CN114113013B - 一种荧光适配体探针及其在microRNA/ctDNA核酸分子检测中的应用 - Google Patents
一种荧光适配体探针及其在microRNA/ctDNA核酸分子检测中的应用 Download PDFInfo
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Abstract
本发明公开了一种荧光适配体探针及其在microRNA/ctDNA核酸分子检测中的应用。所述荧光适配体探针Dye‑dsDNA‑Aps的结构为：在microRNA或ctDNA的适配体（Ap）上连接附加链dsDNA，并在附加链dsDNA的双链结构中嵌入大量荧光分子（Dye）。Dye‑dsDNA‑Aps中的Aps端可通过氢键和π‑π键等相互作用，吸附于纳米猝灭剂表面，并导致其Dye‑dsDNA端发生荧光猝灭；当目标microRNA或ctDNA存在时，Dye‑dsDNA‑Aps中的Aps端与microRNA或ctDNA形成双链结构，进而导致双链结构的Dye‑dsDNA‑Aps从纳米荧光淬灭剂表面释放，并使Dye‑dsDNA端的荧光信号复燃。本发明操作方便，具有高灵敏性特点，在核酸检测应用方面具有良好的前景。
Description
技术领域
本发明属于核酸检测技术领域，具体涉及一种用于检测microRNA/ctDNA核酸分子的荧光适配体探针。
背景技术
循环游离核酸（包括循环肿瘤DNA和microRNA），已被证实密切相关许多生命过程，包括细胞增殖、分化、衰老和凋亡。因此，循环游离核酸可作为各种疾病（如癌症、神经退行性疾病、糖尿病和免疫系统病理学）的诊断和预后生物标志物。例如，miR-21与肿瘤的增殖、凋亡和侵袭有关，已被用作肿瘤治疗和诊断的生物标志物；持续上调的miR-155不仅导致持续的炎症反应，又可促进肿瘤的发生。而该类核酸物质在血液系统中的表达水平普遍很低。到目前为止，已经开发出检测 miRNA 的技术，包括聚合酶链反应 (PCR)、微阵列分析和使用放射性标记探针的RNA印迹分析。然而，由于miRNA序列短，序列相似度高，表达水平低，miRNA 的检测仍然面临挑战。开发简单、灵敏、特异的microRNA和ctDNA定量检测方法仍然是一个挑战。
对于miRNA/ctDNA的荧光传感，核酸适配体通常用于设计基于纳米荧光淬灭剂的荧光传感器。荧光团标记的核酸适配体通过静电或π-π相互作用吸附在纳米荧光淬灭剂上，导致荧光团的荧光猝灭（信号“关闭”）；在存在目标miRNA/ctDNA的情况下，由于双链寡核苷酸的形成，适体从纳米荧光淬灭剂中释放出来，从而允许荧光团的荧光恢复（信号“开启”）。但是，由于通常情况下设计的核酸适配体探针只标记一个荧光基团，所以其荧光强度较弱，不利于检测。
发明内容
本发明的目的是提供了一种用于检测microRNA/ctDNA核酸分子的荧光适配体探针（Dye-dsDNA-Aps），通过在microRNA或ctDNA的适配体上连接附加链dsDNA，由于附加链dsDNA可以嵌入大量荧光分子，从而增强适配体探针的荧光强度。
为了实现上述发明目的，本发明采用以下技术方案：
一种荧光适配体探针，包括核酸适配体和附加链dsDNA，所述附加链dsDNA连接在核酸适配体的一端或两端；
所述附加链dsDNA的双链结构内嵌入有荧光分子。
进一步地，所述附加链dsDNA为碱基数目在5-20之间的双链DNA。
更进一步地，所述附加链dsDNA为5′-3′TCTCAGAG、5′-3′TCTCAGAGCG或5′-3′CCTCTCAGAG。
进一步地，所述荧光分子选自SYBR Green I、TOTO-1、YoYo-1或吖啶橙。
上述荧光适配体探针在microRNA或ctDNA检测中的应用，所述应用为非疾病诊断目的。
进一步地，所述应用的具体方法为：将荧光适配体探针与纳米荧光猝灭剂组装后，加至含有待测microRNA/ctDNA的样品中，混合5-10 min后离心，通过分析上清液荧光强度，实现microRNA/ctDNA的高灵敏检测。
进一步地，所述纳米荧光猝灭剂选自氧化石墨烯、金纳米颗粒或金属有机框架材料。
