Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

USDA-ARS?s Scientific Manuscript database

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal and polyclonal anti...

Fluorescent Cell Barcoding for Multiplex Flow Cytometry

PubMed Central

Krutzik, Peter O.; Clutter, Matthew R.; Trejo, Angelica; Nolan, Garry P.

2011-01-01

Fluorescent Cell Barcoding (FCB) enables high throughput, i.e. high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10 to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines as well as primary peripheral blood samples. Important technical considerations such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis are discussed. PMID:21207359

Linkage analysis of chromosome 22q12-13 in a United Kingdom/Icelandic sample of 23 multiplex schizophrenia families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalsi, G.; Read, T.; Butler, R.

A possible linkage to a genetic subtype of schizophrenia and related disorders has been reported on the long arm of chromosome 22 at q12-13. However formal statistical tests in a combined sample could not reject homogeneity and prove that there was linked subgroup of families. We have studied 23 schizophrenia pedigrees to test whether some multiplex schizophrenia families may be linked to the microsatellite markers D22S274 and D22S283 which span the 22q12-13 region. Two point followed by multipoint lod and non-parametric linkage analyses under the assumption of heterogeneity provided no evidence for linkage over the relevant region. 16 refs., 4more » tabs.« less

Multiplex and high-throughput DNA detection using surface plasmon mediated fluorescence

NASA Astrophysics Data System (ADS)

Mei, Zhong

The overall objective of this research project was to develop a user-friendly and sensitive biosensor for nucleic acid aptamers with multiplexing and high-throughput capability. The sensing was based on the fluorescence signals emitted by the fluorophores coupling with plamonic nanoparticle (gold nanorod) deposited on a patterned substrate. Gold nanorods (GNRs) were synthesized using a binary mixture of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL) in seed mediated growth method. Polytetrafluoroethylene (PTFE) printed glass slides were selectively coated with a gold thin-film to define hydrophilic areas for GNR deposition. Due to the wettablity contrast, GNR solution dropped on the slide was induced to assemble exclusively in the hydrophilic spots. By controlling temperature and humidity of the evaporation process, vertically-standing GNR arrays were achieved on the pattered slide. Fluorescence was conjugated to GNR surface via DNA double strand with tunable length. Theoretical simulation predicted a flat layer ( 30 nm thick) of uniform "hot spots" presented on the GNR tips, which could modify the nearby fluorescence. Experimentally, the vertical GNR arrays yielded metallic enhanced fluorescence (MEF) effect, which was dependent on the spectrum overlap and GNR-fluorophore distance. Specifically, the maximum enhancement of Quasar 670 and Alexa 750 was observed when it was coupled with GNR664 (plasmonic wavelength 664 nm) and GNR778 respectively at a distance of 16 nm, while the carboxyfluorescein (FAM) was at maximal intensity when attached to gold nanosphere520. This offers an opportunity for multiplexed DNA sensing. Based on this, we developed a novel GNR mediated fluorescence biosensor for DNA detection. Fluorescence labeled haipin-DNA probes were introduced to designated spots of GNR array with the matching LSPR wavelengths on the substrate. The fluorescence was quenched originally because of Forster resonance energy transfer (FRET) effect

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

PubMed Central

Chan, Jacky C. K.; Diebold, Eric D.; Buckley, Brandon W.; Mao, Sien; Akbari, Najva; Jalali, Bahram

2014-01-01

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat’s-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures. PMID:25574449

High-speed bioimaging with frequency-division-multiplexed fluorescence confocal microscopy

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Harmon, Jeffrey; Ozeki, Yasuyuki; Goda, Keisuke

2017-04-01

We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth ( 400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.

SERS-fluorescence joint spectral encoded magnetic nanoprobes for multiplex cancer cell separation.

PubMed

Wang, Zhuyuan; Zong, Shenfei; Chen, Hui; Wang, Chunlei; Xu, Shuhong; Cui, Yiping

2014-11-01

A new kind of cancer cell separation method is demonstrated, using surface-enhanced Raman scattering (SERS) and fluorescence dual-encoded magnetic nanoprobes. The designed nanoprobes can realize SERS-fluorescence joint spectral encoding (SFJSE) and greatly improve the multiplexing ability. The nanoprobes have four main components, that is, the magnetic core, SERS generator, fluorescent agent, and targeting antibody. These components are assembled with a multi-layered structure to form the nanoprobes. Specifically, silica-coated magnetic nanobeads (MBs) are used as the inner core. Au core-Ag shell nanorods (Au@Ag NRs) are employed as the SERS generators and attached on the silica-coated MBs. After burying these Au@Ag NRs with another silica layer, CdTe quantum dots (QDs), that is, the fluorescent agent, are anchored onto the silica layer. Finally, antibodies are covalently linked to CdTe QDs. SFJSE is fulfilled by using different Raman molecules and QDs with different emission wavelengths. By utilizing four human cancer cell lines and one normal cell line as the model cells, the nanoprobes can specifically and simultaneously separate target cancer cells from the normal ones. This SFJSE-based method greatly facilitates the multiplex, rapid, and accurate cancer cell separation, and has a prosperous potential in high-throughput analysis and cancer diagnosis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR

PubMed Central

Schrell, Adrian M.; Roper, Michael G.

2014-01-01

A frequency-modulated fluorescence encoding method was used as a means to increase the number of fluorophores monitored during infrared-mediated polymerase chain reaction. Laser lines at 488-nm and 561-nm were modulated at 73- and 137-Hz, respectively, exciting fluorescence from the dsDNA intercalating dye, EvaGreen, and the temperature insensitive dye, ROX. Emission was collected in a color-blind manner using a single photomultiplier tube for detection and demodulated by frequency analysis. The resulting frequency domain signal resolved the contribution from the two fluorophores as well as the background from the IR lamp. The detection method was successfully used to measure amplification of DNA samples containing 104 – 107 starting copies of template producing an amplification efficiency of 96%. The utility of this methodology was further demonstrated by simultaneous amplification of two genes from human genomic DNA using different color TaqMan probes. This method of multiplexing fluorescence detection with IR-qPCR is ideally suited as it allowed isolation of the signals of interest from the background in the frequency domain and is expected to further reduce the complexity of multiplexed microfluidic IR-qPCR instrumentation. PMID:24448431

RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

PubMed Central

Moffitt, Jeffrey R.; Zhuang, Xiaowei

2016-01-01

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

[Detection of large deletions in X linked Alport syndrome using competitive multiplex fluorescence polymerase chain reaction].

PubMed

Wang, F; Zhang, Y Q; Ding, J; Yu, L X

2017-10-18

To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome. Clinical diagnosis of X-linked Alport syndrome was based on either abnormal staining of type IV collagen α5 chain in the epidermal basement membrane alone or with abnormal staining of type IV collagen α5 chain in the glomerular basement membrane and Bowman's capsule/ultrastructural changes in the glomerular basement membrane typical of Alport syndrome. A total of 20 unrelated Chinese patients (13 males and 7 females) clinically diagnosed as X-linked Alport syndrome were included in the study. Their genotypes were unknown. Control subjects included a male patient with other renal disease and two patients who had large deletions in COL4A5 gene detected by multiplex ligation-dependent probe amplification. Genomic DNA was isolated from peripheral blood leukocytes in all the participants. Multiplex competitive fluorescence polymerase chain reaction was used to coamplify 53 exons of COL4A5 gene and four reference genes in a single reaction. When a deletion removed exon 1 of COL4A5 gene was identified, the same method was used to coamplify the first 4 exons of COL4A5 and COL4A6 genes, a promoter shared by COL4A5 and COL4A6 genes, and three reference genes in a single reaction. Any copy number loss suggested by this method was verified by electrophoresis of corresponding polymerase chain reaction amplified products or DNA sequencing to exclude possible DNA variations in the primer regions. Genotypes of two positive controls identified by multiplex competitive fluorescence polymerase chain reaction were consistent with those detected by multiplex ligation-dependent probe amplification. Deletions were identified in 6 of the 20 patients, including two large deletions removing the 5' part of both COL4A5 and COL4A6 genes with the breakpoint located in the second intron of COL4A6, two large deletions

Multiplexed phase-space imaging for 3D fluorescence microscopy.

PubMed

Liu, Hsiou-Yuan; Zhong, Jingshan; Waller, Laura

2017-06-26

Optical phase-space functions describe spatial and angular information simultaneously; examples of optical phase-space functions include light fields in ray optics and Wigner functions in wave optics. Measurement of phase-space enables digital refocusing, aberration removal and 3D reconstruction. High-resolution capture of 4D phase-space datasets is, however, challenging. Previous scanning approaches are slow, light inefficient and do not achieve diffraction-limited resolution. Here, we propose a multiplexed method that solves these problems. We use a spatial light modulator (SLM) in the pupil plane of a microscope in order to sequentially pattern multiplexed coded apertures while capturing images in real space. Then, we reconstruct the 3D fluorescence distribution of our sample by solving an inverse problem via regularized least squares with a proximal accelerated gradient descent solver. We experimentally reconstruct a 101 Megavoxel 3D volume (1010×510×500µm with NA 0.4), demonstrating improved acquisition time, light throughput and resolution compared to scanning aperture methods. Our flexible patterning scheme further allows sparsity in the sample to be exploited for reduced data capture.

Frequency division multiplexed multi-color fluorescence microscope system

NASA Astrophysics Data System (ADS)

Le, Vu Nam; Yang, Huai Dong; Zhang, Si Chun; Zhang, Xin Rong; Jin, Guo Fan

2017-10-01

Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video's frame

Enhanced speed in fluorescence imaging using beat frequency multiplexing

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

2016-03-01

Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

Multicolor fluorescent biosensor for multiplexed detection of DNA.

PubMed

Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Huan, Shuang-Yan; Wu, Cuichen; Fu, Ting; Tan, Weihong

2014-05-20

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

TiO2 Nanolayer-Enhanced Fluorescence for Simultaneous Multiplex Mycotoxin Detection by Aptamer Microarrays on a Porous Silicon Surface.

PubMed

Liu, Rui; Li, Wei; Cai, Tingting; Deng, Yang; Ding, Zhi; Liu, Yan; Zhu, Xuerui; Wang, Xin; Liu, Jie; Liang, Baowen; Zheng, Tiesong; Li, Jianlin

2018-05-02

A new aptamer microarray method on the TiO 2 -porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO 2 nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO 2 -PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B 1 (AFB 1 ), and 0.001-10 ng/mL for fumonisin B 1 (FB 1 ) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB 1 , and FB 1 , respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma.

PubMed

Tang, Dong-ling; Li, Yan; Zhou, Xin; Li, Xia; Zheng, Fang

2009-05-01

To develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. The DNA template was extracted from 47 pregnant women and the whole blood samples from the stated biological fathers were used to detect genotype. Using multiplex fluorescent PCR at 16 different polymorphic short tandem repeat (STR) loci, maternal DNA extracted from plasma samples at early pregnancy, medium pregnancy and late pregnancy were used to detect genotype. Their husbands' DNA was also used for fetal genotype ascertainment. Multiplex fluorescent PCR with 16 polymorphic short tandem repeats revealed the presence of fetal DNA in all cases. Every pregnant women/husband pair was informative in at least 3 of 16 loci. The chances of detecting paternally inherited fetal alleles ranged from 66.67 to 94.12%. They are 66.67% in early pregnancy, 85.71% in medium pregnancy and 94.12% in late pregnancy. The accuracy of Multiplex PCR assay to detect fetal DNA was 100%. Circulating fetal DNA analysis can be used as a possible alternative tool in routine laboratory prenatal diagnosis in the near future; this highly polymorphic STR multiplex has greatly improved the chances of detecting paternally inherited fetal alleles compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma. Our proposed technique can be applied to both female and male fetuses, which provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

The Statistical Value of Raw Fluorescence Signal in Luminex xMAP Based Multiplex Immunoassays

PubMed Central

Breen, Edmond J.; Tan, Woei; Khan, Alamgir

2016-01-01

Tissue samples (plasma, saliva, serum or urine) from 169 patients classified as either normal or having one of seven possible diseases are analysed across three 96-well plates for the presences of 37 analytes using cytokine inflammation multiplexed immunoassay panels. Censoring for concentration data caused problems for analysis of the low abundant analytes. Using fluorescence analysis over concentration based analysis allowed analysis of these low abundant analytes. Mixed-effects analysis on the resulting fluorescence and concentration responses reveals a combination of censoring and mapping the fluorescence responses to concentration values, through a 5PL curve, changed observed analyte concentrations. Simulation verifies this, by showing a dependence on the mean florescence response and its distribution on the observed analyte concentration levels. Differences from normality, in the fluorescence responses, can lead to differences in concentration estimates and unreliable probabilities for treatment effects. It is seen that when fluorescence responses are normally distributed, probabilities of treatment effects for fluorescence based t-tests has greater statistical power than the same probabilities from concentration based t-tests. We add evidence that the fluorescence response, unlike concentration values, doesn’t require censoring and we show with respect to differential analysis on the fluorescence responses that background correction is not required. PMID:27243383

Optimization and Standardization of Fluorescent Cell Barcoding for Multiplexed Flow Cytometric Phenotyping

PubMed Central

Giudice, Valentina; Feng, Xingmin; Kajigaya, Sachiko; Young, Neal S.; Biancotto, Angélique

2017-01-01

Fluorescent cell barcoding (FCB) is a cell-based multiplexing technique for high-throughput flow cytometry. Barcoded samples can be stained and acquired collectively, minimizing staining variability and antibody consumption, and decreasing required sample volumes. Combined with functional measurements, FCB can be used for drug screening, signaling profiling, and cytokine detection, but technical issues are present. We optimized the FCB technique for routine utilization using DyLight 350, DyLight 800, Pacific Orange, and CBD500 for barcoding six, nine, or 36 human peripheral blood specimens. Working concentrations of FCB dyes ranging from 0 to 500 μg/ml were tested, and viability dye staining was optimized to increase robustness of data. A five-color staining with surface markers for Vβ usage analysis in CD4+ and CD8+ T cells was achieved in combination with nine sample barcoding. We provide improvements of the FCB technique that should be useful for multiplex drug screening and for lymphocyte characterization and perturbations in the diagnosis and during the course of disease. PMID:28692789

Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

PubMed

Beloglazova, Natalia V; Sobolev, Aleksander M; Tessier, Mickael D; Hens, Zeger; Goryacheva, Irina Yu; De Saeger, Sarah

2017-03-01

A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO 2 ). Then we applied the QD@SiO 2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO 2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500μgkg -1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of short tandem repeat polymorphisms using infrared fluorescence with M18 tailed primers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oetting, W.S.; Wiesner, G.; Laken, S.

The use of short tandem repeat polymorphisms (STRPs) are becoming increasingly important as markers for linkage analysis due to their large numbers of the human genome and their high degree of polymorphism. Fluorescence based detection of the STRP pattern using the LI-COR model 4000S automated DNA sequencer eliminates the need for radioactivity and produces a digitized image that can be used for the analysis of the polymorphisms. In an effort to reduce the cost of STRP analysis, we have synthesized primers with a 19 bp extension complementary to the sequence of the M13 primer on the 5{prime} end of onemore » of the two primers used in the amplification of the STRP instead of using primers with direct conjugation of the infrared fluorescent dye. Up to 5 primer pairs can be multiplexed together with the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye. Comparisons between primers that have been directly conjugated to the fluor with those having the M13 sequence extension show no difference in the ability to determine the STRP pattern. At present, the entire Weber 4A set of STRP markers is available with the M13 5{prime} extension. We are currently using this technique for linkage analysis of familial breast cancer and asthma. The combination of STRP analysis using fluorescence detection will allow this technique to be fully automated for allele scoring and linkage analysis.« less

A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.

PubMed

Tewson, Paul H; Quinn, Anne Marie; Hughes, Thomas E

2013-08-01

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca(2+)). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca(2+) sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

Multiplexed detection of tumor markers with multicolor quantum dots based on fluorescence polarization immunoassay.

PubMed

Tian, Jianniao; Zhou, Liujin; Zhao, Yanchun; Wang, Yuan; Peng, Yan; Zhao, Shulin

2012-04-15

A multicolor quantum dot (QD)-based nanosensor for multiplex detection of two tumor markers in a homogeneous format based on fluorescence polarization immunoassay was proposed. QDs520 and QDs620 were labeled alpha-fetoprotein(α-AFP) and carcinoembryonic antigen (CEA), respectively. After separated and purified by ultrafiltration, they were used in fluorescence polarization immunoassay for the simultaneous detection of human serum alpha-fetoprotein and carcinoembryonic antigen. Under the optimal conditions, the multi-analyte immunosensor had a wide linear range (from 0.5 ng mL(-1) to 500 ng mL(-1)) for both two tumor markers and good correlation (0.996 for α-AFP and 0.993 for CEA). The detection limits (LOD) were 0.36 ng mL(-1) for CEA and 0.28 ng mL(-1) for α-AFP (S/N=3). The carcinoembryonic antigen and fetoprotein in clinical serum samples were simultaneously detected. The results from 28 serum samples had a good agreement with enzyme-linked immunosorbent assay (ELISA). The relative standard deviation and the recovery suggested that the precision and the accuracy of this analytical method were satisfactory. This strategy with high sensitivity, good specificity, easy procedures and short analysis time shows great promise for clinical diagnoses and basic discovery. The application of QDs with longer fluorescence lifetime and small fluorescence polarization can be used for the determination of high molecular-weight substances which cannot be analyzed using dye fluorescence polarization immunoassay. Copyright © 2012 Elsevier B.V. All rights reserved.

Optofluidic wavelength division multiplexing for single-virus detection

PubMed Central

Ozcelik, Damla; Parks, Joshua W.; Wall, Thomas A.; Stott, Matthew A.; Cai, Hong; Parks, Joseph W.; Hawkins, Aaron R.; Schmidt, Holger

2015-01-01

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context––the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine. PMID:26438840

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

PubMed

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein Multiplexed Immunoassay Analysis with R.

PubMed

Breen, Edmond J

2017-01-01

Plasma samples from 177 control and type 2 diabetes patients collected at three Australian hospitals are screened for 14 analytes using six custom-made multiplex kits across 60 96-well plates. In total 354 samples were collected from the patients, representing one baseline and one end point sample from each patient. R methods and source code for analyzing the analyte fluorescence response obtained from these samples by Luminex Bio-Plex ® xMap multiplexed immunoassay technology are disclosed. Techniques and R procedures for reading Bio-Plex ® result files for statistical analysis and data visualization are also presented. The need for technical replicates and the number of technical replicates are addressed as well as plate layout design strategies. Multinomial regression is used to determine plate to sample covariate balance. Methods for matching clinical covariate information to Bio-Plex ® results and vice versa are given. As well as methods for measuring and inspecting the quality of the fluorescence responses are presented. Both fixed and mixed-effect approaches for immunoassay statistical differential analysis are presented and discussed. A random effect approach to outlier analysis and detection is also shown. The bioinformatics R methodology present here provides a foundation for rigorous and reproducible analysis of the fluorescence response obtained from multiplexed immunoassays.

Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

NASA Astrophysics Data System (ADS)

Shikha, Swati; Zheng, Xiang; Zhang, Yong

2018-06-01

Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser induced fluorescence detection

PubMed Central

Nikcevic, Irena; Piruska, Aigars; Wehmeyer, Kenneth R.; Seliskar, Carl J.; Limbach, Patrick A.; Heineman, William R.

2010-01-01

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser induced fluorescence detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be analyzed on parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pKa determination of small molecule analytes is demonstrated with the multilane microchip. PMID:20737446

Irish study of high-density Schizophrenia families: Field methods and power to detect linkage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kendler, K.S.; Straub, R.E.; MacLean, C.J.

Large samples of multiplex pedigrees will probably be needed to detect susceptibility loci for schizophrenia by linkage analysis. Standardized ascertainment of such pedigrees from culturally and ethnically homogeneous populations may improve the probability of detection and replication of linkage. The Irish Study of High-Density Schizophrenia Families (ISHDSF) was formed from standardized ascertainment of multiplex schizophrenia families in 39 psychiatric facilities covering over 90% of the population in Ireland and Northern Ireland. We here describe a phenotypic sample and a subset thereof, the linkage sample. Individuals were included in the phenotypic sample if adequate diagnostic information, based on personal interview and/ormore » hospital record, was available. Only individuals with available DNA were included in the linkage sample. Inclusion of a pedigree into the phenotypic sample required at least two first, second, or third degree relatives with non-affective psychosis (NAP), one of whom had schizophrenia (S) or poor-outcome schizoaffective disorder (PO-SAD). Entry into the linkage sample required DNA samples on at least two individuals with NAP, of whom at least one had S or PO-SAD. Affection was defined by narrow, intermediate, and broad criteria. 75 refs., 6 tabs.« less

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

PubMed Central

Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

2007-01-01

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform.

PubMed

Cheow, Lih Feng; Viswanathan, Ramya; Chin, Chee-Sing; Jennifer, Nancy; Jones, Robert C; Guccione, Ernesto; Quake, Stephen R; Burkholder, William F

2014-10-07

Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.

Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis.

PubMed

Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Maleewong, Wanchai

2009-01-01

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections.

PubMed

Wegner, Kyle A; Keikhosravi, Adib; Eliceiri, Kevin W; Vezina, Chad M

2017-08-01

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

2013-07-25

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples.

PubMed

Sanchis, Ana; Salvador, J-Pablo; Campbell, Katrina; Elliott, Christopher T; Shelver, Weilin L; Li, Qing X; Marco, M-Pilar

2018-07-01

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal or polyclonal antibodies raised against important families of chemical pollutants such as triazine biocide (i.e. Irgarol 1051®), sulfonamide and chloramphenicol antibiotics, polybrominated diphenyl ether flame-retardant (PBDE, i.e. BDE-47), hormone (17β-estradiol), and algae toxin (domoic acid). These contaminants were selected as model analytes, however, the platform developed has the potential to detect a broader group of compounds based on the cross-reactivity of the immunoreagents used. The microarray chip is able to simultaneously determine these families of contaminants directly in seawater samples reaching limits of detection close to the levels found in contaminated areas (Irgarol 1051®, 0.19 ± 0,06 µg L -1 ; sulfapyridine, 0.17 ± 0.07 µg L -1 ; chloramphenicol, 0.11 ± 0.03 µg L -1 ; BDE-47, 2.71 ± 1.13 µg L -1 ; 17β-estradiol, 0.94 ± 0.30 µg L -1 and domoic acid, 1.71 ± 0.30 µg L -1 ). Performance of the multiplexed microarray chip was assessed by measuring 38 blind spiked seawater samples containing either one of these contaminants or mixtures of them. The accuracy found was very good and the coefficient of variation was < 20% in all the cases. No sample pre-treatment was necessary, and the results could be obtained in just 1 h 30 min. The microarray shows high sample throughput capabilities, being able to measure simultaneously more than 68 samples and screen them for a significant number of chemical contaminants of interest in environmental screening programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

Hyperspectral fluorescence imaging with multi wavelength LED excitation

NASA Astrophysics Data System (ADS)

Luthman, A. Siri; Dumitru, Sebastian; Quirós-Gonzalez, Isabel; Bohndiek, Sarah E.

2016-04-01

Hyperspectral imaging (HSI) can combine morphological and molecular information, yielding potential for real-time and high throughput multiplexed fluorescent contrast agent imaging. Multiplexed readout from targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. There remains, however, a need for compact and cost effective implementations of the technology. We have implemented a low-cost wide-field multiplexed fluorescence imaging system, which combines LED excitation at 590, 655 and 740 nm with a compact commercial solid state HSI system operating in the range 600 - 1000 nm. A key challenge for using reflectance-based HSI is the separation of contrast agent fluorescence from the reflectance of the excitation light. Here, we illustrate how it is possible to address this challenge in software, using two offline reflectance removal methods, prior to least-squares spectral unmixing. We made a quantitative comparison of the methods using data acquired from dilutions of contrast agents prepared in well-plates. We then established the capability of our HSI system for non-invasive in vivo fluorescence imaging in small animals using the optimal reflectance removal method. The HSI presented here enables quantitative unmixing of at least four fluorescent contrast agents (Alexa Fluor 610, 647, 700 and 750) simultaneously in living mice. A successful unmixing of the four fluorescent contrast agents was possible both using the pure contrast agents and with mixtures. The system could in principle also be applied to imaging of ex vivo tissue or intraoperative imaging in a clinical setting. These data suggest a promising approach for developing clinical applications of HSI based on multiplexed fluorescence contrast agent imaging.

Development and potential applications of microarrays based on fluorescent nanocrystal-encoded beads for multiplexed cancer diagnostics

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Grinevich, Regina; Efimov, Anton E.; Nabiev, Igor; Sukhanova, Alyona

2014-05-01

Advanced multiplexed assays have recently become an indispensable tool for clinical diagnostics. These techniques provide simultaneous quantitative determination of multiple biomolecules in a single sample quickly and accurately. The development of multiplex suspension arrays is currently of particular interest for clinical applications. Optical encoding of microparticles is the most available and easy-to-use technique. This technology uses fluorophores incorporated into microbeads to obtain individual optical codes. Fluorophore-encoded beads can be rapidly analyzed using classical flow cytometry or microfluidic techniques. We have developed a new generation of highly sensitive and specific diagnostic systems for detection of cancer antigens in human serum samples based on microbeads encoded with fluorescent quantum dots (QDs). The designed suspension microarray system was validated for quantitative detection of (1) free and total prostate specific antigen (PSA) in the serum of patients with prostate cancer and (2) carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) in the serum of patients with breast cancer. The serum samples from healthy donors were used as a control. The antigen detection is based on the formation of an immune complex of a specific capture antibody (Ab), a target antigen (Ag), and a detector Ab on the surface of the encoded particles. The capture Ab is bound to the polymer shell of microbeads via an adapter molecule, for example, protein A. Protein A binds a monoclonal Ab in a highly oriented manner due to specific interaction with the Fc-region of the Ab molecule. Each antigen can be recognized and detected due to a specific microbead population carrying the unique fluorescent code. 100 and 231 serum samples from patients with different stages of prostate cancer and breast cancer, respectively, and those from healthy donors were examined using the designed suspension system. The data were validated by comparing with the results of

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

PubMed

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

2017-04-15

Solid-phase PCR (SP-PCR) has become increasingly popular for molecular diagnosis and there have been a few attempts to incorporate SP-PCR into lab-on-a-chip (LOC) devices. However, their applicability for on-line diagnosis is hindered by the lack of sensitive and portable on-chip optical detection technology. In this paper, we addressed this challenge by combining the SP-PCR with super critical angle fluorescence (SAF) microlens array embedded in a microchip. We fabricated miniaturized SAF microlens array as part of a microfluidic chamber in thermoplastic material and performed multiplexed SP-PCR directly on top of the SAF microlens array. Attribute to the high fluorescence collection efficiency of the SAF microlens array, the SP-PCR assay on the LOC platform demonstrated a high sensitivity of 1.6 copies/µL, comparable to off-chip detection using conventional laser scanner. The combination of SP-PCR and SAF microlens array allows for on-chip highly sensitive and multiplexed pathogen detection with low-cost and compact optical components. The LOC platform would be widely used as a high-throughput biosensor to analyze food, clinical and environmental samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Graphene-based aptamer logic gates and their application to multiplex detection.

PubMed

Wang, Li; Zhu, Jinbo; Han, Lei; Jin, Lihua; Zhu, Chengzhou; Wang, Erkang; Dong, Shaojun

2012-08-28

In this work, a GO/aptamer system was constructed to create multiplex logic operations and enable sensing of multiplex targets. 6-Carboxyfluorescein (FAM)-labeled adenosine triphosphate binding aptamer (ABA) and FAM-labeled thrombin binding aptamer (TBA) were first adsorbed onto graphene oxide (GO) to form a GO/aptamer complex, leading to the quenching of the fluorescence of FAM. We demonstrated that the unique GO/aptamer interaction and the specific aptamer-target recognition in the target/GO/aptamer system were programmable and could be utilized to regulate the fluorescence of FAM via OR and INHIBIT logic gates. The fluorescence changed according to different input combinations, and the integration of OR and INHIBIT logic gates provided an interesting approach for logic sensing applications where multiple target molecules were present. High-throughput fluorescence imagings that enabled the simultaneous processing of many samples by using the combinatorial logic gates were realized. The developed logic gates may find applications in further development of DNA circuits and advanced sensors for the identification of multiple targets in complex chemical environments.

Linkage and related analyses of Barrett's esophagus and its associated adenocarcinomas.

PubMed

Sun, Xiangqing; Elston, Robert; Falk, Gary W; Grady, William M; Faulx, Ashley; Mittal, Sumeet K; Canto, Marcia I; Shaheen, Nicholas J; Wang, Jean S; Iyer, Prasad G; Abrams, Julian A; Willis, Joseph E; Guda, Kishore; Markowitz, Sanford; Barnholtz-Sloan, Jill S; Chandar, Apoorva; Brock, Wendy; Chak, Amitabh

2016-07-01

Familial aggregation and segregation analysis studies have provided evidence of a genetic basis for esophageal adenocarcinoma (EAC) and its premalignant precursor, Barrett's esophagus (BE). We aim to demonstrate the utility of linkage analysis to identify the genomic regions that might contain the genetic variants that predispose individuals to this complex trait (BE and EAC). We genotyped 144 individuals in 42 multiplex pedigrees chosen from 1000 singly ascertained BE/EAC pedigrees, and performed both model-based and model-free linkage analyses, using S.A.G.E. and other software. Segregation models were fitted, from the data on both the 42 pedigrees and the 1000 pedigrees, to determine parameters for performing model-based linkage analysis. Model-based and model-free linkage analyses were conducted in two sets of pedigrees: the 42 pedigrees and a subset of 18 pedigrees with female affected members that are expected to be more genetically homogeneous. Genome-wide associations were also tested in these families. Linkage analyses on the 42 pedigrees identified several regions consistently suggestive of linkage by different linkage analysis methods on chromosomes 2q31, 12q23, and 4p14. A linkage on 15q26 is the only consistent linkage region identified in the 18 female-affected pedigrees, in which the linkage signal is higher than in the 42 pedigrees. Other tentative linkage signals are also reported. Our linkage study of BE/EAC pedigrees identified linkage regions on chromosomes 2, 4, 12, and 15, with some reported associations located within our linkage peaks. Our linkage results can help prioritize association tests to delineate the genetic determinants underlying susceptibility to BE and EAC.

Genome-wide linkage meta-analysis identifies susceptibility loci at 2q34 and 13q31.3 for genetic generalized epilepsies.

PubMed

Leu, Costin; de Kovel, Carolien G F; Zara, Federico; Striano, Pasquale; Pezzella, Marianna; Robbiano, Angela; Bianchi, Amedeo; Bisulli, Francesca; Coppola, Antonietta; Giallonardo, Anna Teresa; Beccaria, Francesca; Trenité, Dorothée Kasteleijn-Nolst; Lindhout, Dick; Gaus, Verena; Schmitz, Bettina; Janz, Dieter; Weber, Yvonne G; Becker, Felicitas; Lerche, Holger; Kleefuss-Lie, Ailing A; Hallman, Kerstin; Kunz, Wolfram S; Elger, Christian E; Muhle, Hiltrud; Stephani, Ulrich; Møller, Rikke S; Hjalgrim, Helle; Mullen, Saul; Scheffer, Ingrid E; Berkovic, Samuel F; Everett, Kate V; Gardiner, Mark R; Marini, Carla; Guerrini, Renzo; Lehesjoki, Anna-Elina; Siren, Auli; Nabbout, Rima; Baulac, Stephanie; Leguern, Eric; Serratosa, Jose M; Rosenow, Felix; Feucht, Martha; Unterberger, Iris; Covanis, Athanasios; Suls, Arvid; Weckhuysen, Sarah; Kaneva, Radka; Caglayan, Hande; Turkdogan, Dilsad; Baykan, Betul; Bebek, Nerses; Ozbek, Ugur; Hempelmann, Anne; Schulz, Herbert; Rüschendorf, Franz; Trucks, Holger; Nürnberg, Peter; Avanzini, Giuliano; Koeleman, Bobby P C; Sander, Thomas

2012-02-01

Genetic generalized epilepsies (GGEs) have a lifetime prevalence of 0.3% with heritability estimates of 80%. A considerable proportion of families with siblings affected by GGEs presumably display an oligogenic inheritance. The present genome-wide linkage meta-analysis aimed to map: (1) susceptibility loci shared by a broad spectrum of GGEs, and (2) seizure type-related genetic factors preferentially predisposing to either typical absence or myoclonic seizures, respectively. Meta-analysis of three genome-wide linkage datasets was carried out in 379 GGE-multiplex families of European ancestry including 982 relatives with GGEs. To dissect out seizure type-related susceptibility genes, two family subgroups were stratified comprising 235 families with predominantly genetic absence epilepsies (GAEs) and 118 families with an aggregation of juvenile myoclonic epilepsy (JME). To map shared and seizure type-related susceptibility loci, both nonparametric loci (NPL) and parametric linkage analyses were performed for a broad trait model (GGEs) in the entire set of GGE-multiplex families and a narrow trait model (typical absence or myoclonic seizures) in the subgroups of JME and GAE families. For the entire set of 379 GGE-multiplex families, linkage analysis revealed six loci achieving suggestive evidence for linkage at 1p36.22, 3p14.2, 5q34, 13q12.12, 13q31.3, and 19q13.42. The linkage finding at 5q34 was consistently supported by both NPL and parametric linkage results across all three family groups. A genome-wide significant nonparametric logarithm of odds score of 3.43 was obtained at 2q34 in 118 JME families. Significant parametric linkage to 13q31.3 was found in 235 GAE families assuming recessive inheritance (heterogeneity logarithm of odds = 5.02). Our linkage results support an oligogenic predisposition of familial GGE syndromes. The genetic risk factor at 5q34 confers risk to a broad spectrum of familial GGE syndromes, whereas susceptibility loci at 2q34 and 13q31

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

1998-04-21

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

1996-12-10

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Li, Q.; Lu, X.

1998-04-21

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

1996-12-10

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

PubMed Central

Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

2015-01-01

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

PubMed

Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

2016-06-22

In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

Multi-Channel Hyperspectral Fluorescence Detection Excited by Coupled Plasmon-Waveguide Resonance

PubMed Central

Du, Chan; Liu, Le; Zhang, Lin; Guo, Jun; Guo, Jihua; Ma, Hui; He, Yonghong

2013-01-01

We propose in this paper a biosensor scheme based on coupled plasmon-waveguide resonance (CPWR) excited fluorescence spectroscopy. A symmetrical structure that offers higher surface electric field strengths, longer surface propagation lengths and depths is developed to support guided waveguide modes for the efficient excitation of fluorescence. The optimal parameters for the sensor films are theoretically and experimentally investigated, leading to a detection limit of 0.1 nM (for a Cy5 solution). Multiplex analysis possible with the fluorescence detection is further advanced by employing the hyperspectral fluorescence technique to record the full spectra for every pixel on the sample plane. We demonstrate experimentally that highly overlapping fluorescence (Cy5 and Dylight680) can be distinguished and ratios of different emission sources can be determined accurately. This biosensor shows great potential for multiplex detections of fluorescence analytes. PMID:24129023

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

PubMed Central

Bray, Mark-Anthony; Singh, Shantanu; Han, Han; Davis, Chadwick T.; Borgeson, Blake; Hartland, Cathy; Kost-Alimova, Maria; Gustafsdottir, Sigrun M.; Gibson, Christopher C.; Carpenter, Anne E.

2016-01-01

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks. PMID:27560178

Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection

PubMed Central

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2008-01-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive and multiplex format, an alternative surface enhanced Raman scattering (SERS) based probe was designed and fabricated to covalently attach both DNA probing sequence and non-fluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the non-fluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA (ssDNA) to its complementary targets was successfully accomplished with a long-term goal to use non-fluorescent RTags in a Raman-based DNA microarray platform. PMID:17465531

3D multiplexed immunoplasmonics microscopy

NASA Astrophysics Data System (ADS)

Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

2016-07-01

Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

Multiplexing a high-throughput liability assay to leverage efficiencies.

PubMed

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Fluorescent Quantification of DNA Based on Core-Shell Fe3O4@SiO2@Au Nanocomposites and Multiplex Ligation-Dependent Probe Amplification.

PubMed

Fan, Jing; Yang, Haowen; Liu, Ming; Wu, Dan; Jiang, Hongrong; Zeng, Xin; Elingarami, Sauli; Ll, Zhiyang; Li, Song; Liu, Hongna; He, Nongyue

2015-02-01

In this research, a novel method for relative fluorescent quantification of DNA based on Fe3O4@SiO2@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation- dependent probe amplification (MLPA) has been developed. With the help of self-assembly, seed-mediated growth and chemical reduction method, core-shell Fe3O4@SiO2@Au GMNPs were synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which can capture the biotinylated probes. Then we designed MLPA probes which were tagged with biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed our method can be used for the relative quantitative analysis of the target DNA in the concentration range of 03004~0.5 µM.

Multiplex fluorescent immunoassay device based on magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Godjevargova, T. I.; Ivanov, Y. L.; Dinev, D. D.

2017-02-01

Immunofluorescent analyzer based compact disc for simultaneous detection of 3 antibiotics in the same milk sample is consisting of two parts: CD-based immunofluorescence kit and optoelectronic fluorometer. Kit consists of 2 parts: Lyophilized immobilized antibodies on supermagnetic nanoparticles in Eppendorf tubes and CD-based microfluidic disk, in which are formed five chamber systems for simultaneous detecting of 5 separate samples. Each system consists of 2 chambers connected by a special micro channel acting as a hydrophobic valve. In the first chamber lyophilised conjugates of 3 antibiotics with accordingly 3 different fluorescent dyes are placed. The second chamber is for detection of fluorescent signal. The optoelectronic fluorometer is comprising of: integrated thermostatic block; mechanical-detecting unit (fluorometer) and block with controlling and visualizing electronics.The disc gets into a second block of the analyzer, where centrifugation is performed and also reporting of the fluorescent signals. This unit comprises a rotor on which the disc is fixed, permanent electromagnet in the form of a ring inserted under the disc and module of 3 LED diodes with emission filters for the relevant wavelengths corresponding to the used fluorescent dyes and 1 integrated photodiode, in front of which is mounted filter with 3 spectral peaks.The signal from the photodiode is detected by the electronic unit which is sensitive "lock-in" amplifier, the engine rotor management, control of thermostatic device and management of periphery of the analyzer, consisting of display and communications with computer.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

PubMed

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

PubMed

Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

2015-07-07

Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Exploiting fluorescence for multiplex immunoassays on protein microarrays

NASA Astrophysics Data System (ADS)

Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

2014-09-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Linkage and association studies identify a novel locus for Alzheimer disease at 7q36 in a Dutch population-based sample.

PubMed

Rademakers, Rosa; Cruts, Marc; Sleegers, Kristel; Dermaut, Bart; Theuns, Jessie; Aulchenko, Yurii; Weckx, Stefan; De Pooter, Tim; Van den Broeck, Marleen; Corsmit, Ellen; De Rijk, Peter; Del-Favero, Jurgen; van Swieten, John; van Duijn, Cornelia M; Van Broeckhoven, Christine

2005-10-01

We obtained conclusive linkage of Alzheimer disease (AD) with a candidate region of 19.7 cM at 7q36 in an extended multiplex family, family 1270, ascertained in a population-based study of early-onset AD in the northern Netherlands. Single-nucleotide polymorphism and haplotype association analyses of a Dutch patient-control sample further supported the linkage at 7q36. In addition, we identified a shared haplotype at 7q36 between family 1270 and three of six multiplex AD-affected families from the same geographical region, which is indicative of a founder effect and defines a priority region of 9.3 cM. Mutation analysis of coding exons of 29 candidate genes identified one linked synonymous mutation, g.38030G-->C in exon 10, that affected codon 626 of the PAX transactivation domain interacting protein gene (PAXIP1). It remains to be determined whether PAXIP1 has a functional role in the expression of AD in family 1270 or whether another mutation at this locus explains the observed linkage and sharing. Together, our linkage data from the informative family 1270 and the association data in the population-based early-onset AD patient-control sample strongly support the identification of a novel AD locus at 7q36 and re-emphasize the genetic heterogeneity of AD.

Fluorescent metallacycle-cored polymers via covalent linkage and their use as contrast agents for cell imaging.

PubMed

Zhang, Mingming; Li, Shuya; Yan, Xuzhou; Zhou, Zhixuan; Saha, Manik Lal; Wang, Yu-Cai; Stang, Peter J

2016-10-04

The covalent linkage of supramolecular monomers provides a powerful strategy for constructing dynamic polymeric materials whose properties can be readily tuned either by the selection of monomers or the choice of functional linkers. In this strategy, the stabilities of the supramolecular monomers and the reactions used to link the monomers are crucial because such monomers are normally dynamic and can disassemble during the linking process, leading to mixture of products. Therefore, although noncovalent interactions have been widely introduced into metallacycle structures to prepare metallosupramolecular polymers, metallacycle-cored polymers linked by covalent bonds have been rarely reported. Herein, we used the mild, highly efficient amidation reaction between alkylamine and N-hydroxysuccinimide-activated carboxylic acid to link the pendent amino functional groups of a rhomboidal metallacycle 10 to give metallacycle-cored polymers P1 and P2, which further yielded nanoparticles at low concentration and transformed into network structures as the concentration increased. Moreover, these polymers exhibited enhanced emission and showed better quantum yields than metallacycle 10 in methanol and methanol/water (1/9, vol/vol) due to the aggregation-induced emission properties of a tetraphenylethene-based pyridyl donor, which serves as a precursor for metallacycle 10. The fluorescence properties of these polymers were further used in cell imaging, and they showed a significant enrichment in lung cells after i.v. injection. Considering the anticancer activity of rhomboidal Pt(II) metallacycles, this type of fluorescent metallacycle-cored polymers can have potential applications toward lung cancer treatment.

Novel Multiplex Fluorescent PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of β-Thalassemia.

PubMed

Khosravi, Sharifeh; Salehi, Mansour; Ramezanzadeh, Mahboobeh; Mirzaei, Hamed; Salehi, Rasoul

2016-05-01

Thalassemia is curable by bone marrow transplantation; however, finding suitable donors with defined HLA combination remains a major challenge. Cord blood stem cells with preselected HLA system through preimplantation genetic diagnosis (PGD) proved very useful for resolving scarce HLA-matched bone marrow donors. A thalassemia trait couple with an affected child was included in this study. We used informative STR markers at the HLA and beta globin loci to develop a single cell multiplex fluorescent PCR protocol. The protocol was extensively optimized on single lymphocytes isolated from the couple's peripheral blood. The optimized protocol was applied on single blastomeres biopsied from day 3 cleavage stage IVF embryos of the couple. Four IVF embryos biopsied on day 3 and a single blastomere of each were provided for genetic diagnosis of combined β-thalassemia mutations and HLA typing. Of these, one embryo was diagnosed as homozygous normal for the thalassemia mutation and HLA matched with the existing affected sibling. The optimized protocol worked well in PGD clinical cycle for selection of thalassemia-unaffected embryos with the desired HLA system. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

Parallel-multiplexed excitation light-sheet microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Laser scanning light-sheet imaging allows fast 3D image of live samples with minimal bleach and photo-toxicity. Existing light-sheet techniques have very limited capability in multi-label imaging. Hyper-spectral imaging is needed to unmix commonly used fluorescent proteins with large spectral overlaps. However, the challenge is how to perform hyper-spectral imaging without sacrificing the image speed, so that dynamic and complex events can be captured live. We report wavelength-encoded structured illumination light sheet imaging (λ-SIM light-sheet), a novel light-sheet technique that is capable of parallel multiplexing in multiple excitation-emission spectral channels. λ-SIM light-sheet captures images of all possible excitation-emission channels in true parallel. It does not require compromising the imaging speed and is capable of distinguish labels by both excitation and emission spectral properties, which facilitates unmixing fluorescent labels with overlapping spectral peaks and will allow more labels being used together. We build a hyper-spectral light-sheet microscope that combined λ-SIM with an extended field of view through Bessel beam illumination. The system has a 250-micron-wide field of view and confocal level resolution. The microscope, equipped with multiple laser lines and an unlimited number of spectral channels, can potentially image up to 6 commonly used fluorescent proteins from blue to red. Results from in vivo imaging of live zebrafish embryos expressing various genetic markers and sensors will be shown. Hyper-spectral images from λ-SIM light-sheet will allow multiplexed and dynamic functional imaging in live tissue and animals.

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

PubMed

Berger, Sanne Schou; Lauritsen, Klara Tølbøll; Boas, Ulrik; Lind, Peter; Andresen, Lars Ole

2017-11-01

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

Functionalization of optical nanotip arrays with an electrochemical microcantilever for multiplexed DNA detection.

PubMed

Descamps, Emeline; Duroure, Nathalie; Deiss, Frédérique; Leichlé, Thierry; Adam, Catherine; Mailley, Pascal; Aït-Ikhlef, Ali; Livache, Thierry; Nicu, Liviu; Sojic, Neso

2013-08-07

Optical nanotip arrays fabricated on etched fiber bundles were functionalized with DNA spots. Such unconventional substrates (3D and non-planar) are difficult to pattern with standard microfabrication techniques but, using an electrochemical cantilever, up to 400 spots were electrodeposited on the nanostructured optical surface in 5 min. This approach allows each spot to be addressed individually and multiplexed fluorescence detection is demonstrated. Finally, remote fluorescence detection was performed by imaging through the optical fiber bundle itself after hybridisation with the complementary sequence.

