CN115184431A - 一种双信号探针策略的分子印迹电化学传感器的制备方法及其应用 - Google Patents
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Abstract
本发明属于分析化学和电化学技术领域,公开了一种双信号探针策略的分子印迹电化学传感器的制备方法及其应用。将第一种模板分子IgG通过金硫键固定到普鲁士蓝@金纳米粒子/氧化石墨烯/丝网印刷碳电极上,得到,将第二种模板分子IgM通过金硫键固定到多壁碳纳米管‑硫堇‑金纳米粒子/牛血清白蛋白/IgG/普鲁士蓝@金纳米粒子/氧化石墨烯/氧化石墨烯/丝网印刷碳电极上,电化学聚合吡咯,用洗脱液将IgG、IgM洗脱后,形成与这两种模板分子在三维空间上互补的印迹空腔,得到分子印迹电化学传感器。本发明所述的分子印迹电化学传感器能够同时检测IgG和IgM。
Description
技术领域
本发明属于分析化学和电化学技术领域,具体涉及一种双信号探针策略的分子印迹电化学传感器的制备方法及其应用。
背景技术
免疫球蛋白(Ig)在生物体对抗各种潜在破坏性物体(如病毒或细菌)的许多防御机制中发挥着重要作用。已有报道在人体循环***中存在五类常见的免疫球蛋白,即免疫球蛋白A、G、M、E和D。Ig水平过高会引起类风湿关节炎、肝病和感染性疾病,但低Ig水平也会引起体液免疫缺陷和代谢性疾病。自2019年12月以来,由于新冠疫情的爆发,IgG和IgM已成为评估早期或感染阶段的重要指标。酶联免疫分析已普遍用于IgG和IgM的检测,但其存在仪器昂贵、操作复杂和测定耗时等缺点。电化学检测具有灵敏度高、成本低、操作简单等优点,因而在医疗方面具有巨大的潜力。
分子印迹电化学传感器将分子印迹技术与电化学传感技术相结合,采用分子印迹聚合物(MIP)作为特异性分子识别元件,增强电化学传感器的选择性识别性能。分子印迹电化学传感器具有灵敏度高、选择性好、分析速度快、易于小型化等优点,已被广泛应用于血清中多靶点的检测。然而,到目前为止,MIP的研究主要针对小分子。由于蛋白质属于生物大分子,其具有相对较大的分子量、复杂的表面形态和多变的分子构象,因此需要寻求一种合适的方法用于蛋白质的分子印迹。
发明内容
为同时检测IgG和IgM,本发明提供一种分子印迹电化学传感器的制备方法及其应用。
技术方案:为解决上述技术问题,本发明采用的技术方案为:
一种双信号探针策略的分子印迹电化学传感器,能够对IgG和IgM进行同时检测。以丝网印刷碳电极(SPCE)作为基底电极,用氧化石墨烯(GO)修饰SPCE,一步电沉积普鲁士蓝@金纳米粒子(PB@AuNPs)在GO/SPCE表面,将免疫球蛋白G(IgG)作为第一种模板分子修饰在PB@AuNPs/GO/SPCE表面,PB为指示IgG信号变化的媒介物。用牛血清白蛋白(BSA)封闭剩余的结合位点。制备多壁碳纳米管-硫堇-金纳米粒子(MWCNTs-TH-AuNPs),将其修饰在BSA/IgG/PB@AuNPs/GO/SPCE表面,将免疫球蛋白M(IgM)作为第二种模板分子修饰在MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE表面,TH为指示IgM信号变化的媒介物。接着,电聚合吡咯(Py)形成聚合物层将IgG和IgM进一步包埋,最后用洗脱液将IgG、IgM模板分子洗脱,形成印迹空腔,成功制备分子印迹电化学传感器。
一种双信号探针策略的分子印迹电化学传感器,将第一种模板分子IgG通过金硫键固定到普鲁士蓝@金纳米粒子/氧化石墨烯/丝网印刷碳电极PB@AuNPs/GO/SPCE上,得到IgG/PB@AuNPs/GO/SPCE,将第二种模板分子IgM通过金硫键固定到多壁碳纳米管-硫堇-金纳米粒子/牛血清白蛋白/IgG/普鲁士蓝@金纳米粒子/氧化石墨烯/氧化石墨烯/丝网印刷碳电极MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE上,得到IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE;最后,电化学聚合吡咯得到PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE,用洗脱液将IgG、IgM洗脱后,形成与这两种模板分子在三维空间上互补的印迹空腔,得到分子印迹电化学传感器。
