High-throughput Sample Analysis Research Articles

Miniaturized two‐dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.

Advances in molecular techniques for the detection and quantification of genetically modified organisms

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.

Identification of hepatotoxin‐producing cyanobacteria by DNA‐chip

We developed a new tool to detect and identify hepatotoxin-producing cyanobacteria of the genera Anabaena, Microcystis, Planktothrix, Nostoc and Nodularia. Genus-specific probe pairs were designed for the detection of the microcystin (mcyE) and nodularin synthetase genes (ndaF) of these five genera to be used with a DNA-chip. The method couples a ligation detection reaction, in which the polymerase chain reaction (PCR)-amplified mcyE/ndaF genes are recognized by the probe pairs, with a hybridization on a universal microarray. All the probe pairs specifically detected the corresponding mcyE/ndaF gene sequences when DNA from the microcystin- or nodularin-producing cyanobacterial strains were used as template in the PCR. Furthermore, the strict specificity of detection enabled identification of the potential hepatotoxin producers. Detection of the genes was very sensitive; only 1-5 fmol of the PCR product were needed to produce signal intensities that exceeded the set background threshold level. The genus-specific probe pairs also reliably detected potential microcystin producers in DNA extracted from six lake and four brackish water samples. In lake samples, the same microcystin producers were identified with quantitative real-time PCR analysis. The specificity, sensitivity and ability of the DNA-chip in simultaneously detecting all the main hepatotoxin producers make this method suitable for high-throughput analysis and monitoring of environmental samples.

High-Throughput Elemental Analysis of Small Aqueous Samples by Emission Spectrometry with a Compact, Atmospheric-Pressure Solution-Cathode Glow Discharge

A miniaturized version of an atmospheric-pressure glow discharge using a solution as the cathode was recently evaluated for elemental analysis of continuously sampled aqueous solutions. Although continuous sampling is useful, transient analysis is required for certain applications, including chromatographic or similar separations, small-volume sampling, high-throughput sampling, and on-line preconcentration. The miniaturized solution-cathode glow discharge seems particularly well suited to transient analysis by virtue of its low dead volume and high sensitivity. Two benefits of transient analysis were exploited here: high throughput and small sample volume. Sampling 25-microL volumes at 1000 samples/h, the discharge achieved detection limits ranging from 5 pg (0.2 ppb) for Li to 6 ng (270 ppb) for Hg.

Reverse transcription-polymerase chain reaction (RT-PCR) in flow-through micro-reactors: Thermal and fluidic concepts

This paper presents a novel flow-through reverse transcription-polymerase chain reaction (RT-PCR) micro-reactor with optimized thermal and fluidic characteristics. We have designed a micro-reactor comprising a heating plate for different temperature zones and a fluidic chip with serpentine micro-channels. One feature of this chip system is the generation of a segmented flow for high-throughput analysis of PCR samples. We show the first successful reactions on the chip with different flow regimes and variations of PCR methods from a standard PCR to one-step RT-PCR reactions. The aim of the experiments was the detection of the HPV 16 DNA genome and of viral oncogene transcripts, respectively. Both markers are of importance in medical diagnostics.

Linear combinatorial approach to thin film research

We describe high-throughput experimentation of film synthesis by use of a linear tape transport system (similar to a web-coating system). Metal tape is fed continuously in a reel-to-reel transport system inside the vacuum deposition chamber. Ion-beam assisted deposition (IBAD) texturing is used to enable the growth of epitaxial films on flexible, polycrystalline metal tapes which further enhances the capability of this research. The tape that is continuously fed can be used as a sample itself, via the use of IBAD-textured templates on the tape, or can be a carrier of other smaller substrates (even nonflexible ones). Characterization of samples is done by means of in situ monitoring as well as ex situ sequential analysis. We utilize in situ reflection high-energy electron diffraction for high-throughput analysis of samples. Epitaxial films are deposited on heated samples by evaporation and by pulsed laser deposition. Here, we explain the techniques and the methodologies developed for this type of combinatorial experimentation and show some examples of the material research completed.

