摘要
30年前人们首次发现了表面等离子共振现象(SPR),这种方法使我们在传感仪器设备和应用上得到了快速迅猛的发展。SPR传感器现在已经商业化并且SPR生物传感器已经成为主要研究和表征生物分子相互作用的有力工具。本论文主要阐述了SPR传感器在抗蛋白非特异性吸附和研究生物分子相互作用方面的应用。同时我们还研究了SPR与电化学技术的关系,并且用EC-SPR技术检测了化学反应过程中产生的中间体。最后,我们还研究了自合成的新型吸附材料对痕量组分进行预富集和选择性的分离。具体工作如下：
1.研究了通过在金片表面固定不同聚合度的聚乙二醇(n=3,4,6)自组装单分子膜(PEG-SAMs)来阻止蛋白在表面的非特异性吸附的方法。在SAMs紧密有序的排列的情况下,末端为羟基的PEG-SAMs有良好的亲水性。分别用原子力显微镜,偏振椭圆光度法和红外光谱技术对裸金和PEG-SAMs进行了表征。用SPR技术测量了蛋白在PEG-SAMs表面的吸附情况,相对于PEG2-和PEG4-SAMs来说,PEG6-SAMs具有最好的抗蛋白非特异性吸附的能力。
2.研究讨论了在不同比例的混合巯基烷烃自组装单分子膜表面固定IgG的方法。固定蛋白的步骤主要包括2步：首先用N-乙基二甲氨基丙基碳化二亚胺(EDC)和羟基琥珀酸亚胺(NHS)活化SAM表面的羧基,形成的NHS-酯再与蛋白的氨基发生偶联反应。因为这种方法的活性反应中间体很容易制备,所以它为在SAMs表面固定蛋白提供了一种新的途径。更重要的是这种混合SAMs具有良好的抗蛋白非特异性吸附的能力。同时还研究了蛋白溶液的pH值在偶联反应中对表面固定密度的影响。最后计算和分析了IgG和Anti-IgG反应的动力学。
3.通过研究电化学(EC)反应,建立了一种电化学和SPR定量关系。反应的EC-SPR信号来自于电化学电流的卷积积分。因此EC-SPR是一种可以提供类似于传统电流信息的有力工具。我们以扫描电势EC-SPR为例分析了各种情况,发现了一种不需要进行积分计算来测量卷积积分伏安法的新途径。除此之外,EC-SPR还有许多独特的优点：1)具有很好的空间分辨率可以用来研究非均相反应；2)反应体系光学性质的研究有利于理解反应机理；3)高灵敏度。我们以三氯六铵合钌体系为模板进行了理论和实验的研究,并对实验结果进行了模拟,二者非常一致。
4.用流动EC-SPR技术测量了乙腈溶液中对苯醌-对苯二酚体系的电化学反应。半醌自由基第一次通过EC-SPR技术被检测到具有很大的负SPR信号。实时的SPR信号监测到了半醌自由基从产生、转化到消失变化的全过程。同时还计算了半醌自由基的半衰期。这个研究证明流动EC-SPR技术不仅仅能检测物质在表面发生结合,也能检测溶液的化学反应。此外,它还能用于检测传统方法很难检测到的不稳定的反应中间体产物。
5.合成了一种新型的三乙基氨基胺修饰的硅胶吸附剂,并通过电感耦合等离子体发射光谱(ICP-AES)研究了该吸附剂对痕量Cr(Ⅲ).Cd(Ⅱ)和Pb(Ⅱ)的选择性固相萃取。用红外光谱对其表面结构进行了表征,并且详细讨论了溶液pH值、振荡时间、流速、溶液体积、洗脱和离子干扰等条件。最后将此方法应用到自然水样品的检测中,取得了良好的结果。
Since the first application of the surface plasmon resonance (SPR) phenomenon for sensing almost three decades ago, this method has made great strides both in terms of instrumentation development and applications. SPR sensor technology has been commercialized and SPR biosensors have become a central tool for characterizing and quantifying biomolecular interactions. This paper mainly described the application of SPR on resistance of protein non-specific adsorption and the interaction between bio molecules. Moreover, we studied the relationship of SPR and electrochemistry, and detected the intermediate in the reaction by EC-SPR. At the last chapter, we focused on the preparation of the adsorption material for the pre-concentration and separation of trace components. The more detailed novelty of this work can be categorized as following:
1. A study of protein resistance of poly (ethylene glycol) (PEG), HS(CH2)11(OCH2CH2)nOH (n=3,4, and 6), self-assembled mono layers (SAMs) on gold surfaces was presented here. Hydroxyl-terminated PEG-SAMs were chosen to avoid the hydrophobic effect, particularly at high packing densities. The structure of the bare gold and PEG-SAM surfaces were characterized by atomic force microscopy (AFM), Ellipsometry and FT-IR Protein adsorption on these surfaces was investigated by surface plasmon resonance (SPR). Compared with PEG2-and PEG4-SAMs, PEG6-SAMs show great resistance of protein adsorption.
