多种分子识别生物传感活性界面的构建及其应用研究
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摘要
经过半个世纪的快速发展,生物传感器已成为一种先进的检测方法和分析手段,在药物研发、食品安全、工业控制、临床诊断以及环境保护等领域中具有广阔的应用前景。在生物传感器构建过程中,最关键的环节是如何构建分子识别生物传感活性界面,它直接影响着生物传感器的检测灵敏度、最低检测限、线性范围以及稳定性等方面。因此,本文致力于采用不同的新型功能化材料和固定化技术,构建出基于压电石英晶体微天平的亲和型生物传感界面,以及能够检测过氧化氢、葡萄糖等重要小分子物质的电化学生物传感界面,并进一步对所制备的生物传感器在生物分析中的应用进行探讨。本文的主要研究内容如下：
(一)利用羧基化的多壁碳纳米管(MWCNTs)修饰石英晶体微天平铂基片(QCMPt基片),构建出能够实时监测白细胞介素-6(IL-6)及其可溶性受体(sIL-6R)之间相互作用的亲和型生物传感活性界面。通过实验分析可以获知IL-6与sIL-6R之间相互作用的动力学过程,进一步计算出两者的结合平衡常数(KA)和解离平衡常数(KD)分别为3.37×106M-1和2.97x10-7M。此结果与传统检测方法报道的一致,证实了该方法的可靠性和科学性。此外,已有报道证实IL-6及其受体是活血化瘀中药的潜在作用靶点之一,因此研究二者之间结合动力学过程可为我国传统中药作用机理探究提供一种新的研究方法,扩展了生物传感器的应用范围。
(二)将铂纳米颗粒(PtNPs)有效固定于经聚乙烯醇(PVA)分散的MWCNTs表面,制备出PVA-MWCNTs-PtNPs复合纳米材料,用于构建无酶过氧化氢(H2O2)生物传感活性界面。该传感器对H202的检测表现出宽的线性范围(0.002～3.8mM),显著的灵敏度(122.63μAmM-1cm-2),较低的检测限(0.7μM,S/N=3)以及快速的响应时间(5s内)。此外,传感器也表现出良好的重复性、长时间稳定性和较强的抗干扰性能。实验证明该构建方法可为其他类型的无酶电化学传感技术的创新开发提供可行的技术手段。
(三)以硫酸铜作为铜源,利用电化学沉积法在不同外界条件下制备一系列具有不同形貌结构的铜纳米材料,包括铜纳米颗粒、铜纳米块状、铜纳米树枝以及铜纳米花等,整个制备过程无需添加任何模板分子或催化剂,操作简单易行。分别用透射电子显微镜(TEM)和扫描电子显微镜(SEM)对多种纳米铜结构进行形态表征,并详细阐明了其形成机制。将铜纳米树枝修饰于玻碳(GC)电极表面,在碱性条件下研究了铜纳米树枝自身的氧化还原特征及其对葡萄糖的无酶催化行为。检测结果表明：铜纳米树枝对葡萄糖具有良好的电催化活性,在工作电压为0.55V vs. Ag/AgCl的条件下,所制备的传感器对葡萄糖检测有快速的电流响应、较低检测限和较宽的线性范围,并具有良好的稳定性、抗干扰性和重复性。
(四)以氯化铜作为铜前体,通过简单易行的两步电化学沉积法将氧化铜(CuO)纳米叶修饰于GC电极表面,构建基于CuO/GC电极的无酶传感器,并通过循环伏安法和计时电流法来研究其自身在0.1MNaOH溶液中的电化学响应特征,以及对葡萄糖和过氧化氢的电催化性能。结果表明,CuO/GC电极催化葡萄糖的氧化峰电位为0.55V,在此工作电压下对葡萄糖进行i-t曲线检测,得到较高的灵敏度(53.19NμAmM-1)、较宽的线性范围(12.5μM-4.29mM)以及较低的检测限(4.171xM)。该传感器对于过氧化氢的还原同样表现出优异的催化性能。
(五)采用高温还原法将金纳米颗粒(AuNPs)固定于经壳聚糖(CS)还原的还原型氧化石墨烯(RGO)表面,制备出CS-RGO-AuNPs纳米复合材料,将其作为葡萄糖氧化酶(GOD)的固定载体,构建检测葡萄糖的分子识别生物传感活性界面。结果表明,所制备的基于GOD/CS-RGO-AuNPs/Pt电极的传感器,在工作电压为0.5V vs. Ag/AgCl条件下,对葡萄糖的检测灵敏度为102.4μA.mM-1cm-2,最低检测限为1.7μM(S/N=3),线性范围为0.015～2.13mM,线性相关系数R2为0.988。此外,该传感器还具有良好的稳定性和抗干扰能力。
As an advanced monitoring method to develop biotechnology, biosensor is a multidisciplinary approach which is composed of molecular recognition component and transduction element. It has wide application prospects in pharmaceutical analysis, foodsafety, industry control, clinical diagnosis,biochip technology and environmental protection. The biosensor interface fabrication is a crucial step in biosensor design, which directly influences the electrochemical performance of biosensor, such as sensitivity, limit of detection, linearly range and stability etc. This thesis focus on developing new functional materials and immobilization technology for constructing quartz crystal microbalance (QCM) affinity biosensor and hydrogen peroxide, glucose biosensor, and investigate their applications in bioanalysis field. The main Research contents and results are as follows:
(a)A novel QCM affinity biosensor based carboxylated multi-walledcarbon nanotubes(MWCNTs) immobilized onto the platinum electrode for real time monitoring the interaction between interleukin-6(IL-6) and solubleinterleukin-6receptor (sIL-6R). The results showed that the apparent equilibrium association constant (Ka) and Dissociation Constant (KD) are respectively. This results are consist with traditional way's, revealing the reliability and scientificity of this method. Moreover, as a simple technology, this method not only provides a possibility for monitoring the interaction of biomacromolecules more efficiently, but also has broad application prospects in studies on the action mechanism of traditional Chinese medicine, which will greatly extend the biosensor's application range.
