纳米增敏与流动注射电化学发光分析研究
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摘要
本论文主要涉及纳米增敏电化学发光分析与流动注射电化学发光分析系统研制,共包括以下几部分内容:
1纳米增敏鲁米诺电化学发光
(1)纳米Pt-Au合金增敏鲁米诺的电化学发光用化学还原法制备了不同比例及不同粒径的纳米Pt-Au合金,并用UV-Vis、TEM、激光粒径、XRD等方法进行了表征,确认所合成物质确系双金属合金纳米粒子而非两种金属纳米粒子的混合物,通过改变合成方法(如直接滴加法、雾化与超声结合、超声滴加法、氢气还原法、氢气还原与雾化结合)和条件,可以得到一系列不同含量比和粒径范围在4.03-92.33nm之间的Pt-Au合金纳米溶胶。采用电化学沉积法可将所制备纳米粒子修饰到铂盘电极上,在碱性介质(pH=12)中,随着合金比例的改变和合金粒径的减小,鲁米诺的电化学发光强度显著增强,当合金中Pt:Au =6:1,粒子粒径为最小4.03nm时,所获得修饰电极上鲁米诺的电化学发光强度较裸电极增强近1个数量级。研究了纳米Pt-Au合金修饰电极对鲁米诺电化学发光增敏的机理。深入分析了纳米合金晶胞中的原子排布,纳米Pt-Au合金晶胞为面心立方结构,一个合金晶胞中有10个Pt原子和4个Au原子,这四个Au原子占据着晶胞中呈对角线的四个顶点,且分两次取代晶胞中的Pt原子。研究了纳米合金与鲁米诺的相互作用,纳米合金与鲁米诺之间存在吸附作用,易发生能量转移,从而增敏鲁米诺的电化学发光,具体原因可能是:一、纳米Pt-Au合金作为一种纳米粒子,本身具有小尺寸效应、表面效应等催化特性,延长了OH.和Luminol阴离子自由基的作用时间,从而增强了发光信号;二、当Pt与Au合金化后,Pt的d空穴增多,空的d空穴成为溶液中各种自由基的受体,使得电极表面吸附了更多的OH.与Luminol阴离子等,从而增强了鲁米诺发光强度。
(2)纳米金属氧化物增敏铂电极上的电化学发光研究了纳米金属氧化物(ZnO、MnO_2、TiO_2)对鲁米诺在铂电极上电化学发光的影响。实验发现所研究的三种金属氧化物在溶液中对鲁米诺的电化学发光有明显的增敏作用,当用溶胶-凝胶法将纳米氧化物修饰固定在电极后,对鲁米诺的电化学发光亦有明显的增敏作用,在修饰电极上可以获得更稳定的发光信号和更高的信噪比。在此基础上采用溶胶-凝胶法把纳米金属氧化物和鲁米诺同时修饰于铂电极表面,制得纳米金属氧化物修饰的ECL电极。实验结果表明,纳米金属氧化物修饰ECL电极对H_2O_2均有响应,其中纳米TiO_2修饰的ECL电极的发光强度与过氧化氢在1.0×10~(-7)至1.0×10~(-5)mol/L浓度范围内成线性关系,检测下限可达1×10~(-8)mol/L。该ECL电极可用于葡萄抗氧化能力的综合评估,以每克水果消耗过氧化氢毫克数mgH2O_2/g为单位,葡萄肉汁为1.57,葡萄籽为4.72,即葡萄籽的抗氧化能力要显著强于葡萄肉。用循环伏安法和紫外-可见吸收光谱法研究了纳米金属氧化物的增敏机理,研究表明增敏原因有两点:一、鲁米诺分子被吸附在纳米金属氧化物表面,近距离接触使得能量传递成为可能。二、在外加电压的情况下,纳米金属氧化物催化产生活性氧,而活性氧可有效增强鲁米诺电化学发光。
(3)纳米ITO增敏鲁米诺电化学发光
纳米ITO为锡掺杂的氧化铟,纳米ITO粉末中氧化铟与氧化锡的含量比为In2O3:SnO_2=9:1。采用微乳液法、溶胶-凝胶法以及共沸蒸馏法合成了不同粒径的纳米In2O3并进行了透射电镜表征,考察了碱性条件下纳米In2O3对鲁米诺电化学发光的影响。实验结果表明,在碱性条件下纳米In2O3在溶液中对鲁米诺电化学发光有明显的增敏作用,并进一步研究了粒径大小与这种增敏作用的关系,采用紫外-可见吸收光谱和荧光光谱技术讨论了增敏机理。
采用溶胶-凝胶法制备粒径为10nm左右的纳米SnO_2,该纳米粒子对碱性溶液中的鲁米诺-O_2化学发光有显著的增强,这种增敏作用与纳米SnO_2的加入量以及体系中溶解氧的浓度有关系,基于此得出了纳米SnO_2存在下溶解氧浓度与鲁米诺化学发光强度之间的线性关系,可用于溶解氧测定,检测下限可达0.3mg/L。采用紫外-可见吸收光谱和荧光光谱技术研究了纳米SnO_2增敏鲁米诺化学发光机理。
基于上述研究合成了粒径为10nm左右的ITO纳米粒子,当溶液中纳米ITO含固量达到0.3mg/mL左右时,对鲁米诺的ECL强度增敏作用最强。当用溶胶—凝胶法将其固定修饰到铂电极表面后对鲁米诺的电化学发光有明显的增敏作用,考察了三种溶胶—凝胶成膜方式(红外灯烘干、烘箱烘干、自然晾干)固定纳米ITO及对增敏作用的影响,结果表明自然晾干成膜的纳米ITO修饰电极上鲁米诺的ECL强度高,增敏效果可以达到四倍左右,可以使用五天以上。实验对比发现溶胶-凝胶法修饰电极时成膜条件越缓和修饰效果越好,控制湿度在60%左右,温度低于30℃,隔夜自然晾干成膜效果最好。
2 ITO玻璃上鲁米诺电化学发光行为及机理研究
