CN113624811A - 一种用于特异性检测丙溴磷的电化学发光适配体传感器及其制备方法和应用 - Google Patents
一种用于特异性检测丙溴磷的电化学发光适配体传感器及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种用于特异性检测丙溴磷的电化学发光适配体传感器及其制备方法和应用,具体属于电化学发光检测领域。包括:(1)纳米金多壁碳纳米管(AuNPs@MWCNTs)和具有核壳结构的纳米金银复合材料(Au@AgNPs)的制备;(2)电化学发光适配体传感器的制备;(3)将AuNPs@MWCNTs与Au@AgNP共同修饰到铂金电极表面,提高电化学发光的灵敏度和稳定性,随后负载适配体即可获得电化学发光适配体传感器,该传感器可特异性识别丙溴磷,检测范围为100 fg/mL~1µg/mL,最低检测限为5.32 fg/mL。本发明检测丙溴磷的灵敏度高、特异性强、操作简单。
Description
技术领域
本发明属于电化学发光检测领域,涉及一种用于特异性检测丙溴磷的电化学发光适配体传感器及其检测方法。
背景技术
丙溴磷是一种广谱高效、中等毒性、低残留的有机磷杀虫剂,它具有触杀和胃毒作用的非内吸性杀虫、杀螨剂,有传导作用,具有杀卵活性,主要用于棉花、玉米、甜菜、大豆、马铃薯、蔬菜、烟草等作物,防治害虫(特别是鳞翅目害虫)和螨;有机磷农药可以抑制体内的乙酰胆碱酯酶活性并可导致神经元功能紊乱和死亡,进而造成神经系统损伤,长期接触或长期通过饮食摄入低剂量的有机磷农药,就会出现脏器损伤、致畸、致癌、致突变等严重疾病。
目前,已有报道的检测丙溴磷的方法包括微生物法,气相色谱-质谱法(GC-MS),酶联免疫吸附测定法(ELISA),高效液相色谱-质谱法(HPLC-MS),光电化学分析法(PEC)及电化学法等;但大多数分析法的仪器操作专业度高,前处理过程复杂,耗时长,尤其是微生物法的结果容易出现假阳性、色谱法的检测费用昂贵,因此它们难以推广到市场中进行现场检测;基于此,开发一种简单快速、选择性好的检测丙溴磷的方法是必要且极其重要的。
核酸适配体作为一种新型识别元件,合成简单快速、成本低、选择性好、性质稳定且易于修饰标记,是一种优良的抗体替代识别元件;电化学发光(ECL)也称为电致化学发光,是化学发光与电化学的结合和延伸,因而它具有化学发光方法优点如灵敏度高、线性范围宽、观察方便和仪器简单;同时具有许多化学发光方法无法比拟的优点,如重现性好、试剂稳定、控制容易等优点;并且,无需引入外部光源,在光电倍增管等光学仪器的辅助下采集发光强度图谱,并建立其与待测物关系从而实现微量分析的一种方法。
发明内容
本发明的目的是提供一种检测丙溴磷的电化学发光适配体传感器,具有灵敏度高、重现性好、选择性好和线性范围宽的优点。
本发明是基于适配体负载在由纳米复合材料修饰铂金电极表面而成,充分利用纳米金多壁碳纳米管(AuNPs@MWCNTs)和具有核壳结构的纳米金银复合材料(Au@AgNPs)的静电相互作用共同修饰到铂金电极表面,使得电化学发光的灵敏度和稳定性显著提高;该传感器的灵敏度提高,是由于导电性、电化学性能改善导致的,并且AuNPs@MWCNTs和Au@AgNPs之间具有协同作用;
该传感器的制备过程如下:
1 µL AuNPs@MWCNTs溶液修饰在预处理后的铂金电极(PE)表面室温下干燥;再将1µL 0.01 M鲁米诺滴在铂金电极表面,室温自然干燥。随后在铂金电极表面滴上1 µL Au@AgNPs溶液,在空气中干燥;接着在铂金电极表面滴上1 µL丙溴磷适配体传感器,在空气中干燥;然后将修饰的PE电极浸泡在0.5 %的BSA中阻断非特异性位点,得到电化学发光适配体传感器:BSA/适配体/Au@AgNPs/鲁米诺/AuNPs@MWCNTs/PE。
