功能化金纳米材料的合成、性质及其在化学发光传感器中的应用研究
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摘要
论文首先综述了化学发光(CL)以及功能化金纳米材料的制备、性质及其在传感器中的应用的研究现状。虽然化学发光的基础理论和分析应用有着多年的研究历史,但是一直以来有关化学发光的研究局限于分子和离子水平。尽管最近半导体量子点的化学发光、金属纳米材料直接和间接参与的化学发光已经逐渐受到研究者的关注,但目前的研究主要集中于未修饰的纳米材料直接参与、或在其他条件诱导下参与化学发光,改性或功能化金纳米材料在化学发光理论及分析应用中的研究工作则鲜见报道。基于此,本论文以金纳米材料为研究对象,合成了两种具有化学发光活性的功能化金纳米结构,研究了其表面化学组成、表面等离子共振吸收(SPR)、荧光性质和化学发光活性,并探讨了其在化学发光和电致化学发光(ECL)传感器中的应用;研究了适配体功能化的金纳米粒子对化学发光反应的催化作用,并基于适配体—靶分子相互作用对其催化活性的影响建立了对该靶分子的特异性识别和定量分析方法。主要研究内容如下:
1.发现发光试剂鲁米诺能够作为还原剂和保护剂还原氯金酸制备金纳米粒子。此方法能够合成粒径在14～35nm之间的球形纳米粒子(1umAuNPs),且其粒径随着鲁米诺浓度的增大而降低。利用紫外—可见吸收光谱、X射线光电子能谱和热重分析等手段对金纳米粒子的表面化学组成进行了细致研究。结果表明鲁米诺及其氧化产物3-氨基邻苯二甲酸根离子(AP~(2-))以Au-N弱共价相互作用共存于金纳米粒子表面。实验发现,鲁米诺的存在使得该金纳米粒子具有ECL活性。随后,以半胱氨酸为桥联分子,该金纳米粒子得以在金电极表面固载,从而得到了表面固载有ECL分子的修饰电极。实验发现,在一定电极电位下,该修饰电极能够在碱性溶液中直接产生发光现象,且发光强度随溶液中H_2O_2浓度的增大而增强。基于这一原理,该修饰电极能够作为H_2O_2的ECL传感器在3×10~(-7)～1×10~(-3) mol/L范围内对其进行定量测定。
2.考虑到上述功能化金纳米粒子表面为具有荧光活性的AP~(2-)和具有ECL活性的鲁米诺分子所保护,继而深入研究了其荧光和ECL性质。结果表明,虽然一个粒径为14nm的金纳米颗粒表面约连接有765个AP~(2-)荧光分子,但是在直接相连的金核的粒子内猝灭效应和附近共存的金核的粒子间猝灭效应的共同作用下,只有约70个荧光分子具有荧光活性。其粒子内和粒子间猝灭效率分别为81%和52%。此外,实验发现出于氧化性碳酸根自由基的生成,AP~(2-)分子在碳酸介质中遭受严重的光漂白。相同条件下结合在金纳米粒子表面的AP~(2-)光漂白程度则显著减弱。研究认为,其光稳定性的增强来自于金纳米内核对其表面附近的氧化性自由基的清除作用,从而保护纳米表面结合的有机荧光分子免遭氧化失去荧光活性。这一自由基清除机理与为人们所熟知的SiO_2纳米粒子的自由基阻碍机理大不相同,从而为有机荧光分子光稳定性的提高指出了新的研究方向。最后发现半胱氨酸的存在能显著提高1umAuNPs的ECL强度。研究认为金纳米粒子的平台效应是导致ECL增强的主要原因。即鲁米诺与半胱氨酸分子同时结合到金纳米粒子表面,有利于克服反应物分子的静电排斥力,从而提高反应速率并增强ECL强度。这一工作首次将ECL分子与共反应物分子同时结合于金纳米粒子上,实现了微平台上的ECL反应,并观察到由此而导致的ECL增强效应。
3.实验发现,在亲水聚合物壳聚糖存在下以鲁米诺还原氯金酸能够得到具有CL活性的花状三维纳米结构,其粒径在90～200 nm之间可调,且在可见光和近红外区都有明显的SPR吸收。透射电镜、扫描电镜和X射线粉末衍射的结果表明,该纳米花由大量小粒径纳米球以三维聚集的方式形成。研究了鲁米诺和壳聚糖浓度对纳米花形貌和粒径的影响,以及相应的SPR吸收特征。基于上述形貌表征,本文提出了该金纳米花的二次生长机理,认为鲁米诺首先还原氯金酸生成大量粒径10～20 nm的纳米点。这些纳米点在富含氨基且具有柔曲空间构象的壳聚糖分子作用下受控聚集为初级聚集体。随后剩余的氯金酸在其表面还原,使得聚集体经过二次生长由新生的金原子连接、融合为牢固的一体化花状纳米结构。以此二次生长机理为指导,本文得以通过数值方法对纳米花的空间结构进行描述,并以离散偶极近似这一模拟方法从电磁相互作用的角度建立了该纳米花的形貌与SPR光谱之间的关系。计算结果与实验观察十分吻合。纳米材料的形貌尺寸与SPR性质之间的关系是其光学性质的重要研究内容。以往的研究多关注球形、棒状、核壳结构及多边形等几何外形较为规则的纳米结构。本工作则首次从实验和理论两方面对纳米花这一几何外形不规则的纳米结构这一规律进行了系统研究,为不规则纳米结构光学性质的研究提供了参考。最后,利用成膜聚合物壳聚糖将具有CL活性的纳米花固载在玻璃表面,并研究了这一CL功能膜对H_2O_2的响应规律。结果表明,在3×10~(-5)～3×10~(-3)mol/L浓度范围内,含有功能化纳米花的壳聚糖膜的CL强度随H_2O_2浓度线性增加,从而得以构建了一个无需外加发光试剂的CL传感器用于H_2O_2的定量分析。
