CN113624812A - 一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法 - Google Patents
一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法 Download PDFInfo
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Abstract
一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,涉及一种新的生物传感器制备方法。本发明利用铜金双金属核壳结构纳米粒子对电化学发光的增强作用,构建了一种检测有机磷农药的适配体传感器。具体制备过程如下:将发光试剂氯化三(2,2'‑联吡啶)钌(II)六水合物滴加在多壁碳纳米管‑壳聚糖改性的电极表面,然后加入水相中合成的铜金双金属核壳结构纳米粒子。铜金双金属核壳结构纳米粒子表面的纳米金可以催化三正丙胺的分解显著增加体系的电化学发光强度,同时,借助纳米铜大的比表面积特性有利于纳米金的固载。本发明适配体传感器制备方法简单、检测速度快、稳定性好、选择性高,可用于蔬菜中有机磷农药残留的高灵敏度检测。
Description
技术领域
本发明涉及一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,属于电化学发光生物传感器领域。
背景技术
有机磷农药是一类含有磷元素的化合物农药,在保护作物和防治害虫方面具有成本低、效率高的优点,因此被广泛应用于农业生产中,有机磷农药具有很好的水溶性和易吸收性,环境中的农药成分短时间内无法完全清除,这些残留在蔬菜中的农药最终通过生物富集作用进入人体,即使农药含量低,同样会对人体的呼吸系统和神经系统造成一定的危害,所以,对蔬菜中的有机磷农药含量进行检测是非常有价值的,适配体是一种通过体外筛选的短寡核苷酸单链,能够与目标物高亲和力的结合,而且具有很强的特异性,与酶和抗体相比,适配体具有成本低、易合成、稳定性好、易于修饰等优点,是构建快速、稳定的生物传感器的理想识别原件。
在过去的十年中,开发了很多检测农药的新方法,它们包括电化学传感器、荧光传感器、比色传感器、表面增强拉曼散射传感器等,电化学发光是由电化学和化学发光结合的一种新型技术,由于易于控制、检测灵敏度高、响应速度快等优点,使它迅速成为生物分析领域的研究热点,氯化三(2,2'-联吡啶)钌(II) 六水合物是电化学发光中常用的发光试剂之一,由于其化学性质稳定、发光效率高、电化学发光可逆的特点,为构建灵敏度高、稳定性好的传感器提供了可能。
随着纳米技术的不断发展,各种具有不同空间结构、化学性质的纳米材料被应用于传感器的开发,多壁碳纳米管是一类具有良好电子传递能力、大的比表面积、极高化学稳定性的碳材料,广泛应用于传感器的构建中,金属纳米粒子由于其具有良好的催化性能、导电能力、表面等离子体共振效应等特点受到了广泛的关注,其中,对单金属纳米颗粒合成与应用的研究比较多,近几年,人们发现双金属纳米材料由于协同作用的原因,在催化、导电、发光方面具有比单金属更好的效果。
发明内容
本发明将铜金双金属核壳结构纳米粒子应用于电化学发光系统中,显著提高了体系的电化学发光强度,构建了一种新型电化学发光适配体传感器,并成功实现对蔬菜中有机磷农药的检测。
本发明的技术方案为:通过层层自组装技术先后将纳米材料、发光试剂、识别原件、封闭剂、目标物涂覆于电极表面,具体制备过程如下:(1)多壁碳纳米管-壳聚糖复合材料改性电极表面;(2)滴涂发光试剂;(3)滴加铜金双金属核壳结构纳米粒子;(4)适配体的修饰;(5)涂覆一层牛血清白蛋白溶液封闭非特异性结合位点,超纯水清洗表面,氮气干燥,适配体传感器制备完成;(6)有机磷农药的检测。
