CN113373216B - 一种同时定量检测血清中3种急性心肌梗死相关microRNA荧光探针的制备方法 - Google Patents
一种同时定量检测血清中3种急性心肌梗死相关microRNA荧光探针的制备方法 Download PDFInfo
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Abstract
本发明公开了一种同时定量检测血清中3种急性心肌梗死相关microRNA荧光探针的制备方法：合成金属有机骨架Fe‑MIL‑88；发夹探针H1～H9与Fe‑MIL‑88发生荧光共振能量转移作用，发夹探针上标记的FAM(H1‑H3),TAMRA(H4‑H6),Cy5(H7‑H9)的荧光被猝灭，当存在目标物时，520nm处FAM的荧光随着目标物miR‑21浓度增加而增强，580nm处TAMRA的荧光随着目标物miR‑499浓度增加而增强，680nm处Cy5的荧光随着目标物miR‑133a浓度增加而增强；根据相应荧光强度对目标物的浓度进行定量检测。该方法灵敏度高、选择性好、检测简便。
Description
技术领域
本发明属纳米材料、荧光传感技术和生物分析检测领域，具体涉及基于金属有机骨架的信号放大荧光传感平台同时定量测定血清中3种急性心肌梗死相关microRNA的方法。
背景技术
急性心肌梗死(AMI)伴有血管腔狭窄、急性持续缺氧缺血性心肌缺血和损伤，且可导致严重残疾和超高死亡率，是最常见的一类冠状动脉综合征。目前，高灵敏性心肌肌钙蛋白(hs-cTnT)被认为是诊断AMI的金标准。然而，在慢性稳定型冠状动脉疾病(应激性心肌病和血管痉挛型心绞痛)和终末期肾病(肾功能衰竭)中，血浆hs-cTnT也会有明显的升高。此外，在最新的高灵敏免疫测定法下，cTnT在6小时67左右才能有显著升高。因此，寻找更好的潜在生物标志物对于医学研究中早期快速、准确诊断AMI显得尤为重要。
MicroRNA是一种短的内源性非编码RNA(17-25个核苷酸)，通过调节基因转录后水平的表达，在生物过程中发挥重要作用。越来越多的证据表明，心肌组织特异性表达microRNA，如microRNA-21(miR-21)、microRNA-499(miR-499)、microRNA-208a和microRNA-133a(miR-133a)，且它们在循环中也高度稳定。值得注意的是，miR-21能够增强AMI中干细胞和祖细胞的功能，导致AMI后纤维形成和细胞肥大。而心肌和骨骼肌中的miR-499在生理上与心脏分化和I/R损伤相关，是再灌注损伤潜在的生物标志物。并且，miR-133a在早期分化、心脏发育和介导心脏传导、自律性、肥厚和心脏重构等过程中发挥着重要作用。此外，甚至在AMI事件后的1小时内，血浆和血清中相关microRNA的表达水平在患者血流中迅速显著上调。在这个意义上，这些microRNA可以被认为是AMI的早期诊断和快速预后潜在的生物标志物。目前，已有多种检测AMI相关microRNA的定量分析方法报道，如实时聚合酶链反应、微阵列、northern blot、表面增强拉曼光谱等。然而，由于microRNA尺寸小，丰度低和同源性高等特点，导致这些技术存在耗时及缺乏灵敏度的不足。另一方面，传统方法仅基于单一的AMI相关microRNA检测，不可避免地会产生假阳性或假阴性结果。为了克服这些障碍，迫切需要将信号放大技术与多路复用技术相结合。
为了进一步提高同时定量检测多个AMI相关microRNA的灵敏度，我们引入了纳米材料。近年来，由于纳米材料如金纳米颗粒、碳纳米管、氧化石墨烯、二硫化钼纳米片、金属有机框架(MOFs)等具有良好的结构和优异的光学性能而受到越来越广泛的关注。特别是MOFs，一种独特的新型纳米多孔有机-无机材料，具有超高的表面积、柔性孔隙率和良好的热稳定性。MOFs在催化、气体储存与分离、化学传感和药物递送等方面的应用已被广泛报道。此外，一些MOFs包括UiO-66和MIL-101已经被设计用于DNA传感。这些MOF对各种染料标记的单链DNA展现出极好的荧光猝灭能力以及对单链DNA和双链DNA有较好的区分能力。但是，基于MOFs和染料标记的DNA链的一步杂交的检测方法信号较弱，限制了其灵敏度。然而，借助催化发夹组装(CHA)辅助的目标物循环，可以有效放大检测信号，这有利于进一步提高灵敏度。据我们所知，通过结合CHA放大技术和金属有机骨架的荧光测定技术用于多路microRNA分析，具有极好的灵敏度和特异性，目前仍未被广泛探索。
