基于纳米材料构建过氧化氢生物传感器的研究
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摘要
过氧化氢不仅是许多高选择性氧化酶的催化反应产物,又是食品、药物、环境分析中的重要成份。因此,快速、准确的检测过氧化氢具有非常重要的意义。目前用于检测过氧化氢的方法很多,如滴定法、分光光度法、化学发光法、高效液相色谱法以及电化学方法。其中,电流型酶生物传感器,由于其方法简单、灵敏度高以及选择性高等优点而被广泛应用于过氧化氢的测定。酶的氧化还原活性中心与电极间的电子传递是制备酶电化学传感器的基础,可以通过两种不同的途径得以实现。其一是借助电子介体,如何将电子介体有效固定于电极上,而不从电极的修饰层渗漏出来是非常重要的;其二是借助适合的材料实现酶与电极之间的直接电子传递。另一方面,生物活性分子的固定化是构筑生物传感器最关键的步骤,是影响生物传感器的稳定性、灵敏度和选择性的关键因素。基于此,本文利用比表面积大、表面自由能高的纳米金和甲苯胺蓝层层组装,利用戊二醛作交联剂使牛血清白蛋白与硫堇共价结合以实现了电子介体的有效固定,从而构建了性能优良的介体型过氧化氢传感器。同时,还对碳纳米管与纳米金、碳纳米管与核—壳型纳米球的复合物作为固酶基质在生物传感器领域的应用做了初步的探索和研究,基于酶在复合纳米材料基质上的直接电化学构建了无介体型的第三代过氧化氢传感器。具体研究工作如下:
1.基于层层自组装纳米金/甲苯胺蓝膜修饰的过氧化氢生物传感器的研究
在金电极表面自组装一层半胱氨酸,然后吸附纳米金(nano-Au),以此为基底,通过静电吸附作用和金—氮共价键合作用层层自组装nano-Au和甲苯胺蓝(TB),构建了以多层{nano-Au/TB}_n膜为载体固定辣根过氧化物酶(HRP)的过氧化氢生物传感器。用循环伏安法和石英晶体微天平(QCM)技术对{nano-Au/TB}_n膜的组装过程进行了表征,并用原子力显微镜(AFM)对膜的表面形貌进行了表征。探讨了工作电位、温度、pH值对电极响应的影响,考察了电极的重现性、稳定性及抗干扰能力。该传感器具有响应快、线性范围宽、检出限低等特点。对H_2O_2响应的线性范围为1.5x10~(-7)mol/L～8.6×10~(-3)mol/L,检测限为7.0x10~(-8)mol/L(S/N=3)。
2.基于硫堇-牛血清白蛋白复合物和纳米金固定HRP的过氧化氢传感器的研究
本文以戊二醛(GA)为交联剂,首先将硫堇(Th)与牛血清白蛋白(BSA)共价交联制备硫堇-牛血清白蛋白复合物(Th-BSA),然后用硫堇的自由氨基(-NH_2)固定纳米金(nano-Au),进一步再吸附HRP,从而构建出一种新型的过氧化氢生物传感器。nano-Au/Th-BSA复合物具有良好的生物相容性,可提供良好的微环境保持酶的生物活性。固定于复合物中的硫堇能在HRP与电极间有效地传递电子。利用原子力显微镜(AFM)、X射线光电子能谱(XPS)、红外光谱仪(IR)和电化学交流阻抗(EIS)表征了整个组装过程。该传感器对H_2O_2具有好的催化响应,且响应快。在优化的实验条件下,所制备的传感器对H_2O_2的线性范围为4.9x10~(-7)～1.6x10~(-3)mol/L,检测限为2.1×10~(-7)mol/L(S/N=3),表观米氏常数为0.023 mmol/L。
3.基于多壁碳纳米管和纳米金固定血红蛋白的第三代过氧化氢传感器的研究
本文以蛋白质为联接剂,通过静电吸附作用制得了碳纳米管和纳米金的复合材料。首先将纯化的多壁碳纳米管(MWNTs)修饰于玻碳电极(GC)表面,构建负电荷的修饰界面,基于静电吸附作用固定血红蛋白(Hb),然后再吸附纳米金(nano-Au),最后利用nano-Au再固定一层Hb,从而成功构建了基于MWNTs和nano-Au固定Hb的第三代过氧化氢传感器(Hb/nano-Au/Hb/MWNTs/GC)。用循环伏安法(CV)、电化学交流阻抗(EIS)以及透射电子显微镜(TEM)对修饰电极进行了表征。Hb在修饰电极上实现了直接电子转移。与仅基于碳纳米管构建的H_2O_2传感器相比,该传感器对H_2O_2的响应具有更宽的线性范围和更低的检测下限。其线性范围为2.1×10~(-7)～3.0x10~(-3)mol/L,检测限为8.0x10~(-8)mol/L(S/N=3),表观米氏常数为0.26mmol/L。此外,该传感器还具有较快的响应速率、较好的稳定性和重现性。
4.基于多壁碳纳米管/壳聚糖复合物及多层纳米金,血红蛋白修饰的过氧化氢传感器
将多壁纳米碳管(MWNTs)分散于壳聚糖(CS)中得到稳定的CS-MWNTs复合物,然后将其滴涂于玻碳(GC)电极表面。利用CS丰富的氨基固定nano-Au,进一步静电吸附血红蛋白(Hb)。利用层层自组装技术将带相反电荷的nano-Au和Hb固定于CS-MWNTs复合物上制备了基于MWNTs和多层Hb/nano-Au膜修饰电极。用循环伏安法(CV)、紫外可见(UV-vis)吸收光谱法以及透射电子显微镜(TEM)对修饰电极进行了表征。研究了传感器对H_2O_2的响应及动力学性质,表观米氏常数为0.19 mmol/L,线性响应范围为5.0x10~(-7)～2.0x10~(-3)mol/L,检测限为2.1×10~(-7)mol/L(S/N=3)。同时研究了pH值及应用电位对H_2O_2传感器的影响。
5.基于有机硅/壳聚糖核-壳纳米球和多壁碳纳米管复合物固定HRP的过氧化氢传感器
本文以多壁碳纳米管(MWNTs)和核-壳结构的有机硅/壳聚糖纳米球(organosilica@chitosan)复合物作为固酶基质,构建了一种新型的无介体型过氧化氢生物传感器。首先将MWNTs分散于荷正电荷的organosilica@chitosan的HAc悬浮液中,制备organosilica@chitosan/MWNTs复合材料,并将其直接滴涂在玻碳电极(GCE)表面。然后通过organosilica@chitosan与带相反电荷的辣根过氧化物酶(HRP)的静电吸附作用固定HRP制得HRP/organosilica@chitosan/MWNTs修饰电极。该传感器对H_2O_2具有较好的电催化活性。其线性范围为7.0x10~(-7)～2.8×10~(-3)mol/L,检测限为2.5x10~(-7)mol/L(S/N=3),表观米式常数为0.32 mmol/L。另外,该传感器对H_2O_2还具有较快的响应速度,较好的稳定性和重现性。将其应用于消毒液的检测时,其结果也令人满意。