进一步的,铂金电极预处理步骤如下:电极需要用0.05 mm的氧化铝粉进行抛光,在98%乙醇与超纯水1:1的混合液中超声清洗3 min,再用超纯水彻底清洗,用氮气吹干。
进一步的,该电化学发光适配体传感器所用适配体核苷酸序列为5’-SH-AAGCTTGCTTTATAGCCTGCAGCGATTCTTGATCGGAAAAGGCTGAGAGCTACGC-3’。
进一步的,纳米金多壁碳纳米管(AuNPs@MWCNTs)的制备方法如下:将2.0 mg羧基功能化的多壁碳纳米管加入到4 mL 1%聚乙烯亚胺溶液中(PEI),超声分散,离心10 min以去除多余的PEI,用2 mL超纯水将剩余的沉淀物重新分散,制备PEI功能化的MWCNTs;将1mL纳米金溶液(AuNPs)滴加到1mL制备的PEI功能化MWCNTs的溶液中,搅拌过夜,离心得到的黑色固体再分散于2 mL 磷酸盐缓冲液(pH=7.4)中,此时就得到AuNPs@MWCNTs溶液。
进一步的,具有核壳结构的纳米金银复合材料(Au@AgNPs)的制备方法如下:首先,将12 µL L-抗坏血酸(AA:100 mM)、3 µL 硝酸银(AgNO3:100 mM)和15 µL氢氧化钠(NaOH:100 mM)分别加入到2 mL制备好的AuNPs (15 nm)溶液中,将混合溶液的pH值调整为为8.5左右;然后将上述溶液用磁力搅拌器匀速搅拌30 min,然后以1800 rpm离心20 min;除去上清液后,将剩余的液体重新分散到10 mL超纯水中,此时完成第一次添加;重复上述步骤再添加四次完成Au@AgNPs的合成。
一种基于电化学发光适配体传感器检测丙溴磷的方法,所述方法为:以如上所述的电化学发光适配体传感器作为工作电极,饱和甘汞为参比电极,铂丝电极为对电极组成三电极体系,样品中的丙溴磷被定量捕捉到传感器的表面,通过产生的发光信号用于检测丙溴磷。
进一步的,具体步骤为:
步骤1,含过氧化氢(H2O2)的PBS缓冲溶液的配制:用pH为9.0的0 .1mol/L的PBS缓冲溶液配制含10 nM H2O2的PBS缓冲溶液;
步骤2,不同浓度的丙溴磷标准溶液的配制:准确称取一定量的丙溴磷,用PBS(pH=7.4)配制10 μg/mL丙溴磷溶液,再讲上述溶液稀释得到一系列不同浓度的丙溴磷标准溶液,浓度范围为100 fg/mL~1µg/mL。
步骤3,标准曲线的绘制:将所述电化学发光适配体传感器置于步骤2配制的不同浓度的丙溴磷标准溶液中浸泡相同时间,使电化学发光适配体传感器结合丙溴磷,然后取出并淋洗,作为工作电极,饱和甘汞为参比电极,铂丝电极为对电极组成三电极体系,以步骤1中的含过氧化氢(H2O2)的PBS缓冲溶液为电解液在电化学发光工作站进行测量;记录发光强度-时间曲线,建立电化学发光适配体传感器结合丙溴磷的发光强度与丙溴磷标准溶液中的丙溴磷浓度对数值的线性关系,得到相应的线性回归方程。
步骤4,样品中丙溴磷的检测:所述样品先经过预处理再放入电化学发光适配体传感器浸泡相同时间,使电化学发光适配体传感器结合丙溴磷,然后取出并冲洗,作为工作电极,再采用步骤3方法检测发光强度,再根据线性回归方程计算出样品中丙溴磷的浓度。
本发明充分利用了适配体和电化学发光传感器的优势,通过丙溴磷对该体系电化学发光提醒信号强度的增强的机理,成功实现对丙溴磷的灵敏检测,该传感平台可特异性识别检测物丙溴磷,具有高选择性。本发明的检测范围为100 fg/mL~1µg/mL,最低检测限为5.32 fg/mL(S/N = 3)。本发明检测丙溴磷的操作简单、选择性好、检测成本低、灵敏度高。本发明对推广适配体传感器在环境及食品安全方面的实际应用具有重要的意义。
附图说明
图1 实施例1加入丙溴磷传感器发光强度与丙溴磷胺浓度对数值的标准曲线。
图2 实施例1传感器对丙溴磷的特异性检测。
图2中a是倍硫磷,b是毒死蜱,c是啶虫脒,d是辛硫磷,e是马拉硫磷,f是倍硫磷、毒死蜱、啶虫脒、辛硫磷、马拉硫磷的混合农药,g是丙溴磷,h是所有农药混合。
