CN111965355A - 一种阴极光电化学免疫传感器及其制备方法与应用 - Google Patents
一种阴极光电化学免疫传感器及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种阴极光电化学免疫传感器及其制备方法与应用,属于生物传感器技术领域。本发明以P型半导体材料作为光电化学基底,并公开将铂纳米催化剂作为信号放大元件,标记于信号抗体上,通过捕获抗体探针、目标抗原和信号抗体之间的夹心免疫反应,实现阴极光电流检测信号的显著放大和对目标抗原的灵敏检测。本发明不仅为阴极光电化学免疫传感器提供一种高效的信号放大策略,还能有效提升对抗原类疾病标志物的检测灵敏度,适于市面推广与应用。
Description
技术领域
本发明属于生物传感器技术领域,涉及一种用于疾病体外诊断的方法策略。更具体地,涉及一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器及其制备方法。
背景技术
随着经济社会的快速发展和生活质量的提高,人们对身体健康越来越重视。对常见重大疾病准确的诊断,有利于疾病的早发现、早治疗,从而有力保障人民的健康。生物传感器是重大疾病体外诊断的重要方式,它主要由分子识别元件与信号转换元件构成的分析检测器件。其中,光电化学生物传感是将光电化学技术与电化学分析法有机结合而发展起来的新一代传感技术。它不仅继承了电化学生物传感具有装置简单、操作方便、花费低、易于集成化和微型化的优点,还使得背景干扰低;并且体系能够实现自供能,更易于实时快速的现场检测。
光电化学生物传感按传感类别分为阳极传感和阴极传感两种。虽然阳极光电化学生物传感输出的光电流信号明显,灵敏度较高,但由于阳极界面发生的是电子氧化反应,实际生物样品中多组分还原性物质如抗坏血酸、多巴胺、谷胱甘肽等对检测结果的准确性有一定的干扰;然而阴极界面发生的是电子还原反应,使得阴极光电化学生物传感具有优良的抗实际生物样品中多组分还原性物种干扰的能力,以致光电化学生物传感更具有在实际复杂生物样品中准确检测的潜力。然而,当前对高灵敏阴极光电化学生物传感器的研制还处于初级阶段,尤其是针对高效信号放大策略的设计和开发报道很少。
铂(Pt)纳米催化剂是目前催化氧还原反应(ORR)最有效的材料之一。多年来,人们对Pt催化剂进行了各种改性,以有效利用ORR中的Pt原子。且为了获得高催化活性,Pt催化剂颗粒应具有纳米尺寸,通过化学或电化学还原催化剂前驱体,使其较为均匀地分布于碳材料载体表面,并具有适当的颗粒间距离。石墨烯是Pt纳米催化剂理想的碳材料载体之一,因为其具有比表面积大、电子传递性能优异、生物相容性好等显著优点。选择石墨烯作为Pt纳米催化剂的载体,能够明显增加Pt纳米催化剂的负载量,显著提升ORR催化反应效率。
此外,Pt纳米催化剂作为高效氧还原反应催化剂,能够加速阴极光电化学传感系统的电荷流动,从而明显增强阴极光电流检测信号。虽然利用Pt纳米催化剂高效催化氧还原反应,在电催化领域应用较为广泛,但以Pt纳米催化剂催化氧还原反应作为高效的信号放大策略在阴极光电化学免疫传感领域尚无任何相关应用的报道。
因此,开发一种灵敏度高、利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器,不仅为阴极光电化学免疫传感器提供一种高效的信号放大策略,还能有效提升对抗原类疾病标志物的检测灵敏度,其对于疾病体外诊断具有十分深远的意义。
发明内容
有鉴于此,本发明的目的是针对现有技术中存在的问题,提供一种灵敏度高、利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器。
为了实现上述目的,本发明的技术方案如下:
一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器,所述阴极光电化学免疫传感器是以P型半导体材料作为光电化学基底,将铂纳米催化剂作为信号放大元件,标记于信号抗体,通过捕获抗体探针、目标抗原和信号抗体之间的夹心免疫反应,实现阴极光电流检测信号的显著放大和对目标抗原的灵敏检测。
