Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase

Takayanagi, Toshio; Mine, Masanori; Mizuguchi, Hitoshi

doi:10.2116/analsci.19p471

Cited by 11 publications

(9 citation statements)

References 40 publications

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“…The measurements of pNP taken in a fused-silica capillary with an effective length of 15 cm and a diameter of 50 μm displayed peaks at migration time of 5 min. This also was confirmed by other studies [ 45 , 46 , 47 ]. The separation peaks were affected by the length of incubation time and by applied voltage [ 45 ].…”

Section: Resultssupporting

confidence: 91%

Electrochemical Detection and Capillary Electrophoresis: Comparative Studies for Alkaline Phosphatase (ALP) Release from Living Cells

2020

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Alkaline phosphatase (ALP) is one of the main biomarkers that is clinically detected in bone and liver disorders using optical assays. The electrochemical principle is important because point-of-care testing is increasing dramatically and absorbance techniques hardly compete with the medical revolution that is occurring. The detection of ALP using electrochemical detection is contributing to the integration systems field, and hence enhancing the detection of biological targets for pharmaceutical research and design systems. Moreover, in vitro electrochemical measurements use cost effective materials and simple techniques. Graphite screen-printed electrodes and linear sweep voltammetry were used to optimize the electrochemistry of the enzymatic product p-aminophenol using the enzyme kinetic assay. ALP release from embryonic and cancer cells was determined from adhesion cell culture. Additionally, capillary electrophoresis and colorimetric methods were applied for comparison assays. The resulting assays showed a dynamic range of ALP ranging from 1.5 to 1500 U/L, and limit of detection of 0.043 U/L. This was achieved by using 70 μL of the sample and an incubation time of 10 min at an optimal substrate concentration of 9.6 mM of p-aminophenol phosphate. A significant difference (p < 0.05) was measured between the absorbance assays. This paper demonstrates the advantages of the electrochemical assay for ALP release from cells, which is in line with recent trends in gene expression systems using microelectrode array technologies and devices for monitoring electrophysiological activity.

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Section: Resultssupporting

confidence: 91%

Electrochemical Detection and Capillary Electrophoresis: Comparative Studies for Alkaline Phosphatase (ALP) Release from Living Cells

2020

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“…As a result of the constant reaction rate and the continuous CE resolution, a plateau signal would be detected with the product (Fig. 13) [68]. This plateau signal of the product is pulled out from the short zone of the substrate by the dynamic enzymatic reaction, and this format is comparable to CE/FA.…”

Section: Michaelis-menten Analysis Through Ce/dfamentioning

confidence: 93%

“…A new mode of EMMA has been proposed as capillary electrophoresis/dynamic frontal analysis (CE/DFA) [68]. When a capillary is filled with an enzyme solution and a…”

Section: Michaelis-menten Analysis Through Ce/dfamentioning

confidence: 99%

Development of Novel Analysis and Characterization Methods Utilizing Reaction Dynamics in a Separation Capillary

Takayanagi

2022

Chromatography

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Electrophoretic migration of an analyte in capillary electrophoresis (CE) reflects reaction dynamics of the analyte in solution.In affinity CE, an analyte of interest interacts with a modifier added in the separation buffer in fast equilibrium, and effective electrophoretic mobility of the analyte is contributed from its equilibrium species. Precise measurement of effective electrophoretic mobility allows analyzing the equilibrium. Analysis of equilibria under CE separation possesses several advantages against traditional analyses in homogeneous solution; coexisting substances including impurities and kinetically generated substances are resolved by CE from the equilibrium species of interest. Characteristics of the CE analysis have been applied to analyses of acid-base equilibria of degradable substances and ion-association equilibria in an aqueous solution. Since CE is operated in an open-tubular capillary, it is also suitable for the characterization of carbon nanoclusters such as graphene and carbon nanotube, and measurement of effective electrophoretic mobility helps characterization of nanoclusters. A novel analysis technique of capillary electrophoresis/dynamic frontal analysis (CE/DFA) has also been proposed for the analysis of such reactions as involving equilibria and kinetic reactions. In CE/DFA, kinetically generated product is continuously resolved from the equilibrium species, and a plateau signal would be detected when the reaction rate is constant. Michaelis-Menten constants have successfully been determined through the plateau height by CE/DFA. In this review, analysis and characterization methods utilizing reaction dynamics in a separation capillary are summarized.

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“…The height of this plateau is related to the enzymatic reaction rate. In this setup, the product cannot inhibit the enzymatic reaction since it is moving ahead of the substrate [37].…”

Section: Homogeneous Online Enzyme Reaction Studiesmentioning

confidence: 99%

Overview of in‐capillary enzymatic reactions using capillary electrophoresis

2021

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This review summarizes the research that has recently been performed on in‐capillary enzymatic reactions integrated with capillary electrophoresis. The manuscript is subdivided in homogeneous and heterogeneous approaches. The main homogeneous techniques are Electrophoretically Mediated Microanalysis, At‐inlet and Transverse Diffusion of Laminar Flow Profiles. The main heterogeneous ones are Immobilized MicroEnzyme Reactors with enzymes grafted on either non‐magnetic or magnetic particles. The overview covers the period from 2018 to early 2021. The applications range from drug discovery over natural products to food, beverage and pesticide analysis.

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Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase

Cited by 11 publications

References 40 publications

Electrochemical Detection and Capillary Electrophoresis: Comparative Studies for Alkaline Phosphatase (ALP) Release from Living Cells

Electrochemical Detection and Capillary Electrophoresis: Comparative Studies for Alkaline Phosphatase (ALP) Release from Living Cells

Development of Novel Analysis and Characterization Methods Utilizing Reaction Dynamics in a Separation Capillary

Overview of in‐capillary enzymatic reactions using capillary electrophoresis

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