免疫传感器的发展与制作

本文介绍了几种常见免疫传感器及其换能器的发展概况,同时也对免疫传感器制作中的固定方法和再生性等问题进行了探讨。     

压电石英晶体免疫传感器及其应用

压电石英晶体免疫传感器是利用压电石英晶体振荡频率对表面质量负载的高度敏感性与免疫反应的高度特异性相结合发展起来的一种新型生物传感器。它具有灵敏度高、特异性好；操作简单，不需要任何标记；检测速度快，成本低廉等特点，有望成为临床实验诊断大规模应用的方法。仪器体积小，重量轻，能实时检测，特别适于环境监测、食品卫生监督、军事监视等野外流动作业。本就压电石央晶体免疫传感器的原理、基本结构、特点及其应用等方面进行综述。     

电化学免疫传感器的研究进展

近年来，随着电化学免疫传感器在各方面的广泛应用，关于提高它的性能、优化其检测方法的研究也越来越多。根据检测信号的不同，将其分为电位型、电流型、电导型、电容型免疫传感器。并逐一介绍了它们有关国内外研究进展。     

DNA生物传感器

本文介绍了DNA生物传感器的原理、构造,同时对DNA生物传感器的研究现状进行了论述.     

基于压电谐振检测技术的癌胚抗原免疫传感器的实验研究

为了建立一种新型压电石英晶体癌胚抗原(CEA)免疫传感器,传感器检测池用AT切向、基频10 MH z的石英晶体通过金属夹具和乳胶套圈固定组成,采用巯基化方法把抗人CEA单克隆抗体固定在金膜电极表面制成抗体敏感膜,构成的压电CEA免疫传感器应用于临床检测。实验结果显示:构建的压电CEA免疫传感器对CEA的响应特性良好,其线性检测范围为1.56~50.00 ng/m l,甲胎蛋白(AFP)、前列腺特异抗原(PSA)、人绒毛膜促性腺激素(hCG)等对CEA的检测基本无干扰;传感器可再生后重复使用5次;50例临床标本检测结果与放射免疫检测法符合(P>0.05),两种方法的相关系数为0.90。研制的压电CEA免疫传感器具有灵敏度高、特异性好、不需标记、操作简单、省时、能实时在线检测和重复使用等优点,对比分析表明其检测结果准确、可靠,可用于临床检验。     

血清中丙肝病毒的压电免疫传感器检测

构建一种新型的丙肝压电免疫传感器.采用AT切型、基频为10MHz石英晶体作为换能器,在清洗后的晶体金电极表面固定蛋白A之后固定丙肝的单克隆抗体,最后检测血清样品中的丙肝病毒.研究了不同固定方法的固定效果,并采用最佳方法做了阴阳对照实验、清洗实验、封阻实验、温度实验等,得到了初步的结果.此传感器具有特异性好、不需标记、操作简单、能实时在线检测和重复使用等优点,在临床实验诊断应用中具有潜在前途.     

乙肝表面抗原压电免疫传感器阵列的研制

采用戊二醛交联法,将乙肝表面抗体固定于石英晶体表面,研制成HBsAg压电免疫传感器阵列.根据频率改变值考察了传感器的固定化过程和传感器对乙肝表面抗原的响应特性.该传感器对乙肝表面抗原进行定量分析,线性范围为1～25 μg/ml,线性相关系数为0.9977.取临床血清进行检测,将结果与临床常用的ELISA法比较,探索了该传感器应用于临床检测的可行性.     