本发明通过将microRNA或ctDNA的适配体连接上附加链dsDNA，并将荧光分子（如SYBR Green I等）嵌入到该dsDNA的双链结构中。所制备的荧光适配体探针（Dye-dsDNA-Aps）可与荧光猝灭剂（如氧化石墨烯、金纳米颗粒和金属有机框架材料等）组装，作为microRNA或ctDNA的荧光传感器。首先，Dye-dsDNA-Aps吸附于荧光猝灭剂表面，并发生荧光猝灭；在相应的microRNA或ctDNA存在下，Dye-dsDNA-Aps中的适配体部分形成双链DNA结构，导致Dye-dsDNA-Aps从荧光猝灭剂表面释放，并使荧光复燃。该荧光信号变化过程，可用于microRNA或ctDNA的体外检测。结果显示，该方法中设计的Dye-dsDNA-Aps荧光适配体探针具有较高的灵敏度，为体液中microRNA或ctDNA的高灵敏检测提供了新的方法。
本发明的有益效果在于：
（1）本发明的microRNA检测方法简单，且成本低。
（2）本发明的荧光适配体探针Dye-dsDNA-Aps可标记不同发射波长的荧光分子，对多种microRNA和/或ctDNA进行检测。
（3）本发明的荧光适配体探针Dye-dsDNA-Aps灵敏度较高，该探针的荧光强度取决于dsDNA片段的长度和嵌入的荧光分子的数量。
附图说明
图1为本发明荧光适配体探针的检测原理图。
图2为本发明实施例1中SYBR-dsDNA-Aps+ AuNCs@ZIF-8检测miR-21的原理。
图3 为实施例1中AuNCs和AuNCs@ZIF-8材料的扫描电镜图。其中：(a)为AuNCs，(b)为AuNCs@ZIF-8。
图4为实施例1中SYBR-dsDNA-Aps+AuNCs@ZIF-8用于检测miR-21的荧光光谱图（FI为荧光强度）。
图5为实施例1中SYBR-dsDNA-Aps+AuNCs@ZIF-8对miR-21的检测的线性关系。
图6为实施例1中 SYBR-dsDNA-Aps+AuNCs@ZIF-8对miR-21检测的选择性。
图7为实施例2中超声破碎后的Zn2Ph2Da晶体和Zn2Ph2Da-100材料的扫描电镜图。其中：C1为Zn2Ph2Da晶体，C2为Zn2Ph2Da-100材料。
图8为实施例3中SYBR-dsDNA-Aps’+ Zn2Ph2Da-100用于检测miR-155的荧光光谱图（FI为荧光强度）。
图9为实施例3中SYBR-dsDNA-Aps’+ Zn2Ph2Da-100对miR-155的线性关系。
图10为实施例3中SYBR-dsDNA-Aps’+ Zn2Ph2Da-100对血清中miR-155的线性关系。
具体实施方式
本发明通过设计荧光适配体探针Dye-dsDNA-Aps，与荧光猝灭剂组装后可作为microRNA或ctDNA的高灵敏度的荧光传感器。
所述荧光适配体探针Dye-dsDNA-Aps的结构为：在microRNA或ctDNA的适配体（Ap）上连接附加链dsDNA，并在附加链dsDNA的双链结构中嵌入大量荧光分子（Dye）。如图1所示，Dye-dsDNA-Aps中的适配体端Aps可通过氢键和π-π键等相互作用，吸附于纳米猝灭剂表面，并导致其Dye-dsDNA端发生荧光猝灭；当目标microRNA或ctDNA存在时，Dye-dsDNA-Aps中的Aps端与microRNA或ctDNA形成双链结构，进而导致双链结构的Dye-dsDNA-Aps从纳米荧光淬灭剂表面释放，从而Dye-dsDNA端的荧光信号复燃。由于Dye-dsDNA端的荧光信号可通过调节dsDNA的链长和嵌入其中荧光分子（Dye）的数量决定。因此，通过适当增加dsDNA的链长和嵌入其中荧光分子（Dye）的数量可得到高荧光发射强度的适配体探针（即Dye-dsDNA-Aps）。
