CN109444098A - 一种基于循环扩增技术和羧基碳量子点的荧光生物传感器及其制法和应用 - Google Patents
一种基于循环扩增技术和羧基碳量子点的荧光生物传感器及其制法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于酶催化靶标循环扩增技术并结合羧基功能化碳量子点的荧光生物传感器及其制法和应用。本发明的技术方案是以肿瘤相关基因miRNA34c为燃料分子，设计了一种由无标记的茎环结构1(HP1)和茎环结构2(HP2)和驱动引物(DP)组成的多轮驱动型DNA纳米机器，从而对痕量靶标进行循环放大。同时，我们合成生物相容性良好的羧基碳量子点(cCQD)首先与荧光探针结合后使得荧光基团淬灭，将由靶标刺激循环扩增过程的S1循环产物加入，室温下放置20min，致使荧光恢复，然后测定混合物的荧光强度，实现对目标物miRNA34c的灵敏检测。该研究思路为实现miRNA34c的灵敏检测提供了新的策略，有望用于肿瘤的早期的诊断中。
Description
技术领域：
本发明涉及一种基于酶催化靶标循环扩增技术并结合羧基功能化的碳量子点荧光探针的生物传感器；本发明还涉及所述生物传感器的制备方法及其检测miRNA的分析应用。
背景技术：
MicroRNAs(miRNAs)是一类内源性的非编码RNA小分子(20～23nt)，通过翻译抑制或通过与目标信使RNA形成RNA诱导沉默复合物(RISC)的靶向降解，miRNAs在多种生物学中的增殖、发育、新陈代谢、免疫反应、肿瘤和病毒感染等生理过程产生着至关重要的影响[Lee I,Ajay S S,Chen H,et al.Nucleic Acids Research,2008,36(5):e27.]。由于miRNA丰度低且易降解，直接检测只能是痕量检测，因此，迫切需要开发灵敏、高选择性、成本效益高和快速的核酸检测系统[Li H,Zhang Y,Wang L,et al.ChemicalCommunications,2011,47(3):961-963.][Qiang W,Li W,Li X,et al.Chemical Science,2014,5(8):3018-3024.][Gresham D,Ruderfer D M,Pratt S C,et al.Science,2006,311(5769):1932-1936.]。
近年来，由于荧光传感器所具有的固有优点，包括操作简单、灵敏度高、成像性能好等[Yuan L,Lin W,Zheng K,et al.Chemical Society Reviews,2013,42(2):622-661.][Yang Y,Zhao Q,Feng W,et al.Chemical Reviews,2012,113(1):192-270.]，在核酸检测方面取得了相当大的进展。值得注意的是，这一类的荧光传感器通常是基于荧光共振能量转移(FRET)或用于检测的淬灭机制[Ray P C,Darbha G K,Ray A,et al.Plasmonics,2007,2(4):173-183.][Li H,Zhang Y,Luo Y,et al.Small,2011,7(11):1562-1568.]，它们通常由一个荧光团和一个位于DNA探针分子相反端的淬灭分子组成。在没有目标存在的情况下，荧光团分子会和淬灭分子接近，荧光被抑制，当与目标分子发生杂交时，淬灭分子与荧光团分子分离距离变远，致使荧光信号被释放出来。然而，尽管荧光传感器在多种应用方面被广泛利用[Holland P M,Abramson R D,Watson R,et al.Proceedings of theNational Academy of Sciences,1991,88(16):7276-7280.][Tyagi,S.；Kramer,F.R.Biotechnol.1996,14,303-308.][Yang C J,Medley C D,Tan W.Currentpharmaceutical biotechnology,2005,6(6):445-452.]，传感器的设计仍然存在缺点，需要挑选荧光团和与之对应的淬灭剂以确保最佳检测效率。近年来纳米材料经常被用作淬灭器，因为它们能够淬灭不同发射波长的各种荧光基团，提高信噪比[Yang R,Tang Z,Yan J,et al.Analytical chemistry,2008,80(19):7408-7413.]。到目前为止，金纳米粒子[Dubertret B,Calame M,Libchaber A J.Nature biotechnology,2001,19(4):365.]，过渡金属双卤代烷[Zhu C,Zeng Z,Li H,et al.Journal of the American ChemicalSociety,2013,135(16):5998-6001.]，碳纳米管[Yang R,Jin J,Chen Y,et al.Journalof the American Chemical Society,2008,130(26):8351-8358.]，氧化石墨烯[Lu C H,Yang H H,Zhu C L,et al.Angewandte Chemie,2009,121(26):4879-4881.]以及金属-有机骨架等[Zhu X,Zheng H,Wei X,et al.Chemical Communications,2013,49(13):1276-1278.]已经被用作高效的纳米淬灭器来开发荧光传感器。虽然这些纳米材料已经成功地作为纳米淬灭剂用于核酸的检测，但其中一些纳米材料的制备往往是复杂而严格的，难以保证其中一些纳米材料具有良好的生物相容性[Liao K H,Lin Y S,Macosko C W,et al.ACSapplied materials&interfaces,2011,3(7):2607-2615.]。因此，需要寻找新的纳米材料作为替代物。
