【作者】 高霞；

【导师】 叶建山；

【作者基本信息】 华南理工大学 ， 应用化学， 2014， 博士

【摘要】 脑研究已经成为了当代科学研究的热点，随着自然科学的不断发展和多学科交叉，对脑的认识和研究的手段也逐步地深入，并取得了长足进展。脑功能相关的神经化学物质的活体分析对认识和了解生理、病理过程中的化学信息以及相关疾病的诊断和治疗有着极为重要的意义，一直是人们关注的热点问题。美国政府更是在2013年10月提出了脑研究的长期计划。脑神经化学是涉及到分析化学和生命科学等多种学科相互交叉的前沿研究领域，分析化学的发展促进了脑神经化学的发展，而脑神经化学的发展也给分析化学提供了更多的机遇和挑战。目前虽然微透析活体取样-在线检测的方法已经比较成熟，在脑神经化学研究中的应用也最多，但随着脑神经科学的发展，对于分析方法的要求也将更加苛刻。脑神经化学过程的分析科学研究中的瓶颈问题在于新型分析体系的提出和建立，而新的分析体系的建立可以通过两种途径来实现：其一是分析方法本身的进一步发展，其二是基于其它学科如材料科学、生物化学等的最新进展所构建的新化学体系以及分析化学新原理和新技术。本论文针对上述瓶颈问题，主要探讨可用于活体分析的新型分析体系。通过探讨Fe基无限配位聚合物（FeCP）和聚二甲基硅氧烷（PDMS）型微流控芯片等新材料新技术在活体分析化学中的应用，构建了基于FeCP配位聚合物的葡萄糖活体比色检测法和基于PDMS型微流控芯片的抗坏血酸和Mg2+的活体在线检测法。本论文的主要研究工作如下：（1）基于材料化学的最新进展，我们利用具有电化学活性的二羧酸二茂铁（H2FcDC）作为配体，通过其上的羧酸根与不同金属离子M2+/3+之间的配位作用制得了一系列无限配位聚合物（ICPs）。对该系列ICPs材料的形貌及结构进行了表征，最终选取中心离子为Fe3+的FeCP配位聚合物为代表性产物，并对其电化学及化学性能进行了研究。研究发现，FeCP保持了配体H2FcDC的电化学活性，可实现对H2O2的电化学双催化，也就是既可以催化氧化H2O2，也可以催化还原H2O2。同时研究发现FeCP在不同的pH环境下表现出了不同的模拟酶性质，比如在中性pH环境下FeCP可以直接化学歧化H2O2，具备过氧化氢酶的性质；而在酸性pH环境下，FeCP可以在H2O2存在时将过氧化物酶底物TMB催化氧化，具备过氧化物酶的性质。FeCP这一pH依赖的模拟酶性质使其在活体分析的研究中具有很大的潜在应用。（2）基于FeCP配位聚合物在不同pH下的不同模拟酶行为，着重研究了FeCP配位聚合物的过氧化氢模拟酶性质并对其进行了应用探讨。实验结果表明，在中性环境下FeCP确实具有过氧化氢酶的性质，可以直接化学歧化H2O2生成H2O和O2。我们推测FeCP这种固有的模拟酶性质可能是来自于其本身的Fe2+/Fe3+活性中心。基于FeCP的这一性质，我们将可以把分子O2两电子还原为H2O2的钴卟啉（CoP）作为氧还原的第一电化学催化剂，和可将生成的H2O2化学歧化为H2O和O2的FeCP作为氧还原的第二化学催化剂，同时掺杂在离子液体（Ionic liquids，IL）型的碳纳米管凝胶中，CoP与FeCP连续的发挥作用，提高了O2的利用率，实现了中性环境下氧气的表观四电子还原。（3）基于FeCP配位聚合物在不同pH下的不同模拟酶行为，着重研究了FeCP配位聚合物固有的过氧化物模拟酶性质并对其进行了应用探讨。实验结果表明，在酸性pH值下FeCP确实具有过氧化物酶的性质，并排除了可能来自FeCP泄漏的Fe离子（Fe2+/Fe3+）的干扰。对不同条件下FeCP配位聚合物的过氧化物酶活性进行了考察，得出最优条件为在25mM Tris-HCl和0.1M KCl，pH=4.0的缓冲溶液中，于40oC进行反应。在最优条件下，我们利用H2O2存在下FeCP催化氧化过氧化物酶底物ABTS的显色反应实现了H2O2的比色法检测，进一步的利用葡萄糖氧化酶实现了葡萄糖的比色法检测。实验结果证明基于ABTS显色的比色检测法对葡萄糖具有很好的选择性和线性，并最终实现了鼠脑内葡萄糖的检测。（4）通过将PDMS型的微流控芯片与在线微透析采样技术相结合，我们成功的制备了一种简单有效的可用于同时检测鼠脑内抗坏血酸和镁离子的在线电化学检测系统。虽然微透析活体取样-电化学在线检测的方法已经比较成熟，但其中承载电化学传感器的商用微流动电解池的结构却固定统一，不可更改，这极大的限制了其在双组份或多组分脑化学物质检测中的应用。本工作利用微流控芯片结构上的可设计性与灵活性构建了新型的电化学检测器代替商用的微流动电解池，成功地用于了抗坏血酸与镁离子无交叉干扰的同时在线检测。该基于微流控芯片的在线电化学检测系统具有响应好，选择性高，稳定性好和可重复性等优点，因此可用于鼠脑中抗坏血酸与镁离子的连续同时检测。这一结果对实现鼠脑内多组分的检测具有技术上及实验上的借鉴意义，有望发现其在生理和病理研究上的其他应用。更多还原