(1)研究了碱性溶液中氧化铟锡玻璃上活性氧的电化学发光行为。实验结果表明,以ITO玻璃为工作电极,在碱性溶液中观察到的电化学发光是活性氧系分子所发出的。该体系的电化学发光的可能机理是:在外加脉冲电压下ITO玻璃表面的氧化铟锡纳米粒子被激发至一定的能级,OH-、O_2及H2O_2分子在ITO玻璃电极表面发生氧化还原反应产生活性氧分子,处于一定能级的纳米ITO粒子吸附活性氧系分子并将能量转移给活性氧系分子使其到达激发态,当返回基态时能量以光子的形式释放而发光。
(2)研究了在碱性溶液中鲁米诺在ITO玻璃电极上的电化学发光机理。鲁米诺在ITO电极上的电化学发光能在较低的电位下发生主要是由于鲁米诺分子与纳米ITO粒子的相互作用,这主要是鲁米诺分子被吸附在纳米ITO粒子表面,近距离接触使得能量传递成为可能。在较低的电压下,纳米ITO粒子被激发到一定能级,当电极表面鲁米诺被氧化至中间态时,两者之间发生能量转移导致鲁米诺氧化至激发态,当返回基态时能量以光子的形式释放而发光。
(3)研究了鲁米诺在ITO玻璃上的电聚合,实验结果表明,在酸性条件下可以将鲁米诺电聚合修饰于ITO电极的表面,聚合在ITO玻璃表面的鲁米诺保持其良好的电化学发光性能,考察了此修饰电极的性能以及相关因素对聚合膜的电化学发光强度的影响。在碱性介质中该电聚合鲁米诺修饰ECL电极对碘离子、硫离子和双氧水均有响应。在选定条件下,上述物质显著增敏聚鲁米诺的ECL。在1.0×10~(-6)mol/L-8.0×10~(-6)mol/L范围内,电化学发光强度与碘离子浓度有良好的线性关系,r=0.9896;在8.0×10-7mol/L—1.0×10~(-5)mol/L范围内,电化学发光强度与硫离子浓度有良好的线性关系,r=0.9903;在8.0×10~(-6)- 6.0×10~(-5)mol/L范围内,电化学发光强度与双氧水浓度有良好的线性关系,r=0.9939.
(4)进一步将具有较高增敏效率的纳米TiO_2修饰于ITO玻璃表面,研究其对鲁米诺ECL的增敏作用,通过Nafion作为载体可以均匀吸附于ITO表面,且具有较大的修饰量。修饰电极经650°C高温灼烧后去除Nafion,纳米TiO_2被烧结固定于ITO电极表面同时发生晶型改变得到锐钛矿相与金红石相的混晶,两相比例为43.64?56.36时,其对鲁米诺电化学发光的增敏最明显。结果表明,当粒径较小,经650°C煅烧处理形成混晶时,纳米TiO_2/ ITO修饰电极对鲁米诺电化学发光的增敏效果最明显,为裸电极的7.5倍。由于先将纳米二氧化钛均匀修饰在ITO电极表面,再对电极进行煅烧,有效避免了煅烧过程中TiO_2粒子间的相互烧结团聚,在有效控制TiO_2粒径的同时得到混晶。考察7支混晶TiO_2/ITO电极对浓度为4×10-7mol/L的碱性鲁米诺溶液的电化学发光响应,其ECL强度的相对标准偏差为1.88%,在连续7天使用中发光强度仅衰减5.88%,表明用该方法制备的修饰电极可以提供满意的重现性和稳定性。
3流动注射电化学发光分析系统研制
(1)研制了一套流动注射电化学发光分析系统,结合了电化学发光的高灵敏度和流动注射的实用性,并采用光纤传输模式工作,增强了仪器的连接柔软性,以计算机嵌入式设计,操作方便、数据处理功能强,经优化形成高性能的电化学发光分析系统。在此基础上研究了纳米ITO对流动注射体系中鲁米诺电化学发光的影响,考察了流量对ECL强度的影响,当流量为10mL/h时研究体系的发光强度最大。
(2)为了克服现有的电化学发光池存在的不足,应用氧化铟锡(ITO)玻璃和有机玻璃等材料,研制出一种可以产生并检测电化学发光信号的流动注射电化学发光池,该池较好地解决了现有电化学发光池存在的一些问题,也为实验室以后开展此方面的实验工作提供硬件方面的准备。基于流动注射分析技术和电化学发光分析技术的综合应用,采用ITO玻璃作为工作电极,通过设计新的流通式电化学发光池,进行动态对流传质为主条件下ITO玻璃电极表面反应层中电化学发光反应的特征和分析特性的研究,探讨所设计的电化学发光池中鲁米诺发光体系的发光行为,考察相关因素对鲁米诺电化学发光分析特性的影响。通过对微型流动电化学发光池及ITO玻璃工作电极上鲁米诺电化学发光的相关因素的优化,提高电化学发光池的分析性能和应用范围。基于葡萄提取物对鲁米诺电化学发光的猝灭作用,考察了该流动注射电化学发光池的性能,结果表明猝灭效率与所加入水果提取液的量成线性相关,据此可评估样品的抗氧化性能,同时表明该电化学发光检测池具有良好的可实用性。在实际样品测定中,该流动注射电化学发光池具有较好的重现性,其设计和功能完全能满足电化学发光分析技术的要求,还可进一步开发与其它技术如毛细管电泳(CE)或高效液相色谱(HPLC)等联用,在实际应用研究分析中将有广阔的应用前景。
This thesis focuses on the sensitization of electrochemiluminescence by nanoparticles and related development of flow-injection analysis system with electrochemiluminescent detection. It consists of the following sections.