The rapid and accurate determination of hydrogen peroxide(H_2O_2)is of great importance because it is not only the product of the reactions catalyzed by many highly selective oxidases but also an essential compound in food,pharmaceutical and environmental analyses.Among these techniques employed for hydrogen peroxide analysis,such as titrimetry,photometry, chemiluminescence,high performance liquid chromatography and electrochemistry,amperometric enzyme-based biosensors have received considerable interest,because this class of technique is characterized by sensitivity,convenience and high selectivity.Electron transfer between an electrode and the redox activity center of an enzy me is the basis for developing various enzyme-based biosensors.It can be achieved through two different pathways.One involves electron-shuttling mediators to establish an electrical communication between redox proteins and the underlying electrodes.In this scheme,there are still several challenges to keep mediators from diffusing away from the electrode surface into the bulk solution.The other pathway involves direct electron transfer between redox proteins and the electrode.On the other hand,the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability,sensitivity and selectivity of biosensors.In this paper,nano-Au,with large surface area,high-surface free energy, was chosen to immobilize toluidine blue by layer-by-layer(LBL)assembly technique,and thionine was covalently bound onto the bovine serum albumin film with glutaraldehyde as cross-linker to achieve the immobilization of electron-shuttling mediators.For the leakage significantly decreases, the proposed mediator-based hydrogen peroxide biosensors exhibit good analytical performance.In addition,attention has been paid to the nano-Au/MWNTs composite and core-shell organosilica@chitosan/MWNTs composite.The application of these composites as an immobilization matrix for fabricating mediator-free hydrogen peroxide biosensors has been preliminarily investigated.The main points of this dissertation are summarized as follows:
Part one:Study of the hydrogen peroxide biosensor based on the layer-by-layer assembly films of gold colloidal nanoparticles and toluidine blue
The precursor film was first formed on the Au electrode surface based on the self-assembly of L-cysteine and the adsorption of gold colloidal nan oparticles(nano-Au).Layer-by-layer(LBL) assembly films of toluidine blue(TB)and nano-Au were fabricated by Au-N covalent bond and electrostatic adsorption between TB and nano-Au.Finally,horseradish peroxidase(HRP)was assembled onto {nano-Au/TB}_nmultilayer films to fabricate a novel hydrogen peroxide biosensor. Cyclic voltammeter(CV)and quartz crystal microbalance(QCM)were adopted to monitor the regular growth of {nano-Au/TB} bilayer films.Morphologies of the films were characterized with atomic force microscopy(AFM).The effects of applied potential,temperature and pH on the current response were investigated.Repeatability,stability and anti-interference were also researched.The proposed biosensor responds rapidly to H_2O_2 in the linear range from 1.5×10~(-7)mol/L to 8.6×10~(-3) mol/L with a detection limit of 7.0×10~(-8)mol/L(S/N=3).
