High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS |
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Authors: | Yang Liu Yan Li Junyan Liu Chunhui Deng Xiangmin Zhang |
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Institution: | (1) Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China;(2) Department of Pharmaceutical Analysis, School of Pharmacy, Fudan University, Shanghai, China; |
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Abstract: | 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 (R2 = 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 (R2 = 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. |
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