH stress and labelled with fluorescent probes, carboxyfluorescein diacetate (CFDA) and propidium iodine (PI), which provided the insight into esterase activity and membrane integrity of cells. Among isolates, fluorescence in situ hybridization (FISH) enabled us to specifically detect L. plantarum and L. brevis. Interestingly, FCM analysis revealed that at pHs between 7.0 and 4.0 the cell membrane was intact, while after the exposure at pH 3.0, and 2.0 became perturbed or impaired. Finally, L. brevis and L. plantarum differed from each other in fluorescence labeling behaviour and culturability. However, the results showed that the same standard protocol for labeling enables discrimination of subpopulations of tested species. Depending on the species, the substantial culturability loss was observed at pH 3.0 and 2.0. These results suggest that the taxonomic and physiological fluorescent probes could be suitable for in situ detection of specific bacteria and rapid assessment of the physiological status of cells.

  8. Introduction to Pesticide Labels

    EPA Pesticide Factsheets

    Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

  9. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  10. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  11. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  12. Endocavity Ultrasound Probe Manipulators.

    PubMed

    Stoianovici, Dan; Kim, Chunwoo; Schäfer, Felix; Huang, Chien-Ming; Zuo, Yihe; Petrisor, Doru; Han, Misop

    2013-06-01

    We developed two similar structure manipulators for medical endocavity ultrasound probes with 3 and 4 degrees of freedom (DoF). These robots allow scanning with ultrasound for 3-D imaging and enable robot-assisted image-guided procedures. Both robots use remote center of motion kinematics, characteristic of medical robots. The 4-DoF robot provides unrestricted manipulation of the endocavity probe. With the 3-DoF robot the insertion motion of the probe must be adjusted manually, but the device is simpler and may also be used to manipulate external-body probes. The robots enabled a novel surgical approach of using intraoperative image-based navigation during robot-assisted laparoscopic prostatectomy (RALP), performed with concurrent use of two robotic systems (Tandem, T-RALP). Thus far, a clinical trial for evaluation of safety and feasibility has been performed successfully on 46 patients. This paper describes the architecture and design of the robots, the two prototypes, control features related to safety, preclinical experiments, and the T-RALP procedure.

  13. Endocavity Ultrasound Probe Manipulators

    PubMed Central

    Stoianovici, Dan; Kim, Chunwoo; Schäfer, Felix; Huang, Chien-Ming; Zuo, Yihe; Petrisor, Doru; Han, Misop

    2014-01-01

    We developed two similar structure manipulators for medical endocavity ultrasound probes with 3 and 4 degrees of freedom (DoF). These robots allow scanning with ultrasound for 3-D imaging and enable robot-assisted image-guided procedures. Both robots use remote center of motion kinematics, characteristic of medical robots. The 4-DoF robot provides unrestricted manipulation of the endocavity probe. With the 3-DoF robot the insertion motion of the probe must be adjusted manually, but the device is simpler and may also be used to manipulate external-body probes. The robots enabled a novel surgical approach of using intraoperative image-based navigation during robot-assisted laparoscopic prostatectomy (RALP), performed with concurrent use of two robotic systems (Tandem, T-RALP). Thus far, a clinical trial for evaluation of safety and feasibility has been performed successfully on 46 patients. This paper describes the architecture and design of the robots, the two prototypes, control features related to safety, preclinical experiments, and the T-RALP procedure. PMID:24795525

  14. Label-free molecular imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Junqi; Li, Qi; Fu, Rongxin; Wang, Tongzhou; Wang, Ruliang; Huang, Guoliang

    2014-03-01

    Optical microscopy technology has achieved great improvements in the 20th century. The detection limit has reached about twenty nanometers (with near-field optics, STED, PALM and STORM). But in the application areas such as life science, medical science, clinical treatment and especially in vivo dynamic measurement, mutual restrictions still exist between numeric aperture/magnification and working distance, fluorescent dependent, and between resolution and frame rate/field size, etc. This paper explores a hyperspectral scanning super-resolution label free molecules imaging method based on the white light interferometry. The vertical detection resolution was approximate to 1 nm which is the thickness of a single molecular layer and dynamic measuring range of thickness reaches to 10 μm. The spectrum-shifting algorithm is developed for robust restructure of images when the pixels are overlapped. Micro-biochip with protein binding and DNA amplification could be detected by using this spectral scanning super-resolution molecules imaging in label free. This method has several advantages as following: Firstly, the decoding and detecting steps are combined into one step. It makes tests faster and easier. Secondly, we used thickness-coded, minimized chips instead of a large microarray chip to carry the probes. This accelerates the interaction of the biomolecules. Thirdly, since only one kind of probes are attached to our thickness-coded, minimized chip, users can only pick out the probes they are interested in for a test without wasting unnecessary probes and chips.

  15. Automated data extraction from in situ protein stable isotope probing studies

    SciTech Connect

    Slysz, Gordon W.; Steinke, Laurey A.; Ward, David M.; Klatt, Christian G.; Clauss, Therese RW; Purvine, Samuel O.; Payne, Samuel H.; Anderson, Gordon A.; Smith, Richard D.; Lipton, Mary S.