进一步的,所述普鲁士蓝@金纳米粒子/氧化石墨烯/丝网印刷碳电极PB@AuNPs/GO/SPCE的制备方法为:将无水三氯化铁、铁***和氯化钾溶解于盐酸溶液中,随后在溶液中加入氯金酸溶液,得到PB@AuNPs溶液;用移液枪移取GO溶液滴涂于SPCE表面,红外灯下烘干,得到GO/SPCE;用循环伏安法在0~1V的电位范围内扫描,在GO/SPCE表面电沉积PB@AuNPs层,形成PB@AuNPs/GO/SPCE。
进一步的,所述多壁碳纳米管-硫堇-金纳米粒子/牛血清白蛋白/IgG/普鲁士蓝@金纳米粒子/氧化石墨烯/氧化石墨烯/丝网印刷碳电极MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE的制备方法包括:
将IgG溶液滴涂于PB@AuNPs/GO/SPCE表面,晾干后再移取BSA作为封闭液滴涂于其表面,得到BSA/IgG/PB@AuNPs/GO/SPCE;将MWCNTs溶解于聚乙烯亚胺水溶液中,超声分散;将TH水溶液与上述溶液混合在一起,剧烈搅拌,待搅拌结束,将离心分离后得到的固体物质用超纯水洗涤数次,分散在水中,得到MWCNTs-TH溶液,将MWCNTs-TH溶液加入到AuNPs溶液中,搅拌均匀后,离心所得固体即为MWCNTs-TH-AuNPs复合材料;将MWCNTs-TH-AuNPs复合材料超声分散于水中,用移液枪移将其涂于BSA/IgG/PB@AuNPs/GO/SPCE表面,得到MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE。
进一步的,洗脱步骤为:用移液枪移取含有质量分数为10%十二烷基硫酸钠的体积分数为10%乙酸溶液滴涂于PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE表面,洗脱0.5~2.5h,脱去模板分子IgG和IgM。
进一步的,在GO/SPCE表面电沉积PB@AuNPs层时,循环伏安法扫描速率为90~110mV s–1,扫描圈数为10~30圈。
进一步的,将吡咯的磷酸盐缓冲溶液滴加在IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE表面,循环伏安法扫描速率为25~125mV s–1,优选为75mV s–1,沉积圈数为3~15圈,形成PPy聚合层。
进一步的,所述IgG的浓度为0.5~2.0μg mL–1;IgM的浓度为400~600ng mL–1。
进一步的,盐酸溶液的浓度为0.1mM;氯金酸溶液的浓度为0.2mM。
本发明还提供上述双信号探针策略的分子印迹电化学传感器在检测IgG和IgM中的应用。
进一步的,将分子印迹电化学传感器在含有IgG和IgM的溶液中孵育1~6h,在–0.5~0.5V的电位范围内记录差分脉冲伏安图,实现对IgG和IgM的同时检测。本发明还提供所述的双信号探针策略的分子印迹电化学传感器的制备方法,包括:
步骤a、配置PB@AuNPs溶液:称取无水三氯化铁、铁***和氯化钾溶解于0.1M的盐酸溶液中,随后将0.2mM的氯金酸溶液加入到上述溶液中,即可得PB@AuNPs溶液;
步骤b、制备PB@AuNPs/GO/SPCE:用移液枪移取10μL GO溶液滴涂于SPCE表面,红外灯下烘干,即可得GO/SPCE;用循环伏安法在0~1V的电位范围内以一定扫描速率扫描15圈,在GO/SPCE表面电沉积PB@AuNPs层,形成PB@AuNPs/GO/SPCE;