选优地,所述电极为铂电极,多壁碳纳米管-壳聚糖复合材料制备方法:将0.05 g壳聚糖粉末溶解于1 mL 冰乙酸和99 mL 超纯水的混合溶液中,搅拌3 h以上使壳聚糖粉末充分溶解,配制成0.05%的壳聚糖溶液,称取25 mg的多壁碳纳米管分散于5 mL上述配置好的壳聚糖溶液中,完成浓度为5 mg/mL多壁碳纳米管-壳聚糖复合材料的配制,滴在裸电极表面进行改性。
选优地,发光试剂制备方法:称取7.5 mg 氯化三(2,2'-联吡啶)钌(II) 六水合物粉末溶于5 mL配置好的壳聚糖溶液,充分溶解后静置3天,将其滴涂在多壁碳纳米管-壳聚糖复合材料改性后的电极表面。
选优地,铜金双金属核壳结构纳米粒子制备方法:在冰浴条件下,将0.6 mM硼氢化钠滴加到100 ml浓度为1 mM 硫酸铜和1 mM碘化钾的混合溶液中,剧烈搅拌,得到纳米铜胶体,向制备好的纳米铜胶体中加入25 ml浓度为1 mM的柠檬酸三钠溶液混匀,然后将10 ml浓度为1 mM氯金酸缓慢的加入到上述溶液中,加入过程中需要剧烈搅拌,待反应完成后,将溶液离心纯化,将制备好的铜金双金属核壳结构纳米粒子滴加在涂有发光试剂的电极表面。
选优地,适配体为一端修饰有巯基的广谱性适配体,可特异性识别丙溴磷、水胺硫磷、甲拌磷以及氧化乐果四种农药,将适配体修饰于滴加有铜金双金属核壳结构纳米粒子的电极表面,作用时间为1.5 h。
选优地,封闭非特异性结合位点条件:在修饰有适配体的电极表面涂覆一层牛血清白蛋白溶液,反应时间为30 min,超纯水清洗表面,氮气干燥,适配体传感器制备完成。
选优地,有机磷农药的检测过程:将待测有机磷农药样品滴加在制备好的传感器上面,在含有三正丙胺的缓冲溶液中测试电化学发光强度,根据电化学发光强度的大小反应有机磷农药的浓度。
与现有技术相比,本发明的有益效果在于:本发明将铜金双金属核壳结构纳米粒子用于构建电化学发光适配体传感器,铜金双金属核壳结构纳米粒子充分利用了纳米铜和纳米金两种纳米粒子的空间结构特性以及化学性质,在保证纳米金高效催化作用的同时,借助纳米铜大的比表面积实现了更多纳米金的固载,增强了电化学发光强度,提高了适配体传感器对有机磷农药的检测灵敏度。
附图说明
图1适配体传感器的制备过程。
图2纳米材料的SEM表征。
图3纳米材料的TEM表征。
图4纳米材料的AFM表征。
图5纳米材料的CV表征。
图6纳米材料的ECL表征。
图7适配体传感器的条件优化。
图8不同浓度的农药所对应的ECL强度。
图9适配体传感器检测有机磷农药的标准曲线图。
图10传感器检测不同农药的主要参数。
图11实际样品加标回收率。
图12适配体感器的稳定性分析。
图13适配体感器的重现性分析。
图14适配体感器的特异性分析。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但实施例并不对本发明做任何形式的限定。
实施例1:适配体传感器的制备
图1是适配体传感器的制备过程,首先,在裸电极表面滴加3 uL的多壁碳纳米管–壳聚糖溶液,晾干,接下来取3 uL的发光试剂滴加到电极上,温室干燥,第三,将3 uL的铜金双金属核壳结构纳米粒子滴加到电极上,随后,在电极上加入3 uL一端修饰有巯基的适配体溶液,让反应作用1.5 h,最后,在电极表面涂覆一层牛血清白蛋白溶液反应30 min,用超纯水轻轻冲洗掉未结合的材料,验证适配体传感器的性能,取3 uL的有机磷农药滴加到电极上测试发光效果。
实施例2:纳米材料的表征
(1)SEM表征
利用扫描电子显微镜对纳米材料的表面形貌进行了表征(图2),多壁碳纳米管-壳聚糖由大量的细管状结构组成(图2a),铜金双金属核壳结构纳米粒子均匀的分布在多壁碳纳米管材料的表面(图2b)
(2)TEM表征
图3是铜金双金属核壳结构纳米粒子的透射电子显微镜图,从图中可以清楚的看出小粒径的纳米金颗粒生长在纳米铜的表面
(3)AFM表征
