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。     

基于巯基和聚电解质双层自组装膜的压电免疫传感器检测小鼠IgG气溶胶

本文结合自组装单分子层膜(SAMs)和聚电解质静电吸附组装技术,提出了一种新的用于气相压电免疫检测的生物分子固定化方法,研制了一种用于检测小鼠IgG抗体的压电免疫传感器。首先在石英晶片的金电极表面自组装了一层L-胱氨酸SAMs,再在膜上组装带相反电荷的海藻酸钠,最后通过调节pH值定向固定羊抗鼠纯化抗体,优化了固定条件。通过超声雾化法产生的小鼠IgG气溶胶,研制成了直接气相检测小鼠IgG的压电免疫系统。结果表明,该方法对所固定的生物分子活性影响较小,传感器对小鼠IgG的响应快,灵敏度高,在0.14~6μg.μL-1范围内具有良好的线性关系,精密度好,再生方便。     

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。     

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。     

基于等离子体聚合膜的日本血吸虫压电免疫传感器的研究

提出了一种测定日本血吸虫抗体的可逆压电免疫传感器。先在石英晶振上沉积正丁胺等离子体聚合膜,再自组装聚电解质,用以静电吸附固定日本血吸虫抗原。然后采用BSA和NRS作封闭剂,以封闭晶振上非特异必吸附位点,实现对日本血吸虫感染兔血清的测定。探讨了聚电解质(PSS和AASS)自组装、抗原包被和免疫反应等实难条件的影响;考察了该传感器的响应特性与再生性能,并与采用戊二醛共价键合固定法进行比较。发现该传感器具有灵敏度高、重现性好、非物异性吸附低、再生简便等优点。将它用于测定一系列不同感染程度的兔血清样本,结果表明,该传感系统是临床定性和定量诊断日本血吸虫病的一种有效工具。     

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 ,…     

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．     

一种高灵敏的压电免疫传感器用于高密度脂蛋白的测定

采用自组装单层膜(SAMs)技术在压电石英晶体的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,以共价键合方式固定高密度脂蛋白抗体,研制成一种高灵敏的压电免疫传感器用于检测人血清中低含量的高密度脂蛋白。利用压电装置的实时监测功能,考察了巯基丙酸在金电极表面的自组装成膜过程与机制;研究了晶振固定抗体及其反应结合抗原的液相振荡行为和传感特性。传感器采用初始速率法测定高密度脂蛋白的线性浓度范围为1.63~18.8 mg.L-1,检出限为0.82 mg.L-1。     

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.     

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…     

Development of a Quartz Piezoelectric Crystal Immunosensor Based on Self-Assembled Monolayer

A novel method of immobilizing antibodies on piezoelectric crystal (P/Z) sensor was developed based on self-assembled monolayer (SAM) using alkanethiols for immunosensing. The assembly of SAMs on metal surface is relatively easy and they are closely packed, well ordered and stable in ambient conditions. Thus,SAMs are good candidates for immobilizing biomolecules onto ultrathin layers.     

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.     

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.     

Immobilization of proteins on carboxylic acid functionalized nanopatterns

The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance–absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.     

Mechanism underlying bioinertness of self-assembled monolayers of oligo(ethyleneglycol)-terminated alkanethiols on gold: protein adsorption, platelet adhesion, and surface forces

The mechanism underlying the bioinertness of the self-assembled monolayers of oligo(ethylene glycol)-terminated alkanethiol (OEG-SAM) was investigated with protein adsorption experiments, platelet adhesion tests, and surface force measurements with an atomic force microscope (AFM). In this work, we performed systematic analysis with SAMs having various terminal groups (-OEG, -OH, -COOH, -NH(2), and -CH(3)). The results of the protein adsorption experiment by the quartz crystal microbalance (QCM) method suggested that having one EG unit and the neutrality of total charges of the terminal groups are essential for protein-resistance. In particular, QCM with energy dissipation analyses indicated that proteins absorb onto the OEG-SAM via a very weak interaction compared with other SAMs. Contrary to the protein resistance, at least three EG units as well as the charge neutrality of the SAM are found to be required for anti-platelet adhesion. When the identical SAMs were formed on both AFM probe and substrate, our force measurements revealed that only the OEG-SAMs possessing more than two EG units showed strong repulsion in the range of 4 to 6 nm. In addition, we found that the SAMs with other terminal groups did not exhibit such repulsion. The repulsion between OEG-SAMs was always observed independent of solution conditions [NaCl concentration (between 0 and 1 M) and pH (between 3 and 11)] and was not observed in solution mixed with ethanol, which disrupts the three-dimensional network of the water molecules. We therefore concluded that the repulsion originated from structured interfacial water molecules. Considering the correlation between the above results, we propose that the layer of the structured interfacial water with a thickness of 2 to 3 nm (half of the range of the repulsion observed in the surface force measurements) plays an important role in deterring proteins and platelets from adsorption or adhesion.     