需要说明的是,糖抗原19-9(CA19-9)是一种与粘蛋白1相关的细胞表面的路易斯抗原,它是迄今报道的对胰腺癌敏感性最高的标志物。血清中高含量的CA19-9也与其他一些癌症紧密相连,如胃癌、泌尿上皮癌、结直肠癌等。准确检测CA19-9的表达水平对相关疾病的早期诊断和治疗非常重要。因此,本发明以CA19-9作为目标检测物,具有一定的代表性。
本发明的另一目的是提供一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法。
为了实现上述目的,本发明采用如下技术方案:
一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,具体步骤包括:
(1)制备Au/CuBi2O4光阴极:以P型半导体材料CuBi2O4作为阴极光电化学基底,在所述基底表面修饰增敏剂金纳米颗粒制备Au/CuBi2O4光阴极;
(2)制备免疫传感电极:用CA19-9捕获抗体Ab1修饰步骤(1)制备的Au/CuBi2O4光阴极,并用牛血清白蛋白封闭电极活性位点,室温孵育,得到免疫传感电极;
(3)制备Ab2-Pt/GR:将Pt前驱体试剂与氧化石墨烯共混,采用一锅还原反应制备石墨烯GR负载Pt纳米催化剂的Pt/GR复合物,随后将CA19-9信号抗体Ab2修饰于Pt/GR上,即得到Ab2-Pt/GR;
(4)将所述免疫传感电极在室温下孵育目标Ag,以使Ab1与Ag发生特异性免疫反应;随后所述免疫传感电极继续在室温下孵育所述Ab2-Pt/GR,使得Ag与Ab2发生特异性免疫反应,以在所述免疫传感电极上引入Pt/GR复合物,即得所述阴极光电化学免疫传感器。
通过采用上述技术方案,本发明的有益效果如下:
本发明公开的制备方法简单、易于操作,适于推广与应用。
优选的,所述步骤(1)中,采用恒电位法在氧化铟锡电极上沉积CuBi2O4纳米膜,随后煅烧、冷却,得到CuBi2O4纳米膜修饰电极;配置HAuCl4溶液并加热煮沸,随后加入柠檬酸钠溶液加热后,得到Au纳米颗粒溶液;取所述Au纳米颗粒溶液滴加分散至CuBi2O4修饰电极上,最终得到Au/CuBi2O4光阴极。
需要说明的是,Au/CuBi2O4光阴极使用的电极材料环境友好,阴极电流信号响应明显、光化学稳定性好。
进一步优选的,制备所述CuBi2O4纳米膜的沉积时间为40~80s;及制备所述Au/CuBi2O4光阴极时,所述Au纳米颗粒溶液的滴加体积为5~15μL。
优选的,所述步骤(2)中,在所述Au/CuBi2O4光阴极上滴加Ab1,低温孵育,随后用磷酸盐缓冲液冲洗干净后,滴加牛血清白蛋白溶液,室温孵育以封闭电极活性位点,最终得到免疫传感电极。
需要说明的是,上述阴极免疫传感电极制备过程简单快速、样品消耗量小。
进一步优选的,所述Ab1低温孵育的浓度为100~200μg/mL。
优选的,所述步骤(3)中,在去离子水中依次加入氧化石墨烯和聚乙烯吡咯烷酮,超声处理后,加入H2PtCl6溶液搅拌均匀,随后滴加NaBH4溶液搅拌后,离心洗涤、干燥,得到Pt/GR复合物;配置含有所述Pt/GR复合物的分散液,随后滴加入Ab2,低温孵育,即得到Ab2-Pt/GR。
优选的,所述步骤(4)中,在所述免疫传感电极上滴加目标Ag室温孵育后,在所述免疫传感电极上继续滴加所述Ab2-Pt/GR室温孵育,以最终在免疫传感电极上引入Pt/GR复合物。
需要说明的是,上述公开保护的Pt/GR复合物制备方法简单、信号放大效果显著;以及所述免疫传感电极对目标Ag的检测步骤简单、无需提纯、灵敏度高、准确、方便、快速。
示范性的,本发明优选的制备方案为:
(1)以P型半导体材料CuBi2O4作为阴极光电化学基底,在其表面修饰增敏剂金(Au)纳米颗粒后,制备Au/CuBi2O4光阴极:
