纳米金聚集复合物放大的计时电位法测定补体C3

报道了一种用金标复合物放大的计时电位法测定补体C₃的电化学免疫检测方法. 对影响传感器响应的因素如蛋白A固定浓度、抗体包被过程的pH及浓度、金标物混合比等进行了考察. 在优化的实验条件下, 传感器的信号响应和补体C₃的浓度在0.12～117.3 ng/mL范围内具有良好的线性关系, 检出限达0.02 ng/mL.     

基于巯基自组装单层膜技术的补体C3压电免疫传感器的研究

本文采用自组装单层膜(SAMs)技术在压电石英晶振金电极表面组装巯基丙酸SAMs,以盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)作偶联剂共价固定补体C3抗体,研制了一种检测人血清中补体C3成分的压电免疫传感器。研究了巯基丙酸的自组装和抗体的固定化条件,考察了晶振固定抗体后的液相振荡行为和检测特性。并利用压电传感装置的实时监测功能,研究了巯基丙酸在金电极表面的自组装成膜过程和补体C3免疫反应动力学,获得了重要的动力学依据和参数。传感器检测补体C3的线性范围为2．34～23．2μg／mL。将传感器用于临床样品的检测,所得结果与酶联免疫吸附法基本一致。     

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。     

一种基于静电吸附作用的直接检测补体C3新型可再生电容型免疫传感器

通过壳聚糖/褐藻酸钠体系实现抗体在电极上的固定,制得可多次再生的电容型免疫传感器,用于补体C₃的检测.先在金电极表面上组装一层半胱胺单分子层,通过戊二醛把壳聚糖修饰在金电极上,再用十二硫醇封闭电极,最后,利用壳聚糖与褐藻酸钠之间的强静电相互作用实现抗体的固定.使用交流阻抗法研究了溶液pH值和离子强度对电极膜层稳定性及对抗体固定性的影响.结果表明,所制备的传感器操作简便,易于再生,电容响应与补体C₃浓度在18.2～292.5ng/mL范围内呈线性关系,检出限为9.1ng/mL.     

Love波免疫传感器在免疫分析中的研究

Love波免疫传感器是一种以免疫反应为识别方式的新型声表面波传感器。与其它压电声波免疫传感技术相比,水平剪切型表面波、高的压电晶体基频以及声波导层的存在使Love波免疫传感器在可用于气液两相检测的同时具有更高的灵敏度,从而可以成为免疫分析中的一种重要的工具。本文分别从Love波传感器的构造、原理、发展现状和以抗体作为探针在传感器表面的固定化方法两方面综述了Love波免疫传感器的研究进展。     

基于网状混合自组装膜的压电免疫传感器及其应用

结合纳米金及混合自组装技术, 制备了一种新型网状混合膜, 提出了一种新的生物分子固定化方法, 研制了一种用于检测人血清抗精子抗体的压电免疫传感器. 首先, 将纳米金溶胶、巯基丙酸和1,6-二巯基己烷按一定的比例混合制得网状混合自组装膜, 然后将此膜组装到压电石英晶振的金电极表面, 经EDC/NHS活化后, 再将抗原固定到电极上, 实现对抗精子抗体的检测. 结果表明, 该方法能明显提高抗体抗原结合效率, 从而提高传感器的灵敏度, 并降低传感界面的非特异性吸附. 将此传感器应用于人血清抗精子抗体的检测, 线性范围为10~800 mU/mL, 检出限为7 mU/mL. 此传感器为抗精子抗体的临床检测提供了新平台.     

基于金属离子螯合的压电免疫传感器新型固定化方法

提出了一种基于金属离子螯合作用的压电免疫传感器新型固定化方法. 先在压电石英晶振表面沉积正丁胺等离子体聚合膜(BA-PPF), 再在BA-PPF表面修饰可与金属离子螯合的氨三乙酸基团, 用金属铜离子活化后, 修饰了二乙三胺五乙酸基团的IgG抗体蛋白质分子即可螯合固定于BA-PPF上. 将固定了抗体的压电石英传感器用于正常人免疫球蛋白IgG (NHIgG)的测定, 其频率响应与NHIgG浓度在0.36～63.8 μg/mL范围内存在良好的线性关系. 这种新型压电免疫传感器固定化方法简单快速, 具有良好的通用性.     