原子力显微镜表征了电极改性过程中的表面形态变化,多壁碳纳米管-壳聚糖涂覆的裸电极具有较大的起伏,表面粗糙度为0.745 nm(图4a),图4b是铜金双金属核壳结构纳米粒子滴在多壁碳纳米管-壳聚糖表面的原子力显微镜图像,与多壁碳纳米管-壳聚糖涂层相比,表面粗糙度进一步增加到 6.528 nm
(4)CV表征
如图5所示,用循环伏安法对适配体传感器的电化学性质进行了分析,裸电极具有一对可逆的氧化还原峰(曲线a);将多壁碳纳米管–壳聚糖复合材料滴加到电极表面,电极的电流峰值显著增大(曲线b);继续滴加发光试剂溶液,电极的电流峰值降低(曲线c);在电极上修饰了铜金双金属核壳结构纳米粒子后,电极的电流峰值得到提高(曲线d);电极表面修饰适配体后,电流响应显著降低(曲线e);添加牛血清白蛋白后,电极的电流峰值继续下降(曲线f),牛血清白蛋白成功阻碍了电极表面的非特异性位点;滴加有机磷农药后电极的电流峰值再一次下降(曲线g)
(5)ECL表征
图6是不同纳米材料修饰电极的电化学发光行为表征,在电极表面滴加发光试剂后,体系中开始出现电化学发光信号(曲线a),当电极上修饰了多壁碳纳米管–壳聚糖纳米材料后,电化学发光信号获得了很大的提高(曲线b),分别将单独的纳米金和铜金双金属核壳结构纳米粒子修饰到电极表面,发现两种材料都对发光体系有一定的促进作用,但是,铜金双金属核壳结构纳米粒子(曲线d)的促进作用要高于单独的纳米金粒子(曲线c)。
实施例3:适配体传感器的条件优化
适配体浓度、孵育时间、底液pH值、三正丙胺浓度是影响适配体传感器电化学发光强度的重要因素,如图7a所示,电化学发光强度随着适配体浓度的升高而减小,当适配体浓度为1 uM时电化学发光强度降低到最低值,之后发光强度随着适配体浓度的升高而升高,因此,适配体浓度选用1 uM继续后面的实验,孵育时间对适配体传感器发光强度的影响如图7b,电化学发光强度随着孵育时间的变长而逐渐下降,但是50 min后变化开始变得不明显,说明适配体与农药完全结合需要50 min左右,底液的pH值也是影响电化学发光强度的关键因素之一,如图7c所示,在pH8.0时得到了最佳响应,三正丙胺作为发光体系的共反应物,它的浓度对电化学发光强度的影响较大,随着溶液中三正丙胺浓度的提高,电化学发光强度显著增强(图7d),当它的浓度增加到8 mM的时候,电化学发光强度达到最大,之后保持不变,所以选择三正丙胺浓度为8 mM。
实施例4:适配体传感器对有机磷农药的检测
在最佳实验条件下,用所制备的传感器对丙溴磷、水胺硫磷、甲拌磷、氧化乐果四种有机磷农药进行检测,图8是农药浓度与电化学发光强度的关系(图8a:丙溴磷;图8b:水胺硫磷;图8c:甲拌磷;图8d:氧化乐果),随着农药浓度的增加,电化学发光强度逐渐减小,图9显示(图9e:丙溴磷;图9f:水胺硫磷;图9g:甲拌磷;图9h:氧化乐果),在一定的浓度范围内,电化学发光强度与农药浓度的对数成良好的线性关系,且具有较低的检出限(S/N=3),适配体传感器的相关检测参数如图10,利用该传感平台对蔬菜中的水胺硫磷进行检测,检测结果如图11,回收率在89.43%-126.15%,相对标准偏差小于5.48%。
实施例5:适配体传感器的性能分析
(1)稳定性测试
图12是传感器的稳定性测试结果,在相同条件下制备了12根电极,并立即将浓度为1×105 ng/L的水胺硫磷孵育在4根电极表面,并对其电化学发光强度进行检测,将其余8根电极保存于零下四度的冰箱中,每隔一周取出4根电极对其进行检测,发现七天后电极的发光强度为七天前电极的97.95%(RSD=3.33%),十四天后的4根电极发光强度仅下降7.32%(RSD=6.95%)
(2)重现性测试
传感器重现性测试结果如图13所示,在相同条件下制备4根电极,用于检测浓度为1×103 ng/L的水胺硫磷,4个电极之间的相对标准偏差为2.71%
(3)特异性测试
