功能化金纳米修饰电极自组装及其在固定化酶生物传感器中应用

功能化金纳米修饰电极是化学修饰电极,不仅具有特定功能团性能,且能提供电化学信号,可用于与待测物的电子传递,电子捕获,判定某化学反应是否发生.功能化金纳米修饰电极检测待测物,具有灵敏度高、检测限低及长久使用的优势.我们就功能化金纳米修饰电极自组装制备、电化学表征方法及其在固定化酶生物传感器方面的应用研究,进行综述报道.     

硫辛酸在金电极上自组装成膜动力学研究

自组装单层膜 (ＳＡＭ)应用广泛[1～ 3] 。但对于硫辛酸 (ＴＡ)自组装成膜动力学过程的研究尚未见报道。本研究利用石英晶体微天平 (ＱＣＭ)对ＴＡ自组装成膜过程进行在线监测 ,研究成膜动力学特征 ,并探讨浓度、温度对成膜过程的影响 ,以及在不同ｐＨ值下 ,ＳＡＭ的稳定性 ,推算了不同温度下的速率常数及成膜的活化能。1　实验部分1 1　基本原理通过监测成膜过程中石英晶体微天平 (ＱＣＭ)的振荡频率的变化 ,据Ｓａｕｅｒｂｒｅｙ方程 ,可推知在石英谐振器 (ＱＣＭ)的金电极表面 ,ＴＡ的吸附质量Γ(Γ =Δｍ Ａ ,ｇ ｃｍ2 )与ＱＣＭ的频率…     

巯基修饰单链DNA在纳米金薄膜电极上自组装、杂交和取向的电化学交流阻抗谱

利用电化学交流阻抗技术对SH-ssDNA在纳米金薄膜电极表面的自组装、杂交和取向进行了系统表征。 探讨了SH-ssDNA的组装时间、浓度和链长对其自组装的影响,自组装15 h时电荷传递电阻Rct最大,表面覆盖率最高;研究了SH-ssDNA的浓度、链长以及与互补DNA的杂交方式对杂交反应的影响。 结果发现,随着单链浓度的增加,杂交后R_ct的变化值逐渐降低,当SH-ssDNA为5 μmol/L时Rct值比杂交前增加了16%。 通过对阻抗谱数据模拟和分析,表明SH-ssDNA以垂直竖立取向在金电极表面形成均匀致密单分子层,杂交效率与SH-ssDNA的覆盖率密切相关。     

层层自组装纳米金与乙酰胆碱酯酶电化学生物传感器检测有机磷农药

采用层层自组装技术制备了快速检测有机磷农药的生物传感器,利用带正电荷的高分子聚电解质聚二烯丙基二甲基氯化铵(PDDA)将乙酰胆碱酯酶(AChE)和金纳米粒子(AuNPs)通过静电力逐层固定到玻碳电极(GCE)表面,并采用交流阻抗和微分脉冲伏安法研究了此生物传感器的电化学行为。由于金纳米粒子优异的电催化性能和良好的生物相容性,使固定化的乙酰胆碱酯酶对其底物具有更高的亲和力和更快的响应速度。实验结果表明:修饰金纳米粒子后,传感器的氧化电流明显增大,在4.6×10-5～5.3×10-3mol/L范围内,固定化酶的抑制率与甲基对硫磷浓度的对数成正比,检出限为7.6×10-6mol/L。该生物传感器具有制备方法简便、成本低、灵敏度高等优点,已成功用于蔬菜样品中甲基对硫磷含量的测定。     

金表面自组装化学发光免疫传感器

用Ｎ－乙酰半胱氨酸金表面自组装技术及ＥＤＣ、ＮＨＳ偶联剂将兔ＩｇＧ固定于金表面制成免疫传感器探头，用碱性磷酸酯酶标记山羊抗兔ＩｇＧ加入待测的含有山羊抗兔ＩｇＧ的样品中，采用竞争头测定山羊抗兔ＩｇＧ含量，标记的碱性磷酸酯酶可催化底物ＡＭＰＰＤ产生化学发光，其发光强度与样品浓度成反比。测得山羊抗兔ＩｇＧ的最低检出限为０．８ｍｇ／Ｌ，线性测定范围为０．８－４０ｍｇ／Ｌ。     

