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 (R² = 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.