A novel enzyme-immobilized flow cell used as end-column chemiluminescent detection interface in open-tubular capillary electrochromatography

Abstract

A novel enzyme-immobilized flow-through interface was designed for sensitive end-column chemiluminescent (CL) detection in open-tubular capillary electrochromatography (OTCEC). Enzyme was covalently bound on an aldehyde-activated polymer membrane immobilized in a flow cell to catalyze the CL reaction that occurred in it. Using glycine as the model analyte, N-(4-aminobutyl)-N-ethylisoluminol-derivatized glycine effused from the OTCEC column and triggered a horseradish peroxidase (HRP)-catalyzed CL reaction, to produce an enhanced detection signal. To obtain a satisfying result for complex biological sample analysis, a thiolated β-cyclodextrin-modified gold nanoparticles-coated OTCEC column was adopted to improve the separation efficiency. Glycine can be assayed in the range of 0.50-200 μM (R(2) = 0.9921) with a detection limit of 0.12 μM (S/N of 3). The whole analysis process can be completed within 13 min with a theoretical plate number of 22,500. Compared to the previously reported solution-phase enzyme catalysis, pre-column and on-column immobilized enzyme catalysis for capillary electrophoresis detection, a significantly reduced enzyme consumption and greatly improved enzyme stability can be achieved with the use of this end-column enzyme-immobilized detection interface. The novel flow cell can be further applied in other capillary electrophoresis modes including capillary zone electrophoresis, capillary gel electrophoresis, and micellar electrokinetic chromatography. It is also suitable for some other detectors such as fluorimetric, ultraviolet-visible absorption spectrometric and electrochemical detectors.

Advances in enzyme substrate analysis with capillary electrophoresis

“…This is critical for developing assays that are high throughput [86,87] and can be automated [86] while still consuming small volumes of reagents for the assay. Immobilized enzymes allow for enzyme preparations to be reused [13] and may exhibit higher stability than the enzymes that are free in solution [83,87,88]. Immobilized enzyme reactors enable rapid analysis times through the use of high concentrations of enzymes in small volumes [89], which cannot be achieved with benchtop reactions due to practical reasons, such as limitations in manually handling nanoliter volumes.…”

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…[128][129][130][131][132][133][134][135]. Another suitable approach was the adsorption of either the enzyme [136][137][138][139][140][141] or enzyme previously immobilized to gold nanoparticles [142][143][144] to a capillary which had been coated by polybrene or poly(ethyleneimine). Alginate encapsulation of the enzymes prior to adsorption to the capillary has also been applied [145].…”

“…Using this approach, the determination of enzyme kinetic data and inhibitor constants (IC 50 values) as well as the screening of inhibitors including herbal extracts has been accomplished. However, an IMER has also been developed as a novel enzyme-catalyzed detection system in combination with chemiluminescence as a post-column reactor [144]. Horseradish peroxidase was immobilized to an aldehyde-activated membrane which was subsequently fixed in a flow through cell that also allowed the addition of bromophenol blue as chemiluminescence enhancer.…”

Advance of CE and CEC in phytochemical analysis (2012–2013)

“…The novel flow cell can be further applied in other CE modes including CZE, CGE, and MEKC. It is also suitable for some other detectors such as fluorimetric, UV‐visible absorption spectrometric, and electrochemical detectors .…”