CN116333721A - 一种用于同步检测PTK7和miRNA-21的比率荧光探针、制备方法及其应用 - Google Patents
一种用于同步检测PTK7和miRNA-21的比率荧光探针、制备方法及其应用 Download PDFInfo
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Abstract
本发明提供一种用于同步检测PTK7和miRNA‑21的比率荧光探针、制备方法及其应用,其制备方法,包括以下步骤:步骤1,羧化四氧化三铁纳米颗粒的制备;步骤2,Fe3O4MNPs与氨基化适配体互补链的结合;步骤3,绿色荧光CdSe/ZnS量子点与氨基化适配体1的结合;步骤4,红色荧光CdSe/ZnS量子点与适配体2的结合;步骤5,荧光探针的制备。本发明基于碳点、量子点和磁性纳米颗粒构建荧光探针用于双目标物的同步检测,并将其应用于实际人血清样品的检测,提高了检测结果的准确度,且合成简单、成本低、有良好的灵敏度和选择性。
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种用于同步检测PTK7和miRNA-21的比率荧光探针、制备方法及其应用。
背景技术
酪氨酸蛋白激酶7(PTK7)和miRNA-21均为明确的乳腺癌标志物。目前,同步检测多个肿瘤标志物的研究仍比较少,故有必要开发新检测方法用于多个肿瘤标志物的同步检测,以提高检测结果的准确性和可靠性。
迄今为止,多个肿瘤标志物检测方法有电化学法、表面增强拉曼散射法、光致发光法、荧光法等,其中荧光法具有灵敏度高、准确度好、使用简便的优点,受到广泛关注。荧光法检测多个肿瘤标志物主要是基于配体-受体的相互作用,以各种荧光标记物与蛋白质或核酸片段等形成生物分子探针,由荧光发射基团、荧光淬灭基团以及识别和结合目标物的识别基团组成。荧光发射基团主要是一些荧光物质(有机荧光染料、荧光纳米材料等),荧光淬灭基团是能够淬灭荧光的物质(贵金属纳米粒、聚多巴胺纳米微粒、磁性纳米粒等),识别基团则是适配体,一小段经体外筛选得到的寡核苷酸序列或者短的多肽,对配体有高亲和力,能特异性识别配体并与之结合。适配体易于合成和修饰、强特异性、稳定性好、生物相容性好,因而在生物传感领域应用广泛。
应用荧光法对多个癌症标志物进行测定时,由于仅基于一种荧光信号,荧光信号易受外界环境的干扰,检测结果准确度易受影响。而基于两种荧光信号的比率荧光,是通过两个波长处的荧光强度比值作为响应信号以确定目标物的含量,它不受外界环境光源强度和仪器灵敏度的影响,从而克服了荧光法的缺点,是一种新型抗干扰高灵敏度的检测。因此,可基于比率荧光对多个肿瘤标志物进行同步检测。
近年来,新型荧光纳米材料迅速发展,相较于传统的有机荧光染料(如荧光素类、罗丹明类等)有着极大的优势。有机荧光染料存在斯托克斯红移小发射光谱易受激发光谱干扰、荧光易被漂白影响重复使用等缺陷,而荧光纳米材料(碳量子点、石墨烯量子点、贵金属纳米团簇等)具有激发光谱窄、发射峰半峰宽小且对称、光谱可调等特性,可替代传统荧光物质并被广泛应用于生物传感和生物成像等领域。
碳点作为零维碳纳米材料,粒径小于10nm,具有稳定性好、水溶性好、生物相容好、单粒子荧光强、斯托克斯红移大、耐光漂白等特点,已被广泛应用于生物传感和生物成像等领域。
量子点是一种零维半导体纳米粒子,粒径通常在2-20,nm,具有光稳定性好、荧光寿命长、耐光漂白、表面易于修饰、激发谱宽而发射谱窄等特点,是理想的用于构建比率荧光探针的荧光基团。
四氧化三铁磁性纳米粒(Fe3O4,Magnetic,Nanoparticles,Fe3O4 MNPs)在200-800nm波长范围内有广泛的紫外吸收,与多种荧光基团的发射光谱重叠,是淬灭性能良好的荧光淬灭剂。Fe3O4MNPs不仅能够作为荧光淬灭剂还能作为磁性分离器,在磁场作用下易于进行磁性分离,能够将探针从复杂基质中分离出来,这是其他荧光淬灭剂所不具有的。
基于以上研究背景,本课题拟构建一种基于碳点量子点的荧光探针用于两个肿瘤标志物的检测。
发明内容
为解决上述技术问题,本发明的目的在于提供一种用于同步检测PTK7和miRNA-21的比率荧光探针及制备方法和应用。
为实现上述目的,本发明的技术方案如下。
一种用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,包括以下步骤:
步骤1,制备羧化四氧化三铁纳米颗粒
以三氯化铁、乙酸钠和乙二醇为原料,通过水热法合成四氧化三铁纳米颗粒(Fe3O4MNPs),然后加入到柠檬酸溶液中,合成羧化四氧化三铁纳米颗粒(羧化Fe3O4 MNPs);
步骤2,制备羧化四氧化三铁纳米颗粒(羧化Fe3O4MNPs)与氨基化适配体(apt)互补链(cDNA)的复合物
将羧化四氧化三铁纳米颗粒在EDC/NHS催化下,分别与两个氨基化适配体互补链(cDNA1,cDNA2)通过酰胺键联结,合成Fe3O4-cDNA1复合物和Fe3O4-cDNA2复合物;
步骤3,制备绿色荧光CdSe/ZnS量子点(gQDs)与氨基化适配体apt1的复合物
将羧基修饰的绿色荧光CdSe/ZnS量子点在EDC/NHS催化下,与氨基化适配体apt1通过酰胺键联结,合成gQDs-apt1复合物;
步骤4,制备红色荧光CdSe/ZnS量子点(rQDs)与适配体apt2的复合物
