基于有序Au纳米线阵列的葡萄糖电化学生物传感器的制备及性能研究
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摘要
本论文以高度有序Au纳米线阵列为平台,采用不同的制备技术将葡萄糖氧化酶负载于Au纳米线阵列表面构筑高灵敏度的葡萄糖电化学生物传感器。一方面,选取合适的AAO模板和电解液,以AAO模板结合电沉积技术制备高度有序Au纳米线阵列；结果表明,相对于由H3B03和HAuCl4组成酸液电解液而言,以EDTA,Na2SO3,K2HPO4和]HAuCl4所组成的碱性电解液所制备的Au纳米线具有规则的形貌和粗糙的表面,更加有利于葡萄糖氧化酶的负载和所制备的葡萄糖电化学生物传感器的稳定性。另一方面,研究了不同制备技术对所制备的葡萄糖电化学生物传感器性能的影响,对不同制备技术条件下影响生物传感器性能的参数进行优化与分析,从而提高生物传感器对葡萄糖检测的性能,并结合FIA技术进一步提高传感器的检测效率,其具体研究内容如下：
首先,利用Au纳米线阵列大比表面积的特性,采用物理吸附法将葡萄糖氧化酶(GOx)吸附于Au纳米线阵列表面,并以全氟磺酸树脂(Nafion)覆盖于GOx修饰后的Au纳米线阵列表面以提高葡萄糖电化学生物传感器的稳定性。分别通过SEM、TEM、XRD以及CV对Au纳米线阵列的形貌、微观结构和电活性表面积进行表征,利用FITR对以Nafion包覆的GOx进行表征。对浸渍时间、GOx浓度、Nafion浓度以及电沉积时间等制备参数以及其测试参数进行优化,改善所制备的生物传感器对葡萄糖的电流响应。实验结果表明,高度有序Au纳米线阵列具有很高的电活性表面积,能够有效地加速反应过程中的电子传递。基于Nafion-GOx-AuNWAs的葡萄糖生物传感器对葡萄糖的检测具有高灵敏度(258.8μA·cm-2·mM-1),线性范围宽(10-3270μmol/L),低检测极限(0.2μmol/L),高的稳定性及抗干扰能力好等性能；同时采用物理吸附法制备的葡萄糖生物传感器具有较好的酶动力学响应,其Michaelis-Menten常数Kmapp为5.8mM。
其次,以电聚合吡咯的方式将GOx包埋于Au纳米线阵列表面的聚吡咯膜中制备葡萄糖电化学生物传感器。采用SEM以及TEM对Au纳米线的形貌进行观察,通过电聚合吡咯过程中的电势-时间曲线以及电化学交流阻抗谱(EIS)对GOx在聚吡咯膜中的负载情况进行分析,以吡咯单体为电解液和吡咯单体/GOx混合液为电解液进行电聚合生长曲线可以判断GOx被成功包埋于聚吡咯膜中,同时,不同电极的电化学交流阻抗谱对此进一步确认了GOx可以通过电聚合的方式而被负载于Au纳米线阵列表面。在对其制备参数优化的基础之上,在一定程度改善了生物传感器的性能,基于电聚合法制备的葡萄电化学生物传感器的灵敏度可达183.3pA·cm-2·mM-1,线性范围较宽为10-6140μmol/L,检测极限为0.5μmol/L.
