High throughput enzyme inhibitor screening by functionalized magnetic carbonaceous microspheres and graphene oxide-based MALDI-TOF-MS.

In this work, a high throughput methodology for screening enzyme inhibitors has been demonstrated by combining enzyme immobilized magnetic carbonaceous microspheres and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection (LOD) for ACh was 0.25 fmol/μL, and excellent linearity (R(2)=0.9998) was maintained over the range of 0.5 and 250 fmol/μL. Choline was quantified over the range of 0.05 and 15 pmol/μL, also with excellent linearity (R(2)=0.9994) and low LOD (0.15 fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together, eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.