三种纳米材料和离子液体修饰的电化学生物传感器的制备与应用
摘要
电化学生物传感器是一类以生物材料(生物分子、细胞、组织、器官等)作为生物敏感元件,以电极作为换能器,以电化学信号(如电流、阻抗、电容等)作为检测信号的分析装置。它具有操作简便、制作简单、分析快速等优点。本文以高导电率的碳离子液体电极(carbon ionic liquid electrode,CILE)为基体电极,结合纳米材料的特殊性质,制备了三种新型的电化学生物传感器;分别研究了肌红蛋白(myoglobin,Mb)和血红蛋白(hemoglobin,Hb)在两种不同金属纳米粒子修饰电极上的直接电化学和电催化行为;制备了一种基于V_2O_5纳米带-多壁碳纳米管(MWCNTs)-壳聚糖(chitosan,CTS)复合膜修饰碳离子液体电极的电化学DNA生物传感器。本论文共分三个部分:
I.使用离子液体1-乙基-3-甲基咪唑四氟硼酸盐(1-ethyl-3-methylimidazolium tetrafluoroborat,EMIMBF4)修饰碳糊电极为基体电极,使用循环伏安法将Co纳米粒子同位电沉积于电极表面,进一步涂布Mb和Nafion制得Nafion/Mb/Co/CILE;研究了肌红蛋白在该电极上的直接电化学行为,同时考察了Nafion/Mb/Co/CILE对H_2O_2和TCA的电化学催化研究。实验结果表明,Co纳米粒子已成功的修饰在CILE表面,Co纳米粒子的存在能够极大地提高Mb的电化学和电催化活性。
II.以离子液体1-乙基-3-甲基咪唑乙基磺酸盐(1-ethyl-3-methylimidazolium ethylsulphate,EMIMEtOSO3)修饰电极为基体电极,利用恒电位电沉积法制备了金纳米粒子修饰电极,并使用SEM等方法对制备的电极进行了表征;而且研究了Hb在Nafion/Hb/Au/CILE上的直接电化学行为;同时对TCA在Nafion/Hb/Au/CILE上电化学催化还原进行了研究。
III.构建了一种基于V_2O_5纳米带-多壁碳纳米管(MWCNTs)-壳聚糖(chitosan,CTS)复合膜修饰CILE的DNA生物传感器。将离子液体N-己基吡啶六氟磷酸盐(N-hexylpyridinium hexafluorophosphate,HPPF6)代替石蜡作为粘合剂与石墨粉一起混合制得CILE。首先将单链DNA(ssDNA)探针固定在CTS-V_2O_5-MWCNTs/CILE上,然后与目标ssDNA序列杂交,接着使用亚甲基蓝(methylene blue,MB)为指示剂、微分脉冲伏安法(differential pulse voltammetry,DPV)来检测杂交反应的发生。实验结果表明,V_2O_5纳米带与MWCNTs之间的协同效应使ssDNA探针的负载量大大增加,从而间接地提高了电化学响应信号。并且这种传感器对单碱基和三碱基错配序列具有良好的识别能力。使用这种DNA生物传感器对猪肉样品中的耶尔森菌(Yersinia enterocolitica)DNA序列的环介导等温扩增(loop-mediated isothermal amplification,LAMP)产物进行了检测,具有良好的灵敏度和选择性。
Electrochemistry biosensor is a kind of analytical devices with many advantages such as convenient, simply, cheap and fast, which employ biomaterials (such as biomolecules, cells, tissue and organs) as the identification module, electrodes as the transducer and electrochemistry responses (such as current, impedance and capacitance) as the detection signal. In this paper, three kinds of novel biosensors combined the nanomaterials and ionic liquids were fabricated. Direct electrochemistry and electrocatalysis of myoglobin (Mb) and hemoglobin (Hb) was studied on two kinds of metal nanoparticles modified carbon ionic liquid electrode (CILE). Moreover a novel electrochemiscal DNA biosensor was prepared based on V_2O_5 nanobelts-multiwalled carbon nanotubes (MWCNTs)-chitosan (CTS) composite modified CILE. The main contributions are summrized as below:
I. The electrochemical deposition of Co nanoparticles on CILE was described and further used as the platform to construct a Mb electrochemical biosensor. CILE was prepared by mixing a certain ratio of carbon powder, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4) and liquid paraffin together. A layer of Co nanoparticles was deposited on the surface of CILE with the average diameter of 300 nm after the cyclic voltammetic scan in the CoCl2 solution. Mb molecules were further cast on the surface of Co/CILE and immobilized with Nafion film and the formed Co/CILE was used as a new basal electrode for the investigation on the direct electrochemistry of protein. The Mb electrochemical biosensor showed good electrocatalytic activity to the reduction of hydrogen peroxide (H_2O_2) and trichloroacetic acid (TCA).
