学科：物理化学. 组装体与纳米结构谱学,出版时间：2009,导师：赵冰指导,学位授予单位：吉林大学,论文作者：韩晓霞著,副题名：,学科专业：,关键词：,馆藏号：,
中文
作为一种超灵敏的分析技术，表面增强拉曼散射(SERS)在生命科学领域得到了越来越多的关注，建立基于SERS的蛋白质检测方法是本论文的研究重点。利用SERS在高灵敏度、光稳定性及光谱的多重检测方面的显著优势，通过SERS与生物技术的结合，成功地建立了几种基于SERS的蛋白质检测方法。这些方法可用于蛋白质鉴定，蛋白质与蛋白质，蛋白质与药物或其他小分子的相互作用检测等研究领域。主要研究成果归纳如下： (1)银溶胶蛋白质染色和蛋白质介导的SERS活性基底 利用SERS基底银溶胶做银染试剂，发展了一种新的印迹后总蛋白银染方法。该方法操作简便，显色迅速，灵敏度高，更重要的是，染色之后通过SERS可以提供蛋白质或标记分子在分子水平上的振动信息，鉴定蛋白质或者检测蛋白质及其配体间的相互作用。 传统的SERS研究大都是先制备好SERS活性基底，之后再将待测分子组装到该基底上进行SERS探测。在我们的研究中，采取了一种反向路线，即先在固相基底上组装好蛋白质或蛋白质和其配体复合物，然后再组装上SERS基底，这种组装方式主要依靠蛋白质与金属纳米粒子间强烈的相互作用(静电，疏水和共价相互作用)。我们的检测方法的最大特点是不用借助外力，依靠待测蛋白质和金属纳米粒子自组装SERS基底，组装好的基底反过来又能够检测待测蛋白质。 (2)印迹膜上非标记多蛋白质检测 将SERS通过蛋白质银溶胶染色与蛋白质印迹技术结合起来，建立了一种非标记多蛋白质检测方法。蛋白质被转移在硝酸纤维素膜上后利用银溶胶对膜上总蛋白染色，之后在不同的蛋白质条带上得到了不同蛋白质分子特征的拉曼指纹图谱。与其他基于SERS的蛋白质检测方法相比，该方法最大的优点是它与蛋白质分离纯化技术相结合，从而能够在一张印迹膜上实现非标记多蛋白质检测。另外，与传统的蛋白质印迹相比，我们的方法简化了原有繁琐步骤的同时兼顾了SERS的超灵敏性，从而为蛋白质提供了更简便更高效的鉴定手段。 (3)溶液中高灵敏度非标记蛋白质检测 在溶液条件下，SERS光谱的重复性较好，但灵敏度很低，这主要是由于目前常用卤化物为聚集剂，Cl⁻在银纳米粒子上有强烈的吸附，大大减少了待检测分子的进一步吸附。根据SO₄²⁻在银纳米粒子上的弱吸附从而可以诱导出强的SERS效应，我们利用酸化的硫酸盐做聚集剂，在溶液中得到了高灵敏度非标记蛋白质的SERS。在较低pH条件下，大多数蛋白质带正电荷，有利于蛋白质与带负电的银纳米粒子结合，形成H+和蛋白质介导的银聚集体，蛋白质被夹在银纳米粒子之间，从而可以得到整个蛋白质整体的SERS指纹，避免了因蛋白质的不同定位而引起的SERS光谱的不重复。该方法在快速高灵敏度检测蛋白质提取液方面有很大的应用潜力。 (4)荧光分子连接的免疫吸附试验 结合SERS和酶联免疫吸附试验(ELISA)的基本理论，我们建立了一种基于SERS的免疫吸附试验，利用荧光探针代替酶促反应产物，以SERS检测取代吸光度检测。抗原包被后，封闭未结合蛋白的部位，之后连接荧光分子的抗体与待测抗原发生免疫反应。银纳米粒子通过疏水静电等相互作用吸附到免疫复合物上，自组装形成银聚集体。通过这种方式，可以在酶标板底部直接检测到标记分子的表面增强共振拉曼散射(SERRS)，从而间接地检测出待测抗原，该方法操作简单，节约试剂，而且灵敏度很高，在高通量高灵敏度免疫检测领域有很大应用潜力。 (5)蛋白质与其配体相互作用检测 蛋白质芯片是高通量蛋白质检测方法，可以筛选出蛋白质的各种配体，目前主要利用荧光对作用结果进行检测。我们将银溶胶染色和SERS引入到了蛋白质芯片检测体系中。蛋白质和其配体依次组装到醛基化的玻璃片上后，依靠银纳米粒子在蛋白质上自组装形成SERS活性基底。通过标记在蛋白质配体上的荧光分子的SERRS和表面增强荧光(SEF)可以间接地检测出蛋白质与其配体间的相互作用。结果发现，SERRS和SEF在检测灵敏度和光谱稳定方面明显优越于传统的荧光方法。因此，利用SERRS／SEF可以有效地促进蛋白质芯片技术的发展。 (6)金银三明治SERS活性基底用于多目的蛋白质检测 为了解决以上研究(5)在光谱重复性方面的弱势，我们发展了一种蛋白质介导的三明治SERS基底，并把它应用于蛋白质鉴定，免疫检测及药物筛选中。这种三明治结构的最大特点在于，第一层自组装金单层膜既能够吸附目标蛋白质分子，又能够与第二层银纳米粒子一起诱导SERS效应，从而使蛋白质及标记分子的SERS兼顾金银两种基底的优越性。对于同一种目标分子，来自金银三明治的SERRS强度是来自金金三明治基底强度的7倍左右。同时发现，从金金和金银三明治基底中得到的SERS光谱的重复性相当，而与我们以前从银聚集体中得到的SERRS相比有很大改善。该方法不仅可以用于标记免疫检测，而且还可以用在非标记蛋白质的鉴定以及药物筛选等研究领域。 关键词：蛋白质，SERS，SERRS，非标记，拉曼标记，检测方法
英文
As an ultrasensitive analytical technique, suface-enhanced Raman scattering has been attracted increasing attention in biological science. SERS-based methods have salient advantages of high sensitivity, photostability and spectral multiplexing over conventional methods, based on which we successfully developed several SERS-based detection methods for protein identification, detection of protein-protein and protein-small molecule interactions. The major contributions of this work are as follows: (1)Silver colloid staining and protein-mediated SERS-active substrates We developed a new staining method by using silver colloid, which is an excellent SERS-active substrate. The proposed method is facile and sensitive, and the most important thing is after staining, we can determine different proteins or protein-ligand interactions by SERS, wich is unaccessible for the conventional silver staining. In most SERS-based studies, SERS-active