基于多壁碳纳米管和4-SPCE的福氏志贺氏菌酶免疫传感器的研制
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摘要
志贺氏菌是引起人类传染性疾病的主要病原体之一,平均每年都有上百万人感染痢疾。由志贺氏菌感染而引发的痢疾每年造成100多万人死亡,其中大多数是发展中国家的儿童。因此,志贺氏菌的检测对全球性痢疾和死亡的威胁非常重要,也对公众健康非常重要。
过去几十年里,基于抗原-抗体特异性反应的电化学免疫传感器直是研究热点,广泛应用于食品分析、临床诊断、环境监测等研究领域。新型纳米材料和纳米技术的迅速发展为电分析化学提供了更多新的机会,多壁碳纳米管具有生物电化学稳定性和明显促进生物分子的电子传递作用,能作为生物活性物质的固定基质和构建生物传感器,在生物传感器领域具有较大的应用前景。
本论文主要围绕以下几个方面开展了一些研究工作：
1福氏志贺氏菌抗原的制备与灭活
本试验成功制备了福氏志贺氏菌抗原,平板计数得抗原浓度为1011cfu/mL,然后以0.4%(v／v)甲醛灭活12 h。抗原形态学与文献报道一致,具有良好的生物活性和特异性,为后续工作的顺利开展奠定了基础。
2多壁碳纳米管修饰的4-SPCE直接检测过氧化氢的研究
构建用于过氧化氢检测的多壁碳纳米管修饰四通道丝网印刷碳电极,循环伏安阳极最大电流法测试结果表明：多壁碳纳米管能提高电极的有效表面积和加速电子的传递,循环伏安阳极最大电流与过氧化氢的浓度变化成线性关系,灵敏度和线性相关系数分别为0.3192μA/(mmol/L)和0.9979；检测方法过程简单,结果令人满意,为后续免疫传感器的研究打下了很好的基础。
3基于多壁碳纳米管／DMF复合物和4-SPCE的福氏志贺氏菌酶免疫传感器的研制
利用戊二醛交联法将辣根过氧化物酶标记的福氏志贺氏菌抗体固定在多壁碳纳米管修饰的四通道丝网印刷碳电极表面,制备了可用于检测福氏志贺氏菌的酶免疫传感器。采用循环伏安法对不同修饰电极进行电化学表征。根据抗原-抗体特异性结合形成的免疫复合物使敏感膜有效扩散截面积减小的特性,采用循环伏安法检测样品中的福氏志贺氏菌。在优化的实验条件下,该酶免疫传感器对福氏志贺氏菌的检测范围为104～109cfu/mL,检出限为3.4×103cfu/mL(S/N=3)。该酶传感器还具有较好的选择性、重现性、稳定性和准确性
4基于多壁碳纳米管／壳聚糖复合物和4-SPCE的福氏志贺氏菌酶免疫传感器的研制
为研究快速检测福氏志贺氏菌的电化学免疫传感器,将羧基化多壁碳纳米管与壳聚糖制备成复合物,应用此复合物将辣根过氧化物酶标记的福氏志贺氏菌抗体一步直接固定在四通道丝网印刷碳电极表面,制成快速检测福氏志贺氏菌的酶免疫传感器。采用原子力显微镜表征不同修饰电极的表面形态,循环伏安法考察不同电极的电化学特性和监测酶促反应,利用免疫反应前后还原峰峰电流的减小来测定福氏志贺氏菌。在优化的测定条件下,免疫电极对福氏志贺氏菌的检测范围为104～109cfu/mL,检出限为2.3×104 cfu/mL(S/N=3)。而且该酶免疫传感器具有较好的特异性、重现性、稳定性和准确性,该方法还具有快速、简便、易于操作和价格低廉等优点,具有用十福氏志贺氏菌快速筛检的潜力。
5基于多壁碳纳米管／海藻酸钠复合物和4-SPCE的福氏志贺氏菌酶免疫传感器的研制
为快速检测福氏志贺氏菌,将辣根过氧化物酶标记的福氏志贺氏菌抗体吸附在多壁碳纳米管／海藻酸钠复合物修饰的四通道丝网印刷碳电极表面,制得快速检测福氏志贺氏菌的酶免疫传感器。采用原子力显微镜表征不同修饰电极的表面形态,循环伏安法考察不同电极的电化学特性,采用一步免疫法检测福氏志贺氏菌和循环伏安法监测酶促反应,根据免疫反应前后还原峰峰电流的降低值来检测样品中的福氏志贺氏菌。在优化的实验条件下,酶免疫传感器与福氏志贺氏菌浓度的对数在104～1010 cfu/mL范围内保持良好的线性关系,检出限为3.1×103 cfu/mL(S/N=3)。该酶免疫传感器具有较好的特异性、重现性、稳定性和准确性,可望初步用于福氏志贺氏菌的快速筛检。
Shigella spp is one of the major causes of human infectious diseases and is responsible for millions of cases of diarrhea worldwide every year. More than one million deaths occur yearly due to infections with Shigella spp. and the victims are mostly children of the developing world. So as to deal with the global threat of disease and death caused by Shigella, its detection is very important for public health.
