基于磁性纳米粒子和量子点的电化学生物传感器及修饰电极的研究
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摘要
纳米材料是当今材料科学的研究热点,其在化学修饰电极和电化学生物传感器中的应用近年来引起了人们的重视。本论文旨在合成磁性纳米材料(如纳米Fe_3O_4等)和量子点(如CdS),并将上述纳米材料修饰于电极表面,用于药物分子的测定。并研制出了新型的电化学生物传感器,已用于实际生化样品的测定。
本论文的主要内容如下:
(1)制备了壳聚糖(CS)包裹四氧化三铁的磁性微球(Fe_3O_4/CS),并将其用于固定辣根过氧化物酶(HRP),制得了测定过氧化氢的电化学生物传感器(HRP-Fe_3O_4/CS-GCE)。考察了各种实验变量如支持电解质浓度、pH、酶的用量、媒介体亚甲基蓝浓度、工作电位等对生物传感器性能的影响。对H_2O_2进行测定,线性范围为2.0×10~(-4)～1.2×10-2 mol/L,检出限为1.0×10~(-4) mol/L(S/N=3),响应时间小于12 s。该传感器的表观米氏常数为21.4 mmol/L,表明所固定的酶具有较高的生物活性。该生物传感器具有高的稳定性和良好的重现性,用该法对实际样品进行了测定。
(2)制备了壳聚糖(CS)包裹四氧化三铁的磁性微球(Fe_3O_4/CS),利用吸附交联作用,制得了磁性纳米微球-血红蛋白夹心法修饰的玻碳电极(Fe_3O_4/CS-Hb-Fe_3O_4/CS-GCE)。以对苯二酚(HQ)为电子媒介体,考察了各种实验变量如支持电解质浓度、pH、酶的用量、戊二醛的用量、对苯二酚浓度、工作电位等对生物传感器性能的影响。用计时电流法对H_2O_2进行测定,线性范围分别为5.0×10-5～1.8×10~(-3) mol/L和1.8×10~(-3)～6.8×10~(-3) mol/L,检出限为4.0×10~(-6) mol/L(S/N=3),响应时间小于10 s。表观米氏常数Km为0.29 mmol/L,表明固定的Hb有较好的生物活性。该生物传感器具有高的稳定性和良好的重现性,已用于实际样品测定。
(3)研究了纳米四氧化三铁化学修饰电极对非甾体抗炎药尼美舒利(nimesulide)的测定。实验结果表明,在0.4 mol/L HAc-NaAc(pH 5.0)溶液中,尼美舒利在裸玻碳电极或纳米四氧化三铁修饰电极上均发生不可逆还原反应,但与裸玻碳电极相比,纳米四氧化三铁修饰电极对尼美舒利具有明显的催化和增敏作用,峰电位由-0.707 V(裸电极)正移到-0.682 V(vs. Ag/AgCl)(修饰电极),峰电位正移25 mV,灵敏度增加约3倍。对各种实验条件进行了优化,测得峰电流Ip与尼美舒利浓度在2.6×10~(-6)～1.0×10~(-4) mol/L范围内呈良好的线性关系,相关系数为0.993,检出限为1.3×10~(-7) mol/L。利用优化后的条件对尼美舒利药片进行了测定,测量值与标示值符合,回收率在96.9%～101.9%,并与紫外分光光度法进行了比较,无显著性差异。
(4)研究了氯霉素在纳米钴修饰玻碳电极上的电化学行为及测定。实验结果表明,在0.3 mol/L NH3-NH4Cl(pH=10.0)缓冲液中,氯霉素在裸玻碳电极或纳米钴修饰电极上均发生不可逆还原反应,但与裸玻碳电极相比,纳米钴修饰电极对氯霉素的还原具有明显的增敏作用,灵敏度增加约6倍。对支持电解质、修饰剂用量等各种实验条件进行了优化。测得峰电流Ip与氯霉素浓度在5.0×10~(-6)～1.2×10~(-4) mol/L范围内呈良好的线性关系,相关系数为0.997,检出限为3.0×10~(-7) mol/L。利用优化后的条件对氯霉素滴眼液进行了测定,测量值与标示值符合,回收率在98.7%～102.2%。
(5)将血红蛋白吸附在巯基乙酸包覆的硫化镉/羧基化多壁碳纳米管膜上,制得了测定过氧化氢的电化学生物传感器(Hb/CdS/MWNTs/GCE)。实验表明在pH 8.0的磷酸盐缓冲溶液中循环伏安扫描出现一对峰形良好的准可逆氧化还原峰,为Hb Fe(Ⅲ)/Fe(Ⅱ)电对的特征峰,其式量电位(E0')为-0.230 V(vs. Ag/AgCl)。该修饰电极对H_2O_2表现出较好的电催化行为,线性范围为2.0×10~(-6)～2.7×10~(-3) mol/L和2.7×10~(-3)～7.7×10~(-3) mol/L,检出限为3.0×10~(-7) mol/L(S/N=3),响应时间小于2 s。该传感器的表观米氏常数为1.324 mmol/L,表明所固定的血红蛋白具有较高的生物活性。
(6)研究了以Nafion分散功能化多壁碳纳米管化学修饰电极对尼美舒利的测定。实验结果表明,在0.2 mol/L PBS(pH 6.6)溶液中,碳纳米管修饰电极对尼美舒利具有明显的催化和增敏作用。峰电位由-0.665 V(裸电极)正移到-0.553 V(修饰电极),峰电位正移112 mV,灵敏度增加约7倍。峰电流Ip与尼美舒利浓度在3.2×10~(-7)～6.5×10-5 mol/L范围内呈良好的线性关系,检出限为1.6×10~(-7) mol/L。研究了尼美舒利的电化学行为,利用优化后的条件对尼美舒利药片进行了测定,测量值与标示值符合。并且与紫外分光光度法比较进行了比较,无显著性差异。