基于纳米材料的电化学发光免疫传感器

电化学发光传感器是人们研究比较活跃的一个领域,本文就近5年基于纳米颗粒、碳纳米管、量子点等纳米材料构建的电化学发光免疫传感器新技术做一综述。纳米颗粒、碳纳米管的高比表面积、生物相容性及高导电活性,不仅可以降低电化学发光的初始电位,还能加快电极与免疫材料间的电子传递并增强电化学发光强度,提高电化学发光检测的灵敏度及稳定性,已经广泛用于化学及生物化学分析。另外,量子点独特的光学性质,与碳纳米管、纳米颗粒等材料组成的纳米混合物,为开发新型电化学发光免疫传感器提供了一种新思路。     

Development of a Biosensor-based Immunoassay for Screening of Chloramphenicol Residues in Milk

Biosensor immunoassays were developed recently for antibiotics with an established maximum residue limit (MRL). In this study, according to the regulatory banning of chloramphenicol (CAP) use for food producing animals, the main objectives were: the specificity of the biosensor assay and the lowest detection limit possible. The assay was based on the inhibition of the binding of polyclonal antibodies against CAP to immobilized CAP on a sensor chip by CAP in solution. The response varied inversely with the antibiotic concentration in the sample. Two different antibodies and two immobilization protocols were tested. As in ELISA tests the antibody influenced the assay performances. Moreover, we showed that particular care should be concentrated on the immobilization step because it is a critical point in the assay development. Three different protocols were developed in milk. The best assay was obtained with antibody 1 in milk on the CAP base surface because of its very low detection limit (0.1 μg l^-1) and the decreased consumption of antibody (four times less than on the CAP surface). This assay is rapid (3 min/run), sensitive, and specific for CAP and CAP glucuronide. It could be integrated in a multi-residue screening test and applied to other matrices (bile, urine, meat).     

A Biosensor-based Immunoassay for Rapid Screening of Deoxynivalenol Contamination in Wheat

A surface plasmon resonance (SPR) based indirect inhibitive immunoassay was developed for the rapid quantification of concentrations of the trichothecene mycotoxin deoxynivalenol (DON). A DON-biotin conjugate was synthesized and immobilized to a streptavidin coated SPR sensor surface to measure free anti-DON antibody added to a sample. For analysis, ground wheat was extracted with 10% (v/v) methanol in water with 6% (w/v) polyvinyl-pyrrolidone, filtered and cleaned up with MycoSep™ columns. Extracts were mixed with a polyclonal anti-DON antibody and pre-incubated prior to injection into a BIAcore® device. The sensor surface was regenerated with a rinse of 6 M-guanidinechloride in 10 mm-glycine (pH 2.9). The assay had a working range between 0.13 and 10.0 μg ml -1 of DON, a 50% inhibition concentration (IC 50 ) of 0.72 μg ml -1 , and a detection limit of 2.5 pg μl -1 . Recovery of DON in spiked wheat was 104 ±15%. The system enables analysis of a sample to be completed in 15 min including sample preparation (10 min) and quantification (5 min). The assay was applied to the analysis of wheat samples with different levels of DON contamination. Statistical analysis revealed a positive, linear correlation between concentrations of DON measured with the new biosensor and GC/MS or HPLC as reference methods. Coefficients of correlation of R 2 = 0.9464 (GC/MS data) and R 2 = 0.9066 (HPLC data) were found.     

A Direct (Non-Competitive) Immunoassay for Gentamicin Residues with an Optical Biosensor

Using sensor chip immobilized anti-gentamicin monoclonal antibodies in an optical biosensor (BIACORE 3000) resulted in the first single antibody based and label free noncompetitive immunoassay for the direct detection of residues of a low molecular weight compound in a food product. Calibration curves for gentamicin (Mw 466 Da) in buffer and milk showed 50% binding at 20 and 35 ng ml^-1, respectively, which is well below the maximum residue limit of 100 ng ml^-1.     

Direct Versus Competitive Biosensor Immunoassays for the Detection of (Dihydro)Streptomycin Residues in Milk

