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鼠脑内几种生物小分子的活体在线检测分析
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摘要
脑神经化学过程的研究一直是分析化学、生命科学和脑神经生理学领域最热点和最前沿的研究课题之一,对脑神经现象过程中的化学物质进行实时定量监测和分析一直是科学家殚精竭虑研究和解决的重大问题,因为在脑神经过程中的信息传递或者在各种生理和病理过程中,都有相关的化学物质的参与。为了了解脑神经过程中生物分子的作用,对这些物质进行活体、实时、动态分析是了解神经活动的基本过程、研究脑功能和脑神经系统的生理过程以及调查很多重大神经系统疾病的发病机制和病理过程的前提和基础。因此,发展高灵敏的、高选择性的新型活体在线分析方法,实现脑内生理活性化学物质的有效检测,将为从分子层次上认识和研究脑神经过程中的化学本质提供可能。微透析活体取样-在线化学检测方法由于具有保持了大脑的完整性、对大脑的损伤较小、选择性好、分析步骤简便易行、近实时的时间分辨和可以连续检测脑神经活动中的化学物质的动态变化等优点,在脑神经化学的研究中具有独特的优势。本论文针对活体在线-化学分析方法研究中存在的瓶颈问题,基于光、电化学基本原理和生物化学传感技术,提出并建立了重要生理活性物质包括葡萄糖、镁离子和过氧化氢等的活体在线化学分析新原理和新方法,取得了良好的检测效果。具体工作可以概括如下:
     一、基于调控介体电位的活体在线电化学分析:通过设计并合成了基于咪唑类聚阳离子,其不但可以很好的分散碳纳米管,而且与铁氰化钾具有强烈的相互作用。这种相互作用不但可以将铁氰化钾固定在电极表面,而且可以导致铁氰化钾的式电位负移0.10V至0.16V (vs. Ag/AgCl),这个电位可以避开常规电化学生理小分子的干扰,从而实现了基于酶的底物的选择性测定。我们以葡萄糖为例,通过在电极表面修饰葡萄糖氧化酶,成功实现了豚鼠脑内葡萄糖的选择性活体在线测定。该工作为研究基于相互作用研制新型传感器奠定了基础。
     二、鼠脑内镁离子的活体比色法检测:利用1,4-二硫苏糖醇(DTT)和镁离子之间的强螯合作用,建立了鼠脑内镁离子简单但高选择性的活体比色检测的新方法。该方法通过用DTT和半胱氨酸(Cys)合理的修饰金纳米粒子(Au-NPs),在镁离子加入溶液后,可以高选择性的使得Au-NPs聚集,从而引起宏观上颜色从酒红色到蓝紫色的变化。一方面,由于Cys的加入,从而避免了加入DTT引起的Au-NPs的自聚集。另一方面,在Au-NPs表面修饰上DTT,它可以与镁离子之间发生强螯合作用,所以可以有效的高选择性检测镁离子。而且,除了钙离子外,鼠脑内共存的其他物质对检测结果没有明显的影响。我们加入乙二醇二乙醚二胺四乙酸(EGTA)为钙离子选择性的掩蔽剂,从而消除了钙离子的干扰。该方法简单有效,通过结合微透析技术,可以实现鼠脑内镁离子生理水平值的测定。本研究为与镁离子相关的生理病理的研究提供了新的有效方法。
     三、鼠脑内过氧化氢的比率型荧光检测:利用量子点优越的光学性质的优势,设计并构建检测过氧化氢浓度的量子点比率型荧光探针。选择两种不同颜色的量子点,分别包埋在氧化硅内部和共价连接在氧化硅表面,构筑出双发射量子点比率型荧光探针。该探针内部的发红光量子点的荧光并不受到干扰,但其外部的量子点可以通过与过氧化氢发生荧光共振能量转移而发生荧光淬灭,选择性地检测过氧化氢。随过氧化氢量的变化,两个发射峰的强度比值也发生变化,并伴随着荧光颜色由黄绿色到红色的转变,从而可以检测鼠脑中过氧化氢的含量。
The research of neurochemistry process has been one of the heated research fields of analytical chemistry for life sciences, because the transmission of information in brain and various process related to physiology and pathology all have participation of chemicals, so, developing selective and sensitive analytical method for in vivo monitoring of neurochemicals in the brain has paved a straightforward approach to understanding of the molecular basis of neurochemistry process. By take advantages of simple procedure, high selectivity and near real-time temporal resolution, etc., on-line detection combined with in vivo microdialysis was widely used in investigation on research of chemical process of brain research. Arming at the bottleneck problems and key problems in in vivo on-line analytical method, this dissertation focuses to highlight the development of new analytical methods for in vivo monitoring of physiological important species, such as glucose, Mg2+, based on electro-chemical and photochemistry fundamentals and biosensor. The work undertaken here can be summarized as follows:
     (1) Strong Interaction between Imidazolium-Based Polycationic Polymer and Ferricyanide:Toward Redox Potential Regulation for Selective In Vivo Electro-chemical Measurements. This chapter is based on regulation of redox potential of ferricyanide mediator by carefully controlling the different adsorption ability of ferricyanide (Fe(CN)63-) and ferrocyanide (Fe(CN)64-) onto electrode surface. To realize the negative shift of the redox potential of Fe(CN)63-/4-, imidazolium-based polymer (Pim) is synthesized and used as a matrix for surfaceadsorption of Fe(CN)63-/4-due to its stronger interaction with Fe(CN)63than with Fe(CN)64-. The different adsorption ability of Fe(CN)63-and Fe(CN)64-onto electrodes modified with a composite of Pim and multiwalled carbon nanotubes (MWNTs) eventually enables the