CN111235229A - 一种用于检测ptk7的比率荧光探针及其制备方法 - Google Patents
一种用于检测ptk7的比率荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种用于检测PTK7的比率荧光探针及其制备方法,探针采用Fe3O4纳米粒作为淬灭剂和磁性分离剂,在表面交联PTK7适配体(APT)的互补链(cDNA)以特异性结合黄色荧光发射碳点(y‑CDs)标记的APT并淬灭其荧光,PTK7与cDNA竞争结合APT使其脱离Fe3O4纳米粒恢复荧光,并通过荧光内滤效应淬灭蓝色荧光发射碳点(b‑CDs),形成比率荧光检测PTK7。核酸外切酶I增大y‑CDs与b‑CDs的荧光比值,两者荧光强度的比值I580/I460与待测物浓度log10呈线性关系,根据两者荧光强度的比值,定量测定PTK7生物分子,灵敏度高、检测简便,具有免疫反应的高选择性、高亲和力。
Description
技术领域
本发明属纳米材料、荧光比率技术和生物分析检测领域,具体是基于荧光内滤效应和酶切循环放大技术,将黄色荧光的碳点和蓝色荧光的碳点混合,发生内滤效应而使蓝色荧光淬灭,形成比率荧光探针定量生物分子,用于检测PTK7的比率荧光探针,灵敏度高于紫外吸收,具有良好的选择性。
背景技术
据统计,肺癌已经成为中国癌症发病率排行榜第一位,其高发病率对早期诊断技术的革新提出了新的要求。血清中肿瘤标志物检测以其创伤小、灵敏度高的特性,在早期诊断中展现出重要作用。荧光检测方法作为一种简单、灵敏的检测方法,在临床检验中得到广泛应用,新型荧光纳米材料的发展拓宽了荧光检测的应用范围。比率荧光探针通过两个荧光信号的比值表示待测物的含量,克服单荧光探针易受干扰的缺陷,同时放大检测信号。酪氨酸蛋白激酶7(PTK7)是一种膜蛋白,与肺癌的发生发展具有直接相关性,其在血清中的含量测定对于肺癌诊断具有指征意义,可以作为一种肿瘤标志物。但是PTK7在肺癌发生早期血清含量低,为提高探针的灵敏度,使用酶促循环放大荧光信号。
适配体是人工合成的短的单链DNA或折叠成独特的三维形状的RNA寡核苷酸。与抗体相比,这些结构能够以高亲和力、高选择性和特异性靶向分子。相对于抗体,小尺寸和相当简单的结构使得它们易于合成和化学修饰。而且,除此之外,它们显示出较低的免疫原性。因此,适配体已经成为临床医学中检测和分离蛋白质以及充当靶向剂和治疗剂的新分子工具。已经合成了DNA适配体序列sgc8以特异性识别PTK7,其中已知其与PTK7结合具有高亲和力。已经发现PTK7在多种其他癌症类型中过表达,包括肺癌和结肠癌,前列腺癌,淋巴结癌和乳腺癌。
作为一类新型的荧光纳米材料,碳点(C-dots)因其优异的特性(例如出色的光稳定性和低碳性)而引起了极大的关注。由于其出色的物理和化学特性、低毒性、优异的生物相容性和良好的水溶性,碳点已用于构建检测蛋白质、金属离子和其它小分子的新型检测方法。然而,尽管具有优异的光学性能,但由于缺乏选择性而限制了荧光测定的发展。
发明内容
本发明的目的在于针对上面所述的问题,提出一种用于检测PTK7的比率荧光探针,该比率荧光探针基于荧光内滤效应和酶切循环放大技术,是基于在Fe3O4纳米粒表面交联PTK7适配体(APT)的互补链(cDNA)以特异性结合黄色荧光发射碳点(y-CDs)标记的APT的探针进行,具有灵敏度高、选择性高、检测简便、测定范围广的特点。
PTK7作为一种肿瘤标志物,被广泛应用于肿瘤的早期诊断(肺癌和结肠癌,前列腺癌,淋巴结癌和乳腺癌等)。因此,将该探针制备成比率荧光通用检测平台,可广泛应用于该类生物分子的检测。
该探针可用于生物样本中PTK7含量的测定,进而检测多种癌症,如肺癌和结肠癌,前列腺癌,淋巴结癌和乳腺癌等。而且将适配体引入到荧光测定中,克服碳点选择性低的缺点。
本发明的目的还在于提供一种用于检测PTK7的比率荧光探针的制备方法。
本发明的比率荧光探针具体说明如下:
