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PubMed2007

Multiplex biosensor using gold nanorods.

Gold nanorods (GNRs) with different aspect ratios were fabricated through seed-mediated growth and surface activation by alkanethiols for the attachment of antibodies to yield gold nanorod molecular probes (GNrMPs). Multiplex sensing was demonstrated by the distinct response of the plasmon spectra of the GNrMPs to binding events of three targets (goat anti-human IgG1 Fab, rabbit anti-mouse IgG1 Fab, rabbit anti-sheep IgG (H+L)). Plasmonic sensors are highly specific and sensitive and can be used to monitor refractive index changes caused by molecular interactions in their immediate vicinity with potential to achieve single-particle biosensing. This technique can play a key role in developing novel optical biosensors for both in vivo and in vitro detection and single-receptor kinetics.

Animals↗
PubMed2007

Gradient elution moving boundary electrophoresis for high-throughput multiplexed microfluidic devices.

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterflow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a micro-fluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in.2 in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses.

Journal Article↗
PubMed1998

Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing.

The steps of cell lysis, multiplex PCR amplification, and electrophoretic analysis are executed sequentially on a monolithic microchip device. The entire microchip is thermally cycled to lyse cells and to amplify DNA, and the products are then analyzed using a sieving medium for size separation and an intercalating dye for fluorescence detection. Using a standard PCR protocol, a 500-base pair (bp) region of bacteriophage lambda DNA and 154-, 264-, 346-, 410-, and 550-bp regions of E. coli genomic and plasmid DNAs are amplified. The electrophoretic analysis of the products is executed in <3 min following amplification using hydroxyethyl cellulose or poly(dimethylacrylamide) sieving gels. Product sizing is demonstrated by proportioning the amplified product with a DNA sizing ladder.

DNA, Bacterial↗
PubMed1998

On-the-fly fluorescence lifetime detection of dye-labeled DNA primers for multiplex analysis.

Mixtures of dye-labeled, M13-forward DNA primers were separated by capillary gel electrophoresis and detected on-the-fly, using fluorescence lifetime measurements, to evaluate four-decay detection for multiplex DNA sequencing. Three different four-dye systems were used, two that were excited at 488 nm and one that was excited at 514 nm. Each dye-labeled primer was identified on the basis of the lifetime of the conjugated dye using nonlinear least squares or the maximum entropy method to analyze the lifetime data. Overlapping electrophoretic peaks were generated by making multiple injections of mixtures of the dye-labeled primers. The overlapping peaks were resolved by fitting the data to two-, three- or four-component lifetime models used in nonlinear least-squares analysis in which each lifetime component was fixed to the predetermined lifetime of the corresponding dye-labeled primer. In two of the dye systems, the lifetimes of the four dye-labeled primers were sufficiently different to allow peak resolution. In the other dye system, addition of 10% DMSO to the run buffer changed the lifetime of one dye-labeled primer, allowing it to be resolved from another dye-labeled primer with similar lifetime.

DNA Primers↗
PubMed1998

Multiplex dye DNA sequencing in capillary gel electrophoresis by diode laser-based time-resolved fluorescence detection.

A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of > 90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods.

DNA↗
PubMed1999

BIA/MS of epitope-tagged peptides directly from E. coli lysate: multiplex detection and protein identification at low-femtomole to subfemtomole levels.

The use of biomolecular interaction analysis mass spectrometry to selectively isolate, detect, and characterize epitope-tagged peptides present in total cell lysates is demonstrated. Epitope-tagged tryptic peptides were captured via affinity interactions with either chelated Ni2+ or monoclonal antibodies and detected using surface plasmon resonance biomolecular interaction analysis (SPR-BIA). After SPR-BIA the tagged peptides were either eluted from the biosensor chips for mass spectrometric analysis or analyzed directly from the biosensor chip using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Protein database searches were performed using the masses of the tagged tryptic peptides, resulting in identification of the protein into which the epitope tag was inserted. Detection limits for both SPR-BIA and MALDI-TOF were at the low-femtomole to subfemtomole level. The approach represents a (multiplexed) high-sensitivity chip-based technique capable of identifying epitope-tagged proteins as they are present in complex mixtures.

