CN117368284A - 一种检测外泌体miRNA的电化学传感器及其制备方法与应用 - Google Patents
一种检测外泌体miRNA的电化学传感器及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于电化学传感器技术领域,具体公开了一种检测外泌体miRNA的电化学传感器及其制备方法与应用。本发明将巯基和亚甲蓝标记的单链信号报告探针固定至金电极表面;将外泌体miRNA、模板链T1、模板链T2、KF聚合酶、Nt.BbvCI限制性内切酶、dNTP混合,孵育得MC‑SDA扩增产物;将MC‑SDA扩增产物与Cas12a/crRNA复合物混合,形成Cas12a激活体;将Cas12a激活体滴加到固定有信号报告探针的金电极表面,Cas12a激活体反式切割信号报告探针,检测电化学DPV响应信号即可获知相应外泌体miRNA含量。本发明成功构建了一种MC‑SDA介导CRISPER/Cas12a的检测外泌体miRNA电化学传感器,具有成本低、操作简单、反应速度快、灵敏度高、特异性好的优势。
Description
技术领域
本发明涉及外泌体miRNA检测技术领域,特别是涉及一种检测外泌体miRNA的电化学传感器及其制备方法与应用。
背景技术
作为液体活检重要组成部分的外泌体(exosomes)是由细胞分泌的具有磷脂双分子层的纳米囊泡(直径30-100nm),其能够通过其携带的生物大分子,介导细胞间通讯,参与机体的生理和病理过程。同时,受外泌体的脂质双层膜结构保护的蛋白、核酸等标志物更稳定,更易于储存检测。近年来多项研究证实肿瘤细胞来源外泌体miRNA在肿瘤发生、发展过程中发挥重要作用,如miRNA-21与乳腺癌转移密切相关,miRNA-375可用于诊断乳腺癌,miRNA-124介导肿瘤免疫逃逸等。因此,外泌体miRNA可作为肿瘤液体活检的潜在新型生物标志物,辅助肿瘤早期诊断和个体化精准医疗。
目前,qRT-PCR是外泌体miRNA定量检测的“金标准”,但qRT-PCR仍然存在耗时长,检测仪器昂贵和易导致假阳性等缺陷。近年来,已经开发出多种无需PCR反应的检测外泌体miRNA生物传感方法,例如纳米材料辅助信号扩增方法,无酶DNA纳米自组装,蛋白酶辅助的等温扩增方法等。其中,基于CRISPR/Cas系统的外泌体miRNA检测方法尤为受到关注。CRISPR是来源于细菌和古细菌中针对入侵核酸成分的适应性免疫系统,由于该系统的可编程性,现如今已成为转录调控、基因组编辑和核酸检测等方面的重要工具。特别是在CRISPR相关蛋白Cas12a、Cas13a的反式切割活性被发现之后,拓宽了其在核酸检测中的应用。CRISPR/Cas12a,属于II类V-A CRISPR系统,能够利用CRISPR RNA(crRNA)特异性识别单链DNA(ssDNA)或含有原间隔子相邻基序(PAM)的双链DNA(dsDNA),激活Cas12a的反式切割活性并非特异性地切割ssDNA。
尽管CRISPR/Cas12a在核酸检测方面具有很大潜力,但仍存在某些限制。首先,CRISPR效应器严格要求在dsDNA目标序列上具有特征性的PAM,从而限制了核酸分析的通用性。此外,单独的CRISPR/Cas系统检测灵敏度不能满足外泌体低丰度miRNA的检测需求,当前大多数方法依赖于PCR或RPA来扩增靶物质,需要复杂的引物设计和热循环等严格的反应条件,不利于即时检测。
发明内容