Determination of Olive Oil Oxidative Status by Selected Ion Flow Tube Mass Spectrometry

The emergence of primary and secondary oxidation products in New Zealand extra virgin olive oil during accelerated thermal oxidation was measured and correlated with the concentrations of 13 headspace volatile compounds measured by selected ion flow tube mass spectrometry (SIFT-MS). SIFT-MS is a mass spectrometric technique that permits qualitative and absolute quantitative measurements to be made from whole air, headspace, or breath samples in real-time down to several parts per billion (ppb). It is well-suited to high-throughput analysis of headspace samples. Propanal, hexanal, and acetone were found at high concentrations in a rancid standard oil, while propanal, acetone, and acetic acid showed marked increases with oxidation time for the oils used in this study. A partial least-squares (PLS) regression model was constructed, which allowed the prediction of peroxide values (PV) for three separate oxidized oils. Sensory rancidity was also measured, although the correlations of headspace volatile compounds with sensory rancidity score were less satisfactory, and too few results were available for the construction of a PLS regression model. A fast (approximately 1 min), reliable method for prediction of olive oil PVs by SIFT-MS was developed.

High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC–MS/MS

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine ( mCPP) in human plasma. The method utilized d 7-nefazodone, d 7-hydroxynefazodone, d 4-BMS-180492 and d 4- mCPP as internal standards (IS). The plasma samples were injected into the LC–MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis ® HLB column (on-line extraction column, 1 mm × 50 mm, 30 μm) and a conventional Luna C8 column (analytical column, 4.6 mm × 50 mm, 5 μm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC–MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.

NanoLC‐FT‐ICR MS improves proteome coverage attainable for ∼3000 laser‐microdissected breast carcinoma cells

Proteomics assays hold great promise for unraveling molecular events that underlie human diseases. Effective analysis of clinical samples is essential, but this task is considerably complicated by tissue heterogeneity. Laser capture microdissection (LCM) can be used to selectively isolate target cells from their native tissue environment. However, the small number of cells that is typically procured by LCM severely limits proteome coverage and biomarker discovery potential achievable by conventional proteomics platforms. Herein, we describe the use of nanoLC-FT-ICR MS for analyzing protein digests of 3000 LCM-derived tumor cells from breast carcinoma tissue, corresponding to 300 ng of total protein. A total of 2282 peptides were identified by matching LC-MS data to accurate mass and time (AMT) tag databases that were previously established for human breast (cancer) cell lines. One thousand and three unique proteins were confidently identified with two or more peptides. Based on gene ontology categorization, identified proteins appear to cover a wide variety of biological functions and cellular compartments. This work demonstrates that a substantial number of proteins can be detected and identified from limited number of cells using the AMT tag approach, and opens doors for high-throughput in-depth proteomics analysis of clinical samples.

A Rapid Single-Step Multiplex Method for Discriminating Between Trichogramma (Hymenoptera: Trichogrammatidae) Species in Australia

Inaccurate species identification confounds insect ecological studies. Examining aspects of Trichogramma ecology pertinent to the novel insect resistance management strategy for future transgenic cotton, Gossypium hirsutum L., production in the Ord River Irrigation Area (ORIA) of Western Australia required accurate differentiation between morphologically similar Trichogramma species. Established molecular diagnostic methods for Trichogramma identification use species-specific sequence difference in the internal transcribed spacer (ITS)-2 chromosomal region; yet, difficulties arise discerning polymerase chain reaction (PCR) fragments of similar base pair length by gel electrophoresis. This necessitates the restriction enzyme digestion of PCR-amplified ITS-2 fragments to readily differentiate Trichogramma australicum Girault and Trichogramma pretiosum Riley. To overcome the time and expense associated with a two-step diagnostic procedure, we developed a "one-step" multiplex PCR technique using species-specific primers designed to the ITS-2 region. This approach allowed for a high-throughput analysis of samples as part of ongoing ecological studies examining Trichogramma biological control potential in the ORIA where these two species occur in sympatry.

Determination of five phenolic antioxidants in edible oils: Method validation and estimation of measurement uncertainty

An analytical protocol and the measurement uncertainty for determination of butylated hydroxylanisole, butylated hydroxyltoluene propyl gallate, octyl gallate and dodecyl gallate in edible oils were reported. Details of the validation including application of certified reference materials and participation in proficiency tests were discussed. Traceability of measurement was established through traceable standards and calibrated volumetric equipment and measuring instruments. The protocol, comprising a TLC screening step, quantification by gas chromatography-flame ionization detection (GC-FID) and high performance liquid chromatography-photodiode array detection (HPLC-PDA) techniques, and unequivocal confirmation by mass spectrometry or a second chromatographic technique, provides a reliable and validated means for fast turnaround and high sample throughput analysis of the five antioxidants in edible oils. This protocol was also found to be applicable to other fat-containing food matrices.