碳量子点(CQDs)作为一种新型的碳纳米材料，因其化学惰性和稳定性、可调谐激发和发射光谱、良好的生物相容性等优点而受到越来越多的关注[Loo AH,Sofer Z,D,et al.ACS applied materials&interfaces,2016,8(3):1951-1957.]。人们对碳量子点(CQD)的兴趣在于其优越而独特的性质，这些特性包括：功能化和制备容易，制备粒径小，生物相容性好，细胞毒性低，抗光漂白稳定性好。CQD的另一个重要特点是它们的高水溶性，从而使均相传感分析成为核酸检测的首选方法。
在本发明设计中，针对miRNA序列较短、单链和丰度低等特点，我们构建了DNA纳米分子机器，对目标进行循环放大并合成生物相容性良好的羧基碳量子点(cCQD)，作为荧光淬灭剂，用于核酸检测的荧光传感平台。本发明以肿瘤相关基因miRNA34c为燃料分子，设计了一种由无标记的茎环结构1(HP1)和茎环结构2(HP2)和驱动引物(DP)组成的多轮驱动型DNA纳米机器，从而对痕量靶标进行循环放大。同时，我们合成生物相容性良好的羧基碳量子点(cCQD)，首先与荧光探针结合后使得荧光基团淬灭，DNA与cCQD的相互作用是静电斥力和疏水π-π键相互作用之间的不断竞争的作用结果。在疏水作用大于静电斥力的条件下，ssDNA被吸附在cCQD表面上，反之，在静电斥力大于疏水作用的条件下，dsDNA就会远离cCQD表面。因此，cCQD表现出良好的检测范围和潜在的选择性。此外，CQD表面羧基的存在提供了多功能的锚定点，可以通过多种途径加以利用。
发明内容：
本发明的目的之一是提供一种新型的碳纳米材料cCQD作为探针，为检测提供淬灭信号来源。具体包括以下步骤：
羧基碳量子点的合成：称取6g柠檬酸放入带有盖子的干燥玻璃烧杯里，在烘箱中200℃下加热，直到白色固体颗粒完全融化呈淡黄色，再保持该温度15min，呈棕黄色，然后取出冷却至室温。在上述反应物中加入50ml 0.25mol NaOH溶液，玻璃棒搅拌，直至全部溶解，然后用0.05M NaOH溶液调节其pH值为6.0为止。然后使用截留分子量为1kDa的透析膜进行纯化透析3天后取出杂质后再使用，最后用真空蒸发器在40℃下将羧基碳量子点(cCQD)胶体溶液蒸发至原溶液的四分之一，即得到淡黄色羧基碳量子点溶液，置于4℃下避光保存备用。
本发明的目的之二是提供一种基于酶催化靶标循环扩增技术并结合羧基功能化的碳量子点荧光探针的生物传感器，以及利用该生物传感器检测miR34c的分析应用。它由以下步骤组成：
生物传感器的制备：
步骤1.传感器的构建：先在浓度为40nM FAM-probe荧光探针中加入40μL cCQD，在50℃下孵育30min。cCQD作为纳米淬灭剂，能够淬灭FAM-probe荧光探针上所携带的荧光基团，导致荧光淬灭。培养后，将由靶标刺激循环扩增过程的S1循环产物加入，室温下放置20min，致使荧光恢复，然后测定混合物的荧光强度。
本发明利用羧基功能化的碳量子点作为荧光信号淬灭探针，并采用酶催化靶标循环扩增技术研制了荧光生物传感器，使荧光恢复，成功实现了对miR34c的高灵敏、高选择性检测。该研究有望用于肿瘤早期诊断中。
本发明的荧光传感器表现出了优良的准确性、高灵敏性、高选择性、稳定性与重现性，分析检测迅速、方便，该生物传感器在生物医学分析检测和早期临床诊断中具有巨大的应用潜力，可用于实际样品的检测。
附图说明：
图1(A)cCQD的透射电子显微镜(TEM)图，(B)cCQD的荧光光谱图。
图2基于酶辅助靶标多重循环扩增原理图。
图3电泳表征：(a)Mark，(b)HP2，(c)DP，(d)target，(e)DP+HP2，(f)DP+HP2+target，(g)DP+HP2+target+HP1。
图4研究FAM-probe在不同实验条件下的荧光发射光谱：(a)Tris-HCl，(b)FAM-probe+cCQD，(c)FAM-probe+cCQD+1.0×10-9M target，(d)FAM-probe。
图5phi29聚合酶和Nt.BbvCI切刻内切酶对荧光传感平台FL响应的影响。
图6不同浓度的miR34c产生的荧光信号：(a)10nM，(b)1.0nM，(c)0.1nM，(d)10pM，(e)1.0pM，(f)0.1pM，(g)10fM，(h)blank。
图7荧光信号与目标miR34c浓度变化关系图，插图为对目标miR34c检测的标准校正曲线。
图8不同miRNA对应的FL信号；MicroRNA浓度均为5.0nmol·L-1。(a)blank，(b)miR34c(target miRNA)，(c)miR34a(mismatch miRNA)，(d)miR34b*(mismatch miRNA)。
