CN106591437A - 一种基于铂‑金‑三维石墨烯纳米复合材料的电化学基因传感器件的制备与应用 - Google Patents
一种基于铂‑金‑三维石墨烯纳米复合材料的电化学基因传感器件的制备与应用 Download PDFInfo
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Abstract
本发明采用水热法合成铂‑金‑三维石墨烯(Pt‑Au‑3DGR)纳米复合材料,以离子液体N‑己基吡啶六氟磷酸盐为粘合剂和修饰剂制备的离子液体碳糊电极(CILE)为基底电极,修饰Pt‑Au‑3DGR纳米复合材料后在该界面上固定探针ssDNA,得到一种新型的电化学基因传感器件。采用示差脉冲伏安(DPV)技术对所构建的电化学基因传感器件进行电化学检测。由于Pt‑Au‑3DGR纳米复合材料具有高的导电性和大的比表面积,在增大探针ssDNA负载量的同时能够提高电化学响应,所以本发明所构建的电化学基因传感器件表现出良好的选择性和较高的灵敏度。
Description
技术领域
本发明涉及电化学和电分析化学方面的化学修饰电极领域,以及电化学基因传感器件领域。
背景技术
二维的石墨烯片可以组装成三维石墨烯(3DGR)宏观材料,并在能源、环境、敏感器件和生物领域得到应用(Li C, Shi G Q, Three-dimensional graphene architectures,Nanoscale, 2012, 4: 5549)。三维结构赋予了石墨烯材料较大的比表面积、较高的力学强度以及快的电子迁移率。目前三维石墨烯的制备有自组装法、模板法等。Sun等人用溶剂热还原氧化石墨烯(GO)分散液合成具有3D结构和高导电性的水凝胶,经冷冻干燥或临界二氧化碳干燥就可以得到3D石墨烯气凝胶(Sun Y Q, Wu Q, Shi G Q, Supercapacitorsbased on self-assembled graphene organogel, Phys. Chem. Chem. Phys., 2011,13: 17249);Jiang等将氧化石墨烯在120℃下加热10 h得到3D石墨烯(Jiang X, Ma Y, LiJ et al., Self-assemblely of reduced graphene oxide into three-dimensionalarchitecture by divalent ion linkage, J. Phys. Chem. C, 2010, 114: 22462);Gao等人利用间接冷模板法合成了3D多孔还原石墨烯,随后与银纳米粒子形成3D Ag/rGR (HeY Q, Zhang N N, Gong Q J, Li Z L et al., Metal nanoparticles supportedgraphene oxide 3D porous monoliths and their excellent catalytic activity,Mater. Chem. Phys., 2012, 134: 585)。
三维石墨烯已经成为制备复合材料的理想载体,将三维石墨烯和其他活性材料,如与金属纳米粒子、过渡族金属氧化物、半导体纳米粒子等复合,制备的复合材料能将两种或两种以上的材料的性能结合起来,并且发挥它们的协同作用,在电化学传感器、超级电容器、催化和锂离子电池等方面显示出广阔的应用前景。
DNA的检测对于诊断各种疾病有着重要的意义。电化学DNA生物传感器为DNA检测技术提供了一种全新途径,它具有制作简单、灵敏度高、重现性好、成本低、选择性好、可用于活体检测和易于实现微型化等优点而被广泛应用。
发明内容
本发明构建了一种新型电化学基因传感器件。以铂-金-三维石墨烯纳米复合材料作为电极修饰材料,在增大电极表面的导电性和比表面积的同时,增加探针ssDNA的负载量。所以本发明构建的电化学基因传感器件具有良好的选择性和灵敏度。
为了实现上述任务,本发明采取如下的技术解决方案:
1. 一种基于Pt-Au-3DGR纳米复合材料修饰电极的电化学基因传感器件的制备,其步骤如下:
(1) Pt-Au-3DGR纳米复合材料的制备
配制浓度比为1:1的HAuCl4和HPtCl6溶液,超声混合15 min后加入一定量的GO溶液,用二次蒸馏水定容后继续超声15 min,随后将混合液转入50 mL的聚四氟乙烯反应釜中,于适当的温度下加热反应一定时间,冷却至室温后取出沉淀物,用二次蒸馏水清洗三次后并用蒸馏水浸泡24 h,冷冻干燥后即得Pt-Au-3DGR纳米复合材料;
(2) 离子液体碳糊电极的制备
由一定比例的石墨粉、离子液体和液体石蜡充分混合研磨后作为填充物,装入内插细铜丝的玻璃电极管内,用铁棒将其压实,即得离子液体碳糊电极(CILE),使用前在抛光纸上打磨至镜面;
