化学发光功能化纳米材料在新型生物传感器中的应用
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摘要
论文首先综述了化学发光、化学发光生物分析以及发光功能化纳米材料在生物分析中的应用的研究现状。近年来,一系列固载和富集发光试剂的化学发光功能化纳米材料被制备出来,该材料具有很好的信号放大作用,已成为化学发光生物分析发展的新趋势。我们实验室前期的研究工作中,发展了一系列新的合成方法,制备了一批新型的发光功能化纳米材料,包括鲁米诺及其类似物功能化的纳米金、鲁米诺功能化纳米银、鲁米诺功能化纳米银／氧化石墨烯和鲁米诺衍生物及血红素双功能化石墨烯等。这些发光功能化纳米材料为核酸分析和免疫分析提供了理想的具有放大功能的信号探针,能够有效提高传感器的灵敏度。基于此,本论文围绕着发光功能化纳米材料在化学发光生物传感器中的应用这一研究主题,开展了一系列研究工作。基于N-(4-氨基丁基)-N-乙基异鲁米诺功能化的纳米金,构建了一个三明治结构DNA传感器用于结核病(MTB)的检测和一个无标记适配体传感器用于凝血酶的检测；基于鲁米诺功能化纳米银,构建了一个竞争性的免疫分析传感器用于小分子抗生素氯霉素的检测；基于鲁米诺功能化纳米银／氧化石墨烯复合材料和ABEI血红素双功能化石墨烯复合材料构建了无标记适配体传感器用于2,4,6-三硝基甲苯(TNT)的检测。主要研究内容如下：
1.利用N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金作为标记物构建了一个有效的DNA传感器用于MTB的特异DNA序列的检测。生物素化的DNA捕获探针通过电极表面修饰的链霉亲和素包被的纳米金直接固载到电极上,目标DNA和N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金标记的DNA序列随后通过DNA特异性杂交反应连接在电极表面形成三明治结构的DNA传感器。该DNA传感器在含有1.75×10-3mol/L H2O2的CBS缓冲中施加双脉冲电压能得到很好的电致化学发光信号。该方法能检测到1.0×10-15-1.0×10-12mol/L范围内的MTB的特异DNA序列,检测限低至3.3×10-16mol/L,优于之前报道的无需PCR放大的MTB分析方法。在实际样品血清样中,该方法对于检测MTB的特异DNA序列也有很好的测定能力。该传感器简单,快速,可靠。因为N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金的信号放大作用,简单的标记过程和传感器的构建过程简单,使得该方法在灵敏度和成本方面有很大的优势。该方法在临床上快速测定MTB具有巨大的应用前景。
2.基于鲁米诺功能化纳米银作为纳米标记物我们首次开发了一个竞争的免疫分析传感器用于小分子氯霉素的灵敏检测。在1.0－10-8-1.0×10-6g/mL范围内可以根据发光强度测定氯霉素含量,检测限低至7.6×10-9g/mL。标记过程简单快速,优于所有文献报道的标记方法。由于小分子的标记是非常困难的工作,此项工作提供了一种新颖而有效的氯霉素标记策略。该免疫分析方法简单、快捷、有效、灵敏度和选择性好,且简捷不需要复杂的仪器设备,使之很有希望用于实际样品中氯霉素的快速检测。
3.基于适配体,凝血酶和N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金的动态组装构建了一个电致化学发光生物分析方法用于凝血酶的检测。所提出电致化学发光生物分析方法灵敏、特异、简便、快捷。该方法在1.0×10-12-1.0×10-9mol/L较宽的范围内具有线性关系,检测限为3.8×10-13mol/L,优于大多数文献报道的基于适配体的无标记生物分析方法。另外,在动态组装过程中基质中的干扰物质可以通过简单的清洗电极很容易地从检测系统分离。因此,该方法具有较高的灵敏度和很小的基体效应,因为基质中的很多物质可增强和抑制鲁米诺和类似物的发光导致干扰。该方法已成功地应用于实际人血清样品中凝血酶的检测。这项工作为设计基于适配体检测重要生物分子提供了一个新的思路。在原理上,使用相应的适配体,本章提出的分析方法也适用于其它重要生物分子的测定。
4.基于两种新型的功能化石墨烯复合材料和适配体构建了一种无标记的电致化学发光适配体传感器用于TNT的检测。该传感器在1.0×10-12-1.0×10-9g/mL较宽的范围内具有良好的线性,检测限低至6.3×10-13g/mL,优于大多数文献报道其他TNT传感器。课题组新开发出的两种新型石墨烯复合材料首次被用于无标记传感器的构建,表现出了良好的发光性能,有效提高了该方法的灵敏度。我们开发的传感器构建过程简单、耗时短,目标物不需要标记和纯化,线性范围宽且检测限优于文献报道的TNT传感器。理论上,基于该传感器的构建原理,可用于其它具有适配体的各种目标分析物的测定。
In this dissertation, the state of arts in the field of chemiluminescence (CL), CL biosensors, and the applications of CL functionalized nanomaterials in biosensors were reviewed. In recent years, chemiluminescence (CL) functionalized nanomaterials have received more and more attention because a single functionalized nanoparticle could bind thousands of CL reagent molecules, leading to excellent signal amplification. In our research group, a series of novel methods was developed for the preparation of various CL functionalized nanomaterials and nanocomposites including luminol and its derivatives functionalized gold nanomaterials, luminol functionalized silver nanoparticles, luminol functionalized silver/graphene oxide composite(Ag/GO), N-(Aminobutyl)-N-(ethylisoluminol) and hemin dual-functionalized graphene hybrids. This new type of various CL functionalized nanomaterials and nanocomposites would be used as signal reporters for nucleic acid analysis and immunoassays with excellent signal amplification, improving the sensitivity of the bioassays. The aim of this dissertation is to explore the applications of chemiluminescence functionalized nanomaterials in novel DNA sensors and immunosensors. Based on N-(Aminobutyl)-N-(ethylisoluminol) functionalized