新型纳米材料用于电化学生物传感界面的构建
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摘要
电化学生物传感器是近年来迅速发展起来的一种全新的生物传感器。该类传感器具有灵敏度高、成本低、选择性好、易于微型化等优点,在临床医学检验、环境监测和食品工程等领域有广阔的应用前景。电化学生物传感器构制的一个关键技术在于如何将生物组分稳定、高效的固定到基体上面。本研究论文通过发展新型生物材料固定方法,构制了一系列性能良好的电化学DNA生物传感器和适体传感器以达到改进固定生物组分活性、延长传感器的使用寿命等目的。本论文主要研究工作包括:
1、以室温固相法制备纳米MnO_2,通过壳聚糖(CHIT)的成膜效应将纳米MnO_2固定在玻碳电极表面。DNA在MnO_2/CHIT膜上的固定和杂交通过循环伏安和电化学交流阻抗进行表征。固定于电极表面的DNA探针与目标DNA杂交后使电极表面的电子传递电阻增大,以此作为检测信号可以高灵敏度地测定目标DNA。电化学阻抗谱检测大肠杆菌基因片段的线性范围为2.0×10~(-11) mol/L～2.0×10~(-6) mol/L,检测限为1.0×10~(-12) mol/L。
2、以室温固相法制备纳米ZnO,通过CHIT的成膜效应将纳米ZnO固定在玻碳电极表面,制得的ZnO/CHIT/GCE电极成为DNA固定和杂交的良好平台。以电化学交流阻抗免标记法检测人类免疫缺陷病毒(HIV)基因片段的线性范围为2.0×10~(-11)～2.0×10~(-6) mol/L,检测限为2.0×10~(-12) mol/L。
3、通过CHIT的成膜效应将腺苷适体固定在MnO_2/CHIT修饰电极表面,腺苷适体与目标腺苷杂交后使电极表面的电子传递电阻增大,杂交前后阻抗值的变化与样品中腺苷的量是成比例的,以此可以高灵敏度地测定目标腺苷。电化学阻抗谱检测目标腺苷的线性范围为1.0×10-8～5.0×10~(-6) mol/L,相关系数r = 0.9990,检测限为1.0×10~(-9) mol/L。
4、以MnO_2为传感基底构制了基于目标诱导适体置换的非标记型电化学腺苷传感器。通过氧化还原探针[Fe(CN)_6]~(3-/4-)监测传感器界面表面电子传递电阻的变化。应用此电化学适体传感器,以电化学阻抗法对腺苷进行免标记检测,其线性范围为1.0×10~(-9)～1.0×10~(-6) mol/L,检测限为8.0×10~(-10) mol/L。
Electrochemical biosensor, which has been developed rapidly in recent years, is one new type of biosensors. This kind of biosensors has many superior advantages such as high sensitivity, nice selectivity, low cost and easy miniaturization, which has been widely used in clinical medical analysis, environmental monitoring and food engineering. The key aspect in the construction of the electrochemical biosensor is the immobilization procedure of biomolecules on the sensor surface. The aim of this dissertation is to develop simple and sensitive immobilization strategies of biomaterials in order to improve the performance and long-term stability of the electrochemical biosensors. The main works of the research paper are as follows:
1. MnO_2 nanoparticle was synthesized by solid state reaction at room temperature and was immobilized on glass carbon electrode (GCE) by way of the film forming of chitosan (CHIT).The immobilization and hybridization of DNA on the MnO_2/CHIT film were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) method.The results indicated that the electron transfer resistance (Ret) of the electron surface was increased after the hybridization of probe DNA with target DNA.Based on this,a sensitive label-free DNA electrochemical biosensor was fabricated. The DNA biosensor showed a wide linear response to the logarithm values of Escherichia coli concentration in the range of 2.0×10~(-11) mol/L～2.0×10~(-6) mol/L, with a detection limit of 1.0×10~(-12) mol/L.
2. ZnO was synthesized by solid state reaction and was immobilized on GCE via the film forming by chitosan (CHIT). The developed ZnO/CHIT/GCE electrode was a good platform of DNA immobilization and hybridization. Under optimal conditions, the DNA biosensor showed a wide linear response to human immunodeficiency virus (HIV) gene in the range of 2.0×10~(-11) ~ 2.0×10~(-6) mol/L, with a detection limit of 2.0×10~(-12) mol/L of complementary target.
3. Adenosine aptamer was immobilized on GCE via the film forming property of chitosan (CHIT). The results indicated that the electron transfer resistance (Ret) of the electron surface was increased after the hybridization of probe aptamer with target adenosine. The response impedance change is proportional to the amount of adenosine in sample. The linear range of the signal was observed between 1.0×10-8 mol/L and 5.0×10~(-6) mol/L of adenosine with 0.9990 correlation factor. This method was able to linearly and selectively detect adenosine with a detection limit of 1.0×10~(-9) mol/L.
