基于巯基自组装单层膜的日本血吸虫石英晶体微天平免疫传感器

应用自组装膜技术在压电石英晶振金电极表面自组装一带羧基的巯基丙酸单层膜,通过盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺及N-羟基琥珀酰亚胺共价固定32KD的日本血吸虫分子抗原(SjAg32),设计了石英晶振微天平免疫传感器,用于测定日本血吸虫抗体.比较了巯基自组装单层膜与HEMA-MMA共聚物涂层修饰的石英晶振在溶液中的振荡行为,发现巯基自组装单层膜修饰的石英晶振稳定快,且稳定性好.在优化条件下,测得IRS(49-2000)的滴度为1:1500.此外,对不同程度血吸虫感染的兔血清进行了测试,结果表明,该传感器能较好地定量区别血吸虫感染程度.     

一种新的转铁蛋白压电免疫传感器的研究

报道了一种基于等离子体聚合膜、结合聚电解质设计的转铁蛋白压电免疫传感器.采用辉光放电等离子体聚合技术,在石英晶体上沉积一层正丁胺聚合膜,再在膜上自组装一层易再生的、带负电的聚电解质,调节抗体溶液的pH值使其带正电,经静电吸附包被抗体后用以测定抗原.探讨了自组装聚电解质的浓度和自组装时间,抗体的包被浓度、包被时间和pH值以及免疫反应的酸度、温度及响应频率与时间的关系等实验条件的影响.考察了传感器的灵敏度、选择性、重现性和再生性能.用传感器测定人血清中转铁蛋白的线性范围为0.10～12.65μg/mL.将其用于实际样品中转铁蛋白的测定,结果与酶联免疫法基本一致.     

一种新的转铁蛋白压电负免传感器的研究

报道了一种基于等离子体聚合膜、结合聚电解设计的转铁蛋白压电免疫传感器，采用辉光放电等离子体聚合技术，在石英晶体上沉积一层正丁胺聚合膜，再在膜上自组装一层易再生的，带负电压的聚电解抽，调节抗体溶液的P 带正电，经静电吸附包被抗体后用以测定抗原，探讨了自组装聚电解质的浓度和自组装时间，抗体的包被浓度，包被时间和PH值以及免疫反应的酸度，温度及响应频率与时间的关系等实验条件的影响，考察了传感器的灵敏度、选择性、重现性和再生性能，用传感器测定人血清中转铁蛋白的线性范围为0．10－12．65μg/mL。将其用于实际样品中转铁蛋白的测定，结果与酶联免疫法基本一致。     

一种新的压电免疫传感器中生物分子固定化方法的研究

生物分子固定化或传感界面设计技术是研制压电免疫传感器的关键之一。本文 结合自组装单分子膜（SAMs）和聚电解质静电吸附组装技术，提出了一种新的压电 免疫传感器中生物分子固定化方法，研制成一种检测补体C_3的压电免疫传感器。 先在石英晶振的金电极表面组装一层胱胺SAMs，再在膜上组装带相反电荷的聚苯磺 酸钠（PSS）单层膜，通过静电吸附作用固定抗体（抗原），实现对相应抗原（抗 体）的检测。利用扫描电镜技术，从形态上考察了晶振组装胱氨SAMs与PSS及固定 补体C_3抗体后的表面形貌。研究了抗体的固定化条件，探讨了传感器采用这种固 定化方法的响应与再生性能，并与戊二醛键合固定法进行比较。结果表明，这种固 定化方法不仅对蛋白质类生物分子的固定化具有普适性，而且对所固定的生物分子 的活性影响小，传感器的响应的频移值大，灵敏度高，选择性和再生性能均较好。     