将羧基修饰的红色荧光CdSe/ZnS量子点在EDC/NHS催化下,与氨基化适配体apt2通过酰胺键联结,合成rQDs-apt2复合物;
步骤5,制备荧光探针
将步骤2的Fe3O4-cDNA1复合物与步骤3的gQDs-apt1复合物通过碱基互补配对形成Fe3O4-cDNA1-apt1-gQDs;
将步骤2的Fe3O4-cDNA2复合物与步骤4的rQDs-apt2复合物通过碱基互补配对形成Fe3O4-cDNA2-apt2-rQD;
将Fe3O4-cDNA1-apt1-gQDs和Fe3O4-cDNA2-apt2-rQD混合,然后加入蓝色荧光碳点(bCDs),得到荧光探针bCDs/(Fe3O4-cDNA1-apt1-gQDs)/(Fe3O4-cDNA2-apt2-rQD)。
进一步,步骤1中,水热法的条件为:反应温度200℃,反应时间4~8h。
进一步,步骤1中,三氯化铁和乙酸钠的质量比为0.54:1.44;
柠檬酸与四氧化三铁纳米颗粒的质量比为5:0.2。
进一步,步骤2中,羧化四氧化三铁纳米颗粒、EDC和NHS的质量比为0.2:19:22;羧化四氧化三铁纳米颗粒与cDNA1的用量比为0.2mg:10μL;
羧化四氧化三铁纳米颗粒与cDNA2的用量比为0.2mg:10μL。
进一步,步骤3中羧基修饰的绿色荧光CdSe/ZnS量子点(gQDs)、EDC和NHS的质量比为0.2:19:22;
羧基修饰的绿色荧光CdSe/ZnS量子点(gQDs)与氨基化适配体apt1的用量比为0.2mg:10μL。
进一步,步骤4中,羧基修饰的红色荧光CdSe/ZnS量子点(rQDs)、EDC和NHS的质量比为0.2:19:22;
羧基修饰的红色荧光CdSe/ZnS量子点(rQDs)与氨基化适配体apt2的用量比为0.2mg:10μL。
进一步,步骤5中,Fe3O4-cDNA1复合物与gQDs-apt1复合物的体积比为1:1;Fe3O4-cDNA2复合物与rQDs-apt2复合物的体积比为1:1;
Fe3O4-cDNA1-apt1-gQDs、Fe3O4-cDNA2-apt2-rQD和蓝色荧光碳点(bCDs)的体积比为1:1:1。
本发明提供一种采用上述方法制备得到的用于同步检测PTK7和miRNA-21的比率荧光探针。
本发明还提供用于同步检测PTK7和miRNA-21的比率荧光探针的应用,所述荧光探针应用于PTK7和miRNA-21的同步检测。
本发明首先利用适配体和适配体互补链将两个绿色荧光量子点和红色荧光量子点分别连接在Fe3O4 MNPs上,背景中引入蓝色参比荧光。当体系中不存在PTK7和miRNA-21时,绿、红荧光均淬灭,体系中仅有蓝色参比荧光;当体系中存在PTK7时,绿色荧光恢复,蓝色参比荧光不变,利用绿色和蓝色荧光强度比值(I530/I430)对PTK7进行定量检测;当体系中存在miRNA-21时,红色荧光恢复,蓝色参比荧光不变,利用红色和蓝色荧光强度比值(I640/I430)对miRNA-21进行定量检测;当体系中PTK7和miRNA-21均存时,绿色和红色荧光都恢复,蓝色参比荧光不变,利用荧光强度比值(I530/I430)和(I640/I430)分别对PTK7和miRNA-21进行定量检测。
本发明的有益效果:
1、本发明制备的荧光探针可对两种目标物PTK7和miRNA-21进行同步检测,由于两种荧光不会相互影响,当存在PTK7和miRNA-21时,PTK7竞争结合apt1,miRNA-21竞争结合apt2,从而使gQDs-apt1和rQDs-apt2分别从Fe3O4-cDNA1和Fe3O4-cDNA2上脱离。gQDs和rQDs与Fe3O4 MNPs距离增大,从而不产生内滤效应,绿色和红色荧光恢复,随着两目标物加入的量增大,荧光恢复增多,从而实现对PTK7和miRNA-21的同步检测。
2、本发明的荧光探针bCDs/(Fe3O4-cDNA1-apt1-gQDs)/(Fe3O4-cDNA2-apt2-rQDs)能同步检测两种乳腺标志物PTK7和miRNA-21,提高了检测结果的准确度,且合成简单、成本低、有良好的灵敏度和选择性,并且成功应用于血清样品中PTK7和miRNA-21的测定。
附图说明
图1是Fe3O4MNPs、bCDs、gQDs和rQDs的TEM图。其中,A图为Fe3O4MNPs的TEM图;B、C、D图分别为bCDs、gQDs、rQDs的TEM图;内插图分别为其高分辨率TEM图。
图2是Fe3O4MNPs的磁滞回线图和经磁铁分离再震摇后的日光下图像。其中,A图是Fe3O4MNPs的磁滞回线图;B图是Fe3O4MNPs经磁铁分离再震摇后的日光下图像。
图3中图A、B、C分别是bCDs、gQDs、rQDs紫外吸收和荧光发射图,内插图从左到右依次为其日光和紫外灯下拍摄的图像。
图4是本发明实施例1的荧光探针用于双目标物检测原理图。
图5中A图是Fe3O4MNPs紫外吸收图和gQDs、rQDs荧光发射图;B图是gQDs和Fe3O4-cDNA1-apt1-gQDs荧光寿命图;C图是rQDs和Fe3O4-cDNA2-apt2-rQDs荧光寿命图。
图6中A图是bCDs、gQDs、rQDs以及三者混合后的荧光图,B图是bCDs、gQDs-apt1、rQDs-apt2以及三者混合后的荧光图。
图7中A图是加入不同浓度PTK7荧光图,B图是加入不同浓度miRNA-21荧光图,C图是同时加入不同浓度PTK7、miRNA-21荧光图。