1)采用恒电位法在修饰面积为0.25cm2氧化铟锡(ITO)电极上沉积CuBi2O4纳米膜;电解液为乙二醇溶液,其中含有30mM Cu(NO3)2和100mM Bi(NO3)3,沉积过程以恒定电位E=-1.8V vs Hg/Hg2Cl2进行,持续时间为60s;在450℃空气氛中煅烧3h,自然冷却至室温,得到CuBi2O4纳米膜修饰电极;
2)配置质量分数为0.01%的HAuCl4溶液,取100mL该溶液至烧瓶,加热煮沸;在搅拌条件下,快速加入4mL质量分数为1%的柠檬酸钠溶液;溶液持续沸腾10min后,除去热源自然冷却至室温,得到Au纳米颗粒溶液;
3)将10μL纯化的Au纳米颗粒溶液分散至CuBi2O4修饰电极上,得到Au/CuBi2O4光阴极。
(2)将CA19-9捕获抗体(Ab1)修饰于步骤(1)制备的Au/CuBi2O4光阴极,用牛血清白蛋白封闭电极活性位点后,完成免疫传感电极的制备:
在Au/CuBi2O4光阴极上滴加20μL 100μg/mL的Ab1,4℃冰箱中孵育过夜;用磷酸盐缓冲液(10mM,pH 7.4)将电极洗净后,滴加20μL质量分数1%的牛血清白蛋白溶液,室温孵育1h,封闭电极活性位点。
(3)将Pt前驱体试剂与氧化石墨烯共混,采用一锅还原反应制备石墨烯(GR)负载Pt纳米催化剂的Pt/GR复合物,随后将CA19-9信号抗体(Ab2)修饰于Pt/GR上,制备成Pt/GR标记的Ab2(Ab2-Pt/GR):
在20mL去离子水中依次加入4mL 5.5mg/mL氧化石墨烯(GO)和0.01g聚乙烯吡咯烷酮(PVP),超声处理20min后,加入50.16mL(0.73mM)H2PtCl6溶液;室温搅拌1h后,将5mL2.50M新制的NaBH4溶液缓慢滴入,继续搅拌12h;用去离子水和无水乙醇离心洗涤,并在25℃真空干燥箱干燥24h后,得到Pt/GR复合物;将Pt/GR复合物用磷酸盐缓冲液(0.1M,pH=7.4)配置1mg/mL分散液,随后滴加入100μL 200μg/mL的Ab2,振荡均匀后,4℃冰箱中孵育12h,即得到Ab2-Pt/GR。
(4)对CA19-9目标抗原(Ag)的检测采用夹心免疫反应:
将步骤(2)制备的免疫传感电极首先在室温下孵育20μL不同浓度的目标Ag1h,让Ab1与Ag发生特异性免疫反应,随后免疫传感电极继续在室温下孵育20μL步骤(3)制备的Ab2-Pt/GR 1h,让Ag与Ab2发生特异性免疫反应,从而在免疫传感电极上引入Pt/GR复合物,即得所述阴极光电化学免疫传感器。
进一步的,参见说明书附图3~4,本发明通过扫描电镜及XRD的表征测定,表明所述Au/CuBi2O4光阴极制备成功。
本发明还有一个目的,就是提供上述利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器在体外诊断产品中的应用。
经由上述的技术方案可知,与现有技术相比,本发明提供了一种阴极光电化学免疫传感器及其制备方法与应用,具有如下优异效果:
1)传感器具有装置简单、操作方便、花费低、背景干扰低、体系自供能的显著特点,同时具有对目标Ag检测光电流信号响应明显、灵敏度高、抗干扰能力强的独特优势。
2)本发明公开利用Pt纳米催化剂高效的催化氧还原反应活性,能够加速阴极光电化学传感系统的电荷流动,从而明显增强阴极光电流检测信号,其不仅为阴极光电化学免疫传感器提供一种高效的信号放大策略,还能有效提升对抗原类疾病标志物的检测灵敏度,适于市面推广与应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1为不同沉积时间对应CuBi2O4纳米膜修饰电极的光电流响应图。
图2为不同Au纳米颗粒体积对应Au/CuBi2O4光阴极的光电流响应图。
图3为不同Ab1孵育浓度条件下Ab1修饰阴极的光电流响应图。
图4为CuBi2O4纳米膜的扫描电子显微镜图。
图5为Au纳米颗粒的透射电子显微镜图。
图6为Au/CuBi2O4光阴极的扫描电子显微镜图。
图7为Au/CuBi2O4光阴极的X射线衍射图。
图8为阴极光电化学免疫传感电极制备过程的光电流响应图。