再者,利用GLA和BSA共交联法将GOx固定于Au纳米线阵列表面构筑葡萄糖电化学生物传感器。分别利用SEM和TEM对Au纳米线阵列进行表征,并通过CV和EIS对基于交联法制备的葡萄糖电化学生物传感器的传质特性和界面结构进行研究。以计时电流法对不同的GLA浓度、BSA浓度、GOx浓度等参数条件下制备的葡萄糖电化学生物传感器的性能进行优化,电流响应结果显示,GLA-BSA-GOx-AuNWAs葡萄糖生物传感器对葡萄糖检测显示了较高的检测性能,其灵敏度高达379.0μA·cm-2·mM-1,线性范围为5-5000μmol/L,检测极限达0.05μmol/L。传感器稳定性研究结果显示,经过一个月的保存之后,该葡萄糖生物传感器仍保留90%的原始电流响应,显示传感器具有较好的稳定性。为了克服葡萄糖电化学生物传感器对测试溶液中溶氧的依赖,改善葡萄糖电化学生物传感器的电流响应以及稳定性,以交联法制备葡萄糖生物传感器过程中引入电子介体,将GOx与K3Fe(CN)6共同固定子Au纳米线阵列表面与纳米线之间。通过在常规测试环境和无氧测试环境中对不同条件下制备的葡萄糖电化学生物传感器的电流响应对K3Fe(CN)6的引入对生物传感器性能的作用进行分析,葡萄糖电化学生物传感器的构筑过程中引入铁氰化钾。一方面K3Fe(CN)6与O2共同作为葡萄糖催化反应过程中的电子受体,提高生物传感器的电流响应,另一方面,当测试溶液中的溶氧浓度不足或受到外界干扰时,K3Fe(CN)6将代替溶氧作为葡萄糖氧化酶催化葡萄糖反应过程中的电子受体,保证对葡萄糖检测的稳定性。由于K3Fe(CN)6的引入改变了GOx在Au纳米线阵列中存在的微环境,从而使制备参数对传感器性能的影响发生了较大的改变,在参数重新优化的基础之上,相对于无电子介体的葡萄糖生物传感器而言,基于电子介体的葡萄糖电化学生物传感的性能获得极大改善,所制备传感器的灵敏度高达548.1μA·cm-2·mM-1,线性范围为2.5-5400μmol/L,其理论检测极限为0.04umol/L。与此同时,传感器具有良好的酶动力学响应,其Michaelis-Menten常数Kmapp为5.6mM。
最后将基于Au纳米线阵列的葡萄糖生物传感器与流动注射分析(FIA)技术相结合,一方面基于Au纳米线阵列的葡萄糖生物传感器自身具有良好的综合性能,另一方面,FIA技术是一种非平衡态、快速测量的技术；将二者的优势结合之后,实现了对不同浓度葡萄糖的批量测试,大大提高了对葡萄糖浓度检测的效率。基于Au纳米线阵列的生物传感器对葡萄糖实际样品分析具有较高的回收率和较低的相对标准偏差,说明所制备的葡萄糖电化学生物传感器对葡萄糖的检测具有较高的可靠性。
In this dissertation, glucose oxidase is immobilized onto the surface of gold nanowire arrays by different methods to construct highly sensitive electrochemical biosensors for glucose detection. On the one hand, well aligned gold nanowire arrays have been synthesized by direct electrodeposition in conjunction with AAO template based on appropriate AAO templates and electrolytes. The results indicate that the gold nanowire arrays fabricated in alkaline solution containing EDTA, Na2SO3, K2HPO4and HAuCl4are prone to obtain uniform morphology and rough surface compared with acidic solution composing of H3BO3and HAuCl4, which is in favour of immobilization of glucose oxidase and the stability of glucose electrochemical biosensors. On the other hand, the influences of different techniques for immobilization of glucose oxidase on the performance of glucose electrochemical biosensors are also investigated and the parameters for constructing glucose electrochemical biosensors with different techniques are optimized to improve the performance of as-prepared biosensors. In addition, FIA is employed to determine glucose concentration in combination with the as-prepared glucose nanobiosensors to improve the efficiency for glucose detection. Glucose bisensors based on different techniques are elucidated in detail as follows.
Firstly, a great amount of glucose oxidase is adsorbed onto the surface of gold nanowire arrays due to their large specific surface area, a thin Nafion film is then cast onto the surface of GOx-modified gold nanowire arrays to improve the stability of as-prepard glucose biosensors in case of glucose oxidase loss during storage and measurements. SEM, TEM, XRD and CV are employed to characterize the morphology, microstructure and electroactive surface area of gold nanowire arrays, respectively. Furthermore, FTIR is also used to confirm whether GOx can be effectively immobilized or not by physical adsorption. Based on the optimum parameters such as immersing time, GOx solution concentration, Nafion solution concentration and electrodeposition time, as-prepared glucose biosensors achieve good performance with a high sensitivity of258.8μA·cm-2·mM-1for amperometric detection of glucose, while also achieving a low detection limit of0.2μM, and a wide linear range of10-3270μM, resulting from the accelaration of electron relay between active sites of GOx and gold nanowire arrays. Meanwhile, the presence of the two common interferants, uric acid (UA) and ascorbic acid (AA), has no effect on the performance of the glucose biosensor owing to selectivity of Nafion film. The apparent Michaelis-Menten constant Kmapp is calculated to be5.8mM, indicating an excellent affinity between immobilized GOx in the Nafion-GOx-AuNWA biosensor and the glucose in solution.
