基于MWNT、CS-PB和GNPs共修饰检测前列腺特异性抗原电流型免疫传感器的研究

目的采用多壁碳纳米管(MWNT)、普鲁士蓝-壳聚糖(CS-PB)纳米复合物及纳米金(GNPs)共修饰于氧化铟锡(ITO)电极表面固载抗体制得高灵敏前列腺特异性抗原电流型免疫传感器,并对传感器性能参数进行实验测定。方法先在ITO电极表面电沉积一层纳米金,随后将浓硝酸处理过的MWNT滴涂在ITO玻璃电极的纳米金膜上,然后通过静电吸附固定CS-PB纳米复合物,利用壳聚糖中大量的氨基固定GNPs并吸附前列腺特异性抗体(anti-PSA),制得电流型免疫传感器,同时测定传感器检测灵敏度、线性响应范围等。结果在最佳实验条件下,该传感器响应的峰电流值与前列腺特异性抗原(PSA)浓度在1～15ng/mL和15～150ng/mL的范围内保持良好的线性关系,检测下限为0.8ng/mL。结论成功建立了前列腺特异性抗体免疫传感器,该方法制备简单,操作便捷,灵敏度高。     

乙肝电化学免疫传感器阵列电极的研制

研制了以乙肝表面抗原为检测对象的电化学免疫传感器阵列,采用微电子平面加工技术制备了八通道金电极阵列,在金电极表面自组装半胱胺分子,戊二醛进行醛基化,最后通过竞争组装同时固定乙肝抗体和硫堇,以硫堇的还原峰电流的下降百分比对乙肝表面抗原进行定性和定量的检测.该传感器阵列可同步检测乙肝表面抗原,大大提高了检测的准确度和可靠性.应用于临床血清样品的检测,其结果与ELISA法相一致.     

基于巯基和纳米金固定抗体的免疫传感器的初步应用研究

目的 制得乙肝免疫传感器,用于临床检验.方法 以金电极为基底,自组装半胱胺,通过纳米金静电竞争吸附硫堇分子和乙肝过氧化酶酶标抗体,制得乙肝免疫传感器.采用循环伏安法和线形扫描伏安法考察了传感器的组装过程, 响应电流的性质,传感器对乙肝病毒的响应特性.结果 对乙肝抗原进行了定量分析,线形范围为1∶30～ 1∶200 (稀释浓度V);线性相关系数 R 为0.994.取临床血清进行检测,将结果与临床常用的 ELISA 法比较,符合要求.结论 用半胱胺/纳米金方法固定乙肝抗体制得免疫传感器能应用于临床检验.     

石英晶体制备工艺对压电免疫传感芯片的影响

目的 评估石英晶体制备工艺对压电免疫传感芯片的影响.方法 采用多壁碳纳米管-纳米金技术在四种类型的石英晶体（镀镍层金电极、无镍层金电极、镀镍层银电极和无镍层银电极）上固定梅毒螺旋体（Treponema pallidum,TP）基因工程抗原,重复检测抗-TP阳性血清和阴性血清各8次.以压电免疫传感芯片制备过程中的清洗漂移、表面水珠附着、抗原固定量、测试稳定时间、抗原脱落状况、响应值及变异系数（CV）值七项作为测评指标,整体评估石英晶体制备工艺对梅毒压电免疫传感芯片的影响.结果 真空镀镍层金电极石英晶体制备的梅毒自组装压电免疫传感芯片各项指标综合评价最佳.结论 镀镍层与金属电极是制备压电免疫传感芯片的两项重要指标.     