4.由于鲁米诺并非用于化学合成的常规还原剂,本文随即以抗坏血酸等其它还原剂代替鲁米诺进行了更具一般性的花状纳米结构的合成研究。提出了一种壳聚糖存在时以抗坏血酸等常规还原剂还原HAuCl_4从而在室温下合成金纳米花的简单方法,并研究了金纳米花形貌和尺寸与还原剂用量之间的关系。通过对五种还原剂的系统研究和比较发现,三类不同的还原剂以其还原能力的不同展现出明显不同的合成效果。此外,以还原剂抗坏血酸为模型,本章还利用实时表面等离子共振(SPR)光谱和共振瑞利散射(RRS)强度方法对其生长过程进行监测,并进一步验证和完善了金纳米花的二次生长机理。结果表明,利用常规荧光计对纳米材料的RRS强度进行分析即可获得纳米合成过程中的动力学信息,并有助于对其生长机理进行研究。相比于SPR光谱而言,RRS方法具有灵敏度高、实验设备简单等优点。值得一提的是,由于光散射主要取决于粒子尺寸,因此RRS方法也可以用于许多没有特征SPR吸收带的纳米材料如SiO_2及聚合物纳米球等的生长过程研究。这一点也是SPR方法所不足的。
5.利用适配体功能化金纳米粒子催化的化学发光反应作为金纳米粒子表面状态的探针,以定量检测由于适配体—靶分子特异性相互作用引起的金纳米粒子表面状态及催化活性的改变。未修饰的金纳米粒子能够催化鲁米诺和AgNO_3之间的化学发光反应。金纳米粒子表面经过适配体修饰后,其催化能力将受到强烈抑制而只能获得较弱的化学发光。钾离子的存在则能够使金纳米粒子的催化活性得到一定程度的恢复,并增强其化学发光强度。这一工作首次尝试利用适配体功能化金纳米粒子的催化活性而不是传统的静电稳定性来反映表面状态的改变。结果表明,以钾离子适配体为模型化合物,这一方法能够对Na~+、Mg~(2+)和Ca~(2+)混合样品中0.7～40 mM范围内的钾离子进行定量测定。此外,由于无需利用复杂且昂贵的端基修饰适配体,此方法具有方便、省时等优点。因此,这一基于金纳米粒子催化活性的钾离子识别方法为适配体传感器的开发提供了新的思路。
6.在利用循环伏安法研究鲁米诺的ECL性质时,我们注意到一个常见的对峰现象。即,如果在电位初始扫描方向存在一个发光峰,那么在电位反向扫描回来的过程中该电位区间附近通常会观察到另一个ECL发光峰。本文以一组良好分辨的鲁米诺ECL对峰为研究对象探讨了这一对峰现象的产生机理。结果表明,其发光机理为鲁米诺电氧化过程中的醌式中间体和析氧过程中产生的过氧化氢发生反应并产生化学发光。在正向扫描过程中,当电位继续增加时,两个化学发光反应物都将遭受进一步电氧化而使其浓度降低,导致正扫发光峰的降低直至消失。在回扫过程中,其电氧化速率有所降低但同时仍在不断生成,因此其浓度将得到一定的恢复并引起化学发光强度的提高,即形成了回扫过程的对峰。电化学数值模拟技术进一步验证了这一机理的可能性。这表明这一具体机理可以推广到更一般的情形,即如果化学发光反应的某一个反应物是连续电化学过程的中间体,就有可能在回扫过程中观察到对峰的产生。这一结论被认为是ECL对峰产生的一般性机理,并在鲁米诺ECL体系中得到了确认。根据上述机理,本文提出转折电位、ECL峰宽度和共存电活性物种是影响ECL回扫峰的三个重要因素,并逐一为实验研究所证实。
In this dissertation,the state of arts in the field of chemiluminescence(CL) and the synthesis and properties of nano-materials and their applications in the fabrications of sensors were reviewed.Recently,nanoparticle-involved CL became one of the most attractive and valuable aspects in these fields.However,most reports focused on the CL reactions initiated by unmodified nano-materials.There were few reports about the CL systems involved by functionalized nanomaterials.Therefore,the present dissertation was engaged in the synthesis and optical properties of functionalized gold nanomaterials,which were of CL activity or could initiate CL reactions through a controllable way,and their applications in CL sensors.The main results are as follows:
1.It was found that chloroauric acid(HAuCl_4) could be directly reduced by luminescent reagent luminol in aqueous solution to form gold nanoparticles(AuNPs) and the size of AuNPs depended on the amount of luminol.The morphology and surface state of as-prepared AuNPs were characterized by transmission electron microscopy,UV-visible spectroscopy,X-ray photoelectron spectroscopy,and thermal gravimetric analysis.All results indicated that residual luminol and its oxidation product 