    2014-01-27

    Protein stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism, a key application will be in situ studies of microbial communities under conditions that result in small degrees of partial labeling. One hurdle restricting large scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large scale extraction and visualization of data from short term (3 h) protein-SIP experiments performed in situ on Yellowstone phototrophic bacterial mats. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification.

  16. Automated data extraction from in situ protein-stable isotope probing studies.

    PubMed

    Slysz, Gordon W; Steinke, Laurey; Ward, David M; Klatt, Christian G; Clauss, Therese R W; Purvine, Samuel O; Payne, Samuel H; Anderson, Gordon A; Smith, Richard D; Lipton, Mary S

    2014-03-07

    Protein-stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), a key application will be in situ studies of microbial communities for short periods of time under natural conditions that result in small degrees of partial labeling. One hurdle restricting large-scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large-scale extraction and visualization of data from short-term (3 h) protein-SIP experiments performed in situ on phototrophic bacterial mats isolated from Yellowstone National Park. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification.

  17. Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

    PubMed

    Keiderling, Timothy A

    2017-10-04

    Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

  18. Dendrimer Probes for Enhanced Photostability and Localization in Fluorescence Imaging

    PubMed Central

    Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A.; Schroeder, Charles M.

    2013-01-01

    Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. PMID:23561533

  19. Labeling the plasma membrane with TMA-DPH.

    PubMed

    Chazotte, Brad

    2011-05-01

    INTRODUCTION TMA-DPH (trimethylamine-diphenylhexatriene) is a fluorescent membrane probe that has classically been used to label the outer leaflet of a membrane bilayer, to label the outer leaflet of the plasma membrane in cells, and to report on membrane dynamics using the techniques of fluorescence polarization and/or fluorescence lifetime. This probe has also been used to follow exocytosis and endocytosis of labeled plasma membranes. The interaction of the aqueous environment with mitochondrial inner membrane dynamics has also been studied following the fluorescence polarization and the lifetime of TMA-DPH. This protocol describes the use of TMA-DPH to label the plasma membrane.

  20. Detection of human neutrophil elastase by aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence using specified site fluorescently labeled aptamer.

    PubMed

    Bai, Yunlong; Wang, Hailin; Zhao, Qiang

    2017-09-29

    As a multifunctional serine protease, human neutrophil elastase (HNE) plays critical roles in a variety of physiopathological processes, such as acute lung injury, emphysema, atherosclerosis, and arthritis. The quantification of HNE is important in many applications. In this paper, we report an aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) assay for detection of HNE using a tetramethylrhodamine (TMR)-labeled DNA aptamer probe. The affinity complex of HNE and DNA aptamer probe was well separated from the unbound aptamer probe in CE separation based on the difference of electrophoretic mobility. Broad complex peaks appeared due to possible multiple binding. The 45-mer aptamer having TMR labeling on the 40th T base was used as affinity probe, as larger complex peaks were obtained. We investigated the effects of various metal cations (Na(+), K(+), and Mg(2+)) in sample buffer on the binding of HNE and the aptamer in CE-LIF analysis. The presence of Na(+), K(+), or Mg(2+) in sample buffer caused a decrease of complex peaks, and Mg(2+) showed a larger effect. Under optimized conditions, this aptamer CE-LIF assay enabled the detection of HNE at 0.5 nM. This assay showed good specificity and allowed for detection of HNE spiked in diluted human serum sample. Graphical abstract The complex of HNE and DNA aptamer probe was isolated from the unbound aptamer probe in CE separation due to difference of electrophoretic mobility, allowing a CE-LIF assay for HNE.

  1. Optical probe

    DOEpatents

    Hencken, Kenneth; Flower, William L.

    1999-01-01

    A compact optical probe is disclosed particularly useful for analysis of emissions in industrial environments. The instant invention provides a geometry for optically-based measurements that allows all optical components (source, detector, rely optics, etc.) to be located in proximity to one another. The geometry of the probe disclosed herein provides a means for making optical measurements in environments where it is difficult and/or expensive to gain access to the vicinity of a flow stream to be measured. Significantly, the lens geometry of the optical probe allows the analysis location within a flow stream being monitored to be moved while maintaining optical alignment of all components even when the optical probe is focused on a plurality of different analysis points within the flow stream.

  2. Technology Enabled Learning. Symposium.

    ERIC Educational Resources Information Center

    2002

    This document contains three papers on technology-enabled learning and human resource development. Among results found in "Current State of Technology-enabled Learning Programs in Select Federal Government Organizations: a Case Study of Ten Organizations" (Letitia A. Combs) are the following: the dominant delivery method is traditional…

  3. Outcomes from Enabling Courses.

    ERIC Educational Resources Information Center

    Phan, Oanh; Ball, Katrina

    The outcomes of enabling courses offered in Australia's vocational education and training (VET) sector were examined. "Enabling course" was defined as lower-level preparatory and prevocational courses covering a wide range of areas, including remedial education, bridging courses, precertificate courses, and general employment preparation…

  4. Improvement of inhibitor identification for heat shock protein 90α by utilizing a red-shifted fluorescence polarization probe.