(b) The amperometric non-enzymatic hydrogen peroxide biosensor based on poly(vinylalcohol)(PVA)/MWCNTs/Platinum nanoparticles (PtNPs) nano-composite film was constructed. In this hybrid film, the PtNPs were electrochemical deposited onto the surface of MWCNTs which were non-covalentlyfunctionalizedby freezing-thawingPVA. Since the synergic effect of MWCNTs and PtNPs, the PVA-MWCNTs-PtNPs film modified electrode exhibited excellent electrocatalytic performance for H2O2, such as awide linear range(0.002-3.8mM), a remarkable sensitivity(122.63μA mM-1cm-2), a low detection limit (0.7μM) at the signal-to-noiseratio of3and a fast response time (within5s). In addition, the as-prepared sensor also showed long-termstability, excellentreproducibility and anti-interference performance. The current construction method canprovidea feasiblemeanand development platform to fabricate other non-enzymatic sensors.
(c) Aseriesof different morphology of copper nanomaterials, such as copper nano-particles, bulks, dendrites and flowers were prepared by direct electrochemical position process from coppersulfate (CuSO4)precursor through the change of reaction conditions. The morphology was characterized by transmission electron microscopy (TEM), scanning electron microscope (SEM), and X ray diffracmeter (XRD). The growth mechanism of copper structures was also illustrated in detail. The electrochemical performance of the copper nanodendrites and its oxidation catalytic activity towards glucose in NaOH solution were detected by electrochemical workstation. The results showed that the copper nanodendrites modified electrode exhibited good catalytic activity towards glucose in the absence of enzyme. At the work potential of0.55Vvs. Ag/AgCl, it displayed fast current response, low detection limit and wide detection range. Meanwhile, it possessed good stability, anti-interference, and repeatability.
(d) A novel enzyme-free biosensor based on copper oxide nanoleaves which were prepared through a film plating/potential cycling method (consecutive two-step electrodeposition). The resulting sensor showed excellent electrocatalytic activity to glucose and H2O2in0.1M NaOH solution. For the glucose detection, the sensor showed a high sensitivity of53.19μA mM-1, a wide linear range of12.5μM-4.29mM, and a low detection limit of4.17μM. Furthermore, the as-prepared sensor exhibited long-time stability, good anti-interference, and reproducibility.
(e) The reduction graphene oxide (RGO) was obtained through the reaction of chitosan (CS) and graphene oxide (GO) in the high temperature of90℃for10hours. Then the gold nanoparticles (AuNPs) were modified onto the surface of RGO through high temperature reduction. The resulted CS-RGO-AuNPs hybrids were employed to immobilize glucose oxidase (GOD). The glucose biosensor based on GOD/CS-RGO-AuNPs modified Pt electrode was developed and its electrochemical properties were detected by cyclicvoltammetry and chronoamperometry methods. At the work potential of0.5V vs. Ag/AgCl, it showed a high sensitivity of102.4μA mM"1cm-2, a low detection limit of1.7μM (S/N=3), and a wide linear range of0.015-2.13mM. This good performance is attributed to synergic effect of RGO and AuNPs, such as large specific surface area, rapid electron transferring etc. Moreover, the CS provided a suitable microenvironment to keep GOD bioactivity.