基于磁性纳米颗粒的日本血吸虫抗体荧光免疫分析

研究了一种基于磁性纳米颗粒的日本血吸虫荧光免疫分析方法。日本血吸虫抗原通过共价吸附到核壳结构的磁性颗粒表面,与待测抗体结合后,再与酶标二抗夹心反应,最后以3,3′,5,5′-四甲基联苯胺(TMB)为底物,通过测定酶催化下TMB氧化生成无荧光的二聚体化合物,使溶液荧光强度降低来间接测定日本血吸虫抗体的浓度。应用本方法测定了兔血清中日本血吸虫抗体,荧光强度(If)与抗体浓度(C)在5.0~100μg/L之间呈线性关系,线性回归方程为If=225.8-1.6C(r=0.9976),检出限达1.5μg/L。方法简单实用,具有良好的检测灵敏度和重现性。     

基于壳聚糖-溴化氰改性褐藻酸钠凝集作用的日本血吸虫安培免疫传感器

研制了一种基于壳聚糖和溴化氰改性褐藻酸钠凝集作用的日本血吸虫安培免疫传感器.褐藻酸钠-抗体复合物通过静电吸附作用被凝集到含石墨-石蜡-壳聚糖组分的电极表面,然后与抗原和酶标抗原进行竞争反应,以邻氨基酚(o-AP)为电子媒介,通过测定酶催化下双氧水对其氧化的电流大小来间接测定抗原的浓度.研究表明这种免疫传感器具有很低的非特异吸附性能,而且在经过简单的处理后可以重复使用,其重现性和灵敏度良好.传感器对日本血吸虫抗原的响应在0.64～40μg/mL之间呈线性关系.检测限为0.64μg/mL.将电极应用于兔血清中日本血吸虫抗原的测定,取得了满意结果.     

基于胱氨自组装膜的压电免疫传感器检测抗凝血酶Ⅲ

提出了一种基于胱氨自组装膜和采用聚电解质吸附法固定活性物质的压电免疫传感器，用于检测人血浆中抗凝血酶Ⅲ（AT－Ⅲ）。先在压电石英晶振的金电极表面自组装一层带疏基的胱氨单分子膜，再在膜上组装一层聚电解质褐藻酸钠（AAS），通过静电吸附作用，将AT－Ⅲ抗体固定于石英晶体表面，在含有3.5%聚乙二醇（PEG）的缓冲溶液中检测AT－Ⅲ。比较了传感器分别采用AAS吸附法和戊二醛键合法固定AT－Ⅲ抗体的响应性能，发现前者固定的抗体的活性较高，反应响应的频移值较大，检测的线性范围也较宽。实验采用PEG作免疫反应的促进剂，进一步改善了传感器的检测灵敏度和检测限。采用Piranha试剂作洗脱液可实现传感器的反复再生。干扰与回收率实验结果表明，该传感系统可用于人血浆中AT－Ⅲ的临床检测。     

基于Nafion膜修饰的血吸虫抗体压电免疫传感器研究

将Nafion膜包埋血吸虫抗原固定在石英晶体微天平表面上,构制了用于测定血清中血吸虫抗体的压电免疫传感器.这种生物敏感膜由涂敷在石英晶片表面上的一滴Nafion+血吸虫抗原磷酸盐缓冲液干燥而成,它的构制和再生过程具有良好的重现性.用扫描电镜观察了这种生物敏感膜的形貌,并对传感器的构制参数、测量条件进行了优化,优化的传感器对血吸虫抗体的检测范围为0.2～6.0 mg/L.本传感器具有仪器简单、操作方便、灵敏度高、重现性好等优点,可判断血吸虫感染情况.     

基于Nafion膜修饰的血吸虫抗体压电免疫传感器研究

将Nafion膜包埋血吸虫抗原固定在石英晶体微天平表面上，构制了用于测定血清中血吸虫抗体的压电免疫传感器。这种生物敏感膜由涂敷在石英晶片表面上的一滴Nafion＋血吸虫抗原磷酸盐缓冲液干燥而成，它的构制和再生过程具有良好的重现性。用扫描电镜观察了这种生物敏感膜的形貌，并对传感器的构制参数、测量条件进行了优化，优化的传感器埘血吸虫抗体的检测范围为0．2～6．0mg／L。本传感器具有仪器简单、操作方便、灵敏度高、重现性好等优点，可判断血吸虫感染情况。     

