生物素化高分子涂层的制备与评价

制备了一种能固载目标蛋白质, 却没有非特异性蛋白质吸附的高分子涂层. 该涂层是可生物降解的油水两亲性的三嵌段聚合物, 即生物素偶联的聚乙二醇-聚丙交酯-聚赖氨酸共聚物. 将高分子溶解于N,N-二甲基甲酰胺中, 并涂布在预先包被了聚赖氨酸的脱脂玻片基质上, 形成高分子涂层, 在其表面包被一层由明胶和聚N-乙烯基吡咯烷酮组成的封闭剂. 使用酶标免疫分析法, 对高分子涂层表面的生物活性进行评价. 依次将辣根过氧化物酶标记的链亲和素和生物素偶联的小鼠球蛋白抗原和碱性磷酸酯酶标记的马抗小鼠抗体固载在高分子涂层表面上, 通过标记酶与底物作用生色. 分析结果表明, 经过封闭以后, 生物素化的高分子涂层表面能够排斥非特异性的蛋白质; 同时特异性蛋白质之间(如生物素和链亲和素之间、抗原和抗体之间)的相互作用依然保留, 并且固定在表面的蛋白质依然保留其生物活性. 因此生物素化的聚乙二醇-聚丙交酯-聚赖氨酸三嵌段高分子可以作为生物活性材料, 用于蛋白质固载和蛋白质分离及分析.     

Ultrasensitive Eletrogenerated Chemiluminescence Immunoassay by Magnetic Nanobead Amplification

An ultrasensitive electrogenerated chemiluminescence (ECL) immunoassay was proposed by using magnetic nanobeads (MNBs) as the carrier of ECL labels for ECL emission amplification. Carcinoembryonic antigen (CEA) and MNBs were initially immobilized on a platform in 1 : 1 molar ratio via sandwich immunoreaction. Subsequently, the MNBs were released from the platform and labeled with Ru(bpy)₃²⁺ species. After the MNBs with Ru(bpy)₃²⁺ were immobilized on an Au electrode, ECL of the Ru(bpy)₃²⁺ was measured for CEA determination. A linear relation between the ECL intensity and CEA concentration was obtained in a range of 1×10^?14 to 3×10^?13 mol/L (2.0 to 60 pg/mL) with a limit of detection of 8.0×10^?15 mol/L (1.6 pg/mL).     

Novel Concepts for the Immunological Detection of Bound Residues

Abstract

Immunoassays could be applied successfully to the determination of bound residues in soil and other complex matrices. Nevertheless, there was some doubt whether these assays could be regarded as quantitative and selective. We present some results, which imply that several approaches are quite promising. One approach is based on a non-competitive saturation immunoassay, which evens out the different cross-reactivities of the bound species. This may lead to a true molar sum value, which is not an equivalent concentration. For competitive assays, a method for the determination of the affinity constant(s) of the bound species is discussed. This would enable a correction for the cross-reactivity. The selectivity problem could be diminished very much, too. The application of special blocking reagents and the use of inhibition tests essentially solved the problem of non-specific binding (NSB). In addition, it could be proven experimentally that adsorbed analytes do not disturb the non-competitive assays, which have been suspected to be highly selective for covalently bound residues.     

Electrochemical Dissolution of Silver Nanoparticles and Its Application in Metalloimmunoassay

This work demonstrates the use of silver nanoparticles as a simple electrochemical biolabel to induce 10⁶ signal enhancement. We propose a mechanism of measuring the silver nanoparticles on a specific screen‐printed planar carbon electrode, without the requirement for the harsh oxidant or toxic reagents described in prior‐art for gold sol methods. The possible measurement process was validated with orthogonal techniques such as UV/Vis spectroscopy, dynamic light scattering and by anodic stripping voltammetry (ASV). The findings were utilized to develop a novel electrochemical sandwich immunoassay where the analyte concentration is directly proportional to ASV oxidation peak of silver. This technique in the future is envisaged to form the foundation of a generic Point of Care platform. The assay was applied to cardiac marker: myoglobin with detection limit of 3 ng/mL.     

Monitoring Chemical Warfare Agents: A New Method for the Detection of Methylphosphonic Acid

Methylphosphonic acid (MPA) is the degradation product of many chemical warfare agents. The convenient detection of this substance would aid in field testing to confirm illicit manufacture and use of banned chemical weapons. Efficient functionalization of MPA with an aromatic diazo compound allowed binding by monoclonal antibodies elicited by using an analogous hapten (see scheme). An ELISA assay was rapid, sensitive, and specific.     

