微渗析活体取样—电化学在线分析系统对生命信息分子实时检测的研究与应用
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摘要
生命科学是研究生命活动过程以及生命体与环境相互作用规律的一门科学。在当今社会中,人类面对的诸多重大问题如人类生存、身体健康、人口膨胀以及食品安全等,皆与生命科学有着必然和密不可分的联系。同时,生命科学的进步与发展,也极大地促进了多学科之间的交叉研究,从而促使生命科学向着更高水平和更深层次发展。通过生命科学领域一系列重大突破技术的实现,如上世纪50年代DNA螺旋结构的发现、80年代PCR技术的发明以及90年代克隆动物的无性繁殖等,展示了生命科学作为未来前沿研究领域中先导科学的美好前景。
进入21世纪以来,脑科学研究作为生命科学探索中的重要部分正日益成为世界关注的热点之一。人们对脑神经中神经系统机理的探索一直是当今脑科学面临的最具挑战性的领域之一。小分子生理活性物质(如神经递质、调质等)是各种脑神经活动的物质基础。尤其是各种神经递质,作为调控生理活动的重要生命信息分子,神经递质在神经元细胞之间的信息传递,实现了大脑的诸多高级神经活动,如学习、记忆、感觉及运动控制等。递质分子是在突触内经突触前神经元内的递质前体和酶系统经反应后合成的,并储存于突触前不同的囊泡里。当突触前神经元的兴奋传递到神经末梢时,突触前膜发生去极化,从而打开前膜上的Ca2+通道,激活蛋白激酶Ⅱ,使得储存于突触前囊泡内的递质释放并进入突触间隙,作用于突触后膜上的特异性受体,最终产生信息传递效应并发生相应的生理反应。
神经递质传导的紊乱和平衡状态的打破,已被证实是导致众多中枢神经系统疾病的重要原因之一。因此,实现对生命信息分子如神经递质分子的可控、实时、连续、高效的活体测定,可以便于我们更好地了解整个神经元网络中神经传递过程及其作用机理,进一步了解大脑中的生命信息分子与行为学的关系,同时也为从分子机制层面上开展脑科学的研究提供非常重要的科学研究价值。不仅如此,从伦理道德上来说,实现神经递质的活体检测还可以在很大程度上减少对实验动物的需求量。因此,探索具有高选择性、高灵敏度、高时间和空间分辨率的分析新原理、新方法,并将其应用于对神经递质分子的实时、在体、连续的检测中,是本论文研究工作的主要内容。全文共分为七个部分,具体内容如下：
1绪论(第一章)
本章节内容主要介绍了微渗析活体取样技术的发展及其与电化学生物传感技术联用,应用于对大鼠脑中谷氨酸等重要神经递质的实时、在线、连续检测中。此外,在回顾了电化学生物传感及纳米材料各自的发展历程后,我们重点介绍了功能型纳米材料与电化学生物传感有机结合后的新原理、新方法的研究及其在生物-化学交叉领域中的应用；最后阐述了本论文的研究目的和意义,同时指出本论文的创新之处与主要研究内容。
2微渗析活体取样-电化学生物在线传感系统的建立(第二章)
为了能够达到实时、动态、连续地监测实验动物模型各器官如脑内生命活性物质水平变化的目的,我们建立了用于生命活性物质在线测定的活体在线取样-分析系统。该系统主要由微渗析取样和电化学生物传感两部分构成。以微渗析泵为动力,在一定流速的灌流液的带动下,通过微渗析管对动物的特定脑区进行灌流,最后通过电化学传感器系统实现活体动物的取样分析。该装置的取样过程是在完全封闭的情况下进行的,取样过程和分析测定过程均是连续不断地进行的,因此可以反映实验动物脑中每一个时间点的生命活性物质的瞬时、连续变化。在电化学传感部分,我们利用生物氧化酶对底物的高度特异性以及各种电子媒介体如功能型金属纳米材料、介孔碳、石墨烯等纳米材料对电子传递的促进作用,从而实现了对多种重要生命活性物质如谷氨酸、葡萄糖、乳酸等的高选择、高灵敏的检出。该在线取样-分析系统解决了生物样品量少、浓度低、容易变质等缺点。微渗析活体取样-电化学生物传感联用技术为快速、准确、科学地研究实验动物各器官尤其是脑部各区域中的生命活性物质的水平及其变化提供了有效的手段,在神经系统分子机制以及相应疾病病理的探索等方面具有很好的科学研究价值,在临床医学、药物筛选等方面也具有广阔的应用前景。
3形貌和粒径可控的石墨烯/TiO2/Pd复合纳米材料的制备及其电催化行为的研究(第三章)
本工作中,我们通过一锅水相合成法结合紫外光激发等手段,制备了可控的石墨烯/TiO2/Pd复合材料。通过石墨烯和TiCl3之间的氧化还原反应,得到还原型石墨烯/TiO2复合型纳米材料。实验结果表明,以还原石墨烯为载体,通过改变还原齐(?)TiCl3的加入量(0.6,1.2,1.8,2.4,3.0m1,TiCl3/GO的重量比分别为9.4,18.8,28.2,37.6,47.0),Ti02的形貌从颗粒状逐渐转变为针状结构为主。通过SEM、TEM等方法对Ti02纳米粒子和纳米针的直径、长度以及在石墨烯表面的分散性等参数进行了表征。同时,在紫外光激发下,该五种石墨烯/Ti02复合纳米材料能够将Pd2+还原至Pd纳米颗粒并且Pd纳米颗粒的尺寸也随着TiCl3的加入量的不同而改变。电化学研究结果表明,以该五种石墨烯/Ti02复合纳米材料为基底构筑的修饰电极对Fe(CN)63-/4-的电化学行为(峰电流,氧化还原电势差ΔEp)和对H2O2的电催化还原性质也有着明显的差异。本工作的开展为纳米材料的可控性制备和对某些底物的可控性电化学行为的研究提供了一种新的方法,为石墨烯纳米材料在生物传感领域中的进一步广泛应用提供了新的思路。
4微渗析活体取样-PAMAM/Pt修饰电化学在线传感联用在大鼠纹状体中谷氨酸检测中的研究与应用(第四章)
本章通过基于树枝状大分子PAMAM/Pt复合纳米材料的谷氨酸传感器的研制,实现了对大鼠脑内重要神经递质-谷氨酸的定量检测。首先,我们利用端基为氨基的3.0代聚酰胺-胺型(IPAMAM)树枝大分子为稳定剂,通过金属盐溶液的化学还原法,制备了PAMAM包裹Pt纳米粒子的PAMAM/Pt复合纳米材料,同时以MWCNTs为电子媒介体,构筑了MWCNTs/PAMAM/Pt生物传感器。论文中通过线性扫描伏安(LSV)和电流-时间(i-t)等电化学表征手段,对该传感器的电化学性能进行了考察,发现该修饰电极对H202有着良好的电催化响应。此外,我们还以谷氨酸氧化酶(GlutaOx)为识别元件,通过谷氨酸氧化酶在MWCNTs/PAMAM/Pt表面的有效固定和修饰,结合第二章中构建的微渗析活体取样-电化学生物传感在线体系,实现了对谷氨酸的特异性、高灵敏的在线检测,其响应电流与谷氨酸浓度在1.0～50.0μM范围内呈现良好的线性关系,检测下限为0.5μM(S/N=3)。最后,我们将该GlutaOx/MWCNTs/PAMAM/Pt/Nafion传感器成功地用于对大鼠纹状体中谷氨酸的在线检测中,测得大鼠纹状体中谷氨酸的基础含量为5.80±0.12μM (mean±s.d., n=3),与文献报道数据基本一致。该方法的建立,实现了对神经递质的实时、在线、连续检测,为生命科学及临床医学等领域的相关研究提供了一种有效的手段。