Nanomaterials are the research focus in modern materials science. Considerable research efforts are being devoted toward the development of the chemical modified electrodes and electrochemical biosensors based on the nanomaterials. The main work of this paper is focus on the synthesis of magnetic nanoparticles (Fe_3O_4 nanoparticles) and quantum dots (CdS) and the fabrication of the modified electrodes based on the nanomaterials. Besides, some electrochemical biosensors have been developed. Also, the biosensors have been used to determine the real samples. The main results are listed below:
(1) A new type of amperometric hydrogen peroxide biosensor was constructed based on horseradish peroxidase (HRP) immobilized on Fe_3O_4/chitosan modified glass carbon electrode. The effects of some experimental variables such as the concentration of supporting electrolyte, pH, enzyme loading, the concentration of the mediator of methylene blue (MB) and the applied potential were investigated. The linear range of the calibration curve for H_2O_2 was 2.0×10~(-4)～1.2×10-2 mol/L and the detection limit was 1.0×10~(-4) mol/L (S/N=3). The response time was less than 12 s. The apparent Michaelis-Menten constant Km was 21.4 mmol/L and it illustrated the excellent biological activity of the fixed enzyme. In addition, the biosensor had long-time stability and good reproducibility. And this method has been used to determine H_2O_2 concentration in the real sample.
(2) A novel magnetic microsphere (Fe_3O_4/chitosan) was prepared using Fe_3O_4 magnetic nanoparticles and natural macromolecule (chitosan). Then an innovative biosensor was constructed based on an easy and effective Hemoglobin (Hb) immobilization method with the“sandwich”configuration of Fe_3O_4/CS-Hb-Fe_3O_4/CS. The effects of some experimental variables such as amount of glutaraldehyde, the concentration of HQ, and the working potential were investigated in the presence of the mediator of hydroquinone (HQ). This biosensor had a fast response to H_2O_2 less than 10 s and excellent linear relationships were obtained in the concentration range of 5.0×10-5 to 1.8×10~(-3) mol/L and 1.8×10~(-3) to 6.8×10~(-3) mol/L with the detection limit of 4.0×10~(-6) mol/L (S/N=3) under the optimum conditions. The apparent Michaelis-Menten constant Km was 0.29 mmol/L and it illustrated the excellent biological activity of the fixed Hb. Moreover, the biosensor had long-time stability and good reproducibility. And this method has been used to determine H_2O_2 concentration in the real sample.
