基于鲁米诺及其功能化纳米材料的新型电化学发光传感器研究

发布时间：2018-01-17 17:36

本文关键词：基于鲁米诺及其功能化纳米材料的新型电化学发光传感器研究　出处：《西南大学》2017年博士论文　论文类型：学位论文

【摘要】：电化学发光分析技术(Electrochemiluminescence,ECL)是在化学发光和电化学基础上发展起来的一种新的分析方法,它是通过电驱动促使某些物质发生电化学反应形成激发态并通过辐射光子返回基态,并对发光强度进行测量的一种分析方法。电化学发光分析技术兼具了化学发光和电化学技术的一些特点,比如灵敏度高、选择性好、背景信号低、线性范围宽,仪器设备简单、易操控。其中以鲁米诺为发光试剂的电化学发光传感器在生物分子的检测中表现出巨大的优越性,鲁米诺的电化学发光强度可以被过氧化氢(H_2O_2)强烈增敏,在生命体系中,多种代谢中间产物在相应氧化酶的催化下生成H_2O_2,据此建立起的基于酶-鲁米诺体系的电发光传感器可实现包括葡萄糖、乳酸、胆碱等在内的多种活性物质的灵敏检测。随着生命科学的快速发展,生物活性分子分析逐渐成为生物学家和分析家的的重要研究内容,成为揭开生命奥秘、监控生命体征、征服疾病的重要手段之一。因此,利用电化学发光开展对生物分子的定量分析已然成为研究的热点。但是,电化学生物传感器的蓬勃发展是近十年的时间,因此还具有巨大的上升空间。基于鲁米诺和酶构建的电化学发光生物传感器,其主要构建模式目前还比较简单,主要通过一些聚合膜对酶进行简单封装,电阻高,酶活性中心受阻,因而其灵敏度也有待进一步提高。同时,由于鲁米诺分子是加入到待测溶液中,也会对样品会产生一定的干扰和污染,对活体物质可能有毒害作用,操作也相对复杂,因此所构建的传感器在使用寿命、稳定性、长效性、灵敏度上均还有待提高。本研究即针对基于鲁米诺电致发光传感器的重要性及存在的问题,借助纳米科技的发展和应用,致力于改善和提高鲁米诺电发光传感器的性能,一方面开发高效的响应界面;另一方面以鲁米诺为前驱体合成鲁米诺功能化的电化学发光新材料,实现鲁米诺的固定化制备固相电化学发光电极,从而对生物活性分子可以快速、灵敏、无污染的检测。通过两方面的改进,为生物活性分子提供更有效的,充满前途的分析方法。其主要研究内容如下:(1)基于三维响应界面的葡萄糖电化学发光传感器构建及用于葡萄糖的超灵敏检测。葡萄糖是重要的生物活性分子,在近几年中,采用ECL法构建葡萄糖生物传感器取得了一定进展,但是在传统的葡萄糖ECL传感器的构建当中,葡萄糖氧化酶(GOD)主要通过nafion、壳聚糖等聚合膜封装,电阻高,酶活性中心被封闭,因此灵敏度并不高。本工作将导电聚合物引入到葡萄糖ECL传感器的构建当中,通过电聚合聚苯胺,在玻碳电极表面形成一层疏松、多孔的三维网状聚苯胺纳米线(PANi),然后制备带负电荷的16nm的纳米金颗粒,并通过静电作用吸附到表面富含氨基的聚苯胺纳米线上,然后通过酶与纳米金之间的静电吸附及共价键合作用将GOD自组装到纳米金,最终构建成三维的GOD/Au NPs/PANi响应平台。由于该结构疏松多孔、导电性好、比表面积高,GOD负载量大,因此在以鲁米诺为发光试剂进行葡萄糖的ECL检测时,表现出超灵敏的响应,其线性范围为0.1-100μM,检测限0.05μM(S/N=3)。通过比较前人的工作,本实验所设计的电化学发光传感器具有更宽的线性范围和更低的检测限。在对血清样品中的葡萄糖进行检测时,也表现出良好的选择性、稳定性和灵敏度。(2)新型电化学发光材料鲁米诺还原纳米金/还原氧化石墨烯复合物的合成及用于细胞释放H_2O_2的检测。在传统的鲁米诺电化学发光传感器构建当中,鲁米诺作为发光试剂是加入到待测溶液中,模式虽然简单,但是鲁米诺会对样品、电极带来一定的污染和干扰,对活体待测物可能有毒害,同时也会增加操作步骤,增大误差。因此实现鲁米诺在电极上的固定,进行无试剂的检测,是该类传感器构建的重要发展目标。石墨烯具有众多非比寻常的物理、化学、光学性质,在本实验中,我们采用鲁米诺还原氯金酸的办法,在水热条件下制备出鲁米诺修饰的纳米金粒子,并在制备的过程中,以片状的还原氧化石墨烯为载体,一步合成出具有优秀电化学发光特性的鲁米诺还原纳米金/还原氧化石墨烯复合物(Lu-Au NPs/r GO),用以制备性能优良的固相电化学发光传感器。过氧化氢(H_2O_2)是细胞中重要的生物活性小分子之一,对其进行活细胞的实时检测具有非常重要的意义。我们进一步利用合成的新材料、二甲基硅氧烷以及氧化铟锡玻璃构建出即可用于直接培养细胞,又能够进行原位检测细胞释放H_2O_2的电化学发光传感器,实现了对人肝癌细胞Hep G2所释放的H_2O_2的定量检测。由于细胞与电极响应界面的直接接触以及电化学发光自身的高灵敏响应特性,该传感器对细胞释放H_2O_2的检测取得良好效果,而且发光试剂的固定也避免了鲁米诺分子在溶液中对细胞的影响。(3)爆米花状聚鲁米诺包裹纳米金复合物的可控合成及用于尿酸的固相电化学发光检测。虽然鲁米诺分子固定化前人做了一定的工作,包括本人合成高效的Lu-Au NPs/r GO材料,但是这些材料在长效性和稳定性上还有待提高,因为从本质上这些方法均是鲁米诺分子在纳米材料上的吸附,鲁米诺分子有限导致在使用几次后信号发生衰减。因此寻找发光更持久、稳定的鲁米诺纳米材料非常具有吸引力,聚鲁米诺是有效的选择之一。在本实验中,我们采用水热法,通过调节鲁米诺与氯金酸的反应比例,合成出聚鲁米诺包裹纳米金的核壳结构纳米复合材料(Au@Polyluminol),该复合物呈爆米花状,形状均一,分散度好,直径在200nm左右。由于聚鲁米诺聚合了大量的鲁米诺分子,因此比鲁米诺吸附的材料具有更稳定和持久的发光性能,而且由于纳米金增强电化学发光的作用,该材料对H_2O_2表现出高灵敏的ECL响应。我们将该材料与尿酸酶结合制备用于尿酸检测的固相电化学发光传感器,结果对尿酸检测表现出良好的线性范围、检测限、稳定性以及选择性。可以预见该复合物作为一种新的电化学发光材料在构建其他ECL传感器方面也将会发挥巨大作用。(4)红毛丹果状葡萄糖氧化酶/聚鲁米诺/纳米金复合物的合成及用于葡萄糖检测。在以聚鲁米诺包裹纳米金为电化学发光材料构建酶生物传感器的过程中,酶与材料之间的比例需要优化和调节,增加了传感器制备的过程,也加大了检测误差,如果能够制备出即含有活性酶,又具有电化学发光性能的双功能材料,那么将能够直接实现各种生物分子的检测,简化传感器制作步骤,增加检测的稳定性和重复性,具有非常大的应用价值。在本实验中,我们发现即使在常温下,只要有足够的时间,氯金酸也可缓慢氧化鲁米诺形成聚鲁米诺包裹的纳米金粒子。当加入适量的葡萄糖氧化酶后,不但没有阻止氧化的进行,反而在酶的调节下生成红毛丹果状、形状均一、分散度好、直径400nm左右的纳米绒球聚合物,经成分分析其组成为葡萄糖氧化酶/聚鲁米诺/纳米金(Au/PLUM/GOD)。由于在常温下合成,酶仍然保持其活性,基于该复合物构建的生物传感器后对葡萄糖表现出显著的电化学发光响应,并具有较好的灵敏度和很宽的线性范围,在葡萄糖检测上具有很大的利用价值。
