【作者】 黄茜；

【导师】 张爱东； 杜丹； 涂海洋；

【作者基本信息】 华中师范大学 ， 有机化学， 2009， 博士

【摘要】 农药在为农业生产提供保障的同时其残留物也对环境和人类健康造成危害,这就对当前农药研究提出了两方面的研究课题:一方面,发展快速、可靠的农药分析检测方法保障食品安全和品质:另一方面,开发高效、低毒、低残留的农药新品种,从源头上降低农药残留带来的副作用。将靶标蛋白与底物结合的高选择性和分子识别能力与电化学检测的灵敏、快速等特点结合,成为研究高效、便捷的农药分析检测新方法的发展趋势。利用靶标蛋白与底物的分子识别能力建立抑制剂离体生物活性分析方法,同样对实现新型农药候选化合物的初期筛选和先导结构化合物验证具有重要意义。无论是农残检测还是药物筛选,界面构建和生物大分子的固定是影响传感分析信号灵敏度和稳定性的关键因素。本文利用电化学和表面等离子体共振技术,以乙酰胆碱酯酶（AChE）和D1蛋白酶（CtpA）为靶标蛋白构建了几种生物兼容的界面,围绕发展基于靶标蛋白分子识别的农药分子检测和生物活性研究新方法这一中心,开展了以下几个方面的工作:1.多壁碳纳米管-壳聚糖-乙酰胆碱酯酶复合界面的构建及其与底物的分子识别利用戊二醛作为交联剂将AChE固定在壳聚糖与多壁碳纳米管组成的复合界面上。壳聚糖作为载体固定酶,为AChE提供一个理想的微观环境以保持酶的活性,碳纳米管的引入能有效降低酶催化产物硫代胆碱在电极上的氧化电位。固载的AChE能快速灵敏的催化溶液中的底物氯化乙酰硫代胆碱（ATCl）,定量检测ATCl在2.0-20.0μM和20.0-400.0μM两个浓度区间内与电流响应有良好线性关系,AChE的表观米氏常数为132μM。该方法重现性好、灵敏度高、响应迅速、稳定可靠,有望为酶抑制剂的检测提供一个经济、便利的检测方法。2.构建多壁碳纳米管-交联壳聚糖-乙酰胆碱酯酶复合界面用于杀虫剂检测及生物活性分析以多壁碳纳米管交联壳聚糖复合膜固定AChE,采用竞争结合方法,分析溶液中的杀虫剂与ATCl竞争结合表面固定的酶。电化学信号的降低与杀虫剂三唑磷的浓度在0.03-7.8μM和7.8-32.0μM浓度范围内呈现良好线性,并定性分析比较了西维因、马拉硫磷、乐果对AChE的抑制效果,建立了基于AChE抑制的农药药效快速分析电化学方法。改变表面固载的靶标酶蛋白,可用于其它酶底物或模拟底物水解产物具有电化学活性的酶抑制剂的活性比较。3.银纳米粒子-生物素复合界面固定乙酰胆碱酯酶检测有机磷农药将自主合成的biotin-聚醚链-硫辛酸与11-巯基-1-十一醇混合组装于金电极表面,以avidin作为桥接将biotin衍生化的乙酰胆碱酯酶（ACHE）固定于表面,银纳米通过与avidin之间的静电引力结合,进而形成银纳米粒子-avidin-AChE复合界面。以ATCl为底物,根据氧化峰电流的减少,检测到乐果在0.05-10.0μM浓度范围内与酶抑制率呈线性关系,线性拟合的相关系数分别0.9983,检测限为0.01μM。4.金纳米粒子标记的氨基甲酸酯与乙酰胆碱酯酶相互作用的表面等离子体共振研究AChE通过共价键固定在11-巯基-1-十一酸的自组装膜上,将本课题组设计合成的两种抑制剂先导化合物（HY1和HY2）通过硫金键固定于金纳米粒子表面实现标记（记作ALC1和ALC2）。AChE与抑制剂之间的相互作用会引起表面等离子体（SPR）信号的变化,金纳米特殊的光学性质放大SPR检测信号,提高检测灵敏度,得到AChE与HY1、HY2的结合动力学数据:结合速率常数（K_a）分别为1.46×10⁵（Ms）^-1和7.73×104（Ms）^-1,解离速率常数（K_d）分别为4.66×10^-2s^-1和1.21×10^-1s^-1,计算得到亲合常数（K_A）分别为3.13×10⁶和6.39×10⁵ M^-1。此体系结合竞争模式可以实现其它小分子农药类似物的免标记活性比较分析。5.芯片表面原位形成金纳米粒子放大表面等离子体共振信号检测酶抑制剂固定反应时间,SPR信号与AChE的活性有对应关系,根据酶活性的抑制程度建立了有机磷农药三唑磷的高灵敏检测方法,在0.5-14.0μM浓度范围内,三唑磷浓度与SPR信号呈线性关系,检测限为0.05μM。在这个体系中,无论是酶、底物还是分析物都无需标记和固定,反应在溶液中进行,为农药检测提供了一种简单、无标记、实时、灵敏的检测方法。6.D1蛋白酶与二茂铁标记24肽相互作用的电化学检测及抑制剂活性分析利用一步沉积法将氯金酸还原成金纳米原位沉积于电极表面构成金纳米-壳聚糖的复合界面固定CtpA。二茂铁（Fc）标记的24肽作为底物,通过与界面固定的CtpA相互作用结合到电极表面。结果表明,溶液中的Fc-24P在2.0-65.0μM的浓度范围内与电流响应呈现良好线性,通过测量小分子抑制剂与Fc-24P对D1蛋白酶的竞争反应,对三种CtpA抑制剂先导化合物HY1、HY2和NA1进行了生物活性分析。7.D1蛋白酶与金纳米粒子标记24肽相互作用动力学的表面等离子体共振研究及其应用金片表面修饰生成羧基化葡聚糖界面后将羧基活化,利用酰胺键将CtpA固定在芯片上。利用SPR技术研究金纳米标记的24肽与表面固定的CtpA相互作用,得到动力学数据K_a=3.08×10²（Ms）^-1,K_d=6.24×10^-3s^-1。当溶液中有酶的抑制剂存在时,抑制剂与底物竞争性结合界面固定的CtpA。选取竞争性拟合模型拟合了抑制剂存在条件下的SPR数据,获得了抑制剂与酶的结合速率常数、解离速率常数及亲合常数等动力学数据,为实现D1蛋白酶抑制剂的生物活性筛选提供了新方法。更多还原

【Abstract】 Use of pesticides contributes positively to agricultural development.However,excessive use is associated with serious risks to environment and human health.Hence,scientific researchers are facing by these two challenges:Firstly,development of rapid,effective and credible pesticide-residual analytical methods to monitor food quality process.Secondly,to discover high potent pesticides of low toxicity and residue to control the environmental and health risks.Combining high selectivity and molecular recognization ability of interaction between target protein and its substrate with sensitivity and convenience of electrochemical detection techniques,biosensor is considered to be hot topics of current research in pesticide residue detection.It is significant to develope