5功能化离子液体的设计及其对Au/Pt复合纳米粒子粒径和电催化活性的可控性研究(第五章)
在本研究工作中,我们以-OH,-CN,-COOH,-SH为阳离子功能化基团,通过与四种阴离子基团,Cl-, BF4-, PF6-,Tf2N-之间不同的组合方式,合成了九种新型的功能化咪唑环离子液体(RTIL)。探讨了九种功能化离子液体的热稳定性、导电能力、黏度等物理性质。将该九种离子液体作为模板和稳定剂,结合电化学沉积的方法,制备了九种粒径各异的Au/Pt复合纳米粒子。通过SEM、AFM等方法对制得的Au/Pt纳米粒子的粒径及分布进行了表征。结果显示,离子液体结构中的功能化基团对Au/Pt纳米粒子的大小等物理性质有着显著的影响,制得的Au/Pt纳米粒子的粒径分布范围为2.8～84.7nm。同时,线性扫描伏安(LSV)、电流-时间(i-t)等电化学研究结果表明,以该九种离子液体为基底构筑的RTIL-Au/Pt修饰电极对Fe(CN)63-/4-的电化学行为(峰电流Ip,氧化还原电势差ΔEp等)和对H2O2的电催化还原性质也有着明显的差异。我们结合离子液体的物理性质和电化学表征结果,对功能化离子液体对Au/Pt纳米粒子粒径及电催化活性的调控机制进行了初步的探讨。本工作的开展,为特定的功能化离子液体的设计和不同尺寸纳米颗粒的合成等方面的研究提供了坚实的理论基础。
6[C3(OH)2mim][BF4]-Au/Pt与微渗析活体取样联用技术在大鼠纹状体中谷氨酸在线检测中的应用(第六章)
本工作是基于前一章中系列性功能化离子液体对Au/Pt纳米粒子粒径及电催化活性的调控的研究而进一步展开。通过基于功能化离子液体-Au/Pt复合纳米材料的谷氨酸传感器的研制,实现了对大鼠脑内重要神经递质等生命信息分子的检测。我们结合九种功能化离子液体-Au/Pt复合纳米材料对H202不同的电催化还原行为,从其中挑选出具有最高电催化活性的离子液体[C3(OH)2mim][BF4],并采用原位电化学沉积的方法,在电极表面制备了平均粒径为2.5nm的Au/Pt合金纳米粒子,以谷氨酸氧化酶(GlutaOx)为识别元件,构筑了GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion生物传感器并研究了该传感器的电催化还原性能。结果显示,该传感器对谷氨酸有着良好的电催化响应,线性范围为0.520.0μM,检测限达到0.17μM(S/N=3)。利用第二章中构建的微渗析活体取样-电化学生物传感在线体系,我们对大鼠进行了腹腔注射100mM KCl溶液和电刺激丘脑底核(STN)实验,同时对给予刺激后引起的谷氨酸含量的变化进行了实时、在线、连续的测定。结果显示上述两种刺激均能引起谷氨酸含量的上升,相对于纹状体内谷氨酸的正常水平,上升百分比分别67.7%和155.5%(mean±s.d.,n=3)。该方法的建立,实现了对神经递质的实时、在线、连续检测,为生命科学及临床医学等领域的相关研究提供了一种有效的手段。
7功能化介孔碳/Pt复合纳米材料电化学性质研究及其对大鼠纹状体中葡萄糖及乳酸水平的在线同时测定(第七章)
本文利用微渗析活体取样-电化学生物传感联用技术实现了对大鼠纹状体中葡萄糖和乳酸的同时和选择性在线检测。首先,我们利用带正电荷的聚合物PDDA与介孔碳(CMM)之间的非共价作用力,对CMM进行了有效的功能化(PDDA-CMM),并将其作为模板制备了不同Pt负载量(5%,10%,20%,30%,40%和50%)的PDDA-CMM/Pt复合纳米材料。通过扫描电镜(SEM)和透射电镜(TEM)等表征手段,我们对PDDA-CMM和掺杂不同含量Pt的PDDA-CMM/Pt复合纳米材料进行了形貌和粒径的表征,同时研究了金属Pt在PDDA-CMM表面的负载量对Pt纳米颗粒粒径的影响。结果表明,当增加Pt在PDDA-CMM表面的负载量时,Pt纳米粒子的粒径及粒子之间的交联度均随之增加。Pt纳米粒子的粒径分布范围3.2±0.4～6.8±1.4nm,粒子之间的交联度分布为0.5～5.6。此外,我们还对五种基于不同Pt负载量的PDDA-CMM/Pt复合纳米材料对H202的电催化还原行为进行了探讨,发现30%Pt掺杂的PDDA-CMM/Pt对H202具有最高的电催化活性。基于以上结果,我们以葡萄糖氧化酶(GOx)和乳酸氧化酶(LOD)在电极表面的进一步修饰,研制了葡萄糖和乳酸同时在线电化学检测的双电极工作体系。结合微渗析活体取样技术,对大鼠纹状体内的葡萄糖和乳酸含量进行了同时、选择性在线检测。经校准,大鼠纹状体内葡萄糖和乳酸的基础浓度分别为0.27±0.03和0.71±0.05mM(mean±s.d.,n=3),与文献报道相符。该传感器具有较高的灵敏度和选择性,且双工作电极之间无明显的相互干扰,为今后进一步开展缺血性脑疾病的分子机制研究以及用于多成分测定的阵列电极研制提供了科学有效的方法。
Life science is a subject which focuses upon the researches of life process and the mutual relationships of life organisms and its neighboring environment. Nowadays, a series of vital problems we pay attention to, such as, human survive, health, population explosion, food safety, etc, have been demonstrated to be tightly associated with the development of life science. Meanwhile, the rapid progress in life science has also boosted the intersection of some related disciplines, including mathematics, chemistry and physics. What's more important, the successive technology breakthroughs in life science area like the discovery of DNA helical structure in the1950s, the innovation of PCR technique and the asexual reproduction in the90s, further confirms the leading position of life science which will definitely become the most important part in the future frontier research fields.