Secondly, glucose oxidase is entrapped into polypyrrole (PPy) film formed on the surface of gold nanowire arrays in the process of electropolymerization to fabricate glucose electrochemical biosensors. SEM and TEM are employed to observe the morphology of gold nanowire arrays, potential-time curves of polymerization and EIS are utilized to analyze glucose oxidase loading in the polypyrrole film. It is apparent that the potential-time curves for pyrrole only and pyrrole/GOx mixture solution are completely different, implying that GOx is successfully entrapped into polypyrrole film in the process of electropolymerization, and this is further confirmed by EIS for different electrodes. PPy-GOx-AuNWA-based glucose biosensors also obtain good performance towards glucose detection with optimized parameters. The sensitivity of as-prepared glucose biosensor is183.3μA·cm-2·mM-1, linear range and detection limit achieve10-6140μM and0.5μM, respectively.
Thirdly, cross-linking method is one of the most popular techniques to construct biosensors. In this thesis, two different kinds of glucose biosensors, the GLA-BSA-GOx-AuNWAs glucose biosensor and GLA-BSA-GOx-Med-AuNWAs glucose biosensor based on cross-linking method are introduced. As for GLA-BSA-GOx-AuNWAs glucose biosensor, the morphology of gold nanowire arrays is characterized by SEM and TEM, respectively. The mass transport through the biofilm made of GLA and BSA and interface features of GLA-BSA-GOx-AuNWAs glucose biosensor are also investigated by CV and EIS, respectively. Effect of parameters, such as GLA concentration, BSA concentration, GOx concentration etc. on the performance of GLA-BSA-GOx-AuNWAs glucose biosensors are studied and these parameters are optimized. GLA-BSA-GOx-AuNWAs glucose biosensors are sensitive to glucose, the sensitivity is as high as379.0μA·cm-2·mM-1. Linear range and limit of detection of GLA-BSA-GOx-AuNWAs glucose biosensors are5-5000μM and0.05μM, respectively. Furthermore,90%of the original amperometric response of the GLA-BSA-GOx-AuNWAs glucose biosensors can be maintained for over1month, indicating an excellent stability, due mainly to the3D nanostructure and the rough surface of the AuNWs.
In order to overcome the disadvantage of O2-dependence for aforementioned glucose biosensors, and to improve the amperometric responses and enhance stability of glucose biosensors, mediator K3Fe(CN)6is incorporated into GLA-BSA-GOx-AuNWAs glucose biosensors. Functions of K3Fe(CN)6are investigated by amperometric measurement under O2and N2atomsphere. The results indicate that both K3Fe(CN)6and O2accept electrons from FADH2due to the synergisitic effect so that amperometric responses of GLA-BSA-GOx-Med-AuNWAs glucose biosensors increase drastically. In addition, K3Fe(CN)6also accepts electrons from FADH2instead of O2once the dissolved O2is insufficient. Parameters for constructing GLA-BSA-GOx-Med-AuNWAs glucose biosensors change greatly due to the change of microenvironment for GOx on the surface of AuNWAs after incorporation of K3Fe(CN)6. Compared with GLA-BSA-GOx-AuNWAs glucose biosensors, the performance of the GLA-BSA-GOx-Med-AuNWAs glucose biosensors is much improved because of the incorporation of K3Fe(CN)6after parameter re-optimization. GLA-BSA-GOx-Med-AuNWAs glucose biosensors achieve a high sensitivity as much as548.1"1, a wider linear range between2.5and5400μM and a lower detection limit of0.04 μM. The apparent Michaelis-Menten constant Kmapp is calculated to be5.6mM, indicating an excellent affinity between immobilized GOx in the GLA-BSA-GOx-Med-AuNWAs biosensors and the glucose in solution and good enzymatic dynamic response.