1 Sensitization of Electrochemiluminescence by Nanoparticles
(1) Sensitization of Luminol’s ECL by Pt-Au Bimetallic Nanoparticles Modified Electrode
The platinum-gold bimetallic nanoparticles with different component ratio and size were prepared by chemical reduction. The methods such as UV-Vis spectra, TEM and XRD were applied to characterize the properties of the nanoparticles. The information from these methods revealed that the prepared bimetallic nanoparticles were truly of alloy structure and absolutely not the mixture of two kinds of metallic nanoparticles. The component of the nanoparticles could be regulated for a series of Pt/Au ratio and the diameter of nanoparticles, which were determined by Laser-granulometer, could be regulated from 4.03-92.33nm by the method or controlled condition. In alkaline medium of pH=12.0, the bimetallic Pt-Au nanoparticle modified electrode, which was modified on a Pt disk electrode by electrodeposition, sensitized the electrochemiluminescence of luminol. With the variation in the composition and size of the bimetallic nanoparticles, the sensitization efficiency varied as well, with a highest at a Pt/Au ratio of 6:1 and diameter of 4.03nm, at which the electrochemiluminescence intensity was an order of magnitude better than that obtained from unmodified electrode.
The atom distribution in the cell of Pt-Au bimetallic nanoparticles was studied. Then found that the crystalline structure is face--centered cubic. A cell contains 10 Pt atoms and 4 Au atoms, The 4 Au atoms hold the 4 corners that are in the two diagonals, and they substitute Pt atoms in two steps. The paper also studied the interaction between bimetallic Pt-Au nanoparticle and luminol. Adsorption occurred between them, and energy transfers from one to the other, thereby Pt-Au bimetallic nanoparticles enhance the intensity of luminol. There are two reasons for sensitization for electrochemiluminescence of luminol from Platinum-Gold bimetallic nanoparticles modified electrode. One is as nanoparticle, the Pt-Au bimetallic nanoparticle has catalysis such as size effect, surface effect and etc. It prolongs the interaction time of OH. and luminol-.,thereby enhances the intensity of luminol. The other reason is when Pt and Au were alloyed; the d band cavity in Pt was increased. The cavity became the adsorption of radicals in the solution. So the electrode surface adsorped more OH. and Luminol-. , thereby enhances the intensity of luminol.
(2) Sensitization of Luminol ECL by Metallic Oxide Nanoparticles on Pt Electrode
In this work, the sensitization of luminol electrochemiluminescence by metallic oxide nanoparticles, as ZnO, MnO_2 and TiO_2, under alkaline condition was reported and the related mechanism was studied. It was found that all three types of nanoparticles exhibited similar enhancement toward the electrochemiluminescence reaction. Furthermore a sol-gel method was taken for the immobilization of the metallic oxide nanoparticles onto platinum electrodes. The so-obtained modified electrodes also showed enhanced electrochemiluminescence and better signal/noise ratio, and the stability of the signal was improved as well. Luminol together with the nanoparticles were directly immobilized onto the electrode surface and the performance of the sensors greatly improved. Also good linear response was obtained toward hydrogen peroxide. It was found that the developed modified ECL electrodes provided responses to H2O_2. For the TiO_2 modified one, a linear response was obtained with the range 1.0×10~(-7)~1.0×10-5mol/L. This could be used to measure the concentration of H2O_2 and the detection limit was low to 1×10~(-8)mol/L. This ECL electrode was applied to test the antioxidant capacity of grapes. The result was that the antioxidant capacity of the grape seed was much better than grape meat. Cyclic voltammetry and UV-visible absorption methods were taken for the study of the mechanism. It was proposed that the nanoparticles could enhance the production of reactive oxygen species, as well as the adsorption of luminescent reagent on nanoparticle surface. These two factors could give an enhancement on the electrochemiluminescent reaction.
(3) Sensitization of Luminol ECL by ITO Nanoparticle on ITO Electrode
Nanosized ITO particles were prepared from In2O3 and SnO_2 with an amount ratio at 9:1. A series of In2O3 nanoparticles at different sizes were synthesized by microemulsion, sol-gel or azeotropic distillation methods, and characterized by a transmission electron microscope (TEM). In alkaline solution of pH12.5, the influence of In2O3 nanoparticles on the ECL of luminol was studied. The results showed that the ECL intensity of luminol was enhanced obviously in the presence of In2O3 nanoparticles. The sensitization efficiency was related to the content and the size of In2O3 nanoparticles. The paper also discussed the mechanism of the sensitization by UV-Vis spectra and fluorescence spectra.
Nanosized SnO2 were synthesized by sol-gel methods and characterized by a transmission electron microscope (TEM). The result showed that the average particle sizes of nanosized SnO2 was about 10nm.In the chemiluminescence system of luminol-O2,the CL intensity of luminol was enhanced obviously in the presence of nanosized SnO2.This sensitization efficiency was related to the content of nanosized SnO2 and the dissolved oxygen. Based on this, it was educed that the CL intensity of luminol was linear with the content of the dissolved oxygen in the presence of nanosized SnO2.This linear relationship could be used to determine the dissolved oxygen and the detection limit was 0.3mg/L. The paper also discussed the mechanism of the sensitization by UV-Vis spectrum and fluorescence spectrum.