All brain functions, including memory, movement, emotion, etc, have always involved the participation of various chemicals. The existence of the small molecular biological active substances (neurotransmitters and neuromodulators) provides the essential preconditions for these mentioned brain functions. Neurotransmitters are one of these chemicals that are secreted by neurons and relay messages to the target cells. The brain contains a vast network of neurons that are connected with each other at specialized junctions called synapses. Chemicals that are released into the synaptic gap interact with its corresponding receptors, which then lead to the intracellular changes in the postsynaptic neuron, manifested by the altered membrane potential or gene expression. The chemical signal is terminated by transport proteins that transfer transmitter molecules across the membrane to the intracellular space. This is just how the intact network operates and how the behaviors happen.
On-line analytical techniques, consisting of microdialysis sampling and direct on-line detector, without any sample separation or pretreatment, are becoming increasingly common for continuous measurement of glucose and glutamate etc. in biological samples. Due to its short analytical time, high sensitivity and specificity to provide near real-time measurements, microdialysis sampling can provide both the temporal and chemical information that we need to fully elucidate the biochemical processes. Compared with various detection techniques coupled with microdialysis, electrochemical biosensors have been proved more efficient in view of its high time and spatial revolution, easy-to-prepare, and excellent selectivity and sensitivity. With these advantages, microdialysis-electrochemical biosensor has become a facile and reliable tool for the real-time determination of neurotransmitters in vivo.
The main work of this paper focuses upon novel analytical methods and technique design that is suitable for life science. A series of promising nanomaterials based on metal nanoparticles and carbon materials are prepared and employed in the construction of enzyme biosensors. Combined with microdialysis sampling technique, the effective on-line detection system is developed in our group and applied for the real-time, continuous on-line measurement of physiologically important species such as glutamate, glucose and lactate. The method demonstrated here has been proved to be sensitive and reproducible in this paper, which enables its promising application in physiology and pathology. The primary research work is as followed:
Chapter1Overview
In chapter1, we mainly elaborated the development of microdialysis sampling technique and its combination with electrochemical biosensor, which is then applied for the real-time on-line determination of neurotransmitters in vivo. Moreover, we also pay more attention to the introduction of novel analytical principle and method into biology and chemistry based on the integration of functionalized nanomaterials with electrochemical biosensors. In the end, we also emphatically pointed out the purpose and significance of our research, as well as the innovation spot and contents.
Chapter2Construction of on-line microdialysis-electrochemical biosensor system
For the purpose of real-time, on-line, continuous determination of neurotransmitters in vivo, an effective on-line microdialysis-electrochemical biosensor system was constructed in our work. This system consisted of two parts:microdialysis sampling and electrochemical detection. The microdialysis sampling was powered by a microdialysis pump, and a perfusion fluid could be pumped into the rat brain through a microdialysis probe at a very low perfusion rate, which was then detected by the electrochemical biosensor. It was performed under a totally sealed condition, and both the sampling and detection procedure was continuously performed, so every time point change of the extracellular concentrations of neurotransmitters in brain can be detected in real time. For the preparation of electrochemical biosensors, various namomaterials including metal nanoparticles and carbon materials have been involved, which achieved a high sensitivity and selectivity. Through the integration of microdialysis with electrochemical biosensors, the on-line, real-time measurement of some physiologically important species such as, glutamate, glucose and lactate have been realized in vivo.
Chapter3Morphology-tunable TiO2Nanostructure self-assembled on Graphene nanosheet for the Preparation of Palladium Nanoparticle and their Electrochemical application in Hydrogen Peroxide Reduction
Morphology-tunable TiO2nanostructure synthesis using graphite oxide and TiCl3as the starting materials is demonstrated for the first time. Our results elucidate that both weight deposition ratio and morphology-controllable TiO2nanostructures can be facilely achieved on graphene nanosheet by simply manipulating the feeding volume of TiCl3, from0.6,1.2,1.8,2.4to3.0ml (i.e., TiCl3/GO weight ratio=9.4,18.8,28.2,37.6,47.0), forming five GTx nanocomposites (x=0.6,1.2,1.8,2.4to3.0). The nanostructures of TiO2are crystalline and vary from spherical to needle-like with the increase of TiCl3dosage. Moreover, these tunable GTx nanocomposites are further successfully employed as supporting materials for the reduction and dispersion of Pd nanoparticles (NPs). The photogenerated electrons from UV-irradiated TiO2are transported across the GTx composites to stepwise reduce Pd2+into Pd NPs. The diameters of these as-prepared Pd NPs show a similar GTx-dependent behavior. The five GTx-Pd hybrid nanocomposites are also observed to play a key role in deciding the electrochemical performance of H2O2, thereby tuning their electrocatalytic activities. Hence, this study demonstrates us a novel and facile method for constructing high-quantity graphene/metal oxide semiconductor/metallic NP hybrids with tunable morphology, size as well as electrocatalytic activity via an effective and low-cost method and their potential application in biocatalysis.