采用Fe3O4纳米粒作为淬灭剂和磁性分离剂,在表面交联PTK7适配体(APT)的互补链(cDNA)以特异性结合黄色荧光发射碳点(y-CDs)标记的APT并淬灭其荧光;PTK7与cDNA竞争结合APT使其脱离Fe3O4纳米粒恢复荧光,并通过荧光内滤效应淬灭蓝色荧光发射碳点(b-CDs),形成比率荧光检测PTK7。
在体系中加入核酸外切酶I (DNase I),剪切与PTK7结合的y-CDs标记的APT,使PTK7再次游离,与cDNA竞争结合APT,增大y-CDs与b-CDs的荧光比值,其中b-CDs的荧光值在一定值维持不变,而y-CDs的荧光值随PTK7浓度的增加而提升,使该探针对目标物的检测灵敏度大幅度提升,使其具有更高的临床应用价值。
为了实现上述发明目的,本发明采用的技术方案为:
一种用于检测PTK7的比率荧光探针,步骤如下:
步骤1、制备y-CDs、b-CDs和Fe3O4纳米粒:三种纳米材料均采用一锅水热法合成。
(1)、蓝色荧光发射碳点(b-CDs)的制备如下:(“On-off-on_ fluorescent systemfor detection of Zn2+ in biological sample using quantum dots-carbon dotsratiometric nanosensor”,《Journal of Colloid and Interface Science》,516(2018)522-528)是以一水合柠檬酸、二乙烯三胺为原料,二者的摩尔比为1:1,以去离子水为溶剂,在180 ℃反应4 h后获得。
(2)、黄色荧光发射碳点(y-CDs)的制备如下:(Lu, W.; Jiao, Y.; Gao, Y., etal., Bright Yellow Fluorescent Carbon Dots as a Multifunctional SensingPlatform for the Label-Free Detection of Fluoroquinolones and Histidine. 《ACSAppl Mater Interfaces》 2018, 10 (49): 42915-42924),是以邻苯二胺、4-氨基丁酸为原料,二者的摩尔比为1:1,以去离子水为溶剂,180 ℃反应8 h。
(3)、Fe3O4纳米粒的合成如下,首先,乙酸钠、无水三氯化铁分别溶解于乙二醇中,在200 ℃反应8 h。然后,加入一水合柠檬酸,以水为溶剂,室温搅拌反应4 h。
步骤2、制备在Fe3O4纳米粒表面交联PTK7适配体(APT)的互补链(cDNA)以特异性结合黄色荧光发射碳点(y-CDs)标记的APT的探针;
步骤(2-1)、Fe3O4纳米粒表面交联PTK7适配体(APT)的互补链(cDNA)形成Fe3O4-cDNA复合物:
称取Fe3O4纳米粒0.20g,溶于1 mL PBS(pH7.2-7.4)缓冲液。之后,加入EDC 0.0192 g和NHS 0.0223 g,室温摇床30 min后调节pH7.5;将cDNA在4000 rpm下离心1 min后加入37μL PBS(pH7.2-7.4)缓冲液配制成100 μM,随后100 μM cDNA于95 ℃加热4 min,冰浴冷却4min,室温放置一段时间。
将5 μL的100 uM cDNA加入上述溶液中后室温摇床过夜。未反应的cDNA通过磁性分离。最终,重新分散于PBS(pH7.2-7.4)缓冲液中,4 ℃保存,形成Fe3O4-cDNA复合物。
步骤(2-2)、制备黄色荧光发射碳点(y-CDs)标记的APT:
首先,量取500 μL的黄色荧光发射碳点溶液(y-CDs)和500 μL的PBS(pH7.2-7.4)缓冲液,充分震摇使混匀。随后,加入EDC 0.0193 g和NHS 0.0226 g,室温摇床30 min后调节pH7.5;将APT在4000 rpm下离心1 min后加入25 μL PBS(pH7.2-7.4)缓冲液配制成100 μM,随后100 μM cDNA 于95 ℃加热4 min,冰浴冷却4 min,室温放置一段时间。将5 μL的100 μM APT加入上述溶液中后室温摇床过夜,得到y-CDs标记的APT(y-CDs-APT)。
步骤(2-3)、 探针的制备