(3) 修饰电极的制备
采用滴涂法将Pt-Au-3DGR纳米复合材料修饰在CILE表面,室温晾干后即得修饰电极Pt-Au-3DGR/CILE;
(4) 电化学基因传感器件的制备
采用恒电位吸附法将探针ssDNA固定在修饰电极Pt-Au-3DGR/CILE表面,用5% SDS溶液和二次蒸馏水冲洗,记作ssDNA/Pt-Au-3DGR/CILE;
(5) 分子杂交与电化学检测
采用滴涂法将10 μL含有目标ssDNA序列的溶液滴加在电极表面,使目标ssDNA序列与探针ssDNA序列在室温下发生分子杂交反应,30 min后用5% SDS溶液和二次蒸馏水冲洗,杂交后的电极记作dsDNA/Pt-Au-3DGR/CILE;
所述电化学基因传感器件构建,步骤(1)中HAuCl4和HPtCl6溶液的体积和浓度均为2.5mL 和2.426×10-2 mol/L;加热的条件为180℃下加热反应12 h;冷冻干燥的条件为-80℃下冷冻干燥12 h;
所述电化学基因传感器件构建,步骤(4)中ssDNA的储备液为50 mmol/L pH 8.0的TE缓冲溶液;
所述电化学基因传感器件构建,步骤(4)中探针ssDNA浓度为1.0×10-6 mol/L,步骤(5)中目标ssDNA浓度在1.0×10-13 mol/L~1.0×10-6 mol/L范围内;
所述电化学基因传感器件构建,步骤(4)中探针ssDNA的恒电位吸附条件为恒电位+0.5V下吸附300 s;
所述电化学基因传感器件构建,步骤(1)中的纳米复合材料利用SEM表征其微观形貌结构。
2. 基于Pt-Au-3DGR纳米复合材料修饰电极的电化学基因传感器件的应用,其特征在于对一种基因序列的检测。采用CV法考察了不同修饰电极在电解质溶液中的电化学行为以及利用DPV技术对所构建的电化学基因传感器件进行电化学测试;
所述电化学基因传感器件的应用,电化学测试溶液为10.0 mmol/L K3[Fe(CN)6]和0.1mol/L KCl的混合溶液。
附图说明
图1为电极修饰材料的SEM图,A、B为不同放大尺寸下Pt-Au-3DGR纳米复合材料。
图2为不同修饰电极的CV曲线。a到d分别为CILE、dsDNA/Pt-Au-3DGR/CILE、ssDNA/Pt-Au-3DGR/CILE和Pt-Au-3DGR/CILE.。
图3为与不同目标序列杂交后电极上的DPV曲线。a到e分别为与目标序列杂交、单碱基错配序列杂交、与三碱基错配序列杂交、与非互补序列杂交和杂交前。内嵌图为与不同目标序列杂交后的还原峰电流值。
图4为探针序列与不同浓度的目标序列杂交后的DPV曲线。a到i的目标序列浓度依次为1.0×10-6, 1.0×10-7, 1.0×10-8, 1.0×10-9, 1.0×10-10, 1.0×10-11, 1.0×10-12,1.0×10-13和0 mol/L。(内嵌图为铁氰化钾的还原峰电流与目标序列浓度间的线性关系)。
具体实施方式
下面给出的实施例对本发明作进一步说明,但不超出本发明保护范围的限制。
实施例1
电极修饰材料的电镜表征
图1A和B展示了不同放大尺寸下的Pt-Au-3DGR纳米复合材料,从SEM所示的纳米材料形貌图可以看到,颗粒状的铂-金纳米材料均匀分散在呈卷曲、褶皱状的三维石墨烯表面,因此具有大的比表面积的三维石墨烯为铂-金的负载提供了更多空间。
实施例2
不同修饰电极的电化学行为
为了考察不同修饰电极在10.0 mmol/L K3[Fe(CN)6]和0.1 mol/L KCl的混合溶液中的电化学行为,对其进行了CV检测,结果如图2所示。在CILE (曲线a)上的氧化还原峰电流分别为Ipc=272.2 μA、Ipa=-233.9 μA;Pt-Au-3DGR/CILE (曲线d)上的氧化还原峰电流分别为Ipc=528.6 μA、Ipa=-501.9 μA;ssDNA/Pt-Au-3DGR/CILE (曲线c)上的氧化还原峰电流分别为Ipc=457.1 μA、Ipa=-414.6 μA;dsDNA/Pt-Au-3DGR/CILE (曲线b)上的氧化还原峰电流分别为Ipc=383.9 μA、Ipa=-326.9 μA。可以看出从曲线d到曲线b的氧化还原峰电流逐渐减小,这是由于探针ssDNA被固定在电极表面后,ssDNA的磷酸骨架带有负电,与带负电的氧化还原电子对之间发生静电排斥,阻碍其在电极表面的电子传递,同时dsDNA的磷酸骨架数量是ssDNA磷酸骨架数量的二倍,阻碍的效果更为明显,因此充分地解释了上述实验现象的原因。由于Pt-Au-3DGR纳米复合材料具有良好的导电能力,所以有效地增加了修饰电极界面导电性。
实施例3
电化学基因传感器件的选择性