为了解决现有技术中的问题,本发明的目的在于提供一种检测外泌体miRNA的电化学传感器及其制备方法与应用。本方案创新地提出了一种多重级联链置换反应(multiplecascaded strand displacement amplification,MC-SDA)介导CRISPR/Cas12a系统的电化学传感器,外泌体miRNA识别触发MC-SDA扩增,生成大量单链激活DNA,激活DNA与Cas12a-crRNA复合物中crRNA特异性结合后形成Cas12a激活体,Cas12a激活体反式切割金电极表面单链报告探针,通过检测电化学DPV信号实现对外泌体miRNA的快速、高灵敏、高特异检测。将MC-SDA的灵活可编程性与Cas12a的功能相结合,成功构建了一个新的灵敏度高、特异性高、具有普适性的等温核酸电化学检测平台。
为实现本发明的目的,第一方面,本发明提供一种检测外泌体miRNA的电化学传感器。
一种检测外泌体miRNA的电化学传感器,其特征在于:将巯基和亚甲蓝标记的单链信号报告探针固定至金电极表面;将外泌体miRNA、模板链T1、模板链T2、KF聚合酶、Nt.BbvCI限制性内切酶混合,孵育得MC-SDA扩增产物;将MC-SDA扩增产物与Cas12a/crRNA复合物混合,形成Cas12a激活体;将Cas12a激活体滴加到固定有信号报告探针的金电极表面,Cas12a激活体反式切割信号报告探针,检测电化学DPV响应信号即可获知相应外泌体miRNA含量。
本发明包括电化学传感界面构建:裸金电极表面固定亚甲蓝标记的单链DNA信号报告探针;信号识别扩增:外泌体miRNA识别触发MC-SDA扩增出大量单链激活DNA,与Cas12a/crRNA复合物结合形成Cas12a激活体;信号转换输出:Cas12a激活体反式切割金电极表面信号报告探针,检测电化学DPV信号变化即可获知相应外泌体miRNA的含量。
进一步,所述电化学传感界面构建部分为先在裸金电极表面固定单链信号报告探针,而后进行封闭金电极非特异吸附位点所得。
进一步,信号识别扩增部分包括外泌体miRNA、模板链T1、模板链T2、KF聚合酶、Nt.BbvCI限制性内切酶,模板链T1、T2上均设计两个Nt.BbvCI酶识别位点(b),外泌体miRNA(a)为引物,与T1结合后,在KF聚合酶和Nt.BbvCI限制性内切酶的作用下发生级联SDA(C-SDA),生成c*和d*(单链激活DNA);c*又可作为引物与模板链T1结合,通过SDA反应,生成大量d*;同时,c*也可与模板链T2结合,触发新一轮C-SDA,生成大量a*和d*,a*与靶物质a的序列相同,又与模板链T1结合触发C-SDA,从而实现多重级联SDA(MC-SDA),并生成大量d*;d*与Cas12a/crRNA复合物中crRNA特异性结合形成Cas12a激活体。
进一步,信号转换输出部分包括Cas12a激活体反式切割金电极表面亚甲蓝标记信号报告探针,亚甲蓝从电极表面分离,电极表面电活性物质减少,检测电化学DPV信号即可获知相应外泌体miRNA含量。
进一步,所述单链信号报告探针的核苷酸序列为:
5'-TCAGCGTTCCTTTACCATTTTTTTAACTTATTTGGTTTTTTTTTT-3'(SEQ ID NO.1);
所述信号报告探针的5'标记巯基基团,3'端标记亚甲蓝基团。
进一步,所述模板链T1探针的核苷酸序列为:
5’-AAAAAAAAAAAAAAAAAAAAAAAAGCTGAGGAACCGCTGAGGTCAACATCAGTCTGATAAGCTA-3’(SEQ ID NO.2);
