蝰蛇毒压电免疫传感器固定方法的研究

目的：为研制蝰蛇毒压电免疫传感器，研究抗蛇毒抗体固定于石英晶体银电极表面的固定技术。方法：采用马抗蝰蛇毒血清抗体和抗蝰蛇毒鸡卵黄抗体作为生物敏感材料，对比研究了胱胺自组装-PSS反相吸附法和PEI粘附-戊二醛交联法：比较了采用两种固定方法所制的压电免疫传感器的性能。结果：鸡卵黄抗体采用PEI粘附-戊二醛交联法效果较好，其制备的IgY压电免疫传感器检测蝰蛇毒灵敏度为0．5ug／mL；而马血清抗体用胱胺自组装-PSS反相吸附法较好，其制备的IgG’免疫传感器检测蝰蛇毒灵敏度为10ug／mL。结论：以PEI粘附-戊二醛交联法固定抗蝰蛇毒鸡卵黄抗体所制备的蝰蛇毒压电免疫传感器的性能稳定，特异性好，可实现蛇毒的快速检测。     

The immune system as the sixth sense

One of the truly remarkable discoveries in modern biology is the finding that the nervous system and immune system use a common chemical language for intra- and inter-system communication. This review will discuss some of the pivotal results that deciphered this chemical language. Specifically the nervous and immune systems produce a common set of peptide and nonpeptide neurotransmitters and cytokines that act on a common repertoire of receptors in the two systems. The paper will also review more recent studies that have delineated hardwired and humoral pathways for such bidirectional communication. This is discussed in the context of the idea that the sharing of ligands and receptors allows the immune system to serve as the sixth sense that notifies the nervous system of the presence of entities, such as viruses and bacteria, that are imperceptible to the classic senses. Lastly, this review will suggest ways to apply the newfound knowledge of the sixth sense to understand a placebo effect and to treat human disease.     

Paper-based immunosensor with signal amplification by enzyme-labeled anti-p16INK4a multifunctionalized gold nanoparticles for cervical cancer screening

The aim of this study was to develop a paper-based immunosensor for cervical cancer screening, with signal amplification by multifunctionalized gold nanoparticles (AuNPs). The AuNPs were functionalized with a highly specific antibody to the p16^INK4a cancer biomarker. The signal was amplified using a combination of the peroxidase activity of horseradish peroxidase (HRP) enzyme-antibody conjugate and the peroxidase-like activity of the AuNPs. The immune complex of p16^INK4a protein and multifunctionalized AuNPs was deposited on the nitrocellulose membrane, and a positive result was generated by catalytic oxidation of peroxidase enzyme substrate 3,3’,5,5’-Tetramethylbenzidine (TMB). The entire reaction occurred on the membrane within 30 min. Evaluation in clinical samples revealed 85.2% accuracy with a kappa coefficient of 0.69. This proof of concept study demonstrates the successful development of a highly accurate, paper-based immunosensor that is easy to interpret using the naked eye and that is suitable for cervical cancer screening in low-resource settings.     

A computerised enzyme immunosensor: application for the determination of antigens

The present report gives preliminary results of a new sensitive method for the amperometric determination of antigens in serum. This method, developed from the biological model 'hepatitis B surface antigen antibodies' is less time-consuming than most immunochemical techniques, and eliminates many inconveniences arising from use of isotopes. Specific antibodies immobilised onto a gelatin membrane are applied in a solid phase 'sandwich' procedure. The antibodies are labelled with glucose oxidase. The measurement consists of an immunological process and an enzymatic reaction. The second part is carried out by fixing the active membrane onto a pO2 electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen due to the enzyme reaction. This response is correlated to the antigen concentration of the sample. It is a function of both the diffusional and the reactional characteristics of the active layer. Under software conditions, the signal is sampled when the stationary state is obtained. The difference between initial signal and the stationary state signal is measured and compared with the pre-set calibration curve. Use of the computerised enzyme immunosensor could easily be extended to assay of other antigens and haptens that are usually determined by radioimmunoassay.     

无标记检测CD4~+T淋巴细胞的免疫传感器构建

目的构建一种无标记检测CD4~+T淋巴细胞的免疫传感器。方法采用葡萄球菌A蛋白(SPA)法定向固定单克隆抗体于金叉指微阵列电极表面以捕获CD4~+T淋巴细胞,并用循环伏安法(CV)扫描对金叉指微阵列电极表面的修饰情况进行表征,最后通过电化学交流阻抗谱法(EIS)对免疫传感器捕获的CD4~+T淋巴细胞进行阻抗检测,通过等效电路拟合获得的阻抗值变化绘制标准曲线。结果该免疫传感器检测CD4~+T淋巴细胞的线性范围是(5.0×103-5.0×106)/mL,检测下限为5.0×102/mL。结论该免疫传感器的结果准确可靠,检测过程简便,价格低廉,有望用于实时检测系统,为实现快速、准确、价廉的CD4~+T淋巴细胞分类计数提供帮助。     