具体实施方式：
实施例1.荧光生物传感器的制备及对miR34c的检测
目标辅助循环放大过程:发夹结构在使用之前，发夹1(HP1)和发夹2(HP2)在90℃水浴加热作退火处理5min后形成发夹结构，缓慢自然冷却到室温形成茎环DNA结构。分别取6μL Tris缓冲液、2μL HP1(10μM)、2μL HP2(10μM)、2μL DP1(10μM)和2μL目标(MicroRMA-34)(不同的浓度)，加入2μL 10×phi29聚合酶Buffer，2μL 10×NEB Cutsmart Buffer，0.5μL phi29(10,000U/ml)的聚合酶、0.5μL Nt.BbvCI(10U/μL)切刻内切酶与1μL dNTPs(10mM)构成20μL循环扩增反应体系中。将上述反应体系置于恒温振荡器中37℃反应3.5h。最后，在80℃下放置20min终止反应以获得S1。
荧光淬灭过程:为实现荧光淬灭，用Tris-HCl缓冲液将荧光DNA寡核苷酸(FAM-probe)稀释至40nM左右，制得荧光DNA寡核苷酸(FAM-probe)的溶液。然后将最适体积的cCQD溶液加入到含有FAM-probe的Tris-HCl缓冲溶液中并使其在室温下孵育30min。cCQD作为荧光淬灭剂，用作荧光传感平台检测核酸。
基于羧基碳量子点构建荧光传感平台:先在浓度为40nM FAM-probe荧光探针中加入40μL cCQD，在50℃下孵育30min。cCQD作为纳米淬灭剂，能够淬灭FAM-probe荧光探针上所携带的荧光基团，导致荧光淬灭。培养后，将由靶标刺激循环扩增过程的S1循环产物加入，室温下放置20min，致使荧光恢复，然后测定混合物的荧光强度。
实施例2.荧光生物传感器的制备及对miR34c的检测
将“先在浓度为40nM FAM-probe荧光探针中加入40μL cCQD，在50℃下孵育30min。”改为“先在浓度为40nM FAM-probe荧光探针中加入30μL cCQD，在50℃下孵育30min。”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miR34c检测的结果同实施例1。
实施例3.荧光生物传感器的制备及对miR34c的检测
将“分别取6μL Tris缓冲液、2μL HP1(10μM)、2μL HP2(10μM)、2μL DP1(10μM)和2μL目标(MicroRMA-34)(不同的浓度)，加入2μL 10×phi29聚合酶Buffer，2μL 10×NEBCutsmart Buffer，0.5μL phi29(10,000U/ml)的聚合酶、0.5μL Nt.BbvCI(10U/μL)切刻内切酶与1μL dNTPs(10mM)构成20μL循环扩增反应体系中。”改为“分别取6μL Tris缓冲液、2μL HP1(10μM)、2μL HP2(10μM)、2μL DP1(10μM)和2μL目标(MicroRMA-34)(不同的浓度)，加入2μL 10×phi29聚合酶Buffer，2μL 10×NEB Cutsmart Buffer，0.4μL phi29(10,000U/ml)的聚合酶、0.4μL Nt.BbvCI(10U/μL)切刻内切酶与1μL dNTPs(10mM)构成20μL循环扩增反应体系中。”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miR34c检测的结果同实施例1。
实施例4.荧光生物传感器的制备及对miR34c的检测
将“上述反应体系置于恒温振荡器中37℃反应3.5h”改为“上述反应体系置于恒温振荡器中37℃反应2.5h”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miR34c检测的结果同实施例1。
Claims (2)
1.一种基于酶催化靶标循环扩增技术并结合羧基碳量子点的荧光生物传感器，其特征是：利用DNA纳米分子机器，对目标进行循环放大，并合成生物相容性良好的羧基碳量子点(cCQD)，作为荧光淬灭剂发挥作用，构建用于核酸检测的荧光传感平台。
2.一种制备权利要求1所述的基于酶催化靶标循环扩增技术并结合羧基功能化的碳量子点的荧光生物传感器的制法和应用，其特征方法由下列步骤组成：
步骤1.荧光淬灭过程：为实现荧光淬灭，用Tris-HCl缓冲液将荧光DNA寡核苷酸(FAM-probe)稀至40nM左右，制得荧光DNA寡核苷酸(FAM-probe)的溶液。然后将最适体积的cCQD溶液加入到含有FAM-probe的TrisHCl缓冲溶液中并使其在室温下孵育30min。cCQD作为纳米淬灭剂，可以被用作荧光传感平台来检测核酸。
步骤2 生物传感器的制备：先在浓度为40nM FAM-probe荧光探针中加入40μL cCQD，在50℃下孵育30min。cCQD作为纳米淬灭剂，能够淬灭FAM-probe荧光探针上所携带的荧光基团，导致荧光淬灭。培养后，将由靶标刺激循环扩增过程的S1循环产物加入，室温下放置20min，致使荧光恢复，然后测定混合物的荧光强度。
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