进一步,所述模板链T2探针的核苷酸序列为:
5’-AAAAAAAAAAAAAAAAAAAAAAAAGCTGAGGTCAACATCAGTCTGATAAGCTGAGGAACCGCTGA-3’(SEQ ID NO.3);
进一步,所述外泌体miRNA(miR21)的核苷酸序列为:
5'-UAGCUUAUCAGACUGAUGUUGA-3'(SEQ ID NO.4);
进一步,所述crRNA的核苷酸序列为:
5'-UAAUUUCUACUAAGUGUAGAUAAAAAAAAAAAAAAAAAAAAAAA-3'(SEQ ID NO.5)。
本发明第二方面提供一种如第一方面所述的检测外泌体miRNA的电化学传感器的制备方法,包括如下步骤:
(1)裸金电极表面处理:将裸金电极抛光,洗涤,然后浸入食人鱼溶液中10min,再取出清洗、干燥;
(2)固定信号报告探针:将5'巯基标记,3'端亚甲蓝标记的信号报告探针滴加于上述金电极上,置于4℃冰箱中过夜;
(3)封闭金电极:将步骤2中的金电极取出,PBS冲洗后用6-巯基-1-己醇(MCH)封闭1h,再次用PBS冲洗金电极,即构建成功电化学传感界面。
(4)取待测外泌体miRNA、模板链T1、模板链T2探针、KF聚合酶、Nt.BbvCI限制性内切酶,构建MC-SDA体系,混合,37℃孵育扩增,得到单链激活DNA;
(5)将单链激活DNA与预先准备的Cas12a/crRNA复合物混合形成Cas12a激活体,滴加于步骤3的金电极表面,37℃孵育,构建得到电化学反应体系。
(6)将步骤5中的金电极取出冲洗后置入PBS溶液中进行电化学DPV信号检测,所得信号用标准曲线法即可获知待检样品中外泌体miRNA的浓度。
进一步,所述步骤(1)中金电极抛光用粒度为0.05μm的氧化铝粉,所述的食人鱼溶液为浓H2SO4:H2O2,体积比为3:1。
进一步,所述步骤(2)中,信号报告探针用报告探针用10mM TCEP溶液还原巯基,通过巯基共价链接固定在裸金电极表面;信号报告探针的浓度为100nM~500nM,优选为300nM。
进一步,所述步骤(3)中,MCH浓度为1mM。
进一步,所述步骤(4)中,MC-SDA反应体系包括:1μL不同浓度的靶物质、1μL模板链T1探针、1μL模板链T2探针、1μL 10×NEBuffer 2.0缓冲液、1μL 10×rCutSart缓冲液、1μL25mM dNTP、0.2μL 5U/μL KF聚合酶、0.2μL 10U/μL Nt.BbvCI限制性内切酶、0.5μL10U/μLRNase Inhabitor、3.1μL DEPC水,共10μL。MC-SDA扩增反应时间为20min~100min,优选为60min;模板链T1的浓度为50nM~500nM,优选为200nM。
进一步,所述步骤(5)中,反应体系包括:由步骤4扩增得到的10μL单链激活DNA、4μL Cas12a/crRNA复合物、2μL 10×NEBuffer3.0缓冲液、0.5μL 10U/μL RNase Inhabitor、3.5μL DEPC水。
进一步,所述步骤(5)中,孵育在避光下进行,孵育反应条件为:37℃,孵育时间为10min~60min,优选为30min。
本发明第三方面提供一种检测外泌体miRNA的检测系统,采用第一方面所述的电化学传感器和/或第二方面所述方法制备得到的电化学传感器,用电化学工作站检测DPV信号,获得外泌体miRNA的含量。
进一步,所述方法包括如下步骤:
(1)电解液准备:准备4mL 10mM PBS(PH 7.4),其中含0.5M NaCl、5mM MgCl2。
(2)设置参数:电化学DPV检测的调制时间为0.05s,间隔时为0.017s,阶跃电位为5mV,调制幅值为50mV,电位扫描范围为-0.5V~0.0V。
(3)进行检测:将工作电极、参比电极以及对电极连接到电化学工作站上,用PBS作为反应基底液,点击检测,即可获得电化学DPV信号。
有益效果:
本发明创新地提出了一种MC-SDA介导CRISPER/Cas12a系统的外泌体miRNA电化学检测传感器,通过外泌体miRNA识别触发MC-SDA扩增生成大量可被Cas12a/crRNA复合物识别的单链激活DNA,Cas12a激活体反式切割金电极上亚甲蓝标记单链信号报告探针,亚甲蓝从电极表面分离,电极表面电活性物质减少,检测电化学DPV信号即可获知相应外泌体miRNA含量。本发明通过将MC-SDA的灵活可编程性与Cas12a的功能相结合,成功构建了一种新的等温核酸检测平台。本发明主要有以下几点优势:(1)高灵敏度,MC-SDA可以循环利用靶物质,扩增靶信号,灵活与CRISPR/Cas12a系统整合,双重扩增系统提高了检测的灵敏度;(2)MC-SDA中KF聚合酶和Nt.BbvCI限制性内切酶能够进行高保真识别,MC-SDA扩增的单链激活DNA特异性识别激活Cas12a/crRNA复合物,均能确保检测具有高特异性的目标;(3)具有普适性:MC-SDA扩增生成的单链激活DNA克服了Cas12a的PAM序列限制,扩展了其检测范围和通用性;(4)等温反应,适用于现场即时检测。
附图说明
图1显示为本发明实施例1中电化学传感器检测外泌体miRNA的原理图。
图2(A)显示为本发明实施例2中电化学传感器在含0.4M KCl的5×10-3M[Fe(CN)6]3-/4中,不同修饰电极(a)GE,(b)MCH/CP/GE,(c)Cas 12激活体剪切金电极表面信号报告探针后的电化学阻抗谱;(B)显示为检测外泌体miRNA电化学传感器可行性的DPV响应曲线图(a)实验组,(b)缺乏靶物质对照组,(c)缺乏MC-SDA扩增体系对照组,(d)缺乏Cas12a/crRNA复合物对照组的电化学DPV响应曲线图;(C)外泌体miRNA识别触发MC-SDA的PAGE电泳结果图。
图3显示显示为本发明实施例2中外泌体的表征结果图(A)透射电子显微镜表征,(B)纳米粒子追踪分析粒径图。
图4显示为本发明实施例3中电化学传感器的使用条件优化,即信号报告探针浓度、模板链T1浓度、MC-SDA反应时间和Cas12a激活体反应时间的优化结果图。
图5显示为本发明实施例4中电化学传感器检测不同浓度外泌体miRNA的电化学DPV响应曲线图。
图6为本发明制备的电化学传感器的特异性分析实验结果。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。本发明所使用的Klenow Fragment DNA聚合酶、Nt.BbvCI限制性内切酶均为本领域所常用的生物化学工具酶,KF聚合酶参考文献1:STARProtoc.2023Jun16;4(2):102267.;KF聚合酶参考文献2:Nucleic Acids Research,2022,50(21),12149-12165.Nt.BbvCI限制性内切酶酶切位点:CCTCAGC(-5/-7),Nt.BbvCI限制性内切酶的参考文献ACSNano 2021,15,7,11573-11584.