Genomewide scan for gout in taiwanese aborigines reveals linkage to chromosome 4q25.

PubMed

Cheng, Li Shu-Chuan; Chiang, Shang-Lun; Tu, Hung-Pin; Chang, Shun-Jen; Wang, Tsu-Nai; Ko, Allen Min-Jen; Chakraborty, Ranajit; Ko, Ying-Chin

2004-09-01

Gout is a disorder of uric-acid metabolism. The Pacific Austronesian population, including Taiwanese aborigines, has a remarkably high prevalence of hyperuricemia and gout, which suggests a founder effect across the Pacific region. We report here a genomewide linkage study of 21 multiplex pedigrees with gout from an aboriginal tribe in Taiwan. From observations of familial clustering, early onset of gout, and clinically severe manifestations, we hypothesized that a major gene plays a role in this trait. Using 382 random polymorphic markers spread across 22 autosomes, we demonstrated a highly significant linkage for gout at marker D4S2623 on chromosome 4q25 (P=.0002 by nonparametric linkage [the NPL(all) statistic]; empirical P=.0006; LOD=4.3, P=4.4x10-6 by logistic regression). When alcohol consumption was included as a covariate in the model, the LOD score increased to 5.66 (P=1.3x10-6). Quantitative traits, including serum uric acid and creatinine, also showed a moderate linkage to this region. To our knowledge, this is the first genome-scan report to identify a genetic locus harboring a gout-susceptibility gene.

Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

PubMed

Dong, Juyao; Salem, Daniel P; Sun, Jessica H; Strano, Michael S

2018-04-24

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm 2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.

Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk.

PubMed

Chen, Yiqiang; Chen, Qian; Han, Miaomiao; Liu, Jiangyang; Zhao, Peng; He, Lidong; Zhang, Yuan; Niu, Yiming; Yang, Wenjun; Zhang, Liying

2016-05-15

In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk. Copyright © 2015 Elsevier B.V. All rights reserved.

An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

PubMed

Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin

2014-08-15

Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiplexed aberration measurement for deep tissue imaging in vivo

PubMed Central

Wang, Chen; Liu, Rui; Milkie, Daniel E.; Sun, Wenzhi; Tan, Zhongchao; Kerlin, Aaron; Chen, Tsai-Wen; Kim, Douglas S.; Ji, Na

2014-01-01

We describe a multiplexed aberration measurement method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine their phase gradients. Applicable to fluorescent-protein-labeled structures of arbitrary complexity, it allows us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improves structural and functional imaging of fine neuronal processes over a large imaging volume. PMID:25128976

Dual-excitation upconverting nanoparticle and quantum dot aptasensor for multiplexed food pathogen detection.

PubMed

Kurt, Hasan; Yüce, Meral; Hussain, Babar; Budak, Hikmet

2016-07-15

In this report, a dual-excitation sensing method was developed using aptamer-functionalized quantum dots and upconverting nanoparticles, exhibiting Stokes and anti-Stokes type excitation profiles, respectively. Conjugation of the aptamer-functionalized luminescent nanoparticles with the magnetic beads, comprising short DNA sequences that were partially complementary to the aptamer sequences, enabled facile separation of the analyte-free conjugates for fluorescent measurement. UV-Visible spectroscopy, Circular Dichroism spectroscopy, Dynamic Light Scattering and Polyacrylamide Gel Electrophoresis techniques were used to characterize the aptamer probes developed. The target-specific luminescent conjugates were applied for multiplex detection of model food pathogens, Salmonella typhimurium, and Staphylococcus aureus, in which the fluorescent emission spectra were obtained under UV excitation at 325nm for quantum dots and NIR excitation at 980nm for upconverting nanoparticles, respectively. The dual-excitation strategy was aimed to minimize cross-talk between the luminescent signals for multiplexed detection, and yielded limit of detection values of 16 and 28cfumL(-1) for Staphylococcus aureus, and Salmonella typhimurium, respectively. By employing a greater number of quantum dots and upconverting nanoparticles with non-overlapping fluorescent emissions, the proposed methodology might be exploited further to detect several analytes, simultaneously. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescence-based ion-sensing with colloidal particles.

PubMed

Ashraf, Sumaira; Carrillo-Carrion, Carolina; Zhang, Qian; Soliman, Mahmoud G; Hartmann, Raimo; Pelaz, Beatriz; Del Pino, Pablo; Parak, Wolfgang J

2014-10-01

Particle-based fluorescence sensors for the quantification of specific ions can be made by coupling ion-sensitive fluorophores to carrier particles, or by using intrinsically fluorescent particles whose fluorescence properties depend on the concentration of the ions. Despite the advantages of such particle-based sensors for the quantitative detection of ions, such as the possibility to tune the surface chemistry and thus entry portal of the sensor particles to cells, they have also some associated problems. Problems involve for example crosstalk of the ion-sensitive fluorescence read-out with pH, or spectral overlap of the emission spectra of different fluorescent particles in multiplexing formats. Here the benefits of using particle-based fluorescence sensors, their limitations and strategies to overcome these limitations will be described and exemplified with selected examples. Copyright © 2014 Elsevier Ltd. All rights reserved.

A universal multi-wavelength fluorescence polarization immunoassay for multiplexed detection of mycotoxins in maize.

PubMed

Li, Chenglong; Wen, Kai; Mi, Tiejun; Zhang, Xiya; Zhang, Huiyan; Zhang, Suxia; Shen, Jianzhong; Wang, Zhanhui

2016-05-15

Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0 μg kg(-1) for DON, 17.8 μg kg(-1) for T-2 toxin and 331.5 μg kg(-1) for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5-106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC-MS/MS, with correlation coefficients (R(2)) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel multiplexed fluorescence polarisation immunoassay based on a recombinant bi-specific single-chain diabody for simultaneous detection of fluoroquinolones and sulfonamides in milk.

PubMed

Chen, Min; Wen, Kai; Tao, Xiaoqi; Ding, Shuangyang; Xie, Jie; Yu, Xuezhi; Li, Jiancheng; Xia, Xi; Wang, Yang; Xie, Sanlei; Jiang, Haiyang

2014-01-01

Major research efforts are focusing on the development of simultaneous multiplexed immunoassays. In this study, a novel dual-binding fluorescence polarisation immunoassay (DB-FPIA) using a broad-specificity bi-specific single-chain diabody (scDb) and two fluorescent-labelled tracers (sulfamethoxypyridazine-fluorescein isothiocyanate (SMP-FITC) and sarafloxacin-Texas Red (SAR-TR)) with different excitation and emission wavelengths was developed for simultaneous and high-throughput detection of 19 fluoroquinolones (FQs) and 13 sulfonamides (SAs) at the maximum residue limits in milk samples. Recoveries for spiked milk samples were from 76.4% to 128.4%, with a relative standard deviation lower than 13.9%. The developed DB-FPIA was then applied to field samples, followed by confirmation by LC-MS/MS. All three instances in which FQs and SAs were present at concentrations near or above the assay limit of detection were identified as positive by the developed DB-FPIA, demonstrating that the method is suitable for rapid screening of FQs and SAs contamination. The novel methodology combines the advantage of the FPIA and the broad sensitivity of scDb and shows great promise for fast multi-analyte screening of low-molecular weight chemical residues in food samples.

A Genetic Linkage Map of the Male Goat Genome

PubMed Central

Vaiman, D.; Schibler, L.; Bourgeois, F.; Oustry, A.; Amigues, Y.; Cribiu, E. P.

1996-01-01

This paper presents a first genetic linkage map of the goat genome. Primers derived from the flanking sequences of 612 bovine, ovine and goat microsatellite markers were gathered and tested for amplification with goat DNA under standardized PCR conditions. This screen made it possible to choose a set of 55 polymorphic markers that can be used in the three species and to define a panel of 223 microsatellites suitable for the goat. Twelve half-sib paternal goat families were then used to build a linkage map of the goat genome. The linkage analysis made it possible to construct a meiotic map covering 2300 cM, i.e., >80% of the total estimated length of the goat genome. Moreover, eight cosmids containing microsatellites were mapped by fluorescence in situ hybridization in goat and sheep. Together with 11 microsatellite-containing cosmids previously mapped in cattle (and supposing conservation of the banding pattern between this species and the goat) and data from the sheep map, these results made the orientation of 15 linkage groups possible. Furthermore, 12 coding sequences were mapped either genetically or physically, providing useful data for comparative mapping. PMID:8878693

Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Peng, Leilei

2016-03-01

Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.

Capillaries for use in a multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.

1997-12-09

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Capillaries for use in a multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Chang, H.T.; Fung, E.N.

1997-12-09

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

PubMed

Alcaine, S D; Tilton, L; Serrano, M A C; Wang, M; Vachet, R W; Nugen, S R

2015-10-01

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

PubMed

Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

2018-01-01

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

A Novel Multiplex HRM Assay to Detect Clopidogrel Resistance.

PubMed

Zhang, Lichen; Ma, Xiaowei; You, Guoling; Zhang, Xiaoqing; Fu, Qihua

2017-11-22

Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5' end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

PubMed

Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

2012-08-01

Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

Equivalence of time-multiplexed and frequency-multiplexed signals in digital communications.

NASA Technical Reports Server (NTRS)

Timor, U.

1972-01-01

In comparing different techniques for multiplexing N binary data signals into a single channel, time-division multiplexing (TDM) is known to have a theoretic efficiency of 100 percent (neglecting sync power) and thus seems to outperform frequency-division multiplexing systems (FDM). By considering more general FDM systems, we will show that both TDM and FDM are equivalent and have an efficiency of 100 percent. The difference between the systems is in the multiplexing and demultiplexing subsystems, but not in the performance or in the generated waveforms.

Toward photostable multiplex analyte detection on a single mode planar optical waveguide

NASA Astrophysics Data System (ADS)

Mukundan, Harshini; Xie, Hongzhi; Anderson, Aaron; Grace, W. Kevin; Martinez, Jennifer S.; Swanson, Basil

2009-02-01

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

Self-calibrating multiplexer circuit

DOEpatents

Wahl, Chris P.

1997-01-01

A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

Multiplex PageRank.

PubMed

Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

2013-01-01

Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

Combined genome-wide linkage and targeted association analysis of head circumference in autism spectrum disorder families.

PubMed

Woodbury-Smith, M; Bilder, D A; Morgan, J; Jerominski, L; Darlington, T; Dyer, T; Paterson, A D; Coon, H

2017-01-01

It has long been recognized that there is an association between enlarged head circumference (HC) and autism spectrum disorder (ASD), but the genetics of HC in ASD is not well understood. In order to investigate the genetic underpinning of HC in ASD, we undertook a genome-wide linkage study of HC followed by linkage signal targeted association among a sample of 67 extended pedigrees with ASD. HC measurements on members of 67 multiplex ASD extended pedigrees were used as a quantitative trait in a genome-wide linkage analysis. The Illumina 6K SNP linkage panel was used, and analyses were carried out using the SOLAR implemented variance components model. Loci identified in this way formed the target for subsequent association analysis using the Illumina OmniExpress chip and imputed genotypes. A modification of the qTDT was used as implemented in SOLAR. We identified a linkage signal spanning 6p21.31 to 6p22.2 (maximum LOD = 3.4). Although targeted association did not find evidence of association with any SNP overall, in one family with the strongest evidence of linkage, there was evidence for association (rs17586672, p  = 1.72E-07). Although this region does not overlap with ASD linkage signals in these same samples, it has been associated with other psychiatric risk, including ADHD, developmental dyslexia, schizophrenia, specific language impairment, and juvenile bipolar disorder. The genome-wide significant linkage signal represents the first reported observation of a potential quantitative trait locus for HC in ASD and may be relevant in the context of complex multivariate risk likely leading to ASD.

High resolution multiplexed functional imaging in live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

Ultrafast FADC multiplexer

NASA Astrophysics Data System (ADS)

Mirzoyan, R.; Cortina, J.; Lorenz, E.; Martinez, M.; Ostankov, A.; Paneque, D.

2002-10-01

Ultrafast Flash amplitude-to-digital converters (FADCs) are still very expensive. Here we propose a multiplexing scheme allowing one in common trigger mode to read out multiple signal sources by using a single FADC channel. Usual coaxial cables can be used in the multiplexer as analog signal delay elements. The limited bandwidth of the coaxial cable, depending on its type and length will set an upper limit to the number of multiplexed channels. Better bandwidth and the correspondingly higher number of multiplexed channels one can obtain when using the technique of transmission of analog signals via optical fibers. Low-cost vertical cavity surface emitting laser (VCSEL) diodes can be used as converters of fast electrical signals into near infrared light. Multiplexing can be an economically priced solution when one needs ultrafast digitization of hundreds of fast signal channels.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

A GENOME-WIDE LINKAGE AND ASSOCIATION SCAN REVEALS NOVEL LOCI FOR AUTISM

PubMed Central

Weiss, Lauren A.; Arking, Dan E.

2009-01-01

Summary Although autism is a highly heritable neurodevelopmental disorder, attempts to identify specific susceptibility genes have thus far met with limited success 1. Genome-wide association studies (GWAS) using half a million or more markers, particularly those with very large sample sizes achieved through meta-analysis, have shown great success in mapping genes for other complex genetic traits (http://www.genome.gov/26525384). Consequently, we initiated a linkage and association mapping study using half a million genome-wide SNPs in a common set of 1,031 multiplex autism families (1,553 affected offspring). We identified regions of suggestive and significant linkage on chromosomes 6q27 and 20p13, respectively. Initial analysis did not yield genome-wide significant associations; however, genotyping of top hits in additional families revealed a SNP on chromosome 5p15 (between SEMA5A and TAS2R1) that was significantly associated with autism (P = 2 × 10−7). We also demonstrated that expression of SEMA5A is reduced in brains from autistic patients, further implicating SEMA5A as an autism susceptibility gene. The linkage regions reported here provide targets for rare variation screening while the discovery of a single novel association demonstrates the action of common variants. PMID:19812673

Multiplexity and multireciprocity in directed multiplexes.

PubMed

Gemmetto, Valerio; Squartini, Tiziano; Picciolo, Francesco; Ruzzenenti, Franco; Garlaschelli, Diego

2016-10-01

Real-world multilayer networks feature nontrivial dependencies among links of different layers. Here we argue that if links are directed, then dependencies are twofold. Besides the ordinary tendency of links of different layers to align as the result of "multiplexity," there is also a tendency to antialign as a result of what we call "multireciprocity," i.e., the fact that links in one layer can be reciprocated by opposite links in a different layer. Multireciprocity generalizes the scalar definition of single-layer reciprocity to that of a square matrix involving all pairs of layers. We introduce multiplexity and multireciprocity matrices for both binary and weighted multiplexes and validate their statistical significance against maximum-entropy null models that filter out the effects of node heterogeneity. We then perform a detailed empirical analysis of the world trade multiplex (WTM), representing the import-export relationships between world countries in different commodities. We show that the WTM exhibits strong multiplexity and multireciprocity, an effect which is, however, largely encoded into the degree or strength sequences of individual layers. The residual effects are still significant and allow us to classify pairs of commodities according to their tendency to be traded together in the same direction and/or in opposite ones. We also find that the multireciprocity of the WTM is significantly lower than the usual reciprocity measured on the aggregate network. Moreover, layers with low (high) internal reciprocity are embedded within sets of layers with comparably low (high) mutual multireciprocity. This suggests that, in the WTM, reciprocity is inherent to groups of related commodities rather than to individual commodities. We discuss the implications for international trade research focusing on product taxonomies, the product space, and fitness and complexity metrics.

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

NASA Astrophysics Data System (ADS)

Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

2016-05-01

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

Polymeric nanoparticles with sequential and multiple FRET cascade mechanisms for multicolor and multiplexed imaging.

PubMed

Wagh, Anil; Jyoti, Faidat; Mallik, Sanku; Qian, Steven; Leclerc, Estelle; Law, Benedict

2013-06-24

The ability to map multiple biomarkers at the same time has far-reaching biomedical and diagnostic applications. Here, a series of biocompatible poly(D,L-lactic-co-glycolic acid) and polyethylene glycol particles for multicolor and multiplexed imaging are reported. More than 30 particle formulations that exhibit distinct emission signatures (ranging from the visible to NIR wavelength region) are designed and synthesized. These particles are encapsulated with combinations of carbocyanine-based fluorophores DiO, Dil, DiD, and DiR, and are characterized as <100 nm in size and brighter than commercial quantum dots. A particle formulation is identified that simultaneously emits fluorescence at three different wavelengths upon a single excitation at 485 nm via sequential and multiple FRET cascade events for multicolor imaging. Three other particles that display maximum fluorescence intensities at 570, 672, or 777 nm for multiplexed imaging are also identified. These particles are individually conjugated with specific (Herceptin or IgG2A11 antibody) or nonspecific (heptaarginine) ligands for targeting and, thus, could be applied to differentiate different cancer cells from a cell mixture according to the expressions of cell-surface human epidermal growth factor receptor 2 and the receptor for advanced glycation endproducts. Using an animal model subcutaneously implanted with the particles, it is further demonstrated that the developed platform could be useful for in vivo multiplexed imaging. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

PubMed

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

On-chip wavelength multiplexed detection of cancer DNA biomarkers in blood

PubMed Central

Cai, H.; Stott, M. A.; Ozcelik, D.; Parks, J. W.; Hawkins, A. R.; Schmidt, H.

2016-01-01

We have developed an optofluidic analysis system that processes biomolecular samples starting from whole blood and then analyzes and identifies multiple targets on a silicon-based molecular detection platform. We demonstrate blood filtration, sample extraction, target enrichment, and fluorescent labeling using programmable microfluidic circuits. We detect and identify multiple targets using a spectral multiplexing technique based on wavelength-dependent multi-spot excitation on an antiresonant reflecting optical waveguide chip. Specifically, we extract two types of melanoma biomarkers, mutated cell-free nucleic acids —BRAFV600E and NRAS, from whole blood. We detect and identify these two targets simultaneously using the spectral multiplexing approach with up to a 96% success rate. These results point the way toward a full front-to-back chip-based optofluidic compact system for high-performance analysis of complex biological samples. PMID:28058082

A Spread-Spectrum SQUID Multiplexer

NASA Astrophysics Data System (ADS)

Irwin, K. D.; Chaudhuri, S.; Cho, H.-M.; Dawson, C.; Kuenstner, S.; Li, D.; Titus, C. J.; Young, B. A.

2018-06-01

The transition-edge sensor (TES) is a mature, high-resolution x-ray spectrometer technology that provides a much higher efficiency than dispersive spectrometers such as gratings and crystal spectrometers. As larger arrays are developed, time-division multiplexing schemes operating at MHz frequencies are being replaced by microwave SQUID multiplexers using frequency-division multiplexing at GHz frequencies. However, the multiplexing factor achievable with microwave SQUIDs is limited by the high slew rate on the leading edge of x-ray pulses. In this paper, we propose a new multiplexing scheme for high-slew-rate TES x-ray calorimeters: the spread-spectrum SQUID multiplexer, which has the potential to enable higher multiplexing factors, especially in applications with lower photon-arrival rates.

Sequential strategy to identify a susceptibility gene for schizophrenia: Report of potential linkage on chromosome 22q12-q13.1: Part 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pulver, A.E.; Wolyniec, P.S.; Lasseter, V.K.

To identify genes responsible for the susceptibility for schizophrenia, and to test the hypothesis that schizophrenia is etiologically heterogeneous, we have studied 39 multiplex families from a systematic sample of schizophrenic patients. Using a complex autosomal dominant model, which considers only those with a diagnosis of schizophrenia or schizoaffective disorder as affected, a random search of the genome for detection of linkage was undertaken. Pairwise linkage analyses suggest a potential linkage (LRH = 34.7 or maximum lod score = 1.54) for one region (22q12-q13.1). Reanalyses, varying parameters in the dominant model, maximized the LRH at 660.7 (maximum lod score 2.82).more » This finding is of sufficient interest to warrant further investigation through collaborative studies. 72 refs., 5 tabs.« less

Efficiency of semi-automated fluorescent multiplex PCRs with 11 microsatellite markers for genetic studies of deer populations.

PubMed

Bonnet, A; Thévenon, S; Maudet, F; Maillard, J C

2002-10-01

Thirty bovine and eight ovine microsatellite primer pairs were tested on four tropical deer species: Eld's and Swamp deer (highly threatened) and Rusa and Vietnamese Sika deer (economically important). Thirty markers gave an amplified product in all four species (78.9%). The number of polymorphic microsatellite markers varied among the species from 14 in Eld's deer (47%) to 20 in Swamp deer (67%). Among them, 11 microsatellite loci were multiplexed in three polymerase chain reactions (PCRs) and labelled with three different fluorochromes that can be loaded in one gel-lane. To test the efficiency of the multiplex, primary genetic studies (mean number of alleles, expected heterozygosities and Fis values) were carried out on four deer populations. Parentage exclusion probability and probability of identity were computed and discussed on a Swamp deer population. These multiplexes PCRs were also tested on several other deer species and subspecies. The aim of this study is to establish a tool useful for genetic studies of population structure and diversity in four tropical deer species which with few modifications can be applied to other species of the genus Cervus.

Generating multiplex gradients of biomolecules for controlling cellular adhesion in parallel microfluidic channels.

PubMed

Didar, Tohid Fatanat; Tabrizian, Maryam

2012-11-07

Here we present a microfluidic platform to generate multiplex gradients of biomolecules within parallel microfluidic channels, in which a range of multiplex concentration gradients with different profile shapes are simultaneously produced. Nonlinear polynomial gradients were also generated using this device. The gradient generation principle is based on implementing parrallel channels with each providing a different hydrodynamic resistance. The generated biomolecule gradients were then covalently functionalized onto the microchannel surfaces. Surface gradients along the channel width were a result of covalent attachments of biomolecules to the surface, which remained functional under high shear stresses (50 dyn/cm(2)). An IgG antibody conjugated to three different fluorescence dyes (FITC, Cy5 and Cy3) was used to demonstrate the resulting multiplex concentration gradients of biomolecules. The device enabled generation of gradients with up to three different biomolecules in each channel with varying concentration profiles. We were also able to produce 2-dimensional gradients in which biomolecules were distributed along the length and width of the channel. To demonstrate the applicability of the developed design, three different multiplex concentration gradients of REDV and KRSR peptides were patterned along the width of three parallel channels and adhesion of primary human umbilical vein endothelial cell (HUVEC) in each channel was subsequently investigated using a single chip.

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

PubMed

Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

2018-04-01

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Polarization Multiplexing of Fluorescent Emission Using Multiresonant Plasmonic Antennas.

PubMed

De Leo, Eva; Cocina, Ario; Tiwari, Preksha; Poulikakos, Lisa V; Marqués-Gallego, Patricia; le Feber, Boris; Norris, David J; Prins, Ferry

2017-12-26

Combining the ability to localize electromagnetic fields at the nanoscale with a directional response, plasmonic antennas offer an effective strategy to shape the far-field pattern of coupled emitters. Here, we introduce a family of directional multiresonant antennas that allows for polarization-resolved spectral identification of fluorescent emission. The geometry consists of a central aperture surrounded by concentric polygonal corrugations. By varying the periodicity of each axis of the polygon individually, this structure can support multiple resonances that provide independent control over emission directionality for multiple wavelengths. Moreover, since each resonant wavelength is directly mapped to a specific polarization orientation, spectral information can be encoded in the polarization state of the out-scattered beam. To demonstrate the potential of such structures in enabling simplified detection schemes and additional functionalities in sensing and imaging applications, we use the central subwavelength aperture as a built-in nanocuvette and manipulate the fluorescent response of colloidal-quantum-dot emitters coupled to the multiresonant antenna.

Polarization Multiplexing of Fluorescent Emission Using Multiresonant Plasmonic Antennas

PubMed Central

2017-01-01

Combining the ability to localize electromagnetic fields at the nanoscale with a directional response, plasmonic antennas offer an effective strategy to shape the far-field pattern of coupled emitters. Here, we introduce a family of directional multiresonant antennas that allows for polarization-resolved spectral identification of fluorescent emission. The geometry consists of a central aperture surrounded by concentric polygonal corrugations. By varying the periodicity of each axis of the polygon individually, this structure can support multiple resonances that provide independent control over emission directionality for multiple wavelengths. Moreover, since each resonant wavelength is directly mapped to a specific polarization orientation, spectral information can be encoded in the polarization state of the out-scattered beam. To demonstrate the potential of such structures in enabling simplified detection schemes and additional functionalities in sensing and imaging applications, we use the central subwavelength aperture as a built-in nanocuvette and manipulate the fluorescent response of colloidal-quantum-dot emitters coupled to the multiresonant antenna. PMID:29161502

Optofluidic devices for biomolecule sensing and multiplexing

NASA Astrophysics Data System (ADS)

Ozcelik, Damla

Optofluidics which integrates photonics and microfluidics, has led to highly compact, sensitive and adaptable biomedical sensors. Optofluidic biosensors based on liquid-core anti-resonant reflecting optical waveguides (LC-ARROWs), have proven to be a highly sensitive, portable, and reconfigurable platform for fluorescence spectroscopy and detection of single biomolecules such as proteins, nucleic acids, and virus particles. However, continued improvements in sensitivity remain a major goal as we approach the ultimate limit of detecting individual bio-particles labeled by single or few fluorophores. Additionally, the ability to simultaneously detect and identify multiple biological particles or biomarkers is one of the key requirements for molecular diagnostic tests. The compactness and adaptability of these platforms can further be advanced by introducing tunability, integrating off-chip components, designing reconfigurable and customizable devices, which makes these platforms very good candidates for many different applications. The goal of this thesis was to introduce new elements in these LC-ARROW optofluidics platforms that provide major enhancements in their functionality, making them more sensitive, compact, customizable and multiplexed. First, a novel integrated tunable spectral filter that achieves effective elimination of background noise on the ARROW platform was demonstrated. A unique dual liquid-core design enabled the independent multi-wavelength tuning of the spectral filter by adjusting the refractive index and chemical properties of the liquid. In order to enhance the detection sensitivity of the platform, Y-splitter waveguides were integrated to create multiple excitation spots for each target molecule. A powerful signal processing algorithm was used to analyze the data to improve the signal-to-noise ratio (SNR) of the collected data. Next, the design, optimization and characterization of the Y-splitter waveguides are presented; and single

Linkage Analysis in Autoimmune Addison's Disease: NFATC1 as a Potential Novel Susceptibility Locus.

PubMed

Mitchell, Anna L; Bøe Wolff, Anette; MacArthur, Katie; Weaver, Jolanta U; Vaidya, Bijay; Erichsen, Martina M; Darlay, Rebecca; Husebye, Eystein S; Cordell, Heather J; Pearce, Simon H S

2015-01-01

Autoimmune Addison's disease (AAD) is a rare, highly heritable autoimmune endocrinopathy. It is possible that there may be some highly penetrant variants which confer disease susceptibility that have yet to be discovered. DNA samples from 23 multiplex AAD pedigrees from the UK and Norway (50 cases, 67 controls) were genotyped on the Affymetrix SNP 6.0 array. Linkage analysis was performed using Merlin. EMMAX was used to carry out a genome-wide association analysis comparing the familial AAD cases to 2706 UK WTCCC controls. To explore some of the linkage findings further, a replication study was performed by genotyping 64 SNPs in two of the four linked regions (chromosomes 7 and 18), on the Sequenom iPlex platform in three European AAD case-control cohorts (1097 cases, 1117 controls). The data were analysed using a meta-analysis approach. In a parametric analysis, applying a rare dominant model, loci on chromosomes 7, 9 and 18 had LOD scores >2.8. In a non-parametric analysis, a locus corresponding to the HLA region on chromosome 6, known to be associated with AAD, had a LOD score >3.0. In the genome-wide association analysis, a SNP cluster on chromosome 2 and a pair of SNPs on chromosome 6 were associated with AAD (P <5x10-7). A meta-analysis of the replication study data demonstrated that three chromosome 18 SNPs were associated with AAD, including a non-synonymous variant in the NFATC1 gene. This linkage study has implicated a number of novel chromosomal regions in the pathogenesis of AAD in multiplex AAD families and adds further support to the role of HLA in AAD. The genome-wide association analysis has also identified a region of interest on chromosome 2. A replication study has demonstrated that the NFATC1 gene is worthy of future investigation, however each of the regions identified require further, systematic analysis.

Multiplexed quantitation of protein expression and phosphorylation based on functionalized soluble nanopolymers

PubMed Central

Pan, Li; Iliuk, Anton; Yu, Shuai; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

We report here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. The soluble nanopolymer, pIMAGO, is functionalized with Ti (IV) ions for chelating phosphoproteins in high specificity, and with infrared fluorescent tags for direct, multiplexed assays. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. The new strategy has a great potential to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and discovering new signaling events. PMID:23088311

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

PubMed Central

Xu, Hongxia; Sha, Michael Y.; Wong, Edith Y.; Uphoff, Janet; Xu, Yanzhang; Treadway, Joseph A.; Truong, Anh; O’Brien, Eamonn; Asquith, Steven; Stubbins, Michael; Spurr, Nigel K.; Lai, Eric H.; Mahoney, Walt

2003-01-01

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications. PMID:12682378

Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

PubMed

Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

2017-01-01

Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

Evaluation of the impact of genetic linkage in forensic identity and relationship testing for expanded DNA marker sets.

PubMed

Tillmar, Andreas O; Phillips, Chris

2017-01-01

Advances in massively parallel sequencing technology have enabled the combination of a much-expanded number of DNA markers (notably STRs and SNPs in one or combined multiplexes), with the aim of increasing the weight of evidence in forensic casework. However, when data from multiple loci on the same chromosome are used, genetic linkage can affect the final likelihood calculation. In order to study the effect of linkage for different sets of markers we developed the biostatistical tool ILIR, (Impact of Linkage on forensic markers for Identity and Relationship tests). The ILIR tool can be used to study the overall impact of genetic linkage for an arbitrary set of markers used in forensic testing. Application of ILIR can be useful during marker selection and design of new marker panels, as well as being highly relevant for existing marker sets as a way to properly evaluate the effects of linkage on a case-by-case basis. ILIR, implemented via the open source platform R, includes variation and genomic position reference data for over 40 STRs and 140 SNPs, combined with the ability to include additional forensic markers of interest. The use of the software is demonstrated with examples from several different established marker sets (such as the expanded CODIS core loci) including a review of the interpretation of linked genetic data. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Probabilistic record linkage

PubMed Central

Sayers, Adrian; Ben-Shlomo, Yoav; Blom, Ashley W; Steele, Fiona

2016-01-01

Abstract Studies involving the use of probabilistic record linkage are becoming increasingly common. However, the methods underpinning probabilistic record linkage are not widely taught or understood, and therefore these studies can appear to be a ‘black box’ research tool. In this article, we aim to describe the process of probabilistic record linkage through a simple exemplar. We first introduce the concept of deterministic linkage and contrast this with probabilistic linkage. We illustrate each step of the process using a simple exemplar and describe the data structure required to perform a probabilistic linkage. We describe the process of calculating and interpreting matched weights and how to convert matched weights into posterior probabilities of a match using Bayes theorem. We conclude this article with a brief discussion of some of the computational demands of record linkage, how you might assess the quality of your linkage algorithm, and how epidemiologists can maximize the value of their record-linked research using robust record linkage methods. PMID:26686842

Extracting information from multiplex networks

NASA Astrophysics Data System (ADS)

Iacovacci, Jacopo; Bianconi, Ginestra

2016-06-01

Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ ˜ S for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

PubMed

Zhong, Qun; Bhattacharya, Smiti; Kotsopoulos, Steven; Olson, Jeff; Taly, Valérie; Griffiths, Andrew D; Link, Darren R; Larson, Jonathan W

2011-07-07

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented. This journal is © The Royal Society of Chemistry 2011

Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes.

PubMed

Schuler, Friedrich; Trotter, Martin; Zengerle, Roland; von Stetten, Felix

2016-03-01

Multiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2-plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays.

Improving record linkage performance in the presence of missing linkage data.

PubMed

Ong, Toan C; Mannino, Michael V; Schilling, Lisa M; Kahn, Michael G

2014-12-01

Existing record linkage methods do not handle missing linking field values in an efficient and effective manner. The objective of this study is to investigate three novel methods for improving the accuracy and efficiency of record linkage when record linkage fields have missing values. By extending the Fellegi-Sunter scoring implementations available in the open-source Fine-grained Record Linkage (FRIL) software system we developed three novel methods to solve the missing data problem in record linkage, which we refer to as: Weight Redistribution, Distance Imputation, and Linkage Expansion. Weight Redistribution removes fields with missing data from the set of quasi-identifiers and redistributes the weight from the missing attribute based on relative proportions across the remaining available linkage fields. Distance Imputation imputes the distance between the missing data fields rather than imputing the missing data value. Linkage Expansion adds previously considered non-linkage fields to the linkage field set to compensate for the missing information in a linkage field. We tested the linkage methods using simulated data sets with varying field value corruption rates. The methods developed had sensitivity ranging from .895 to .992 and positive predictive values (PPV) ranging from .865 to 1 in data sets with low corruption rates. Increased corruption rates lead to decreased sensitivity for all methods. These new record linkage algorithms show promise in terms of accuracy and efficiency and may be valuable for combining large data sets at the patient level to support biomedical and clinical research. Copyright © 2014 Elsevier Inc. All rights reserved.

Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Holst-Jensen, Arne; Žel, Jana

2015-08-18

Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

PubMed Central

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-01-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets. PMID:27739510

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

NASA Astrophysics Data System (ADS)

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.

PubMed

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-14

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors.

PubMed

Bereza-Malcolm, Lara; Aracic, Sanja; Kannan, Ruban; Mann, Gülay; Franks, Ashley E

2017-08-15

Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10µgml -1 , as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20-40min following exposure to 3µgml -1 cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera. Copyright © 2017 Elsevier B.V. All rights reserved.

Sequencing of oligosaccharides using enzyme array digestion with electrochemical and fluorescent detections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, M.; Lee, C.S.

1997-12-31

The objective of this study is to develop a rapid and sensitive method for oligosaccharide sequencing. The oligosaccharides are subjected to the enzyme array digestion with exoglycosidases of known and well-defined specificities. The enzyme array method involves the division of oligosaccharide sample into aliquots, and the incubation of each aliquot with a precisely defined mixture of exoglycosidases. In the enzyme array method, the presence of a specific linkage anywhere in the oligosaccharide is determined by the inability of an enzyme mixture lacking a given enzyme to cleave that linkage ( a stop point) and the ability of the other enzymesmore » to cleave the linkage up to that point. The direct quantification of released monosaccharides from the enzyme array can be achieved by using pulsed amperometric detection (PAD) or by fluorescent derivatization with a fluorophoric agent. The measured monosaccharide concentrations in combination with the enzyme array analysis provide detail characterization of oligosaccharides with their sugar composition, configuration, and linkage information, The released monosaccharides are further quantified by anion exchange chromatography and capillary electrophoresis for the comparison with the results obtained from PAD and fluorescence measurements. Our enzyme array-electrochemical (or fluorescent) detection method does not require any separation procedure and any prior labeling of oligosaccharide and have several practical advantages over the current carbohydrate sequencing techniques including simplicity, speed, and the ability to use small amounts of starting material.« less

Metal Stable Isotope Tagging: Renaissance of Radioimmunoassay for Multiplex and Absolute Quantification of Biomolecules.

PubMed

Liu, Rui; Zhang, Shixi; Wei, Chao; Xing, Zhi; Zhang, Sichun; Zhang, Xinrong

2016-05-17

The unambiguous quantification of biomolecules is of great significance in fundamental biological research as well as practical clinical diagnosis. Due to the lack of a detectable moiety, the direct and highly sensitive quantification of biomolecules is often a "mission impossible". Consequently, tagging strategies to introduce detectable moieties for labeling target biomolecules were invented, which had a long and significant impact on studies of biomolecules in the past decades. For instance, immunoassays have been developed with radioisotope tagging by Yalow and Berson in the late 1950s. The later languishment of this technology can be almost exclusively ascribed to the use of radioactive isotopes, which led to the development of nonradioactive tagging strategy-based assays such as enzyme-linked immunosorbent assay, fluorescent immunoassay, and chemiluminescent and electrochemiluminescent immunoassay. Despite great success, these strategies suffered from drawbacks such as limited spectral window capacity for multiplex detection and inability to provide absolute quantification of biomolecules. After recalling the sequences of tagging strategies, an apparent question is why not use stable isotopes from the start? A reasonable explanation is the lack of reliable means for accurate and precise quantification of stable isotopes at that time. The situation has changed greatly at present, since several atomic mass spectrometric measures for metal stable isotopes have been developed. Among the newly developed techniques, inductively coupled plasma mass spectrometry is an ideal technique to determine metal stable isotope-tagged biomolecules, for its high sensitivity, wide dynamic linear range, and more importantly multiplex and absolute quantification ability. Since the first published report by our group, metal stable isotope tagging has become a revolutionary technique and gained great success in biomolecule quantification. An exciting research highlight in this area

Super-multiplex vibrational imaging

PubMed Central

Wei, Lu; Chen, Zhixing; Shi, Lixue; Long, Rong; Anzalone, Andrew V.; Zhang, Luyuan; Hu, Fanghao; Yuste, Rafael; Cornish, Virginia W.; Min, Wei

2017-01-01

The ability to directly visualize a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ, reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly1–4, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm−1) and featureless nature of fluorescence spectra5 that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis)6–8. Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width ~10 cm−1) and thus doesn’t suffer this problem, but its feeble signals make many demanding bio-imaging applications impossible. And while surface-enhanced Raman scattering offers remarkable sensitivity and multiplicity, it cannot be readily used to quantitatively image specific molecular targets inside live cells9. Here we show that carrying out stimulated Raman scattering under electronic pre-resonance conditions (epr-SRS) enables imaging with exquisite vibrational selectivity and sensitivity (down to 250 nM with 1-ms) in living cells. We also create a palette of triple-bond-conjugated near-infrared dyes that each display a single epr-SRS peak in the cell-silent spectral window, and that with available fluorescent probes give 24 resolvable colors with potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this super-multiplex optical imaging approach for untangling intricate

Super-multiplex vibrational imaging

NASA Astrophysics Data System (ADS)

Wei, Lu; Chen, Zhixing; Shi, Lixue; Long, Rong; Anzalone, Andrew V.; Zhang, Luyuan; Hu, Fanghao; Yuste, Rafael; Cornish, Virginia W.; Min, Wei

2017-04-01

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a ‘colour barrier’, owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the

Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

NASA Astrophysics Data System (ADS)

Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

2017-02-01

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

PubMed

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

Multiplexer and time duration measuring circuit

DOEpatents

Gray, Jr., James

1980-01-01

A multiplexer device is provided for multiplexing data in the form of randomly developed, variable width pulses from a plurality of pulse sources to a master storage. The device includes a first multiplexer unit which includes a plurality of input circuits each coupled to one of the pulse sources, with all input circuits being disabled when one input circuit receives an input pulse so that only one input pulse is multiplexed by the multiplexer unit at any one time.

Delay grid multiplexing: simple time-based multiplexing and readout method for silicon photomultipliers

NASA Astrophysics Data System (ADS)

Won, Jun Yeon; Ko, Guen Bae; Lee, Jae Sung

2016-10-01

In this paper, we propose a fully time-based multiplexing and readout method that uses the principle of the global positioning system. Time-based multiplexing allows simplifying the multiplexing circuits where the only innate traces that connect the signal pins of the silicon photomultiplier (SiPM) channels to the readout channels are used as the multiplexing circuit. Every SiPM channel is connected to the delay grid that consists of the traces on a printed circuit board, and the inherent transit times from each SiPM channel to the readout channels encode the position information uniquely. Thus, the position of each SiPM can be identified using the time difference of arrival (TDOA) measurements. The proposed multiplexing can also allow simplification of the readout circuit using the time-to-digital converter (TDC) implemented in a field-programmable gate array (FPGA), where the time-over-threshold (ToT) is used to extract the energy information after multiplexing. In order to verify the proposed multiplexing method, we built a positron emission tomography (PET) detector that consisted of an array of 4  ×  4 LGSO crystals, each with a dimension of 3  ×  3  ×  20 mm3, and one- to-one coupled SiPM channels. We first employed the waveform sampler as an initial study, and then replaced the waveform sampler with an FPGA-TDC to further simplify the readout circuits. The 16 crystals were clearly resolved using only the time information obtained from the four readout channels. The coincidence resolving times (CRTs) were 382 and 406 ps FWHM when using the waveform sampler and the FPGA-TDC, respectively. The proposed simple multiplexing and readout methods can be useful for time-of-flight (TOF) PET scanners.

Simultaneous detection of eight avian influenza A virus subtypes by multiplex reverse transcription-PCR using a GeXP analyser.

PubMed

Li, Meng; Xie, Zhixun; Xie, Zhiqin; Liu, Jiabo; Xie, Liji; Deng, Xianwen; Luo, Sisi; Fan, Qing; Huang, Li; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng

2018-04-18

Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.

Navigability of multiplex temporal network

NASA Astrophysics Data System (ADS)

Wang, Yan; Song, Qiao-Zhen

2017-01-01

Real world complex systems have multiple levels of relationships and in many cases, they need to be modeled as multiplex networks where the same nodes can interact with each other in different layers, such as social networks. However, social relationships only appear at prescribed times so the temporal structures of edge activations can also affect the dynamical processes located above them. To consider both factors are simultaneously, we introduce multiplex temporal networks and propose three different walk strategies to investigate the concurrent dynamics of random walks and the temporal structure of multiplex networks. Thus, we derive analytical results for the multiplex centrality and coverage function in multiplex temporal networks. By comparing them with the numerical results, we show how the underlying topology of the layers and the walk strategy affect the efficiency when exploring the networks. In particular, the most interesting result is the emergence of a super-diffusion process, where the time scale of the multiplex is faster than that of both layers acting separately.

Simultaneous two-dimensional laser-induced-fluorescence measurements of argon ions.

PubMed

Hansen, A K; Galante, Matthew; McCarren, Dustin; Sears, Stephanie; Scime, E E

2010-10-01

Recent laser upgrades on the Hot Helicon Experiment at West Virginia University have enabled multiplexed simultaneous measurements of the ion velocity distribution function at a single location, expanding our capabilities in laser-induced fluorescence diagnostics. The laser output is split into two beams, each modulated with an optical chopper and injected perpendicular and parallel to the magnetic field. Light from the crossing point of the beams is transported to a narrow-band photomultiplier tube filtered at the fluorescence wavelength and monitored by two lock-in amplifiers, each referenced to one of the two chopper frequencies.

Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

PubMed

Luo, Yuan; Gelsinger-Austin, Paul J; Watson, Jonathan M; Barbastathis, George; Barton, Jennifer K; Kostuk, Raymond K

2008-09-15

A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously.

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.).

PubMed

Lee-Montero, I; Navarro, A; Borrell, Y; García-Celdrán, M; Martín, N; Negrín-Báez, D; Blanco, G; Armero, E; Berbel, C; Zamorano, M J; Sánchez, J J; Estévez, A; Ramis, G; Manchado, M; Afonso, J M

2013-08-01

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Multiplexed DNA detection using spectrally encoded porous SiO2 photonic crystal particles.

PubMed

Meade, Shawn O; Chen, Michelle Y; Sailor, Michael J; Miskelly, Gordon M

2009-04-01

A particle-based multiplexed DNA assay based on encoded porous SiO(2) photonic crystal disks is demonstrated. A "spectral barcode" is generated by electrochemical etch of a single-crystal silicon wafer using a programmed current-time waveform. A lithographic procedure is used to isolate cylindrical microparticles 25 microm in diameter and 10 microm thick, which are then oxidized, modified with a silane linker, and conjugated to various amino-functionalized oligonucleotide probes via cyanuric chloride. It is shown that the particles can be decoded based on their reflectivity spectra and that a multiple analyte assay can be performed in a single sample with a modified fluorescence microscope. The homogeneity of the reflectivity and fluorescence spectra, both within and across the microparticles, is also reported.

Multiplex-Ready Technology for mid-throughput genotyping of molecular markers.

PubMed

Bonneau, Julien; Hayden, Matthew

2014-01-01

Screening molecular markers across large populations in breeding programs is generally time consuming and expensive. The Multiplex-Ready Technology (MRT) (Hayden et al., BMC genomics 9:80, 2008) was created to optimize polymorphism screening and genotyping using standardized PCR reaction conditions. The flexibility of this method maximizes the number of markers (up to 24 markers SSR or SNP, ideally small PCR product <500 bp and highly polymorphic) by using fluorescent dye (VIC, FAM, NED, and PET) and a semiautomated DNA fragment analyzer (ABI3730) capillary electrophoresis for large numbers of DNA samples (96 or 384 samples).

Linkage of the VNTR/insulin-gene and type I diabetes mellitus: Increased gene sharing in affected sibling pairs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Owerbach, D.; Gabbay, K.H.

