CN116445585A - 一种基于expar的无酶指数扩增生物传感器及其应用 - Google Patents
一种基于expar的无酶指数扩增生物传感器及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于EXPAR的无酶指数扩增生物传感器及其应用。该传感器由第一部分EXPAR和第二部分AT‑HCR组成;第一部分EXPAR包括:用于指数放大反应的模板链TP,启动指数放大反应的启动子链P1,NEBuffer缓冲液,ThermoPol缓冲液,Nt.BstNBI切刻内切酶,DNA聚合酶,dNTPs;第二部分AT‑HCR包括:发卡H1,发卡H2,发卡H3,Tris‑HCl缓冲液。该传感器通过第一部分的扩增反应扩增出大量的目标物,然后通过第二部分的杂交链式HCR反应,结合扩增出来的目标物将发夹H1和H2无酶聚合连接形成长链,然后发夹H2的DNAzyme亚基接枝形成未被激活的DNAzyme,裂解底物H3被裂解后的一端用于反向触发激活HCR形成更多的核酸长链,两次放大检测信号,一次循环检测信号,实现了血清中肿瘤标志物的超灵敏检测。
Description
技术领域
本发明属于生物传感技术领域,具体涉及一种基于EXPAR的无酶指数扩增生物传感器及其应用。
背景技术
一直以来,聚合酶链式反应(Polymerase chainreaction,PCR)技术是微量DNA检测的“金标准”,被广泛用于DNA检测,尽管有着高灵敏度的优势,但是也存在着操作复杂、费用昂贵、易污染等问题。近年来,基于无酶链取代反应、新型纳米材料辅助信号放大反应、基于酶信号放大的电化学检测等信号放大策略,构建了包括光学、电化学在内的多种检测DNA的方法。其中光学包括了荧光分析法、比色法、化学发光法以及比较新的比率荧光法,具有简便快速、灵敏度高、特异性高等特点。在已报道的方法中,无酶链取代反应,如杂交链式反应(HybridizationChainReaction,HCR)、级联杂交反应、金属辅助脱氧核酶催化剂(DNAzyme),被广泛用于细胞内miRNA成像。传统的杂交链式反应(HCR),两个不能相互打开的发夹在引发剂的触发下依次交叉打开形成超长双链。然而,HCR的线性放大效率很低,不利于分析微量小分子。恒温指数扩增反应(Exponential Amplification Reaction,EXPAR),在模板上设计好序列,通过酶切,触发扩增反应,可以在30min内测出10aM的miRNA。脱氧核酶(Deoxyribozyme,DRz)是一种具有高效催化活性和特定识别序列的单链DNA,能识别特定序列的切割反应,可作为基因分析和诊断的工具,还可以通过切割双荧光标记底物,放大识别作用。然而,受到其固有的低扩增效率的限制,需要特定的金属离子的催化。不过DNAzyme依然是检测微量DNA的有力手段,依然有可能成为分子生物学的分析工具。
为了克服这些难题,近年来,人们已经把HCR与DNAzyme巧妙结合,开发了一种指数扩增效率的无酶扩增方法。通过目标物引发发夹DNA依次打开形成具有未激活DNAzyme的超长双链,随后在特定金属离子的催化下,切割标记好的荧光底物。然而,在活细胞检测中微量的miRNA难以被捕捉,会出现荧光信号弱甚至没有荧光信号的情况。因此,迫切需要能够将活细胞中的miRNA大量扩增到能被轻易检测到的方法。
发明内容
为了解决上述问题,本发明提供了一种基于EXPAR的无酶指数扩增生物传感器及其应用。通过利用EXPAR扩增出大量原本痕量的目标物,再利用目标物作为引发链,引发发夹H1、H2进行HCR反应,再将HCR反应与DNAzyme结合起来,形成一个内循环,持续不断的引发HCR反应直至体系中的H1被反应完全;利用放大反应将目标物富集并利用碱基配对使目标物特异性识别,将识别到的目标物转化为FRET信号输出,从而实现对肿瘤标志物的高灵敏度、高特异性检测。
为实现上述目的,本发明采取了以下技术方案:
