CN113621688B - SERS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
SERS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
本发明涉及SERS生物传感器,其是将发卡探针DNA修饰在Fe3O4的表面;加MCH孵育防止非特异性结合;加入miRNA、辅链DA、辅链DB产生酶切产物;预先将发夹DNA 1、发夹DNA 2修饰在AuNP的表面;将分散好的发夹DNA 1、发夹DNA 2修饰的AuNP加入孵育好的Fe3O4进行杂化链式扩增反应,反应过夜,离心洗涤。本发明在纳米组装SERS检测体系中,针对没有反应的金纳米簇元件以及非触发产生的聚集体,采用Fe3O4作为磁性分离基底,通过表面功能化,排除非特异性吸附,快速分离出杂化链式扩增触发的组装体,得到只含有Fe3O4表面金纳米粒子的聚集体,具有很高的SERS信号,实现了心梗miRNA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SERS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
MicroRNA(miRNA)是一类18–25个碱基的单链RNA分子。目前的研究证实,血液循环系统中存在与疾病相关的miRNA,这些循环miRNA并不是保持不变的,通常会随着疾病的发展阶段发生改变,在不同器官、组织、细胞类型的不同生命活动中,miRNA的种类及表达也有很大差异。越来越多的研究表明,随着癌症、心肌梗死等重大疾病的发展阶段变化,循环miRNA的表达会发生失调。因此,血液中的miRNA被证实是疾病诊断和防治的重要标志物。分析循环miRNA是一个热点问题,因此许多基于多种光学技术的光学纳米传感器,包括荧光,化学发光,电化学等被设计用于分析疾病生物学中的循环miRNA。但是,大多数此类光学纳米传感器无法将生物学条件下的所有因素都考虑在内,而且miRNA在生物体内的浓度很低,因此,在构建用于miRNA检测的新型光学纳米传感器中,出现了一些结合了先进光谱的新型纳米材料。在这些新的纳米传感器中,基于表面增强拉曼光谱(SERS)的传感器在生物学领域被认为是比较新颖的。
SERS是一种主要由电磁机制产生的纳米光学现象。SERS的电磁增强发生在光束照射金属纳米结构时,这一过程诱导了自由传导电子的集体振荡,称为“表面等离子体子”,从而在纳米结构周围产生强局域电磁场。近年来,随着纳米技术和纳米加工技术的进步,各种等离子体活性纳米结构和纳米颗粒的发展,其拉曼信号的增强幅度达到了几百个量级。通过两个或更多紧密分离的纳米粒子之间所谓的等离子体耦合效应,这种增强甚至可以达到一千多。与荧光相比,拉曼散射具有以下优点。首先,拉曼散射可抵抗光漂白,这使得拉曼信号高度稳定,可用于长时间分析。其次,拉曼信号的峰值宽度很窄,使其易于进行多重检测。然而,由于拉曼光谱的横截面非常小,因此其灵敏度非常低。但是,表面增强的拉曼效应以及共振增强效应可以显着提高该技术的灵敏度,甚至可以达到单分子水平。由此人们制造表面-拉曼效应,用于生物分子检测的增强拉曼散射。SERS具有很高的特异性,已被广泛用于有机污染物分析,重金属离子检测,蛋白质测量等。随着拉曼光谱学的迅速发展和对疾病诊断的迫切需求,SERS被广泛应用于分析与疾病相关的miRNA。
发明内容
本发明设计基于杂化链式扩增反应的纳米聚集SERS检测策略,构建了用于检测心梗miRNA的检测体系。
本发明的SERS生物传感器,采用下述方法制备得到:(1)将发卡探针DNA修饰在Fe3O4的表面;(2)加MCH孵育防止非特异性结合;(3)加入miRNA、辅链DA、辅链DB产生酶切产物;(4)预先将发夹DNA 1、发夹DNA 2修饰在AuNP的表面;(5)将分散好的发夹DNA 1、发夹DNA 2修饰的AuNP加入经步骤(3)孵育好的Fe3O4进行杂化链式扩增反应,反应过夜,离心洗涤。
