基于蛋白A定向固定的Love波免疫传感技术研究

报道了一种基于蛋白A固定技术的新型Love波免疫传感器,可用于检测B型葡萄球菌肠毒素(SEB).本传感器由压电石英基片、叉指换能器和SiO2声波导层构成.为提高传感器对SEB分子的响应能力和检测灵敏度,设计了抗体分子的蛋白A定向固定路线,通过APTES和戊二醛层交联蛋白A,随即用100mmol/LNaBH4还原蛋白A与戊二醛层之间形成的Schiff's base结构,实现抗体分子在Love波器件表面高效率的定向固定.该固定化方案使传感器的再生能力和检测灵敏度都得到显著提高,对10 mg/L SEB进行连续5次检测后,其响应能力没有明显下降;而对SEB的浓度检出限度达到了10μg/L.原子力显微镜对传感器表面形态的成像结果表明,与戊二醛共价交联法相比,蛋白A结合法使抗体分子探针在传感器表面拥有更高的覆盖度和活性,对SEB具有更强的结合能力.     

基于蛋白A定向固定的Love波免疫传感技术研究

报道了一种基于蛋白A固定技术的新型Love波免疫传感器，可用于检测B型葡萄球菌肠毒素（SEB）。本传感器由压电石英基片、叉指换能器和SiO2声波导层构成。为提高传感器对SEB分子的响应能力和检测灵敏度，设计了抗体分子的蛋白A定向固定路线，通过APTES和戊二醛层交联蛋白A，随即用100mmol／L NaBH4还原蛋白A与戊二醛层之间形成的Schiff＇s base结构，实现抗体分子在Love波器件表面高效率的定向固定。该固定化方案使传感器的再生能力和检测灵敏度都得到显著提高，对1．0mg／L SEB进行连续5次检测后，其响应能力没有明显下降；而对SEB的浓度检出限度达到了1．0μg／L。原子力显微镜对传感器表面形态的成像结果表明，与戊二醛共价交联法相比，蛋白A结合法使抗体分子探针在传感器表面拥有更高的覆盖度和活性，对SEB具有更强的结合能力。     

检测大肠杆菌的免疫生物传感器研究进展

大肠杆菌是人体内常见的食源性致病菌,对其检测极为重要。免疫生物传感器技术作为目前研究较热的一种技术,在大肠杆菌的检测方面有不错的进展。本文综述了电化学、光学、压电等类型免疫传感器近十年的研究进展,从检测原理、抗体固定方式、修饰材料、检测能力等方面进行了综述,并对各自的优劣势进行了讨论,对免疫传感器的发展趋势进行了总结与展望。     

新型压电肿瘤标志物微阵列免疫传感器的研究

采用螺旋固定夹具构建一种新型插拔式压电石英晶体传感器,巯基化自组装技术固定抗人甲胎蛋白(AFP)、癌胚抗原(CEA)和前列腺特异抗原(PSA)的单克隆抗体,并组装成2×5型压电肿瘤标志物微阵列免疫传感器.研究了压电肿瘤标志物微阵列免疫传感器的响应特性及其影响因素.该微阵列传感器在AFP、CEA和PSA浓度分别为20～640 μg/L、1.56～50 μg/L和1.25～50 μg/L,压电石英晶体振荡频率偏移值对肿瘤标志物浓度呈现良好的响应特性.微阵列传感器用于68例临床血清标本的测定,结果与免疫化学发光法一致;相关系数分别为0.92、0.90和0.91.     

插拔式压电肿瘤标志物微阵列免疫传感器的研制

以AT切型、基频10 MHz的金膜石英晶体作为换能器,通过螺旋检测池固定夹具构建一种新型插拔式压电石英晶体传感器,并组装成2×5型压电肿瘤标志物微阵列免疫传感器。研究了传感器的响应特性及参数。该微阵列传感器在甲胎蛋白(AFP)、癌胚抗原(CEA)、前列腺特异性抗原(PSA)和人绒毛膜促性腺激素(hCG)质量浓度分别为20~640μg/L、1.56~50μg/L、1.25~50μg/L、2.5~250 mIU/mL的范围内,压电石英晶体振荡频率偏移值对肿瘤标志物浓度均呈现良好的响应特性。应用微阵列传感器测定68例临床血清标本,结果与化学发光免疫分析法符合(相关系数分别为0.92、0.90、0.91、0.94)。该压电肿瘤标志物传感器微阵列具有结构简单、操作方便、稳定性好、灵敏度和特异性高,不需标记,能实时检测和重复使用等优点,可用于临床实验诊断,具有临床推广应用价值。     