具体实施例
本发明结合下面实施例作进一步详述:
实施例1:
(1)纳米金多壁碳纳米管(AuNPs@MWCNTs)的制备:
将2.0 mg羧基功能化的多壁碳纳米管加入到4 mL 1%聚乙烯亚胺溶液中(PEI),超声分散,离心10 min以去除多余的PEI,再用2 mL超纯水将剩余的沉淀物重新分散,制备PEI功能化的MWCNTs;将1mL 纳米金溶液(AuNPs)滴加到1mL制备的PEI功能化MWCNTs的溶液中,搅拌过夜,离心得到的黑色固体再分散于2 mL磷酸盐缓冲液(pH=7.4)中,此时就得到AuNPs@MWCNTs溶液。
(2)具有核壳结构的纳米金银复合材料(Au@AgNPs)的制备:
首先,将12 µL L-抗坏血酸(AA:100 mM)、3 µL 硝酸银(AgNO3:100 mM)和15 µL氢氧化钠(NaOH:100 mM)分别加入到2 mL制备好的AuNPs (15 nm)溶液中,将混合溶液的pH值调整为为8.5左右;然后将上述溶液用磁力搅拌器匀速搅拌30 min,然后以1800 rpm离心20min;除去上清液后,将剩余的液体重新分散到10 mL超纯水中,此时完成第一次添加;重复上述步骤再添加四次完成Au@AgNPs的合成。
(3)传感器的构建
将珀金电极抛光,依次用无水乙醇和去离子水混合液超声,用氮气吹干;1 µLAuNPs@MWCNTs溶液修饰在预处理后的铂金电极表面室温下干燥;再将1 µL 0.01 M鲁米诺滴在铂金电极表面,室温自然干燥;随后在铂金电极表面滴上1 µL Au@AgNPs溶液,在空气中干燥;接着在铂金电极表面滴上1 µL丙溴磷适配体传感器,在空气中干燥;然后将修饰的铂金电极浸泡在0.5 %的BSA中阻断非特异性位点,得到电化学发光适配体传感器;所用适配体序列为5’-SH-AAGCTTGCTTTATAGCCTGCAGCGATTCTTGATCGGAAAAGGCTGAGAGCTACGC-3’。
(4)传感器条件的优化
为了使适配体传感器的性能在最佳实验条件下得到最大程度的发挥,对适配体的浓度、测试底液的pH值、丙溴磷与适配体的孵育时间分别进行了优化,结果表明,当适配体的浓度为100nM、测试pH值为9.0、丙溴磷与适配体的孵育时间为40分钟时,该传感器的到达了最佳的实验条件。
(5)标准曲线的绘制
将修饰电极BSA/适配体/Au@AgNPs/鲁米诺/AuNPs@MWCNTs/PE作为工作电极,铂电极作为辅助电极,饱和甘汞为参比电极,铂丝电极为对电极组成三电极体系,以将三电极体系置于一系列丙溴磷浓度,含有10 nM的H2O2的pH=9.0的0.1mol/L PBS的缓冲溶液中得到在电化学发光工作站进行测量;记录发光强度-时间曲线,建立电化学发光适配体传感器结合丙溴磷的发光强度与丙溴磷标准溶液中的丙溴磷浓度对数值的线性关系,得到相应的相应的线性回归方程为:IECL=2815.56058- 597.11635 logC(ng/mL),相关系数(R2)为0.99941,计算出检出限为5.32 fg/mL (S/N = 3),线性回归方程的检测范围为100 fg/mL~1µg/mL。
(6)样品的检测
首先,将蔬菜清洗,除去土壤,蔬菜晾干后,切成2×2毫米左右(200 mg);然后,在蔬菜表面喷洒不同浓度的2 mL的丙溴磷和加入2 mL水,室温放置24小时;再将9 mL PBS(0.1 M, pH 7.5)和1mL丙酮加入进去;最后,在离心机里以10000 rpm离心10 min;将上清液用0.22mm的滤膜过滤,得到的滤液加入含有10 nM的0.1mol/L的PBS缓冲溶液 pH至9.0,取9 mL所得溶液用于电化学发光分析,记录发光强度,按步骤(5)所得的线性回归方程计算出待检测样品中丙溴磷的浓度。