in-vitro sensitive analyical methods for pesticides screening and discovery.For both pesticide-residual detection and drug screening,a construction of interface for immobilization of biomolecules is a key factor to influence sensitivity and stability of sensing signal.Several bionic interfaces were constructed in this paper,using acetylcholinesterase（ACHE） and D1 protease（CtpA） as target proteins.Focusing on development of new methods for pesticide residue detection and drug sensitivity comparison based on target protein molecular recognization by the technology of surface plasmon resonance（SPR） and electrochemistry,we carried out some researches as follow.1.Molecular recognization of acetylthiocholine to immobilized AChE on multiwall carbon nanotube-cross linker chitosan compositeChitosan provided a satisfied microenvironment to retain the biological activity of AChE with glutaraldehyde as cross-linker.Because of the introduction of multiwall carbon nanotube,the oxidation potential of thiocholine was much reduced.The immobilized AChE had greater affinity for acetylthiocholine（ATCI） and excellent catalytic effect in the hydrolysis of ATCI,with a K_m^app value of 132μM.Under optimum conditions the amperometric current increased linearly with increasing concentration of ATC1 in the range 2.0-400.0μM.This method showed very good reproducibility, sensitivity and acceptable stability.It is a promising new tool for characterization of enzyme inhibitors and for pesticide analysis.2.An composite interface based on immobilization of acetylcholinesterase on a multiwall carbon nanotube-cross-linked chitosan for pesticide detection and sensitivity comparisonPresence of pesticides in solution will reduce the interaction between ATCI and immobilized AChE on a multiwall carbon nanotube-cross-linked chitosan composite,which resulted in an electrochemical signal reduction caused by thiocholine.Based on competition strategy,the inhibition of triazophos was proportional to its concentration in two ranges,from 0.03 to 7.8μM and 7.8 to 32 μM.Pesticides of carbaryl,malathion and dimethoate were selected to study their inhibition efficiencies to ACHE,which is illuminated the establishment of a speedy comparison for pesticide sensitivity by electrochemical technique.The constructed biosensor processing prominent characteristics and performance such as simple fabrication,fast response,acceptable stability and accuracy has potential application in the characterization of enzyme inhibitors and detection of toxic compounds to enzyme.3.Composite assembly of silver nanoparticles with biotinylated AChE for pesticidal sensingUsing avidin as a linker,a biosensor has been devised by immobilization of biotinylated AChE on a biotin-terminated and 11-mercapto-l-undecanolmixed self-ssembly monolayer.silver nanoparticles was introduced by the electrostatic interaction between silver nanoparticles and avidin.Under the optimum conditions a quantitative measurement of organophosphate pesticide dimethoate was achieved with the linear range of 0.05μM to10.0μM and the correlation coefficients of 0.9983.The detection limit was 0.01μM,which