S

Fig. 5.9 Design of the chip-based enzyme ESI-MS assay. MS instrument: Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). I: Sample components/inhibitors injected by flow injection or eluting from capillary HPLC column. E: Infusion pump delivering the enzyme cathepsin B. S: infusion pump delivering the substrate Z-FR-AMC. Micro-chip design: Vrije Universiteit Amsterdam. Micro-chip production: Micronit Microfluidics BV (Enschede, The Netherlands).

Fig. 5.9 Design of the chip-based enzyme ESI-MS assay. MS instrument: Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). I: Sample components/inhibitors injected by flow injection or eluting from capillary HPLC column. E: Infusion pump delivering the enzyme cathepsin B. S: infusion pump delivering the substrate Z-FR-AMC. Micro-chip design: Vrije Universiteit Amsterdam. Micro-chip production: Micronit Microfluidics BV (Enschede, The Netherlands).

much larger, like glass, silicon, plastic, quartz, and fused silica. The design of the chip (see Fig. 5.9) is mainly dictated by the flow rates compatible with electro-spray MS. In order to achieve proper mixing on the microchip, flow rates of 2 mL min-1 for capillary LC and 1 mL min-1 for both enzyme and substrate solutions were chosen. The choice of a total flow rate in the chip of 4 mL min-1 resulted in reaction times of 32 s and 36 s in the two reactors, respectively. In comparison with the macro-scale system, the flow rates of both enzyme and substrate were reduced by a factor of 25. Employing the optimum concentrations of the macro-scale system did not result in sufficient product formation for screening. For that reason, the enzyme concentration was increased 5-fold, having an overall decrease in enzyme and substrate consumption of 5x and 25 x, respectively.

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