CN113073132A - ECL生物传感器及其在制备用于检测心梗miRNA的检测体系中的应用 - Google Patents
ECL生物传感器及其在制备用于检测心梗miRNA的检测体系中的应用 Download PDFInfo
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Abstract
本发明设计合成基于DNAzyme的能够精准捕获目标miRNA并能实现循环放大的探针,再将AgNCs的ECL信号与目标循环和杂化链式扩增的信号放大策略相结合,构建了用于心肌梗死中miRNA检测的超灵敏ECL生物传感器,同时ECL生物传感器可用于检测心肌梗死相关的miRNA。本发明通过以DNA‑AgNCs为ECL发射体,以DNA‑AgNCs为发光体的ECL具有低毒性,避免了标记过程,有利于其进一步的传感应用。此外,将循环扩增技术与杂化链式扩增反应相结合,成功地构建了一种灵敏的通用型ECL生物传感器,可用于同时检测心肌梗死相关的miRNA,其线性范围从1nM到0.1fM,可以应用于实际样品的检测。
Description
技术领域
本发明涉及生物分析检测领域,具体涉及一种通用型、高灵敏的ECL生物传感器及其在制备用于检测心梗miRNA的检测体系中的应用。
背景技术
急性心肌梗死(AMI)是指急性心肌缺血性坏死,在全世界范围内发病率居高不下。因为其独有特点,发病时突然出现持久的胸骨后剧烈疼痛、心悸、呼吸困难、发热等不适,并且难以及时诊断一直成为研究者们关注的重点。在急性心肌梗死的研究中也一直有关于miRNA的报道,miRNA是一类小型非转录RNA,核苷酸长度约在21-25之间,它的生物学功能主要通过转录物降解或转录后翻译水平来调节基因表达,因此对于心脏生长发育和应激反应具有关键作用,为AMI提供了潜在预测、诊断及治疗方向。
目前miRNA的检测方法有许多,传统的miRNA的检测方法包括Northern Blot、微阵列芯片、微球、实时荧光定量PCR等技术。然而由于miRNA序列短,使得探针碱基序列的设计受限。其次,由于miRNA的序列存在高度相似性,所以检测的特异性往往难以保证。此外,在循环血中,心梗相关miRNA的含量极低。针对这些问题研究者们将先进的光谱技术,例如电致化学发光、化学发光、表面增强拉曼光谱、荧光检测等,与核酸放大技术及先进的材料相结合,为miRNA检测提供了新思路。
电化学发光(ECL)又称电致化学发光,是指在电极表面产生的物质经过电子转移反应形成激发态发光的发光过程。当ECL通过电致自由基的双分子复合而发光时,根据自由基的来源,其作用机理可分为两类,即湮灭机理和协同作用机理。对于前者,自由基物种是由单个发射体产生的,而后者涉及发射体与合适的共反应物之间的一组双分子电化学反应。发射体在电能向辐射能的转化中起着关键作用。钌(II)配合物、鲁米诺和量子点(QDs)等三种发光体在ECL研究中得到了广泛的应用。从某种意义上说,电致化学发光是电化学方法和光谱方法的理想结合。因此,电化学发光不仅保持了传统化学发光方法的灵敏度和宽的线性范围,而且显示出电化学方法的一些优点,包括简单、稳定和便携。另一方面,作为一种发光技术,与光致发光(PL)和化学发光(CL)等其它发光方法相比,ECL具有独特的优越性,特别是与化学发光(CL)相比,ECL对发光的时间和空间控制更为优越。因此,ECL已成为一种强有力的分析技术,并在大量生物检测中得到了广泛的应用,从基础研究到探测微量目标分子的实际应用。电化学发光(ECL)以其低背景信号、简化光学装置、高灵敏度等独特的优点,更适于心梗miRNA的临床分析。