纸基电化学免疫传感器的研制及初步应用

目的研制1种以纸为基底材料的免疫传感器,并探讨其在临床检验中的应用价值。方法本实验选用人IgG为靶抗原,以纳米金修饰的醋酸纤维素滤膜为基底电极,将单克隆鼠抗人IgG抗体固定其表面,利用夹心法结合HRP标记的多克隆兔抗人IgG抗体,方波伏安法检测HRP催化底物所产生的还原电流来检测人IgG含量。结果该免疫传感器对IgG响应良好,其线性范围为10~200 ng/ml,相关系数为0.996,检出限为10 ng/ml,并成功应用于检测人血清中IgG。结论纸基电化学免疫传感器具有检测范围宽、灵敏度高和稳定性好等优点,在临床检验中有广阔的应用前景。     

多层纳米金修饰膜的HCG表面等离子体传感器

探索一种可以高密度固定抗原的新方法,并用于研究多层纳米金修饰金膜表面包被抗原的新型人绒毛膜促性腺激素(HCG)表面等离子体共振(SPR)传感器。3-巯丙基甲基二甲氧基硅烷(MPTMS)与纳米金粒子(AuNPs)可通过Au-S键在传感器金膜表面交替组装形成三维网络,膜表面空间结构中的纳米金经静电作用可吸附大量的小分子抗原HCG,从而获得多层膜修饰的HCG免疫传感器用于检测抗原抗体结合反应。用所构建的HCG传感器,直接检测不同浓度的anti-HCG抗体有良好的线性关系,且修饰两层胶体金膜比修饰一层时抗体响应的共振峰移动幅度提高了1.8倍,检测下限降低至1.5μg/mL。间接法测定了不同浓度的HCG,其浓度与SPR共振峰的位移呈负相关。实验通过修饰两层胶体金膜大大提高了金膜表面固定抗原的量,提高了检测抗体的灵敏度,并且进而用竞争法可检测HCG抗原而不需要用第二抗体进行信号放大。有望用于临床实验诊断。     

DNA电化学传感器中电极修饰条件的探讨

目的 探讨ssDNA单层膜的制备的最佳条件.方法利用自组装单分子膜技术,将人工合成并修饰的含22个碱基的特异性单链DNA序列固定到金电极上制备成DNA修饰的金电极,通过控制DNA溶液的浓度、作用时间,以及改变修饰基团探讨最佳的修饰条件,并利用循环伏安法初步研究了该修饰电极的电化学行为.结果 通过对不同标记探针、不同探针浓度及不同标记时间的表征,结果显示巯基化ssDNA和二硫键修饰的ssDNA探针浓度为80 μmol/L,自组装固定时间为12 h时对DNA探针的固定效率最佳.结论 巯基化ssDNA和二硫键修饰的ssDNA通过自组装方法可在金电极表面形成稳定、有序的自组装单分子膜(self-assembled monolayer,SAM),可应用于SAM为基底的DNA传感器的设计与应用.     

基于纳米材料的电化学发光免疫传感器

电化学发光传感器是人们研究比较活跃的一个领域,本文就近5年基于纳米颗粒、碳纳米管、量子点等纳米材料构建的电化学发光免疫传感器新技术做一综述。纳米颗粒、碳纳米管的高比表面积、生物相容性及高导电活性,不仅可以降低电化学发光的初始电位,还能加快电极与免疫材料间的电子传递并增强电化学发光强度,提高电化学发光检测的灵敏度及稳定性,已经广泛用于化学及生物化学分析。另外,量子点独特的光学性质,与碳纳米管、纳米颗粒等材料组成的纳米混合物,为开发新型电化学发光免疫传感器提供了一种新思路。     