(3) A novel type of Fe_3O_4 nanoparticles modified glass carbon electrode (Fe_3O_4/GCE) was constructed and the electrochemical properties of N-(4-nitro-2-phenoxyphenyl) methanesulfonamide (nimesulide) were studied on the Fe_3O_4/GCE. In 0.4 mol/L HAc-NaAc (pH 5.0) buffer solution, the electrode process of the nimesulide was irreversible at bare GCE or Fe_3O_4/GCE. The Fe_3O_4/GCE exhibited a remarkable catalytic and enhancement effect on reductin of the nimesulide. The reduction peak potential of nimesulide shifted positively from -0.707 V at bare GCE to -0.682 V at Fe_3O_4/GCE, and the sensitivity increased ca 3 times. Some experimental conditions were optimized. The linear range between the peak current and the concentration of nimesulide was 2.6×10~(-6)～1.0×10~(-4) mol/L (R=0.993) with a detection limit of 1.3×10~(-7) mol/L. This method has been used to determine the content of nimesulide in medical tablets. The recovery was determined to be 96.9%～101.9% by means of standard addition method. Compared with ultraviolet spectrometry, the method is not remarkable difference.
(4) A novel type of cobalt nanoparticles modified glass carbon electrode (CoNP/GCE) was constructed and the electrochemical properties of chloramphenicol (CAP) were studied on the CoNP/GCE. In 0.3 mol/L NH3-NH4Cl (pH 10.0) buffer solution, the electrode process of CAP was irreversible at bare GCE or CoNP/GCE. The CoNP/GCE exhibited a remarkable enhancement effect on reductin of CAP. The sensitivity increased ca 6 times. Some experimental conditions were optimized. The linear range between the peak current and the concentration of CAP was 5.0×10~(-6)～1.2×10~(-4) mol/L (R=0.997) with a detection limit of 3.0×10~(-7) mol/L. This method has been used to determine the content of CAP in the real sample. The recovery was determined to be 98.7%～102.2% by means of standard addition method.
(5) A novel third-generation hydrogen peroxide (H_2O_2) biosensor (Hb/CdS/MWNTs/GCE) was fabricated through hemoglobin (Hb) adsorbed onto the mercaptoacetic acid modified CdS QDs/carboxyl multiwall carbon nanotubes (MWNTs) films. Cyclic voltammogram of Hb/CdS/MWNTs/GCE showed a pair of well-defined and quasi-reversible redox peaks with the formal potential (E0') of -0.230 V (vs. Ag/AgCl) in 0.1 mol/L pH 8.0 phosphate buffer solution (PBS), which was the characteristic of the Hb heme Fe(Ⅲ)/Fe(Ⅱ) redox couples. The biosensor showed excellent electrocatalytic activity to the reduction of H_2O_2. The response time to H_2O_2 of the designed biosensor at a potential of -0.30 V was less than 2 s and linear relationships were obtained in the concentration range of 2.0×10~(-6)～2.7×10~(-3) mol/L and 2.7×10~(-3)～7.7×10~(-3) mol/L with the detection limit of 3.0×10~(-7) mol/L (S/N=3). The apparent Michaelis-Menten constant Km was estimated to be 1.324 mmol/L and it illustrated the excellent biological activity of the fixed Hb.
(6) A new method for the determination of Nimesulide was established based on the multiwalled carbon nanotubes (MWCNTs) modified glassy carbon electrode (MWCNTs/GCE). In 0.2 mol/L PBS (pH 6.6) buffer solution, the MWCNTs/GCE showed a remarkable catalytic and enhancement effect on reduction of the nimesulide. The reductoin peak potential of nimesulide shifted positively from -0.665 V at bare GCE to -0.553 V at MWCNTs/GCE, and the sensitivity increased ca 7 times. A linear dynamic range of 3.2×10~(-7) mol/L to 6.5×10-5 mol/L (R=0.999) with a detection limit of 1.6×10~(-7) mol/L was obtained. The electrochemical behaviors of nimesulide were studied and this method has been used to determine the content of nimesulide in medical tablets. Compared with UV-Vis spectrometry, the method was not remarkable difference.