图9为Pt/GR复合物的透射电子显微镜图。
图10为Pt/GR复合物的X射线衍射图。
图11为Ab2-Pt/GR紫外-可见吸收谱图。
图12为阴极光电化学免疫传感器对目标Ag检测的光电流信号图。
图13为阴极光电化学免疫传感器对目标Ag检测的标准曲线图。
图14为阴极光电化学免疫传感器的抗干扰实验数据图。
具体实施方式
下面将结合本发明说明书附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实施例公开了一种灵敏度高、以P型半导体材料作为光电化学基底并利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器。
为更好地理解本发明,下面通过以下实施例对本发明作进一步具体的阐述,但不可理解为对本发明的限定,对于本领域的技术人员根据上述发明内容所作的一些非本质的改进与调整,也视为落在本发明的保护范围内。
下面,将结合具体实施例,对本发明的技术方案进行进一步的说明。
实施例1
因Au/CuBi2O4光阴极的光电流输出大小对最终制备的阴极光电化学免疫传感器的检测灵敏度有重要影响,所以下述对Au/CuBi2O4光阴极的制备工艺参数进行了优化:
1)因CuBi2O4的沉积时间可以反映在其电极上的沉积量,所以对CuBi2O4的沉积时间进行优化,具体如下:
采用恒电位法在ITO电极上沉积CuBi2O4纳米膜,电解液为乙二醇溶液,其中含有30mM Cu(NO3)2和100mM Bi(NO3)3,沉积过程以恒定电位E=-1.8V vs Hg/Hg2Cl2进行,持续时间分别选择为20s、40s、60s、80s、100s;随后在450℃空气氛中煅烧3h,自然冷却至室温,得到不同沉积时间的CuBi2O4纳米膜修饰电极。
通过进行光电流表征测试可得,如附图1所示。当沉积时间为60s时,CuBi2O4纳米膜修饰电极的光电流响应最佳,因此选择60s作为CuBi2O4最佳的制备工艺参数。
2)因Au纳米颗粒的修饰量可以通过在电极上的加入体积来体现,所以下述对Au纳米颗粒分散至CuBi2O4修饰电极的体积进行了优化,具体如下:
在优化制备的CuBi2O4修饰电极上,分别滴加5μL、10μL、15μL、20μL纯化后的Au纳米颗粒,空气氛中自然干燥后,得到Au/CuBi2O4光阴极。
通过进行光电流表征测试可得,如附图2所示。当Au纳米颗粒滴加体积为10μL时,Au/CuBi2O4光阴极对应的光电流响应最佳,因此选择10μL作为Au/CuBi2O4光阴极最佳的制备工艺参数。
实施例2:
因免疫传感电极上CA19-9捕获抗体探针(Ab1)的修饰量,对阴极光电化学免疫传感器的定量检测范围有显著影响,所以对修饰Ab1的制备工艺参数进行了优化:
且因Ab1在免疫传感电极上的修饰量,可以通过其在电极上的孵育浓度体现,所以下述对Ab1的孵育浓度进行了优化,具体如下:
通过在优化的Au/CuBi2O4光阴极滴加20μL浓度分别为50μg/mL、100μg/mL、150μg/mL、200μg/mL、250μg/mL的CA19-9捕获抗体(Ab1),在4℃冰箱中孵育过夜,并用磷酸盐缓冲液(10mM,pH 7.4)将电极洗净后,得到Ab1修饰电极。
通过进行光电流表征测试可得,如附图3所示,Ab1的孵育浓度需要大于等于100μg/mL,这样才能保证Ab1在传感电极上的充分固定,以获得最佳的定量检测范围,因此选择大于等于100μg/mL的Ab1作为最优孵育浓度。
本发明内容不仅限于上述各实施例的内容,其中一个或几个实施例的组合同样也可以实现本发明目的。
为了进一步验证本发明的优异效果,发明人还进行了如下实验:
首先需要说明的是,本发明下述实验中光电流信号是在光电化学系统上测试完成的,150W氙灯作为激发光源,光强度约为300mW/cm2,每10s开/关光源一次,光电流的记录由电化学工作站完成。
且使用的三电极体系为:修饰面积为0.25cm2的传感电极作为工作电极,铂丝电极作为对电极,Ag/AgCl电极作为参比电极;以及系统外加电压为0.0V。
