CN111562296A - 一种以纳米金/氧化锌-石墨烯复合材料为光电敏感元件的适配体传感器的构建及应用 - Google Patents
一种以纳米金/氧化锌-石墨烯复合材料为光电敏感元件的适配体传感器的构建及应用 Download PDFInfo
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Abstract
本发明涉及一种以纳米金/氧化锌‑还原氧化石墨烯三元复合材料为光电敏感元件的光电化学适配体传感器的构建及应用。以导电玻璃(ITO)为基底电极,制备了还原氧化石墨烯(rGO)、氧化锌(ZnO)和纳米金(Au)修饰的光电极Au/ZnO‑rGO/ITO,并修饰适配体S1及互补链S2,形成双链DNA(dsDNA),光敏剂亚甲基蓝(MB)与dsDNA结合,构建了检测信号放大的传感器。当该传感界面感受Cd(II)后,S2特异性识别Cd(II)形成发卡结构,并伴随dsDNA解旋和MB从dsDNA脱离,根据识别后传感器的光电流响应,实现Cd(II)定量分析。测试采用双工作电极方法,以传感器和玻碳电极为第一和第二工作电极,电解液中添加多巴胺(DA)为电子供体,实现DA在双工作电极表面的氧化还原循环,提高信号的稳定性。该方法应具有较高的灵敏度和较低的检测限。
Description
技术领域
本发明涉及功能纳米复合材料和生物传感分析技术领域,提供了一种以纳米金/氧化锌-还原氧化石墨烯三元复合材料为光电敏感元件的光电化学适配体传感器的构建方法及应用。
背景技术
镉离子Cd(II)是一种具有较强生物毒性的重金属离子,主要来自金属矿山的采选、冶炼、电解、农药、医药、油漆、纺织印染等工业,不但对当地的生态环境造成严重的破坏,而且会随着食物链进入人体内,代换骨骼中的钙引起骨质疏松,骨质软化等病症,使人感觉骨骼疼痛、疲倦无力、头疼头晕等,而且还会在肝脏、肾脏中富集,造成累积性中毒,对人类健康产生不可逆的危害。世界健康组织(WHO)规定,饮用水中Cd(II)总含量不能超过3.0 μg/L。目前,对Cd(II)的检测方法有很多,包括原子吸收光谱法,紫外分光光度法,电感耦合等离子体质谱法和电感耦合等离子体原子发射光谱法等,这些方法具有较高的灵敏度和准确性,但设备昂贵,检测成本高,样品的前处理过程复杂,难以实现快速检测的目的。因此,设计一种绿色环保、成本低廉、快速响应、准确性高的传感方法应用于Cd(II)的检测,是极其必要的。
适配体(Aptamer)是经过体外筛选技术而获得的一段寡核苷酸序列片段,能够与相应的靶标物高特异性和强作用力结合,具有目标物种类多样性、易于体外合成与修饰、稳定性好、便于保存等优势。基于此,适配体在分析化学、临床检测、环境科学、分子识别及药物筛选等方面都表现出极好的应用前景。
电化学测试的常用方法是三电极检测系统,即工作电极、对电极和参比电极,待测物在工作电极(即修饰传感器)表面发生直接或间接反应并产生相应的检测信号。当待测物在该界面的反应不显著或者浓度较低时,所产生的检测信号则是微弱的。如何提高传感器的灵敏度,提高检测信号的响应值,实现痕量检测的目的,也是现阶段研究中亟待解决的技术问题。
发明内容
针对现有的Cd(II)检测技术问题,本发明的目的在于构建一种用于Cd(II)检测的高灵敏度光电化学适配体传感器,采用光电化学的分析方法,引入双通道-双工作电极(即四电极)检测系统,实现Cd(II)高灵敏度的快速检测。
本发明的具体实施措施如下:
将ITO导电玻璃分别浸入丙酮、乙醇和超纯水中超声30 min,高纯氮吹扫干燥后备用;将ITO导电玻璃浸入0.2 mg/mL的氧化石墨烯(GO)水溶液中,采用恒电流沉积法,电流密度为0.25 mA/cm2,沉积时间为100 s,制备还原氧化石墨烯(rGO)均匀负载的电极rGO/ITO;