Amino Acid Sequence↗
PubMed1999

Multiplexed automated DNA sequencing directly from single bacterial colonies.

Sample preparation has been one of the major bottlenecks for large-scale DNA sequencing projects in terms of time and cost. To improve sample throughput and to integrate the front-end tasks to capillary-array DNA sequencers, protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony is picked, lysis is accomplished in situ in the plastic sample tube using either a thermocycler or a heating block. Upon heating, the plasmids are released while chromosomal DNA and membrane proteins are denatured and precipitate to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed online DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. By the marriage of colony sequencing with the capillary array sequencer, both the front end and the back end of DNA sequencing are combined in a miniaturized format. This protocol will ultimately reduce the cost of sequencing to well below current levels.

DNA, Bacterial↗
PubMedDate not supplied

Multiplex analysis with peptide-encoded beads.

Paramagnetic beads have considerable potential as identification tags in biological analysis. For example, magnetic sensor-based arrays using the magnetic field generated by paramagnetic beads to test hybridization between interacting molecules have attracted widespread interest in recent years. However, application of paramagnetic beads as identification tags is still limited, since they do not permit differentiation between samples for multiplex analysis. Here, we report the application of a novel encoding of paramagnetic beads with peptide sequences. This strategy allows DNA samples labeled with peptide-encoded paramagnetic beads to be identified by the selective enzymatic cleavage of each peptide cross-linker.

Cross-Linking Reagents↗
PubMed2003

RAMPED-UP NMR: multiplexed NMR-based screening for drug discovery.

The crucial step in drug discovery is the identification of a lead compound from a vast chemical library by any number of screening techniques. NMR-based screening has the advantage of directly detecting binding of a compound to the target. The spectra resulting from these screens can also be very complex and difficult to analyze, making this an inefficient process. We present here a method, RAMPED-UP NMR, (Rapid Analysis and Multiplexing of Experimentally Discriminated Uniquely Labeled Proteins using NMR) which generates simple spectra which are easy to interpret and allows several proteins to be screened simultaneously. In this method, the proteins to be screened are uniquely labeled with one amino acid type. There are several benefits derived from this unique labeling strategy: the spectra are greatly simplified, resonances that are most likely to be affected by binding are the only ones observed, and peaks that yield little or no information upon binding are eliminated, allowing the analysis of multiple proteins easily and simultaneously. We demonstrate the ability of three different proteins to be analyzed simultaneously for binding to two different ligands. This method will have significant impact in the use of NMR spectroscopy for both the lead generation and lead optimization phases of drug discovery by its ability to increase screening throughput and the ability to examine selectivity. To the best of our knowledge, this is the first time in any format that multiple proteins can be screened in one tube.

Combinatorial Chemistry Techniques↗
PubMed2005

Quantum dot-based multiplexed fluorescence resonance energy transfer.

We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured. However, data analysis was simpler for the configuration where multiple QD donors are used in conjunction with one acceptor. Steady-state fluorescence results were corroborated by time-resolved measurements where selective shortening of QD lifetime was measured only for populations that were selectively engaged in nonradiative energy transfer.

Carrier Proteins↗
PubMed2025

Boosting Quantification of N-Glycans by an Enhanced Isobaric Multiplex Reagents for Carbonyl-Containing Compound (SUGAR) Tagging Strategy.

Glycans are complex molecules composed of various monosaccharides and exhibit diverse, branched polymer structures. Extensive research has been conducted on mass spectrometry (MS)-based qualitative and quantitative glycan analysis due to their critical biological functions. However, traditional data-dependent acquisition (DDA) in MS analysis primarily selects a limited subset of abundant ions during MS1 scans for fragmentation in subsequent MS2 stages. In this study, we introduce an advanced isobaric labeling strategy that incorporates a large amount of content-relevant sample labeled with one isobaric tag channel as an additional boosting channel. This innovation enhances the efficiency of isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tagging in quantitative glycomics. Notably, this approach significantly improves the characterization of low-abundance N-glycans and enables the detection of subtle quantitative differences in N-glycan profiling.

