基于碳纳米管的生物传感器和液/液界面离子转移的研究
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摘要
本文主要采用透射电子显微镜(TEM)、X射线衍射(XRD)、紫外可见吸收光谱法(UV-Vis)、电化学交流阻抗法(EIS)、循环伏安法(CV)、计时电流法(i-t)、差式脉冲法(DPV)等研究了基于碳纳米管的生物传感器和液/液界面上的离子转移过程。
1.合成了PAMAM-MWNTs-AuNPs纳米复合物,并将血红蛋白通过静电相互作用固载到带有正电的PAMAM-MWNTs-AuNPs纳米复合物修饰的玻碳电极上,实现了血红蛋白在该修饰电极上的直接电化学。并在此基础上研究了该修饰电极对过氧化氢的催化还原能力。
2.将由表面活性剂SDS分散的羧基化的碳纳米管与血红蛋白通过共价键结合到玻碳电极表面,实现了血红蛋白与电极之间的直接电子转移。并探讨了该传感器对亚硝酸根催化还原能力。
3.利用三电极体系,采用循环伏安法研究了稀土金属离子Yb3+在水/1,2-二氯乙烷界面的转移,在电位窗内实现了Yb3+的加速离子转移。探讨了影响Yb3+转移的因素,如冠醚的浓度、Yb3+的浓度和扫速等,并讨论了Yb3+和DB18C6在界面上形成络合物的络合比。
4.使用内壁硅烷化的微管研究了K+在微液/液界面上的离子转移过程。探讨了影响K+转移的因素,同时讨论了K+在微水/1,2-二氯乙烷界面转移的转移机理。
Biosensor is an interdisciplinary area which was developed in 1960’s. After over 40 years of development, it has been achieved remarkable progress. In recent years, with the development of nano-technology, some nano-materials which have unique property began to be used to fabricate the third generation biosensor based on the redox protein or enzyme.Study the third-generation biosensors based on redox protein or enzyme can help us understand the electron transfer process, the material metabolism and the energy metabolism. The biosensors based on redox protein or enzyme is a valuable system to study the electrochemical process.
Liquid/Liquid interface electrochemistry is a branch of electrochemistry and electroanalytical chemistry between the traditional electrochemistry and chemical sensors. Charge transfer across the Liquid/Liquid interface is fundamental to a variety of industrial applications including separation and extraction processes, phase transfer catalysis, electrochemical processes, and drug delivery in pharmacology.
In this thesis, we fabricated several carbon nanotube-based redox protein biosensor, and investigate their electrocatalytic response. Then, we studied the facilitate ion transfer across the Liquid/Liquid interface.The details are summarized as follows:
1. In Chapter II, we synthesis the nanocomposites based on PAMAM, MWNTs, and AuNPs. The method is simple. In order to investigate the PAMAM-MWNTs-AuNPs composite, we employed the TEM and XRD. Hb have been immobilized onto the positive charged PAMAM-MWNTs-AuNPs composite film modified glassy carbon electrode through electrostatic interaction. The UV-Vis result showed that the immobilized Hb retains its original conformation. EIS was carried out to investigate the impedance changes of the electrode surface during the fabrication process. Experimental Results indicated that PAMAM-MWNTs-AuNPs composite film can enhance the rate of electron transfer, and the Hb had been immobilized on the electrode. Direct electron transfer between Hb and the modified electrode was observed by cyclic voltammety and the PAMAM-MWNTs-AuNPs composite is a suitable matrix for the immobilization of Hb. The formal potential (E°') of HbFe(III)/Fe(II) is -0.370 V. The electron transfer rate constant (ks) is 4.66 s-1, the faster ks indicated that the PAMAM-MWNTs-AuNPs composite film was an excellent promoter for the electron transfer between Hb and the underlying electrode. E°' of the HbFe(III)/Fe(II) shifted linearly with pH with a slope of -49.2 mV pH-1, indicating that an electron transfer accompanies single-proton transportation. The modified electrode exhibited excellent electrocatalytic response to the reduction of H2O2. The linear range for H2O2 determination was from 1.0×10?6 to 2.2×10?3 mol L-1 (r =0.999) with a detection limit of 2.0×10?7M at a signal-to-noise ratio of 3. The apparent Michaelis–Menten constant (Kmapp), which gives an indication of the enzyme–substrate kinetics, is 2.95 mM as calculated by Lineweaver–Burk equation. The resulted biosensor showed a low detection limit, good stability, high reproducibility, good selectivity and fast response time. The work reported a new platform for preparing the third-generation electrochemical biosensors.