Chapter4Development of On-line Microdialysis System with Poly(amidoamine)-encapsulated Pt Nanoparticles Biosensor for Glutamate Sensing in Vivo
In this work, on-line microdialysis system with amperometric detection was constructed for in vivo glutamate measurement in rat's brain successfully. Firstly, an amine-terminated poly (amidoamine) dendrimer containing Pt nanoparticles (PAMAM/Pt) was synthesized and its electrochemical behaviors were investigated by linear sweep voltammetry (LSV) and amperometric i-t curve. Combined with MWCNTs fabrication, MWCNTs/PAMAM/Pt/Nafion modified electrode was prepared. It demonstrated excellent electrocatalytic responses to the reduction of H2O2at a potential of-200mV without HRP participation. Then, glutamate oxidase (GlutaOx) was immobilized on the MWCNTs/PAMAM/Pt films for glutamate measurement with on-line microdialysis system, which was powered by a microdialysis pump. The glutamate biosensor in the on-line microdialysis system showed good linear range from1.0μM to50.0μM with the detection limit of0.5μM (S/N=3). Under the above optimal conditions, the content of glutamate in the striatum of rat was detected in vivo continuously with this on-line system by implanting of microdialysis probe. The concentration of glutamate was5.80±0.12μM (mean±s.d., n=3) in the striatum of rats. It was proved to be sensitive and reproducible with wider linear range. These properties enabled its promising application in physiology and pathology.
Chapter5Size-controllable Gold-Platinum Alloy Nanoparticles on Nine Functionalized Ionic Liquid Surfaces and Their Application as Electrocatalysts for Hydrogen Peroxide Reduction
A series of room temperature ionic liquids (RTILs) containing different functional groups, such as hydroxyl, nitrile, carboxyl, and thiol attached to imidazolium cations, combined with various anions, e.g., chloride [Cl], tetrafluoroborate [BF4], hexafluorophosphate [PF6], bis[(trifluoromethyl)sulfonyl]imide [Tf2N], are successfully synthesized. Dissolved in chitosan (Chi), the RTILs are employed as flexible templates for the preparation of Au/Pt nanostructures. These Au/Pt nanostructures can be facilely in situ deposited on the surface RTILs through electrodeposition. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) results demonstrate that the alloy size is significantly dependent on the structure of RTILs, with their sizes ranging from2.8nm to84.7nm. Based upon the functionalized RTILs, we fabricate nine RTIL-Au/Pt biosensors and the size-dependent electrochemistry of RTIL-Au/Pt is firstly investigated using potassium ferricyanide as the probe. Reversible electron transfer of Fe(CN)63-/4-redox couple is realized at nine biosensors while the peak current, as well as the peak-to-peak separation (⊿Ep) and electron transfer rate, differs greatly from each other, which is mainly caused by the diversity of RTILs. Further electrochemical researches reveal that the functional groups of these RTILs exert an obvious influence on the reduction behavior of H2O2, which in turn illustrates that the electrocatalytical activity of RTIL-Au/Pt nanocomposites can be tuned by means of employing RTILs with different functional groups and an appropriate combination of cations and anions could produce a higher activity. The facilitated electron transfer and the intrinsic catalytical activity of Au/Pt NPs provide us a facile way to construct a third generation H2O2biosensor with high sensitivity, lower detection limit, quick response time and excellent selectivity.
Chapter6[C3(OH)2mim][BF4]-Au/Pt Biosensor for Glutamate Sensing in Vivo Integrated with On-line Microdialysis System
A new type of hydroxyl functionalized room temperature ionic liquid (RTIL),[C3(OH)2mim][BF4], was synthesized herein and a novel H2O2biosensor was fabricated with [C3(OH)2mim][BF4] as the substrate and ultrasonically electrodepositing bimetallic Au/Pt nanoparticles (NPs) onto the [C3(OH)2mim][BF4] film. It was found that the functionalization of RTIL with hydroxyl groups provides an appropriate environment for the preparation of more uniform and smaller Au/Pt NPs, of which the average diameter was2.5run. Immobilized with glutamate oxidase (GlutaOx), the resulting GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion biosensor displayed excellent electrocatalytic responses to glutamate at a potential of-200mV. An effective on-line microdialysis system, which was powered by a microdialysis pump, was successively set up and used for the detection of glutamate in the striatum of rats. The glutamate biosensor in the on-line microdialysis system showed good linear range from0.5μM to20.0μM with the detection limit of0.17μM (S/N=3). The application of the GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion electrode is demonstrated for in vivo sensing of the variation of glutamate level in the striatum when rats received intraperitoneal (i.p.) injection of100mM KCl and brain electrical stimulation of the subthalamic nucleus area (STN). Both of the two kinds of stimulation resulted in an increase in the extracellular concentration of glutamate. The method demonstrated here was proved to be sensitive and reproducible, which enabled its promising application in physiology and pathology.
Chapter7Size-Tunable Pt nanoparticles assembled on ordered mesoporous carbons for the Simultaneous and On-line detection of Glucose and Lactate in Brain microdialysate
This study presents a facile electrochemical method for simultaneous and selective on-line detection of glucose and lactate in the striatum of anaesthetic rats through the integration of selective electrochemical detection with in vivo microdialysis system. A positively-charged polyelectrolyte,(diallyldimethylammonium chloride)(PDDA), was attached onto carbon mesoporous material (CMM) through non-covalent interaction, which provides an ideal environment for the dispersion of nanoparticle electrocatalyst. Platinum nanoparticles with uniform distribution and wide Pt loadings from5to50wt%are successfully self-assembled on PDDA-functionalized CMM via electrostatic interaction. TEM results show that with the increase in the Pt loadings on CMM, both the size and interconnectivity between particles increase, with Pt sizes ranging from3.2±0.4to6.8±1.4nm and interconnectivity from0.5～5.6. Moreover, the electrocatalytic activities of the as-prepared six Pt/PDDA-CMM hybrid nanocomposites are also observed to show an inverted V-shaped profile as a function of loading amount of Pt NPs. Integrated with glucose oxidase (GOx), lactate oxidase (LOD) and the in vivo microdialysis system, the constructed dual Oxidase/PDDA-CMM/Pt biosensors were successfully applied for the simultaneous and on-line detection of glucose and lactate. After post-calibration, the basal level of glucose and lactate in the striatum of anaesthetic rats was calculated to be0.27±0.03and0.71±0.05mM (mean±s.d., n=3), respectively. What's more important, the dual oxidase biosensors almost suffer from little cross-talk, which is characteristic of an excellent sensor with high performance. This property, along with the good linearity and a high stability toward glucose and lactate substantially enables this method elaborated in this work promising application in physiology and pathology.