压电免疫传感器用于乙肝表面抗原的测定

乙肝表面抗原 ( HBs Ag)的检测是临床诊断乙型肝炎的一项重要指标 .目前常用酶联免疫法和放射免疫法检测 [1] ,但酶本身性质不稳定且价格昂贵、操作繁琐 ;而放射免疫法存在放射性废物难处理的局限性 .压电免疫传感器具有装置简单、价格便宜、灵敏度高、实时快速和无需标记等优点 ,广泛应用于环境监测、药物分析及微生物检测等多个领域 [2～ 4 ] .本文应用自组装单分子膜技术 ,在压电石英晶体表面形成致密有序的半胱胺单分子膜 ,通过戊二醛共价交联 ,将乙肝表面抗原单克隆抗体分子固定于晶体电极表面 ,研制成 HBs Ag压电免疫传感器 ,用于…     

甲胎蛋白压电免疫传感器的研究

以合成的甲基丙烯酸甲酯和甲基丙烯酸羟乙酯共聚作载体，在压电石英晶体表面涂覆共聚物涂层，经ＣＮＢｒ活化后固定ＡＦＰ抗体，研制成ＡＦＰ压电晶体免疫传感器，研究了固定化方法，载体量，抗原抗体结合时间，重现性选择性等实验条件和参数的影响，压电晶体的频移值与ＡＦＰ浓度在１００ｎｇ／ｍＬ～８００ｎｇ／ｍＬ的范围内有良好的线性关系，将此传感器初步用于人体血清样品的测定，并与放射免疫法对照，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。     

一种基于纳米二氧化硅增强凝集反应的压电免疫传感器

本文提出了一种基于抗体包被纳米粒子的简单快速的压电免疫凝集法,用于蛋白质检测。该方法原理是利用羊抗人IgG（G-anti-hIgG）包被的二氧化硅（或金）纳米粒子和人IgG（hIgG）发生免疫凝集反应而使得压电晶体频率发生改变进行测定。当凝集反应发生时,修饰在探针表面的G-anti-hIgG通过hIgG与G-anti-hIgG包被的纳米粒子结合,将质量效应和粘弹性因素叠加作用于压电晶体。结果表明这使得背景值大幅减小而信号明显增强。另外,对修饰后了抗体及结合免疫复合物的探针表面进行了SEM表征,对使用聚乙二醇作为增敏剂和实验最佳离子强度、pH值进行了优化选择。该传感器检测hIgG线性范围是0.26-16.7 mg mL-1,最低检出限为84 ng mL-1。     

用于液相中检测的压电免疫传感器研究

利用聚乙烯亚胺法,将Ｃ２型葡萄菌肠毒素（ＳＥ）抗体固化在未抛光的压电晶体的金电极上,利用双调式振荡电路制作的振荡器,构建压电免疫传感器,其稳定性,特异性都较好。     

Love waves in SiO(2) layers on STW-resonators based on LiTaO(3)

We present first studies of sensitivity increase of commercially available Murata SAF 380-type surface acoustic wave (SAW) devices by the excitation of Love waves. Sputtered SiO₂ is studied as wave-guiding layer. Excitation of Love waves on such devices is investigated both theoretically and experimentally. It is demonstrated that the application of an optimized wave-guiding layer increases the sensitivity. Both theoretical predictions and experiments yield an optimum layer thickness for maximum mass sensitivity between 3 and 4 μm for the given system.     

Label-free detection of HIV-2 antibodies in serum with an ultra-high frequency acoustic wave sensor

Herein is described a label-free immunosensor dedicated to the detection of HIV-2. The biosensor platform is constructed as a mixed self-assembled monolayer-coated quartz wafer onto which HIV-2 immunodominant epitopes are immobilized. The biosensing properties, in terms of specific vs. non-specific antigen-antibody interactions, are evaluated with the electromagnetic piezoelectric acoustic sensor (EMPAS) using equimolar serum solutions of HIV-2 or HIV-1 monoclonal antibodies, respectively. This immunosensor constitutes the first real-world application of the EMPAS technology in the bioanalytical field.     

On-line monitoring of polymer deposition for tailoring the waveguide characteristics of love-wave biosensors

The Love-wave sensor is an acoustic sensing device which is particularly suitable for sensing in a liquid environment. The superior characteristics of the device are achieved by the use of an acoustic waveguide, consisting of a thin layer deposited on the surface of the substrate material. The exact thickness and material properties of the layer will not only determine sensitivity and sensing performance of the resulting device but can also be adjusted to generate higher-order Love modes. Thus, to obtain a sensing device with the desired specifications, precise control over the process of waveguide deposition is required. This has been realized by implementation of a vapor deposition polymerization system where the transmission curve (amplitude vs frequency) of one of the sensing devices is continuously monitored during deposition. As soon as the desired device specifications are reached, the deposition can be interrupted immediately. From the recorded transmission curves, information about the sensitivity of the device can be deduced, and the formation of higher-order Love modes can be visualized. The system has been used to produce biosensors based on various Love modes. It is shown that sensors operated on higher-order Love modes have a high mass sensitivity which, together with their excellent shielding properties, makes them advantageous for biosensing in conducting buffer solutions.     