Polysaccharides↗
PubMed2025

SynchroSep-MS: Parallel LC Separations for Multiplexed Proteomics.

Achieving high throughput remains a challenge in MS-based proteomics for large-scale applications. We introduce SynchroSep-MS, a novel method for parallelized, label-free proteome analysis that leverages the rapid acquisition speed of modern mass spectrometers. This approach employs multiple liquid chromatography columns, each with an independent sample, simultaneously introduced into a single mass spectrometer inlet. A precisely controlled retention time offset between sample injections creates distinct elution profiles, facilitating unambiguous analyte assignment. We modified the DIA-NN workflow to effectively process these unique parallelized data, accounting for retention time offsets. Using a dual-column setup with mouse brain peptides, SynchroSep-MS detected approximately 16,700 unique protein groups, nearly doubling the peptide information obtained from a conventional single proteome analysis. The method demonstrated excellent precision and reproducibility (median protein %RSDs less than 4%) and high quantitative linearity (median R2 greater than 0.96) with minimal matrix interference. SynchroSep-MS represents a new paradigm for data collection and the first example of label-free multiplexed proteome analysis via parallel LC separations, offering a direct strategy to accelerate throughput for demanding applications such as large-scale clinical cohorts and single-cell analyses without compromising peak capacity or causing ionization suppression.

Proteomics↗
PubMed2001

Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.

Animals↗
PubMed2004

Polyozellin isolated from Polyozellus multiplex induces phase 2 enzymes in mouse hepatoma cells and differentiation in human myeloid leukaemic cell lines.

Induction of cellular phase 2 detoxifying enzymes is associated with cancer preventive potential. Quinone reductase (QR) has been used as a prototype for anticarcinogenic phase 2 enzymes because of its widespread distribution in mammalian systems, large amplitude of inducer response, and ease of measurement in murine hepatoma cells. Methanol extract of Polyozellus multiplex, which shows a strong QR induction potential, was subjected to bioassay-guided fractionation, and polyozellin (PM1) appeared to be a major active component. In in vitro cultured cells (hepa1c1c7 and BPRc1 cells), polyozellin enhanced QR, glutathione S-transferase (GST) activities, and glutathione (GSH) content in a dose-dependent manner. The compound also significantly promoted differentiation of HL-60 human promyelocytic emia cells. In conclusion, polyozellin deserves further in vivo study to evaluate its potential as a cancer preventive agent.

Agaricales↗
PubMed2004

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods.

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.

Animal Feed↗
PubMed2005

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

Animals↗
PubMed2006

Three-dimensional vibrational imaging of a microcrystalline J-aggregate using supercontinuum-based ultra-broadband multiplex coherent anti-stokes Raman scattering microscopy.

Vibrational properties of a porphyrin J-aggregate microcrystal have been investigated by ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy using a supercontinuum light source generated from a photonic crystal fiber. Owing to a strong resonance effect due to an excitonic transition, clear spectral and spatial profiles of the CARS signal have been successfully obtained. On the basis of the comparison between the CARS and the fluorescence images, the spatial dependence of the CARS signal can be explained by the spatial inhomogeneity of the excitonic transition energy in the single J-aggregate microcrystal.

Journal Article↗
PubMed2001

Multiplolides A and B, new antifungal 10-membered lactones from Xylaria multiplex.

Two new 10-membered lactones, namely, multiplolides A (1) and B (2), were isolated from the broth extract of the fungus Xylaria multiplex BCC 1111. Chemical structures of 1 and 2 were elucidated on the basis of their spectral data. Multiplolides A (1) and B (2) exhibited antifungal activity against Candida albicans with IC(50) values of 7 and 2 microg/mL, respectively. Both 1 and 2 were inactive in the screening systems toward the malarial parasite Plasmodium falciparum (at 20 microg/mL) and were not cytotoxic to BC-1 and KB cell lines (at 20 microg/mL).

Animals↗

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