We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.
We describe a method of performing multiple enzyme assays in a single reaction vessel. The resolving power of capillary electrophoresis enables several enzyme assays to be analyzed at high speed in microfluidic arrays. Multiplexed measurement can increase throughput significantly without requiring highly dense microfluidic arrays. Enzyme assays in a multiplexed format for selected kinases in this work show essentially identical performance to assays performed individually. This establishes an approach for screening one compound against multiple enzyme targets simultaneously. Another potential application for performing multiplexed enzyme assay is to study protein-protein (especially enzyme-enzyme) interaction by monitoring the enzymatic activity changes.
An automated, sheathless capillary electrophoresis-mass spectrometry platform for discovery of biomarkers in human serumA capillary electrophoresis-mass spectrometry (CE-MS) method has been developed to perform routine, automated analysis of low-molecular-weight peptides in human serum. The method incorporates transient isotachophoresis for in-line preconcentration and a sheathless electrospray interface. To evaluate the performance of the method and demonstrate the utility of the approach, an experiment was designed in which peptides were added to sera from individuals at each of two different concentrations, artificially creating two groups of samples. The CE-MS data from the serum samples were divided into separate training and test sets. A pattern-recognition/feature-selection algorithm based on support vector machines was used to select the mass-to-charge (m/z) values from the training set data that distinguished the two groups of samples from each other. The added peptides were identified correctly as the distinguishing features, and pattern recognition based on these peptides was used to assign each sample in the independent test set to its respective group. A twofold difference in peptide concentration could be detected with statistical significance (pvalue , 0.0001). The accuracy of the assignment was 95%, demonstrating the utility of this technique for the discovery of patterns of biomarkers in serum.
Abstract. Separations by capillary zone electrophoresis (CZE) in plain, uncoated fused silica capillaries were compared with those in electroendosmosis-free coated capillaries. For small ions, exemplified by some anti-inflammatory drugs, resolution and analysis times were comparable in the two types of capillaries. The advantage of the coated capillary was that it predictably eluted all negative analytes regardless of electrolyte pH. The uncoated capillary, on the other hand, allowed separation of both positive and negative species in one run, provided that the electroendosmotic flow was sufficiently high to elute all negatively charged analytes. Because electrolyte conditions were established that eluted all sample analytes, subsequent analyses were performed in uncoated capillaries.Antibiotics such as sulfonamides, cephalosporins, and penicillins were separated by zone electrophoresis. Optimal separation conditions were established by varying the pH and ionic strength of the electrolyte. For the separation of structurally similar peptides and barbiturates, micellar electrokinetic capillary chromatography (MECC) was the method of choice because the structural differences conferred differences in hydrophobicity.The feasibility of using CZE in pharmaceutical analysis was shown in the evaluation of over-the-counter pain, cold, and allergy medications. These typically gave relative standard deviations of 1% for retention times and 3% for peak areas.
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