基于多层酶/纳米金固定甲胎蛋白免疫传感器的研究

>利用自组装技术和静电吸附作用, 将甲胎蛋白抗体(anti-AFP)固定在多层辣根过氧化物酶/纳米金及L-半胱胺酸修饰的金电极表面, 制备出用于检测甲胎蛋白抗原(AFP)的无试剂型免疫传感器. 通过交流阻抗技术、循环伏安法和计时电流法考察了电极的电化学特性, 并对该免疫传感器的作用机理及性能进行了详细的研究. 用计时电流法测得AFP的线性范围为1.0～10.0和10～200 ng•mL^－1, 检出限为0.5 ng•mL^－1. 实验结果表明, 该方法提高了抗体的固定量, 增强了传感器的灵敏度和稳定性, 且该传感器响应迅速、选择性好, 血清中常见抗原不干扰测定. 将其用于临床血清检验, 与放射免疫测定法(RIA)的符合率为86.7%.     

基于HGB/PTh/AuNCs无标记癌胚抗原免疫传感器的研究

本文研制了一种基于中空石墨烯球(HGB)/聚硫堇/金纳米笼的无标记型癌胚抗原传感器。将合成的中空石墨烯球修饰于玻碳电极(GCE)表面,在其上电聚合硫堇形成多孔聚硫堇(PTh)复合膜后吸附固定金纳米笼,然后将癌胚抗体(anti-CEA)固定到电极表面,制得无标记型癌胚抗原(CEA)免疫传感器。当抗体与抗原发生免疫反应时,形成的复合物阻碍了电子传递,从而降低聚硫堇的电催化活性。通过示差脉冲伏安法(DPV)检测聚硫堇的响应电流信号的降低,实现对CEA的无标记检测。在最优的实验条件下,检测癌胚抗原的线性范围为0.5~80 ng·mL-1,线性相关系数为0.9932,检测限为0.17 ng·mL-1。该免疫传感器可用于临床上CEA的检测。在血清样品中进行了CEA的检测,结果令人满意。     

基于纳米金/硫堇层层自组装的新型电流型酶-癌胚抗原免疫传感器

将Nafion 膜固定在金电极(Au)表面, 通过静电吸附和共价键合作用将硫堇(Thi)和纳米金颗粒(nano-Au)层层自组装到Nafion膜修饰的金电极表面. 再通过形成的纳米金单层吸附癌胚抗体(anti-CEA), 最后用辣根过氧化物酶(HRP)代替牛血清白蛋白(BSA)封闭电极上的非特异性吸附位点, 并同时起到放大响应电流信号的作用, 从而制得高灵敏、高稳定电流型酶-癌胚抗原(CEA)免疫传感器. 通过循环伏安和交流阻抗考察了电极表面的电化学特性, 并对该免疫传感器的性能进行了详细的研究. 该传感器对CEA检测的线性范围为2.5~80.0 ng/mL, 检测限为0.90 ng/mL.     

可检测有机磷农药残留的丝网印刷酶电极

制备了用于有机磷农药检测的电化学生物酶电极, 并从工作电极上修饰物的选择、测定电压的确定、生物酶电极预活化时间的选定、测定时间的选定、温度的影响、pH值的影响、底物浓度的影响、固定方法的选择、固定酶时温度和时间的影响等多方面对丝网印刷的生物酶电极进行了较为全面的研究, 应用经过优化的生物酶电极对有机磷农药乙基对氧磷进行了检测. 结果表明: 抑制率与乙基对氧磷浓度的常用对数在1.00×10^－7～1.00×10^－5 g•mL^－1范围内成线性关系, 线性回归方程为A/%＝267.2＋37.20lg[c/(g•mL^－1)], 相关系数r＝0.9882. 当信噪比为3时, 检出限为2.10 ng•mL^－1, 低于国家标准所要求的最低残留量.     