选取啶虫脒、克百威、马拉硫磷和久效磷四种农药对传感器的特异性进行了分析,如图14所示,在相同的浓度下,四种非检测农药无论是单一体系还是混合体系,都不能有效抑制电化学发光的强度,发光强度很高(图14a:啶虫脒;图14b:克百威;图14c:马拉硫磷;图14d:久效磷;图14e:啶虫脒+克百威+马拉硫磷+久效磷),而体系中一旦有目标物的成分,电化学发光强度就会显著下降(图14f:丙溴磷;图14g:水胺硫磷;图14h:甲拌磷;图14i:氧化乐果;图14j:丙溴磷+水胺硫磷+甲拌磷+氧化乐果;图14k:丙溴磷+水胺硫磷+甲拌磷+氧化乐果+啶虫脒+克百威+马拉硫磷+久效磷)。
Claims (6)
1.一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,其特征在于,在水相中合成了铜金双金属核壳结构纳米粒子,纳米铜大的比表面积特性有利于纳米金的固载,同时,纳米金通过催化三正丙胺的氧化过程可以增强体系的电化学发光强度,利用铜金双金属核壳结构纳米粒子构建了一种适配体传感器,具体制备过程如下:
(1)多壁碳纳米管-壳聚糖复合材料改性电极表面;
(2)滴涂发光试剂;
(3)滴加铜金双金属核壳结构纳米粒子;
(4)适配体的修饰;
(5)涂覆一层牛血清白蛋白溶液封闭非特异性结合位点,超纯水清洗表面,氮气干燥,适配体传感器制备完成。
2.根据权利要求1所述的一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,其特征在于,所述电极为铂电极,多壁碳纳米管-壳聚糖复合材料制备方法:将0.05g 壳聚糖粉末溶解于1 mL 冰乙酸和99 mL 超纯水的混合溶液中,搅拌3 h以上使壳聚糖粉末充分溶解,配制成0.05%的壳聚糖溶液,称取25 mg的多壁碳纳米管分散于5 mL上述配置好的壳聚糖溶液中,完成浓度为5 mg/mL多壁碳纳米管-壳聚糖复合材料的配制,滴在裸电极表面进行改性。
3.根据权利要求1所述的一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,其特征在于,发光试剂制备方法:称取7.5 mg 氯化三(2,2'-联吡啶)钌(II) 六水合物粉末溶于5 mL配置好的壳聚糖溶液,充分溶解后静置3天,将其滴涂在多壁碳纳米管-壳聚糖复合材料改性后的电极表面。
4.根据权利要求1所述的一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,其特征在于,铜金双金属核壳结构纳米粒子制备方法:在冰浴条件下,将0.6 mM硼氢化钠滴加到100 ml浓度为1 mM 硫酸铜和1 mM碘化钾的混合溶液中,剧烈搅拌,得到纳米铜胶体,向制备好的纳米铜胶体中加入25 ml浓度为1 mM的柠檬酸三钠溶液混匀,然后将10ml浓度为1 mM氯金酸缓慢的加入到上述溶液中,加入过程中需要剧烈搅拌,待反应完成后,将溶液离心纯化,将制备好的铜金双金属核壳结构纳米粒子滴加在涂有发光试剂的电极表面。
5.根据权利要求1所述的一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,其特征在于,适配体为一端修饰有巯基的广谱性适配体,可特异性识别丙溴磷、水胺硫磷、甲拌磷以及氧化乐果四种农药,将适配体修饰于滴加有铜金双金属核壳结构纳米粒子的电极表面,作用时间为1.5 h。
6.根据权利要求1所述的一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,其特征在于,封闭非特异性结合位点条件:在修饰有适配体的电极表面涂覆一层牛血清白蛋白溶液,反应时间为30 min,超纯水清洗表面,氮气干燥,适配体传感器制备完成。
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