纳米磁性微球免疫伏安法测定乙肝表面抗原

采用化学键合法将乙肝抗体固化在自行制备的纳米磁性高分子功能微球表面 ,利用免疫夹心反应原理 ,捕获溶液中的乙肝表面抗原和标记有辣根过氧化物酶的乙肝第二抗体 .在外加磁场的作用下 ,抗体抗原结合物从样品溶液中分离 ,在含有邻氨基苯酚和过氧化氢的底液中 ,生成具有电活性的化合物 3 氨基吩呃嗪 ,用示差脉冲伏安法进行测定 .响应电流与乙肝表面抗原浓度分别在 0 2～ 1 0和 1 0～ 5 0 0ng·mL-1成线性关系 ,检出限达 0 0 60ng·mL-1.采用本方法检测血清中乙肝表面抗原 ,灵敏度大大高于目前临床采用的酶联免疫吸附法 .     

基于自动磁珠转运的转基因蛋白Cry1Ab检测

基于自动磁珠转运,建立了转基因蛋白Cry1Ab免疫检测的新方法.利用水热法制备了粒径约400 nm的纳米磁球,并进行电镜表征,通过溶胶法对磁球表面进行氨基修饰,采用戊二醛偶联对磁珠实现抗体包被,在核酸提取仪中进行酶联免疫反应,采用分光光度法进行检测.本方法对转基因蛋白Cry1Ab的检出限低于1 μg/L,与商品化酶联免...     

超支化聚合物作为放大信号的纳米磁性微球电化学免疫分析法

提出了一种基于超支化聚合物(HBP)固化酶标二抗作为放大信号和纳米磁球相结合的超灵敏的免疫分析新方法。首先羧基纳米磁性微球共价键合乙肝抗体(HBsAb)形成免疫磁性微球,然后与待测乙肝表面抗原(HBsAg)发生特异性结合,加入HBP标记的酶标二抗(HBPS)与结合的抗原结合发生夹心反应。在外加磁场的作用下,抗体抗原免疫复合物易从样品溶液中分离,在含有邻氨基苯酚和H2O2的底液中,快速生成具有电活性的化合物3-氨基吩呃嗪,用示差脉冲伏安法(DPV)测定响应电流,电流强度(I)与乙肝表面抗原浓度(c)在0.05～10.0μg/L范围内呈线性关系,线性回归方程为I(μA)=0.140+16.80 c(μg/L),相关系数r=0.9995,检出限达0.008μg/L,并用于实际样品的测定。     

纤维素磁性微球在病原微生物检测中的应用研究

采用反向悬浮包埋技术和高速匀浆法制备了不同粒径的纤维素磁性微球,扫描电镜和铁离子分析结果表明,最佳粒径为5.82μm。后采用自行构建并表达的融合蛋白CBD-ProA和沙门氏菌多价抗体对最佳粒径的纤维素磁性微球进行活化,获得免疫磁性微球,其抗体偶联量为186.8mg/mL。最后采用致敏的免疫磁性微球分别对污染沙门氏菌的饮用水、牛奶和花生酱等样品进行免疫磁性捕获及分子鉴定,其检测限均可达10cfu/100mL。本研究开发的免疫磁性捕获技术,具有微球活化速度快、工艺简单、使用便捷等优势,同时,建立的免疫磁性捕获-PCR的方法与国标法相比,检测时间缩短48h,在食品安全、环境检测等方面具有实用价值。     

免疫磁性纳米微球的制备与表征

成功制备了Fe3O4磁性纳米颗粒及二甲基丙烯酸乙二醇酯-甲基丙烯酸(EGDMA-MAA)共聚物包覆的Fe3O4磁性复合微球。将吲哚美辛抗体固定在复合微球表面,形成了Fe3O4(核)/聚合物-抗体(壳)的复合免疫磁性颗粒。XRD结果表明,制备的Fe3O4的晶型为反立方尖晶石型且纯度较高;TEM表征表明Fe3O4粒径较为均匀,平均粒径为12nm;磁性复合微球的平均直径为460nm。制备的Fe3O4磁性纳米颗粒和磁性复合微球有较强的磁响应强度,其饱和磁化率分别为49.16和8.38emu/g,能够满足磁性分离的要求。FT IR验证了磁性复合微球中羧基特征峰的存在,表明羧基成功连接在磁性微球上面。通过碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)活化法将微球表面羧基活化并成功与抗吲哚美辛抗体交联。     