进入21世纪以来,脑科学研究作为生命科学探索中的重要部分正日益成为世界关注的热点之一。人们对脑神经中神经系统机理的探索一直是当今脑科学面临的最具挑战性的领域之一。小分子生理活性物质(如神经递质、调质等)是各种脑神经活动的物质基础。尤其是各种神经递质,作为调控生理活动的重要生命信息分子,神经递质在神经元细胞之间的信息传递,实现了大脑的诸多高级神经活动,如学习、记忆、感觉及运动控制等。递质分子是在突触内经突触前神经元内的递质前体和酶系统经反应后合成的,并储存于突触前不同的囊泡里。当突触前神经元的兴奋传递到神经末梢时,突触前膜发生去极化,从而打开前膜上的Ca2+通道,激活蛋白激酶Ⅱ,使得储存于突触前囊泡内的递质释放并进入突触间隙,作用于突触后膜上的特异性受体,最终产生信息传递效应并发生相应的生理反应。
神经递质传导的紊乱和平衡状态的打破,已被证实是导致众多中枢神经系统疾病的重要原因之一。因此,实现对生命信息分子如神经递质分子的可控、实时、连续、高效的活体测定,可以便于我们更好地了解整个神经元网络中神经传递过程及其作用机理,进一步了解大脑中的生命信息分子与行为学的关系,同时也为从分子机制层面上开展脑科学的研究提供非常重要的科学研究价值。不仅如此,从伦理道德上来说,实现神经递质的活体检测还可以在很大程度上减少对实验动物的需求量。因此,探索具有高选择性、高灵敏度、高时间和空间分辨率的分析新原理、新方法,并将其应用于对神经递质分子的实时、在体、连续的检测中,是本论文研究工作的主要内容。全文共分为七个部分,具体内容如下：
1绪论(第一章)
本章节内容主要介绍了微渗析活体取样技术的发展及其与电化学生物传感技术联用,应用于对大鼠脑中谷氨酸等重要神经递质的实时、在线、连续检测中。此外,在回顾了电化学生物传感及纳米材料各自的发展历程后,我们重点介绍了功能型纳米材料与电化学生物传感有机结合后的新原理、新方法的研究及其在生物-化学交叉领域中的应用；最后阐述了本论文的研究目的和意义,同时指出本论文的创新之处与主要研究内容。
2微渗析活体取样-电化学生物在线传感系统的建立(第二章)
为了能够达到实时、动态、连续地监测实验动物模型各器官如脑内生命活性物质水平变化的目的,我们建立了用于生命活性物质在线测定的活体在线取样-分析系统。该系统主要由微渗析取样和电化学生物传感两部分构成。以微渗析泵为动力,在一定流速的灌流液的带动下,通过微渗析管对动物的特定脑区进行灌流,最后通过电化学传感器系统实现活体动物的取样分析。该装置的取样过程是在完全封闭的情况下进行的,取样过程和分析测定过程均是连续不断地进行的,因此可以反映实验动物脑中每一个时间点的生命活性物质的瞬时、连续变化。在电化学传感部分,我们利用生物氧化酶对底物的高度特异性以及各种电子媒介体如功能型金属纳米材料、介孔碳、石墨烯等纳米材料对电子传递的促进作用,从而实现了对多种重要生命活性物质如谷氨酸、葡萄糖、乳酸等的高选择、高灵敏的检出。该在线取样-分析系统解决了生物样品量少、浓度低、容易变质等缺点。微渗析活体取样-电化学生物传感联用技术为快速、准确、科学地研究实验动物各器官尤其是脑部各区域中的生命活性物质的水平及其变化提供了有效的手段,在神经系统分子机制以及相应疾病病理的探索等方面具有很好的科学研究价值,在临床医学、药物筛选等方面也具有广阔的应用前景。
3形貌和粒径可控的石墨烯/TiO2/Pd复合纳米材料的制备及其电催化行为的研究(第三章)
本工作中,我们通过一锅水相合成法结合紫外光激发等手段,制备了可控的石墨烯/TiO2/Pd复合材料。通过石墨烯和TiCl3之间的氧化还原反应,得到还原型石墨烯/TiO2复合型纳米材料。实验结果表明,以还原石墨烯为载体,通过改变还原齐(?)TiCl3的加入量(0.6,1.2,1.8,2.4,3.0m1,TiCl3/GO的重量比分别为9.4,18.8,28.2,37.6,47.0),Ti02的形貌从颗粒状逐渐转变为针状结构为主。通过SEM、TEM等方法对Ti02纳米粒子和纳米针的直径、长度以及在石墨烯表面的分散性等参数进行了表征。同时,在紫外光激发下,该五种石墨烯/Ti02复合纳米材料能够将Pd2+还原至Pd纳米颗粒并且Pd纳米颗粒的尺寸也随着TiCl3的加入量的不同而改变。电化学研究结果表明,以该五种石墨烯/Ti02复合纳米材料为基底构筑的修饰电极对Fe(CN)63-/4-的电化学行为(峰电流,氧化还原电势差ΔEp)和对H2O2的电催化还原性质也有着明显的差异。本工作的开展为纳米材料的可控性制备和对某些底物的可控性电化学行为的研究提供了一种新的方法,为石墨烯纳米材料在生物传感领域中的进一步广泛应用提供了新的思路。
4微渗析活体取样-PAMAM/Pt修饰电化学在线传感联用在大鼠纹状体中谷氨酸检测中的研究与应用(第四章)
本章通过基于树枝状大分子PAMAM/Pt复合纳米材料的谷氨酸传感器的研制,实现了对大鼠脑内重要神经递质-谷氨酸的定量检测。首先,我们利用端基为氨基的3.0代聚酰胺-胺型(IPAMAM)树枝大分子为稳定剂,通过金属盐溶液的化学还原法,制备了PAMAM包裹Pt纳米粒子的PAMAM/Pt复合纳米材料,同时以MWCNTs为电子媒介体,构筑了MWCNTs/PAMAM/Pt生物传感器。论文中通过线性扫描伏安(LSV)和电流-时间(i-t)等电化学表征手段,对该传感器的电化学性能进行了考察,发现该修饰电极对H202有着良好的电催化响应。此外,我们还以谷氨酸氧化酶(GlutaOx)为识别元件,通过谷氨酸氧化酶在MWCNTs/PAMAM/Pt表面的有效固定和修饰,结合第二章中构建的微渗析活体取样-电化学生物传感在线体系,实现了对谷氨酸的特异性、高灵敏的在线检测,其响应电流与谷氨酸浓度在1.0～50.0μM范围内呈现良好的线性关系,检测下限为0.5μM(S/N=3)。最后,我们将该GlutaOx/MWCNTs/PAMAM/Pt/Nafion传感器成功地用于对大鼠纹状体中谷氨酸的在线检测中,测得大鼠纹状体中谷氨酸的基础含量为5.80±0.12μM (mean±s.d., n=3),与文献报道数据基本一致。该方法的建立,实现了对神经递质的实时、在线、连续检测,为生命科学及临床医学等领域的相关研究提供了一种有效的手段。
5功能化离子液体的设计及其对Au/Pt复合纳米粒子粒径和电催化活性的可控性研究(第五章)
