基于碳纳米材料的一氧化氮电化学传感器的制备及生物医学应用研究
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摘要
一氧化氮(NO)既是一种神经递质又是一种重要的信使分子,它广泛存在于人体的各种组织器官中,有很强的生物活性,在神经信息传递、胃肠保护和动力调控、心肺功能、脏器血流调节、内分泌调节、机体防御免疫功能以及细胞凋亡等很多方面都起着重要的作用。电化学方法灵敏度高,响应快,可以对NO进行实时、在线检测,而且微电极及超微电极又可以实现活体检测和单细胞分析,因此电化学方法受到了科学家的高度关注。依赖于各种化学修饰电极的电化学传感器,是目前最受重视的电化学方法之一,因此,设计优异的电极界面,开发研制灵敏度高、选择性好的NO电化学传感器,以及深入探讨化学与生物分子的传感机理,其关键就在于寻找合适的分子敏感与高效换能的电极修饰材料。
碳纳米材料比表面积大、导电性好、电催化性能好、吸附能力强、表面活性高,生物相容性好,因此被广泛地用作电极的修饰材料来制备电化学传感器。本论文中,基于碳纳米材料的高表面活性,我们将其与其他功能材料进行结合,制备出了选择性好、灵敏度高、生物相容性好的新型NO电化学传感器,并对这些NO电化学传感器在生物医学方面的应用做了研究。主要内容如下：
(1)利用偶氮胭脂红(ACB)实现了对多壁碳纳米管(MWCNTs)的非共价功能化,制得了水溶性良好的MWCNTs.通过简单的电聚合方法,制得了一种新型的基于聚偶氮胭脂红(PACB)-水溶性多壁碳纳米管(MWCNTs)纳米膜修饰玻碳电极(GCE)的NO电化学传感器。该传感器对NO的电化学氧化有很好的催化作用。我们对PACB-MWCNTs复合膜进行了多种表征并对NO的检测条件进行了优化。该传感器具有重现性好、稳定性好、灵敏度高以及抗干扰能力强等特点,并能成功应用于小鼠肝细胞释放NO的检测。
(2)碳纳米纤维(CNFs)经茜素红(AR)非共价功能化后,可以稳定地分散于水中,通过简单的在线电聚合法,制得了聚合茜素红(PAR)-CNFs复合膜修饰碳纤维电极(CFME),可用作NO电化学微传感器。用红外光谱表征技术研究了AR的电聚合机理,并用扫描电镜对PAR-CNFs复合膜的形貌进行了表征。PAR-CNFs复合膜疏松多孔的结构能够增大NO微传感器的有效面积,提高了NO检测的灵敏度。NO微传感器已成功应用于巨噬细胞NO释放的检测,说明该微传感器在生物医学分析领域有很大的应用潜力。
(3)将铂纳米颗粒(PtNPs)通过恒电位法电沉积到乙炔黑(AB)修饰玻碳电极表面,制得了一种基于PtNPs-AB复合膜的NO传感器。用扫描电镜、X荧光光谱,电化学交流阻抗技术对PtNPs-AB复合膜进行了表征,并对NO在该传感器上的电化学反应机理进行了探讨。电化学实验表明,该PtNPs-AB复合膜对NO的电化学氧化有很好的催化作用。用该NO电化学传感器对NO进行检测,其线性范围为：0.18μM～120.0 gM,检出限为50 nM。将该NO电化学传感器用于豚鼠肝组织中NO的释放检测,取得了满意的效果。
(4)自制了一种碳纳米纤维糊微电极(CNFPME),并制备了一种高灵敏的基于十六烷基三甲基溴化铵(CTAB)-Nafion复合膜修饰CNFPME的NO微传感器。扫描电镜和电化学技术表征表明,CTAB-Nafion复合膜具有较大的表面积和良好的导电性,所以可以大大提高NO微传感器的灵敏度。Nafion的抗阴离子干扰及防止电极表面钝化的作用又使得NO微传感器有很好的抗干扰作用。该NO微传感器还具有稳定性好、重现性好以及检出限低等特点,并成功用于了小鼠肝细胞中NO释放的检测。
(5)利用香兰素(vanillin)实现了多壁碳纳米管(MWCNTs)在水中的稳定分散,并通过电聚合的方式将聚香兰素(PVN)-MWCNTs修饰于碳纤维微电极(CFME)表面。借助于扫描电镜、红外光谱及伏安法对PVN-MWCNTs复合膜进行了表征,结果表明,PVN-MWCNTs复合膜具有大面积的致密多孔结构。PVN-MWCNT/CFME对亚硝酸盐(NO2-)的电化学氧化有很好的电催化作用。我们将PVN-MWCNT/CFME用于了湖水中N02-浓度的检测。较好的回收率表明该修饰电极可以用于实际样品中NO2-的测定。
(6)利用酵母细胞生物合成了纳米金银合金(Au-AgNPs)并将其用于高灵敏香兰素(vanillin)电化学传感器的制备。荧光电子显微镜和透射电子显微镜的表征结果表明,Au-AgNPs是由酵母细胞通过细胞外方式生物合成的,而且呈现出不规则的多角形。电化学研究表明,在基于Au-AgNPs修饰玻碳电极(GCE)的vanillin电化学传感器上,vanillin的电化学响应较在裸GCE上提高了近5倍。该vanillin电化学传感器成功用于了香荚兰豆和香草茶中vanillin含量的准确测定,说明它具有较好的实际应用价值。
Nitric oxide (NO) is a neurotransmitter and an important messenger molecule. It exists widely in human's organs and has strong biological activity. It also plays important roles in neural information transmitting, gastrointestinal protection, power control, cardiopulmonary, organ blood flow regulation, endocrine regulation, organism defense immune ability and apoptosis et al. Electrochemical methods have the merits of high sensitivity and quick response, and can realize the real time and online determination of NO; moreover, microelectrode and ultramicroelectrode