随着纳米材料的发展,银纳米团簇(AgNCs),因其低毒性、良好的生物相容性和良好稳定性等特点,可用于电化学发光生物传感分析中进行生物分子检测。DNA模板银纳米簇(DNA AgNCs)是一类相对较新的发射体,由2–30个银原子嵌入一个或多个单链DNA寡聚体中形成。由于其简单的合成和激发和发射波长的可调性,DNA模板银纳米簇(DNA AgNCs)可用于生物传感和荧光成像中。DNA稳定的AgNCs结合了AgNCs的电化学发光特性和柔性DNA的生物识别特性,具有多种优越的特性,例如无标记、低毒性和稳定性,已被广泛用作各种生物测定中的ECL发光体。
催化核酸DNAzyme作为传感器的放大元件在核酸分析中应用广泛。与蛋白酶类似,DNAzyme具有高选择性和强催化活性的特点。同时,与蛋白酶相比,DNAzyme具有易合成、易改性、稳定性好、生产成本低等优点。为了提高传感器的性能,DNAzyme常被用作开发生物分析平台的理想生物敏感元件。活性DNAzyme的组装方案为心肌梗死miRNAs的分析开辟了新的思路。通过适当的DNAzyme裂解和合理的miRNA循环结构设计,可以实现对miRNA的准确识别和多次循环放大。将该放大元件与其他放大技术相结合进行多电平信号放大,可以提高极端条件下分析的灵敏度。此外,通过进一步的结构设计和探针加载,该扩增方案将为心肌miRNA的ECL传感平台提供更多的思路。
发明内容
本发明设计合成基于DNAzyme的能够精准捕获目标miRNA并能实现循环放大的纳米探针,再将AgNCs的ECL信号与目标循环和杂化链式扩增的信号放大策略相结合,构建了用于心肌梗死中miRNA检测的超灵敏ECL平台。
本发明的一种ECL生物传感器,采用下述方法制备得到:(1)、预处理电极;(2)、将发夹DNA修饰于电极表面;(3)、加MCH(巯基聚乙二醇SH-PEG)防止非特异性结合;(4)、加入miRNA、辅链DA、辅链DB形成信号循环放大单元进而产生酶切产物;(5)、加入发夹DNA1、发夹DNA2进行杂化链式扩增反应;(6)、将AgNO3溶液滴到修饰好电极的表面,再用NaBH4进行还原。
作为优选方案,上述所述ECL生物传感器中,步骤(1)预处理包括抛光、清洗、干燥。
作为优选方案,上述所述ECL生物传感器中,步骤(2)修饰于电极表面是指将发夹DNA即S1-HP于电极上孵育;所述S1-HP序列自行设计,为序列表中SEQ NO.1,如下:
S1-HP:
5’-CTGATAAGCTACAGGACATCGAATAGTCTTTTTTGAGCGACACACTATrAGGAAGAGATACTTTTTTGACTATTCGA-3’。
作为优选方案,上述所述ECL生物传感器中,步骤(2)加金纳米颗粒(AuNPs)修饰于电极表面,粒径10-20nm。
作为优选方案,上述所述ECL生物传感器中,步骤(4)加DA、DB、miRNA一起退火滴在电极表面孵育,再加Mg2+孵育过夜;所述辅链DA、辅链DB序列分别为序列表中SEQ NO.2和SEQNO.3,如下:
DA:5’-GTATCTCTTCCGCGATTAACCAAGTCTTAA-3’
DB:5’-AAACATCACTGGTTAGACCCATGTTAGTGTGTCGCTC-3’
作为优选方案,上述所述ECL生物传感器中,步骤(5)发夹DNA 1即S2-HP1、发夹DNA2即S2-HP2一起退火再加Mg2+滴在电极表面孵育。所述S2-HP1、S2-HP2分别为序列表中SEQNO.4和SEQ NO.5,自行设计并包含合成银纳米簇所需长链C模板,如下:
S2-HP1:
5’-TTTTTTTCATCGAATAGTCCTGACTGACTATTCGATGTCCTGTCCCCCCCCCCCCCCC-3’
S2-HP2:
5’-AGTCAGGACTATTCGATGACAGGACATCGAATAGTCTTTTTTTCCCCCCCCCCCCCCC-3’