优选的,步骤(1)Fe3O4预先进行活化:取0.1-0.3mg/mL的Fe3O4,加入EDC和NHS振摇过夜。Fe3O4可采用常用方法,如将FeCl3、聚乙二醇、醋酸钠、在乙二醇溶液中搅拌至均匀黄色溶液,再将溶液转移至反应釜中,200℃反应8小时,离心洗涤干燥。配置0.1-0.3mg/mL备用。EDC:1-乙基-3-(3-二甲基氨基丙基碳),NHS:N-羟基琥珀酰亚胺。加入EDC和NHS可使Fe3O4表面羧基化,带有羧基,可以和带氨基的DNA发生酰胺反应,从而将DNA链连接在Fe3O4的表面。优选的,可取500μL、0.2mg/mL的Fe3O4,加入10mM EDC 5μL,1mM NHS 5μL,振摇过夜,从而能够将Fe3O4更好的活化。
所述发卡探针DNA即S1-HP序列自行设计,如下:
5’-CTGATAAGCTACAGGACATCGAATAGTCTTTTTTGAGCGACACACTATrAGGAAGAGATACTTTTTTGACTATTCGA-3’。
步骤(2)MCH为氨基聚乙二醇NH-PEG。
优选的,步骤(1)是将发卡探针和Fe3O4添加到pH为7.0-7.5的PBS缓冲液中,室温孵育,借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中。
优选的,步骤(3)加Mg2+孵育过夜。所述辅链DA、辅链DB序列分别如下:
DA:5’-GTATCTCTTCCGCGATTAACCAAGTCTTAA-3’
DB:5’-AAACATCACTGGTTAGACCCATGTTAGTGTGTCGCTC-3’。
经步骤(3)酶切后的产物链S1-HP2序列如下:
CTGATAAGCTACAGGACATCGAATAGTCTTTTTTGAGCGACACACTAT
优选的,步骤(4)发夹DNA1、发夹DNA 2修饰在AuNP的表面。所述发夹DNA 1即S2-HP1、发夹DNA 2即S2-HP2,其序列自行设计,分别如下:
S2-HP1:
5’-TTTTTTTTTTTTTTTTTTTCATCGAATAGTCCTGACTGACTATTCGATGTCCTGT-3’
S2-HP2:
5’-AGTCAGGACTATTCGATGACAGGACATCGAATAGTCTTTTTTTTTTTTTTTTTTT-3’
所述的SERS生物传感器,具体的可采用下述方法制备得到:
(1)取500μL,0.2mg/mL的Fe3O4,加入10mM EDC 5μL,1mM NHS 5μL,振摇过夜;
(2)再将发卡探针DNA和上述活化的Fe3O4添加到1mLpH为7.4的PBS缓冲液中,室温振摇孵育10h以上,借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中;
(3)取上述步骤(2)溶液加1mM的MCH孵育半小时,孵育后用PBS洗涤;
(4)将1μM辅链DA、1μM辅链DB、和miRNA一起退火冷却后加入上述溶液中,再加10mMMg2+孵育过夜;
(5)将发夹DNA 1、发夹DNA 2单独修饰在10-20nm的AuNP上,然后,取步骤(4)孵育好的有酶切产物发生的Fe3O4加入其中,再加10mM Mg2+孵育过夜,借助磁铁用H2O洗涤。
上述所述的SERS生物传感器,优选的,步骤(4)孵育完成后借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中。
上述所述的SERS生物传感器,优选的,步骤(5)将发夹DNA 1、发夹DNA 2预先分别修饰在AuNP的表面,修饰好后分别加入10-5M的拉曼染料浸染30min,再离心洗涤,分散在TAE溶液中,再分别加入MCH孵育30min,离心洗涤再分散在TAE溶液中。所述将两个发卡环预先修饰在AuNP的表面,是常用修饰方法,其一般是先将发夹DNA 1、发夹DNA 2、AuNP和柠檬酸一起孵育,然后再加NaCl孵育,通过DNA间杂交可把AuNP带上去。另外,Fe3O4的粒径没有太大要求,AuNP的粒径一般选择十几纳米,组装效果较好,而AuNP的粒径15-17nm时组装效果最佳。