are adequate for in vivo analysis and single cell analysis. So scientists have paid more attention to electrochemical methods. Electrochemical sensor depended on kinds of modified electrodes is one of the most important electrochemical methods. Therefore, looking for suitable electrode modifier that is molecular sensitive and energy transfer effective is essential for the excellent engineering of electrode interface, developing sensitive and selective NO electrochemical sensors and investigating the sensing mechanism of chemical and biological molecules.
Carbon nano-materials are widely used as electrode modifier in fabricating electrochemical sensors due to their large surface area, good conductive ability, excellent electro-catalytic ability, strong adsorption ability, high surface activity and good biocompatibility. In this thesis, we combined carbon nano-materials with other function materials based on their high surface activity to fabricate some novel NO electrochemical sensors with good selectivity, sensitivity and biocompatibility. Moreover, the application of the NO electrochemical sensors in biomedicine had been investigated. The main results are summarized as follows:
(1) A new noncovalent approach for the dissolution of MWCNTs in water by azocarmine B (ACB) was reported. Through a simple electropolymerization procedure, a novel electrochemical NO sensor based on water-soluble MWCNTs and polyazocarmine B (PACB) nanofilm electrode was prepared, which showed excellent electrocatalytic activity towards the oxidation of nitric oxide (NO). PACB-MWNTs composite film was characterized through many techniques and the NO detection conditions were optimized. The sensor has the merits of good stability, reproducibility, high sensitivity and selectivity, and it can be used to monitor NO released from rat liver cells effectively.
(2) Carbon naofibers (CNFs) were functionalized by Alizarin Red (AR) through new noncovalent approach and could be dispersed in water stably. PAR-CNFs composite film modified carbon fiber miro-electrode (CFME) denoted as NO microsensor was fabricated through a simple in-situ electropolymerization. The electropolymerization mechanism of AR was studied with infrared spectrum and the surface morphology of PAR-CNFs composite film was characterized by scanning electron microscope. The PAR-CNFs composite film has loose structure with lots of nanopores which can improve the effect area and sensitivity of the NO micosensor. The NO microsensor was successfully used to detect the NO released from macrophage, which indicated its potential application in biomedicine.