[Abstract]:Electrochemiluminescence Technology (Electrochemiluminescence, ECL) is a new analysis method is developed based on Cl and electrochemistry, it is through the electric drive prompted some substances formed by electrochemical reaction by radiation photon excited state and return to the ground state, and an analysis method for measuring the luminous intensity of electrochemiluminescence technique with some characteristics of chemiluminescence and electrochemical techniques, such as high sensitivity, good selectivity, low background signal, wide linear range, simple equipment, easy operation. The ECL reagent for luminol chemiluminescence sensor shows great advantages in the detection of biological molecules, the electrochemical luminescence intensity can be Lumino hydrogen peroxide (H_2O_2) strong sensitization, in life system, a variety of metabolic intermediates in the catalytic formation of H_2O_2 oxidase under the corresponding, According to the established El Lumino system based on enzyme sensor can be realized including glucose, lactic acid, choline, sensitive detection of various active substances. With the rapid development of life science, molecular biological activity analysis has gradually become an important research content of biologists and analysts, is to open the mystery of life, monitoring of vital signs, one of the most important means to conquer disease. Therefore, to carry out the quantitative analysis by using electrochemiluminescence of biological molecules have become a hot research topic. However, the vigorous development of electrochemical biosensor is nearly ten years, it also has a huge rise in space. Lumino and the construction of electrochemical enzyme biosensor based on light, its main mode is simple, mainly through some simple encapsulation of polymeric membrane, enzyme resistance of a high center of enzyme activity is blocked, because of its sensitivity Also needs to be further improved. At the same time, because the Lumino elements are added to the test solution, will also have certain interference and pollution to the sample, may have toxic effects on living matter, the operation is relatively complicated, therefore the service life of the sensor, long-term stability, sensitivity, both need to be improved. This study focuses on the importance of Lumino electric luminescence sensor and based on the existing problems, with the development and application of nano science and technology, is committed to improve and enhance the performance of Lumino electric light sensor, a high efficiency response interface development; on the other hand, the electrochemical synthesis of precursor Lumino functional luminescent new material by Lumino. The immobilized system of Lumino using solid state electrochemiluminescence, and biological activity of molecules can be rapid, sensitive detection, no pollution. The improvement of two aspects, for the students Active molecules provide more effective and promising analysis method. The main research contents are as follows: (1) ultra sensitive detection of glucose electrochemical luminescence sensor response three-dimensional interface construction and for glucose based on glucose is the important bioactive molecule, in recent years, using the ECL method to construct glucose biosensor has been some progress, but in the construction of the traditional ECL sensor of glucose, glucose oxidase (GOD) by Nafion, chitosan polymer film packaging, high resistance, enzyme active center was closed, so the