构建基于纳米材料的电化学生物传感器
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摘要
自从微观特别是纳米尺度上材料诞生以来,对它们的研究及应用一直受到人们的高度关注,并且一直处在高速发展阶段,各种具有特殊功能的新型纳米材料层出不穷,而且对它们的研究和应用领域也在快速的扩展。电化学传感技术由于具有灵敏性好、仪器简单易于操作、可控性好、检测成本低及易于微型化等特点,同样受到广泛关注。如今,人们研究发现：将纳米技术引入到电化学传感领域后,在使得电化学传感技术得到了革命性发展的同时,也促进了纳米技术的变革。基于此,在本论文中,我们研究了具有较高电催化和生物相容性的碳材料和贵金属纳米材料的制备及其在电分析传感领域的应用。
(1)在第2章中,我们研究发现双螺旋DNA的磷酸骨架可以作为多价金属离子或复合物的结合位点,并且在结合的过程中,这些多价离子会改变DNA的构象,形成压缩结构。利用这一性质,我们首先让氯金酸盐与双螺旋DNA通过静电力作用形成Au(Ⅲ)一DNA网状结构的复合物；然后,通过化学还原Au(Ⅲ)最终形成的Au.DNA网状结构的纳米复合物。该杂交材料有较高的生物相容性以及电子传导能力,有利于酶的固定。因此,我们将HRP固定在该复合材料上进行电化学研究,发现固载在该复合材料上的HRP表现出良好的直接电化学行为和电催化能力。
(2)在第3章中,我们利用碳化技术,制备了具有生物形态的有序多孔碳(OPC),并且通过电沉积技术在OPC的表面和孔穴内沉积金属Pt,形成OPC-Pt复合材料。详细地研究了OPC及OPC-Pt的结构形貌等特征,并通过对其电化学行为的研究发现,该复合材料具有较高的电催化性能并对H202具有明显的电催化活性,因此该复合材料可用于构建非酶传感器。
(3)在第4章中,同样以加工好的椴木块为原材料,通过碳化工艺,制备出了具有良好生物形态结构的椴木多孔碳(BBPC).我们利用XRD、红外光谱(FT-IR).扫描电镜(SEM)等研究手段对该材料的微观三维形态结构进行了详细的研究。同时在电化学实验中,我们意外的发现该多孔碳材料具有良好的酶促催化性能,并可以促进电子传递。另外,动力学研究发现该多孔碳材料对H202有良好的亲和性,这也就进一步证明了BBPC构建非酶传感器的可行性。因此,我们用BBPC代替HRP构建了一个检测H202的非酶电化学生物传感器。实验结果表明,该非酶传感器能够对H202进行快速灵敏的检测,同时具有较宽的线性范围和较低的检测限。
(4)在第5章中,我们设计了利用电化学发光技术(ECL)来对蛋白激酶活性及其抑制剂进行高灵敏检测的生物传感器。在共反应剂巯基化三磷酸腺苷(ATP-γ-s)的共在下,激酶催化磷酸化反应,然后以金纳米粒子为标记,与磷酸化过程中巯基化的磷酸根特异结合。由于金纳米粒子具有高的电导性、大的比表面积以及对鲁米诺(luminol)氧化的高电催化活性,因此可以极大的催化鲁米诺的电化学发光(ECL)信号增强,实现对激酶活性的高灵敏检测。蛋白激酶A(PKA)在人体的诸多生理及新陈代谢过程中发挥着重要作用,因此该酶被作为本研究的模型激酶进行理论上的检测分析。实验结果表明,该ECL传感器对PKA有较低的检测限(0.07UmL-1),宽的线性检测范围(0.07-32U mL-1)及良好的检测稳定性。除此之外,该ECL传感器还可用于激酶抑制剂的量化分析检测。在本实验中,我们测定了PKA抑制剂鞣花酸的IC50,发现该值与用传统分析技术所获得的检测结果相符。同时我们利用酪氨酸激酶抑制剂(Tyrphostin AG1478)重复上述检测过程,发现ECL响应几乎没有变化,这些结果说明ECL传感器可以用于对激酶抑制剂的筛选。
(5)在第6章中,通过研究我们首次发现,石墨烯(graphene)可以诱导鲁米诺在正电位(ca.0.05V vs.Ag/AgCl)产生较强而稳定的阴极ECL现象。同时,石墨烯修饰的玻碳电极是构建高性能生物传感器的优良平台。基于以上考虑,我们利用石墨烯修饰的玻碳电极构建了低电位下阴极ECL检测癌症标识物的夹心型ECL免疫传感器。葡萄糖氧化酶(GOx)和检测抗体(Ab2)修饰的金纳米棒(GNRs)作为该传感器的生物探针。功能化的石墨烯能够促进电极表面的电子转移；同时由于其大的比表面积,因此用于固载捕获抗体(Ab1)；功能化的GNRs不仅作为酶和抗体的固定载体,其自身对鲁米诺的ECL也具有增强作用,另外固载其上的GOx在反应体系中含有葡萄糖和氧气时,通过催化底物反应又可以进一步增强ECL信号。在以前列腺抗原(PSA)作为研究模型的条件下,我们所构建的低电位ECL免疫传感器对PSA能够进行高灵敏、专一性的检测,获得的线性检测范围为10pgmL-1-8ng mL-1,最低检测限为8pg mL-1,同时具有较高的稳定性和重复性。而且该传感器还被用于对10种不同PSA浓度的临床血清样品进行检测,分析结果与标准试剂盒检测获得结果进行对比发现,所构建的ECL免疫传感器可直接用于临床诊断分析。
Since the birth of micro-scale especially for nanomaterials, they are a hotspot in research and applications field all the time, which are now in the stage of rapid development. A variety of new nanomaterials with special functions are emerging in an endless stream, which expands further research and applications of these materials. Electrochemical apparatus because of some advantages such as sensitive, simple and easy to operate, good controllability, low cost and easy to be minisized, has received extensive attention. Nowadays, people found that when combining nanotechnology with electrochemical sensing technology, it not only made sensors in a revolutionary development but also promoted the development of nanotechnology. For those reasons, we investigate the preparation of carbon materials and noble metal nanomaterials, with electrocatalytic activity and high biocompatibility, and their applications in electroanalysis and sensing field.