本论文的主要内容如下:
(1)制备了壳聚糖(CS)包裹四氧化三铁的磁性微球(Fe_3O_4/CS),并将其用于固定辣根过氧化物酶(HRP),制得了测定过氧化氢的电化学生物传感器(HRP-Fe_3O_4/CS-GCE)。考察了各种实验变量如支持电解质浓度、pH、酶的用量、媒介体亚甲基蓝浓度、工作电位等对生物传感器性能的影响。对H_2O_2进行测定,线性范围为2.0×10~(-4)～1.2×10-2 mol/L,检出限为1.0×10~(-4) mol/L(S/N=3),响应时间小于12 s。该传感器的表观米氏常数为21.4 mmol/L,表明所固定的酶具有较高的生物活性。该生物传感器具有高的稳定性和良好的重现性,用该法对实际样品进行了测定。
(2)制备了壳聚糖(CS)包裹四氧化三铁的磁性微球(Fe_3O_4/CS),利用吸附交联作用,制得了磁性纳米微球-血红蛋白夹心法修饰的玻碳电极(Fe_3O_4/CS-Hb-Fe_3O_4/CS-GCE)。以对苯二酚(HQ)为电子媒介体,考察了各种实验变量如支持电解质浓度、pH、酶的用量、戊二醛的用量、对苯二酚浓度、工作电位等对生物传感器性能的影响。用计时电流法对H_2O_2进行测定,线性范围分别为5.0×10-5～1.8×10~(-3) mol/L和1.8×10~(-3)～6.8×10~(-3) mol/L,检出限为4.0×10~(-6) mol/L(S/N=3),响应时间小于10 s。表观米氏常数Km为0.29 mmol/L,表明固定的Hb有较好的生物活性。该生物传感器具有高的稳定性和良好的重现性,已用于实际样品测定。
(3)研究了纳米四氧化三铁化学修饰电极对非甾体抗炎药尼美舒利(nimesulide)的测定。实验结果表明,在0.4 mol/L HAc-NaAc(pH 5.0)溶液中,尼美舒利在裸玻碳电极或纳米四氧化三铁修饰电极上均发生不可逆还原反应,但与裸玻碳电极相比,纳米四氧化三铁修饰电极对尼美舒利具有明显的催化和增敏作用,峰电位由-0.707 V(裸电极)正移到-0.682 V(vs. Ag/AgCl)(修饰电极),峰电位正移25 mV,灵敏度增加约3倍。对各种实验条件进行了优化,测得峰电流Ip与尼美舒利浓度在2.6×10~(-6)～1.0×10~(-4) mol/L范围内呈良好的线性关系,相关系数为0.993,检出限为1.3×10~(-7) mol/L。利用优化后的条件对尼美舒利药片进行了测定,测量值与标示值符合,回收率在96.9%～101.9%,并与紫外分光光度法进行了比较,无显著性差异。
(4)研究了氯霉素在纳米钴修饰玻碳电极上的电化学行为及测定。实验结果表明,在0.3 mol/L NH3-NH4Cl(pH=10.0)缓冲液中,氯霉素在裸玻碳电极或纳米钴修饰电极上均发生不可逆还原反应,但与裸玻碳电极相比,纳米钴修饰电极对氯霉素的还原具有明显的增敏作用,灵敏度增加约6倍。对支持电解质、修饰剂用量等各种实验条件进行了优化。测得峰电流Ip与氯霉素浓度在5.0×10~(-6)～1.2×10~(-4) mol/L范围内呈良好的线性关系,相关系数为0.997,检出限为3.0×10~(-7) mol/L。利用优化后的条件对氯霉素滴眼液进行了测定,测量值与标示值符合,回收率在98.7%～102.2%。
(5)将血红蛋白吸附在巯基乙酸包覆的硫化镉/羧基化多壁碳纳米管膜上,制得了测定过氧化氢的电化学生物传感器(Hb/CdS/MWNTs/GCE)。实验表明在pH 8.0的磷酸盐缓冲溶液中循环伏安扫描出现一对峰形良好的准可逆氧化还原峰,为Hb Fe(Ⅲ)/Fe(Ⅱ)电对的特征峰,其式量电位(E0')为-0.230 V(vs. Ag/AgCl)。该修饰电极对H_2O_2表现出较好的电催化行为,线性范围为2.0×10~(-6)～2.7×10~(-3) mol/L和2.7×10~(-3)～7.7×10~(-3) mol/L,检出限为3.0×10~(-7) mol/L(S/N=3),响应时间小于2 s。该传感器的表观米氏常数为1.324 mmol/L,表明所固定的血红蛋白具有较高的生物活性。
(6)研究了以Nafion分散功能化多壁碳纳米管化学修饰电极对尼美舒利的测定。实验结果表明,在0.2 mol/L PBS(pH 6.6)溶液中,碳纳米管修饰电极对尼美舒利具有明显的催化和增敏作用。峰电位由-0.665 V(裸电极)正移到-0.553 V(修饰电极),峰电位正移112 mV,灵敏度增加约7倍。峰电流Ip与尼美舒利浓度在3.2×10~(-7)～6.5×10-5 mol/L范围内呈良好的线性关系,检出限为1.6×10~(-7) mol/L。研究了尼美舒利的电化学行为,利用优化后的条件对尼美舒利药片进行了测定,测量值与标示值符合。并且与紫外分光光度法比较进行了比较,无显著性差异。