图8中A图是加入PTK7及PTK7掺杂miRNA-21荧光图;B图是加入miRNA-21及miRNA-21掺杂PTK7荧光图。
图9中A图是Fe3O4 MNPs、cDNA1、cDNA2、Fe3O4-cDNA1和Fe3O4-cDNA2的紫外吸收图,B图是Fe3O4 MNPs、Fe3O4-cDNA1和Fe3O4-cDNA2的红外光谱图。
图10中A、B、C图分别为Fe3O4 MNPs、Fe3O4-cDNA1和Fe3O4-cDNA2的TEM mapping图。
图11是Fe3O4 MNPs、Fe3O4-cDNA1和Fe3O4-cDNA2的zeta电位图。
图12是Fe3O4 MNPs、Fe3O4-cDNA1和Fe3O4-cDNA2的磁滞回线图。
图13中A图是gQDs、apt1和gQDs-apt1琼脂糖凝胶电泳图,B图是rQDs、apt2和gQDs-apt2琼脂糖凝胶电泳图,C图是gQDs、rQDs、gQDs-apt1、rQDs-apt2红外光谱图。
图14是gQDs、rQDs、gQDs-apt1、rQDs-apt2的Zeta电位图。
图15中A图是加入不同浓度PTK7荧光变化图;B图是荧光强度比值(I530/I430)与lg[PTK7]的线性拟合曲线。
图16是荧光探针对PTK7及干扰离子、氨基酸、蛋白质的选择性(其中PTK7浓度为100ng mL-1,干扰物浓度为1000ng mL-1)。
图17中A图是加入不同浓度miRNA-21荧光变化图;B图是荧光强度比值(I640/I430)与lg[miRNA-21]的线性拟合曲线。
图18是荧光探针对miRNA-21及干扰RNA单碱基错配、双碱基错配、BASE1-AS、UUUUU、AAAAA选择性(其中miRNA-21浓度为10nM,其余RNA浓度为20nM)。
图19中A图是加入不同浓度PTK7和miRNA-21荧光变化图;B图是荧光强度比值(I530/I430)与lg[PTK7]的线性拟合曲线;C图是荧光强度比值(I640/I430)与lg[miRNA-21]的线性拟合曲线。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
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。
下述各实施例中所述的试剂来源如下:
脱氧核糖核酸酶I(DNaseI)、牛血清白蛋白Ⅴ(Albumin BovineⅤ,BSA)和肌红蛋白(Myoglobin)索莱宝生物科技有限公司。
干粉apt1、apt2、cDNA1、cDNA2,索莱宝生物科技有限公司。
下述各实施例中所述实验方法如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可在市场上购买得到。
实施例1
用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,包括以下步骤:
S1、蓝色荧光碳点(bCDs)的制备
称取1.2g CA(柠檬酸),加入600μL DETA(二乙烯三胺),再加入超纯水至体积20mL超声使之溶解并置于振荡器混合均匀。将混合溶液转移至聚四氟乙烯为内衬的不锈钢高压反应釜中,200℃反应4h,冷却至室温后,用丙酮洗涤三次,50℃真空干燥12h,得淡黄色固体粉末(bCDs)。
S2、羧化四氧化三铁纳米颗粒(羧化Fe3O4 MNPs)的制备
称取0.54g FeCl3和1.44g乙酸钠,分别用5mL乙二醇超声溶解,得到FeCl3-乙二醇溶液和乙酸钠-乙二醇溶液;在磁力搅拌下将乙酸钠-乙二醇溶液缓慢滴加至FeCl3-乙二醇溶液中,全部加入后持续搅拌30min,转移至聚四氟乙烯为内衬的不锈钢高压反应釜中,200℃反应8h,冷却至室温后,用乙醇洗涤三次,50℃真空干燥12h,得到干燥的四氧化三铁纳米颗粒(Fe3O4MNPs)。
称取5g柠檬酸溶于50mL超纯水中,再加入0.2g干燥Fe3O4 MNPs,超声20min,机械搅拌4h,产物用超纯水和乙醇洗涤三次,50℃真空干燥,得羧化四氧化三铁纳米颗粒(羧化Fe3O4 MNPs)。
S3、羧化Fe3O4 MNPs与适配体互补链(cDNA1,cDNA2)的结合
S3.1、羧化Fe3O4 MNPs与适配体互补链cDNA1的结合
将0.2mg羧化Fe3O4MNPs,溶于1mL PBS缓冲溶液(pH 6.0)中加入19mg EDC(1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺)和22mg NHS(N-羟基琥珀酰亚胺),超声使之溶解,室温振荡活化20min,得到活化后的羧化Fe3O4MNPs。
对cDNA1进行预处理:将cDNA1置于4℃4000rpm离心1min,加入PBS缓冲溶液(pH7.4)使之溶解成100μM溶液,95℃加热4min,再冰浴冷却4min,得到预处理好的cDNA1。
将活化后的羧化Fe3O4 MNPs用1M NaOH调节PH至7.4,再加入10μL预处理好的cDNA1,室温振荡过夜,再用PBS缓冲溶液(pH 7.4)磁洗三次,除去未连接在Fe3O4 MNPs上的cDNA1,得到Fe3O4-cDNA1复合物。
S3.2、羧化Fe3O4MNPs与适配体互补链cDNA2的结合(方法同S3.1)
将0.2mg羧化Fe3O4 MNPs,溶于1mL PBS缓冲溶液(pH 6.0)中加入19mg EDC和22mgNHS,超声使之溶解,室温振荡活化20min,得到活化后的羧化Fe3O4 MNPs。