3-aminophthalate(AP~(2-)) coexisted on the surface of AuNPs through the weak covalent interaction between gold and nitrogen atoms in their amino groups. Subsequently,a luminol-capped gold nanoparticle(lumAuNPs) modified electrode was fabricated via the immobilization of AuNPs on a gold electrode by virtue of cysteine molecules and then immersion in luminol solution.The modified electrode was characterized by cyclic voltammetry,electrochemical impedance spectroscopy and scanning electron microscopy.The as-prepared modified electrode exhibited an electrochemiluminescence(ECL) response in alkaline aqueous solution under a double-step potential.H_2O_2 was found to enhance the ECL intensity.On this basis,an ECL sensor for the detection of H_2O_2 was developed.The method is simple,fast and reagentless.It is applicable for the determination of hydrogen peroxide in the range of 3×10~(-7) mol/L～1×10~(-3) mol/L with the detection limit of 1×10~(-7) mol/L(S/N=3).
2.Fluorescent(FL) and ECL properties of functionalized lumAuNPs synthesized above were investigated.It was observed that the FL intensity of a single gold nanoparticle was 70 times as bright as that of one free APA molecule,even though 91%FL emission of APA molecules on the surface of AuNPs were inhibited by gold cores through both intra- and interparticle quenching effects.Moreover,the photobleaching of surface-bound APA molecules was found to be dramatically inhibited compared with that of free ones in carbonate buffer.The improvement of photostability was attributed to the reactive AuNPs which acted as radical scavengers to protect the surface-bound APA molecules from oxidation by carbonate radicals. Furthermore,as-prepared lumAuNPs could react with cysteine to produce strong ECL, which was enhanced by 20 fold compared with that in the absence of cysteine.The experimental results suggested that luminol and cysteine were co-adsorbed on the gold nanoparticle platform via Au-N and Au-S interactions,respectively.The shorter distance between reactant molecules by overcoming the electrostatic repulsion,i.e., platform effect,was proposed to be responsible for the ECL enhancement.Combined with the biocompatibility of gold cores,the brighter FL emission,enhanced photostability and stronger ECL intensity may make as-prepared lumAuNPs promising FL and ECL biomarkers for their applications in biosensors and bio-imaging.