本发明是在重庆市自然科学基金项目(cstc2019jcyj-msxmX0179)和重庆市科卫联合医学科研项目(2023QNXM026)的资助下完成的。
本发明创新地提出了一种MC-SDA介导CRISPER/Cas系统的外泌体miRNA生物传感器,通过靶标识别触发MC-SDA扩增生成大量与Cas12a/crRNA复合物结合形成Cas12a激活体的单链激活DNA,Cas12a激活体反式切割金电极上亚甲蓝标记信号报告探针。本发明的电化学传感器是首次将MC-SDA和CRISPR/Cas12a相结合并且用于外泌体miRNA的检测,通过MC-SDA结合CRISPR系统本身的信号放大作用,实现级联信号放大,从而实现对外泌体miRNA的高灵敏检测。
MC-SDA是一种高效的等温扩增反应,同时,高度保真的KF聚合酶和特异性Nt.BbvCI限制性内切酶具有降低背景噪声的作用,有助于构建一个简便、低成本、高效的检测平台。因此将MC-SDA与CRISPR/Cas连用能解决现有技术中存在的问题,可构建一种高效的外泌体miRNA检测体系。
本发明构建的电化学传感器制备及检测方法简便、成本低,灵敏度高,特异性良好,有望在外泌体miRNA的检测分析及应用研究方面推广使用。
实施例1
制备电化学传感器并检测外泌体miRNA
1.材料与方法
1.1材料
HPLC纯化的DNA由生工合成(上海,中国),crRNA由Takara合成(大连,中国)。Klenow Fragment DNA聚合酶、Nt.BbvCI限制性内切酶、EnGen Lba Cas12a购自NewEnglandBiolabs公司(北京,中国)。DEPC水和RNA酶抑制剂购于生工(上海,中国)。人乳腺癌细胞系(MCF-7)购自Procell(武汉,中国)。磷酸盐缓冲液(PBS)和牛血清白蛋白(BSA)购自Thermo Fisher Scientific(威尔明顿,美国)。Dulbecco的改良版Eagle培养基(DMEM)和胎牛血清(FBS)购自Gibco(中国上海)。通用miRNA纯化试剂盒购自EZBioscience(中国北京)。
1.2检测仪器
上海辰华CHI 660D电化学工作站。
1.3检测原理
如图1所示,模板链T1、T2上均设计两个Nt.BbvCI酶识别位点(b),靶物质a为引物,与T1结合后,在KF聚合酶和Nt.BbvCI限制性内切酶的作用下发生级联SDA(C-SDA),生成c*和d*(单链激活DNA);c*又可作为引物与模板链T1结合,通过SDA反应,生成大量d*;同时,c*也可与模板链T2结合,触发新一轮C-SDA,生成大量a*和d*,a*与靶物质a的序列相同,又与模板链T1结合触发C-SDA,从而实现多重级联SDA(MC-SDA),并生成大量d*;d*与Cas12a/crRNA复合物结合形成Cas12a激活体;Cas12a激活体反式切割金电极上亚甲蓝标记信号报告探针,亚甲蓝从电极表面分离,电极表面电活性物质减少,检测电化学DPV信号的改变即可获知相应靶物质含量。
1.4电化学传感界面构建
(1)裸金电极表面处理:将裸金电极抛光,洗涤,然后浸入食人鱼溶液中10min,再取出清洗、干燥;
(2)固定信号报告探针:10mM TCEP还原信号报告探针,然后将10μL 300nM信号报告探针滴加于上述金电极上,置于4℃冰箱中过夜;
(3)封闭金电极:将步骤2中的金电极取出,PBS冲洗后用6-巯基-1-己醇(MCH)封闭1h,再次用PBS冲洗金电极,即构建成功电化学传感界面。
信号报告探针的核苷酸序列为:
5'-TCAGCGTTCCTTTACCATTTTTTTAACTTATTTGGTTTTTTTTTT-3'(SEQ ID NO.1);
所述信号报告探针的5'标记巯基基团,3'端标记亚甲蓝基团。
1.5构建靶物质识别触发的多重级联链置换反应(MC-SDA)
