基于对烟酰胺腺嘌呤二核苷酸光致电化学响应的胆汁酸传感器

doi:10.11944/j.issn.1000-0518.2016.01.150239

摘要/Abstract

摘要：

将硫堇电聚合在多壁碳纳米管修饰电极上,然后在其表面固定3α-羟类固醇脱氢酶制备了胆汁酸传感器。由于该传感器具有光敏和电子受体功能的光电界面,同时偶合了光致电化学效应和酶促反应,能与3α-羟类固醇脱氢酶催化胆汁酸产生的电子供体NADH(烟酰胺腺嘌呤二核苷酸)产生光致电化学反应,通过测量反应产生的光电流实现了对胆汁酸的检测。探讨了胆汁酸传感器对胆汁酸的光致电化学响应机理,研究了多壁碳纳米管、酶固定量和硫堇初始浓度对光电极性能的影响。在优化了电解质溶液pH值、辅酶用量和偏压的测试条件下,该传感器对胆汁酸的测定范围为1.80~40.0 μmol/L,检出限为0.67 μmol/L。应用该传感器对药品胆酸钠片和人尿中胆汁酸进行的检测结果显示,相对标准偏差小于5.64%,加标回收率为96%~104%。该传感器的制备和对胆汁酸的检测不需要除氧,有经济、简便等优点。

关键词: 胆汁酸传感器, 光致电化学, 多壁碳纳米管, 硫堇, 烟酰胺腺嘌呤二核苷酸

Abstract:

Poly(thionine)/multi-walled carbon nanotubes(p-Th/MWNTs) photoelectrode was prepared by electropolymerizing thionine(Th) on the surface of MWNTs-modified ITO electrode. A novel photoelectrochemical bile acid sensor was fabricated by immobilizing 3α-hydroxysteroid dehydrogenase(3α-HSD) on the surface of the photoelectrode. With this photoelectrode as an electron acceptor, and nicotinamide adenine dinucleotide(NADH) from enzymatic reaction as an electron donor, bile acid was determined by the photocurrent from photoelectrochemical effect between p-Th on the photoelectrode and NADH produced in 3α-HSD catalyzed reaction of bile acid. The preparing condition of the photoelectrode, such as MWNTs coating amount, initial concentration of Th and the enzyme loading were optimized. The effects of the electrolyte pH, the concentration of NAD⁺ and the bias voltage on detecting the substrate were investigated at optimized conditions. The measuring range of bile acid sensor is from 1.80 to 40.0 μmol/L with a sensitivity of 5.86 μA/(mol·L), and the detection limit is estimated to be 0.67 μmol/L. The recovery is in the range of 96%~104% for the determination of cholic acid in sodinm cholate tablets and bile acid in human urine.

Key words: bile acid sensor, photoelectrochemistry, multi-walled carbon nanotubes, thionine, nicotinamide adenine dinucleotide

赵常志, 孔燕云, 何艳艳, 张兆霞. 基于对烟酰胺腺嘌呤二核苷酸光致电化学响应的胆汁酸传感器[J]. 应用化学, 2016, 33(1): 116-122.

ZHAO Changzhi, KONG Yanyun, HE Yanyan, ZHANG Zhaoxia. Bile Acid Sensor Based on Photoelectrochemical Response to Nicotinamide Adenine Dinucleotide[J]. Chinese Journal of Applied Chemistry, 2016, 33(1): 116-122.

参考文献

[1]	Ridlon J M,Kang D J,Hylemon P B. Bile Salt Biotransformations by Human Intestinal Bacteria[J]. J Lipid Res,2006,47(2):241-259.
[2]	Pellicoro A,Faber K N. Review article:The Function and Regulation of Proteins Involved in Bile Salt Biosynthesis and Transport[J]. Alim Pharmacol Ther,2007,26(Suppl 2):149-160.
[3]	Zollner G,Trauner M. Mechanisms of Cholestasis[J]. Clin Liver Dis,2008,12(1):1-26.
[4]	Chanudhury S,Chaplin M F. Determination of Bile Acids in Human Faecal Samples Using Supercritical Fluid Extraction and High-Performance Liquid Chromatography[J]. J Chromatogr B,1999,726(1-2):71-78.
[5]	Xiang X,Han Y,Neuvonen M,et al.High Performance Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Bile Acid Concentrations in Human Plasma[J]. J Chromatogr B,2010,878(1):51-60.
[6]	Zhang G H,Cong A R,Xu G B,et al.An Enzymatic Cycling Method for the Determination of Serum Total Bile Acids with Recombinant 3alpha-Hydroxysteroid Dehydrogenase[J]. Biochem Biophys Res Commun,2005,326(1):87-92.
[7]	Campanella L,Tomassetti M,Cordatore M. Application of a New Cholate Liquid Membrane Electrode to the Determination of the Cholic Acids Pool in Human Bile[J]. J Pharm Biomed Anal,1986,4(2):155-162.
[8]	Gültekin A,Karanfil G,Sönmezoğlu S,et al.Development of a Highly Sensitive MIP Based-QCM Nanosensor for Selective Determination of Cholic Acid Level in Body Fluids[J]. Mater Sci Eng C,2014,42:436-442.
[9]	Ueda S,Oda M,Imamura S,Ohnishi M. Kinetic Study of the Enzymatic Cycling Reaction Conducted with 3alpha-Hydroxysteroid Dehydrogenase in the Presence Of Excessive Thio-NAD(+) and NADH[J]. Anal Biochem,2004,332(1):84-89.
[10]	Zhao W W,Xu J J,Chen H Y. Photoelectrochemical DNA Biosensors[J]. Chem Rev,2014,114(15):7421-7441.
[11]	ZHANG Zhaoxia,ZHAO Changzhi. Progress of Photoelectrochemical Analysis and Sensors[J]. Chinese J Anal Chem,2013,41(3):436-444(in Chinese). 张兆霞,赵常志. 光致电化学分析及其传感器的研究进展[J]. 分析化学,2013,41(3):436-444.
[12]	Jafari F,Salimi A,Navaee A. Electrochemical and Photoelectrochemical Sensing of NADH and Ethanol Based on Immobilization of Electrogenerated Chlorpromazine Sulfoxide onto Graphene-CdS Quantum Dot/Ionic Liquid Nanocomposite[J]. J Electroanal Chem,2014,26(3):530-540.
[13]	Zhao C, Zhang Z, Zhao Y, et al. Photoelectrochemical Detection of Glucose by Using an Enzyme-Modified Photoelectrode[J]. Chinese J Chem,2012,30(8):1851-1856.
[14]	Reid G D,Whittaker D J,Day M A,et al.Femtosecond Electron-Transfer Reactions in Mono- and Polynucleotides and in DNA[J]. J Am Chem Soc,2002,124(19):5518-5527.
[15]	Musameh M,Wang J,Merkoci A,et al.Low-Potential Stable NADH Detection at Carbon-Nanotube-Modified Glassy Carbon Electrodes[J]. Electrochem Commun,2002,4(10):743-746.
[16]	LUO Hongxia,SHI Zujin J,LI Nanqiang,et al.Investigation on the Electrochemical and Electrocatalytic Behavior of Chemically Modified Electrode of Single Wall Carbon Nanotube Functionalized with Carboxylic Acid Group[J]. Chem J Chinese Univ,2000,21(9):1372-1374 (in Chinese). 罗红霞,施祖进,李南强,等. 羧基化单层碳纳米管修饰电极的电化学表征及其电催化作用[J]. 高等学校化学学报,2000,21(9):1372-1374.