2. This chapter describes the immobilization of IgG on mixed self assembled monolayers (SAMs) of alkanethiolates with different ratio on gold generated from HS(CH2)11(OCH2CH2)6OH and the HS(CH2)11(OCH2CH2)6CH2COOH. The immobilization was achieved by a two-step procedure:generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups in the SAM and coupling of these groups with amines on the protein or ligand. Because this method involves a common reactive intermediate that is easily prepared, it provides a convenient method for attaching ligands to SAMs using surface plasmon resonance spectroscopy. These SAMs were highly resistant to nonspecific adsorption of proteins. The pH of the protein buffer solution that influenced the amount of the protein immobilized was investigated. The kinetics of IgG and Anti-IgG binding was calculated and analysed.
3. A quantitative formalism of electrochemical Surface Plasmon Resonance (EC-SPR) was developed for studying electrochemical reactions. The EC-SPR signal from the reactions was found to be a convolution function of electrochemical current, and therefore, EC-SPR is a powerful tool that can provide information similar to the conventional current-based electrochemical techniques. As an example, potential-sweep EC-SPR was analyzed in details and found to provide a new way to measure convolution voltammetry without numerical integration. In addition to the benefits provided by the conventional convolution voltammetry, the EC-SPR possesses several unique advantages, including 1) spatial resolution that is particularly attractive for studying heterogeneous reactions,2) optical properties of the reactions species that may assist identification of reaction mechanisms; and 3) high surface sensitivity for studying surface binding of the reaction species. Experiments and numerical simulations were carried out for a model system, hexaammineruthenium (Ⅲ) chloride. The simultaneously measured electrochemical current and SPR response confirmed the relationship between the two quantities, and the numerical simulations were in excellent agreement with the measurements.
4. The electrochemical reaction of Hydroquinone-Benzoquinone (HQ-BQ) system in acetonitrile was studied by a flow-through Electrochemical Surface Plasmon Resonance (EC-SPR) technique. The semiquinone radical anion (BQ*-) was detected as a large negative SPR shift for the first time by the hyphenated technique. The real time SPR signal shows the entire reaction process of the BQ including generation, transformation and disappearance. The lifetime of the BQ in the reaction was extracted by comparing the SPR signals along the flow stream The study demonstrates that the flow-through EC-SPR is a powerful tool for detecting not only surface bindings, but also solution phase chemical reactions. In addition, it is especially suitable for detecting unstable intermediate reaction products, which are difficult to measure using conventional analytical methods.
5. A new tris(2-aminoethyl) amine (TREN) functionalized silica gel (SG-TREN) was prepared and investigated for selective solid-phase extraction (SPE) of trace Cr(Ⅲ), Cd(Ⅱ) and Pb(Ⅱ) prior to its determination by inductively coupled plasma atomic emission spectrometry (ICP-AES). Identification of the surface modification was characterized and performed on the basis of FT-IR The separation/ preconcentration conditions of analytes were investigated, including effects of pH, the shaking time, the sample flow rate and volume, the elution condition and the interfering ions. The application of this modified silica gel to preconcentration trace Cr(Ⅲ), Cd(Ⅱ) and Pb(Ⅱ) of two water samples gave high accurate and precise results.
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