进一步方案取孵育好的有酶切产物发生的Fe3O4加入预先修饰好带有发卡DNA-AuNP(包括DNA1-AuNP、DNA2-AuNP)以及染料中,再加10mM Mg2+孵育过夜,孵育完成后借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中进行SERS检测。
本发明中,由于Fe3O4具有磁性,用磁铁可吸住触发产生的聚集体,去掉上清液,就是去掉没有反应的聚集体、金纳米颗粒,有聚集体产生用于检测。故可借助磁铁用H2O洗涤。
本发明所述SERS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miRNA-499、miRNA-208、miRNA-328,但不限于这些。
在进行琼脂糖凝胶验证杂化链式扩增反应策略时,由于miRNA反应出来的产物链量很少,杂化链式扩增反应的现象会不够明显,故可以直接采用定制购买的酶切后的产物链S1-HP2进行触发反应,不加入miRNA。定制的酶切后的产物链S1-HP2可购自专业的生物技术供应商如宁波康贝生化有限公司等等。
本发明的原理分析如下:
1、miRNA的识别及信号放大
根据miRNA特点,采用基于DNAzyme的能够精准捕获目标miRNA并能实现循环放大的纳米探针。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。然后,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、基于杂化链式扩增反应的纳米组装SERS检测策略的依据
基于杂化链式扩增反应的热点组装SERS检测策略,主要考虑三点。第一,设计杂化链式扩增所需的发夹结构序列:针对DNAzyme发夹探针上的酶切产物序列,设计能够被酶切产物触发的发夹单元,在防止背景干扰的同时,优化发夹单元提高组装效率。第二,纳米粒子组装条件的探索:纳米粒子的大小、形貌、发夹结构的修饰密度以及离子条件等都能影响组装的效果,为能实现超灵敏SERS检测,需要优化条件使纳米粒子高效组装以产生足够的热点。第三,分离方法的建立:在纳米组装体系中,针对没有反应的纳米元件以及非触发产生的聚集体,设计合成多功能分离基底,建立快速的分离方法。
3、磁性材料分离检测miRNA
SERS信号强度依赖于电磁场中两个或多个相邻金属纳米粒子间隙处的等离子体耦合,金纳米粒子可以算得上比较好的等离子体增强,通过杂化链式扩增反应聚集的Au聚集体具有更强的等离子体增强。本发明在纳米组装SERS检测体系中,针对没有反应的金纳米簇元件以及非触发产生的聚集体,采用Fe3O4作为磁性分离基底,通过表面功能化,排除非特异性吸附,快速分离出杂化链式扩增触发的组装体,得到只含有Fe3O4表面金纳米粒子的聚集体,具有很高的SERS信号,实现miRNA的检测。
本发明基于DNAzyme的miRNA循环放大探针设计,融入通用型DNAzyme发夹探针,发展出了用于分析多种心梗miRNA 的SERS检测策略。具体的,本发明在纳米组装SERS检测体系中,针对没有反应的金纳米簇元件以及非触发产生的聚集体,采用Fe3O4作为磁性分离基底,通过表面功能化,排除非特异性吸附,快速分离出杂化链式扩增触发的组装体,得到只含有Fe3O4表面金纳米粒子的聚集体,具有很高的SERS信号,实现了心梗miRNA的检测。
附图说明
图1SERS法检测microRNA的原理示意图;
图2为通过琼脂糖凝胶电泳实验验证了杂化链式扩增策略;
图3聚集体透射电镜图;
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的浓度与拉曼强度的关系图及线性关系;
图6是实施例2中miRNA-328的浓度与拉曼强度的关系图及线性关系;
图7是实施例3中miRNA-208的浓度与拉曼强度的关系图及线性关系;
具体实施方式
下述实施例是对于本发明内容的进一步说明以作为对本发明技术内容的阐释,但本发明的实质内容并不仅限于下述实施例所述,本领域的普通技术人员可以且应当知晓任何基于本发明实质精神的简单变化或替换均应属于本发明所要求的保护范围。
