新型生物传感器的构建及其在环境和生物检测中的应用研究
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摘要
生物传感器是一种由生物感应元件和信号处理元件组成，用于对各种生物、化学物质进行检测的分析系统，其基本原理是生物感应元件与检测目标物相互作用后产生响应信号，信号处理元件对响应信号进行接收、加工、转换、输出，以此实现对目标物的定性、定量检测。鉴于生物传感器具有传统分析方法不可比拟的优势，生物传感器已成为分析检测领域的重要技术,被广泛应用于环境检测、生物医学研究、食品检测、发酵工业生产等众多领域。
近年来，对环境污染物的检测成为环境评估、环境治理的重要手段，对生物分子的检测成为生物学研究的重要内容，对环境污染物和生物分子进行快速、灵敏检测的生物传感器的研制开发具有重要的理论价值和广阔的应用前景。本论文成功构建了5种新颖的生物传感器分别用于检测环境污染物重金属二价铅离子、三硝基甲苯、苯并（а）芘及生物小分子含巯基氨基酸。
1、基于谷胱甘肽能够保护银纳米颗粒抵抗盐诱导的聚集原理构建了检测重金属Pb（Ⅱ）离子的生物传感器。谷胱甘肽通过Ag-S键结合到银纳米颗粒表面能够增强银纳米颗粒稳定性，协助银纳米颗粒抵抗盐诱导的聚集；重金属Pb（Ⅱ）离子可竞争性与谷胱甘肽结合，以此削弱谷胱甘肽对银纳米颗粒的保护作用从而导致银纳米颗粒在盐诱导下发生聚集。银纳米颗粒分散、聚集的不同状态导致其紫外可见吸收值的不同即可定性、定量反映不同浓度的Pb（Ⅱ）离子。该生物传感器实现了对Pb（Ⅱ）离子的特异性检测，检测线性范围在0.5-4μM，检测下限为0.5μM。同时该生物传感器实现了对实际水样中Pb（Ⅱ）离子的检测，说明该生物传感器具有良好的实际应用前景。本论文首次发现谷胱甘肽能够保护银纳米颗粒抵抗盐诱导的聚集，增强银纳米颗粒的稳定性，并将这一原理应用于构建可视化、定量检测Pb（Ⅱ）离子的生物传感器。该生物传感器利用盐溶液放大检测信号，可以实现高灵敏度、特异性检测Pb（Ⅱ）离子。该研究方法为检测环境中的重金属离子提供了一条可能的思路：通过寻找与不同重金属特异性结合的配体，结合银纳米等纳米材料在分散、聚集条件下呈现不同颜色所带来的紫外可见吸收值的变化，实现对环境中重金属的特异性检测。
2、利用TNT能够淬灭银纳米簇荧光的原理构建了检测TNT的生物传感器。银离子与单链DNA中胞嘧啶结合后，在硼氢化钠还原作用下生成高质量、稳定的荧光银纳米簇。三硝基甲苯（TNT）是一种较强电子受体，可以有效淬灭银纳米簇的荧光。TNT通过疏水作用结合到银纳米簇表面，淬灭其荧光，随着TNT浓度的增加，TNT荧光淬灭效率增加，银纳米簇荧光强度减小，以此可以实现TNT检测。该生物传感器检测TNT线性范围在0.5-6μg/mL，检测限为0.5μg/mL。通过对土壤和河水添加样中TNT的检测，证明该生物传感器能够实现对环境实际样品中TNT的检测。本论文首次发现三硝基甲苯可以淬灭ssDNA保护的银纳米簇荧光效应，并将其应用于构建检测TNT的生物传感器，为将荧光纳米材料应用于TNT检测生物传感器的构建提供可借鉴的经验。
3、基于斑点杂交原理构建了可视化、半定量检测多环芳烃代表物苯并（а）芘的生物传感器。该生物传感器利用苯并（а）芘与苯并（а）芘抗体、吸附于硝酸纤维素膜上的苯并（а）芘抗原之间发生竞争性免疫反应特性，使用感光胶片对检测信号进行收集。通过对感光胶片的曝光、显影，该生物传感器可以对浓度低至5ng/mL的苯并（а）芘进行可视化检测。使用专业分析软件对检测信号强度进行分析，该生物传感器可以实现半定量检测苯并（а）芘。使用该生物传感器对固相萃取法富集的河水样品中的苯并（а）芘进行检测，检测结果说明该生物传感器能够应用于实际检测。本论文通过将斑点杂交原理应用于构建检测苯并（а）芘的生物传感器，在不使用任何检测仪器的条件下，可以实现对环境样品中苯并（а）芘的定性、半定量检测。该生物传感器操作简单、检测特异性强，为环境有毒污染物检测方法的研究提供了可行的思路。