在本研究工作中,我们以-OH,-CN,-COOH,-SH为阳离子功能化基团,通过与四种阴离子基团,Cl-, BF4-, PF6-,Tf2N-之间不同的组合方式,合成了九种新型的功能化咪唑环离子液体(RTIL)。探讨了九种功能化离子液体的热稳定性、导电能力、黏度等物理性质。将该九种离子液体作为模板和稳定剂,结合电化学沉积的方法,制备了九种粒径各异的Au/Pt复合纳米粒子。通过SEM、AFM等方法对制得的Au/Pt纳米粒子的粒径及分布进行了表征。结果显示,离子液体结构中的功能化基团对Au/Pt纳米粒子的大小等物理性质有着显著的影响,制得的Au/Pt纳米粒子的粒径分布范围为2.8～84.7nm。同时,线性扫描伏安(LSV)、电流-时间(i-t)等电化学研究结果表明,以该九种离子液体为基底构筑的RTIL-Au/Pt修饰电极对Fe(CN)63-/4-的电化学行为(峰电流Ip,氧化还原电势差ΔEp等)和对H2O2的电催化还原性质也有着明显的差异。我们结合离子液体的物理性质和电化学表征结果,对功能化离子液体对Au/Pt纳米粒子粒径及电催化活性的调控机制进行了初步的探讨。本工作的开展,为特定的功能化离子液体的设计和不同尺寸纳米颗粒的合成等方面的研究提供了坚实的理论基础。
6[C3(OH)2mim][BF4]-Au/Pt与微渗析活体取样联用技术在大鼠纹状体中谷氨酸在线检测中的应用(第六章)
本工作是基于前一章中系列性功能化离子液体对Au/Pt纳米粒子粒径及电催化活性的调控的研究而进一步展开。通过基于功能化离子液体-Au/Pt复合纳米材料的谷氨酸传感器的研制,实现了对大鼠脑内重要神经递质等生命信息分子的检测。我们结合九种功能化离子液体-Au/Pt复合纳米材料对H202不同的电催化还原行为,从其中挑选出具有最高电催化活性的离子液体[C3(OH)2mim][BF4],并采用原位电化学沉积的方法,在电极表面制备了平均粒径为2.5nm的Au/Pt合金纳米粒子,以谷氨酸氧化酶(GlutaOx)为识别元件,构筑了GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion生物传感器并研究了该传感器的电催化还原性能。结果显示,该传感器对谷氨酸有着良好的电催化响应,线性范围为0.520.0μM,检测限达到0.17μM(S/N=3)。利用第二章中构建的微渗析活体取样-电化学生物传感在线体系,我们对大鼠进行了腹腔注射100mM KCl溶液和电刺激丘脑底核(STN)实验,同时对给予刺激后引起的谷氨酸含量的变化进行了实时、在线、连续的测定。结果显示上述两种刺激均能引起谷氨酸含量的上升,相对于纹状体内谷氨酸的正常水平,上升百分比分别67.7%和155.5%(mean±s.d.,n=3)。该方法的建立,实现了对神经递质的实时、在线、连续检测,为生命科学及临床医学等领域的相关研究提供了一种有效的手段。
7功能化介孔碳/Pt复合纳米材料电化学性质研究及其对大鼠纹状体中葡萄糖及乳酸水平的在线同时测定(第七章)
本文利用微渗析活体取样-电化学生物传感联用技术实现了对大鼠纹状体中葡萄糖和乳酸的同时和选择性在线检测。首先,我们利用带正电荷的聚合物PDDA与介孔碳(CMM)之间的非共价作用力,对CMM进行了有效的功能化(PDDA-CMM),并将其作为模板制备了不同Pt负载量(5%,10%,20%,30%,40%和50%)的PDDA-CMM/Pt复合纳米材料。通过扫描电镜(SEM)和透射电镜(TEM)等表征手段,我们对PDDA-CMM和掺杂不同含量Pt的PDDA-CMM/Pt复合纳米材料进行了形貌和粒径的表征,同时研究了金属Pt在PDDA-CMM表面的负载量对Pt纳米颗粒粒径的影响。结果表明,当增加Pt在PDDA-CMM表面的负载量时,Pt纳米粒子的粒径及粒子之间的交联度均随之增加。Pt纳米粒子的粒径分布范围3.2±0.4～6.8±1.4nm,粒子之间的交联度分布为0.5～5.6。此外,我们还对五种基于不同Pt负载量的PDDA-CMM/Pt复合纳米材料对H202的电催化还原行为进行了探讨,发现30%Pt掺杂的PDDA-CMM/Pt对H202具有最高的电催化活性。基于以上结果,我们以葡萄糖氧化酶(GOx)和乳酸氧化酶(LOD)在电极表面的进一步修饰,研制了葡萄糖和乳酸同时在线电化学检测的双电极工作体系。结合微渗析活体取样技术,对大鼠纹状体内的葡萄糖和乳酸含量进行了同时、选择性在线检测。经校准,大鼠纹状体内葡萄糖和乳酸的基础浓度分别为0.27±0.03和0.71±0.05mM(mean±s.d.,n=3),与文献报道相符。该传感器具有较高的灵敏度和选择性,且双工作电极之间无明显的相互干扰,为今后进一步开展缺血性脑疾病的分子机制研究以及用于多成分测定的阵列电极研制提供了科学有效的方法。
Life science is a subject which focuses upon the researches of life process and the mutual relationships of life organisms and its neighboring environment. Nowadays, a series of vital problems we pay attention to, such as, human survive, health, population explosion, food safety, etc, have been demonstrated to be tightly associated with the development of life science. Meanwhile, the rapid progress in life science has also boosted the intersection of some related disciplines, including mathematics, chemistry and physics. What's more important, the successive technology breakthroughs in life science area like the discovery of DNA helical structure in the1950s, the innovation of PCR technique and the asexual reproduction in the90s, further confirms the leading position of life science which will definitely become the most important part in the future frontier research fields.
