CN114574553A - 一种碳点-纳米金球形核酸及其制备方法和应用 - Google Patents
一种碳点-纳米金球形核酸及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种碳点‑纳米金球形核酸及其制备方法和应用，所述碳点‑纳米金球形核酸以纳米金为核，其表面偶联DNAzyme链和末端标记碳点的底物。本发明制备的碳点‑纳米金球形核酸在检测外泌体miRNA时，金属离子存在的条件下，靶标miRNA识别触发DNAzyme行走，产生荧光；采用荧光分光光度计测量行走结束后CDs的荧光信号，实现对miRNA的检测分析。同时，本发明中基于双色CDs‑SNA用于两种外泌体miRNAs检测，采用DNAzyme介导CDs荧光信号放大，实现miRNAs高灵敏检测；采用双色CDs‑SNA实现miRNAs的同时检测，为外泌体miRNA精确检测提供了新方法。
Description
技术领域
本发明属于生物医学领域，具体涉及一种碳点-纳米金球形核酸及其制备方法和应用。
背景技术
外泌体是由细胞主动分泌的直径为30-150nm的细胞外囊泡，存在于多种体液中，研究发现其携带的microRNAs(miRNAs)可以提供很多关于疾病进展的信息。miRNA是由19-25个核苷酸组成的非编码RNA，可通过抑制靶向mRNA的表达来调控基因转录后的蛋白质表达，进而调控细胞发生、细胞分化、免疫应答等重要的生理过程，也可作为原癌基因或抑癌基因在不同的癌细胞中异常表达以调控相应的病理过程。由于外泌体中miRNAs的高稳定性，有望作为液体活检中具有巨大潜力的肿瘤标志物，一些特定的外泌体miRNA，如膀胱癌血清外泌体miRNA在疾病诊断、病程监测等方面具有重要临床价值。
传统的外泌体miRNA检测方法为qPCR法等。qPCR法检测外泌体miRNA过程中引物设计、温度调控等相对复杂。荧光检测具有制备简单和检测灵敏度高等优点，以球形核酸为代表的纳米材料结合荧光信号放大策略已在多项研究中用于miRNA的检测。纳米金能够实现荧光的高效淬灭，具有比表面积大、表面易修饰的特点。碳点作为一种零维球形纳米颗粒，荧光多色、稳定性高，且原料丰富、制备简单，成为有机染料及量子点的补充和替代，可提高检测的稳定性和安全性。
外泌体提取步骤复杂，含量低，使得外泌体miRNA的获取量极低。外泌体miRNA在体液中含量低的特性，对检测方法的灵敏度提出了更高的要求。目前发展的多种等温扩增技术如链式杂交反应(HCR)、滚环扩增反应(RCA)、链式取代反应(SDA)、催化发卡自组装(CHA)和DNA纳米机器(如DNAzyme walker)等均可实现有效的信号放大。
发明内容
发明目的：针对现有技术存在的问题，本发明提供一种碳点-纳米金球形核酸，该碳点-纳米金球形核酸使用碳点作为荧光基团，具有荧光强度高，光稳定性好等优点，进一步利用DNAzyme信号放大来提高检测的灵敏度。
本发明还提供了所述的碳点-纳米金球形核酸制备方法和应用。
技术方案：为了实现上述目的，本发明提出一种碳点-纳米金球形核酸，所述碳点-纳米金球形核酸是以纳米金为核，其表面偶联DNAzyme链和末端标记碳点的底物，所述DNAzyme链为靶标互补序列-DNAzyme的双功能核酸，其包括靶标互补序列、两条臂、酶的活性中心；所述末端标记碳点的底物是包含DNAzyme剪切位点，末端偶联碳点的单链核酸。
作为优选，所述靶标互补序列-DNAzyme双功能核酸包括DNAzyme-133b或者DNAzyme-135b，其序列分别为SEQ ID NO.1和2：
HS-T(10)TAGCTGGTTGAAGGGGACCAAATTTTTTTTTTTTTTTCAACCAGCTCCGAGCCGGTCGAAATAGT
HS-T(10)TCACATAGGAATGAAAAGCCATATTTTTTTAAAAAATTCCTATGTCCGAGCCGGTCGAAATAGT。
作为优选，所述包含DNAzyme剪切位点，末端可以偶联碳点的单链核酸为底物包括Substrate-133b或者Substrate-135b，其序列分别为SEQ ID NO.3和4：