基于电聚合膜和纳米金自组装的生物分子固定化新方法的研究

结合电聚合膜和纳米金自组装技术,提出了一种新的生物分子固定化方法,研制成一种检测抗胰蛋白酶的压电免疫传感器。通过在石英晶振金电极表面电聚合邻苯二胺膜,再在膜表面自组装一层纳米金粒．以静电吸附作用固定抗体(抗原),实现对相应抗原(抗体)的检测。利用扫描电镜技术,从形态上考察了晶振金电极上自组装纳米金后的表面形貌。研究了抗体的固定化条件,探讨了传感器的响应与再生性能结果表日月．这种固定化方法对所固定的生物分子的生物活性影响小,传感器的测定灵敏度高．响应性能和再生性能较好。     

Quartz-crystal microbalance immunosensor for Schistsoma-japonicum-infected rabbit serum.

A quartz-crystal microbalance immunosensor (QCM) has been developed for the direct determination of Schistosoma-japonicum-infected rabbit serum. A self-assembled monolayer with carboxyl groups was first coated on a gold electrode of a quartz-crystal resonator by the spontaneous adsorption of 3-mercaptopropionic acid. Schistosoma-japonicum molecular antigen of 32 kD molecular weight was then covalently attached to the crystal surface. The QCM immunosensor was used to detect infected rabbit serum (IRS49-2000); a maximum titer of 1:800 was achieved.     

基于邻苯二胺电聚合膜的抗体固定化方法研究

Si等 [1] 在压电石英晶体金电极表面先电聚合了一层聚苯胺膜 (PAn) ,再于 PAn膜上电聚合一层聚间苯二胺膜 (Pm PD) ,形成一双层膜 (Pm PD和 PAn) ,而后通过戊二醛共价键合固定化方法 ,实现对生物蛋白质分子的固定和对生物细胞的测定 .但在上述方法中 ,传感器难以再生且蛋白质分子的固定量较少 .参照文献 [2 ],本文提出了一种在电聚合邻苯二胺薄膜上进行可逆的抗体固定化的新方法 .通过控制溶液的 p H值 ,在带正电的电聚合邻苯二胺膜表面先自组装一层聚阴离子聚苯磺酸根 (PSS)层 ,使传感器得到一个带负电的载体表面 ,再通过静电吸附 ,…     

An amperometric immunosensor based on a conducting immunocomposite electrode for the determination of Schistosoma japonicum antigen.

A renewable amperometric immunosensor based on a graphite-paraffin-Schistosoma japonicum antibody (SjAb) biocomposite electrode has been prepared for the detection of Schistosoma japonicum antigen (SjAg). Competitive ELISA was employed involving HRP-SjAg as a tracer and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The product of an enzyme catalytic reaction was detected at +0.1 V (vs. Ag/AgCl reference electrode) for measuring the amount of HRP-labeled SjAg binding to the electrode surface. The assay conditions were optimized, including the amount of SjAb loading in the electrode and HRP-SjAg in the incubation solution, the pH of the measuring solution and the incubation time. The measuring range was 0.5-30 microg/ml under the optimum conditions. Rabbit serum samples of different infection degree were measured, which demonstrated that the immunosensor meets the demands of clinical analysis. It exhibits some advantages, such as simplicity of fabrication, rapidity of measurement, and satisfactory sensitivity and reproducibility.     

A piezoelectric immunoassay based on self-assembled monolayers of cystamine and polystyrene sulfonate for determination of Schistosoma japonicum antibodies

