基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

金增强多重胶体金标记树枝状复合物放大的电化学免疫分析新方法

报道了一种电化学免疫分析新方法用于痕量(amol/L级)蛋白质的高灵敏检测. 该法采用了两级信号放大策略. 先以金标蛋白A与金标抗体形成的树枝状复合物作为一级信号放大, 随后使用金增强溶液催化捕获在此复合物上的大量的胶体金纳米颗粒的增大以实现二级信号放大. 这种两级信号放大步骤进而结合灵敏的金属溶出伏安分析法以有效增强抗原-抗体免疫反应响应. 实验还通过引入免疫复合物的磁分离操作来降低非特异性吸附的影响. 结果表明, 在优化的实验条件下, 开发的新方法在用于人IgG模型分析物测定时的检测下限可低至5.2 fg•mL^－1或34.7 amol/L (S/N＝3). 这种基于胶体金的金属免疫分析新技术有望用于临床诊断、环境监测、生化防预等诸多领域中目标蛋白质的高灵敏检测.     

金纳米簇信号放大的电化学免疫传感器

通过抗原抗体的特异性识别作用以及金纳米簇(AuNCs)探针和金标银染的双重信号放大作用,构建了一种新的电化学免疫传感器,对人的免疫球蛋白(IgG)进行了检测。受贻贝分泌的黏附蛋白启示,首先将聚多巴胺薄膜修饰在铟锡氧化物电极(ITO)上,并对一抗抗体进行固定,通过观察电化学阻抗的变化来监控免疫传感器的构建过程。将待检测的IgG抗原组装在该电极上并与AuNCs标记的二抗反应,最后经银染反应,用溶出伏安法对IgG的含量进行定量检测,其灵敏度达到0.5 ng/L。该方法可应用于实际血清样品中IgG含量的测定。     

板式磁颗粒化学发光免疫分析在癌胚抗原测定中的应用

开发了一种板式磁颗粒化学发光免疫分析方法并将其用于人血清中癌胚抗原(CEA)的测定,该方法利用HRP标记的CEA抗体以及FITC标记的CEA抗体与CEA抗原在微孔板中进行夹心反应,使用抗FITC抗体包被的磁性微粒子溶液作为分离固相,利用了一个特殊的适用于微孔板的磁性分离器进行分离,最后加入鲁米诺和H2O2底物进行发光反应.该方法采用微孔板代替透明管作为反应容器,磁颗粒作为分离固相在微孔里与免疫试剂反应,提前用BSA将微孔板封闭,很好地解决了管式磁颗粒方法非特异吸附高以及包被板方法线性范围小的局限性,同时避免了管间差异以及包被不均问题.该方法结合了微孔板作为反应容器以及磁颗粒分离体系的优点,使用夹心法对人血清中的CEA在0～250ng/mL浓度范围进行了测定,检测灵敏度为0.57ng/mL,批内和批间变异分别小于7%和11%.与市售CLIA试剂盒测定结果做了比对,两者显示了很好的相关性.该法经济、优越,有望实现商品化.     

基于酪胺信号放大的新型免疫传感器

将酪胺应用于酶联免疫分析,建立了一种新的高灵敏伏安型免疫传感器。利用纳米金的静电吸咐和己二硫醇、巯基乙胺的自组装,将羊抗人IgG抗体固定到金电极表面上,以辣根过氧化物酶标记羊抗人IgG抗体为酶标抗体,以生物素化酪胺为酶底物,利用催化酪胺沉积反应,在传感界面沉积大量生物素,使原始信号得到几何级数的放大。结果表明,通过生物素化酪胺催化放大后,制得的免疫传感器对H2O2的催化能力增大近20倍,检测hIgG在1.5μg/L～22 mg/L范围内有良好的线性关系,检出限为0.1μg/L。用于实际试样的回收率的测定,结果良好。     

铜离子掺杂双金属纳米球免疫传感器检测前列腺特异性抗原

以比表面积大、结合位点多的金铂纳米球杂化二氧化锡石墨烯(GS-SnO₂@Au-Pt)修饰玻碳电极,作为传感平台固定捕获抗体(Ab₁),铜离子掺杂金银纳米球(Au-Ag@Cu²⁺)与检测抗体(Ab₂)结合作为免疫探针,构建夹心型电化学免疫传感器,并用于检测前列腺特异性抗原(PSA)。基于Cu²⁺和Cu⁺之间的电子转移,以及金银双金属协同效应增强检测信号,通过方波伏安法(SWV)检测,在0.25 V处获得尖锐信号峰。结果表明,该免疫传感器具有较宽的线性范围(1 fg/mL～10 ng/mL)和较低的检出限(0.34 fg/mL),在PSA临床检测中有潜在应用前景。     