基于纳米金与碳纳米管-纳米铂-壳聚糖纳米复合物固定癌胚抗原免疫传感器的研究

采用纳米金(nano-Au)、多壁碳纳米管-纳米铂-壳聚糖的纳米复合物(MWNT-Pt-CS)及电子媒介体硫堇(Th)固载抗体制得高灵敏癌胚抗原免疫传感器．首先, 于壳聚糖溶液中用NaBH4还原H2PtCl6, 并将多壁碳纳米管分散于其中制得碳纳米管-纳米铂-壳聚糖纳米复合物, 并将其滴涂在玻碳电极上成膜; 然后, 吸附电子媒介体硫堇制得硫堇/碳纳米管-纳米铂-壳聚糖(Th/MWNT-Pt-CS)修饰电极．利用壳聚糖和硫堇分子中大量的氨基固定纳米金并吸附癌胚抗体(anti-CEA); 最后, 用辣根过氧化物酶(HRP)封闭活性位点从而制得高灵敏电流型免疫传感器．在优化的实验条件下, 该传感器响应的峰电流值与癌胚抗原(carcinoembryonic antigen)浓度在0.5～10和10～120 ng/mL的范围内保持良好的线性关系, 检测限为0.2 ng/mL．     

癌胚抗原快速无分离电化学免疫检测

通过同时固定硫堇和HRP标记癌胚抗原(CEA)抗体于电化学预处理的玻碳电极表面,制备了新型无需分离的CEA快速电化学检测免疫传感器.CEA测定的两段线性范围为0.5～3.0和3.0～167 ng/mL,检测下限为0.1 ng/mL.该传感器具有良好的准确性、精密度、制备重复性和稳定性.该方法缩短了分析时间,降低了测定成本,适用于临床CEA的快速测定.     

基于聚2,6-二氨基吡啶膜及纳米金修饰的癌胚抗原免疫传感器研究

在玻碳电极表面电聚合2,6-二氨基吡啶(pPA), 利用硫堇(Thi)、纳米金(nano-Au)固载癌胚抗体, 制得稳定性好、灵敏度较高、线性范围宽的电流型免疫传感器. 通过循环伏安法考察了该免疫传感器的电化学特性, 在优化的实验条件下, 该免疫传感器的峰电流随着检测溶液中癌胚抗原(CEA)浓度的增大而减小, 并在0.5～20和20～160 ng/mL CEA范围内呈现出良好的线性关系, 检测下限为0.2 ng/mL. 该免疫传感器具有制作简单、重现性好、线性范围宽等优点, 可用于临床上对CEA的检测.     

基于脂质体信号增强癌胚抗原电化学免疫传感器的研究

研究了在玻碳电极利用巯基乙胺固定纳米金、然后纳米金固载癌胚抗体(Ab1),采用脂质体包裹电子媒介体硫堇,脂质体周围联接标记辣根过氧化物酶(HRP)的癌胚抗体(Ab2)对其传感器进行信号放大,通过循环伏安法考察了该免疫传感器的电化学特性,在优化的实验条件下,该免疫传感器的峰电流随着检测溶液中癌胚抗原(CEA)浓度的增大而增大,并在0.05～200 ng/mL CEA范围内呈现线性关系,回归方程为:Δi=0.20+0.24ρ(ng/mL);检测限为:18pg/mL(R=0.9947)。该免疫传感器可用于临床上对CEA的检测。     

基于聚硫堇和纳米银固定酶的葡萄糖生物传感器

用循环伏安法将电子媒介体硫堇电聚合在玻碳电极表面上,使其表面形成均匀的带负电的聚硫堇膜,通过静电吸附作用吸附表面带正电荷的纳米银溶胶,接着通过静电吸附带负电的葡萄糖氧化酶,最后用聚硫堇包埋电极,从而制得性能优良的葡萄糖氧化酶(GOD)生物传感器。实验发现传感器氧化峰电流与葡萄糖的浓度在1.0×10-8~5.0×10-6mol/L(r=0.9963)范围内呈良好线性关系,检出限为5.0×10-9mol/L(S/N=3)。     