Proteomics in Pulmonary Medicine

Proteomics is the study of the entire protein complement of the genome (the proteome) in a biological system. Proteomic studies require a multidisciplinary approach and have only been practical with the convergence of technical and methodologic improvements including the following: advances in mass spectrometry and genomic sequencing that now permit the identification and relative quantization of small amounts (femtomole) of nearly any single protein; new methods in gel electrophoresis that allow the detection of subtle changes in protein expression, including posttranslational modifications; automation and miniaturization that permit high-throughput analysis of clinical samples; and new bioinformatics and computational methods that facilitate analysis and interpretation of the abundant data that are generated by proteomics experiments. This convergence makes proteomics studies practical for pulmonary researchers using BAL fluid, lung tissue, blood, and exhaled breath condensates, and will facilitate the research of complex, multifactorial lung diseases such as acute lung injury and COPD. This review describes how proteomics experiments are conducted and interpreted, their limitations, and how proteomics has been used in clinical pulmonary medicine.

Real-time GUS analysis using Q-PCR instrumentation

The development of new technology within biological sciences has resulted in a number of real-time PCR instruments that have become essential tools within molecular biology. This equipment has facilitated high throughput analysis of samples and optimal information gathering of completed PCR reactions for example estimating the copy number of a gene of interest that is inserted into particular genomes. Real-time PCR instruments frequently come with optional filter sets, e.g. the ALEXA filter set which has parameters in common with excitation and emission wavelengths of sodium methyl umbelliferone (NaMU) widely used in beta-glucuronidase reporter gene assays. Using these filter sets it has been possible to quantify and measure gus A activity of Ulmus procera SR4 in real-time removing the necessity for aliquots of reactions to be stopped by pipetting into carbonate buffer for each time point. The introduction of real-time GUS analysis leads to faster, more accurate and reproducible assays with reduced potential for pipetting errors, requires fewer manipulations and encourages high throughput analysis of inter-individual gene expression variation.

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.

Characterization of must and wine of six varieties of grapes by direct infusion electrospray ionization mass spectrometry

Samples of must derived from six different varieties of grapes taken during the fermentation process, as well as the respective wine samples directly after the end of the malolactic fermentation, were analyzed by direct infusion negative ion mode electrospray ionization mass spectrometry (ESI-MS). Diagnostic ions for must were different from those of wine samples, although small variations for each of the grape varieties were also detected. The addition of unfermented must or sugar to wine could also be clearly detected. The spectra were acquired in a few minutes per sample, indicating that ESI-MS can be used for high-throughput analysis of samples and should prove useful for quality control during and after the fermentation process.

A Pilot Application of SELDI-TOF Mass Spectrometry in Bone Marrow Failure Syndromes.

Myelodysplastic syndrome (MDS), aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are a heterogeneous group of disorders with significant clinical overlap. Current nosologic classifications schemes based mostly on morphology may not reflect pathophysiologic relationship between individual entities. The recent refinement of surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) technology allows for a high-throughput protein analysis of various biologic samples. Plasma samples from patients with MDS (N=134), AA (N=41), PNH (N=34), and healthy controls (N=75) were subjected to SELDI analyses using H4 chips. We limited analysis to spectra with more than 80 peaks. Reproducibility for each array run was monitored by repeating measurement of one standard sample on each chip. All spectra with peaks characterized by mass-to-charge ratios between 1.5–30 kDa were examined. Peaks with amplitude of at least > 3 times the average background noise were considered as meaningful. We had previously attempted SELDI analysis in bone marrow failure in a direct, manual, non-parametric fashion; this approach did not result in satisfactory discrimination. Here we have designed a new method utilizing a "binning" algorithm of peaks fulfilling selection criteria. A non-parametric approach was applied to select peaks within the bin defined by specific m/z amplitude. For each condition the "binned" signals were screened using Fisher's exact test to determine significant peaks; p<0.001 was used as the cutoff for selecting signals for further study. Logistic regression models with step-wise and backward variable selection was then used to determine peaks that had independent prognostic value and could therefore discriminate between the patient groups. The resulting model was validated internally using a bootstrap re-sampling procedure; random samples from the original population were drawn with replacement in such a way that the resulting "bootstrap population" had the same number of subjects as the original study population. This process was repeated 500 times and the frequency of each factor's inclusion in the resulting models was calculated. Factors present in ≥ 50% of the models were considered significant and used to build a final "bootstrap-based" model. Discriminating signals were identified and a scoring system was used to derive a score for each condition relative to controls (+1 for the presence of the signal in a bin being associated with the condition and −1 for the absence of the signal being associated with the condition). A score of 3 was used as a cutoff in MDS and a score of 2 in other diseases to differentiate from normal controls (Table 1). The observed discrimination obtained using only one chip surface and crude disease definitions encourages future studies. Currently non-overlapping sample sets are being generated for further analysis to identify proteomic patterns in specific cytogenetic defects and morphological categories; thereby validating the pilot results in more stringently defined disease phenotypes.Disease versus normal controlsGroupNumberScore + in %Score - in %psensitivity/specificityControl758317MDS1341783<0.00183%/83%Control758812AA412476<0.00176%/88%Control75928PNH342179<0.00179%/92%