All brain functions, including memory, movement, emotion, etc, have always involved the participation of various chemicals. The existence of the small molecular biological active substances (neurotransmitters and neuromodulators) provides the essential preconditions for these mentioned brain functions. Neurotransmitters are one of these chemicals that are secreted by neurons and relay messages to the target cells. The brain contains a vast network of neurons that are connected with each other at specialized junctions called synapses. Chemicals that are released into the synaptic gap interact with its corresponding receptors, which then lead to the intracellular changes in the postsynaptic neuron, manifested by the altered membrane potential or gene expression. The chemical signal is terminated by transport proteins that transfer transmitter molecules across the membrane to the intracellular space. This is just how the intact network operates and how the behaviors happen.
On-line analytical techniques, consisting of microdialysis sampling and direct on-line detector, without any sample separation or pretreatment, are becoming increasingly common for continuous measurement of glucose and glutamate etc. in biological samples. Due to its short analytical time, high sensitivity and specificity to provide near real-time measurements, microdialysis sampling can provide both the temporal and chemical information that we need to fully elucidate the biochemical processes. Compared with various detection techniques coupled with microdialysis, electrochemical biosensors have been proved more efficient in view of its high time and spatial revolution, easy-to-prepare, and excellent selectivity and sensitivity. With these advantages, microdialysis-electrochemical biosensor has become a facile and reliable tool for the real-time determination of neurotransmitters in vivo.
The main work of this paper focuses upon novel analytical methods and technique design that is suitable for life science. A series of promising nanomaterials based on metal nanoparticles and carbon materials are prepared and employed in the construction of enzyme biosensors. Combined with microdialysis sampling technique, the effective on-line detection system is developed in our group and applied for the real-time, continuous on-line measurement of physiologically important species such as glutamate, glucose and lactate. The method demonstrated here has been proved to be sensitive and reproducible in this paper, which enables its promising application in physiology and pathology. The primary research work is as followed:
Chapter1Overview
In chapter1, we mainly elaborated the development of microdialysis sampling technique and its combination with electrochemical biosensor, which is then applied for the real-time on-line determination of neurotransmitters in vivo. Moreover, we also pay more attention to the introduction of novel analytical principle and method into biology and chemistry based on the integration of functionalized nanomaterials with electrochemical biosensors. In the end, we also emphatically pointed out the purpose and significance of our research, as well as the innovation spot and contents.
Chapter2Construction of on-line microdialysis-electrochemical biosensor system
For the purpose of real-time, on-line, continuous determination of neurotransmitters in vivo, an effective on-line microdialysis-electrochemical biosensor system was constructed in our work. This system consisted of two parts:microdialysis sampling and electrochemical detection. The microdialysis sampling was powered by a microdialysis pump, and a perfusion fluid could be pumped into the rat brain through a microdialysis probe at a very low perfusion rate, which was then detected by the electrochemical biosensor. It was performed under a totally sealed condition, and both the sampling and detection procedure was continuously performed, so every time point change of the extracellular concentrations of neurotransmitters in brain can be detected in real time. For the preparation of electrochemical biosensors, various namomaterials including metal nanoparticles and carbon materials have been involved, which achieved a high sensitivity and selectivity. Through the integration of microdialysis with electrochemical biosensors, the on-line, real-time measurement of some physiologically important species such as, glutamate, glucose and lactate have been realized in vivo.
Chapter3Morphology-tunable TiO2Nanostructure self-assembled on Graphene nanosheet for the Preparation of Palladium Nanoparticle and their Electrochemical application in Hydrogen Peroxide Reduction
Morphology-tunable TiO2nanostructure synthesis using graphite oxide and TiCl3as the starting materials is demonstrated for the first time. Our results elucidate that both weight deposition ratio and morphology-controllable TiO2nanostructures can be facilely achieved on graphene nanosheet by simply manipulating the feeding volume of TiCl3, from0.6,1.2,1.8,2.4to3.0ml (i.e., TiCl3/GO weight ratio=9.4,18.8,28.2,37.6,47.0), forming five GTx nanocomposites (x=0.6,1.2,1.8,2.4to3.0). The nanostructures of TiO2are crystalline and vary from spherical to needle-like with the increase of TiCl3dosage. Moreover, these tunable GTx nanocomposites are further successfully employed as supporting materials for the reduction and dispersion of Pd nanoparticles (NPs). The photogenerated electrons from UV-irradiated TiO2are transported across the GTx composites to stepwise reduce Pd2+into Pd NPs. The diameters of these as-prepared Pd NPs show a similar GTx-dependent behavior. The five GTx-Pd hybrid nanocomposites are also observed to play a key role in deciding the electrochemical performance of H2O2, thereby tuning their electrocatalytic activities. Hence, this study demonstrates us a novel and facile method for constructing high-quantity graphene/metal oxide semiconductor/metallic NP hybrids with tunable morphology, size as well as electrocatalytic activity via an effective and low-cost method and their potential application in biocatalysis.