基于二氧化硅-硫堇纳米复合物构建高灵敏癌胚抗原电流型免疫传感器

利用电沉积纳米金(AuNPs)修饰玻碳电极(GCE)表面并通过AuNPs固定癌胚抗原(CEA)的捕获抗体(Ab1),以牛血清白蛋白(BSA)封闭非特异性吸附位点;以γ-(2,3环氧丙氧)丙基三甲氧基硅烷(GPMS)作交联剂,将单分散的SiO_2纳米粒子与电子媒介体硫堇(Thi)结合成SiO_2-Thi纳米复合物,偶联辣根过氧化物酶(HRP)标记的CEA二抗(HRP-Ab2)作为电化学免疫检测信号,构建了具有信号放大效应的电流型免疫传感器并用于CEA的高灵敏检测。在CEA存在下,进行电化学酶联夹心免疫反应。在含有H2O2的磷酸盐缓冲溶液(PBS)中,标记在SiO_2-Thi纳米复合物上的HRP能催化H_2O_2氧化电子媒介体Thi,产生增强的还原峰电流,从而提高检测CEA的峰电流响应信号,进而实现对CEA的高灵敏电化学酶联夹心免疫分析。在最优实验条件下,该免疫传感器的差分脉冲伏安(DPV)还原峰电流与CEA质量浓度的对数在0.01~20ng/mL范围内呈良好的线性关系,检出限为3pg/mL(S/N=3)。该传感器对血清样品进行加标回收实验,回收率为97.3%~105.7%,可初步用于临床对CEA的检测。     

Ultrasensitive amperometric immunosensor for the determination of carcinoembryonic antigen based on a porous chitosan and gold nanoparticles functionalized interface

An immunosensor has been fabricated for direct amperometric determination of carcinoembryonic antigen. It is based on a biocompatible composite film composed of porous chitosan (pChit) and gold nanoparticles (GNPs). Firstly, a pChit film was formed on a glassy carbon electrode by means of electrodeposition. Then, thionine as a redox probe was immobilized on the pChit film modified electrode using glutaraldehyde as a cross-linker. Finally, GNPs were adsorbed on the electrode surface to assemble carcinoembryonic antibody (anti-CEA). The surface morphology of the pChit films was studied by means of a scanning electron microscope. The immunosensor was further characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical behaviors and factors influencing the performance of the resulting immunosensors were studied in detail. Results showed that the pChit films can enhance the surface coverage of antibodies and improve the sensitivity of the immunosensor. Under optimal conditions, the immunosensor was highly sensitive to CEA with a detection limit of 0.08 ng·mL^?1 at three times the background noise and linear ranges of 0.2～10.0 ng·mL^?1 and 10.0～160 ng·mL^?1. Moreover, the immunosensor exhibited high selectivity, good reproducibility and stability.     

四环素类抗生素-钨酸钠-乙基紫体系的共振瑞利散射光谱及其分析应用

在pH为9.0的Clark-Lubs缓冲溶液中, 强力霉素、土霉素、四环素和金霉素等四环素类抗生素与钨酸钠反应形成1∶1的阴离子螯合物, 它仅能引起吸收光谱的变化, 不能引起共振瑞利散射(RRS)的增强, 但是当该螯合物进一步与乙基紫反应形成三元离子缔合物时, RRS显著增强并产生新的RRS光谱, 它们具有相似的光谱特征, 最大RRS波长均位于328 nm处. 4种抗生素的线性范围和检出限分别为0.047～4.8 μg•mL^－1和14.1 ng•mL^－1(强力霉素); 0.078～5.0 μg•mL^－1和23.5 ng•mL^－1(土霉素); 0.081～5.7 μg•mL^－1和24.4 ng•mL^－1(四环素); 0.122～7.7 μg•mL^－1和36.6 ng•mL^－1(金霉素). 考察了三元离子缔合配合物的组成, 讨论了配合物的结构和反应机理, 并发展了一种高灵敏、简便快速测定四环素类抗生素的新方法.     