3.By reducing HAuCl_4 with CL reagent luminol in the presence of hydrophilic polymer chitosan,three-dimensional(3-D) flower-like gold nanostructures(AuNFs) were synthesized via a convenient one-pot method.As-prepared stable and monodisperse AuNFs were consisted of smaller-sized nanodots according to subsequent characterizations by high-resolution transmission electron microscopy, scanning electron microscopy and powder X-ray diffraction.The size and morphology of AuNFs could be tailored by varying the amount of luminol or chitosan,which further influenced their SPR properties in both visible and near-infrared regions. Based on the characterizations,a chitosan-assisted second-growth mechanism was proposed to explain their formations and morphology evolutions with the amount of reactants.Moreover,an electromagnetic simulation method,discrete dipole approximation,was introduced to calculate the morphology-dependent extinction spectra of geometrically irregular AuNFs.The simulations were well consistent with the experimental results.Finally,because luminol was attached on the surface of AuNFs,as-prepared AuNFs could react with H_2O_2 to generate CL.The functionalized AuNFs were immobilized on the solid supports by virtue of the film-forming property of chitosan solution to fabricate a reagent-free CL sensor for the determination of H_2O_2.Due to their shape-dependent SPR properties and specific surface structures, these AuNFs might also have great potential for the applications in biosensors and surface enhanced Raman scattering.
4.A facile method is proposed for the room-temperature synthesis of flower-like AuNFs with the size of 50～115nm by reducing HAuCl_4 with ascorbic acid(AA) in the presence of chitosan.It was found that the concentration of chitosan controlled the size,while that of AA influenced the morphology of as-prepared AuNFs.With higher concentration of AA,flower-like nanostructures were produced,whereas,with lower concentration of AA,quasi-spherical nanoparticles were formed.Time-dependent surface plasmon resonance(SPR) absorption spectroscopy and resonance Rayleigh scattering(RRS) technique were used to monitor the growth processes.According to the temporal evolutions of SPR maximum absorption wavelength and RRS intensity,a second-growth mechanism is proposed to explain the effect of AA concentration on the morphology and the effect of chitosan concentration on the size of obtained gold nanostructures.In order to determine whether or not the present method is suitable for the synthesis of flower-like gold nanostructures by use of other reductants in the presence of chitosan,four other conventional reductants,including gallic acid,oxalate acid,tartaric acid and sodium citrate,instead of AA were examined.The intrinsic reason for the different performances of these reductants was further investigated,and the results also supported the proposed second-growth mechanism.
5.It was found that the catalytic activity of aptamer-functionalized AuNPs could be improved after the target-binding to surface-bound aptamers in the presence of specific targets.When AuNP-catalyzed CL reaction between luminol and AgNO_3 was used as an indicator,the change in the catalytic activity of AuNPs induced by specific target could be quantitatively investigated by CL intensity.By taking anti-K~+ aptamer as a model system,the proposed homogeneous method could determine K~+ ion with the concentration as low as 0.7 mM in the presence of high concentration Na~+,Mg~(2+) and Ca~(2+) ions.Moreover,unmodified aptamers were used in the present strategy to avoid complex and expensive terminal modifications of aptamers.
6.The curves of ECL intensity(I_(ECL)) versus potential(E)(I_(ECL)-E curves) were obtained when the ECL of luminol was induced by cyclic voltammetry(CV).In the I_(ECL)-E curves,if there was an ECL peak during initial scan,a corresponding ECL peak called as a counter-peak was usually observed around the similar potential during the reversal scan.In the present work,a couple of strong and well-resolved ECL peaks were found in a model luminol ECL system.The effects of various factors on this couple of ECL peaks,including electrolyte,buffer solution,electrode material,pH, N_2/O_2/air atmosphere,scan rate,and electrochemical technique,were studied.On this basis,a continuous electron transfer coupled with a competitive chemical reaction (E-E/C) mechanism involved in an accumulation and consumption model has been proposed for the formation of the ECL counter peak.Electrochemical digital simulation method was used to simulate the formation of the ECL counter-peak according to the proposed E-E/C mechanism.The results also supported the mechanism.Subsequently,it was further validated that ECL peak width,reversal potential and coexisted electro-active species were three important factors affecting the emergence and intensity of the ECL counter-peak.The proposed E-E/C mechanism is considered to be the general explanation of usual counter-peak phenomenon in the luminol ECL under CV conditions since the mechanism is involved in most pathways of the luminol ECL.