In the past decades, electrochemical immunosensors based on the specificity of antigen-antibody interactions with electrochemical transduction, have become an attractive subject for food analysis, clinical diagnosis, environmental monitoring and other fields.
The rapid development of new nanomaterials and nanotechnologies has provided many new opportunities for electroanalysis. Particularly, multi-wall carbon nanotubes (MWCNT) exhibit promising potential within the realm of bioelectrochemistry as the matrix to incorporate biorecognition elements and construct biosensors.
The comments of this paper was described as follows:
1 Preparation of Shigella flexneri
In this experiment, Shigella flexneri (S. flexneri) was successfully prepared. The concentration of S. flexneri (1011 cfu/mL) was acquired by plate colony-counting method. Then inactivated with formaldehyde at a concentration of 0.4%(v/v) for 12 h at room temperature. Experimental results showed that S. flexneri possessed good bioactivity. Following experiment could be successfully carried on because of the supportment of the above work.
2 MWCNT modified four-channel screen-printed carbon electrode for hydrogen peroxide determination
A MWCNT modified four-channel screen-printed carbon electrode was successfully constructed to be used to detect hydrogen peroxide. Cyclic voltammetry (CV) was used to measure the concentration of hydrogen peroxide. The results showed that the MWCNT could enlarge the effective surface area of four-channel screen-printed carbon electrode and could enormously enhance the electron transfer. The sensitivity and correlation coefficient for hydrogen peroxide detection were 0.3192μA/(mmol/L) and 0.9979. The procedure is simple and the results obtained are satisfied.
3 Fabrication of enzymatic immunosensor for Shigella flexneri detection based on MWCNT/DMF composite and four-channel screen-printed carbon electrode
An immunosensor based on MWCNT/DMF composite was successfully constructed. The horseradish peroxidase labeled antibodies to S. flexneri (HRP-anti-S. flexneri) was co-immobilized on the four-channel screen-printed carbon electrode surface by crosslinking with glutaraldehyde to form a combined sensing and electron transfer system. CV was carried out to characterize the electrochemical properties of different electrodes and to detect S. flexneri. When the antigen (S. flexneri) was bound to the antibody on the surface of the sensing film, a "barrier" occurred between the electrode and the redox centres in the bound HRP. Under optimal conditions, concentrations of S. flexneri from 104 to 109 cfu/mL could be detected, with a detection limit of 3.4×103 cfu/mL (S/N=3).The immunosensor showed satisfactory selectivity, reproducibility, stability and accuracy.
4 Rapid detection of Shigella flexneri with MWCNT/chitosan modified four-channel screen-printed carbon electrode
A disposable S. flexneri immunosensor based on MWCNT/chitosan/ HRP-anti-S. flexneri biocomposite on four-channel screen-printed carbon electrode without any other cross-linked reagents has been developed. The MWCNT/chitosan matrix provided a congenial microenvironment for the immobilization of HRP-anti-S. flexneri and promoted the electron transfer. Due to the strong electrocatalytic properties of HRP and MWCNT toward H2O2, the current signal was significantly amplified. The atomic force microscope (AFM) technology was used to characterize the surface morphologies of different electrodes. And CV was carried out to characterize the electrochemical properties of different electrodes and detect S. flexneri. Under optimal conditions, concentrations of S. flexneri from 104 to 109 cfu/mL could be detected, with a detection limit of 2.3×104 cfu/mL (S/N=3). The proposed immunosensor displayed a satisfactory stability, reproducibility and accuracy. Moreover, the immunosensor was inexpensive, relatively simple to fabricate and had a potential possibility for early assessment of S. flexneri.
5 A disposable immunosensor for Shigella flexneri based on MWCNT/sodium alginate composite and four-channel screen-printed carbon electrode
A novel S. flexneri immunosensor based on HRP-anti-S. flexneri immobilized by physical adsorption on the MWCNT/sodium alginate (MWCNT/SA) composite modified four-channel screen-printed carbon electrode surface was successfully fabricated. In this strategy, MWCNT/SA biocomposite acted as the matrix to adsorb and immobilize HRP-anti-S. flexneri. The preparation process of modified electrodes was characterized with AFM and CV. The analytical performance of proposed immunosensor toward S. flexneri was investigated by CV. Under optimal conditions, the concentration of S. flexneri from 104 to 1010 cfu mL-1 could be detected, with a detection limit of 3.1×103 cfu/mL (S/N=3). The specificity, reproducibility, stability and accuracy of the proposed immunosensor were also evaluated. The proposed immunosensor showed simply fabricative, economical, efficient and potential application for early assessment of S. flexneri.
