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Electrophoresis is integral to analytical and biochemistry experiences in undergraduate education; however, fundamental principles of the method are often taught in upper-level laboratories through hands-on experiences. A laboratory activity is reported that teaches the concepts of electrophoretic mobility and electroosmotic flow. A single reuseable instrument, called a mini-E, costs 37 USD and consists of a DC power supply, a voltmeter, platinum electrodes, and a chip cast in polydimethylsiloxane. This activity uses common reagents costing only 0.02 USD per student. Experiments are devised that allow students to investigate the properties of electrophoretic flow and electroosmotic flow by separating the two commonly used food dyeing agents Brilliant Blue FCF and Allura Red AC in vinegar and in a solution of ammonium hydroxide. A dark-purple mixture of these dyes is separated into red and blue bands that are easily visualized. The migration order of the dyes differs when the separation is performed under conditions of reversed polarity and suppressed electroosmotic flow (vinegar) compared to conditions of normal polarity and active electroosmotic flow (ammonium hydroxide). When delivered to chemistry majors, students had a significant gain in their ability to apply the concepts of electroosmosis and electrophoresis to predict analyte migration. Although this activity targets upper-level chemistry content, it can also be adapted for other laboratory experiences.
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Fluorescence labeling of biomolecules and fluorescence detection platforms provide a powerful approach to high-sensitivity bioanalysis. Reactive probes can be chosen to target specific functional groups to enable selective analysis of a chosen class of analytes. Particularly, when targeting trace levels of analytes, it is important to optimize the reaction chemistry to maximize the labeling efficiency and minimize the background. Here, we develop methods to optimize the labeling and detection of Pacific Blue (PB)-tagged amino acids. A model is developed to quantitate labeling kinetics and completeness in the circumstance where analyte labeling and reactive probe hydrolysis are in competition. The rates of PB hydrolysis and amino acid labeling are determined as a function of pH. Labeling kinetics and completeness as a function of PB concentration are found to depend on the ratio of the hydrolysis time to the initial labeling time, which depends on the initial PB concentration. Finally, the optimized labeling and detection conditions are used to perform capillary electrophoresis analysis demonstrating 100 pM sensitivities and high-efficiency separations of an 11 amino acid test set. This work provides a quantitative optimization model that is applicable to a variety of reactive probes and targets. Our approach is particularly useful for the analysis of trace amine and amino acid biosignatures in extraterrestrial samples. For illustration, our optimized conditions (reaction at 4 °C in a pH 8.5 buffer) are used to detect trace amino acid analytes at the 100 pM level in an Antarctic ice core sample.
Assuntos
Aminoácidos , Eletroforese Capilar , Aminas/análise , Aminoácidos/análise , Eletroforese Capilar/métodos , Hidrólise , Indicadores e ReagentesRESUMO
Neuraminidase inhibitors modulate infections that involve sialic acids, making quantitative analyses of this inhibitory effect important for selecting and designing potential therapeutics. An automated nanogel capillary electrophoresis system is developed that integrates a 5 nL enzyme inhibition reaction in line with a 5 min separation-based assay of the enzymatic product to quantify inhibition as the half maximal inhibitory concentration (IC50) and inhibitor constant (Ki). A neuraminidase enzyme from Clostridium perfringens is non-covalently immobilized in a thermally tunable nanogel positioned in the thermally controlled region of the capillary by increasing the capillary temperature to 37 °C. Aqueous inhibitor solutions are loaded into the capillary during the nanogel patterning step to surround the enzyme zone. The capillary electrophoresis separation provides a means to distinguish the de-sialylated product, enabling the use of sialyllactose which contains the trisaccharide motif observed on serine/threonine-linked (O-linked) glycans. A universal nanogel patterning scheme is developed that does not require pre-mixing of enzymes with inhibitors when an automated capillary electrophoresis instrument is used, thus reducing the consumption of enzymes and enabling adaption of the method to different inhibitors. The universal approach is successfully applied to two classical neuraminidase inhibitors with different electrophoretic mobilities. The IC50 and Ki values obtained for N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (DANA) are 13 ± 3 and 5.0 ± 0.9 µM, respectively, and 28 ± 3 and 11 ± 1 µM, respectively, for Siastatin B. These values agree with literature reports and reflect the weaker inhibition anticipated for Siastatin B in comparison to DANA.
Assuntos
Eletroforese Capilar , Neuraminidase , Nanogéis , Eletroforese Capilar/métodos , Polietilenoimina , Inibidores EnzimáticosRESUMO
BACKGROUND: Enzyme inhibitors comprise the largest class of pharmaceutical compounds. The discovery and development of new enzyme inhibitor drug candidates depends on sensitive tools to quantify inhibition constants, Ki, for the most promising candidates. A high throughput, automated, and miniaturized approach to measure inhibition is reported. In this technique enzyme inhibition occurs within a 16 nL nanogel reaction zone that is integrated into a capillary. The reaction and electrophoresis separation are completed in under 10 min. The nanoliter enzyme reaction zones are easily positioned inside a standard separation capillary by pseudo-immobilizing enzymes within a thermally reversible nanogel. RESULTS: This report optimizes and validates a capillary nanogel electrophoresis reaction and separation with a multi-capillary array instrument. Inhibitor constants are determined for the neuraminidase enzyme to quantify the effect of the transition state analog, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), as well as the inhibitor Siastatin B. With the multi-capillary array assay replicate Ki values are determined to be 5.7 ± 0.1 µM (n = 3) and 9.2 ± 0.2 µM (n = 3) for DANA and Siastatin B, respectively. The enzyme reaction in each separation capillary converts the substrate to a product in real time. The nanogel is used under suppressed electroosmotic flow, sustains enzyme function, and is easily filled and replaced by changing the capillary temperature. Using laser-induced fluorescence allows the determination to be achieved with substrate concentrations well below the Michaelis-Menten constant, making the method independent of the substrate concentration and therefore a more easily implemented assay. SIGNIFICANCE: A lower measurement cost is realized when the reaction volume is miniaturized because the amounts of enzyme, substrate and inhibitor are reduced. Fast enzyme reactions are possible because of the small reaction volume. With a multi-capillary array, the inhibition assay is achieved in a fraction of the time required for traditional methods. The separation-based assay can even be applied to labeled substrates not cleaned up following the labeling reaction.
Assuntos
Eletroforese Capilar , Inibidores Enzimáticos , Polietilenoglicóis , Polietilenoimina , Nanogéis , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Eletroforese Capilar/métodos , Neuraminidase/químicaRESUMO
Gel matrices are fundamental to electrophoresis analyses of biopolymers in microscale channels. Both capillary gel and microchannel gel electrophoresis systems have produced fundamental advances in the scientific community. These analytical techniques remain as foundational tools in bioanalytical chemistry and are indispensable in the field of biotherapeutics. This review summarizes the current state of gels in microscale channels and provides a brief description of electrophoretic transport in gels. In addition to the discussion of traditional polymers, several nontraditional gels are introduced. Advances in gel matrices highlighted include selective polymers modified to contain added functionality as well as thermally responsive gels formed through self-assembly. This review discusses cutting-edge applications to challenging areas of discovery in DNA, RNA, protein, and glycan analyses. Finally, emerging techniques that result in multifunctional assays for real-time biochemical processing in capillary and three-dimensional channels are identified.