A novel immunosensor for carcinoembryonic antigen based on poly(diallyldimethylammonium chloride) protected prussian blue nanoparticles and double-layer nanometer-sized gold particles

A highly sensitive amperometric immunosensor has been developed for the detection of carcinoembryonic antigen (CEA). It is based on (a) Prussian Blue nanoparticles coated with poly(diallyldimethylammonium chloride) (P-PB) and (b) double-layer gold nanocrystals. The sensor was obtained by first electrodepositing porous gold nanocrystals on the glassy carbon electrode (GCE), and then by modifying the electrode with the coated P-PB. Subsequently, colloidal gold nanoparticles (nano-Au) were adsorbed onto the GCE by electrostatic interactions between the negatively charged nano-Au and the positively charged P-PB to immobilize CEA antibodies. Finally, bovine serum albumin was employed to block possible remaining active sites and to prevent the non-specific adsorption on the nano-Au. This immunosensor was characterized by cyclic voltammetry and scanning electron microscopy. The working range was adjusted to two concentration ranges, viz. from 0.5 to 10 ng.mL^?1, and from 10 to 120 ng.mL^?1 of CEA, with a detection limit of 0.2 ng.mL^?1 at three times the background noise.     

Electrochemical immunosensor for the carcinoembryonic antigen based on a nanocomposite consisting of reduced graphene oxide,gold nanoparticles and poly(indole-6-carboxylic acid)

We describe a label-free electrochemical immunosensor for the carcinoembryonic antigen (CEA). It is based on a nanocomposite consisting of electrochemically reduced graphene oxide, gold nanoparticles (AuNPs), and poly(indole-6-carboxylic acid). Coupled to nanoparticle-amplification techniques and modified with ionic liquid (IL), this immunoassay shows high sensitivity and good selectivity for CEA. At the best working voltage of 0.95 V (vs. Ag/AgCl), the lower detection limit is 0.02 ng·mL^?1, and the response to CEA is linear in the range from 0.02 to 90 ng·mL^?1. The method was applied to the determination of CEA in spiked serum samples and gave recoveries in the range from 98.5 % to 102 %.

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Graphical abstract A label-free electrochemical immunosensor was fabricated for the carcinoembryonic antigen (CEA) with a detection limit of 0.02 ng·mL^?1. It is based on a nanocomposite consisting of electrochemically reduced graphene oxide (erGO), gold nanoparticles (Au NP), and poly(indole-6-carboxylic acid) (PICA).

     

A Novel Electrochemical Immunosensor for Prostate-Specific Antigen Based on Noncovalent Nanocomposite of Ferrocene Monocarboxylic Acid with Graphene Oxide

A novel electrochemical immunosensor was developed for the determination of prostate-specific antigen based on immobilization of appropriate antibodies on gold nanoparticles and a poly-(2,6-pyridinediamine) modified electrode. The nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide was prepared by a π-π stacking interaction and was used as the electrochemical probe. A sandwich-type complex immunoassay was applied with polyclonal prostate-specific antigen antibodies labeled with the nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide. In order to improve the sensitivity, a potentiostatic method was used to reduce graphene oxide. Cyclic voltammetry and differential pulse voltammetry were employed to characterize the assembly process and the performance of the immunosensor. Under optimal conditions, the peak current of the immunosensor increased with concentration, showing a linear relationship between the peak current and the logarithm of the prostate-specific antigen concentrations in a wide range of 2.0 pg mL^?1 to 10.0 ng mL^?1 with a low detection limit of 0.5 pg mL^?1. The immunosensor was used for the determination of prostate-specific antigen in serum.     