(3) A NO electrochemical sensor was fabricated through constant-potential electrodepositing Pt nanoparticles (PtNPs) onto the film of acetylene black (AB) modified glassy carbon electrode (GCE). Scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDX) and electrochemical impedance spectroscopy (EIS) were used to characterize the PtNPs-AB. Electrochemical experiments revealed that the NO electrochemical sensor showed high electrocatalytic activity for the oxidation of nitric oxide. The linear range of the determination of nitric oxide was from 0.18μM to 120.0μM and the detection limit was low to 50 nM (S/N=3). This NO electrochemical sensor was applied to the determination of NO released from rat liver tissue and the result was satisfied.
(4) We fabricated a novel carbon nanofiber paste microelectrode (CNFPME) and reported a sensitive NO microsensor based on cetyltrimethyl ammonium bromide (CTAB)-Nafion composite film modified CNFPME. The results from scanning electron microscope (SEM) and electrochemical technique showed that the CTAB-Nafion composite has large surface area and good conductive ability, which can improve the sensitivity of the NO microsensor. The NO microsensor also has good anti-interference ability because of the function of Nafion. The NO microsensor has good stability, reproducibility and low detection limit, and was successfully applied in the determination of NO released from rat liver cells.
(5) We reported a simple method for the stable dispersion of multi-walled carbon nanotubes (MWNTs) in water by vanillin and controllable surface addition onto carbon fiber microelectrodes (CFME) via electropolymerization. We had characterized these polyvanillin-carbon nanotube (PVN-MWCNT) composite films, with techniques including scanning electron microscopy (SEM), infrared spectroscopy (IR) and voltammetry. These investigations showed that the film has a uniform porous nanostructure with a large surface area. This PVN-MWCNT composite-modified CFME (PVN-MWCNT/CFME) exhibited a sensitive response to the electrochemical oxidation of nitrite. We successfully applied the PVN-MWCNT/CFME system to the determination of nitrite from lake water. The efficient recovery of nitrite indicated that this electrode was able to detect nitrite in real samples.
(6) In this work, Au-Ag alloy nanoparticles were biosynthesized by yeast cells and applied to fabricate a sensitive electrochemical vanillin sensor. Fluorescence microscopic and transmission electron microscopic characterizations indicated that the Au-Ag alloy nanoparticles were mainly synthesized via an extracellular approach and generally existed in the form of irregular polygonal nanoparticles. Electrochemical investigations revealed that the vanillin sensor based on Au-Ag alloy nanoparticles modified glassy carbon electrode was able to enhance the electrochemical response of vanillin for at least five times. This vanillin sensor was successfully applied to the determination of vanillin from vanilla bean and vanilla tea sample, suggesting that it may have practical applications in vanillin monitoring system.