(1) In chapter2, a novel DNA-templated Au nanoparticles (Au-DNA) nanoconjugate was prepared by using the combination of metallization and DNA compaction. The electrostatic interaction between Au(Ⅲ) and the phosphate backbone of DNA formed the netlike coordination compound of Au(Ⅲ)-DNA, and then the complex was chemically reduced to form Au nanoparticles in this network-like DNA conformation. The negatively charged nanoconjugate was used as the matrix for immobilization of horseradish peroxidase (HRP). A stable and well-defined redox peaks of HRP were observed on the Au-DNA nanoconjugate modified glassy carbon (GC) electrode, which indicated that the modified enzyme electrode displayed good direct electron transfer behavior and excellent reducing ability toward hydrogen peroxide (H2O2) with the apparent Michaelis-Menten constant (Km) estimated to be0.147mM.
(2) In chapter3, a biomorphic carbon, retained its anatomical features, was prepared by carbonizing wood through controlled heated rates. Then Pt particles were electrodeposited onto the synthesized carbon materials. The OPC/Pt composite materials were modified onto the surface of GC electrode by means of chitosan. The modified electrode shows good response to H2O2due to the good electrochemical properties of the hybrid.
(3) In chapter4, natural basswood biomorphic porous carbon (BBPC) materials with unidirectional ordered pores have been successfully prepared by carbonization in an inert atmosphere. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM) were employed to characterize the phase identification, microstructure and morphology analysis. The carbon materials were used to fabricate biosensor to detect hydrogen peroxide (H2O2) without any assistance of enzymes because of their satisfying electrocatalytic properties. It was immobilized on glassy carbon electrode (GCE) with chitosan (CHIT) to fabricate a new kind of biosensor, BBPC/CHIT/GCE, which showed excellent electrocatalytic activity to the reduction of H2O2. Meanwhile, BBPC also could promote electron transfer with the help of hydroquinone. The simple and low-cost biosensor exhibited high sensitivity, good operational and long-term stability.
(4) In chapter5, a novel electrogenerated chemiluminescence (ECL) biosensor using gold nanoparticle as signal transduction probes was described for the detection of kinase activity. The gold nanoparticles were specifically conjugated to the thiophosphate group after phosphorylation process in the presence of adenosine59-[y-thio] triphosphate (ATP-y-s) co-substrate. Due to its good conductivity, large surface area and excellent electroactivity to luminol oxidization, the gold nanoparticles extremely amplified the ECL signal of luminol, offering a highly sensitive ECL biosensor for kinase activity detection. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar and lipid metabolism in human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of0.07U mL"1, wide linear rang (from0.07to32U mL"1) and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. Based on the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC50of ellagic acid, a PICA inhibitor, was estimated, which was in agreement with those characterized with conventional kinase assay. While nearly no ECL signal change can be observed in the presence of Tyrphostin AG1478, a tyrosine kinase inhibitor but not PKA inhibitor, showing its excellent performance in kinase inhibitor screening.
