基于功能树状高分子修饰电极快速检测禽流感病毒的研究
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摘要
DNA电化学生物传感器是用电化学手段进行DNA结构分析和检测的生物传感器,具有高度的分子识别功能、准确性好、灵敏度高、易于集成化和自动化等优点,特别适用于表面过程、修饰电极、电子转移过程等的研究,已经广泛应用在基因诊断、传染病检测、基因表达检测、法医学和环境监控等领域中。在传感器的构建过程中,如何有效利用生物分子的固定技术及选择合适的固定材料决定着DNA电化学生物传感器的稳定性、选择性和灵敏性等主要性能。本论文探讨设计了三种新型的电化学生物传感器,根据鸟嘌呤G氧化信号的变化来初步检测禽流感病毒DNA的固定与杂交的方法,使用循环伏安法、示差脉冲伏安法和电化学阻抗谱对其进行表征,并对DNA电化学生物传感器在禽流感病毒检测中的应用进行了研究。研究内容主要分为以下三部分:
(1)利用铁氰化钴-树状高分子(CoHCF-PAMAM)复合材料修饰玻碳电极(GCE),制备了免标记检测基因突变的新型DNA电化学传感器。传感器中树状高分子层明显增加了单链DNA探针的固定量,铁氰化钴层增大了鸟嘌呤的氧化信号,该传感器可以灵敏识别单碱基错配的基因序列,具有良好的选择性和灵敏度。在7.6×10~(-11)～3.05×10~(-8) mol/L浓度范围内,鸟嘌呤(G)的氧化峰电流差值与突变基因浓度呈良好的线性关系,检出限为1.0×10~(-11 )mol/L (66 pg/mL) (S/N=3)。
(2)利用二氧化铅(PbO_2)、多壁碳纳米管(MWNTs)与疏水性室温离子液体(RTIL)1-丁基-3-甲基咪唑六氟磷酸盐(BMIMPF6)复合材料修饰玻碳电极(PbO_2-MWNT-RTIL/GCE),制备了一种新型电化学传感器,用于同时或分别检测鸟嘌呤(G)和腺嘌呤(A)。当修饰电极在外加电压+0.30 V下于pH 7.0的磷酸盐缓冲溶液中富集120 s后,进行微分脉冲伏安法(DPV)检测,鸟嘌呤和腺嘌呤分别在+0.60 V和+0.89 V处得到良好的氧化峰。与裸电极相比,PbO_2-MWNT-RTIL/GCE不仅明显地提高了鸟嘌呤和腺嘌呤的电化学氧化峰电流,而且降低了它们的氧化峰电位,这表明PbO_2, MWNT和RTIL的协同效应可以很好的提高检测鸟嘌呤和腺嘌呤的灵敏度。实验结果表明PbO_2-MWNT-RTIL/GCE具有良好的稳定性,优异的电催化性能和富集效应,并能同时检测鸟嘌呤和腺嘌呤(氧化峰电位差为0.29 V)。该修饰电极在最佳条件下,鸟嘌呤和腺嘌呤的检测限分别为1.0 ng/mL和4.0 ng/mL (S/N=3,信噪比)。用这种方法检测鲱鱼精DNA中的鸟嘌呤和腺嘌呤也取得了令人满意的结果。
(3)制备了一种利用免标记法检测禽流感病毒DNA序列的新型DNA电化学生物传感器。首先,将多壁碳纳米管和酞菁钴纳米复合材料(MWNTs-CoPc)以及4.0G树状高分子(PAMAM G4.0)修饰到玻碳电极上,然后以G4 PAMAM为桥梁将DNA探针固定到修饰电极。通过示差脉冲伏安法观察鸟嘌呤氧化信号的变化,来检测杂交反应的进行,避免了外加指示剂的使用。在最佳条件下,互补靶基因浓度在0.01到500 ng/mL范围内,修饰电极上获得的鸟嘌呤氧化信号差值(ΔI_p)与互补靶基因浓度的对数成线性关系,相关系数为0.998,检测限为1.0 pg/mL。
DNA electrochemical biosensor is noted widely in recent years for rapid development. It has been used in many fields such as food industry, disease diagnosis, pharmaceutical analysis and environmental monitoring due to the high sensitivity, better specificity, rapid response, easy handling, no pollution and low cost. However, the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation. Three New-typed DNA electrochemical biosensors were prepared, and the detection of Avian Influenza Virus genotype based on the guanine oxidation signal was studied in this paper. The dissertation is divided into three main parts:
(1) A novel electrochemical hybridization biosensor for label-free determination of sequence-specific DNA was described in this paper. First, cobalt hexacyanoferrate films and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. This biosensor exhibited a superior electrochemical stability, selectivity and sensitivity in detection of the oligonucleotide sequence. Under the optimal conditions, the guanine oxidation signal was directly proportional to the mutant gene sequence concentration from 7.6×10~(-11) to 3.05×10~(-8) mol/L with a correlation coefficient of 0.9975 and a detection limit of 1.0×10~(-11)mol/L (6.6×10~(-2) ng/mL)(S/N=3).