实验例一:
(1)采用恒电位法在ITO电极上沉积CuBi2O4纳米膜,具体制备步骤如下所示:
电解液为乙二醇溶液,其中含有30mM Cu(NO3)2和100mM Bi(NO3)3,沉积过程以恒定电位E=-1.8V vs Hg/Hg2Cl2进行,持续时间为60s。在450℃空气氛中煅烧3h,自然冷却至室温,得到CuBi2O4纳米膜修饰的ITO电极。
其中,扫描电子显微镜如附图4所示,CuBi2O4纳米膜是由大量的尺寸为80-120nm的光滑颗粒组成的互联结构,具有较大的比表面积,有利于后续Au纳米颗粒的修饰。
(2)通过水相合成法制备金(Au)纳米颗粒,具体制备步骤如下所示:
(3)配置质量分数为0.01%的HAuCl4溶液,取100mL该溶液至烧瓶,加热煮沸。然后在搅拌条件下,快速加入4mL质量分数为1%的柠檬酸钠溶液。溶液持续沸腾10min后,颜色变成酒红色,表明Au纳米颗粒的形成,随后除去热源自然冷却至室温,所得Au纳米颗粒溶液经离心纯化后,重新分散于去离子水中。
其中,透射电子显微镜如附图5所示,所制备Au纳米颗粒的水分散性优良,颗粒尺寸均一,直径约15nm。
实验例二:
阴极光电化学免疫传感电极的制备步骤如下:
将实验例一中10μL纯化后的Au纳米颗粒分散至CuBi2O4修饰电极上,空气氛中自然干燥后,得到Au/CuBi2O4光阴极。
扫描电子显微镜如附图6所示,许多直径约15nm的纳米颗粒较为均匀地分布在CuBi2O4纳米膜表面,表明Au纳米颗粒在CuBi2O4修饰电极上成功修饰。
X射线衍射如附图7所示,CuBi2O4的特征衍射峰在2θ=28.67°、30.83°、35.73°、40.44°、45.93°和55.96°,分别对应于纯CuBi2O4相(PDFno.48-1886)的晶面(211)、(002)、(202)、(400)、(312)和(332);Au的特征衍射峰在2θ=38.18°、65.97°和77.73°,对应于纯Au相(PDFno.65-2876)的晶面(111)、(220)和(311);其他衍射峰2θ=29.78°、34.13°、51.0°和60.65°来自于ITO基底纯氧化铟锡(222)、(400)、(441)和(622)的晶面。由此证明了,CuBi2O4、Au以及Au/CuBi2O4的成功制备。
在Au/CuBi2O4光阴极上滴加20μL 100μg/mL的CA19-9捕获抗体(Ab1),在4℃冰箱中孵育过夜;用磷酸盐缓冲液(10mM,pH 7.4)将电极洗净后,滴加20μL质量分数1%的牛血清白蛋白溶液,室温孵育1h后,完成传感电极构建。
其中,光电流响应如附图8所示,CuBi2O4修饰电极具有较明显的阴极光电流响应(曲线a);修饰Au纳米颗粒后,阴极光电流增加(曲线b),这是由于Au纳米颗粒的增敏作用;依次修饰Ab1和BSA后,光电流逐渐减小(曲线c和d),这是由于Ab1和BSA是蛋白质属性,它们的空间位阻阻碍了电荷交换反应。由此,证明了阴极光电化学免疫传感电极成功制备。
实验例三:
石墨烯负载Pt纳米催化剂(Pt/GR)复合物的制备步骤如下所示:
以NaBH4为还原剂,采用一锅法制备Pt/GR复合物。在20mL去离子水中依次加入4mL5.5mg/mL氧化石墨烯(GO)和0.01g聚乙烯吡咯烷酮(PVP),超声处理20min。然后,加入50.16mL(0.73mM)H2PtCl6溶液,室温下搅拌1h后,将5mL 2.50M新制的NaBH4溶液缓慢滴入,继续搅拌12h。随后,用去离子水和无水乙醇离心洗涤数次,之后在真空干燥箱25℃干燥24h,即得到所需的Pt/GR复合物。
具体地,如附图9所示的透射电子显微镜照片,许多尺寸为5-7nm的Pt纳米颗粒较为均匀地分散在具有皱褶的GR上;且GR为非常薄的纳米片结构,边长为600-800nm。
如附图10所示的X射线衍射谱,GR在2θ=22.7°处的特征衍射峰对应于(002)晶面;Pt的特征衍射峰在2θ=39.7°、46.1和66.7°,对应于纯Pt相(PDF no.87-0640),表明Pt/GR复合物成功制备。