【Abstract】 Brain research has become one of the most hottest research fields and along with thedevelopment of natural science and multidisciplinary, it has been studied deeper and knownbetter and as a result, considerable progress has been made. The U.S. government hasproposed a brain research program in October,2013. The in vivo analysis of neurochemicalsrelated to brain function has drawn extensive attention because it plays an important role inknowing the chemical information in physiological and pathological processes and in thediagnosis and treatment of related diseases. The research of neurochemistry is in relation toanalytical chemistry, life sciences and other subjects. The development of analytical chemistryhas promoted the development of neuroscience, which also returns more opportunities andchallenges to analytical chemistry. Although in vivo microdialysis sampling-onlinevoltammetry methods are relatively mature and have been used most for the neurochemistrystudy, however, there still needs more novel analytical approaches due to the fast developmentof neuroscience. In our opinion, the bottlenecks of analytical science in neurochemistry are toestablish new analytical systems, which can be achieved by the two ways: the innovation ofanalytical method and building new chemical systems, analytical chemistry principles andnew technology based on the latest developments in other subjects, such as materials scienceand biochemistry.Aiming at the key problems mentioned above, this dissertation focuses on thedevelopment of new in vivo analytical approaches. By exploring the application of Fe-basedinfinite coordination polymer (FeCP) and PDMS-based microfluidic technique, we fabricateda colorimetric method based on the FeCP for detecting the glucose in rat brain and also a newmicrofluidic-based online detecting system for the measurements of ascorbate and Mg2+. Thework undertaken here can be summarized as follows:(1) In this work, based on the latest development in materials science, we have prepareda series of electrochemically active infinite coordination polymers (ICPs) by a reaction ofdifferent metal ions with1,1’-ferrocenedicarboxylic acid in an aqueous solution. Themorphologies and structures of the resulting ICPs were characterized and finally FeCP waschosen to be studied further. The electrochemical and chemical properties of FeCP have beeninvestigated carefully and the research finds out FeCP shows bifunctional mediation of H2O2for electrochemical oxidation of H2O2to O2and reduction to H2O. Moreover, FeCP showsdifferent mimetic properties depending on pH. In neutral solution, FeCP exhibits thecatalase-like activity and can catalyze the disproportionation reaction of H2O2, while in acidic solution FeCP has the peroxidase-like activity and can catalytically oxidize the enzymesubstrate TMB. The different mimetic properties of FeCP make it have potential applicationsin the study of in vivo analysis.(2) Based on the different mimetic properties in different pH solution, this work focuseson the catalase-like activity and related application of FeCP in neutral solution. The researchproves that FeCP do have the catalase-like activity and can catalyze the disproportionationreaction of H2O2to H2O and O2. We speculate that the couple of Fe2+/Fe3+in FeCP plays akey role in the mimetic property. Based on the catalase-like activityof FeCP, cobalt porphyrin(CoP) is embedded into multiwalled carbon nanotube/ionic liquid (IL) bucky gel to serve asthe first electrocatalyst to reduce O2to H2O2while FeCP is also embedded into the gel as thesecond catalyst to disproportionate H2O2to H2O and O2. The new born O2was reduced againand as a result, the utilization of O2was greatly improved, evoking an apparent4e-reductionof O2into H2O in neutral media.(3) Based on the different mimetic properties in different pH solution, this work focuseson the peroxidase-like activity and related application of FeCP in acidic solution. Theresearch finds out that FeCP do have the peroxidase-like activity and has excluded thepossible interference from the leaching solution (Fe2+/Fe3+). The optimal condition for theactivity of FeCP is25mM Tris-HCl and0.1M KCl (pH4.0) and the reaction temperature is40oC. Under the optimal conditions, the FeCP as peroxidase mimetic provides a colorimetricassay for H2O2based on the catalytic oxidation of peroxidase substrate ABTS and moreperfect, an analytical platform for glucose detection was fabricated using glucose oxidase andthe as-prepared FeCP. The colorimetric detection method for glucose has good selectivity andlinearity and thus realized the measurements of glucose in rat brain.(4) By integrating microfluidic chip with in vivo microdialysis, we have successfullydeveloped a facile yet effective online electrochemical detecting system for continuous andsimultaneous monitoring of ascorbate and Mg2+in rat brain. Although in vivo microdialysissampling-online voltammetry methods are relatively mature, however, the commerciallyavailable flow cell used in these methods have been limited in the detection of two or morethan two components due to its uniform and unchangeable electrode structure and alignment.This work takes advantage of the designable and flexible cell structures of the microfluidicchip instead of the commercially available flow cell to establish a new system which enablesthe simultaneous measurements of ascorbate and Mg2+to be successfully achieved withoutcrosstalk. The microfluidic chip-based online electrochemical system is very responsive,highly selective, stable, and reproducible and is thus reliable and durable for the continuous and simultaneous measurements of ascorbate and Mg2+in cerebral systems. This study pavesa new avenue to in vivo multiple-neurochemical monitoring in a technically simple andexperimentally designable fashion, which is envisaged to find interesting applications inphysiological and pathological applications.更多还原