corresponds to a 10%decrease in signal.4.Interaction research between gold nanoparticles labeled carbamate and AChE by SPRTwo carbamate inhibitors with different ether linkages and the terminal lipoate were synthesized and labeled with gold nanoparticles.With the signal amplification of AuNPs,the specific interactions between the AuNPs labeled carbamate inhibitors and the immobilized ACHE on sensor chip surface were readily examined by SPR.Using 1:1 fitting model,the association/dissociation rate constants were first obtained for the binding interaction between carbamate inhibitors and ACHE.This AuNPs labeling strategy is versatile and may be applicable for the competitive SPR kinetic assay of the interaction between small molecule inhibitors and their target proteins with a high sensitivity.5.Signal enhancement by AChE stimulated in situ growth of gold nanoparticles for SPR technology-based sensing of inhibitor.The hydrolysis of acetylthiocholine chloride（ATC1） catalyzed by AChE can yeild a reducing agent thiocholine that stimulates the formation of AuNPs in the presence of HAuCI4,which caused a SPR signal.The formation of the AuNPs was inhibited by triazophos,thus enabling a sensitive determination for AChE inhibitor by SPR technology.The inhibition of methomyl on AChE was proportional to its concentration in the range of 0.5-14.0μM,with detection limit of 0.05μM. Inhibitor determination was achieved without labelling or modification for target protein ACHE,which is appropriate for enzyme-based detection.Such a simple and convenient strategy may find wide potential applications in biosensors,biocatalysis and durg screen.6.Interaction research between D1 protease and ferrocene labeled 24 peptide and application in inhibitor sensitivity comparison by electrochemical technique. A novel interface embedded in situ gold nanoparticles in chitosan hydrogel was constructed for CtpA immobilization by one-step electrochemical deposition.Ferrocene labeled 24 peptide was acted as electrochemical probe because of its redox activity.The current response that arose by ferrocen displayed interaction between immobilized CtpA and 24 peptide.The current response was proportional to concentration of ferrocene labeled 24 peptide in the range of 2.0-65.0μM.Inhibitor sensitivity comparison was achieved by comparing inhibition rate of three kinds of CtpA inhibitors at the same concentration.7.Interaction research between D1 protease and gold nanopartivcle labeled 24 peptide by SPR: Kinetic analysisCtpA was immobilized on a carboxyl methyl dextran interface by acylamide bond to study the interaction between CtpA and gold nanoparticle labeled 24 peptide by SPR.The kinetic data was obtained with K_a= 3.08×10²（Ms）^-1,K_d=6.24×10^-3 s^-1.Presence of inhibitor in solution will reduce the interaction between CtpA and 24 peptide on a multiwall carbon nanotube-cross-linked chitosan. composite,resulting in SPR signal reduction.Based on competition fitting model,the kinetic data including association rate constant,dissociation rate constant were obtained,which provided a new method for inhibitor sensitivity comparison and screening.更多还原