基于纳米氧化物和碳纳米管的免疫传感器的研究
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摘要
电化学免疫传感器在临床医学、环境和食品工业等方面都有重要应用,并以其体积小、专一性强、灵敏度高、检测快速方便、成本低和容易实现实时在线活体检测等优点,成为当前研究的热点之一。而传感器的性能主要取决于生物活性物质的固定方法,因此如何将生物活性组分有效地固定在电极表面上并保持其良好的生物活性是传感器研究和开发中最为重要的工作。本文利用二氧化硅-亚甲基蓝纳米复合物、纳米二氧化锰-壳聚糖和纳米金及碳纳米管等新型材料作为构建免疫传感器的基底,运用包埋、吸附、沉积、聚合等固定方法,直接测定抗原与抗体反应前后的电流的变化。
1.基于壳聚糖包埋二氧化硅-亚甲基蓝纳米复合的电流型癌胚抗原免疫传感器的研究
提出了一种新型的基于二氧化硅-亚甲基蓝纳米复合物和壳聚糖制备的一种新型的电流型癌胚抗原免疫传感器。利用溶胶-凝胶法形成二氧化硅-亚甲基蓝-壳聚糖复合膜,再利用壳聚糖丰富的氨基吸附纳米金,从而通过比表面积大,生物相容性好的纳米金胶吸附大量抗体,制得稳定性较好的免疫传感器。采用循环伏安法(CV)和透射电子显微镜对电极的层层组装过程进行了考察,并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是1.0-80.0 ng·mL~(-1),线性相关系数为0.9978,检出限为0.333 ng·mL~(-1)。
2.基于纳米金/纳米二氧化锰-壳聚糖复合物/普鲁士蓝修饰的金电极的免疫传感器研究
采用电沉积普鲁士蓝在电极表面形成一层均匀的电子媒介体层,增大电极的电流响应信号,再利用静电吸附将制备的纳米二氧化锰-壳聚糖固定在电极上,然后利用壳聚糖上丰富的氨基吸附纳米金,最后通过吸附性能强,生物兼容性好的双层纳米金吸附癌胚抗体,从而制得癌胚免疫传感器。采用循环伏安法(CV)对修饰电极进行了考察,该免疫传感器线性范围是0.25-8.0 ng·mL~(-1)和8.0-100 ng·mL~(-1),线性相关系数分别为0.9982,检出限为0.083 ng·mL~(-1),将其用于病人的血清样品分析,与现行的ELISA法的结果相比较,有很好的相关性。
3.基于聚苯乙烯磺酸钠接枝的多壁碳纳米管/铁氰化镍/纳米金修饰的电流型前列腺抗原免疫传感器的研究
将聚乙烯磺酸钠均匀分散在多壁碳纳米管中,并采用物理吸附的方法固定在洁净的金电极表面形成一层有别于经典的十二磺酸钠分散的多壁碳纳米管,其优点在于可以减小背景电流,而且电流的循环伏安电位也向正的方向偏移,有利于我们实验研究的考察,再利用电聚合铁氰化镍将其固定在致密的碳纳米管层表面,最后通过电还原氯金酸制得均匀的纳米金层以吸附前列腺癌抗体,从而制得前列腺癌免疫传感器。采用循环伏安法(CV)对修饰电极进行了考察,研究了电极修饰过程中的测试液pH值、温度、孵育时间等实验条件和参数对传感器性能的影响。该传感器在PSA浓度为1.5-35 ng·mL~(-1)的范围内有良好的线性关系,并且制作简单,响应快,能满足实际测定的要求。
Electrochemical immunosensor are widely used for clinical diagnosis,environmental inspection and food analysis,and have been have gained considerable attention due to their small volume,intrinsic selectivity,fast analytical time,simple measurement,high sensitivity and low cost, It has been reported that the performance of biosensors largely contact with the fabrication of biorecognition molecule.Thus,it's important work how to immobilize effectively on the cleaned electrode with biorecognition molecule in high amounts with retention of their bioactivity.In this article,we have constructed several immunoelectrodes immobilized antibody onto substrate electrode based on SiO_2 and methylene blue(MB) nanocomposite,nano-MnO_2 and chitosan(CS) composite,gold nanoparticles(nano-Au) and multi-walled carbon nanotube(MWNT) as matrixes. The detailed materials are shown as follows:
1.Amperometrie immunosensor for carcinoembryonic antigen based on ehitosan entrapped SiO_2 and methylene blue nanoeomposite
An amperometric immunosensor for the detection of carcinoembryonic antigen(CEA) has been developed by means of sol-gel techniques.SiO_2+MB-CS nanocomposite membrane was combined.Subsequently,nano-Au was adsorbed by CS with a large amount of amido matrixes, which owned a high surface area,good bioconsistent.Carcinoembryonic antibody(anti-CEA) was adsorbed by nano-Au firmly.Then a potentiometric immunosensor with good sensitivity and stability was prepared.The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry(CV) and transmission electron microscope (TEM).The resulting immunosensor exhibits wide linear range from 1.0 to 80.0 ng·mL~(-1) with a detection limit of 0.333 ng·mL~(-1).The correlation coefficient is 0.9978.