对cDNA2进行预处理:将cDNA2置于4℃4000rpm离心1min,加入PBS缓冲溶液(pH7.4)使之溶解成100μM溶液,95℃加热4min,再冰浴冷却4min,得到预处理好的cDNA2。
将活化后的羧化Fe3O4 MNPs用1M NaOH调节PH至7.4,再加入10μL预处理好的cDNA2,室温振荡过夜,再用PBS缓冲溶液(pH 7.4)磁洗三次,除去未连接在Fe3O4 MNPs上的cDNA2,得到Fe3O4-cDNA2复合物。
S4、绿色荧光CdSe/ZnS量子点(gQDs)与适配体1(apt1)的结合
将0.2mg gQDs溶于1mL PBS缓冲溶液(pH 6.0)中加入19mg EDC和22mg NHS,超声使之溶解,室温振荡活化20min,得到活化后的gQDs。
对apt1进行预处理:将apt1置于4℃4000rpm离心1min,加入PBS缓冲溶液(pH 7.4)使之溶解成100μM溶液,95℃加热4min,再冰浴冷却4min,得到预处理好的apt1。
将活化后的gQDs用1M NaOH调节PH至7.4,再加入10μL 100μM预处理好的apt1,室温振荡过夜,得到gQDs-apt1复合物。
S5、红色荧光CdSe/ZnS量子点(rQDs)与适配体2(apt2)的结合(方法同S4)
将0.2mg rQDs溶于1mL PBS缓冲溶液(pH 6.0)中加入19mg EDC和22mg NHS,超声使之溶解,室温振荡活化20min,得到活化后的rQDs。
对apt2进行预处理:将apt2置于4℃,4000,rpm离心1min,加入PBS缓冲溶液(pH7.4)使之溶解成100μM溶液,95℃加热4min,再冰浴冷却4min,室温静置备用,得到预处理好的apt2。
将活化后的rQDs用1M NaOH调节PH至7.4,再加入10μL 100μM预处理好的apt2,室温振荡过夜,得到rQDs-apt1复合物。
S6、荧光探针的制备
将50μL Fe3O4-cDNA1复合物与50μL gQDs-apt1复合物在37℃孵育2h,采用PBS缓冲溶液(pH 7.4)磁洗三次,除去多余的gQDs-apt1,得到Fe3O4-cDNA1-apt1-gQDs溶液。
将50μL Fe3O4-cDNA2复合物与50μL rQDs-apt2复合物在37℃孵育2h,采用PBS缓冲溶液(pH 7.4)磁洗三次,除去多余的rQDs-apt2,得到Fe3O4-cDNA2-apt2-rQD溶液。
将Fe3O4-cDNA1-apt1-gQDs溶液和Fe3O4-cDNA2-apt2-rQD溶液按照体积比1:1混合,得到(Fe3O4-cDNA1-apt1-gQDs)/(Fe3O4-cDNA2-apt2-rQDs)溶液,然后加入1μL蓝色荧光碳点(bCDs,3.125μg·mL-1),37℃恒温振荡1h,得到用于同步检测PTK7和miRNA-21的比率荧光探针bCDs/(Fe3O4-cDNA1-apt1-gQDs)/(Fe3O4-cDNA2-apt2-rQD)。
应用实施例1
将实施例1的荧光探针用于PTK7和miRNA-21同步检测,具体方法如下:
取实施例1的荧光探针bCDs/(Fe3O4-cDNA1-apt1-gQDs)/(Fe3O4-cDNA2-apt2-rQD)加入25μL PTK7和25μL miRNA-21,使PTK7终浓度为0、0.5、1、2、5、10、20、50、100ng·mL-1,使miRNA-21终浓度为0、0.1、0.2、0.5、1、2、5、10、20nM,加入2.5μL 20U·μL-1DNaseI,37℃恒温振荡1h。经磁分离,在360nm激发下测上清荧光强度。并根据荧光探针的荧光强度比值(I530/I430)的变化程度对PTK7浓度绘制标准曲线;根据荧光探针的荧光强度比值(I640/I430)的变化程度对miRNA-21浓度绘制标准曲线。
应用实施例2
将实施例1的荧光探针用于血清中PTK7和miRNA-21同步检测,具体方法如下:
血清样本来自南京鼓楼医院。
(1)采用标准加入法对血清1中PTK7和miRNA-21的同步检测
25μL血清1中加入12.5μL PTK7,使其终浓度为0、0.5、1、1.5ng·mL-1。再加入12.5μL miRNA-21,使其终浓度为0、1.5、3、4.5nM。将加入标准溶液的血清,按照“应用实施例3的PTK7和miRNA-21同步检测”操作,测定血清1中PTK7和miRNA-21的含量。
(2)采用标准加入法对血清2中PTK7和miRNA-21的同步检测
1mL血清中加入200μL PBS缓冲溶液(pH7.4),然后取25μL血清-PBS溶液中加入12.5μL PTK7,使其终浓度为0、1、2、3ng·mL-1;再加入12.5μL miRNA-21,使其终浓度为0、0.5、1、1.5nM。将加入标准溶液的血清,按照“应用实施例3的PTK7和miRNA-21同步检测”操作,测定血清2中PTK7和miRNA-21的含量。
下面对实施例1的荧光探针以及应用实施例1~2的方法进行性能表征。
1、Fe3O4MNPs、bCDs、gQDs和rQDs的性能表征
1.1、Fe3O4 MNPs、bCDs、gQDs和rQDs的微观形态表征