步骤c、制备BSA/IgG/PB@AuNPs/GO/SPCE:用移液枪先移取10μL一定浓度的IgG溶液滴涂于PB@AuNPs/GO/SPCE表面,4℃下晾干,再移取10μL BSA作为封闭液滴涂于该电极表面,即可得BSA/IgG/PB@AuNPs/GO/SPCE;
步骤d、制备MWCNTs-TH-AuNPs复合材料:称取10mg MWCNTs溶解于聚乙烯亚胺(PEI)水溶液中,超声分散;将一定浓度的TH水溶液与上述溶液混合在一起,剧烈搅拌,待搅拌结束,将离心分离后得到的固体物质用超纯水洗涤数次,分散在水中,即可得MWCNTs-TH溶液,用移液枪移取0.5mL MWCNTs-TH溶液加入到2.5mL AuNPs溶液中,搅拌一定时间后,离心所得固体即为MWCNTs-TH-AuNPs复合材料。
步骤e、制备IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE:将步骤d制得的MWCNTs-TH-AuNPs复合材料超声分散于4mL水中,用移液枪移取10μL滴涂于步骤c电极表面,自然晾干,再用移液枪移取10μL一定浓度的IgM溶液滴在电极表面,4℃晾干,即得IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE;
步骤f、制备PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE:用移液枪移取100μL含有0.1M吡咯的0.01M磷酸盐缓冲溶液滴加在步骤e制得的电极表面,用循环伏安法在–0.3~0.8V的电位范围内以一定扫描速率,聚合一定圈数,即可得PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE;
步骤g、制备分子印迹电化学传感器:用移液枪移取100μL含有10%(w%)十二烷基硫酸钠的10%(v/v)乙酸溶液滴涂于步骤f制得的电极表面,洗脱一定时间以脱去模板分子IgG和IgM,即可得分子印迹电化学传感器。
进一步的,步骤a中,无水三氯化铁、铁***和氯化钾的质量比为0.04~0.05:0.08~0.09:0.7~0.8,0.1M盐酸和0.2mM氯金酸的体积比为10~50:0.6~0.7。
进一步的,步骤b中,移液枪移取GO溶液的体积为8~12μL;循环伏安法扫描速率为90~110mV s–1,扫描圈数为10~30圈对电极进行修饰,可在GO/SPCE表面形成PB@AuNPs层。
进一步的,步骤c中,移液枪移取IgG的体积为8~12μL,浓度为0.5~2.0μg mL–1,移液枪移取BSA封闭液的体积为10~30μL,孵育时间为0.5~2h。
进一步的,步骤d中,称取PEI的质量为5~15mg,MWCNTs的质量为5~15mg,用于溶解MWCNTs的PEI水溶液体积为5~15mL,移液管移取TH溶液的体积为0.5~1.5mL,浓度为1~3mg mL–1,MWCNTs-TH的体积为0.25~1.0mL,AuNPs溶液的体积为1.5~3.0mL,搅拌的时间为10~14h。其中,PEI和MWCNTs的质量比为5~15:5~15,PEI水溶液、TH溶液、MWCNTs-TH和AuNPs溶液的体积比为5~15:0.5~1.5:0.25~1.0:1.5~3.0。
进一步的,步骤e中,移液枪移取MWCNTs-TH-AuNPs溶液的体积为8~12μL,移取IgM的浓度为400~600ng mL–1。
进一步的,步骤f中,移取含有0.1M吡咯的0.01M pH为7.4的磷酸盐缓冲溶液的体积为50~150μL,循环伏安法扫描速率为25~125mV s–1,优选为75mV s–1,沉积圈数为3~15圈,优选为7圈,形成PPy聚合层。
进一步的,步骤g中,移液枪移取含有质量分数为10%十二烷基硫酸钠的体积分数为10%乙酸溶液的体积为100~200μL,洗脱时间为0.5~2.5h,优选为2h。