1994-05-01

Ninety-six multiplex type I diabetic families were typed at the 5' flanking region of the insulin gene by using a PCR assay that better resolves the VNTR into multiple alleles. Affected sibling pairs shared 2, 1, and 0 VNTR alleles - identical by descent - at a frequency of .47, .45, and .08, respectively, a ratio that deviated from the expected 1:2:1 ratio (P<.001). These results confirm linkage of the chromosome 11p15.5 region with type I diabetes mellitus susceptibility. 20 refs., 2 tabs.

Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device

PubMed Central

Barbee, Kristopher D.; Hsiao, Alexander P.; Roller, Eric E.; Huang, Xiaohua

2011-01-01

We report the development of a microfabricated electrophoretic device for assembling high-density arrays of antibody-conjugated microbeads for chip-based protein detection. The device consists of a flow cell formed between a gold-coated silicon chip with an array of microwells etched in a silicon dioxide film and a glass coverslip with a series of thin gold counter electrode lines. We have demonstrated that 0.4 and 1 μm beads conjugated with antibodies can be rapidly assembled into the microwells by applying a pulsed electric field across the chamber. By assembling step-wise a mixture of fluorescently labeled antibody-conjugated microbeads, we incorporated both spatial and fluorescence encoding strategies to demonstrate significant multiplexing capabilities. We have shown that these antibody-conjugated microbead arrays can be used to perform on-chip sandwich immunoassays to detect test antigens at concentrations as low as 40 pM (6 ng/mL). A finite element model was also developed to examine the electric field distribution within the device for different counter electrode configurations over a range of line pitches and chamber heights. This device will be useful for assembling high-density, encoded antibody arrays for multiplexed detection of proteins and other types of protein-conjugated microbeads for applications such as the analysis of protein-protein interactions. PMID:20820631

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

PubMed

Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

2016-02-01

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification.

PubMed

Heo, Hyun Young; Chung, Soyi; Kim, Yong Tae; Kim, Do Hyun; Seo, Tae Seok

2016-04-15

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Genetic linkage analysis using pooled DNA and infrared detection of tailed STRP primer patterns

NASA Astrophysics Data System (ADS)

Oetting, William S.; Wildenberg, Scott C.; King, Richard A.

1996-04-01

The mapping of a disease locus to a specific chromosomal region is an important step in the eventual isolation and analysis of a disease causing gene. Conventional mapping methods analyze large multiplex families and/or smaller nuclear families to find linkage between the disease and a chromosome marker that maps to a known chromosomal region. This analysis is time consuming and tedious, typically requiring the determination of 30,000 genotypes or more. For appropriate populations, we have instead utilized pooled DNA samples for gene mapping which greatly reduces the amount of time necessary for an initial chromosomal screen. This technique assumes a common founder for the disease locus of interest and searches for a region of a chromosome shared between affected individuals. Our analysis involves the PCR amplification of short tandem repeat polymorphisms (STRP) to detect these shared regions. In order to reduce the cost of genotyping, we have designed unlabeled tailed PCR primers which, when combined with a labeled universal primer, provides for an alternative to synthesizing custom labeled primers. The STRP pattern is visualized with an infrared fluorescence based automated DNA sequencer and the patterns quantitated by densitometric analysis of the allele pattern. Differences in the distribution of alleles between pools of affected and unaffected individuals, including a reduction in the number of alleles in the affected pool, indicate the sharing of a region of a chromosome. We have found this method effective for markers 10 - 15 cM away from the disease locus for a recessive genetic disease.

Optimal percolation on multiplex networks.

PubMed

Osat, Saeed; Faqeeh, Ali; Radicchi, Filippo

2017-11-16

Optimal percolation is the problem of finding the minimal set of nodes whose removal from a network fragments the system into non-extensive disconnected clusters. The solution to this problem is important for strategies of immunization in disease spreading, and influence maximization in opinion dynamics. Optimal percolation has received considerable attention in the context of isolated networks. However, its generalization to multiplex networks has not yet been considered. Here we show that approximating the solution of the optimal percolation problem on a multiplex network with solutions valid for single-layer networks extracted from the multiplex may have serious consequences in the characterization of the true robustness of the system. We reach this conclusion by extending many of the methods for finding approximate solutions of the optimal percolation problem from single-layer to multiplex networks, and performing a systematic analysis on synthetic and real-world multiplex networks.

Development of an integrated endoscopic device for multiplexed low coherence interferometry measurements of microbicide gel coating thickness

NASA Astrophysics Data System (ADS)

Drake, Tyler K.; Robles, Francisco E.; DeSoto, Michael; Henderson, Marcus H.; Katz, David F.; Wax, Adam P.

2009-02-01

Microbicide gels are topical products that have recently been developed to combat sexually transmitted diseases including HIV/AIDS. The extent of gel coverage, thickness, and structure are crucial factors in gel effectiveness. It is necessary to be able to monitor gel distribution and behavior under various circumstances, such as coatis, and over an extended time scale in vivo. We have developed a multiplexed, Fourier-domain low coherence interferometry (LCI) system as a practical method of measuring microbicide gel distribution, with precision and accuracy comparable to currently used fluorometric techniques techniques. The multiplexed system achieved a broad scanning area without the need for a mechanical scanning device, typical of OCT systems, by utilizing six parallel channels with simultaneous data collection. We now propose an imaging module which will allow the integration of the multiplexed LCI system into the current fluorescence system in conjunction with an endoscope. The LCI imaging module will meet several key criteria in order to be compatible with the current system. The fluorescent system features a 4-mm diameter rigid endsoscope enclosed in a 27-mm diameter polycarbonate tube, with a water immersion tip. Therefore, the LCI module must be low-profile as well as water-resistant to fit inside the current design. It also must fulfill its primary function of delivering light from each of the six channels to the gel and collecting backscattered light. The performance of the imaging module will be characterized by scanning a calibration socket which contains grooves of known depths, and comparing these measurements to the fluorometric results.

Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

USDA-ARS?s Scientific Manuscript database

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags

PubMed Central

Wee, Eugene J.H.; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research. PMID:27446486

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags.

PubMed

Wee, Eugene J H; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.

Chopped molecular beam multiplexing system

NASA Technical Reports Server (NTRS)

Adams, Billy R. (Inventor)

1986-01-01

The integration of a chopped molecular beam mass spectrometer with a time multiplexing system is described. The chopping of the molecular beam is synchronized with the time intervals by a phase detector and a synchronous motor. Arithmetic means are generated for phase shifting the chopper with respect to the multiplexer. A four channel amplifier provides the capacity to independently vary the baseline and amplitude in each channel of the multiplexing system.

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

PubMed Central

Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Johnston, Ian C. D.; Bosio, Andreas; Schauss, Astrid; Wild, Stefan

2016-01-01

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions. PMID:26901056

Polarization-multiplexing ghost imaging

NASA Astrophysics Data System (ADS)

Dongfeng, Shi; Jiamin, Zhang; Jian, Huang; Yingjian, Wang; Kee, Yuan; Kaifa, Cao; Chenbo, Xie; Dong, Liu; Wenyue, Zhu

2018-03-01

A novel technique for polarization-multiplexing ghost imaging is proposed to simultaneously obtain multiple polarimetric information by a single detector. Here, polarization-division multiplexing speckles are employed for object illumination. The light reflected from the objects is detected by a single-pixel detector. An iterative reconstruction method is used to restore the fused image containing the different polarimetric information by using the weighted sum of the multiplexed speckles based on the correlation coefficients obtained from the detected intensities. Next, clear images of the different polarimetric information are recovered by demultiplexing the fused image. The results clearly demonstrate that the proposed method is effective.

Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay.

PubMed

Wang, Hong; Li, Yanbin; Wang, Andrew; Slavik, Michael

2011-12-01

Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths.

The Microwave SQUID Multiplexer

NASA Astrophysics Data System (ADS)

Mates, John Arthur Benson

2011-12-01

This thesis describes a multiplexer of Superconducting Quantum Interference Devices (SQUIDs) with low-noise, ultra-low power dissipation, and great scalability. The multiplexer circuit measures the magnetic flux in a large number of unshunted rf SQUIDs by coupling each SQUID to a superconducting microwave resonator tuned to a unique resonance frequency and driving the resonators from a common feedline. A superposition of microwave tones measures each SQUID simultaneously using only two coaxial cables between the cryogenic device and room temperature. This multiplexer will enable the instrumentation of arrays with hundreds of thousands of low-temperature detectors for new applications in cosmology, materials analysis, and nuclear non-proliferation. The driving application of the Microwave SQUID Multiplexer is the readout of large arrays of superconducting transition-edge sensors, by some figures of merit the most sensitive detectors of electromagnetic signals over a span of more than nine orders of magnitude in energy, from 40 GHz microwaves to 200 keV gamma rays. Modern transition-edge sensors have noise-equivalent power as low as 10-20 W / Hz1/2 and energy resolution as good as 2 eV at 6 keV. These per-pixel sensitivities approach theoretical limits set by the underlying signals, motivating a rapid increase in pixel count to access new science. Compelling applications, like the non-destructive assay of nuclear material for treaty verification or the search for primordial gravity waves from inflation use arrays of these detectors to increase collection area or tile a focal plane. We developed three generations of SQUID multiplexers, optimizing the first for flux noise 0.17 muPhi0 / Hz1/2, the second for input current noise 19 pA / Hz1/2, and the last for practical multiplexing of large arrays of cosmic microwave background polarimeters based on transition-edge sensors. Using the last design we demonstrated multiplexed readout of prototype polarimeters with the

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics.

PubMed

Levine, Peter M; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L

2009-03-15

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

An ion quencher operated lamp for multiplexed fluorescent bioassays.

PubMed

Qing, Taiping; Sun, Huanhuan; He, Xiaoxiao; Huang, Xiaoqin; He, Dinggeng; Bu, Hongchang; Qiao, Zhenzhen; Wang, Kemin

2018-02-01

A novel and adjustable lamp based on competitive interaction among dsDNA-SYBR Green I (SGI), ion quencher, and analyte was designed for bioanalysis. The "filament" and switch of the lamp could be customized by employing different dsDNA and ion quencher. The poly(AT/TA) dsDNA was successfully screened as the most effective filament of the lamp. Two common ions, Hg 2+ and Fe 3+ , were selected as the model switch, and the corresponding ligand molecules cysteine (Cys) and pyrophosphate ions (PPi) were selected as the targets. When the fluorescence-quenched dsDNA/SGI-ion complex was introduced into a target-containing system, ions could be bound by competitive molecules and separate from the complex, thereby lighting the lamp. However, no light was observed if the biomolecule could not snatch the metal ions from the complex. Under the optimal conditions, sensitive and selective detection of Cys and PPi was achieved by the lamp, with practical applications in fetal bovine serum and human urine. This ion quencher regulated lamp for fluorescent bioassays is simple in design, fast in operation, and is more convenient than other methods. Significantly, as many molecules could form stable complexes with metal ions selectively, this ion quencher operated lamp has potential for the detection of a wide spectrum of analytes. Graphical abstract A novel and adjustable lamp on the basis of competitive interaction among dsDNA-SYBR Green I, ions quencher and analyte was designed for bioanalysis. The filament and switch of lamp could be customized by employing different dsDNA and ions quencher.

Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay.

PubMed

Maksyutov, Rinat A; Gavrilova, Elena V; Shchelkunov, Sergei N

2016-10-01

A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species. Copyright © 2016 Elsevier B.V. All rights reserved.

Near-infrared laser-induced fluorescence detection in capillary electrophoresis.

PubMed

McWhorter, S; Soper, S A

2000-04-01

As capillary electrophoresis continues to focus on miniaturization, either through reducing column dimensions or situating entire electrophoresis systems on planar chips, advances in detection become necessary to meet the challenges posed by these electrophoresis platforms. The challenges result from the fact that miniaturization requires smaller load volumes, demanding highly sensitive detection. In addition, many times multiple targets must be analyzed simultaneously (multiplexed applications), further complicating detection. Near-infrared (NIR) fluorescence offers an attractive alternative to visible fluorescence for critical applications in capillary electrophoresis due to the impressive limits of detection that can be generated, in part resulting from the low background levels that are observed in the NIR. Advances in instrumentation and fluorogenic labels appropriate for NIR monitoring have led to a growing number of examples of the use of NIR fluorescence in capillary electrophoresis. In this review, we will cover instrumental components used to construct ultrasensitive NIR fluorescence detectors, including light sources and photon transducers. In addition, we will discuss various types of labeling dyes appropriate for NIR fluorescence and finally, we will present several applications that have used NIR fluorescence in capillary electrophoresis, especially for DNA sequencing and fragment analysis.

Populational analysis of suspended microtissues for high-throughput, multiplexed 3D tissue engineering

PubMed Central

Chen, Alice A.; Underhill, Gregory H.; Bhatia, Sangeeta N.

2014-01-01

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, approaches for functional assessment have been relatively limited. Here, we describe the fabrication of microtissue (μ-tissue) suspensions and their quantitative evaluation with techniques capable of analyzing large sample numbers and performing multiplexed parallel analysis. We applied this platform to 3D μ-tissues representing multiple stages of liver development and disease including: embryonic stem cells, bipotential hepatic progenitors, mature hepatocytes, and hepatoma cells photoencapsulated in polyethylene glycol hydrogels. Multiparametric μ-tissue cytometry enabled quantitation of fluorescent reporter expression within populations of intact μ-tissues (n≥102-103) and sorting-based enrichment of subsets for subsequent studies. Further, 3D μ-tissues could be implanted in vivo, respond to systemic stimuli, retrieved and quantitatively assessed. In order to facilitate multiplexed ‘pooled’ experimentation, fluorescent labeling strategies were developed and utilized to investigate the impact of μ-tissue composition and exposure to soluble factors. In particular, examination of drug/gene interactions on collections of 3D hepatoma μ-tissues indicated synergistic influence of doxorubicin and knockdown of the anti-apoptotic gene BCL-XL. Collectively, these studies highlight the broad utility of μ-tissue suspensions as an enabling approach for high n, populational analysis of 3D tissue biology in vitro and in vivo. PMID:20820630

Fluorescent imaging of cancerous tissues for targeted surgery

PubMed Central

Bu, Lihong; Shen, Baozhong; Cheng, Zhen

2014-01-01

To maximize tumor excision and minimize collateral damage is the primary goal of cancer surgery. Emerging molecular imaging techniques have to “image-guided surgery” developing into “molecular imaging-guided surgery”, which is termed “targeted surgery” in this review. Consequently, the precision of surgery can be advanced from tissue-scale to molecule-scale, enabling “targeted surgery” to be a component of “targeted therapy”. Evidence from numerous experimental and clinical studies has demonstrated significant benefits of fluorescent imaging in targeted surgery with preoperative molecular diagnostic screening. Fluorescent imaging can help to improve intraoperative staging and enable more radical cytoreduction, detect obscure tumor lesions in special organs, highlight tumor margins, better map lymph node metastases, and identify important normal structures intraoperatively. Though limited tissue penetration of fluorescent imaging and tumor heterogeneity are two major hurdles for current targeted surgery, multimodality imaging and multiplex imaging may provide potential solutions to overcome these issues, respectively. Moreover, though many fluorescent imaging techniques and probes have been investigated, targeted surgery remains at a proof-of-principle stage. The impact of fluorescent imaging on cancer surgery will likely be realized through persistent interdisciplinary amalgamation of research in diverse fields. PMID:25064553

Time domain multiplexed spatial division multiplexing receiver.

PubMed

van Uden, Roy G H; Okonkwo, Chigo M; Chen, Haoshuo; de Waardt, Hugo; Koonen, Antonius M J

2014-05-19

A novel time domain multiplexed (TDM) spatial division multiplexing (SDM) receiver which allows for the reception of >1 dual polarization mode with a single coherent receiver, and corresponding 4-port oscilloscope, is experimentally demonstrated. Received by two coherent receivers and respective 4-port oscilloscopes, a 3 mode transmission of 28GBaud QPSK, 8, 16, and 32QAM over 41.7km of few-mode fiber demonstrates the performance of the TDM-SDM receiver with respect to back-to-back. In addition, by using carrier phase estimation employing one digital phase locked loop per output, the frequency offset between the transmitter laser and local oscillator is shown to perform similar to previous work which employs 3 coherent receivers and 4-port oscilloscopes which are dedicated to the reception of each the three modes.

Downlink data multiplexer

NASA Technical Reports Server (NTRS)

Holland, S. Douglas (Inventor); Steele, Glen F. (Inventor); Romero, Denise M. (Inventor); Koudelka, Robert David (Inventor)

2008-01-01

A data multiplexer that accommodates both industry standard CCSDS data packets and bits streams and standard IEEE 1394 data is described. The multiplexer provides a statistical allotment of bandwidth to the channels in turn, preferably four, but expandable in increments of four up to sixteen. A microcontroller determines bandwidth requested by the plurality of channels, as well as the bandwidth available, and meters out the available bandwidth on a statistical basis employing flow control to the input channels.

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

PubMed Central

Gerdes, Michael J.; Sevinsky, Christopher J.; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O.; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J.; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Lowes, Christina; McCulloch, Colin C.; McDonough, Elizabeth; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D.; Seel, Maximilian L.; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-01-01

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics. PMID:23818604

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

PubMed

Gerdes, Michael J; Sevinsky, Christopher J; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Li, Qing; Lowes, Christina; McCulloch, Colin C; McDonough, Elizabeth; Montalto, Michael C; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D; Seel, Maximilian L; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-07-16

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

PubMed

Song, Yunke; Zhang, Yi; Wang, Tza-Huei

2013-04-08

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetics Home Reference: steatocystoma multiplex

MedlinePlus

... Genetic Changes Steatocystoma multiplex can be caused by mutations in the KRT17 gene. This gene provides instructions ... skin, nails, and other tissues. The KRT17 gene mutations that cause steatocystoma multiplex alter the structure of ...

Measuring and modeling correlations in multiplex networks.

PubMed

Nicosia, Vincenzo; Latora, Vito

2015-09-01

The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

All-fiber-based selective mode multiplexer and demultiplexer for weakly-coupled mode-division multiplexed systems

NASA Astrophysics Data System (ADS)

Igarashi, Koji; Park, Kyung Jun; Tsuritani, Takahiro; Morita, Itsuro; Kim, Byoung Yoon

2018-02-01

We show all-fiber-based selective mode multiplexers and demultiplexers for weakly-coupled mode-division multiplexed systems. We fabricate a set of six-mode multiplexer and demultiplexer based on fiber mode selective couplers, and experimentally evaluate the performance for the six-mode dual-polarization (DP) quadrature phase shift keying (QPSK) optical signals. In the mode multiplexer and demultiplexer, the mode couplings between the lower three modes and the higher three modes are suppressed to be less than -20 dB, which enables us to apply partial 6 ×6 MIMO equalizers even for the six-mode demultiplexing. For the six-mode DP-QPSK signals, the penalty of optical signal-to-noise ratio by replacing the full 12 ×12MIMO to the partial 6 ×6 MIMO is suppressed by less than 1 dB.

Seeing biomass recalcitrance through fluorescence.

PubMed

Auxenfans, Thomas; Terryn, Christine; Paës, Gabriel

2017-08-18

Lignocellulosic biomass is the only renewable carbon resource available in sufficient amount on Earth to go beyond the fossil-based carbon economy. Its transformation requires controlled breakdown of polymers into a set of molecules to make fuels, chemicals and materials. But biomass is a network of various inter-connected polymers which are very difficult to deconstruct optimally. In particular, saccharification potential of lignocellulosic biomass depends on several complex chemical and physical factors. For the first time, an easily measurable fluorescence properties of steam-exploded biomass samples from miscanthus, poplar and wheat straw was shown to be directly correlated to their saccharification potential. Fluorescence can thus be advantageously used as a predictive method of biomass saccharification. The loss in fluorescence occurring after the steam explosion pretreatment and increasing with pretreatment severity does not originate from the loss in lignin content, but rather from a decrease of the lignin β-aryl-ether linkage content. Fluorescence lifetime analysis demonstrates that monolignols making lignin become highly conjugated after steam explosion pretreatment. These results reveal that lignin chemical composition is a more important feature to consider than its content to understand and to predict biomass saccharification.

Universal fluorescent multiplex PCR and capillary electrophoresis for evaluation of gene conversion between SMN1 and SMN2 in spinal muscular atrophy.

PubMed

Wang, Chun-Chi; Jong, Yuh-Jyh; Chang, Jan-Gowth; Chen, Yen-Ling; Wu, Shou-Mei

2010-07-01

We have developed a capillary electrophoresis (CE) method with universal fluorescent multiplex PCR to simultaneously detect the SMN1 and SMN2 genes in exons 7 and 8. Spinal muscular atrophy (SMA) is a very frequent inherited disease caused by the absence of the SMN1 gene in approximately 94% of patients. Those patients have deletion of the SMN1 gene or gene conversion between SMN1 and SMN2. However, most methods only focus on the analysis of whole gene deletion, and ignore gene conversion. Simultaneous quantification of SMN1 and SMN2 in exons 7 and 8 is a good strategy for estimating SMN1 deletion or SMN1 to SMN2 gene conversion. This study established a CE separation allowing differentiation of all copy ratios of SMN1 to SMN2 in exons 7 and 8. Among 212 detected individuals, there were 23 SMA patients, 45 carriers, and 144 normal subjects. Three individuals had different ratios of SMN1 to SMN2 in two exons, including an SMA patient having two SMN2 copies in exon 7 but one SMN1 copy in exon 8. This method could provide more information about SMN1 deletion or SMN1 to SMN2 gene conversion for SMA genotyping and diagnosis.

Percolation in real multiplex networks

NASA Astrophysics Data System (ADS)

Bianconi, Ginestra; Radicchi, Filippo

2016-12-01

We present an exact mathematical framework able to describe site-percolation transitions in real multiplex networks. Specifically, we consider the average percolation diagram valid over an infinite number of random configurations where nodes are present in the system with given probability. The approach relies on the locally treelike ansatz, so that it is expected to accurately reproduce the true percolation diagram of sparse multiplex networks with negligible number of short loops. The performance of our theory is tested in social, biological, and transportation multiplex graphs. When compared against previously introduced methods, we observe improvements in the prediction of the percolation diagrams in all networks analyzed. Results from our method confirm previous claims about the robustness of real multiplex networks, in the sense that the average connectedness of the system does not exhibit any significant abrupt change as its individual components are randomly destroyed.

Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.

PubMed

Michel, Martin A; Swatek, Kirby N; Hospenthal, Manuela K; Komander, David

2017-10-05

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Percolation in multiplex networks with overlap.

PubMed

Cellai, Davide; López, Eduardo; Zhou, Jie; Gleeson, James P; Bianconi, Ginestra

2013-11-01

From transportation networks to complex infrastructures, and to social and communication networks, a large variety of systems can be described in terms of multiplexes formed by a set of nodes interacting through different networks (layers). Multiplexes may display an increased fragility with respect to the single layers that constitute them. However, so far the overlap of the links in different layers has been mostly neglected, despite the fact that it is an ubiquitous phenomenon in most multiplexes. Here, we show that the overlap among layers can improve the robustness of interdependent multiplex systems and change the critical behavior of the percolation phase transition in a complex way.

Multiplexed fibre optic sensing in the distal lung (Conference Presentation)

NASA Astrophysics Data System (ADS)

Choudhary, Tushar R.; Tanner, Michael G.; Megia-Fernandez, Alicia; Harrington, Kerrianne; Wood, Harry A.; Chankeshwara, Sunay; Zhu, Patricia; Choudhury, Debaditya; Yu, Fei; Thomson, Robert R.; Duncan, Rory R.; Dhaliwal, Kevin; Bradley, Mark

2017-02-01

We present a toolkit for a multiplexed pH and oxygen sensing probe in the distal lung using multicore fibres. Measuring physiological relevant parameters like pH and oxygen is of significant importance in understanding changes associated with disease pathology. We present here, a single multicore fibre based pH and oxygen sensing probe which can be used with a standard bronchoscope to perform in vivo measurements in the distal lung. The multiplexed probe consists of fluorescent pH sensors (fluorescein based) and oxygen sensors (Palladium porphyrin complex based) covalently bonded to silica microspheres (10 µm) loaded on the distal facet of a 19 core (10 µm core diameter) multicore fibre (total diameter of 150 µm excluding coating). Pits are formed by selectively etching the cores using hydrofluoric acid, multiplexing is achieved through the self-location of individual probes on differing cores. This architecture can be expanded to include probes for further parameters. Robust measurements are demonstrated of self-referencing fluorophores, not limited by photobleaching, with short (100ms) measurement times at low ( 10µW) illumination powers. We have performed on bench calibration and tests of in vitro tissue models and in an ovine whole lung model to validate our sensors. The pH sensor is demonstrated in the physiologically relevant range of pH 5 to pH 8.5 and with an accuracy of ± 0.05 pH units. The oxygen sensor is demonstrated in gas mixtures downwards from 20% oxygen and in liquid saturated with 20% oxygen mixtures ( 8mg/L) down to full depletion (0mg/L) with 0.5mg/L accuracy.

Convergent linkage evidence from two Latin-American population isolates supports the presence of a susceptibility locus for bipolar disorder in 5q31-34.

PubMed

Herzberg, Ibi; Jasinska, Anna; García, Jenny; Jawaheer, Damini; Service, Susan; Kremeyer, Barbara; Duque, Constanza; Parra, María V; Vega, Jorge; Ortiz, Daniel; Carvajal, Luis; Polanco, Guadalupe; Restrepo, Gabriel J; López, Carlos; Palacio, Carlos; Levinson, Matthew; Aldana, Ileana; Mathews, Carol; Davanzo, Pablo; Molina, Julio; Fournier, Eduardo; Bejarano, Julio; Ramírez, Magui; Ortiz, Carmen Araya; Araya, Xinia; Sabatti, Chiara; Reus, Victor; Macaya, Gabriel; Bedoya, Gabriel; Ospina, Jorge; Freimer, Nelson; Ruiz-Linares, Andrés

2006-11-01

We performed a whole genome microsatellite marker scan in six multiplex families with bipolar (BP) mood disorder ascertained in Antioquia, a historically isolated population from North West Colombia. These families were characterized clinically using the approach employed in independent ongoing studies of BP in the closely related population of the Central Valley of Costa Rica. The most consistent linkage results from parametric and non-parametric analyses of the Colombian scan involved markers on 5q31-33, a region implicated by the previous studies of BP in Costa Rica. Because of these concordant results, a follow-up study with additional markers was undertaken in an expanded set of Colombian and Costa Rican families; this provided a genome-wide significant evidence of linkage of BPI to a candidate region of approximately 10 cM in 5q31-33 (maximum non-parametric linkage score=4.395, P<0.00004). Interestingly, this region has been implicated in several previous genetic studies of schizophrenia and psychosis, including disease association with variants of the enthoprotin and gamma-aminobutyric acid receptor genes.

Multiplex Immunoassay Profiling.

PubMed

Stephen, Laurie

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex ® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.

Linkages: Effective Keys to Success.

ERIC Educational Resources Information Center

Tindall, Lloyd W.

1986-01-01

Discusses various elements of linkages: informal agreements; formal written agreements; making agreements work; the non-financial agreement; how linkages work at the elementary, secondary, and postsecondary levels in Wisconsin; mandated linkages; and the importance of linkages in the vocational education and training arena. (CT)

TES Detector Noise Limited Readout Using SQUID Multiplexers

NASA Technical Reports Server (NTRS)

Staguhn, J. G.; Benford, D. J.; Chervenak, J. A.; Khan, S. A.; Moseley, S. H.; Shafer, R. A.; Deiker, S.; Grossman, E. N.; Hilton, G. C.; Irwin, K. D.

2004-01-01

The availability of superconducting Transition Edge Sensors (TES) with large numbers of individual detector pixels requires multiplexers for efficient readout. The use of multiplexers reduces the number of wires needed between the cryogenic electronics and the room temperature electronics and cuts the number of required cryogenic amplifiers. We are using an 8 channel SQUID multiplexer to read out one-dimensional TES arrays which are used for submillimeter astronomical observations. We present results from test measurements which show that the low noise level of the SQUID multiplexers allows accurate measurements of the TES Johnson noise, and that in operation, the readout noise is dominated by the detector noise. Multiplexers for large number of channels require a large bandwidth for the multiplexed readout signal. We discuss the resulting implications for the noise performance of these multiplexers which will be used for the readout of two dimensional TES arrays in next generation instruments.

Apollo Multiplexer operations manual

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, M.M.

1985-04-01

This report describes the operation of the the Apollo Multiplexer, a microprocessor based communications device designed to process data between an Apollo computer and up to four Gandalf PACXIV data switches. Details are given on overall operation, hardware, and troubleshooting. The reader should gain sufficient knowledge from this report to understand the operation of the multiplexer and effectively analyze and correct any problems that might occur.

Free-space optical communications using orbital-angular-momentum multiplexing combined with MIMO-based spatial multiplexing.

PubMed

Ren, Yongxiong; Wang, Zhe; Xie, Guodong; Li, Long; Cao, Yinwen; Liu, Cong; Liao, Peicheng; Yan, Yan; Ahmed, Nisar; Zhao, Zhe; Willner, Asher; Ashrafi, Nima; Ashrafi, Solyman; Linquist, Roger D; Bock, Robert; Tur, Moshe; Molisch, Andreas F; Willner, Alan E

2015-09-15

We explore the potential of combining the advantages of multiple-input multiple-output (MIMO)-based spatial multiplexing with those of orbital angular momentum (OAM) multiplexing to increase the capacity of free-space optical (FSO) communications. We experimentally demonstrate an 80 Gbit/s FSO system with a 2×2 aperture architecture, in which each transmitter aperture contains two multiplexed data-carrying OAM modes. Inter-channel crosstalk effects are minimized by the OAM beams' inherent orthogonality and by the use of 4×4 MIMO signal processing. Our experimental results show that the bit-error rates can reach below the forward error correction limit of 3.8×10(-3) and the power penalties are less than 3.6 dB for all channels after MIMO processing. This indicates that OAM and MIMO-based spatial multiplexing could be simultaneously utilized, thereby providing the potential to enhance system performance.

Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis.

PubMed

Lindstedt, Bjørn-Arne; Vardund, Traute; Kapperud, Georg

2004-08-01

The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison.

Advanced Code-Division Multiplexers for Superconducting Detector Arrays

NASA Astrophysics Data System (ADS)

Irwin, K. D.; Cho, H. M.; Doriese, W. B.; Fowler, J. W.; Hilton, G. C.; Niemack, M. D.; Reintsema, C. D.; Schmidt, D. R.; Ullom, J. N.; Vale, L. R.

2012-06-01

Multiplexers based on the modulation of superconducting quantum interference devices are now regularly used in multi-kilopixel arrays of superconducting detectors for astrophysics, cosmology, and materials analysis. Over the next decade, much larger arrays will be needed. These larger arrays require new modulation techniques and compact multiplexer elements that fit within each pixel. We present a new in-focal-plane code-division multiplexer that provides multiplexing elements with the required scalability. This code-division multiplexer uses compact lithographic modulation elements that simultaneously multiplex both signal outputs and superconducting transition-edge sensor (TES) detector bias voltages. It eliminates the shunt resistor used to voltage bias TES detectors, greatly reduces power dissipation, allows different dc bias voltages for each TES, and makes all elements sufficiently compact to fit inside the detector pixel area. These in-focal plane code-division multiplexers can be combined with multi-GHz readout based on superconducting microresonators to scale to even larger arrays.

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay.

PubMed

Tanigawa, Chihiro; Fujii, Yoshito; Miura, Masashi; Nzou, Samson Muuo; Mwangi, Anne Wanjiru; Nagi, Sachiyo; Hamano, Shinjiro; Njenga, Sammy M; Mbanefo, Evaristus Chibunna; Hirayama, Kenji; Mwau, Matilu; Kaneko, Satoshi

2015-01-01

Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance. Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman's rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006). Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system.

Preliminary study of visual effect of multiplex hologram

NASA Astrophysics Data System (ADS)

Fu, Huaiping; Xiong, Bingheng; Yang, Hong; Zhang, Xueguo

2004-06-01

The process of any movement of real object can be recorded and displayed by a multiplex holographic stereogram. An embossing multiplex holographic stereogram and a multiplex rainbow holographic stereogram have been made by us, the multiplex rainbow holographic stereogram reconstructs the dynamic 2D line drawing of speech organs, the embossing multiplex holographic stereogram reconstructs the process of an old man drinking water. In this paper, we studied the visual result of an embossing multiplex holographic stereogram made with 80 films of 2-D pictures. Forty-eight persons of aged from 13 to 67 were asked to see the hologram and then to answer some questions about the feeling of viewing. The results indicate that this kind of holograms could be accepted by human visual sense organ without any problem. This paper also discusses visual effect of the multiplex holography stereograms base on visual perceptual psychology. It is open out that the planar multiplex holograms can be recorded and present the movement of real animal and object. Not only have the human visual perceptual constancy for shape, just as that size, color, etc... but also have visual perceptual constancy for binocular parallax.

Development of a 20-locus fluorescent multiplex system as a valuable tool for national DNA database.

PubMed

Jiang, Xianhua; Guo, Fei; Jia, Fei; Jin, Ping; Sun, Zhu

2013-02-01

The multiplex system allows the detection of 19 autosomal short tandem repeat (STR) loci [including all Combined DNA Index System (CODIS) STR loci as well as D2S1338, D6S1043, D12S391, D19S433, Penta D and Penta E] plus the sex-determining locus Amelogenin in a single reaction, comprising all STR loci in various commercial kits used in the China national DNA database (NDNAD). Primers are designed so that the amplicons are distributed ranging from 90 base pairs (bp) to 450 bp within a five-dye fluorescent design with the fifth dye reserved for the internal size standard. With 30 cycles, 125 pg to 2 ng DNA template showed optimal profiling result, while robust profiles could also be achieved by adjusting the cycle numbers for the DNA template beyond that optimal DNA input range. Mixture studies showed that 83% and 87% of minor alleles were detected at 9:1 and 1:9 ratios, respectively. When 4 ng of degraded DNA was digested by 2-min DNase and 1 ng undegraded DNA was added to 400 μM haematin, the complete profiles were still observed. Polymerase chain reaction (PCR)-based procedures were examined and optimized including the concentrations of primer set, magnesium and the Taq polymerase as well as volume, cycle number and annealing temperature. In addition, the system has been validated by 3000 bloodstain samples and 35 common case samples in line with the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The total probability of identity (TPI) can reach to 8×10(-24), where DNA database can be improved at the level of 10 million DNA profiles or more because the number of expected match is far from one person (4×10(-10)) and can be negligible. Further, our system also demonstrates its good performance in case samples and it will be an ideal tool for forensic DNA typing and databasing with potential application. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Demonstration of hybrid orbital angular momentum multiplexing and time-division multiplexing passive optical network.

PubMed

Wang, Andong; Zhu, Long; Liu, Jun; Du, Cheng; Mo, Qi; Wang, Jian

2015-11-16

Mode-division multiplexing passive optical network (MDM-PON) is a promising scheme for next-generation access networks to further increase fiber transmission capacity. In this paper, we demonstrate the proof-of-concept experiment of hybrid mode-division multiplexing (MDM) and time-division multiplexing (TDM) PON architecture by exploiting orbital angular momentum (OAM) modes. Bidirectional transmissions with 2.5-Gbaud 4-level pulse amplitude modulation (PAM-4) downstream and 2-Gbaud on-off keying (OOK) upstream are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties for downstream and upstream transmissions at a bit-error rate (BER) of 2 × 10(-3) are less than 2.0 dB and 3.0 dB, respectively.

Exclusion of linkage between cleft lip with or without cleft palate and markers on chromosomes 4 and 6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blanton, S.H.; Malcolm, S.; Winter, R.

1996-01-01

Nonsyndromic cleft lip with or without associate cleft palate (CLP) is a common craniofacial defect, occurring in {approximately}1/1,000 live births. While the defect generally occurs sporadically, multiplex families have been reported. Segregation analyses have demonstrated that, in some families, CLP is inherited as an autosomal dominant/codominant disorder with low penetrance. Several clefting loci have been proposed on multiple chromosomes, including 6p24, 4q, and 19q13.1. Association studies and linkage studies suggested a locus that mapped to 6p24. We were unable to confirm this in a linkage study of 12 multigenerational families. A subsequent linkage study by Carinci et al., however, foundmore » evidence for linkage to this region in 14 of 21 clefting families. Additionally, Davies et al. studied the chromosomes of three individuals with cleft lip and palate, all of whom had a rearrangement involving 6p24. Their investigation supported a locus at 6p24. Carinci et al. reported that the most likely position for a clefting locus was at D6S89, which is centromeric to EDN1. This is in contrast to the findings of Davies et al., who suggested a placement telomeric to EDN1. F13A, which had been implicated in the initial association studies, is telomeric to EDN1. Thus, the region between F13A and D6S89 encompasses the regions proposed by both Davies et al. and Carinci et al. A second clefting locus, at 4q, was proposed by Beiraghi et al., who studied a single multigenerational family by linkage analysis. Their data suggested a locus near D4S175 and D4S192. 10 refs., 1 tab.« less

Developing a novel fiber optic fluorescence device for multiplexed high-throughput cytotoxic screening.

PubMed

Lee, Dennis; Barnes, Stephen

2010-01-01

The need for new pharmacological agents is unending. Yet the drug discovery process has changed substantially over the past decade and continues to evolve in response to new technologies. There is presently a high demand to reduce discovery time by improving specific lab disciplines and developing new technology platforms in the area of cell-based assay screening. Here we present the developmental concept and early stage testing of the Ab-Sniffer, a novel fiber optic fluorescence device for high-throughput cytotoxicity screening using an immobilized whole cell approach. The fused silica fibers are chemically functionalized with biotin to provide interaction with fluorescently labeled, streptavidin functionalized alginate-chitosan microspheres. The microspheres are also functionalized with Concanavalin A to facilitate binding to living cells. By using lymphoma cells and rituximab in an adaptation of a well-known cytotoxicity protocol we demonstrate the utility of the Ab-Sniffer for functional screening of potential drug compounds rather than indirect, non-functional screening via binding assay. The platform can be extended to any assay capable of being tied to a fluorescence response including multiple target cells in each well of a multi-well plate for high-throughput screening.

Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

NASA Astrophysics Data System (ADS)

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-10-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ˜40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

Thermally multiplexed polymerase chain reaction.

PubMed

Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

2015-07-01

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.

Privacy preserving interactive record linkage (PPIRL).

PubMed

Kum, Hye-Chung; Krishnamurthy, Ashok; Machanavajjhala, Ashwin; Reiter, Michael K; Ahalt, Stanley

2014-01-01

Record linkage to integrate uncoordinated databases is critical in biomedical research using Big Data. Balancing privacy protection against the need for high quality record linkage requires a human-machine hybrid system to safely manage uncertainty in the ever changing streams of chaotic Big Data. In the computer science literature, private record linkage is the most published area. It investigates how to apply a known linkage function safely when linking two tables. However, in practice, the linkage function is rarely known. Thus, there are many data linkage centers whose main role is to be the trusted third party to determine the linkage function manually and link data for research via a master population list for a designated region. Recently, a more flexible computerized third-party linkage platform, Secure Decoupled Linkage (SDLink), has been proposed based on: (1) decoupling data via encryption, (2) obfuscation via chaffing (adding fake data) and universe manipulation; and (3) minimum information disclosure via recoding. We synthesize this literature to formalize a new framework for privacy preserving interactive record linkage (PPIRL) with tractable privacy and utility properties and then analyze the literature using this framework. Human-based third-party linkage centers for privacy preserving record linkage are the accepted norm internationally. We find that a computer-based third-party platform that can precisely control the information disclosed at the micro level and allow frequent human interaction during the linkage process, is an effective human-machine hybrid system that significantly improves on the linkage center model both in terms of privacy and utility.

Privacy preserving interactive record linkage (PPIRL)

PubMed Central

Kum, Hye-Chung; Krishnamurthy, Ashok; Machanavajjhala, Ashwin; Reiter, Michael K; Ahalt, Stanley

2014-01-01

Objective Record linkage to integrate uncoordinated databases is critical in biomedical research using Big Data. Balancing privacy protection against the need for high quality record linkage requires a human–machine hybrid system to safely manage uncertainty in the ever changing streams of chaotic Big Data. Methods In the computer science literature, private record linkage is the most published area. It investigates how to apply a known linkage function safely when linking two tables. However, in practice, the linkage function is rarely known. Thus, there are many data linkage centers whose main role is to be the trusted third party to determine the linkage function manually and link data for research via a master population list for a designated region. Recently, a more flexible computerized third-party linkage platform, Secure Decoupled Linkage (SDLink), has been proposed based on: (1) decoupling data via encryption, (2) obfuscation via chaffing (adding fake data) and universe manipulation; and (3) minimum information disclosure via recoding. Results We synthesize this literature to formalize a new framework for privacy preserving interactive record linkage (PPIRL) with tractable privacy and utility properties and then analyze the literature using this framework. Conclusions Human-based third-party linkage centers for privacy preserving record linkage are the accepted norm internationally. We find that a computer-based third-party platform that can precisely control the information disclosed at the micro level and allow frequent human interaction during the linkage process, is an effective human–machine hybrid system that significantly improves on the linkage center model both in terms of privacy and utility. PMID:24201028

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Sub-micrometer Geometrically Encoded Fluorescent Barcodes Self-Assembled from DNA

PubMed Central

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-01-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here we use DNA-origami technology to construct sub-micrometer nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be unambiguously decoded using epifluorescence or total internal reflection fluorescence (TIRF) microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ~40 nm. One species of the barcodes was used to tag yeast surface receptors, suggesting their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments. PMID:23000997

Simultaneous fluorescence and quantitative phase microscopy with single-pixel detectors

NASA Astrophysics Data System (ADS)

Liu, Yang; Suo, Jinli; Zhang, Yuanlong; Dai, Qionghai

2018-02-01

Multimodal microscopy offers high flexibilities for biomedical observation and diagnosis. Conventional multimodal approaches either use multiple cameras or a single camera spatially multiplexing different modes. The former needs expertise demanding alignment and the latter suffers from limited spatial resolution. Here, we report an alignment-free full-resolution simultaneous fluorescence and quantitative phase imaging approach using single-pixel detectors. By combining reference-free interferometry with single-pixel detection, we encode the phase and fluorescence of the sample in two detection arms at the same time. Then we employ structured illumination and the correlated measurements between the sample and the illuminations for reconstruction. The recovered fluorescence and phase images are inherently aligned thanks to single-pixel detection. To validate the proposed method, we built a proof-of-concept setup for first imaging the phase of etched glass with the depth of a few hundred nanometers and then imaging the fluorescence and phase of the quantum dot drop. This method holds great potential for multispectral fluorescence microscopy with additional single-pixel detectors or a spectrometer. Besides, this cost-efficient multimodal system might find broad applications in biomedical science and neuroscience.

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii)

PubMed Central

2011-01-01

Background The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species. Results A second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a) sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH), (b) End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c) tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers) to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map. Conclusions Construction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby genome and considerably

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii).

PubMed

Wang, Chenwei; Webley, Lee; Wei, Ke-jun; Wakefield, Matthew J; Patel, Hardip R; Deakin, Janine E; Alsop, Amber; Marshall Graves, Jennifer A; Cooper, Desmond W; Nicholas, Frank W; Zenger, Kyall R

2011-08-19

The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species. A second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a) sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH), (b) End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c) tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers) to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map. Construction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby genome and considerably extends that of the first

Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency

PubMed Central

Lin, Yii-Lih; Huang, Yen-Jun; Teerapanich, Pattamon; Leïchlé, Thierry

2016-01-01

Nanofluidic devices promise high reaction efficiency and fast kinetic responses due to the spatial constriction of transported biomolecules with confined molecular diffusion. However, parallel detection of multiple biomolecules, particularly proteins, in highly confined space remains challenging. This study integrates extended nanofluidics with embedded protein microarray to achieve multiplexed real-time biosensing and kinetics monitoring. Implementation of embedded standard-sized antibody microarray is attained by epoxy-silane surface modification and a room-temperature low-aspect-ratio bonding technique. An effective sample transport is achieved by electrokinetic pumping via electroosmotic flow. Through the nanoslit-based spatial confinement, the antigen-antibody binding reaction is enhanced with ∼100% efficiency and may be directly observed with fluorescence microscopy without the requirement of intermediate washing steps. The image-based data provide numerous spatially distributed reaction kinetic curves and are collectively modeled using a simple one-dimensional convection-reaction model. This study represents an integrated nanofluidic solution for real-time multiplexed immunosensing and kinetics monitoring, starting from device fabrication, protein immobilization, device bonding, sample transport, to data analysis at Péclet number less than 1. PMID:27375819

Monte Carlo based method for fluorescence tomographic imaging with lifetime multiplexing using time gates

PubMed Central

Chen, Jin; Venugopal, Vivek; Intes, Xavier

2011-01-01

Time-resolved fluorescence optical tomography allows 3-dimensional localization of multiple fluorophores based on lifetime contrast while providing a unique data set for improved resolution. However, to employ the full fluorescence time measurements, a light propagation model that accurately simulates weakly diffused and multiple scattered photons is required. In this article, we derive a computationally efficient Monte Carlo based method to compute time-gated fluorescence Jacobians for the simultaneous imaging of two fluorophores with lifetime contrast. The Monte Carlo based formulation is validated on a synthetic murine model simulating the uptake in the kidneys of two distinct fluorophores with lifetime contrast. Experimentally, the method is validated using capillaries filled with 2.5nmol of ICG and IRDye™800CW respectively embedded in a diffuse media mimicking the average optical properties of mice. Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation. PMID:21483610

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser-induced fluorescence detection.

