CN115932001A - 一种基于AgInS2量子点-DNA纳米线的光致电化学生物传感器及其检测应用 - Google Patents
一种基于AgInS2量子点-DNA纳米线的光致电化学生物传感器及其检测应用 Download PDFInfo
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Abstract
本发明公开了一种基于AgInS2量子点‑DNA纳米线的光致电化学生物传感器及检测Hg2+和黄曲霉素B1的分析应用。本发明的技术方案是利用AgInS2量子点标记DNA纳米线放大信号,结合AgInS2量子点反转NPC‑ZnO纳米多面体的光电流信号,研制了光致电化学生物传感器,通过光电信号“on‑off”检测模式,实现了对Hg2+和黄曲霉素B1双目标高灵敏检测。该发明中AgInS2QDs‑DNA纳米线为研制光电生物传感器检测多种目标开发了新的信号放大技术,提高了检测灵敏度;同时,AgInS2QDs反转NPC‑ZnO光电流信号,极大提高了检测准确度,降低了假阳性,在生物检测和实际应用中具有很高的潜力。
Description
技术领域:
本发明涉及一种基于AgInS2量子点-DNA纳米线的多功能光致电化学生物传感器;以及所述生物传感器的制备方法及其检测Hg2+和黄曲霉素B1的分析应用。
背景技术:
近几年,无毒量子点如CuInS2、AgInS2因具有光电活性而被重点研究[Zhang,Y.;Ma,Q.;Yan,Y.et al.Anal.Chem.2020,92:15679-15684.],它们具有宽带发射,荧光量子产率高[Baimuratov,A.S.;Martynenko,I.V.;Baranov,A.V.et al.Phys.Chem.C,2019,123,16430.],在可见光到近红外区具有可调的光学带隙,已广泛用于光电传感检测中[Shi,H.;Jia,L.;Wang,C.etal.OpticalMaterials,2020,99:109549]。金属有机骨架具有高比表面积、高孔隙率,有利于光电子传输,在光电化学分析中应用越来越多[Shen,K.;Chen,X.etal.ACSCatal,2016,6,5887-5903.]。掺氮多孔炭(NPC)具有多孔结构和高电子电导率,在抗坏血酸(AA)氧化和还原反应中表现出优异的电化学性能。
然而,许多光敏材料在应用过程易自发性团聚、利用率低,为此研究人员开发DNA纳米结构负载光敏材料。例如:Chen的团队提出一种DNA纳米丛林结构负载[Ru(NH3)6 3+,增强传感信号[Chen,X.;Liu,Y.;Xu,L.et al.Anal.Chem.2019,91,13712-13719.]。在我们先前工作中,通过3D DNA纳米网状结构组装CdSe量子点[Cai,Q.;Wu,D.;Li,H.etal.Biosensors&Bioelectronics,2021:113455.]。
本工作将AgInS2 QDs组装到DNA纳米线上构建放大信号的光电探针,增加AgInS2QDs负载量,提高光电流信号。基于AgInS2 QDs-DNA纳米线对NPC-ZnO多面体光电流的极性反转,构建了一种超灵敏的多功能光电生物传感器,用于双目标检测。目标Hg2+诱导循环放大产生大量RDNA,将纳米线信号探针修饰到电极上,产生极强的光电流信号,实现对目标一Hg2+的灵敏检测;当目标二AFB1与电极上的适体特异性结合时,将纳米线信号探针取代下来,减弱光电流信号,对AFB1进行检测。
发明内容:
本发明的目的是提供一种基于AgInS2QDs-DNA纳米线的光致电化学生物传感器;以及所述生物传感器的制备方法及其检测Hg2+和黄曲霉素B1的分析应用。
具体包括以下步骤:
步骤1.合成NPC-ZnO:将1.485g硝酸锌和3.28g 2-甲基咪唑分别溶解、混合,常温搅拌2h得到晶体。将产物离心、无水甲醇洗涤,冷冻干燥过夜得ZIF-8。随后放于管式炉中,保持N2氛围,在600℃下直接碳化3h,得到NPC-ZnO。将0.5mL3-氨丙基三乙氧基硅烷分散在5mL乙醇中,用盐酸调pH至5,搅拌下添加0.125g NPC-ZnO,搅拌14h,离心洗涤得NPC-ZnO-NH2。
步骤2.AgInS2 QDs制备:在100mL烧瓶中加入50mL含有0.1mmolAgNO3、0.4mmol In(NO)3和0.1mmol MPA的水溶液。在强力搅拌下,将3mL 0.2M的Na2S溶液快速注入烧瓶中。将混合溶液加热到100℃,保温2h,让AgInS2QDs生长。
步骤3.AgInS2 QDs-DNA纳米线的制备:取50μL 1μM DNA1和50μL 1μM DNA2加入到离心管中,在95℃下加热5min,自然冷却到室温,得到DNA1与DNA2的杂交产物。取70μL纯化的AgInS2QDs,加入15μL EDC/NHS混合液(含0.1M EDC和0.025M NHS)活化40min,再加入15μL 10μM的DNA3,将混合溶液于37℃摇床反应6h,得AgInS2QDs-DNA3混合物,离心后分散在100μL水。然后将DNA1和DNA2的杂交产物加入到AgInS2QDs-DNA3的混合溶液中,孵育2h,重新离心(2000rpm,5min)分散在200μL水中,得AgInS2QDs-DNA纳米线。
步骤4.目标Hg2+诱导扩增过程:在含有20mM Tris-HCl、100mM NaCl、1mM EDTA和5mM MgCl2的40μLTris-HCl缓冲液(pH=7.4)中,加入30μLHP(5μM)、6U的ExoⅢ和30μL不同浓度的Hg2+溶液,在37℃下温育2.5h制备RDNA。然后将溶液加热到80℃,时间10min,然后冷却到室温。最后,将RDNA保存在4℃中,以备进一步使用。
步骤5.PEC生物传感器的制备:用10mM的EDC/NHS混合液活化AFB1适体。取20μL0.5mg·mL-1氨基化NPC-ZnO滴到ITO电极上,自然干燥后,将活化好的20μLAFB1适体滴到电极上,孵育2h,再用2mM巯基己醇封闭电极1h。随后向电极上滴加不同浓度的RDNA,孵育2h后再滴加20μLAgInS2 QD-DNA纳米线,反应2h。用超纯水轻微冲洗以除去未连接上的反应物,最后一步冲洗后在空气中干燥,待测ECL检测Hg2+。
构建检测黄曲霉素B1的传感平台,只需在上述步骤完成后,再滴加不同浓度的黄曲霉素B1,孵育2h后,用PBS冲洗并在空气中干燥、待测ECL。
步骤6.PEC检测:PEC检测在含有10mM抗坏血酸(AA)的PBS(pH=7.4,100mM)中进行光电流测量,利用CHI660e电化学工作站,蓝光作激发光源。本实验使用三电极系统:ITO作为工作电极,Pt丝作为对电极,Ag/AgCl电极作为参比电极。
本发明研制了多功能光致电化学生物传感器,用于Hg2+和黄曲霉素B1的双目标分析。本发明与现有技术相比,主要优点在于:AgInS2 QDs-DNA纳米线能够负载大量的AISQDs信号分子,极大地放大了信号,提高了检测灵敏度;同时,利用AgInS2 QDs-DNA纳米线反转NPC-ZnO的光电流信号,极大提高了检测准确度,降低了假阳性;利用Hg2+引发酶切循环实现了目标的放大,以及黄曲霉素B1与适体特异性结合检测两种目标,具有很高的选择性。该传感器线性范围宽、检测限低、灵敏度高,在生物检测和实际应用中具有很高的潜力。
表1 DNA序列
本发明所用到的DNA序列均购自上海生工生物有限公司。
附图说明:
图1基于AgInS2 QDs-DNA纳米线反转NPC-ZnO光电流信号的光电化学传感器原理图。
