Ultrasensitive analysis of glucose in serum by capillary electrophoresis with LIF detection in combination with signal amplification strategies and on‐column enzymatic assay

Abstract

A highly specific and sensitive method for glucose quantification in human serum samples based on on-column enzymatic assay is described. In this method, the head of the capillary was used as a nanoliter-microreactor, the diluted samples spiked with a novel fluorogenic reagent named 2-[6-(4'-amino) phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), and the mixed enzyme solutions of glucose oxidase (GOx) and horseradish peroxidase (HRP), were individually injected into the capillary. Hydrogen peroxide (H2 O2 ) generated in situ by catalytic reaction between GOx and glucose, activates APF in the presence of HRP to form a highly fluorescent product, which was electrophoretically separated from the unreacted APF and detected by the LIF detector. The proposed method allowed the determination of glucose down to 10 nM in real samples, with RSD values lower than 3.5%, which also has the potential for measurements of multicomponents in many other systems including measurement of α-glucosidase activity and screening for its inhibitors.

Capillary Electrophoresis: Trends and Recent Advances

“…Guan et al developed a CE method for the precise and accurate determination of glucose levels in blood. 165 The first part of the capillary is used as a microreactor for the coupled enzymatic assay of glucose oxidase and horseradish peroxidase. These enzymes are separately injected and govern two sequential reactions, where through peroxide formation a fluorogenic reagent is converted into fluorescein.…”

Advances in Capillary Electrophoretically Mediated Microanalysis for On‐line Enzymatic and Derivatization Reactions

“…It is possible to apply the sandwich mode to improve the quality of mixing . This method needs a double injection of reactants, which improves the mixing efficiency and overlapping area as well as the distribution throughout the capillary.…”

Derivatisation for separation and detection in capillary electrophoresis (2015–2017)

“…There were 12 papers on in‐capillary derivatisation . The derivatisation was performed by adding the reagents to the separation buffer or by zone injections of reagents and sample.…”

“…The addition of γ‐cyclodextrin to the separation buffer enabled baseline separation of the derivatised amino acids prior to detection at 238 nm. Guan and Zhou performed enzyme‐mediated fluorescence amplification of glucose via APF labelling . AFP was added to the sample and the sample was sandwiched between the enzyme solutions.…”

“…This is often the case for fluorescence detection where native fluorescence is rather uncommon and only the labelled species provide a strong signal. There were 24 articles on LIF , 16 on UV , 3 on electrochemical detection , and 4 on MS . In MS detection, derivatisation was used to improve the separation (see Section ) and to enable MS detection in favourable positive ion ESI mode .…”