取 等体积的Fe3O4-cDNA复合物和 y-CDs-APT在5 ℃摇床反应60 min,得到探针Fe3O4-cDNA-APT-y-CDs复合物。磁性分离并用PBS(pH7.2-7.4)缓冲液洗涤除去未反应的Fe3O4-cDNA复合物和y-CDs-APT。
步骤3、根据标准曲线法测定PTK7含量
步骤(3-1)、将不同浓度的PTK7和DNase I加入到步骤(2)的反应体系中,制备标准曲线;
PTK7与cDNA竞争结合探针(Fe3O4-cDNA-APT-y-CDs复合物)表面的APT使其脱离Fe3O4纳米粒,荧光由复合后的淬灭状态改变为恢复荧光,并通过荧光内滤效应淬灭蓝色荧光发射碳点(b-CDs),形成比率荧光检测PTK7。
另外,在体系中加入核酸外切酶I (DNase I)以及b-CDs,其中DNase I剪切探针(Fe3O4-cDNA-APT-y-CDs复合物)表面与PTK7结合的y-CDs标记的APT,使PTK7再次游离,与cDNA竞争结合APT;b-CDs的荧光值维持不变,而y-CDs的荧光值随PTK7浓度的增加而提升,因此增大y-CDs与b-CDs的荧光比值,形成比率荧光,在一定范围内,y-CDs与b-CDs的荧光比值随生物分子浓度增大而增强,而且y-CDs(I580)与b-CDs(I460)的荧光比值I580/I460与PTK7的浓度呈线性关系,通过测定y-CDs(I580)与b-CDs(I460)的荧光比值I580/I460。
步骤(3-1)、根据标准曲线法,通过检测荧光,获得样品中PTK7的含量。
有益效果
本发明建立了一个对PTK7灵敏传感的比率荧光探针,该探针以两种荧光发射的荧光纳米材料b-CDs和y-CDs作为信号来源,以四氧化三铁纳米粒(Fe3O4)为磁性分离材料和荧光淬灭剂,通过适配体与PTK7之间的高度亲和力,使Fe3O4表面的y-CDs荧光恢复。并通过加入DNase Ι循环放大检测信号,使该探针具有高灵敏度和高选择性。b-CDs和y-CDs之间相互作用推断为内滤效应,并无能量转移现象发生;其中b-CDs被淬灭时,荧光值在一定值维持不变,而y-CDs的荧光值随PTK7浓度的增加而提升。该探针在体系中存在不同浓度的PTK7时,呈现580 nm荧光发射峰增强、460 nm处荧光发射峰减弱,以I580/I460与PTK7的浓度进行相关性拟合,定量体系中的浓度。具有较高的灵敏度。
附图说明
图1是实施例1制备得到的y-CDs的紫外吸收和荧光发射图;如图所示,y-CDs在280nm和430 nm处有紫外吸收,后者的吸收较弱;激发为380 nm,其荧光发射为560 nm。
图2是实施例1制备得到的b-CDs的紫外吸收和荧光发射图;如图所示,b-CDs在360nm处有紫外吸收;激发为380 nm,荧光发射为460 nm。
图3是实施例1制备得到的b-CDs的紫外吸收和y-CDs荧光发射图;如图所示,b-CDs的紫外吸收和y-CDs荧光发射图谱有一定程度的重叠,说明b-CDs的荧光可部分被y-CDs淬灭。
图4是实施例2制备得到的Fe3O4-cDNA复合物的紫外吸收图;如图所示,Fe3O4纳米粒表面交联PTK7适配体(APT)的互补链(cDNA)的紫外吸收图具有Fe3O4纳米粒的广泛吸收和cDNA在280 nm处的紫外吸收,证明制备成功。
图5是实施例2制备得到的y-CDs-APT的凝胶电泳图;如图所示,左边代表黄色荧光发射碳点(y-CDs)标记的APT,右边代表y-CDs;y-CDs标记的APT比y-CDs迁移的慢,证明APT成功结合y-CDs。
图6是实施例3中加入不同浓度的PTK7后的I580/I460比值变化趋势图;如图所示,在0-200 ng mL-1范围内,随着PTK7浓度的增加,y-CDs的荧光强度增大,b-CDs的荧光强度稍微降低,其I580/I460比值逐渐增大。
图7是实施例3制备得到的I580/I460比值与log10([PTK7])的线性关系图;如图所示,在0.1-100 ng mL-1范围内,I580/I460比值与log10([PTK7])成线性相关,其线性方程是y=0.92258+0.46653x,R2=0.98442。
