微流控芯片在表面等离子体共振生物传感器中的应用

作为众所周知的生物传感器技术,表面等离子体共振（SurfacePlasmonResonance,SPR）正在被越来越普遍地用于实现各种生物化学检测方法,特别是用途广泛的固相表面生物检测（Sol—id—PhaseBioassay）。SPR对样品进行非标记检测,能够用于测量生物化学反应全过程的反应动力学。为了提高SPR的检测效率,通常将微流控技术（Microfluidics）与SPR相结合,即在SPR生物传感器中使用微流控芯片（MicrofluidicChip）作为反应装置。基于微型化带来的优势,使用微流控芯片作为反应装置可以有效地缩短生物化学检测方法的反应时间,并减少样品消耗。微流控芯片还可以平行排布相同的结构单元,提高SPR生物传感器的检测通量。因此,使用微流控芯片作为反应装置是SPR生物传感器,特别是商品化的SPR生物传感器的发展趋势。     

反射式光纤银膜SPR传感器的湿法构建及应用

采用银镜反应湿法制备了反射式光纤表面等离子体共振(Surface plasmon resonance,SPR)传感器,并结合多巴胺自聚合功能对光纤表面银膜进行快速生物功能化修饰,实现了抗体抗原相互结合的在线监测。通过自行搭建SPR仪器,考察了传感区长度的影响,跟踪测定了传感器各个修饰步骤后SPR信号的变化。测试结果表明,该法制备的光纤SPR传感器具有很高的灵敏度。随着传感区长度的增加,基于SPR峰位的检测灵敏度随之增大,而基于SPR峰强的检测灵敏度无明显变化。当传感区长度为1.5 cm时,基于SPR峰位对蔗糖溶液折射率的检测灵敏度为4 166 nm/RIU,而基于SPR峰强的检测灵敏度为99%/RIU。进一步利用该传感器监测了Ig G的连接过程、Ig G抗体-抗原的相互结合过程,结果显示其可应用于生物传感检测领域。     

表面等离子体共振传感技术和生物分析仪

表面等离子体共振（surface plasmon resonance, SPR）传感技术是生物化学分析领域常用的分析手段和研究工具,与其相关的研究不计其数,发展日新月异。本研究小组从事SPR传感技术研究近十年,从初期的理论研究、仿真计算、传感器设计以及全自动SPR生物分析仪开发与应用研究,到目前的传感器性能提高、应用拓展,时刻关注着该项技术的最新动态。本文系统综述了SPR传感技术和生物分析仪的原理、结构以及主要功能模块,SPR传感器的调制类型、耦合方式以及SPR成像传感器;介绍了结合局域表面等离子体共振（local surface plasmon resonance, LSPR）技术、改进金属膜系设计、优化数据处理算法等提高SPR生物分析仪性能的方法;阐述了SPR传感技术和生物分析仪的最新进展,包括SPR技术和微流控芯片、电化学技术、表面增强拉曼散射技术（SERS）的联用;列举了SPR生物分析仪在临床诊断、药物筛选、生物分子研究、食品安全和环境监测等领域的应用实例;最后,分析了SPR生物分析仪面临的主要问题以及未来的发展趋势。     

基于分子印迹聚合物的SPR传感器用于牛血清蛋白的检测

以牛血清蛋白（BSA）为模板分子、多巴胺为功能单体和交联剂、高碘酸钠为氧化剂，制备了BSA表面印迹表面等离子共振（SPR）生物传感器。通过聚丙烯酸（PAA）与BSA的氢键作用预先固定BSA，增加印迹效率，同时在聚多巴胺表面非印迹区域修饰部分水解的聚（2-甲基-2-噁唑啉）（PMOXA-EI）抵抗蛋白质的非特异性吸附。通过X射线光电子能谱、原子力显微镜、可变角光谱椭偏仪和静态水接触角对制备的BSA表面印迹SPR生物传感器进行表征。对质量浓度为0.1～10μg/mL的BSA水溶液进行SPR吸附研究，检测限和定量限分别达到了53 ng/mL和161 ng/mL。以β-乳球蛋白、卵白蛋白、溶菌酶和细胞色素C为参比蛋白进行选择性研究，相应的选择性系数分别达到了4.43、3.45、3.17和3.64。上述5种蛋白混合溶液中BSA的检测回收率为97.5%～102.5%。     

表面等离子体共振生物传感器及其应用

表面等离子体共振(Surface Plasmon Resonance,SPR)生物传感器在检测生物分子特异性结合方面具有的高灵敏度、免标记及检测快速等优点,使其在过去的20年中在生命科学、医药科学、食品安全等领域取得了快速发展。本文对SPR生物传感器进行了简要介绍,并着重对其在生命科学、医药学、环境分析、食品安全等领域的应用进行了综述,最后对该领域的研究前景进行了展望。     

表面等离子体共振免疫传感器在蛋白质检测中的应用及其研究进展

表面等离子体共振(SPR)技术是20世纪90年代发展起来的一种新型技术,应用SPR原理可检测生物传感芯片上配位体与分析物之间的相互作用情况,在生命科学、医疗检测、药物筛选、食品检测及环境监测等领域具有广泛的应用需求.SPR技术可与免疫传感器结合,利用抗原抗体的特异性反应可用于各种蛋白质抗原的检测.本文重点总结了SPR免疫传感器在食品及医疗领域蛋白质检测的应用,综述了近年来SPR免疫传感技术在这该领域的研究热点及进展.     

基于金银合金薄膜的波长检测型表面等离子体共振传感器

从实验和理论两方面详细研究了金银合金膜表面等离子体共振（SPR）传感器在可见光波段的敏感特性. 实验方面,通过在玻璃基底上溅射50 nm厚的金银合金薄膜制备了一种新型的SPR传感芯片,并且自行搭建了基于Kretschmann 结构的波长检测型SPR传感器测试平台. 利用不同浓度的氯化钠（NaCl）水溶液和浓度为10 μmol·L^-1的牛血清蛋白（BSA）水溶液分别作为折射率样品和分子吸附样品,研究了传感器的折射率灵敏度和吸附灵敏度,并与金膜和银膜SPR传感器进行了对比研究. 结果表明,对于折射率灵敏度的测试,金银合金膜SPR传感器大幅高于金膜SPR传感器,略低于银膜SPR传感器;而对于吸附敏感的研究,金银合金膜SPR传感器的灵敏度与银膜SPR传感器相近,是金膜SPR传感器的3倍. 理论方面,利用菲涅尔公式和等效折射率计算公式仿真计算了这三种薄膜结构的SPR传感器的灵敏度和精确度,结果指出金银合金膜SPR传感器的灵敏度与银膜SPR传感器接近,是常规金膜SPR传感器的2.31倍,而半高峰宽仅为金膜和银膜SPR传感器的1.36 倍. 在稳定性方面,金银合金膜SPR传感器与金膜SPR传感器均具有良好的化学稳定性,而银膜SPR传感器较易氧化,使用寿命低,不常被采用. 综上,金银合金膜在改善传感器灵敏度的同时,不会降低精度,是一种高灵敏、低成本、良好稳定性的SPR传感器敏感材料.     

表面等离子体共振光谱技术与电化学方法联用及其应用

表面等离子体共振（Surface Plasmon Resonance,SPR）技术是利用金属薄膜光学耦合产生的物理光学现象建立的一种非常灵敏的光学分析手段. 近年发展的电化学表面等离子体共振（Electrochemical Surface Plasmon Resonance,EC-SPR）是将时间分辨表面等离子体共振光谱技术与电化学方法联用的一种新技术. 本文介绍了SPR和EC-SPR的基本原理,并重点阐述了时间分辨SPR光谱技术与电化学方法联用及应用,该技术已广泛地应用于反应动态过程研究、生物化学传感器、电极/溶液界面的表征、动力学常数的测定以及生物分子相互作用等领域.     

利用表面等离子体谐振生物传感器实时监测酶促反应的新方法

在理论上，基于表面等离子体谐振(surface plasmon resonance，SPR)生物传感器的酶促反应检测分析方法被证明是可行的。这种方法与通常SPR传感器检测技术不同，它消除通常当成检测目标的表面吸附信号，而检测通常认为是噪声的本体折射率变化信号，以此实时检测酶促反应的动态过程。利用自产的SPR2000生化分析仪，在检测胶原酶I降解胶原I的实验中检测到了反应引起的反应液本体折射率的上升变化，从而证明了这种新颖的检测方法实际上也是可行的。这种技术一旦成熟，可以用来进行酶学分析及酶靶标的药物筛选研究开发。     

基于氨基与表面乙烯砜基反应动力学调控配基表面密度

配基表面密度可控为定量研究生物分子相互作用提供了精准的分子基础。然而,经典混合自组装的方法控制配基密度对于不同自组装体系不具有普适性。本文报道了一种基于表面乙烯砜基反应动力学的配基表面密度调控方法。以Nα,Nα-二（羧甲基）-L-赖氨酸（ab-NTA）为生物配基模型,对该表面反应进行了催化剂筛选并利用X射线光电子能谱（XPS）和表面膜电位对该表面反应进行了表征。采用静态水接触角的方法对表面反应的动力学进行了定量表征,计算得到反应速率常数为0.0012 min^-1。采用表面等离子体共振（SPR）分析了该生物功能表面结合组氨酸标签蛋白（SA-6His）的能力,结果表明该表面比传统NHS-NTA表面具有更高的蛋白结合量和结合强度。通过控制反应时间和催化剂种类制备了四种配基密度不同的生物功能表面,并利用SPR对四种表面进行了蛋白质静态吸附实验。实验结果表明通过控制反应时间和催化剂类型均能够实现配基表面密度的调控,并且可以实现表面多价态的调控。     

Kinetic Analysis of the Interaction between Nonsteroidal Anti-inflammatory Drugs and Cyclooxygenase-2Using Wavelength Modulation Surface Plasmon Resonance

A surface plasmon resonance (SPR) biosensor based on wavelength modulation technology was developed and validated for the kinetic analysis of the interactions between two nonsteroidal anti‐inflammatory drugs (NSAIDs) and cyclooxygenase‐2 (COX‐2). After the effect of different concentration COX‐2 on the binding capacity of the SPR biosensor surface was studied, the COX‐2 was immobilized covalently onto the biosensor surface using a standard amine coupling method. The affinity constants for indomethacin, ketoprofen binding to COX‐2 are 7.5×10³ L/mol and 9.25×10³ L/mol, respectively. The biosensor surface can be regenerated after being rinsed with 0.01 mol/L NaOH, and the biosensor can be used repeatedly. These indicated that the wavelength modulation SPR biosensor has the potential application in the fields of pharmacokinetics, pharmacodynamics and drug discovery.     

纳米金颗粒增强信号的表面等离子体共振生物传感器用于甲氧檗因高灵敏检测的研究

报道了一种基于表面等离子体共振(SPR)生物传感器的高灵敏检测抗癌药物甲氧檗因的新方法. 分别在纳米金颗粒和金膜表面修饰富含腺嘌呤(A)的DNA链, 当存在甲氧檗因时, 由于一个甲氧檗因分子可与4个A碱基相结合, 从而使得修饰在纳米金颗粒和金膜表面的DNA形成稳定的双链结构, 进而将功能化纳米金颗粒捕获在金膜表面. 由于纳米金颗粒与金膜之间的电场耦合效应可增强SPR信号, 从而可实现对小分子甲氧檗因的高灵敏、特异性检测. 本方法的检测下限低至0.07 pmol/L, 相对比色法和荧光法而言, 降低了约5～6个数量级. 以4种药物(盐酸小檗碱、青霉素G、硫酸庆大霉素、5-氟尿嘧啶)作为对照考察了该传感器的选择性, 结果表明该传感器具有较好的选择性.     

Continuous Immunoassay for Sulfamethazine by Surface Plasmon Resonance-Based Biosensor

Regeneration of the sensor chip surface is difficult in many surface plasmon resonance (SPR) biosensor assays. Improper regeneration will reduce life span of the sensor chip and decrease the quality of the data. Considering the advantages of reducing the regeneration frequency, a theoretically feasible continuous SPR biosensor immunoassay for sulfamethazine (SMT) was developed. In the continuous inhibitive immunoassay, the sensor chip surface is regenerated only once after a definite number of tests instead of every test. The SMT-bovine serum albumin (BSA) conjugate was covalently immobilized to a carboxymethyldextran modified gold film. The immobilization conditions of the antigen were studied and the working dilution of the antibody was optimized. The antibody was mixed with SMT of different concentrations prepared with PBS buffer to construct the calibration curve. The limit of detection was 0.5 ng mL^?1. The continuous SPR biosensor assay was proved to be simpler and more practical than a normal one.     

表面等离子体共振生物传感器研究人参皂甙与血清白蛋白的相互作用

To use a newly developed wavelength modulation surface plasmon resonance （SPR） biosensor, an experimental protocol was developed to investigate the interaction of ginsenosides with serum albumin. With a known concentration of the ginsenosides, bound percentages of the ginsenosides with human serum albumin （HSA） or bovine serum albumin （BSA） were obtained. SPR technique could require no labeling and this method provided the detailed information on association and disassociation of molecules in real time. The results indicate that the sensitivity of wavelength modulation SPR biosensor is sufficient for detection and characterization of binding events involving low-molecular weight compounds and their immobilized protein targets.     

A Single and a Dual-Fractal Analysis of Analyte-Receptor Binding Kinetics for Surface Plasmon Resonance Biosensor Applications

The diffusion-limited binding kinetics of analyte in solution to either a receptor immobilized on a surface or to a receptorless surface is analyzed within a fractal framework for a surface plasmon resonance biosensor. The data is adequately described by a single- or a dual-fractal analysis. Initially, the data was modeled by a single-fractal analysis. If an inadequate fit was obtained then a dual-fractal analysis was utilized. The regression analysis provided by Sigmaplot (32) was used to determine if a single fractal analysis is sufficient or if a dual-fractal analysis is required. In general, it is of interest to note that the binding rate coefficient and the fractal dimension exhibit changes in the same direction (except for a single example) for the analyte-receptor systems analyzed. Binding rate coefficient expressions as a function of the fractal dimension developed for the analyte-receptor binding systems indicate, in general, the high sensitivity of the binding rate coefficient on the fractal dimension when both a single- and a dual-fractal analysis is used. For example, for a single-fractal analysis and for the binding of human endothelin-1 (ET-1) antibody in solution to ET-115-21.BSA immobilized on a surface plasmon resonance (SPR) surface (33), the order of dependence of the binding rate coefficient, k, on the fractal dimension, Df, is 6.4405. Similarly, for a dual-fractal analysis and for the binding of 10(-6) to 10(-4) M bSA in solution to a receptorless surface (direct binding to SPR surface) (41) the order of dependence of k1 and k2 on Df1 and Df2 were -2.356 and 6.241, respectively. Binding rate coefficient expressions are also developed as a function of the analyte concentration in solution. The binding rate coefficient expressions developed as a function of the fractal dimension(s) are of particular value since they provide a means to better control SPR biosensor performance by linking it to the degree of heterogeneity that exists on the SPR biosensor surface. Copyright 1999 Academic Press.     

Surface Plasmon Resonance Immunoassay for Ochratoxin A Based on Nanogold Hollow Balls with Dendritic Surface

Abstract

A surface plasmon resonance (SPR)‐immunosensor based on nano‐size gold hollow ball (GHB) with dendritic surface has been developed for detection of Ochratoxin A (OTA). A thionine thin film was initially electropolymerized onto the SPR‐probe surface, and then anti‐OTA monoclonal antibody (anti‐OTA) was immobilized onto the SPR‐probe surface by means of GHB conjugation. The binding of target molecules onto the immobilized antibodies causes an increase in the resonant angle of the sensor chip, and the resonant angle shift was proportional to the OTA concentration in the range of 0.05–7.5 ng/ml with a detection limit of 0.01 ng/ml at a signal/noise ration of 3. A glycine‐HCl solution (pH 2.8) was used to release antigen‐antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of OTA into three milk samples was assayed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable. Compared with the conventional enzyme‐linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

表面等离子体共振技术在蛋白质-蛋白质相互作用研究中的应用

表面等离子共振(SPR)近年来迅速发展为用于分析生物分子相互作用的一项技术.该技术无需标记、特异性强、灵敏度高、样品用量小,可实现在线连续实时检测.目前SPR已被广泛应用于免疫学、蛋白质组学、药物筛选、细胞信号转导、受体/配体垂钓等领域.该文阐述了基于表面等离子体共振技术生物传感器的基本原理和技术流程,综述了SPR在蛋白质-蛋白质相互作用动力学研究、蛋白质结构及功能研究、蛋白质突变和碎片分析、信号转导中的应用以及SPR在蛋白质-蛋白质相互作用研究中的多项关键技术.指出SPR通过与光谱、电化学等多技术联用后,可以获得更加详实的信息.     

Detection of bisphenol A using a novel surface plasmon resonance biosensor

We present a compact surface plasmon resonance (SPR) biosensor for the detection of bisphenol A (BpA), an endocrine-disrupting chemical. The biosensor is based on an SPR sensor platform (SPRCD) and the binding inhibition detection format. The detection of BpA in PBS and wastewater was performed at concentrations ranging from 0.05 to 1,000 ng/ml. The limit of detection for BpA in PBS and wastewater was estimated to be 0.08 and 0.14 ng/ml, respectively. It was also demonstrated that the biosensor can be regenerated for repeated use. Results achieved with the SPR biosensor are compared with those obtained using ELISA and HPLC methods.     

纳米金增强SPR检测产毒赭曲霉中PKS基因的特异性碱基

基于双通道表面等离子体子共振(surface plasmon resonance,SPR)传感器,分别采用直接法和金纳米粒子作为传感层的方法,通过检测赭曲霉毒素A(ochratoxin A,OTA)合成初期阶段表达的聚酮合成酶(polyketide synthase,PKS)基因的25个特异性碱基的寡核苷酸链,建立了一种高灵敏、间接检测OTA的新方法.同时考察了6-巯基己醇(6-mercapto-1-hexanol,MCH)作为封闭液对SPR响应信号的影响.结果表明,直接法的检测限为12.5 nmol/L,MCH的加入可使响应信号有所增强,使用金纳米粒子作为传感层的检测下限为0.25 nmol/L,与直接法相比较灵敏度提高了50倍,与以往使用金纳米粒子标记抗体或抗原相比,其作为传感层也能大大提高SPR检测灵敏度,且操作简单易行.     

Preparation of Anti—Cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

IntroductionIn fast and slow skeletal and cardiac muscles,troponin I,the inhibitory protein of the troponin-tropomyosin complex,exists in three isotype formsencoded by three separated genes.The amino acidsequences of the two skeletal and one cardiac Tn Iforms( s Tn I and c Tn I,respectively) exhibit40 %dissimilarity[1] .Moreover,human cardiac Tn I has31 additional residues on the N - terminal end,which do not exist in skeletal forms,thus it pro-vides a high potential for obtaining cardiac-…