    PubMed

    Qian, Jie; Holskin, Beverly P; Theroff, Jay; Underiner, Ted; Meyer, Sheryl L; Angeles, Thelma S

    2012-08-01

    Heat shock protein-90 (HSP90) is an ATP-dependent molecular chaperone with intrinsic ATPase activity. HSP90 is required for the stability and function of client proteins, many of which are involved in oncogenesis. Thus, identification of HSP90 inhibitors would potentially lead to the discovery of cancer therapeutics. Here, we present a high-throughput screening campaign utilizing two geldanamycin (GM)-labeled probes in a fluorescence polarization (FP) assay. For the primary screen, a previously reported green BODIPY-labeled GM (GM-BODIPY) was used to evaluate a library collection of about 400,000 compounds. From this screen, 3058 compounds showed >30% inhibition. To distinguish true positives from compound interference, a confirmatory screen was deemed necessary. Accordingly, a red-shifted FP binding assay was developed using GM labeled with red BODIPY. This tool enabled reliable identification of promising HSP90α inhibitors.

  5. Combining Perfluorocarbon and Superparamagnetic Iron-oxide Cell Labeling for Improved and Expanded Applications of Cellular MRI

    PubMed Central

    Hitchens, T. Kevin; Liu, Li; Foley, Lesley M.; Simplaceanu, Virgil; Ahrens, Eric T.; Ho, Chien

    2014-01-01

    Purpose The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI. Methods We characterized 19F-NMR relaxation properties of PFC-labeled cells in the presence of SPIO and imaged cells both ex vivo and in vivo in a rodent inflammation model to demonstrate selective visualization of cell populations. Results We show that with UTE3D, RARE and FLASH 19F images one can uniquely identify PFC-labeled cells, co-localized PFC- and SPIO-labeled cells, and PFC/SPIO co-labeled cells. Conclusion This new methodology has the ability to improve and expand applications of MRI cell tracking. Combining PFC and SPIO strategies can potentially provide a method to quench PFC signal transferred from dead cells to macrophages, thereby eliminating false positives. In addition, combining these techniques could also be used to track two cell types simultaneously and probe cell-cell proximity in vivo with MRI. PMID:24478194

  6. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

  7. Self-referenced label free biosensors based on differential fiber optic interferometry

    NASA Astrophysics Data System (ADS)

    Queirós, R. B.; Gouveia, C.; Martins, M. Cristina L.; Jorge, P. A. S.

    2014-02-01

    The measurement of refractive index (RI) is an important tool for label free biosensing in biomedical applications [1,2]. In this work, a LPG based fiber optic interferometric probe is used for thrombin detection. The aptamer raised against the thrombin was immobilized through an electrostatic immobilization method, using poly-L-lysine as cationic polymer. The functionalized probe was characterized and tested against thrombin. The system was validated with the detection of thrombin using an aptamer based probe (5'-[amine]GGTTGGTGTGGTTGG-3') as a model system for protein detection. The shift corresponding to the affinity-assay between TBA and the thrombin was of about 56 pm. A differential readout interferometer based on a white light Mach-Zehnder configuration, with pseudo-heterodyne phase modulation is described. The system can be used to interrogate two similar LPGs based interferometers in a differential scheme. Considering the configuration where both devices are functionalized being one active (sensor) and the other one passive (reference) it is possible to accurately measure the behavior of the analyte of interest independent of non-specific binding events, bulk refractive index changes and temperature. Signal processing with low cost digital instrumentation developed in Labview environment allows a detectable change in refractive index of Δn ≍ 2x10-6 [3]. Coupling the sensing probe together with a passively functionalized reference probe in a differential system will enable pseudo-heterodyne interrogation and extremely sensitive phase detection of biological species.

  8. Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

    PubMed

    Neves, André A; Wainman, Yéléna A; Wright, Alan; Kettunen, Mikko I; Rodrigues, Tiago B; McGuire, Sarah; Hu, De-En; Bulat, Flaviu; Geninatti Crich, Simonetta; Stöckmann, Henning; Leeper, Finian J; Brindle, Kevin M

    2016-01-22

    Glycosylation is a ubiquitous post-translational modification, present in over 50% of the proteins in the human genome, with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases, including cancer. We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe. Significant N-azidoacetylgalactosamine dependent T1  contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10-fold). This approach has the potential to enable the rapid and non-invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease.

  9. Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

    PubMed

    Neves, André A; Wainman, Yéléna A; Wright, Alan; Kettunen, Mikko I; Rodrigues, Tiago B; McGuire, Sarah; Hu, De-En; Bulat, Flaviu; Geninatti Crich, Simonetta; Stöckmann, Henning; Leeper, Finian J; Brindle, Kevin M

    2016-01-22

    Glycosylation is a ubiquitous post-translational modification, present in over 50 % of the proteins in the human genome,1 with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases,2 including cancer.3 We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe. Significant N-azidoacetylgalactosamine dependent T1 contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10-fold). This approach has the potential to enable the rapid and non-invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease.