发明内容
针对现有技术的问题，本发明的目的在于提供一种用于同时定量检测血清中3种急性心肌梗死相关microRNA(microRNA-21,microRNA-499,microRNA-133a)荧光探针的制备方法，基于金属有机骨架和信号放大技术的荧光传感平台，具有灵敏度高、选择性好、检测简便的优点，可以直接应用于血清中3种急性心肌梗死相关microRNA的同时检测。
为了实现上述发明目的，本发明采用的技术方案为：
一种用于同时定量检测血清中3种急性心肌梗死相关microRNA荧光探针的制备方法，步骤如下：
(1)、合成金属有机骨架Fe-MIL-88：将1,4-二羧基苯，六水合氯化铁与N,N-二甲基甲酰胺(DMF)混合，然后加入乙酸。混合物在120℃下反应4h，离心后获得的产物分别用DMF、乙醇和水洗涤。(参考文献：S.Xie,J.Ye,Y.Yuan,Y.Chai,R.Yuan,A multifunctionalhemin@metal-organic framework and its application to construct anelectrochemical aptasensor for thrombin detection,Nanoscale 7(2015)18232-18238)。
(2)、制备发夹探针H1,H2,H3,H4,H5,H6,H7,H8,H9(其中H1，H2，H3序列设计参照文献：D.Zhu,J.X.Huang,B.Lu,Y.Zhu,Y.Q.Wei,Q.Zhang,X.X.Guo,L.H.Yuwen,S.Su,J.Chao,L.H.Wang,Intracellular microRNA imaging with MoS2-supported nonenzymaticcatassembly of DNA hairpins，ACS Appl.Mater.Interfaces 11(2019)20725-20733。其中H1的序列为：TCGTTTCAACATCAGTCTGATAAGCTATGATCAGATCGTTAGCTTATCAGACTG；H2的序列为：CAGTCTGATAAGCTAACGATCTGATCACTGATGTTGAAATGATCAGATCGTTAG；H3的序列为：CTAACGATCTGATCGTTTCAACATCAGTAGCTTATCAGACTGATGTTGAAACGA。
H4-H9设计序列具体如下：
H4：
TAMRA-TCGTTAAACATCACTGCAAGTCTTAAGAATCAGATCGTTTAAGACTTGCAGT
H5：
TAMRA-ACTGCAAGTCTTAAACGATCTGATTCGTGATGTTTAACGAATCAGATCGTTT
H6：
TAMRA-AAACGATCTGATTCGTTAAACATCACTTAAGACTTGCAGTGATGTTTAACGA
H7：
Cy5-TCGTTATTTGGTTCCATTTTACCAGCTCGATCAGATCGTAGCTGGTAAAATGGA
H8：
Cy5-TCCATTTTACCAGCTACGATCTGATCGGGAACCAAATAACGATCAGATCGTAGC
H9：
Cy5-GCTACGATCTGATCGTTATTTGGTTCCAGCTGGTAAAATGGAACCAAATAACGA))
将H1-H9于95℃反应5分钟，然后自然冷却至室温。
(3)、制备荧光探针：将步骤(2)所得的发夹探针与步骤(1)所得的Fe-MIL-88混合，发夹探针的FAM(H1-H3),TAMRA(H4-H6),Cy5(H7-H9)的荧光被猝灭，随着目标物的加入，520nm处FAM的荧光随着目标物miR-21浓度逐渐增加而逐渐增强，580nm处TAMRA的荧光随着目标物miR-499浓度逐渐增加而逐渐增强，680nm处Cy5的荧光随着目标物miR-133a浓度逐渐增加而逐渐增强。