A piezoelectric immunosensor based on an improved immobilization strategy combining self-assembled monolayers (SAM) of cystamine (Cys) and polystyrene sulfonate (PSS) has been developed for the determination of Schistosoma japonicum antibodies (SjAb) in rabbit serum. Cys SAM were first applied to the gold electrode surface of the crystal, serving as a positively-charged base. Schistosoma japonicum antigen (SjAg) was then electrostatically immobilized on the crystal by means of a negatively-charged PSS layer. When sealed by use of an appropriately selected blocking reagent for BSA and normal rabbit serum (NRS), non-specific adsorption could be substantially reduced.The immunosensor was used to determine SjAb in optimized buffer medium with addition of poly(ethylene glycol) (PEG), which served as an immunoreaction enhancer. It was shown experimentally that SjAg immobilized by the Cys-PSS adsorption procedure had higher immunological activity or binding efficiency than those immobilized by the glutaraldehyde (GLU) binding or direct attachment procedures. The immunosensor developed had satisfactory sensitivity and detection limit, and regeneration of the piezoelectric quartz-crystal was easy. Analytical results obtained with infected rabbit serum samples indicated that the proposed immunosensor is a promising alternative for qualitative and quantitative determination of SjAb in clinical diagnosis of infection with Schistosoma japonicum.     

Electrochemical Detection of Schistosoma Japonicum Antibody Using Biocatalytic Deposition

Abstract

An ultrasensitive electrochemical immunoassay based on biocatalytic deposition has been proposed for the detection of Schistosoma japonicum antibody (SjAb) in infected rabbit serum. Schistosoma japonicum antigen (SjAg) was immobilized on the gold electrode surface via glutaraldehyde crosslink and then incubated with infected rabbit serum containing SjAb; finally, the goat anti-rabbit IgG labeled with alkaline phosphatase was sandwiched to form the immunocomplex on the gold electrode surface. The alkaline phosphatase converted nonelectroactive substrate into the reducing agent and the latter, in turn, reduced metal ions to form electroactive metallic product on the electrode surface. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for SjAb. Assay conditions such as the antigen concentration and enzymatic silver deposition time were optimized. The electrochemical immunosensor was able to realize a reliable determination of SjAb in the dilution range from 1:5000 to 1:100 with a detection limit of 1:6457 of dilution ratio. The feasibility of the proposed immunosensor for possible clinical applications was also investigated by analyzing real serum samples.     

Silica sol-gel amperometric immunosensor for Schistosoma japonicum antibody assay.

An amperometric immunosensor was constructed by dispersing graphite, schistosoma-japonicum antigen (SjAg) and silica sol-gel at low temperature. The performance characteristics of the prepared immunosensor were examined in the buffered solution of o-aminophenol (o-AP) used as a substrate. It exhibited excellent physical and electrochemical stability with a renewable external surface. A competitive binding assay was employed to determine schistosoma-japonicum antibody (SjAb) with the aid of horseradish peroxidase labeled SjAb (HRP-SjAb). The experimental parameters for SjAb assay were optimized, including the amount of labeled SjAb in incubation solution, incubation time, temperature and the pH of solution. The use of o-AP substrate and amperometric detection at -250 mV (vs. SCE) results in a determination limit of 0.32 microg/ml and a linear range extending up to 0.18 microg/ml. The results of SjAb assay in serum samples demonstrate the feasibility of using the proposed immunosensor for clinical analysis.     

Quartz crystal microbalance immunoassay for carcinoma antigen 125 based on gold nanowire-functionalized biomimetic interface

Gold nanowires with of designed length on a solid substrate have been proven as an efficiently immobilized affinity support for the detection of carcinoma antigen 125 (CA 125) in this study. The presence of gold nanowires provides a well-defined three-dimensional structure, and greatly amplifies the coverage of the anti-CA 125 protein on the probe surface. Moreover, the amount of anti-CA 125 varied with the change of the morphology of the probe, and achieved an optimal quartz crystal microbalance (QCM) response towards anti-CA 125 adsorption at the number of gold nanolayers of 5. The formed immune-probe exhibits good QCM responses for the detection of CA 125, and allows the detection of CA 125 at concentrations as low as 0.5 U ml(-1). The QCM immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. The as-prepared immunosensors were used to analyze CA 125 in human serum specimens. Analytical results suggest that the developed immunoassay method is a promising alternative approach for detecting CA 125 in the clinical diagnosis. Compared with conventional ELISA, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the route provides an alternative approach to incorporate multiple gold nanolayers onto the solid matrix for biosensing applications.