sensitivity is not high. The conductive polymer is introduced to construct ECL glucose sensor, formed by electropolymerization of polyaniline. A layer of loose on the surface of glassy carbon electrode, three-dimensional porous polyaniline nanowires (PANi), gold nanoparticles and preparation of negatively charged 16nm, and through the static Electric adsorption to polyaniline nano line surface rich in amino, followed by electrostatic adsorption and covalent bond cooperation between enzyme and gold nanoparticles with GOD self-assembled onto gold nanoparticles, to build up the 3D GOD/Au NPs/PANi response platform. Due to the porous structure, good conductivity, high surface area, large load capacity GOD therefore, the ECL for the detection of glucose reagent in response to Lumino, showed a super sensitive, the linear range is 0.1-100 ~ M, the detection limit of 0.05 M (S/N=3). Through the comparison of previous work, detection of electrical chemiluminescence sensor designed in this experiment has a wider linear range and lower limit in the detection of glucose in blood serum samples, also exhibited good selectivity, stability and sensitivity. (2) the new type of electrochemical luminescence materials Lumino synthesis of reduced gold nanoparticles / graphene Composites And for the detection of cells to release H_2O_2. Lumino in the traditional electrochemical sensor light construction, Lumino as a light reagent is added to the test solution, although the mode is simple, but Lumino will bring some of the sample, the electrode pollution and interference on the in vivo analyte may be toxic, will also increase the operation steps. Increase the error. So the realization of Lumino on the electrode fixed, no detection reagent, is an important goal of this kind of sensor construction. Physics, graphene has many unusual chemical and optical properties, in this experiment, we use the Lumino reduction of HAuCl4 to prepare gold nanoparticles Lumino modified under hydrothermal conditions and in the preparation process, the reduction of graphene oxide sheets as the carrier, one step synthesis has excellent electrochemical luminescence properties of Lumino Na reduction Nano / reduced graphene oxide (Lu-Au NPs/r GO), with excellent performance by using solid state electrochemical luminescent sensor. Hydrogen peroxide (H_2O_2) is one of the important biological activity of small molecules in the cell, real-time detection of living cells on it has very important significance. We further use of new synthetic materials. Two methyl siloxane and indium tin oxide glass can be used to construct directly cultured cells, but also be able to detect in situ electrochemical cells release H_2O_2 luminescence sensor, realizes the quantitative detection of the release of human hepatocellular carcinoma cell line Hep G2 H_2O_2. Due to the direct contact with the electrode cell response interface and ECL its high sensitive response characteristics detection of the sensor on the release of H_2O_2 cells achieved good results, but also avoid the fixed reagent Lumino molecule in solution on the cell. Ring. (3) for solid phase controlled synthesis and electrochemical luminescence detection of uric acid popcorn poly Lumino wrapped gold