摘要
生物大分子如何被持久、高效并保持活性状态固定在生物传感表面,同时保证生化反应信号迅速而充分地被信号转换元件所采集,是生物传感器研究的重要课题。氧化还原酶类催化底物分子的氧化还原反应,反应过程中转移的电子可被电化学检测仪器捕获,或者产生颜色变化可通过紫外-可见分光光度计来检测,易于获得可定量测定的信号变化,因而适合充当生物传感器的分子识别元件,并作为量化评价酶固定化效果的模式分子。
本论文选择三种在生物传感器中广泛应用的氧化还原酶蛋白:胆碱氧化酶(ChOx)、葡萄糖氧化酶(GOx)和辣根过氧化物酶(HRP)来研究两类蛋白质固定化技术,分别为疏水蛋白自组装薄膜固定化ChOx和GOx,以及聚电解质层层累积微胶囊固定化HRP。
疏水蛋白是一类由丝状真菌产生的外分泌蛋白,包含约100个氨基酸残基。其分子的表面结构包括一个4 nm~2大小的平整的连续疏水区域,其余部分为亲水性,是一个典型的两亲性分子,具有极强的界面活性,能够显著降低水的表面张力,并在多种表面自组装形成高度有序的纳米级结构薄膜,同时改变表面对水的湿润性,帮助真菌在多种环境条件下完成表面吸附或冲破水面生长。根据氨基酸序列疏水模式及蛋白质生物物理特点的差异,疏水蛋白分为Ⅰ型和Ⅱ型,本论文研究了来源于瑞氏木霉Trichoderma reesei的Ⅱ型疏水蛋白HFBI在金属表面的自组装行为,及其用作电流型生物传感器的酶固定化基质的可行性,致力于构建完全由蛋白质修饰、能够最大程度利用固定化酶的催化活力的酶电极。
石英晶体微天平(QCM)实验结果证实了HFBI在铂表面的自组装行为,并显示HFBI自组装量受酸碱度影响不明显,而随HFBI浓度增加呈非线性增长。通过循环伏安法检测铂电极表面HFBI自组装薄膜的通透性,验证了QCM实验结果,即浓度较高的HFBI溶液能够在铂表面自组装形成更为致密的疏水蛋白薄膜,表现为较高的蛋白质自组装量和较低的通透性。筛选出了适于构建生物传感器的适当HFBI浓度,由20μg/mL HFBI自组装形成的疏水蛋白薄膜,能够允许信号分子过氧化氢通过,同时有效屏蔽电活性干扰物质抗坏血酸、尿酸和醋氨酚到达电极表面。
在生物传感表面构造亲水性微环境有利于酶蛋白保持活性构象,通过测定HFBI自组装薄膜修饰前后金表面与水的接触角变化,证明该薄膜能够显著而且稳定地提高金表面的亲水性。在HFBI自组装薄膜修饰的金表面通过物理吸附成功地固定化了ChOx,酶的表面覆盖率为3366 ng·cm~(-2),固定化后ChOx的最适pH值由游离酶的pH 8.0转变为pH 7.6,表观米氏常数Km~(app)为1.27 mM,与游离酶Km值接近。在+400 mV工作电位下,基于Au/HFBI/ChOx电极的胆碱生物传感器各项性能指标良好,最低检测限为0.01 mM(信噪比=3),线性范围0.01-1.0mM,灵敏度2184.06458 nA·mM~(-1),表现出抗干扰性能和较长的使用寿命,对酶活力的利用效率显著优于已报道的同类胆碱生物传感器。
在HFBI自组装薄膜修饰的铂表面通过静电吸附成功固定化了GOx,酶的表面覆盖率为859 ng·cm~(-2),固定化后GOx的表观米氏常数Km~(app)等于14.8 mM,略低于游离酶Km值。在+400 mV工作电位下,基于Pt/HFBI/GOx电极的葡萄糖生物传感器各项性能指标良好,最低检测限为0.12 mM(信噪比=3),线性范围0.5-20 mM,灵敏度0.29806μA·mM~(-1),表现出抗干扰性能和较长的使用寿命,对酶活力的利用效率显著优于已报道的同类葡萄糖生物传感器。
本论文首次将Ⅱ型疏水蛋白应用于电流型生物传感器,获得了高性能的胆碱和葡萄糖生物传感器,并提供了两种基于疏水蛋白HFBI自组装薄膜的、简单高效的酶蛋白固定化策略。
本论文还报道了一种操作流程简化、条件温和、能够稳定固定化酶蛋白的微胶囊制备技术。使用包含HRP并带有负电荷的碳酸钙微粒作为模板,通过带有相反电荷的两种聚电解质:聚烯丙基胺盐酸盐(PAH)和聚苯乙烯磺酸钠(PSS)交替累积,在模板表面形成(PAH/PSS)_5自组装薄膜,溶解模板后获得了形态大小均一、平均直径约6μm、分散度较好的HRP-(PAH/PSS)_5微胶囊,50天内HRP向微胶囊外释放量维持在总固定化量的6%以内。应用紫外-可见分光光度法测定微胶囊对HRP显色底物的催化活性,30天内对邻苯三酚的催化活力降低不超过20%。所制备的微胶囊表现出一定程度的选择透过性,有可能在用于生物传感器构建时提高对底物的专一性,并屏蔽部分干扰物质,应用于酚类环境污染控制。
It's an exciting challenge in the research of biosensors that how bio-macromolecules could be efficiently immobilized on kinds of sensing surfaces with their active conformations during a relatively long period,while facilitating the collection of biochemical reaction signals by transducers promptly and adequately. Redox enzymes catalyze oxidation-reduction reactions,during which measurable signals are easy to be obtained,for example,the transferred electrons are able to be detected by electrochemical instruments,or the color changes are able to be detected by UV-visible spectrum.As a result,redox enzymes are suitable to serve as sensitive biological elements for biosensors,also as model molecules for quantitative evaluation to the effect of enzyme immobilization.