stable surface adsorption of both species to generate integrated biosensors and, more importantly, leads to a negative shift of the redox potential of the surface-confined redox mediator. Using glucose oxidase (GOD) as the model biorecognition units, we demonstrate the validity of the ferricyanide-based second-generation biosensors for selective in vivo neurochemical measurements. We find that the biosensors developed with the strategy demonstrated in this study can be used well as the selective detector for continuous online detection of striatum glucose of guinea pigs, by integration with in vivo microdialysis.
     (2) Cysteine-Modulated Colorimetric Sensing of Extracellular Mg2+in Rat Brain Based on the Strong Chelation Interaction between Dithiothreitol and Mg2+. In this chapter, we report a facile yet highly selective colorimetric method for effective sensing of cerebral Mg2+. The method is based on rational design of surface chemistry of gold nanoparticles (Au-NPs) with functional molecules including1,4-dithiothreitol (DTT) and cysteine, enabling the fine tuning of the surface chemistry of Au-NPs in such a way that the addition of Mg2+into the Au-NPs dispersion could selectively trigger the change of the dispersion/aggregation states of Au-NPs. The strong chelation interaction between Mg2+and the hydroxyls in1,4-dithiothreitol and the co-existence of cysteine on the surface of Au-NPs could, on one hand, enable the selective colorimetric detection of Mg2+and, on the other hand, avoid the aggregation of Au-NPs induced by DTT itself. As a result, the addition of Mg2+into the dispersion of the Au-NPs containing both cysteine and DTT results in the changes in both the color and the UV-vis spectra of the Au-NPs dispersion. The signal readout shows a linear relationship of Mg2+within the concentration range from1μM to40μM with a detection limit of800nM (S/N=3). Moreover, the assay demonstrated here is free from the interference of some physiological species commonly existing in rat brain. Although Ca2+could interfere with the detection of Mg2+because of its strong chelation with DTT, it could be selectively masked by masking agent (i.e., ethyleneglcolbis (2-aminoethylether) tetraacetic acid). By combining the microdialysis technique, the basal dialysate level of Mg2+is determined to be299.2±41.1μM (n=3) in the cerebral systems. The method essentially offers a new method for the detection of Mg2+in the cerebral system.
     (3) Visual Detection of H2O2in Rat Brain by Ratiometric Fluorescence of Dual-Emission Quantum Dots Hybrid. This chapter demonstrates a new concept for fluorescence detection of H2O2in rat brain. The concept takes advantages of the superior fluorescent properties of quantum dots (QDs) via ratiometric fluorescence. Two-sized CdTe QDs emitting red and green fluorescences have been hybridized by embedding in silica nanoparticles and covalently linking on the silica surfaces, respectively, to form a dual-emissive fluorescent hybrid nanoparticle. The fluorescence of red QDs in the silica nanoparticles keeps constant, whereas the green QDs can selectively quench by H2O2due to resonance energy transfer. The variations of the two fluorescence intensity ratios display continuous color change from yellow-green to red upon exposure to different amounts of glucose, indicating this method can apply for fluorescence detection of H2O2in rat brain.
引文
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