纳米金自组装膜的IgM压电免疫传感器的研究

利用等离子体聚合膜沉积技术和纳米金亚单层自组装技术设计传感器界面，用 于固定羊抗人M抗体，研制了一种新的M压电免疫传感器．先在石英品振上沉积正丁 胺等离子体聚合膜，通过戊二醛交联结合一半肮胺单层膜，利用膜上流基与纳米金 键合组装纳米金亚单层，得到可用于固定18kI抗体的界面，再以牛血清白蛋白 (BSA)和聚乙二醇(PEG)封闭晶振上的非特异性吸附位点．实验探讨了影响纳米金自 组装和抗体包被等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，与戊二醛共价交联固定法和金电极表面直接吸附固定法进行了比较．结果表明 ，以纳米金单层作界面固定抗体时，具有传感界面不需活化、固定抗体的活性高、 检测时的非特异性吸附小、传感器能反复再生等优点．将传感器用于实际样品的检 测，结果令人满意．     

补体C1q压电免疫传感器的研究

研制了一种可重复使用的压电免疫传感器用于检测补体C1q.比较了聚乙烯亚胺粘附、戊二醛交联法,物理吸附法以及蛋白A法三种固定化抗体蛋白的方法.使用蛋白A法,补体C1q在所测浓度范围内有良好的线性关系.使用过的晶片用0.1 mol/L柠檬酸盐缓冲溶液(pH 3.0)解吸,可重复使用4次.     

基于酶催化银沉积质量放大的血吸虫压电免疫传感器的研究

发展了一种基于酶催化金属银沉积信号放大的新型高灵敏气相压电免疫传感检测技术.先将血吸虫抗原(SjAg)共价固定在石英晶体表面,制备得到血吸虫压电免疫传感器.检测时,在晶振上滴加不同浓度的待测血吸虫抗体,再将碱性磷酸酶标记的二抗通过夹心方式结合到传感器表面.然后利用碱性磷酸酶催化磷酸化的抗坏血酸酯水解从而还原硝酸银,使金属银沉积在晶振表面上,放大传感器的质量响应信号.实验结果表明该传感检测方法可显著提高气相压电免疫传感器的检测灵敏度,传感器对血吸虫抗体的响应线性范围在1～225 ng/mL,检测下限为1 ng/mL.     

A novel biosensing interfacial design based on the assembled multilayers of the oppositely charged polyelectrolytes

A novel biosensing interfacial design strategy has been produced by the alternate adsorption of the oppositely charged polyelectrolytes. A quartz-crystal microbalance (QCM) as a model transducer was modified by use of mercaptoacetic acid (MAA) self-assembled monolayer (SAM) and the adsorption multilayers of the oppositely charged polyelectrolytes. MAA-SAM was first applied to the gold electrode surface of the crystal, and the positively charged chitosan was used as a double-sided linker to attach the negatively charged alginate-HSA antibodies to the negatively charged MAA-SAM layer. The assembly process and conditions were studied using the real-time output device and the surface topologies of the resulting crystals were characterized by atomic force microscopy (AFM) imaging. It is discovered that the optimal pH of immobilizing antibodies was 7.2 and the suited dilution ratio of antibodies was 10:30. The proposed immunosensor in optimal conditions has a linear detection range of 12.3-184.5 μg/mL for HSA detection. Comparing with the direct immobilization method of antibodies, the immunosensor with the proposed immobilization procedure shows some advantages, such as improved sensitivity due to the well-retained antibody activity and the significantly extended detection range. In particular, the regeneration of the developed immunosensor was simple and fast. Analytical results indicate that the developed immobilization procedure is a promising alternative for the immobilization of biorecognition element on the electrode surface.     

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 ,…     

Poly-L-lysine/hydroxyapatite/carbon nanotube hybrid nanocomposite applied for piezoelectric immunoassay of carbohydrate antigen 19-9

Hybrid composites are of special scientific interest for biochemical applications wherein the abilities to modulate the morphology and property of the hybrid material are important. In this paper, the formation of poly-L-lysine/hydroxyapatite/carbon nanotube (PLL/HA/CNT) hybrid nanoparticles is described and a general design strategy for an immunosensing platform has been proposed on the basis of PLL/HA/CNT nanocomposite adsorption of antibodies. Quartz crystal microbalance (QCM) used as a model transducer and the detection performances of the resulting immunosensor were investigated by use of the immuno-system of carbohydrate antigen 19-9 (CA19-9), an important indicator in the diagnosis of clinical cancers. The hybrid nanocomposite was characterized by the transmission electron microscope (TEM), scanning electron microscope (SEM) and Fourier transform-infrared (FT-IR) spectrum measurements. The frequency response characteristics for the processes of immobilization and immunoreaction of anchored anti-CA19-9 antibodies were studied in detail. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of CA19-9 in the concentration range of around 12.5-270.0 U ml(-1). Such an interface design with the hybrid nanocomposite should be tailored as a new alternative used for biosensor design.     