一种基于EXPAR的无酶指数扩增生物传感器,其由第一部分EXPAR和第二部分AT-HCR组成;第一部分EXPAR包括:用于指数放大反应的模板链TP,启动指数放大反应的启动子链P1,NEBuffer缓冲液,ThermoPol缓冲液,Nt.BstNBI切刻内切酶,DNA聚合酶,dNTPs;第二部分AT-HCR包括:发卡H1,发卡H2,发卡H3,Tris-HCl缓冲液;
其中,模板链TP:
5'-CCTGCTCCAAAAATCCATTAACAGACTCTCTGTTAGGAACTGTATCCGAA-P-3',启动子链P1:5'-TCGGATACAGTTCCTAACAGA-3',
发卡H1:5'-ATTTTTGGAGCAGGCACACCCTGCTCCAAAAATCCATT-cy5-3',发卡H2:
5'-
AAACCGAGGCTAGCCGTGTGCCTGCTCCAAAAA/iCy3dT/AATGGATTTTTGGAGCAGGGTACAACGACGCCTGC-3',发卡H3:5'-AATGGATTTTTGGAGCAGGCG/rA//rU/CGGTTTTCCAAATATCCATT-3';
其中,所述NEBuffer缓冲液配方为:1000mM NaCl,500mM Tris-HCl,100mM MgCl2,1000μg/mL BSA,pH=7.9@25℃;所述ThermoPol缓冲液配方为:200mM Tris-HCl,100mMKCl,100mM(NH4)2SO4、20mM MgSO4、1%Triton X-100、pH=8.8@25℃;所述Tris-HCl缓冲液配方为:100mM NaCl,20mM MgCl2@pH7.4。
上述一种基于EXPAR的无酶指数扩增生物传感器的制备方法,包括以下步骤:
(1)将1μL 5μM模板链TP与1μL 50nM启动子链P1溶于23μLNEBuffer缓冲液中,混匀后于95℃金属浴条件下加热反应5min,自热冷却至室温,得到TP-P1杂交溶液A;
(2)将18μL ThermoPol缓冲液、1μL 15U/μLNt.BstNBI切刻内切酶、1μL 3U/μL DNA聚合酶、5μL 3000mM dNTPs混匀,得到溶液B;
(3)将25μLTP-P1杂交溶液A与25μL溶液B混匀,然后立即在55℃条件下孵育40min,之后于80℃条件下孵育20min,以灭活DNA聚合酶和Nt.BstNBI切刻内切酶,得到混合溶液C;
(4)将1μL 100μM发卡H1溶于9μL超纯水中得到浓度为10μM的H1溶液,95℃条件下退火5min,自然冷却至室温;将1μL 100μM发卡H2溶于9μL超纯水中得到浓度为10μM的H2溶液,95℃条件下退火5min,自然冷却至室温;取2μL退火后的10μM H1溶液和2μL退火后的10μM H2溶液,与50μL混合溶液C混匀,于37℃条件下反应1h,得到混合溶液D;
(5)将1μL 100μM发卡H3溶于9μL超纯水中得到浓度为10μM的H3溶液,95℃条件下退火5min,自然冷却至室温;取2.5μL退火后的10μM H3溶液溶于43.5μL 200mM Tris-HCl缓冲液中,得到溶液E;
(6)将56.5μL混合溶液D与43.5μL溶液E混匀,然后在37℃条件下避光反应2h,得到总体积为100μL的待测溶液F;在528nm的激发光波长下测量,记录在550-700nm范围内的荧光强度。
上述一种基于EXPAR的无酶指数扩增生物传感器在制备检测乳腺癌生物标志物的产品中的应用。
进一步的,上述乳腺癌生物标志物为microRNA-1246。
本发明的显著优点在于:
(1)本发明将微量或不可见的生物标志物通过EXPAR扩增富集成可轻易检测的范围,大大增加了检测范围;
(2)本发明具有响应时间快的特点,并且具有高度的特异性,使其在快速及准确检测方面具有一定潜力;
(3)本发明将EXPAR与HCR、DNAzyme结合,使信号得到两次扩增,一次循环,从而使检测限更低,灵敏度更高;
(4)本发明所构建的EXPAR-HCR@DNAzyme生物传感器,可实现肿瘤标志物的超灵敏检测。
附图说明
图1是基于EXPAR的无酶指数扩增生物传感器的原理图。
图2是实施例1中加入不同浓度目标物后的荧光响应图。
图3是实施例2中加入不同干扰物与目标物的荧光响应图。