为了证明Fe3O4 MNPs、bCDs、gQDs和rQDs的成功合成,对其进行了TEM表征,见图1。图1的A图为Fe3O4 MNPs的TEM图,可以看出Fe3O4MNPs呈球形,粒径约为400nm,证明Fe3O4MNPs的成功合成。图1的B图为bCDs的TEM图,内插图为高分辨率TEM图像,图中可以看出bCDs呈球形,分散均匀,粒径约3nm,晶格大小为0.23nm,与文献一致,证明bCDs的成功合成。图1的C图为gQDs的TEM图,内插图为高分辨率TEM图像,晶格大小为0.28nm,图中可以看出gQDs呈近球形,分散均匀,粒径约8nm。图1的D图为rQDs的TEM图,内插图为高分辨率TEM图像,图中可以看出rQDs呈近球形,分散均匀,粒径约10nm,晶格大小为0.26nm。
1.2、Fe3O4 MNPs磁性表征
对Fe3O4 MNPs的磁性进行表征,见图2。图2中,图A磁滞回线数据表明,Fe3O4 MNPs有良好的磁性;图B为Fe3O4 MNPs经磁铁分离再震摇后的日光下图像,结果表明,经磁铁分离10S溶液澄清,表明Fe3O4 MNPs具有良好的磁性,再经震摇后重新均匀分散,表明Fe3O4MNPs在水溶液中有良好的分散性,可作为磁性分离剂用于荧光探针的构建。
1.3、bCDs、gQDs和rQDs的光谱性能表征
对bCDs、gQDs和rQDs进行了紫外和荧光表征,见图3。图3中,图A为bCDs的紫外和荧光光谱,bCDs分别在240nm和352nm处有明显吸收,它们分别归属于bCDs中C=C芳环结构的π-π*跃迁和C=O的n-π*跃迁,在360nm激发下,bCDs在430nm处有最大荧光发射波长,内插图所示bCDs在日光下呈无色溶液,在紫外灯下呈较强的蓝色荧光,证明bCDs的成功合成。图B为gQDs的紫外和荧光光谱gQDs在530nm处有明显吸收,归属于跃迁,在360nm激发下gQDs在530nm处有最大荧光发射波长,内插图所示gQDs在日光下呈淡黄色溶液,在紫外灯下呈较强的绿色荧光。结果表明gQDs有良好的荧光性能,可作为荧光供体构建荧光探针。图C为rQDs的紫外和荧光光谱,rQDs在640nm处有明显吸收,归属于跃迁,在360nm激发下gQDs在640nm处有最大荧光发射波长,内插图所示rQDs在日光下淡黄色溶液,在紫外灯下呈较强的红色荧光,结果表明rQDs有良好的荧光性能,可作为荧光供体构建荧光探针。
2、gQDs、rQDs和Fe3O4MNPs之间的相互作用研究
图4是荧光探针用于双目标物检测原理图;图5中,A图为Fe3O4 MNPs的紫外吸收图以及gQDs和rQDs的荧光发射图;B图为gQDs和Fe3O4-cDNA1-apt1-gQDs荧光寿命图;C图为rQDs和Fe3O4-cDNA2-apt2-rQDs荧光寿命图。
从图5中图A紫外荧光光谱可以看出,Fe3O4MNPs具有广泛的紫外吸收,并与gQDs和rQDs荧光光谱有较大的重叠,由此推断gQDs和rQDs为荧光供体,Fe3O4 MNPs为荧光受体淬灭荧光供体的荧光。
对gQDs和Fe3O4-cDNA1-apt1-gQDs,rQDs和Fe3O4-cDNA2-apt2-rQDs的荧光寿命进行了测定(图5中B、C),gQDs和Fe3O4-cDNA1-apt1-gQDs、rQDs和Fe3O4-cDNA2-apt2-rQDs的荧光寿命几乎重合,说明gQDs、rQDs被Fe3O4MNPs猝灭并不影响其荧光寿命,gQDs、rQDs和Fe3O4MNPs之间的相互作用并不涉及电荷或能量转移。由此表明gQDs和rQDs荧光强度猝灭的机理是基于内滤效应(IFE)的静态猝灭机制。
3、bCDs、gQDs、rQDs之间的相互作用研究
bCDs、gQDs、rQDs三者荧光相互关系,见图6。从图6可以看出,三者混合后荧光下降(A图),而将bCDs与连有适配体的gQDs-apt1、rQDs-apt2三者混合后荧光基本不变(B图),说明连有适配体的gQDs和rQDs与bCDs荧光不会互相影响,这是因为bCDs与连有适配体的gQDs-apt1和rQDs-apt2距离增大,不产生荧光淬灭。说明bCDs与gQDs-apt1、rQDs-apt2三者荧光不会互相影响,为检测目标物提供可能性。
基于bCDs、gQDs、rQDs构建的荧光探针进行两种肿瘤标志物同步检测的结果,见图7。从图7可以看出,加入不同浓度的PTK7,530nm处的绿色荧光恢复,430nm处的蓝色参比信号保持不变(A图);加入不同浓度的miRNA-21640nm处的红色荧光恢复,430nm处的蓝色参比信号保持不变(B图);当两种目标物PTK7和miRNA-21同时加入后,530nm处的绿色荧光和640nm处的红色荧光恢复,蓝色参比信号保持不变(C图)。结果表明,基于bCDs、gQDs、rQDs构建的荧光探针可用于PTK7和miRNA-21的同步检测。
对两种目标物进行同步检测的相互干扰试验,见图8。将基于bCDs、gQDs、rQDs构建的荧光探针用于PTK7(10ng.mL-1)单独检测,同时掺杂大量的miRNA-21(10nM),结果表明,掺杂前、后对PTK7的检测荧光信号几乎无影响(A图);同理,将基于bCDs、gQDs、rQDs构建的荧光探针用于miRNA-21(1nM)单独检测,同时掺杂大量的PTK7(100ng.mL-1),结果表明,掺杂前、后对miRNA-21的检测荧光信号几乎无影响,两种检测荧光信号不会互相干扰(B图)。说明该荧光探针可用于PTK7和miRNA-21的同步检测。
4、Fe3O4-cDNA1、Fe3O4-cDNA2、gQDs-apt1和rQDs-apt2的性能表征