电沉积纳米金修饰的16通道电流型PSA免疫传感器的制备

近年来有关电化学免疫传感器的研究报道虽然很多,但普遍存在如预处理操作繁琐、灵敏度低、特异性差和成本昂贵等不足。本研究通过电沉积纳米金修饰16通道丝网印刷电极,以提高免疫传感器的性能。采用恒电位沉积法将氯金酸(HAu Cl4)直接还原成纳米金,并修饰于16通道丝网印刷碳电极(16-SPCE)表面;以浓度为0.05mg/m L的HAu Cl4溶液和150 s电沉积时间为电沉积纳米金修饰16-SPCE的最优条件。利用静电吸附原理将前列腺抗原(PSA)抗体固定在电极表面,结合双抗夹心法制备免疫电极。通过循环伏安法和稳态时间电流曲线法表征免疫电极的性能。对30份前列腺癌患者和3份正常人血清进行PSA检测,与标准ELISA方法对照。结果表明:获得的免疫电极线性检测范围为0.2~100 ng/m L,其线性回归方程为Y=134.558X+72.705,线性相关系数为1,检测限为0.14 ng/m L。采用免疫传感器检测到的前列腺癌患者血清PSA水平与标准ELISA方法检测结果具有良好的线性相关性(R=0.944,P0.001),正常人的检测值符合正常人血清中PSA的含量。所研制的免疫传感器具有灵敏度高、特异性强、成本低和反应时间短等特点,具有应用前景。     

层层自组装薄膜改性的电化学传感器应用

电化学传感器具有优异的灵敏度、检测限、选择性和响应速度，被广泛用于各种生物化学物质的检测。电极作为电化学传感器的敏感元件，合理的表面改性涂层对提高传感器的检测精度和稳定性尤为重要。层层自组装技术是通过在电极基底上交替沉积具有相互作用的物质，以纳米级精度构建参数可控的三维材料体系，在电化学传感器领域已得到广泛应用。综述了层层自组装技术的成膜材料(包括聚电解质和纳米颗粒等)、成膜作用力(包括静电作用、共价键、氢键等)及其性能优化与技术改进，并介绍了基于纳米材料构建改性的层层自组装薄膜电化学传感器，展示了其在生物标志物检测、空气监测和金属离子检测等方面的应用实例。     

An amperometric immunosensor for separation-free immunoassay of CA125 based on its covalent immobilization coupled with thionine on carbon nanofiber

A carbon nanomaterial, soluble carbon nanofiber, was used for the first time to construct an immunosensor for a rapid separation-free immunoassay. The acidic oxidation of the carbon nanofiber provided its solubility and wettability for convenient preparation of a porous carbon nanofiber membrane and a larger number of active sites for covalent binding of carcinoma antigen-125 (CA125) and thionine as electron transfer mediator. This matrix was a suitable environment for the immobilized protein. The immobilized HRP-labeled immunoconjugate showed good enzymatic activity for the oxidation of thionine by hydrogen peroxide. With a competitive mechanism, the differential pulse voltammetric peak current of this system decreased linearly with increasing CA125 concentration (from 2 to 75 U/ml) in the incubation solution. The CA125 immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility with a detection limit of 1.8 U/ml. The soluble carbon nanofiber is a novel method for preparation of immunosensors.     

Highly sensitive amperometric immunosensor for detection of Plasmodium falciparum histidine-rich protein 2 in serum of humans with malaria: comparison with a commercial kit

A disposable amperometric immunosensor was developed for the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) in the sera of humans with P. falciparum malaria. For this purpose, disposable screen-printed electrodes (SPEs) were modified with multiwall carbon nanotubes (MWCNTs) and Au nanoparticles. The electrodes were characterized by cyclic voltammetry, scanning electron microscopy, and Raman spectroscopy. In order to study the immunosensing performances of modified electrodes, a rabbit anti-PfHRP-2 antibody (as the capturing antibody) was first immobilized on an electrode. Further, the electrode was exposed to a mouse anti-PfHRP-2 antibody from a serum sample (as the revealing antibody), followed by a rabbit anti-mouse immunoglobulin G-alkaline phosphatase conjugate. The immunosensing experiments were performed on bare SPEs, MWCNT-modified SPEs, and Au nanoparticle- and MWCNT-modified SPEs (Nano-Au/MWCNT/SPEs) for the amperometric detection of PfHRP-2 in a solution of 0.1 M diethanolamine buffer, pH 9.8, by applying a potential of 450 mV at the working electrode. Nano-Au/MWCNT/SPEs yielded the highest-level immunosensing performance among the electrodes, with a detection limit of 8 ng/ml. The analytical results of immunosensing experiments with human serum samples were compared with the results of a commercial Paracheck Pf test, as well as the results of microscopy. The specificities, sensitivities, and positive and negative predictive values of the Paracheck Pf and amperometric immunosensors were calculated by taking the microscopy results as the “gold standard.” The Paracheck Pf kit exhibited a sensitivity of 79% (detecting 34 of 43 positive samples; 95% confidence interval [CI], 75 to 86%) and a specificity of 81% (correctly identifying 57 of 70 negative samples; 95% CI, 76 to 92%), whereas the developed amperometric immunosensor showed a sensitivity of 96% (detecting 41 of 43 positive samples; 95% CI, 93 to 98%) and a specificity of 94% (correctly identifying 66 of 70 negative samples; 95% CI, 92 to 99%). The developed method is more sensitive and specific than the Paracheck Pf kit.     