过去几十年里,基于抗原-抗体特异性反应的电化学免疫传感器直是研究热点,广泛应用于食品分析、临床诊断、环境监测等研究领域。新型纳米材料和纳米技术的迅速发展为电分析化学提供了更多新的机会,多壁碳纳米管具有生物电化学稳定性和明显促进生物分子的电子传递作用,能作为生物活性物质的固定基质和构建生物传感器,在生物传感器领域具有较大的应用前景。
本论文主要围绕以下几个方面开展了一些研究工作：
1福氏志贺氏菌抗原的制备与灭活
本试验成功制备了福氏志贺氏菌抗原,平板计数得抗原浓度为1011cfu/mL,然后以0.4%(v／v)甲醛灭活12 h。抗原形态学与文献报道一致,具有良好的生物活性和特异性,为后续工作的顺利开展奠定了基础。
2多壁碳纳米管修饰的4-SPCE直接检测过氧化氢的研究
构建用于过氧化氢检测的多壁碳纳米管修饰四通道丝网印刷碳电极,循环伏安阳极最大电流法测试结果表明：多壁碳纳米管能提高电极的有效表面积和加速电子的传递,循环伏安阳极最大电流与过氧化氢的浓度变化成线性关系,灵敏度和线性相关系数分别为0.3192μA/(mmol/L)和0.9979；检测方法过程简单,结果令人满意,为后续免疫传感器的研究打下了很好的基础。
3基于多壁碳纳米管／DMF复合物和4-SPCE的福氏志贺氏菌酶免疫传感器的研制
利用戊二醛交联法将辣根过氧化物酶标记的福氏志贺氏菌抗体固定在多壁碳纳米管修饰的四通道丝网印刷碳电极表面,制备了可用于检测福氏志贺氏菌的酶免疫传感器。采用循环伏安法对不同修饰电极进行电化学表征。根据抗原-抗体特异性结合形成的免疫复合物使敏感膜有效扩散截面积减小的特性,采用循环伏安法检测样品中的福氏志贺氏菌。在优化的实验条件下,该酶免疫传感器对福氏志贺氏菌的检测范围为104～109cfu/mL,检出限为3.4×103cfu/mL(S/N=3)。该酶传感器还具有较好的选择性、重现性、稳定性和准确性
4基于多壁碳纳米管／壳聚糖复合物和4-SPCE的福氏志贺氏菌酶免疫传感器的研制
为研究快速检测福氏志贺氏菌的电化学免疫传感器,将羧基化多壁碳纳米管与壳聚糖制备成复合物,应用此复合物将辣根过氧化物酶标记的福氏志贺氏菌抗体一步直接固定在四通道丝网印刷碳电极表面,制成快速检测福氏志贺氏菌的酶免疫传感器。采用原子力显微镜表征不同修饰电极的表面形态,循环伏安法考察不同电极的电化学特性和监测酶促反应,利用免疫反应前后还原峰峰电流的减小来测定福氏志贺氏菌。在优化的测定条件下,免疫电极对福氏志贺氏菌的检测范围为104～109cfu/mL,检出限为2.3×104 cfu/mL(S/N=3)。而且该酶免疫传感器具有较好的特异性、重现性、稳定性和准确性,该方法还具有快速、简便、易于操作和价格低廉等优点,具有用十福氏志贺氏菌快速筛检的潜力。
5基于多壁碳纳米管／海藻酸钠复合物和4-SPCE的福氏志贺氏菌酶免疫传感器的研制
为快速检测福氏志贺氏菌,将辣根过氧化物酶标记的福氏志贺氏菌抗体吸附在多壁碳纳米管／海藻酸钠复合物修饰的四通道丝网印刷碳电极表面,制得快速检测福氏志贺氏菌的酶免疫传感器。采用原子力显微镜表征不同修饰电极的表面形态,循环伏安法考察不同电极的电化学特性,采用一步免疫法检测福氏志贺氏菌和循环伏安法监测酶促反应,根据免疫反应前后还原峰峰电流的降低值来检测样品中的福氏志贺氏菌。在优化的实验条件下,酶免疫传感器与福氏志贺氏菌浓度的对数在104～1010 cfu/mL范围内保持良好的线性关系,检出限为3.1×103 cfu/mL(S/N=3)。该酶免疫传感器具有较好的特异性、重现性、稳定性和准确性,可望初步用于福氏志贺氏菌的快速筛检。
Shigella spp is one of the major causes of human infectious diseases and is responsible for millions of cases of diarrhea worldwide every year. More than one million deaths occur yearly due to infections with Shigella spp. and the victims are mostly children of the developing world. So as to deal with the global threat of disease and death caused by Shigella, its detection is very important for public health.
