新型纳米生物传感界面的构建及其电化学行为研究
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摘要
电化学传感器是化学传感器中研究较为成熟的一种,它是采用电化学效应将检测信号转变为电信号的化学传感器,电化学传感器在分析测定中有广泛的应用。含酶或蛋白质的生物传感器是利用生物活性物质的亲和性,如酶-底物、抗原-抗体、激素-受体等的分子识别功能来检测待测物,因其具有较高的选择性而备受关注,纳米微粒具有庞大的比表面积、较强的催化活性和较好的生物相容性等优点,研究蛋白质及纳米粒子在纳米修饰电极的电化学行为是本论文的主要工作。随着生物传感器的发展,酶及蛋白质生物活性物质的活性受温度以及pH值等环境因素的影响大大限制了含酶生物传感器的应用,随之发展起来的无酶生物传感器的制备和应用成为一个新的研究热点,本论文还构建了基于纳米材料的无酶电化学生物传感,并研究了修饰电极材料的其它电化学性质,取得了一定的研究进展。
第一章主要介绍了电化学生物传感器的概念、种类、研究进展情况以及电化学传感器中电极的修饰方法。简单介绍了纳米材料及其在电化学生物传感器中的应用和发展前景。分别对导电聚合物和石墨烯做了概括性的介绍,并论述了导电聚合物与石墨烯复合材料的研究现状及发展前景。
第二章中,我们利用高压水热合成法,制备了刺球状的CeO2纳米粒子(CeO2NPs),将其与肌红蛋白(Mb)混合后固定在玻碳电极(GCE)上,构建了无媒介第三代生物传感器。采用TEM、UV-vis和XRD对制备的CeO2 NPs进行表征,结果表明CeO2 NPs表面的独特的刺球状结构,能够增大Mb在电极表面的有效负载量。循环伏安法研究表明CeO2 NPs对肌红蛋白直接电子转移(DET)有促进作用,CeO2NPs的独特电子传导性能加快Mb与电极之间的电子传递速率,且具有优良生物相容性的CeO2 NPs为所固定的蛋白质提供了一个良好的生物微环境。构建的Mb/CeO2 NPs/GCE生物传感器对双氧水(H2O2)具有良好的电催化还原作用,其检测限为0.3μM(S/N=3),线性响应范围：0.7-194μM,并且有一个较低的KMapp值,约为0.048 mM。表明该生物传感器具有灵敏度高,稳定性强、能够增强Mb对H202的亲和性,促进其对H202的催化还原等特点。
第三章是根据柯肯达尔效应,利用高压水热合成法,制备了CeO2-ZrO2空心复合纳米球,将肌红蛋白(Mb)固定在CeO2-ZrO2修饰的玻碳电极(GCE)上,构建了一种新的第三代无媒介生物传感器。采用TEM、UV-vis对制备的复合物进行表征,结果表明CeO2-ZrO2复合物具有独特的介孔结构和良好的生物相容性,可增大Mb在电极表面的有效负载量。通过循环伏安法研究了该复合物对肌红蛋白直接电子转移的促进作用,Mb/CeO2-ZrO2修饰电极为蛋白质和基底电极的直接电子传输提供了一个有利的微环境,因此加快了Mb与电极之间的电子传递速率。实验结果表明构建的Mb/CeO2-ZrO2/GCE生物传感器对双氧水(H202)具有良好的电催化,其检测限为1.1μM,KMapp为0.128 mM,线性响应范围：8.5-330μM,ks约为2.65 s-1。CeO2-ZrO2复合材料为固定蛋白质和制备生物传感器提供了一种很好的基体材料,有利于推动生物传感器及其它生物电化学器件的发展。
第四章用水热法制备了Fe3O4@ZrO2核壳纳米微球,将肌红蛋白(myoglobin,Mb)通过静电吸附固定在Fe3O4@ZrO2核壳纳米微球表面上,利用磁性玻碳电极(MGCE)的外磁场磁力将此复合材料固定于MGCE上研究Mb的直接电化学,实现了材料在电极上的快速固定化,只需要几秒钟的时间。实验结果表明,Mb在电极上的平均表面覆盖度约为1.3×10-10 mol·cm-2,在-326 mV和-363 mV左右有一对血红素类蛋白的特征氧化还原峰,峰电位差约为37 mV。该传感器对H202有良好的电流响应,速率常数ks为1.4 s-1信噪比为3时(S/N=3)其检测限为0.32μM。在较大的检测范围内,传感器表现出Michaelis-Menten行为,其米氏常数(KMapp)计算值约为40μM,较小的KMapp表明固定在电极上的Mb很好的保持了它的生物活性。
第五章中我们首先制备了聚苯胺／氧化石墨烯(PANI/GO)复合材料,然后在PANI/GO表面原位生长铂纳米粒子(Pt NPs)进而制备了铂／聚苯胺／氧化石墨烯(Pt/PANI/GO)纳米复合材料,用扫描电子显微镜(SEM)、透射电子显微镜(TEM)、x-射线衍射(XRD)、红外光谱(FT-IR)等表征手段研究了材料的结构和性质,研究表明所制备的Pt NPs粒径小,在PANI/GO的表面负载均匀、负载密度高。将所制备的材料修饰在玻碳电极(GCE)表面,研究了材料的各种电化学性质。实验结果表明,负载在PANI/GO复合材料上的Pt NPs对氧气和甲醇有良好的催化作用有望用于燃料电池的应用,所制备的传感器在优化的条件下对H202和葡萄糖也有很好的电流响应。该传感器还表现出了优良的稳定性和重现性。