II. A carbon ionic liquid electrode (CILE) was fabricated by using ionic liquid 1-ethyl-3-methylimidazolium ethylsulfate (EMIMEtOSO_3) as modifier and further gold nanoparticles were in situ electrodeposited on the surface of CILE. The fabricated Au/CILE was used as a new platform for the immobilization of Hb with the help of a Nafion film. Electrochemical experimental results indicated that direct electron transfer of Hb was realized on the surface of Au/CILE with a pair of well-defined quasi-reversible redox peaks appeared. The fabricated Nafion/Hb/Au/CILE showed excellent electrocatalytic activity to the reduction of trichloroacetic acid (TCA). The proposed electrode showed good stability and reproducibility, and it had the potential application as a new third-generation electrochemical biosensor.
III. A new electrochemical DNA biosensor was fabricated by using a V_2O_5 nanobelts (nano-V_2O_5), multiwalled carbon nanotubes (MWCNTs) and chitosan (CTS) nanocomposite materials modified carbon ionic liquid electrode (CILE) as the working electrode. The CILE was prepared by using N-hexylpyridinium hexafluorophosphate (HPPF6) as the binder with the graphite powder. The CTS-V_2O_5-MWCNTs/CILE was used as the basal electrode for the immobilization of the single-stranded DNA (ssDNA) probe. After the hybridization with the target ssDNA sequence, the electrochemical indicator of methylene blue (MB) was used to monitor the hybridization reaction. Experimental data indicated that the synergistic effect of nano-V_2O_5 and MWCNTs increased the amounts of ssDNA adsorbed on the electrode surface and resulted in the corresponding increase of the electrochemical responses. This DNA biosensor combined the advantages such as the biocompatibility of V_2O_5 nanobelt, the excellent electron transfer ability of MWCNTs, the good film-forming ability of CTS and the high conductivity of CILE. The DNA biosensor showed good stability and discrimination ability to the one-base and three-base mismatched ssDNA sequence. The loop-mediated isothermal amplification (LAMP) product of Yersinia enterocolitica gene sequence in pork meat was detected by the proposed method with satisfactory result, suggesting that the CTS-V_2O_5-MWCNTs/CILE had the potential for the sensitive detection of specific gene sequence.
I.使用离子液体1-乙基-3-甲基咪唑四氟硼酸盐(1-ethyl-3-methylimidazolium tetrafluoroborat,EMIMBF4)修饰碳糊电极为基体电极,使用循环伏安法将Co纳米粒子同位电沉积于电极表面,进一步涂布Mb和Nafion制得Nafion/Mb/Co/CILE;研究了肌红蛋白在该电极上的直接电化学行为,同时考察了Nafion/Mb/Co/CILE对H_2O_2和TCA的电化学催化研究。实验结果表明,Co纳米粒子已成功的修饰在CILE表面,Co纳米粒子的存在能够极大地提高Mb的电化学和电催化活性。
II.以离子液体1-乙基-3-甲基咪唑乙基磺酸盐(1-ethyl-3-methylimidazolium ethylsulphate,EMIMEtOSO3)修饰电极为基体电极,利用恒电位电沉积法制备了金纳米粒子修饰电极,并使用SEM等方法对制备的电极进行了表征;而且研究了Hb在Nafion/Hb/Au/CILE上的直接电化学行为;同时对TCA在Nafion/Hb/Au/CILE上电化学催化还原进行了研究。
III.构建了一种基于V_2O_5纳米带-多壁碳纳米管(MWCNTs)-壳聚糖(chitosan,CTS)复合膜修饰CILE的DNA生物传感器。将离子液体N-己基吡啶六氟磷酸盐(N-hexylpyridinium hexafluorophosphate,HPPF6)代替石蜡作为粘合剂与石墨粉一起混合制得CILE。首先将单链DNA(ssDNA)探针固定在CTS-V_2O_5-MWCNTs/CILE上,然后与目标ssDNA序列杂交,接着使用亚甲基蓝(methylene blue,MB)为指示剂、微分脉冲伏安法(differential pulse voltammetry,DPV)来检测杂交反应的发生。实验结果表明,V_2O_5纳米带与MWCNTs之间的协同效应使ssDNA探针的负载量大大增加,从而间接地提高了电化学响应信号。并且这种传感器对单碱基和三碱基错配序列具有良好的识别能力。使用这种DNA生物传感器对猪肉样品中的耶尔森菌(Yersinia enterocolitica)DNA序列的环介导等温扩增(loop-mediated isothermal amplification,LAMP)产物进行了检测,具有良好的灵敏度和选择性。