HS-T(3)ACTAT rA GGCTGGTT-NH2，HS-T(3)ACTAT rA GCATAGGA-NH2；
作为优选，所述碳点包括绿色碳点或者蓝色碳点。
本发明所述的碳点-纳米金球形核酸的制备方法，包括如下步骤：
(1)制备绿色碳点或者蓝色碳点；
(2)制备纳米金；
(3)碳点-纳米金球形核酸的制备：在纳米金表面偶联核酸，将两种核酸双功能核酸、单链核酸混合，加入TCEP进行孵育，随后加入纳米金溶液静置过夜，加入SDS进行孵育后多次加入氯化钠溶液静置过夜，离心洗涤得到AuNPs-DNA溶液；取碳点，加入EDC、NHS活化表面的羧基，孵育后加入到AuNPs-DNA溶液中孵育后离心洗涤重悬得到CDs-SNA溶液即为碳点-纳米金球形核酸。
其中，所述DNAzyme链和底物的摩尔比为1-4:6-9。
作为优选，所述DNAzyme链和底物两种核酸的摩尔比为2：8。
其中，所述核酸为DNAzyme链和底物，所述碳点与底物的氨基端偶联。
本发明所述的碳点-纳米金球形核酸在外泌体miRNA检测中的应用。
其中，所述外泌体miRNA，包括常规体液中的外泌体miRNA，以及特殊的外泌体miRNA如膀胱癌血清外泌体miRNA。
其中，包括如下步骤：金属离子存在的条件下，靶标miRNA识别触发碳点-纳米金球形核酸中DNAzyme行走，产生荧光；采用荧光分光光度计测量行走结束后碳点的荧光信号，实现对miRNA的检测分析。
本发明所述的碳点-纳米金球形核酸在制备外泌体miRNA检测试剂或者试剂盒中的应用。
本发明制备的用于外泌体miRNA检测的碳点-纳米金球形核酸(CDs-SNA)，碳点-纳米金球形核酸(CDs-SNA)包括纳米金及其表面偶联的DNAzyme和末端标记碳点的底物；基于碳点-纳米金球形核酸用于检测外泌体miRNA时，在金属离子存在的条件下，靶标miRNA识别触发碳点-纳米金球形核酸上DNAzyme在纳米金表面自动化行走，产生荧光，具体由靶标与靶标互补序列-DNAzyme杂交将DNAzyme的上下臂释放出来，识别邻近的底物，结合金属离子激活DNAzyme活性特异性剪切底物上的rA位点，剪切后碳点被释放；持续剪切驱动DNAzyme沿纳米金表面行走，导致大量碳点脱离纳米金表面，实现信号放大。采用荧光分光光度计测量DNAzyme行走结束后CDs的荧光信号，实现对miRNA的检测分析。本发明中还提出了基于双色CDs-SNA用于两种外泌体miRNAs检测的方法，采用DNAzyme介导CDs荧光信号放大，碳点荧光发射波长标记两种靶标miRNA，碳点荧光强度标定靶标miRNA数量，实现miRNAs高灵敏检测；采用双色CDs-SNA实现miRNAs的同时检测，为外泌体miRNA精确检测提供了新方法。
本发明中的碳点-纳米金球形核酸(CDs-SNA)包括两种碳点-纳米金球形核酸，具体包括两种颜色的碳点、两种靶标、两种DNAzyme及其对应底物和金属离子；所述的两种颜色碳点分别为绿色和蓝色；所述两种靶标分子为血清外泌体中的两种miRNA；所述两种DNAzyme包括DNAzyme-133b、DNAzyme-135b，其5’端均修饰巯基；所述两种底物包括Substrate-133b、Substrate-135b，其5’端修饰巯基，3’端修饰氨基；所述金属离子为镁离子。
其中，本发明所述的两种外泌体miRNA为膀胱癌相关外泌体miR-133b和外泌体miR-135b。
作为优选，所述的碳点-纳米金球形核酸(CDs-SNA)包括基于绿色和蓝色碳点，设计不同的靶标互补序列-DNAzyme发卡结构，用于不同miRNA的特异性识别。两种靶标分子为血清外泌体中的两种miRNA，如膀胱癌相关外泌体miR-133b和外泌体miR-135b；两种靶标互补序列-DNAzyme和对应的底物序列分别为DNAzyme-133b/Substrate-133b、DNAzyme-135b/Substrate-135b，DNAzyme的5’端修饰巯基，Substrate的5’端和3’端分别修饰巯基和氨基。