基于石墨烯复合界面的DNA电化学传感研究
摘要
本论文围绕纳米材料领域的研究热点,结合石墨烯、离子液体、纳米金、纳米导电聚合物和金属氧化物等,设计合成了具有不同结构和性质的石墨烯复合界面,并将其应用于电化学传感,得到具有高灵敏度和高选择性的新型DNA电化学生物传感器。主要内容如下：
(1)研究了离子液体功能化的石墨烯对多巴胺和嘌呤的电化学催化。离子液体功能化的石墨烯修饰电极对多巴胺和嘌呤具有很好的电催化性能,能够极大地降低氧化电位,对多巴胺和嘌呤具有较好的线性响应和较低的检测限。该传感器能够检测特定序列DNA的单碱基变异。
(2)分别利用电化学还原法和化学还原法制得石墨烯。与化学还原法相比,电化学还原法更加简单,高效,低耗费,绿色环保,并且制得的石墨烯拥有更强的电化学催化活性。纳米金/石墨烯电极能够在中性条件下同时检测DNA的四种碱基,并且在免杂交和免标记的情况下,实现了对人工合成DNA碱基变异的识别检测。
(3)利用DNA与石墨烯的非共价π-π堆积作用,将DNA探针固定在石墨烯/聚苯胺复合界面上。当互补DNA存在时,互补DNA与DNA探针的杂交反应会削弱DNA探针与石墨烯之间的作用力,使DNA探针(部分或全部)从石墨烯/聚苯胺复合界面脱离。通过监测DNA探针上结合的[Ru(NH3)6]3+的电化学信号变化,能够实现对花椰菜花叶病毒CaMV35S基因片段的检测。
(4)将Fe203/石墨烯复合界面应用于蛋白质的检测。用恒电位法将Fe203沉积到石墨烯电极上。把复合电极作为固载平台,溶菌酶适体通过静电吸附作用固定到电极表面,并用电化学交流阻抗的方法检测溶菌酶。当溶菌酶存在的情况下,电极表面的适体能够结合溶菌酶,引起电极电化学阻抗信号的增强。
(5)以组氨酸为还原剂制备水溶性石墨烯,制得的石墨烯用作一种双组分的适体传感平台。由两种蛋白质适体(凝血酶适体和溶菌酶适体)组成的DNA序列与石墨烯以π-π堆积的方式结合,将目标物溶菌酶和凝血酶作为输入,DNA上静电结合的[Ru(NH3)6]3+的电化学信号作为输出。不同目标物的加入会改变DNA/石墨烯的空间结构,引起输出信号的变化,模拟了NAND逻辑门。
(6)用电化学共沉积的方法制备了Al3+/石墨烯的复合界面。把双链DNA固定在Al3+/石墨烯上,将低毒性的黄尿酸嵌插在双链DNA里作为电化学指示剂,通过研究黄尿酸的电化学信号变化监测DNA的损伤。实验结果表明,氧化石墨烯和一些金属离子的混合体系可以引起双链DNA的解链。
In the scope of nanomaterials fields, the nanocomposite interfaces based on graphene, ionic liquids (IL), gold nanoparticles, polymer, and metal oxides were synthesized and applied in electrochemical biosensing. The resulted novel DNA electrochemical biosensors show good performances and exhibit potential application in bioanalysis. The main points of this dissertation are addressed as follows:
(1) Electrochemical activities of typically electrochemical targets at three kinds of modified carbon electrodes, i.e. carbon ionic liquid electrode (CILE), graphene/carbon paste electrode (CPE), and ionic liquid-functionalized graphene (IL-graphene)/CPE, were compared. The redox processes of the targets at IL-graphene/CPE were faster than those at CILE and graphene/CPE. Single base mutation of sequence-specific DNA could be discriminated by IL-graphene/CPE.
(2) A direct electrochemical DNA sensor was constructed based on gold nanoparticles/graphene film. A precursor graphene film was fabricated on the glassy carbon electrode (GCE) using both electrochemically reduced graphene oxide (ERGNO) and chemically reduced graphene oxide (CRGNO). Electrochemical approach was green and fast, and would not result in contamination of the reduced material compared with chemical reduction, and at highly negative potential could reduce the oxygen functionalities of graphene oxide more efficiently than chemical reduction. ERGNO exhibited better electrochemical and electrocatalytic performances than CRGNO. The gold nanoparticles (AuNPs) were electrodeposited on the ERGNO/GCE to amplify the electrochemical signals. The electrochemical responses of DNA bases were investigated at AuNPs/ERGNO/GCE, which showed more favorable electron transfer kinetics than at ERGNO/GCE, demonstrating the significantly synergistic electrocatalytic effect of ERGNO and AuNPs. The synthetic sequence-specific DNA was successfully detected.