  10. Nanocrystal clusters in combination with spectral imaging to improve sensitivity in antibody labeling applications of fluorescent nanocrystals

    NASA Astrophysics Data System (ADS)

    Maier, John S.; Panza, Janice L.; Bootman, Matt

    2007-02-01

    Composition-tunable nanocrystals are fluorescent nanoparticles with a uniform particle size and with adjustable optical characteristics. When used for optical labeling of biomolecular targets these and other nanotechnology solutions have enabled new approaches which are possible because of the high optical output, narrow spectral signal, consistent quantum efficiency across a broad emission range and long lived fluorescent behavior of the nanocrystals. When coupled with spectral imaging the full potential of multiplexing multiple probes in a complex matrix can be realized. Spectral imaging can be used to improve sensitivity of narrowband fluorophores through application of chemometric image processing techniques used to reduce the influence of autofluorescence background. Composition-tunable nanocrystals can be complexed together to form nanoclusters which have the advantage of significantly stronger signal and therefore a higher sensitivity. These nanoclusters can be targeted in biomolecular systems using standard live-cell labeling and immunohistochemistry based techniques. Composition-tunable nanocrystals and nanoclusters have comparable mass and brightness across a wide emission range. This enables the production of nanocrystal-based probes that have comparable reactivity and sensitivity over a large color range. We present spectral imaging results of antibody targeted nanocrystal cluster labeling of target proteins in cultured cells and a Western blot experiment. The combination of spectral imaging with the use of clusters of nanocrystals further improves the sensitivity over either of the approaches independently.

  11. Mechanosensitive membrane probes.

    PubMed

    Dal Molin, Marta; Verolet, Quentin; Soleimanpour, Saeideh; Matile, Stefan

    2015-04-13

    This article assembles pertinent insights behind the concept of planarizable push-pull probes. As a response to the planarization of their polarized ground state, a red shift of their excitation maximum is expected to report on either the disorder, the tension, or the potential of biomembranes. The combination of chromophore planarization and polarization contributes to various, usually more complex processes in nature. Examples include the color change of crabs or lobsters during cooking or the chemistry of vision, particularly color vision. The summary of lessons from nature is followed by an overview of mechanosensitive organic materials. Although often twisted and sometimes also polarized, their change of color under pressure usually originates from changes in their crystal packing. Intriguing exceptions include the planarization of several elegantly twisted phenylethynyl oligomers and polymers. Also mechanosensitive probes in plastics usually respond to stretching by disassembly. True ground-state planarization in response to molecular recognition is best exemplified with the binding of thoughtfully twisted cationic polythiophenes to single- and double-stranded oligonucleotides. Molecular rotors, en vogue as viscosity sensors in cells, operate by deplanarization of the first excited state. Pertinent recent examples are described, focusing on λ-ratiometry and intracellular targeting. Complementary to planarization of the ground state with twisted push-pull probes, molecular rotors report on environmental changes with quenching or shifts in emission rather than absorption. The labeling of mechanosensitive channels is discussed as a bioengineering approach to bypass the challenge to create molecular mechanosensitivity and use biological systems instead to sense membrane tension. With planarizable push-pull probes, this challenge is met not with twistome screening, but with "fluorescent flippers," a new concept to insert large and bright monomers into oligomeric

  12. Human papillomavirus 35 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorincz, A.T.

    1989-07-18

    This patent describes an HPV 35 hybridization probe comprising a member selected from the group consisting of (i) HPV 35 DNA or fragments thereof labelled with a marker and (ii) HPV 35 RNA or fragments thereof labelled with a marker.

  13. Human papillomavirus 43 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorincz, A.T.

    1989-07-18

    This patent describes an HPV 43 hybridization probe comprising a member selected from the group consisting of (i) HPV 43 DNA or fragments thereof labelled with a marker and (ii) HPV 43 RNA or fragments thereof labelled with a marker.

  14. Human papillomavirus 56 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorinez, A.T.

    1990-03-13

    This patent describes an HPV 56 hybridization probe. It comprises: a member selected from the group consisting of HPV 56 DNA or fragments thereof labelled with a marker and HPV 56 RNA or fragments thereof labelled with a marker.

  15. Human papillomavirus 44 nucleic acid hybridization probes and methods for employing the same

    SciTech Connect

    Lorincz, A.T.

    1989-07-18

    This patent describes an HPV 44 hybridization probe comprising a member selected from the group consisting of (1) HPV 44 DNA or fragments thereof labelled with a marker and (ii) HPV 44 RNA or fragments thereof labelled with a marker.

