Abstract
A successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50% of its activity, promising thermal stability up to 90°C, reusability for up to three cycles, pH stability over a broad pH of 4-9 and a shelf-life of 44days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness.
Highlights
Electrochemical etching of single crystalline silicon in hydrofluoric acid (HF)-based electrolytic solutions leads to the formation of various pore arrays, known as porous silicon [1]
The presence of enzyme in the porous silicon material was indicated in the Fourier transform IR spectroscopy (FT-IR) spectra by the appearance of a new broad signal at 3364 cm − 1, assigned to acetylcholinesterase’s free hydroxyl stretching mode, whereas no such signal was detected in the highlighted area on the bare porous silicon spectrum
There was an appreciable appearance of new elements, including chlorine, magnesium and sodium, which are attributed to the enzyme skeleton adsorbed on to the porous silicon surface
Summary
Electrochemical etching of single crystalline silicon in hydrofluoric acid (HF)-based electrolytic solutions leads to the formation of various pore arrays, known as porous silicon [1]. The assay solution consisted of 180 μl of 50 mM Tris/HCl buffer, pH 8.0, containing 0.1 M sodium chloride, 0.02 M magnesium chloride, 20 μl of enzyme and 15 μl of 14.9 μM neostigmine methyl sulfate, and pre-incubated for 30 min at 4 ◦C To this reaction mixture 5,5 -dithio-bis(2-nitrobenzoic acid) (0.3 mM, 20 μl) and acetylthiocholine iodide (1.8 mM, 20 μl) were added and incubated for 10 min at 37 ◦C, followed by measurement of the absorbance at 412 nm. Porous silicon samples obtained by varying the etching time from 5 min to 35 min, with constant current density, were tested for immobilization of acetylcholinesterase, and the efficiency of the immobilization was confirmed by studying the retention of activity on the surface of the porous silicon spectrophotometrically. The samples prepared with a current density of 20 mA/cm for 30 min showed optimum results and were used for further measurements
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