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

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 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.

Recent developments in the rapid analysis of plants and tracking their bioactive constituents

“…LC column with a flow of 1 lL/min may solve some of these issues. The mixing and post-column reaction coil can be constructed on a chip (de Boer et al 2005a). In addition, the much lower total flow will allow higher enzyme concentrations to be used at reduced costs.…”

Real-time ESI-MS of Enzymatic Conversion: Impact of Organic Solvents and Multiplexing

“…16 Studies with continuous-flow microfluidic assay systems require the systematical research of the impact of organic solvents. 17 Additionally, for several applications in biotechnology and analytical chemistry, the use of organic solvents offers advantages and enables several new applications in industry, these have been discussed in detail in the literature. 12,18 Another promising approach to studying enzymatic reactions is the development of multiplexed assays with two or more different enzymes and their substrates in one single experiment.…”

Microfluidic Chip–Based Online Screening Coupled to Mass Spectrometry

“…We have developed fluorescence-based online screening methods coupled with mass spectrometry in miniaturized formats for bioaffinity profiling of AChBP ligands, cathepsin B inhibitors, and inhibitors of p38α mitogen-activated protein kinase. [15][16][17][18][19] In this study, we describe the development of an analytical platform for profiling of thrombin and FXa inhibitors.…”

“…We have also developed a microfluidic chip-based method for screening of cathepsin B inhibitors and an HRS liquid chromatography-mass spectrometry (LC-MS) method for detection and identification of small-molecule inhibitors of p38α mitogen-activated protein kinase. 15,19 Here, we describe a microfluidic postcolumn method for identifying thrombin and FXa inhibitors from a mixture of compounds. Since both enzymes prefer to hydrolyze an arginyl bond, we have used the same fluorogenic substrate, R22124 (Rhodamine 110, bis-(p-Tosyl-L-Glycyl-L-Prolyl-L-Arginine Amide)), for both enzymes to keep the method simple.…”

“…15 Moreover, when coupled to a sensitive nano-spray electrospray ionization mass spectrometry (ESI-MS), these platforms also allow parallel mass determination and further peptide analysis from minute amounts of starting material that macro-scale systems fail to handle.…”