(3) A novel DNA electrochemical biosensor was described for the detection of specific gene sequences. ERGNO was prepared on polyaniline (PAN) nanofibers modified GCE. Compared with the electrochemical reduction of graphene oxide directly on bare GCE, more positive reduction potential for graphene oxide was observed with the PAN membrane existing. The formed ERGNO/PAN nanocomposites were applied to bind single-stranded DNA (ssDNA) probe via the non-covalent assembly. After the hybridization of ssDNA probe with complementary DNA, the response of surface-bound [Ru(NH3)6]3+changed obviously, which could been adopted to recognize the DNA hybridization. This biosensor could be used to detect the sequence-specific DNA of cauliflower mosaic virus (CaMV35S) gene.
(4) An electrochemical platform was employed for rapid detection of protein, where Fe2O3was fabricated on graphene surface using electrochemical deposition. The negatively charged lysozyme-binding aptamer (LBA) was immobilized on the positively charged Fe2O3via electrostatic interaction. Electrochemical impedance spectroscopy was adopted for the indicator-free detection of lysozyme. The LBA on the outermost layer would catch lysozyme in solution by physical affinity, which induced an increase of impedimetric signals. The results showed that the indicator-free impedimetric aptasensing strategy had good sensitivity and selectivity.
(5) Graphene was obtained by the nontoxic reductant L-histidine, and demonstrated as a dual-analyte biosensing platform that functioned as electrochemical logic gate. Thrombin and lysozyme were used as inputs to activate the change of aptamer configuration. The electrochemical signals of redox cation [Ru(NH3)e]3+bound to the aptamer were read out as outputs in the presence of the appropriate inputs. The fabricated logic aptasensor could determine whether both specific targets were present through the built-in NAND logic gate.
(6) The electrochemical co-deposition of Al3+/graphene composites directly from an aqueous mixture containing graphene oxide (GNO) and Al3+was presented. The obtained Al3+/graphene composites with good electrochemical activity were regarded as the appropriate immobilization platform for double-stranded DNA (dsDNA). The nontoxic redox probe xanthurenic acid (XA) was successfully applied to recognize ssDNA and dsDNA. The scission of dsDNA caused by GNO combining with some metal ions could be detected by monitoring the electrochemical signals of XA.
(1)研究了离子液体功能化的石墨烯对多巴胺和嘌呤的电化学催化。离子液体功能化的石墨烯修饰电极对多巴胺和嘌呤具有很好的电催化性能,能够极大地降低氧化电位,对多巴胺和嘌呤具有较好的线性响应和较低的检测限。该传感器能够检测特定序列DNA的单碱基变异。
(2)分别利用电化学还原法和化学还原法制得石墨烯。与化学还原法相比,电化学还原法更加简单,高效,低耗费,绿色环保,并且制得的石墨烯拥有更强的电化学催化活性。纳米金/石墨烯电极能够在中性条件下同时检测DNA的四种碱基,并且在免杂交和免标记的情况下,实现了对人工合成DNA碱基变异的识别检测。
(3)利用DNA与石墨烯的非共价π-π堆积作用,将DNA探针固定在石墨烯/聚苯胺复合界面上。当互补DNA存在时,互补DNA与DNA探针的杂交反应会削弱DNA探针与石墨烯之间的作用力,使DNA探针(部分或全部)从石墨烯/聚苯胺复合界面脱离。通过监测DNA探针上结合的[Ru(NH3)6]3+的电化学信号变化,能够实现对花椰菜花叶病毒CaMV35S基因片段的检测。
(4)将Fe203/石墨烯复合界面应用于蛋白质的检测。用恒电位法将Fe203沉积到石墨烯电极上。把复合电极作为固载平台,溶菌酶适体通过静电吸附作用固定到电极表面,并用电化学交流阻抗的方法检测溶菌酶。当溶菌酶存在的情况下,电极表面的适体能够结合溶菌酶,引起电极电化学阻抗信号的增强。
(5)以组氨酸为还原剂制备水溶性石墨烯,制得的石墨烯用作一种双组分的适体传感平台。由两种蛋白质适体(凝血酶适体和溶菌酶适体)组成的DNA序列与石墨烯以π-π堆积的方式结合,将目标物溶菌酶和凝血酶作为输入,DNA上静电结合的[Ru(NH3)6]3+的电化学信号作为输出。不同目标物的加入会改变DNA/石墨烯的空间结构,引起输出信号的变化,模拟了NAND逻辑门。
(6)用电化学共沉积的方法制备了Al3+/石墨烯的复合界面。把双链DNA固定在Al3+/石墨烯上,将低毒性的黄尿酸嵌插在双链DNA里作为电化学指示剂,通过研究黄尿酸的电化学信号变化监测DNA的损伤。实验结果表明,氧化石墨烯和一些金属离子的混合体系可以引起双链DNA的解链。
In the scope of nanomaterials fields, the nanocomposite interfaces based on graphene, ionic liquids (IL), gold nanoparticles, polymer, and metal oxides were synthesized and applied in electrochemical biosensing. The resulted novel DNA electrochemical biosensors show good performances and exhibit potential application in bioanalysis. The main points of this dissertation are addressed as follows:
(1) Electrochemical activities of typically electrochemical targets at three kinds of modified carbon electrodes, i.e. carbon ionic liquid electrode (CILE), graphene/carbon paste electrode (CPE), and ionic liquid-functionalized graphene (IL-graphene)/CPE, were compared. The redox processes of the targets at IL-graphene/CPE were faster than those at CILE and graphene/CPE. Single base mutation of sequence-specific DNA could be discriminated by IL-graphene/CPE.
