甲状腺素T4免疫传感器的研究

报道以甲基丙烯酸羟乙酯与甲基丙烯酸甲酯的共聚物为载体膜材料，研制成非酶标记的Ｔ４免疫传感器，并对载体膜材料的共聚方法及共聚物共聚比与传感器灵敏度的关系进行了探讨。     

新型乙肝压电免疫传感器的研制

通过自组装半胱胺在压电石英金电极上,用缩合剂（EDC和NHS）将羧基化碳纳米管和乙肝表面抗体连接到半胱胺膜,构建乙肝表面抗原的一种新的生物压电免疫传感器。传感器的灵敏度为17.268 Hz/（μg/mL）,线性范围为0.05μg/mL-15μg/mL,并能实时检测。     

适用于鱼肉中氯霉素检测的抗体包被金磁纳米微粒修饰安培免疫传感器研究

研制了基于氯霉素抗体包被Fe3O4/Au金磁纳米微粒(GMP)和三乙撑四胺铜(II)(CuL)共固定修饰平面热解石墨电极的安培免疫传感器(PRG|CuL / anti CAP-GMP),用于测定鱼肉中CAP含量.该免疫传感器是利用外加磁场,将anti CAP-GMP吸引到CuL修饰的PRG电极(PRG|CuL)表面制备而成.CuL对H2O2还原具有良好的电催化能力,当该传感器在含CAP样品液中温育后,CAP与电极表面的anti CAP的免疫结合物导致CuL对H2O2的催化还原电流(I)降低,电流下降值(△I)和CAP浓度成正比,可用于CAP定量测定.在25℃的pH=6.5磷酸盐缓冲液(PBS)中温育30 min,该传感器对CAP的检测线性范围为0.6~110 ng/mL,检出下限为0.092 ng/mL(3σ法).应用于鱼肉中CAP检测并与传统的液相色谱法(HPLC)比较,结果一致,其添加回收率在97%~104%之间.该免疫传感器集分离、富集为一体,电极表面可更新,检测灵敏快速,对于水产品中痕量氯霉素分析提供了一种新颖的方法.     

Graphene-Au nanoparticle based electrochemical immunosensor for fish pathogen Aphanomyces invadans detection

Epizootic ulcerative syndrome (EUS) that is primarily caused by an invasive oomycete fungus Aphanomyces invadans is a devastating fish disease. Rapid diagnosis of EUS is significantly important for the control and treatment of this highly invasive disease. In our study, a label-free immunosensor constructed with G-AuNPs/SAM-Ab-BSA/GCE was proposed for the determination of Aphanomyces invadans. The electrode was prepared by the immobilization of anti-mycelium antibodies on graphene-AuNPs nanocomposite-cysteamine monolayers modified GCE. The optimized parameters were as follows: 90 min as the immersion time of SAM modified electrode in the anti-mycelium solution, 0.20 µg/mL as the concentration of anti-mycelium solution and 10 min as the interaction time of immunoreaction. The immunosensors exhibited low limit detection of 309 ng/mL and good reproducibility.     

Multi-enzyme layer-by-layer assembly for dual amplified ultrasensitive electronic detection of cancer biomarkers

The preparation and use of multi-enzyme layer-by-layer (LBL) assembled single wall carbon nanotube (SWCNT) composite labels for amplified ultrasensitive electrochemical detection of a cancer biomarker is described. The target protein, carcinoembryonic antigen (CEA), is sandwiched between an electrode surface-confined capture anti-CEA antibody and the secondary signal anti-CEA/enzyme-LBL/SWCNT bioconjugate. The dual biocatalytic signal amplification for CEA monitoring is achieved through both the numerous enzymes loaded on the CNTs and redox-recycling of the enzymatic products in the presence of the secondary enzyme and the corresponding substrate. Our novel dramatic signal amplification strategy, with a detection limit of 0.04 pg mL⁻¹, shows about 2-4 orders of magnitude improvement in sensitivity for CEA detection compared with other universal signal amplified assays, which makes our signal amplification approach hold great potential applications in detection of ultra-trace protein biomarkers.     

日本血吸虫抗体电化学免疫传感器研究

该文研制了一种高灵敏的基于辣根过氧化物酶(HRP)放大的电化学免疫传感器用于日本血吸虫抗体的检测.实验采用夹心法的模式,在电极表面固定了日本血吸虫抗原(SjAg),用以捕获血清中的日本血吸虫抗体(SjAb),然后再与辣根过氧化物酶(HRP)标记的羊抗兔IgG二抗形成免疫复合物.用计时安培法检测HRP酶催化H2O2还原对苯二酚底物产牛的电流,其大小与日本血吸虫抗体(SjAb)浓度在一定范围内成正比.对免疫反应的实验条件如电极表面固定抗原的浓度,HRP酶标二抗的浓度进行了优化.此外,还考察了其它蛋白质对测定结果的影响.结果表明,该方法操作简单,灵敏度高,线性范围宽,检测下限低.     