PubMed

Nikcevic, Irena; Piruska, Aigars; Wehmeyer, Kenneth R; Seliskar, Carl J; Limbach, Patrick A; Heineman, William R

2010-08-01

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK(a) determination of small molecule analytes is demonstrated with the multilane microchip.

Multiplexed chirp waveform synthesizer

DOEpatents

Dudley, Peter A.; Tise, Bert L.

2003-09-02

A synthesizer for generating a desired chirp signal has M parallel channels, where M is an integer greater than 1, each channel including a chirp waveform synthesizer generating at an output a portion of a digital representation of the desired chirp signal; and a multiplexer for multiplexing the M outputs to create a digital representation of the desired chirp signal. Preferably, each channel receives input information that is a function of information representing the desired chirp signal.

Multiplexed EFPI sensors with ultra-high resolution

NASA Astrophysics Data System (ADS)

Ushakov, Nikolai; Liokumovich, Leonid

2014-05-01

An investigation of performance of multiplexed displacement sensors based on extrinsic Fabry-Perot interferometers has been carried out. We have considered serial and parallel configurations and analyzed the issues and advantages of the both. We have also extended the previously developed baseline demodulation algorithm for the case of a system of multiplexed sensors. Serial and parallel multiplexing schemes have been experimentally implemented with 3 and 4 sensing elements, respectively. For both configurations the achieved baseline standard deviations were between 30 and 200 pm, which is, to the best of our knowledge, more than an order less than any other multiplexed EFPI resolution ever reported.

Reflection holograms using peristrophic multiplexing

NASA Astrophysics Data System (ADS)

Sayeh, Mohammed R.; Jeong, Y.

2000-07-01

In this paper, we consider a peristrophic multiplexing for reflection holograms. This type of multiplexing the rotation of either the material or the reference beam causes the grating vector to be off the plane of the reference and image beams. In the case of reflection hologram, we developed a relationship for the angular selectivity which is verified experimentally.

Time Division Multiplexing of Semiconductor Qubits

NASA Astrophysics Data System (ADS)

Jarratt, Marie Claire; Hornibrook, John; Croot, Xanthe; Watson, John; Gardner, Geoff; Fallahi, Saeed; Manfra, Michael; Reilly, David

Readout chains, comprising resonators, amplifiers, and demodulators, are likely to be precious resources in quantum computing architectures. The potential to share readout resources is contingent on realising efficient means of time-division multiplexing (TDM) schemes that are compatible with quantum computing. Here, we demonstrate TDM using a GaAs quantum dot device with multiple charge sensors. Our device incorporates chip-level switches that do not load the impedance matching network. When used in conjunction with frequency multiplexing, each frequency tone addresses multiple time-multiplexed qubits, vastly increasing the capacity of a single readout line.

Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.

PubMed

Woo, Nain; Kim, Su-Kang; Sun, Yucheng; Kang, Seong Ho

2018-01-01

Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

PubMed

Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

2004-12-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

PubMed Central

Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

2004-01-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

Platform for Quantitative Evaluation of Spatial Intratumoral Heterogeneity in Multiplexed Fluorescence Images.

PubMed

Spagnolo, Daniel M; Al-Kofahi, Yousef; Zhu, Peihong; Lezon, Timothy R; Gough, Albert; Stern, Andrew M; Lee, Adrian V; Ginty, Fiona; Sarachan, Brion; Taylor, D Lansing; Chennubhotla, S Chakra

2017-11-01

We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR . ©2017 American Association for Cancer Research.

Improvement for identification of heterophile antibody interference and AFP hook effect in immunoassays with multiplex suspension bead array system.

PubMed

Wang, Yajie; Yu, Jinsheng; Ren, Yuan; Liu, Li; Li, Haowen; Guo, Anchen; Shi, Congning; Fang, Fang; Juehne, Twyla; Yao, Jianer; Yang, Enhuan; Zhou, Xuelei; Kang, Xixiong

2013-11-15

A variety of immunoassays including multiplex suspension bead array have been developed for tumor marker detections; however, these assays could be compromised in their sensitivity and specificity by well-known heterophile antibody interference and hook effect. Using Luminex® multiplex suspension bead arrays, we modified protocols with two newly-developed solutions that can identify heterophile antibody interference and AFP hook effect. Effectiveness of the two solutions was assessed in serum samples from patients. Concentrations of 9 tumor markers in heterophile antibody positive samples assayed with Solution A, containing murine monoclonal antibodies and mouse serum, were significantly reduced when compared with those false high signals assayed without Solution A (all p<0.01). With incorporation of Solution H (fluorescent beads linked with AFP antigen), a new strategy for identification of AFP hook effect was established, and with this strategy AFP hook effect was identified effectively in serum samples with very high levels of AFP. Two proprietary solutions improve the identification of heterophile antibody interference and AFP hook effect. With these solutions, multiplex suspension bead arrays provide more reliable testing results in tumor marker detection where complex clinical serum samples are used. © 2013.

Multiplexed image storage by electromagnetically induced transparency in a solid

NASA Astrophysics Data System (ADS)

Heinze, G.; Rentzsch, N.; Halfmann, T.

2012-11-01

We report on frequency- and angle-multiplexed image storage by electromagnetically induced transparency (EIT) in a Pr3+:Y2SiO5 crystal. Frequency multiplexing by EIT relies on simultaneous storage of light pulses in atomic coherences, driven in different frequency ensembles of the inhomogeneously broadened solid medium. Angular multiplexing by EIT relies on phase matching of the driving laser beams, which permits simultaneous storage of light pulses propagating under different angles into the crystal. We apply the multiplexing techniques to increase the storage capacity of the EIT-driven optical memory, in particular to implement multiplexed storage of larger two-dimensional amounts of data (images). We demonstrate selective storage and readout of images by frequency-multiplexed EIT and angular-multiplexed EIT, as well as the potential to combine both multiplexing approaches towards further enhanced storage capacities.

Genome scan for linkage to asthma using a linkage disequilibrium-lod score test.

PubMed

Jiang, Y; Slager, S L; Huang, J

2001-01-01

We report a genome-wide linkage study of asthma on the German and Collaborative Study on the Genetics of Asthma (CSGA) data. Using a combined linkage and linkage disequilibrium test and the nonparametric linkage score, we identified 13 markers from the German data, 1 marker from the African American (CSGA) data, and 7 markers from the Caucasian (CSGA) data in which the p-values ranged between 0.0001 and 0.0100. From our analysis and taking into account previous published linkage studies of asthma, we suggest that three regions in chromosome 5 (around D5S418, D5S644, and D5S422), one region in chromosome 6 (around three neighboring markers D6S1281, D6S291, and D6S1019), one region in chromosome 11 (around D11S2362), and two regions in chromosome 12 (around D12S351 and D12S324) especially merit further investigation.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

PubMed Central

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-01

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

PubMed

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Immunization of Epidemics in Multiplex Networks

PubMed Central

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks. PMID:25401755

Immunization of epidemics in multiplex networks.

PubMed

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks.

Multiplexed interfacial transduction of nucleic acid hybridization using a single color of immobilized quantum dot donor and two acceptors in fluorescence resonance energy transfer.

PubMed

Algar, W Russ; Krull, Ulrich J

2010-01-01

A multiplexed solid-phase assay for the detection of nucleic acid hybridization was developed on the basis of a single color of immobilized CdSe/ZnS quantum dot (QD) as a donor in fluorescence resonance energy transfer (FRET). This work demonstrated that two channels of detection did not necessitate two different QD donors. Two probe oligonucleotides were coimmobilized on optical fibers modified with QDs, and a sandwich assay was used to associate the acceptor dyes with interfacial hybridization events without target labeling. FRET-sensitized acceptor emission provided an analytical signal that was concentration dependent down to 10 nM. Changes in the ratio of coimmobilized probe oligonucleotides were found to yield linear changes in the relative amounts of acceptor emission. These changes were compared to previous studies that used mixed films of two QD donors for two detection channels. The analysis indicated that probe dilution effects were primarily driven by changes in acceptor number density and that QD dilution effects or changes in mean donor-acceptor distance were secondary. Hybridization kinetics were found to be consistent between different ratios of coimmobilized probes, suggesting that hybridization in this type of system occurred via the accepted model for solid-phase hybridization, where adsorption and then diffusion at the solid interface drove hybridization.

Multiplexed detection of mycotoxins in foods with a regenerable array.

PubMed

Ngundi, Miriam M; Shriver-Lake, Lisa C; Moore, Martin H; Ligler, Frances S; Taitt, Chris R

2006-12-01

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

PubMed

Petryayeva, Eleonora; Algar, W Russ

2014-03-18

Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

PubMed

Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Exclusion of close linkage between the synaptic vesicular monoamine transporter locus and schizophrenia spectrum disorders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Persico, A.M.; Uhl, G.R.; Wang, Zhe Wu

The principal brain synaptic vesicular monoamine transporter (VMAT2) is responsible for the reuptake of serotonin, dopamine, norepinephrine, epinephrine, and histamine from the cytoplasm into synaptic vesicles, thus contributing to determination of the size of releasable neurotransmitter vesicular pools. Potential involvement of VMAT2 gene variants in the etiology of schizophrenia and related disorders was tested using polymorphic VMAT2 gene markers in 156 subjects from 16 multiplex pedigrees with schizophrenia, schizophreniform, schizoaffective, and schizotypal disorders and mood incongruent psychotic depression. Assuming genetic homogeneity, complete ({theta} = 0.0) linkage to the schizophrenia spectrum was excluded under both dominant and recessive models. Allelic variantsmore » at the VMAT2 locus do not appear to provide major genetic contributions to the etiology of schizophrenia spectrum disorders in these pedigrees. 16 refs.« less

Prototype data terminal-multiplexer/demultiplexer

NASA Technical Reports Server (NTRS)

Leck, D. E.; Goodwin, J. E.

1972-01-01

The design and operation of a quad redundant data terminal and a multiplexer/demultiplexer (MDU) is described. The most unique feature is the design of the quad redundant data terminal. This is one of the few designs where the unit is fail/op, fail/op, fail/safe. Laboratory tests confirm that the unit will operate satisfactorily with the failure of three out of four channels. Although the design utilizes state-of-the-art technology, the waveform error checks, the voting techniques, and the parity bit checks are believed to be used in unique configurations. Correct word selection routines are also novel. The MDU design, while not redundant, utilizes, the latest state-of-the-art advantages of light coupler and interested amplifiers. Much of the technology employed was an evolution of prior NASA contracts related to the Addressable Time Division Data System. A good example of the earlier technology development was the development of a low level analog multiplexer, a high level analog multiplexer, and a digital multiplexer. A list of all drawings is included for reference and all schematic, block and timing diagrams are incorporated.

Weak percolation on multiplex networks

NASA Astrophysics Data System (ADS)

Baxter, Gareth J.; Dorogovtsev, Sergey N.; Mendes, José F. F.; Cellai, Davide

2014-04-01

Bootstrap percolation is a simple but nontrivial model. It has applications in many areas of science and has been explored on random networks for several decades. In single-layer (simplex) networks, it has been recently observed that bootstrap percolation, which is defined as an incremental process, can be seen as the opposite of pruning percolation, where nodes are removed according to a connectivity rule. Here we propose models of both bootstrap and pruning percolation for multiplex networks. We collectively refer to these two models with the concept of "weak" percolation, to distinguish them from the somewhat classical concept of ordinary ("strong") percolation. While the two models coincide in simplex networks, we show that they decouple when considering multiplexes, giving rise to a wealth of critical phenomena. Our bootstrap model constitutes the simplest example of a contagion process on a multiplex network and has potential applications in critical infrastructure recovery and information security. Moreover, we show that our pruning percolation model may provide a way to diagnose missing layers in a multiplex network. Finally, our analytical approach allows us to calculate critical behavior and characterize critical clusters.

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

PubMed

Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

2009-01-01

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

Dynamic Optically Multiplexed Imaging

DTIC Science & Technology

2015-07-29

Direction LENS.ZMX Configuration 1 of 1 3D Layout 10/21/2014 Scale: 1.000020.00 Millimeters X Y Z Parent Lens (a) (b) 0 20 40 60 80 100 0 20 40 60 80 100...V. Shah, and T. Shih “Design Architectures for Optically Multiplexed Imaging,” in submission 9 R. Gupta, P . Indyk, E. Price, and Y . Rachlin...the number of multiplexed images. As a result, measurements from a sufficiently fast sampling sensor can be processed to yield a low distortion image

Multiplexing 200 spatial modes with a single hologram

NASA Astrophysics Data System (ADS)

Rosales-Guzmán, Carmelo; Bhebhe, Nkosiphile; Mahonisi, Nyiku; Forbes, Andrew

2017-11-01

The on-demand tailoring of light's spatial shape is of great relevance in a wide variety of research areas. Computer-controlled devices, such as spatial light modulators (SLMs) or digital micromirror devices, offer a very accurate, flexible and fast holographic means to this end. Remarkably, digital holography affords the simultaneous generation of multiple beams (multiplexing), a tool with numerous applications in many fields. Here, we provide a self-contained tutorial on light beam multiplexing. Through the use of several examples, the readers will be guided step by step in the process of light beam shaping and multiplexing. Additionally, we provide a quantitative analysis on the multiplexing capabilities of SLMs to assess the maximum number of beams that can be multiplexed on a single SLM, showing approximately 200 modes on a single hologram.

Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification.

PubMed

Houston, Rachel; Birck, Matthew; Hughes-Stamm, Sheree; Gangitano, David

2016-05-01

Marijuana (Cannabis sativa) is the most commonly used illicit substance in the USA. The development of a validated method using Cannabis short tandem repeats (STRs) could aid in the individualization of samples as well as serve as an intelligence tool to link multiple cases. For this purpose, a modified 13-loci STR multiplex method was optimized and evaluated according to ISFG and SWGDAM guidelines. A real-time PCR quantification method for C. sativa was developed and validated, and a sequenced allelic ladder was also designed to accurately genotype 199 C. sativa samples from 11 U.S. Customs and Border Protection seizures. Distinguishable DNA profiles were generated from 127 samples that yielded full STR profiles. Four duplicate genotypes within seizures were found. The combined power of discrimination of this multilocus system is 1 in 70 million. The sensitivity of the multiplex STR system is 0.25 ng of template DNA. None of the 13 STR markers cross-reacted with any of the studied species, except for Humulus lupulus (hops) which generated unspecific peaks. Phylogenetic analysis and case-to-case pairwise comparison of 11 cases using F st as genetic distance revealed the genetic association of four groups of cases. Moreover, due to their genetic similarity, a subset of samples (N = 97) was found to form a homogeneous population in Hardy-Weinberg and linkage equilibrium. The results of this research demonstrate the applicability of this 13-loci STR system in associating Cannabis cases for intelligence purposes.

Optimizing diffusion in multiplexes by maximizing layer dissimilarity

NASA Astrophysics Data System (ADS)

Serrano, Alfredo B.; Gómez-Gardeñes, Jesús; Andrade, Roberto F. S.

2017-05-01

Diffusion in a multiplex depends on the specific link distribution between the nodes in each layer, but also on the set of the intralayer and interlayer diffusion coefficients. In this work we investigate, in a quantitative way, the efficiency of multiplex diffusion as a function of the topological similarity among multiplex layers. This similarity is measured by the distance between layers, taken among the pairs of layers. Results are presented for a simple two-layer multiplex, where one of the layers is held fixed, while the other one can be rewired in a controlled way in order to increase or decrease the interlayer distance. The results indicate that, for fixed values of all intra- and interlayer diffusion coefficients, a large interlayer distance generally enhances the global multiplex diffusion, providing a topological mechanism to control the global diffusive process. For some sets of networks, we develop an algorithm to identify the most sensitive nodes in the rewirable layer, so that changes in a small set of connections produce a drastic enhancement of the global diffusion of the whole multiplex system.

Available number of multiplexed holograms based on signal-to-noise ratio analysis in reflection-type holographic memory using three-dimensional speckle-shift multiplexing.

PubMed

Nishizaki, Tatsuya; Matoba, Osamu; Nitta, Kouichi

2014-09-01

The recording properties of three-dimensional speckle-shift multiplexing in reflection-type holographic memory are analyzed numerically. Three-dimensional recording can increase the number of multiplexed holograms by suppressing the cross-talk noise from adjacent holograms by using depth-direction multiplexing rather than in-plane multiplexing. Numerical results indicate that the number of multiplexed holograms in three-layer recording can be increased by 1.44 times as large as that of a single-layer recording when an acceptable signal-to-noise ratio is set to be 2 when NA=0.43 and the thickness of the recording medium is 0.5 mm.

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

PubMed

Verhoef, Lisette G G C; Mattioli, Michela; Ricci, Fernanda; Li, Yao-Cheng; Wade, Mark

2016-02-01

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

Modulation-frequency encoded multi-color fluorescent DNA analysis in an optofluidic chip.

PubMed

Dongre, Chaitanya; van Weerd, Jasper; Besselink, Geert A J; Vazquez, Rebeca Martinez; Osellame, Roberto; Cerullo, Giulio; van Weeghel, Rob; van den Vlekkert, Hans H; Hoekstra, Hugo J W M; Pollnau, Markus

2011-02-21

We introduce a principle of parallel optical processing to an optofluidic lab-on-a-chip. During electrophoretic separation, the ultra-low limit of detection achieved with our set-up allows us to record fluorescence from covalently end-labeled DNA molecules. Different sets of exclusively color-labeled DNA fragments-otherwise rendered indistinguishable by spatio-temporal coincidence-are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a single ultrasensitive, albeit color-blind photomultiplier, and Fourier analysis decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed.

Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

PubMed Central

Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

2016-01-01

Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

DNA sequencing using fluorescence background electroblotting membrane

DOEpatents

Caldwell, Karin D.; Chu, Tun-Jen; Pitt, William G.

1992-01-01

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.

DNA sequencing using fluorescence background electroblotting membrane

DOEpatents

Caldwell, K.D.; Chu, T.J.; Pitt, W.G.

1992-05-12

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through amino groups contained on the surface. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to the target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membranes may be reprobed numerous times. No Drawings

Flexible wavelength de-multiplexer for elastic optical networking.

PubMed

Zhou, Rui; Gutierrez Pascual, M Deseada; Anandarajah, Prince M; Shao, Tong; Smyth, Frank; Barry, Liam P

2016-05-15

We report an injection locked flexible wavelength de-multiplexer (de-mux) that shows 24-h frequency stability of 1 kHz for optical comb-based elastic optical networking applications. We demonstrate 50 GHz, 87.5 GHz equal spacing and 6.25G-25G-50 GHz, 75G-50G-100 GHz unequal spacing for the de-multiplexer outputs. We also implement an unequally spaced (75G-50G-100 GHz), mixed symbol rate (12.5 GBaud and 40 GBaud) and modulation format (polarization division multiplexed quadrature phase shift keying and on-off keying) wavelength division multiplexed transmission system using the de-multiplexer outputs. The results show 0.6 dB receiver sensitivity penalty, at 7% hard decision forward error correction coding limit, of the 100 km transmitted de-mux outputs when compared to comb source seeding laser back-to-back.

Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

PubMed

Liu, Yacui; Zhang, Jiangyan; Tian, Jingxiao; Fan, Xiaofei; Geng, Hao; Cheng, Yongqiang

2017-01-01

A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

Light-Addressed Electrodeposition of Enzyme-Entrapped Chitosan Membranes for Multiplexed Enzyme-Based Bioassays Using a Digital Micromirror Device

PubMed Central

Huang, Shih-Hao; Wei, Lu-Shiuan; Chu, Hsiao-Tzu; Jiang, Yeu-Long

2013-01-01

This paper describes a light-addressed electrolytic system used to perform an electrodeposition of enzyme-entrapped chitosan membranes for multiplexed enzyme-based bioassays using a digital micromirror device (DMD). In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-cathode to electrolytically produce hydroxide ions, which leads to an increased pH gradient. The high pH generated at the cathode can cause a local gelation of chitosan through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressed electrodeposition of chitosan membranes with different shapes and sizes, as well as multiplexed micropatterning, was performed. The effect of the illumination time of the light pattern on the dimensional resolution of chitosan membrane formation was examined experimentally. Moreover, multiplexed enzyme-based bioassay of enzyme-entrapped chitosan membranes was also successfully demonstrated through the electrodeposition of the chitosan membranes with various shapes/sizes and entrapping different enzymes. As a model experiment, glucose and ethanol were simultaneously detected in a single detection chamber without cross-talk using shape-coded chitosan membranes entrapped with glucose oxidase (GOX), peroxidase (POD), and Amplex Red (AmR) or alcohol oxidase (AOX), POD, and AmR by using same fluorescence indicator (AmR). PMID:23959236

Light-addressed electrodeposition of enzyme-entrapped chitosan membranes for multiplexed enzyme-based bioassays using a digital micromirror device.

PubMed

Huang, Shih-Hao; Wei, Lu-Shiuan; Chu, Hsiao-Tzu; Jiang, Yeu-Long

2013-08-16

This paper describes a light-addressed electrolytic system used to perform an electrodeposition of enzyme-entrapped chitosan membranes for multiplexed enzyme-based bioassays using a digital micromirror device (DMD). In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-cathode to electrolytically produce hydroxide ions, which leads to an increased pH gradient. The high pH generated at the cathode can cause a local gelation of chitosan through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressed electrodeposition of chitosan membranes with different shapes and sizes, as well as multiplexed micropatterning, was performed. The effect of the illumination time of the light pattern on the dimensional resolution of chitosan membrane formation was examined experimentally. Moreover, multiplexed enzyme-based bioassay of enzyme-entrapped chitosan membranes was also successfully demonstrated through the electrodeposition of the chitosan membranes with various shapes/sizes and entrapping different enzymes. As a model experiment, glucose and ethanol were simultaneously detected in a single detection chamber without cross-talk using shape-coded chitosan membranes entrapped with glucose oxidase (GOX), peroxidase (POD), and Amplex Red (AmR) or alcohol oxidase (AOX), POD, and AmR by using same fluorescence indicator (AmR).

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

PubMed

Bolognesi, Maddalena Maria; Manzoni, Marco; Scalia, Carla Rossana; Zannella, Stefano; Bosisio, Francesca Maria; Faretta, Mario; Cattoretti, Giorgio

2017-08-01

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

Synthesis and fluorescence studies of multiple labeled oligonucleotides containing dansyl fluorophore covalently attached at 2'-terminus of cytidine via carbamate linkage.

PubMed

Misra, Arvind; Mishra, Satyendra; Misra, Krishna

2004-01-01

Synthesis of modified oligonucleotides in which the specific cytidine nucleoside analogues linked at 2'-OH position via a carbamate bond with an amino ethyl derivative of dansyl fluorophore is reported. For the multiple labeling of oligonucleotides, a strategy involving prelabeling at the monomeric level followed by solid phase assembly of oligonucleotides to obtain regiospecifically labeled probes has been described. The labeled monomer was phosphitylated using 2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoramidite (Bis-reagent) and pyridiniumtrifluoro acetate (Py.TFA) as an activator. To ascertain the minimal number of labeled monomers required for a specific length of oligonucleotide for detection and also to assess the effect of carbamate linkage on hybridization, hexamer and 20-mer sequences were selected. Both were labeled with 1, 2, and 3 monomers at the 5'-end and hybridized with normal (unmodified) complementary sequences. As compared to midsequence or 3'-terminal labeling reported earlier, the 5'-terminal labeling has been found to have minimal contact-mediated quenching on duplex formation. This may be due to complementary deoxyguanosine (dG) rich oligonucleotide sequences or CG base pairs at a terminus that is known to yield stronger binding. This is one reason for selecting cytidine for labeling. The results may aid rational design of multiple fluorescent DNA probes for nonradioactive detection of nucleic acids.

Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

NASA Astrophysics Data System (ADS)

Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

2015-01-01

MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of

Multiplexed flow cytometric sensing of blood electrolytes in physiological samples using fluorescent bulk optode microspheres.

PubMed

Xu, Chao; Wygladacz, Katarzyna; Retter, Robert; Bell, Michael; Bakker, Eric

2007-12-15

Polymeric bulk optode microsphere ion sensors in combination with suspension array technologies such as analytical flow cytometry may become a power tool for measuring electrolytes in physiological samples. In this work, the methodology for the direct measurement of common blood electrolytes in physiological samples using bulk optode microsphere sensors was explored. The simultaneous determination of Na(+), K(+), and Ca(2+) in diluted sheep blood plasma was demonstrated for the first time, using a random suspension array containing three types of mixed microsphere bulk optodes of similar size, fabricated from the same chromoionophore without additional labeling. Sodium ionophore X, potassium ionophore III, and grafted AU-1 in poly(butyl acrylate) were the ionophores used in the bulk optode microsphere ion sensors for Na(+), K(+), and Ca(2+), respectively, in combination with the cation-exchanger NaTFPB (sodium tetrakis-[3,5-bis(trifluoromethyl)phenyl]borate) and the same concentration of the chromoionophore ETH 5294 (9-(di-ethylamino)-5-octadecanoylimino-5H-benzo[a]phen-oxazine) in plasticized poly(vinyl chloride). Excellent reproducibility was achieved for the sensing of potassium ions. The effect of sample pH was relatively small at near-physiological pH and followed theoretical predictions, yet the sample temperature was found to influence the sensor response to a larger extent. Multiplexed ion sensing was achieved by taking advantage of the chemical tunability of the sensor response, adjusting the sensor compositions so that the three types of ion sensors responded with distinct levels of protonation of the chromoionophore. Consequently, three well-resolved peaks were simultaneously observed in the single-channel histogram during the multiplexed calibration as well as in the subsequent measurement of the three cations in 10-fold-diluted sheep plasma. The assigned peak positions corresponded very well to the physiological range of the measured ions.

Growing multiplex networks with arbitrary number of layers

NASA Astrophysics Data System (ADS)

Momeni, Naghmeh; Fotouhi, Babak

2015-12-01

This paper focuses on the problem of growing multiplex networks. Currently, the results on the joint degree distribution of growing multiplex networks present in the literature pertain to the case of two layers and are confined to the special case of homogeneous growth and are limited to the state state (that is, the limit of infinite size). In the present paper, we first obtain closed-form solutions for the joint degree distribution of heterogeneously growing multiplex networks with arbitrary number of layers in the steady state. Heterogeneous growth means that each incoming node establishes different numbers of links in different layers. We consider both uniform and preferential growth. We then extend the analysis of the uniform growth mechanism to arbitrary times. We obtain a closed-form solution for the time-dependent joint degree distribution of a growing multiplex network with arbitrary initial conditions. Throughout, theoretical findings are corroborated with Monte Carlo simulations. The results shed light on the effects of the initial network on the transient dynamics of growing multiplex networks and takes a step towards characterizing the temporal variations of the connectivity of growing multiplex networks, as well as predicting their future structural properties.

Efficient Record Linkage Algorithms Using Complete Linkage Clustering

PubMed Central

Mamun, Abdullah-Al; Aseltine, Robert; Rajasekaran, Sanguthevar

2016-01-01

Data from different agencies share data of the same individuals. Linking these datasets to identify all the records belonging to the same individuals is a crucial and challenging problem, especially given the large volumes of data. A large number of available algorithms for record linkage are prone to either time inefficiency or low-accuracy in finding matches and non-matches among the records. In this paper we propose efficient as well as reliable sequential and parallel algorithms for the record linkage problem employing hierarchical clustering methods. We employ complete linkage hierarchical clustering algorithms to address this problem. In addition to hierarchical clustering, we also use two other techniques: elimination of duplicate records and blocking. Our algorithms use sorting as a sub-routine to identify identical copies of records. We have tested our algorithms on datasets with millions of synthetic records. Experimental results show that our algorithms achieve nearly 100% accuracy. Parallel implementations achieve almost linear speedups. Time complexities of these algorithms do not exceed those of previous best-known algorithms. Our proposed algorithms outperform previous best-known algorithms in terms of accuracy consuming reasonable run times. PMID:27124604

Efficient Record Linkage Algorithms Using Complete Linkage Clustering.

PubMed

Mamun, Abdullah-Al; Aseltine, Robert; Rajasekaran, Sanguthevar

2016-01-01

Data from different agencies share data of the same individuals. Linking these datasets to identify all the records belonging to the same individuals is a crucial and challenging problem, especially given the large volumes of data. A large number of available algorithms for record linkage are prone to either time inefficiency or low-accuracy in finding matches and non-matches among the records. In this paper we propose efficient as well as reliable sequential and parallel algorithms for the record linkage problem employing hierarchical clustering methods. We employ complete linkage hierarchical clustering algorithms to address this problem. In addition to hierarchical clustering, we also use two other techniques: elimination of duplicate records and blocking. Our algorithms use sorting as a sub-routine to identify identical copies of records. We have tested our algorithms on datasets with millions of synthetic records. Experimental results show that our algorithms achieve nearly 100% accuracy. Parallel implementations achieve almost linear speedups. Time complexities of these algorithms do not exceed those of previous best-known algorithms. Our proposed algorithms outperform previous best-known algorithms in terms of accuracy consuming reasonable run times.

Multiplex Detection of IgG and IgM to Rift Valley Fever Virus Nucleoprotein, Nonstructural Proteins, and Glycoprotein in Ovine and Bovine.

PubMed

Hossain, Mohammad M; Wilson, William C; Faburay, Bonto; Richt, Jürgen; McVey, David S; Rowland, Raymond R

2016-08-01

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were assembled into a multiplex and tested in serum samples from infected wild-type RVFV or MP12, a modified live virus vaccine. As expected, the N protein was immunodominant and the best target for early detection of infection. Antibody activity against the other targets was also detected. The experimental results demonstrate the capabilities of FMIA for the detection of antibodies to RVFV structural and nonstructural proteins, which can be applied to future development and validation of diagnostic tests that can be used to differentiate vaccinated from infected animals.

47 CFR 73.319 - FM multiplex subcarrier technical standards.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 4 2011-10-01 2011-10-01 false FM multiplex subcarrier technical standards. 73... SERVICES RADIO BROADCAST SERVICES FM Broadcast Stations § 73.319 FM multiplex subcarrier technical standards. (a) The technical specifications in this Section apply to all transmissions of FM multiplex...

Multiplexed lasing in tissues

NASA Astrophysics Data System (ADS)

Chen, Yu-Cheng; Chen, Qiushu; Fan, Xudong

2017-02-01

Biolasers are an emerging technology for next generation biochemical detection and clinical applications. Progress has recently been made to achieve lasing from biomolecules and single living cells. Tissues, which consist of cells embedded in extracellular matrix, mimic more closely the actual complex biological environment in a living body and therefore are of more practical significance. Here, we developed a highly versatile tissue laser platform, in which tissues stained with fluorophores are sandwiched in a high-Q Fabry-Pérot microcavity. Distinct lasing emissions from muscle and adipose tissues stained respectively with fluorescein isothiocyanate (FITC) and boron-dipyrromethene (BODIPY), and hybrid muscle/adipose tissue with dual-staining were achieved with a threshold of only 10 μJ/mm2. Additionally, we investigated how tissue structure/geometry, tissue thickness, and staining dye concentration affect the tissue laser. It is further found that, despite large fluorescence spectral overlap between FITC and BODIPY in tissues, their lasing emissions could be clearly distinguished and controlled due to their narrow lasing bands and different lasing thresholds, thus enabling highly multiplexed detection. Our tissue laser platform can be broadly applicable to various types of tissues/diseases. It provides a new tool for a wide range of biological and biomedical applications, such as diagnostics/screening of tissues and identification/monitoring of biological transformations in tissue engineering.

Automated methods for multiplexed pathogen detection.

PubMed

Straub, Timothy M; Dockendorff, Brian P; Quiñonez-Díaz, Maria D; Valdez, Catherine O; Shutthanandan, Janani I; Tarasevich, Barbara J; Grate, Jay W; Bruckner-Lea, Cynthia J

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However

A new design approach to MMI-based (de)multiplexers

NASA Astrophysics Data System (ADS)

Yueyu, Xiao; Sailing, He

2004-09-01

A novel design method of the wavelength (de)multiplexer is presented. The output spectral response of a (de)multiplexer is designed from the view of FIR filters. Avoiding laborious mathematic analysis, the (de)multiplexer is analyzed and designed in this explicit and simple method. A four channel (de)multiplexer based on multimode interference (MMI) is designed as an example. The result obtained agrees with that of the commonly used method, and is verified by a finite difference beam propagation method (FDBPM) simulation.

Time-division multiplexer uses digital gates

NASA Technical Reports Server (NTRS)

Myers, C. E.; Vreeland, A. E.

1977-01-01

Device eliminates errors caused by analog gates in multiplexing a large number of channels at high frequency. System was designed for use in aerospace work to multiplex signals for monitoring such variables as fuel consumption, pressure, temperature, strain, and stress. Circuit may be useful in monitoring variables in process control and medicine as well.

A multiplexed quantum memory.

PubMed

Lan, S-Y; Radnaev, A G; Collins, O A; Matsukevich, D N; Kennedy, T A; Kuzmich, A

2009-08-03

A quantum repeater is a system for long-distance quantum communication that employs quantum memory elements to mitigate optical fiber transmission losses. The multiplexed quantum memory (O. A. Collins, S. D. Jenkins, A. Kuzmich, and T. A. B. Kennedy, Phys. Rev. Lett. 98, 060502 (2007)) has been shown theoretically to reduce quantum memory time requirements. We present an initial implementation of a multiplexed quantum memory element in a cold rubidium gas. We show that it is possible to create atomic excitations in arbitrary memory element pairs and demonstrate the violation of Bell's inequality for light fields generated during the write and read processes.

Image Decoding of Photonic Crystal Beads Array in the Microfluidic Chip for Multiplex Assays

PubMed Central

Yuan, Junjie; Zhao, Xiangwei; Wang, Xiaoxia; Gu, Zhongze

2014-01-01

Along with the miniaturization and intellectualization of biomedical instruments, the increasing demand of health monitoring at anywhere and anytime elevates the need for the development of point of care testing (POCT). Photonic crystal beads (PCBs) as one kind of good encoded microcarriers can be integrated with microfluidic chips in order to realize cost-effective and high sensitive multiplex bioassays. However, there are difficulties in analyzing them towards automated analysis due to the characters of the PCBs and the unique detection manner. In this paper, we propose a strategy to take advantage of automated image processing for the color decoding of the PCBs array in the microfluidic chip for multiplex assays. By processing and alignment of two modal images of epi-fluorescence and epi-white light, every intact bead in the image is accurately extracted and decoded by PC colors, which stand for the target species. This method, which shows high robustness and accuracy under various configurations, eliminates the high hardware requirement of spectroscopy analysis and user-interaction software, and provides adequate supports for the general automated analysis of POCT based on PCBs array. PMID:25341876

Cavity enhanced eigenmode multiplexing for volume holographic data storage

NASA Astrophysics Data System (ADS)

Miller, Bo E.; Takashima, Yuzuru

2017-08-01

Previously, we proposed and experimentally demonstrated enhanced recording speeds by using a resonant optical cavity to semi-passively increase the reference beam power while recording image bearing holograms. In addition to enhancing the reference beam power the cavity supports the orthogonal reference beam families of its eigenmodes, which can be used as a degree of freedom to multiplex data pages and increase storage densities for volume Holographic Data Storage Systems (HDSS). While keeping the increased recording speed of a cavity enhanced reference arm, image bearing holograms are multiplexed by orthogonal phase code multiplexing via Hermite-Gaussian eigenmodes in a Fe:LiNbO3 medium with a 532 nm laser at two Bragg angles for expedited recording of four multiplexed holograms. We experimentally confirmed write rates are enhanced by an average factor of 1.1, and page crosstalk is about 2.5%. This hybrid multiplexing opens up a pathway to increase storage density while minimizing modifications to current angular multiplexing HDSS.

Multiplex Detection of Toxigenic Penicillium Species.

PubMed

Rodríguez, Alicia; Córdoba, Juan J; Rodríguez, Mar; Andrade, María J

2017-01-01

Multiplex PCR-based methods for simultaneous detection and quantification of different mycotoxin-producing Penicillia are useful tools to be used in food safety programs. These rapid and sensitive techniques allow taking corrective actions during food processing or storage for avoiding accumulation of mycotoxins in them. In this chapter, three multiplex PCR-based methods to detect at least patulin- and ochratoxin A-producing Penicillia are detailed. Two of them are different multiplex real-time PCR suitable for monitoring and quantifying toxigenic Penicillium using the nonspecific dye SYBR Green and specific hydrolysis probes (TaqMan). All of them successfully use the same target genes involved in the biosynthesis of such mycotoxins for designing primers and/or probes.

Multiplexing in the primate motion pathway.

PubMed

Huk, Alexander C

2012-06-01

This article begins by reviewing recent work on 3D motion processing in the primate visual system. Some of these results suggest that 3D motion signals may be processed in the same circuitry already known to compute 2D motion signals. Such "multiplexing" has implications for the study of visual cortical circuits and neural signals. A more explicit appreciation of multiplexing--and the computations required for demultiplexing--may enrich the study of the visual system by emphasizing the importance of a structured and balanced "encoding/decoding" framework. In addition to providing a fresh perspective on how successive stages of visual processing might be approached, multiplexing also raises caveats about the value of "neural correlates" for understanding neural computation.

In vivo tomographic imaging of deep seated cancer using fluorescence lifetime contrast

PubMed Central

Rice, William L.; Shcherbakova, Daria M; Verkusha, Vladislav V.; Kumar, Anand T.N.

2015-01-01

Preclinical cancer research would benefit from non-invasive imaging methods that allow tracking and visualization of early stage metastasis in vivo. While fluorescent proteins revolutionized intravital microscopy, two major challenges which still remain are tissue autofluorescence and hemoglobin absorption, which act to limit intravital optical techniques to large or subcutaneous tumors. Here we employ time-domain technology for the effective separation of tissue autofluorescence from extrinsic fluorophores, based on their distinct fluorescence lifetimes. Additionally, we employ cancer cells labelled with near infra-red fluorescent proteins (iRFP) to allow deep-tissue imaging. Our results demonstrate that time-domain imaging allows the detection of metastasis in deep-seated organs of living mice with a more than 20-fold increase in sensitivity compared to conventional continuous wave techniques. Furthermore, the distinct fluorescence lifetimes of each iRFP enables lifetime multiplexing of three different tumors, each expressing unique iRFP labels in the same animal. Fluorescence tomographic reconstructions reveal 3D distributions of iRFP720-expressing cancer cells in lungs and brain of live mice, allowing ready longitudinal monitoring of cancer cell fate with greater sensitivity than otherwise currently possible. PMID:25670171

FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging

PubMed Central

Zhao, Ming; Li, Yu; Peng, Leilei

2014-01-01

We report a fast non-iterative lifetime data analysis method for the Fourier multiplexed frequency-sweeping confocal FLIM (Fm-FLIM) system [ Opt. Express22, 10221 ( 2014)24921725]. The new method, named R-method, allows fast multi-channel lifetime image analysis in the system’s FPGA data processing board. Experimental tests proved that the performance of the R-method is equivalent to that of single-exponential iterative fitting, and its sensitivity is well suited for time-lapse FLIM-FRET imaging of live cells, for example cyclic adenosine monophosphate (cAMP) level imaging with GFP-Epac-mCherry sensors. With the R-method and its FPGA implementation, multi-channel lifetime images can now be generated in real time on the multi-channel frequency-sweeping FLIM system, and live readout of FRET sensors can be performed during time-lapse imaging. PMID:25321778

Nanoscale Test Strips for Multiplexed Blood Analysis

NASA Technical Reports Server (NTRS)

Chan, Eugene

2015-01-01

A critical component of the DNA Medicine Institute's Reusable Handheld Electrolyte and Lab Technology for Humans (rHEALTH) sensor are nanoscale test strips, or nanostrips, that enable multiplexed blood analysis. Nanostrips are conceptually similar to the standard urinalysis test strip, but the strips are shrunk down a billionfold to the microscale. Each nanostrip can have several sensor pads that fluoresce in response to different targets in a sample. The strips carry identification tags that permit differentiation of a specific panel from hundreds of other nanostrip panels during a single measurement session. In Phase I of the project, the company fabricated, tested, and demonstrated functional parathyroid hormone and vitamin D nanostrips for bone metabolism, and thrombin aptamer and immunoglobulin G antibody nanostrips. In Phase II, numerous nanostrips were developed to address key space flight-based medical needs: assessment of bone metabolism, immune response, cardiac status, liver metabolism, and lipid profiles. This unique approach holds genuine promise for space-based portable biodiagnostics and for point-of-care (POC) health monitoring and diagnostics here on Earth.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

PubMed

Kamburova, E G; Wisse, B W; Joosten, I; Allebes, W A; van der Meer, A; Hilbrands, L B; Baas, M C; Spierings, E; Hack, C E; van Reekum, F E; van Zuilen, A D; Verhaar, M; Bots, M L; Drop, A C A D; Plaisier, L; Seelen, M A J; Sanders, J S F; Hepkema, B G; Lambeck, A J; Bungener, L B; Roozendaal, C; Tilanus, M G J; Vanderlocht, J; Voorter, C E; Wieten, L; van Duijnhoven, E M; Gelens, M; Christiaans, M H L; van Ittersum, F J; Nurmohamed, A; Lardy, N M; Swelsen, W; van der Pant, K A; van der Weerd, N C; Ten Berge, I J M; Bemelman, F J; Hoitsma, A; van der Boog, P J M; de Fijter, J W; Betjes, M G H; Heidt, S; Roelen, D L; Claas, F H; Otten, H G

2016-09-01

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Detection of Neisseria meningitidis in cerebrospinal fluid using a multiplex PCR and the Luminex detection technology.

PubMed

Møller, Jens Kjølseth

2012-01-01

Rapid clinical and laboratory diagnoses are the foundation for a successful management of serious infections with Neisseria meningitidis. A species-specific multiplex polymerase chain reaction (PCR) coupled with fluidic microarrays using microbeads (the Luminex xMAP™ Technology) can detect pathogens most frequently found in the cerebrospinal fluid of patients. The Luminex suspension array system uniquely combines flow cytometry, microspheres, laser technology, digital signal processing, and traditional chemistry. In this method, the reaction is carried out in one vessel, in which distinctly color-coded bead sets, each conjugated with a different specific nucleic acid reactant, are hybridized with the PCR products, and a reporter molecule is used to quantify the interaction. The flow-based Luminex array reader identifies each reaction (bead set) after excitation by a red classification laser. Reporter signals from each reaction are simultaneously quantified by fluorescence generated by a green reporter laser. This nonculture, multiplex assay may prove to be an important tool for optimal laboratory diagnosis, not only of meningococcal meningitis, but also of meningitis caused by other bacterial or viral pathogens.

Three-mode all-optical (de)multiplexing on a SOI chip

NASA Astrophysics Data System (ADS)

Le, Yan-Si; Wang, Zhi; Li, Zhi-Yong; Li, Ying; Li, Qiang; Cui, Can; Wu, Chong-Qing

2018-01-01

An on-chip three-mode division multiplexing circuit using a simple ADC-based TE0 & TE1 & TE2 (de)multiplexer is demonstrated to improve the link capacity of on-chip optical interconnects. The proposed (de)multiplexer does not contain any tapered waveguide which is different from the previous mode (de)multiplexer based on ADCs. Here, we choose multimode waveguide width first and then confirm corresponding width of the other two waveguides. Thus the bus waveguide without any tapers can not only reduce complexity of (de)multiplexer but also reduce difficulty of the fabrication. Our simulation results show that the hybrid multiplexer has relatively low loss and low crosstalk about -40 dB, -26.99 dB and -28.72 dB for each mode around 1550 nm with a width-variation w =± 25 nm. These properties make the proposed mode-(de)multiplexer suitable for application in high-capacity data transmission.

Multiplexed 3D FRET imaging in deep tissue of live embryos

PubMed Central

Zhao, Ming; Wan, Xiaoyang; Li, Yu; Zhou, Weibin; Peng, Leilei

2015-01-01

Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca2+ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms. PMID:26387920

Interlaboratory Evaluation of a Multiplexed High Information Content In Vitro Genotoxicity Assay

PubMed Central

Bryce, Steven M.; Bernacki, Derek T.; Bemis, Jeffrey C.; Spellman, Richard A.; Engel, Maria E.; Schuler, Maik; Lorge, Elisabeth; Heikkinen, Pekka T.; Hemmann, Ulrike; Thybaud, Véronique; Wilde, Sabrina; Queisser, Nina; Sutter, Andreas; Zeller, Andreas; Guérard, Melanie; Kirkland, David; Dertinger, Stephen D.