作为优选方案,上述所述ECL生物传感器中,步骤(6)先将AgNO3溶液滴到修饰好的电极表面,在黑暗中孵育,再将新鲜制备的NaBH4溶液滴到上述电极表面,于黑暗中孵育。
作为优选方案,上述所述ECL生物传感器可采用下述方法制备得到:
(1)用氧化铝浆抛光电极,再用超纯水彻底冲洗,并在乙醇和水中超声处理,室温干燥;
(2)加10μL AuNP于电极上,再加10μL 3μM S1-HP于电极上孵育4h-6h;
(3)加1mM MCH孵育半小时;
(4)加1μM DA、1μM DB、不同浓度的miRNA-499一起退火滴在电极表面孵育6h-10h以上,再加10mM Mg2+孵育过夜;
(5)加10μL1μM S2-HP1、1μM S2-HP2一起退火再加1μL 10mM Mg2+滴在电极表面孵育5h-8h以上;
(6)将8μL的100μM AgNO3溶液滴到修饰好的电极表面,在黑暗中孵育30分钟,然后将8μL的100μM新鲜制备的NaBH4溶液滴到上述电极表面并在环境温度下于黑暗中孵育2小时。
本发明所述ECL生物传感器可在制备用于检测心梗miRNA的检测体系中应用,其中,所述miRNA包括但不限于miRNA-499、miRNA-208、miRNA-328。
本发明的原理分析如下:
1、基于DNAzyme的miRNA循环放大探针设计的依据
针对miRNA丰度低、序列短、同源性等特点,设计合成基于DNAzyme的能够精准捕获目标miRNA并能实现循环放大的纳米探针。首先,设计能够精准捕获目标miRNA的DNAzyme辅助酶链,辅助酶链要在目标miRNA存在时既能精准捕获目标,又能形成稳定的DNAzyme循环单元;其次,提高探针的特异性,以心梗疾病相关的多条miRNA为分析对象,调整DNAzyme辅助酶链与miRNA的互补序列,避免相似度较高的miRNA与两条辅助酶链甚至一条辅助酶链发生杂化,产生假阳性信号。第三,设计具有酶切位点及后续纳米粒子触发链的发夹探针序列,由于该发夹探针不直接与目标miRNA互补配对,故可作为DNAzyme通用型发夹探针于各种DNA扩增技术。
2、纳米探针对心梗miRNA的捕获和信号放大研究的依据
根据多种miRNA同时放大检测的设计思路,开展多种心梗miRNA的同时抓捕及信号放大研究。利用数据库(http://www.mirbase.org),筛选出3-4条心梗相关miRNA作为分析对象,针对不同miRNA分析对象,研究DNAzyme探针对不同miRNA的放大效果,实现多种miRNA的高效放大检测。
3、杂化链式扩增发夹结构设计的依据
杂化链式扩增反应可以将大量的纳米粒子聚集在一起,同时反应后发夹单元互补段使纳米粒子聚集,进一步实现信号放大。miRNA含量低,很难从血液中提取出来。为实现多种miRNA循环放大,在DNAzyme探针的设计中引入通用型DNAzyme发夹结构。在此通用型发夹结构中包埋一段序列作为杂化链式扩增的通用型引发物。进一步设计两个DNA发夹结构(S2-HP1和S2-HP2),作为纳米粒子的原料,以便在酶切产物的触发下,从发夹环粘性末端打开发夹进行DNA自组装。针对DNAzyme发夹探针上的触发序列,设计时既要避免发夹结构与DNAzyme发夹探针发生反应,又要保证扩增反应能被酶切产物触发,防止背景干扰的同时,提高结合效率。
4、DNA模板化AgNCs设计的依据
AgNCs是通过使用化学还原剂还原银离子来合成的,导致AgNCs聚集并形成大的纳米颗粒,可以识别和合成序列可以自然地整合到一个DNA探针中。所设计的两个DNA发夹结构(S2-HP1和S2-HP2)是富含C碱基的,通过还原法生成AgNCs。在AgNCs/S2O8 2-的ECL体系中,随着靶miRNA浓度的增加,ECL强度增加。