基于酶催化沉积质量放大的压电免疫传感器的研究

提出了一种酶催化沉积质量放大的高灵敏压电免疫传感器. 采用1,6-二巯基己烷在石英晶振上自组装一单分子层, 再通过另一端巯基连接胶体金, 利用胶体金的高比表面积和强吸附作用力增加抗IgG抗体的固定量, 同时借助胶体金优良的生物亲和性保持抗IgG抗体的活性. 在H2O2存在下, 通过标记在抗人IgG抗体上的HRP酶催化底物DAB(3,3′-联苯二胺), 反应中生成的不溶性产物沉积到石英晶振的Au电极表面, 达到质量放大的目的. 结果表明, 检测人IgG在16 ng/mL-100 μg/mL范围内有很好的线性关系, 检测下限为10 ng/mL, 在用于实际试样的回收率测定中, 结果良好.     

三组分抗原的磁分离及分离效率的SERS研究

利用种子生长法制备了磁性γ-Fe₂O₃@Au核壳纳米粒子, 通过修饰抗体实现表面功能化, 利用抗原抗体间的特异性作用, 通过外加磁场对三组分抗原进行了逐个以及双抗原的磁分离, 采用基于表面增强拉曼光谱(SERS)技术的免疫检测方法对磁分离效率进行了评价, 并且研究了该磁分离和效率评估方法的极限工作浓度. 研究结果表明, 该磁免疫分离法能对三组分混合抗原中的任意组分进行很好的选择性分离, 而不影响其它抗原的存在, 使其分离后溶液中被分离抗原的浓度降低到SERS免疫检测限, 分离所能达到的极限抗体浓度约0.1 pg/mL.     

基于Fe3O4@Au复合纳米粒子标记抗体的电化学免疫方法用于水体中大肠杆菌的检测

合成了Fe₃O₄@Au复合纳米粒子作为辣根过氧化酶标记抗体的载体, 并将该复合纳米粒子标记物应用于电化学放大免疫分析. 将电子媒介体硫堇聚合在玻碳电极表面, 以纳米金作为固定大肠杆菌抗体的基底, 通过辣根过氧化酶催化溶液中H₂O₂产生的电流信号来测定大肠杆菌. 实验结果表明, 该方法对水体中大肠杆菌检测的线性范围为50～1×10⁵ cfu/mL, 检出限为20 cfu/mL. 对过富集后的实际水样进行测定, 该法结果表明, 对水体中大肠杆菌的检测灵敏度达到2 cfu/mL.     

基于免疫磁分离的荧光微球免疫层析法检测猪霍乱沙门氏菌

建立了基于免疫磁分离的荧光微球免疫层析法,检测猪霍乱沙门氏菌.待检样品经免疫磁分离富集和热洗脱处理后,用荧光微球免疫层析试纸条进行检测.每毫克纳米磁珠标记30μg抗体制备的免疫磁珠,对浓度为102 ～ 106 CFU/mL的猪霍乱沙门氏菌的捕获率均大于90％,特异性好;在pH=6时,以300μ,g/mg猪霍乱沙门氏菌单抗11D8-D4标记荧光微球,制备免疫荧光微球;以2.0 mg/mL猪霍乱沙门氏菌单抗5F11-B11喷涂检测线(T线),以1.0 mg/mL驴抗鼠IgG喷涂质控线(C线),制备免疫层析试纸条.采用建立的基于免疫磁分离的荧光微球免疫层析方法检测猪霍乱沙门氏菌,在PBS缓冲液中检出限为1.5×105 CFU/mL,牛奶中检出限为7.6×105 CFU/mL,与直接采用荧光微球免疫层析方法检测相比,检出限分别降低了10倍和200倍.本方法可有效富集牛奶中的沙门氏菌,避免了基质干扰,灵敏度大大提高,具有较好的应用前景.     

基于微机电系统技术和纳米金自组装膜的安培型免疫传感器研究

基于微机电系统(MEMS)技术制备安培型免疫传感器,并利用基于硫醇单层膜的纳米金单层膜自组装技术设计传感器界面,用于固定人免疫球蛋白(IgG)抗体,研制了一种新型的安培型免疫传感器。采用MEMS技术,在硅片上制备微型的三电极系统以及SU-8反应池。基于自组装技术,先在金电极上自组装巯基乙胺单层膜,利用膜上氨基与纳米金共价结合组装纳米金单层膜,得到可用于固定抗体的界面。实验探讨了影响抗体固定的主要实验参数和条件;考察了采用此固定化方法传感器的响应性能,与金电极直接吸附固定法和戊二醛共价交联固定法进行了比较。对IgG检测的实验结果表明,采用纳米金自组装膜固定抗体,具有活性高、非特异性吸附小、检测线性范围宽等优点。并且,基于MEMS技术的安培型免疫传感器具有微型化、与集成电路工艺相兼容、易于实现传感器的阵列化和实时多参数检测等优点。     