2017-01-01

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. PMID:28370322

Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

PubMed Central

2016-01-01

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions. PMID:25555111

A duplicated PLP gene causing Pelizaeus-Merzbacher disease detected by comparative multiplex PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inoue, K.; Sugiyama, N.; Kawanishi, C.

1996-07-01

Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused by abnormalities in the proteolipid protein (PLP) gene, which is essential for oligodendrocyte differentiation and CNS myelin formation. Although linkage analysis has shown the homogeneity at the PLP locus in patients with PMD, exonic mutations in the PLP gene have been identified in only 10% - 25% of all cases, which suggests the presence of other genetic aberrations, including gene duplication. In this study, we examined five families with PMD not carrying exonic mutations in PLP gene, using comparative multiplex PCR (CM-PCR) as a semiquantitative assay of gene dosage. PLP genemore » duplications were identified in four families by CM-PCR and confirmed in three families by densitometric RFLP analysis. Because a homologous myelin protein gene, PMP22, is duplicated in the majority of patients with Charcot-Marie-Tooth 1A, PLP gene overdosage may be an important genetic abnormality in PMD and affect myelin formation. 38 ref., 5 figs., 2 tabs.« less

The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae, Lamiaceae, Rosaceae.

PubMed

Sytar, Oksana; Bruckova, Klaudia; Hunkova, Elena; Zivcak, Marek; Konate, Kiessoun; Brestic, Marian

2015-01-16

The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to family Rosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R) 3 (Force-A, France) for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units), that is deduced from flavonoids UV absorbing properties. Among observed plant species, the highest amount of total flavonoids has been found in leaves of Helianthus multiflorus (1.65 RU) and Echinops ritro (1.27 RU), Rudbeckia fulgida (1.13 RU) belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis) (0.14 RU) also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU) and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU). This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05) in flavonoids content were observed not only between families, but also among individual plant species within one family.

Helicity multiplexed broadband metasurface holograms.

PubMed

Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Pun, Edwin Yue Bun; Zhang, Shuang; Chen, Xianzhong

2015-09-10

Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices.

Helicity multiplexed broadband metasurface holograms

PubMed Central

Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Yue Bun Pun, Edwin; Zhang, Shuang; Chen, Xianzhong

2015-01-01

Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices. PMID:26354497

Linkage analysis of alternative anxiety phenotypes in multiply affected panic disorder families

PubMed Central

Fyer, Abby J.; Costa, Ramiro; Haghighi, Fatemeh; Logue, Mark W.; Knowles, James A.; Weissman, Myrna M.; Hodge, Susan E.; Hamilton, Steven P.

2013-01-01

Background The choice of phenotype definitions for genetic studies of panic and phobic disorders is complicated by family, twin and neurobiological data indicating both distinct and shared risk factors as well as heterogeneity within categories. We previously reported a genome scan in 120 multiplex panic disorder (PD) families using a phenotype that closely adhered to the DSM IV PD definition. Here we extend this work by conducting exploratory linkage analyses in this same pedigree set using ten additional literature- based panic and phobia-related phenotypes that take into account aspects of these hypothesized complexities. Methods Multiply affected families (> 2 individuals with PD) were recruited from clinical and non-clinical sources, evaluated by clinician administered semi-structured interview and subsequent blind consensus best estimate procedure. Each phenotype was analyzed under dominant and recessive models using parametric 2-point (homogeneity and heterogeneity), multipoint, and non-parametric methods. Empirically based permutations were used to estimate model specific and global (across all phenotypes) p-values. Results The highest score was a 2-point lod (4.27, global p < 0.08) on chromosome 13 (D13S793, 76cM) for the phenotype “specific or social phobia” under a recessive model and conditions of homogeneity. There was minimal support for linkage to any of the remaining nine phenotypes. Conclusions Though interpretation of findings is limited by sample size and the large number of phenotypes and models analyzed these data suggest a region on chromosome 13 as a potential site for further exploration in relation to risk for specific and social phobias. PMID:22525237

Transmission of multiplexed video signals in multimode optical fiber systems

NASA Technical Reports Server (NTRS)

White, Preston, III

1988-01-01

Kennedy Space Center has the need for economical transmission of two multiplexed video signals along multimode fiberoptic systems. These systems must span unusual distances and must meet RS-250B short-haul standards after reception. Bandwidth is a major problem and studies of the installed fibers, available LEDs and PINFETs led to the choice of 100 MHz as the upper limit for the system bandwidth. Optical multiplexing and digital transmission were deemed inappropriate. Three electrical multiplexing schemes were chosen for further study. Each of the multiplexing schemes included an FM stage to help meet the stringent S/N specification. Both FM and AM frequency division multiplexing methods were investigated theoretically and these results were validated with laboratory tests. The novel application of quadrature amplitude multiplexing was also considered. Frequency division multiplexing of two wideband FM video signal appears the most promising scheme although this application requires high power highly linear LED transmitters. Futher studies are necessary to determine if LEDs of appropriate quality exist and to better quantify performance of QAM in this application.

Optical switch compatible with wavelength division multiplexing and mode division multiplexing for photonic networks-on-chip.

PubMed

Jia, Hao; Zhou, Ting; Zhang, Lei; Ding, Jianfeng; Fu, Xin; Yang, Lin

2017-08-21

We propose a 2 × 2 multimode optical switch, which is composed of two mode de-multiplexers, n 2 × 2 single-mode optical switches where n is the number of the supported spatial modes, and two mode multiplexers. As a proof of concept, asymmetric directional couplers are employed to construct the mode multiplexers and de-multiplexers, balanced Mach-Zehnder interferometer is utilized to construct the 2 × 2 single-mode optical switches. The fabricated silicon 2 × 2 multimode optical switch has a broad optical bandwidth and can support four spatial modes. The link-crosstalk for all four modes is smaller than -18.8 dB. The inter-mode crosstalk for the same optical link is less than -22.1 dB. 40 Gbps data transmission is performed for all spatial modes and all optical links. The power penalties for the error-free switching (BER<10 -9 ) at 25 Gbps are less than 1.8 dB for all channels at the wavelength of 1550 nm. The power consumption of the device is 117.9 mW in the "cross" state and 116.2 mW in the "bar" state. The switching time is about 21 μs. This work enables large-capacity multimode photonic networks-on-chip.

High-volume optical vortex multiplexing and de-multiplexing for free-space optical communication.

PubMed

Wang, Zhongxi; Zhang, N; Yuan, X-C

2011-01-17

We report an approach to the increase of signal channels in free-space optical communication based on composed optical vortices (OVs). In the encoding process, conventional algorithm employed for the generation of collinearly superimposed OVs is combined with a genetic algorithm to achieve high-volume OV multiplexing. At the receiver end, a novel Dammann vortex grating is used to analyze the multihelix beams with a large number of OVs. We experimentally demonstrate a digitized system which is capable of transmitting and receiving 16 OV channels simultaneously. This system is expected to be compatible with a high-speed OV multiplexing technique, with potentials to extremely high-volume information density in OV communication.

The use of analytical sedimentation velocity to extract thermodynamic linkage

PubMed Central

Cole, James L.; Correia, John J.; Stafford, Walter F.

2011-01-01

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980’s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS. PMID:21703752

Automated Methods for Multiplexed Pathogen Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cyclermore » where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities

Superconducting Digital Multiplexers for Sensor Arrays

NASA Technical Reports Server (NTRS)

Kadin, Alan M.; Brock, Darren K.; Gupta, Deepnarayan

2004-01-01

Arrays of cryogenic microbolometers and other cryogenic detectors are being developed for infrared imaging. If the signal from each sensor is amplified, multiplexed, and digitized using superconducting electronics, then this data can be efficiently read out to ambient temperature with a minimum of noise and thermal load. HYPRES is developing an integrated system based on SQUID amplifiers, a high-resolution analog-to-digital converter (ADC) based on RSFQ (rapid single flux quantum) logic, and a clocked RSFQ multiplexer. The ADC and SQUIDs have already been demonstrated for other projects, so this paper will focus on new results of a digital multiplexer. Several test circuits have been fabricated using Nb Josephson technology and are about to be tested at T = 4.2 K, with a more complete prototype in preparation.

Metric projection for dynamic multiplex networks.

PubMed

Jurman, Giuseppe

2016-08-01

Evolving multiplex networks are a powerful model for representing the dynamics along time of different phenomena, such as social networks, power grids, biological pathways. However, exploring the structure of the multiplex network time series is still an open problem. Here we propose a two-step strategy to tackle this problem based on the concept of distance (metric) between networks. Given a multiplex graph, first a network of networks is built for each time step, and then a real valued time series is obtained by the sequence of (simple) networks by evaluating the distance from the first element of the series. The effectiveness of this approach in detecting the occurring changes along the original time series is shown on a synthetic example first, and then on the Gulf dataset of political events.

Suspension arrays based on nanoparticle-encoded microspheres for high-throughput multiplexed detection

PubMed Central

Leng, Yuankui

2017-01-01

Spectrometrically or optically encoded microsphere based suspension array technology (SAT) is applicable to the high-throughput, simultaneous detection of multiple analytes within a small, single sample volume. Thanks to the rapid development of nanotechnology, tremendous progress has been made in the multiplexed detecting capability, sensitivity, and photostability of suspension arrays. In this review, we first focus on the current stock of nanoparticle-based barcodes as well as the manufacturing technologies required for their production. We then move on to discuss all existing barcode-based bioanalysis patterns, including the various labels used in suspension arrays, label-free platforms, signal amplification methods, and fluorescence resonance energy transfer (FRET)-based platforms. We then introduce automatic platforms for suspension arrays that use superparamagnetic nanoparticle-based microspheres. Finally, we summarize the current challenges and their proposed solutions, which are centered on improving encoding capacities, alternative probe possibilities, nonspecificity suppression, directional immobilization, and “point of care” platforms. Throughout this review, we aim to provide a comprehensive guide for the design of suspension arrays, with the goal of improving their performance in areas such as multiplexing capacity, throughput, sensitivity, and cost effectiveness. We hope that our summary on the state-of-the-art development of these arrays, our commentary on future challenges, and some proposed avenues for further advances will help drive the development of suspension array technology and its related fields. PMID:26021602

Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters

PubMed Central

Lai, Charles P.; Kim, Edward Y.; Badr, Christian E.; Weissleder, Ralph; Mempel, Thorsten R.; Tannous, Bakhos A.; Breakefield, Xandra O.

2015-01-01

Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA. PMID:25967391

Multiplexed Detection of Cytokines Based on Dual Bar-Code Strategy and Single-Molecule Counting.

PubMed

Li, Wei; Jiang, Wei; Dai, Shuang; Wang, Lei

2016-02-02

Cytokines play important roles in the immune system and have been regarded as biomarkers. While single cytokine is not specific and accurate enough to meet the strict diagnosis in practice, in this work, we constructed a multiplexed detection method for cytokines based on dual bar-code strategy and single-molecule counting. Taking interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) as model analytes, first, the magnetic nanobead was functionalized with the second antibody and primary bar-code strands, forming a magnetic nanoprobe. Then, through the specific reaction of the second antibody and the antigen that fixed by the primary antibody, sandwich-type immunocomplex was formed on the substrate. Next, the primary bar-code strands as amplification units triggered multibranched hybridization chain reaction (mHCR), producing nicked double-stranded polymers with multiple branched arms, which were served as secondary bar-code strands. Finally, the secondary bar-code strands hybridized with the multimolecule labeled fluorescence probes, generating enhanced fluorescence signals. The numbers of fluorescence dots were counted one by one for quantification with epi-fluorescence microscope. By integrating the primary and secondary bar-code-based amplification strategy and the multimolecule labeled fluorescence probes, this method displayed an excellent sensitivity with the detection limits were both 5 fM. Unlike the typical bar-code assay that the bar-code strands should be released and identified on a microarray, this method is more direct. Moreover, because of the selective immune reaction and the dual bar-code mechanism, the resulting method could detect the two targets simultaneously. Multiple analysis in human serum was also performed, suggesting that our strategy was reliable and had a great potential application in early clinical diagnosis.

A color-corrected strategy for information multiplexed Fourier ptychographic imaging

NASA Astrophysics Data System (ADS)

Wang, Mingqun; Zhang, Yuzhen; Chen, Qian; Sun, Jiasong; Fan, Yao; Zuo, Chao

2017-12-01

Fourier ptychography (FP) is a novel computational imaging technique that provides both wide field of view (FoV) and high-resolution (HR) imaging capacity for biomedical imaging. Combined with information multiplexing technology, wavelength multiplexed (or color multiplexed) FP imaging can be implemented by lighting up R/G/B LED units simultaneously. Furthermore, a HR image can be recovered at each wavelength from the multiplexed dataset. This enhances the efficiency of data acquisition. However, since the same dataset of intensity measurement is used to recover the HR image at each wavelength, the mean value in each channel would converge to the same value. In this paper, a color correction strategy embedded in the multiplexing FP scheme is demonstrated, which is termed as color corrected wavelength multiplexed Fourier ptychography (CWMFP). Three images captured by turning on a LED array in R/G/B are required as priori knowledge to improve the accuracy of reconstruction in the recovery process. Using the reported technique, the redundancy requirement of information multiplexed FP is reduced. Moreover, the accuracy of reconstruction at each channel is improved with correct color reproduction of the specimen.

Integrated multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Tan, Hongdong

2002-05-14

The present invention provides an integrated multiplexed capillary electrophoresis system for the analysis of sample analytes. The system integrates and automates multiple components, such as chromatographic columns and separation capillaries, and further provides a detector for the detection of analytes eluting from the separation capillaries. The system employs multiplexed freeze/thaw valves to manage fluid flow and sample movement. The system is computer controlled and is capable of processing samples through reaction, purification, denaturation, pre-concentration, injection, separation and detection in parallel fashion. Methods employing the system of the invention are also provided.

[MapDraw: a microsoft excel macro for drawing genetic linkage maps based on given genetic linkage data].

PubMed

Liu, Ren-Hu; Meng, Jin-Ling

2003-05-01

MAPMAKER is one of the most widely used computer software package for constructing genetic linkage maps.However, the PC version, MAPMAKER 3.0 for PC, could not draw the genetic linkage maps that its Macintosh version, MAPMAKER 3.0 for Macintosh,was able to do. Especially in recent years, Macintosh computer is much less popular than PC. Most of the geneticists use PC to analyze their genetic linkage data. So a new computer software to draw the same genetic linkage maps on PC as the MAPMAKER for Macintosh to do on Macintosh has been crying for. Microsoft Excel,one component of Microsoft Office package, is one of the most popular software in laboratory data processing. Microsoft Visual Basic for Applications (VBA) is one of the most powerful functions of Microsoft Excel. Using this program language, we can take creative control of Excel, including genetic linkage map construction, automatic data processing and more. In this paper, a Microsoft Excel macro called MapDraw is constructed to draw genetic linkage maps on PC computer based on given genetic linkage data. Use this software,you can freely construct beautiful genetic linkage map in Excel and freely edit and copy it to Word or other application. This software is just an Excel format file. You can freely copy it from ftp://211.69.140.177 or ftp://brassica.hzau.edu.cn and the source code can be found in Excel's Visual Basic Editor.

Constructing STR multiplex assays.

PubMed

Butler, John M

2005-01-01

Multiplex polymerase chain reaction (PCR) refers to the simultaneous amplification of multiple regions of deoxyribonucleic acid (DNA) using PCR. Commercial short tandem repeat (STR) assays that can coamplify as many as 16 different loci have become widely used in forensic DNA typing. This chapter will focus on some of the aspects of constructing robust STR multiplex assays, including careful design and quality control of PCR primers. Examples from the development of a cat STR 12plex and a human Y chromosome STR 20plex are used to illustrate the importance of various parts of the protocol. Primer design parameters and Internet-accessible resources are discussed, as are solutions to problems with residual dye artifacts that result from impure primers.

21 CFR 862.2570 - Instrumentation for clinical multiplex test systems.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Instrumentation for clinical multiplex test... Laboratory Instruments § 862.2570 Instrumentation for clinical multiplex test systems. (a) Identification. Instrumentation for clinical multiplex test systems is a device intended to measure and sort multiple signals...

21 CFR 862.2570 - Instrumentation for clinical multiplex test systems.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Instrumentation for clinical multiplex test... Laboratory Instruments § 862.2570 Instrumentation for clinical multiplex test systems. (a) Identification. Instrumentation for clinical multiplex test systems is a device intended to measure and sort multiple signals...

21 CFR 862.2570 - Instrumentation for clinical multiplex test systems.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Instrumentation for clinical multiplex test... Laboratory Instruments § 862.2570 Instrumentation for clinical multiplex test systems. (a) Identification. Instrumentation for clinical multiplex test systems is a device intended to measure and sort multiple signals...

21 CFR 862.2570 - Instrumentation for clinical multiplex test systems.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Instrumentation for clinical multiplex test... Laboratory Instruments § 862.2570 Instrumentation for clinical multiplex test systems. (a) Identification. Instrumentation for clinical multiplex test systems is a device intended to measure and sort multiple signals...

Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma

PubMed Central

Becker, Aline; Elder, Brad; Puduvalli, Vinay; Winter, Jessica; Gurcan, Metin

2017-01-01

Multiplexed immunofluorescent testing has not entered into diagnostic neuropathology due to the presence of several technical barriers, amongst which includes autofluorescence. This study presents the implementation of a methodology capable of overcoming the visual challenges of fluorescent microscopy for diagnostic neuropathology by using automated digital image analysis, with long term goal of providing unbiased quantitative analyses of multiplexed biomarkers for solid tissue neuropathology. In this study, we validated PTBP1, a putative biomarker for glioma, and tested the extent to which immunofluorescent microscopy combined with automated and unbiased image analysis would permit the utility of PTBP1 as a biomarker to distinguish diagnostically challenging surgical biopsies. As a paradigm, we utilized second resections from patients diagnosed either with reactive brain changes (pseudoprogression) and recurrent glioblastoma (true progression). Our image analysis workflow was capable of removing background autofluorescence and permitted quantification of DAPI-PTBP1 positive cells. PTBP1-positive nuclei, and the mean intensity value of PTBP1 signal in cells. Traditional pathological interpretation was unable to distinguish between groups due to unacceptably high discordance rates amongst expert neuropathologists. Our data demonstrated that recurrent glioblastoma showed more DAPI-PTBP1 positive cells and a higher mean intensity value of PTBP1 signal compared to resections from second surgeries that showed only reactive gliosis. Our work demonstrates the potential of utilizing automated image analysis to overcome the challenges of implementing fluorescent microscopy in diagnostic neuropathology. PMID:28282372

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

PubMed

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

A new OTDR based on probe frequency multiplexing

NASA Astrophysics Data System (ADS)

Lu, Lidong; Liang, Yun; Li, Binglin; Guo, Jinghong; Zhang, Xuping

2013-12-01

Two signal multiplexing methods are proposed and experimentally demonstrated in optical time domain reflectometry (OTDR) for fault location of optical fiber transmission line to obtain high measurement efficiency. Probe signal multiplexing is individually obtained by phase modulation for generation of multi-frequency and time sequential frequency probe pulses. The backscattered Rayleigh light of the multiplexing probe signals is transferred to corresponding heterodyne intermediate frequency (IF) through heterodyning with the single frequency local oscillator (LO). Then the IFs are simultaneously acquired by use of a data acquisition card (DAQ) with sampling rate of 100Msps, and the obtained data are processed by digital band pass filtering (BPF), digital down conversion (DDC) and digital low pass filtering (BPF) procedure. For each probe frequency of the detected signals, the extraction of the time domain reflecting signal power is performed by parallel computing method. For a comprehensive performance comparison with conventional coherent OTDR on the probe frequency multiplexing methods, the potential for enhancement of dynamic range, spatial resolution and measurement time are analyzed and discussed. Experimental results show that by use of the probe frequency multiplexing method, the measurement efficiency of coherent OTDR can be enhanced by nearly 40 times.

Burst noise in the HAWAII-1RG multiplexer

NASA Astrophysics Data System (ADS)

Bacon, Candice M.; McMurtry, Craig W.; Pipher, Judith L.; Forrest, William J.; Garnett, James D.

2005-08-01

Burst noise (also known as popcorn noise and random telegraph signal/noise) is a phenomenon that is understood to be a result of defects in the vicinity of a p-n junction. It is characterized by rapid level shifts in both positive and negative directions and can have varying magnitudes. This noise has been seen in both HAWAII-1RG and HAWAII-2RG multiplexers and is under investigation. We have done extensive burst noise testing on a HAWAII-1RG multiplexer, where we have determined a significant percentage of pixels exhibit the phenomenon. In addition, the prevalence of small magnitude transitions make sensitivity of detection the main limiting factor. Since this is a noise source for the HAWAII-1RG multiplexer, its elimination would make the HAWAII-1RG and the HAWAII-2RG even lower noise multiplexers.

Realizing up-conversion fluorescence tuning in lanthanide-doped nanocrystals by femtosecond pulse shaping method

PubMed Central

Zhang, Shian; Yao, Yunhua; Shuwu, Xu; Liu, Pei; Ding, Jingxin; Jia, Tianqing; Qiu, Jianrong; Sun, Zhenrong

2015-01-01

The ability to tune color output of nanomaterials is very important for their applications in laser, optoelectronic device, color display and multiplexed biolabeling. Here we first propose a femtosecond pulse shaping technique to realize the up-conversion fluorescence tuning in lanthanide-doped nanocrystals dispersed in the glass. The multiple subpulse formation by a square phase modulation can create different excitation pathways for various up-conversion fluorescence generations. By properly controlling these excitation pathways, the multicolor up-conversion fluorescence can be finely tuned. This color tuning by the femtosecond pulse shaping technique is realized in single material by single-color laser field, which is highly desirable for further applications of the lanthanide-doped nanocrystals. This femtosecond pulse shaping technique opens an opportunity to tune the color output in the lanthanide-doped nanocrystals, which may bring a new revolution in the control of luminescence properties of nanomaterials. PMID:26290391

Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.

PubMed

Hontani, Yusaku; Shcherbakova, Daria M; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav V; Kennis, John T M

2016-11-18

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Thirty-Four Megabit Four-Channel Multiplexer

DTIC Science & Technology

1985-10-01

person or conveying any rights or permis- sion to manufacture , use, or sell any patented invention thai ma> in any way be related thereto. WARNING...System (DCS) in Europe. The system is evolving from frequency division multiplex ( FDM ) to time division multiplex (TDM) equipment. The bulk of the...channel multi- plexer permits expansion by providing the spare MBS and 2.048~Mb/s data channels, and will do so with no additional cost

Multiplexing and Filtering of Optical Signals.

DTIC Science & Technology

1977-06-01

A0-A017 22« SPERRY RESEARCH CENTER SUOBURY MASS MULTIPLEXING ANO FILTERING OF OPTICAL SIGNALS.(U) JUN 77 A R NELSON UNCLASSIFIED SCRC-CR-77...0 F/G 20/6 Nil <r Research and Development Technical Report ECOM -76-1343-F MULTIPLEXING AND FILTERING OF OPTICAL SIGNALS A. R...Nelson SPERRY RESEARCH CENTER 100 North Road Sudbury, MA 01776 June 1977 Final Report for Period 29 April 1976 - 29 April 1977 DISTRIBUTION

Magnetically multiplexed heating of single domain nanoparticles

NASA Astrophysics Data System (ADS)

Christiansen, M. G.; Senko, A. W.; Chen, R.; Romero, G.; Anikeeva, P.

2014-05-01

Selective hysteretic heating of multiple collocated types of single domain magnetic nanoparticles (SDMNPs) by alternating magnetic fields (AMFs) may offer a useful tool for biomedical applications. The possibility of "magnetothermal multiplexing" has not yet been realized, in part due to prevalent use of linear response theory to model SDMNP heating in AMFs. Dynamic hysteresis modeling suggests that specific driving conditions play an underappreciated role in determining optimal material selection strategies for high heat dissipation. Motivated by this observation, magnetothermal multiplexing is theoretically predicted and empirically demonstrated by selecting SDMNPs with properties that suggest optimal hysteretic heat dissipation at dissimilar AMF driving conditions. This form of multiplexing could effectively offer multiple channels for minimally invasive biological signaling applications.

Eigenmode multiplexing with SLM for volume holographic data storage

NASA Astrophysics Data System (ADS)

Chen, Guanghao; Miller, Bo E.; Takashima, Yuzuru

2017-08-01

The cavity supports the orthogonal reference beam families as its eigenmodes while enhancing the reference beam power. Such orthogonal eigenmodes are used as additional degree of freedom to multiplex data pages, consequently increase storage densities for volume Holographic Data Storage Systems (HDSS) when the maximum number of multiplexed data page is limited by geometrical factor. Image bearing holograms are multiplexed by orthogonal phase code multiplexing via Hermite-Gaussian eigenmodes in a Fe:LiNbO3 medium with a 532 nm laser at multiple Bragg angles by using Liquid Crystal on Silicon (LCOS) spatial light modulators (SLMs) in reference arms. Total of nine holograms are recorded with three angular and three eigenmode.

Colorimetric and Fluorescent Biosensors Based on Directed Assembly of Nanomaterials with Functional DNA

NASA Astrophysics Data System (ADS)

Liu, Juewen; Lu, Yi

This chapter reviews recent progress in the interface between functional nucleic acids and nanoscale science and technology, and its analytical applications. In particular, the use of metallic nanoparticles as the color reporting groups for the action (binding, catalysis, or both) of aptamers, DNAzymes, and aptazymes is described in detail. Because metallic nanoparticles possess high extinction coefficients and distance-dependent optical properties, they allow highly sensitive detections with minimal consumption of materials. The combination of quantum dots (QDs) with functional nucleic acids as fluorescent sensors is also described. The chapter starts with the design of colorimetric and fluorescent sensors responsive to single analytes, followed by sensors responsive to multiple analytes with controllable cooperativity and multiplex detection using both colorimetric and fluorescent signals in one pot, and ends by transferring solution-based detections into litmus paper type of tests, making them generally applicable and usable for a wide range of on-site and real-time analytical applications such as household tests, environmental monitoring, and clinical diagnostics.

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

PubMed Central

Cole, Russell H.; Gartner, Zev J.; Abate, Adam R.

2016-01-01

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein. PMID:27214249

Spectral Demultiplexing in Holographic and Fluorescent On-chip Microscopy

NASA Astrophysics Data System (ADS)

Sencan, Ikbal; Coskun, Ahmet F.; Sikora, Uzair; Ozcan, Aydogan

2014-01-01

Lensfree on-chip imaging and sensing platforms provide compact and cost-effective designs for various telemedicine and lab-on-a-chip applications. In this work, we demonstrate computational solutions for some of the challenges associated with (i) the use of broadband, partially-coherent illumination sources for on-chip holographic imaging, and (ii) multicolor detection for lensfree fluorescent on-chip microscopy. Specifically, we introduce spectral demultiplexing approaches that aim to digitally narrow the spectral content of broadband illumination sources (such as wide-band light emitting diodes or even sunlight) to improve spatial resolution in holographic on-chip microscopy. We also demonstrate the application of such spectral demultiplexing approaches for wide-field imaging of multicolor fluorescent objects on a chip. These computational approaches can be used to replace e.g., thin-film interference filters, gratings or other optical components used for spectral multiplexing/demultiplexing, which can form a desirable solution for cost-effective and compact wide-field microscopy and sensing needs on a chip.

Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Pian, Qi; Yao, Ruoyang; Sinsuebphon, Nattawut; Intes, Xavier

2017-07-01

Spectrally resolved fluorescence lifetime imaging and spatial multiplexing have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (∼40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

The robustness of multiplex networks under layer node-based attack

PubMed Central

Zhao, Da-wei; Wang, Lian-hai; Zhi, Yong-feng; Zhang, Jun; Wang, Zhen

2016-01-01

From transportation networks to complex infrastructures, and to social and economic networks, a large variety of systems can be described in terms of multiplex networks formed by a set of nodes interacting through different network layers. Network robustness, as one of the most successful application areas of complex networks, has attracted great interest in a myriad of research realms. In this regard, how multiplex networks respond to potential attack is still an open issue. Here we study the robustness of multiplex networks under layer node-based random or targeted attack, which means that nodes just suffer attacks in a given layer yet no additional influence to their connections beyond this layer. A theoretical analysis framework is proposed to calculate the critical threshold and the size of giant component of multiplex networks when nodes are removed randomly or intentionally. Via numerous simulations, it is unveiled that the theoretical method can accurately predict the threshold and the size of giant component, irrespective of attack strategies. Moreover, we also compare the robustness of multiplex networks under multiplex node-based attack and layer node-based attack, and find that layer node-based attack makes multiplex networks more vulnerable, regardless of average degree and underlying topology. PMID:27075870

The robustness of multiplex networks under layer node-based attack.

PubMed

Zhao, Da-wei; Wang, Lian-hai; Zhi, Yong-feng; Zhang, Jun; Wang, Zhen

2016-04-14

From transportation networks to complex infrastructures, and to social and economic networks, a large variety of systems can be described in terms of multiplex networks formed by a set of nodes interacting through different network layers. Network robustness, as one of the most successful application areas of complex networks, has attracted great interest in a myriad of research realms. In this regard, how multiplex networks respond to potential attack is still an open issue. Here we study the robustness of multiplex networks under layer node-based random or targeted attack, which means that nodes just suffer attacks in a given layer yet no additional influence to their connections beyond this layer. A theoretical analysis framework is proposed to calculate the critical threshold and the size of giant component of multiplex networks when nodes are removed randomly or intentionally. Via numerous simulations, it is unveiled that the theoretical method can accurately predict the threshold and the size of giant component, irrespective of attack strategies. Moreover, we also compare the robustness of multiplex networks under multiplex node-based attack and layer node-based attack, and find that layer node-based attack makes multiplex networks more vulnerable, regardless of average degree and underlying topology.

System for Multiplexing Acoustic Emission (AE) Instrumentation

NASA Technical Reports Server (NTRS)

Prosser, William H. (Inventor); Perey, Daniel F. (Inventor); Gorman, Michael R. (Inventor); Scales, Edgar F. (Inventor)

2003-01-01

An acoustic monitoring device has at least two acoustic sensors with a triggering mechanism and a multiplexing circuit. After the occurrence of a triggering event at a sensor, the multiplexing circuit allows a recording component to record acoustic emissions at adjacent sensors. The acoustic monitoring device is attached to a solid medium to detect the occurrence of damage.

Dual-channel-coded microbeads for multiplexed detection of biomolecules using assembling of quantum dots and element coding nanoparticles.

PubMed

Lu, Bangrong; He, Qinghua; He, Yonghong; Chen, Xuejing; Feng, Guangxia; Liu, Siyu; Ji, Yanhong

2018-09-18

To achieve the dual-channel (analog and digital) encoding, microbeads assembled with quantum dots (QDs) and element coding nanoparticles (ECNPs) have been prepared. Dual-spectra, including fluorescence generated from quantum dots (QDs) and laser induced breakdown spectrum obtained from the plasma of ECNPs, including AgO, MgO and ZnO nanoparticles, has been adopted to provide more encoding amounts and more accurate dual recognition for encoded microbeads in multiplexed utilization. The experimental results demonstrate that the single microbead can be decoded in two optical channels. Multiplexed analysis and contrast adsorption experiment of anti-IgG verified the availability and specificity of dual-channel-coded microbeads in bioanalysis. In gradient detection of anti-IgG, we obtained the linear concentration response to target biomolecules from 3.125 × 10 -10  M to 1 × 10 -8  M, and the limit of detection was calculated to be 2.91 × 10 -11  M. Copyright © 2018 Elsevier B.V. All rights reserved.

Multiplexing of miniaturized planar antibody arrays for serum protein profiling--a biomarker discovery in SLE nephritis.

PubMed

Petersson, Linn; Dexlin-Mellby, Linda; Bengtsson, Anders A; Sturfelt, Gunnar; Borrebaeck, Carl A K; Wingren, Christer

2014-06-07

In the quest to decipher disease-associated biomarkers, miniaturized and multiplexed antibody arrays may play a central role in generating protein expression profiles, or protein maps, of crude serum samples. In this conceptual study, we explored a novel, 4-times larger pen design, enabling us to, in a unique manner, simultaneously print 48 different reagents (antibodies) as individual 78.5 μm(2) (10 μm in diameter) sized spots at a density of 38,000 spots cm(-2) using dip-pen nanolithography technology. The antibody array set-up was interfaced with a high-resolution fluorescent-based scanner for sensitive sensing. The performance and applicability of this novel 48-plex recombinant antibody array platform design was demonstrated in a first clinical application targeting SLE nephritis, a severe chronic autoimmune connective tissue disorder, as the model disease. To this end, crude, directly biotinylated serum samples were targeted. The results showed that the miniaturized and multiplexed array platform displayed adequate performance, and that SLE-associated serum biomarker panels reflecting the disease process could be deciphered, outlining the use of miniaturized antibody arrays for disease proteomics and biomarker discovery.

Validating multiplexes for use in conjunction with modern interpretation strategies.

PubMed

Taylor, Duncan; Bright, Jo-Anne; McGoven, Catherine; Hefford, Christopher; Kalafut, Tim; Buckleton, John

2016-01-01

In response to requests from the forensic community, commercial companies are generating larger, more sensitive, and more discriminating STR multiplexes. These multiplexes are now applied to a wider range of samples including complex multi-person mixtures. In parallel there is an overdue reappraisal of profile interpretation methodology. Aspects of this reappraisal include 1. The need for a quantitative understanding of allele and stutter peak heights and their variability, 2. An interest in reassessing the utility of smaller peaks below the often used analytical threshold, 3. A need to understand not just the occurrence of peak drop-in but also the height distribution of such peaks, and 4. A need to understand the limitations of the multiplex-interpretation strategy pair implemented. In this work we present a full scheme for validation of a new multiplex that is suitable for informing modern interpretation practice. We predominantly use GlobalFiler™ as an example multiplex but we suggest that the aspects investigated here are fundamental to introducing any multiplex in the modern interpretation environment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Integrated spatial multiplexing of heralded single-photon sources

PubMed Central

Collins, M.J.; Xiong, C.; Rey, I.H.; Vo, T.D.; He, J.; Shahnia, S.; Reardon, C.; Krauss, T.F.; Steel, M.J.; Clark, A.S.; Eggleton, B.J.

2013-01-01

The non-deterministic nature of photon sources is a key limitation for single-photon quantum processors. Spatial multiplexing overcomes this by enhancing the heralded single-photon yield without enhancing the output noise. Here the intrinsic statistical limit of an individual source is surpassed by spatially multiplexing two monolithic silicon-based correlated photon pair sources in the telecommunications band, demonstrating a 62.4% increase in the heralded single-photon output without an increase in unwanted multipair generation. We further demonstrate the scalability of this scheme by multiplexing photons generated in two waveguides pumped via an integrated coupler with a 63.1% increase in the heralded photon rate. This demonstration paves the way for a scalable architecture for multiplexing many photon sources in a compact integrated platform and achieving efficient two-photon interference, required at the core of optical quantum computing and quantum communication protocols. PMID:24107840

Social contagions on correlated multiplex networks

NASA Astrophysics Data System (ADS)

Wang, Wei; Cai, Meng; Zheng, Muhua

2018-06-01

The existence of interlayer degree correlations has been disclosed by abundant multiplex network analysis. However, how they impose on the dynamics of social contagions are remain largely unknown. In this paper, we propose a non-Markovian social contagion model in multiplex networks with inter-layer degree correlations to delineate the behavior spreading, and develop an edge-based compartmental (EBC) theory to describe the model. We find that multiplex networks promote the final behavior adoption size. Remarkably, it can be observed that the growth pattern of the final behavior adoption size, versus the behavioral information transmission probability, changes from discontinuous to continuous once decreasing the behavior adoption threshold in one layer. We finally unravel that the inter-layer degree correlations play a role on the final behavior adoption size but have no effects on the growth pattern, which is coincidence with our prediction by using the suggested theory.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

The score statistic of the LD-lod analysis: detecting linkage adaptive to linkage disequilibrium.

PubMed

Huang, J; Jiang, Y

2001-01-01

We study the properties of a modified lod score method for testing linkage that incorporates linkage disequilibrium (LD-lod). By examination of its score statistic, we show that the LD-lod score method adaptively combines two sources of information: (a) the IBD sharing score which is informative for linkage regardless of the existence of LD and (b) the contrast between allele-specific IBD sharing scores which is informative for linkage only in the presence of LD. We also consider the connection between the LD-lod score method and the transmission-disequilibrium test (TDT) for triad data and the mean test for affected sib pair (ASP) data. We show that, for triad data, the recessive LD-lod test is asymptotically equivalent to the TDT; and for ASP data, it is an adaptive combination of the TDT and the ASP mean test. We demonstrate that the LD-lod score method has relatively good statistical efficiency in comparison with the ASP mean test and the TDT for a broad range of LD and the genetic models considered in this report. Therefore, the LD-lod score method is an interesting approach for detecting linkage when the extent of LD is unknown, such as in a genome-wide screen with a dense set of genetic markers. Copyright 2001 S. Karger AG, Basel

Multivariate optical element platform for compressed detection of fluorescence markers

NASA Astrophysics Data System (ADS)

Priore, Ryan J.; Swanstrom, Joseph A.

2014-05-01

The success of a commercial fluorescent diagnostic assay is dependent on the selection of a fluorescent biomarker; due to the broad nature of fluorescence biomarker emission profiles, only a small number of fluorescence biomarkers may be discriminated from each other as a function of excitation source. Multivariate Optical Elements (MOEs) are thin-film devices that encode a broad band, spectroscopic pattern allowing a simple broadband detector to generate a highly sensitive and specific detection for a target analyte. MOEs have historically been matched 1:1 to a discrete analyte or class prediction; however, MOE filter sets are capable of sensing projections of the original sparse spectroscopic space enabling a small set of MOEs to discriminate a multitude of target analytes. This optical regression can offer real-time measurements with relatively high signal-to-noise ratios that realize the advantages of multiplexed detection and pattern recognition in a simple optical instrument. The specificity advantage of MOE-based sensors allows fluorescent biomarkers that were once incapable of discrimination from one another via optical band pass filters to be employed in a common assay panel. A simplified MOE-based sensor may ultimately reduce the requirement for highly trained operators as well as move certain life science applications like disease prognostication from the laboratory to the point of care. This presentation will summarize the design and fabrication of compressed detection MOE filter sets for detecting multiple fluorescent biomarkers simultaneously with strong spectroscopic interference as well as comparing the detection performance of the MOE sensor with traditional optical band pass filter methodologies.

Linkage Analyses of Stimulant Dependence, Craving and Heavy Use in American Indians

PubMed Central

Ehlers, Cindy L.; Gizer, Ian R.; Gilder, David A.; Wilhelmsen, Kirk C.

2011-01-01

Amphetamine-type substances are the second most widely used illicit drugs in the United States. There is evidence to suggest that stimulant use (cocaine and methamphetamine) has a heritable component, yet the areas of the genome underlying these use disorders are yet to be identified. This study’s aims were to map loci linked to stimulant dependence, heavy use, and craving in an American Indian community at high risk for substance dependence. DSM diagnosis of stimulant dependence, as well as indices of stimulant “craving” and “heavy use”, were obtained using the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA). Genotypes were determined for a panel of 791 micro-satellite polymorphisms in 381 members of multiplex families using SOLAR. Stimulant dependence, stimulant “craving” and “heavy stimulant use”, were all found to be heritable. Analyses of multipoint variance component LOD scores, failed to yield evidence of linkage for stimulant dependence. For the stimulant “craving” phenotype, linkage analysis revealed a locus that had a LOD score of 3.02 on chromosome 15q25.3-26.1 near the nicotinic receptor gene cluster. A LOD score of 2.05 was found at this same site for “heavy stimulant use”. Additional loci with LOD scores above 2.00 were found for stimulant “craving” on chromosomes 12p13.33-13.32 and 18q22.3. These results corroborate the importance of “craving” as an important phenotype that is associated with regions on chromosome 12, 15 and 18, that have been highlighted in prior segregation studies in this and other populations for substance dependence-related phenotypes. PMID:21812097

Moving through a multiplex holographic scene

NASA Astrophysics Data System (ADS)

Mrongovius, Martina

2013-02-01

This paper explores how movement can be used as a compositional element in installations of multiplex holograms. My holographic images are created from montages of hand-held video and photo-sequences. These spatially dynamic compositions are visually complex but anchored to landmarks and hints of the capturing process - such as the appearance of the photographer's shadow - to establish a sense of connection to the holographic scene. Moving around in front of the hologram, the viewer animates the holographic scene. A perception of motion then results from the viewer's bodily awareness of physical motion and the visual reading of dynamics within the scene or movement of perspective through a virtual suggestion of space. By linking and transforming the physical motion of the viewer with the visual animation, the viewer's bodily awareness - including proprioception, balance and orientation - play into the holographic composition. How multiplex holography can be a tool for exploring coupled, cross-referenced and transformed perceptions of movement is demonstrated with a number of holographic image installations. Through this process I expanded my creative composition practice to consider how dynamic and spatial scenes can be conveyed through the fragmented view of a multiplex hologram. This body of work was developed through an installation art practice and was the basis of my recently completed doctoral thesis: 'The Emergent Holographic Scene — compositions of movement and affect using multiplex holographic images'.

Multiplex biomarker approach to cardiovascular diseases.

PubMed

Adamcova, Michaela; Šimko, Fedor

2018-04-12

Personalized medicine is partly based on biomarker-guided diagnostics, therapy and prognosis, which is becoming an unavoidable concept in modern cardiology. However, the clinical significance of single biomarker studies is rather limited. A promising novel approach involves combining multiple markers into a multiplex panel, which could refine the management of a particular patient with cardiovascular pathology. Two principally different assay formats have been developed to facilitate simultaneous quantification of multiple antigens: planar array assays and microbead assays. These approaches may help to better evaluate the complexity and dynamic nature of pathologic processes and offer substantial cost and sample savings compared with traditional enzyme-linked immunosorbent assay (ELISA) measurements. However, a multiplex multimarker approach cannot become a generally disseminated method until analytical problems are solved and further studies confirming improved clinical outcomes are accomplished. These drawbacks underlie the fact that a limited number of systematic studies are available regarding the use of a multiplex biomarker approach in cardiovascular medicine to date. Our perspective underscores the significant potential of the use of the multiplex approach in a wider conceptual framework under the close cooperation of clinical and experimental cardiologists, pathophysiologists and biochemists so that the personalized approach based on standardized multimarker testing may improve the management of various cardiovascular pathologies and become a ubiquitous partner of population-derived evidence-based medicine.

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

PubMed Central

Medintz, Igor; Wong, Wendy W.; Berti, Lorenzo; Shiow, Lawrence; Tom, Jennifer; Scherer, James; Sensabaugh, George; Mathies, Richard A.

2001-01-01

An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population. PMID:11230165

Multiplexing readout channels in proportional counters

NASA Technical Reports Server (NTRS)

Caristi, James

1991-01-01

Proportional counters are important instruments used in sensing hard x-rays. The possibility is described of doubling the number of readout channels in the detector without increasing the electronics needed to amplify channel signals. This suggests that it should be possible, conversely, to reduce the number of amplifiers, thereby reducing the weight and energy budget of the instrument. Various numerical multiplexing schemes are analyzed, and a computer program is presented that can reconstruct multiplexed channel outputs with very good accuracy.

The use of analytical sedimentation velocity to extract thermodynamic linkage.

PubMed

Cole, James L; Correia, John J; Stafford, Walter F

2011-11-01

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS. Copyright © 2011 Elsevier B.V. All rights reserved.

An integrated passive micromixer-magnetic separation-capillary electrophoresis microdevice for rapid and multiplex pathogen detection at the single-cell level.

PubMed

Jung, Jae Hwan; Kim, Gha-Young; Seo, Tae Seok

2011-10-21

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (∼10(4)) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.

Multiplexing technique for computer communications via satellite channels

NASA Technical Reports Server (NTRS)

Binder, R.

1975-01-01

Multiplexing scheme combines technique of dynamic allocation with conventional time-division multiplexing. Scheme is designed to expedite short-duration interactive or priority traffic and to delay large data transfers; as result, each node has effective capacity of almost total channel capacity when other nodes have light traffic loads.

Apprenticeship - School Linkage Implementation Manual.

ERIC Educational Resources Information Center

Martin, Sharon T.; And Others

Developed to assist interested sponsors in implementing apprenticeship-school linkage projects, this guide is intended to organize the collective experiences of those who have implemented the demonstration projects to highlight the day-to-day mechanics involved. Section 1 overviews apprenticeship-school linkage. In section 2 factors are described…

Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

NASA Astrophysics Data System (ADS)

Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong

2013-10-01

The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to

Structural Determinants of Improved Fluorescence in a Family of Bacteriophytochrome-Based Infrared Fluorescent Proteins: Insights from Continuum Electrostatic Calculations and Molecular Dynamics Simulations.

