[Analytical Biochemistry] Continuous-flow injection microfluidic thrombin assays: The effect of bind
A microfluidic assay for monitoring the inhibition of thrombin peptidase activity was developed. The system, which utilised soluble reagents in continuous-flow injection mode, was configured so as to allow inhibitor titrations via gradient formation. This microfluidic continuous-flow injection titration assay (CFITA) enabled the potency of a set of small-molecule serine peptidase inhibitors (SPIs) to be evaluated. The results, compared to standard microtiter plate (MTP) data, indicated that a microfluidic CFITA provided an efficient and effective method for evaluating compound potency. Crucially, whereas for fast-acting compounds the rank order of potency between the CFITA and MTP methods was preserved, for slow-acting compounds the observed CFITA potencies were significantly lower. These results, in conjunction with data from computer simulations, clearly demonstrated that continuous-flow assays, and perhaps microfluidic assays in general, must take into account binding kinetics when used to assess reaction criteria.
Manoj K. Ramjee, Sital Patel Cyclofluidic Limited, BioPark, Broadwater Road, Welwyn Garden City AL7 3AX, United Kingdom Received 3 March 2017, Revised 24 April 2017, Accepted 25 April 2017, Available online 27 April 2017 https://doi.org/10.1016/j.ab.2017.04.016