Nanomaterials are the research focus in modern materials science. Considerable research efforts are being devoted toward the development of the chemical modified electrodes and electrochemical biosensors based on the nanomaterials. The main work of this paper is focus on the synthesis of magnetic nanoparticles (Fe_3O_4 nanoparticles) and quantum dots (CdS) and the fabrication of the modified electrodes based on the nanomaterials. Besides, some electrochemical biosensors have been developed. Also, the biosensors have been used to determine the real samples. The main results are listed below:
(1) A new type of amperometric hydrogen peroxide biosensor was constructed based on horseradish peroxidase (HRP) immobilized on Fe_3O_4/chitosan modified glass carbon electrode. The effects of some experimental variables such as the concentration of supporting electrolyte, pH, enzyme loading, the concentration of the mediator of methylene blue (MB) and the applied potential were investigated. The linear range of the calibration curve for H_2O_2 was 2.0×10~(-4)～1.2×10-2 mol/L and the detection limit was 1.0×10~(-4) mol/L (S/N=3). The response time was less than 12 s. The apparent Michaelis-Menten constant Km was 21.4 mmol/L and it illustrated the excellent biological activity of the fixed enzyme. In addition, the biosensor had long-time stability and good reproducibility. And this method has been used to determine H_2O_2 concentration in the real sample.
(2) A novel magnetic microsphere (Fe_3O_4/chitosan) was prepared using Fe_3O_4 magnetic nanoparticles and natural macromolecule (chitosan). Then an innovative biosensor was constructed based on an easy and effective Hemoglobin (Hb) immobilization method with the“sandwich”configuration of Fe_3O_4/CS-Hb-Fe_3O_4/CS. The effects of some experimental variables such as amount of glutaraldehyde, the concentration of HQ, and the working potential were investigated in the presence of the mediator of hydroquinone (HQ). This biosensor had a fast response to H_2O_2 less than 10 s and excellent linear relationships were obtained in the concentration range of 5.0×10-5 to 1.8×10~(-3) mol/L and 1.8×10~(-3) to 6.8×10~(-3) mol/L with the detection limit of 4.0×10~(-6) mol/L (S/N=3) under the optimum conditions. The apparent Michaelis-Menten constant Km was 0.29 mmol/L and it illustrated the excellent biological activity of the fixed Hb. Moreover, the biosensor had long-time stability and good reproducibility. And this method has been used to determine H_2O_2 concentration in the real sample.