nanocomposites. Although Lumino molecules immobilized predecessors have done some work, including the Lu-Au NPs/r GO I synthesis of highly efficient materials, but these materials in the long-term and stability need to be improved, because from essentially, these methods are Lumino adsorption molecules in nano materials, Lumino molecular Co. resulted in the use of several times after the signal attenuation. So looking for more durable luminescent nano materials, Lumino stability is very attractive, poly Lumino is one of the effective options. In this experiment, we used the hydrothermal method. Lumino and the proportion of adjusting the reaction of chloroauric acid, synthesis of core-shell structure nano composite material poly Lumino wrapped gold nanoparticles (Au@Polyluminol), the compound is popcorn shaped shape. The shape of uniform, good dispersion degree, the diameter of around 200nm. The Lumino Lumino molecular mass poly polymerization, so the adsorption material than Lumino emission is more stable and persistent, and because the effect of gold nanoparticles enhanced electrochemical luminescence, the material of H_2O_2 exhibits a high sensitive ECL response. We use the materials and uricase combined with the preparation for the electrochemical detection of uric acid solid luminescence sensor, the determination of uric acid showed a good linear range, detection limit, stability and selectivity. The composite can be predicted as a new electrochemical luminescent materials in the construction of other ECL sensors will also play a significant role. (4) the fruiting of glucose oxidase Lumino / / poly rambutan complexes and synthesis of gold nanoparticles for glucose detection. On poly Lumino wrapped gold nanoparticles as electrochemical luminescence materials to construct enzyme The sensor in the process between the enzyme and the proportion of materials need to be optimized and adjusted, increasing the process of sensor preparation, but also increased the detection error, if can be prepared with enzyme activity, and has the dual function materials of electrochemiluminescence detection, then it will be able to directly realize all kinds of biological molecules, simplified sensor the production steps, increase the detection stability and repeatability, has great application value. In this experiment, we found that even at room temperature, as long as there is enough time, chloroauric acid can also slow oxidation of luminol formation of gold nanoparticles Polyluminol package. When the adding amount of glucose oxidase, not only did not stop oxidation, but produced rambutan, in the regulation of the enzyme under the uniform shape, good dispersion, nano polymer ball diameter of about 400nm, the composition is composed of glucose and oxygen Enzyme / Polyluminol / gold nanoparticles (Au/PLUM/GOD). The synthesis at room temperature, the enzyme still maintain its biological activity, the compound sensor constructed of glucose showed a significant response based on electrochemiluminescence, and has good sensitivity and linear range is very wide, has great application value in the detection of glucose..

【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3;TP212

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