并将实施例1制备的用于检测丙溴磷的电化学发光传感器进行抗干扰检测,其中将适配体孵育后的工作电极分别在倍硫磷、毒死蜱、啶虫脒、辛硫磷、马拉硫磷、丙溴磷农药标准液中测试,并再将工作电极在上述物质的混合物中检测,检测结果见图2,从图2可知,具有优异电化学性能的修饰电极上孵育适配体后对丙溴磷起到选择性识别效果,其他农药干扰物对丙溴磷的检测影响也很微小,因此该工作电极可以实现丙溴磷抗干扰的选择性检测。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,当可利用上述揭示的技术内容做出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (5)
1.一种用于特异性检测丙溴磷的电化学发光适配体传感器,其特征在于:所述的电化学发光适配体传感器由适配体负载于复合材料Au@AgNPs/AuNPs@MWCNTs修饰铂金电极的表面而成,用于电化学发光检测丙溴磷。
2.根据权利要求1所述的用于特异性检测丙溴磷的电化学发光适配体传感器,其特征在于:所述丙溴磷适配体核苷酸序列如下所示5’-SH-AAGCTTGCTTTATAGCCTGCAGCGATTCTTGATCGGAAAAGGCTGAGAGCTACGC-3’。
3.根据权利要求1所述用于特异性检测丙溴磷的电化学发光适配体传感器的制备方法,其特征在于:所述的制备方法如下:
S1.纳米金多壁碳纳米管(AuNPs@MWCNTs)的制备:将2.0 mg羧基功能化的多壁碳纳米管加入到4 mL 1%聚乙烯亚胺溶液中(PEI),超声分散;离心10 min以去除多余的PEI;然后用2 mL超纯水将剩余的沉淀物重新分散,制备PEI功能化的MWCNTs;将1mL 纳米金溶液(AuNPs)滴加到1mL制备的PEI功能化MWCNTs的溶液中,搅拌过夜;离心得到的黑色固体再分散于2 mL 磷酸盐缓冲液(pH=7.4)中,此时就得到AuNPs@MWCNTs溶液;
S2. 具有核壳结构的纳米金银复合材料(Au@AgNPs)的制备:首先,将12 µL L-抗坏血酸(AA:100 mM)、3 µL 硝酸银(AgNO3:100 mM)和15 µL氢氧化钠(NaOH:100 mM)分别加入到2 mL制备好的AuNPs (15 nm)溶液中,将混合溶液的pH值调整为8.5左右;然后将上述溶液用磁力搅拌器缓慢匀速搅拌30 min,然后以1800 rpm离心20 min;除去上清液后,将剩余的液体重新分散到10 mL超纯水中,此时完成一次添加;重复上述步骤再添加四次完成Au@AgNPs的合成;
S3. 传感器的构建
将珀金电极抛光,依次用无水乙醇和去离子水混合液超声,用氮气吹干;1 µL AuNPs@MWCNTs溶液修饰在预处理后的铂金电极表面室温下干燥;再将1 µL 0.01 M鲁米诺滴在铂金电极表面,室温自然干燥;随后在铂金电极表面滴上1 µL Au@AgNPs溶液,在空气中干燥;接着在铂金电极表面滴上1 µL丙溴磷适配体传感器,在空气中干燥;然后将修饰的铂金电极浸泡在0.5 %的BSA中阻断非特异性位点,得到BSA/适配体/Au@AgNPs/鲁米诺/AuNPs@MWCNTs/PE。
4.一种基于电化学发光适配体传感器检测丙溴磷的方法,其特征在于:以权利要求1-3任一项所述的电化学发光适配体传感器作为工作电极,饱和甘汞为参比电极,铂丝电极为对电极组成三电极体系,样品中的丙溴磷被定量捕捉到传感器的表面,通过产生的发光信号用于检测丙溴磷。
5.根据权利要求4所述的检测丙溴磷的电化学发光适配体传感器的应用,其特征在于,具体步骤为:
A1.含过氧化氢(H2O2)的PBS缓冲溶液的配制:用pH为9.0的0 .1mol/L的PBS 缓冲溶液配制含10 nM H2O2的PBS缓冲溶液
A2.不同浓度的丙溴磷标准溶液的配制:准确称取一定量的丙溴磷,用PBS(pH=7.4)配制10 μg/mL丙溴磷溶液,再将上述溶液稀释得到一系列不同浓度的丙溴磷标准溶液,浓度范围为100 fg/mL~1µg/mL