Pyrosequencing: nucleotide sequencing technology with bacterial genotyping applications

Pyrosequencing is a relatively new method for real-time nucleotide sequencing. It has rapidly found applications in DNA sequencing, genotyping, single nucleotide polymorphism analysis, allele quantification and whole-genome sequencing within the areas of microbiology, clinical genetics and pharmacogenetics. It is fast becoming a real alternative to the traditional Sanger sequencing method although, at present, read lengths are normally limited to approximately 70 nucleotides. The pyrosequencing method involves four main stages: first, target DNA is amplified using PCR; second, double-stranded DNA is converted to single-stranded DNA templates; third, oligonucleotide primers are hybridized to a complementary sequence of interest; and, finally, the pyrosequencing reaction itself, in which a reaction mixture of enzymes and substrates catalyses the synthesis of complementary nucleotides. Data are shown as a collection of signal peaks in a pyrogram. Pyrosequencing is increasingly used for bacterial detection, identification and typing, and, recently, a commercial system became available for the identification of bacterial isolates. Pyrosequencing can also be partially or fully automated, thus enabling the high-throughput analysis of samples. Wider use of pyrosequencing may occur in the future if longer nucleotide reads are made possible, which will enable its expansion into larger nucleotide sequencing such as multilocus sequence typing and whole-genome sequencing.

Desorption Electrospray Ionization Mass Spectrometry for High-Throughput Analysis of Pharmaceutical Samples in the Ambient Environment

Desorption electrospray ionization (DESI) allows mass spectrometry to be used for on-line high-throughput monitoring of pharmaceutical samples in the ambient environment, without prior sample preparation. Positive and negative ion DESI are used to characterize the active ingredients in pharmaceutical samples formulated as tablets, ointments, and liquids. Compounds of a wide variety of chemical types are detected in these complex matrices. The effects on analytical performance of operating parameters, including the electrospray high voltage, heated capillary temperature, solvent infusion rate, and solvent composition, are evaluated and optimized. In addition to experiments in which a simple solvent is sprayed onto the solid analyte samples, reactive desorption is performed by adding reagents to the solvent spray to generate particularly stable or characteristic ions with the analytes of interest. A variable-speed moving belt was built for high-throughput sampling and used to provide rapid qualitative and semiquantitative information on drug constituents in tablets. Sampling rates as high as 3 samples/s are achieved in the ambient environment. Relative standard deviations of the relative ion abundances for major components in the mass spectra are in the range of 2-8%. Impurities and components present at levels as low as approximately 0.1% are identified and carryover effects are minimized in high-throughput on-line analysis of pharmaceutical samples.

Utility of lab-on-a-chip technology for high-throughput nucleic acid and protein analysis

On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis to achieve rapid analysis times (<120 s). This work describes the utility of LoaC systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of multiplex PCR samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis are illustrated.

High-throughput capillary electrophoresis for the identification and differentiation of seven species ofEimeria from chickens

A capillary electrophoretic approach has been evaluated for the identification of seven currently recognised species of Eimeria infecting chickens. The second internal transcribed spacer of ribosomal DNA is PCR-amplified from any of the seven species using a single set of oligonucleotide primers (one of which is fluorescently labelled). The amplicons are heat-denatured and subjected to capillary electrophoresis in a MegaBACE 1000 (Amersham). The chromatograms captured are stored electronically and then analysed using MegaBACE Fragment Profiler software. Using control DNA samples representing monospecific lines of Eimeria, specific peaks in the chromatograms were defined for the unequivocal identification of each of the seven species and their differentiation. Electrophoretic reading and analysis are carried out automatically, thus making it a time- and cost-effective method. This procedure should find applicability as a tool for the quality control of Eimeria vaccines, the monitoring of coccidiosis outbreaks and the high-throughput analysis of oocyst samples for epidemiological surveys.