Electrochemical sensor, the chemical sensor that translate detecting signal into electric signal by electrochemical effect, is the maturest one in chemical sensors. Electrochemical sensor was widely used in the analytical determination. Enzymatic or protein biosensors, which can detect analytes using the specific reaction of bioactive materials such as the enzyme-substrate, enzyme-coenzyme, antigen-antibody, incretion-acceptor and so on, have attracted much attentions due to their good selectivity. Nanoparticles have many extraordinary features such as huge specific surface area, strong catalytic activity and good bio-compatibility. Study on the electrochemical behavior of protein on the nanoparticles modified electrode is one of the main research works of the thesis. Howerver, the enzymatic or protein biosensors are limited by the stability of the enzyme to both pH value and temperature with the development of biosensor. As such recent effort has been put into producing nonenzymatic biosensors. we also used the nanoparticles to fabricate nonenzymatic biosensors and studied the other electrochemical behaviors of the nanoparticles modified electrode in this paper.
This section described the concept, classification and actuality of electrochemical biosensor, the nanoparticles and it's application in electrochemical biosensor, the polymer and graphene and the study of their compounds.
In part two, monodispersed urchinlike spherical ceria nanoparticles (CeO2 NPs) with rough surface were prepared via a simple one step hydrolysis process in glycol and have been utilized for immobilization of proteins to fabricate the third biosensor. Those as-prepared CeO2 NPs have good biocompatibility, favorable conductivity, large surface area, as verified by transmission electron microscopy (TEM) and X-ray powder diffraction spectroscopy. UV-vis spectroscopy analysis of the Mb/CeO2 NPs film suggested that the immobilized Mb could retain its natural structure, which is illuminated that the prepared CeO2 NPs have excellent biocompatible. The modified glassy carbon electrode (GCE) by Mb and CeO2 NPs exhibited direct electrochemistry with a fast electron transfer rate (1.03 s-1) and good electrochemical performance to reduce hydrogen peroxide (H2O2). The proposed biosensor shows a linear response to H2O2 concentrations ranging from 0.7μM to 194μM with a detection limit of 0.3μM at a signal-to-noise ratio of 3 and the low value of apparent Michaelis-Menten constant (0.048 mM) indicates enhanced enzyme affinity of Mb to H2O2. Therefore, our experiments implemented the fast direct electron transfer (DET) of proteins indicated the present CeO2 NPs may provide a favorable bio-microenvironment for proteins immobilized on the electrode surface and enhance electron transfer between proteins and electrodes.