  16. Raman tags: Novel optical probes for intracellular sensing and imaging.

    PubMed

    Li, Yuee; Wang, Zhong; Mu, Xijiao; Ma, Aning; Guo, Shu

    Optical labels are needed for probing specific target molecules in complex biological systems. As a newly emerging category of tags for molecular imaging in live cells, the Raman label attracts much attention because of the rich information obtained from targeted and untargeted molecules by detecting molecular vibrations. Here, we list three types of Raman probes based on different mechanisms: Surface Enhanced Raman Scattering (SERS) probes, bioorthogonal Raman probes, and Resonance Raman (RR) probes. We review how these Raman probes work for detecting and imaging proteins, nucleic acids, lipids, and other biomolecules in vitro, within cells, or in vivo. We also summarize recent noteworthy studies, expound on the construction of every type of Raman probe and operating principle, sum up in tables typically targeting molecules for specific binding, and provide merits, drawbacks, and future prospects for the three Raman probes. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

    PubMed

    Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

    2016-05-01

    Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Pesticide Label Review Training

    EPA Pesticide Factsheets

    This training will help ensure that reviewers evaluate labels according to four core principles. It also will help pesticide registrants developing labels understand what EPA expects of pesticide labels, and what the Agency generally finds acceptable.

  19. Probe-rotating atomic force microscopy for determining material properties

    SciTech Connect

    Lee, Sang Heon

    2014-03-15

    In this paper, we propose a probe-rotating atomic force microscope that enables scan in an arbitrary direction in the contact imaging mode, which is difficult to achieve using a conventional atomic force microscope owing to the orientation-dependent probe and the inability to rotate the probe head. To enable rotation of the probe about its vertical axis, we employed a compact and light probe head, the sensor of which is made of an optical disk drive pickup unit. Our proposed mechanical configuration, operating principle, and control system enables axial and lateral scan in various directions.

  20. Development and Application of Multiple-Probe Scanning Probe Microscopes

    SciTech Connect

    Nakayama, T.; Kubo, O.; Shingaya, Y.; Higuchi, S.; Hasegawa, T.; Jiang, C. S.; Okuda, T.; Kuwahara, Y.; Takami, K.; Aono, M.

    2012-04-03

    the research of advanced materials based on nanoscience and nanotechnology, it is often desirable to measure nanoscale local electrical conductivity at a designated position of a given sample. For this purpose, multiple-probe scanning probe microscopes (MP-SPMs), in which two, three or four scanning tunneling microscope (STM) or atomic force microscope (AFM) probes are operated independently, have been developed. Each probe in an MP-SPM is used not only for observing high-resolution STM or AFM images but also for forming an electrical contact enabling nanoscale local electrical conductivity measurement. The world's first double-probe STM (DP-STM) developed by the authors, which was subsequently modified to a triple-probe STM (TP-STM), has been used to measure the conductivities of one-dimensional metal nanowires and carbon nanotubes and also two-dimensional molecular films. A quadruple-probe STM (QP-STM) has also been developed and used to measure the conductivity of two-dimensional molecular films without the ambiguity of contact resistance between the probe and sample. Moreover, a quadruple-probe AFM (QP-AFM) with four conductive tuning-fork-type self-detection force sensing probes has been developed to measure the conductivity of a nanostructure on an insulating substrate. A general-purpose computer software to control four probes at the same time has also been developed and used in the operation of the QP-AFM. These developments and applications of MP-SPMs are reviewed in this paper.

  1. Development and application of multiple-probe scanning probe microscopes.

    PubMed

    Nakayama, Tomonobu; Kubo, Osamu; Shingaya, Yoshitaka; Higuchi, Seiji; Hasegawa, Tsuyoshi; Jiang, Chun-Sheng; Okuda, Taichi; Kuwahara, Yuji; Takami, Kazuhiro; Aono, Masakazu

    2012-04-03

    In the research of advanced materials based on nanoscience and nanotechnology, it is often desirable to measure nanoscale local electrical conductivity at a designated position of a given sample. For this purpose, multiple-probe scanning probe microscopes (MP-SPMs), in which two, three or four scanning tunneling microscope (STM) or atomic force microscope (AFM) probes are operated independently, have been developed. Each probe in an MP-SPM is used not only for observing high-resolution STM or AFM images but also for forming an electrical contact enabling nanoscale local electrical conductivity measurement. The world's first double-probe STM (DP-STM) developed by the authors, which was subsequently modified to a triple-probe STM (TP-STM), has been used to measure the conductivities of one-dimensional metal nanowires and carbon nanotubes and also two-dimensional molecular films. A quadruple-probe STM (QP-STM) has also been developed and used to measure the conductivity of two-dimensional molecular films without the ambiguity of contact resistance between the probe and sample. Moreover, a quadruple-probe AFM (QP-AFM) with four conductive tuning-fork-type self-detection force sensing probes has been developed to measure the conductivity of a nanostructure on an insulating substrate. A general-purpose computer software to control four probes at the same time has also been developed and used in the operation of the QP-AFM. These developments and applications of MP-SPMs are reviewed in this paper.

  2. Aptamer and 5-fluorouracil dual-loading Ag2S quantum dots used as a sensitive label-free probe for near-infrared photoluminescence turn-on detection of CA125 antigen.