(一)利用羧基化的多壁碳纳米管(MWCNTs)修饰石英晶体微天平铂基片(QCMPt基片),构建出能够实时监测白细胞介素-6(IL-6)及其可溶性受体(sIL-6R)之间相互作用的亲和型生物传感活性界面。通过实验分析可以获知IL-6与sIL-6R之间相互作用的动力学过程,进一步计算出两者的结合平衡常数(KA)和解离平衡常数(KD)分别为3.37×106M-1和2.97x10-7M。此结果与传统检测方法报道的一致,证实了该方法的可靠性和科学性。此外,已有报道证实IL-6及其受体是活血化瘀中药的潜在作用靶点之一,因此研究二者之间结合动力学过程可为我国传统中药作用机理探究提供一种新的研究方法,扩展了生物传感器的应用范围。
(二)将铂纳米颗粒(PtNPs)有效固定于经聚乙烯醇(PVA)分散的MWCNTs表面,制备出PVA-MWCNTs-PtNPs复合纳米材料,用于构建无酶过氧化氢(H2O2)生物传感活性界面。该传感器对H202的检测表现出宽的线性范围(0.002～3.8mM),显著的灵敏度(122.63μAmM-1cm-2),较低的检测限(0.7μM,S/N=3)以及快速的响应时间(5s内)。此外,传感器也表现出良好的重复性、长时间稳定性和较强的抗干扰性能。实验证明该构建方法可为其他类型的无酶电化学传感技术的创新开发提供可行的技术手段。
(三)以硫酸铜作为铜源,利用电化学沉积法在不同外界条件下制备一系列具有不同形貌结构的铜纳米材料,包括铜纳米颗粒、铜纳米块状、铜纳米树枝以及铜纳米花等,整个制备过程无需添加任何模板分子或催化剂,操作简单易行。分别用透射电子显微镜(TEM)和扫描电子显微镜(SEM)对多种纳米铜结构进行形态表征,并详细阐明了其形成机制。将铜纳米树枝修饰于玻碳(GC)电极表面,在碱性条件下研究了铜纳米树枝自身的氧化还原特征及其对葡萄糖的无酶催化行为。检测结果表明：铜纳米树枝对葡萄糖具有良好的电催化活性,在工作电压为0.55V vs. Ag/AgCl的条件下,所制备的传感器对葡萄糖检测有快速的电流响应、较低检测限和较宽的线性范围,并具有良好的稳定性、抗干扰性和重复性。
(四)以氯化铜作为铜前体,通过简单易行的两步电化学沉积法将氧化铜(CuO)纳米叶修饰于GC电极表面,构建基于CuO/GC电极的无酶传感器,并通过循环伏安法和计时电流法来研究其自身在0.1MNaOH溶液中的电化学响应特征,以及对葡萄糖和过氧化氢的电催化性能。结果表明,CuO/GC电极催化葡萄糖的氧化峰电位为0.55V,在此工作电压下对葡萄糖进行i-t曲线检测,得到较高的灵敏度(53.19NμAmM-1)、较宽的线性范围(12.5μM-4.29mM)以及较低的检测限(4.171xM)。该传感器对于过氧化氢的还原同样表现出优异的催化性能。
(五)采用高温还原法将金纳米颗粒(AuNPs)固定于经壳聚糖(CS)还原的还原型氧化石墨烯(RGO)表面,制备出CS-RGO-AuNPs纳米复合材料,将其作为葡萄糖氧化酶(GOD)的固定载体,构建检测葡萄糖的分子识别生物传感活性界面。结果表明,所制备的基于GOD/CS-RGO-AuNPs/Pt电极的传感器,在工作电压为0.5V vs. Ag/AgCl条件下,对葡萄糖的检测灵敏度为102.4μA.mM-1cm-2,最低检测限为1.7μM(S/N=3),线性范围为0.015～2.13mM,线性相关系数R2为0.988。此外,该传感器还具有良好的稳定性和抗干扰能力。
As an advanced monitoring method to develop biotechnology, biosensor is a multidisciplinary approach which is composed of molecular recognition component and transduction element. It has wide application prospects in pharmaceutical analysis, foodsafety, industry control, clinical diagnosis,biochip technology and environmental protection. The biosensor interface fabrication is a crucial step in biosensor design, which directly influences the electrochemical performance of biosensor, such as sensitivity, limit of detection, linearly range and stability etc. This thesis focus on developing new functional materials and immobilization technology for constructing quartz crystal microbalance (QCM) affinity biosensor and hydrogen peroxide, glucose biosensor, and investigate their applications in bioanalysis field. The main Research contents and results are as follows:
(a)A novel QCM affinity biosensor based carboxylated multi-walledcarbon nanotubes(MWCNTs) immobilized onto the platinum electrode for real time monitoring the interaction between interleukin-6(IL-6) and solubleinterleukin-6receptor (sIL-6R). The results showed that the apparent equilibrium association constant (Ka) and Dissociation Constant (KD) are respectively. This results are consist with traditional way's, revealing the reliability and scientificity of this method. Moreover, as a simple technology, this method not only provides a possibility for monitoring the interaction of biomacromolecules more efficiently, but also has broad application prospects in studies on the action mechanism of traditional Chinese medicine, which will greatly extend the biosensor's application range.