在MC-SDA体系中分别加入待测靶物质、模板链T1、T2、10×NEBuffer 2.0、10×rCutSart Buffer、dNTP、KF聚合酶、Nt.BbvCI限制性内切酶、RNase Inhabitor和DEPC水,37℃条件下反应60min。
模板链T1的核苷酸序列为:
5’-AAAAAAAAAAAAAAAAAAAAAAAAGCTGAGGAACCGCTGAGGTCAACATCAGTCTGATAAGCTA-3’(SEQ ID NO.2);
模板链T2的核苷酸序列为:
5’-AAAAAAAAAAAAAAAAAAAAAAAAGCTGAGGTCAACATCAGTCTGATAAGCTGAGGAACCGCTGA-3’(SEQ ID NO.3);
靶物质miRNA的核苷酸序列为:
5'-UAGCUUAUCAGACUGAUGUUGA-3'(SEQ ID NO.4);
1.6构建Cas12a激活体系
激活单链DNA与Cas12a/crRNA复合物结合形成Cas12a激活体。在体系中加入MC-SDA扩增形成的单链激活DNA、预先配制的Cas12a/crRNA复合物、NEBuffer 3.0、RNaseInhabitor和DEPC水,混匀后取10μL滴加于制备好的金电极表面,37℃条件下避光反应30min。
所述crRNA的核苷酸序列为:
5'-UAAUUUCUACUAAGUGUAGAUAAAAAAAAAAAAAAAAAAAAAAA-3'(SEQ ID NO.5)。
1.7检测电化学DPV信号
Cas12a激活体反式切割电极表面信号报告探针后,冲洗金电极,置入PBS溶液中,利用电化学工作站进行电化学DPV信号检测。
实施例2
验证外泌体miRNA电化学传感器的可行性
1、电化学阻抗谱(EIS)和差分脉冲伏安法(DPV)验证了外泌体miRNA传感器策略的可行性。首先,在含有[Fe(CN)6]3-/4-的溶液中,用电化学阻抗谱(EIS)表征了裸金电极的逐步修饰过程,半圆形直径等于电子转移电阻(Ret)的曲线。图2A中,曲线a为裸金电极,几乎呈一条直线,表明了良好的电子转移能力。曲线b为电极表面固定信号报告探针且经MCH封闭,由于信号报告探针和MCH阻碍了电子从溶液到电极表面的传输,Ret增加。图2B中,曲线a为实验组;曲线b为缺乏靶物质的对照组;曲线c为缺乏MC-SDA扩增体系;曲线d为缺乏Cas12a/crRNA复合物的对照组。由图2B可知,只有当靶物质存在时,才能启动MC-SDA介导的CRISPER/Cas系统反式剪切活性,剪切电极表面信号报告探针,导致电化学DPV信号减小。
2、聚丙烯酰氨凝胶电泳(PAGE)电泳表征MC-SDA扩增
图2C中,泳道4为外泌体miRNA识别触发MC-SDA扩增生成大量单链激活DNA(d*),可以看到明亮的d*条带,证实了MC-SDA能够实现高效扩增;泳道5为空白对照,几乎没有单链激活DNA生成,证实当体系中不存在外泌体miRNA时,不能触发MC-SDA扩增反应。
3、外泌体的表征
从MCF-7细胞培养上清液中收集Exosome。收集到的外泌体通过透射电子显微镜(TEM)和纳米粒子追踪分析(NTA)进行表征(图3)。如图3A所示,TEM图像清楚地表明纯化的外泌体具有典型的碟状结构。图3B中,NTA评估所收集的外泌体的平均大小和浓度与文献报道一致(Nature,2018,560(7718):382-386.),表明外泌体分离成功。
实施例3
检测外泌体miRNA电化学传感器及其使用条件的优化
为提高电化学传感器的检测灵敏度和特异性,本实施例对实验过程中几个重要的条件即信号报告探针的浓度、模板链T1的浓度、MC-SDA的反应时间和Cas12a激活体切割信号报告探针的时间进行了进一步的优化,并对每一个优化条件都由低值到高值分别选取至少五个点进行了一系列实验。