具体实施方式
以下将结合具体实施例说明本发明的技术方案:
以下实施例所采用试剂的说明:一水合柠檬酸、二乙烯三胺、丙酮均从国药集团化学试剂有限公司购得;邻苯二胺、4-氨基丁酸、EDC、NHS从上海阿拉丁生化科技股份有限公司购得;乙酸钠、无水三氯化铁、乙二醇从国药集团化学试剂有限公司获得;酪氨酸蛋白激酶(PTK7)重组蛋白(武汉优尔生商贸有限公司);核酸外切酶I(10 U/μL)、PTK7适配体(APT)和互补链(cDNA)从生工生物工程(上海)股份有限公司购得。
实施例1
制备y-CDs、b-CDs和Fe3O4纳米粒,步骤如下:
b-CDs是取1.2 g一水合柠檬酸与600 μL二乙烯三胺溶解于20 mL去离子水中,超声20min使其充分溶解;将上述溶液转移至30 mL高压反应釜内,180 ℃反应4h;反应完成后,将其冷却至室温,取出产物交替加入乙醇、丙酮直至析出沉淀,8000 rpm离心10 min,弃去上清液、固体60 ℃真空干燥得到浅黄色粉末,封闭保存备用得到b-CDs。其TEM图如图2所示,其荧光与紫外吸收图如图2。
y-CDs是以邻苯二胺(0.32g)、4-氨基丁酸(0.31g)为原料,加入去离子水20 mL,超声20 min溶解,将上述溶液转移至30 mL高压反应釜内,180 ℃反应8 h;反应完成后,将其冷却至室温,得到黄褐色溶液,紫外吸收和荧光发射图见图1所示。
Fe3O4纳米粒的合成如下:首先,1.5 g乙酸钠、0.5 g无水三氯化铁分别溶解于10mL乙二醇中,超声溶解后将乙酸钠溶液缓慢滴加到三氯化铁溶液中;将上述溶液转移至30mL高压反应釜内,200 ℃反应8 h,将其冷却至室温,得到黑色溶液,溶液用乙醇、水交替洗涤,磁性分离后固体在60 ℃真空干燥。然后,取0.1 g固体加入2.1 g一水合柠檬酸、20 mL水,室温搅拌反应4 h,溶液用乙醇、水交替洗涤,磁性分离后固体在60 ℃真空干燥,得到Fe3O4纳米粒。
实施例2
制备在Fe3O4纳米粒表面交联PTK7适配体(APT)的互补链(cDNA)以特异性结合黄色荧光发射碳点(y-CDs)标记的APT的探针,步骤如下:
步骤一、y-CDs标记的APT(y-CDs-APT)的制备
步骤(1),量取500 μL实施例1制备的黄色碳点溶液(y-CDs)和500 μL的PBS(pH7.2-7.4)缓冲液。
步骤(2),加入EDC 0.0193 g和NHS 0.0226 g,室温摇床30 min后用1M NaOH调节pH7.5;
步骤(3)、将APT在4000 rpm下离心1 min后加入25 μL PBS(pH7.2-7.4)缓冲液配制成100 μM,随后100 μM cDNA 于95 ℃加热4 min,冰浴冷却4 min,室温放置一段时间。
步骤(4)、将5 μL的100 μM APT加入步骤(2)的溶液中后室温摇床过夜,得到y-CDs标记的APT(y-CDs-APT),结果如图5所示。
步骤二、制备Fe3O4纳米粒表面交联PTK7适配体(APT)的互补链(cDNA)(Fe3O4-cDNA复合物),结果如图4所示。
步骤三、取250 μL Fe3O4-cDNA复合物和250 μLy-CDs-APT反应得到探针Fe3O4-cDNA-APT-y-CDs复合物,反应体系是5 ℃反应60 min。
实施例3 标准曲线的制备PTK7:
步骤如下:取600 μL不同浓度的PTK7(0、0.1、0.2、0.5、1.0、2.0、5.0、10、20、50、100、200 ng mL-1),各加入20 U的DNase I,反应体系是在37 ℃孵育30 min。加入20 μL b-CDs,充分震荡混合。每个浓度平行操作三份,荧光测定激发波长为380 nm,y-CDs发射波长580nm,b-CDs发射波长460 nm,以y-CDs(I580)与b-CDs(I460)的荧光比值I580/I460与PTK7的浓度相关关系定量其浓度。结果见图6、7所示。
实施例4 血清样品中PTK7的检测方法:
步骤如下:使用标准加入法检测人血清中的PTK7含量。取5 μL不同浓度的PTK7溶液加入到600 μL血清样本中,使其加入浓度分别为0.1、0.2、0.3 ngmL-1,其它步骤按照“实施例3”操作,测定血清中PTK7的含量。结果见表1所示:
表1 健康人血清样本中PTK7含量的测定(n=3)
根据表1可知,健康人血清样本低于LOD,PTK7的回收率在90.23 – 96.42%,且RSD小于6.7%,符合生物样品检测要求。以上结果证实了本发明所合成的探针可应用于临床血清样本的检测。
Claims (7)
1.一种用于检测PTK7的比率荧光探针,其特征在于,是采用Fe3O4纳米粒作为淬灭剂和磁性分离剂,在Fe3O4纳米粒表面交联PTK7适配体(APT)的互补链(cDNA)以特异性结合黄色荧光发射碳点y-CDs标记的APT并淬灭其荧光;PTK7与cDNA竞争结合APT使其脱离Fe3O4纳米粒恢复荧光,并通过荧光内滤效应淬灭蓝色荧光发射碳点b-CDs,形成比率荧光检测PTK7。
2.权利要求1所述的一种用于检测PTK7的比率荧光探针的制备方法,其特征在于步骤如下:
步骤(1)、分别制备黄色荧光发射碳点y-CDs、蓝色荧光发射碳点b-CDs和Fe3O4纳米粒;
步骤(2)、在Fe3O4纳米粒表面交联PTK7适配体的互补链cDNA形成Fe3O4-cDNA复合物;
步骤(3)、制备黄色荧光发射碳点y-CDs标记的APT,获得y-CDs-APT复合物;
步骤(4)、等体积的 Fe3O4-cDNA复合物和 y-CDs-APT在5 ℃摇床反应60 min,得到探针Fe3O4-cDNA-APT-y-CDs复合物;
步骤(5)、将DNase I、b-CDs以及不同浓度的PTK7加入步骤(4)的反应体系中;PTK7与cDNA竞争结合Fe3O4-cDNA-APT-y-CDs复合物表面的APT使其脱离Fe3O4纳米粒,荧光由复合后的淬灭状态改变为恢复荧光;通过荧光内滤效应淬灭蓝色荧光发射碳点(b-CDs),b-CDs被淬灭时荧光值维持不变,而y-CDs的荧光值随PTK7浓度的增加而提升,y-CDs与b-CDs的荧光比值增大,在一定范围内,y-CDs与b-CDs的荧光比值随生物分子浓度增大而增强,而且y-CDs(I580)与b-CDs(I460)的荧光比值I580/I460与PTK7的浓度呈线性关系,
步骤(6)、通过测定生物样品中y-CDs(I580)与b-CDs(I460)的荧光比值I580/I460获得PTK7的浓度。
3.根据权利要求2所述的用于检测PTK7的比率荧光探针的制备方法,其特征在于步骤(1)中,蓝色荧光发射碳点b-CDs的制备是以一水合柠檬酸、二乙烯三胺为原料,二者的摩尔比为1:1,以去离子水为溶剂,在180 ℃反应4 h后获得。
4.根据权利要求2所述的用于检测PTK7的比率荧光探针的制备方法,其特征在于步骤(1)中,黄色荧光发射碳点y-CDs的制备是以邻苯二胺、4-氨基丁酸为原料,二者的摩尔比为1:1,以去离子水为溶剂,180 ℃反应8 h。
5.根据权利要求2所述的用于检测PTK7的比率荧光探针的制备方法,其特征在于步骤(1)中,Fe3O4纳米粒的合成如下,首先,乙酸钠、无水三氯化铁分别溶解于乙二醇中,在200℃反应8 h;
然后,加入一水合柠檬酸,以水为溶剂,室温搅拌反应4 h。
6.根据权利要求2所述的用于检测PTK7的比率荧光探针的制备方法,其特征在于步骤(5)中DNase I的加入浓度为20 U,PTK7的加入体积是600 μL;
反应温度为37 ℃,反应时间为30 min。
7.根据权利要求2所述的用于检测PTK7的比率荧光探针的制备方法,其特征在于步骤(5)中:b-CDs的加入量是20 μL。
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CN116333721A (zh) * | 2023-02-16 | 2023-06-27 | 徐州医科大学科技园发展有限公司 | 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 |
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