新型游离三碘甲腺原氨酸人工抗原的合成与表征

利用快速、温和的“Click Chemistry(点击化学)”反应,在游离三碘甲腺原氨酸(FT3)的羧基上引入一个带有三唑环的羧基“间接臂”,生成半抗原(FT3C)。 再用N-羟基琥珀酰亚胺活性酯法,将半抗原(FT3C)分别与牛血清白蛋白(BSA)和钥孔戚血蓝素(KLH)偶联,合成了免疫抗原(FT3C-BSA) 和包被抗原(FT3C-KLH)。 免疫抗原(FT3C-BSA)的紫外吸收光谱相对于FT3C和BSA有明显差异,表明成功实现了偶联,经计算得到偶联比为7∶1;用FT3C-BSA免疫Balb/c小鼠,获得高效价的抗FT3血清。 采用间接ELISA法检测抗体的IC50值为0.37 mg/L,抗血清效价可达6.24×104,是制备FT3人工抗原的一种新方法。     

基于酶标噬菌体抗体的磁分离免疫分析方法

以磁微粒偶联多抗为磁性捕获探针,酶标噬菌体抗体为特异信号检测探针,采用"磁性捕获探针-待测物-酶标噬菌体抗体探针"的检测模式,成功建立了一种基于酶标噬菌体抗体的磁分离免疫分析方法。本方法检测β-银环蛇毒素线性范围为0.016~62.5μg/L,回归方程为Y=0.641X+1.355(R=0.9925,n=13,p<0.0001),检出限为0.016μg/L。本方法比传统ELISA法检测灵敏度提高了10倍,与采用酶标单抗复合物探针的双抗体夹心磁分离免疫分析法相比,检测灵敏度提高4倍。本方法灵敏度高,具有较好重现性与特异性,在毒素的痕量检测方面具有广阔的应用前景。     

基于噬菌体展示抗体的电化学发光免疫传感器检测相思子毒素研究

以多克隆抗体包被磁性微球制备捕获探针,以表面负载大量三联吡啶钌标记物的噬菌体展示抗体为发光探针有效放大信号,成功建立了相思子毒素电化学发光免疫传感检测方法。利用本方法检测相思子毒素,浓度在0.005～100μg/L范围内与电化学发光强度呈良好的对数线性关系,拟合方程为lgY=0.701lgX+1.767(R=0.9964,N=7,p<0.0001),检测限达0.005μg/L(S/N=3)。与采用单克隆抗体制备标记探针相比,检测灵敏度提高20倍,可用于相思子毒素的痕量检测。     

应用纳米磁性球电化学检测特定序列DNA

采用分散聚合法制备纳米磁性羧基球,利用化学偶联法将末端修饰氨基的寡聚核苷酸固定在纳米磁性球表面,制成新型核酸探针,该探针可特异性结合目标单链寡聚核苷酸.在磁场作用下,将纳米磁珠与本体溶液分离并富集在电极表面,以中性红为嵌合指示剂,用示差脉冲伏安法测定杂交结果.经过条件优化,本法测定DNA的浓度线性范围为1.0×10-6～5.0×10-9mol/L,检出限为8.6×10-10mol/L.     

磁性明胶微球的制备及表征

生物高分子磁性微球作为性能优异的功能高分子材料在固定化酶、靶向药物、细胞分离和免疫分析等方面显示出强大的生命力。我们曾用凝胶-微乳液法化学剪裁技术制备了明胶包裹的复合磁性微粒，本文用共沉淀法制备磁性Fe3O4微晶作为磁性内核，明胶为包裹材料，     

Preparation of Functional Magnetic Nanoparticles and its Application in Diagnostic Analysis

The magnetic nanoparticles modified with carboxyl functional group were synthesized and characterized. These nanoparticles covalently bound with hepatitis B surface antibody（HBsAb）, were used to detect hepatitis B surface antigen （HBsAg） in immunovoltammetry. The detection limit was found to be 0.06 ng/mL, which is much higher than that of enzyme-linked immunosorbent assay （ELISA） used in clinical analysis.     

亚微米级免疫磁球及其在细菌分离中的应用

以亚微米级的单分散磁性微球为基础,制备出了表面包被有沙门氏菌抗体的免疫磁球. 利用表面电位、荧光和ELISA等方法研究了抗体在磁性微球表面的吸附行为. 在沙门氏菌磁分离实验中,通过调节投料抗体的浓度,研究了免疫磁球表面抗体浓度和磁分离效率的相关性,与微米级商品化免疫磁球的对比中,亚微米级的免疫磁球表现出了更高的磁分离效率.     