PubMed

Feliks, Mikolaj; Lafaye, Céline; Shu, Xiaokun; Royant, Antoine; Field, Martin

2016-08-09

Using X-ray crystallography, continuum electrostatic calculations, and molecular dynamics simulations, we have studied the structure, protonation behavior, and dynamics of the biliverdin chromophore and its molecular environment in a series of genetically engineered infrared fluorescent proteins (IFPs) based on the chromophore-binding domain of the Deinococcus radiodurans bacteriophytochrome. Our study suggests that the experimentally observed enhancement of fluorescent properties results from the improved rigidity and planarity of the biliverdin chromophore, in particular of the first two pyrrole rings neighboring the covalent linkage to the protein. We propose that the increases in the levels of both motion and bending of the chromophore out of planarity favor the decrease in fluorescence. The chromophore-binding pocket in some of the studied proteins, in particular the weakly fluorescent parent protein, is shown to be readily accessible to water molecules from the solvent. These waters entering the chromophore region form hydrogen bond networks that affect the otherwise planar conformation of the first three rings of the chromophore. On the basis of our simulations, the enhancement of fluorescence in IFPs can be achieved either by reducing the mobility of water molecules in the vicinity of the chromophore or by limiting the interactions of the nearby protein residues with the chromophore. Finally, simulations performed at both low and neutral pH values highlight differences in the dynamics of the chromophore and shed light on the mechanism of fluorescence loss at low pH.

Single particle tracking through highly scattering media with multiplexed two-photon excitation

NASA Astrophysics Data System (ADS)

Perillo, Evan; Liu, Yen-Liang; Liu, Cong; Yeh, Hsin-Chih; Dunn, Andrew K.

2015-03-01

3D single-particle tracking (SPT) has been a pivotal tool to furthering our understanding of dynamic cellular processes in complex biological systems, with a molecular localization accuracy (10-100 nm) often better than the diffraction limit of light. However, current SPT techniques utilize either CCDs or a confocal detection scheme which not only suffer from poor temporal resolution but also limit tracking to a depth less than one scattering mean free path in the sample (typically <15μm). In this report we highlight our novel design for a spatiotemporally multiplexed two-photon microscope which is able to reach sub-diffraction-limit tracking accuracy and sub-millisecond temporal resolution, but with a dramatically extended SPT range of up to 200 μm through dense cell samples. We have validated our microscope by tracking (1) fluorescent nanoparticles in a prescribed motion inside gelatin gel (with 1% intralipid) and (2) labeled single EGFR complexes inside skin cancer spheroids (at least 8 layers of cells thick) for ~10 minutes. Furthermore we discuss future capabilities of our multiplexed two-photon microscope design, specifically to the extension of (1) simultaneous multicolor tracking (i.e. spatiotemporal co-localization analysis) and (2) FRET studies (i.e. lifetime analysis). The high resolution, high depth penetration, and multicolor features of this microscope make it well poised to study a variety of molecular scale dynamics in the cell, especially related to cellular trafficking studies with in vitro tumor models and in vivo.

Multimode fiber optic wavelength division multiplexing

NASA Technical Reports Server (NTRS)

Spencer, J. L.

1982-01-01

Optical wavelength division multiplexing (WDM) systems, with signals transmitted on different wavelengths through a single optical fiber, can have increased bandwidth and fault isolation properties over single wavelength optical systems. Two WDM system designs that might be used with multimode fibers are considered and a general description of the components which could be used to implement the system are given. The components described are sources, multiplexers, demultiplexers, and detectors. Emphasis is given to the demultiplexer technique which is the major developmental component in the WDM system.

Simple Multiplexing Hand-Held Control Unit

NASA Technical Reports Server (NTRS)

Hannaford, Blake

1989-01-01

Multiplexer consists of series of resistors, each shunted by single-pole, single-throw switch. User operates switches by pressing buttons or squeezing triggers. Prototype includes three switches operated successfully in over 200 hours of system operations. Number of switches accommodated determined by signal-to-noise ratio of current source, noise induced in control unit and cable, and number of bits in output of analog-to-digital converter. Because many computer-contolled robots have extra analog-to-digital channels, such multiplexer added at little extra cost.

Sexual selection and sex linkage.

PubMed

Kirkpatrick, Mark; Hall, David W

2004-04-01

Some animal groups, such as birds, seem prone to extreme forms of sexual selection. One contributing factor may be sex linkage of genes affecting male displays and female preferences. Here we show that sex linkage can have substantial effects on the genetic correlation between these traits and consequently for Fisher's runaway and the good-genes mechanisms of sexual selection. Under some kinds of sex linkage (e.g. Z-linked preferences), a runaway is more likely than under autosomal inheritance, while under others (e.g., X-linked preferences and autosomal displays), the good-genes mechanism is particularly powerful. These theoretical results suggest empirical tests based on the comparative method.

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

Photonic Crystal Enhanced Fluorescence for Early Breast Cancer Biomarker Detection

PubMed Central

Cunningham, Brian T.; Zangar, Richard C.

2013-01-01

Photonic crystal surfaces offer a compelling platform for improving the sensitivity of surface-based fluorescent assays used in disease diagnostics. Through the complementary processes of photonic crystal enhanced excitation and enhanced extraction, a periodic dielectric-based nanostructured surface can simultaneously increase the electric field intensity experienced by surface-bound fluorophores and increase the collection efficiency of emitted fluorescent photons. Through the ability to inexpensively fabricate photonic crystal surfaces over substantial surface areas, they are amenable to single-use applications in biological sensing, such as disease biomarker detection in serum. In this review, we will describe the motivation for implementing high-sensitivity, multiplexed biomarker detection in the context of breast cancer diagnosis. We will summarize recent efforts to improve the detection limits of such assays though the use of photonic crystal surfaces. Reduction of detection limits is driven by low autofluorescent substrates for photonic crystal fabrication, and detection instruments that take advantage of their unique features. PMID:22736539

Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-01

Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1β) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing.

PubMed

Shang, Ying; Xu, Wentao; Wang, Yong; Xu, Yuancong; Huang, Kunlun

2017-12-15

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events. Copyright © 2017. Published by Elsevier Ltd.

Metal-enhanced fluorescence/visual bimodal platform for multiplexed ultrasensitive detection of microRNA with reusable paper analytical devices.

PubMed

Liang, Linlin; Lan, Feifei; Yin, Xuemei; Ge, Shenguang; Yu, Jinghua; Yan, Mei

2017-09-15

Convenient biosensor for simultaneous multi-analyte detection was increasingly required in biological analysis. A novel flower-like silver (FLS)-enhanced fluorescence/visual bimodal platform for the ultrasensitive detection of multiple miRNAs was successfully constructed for the first time based on the principle of multi-channel microfluidic paper-based analytical devices (µPADs). Fluorophore-functionalized DNA 1 (DNA 1 -N-CDs) was combined with FLS, which was hybridized with quencher-carrying strand (DNA 2 -CeO 2 ) to form FLS-enhanced fluorescence biosensor. Upon the addition of the target miRNA, the fluorescent intensity of DNA 1 -N-CDs within the proximity of the FLS was strengthened. The disengaged DNA/CeO 2 complex could result in color change after joining H 2 O 2 , leading to real-time visual detection of miRNA firstly. If necessary, then the fluorescence method was applied for a accurate determination. In this strategy, the growth of FLS in µPADs not only reduced the background fluorescence but also provided an enrichment of "hot spots" for surface enhanced fluorescence detection of miRNAs. Results also showed versatility of the FLS in the enhancement of sensitivity and selectivity of the miRNA biosensor. Remarkably, this biosensor could detect as low as 0.03fM miRNA210 and 0.06fM miRNA21. Interestingly, the proposed biosensor also possessed good capability of recycling in three cycles upon change of the supplementation of DNA 2 -CeO 2 and visual substitutive device. This method opened new opportunities for further studies of miRNA related bioprocesses and will provide a new instrument for simultaneous detection of multiple low-level biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiplex PCR identification of Taenia spp. in rodents and carnivores.

PubMed

Al-Sabi, Mohammad N S; Kapel, Christian M O

2011-11-01

The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

Characterization of highly multiplexed monolithic PET / gamma camera detector modules.

PubMed

Pierce, L A; Pedemonte, S; DeWitt, D; MacDonald, L; Hunter, W C J; Van Leemput, K; Miyaoka, R

2018-03-29

PET detectors use signal multiplexing to reduce the total number of electronics channels needed to cover a given area. Using measured thin-beam calibration data, we tested a principal component based multiplexing scheme for scintillation detectors. The highly-multiplexed detector signal is no longer amenable to standard calibration methodologies. In this study we report results of a prototype multiplexing circuit, and present a new method for calibrating the detector module with multiplexed data. A [Formula: see text] mm 3 LYSO scintillation crystal was affixed to a position-sensitive photomultiplier tube with [Formula: see text] position-outputs and one channel that is the sum of the other 64. The 65-channel signal was multiplexed in a resistive circuit, with 65:5 or 65:7 multiplexing. A 0.9 mm beam of 511 keV photons was scanned across the face of the crystal in a 1.52 mm grid pattern in order to characterize the detector response. New methods are developed to reject scattered events and perform depth-estimation to characterize the detector response of the calibration data. Photon interaction position estimation of the testing data was performed using a Gaussian Maximum Likelihood estimator and the resolution and scatter-rejection capabilities of the detector were analyzed. We found that using a 7-channel multiplexing scheme (65:7 compression ratio) with 1.67 mm depth bins had the best performance with a beam-contour of 1.2 mm FWHM (from the 0.9 mm beam) near the center of the crystal and 1.9 mm FWHM near the edge of the crystal. The positioned events followed the expected Beer-Lambert depth distribution. The proposed calibration and positioning method exhibited a scattered photon rejection rate that was a 55% improvement over the summed signal energy-windowing method.

Characterization of highly multiplexed monolithic PET / gamma camera detector modules

NASA Astrophysics Data System (ADS)

Pierce, L. A.; Pedemonte, S.; DeWitt, D.; MacDonald, L.; Hunter, W. C. J.; Van Leemput, K.; Miyaoka, R.

2018-04-01

PET detectors use signal multiplexing to reduce the total number of electronics channels needed to cover a given area. Using measured thin-beam calibration data, we tested a principal component based multiplexing scheme for scintillation detectors. The highly-multiplexed detector signal is no longer amenable to standard calibration methodologies. In this study we report results of a prototype multiplexing circuit, and present a new method for calibrating the detector module with multiplexed data. A 50 × 50 × 10 mm3 LYSO scintillation crystal was affixed to a position-sensitive photomultiplier tube with 8 × 8 position-outputs and one channel that is the sum of the other 64. The 65-channel signal was multiplexed in a resistive circuit, with 65:5 or 65:7 multiplexing. A 0.9 mm beam of 511 keV photons was scanned across the face of the crystal in a 1.52 mm grid pattern in order to characterize the detector response. New methods are developed to reject scattered events and perform depth-estimation to characterize the detector response of the calibration data. Photon interaction position estimation of the testing data was performed using a Gaussian Maximum Likelihood estimator and the resolution and scatter-rejection capabilities of the detector were analyzed. We found that using a 7-channel multiplexing scheme (65:7 compression ratio) with 1.67 mm depth bins had the best performance with a beam-contour of 1.2 mm FWHM (from the 0.9 mm beam) near the center of the crystal and 1.9 mm FWHM near the edge of the crystal. The positioned events followed the expected Beer–Lambert depth distribution. The proposed calibration and positioning method exhibited a scattered photon rejection rate that was a 55% improvement over the summed signal energy-windowing method.

Lab on a chip for multiplexed immunoassays to detect bladder cancer using multifunctional dielectrophoretic manipulations.

PubMed

Chuang, Cheng-Hsin; Wu, Ting-Feng; Chen, Cheng-Ho; Chang, Kai-Chieh; Ju, Jing-Wei; Huang, Yao-Wei; Van Nhan, Vo

2015-07-21

A multiplexed immunosensor has been developed for the detection of specific biomarkers Galectin-1 (Gal-1) and Lactate Dehydrogenase B (LDH-B) present in different grades of bladder cancer cell lysates. In order to immobilize nanoprobes with different antibodies on a single chip we employed three-step programmable dielectrophoretic manipulations for focusing, guiding and trapping to enhance the fluorescent response and reduce the interference between the two antibody arrays. The chip consisted of a patterned indium tin oxide (ITO) electrode for sensing and a middle fish bone shaped gold electrode for focusing and guiding. Using ITO electrodes for the sensing area can effectively eliminate the background noise of fluorescence response as compared to metal electrodes. It was also observed that the three step manipulation increased fluorescence response after immunosensing by about 4.6 times as compared to utilizing DEP for just trapping the nanoprobes. Two different-grade bladder cancer cell lysates (grade I: RT4 and grade III: T24) were individually analyzed for detecting the protein expression levels of Gal-1 and LDH-B. The fluorescence intensity observed for Gal-1 is higher than that of LDH-B in the T24 cell lysate; however the response observed in RT4 is higher for LDH-B as compared to Gal-1. Thus we can effectively identify the different grades of bladder cancer cells. In addition, the platform for DEP manipulation developed in this study can enable real time detection of multiple analytes on a single chip and provide more practical benefits for clinical diagnosis.

Multiplex lexical networks reveal patterns in early word acquisition in children

NASA Astrophysics Data System (ADS)

Stella, Massimo; Beckage, Nicole M.; Brede, Markus

2017-04-01

Network models of language have provided a way of linking cognitive processes to language structure. However, current approaches focus only on one linguistic relationship at a time, missing the complex multi-relational nature of language. In this work, we overcome this limitation by modelling the mental lexicon of English-speaking toddlers as a multiplex lexical network, i.e. a multi-layered network where N = 529 words/nodes are connected according to four relationship: (i) free association, (ii) feature sharing, (iii) co-occurrence, and (iv) phonological similarity. We investigate the topology of the resulting multiplex and then proceed to evaluate single layers and the full multiplex structure on their ability to predict empirically observed age of acquisition data of English speaking toddlers. We find that the multiplex topology is an important proxy of the cognitive processes of acquisition, capable of capturing emergent lexicon structure. In fact, we show that the multiplex structure is fundamentally more powerful than individual layers in predicting the ordering with which words are acquired. Furthermore, multiplex analysis allows for a quantification of distinct phases of lexical acquisition in early learners: while initially all the multiplex layers contribute to word learning, after about month 23 free associations take the lead in driving word acquisition.

Accommodating Chromosome Inversions in Linkage Analysis

PubMed Central

Chen, Gary K.; Slaten, Erin; Ophoff, Roel A.; Lange, Kenneth

2006-01-01

This work develops a population-genetics model for polymorphic chromosome inversions. The model precisely describes how an inversion changes the nature of and approach to linkage equilibrium. The work also describes algorithms and software for allele-frequency estimation and linkage analysis in the presence of an inversion. The linkage algorithms implemented in the software package Mendel estimate recombination parameters and calculate the posterior probability that each pedigree member carries the inversion. Application of Mendel to eight Centre d'Étude du Polymorphisme Humain pedigrees in a region containing a common inversion on 8p23 illustrates its potential for providing more-precise estimates of the location of an unmapped marker or trait gene. Our expanded cytogenetic analysis of these families further identifies inversion carriers and increases the evidence of linkage. PMID:16826515

Preliminary Assessment of Microwave Readout Multiplexing Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Croce, Mark Philip; Koehler, Katrina Elizabeth; Rabin, Michael W.

2017-01-23

Ultra-high resolution microcalorimeter gamma spectroscopy is a new non-destructive assay technology for measurement of plutonium isotopic composition, with the potential to reduce total measurement uncertainty to a level competitive with destructive analysis methods [1-4]. Achieving this level of performance in practical applications requires not only the energy resolution now routinely achieved with transition-edge sensor microcalorimeter arrays (an order of magnitude better than for germanium detectors) but also high throughput. Microcalorimeter gamma spectrometers have not yet achieved detection efficiency and count rate capability that is comparable to germanium detectors, largely because of limits from existing readout technology. Microcalorimeter detectors must bemore » operated at low temperature to achieve their exceptional energy resolution. Although the typical 100 mK operating temperatures can be achieved with reliable, cryogen-free systems, the cryogenic complexity and heat load from individual readout channels for large sensor arrays is prohibitive. Multiplexing is required for practical systems. The most mature multiplexing technology at present is time-division multiplexing (TDM) [3, 5-6]. In TDM, the sensor outputs are switched by applying bias current to one SQUID amplifier at a time. Transition-edge sensor (TES) microcalorimeter arrays as large as 256 pixels have been developed for X-ray and gamma-ray spectroscopy using TDM technology. Due to bandwidth limits and noise scaling, TDM is limited to a maximum multiplexing factor of approximately 32-40 sensors on one readout line [8]. Increasing the size of microcalorimeter arrays above the kilopixel scale, required to match the throughput of germanium detectors, requires the development of a new readout technology with a much higher multiplexing factor.« less

Structure-function clustering in multiplex brain networks

NASA Astrophysics Data System (ADS)

Crofts, J. J.; Forrester, M.; O'Dea, R. D.

2016-10-01

A key question in neuroscience is to understand how a rich functional repertoire of brain activity arises within relatively static networks of structurally connected neural populations: elucidating the subtle interactions between evoked “functional connectivity” and the underlying “structural connectivity” has the potential to address this. These structural-functional networks (and neural networks more generally) are more naturally described using a multilayer or multiplex network approach, in favour of standard single-layer network analyses that are more typically applied to such systems. In this letter, we address such issues by exploring important structure-function relations in the Macaque cortical network by modelling it as a duplex network that comprises an anatomical layer, describing the known (macro-scale) network topology of the Macaque monkey, and a functional layer derived from simulated neural activity. We investigate and characterize correlations between structural and functional layers, as system parameters controlling simulated neural activity are varied, by employing recently described multiplex network measures. Moreover, we propose a novel measure of multiplex structure-function clustering which allows us to investigate the emergence of functional connections that are distinct from the underlying cortical structure, and to highlight the dependence of multiplex structure on the neural dynamical regime.

A portable time-domain LED fluorimeter for nanosecond fluorescence lifetime measurements

NASA Astrophysics Data System (ADS)

Wang, Hongtao; Qi, Ying; Mountziaris, T. J.; Salthouse, Christopher D.

2014-05-01

Fluorescence lifetime measurements are becoming increasingly important in chemical and biological research. Time-domain lifetime measurements offer fluorescence multiplexing and improved handling of interferers compared with the frequency-domain technique. In this paper, an all solid-state, filterless, and highly portable light-emitting-diode based time-domain fluorimeter (LED TDF) is reported for the measurement of nanosecond fluorescence lifetimes. LED based excitation provides more wavelengths options compared to laser diode based excitation, but the excitation is less effective due to the uncollimated beam, less optical power, and longer latency in state transition. Pulse triggering and pre-bias techniques were implemented in our LED TDF to improve the peak optical power to over 100 mW. The proposed pulsing circuit achieved an excitation light fall time of less than 2 ns. Electrical resetting technique realized a time-gated photo-detector to remove the interference of the excitation light with fluorescence. These techniques allow the LED fluorimeter to accurately measure the fluorescence lifetime of fluorescein down to concentration of 0.5 μM. In addition, all filters required in traditional instruments are eliminated for the non-attenuated excitation/emission light power. These achievements make the reported device attractive to biochemical laboratories seeking for highly portable lifetime detection devices for developing sensors based on fluorescence lifetime changes. The device was initially validated by measuring the lifetimes of three commercial fluorophores and comparing them with reported lifetime data. It was subsequently used to characterize a ZnSe quantum dot based DNA sensor.

A portable time-domain LED fluorimeter for nanosecond fluorescence lifetime measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hongtao; Salthouse, Christopher D., E-mail: salthouse@ecs.umass.edu; Center for Personalized Health Monitoring, University of Massachusetts, Amherst, Massachusetts 01003

2014-05-15

Fluorescence lifetime measurements are becoming increasingly important in chemical and biological research. Time-domain lifetime measurements offer fluorescence multiplexing and improved handling of interferers compared with the frequency-domain technique. In this paper, an all solid-state, filterless, and highly portable light-emitting-diode based time-domain fluorimeter (LED TDF) is reported for the measurement of nanosecond fluorescence lifetimes. LED based excitation provides more wavelengths options compared to laser diode based excitation, but the excitation is less effective due to the uncollimated beam, less optical power, and longer latency in state transition. Pulse triggering and pre-bias techniques were implemented in our LED TDF to improve themore » peak optical power to over 100 mW. The proposed pulsing circuit achieved an excitation light fall time of less than 2 ns. Electrical resetting technique realized a time-gated photo-detector to remove the interference of the excitation light with fluorescence. These techniques allow the LED fluorimeter to accurately measure the fluorescence lifetime of fluorescein down to concentration of 0.5 μM. In addition, all filters required in traditional instruments are eliminated for the non-attenuated excitation/emission light power. These achievements make the reported device attractive to biochemical laboratories seeking for highly portable lifetime detection devices for developing sensors based on fluorescence lifetime changes. The device was initially validated by measuring the lifetimes of three commercial fluorophores and comparing them with reported lifetime data. It was subsequently used to characterize a ZnSe quantum dot based DNA sensor.« less

Mapping a multiplexed zoo of mRNA expression

PubMed Central

Choi, Harry M. T.; Calvert, Colby R.; Husain, Naeem; Huss, David; Barsi, Julius C.; Deverman, Benjamin E.; Hunter, Ryan C.; Kato, Mihoko; Lee, S. Melanie; Abelin, Anna C. T.; Rosenthal, Adam Z.; Akbari, Omar S.; Li, Yuwei; Hay, Bruce A.; Sternberg, Paul W.; Patterson, Paul H.; Davidson, Eric H.; Mazmanian, Sarkis K.; Prober, David A.; van de Rijn, Matt; Leadbetter, Jared R.; Newman, Dianne K.; Readhead, Carol; Bronner, Marianne E.; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E.

2016-01-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. PMID:27702788

Infrared Avionics Signal Distribution using Wavelength Division Multiplexing

NASA Technical Reports Server (NTRS)

Atiquzzaman, Mohammed; Sluss, Jim; Nguyen, Hung; Ngo, Duc

2003-01-01

Pilots in the cockpits of aircrafts currently communicate with ground stations using Radio Frequency (RF) signals. Antennas mounted outside the aircraft receive and transmit RF signals from and to the ground stations. The RF signals received at the antennas are sent to the cockpit using coaxial cables. As the number of antennas needed to provide more than one frequency band in aircrafts increases, RF distribution media (such as coaxial cable) adds to the complexity and weight of the cockpit wiring. Concomitantly, the safety and signal to noise ratio also decreases due to the use of RF signals. The University of Oklahoma is collaborating with the National Aeronautics and Space Administration to develop optical fiber based schemes to replace the coaxial cable used for RF signal distribution within an aircraft. The project aims at exploiting emerging Wavelength Division Multiplexing (WDM) techniques to reduce the weight of cabling, and increase the signal to noise ratio and reliability. This will be achieved by wavelength division multiplexing the signals from the various antennas and then demultiplexing the signals to recover the original signals at the cockpit. This paper will show that (i) RF signals can not only be wavelength multiplexed at the end of a fiber, but additional signals can be inserted into the middle of the fiber using WDM technology, and (ii) the signals can also be successfully extracted by tapping into the middle of the fiber. We are currently extending our previous laboratory prototype (which could multiplex signals only at the end of the fiber) to include additional multiplexing and demultiplexing of RF signals from the middle of the optical backbone with a view to validating the proof of concept, and carrying out measurements to determine the effectiveness of Wavelength Division Multiplexing for avionics applications. A test bed to perform measurements of several relevant parameters for various modulation schemes and frequencies (such as VHF

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.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Two-dimensional simulation of holographic data storage medium for multiplexed recording.

PubMed

Toishi, Mitsuru; Takeda, Takahiro; Tanaka, Kenji; Tanaka, Tomiji; Fukumoto, Atsushi; Watanabe, Kenjiro

2008-02-18

In this paper, we propose a new analysis model for photopolymer recording processes that calculate the two-dimensional refractive index distribution of multiplexed holograms. For the simulation of the photopolymer medium, time evolution of monomer diffusion and polymerization need to be calculated simultaneously. The distribution of the refractive index inside the medium is induced by these processes. By evaluating the refractive index pattern on each layer, the diffraction beams from the multiplexed hologram can be read out by beam propagation method (BPM). This is the first paper to determine the diffraction beam from a multiplexed hologram in a simulated photopolymer medium process. We analyze the time response of the multiplexed hologram recording processes in the photopolymer, and estimate the degradation of diffraction efficiency with multiplexed recording. This work can greatly contribute to understanding the process of hologram recording.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

PubMed

Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

2018-05-31

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

The Application of Fluorescent Quantum Dots to Confocal, Multiphoton, and Electron Microscopic Imaging

PubMed Central

Deerinck, Thomas J.

2009-01-01

Fluorescent quantum dots are emerging as an important tool for imaging cells and tissues, and their unique optical and physical properties have captured the attention of the research community. The most common types of commercially available quantum dots consist of a nanocrystalline semiconductor core composed of cadmium selenide with a zinc sulfide capping layer and an outer polymer layer to facilitate conjugation to targeting biomolecules such as immunoglobulins. They exhibit high fluorescent quantum yields and have large absorption cross-sections, possess excellent photostability, and can be synthesized so that their narrow-band fluorescence emission can occur in a wide spectrum of colors. These properties make them excellent candidates for serving as multiplexing molecular beacons using a variety of imaging modalities including highly correlated microscopies. Whereas much attention has been focused on quantum-dot applications for live-cell imaging, we have sought to characterize and exploit their utility for enabling simultaneous multiprotein immunolabeling in fixed cells and tissues. Considerations for their application to immunolabeling for correlated light and electron microscopic analysis are discussed. PMID:18337229

Privacy-preserving record linkage using Bloom filters.

PubMed

Schnell, Rainer; Bachteler, Tobias; Reiher, Jörg

2009-08-25

Combining multiple databases with disjunctive or additional information on the same person is occurring increasingly throughout research. If unique identification numbers for these individuals are not available, probabilistic record linkage is used for the identification of matching record pairs. In many applications, identifiers have to be encrypted due to privacy concerns. A new protocol for privacy-preserving record linkage with encrypted identifiers allowing for errors in identifiers has been developed. The protocol is based on Bloom filters on q-grams of identifiers. Tests on simulated and actual databases yield linkage results comparable to non-encrypted identifiers and superior to results from phonetic encodings. We proposed a protocol for privacy-preserving record linkage with encrypted identifiers allowing for errors in identifiers. Since the protocol can be easily enhanced and has a low computational burden, the protocol might be useful for many applications requiring privacy-preserving record linkage.

Quantitative multiplex detection of biomarkers on a waveguide-based biosensor using quantum dots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Hongzhi; Mukundan, Harshini; Martinez, Jennifer S

2009-01-01

multiple color imaging of live cells using QD-bioconjugates [Jaiswal 2003]. Gao [Gao 2004] and So [So 2006] have used QDs as probes for in-vivo cancer targeting and imaging. Medintz et al. reported self-assembled QD-based biosensors for detection of analytes based on energy transfer [Medintz 2003]. Others have developed an approach for multiplex optical encoding of biomolecules using QDs [Han 2001]. Immunoassays have also benefited from the advantages of QDs. Recently, dihydrolipoic acid (DHLA) capped-QDs have been attached to antibodies and used as fluorescence reporters in plate-based multiplex immunoassays [Goodman 2004]. However, DHLA-QDs are associated with low quantum efficiency and are unstable at neutral pH. These problems limit the application of this technology to the sensitive detection of biomolecules, especially in complex biological samples. Thus, the development of a rapid, sensitive, quantitative, and specific multiplex platform for the detection of biomarkers in difficult samples remains an elusive target. The goal stated above has applications in many fields including medical diagnostics, biological research, and threat reduction. The current decade alone has seen the development of a need to rapidly and accurately detect potential biological warfare agents. For example, current methods for the detection of anthrax are grossly inadequate for a variety of reasons including long incubation time (5 days from time of exposure to onset of symptoms) and non-specific ('flu-like') symptoms. When five employees of the United State Senate were exposed to B. anthracis in the mail (2001), only one patient had a confirmed diagnosis before death. Since then, sandwich immunoassays using both colorimetric and fluorescence detectors have been developed for key components of the anthrax lethal toxin, namely protective antigen (PA), lethal factor (LF), and the edema factor [Mourez 2001]. While these platforms were successful in assays against anthrax toxins, the sensitivity

Control of Angular Intervals for Angle-Multiplexed Holographic Memory

NASA Astrophysics Data System (ADS)

Kinoshita, Nobuhiro; Muroi, Tetsuhiko; Ishii, Norihiko; Kamijo, Koji; Shimidzu, Naoki

2009-03-01

In angle-multiplexed holographic memory, the full width at half maximum of the Bragg selectivity curves is dependent on the angle formed between the medium and incident laser beams. This indicates the possibility of high density and high multiplexing number by varying the angular intervals between adjacent holograms. We propose an angular interval scheduling for closely stacking holograms into medium even when the angle range is limited. We obtained bit error rates of the order of 10-4 under the following conditions: medium thickness of 1 mm, laser beam wavelength of 532 nm, and angular multiplexing number of 300.

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection.

PubMed

Pravadali-Cekic, Sercan; Kocic, Danijela; Hua, Stanley; Jones, Andrew; Dennis, Gary R; Shalliker, R Andrew

2015-12-15

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

Multiplexing Technology for Acoustic Emission Monitoring of Aerospace Vehicles

NASA Technical Reports Server (NTRS)

Prosser, William; Percy, Daniel

2003-01-01

The initiation and propagation of damage mechanisms such as cracks and delaminations generate acoustic waves, which propagate through a structure. These waves can be detected and analyzed to provide the location and severity of damage as part of a structural health monitoring (SHM) system. This methodology of damage detection is commonly known as acoustic emission (AE) monitoring, and is widely used on a variety of applications on civil structures. AE has been widely considered for SHM of aerospace vehicles. Numerous successful ground and flight test demonstrations have been performed, which show the viability of the technology for damage monitoring in aerospace structures. However, one significant current limitation for application of AE techniques on aerospace vehicles is the large size, mass, and power requirements for the necessary monitoring instrumentation. To address this issue, a prototype multiplexing approach has been developed and demonstrated in this study, which reduces the amount of AE monitoring instrumentation required. Typical time division multiplexing techniques that are commonly used to monitor strain, pressure and temperature sensors are not applicable to AE monitoring because of the asynchronous and widely varying rates of AE signal occurrence. Thus, an event based multiplexing technique was developed. In the initial prototype circuit, inputs from eight sensors in a linear array were multiplexed into two data acquisition channels. The multiplexer rapidly switches, in less than one microsecond, allowing the signals from two sensors to be acquired by a digitizer. The two acquired signals are from the sensors on either side of the trigger sensor. This enables the capture of the first arrival of the waves, which cannot be accomplished with the signal from the trigger sensor. The propagation delay to the slightly more distant neighboring sensors makes this possible. The arrival time from this first arrival provides a more accurate source location

Demonstration of Time Domain Multiplexed Readout for Magnetically Coupled Calorimeters

NASA Technical Reports Server (NTRS)

Porst, J.-P.; Adams, J. S.; Balvin, M.; Bandler, S.; Beyer, J.; Busch, S. E.; Drung, D.; Seidel, G. M.; Smith, S. J.; Stevenson, T. R.

2012-01-01

Magnetically coupled calorimeters (MCC) have extremely high potential for x-ray applications due to the inherent high energy resolution capability and being non-dissipative. Although very high energy-resolution has been demonstrated, until now there has been no demonstration of multiplexed read-out. We report on the first realization of a time domain multiplexed (TDM) read-out. While this has many similarities with TDM of transition-edge-sensors (TES), for MGGs the energy resolution is limited by the SQUID read-out noise and requires the well established scheme to be altered in order to minimize degradation due to noise aliasing effects. In cur approach, each pixel is read out by a single first stage SQUID (SQ1) that is operated in open loop. The outputs of the SQ1 s are low-pass filtered with an array of low cross-talk inductors, then fed into a single-stage SQUID TD multiplexer. The multiplexer is addressed from room temperature and read out through a single amplifier channel. We present results achieved with a new detector platform. Noise performance is presented and compared to expectations. We have demonstrated multiplexed X-ray spectroscopy at 5.9keV with delta_FWHM=10eV. In an optimized setup, we show it is possible to multiplex 32 detectors without significantly degrading the Intrinsic detector resolution.

Incorporation of antigens from whole cell lysates and purified virions from MP12 into fluorescence microsphere immunoassays for the detection of antibodies against Rift Valley fever virus

USDA-ARS?s Scientific Manuscript database

Background: The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of Rift Valley fever virus (RVFV) IgG and IgM antibodies by incorporation of antigens from whole cell lysates and purified virions from MP12. Methods and Findings: Vir...

Some methods for blindfolded record linkage.

PubMed

Churches, Tim; Christen, Peter

2004-06-28

The linkage of records which refer to the same entity in separate data collections is a common requirement in public health and biomedical research. Traditionally, record linkage techniques have required that all the identifying data in which links are sought be revealed to at least one party, often a third party. This necessarily invades personal privacy and requires complete trust in the intentions of that party and their ability to maintain security and confidentiality. Dusserre, Quantin, Bouzelat and colleagues have demonstrated that it is possible to use secure one-way hash transformations to carry out follow-up epidemiological studies without any party having to reveal identifying information about any of the subjects - a technique which we refer to as "blindfolded record linkage". A limitation of their method is that only exact comparisons of values are possible, although phonetic encoding of names and other strings can be used to allow for some types of typographical variation and data errors. A method is described which permits the calculation of a general similarity measure, the n-gram score, without having to reveal the data being compared, albeit at some cost in computation and data communication. This method can be combined with public key cryptography and automatic estimation of linkage model parameters to create an overall system for blindfolded record linkage. The system described offers good protection against misdeeds or security failures by any one party, but remains vulnerable to collusion between or simultaneous compromise of two or more parties involved in the linkage operation. In order to reduce the likelihood of this, the use of last-minute allocation of tasks to substitutable servers is proposed. Proof-of-concept computer programmes written in the Python programming language are provided to illustrate the similarity comparison protocol. Although the protocols described in this paper are not unconditionally secure, they do suggest the

Some methods for blindfolded record linkage

PubMed Central

Churches, Tim; Christen, Peter

2004-01-01

Background The linkage of records which refer to the same entity in separate data collections is a common requirement in public health and biomedical research. Traditionally, record linkage techniques have required that all the identifying data in which links are sought be revealed to at least one party, often a third party. This necessarily invades personal privacy and requires complete trust in the intentions of that party and their ability to maintain security and confidentiality. Dusserre, Quantin, Bouzelat and colleagues have demonstrated that it is possible to use secure one-way hash transformations to carry out follow-up epidemiological studies without any party having to reveal identifying information about any of the subjects – a technique which we refer to as "blindfolded record linkage". A limitation of their method is that only exact comparisons of values are possible, although phonetic encoding of names and other strings can be used to allow for some types of typographical variation and data errors. Methods A method is described which permits the calculation of a general similarity measure, the n-gram score, without having to reveal the data being compared, albeit at some cost in computation and data communication. This method can be combined with public key cryptography and automatic estimation of linkage model parameters to create an overall system for blindfolded record linkage. Results The system described offers good protection against misdeeds or security failures by any one party, but remains vulnerable to collusion between or simultaneous compromise of two or more parties involved in the linkage operation. In order to reduce the likelihood of this, the use of last-minute allocation of tasks to substitutable servers is proposed. Proof-of-concept computer programmes written in the Python programming language are provided to illustrate the similarity comparison protocol. Conclusion Although the protocols described in this paper are not

47 CFR 73.293 - Use of FM multiplex subcarriers.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 4 2011-10-01 2011-10-01 false Use of FM multiplex subcarriers. 73.293 Section 73.293 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES RADIO BROADCAST SERVICES FM Broadcast Stations § 73.293 Use of FM multiplex subcarriers. Licensees of FM broadcast...

47 CFR 73.293 - Use of FM multiplex subcarriers.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 4 2010-10-01 2010-10-01 false Use of FM multiplex subcarriers. 73.293 Section 73.293 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES RADIO BROADCAST SERVICES FM Broadcast Stations § 73.293 Use of FM multiplex subcarriers. Licensees of FM broadcast...

Multiplexed operation of a micromachined ultrasonic droplet ejector array.

PubMed

Forbes, Thomas P; Degertekin, F Levent; Fedorov, Andrei G

2007-10-01

A dual-sample ultrasonic droplet ejector array is developed for use as a soft-ionization ion source for multiplexed mass spectrometry (MS). Such a multiplexed ion source aims to reduce MS analysis time for multiple analyte streams, as well as allow for the synchronized ejection of the sample(s) and an internal standard for quantitative results and mass calibration. Multiplexing is achieved at the device level by division of the fluid reservoir and separating the active electrodes of the piezoelectric transducer for isolated application of ultrasonic wave energy to each domain. The transducer is mechanically shaped to further reduce the acoustical crosstalk between the domains. Device design is performed using finite-element analysis simulations and supported by experimental characterization. Isolated ejection of approximately 5 microm diameter water droplets from individual domains in the micromachined droplet ejector array at around 1 MHz frequency is demonstrated by experiments. The proof-of-concept demonstration using a dual-sample device also shows potential for multiplexing with larger numbers of analytes.

Multiplexing of spatial modes in the mid-IR region

NASA Astrophysics Data System (ADS)

Gailele, Lucas; Maweza, Loyiso; Dudley, Angela; Ndagano, Bienvenu; Rosales-Guzman, Carmelo; Forbes, Andrew

2017-02-01

Traditional optical communication systems optimize multiplexing in polarization and wavelength both trans- mitted in fiber and free-space to attain high bandwidth data communication. Yet despite these technologies, we are expected to reach a bandwidth ceiling in the near future. Communications using orbital angular momentum (OAM) carrying modes offers infinite dimensional states, providing means to increase link capacity by multiplexing spatially overlapping modes in both the azimuthal and radial degrees of freedom. OAM modes are multiplexed and de-multiplexed by the use of spatial light modulators (SLM). Implementation of complex amplitude modulation is employed on laser beams phase and amplitude to generate Laguerre-Gaussian (LG) modes. Modal decomposition is employed to detect these modes due to their orthogonality as they propagate in space. We demonstrate data transfer by sending images as a proof-of concept in a lab-based scheme. We demonstrate the creation and detection of OAM modes in the mid-IR region as a precursor to a mid-IR free-space communication link.

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

PubMed Central

Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD). PMID:25083509

Shift-phase code multiplexing technique for holographic memories and optical interconnection

NASA Astrophysics Data System (ADS)

Honma, Satoshi; Muto, Shinzo; Okamoto, Atsushi

2008-03-01

Holographic technologies for optical memories and interconnection devices have been studied actively because of high storage capacity, many wiring patterns and high transmission rate. Among multiplexing techniques such as angular, phase code and wavelength-multiplexing, speckle multiplexing technique have gotten attention due to the simple optical setup having an adjustable random phase filter in only one direction. To keep simple construction and to suppress crosstalk among adjacent page data or wiring patterns for efficient holographic memories and interconnection, we have to consider about optimum randomness of the phase filter. The high randomness causes expanding an illumination area of reference beam on holographic media. On the other hands, the small randomness causes the crosstalk between adjacent hologram data. We have proposed the method of holographic multiplexing, shift-phase code multiplexing with a two-dimensional orthogonal matrix phase filter. A lot of orthogonal phase codes can be produced by shifting the phase filter in one direction. It is able to read and record the individual holograms with low crosstalk. We give the basic experimental result on holographic data multiplexing and consider the phase pattern of the filter to suppress the crosstalk between adjacent holograms sufficiently.

Steatocystoma multiplex generalisata partially suppurativa--case report.

PubMed

Fekete, Gyula László; Fekete, Júlia Edit

2010-01-01

Steatocystoma multiplex is a rare inherited disorder with an autosomal dominant mode of transmission, but sometimes it may appear sporadically. Usually the onset tends to occur during adolescence or early adult life. A clinical case of a 27-year-old male patient is presented. Since the age of 8, he had been presenting with multiple, asymptomatic, round-to-oval, well-defined, smooth-surfaced, yellow to skin-colored, 5- to 22-mm diameter cysts and nodules initially scattered on the trunk and lately disseminated all over the body with less lesions on the lower extremities. At one of follow-up visits, he presented with high fever, pain with tumefaction of the small and medium size joints of the palms and soles, and deteriorated general status with polymorphous skin lesions. Based on the clinical and paraclinical features, the diagnosis of steatocystoma multiplex generalisata partially suppurativa was made. He was treated with oral isotretinoin (1 mg/kg per day) for 14 weeks, antibiotics and local treatments. The lesions healed slowly, with local disfigurement, hyperpigmentation and unpleasant scars. Isotretinoin usually does not eradicate the condition but could be effective in suppurative abscesses. Steatocystoma multiplex generalisata is considered rare; the true incidence of the disease is unknown. In the disease evolution, the severe inflammatory variant, steatocystoma multiplex suppurativa, may appear at any time.

Determinants of public cooperation in multiplex networks

NASA Astrophysics Data System (ADS)

Battiston, Federico; Perc, Matjaž; Latora, Vito

2017-07-01

Synergies between evolutionary game theory and statistical physics have significantly improved our understanding of public cooperation in structured populations. Multiplex networks, in particular, provide the theoretical framework within network science that allows us to mathematically describe the rich structure of interactions characterizing human societies. While research has shown that multiplex networks may enhance the resilience of cooperation, the interplay between the overlap in the structure of the layers and the control parameters of the corresponding games has not yet been investigated. With this aim, we consider here the public goods game on a multiplex network, and we unveil the role of the number of layers and the overlap of links, as well as the impact of different synergy factors in different layers, on the onset of cooperation. We show that enhanced public cooperation emerges only when a significant edge overlap is combined with at least one layer being able to sustain some cooperation by means of a sufficiently high synergy factor. In the absence of either of these conditions, the evolution of cooperation in multiplex networks is determined by the bounds of traditional network reciprocity with no enhanced resilience. These results caution against overly optimistic predictions that the presence of multiple social domains may in itself promote cooperation, and they help us better understand the complexity behind prosocial behavior in layered social systems.

Privacy-preserving record linkage using Bloom filters

PubMed Central

2009-01-01

Background Combining multiple databases with disjunctive or additional information on the same person is occurring increasingly throughout research. If unique identification numbers for these individuals are not available, probabilistic record linkage is used for the identification of matching record pairs. In many applications, identifiers have to be encrypted due to privacy concerns. Methods A new protocol for privacy-preserving record linkage with encrypted identifiers allowing for errors in identifiers has been developed. The protocol is based on Bloom filters on q-grams of identifiers. Results Tests on simulated and actual databases yield linkage results comparable to non-encrypted identifiers and superior to results from phonetic encodings. Conclusion We proposed a protocol for privacy-preserving record linkage with encrypted identifiers allowing for errors in identifiers. Since the protocol can be easily enhanced and has a low computational burden, the protocol might be useful for many applications requiring privacy-preserving record linkage. PMID:19706187

Mapping a multiplexed zoo of mRNA expression.

PubMed

Choi, Harry M T; Calvert, Colby R; Husain, Naeem; Huss, David; Barsi, Julius C; Deverman, Benjamin E; Hunter, Ryan C; Kato, Mihoko; Lee, S Melanie; Abelin, Anna C T; Rosenthal, Adam Z; Akbari, Omar S; Li, Yuwei; Hay, Bruce A; Sternberg, Paul W; Patterson, Paul H; Davidson, Eric H; Mazmanian, Sarkis K; Prober, David A; van de Rijn, Matt; Leadbetter, Jared R; Newman, Dianne K; Readhead, Carol; Bronner, Marianne E; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E; Pierce, Niles A

2016-10-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. © 2016. Published by The Company of Biologists Ltd.

Linkage map of the peppered moth, Biston betularia (Lepidoptera, Geometridae): a model of industrial melanism

PubMed Central

Van't Hof, A E; Nguyen, P; Dalíková, M; Edmonds, N; Marec, F; Saccheri, I J

2013-01-01

We have constructed a linkage map for the peppered moth (Biston betularia), the classical ecological genetics model of industrial melanism, aimed both at localizing the network of loci controlling melanism and making inferences about chromosome dynamics. The linkage map, which is based primarily on amplified fragment length polymorphisms (AFLPs) and genes, consists of 31 linkage groups (LGs; consistent with the karyotype). Comparison with the evolutionarily distant Bombyx mori suggests that the gene content of chromosomes is highly conserved. Gene order is conserved on the autosomes, but noticeably less so on the Z chromosome, as confirmed by physical mapping using bacterial artificial chromosome fluorescence in situ hybridization (BAC-FISH). Synteny mapping identified three pairs of B. betularia LGs (11/29, 23/30 and 24/31) as being orthologous to three B. mori chromosomes (11, 23 and 24, respectively). A similar finding in an outgroup moth (Plutella xylostella) indicates that the B. mori karyotype (n=28) is a phylogenetically derived state resulting from three chromosome fusions. As with other Lepidoptera, the B. betularia W chromosome consists largely of repetitive sequence, but exceptionally we found a W homolog of a Z-linked gene (laminin A), possibly resulting from ectopic recombination between the sex chromosomes. The B. betularia linkage map, featuring the network of known melanization genes, serves as a resource for melanism research in Lepidoptera. Moreover, its close resemblance to the ancestral lepidopteran karyotype (n=31) makes it a useful reference point for reconstructing chromosome dynamic events and ancestral genome architectures. Our study highlights the unusual evolutionary stability of lepidopteran autosomes; in contrast, higher rates of intrachromosomal rearrangements support a special role of the Z chromosome in adaptive evolution and speciation. PMID:23211790

Investigation into constant envelope orthogonal frequency division multiplexing for polarization-division multiplexing coherent optical communication

NASA Astrophysics Data System (ADS)

Li, Yupeng; Ding, Ding

2017-09-01

Benefiting from the high spectral efficiency and low peak-to-average power ratio, constant envelope orthogonal frequency division multiplexing (OFDM) is a promising technique in coherent optical communication. Polarization-division multiplexing (PDM) has been employed as an effective way to double the transmission capacity in the commercial 100 Gb/s PDM-QPSK system. We investigated constant envelope OFDM together with PDM. Simulation results show that the acceptable maximum launch power into the fiber improves 10 and 6 dB for 80- and 320-km transmission, respectively (compared with the conventional PDM OFDM system). The maximum reachable distance of the constant envelope OFDM system is able to reach 800 km, and even 1200 km is reachable if an ideal erbium doped fiber amplifier is employed.