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,序列分别如下:
miRNA-499:UUA AGA CUU GCA GUG AUG UUU
miRNA-208:AAG CUU UUU GCU CGAAUU AUG U
miRNA-328:CUG GCC CUC UCU GCC CUU CCG U
实施例1
杂化链式扩增策略是在1.5%琼脂糖凝胶上证实。首先将两个发卡环S2-HP1、S2-HP2单独修饰在16nm左右的AuNP上,然后,将含有不同浓度的S1-HP2(定制,与酶切后的产物链相同)和10mM MgAc2的混合物在37℃下培养3小时以形成杂化链式扩增反应。在0.5×TBE缓冲液中以8V/cm 3进行电泳。最终的电泳结果由智能手机捕获,如图2。
SERS传感器制备:首先取500μL、0.2mg/mL的Fe3O4,加入EDC和NHS振摇过夜(10mMEDC 5μL,1mM NHS 5μL),将Fe3O4活化。再将发卡探针S1-HP和上述活化的Fe3O4添加到1mLPH为7.4的PBS缓冲液中,室温振摇孵育10h以上,借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中。取上述溶液加MCH孵育半小时,孵育后用PBS洗涤。将1μM辅链DA、1μM辅链DB、和不同浓度的miRNA一起退火冷却后加入上述溶液中,再加10mM Mg2+孵育过夜。孵育完成后借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中。将触发HCR反应(杂交链反应)的两个发卡环预先修饰在16nm AuNP(柠檬酸钠还原法合成)的表面,修饰好后分别加入10-5M的拉曼染料4-硝基苯硫酚浸染30min,再离心洗涤,分散在TAE(三羟甲基氨基甲烷)溶液中,再分别加入MCH孵育30min,离心洗涤再分散在TAE溶液中。取孵育好的有酶切产物发生的Fe3O4加入上述溶液中,再加10mM Mg2+孵育过夜,孵育完成后借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中进行SERS检测。
如图2所示是琼脂糖凝胶电泳分析AuNP上触发的杂化链式扩增策略。裸的AuNP(孔道1),S2-HP1修饰的AuNP(孔道2)和S2-HP2修饰的AuNP(孔道3)作为参照,S2-HP1修饰的AuNP和S2-HP2修饰的AuNP混合物(孔道4)显示出和单个发卡修饰的AuNP的原始条带,表明没有酶切后的产物,两个发卡是不会发生反应。当与酶切后的产物(1pM)孵育时,其纳米带的速度变慢(孔道5),表明形成了部分的金纳米粒子的聚集体。孔道6中为酶切后的产物,浓度是1nM,可用作对比,其纳米带的速度变得更慢,证明酶切后的产物触发了杂化链式扩增,形成更多的金纳米粒子的聚集体。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TEM图像,从TEM图上可以直观的看到金纳米粒子(AuNP)的聚集体聚体通过HCR反应聚集在Fe3O4的表面。证明这种组装方法是可行的。图中,大圆球是Fe3O4,大圆球周边的众多小球就是金纳米粒子。金纳米AuNP触发产生的聚集体,Fe3O4作为磁性分离基底,快速分离出杂化链式扩增触发的组装体,四氧化三铁与金纳米形成团簇结构,纳米粒子间产生间隙,从而产生大量热点,形成较强的SERS信号。
(以检测miRNA-499为例):如图4所示为了评价该方法的实际应用情况,研究了10%急性心肌梗死患者血清中miRNA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的浓度与拉曼强度的关系图及线性关系,在加入一系列浓度范围为1fM至1nM的miRNA-499可显著增强拉曼强度,这使得拉曼强度与miRNA-499浓度的对数之间呈线性关系。
实施例2