A Highly Sensitive Electrochemical Impedance Spectroscopy Immunosensor for Determination of 1‐Pyrenebutyric Acid Based on the Bifunctionality of Nafion/Gold Nanoparticles Composite Electrode

A simple, highly sensitive and label‐free electrochemical impedance spectroscopy (EIS) immunosensor was developed using Nafion and gold nanoparticles (nano‐Au/Nafion) composites for the determination of 1‐pyrenebutyric acid (PBA). Under the optimal conditions, the amount of immobilized antibody was significantly improved on the nano‐Au/Nafion electrode due to the synergistic effect and biocompatibility of Nafion film and gold nanoparticles composites. The results showed that the sensitivity and stability of nano‐Au/Nafion composite electrode for PBA detection were much better than those of nano‐Au modified glassy carbon electrode (nano‐Au/GCE). The plot of increased electron transfer resistances (R_ets) against the logarithm of PBA concentration is linear over the range from 0.1 to 150 ng·mL^?1 with the detection limit of 0.03 ng·mL^?1. The selectivity and accuracy of the proposed EIS immunosensor were evaluated with satisfactory results.     

Electrochemical Immunoanalysis for Carcinoembryonic Antigen Based on Multilayer Architectures of Gold Nanoparticles and Polycation Biomimetic Interface on Glassy Carbon Electrode

This paper describes a layer‐by‐layer (LBL) self‐assembly process of chitosan (CTS) and gold nanoparticles (Au) on the pretreated negatively charged glassy carbon (GC) electrode to fabricate electrochemistry immunosensor with a nontoxic biomimetic interface, which provided an environment similar to a native system and allowed more freedom in orientation for immobilization of carcinoembryonic antibody (anti‐CEA) to monitor carcinoembryonic antigen (CEA). UV‐vis spectroscope, atomic force microscopy (AFM), and cyclic voltammetric (CV) measurements were used to follow the multilayer film formation. The performance of the biominetic interface and factors influencing the assay system were investigated in detail. The differential pulse voltammetry (DPV) current response is used for the CEA concentration assay. The dynamic range was from 0.50 to 80.00 ng mL^?1 with a detection limit of 0.27 ng mL^?1 at 3σ. In addition, the experiment results indicate that immobilization described in this proposed method exhibits a good sensitivity, selectivity, and stability.     

Study on an immunosensor based on gold nanoparticles and a nano-calcium carbonate/Prussian blue modified glassy carbon electrode

An amperometric carcinoembryonic antigen (CEA) immunosensor was fabricated based on Prussian blue (PB), nano-calcium carbonate (nano-CaCO₃) and nano-gold modified glassy carbon electrode. First, PB as a mediator was deposited on glassy carbon electrode to obtain a negatively charged surface. Then, positive nano-CaCO₃ was adsorbed on the PB modified electrode through electrostatic interaction. Subsequently, gold nanoparticles were deposited on the nano-CaCO₃/PB modified electrode. The use of two kinds of nanomaterials (nano-CaCO₃ and nano-gold) with good biocompatibility as immobilization matrixes not only provides a biocompatible surface for protein loading but also avoids the leaking of PB. The size of nano-CaCO₃ was characterized by transmission electron microscopy (TEM). The factors influencing the performance of the immunosensor presented were studied in detail. Under the optimized conditions, cyclic voltammograms (CV) determination of CEA showed a specific response in two concentration ranges from 0.3 to 20 ng mL^?1 and from 20 to 100 ng mL^?1 with a detection limit of 0.1 ng mL^?1 at a signal-to-noise ratio of 3. The immunosensor presented exhibited high selectivity, sensitivity and good stability.     

Electrochemical Biosensor Based on Nanocomposites Film of Thiol Graphene‐Thiol Chitosan/Nano Gold for the Detection of Carcinoembryonic Antigen

In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL^?1 and 8.0–100 ng mL^?1) with a detection limit of 0.03 ng mL^?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.