(2) In this paper, a novel and reliable electrochemical sensor based on PbO_2-carbon nanotubes-room temperature ionic liquid (i.e., 1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) composites film modified glassy carbon electrode (GCE) (PbO_2-MWNT-RTIL/GCE) was proposed for simultaneous and individual determination of guanine and adenine. The guanine and adenine oxidation responses were monitored by differential pulse voltammetry (DPV) measurement. Compared with the bare electrode, the PbO_2-MWNT-RTIL/GCE not only significantly enhanced the oxidation peak currents of guanine and adenine, but also lowered their oxidation over potentials, suggesting that the synergistic effect of PbO_2, MWNT and RTIL could dramatically improve the determining sensitivity of guanine and adenine. The results demonstrated that the PbO_2-MWNT- RTIL/GCE showed good stability, high accumulation efficiency and enhanced electrocatalytic ability for the detection of guanine and adenine. Besides, the modified electrode also exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.29 V in 0.1 M pH 7.0 phos/phate buffer solutions (PBS). Under the optimal conditions, the detection limit for individual determination of guanine and adenine was 1.0 ng/mL and 4.0 ng/mL (S/N=3), respectively. The proposed method for the measurements of guanine and adenine in herring sperm DNA were successfully applied with satisfactory results.
(3) A novel DNA electrochemical biosensor for label-free determination of DNA sequence related to the Avian Influenza Virus (AIV) genotype was demonstrated in this paper. First, the multi-walled carbon nanotubes-cobalt phthalocyanine (MWNTs-CoPc) nanocomposite and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. Under the optimal conditions, the difference of guanine oxidation signal of the probe modified GCE in the absence and presence of complementary target (ΔI_p) was linear with the logarithmic value of the complementary target concentration from 0.01 to 500 ng/mL with a correlation coefficient of 0.998 and a detection limit of 1.0 pg/mL.
(1)利用铁氰化钴-树状高分子(CoHCF-PAMAM)复合材料修饰玻碳电极(GCE),制备了免标记检测基因突变的新型DNA电化学传感器。传感器中树状高分子层明显增加了单链DNA探针的固定量,铁氰化钴层增大了鸟嘌呤的氧化信号,该传感器可以灵敏识别单碱基错配的基因序列,具有良好的选择性和灵敏度。在7.6×10~(-11)～3.05×10~(-8) mol/L浓度范围内,鸟嘌呤(G)的氧化峰电流差值与突变基因浓度呈良好的线性关系,检出限为1.0×10~(-11 )mol/L (66 pg/mL) (S/N=3)。
(2)利用二氧化铅(PbO_2)、多壁碳纳米管(MWNTs)与疏水性室温离子液体(RTIL)1-丁基-3-甲基咪唑六氟磷酸盐(BMIMPF6)复合材料修饰玻碳电极(PbO_2-MWNT-RTIL/GCE),制备了一种新型电化学传感器,用于同时或分别检测鸟嘌呤(G)和腺嘌呤(A)。当修饰电极在外加电压+0.30 V下于pH 7.0的磷酸盐缓冲溶液中富集120 s后,进行微分脉冲伏安法(DPV)检测,鸟嘌呤和腺嘌呤分别在+0.60 V和+0.89 V处得到良好的氧化峰。与裸电极相比,PbO_2-MWNT-RTIL/GCE不仅明显地提高了鸟嘌呤和腺嘌呤的电化学氧化峰电流,而且降低了它们的氧化峰电位,这表明PbO_2, MWNT和RTIL的协同效应可以很好的提高检测鸟嘌呤和腺嘌呤的灵敏度。实验结果表明PbO_2-MWNT-RTIL/GCE具有良好的稳定性,优异的电催化性能和富集效应,并能同时检测鸟嘌呤和腺嘌呤(氧化峰电位差为0.29 V)。该修饰电极在最佳条件下,鸟嘌呤和腺嘌呤的检测限分别为1.0 ng/mL和4.0 ng/mL (S/N=3,信噪比)。用这种方法检测鲱鱼精DNA中的鸟嘌呤和腺嘌呤也取得了令人满意的结果。
(3)制备了一种利用免标记法检测禽流感病毒DNA序列的新型DNA电化学生物传感器。首先,将多壁碳纳米管和酞菁钴纳米复合材料(MWNTs-CoPc)以及4.0G树状高分子(PAMAM G4.0)修饰到玻碳电极上,然后以G4 PAMAM为桥梁将DNA探针固定到修饰电极。通过示差脉冲伏安法观察鸟嘌呤氧化信号的变化,来检测杂交反应的进行,避免了外加指示剂的使用。在最佳条件下,互补靶基因浓度在0.01到500 ng/mL范围内,修饰电极上获得的鸟嘌呤氧化信号差值(ΔI_p)与互补靶基因浓度的对数成线性关系,相关系数为0.998,检测限为1.0 pg/mL。