Chapter4Development of On-line Microdialysis System with Poly(amidoamine)-encapsulated Pt Nanoparticles Biosensor for Glutamate Sensing in Vivo
In this work, on-line microdialysis system with amperometric detection was constructed for in vivo glutamate measurement in rat's brain successfully. Firstly, an amine-terminated poly (amidoamine) dendrimer containing Pt nanoparticles (PAMAM/Pt) was synthesized and its electrochemical behaviors were investigated by linear sweep voltammetry (LSV) and amperometric i-t curve. Combined with MWCNTs fabrication, MWCNTs/PAMAM/Pt/Nafion modified electrode was prepared. It demonstrated excellent electrocatalytic responses to the reduction of H2O2at a potential of-200mV without HRP participation. Then, glutamate oxidase (GlutaOx) was immobilized on the MWCNTs/PAMAM/Pt films for glutamate measurement with on-line microdialysis system, which was powered by a microdialysis pump. The glutamate biosensor in the on-line microdialysis system showed good linear range from1.0μM to50.0μM with the detection limit of0.5μM (S/N=3). Under the above optimal conditions, the content of glutamate in the striatum of rat was detected in vivo continuously with this on-line system by implanting of microdialysis probe. The concentration of glutamate was5.80±0.12μM (mean±s.d., n=3) in the striatum of rats. It was proved to be sensitive and reproducible with wider linear range. These properties enabled its promising application in physiology and pathology.
Chapter5Size-controllable Gold-Platinum Alloy Nanoparticles on Nine Functionalized Ionic Liquid Surfaces and Their Application as Electrocatalysts for Hydrogen Peroxide Reduction
A series of room temperature ionic liquids (RTILs) containing different functional groups, such as hydroxyl, nitrile, carboxyl, and thiol attached to imidazolium cations, combined with various anions, e.g., chloride [Cl], tetrafluoroborate [BF4], hexafluorophosphate [PF6], bis[(trifluoromethyl)sulfonyl]imide [Tf2N], are successfully synthesized. Dissolved in chitosan (Chi), the RTILs are employed as flexible templates for the preparation of Au/Pt nanostructures. These Au/Pt nanostructures can be facilely in situ deposited on the surface RTILs through electrodeposition. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) results demonstrate that the alloy size is significantly dependent on the structure of RTILs, with their sizes ranging from2.8nm to84.7nm. Based upon the functionalized RTILs, we fabricate nine RTIL-Au/Pt biosensors and the size-dependent electrochemistry of RTIL-Au/Pt is firstly investigated using potassium ferricyanide as the probe. Reversible electron transfer of Fe(CN)63-/4-redox couple is realized at nine biosensors while the peak current, as well as the peak-to-peak separation (⊿Ep) and electron transfer rate, differs greatly from each other, which is mainly caused by the diversity of RTILs. Further electrochemical researches reveal that the functional groups of these RTILs exert an obvious influence on the reduction behavior of H2O2, which in turn illustrates that the electrocatalytical activity of RTIL-Au/Pt nanocomposites can be tuned by means of employing RTILs with different functional groups and an appropriate combination of cations and anions could produce a higher activity. The facilitated electron transfer and the intrinsic catalytical activity of Au/Pt NPs provide us a facile way to construct a third generation H2O2biosensor with high sensitivity, lower detection limit, quick response time and excellent selectivity.
Chapter6[C3(OH)2mim][BF4]-Au/Pt Biosensor for Glutamate Sensing in Vivo Integrated with On-line Microdialysis System
A new type of hydroxyl functionalized room temperature ionic liquid (RTIL),[C3(OH)2mim][BF4], was synthesized herein and a novel H2O2biosensor was fabricated with [C3(OH)2mim][BF4] as the substrate and ultrasonically electrodepositing bimetallic Au/Pt nanoparticles (NPs) onto the [C3(OH)2mim][BF4] film. It was found that the functionalization of RTIL with hydroxyl groups provides an appropriate environment for the preparation of more uniform and smaller Au/Pt NPs, of which the average diameter was2.5run. Immobilized with glutamate oxidase (GlutaOx), the resulting GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion biosensor displayed excellent electrocatalytic responses to glutamate at a potential of-200mV. An effective on-line microdialysis system, which was powered by a microdialysis pump, was successively set up and used for the detection of glutamate in the striatum of rats. The glutamate biosensor in the on-line microdialysis system showed good linear range from0.5μM to20.0μM with the detection limit of0.17μM (S/N=3). The application of the GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion electrode is demonstrated for in vivo sensing of the variation of glutamate level in the striatum when rats received intraperitoneal (i.p.) injection of100mM KCl and brain electrical stimulation of the subthalamic nucleus area (STN). Both of the two kinds of stimulation resulted in an increase in the extracellular concentration of glutamate. The method demonstrated here was proved to be sensitive and reproducible, which enabled its promising application in physiology and pathology.
Chapter7Size-Tunable Pt nanoparticles assembled on ordered mesoporous carbons for the Simultaneous and On-line detection of Glucose and Lactate in Brain microdialysate
This study presents a facile electrochemical method for simultaneous and selective on-line detection of glucose and lactate in the striatum of anaesthetic rats through the integration of selective electrochemical detection with in vivo microdialysis system. A positively-charged polyelectrolyte,(diallyldimethylammonium chloride)(PDDA), was attached onto carbon mesoporous material (CMM) through non-covalent interaction, which provides an ideal environment for the dispersion of nanoparticle electrocatalyst. Platinum nanoparticles with uniform distribution and wide Pt loadings from5to50wt%are successfully self-assembled on PDDA-functionalized CMM via electrostatic interaction. TEM results show that with the increase in the Pt loadings on CMM, both the size and interconnectivity between particles increase, with Pt sizes ranging from3.2±0.4to6.8±1.4nm and interconnectivity from0.5～5.6. Moreover, the electrocatalytic activities of the as-prepared six Pt/PDDA-CMM hybrid nanocomposites are also observed to show an inverted V-shaped profile as a function of loading amount of Pt NPs. Integrated with glucose oxidase (GOx), lactate oxidase (LOD) and the in vivo microdialysis system, the constructed dual Oxidase/PDDA-CMM/Pt biosensors were successfully applied for the simultaneous and on-line detection of glucose and lactate. After post-calibration, the basal level of glucose and lactate in the striatum of anaesthetic rats was calculated to be0.27±0.03and0.71±0.05mM (mean±s.d., n=3), respectively. What's more important, the dual oxidase biosensors almost suffer from little cross-talk, which is characteristic of an excellent sensor with high performance. This property, along with the good linearity and a high stability toward glucose and lactate substantially enables this method elaborated in this work promising application in physiology and pathology.
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