The role played by hydration forces in the stability of protein-coated particles: non-classical DLVO behaviour

An anomalous colloidal stability is observed when protein-covered particles are exposed to high salt concentrations, in opposite to the classical theory (DLVO) predictions. In hydrophilic systems some other discrepancies with respect to this theory has also been described in the literature and hydration forces are invoked to rationalize these phenomena. In our case, the dependence of the anomalous behaviour with pH and electrolyte ion concentration points to the specific adsorption of cations as responsible. An extension to the DLVO theory including hydration forces and its dependence with salt concentration is proposed. From the practical point of view, this stabilization mechanism is of great interest in the development of clinical latex immunoassays, which often suffer from colloidal stability problems.     

微流控免疫芯片检测方法的研究进展

微流控免疫芯片以其微型化、高通量、快速检测及低消耗等优点成为近年来分析领域的研究热点. 检测技术是微流控芯片的重要组成部分之一. 本文重点综述了近年来微流控免疫芯片的微系统研究及相应的检测方法和技术, 包括电化学检测及荧光检测、紫外-可见吸收光谱检测、化学发光和生物发光检测、表面增强拉曼散射检测、光纤检测、表面等离子体共振谱检测、热透镜显微镜检测和比色检测等光学检测及其它新型检测方面的进展, 并展望了其发展前景.     

Immunoassays using capillary electrophoresis laser induced fluorescence detection for DNA adducts

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage. The critical role of DNA damage in cancer development makes it essential to develop highly sensitive and specific assays for DNA lesions. We describe here ultrasensitive assays for DNA damage, which incorporate immuno-affinity with capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection. Both competitive and non-competitive assays using CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol epoxide (BPDE). A fluorescently labeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay. Binding between this synthetic oligonucleotide and a monoclonal antibody (MAb) showed both 1:1 and 1:2 complexes between the MAb and the oligonucleotide. The 1:1 and 1:2 complexes were separated by CE and detected with LIF, revealing binding stoichiometry information consistent with the bidentate nature of the immunoglobulin G antibody. For non-competitive assay, a fluorescently labeled secondary antibody fragment F(ab′)₂ was used as an affinity probe to recognize a primary antibody that was specific for the BPDE-DNA adducts. The ternary complex of BPDE-DNA adducts with the bound antibodies was separated from the unbound antibodies using CE and detected with LIF for quantitation of the DNA adducts. The assay was used for the determination of trace levels of BPDE-DNA adducts in human cells. Analysis of cellular DNA from A549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM–1 μM) showed a clear dose–response relationship. BPDE is a potent environmental carcinogen, and the ultrasensitive assays for BPDE-DNA adducts are potentially useful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage.     

Comparative Analysis of Proline Enantiomer in Chiral Recognition of Biological Macromolecule in Immunoassay

An interesting phenomenon of the stereoselective interaction between biological macromolecule and amino acid enantiomorphous was described. Firstly, proline enantiomer (L‐ and D‐proline) was assembled on the glassy carbon electrode surface. Then carcinoembryonic antibody (anti‐CEA) was loaded to the enantiomer surface, electrochemical impedance spectroscopy (EIS) was used to monitor the growth of amino acid film. The assembly process was characterized by cyclic voltammetry (CV) and atomic force microscopy (AFM) was applied to image the chiral films. Finally, the developed electrodes had interacted with carcinoembryonic antigen (CEA) in varying concentration solutions. The AFM and amperometric results revealed that the proline enantioner had the chiral recognition function to antibody.     

Multichannel SPR biosensor for detection of endocrine-disrupting compounds

A surface plasmon resonance (SPR) biosensor for simultaneous detection of multiple organic pollutants exhibiting endocrine-disrupting activity, namely atrazine, benzo[a]pyrene, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-nonylphenol, is reported. The biosensor utilizes a multichannel SPR sensor based on wavelength modulation of SPR and wavelength division multiplexing (WDM) of sensing channels, antibodies as biorecognition element and a competitive immunoassay detection format. An analysis time of 45 min (including 30-min incubation of the sample with antibodies) and limits of detection as low as 0.05, 0.07, 0.16 and 0.26 ng mL⁻¹ are demonstrated for benzo[a]pyrene, atrazine, 2,4-D and 4-nonylphenol, respectively. The biosensor is also shown to be regenerable and suitable for repeated use.