为了得到快速、便捷、准确的食品中胆碱含量的检测方法,该文探究用于测定胆碱含量的光敏电极上胆碱氧化酶(choline oxidase,ChOx)固定化方法,采用壳聚糖包埋和戊二醛交联相结合固定化ChOx,通过单因素试验和响应面分析研究壳聚糖浓度、戊二醛浓度以及固定时间对ChOx活力的影响,优化出最佳的ChOx固定化条件,研究结果表明,在壳聚糖质量分数1%、戊二醛质量分数0.55%、固定时间1 h时,固定化ChOx的酶活力最高,可达17.2 U。扫描电镜表明光敏电极表面成功固定ChOx,此外固定于光敏电极的ChOx重复利用10次后酶活力保持在83%,贮存20 d后酶活力保持在86%,具有良好的重复利用性和贮存稳定性。该方法初步探究了测定食品中胆碱的光敏电极研制过程,为食品中胆碱的快速检测奠定一定的理论基础。
In order to get a rapid, convenient and accurate method for the determination of choline in food, this paper explored the immobilization method of enzyme on photosensitive electrode for the determination of choline content. Choline oxidase was immobilized by chitosan embedding and glutaraldehyde crosslinking. The effects of chitosan concentration, glutaraldehyde concentration and immobilization time on the activity of choline oxidase were studied by single factor experiment and response surface analysis. The immobilization conditions of choline oxidase were optimized. When mass fraction of chitosan was 1%, mass fraction of glutaraldehyde was 0.55% and immobilization time was 1 h, the activity of immobilized choline oxidase was the highest, reaching 17.2 U. Scanning electron microscopy showed that choline oxidase was successfully immobilized on the surface of the photosensitive electrode. In addition, the activity of choline oxidase immobilized on the photosensitive electrode remained at 83% after 10 times of reuse and 86% after 20 days of storage. The choline oxidase immobilized on the photosensitive electrode had good reusability and storage stability. The method preliminarily explored the development process of the photosensitive electrode for the determination of choline in food, which lays a theoretical foundation for the rapid determination of choline in food.
[1] ZHAO Y Y, XIONG Y P, CURTIS J M.Measurement of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry:The determination of choline containing compounds in foods[J].Journal of Chromatography A, 2011, 1 218(32):5 470-5 479.
[2] MICHEL V, YUAN Z F, RAMSUBIR S, et al.Choline transport for phospholipid synthesis[J].Experimental Biology & Medicine, 2006, 231(5):490-504.
[3] PATI S, PALMISANO F, QUINTO M, et al.Quantitation of major choline fractions in milk and dietary supplements using a phospholipase D bioreactor coupled to a choline amperometric biosensor[J].Journal of Agricultural & Food Chemistry, 2005, 53(18):6 974-6 979.
[4] BAI Y H, DU Y, XU J J, et al.Choline biosensors based on a bi-electrocatalytic property of MnO2 nanoparticles modified electrodes to H2O2[J].Electrochemistry Communications, 2007, 9(10):2 611-2 616.
[5] SZILAGYI P I, SCHMIDT D E, GREEN J P.Microanalytical determination of acetylcholine, other choline esters, and choline by pyrolysis-gas chromatography[J].Analytical Chemistry, 1968, 40(13):2 009-2 013.
[6] HEFNI M, MCENTYRE C, LEVER M, et al.A simple HPLC method with fluorescence detection for choline quantification in foods[J].Food Analytical Methods, 2015, 8(9):2 401-2 408.
[7] LAIKHTMAN M, ROHRER J S.Determination of choline in infant formula by ion chromatography[J].Journal of Aoac International, 1999, 82(5):1 156-1 162.
[8] ANDEIEUX P, KILINC T, PERRIN C, et al.Simultaneous determination of free carnitine and total choline by liquid chromatography/mass spectrometry in infant formula and health-care products:Single-laboratory validation[J].Journal of Aoac International, 2008, 91(4):777-785.