    PubMed

    Jin, Hui; Gui, Rijun; Gong, Jun; Huang, Wenxue

    2017-06-15

    In this article, Ag2S quantum dots (QDs) were prepared by a facile aqueous synthesis method, using thiourea as a new sulfur precursor. Based on electrostatic interactions, 5-fluorouracil (5-Fu) was combined with the aptamer of CA125 antigen to fabricate aptamer/5-Fu complex. The surface of as-prepared Ag2S QDs was modified with polyethylenimine, followed by combination with the aptamer/5-Fu complex to form Ag2S QDs/aptamer/5-Fu hybrids. During the combination of Ag2S QDs with aptamer/5-Fu complex, near-infrared (NIR) photoluminescence (PL) of QDs (peaked at 850nm) was markedly reduced under excitation at 625nm, attributed to photo-induced electron transfer from QDs to 5-Fu. However, the addition of CA125 induced obvious NIR PL recovery, which was ascribed to the strong binding affinity of CA125 with its aptamer, and the separation of aptamer/5-Fu complex from the surface of QDs. Hence, the Ag2S QDs/aptamer/5-Fu hybrids were developed as a novel NIR PL turn-on probe of CA125. In the concentration range of [CA125] from 0.1 to 10(6)ngmL(-1), there were a good linear relationship between NIR PL intensities of Ag2S QDs and Log[CA125], and a low limit of detection of 0.07ngmL(-1). Experimental results revealed the highly selective and sensitive NIR PL responses of this probe to CA125, over other potential interferences. In real human body fluids, this probe also exhibited superior analytical performance, together with high detection recoveries. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. An Ultrasensitive Electrochemiluminescence Immunoassay for Carbohydrate Antigen 19-9 in Serum Based on Antibody Labeled Fe3O4 Nanoparticles as Capture Probes and Graphene/CdTe Quantum Dot Bionanoconjugates as Signal Amplifiers

    PubMed Central

    Gan, Ning; Zhou, Jing; Xiong, Ping; Li, Tianhua; Jiang, Shan; Cao, Yuting; Jiang, Qianli

    2013-01-01

    The CdTe quantum dots (QDs), graphene nanocomposite (CdTe-G) and dextran–Fe3O4 magnetic nanoparticles have been synthesized for developing an ultrasensitive electrochemiluminescence (ECL) immunoassay for Carcinoembryonic antigen 19-9 (CA 19-9) in serums. Firstly, the capture probes (CA 19-9 Ab1/Fe3O4) for enriching CA 19-9 were synthesized by immobilizing the CA 19-9’s first antibody (CA 19-9 Ab1) on magnetic nanoparticles (dextran-Fe3O4). Secondly, the signal probes (CA 19-9 Ab2/CdTe-G), which can emit an ECL signal, were formed by attaching the secondary CA 19-9 antibody (CA 19-9 Ab2) to the surface of the CdTe-G. Thirdly, the above two probes were used for conjugating with a serial of CA 19-9 concentrations. Graphene can immobilize dozens of CdTe QDs on their surface, which can emit stronger ECL intensity than CdTe QDs. Based on the amplified signal, ultrasensitive antigen detection can be realized. Under the optimal conditions, the ECL signal depended linearly on the logarithm of CA 19-9 concentration from 0.005 to 100 pg/mL, and the detection limit was 0.002 pg/mL. Finally, five samples of human serum were tested, and the results were compared with a time-resolved fluorescence assay (TRFA). The novel immunoassay provides a stable, specific and highly sensitive immunoassay protocol for tumor marker detection at very low levels, which can be applied in early diagnosis of tumor. PMID:23685872

  4. Advances in the chemistry of small molecule fluorescent probes.

    PubMed

    Wysocki, Laura M; Lavis, Luke D

    2011-12-01

    Small molecule fluorophores are essential tools for chemical biology. A benefit of synthetic dyes is the ability to employ chemical approaches to control the properties and direct the position of the fluorophore. Applying modern synthetic organic chemistry strategies enables efficient tailoring of the chemical structure to obtain probes for specific biological experiments. Chemistry can also be used to activate fluorophores; new fluorogenic enzyme substrates and photoactivatable compounds with improved properties have been prepared that facilitate advanced imaging experiments with low background fluorescence. Finally, chemical reactions in live cells can be used to direct the spatial distribution of the fluorophore, allowing labeling of defined cellular regions with synthetic dyes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Pollution Probe.

    ERIC Educational Resources Information Center

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  6. Pollution Probe.