一种新的基于正丁胺等离子体聚合膜的免疫传感器抗体(抗原)固定化方法

等离子体聚合膜 ( Plasma- polymerized film)是由有机蒸气在辉光放电下聚合而成 ,这种高度交联的膜具有均匀、超薄、附着力强、化学稳定、机械性能良好、基质类型多样以及成膜有机物品种多样等优点 ,因此已引起了传感器工作者的兴趣 ,目前 ,所研制的传感器已用于有机气体的测定 [1 ,2 ] .Karube小组报道了乙烯二胺等离子体聚合膜在免疫传感器方面的应用[3,4] .但由于抗体直接共价键合于等离子体聚合膜上 ,无法洗脱 ,使等离子体聚合膜修饰的传感器不能再生 ,而不同批次沉积的等离子体聚合膜其交联度、活性基团含量等又难以一致 ,严重影响了…     

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69+/-8% in optimal conditions. When avidin-biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.     

A novel biosensing interfacial design produced by assembling nano-Au particles on amine-terminated plasma-polymerized films

A novel biosensing interfacial design strategy has, for the first time, been produced by assembling nano-Au particles on amine-terminated plasma-polymerized films (PPF). A quartz-crystal microbalance (QCM) as a model transducer was deposited with PPF of n-butylamine by use of a glow discharge and then treated with nano-Au particles. The kinetic assembly process and conditions were studied using the real-time-output device and the surface topology of the resulting crystal was characterized by atomic force microscopy (AFM) imaging. Based on analysis of the experimental data, including the association constant of Au–amine interaction, the assembly mechanism is considered to be partly or even mainly chemical adsorption. Moreover, immobilization of anti-human IgM antibody (IgM Ab), as an example, on the developed PPF-Au interface was investigated. It was found that antibody molecules immobilized by the proposed procedure had higher immunological activity than those immobilized by the conventional glutaraldehyde (GLU) cross-linking procedure or the direct gold-attachment procedure. The newly developed sensor had a better response, with a detection limit of IgM concentration as low as ~1.00 g mL^–1. In particular, the extremely high stability of both PPF and nano-Au monolayer formulated allows the designed biosensing interface to withstand harsh regeneration treatment, making it reusable.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

An impedimetric immunosensor for the detection of autoantibodies directed against gliadins

An immunosensor has been developed for the detection of autoantibodies directed against wheat gliadin, a protein fraction of cereal gluten which is involved in celiac disease. The immunosensor is based on the immobilization of gliadins onto gold electrodes covered with a polyelectrolyte layer of poly(4-styrenesulfonic acid sodium salt). The immobilization was monitored by quartz crystal microbalance (QCM) analysis. The antigen-antibody interaction signal was amplified by an incubation step with peroxidase-labeled immunoglobulins and subsequent peroxidase-catalyzed oxidation of 3-amino-9-ethylcarbazole (AEC). Changes in the insulating properties of the electrode layer were measured by electrochemical impedance spectroscopy (EIS) in the presence of ferri/ferro-cyanide. Impedance spectra could be fitted to a Randles equivalent circuit with high accuracy. Exposing the sensor electrodes to various antigliadin antibody concentrations resulted in proportional changes in the charge transfer resistance. A calibration graph for the detection of antigliadin antibodies was established for antibody concentrations between 10(-8) and 10(-6) M. Finally, the sensor was used for the determination of antigliadin autoantibodies of the IgG and IgA type in several human sera.     

Supramolecular Protein Immobilization on Lipid Bilayers

Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and obviates the use of harsh ligation conditions that could denature fragile proteins. In the work presented here, reversible supramolecular immobilization of proteins on lipid bilayer surfaces was achieved by using the host–guest interaction of the macrocyclic molecule cucurbit[8]uril. A fluorescent protein was successfully immobilized on the lipid bilayer by making use of the property of cucurbit[8]uril to host together a methylviologen and the indole of a tryptophan positioned on the N‐terminal of the protein. The supramolecular complex was anchored to the bilayer through a cholesterol moiety that was attached to the methylviologen tethered with a small polyethylene glycol spacer. Protein immobilization studies using a quartz crystal microbalance (QCM) showed the assembly of the supramolecular complexes on the bilayer. Specific immobilization through the protein N‐terminus is more efficient than through protein side‐chain events. Reversible surface release of the proteins could be achieved by washing with cucurbit[8]uril or buffer alone. The described system shows the potential of supramolecular assembly of proteins and provides a method for site‐specific protein immobilization under mild conditions in a reversible manner.