将rGO/ITO浸入0.02 mol/L Zn(NO3)2水溶液中,于80℃恒温水浴条件下恒电位沉积,沉积电位为-1.4 V,沉积时间为30 min,制得均匀负载ZnO纳米棒的修饰电极ZnO-rGO/ITO;
将ZnO-rGO/ITO浸入2.0 mmol/L HAuCl4和0.5 mol/L KCl混合溶液中,采用恒电位还原法制备负载Au纳米粒子的修饰电极Au/ZnO-rGO/ITO,还原电位为-0.4 V,时间为50 s;
取20 μL 1.0 μmol/L巯基预修饰的适配体 S1溶液,滴涂在上述光电极Au/ZnO-rGO/ITO表面,置于室温恒湿孵育2 h,将S1通过Au-S共价键自组装于光电极表面,然后滴加20 μL 0.2 mmol/L巯基丙酸(MPA)作为封闭剂孵育30 min,以封闭光电极表面的活性吸附位点,即得修饰电极S1/Au/ZnO-rGO/ITO;
取20 μL 1.0 μmol/L适配体S2溶液,滴涂于S1/Au/ZnO-rGO/ITO表面,置于室温恒湿孵育2 h,S1与S2互补配对形成双链DNA (dsDNA)结构,得到修饰电极S2/S1/Au/ZnO-rGO/ITO;
取20 μL 10.0 μmol/L亚甲基蓝(MB)溶液,滴涂于S2/S1/Au/ZnO-rGO/ITO表面,作为光敏剂的MB通过嵌插或静电键合作用与dsDNA结合,得到适配体传感器MB/S2/S1/Au/ZnO-rGO/ITO。
本发明还提供了一种上述光电化学适配体传感器在Cd(II)检测中的应用,将20 μL一定浓度的Cd(II)溶液孵育于传感器MB/S2/S1/Au/ZnO-rGO/ITO表面,置于室温恒湿1 h,记作Cd/MB/S2/S1/Au/ZnO-rGO/ITO。
本发明的目的还可以通过如下测试技术来实现:
采用双通道-双工作电极(四电极)系统,以上述孵育Cd(II)前后的适配体传感器为第一工作电极,玻碳电极(GCE)为第二工作电极,Ag/AgCl为参比电极,铂丝电极为对电极,对Cd(II)进行定量检测。电解液为含0.1 mol/L多巴胺(DA)的磷酸盐缓冲溶液(pH=7.0),测试偏压为0 V,LED灯波长为365 nm,DA作为电子供体,在第一工作电极和第二工作电极表面实现氧化还原循环,提高光电响应的同时,有利于检测信号的稳定性。
和现有技术相比,本发明方法的优点在于:
本发明采用电还原和电沉积的方法,制备了纳米Au、ZnO纳米棒和rGO均匀负载的修饰光电极,不仅有利于改善传感界面的生物相容性和提高光电子传递速率,而且有利于增大比表面积,为适配体的自组装提供位点。MB作为光电信号的放大剂嵌入互补的DNA双链结构中的,能够有效放大检测信号,提高该适配体传感器检测的灵敏度。检测采用双工作电极(四电极)检测系统,能够实现电子供体DA在双工作电极表面的氧化还原循环,提高检测信号的稳定性。此外,本发明的光电化学适配体传感器,制备工艺简单、成本低廉、操作方便,且具有良好的稳定性和灵敏度。
附图说明
图1为不同修饰电极的SEM图,其中(A)为rGO/ITO,(B)为ZnO-rGO/ITO,(C)为Au/ZnO-rGO/ITO。
图2为不同修饰电极的XRD谱图,其中(a)为rGO/ITO,(b)为ZnO-rGO/ITO,(c)为Au/ZnO-rGO/ITO。
图3为不同材料修饰电极的光电响应曲线,其中(a)为工作电极ZnO/ITO,(b)为工作电极ZnO-rGO/ITO,(c)为工作电极Au/ZnO-rGO/ITO。
图4为修饰电极Au/ZnO-rGO/ITO采用不同的测试方法时的光电响应曲线,其中(a)为三电极检测系统,电解液中不含DA,(b)为三电极检测系统,电解液中含有0.1 mol/L DA,(c)为四电极检测系统,电解液中含有0.1 mol/L DA。
图5为以DA作为电子供体,在双工作电极表面的氧化还原循环示意图。
