The sensitivity of the microfluidic system was determined by measuring calibration curves of four cathepsin B inhibitors. The inhibitors caused negative peaks in the product mass chromatograms by inhibiting cathepsin B and thus the substrate turnover. The measured order of affinities of the four inhibitors is in agreement with the affinities determined in microtiter plate assays and the macro-scale system.
LODs and IC50 values were derived from the calibration measurements and compared with the conventional system. The concentration LODs of the micro-fluidic system were six times higher under similar experimental conditions, while the IC50 values were four times higher. These differences could be caused by less efficient mixing of sample and reagents in the chip compared with the macro-scale system. Despite the more unfavorable detection limit compared to the macro-scale system, the LODs are still in the concentration range for bioactivity screening, while the complete system is miniaturized to a micro-scale level. In addition, the absolute LODs and IC50 values with the chip were four times and six times lower, respectively, which means that less sample is required for screening.
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