ITO nanoparticles with diameter around 10nm were prepared and the highest enhancement of luminol ECL was obtained with ITO content of 0.3mg/mL. When the nanosized ITO was immobilized to the surface of the Pt electrode by using the method of sol-gel, the ECL intensity of luminol is obviously enhanced. The work studied three different ways of sol-gel becoming film(dried by infrared radiation、dried in oven and drying naturally) to immobilize the nanosized ITO to the electrode and the effect to the enhancement of the ECL intensity .The research showed that when the sol-gel became film naturally, the ECL of luminol on the surface of nano ITO modified electrode is strong, the light emission is enhanced about four times and the electrode can be used for about five days. Through experiments we found that the milder the condition the better the film will be, so when the humidity is 60%, the temperature is under 30℃and drying the sol-gel for one night the film is the best .
2 The ECL Behavior and Mechanism of Luminol on ITO Electrode
(1)The ECL behavior of reactive oxygen species(ROS) on ITO electrode in the alkaline solution was studied. The results showed that when ITO glass as working electrode,the ECL observed was produced by reactive oxygen species in the alkaline solution.The possible mechanism was given: under the pulse voltage, ITO nanoparticles on the surface of ITO glass were excited to a level of energy and the redox reactions of OH-,O2 and H_2O_2 occurred producing ROS. Meanwhile ITO nanoparticles at a level of energy adsorbed ROS and translated the energy to them so that the active oxygen molecules could reach the state of excited and the light was relieved when they returned to the ground state.
(2)The ECL mechanism of luminol on ITO electrode in the alkaline solution was studied. The results indicated that the phenomenon that the ECL of luminol on the ITO electrode could occur under the lower voltage was mainly related to the interaction between luminol molecules and ITO nanoparticles. Luminol molecules could adsorb onto the surface of ITO nanoparticles and it was possible that energy transferred during this process. Under the lower voltage, ITO nanoparticles were excited to a level of energy and when luminol molecules on the surface of ITO electrode were oxidized to a transitional state and the energy was translated from ITO nanoparticles to the middle state luminol. So the luminol molecules could reach the state of excited and the light was relieved when they returned to the ground state.
(3) The indium tin oxide (ITO) glass was applied as the electrode to study the electrochemical polymerization and electrochemiluminescent behavior of luminol on its surface. The experimental results had indicated that the luminol could be polymerized on the surface of ITO glass in acidic solution. The cyclic voltammetry and UV-Vis spectrometry were applied to confirm the polymerization of luminol. The polymerized luminol on ITO electrode kept the ECL property and some important effecting factors had been investigated. The influence of I-, S_2~- and H_2O_2 on the ECL of polymerized luminol on ITO electrode in alkaline medium has been studied. The iodide could be sensitively determined in the select conditions. The ECL intensity responded linearly to the concentration of iodide within the range from 1.0×10~(-6)mol/L to 8.0×10~(-6)mol/L(r=0.9896), of S_2~- within the range from 8.0×10-7mol/L to 1.0×10~(-5)mol/L(r=0.9903) and of H_2O_2 within the range from 8.0×10~(-6)mol/L to 6.0×10~(-5)mol/L(r=0.9939).
(4) The nanosized TiO2 particles were further modified onto ITO electrode and their performance towards the enhancement of luminol ECL was investigated. Nafion was adopted as the support and homogenously adsorbed onto ITO surface, which provided a good platform for large amount of modification. After calcination under 650°C, nafion was removed and TiO_2 particles were tightly held on ITO electrode surface. At the same time, the crystalline of ITO changed into a mixture of anatase and rutile with ratio of 43.64-56.36, which gave the highest sensitization of luminol ECL. The ECL intensity of luminol was enhanced 7.5 times on this electrode compared with the bare ITO glass electrode. The aggregation has been avoided effectively because TiO_2 was modified previously and than send to be calcined, so the mixed crystal was obtained while the size of the particles was controlled. A group of seven mixed crystal TiO_2/ITO electrodes were used to the detection of ECL and the RSD was 1.88%. One electrode has been used for the detection of ECL for 7 days and the damping is 5.88%. The result shows that this kind of electrode has good stability and reproducibility.
3 Development of Flow-injection Analysis Electrochemiluminescent System
(1) A self-designed flow-injection analysis electrochemiluminescent (FIA-ECL) system was fabricated. The ECL analysis is a new method with high sensitivity and has the wider application on many special fields. But there is lack of high quality special instrument for research or practice of this method in our country. A specimen was designed and fabricated, which colligated the advantages of sensitivity of ECL analysis, the practicability of flow-injection and the connection feasibility of optical fiber transmission. It has been designed as a computer embedded one with facility of operation and powerful datum processing function. The work studied the effect of flux to the intensity of the ECL, when the flow was 10mL/h there will be a strongest light emission in the FIA-ECL system.
(2) In order to get over disadvantages of existing electrochemiluminescent (ECL) cell, a novel micro-electrochemiluminescent (μ-ECL) cell based on flow injection was designed and fabricated. Made of indium tin oxide (ITO) and PDMS, it had solved the problems well and provided hardware for further study. The cell has many advantages and innovations. In this paper, the ECL behavior of luminol on the ITO glass based on the flow cell was studied. The influences of main effect factors on the cell were studied and optimized. The performance and application of the cell were improved after optimization of main effect factors and structure of the cell. Application of the cell to real samples was studied on anti-oxygen ability evaluation of fruits. The ECL intensity of detection cell decreased linearly with the concentration of red grape. The anti-oxygen ability of red grape seed was stronger than red grape flesh, which showed the practical applicability of developed ECL detection cell. The cell has good reproducibility in real sample determination. Its design and function have satisfied the ECL analysis. It also can couple with other technologies, such as HPLC and CE et al. It has a great potential for further application in the real research and analysis.