Pt/GR标记的CA19-9信号抗体(Ab2-Pt/GR)的制备步骤如下:
首先用磷酸盐缓冲液(0.1M,pH=7.4)配置1mg/mL的Pt/GR分散液,随后滴加入100μL 200μg/mL的Ab2;振荡30min后,4℃冰箱中孵育12h,随后离心洗涤;将所得的Ab2-Pt/GR分散到1.0mL磷酸盐缓冲液(0.1M,pH=7.4)中,4℃保存备用。
其中,如附图11所示的紫外-可见吸收谱可知,Ab2在280nm处有一明显的特征峰(曲线a),来自于色氨酸和酪氨酸残基π-π*共振所产生;Pt/GR在201nm和267nm处有明显吸收峰(曲线b),分别对应Pt纳米颗粒的等离子体共振特征吸收和石墨烯的特征吸收。Ab2用Pt/GR标记后,Pt/GR的两个原始吸收峰出现,并相应红移至207nm和271nm,表明Ab2-Pt/GR成功制备。
实验例四:
基于Pt纳米催化剂信号放大对CA19-9目标抗原(Ag)的检测:
将实验例二制备的阴极光电化学免疫传感电极首先在室温下孵育20μL不同浓度的目标Ag 1h,磷酸盐缓冲液(10mM,pH=7.4)清洗后,传感电极继续在室温下孵育20μL实验例三制备的Ab2-Pt/GR 1h,使得Ag与Ab2发生特异性免疫反应,以在所述免疫传感电极上引入Pt/GR复合物,即得所述阴极光电化学免疫传感器。
最终的免疫传感电极在含有溶解氧(O2)的磷酸盐缓冲液(pH 7.4,0.1M)中进行光电流信号测量。
检测结果表明:随着目标Ag浓度的增加,阴极光电流信号逐渐增强,如附图12所示;
且在目标Ag浓度为0.1pg/mL到1ng/mL范围内,阴极光电流信号变化值与目标Ag浓度的对数成线性关系,如附图13所示,线性相关系数为0.9982,实验最低检测限为0.1pg/mL,得以表明通过本发明公开制备的纳米孔道光电化学DNA传感器对目标检测物质具有较高的灵敏度。
实验例五:
为了证明上述阴极光电化学免疫传感器具有优良的抗干扰能力,包括对生物大分子的干扰以及对还原性小分子的干扰,而选择常见的其他疾病标志物抗原:甲胎蛋白(AFP)、人免疫球蛋白(HIgG)、前列腺特异性抗原(PSA),以及常见的还原性小分子:葡萄糖(Glu)、抗坏血酸(AA)、多巴胺(DA)、谷胱甘肽(GSH)作为典型的干扰物,具体操作如下:
取100pg/mL的AFP、HIgG、PSA和10mM的Glu、AA、DA、GSH分别加入含有10pg/mLCA19-9标准样品的稀释10倍的血清中。并利用本发明制备的阴极光电化学免疫传感器按上述方法分别检测,光电流信号响应如附图14所示。
结果表明,具有潜在干扰物质如AFP、HIgG、PSA、Glu、AA、DA、GSH的测试结果与仅有目标物CA19-9的光电流信号没有明显差距,误差保持在6%以内。由此证明了本发明制备的阴极光电化学免疫传感器不仅具有高灵敏度,亦同时具有抗生物大分子及还原性小分子干扰的能力,在实际复杂生物基质中具有优良的应用潜力。
此外,为了进一步验证上述阴极光电化学免疫传感器的正确性及实用性,取已知浓度分别为10pg/mL、100pg/mL、500pg/mL的标准样品加入稀释10倍的血清中,利用本发明制备的阴极光电化学免疫传感器按上述方法分别检测、计算各样品的浓度,结果依次为9.5pg/mL、102.3pg/mL、526pg/mL,通过数值对比,可以得到对10pg/mL、100pg/mL、500pg/mL的标准样品的回收率分别为95.0%、102.3%、105.2%检测结果的误差范围均在6%以内,由此进一步证明了本发明制备的阴极光电化学免疫传感器对目标Ag能够实现快速、灵敏、准确及高效的检测。
对所公开的实施例及实验例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (9)