其中DNAzyme-133b/Substrate-133b/绿色CDs用于检测miR-133b；DNAzyme-135b/Substrate-135b/蓝色CDs用于检测miR-135b，分别偶联至纳米金表面。由于CDs靠近纳米金表面，荧光处于猝灭状态。本发明检测时在金属离子存在的条件下，靶标miRNA识别触发DNAzyme沿纳米金表面行走。在无miRNA存在条件下，靶标互补序列-DNAzyme为发卡结构，无剪切活性。miRNA与靶标互补序列杂交，导致发卡结构构象发生改变，形成DNAzyme结构；进一步添加Mg2+，DNAzyme剪切活性激活，可剪切单链核酸；持续剪切活性驱动DNAzyme沿纳米金表面行走，导致大量碳点脱离纳米金表面，实现信号放大。可采用荧光分光光度计测量碳点荧光强度与添加靶标浓度之间的定量关系，计算检测限与线性检测范围。
本发明设计特定的DNAzyme链，包括靶标互补序列、两条臂、酶的活性中心。本发明的DNAzyme链为单分子设计，与普通DNAzyme的不同在于，将靶标互补序列设计到其中，分子内杂交形成发夹结构，无靶标存在时，保持发卡结构，DNAzyme无活性。
本发明将碳点、DNAzyme、底物整合到纳米金表面，利用纳米金的高淬灭性能，碳点的高荧光强度、高灵光稳定性，以及DNAzyme信号放大功能，实现对miRNA的高灵敏检测。本发明采用碳点作为荧光基团，取代先前研究中的有机荧光染料和传统的量子点。有机荧光染料容易淬灭，传统的量子点一般有毒，对环境也有很大危害，且粒径较大。而碳点不仅合成简便、成本低、具有高荧光强度和荧光稳定性、低毒、粒径小，有利于实现检测的准确性，而且可以实现使用同一激发波长激发，使同时、多元检测更加便捷。
本发明中DNAzyme序列设计灵活，可对任意给定的miRNA设计相应的DNAzyme进行检测，可以实现靶标miRNA的特异性检测。
此外，使用本发明的碳点-纳米金球形核酸检测外泌体该实验反应条件单一，在37℃即可完成所有反应，无需复杂的操作步骤及较高的反应温度。
本发明为了区分两种球形核酸，设计两种碳点颜色的球形核酸分别用来检测两种靶标，两种颜色的碳点可以相互互换对应的DNAzyme和Substrate。同时本发明制备的CDs-SNA可用于两种miRNA同时检测。
有益效果：与现有技术相比，本发明具有如下优点：
本发明制备了负载DNAzyme的碳点-纳米金球形核酸(CDs-SNA)，通过靶标识别触发DNAzyme自动行走介导的CDs荧光信号增强，实现对外泌体miRNA的高灵敏度和同时检测。
采用碳点作为荧光基团，取代常规的有机荧光染料，更有利于实现检测的准确性，且多色碳点可以使用同一激发波长激发，使同时、多元检测更加便捷。
附图说明
图1为基于DNAzyme行走的CDs-SNA检测外泌体miRNA示意图；
图2为CDs-SNA的性质表征，其中，A图为透射电子显微镜照片；B图为纳米金(AuNPs)、纳米金表面偶联DNA(AuNP-DNA)及CDs-SNA的水动力尺寸；C图为纳米金、纳米金表面偶联DNA及CDs-SNA的UV-vis光谱图；
图3为PAGE电泳验证DNAzyme行走的可行性；
图4为CDs-SNA检测miRNA的灵敏度和标准曲线；
图5为CDs-SNA检测miRNA的特异性，***P<0.001,**P<0.01,*P<0.05,ns:无明显差异(P>0.05)。
图6为CDs-SNA同时检测两种靶标能力评估；
图7为以miR-133b和miR-135b标准品为模板进行qPCR得到的标准曲线；
图8为CDs-SNA检测血清外泌体miRNA，NC-SNA表示用碳点-纳米金球形核酸方法检测正常对照组，NC-PCR表示用qPCR方法检测正常对照组，BC-SNA表示用碳点-纳米金球形核酸方法检测膀胱癌组，BC-PCR表示用qPCR方法检测膀胱癌组，***P<0.001。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
实施例1
靶标互补序列-DNAzyme及底物的序列设计