通过ECL快速、灵敏的优势,将核酸扩增、纳米合成等策略与之融合,发展心梗相关循环miRNA精准识别和高效检测的传感原理与方法。针对循环miRNA含量低、序列短、同源性等特点,发展基于DNAzyme的通用型miRNA放大及识别探针原理,建立基于杂化链式扩增的ECL体系,进一步提高检测的灵敏性及重复性,构建稳定、可靠的miRNA检测平台。
本发明通过以DNA模板银纳米团簇(DNA-AgNCs)为ECL发射体,金纳米颗粒(AuNPs)以辅助,扩大反应的接触面,以DNA-AgNCs为发光体的ECL具有低毒性,避免了标记过程,有利于其进一步的传感应用。此外,将循环扩增技术与杂化链式扩增反应相结合,成功地构建了一种灵敏的通用型ECL生物传感器,可用于同时检测心肌梗死相关的miRNA,其线性范围从1nM到0.1fM,可以应用于实际样品的检测,结果理想。
附图说明
图1为DNA模板银纳米团簇的电化学发光检测miRNA的原理示意图;
图2为通过凝胶电泳实验验证了DNAzyme和杂化链式扩增策略;
图3形成DNA模板银纳米团簇的透射电镜图;
图4为10%急性心肌梗死患者血清和缓冲液中miRNA的分析能力的对比
图5是实施例1中miRNA-499的浓度与ECL强度的关系图及线性关系;
图6是实施例2中miRNA-208的浓度与ECL强度的关系图及线性关系;
图7是实施例3中miRNA-328的浓度与ECL强度的关系图及线性关系。
具体实施方式
利用mirbase数据库,筛选出3条心梗相关miRNA(miRNA-499、miRNA-208、miRNA-328)作为分析对象,并由苏州吉玛基因股份有限公司提供,序列分别为序列表中SEQ NO.6-SEQ NO.8,如下:
miRNA-499:UUA AGA CUU GCA GUG AUG UUU
miRNA-208:AAG CUU UUU GCU CGA AUU AUG U
miRNA-328:CUG GCC CUC UCU GCC CUU CCG U
实施例1
DNAzyme探针的设计、合成及miRNA循环放大是在12%(w/w)聚丙烯酰胺凝胶上证实。首先,将每个单链DNA、miRNA在90℃下退火10分钟。然后,将含有0.1μM DA、0.1μM DB、0.1μM S1-HP和10mM MgAc2的混合物溶液在37℃下培养2小时。然后将靶miRNA-499注入混合物中,并将***温度保持在37℃孵育过夜。最后,将2μL 0.1μM S2-HP1、0.1μM S2-HP2移液到溶液中以形成杂化链式扩增反应。总体积为20μL,样品电泳(10%,w/w)在110V的1×TA缓冲液中电泳3h。然后用凝胶红染色30min,用gel-Doc-EZ成像***成像。
首先,用氧化铝浆在抛光麂皮上进一步抛光至镜面。然后,用超纯水彻底冲洗电极(GCE),并在乙醇和水中超声处理,随后在室温下干燥清洁。加10μLAuNPs溶胶(柠檬酸钠还原法合成)于电极上待干,再加10μL 3μM S1-HP与电极上孵育4h-6h。甩掉电极上溶液,加1mM MCH(国药试剂)孵育半小时,防止非特异性结合。加1μM DA、1μM DB、不同浓度的miRNA-499一起退火滴在电极表面孵育6h-10h以上,再加10mM Mg2+孵育过夜。10μL1μM S2-HP1、S2-HP2一起退火再加1μL 10mM Mg2+滴在电极表面孵育5h-8h以上。甩掉电极上溶液,将8μL的100μM AgNO3溶液滴到修饰好的电极表面,在黑暗中孵育30分钟,然后将8μL的100μM新鲜制备的NaBH4溶液滴到上述电极表面并在环境温度下于黑暗中孵育2小时。最后,获得DNA模板银纳米团簇DNA-AgNCs,在含0.05M K2S2O8和0.1M KCl的PBS缓冲液中进行ECL测量。ECL光电倍增管800V,从-0.6到0.2扫描速率0.1/s。