为考察信号报告探针浓度对本电化学传感器检测结果的影响,本实施例采用了不同浓度的(100nM,200nM,300nM,400nM,500nM)信号报告探针。由图4A可知,ΔI随信号报告探针浓度的增加而增加,当信号报告探针浓度达到300nM以后,再增大信号报告探针的浓度,ΔI值增大不明显。说明当浓度为300nM时,电极表面固定的信号报告探针已经达到饱和。因此,信号报告探针的最佳浓度选为300nM。
为考察模板链T1浓度对外泌体miRNA传感器检测结果的影响,本实验采用了不同浓度的(50nM,100nM,200nM,300nM,500nM)模板链T1。通过计算电化学DPV峰值电流改变比例(ΔI%值)选择最佳的T1浓度,ΔI%=(IBlank-Itarget)/IBlank×100%。由图4B可知,ΔI%随模板链T1浓度的增加而增加,当T1的浓度为200nM时,ΔI%达到平台期,后续继续增加T1浓度,ΔI%并不明显增加。因此,T1的最佳浓度选为200nM。
为考察策略中MC-SDA的反应时间对外泌体miRNA检测结果的影响,本实验采用了不同的MC-SDA的反应时间(20min,40min,60min,80min,100min)构建电化学传感器。如图4C可见,随着MC-SDA反应时间的增加,ΔI%也随之增大;当MC-SDA的反应时间为60min时,ΔI%达到平台期,其后继续增加反应时间,ΔI%并不明显增加。因此,MC-SDA反应的最佳时间选为60min。
为考察策略中Cas12a激活体的反应时间对外泌体miRNA检测结果的影响,本实验采用了不同的Cas12a激活体反应时间(10min,20min,30min,40min,60min)构建电化学传感器。如图4D可见,ΔI%随Cas12a激活体反应时间的增加而增大;当反应时间为30min时,ΔI%达到平台期,说明当反应时间为30min时,Cas12a激活体能完全切割电极表面的信号报告探针。因此,Cas12a激活体的最佳反应时间选为30min。
实施例5
检测外泌体miRNA电化学传感器的性能分析
为了评估检测外泌体miRNA电化学传感器的性能,在实施例4所得最优实验条件下,配制了不同浓度的miRNA-21进行了试验分析。如图5所示,检测实验结果显示,当miRNA-21浓度在0.1fM到1nM之间时,电化学DPV的ΔI%值与miRNA-21浓度的对数呈线性相关,线性方程为Y=10.404g C+21.854,线性相关系数为0.9980。将传感器在空白溶液中重复检测3次,根据空白信号平均值加上3倍标准差所对应的值估计最低检出限,计算得0.08fM。
综上所述,本发明成功构建了一种外泌体miRNA的电化学传感器,应用本发明的传感器,对外泌体miRNA的测定显示了稳定、灵敏度高、特异性好的能力。与现有技术比较,本发明的传感器成本低,检测周期短,灵敏度高,背景干扰小。有望应用于实际样品和临床标本的测定,发展成为具有临床应用价值的传感器。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (10)
1.一种检测外泌体miRNA的电化学传感器,其特征在于:将巯基和亚甲蓝标记的单链信号报告探针固定至金电极表面;将外泌体miRNA、模板链T1、模板链T2、KF聚合酶、Nt.BbvCI限制性内切酶、dNTP混合,孵育得MC-SDA扩增产物;将MC-SDA扩增产物与Cas12a/crRNA复合物混合,形成Cas12a激活体;将Cas12a激活体滴加到固定有信号报告探针的金电极表面,Cas12a激活体反式切割电极表面信号报告探针,检测电化学DPV响应信号即可获知相应外泌体miRNA含量;
所述单链DNA信号报告探针的核苷酸序列为:
5'-TCAGCGTTCCTTTACCATTTTTTTAACTTATTTGGTTTTTTTTTT-3'(SEQ ID NO.1);
所述信号报告探针的5'标记有巯基基团,3'端标记有亚甲蓝基团;
所述模板链T1探针的核苷酸序列为:
5’-AAAAAAAAAAAAAAAAAAAAAAAAGCTGAGGAACCGCTGAGGTCAACATCAG TCTGATAAGCTA-3’(SEQ ID NO.2);