76 FR 48169 - Advancing Regulatory Science for Highly Multiplexed Microbiology/Medical Countermeasure Devices...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-08

...] Advancing Regulatory Science for Highly Multiplexed Microbiology/ Medical Countermeasure Devices; Public... Regulatory Science for Highly Multiplexed Microbiology/Medical Countermeasure Devices.'' The purpose of the public meeting is to discuss performance evaluation of highly multiplexed microbiology/medical...

Experimental demonstration of subcarrier multiplexed quantum key distribution system.

PubMed

Mora, José; Ruiz-Alba, Antonio; Amaya, Waldimar; Martínez, Alfonso; García-Muñoz, Víctor; Calvo, David; Capmany, José

2012-06-01

We provide, to our knowledge, the first experimental demonstration of the feasibility of sending several parallel keys by exploiting the technique of subcarrier multiplexing (SCM) widely employed in microwave photonics. This approach brings several advantages such as high spectral efficiency compatible with the actual secure key rates, the sharing of the optical fainted pulse by all the quantum multiplexed channels reducing the system complexity, and the possibility of upgrading with wavelength division multiplexing in a two-tier scheme, to increase the number of parallel keys. Two independent quantum SCM channels featuring a sifted key rate of 10 Kb/s/channel over a link with quantum bit error rate <2% is reported.

Inter-layer synchronization in multiplex networks of identical layers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sevilla-Escoboza, R.; Sendiña-Nadal, I.; Leyva, I.

2016-06-15

Inter-layer synchronization is a distinctive process of multiplex networks whereby each node in a given layer evolves synchronously with all its replicas in other layers, irrespective of whether or not it is synchronized with the other units of the same layer. We analytically derive the necessary conditions for the existence and stability of such a state, and verify numerically the analytical predictions in several cases where such a state emerges. We further inspect its robustness against a progressive de-multiplexing of the network, and provide experimental evidence by means of multiplexes of nonlinear electronic circuits affected by intrinsic noise and parametermore » mismatch.« less

Nomad Biosensors: A New Multiplexed Technology for the Screening of GPCR Ligands.

PubMed

Mella, Rosa M; Kortazar, Danel; Roura-Ferrer, Meritxell; Salado, Clarisa; Valcárcel, María; Castilla, Amaia; Villacé, Patricia

2018-06-01

Nomad Technology (Innoprot [Innovative Technologies in Biological Systems], Derio, Spain), a novel tool for multiplexing high-throughput cell-based G protein-coupled receptor (GPCR) assays, is described in this work. This new technology comprises a family of fluorescent biosensors called Nomad Biosensors that allow for the measurement of responses mediated by G proteins through their interactions with second-messenger transduction proteins. GPCRs are one of the largest protein families of receptors in eukaryotes, and their signaling mediates important physiological processes within cells. Thus, GPCRs are associated with a wide variety of diseases, and considered major targets in therapeutic research. Nomad constitutes a novel tool for unraveling the mechanism of GPCR signal transduction by simultaneously tracing different pathways. GPCR activation changes the structural folding of the biosensor and promotes its vesicularization, as well as an increase in the fluorescence intensity. Based on this technology, the MPX Nomad cellular model was developed to discriminate between the Ca 2+ -mediated pathway and the cyclic adenosine monophosphate (cAMP)-mediated pathway. To validate this model, endothelin receptor B (ET B R) was coexpressed into the MPX Nomad cell line and assessed with a specific agonist, an antagonist, and a chemical library of compounds. Nomad Technology optimizes the identification of novel GPCR ligands and enables the testing of large numbers of compounds.

Particle swarm optimization with recombination and dynamic linkage discovery.

PubMed

Chen, Ying-Ping; Peng, Wen-Chih; Jian, Ming-Chung

2007-12-01

In this paper, we try to improve the performance of the particle swarm optimizer by incorporating the linkage concept, which is an essential mechanism in genetic algorithms, and design a new linkage identification technique called dynamic linkage discovery to address the linkage problem in real-parameter optimization problems. Dynamic linkage discovery is a costless and effective linkage recognition technique that adapts the linkage configuration by employing only the selection operator without extra judging criteria irrelevant to the objective function. Moreover, a recombination operator that utilizes the discovered linkage configuration to promote the cooperation of particle swarm optimizer and dynamic linkage discovery is accordingly developed. By integrating the particle swarm optimizer, dynamic linkage discovery, and recombination operator, we propose a new hybridization of optimization methodologies called particle swarm optimization with recombination and dynamic linkage discovery (PSO-RDL). In order to study the capability of PSO-RDL, numerical experiments were conducted on a set of benchmark functions as well as on an important real-world application. The benchmark functions used in this paper were proposed in the 2005 Institute of Electrical and Electronics Engineers Congress on Evolutionary Computation. The experimental results on the benchmark functions indicate that PSO-RDL can provide a level of performance comparable to that given by other advanced optimization techniques. In addition to the benchmark, PSO-RDL was also used to solve the economic dispatch (ED) problem for power systems, which is a real-world problem and highly constrained. The results indicate that PSO-RDL can successfully solve the ED problem for the three-unit power system and obtain the currently known best solution for the 40-unit system.

Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophy.

PubMed

Wang, Chun-Chi; Chang, Jan-Gowth; Jong, Yuh-Jyh; Wu, Shou-Mei

2009-04-01

We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease.

Pre-capture multiplexing improves efficiency and cost-effectiveness of targeted genomic enrichment.

PubMed

Shearer, A Eliot; Hildebrand, Michael S; Ravi, Harini; Joshi, Swati; Guiffre, Angelica C; Novak, Barbara; Happe, Scott; LeProust, Emily M; Smith, Richard J H

2012-11-14

Targeted genomic enrichment (TGE) is a widely used method for isolating and enriching specific genomic regions prior to massively parallel sequencing. To make effective use of sequencer output, barcoding and sample pooling (multiplexing) after TGE and prior to sequencing (post-capture multiplexing) has become routine. While previous reports have indicated that multiplexing prior to capture (pre-capture multiplexing) is feasible, no thorough examination of the effect of this method has been completed on a large number of samples. Here we compare standard post-capture TGE to two levels of pre-capture multiplexing: 12 or 16 samples per pool. We evaluated these methods using standard TGE metrics and determined the ability to identify several classes of genetic mutations in three sets of 96 samples, including 48 controls. Our overall goal was to maximize cost reduction and minimize experimental time while maintaining a high percentage of reads on target and a high depth of coverage at thresholds required for variant detection. We adapted the standard post-capture TGE method for pre-capture TGE with several protocol modifications, including redesign of blocking oligonucleotides and optimization of enzymatic and amplification steps. Pre-capture multiplexing reduced costs for TGE by at least 38% and significantly reduced hands-on time during the TGE protocol. We found that pre-capture multiplexing reduced capture efficiency by 23 or 31% for pre-capture pools of 12 and 16, respectively. However efficiency losses at this step can be compensated by reducing the number of simultaneously sequenced samples. Pre-capture multiplexing and post-capture TGE performed similarly with respect to variant detection of positive control mutations. In addition, we detected no instances of sample switching due to aberrant barcode identification. Pre-capture multiplexing improves efficiency of TGE experiments with respect to hands-on time and reagent use compared to standard post-capture TGE

76 FR 71982 - Advancing Regulatory Science for Highly Multiplexed Microbiology/Medical Countermeasure Devices...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-21

...] Advancing Regulatory Science for Highly Multiplexed Microbiology/ Medical Countermeasure Devices; Public... Multiplexed Microbiology/ Medical Countermeasure Devices'' that published in the Federal Register of August 8... the October 13, 2011, meeting, including the performance evaluation of highly multiplexed microbiology...

Turbulence mitigation scheme based on spatial diversity in orbital-angular-momentum multiplexed system

NASA Astrophysics Data System (ADS)

Zou, Li; Wang, Le; Zhao, Shengmei

2017-10-01

Atmospheric turbulence (AT) induced crosstalk can significantly impair the performance of free-space optical (FSO) communication link using orbital angular momentum (OAM) multiplexing. In this paper, we propose a spatial diversity (SD) turbulence mitigation scheme in an OAM-multiplexed FSO communication link. First, we present a SD mitigation model for the OAM-multiplexed FSO communication link under AT. Then we present a SD combining technique based on equal gain to enhance AT tolerance of the OAM-multiplexed FSO communication link. The numerical results show that performance of the OAM-multiplexed communication link has greatly improved by the proposed scheme. When the turbulence strength Cn2 is 5 × 10-15m - 2 / 3, the transmission distance is 1000 m and the channel signal-to-noise ratio (SNR) is 20 dB, the bit-error-rate (BER) performance of four spatial multiplexed OAM modes lm = + 1 , + 2 , + 3 , + 4 are 3 fold increase in comparison with those results without the proposed scheme. The proposed scheme is a promising direction for compensating the interference caused by AT in the OAM-multiplexed FSO communication link.

Multiplex biosensing with highly sensitive magnetic nanoparticle quantification method

NASA Astrophysics Data System (ADS)

Nikitin, M. P.; Orlov, A. V.; Znoyko, S. L.; Bragina, V. A.; Gorshkov, B. G.; Ksenevich, T. I.; Cherkasov, V. R.; Nikitin, P. I.

2018-08-01

Unique properties of magnetic nanoparticles (MNP) have provided many breakthrough solutions for life science. The immense potential of MNP as labels in advanced immunoassays stems from the fact that they, unlike optical labels, can be easily detected inside 3D opaque porous biosensing structures or in colored mediums, manipulated by an external magnetic field, exhibit high stability and negligible background signal in biological samples, etc. In this research, the magnetic nanolabels and an original technique of their quantification by non-linear magnetization have permitted development of novel methods of multiplex biosensing. Several types of highly sensitive multi-channel readers that offer an extremely wide linear dynamic range are developed to count MNP in different recognition zones for quantitative concentration measurements of various analytes. Four approaches to multiplex biosensing based on MNP have been demonstrated in one-run tests based on several 3D porous structures; flat and micropillar microfluidic sensor chips; multi-line lateral flow strips and modular architecture of the strips, which is the first 3D multiplexing method that goes beyond the traditional planar techniques. Detection of cardio- and cancer markers, small molecules and oligonucleotides were used in the experiments. The analytical characteristics of the developed multiplex methods are on the level of the modern time-consuming laboratory techniques. The developed multiplex biosensing platforms are promising for medical and veterinary diagnostics, food inspection, environmental and security monitoring, etc.

Cavity-enhanced eigenmode and angular hybrid multiplexing in holographic data storage systems.

PubMed

Miller, Bo E; Takashima, Yuzuru

2016-12-26

Resonant optical cavities have been demonstrated to improve energy efficiencies in Holographic Data Storage Systems (HDSS). The orthogonal reference beams supported as cavity eigenmodes can provide another multiplexing degree of freedom to push storage densities toward the limit of 3D optical data storage. While keeping the increased energy efficiency of a cavity enhanced reference arm, image bearing holograms are multiplexed by orthogonal phase code multiplexing via Hermite-Gaussian eigenmodes in a Fe:LiNbO₃ medium with a 532 nm laser at two Bragg angles. We experimentally confirmed write rates are enhanced by an average factor of 1.1, and page crosstalk is about 2.5%. This hybrid multiplexing opens up a pathway to increase storage density while minimizing modification of current angular multiplexing HDSS.

Multiplexing of Radio-Frequency Single Electron Transistors

NASA Technical Reports Server (NTRS)

Stevenson, Thomas R.; Pellerano, F. A.; Stahle, C. M.; Aidala, K.; Schoelkopf, R. J.; Krebs, Carolyn (Technical Monitor)

2001-01-01

We present results on wavelength division multiplexing of radio-frequency single electron transistors. We use a network of resonant impedance matching circuits to direct applied rf carrier waves to different transistors depending on carrier frequency. A two-channel demonstration of this concept using discrete components successfully reconstructed input signals with small levels of cross coupling. A lithographic version of the rf circuits had measured parameters in agreement with electromagnetic modeling, with reduced cross capacitance and inductance, and should allow 20 to 50 channels to be multiplexed.

Model-Based Linkage Analysis of a Quantitative Trait.

PubMed

Song, Yeunjoo E; Song, Sunah; Schnell, Audrey H

2017-01-01

Linkage Analysis is a family-based method of analysis to examine whether any typed genetic markers cosegregate with a given trait, in this case a quantitative trait. If linkage exists, this is taken as evidence in support of a genetic basis for the trait. Historically, linkage analysis was performed using a binary disease trait, but has been extended to include quantitative disease measures. Quantitative traits are desirable as they provide more information than binary traits. Linkage analysis can be performed using single-marker methods (one marker at a time) or multipoint (using multiple markers simultaneously). In model-based linkage analysis the genetic model for the trait of interest is specified. There are many software options for performing linkage analysis. Here, we use the program package Statistical Analysis for Genetic Epidemiology (S.A.G.E.). S.A.G.E. was chosen because it also includes programs to perform data cleaning procedures and to generate and test genetic models for a quantitative trait, in addition to performing linkage analysis. We demonstrate in detail the process of running the program LODLINK to perform single-marker analysis, and MLOD to perform multipoint analysis using output from SEGREG, where SEGREG was used to determine the best fitting statistical model for the trait.

Cooperative spreading processes in multiplex networks.

PubMed

Wei, Xiang; Chen, Shihua; Wu, Xiaoqun; Ning, Di; Lu, Jun-An

2016-06-01

This study is concerned with the dynamic behaviors of epidemic spreading in multiplex networks. A model composed of two interacting complex networks is proposed to describe cooperative spreading processes, wherein the virus spreading in one layer can penetrate into the other to promote the spreading process. The global epidemic threshold of the model is smaller than the epidemic thresholds of the corresponding isolated networks. Thus, global epidemic onset arises in the interacting networks even though an epidemic onset does not arise in each isolated network. Simulations verify the analysis results and indicate that cooperative spreading processes in multiplex networks enhance the final infection fraction.

Parallel multiplex laser feedback interferometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Song; Tan, Yidong; Zhang, Shulian, E-mail: zsl-dpi@mail.tsinghua.edu.cn

2013-12-15

We present a parallel multiplex laser feedback interferometer based on spatial multiplexing which avoids the signal crosstalk in the former feedback interferometer. The interferometer outputs two close parallel laser beams, whose frequencies are shifted by two acousto-optic modulators by 2Ω simultaneously. A static reference mirror is inserted into one of the optical paths as the reference optical path. The other beam impinges on the target as the measurement optical path. Phase variations of the two feedback laser beams are simultaneously measured through heterodyne demodulation with two different detectors. Their subtraction accurately reflects the target displacement. Under typical room conditions, experimentalmore » results show a resolution of 1.6 nm and accuracy of 7.8 nm within the range of 100 μm.« less

Differential polarization nonlinear optical microscopy with adaptive optics controlled multiplexed beams.

PubMed

Samim, Masood; Sandkuijl, Daaf; Tretyakov, Ian; Cisek, Richard; Barzda, Virginijus

2013-09-09

Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures.

Screening newborn blood spots for 22q11.2 deletion syndrome using multiplex droplet digital PCR.

PubMed

Pretto, Dalyir; Maar, Dianna; Yrigollen, Carolyn M; Regan, Jack; Tassone, Flora

2015-01-01

The diagnosis of 22q11 deletion syndrome (22q11DS) is often delayed or missed due to the wide spectrum of clinical involvement ranging from mild to severe, often life-threatening conditions. A delayed diagnosis can lead to life-long health issues that could be ameliorated with early intervention and treatment. Owing to the high impact of 22q11DS on public health, propositions have been made to include 22q11DS in newborn screening panels; however, the method of choice for detecting 22q11DS, fluorescent in situ hybridization, requires specialized equipment and is cumbersome for most laboratories to implement as part of their routine screening. We sought to develop a new genetic screen for 22q11DS that is rapid, cost-effective, and easily used by laboratories currently performing newborn screening. We evaluated the accuracy of multiplex droplet digital PCR (ddPCR) in the detection of copy number of 22q11DS by screening samples from 26 patients with 22q11DS blindly intermixed with 1096 blood spot cards from the general population (total n = 1122). Multiplex ddPCR correctly identified all 22q11DS samples and distinguished between 1.5- and 3-Mb deletions, suggesting the approach is sensitive and specific for the detection of 22q11DS. These data demonstrate the utility of multiplex ddPCR for large-scale population-based studies that screen for 22q11DS. The use of samples from blood spot cards suggests that this approach has promise for newborn screening of 22q11DS, and potentially for other microdeletion syndromes, for which early detection can positively impact clinical outcome for those affected. © 2014 American Association for Clinical Chemistry.

Structural synthesis of linkages for quadruped bio-robot legs

NASA Astrophysics Data System (ADS)

Antonescu, O.; Robu, C.; Antonescu, P.

2016-08-01

The paper presents a few kinematic schemes of planar mechanisms with bars (linkages) used as part of the quadruped robot legs. The Dunshee linkage having only four elements as crank-rocker mechanism is analyzed. Further, the Klann linkage, which is accomplished by amplifying the crank-rocker mechanism with a dyadic kinematic chain, is also presented. More than that, the Jansen linkage, which is obtained by extending and amplifying the crank-rocker mechanism with two dyadic kinematic chains, is also analyzed. At the end of the paper, the authors present a novel linkage application consisting of a quadric kinematic chain.

Multiplexed operation of a micromachined ultrasonic droplet ejector array

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forbes, Thomas P.; Degertekin, F. Levent; Fedorov, Andrei G.

2007-10-15

A dual-sample ultrasonic droplet ejector array is developed for use as a soft-ionization ion source for multiplexed mass spectrometry (MS). Such a multiplexed ion source aims to reduce MS analysis time for multiple analyte streams, as well as allow for the synchronized ejection of the sample(s) and an internal standard for quantitative results and mass calibration. Multiplexing is achieved at the device level by division of the fluid reservoir and separating the active electrodes of the piezoelectric transducer for isolated application of ultrasonic wave energy to each domain. The transducer is mechanically shaped to further reduce the acoustical crosstalk betweenmore » the domains. Device design is performed using finite-element analysis simulations and supported by experimental characterization. Isolated ejection of {approx}5 {mu}m diameter water droplets from individual domains in the micromachined droplet ejector array at around 1 MHz frequency is demonstrated by experiments. The proof-of-concept demonstration using a dual-sample device also shows potential for multiplexing with larger numbers of analytes.« less

Precoded spatial multiplexing MIMO system with spatial component interleaver.

PubMed

Gao, Xiang; Wu, Zhanji

In this paper, the performance of precoded bit-interleaved coded modulation (BICM) spatial multiplexing multiple-input multiple-output (MIMO) system with spatial component interleaver is investigated. For the ideal precoded spatial multiplexing MIMO system with spatial component interleaver based on singular value decomposition (SVD) of the MIMO channel, the average pairwise error probability (PEP) of coded bits is derived. Based on the PEP analysis, the optimum spatial Q-component interleaver design criterion is provided to achieve the minimum error probability. For the limited feedback precoded proposed scheme with linear zero forcing (ZF) receiver, in order to minimize a bound on the average probability of a symbol vector error, a novel effective signal-to-noise ratio (SNR)-based precoding matrix selection criterion and a simplified criterion are proposed. Based on the average mutual information (AMI)-maximization criterion, the optimal constellation rotation angles are investigated. Simulation results indicate that the optimized spatial multiplexing MIMO system with spatial component interleaver can achieve significant performance advantages compared to the conventional spatial multiplexing MIMO system.

Compact reflection holographic recording system with high angle multiplexing

NASA Astrophysics Data System (ADS)

Kanayasu, Mayumi; Yamada, Takehumi; Takekawa, Shunsuke; Akieda, Kensuke; Goto, Akiyo; Yamamoto, Manabu

2011-02-01

Holographic memory systems have been widely researched since 1963. However, the size of the drives required and the deterioration of reconstructed data resulting from shrinkage of the medium have made practical use of a hologram memory difficult. In light of this, we propose a novel holographic recording/reconstructing system: a dual-reference beam reflection system that is smaller than conventional systems such as the off-axis or co-axis types, and which is expected to increase the number of multiplexing in angle multiplexed recording. In this multiplex recording system, two laser beams are used as reference beams, and the recorded data are reconstructed stably, even if there is shrinkage of the recording medium. In this paper, a reflection holographic memory system is explained in detail. In addition, the change in angle selectivity resulting from shrinkage of the medium is analyzed using the laminated film three-dimensional simulation method. As a result, we demonstrate that a dual-reference beam multiplex recording system is effective in reducing the influence of medium shrinkage.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

Orbital Angular Momentum Multiplexing over Visible Light Communication Systems

NASA Astrophysics Data System (ADS)

Tripathi, Hardik Rameshchandra

This thesis proposes and explores the possibility of using Orbital Angular Momentum multiplexing in Visible Light Communication system. Orbital Angular Momentum is mainly applied for laser and optical fiber transmissions, while Visible Light Communication is a technology using the light as a carrier for wireless communication. In this research, the study of the state of art and experiments showing some results on multiplexing based on Orbital Angular Momentum over Visible Light Communication system were done. After completion of the initial stage; research work and simulations were performed on spatial multiplexing over Li-Fi channel modeling. Simulation scenarios which allowed to evaluate the Signal-to-Noise Ratio, Received Power Distribution, Intensity and Illuminance were defined and developed.

Time-varying multiplex network: Intralayer and interlayer synchronization

NASA Astrophysics Data System (ADS)

Rakshit, Sarbendu; Majhi, Soumen; Bera, Bidesh K.; Sinha, Sudeshna; Ghosh, Dibakar

2017-12-01

A large class of engineered and natural systems, ranging from transportation networks to neuronal networks, are best represented by multiplex network architectures, namely a network composed of two or more different layers where the mutual interaction in each layer may differ from other layers. Here we consider a multiplex network where the intralayer coupling interactions are switched stochastically with a characteristic frequency. We explore the intralayer and interlayer synchronization of such a time-varying multiplex network. We find that the analytically derived necessary condition for intralayer and interlayer synchronization, obtained by the master stability function approach, is in excellent agreement with our numerical results. Interestingly, we clearly find that the higher frequency of switching links in the layers enhances both intralayer and interlayer synchrony, yielding larger windows of synchronization. Further, we quantify the resilience of synchronous states against random perturbations, using a global stability measure based on the concept of basin stability, and this reveals that intralayer coupling strength is most crucial for determining both intralayer and interlayer synchrony. Lastly, we investigate the robustness of interlayer synchronization against a progressive demultiplexing of the multiplex structure, and we find that for rapid switching of intralayer links, the interlayer synchronization persists even when a large number of interlayer nodes are disconnected.

Time-varying multiplex network: Intralayer and interlayer synchronization.

PubMed

Rakshit, Sarbendu; Majhi, Soumen; Bera, Bidesh K; Sinha, Sudeshna; Ghosh, Dibakar

2017-12-01

A large class of engineered and natural systems, ranging from transportation networks to neuronal networks, are best represented by multiplex network architectures, namely a network composed of two or more different layers where the mutual interaction in each layer may differ from other layers. Here we consider a multiplex network where the intralayer coupling interactions are switched stochastically with a characteristic frequency. We explore the intralayer and interlayer synchronization of such a time-varying multiplex network. We find that the analytically derived necessary condition for intralayer and interlayer synchronization, obtained by the master stability function approach, is in excellent agreement with our numerical results. Interestingly, we clearly find that the higher frequency of switching links in the layers enhances both intralayer and interlayer synchrony, yielding larger windows of synchronization. Further, we quantify the resilience of synchronous states against random perturbations, using a global stability measure based on the concept of basin stability, and this reveals that intralayer coupling strength is most crucial for determining both intralayer and interlayer synchrony. Lastly, we investigate the robustness of interlayer synchronization against a progressive demultiplexing of the multiplex structure, and we find that for rapid switching of intralayer links, the interlayer synchronization persists even when a large number of interlayer nodes are disconnected.

Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Krull, Ulrich J

2013-08-06

A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.

PubMed

Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J

2013-01-01

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (<10 ns) emission signals from organic chromophores or tissue autofluorescence. Here using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by >50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

In Vivo Time-gated Fluorescence Imaging with Biodegradable Luminescent Porous Silicon Nanoparticles

PubMed Central

Gu, Luo; Hall, David J.; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J.; Howell, Stephen B.; Sailor, Michael J.

2014-01-01

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (< 10 ns) emission signals from organic chromophores or tissue autofluorescence.Here, using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by > 50-fold in vitro and by > 20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed. PMID:23933660

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.

Posterior probability of linkage and maximal lod score.

PubMed

Génin, E; Martinez, M; Clerget-Darpoux, F

1995-01-01

To detect linkage between a trait and a marker, Morton (1955) proposed to calculate the lod score z(theta 1) at a given value theta 1 of the recombination fraction. If z(theta 1) reaches +3 then linkage is concluded. However, in practice, lod scores are calculated for different values of the recombination fraction between 0 and 0.5 and the test is based on the maximum value of the lod score Zmax. The impact of this deviation of the test on the probability that in fact linkage does not exist, when linkage was concluded, is documented here. This posterior probability of no linkage can be derived by using Bayes' theorem. It is less than 5% when the lod score at a predetermined theta 1 is used for the test. But, for a Zmax of +3, we showed that it can reach 16.4%. Thus, considering a composite alternative hypothesis instead of a single one decreases the reliability of the test. The reliability decreases rapidly when Zmax is less than +3. Given a Zmax of +2.5, there is a 33% chance that linkage does not exist. Moreover, the posterior probability depends not only on the value of Zmax but also jointly on the family structures and on the genetic model. For a given Zmax, the chance that linkage exists may then vary.

A multiplex PCR for detection of six viruses in ducks.

PubMed

Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

2017-10-01

In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10 2 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

A reference linkage map for Eucalyptus

PubMed Central

2012-01-01

Background Genetic linkage maps are invaluable resources in plant research. They provide a key tool for many genetic applications including: mapping quantitative trait loci (QTL); comparative mapping; identifying unlinked (i.e. independent) DNA markers for fingerprinting, population genetics and phylogenetics; assisting genome sequence assembly; relating physical and recombination distances along the genome and map-based cloning of genes. Eucalypts are the dominant tree species in most Australian ecosystems and of economic importance globally as plantation trees. The genome sequence of E. grandis has recently been released providing unprecedented opportunities for genetic and genomic research in the genus. A robust reference linkage map containing sequence-based molecular markers is needed to capitalise on this resource. Several high density linkage maps have recently been constructed for the main commercial forestry species in the genus (E. grandis, E. urophylla and E. globulus) using sequenced Diversity Arrays Technology (DArT) and microsatellite markers. To provide a single reference linkage map for eucalypts a composite map was produced through the integration of data from seven independent mapping experiments (1950 individuals) using a marker-merging method. Results The composite map totalled 1107 cM and contained 4101 markers; comprising 3880 DArT, 213 microsatellite and eight candidate genes. Eighty-one DArT markers were mapped to two or more linkage groups, resulting in the 4101 markers being mapped to 4191 map positions. Approximately 13% of DArT markers mapped to identical map positions, thus the composite map contained 3634 unique loci at an average interval of 0.31 cM. Conclusion The composite map represents the most saturated linkage map yet produced in Eucalyptus. As the majority of DArT markers contained on the map have been sequenced, the map provides a direct link to the E. grandis genome sequence and will serve as an important reference for

The new challenges of multiplex networks: Measures and models

NASA Astrophysics Data System (ADS)

Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

2017-02-01

What do societies, the Internet, and the human brain have in common? They are all examples of complex relational systems, whose emerging behaviours are largely determined by the non-trivial networks of interactions among their constituents, namely individuals, computers, or neurons, rather than only by the properties of the units themselves. In the last two decades, network scientists have proposed models of increasing complexity to better understand real-world systems. Only recently we have realised that multiplexity, i.e. the coexistence of several types of interactions among the constituents of a complex system, is responsible for substantial qualitative and quantitative differences in the type and variety of behaviours that a complex system can exhibit. As a consequence, multilayer and multiplex networks have become a hot topic in complexity science. Here we provide an overview of some of the measures proposed so far to characterise the structure of multiplex networks, and a selection of models aiming at reproducing those structural properties and quantifying their statistical significance. Focusing on a subset of relevant topics, this brief review is a quite comprehensive introduction to the most basic tools for the analysis of multiplex networks observed in the real-world. The wide applicability of multiplex networks as a framework to model complex systems in different fields, from biology to social sciences, and the colloquial tone of the paper will make it an interesting read for researchers working on both theoretical and experimental analysis of networked systems.

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

PubMed

Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

Photoacoustic and fluorescent imaging GAF2 photoswitchable chromoproteins (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chee, Ryan K.; Li, Yan; Paproski, Robert J.; Campbell, Robert E.; Zemp, Roger J.

2017-03-01

Molecular photoacoustic imaging is hindered by hemoglobin background signal. Photoswitchable chromoproteins can be used to obtain images with significantly reduced background signal. Molecular imaging of multiple biological processes via multiple chromoprotiens is difficult due to overlapping imaging spectra. Using a new rate-of-change imaging methodology, we can obtain molecular images with multiple chromoprotiens with overlapping imaging spectra. We also present a new photoswitchable chromoprotein, GAF2, which is significantly smaller than the BphP1 which has shown promise for photoswitchable photoacoustic imaging [Yao et al., Nat. Meth. 13, 67-73 (2016)]. We use BphP1 and GAF2 with photoacoustic (Vevo LAZR, Fujifilm Visualsonics Inc) and fluorescence (In vivo Xtreme, Bruker) imaging systems to show background-free multiplexed images. We image before, after, and during photoconversion to obtain background-free rate-of-change images and compare our results to difference imaging and spectral demixing. After phantom imaging, we inject mice with different chromoprotein-expressing E. coli bacteria to show multiplexed images of bacterial infections. We show distinguishable differences in the rate-of-change between GAF2 and BphP1. We obtain rate-of-change feasibility images and in vivo images in mice showing the ability to differentiate between GAF2 and BphP1 even though they are spectrally similar. We photoconvert both GAF2 and BphP1 using 550nm and 735nm light. Phantom studies suggest a 10-20dB improvement in the rate-of-change and difference images in comparison to images with background. Multiplexed background-free molecular imaging using chromoproteins could prove to be a promising new imaging methodology especially when combined with spectral demixing.

A Genetic Linkage Map for Cattle

PubMed Central

Bishop, M. D.; Kappes, S. M.; Keele, J. W.; Stone, R. T.; Sunden, SLF.; Hawkins, G. A.; Toldo, S. S.; Fries, R.; Grosz, M. D.; Yoo, J.; Beattie, C. W.

1994-01-01

We report the most extensive physically anchored linkage map for cattle produced to date. Three-hundred thirteen genetic markers ordered in 30 linkage groups, anchored to 24 autosomal chromosomes (n = 29), the X and Y chromosomes, four unanchored syntenic groups and two unassigned linkage groups spanning 2464 cM of the bovine genome are summarized. The map also assigns 19 type I loci to specific chromosomes and/or syntenic groups and four cosmid clones containing informative microsatellites to chromosomes 13, 25 and 29 anchoring syntenic groups U11, U7 and U8, respectively. This map provides the skeletal framework prerequisite to development of a comprehensive genetic map for cattle and analysis of economic trait loci (ETL). PMID:7908653

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

PubMed

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

Multiplexed Energy Coupler for Rotating Equipment

NASA Technical Reports Server (NTRS)

Zhao, Xiaoliang

2011-01-01

A multiplexing antenna assembly can efficiently couple AC signal/energy into, or out of, rotating equipment. The unit only passes AC energy while blocking DC energy. Concentric tubes that are sliced into multiple pieces are assembled together so that, when a piece from an outer tube aligns well with an inner tube piece, efficient energy coupling is achieved through a capacitive scheme. With N outer pieces and M inner pieces, an effective N x M combination can be achieved in a multiplexed manner. The energy coupler is non-contact, which is useful if isolation from rotating and stationary parts is required. Additionally, the innovation can operate in high temperatures. Applications include rotating structure sensing, non-contact energy transmission, etc.

Centrality in earthquake multiplex networks

NASA Astrophysics Data System (ADS)

Lotfi, Nastaran; Darooneh, Amir Hossein; Rodrigues, Francisco A.

2018-06-01

Seismic time series has been mapped as a complex network, where a geographical region is divided into square cells that represent the nodes and connections are defined according to the sequence of earthquakes. In this paper, we map a seismic time series to a temporal network, described by a multiplex network, and characterize the evolution of the network structure in terms of the eigenvector centrality measure. We generalize previous works that considered the single layer representation of earthquake networks. Our results suggest that the multiplex representation captures better earthquake activity than methods based on single layer networks. We also verify that the regions with highest seismological activities in Iran and California can be identified from the network centrality analysis. The temporal modeling of seismic data provided here may open new possibilities for a better comprehension of the physics of earthquakes.

Emergence of Multiplex Communities in Collaboration Networks.

PubMed

Battiston, Federico; Iacovacci, Jacopo; Nicosia, Vincenzo; Bianconi, Ginestra; Latora, Vito

2016-01-01

Community structures in collaboration networks reflect the natural tendency of individuals to organize their work in groups in order to better achieve common goals. In most of the cases, individuals exploit their connections to introduce themselves to new areas of interests, giving rise to multifaceted collaborations which span different fields. In this paper, we analyse collaborations in science and among movie actors as multiplex networks, where the layers represent respectively research topics and movie genres, and we show that communities indeed coexist and overlap at the different layers of such systems. We then propose a model to grow multiplex networks based on two mechanisms of intra and inter-layer triadic closure which mimic the real processes by which collaborations evolve. We show that our model is able to explain the multiplex community structure observed empirically, and we infer the strength of the two underlying social mechanisms from real-world systems. Being also able to correctly reproduce the values of intra-layer and inter-layer assortativity correlations, the model contributes to a better understanding of the principles driving the evolution of social networks.

Multiplex primer prediction software for divergent targets

PubMed Central

Gardner, Shea N.; Hiddessen, Amy L.; Williams, Peter L.; Hara, Christine; Wagner, Mark C.; Colston, Bill W.

2009-01-01

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus. PMID:19759213

DNA linkage studies of degenerative retinal diseases.

PubMed

Daiger, S P; Heckenlively, J R; Lewis, R A; Pelias, M Z

1987-01-01

DNA linkage studies of human genetic diseases have led to rapid characterization of a number of otherwise intractable disease loci. Detection of a linked DNA marker, the first step in "reverse genetics", has permitted cloning of the genes for Duchenne muscular dystrophy, retinoblastoma and chronic granulomatosis disease, among others. Thus, the case for applying these techniques to retinitis pigmentosa and related diseases, and the urgency in capitalizing on molecular developments, is justified and compelling. The first major success regarding RP was in demonstrating linkage of the DNA marker DXS7 (L1.28) to XRP. For autosomal forms of the disease, conventional linkage studies have provided tentative evidence for linkage of ADRP to the Rh blood group on chromosome lp and for linkage of Usher's syndrome to Gc and 4q. These provisional assignments are, at least, an important starting point for DNA analysis. The Support Program for DNA Linkage Studies of Degenerative Retinal Diseases was established to provide access for the scientific community to appropriate families, using the resources of the Human Genetic Mutant Cell Repository to prepare, store and distribute lymphoblast lines. To date, two extensive, well-characterized families are included in the program: the autosomal dominant RP family UCLA-RP01, and the Usher's syndrome families LSU-US01. It is highly likely that rapid progress will be made in mapping and characterizing the inherited retinal dystrophies. We believe the support program will facilitate this progress.

Testing association and linkage using affected-sib-parent study designs.

PubMed

Millstein, Joshua; Siegmund, Kimberly D; Conti, David V; Gauderman, W James

2005-11-01

We have developed a method for jointly testing linkage and association using data from affected sib pairs and their parents. We specify a conditional logistic regression model with two covariates, one that quantifies association (either direct association or indirect association via linkage disequilibrium), and a second that quantifies linkage. The latter covariate is computed based on expected identity-by-descend (ibd) sharing of marker alleles between siblings. In addition to a joint test of linkage and association, our general framework can be used to obtain a linkage test comparable to the mean test (Blackwelder and Elston [1985] Genet. Epidemiol. 2:85-97), and an association test comparable to the Family-Based Association Test (FBAT; Rabinowitz and Laird [2000] Hum. Hered. 50:211-223). We present simulation results demonstrating that our joint test can be more powerful than some standard tests of linkage or association. For example, with a relative risk of 2.7 per variant allele at a disease locus, the estimated power to detect a nearby marker with a modest level of LD was 58.1% by the mean test (linkage only), 69.8% by FBAT, and 82.5% by our joint test of linkage and association. Our model can also be used to obtain tests of linkage conditional on association and association conditional on linkage, which can be helpful in fine mapping. Copyright 2005 Wiley-Liss, Inc.

Multiplex congruence network of natural numbers.

PubMed

Yan, Xiao-Yong; Wang, Wen-Xu; Chen, Guan-Rong; Shi, Ding-Hua

2016-03-31

Congruence theory has many applications in physical, social, biological and technological systems. Congruence arithmetic has been a fundamental tool for data security and computer algebra. However, much less attention was devoted to the topological features of congruence relations among natural numbers. Here, we explore the congruence relations in the setting of a multiplex network and unveil some unique and outstanding properties of the multiplex congruence network. Analytical results show that every layer therein is a sparse and heterogeneous subnetwork with a scale-free topology. Counterintuitively, every layer has an extremely strong controllability in spite of its scale-free structure that is usually difficult to control. Another amazing feature is that the controllability is robust against targeted attacks to critical nodes but vulnerable to random failures, which also differs from ordinary scale-free networks. The multi-chain structure with a small number of chain roots arising from each layer accounts for the strong controllability and the abnormal feature. The multiplex congruence network offers a graphical solution to the simultaneous congruences problem, which may have implication in cryptography based on simultaneous congruences. Our work also gains insight into the design of networks integrating advantages of both heterogeneous and homogeneous networks without inheriting their limitations.

Multiplex congruence network of natural numbers

NASA Astrophysics Data System (ADS)

Yan, Xiao-Yong; Wang, Wen-Xu; Chen, Guan-Rong; Shi, Ding-Hua

2016-03-01

Congruence theory has many applications in physical, social, biological and technological systems. Congruence arithmetic has been a fundamental tool for data security and computer algebra. However, much less attention was devoted to the topological features of congruence relations among natural numbers. Here, we explore the congruence relations in the setting of a multiplex network and unveil some unique and outstanding properties of the multiplex congruence network. Analytical results show that every layer therein is a sparse and heterogeneous subnetwork with a scale-free topology. Counterintuitively, every layer has an extremely strong controllability in spite of its scale-free structure that is usually difficult to control. Another amazing feature is that the controllability is robust against targeted attacks to critical nodes but vulnerable to random failures, which also differs from ordinary scale-free networks. The multi-chain structure with a small number of chain roots arising from each layer accounts for the strong controllability and the abnormal feature. The multiplex congruence network offers a graphical solution to the simultaneous congruences problem, which may have implication in cryptography based on simultaneous congruences. Our work also gains insight into the design of networks integrating advantages of both heterogeneous and homogeneous networks without inheriting their limitations.

Ignoring Intermarker Linkage Disequilibrium Induces False-Positive Evidence of Linkage for Consanguineous Pedigrees when Genotype Data Is Missing for Any Pedigree Member

PubMed Central

Li, Bingshan; Leal, Suzanne M.

2008-01-01

Missing genotype data can increase false-positive evidence for linkage when either parametric or nonparametric analysis is carried out ignoring intermarker linkage disequilibrium (LD). Previously it was demonstrated by Huang et al. [1] that no bias occurs in this situation for affected sib-pairs with unrelated parents when either both parents are genotyped or genotype data is available for two additional unaffected siblings when parental genotypes are missing. However, this is not the case for autosomal recessive consanguineous pedigrees, where missing genotype data for any pedigree member within a consanguinity loop can increase false-positive evidence of linkage. False-positive evidence for linkage is further increased when cryptic consanguinity is present. The amount of false-positive evidence for linkage, and which family members aid in its reduction, is highly dependent on which family members are genotyped. When parental genotype data is available, the false-positive evidence for linkage is usually not as strong as when parental genotype data is unavailable. For a pedigree with an affected proband whose first-cousin parents have been genotyped, further reduction in the false-positive evidence of linkage can be obtained by including genotype data from additional affected siblings of the proband or genotype data from the proband's sibling-grandparents. For the situation, when parental genotypes are unavailable, false-positive evidence for linkage can be reduced by including genotype data from either unaffected siblings of the proband or the proband's married-in-grandparents in the analysis. PMID:18073490

Digital image compression for a 2f multiplexing optical setup

NASA Astrophysics Data System (ADS)

Vargas, J.; Amaya, D.; Rueda, E.

2016-07-01

In this work a virtual 2f multiplexing system was implemented in combination with digital image compression techniques and redundant information elimination. Depending on the image type to be multiplexed, a memory-usage saving of as much as 99% was obtained. The feasibility of the system was tested using three types of images, binary characters, QR codes, and grey level images. A multiplexing step was implemented digitally, while a demultiplexing step was implemented in a virtual 2f optical setup following real experimental parameters. To avoid cross-talk noise, each image was codified with a specially designed phase diffraction carrier that would allow the separation and relocation of the multiplexed images on the observation plane by simple light propagation. A description of the system is presented together with simulations that corroborate the method. The present work may allow future experimental implementations that will make use of all the parallel processing capabilities of optical systems.

Validation of HAV biomarker 2A for differential diagnostic of hepatitis A infected and vaccinated individuals using multiplex serology.

PubMed

Bohm, Katrin; Filomena, Angela; Schneiderhan-Marra, Nicole; Krause, Gérard; Sievers, Claudia

2017-10-13

Worldwide about 1.5 million clinical cases of hepatitis A virus (HAV) infections occur every year and increasingly countries are introducing HAV vaccination into the childhood immunization schedule with a single dose instead of the originally licenced two dose regimen. Diagnosis of acute HAV infection is determined serologically by anti-HAV-IgM detection using ELISA. Additionally anti-HAV-IgG can become positive during the early phase of symptoms, but remains detectable after infection and also after vaccination against HAV. Currently no serological marker allows the differentiation of HAV vaccinated individuals and those with a past infection with HAV. Such differentiation would greatly improve evaluation of vaccination campaigns and risk assessment of HAV outbreaks. Here we tested the HAV non-structural protein 2A, important for the capsid assembly, as a biomarker for the differentiation of the immune status in previously infected and vaccinated individuals. HAV antigens were recombinantly expressed as glutathione-S-transferase (GST) fusion proteins. Using glutathione tagged, magnetic fluorescent beads (Luminex®), the proteins were affinity purified and used in a multiplex serological assay. The multiplex HAV assay was validated using 381 reference sera in which the immune status HAV negative, vaccinated or infected was established using the Abbott ARCHITECT® HAVAb-IgM or IgG, the commercial HAV ELISA from Abnova and documentation in vaccination cards. HAV multiplex serology showed a sensitivity of 99% and specificity of 95% to detect anti-HAV IgG/IgM positive individuals. HAV biomarker 2A allowed the differentiation between previously infected and vaccinated individuals. HAV vaccinated individuals and previously infected individuals could be identified with 92% accuracy. HAV biomarker 2A can be used to differentiate between previously HAV-vaccinated and naturally infected individuals. Within a multiplex serological approach this assay can provide valuable

Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.

PubMed

Guion, Chase E; Ochoa, Theresa J; Walker, Christopher M; Barletta, Francesca; Cleary, Thomas G

2008-05-01

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

Topology-optimized silicon photonic wire mode (de)multiplexer

NASA Astrophysics Data System (ADS)

Frellsen, Louise F.; Frandsen, Lars H.; Ding, Yunhong; Elesin, Yuriy; Sigmund, Ole; Yvind, Kresten

2015-02-01

We have designed and for the first time experimentally verified a topology optimized mode (de)multiplexer, which demultiplexes the fundamental and the first order mode of a double mode photonic wire to two separate single mode waveguides (and multiplexes vice versa). The device has a footprint of ~4.4 μm x ~2.8 μm and was fabricated for different design resolutions and design threshold values to verify the robustness of the structure to fabrication tolerances. The multiplexing functionality was confirmed by recording mode profiles using an infrared camera and vertical grating couplers. All structures were experimentally found to maintain functionality throughout a 100 nm wavelength range limited by available laser sources and insertion losses were generally lower than 1.3 dB. The cross talk was around -12 dB and the extinction ratio was measured to be better than 8 dB.