Poly(dimethylsiloxane)-coated sensor devices for the formation of supported lipid bilayers and the subsequent study of membrane interactions

The development of smooth hydrophilic surfaces that act as substrates for supported lipid bilayers (SLBs) is important for membrane studies in biology and biotechnology. In this article, it is shown that thin films of poly(dimethylsiloxane) (PDMS) formed on a sensor surface can be used as a substrate for the deposition of reproducible and homogeneous zwitterionic SLBs by the direct fusion of vesicles. Poly(dimethylsiloxane) solution (1% w/v) was spin coated on Love acoustic wave and surface plasmon resonance devices to form a thin PDMS layer. Acoustic, fluorescence, and contact angle measurements were used for the optimization of the PDMS film properties as a function of plasma etching time; parameters of interest involve the thickness and hydrophilicity of the film and the ability to induce the formation of homogeneous SLBs without adsorbed vesicles. The application of PDMS-coated sensor devices to the study membrane of interactions was demonstrated during the acoustic and fluorescence detection of the binding of melittin and defensin Crp4 peptides to model supported lipid bilayers.     

Insulin Surface Acoustic Wave Immunosensor Based on Immobilized C60/Anti‐Insulin Antibody

Immobilized fullerene C₆₀/anti‐insulin antibody was prepared and applied in shear horizontal surface acoustic wave (SH‐SAW) immunosensors to detect insulin in aqueous solutions. The immobilizations of anti‐insulin onto fullerene were studied through a C₆₀/PVC coated SH‐SAW sensor system in liquid. The partially irreversible frequency response for an anti‐insulin antibody was observed by the desorption study, which implied that fullerene could chemically react with anti‐insulin. C₆₀/anti‐insulin coating materials were successfully prepared and identified with an FTIR spectrometer. The C₆₀/anti‐insulin coated SH‐SAW immunosensors were developed and applied for detection of insulin in aqueous solutions. Within the range of normal human insulin concentration, the SH‐SAW immunosensors immobilized with C₆₀/anti‐insulin exhibited linear frequency responses to the concentration of insulin with a sensitivity of 130 Hz/pM. The SH‐SAW immunosensor immobilized with C₆₀/anti‐insulin showed a detection limit of 0.58 pM for insulin in aqueous solution. The interference of various common bio‐species in human blood, e.g. urea, ascorbic acid, tyrosine, and metal ions, to the SH‐SAW immunosensor immobilized with C₆₀/anti‐insulin for insulin was investigated. These common bio‐species interferences showed nearly no interference to the SAW immunosensors coated with C₆₀/anti‐insulin. The reproducibility of the SH‐SAW immunosensor immobilized with C₆₀/anti‐insulin for insulin was also investigated and is discussed.     

Fabrication and Packaging Technologies of Love-wave-based Microbalance for Fluid Analysis

Quartz crystal microbalance (QCM) based techniques have been developed for years to address various kinds of biochemical analyses in liquid media. An alternative to this approach based on guided acoustic shear waves, the socalled Love wave devices, has been proved to allow for increasing gravimetric sensitivity. However, this approach reveals more complicated to implement as the surface on which reactions are achieved is the same as the one used for electrical connection. As a consequence, a microfluidic set-up must be implemented to prevent unwanted interactions between the corresponding areas (IDTs and propagation path). The main issue when using SAW Sensors for in-liquid biochemical analyses [1-4], especially in a commercial objective, is the development of a reliable and reproducible fluidic system [5] meeting the main following requirements: i) low acoustic leakage. ii) chemically inert to biological samples. iii) reproducible fabrication at the wafer scale level.In the present work we explore the use of the SU-8 epoxy-based photoresist combined with silicon or quartz machined covers for the fabrication of this fluidic circuit. A first structure is fabricated using deep etch lithography, the cover is then glued to the remaining SU-8 structure using a thin glue layer. The packaging system prevents covering the IDTs with liquids and defines the sensing area in the region in-between the IDTs. Once the fabrication achieved, we evaluate the velocity and propagation loss using a network analyzer to measure the influence of the proposed packaging approaches on the principal wave characteristics.     