1.发现发光试剂鲁米诺能够作为还原剂和保护剂还原氯金酸制备金纳米粒子。此方法能够合成粒径在14～35nm之间的球形纳米粒子(1umAuNPs),且其粒径随着鲁米诺浓度的增大而降低。利用紫外—可见吸收光谱、X射线光电子能谱和热重分析等手段对金纳米粒子的表面化学组成进行了细致研究。结果表明鲁米诺及其氧化产物3-氨基邻苯二甲酸根离子(AP~(2-))以Au-N弱共价相互作用共存于金纳米粒子表面。实验发现,鲁米诺的存在使得该金纳米粒子具有ECL活性。随后,以半胱氨酸为桥联分子,该金纳米粒子得以在金电极表面固载,从而得到了表面固载有ECL分子的修饰电极。实验发现,在一定电极电位下,该修饰电极能够在碱性溶液中直接产生发光现象,且发光强度随溶液中H_2O_2浓度的增大而增强。基于这一原理,该修饰电极能够作为H_2O_2的ECL传感器在3×10~(-7)～1×10~(-3) mol/L范围内对其进行定量测定。
2.考虑到上述功能化金纳米粒子表面为具有荧光活性的AP~(2-)和具有ECL活性的鲁米诺分子所保护,继而深入研究了其荧光和ECL性质。结果表明,虽然一个粒径为14nm的金纳米颗粒表面约连接有765个AP~(2-)荧光分子,但是在直接相连的金核的粒子内猝灭效应和附近共存的金核的粒子间猝灭效应的共同作用下,只有约70个荧光分子具有荧光活性。其粒子内和粒子间猝灭效率分别为81%和52%。此外,实验发现出于氧化性碳酸根自由基的生成,AP~(2-)分子在碳酸介质中遭受严重的光漂白。相同条件下结合在金纳米粒子表面的AP~(2-)光漂白程度则显著减弱。研究认为,其光稳定性的增强来自于金纳米内核对其表面附近的氧化性自由基的清除作用,从而保护纳米表面结合的有机荧光分子免遭氧化失去荧光活性。这一自由基清除机理与为人们所熟知的SiO_2纳米粒子的自由基阻碍机理大不相同,从而为有机荧光分子光稳定性的提高指出了新的研究方向。最后发现半胱氨酸的存在能显著提高1umAuNPs的ECL强度。研究认为金纳米粒子的平台效应是导致ECL增强的主要原因。即鲁米诺与半胱氨酸分子同时结合到金纳米粒子表面,有利于克服反应物分子的静电排斥力,从而提高反应速率并增强ECL强度。这一工作首次将ECL分子与共反应物分子同时结合于金纳米粒子上,实现了微平台上的ECL反应,并观察到由此而导致的ECL增强效应。
3.实验发现,在亲水聚合物壳聚糖存在下以鲁米诺还原氯金酸能够得到具有CL活性的花状三维纳米结构,其粒径在90～200 nm之间可调,且在可见光和近红外区都有明显的SPR吸收。透射电镜、扫描电镜和X射线粉末衍射的结果表明,该纳米花由大量小粒径纳米球以三维聚集的方式形成。研究了鲁米诺和壳聚糖浓度对纳米花形貌和粒径的影响,以及相应的SPR吸收特征。基于上述形貌表征,本文提出了该金纳米花的二次生长机理,认为鲁米诺首先还原氯金酸生成大量粒径10～20 nm的纳米点。这些纳米点在富含氨基且具有柔曲空间构象的壳聚糖分子作用下受控聚集为初级聚集体。随后剩余的氯金酸在其表面还原,使得聚集体经过二次生长由新生的金原子连接、融合为牢固的一体化花状纳米结构。以此二次生长机理为指导,本文得以通过数值方法对纳米花的空间结构进行描述,并以离散偶极近似这一模拟方法从电磁相互作用的角度建立了该纳米花的形貌与SPR光谱之间的关系。计算结果与实验观察十分吻合。纳米材料的形貌尺寸与SPR性质之间的关系是其光学性质的重要研究内容。以往的研究多关注球形、棒状、核壳结构及多边形等几何外形较为规则的纳米结构。本工作则首次从实验和理论两方面对纳米花这一几何外形不规则的纳米结构这一规律进行了系统研究,为不规则纳米结构光学性质的研究提供了参考。最后,利用成膜聚合物壳聚糖将具有CL活性的纳米花固载在玻璃表面,并研究了这一CL功能膜对H_2O_2的响应规律。结果表明,在3×10~(-5)～3×10~(-3)mol/L浓度范围内,含有功能化纳米花的壳聚糖膜的CL强度随H_2O_2浓度线性增加,从而得以构建了一个无需外加发光试剂的CL传感器用于H_2O_2的定量分析。
4.由于鲁米诺并非用于化学合成的常规还原剂,本文随即以抗坏血酸等其它还原剂代替鲁米诺进行了更具一般性的花状纳米结构的合成研究。提出了一种壳聚糖存在时以抗坏血酸等常规还原剂还原HAuCl_4从而在室温下合成金纳米花的简单方法,并研究了金纳米花形貌和尺寸与还原剂用量之间的关系。通过对五种还原剂的系统研究和比较发现,三类不同的还原剂以其还原能力的不同展现出明显不同的合成效果。此外,以还原剂抗坏血酸为模型,本章还利用实时表面等离子共振(SPR)光谱和共振瑞利散射(RRS)强度方法对其生长过程进行监测,并进一步验证和完善了金纳米花的二次生长机理。结果表明,利用常规荧光计对纳米材料的RRS强度进行分析即可获得纳米合成过程中的动力学信息,并有助于对其生长机理进行研究。相比于SPR光谱而言,RRS方法具有灵敏度高、实验设备简单等优点。值得一提的是,由于光散射主要取决于粒子尺寸,因此RRS方法也可以用于许多没有特征SPR吸收带的纳米材料如SiO_2及聚合物纳米球等的生长过程研究。这一点也是SPR方法所不足的。