图2DNA纳米线的原子力显微镜(AFM)图。
图3电泳表征:(A)PEC传感过程的分析,(B)DNA纳米线形成过程的分析。
图4(A)传感器不同阶段光电流响应:(a)裸ITO,(b)ITO/NPC-ZnO,(c)ITO/NPC-ZnO/AFB1适体/RDNA/AIS QDs-DNA纳米线,(d)ITO/NPC-ZnO/AFB1适体/RDNA/AIS QDs-DNA纳米线/AFB1,(B)在连续扫描200s下,传感器检测Hg2+的稳定性,(Hg2+浓度为0.1μM),(C)在连续扫描200s下,传感器检测AFB1的稳定性,(AFB1浓度为10ng·mL-1)。
图5(A)PEC传感平台对不同目标浓度的光电流曲线,(B)线性光电流变化值ΔI与Hg2+浓度之间的线性关系。(1fM-0.1μM)。
图6(A)PEC传感平台对不同AFB1浓度的光电流曲线,(B)线性光电流变化值ΔI与AFB1浓度之间的线性关系。(1fg·mL-1-10ng·mL-1)。
具体实施方式:
实施例1.PEC生物传感器的制备及对目标的检测
AgInS2 QDs-DNA纳米线的制备:取50μL 1μM DNA1和50μL 1μM DNA2加入到离心管中,在95℃下加热5min,自然冷却到室温,得到DNA1与DNA2的杂交产物。
取70μL纯化AgInS2 QDs,加入15μL EDC/NHS混合液(含0.1M EDC)和0.025MNHS),活化40min,再加入15μL 10μM DNA3,将混合溶液放进37℃的摇床里反应6h,得到AgInS2QDs-DNA3混合物,将其重新离心(3000rpm,5min)分散在100μL水。
然后将DNA1和DNA2的杂交产物加入到AgInS2 QDs-DNA3混合溶液中,孵育2h,重新离心(2000rpm,5min)分散在200μL水中,得到AgInS2 QDs-DNA纳米线。
PEC生物传感器的制备及目标检测:依次用丙酮、1M NaOH、超纯水超声处理ITO电极10分钟,使电极修饰上羟基。取20μL 0.5mg·mL-1氨基化NPC-ZnO滴到ITO电极上,自然干燥;同时用10mM的EDC/NHS混合液活化AFB1适体。将20μL活化好的AFB1适体滴到电极上,孵育2h,再用2mM巯基己醇封闭修饰电极1h。随后向电极上滴加不同浓度的RDNA,孵育2h后再滴加20μLAgInS2 QDs-DNA纳米线,反应2h。每一步在电极上孵育完成后,用超纯水轻微冲洗以除去未连接上的反应物,最后一步冲洗后在空气中干燥,通过光电信号检测Hg2+的浓度。
构建检测黄曲霉素B1的传感平台:在上述检测一定浓度Hg2+步骤完成后,再滴加不同浓度的黄曲霉素B1,孵育2h后,用PBS缓冲液轻微冲洗并在空气中干燥,通过光电信号检测黄曲霉素B1的浓度。
实施例2.PEC生物传感器的制备及对目标的检测
将“取70μL纯化后的AgInS2 QDs,向其中加入15μL EDC/NHS混合液,活化羧基40min,再向其中加入15μL 10μM的DNA3,将混合溶液放进37℃的摇床里反应6h”改为“取70μL纯化后的AgInS2 QDs,向其中加入15μL EDC/NHS混合液,活化羧基40min,再向其中加入15μL 10μM的DNA3,将混合溶液放进37℃的摇床里反应4h。”制备的其他条件同实施例1,得到形貌与性质类似于实施例1的生物传感器。对Hg2+和黄曲霉素B1检测的结果同实施例1。
实施例3.PEC生物传感器的制备及对目标的检测
将“用0.1M磷酸盐缓冲液(PBS,pH=7.0)配制不同浓度黄曲霉素B1的标准溶液。构建检测黄曲霉素B1的传感平台,再滴加不同浓度的黄曲霉素B1,孵育2h”改为“用0.1M磷酸盐缓冲液(PBS,pH=7.0)配制不同浓度黄曲霉素B1的标准溶液。构建检测黄曲霉素B1的传感平台,再滴加不同浓度的黄曲霉素B1,孵育1.5h”,制备的其他条件同实施例1,得到形貌与性质类似于实施例1的生物传感器。对Hg2+和黄曲霉素B1检测的结果同实施例1。