(b) The amperometric non-enzymatic hydrogen peroxide biosensor based on poly(vinylalcohol)(PVA)/MWCNTs/Platinum nanoparticles (PtNPs) nano-composite film was constructed. In this hybrid film, the PtNPs were electrochemical deposited onto the surface of MWCNTs which were non-covalentlyfunctionalizedby freezing-thawingPVA. Since the synergic effect of MWCNTs and PtNPs, the PVA-MWCNTs-PtNPs film modified electrode exhibited excellent electrocatalytic performance for H2O2, such as awide linear range(0.002-3.8mM), a remarkable sensitivity(122.63μA mM-1cm-2), a low detection limit (0.7μM) at the signal-to-noiseratio of3and a fast response time (within5s). In addition, the as-prepared sensor also showed long-termstability, excellentreproducibility and anti-interference performance. The current construction method canprovidea feasiblemeanand development platform to fabricate other non-enzymatic sensors.
(c) Aseriesof different morphology of copper nanomaterials, such as copper nano-particles, bulks, dendrites and flowers were prepared by direct electrochemical position process from coppersulfate (CuSO4)precursor through the change of reaction conditions. The morphology was characterized by transmission electron microscopy (TEM), scanning electron microscope (SEM), and X ray diffracmeter (XRD). The growth mechanism of copper structures was also illustrated in detail. The electrochemical performance of the copper nanodendrites and its oxidation catalytic activity towards glucose in NaOH solution were detected by electrochemical workstation. The results showed that the copper nanodendrites modified electrode exhibited good catalytic activity towards glucose in the absence of enzyme. At the work potential of0.55Vvs. Ag/AgCl, it displayed fast current response, low detection limit and wide detection range. Meanwhile, it possessed good stability, anti-interference, and repeatability.
(d) A novel enzyme-free biosensor based on copper oxide nanoleaves which were prepared through a film plating/potential cycling method (consecutive two-step electrodeposition). The resulting sensor showed excellent electrocatalytic activity to glucose and H2O2in0.1M NaOH solution. For the glucose detection, the sensor showed a high sensitivity of53.19μA mM-1, a wide linear range of12.5μM-4.29mM, and a low detection limit of4.17μM. Furthermore, the as-prepared sensor exhibited long-time stability, good anti-interference, and reproducibility.
(e) The reduction graphene oxide (RGO) was obtained through the reaction of chitosan (CS) and graphene oxide (GO) in the high temperature of90℃for10hours. Then the gold nanoparticles (AuNPs) were modified onto the surface of RGO through high temperature reduction. The resulted CS-RGO-AuNPs hybrids were employed to immobilize glucose oxidase (GOD). The glucose biosensor based on GOD/CS-RGO-AuNPs modified Pt electrode was developed and its electrochemical properties were detected by cyclicvoltammetry and chronoamperometry methods. At the work potential of0.5V vs. Ag/AgCl, it showed a high sensitivity of102.4μA mM"1cm-2, a low detection limit of1.7μM (S/N=3), and a wide linear range of0.015-2.13mM. This good performance is attributed to synergic effect of RGO and AuNPs, such as large specific surface area, rapid electron transferring etc. Moreover, the CS provided a suitable microenvironment to keep GOD bioactivity.
引文
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