Characteristics of a label-free piezoelectric immunosensor detecting Pseudomonas aeruginosa

A label-free immunosensor system detecting a psychrophylic bacterium, Pseudomonas aeruginosa was developed as follows. Four types of anti-P. aeruginosa antibody were individually chemisorbed onto one-side gold electrodes of piezoelectric quartz crystals according to a thiolated antibody coupling procedure initiated with a thiol-cleavable heterobifunctional cross-linker, sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate. A flow-type biosensor system was operated optimally at 0.2 M sodium potassium phosphate, pH 7.2 with a minimal matrix effect and the selected flow rate for it was 0.155 ml/min. A biosensor response was detected by measuring a steady-state resonant frequency shift after the response time around 8 min. The frequency shifts obtained were quite specific according to the antibody types and P. aeruginosa strains. The biosensor responses to varying concentrations of the P. aeruginosa cells ranging from 1.3×10⁷ to 1.3×10⁸ CFU/ml were determined as 17–176 Hz and a linear calibration curve (r=0.942) was obtained by plotting the responses in a double-logarithmic scale. The selectivity of the biosensor between P. aeruginosa and Xanthomonas spp. which both belong to the aerobic pseudomonads was, however, not so good owing to the property of the antibody used. The sensor chip could be reused at least seven times without an appreciable decrease in sensitivity.     

基于琼脂糖和纳米金的电流型免疫传感器快速检测副溶血性弧菌

将HRP-anti-VP掺杂于琼脂糖和纳米金中并修饰于丝网印刷电极表面,从而制备副溶血性弧菌免疫电极.采用循环伏安法表征免疫电极和监测酶促反应,根据还原电流的变化来定量测定副溶血性弧菌.在优化的实验条件下,免疫电极线性检测范围为105～109 cfu/mL,检出限为7.4×104 cfu/mL(S/N=3).该免疫电极具备较好的特异性、重现性(RSD＜6%)、稳定性(一周后电流响应为初始值的91%)和准确性(与ELISA符合率为97.5%),可初步用于副溶血性弧菌的快速筛检.     

基于纳米金/硫堇修饰金电极的脱落酸安培免疫传感器的研制

提出了一种基于纳米金/硫堇修饰金电极的ABA安培免疫传感器。该传感器基于H2O2-HRP-硫堇催化波体系构建,其中硫堇为传感介质。当HRP存在时,通过加入H2O2,硫堇的还原电流大幅增加,并且电流的增加依赖于HRP活性。HRP活性又由ABA与HRP酶标抗体结合物调控,产生一个减小的催化波。用BSA封闭硫堇单分子层修饰后可能存在的活性位点以避免非特异性吸附。优化了测定条件,包括酶标抗体和硫堇的最佳比例、培育时间、缓冲液的pH值和H2O2浓度。此传感器的还原电流在ABA浓度0.5~1000ng/mL范围内呈线性下降,回归方程为y=0.0209x 17.071,相关系数为0.9922,检测限为0.2ng/mL。     

Preparation of immunomagnetic beads coupled with a rhodamine hydrazine immunosensor for the detection of Mycobacterium avium subspecies paratuberculosis in bovine feces,milk, and colostrum

The aim of this study was to develop and evaluate a method for detecting Mycobacterium avium ssp. paratuberculosis (MAP) bacteria in bovine fecal, milk, and colostrum samples using immunomagnetic beads (IMB) and a rhodamine hydrazone immunosensor. Immunomagnetic beads were prepared by using purified antibodies from hyperimmunized sera that were coupled to Fe nanoparticles with diethylene triamine pentaacetic acid (DTPA) or ethyl (dimethyl aminopropyl) carbodiimide (EDC)-N-hydroxy succinimide (NHS) as linkers. Rhodamine hydrazone particles were synthesized and coupled to IgY anti-MAP antibodies using DTPA or EDC-NHS linkers. Separation efficiency of the IMB was tested on bovine fecal, milk, and colostrum samples experimentally contaminated with MAP. The studied methods were evaluated on their ability to detect MAP and separate bacteria in complex mediums. The ELISA results indicated 95% efficacy in antibody coupling to IMB, with the DTPA-IMB method being more efficient than the EDC-NHS-IMB method. By using the DTPA-IMB method, MAP bacteria were successfully recovered from fecal, milk, and colostrum samples. The DTPA-IMB method used in combination with the rhodamine hydrazone immunosensor had a limit of detection equal to 30 and 30,000 MAP cells/mL using chromogenic and fluorescent properties, respectively. Combining the DTPA-IMB separation method with the rhodamine hydrazone immunosensor provides a fast, sensitive, and cost-beneficial method for detecting MAP in bovine feces, milk, and colostrum.