所用Fe-MIL-88与探针反应时间0-40min；
所用目标物与荧光探针反应时间为0-120min；
所用Fe-MIL-88浓度为175-525μg/mL。
(4)、根据荧光强度对相应目标物的浓度绘制标准曲线；根据520nm处荧光强度对miR-21的浓度绘制标准曲线，根据580nm处荧光强度对miR-499的浓度绘制标准曲线，根据680nm处荧光强度对miR-133a的浓度绘制标准曲线。
(5)、根据标准曲线，获得样品中miR-21,miR-499,
miR-133a的浓度，完成对3种microRNA的定量检测。
本发明同时定量检测血清中3种急性心肌梗死相关microRNA的原理是：
首先，我们根据miR-21序列，设计修饰FAM的发夹DNA探针H1、H2、H3；根据miR-499序列，设计修饰TAMRA的发夹DNA探针H4、H5、H6；根据miR-133a序列，设计修饰Cy5的发夹DNA探针H7、H8、H9。发夹探针可通过范德华力和π-π堆积作用紧密吸附在Fe-MIL-88表面，从而淬灭了荧光染料的荧光。当目标物存在时，以miR-21为例，每个miR-21都可以与H1的悬垂部分杂交，打开H1的发夹结构，产生与H2部分互补的粘性末端。伴随着H2的开放，暴露的粘性末端引发了H2和H3的杂交。最终，由于H3和H1之间形成了更稳定的状态，形成了“Y”形双工纳米结构而释放miR-21。随后，释放的miR-21反复引发新一轮的杂交。最终，一个miR-21分子通过产生几个带有FAM荧光团的“Y”型纳米结构而放大信号。由于“Y”型纳米结构的双链DNA与Fe-MIL-88之间的弱相互作用，“Y”型纳米结构被释放，荧光得到明显增强。
同样，miR-499可以触发H4、H5、H6之间的催化发夹自组装，miR-133a可以特异性触发H7、H8、H9之间的催化发夹自组装，因此TAMRA和Cy5的荧光强度分别与miR-499和miR-133a浓度密切相关。最后，根据520nm处荧光强度对miR-21的浓度进行定量检测，根据580nm处荧光强度对miR-499的浓度进行定量检测，根据680nm处荧光强度对miR-133a的浓度进行定量检测。
有益效果：本发明利用金属有机骨架Fe-MIL-88具有广吸收及可区分单链DNA及双链DNA的特性，结合催化发夹自组装的信号放大技术，构建了一种基于Fe-MIL-88的荧光探针，用于同时定量检测血清中3种急性心肌梗死相关microRNA的浓度。与传统的检测方法相比，不仅具有灵敏度高、选择性好、检测简便的优点，并且可以减少实际检测中假阳性假阴性的出现。
附图说明
图1是实施例1制备得到的Fe-MIL-88的理化性能图谱，其中：
图1A是实施例1制备得到的Fe-MIL-88的透射电子显微镜图；如图所示，Fe-MIL-88为均一的八面体结构，粒径约在200nm(图中标尺为500nm)。
图1B是实施例1制备得到的Fe-MIL-88的紫外吸收光谱图；如图所示，Fe-MIL-88在400-800nm均存在紫外吸收。
图2是实施例3中的Fe-MIL-88与探针的反应时间优化图；如图所示，随着反应时间的增加，荧光强度先减后维持不变。
图3是实施例3中目标物与荧光探针的反应时间优化图；如图所示，随着反应时间的增加，荧光强度逐渐增强然后趋于稳定。
图4是实施例3中Fe-MIL-88的浓度优化图。如图所示，随着Fe-MIL-88浓度的增大，FAM,TAMRA,Cy5的荧光强度均逐渐增大然后再减小。
图5是实施例4中miR-21,miR-499,miR-133a浓度与荧光探针的荧光变化荧光图；如图所示，随着miR-21浓度的增大，520nm处的荧光逐渐增强；随着miR-499浓度的增大，580nm处的荧光逐渐增强；随着miR-133a浓度的增大，680nm处的荧光逐渐增强。
图6是实施例4中miR-21,miR-499,miR-133a浓度与荧光探针的荧光强度的相关图；如图所示，随着miR-21浓度的增大，荧光强度逐渐增强，在0.05-30nM范围内，miR-21的浓度与荧光强度呈线性相关，线性回归方程为F＝16.256cmiR-21+164.77，相关系数R2＝0.9843；