实施例4.PEC生物传感器对实际样品的检测
从发霉的食物样品花生中提取AFB1:用刀切碎干净的花生,并在37℃的潮湿环境中储存7天。然后通过相同的程序提取干净和发霉的样品:将1g花生样品浸入2mL甲醇中,摇动1h后,将其以3000rpm离心5min,将上述提取步骤重复三次,收集所有提取剂并转移至相同的离心管中。最后,将100μL提取的样品溶液用于光电化学检测。
将“黄曲霉素B1的标准溶液。”改为“黄曲霉素B1样品提取溶液”,制备的其他条件同实施例1,得到形貌与性质类似于实施例1的光电传感器,对Hg2+和黄曲霉素B1进行检测。
Claims (1)
1.一种基于DNA-量子点纳米线的光致电化学生物传感器在检测Hg2+和黄曲霉素B1中的应用,其特征是:利用AgInS2量子点标记的DNA纳米线放大信号,结合AgInS2量子点反转NPC-ZnO纳米多面体的光电流信号,研制了光致电化学生物传感器,通过光电信号“on-off”检测模式,实现了对Hg2+和黄曲霉素B1双目标高灵敏检测;目标一Hg2+引发了DNA循环放大反应,产生大量RDNA,将AgInS2量子点标记的DNA纳米线组装到电极上,实现了光电化学信号“on”检测Hg2+;进一步利用目标二黄曲霉素B1竞争结合电极表面的适体,使AgInS2量子点标记的DNA纳米线从电极表面脱落,实现了光电化学信号“off”检测黄曲霉素B1;
具体如下:
步骤1.AgInS2量子点-DNA纳米线的制备:
取50μL 1μM DNA1和50μL 1μM DNA2加入到离心管中,在95℃下加热5min,自然冷却到室温,得到DNA1与DNA2的杂交产物;
取70μL纯化的AgInS2量子点,向其中加入15μL含有0.1M的EDC和0.025M NHS的混合溶液,活化羧基40min,再向其中加入15μL 10μM的DNA3,将混合溶液放进37℃的摇床里反应6h,得到AgInS2量子点-DNA3混合物,于3000rpm下重新离心5min,之后分散在100μL水里;然后将DNA1和DNA2的杂交产物加入到AgInS2量子点-DNA3的混合溶液中,孵育2h,于2000rpm下重新离心5min,分散在200μL水中,得到AgInS2量子点-DNA纳米线;
步骤2.目标Hg2+诱导扩增过程:
在含有20mM Tris-HCl、100mM NaCl、1mM EDTA和5mM MgCl2的40μL Tris-HCl缓冲液中加入30μL 5μM发夹DNA HP、6U的ExoⅢ和30μL的不同浓度的Hg2+溶液,将混合物于37℃反应150min得扩增产物RDNA;然后将溶液加热到80℃,时间10min,然后冷却到室温;最后,将RDNA保存在4℃中,以备进一步使用;
步骤3.光致电生物传感器的制备及目标检测:
取20μL 0.5mg·mL-1的氨基化NPC-ZnO滴到ITO电极上,自然干燥后,将活化好的20μLAFB1适体滴到电极上,孵育2h,接着用2mM巯基己醇封闭修饰电极1h;随后向电极上滴加不同浓度的RDNA,孵育2h后再滴加20μLAgInS2量子点-DNA纳米线,反应2h,构建检测Hg2+的光电生物传感平台;
表1 DNA序列
上述检测Hg2+步骤完成后,再于电极上滴加不同浓度的黄曲霉素B1,孵育2h后,用PBS缓冲液轻微冲洗并在空气中干燥,构建检测黄曲霉素B1的光电传感平台;在含有10mM抗坏血酸的pH 7.4、100mM PBS中进行光电流测量,蓝光作激发光源,ITO作为工作电极,Pt丝作为对电极,Ag/AgCl电极作为参比电极。
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