Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa

Journal of Biomolecular Screening
Janaki Krishnamoorthy IyerR Manjunatha Kini

Abstract

Thrombin and factor Xa (FXa) are critical enzymes of the blood coagulation cascade and are excellent targets of anticoagulant agents. Natural sources present an array of anticoagulants that can be developed as antithrombotic drugs. High-resolution, online screening techniques have been developed for the identification of drug leads from complex mixtures. In this study, we have developed and optimized a microfluidic online screening technique coupled to nano-liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are suitable for the identification of inhibitors from sample amounts as low as sub-microliter volumes.

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Citations

Feb 13, 2016·Biomicrofluidics·P EribolK O Ulgen
Jan 17, 2016·Journal of Biomolecular Screening·Jonathan Wingfield, Ian D Wilson
Feb 23, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Heta Jigar PanchalLeanne F Harris
Feb 18, 2017·Wiley Interdisciplinary Reviews. Systems Biology and Medicine·Nauman KhalidMitsutoshi Nakajima

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Methods Mentioned

BETA
fluorescence enhancement assay
ELISA
chip
Assay

Software Mentioned

GraphPad Prism

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