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At least 919 records · Page 51Linked to original sources
PubMed2002

High-throughput mass-directed parallel purification incorporating a multiplexed single quadrupole mass spectrometer.

We report on the development of a parallel HPLC/MS purification system incorporating an indexed (i.e., multiplexed) ion source. In the method described, each of the flow streams from a parallel array of HPLC columns is directed toward the multiplexed (MUX) ion source and sampled in a time-dependent, parallel manner. A visual basic application has been developed and monitors in real-time the extracted ion current from each sprayer channel. Mass-directed fraction collection is initiated into a parallel array of fraction collectors specific for each of the spray channels. In the first embodiment of this technique, we report on a four-column semipreparative parallel LC/MS system incorporating MUX detection. In this parallel LC/MS application (in which sample loads between 1 and 10 mg on-column are typically made), no cross talk was observed. Ion signals from each of the channels were found reproducible over 192 injections, with interchannel signal variations between 11 and 17%. The visual basic fraction collection application permits preset individual start collection and end collection thresholds for each channel, thereby compensating for the slight variation in signal between sprayers. By incorporating postfraction collector UV detection, we have been able to optimize the valve-triggering delay time with precut transfer tubing between the mass spectrometer and fraction collectors and achieve recoveries greater than 80%. Examples of the MUX-guided, mass-directed fraction purification of both standards and real library reaction mixtures are presented within.

Chromatography, High Pressure Liquid↗
PubMed2003

Pulsed excitation source multiplexed fluorometry for the simultaneous measurement of multiple analytes. Continuous measurement of atmospheric hydrogen peroxide and methyl hydroperoxide.

Presently, solid-state sources such as light-emitting diodes (LEDs) provide for intense, nearly monochromatic light. They are available over a broad range of emission wavelengths. Unlike incandescent and discharge lamps, LEDs can be turned on and off at high speeds. The resulting light pulses are highly reproducible. This allows the use of a single photomultiplier tube (PMT), often the most expensive component in a high-sensitivity measurement system, as a multiplexed detector with multiple, fiber-optic-coupled, fluorescence-detection cells excited by solid-state sources. A time resolution of 1 min is adequate in many continuous detection schemes. This enables multiple-channel single-detector multiplexed measurement without any loss of S/N. On the basis of this principle, we describe a new automated continuous instrument for the simultaneous measurement of atmospheric hydrogen peroxide and methyl hydroperoxide (MHP). A Nafion membrane diffusion scrubber (DS) is used with hematin-catalyzed oxidation of thiamine to thiochrome for the measurement of H2O2, and an expanded poly(tetrafluoroethylene) (ePTFE) DS is used with a H2O2 destruction catalyst and horseradish peroxidase-catalyzed oxidation of thiamine to thiochrome for the measurement of MHP. The respective limits of detection are 25 pptv and 15 pptv. Design, performance details, and illustrative results from a field campaign (Philadelphia NEO3PS study, 2001) are presented.

Journal Article↗
PubMed2003

Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents.

Liquid array-based multiplexed immunoassays designed for rapid, sensitive, specific, and simultaneous detection of multiple simulants of biological warfare agents have been developed. In both blind and standard laboratory trials, we demonstrate the simultaneous detection of four simulant agents from a single sample. The challenge agents comprise broad classes of pathogens (virus, protein toxins, bacterial spores, vegetative cells). Assay performance of each analyte was optimized, and dose-response curves and the limits of detection (LODs) for individual analytes are presented. Assay performance, including dynamic range, sensitivity, and LODs for liquid arrays and enzyme-linked immunosorbant assay were compared and are shown to be similar. Maximum assay sensitivity is obtained in approximately 1 h, and good sensitivity is achieved in as little as 30 min. Although the sample matrixes are very complex, even for highly multiplexed assays the samples do not exhibit evidence of nonspecific binding, demonstrating that the assays also have high specificity.

Bacteria↗
PubMed2003

Micromachining microcarrier-based biomolecular encoding for miniaturized and multiplexed immunoassay.

Micromachining techniques, which originated in the microelectronics industry, have been employed to manufacture microparticles bearing an engraved dot-type signature for biomolecular encoding. These metallic microstructures are photolithographically defined and manufactured in a highly reproducible manner. In addition, the code introduced on the particle face is a straightforward visible feature that is easily recognizable with the use of optical microscopy. The number of distinct codes theoretically could be many thousands, depending on the coding element numbers. Such microparticles are, thus, with appropriate surface organic functionalizations, ideal for encoding biomolecular libraries and serving as a platform for developing high-throughput multiplexed bioassay schemes based on suspension array technology. As proof of this statement, we demonstrated that encoded microparticles tagged with antibodies to human immunoglobulin classes are capable, using imaging detection as the interrogating approach, of high sensitivity and high specificity, as well as multiplexed detection of the respective antigens in a microliter-sample volume.

Antigens↗
PubMed2004

Multiplexed toxin analysis using four colors of quantum dot fluororeagents.