倏逝波光纤免疫传感器探头的修饰及表征

光纤探头上修饰生物识别分子是倏逝波光纤免疫传感器实现目标物检测的关键步骤. 以微囊藻毒素-LR (microcystin-LR, MC-LR)为例, 采用先将小分子半抗原MC-LR与经戊二醛活化的惰性蛋白(OVA)反应生成复合物MC-LR-OVA, 然后将该复合物通过双功能试剂连接到硅烷化后的光纤探头表面, 并采用XPS和倏逝波全光纤免疫传感器对其修饰效果进行表征. 结果表明, 修饰后探头表面化学元素组成随不同修饰步骤发生了显著的变化, MC-LR-OVA被共价键合到探头表面上. 探头对MC-LR抗体表现出强烈的特异性响应, 而对其它抗体蛋白的非特异性吸附很弱, 并且具有良好的再生性能. 因此, 该修饰方案适用于环境小分子污染物的检测.     

Detection of cortisol at a gold nanoparticle|Protein G-DTBP-scaffold modified electrochemical immunosensor

An ultrasensitive electrochemical immmunosensor was demonstrated to be capable of detecting the hormone cortisol down to concentrations as low as 16 pg mL(-1). In addition, the immunosensor displayed a sensitivity of 1.6 μA pg(-1) mL(-1) and a linear range up to ～2500 pg mL(-1) of cortisol. This immunosensor was constructed based on a Au nanoparticle|dimethyl 3,3'-dithiobispropionimidate·2HCl (DTBP)-Protein G scaffold-modified Au electrode. In this work, the Au nanoparticles were used to increase the electrochemically active surface area by 28% (with a standard deviation of 3%) to enhance the quantity of the Protein G scaffold on the electrode. Thiolation of Protein G by DTBP aided in avoiding the confirmation change of Protein G, while this Protein G-DTBP component offered an orientation-controlled immobilisation of the capture antibody on the Au electrode. In this immunosensor, a monoclonal anti-cortisol capture antibody was optimally aligned by the scaffold before a competitive immunoassay between sample cortisol and a horseradish peroxidase-labelled cortisol conjugate was conducted. For quantitative analysis, square wave voltammetry was used to monitor the reduction current of benzoquinone produced from a horseradish peroxidase catalysed reaction. The improved analytical performance of our immunosensor was attributed to the synergetic effect of Au nanoparticles and the Protein G-DTBP scaffold.     

Human immunoglobulin adsorption investigated by means of quartz crystal microbalance dissipation, atomic force microscopy, surface acoustic wave, and surface plasmon resonance techniques

Time-resolved adsorption behavior of a human immunoglobin G (hIgG) protein on a hydrophobized gold surface is investigated using multitechniques: quartz crystal microbalance/dissipation (QCM-D) technique; combined surface plasmon resonance (SPR) and Love mode surface acoustic wave (SAW) technique; combined QCM-D and atomic force microscopy (AFM) technique. The adsorbed hIgG forms interfacial structures varying in organization from a submonolayer to a multilayer. An "end-on" IgG orientation in the monolayer film, associated with the surface coverage results, does not corroborate with the effective protein thickness determined from SPR/SAW measurements. This inconsistence is interpreted by a deformation effect induced by conformation change. This conformation change is confirmed by QCM-D measurement. Combined SPR/SAW measurements suggest that the adsorbed protein barely contains water after extended contact with the hydrophobic surface. This limited interfacial hydration also contributed to a continuous conformation change in the adsorbed protein layer. The viscoelastic variation associated with interfacial conformation changes induces about 1.5 times overestimation of the mass uptake in the QCM-D measurements. The merit of combined multitechnique measurements is demonstrated.     

BSA‐templated Pb Nanocluster as a Biocompatible Signaling Probe for Electrochemical EGFR Immunosensing

We report here a new electrochemical probe for the development of a sensitive, and selective sandwich‐type electrochemical immunosensor for the detection of epidermal growth factor receptor (EGFR). The probe is a newly synthesized bovine serum albumin (BSA)‐templated Pb nanocluster (Pb_NC@BSA). For fabrication of the immunosensor, we employed streptavidin‐coated magnetic beads (MB) as a platform for immobilization of the biotinylated primary antibody (Ab₁), and utilized the Pb_NC@BSA conjugated to secondary antibody (Ab₂) as a signaling probe. After sandwiching the target protein between Ab₁ and Ab₂, we dissolved Pb_NC@BSA into an acid, and recorded square wave anodic stripping voltammetric (SWASV) signal of the Pb ions as an analytical signal for quantification of the EGFR. The immunosensor responded linearly towards EGFR within the range of 0.4 ng/mL to 35 ng/mL, with a detection limit of 8 pg/mL. The immunosensor displayed good sensitivity, selectivity, stability, and reproducibility, and proved suitable for direct measurement of EGFR in human serum samples. Moreover, we used the as‐synthesized Pb_NC@BSA as a fluorescence label for in vitro cell viability analysis as well as bioimaging of cancerous HeLa and non‐cancerous HUVEC cells. Pb_NC@BSA exhibited low cytotoxicity and high biocompatibility in living cells, and was a suitable fluorescent probe for live cell imaging, with potential therapeutic applications.