具体地，所述荧光适配体探针包括microRNA或ctDNA的适配体连接上附加链dsDNA（如5′-3′: TCTCAGAG；5′-3′: TCTCAGAGCG；5′-3′: CCTCTCAGAG等碱基数目在5-20之间dsDNA），并将荧光分子嵌入到该dsDNA附加链中。所述的附加链dsDNA可以连接在microRNA或ctDNA适配体的一端或两端。所述荧光分子是SYBR Green I 、TOTO-1、YoYo-1或吖啶橙（AO）等。与传统的单个荧光基团标记的适配体探针相比，检测灵敏度逐渐增加。所制备的荧光适配体探针（Dye-dsDNA-Aps）可与荧光猝灭剂（如氧化石墨烯、金纳米颗粒和金属有机框架材料等）组装，作为microRNA或ctDNA的荧光传感器。在本发明中，所述荧光淬灭剂一种为以多孔材料沸石咪唑酯框架ZIF-8为平台，金纳米簇作为客体发光物质构建的AuNCs@ZIF-8荧光复合材料，另一种为2，2’-二硫代二苯甲酸（dtba）和1，10-菲罗啉（phen）为配体，Zn2+为金属中心，通过溶剂热方法制备的MOF（Zn2Ph2Da）。
在本发明的一个实施例中，选用荧光适配体探针SYBR-dsDNA-Aps吸附在AuNCs@ZIF-8材料表面，然后将SYBR-dsDNA-Aps+ AuNCs@ZIF-8用于miR-21检测。所述应用是在AuNCs@ZIF-8中先加入SYBR-dsDNA-Aps，再将其混合溶液加入待测miR-21样品中并进行荧光检测。具体地：将荧光适配体探针SYBR-dsDNA-Aps通过π-π 相互作用或静电吸附作用，吸附于AuNCs@ZIF-8表面，形成SYBR-dsDNA-Aps+ AuNCs@ZIF-8复合材料；当与样品中miR-21作用后，SYBR-dsDNA-Aps中Ap部分与miR-21形成双链结构，该结构不再吸附于AuNCs@ZIF-8表面，通过检测上清液中双链SYBR-dsDNA-Aps的荧光强度，可对miR-21进行定量。检测原理如图2所示。
同样地，在本发明的另一个实施例中，将荧光适配体探针SYBR-dsDNA-Aps’吸附在Zn2Ph2Da-100表面，然后用于miR-155的检测。
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
miR-21的荧光检测
（一）荧光传感材料的制备：
1. AuNCs@ZIF-8的制备
① 金纳米簇的合成
将 2.0 mL，40 mM 的氯金酸水溶液和 1.20 mL, 100 mM 谷胱甘肽水溶液与36.8 mL H2O 在 25℃下混合。在温和搅拌（500 rpm）下将溶液加热到 70℃ 24 h，然后冷却到室温。使用透析袋（3000 da 分子量截止）纯化，并在4℃下储存以供以后使用。
② AuNCs@ZIF-8 的制备
AuNCs@ZIF-8的制备：取5.6 mg的Zn（NO3）2·6H2O 溶于 80 μL的 H2O 溶液，加入1.0 mL 的金纳米簇水溶液，然后将 pH 调至 5.0，离心水洗得金纳米簇沉淀。将沉淀分散于 1.0 mL 甲醇中，与 Zn（NO3）2·6H2O（25 mM，7.5 mL）和 2-甲基咪唑（25 mM，7.5 mL）的甲醇溶液混合，室温静置 12 小时，离心收集得 AuNCs@ZIF-8，用甲醇洗涤数次，保存于甲醇溶液。
图3（a和b）为AuNCs纳米粒子和AuNCs@ZIF-8材料的透射电镜图，由电镜图可知AuNCs成功包裹在了ZIF-8中。
2. miR-21荧光适配体探针在AuNCs@ZIF-8上的组装
将标记荧光分子SYBR Green I的miR-21适配体（SYBR-dsDNA-Aps）（50 nM）（dsDNA：5′-3′: TCTCAGAG；5′-3′: CTCTGAGA）与AuNCs@ZIF-8在1 mL磷酸缓冲液中混合，15min后形成SYBR-dsDNA-Aps与AuNCs@ZIF-8形成的复合物（SYBR-dsDNA-Aps+AuNCs@ZIF-8）。
（二）miR-21的荧光检测
取SYBR-dsDNA-Aps+AuNCs@ZIF-8的分散液(0.2 mg·mL-1)，加入含miR-21的血清样品中，5 min后通过荧光法检测上清液荧光强度。
图4为检测miR-21的荧光光谱图观察到探针SYBR-dsDNA-Aps在524 nm处的荧光强度随miR-21浓度从5 pM到100 pM增加，而AuNCs@ZIF-8-2在647 nm处的荧光强度变化不明显；由图5可知，SYBR-dsDNA-Aps+AuNCs@ZIF-8对miR-21的检测具有较好的线性关系。
（三）检测方法的选择性
