Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System.
Ontology highlight
ABSTRACT:
SUBMITTER: Nagy C
PROVIDER: S-EPMC8513095 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.
| S-EPMC6098732 | biostudies-literature
Project description:A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed.
| S-EPMC1592466 | biostudies-other
Project description:Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. Eighty-eight percent of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.
| S-EPMC3696430 | biostudies-literature
Project description:We developed a two-layer, four-channel polymerase chain reaction (PCR)-capillary electrophoresis microdevice that integrates nucleic acid amplification, sample cleanup and concentration, capillary electrophoretic separation, and detection for multiplex analysis of four human respiratory viral pathogens, influenza A, influenza B, coronavirus OC43, and human metapneumovirus. Biotinylated and fluorescently labeled double-stranded (ds) deoxyribonucleic acid (DNA) amplification products are generated in a 100 nL PCR reactor incorporating an integrated heater and a temperature sensor. After amplification, the products are captured and concentrated in a cross-linked acrylamide gel capture matrix copolymerized with acrydite-functionalized streptavidin-capture agents. Thermal dehybridization releases the fluorescently labeled DNA strand for capillary electrophoresis injection, separation, and detection. Using plasmid standards containing the viral genes of interest, each target can be detected starting from as few as 10 copies/reactor. When a two-step reverse transcription PCR amplification is employed, the device can detect ribonucleic acid (RNA) analogues of all four viral targets with detection limits in the range of 25-100 copies/reactor. The utility of the microdevice for analyzing samples from nasopharyngeal swabs is demonstrated. When size-based separation is combined with four-color detection, this platform provides excellent product discrimination, making it readily extendable to higher-order multiplex assays. This portable microsystem is also suitable for performing automated assays in point-of-care diagnostic applications.
| S-EPMC3076062 | biostudies-literature
Project description:In this work, a method for species-specific isotopic analysis of sulfur via capillary electrophoresis hyphenated on-line with multicollector ICP-MS (CE/MC-ICP-MS) was developed. Correction for the mass bias caused by instrumental mass discrimination was realized via external correction with multiple-injection sample-standard bracketing. By comparing the isotope ratio measurement results obtained using the newly developed on-line CE/MC-ICP-MS method with those obtained via traditional MC-ICP-MS measurement after analyte/matrix separation by anion exchange chromatography for isotopic reference materials and an in-house bracketing standard, the most suitable data evaluation method could be identified. The repeatability for the sulfate-δ34S value (calculated from 18 measurements of a standard conducted over seven measurement sessions) was 0.57‰ (2SD) and thereby only twice that obtained with off-line measurements (0.30‰, n = 68). As a proof of concept for analysis of samples with a real matrix, the determination of the sulfur isotopic composition of naturally present sulfate was performed for different river systems. The CE/MC-ICP-MS results thus obtained agreed with the corresponding off-line MC-ICP-MS results within the 2SD ranges, and the repeatability of consecutive δ34S measurements (n = 3) was between 0.3‰ and 1.3‰ (2SD). Finally, the isotopic analysis of two different S-species in a river water sample spiked with 2-pyridinesulfonic acid (PSA) was also accomplished. Graphical abstract The CE/MC-ICP-MS method developed allows for species-specific S isotopic analysis in samples containing multiple species. Mass bias is corrected for by multiple-injection sample-standard bracketing, while the repeatability (2SD) of the resulting 34δ-values is <1‰.
| S-EPMC8236454 | biostudies-literature
Project description:We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and ?-casein as model proteins.
| S-EPMC3038187 | biostudies-literature
Project description:Various compound markers encompassing two or more variants within a small region can be regarded as generalized microhaplotypes. Many of these markers have been investigated for various forensic purposes, such as individual identification, deconvolution of DNA mixtures, or forensic ancestry inference. SNP-STR is a compound biomarker composed of a single nucleotide polymorphism (SNP) and a closely linked short tandem repeat polymorphism (STR), and possess the advantages of both SNPs and STRs. In addition, in conjunction with a polymerase chain reaction (PCR) technique based on the amplification refractory mutation system (ARMS), SNP-STRs can be used for forensic unbalanced DNA mixture analysis based on capillary electrophoresis (CE), which is the most commonly used platform in worldwide forensic laboratories. Our previous research reported 11 SNP-STRs, but few of them are derived from the commonly used STR loci, for which existing STR databases can be used as a reference. For maximum compatibility with existing DNA databases, in this study, we screened 18 SNP-STR loci, of which 14 were derived from the expanded CODIS core loci set. Stable and sensitive SNP-STR multiplex PCR panels based on the CE platform were established. Assays on simulated two-person DNA mixtures showed that all allele-specific primers could detect minor DNA components in 1:500 mixtures. Population data based on 113 unrelated Chengdu Han individuals were investigated. A Bayesian framework was developed for the likelihood ratio (LR) evaluation of SNP-STR profiling results obtained from two-person mixtures. Furthermore, we report on the first use of SNP-STRs in casework to show the advantages and limitations for use in practice. Compared to 2.86 × 103 for autosomal STR kits, the combined LR reached 7.14 × 107 using the SNP-STR method in this casework example.
| S-EPMC7893108 | biostudies-literature
Project description:[This corrects the article DOI: 10.3389/fgene.2021.636821.].
| S-EPMC9648558 | biostudies-literature
Project description:An integrated microfluidic chip is proposed for rapid DNA digestion and time-resolved capillary electrophoresis (CE) analysis. The chip comprises two gel-filled chambers for DNA enrichment and purification, respectively, a T-form micromixer for DNA/restriction enzyme mixing, a serpentine channel for DNA digestion reaction, and a CE channel for on-line capillary electrophoresis analysis. The DNA and restriction enzyme are mixed electroomostically using a pinched-switching DC field. The experimental and numerical results show that a mixing performance of 97% is achieved within a distance of 1 mm from the T-junction when a driving voltage of 90 V/cm and a switching frequency of 4 Hz are applied. Successive mixing digestion and capillary electrophoresis operation clearly present the changes on digesting φx-174 DNA in different CE runs. The time-resolved electropherograms show that the proposed device enables a φx-174 DNA sample comprising 11 fragments to be concentrated and analyzed within 24 min. Overall, the results presented in this study show that the proposed microfluidic chip provides a rapid and effective tool for DNA digestion and CE analysis applications.
| S-EPMC3365337 | biostudies-other
Project description:Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.
| S-EPMC3129339 | biostudies-literature