Flow-injection chemiluminescent immunoassay for alpha-fetoprotein based on epoxysilane modified glass microbeads

A flow-injection chemiluminescent immunoassay system based on a novel transparent immunoaffinity reactor is proposed for the quantitation of alpha-fetoprotein. The reactor prepared with alpha-fetoprotein immobilized epoxysilane modified glass microbeads was used as an immunosensor for chemiluminescent detection. With a non-competitive immunoassay format, the proposed immunosensor system is a low cost, flexible and rapid assay for alpha-fetoprotein. After an off-line incubation of the analyte alpha-fetoprotein with horseradish peroxidase-labeled alpha-fetoprotein antibody as enzyme tracer, the mixture was injected into the reactor, which led to trapping of the free enzyme tracer by the reactor. The trapped enzyme tracer was detected by the p-iodophenol-luminol-H2O2 chemiluminescence system. Under optimal conditions, the decrease in chemiluminescence intensity was proportional to the alpha-fetoprotein concentration in the range of 5.0-100 ng/ml with a detection limit of 2.7 ng/ml at a signal/noise ratio of 3. The immunosensor system showed an acceptable reproducibility and stability. Clinical serum samples were assayed with this method and the results were in acceptable agreement with those obtained from immunoradiometric assay.     

A conductive ormosil encapsulated with ferrocene conjugate and multiwall carbon nanotubes for biosensing application

Highly non-toxic and conductive ormosil composite film was prepared using (3-aminopropyl)triethoxysilane and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane by doping with ferrocenemonocarboxylic acid-bovine serum albumin (FMC-BSA) conjugate and multiwall carbon nanotubes (MWNTs). With glucose oxidase (GOD) as a model enzyme this film could be used to design an amperometric biosensor for glucose determination. The entrapped FMC-BSA conjugate performed excellent redox electrochemistry and the immobilized GOD was highly stable. Under optimal conditions this biosensor was able to detect glucose with a detection limit of 20 microm (S/N=3) in the linear range of 0.05-20.0 mm in flow system, which was wider than the batch amperometric mode, with an analysis time of 25 s for each sample. The value of K(M)(app) was 6.6 mm. The proximity of these three components FMC-BSA, MWNTs and GOD enhanced the electron transfer between the film and electrode. This film could be used efficiently for the entrapment of other redox bioactive compounds and biosensing/bioelectrochemical applications.     

AFP电化学发光免疫分析方法的建立

目的建立一种快速、特异、灵敏的检测人甲胎蛋白（AFP）的电化学发光免疫分析法。方法用链霉亲和素包被的磁性微粒、生物素标记的抗AFP单克隆抗体、钌复合物标记配对的抗AFP单克隆抗体组成AFP电化学发光免疫分析法试剂,在电化学发光免疫分析仪上对其准确性、灵敏度、特异性等效能进行方法学评价,同时用所建方法与进口的同类电化学发光免疫分析法试剂（Roche）对80例肝癌患者血清AFP检测结果进行相关性分析。对218名健康志愿者血清AFP进行了正常值调查。结果自建电化学发光免疫分析法检测AFP的批内精密度在2.8%～4.5%之间,批间精密度在3.2%～9.8%之间,分析灵敏度为0.605ng/ml。与进口同类试剂比较,直线回归方程为y=0.9936x-0.4566（r=0.9877,P〈0.05）。分析测量范围为0.605～1452.0ng/ml,自建试剂与CEA和CA199无交叉反应。自建电化学发光免疫分析法检测AFP的正常参考值为〈6.7ng/ml。结论自建AFP电化学发光免疫分析法特异性高,灵敏度好,与进口同类试剂检测结果相关性达0.9877。确定的实验室正常参考范围接近进口同类试剂,具备产业化的潜能。     