Finally, glucose nanobiosensors in combination with FIA technique are employed to determine glucose concentration. On the one hand, glucose nanobiosensors have excellent performance with high sensitivity, wide linear range and low detection limit. On the other hand, FIA technique can analyze the concentration of analyte quickly and continuously. By combining the advantages of glucose nanobiosensors and FIA technique, efficiency for glucose detection is improved significantly. Experimental results indicate that Au-based glucose biosensors show high recovery rate and low relative stardard deviation (RSD), suggesting good reliability of as-prepard glucose biosensors towards glucose detection.
首先,利用Au纳米线阵列大比表面积的特性,采用物理吸附法将葡萄糖氧化酶(GOx)吸附于Au纳米线阵列表面,并以全氟磺酸树脂(Nafion)覆盖于GOx修饰后的Au纳米线阵列表面以提高葡萄糖电化学生物传感器的稳定性。分别通过SEM、TEM、XRD以及CV对Au纳米线阵列的形貌、微观结构和电活性表面积进行表征,利用FITR对以Nafion包覆的GOx进行表征。对浸渍时间、GOx浓度、Nafion浓度以及电沉积时间等制备参数以及其测试参数进行优化,改善所制备的生物传感器对葡萄糖的电流响应。实验结果表明,高度有序Au纳米线阵列具有很高的电活性表面积,能够有效地加速反应过程中的电子传递。基于Nafion-GOx-AuNWAs的葡萄糖生物传感器对葡萄糖的检测具有高灵敏度(258.8μA·cm-2·mM-1),线性范围宽(10-3270μmol/L),低检测极限(0.2μmol/L),高的稳定性及抗干扰能力好等性能；同时采用物理吸附法制备的葡萄糖生物传感器具有较好的酶动力学响应,其Michaelis-Menten常数Kmapp为5.8mM。
其次,以电聚合吡咯的方式将GOx包埋于Au纳米线阵列表面的聚吡咯膜中制备葡萄糖电化学生物传感器。采用SEM以及TEM对Au纳米线的形貌进行观察,通过电聚合吡咯过程中的电势-时间曲线以及电化学交流阻抗谱(EIS)对GOx在聚吡咯膜中的负载情况进行分析,以吡咯单体为电解液和吡咯单体/GOx混合液为电解液进行电聚合生长曲线可以判断GOx被成功包埋于聚吡咯膜中,同时,不同电极的电化学交流阻抗谱对此进一步确认了GOx可以通过电聚合的方式而被负载于Au纳米线阵列表面。在对其制备参数优化的基础之上,在一定程度改善了生物传感器的性能,基于电聚合法制备的葡萄电化学生物传感器的灵敏度可达183.3pA·cm-2·mM-1,线性范围较宽为10-6140μmol/L,检测极限为0.5μmol/L.
再者,利用GLA和BSA共交联法将GOx固定于Au纳米线阵列表面构筑葡萄糖电化学生物传感器。分别利用SEM和TEM对Au纳米线阵列进行表征,并通过CV和EIS对基于交联法制备的葡萄糖电化学生物传感器的传质特性和界面结构进行研究。以计时电流法对不同的GLA浓度、BSA浓度、GOx浓度等参数条件下制备的葡萄糖电化学生物传感器的性能进行优化,电流响应结果显示,GLA-BSA-GOx-AuNWAs葡萄糖生物传感器对葡萄糖检测显示了较高的检测性能,其灵敏度高达379.0μA·cm-2·mM-1,线性范围为5-5000μmol/L,检测极限达0.05μmol/L。传感器稳定性研究结果显示,经过一个月的保存之后,该葡萄糖生物传感器仍保留90%的原始电流响应,显示传感器具有较好的稳定性。为了克服葡萄糖电化学生物传感器对测试溶液中溶氧的依赖,改善葡萄糖电化学生物传感器的电流响应以及稳定性,以交联法制备葡萄糖生物传感器过程中引入电子介体,将GOx与K3Fe(CN)6共同固定子Au纳米线阵列表面与纳米线之间。通过在常规测试环境和无氧测试环境中对不同条件下制备的葡萄糖电化学生物传感器的电流响应对K3Fe(CN)6的引入对生物传感器性能的作用进行分析,葡萄糖电化学生物传感器的构筑过程中引入铁氰化钾。一方面K3Fe(CN)6与O2共同作为葡萄糖催化反应过程中的电子受体,提高生物传感器的电流响应,另一方面,当测试溶液中的溶氧浓度不足或受到外界干扰时,K3Fe(CN)6将代替溶氧作为葡萄糖氧化酶催化葡萄糖反应过程中的电子受体,保证对葡萄糖检测的稳定性。由于K3Fe(CN)6的引入改变了GOx在Au纳米线阵列中存在的微环境,从而使制备参数对传感器性能的影响发生了较大的改变,在参数重新优化的基础之上,相对于无电子介体的葡萄糖生物传感器而言,基于电子介体的葡萄糖电化学生物传感的性能获得极大改善,所制备传感器的灵敏度高达548.1μA·cm-2·mM-1,线性范围为2.5-5400μmol/L,其理论检测极限为0.04umol/L。与此同时,传感器具有良好的酶动力学响应,其Michaelis-Menten常数Kmapp为5.6mM。
最后将基于Au纳米线阵列的葡萄糖生物传感器与流动注射分析(FIA)技术相结合,一方面基于Au纳米线阵列的葡萄糖生物传感器自身具有良好的综合性能,另一方面,FIA技术是一种非平衡态、快速测量的技术；将二者的优势结合之后,实现了对不同浓度葡萄糖的批量测试,大大提高了对葡萄糖浓度检测的效率。基于Au纳米线阵列的生物传感器对葡萄糖实际样品分析具有较高的回收率和较低的相对标准偏差,说明所制备的葡萄糖电化学生物传感器对葡萄糖的检测具有较高的可靠性。