具体实施方式
为了使本发明的内容更容易理解,以下结合了具体实施方式对本发明所述的技术方案进行进一步的说明,但本发明不仅限于此。
本申请中,microRNA-1246序列为:5’-AAUGGAUUUUUGGAGCAGG-3’,microRNA-141序列为:5’-UAACACUGUCUGGUAAAGAUGG-3’,microRNA-21序列为:5’-UAGCUUAUCAGACUGAUGUUGA-3’。
本申请中,所述一种用于乳腺癌生物标志物超灵敏检测的基于EXPAR的无酶指数扩增生物传感器EXPAR-AT-HCR,其由两部分组成:第一部分(EXPAR),包括:用于指数放大反应的模板链TP、用于启动指数放大反应的启动子链P1以及NEBuffer缓冲液形成的TP-P1杂交溶液A,含ThermoPol缓冲液、DNA聚合酶、Nt.BstNBI切刻内切酶以及dNTPs的混合溶液B;第二部分(AT-HCR),包括:发生HCR反应的发卡H1(含有cy5)、发卡H2(含有cy3和接枝DNAzyme的亚基)、发卡H3(DNAzyme裂解底物),Tris-HCl缓冲液。
其中,TP:5'-CCTGCTCCAAAAATCCATTAACAGACTCTCTGTTAGGAACTGTATCCGAA-P-3';
P1:5'-TCGGATACAGTTCCTAACAGA-3';
H1:5'-ATTTTTGGAGCAGGCACACCCTGCTCCAAAAATCCATT-cy5-3';
H2:5'-AAACCGAGGCTAGCCGTGTGCCTGCTCCAAAAA/iCy3dT/AATGGATTTTTGGAGCAGGGTACAACGACGCCTGC-3';
H3:5'-AATGGATTTTTGGAGCAGGCG/rA//rU/CGGTTTTCCAAATATCCATT-3';
NEBuffer缓冲液配方为:1000mM NaCl,500mM Tris-HCl,100mM MgCl2,1000μg/mLBSA,pH=7.9@25℃;
ThermoPol缓冲液配方为:200mM Tris-HCl,100mM KCl,100mM(NH4)2SO4、20mMMgSO4、1%Triton X-100、pH=8.8@25℃;
Tris-HCl缓冲液配方为:100mM NaCl,20mM MgCl2@pH7.4。
本发明所述一种用于乳腺癌生物标志物超灵敏检测的基于EXPAR的无酶指数扩增生物传感器,其原理如图1所示。等温指数扩增(EXPAR)反应中,若启动子链P1不存在,在DNA聚合酶的作用下不会形成具有切刻酶识别位点的粘性末端,扩增反应就会停止。只有在启动子链P1存在的情况下,才能形成具有切刻酶位点的5'粘性末端,扩增反应才会持续不断,从而产生大量游离的目标物C*,因为人体内的目标物是很少的,可以明显的将检测限放大。由于发夹H1和H2在目标物C*不存在的情况下不会相互打开结合,所以EXPAR扩增产生的大量游离C*能与发夹H1的粘性末端结合形成双链,与目标物C*结合的H1在熵驱动下能与发夹H2的粘性末端结合,C*-H1-H2又能与发夹H1的粘性末端结合,如此循环,形成了未激活的HCR-DNAzyme长链,完成了检测过程的第一次放大,即发生了HCR反应。接着未激活的HCR-DNAzyme长链与发卡H3(DNAzyme裂解底物)结合并在Mg+的催化下裂解底物H3。发夹H3含有目标物片段的一部分,被裂解且含有目标物片段的一部分可以用于反向触发HCR,实现了检测过程的第二次放大,可以得到显著增强的荧光信号。
实施例1
一种以乳腺癌生物标志物microRNA-1246为目标物进行超灵敏检测的基于EXPAR的无酶指数扩增生物传感器的制备方法,包括以下步骤:
(1)将1μL 5μM模板链TP与1μL 50nM启动子链P1溶于23μLNEBuffer缓冲液中,混匀后于95℃金属浴条件下加热反应5min,自热冷却至室温,得到TP-P1杂交溶液A。
(2)将18μL ThermoPol缓冲液、1μL 15U/μL Nt.BstNBI切刻内切酶、1μL 3U/μLVent(exoˉ)DNA聚合酶、5μL 3000mM dNTPs混匀,得到溶液B。