Part two:Study of the hydrogen peroxide biosensor based on immobilizing HRP on thionine-bovine serum albumin conjugate and gold colloidal nanoparticles
A novel enzyme immobilization technique based on thionine-bovine serum albumin conjugate (Th-BSA)and gold colloidal nanoparticles(nano-Au)was developed.Thionine was covalently bound onto the BSA film with glutaraldehyde(GA)as cross-linker to achieve Th-BSA conjugate. The free amino groups of thionine were then used to attach nano-Au for the immobilization of horseradish peroxidase(HRP).Such nano-Au/Th-BSA matrix shows a favorable microenviment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme.Several techniques,including atomic force microscopy(AFM),X-ray photoelectron spectroscopy(XPS),Infrared spectra(IR)and electrochemical impedance spectroscopy(EIS)have been employed to characterize the assembly process.The proposed biosensor displays excellent catalytic activity and rapid response for H_2O_2. The linear range for the determination of H_O_2 is from 4.9×10~(-7)to 1.6×10~(-3)mol/L with a detection limit of 2.1×10~(-7)mol/L at 30 and a Michaelies-Menten constant value of 0.023 mmol/L.
Part three:Amperometric third-generation hydrogen peroxide biosensors based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles
A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes(MWNTs)and gold colloidal nanoparticles(nano-Au)by using proteins as linker is proposed.In such a strategy,Hemoglobin(Hb)was selected as model protein to fabricate third-generation H_2O_2 biosensor based on MWNTs and nano-Au.Acid-pretreated,negatively charged MWNTs was first modified on the surface of glassy carbon(GC)electrode,then,positively charged Hb was adsorbed onto MWNTs films by electrostatic interaction.Finally,nano-Au and Hb were successively assembled onto the modified electrode to obtain the Hb/nano-Au/Hb/MWNTs/GC electrode.The assembly of Hb and nano-Au was characterized with cyclic voltammetry(CV), electrochemical impedance spectroscopy(EIS)and transmission electron microscopy(TEM).The direct electron transfer of Hb is observed on the Hb/nano-Au/Hb/MWNTs/GC electrode,which exhibits excellent electrocatalytic activity for the reduction of H_2O_2 to construct a third generation mediator-free H_2O_2 biosensor.As compared to those H_2O_2 biosensors only based on carbon nanotubes,the proposed biosensor modified with MWNTs and nano-Au displays a broader linear range and a lower detection limit for H_2O_2 determination.The linear range is from 2.1×10~(-7)to 3.0×10~(-3)mol/L with a detection limit of 8.0×10~(-8)mol/L at 3σ.The Michaelies-Menten constant K_M~(app)value is estimated to be 0.26 mmol/L.Moreover,this biosensor displays rapid response to H_2O_2 and possesses good stability and reproducibility.