图6为修饰电极(a) ZnO-rGO/ITO,(b) Au/ZnO-rGO/ITO,(c) S1/Au/ZnO-rGO/ITO,(d) S2/S1/Au/ZnO-rGO/ITO,(e) MB/S2/S1/Au/ZnO-rGO/ITO,(f) Cd/MB/S2/S1/Au/ZnO-rGO/ITO的光电响应曲线。
图7(A)为Cd(II)浓度依次为5.0×10-12, 6.0×10-11, 3.0×10-10, 1.0×10-9,6.0×10-9, 2.0×10-8 mol/L (曲线a至f)时,传感器的光电流响应曲线,(B)为光电流响应与Cd(II)浓度的线性关系曲线。
图8为不同干扰离子(浓度为6.0×10-9 mol/L)对该光电化学适配体传感器检测的影响。
具体实施方式
以下结合实施例对本发明的技术方案做进一步地详细介绍,但本发明的保护范围并不局限于此。
下述实施例中,所用的适配体S1(5’-SH-CAT ACT GCA CAA CCA AAA ATA ATA CCACAA CAG TCC-3’)和适配体S2(5’-GGA CTG TTG TGG TAT TAT TTT TGG TTG TGC AGT ATG-3’)均购自生工生物工程上海股份有限公司。
实施例1:
一种以纳米金/氧化锌-还原氧化石墨烯三元复合材料为光电敏感元件的光电化学适配体传感器的构建方法,其包括如下步骤:
(1)光电极的制备:
(1.1)修饰电极rGO/ITO的制备:
将ITO导电玻璃分别浸入丙酮、乙醇和超纯水中超声30 min,高纯氮吹扫干燥后备用;将ITO导电玻璃浸入0.2 mg/mL的氧化石墨烯(GO)水溶液中,采用恒电流沉积法,电流密度为0.25 mA/cm2,沉积时间为100 s,制备还原氧化石墨烯(rGO)均匀负载的电极rGO/ITO;
(1.2)修饰电极ZnO-rGO/ITO的制备:
将rGO/ITO浸入0.02 mol/L Zn(NO3)2水溶液中,于80℃恒温水浴条件下恒电位沉积,沉积电位为-1.4 V,沉积时间为30 min,制得ZnO纳米棒均匀负载的修饰电极ZnO-rGO/ITO;
为了做对比,使用上述同样的方法,在洁净的ITO导电玻璃表面,直接电沉积ZnO,制得修饰电极ZnO/ITO;
(1.3)修饰电极Au/ZnO-rGO/ITO的制备:
将ZnO-rGO/ITO浸入2.0 mmol/L HAuCl4和0.5 mol/L KCl混合溶液中,采用恒电位还原法制备Au纳米粒子负载的修饰电极Au/ZnO-rGO/ITO,还原电位为-0.4 V,时间为50 s;
(2)适配体传感器的制备:
取20 μL 1.0 μmol/L巯基预修饰的适配体S1溶液,滴涂在上述光电极Au/ZnO-rGO/ITO表面,置于室温恒湿孵育2 h,将S1通过Au-S共价键自组装于光电极表面,然后滴加20 μL0.2 mmol/L巯基丙酸(MPA)作为封闭剂孵育30 min,以封闭光电极表面的活性吸附位点,即得修饰电极S1/Au/ZnO-rGO/ITO;
取20 μL 1.0 μmol/L适配体S2溶液,滴涂于S1/Au/ZnO-rGO/ITO表面,置于室温恒湿孵育2 h,S1与S2互补配对形成双链DNA (dsDNA)结构,得到修饰电极S2/S1/Au/ZnO-rGO/ITO;
取20 μL 10.0 μmol/L亚甲基蓝(MB)溶液,滴涂于S2/S1/Au/ZnO-rGO/ITO表面,作为光敏剂的MB通过嵌插或静电键合作用与dsDNA结合,得到适配体传感器MB/S2/S1/Au/ZnO-rGO/ITO,将所制备好的修饰电极储存于4℃冰箱内保存,备用。
对实施例1的修饰电极rGO/ITO、ZnO-rGO/ITO和Au/ZnO-rGO/ITO进行形貌表征。如图1所示,图A为rGO/ITO的SEM图,可观察到rGO的褶皱结构,图B为ZnO-rGO/ITO的SEM图,可看到规则的六边形短柱状结构,图C为Au/ZnO-rGO/ITO的SEM图,其表面均匀负载Au纳米粒子。因此可知,光电极Au/ZnO-rGO/ITO表面达到理想且均一的形貌。