1纳米增敏鲁米诺电化学发光
(1)纳米Pt-Au合金增敏鲁米诺的电化学发光用化学还原法制备了不同比例及不同粒径的纳米Pt-Au合金,并用UV-Vis、TEM、激光粒径、XRD等方法进行了表征,确认所合成物质确系双金属合金纳米粒子而非两种金属纳米粒子的混合物,通过改变合成方法(如直接滴加法、雾化与超声结合、超声滴加法、氢气还原法、氢气还原与雾化结合)和条件,可以得到一系列不同含量比和粒径范围在4.03-92.33nm之间的Pt-Au合金纳米溶胶。采用电化学沉积法可将所制备纳米粒子修饰到铂盘电极上,在碱性介质(pH=12)中,随着合金比例的改变和合金粒径的减小,鲁米诺的电化学发光强度显著增强,当合金中Pt:Au =6:1,粒子粒径为最小4.03nm时,所获得修饰电极上鲁米诺的电化学发光强度较裸电极增强近1个数量级。研究了纳米Pt-Au合金修饰电极对鲁米诺电化学发光增敏的机理。深入分析了纳米合金晶胞中的原子排布,纳米Pt-Au合金晶胞为面心立方结构,一个合金晶胞中有10个Pt原子和4个Au原子,这四个Au原子占据着晶胞中呈对角线的四个顶点,且分两次取代晶胞中的Pt原子。研究了纳米合金与鲁米诺的相互作用,纳米合金与鲁米诺之间存在吸附作用,易发生能量转移,从而增敏鲁米诺的电化学发光,具体原因可能是:一、纳米Pt-Au合金作为一种纳米粒子,本身具有小尺寸效应、表面效应等催化特性,延长了OH.和Luminol阴离子自由基的作用时间,从而增强了发光信号;二、当Pt与Au合金化后,Pt的d空穴增多,空的d空穴成为溶液中各种自由基的受体,使得电极表面吸附了更多的OH.与Luminol阴离子等,从而增强了鲁米诺发光强度。
(2)纳米金属氧化物增敏铂电极上的电化学发光研究了纳米金属氧化物(ZnO、MnO_2、TiO_2)对鲁米诺在铂电极上电化学发光的影响。实验发现所研究的三种金属氧化物在溶液中对鲁米诺的电化学发光有明显的增敏作用,当用溶胶-凝胶法将纳米氧化物修饰固定在电极后,对鲁米诺的电化学发光亦有明显的增敏作用,在修饰电极上可以获得更稳定的发光信号和更高的信噪比。在此基础上采用溶胶-凝胶法把纳米金属氧化物和鲁米诺同时修饰于铂电极表面,制得纳米金属氧化物修饰的ECL电极。实验结果表明,纳米金属氧化物修饰ECL电极对H_2O_2均有响应,其中纳米TiO_2修饰的ECL电极的发光强度与过氧化氢在1.0×10~(-7)至1.0×10~(-5)mol/L浓度范围内成线性关系,检测下限可达1×10~(-8)mol/L。该ECL电极可用于葡萄抗氧化能力的综合评估,以每克水果消耗过氧化氢毫克数mgH2O_2/g为单位,葡萄肉汁为1.57,葡萄籽为4.72,即葡萄籽的抗氧化能力要显著强于葡萄肉。用循环伏安法和紫外-可见吸收光谱法研究了纳米金属氧化物的增敏机理,研究表明增敏原因有两点:一、鲁米诺分子被吸附在纳米金属氧化物表面,近距离接触使得能量传递成为可能。二、在外加电压的情况下,纳米金属氧化物催化产生活性氧,而活性氧可有效增强鲁米诺电化学发光。
(3)纳米ITO增敏鲁米诺电化学发光
纳米ITO为锡掺杂的氧化铟,纳米ITO粉末中氧化铟与氧化锡的含量比为In2O3:SnO_2=9:1。采用微乳液法、溶胶-凝胶法以及共沸蒸馏法合成了不同粒径的纳米In2O3并进行了透射电镜表征,考察了碱性条件下纳米In2O3对鲁米诺电化学发光的影响。实验结果表明,在碱性条件下纳米In2O3在溶液中对鲁米诺电化学发光有明显的增敏作用,并进一步研究了粒径大小与这种增敏作用的关系,采用紫外-可见吸收光谱和荧光光谱技术讨论了增敏机理。
采用溶胶-凝胶法制备粒径为10nm左右的纳米SnO_2,该纳米粒子对碱性溶液中的鲁米诺-O_2化学发光有显著的增强,这种增敏作用与纳米SnO_2的加入量以及体系中溶解氧的浓度有关系,基于此得出了纳米SnO_2存在下溶解氧浓度与鲁米诺化学发光强度之间的线性关系,可用于溶解氧测定,检测下限可达0.3mg/L。采用紫外-可见吸收光谱和荧光光谱技术研究了纳米SnO_2增敏鲁米诺化学发光机理。
基于上述研究合成了粒径为10nm左右的ITO纳米粒子,当溶液中纳米ITO含固量达到0.3mg/mL左右时,对鲁米诺的ECL强度增敏作用最强。当用溶胶—凝胶法将其固定修饰到铂电极表面后对鲁米诺的电化学发光有明显的增敏作用,考察了三种溶胶—凝胶成膜方式(红外灯烘干、烘箱烘干、自然晾干)固定纳米ITO及对增敏作用的影响,结果表明自然晾干成膜的纳米ITO修饰电极上鲁米诺的ECL强度高,增敏效果可以达到四倍左右,可以使用五天以上。实验对比发现溶胶-凝胶法修饰电极时成膜条件越缓和修饰效果越好,控制湿度在60%左右,温度低于30℃,隔夜自然晾干成膜效果最好。
2 ITO玻璃上鲁米诺电化学发光行为及机理研究
(1)研究了碱性溶液中氧化铟锡玻璃上活性氧的电化学发光行为。实验结果表明,以ITO玻璃为工作电极,在碱性溶液中观察到的电化学发光是活性氧系分子所发出的。该体系的电化学发光的可能机理是:在外加脉冲电压下ITO玻璃表面的氧化铟锡纳米粒子被激发至一定的能级,OH-、O_2及H2O_2分子在ITO玻璃电极表面发生氧化还原反应产生活性氧分子,处于一定能级的纳米ITO粒子吸附活性氧系分子并将能量转移给活性氧系分子使其到达激发态,当返回基态时能量以光子的形式释放而发光。