Interfacial supramolecular self-assembled monolayers of C(60) by thiolated beta-cyclodextrin on gold surfaces via monoanionic C(60)

The supramolecular self-assembled monolayers (SAMs) of C(60) by thiolated beta-cyclodextrin (CD) on gold surfaces were constructed for the first time using C(60) monoanion. The results indicate that monoanionic C(60) plays a crucial role in the formation of the C(60)-containing self-assembled monolayers. The generation of C(60) monoanion and the formation process of C(60) SAMs were monitored in-situ by UV-visible and near-IR spectroscopy. The resulting C(60) SAMs were fully characterized by spectroscopic ellipsometry (SE), cyclic voltammetry, X-ray photoelectron spectroscopy (XPS), and water contact angle measurements. After the immobilization of C(60) by the SAMs of thiolated beta-CD, the film thickness increased by approximately 1 nm from 0.8 to 1.8 nm as determined by SE, demonstrating the formation of the supramolecular self-assembled monolayers of thiolated beta-CD/C(60). The new C(60) SAMs exhibited one quasi-reversible redox couple at half wave potential of -0.57 V vs SCE in aqueous solution containing 0.1 M KCl. The surface coverage of C(60) on the gold surfaces was estimated to be 1.1 x 10(-10) mol cm(-2). The XPS showed the assembly of C(60) over the thiolated beta-CD SAMs. The surface hydrophobicity increased greatly upon the formation of the C(60)-containing SAMs as analyzed by water contact angle measurements. The results are in agreement with the formation of 1:1 complex of C(60) and cyclodextrin on gold surfaces, though it also reveals some non-homogeneous features of the monolayers.     

Site-Directed Antibody Immobilization by Resorc[4]arene-Based Immunosensors

One of the main problems in the development of immunosensors is to overcome the complexity of binding antibodies to the sensor surface. Most immobilizing methods lead to a random orientation of antibodies with a lower binding site density and immunoaffinity. In order to control the orientation of antibody immobilization, several resorc[4]arene derivatives were designed and synthesized. After the spectroscopic characterization of resorc[4]arene self-assembled monolayers (SAMs) onto gold films, the surface coverage and the orientation of insulin antibody (Ab-Ins) were assessed by a surface plasmon resonance (SPR) technique and compared with a random immobilization method. Experimental results combined with theoretical studies confirmed the dipole–dipole interaction as an important factor in antibody orientation and demonstrated the importance of the upper rim functionalization of resorcarenes. Accordingly, resorcarene 5 showed a major binding force towards Ab-Ins thanks to the H-bond interactions with the amine protein groups. Based on these findings, the resorcarene-based immunosensor is a powerful system with improved sensitivity providing new insight into sensor development.     

3-氨基-1,2,4-三氮唑自组装膜对黄铜的缓蚀作用

3-氨基-1,2,4-三氮唑(ATA)是一种环境友好型金属处理剂, 以其在黄铜表面制备了自组装单分子膜(SAMs), 用电化学方法研究ATA SAMs对黄铜的缓蚀作用及其吸附行为. 结果表明, ATA分子易在黄铜表面形成稳定的ATA SAMs, SAMs抑制了黄铜的阳极氧化过程, 改变了电极表面的双电层结构, 固/液界面双电层电容明显降低, 有良好的缓蚀效果. 研究结果还表明, ATA的吸附行为符合Langmuir吸附等温式, 吸附机理是典型的化学吸附.