4、应用单链DNA能够通过碱基与银纳米颗粒之间形成的范德华力吸附到银纳米颗粒表面帮助银纳米颗粒抵抗盐诱导的聚集原理构建了检测含巯基氨基酸的生物传感器。含巯基氨基酸通过巯基结合到银纳米颗粒表面，阻碍了单链DNA的吸附，失去单链DNA保护的银纳米颗粒在盐的诱导下发生聚集，纳米颗粒的紫外可见吸收值发生改变，以此可以实现检测含巯基氨基酸。该生物传感器对同型半胱氨酸、半胱氨酸、谷胱甘肽的检测线性范围分别在在10-500nM、50-500nM、100-500nM，检测限分别为10nM、50nM、100nM。对模拟血清和尿液样品中同型半胱氨酸的成功检测证明该生物传感器具有良好的实际应用前景。本论文基于单链DNA保护银纳米颗粒抵抗盐诱导的聚集原理，根据含巯基氨基酸通过Ag-S键结合到银纳米颗粒表面替代单链DNA的实验现象，创新性的将两者有机结合构建了特异性检测含巯基氨基酸的生物传感器。通过盐溶液放大信号作用，该生物传感器能够实现高灵敏度检测含巯基氨基酸。通过分析银纳米颗粒分散、聚集状态下不同紫外可见吸收值，该生物传感器可以实现可视化、定量检测含巯基氨基酸。该生物传感器设计简单、新颖，检测快速、高效，为生物小分子物质检测提供了新颖的研究思路。
5、基于单链DNA保护的金纳米颗粒的稳定性增强并能够有效抵抗盐诱导的聚集原理构建了检测半胱氨酸的生物传感器。单链DNA通过碱基与金纳米颗粒之间形成的范德华力吸附到金纳米颗粒表面，增强金纳米颗粒的稳定性。半胱氨酸通过巯基以Au-S键结合到金纳米颗粒表面将单链DNA从金纳米颗粒表面排开，失去单链DNA保护的金纳米颗粒在盐诱导条件下发生聚集，导致纳米颗粒的紫外可见吸收值发生改变，以此实现检测半胱氨酸。该生物传感器检测半胱氨酸线性范围在0.1-5μM，检测限为0.1μM。通过对模拟血清样品中半胱氨酸的检测，证明该生物传感器具有良好的实际应用前景。该生物传感器简单、高效，检测灵敏度高，为将纳米材料用于构建检测生物小分子物质的生物传感器提供了可借鉴的研究思路。
Biosensor is an analytic device composed of biological sensor and signalprocessing components for biological, chemical analysis detection. On binding ofanalyte to biological sensor, the consequent signal was received, processed, converedand output by signal processing components, and as a result, the qualitative,quantitative analysis to the analyte are achieved. In view of the incomparableadvantage compared with conventional analysis methods, biosensor has alreadyevolved as an important technology for analysis and detection, and it has been widelyused in environmental monitoring, medicine, biology research, food testing,fermentation industrial production etc.
Recently, the detection for environmental pollutant has become the key indicatorfor environmental evaluation and protection. The detection for biological moleculehas also become the effective reference for biology research. The design of thebiosensor to detect environmental pollutant and biological molecule is of greatscientific significance and promising practical application. Here,5novel biosensorsfor the determination of heavy metal lead ions, benzene(а)pyrene, TNT or thiol aminoacids are described.