Highly sensitive electrochemical immunoassay for human IgG using double-encoded magnetic redox-active nanoparticles

A new sandwich-type electrochemical immunoassay was developed for the detection of human IgG using doubly-encoded and magnetic redox-active nanoparticles as recognition elements on the surface of a glassy carbon electrode modified with anti-IgG on nanogold particles. The recognition elements were synthesized by coating magnetic Fe₃O₄ nanoparticles with Prussian blue nanoparticles and then covered with peroxidase-labeled anti-IgG antibodies (POx-anti-IgG) on Prussian blue nanoparticles. The immunoelectrode displays very good electrochemical properties towards detection of IgG via using double-encoded magnetic redox-active nanoparticles as trace and hydrogen peroxide as enzyme substrate. Its limit of detection (10 pmol·L^?1) is 10-fold better than that of using plain POx-anti-IgG secondary antibodies. The method was applied to the detection of IgG in serum samples, and an excellent correspondence with the reference values was found.     

Free-standing nanogold membranes as scaffolds for enzyme immobilization

We demonstrate herein the formation of a free-standing gold nanoparticle membrane and its use in the immobilization of the enzyme, pepsin. The nanogold membrane is synthesized by the spontaneous reduction of aqueous chloroaurate ions at the liquid-liquid interface by the bifunctional molecule bis(2-(4-aminophenoxy)ethyl) ether (DAEE) taken in chloroform. This process results in the formation of a robust, malleable free-standing nanogold membrane consisting of gold nanoparticles embedded in a polymeric background. Recognizing that gold nanoparticles are excellent candidates for immobilization of enzymes, we have immobilized pepsin on the nanogold membrane, leading to a new class of biocatalyst. A highlight of the new pepsin-nanogold biocatalyst is the ease with which separation from the reaction medium may be achieved. The catalytic activity of pepsin in the bioconjugate was comparable to that of the free enzyme in solution. The pepsin-nanogold membrane bioconjugate material exhibited excellent biocatalytic activity over 10 successive reuse cycles as well as enhanced pH, temperature, and temporal stability.     

纳米金标记-膜过滤分光光度法检测免疫球蛋白G

采用10 nm的纳米金标记羊抗人免疫球蛋白G获得免疫球蛋白G(IgG)的探针(AuIgG)。在pH 6.8的NaH2PO4-Na2HPO4磷酸盐缓冲溶液及聚乙二醇6000、KCl溶液存在下,IgG与AuIgG探针发生免疫反应,用0.15μm滤膜过滤反应生成的免疫复合物溶液,滤液在524 nm处有一最大吸收峰。其降低值ΔA524 nm随着IgG浓度的增加线性增加,据此建立了一种测定IgG的分光光度法。在最佳实验条件下,免疫球蛋白G浓度在0.025~0.375μg/mL范围内与ΔA呈良好的线性关系,其线性回归方程为ΔA=0.783ρ+0.0232,相关系数为0.9927,检出限(3σ)为0.0082μg/mL。该法用于分析人血清中免疫球蛋白G,结果与免疫透射比浊法结果一致,相对标准偏差在2.0%~5.6%之间。     

Spectral and fluorescent kinetics features of Nd3+ ion in Nb2O5, Ta2O5 and La2O3 mixed lithium zirconium silicate glasses

A sensitive competitive flow injection chemiluminescence (CL-FIA) immunoassay for immunoglobulin G (IgG) was developed using gold nanoparticle as CL label. In the configuration, anti-IgG antibody was immobilized on a glass capillary column surface by 3-(aminopropyl)-triethoxysilane and glutaraldehyde to form immunoaffinity column. Analyte IgG and gold nanoparticle labeled IgG were passed through the immunoaffinity column mounted in a flow system and competed for the surface-confined anti-IgG antibody. CL emission was generated from the reaction between luminol and hydrogen peroxide in the presence of Au (III), generated from chemically oxidative dissolution of gold nanoparticle by an injection of 0.10 mol L⁻¹ HCl–0.10 mol L⁻¹ NaCl solution containing 0.10 mmol L⁻¹ Br₂. The concentration of analyte IgG was inversely related to the amount of bound gold nanoparticle labeled IgG and the CL intensity was linear with the concentration of analyte IgG from 1.0 ng mL⁻¹ to 40 ng mL⁻¹ with a detection limit of 5.2 × 10⁻¹⁰ g mL⁻¹. The whole assay time including the injections and washing steps was only 30 min for one sample, which was competitive with CL immunoassays based on a gold nanoparticle label and magnetic separation. This work demonstrates that the CL immunoassay incorporation of nanoparticle label and flow injection is promising for clinical assay with sensitivity and high-speed.     