In the past decades, electrochemical immunosensors based on the specificity of antigen-antibody interactions with electrochemical transduction, have become an attractive subject for food analysis, clinical diagnosis, environmental monitoring and other fields.
The rapid development of new nanomaterials and nanotechnologies has provided many new opportunities for electroanalysis. Particularly, multi-wall carbon nanotubes (MWCNT) exhibit promising potential within the realm of bioelectrochemistry as the matrix to incorporate biorecognition elements and construct biosensors.
The comments of this paper was described as follows:
1 Preparation of Shigella flexneri
In this experiment, Shigella flexneri (S. flexneri) was successfully prepared. The concentration of S. flexneri (1011 cfu/mL) was acquired by plate colony-counting method. Then inactivated with formaldehyde at a concentration of 0.4%(v/v) for 12 h at room temperature. Experimental results showed that S. flexneri possessed good bioactivity. Following experiment could be successfully carried on because of the supportment of the above work.
2 MWCNT modified four-channel screen-printed carbon electrode for hydrogen peroxide determination
A MWCNT modified four-channel screen-printed carbon electrode was successfully constructed to be used to detect hydrogen peroxide. Cyclic voltammetry (CV) was used to measure the concentration of hydrogen peroxide. The results showed that the MWCNT could enlarge the effective surface area of four-channel screen-printed carbon electrode and could enormously enhance the electron transfer. The sensitivity and correlation coefficient for hydrogen peroxide detection were 0.3192μA/(mmol/L) and 0.9979. The procedure is simple and the results obtained are satisfied.
3 Fabrication of enzymatic immunosensor for Shigella flexneri detection based on MWCNT/DMF composite and four-channel screen-printed carbon electrode
An immunosensor based on MWCNT/DMF composite was successfully constructed. The horseradish peroxidase labeled antibodies to S. flexneri (HRP-anti-S. flexneri) was co-immobilized on the four-channel screen-printed carbon electrode surface by crosslinking with glutaraldehyde to form a combined sensing and electron transfer system. CV was carried out to characterize the electrochemical properties of different electrodes and to detect S. flexneri. When the antigen (S. flexneri) was bound to the antibody on the surface of the sensing film, a "barrier" occurred between the electrode and the redox centres in the bound HRP. Under optimal conditions, concentrations of S. flexneri from 104 to 109 cfu/mL could be detected, with a detection limit of 3.4×103 cfu/mL (S/N=3).The immunosensor showed satisfactory selectivity, reproducibility, stability and accuracy.
4 Rapid detection of Shigella flexneri with MWCNT/chitosan modified four-channel screen-printed carbon electrode
A disposable S. flexneri immunosensor based on MWCNT/chitosan/ HRP-anti-S. flexneri biocomposite on four-channel screen-printed carbon electrode without any other cross-linked reagents has been developed. The MWCNT/chitosan matrix provided a congenial microenvironment for the immobilization of HRP-anti-S. flexneri and promoted the electron transfer. Due to the strong electrocatalytic properties of HRP and MWCNT toward H2O2, the current signal was significantly amplified. The atomic force microscope (AFM) technology was used to characterize the surface morphologies of different electrodes. And CV was carried out to characterize the electrochemical properties of different electrodes and detect S. flexneri. Under optimal conditions, concentrations of S. flexneri from 104 to 109 cfu/mL could be detected, with a detection limit of 2.3×104 cfu/mL (S/N=3). The proposed immunosensor displayed a satisfactory stability, reproducibility and accuracy. Moreover, the immunosensor was inexpensive, relatively simple to fabricate and had a potential possibility for early assessment of S. flexneri.
5 A disposable immunosensor for Shigella flexneri based on MWCNT/sodium alginate composite and four-channel screen-printed carbon electrode
A novel S. flexneri immunosensor based on HRP-anti-S. flexneri immobilized by physical adsorption on the MWCNT/sodium alginate (MWCNT/SA) composite modified four-channel screen-printed carbon electrode surface was successfully fabricated. In this strategy, MWCNT/SA biocomposite acted as the matrix to adsorb and immobilize HRP-anti-S. flexneri. The preparation process of modified electrodes was characterized with AFM and CV. The analytical performance of proposed immunosensor toward S. flexneri was investigated by CV. Under optimal conditions, the concentration of S. flexneri from 104 to 1010 cfu mL-1 could be detected, with a detection limit of 3.1×103 cfu/mL (S/N=3). The specificity, reproducibility, stability and accuracy of the proposed immunosensor were also evaluated. The proposed immunosensor showed simply fabricative, economical, efficient and potential application for early assessment of S. flexneri.
引文
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