1. A type of Pb (Ⅱ) ion biosensor has been generated based on glutathioneprotection of the silver nanoparticles(AgNPs). Glutathione can be conjugated toAgNPs via Ag–S bond, helping AgNPs to resist against aggregation induced by salt.However, the interaction between glutathione and AgNPs can be compromised by Pb(Ⅱ) ion binding to glutathione. As a result, Pb (Ⅱ) mediated-aggregation of AgNPscan be reflected by the UV-Visible spectrum change, and the qualitative andquantitative detection for Pb (Ⅱ) ion is accomplished, with the detection range0.5-4μM and a detection limit of0.5μM. Pb (Ⅱ) ion in water sample has also beendetermined by this biosensor. The principle that glutathione is capable of enhancingthe stability of the AgNPs is first reported in this thesis. Furthermore, thevisualization and quantitative detection for Pb (Ⅱ) ion biosensor was constructedbased on this principle. This study implies that a ligand which combines heavy metalions and silver nanomaterials can be used to construct biosensors to achieve thespecific detection.
2. A type of trinitrotoluene (TNT) biosensor using fluorescence silver nanocluster has been proposed. In the presence of interaction between Ag+and cytosine,fluorescence silver nanocluster can be generated under the reduction effect of sodiumborohydride. TNT is a strong electronic receptor and an effective fluorescent quencher.The interaction between TNT and silver nanocluster leads to fluorescence quenching,thus the detection of TNT is realized, with detection linear range of0.5-6μg/mL anddetection limit of0.5μg/mL. TNT in the soil sample can be detected using thebiosensor, indicating its practical application. Moreover, the successful application offluorescence silver nanocluster in TNT biosensor provides referential experience inthe construction of biosensor using fluorescence nanomaterial.
3. A type of benzene(а)pyrene biosensor based on dot blot has been proposed.Competitive immune response occurs between benzene(а)pyrene, benzene(а)pyreneantibody and benzene(а)pyrene antigen absorbed on nitrocellulose membrane.Detection signal can be collected by film. The biosensor can analyzed as low as5ng/mL of benzene(а)pyrene through a visualization detection. A semi-quantitativebenzene(а)pyrene detection can be achieved by using a professional analysis software.The water sample containing benzene(а)pyrene can be analyzed, imply the potentialpractical application. The principle of dot blot was firstly employed to designbiosensors, and provided qualitative and semi-quantitative detection without any useof instrument. The simplicity and high specificity of benzene(а)pyrene biosensorsheds new light on the development of environmental pollutant biosensor.
4. A type of biological thiols biosensor for using ssDNA/sliver nanoparticlessystem has been proposed. The adsorbing ssDNA provides AgNPs high density chargeto prevent nanoparticles from aggregation induced by salt. However, homocysteine(cysteine or glutathione) can conjugate to AgNPs via Ag-S bond more powerfully thanssDNA, which restrain ssDNA from binding to AgNPs surface. In the presense ofhomocysteine, the salt induced AgNPs aggregation and the corresponding UV-Visiblespectrum change. Homocysteine,cysteine and glutathione can be determined in therange of10-500nM、50-500nM、100-500nM, respectively, with the detection limitof10nM、50nM、100nM, respectively. The method was successfully applied todetermine stimulant serum samples or urine sample containing homocysteine.Through the AgNPs color change, biological thiols biosensor provide simple, rapid,efficient, specific and visual detection for thiol amino acids, which may be instructiveto the biological molecule biosensor development.
5. A type of cysteine biosensor based on ssDNA-stabilized gold nanoparticles(AuNPs) has been proposed. The adsorbing ssDNA provides AuNPs high density charge to prevent nanoparticles from aggregation induced by salt. In the presence ofcysteine, ssDNA is moved away by cysteine, resulting in the AuNPs aggregation viathe introduction of salt. Therefore, cysteine can be detected based on UV-Visiblespectrum change. A favorable linear correlation in the range of0.1-5μM is obtainedwith a detection limit of0.1μM. The biosensor is successfully applied to determinestimulant serum samples containing cysteine, suggesting the promising practicalapplication. The cysteine biosensor is simple, efficient, highly sensitive, which willpromote the biological molecule biosensor designed with nanomaterial.