2.Study on immunosensor based on gold nanoparticles/chitosan and MnO_2 nanoparticles composite membrane/prussian blue modified gold electrode
A novel amperometric immunosensor for the detection of carcinoembryonic antigen(CEA) has been developed by means of electrodeposition and electrostatic adsorption techniques.In this study,Prussian blue(PB) was used as a mediator to amplify the current signal.And nano-MnO_2/CS composite with positively charged was adsorbed on the negatively charged PB.Subsequently, nano-Au was immobilized by CS.A large amount of carcinoembryonic antibody(anti-CEA) was adsorbed by nano-Au.Then an amPerometric immunosensor with good sensitivity and stability was made.The characteristics of the modified electrode at different stages of modification were studied by using cyclic voltammetry(CV)and scanning electron microscope(SEM).The performances of the immunosensor were studied in detail.The immunosensor showed a specific response to CEA in the range 0.25 to 8.0 ng·mL~(-1) and 8.0 to 100 ng·mL~(-1) with a detection limit of 0.083 ng·mL~(-1).The correlation coefficient is 0.9982.The immunosensor was used to analyze the serum samples with satisfactory results.
3.An immunosensor based on poly(sodium 4-styresulfonate) grated-carbon nanotube/niekel hexacyanoferrate/gold nanoparticles modified gold electrode.
A novel immunosensor has been developed by electropolymerizing Nickel Hexacyanoferrate (NiHCF) on to a Poly(sodium 4-styrenesulfonate)(PSS)-grafted Multi-walled Carbon Nanotube (MWNT) and electro-depositing nano-Au for determination of prostate specific antigen(PSA). After the developed immunosensor was incubated with different concentrations of PSA samples at 25℃for 35 min,the current response decreased with an increasing PSA concentration in the sample solution.The decreased percentage of the current was proportional to PSA concentration ranging froml.5 to 35 ng·mL~(-1) PSA with a detection limit of 0.51 ng·mL~(-1) PSA(S/N = 3). Analytical results of 50 specimens using the developed immunosensor showed satisfactory agreement with those using ELISA,indicating the method to be a promising alternative for detecting PSA in clinical diagnosis.
1.基于壳聚糖包埋二氧化硅-亚甲基蓝纳米复合的电流型癌胚抗原免疫传感器的研究
提出了一种新型的基于二氧化硅-亚甲基蓝纳米复合物和壳聚糖制备的一种新型的电流型癌胚抗原免疫传感器。利用溶胶-凝胶法形成二氧化硅-亚甲基蓝-壳聚糖复合膜,再利用壳聚糖丰富的氨基吸附纳米金,从而通过比表面积大,生物相容性好的纳米金胶吸附大量抗体,制得稳定性较好的免疫传感器。采用循环伏安法(CV)和透射电子显微镜对电极的层层组装过程进行了考察,并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是1.0-80.0 ng·mL~(-1),线性相关系数为0.9978,检出限为0.333 ng·mL~(-1)。
2.基于纳米金/纳米二氧化锰-壳聚糖复合物/普鲁士蓝修饰的金电极的免疫传感器研究
采用电沉积普鲁士蓝在电极表面形成一层均匀的电子媒介体层,增大电极的电流响应信号,再利用静电吸附将制备的纳米二氧化锰-壳聚糖固定在电极上,然后利用壳聚糖上丰富的氨基吸附纳米金,最后通过吸附性能强,生物兼容性好的双层纳米金吸附癌胚抗体,从而制得癌胚免疫传感器。采用循环伏安法(CV)对修饰电极进行了考察,该免疫传感器线性范围是0.25-8.0 ng·mL~(-1)和8.0-100 ng·mL~(-1),线性相关系数分别为0.9982,检出限为0.083 ng·mL~(-1),将其用于病人的血清样品分析,与现行的ELISA法的结果相比较,有很好的相关性。
3.基于聚苯乙烯磺酸钠接枝的多壁碳纳米管/铁氰化镍/纳米金修饰的电流型前列腺抗原免疫传感器的研究
将聚乙烯磺酸钠均匀分散在多壁碳纳米管中,并采用物理吸附的方法固定在洁净的金电极表面形成一层有别于经典的十二磺酸钠分散的多壁碳纳米管,其优点在于可以减小背景电流,而且电流的循环伏安电位也向正的方向偏移,有利于我们实验研究的考察,再利用电聚合铁氰化镍将其固定在致密的碳纳米管层表面,最后通过电还原氯金酸制得均匀的纳米金层以吸附前列腺癌抗体,从而制得前列腺癌免疫传感器。采用循环伏安法(CV)对修饰电极进行了考察,研究了电极修饰过程中的测试液pH值、温度、孵育时间等实验条件和参数对传感器性能的影响。该传感器在PSA浓度为1.5-35 ng·mL~(-1)的范围内有良好的线性关系,并且制作简单,响应快,能满足实际测定的要求。