(3) A novel type of Fe_3O_4 nanoparticles modified glass carbon electrode (Fe_3O_4/GCE) was constructed and the electrochemical properties of N-(4-nitro-2-phenoxyphenyl) methanesulfonamide (nimesulide) were studied on the Fe_3O_4/GCE. In 0.4 mol/L HAc-NaAc (pH 5.0) buffer solution, the electrode process of the nimesulide was irreversible at bare GCE or Fe_3O_4/GCE. The Fe_3O_4/GCE exhibited a remarkable catalytic and enhancement effect on reductin of the nimesulide. The reduction peak potential of nimesulide shifted positively from -0.707 V at bare GCE to -0.682 V at Fe_3O_4/GCE, and the sensitivity increased ca 3 times. Some experimental conditions were optimized. The linear range between the peak current and the concentration of nimesulide was 2.6×10~(-6)～1.0×10~(-4) mol/L (R=0.993) with a detection limit of 1.3×10~(-7) mol/L. This method has been used to determine the content of nimesulide in medical tablets. The recovery was determined to be 96.9%～101.9% by means of standard addition method. Compared with ultraviolet spectrometry, the method is not remarkable difference.
(4) A novel type of cobalt nanoparticles modified glass carbon electrode (CoNP/GCE) was constructed and the electrochemical properties of chloramphenicol (CAP) were studied on the CoNP/GCE. In 0.3 mol/L NH3-NH4Cl (pH 10.0) buffer solution, the electrode process of CAP was irreversible at bare GCE or CoNP/GCE. The CoNP/GCE exhibited a remarkable enhancement effect on reductin of CAP. The sensitivity increased ca 6 times. Some experimental conditions were optimized. The linear range between the peak current and the concentration of CAP was 5.0×10~(-6)～1.2×10~(-4) mol/L (R=0.997) with a detection limit of 3.0×10~(-7) mol/L. This method has been used to determine the content of CAP in the real sample. The recovery was determined to be 98.7%～102.2% by means of standard addition method.
(5) A novel third-generation hydrogen peroxide (H_2O_2) biosensor (Hb/CdS/MWNTs/GCE) was fabricated through hemoglobin (Hb) adsorbed onto the mercaptoacetic acid modified CdS QDs/carboxyl multiwall carbon nanotubes (MWNTs) films. Cyclic voltammogram of Hb/CdS/MWNTs/GCE showed a pair of well-defined and quasi-reversible redox peaks with the formal potential (E0') of -0.230 V (vs. Ag/AgCl) in 0.1 mol/L pH 8.0 phosphate buffer solution (PBS), which was the characteristic of the Hb heme Fe(Ⅲ)/Fe(Ⅱ) redox couples. The biosensor showed excellent electrocatalytic activity to the reduction of H_2O_2. The response time to H_2O_2 of the designed biosensor at a potential of -0.30 V was less than 2 s and linear relationships were obtained in the concentration range of 2.0×10~(-6)～2.7×10~(-3) mol/L and 2.7×10~(-3)～7.7×10~(-3) mol/L with the detection limit of 3.0×10~(-7) mol/L (S/N=3). The apparent Michaelis-Menten constant Km was estimated to be 1.324 mmol/L and it illustrated the excellent biological activity of the fixed Hb.
(6) A new method for the determination of Nimesulide was established based on the multiwalled carbon nanotubes (MWCNTs) modified glassy carbon electrode (MWCNTs/GCE). In 0.2 mol/L PBS (pH 6.6) buffer solution, the MWCNTs/GCE showed a remarkable catalytic and enhancement effect on reduction of the nimesulide. The reductoin peak potential of nimesulide shifted positively from -0.665 V at bare GCE to -0.553 V at MWCNTs/GCE, and the sensitivity increased ca 7 times. A linear dynamic range of 3.2×10~(-7) mol/L to 6.5×10-5 mol/L (R=0.999) with a detection limit of 1.6×10~(-7) mol/L was obtained. The electrochemical behaviors of nimesulide were studied and this method has been used to determine the content of nimesulide in medical tablets. Compared with UV-Vis spectrometry, the method was not remarkable difference.
引文
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