2. In Chapter III, the SDS dispersed MWNTs-COOH and Hb was immobilized on the glass carbon electrode through covalent bond. Direct electron transfer between Hb and the modified electrode was observed by cyclic voltammety. E°' of the HbFe(III)/Fe(II) shifted linearly with pH with a slope of -53.07 mV pH-1, indicating that an electron transfer accompanies single-proton transportation. The UV-Vis result showed that the immobilized Hb retains its original conformation.EIS was carried out to investigate the impedance changes of the electrode surface during the fabrication process. Experimental Results indicated that MWNTs have excellent electrical conductivity. It can enhance the rate of electron transfer between Fe(CN)3-/4- and the electrode, and the Hb had been immobilized on the electrode. The modified electrode exhibited excellent electrocatalytic response to the reduction of NO2-. The linear range for NO2- determination was from 2×10-6 to 9×10-5 mol L-1 with a detection limit of 3.2×10-7mol L-1 at a signal-to-noise ratio of 3. The fabricated biosensor showed a low detection limit, good stability, high reproducibility, good selectivity and the method is simple.
3. In Chapter IV, we employ the three electrode system, using cyclic voltammetry, studied the rare earth metal ion Yb3+ transfer across the water/1,2-dichloroethane interface first time. We observed the facilitated ion transfer in the potential window by DB18C6, indicating DB18C6 complexed with Yb3+, which reduced the Gibbs free energy. Under Yb3+ diffusion-controlled conditions, the peak current increases with scan rate, the peak current is proportional to the square root of the scan rate, the peak current increases with the concentration of Yb3+, the half-wave potential shifted negatively with the concentration of DB18C6, there exist a linear relationship between E1/2 and logcDB18C6 with the slope of -18.16mV/decade the 1:1 complexes [Yb(DB18C6)]3+ was formed at the water/1,2-dichloroethane interface. Under DB18C6 diffusion-controlled conditions, the peak current increases with scan rate, the peak current is proportional to the square root of the scan rate, the peak current increases with the concentration of DB18C6, the half-wave potential shifted negatively with the concentration of Yb3+, there exist a linear relationship between E1/2 and logcYb3+ with the slope of -27mV/decade, the 1:1 complexes [Yb(DB18C6)]3+ was formed at the water/1,2-dichloroethane interface.
4. In Chapter V, the pipets with radii of 10μm were made from borosilicate capillaries from Sutter Inatrument Co., using a laster-based pipet puller (P-2000 Sutter Inatrument Co.). The inner wall of the pipet was silanized to render it hydrophobic. So organic solvents can be injected into the pipet, it can reduce the toxicity of the organic solvents, and is beneficial to organism detection. Study the liquid-liquid interface with micropipet can reduce the impact of iR drop and charging current. We studied DB18C6 faciliated the K+ transfer across the micro-water/1, 2-dichloethane interface with inner wall silanized micropipet by cyclic voltammetry. If the organic phase only exists the supporting electrolyte, there only the ion transfer peak of supporting electrolyte transfer between the organic phase and water phase, but no the peak of K+, this is because the K+ is strong hydrophilic, and has a high Gibbs free energy. We observed the facilitated K+ transfer in the potential window by DB18C6, indicating DB18C6 complexed with K+, which reduced the Gibbs free energy. The forward scan is the K+ transfer to the micro-water/1, 2-dichloethane interface, behave a peak current, this is because if the concentration of K+ is much higher than DB18C6, the process is controlled by the linear diffusion of DB18C6 in the micropipet transfer to the micro-water/1, 2-dichloethane interface, the mechanism can be seen as the process of interface complexation (TIC). The Reverse scan is the complex dissociation process at the micro-water/1, 2-dichloethane interface, also behave a peak current, this is because the process is controlled by the linear diffusion of the complex in the micropipet dissociate to the micro-water/1, 2-dichloethane interface, the mechanism can be seen as the process of interface dissociation(TID). Under DB18C6 diffusion-controlled conditions, the peak current is proportional to the square root of the scan rate, the peak current increases with the concentration of DB18C6, the half-wave potential shifted negatively with the concentration of K+, there exist a linear relationship between E1/2 and logcK+ with the slope of -65mV /decade, the 1:1 complexes [K(DB18C6)]+ was formed at the micro-water/1,2-dichloroethane interface.
