Microfluidic Chip–Based Online Screening Coupled to Mass Spectrometry

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.

Detection of Enzyme Inhibitors in Crude Natural Extracts Using Droplet-Based Microfluidics Coupled to HPLC

“…This system also tackles some of the main limitations of current online HPLC bioassays , and gives some important advantages over other biochemical assays performed online in microfluidic chips coupled to LC that do not have droplets. − The first advantage over traditional online methods is the reduction in the amount of sample employed and ∼50-fold less volume in enzyme and substrate solutions. , The second one is that by having a frequency of droplet formation of 8 Hz, the chromatographic peaks are fractionated in 0.125 s intervals, diminishing the dispersion and cross-contamination of the eluting HPLC components. Employing columns with higher efficiency, increasing the droplet frequency, reducing the internal diameter of the tubing and the distance between the UV–vis detector and the droplet formation zone, can further increase the resolution.…”

Microfluidics in Haemostasis: A Review

“…The microchannels of the first micro-reactor provided an environment to carry out the reaction of the enzyme-inhibitor reaction, which was quantified in the presence of a fluorogenic substrate in the second microreactor. This system provided better screening of the compounds when compared to the traditional multi-well plate method [45].…”

Recent lab‐on‐chip developments for novel drug discovery

“…In another study, blood-coagulating enzymes were identified using a microfluidic chip integrated with nano-liquid chromatography (Figure 3(c)) in parallel with a mass spectrometer. 61…”

“…The above model can screen the anti-cancer drug-induced apoptosis in HepG2 cells 60. (c) A CGG microfluidic chip integrated with nanoliquid chromatography (nano-LC) in parallel with a mass spectrometer to analyze the blood coagulating enzymes Thrombin and factor Xa; this chip had three inlets connected to (1) nano-LC, (2) enzyme inlet, (3) substrate, and (4) laser induced florescence (LIF) detector to detect the fluorescent products 61.…”