靶标选择为膀胱癌相关的两个外泌体miRNA：miR-133b和miR-135b。如图1所示，针对这两个靶标，分别设计合成了相应的DNAzyme-133b和DNAzyme-135b和相应的底物(substrate-133b和substrate-135b)，其序列列于表1。如表1所示，DNAzyme-133b和DNAzyme-135b中的加粗部分为对应的靶标互补序列，下划线部分为DNAzyme的上下臂，斜体部分为DNAzyme的活性中心序列；Substrate-133b和substrate-135b中的rA为对应的DNAzyme剪切位点，斜体部分为对应的DNAzyme的上下臂互补序列。miR-133b-1mut、3mut、5mut，miR-135b-1mut、3mut、5mut分别为靶标miRNA的1、3、5个碱基突变序列。
表1靶标miRNA、DNAzyme、底物及碱基突变核酸序列设计
实施例2
碳点的制备
绿色碳点的制备：首先，将柠檬酸3g和尿素3g加入10mL蒸馏水中，形成透明溶液。然后在750W微波炉中加热4-5min，溶液由无色液体变为棕色，最后变成深棕色固体，表明碳点的形成。将碳点的水溶液进行离心(3000rpm，20min)，以去除大的或凝聚的颗粒。将上清液用0.45μm滤膜过滤后用100-500D透析袋透析48h，由此得到的溶液即为绿色荧光碳点溶液。
蓝色碳点的制备：将0.5g柠檬酸和0.5g硫脲加入25mL水中搅拌。然后750W微波炉中加热7min，直至溶液由无色变为深褐色，表明反应物已经碳化。冷却至室温后，用25mL水溶解。将得到的深褐色溶液在10000rpm下离心5min后，取上清经0.45μm滤膜过滤后用100-500D透析袋透析48h，得到蓝色碳点溶液。
实施例3
纳米金的制备
采用柠檬酸盐还原法合成金纳米颗粒，所用的玻璃器皿全部用铬酸浸泡并用超纯水冲洗多次，烘干使用。首先配制1％(g/mL)柠檬酸钠溶液，称取1g柠檬酸三钠，溶于100mLH2O，用前摇匀。向圆底烧瓶中加入96mL H2O与1mL HAuCl4(25mM)溶液，加热至沸腾，并剧烈搅拌，随后加入3mL新配制的柠檬酸钠溶液，计时30min。过程中，溶液开始为深蓝色随后迅速变为红色。待加热结束，继续剧烈搅拌至溶液恢复至室温，保存于4℃备用。
实施例4
碳点-纳米金球形核酸(CDs-SNA)的制备
首先在纳米金表面偶联核酸，按将实施例1中的DNAzyme-133b(1μM，20μL)和Substrate-133b(1μM，80μL)，混合，然后加入100μL TCEP(2.5μM)，孵育1h。随后加入150μL纳米金溶液(实施例3制备)，PBS(pH 7.4)补足体积至400μL，静置过夜。次日加入4μL 1％SDS，孵育20min后加入2M氯化钠溶液，每隔30min加一次，每次加入后均超声10s，以防氯化钠结块，每次使得体系中氯化钠终浓度增加0.1M，直至体系氯化钠终浓度达0.7M，静置过夜。次日离心，用纯水洗三次，得到AuNPs-DNA溶液。取实施例2制备的绿色碳点8μL，加入EDC、NHS(终浓度为0.02M)活化表面的羧基，孵育30min，加入到上述AuNPs-DNA溶液中，室温孵育4h。离心水洗三次，用100μL Tris-HCl(10mM,pH 7.4)重悬得到CDs-SNA溶液。
对制备的CDs-SNA的形貌尺寸及光学性质进行表征，结果见图2。图2a为CDs-SNA的透射电镜图片，结果显示纳米金表面覆盖了一层碳点，呈现卫星结构，且单分散性良好。图2b为AuNPs、AuNPs-DNA及CDs-SNA的水动力尺寸对比图，结果显示，与纳米金尺寸相比，偶联核酸后增加了约14nm，进一步偶联碳点，尺寸再次增加了约24nm。图2c为紫外-可见吸收光谱对比图，结果显示纳米金偶联核酸后吸光度红移5nm，由此表明高密度核酸的成功偶联，进一步偶联碳点，吸光度进一步红移17nm，说明碳点偶联成功。以上均表明碳点-纳米金球形核酸(CDs-SNA)的成功制备。
而采用DNAzyme-135b和Substrate-135b以及蓝色碳点，其制备过程与上述相同，结果类似。
实施例5
DNAzyme作为信号放大器的可行性