    ERIC Educational Resources Information Center

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  7. Deep Label Distribution Learning With Label Ambiguity

    NASA Astrophysics Data System (ADS)

    Gao, Bin-Bin; Xing, Chao; Xie, Chen-Wei; Wu, Jianxin; Geng, Xin

    2017-06-01

    Convolutional Neural Networks (ConvNets) have achieved excellent recognition performance in various visual recognition tasks. A large labeled training set is one of the most important factors for its success. However, it is difficult to collect sufficient training images with precise labels in some domains such as apparent age estimation, head pose estimation, multi-label classification and semantic segmentation. Fortunately, there is ambiguous information among labels, which makes these tasks different from traditional classification. Based on this observation, we convert the label of each image into a discrete label distribution, and learn the label distribution by minimizing a Kullback-Leibler divergence between the predicted and ground-truth label distributions using deep ConvNets. The proposed DLDL (Deep Label Distribution Learning) method effectively utilizes the label ambiguity in both feature learning and classifier learning, which help prevent the network from over-fitting even when the training set is small. Experimental results show that the proposed approach produces significantly better results than state-of-the-art methods for age estimation and head pose estimation. At the same time, it also improves recognition performance for multi-label classification and semantic segmentation tasks.

  8. Enhanced Fluorescence Imaging of Live Cells by Effective Cytosolic Delivery of Probes

    PubMed Central

    Massignani, Marzia; Canton, Irene; Sun, Tao; Hearnden, Vanessa; MacNeil, Sheila; Blanazs, Adam; Armes, Steven P.; Lewis, Andrew; Battaglia, Giuseppe

    2010-01-01

    Background Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. Principal Findings We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. Significance We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation. PMID:20454666

  9. Multifunctional Concentric FRET-Quantum Dot Probes for Tracking and Imaging of Proteolytic Activity.

    PubMed

    Massey, Melissa; Li, Jia Jun; Algar, W Russ

    2017-01-01

    Proteolysis has many important roles in physiological regulation. It is involved in numerous cell signaling processes and the pathogenesis of many diseases, including cancers. Methods of visualizing and assaying proteolytic activity are therefore in demand. Förster resonance energy transfer (FRET) probes offer several advantages in this respect. FRET supports end-point or real-time measurements, does not require washing or separation steps, and can be implemented in various assay or imaging formats. In this chapter, we describe methodology for preparing self-assembled concentric FRET (cFRET) probes for multiplexed tracking and imaging of proteolytic activity. The cFRET probe comprises a green-emitting semiconductor quantum dot (QD) conjugated with multiple copies of two different peptide substrates for two target proteases. The peptide substrates are labeled with different fluorescent dyes, Alexa Fluor 555 and Alexa Fluor 647, and FRET occurs between the QD and both dyes, as well as between the two dyes. This design enables a single QD probe to track the activity of two proteases simultaneously. Fundamental cFRET theory is presented, and procedures for using the cFRET probe for quantitative measurement of the activity of two model proteases are given, including calibration, fluorescence plate reader or microscope imaging assays, and data analysis. Sufficient detail is provided for other researchers to adapt this method to their specific requirements and proteolytic systems of interest.

  10. Physician Enabling Skills Questionnaire

    PubMed Central

    Hudon, Catherine; Lambert, Mireille; Almirall, José

    2015-01-01

    Abstract Objective To evaluate the reliability and validity of the newly developed Physician Enabling Skills Questionnaire (PESQ) by assessing its internal consistency, test-retest reliability, concurrent validity with patient-centred care, and predictive validity with patient activation and patient enablement. Design Validation study. Setting Saguenay, Que. Participants One hundred patients with at least 1 chronic disease who presented in a waiting room of a regional health centre family medicine unit. Main outcome measures Family physicians’ enabling skills, measured with the PESQ at 2 points in time (ie, while in the waiting room at the family medicine unit and 2 weeks later through a mail survey); patient-centred care, assessed with the Patient Perception of Patient-Centredness instrument; patient activation, assessed with the Patient Activation Measure; and patient enablement, assessed with the Patient Enablement Instrument. Results The internal consistency of the 6 subscales of the PESQ was adequate (Cronbach α = .69 to .92). The test-retest reliability was very good (r = 0.90; 95% CI 0.84 to 0.93). Concurrent validity with the Patient Perception of Patient-Centredness instrument was good (r = −0.67; 95% CI −0.78 to −0.53; P < .001). The PESQ accounts for 11% of the total variance with the Patient Activation Measure (r2 = 0.11; P = .002) and 19% of the variance with the Patient Enablement Instrument (r2 = 0.19; P < .001). Conclusion The newly developed PESQ presents good psychometric properties, allowing for its use in practice and research. PMID:26889507

  11. Dust control for Enabler

    NASA Technical Reports Server (NTRS)

    Hilton, Kevin; Karl, Chad; Litherland, Mark; Ritchie, David; Sun, Nancy

    1992-01-01

    The dust control group designed a system to restrict dust that is disturbed by the Enabler during its operation from interfering with astronaut or camera visibility. This design also considers the many different wheel positions made possible through the use of artinuation joints that provide the steering and wheel pitching for the Enabler. The system uses a combination of brushes and fenders to restrict the dust when the vehicle is moving in either direction and in a turn. This design also allows for ease of maintenance as well as accessibility of the remainder of the vehicle.