In this dissertation, glucose oxidase is immobilized onto the surface of gold nanowire arrays by different methods to construct highly sensitive electrochemical biosensors for glucose detection. On the one hand, well aligned gold nanowire arrays have been synthesized by direct electrodeposition in conjunction with AAO template based on appropriate AAO templates and electrolytes. The results indicate that the gold nanowire arrays fabricated in alkaline solution containing EDTA, Na2SO3, K2HPO4and HAuCl4are prone to obtain uniform morphology and rough surface compared with acidic solution composing of H3BO3and HAuCl4, which is in favour of immobilization of glucose oxidase and the stability of glucose electrochemical biosensors. On the other hand, the influences of different techniques for immobilization of glucose oxidase on the performance of glucose electrochemical biosensors are also investigated and the parameters for constructing glucose electrochemical biosensors with different techniques are optimized to improve the performance of as-prepared biosensors. In addition, FIA is employed to determine glucose concentration in combination with the as-prepared glucose nanobiosensors to improve the efficiency for glucose detection. Glucose bisensors based on different techniques are elucidated in detail as follows.
Firstly, a great amount of glucose oxidase is adsorbed onto the surface of gold nanowire arrays due to their large specific surface area, a thin Nafion film is then cast onto the surface of GOx-modified gold nanowire arrays to improve the stability of as-prepard glucose biosensors in case of glucose oxidase loss during storage and measurements. SEM, TEM, XRD and CV are employed to characterize the morphology, microstructure and electroactive surface area of gold nanowire arrays, respectively. Furthermore, FTIR is also used to confirm whether GOx can be effectively immobilized or not by physical adsorption. Based on the optimum parameters such as immersing time, GOx solution concentration, Nafion solution concentration and electrodeposition time, as-prepared glucose biosensors achieve good performance with a high sensitivity of258.8μA·cm-2·mM-1for amperometric detection of glucose, while also achieving a low detection limit of0.2μM, and a wide linear range of10-3270μM, resulting from the accelaration of electron relay between active sites of GOx and gold nanowire arrays. Meanwhile, the presence of the two common interferants, uric acid (UA) and ascorbic acid (AA), has no effect on the performance of the glucose biosensor owing to selectivity of Nafion film. The apparent Michaelis-Menten constant Kmapp is calculated to be5.8mM, indicating an excellent affinity between immobilized GOx in the Nafion-GOx-AuNWA biosensor and the glucose in solution.