碳纳米材料比表面积大、导电性好、电催化性能好、吸附能力强、表面活性高,生物相容性好,因此被广泛地用作电极的修饰材料来制备电化学传感器。本论文中,基于碳纳米材料的高表面活性,我们将其与其他功能材料进行结合,制备出了选择性好、灵敏度高、生物相容性好的新型NO电化学传感器,并对这些NO电化学传感器在生物医学方面的应用做了研究。主要内容如下：
(1)利用偶氮胭脂红(ACB)实现了对多壁碳纳米管(MWCNTs)的非共价功能化,制得了水溶性良好的MWCNTs.通过简单的电聚合方法,制得了一种新型的基于聚偶氮胭脂红(PACB)-水溶性多壁碳纳米管(MWCNTs)纳米膜修饰玻碳电极(GCE)的NO电化学传感器。该传感器对NO的电化学氧化有很好的催化作用。我们对PACB-MWCNTs复合膜进行了多种表征并对NO的检测条件进行了优化。该传感器具有重现性好、稳定性好、灵敏度高以及抗干扰能力强等特点,并能成功应用于小鼠肝细胞释放NO的检测。
(2)碳纳米纤维(CNFs)经茜素红(AR)非共价功能化后,可以稳定地分散于水中,通过简单的在线电聚合法,制得了聚合茜素红(PAR)-CNFs复合膜修饰碳纤维电极(CFME),可用作NO电化学微传感器。用红外光谱表征技术研究了AR的电聚合机理,并用扫描电镜对PAR-CNFs复合膜的形貌进行了表征。PAR-CNFs复合膜疏松多孔的结构能够增大NO微传感器的有效面积,提高了NO检测的灵敏度。NO微传感器已成功应用于巨噬细胞NO释放的检测,说明该微传感器在生物医学分析领域有很大的应用潜力。
(3)将铂纳米颗粒(PtNPs)通过恒电位法电沉积到乙炔黑(AB)修饰玻碳电极表面,制得了一种基于PtNPs-AB复合膜的NO传感器。用扫描电镜、X荧光光谱,电化学交流阻抗技术对PtNPs-AB复合膜进行了表征,并对NO在该传感器上的电化学反应机理进行了探讨。电化学实验表明,该PtNPs-AB复合膜对NO的电化学氧化有很好的催化作用。用该NO电化学传感器对NO进行检测,其线性范围为：0.18μM～120.0 gM,检出限为50 nM。将该NO电化学传感器用于豚鼠肝组织中NO的释放检测,取得了满意的效果。
(4)自制了一种碳纳米纤维糊微电极(CNFPME),并制备了一种高灵敏的基于十六烷基三甲基溴化铵(CTAB)-Nafion复合膜修饰CNFPME的NO微传感器。扫描电镜和电化学技术表征表明,CTAB-Nafion复合膜具有较大的表面积和良好的导电性,所以可以大大提高NO微传感器的灵敏度。Nafion的抗阴离子干扰及防止电极表面钝化的作用又使得NO微传感器有很好的抗干扰作用。该NO微传感器还具有稳定性好、重现性好以及检出限低等特点,并成功用于了小鼠肝细胞中NO释放的检测。
(5)利用香兰素(vanillin)实现了多壁碳纳米管(MWCNTs)在水中的稳定分散,并通过电聚合的方式将聚香兰素(PVN)-MWCNTs修饰于碳纤维微电极(CFME)表面。借助于扫描电镜、红外光谱及伏安法对PVN-MWCNTs复合膜进行了表征,结果表明,PVN-MWCNTs复合膜具有大面积的致密多孔结构。PVN-MWCNT/CFME对亚硝酸盐(NO2-)的电化学氧化有很好的电催化作用。我们将PVN-MWCNT/CFME用于了湖水中N02-浓度的检测。较好的回收率表明该修饰电极可以用于实际样品中NO2-的测定。
(6)利用酵母细胞生物合成了纳米金银合金(Au-AgNPs)并将其用于高灵敏香兰素(vanillin)电化学传感器的制备。荧光电子显微镜和透射电子显微镜的表征结果表明,Au-AgNPs是由酵母细胞通过细胞外方式生物合成的,而且呈现出不规则的多角形。电化学研究表明,在基于Au-AgNPs修饰玻碳电极(GCE)的vanillin电化学传感器上,vanillin的电化学响应较在裸GCE上提高了近5倍。该vanillin电化学传感器成功用于了香荚兰豆和香草茶中vanillin含量的准确测定,说明它具有较好的实际应用价值。
Nitric oxide (NO) is a neurotransmitter and an important messenger molecule. It exists widely in human's organs and has strong biological activity. It also plays important roles in neural information transmitting, gastrointestinal protection, power control, cardiopulmonary, organ blood flow regulation, endocrine regulation, organism defense immune ability and apoptosis et al. Electrochemical methods have the merits of high sensitivity and quick response, and can realize the real time and online determination of NO; moreover, microelectrode and ultramicroelectrode are adequate for in vivo analysis and single cell analysis. So scientists have paid more attention to electrochemical methods. Electrochemical sensor depended on kinds of modified electrodes is one of the most important electrochemical methods. Therefore, looking for suitable electrode modifier that is molecular sensitive and energy transfer effective is essential for the excellent engineering of electrode interface, developing sensitive and selective NO electrochemical sensors and investigating the sensing mechanism of chemical and biological molecules.