进一步的,包括:将分子印迹电化学传感器应用于IgG和IgM进行同时检测,重新孵育时间为1~6h,优选为4h,在–0.5~0.5V的电位范围内记录差分脉冲伏安图实现对IgG和IgM的同时检测。
一种双信号探针策略的分子印迹电化学传感器,由上述的制备方法制备而成。
第三方面,所述的双信号探针策略的分子印迹电化学传感器在同时检测IgG和IgM中的应用。
在一些实施例中,所述的应用,包括:将分子印迹电化学传感器置于含有1μg mL– 1IgG和500ng mL–1IgM的溶液中进行孵育。
进一步的,所述的应用,还包括:在–0.6~0.4V的电位范围内记录重新结合了IgG和IgM的分子印迹传感器的循环伏安图,在–0.5~0.5V的电位范围内记录重新结合了IgG和IgM的分子印迹传感器的差分脉冲伏安图,通过PB和TH在重新结合IgG和IgM前后峰电流的变化实现对IgG和IgM的同时检测。
有益效果
表面分子印迹是合成蛋白质印迹聚合物的有效方法,它不仅可以改善蛋白质分子的传质过程,避免其残留,还可以在聚合物基体中获得具有较高亲和力的结合位点,从而提高识别效率。电聚合是一种可用于形成蛋白质印迹基底的简单方法,聚吡咯(PPy)由于其高的稳定性、生物相容性和导电性,且厚度可控,常被用作蛋白质印迹的聚合物基底材料。
金纳米粒子(AuNPs)、多壁碳纳米管(MWCNTs)、氧化石墨烯(GO)等纳米材料具有比表面大、导电能力强和生物相容性高等优良特性,常被用于修饰电极,可显著提高电化学传感器的分析性能如较高的灵敏度和较快的电子传递速率。当这些纳米材料用于分子印迹电化学传感器时,还可以为结合模板分子提供丰富的活性位点。
电化学检测时,当待测物电化学信号不明显,常需要借助电化学媒介物来指示待测物浓度的变化从而实现对待测物浓度的检测。普鲁士蓝(PB)和硫堇(TH)因具有良好的氧化还原可逆性,因此常被选作电化学媒介物。
本发明双信号探针策略的分子印迹电化学传感器的制备方法及其应用,将聚吡咯层中的IgG和IgM模板分子洗脱后,留下了与它们在三维空间上互补的印迹空腔,印迹空腔有利于PB和TH在氧化还原过程中电子的传输。重新结合IgG和IgM分子后,印迹空腔再次被占据,导致PB和TH的氧化峰电流下降,从而实现对IgG和IgM的同时检测。
附图说明
图1为实施例一中制备的SPCE、GO/SPCE、PB@AuNPs/GO/SPCE、IgG/PB@AuNPs/GO/SPCE、BSA/IgG/PB@AuNPs/GO/SPCE的差分脉冲伏安图;
图2为实施例一中制备的BSA/IgG/PB@AuNPs/GO/SPCE、MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE、IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE的差分脉冲伏安图;
图3为实施例一中制备的分子印迹电化学传感器聚合吡咯、洗脱后和在IgG和IgM溶液中孵育后的循环伏安图;
图4为实施例一中制备的分子印迹电化学传感器对含有不同浓度的IgG和IgM溶液进行检测的差分脉冲伏安图;
图5为实施例一中制备的分子印迹电化学传感器用于对IgG和IgM同时检测时,TH峰电流的降低值(ΔITH)与IgG浓度对数值(lgC)的线性关系图(A)和PB峰电流的降低值(ΔIPB)与IgM浓度对数值(lgC)的线性关系图(B);
图6为实施例一中制备的MWCNTs-TH-AuNPs的紫外可见吸收光谱图。
图7为实施例一中制备的分子印迹电化学传感器的特异性结果。
图8为对比例一中制备的非印迹电化学传感器聚合吡咯、洗脱后和在IgG和IgM溶液中孵育后的循环伏安图。
具体实施方式
下面结合附图及具体实施例对本发明作进一步说明,但本发明并不限于以下实施例。
实施例一:
一种双信号探针策略的分子印迹电化学传感器的制备方法,包括以下步骤:
(1)称取0.0405g无水三氯化铁、0.0823g铁***和0.7455g氯化钾溶解于100mL的0.1M盐酸溶液中,随后将0.689mL的29mM氯金酸溶液加入到该溶液中,即可得PB@AuNPs溶液。