4.1、Fe3O4-cDNA1和Fe3O4-cDNA2的光谱性能表征
对互补链cDNA1和cDNA2是否成功连接在Fe3O4 MNPs上进行紫外和红外表征,见图9。图9中,从图A紫外吸收光谱图中可以看出,Fe3O4 MNPs有较宽的吸收峰,cDNA1和cDNA2在258nm处有一个特征吸收峰,连有互补链的Fe3O4-cDNA1和Fe3O4-cDNA2既有较宽的吸收峰,也有258nm处的特征吸收峰,说明cDNA1和cDNA2成功结合在Fe3O4 MNPs上。从图B红外谱图可以看出,590cm-1对应于Fe-O的伸缩振动,1408cm-1和1625cm-1分别为C-O和C=O的伸缩振动,3400cm-1为O-H的伸缩振动,说明Fe3O4MNPs表面有丰富的-COOH,有利于氨基化cDNA1和cDNA2通过酰胺键结合;Fe3O4-cDNA1和Fe3O4-cDNA2的红外谱图在1520cm-1和1625cm-1有峰,分别对应于C=O的伸缩振动和N-H的弯曲振动,表明cDNA1和cDNA2通过酰胺键结合在Fe3O4MNPs上,说明Fe3O4-cDNA1和Fe3O4-cDNA2的成功制备。
4.2、Fe3O4-cDNA1和Fe3O4-cDNA2的mapping表征
通过mapping对cDNA1和cDNA2结合在Fe3O4MNPs上进行表征,见图10和表1。图10中,图A、B、C分别为Fe3O4 MNPs、Fe3O4-cDNA1和Fe3O4-cDNA2的mapping结果,从图中可以看出,三者均含有大量的Fe、O元素,说明Fe3O4 MNPs的成功合成。而连有互补链的Fe3O4-cDNA1和Fe3O4-cDNA2,P含量较Fe3O4MNPs多,是因为cDNA含有P元素,通过酰胺连接将cDNA连接在Fe3O4 MNPs表面,从而将P元素引入Fe3O4MNPs表面。Mapping结果也表明Fe3O4-cDNA1和Fe3O4-cDNA2的成功制备。
表1 Fe3O4、Fe3O4-cDNA1、Fe3O4-cDNA2的原子分数
4.3、Fe3O4-cDNA1和Fe3O4-cDNA2的电势表征
从图11的zeta电位图可以看出,Fe3O4MNPs电位值为-10.21,在结合的负电荷的cDNA1和cDNA2后,Fe3O4-cDNA1和Fe3O4-cDNA2的电位分别变为-29.18和-26.19,说明cDNA的成功连接。
4.4、Fe3O4-cDNA1和Fe3O4-cDNA2的磁性表征
通过振动样品磁强计考察Fe3O4 MNPs连接互补链前后磁性是否发生变化,见图12。图12结果表明,Fe3O4 MNPs与Fe3O4-cDNA1和Fe3O4-cDNA2磁滞回线几乎重合,表现出超顺磁行为,表明cDNA的连接对Fe3O4 MNPs磁性几乎没有影响,Fe3O4 MNPs可在磁场作用下作为复杂基质的高效分离器。
4.5、gQDs-apt1和rQDs-apt2的电泳、红外表征
对apt1、apt2是否成功连接在gQDs和rQDs上进行电泳、红外表征,见图13。由图13的琼脂糖凝胶电泳图A、B可以看出,连有apt的gQDs-apt1、rQDs-apt2介于gQDs、rQDs和apt1、apt2之间,是因为gQDs-apt1、rQDs-apt2的分子量介于二者之间,说明apt1、apt2成功结合在gQDs、rQDs上。由图C红外也可以看出,1408cm-1和1625cm-1分别为C-O和C=O的伸缩振动,3400cm-1为O-H的伸缩振动,说明gQDs和rQDs表面还有丰富的COOH,有利于氨基化的apt与其通过酰胺键连接;gQDs-apt1和rQDs-apt2的红外谱图在1520cm-1和1625cm-1有峰,分别对应于C=O的伸缩振动和N-H的弯曲振动,表明apt1和apt2通过酰胺键分别结合在gQDs和rQDs上,从而说明gQDs-apt1和rQDs-apt2的成功制备。
4.6、gQDs-apt1和rQDs-apt2的电势表征
从图14的zeta电位图可以看出gQDs电位值为-8.20,在结合的负电荷的apt1后gQDs-apt1的电位分别变为-14.59,说明apt1的成功连接,同理,在连接了负电位的apt2后,rQDs的电位由-8.97变为-14.99,说明apt2的成功连接。
5、荧光探针用于PTK7和miRNA-21的检测
5.1、PTK7的检测
5.1.1、检测限
在最优反应条件下,将实施例1制备的荧光探针用于PTK7定量检测,见图15。由图15中图A可以看出,随着PTK7浓度的增大,530nm处的绿色荧光强度逐渐增强,说明该荧光探针对PTK7有良好的荧光响应;利用I530/I430比值可对PTK7浓度进行量化,并用比值对浓度对数做线性拟合,在0.5-100ng.mL-1范围内有良好的的线性关系,线性方程为I530/I430=0.31158lg[PTK7]+0.11617,R值为0.997,检测限按3σ/k计算为0.065ng.mL-1(其中σ为空白样品的标准偏差(n=10),k为校正曲线的斜率)。结果表明本发明实施例1所制备的荧光探针具有检测PTK7的能力。
5.1.2、检测的灵敏性
为了探究该荧光探针对PTK7检测的灵敏性,我们将本发应用实施例1的LOD与现有的PTK7检测方法的LOD进行了比较,结果如表2所示,该荧光探针相较于其他检测方法LOD更低,并且该探针制备更简便,操作更容易检测PTK7具有更大的优势。
