High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

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 (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.

Biosensors based on graphene oxide and its biomedical application

“…For label-free enzyme activity assay, GO was employed as matrix to analyze substrates and products of enzyme, detecting molecular weight changes before and after enzyme reactions. Liu et al suggested the highly efficient acetylcholinesterase (AChE) assay platform by utilizing GO and functionalized magnetic carbonaceous (MC) microspheres [136]. The substrate and product were easily separated from AChE immobilized onto the MC microspheres and analyzed by LDI-MS using GO as matrix.…”

“…Liu et al established a high-throughput methodology for inhibitor screening acetylcholinesterase (AChE) using GO-based LDI-MS analysis [136]. By combining the magnetic microspheres for AChE immobilization and GO as matrix, this platform presented simple, cost effective, and efficient performance, which are important characteristics to be utilized for large-scale inhibitor screening.…”

Recent progress in application of carbon nanomaterials in laser desorption/ionization mass spectrometry

“…Notably, GO is easier to obtain than oxidized CNTs because it is actually an intermediate in the synthesis of graphene from graphite by chemical methods. Therefore, a number of papers have reported the application of GO as a matrix or a probe for MALDI or SELDI MS [66,[73][74][75][81][82][83]. Whether graphene or GO shows better performance in LDI MS seems to be associated with the nature of the target analytes [66,[73][74][75].…”

Single-step Trypsin Inhibitor Assay on a Microchannel Array Device Immobilizing Enzymes and Fluorescent Substrates by Inkjet Printing

“…[1][2][3] To achieve these objectives, several enzyme inhibitor assay-related approaches have been reported. [4][5][6][7][8][9][10][11][12][13] In enzyme inhibitor assays, high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection, and capillary electrophoresis (CE) are widely used as analytical methods. 14 The HPLC-UV methods [15][16][17] require a complex operation, long elution time, and a large volume of organic solvents.…”