4. A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on nano-MnO_2 as a platform for the immobilization of the aptamer was developed for the determination of adenosine. In the measurement of adenosine, the change in interfacial electron transfer resistance (Ret) of the biosensor using a redox couple of [Fe(CN)6]3-/4- as the probe was monitored. The change of the electron transfer resistance (ΔRet) of the biosensor was linear with the concentration of adenosine in the range from 1.0×10~(-9) mol/L to 1.0×10~(-6)mol/L. The detection limit was 8.0×10-10 mol/L.
1、以室温固相法制备纳米MnO_2,通过壳聚糖(CHIT)的成膜效应将纳米MnO_2固定在玻碳电极表面。DNA在MnO_2/CHIT膜上的固定和杂交通过循环伏安和电化学交流阻抗进行表征。固定于电极表面的DNA探针与目标DNA杂交后使电极表面的电子传递电阻增大,以此作为检测信号可以高灵敏度地测定目标DNA。电化学阻抗谱检测大肠杆菌基因片段的线性范围为2.0×10~(-11) mol/L～2.0×10~(-6) mol/L,检测限为1.0×10~(-12) mol/L。
2、以室温固相法制备纳米ZnO,通过CHIT的成膜效应将纳米ZnO固定在玻碳电极表面,制得的ZnO/CHIT/GCE电极成为DNA固定和杂交的良好平台。以电化学交流阻抗免标记法检测人类免疫缺陷病毒(HIV)基因片段的线性范围为2.0×10~(-11)～2.0×10~(-6) mol/L,检测限为2.0×10~(-12) mol/L。
3、通过CHIT的成膜效应将腺苷适体固定在MnO_2/CHIT修饰电极表面,腺苷适体与目标腺苷杂交后使电极表面的电子传递电阻增大,杂交前后阻抗值的变化与样品中腺苷的量是成比例的,以此可以高灵敏度地测定目标腺苷。电化学阻抗谱检测目标腺苷的线性范围为1.0×10-8～5.0×10~(-6) mol/L,相关系数r = 0.9990,检测限为1.0×10~(-9) mol/L。
4、以MnO_2为传感基底构制了基于目标诱导适体置换的非标记型电化学腺苷传感器。通过氧化还原探针[Fe(CN)_6]~(3-/4-)监测传感器界面表面电子传递电阻的变化。应用此电化学适体传感器,以电化学阻抗法对腺苷进行免标记检测,其线性范围为1.0×10~(-9)～1.0×10~(-6) mol/L,检测限为8.0×10~(-10) mol/L。
Electrochemical biosensor, which has been developed rapidly in recent years, is one new type of biosensors. This kind of biosensors has many superior advantages such as high sensitivity, nice selectivity, low cost and easy miniaturization, which has been widely used in clinical medical analysis, environmental monitoring and food engineering. The key aspect in the construction of the electrochemical biosensor is the immobilization procedure of biomolecules on the sensor surface. The aim of this dissertation is to develop simple and sensitive immobilization strategies of biomaterials in order to improve the performance and long-term stability of the electrochemical biosensors. The main works of the research paper are as follows:
1. MnO_2 nanoparticle was synthesized by solid state reaction at room temperature and was immobilized on glass carbon electrode (GCE) by way of the film forming of chitosan (CHIT).The immobilization and hybridization of DNA on the MnO_2/CHIT film were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) method.The results indicated that the electron transfer resistance (Ret) of the electron surface was increased after the hybridization of probe DNA with target DNA.Based on this,a sensitive label-free DNA electrochemical biosensor was fabricated. The DNA biosensor showed a wide linear response to the logarithm values of Escherichia coli concentration in the range of 2.0×10~(-11) mol/L～2.0×10~(-6) mol/L, with a detection limit of 1.0×10~(-12) mol/L.
2. ZnO was synthesized by solid state reaction and was immobilized on GCE via the film forming by chitosan (CHIT). The developed ZnO/CHIT/GCE electrode was a good platform of DNA immobilization and hybridization. Under optimal conditions, the DNA biosensor showed a wide linear response to human immunodeficiency virus (HIV) gene in the range of 2.0×10~(-11) ~ 2.0×10~(-6) mol/L, with a detection limit of 2.0×10~(-12) mol/L of complementary target.
3. Adenosine aptamer was immobilized on GCE via the film forming property of chitosan (CHIT). The results indicated that the electron transfer resistance (Ret) of the electron surface was increased after the hybridization of probe aptamer with target adenosine. The response impedance change is proportional to the amount of adenosine in sample. The linear range of the signal was observed between 1.0×10-8 mol/L and 5.0×10~(-6) mol/L of adenosine with 0.9990 correlation factor. This method was able to linearly and selectively detect adenosine with a detection limit of 1.0×10~(-9) mol/L.
4. A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on nano-MnO_2 as a platform for the immobilization of the aptamer was developed for the determination of adenosine. In the measurement of adenosine, the change in interfacial electron transfer resistance (Ret) of the biosensor using a redox couple of [Fe(CN)6]3-/4- as the probe was monitored. The change of the electron transfer resistance (ΔRet) of the biosensor was linear with the concentration of adenosine in the range from 1.0×10~(-9) mol/L to 1.0×10~(-6)mol/L. The detection limit was 8.0×10-10 mol/L.
引文
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