(2)称取2mg GO粉末溶解于1mL超纯水中超声分散均匀后,滴涂于SPCE表面,红外灯下烘干,即可得GO/SPCE。如图1,GO/SPCE没有氧化峰;用移液枪移取10μL PB@AuNPs溶液于GO/SPCE上,用循环伏安法在0~1V的电位范围内以100mV s–1的扫描速率,在GO/SPCE表面电沉积PB@AuNPs层,形成PB@AuNPs/GO/SPCE。如图1,PB@AuNPs/SPCE在0.1V处出现了PB的氧化峰,这说明PB是一种优良的电化学媒介物。本发明将PB作为第一种电化学探针,指示IgG的信号变化。
(3)用移液枪先移取10μL的1.0μg mL–1IgG溶液修饰在步骤(2)所得电极表面,在4℃下孵育。如图1,相比于PB@AuNPs/SPCE,IgG/PB@AuNPs/GO/SPCE氧化峰电流显著降低,这是由于IgG是一种导电性能较差的生物大分子,通过巯基与AuNPs结合后抑制了电极表面的电子传递;再移取10μL BSA作为封闭液在4℃下孵育电极1h,即可得BSA/IgG/PB@AuNPs/GO/SPCE,如图1,电流继续降低,归因于BSA本身导电性较差。
(4)移取10μL的MWCNTs-TH-AuNPs溶液滴涂于步骤(3)电极表面,室温下干燥,得到MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE。如图2,MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE在–0.2V处出现了TH的氧化峰,这说明TH也是一种优良的电化学媒介物,而PB的氧化峰位置几乎不变。TH作为第二种电化学探针,指示IgM的信号变化;用移液枪移取10μL的500ng mL–1IgM溶液,在4℃下孵育电极,即可得IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE。如图2,TH的氧化峰减小,归因于导电性能差的生物大分子IgM通过巯基与MWCNTs-TH-AuNPs中的AuNPs结合,阻碍了电子传输。
(5)移取100μL含有0.1M吡咯的0.01M磷酸盐缓冲溶液滴加在步骤(4)制得的电极表面,用循环伏安法在–0.3~0.8V的电位范围内以75mV s–1的扫描速率,聚合7圈,即可得PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE。如图3,电聚合吡咯后,PB和TH的峰消失,这是由于聚吡咯层在电极表面形成后,阻碍了PB和TH的氧化还原过程。
(6)移取100μL含有10%十二烷基硫酸钠的10%乙酸洗脱溶液滴涂于步骤(5)制得的电极表面用于洗脱模板分子,洗脱2h后,用超纯水和磷酸盐缓冲溶液交替多次冲洗电极以除去表面残余的IgG和IgM,即可得分子印迹电化学传感器。如图3,出现了PB和TH的氧化还原峰,归因于酸性洗脱液能破环蛋白结构,IgG和IgM可从聚合层中被洗脱,从而形成了印迹空腔,有利于PB和TH的氧化还原反应的发生。移取10μL含有1.0μg mL–1IgG和500ng mL– 1IgM的溶液在4℃下孵育分子印迹电化学传感器4h。如图3,PB和TH的峰电流明显下降,这是因为IgG和IgM重新占据了印迹空腔,从而抑制了PB和TH的氧化还原过程。
(7)制备好的分子印迹电化学传感器,在含有IgG(0.05ng mL–1~100ng mL–1)和IgM(0.05ng mL–1~100ng mL–1)的溶液中孵育4h。如图4,TH的氧化峰出现在约–0.23V,可用于指示IgM浓度的变化,PB的氧化峰出现在约0.11V,可用于指示IgG浓度的变化。随着IgG和IgM浓度逐渐增大,PB和TH的峰电流逐渐减小,归因于IgG和IgM会占据对应的印迹空腔,从而抑制了PB和TH氧化还原过程中产生的电子的传输。