将SYBR-dsDNA-Aps+AuNCs@ZIF-8分散于磷酸缓冲液中（0.2 mg·mL-1），然后加入miR-21、R1（5′-3′: UAGCUCUUCAGACUGGAGUUGA, SEQ ID NO.1）R2（5′-3′:UAGCAAAUCAGGCUGAUGAAGA, SEQ ID NO.2）、牛血清白蛋白（BSA）、谷胱甘肽（GSH）、三磷酸腺苷（ATP）、三磷酸尿苷（UTP）、三磷酸鸟苷 （GTP）和三磷酸胞苷（CTP）等分子，按上述方法进行荧光检测。由图6可知，SYBR-dsDNA-Aps+AuNCs@ZIF-8对miR-21具有专一性。
实施例2
miR-155的荧光检测
（一）荧光传感材料的制备：
1．Zn2Ph2Da晶体的合成。
将dtba（0.1mmol）、phen（0.2mmol）和ZnAc·2H2O（0.2mmol）混合分散于20mL乙醇中。然后向混合物中添加120 μLNaOH溶液（0.7 M），并将其转移到聚四氟乙烯高压釜中200℃下加热24小时，用乙醇和水分别洗涤三次，离心收集沉淀。
2.Zn2Ph2Da-100的制备。
将dtba（0.1 mmol）、phen（0.2 mmol）、ZnAc·2H2O（0.2 mmol）和NaOH（0.1 mmol）分散于20 mL乙醇中。然后将混合物在聚四氟乙烯高压釜中100℃下加热10小时。用乙醇洗涤三次，离心收集沉淀。
图7（C1和C2）为超声破碎后的Zn2Ph2Da晶体和Zn2Ph2Da-100材料的扫描电镜图。
3. miR-155荧光适配体探针在Zn2Ph2Da-100上的组装
将标记荧光分子SYBR Green I的miR-155适配体（SYBR-dsDNA-Aps’）（20 nM）（dsDNA：5′-3′: TCTCAGAG；5′-3′: CTCTGAGA）与Zn2Ph2Da-100（0.05 mg·mL-1）在磷酸缓冲液中混合，5 min后形成SYBR-dsDNA-Aps’与Zn2Ph2Da-100形成的复合物（SYBR-dsDNA-Aps’+Zn2Ph2Da-100）。
（二）miR-155的荧光检测
取SYBR-dsDNA-Aps’+ Zn2Ph2Da-100的分散液，加入含miR-155的血清样品中，5min后通过荧光法检测上清液荧光强度。
图8为检测miR-155的荧光光谱图，SYBR-dsDNA-Ap’探针在524 nm处的荧光强度随miR-155浓度的增加而恢复。而Zn2Ph2Da-100在366 nm处的荧光强度大致保持不变；由图9可知，SYBR-dsDNA-Aps’+ Zn2Ph2Da-100对miR-155的检测具有较好的线性关系；图10为血清中检测miR-155的线性关系。
序列表
<110> 中国药科大学
<120> 一种荧光适配体探针及其在microRNA/ctDNA核酸分子检测中的应用
<130> 20211112
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Claims (6)
1.一种荧光适配体探针，其特征在于：包括核酸适配体和附加链dsDNA，所述附加链dsDNA连接在核酸适配体的一端或两端；
所述附加链dsDNA的双链结构内嵌入有荧光分子；
所述附加链dsDNA为碱基数目在5-20之间的双链DNA。
2.根据权利要求1所述的荧光适配体探针，其特征在于：所述附加链dsDNA为5′-3′TCTCAGAG、5′-3′TCTCAGAGCG或5′-3′CCTCTCAGAG。
3. 根据权利要求1所述的荧光适配体探针，其特征在于：所述荧光分子选自SYBRGreen I、TOTO-1、YoYo-1或吖啶橙。
4.权利要求1所述的荧光适配体探针在microRNA或ctDNA核酸分子检测中的应用，所述应用为非疾病诊断目的。
5. 根据权利要求4所述的应用，其特征在于：所述应用的具体方法为：将荧光适配体探针与纳米荧光猝灭剂组装后，加至含有待测microRNA或ctDNA的样品中，混合5-10 min后离心，通过检测上清液的荧光强度，实现microRNA或ctDNA的检测。
6.根据权利要求5所述的应用，其特征在于：所述纳米荧光猝灭剂选自氧化石墨烯、金纳米颗粒或金属有机框架材料。
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