5.利用适配体功能化金纳米粒子催化的化学发光反应作为金纳米粒子表面状态的探针,以定量检测由于适配体—靶分子特异性相互作用引起的金纳米粒子表面状态及催化活性的改变。未修饰的金纳米粒子能够催化鲁米诺和AgNO_3之间的化学发光反应。金纳米粒子表面经过适配体修饰后,其催化能力将受到强烈抑制而只能获得较弱的化学发光。钾离子的存在则能够使金纳米粒子的催化活性得到一定程度的恢复,并增强其化学发光强度。这一工作首次尝试利用适配体功能化金纳米粒子的催化活性而不是传统的静电稳定性来反映表面状态的改变。结果表明,以钾离子适配体为模型化合物,这一方法能够对Na~+、Mg~(2+)和Ca~(2+)混合样品中0.7～40 mM范围内的钾离子进行定量测定。此外,由于无需利用复杂且昂贵的端基修饰适配体,此方法具有方便、省时等优点。因此,这一基于金纳米粒子催化活性的钾离子识别方法为适配体传感器的开发提供了新的思路。
6.在利用循环伏安法研究鲁米诺的ECL性质时,我们注意到一个常见的对峰现象。即,如果在电位初始扫描方向存在一个发光峰,那么在电位反向扫描回来的过程中该电位区间附近通常会观察到另一个ECL发光峰。本文以一组良好分辨的鲁米诺ECL对峰为研究对象探讨了这一对峰现象的产生机理。结果表明,其发光机理为鲁米诺电氧化过程中的醌式中间体和析氧过程中产生的过氧化氢发生反应并产生化学发光。在正向扫描过程中,当电位继续增加时,两个化学发光反应物都将遭受进一步电氧化而使其浓度降低,导致正扫发光峰的降低直至消失。在回扫过程中,其电氧化速率有所降低但同时仍在不断生成,因此其浓度将得到一定的恢复并引起化学发光强度的提高,即形成了回扫过程的对峰。电化学数值模拟技术进一步验证了这一机理的可能性。这表明这一具体机理可以推广到更一般的情形,即如果化学发光反应的某一个反应物是连续电化学过程的中间体,就有可能在回扫过程中观察到对峰的产生。这一结论被认为是ECL对峰产生的一般性机理,并在鲁米诺ECL体系中得到了确认。根据上述机理,本文提出转折电位、ECL峰宽度和共存电活性物种是影响ECL回扫峰的三个重要因素,并逐一为实验研究所证实。
In this dissertation,the state of arts in the field of chemiluminescence(CL) and the synthesis and properties of nano-materials and their applications in the fabrications of sensors were reviewed.Recently,nanoparticle-involved CL became one of the most attractive and valuable aspects in these fields.However,most reports focused on the CL reactions initiated by unmodified nano-materials.There were few reports about the CL systems involved by functionalized nanomaterials.Therefore,the present dissertation was engaged in the synthesis and optical properties of functionalized gold nanomaterials,which were of CL activity or could initiate CL reactions through a controllable way,and their applications in CL sensors.The main results are as follows:
1.It was found that chloroauric acid(HAuCl_4) could be directly reduced by luminescent reagent luminol in aqueous solution to form gold nanoparticles(AuNPs) and the size of AuNPs depended on the amount of luminol.The morphology and surface state of as-prepared AuNPs were characterized by transmission electron microscopy,UV-visible spectroscopy,X-ray photoelectron spectroscopy,and thermal gravimetric analysis.All results indicated that residual luminol and its oxidation product 3-aminophthalate(AP~(2-)) coexisted on the surface of AuNPs through the weak covalent interaction between gold and nitrogen atoms in their amino groups. Subsequently,a