多巴胺修饰自组装金电极作为生物传感器测定核酸

采用衍生化接枝法制备了多巴胺(DA)-半胱氨(Cys)自组装修饰金电极(DA-Cys-SAM),研究了其电化学性质。在pH 7.5的Tris-HCl底液中,采用循环伏安法测定其Epa=218 mV,Epc=120 mV,ΔE=98 mV,ipa/ipc≈1,电极反应准可逆。采用示差脉冲法(DPV)研究发现DA氧化峰电流随DNA质量浓度提高而显著变大且峰电位不变,峰电流的增加值与DNA质量浓度在1×10-7~1.2×10-5g/mL范围内成正比,检出限达5×10-8g/mL,相对标准偏差为3.2%(n=8,5×10-7g/mL DNA)。该电极对DNA测定具有特异性,对实际样品进行了测定。     

基于HDT自组装金纳米粒子修饰电极的微囊藻毒素-LR电化学免疫传感器研究

构建了一种新型的基于金纳米粒子(Au NPs)修饰金电极的微囊藻毒素-亮氨酸-精氨酸(MCLR)电化学免疫传感器。采用柠檬酸钠还原法制备了Au NPs溶胶,分别用透射电子显微镜和紫外-可见吸收光谱对其进行表征。将Au NPs组装到1,6-己二硫醇(HDT)自组装单分子层修饰的金电极表面,再将MCLR抗体(anti-MCLR)固定于该修饰电极上,利用扫描探针显微镜法、循环伏安法和电化学交流阻抗法(EIS)表征了自制化学修饰电极表面的形貌特征和电化学免疫传感器的电化学特征。通过辣根过氧化物酶标记的MCLR(MCLR-HRP)与MCLR竞争结合抗体,建立了检测MCLR的差分脉冲伏安法(DPV)。在最佳实验条件下,用DPV对MCLR检测的线性范围为0.01~25μg/L,检出限为0.005μg/L。对构建的免疫传感器的重现性、稳定性和选择性进行了考察。该方法对实际水样中MCLR的加标回收率为100%~102%,测定结果与高效液相色谱法的测定结果一致。     

层层自组装制备基于多壁碳纳米管的胆碱生物传感器

以经混酸处理的多壁碳纳米管(MWCNTs)修饰铂(Pt)电极,在此基础上固定(PAA/PVS)3复合膜,采用层层自组装技术将高分子聚电解质PDDA与胆碱氧化酶交替组装在已修饰的电极上,构建了电流型胆碱生物传感器。实验结果表明,MWCNTs的引入使电极对H2O2的催化电流明显增大,制成的酶电极可以有效控制酶量的使用,酶膜组装层数为8时最优,对胆碱的线性响应范围为5×10-7～1×10-4mol/L;灵敏度为12.53μA/mmol;响应时间为7.60s;检出限为2×10-7mol/L(S/N=3)。传感器的抗干扰能力强,稳定性好,30d时的响应电流值仍保持最初的89.5%。3次平行实验的RSD为3.64%。     

一种基于巯基化聚丙烯胺自组装膜的免疫传感器固定方法

提出了一种基于巯基自组装的新型蛋白质固定化方法。应用偶联试剂碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)使聚丙烯胺盐酸盐(PAH)与3-巯基丙酸(MPA)偶合，在石英晶振表面自组装，形成一带多氨基的巯基自组装膜。应用该方法成功地固定了羊抗人IgM抗体，并用于人血清免疫球蛋白M(IgM)的测定。详细考察了自组装条件、抗体包被和免疫反应的主要实验条件以及传感器的响应性能。与MPA法比较，应用该方法可在传感器表面固定更多的抗体分子，传感器的响应灵敏度亦更好。在优化的实验条件下，反应的线性范围0．66-26．4mg／L；回归方程Y=92．44 12．40X；相关系数r=0．9920。     