如图2所示是用聚丙烯酰胺凝胶电泳(PAGE)来单独分析DNA结构。
图2A中:孔道1,miRNA-499;孔道2,DA;孔道3,DB;孔道4miRNA-499+DA+DB;孔道5,S1-HP;孔道6,DA+S1-HP;孔道7,DB+S1-HP;孔道8,DA+DB+S1-HP;孔道9,S1-HP劈开的右部分;孔道10,miRNA-499+DA+DB+S1-HP(RNA);孔道11,miRNA-499+DA+DB+S1-HP.;
图2B中:孔道12,S1-HP劈开的右部分;孔道13,S2-HP1;孔道14,S2-HP2;孔道15,S1-HP劈开的右部分+S2-HP1;孔道16,S2-HP1+S2-HP2;孔道17,S1-HP劈开的右部分+S2-HP1+S2-HP2。
miRNA-499(孔道1),DA(孔道2)和DB(孔道3)作为参照,miRNA-499、DA、和DB混合物(孔道4)显示出不同于miRNA-499、DA、和DB单体的原始条带,表明信号循环放大单元成功的建立。当与S1-HP(孔道5)孵育时,DA和DB的条带消失,同时出现新的缓慢移动的条带(孔道6-8),表明S1-HP与DA和DB的稳定杂交。9孔道中为酶切后的产物条带,可用作对比,当在Mg2+存在下添加miRNA-499时,10孔道中有一条比8孔道跑的慢带,其迁移率与9通道中酶切后的产物相同。但是在没有RNA切割位点中,酶切后的产物链相对应的条带不会出现(孔道11)。说明DNAzyme和探针成功的建立。如图2B所示由新生成的条带移动速度比酶切后的产物慢(孔道15),证明酶切后的产物能够打开S2-HP1发卡。S2-HP1和S2-HP2的混合物显示出一定的背景,且没有生成新的条带,各自保持稳定,证明单独的S2-HP1发卡不能打开S2-HP2发卡(孔道16)。加入酶切后的产物后,杂交扩增反应的特征条带显著增加(孔道17)。证明完成了扩增。以上结果表明,所设计的生物传感器组装成功。
如图3所示是AgNCs纳米粒子的TEM图像,平均直径约3-5nm,均匀的分布在视线中。
如图4所示为了评价该方法的实际应用情况,研究了10%急性心肌梗死患者(马鞍山市人民医院)血清中miRNA-499的分析能力。从图可以看出,患者血清样品(serum)中的分析结果与Tris缓冲液(buffer)中的结果基本保持一致(图中左柱是buffer右柱是serum),说明设计的传感器平台具有良好的抗干扰性能。对真实患者血清中的miRNA具有良好的分析性能。
如图5为miRNA-499的浓度与ECL强度的关系图及线性关系,在加入一系列浓度范围为0.1fM至1nM的miRNA-499可显著增强ECL强度,这使得ECL强度与miRNA-499浓度的对数之间呈线性关系。
实施例2
DNA模板银纳米团簇的电化学发光检测miRNA-208。在实施例1的基础上实施例2,把检测目标miRNA-499换成检测目标miRNA-208,步骤和方法与实施例1的相同。如图6为miRNA-208的浓度与ECL强度的关系图及线性关系,在加入一系列浓度范围为0.1fM至1nM的miRNA-208可显著增强ECL强度,这使得ECL强度与miRNA-208浓度的对数之间呈线性关系。
实施例3
DNA模板银纳米团簇的电化学发光检测miRNA-328。在实施例1的基础上进行实施例3,把检测目标miRNA-499换成检测目标miRNA-328,步骤和方法与实施例1的相同。如图7为miRNA-328的浓度与ECL强度的关系图及线性关系,在加入一系列浓度范围为0.1fM至1nM的miRNA-208可显著增强ECL强度,这使得ECL强度与miRNA-328浓度的对数之间呈线性关系。