1.一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器,其特征在于,所述阴极光电化学免疫传感器是以P型半导体材料作为光电化学基底。将铂纳米催化剂作为信号放大元件,标记于信号抗体,通过捕获抗体探针、目标抗原和信号抗体之间的夹心免疫反应,实现阴极光电流检测信号的显著放大和对目标抗原的灵敏检测。
2.一种如权利要求1所述的利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,其特征在于,具体步骤包括:
(1)制备Au/CuBi2O4光阴极:以P型半导体材料CuBi2O4作为阴极光电化学基底,在所述基底表面修饰增敏剂金纳米颗粒制备Au/CuBi2O4光阴极;
(2)制备免疫传感电极:用CA19-9捕获抗体Ab1修饰步骤(1)制备的Au/CuBi2O4光阴极,并用牛血清白蛋白封闭电极活性位点,室温孵育,得到免疫传感电极;
(3)制备Ab2-Pt/GR:将Pt前驱体试剂与氧化石墨烯共混,采用一锅还原反应制备石墨烯GR负载Pt纳米催化剂的Pt/GR复合物,随后将CA19-9信号抗体Ab2修饰于Pt/GR上,即得到Ab2-Pt/GR;
(4)将所述免疫传感电极在室温下孵育目标Ag,以使Ab1与Ag发生特异性免疫反应;随后所述免疫传感电极继续在室温下孵育所述Ab2-Pt/GR,使得Ag与Ab2发生特异性免疫反应,以在所述免疫传感电极上引入Pt/GR复合物,即得所述阴极光电化学免疫传感器。
3.根据权利要求2所述的一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,其特征在于,所述步骤(1)中,采用恒电位法在氧化铟锡电极上沉积CuBi2O4纳米膜,随后煅烧、冷却,得到CuBi2O4纳米膜修饰电极;配置HAuCl4溶液并加热煮沸,随后加入柠檬酸钠溶液加热后,得到Au纳米颗粒溶液;取所述Au纳米颗粒溶液滴加分散至CuBi2O4修饰电极上,最终得到Au/CuBi2O4光阴极。
4.根据权利要求3所述的一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,其特征在于,制备所述CuBi2O4纳米膜的沉积时间为40~80s;及制备所述Au/CuBi2O4光阴极时,所述Au纳米颗粒溶液的滴加体积为5~15μL。
5.根据权利要求2所述的一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,其特征在于,所述步骤(2)中,在所述Au/CuBi2O4光阴极上滴加Ab1,低温孵育,随后用磷酸盐缓冲液冲洗干净后,滴加牛血清白蛋白溶液,室温孵育以封闭电极活性位点,最终得到免疫传感电极。
6.根据权利要求5所述的一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,其特征在于,所述Ab1低温孵育的浓度为100~200μg/mL。
7.根据权利要求2所述的一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,其特征在于,所述步骤(3)中,在去离子水中依次加入氧化石墨烯和聚乙烯吡咯烷酮,超声处理后,加入H2PtCl6溶液搅拌均匀,随后滴加NaBH4溶液搅拌后,离心洗涤、干燥,得到Pt/GR复合物;配置含有所述Pt/GR复合物的分散液,随后滴加入Ab2,低温孵育,即得到Ab2-Pt/GR。
8.根据权利要求2所述的一种利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器的制备方法,其特征在于,所述步骤(4)中,在所述免疫传感电极上滴加目标Ag室温孵育后,在所述免疫传感电极上继续滴加所述Ab2-Pt/GR室温孵育,以最终在免疫传感电极上引入Pt/GR复合物。
9.一种如权利要求1所述的利用铂纳米催化剂放大检测信号的阴极光电化学免疫传感器或如权利要求2~8任一所述方法制备的阴极光电化学免疫传感器在体外诊断产品中的应用。
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