金-硫键自组装生物分子膜

应用硫原子与金表面的强烈相互作用,可获得具有特定功能的生物分子自组装膜,在生物学、药理学及仿生学方面有重要的研究意义。本文着重介绍了一些相关的生物分子自组装体系,包括葡萄糖氧化酶、类脂、核酸传感器等在金基底表面的自组装。     

Laccase Biosensor on Monolayer‐Modified Gold Electrode

Laccase enzymes from two different sources, namely, tree laccase from Rhus vernicifera and fungal laccase from Coriolus hirsutus were used for the development of biosensor for catechol. Laccase was immobilized onto the amine terminated thiol monolayers on gold surface by glutaraldehyde coupling. From the different thiol monolayers investigated, cystamine was found to be optimal with respect to sensitivity, stability, reproducibility, and other electrochemical properties of the enzyme electrode. Linear calibration in the range between 1 and 400 μM for catechol was obtained for fungal laccase covalently coupled on the electrode surface. The kinetic parameters determined using the Lineweaver‐Burk and Eadie‐Hofstee plots were K_m=0.65 mM and V_max=24.5 μA for fungal laccase compared to K_m=5.4 mM and V_max=6.6 μA for tree laccase on cystamine monolayer. The electrode showed good stability for 1 month without loosing appreciable activity when stored dry in a refrigerator at ?20 °C.     

Third Generation Horseradish Peroxidase Biosensor Based on Self-assembling Carbon Nanotubes to Gold Electrode Surface

Carbon nanotube (CNT) is a novel carbon material discovered by Iijima1. It had beenfound to have the ability to promote electron transfer reactions when it was used tofabricate electrodes for the oxidation of biomolecules2-4. Just recently, the interest indemonstrating CNT for biosensing applications is now emerging5-10. However, most ofthese biosensors were based on CNT paste electrode or CNT modified glassy carbonelectrode by casting technology. Here, we reported another assembly…     

Enhanced Electrocatalytic Oxidation of Paracetamol at DNA Modified Gold Electrode

Cysteine monolayer has been assembled onto bare gold electrode (SAM/Au), and subsequently deoxyribonucleic acid (DNA) has been successfully immobilized at the SAM/Au electrode. The thus modified electrode is assigned DNA/SAM/Au. Modification steps of the electrode were followed electrochemically using K₄[Fe(CN₆)] electrochemical marker. Also, the build‐up of the modified electrode composition is followed using EDX and the crystallographic orientation is inspected using XRD. The electrochemical behavior of paracetamol (PC) at DNA/SAM/Au electrode is investigated. Interestingly, the sluggish irreversible behavior of PC at the bare gold electrode is converted to a quasi‐reversible one at DNA/SAM/Au electrode pointing to some interaction between the immobilized DNA and PC. The enhanced electrochemical behavior of PC at modified DNA/SAM/Au electrode is successfully used for a sensitive electrochemical determination of PC. Square wave voltammetry (SWV) was used for this purpose. The concentration of PC was in linear relation with the peak current at the optimum conditions within the range 10.0–110.0 μg mL^?1 with correlation coefficient (R²) of 0.998. Also, the standard deviation (SD) and relative standard deviation (RSD) were calculated and found to be 0.817 and 1.52, respectively, indicating the significance of the present method.     

A Penicillamine Biosensor Based on Tyrosinase Immobilized on Nano‐Au/ PAMAM Dendrimer Modified Gold Electrode

Gold electrodes were modified with submonolayers of 3‐mercaptopropionic acid and further reacted with poly(amidoamine) (PAMAM) dendrimers to obtain thin films. The high affinity of PAMAM dendrimer for nano‐Au with its amine groups was used to realize the role of nano‐Au as an intermediator to immobilize the enzyme of tyrosinase. The characterization of the modified electrode was investigated by cyclic voltammetry, electrochemical impedance spectroscopy and atomic force microscopy (AFM). Tyrosinase can catalyze the oxidation of catechol to o‐benzoquinone. When penicillamine was added to the solution, it reacted with o‐benzoquinone to form the corresponding thioquinone derivatives, which resulted in decrease of the reduction current of o‐benzoquinone. Based on this, a new electrochemical sensor for determination of penicillamine has been developed.     