In part three, a novel hollow matrix, CeO2-ZrO2 solid solution nanocages particles which synthesized by hydrothermal method via Kirkendall Effect, were used for immobilizing myoglobin (Mb) to fabricate protein electrodes suitable for studying the direct electron transfer between the redox centers of proteins and the electrode. The transmission electron microscopy (TEM), UV-vis and electrochemical measurements showed that the matrix was well biocompatible and could retain the bioactivity of immobilized protein to a large extent. The direct electron transfer of the immobilized myoglobin (Mb) exhibited a couple of stable and well-defined redox peaks in 0.1 M pH 6.98 PBS. The Mb/CeO2-ZrO2 modified electrode also exhibited excellent catalytic performance for H2O2 with the detection limit of 1.1μM, apparent Michaelis-Menten constant (KMapp) of 0.128 mM and the linear response range was from 8.5μM to 330μM. This matrix could accelerate the electron transfer between Mb and the electrode with a surface controlled process and an electron transfer rate constant ks was 2.65 s-1. CeO2-ZrO2as a good material for immobilization of protein can facilitate the development of electrochemical biosensor.
In part four, direct electron transfer (DET) of myoglobin (Mb) was achieved by its direct immobilization on magnetic glassy carbon electron (MGCE) with the big magnetic Fe3O4@ZrO2 Core-Shell microspheres as binder, the material can fast immobilized on the MGCE. A pair of well-defined redox peaks, characteristic of the protein heme FeⅡ/FeⅢredox couples, was obtained at the Mb/Fe3O4@ZrO2 film on magnetic glassy carbon electrode (MGCE), as a consequence of the direct electron transfer between the protein and the MGCE electrode. The redox peaks were acquired in 0.1 M phosphate buffer solution (pH 6.98) with oxidation potential of-326 mV, reduction potential of-363 mV, the formal potential E0 (E0=(Epa+Epc)/2) at-345 mV and the peak to peak potential separation of 37 mV at 100 mV·s-1. The average surface coverage of the electroactive Mb immobilized on the electrode surface was calculated as 4.82×10-11mol·cm-2. Mb retained its bioactivity on modified electrode and showed excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2). The cathodic peak current of Mb was linear to H2O2 concentration in the range from 1μM to 76μM with a detection limit of 0.32μM (S/N=3). The apparent Michaelis-Menten constant (KMapp) and the electron transfer rate constant (ks) were estimated to be 40μM and 1.4 s-1, respectively. The biosensor achieved the direct electrochemistry of Mb on Fe3O4@ZrO2 Core-Shell microspheres without the help of any supporting film or any electron mediator.
In this section we prepared the PANI/GO firstly by in-situ polymerization and then grow up Pt NPs on both of the surfaces of PANI/GO, the Pt NPs which loaded on the PANI/GO has small size and ultra-high density. The structure and character of different compounds were tested with SEM, TEM, XRD, FT-IR and thermogravimetric and so on. We tested the electrochemical behaviors of different modified GCE, and the Pt/PANI/GO/GCE showed excellent electrocatalytic activity to O2 and methanol, it also performed good electricity response of the H2O2 and glucose under the optimized conditions.