应当说明的是,本发明的上述所述之技术内容仅为使本领域技术人员能够获知本发明技术实质而进行的解释与阐明,故所述之技术内容并非用以限制本发明的实质保护范围。本发明的实质保护范围应以权利要求书所述之为准。本领域技术人员应当知晓,凡基于本发明的实质精神所作出的任何修改、等同替换和改进等,均应在本发明的实质保护范围之内。
序列表
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Claims (10)
1.一种ECL生物传感器,采用下述方法制备得到:
(1)、预处理电极;(2)、将发夹DNA修饰于电极表面;(3)、加MCH防止非特异性结合;(4)、加入miRNA、辅链DA、辅链DB形成信号循环放大单元进而产生酶切产物;(5)、加入发夹DNA 1(S2-HP1)、DNA 2(S2-HP2)进行杂化链式扩增反应;(6)、将AgNO3溶液滴到修饰好电极的表面,再用NaBH4进行还原。
2.如权利要求1所述ECL生物传感器,其特征在于,步骤(1)预处理包括抛光、清洗、干燥。
3.如权利要求1所述ECL生物传感器,其特征在于,步骤(2)修饰于电极表面是指将发夹DNA即S1-HP于电极上孵育;所述S1-HP序列为序列表中SEQ NO.1。
4.如权利要求1所述ECL生物传感器,其特征在于,步骤(2)加AuNPs修饰于电极表面。
5.如权利要求1所述ECL生物传感器,其特征在于,步骤(4)加辅链DA、辅链DB、miRNA一起退火滴在电极表面孵育,再加Mg2+孵育过夜;所述辅链DA、辅链DB序列分别为序列表中SEQNO.2和SEQ NO.3。
6.如权利要求1所述ECL生物传感器,其特征在于,步骤(5)发夹DNA 1即S2-HP1、发夹DNA 2即S2-HP2一起退火再加Mg2+滴在电极表面孵育;所述S2-HP1、S2-HP2序列分别为序列表中SEQ NO.4和SEQ NO.5。
7.如权利要求1所述ECL生物传感器,其特征在于,步骤(6)先将AgNO3溶液滴到修饰好的电极表面,在黑暗中孵育,再将新鲜制备的NaBH4溶液滴到上述电极表面,于黑暗中孵育。
8.如权利要求1所述ECL生物传感器,其特征在于,采用下述方法制备得到:
(1)用氧化铝浆抛光电极,再用超纯水彻底冲洗,并在乙醇和水中超声处理,室温干燥;
(2)加10μLAuNPs于电极上,再加10μL 3μM S1-HP于电极上孵育4h-6h;
(3)加1mM MCH孵育半小时;
(4)加1μM DA、1μM DB、不同浓度的miRNA-499一起退火滴在电极表面孵育6h-10h以上,再加10mM Mg2+孵育过夜;
(5)加10μL 1μM S2-HP1、1μM S2-HP2一起退火再加1μL 10mM Mg2+滴在电极表面孵育5h-8h以上;
(6)将8μL的100μM AgNO3溶液滴到修饰好的电极表面,在黑暗中孵育30分钟,然后将8μL的100μM新鲜制备的NaBH4溶液滴到上述电极表面并在环境温度下于黑暗中孵育2小时。
9.权利要求1-8任一项所述ECL生物传感器在制备用于检测心梗miRNA的检测体系中的应用,其特征在于,所述miRNA包括但不限于miRNA-499、miRNA-208、miRNA-328。
10.如权利要求9所述应用,其特征在于,所述miRNA-499、miRNA-208、miRNA-328序列分别为序列表中SEQ NO.6、SEQ NO.7、SEQ NO.8。
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