Part four:A hydrogen peroxide biosensor based on multi-wall carbon nanotubes/chitosan composite and multilayer films of hemoglobin and colloidal gold nanoparticles
An amperometric biosensor for H_2O_2 was developed based on multilayer assembly of hemoglobin(Hb)and colloidal gold nanoparticles(nano-Au)on multi-wall carbon nanotubes/chitosan composite.Chitosan(CS)was chosen for dispersing multi-wall carbon nanotubes(MWNTs)to form a stable CS-MWNTs composite.This composite was first coated on the surface of glassy carbon electrode to provide a containing amino groups interface for assembling nano-Au,followed by the adsorption of Hb to form a bilayer of {Hb/nano-Au}.Repeating the assembly step of nano-Au and Hb resulted in {Hb/nano-Au}_n multilayers.The resulting system brought a new platform for electrochemical devices by using the synergistic action of the electrocatalytic activity of nano-Au and MWNTs.The assembly of nano-Au onto CS-MWNTs was confirmed by transmission electron microscopy.The consecutive growth of {Hb/nano-Au}_n multilayers was confirmed by cyclic voltammetry and UV-vis absorption spectroscopy.The resulting electrode displays excellent electrocatalytic activity and rapid response for H_2O_2.The linear range for the determination of H_2O_2 is from 5.0×10~(-7)to 2.0×10~(-3)mol/L with a detection limit of 2.1×10~(-7)mol/L at 3σand a Michaelies-Menten constant K_M~(app)value of 0.19 mmol/L.At the same time,the effects of applied potential and pH on the sensor were examined.
Part five:A hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on core-shell organosilica@chitosan nanospheres and multiwall carbon nanotubes composites
The application of a composite of multiwall carbon nanotubes(MWNTs)and core-shell organosilica@chitosan crosslinked nanospheres as an immobilization matrix for a construction of a novel mediator-free amperometric hydrogen peroxide(H_2O_2)biosensor was described.MWNTs were dispersed in a suspension of positively charged organosilica@chitosan nanospheres in acetic acid solution(0.6 wt%)to achieve an organosilica@chitosan/MWNTs composite,which was cast onto a glass carbon electrode(GCE)surface directly.And then,horseradish peroxidase(HRP),as a model enzyme,was immobilized onto it through electrostatic interaction between oppositely charged organosilica@chitosan nanospheres and HRP.The direct electron transfer of HRP is achieved at the HRP/organosilica@chitosan/MWNTs/GCE,which exhibits an excellent electrocatalytic activity for the reduction of H_2O_2.The catalysis currents increased linearly to H_2O_2 concentration in a wide range of 7.0×10~(-7)tO 2.8×10~(-3)mol/L with a detection limit,of 2.5×10~(-7)mol/L at 3σ.A Michaelies-Menten constant K_M~(app)value is estimated to be 0.32 mmol/L,indicating a high-catalytic activity of HRP.Moreover,the proposed biosensor displays rapid response to H_2O_2 and possesses good stability and reproducibility.When used to detect H_2_2 concentration in disinfector sample,it shows satisfactory results.
1.基于层层自组装纳米金/甲苯胺蓝膜修饰的过氧化氢生物传感器的研究