A novel amperometric immunosensor based on three-dimensional sol-gel network and nanoparticle self-assemble technique

A novel hepatitis B surface antigen (HBsAg) immunosensor has been developed by self-assembling gold nanoparticles to a thiol-containing sol-gel network. A cleaned gold electrode was first immersed in a hydrolyzed mercaptopropyltrimethoxysilane (MPS) sol-gel solution to assemble three-dimensional silica gel, and then gold nanoparticles were chemisorbed onto the thiol groups of the sol-gel network. Finally, hepatitis B surface antibody (HBsAb) was adsorbed onto the surface of the gold nanoparticles. Thus, an interfacial design of bare gold electrode (BGE)/MPS/Au/HBsAb was prepared to detect HBsAg in human serum based on the specific reaction of HBsAb and HBsAg. The electrochemistry of ferricyanide redox reaction was used as a marker to probe the interface and as a redox probe to determinate HBsAg. The main conditions of the assembly of MPS sol-gel, gold nanoparticles, the immobilization of HBsAb, and incubation time were investigated in detail. Compared with the glutaraldehyde binding approach, the antibodies immobilized by this method present larger amount and higher immunoactivity. The linearity of HBsAg in the range of 2-360 ng/mL with the correlation coefficient of 0.998 was obtained. This immunosensor system was evaluated on several clinical sample, the analytical results obtained by this method were in agreement with those detected by the enzyme-linked immunosorbent assay (ELISA) method, indicating a promising alternative tool for clinical diagnosis. Moreover, the studied immunosensor exhibited good reproducibility, long-term stability, high sensitivity and specificity.     

Immunomagnetic diffractometry for detection of diagnostic serum markers

We describe an integrated approach for detection of diagnostic markers using in situ assembled optical diffraction gratings in combination with immunomagnetic capture. Folate receptor (FR), a serum protein indicative of various cancers, was chosen as a model system to demonstrate the potential of the method. Magnetic beads coupled to FR antibody were used to capture FR from serum. The FR-bound magnetic beads self-assembled onto microcontact-printed folate-coupled BSA (F-BSA) patterns to form diffraction gratings which served to detect FR by measuring the diffraction intensities caused by laser illumination. The FR-containing beads, upon binding to the F-BSA surface, served as intrinsic signal enhancement agents, circumventing the need for additional enzymatic signal amplification or fluorescent labeling steps. With this approach, a detection sensitivity of 700 fM (20 pg/mL) was achieved. The potential use of this approach in clinical diagnostics was demonstrated by measuring FR concentration in blood samples obtained from cancer patients.     

Potentiometric Immunosensor Based on Immobilization of Hepatitis B Surface Antibody on Platinum Electrode Modified Silver Colloids and Polyvinyl Butyral as Matrixes

A highly sensitive immunosensor based on immobilization of hepatitis B surface antibody (HBsAb) on platinum electrode (Pt) modified silver colloids and polyvinyl butyral (PVB) as matrixes has been developed for potentiometric immunoanalysis to detect hepatitis B surface antigen (HBsAg) in this study. HBsAb molecules were immobilized successfully on nanometer‐sized silver colloid particles associated with polyvinyl butyral on a platinum electrode surface. The modification procedure was electrochemically monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The HBsAb‐silver‐PVB‐modified electrode exhibited direct electrochemical behavior toward HBsAg. The factors influencing the performance of the resulting immunosensor were studied in detail. More than 94.7% of the results of human serum samples obtained by this method were in agreement with those obtained by enzyme‐linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited a sigmoid curve with log HBsAg concentration, high sensitivity (39.8 mV/decade), wide linear range from 16.0 to 800 ng mL^?1 with a detection limit of 3.6 ng mL^?1, fast potentiometric response (<3 min) and long‐term stability (>4 months). The response mechanism of the immunosensors was also studied with AC impedance techniques.     

Polydiacetylene/Anti‐HBs Complexes for Visible and Fluorescent Detection of Hepatitis B Surface Antigen on a Nitrocellulose Membrane

The immunochromatographic assay (ICA) using a nitrocellulose (NC) membrane offers several advantages. This technique is a rapid and straightforward method in contrast to other immunoassays. Polydiacetylene (PDA) vesicles have unique optical properties, displaying red color and red fluorescence at the same time. In this system, red‐phase PDA vesicles are used as a fluorescent dye as well as a surface for immobilized hepatitis B surface antibody (HBsAb). PDA has a remarkable stability compared with other fluorescent dyes. In this study, the most suitable PDA/HBsAb complexes are introduced for detecting hepatitis B surface antigen (HBsAg). Then, the PDA/HBsAb complexes affixed antibody is attached to NC membrane, which has two lines to confirm detection of HBsAg. The main advantage of this system is that the detection of HBsAg can be observed in both visible and fluorescent images due to the optical properties of polydiacetylene. Detection of HBsAg is observed up to 0.1 ng mL⁻¹ by fluorescent analysis and confirmed by red line on the NC membrane up to 1 ng mL⁻¹ (HBsAg) using the naked eye. Consequently, these results show that PDA/HBsAb complexes were successfully applied to ICA for the diagnosis of hepatitis B.     