Secondly, glucose oxidase is entrapped into polypyrrole (PPy) film formed on the surface of gold nanowire arrays in the process of electropolymerization to fabricate glucose electrochemical biosensors. SEM and TEM are employed to observe the morphology of gold nanowire arrays, potential-time curves of polymerization and EIS are utilized to analyze glucose oxidase loading in the polypyrrole film. It is apparent that the potential-time curves for pyrrole only and pyrrole/GOx mixture solution are completely different, implying that GOx is successfully entrapped into polypyrrole film in the process of electropolymerization, and this is further confirmed by EIS for different electrodes. PPy-GOx-AuNWA-based glucose biosensors also obtain good performance towards glucose detection with optimized parameters. The sensitivity of as-prepared glucose biosensor is183.3μA·cm-2·mM-1, linear range and detection limit achieve10-6140μM and0.5μM, respectively.
Thirdly, cross-linking method is one of the most popular techniques to construct biosensors. In this thesis, two different kinds of glucose biosensors, the GLA-BSA-GOx-AuNWAs glucose biosensor and GLA-BSA-GOx-Med-AuNWAs glucose biosensor based on cross-linking method are introduced. As for GLA-BSA-GOx-AuNWAs glucose biosensor, the morphology of gold nanowire arrays is characterized by SEM and TEM, respectively. The mass transport through the biofilm made of GLA and BSA and interface features of GLA-BSA-GOx-AuNWAs glucose biosensor are also investigated by CV and EIS, respectively. Effect of parameters, such as GLA concentration, BSA concentration, GOx concentration etc. on the performance of GLA-BSA-GOx-AuNWAs glucose biosensors are studied and these parameters are optimized. GLA-BSA-GOx-AuNWAs glucose biosensors are sensitive to glucose, the sensitivity is as high as379.0μA·cm-2·mM-1. Linear range and limit of detection of GLA-BSA-GOx-AuNWAs glucose biosensors are5-5000μM and0.05μM, respectively. Furthermore,90%of the original amperometric response of the GLA-BSA-GOx-AuNWAs glucose biosensors can be maintained for over1month, indicating an excellent stability, due mainly to the3D nanostructure and the rough surface of the AuNWs.
In order to overcome the disadvantage of O2-dependence for aforementioned glucose biosensors, and to improve the amperometric responses and enhance stability of glucose biosensors, mediator K3Fe(CN)6is incorporated into GLA-BSA-GOx-AuNWAs glucose biosensors. Functions of K3Fe(CN)6are investigated by amperometric measurement under O2and N2atomsphere. The results indicate that both K3Fe(CN)6and O2accept electrons from FADH2due to the synergisitic effect so that amperometric responses of GLA-BSA-GOx-Med-AuNWAs glucose biosensors increase drastically. In addition, K3Fe(CN)6also accepts electrons from FADH2instead of O2once the dissolved O2is insufficient. Parameters for constructing GLA-BSA-GOx-Med-AuNWAs glucose biosensors change greatly due to the change of microenvironment for GOx on the surface of AuNWAs after incorporation of K3Fe(CN)6. Compared with GLA-BSA-GOx-AuNWAs glucose biosensors, the performance of the GLA-BSA-GOx-Med-AuNWAs glucose biosensors is much improved because of the incorporation of K3Fe(CN)6after parameter re-optimization. GLA-BSA-GOx-Med-AuNWAs glucose biosensors achieve a high sensitivity as much as548.1"1, a wider linear range between2.5and5400μM and a lower detection limit of0.04 μM. The apparent Michaelis-Menten constant Kmapp is calculated to be5.6mM, indicating an excellent affinity between immobilized GOx in the GLA-BSA-GOx-Med-AuNWAs biosensors and the glucose in solution and good enzymatic dynamic response.
Finally, glucose nanobiosensors in combination with FIA technique are employed to determine glucose concentration. On the one hand, glucose nanobiosensors have excellent performance with high sensitivity, wide linear range and low detection limit. On the other hand, FIA technique can analyze the concentration of analyte quickly and continuously. By combining the advantages of glucose nanobiosensors and FIA technique, efficiency for glucose detection is improved significantly. Experimental results indicate that Au-based glucose biosensors show high recovery rate and low relative stardard deviation (RSD), suggesting good reliability of as-prepard glucose biosensors towards glucose detection.
引文
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