A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry

A.R. de Boer, B. Bruyneel, J.G. Krabbe, H. Lingeman, W.M.A. Niessen, H. Irth

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 μL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 μL min
Original languageEnglish
Pages (from-to)1286-92
JournalLab on a Chip
Volume5
Issue number11
DOIs
Publication statusPublished - 2005

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