在金电极表面自组装一层半胱氨酸,然后吸附纳米金(nano-Au),以此为基底,通过静电吸附作用和金—氮共价键合作用层层自组装nano-Au和甲苯胺蓝(TB),构建了以多层{nano-Au/TB}_n膜为载体固定辣根过氧化物酶(HRP)的过氧化氢生物传感器。用循环伏安法和石英晶体微天平(QCM)技术对{nano-Au/TB}_n膜的组装过程进行了表征,并用原子力显微镜(AFM)对膜的表面形貌进行了表征。探讨了工作电位、温度、pH值对电极响应的影响,考察了电极的重现性、稳定性及抗干扰能力。该传感器具有响应快、线性范围宽、检出限低等特点。对H_2O_2响应的线性范围为1.5x10~(-7)mol/L～8.6×10~(-3)mol/L,检测限为7.0x10~(-8)mol/L(S/N=3)。
2.基于硫堇-牛血清白蛋白复合物和纳米金固定HRP的过氧化氢传感器的研究
本文以戊二醛(GA)为交联剂,首先将硫堇(Th)与牛血清白蛋白(BSA)共价交联制备硫堇-牛血清白蛋白复合物(Th-BSA),然后用硫堇的自由氨基(-NH_2)固定纳米金(nano-Au),进一步再吸附HRP,从而构建出一种新型的过氧化氢生物传感器。nano-Au/Th-BSA复合物具有良好的生物相容性,可提供良好的微环境保持酶的生物活性。固定于复合物中的硫堇能在HRP与电极间有效地传递电子。利用原子力显微镜(AFM)、X射线光电子能谱(XPS)、红外光谱仪(IR)和电化学交流阻抗(EIS)表征了整个组装过程。该传感器对H_2O_2具有好的催化响应,且响应快。在优化的实验条件下,所制备的传感器对H_2O_2的线性范围为4.9x10~(-7)～1.6x10~(-3)mol/L,检测限为2.1×10~(-7)mol/L(S/N=3),表观米氏常数为0.023 mmol/L。
3.基于多壁碳纳米管和纳米金固定血红蛋白的第三代过氧化氢传感器的研究
本文以蛋白质为联接剂,通过静电吸附作用制得了碳纳米管和纳米金的复合材料。首先将纯化的多壁碳纳米管(MWNTs)修饰于玻碳电极(GC)表面,构建负电荷的修饰界面,基于静电吸附作用固定血红蛋白(Hb),然后再吸附纳米金(nano-Au),最后利用nano-Au再固定一层Hb,从而成功构建了基于MWNTs和nano-Au固定Hb的第三代过氧化氢传感器(Hb/nano-Au/Hb/MWNTs/GC)。用循环伏安法(CV)、电化学交流阻抗(EIS)以及透射电子显微镜(TEM)对修饰电极进行了表征。Hb在修饰电极上实现了直接电子转移。与仅基于碳纳米管构建的H_2O_2传感器相比,该传感器对H_2O_2的响应具有更宽的线性范围和更低的检测下限。其线性范围为2.1×10~(-7)～3.0x10~(-3)mol/L,检测限为8.0x10~(-8)mol/L(S/N=3),表观米氏常数为0.26mmol/L。此外,该传感器还具有较快的响应速率、较好的稳定性和重现性。
4.基于多壁碳纳米管/壳聚糖复合物及多层纳米金,血红蛋白修饰的过氧化氢传感器
将多壁纳米碳管(MWNTs)分散于壳聚糖(CS)中得到稳定的CS-MWNTs复合物,然后将其滴涂于玻碳(GC)电极表面。利用CS丰富的氨基固定nano-Au,进一步静电吸附血红蛋白(Hb)。利用层层自组装技术将带相反电荷的nano-Au和Hb固定于CS-MWNTs复合物上制备了基于MWNTs和多层Hb/nano-Au膜修饰电极。用循环伏安法(CV)、紫外可见(UV-vis)吸收光谱法以及透射电子显微镜(TEM)对修饰电极进行了表征。研究了传感器对H_2O_2的响应及动力学性质,表观米氏常数为0.19 mmol/L,线性响应范围为5.0x10~(-7)～2.0x10~(-3)mol/L,检测限为2.1×10~(-7)mol/L(S/N=3)。同时研究了pH值及应用电位对H_2O_2传感器的影响。
5.基于有机硅/壳聚糖核-壳纳米球和多壁碳纳米管复合物固定HRP的过氧化氢传感器
本文以多壁碳纳米管(MWNTs)和核-壳结构的有机硅/壳聚糖纳米球(organosilica@chitosan)复合物作为固酶基质,构建了一种新型的无介体型过氧化氢生物传感器。首先将MWNTs分散于荷正电荷的organosilica@chitosan的HAc悬浮液中,制备organosilica@chitosan/MWNTs复合材料,并将其直接滴涂在玻碳电极(GCE)表面。然后通过organosilica@chitosan与带相反电荷的辣根过氧化物酶(HRP)的静电吸附作用固定HRP制得HRP/organosilica@chitosan/MWNTs修饰电极。该传感器对H_2O_2具有较好的电催化活性。其线性范围为7.0x10~(-7)～2.8×10~(-3)mol/L,检测限为2.5x10~(-7)mol/L(S/N=3),表观米式常数为0.32 mmol/L。另外,该传感器对H_2O_2还具有较快的响应速度,较好的稳定性和重现性。将其应用于消毒液的检测时,其结果也令人满意。
The rapid and accurate determination of hydrogen peroxide(H_2O_2)is of great importance because it is not only the product of the reactions catalyzed by many highly selective oxidases but also an essential compound in food,pharmaceutical and environmental analyses.Among these techniques employed for hydrogen peroxide analysis,such as titrimetry,photometry, chemiluminescence,high performance liquid chromatography and electrochemistry,amperometric enzyme-based biosensors have received considerable interest,because this class of technique is characterized by sensitivity,convenience and high selectivity.Electron transfer between an electrode and the redox activity center of an enzy me is the basis for developing various enzyme-based biosensors.It can be achieved through two different pathways.One involves electron-shuttling mediators to establish an electrical communication between redox proteins and the underlying electrodes.In this scheme,there are still several challenges to keep mediators from diffusing away from the electrode surface into the bulk solution.The other pathway involves direct electron transfer between redox proteins and the electrode.On the other