gold nanomaterials, a sandwich-type electrochemiluminescence (ECL) DNA sensor and a label-free ECL aptamer sensor were developed for tuberculosis diagnosis and thrombin determination; Using luminol functionalized silver nanoparticles as labeling, a novel immunoassay based on competitive reaction was developed for sensitive detection of small molecules chloramphenicol; Based on luminol functionalized silver/graphene oxide composite (Ag/GO) and N-(Aminobutyl)-N-(ethylisoluminol) and hemin dual-functionalized graphene hybrids, a novel label-free aptasensor for2,4,6-trinitrotoluene (TNT) detection was developed. The main results are as follows:
1. An effective sensor for detecting sequence-specific DNA from MTB was developed based on ABEI-AuNPs labeling. The DNA sensor exhibited excellent ECL response with a double step potential in CBS containing1.75x10-3M of H2O2. The sequence-specific DNA from MTB in range of1.0x10-15-1.0x10-12M can be detected by use of the ECL intensity with a considerably low detection limit of3.3x10-16M, which is superior to the previously reported methods without PCR amplification for the determination of MTB. Being challenged in real serum samples, this proposed method also shows an excellent capability for the determination of the target DNA from MTB. The proposed sensor is simple, rapid and reliable. It is also of a great advantage in terms of sensitivity and cost because of great signal amplification of ABEI functionalized gold nanoparticles, simple labeling procedure, and easy construction of the sensor. It is of great application potential for rapid diagnosis of TB in clinical fields.
2. A competitive immunoassay for sensitive detection of small molecules CHL was developed using the luminol functionalized silver nanoparticles as nanoprobe for the first time. CHL in a range of1.0x10-8-1.0x10-6g mL-1could be detected by use of the CL intensity with a low detection limit of7.6x10-9mL-1.The labeling procedure is simple and fast, superior to all the previously reported labeling procedures. Since the labeling of small molecules is very difficult work, the present work provides a novel and effective labeling strategy for CHL. The immunoassay is simple, fast, cost-effective, sensitive and selective.
3. An ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs for the detection of thrombin has been developed. The presented ECL bioassay is sensitive, specific, simple and fast. It exhibits a wide dynamic range from1.0x10-12-1.0x10-9M with a low detection limit of3.8x10-13M, which is superior to most previously reported label-free aptamer-based bioassays for thrombin. Moreover, the interfering species in the matrix can be well separated from the detection system by simply washing the electrode during the dynamic interaction. Thus the method is of high sensitivity and small matrix effects since many substances in the matrix could enhance and inhibit the CL of luminol and analogues leading to the interference. It has been successfully applied to the detectionof thrombin in real human serums. This work provides a new way to design aptamer-based protocols for the determination of bio-logically important substances. In principle, the proposed strategy might also be applicable for the determination of other biologically important substances by using corresponding aptamer.