(2)研究了在碱性溶液中鲁米诺在ITO玻璃电极上的电化学发光机理。鲁米诺在ITO电极上的电化学发光能在较低的电位下发生主要是由于鲁米诺分子与纳米ITO粒子的相互作用,这主要是鲁米诺分子被吸附在纳米ITO粒子表面,近距离接触使得能量传递成为可能。在较低的电压下,纳米ITO粒子被激发到一定能级,当电极表面鲁米诺被氧化至中间态时,两者之间发生能量转移导致鲁米诺氧化至激发态,当返回基态时能量以光子的形式释放而发光。
(3)研究了鲁米诺在ITO玻璃上的电聚合,实验结果表明,在酸性条件下可以将鲁米诺电聚合修饰于ITO电极的表面,聚合在ITO玻璃表面的鲁米诺保持其良好的电化学发光性能,考察了此修饰电极的性能以及相关因素对聚合膜的电化学发光强度的影响。在碱性介质中该电聚合鲁米诺修饰ECL电极对碘离子、硫离子和双氧水均有响应。在选定条件下,上述物质显著增敏聚鲁米诺的ECL。在1.0×10~(-6)mol/L-8.0×10~(-6)mol/L范围内,电化学发光强度与碘离子浓度有良好的线性关系,r=0.9896;在8.0×10-7mol/L—1.0×10~(-5)mol/L范围内,电化学发光强度与硫离子浓度有良好的线性关系,r=0.9903;在8.0×10~(-6)- 6.0×10~(-5)mol/L范围内,电化学发光强度与双氧水浓度有良好的线性关系,r=0.9939.
(4)进一步将具有较高增敏效率的纳米TiO_2修饰于ITO玻璃表面,研究其对鲁米诺ECL的增敏作用,通过Nafion作为载体可以均匀吸附于ITO表面,且具有较大的修饰量。修饰电极经650°C高温灼烧后去除Nafion,纳米TiO_2被烧结固定于ITO电极表面同时发生晶型改变得到锐钛矿相与金红石相的混晶,两相比例为43.64?56.36时,其对鲁米诺电化学发光的增敏最明显。结果表明,当粒径较小,经650°C煅烧处理形成混晶时,纳米TiO_2/ ITO修饰电极对鲁米诺电化学发光的增敏效果最明显,为裸电极的7.5倍。由于先将纳米二氧化钛均匀修饰在ITO电极表面,再对电极进行煅烧,有效避免了煅烧过程中TiO_2粒子间的相互烧结团聚,在有效控制TiO_2粒径的同时得到混晶。考察7支混晶TiO_2/ITO电极对浓度为4×10-7mol/L的碱性鲁米诺溶液的电化学发光响应,其ECL强度的相对标准偏差为1.88%,在连续7天使用中发光强度仅衰减5.88%,表明用该方法制备的修饰电极可以提供满意的重现性和稳定性。
3流动注射电化学发光分析系统研制
(1)研制了一套流动注射电化学发光分析系统,结合了电化学发光的高灵敏度和流动注射的实用性,并采用光纤传输模式工作,增强了仪器的连接柔软性,以计算机嵌入式设计,操作方便、数据处理功能强,经优化形成高性能的电化学发光分析系统。在此基础上研究了纳米ITO对流动注射体系中鲁米诺电化学发光的影响,考察了流量对ECL强度的影响,当流量为10mL/h时研究体系的发光强度最大。
(2)为了克服现有的电化学发光池存在的不足,应用氧化铟锡(ITO)玻璃和有机玻璃等材料,研制出一种可以产生并检测电化学发光信号的流动注射电化学发光池,该池较好地解决了现有电化学发光池存在的一些问题,也为实验室以后开展此方面的实验工作提供硬件方面的准备。基于流动注射分析技术和电化学发光分析技术的综合应用,采用ITO玻璃作为工作电极,通过设计新的流通式电化学发光池,进行动态对流传质为主条件下ITO玻璃电极表面反应层中电化学发光反应的特征和分析特性的研究,探讨所设计的电化学发光池中鲁米诺发光体系的发光行为,考察相关因素对鲁米诺电化学发光分析特性的影响。通过对微型流动电化学发光池及ITO玻璃工作电极上鲁米诺电化学发光的相关因素的优化,提高电化学发光池的分析性能和应用范围。基于葡萄提取物对鲁米诺电化学发光的猝灭作用,考察了该流动注射电化学发光池的性能,结果表明猝灭效率与所加入水果提取液的量成线性相关,据此可评估样品的抗氧化性能,同时表明该电化学发光检测池具有良好的可实用性。在实际样品测定中,该流动注射电化学发光池具有较好的重现性,其设计和功能完全能满足电化学发光分析技术的要求,还可进一步开发与其它技术如毛细管电泳(CE)或高效液相色谱(HPLC)等联用,在实际应用研究分析中将有广阔的应用前景。
This thesis focuses on the sensitization of electrochemiluminescence by nanoparticles and related development of flow-injection analysis system with electrochemiluminescent detection. It consists of the following sections.