Choline oxidase(ChOx),glucose oxidase(GOx),and horseradish peroxidase (HRP),all of which are commonly used redox enzymes to fabricate biosensors,have been selected for the study on two types of strategies of protein immobilization. ChOx and GOx have been immobilized on metal surfaces by self-assembled films of hydrophobins,while HRP has been immobilized in microcapsules based on Layer-by-Layer polyelectrolyte deposition.
Hydrophobins are small proteins of about 100 amino acid residues,secreted exclusively by filamentous fungi.There is an approximate 4 nm~2 planar hydrophobic patch on the surface of hydrophobin molecular,and the rest parts are hydrophilic.The characteristic amphiphilic structure contributes to the extremely high surface activity of hydrophobins,which significantly reduce the surface tension of water and self-assemble into strong,highly ordered films at almost any interfaces,helping fungi attach to various surfaces under different environmental conditions or grow into the air from aqueous environment.On the basis of differences in hydropathy patterns of the amino acid sequences and biophysical properties,two different types of hydrophobins are distinguished,namely classⅠand classⅡ.The self assembly behavior of a classⅡhydrophobin,HFBI from Trichoderma reesei has been investigated in this dissertation.Furthermore,the possibility of HFBI to serve as enzyme immobilization matrix for amperometric biosensors has been studied,in order to construct "all-protein modified" enzyme electrodes and to bring all the catalytic activities of immobilized enzymes into play.
Experimental results of quartz crystal microbalance confirmed the self-assembly of HFBI on platinum substrate,indicating that the mass of HFBI self-assembly was barely affected by pH while it increased non-linearly together with the increase of HFBI concentration.The permeability of HFBI films self-assembled on platinum electrode was checked by cyclic voltammetry,whose results were in accordance with those of quartz crystal microbalance.It's believed that solutions of higher HFBI concentrations were apt to form more compact films on platinum surface and exhibited larger self-assembled HFBI mass and stronger block to electrochemical interferences.The optimal HFBI concentration for fabricating biosensors was 20μg/mL.
Hydrophilic microenvironments on the biosensing surfaces would help enzymes keep their bioactive conformations.Water contact angle was measured to evaluate the wettability of gold surfaces before and after HFBI modification.The surface hydrophilicity was remarkably and steadily improved after HFBI processing.ChOx was successfully immobilized on HFBI modified gold surface via physical adsorption, with a coverage density of 3366 ng·cm~(-2).After immobilization,the optimal pH of ChOx slightly changed from pH 8.0 to pH 7.6,while the apparent Michaelis constant Km~(app) was 1.27 mM and close to free ChOx.An amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode,which showed low limits of detection of 0.01mM choline(signal-to-noise ratio=3),wide linear range from 0.01 to 1.0 mM,high sensitivity of 2184.06458 nA·mM~(-1),also well selectivity and lifetime.Comparing with our choline biosensors previously reported,the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, which produced as large as 4718 nA response current by 0.238μg immobilized ChOx, when saturated by choline substrate.
GOx was successfully immobilized on HFBI modified platinum surface via electrostatic adsorption,with a coverage density of 859 ng·cm~(-2).The apparent Michaelis constant Km~(app) of immobilized GOx was 14.8 mM and a little lower than the Km of free ChOx,indicating higher affinity for glucose after immobilization.An amperometric glucose biosensor was constructed based on the Pt/HFBI/GOx electrode,which showed low limits of detection of 0.12 mM glucose(signal-to-noise ratio=3),wide linear range from 0.5 to 20 mM,high sensitivity of 0.29806μA·mM~(-1),also well selectivity and lifetime.Comparing with other glucose biosensors previously reported,the HFBI self-assembled film exhibited especially high efficiency of enzyme utilization,which produced at most 710μA responsive current for per unit activity of GOx,when saturated by glucose substrate.
The dissertation applied a classⅡhydrophobin in amperometric biosensors for the first time.Both of the choline biosensor and the glucose biosensor were of high performances,which thus provided two simple and effective strategies for enzyme immobilization based on hydrophobin HFBI self-assembly.
This dissertation also reported a preparation technique of polyelectrolyte microcapsules for steady immobilization of enzymes,with simplified procedures and mild reaction conditions.Negatively charged CaCO_3 microparticles containing HRP molecules were prepared and subsequently enwrapped with layer-by-layer deposited multilayer films,which were constructed via an alternate electrostatic adsorption of poly(allylamine hydrochloride)(PAH) and poly(styrene sulfonate)(PSS).The CaCO_3 templates were then dissolved,leaving homogeneous and well dispersed HRP-(PAH/PSS)_5 microcapsules with an average diameter of about 6μm.During 50 days,the leakage of HRP out of the microcapsules was no more than 6%of the total immobilized HRP amount.While detected by UV-visible spectrum,the encapsulated HRP showed catalytic activity to pyrogallol substrate,the decrease of which was no more than 20%during 30 days.The selective permeability might help with the substrate specificity and the anti-interference capability of the prepared microcapsules. Thus,HRP-containing microcapsules might be promising in biosensor development for detection and treatment of phenolic compounds in environmental pollution.
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