近年来，对环境污染物的检测成为环境评估、环境治理的重要手段，对生物分子的检测成为生物学研究的重要内容，对环境污染物和生物分子进行快速、灵敏检测的生物传感器的研制开发具有重要的理论价值和广阔的应用前景。本论文成功构建了5种新颖的生物传感器分别用于检测环境污染物重金属二价铅离子、三硝基甲苯、苯并（а）芘及生物小分子含巯基氨基酸。
1、基于谷胱甘肽能够保护银纳米颗粒抵抗盐诱导的聚集原理构建了检测重金属Pb（Ⅱ）离子的生物传感器。谷胱甘肽通过Ag-S键结合到银纳米颗粒表面能够增强银纳米颗粒稳定性，协助银纳米颗粒抵抗盐诱导的聚集；重金属Pb（Ⅱ）离子可竞争性与谷胱甘肽结合，以此削弱谷胱甘肽对银纳米颗粒的保护作用从而导致银纳米颗粒在盐诱导下发生聚集。银纳米颗粒分散、聚集的不同状态导致其紫外可见吸收值的不同即可定性、定量反映不同浓度的Pb（Ⅱ）离子。该生物传感器实现了对Pb（Ⅱ）离子的特异性检测，检测线性范围在0.5-4μM，检测下限为0.5μM。同时该生物传感器实现了对实际水样中Pb（Ⅱ）离子的检测，说明该生物传感器具有良好的实际应用前景。本论文首次发现谷胱甘肽能够保护银纳米颗粒抵抗盐诱导的聚集，增强银纳米颗粒的稳定性，并将这一原理应用于构建可视化、定量检测Pb（Ⅱ）离子的生物传感器。该生物传感器利用盐溶液放大检测信号，可以实现高灵敏度、特异性检测Pb（Ⅱ）离子。该研究方法为检测环境中的重金属离子提供了一条可能的思路：通过寻找与不同重金属特异性结合的配体，结合银纳米等纳米材料在分散、聚集条件下呈现不同颜色所带来的紫外可见吸收值的变化，实现对环境中重金属的特异性检测。
2、利用TNT能够淬灭银纳米簇荧光的原理构建了检测TNT的生物传感器。银离子与单链DNA中胞嘧啶结合后，在硼氢化钠还原作用下生成高质量、稳定的荧光银纳米簇。三硝基甲苯（TNT）是一种较强电子受体，可以有效淬灭银纳米簇的荧光。TNT通过疏水作用结合到银纳米簇表面，淬灭其荧光，随着TNT浓度的增加，TNT荧光淬灭效率增加，银纳米簇荧光强度减小，以此可以实现TNT检测。该生物传感器检测TNT线性范围在0.5-6μg/mL，检测限为0.5μg/mL。通过对土壤和河水添加样中TNT的检测，证明该生物传感器能够实现对环境实际样品中TNT的检测。本论文首次发现三硝基甲苯可以淬灭ssDNA保护的银纳米簇荧光效应，并将其应用于构建检测TNT的生物传感器，为将荧光纳米材料应用于TNT检测生物传感器的构建提供可借鉴的经验。
3、基于斑点杂交原理构建了可视化、半定量检测多环芳烃代表物苯并（а）芘的生物传感器。该生物传感器利用苯并（а）芘与苯并（а）芘抗体、吸附于硝酸纤维素膜上的苯并（а）芘抗原之间发生竞争性免疫反应特性，使用感光胶片对检测信号进行收集。通过对感光胶片的曝光、显影，该生物传感器可以对浓度低至5ng/mL的苯并（а）芘进行可视化检测。使用专业分析软件对检测信号强度进行分析，该生物传感器可以实现半定量检测苯并（а）芘。使用该生物传感器对固相萃取法富集的河水样品中的苯并（а）芘进行检测，检测结果说明该生物传感器能够应用于实际检测。本论文通过将斑点杂交原理应用于构建检测苯并（а）芘的生物传感器，在不使用任何检测仪器的条件下，可以实现对环境样品中苯并（а）芘的定性、半定量检测。该生物传感器操作简单、检测特异性强，为环境有毒污染物检测方法的研究提供了可行的思路。
4、应用单链DNA能够通过碱基与银纳米颗粒之间形成的范德华力吸附到银纳米颗粒表面帮助银纳米颗粒抵抗盐诱导的聚集原理构建了检测含巯基氨基酸的生物传感器。含巯基氨基酸通过巯基结合到银纳米颗粒表面，阻碍了单链DNA的吸附，失去单链DNA保护的银纳米颗粒在盐的诱导下发生聚集，纳米颗粒的紫外可见吸收值发生改变，以此可以实现检测含巯基氨基酸。该生物传感器对同型半胱氨酸、半胱氨酸、谷胱甘肽的检测线性范围分别在在10-500nM、50-500nM、100-500nM，检测限分别为10nM、50nM、100nM。对模拟血清和尿液样品中同型半胱氨酸的成功检测证明该生物传感器具有良好的实际应用前景。本论文基于单链DNA保护银纳米颗粒抵抗盐诱导的聚集原理，根据含巯基氨基酸通过Ag-S键结合到银纳米颗粒表面替代单链DNA的实验现象，创新性的将两者有机结合构建了特异性检测含巯基氨基酸的生物传感器。通过盐溶液放大信号作用，该生物传感器能够实现高灵敏度检测含巯基氨基酸。通过分析银纳米颗粒分散、聚集状态下不同紫外可见吸收值，该生物传感器可以实现可视化、定量检测含巯基氨基酸。该生物传感器设计简单、新颖，检测快速、高效，为生物小分子物质检测提供了新颖的研究思路。