表2应用实施例1的探针性能与现有的PTK7检测方法比较
5.1.3、准确度和精密度
对其检测PTK7的准确度和精密度进行研究。鉴于检测范围为0.5-100ng.mL-1,选取低中高三个浓度(0.75、7.5、80ng.mL-1),计算日内日间精密度和准确度。结果如表3所示,低、中、高三个浓度的日内日间准确度在98.2%-108.0%,符合生物样品的检测要求,日内日间精密度RSD<15%,均符合生物样品检测要求。结果表明,本发明实施例1构建的荧光探针可用于实际样品中PTK7的检测。
表3 PTK7测定的精密度和准确度
5.1.4、抗干扰能力
本发明实施例1所构建的荧光探针用于实际血清样品PTK7检测前,还需验证该荧光探针对血清中复杂基质的抗干扰能力和对PTK7的选择性。血清成分比较复杂,含有蛋白质、氨基酸、金属离子等,可能会对检测结果造成干扰。因此选择常见的基质和金属离子(K+、Fe3+、Mn2+、Ca2+、Cu2+、Glu、Gul、Arg、MB、BSA),对其选择性和抗干扰性进行研究。其中,PTK7浓度为100ng.mL-1,干扰物浓度为1000ng.mL-1。从图16可以看出,对照组(PTK7)与实验组(PTK7+干扰离子/干扰氨基酸)的I530/I430比值变化很小,说明干扰物对PTK7检测的影响小,可以忽略不计,进而说明该荧光探针有良好的抗干扰能力;再将对照组(PTK7)与实验组(MB、BSA)的I530/I430比值进行比较,可以看出该荧光探针对其荧光响应很小,说明该荧光探针对其他蛋白几乎不响应,对PTK7有良好的选择性,可将该荧光探针用于实际血清样品的检测。
5.2、miRNA-21的检测
5.2.1、检测限
在最优反应条件下,将实施例1制备的荧光探针用于miRNA-21定量检测。由图17的图A可以看出,随着miRNA-21浓度的增大,640nm处的红色荧光强度逐渐增强,说明该荧光探针对miRNA-21有良好的荧光响应;利用I640/I430比值可对miRNA-21浓度进行量化,并用比值对浓度对数做线性拟合,在0.1-10nM范围内有良好的的线性关系,线性方程为I640/I430=0.27171lg[miRNA-21]+0.31252,R值为0.991,检测限按3σ/k计算为0.051nM(其中σ为空白样品的标准偏差(n=10),k为校正曲线的斜率)。结果表明本发明实施例1所制备的荧光探针具有检测miRNA-21的能力。
5.2.2、检测的灵敏性
为了探究该荧光探针对miRNA-21检测的灵敏性,我们将应用实施例2的LOD与现有的miRNA-21检测方法的LOD进行了比较,结果如表4所示,该荧光探针相较于其他检测方法LOD更低,并且该探针制备更简便,操作更容易、检测目标物多样性,用于检测miRNA-21具有更大的优势。
表4将本发明实施例1的探针性能与现有的miRNA-21检测方法进行比较
5.2.3、精密度和准确度
并对其检测miRNA-21的准确度和精密度进行研究。鉴于检测范围为0.1-10nM,选取低中高三个浓度(0.2、1、8ng.mL-1),计算日内日间精密度和准确度。结果如表所示,低中高三个浓度的日日日间准确度在100.0%-107.0%,符合生物样品的检测要求,日内日间精密度RSD<15%,均符合生物样品检测要求。结果表明,本发明实施例1构建的荧光探针可用于实际样品中miRNA-21的检测。
表5 miRNA-21测定的精密度和准确度
5.2.4、抗干扰能力
本发明实施例1所构建的荧光探针用于实际血清样品miRNA-21检测前,还需验证该探针对血清中复杂基质的抗干扰能力和对miRNA-21的选择性。miRNA家族成员相似性高,能够区别miRNA家族成员对探针十分重要。选择与miRNA-21单碱基错配、双碱基错配以及三条不同RNA链进行选择性研究。其中,miRNA-21浓度为10nM,干扰物浓度为20nM。从图18可以看出,对照组(miRNA-21)与实验组的I640/I430比值差别很大,说明该荧光探针对其他RNA几乎不响应,对miRNA-21有良好的选择性,可将该荧光探针用于实际血清样品的检测。
5.3、PTK7和miRNA-21的同步检测
在最优反应条件下,将制备的荧光探针用于PTK7和miRNA-21检测。图19中A图是加入不同浓度PTK7和miRNA-21荧光变化图;B图是荧光强度比值(I530/I430)与lg[PTK7]的线性拟合曲线;C图是荧光强度比值(I640/I430)与lg[miRNA-21]的线性拟合曲线。由图A可以看出,随着PTK7和miRNA-21浓度的增大,530nm和640nm处的荧光强度逐渐增强,说明该荧光探针对PTK7和miRNA-21有良好的荧光响应;利用荧光强度比值(I530/I430)和(I640/I430)对PTK7和miRNA-21浓度分别进行量化,并用比值对浓度对数做线性拟合,在0.5-100ng.mL-1和0.1-10nM范围内有良好的的线性关系,PTK7的线性方程为I530/I430=0.30273lg[PTK7]+0.12908,R值为0.996,miRNA-21的线性方程为I640/I430=0.28532lg[miRNA-21]+0.34867,R值为0.995。结果表明本发明实施例1所制备的荧光探针具有检测PTK7和miRNA-21的能力。
6、血清中PTK7和miRNA-21的测定