(3)将25μL TP-P1杂交溶液A与25μL溶液B混匀,然后立即在55℃条件下孵育40min,之后于80℃条件下孵育20min,以灭活Vent(exoˉ)DNA聚合酶和Nt.BstNBI切刻内切酶,得到混合溶液C,混合溶液C中含有C*(C*与目标物microRNA-1246具有相同碱基序列)。将混合溶液C分别稀释对应倍数,并加入超纯水,得到50μL不同浓度混合溶液C·(内含C*浓度10fM至100nM)。
(4)将1μL 100μM发卡H1溶于9μL超纯水中得到浓度为10μM的H1溶液,95℃条件下退火5min,自然冷却至室温;将1μL 100μM发卡H2溶于9μL超纯水中得到浓度为10μM的H2溶液,95℃条件下退火5min,自然冷却至室温;取2μL退火后的10μM H1溶液和2μL退火后的10μM H2溶液,与50μL不同浓度混合溶液C·混匀,于37℃条件下反应1h,得到混合溶液D。
(5)将1μL 100μM发卡H3溶于9μL超纯水中得到浓度为10μM的H3溶液,95℃条件下退火5min,自然冷却至室温;取2.5μL退火后的10μM H3溶液溶于43.5μL 200mM Tris-HCl缓冲液中,得到溶液E。
(6)将56.5μL混合溶液D与43.5μL溶液E混匀,然后在37℃条件下避光反应2h,得到总体积为100μL的待测溶液F;将待测溶液F在528nm的激发光波长下测量,并记录其在550-700nm范围内的荧光强度。
结果如图2所示,图2显示了检测体系中目标物浓度变化与荧光强度之间的关系。图中表明,荧光强度随着目标物浓度(从0nM到100nM之间)的升高而增强。同时,根据空白样品(目标物浓度0nM)的背景信号平均值加3σ(空白样品标准差的3倍)方法,计算出检测限达0.66fM。
实施例2
一种基于EXPAR的无酶指数扩增生物传感器的制备方法,包括以下步骤:
(1)选择性实验:将1μL 100μM目标物(目标物选自miR-21、miR-14、miR-1246中的一种或多种)溶于9μL超纯水中得到得到浓度为10μM的目标物溶液,于95℃退火5min,自然冷却至室温;将1μL 100μM探针H1溶于9μL超纯水中得到浓度为10μM的H1溶液,95℃条件下退火5min,自然冷却至室温;将1μL 100μM探针H2溶于9μL超纯水中得到浓度为10μM的H2溶液,95℃条件下退火5min,自然冷却至室温;取2μL退火后的10μMH1溶液和2μL退火后的10μMH2溶液,与1μL退火后的10μM目标物溶液于92.5μL200mM Tris-HCl缓冲液中混匀,得到混合溶液。
(2)将1μL 100μM探针H3溶于9μL超纯水中得到浓度为10μM的H3溶液,95℃条件下退火5min,自然冷却至室温;取2.5μL退火后的10μM H3溶液溶于92.5μL步骤(1)的混合溶液,于37℃避光反应2h,得到总体积为100μL的待测溶液;将待测溶液在528nm的激发光波长下测量,并记录样品在550-700nm范围内的荧光强度。
结果如图3所示,与空白(blank:未加任何目标物)和其它目标物相比,miR-1246引起的相对荧光信号的增加至少13.8倍(1.52/0.11);另外,在混合其它目标物(mixture1:miR-1246与miR-21混合;mixture2:miR-1246与miR-141混合)的情况下依旧能表现出明显的荧光强度,这表明本发明的传感器对miR-1246具有良好的特异性。
实施例3一种基于EXPAR的无酶指数扩增生物传感器的制备方法,包括以下步骤:将人血清用含200mM NaCl的超纯水稀释10倍代替实施例1中所用到的NEBuffer缓冲液。然后按实施例1中所述步骤,使用标准加样法进行回收实验。对三种不同浓度(100fM,10pM和1pM)的目标物miR-1246进行回收测定。通过三个独立的实验计算相对标准偏差(RSD)。