1.合成了PAMAM-MWNTs-AuNPs纳米复合物,并将血红蛋白通过静电相互作用固载到带有正电的PAMAM-MWNTs-AuNPs纳米复合物修饰的玻碳电极上,实现了血红蛋白在该修饰电极上的直接电化学。并在此基础上研究了该修饰电极对过氧化氢的催化还原能力。
2.将由表面活性剂SDS分散的羧基化的碳纳米管与血红蛋白通过共价键结合到玻碳电极表面,实现了血红蛋白与电极之间的直接电子转移。并探讨了该传感器对亚硝酸根催化还原能力。
3.利用三电极体系,采用循环伏安法研究了稀土金属离子Yb3+在水/1,2-二氯乙烷界面的转移,在电位窗内实现了Yb3+的加速离子转移。探讨了影响Yb3+转移的因素,如冠醚的浓度、Yb3+的浓度和扫速等,并讨论了Yb3+和DB18C6在界面上形成络合物的络合比。
4.使用内壁硅烷化的微管研究了K+在微液/液界面上的离子转移过程。探讨了影响K+转移的因素,同时讨论了K+在微水/1,2-二氯乙烷界面转移的转移机理。
Biosensor is an interdisciplinary area which was developed in 1960’s. After over 40 years of development, it has been achieved remarkable progress. In recent years, with the development of nano-technology, some nano-materials which have unique property began to be used to fabricate the third generation biosensor based on the redox protein or enzyme.Study the third-generation biosensors based on redox protein or enzyme can help us understand the electron transfer process, the material metabolism and the energy metabolism. The biosensors based on redox protein or enzyme is a valuable system to study the electrochemical process.
Liquid/Liquid interface electrochemistry is a branch of electrochemistry and electroanalytical chemistry between the traditional electrochemistry and chemical sensors. Charge transfer across the Liquid/Liquid interface is fundamental to a variety of industrial applications including separation and extraction processes, phase transfer catalysis, electrochemical processes, and drug delivery in pharmacology.
In this thesis, we fabricated several carbon nanotube-based redox protein biosensor, and investigate their electrocatalytic response. Then, we studied the facilitate ion transfer across the Liquid/Liquid interface.The details are summarized as follows:
1. In Chapter II, we synthesis the nanocomposites based on PAMAM, MWNTs, and AuNPs. The method is simple. In order to investigate the PAMAM-MWNTs-AuNPs composite, we employed the TEM and XRD. Hb have been immobilized onto the positive charged PAMAM-MWNTs-AuNPs composite film modified glassy carbon electrode through electrostatic interaction. The UV-Vis result showed that the immobilized Hb retains its original conformation. EIS was carried out to investigate the impedance changes of the electrode surface during the fabrication process. Experimental Results indicated that PAMAM-MWNTs-AuNPs composite film can enhance the rate of electron transfer, and the Hb had been immobilized on the electrode. Direct electron transfer between Hb and the modified electrode was observed by cyclic voltammety and the PAMAM-MWNTs-AuNPs composite is a suitable matrix for the immobilization of Hb. The formal potential (E°') of HbFe(III)/Fe(II) is -0.370 V. The electron transfer rate constant (ks) is 4.66 s-1, the faster ks indicated that the PAMAM-MWNTs-AuNPs composite film was an excellent promoter for the electron transfer between Hb and the underlying electrode. E°' of the HbFe(III)/Fe(II) shifted linearly with pH with a slope of -49.2 mV pH-1, indicating that an electron transfer accompanies single-proton transportation. The modified electrode exhibited excellent electrocatalytic response to the reduction of H2O2. The linear range for H2O2 determination was from 1.0×10?6 to 2.2×10?3 mol L-1 (r =0.999) with a detection limit of 2.0×10?7M at a signal-to-noise ratio of 3. The apparent Michaelis–Menten constant (Kmapp), which gives an indication of the enzyme–substrate kinetics, is 2.95 mM as calculated by Lineweaver–Burk equation. The resulted biosensor showed a low detection limit, good stability, high reproducibility, good selectivity and fast response time. The work reported a new platform for preparing the third-generation electrochemical biosensors.