Prostate-specific antigen detection by using a reusable amperometric immunosensor based on reversible binding and leasing of HRP-anti-PSA from phenylboronic acid modified electrode

BACKGROUND: In recent years, many automated immunoassay analyzers have been developed for accurate diagnosis of various disease states and to improve effective drug administration. Amperometric immunoassay has been increasingly applied to laboratory medicine due to its ease in automation, rapid speed and low detection limits. It is important to develop reusable immunologically-sensitive elements for prostate-specific antigen (PSA) detection. METHODS: The strategy for the immunosensor construction is based on the enzyme-conjugated prostate-specific antibody (HRP-anti-PSA) reversible binding with a self-assembled phenylboronic acid monolayer on gold. RESULTS: After incubating an HRP-anti-PSA modified electrode in a PSA solution, a decrease in the electrocatalytic response of the HRP-anti-PSA modified electrode to the reduction of H(2)O(2) is observed. The photometric activity assays show that this decrease of the electrocatalytic response arises from the formation of immunocomplexes of HRP-conjugated anti-PSA and its antigen, not from the loss of bound HRP-anti-PSA from the electrode surface. Analytical performances and optimal conditions of the described immunosensor are also investigated. Under the optimal conditions, the amperometric immunosensor shows a linear increase of the relative intensity in 2 PSA concentration range from 2 to 15 ng/ml and 15 to 120 ng/ml, respectively. CONCLUSION: This method could be used for rapid analysis of PSA and potentially other antigens.     

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.     

Characterization of the interactions between immobilized parathion and the corresponding recombinant scfv antibody using a piezoelectric biosensor

A piezoelectric quartz crystal sensor with immobilized parathion was used for real‐time kinetic characterization of the interactions with recombinant anti‐parathion single chain Fv antibody IFRN AA01 (scFv). Parathion was linked to the sensor gold electrodes modified with a self‐assembled layer of aminothiophenol using either bovine serum albumin (BSA) or dextran as spacer molecules. The kinetic dissociation rate constant k_d was 8 × 10^?4 s^?1 for both types of sensors, and the association rate constants k_a, were 590 and 260 mol^?1 1 s^?1 for BSA and dextran‐linked parathion, respectively. The regeneration of BSA‐parathion coated crystals was not successful, however. Those crystals with bound scFv were used to study the formation of scFv dimers. Dextran‐parathion modified crystals were successfülly regenerated using proteinase K. The affinity of scFv to dextran‐parathion was 50 X lower when compared with the affinity of the anti‐parathion monoclonal antibody (IgG, IFRN 1701) the sequence of which served for production of scFv.     

基于碳纳米管-硫堇/苝四甲酸二酐衍生物膜/纳米金的新型癌胚抗原电流型免疫传感器的研究

目的探讨采用碳纳米管-硫堇(CNTs-THi)/苝四甲酸二酐衍生物膜(PTC-NH2)/纳米金(nano-Au)构建的新型的高灵敏癌胚抗原(CEA)免疫传感器的性能。方法以氧化铟锡(ITO)电极为基底,在其表面首先电沉积一层纳米金,然后依次修饰CNTs-Thi、PTC-NH2、nano-Au,固载癌胚抗原特异性抗体(anti-CEA)制得新型高灵敏癌胚抗原(CEA)免疫传感器。通过循环伏安法考察电极修饰过程的电化学特性。结果该免疫电极的峰电流响应与癌胚抗原的浓度在1.0～75.0 ng/mL的范围内保持良好的线性关系,相关系数为0.991 5,检测下限为0.5 ng/mL。结论利用该方法制备的免疫传感器具有灵敏度高、线性范围宽、检测下限低等优点。     

表面等离子体免疫传感器同步定量尿液中多种微量蛋白的研究

目的探讨并建立表面等离子体免疫传感器芯片同步定量尿液中多种微量蛋白的方法。方法采用EDC/NHS方法将微量白蛋白(MA)、α-1微球蛋白(A1M)、β-2微球蛋白(B2M)、尿IgG的单克隆抗体固定于免疫传感器芯片之上,并用表面等离子体共振传感器(surface plasmon resonance,SPR)检测样本中微量蛋白的含量。利用4种微量蛋白的标准血清建立标准曲线,然后对其进行灵敏度、特异性等指标的方法学评价。以免疫散射比浊方法为参考方法,检测125例临床患者24 h尿液样本的微量蛋白浓度,比较2种方法检测相同样本时的结果差异。结果该表面等离子体共振免疫传感器芯片具有良好的操作性能和检测特异性。其标准曲线的R2均在0.98以上。其检测灵敏度分别是A1M为5μg/L,B2M为7.3μg/L,MA为11.5μg/L,IgG为22μg/L。所有4项指标可在20 min之内同步完成。与微量蛋白检测的经典方法免疫散射比浊法相比,两者的相关性和一致性较高。结论表面等离子体免疫传感器芯片可实现尿液中多种微量蛋白的快速、准确、同步定量。