选择采用标准加入法来测定人血清样品中PTK7和miRNA-21以消除基质干扰。人体血清中PTK7和miRNA-21回收率测定的结果见表6。如表6所示,血清中PTK7的加标回收率在98.9-104.4%范围内,RSD范围为1.5-5.5%,血清中miRNA-21的加标回收率在87.1-108.7%范围内,RSD范围为1.6-8.8%。由回收率实验结果可知,本方法的回收率和RSD均满足生物样品的检测要求,说明本方法准确可靠,可以用于实际样品测定。
表6人血清测定中PTK 7和miRNA-21的回收率
7、结论
本发明实施例1构建了一种基于碳点量子点的新型荧光探针bCDs/(Fe3O4-cDNA1-apt1-gQDs)/(Fe3O4-cDNA2-apt2-rQDs)实现对两种乳腺标志物PTK7和miRNA-21的同步检测,并将其应用于实际人血清样品的检测。选用猝灭能力良好的Fe3O4MNPs作为能量受体,选用两种荧光颜色的QDs作为能量供体,bCDs作为参比信号,通过单波长激发,可以同时监测两种荧光信号,且不存在光谱重叠现象,利用荧光信号与参比信号荧光比值,实现了PTK7和miRNA-21的同步检测。
以上仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,其特征在于,包括以下步骤:
步骤1,制备羧化四氧化三铁纳米颗粒
以三氯化铁、乙酸钠和乙二醇为原料,通过水热法合成四氧化三铁纳米颗粒,然后加入到柠檬酸溶液中,合成羧化四氧化三铁纳米颗粒;
步骤2,制备羧化四氧化三铁纳米颗粒与氨基化适配体互补链的复合物将羧化四氧化三铁纳米颗粒在EDC/NHS催化下,分别与两个氨基化适配体互补链通过酰胺键联结,合成Fe3O4-cDNA1复合物和Fe3O4-cDNA2复合物;
步骤3,制备绿色荧光CdSe/ZnS量子点与氨基化适配体的复合物
将羧基修饰的绿色荧光CdSe/ZnS量子点在EDC/NHS催化下,与氨基化适配体apt1通过酰胺键联结,合成gQDs-apt1复合物;
步骤4,制备红色荧光CdSe/ZnS量子点与氨基化适配体的复合物
将羧基修饰的红色荧光CdSe/ZnS量子点在EDC/NHS催化下,与氨基化适配体apt2通过酰胺键联结,合成rQDs-apt2复合物;
步骤5,制备荧光探针
将步骤2的Fe3O4-cDNA1复合物与步骤3的gQDs-apt1复合物通过碱基互补配对形成Fe3O4-cDNA1-apt1-gQDs;
将步骤2的Fe3O4-cDNA2复合物与步骤4的rQDs-apt2复合物通过碱基互补配对形成Fe3O4-cDNA2-apt2-rQD;
将Fe3O4-cDNA1-apt1-gQDs和Fe3O4-cDNA2-apt2-rQD混合,然后加入蓝色荧光碳点,得到荧光探针
bCDs/(Fe3O4-cDNA1-apt1-gQDs)/(Fe3O4-cDNA2-apt2-rQD)。
2.根据权利要求1所述的用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,其特征在于,步骤1中,水热法的条件为:反应温度200℃,反应时间4~8h。
3.根据权利要求1所述的用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,其特征在于,步骤1中,三氯化铁和乙酸钠的质量比为0.54:1.44;
柠檬酸与四氧化三铁纳米颗粒的质量比为5:0.2。
4.根据权利要求1所述的用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,其特征在于,步骤2中,羧化四氧化三铁纳米颗粒、EDC和NHS的质量比为0.2:19:22;羧化四氧化三铁纳米颗粒与任意一个氨基化适配体互补链的用量比为0.2mg:10μL。
5.根据权利要求1所述的用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,其特征在于,步骤3中羧基修饰的绿色荧光CdSe/ZnS量子点、EDC和NHS的质量比为0.2:19:22;
羧基修饰的绿色荧光CdSe/ZnS量子点与氨基化适配体apt1的用量比为0.2mg:10μL。
6.根据权利要求1所述的用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,其特征在于,步骤4中,羧基修饰的红色荧光CdSe/ZnS量子点、EDC和NHS的质量比为0.2:19:22;
羧基修饰的红色荧光CdSe/ZnS量子点与氨基化适配体apt2的用量比为0.2mg:10μL。
7.根据权利要求1所述的用于同步检测PTK7和miRNA-21的比率荧光探针的制备方法,其特征在于,步骤5中,Fe3O4-cDNA1复合物与gQDs-apt1复合物的体积比为1:1;Fe3O4-cDNA2复合物与rQDs-apt2复合物的体积比为1:1;
Fe3O4-cDNA1-apt1-gQDs、Fe3O4-cDNA2-apt2-rQD和蓝色荧光碳点的体积比为1:1:1。
8.一种采用权利要求1所述的方法制备得到的用于同步检测PTK7和miRNA-21的比率荧光探针。
9.一种权利要求8所述的用于同步检测PTK7和miRNA-21的比率荧光探针的应用,其特征在于,所述荧光探针应用于PTK7和miRNA-21的同步检测。
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