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe-ZnS core-shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate. Initially the assay performance for the detection of each toxin was examined. We then demonstrated the simultaneous detection of the four toxins from a single sample probed with a mixture of all four QD-antibody reagents. Using a simple linear equation-based algorithm, it was possible to deconvolute the signal from mixed toxin samples, which allowed quantitation of all four toxins simultaneously.

Cadmium↗
PubMed2004

Multiplexed MS/MS in a quadrupole ion trap mass spectrometer.

A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods.

Cytochromes c↗
PubMed2005

Multiplexed flow cytometric immunoassay for influenza virus detection and differentiation.

Microsphere-based immunoassay by flow cytometry has gained popularity lately in protein detection and infectious disease diagnosis due to its capacity for multiplexed analysis and simple assay format. Here, we demonstrated the power of microsphere-based immunoassay for high-sensitivity detection and accurate differentiation of influenza viruses. The effects of sample volume and bead number on the assay sensitivity of viral antigen detection were studied. Compared to enzyme-linked immunosorbent assays, flow-based bead assays provided approximately 10-fold lower detection limit for viral particle detection and performed similarly for recombinant viral hemagglutinin protein detection. A four-plexed assay for influenza virus typing and influenza B virus sublineage characterization was developed to demonstrate the potential for multiplexed viral antigen detection and differentiation.

Flow Cytometry↗
PubMed2006

TNT detection using multiplexed liquid array displacement immunoassays.

The presence of trace contamination of soil and groundwater with explosives is an ongoing concern, for which improved methods are required to facilitate their detection and quantification. This is true both for the monitoring of remediation and for site characterization. Immunosensors have been found effective for solution-phase detection of environmental contaminants. Our work utilized the Luminex100 (flow cytometer) to detect TNT in a multiplexed displacement immunoassay format. The Luminex100 can perform a multiplexed assay by discriminating between up to 100 different bead sets. We used this capability to evaluate four different TNT monoclonal antibodies, two recombinant TNT antibodies, and a control antibody simultaneously for the rapid detection of TNT and other nitroaromatics. TNT could be detected at 0.1 ppb and quantified over the range of 1.0 ppb to 10 ppm. In addition, the assay was shown to be effective in various matrixes such as lake water, seawater, and acetone extracts of soil. Seawater required dilution with two parts buffer to avoid loss of microspheres, while the acetone extracts were diluted 100-fold or more to minimize solvent affects.

Antibodies, Monoclonal↗
PubMed2006

Multiplexed rectilinear ion trap mass spectrometer for high-throughput analysis.

A multichannel mass spectrometer based on the rectilinear ion trap (RIT) analyzer was designed and constructed for simultaneous high-throughput analysis of multiple samples. The instrument features four parallel ion source/mass analyzer/detector channels assembled in a single vacuum chamber and operated using a common set of control electronics, including a single rf amplifier and transformer coil. This multiplexed RIT mass spectrometer employs an array of four millimeter-sized ion traps (x(o) = 5.0 mm and y(o) = 4.0 mm, where x(o) and y(o) are the half-distances in the x and y dimensions, respectively). Mass spectra are acquired from four different samples simultaneously. The available mass/charge range is m/z 15-510 with excellent linearity of the mass calibration (R2 = 0.999 999). The peak width is less than 0.3 mass/charge units at m/z 146, corresponding to a resolution of approximately 500. Simultaneous MS/MS of ions due to four compounds (3-fluoroanisole, 4-fluoroanisole, 2-fluorobenzyl alcohol, 2,6-dimethylcyclohexanone) with the same nominal molecular radical cation but distinctive fragmentation patterns was demonstrated. Isolation and fragmentation efficiencies were approximately 25 and approximately 75%, respectively, measured in the typical case of the molecular radical cation of acetophenone. Preacquisition differential data were obtained by real-time subtraction of the ion signals from two channels of the multiplexed mass spectrometer. The differential experiment presented offers proof of principle of comparative mass spectra in high-throughput screening applications while reducing data storage requirements.

Computer Simulation↗
PubMed1997

Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels.

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.

DNA↗
PubMed2000

Accurate mass multiplexed tandem mass spectrometry for high-throughput polypeptide identification from mixtures.

We report a new tandem mass spectrometric approach for the improved identification of polypeptides from mixtures (e.g., using genomic databases). The approach involves the dissociation of several species simultaneously in a single experiment and provides both increased speed and sensitivity. The data analysis makes use of the known fragmentation pathways for polypeptides and highly accurate mass measurements for both the set of parent polypeptides and their fragments. The accurate mass information makes it possible to attribute most fragments to a specific parent species. We provide an initial demonstration of this multiplexed tandem MS approach using an FTICR mass spectrometer with a mixture of seven polypeptides dissociated using infrared irradiation from a CO2 laser. The peptides were added to, and then successfully identified from, the largest genomic database yet available (C. elegans), which is equivalent in complexity to that for a specific differentiated mammalian cell type. Additionally, since only a few enzymatic fragments are necessary to unambiguously identify a protein from an appropriate database, it is anticipated that the multiplexed MS/MS method will allow the more rapid identification of complex protein mixtures with on-line separation of their enzymatically produced polypeptides.