4. A label-free electrochemiluminescence aptasensor for2,4,6-trinitrotoluene detection was proposed based on an assembly strategy of luminol functionalized silver/graphene oxide composite(Ag/GO) and N-(Aminobutyl)-N-(ethylisoluminol) and hemin dual-functionalized graphene hybrids. It exhibits a wide dynamic range from1.0x10-12-1.0x10-9g/mL with a low detection limit of6.3x10-13g/mL, which is superior to most previously reported bioassays for TNT. The proposed aptasensor has some advantages such as simple operation, high sensitivity, no need of complex labeling and purification steps, making this methods time-saving and easy automation. Due to the wide target recognition range of aptamer, this strategy provides a promising way to develop new aptasensor for other analytes.
1.利用N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金作为标记物构建了一个有效的DNA传感器用于MTB的特异DNA序列的检测。生物素化的DNA捕获探针通过电极表面修饰的链霉亲和素包被的纳米金直接固载到电极上,目标DNA和N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金标记的DNA序列随后通过DNA特异性杂交反应连接在电极表面形成三明治结构的DNA传感器。该DNA传感器在含有1.75×10-3mol/L H2O2的CBS缓冲中施加双脉冲电压能得到很好的电致化学发光信号。该方法能检测到1.0×10-15-1.0×10-12mol/L范围内的MTB的特异DNA序列,检测限低至3.3×10-16mol/L,优于之前报道的无需PCR放大的MTB分析方法。在实际样品血清样中,该方法对于检测MTB的特异DNA序列也有很好的测定能力。该传感器简单,快速,可靠。因为N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金的信号放大作用,简单的标记过程和传感器的构建过程简单,使得该方法在灵敏度和成本方面有很大的优势。该方法在临床上快速测定MTB具有巨大的应用前景。
2.基于鲁米诺功能化纳米银作为纳米标记物我们首次开发了一个竞争的免疫分析传感器用于小分子氯霉素的灵敏检测。在1.0－10-8-1.0×10-6g/mL范围内可以根据发光强度测定氯霉素含量,检测限低至7.6×10-9g/mL。标记过程简单快速,优于所有文献报道的标记方法。由于小分子的标记是非常困难的工作,此项工作提供了一种新颖而有效的氯霉素标记策略。该免疫分析方法简单、快捷、有效、灵敏度和选择性好,且简捷不需要复杂的仪器设备,使之很有希望用于实际样品中氯霉素的快速检测。
3.基于适配体,凝血酶和N-(4-氨基丁基)-N-乙基异鲁米诺功能化纳米金的动态组装构建了一个电致化学发光生物分析方法用于凝血酶的检测。所提出电致化学发光生物分析方法灵敏、特异、简便、快捷。该方法在1.0×10-12-1.0×10-9mol/L较宽的范围内具有线性关系,检测限为3.8×10-13mol/L,优于大多数文献报道的基于适配体的无标记生物分析方法。另外,在动态组装过程中基质中的干扰物质可以通过简单的清洗电极很容易地从检测系统分离。因此,该方法具有较高的灵敏度和很小的基体效应,因为基质中的很多物质可增强和抑制鲁米诺和类似物的发光导致干扰。该方法已成功地应用于实际人血清样品中凝血酶的检测。这项工作为设计基于适配体检测重要生物分子提供了一个新的思路。在原理上,使用相应的适配体,本章提出的分析方法也适用于其它重要生物分子的测定。
4.基于两种新型的功能化石墨烯复合材料和适配体构建了一种无标记的电致化学发光适配体传感器用于TNT的检测。该传感器在1.0×10-12-1.0×10-9g/mL较宽的范围内具有良好的线性,检测限低至6.3×10-13g/mL,优于大多数文献报道其他TNT传感器。课题组新开发出的两种新型石墨烯复合材料首次被用于无标记传感器的构建,表现出了良好的发光性能,有效提高了该方法的灵敏度。我们开发的传感器构建过程简单、耗时短,目标物不需要标记和纯化,线性范围宽且检测限优于文献报道的TNT传感器。理论上,基于该传感器的构建原理,可用于其它具有适配体的各种目标分析物的测定。
In this dissertation, the state of arts in the field of chemiluminescence (CL), CL biosensors, and the applications of CL functionalized nanomaterials in biosensors were reviewed. In recent years, chemiluminescence (CL) functionalized nanomaterials have received more and more attention because a single functionalized nanoparticle could bind thousands of CL reagent molecules, leading to excellent signal amplification. In our research group, a series of novel methods was developed for the preparation of various CL functionalized nanomaterials and nanocomposites including luminol and its derivatives functionalized gold nanomaterials, luminol functionalized silver nanoparticles, luminol functionalized silver/graphene oxide composite(Ag/GO), N-(Aminobutyl)-N-(ethylisoluminol) and hemin dual-functionalized graphene hybrids. This new type of various CL functionalized nanomaterials and nanocomposites would be used as signal reporters for nucleic acid analysis and immunoassays with excellent signal amplification, improving the sensitivity of the bioassays. The aim of this dissertation is to explore the applications of chemiluminescence functionalized nanomaterials in novel DNA sensors and immunosensors. Based on N-(Aminobutyl)-N-(ethylisoluminol) functionalized gold nanomaterials, a sandwich-type electrochemiluminescence (ECL) DNA sensor and a label-free ECL aptamer sensor were developed for tuberculosis diagnosis and thrombin determination; Using luminol functionalized silver nanoparticles as labeling, a novel immunoassay based on competitive reaction was developed for sensitive detection of small molecules chloramphenicol; Based on luminol functionalized silver/graphene oxide composite (Ag/GO) and N-(Aminobutyl)-N-(ethylisoluminol) and hemin dual-functionalized graphene hybrids, a novel label-free aptasensor for2,4,6-trinitrotoluene (TNT) detection was developed. The main results are as follows:
1. An effective sensor for detecting sequence-specific DNA from MTB was developed based on ABEI-AuNPs labeling. The DNA sensor exhibited excellent ECL response with a double step potential in CBS containing1.75x10-3M of H2O2. The sequence-specific DNA from MTB in range of1.0x10-15-1.0x10-12M can be detected by use of the ECL intensity with a considerably low detection limit of3.3x10-16M, which is superior to the previously reported methods without PCR amplification for the determination of MTB. Being challenged in real serum samples, this proposed method also shows an excellent capability for the determination of the target DNA from MTB. The proposed sensor is simple, rapid and reliable. It is also of a great advantage in terms of sensitivity and cost because of great signal amplification of ABEI functionalized gold nanoparticles, simple labeling procedure, and easy construction of the sensor. It is of great application potential for rapid diagnosis of TB in clinical fields.
2. A competitive immunoassay for sensitive detection of small molecules CHL was developed using the luminol functionalized silver nanoparticles as nanoprobe for the first time. CHL in a range of1.0x10-8-1.0x10-6g mL-1could be detected by use of the CL intensity with a low detection limit of7.6x10-9mL-1.The labeling procedure is simple and fast, superior to all the previously reported labeling procedures. Since the labeling of small molecules is very difficult work, the present work provides a novel and effective labeling strategy for CHL. The immunoassay is simple, fast, cost-effective, sensitive and selective.
3. An ECL bioassay based on dynamic interaction of aptamer, thrombin and ABEI-AuNPs for the detection of thrombin has been developed. The presented ECL bioassay is sensitive, specific, simple and fast. It exhibits a wide dynamic range from1.0x10-12-1.0x10-9M with a low detection limit of3.8x10-13M, which is superior to most previously reported label-free aptamer-based bioassays for thrombin. Moreover, the interfering species in the matrix can be well separated from the detection system by simply washing the electrode during the dynamic interaction. Thus the method is of high sensitivity and small matrix effects since many substances in the matrix could enhance and inhibit the CL of luminol and analogues leading to the interference. It has been successfully applied to the detectionof thrombin in real human serums. This work provides a new way to design aptamer-based protocols for the determination of bio-logically important substances. In principle, the proposed strategy might also be applicable for the determination of other biologically important substances by using corresponding aptamer.
4. A label-free electrochemiluminescence aptasensor for2,4,6-trinitrotoluene detection was proposed based on an assembly strategy of luminol functionalized silver/graphene oxide composite(Ag/GO) and N-(Aminobutyl)-N-(ethylisoluminol) and hemin dual-functionalized graphene hybrids. It exhibits a wide dynamic range from1.0x10-12-1.0x10-9g/mL with a low detection limit of6.3x10-13g/mL, which is superior to most previously reported bioassays for TNT. The proposed aptasensor has some advantages such as simple operation, high sensitivity, no need of complex labeling and purification steps, making this methods time-saving and easy automation. Due to the wide target recognition range of aptamer, this strategy provides a promising way to develop new aptasensor for other analytes.
引文
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