luminol-capped gold nanoparticle(lumAuNPs) modified electrode was fabricated via the immobilization of AuNPs on a gold electrode by virtue of cysteine molecules and then immersion in luminol solution.The modified electrode was characterized by cyclic voltammetry,electrochemical impedance spectroscopy and scanning electron microscopy.The as-prepared modified electrode exhibited an electrochemiluminescence(ECL) response in alkaline aqueous solution under a double-step potential.H_2O_2 was found to enhance the ECL intensity.On this basis,an ECL sensor for the detection of H_2O_2 was developed.The method is simple,fast and reagentless.It is applicable for the determination of hydrogen peroxide in the range of 3×10~(-7) mol/L～1×10~(-3) mol/L with the detection limit of 1×10~(-7) mol/L(S/N=3).
2.Fluorescent(FL) and ECL properties of functionalized lumAuNPs synthesized above were investigated.It was observed that the FL intensity of a single gold nanoparticle was 70 times as bright as that of one free APA molecule,even though 91%FL emission of APA molecules on the surface of AuNPs were inhibited by gold cores through both intra- and interparticle quenching effects.Moreover,the photobleaching of surface-bound APA molecules was found to be dramatically inhibited compared with that of free ones in carbonate buffer.The improvement of photostability was attributed to the reactive AuNPs which acted as radical scavengers to protect the surface-bound APA molecules from oxidation by carbonate radicals. Furthermore,as-prepared lumAuNPs could react with cysteine to produce strong ECL, which was enhanced by 20 fold compared with that in the absence of cysteine.The experimental results suggested that luminol and cysteine were co-adsorbed on the gold nanoparticle platform via Au-N and Au-S interactions,respectively.The shorter distance between reactant molecules by overcoming the electrostatic repulsion,i.e., platform effect,was proposed to be responsible for the ECL enhancement.Combined with the biocompatibility of gold cores,the brighter FL emission,enhanced photostability and stronger ECL intensity may make as-prepared lumAuNPs promising FL and ECL biomarkers for their applications in biosensors and bio-imaging.