1 Sensitization of Electrochemiluminescence by Nanoparticles
(1) Sensitization of Luminol’s ECL by Pt-Au Bimetallic Nanoparticles Modified Electrode
The platinum-gold bimetallic nanoparticles with different component ratio and size were prepared by chemical reduction. The methods such as UV-Vis spectra, TEM and XRD were applied to characterize the properties of the nanoparticles. The information from these methods revealed that the prepared bimetallic nanoparticles were truly of alloy structure and absolutely not the mixture of two kinds of metallic nanoparticles. The component of the nanoparticles could be regulated for a series of Pt/Au ratio and the diameter of nanoparticles, which were determined by Laser-granulometer, could be regulated from 4.03-92.33nm by the method or controlled condition. In alkaline medium of pH=12.0, the bimetallic Pt-Au nanoparticle modified electrode, which was modified on a Pt disk electrode by electrodeposition, sensitized the electrochemiluminescence of luminol. With the variation in the composition and size of the bimetallic nanoparticles, the sensitization efficiency varied as well, with a highest at a Pt/Au ratio of 6:1 and diameter of 4.03nm, at which the electrochemiluminescence intensity was an order of magnitude better than that obtained from unmodified electrode.
The atom distribution in the cell of Pt-Au bimetallic nanoparticles was studied. Then found that the crystalline structure is face--centered cubic. A cell contains 10 Pt atoms and 4 Au atoms, The 4 Au atoms hold the 4 corners that are in the two diagonals, and they substitute Pt atoms in two steps. The paper also studied the interaction between bimetallic Pt-Au nanoparticle and luminol. Adsorption occurred between them, and energy transfers from one to the other, thereby Pt-Au bimetallic nanoparticles enhance the intensity of luminol. There are two reasons for sensitization for electrochemiluminescence of luminol from Platinum-Gold bimetallic nanoparticles modified electrode. One is as nanoparticle, the Pt-Au bimetallic nanoparticle has catalysis such as size effect, surface effect and etc. It prolongs the interaction time of OH. and luminol-.,thereby enhances the intensity of luminol. The other reason is when Pt and Au were alloyed; the d band cavity in Pt was increased. The cavity became the adsorption of radicals in the solution. So the electrode surface adsorped more OH. and Luminol-. , thereby enhances the intensity of luminol.
(2) Sensitization of Luminol ECL by Metallic Oxide Nanoparticles on Pt Electrode
In this work, the sensitization of luminol electrochemiluminescence by metallic oxide nanoparticles, as ZnO, MnO_2 and TiO_2, under alkaline condition was reported and the related mechanism was studied. It was found that all three types of nanoparticles exhibited similar enhancement toward the electrochemiluminescence reaction. Furthermore a sol-gel method was taken for the immobilization of the metallic oxide nanoparticles onto platinum electrodes. The so-obtained modified electrodes also showed enhanced electrochemiluminescence and better signal/noise ratio, and the stability of the signal was improved as well. Luminol together with the nanoparticles were directly immobilized onto the electrode surface and the performance of the sensors greatly improved. Also good linear response was obtained toward hydrogen peroxide. It was found that the developed modified ECL electrodes provided responses to H2O_2. For the TiO_2 modified one, a linear response was obtained with the range 1.0×10~(-7)~1.0×10-5mol/L. This could be used to measure the concentration of H2O_2 and the detection limit was low to 1×10~(-8)mol/L. This ECL electrode was applied to test the antioxidant capacity of grapes. The result was that the antioxidant capacity of the grape seed was much better than grape meat. Cyclic voltammetry and UV-visible absorption methods were taken for the study of the mechanism. It was proposed that the nanoparticles could enhance the production of reactive oxygen species, as well as the adsorption of luminescent reagent on nanoparticle surface. These two factors could give an enhancement on the electrochemiluminescent reaction.
(3) Sensitization of Luminol ECL by ITO Nanoparticle on ITO Electrode
Nanosized ITO particles were prepared from In2O3 and SnO_2 with an amount ratio at 9:1. A series of In2O3 nanoparticles at different sizes were synthesized by microemulsion, sol-gel or azeotropic distillation methods, and characterized by a transmission electron microscope (TEM). In alkaline solution of pH12.5, the influence of In2O3 nanoparticles on the ECL of luminol was studied. The results showed that the ECL intensity of luminol was enhanced obviously in the presence of In2O3 nanoparticles. The sensitization efficiency was related to the content and the size of In2O3 nanoparticles. The paper also discussed the mechanism of the sensitization by UV-Vis spectra and fluorescence spectra.
Nanosized SnO2 were synthesized by sol-gel methods and characterized by a transmission electron microscope (TEM). The result showed that the average particle sizes of nanosized SnO2 was about 10nm.In the chemiluminescence system of luminol-O2,the CL intensity of luminol was enhanced obviously in the presence of nanosized SnO2.This sensitization efficiency was related to the content of nanosized SnO2 and the dissolved oxygen. Based on this, it was educed that the CL intensity of luminol was linear with the content of the dissolved oxygen in the presence of nanosized SnO2.This linear relationship could be used to determine the dissolved oxygen and the detection limit was 0.3mg/L. The paper also discussed the mechanism of the sensitization by UV-Vis spectrum and fluorescence spectrum.