substrates are first prepared and then analytes are assembled on these substrates for further SERS detection. In our studies we use a reverse way to produce a SERS-active substrate which is mediated by target proteins and used for detection of these proteins. Silver nanoparticles can adsorb on immobilized target proteins because of strong interactions (i.e., hydrophobic, electrostatic, and covalent interactions) between metal nanoparticles and the proteins, which result in aggregation of silver nanoparticles and a consequent SERS effect. In this way, we combine protein staining with SERS by silver colloid and take advantages of both to detect proteins. (2)Label-free multi-protein detection on a NC membrane Based on Western blot and SERS, we developed a new analytical procedure for label-free protein detection designated “Western-SERS”, consisting of protein electrophoresis, protein blotting, colloidal silver staining, and SERS detection. The most important feature of this method is that it enables multi-protein detection on one nitrocellulose (NC) membrane, which is inaccessible for other SERS-based detection methods. This “Western-SERS” method offers the dual advantages of simplicity and high sensitivity. Compared with Western blot and mass spectrometry, we can detect label-free proteins directly on an NC membrane without time and reagent-consuming procedures. Moreover, the detection limit of Western-SERS is almost consistent with the detection limit of colloidal silver staining (2 ng/band), and SERS signals do not self-quench, unlike fluorescence. Thus, the new method has great potential for identifying proteomic components or proteins of differential expression in some proteomes. (3)Label-free highly sensitive protein detection in aqueous solutions Roughened metal surfaces and dried colloids are difficult to obtain reproducible SERS spectra especially for those proteins with no chromophores because of irreproducible SERS substrates and different orientation of analytes on a metal surface. Otherwise, no previous SERS-based study for proteins allows routine detection of label-free proteins with high sensitivity in an aqueous solution because halide ions, which are commonly used aggregation reagents, can form a strongly bonded surface layer which repels adsorption of proteins. According to weak binding of SO₄²⁻ on silver surfaces that can induce much stronger SERS, we use acidified sulphate as an aggregation agent and obtained strong SERS of targert proteins with high sensitivity. Moreover, when silver nanoparticles are aggregated by the proposed protein involved aggregation agent, target proteins with net positive charges are sandwiched among silver nanoparticles in aqueous solutions, and then vibration information of whole proteins would be probably displayed in their SERS spectra with remarkable enhanced reproducibility. It can be used as a practical method to directly detect label-free proteins with the advantages of rapidness, reproducibility and appropriately high sensitivity. (4)Fluorescence-linked