[9] WEI J F, REN J, LIU J, et al.An eco-friendly, simple, and sensitive fluorescence biosensor for the detection of choline and acetylcholine based on C-dots and the Fenton reaction[J].Biosensors & Bioelectronics, 2014, 52:304-309.
[10] KHAN S M, KHAN T M, WELLS R D, et al.A sensitive enzyme-based colorimetric assay for choline-containing phospholipids[J].Journal of the Science of Food and Agriculture, 1992, 58(3):443-445.
[11] WANG H, HOU W M, LIU Y Z, et al.Janus surface micelles on silica particles:Synthesis and application in enzyme immobilization[J].Macromolecular Rapid Communications, 2020, 42(4):e2000589.
[12] SASSOLAS A, BLUM L J, LECA-BOUVIER B D.Immobilization strategies to develop enzymatic biosensors[J].Biotechnology Advances, 2012, 30(3):489-511.
[13] WANG J W, WANG L P, DI J W, et al.Electrodeposition of gold nanoparticles on indium/tin oxide electrode for fabrication of a disposable hydrogen peroxide biosensor[J].Talanta, 2009, 77(4):1 454-1 459.
[14] ALONSO-LOMILLO M A, DOMíNGUEZ-RENEDO O, MATOS P, et al.Electrochemical determination of levetiracetam by screen-printed based biosensors[J].Bioelectrochemistry, 2009, 74(2):306-309.
[15] NAN C F, ZHANG Y, ZHANG G M, et al.Activation of nylon net and its application to a biosensor for determination of glucose in human serum[J].Enzyme & Microbial Technology, 2009, 44(5):249-253.
[16] ASAV E, YORGANCI E, AKYILMAZ E.An inhibition type amperometric biosensor based on tyrosinase enzyme for fluoride determination[J].Talanta, 2009, 78(2):553-556.
[17] ESSEGHAIER C, BERGAOUI Y, BEN FREDJ H, et al.Impedance spectroscopy on immobilized streptavidin horseradish peroxidase layer for biosensing[J].Sensors & Actuators B Chemical, 2008, 134(1):112-116.
[18] ZHANG Z X, NIWA O, SHIBA S, et al.Electrochemical enzyme biosensor for carnitine detection based on cathodic stripping voltammetry[J].Sensors and Actuators B Chemical, 2020, 321:128473.
[19] LIU Y, YAO L, HE L Z, et al.Electrochemical enzyme biosensor bearing biochar nanoparticle as signal enhancer for bisphenol a detection in water[J].Sensors, 2019, 19(7):1 619.
[20] RAHMAN M A, PARK D S, SHIM Y B.A performance comparison of choline biosensors:Anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer[J].Biosensors & Bioelectronics, 2004, 19(12):1 565-1 571.
[21] HEINZE B, HOVEN N, O′CONNELL T, et al.Recovery of choline oxidase activity by in vitro recombination of individual segments[J].Applied Microbiology & Biotechnology, 2008, 81(2):275-282.
[22] SASSOLAS A, BLUM L J, LECA-BOUVIER B D.Immobilization strategies to develop enzymatic biosensors[J].Biotechnology Advances, 2012, 30(3):489-511.
[23] HUANG M P, WU M, XU Q S, et al.Highly efficient synthesis of fructooligosaccharides by extracellular fructooligosaccharide-producing enzymes and immobilized cells of Aspergillus aculeatus M105 and purification and biochemical characterization of a fructosyltransferase from the fungus[J].Journal of Agricultural and Food Chemistry, 2016, 64(33):6 425-6 432.
[24] PAN C L, HU B, LI W, et al.Novel and efficient method for immobilization and stabilization of β-D-galactosidase by covalent attachment onto magnetic Fe3O4-chitosan nanoparticles[J].Journal of Molecular Catalysis B:Enzymatic, 2009, 61(3):208-215.
[25] SHI H B, YANGY, HUANG J D, et al.Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue modified platinum electrode[J].Talanta, 2006, 70(4):852-858.