A monoclonal antibody (MAb) against dihydrostreptomycin (4G8) was developed and its performance compared with a previously developed MAb against streptomycin (4E2) in biosensor immunoassays (BIAs) using a surface plasmon resonance (SPR)-based biosensor (BIACORE 3000). Direct BIAs for the detection of dihydrostreptomycin (DHS; 583 Da) and streptomycin (STREP; 581 Da) were developed by immobilising the MAbs on the sensor chip (CM5). These direct BIAs were compared with competitive inhibition BIAs, using a STREP- protein conjugate immobilized on the chip. The sensitivities of the direct and competitive BIAs for both drugs in buffer were comparable (10-20 ng ml- 1 at 50% binding or inhibition). With milk, interferences, probably due to the nonspecific binding of proteins to the sensor chips, were observed in both BIAs. These interferences could be largely reduced using ultra filtration (UF) as sample pre-treatment. Another option was the use of a reference flow channel to correct for nonspecific binding. Using this option with five times diluted milk, MAb 4G8 was found to be suited for the direct BIA of both drugs with a limit of detection (LOD) of 20 ng ml- 1 and both MAbs could be applied in the competitive BIA format with similar LODs.     

Epitope mapping and binding kinetics of monoclonal antibodies studied by real time biospecific interaction analysis using surface plasmon resonance

The interaction between human heart myoglobin and ten specific monoclonal antibodies was investigated with a new biosensor technology, real time biospecific interaction analysis (RT BIA), using surface plasmon resonance. Analysis of association and dissociation kinetics was monitored in real time, with unlabelled reactants. Antibody isotyping was rapid and simple. Epitope mapping with RT BIA confirmed, with substantial time saving, the sum of results obtained in conventional labelled systems. Monoclonal antibodies with four different epitope specificities and optimal binding function were selected for a myoglobin sandwich assay with enhanced sensitivity. BIAcore can be used directly as a diagnostic tool, or as an analytical tool in immunoassay development.     

梅毒压电石英晶体免疫传感器的研制

分别用聚乙烯亚胺(PE I)和戊二醛(G lu)溶液在压电石英晶体银电极上固定梅毒抗体来检测不同浓度的标准抗原溶液。抗体在晶体表面固化后通过原子力显微镜(AFM)显示依然保持Y型分子结构。石英晶体的响应线性范围5×10-5~1.25×10-4g/mL,相关系数为0.9976,最佳响应pH值为7.5。在检测时通过和BSA相对照,传感器有良好的选择性。最后,又对传感器重复性作了一定探讨。     

碳纳米管及其复合材料在生物医学领域的研究进展

碳纳米管是一种新型碳材料，具有特殊的纳米结构和优异的力学、电学和磁学性能，在生物医学领域显示出诱人的潜在应用价值和前景，吸引了越来越多的研究者的关注。本文简要介绍了碳纳米管的基本结构与性能，并对近年来碳纳米管在生物大分子的修饰与特异性识别、细胞体外生长支架、植入性生物医学材料、生物医学传感器以及生物医学微电子器件等方面的研究进展和发展趋势进行了综合评述。     

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.     

DNA场效应管传感器的分子设计

为满足临床医学基因诊断的需要,本研究以场效应管为基本传感器,并在栅区构建单链核苷酸敏感栅,实现对目的基因快速、定点、在体检测。本文介绍了ＤＮＡ场效应管传感器的敏感机理和定量依据,并就传感器结构、自组装单分子膜技术、杂交指示剂、多道测量技术等进行设计和探讨。     

Development of Immunochemical Techniques for Detecting Karnal Bunt in Wheat

The protein profile of Tilletia indica, the causal agent of Karnal bunt, was analyzed by SDS-PAGE and compared to protein profiles of T. barclayana, T. tritici, T. controversa and T. laevis. Based on these results, antibodies were developed against a 64 kDa T. indica protein and used to construct both a microwell sandwich ELISA and a dipstick immunoassay. Both of these immunoassays can detect 1.25 ng/well of purified T. indica protein or 40 ng/well of crude spore extract, distinguishing Karnal bunt from all wheat smuts and, to some degree, the rice smut, T. barclayana. To enhance specificity, an innovative approach to species-specific antibody production was utilized to develop antibodies that discern Karnal bunt from all other fungal species. Using these new antibodies, a Karnal bunt specific immunoassay was developed that has all the attributes of the original Karnal bunt immunoassays and detects Karnal bunt in the presence of wheat. This is the first report of immunochemical diagnostic tools that will aid in controlling the spread of this wheat pathogen by accurately and efficiently detecting Karnal bunt teliospores in wheat.