DNA electrochemical biosensor is noted widely in recent years for rapid development. It has been used in many fields such as food industry, disease diagnosis, pharmaceutical analysis and environmental monitoring due to the high sensitivity, better specificity, rapid response, easy handling, no pollution and low cost. However, the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation. Three New-typed DNA electrochemical biosensors were prepared, and the detection of Avian Influenza Virus genotype based on the guanine oxidation signal was studied in this paper. The dissertation is divided into three main parts:
(1) A novel electrochemical hybridization biosensor for label-free determination of sequence-specific DNA was described in this paper. First, cobalt hexacyanoferrate films and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. This biosensor exhibited a superior electrochemical stability, selectivity and sensitivity in detection of the oligonucleotide sequence. Under the optimal conditions, the guanine oxidation signal was directly proportional to the mutant gene sequence concentration from 7.6×10~(-11) to 3.05×10~(-8) mol/L with a correlation coefficient of 0.9975 and a detection limit of 1.0×10~(-11)mol/L (6.6×10~(-2) ng/mL)(S/N=3).
(2) In this paper, a novel and reliable electrochemical sensor based on PbO_2-carbon nanotubes-room temperature ionic liquid (i.e., 1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) composites film modified glassy carbon electrode (GCE) (PbO_2-MWNT-RTIL/GCE) was proposed for simultaneous and individual determination of guanine and adenine. The guanine and adenine oxidation responses were monitored by differential pulse voltammetry (DPV) measurement. Compared with the bare electrode, the PbO_2-MWNT-RTIL/GCE not only significantly enhanced the oxidation peak currents of guanine and adenine, but also lowered their oxidation over potentials, suggesting that the synergistic effect of PbO_2, MWNT and RTIL could dramatically improve the determining sensitivity of guanine and adenine. The results demonstrated that the PbO_2-MWNT- RTIL/GCE showed good stability, high accumulation efficiency and enhanced electrocatalytic ability for the detection of guanine and adenine. Besides, the modified electrode also exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.29 V in 0.1 M pH 7.0 phos/phate buffer solutions (PBS). Under the optimal conditions, the detection limit for individual determination of guanine and adenine was 1.0 ng/mL and 4.0 ng/mL (S/N=3), respectively. The proposed method for the measurements of guanine and adenine in herring sperm DNA were successfully applied with satisfactory results.
(3) A novel DNA electrochemical biosensor for label-free determination of DNA sequence related to the Avian Influenza Virus (AIV) genotype was demonstrated in this paper. First, the multi-walled carbon nanotubes-cobalt phthalocyanine (MWNTs-CoPc) nanocomposite and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. Under the optimal conditions, the difference of guanine oxidation signal of the probe modified GCE in the absence and presence of complementary target (ΔI_p) was linear with the logarithmic value of the complementary target concentration from 0.01 to 500 ng/mL with a correlation coefficient of 0.998 and a detection limit of 1.0 pg/mL.
引文
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