5、基于单链DNA保护的金纳米颗粒的稳定性增强并能够有效抵抗盐诱导的聚集原理构建了检测半胱氨酸的生物传感器。单链DNA通过碱基与金纳米颗粒之间形成的范德华力吸附到金纳米颗粒表面，增强金纳米颗粒的稳定性。半胱氨酸通过巯基以Au-S键结合到金纳米颗粒表面将单链DNA从金纳米颗粒表面排开，失去单链DNA保护的金纳米颗粒在盐诱导条件下发生聚集，导致纳米颗粒的紫外可见吸收值发生改变，以此实现检测半胱氨酸。该生物传感器检测半胱氨酸线性范围在0.1-5μM，检测限为0.1μM。通过对模拟血清样品中半胱氨酸的检测，证明该生物传感器具有良好的实际应用前景。该生物传感器简单、高效，检测灵敏度高，为将纳米材料用于构建检测生物小分子物质的生物传感器提供了可借鉴的研究思路。
Biosensor is an analytic device composed of biological sensor and signalprocessing components for biological, chemical analysis detection. On binding ofanalyte to biological sensor, the consequent signal was received, processed, converedand output by signal processing components, and as a result, the qualitative,quantitative analysis to the analyte are achieved. In view of the incomparableadvantage compared with conventional analysis methods, biosensor has alreadyevolved as an important technology for analysis and detection, and it has been widelyused in environmental monitoring, medicine, biology research, food testing,fermentation industrial production etc.
Recently, the detection for environmental pollutant has become the key indicatorfor environmental evaluation and protection. The detection for biological moleculehas also become the effective reference for biology research. The design of thebiosensor to detect environmental pollutant and biological molecule is of greatscientific significance and promising practical application. Here,5novel biosensorsfor the determination of heavy metal lead ions, benzene(а)pyrene, TNT or thiol aminoacids are described.