A3.传感器条件的优化:对适配体的浓度、PBS缓冲底液的pH值、丙溴磷与适配体的孵育时间分别进行优化;配制不同浓度的丙溴磷适配体,浓度范围为1nM~104nM;配制不同pH值含10 nM过氧化氢(H2O2)的PBS缓冲溶液,pH范围为7.0~10.0;将丙溴磷与适配体的孵育时间控制在20分钟到70分钟范围内;通过对影响传感器的相关条件进行优化得出最佳的实验条件,使适配体传感器的性能得到最大程度的发挥
A4.标准曲线的绘制:将所述电化学发光适配体传感器置于步骤A2配制的不同浓度的丙溴磷标准溶液中浸泡相同时间,使电化学发光适配体传感器结合丙溴磷,然后取出并淋洗,作为工作电极,饱和甘汞为参比电极,铂丝电极为对电极组成三电极体系,以步骤A1中的含过氧化氢(H2O2)的PBS缓冲溶液为电解液在电化学发光工作站进行测量;记录发光强度-时间曲线,建立电化学发光适配体传感器结合丙溴磷的发光强度与丙溴磷标准溶液中的丙溴磷浓度对数值的线性关系,得到相应的线性回归方程
A5.样品中丙溴磷的检测:所述样品先经过预处理再放入电化学发光适配体传感器浸泡相同时间,使电化学发光适配体传感器结合丙溴磷,然后取出并冲洗,作为工作电极,再采用步骤A4方法检测发光强度,再根据线性回归方程计算出样品中丙溴磷的浓度。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110596060A (zh) * | 2019-09-12 | 2019-12-20 | 江南大学 | 一种检测前列腺特异性抗原的光谱分析中荧光传感器的构建方法及其应用 |
CN112505116A (zh) * | 2020-11-05 | 2021-03-16 | 常州大学 | 一种用于特异性检测卡那霉素的电化学发光适配体传感器以及制备方法和应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN112505116A (zh) * | 2020-11-05 | 2021-03-16 | 常州大学 | 一种用于特异性检测卡那霉素的电化学发光适配体传感器以及制备方法和应用 |
Non-Patent Citations (2)
Title |
---|
HUIMIN LIU: "Electrochemiluminescence Aptasensor for Profenofos Detection Based on Silver Nanoparticles Enhanced Luminol Luminescence System", JOURNAL OF THE ELECTROCHEMICAL SOCIETY, vol. 166, no. 15, pages 1562 - 1566 * |
YALING CAO: "Ultrasensitive luminol electrochemiluminescence for protein detection based on in situ generated hydrogen peroxide as coreactant with glucose oxidase anchored AuNPs@MWCNTs labeling", BIOSENSORS AND BIOELECTRONICS, vol. 31, no. 1, pages 305 - 309, XP028353765, DOI: 10.1016/j.bios.2011.10.036 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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