Electrochemical immunosensor are widely used for clinical diagnosis,environmental inspection and food analysis,and have been have gained considerable attention due to their small volume,intrinsic selectivity,fast analytical time,simple measurement,high sensitivity and low cost, It has been reported that the performance of biosensors largely contact with the fabrication of biorecognition molecule.Thus,it's important work how to immobilize effectively on the cleaned electrode with biorecognition molecule in high amounts with retention of their bioactivity.In this article,we have constructed several immunoelectrodes immobilized antibody onto substrate electrode based on SiO_2 and methylene blue(MB) nanocomposite,nano-MnO_2 and chitosan(CS) composite,gold nanoparticles(nano-Au) and multi-walled carbon nanotube(MWNT) as matrixes. The detailed materials are shown as follows:
1.Amperometrie immunosensor for carcinoembryonic antigen based on ehitosan entrapped SiO_2 and methylene blue nanoeomposite
An amperometric immunosensor for the detection of carcinoembryonic antigen(CEA) has been developed by means of sol-gel techniques.SiO_2+MB-CS nanocomposite membrane was combined.Subsequently,nano-Au was adsorbed by CS with a large amount of amido matrixes, which owned a high surface area,good bioconsistent.Carcinoembryonic antibody(anti-CEA) was adsorbed by nano-Au firmly.Then a potentiometric immunosensor with good sensitivity and stability was prepared.The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry(CV) and transmission electron microscope (TEM).The resulting immunosensor exhibits wide linear range from 1.0 to 80.0 ng·mL~(-1) with a detection limit of 0.333 ng·mL~(-1).The correlation coefficient is 0.9978.
2.Study on immunosensor based on gold nanoparticles/chitosan and MnO_2 nanoparticles composite membrane/prussian blue modified gold electrode
A novel amperometric immunosensor for the detection of carcinoembryonic antigen(CEA) has been developed by means of electrodeposition and electrostatic adsorption techniques.In this study,Prussian blue(PB) was used as a mediator to amplify the current signal.And nano-MnO_2/CS composite with positively charged was adsorbed on the negatively charged PB.Subsequently, nano-Au was immobilized by CS.A large amount of carcinoembryonic antibody(anti-CEA) was adsorbed by nano-Au.Then an amPerometric immunosensor with good sensitivity and stability was made.The characteristics of the modified electrode at different stages of modification were studied by using cyclic voltammetry(CV)and scanning electron microscope(SEM).The performances of the immunosensor were studied in detail.The immunosensor showed a specific response to CEA in the range 0.25 to 8.0 ng·mL~(-1) and 8.0 to 100 ng·mL~(-1) with a detection limit of 0.083 ng·mL~(-1).The correlation coefficient is 0.9982.The immunosensor was used to analyze the serum samples with satisfactory results.
3.An immunosensor based on poly(sodium 4-styresulfonate) grated-carbon nanotube/niekel hexacyanoferrate/gold nanoparticles modified gold electrode.
A novel immunosensor has been developed by electropolymerizing Nickel Hexacyanoferrate (NiHCF) on to a Poly(sodium 4-styrenesulfonate)(PSS)-grafted Multi-walled Carbon Nanotube (MWNT) and electro-depositing nano-Au for determination of prostate specific antigen(PSA). After the developed immunosensor was incubated with different concentrations of PSA samples at 25℃for 35 min,the current response decreased with an increasing PSA concentration in the sample solution.The decreased percentage of the current was proportional to PSA concentration ranging froml.5 to 35 ng·mL~(-1) PSA with a detection limit of 0.51 ng·mL~(-1) PSA(S/N = 3). Analytical results of 50 specimens using the developed immunosensor showed satisfactory agreement with those using ELISA,indicating the method to be a promising alternative for detecting PSA in clinical diagnosis.
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