immunoabsorbent assay By combining enzyme-linked immunoabsorbent assay (ELISA) with SERS, we developed a SERRS-based immunoassay on bottom of microtiter plates. In this method, SERRS spectra of FITC are measured after several continuous steps of antigen coating, blocking, antibody adding, and colloidal silver staining. Protein-mediated formation of silver aggregates results in electromagnetic enhancement of resonance Raman scattering. The proposed method has several advantages over ELISA and other immunoassays. First, we can determine the concentration of antigens via the intensity of a SERRS signal of FITC molecules that are attached to antibodies without an enzyme reaction, and thus the process is simple and reagent saving. Second, one can obtain SERRS spectra of FITC directly from silver aggregates on the bottom of a microtiter plate without displacement. Third, by using SERRS of FITC, the present method is highly sensitive. Therefore, it may have great potential as a high-sensitivity and highthroughput immunoassay. (5)Detection of protein-ligand interactions Protein chips with versatile applications play an important role in high-throughput proteomic studies. The purpose of protein arrays is to quickly probe the activity of a given protein against many targets simultaneously, and the currently preferred detection method for protein chips is fluorescence. In our study, we used SERRS instead of fluorescence as detection method for protein chips. Silver nanoparticles were assembled to the protein-ligand complexes via covalent and noncovalent binding, resulting in the formation of SERS-active silver aggregates. We observed both SERRS and Surface-enhanced fluorescence of the target labels, and they are more photostable and sensitive than their corresponding fluorescence. Therefore, the proposed method for detection of protein-ligand interactions has great potential in high-sensitivity and high-throughput chip-based protein function determination. (6)Metal sandwich substrate for versatile protein detection As a more advanced system, we develop a novel type of SERRS-active sandwich substrates bridged by the target proteins for the detection of these proteins. The most significant advantage of the present method over other SERS-based methods for protein detections is that a self-assembling gold nanoparticle monolayer is used for both capturing proteins and forming the SERS-active substrate with the second metal layer. Highly reproducible SERRS and SERS spectra can be obtained by the present gold-protein-gold (Au/Au) and gold-protein-silver (Au/Ag) sandwiches, and we find that the latter yields about seven times stronger SERRS than the former. Moreover, besides labeled immunoassays, the present method can identify label-free proteins and protein-drug interactions without Raman dyes. Because of contributions from the two metal layers to the SERS, this sandwich strategy holds great potential in highly sensitive and reproducible protein detections. Keywords: Protein, SERS, SERRS, label free, Raman-dye label, detection method