Privacy-preserving record linkage on large real world datasets.

PubMed

Randall, Sean M; Ferrante, Anna M; Boyd, James H; Bauer, Jacqueline K; Semmens, James B

2014-08-01

Record linkage typically involves the use of dedicated linkage units who are supplied with personally identifying information to determine individuals from within and across datasets. The personally identifying information supplied to linkage units is separated from clinical information prior to release by data custodians. While this substantially reduces the risk of disclosure of sensitive information, some residual risks still exist and remain a concern for some custodians. In this paper we trial a method of record linkage which reduces privacy risk still further on large real world administrative data. The method uses encrypted personal identifying information (bloom filters) in a probability-based linkage framework. The privacy preserving linkage method was tested on ten years of New South Wales (NSW) and Western Australian (WA) hospital admissions data, comprising in total over 26 million records. No difference in linkage quality was found when the results were compared to traditional probabilistic methods using full unencrypted personal identifiers. This presents as a possible means of reducing privacy risks related to record linkage in population level research studies. It is hoped that through adaptations of this method or similar privacy preserving methods, risks related to information disclosure can be reduced so that the benefits of linked research taking place can be fully realised. Copyright © 2013 Elsevier Inc. All rights reserved.

A Test of Outreach and Drop-in Linkage Versus Shelter Linkage for Connecting Homeless Youth to Services.

PubMed

Slesnick, Natasha; Feng, Xin; Guo, Xiamei; Brakenhoff, Brittany; Carmona, Jasmin; Murnan, Aaron; Cash, Scottye; McRee, Annie-Laurie

2016-05-01

Outreach and service linkage are key for engaging marginalized populations, such as homeless youth, in services. Research to date has focused primarily on engaging individuals already receiving some services through emergency shelters, clinics, or other programs. Less is known about those who are not connected to services and, thus, likely the most vulnerable and in need of assistance. The current study sought to engage non-service-connected homeless youth (N = 79) into a strengths-based outreach and advocacy intervention. Youth were randomly assigned to receive 6 months of advocacy that focused on linking youth to a drop-in center (n = 40) or to a crisis shelter (n = 39). All youth were assessed at baseline and 3, 6, and 9 months post-baseline. Findings indicated that youth prefer drop-in center services to the shelter. Also, the drop-in center linkage condition was associated with more service linkage overall (B = 0.34, SE = 0.04, p < 0.01) and better alcohol-l [B = -0.39, SE = 0.09, t(75) = -4.48, p < 0.001] and HIV-related outcomes [B = 0.62, SE = 0.10, t(78) = 6.34, p < 0.001] compared to the shelter linkage condition. Findings highlight the importance of outreach and service linkage for reconnecting service-marginalized youth, and drop-in centers as a primary service option for homeless youth.

Genotoxic Mode of Action Predictions from a Multiplexed Flow Cytometric Assay and a Machine Learning Approach

PubMed Central

Bryce, Steven M.; Bernacki, Derek T.; Bemis, Jeffrey C.; Dertinger, Stephen D.

2015-01-01

Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, “add and read” type flow cytometric assay. Reagents included a detergent to liberate nuclei, propidium iodide and RNase to serve as a pan-DNA dye, fluorescent antibodies against γH2AX, phospho-histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96 well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4 and 24 hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk-away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non-genotoxic. These mode of action predictions were optimized with a forward-stepping process that considered Wald test p-values, receiver operator characteristic curves, and pseudo R2 values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4hr γH2AX and phospho-histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four-factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave-one-out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short-term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new

Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.

PubMed

Bryce, Steven M; Bernacki, Derek T; Bemis, Jeffrey C; Dertinger, Stephen D

2016-04-01

Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, "add and read" type flow cytometric assay. Reagents included a detergent to liberate nuclei, RNase and propidium iodide to serve as a pan-DNA dye, fluorescent antibodies against γH2AX, phospho-histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96-well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4- and 24-hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk-away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non-genotoxic. These mode of action predictions were optimized with a forward-stepping process that considered Wald test p-values, receiver operator characteristic curves, and pseudo R(2) values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4 hr γH2AX and phospho-histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four-factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave-one-out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short-term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new chemicals

Near-infrared fluorescence amplified organic nanoparticles with aggregation-induced emission characteristics for in vivo imaging

NASA Astrophysics Data System (ADS)

Geng, Junlong; Zhu, Zhenshu; Qin, Wei; Ma, Lin; Hu, Yong; Gurzadyan, Gagik G.; Tang, Ben Zhong; Liu, Bin

2013-12-01

Near-infrared (NIR) fluorescence signals are highly desirable to achieve high resolution in biological imaging. To obtain NIR emission with high brightness, fluorescent nanoparticles (NPs) are synthesized by co-encapsulation of 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenylamino)phenyl)fumaronitrile (TPETPAFN), a luminogen with aggregation-induced emission (AIE) characteristics, and a NIR fluorogen of silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775) using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] as the encapsulation matrix. The good spectral overlap between the emission of TPETPAFN and the absorption of NIR775 leads to efficient energy transfer, resulting in a 47-fold enhancement of the NIR775 emission intensity upon excitation of TPETPAFN at 510 nm as compared to that upon direct excitation of NIR775 at 760 nm. The obtained fluorescent NPs show sharp NIR emission with a band width of 20 nm, a large Stokes shift of 275 nm, good photostability and low cytotoxicity. In vivo imaging study reveals that the synthesized NPs are able to provide high fluorescence contrast in live animals. The Förster resonance energy transfer strategy overcomes the intrinsic limitation of broad emission spectra for AIE NPs, which opens new opportunities to synthesize organic NPs with high brightness and narrow emission for potential applications in multiplex sensing and imaging.Near-infrared (NIR) fluorescence signals are highly desirable to achieve high resolution in biological imaging. To obtain NIR emission with high brightness, fluorescent nanoparticles (NPs) are synthesized by co-encapsulation of 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenylamino)phenyl)fumaronitrile (TPETPAFN), a luminogen with aggregation-induced emission (AIE) characteristics, and a NIR fluorogen of silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775) using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000

Linkage studies in primary open angle glaucoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Avramopoulos, D.; Grigoriadu, M.; Kitsos, G.

1994-09-01

Glaucoma is a leading cause of blindness worldwide. The majority of glaucoma is associated with an open, normal appearing anterior chamber angle and is termed primary open angle glaucoma (POAG, MIM 137760). It is characterized by elevated intraocular pressure and onset in middle age or later. A subset of POAG with juvenile onset has recently been linked to chromosome 1q in two families with autosomal dominant inheritance. Eleven pedigrees with autosomal dominant POG (non-juvenile-onset) have been identified in Epirus, Greece. In the present study DNA samples have been collected from 50 individuals from one large pedigree, including 12 affected individuals.more » Preliminary results of linkage analysis with chromosome 1 microsatellites using the computer program package LINKAGE Version 5.1 showed no linkage with the markers previously linked to juvenile-onset POAG. Further linkage analysis is being pursued, and the results will be presented.« less

Multiplexed lateral flow biosensors: Technological advances for radically improving point-of-care diagnoses.

PubMed

Li, Jia; Macdonald, Joanne

2016-09-15

Lateral flow biosensors are a leading technology in point-of-care diagnostics due to their simplicity, rapidness and low cost. Their primacy in this arena continues through technological breakthroughs such as multiplexing: the detection of more than one biomarker in a single assay. Multiplexing capacity is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases and reducing diagnostic cost. Here we review, for the first time, the various types and strategies employed for creating multiplexed lateral flow biosensors. These are classified into four main categories in terms of specific application or multiplexing level, namely linear, parameter, spatial and conceptual. We describe the practical applications and implications for each approach and compare their advantages and disadvantages. Importantly, multiplexing is still subject to limitations of the traditional lateral flow biosensor, such as sensitivity and specificity. However, by pushing the limitations of the traditional medium into the multiplex arena, several technological breakthroughs are emerging with novel solutions that further expand the utility of lateral flow biosensing for point-of-care applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Encrypted holographic data storage based on orthogonal-phase-code multiplexing.

PubMed

Heanue, J F; Bashaw, M C; Hesselink, L

1995-09-10

We describe an encrypted holographic data-storage system that combines orthogonal-phase-code multiplexing with a random-phase key. The system offers the security advantages of random-phase coding but retains the low cross-talk performance and the minimum code storage requirements typical in an orthogonal-phase-code-multiplexing system.

Multiplex Networks of Cortical and Hippocampal Neurons Revealed at Different Timescales

PubMed Central

Timme, Nicholas; Ito, Shinya; Myroshnychenko, Maxym; Yeh, Fang-Chin; Hiolski, Emma; Hottowy, Pawel; Beggs, John M.

2014-01-01

Recent studies have emphasized the importance of multiplex networks – interdependent networks with shared nodes and different types of connections – in systems primarily outside of neuroscience. Though the multiplex properties of networks are frequently not considered, most networks are actually multiplex networks and the multiplex specific features of networks can greatly affect network behavior (e.g. fault tolerance). Thus, the study of networks of neurons could potentially be greatly enhanced using a multiplex perspective. Given the wide range of temporally dependent rhythms and phenomena present in neural systems, we chose to examine multiplex networks of individual neurons with time scale dependent connections. To study these networks, we used transfer entropy – an information theoretic quantity that can be used to measure linear and nonlinear interactions – to systematically measure the connectivity between individual neurons at different time scales in cortical and hippocampal slice cultures. We recorded the spiking activity of almost 12,000 neurons across 60 tissue samples using a 512-electrode array with 60 micrometer inter-electrode spacing and 50 microsecond temporal resolution. To the best of our knowledge, this preparation and recording method represents a superior combination of number of recorded neurons and temporal and spatial recording resolutions to any currently available in vivo system. We found that highly connected neurons (“hubs”) were localized to certain time scales, which, we hypothesize, increases the fault tolerance of the network. Conversely, a large proportion of non-hub neurons were not localized to certain time scales. In addition, we found that long and short time scale connectivity was uncorrelated. Finally, we found that long time scale networks were significantly less modular and more disassortative than short time scale networks in both tissue types. As far as we are aware, this analysis represents the first

Multiplexed Neurochemical Signaling by Neurons of the Ventral Tegmental Area

PubMed Central

Barker, David J.; Root, David H.; Zhang, Shiliang; Morales, Marisela

2016-01-01

The ventral tegmental area (VTA) is an evolutionarily conserved structure that has roles in reward-seeking, safety-seeking, learning, motivation, and neuropsychiatric disorders such as addiction and depression. The involvement of the VTA in these various behaviors and disorders is paralleled by its diverse signaling mechanisms. Here we review recent advances in our understanding of neuronal diversity in the VTA with a focus on cell phenotypes that participate in ‘multiplexed’ neurotransmission involving distinct signaling mechanisms. First, we describe the cellular diversity within the VTA, including neurons capable of transmitting dopamine, glutamate or GABA as well as neurons capable of multiplexing combinations of these neurotransmitters. Next, we describe the complex synaptic architecture used by VTA neurons in order to accommodate the transmission of multiple transmitters. We specifically cover recent findings showing that VTA multiplexed neurotransmission may be mediated by either the segregation of dopamine and glutamate into distinct microdomains within a single axon or by the integration of glutamate and GABA into a single axon terminal. In addition, we discuss our current understanding of the functional role that these multiplexed signaling pathways have in the lateral habenula and the nucleus accumbens. Finally, we consider the putative roles of VTA multiplexed neurotransmission in synaptic plasticity and discuss how changes in VTA multiplexed neurons may relate to various psychopathologies including drug addiction and depression. PMID:26763116

Multiplex amplification of large sets of human exons.

PubMed

Porreca, Gregory J; Zhang, Kun; Li, Jin Billy; Xie, Bin; Austin, Derek; Vassallo, Sara L; LeProust, Emily M; Peck, Bill J; Emig, Christopher J; Dahl, Fredrik; Gao, Yuan; Church, George M; Shendure, Jay

2007-11-01

A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

Active temporal multiplexing of indistinguishable heralded single photons

PubMed Central

Xiong, C.; Zhang, X.; Liu, Z.; Collins, M. J.; Mahendra, A.; Helt, L. G.; Steel, M. J.; Choi, D. -Y.; Chae, C. J.; Leong, P. H. W.; Eggleton, B. J.

2016-01-01

It is a fundamental challenge in quantum optics to deterministically generate indistinguishable single photons through non-deterministic nonlinear optical processes, due to the intrinsic coupling of single- and multi-photon-generation probabilities in these processes. Actively multiplexing photons generated in many temporal modes can decouple these probabilities, but key issues are to minimize resource requirements to allow scalability, and to ensure indistinguishability of the generated photons. Here we demonstrate the multiplexing of photons from four temporal modes solely using fibre-integrated optics and off-the-shelf electronic components. We show a 100% enhancement to the single-photon output probability without introducing additional multi-photon noise. Photon indistinguishability is confirmed by a fourfold Hong–Ou–Mandel quantum interference with a 91±16% visibility after subtracting multi-photon noise due to high pump power. Our demonstration paves the way for scalable multiplexing of many non-deterministic photon sources to a single near-deterministic source, which will be of benefit to future quantum photonic technologies. PMID:26996317

Estimating parameters for probabilistic linkage of privacy-preserved datasets.

PubMed

Brown, Adrian P; Randall, Sean M; Ferrante, Anna M; Semmens, James B; Boyd, James H

2017-07-10

Probabilistic record linkage is a process used to bring together person-based records from within the same dataset (de-duplication) or from disparate datasets using pairwise comparisons and matching probabilities. The linkage strategy and associated match probabilities are often estimated through investigations into data quality and manual inspection. However, as privacy-preserved datasets comprise encrypted data, such methods are not possible. In this paper, we present a method for estimating the probabilities and threshold values for probabilistic privacy-preserved record linkage using Bloom filters. Our method was tested through a simulation study using synthetic data, followed by an application using real-world administrative data. Synthetic datasets were generated with error rates from zero to 20% error. Our method was used to estimate parameters (probabilities and thresholds) for de-duplication linkages. Linkage quality was determined by F-measure. Each dataset was privacy-preserved using separate Bloom filters for each field. Match probabilities were estimated using the expectation-maximisation (EM) algorithm on the privacy-preserved data. Threshold cut-off values were determined by an extension to the EM algorithm allowing linkage quality to be estimated for each possible threshold. De-duplication linkages of each privacy-preserved dataset were performed using both estimated and calculated probabilities. Linkage quality using the F-measure at the estimated threshold values was also compared to the highest F-measure. Three large administrative datasets were used to demonstrate the applicability of the probability and threshold estimation technique on real-world data. Linkage of the synthetic datasets using the estimated probabilities produced an F-measure that was comparable to the F-measure using calculated probabilities, even with up to 20% error. Linkage of the administrative datasets using estimated probabilities produced an F-measure that was higher

Linkage Analysis of a Model Quantitative Trait in Humans: Finger Ridge Count Shows Significant Multivariate Linkage to 5q14.1

PubMed Central

Medland, Sarah E; Loesch, Danuta Z; Mdzewski, Bogdan; Zhu, Gu; Montgomery, Grant W; Martin, Nicholas G

2007-01-01

The finger ridge count (a measure of pattern size) is one of the most heritable complex traits studied in humans and has been considered a model human polygenic trait in quantitative genetic analysis. Here, we report the results of the first genome-wide linkage scan for finger ridge count in a sample of 2,114 offspring from 922 nuclear families. Both univariate linkage to the absolute ridge count (a sum of all the ridge counts on all ten fingers), and multivariate linkage analyses of the counts on individual fingers, were conducted. The multivariate analyses yielded significant linkage to 5q14.1 (Logarithm of odds [LOD] = 3.34, pointwise-empirical p-value = 0.00025) that was predominantly driven by linkage to the ring, index, and middle fingers. The strongest univariate linkage was to 1q42.2 (LOD = 2.04, point-wise p-value = 0.002, genome-wide p-value = 0.29). In summary, the combination of univariate and multivariate results was more informative than simple univariate analyses alone. Patterns of quantitative trait loci factor loadings consistent with developmental fields were observed, and the simple pleiotropic model underlying the absolute ridge count was not sufficient to characterize the interrelationships between the ridge counts of individual fingers. PMID:17907812

Robustness of linkage strategy that leads to large-scale cooperation.

PubMed

Inaba, Misato; Takahashi, Nobuyuki; Ohtsuki, Hisashi

2016-11-21

One of the most well-known models to characterize cooperation among unrelated individuals is Social dilemma (SD). However there is no consensus about how to solve the SD by itself. Since SDs are often embedded in other social interactions, including indirect reciprocity games (IR), human can coordinate their behaviors across multiple games. Such coordination is called 'linkage'. Recently linkage has been considered as a promising solution to resolve SDs, since excluding SD defectors (i.e. those who defected in SD) from indirectly reciprocal relationships functions as a costless sanction. A previous study performed mathematical modeling and revealed that a linkage strategy, which cooperates in SD and engages in the Standing strategy in IR based on the recipients' behaviors in both SD and IR, was an ESS against a non-linkage strategy which defects in SD and engages in the Standing strategy in IR based on recipients' behaviors only in IR (Panchanathan and Boyd, 2004). In order to investigate the robustness of the linkage strategy, we devised a non-linkage strategy, which cooperates in SD but does not link two games. First, we conducted a mathematical analysis and demonstrated that the linkage strategy was not an ESS against cooperating non-linkage strategy. Second, we conducted a series of agent-based computer simulations to examine how the strategies perform in situations in which various types of errors can occur. Our results showed that the linkage strategy was an ESS only when there are implementation errors in SD. However, the equilibrium of the linkage strategy was unstable when there are perception errors. Since we know that humans are not free from perception errors in their social life, future studies will need to show how perception errors can be overcome in order to provide support for the conclusion that linkage is a plausible solution to SDs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Automated assembly of oligosaccharides containing multiple cis-glycosidic linkages

NASA Astrophysics Data System (ADS)

Hahm, Heung Sik; Hurevich, Mattan; Seeberger, Peter H.

2016-09-01

Automated glycan assembly (AGA) has advanced from a concept to a commercial technology that rapidly provides access to diverse oligosaccharide chains as long as 30-mers. To date, AGA was mainly employed to incorporate trans-glycosidic linkages, where C2 participating protecting groups ensure stereoselective couplings. Stereocontrol during the installation of cis-glycosidic linkages cannot rely on C2-participation and anomeric mixtures are typically formed. Here, we demonstrate that oligosaccharides containing multiple cis-glycosidic linkages can be prepared efficiently by AGA using monosaccharide building blocks equipped with remote participating protecting groups. The concept is illustrated by the automated syntheses of biologically relevant oligosaccharides bearing various cis-galactosidic and cis-glucosidic linkages. This work provides further proof that AGA facilitates the synthesis of complex oligosaccharides with multiple cis-linkages and other biologically important oligosaccharides.

Physicians' Attitudes About Multiplex Tumor Genomic Testing

PubMed Central

Gray, Stacy W.; Hicks-Courant, Katherine; Cronin, Angel; Rollins, Barrett J.; Weeks, Jane C.

2014-01-01

Purpose Although predictive multiplex somatic genomic tests hold the potential to transform care by identifying targetable alterations in multiple cancer genes, little is known about how physicians will use such tests in practice. Participants and Methods Before the initiation of enterprise-wide multiplex testing at a major cancer center, we surveyed all clinically active adult cancer physicians to assess their current use of somatic testing, their attitudes about multiplex testing, and their genomic confidence. Results A total of 160 physicians participated (response rate, 61%): 57% were medical oncologists; 29%, surgeons; 14% radiation oncologists; 37%, women; and 83%, research principal investigators. Twenty-two percent of physicians reported low confidence in their genomic knowledge. Eighteen percent of physicians anticipated testing patients infrequently (≤ 10%), whereas 25% anticipate testing most patients (≥ 90%). Higher genomic confidence was associated with wanting to test a majority of patients (adjusted odds ratio [OR], 6.09; 95% CI, 2.1 to 17.5) and anticipating using actionable (adjusted OR, 2.46; 95% CI, 1.2 to 5.2) or potentially actionable (adjusted OR, 2.89; 95% CI, 1.1 to 7.9) test results to inform treatment recommendations. Forty-two percent of physicians endorsed disclosure of uncertain genomic findings to patients. Conclusion Physicians at a tertiary-care National Cancer Institute–designated comprehensive cancer center varied considerably in how they planned to incorporate predictive multiplex somatic genomic tests into practice and in their attitudes about the disclosure of genomic information of uncertain significance. Given that many physicians reported low genomic confidence, evidence-based guidelines and enhanced physician genomic education efforts may be needed to ensure that genomically guided cancer care is adequately delivered. PMID:24663044

A general model for likelihood computations of genetic marker data accounting for linkage, linkage disequilibrium, and mutations.

PubMed

Kling, Daniel; Tillmar, Andreas; Egeland, Thore; Mostad, Petter

2015-09-01

Several applications necessitate an unbiased determination of relatedness, be it in linkage or association studies or in a forensic setting. An appropriate model to compute the joint probability of some genetic data for a set of persons given some hypothesis about the pedigree structure is then required. The increasing number of markers available through high-density SNP microarray typing and NGS technologies intensifies the demand, where using a large number of markers may lead to biased results due to strong dependencies between closely located loci, both within pedigrees (linkage) and in the population (allelic association or linkage disequilibrium (LD)). We present a new general model, based on a Markov chain for inheritance patterns and another Markov chain for founder allele patterns, the latter allowing us to account for LD. We also demonstrate a specific implementation for X chromosomal markers that allows for computation of likelihoods based on hypotheses of alleged relationships and genetic marker data. The algorithm can simultaneously account for linkage, LD, and mutations. We demonstrate its feasibility using simulated examples. The algorithm is implemented in the software FamLinkX, providing a user-friendly GUI for Windows systems (FamLinkX, as well as further usage instructions, is freely available at www.famlink.se ). Our software provides the necessary means to solve cases where no previous implementation exists. In addition, the software has the possibility to perform simulations in order to further study the impact of linkage and LD on computed likelihoods for an arbitrary set of markers.

Multiplexed Holographic Data Storage in Bacteriorhodopsin

NASA Technical Reports Server (NTRS)

Mehrl, David J.; Krile, Thomas F.

1997-01-01

High density optical data storage, driven by the information revolution, remains at the forefront of current research areas. Much of the current research has focused on photorefractive materials (SBN and LiNbO3) and polymers, despite various problems with expense, durability, response time and retention periods. Photon echo techniques, though promising, are questionable due to the need for cryogenic conditions. Bacteriorhodopsin (BR) films are an attractive alternative recording medium. Great strides have been made in refining BR, and materials with storage lifetimes as long as 100 days have recently become available. The ability to deposit this robust polycrystalline material as high quality optical films suggests the use of BR as a recording medium for commercial optical disks. Our own recent research has demonstrated the suitability of BR films for real time spatial filtering and holography. We propose to fully investigate the feasibility of performing holographic mass data storage in BR. Important aspects of the problem to be investigated include various data multiplexing techniques (e.g. angle- amplitude- and phase-encoded multiplexing, and in particular shift-multiplexing), multilayer recording techniques, SLM selection and data readout using crossed polarizers for noise rejection. Systems evaluations of storage parameters, including access times, memory refresh constraints, erasure, signal-to-noise ratios and bit error rates, will be included in our investigations.

Magnetic Bead Based Immunoassay for Autonomous Detection of Toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Y; Hara, C A; Knize, M G

2008-05-01

As a step towards toward the development of a rapid, reliable analyzer for bioagents in the environment, we are developing an automated system for the simultaneous detection of a group of select agents and toxins. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag{trademark} reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag{trademark} through a cleavable linkage. Aqueous samples are incubatedmore » with the mixture of antibodies along with streptavidin-coated magnetic beads coupled to a photo-activatable porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactivable group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags{trademark}. Released eTags{trademark} are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 ng/mL (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated on a flow-through format with higher LODs of 125 ng/mL (or 2.5 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.« less

Multiplex mass spectrometry imaging for latent fingerprints.

PubMed

Yagnik, Gargey B; Korte, Andrew R; Lee, Young Jin

2013-01-01

We have previously developed in-parallel data acquisition of orbitrap mass spectrometry (MS) and ion trap MS and/or MS/MS scans for matrix-assisted laser desorption/ionization MS imaging (MSI) to obtain rich chemical information in less data acquisition time. In the present study, we demonstrate a novel application of this multiplex MSI methodology for latent fingerprints. In a single imaging experiment, we could obtain chemical images of various endogenous and exogenous compounds, along with simultaneous MS/MS images of a few selected compounds. This work confirms the usefulness of multiplex MSI to explore chemical markers when the sample specimen is very limited. Copyright © 2013 John Wiley & Sons, Ltd.

All Inkjet-Printed Amperometric Multiplexed Biosensors Based on Nanostructured Conductive Hydrogel Electrodes.

PubMed

Li, Lanlan; Pan, Lijia; Ma, Zhong; Yan, Ke; Cheng, Wen; Shi, Yi; Yu, Guihua

2018-06-13

Multiplexing, one of the main trends in biosensors, aims to detect several analytes simultaneously by integrating miniature sensors on a chip. However, precisely depositing electrode materials and selective enzymes on distinct microelectrode arrays remains an obstacle to massively produced multiplexed sensors. Here, we report on a "drop-on-demand" inkjet printing process to fabricate multiplexed biosensors based on nanostructured conductive hydrogels in which the electrode material and several kinds of enzymes were printed on the electrode arrays one by one by employing a multinozzle inkjet system. The whole inkjet printing process can be finished within three rounds of printing and only one round of alignment. For a page of sensor arrays containing 96 working electrodes, the printing process took merely ∼5 min. The multiplexed assays can detect glucose, lactate, and triglycerides in real time with good selectivity and high sensitivity, and the results in phosphate buffer solutions and calibration serum samples are comparable. The inkjet printing process exhibited advantages of high efficiency and accuracy, which opens substantial possibilities for massive fabrication of integrated multiplexed biosensors for human health monitoring.

Evaluation of a rapid single multiplex microsatellite-based assay for use in forensic genetic investigations in dogs.

PubMed

Clark, Leigh Anne; Famula, Thomas R; Murphy, Keith E

2004-10-01

To develop a set of microsatellite markers, composed of a minimal number of these markers, suitable for use in forensic genetic investigations in dogs. Blood, tissue, or buccal epithelial cells from 364 dogs of 85 breeds and mixed breeds and 19 animals from related species in the family Canidae. 61 tetranucleotide microsatellite markers were characterized on the basis of number and size of alleles, ease of genotyping, chromosomal location, and ability to be coamplified. The range in allele size, number of alleles, total heterozygosity, and fixation index for each marker were determined by use of genotype data from 383 dogs and related species. Polymorphism information content was calculated for several breeds of dogs. 7 microsatellite markers could be coamplified. These markers were labeled with fluorescent dyes, multiplexed into a single reaction, and optimized for resolution in a commercial genetic analyzer. The multiplex set was used to identify sires for 2 mixed litters. The test was not species specific; genotype information collected for wolves, coyotes, jackals, New Guinea singing dogs, and an African wild dog could not distinguish between these species. This set of 7 microsatellite markers is useful in forensic applications (ie, identification of dogs and determination of parentage) in closely related animals and is applicable to a wide range of species belonging to the family Canidae.

Tests for linkage and association in nuclear families.

PubMed Central

Martin, E R; Kaplan, N L; Weir, B S

1997-01-01

The transmission/disequilibrium test (TDT) originally was introduced to test for linkage between a genetic marker and a disease-susceptibility locus, in the presence of association. Recently, the TDT has been used to test for association in the presence of linkage. The motivation for this is that linkage analysis typically identifies large candidate regions, and further refinement is necessary before a search for the disease gene is begun, on the molecular level. Evidence of association and linkage may indicate which markers in the region are closest to a disease locus. As a test of linkage, transmissions from heterozygous parents to all of their affected children can be included in the TDT; however, the TDT is a valid chi2 test of association only if transmissions to unrelated affected children are used in the analysis. If the sample contains independent nuclear families with multiple affected children, then one procedure that has been used to test for association is to select randomly a single affected child from each sibship and to apply the TDT to those data. As an alternative, we propose two statistics that use data from all of the affected children. The statistics give valid chi2 tests of the null hypothesis of no association or no linkage and generally are more powerful than the TDT with a single, randomly chosen, affected child from each family. PMID:9311750

Designing robust watermark barcodes for multiplex long-read sequencing.

PubMed

Ezpeleta, Joaquín; Krsticevic, Flavia J; Bulacio, Pilar; Tapia, Elizabeth

2017-03-15

To attain acceptable sample misassignment rates, current approaches to multiplex single-molecule real-time sequencing require upstream quality improvement, which is obtained from multiple passes over the sequenced insert and significantly reduces the effective read length. In order to fully exploit the raw read length on multiplex applications, robust barcodes capable of dealing with the full single-pass error rates are needed. We present a method for designing sequencing barcodes that can withstand a large number of insertion, deletion and substitution errors and are suitable for use in multiplex single-molecule real-time sequencing. The manuscript focuses on the design of barcodes for full-length single-pass reads, impaired by challenging error rates in the order of 11%. The proposed barcodes can multiplex hundreds or thousands of samples while achieving sample misassignment probabilities as low as 10-7 under the above conditions, and are designed to be compatible with chemical constraints imposed by the sequencing process. Software tools for constructing watermark barcode sets and demultiplexing barcoded reads, together with example sets of barcodes and synthetic barcoded reads, are freely available at www.cifasis-conicet.gov.ar/ezpeleta/NS-watermark . ezpeleta@cifasis-conicet.gov.ar. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Fabrication and biological imaging of polyhedral oligomeric silsesquioxane cross-linked fluorescent polymeric nanoparticles with aggregation-induced emission feature

NASA Astrophysics Data System (ADS)

Mao, Liucheng; Liu, Meiying; Xu, Dazhuang; Wan, Qing; Huang, Qiang; Jiang, Ruming; Shi, Yingge; Deng, Fengjie; Zhang, Xiaoyong; Wei, Yen

2017-11-01

Aggregation-induced emission (AIE) dyes based fluorescent polymeric nanoparticles (FNPs) have been intensively explored for biomedical applications. However, many of these AIE-active FNPs are relied on the self-assembly of amphiphilic copolymers, which are not stable in diluted solution. Therefore, the introduction of cross-linkages into these micelles has demonstrated to be an efficient route to overcome this stability problem and endow ultra-low critical micelle concentrations (CMC) of these AIE-active FNPs. In this work, we reported the fabrication of cross-linked AIE-active FNPs through controllable reversible addition fragmentation chain transfer polymerization by using commercially available octavinyl-T8-silsesquioxane (8-vinyl POSS) as the cross-linkage for the first time. The resultant cross-linked amphiphilic copolymers (named as PEG-POSS-PhE) are prone to self-assemble into stable core-shell nanoparticles with well water dispersity, strong red fluorescence and low CMC (0.0069 mg mL-1) in aqueous solution. More importantly, PEG-POSS-PhE FNPs possess some other properties such as high water dispersity, uniform morphology and small size, excellent biocompatibility and cellular internalization, providing great potential of PEG-POSS-PhE FNPs for biological imaging application.

Single-tube, non-isotopic, multiplex PCR/OLA assay and sequence-coded separation for simultaneous screening of 31 cystic fibrosis mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brinson, E.C.; Adriano, T.; Bloch, W.

1994-09-01

We have developed a rapid, single-tube, non-isotopic assay that screens a patient sample for the presence of 31 cystic fibrosis (CF) mutations. This assay can identify these mutations in a single reaction tube and a single electrophoresis run. Sample preparation is a simple, boil-and-go procedure, completed in less than an hour. The assay is composed of a 15-plex PCR, followed by a 61-plex oligonucleotide ligation assay (OLA), and incorporates a novel detection scheme, Sequence Coded Separation. Initially, the multiplex PCR amplifies 15 relevant segments of the CFTR gene, simultaneously. These PCR amplicons serve as templates for the multiplex OLA, whichmore » detects the normal or mutant allele at all loci, simultaneously. Each polymorphic site is interrogated by three oligonucleotide probes, a common probe and two allele-specific probes. Each common probe is tagged with a fluorescent dye, and the competing normal and mutant allelic probes incorporate different, non-nucleotide, mobility modifiers. These modifiers are composed of hexaethylene oxide (HEO) units, incorporated as HEO phosphoramidite monomers during automated DNA synthesis. The OLA is based on both probe hybridization and the ability of DNA ligase to discriminate single base mismatches at the junction between paired probes. Each single tube assay is electrophoresed in a single gel lane of a 4-color fluorescent DNA sequencer (Applied Biosystems, Model 373A). Each of the ligation products is identified by its unique combination of electrophoretic mobility and one of three colors. The fourth color is reserved for the in-lane size standard, used by GENESCAN{sup TM} software (Applied Biosystems) to size the OLA electrophoresis products. The Genotyper{sub TM} software (Applied Biosystems) decodes these Sequence-Coded-Separation data to create a patient summary report for all loci tested.« less

DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR

PubMed Central

Yamasaki, Hiroshi; Allan, James C.; Sato, Marcello Otake; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Qiu, Dongchuan; Mamuti, Wulamu; Craig, Philip S.; Ito, Akira

2004-01-01

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections. PMID:14766815

Highly sensitive and multiplexed platforms for allergy diagnostics

NASA Astrophysics Data System (ADS)

Monroe, Margo R.

Allergy is a disorder of the immune system caused by an immune response to otherwise harmless environmental allergens. Currently 20% of the US population is allergic and 90% of pediatric patients and 60% of adult patients with asthma have allergies. These percentages have increased by 18.5% in the past decade, with predicted similar trends for the future. Here we design sensitive, multiplexed platforms to detect allergen-specific IgE using the Interferometric Reflectance Imaging Sensor (IRIS) for various clinical settings. A microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of patient blood sample. However, conventional fluorescent microarray technology is limited by i) the variation of probe immobilization, which hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics and ii) the use of fluorophore labels, which is not suitable for some clinical applications due to the tendency of fluorophores to stick to blood particulates and require daily calibration methods. This calibrated fluorescence enhancement (CaFE) method integrates the low magnification modality of IRIS with enhanced fluorescence sensing in order to directly correlate immobilized probe (major allergens) density to allergen-specific IgE in patient serum. However, this platform only operates in processed serum samples, which is not ideal for point of care testing. Thus, a high magnification modality of IRIS was adapted as an alternative allergy diagnostic platform to automatically discriminate and size single nanoparticles bound to specific IgE in unprocessed, characterized human blood and serum samples. These features make IRIS an ideal candidate for clinical and diagnostic applications, such a POC testing. The high magnification (nanoparticle counting) modality in conjunction with low magnification of IRIS in a combined instrument

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

On-chip WDM mode-division multiplexing interconnection with optional demodulation function.

PubMed

Ye, Mengyuan; Yu, Yu; Chen, Guanyu; Luo, Yuchan; Zhang, Xinliang

2015-12-14

We propose and fabricate a wavelength-division-multiplexing (WDM) compatible and multi-functional mode-division-multiplexing (MDM) integrated circuit, which can perform the mode conversion and multiplexing for the incoming multipath WDM signals, avoiding the wavelength conflict. An phase-to-intensity demodulation function can be optionally applied within the circuit while performing the mode multiplexing. For demonstration, 4 × 10 Gb/s non-return-to-zero differential phase shift keying (NRZ-DPSK) signals are successfully processed, with open and clear eye diagrams. Measured bit error ratio (BER) results show less than 1 dB receive sensitivity variation for three modes and four wavelengths with demodulation. In the case without demodulation, the average power penalties at 4 wavelengths are -1.5, -3 and -3.5 dB for TE₀-TE₀, TE₀-TE₁ and TE₀-TE₂ mode conversions, respectively. The proposed flexible scheme can be used at the interface of long-haul and on-chip communication systems.

A Fluorescence in Situ Hybridization System for Karyotyping Soybean

PubMed Central

Findley, Seth D.; Cannon, Steven; Varala, Kranthi; Du, Jianchang; Ma, Jianxin; Hudson, Matthew E.; Birchler, James A.; Stacey, Gary

2010-01-01

The development of a universal soybean (Glycine max [L.] Merr.) cytogenetic map that associates classical genetic linkage groups, molecular linkage groups, and a sequence-based physical map with the karyotype has been impeded due to the soybean chromosomes themselves, which are small and morphologically homogeneous. To overcome this obstacle, we screened soybean repetitive DNA to develop a cocktail of fluorescent in situ hybridization (FISH) probes that could differentially label mitotic chromosomes in root tip preparations. We used genetically anchored BAC clones both to identify individual chromosomes in metaphase spreads and to complete a FISH-based karyotyping cocktail that permitted simultaneous identification of all 20 chromosome pairs. We applied these karyotyping tools to wild soybean, G. soja Sieb. and Zucc., which represents a large gene pool of potentially agronomically valuable traits. These studies led to the identification and characterization of a reciprocal chromosome translocation between chromosomes 11 and 13 in two accessions of wild soybean. The data confirm that this translocation is widespread in G. soja accessions and likely accounts for the semi-sterility found in some G. soja by G. max crosses. PMID:20421607

Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging.

PubMed

Ding, Changqin; Zhu, Anwei; Tian, Yang

2014-01-21

, and we emphasize applications in bioimaging and biosensing in live cells, tissues, and animals. In addition, we highlight our work on the design and synthesis of a C-dot ratiometric biosensor for intracellular Cu(2+) detection, and a twophoton fluorescent probe for pH measurement in live cells and tissues. We conclude this Account by outlining future directions in engineering the functional surface of C-dots for a variety of in vivo imaging applications, including dots with combined targeting, imaging and therapeutic-delivery capabilities, or high-resolution multiplexed vascular imaging. With each application C-dots should open new horizons of multiplexed quantitative detection, high-resolution fluorescence imaging, and long-term, real-time monitoring of their target.

Distribution of lod scores in oligogenic linkage analysis.

PubMed

Williams, J T; North, K E; Martin, L J; Comuzzie, A G; Göring, H H; Blangero, J

2001-01-01

In variance component oligogenic linkage analysis it can happen that the residual additive genetic variance bounds to zero when estimating the effect of the ith quantitative trait locus. Using quantitative trait Q1 from the Genetic Analysis Workshop 12 simulated general population data, we compare the observed lod scores from oligogenic linkage analysis with the empirical lod score distribution under a null model of no linkage. We find that zero residual additive genetic variance in the null model alters the usual distribution of the likelihood-ratio statistic.

Data Linkage: A powerful research tool with potential problems

PubMed Central

2010-01-01

Background Policy makers, clinicians and researchers are demonstrating increasing interest in using data linked from multiple sources to support measurement of clinical performance and patient health outcomes. However, the utility of data linkage may be compromised by sub-optimal or incomplete linkage, leading to systematic bias. In this study, we synthesize the evidence identifying participant or population characteristics that can influence the validity and completeness of data linkage and may be associated with systematic bias in reported outcomes. Methods A narrative review, using structured search methods was undertaken. Key words "data linkage" and Mesh term "medical record linkage" were applied to Medline, EMBASE and CINAHL databases between 1991 and 2007. Abstract inclusion criteria were; the article attempted an empirical evaluation of methodological issues relating to data linkage and reported on patient characteristics, the study design included analysis of matched versus unmatched records, and the report was in English. Included articles were grouped thematically according to patient characteristics that were compared between matched and unmatched records. Results The search identified 1810 articles of which 33 (1.8%) met inclusion criteria. There was marked heterogeneity in study methods and factors investigated. Characteristics that were unevenly distributed among matched and unmatched records were; age (72% of studies), sex (50% of studies), race (64% of studies), geographical/hospital site (93% of studies), socio-economic status (82% of studies) and health status (72% of studies). Conclusion A number of relevant patient or population factors may be associated with incomplete data linkage resulting in systematic bias in reported clinical outcomes. Readers should consider these factors in interpreting the reported results of data linkage studies. PMID:21176171

Interferometric space-mode multiplexing based on binary phase plates and refractive phase shifters.

PubMed

Liñares, Jesús; Prieto-Blanco, Xesús; Moreno, Vicente; Montero-Orille, Carlos; Mouriz, Dolores; Nistal, María C; Barral, David

2017-05-15

A Mach-Zehnder interferometer (MZI) that includes in an arm either a reflective image inverter or a Gouy phase shifter (RGPS) can (de)multiplex many types of modes of a few mode fiber without fundamental loss. The use of RGPSs in combination with binary phase plates for multiplexing purposes is studied for the first time, showing that the particular RGPS that shifts π the odd modes only multiplexes accurately low order modes. To overcome such a restriction, we present a new exact refractive image inverter, more compact and flexible than its reflective counterpart. Moreover, we show that these interferometers remove or reduce the crosstalk that the binary phase plates could introduce between the multiplexed modes. Finally, an experimental analysis of a MZI with both an approximated and an exact refractive image inverter is presented for the case of a bimodal multiplexing. Likewise, it is proven experimentally that a RGPS that shifts π/2 demultiplexes two odd modes which can not be achieved by any image inverter.

Identifying Candidate Chemical-Disease Linkages ...

EPA Pesticide Factsheets

Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment. Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment.

Sex-specific Linkage Scans in Opioid Dependence

PubMed Central

Yang, Bao-Zhu; Han, Shizhong; Kranzler, Henry R.; Palmer, Abraham A.; Gelernter, Joel

2017-01-01

Sex influences risk for opioid dependence (OD). We hypothesized that sex might interact with genetic loci that influence the risk for OD. Therefore we performed an analysis to identify sex-specific genomic susceptibility regions for OD using linkage. Over 6000 single nucleotide polymorphism (SNP) markers were genotyped for 1758 African- and European-American (AA and EA) individuals from 739 families, ascertained via affected sib-pairs with OD and/or cocaine dependence. Autosomewide non-parametric linkage scans, stratified by sex and population, were performed. We identified one significant linkage region, segregating with OD in EA men, at 71.1 cM on chromosome 4 (LOD=3.29; point-wise p=0.00005; empirical autosome-wide p=0.042), which significantly differed from the linkage signal at the same location in EA women (empirical p=0.002). Three suggestive linkage signals were identified at 181.3 cM on chromosome 7 (LOD=2.18), 104 cM on chromosome 11 (LOD=1.85), and 60.9 cM on chromosome 16 (LOD=1.93) in EA women. In AA men, four suggestive linkage signals were detected at 201.1 cM on chromosome 3 (LOD=2.32), 152.9 cM on chromosome 6 (LOD=1.86), 16.8 cM on chromosome 7 (LOD=1.95), and 36.1 cM on chromosome 17 (LOD=1.99). The significant region, mapping to 4q12-4q13.1, harbors several OD candidate genes with interconnected functionality, including VEGFR, CLOCK, PDCL2, NMU, NRSF, and IGFBP7. In conclusion, these results provide an evidence for the existence of sex-specific and population-specific differences in OD. Furthermore, these results provide positional information that will facilitate the use of targeted next-generation sequencing to search for genes that contribute to sex-specific differences in OD. PMID:27762075

Fluorescence multiplexing with time-resolved and spectral discrimination using a near-IR detector.

PubMed

Zhu, Li; Stryjewski, Wieslaw; Lassiter, Suzanne; Soper, Steven A

2003-05-15

We report on the design and performance of a two-color, time-resolved detector for the acquisition of both steady-state and time-resolved fluorescence data acquired in real time during the capillary gel electrophoresis separation of DNA sequencing fragments. The detector consisted of a pair of pulsed laser diodes operating at 680 and 780 nm. The diode heads were coupled directly to single-mode fibers, which were terminated into a single fiber mounted via a FC/PC connector to the detector body. The detector contained a dichroic filter, which directed the dual-laser beams to an objective. The objective focused the laser light into a capillary gel column and also collected the resulting fluorescence emission. The dual-color emission was transmitted through the dichroic and focused onto a multimode fiber (core diameter 50 microm), which carried the luminescence to a pair of single-photon avalanche diodes (SPADs). The emission was sorted spectrally using a second dichroic onto one of two SPADs and isolated using appropriate interference filters (710- or 810-nm channel). The dual-color detector demonstrated a time response of 450 and 510 ps (fwhm) for the 710- and 810-nm channels, respectively. The mass detection limits for two near-IR dye-labeled sequencing primers electrophoresed in a capillary gel column were found to be 7.1 x 10(-21) and 3.2 x 10(-20) mol (SNR = 3) for the 710- and 810-nm detector channels, respectively. In addition, no leakage of luminescence excited at 680 nm was observed in the 810-nm channel or 780-nm excited luminescence into the 710-nm channel. An M13mp18 template was sequenced in a single capillary gel column using a two-color, two-lifetime format. The read length was found to be 650 base pairs for the test template at a calling accuracy of 95.1% using a linear poly(dimethylacrylamide) (POP6) gel column, with the read length determined primarily by the electrophoretic resolution produced by the sieving gel.

A microsatellite genetic linkage map of black rockfish ( Sebastes schlegeli)