随着miR-499浓度的增大，荧光强度逐渐增强，在0.08-30nM范围内，miR-499的浓度与荧光强度呈线性相关，线性回归方程为F＝7.9051cmiR-499+314.08，相关系数R2＝0.9836；
随着miR-133a浓度的增大，荧光强度逐渐增强，在0.1-20nM范围内，miR-133a的浓度与荧光强度呈线性相关，线性回归方程为F＝7.5579cmiR-133a+209.84，相关系数R2＝0.9728。
具体实施方式
六水氯化铁、1,4-二羧基苯(西格玛奥德里奇贸易有限公司)；乙酸、N,N-二甲基甲酰胺(DMF)、乙醇、寡核苷酸(生工生物工程(上海)股份有限公司)。
实施例1金属有机骨架Fe-MIL-88的合成(参考文献：S.Xie,J.Ye,Y.Yuan,Y.Chai,R.Yuan,A multifunctional hemin@metal-organic framework and its application toconstruct an electrochemical aptasensor for thrombin detection,Nanoscale 7(2015)18232-18238)，步骤如下：
将1,4-二羧基苯，六水合氯化铁与DMF混合，然后加入乙酸。混合物在120℃下反应4h，离心后获得的产物分别用DMF、乙醇和水洗涤。其透射电镜图如图1A所示，其紫外吸收光谱如图1B所示。
实施例2考察Fe-MIL-88与探针反应时间的影响实验，步骤如下:
将H1(200nM),H2(200nM),H3(200nM)分散在Tris-HCl缓冲溶液中，加入Fe-MIL-88于37℃孵育0、5、10、15、20、25、30、40min后，测定520nm处的荧光强度。考察Fe-MIL-88与探针反应时间的影响实验，结果如图2所示。
实施例3考察目标物与荧光探针的反应时间对目标物响应的影响实验，步骤如下:
将H1(200nM),H2(200nM),H3(200nM)分散在Tris-HCl缓冲溶液中，加入目标物miR-21于37℃孵育0、15、30、45、60、75、90、120min后，加入Fe-MIL-88(200μg/mL)于37℃孵育，测定520nm处的荧光强度。考察目标物与荧光探针的反应时间对目标物响应的影响实验，结果如图3所示。
实施例4考察Fe-MIL-88的浓度对目标物响应的影响实验，步骤如下:
将H1-H9(分别均为200nM)分散在Tris-HCl缓冲溶液中，加入目标物miR-21，miR-499，miR-133a于37℃孵育，加入Fe-MIL-88(175、230、260、300、330、365、400、420、435、450、490、525μg/mL)于37℃孵育，分别测定520nm，580nm，680nm处的荧光强度。考察Fe-MIL-88的浓度对目标物响应的影响实验，结果如图4所示。
实施例5对3种心肌梗死相关microRNA的同时定量检测，步骤如下:
将H1-H9(分别均为200nM)分散在Tris-HCl缓冲溶液中，加入目标物miR-21，miR-499，miR-133a于37℃孵育，加入Fe-MIL-88于37℃孵育，分别测定520nm，580nm，680nm处的荧光强度。所得荧光图如图5所示，荧光强度与相关microRNA浓度的相关性如图6所示。
实施例6实际血清样品中3种心肌梗死相关microRNA的同时定量检测，步骤如下：
将用Tris-HCl缓冲溶液稀释的血清与H1-H9(分别均为200nM)混合，加入不同浓度的目标物miR-21，miR-499，miR-133a于37℃孵育，加入Fe-MIL-88于37℃孵育，分别测定520nm，580nm，680nm处的荧光强度。根据标准曲线计算得到血清中相关microRNA的含量。其回收率结果如表1所示。