第一章主要介绍了电化学生物传感器的概念、种类、研究进展情况以及电化学传感器中电极的修饰方法。简单介绍了纳米材料及其在电化学生物传感器中的应用和发展前景。分别对导电聚合物和石墨烯做了概括性的介绍,并论述了导电聚合物与石墨烯复合材料的研究现状及发展前景。
第二章中,我们利用高压水热合成法,制备了刺球状的CeO2纳米粒子(CeO2NPs),将其与肌红蛋白(Mb)混合后固定在玻碳电极(GCE)上,构建了无媒介第三代生物传感器。采用TEM、UV-vis和XRD对制备的CeO2 NPs进行表征,结果表明CeO2 NPs表面的独特的刺球状结构,能够增大Mb在电极表面的有效负载量。循环伏安法研究表明CeO2 NPs对肌红蛋白直接电子转移(DET)有促进作用,CeO2NPs的独特电子传导性能加快Mb与电极之间的电子传递速率,且具有优良生物相容性的CeO2 NPs为所固定的蛋白质提供了一个良好的生物微环境。构建的Mb/CeO2 NPs/GCE生物传感器对双氧水(H2O2)具有良好的电催化还原作用,其检测限为0.3μM(S/N=3),线性响应范围：0.7-194μM,并且有一个较低的KMapp值,约为0.048 mM。表明该生物传感器具有灵敏度高,稳定性强、能够增强Mb对H202的亲和性,促进其对H202的催化还原等特点。
第三章是根据柯肯达尔效应,利用高压水热合成法,制备了CeO2-ZrO2空心复合纳米球,将肌红蛋白(Mb)固定在CeO2-ZrO2修饰的玻碳电极(GCE)上,构建了一种新的第三代无媒介生物传感器。采用TEM、UV-vis对制备的复合物进行表征,结果表明CeO2-ZrO2复合物具有独特的介孔结构和良好的生物相容性,可增大Mb在电极表面的有效负载量。通过循环伏安法研究了该复合物对肌红蛋白直接电子转移的促进作用,Mb/CeO2-ZrO2修饰电极为蛋白质和基底电极的直接电子传输提供了一个有利的微环境,因此加快了Mb与电极之间的电子传递速率。实验结果表明构建的Mb/CeO2-ZrO2/GCE生物传感器对双氧水(H202)具有良好的电催化,其检测限为1.1μM,KMapp为0.128 mM,线性响应范围：8.5-330μM,ks约为2.65 s-1。CeO2-ZrO2复合材料为固定蛋白质和制备生物传感器提供了一种很好的基体材料,有利于推动生物传感器及其它生物电化学器件的发展。
第四章用水热法制备了Fe3O4@ZrO2核壳纳米微球,将肌红蛋白(myoglobin,Mb)通过静电吸附固定在Fe3O4@ZrO2核壳纳米微球表面上,利用磁性玻碳电极(MGCE)的外磁场磁力将此复合材料固定于MGCE上研究Mb的直接电化学,实现了材料在电极上的快速固定化,只需要几秒钟的时间。实验结果表明,Mb在电极上的平均表面覆盖度约为1.3×10-10 mol·cm-2,在-326 mV和-363 mV左右有一对血红素类蛋白的特征氧化还原峰,峰电位差约为37 mV。该传感器对H202有良好的电流响应,速率常数ks为1.4 s-1信噪比为3时(S/N=3)其检测限为0.32μM。在较大的检测范围内,传感器表现出Michaelis-Menten行为,其米氏常数(KMapp)计算值约为40μM,较小的KMapp表明固定在电极上的Mb很好的保持了它的生物活性。
第五章中我们首先制备了聚苯胺／氧化石墨烯(PANI/GO)复合材料,然后在PANI/GO表面原位生长铂纳米粒子(Pt NPs)进而制备了铂／聚苯胺／氧化石墨烯(Pt/PANI/GO)纳米复合材料,用扫描电子显微镜(SEM)、透射电子显微镜(TEM)、x-射线衍射(XRD)、红外光谱(FT-IR)等表征手段研究了材料的结构和性质,研究表明所制备的Pt NPs粒径小,在PANI/GO的表面负载均匀、负载密度高。将所制备的材料修饰在玻碳电极(GCE)表面,研究了材料的各种电化学性质。实验结果表明,负载在PANI/GO复合材料上的Pt NPs对氧气和甲醇有良好的催化作用有望用于燃料电池的应用,所制备的传感器在优化的条件下对H202和葡萄糖也有很好的电流响应。该传感器还表现出了优良的稳定性和重现性。
Electrochemical sensor, the chemical sensor that translate detecting signal into electric signal by electrochemical effect, is the maturest one in chemical sensors. Electrochemical sensor was widely used in the analytical determination. Enzymatic or protein biosensors, which can detect analytes using the specific reaction of bioactive materials such as the enzyme-substrate, enzyme-coenzyme, antigen-antibody, incretion-acceptor and so on, have attracted much attentions due to their good selectivity. Nanoparticles have many extraordinary features such as huge specific surface area, strong catalytic activity and good bio-compatibility. Study on the electrochemical behavior of protein on the nanoparticles modified electrode is one of the main research works of the thesis. Howerver, the enzymatic or protein biosensors are limited by the stability of the enzyme to both pH value and temperature with the development of biosensor. As such recent effort has been put into producing nonenzymatic biosensors. we also used the nanoparticles to fabricate nonenzymatic biosensors and studied the other electrochemical behaviors of the nanoparticles modified electrode in this paper.
This section described the concept, classification and actuality of electrochemical biosensor, the nanoparticles and it's application in electrochemical biosensor, the polymer and graphene and the study of their compounds.