通过探针序列与不同错配序列的杂交检测来研究所构建的基因传感器件的选择性。即考察了ssDNA/Pt-Au-3DGR/CILE修饰电极与不同错配序列杂交后在10.0 mmol/L K3[Fe(CN)6]和0.1 mol/L KCl的混合溶液中的示差脉冲伏安曲线,结果如图3所示。Pt-Au-3DGR/CILE具有较大比表面积,可以吸附负载探针ssDNA形成ssDNA/Pt-Au-3DGR/CILE,以其为电化学传感器与不同序列的ssDNA序列进行杂交反应,今在电极表面发生部分或全部的分子杂交反应,进而导致电极表面DNA的数量发生变化,相应氧化还原探针的电流也发生变化。在ssDNA/Pt-Au-3DGR/CILE (曲线e)上电流值最大,随着杂交的进行,其值大于与非互补序列杂交(曲线d)、与三碱基错配序列杂交(曲线c)、与单碱基错配序列杂交(曲线b)和与目标序列杂交(曲线a),表明电极界面单双链DNA的数量变化导致界面的带电性发生变化,此信号的变化充分说明本方法具有良好的选择性。
实施例4
电化学基因传感器件的灵敏度
使用DPV技术对电化学基因传感器件的灵敏度进行了检测。考察了ssDNA/Pt-Au-3DGR/CILE与不同浓度目标ssDNA杂交后在10.0 mmol/L K3[Fe(CN)6]和0.1 mol/L KCl混合溶液中的电化学信号的变化。结果如图4所示,铁氰化钾的还原峰电流值随着目标DNA浓度的增大而逐渐减小,且在1.0×10-13 mol/L (曲线h)到1.0×10-6 mol/L (曲线a)的范围内铁氰化钾的还原峰电流的变化与目标序列浓度的对数值呈良好的线性关系,线性回归方程为Ip (µA)=276.78 log C-15.98 (γ=0.999),检测限为2.9×10-14 mol/L (3σ),表明所构建的电化学基因传感器件具有较低的检测限和较宽的检测范围。
Claims (8)
1.一种基于铂-金-三维石墨烯(Pt-Au-3DGR)纳米复合材料的电化学基因传感器件的制备与应用,其特征在于,包括以下步骤:
Pt-Au-3DGR纳米复合材料的制备
配制浓度比为1:1的氯金酸(HAuCl4)和氯铂酸(HPtCl6)溶液,超声混合15 min后加入一定量的氧化石墨烯(GO)溶液,用二次蒸馏水定容后继续超声15 min,随后将混合液转入50mL的聚四氟乙烯反应釜中,于适当的温度下加热反应一定时间,冷却至室温后取出沉淀物,用二次蒸馏水清洗三次后并用蒸馏水浸泡24 h,冷冻干燥后即得Pt-Au-3DGR纳米复合材料;
(2) 离子液体碳糊电极的制备
由一定比例的石墨粉、离子液体和液体石蜡充分混合研磨后作为填充物,装入内插细铜丝的玻璃电极管内,用铁棒将其压实,即得离子液体碳糊电极(CILE),使用前在抛光纸上打磨至镜面;
(3) 修饰电极的制备
采用滴涂法将Pt-Au-3DGR纳米复合材料修饰在CILE表面,室温晾干后即得修饰电极Pt-Au-3DGR/CILE;
(4) 电化学基因传感器件的制备
采用恒电位吸附法将探针ssDNA固定在修饰电极Pt-Au-3DGR/CILE表面,用5% 十二烷基硫酸钠(SDS)溶液和二次蒸馏水冲洗,记作ssDNA/Pt-Au-3DGR/CILE;
分子杂交与电化学检测
采用滴涂法将10 μL含有目标ssDNA序列的溶液滴加在电极表面,使目标ssDNA序列与探针ssDNA序列在室温下发生分子杂交反应,30 min后用5% SDS溶液和二次蒸馏水冲洗,杂交后的电极记作dsDNA/Pt-Au-3DGR/CILE;
根据权利要求1所述的构建方法,步骤(1)中HAuCl4和HPtCl6溶液的体积和浓度均为2.5mL 和2.426×10-2 mol/L;加热的条件为180℃下加热反应12 h;冷冻干燥的条件为-80℃下冷冻干燥12 h。
2.根据权利要求1所述的构建方法,步骤(4)中ssDNA的储备液为50 mmol/L pH 8.0的TE缓冲溶液。
3.根据权利要求1所述的构建方法,步骤(4)中探针ssDNA浓度为1.0×10-6 mol/L,步骤(5)中目标ssDNA浓度在1.0×10-13 mol/L~1.0×10-6 mol/L范围内。
4.根据权利要求1所述的构建方法,步骤(4)中探针ssDNA的恒电位吸附条件为恒电位+0.5 V下吸附300 s。
5.根据权利要求1所述的构建方法,步骤(1)中的纳米复合材料利用扫描电子显微镜(SEM)表征其微观形貌结构。
6.基于Pt-Au-3DGR纳米复合材料修饰电极的电化学基因传感器件的应用,其特征在于对一种基因序列的检测。
7.采用循环伏安法(CV)考察了不同修饰电极在电解质溶液中的电化学行为以及利用DPV技术对所构建的电化学基因传感器件进行电化学测试。
8.根据权利要求7所述,电化学测试溶液为10.0 mmol/L K3[Fe(CN)6]和0.1 mol/L KCl的混合溶液。
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