A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry.
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 microL) 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 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.
de Boer
Arjen R
AR
Vrije Universiteit Amsterdam, Faculty of Sciences, Department of Chemistry and Pharmaceutical Sciences, Section Analytical Chemistry & Applied Spectroscopy, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. ardeboer@gmail.com
Bruyneel
Ben
B
Krabbe
Johannes G
JG
Lingeman
Henk
H
Niessen
Wilfried M A
WM
Irth
Hubertus
H
D002401:Cathepsin B / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004791:Enzyme Inhibitors; D002401:Cathepsin B