Nafion—四硫富瓦烯修饰蜡浸石墨电极的葡萄糖传感器

本文用四硫富瓦烯（ＴＴＦ）作为酶与电极之间的电子传递体，通过牛血清蛋白和戊二醛交联剂，把葡萄糖化氧化酶固定在Ｎａｆｉｏｎ－ＴＴＦ修饰石墨电极上，最后在电极修饰一层Ｎａｆｉｏｎ膜，制备成葡萄糖传感器。Ｎａｆｉｏｎ膜不仅能防止四硫富瓦燃流失，而且能氢抗坏血酸，尿酸等电活生物质阻挡在电极外，防止其干扰，同时具有防污性能。通过实验表明ＴＴＦ＾＋，ＴＴＴ＾２＾＋都能够氧化葡萄糖氧化酶中的辅酶（ＦＡＤＨ２）。     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

Immobilization of Hemoglobin on the Gold Colloid Modified Pretreated Glassy Carbon Electrode for Preparing a Novel Hydrogen Peroxide Biosensor

A novel hydrogen peroxide (H₂O₂) biosensor was developed by immobilizing hemoglobin on the gold colloid modified electrochemical pretreated glassy carbon electrode (PGCE) via the bridging of an ethylenediamine monolayer. This biosensor was characterized by UV-vis reflection spectroscopy (UV-vis), electrochemical impendence spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized Hb exhibited excellent electrocatalytic activity for hydrogen peroxide. The Michaelis–Menten constant (K _m) was 3.6 mM. The currents were proportional to the H₂O₂ concentration from 2.6 × 10⁻⁷ to 7.0 × 10⁻³ M, and the detection limit was as low as 1.0 × 10⁻⁷ M (S/N = 3).     

Nafion－Ferrocene修饰的次黄嘌呤传感器

本文用Ｆｅｒｒｏｃｅｎｅ（ＦＣ）作为黄嘌呤氧化酶与电极之间的电子传递体，通过牛血清白蛋白和戊二醛交联剂，把黄嘌呤氧化酶固定在Ｎａｆｉｏｎ－Ｆｃ修饰电极表面，制备成次黄嘌呤传感器。该传感器的线性范围为５．０×１０－５—７．５×１０－４ｍｏｌ／Ｌ，响应时间小于７ｏｓ。     

甲苯胺蓝修饰石墨电极为基体的乙醇脱氨酶生物传感器

报道了电流型乙醇生物传感器。该传感器以甲苯胺蓝键合修饰浸蜡石墨电极为基 体电极，将醇脱氢酶、烟酰胺腺嘌呤二核苷酸（ＮＡＤ＋）同时固定在蚕丝蛋白膜上，成为无试剂 的醇传感器。在 ｐＨ ８． ５的 Ｔｒｉｓ－ＨＣｌ介质中，该传感器的响应电流与乙醇浓度在 ５． ０× １０－５～ １．１０×１０－３ｍｏｌ／Ｌ范围内有良好的线性关系。响应时间为２０ｓ。本文讨论了影响传感器响应 的各种因素。用该传感器测定了啤酒中乙醇的含量，结果与气相色谱法一致。     

Ultrasensitive electrochemical immunosensor of carcinoembryonic antigen based on gold-label silver-stain signal amplification

A novel gold-label silver-stain electrochemical immunosensor based on polythionine-gold nanoparticles (PTh-Au NPs) modified glassy carbon electrode (GCE) as a platform and secondary antibody labeled Au NPs (Ab₂-Au NPs) as immumoprobe for carcinoembryonic antigen (CEA) detection. The sandwich-type biosensor adopted anodic stripping voltammetry to detect silver stripping signal when the Ab₂-Au NPs of the formed immunocomplexes were stained with silver.