1. A type of Pb (Ⅱ) ion biosensor has been generated based on glutathioneprotection of the silver nanoparticles(AgNPs). Glutathione can be conjugated toAgNPs via Ag–S bond, helping AgNPs to resist against aggregation induced by salt.However, the interaction between glutathione and AgNPs can be compromised by Pb(Ⅱ) ion binding to glutathione. As a result, Pb (Ⅱ) mediated-aggregation of AgNPscan be reflected by the UV-Visible spectrum change, and the qualitative andquantitative detection for Pb (Ⅱ) ion is accomplished, with the detection range0.5-4μM and a detection limit of0.5μM. Pb (Ⅱ) ion in water sample has also beendetermined by this biosensor. The principle that glutathione is capable of enhancingthe stability of the AgNPs is first reported in this thesis. Furthermore, thevisualization and quantitative detection for Pb (Ⅱ) ion biosensor was constructedbased on this principle. This study implies that a ligand which combines heavy metalions and silver nanomaterials can be used to construct biosensors to achieve thespecific detection.
2. A type of trinitrotoluene (TNT) biosensor using fluorescence silver nanocluster has been proposed. In the presence of interaction between Ag+and cytosine,fluorescence silver nanocluster can be generated under the reduction effect of sodiumborohydride. TNT is a strong electronic receptor and an effective fluorescent quencher.The interaction between TNT and silver nanocluster leads to fluorescence quenching,thus the detection of TNT is realized, with detection linear range of0.5-6μg/mL anddetection limit of0.5μg/mL. TNT in the soil sample can be detected using thebiosensor, indicating its practical application. Moreover, the successful application offluorescence silver nanocluster in TNT biosensor provides referential experience inthe construction of biosensor using fluorescence nanomaterial.
3. A type of benzene(а)pyrene biosensor based on dot blot has been proposed.Competitive immune response occurs between benzene(а)pyrene, benzene(а)pyreneantibody and benzene(а)pyrene antigen absorbed on nitrocellulose membrane.Detection signal can be collected by film. The biosensor can analyzed as low as5ng/mL of benzene(а)pyrene through a visualization detection. A semi-quantitativebenzene(а)pyrene detection can be achieved by using a professional analysis software.The water sample containing benzene(а)pyrene can be analyzed, imply the potentialpractical application. The principle of dot blot was firstly employed to designbiosensors, and provided qualitative and semi-quantitative detection without any useof instrument. The simplicity and high specificity of benzene(а)pyrene biosensorsheds new light on the development of environmental pollutant biosensor.
4. A type of biological thiols biosensor for using ssDNA/sliver nanoparticlessystem has been proposed. The adsorbing ssDNA provides AgNPs high density chargeto prevent nanoparticles from aggregation induced by salt. However, homocysteine(cysteine or glutathione) can conjugate to AgNPs via Ag-S bond more powerfully thanssDNA, which restrain ssDNA from binding to AgNPs surface. In the presense ofhomocysteine, the salt induced AgNPs aggregation and the corresponding UV-Visiblespectrum change. Homocysteine,cysteine and glutathione can be determined in therange of10-500nM、50-500nM、100-500nM, respectively, with the detection limitof10nM、50nM、100nM, respectively. The method was successfully applied todetermine stimulant serum samples or urine sample containing homocysteine.Through the AgNPs color change, biological thiols biosensor provide simple, rapid,efficient, specific and visual detection for thiol amino acids, which may be instructiveto the biological molecule biosensor development.
5. A type of cysteine biosensor based on ssDNA-stabilized gold nanoparticles(AuNPs) has been proposed. The adsorbing ssDNA provides AuNPs high density charge to prevent nanoparticles from aggregation induced by salt. In the presence ofcysteine, ssDNA is moved away by cysteine, resulting in the AuNPs aggregation viathe introduction of salt. Therefore, cysteine can be detected based on UV-Visiblespectrum change. A favorable linear correlation in the range of0.1-5μM is obtainedwith a detection limit of0.1μM. The biosensor is successfully applied to determinestimulant serum samples containing cysteine, suggesting the promising practicalapplication. The cysteine biosensor is simple, efficient, highly sensitive, which willpromote the biological molecule biosensor designed with nanomaterial.
引文
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