在液相中进行DNAzyme扩增可行性的验证，通过20％PAGE凝胶在TBE缓冲液中进行电泳。分8组上样，每组上样量为10μL。样品为DNAzyme-133b(2μM，12μL)，靶标miRNA-133b(2μM，12μL)，底物Substrate-133b(2μM，3μL)和Mg2+(5mM，3.5μL)，用Tris-HCl补足体系至35μL。37℃反应3h后上样：泳道1：DNAzyme链(D)；泳道2：靶标Target(T)；泳道3：底物Substrate(S)；泳道4：D+T；泳道5：D+S；泳道6：D+T+S；泳道7：D+S+Zn2+；泳道8：D+T+S+Mg2+。PAGE结果显示(图3)，当靶标与DNAzyme链杂交后，发夹结构打开，导致DNAzyme链的构象的改变，DNAzyme/miRNA的条带(泳道4)明显不同于单独的DNAzyme链条带(泳道1)。当靶标miRNA和Mg2+同时不存在或其中之一不存在时，DNAzyme没有活性，不能触发裂解反应，所以底物条带保持完整(第5、6、7道)。当靶标miRNA和Mg2+同时存在的情况下，DNAzyme与靶标miRNA杂交，对底物进行剪切，底物条带消失(泳道8)。PAGE结果证实了DNAzyme-133b作为信号放大器的可行性。
而采用DNAzyme-135b和Substrate-135b以及靶标miRNA-135b进行PAGE电泳，结果与之相一致。
本实施例首先在液相中验证所设计的DNAzyme链能够在靶标及金属离子存在的条件下对底物进行剪切(结果可用PAGE电泳呈现)，对进一步在纳米金表面偶联DNAzyme链与底物，并实现信号放大奠定基础。
实施例6
miRNAs的灵敏度检测
将实施例4中重悬得到的CDs-SNA溶液分别与不同浓度的靶标miRNA孵育(终浓度分别为0fM、10fM、50fM、100fM、1pM、10pM、100pM、1nM、10nM、100nM)，在37℃条件下充分反应3h后，测量荧光光谱。
实施例4两种不同颜色碳点制备的CDs-SNA分别用于靶标miRNA-133b和miR-135b的灵敏度检测，miR-135b对应蓝色，miR-133b对应绿色。
结果如图4所示，随着靶标miRNA浓度的不断增加，CDs荧光不断增强，荧光强度与相应靶标miRNA浓度的对数存在线性关系。
两miRNA的检测限约为10fM，线性范围均为50fM-10nM，相关系数分别为0.997和0.996。
实施例7
miRNAs的特异性检测
将实施例4重悬得到的两种荧光颜色CDs-SNA溶液，分别与终浓度均为1nM的miR-133b、miR-135b以及它们的单碱基突变序列(1mut)、三个碱基突变序列(3mut)、五个碱基突变序列(5mut)、随机序列(检测miR-135b时使用miR-133b作为随机序列，检测miR-133b时使用miR-135b作为随机序列)37℃溶液共同孵育3h后，将无miRNA加入的空白溶液作为阴性对照，测量荧光光谱。
结果如图5所示，随着突变碱基个数的增加，荧光强度逐渐下降，说明该方法具有较高的miRNA检测特异性。
实施例8
CDs-SNA用于两种miRNA同时检测
基于多色荧光碳点具有可以使用同一激发波长，产生不同发射波长的特性，将实施例4制备的两种CDs-SNA与两种靶标混合孵育，与单独检测进行对比，将无miRNA加入的空白溶液作为阴性对照，测量荧光光谱。
具体是将：将实施例4制备的两种CDs-SNA溶液等体积混合，分别或同时加入两种靶标miRNA(终浓度均为1nM)，将无miRNA加入的空白溶液作为阴性对照，37℃水浴3h，测量荧光光谱。
结果如图6所示，单独检测与同时检测结果相类似，说明该方法可以实现miRNA的同时检测，对结果的分析更加方便、直观。
实施例9
CDs-SNA检测血清外泌体miRNAs