溶胶-凝胶-HBsAb膜免疫传感器的研制与应用

采用溶胶-凝胶(Sol-gel)技术,成功地将乙肝表面抗体(HBsAb)包埋于Sol-gel中,再滴涂于铂盘电极表面,制成溶胶-凝胶-HBsAb膜非标记免疫传感器.根据抗原与抗体特异性结合形成的免疫复合物使敏感膜有效扩散截面积减小的特性,提出了利用铁氰化钾作为氧化还原探针间接检测乙肝表面抗原(HBsAg)的新方法.用循环伏安法(CV)对电极逐层修饰过程进行了表征,并探讨了对HBsAg定量检测的可行性及其响应机理.采用差示脉冲伏安法(DPV)检测人体血清中的HBsAg.线性范围5～320μg/L,线性相关系数r=0.997.该传感器响应迅速,灵敏度高,稳定性好.于4℃干态保存14d,其响应信号基本不变.将其用于108例临床血清检验,与酶联免疫吸附法(ELISA)的符合率为87.5％.     

Gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis B surface antigen in human serum

The application of gold nanoparticle-based electrochemical immunoassays have been extensively studied for the detection of hepatitis B surface antigen (HBsAg), but most often they exhibit low sensitivity. We describe the fabrication of a new electrochemical immunoassay for signal amplification of the antigen-antibody reaction combined with the nanogold-based bio-barcode technique. Hepatitis B surface antibody (HBsAb) was initially immobilized on a nanogold/thionine/DNA-modified gold electrode, and then a sandwich-type immunoassay format was employed for the detection of HBsAg using nanogold-codified horseradish peroxidase-HBsAb conjugates as secondary antibodies. Under optimal conditions, the current response of the sandwich-type immunocomplex relative to the H₂O₂ system was proportional to HBsAg concentration in the range from 0.5 to 650 ng·mL^?1 with a detection limit of 0.1 ng·mL^?1 (S/N?=?3). The precision, reproducibility and stability of the immunosensor were acceptable. Subsequently, the immunosensors were used to assay HBsAg in human serum specimens. Analytical results were in agreement with those obtained by the standard chemiluminescence enzyme-linked immunosorbent assay.     

Selective and rapid immunomagnetic bead-based sample treatment for the liquid chromatography-electrospray ion-trap mass spectrometry detection of Ara h3/4 peanut protein in foods

Complex matrices commonly affect the sensitivity and selectivity of liquid chromatography-mass spectrometry (LC-MS) analysis. Thus, selective sample enrichment strategies are useful particularly to analyze organic biomarkers present in low abundance in samples. A selective immunomagnetic extraction procedure to isolate trace peanut allergen protein Ara h3/4 from breakfast cereals combined with microwave-assisted tryptic digestion and liquid chromatography-electrospray ion-trap tandem mass spectrometry (LC-ESI-IT-MS/MS) measurement was developed. Using protein A-coated magnetic bead (MB) support, anti-Ara h3/4 monoclonal antibodies (Abs) were used as selective capture molecules. The results obtained by LC-ESI-IT-MS/MS in terms of limit of detection (3mg peanuts/kg matrix) and a significantly reduced matrix effect demonstrated that the Ab-coated magnetic bead was very effective to selectively trap Ara h3/4 protein in breakfast cereals. The magnetic bead-based sample treatment followed by LC-IT-MS/MS method here developed can be proposed as very rapid and powerful confirmatory analytical method to verify the reliability of enzyme-linked immunosorbent assay (ELISA) screening methods, since the magnetic bead-LC-IT-MS/MS method combines good sensitivity to the identification capabilities of mass spectrometry.     

基于双层纳米金修饰的高灵敏电位型乙肝表面抗原免疫传感器研究

基于电沉积和层层自组装技术，提出了一种新的生物分子固定化方法，研制成一种高灵敏电位型乙肝表面抗原免疫传感器。利用L-半胱胺酸（LCys）的双官能团结合双层纳米金，从而通过比表面积大，生物相容性好的纳米金胶吸附大量抗体，同时用聚乙烯醇缩丁醛（PVB）薄膜的笼效应把乙肝表面抗体（HBsAb）和纳米金固定在玻碳电极上，从而制得了高灵敏度、高稳定性的电位型免疫传感器。采用循环伏安法（CV）对电极的层层自组装过程进行了考察，并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是8．5～256．0ng／mL，线性相关系数为0．9978，灵敏度为89．0，检出限为3．1ng／mL。已用于病人的血清样品分析。