对实施例1的修饰电极rGO/ITO、ZnO-rGO/ITO和Au/ZnO-rGO/ITO进行表面晶型结构和组成的表征。如图2所示,曲线a为rGO/ITO的XRD谱图,30.2°和35.2°为ITO表面In2O3的特征衍射峰,26.0°处有一个弱且宽的rGO特征衍射峰,曲线b为ZnO-rGO/ITO的XRD谱图,五个明显的特征衍射峰依次对应ZnO的(100),(002),(101),(102)和(103)晶面,曲线c为Au/ZnO-rGO/ITO的XRD谱图, 38.3°,44.6°和77.5°则对应Au的特征衍射峰。表征结果说明,Au纳米粒子均匀地负载在ZnO表面,光电极制备成功。
对实施例1的修饰电极ZnO/ITO、ZnO-rGO/ITO和Au/ZnO-rGO/ITO的光电性能进行比较。采用三电极测试系统,以不同的修饰电极为工作电极,Ag/AgCl为参比电极,铂丝电极为对电极,电解液为pH=7.0的磷酸盐缓冲溶液,激发光波长为365 nm,偏置电压为0 V,分别记录其光电响应曲线。如图3所示,曲线a、b、c分别为修饰电极ZnO/ITO、ZnO-rGO/ITO和Au/ZnO-rGO/ITO的光电响应曲线,ZnO/ITO的光电流最小,而修饰有rGO的ZnO-rGO/ITO电极光电流增大,说明rGO更有利于光生电子-空穴对的分离和光电子的转移,从而获得更高的光电流。当其表面沉积Au后(Au/ZnO-rGO/ITO电极),其局域表面等离子体共振效应进一步增强了光电流响应。
对实施例1的修饰电极Au/ZnO-rGO/ITO在三电极系和四电极检测系统的光电响应测试结果进行比较。如图4所示,曲线a为三电极检测系统(Ag/AgCl为参比电极,铂丝电极为对电极,Au/ZnO-rGO/ITO为工作电极,电解液为pH 7.0的磷酸盐缓冲溶液),光电响应最小,曲线b为三电极检测系统(Ag/AgCl为参比电极,铂丝电极为对电极,Au/ZnO-rGO/ITO为工作电极,电解液为含0.1 mol/L DA的磷酸盐缓冲溶液),光电响应较曲线a增大了近一倍,这是由于电解液中的DA作为电子供体能够促进光生电子-空穴对的分离和光生电子的转移。曲线c为四电极检测系统(Au/ZnO-rGO/ITO为第一工作电极,玻碳电极GCE为第二工作电极,Ag/AgCl为参比电极,铂丝电极为对电极,电解液为含0.1 mol/L DA的磷酸盐缓冲溶液),光电响应较曲线b又进一步增大了近一倍,DA在第一工作电极和第二工作电极表面发生循环的氧化还原反应,示意图如图5所示,这不但有利于增强光电流信号,而且提高了传感器检测信号的稳定性。
实施例2
电流-时间曲线法监测适配体传感器的组装过程
图6为采用四电极检测系统,以含0.1 mol/L DA的磷酸盐缓冲溶液为电解液,测试不同修饰电极的光电响应曲线,曲线a到e分别表示实施例1中修饰电极ZnO-rGO/ITO、Au/ZnO-rGO/ITO、S1/Au/ZnO-rGO/ITO、S2/S1/Au/ZnO-rGO/ITO和MB/S2/S1/Au/ZnO-rGO/ITO的光电响应曲线,ZnO-rGO/ITO在沉积Au纳米粒后(Au/ZnO-rGO/ITO),光电流显著增加,Au纳米粒子良好的导电性和局域表面等离子体共振效应,促进了光生电子-空穴对的分离和电子的传递。当Au/ZnO-rGO/ITO表面自组装适配体S1后(S1/Au/ZnO-rGO/ITO),其光电流显著减小,带负电荷的寡核苷酸序列阻碍了电子的传递,同时其空间位阻效应也阻碍了电子的传递。当适配体S2进一步修饰于S1/Au/ZnO-rGO/ITO后,在电极表面形成dsDNA(S2/S1/Au/ZnO-rGO/ITO),光电流进一步减小。最后将MB嵌入dsDNA后(MB/S2/S1/Au/ZnO-rGO/ITO),光电流显著增加,比S2/S1/Au/ZnO-rGO/ITO提高了四倍,MB的修饰有效放大了检测信号。
实施例3