(8)制备好的分子印迹电化学传感器对IgG和IgM同时检测时,峰电流的降低值与浓度对数值呈线性关系。如图5A,IgM的线性方程为△ITH(μA)=1.662lgC+4.813(R2=0.9962),线性范围为0.05ng mL–1~100ng mL–1,如图5B,IgG的线性方程为△IPB(μA)=2.130lgC+9.802(R2=0.9945),线性范围为0.05ng mL–1~100ng mL–1。根据三倍信噪比,通过计算得出IgM的检测限为28.61pg mL–1,IgG的检测限为23.93pg mL–1。上述结果证明分子印迹电化学传感器能够同时检测IgG和IgM。
(9)移取0.5mL MWCNTs-TH溶液加入到2.5mL AuNPs溶液中,搅拌12h,离心所得固体分散在水中,即可得MWCNTs-TH-AuNPs复合材料的溶液。如图6所示,曲线a为AuNPs的紫外可见光谱,在519nm处出现了AuNPs的特征吸收带,表明AuNPs成功制备,曲线b为MWCNT-TH的紫外可见光谱,在285和601nm处呈现两个特征吸收带,分别属于TH芳香环的π-π*跃迁和C=N键的n-π*跃迁,表明TH分子通过π-π堆积作用非共价附着在MWCNTs表面,曲线c为MWCNT-TH-AuNPs的紫外可见光谱,与MWCNT-TH(曲线b)的光谱相似,但在601nm处的吸收带蓝移到597nm处,这可能是由于TH的N原子与Au发生配位,519nm处吸收带的消失归因于AuNP在MWCNT-TH上的聚集。以上结果表明,MWCNT-TH-AuNPs复合材料已经成功合成。
(10)通过检测100ng mL–1IgG和IgM,以及10倍浓度(1μg mL–1)的牛血清白蛋白(BSA)、血红蛋白(HGB)、L-色氨酸(L-Trp)和D-酪氨酸(D-Tyr)来研究分子印迹电化学传感器的特异性(图7)。测试结果表明该传感器在10倍浓度的BSA、HGB、L-Trp和D-Tyr溶液中孵育4h后,其△IPB和△ITH明显低于检测IgG和IgM时的值,表明该分子印迹电化学传感器具有良好的特异性。
对比例一:
为进一步证明分子印迹电化学传感器的成功制备,另外制备了非印迹电化学传感器,其操作步骤与实施例一步骤(1)~(6)相同,只是未加IgG和IgM模板分子。如图8,没有出现PB和TH的氧化还原峰,归因于聚吡咯层阻碍了PB和TH的氧化还原过程。由于没有IgG和IgM的存在,用酸性洗脱液对电极进行洗脱时,不能形成有利于电子传递的印迹空腔,因此洗脱后的电极仍然没有出现PB和TH的氧化还原峰。移取10μL含有1.0μg mL–1IgG和500ngmL–1IgM的溶液在4℃下孵育分子印迹电化学传感器4h,如图8,电流信号几乎不变,说明没有印迹空腔的存在。通过分子印迹电化学传感器与非印迹电化学传感器的对比,进一步证明分子印迹电化学传感器能够同时检测IgG和IgM。
Claims (10)
1.一种双信号探针策略的分子印迹电化学传感器,其特征在于,将第一种模板分子IgG通过金硫键固定到普鲁士蓝@金纳米粒子/氧化石墨烯/丝网印刷碳电极PB@AuNPs/GO/SPCE上,得到IgG/PB@AuNPs/GO/SPCE,将第二种模板分子IgM通过金硫键固定到多壁碳纳米管-硫堇-金纳米粒子/牛血清白蛋白/IgG/普鲁士蓝@金纳米粒子/氧化石墨烯/氧化石墨烯/丝网印刷碳电极MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE上,得到IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE;最后,电化学聚合吡咯得到PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE,用洗脱液将IgG、IgM洗脱后,形成与这两种模板分子在三维空间上互补的印迹空腔,得到分子印迹电化学传感器。