Carbon nano-materials are widely used as electrode modifier in fabricating electrochemical sensors due to their large surface area, good conductive ability, excellent electro-catalytic ability, strong adsorption ability, high surface activity and good biocompatibility. In this thesis, we combined carbon nano-materials with other function materials based on their high surface activity to fabricate some novel NO electrochemical sensors with good selectivity, sensitivity and biocompatibility. Moreover, the application of the NO electrochemical sensors in biomedicine had been investigated. The main results are summarized as follows:
(1) A new noncovalent approach for the dissolution of MWCNTs in water by azocarmine B (ACB) was reported. Through a simple electropolymerization procedure, a novel electrochemical NO sensor based on water-soluble MWCNTs and polyazocarmine B (PACB) nanofilm electrode was prepared, which showed excellent electrocatalytic activity towards the oxidation of nitric oxide (NO). PACB-MWNTs composite film was characterized through many techniques and the NO detection conditions were optimized. The sensor has the merits of good stability, reproducibility, high sensitivity and selectivity, and it can be used to monitor NO released from rat liver cells effectively.
(2) Carbon naofibers (CNFs) were functionalized by Alizarin Red (AR) through new noncovalent approach and could be dispersed in water stably. PAR-CNFs composite film modified carbon fiber miro-electrode (CFME) denoted as NO microsensor was fabricated through a simple in-situ electropolymerization. The electropolymerization mechanism of AR was studied with infrared spectrum and the surface morphology of PAR-CNFs composite film was characterized by scanning electron microscope. The PAR-CNFs composite film has loose structure with lots of nanopores which can improve the effect area and sensitivity of the NO micosensor. The NO microsensor was successfully used to detect the NO released from macrophage, which indicated its potential application in biomedicine.
(3) A NO electrochemical sensor was fabricated through constant-potential electrodepositing Pt nanoparticles (PtNPs) onto the film of acetylene black (AB) modified glassy carbon electrode (GCE). Scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDX) and electrochemical impedance spectroscopy (EIS) were used to characterize the PtNPs-AB. Electrochemical experiments revealed that the NO electrochemical sensor showed high electrocatalytic activity for the oxidation of nitric oxide. The linear range of the determination of nitric oxide was from 0.18μM to 120.0μM and the detection limit was low to 50 nM (S/N=3). This NO electrochemical sensor was applied to the determination of NO released from rat liver tissue and the result was satisfied.
(4) We fabricated a novel carbon nanofiber paste microelectrode (CNFPME) and reported a sensitive NO microsensor based on cetyltrimethyl ammonium bromide (CTAB)-Nafion composite film modified CNFPME. The results from scanning electron microscope (SEM) and electrochemical technique showed that the CTAB-Nafion composite has large surface area and good conductive ability, which can improve the sensitivity of the NO microsensor. The NO microsensor also has good anti-interference ability because of the function of Nafion. The NO microsensor has good stability, reproducibility and low detection limit, and was successfully applied in the determination of NO released from rat liver cells.
(5) We reported a simple method for the stable dispersion of multi-walled carbon nanotubes (MWNTs) in water by vanillin and controllable surface addition onto carbon fiber microelectrodes (CFME) via electropolymerization. We had characterized these polyvanillin-carbon nanotube (PVN-MWCNT) composite films, with techniques including scanning electron microscopy (SEM), infrared spectroscopy (IR) and voltammetry. These investigations showed that the film has a uniform porous nanostructure with a large surface area. This PVN-MWCNT composite-modified CFME (PVN-MWCNT/CFME) exhibited a sensitive response to the electrochemical oxidation of nitrite. We successfully applied the PVN-MWCNT/CFME system to the determination of nitrite from lake water. The efficient recovery of nitrite indicated that this electrode was able to detect nitrite in real samples.
(6) In this work, Au-Ag alloy nanoparticles were biosynthesized by yeast cells and applied to fabricate a sensitive electrochemical vanillin sensor. Fluorescence microscopic and transmission electron microscopic characterizations indicated that the Au-Ag alloy nanoparticles were mainly synthesized via an extracellular approach and generally existed in the form of irregular polygonal nanoparticles. Electrochemical investigations revealed that the vanillin sensor based on Au-Ag alloy nanoparticles modified glassy carbon electrode was able to enhance the electrochemical response of vanillin for at least five times. This vanillin sensor was successfully applied to the determination of vanillin from vanilla bean and vanilla tea sample, suggesting that it may have practical applications in vanillin monitoring system.
引文
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