In part two, monodispersed urchinlike spherical ceria nanoparticles (CeO2 NPs) with rough surface were prepared via a simple one step hydrolysis process in glycol and have been utilized for immobilization of proteins to fabricate the third biosensor. Those as-prepared CeO2 NPs have good biocompatibility, favorable conductivity, large surface area, as verified by transmission electron microscopy (TEM) and X-ray powder diffraction spectroscopy. UV-vis spectroscopy analysis of the Mb/CeO2 NPs film suggested that the immobilized Mb could retain its natural structure, which is illuminated that the prepared CeO2 NPs have excellent biocompatible. The modified glassy carbon electrode (GCE) by Mb and CeO2 NPs exhibited direct electrochemistry with a fast electron transfer rate (1.03 s-1) and good electrochemical performance to reduce hydrogen peroxide (H2O2). The proposed biosensor shows a linear response to H2O2 concentrations ranging from 0.7μM to 194μM with a detection limit of 0.3μM at a signal-to-noise ratio of 3 and the low value of apparent Michaelis-Menten constant (0.048 mM) indicates enhanced enzyme affinity of Mb to H2O2. Therefore, our experiments implemented the fast direct electron transfer (DET) of proteins indicated the present CeO2 NPs may provide a favorable bio-microenvironment for proteins immobilized on the electrode surface and enhance electron transfer between proteins and electrodes.
In part three, a novel hollow matrix, CeO2-ZrO2 solid solution nanocages particles which synthesized by hydrothermal method via Kirkendall Effect, were used for immobilizing myoglobin (Mb) to fabricate protein electrodes suitable for studying the direct electron transfer between the redox centers of proteins and the electrode. The transmission electron microscopy (TEM), UV-vis and electrochemical measurements showed that the matrix was well biocompatible and could retain the bioactivity of immobilized protein to a large extent. The direct electron transfer of the immobilized myoglobin (Mb) exhibited a couple of stable and well-defined redox peaks in 0.1 M pH 6.98 PBS. The Mb/CeO2-ZrO2 modified electrode also exhibited excellent catalytic performance for H2O2 with the detection limit of 1.1μM, apparent Michaelis-Menten constant (KMapp) of 0.128 mM and the linear response range was from 8.5μM to 330μM. This matrix could accelerate the electron transfer between Mb and the electrode with a surface controlled process and an electron transfer rate constant ks was 2.65 s-1. CeO2-ZrO2as a good material for immobilization of protein can facilitate the development of electrochemical biosensor.
In part four, direct electron transfer (DET) of myoglobin (Mb) was achieved by its direct immobilization on magnetic glassy carbon electron (MGCE) with the big magnetic Fe3O4@ZrO2 Core-Shell microspheres as binder, the material can fast immobilized on the MGCE. A pair of well-defined redox peaks, characteristic of the protein heme FeⅡ/FeⅢredox couples, was obtained at the Mb/Fe3O4@ZrO2 film on magnetic glassy carbon electrode (MGCE), as a consequence of the direct electron transfer between the protein and the MGCE electrode. The redox peaks were acquired in 0.1 M phosphate buffer solution (pH 6.98) with oxidation potential of-326 mV, reduction potential of-363 mV, the formal potential E0 (E0=(Epa+Epc)/2) at-345 mV and the peak to peak potential separation of 37 mV at 100 mV·s-1. The average surface coverage of the electroactive Mb immobilized on the electrode surface was calculated as 4.82×10-11mol·cm-2. Mb retained its bioactivity on modified electrode and showed excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2). The cathodic peak current of Mb was linear to H2O2 concentration in the range from 1μM to 76μM with a detection limit of 0.32μM (S/N=3). The apparent Michaelis-Menten constant (KMapp) and the electron transfer rate constant (ks) were estimated to be 40μM and 1.4 s-1, respectively. The biosensor achieved the direct electrochemistry of Mb on Fe3O4@ZrO2 Core-Shell microspheres without the help of any supporting film or any electron mediator.
In this section we prepared the PANI/GO firstly by in-situ polymerization and then grow up Pt NPs on both of the surfaces of PANI/GO, the Pt NPs which loaded on the PANI/GO has small size and ultra-high density. The structure and character of different compounds were tested with SEM, TEM, XRD, FT-IR and thermogravimetric and so on. We tested the electrochemical behaviors of different modified GCE, and the Pt/PANI/GO/GCE showed excellent electrocatalytic activity to O2 and methanol, it also performed good electricity response of the H2O2 and glucose under the optimized conditions.
引文
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