2.根据权利要求1所述的双信号探针策略的分子印迹电化学传感器的制备方法,其特征在于,所述普鲁士蓝@金纳米粒子/氧化石墨烯/丝网印刷碳电极PB@AuNPs/GO/SPCE的制备方法为:将无水三氯化铁、铁***和氯化钾溶解于盐酸溶液中,随后在溶液中加入氯金酸溶液,得到PB@AuNPs溶液;用移液枪移取GO溶液滴涂于SPCE表面,红外灯下烘干,得到GO/SPCE;用循环伏安法在0~1V的电位范围内扫描,在GO/SPCE表面电沉积PB@AuNPs层,形成PB@AuNPs/GO/SPCE。
3.根据权利要求1所述的双信号探针策略的分子印迹电化学传感器的制备方法,其特征在于,所述多壁碳纳米管-硫堇-金纳米粒子/牛血清白蛋白/IgG/普鲁士蓝@金纳米粒子/氧化石墨烯/氧化石墨烯/丝网印刷碳电极MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE的制备方法包括:
将IgG溶液滴涂于PB@AuNPs/GO/SPCE表面,晾干后再移取BSA作为封闭液滴涂于其表面,得到BSA/IgG/PB@AuNPs/GO/SPCE;将MWCNTs溶解于聚乙烯亚胺水溶液中,超声分散;将TH水溶液与上述溶液混合在一起,剧烈搅拌,待搅拌结束,将离心分离后得到的固体物质用超纯水洗涤数次,分散在水中,得到MWCNTs-TH溶液,将MWCNTs-TH溶液加入到AuNPs溶液中,搅拌均匀后,离心所得固体即为MWCNTs-TH-AuNPs复合材料;将MWCNTs-TH-AuNPs复合材料超声分散于水中,用移液枪移将其涂于BSA/IgG/PB@AuNPs/GO/SPCE表面,得到
MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE。
4.根据权利要求1所述的双信号探针策略的分子印迹电化学传感器的制备方法,其特征在于,洗脱步骤为:用移液枪移取含有质量分数为10%十二烷基硫酸钠的体积分数为10%乙酸溶液滴涂于
PPy/IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE表面,洗脱0.5~2.5h,脱去模板分子IgG和IgM。
5.根据权利要求2所述的双信号探针策略的分子印迹电化学传感器的制备方法,其特征在于,在GO/SPCE表面电沉积PB@AuNPs层时,循环伏安法扫描速率为90~110mV s–1,扫描圈数为10~30圈;将吡咯的磷酸盐缓冲溶液滴加在IgM/MWCNTs-TH-AuNPs/BSA/IgG/PB@AuNPs/GO/SPCE表面,循环伏安法扫描速率为25~125mV s–1,沉积圈数为3~15圈,形成PPy聚合层。
6.根据权利要求1所述的双信号探针策略的分子印迹电化学传感器的制备方法,其特征在于,所述IgG的浓度为0.5~2.0μg mL–1;IgM的浓度为400~600ng mL–1。
7.根据权利要求1所述的双信号探针策略的分子印迹电化学传感器的制备方法,其特征在于,无水三氯化铁、铁***和氯化钾的质量比为0.04~0.05:0.08~0.09:0.7~0.8;PEI和MWCNTs的质量比为5~15:5~15;PEI水溶液、TH溶液、MWCNTs-TH和AuNPs溶液的体积比为5~15:0.5~1.5:0.25~1.0:1.5~3.0。
8.根据权利要求2所述的双信号探针策略的分子印迹电化学传感器的制备方法,其特征在于,盐酸溶液的浓度为0.1mM;氯金酸溶液的浓度为0.2mM;盐酸和氯金酸的体积比为10~50:0.6~0.7。
9.权利要求1所述双信号探针策略的分子印迹电化学传感器在检测IgG和IgM中的应用。
10.根据权利要求9所述的应用,其特征在于,将分子印迹电化学传感器在含有IgG和IgM的溶液中孵育1~6h,在–0.5~0.5V的电位范围内记录差分脉冲伏安图,实现对IgG和IgM的同时检测。
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