一种以纳米金/氧化锌-还原氧化石墨烯三元复合材料为光电敏感元件的光电化学适配体传感器的应用,包括以下步骤:
将20 μL不同浓度的Cd(II)溶液分别孵育于实施例1中的光电化学适配体传感器MB/S2/S1/Au/ZnO-rGO/ITO表面,置于室温恒湿孵育1 h。采用四电极检测系统,对孵育Cd(II)后的传感器进行光电流测试,并根据Cd(II)浓度与光电流响应的关系构建线性回归方程。图7(A)为本发明实施例1中的传感器MB/S2/S1/Au/ZnO-rGO/ITO分别孵育浓度依次为5.0×10-12, 6.0×10-11, 3.0×10-10, 1.0×10-9, 6.0×10-9, 2.0×10-8 mol/L(曲线a至f)的Cd(II)后的光电流响应曲线,(B)为Cd(II)浓度与光电流响应的线性回归图。由图可知,在该浓度范围内,Cd(II)随着浓度的增加,传感器的光电响应信号逐渐减小,线性方程为I (nA)= -(160.70±3.89)logC (mol/L)-(30.65±1.45) (R2=0.997,n=6),检测限为1.8×10-12 mol/L (S/N=3)。
实施例4
考察光电化学适配体传感器的选择性
为了验证该传感器的选择性,将实施例1中的MB/S2/S1/Au/ZnO-rGO/ITO分别孵育浓度为6.0×10-9 mol/L的不同干扰离子,采用四电极检测系统,检测不同干扰离子孵育后的传感器的光电响应,结果如图8所示,该传感器对Cd(II)具有良好的响应,对Ag(I)、Hg(II)、Cu(II)、Fe(III)、Pb(II)、Ni(II)、Se(IV)、Zn(II)不敏感,说明该传感器具有良好的选择性和抗干扰性。
Claims (2)
1.一种以纳米金/氧化锌-还原氧化石墨烯三元复合材料为光电敏感元件的光电化学适配体传感器的构建方法,其特征在于,包括以下步骤:
以ITO导电玻璃为基底电极,依次经丙酮、乙醇和超纯水分别超声清洗30 min,氮气吹扫晾干;将洁净的ITO导电玻璃浸入0.2 mg/mL氧化石墨烯(GO)溶液中,采用恒电流沉积法,电流密度为0.25 mA/cm2,沉积时间为100 s,制备了还原氧化石墨烯(rGO)均匀负载的ITO修饰电极,标记为rGO/ITO;
将上述修饰电极浸入0.02 mol/L Zn(NO3)2水溶液中,于80℃恒温水浴中,-1.4 V恒电位沉积30 min,制得ZnO-rGO/ITO修饰电极;
将ZnO-rGO/ITO电极浸入2.0 mmol/L HAuCl4和0.5 mol/L KCl混合溶液中,于-0.4 V恒电位还原50 s,制得光电极Au/ZnO-rGO/ITO;
取20 μL 1.0 μmol/L巯基预修饰的适配体 S1溶液,滴涂在上述光电极Au/ZnO-rGO/ITO表面,置于室温恒湿孵育2 h,将S1通过Au-S共价键自组装于光电极表面,然后滴加20 μL 0.2 mmol/L巯基丙酸(MPA)作为封闭剂孵育30 min,以封闭光电极表面的活性吸附位点,即得修饰电极S1/Au/ZnO-rGO/ITO;
取20 μL 1.0 μmol/L适配体S2溶液,滴涂于S1/Au/ZnO-rGO/ITO表面,置于室温恒湿孵育2 h,S1与S2互补配对形成双链DNA (dsDNA)结构,得到修饰电极S2/S1/Au/ZnO-rGO/ITO;
取20 μL 10.0 μmol/L亚甲基蓝(MB)溶液,滴涂于S2/S1/Au/ZnO-rGO/ITO表面,作为光敏剂的MB通过嵌插或静电键合作用与dsDNA结合,得到适配体传感器MB/S2/S1/Au/ZnO-rGO/ITO。
2.一种如权利要求1所述的以纳米金/氧化锌-还原氧化石墨烯三元复合材料为光电敏感元件的光电化学适配体传感器的应用,其特征在于,包括以下步骤:
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