Specific immunoreaction-induced controlled release strategy for sensitive impedance immunoassay of a cancer marker

A simple and facile impedance immunoassay strategy for sensitive detection of alpha-fetoprotein (AFP), as a model cancer marker, was developed by using target-induced release of nanogold particle-labelled anti-AFP antibodies from polyvinylpyrrolidone-coated magnetic carbon nanotubes.     

Construction of nanogold hollow balls with dendritic surface as immobilized affinity support for protein adsorption

This contribution introduced the construction of nanosize gold hollow balls (NGB) with dendritic surface as the immobilized affinity support for aflatoxin B(1) antibody (anti-AFB(1)) adsorption, as a model protein, in biorecognition interface. The interaction between nanogold hollow balls and anti-AFB(1) was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and quartz crystal microbalance (QCM) technique. The deposited mass of anti-AFB(1) on the nanogold hollow ball-modified electrodes was more than that on the nanogold particle-modified electrodes via QCM analysis. The cyclic voltammogram tends to be more irreversible with anti-AFB(1) concentration increased. The antigen-antibody interaction was examined by using the nanogold hollow ball-modified QCM probes. Experimental results show that the developed protein assay system is sensitive to the concentration of AFB(1) as low as 0.05 ng mL(-1). Thus, the nanogold hollow ball is a useful matrix, and can be used as the immobilized affinity support for the adsorption of other proteins.     

磁纳米探针检测人绒毛膜促性腺激素

采用链霉亲和素包被磁纳米粒子,将生物素标记的特异性抗体偶联在磁纳米粒子上,制备出高特异性的磁纳米探针;利用此探针对人绒毛膜促性腺激素(HCG)进行测定,建立了定量检测蛋白类激素的化学发光分析方法.利用紫外可见分光光度计、透射电镜及动态光散射仪对磁纳米探针进行表征,同时对化学发光实验条件进行优化.在2×10~(-4) mol/L鲁米诺、8×10~(-4) mol/L H_2O_2, pH=13的优化条件下,将磁纳米探针用于HCG的定量检测.结果表明,所测发光强度与待测HCG浓度之间线性相关,相关系数r为0.9924,线性检测范围由常规板式ELISA的5～200 μg/L扩展到0.5～250 μg/L;相对标准偏差为3.82%.采用本方法和常规ELISA法同时对34份人血清标本HCG进行测试,两者相关性良好.利用制备的磁纳米探针定量测定微量蛋白类激素,具有灵敏、高效、快捷、检测范围宽等优点,有望应用于其它微量蛋白的检测.     

动态磁涂覆混合固定相开管毛细管电色谱中电渗流的特征

将表面分别被改性成C18和氨基的磁性纳米颗粒按照不同比例混合,制备成具有不同分离选择性的混合固定相,进一步采用动态磁涂覆的方法制备开管毛细管电色谱柱。通过考察这种色谱柱中不同种类固定相表面物理化学性质对电渗流的综合影响,从理论上说明了其电渗流的特征。分别在不同固定相配比及不同涂覆长度条件下进行实验,理论与实验结果相符,证实通过调节固定相配比或磁铁对数可以便捷地调节电渗流的大小。     

In situ growth of nanogold on quartz crystal microbalance and its application in the interaction between heparin and antithrombin III

A novel biosensor for detecting antithrombin III (AT III) was constructed based on in situ growth of nanogold on the gold electrode of quartz crystal microbalance (QCM). The growth process of nanogold was monitored by QCM in real time. Heparin was used as the affinity ligand and immobilized onto the nanogold modified gold electrode. A flow injection analysis-quartz crystal microbalance (FIA-QCM) system was used to investigate the relationship between nanogold growth and the AT III response. Along with the nanogold particle growth within initial 5 min, the amount of heparin immobilized onto the nanogold modified electrode increased quickly. Correspondingly, the frequency response to AT III binding increased rapidly at the same time. After that, both the immobilized amount of heparin and the sensor response to AT III decreased gradually. Compared with the directly immobilized large nanogold particles, the in situ grown particles with the same size occupy more sensor surface, resulting in higher frequency shifts to AT III in the interaction study between heparin and AT III. The obtained constants of AT III binding to immobilized heparin are k(ass)=(1.65+/-0.12)x10(3) L/mols, k diss=(2.63+/-0.18)x10(-2) 1/s and K(A)=(6.27+/-0.42)x10(4) L/mol.