Electrochemistry biosensor is a kind of analytical devices with many advantages such as convenient, simply, cheap and fast, which employ biomaterials (such as biomolecules, cells, tissue and organs) as the identification module, electrodes as the transducer and electrochemistry responses (such as current, impedance and capacitance) as the detection signal. In this paper, three kinds of novel biosensors combined the nanomaterials and ionic liquids were fabricated. Direct electrochemistry and electrocatalysis of myoglobin (Mb) and hemoglobin (Hb) was studied on two kinds of metal nanoparticles modified carbon ionic liquid electrode (CILE). Moreover a novel electrochemiscal DNA biosensor was prepared based on V_2O_5 nanobelts-multiwalled carbon nanotubes (MWCNTs)-chitosan (CTS) composite modified CILE. The main contributions are summrized as below:
I. The electrochemical deposition of Co nanoparticles on CILE was described and further used as the platform to construct a Mb electrochemical biosensor. CILE was prepared by mixing a certain ratio of carbon powder, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4) and liquid paraffin together. A layer of Co nanoparticles was deposited on the surface of CILE with the average diameter of 300 nm after the cyclic voltammetic scan in the CoCl2 solution. Mb molecules were further cast on the surface of Co/CILE and immobilized with Nafion film and the formed Co/CILE was used as a new basal electrode for the investigation on the direct electrochemistry of protein. The Mb electrochemical biosensor showed good electrocatalytic activity to the reduction of hydrogen peroxide (H_2O_2) and trichloroacetic acid (TCA).
II. A carbon ionic liquid electrode (CILE) was fabricated by using ionic liquid 1-ethyl-3-methylimidazolium ethylsulfate (EMIMEtOSO_3) as modifier and further gold nanoparticles were in situ electrodeposited on the surface of CILE. The fabricated Au/CILE was used as a new platform for the immobilization of Hb with the help of a Nafion film. Electrochemical experimental results indicated that direct electron transfer of Hb was realized on the surface of Au/CILE with a pair of well-defined quasi-reversible redox peaks appeared. The fabricated Nafion/Hb/Au/CILE showed excellent electrocatalytic activity to the reduction of trichloroacetic acid (TCA). The proposed electrode showed good stability and reproducibility, and it had the potential application as a new third-generation electrochemical biosensor.
III. A new electrochemical DNA biosensor was fabricated by using a V_2O_5 nanobelts (nano-V_2O_5), multiwalled carbon nanotubes (MWCNTs) and chitosan (CTS) nanocomposite materials modified carbon ionic liquid electrode (CILE) as the working electrode. The CILE was prepared by using N-hexylpyridinium hexafluorophosphate (HPPF6) as the binder with the graphite powder. The CTS-V_2O_5-MWCNTs/CILE was used as the basal electrode for the immobilization of the single-stranded DNA (ssDNA) probe. After the hybridization with the target ssDNA sequence, the electrochemical indicator of methylene blue (MB) was used to monitor the hybridization reaction. Experimental data indicated that the synergistic effect of nano-V_2O_5 and MWCNTs increased the amounts of ssDNA adsorbed on the electrode surface and resulted in the corresponding increase of the electrochemical responses. This DNA biosensor combined the advantages such as the biocompatibility of V_2O_5 nanobelt, the excellent electron transfer ability of MWCNTs, the good film-forming ability of CTS and the high conductivity of CILE. The DNA biosensor showed good stability and discrimination ability to the one-base and three-base mismatched ssDNA sequence. The loop-mediated isothermal amplification (LAMP) product of Yersinia enterocolitica gene sequence in pork meat was detected by the proposed method with satisfactory result, suggesting that the CTS-V_2O_5-MWCNTs/CILE had the potential for the sensitive detection of specific gene sequence.
引文
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