(2) A direct electrochemical DNA sensor was constructed based on gold nanoparticles/graphene film. A precursor graphene film was fabricated on the glassy carbon electrode (GCE) using both electrochemically reduced graphene oxide (ERGNO) and chemically reduced graphene oxide (CRGNO). Electrochemical approach was green and fast, and would not result in contamination of the reduced material compared with chemical reduction, and at highly negative potential could reduce the oxygen functionalities of graphene oxide more efficiently than chemical reduction. ERGNO exhibited better electrochemical and electrocatalytic performances than CRGNO. The gold nanoparticles (AuNPs) were electrodeposited on the ERGNO/GCE to amplify the electrochemical signals. The electrochemical responses of DNA bases were investigated at AuNPs/ERGNO/GCE, which showed more favorable electron transfer kinetics than at ERGNO/GCE, demonstrating the significantly synergistic electrocatalytic effect of ERGNO and AuNPs. The synthetic sequence-specific DNA was successfully detected.
(3) A novel DNA electrochemical biosensor was described for the detection of specific gene sequences. ERGNO was prepared on polyaniline (PAN) nanofibers modified GCE. Compared with the electrochemical reduction of graphene oxide directly on bare GCE, more positive reduction potential for graphene oxide was observed with the PAN membrane existing. The formed ERGNO/PAN nanocomposites were applied to bind single-stranded DNA (ssDNA) probe via the non-covalent assembly. After the hybridization of ssDNA probe with complementary DNA, the response of surface-bound [Ru(NH3)6]3+changed obviously, which could been adopted to recognize the DNA hybridization. This biosensor could be used to detect the sequence-specific DNA of cauliflower mosaic virus (CaMV35S) gene.
(4) An electrochemical platform was employed for rapid detection of protein, where Fe2O3was fabricated on graphene surface using electrochemical deposition. The negatively charged lysozyme-binding aptamer (LBA) was immobilized on the positively charged Fe2O3via electrostatic interaction. Electrochemical impedance spectroscopy was adopted for the indicator-free detection of lysozyme. The LBA on the outermost layer would catch lysozyme in solution by physical affinity, which induced an increase of impedimetric signals. The results showed that the indicator-free impedimetric aptasensing strategy had good sensitivity and selectivity.
(5) Graphene was obtained by the nontoxic reductant L-histidine, and demonstrated as a dual-analyte biosensing platform that functioned as electrochemical logic gate. Thrombin and lysozyme were used as inputs to activate the change of aptamer configuration. The electrochemical signals of redox cation [Ru(NH3)e]3+bound to the aptamer were read out as outputs in the presence of the appropriate inputs. The fabricated logic aptasensor could determine whether both specific targets were present through the built-in NAND logic gate.
(6) The electrochemical co-deposition of Al3+/graphene composites directly from an aqueous mixture containing graphene oxide (GNO) and Al3+was presented. The obtained Al3+/graphene composites with good electrochemical activity were regarded as the appropriate immobilization platform for double-stranded DNA (dsDNA). The nontoxic redox probe xanthurenic acid (XA) was successfully applied to recognize ssDNA and dsDNA. The scission of dsDNA caused by GNO combining with some metal ions could be detected by monitoring the electrochemical signals of XA.
引文
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