GP73与AFP联合检测在肝癌诊断中的应用价值

目的 通过检测HCC血清中的GP73和AFP含量,探讨GP73对于肝癌检测的应用价值.方法 通过ELISA方法对81例HCC、72例CH/LC和65例正常对照血清行GP73检测,并通过ROC曲线计算GP73诊断肝癌的敏感性和特异性.结果 HCC组、CH/LC组和正常对照组GP73含量分别为( 152.67±33.59) ng/ml、(93.15±20.02) ng/ml和(58.95±17.29) ng/ml.ROC曲线分析显示以120 ng/ml为Cut-off值,GP73诊断肝癌的敏感性为77.80％,特异性为78.00％.结论 GP73可以作为诊断肝癌的血清标志物,其敏感性好于AFP,同时通过联合检查可以提高肝癌的检出率,有一定的临床意义.     

双标记AFP及Free-β-HCG时间分辨荧光免疫分析法的建立

目的：研制能同时检测人血清AFP和Free-β-HCG的双标记时间分辨荧光免疫分析（TrFIA）试剂盒。方法：铕（Eu3＋）标记抗AFP单克隆抗体,用钐（Sm3＋）标记抗Free-β-HCG单克隆抗体,采用双抗体夹心法建立AFP/Free-β-HCG双标记TrFIA试剂,对试剂的各项性能指标进行评价。结果：AFP分析灵敏度为0.1U/ml,分析内和分析间的精密度分别为1.2%～5.9%和2.8%～6.3%,检测试剂的测量范围为（0.1～500）U/ml;Free-β-HCG分析灵敏度为0.25ng/ml,分析内和分析间的精密度分别为1.5%～6.2%和3.5%～5.6%,检测试剂的测量范围为（0.25～200）ng/ml。孕妇血样用本试剂盒与进口的同类试剂盒同时检测,AFP和Free-β-HCG的相关系数分别为0.987、0.968,具有较好的一致性。结论：自制AFP/Free-β-HCG双标记TrFIA试剂盒的各项性能指标均能达到临床检测要求,可替代国外同类产品。     

A tris(2,2'-bipyridyl)cobalt(III)-bovine serum albumin composite membrane for biosensors

A concept based on a novel redox-biocompatible composite protein membrane fabrication, double enzyme membrane modification technique and antibody immobilization, was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of carcinoembryonic antigen (CEA). In this concept, a solution of bovine serum albumin (BSA) containing horseradish peroxidase (HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved. Then tris(2,2'-bipyridyl) cobalt(III) (Co(bpy)(3)3+), as a mediator, was embedded in BSA-HRP composite membrane vis the electrostatic force and hydrophobe functions. Later a self-assembled conductive nano-Au monolayer was constructed onto the resultant electrode surface by electrostatic interaction between the negatively charged nano-Au and positively charged Co(bpy)3(3+). Protein A is used as a binding material to achieve an adjusted (but not random) orientation of the antibodies surface for efficient combination of antigens. Finally, the HRP, was employed to block the possible remaining active sites and avoid the non-specific adsorption, which acts not only as a blocking reagent instead of the commonly used BSA but also as the conventional enzyme-labeling to amplify the response of the antigen-antibody reaction. The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)(3)3+/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA in the range of 0.50-80.00 ng/mL with a limit of detection of 0.14 ng/mL, as well as good stability and long-term life.