  12. Dust control for Enabler

    NASA Technical Reports Server (NTRS)

    Hilton, Kevin; Karl, Chad; Litherland, Mark; Ritchie, David; Sun, Nancy

    1992-01-01

    The dust control group designed a system to restrict dust that is disturbed by the Enabler during its operation from interfering with astronaut or camera visibility. This design also considers the many different wheel positions made possible through the use of artinuation joints that provide the steering and wheel pitching for the Enabler. The system uses a combination of brushes and fenders to restrict the dust when the vehicle is moving in either direction and in a turn. This design also allows for each of maintenance as well as accessibility of the remainder of the vehicle.

  13. Carbon Nanoparticle-based Fluorescent Bioimaging Probes

    PubMed Central

    Bhunia, Susanta Kumar; Saha, Arindam; Maity, Amit Ranjan; Ray, Sekhar C.; Jana, Nikhil R.

    2013-01-01

    Fluorescent nanoparticle-based imaging probes have advanced current labelling technology and are expected to generate new medical diagnostic tools based on their superior brightness and photostability compared with conventional molecular probes. Although significant progress has been made in fluorescent semiconductor nanocrystal-based biological labelling and imaging, the presence of heavy metals and the toxicity issues associated with heavy metals have severely limited the application potential of these nanocrystals. Here, we report a fluorescent carbon nanoparticle-based, alternative, nontoxic imaging probe that is suitable for biological staining and diagnostics. We have developed a chemical method to synthesise highly fluorescent carbon nanoparticles 1–10 nm in size; these particles exhibit size-dependent, tunable visible emission. These carbon nanoparticles have been transformed into various functionalised nanoprobes with hydrodynamic diameters of 5–15 nm and have been used as cell imaging probes. PMID:23502324

  14. Visual-Inspection Probe For Cryogenic Chamber

    NASA Technical Reports Server (NTRS)

    Friend, Steve; Valenzuela, James; Yoshinaga, Jay

    1990-01-01

    Visual-inspection probe that resembles borescope enables observer at ambient temperature to view objects immersed in turbulent flow of liquid oxygen, liquid nitrogen, or other cryogenic fluid. Design of probe fairly conventional, except special consideration given to selection of materials and to thermal expansion to provide for expected range of operating temperatures. Penetrates wall of cryogenic chamber to provide view of interior. Similar probe illuminates scene. View displayed on video monitor.

  15. Visual-Inspection Probe For Cryogenic Chamber

    NASA Technical Reports Server (NTRS)

    Friend, Steve; Valenzuela, James; Yoshinaga, Jay

    1990-01-01

    Visual-inspection probe that resembles borescope enables observer at ambient temperature to view objects immersed in turbulent flow of liquid oxygen, liquid nitrogen, or other cryogenic fluid. Design of probe fairly conventional, except special consideration given to selection of materials and to thermal expansion to provide for expected range of operating temperatures. Penetrates wall of cryogenic chamber to provide view of interior. Similar probe illuminates scene. View displayed on video monitor.

  16. Chromosome-specific DNA Repeat Probes

    SciTech Connect

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  17. Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET).

    PubMed

    Kosík, Ondřej; Garajová, Soňa; Matulová, Mária; Rehulka, Pavel; Stratilová, Eva; Farkaš, Vladimír

    2011-02-01

    Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc(4)Xyl(3)Gal(2) (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium (Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K(m) for individual acceptors increased in the order [1-(3)H]XLLGXLLG-SR>XLLG-ANTS>[1-(3)H]XLLGol>[1-(3)H]XLLG>XLLG-AA. Catalytic efficiency (expressed as k(cat)/K(m)) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1-(3)H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET. 2010 Elsevier Ltd. All rights reserved.

  18. Component Labeling Algorithm For Video Rate Processing

    NASA Astrophysics Data System (ADS)

    Gotoh, Toshiyuki; Ohta, Yoshiyuki; Yoshida, Masumi; Shirai, Yoshio

    1987-10-01

    In this paper, we propose a raster scanning algorithm for component labeling, which enables processing under pipeline architecture. In the raster scanning algorithm, labels are provisionally assigned to each pixel of components and, at the same time, the connectivities of labels are detected at first scan. Those labels are classified into groups based on the connectivities. Finally provisional labels are updated using the result of classification and a unique label is assigned to each pixel of components. However, in the conventional algorithm, the classification process needs a vast number of operations. This prevents realizing pipeline processing. We have developed a method of preprocessing to reduce the number of provisional labels, which limits the number of label connectivities. We have also developed a new classification method whose operation is proportionate to only the number of label connectivities itself. We have made experiments with computer simulation to verify this algorithm. The experimental results show that we can process 512 x 512 x 8 bit images at video rate(1/30 sec. per 1 image) when this algorithm is implemented on hardware.

  19. Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology.

    PubMed

    von Bergen, Martin; Jehmlich, Nico; Taubert, Martin; Vogt, Carsten; Bastida, Felipe; Herbst, Florian-Alexander; Schmidt, Frank; Richnow, Hans-Hermann; Seifert, Jana

    2013-10-01

    The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying