【作者】 张银堂；

【导师】 周飞艨；

【作者基本信息】 中南大学 ， 应用化学， 2007， 博士

【摘要】 表面等离子体激元共振（surface plasmon resonance,SPR）是一种检测金属表面超薄吸附层厚度和结构变化的光学技术,是研究生物分子与其它分子相互作用的有力工具,具有高灵敏、免标记、实时检测等优点,但是传统的SPR技术难以检测小分子或者弱亲和作用的反应体系,并且它需要将探针分子固定在SPR芯片表面,固定在金膜表面探针分子尤其是生物分子的活性和识别位点是否受到影响值得关注,本文针对这两方面的问题进行了如下研究:为了提高SPR的灵敏度以满足小分子检测的需求,首先,从提高SPR仪器本身灵敏度方面着手,自行搭建了双单元差分型高灵敏的SPR仪,检测灵敏度得到了一定的提高,理想情况下可达10^-5度。其次,从生物分子的组装方法入手,探讨提高SPR灵敏度的途径。用巯基十一酸（MUA）代替羧甲基葡聚糖（CM-Dextran）在中性条件下来固定金属金属硫蛋白（MT）,使MT芯片对金属离子的检测达到了很低的水平,Cd²⁺的检测下限为15ppb或0.1μM,与传统的离子分析技术有很好的可比性。再次,在现有仪器条件下,将酶催化沉淀放大技术用于SPR检测,通过对SPR信号的放大,大大提高了SPR的灵敏度,实现了超痕量DNA的检测,检测下限可达10 fM(1×10^-14M),比其它很多检测DNA方法的检出限都要低。为了研究生物分子的活性是否受表面固定的影响,同时将SPR（着重研究固/液界面）和亲和毛细管电泳（ACE）（溶液方法）两种独立的技术来研究药物（阿魏酸,FA）与蛋白质（牛血清白蛋白,BSA）的相互作用,提出了一个测定结合常数的新方法。SPR测定的结合常数(（5.1±0.6）×10⁴ M^-1)与ACE用迁移率比得到的结果(（5.6±0.4）×10⁴ M^-1)十分吻合,并且与其它方法有很好的可比性。二者的比较研究表明,BSA在SPR芯片表面的固定并没有影响它和药物作用的活性,因此用SPR来研究固液界面亲和作用是行之有效的。最后,本论文探讨不同的固定方法对蛋白质与药物相互活性的影响,分别采用共价偶联、金属螯合和静电吸附三种方法固定脂烯酰基酰基载体蛋白还原酶Ⅰ（FabI）,用SPR测定了FabI与抗菌剂三氯生的结合常数。研究表明,FabI的药物活性受固定方式的影响并不显著。另外,为了对结合在SPR芯片表面的药物分子进行鉴定,利用毛细管电泳（CE）对SPR回收液中的成分进行分析,得到令人满意的结果。通过SPR-CE联用技术研究蛋白质与抗菌剂的相互作用,提供了一个药物筛选的新思路,具有潜在的应用前景。更多还原

【Abstract】 Surface plasmon resonance（SPR）is a powerful optical technique for measuring the thickness and structure of ultrathin adsorbate layers and for evaluating affinity of biomolecules at metal film/solution interface. SPR interrogates biomolecular interactions without the necessity of labeling the analytes.Moreover,the analysis is sensitive and can be conducted in real time.However,there are certain inherent limitations in conventional SPR analysis.On one hand,the analysis becomes very difficult when small molecules are assayed or weak affinity reactions are investigated.On the other hand,at issues are whether the preimmobilized biomolecules onto the sensor surface can retain their native structures and functionality O ur work aims at addressing these two questions.To improve the sensitivity of SPR dectection,three approaches have been implemented.Firstly,much attention was paid to the sensitivity of SPR instrument itself.A highly sensitive SPR spectrometer equipped with a bicell detector was constructed and combined with a microbore flow injection device.As a result,the angle resolution was as small as 10^-5degree.Such a resolution is lower than that available on most commercial SPR instrument.Secondly,optimal immobilization conditions of biomolecules were investigated.For example, 11-mercaptoundecanoic acid（MUA）instead of carboxylmethylated dextran was used to immobilize metallothionein,and the approch afforded a much low concentration detection level for metal ions.The detection level for Cd²⁺（0.1μM or 15 ppb）compares well with other well-established sensitive analytical techniques.The reason is due to compactness of the preformed MUA layer and our highly sensitive SPR. Thirdly,an enzyme-catalyzed precipitation reaction is used to amplify the SPR signal.In the presence of H₂O₂,the horseradish peroxidase enzyme catalyzes oxidation of 4-chloro-1-naphthol（CN）to form a precipitate on the sensor surface.The precipitated film significantly lowers the detection level,allowing detection of oligodeoxynucleotide（ODN）target concentration as low as 10 fM.To verify whether the native structure and function of biomolecules may alter after their immobilization on SPR chip surface,a solution-based,viz,affinity capillary electrophoresis（ACE）,is used to confirm the binding constant K_b obtained by SPR.The K_b value between ferulic acid（FA）and bovine serum albumin（BSA）determined from the SPR measurements(（5.1±0.6）×10⁴ M^-1)is in excellent agreement with the value obtained by the ACE mobility ratio assay(（5.6±0.4）×10⁴ M^-1). The consistency of measurements between ACE and SPR,a technique addressing interfacial processes,confirms that immobilization of the protein onto surfaces does not alter its interaction with other molecules.Finally,three different immobilization methods are carried out to investigate the binding between antimicrobial agent（triclosan）and enoyl-acyl carrier protein（ACP）reductase（FabI）.The similar K_b values indicate that all of the immobilization methods do not alter the interaction of this protein with a potential antimicrobial molecule（triclosan）.CE is utilized for the qualitative and quantitative analysis of the protein/ligand complex recovered after the SPR analysis.Our method provides a new route for screening potential antimicrobial agents.更多还原