2. In Chapter III, the SDS dispersed MWNTs-COOH and Hb was immobilized on the glass carbon electrode through covalent bond. Direct electron transfer between Hb and the modified electrode was observed by cyclic voltammety. E°' of the HbFe(III)/Fe(II) shifted linearly with pH with a slope of -53.07 mV pH-1, indicating that an electron transfer accompanies single-proton transportation. The UV-Vis result showed that the immobilized Hb retains its original conformation.EIS was carried out to investigate the impedance changes of the electrode surface during the fabrication process. Experimental Results indicated that MWNTs have excellent electrical conductivity. It can enhance the rate of electron transfer between Fe(CN)3-/4- and the electrode, and the Hb had been immobilized on the electrode. The modified electrode exhibited excellent electrocatalytic response to the reduction of NO2-. The linear range for NO2- determination was from 2×10-6 to 9×10-5 mol L-1 with a detection limit of 3.2×10-7mol L-1 at a signal-to-noise ratio of 3. The fabricated biosensor showed a low detection limit, good stability, high reproducibility, good selectivity and the method is simple.
3. In Chapter IV, we employ the three electrode system, using cyclic voltammetry, studied the rare earth metal ion Yb3+ transfer across the water/1,2-dichloroethane interface first time. We observed the facilitated ion transfer in the potential window by DB18C6, indicating DB18C6 complexed with Yb3+, which reduced the Gibbs free energy. Under Yb3+ diffusion-controlled conditions, the peak current increases with scan rate, the peak current is proportional to the square root of the scan rate, the peak current increases with the concentration of Yb3+, the half-wave potential shifted negatively with the concentration of DB18C6, there exist a linear relationship between E1/2 and logcDB18C6 with the slope of -18.16mV/decade the 1:1 complexes [Yb(DB18C6)]3+ was formed at the water/1,2-dichloroethane interface. Under DB18C6 diffusion-controlled conditions, the peak current increases with scan rate, the peak current is proportional to the square root of the scan rate, the peak current increases with the concentration of DB18C6, the half-wave potential shifted negatively with the concentration of Yb3+, there exist a linear relationship between E1/2 and logcYb3+ with the slope of -27mV/decade, the 1:1 complexes [Yb(DB18C6)]3+ was formed at the water/1,2-dichloroethane interface.
4. In Chapter V, the pipets with radii of 10μm were made from borosilicate capillaries from Sutter Inatrument Co., using a laster-based pipet puller (P-2000 Sutter Inatrument Co.). The inner wall of the pipet was silanized to render it hydrophobic. So organic solvents can be injected into the pipet, it can reduce the toxicity of the organic solvents, and is beneficial to organism detection. Study the liquid-liquid interface with micropipet can reduce the impact of iR drop and charging current. We studied DB18C6 faciliated the K+ transfer across the micro-water/1, 2-dichloethane interface with inner wall silanized micropipet by cyclic voltammetry. If the organic phase only exists the supporting electrolyte, there only the ion transfer peak of supporting electrolyte transfer between the organic phase and water phase, but no the peak of K+, this is because the K+ is strong hydrophilic, and has a high Gibbs free energy. We observed the facilitated K+ transfer in the potential window by DB18C6, indicating DB18C6 complexed with K+, which reduced the Gibbs free energy. The forward scan is the K+ transfer to the micro-water/1, 2-dichloethane interface, behave a peak current, this is because if the concentration of K+ is much higher than DB18C6, the process is controlled by the linear diffusion of DB18C6 in the micropipet transfer to the micro-water/1, 2-dichloethane interface, the mechanism can be seen as the process of interface complexation (TIC). The Reverse scan is the complex dissociation process at the micro-water/1, 2-dichloethane interface, also behave a peak current, this is because the process is controlled by the linear diffusion of the complex in the micropipet dissociate to the micro-water/1, 2-dichloethane interface, the mechanism can be seen as the process of interface dissociation(TID). Under DB18C6 diffusion-controlled conditions, the peak current is proportional to the square root of the scan rate, the peak current increases with the concentration of DB18C6, the half-wave potential shifted negatively with the concentration of K+, there exist a linear relationship between E1/2 and logcK+ with the slope of -65mV /decade, the 1:1 complexes [K(DB18C6)]+ was formed at the micro-water/1,2-dichloroethane interface.
引文
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