所述模板链T2探针的核苷酸序列为:
5’-AAAAAAAAAAAAAAAAAAAAAAAAGCTGAGGTCAACATCAGTCTGATAAGCTGAGGAACCGCTGA-3’(SEQ ID NO.3);
所述靶物质外泌体miRNA的核苷酸序列为:
5'-UAGCUUAUCAGACUGAUGUUGA-3'(SEQ ID NO.4);
所述crRNA的核苷酸序列为:
5'-UAAUUUCUACUAAGUGUAGAUAAAAAAAAAAAAAAAAAAAAAAA-3'(SEQ ID NO.5)。
2.如权利要求1所述检测外泌体miRNA的电化学传感器的制备方法,包括如下步骤:
1)将裸金电极抛光,洗涤,然后浸入食人鱼溶液中10min,再取出清洗、干燥;
2)将5'巯基标记,3'端亚甲蓝标记的信号报告探针滴加于上述金电极上,置于4℃冰箱中过夜;
3)将步骤2中的金电极取出,PBS冲洗后用6-巯基-1-己醇封闭1h,再次用PBS冲洗金电极;
4)取待测外泌体miRNA、模板链T1、模板链T2探针、KF聚合酶、Nt.BbvCI限制性内切酶、dNTP构建MC-SDA体系,混合,37℃孵育扩增,得到单链激活DNA;
5)将单链激活DNA与预先准备的Cas12a/crRNA复合物混合形成Cas12a激活体,滴加于步骤3的金电极表面,37℃孵育;
6)将步骤5中的金电极取出冲洗后置入PBS溶液中进行示差脉冲伏安法DPV信号检测,所得信号用标准曲线法即可获知待检样品中外泌体miRNA的浓度。
3.如权利要求2所述的方法,其特征在于:所述步骤1)中金电极抛光用粒度为0.05μm的氧化铝粉,所述的食人鱼溶液为浓H2SO4:H2O2,体积比为3:1。
4.如权利要求2所述的方法,其特征在于:所述步骤2)中,亚甲蓝标记信号报告探针的浓度为100-500nM。
5.如权利要求2所述的方法,其特征在于:所述步骤4)中制备MC-SDA所用反应体系包括:1μL不同浓度的靶物质、1μL模板链T1探针、1μL模板链T2探针、1μL 10×NEBuffer 2.0缓冲液、1μL 10×rCutSart缓冲液、1μL 25mM dNTP、0.2μL 5U/μL KF聚合酶、0.2μL 10U/μLNt.BbvCI限制性内切酶、0.5μL 10U/μL RNase Inhabitor、3.1μL DEPC水,共10μL。
6.如权利要求2所述的方法,其特征在于:所述步骤4)中MC-SDA扩增反应时间为20-80min,模板链T1探针的浓度为50-500nM。
7.如权利要求2所述的方法,其特征在于:所述步骤5)中,反应体系包括:由步骤4)扩增得到的10μL单链激活DNA、4μL Cas12a/crRNA复合物、2μL 10×NEBuffer3.0缓冲液、0.5μL10U/μL RNase Inhabitor、3.5μL DEPC水。
8.如权利要求2所述的方法,其特征在于:所述步骤5)中,Cas12a/crRNA的复合物的浓度为50-500nM,孵育时间为10-60min。
9.一种检测外泌体miRNA的检测系统,权利要求1所述的电化学传感器用电化学工作站检测DPV信号,获得外泌体miRNA的含量。
10.如权利要求9所述的检测系统,其特征在于,包括如下步骤:
(1)电解液准备:准备4mL 10mM PBS(PH 7.4),其中含0.5M NaCl、5mM MgCl2;
(2)设置参数:电化学DPV检测的调制时间为0.05s,间隔时为0.017s,阶跃电位为5mV,调制幅值为50mV,电位扫描范围为-0.5V~0.0V;
(3)进行检测:将工作电极、参比电极以及对电极连接到电化学工作站上,点击检测,获得电化学DPV信号。
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