(5) In chapter6, we reported a novel cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca.0.05V vs. Ag/AgCl) with a strong light emission on the graphene modified glass carbon electrode. The resulted graphene modified electrode offers an excellent platform for high performance biosensing applications. Based on the cathodic ECL signal of luminol on the graphene modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab2). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Abi) due to it large surface area. The gold nanorods was not only used as carrier of secondary antibody (Ab2) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from10pg mL-1to8ng mL-1. The detection limit of PSA was8pg mL-1(signal-to-noise ratio of3). Moreover, the as-proposed low potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene based ECL immunosensors accurately detected PSA concentration in10human serum samples from patients demonstrated by excellent correlations with standard chemiluminescene immunoassay.
(1)在第2章中,我们研究发现双螺旋DNA的磷酸骨架可以作为多价金属离子或复合物的结合位点,并且在结合的过程中,这些多价离子会改变DNA的构象,形成压缩结构。利用这一性质,我们首先让氯金酸盐与双螺旋DNA通过静电力作用形成Au(Ⅲ)一DNA网状结构的复合物；然后,通过化学还原Au(Ⅲ)最终形成的Au.DNA网状结构的纳米复合物。该杂交材料有较高的生物相容性以及电子传导能力,有利于酶的固定。因此,我们将HRP固定在该复合材料上进行电化学研究,发现固载在该复合材料上的HRP表现出良好的直接电化学行为和电催化能力。
(2)在第3章中,我们利用碳化技术,制备了具有生物形态的有序多孔碳(OPC),并且通过电沉积技术在OPC的表面和孔穴内沉积金属Pt,形成OPC-Pt复合材料。详细地研究了OPC及OPC-Pt的结构形貌等特征,并通过对其电化学行为的研究发现,该复合材料具有较高的电催化性能并对H202具有明显的电催化活性,因此该复合材料可用于构建非酶传感器。
(3)在第4章中,同样以加工好的椴木块为原材料,通过碳化工艺,制备出了具有良好生物形态结构的椴木多孔碳(BBPC).我们利用XRD、红外光谱(FT-IR).扫描电镜(SEM)等研究手段对该材料的微观三维形态结构进行了详细的研究。同时在电化学实验中,我们意外的发现该多孔碳材料具有良好的酶促催化性能,并可以促进电子传递。另外,动力学研究发现该多孔碳材料对H202有良好的亲和性,这也就进一步证明了BBPC构建非酶传感器的可行性。因此,我们用BBPC代替HRP构建了一个检测H202的非酶电化学生物传感器。实验结果表明,该非酶传感器能够对H202进行快速灵敏的检测,同时具有较宽的线性范围和较低的检测限。
(4)在第5章中,我们设计了利用电化学发光技术(ECL)来对蛋白激酶活性及其抑制剂进行高灵敏检测的生物传感器。在共反应剂巯基化三磷酸腺苷(ATP-γ-s)的共在下,激酶催化磷酸化反应,然后以金纳米粒子为标记,与磷酸化过程中巯基化的磷酸根特异结合。由于金纳米粒子具有高的电导性、大的比表面积以及对鲁米诺(luminol)氧化的高电催化活性,因此可以极大的催化鲁米诺的电化学发光(ECL)信号增强,实现对激酶活性的高灵敏检测。蛋白激酶A(PKA)在人体的诸多生理及新陈代谢过程中发挥着重要作用,因此该酶被作为本研究的模型激酶进行理论上的检测分析。实验结果表明,该ECL传感器对PKA有较低的检测限(0.07UmL-1),宽的线性检测范围(0.07-32U mL-1)及良好的检测稳定性。除此之外,该ECL传感器还可用于激酶抑制剂的量化分析检测。在本实验中,我们测定了PKA抑制剂鞣花酸的IC50,发现该值与用传统分析技术所获得的检测结果相符。同时我们利用酪氨酸激酶抑制剂(Tyrphostin AG1478)重复上述检测过程,发现ECL响应几乎没有变化,这些结果说明ECL传感器可以用于对激酶抑制剂的筛选。
(5)在第6章中,通过研究我们首次发现,石墨烯(graphene)可以诱导鲁米诺在正电位(ca.0.05V vs.Ag/AgCl)产生较强而稳定的阴极ECL现象。同时,石墨烯修饰的玻碳电极是构建高性能生物传感器的优良平台。基于以上考虑,我们利用石墨烯修饰的玻碳电极构建了低电位下阴极ECL检测癌症标识物的夹心型ECL免疫传感器。葡萄糖氧化酶(GOx)和检测抗体(Ab2)修饰的金纳米棒(GNRs)作为该传感器的生物探针。功能化的石墨烯能够促进电极表面的电子转移；同时由于其大的比表面积,因此用于固载捕获抗体(Ab1)；功能化的GNRs不仅作为酶和抗体的固定载体,其自身对鲁米诺的ECL也具有增强作用,另外固载其上的GOx在反应体系中含有葡萄糖和氧气时,通过催化底物反应又可以进一步增强ECL信号。在以前列腺抗原(PSA)作为研究模型的条件下,我们所构建的低电位ECL免疫传感器对PSA能够进行高灵敏、专一性的检测,获得的线性检测范围为10pgmL-1-8ng mL-1,最低检测限为8pg mL-1,同时具有较高的稳定性和重复性。而且该传感器还被用于对10种不同PSA浓度的临床血清样品进行检测,分析结果与标准试剂盒检测获得结果进行对比发现,所构建的ECL免疫传感器可直接用于临床诊断分析。
Since the birth of micro-scale especially for nanomaterials, they are a hotspot in research and applications field all the time, which are now in the stage of rapid development. A variety of new nanomaterials with special functions are emerging in an endless stream, which expands further research and applications of these materials. Electrochemical apparatus because of some advantages such as sensitive, simple and easy to operate, good controllability, low cost and easy to be minisized, has received extensive attention. Nowadays, people found that when combining nanotechnology with electrochemical sensing technology, it not only made sensors in a revolutionary development but also promoted the development of nanotechnology. For those reasons, we investigate the preparation of carbon materials and noble metal nanomaterials, with electrocatalytic activity and high biocompatibility, and their applications in electroanalysis and sensing field.