表1是实施例6中血清样品检测的回收率结果，a、b、c分别代表miR-21、miR-499、miR-133a，如表所示，回收率均在91.7％到108.6％之间，说明复杂基质对同时检测3种心肌梗死相关microRNA不产生明显干扰，该方法具有良好的选择性，可以用于实际样品的测定。
表1
序列表
<110> 南京迈迪森亚生物科技有限公司
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Claims (1)
1.一种同时定量检测血清中3种急性心肌梗死相关microRNA荧光探针的制备方法，其特征在于，步骤如下：
（1）、合成金属有机骨架Fe-MIL-88，其紫外可见吸收波长为400-800 nm；所述金属有机骨架Fe-MIL-88的合成步骤如下：将1 ,4-二羧基苯，六水合氯化铁与N,N-二甲基甲酰胺混合，然后加入乙酸，在120℃下反应4h，离心后获得的产物分别用N,N-二甲基甲酰胺、乙醇和水洗涤；
（2）、制备荧光发夹探针H1,H2,H3,H4,H5,H6,H7,H8,H9：将H1-H9于95 ℃反应，然后自然冷却至室温；其中，所述发夹探针H1-H3标记FAM,所述发夹探针H4-H6标记TAMRA,所述发夹探针H7-H9标记Cy5，其中，
H1的序列为：TCGTTTCAACATCAGTCTGATAAGCTATGATCAGATCGTTAGCTTATCAGACTG；
H2的序列为：CAGTCTGATAAGCTAACGATCTGATCACTGATGTTGAAATGATCAGATCGTTAG；
H3的序列为：CTAACGATCTGATCGTTTCAACATCAGTAGCTTATCAGACTGATGTTGAAACGA；
H4的序列为：TAMRA-TCGTTAAACATCACTGCAAGTCTTAAGAATCAGATCGTTTAAGACTTGCAGT；
H5的序列为：TAMRA-ACTGCAAGTCTTAAACGATCTGATTCGTGATGTTTAACGAATCAGATCGTTT；
H6的序列为：TAMRA-AAACGATCTGATTCGTTAAACATCACTTAAGACTTGCAGTGATGTTTAACGA；
H7的序列为：Cy5-TCGTTATTTGGTTCCATTTTACCAGCTCGATCAGATCGTAGCTGGTAAAATGGA；
H8的序列为：Cy5-TCCATTTTACCAGCTACGATCTGATCGGGAACCAAATAACGATCAGATCGTAGC；
H9的序列为：Cy5-GCTACGATCTGATCGTTATTTGGTTCCAGCTGGTAAAATGGAACCAAATAACGA；
所述3种急性心肌梗死相关microRNA为 microRNA-21、microRNA-499和microRNA-133a，所述H1-H3用于检测microRNA-21、所述H4-H5用于检测microRNA-499；所述H7-H9用于检测microRNA-133a；
（3）、制备荧光淬灭探针：将步骤（2）所得的发夹探针与步骤（1）所得的Fe-MIL-88混合，Fe-MIL-88浓度为175-525 μg/mL，发夹探针与Fe-MIL-88培育时间为0-40 min且大于0，520 nm处FAM的荧光随着目标物microRNA-21浓度逐渐增加而逐渐增强，580 nm处TAMRA的荧光随着目标物microRNA-499浓度逐渐增加而逐渐增强，680 nm处Cy5的荧光随着目标物microRNA-133a浓度逐渐增加而逐渐增强；目标物与发夹探针培育时间为0-120 min且大于0；
（4）、根据荧光强度对相应目标物的浓度绘制标准曲线；根据520 nm处荧光强度对microRNA-21的浓度绘制标准曲线，根据580 nm处荧光强度对microRNA-499的浓度绘制标准曲线，根据680 nm处荧光强度对microRNA-133a的浓度绘制标准曲线；
（5）、根据标准曲线，获得样品中microRNA-21,microRNA-499,microRNA-133a的浓度，完成对3种microRNA的定量检测。
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