Amino Acid Sequence↗
PubMedDate not supplied

Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays.

We succeeded in using 40 nm FRET (fluorescence resonance energy transfer) microspheres conjugated to antibodies as the fluorescent reporters to perform the multiplexing suspension array measurements on two simulants of biological threats, ricin (A chain) and a crude spore preparation of Bacillus globigii (Bg). The microspheres were impregnated with two types of fluorophores in equal number (approximately 140 fluorophores in total per microsphere) and displayed bright PE-like fluorescence via a fluorescence resonance energy transfer mechanism. Activated microspheres (aldehyde groups) were directly coupled to antibodies and used to form sandwich-type immunoassays in a suspension array. For the crude preparations of Bg, the assay sensitivity using antibody-conjugated microspheres is an order of magnitude higher than that using the conventional fluorescent reporter, R-phycoerythrin (PE). Using the microspheres, Bg at the concentration of 5 ng/mL can be easily detected. For ricin, the assay sensitivity was similar to that obtained using PE as the reporter, but washing the reaction mixtures resulted in the fluorescence signals that were 2-3 times higher compared to those using PE. Ricin at a concentration of 1 ng/mL can be readily identified. Importantly, the two simulants do not interfere with each other in the multiplexing experiments. The 40 nm FRET microspheres are a new sensitive alternative as fluorescent reporters for detection in suspension arrays.

Bacillus↗
PubMed2006

Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes.

We have developed the chemistry for preparing a universal probe and the appropriate nano- and microparticle labels that can be used to do highly selective multiplexed detection of three protein cancer markers at low-femtomolar concentration in buffer and serum media. The approach relies on a new multiplexed version of the biobarcode amplification method and offers new opportunities for studying multiple protein markers in a single sample. This could lead to new forms of disease diagnosis and monitoring disease recurrence in a variety of settings.

Biomarkers, Tumor↗
PubMed2006

Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays.

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable no-wash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase DeltaG of folding in 500 mM buffered NaCl of approximately -4 kcal/mol performed better than those with DeltaG > -2 or < -6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 degrees C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with Kd = 1.7 +/- 0.3 nM. The detection limit was approximately 100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

DNA, Viral↗
PubMed2002

Detection and quantification of transgenes in grains by multiplex and real-time PCR.

Multiplex PCR reactions were developed for detecting simultaneously the CryIA(b) and pat genes from events 176, MON810, BT11, and T25 of transgenic maize, using only two pairs of primers, one for the CryIA(b) gene and the other for the pat gene. The Roundup Ready soybean can be precisely detected by a multiplex PCR reaction using known primers, amplifying fragments of the NOS and the epsps sequences simultaneously. Transgenic events such as Roundup Ready soybean and GA21 maize, among others, can be quantified by real-time PCR using a pair of primers and a probe specifically designed for annealing to the NOS ending region. As an alternative to amplifying an endogenous gene, the addition of a foreign gene in a percentage equal to the required level of detection, in a parallel reaction, is proposed. The use of hexane to homogenize large flour samples is suggested.

Amino Acid Sequence↗
PubMed2003

Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis.

Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.

Brassica napus↗
PubMed2005

Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

Base Sequence↗
PubMed2004

Detection and differentiation of several food-spoilage lactic acid bacteria by multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence.

In this work, a complete analytical procedure is investigated to differentiate several food-spoilage lactic acid bacteria. To do that, a method involving multiplex Polymerase Chain Reaction (PCR), capillary gel electrophoresis (CGE), and laser-induced fluorescence (LIF) is developed. The PCR-CGE-LIF protocol allows the simultaneous detection and differentiation of the genera Leuconostoc and Carnobacterium, the nonmotile group of species within the genus Carnobacterium, and the three species of the group individually (C. divergens, C. gallinarum, and C. maltaromicum). The capability of this approach is clearly illustrated through the sensitive and efficient analysis of the two closest amplicons, with sizes equal to 397 and 412 bp, showing very different yields in all of the amplification reactions tested. These two fragments, which could not be resolved by agarose gel electrophoresis (AGE), are clearly distinguishable by CGE-LIF even when very different areas for both peaks are obtained. The PCR-CGE-LIF method also allows the sensitive detection of these bacteria, demonstrating both a significant resolution improvement compared with traditional AGE and the usefulness of this approach to solve real-life analytical challenges. Good reproducibility of the CGE-LIF procedure is shown for the analysis of multiplex PCR samples with percent relative standard deviation values for migration times and corrected peak areas as low as 0.80 and 6.50 for the same sample and three different days (n = 12), respectively.

Bacteria↗