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-328,步骤和方法与实施例1的相同。如图6为miRNA-328的浓度与拉曼强度的关系图及线性关系,在加入一系列浓度范围为1fM至1nM的miRNA-328可显著增强拉曼强度,这使得拉曼强度与miRNA-328浓度的对数之间呈线性关系。效果同miRNA-499。
实施例3
在实施例1的基础上实施例3,把检测目标miRNA-499换成检测目标miRNA-208,如图7为miRNA-208的浓度与拉曼强度的关系图及线性关系,在加入一系列浓度范围为1fM至1nM的miRNA-208可显著增强拉曼强度,这使得拉曼强度与miRNA-208浓度的对数之间呈线性关系。效果同miRNA-499。
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。
Claims (8)
1.SERS生物传感器,采用下述方法制备得到:(1)将发卡探针DNA修饰在Fe3O4的表面;(2)加MCH防止非特异性结合;(3)加入miRNA、辅链DA、辅链DB产生酶切产物;(4)预先将发夹DNA 1、发夹DNA 2修饰在AuNP的表面;(5)将发夹DNA 1、发夹DNA 2修饰的AuNP加入经步骤(3)孵育好的Fe3O4进行杂化链式扩增反应,反应过夜,离心洗涤;
所述辅链DA、辅链DB序列分别如下:
DA:5’-GTATCTCTTCCGCGATTAACCAAGTCTTAA-3’
DB:5’-AAACATCACTGGTTAGACCCATGTTAGTGTGTCGCTC-3’,
所述发夹DNA 1、发夹DNA 2序列分别如下:
5’-TTTTTTTTTTTTTTTTTTTCATCGAATAGTCCTGACTGACTATTCGATGTCCTGT-3’
5’-AGTCAGGACTATTCGATGACAGGACATCGAATAGTCTTTTTTTTTTTTTTTTTTT-3’。
2.如权利要求1所述的SERS生物传感器,其特征在于,步骤(1)Fe3O4预先进行活化:取0.1-0.3mg/mL的Fe3O4,加入EDC和NHS振摇过夜。
3.如权利要求1所述的SERS生物传感器,其特征在于,步骤(1)是将发卡探针和Fe3O4添加到pH为7.0-7.5的PBS缓冲液中孵育,借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中。
4.如权利要求1所述的SERS生物传感器,其特征在于,步骤(3)加Mg2+孵育过夜。
5.如权利要求1所述的SERS生物传感器,其特征在于,采用下述方法制备得到:
(1)取500μL、0.2mg/mL的Fe3O4,加入10mM EDC 5μL,1mM NHS 5μL,振摇过夜;
(2)再将发卡探针DNA和上述活化的Fe3O4添加到1mL、pH为7.4的PBS缓冲液中,室温振摇孵育10h以上,借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中;
(3)取上述步骤(2)溶液加MCH孵育半小时,孵育后用PBS洗涤;
(4)将1μM辅链DA、1μM辅链DB、miRNA一起退火冷却后加入上述步骤(3)溶液中,再加10mM Mg2+孵育过夜;
(5)将两个发夹DNA 1、发夹DNA2单独修饰在10-20nm的AuNP上,然后将步骤(4)溶液加入其中,再加10mM Mg2+孵育过夜,借助磁铁用H2O洗涤。
6.如权利要求5所述的SERS生物传感器,其特征在于,步骤(4)孵育完成后借助磁铁用H2O洗涤一次,再溶于PBS缓冲液中。
7.如权利要求5所述的SERS生物传感器,其特征在于,步骤(5)将两个发夹预先单独修饰在AuNP的表面,修饰好后分别加入10-5M的拉曼染料浸染30min,再离心洗涤,分散在TAE溶液中,再分别加入MCH孵育,离心洗涤再分散在TAE溶液中。
8.权利要求1-7任一项所述SERS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。
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