hand,the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability,sensitivity and selectivity of biosensors.In this paper,nano-Au,with large surface area,high-surface free energy, was chosen to immobilize toluidine blue by layer-by-layer(LBL)assembly technique,and thionine was covalently bound onto the bovine serum albumin film with glutaraldehyde as cross-linker to achieve the immobilization of electron-shuttling mediators.For the leakage significantly decreases, the proposed mediator-based hydrogen peroxide biosensors exhibit good analytical performance.In addition,attention has been paid to the nano-Au/MWNTs composite and core-shell organosilica@chitosan/MWNTs composite.The application of these composites as an immobilization matrix for fabricating mediator-free hydrogen peroxide biosensors has been preliminarily investigated.The main points of this dissertation are summarized as follows:
Part one:Study of the hydrogen peroxide biosensor based on the layer-by-layer assembly films of gold colloidal nanoparticles and toluidine blue
The precursor film was first formed on the Au electrode surface based on the self-assembly of L-cysteine and the adsorption of gold colloidal nan oparticles(nano-Au).Layer-by-layer(LBL) assembly films of toluidine blue(TB)and nano-Au were fabricated by Au-N covalent bond and electrostatic adsorption between TB and nano-Au.Finally,horseradish peroxidase(HRP)was assembled onto {nano-Au/TB}_nmultilayer films to fabricate a novel hydrogen peroxide biosensor. Cyclic voltammeter(CV)and quartz crystal microbalance(QCM)were adopted to monitor the regular growth of {nano-Au/TB} bilayer films.Morphologies of the films were characterized with atomic force microscopy(AFM).The effects of applied potential,temperature and pH on the current response were investigated.Repeatability,stability and anti-interference were also researched.The proposed biosensor responds rapidly to H_2O_2 in the linear range from 1.5×10~(-7)mol/L to 8.6×10~(-3) mol/L with a detection limit of 7.0×10~(-8)mol/L(S/N=3).
Part two:Study of the hydrogen peroxide biosensor based on immobilizing HRP on thionine-bovine serum albumin conjugate and gold colloidal nanoparticles
A novel enzyme immobilization technique based on thionine-bovine serum albumin conjugate (Th-BSA)and gold colloidal nanoparticles(nano-Au)was developed.Thionine was covalently bound onto the BSA film with glutaraldehyde(GA)as cross-linker to achieve Th-BSA conjugate. The free amino groups of thionine were then used to attach nano-Au for the immobilization of horseradish peroxidase(HRP).Such nano-Au/Th-BSA matrix shows a favorable microenviment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme.Several techniques,including atomic force microscopy(AFM),X-ray photoelectron spectroscopy(XPS),Infrared spectra(IR)and electrochemical impedance spectroscopy(EIS)have been employed to characterize the assembly process.The proposed biosensor displays excellent catalytic activity and rapid response for H_2O_2. The linear range for the determination of H_O_2 is from 4.9×10~(-7)to 1.6×10~(-3)mol/L with a detection limit of 2.1×10~(-7)mol/L at 30 and a Michaelies-Menten constant value of 0.023 mmol/L.