ITO nanoparticles with diameter around 10nm were prepared and the highest enhancement of luminol ECL was obtained with ITO content of 0.3mg/mL. When the nanosized ITO was immobilized to the surface of the Pt electrode by using the method of sol-gel, the ECL intensity of luminol is obviously enhanced. The work studied three different ways of sol-gel becoming film(dried by infrared radiation、dried in oven and drying naturally) to immobilize the nanosized ITO to the electrode and the effect to the enhancement of the ECL intensity .The research showed that when the sol-gel became film naturally, the ECL of luminol on the surface of nano ITO modified electrode is strong, the light emission is enhanced about four times and the electrode can be used for about five days. Through experiments we found that the milder the condition the better the film will be, so when the humidity is 60%, the temperature is under 30℃and drying the sol-gel for one night the film is the best .
2 The ECL Behavior and Mechanism of Luminol on ITO Electrode
(1)The ECL behavior of reactive oxygen species(ROS) on ITO electrode in the alkaline solution was studied. The results showed that when ITO glass as working electrode,the ECL observed was produced by reactive oxygen species in the alkaline solution.The possible mechanism was given: under the pulse voltage, ITO nanoparticles on the surface of ITO glass were excited to a level of energy and the redox reactions of OH-,O2 and H_2O_2 occurred producing ROS. Meanwhile ITO nanoparticles at a level of energy adsorbed ROS and translated the energy to them so that the active oxygen molecules could reach the state of excited and the light was relieved when they returned to the ground state.
(2)The ECL mechanism of luminol on ITO electrode in the alkaline solution was studied. The results indicated that the phenomenon that the ECL of luminol on the ITO electrode could occur under the lower voltage was mainly related to the interaction between luminol molecules and ITO nanoparticles. Luminol molecules could adsorb onto the surface of ITO nanoparticles and it was possible that energy transferred during this process. Under the lower voltage, ITO nanoparticles were excited to a level of energy and when luminol molecules on the surface of ITO electrode were oxidized to a transitional state and the energy was translated from ITO nanoparticles to the middle state luminol. So the luminol molecules could reach the state of excited and the light was relieved when they returned to the ground state.
(3) The indium tin oxide (ITO) glass was applied as the electrode to study the electrochemical polymerization and electrochemiluminescent behavior of luminol on its surface. The experimental results had indicated that the luminol could be polymerized on the surface of ITO glass in acidic solution. The cyclic voltammetry and UV-Vis spectrometry were applied to confirm the polymerization of luminol. The polymerized luminol on ITO electrode kept the ECL property and some important effecting factors had been investigated. The influence of I-, S_2~- and H_2O_2 on the ECL of polymerized luminol on ITO electrode in alkaline medium has been studied. The iodide could be sensitively determined in the select conditions. The ECL intensity responded linearly to the concentration of iodide within the range from 1.0×10~(-6)mol/L to 8.0×10~(-6)mol/L(r=0.9896), of S_2~- within the range from 8.0×10-7mol/L to 1.0×10~(-5)mol/L(r=0.9903) and of H_2O_2 within the range from 8.0×10~(-6)mol/L to 6.0×10~(-5)mol/L(r=0.9939).
(4) The nanosized TiO2 particles were further modified onto ITO electrode and their performance towards the enhancement of luminol ECL was investigated. Nafion was adopted as the support and homogenously adsorbed onto ITO surface, which provided a good platform for large amount of modification. After calcination under 650°C, nafion was removed and TiO_2 particles were tightly held on ITO electrode surface. At the same time, the crystalline of ITO changed into a mixture of anatase and rutile with ratio of 43.64-56.36, which gave the highest sensitization of luminol ECL. The ECL intensity of luminol was enhanced 7.5 times on this electrode compared with the bare ITO glass electrode. The aggregation has been avoided effectively because TiO_2 was modified previously and than send to be calcined, so the mixed crystal was obtained while the size of the particles was controlled. A group of seven mixed crystal TiO_2/ITO electrodes were used to the detection of ECL and the RSD was 1.88%. One electrode has been used for the detection of ECL for 7 days and the damping is 5.88%. The result shows that this kind of electrode has good stability and reproducibility.
3 Development of Flow-injection Analysis Electrochemiluminescent System
(1) A self-designed flow-injection analysis electrochemiluminescent (FIA-ECL) system was fabricated. The ECL analysis is a new method with high sensitivity and has the wider application on many special fields. But there is lack of high quality special instrument for research or practice of this method in our country. A specimen was designed and fabricated, which colligated the advantages of sensitivity of ECL analysis, the practicability of flow-injection and the connection feasibility of optical fiber transmission. It has been designed as a computer embedded one with facility of operation and powerful datum processing function. The work studied the effect of flux to the intensity of the ECL, when the flow was 10mL/h there will be a strongest light emission in the FIA-ECL system.
(2) In order to get over disadvantages of existing electrochemiluminescent (ECL) cell, a novel micro-electrochemiluminescent (μ-ECL) cell based on flow injection was designed and fabricated. Made of indium tin oxide (ITO) and PDMS, it had solved the problems well and provided hardware for further study. The cell has many advantages and innovations. In this paper, the ECL behavior of luminol on the ITO glass based on the flow cell was studied. The influences of main effect factors on the cell were studied and optimized. The performance and application of the cell were improved after optimization of main effect factors and structure of the cell. Application of the cell to real samples was studied on anti-oxygen ability evaluation of fruits. The ECL intensity of detection cell decreased linearly with the concentration of red grape. The anti-oxygen ability of red grape seed was stronger than red grape flesh, which showed the practical applicability of developed ECL detection cell. The cell has good reproducibility in real sample determination. Its design and function have satisfied the ECL analysis. It also can couple with other technologies, such as HPLC and CE et al. It has a great potential for further application in the real research and analysis.
引文
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