3.By reducing HAuCl_4 with CL reagent luminol in the presence of hydrophilic polymer chitosan,three-dimensional(3-D) flower-like gold nanostructures(AuNFs) were synthesized via a convenient one-pot method.As-prepared stable and monodisperse AuNFs were consisted of smaller-sized nanodots according to subsequent characterizations by high-resolution transmission electron microscopy, scanning electron microscopy and powder X-ray diffraction.The size and morphology of AuNFs could be tailored by varying the amount of luminol or chitosan,which further influenced their SPR properties in both visible and near-infrared regions. Based on the characterizations,a chitosan-assisted second-growth mechanism was proposed to explain their formations and morphology evolutions with the amount of reactants.Moreover,an electromagnetic simulation method,discrete dipole approximation,was introduced to calculate the morphology-dependent extinction spectra of geometrically irregular AuNFs.The simulations were well consistent with the experimental results.Finally,because luminol was attached on the surface of AuNFs,as-prepared AuNFs could react with H_2O_2 to generate CL.The functionalized AuNFs were immobilized on the solid supports by virtue of the film-forming property of chitosan solution to fabricate a reagent-free CL sensor for the determination of H_2O_2.Due to their shape-dependent SPR properties and specific surface structures, these AuNFs might also have great potential for the applications in biosensors and surface enhanced Raman scattering.
4.A facile method is proposed for the room-temperature synthesis of flower-like AuNFs with the size of 50～115nm by reducing HAuCl_4 with ascorbic acid(AA) in the presence of chitosan.It was found that the concentration of chitosan controlled the size,while that of AA influenced the morphology of as-prepared AuNFs.With higher concentration of AA,flower-like nanostructures were produced,whereas,with lower concentration of AA,quasi-spherical nanoparticles were formed.Time-dependent surface plasmon resonance(SPR) absorption spectroscopy and resonance Rayleigh scattering(RRS) technique were used to monitor the growth processes.According to the temporal evolutions of SPR maximum absorption wavelength and RRS intensity,a second-growth mechanism is proposed to explain the effect of AA concentration on the morphology and the effect of chitosan concentration on the size of obtained gold nanostructures.In order to determine whether or not the present method is suitable for the synthesis of flower-like gold nanostructures by use of other reductants in the presence of chitosan,four other conventional reductants,including gallic acid,oxalate acid,tartaric acid and sodium citrate,instead of AA were examined.The intrinsic reason for the different performances of these reductants was further investigated,and the results also supported the proposed second-growth mechanism.
5.It was found that the catalytic activity of aptamer-functionalized AuNPs could be improved after the target-binding to surface-bound aptamers in the presence of specific targets.When AuNP-catalyzed CL reaction between luminol and AgNO_3 was used as an indicator,the change in the catalytic activity of AuNPs induced by specific target could be quantitatively investigated by CL intensity.By taking anti-K~+ aptamer as a model system,the proposed homogeneous method could determine K~+ ion with the concentration as low as 0.7 mM in the presence of high concentration Na~+,Mg~(2+) and Ca~(2+) ions.Moreover,unmodified aptamers were used in the present strategy to avoid complex and expensive terminal modifications of aptamers.
6.The curves of ECL intensity(I_(ECL)) versus potential(E)(I_(ECL)-E curves) were obtained when the ECL of luminol was induced by cyclic voltammetry(CV).In the I_(ECL)-E curves,if there was an ECL peak during initial scan,a corresponding ECL peak called as a counter-peak was usually observed around the similar potential during the reversal scan.In the present work,a couple of strong and well-resolved ECL peaks were found in a model luminol ECL system.The effects of various factors on this couple of ECL peaks,including electrolyte,buffer solution,electrode material,pH, N_2/O_2/air atmosphere,scan rate,and electrochemical technique,were studied.On this basis,a continuous electron transfer coupled with a competitive chemical reaction (E-E/C) mechanism involved in an accumulation and consumption model has been proposed for the formation of the ECL counter peak.Electrochemical digital simulation method was used to simulate the formation of the ECL counter-peak according to the proposed E-E/C mechanism.The results also supported the mechanism.Subsequently,it was further validated that ECL peak width,reversal potential and coexisted electro-active species were three important factors affecting the emergence and intensity of the ECL counter-peak.The proposed E-E/C mechanism is considered to be the general explanation of usual counter-peak phenomenon in the luminol ECL under CV conditions since the mechanism is involved in most pathways of the luminol ECL.
引文
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