Part three:Amperometric third-generation hydrogen peroxide biosensors based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles
A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes(MWNTs)and gold colloidal nanoparticles(nano-Au)by using proteins as linker is proposed.In such a strategy,Hemoglobin(Hb)was selected as model protein to fabricate third-generation H_2O_2 biosensor based on MWNTs and nano-Au.Acid-pretreated,negatively charged MWNTs was first modified on the surface of glassy carbon(GC)electrode,then,positively charged Hb was adsorbed onto MWNTs films by electrostatic interaction.Finally,nano-Au and Hb were successively assembled onto the modified electrode to obtain the Hb/nano-Au/Hb/MWNTs/GC electrode.The assembly of Hb and nano-Au was characterized with cyclic voltammetry(CV), electrochemical impedance spectroscopy(EIS)and transmission electron microscopy(TEM).The direct electron transfer of Hb is observed on the Hb/nano-Au/Hb/MWNTs/GC electrode,which exhibits excellent electrocatalytic activity for the reduction of H_2O_2 to construct a third generation mediator-free H_2O_2 biosensor.As compared to those H_2O_2 biosensors only based on carbon nanotubes,the proposed biosensor modified with MWNTs and nano-Au displays a broader linear range and a lower detection limit for H_2O_2 determination.The linear range is from 2.1×10~(-7)to 3.0×10~(-3)mol/L with a detection limit of 8.0×10~(-8)mol/L at 3σ.The Michaelies-Menten constant K_M~(app)value is estimated to be 0.26 mmol/L.Moreover,this biosensor displays rapid response to H_2O_2 and possesses good stability and reproducibility.
Part four:A hydrogen peroxide biosensor based on multi-wall carbon nanotubes/chitosan composite and multilayer films of hemoglobin and colloidal gold nanoparticles
An amperometric biosensor for H_2O_2 was developed based on multilayer assembly of hemoglobin(Hb)and colloidal gold nanoparticles(nano-Au)on multi-wall carbon nanotubes/chitosan composite.Chitosan(CS)was chosen for dispersing multi-wall carbon nanotubes(MWNTs)to form a stable CS-MWNTs composite.This composite was first coated on the surface of glassy carbon electrode to provide a containing amino groups interface for assembling nano-Au,followed by the adsorption of Hb to form a bilayer of {Hb/nano-Au}.Repeating the assembly step of nano-Au and Hb resulted in {Hb/nano-Au}_n multilayers.The resulting system brought a new platform for electrochemical devices by using the synergistic action of the electrocatalytic activity of nano-Au and MWNTs.The assembly of nano-Au onto CS-MWNTs was confirmed by transmission electron microscopy.The consecutive growth of {Hb/nano-Au}_n multilayers was confirmed by cyclic voltammetry and UV-vis absorption spectroscopy.The resulting electrode displays excellent electrocatalytic activity and rapid response for H_2O_2.The linear range for the determination of H_2O_2 is from 5.0×10~(-7)to 2.0×10~(-3)mol/L with a detection limit of 2.1×10~(-7)mol/L at 3σand a Michaelies-Menten constant K_M~(app)value of 0.19 mmol/L.At the same time,the effects of applied potential and pH on the sensor were examined.
Part five:A hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on core-shell organosilica@chitosan nanospheres and multiwall carbon nanotubes composites
The application of a composite of multiwall carbon nanotubes(MWNTs)and core-shell organosilica@chitosan crosslinked nanospheres as an immobilization matrix for a construction of a novel mediator-free amperometric hydrogen peroxide(H_2O_2)biosensor was described.MWNTs were dispersed in a suspension of positively charged organosilica@chitosan nanospheres in acetic acid solution(0.6 wt%)to achieve an organosilica@chitosan/MWNTs composite,which was cast onto a glass carbon electrode(GCE)surface directly.And then,horseradish peroxidase(HRP),as a model enzyme,was immobilized onto it through electrostatic interaction between oppositely charged organosilica@chitosan nanospheres and HRP.The direct electron transfer of HRP is achieved at the HRP/organosilica@chitosan/MWNTs/GCE,which exhibits an excellent electrocatalytic activity for the reduction of H_2O_2.The catalysis currents increased linearly to H_2O_2 concentration in a wide range of 7.0×10~(-7)tO 2.8×10~(-3)mol/L with a detection limit,of 2.5×10~(-7)mol/L at 3σ.A Michaelies-Menten constant K_M~(app)value is estimated to be 0.32 mmol/L,indicating a high-catalytic activity of HRP.Moreover,the proposed biosensor displays rapid response to H_2O_2 and possesses good stability and reproducibility.When used to detect H_2_2 concentration in disinfector sample,it shows satisfactory results.
引文
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