(1) In chapter2, a novel DNA-templated Au nanoparticles (Au-DNA) nanoconjugate was prepared by using the combination of metallization and DNA compaction. The electrostatic interaction between Au(Ⅲ) and the phosphate backbone of DNA formed the netlike coordination compound of Au(Ⅲ)-DNA, and then the complex was chemically reduced to form Au nanoparticles in this network-like DNA conformation. The negatively charged nanoconjugate was used as the matrix for immobilization of horseradish peroxidase (HRP). A stable and well-defined redox peaks of HRP were observed on the Au-DNA nanoconjugate modified glassy carbon (GC) electrode, which indicated that the modified enzyme electrode displayed good direct electron transfer behavior and excellent reducing ability toward hydrogen peroxide (H2O2) with the apparent Michaelis-Menten constant (Km) estimated to be0.147mM.
(2) In chapter3, a biomorphic carbon, retained its anatomical features, was prepared by carbonizing wood through controlled heated rates. Then Pt particles were electrodeposited onto the synthesized carbon materials. The OPC/Pt composite materials were modified onto the surface of GC electrode by means of chitosan. The modified electrode shows good response to H2O2due to the good electrochemical properties of the hybrid.
(3) In chapter4, natural basswood biomorphic porous carbon (BBPC) materials with unidirectional ordered pores have been successfully prepared by carbonization in an inert atmosphere. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM) were employed to characterize the phase identification, microstructure and morphology analysis. The carbon materials were used to fabricate biosensor to detect hydrogen peroxide (H2O2) without any assistance of enzymes because of their satisfying electrocatalytic properties. It was immobilized on glassy carbon electrode (GCE) with chitosan (CHIT) to fabricate a new kind of biosensor, BBPC/CHIT/GCE, which showed excellent electrocatalytic activity to the reduction of H2O2. Meanwhile, BBPC also could promote electron transfer with the help of hydroquinone. The simple and low-cost biosensor exhibited high sensitivity, good operational and long-term stability.
(4) In chapter5, a novel electrogenerated chemiluminescence (ECL) biosensor using gold nanoparticle as signal transduction probes was described for the detection of kinase activity. The gold nanoparticles were specifically conjugated to the thiophosphate group after phosphorylation process in the presence of adenosine59-[y-thio] triphosphate (ATP-y-s) co-substrate. Due to its good conductivity, large surface area and excellent electroactivity to luminol oxidization, the gold nanoparticles extremely amplified the ECL signal of luminol, offering a highly sensitive ECL biosensor for kinase activity detection. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar and lipid metabolism in human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of0.07U mL"1, wide linear rang (from0.07to32U mL"1) and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. Based on the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC50of ellagic acid, a PICA inhibitor, was estimated, which was in agreement with those characterized with conventional kinase assay. While nearly no ECL signal change can be observed in the presence of Tyrphostin AG1478, a tyrosine kinase inhibitor but not PKA inhibitor, showing its excellent performance in kinase inhibitor screening.
(5) In chapter6, we reported a novel cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca.0.05V vs. Ag/AgCl) with a strong light emission on the graphene modified glass carbon electrode. The resulted graphene modified electrode offers an excellent platform for high performance biosensing applications. Based on the cathodic ECL signal of luminol on the graphene modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab2). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Abi) due to it large surface area. The gold nanorods was not only used as carrier of secondary antibody (Ab2) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from10pg mL-1to8ng mL-1. The detection limit of PSA was8pg mL-1(signal-to-noise ratio of3). Moreover, the as-proposed low potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene based ECL immunosensors accurately detected PSA concentration in10human serum samples from patients demonstrated by excellent correlations with standard chemiluminescene immunoassay.
引文
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