血清样本为膀胱癌患者(BC)血清和正常对照组(NC)血清，提取血清外泌体试剂盒为Total Exosome Isolation(from serum)。提取外泌体的步骤按照试剂盒中的说明书操作，用TRIzol提取血清外泌体总RNA，溶于10μL DEPC水中，等体积分为两份。将实施例4制备的两种CDs-SNA溶液等体积混合，采用实施例6的方法加入血清外泌体中提取的总RNA溶液，37℃反应3h。用荧光分光光度计测量荧光光谱。根据荧光强度与靶标浓度的关系，计算提取的血清外泌体中的两种miRNA浓度。同时，用qPCR测定另一份总RNA中的miR-133b和miR-135b的Ct值。用miR-133b和miR-135b标准品按梯度稀释后进行qPCR实验，根据拷贝数与Ct值的关系，得到标准曲线(图7)。从而计算两种miRNA的浓度。
根据荧光光谱和标准曲线计算出血清外泌体中2种miRNA浓度，结果如图8所示，采用本发明实施例6的方法(NC-SNA、BC-SNA)中，与正常对照组(NC)相比膀胱癌患者(BC)血清外泌体中miR-135b表达升高，而miR-133b表达下降，这与qPCR方法(NC-PCR、BC-PCR)计算出的浓度类似。因此，该基于多色CDs-SNA的外泌体miRNA检测方法适用于血清外泌体中miRNA的定量检测。同时，与qPCR相比，该方法无需逆转录和扩增等复杂的操作步骤，且能实现同一体系下的同时、多元检测。
序列表
<110> 南京医科大学第二附属医院
<120> 一种碳点-纳米金球形核酸及其制备方法和应用
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Claims (10)
1.一种碳点-纳米金球形核酸，其特征在于，所述碳点-纳米金球形核酸以纳米金为核，其表面偶联DNAzyme链和末端标记碳点的底物，所述DNAzyme链为靶标互补序列-DNAzyme的双功能核酸，其包括靶标互补序列、两条臂、酶的活性中心；所述末端标记碳点的底物是包含DNAzyme剪切位点，末端偶联碳点的单链核酸。
2.根据权利要求1所述的碳点-纳米金球形核酸，其特征在于，所述DNAzyme链包括DNAzyme-133b或者DNAzyme-135b，其序列优选分别为：
HS-T(10)TAGCTGGTTGAAGGGGACCAAATTTTTTTTTTTTTTTCAACCAGCTCCGAGCCGGTCGAAATAGT
HS-T(10)TCACATAGGAATGAAAAGCCATATTTTTTTAAAAAATTCCTATGTCCGAGCCGGTCGAAATAGT。
3.根据权利要求1所述的碳点-纳米金球形核酸，其特征在于，所述底物包括Substrate-133b或者Substrate-135b，其序列分别为：
HS-T(3)ACTAT rA GGCTGGTT-NH2，HS-T(3)ACTAT rA GCATAGGA-NH2。
4.根据权利要求1所述的碳点-纳米金球形核酸，其特征在于，所述碳点包括绿色碳点或者蓝色碳点。
5.一种权利要求1所述的碳点-纳米金球形核酸的制备方法，其特征在于，包括如下步骤：
(1)制备绿色碳点或者蓝色碳点；
(2)制备纳米金；
(3)碳点-纳米金球形核酸的制备：在纳米金表面偶联核酸，将DNAzyme链和底物混合，加入TCEP进行孵育，随后加入纳米金溶液静置过夜，加入SDS进行孵育后多次加入氯化钠溶液静置过夜，离心洗涤得到AuNPs-DNA溶液；取碳点，加入EDC、NHS活化表面的羧基，孵育后加入到AuNPs-DNA溶液中孵育后离心洗涤重悬得到CDs-SNA溶液即为碳点-纳米金球形核酸。
6.根据权利要求5所述的制备方法，其特征在于，所述核酸为DNAzyme链和底物，所述碳点与底物的氨基端偶联。
7.根据权利要求5所述的制备方法，其特征在于，所述DNAzyme链和底物的摩尔比为1-4:6-9。
8.一种权利要求1所述的碳点-纳米金球形核酸在外泌体miRNA检测中的应用。
9.根据权利要求1所述的应用，其特征在于，包括如下步骤：金属离子存在的条件下，靶标miRNA识别触发碳点-纳米金球形核酸中DNAzyme行走，产生荧光；采用荧光分光光度计测量行走结束后碳点的荧光信号，实现对miRNA的检测分析。
10.一种权利要求1所述的碳点-纳米金球形核酸在制备外泌体miRNA检测试剂或者试剂盒中的应用。
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