为了研究所提出的基于EXPAR的无酶指数扩增生物传感器在复杂生物样品中的适用性,使用健康的人血清样品进行回收实验,以确定传感器用于肿瘤标志物miR-1246检测的可靠性。将不同浓度的miR-1246加入人血清样品中。结果如表1,回收率为96.0%~106.0%,由三个独立的实验计算的RSD为4.56%~7.38%。表明本发明提出的基于EXPAR的无酶指数扩增生物传感器EXPAR-AT-HCR在实际样品中具有出色的miR-1246检测能力。
表1
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (5)
1.一种基于EXPAR的无酶指数扩增生物传感器,其特征在于:由第一部分EXPAR和第二部分AT-HCR组成;第一部分EXPAR包括:用于指数放大反应的模板链TP,启动指数放大反应的启动子链P1,NEBuffer缓冲液,ThermoPol缓冲液,Nt.BstNBI切刻内切酶,DNA聚合酶,dNTPs;第二部分AT-HCR包括:发卡H1,发卡H2,发卡H3,Tris-HCl缓冲液;
其中,模板链TP:
5'-CCTGCTCCAAAAATCCATTAACAGACTCTCTGTTAGGAACTGTATCCGAA-P-3',
启动子链P1:5'-TCGGATACAGTTCCTAACAGA-3',
发卡H1:5'-ATTTTTGGAGCAGGCACACCCTGCTCCAAAAATCCATT-cy5-3',
发卡H2:
5'-AAACCGAGGCTAGCCGTGTGCCTGCTCCAAAAA/iCy3dT/AATGGATTTTTGGAGCAGGGTACAACGACGCCTGC-3',
发卡H3:5'-AATGGATTTTTGGAGCAGGCG/rA//rU/CGGTTTTCCAAATATCCATT-3'。
2.根据权利要求1所述的一种基于EXPAR的无酶指数扩增生物传感器,其特征在于:所述NEBuffer缓冲液配方为:1000mM NaCl,500mM Tris-HCl,100mM MgCl2,1000μg/mL BSA,pH=7.9 @ 25℃;所述ThermoPol缓冲液配方为:200mM Tris-HCl,100mM KCl,100mM (NH4)2SO4、20mM MgSO4、1%Triton X-100、pH=8.8 @ 25℃;所述Tris-HCl缓冲液配方为:100mMNaCl,20mM MgCl2 @ pH7.4。
3.如权利要求1所述的一种基于EXPAR的无酶指数扩增生物传感器的制备方法,其特征在于,包括以下步骤:
(1)将1µL 5µM模板链TP与1µL 50nM启动子链P1溶于23µL NEBuffer缓冲液中,混匀后于95℃金属浴条件下加热反应5min,自热冷却至室温,得到TP-P1杂交溶液A;
(2)将18µL ThermoPol缓冲液、1µL 15U/µL Nt.BstNBI切刻内切酶、1µL 3U/µL DNA聚合酶、5µL 3000mM dNTPs混匀,得到溶液B;
(3)将25µL TP-P1杂交溶液A与25µL溶液B混匀,然后立即在55℃条件下孵育40min,之后于80℃条件下孵育20min,以灭活DNA聚合酶和Nt.BstNBI切刻内切酶,得到混合溶液C;
(4)将1µL 100µM发卡H1溶于9µL超纯水中得到浓度为10µM的H1溶液,95℃条件下退火5min,自然冷却至室温;将1µL 100µM发卡H2溶于9µL超纯水中得到浓度为10µM的H2溶液,95℃条件下退火5min,自然冷却至室温;取2µL退火后的10µM H1溶液和2µL退火后的10µM H2溶液,与50µL混合溶液C混匀,于37℃条件下反应1h,得到混合溶液D;
(5)将1µL 100µM发卡H3溶于9µL超纯水中得到浓度为10µM的H3溶液,95℃条件下退火5min,自然冷却至室温;取2.5µL退火后的10µM H3溶液溶于43.5µL 200mM Tris-HCl缓冲液中,得到溶液E;
(6)将56.5µL混合溶液D与43.5µL溶液E混匀,然后在37℃条件下避光反应2h,得到总体积为100µL的待测溶液F;在528nm的激发光波长下测量,记录在550-700nm范围内的荧光强度。
4.如权利要求1所述的一种基于EXPAR的无酶指数扩增生物传感器在制备检测乳腺癌生物标志物的产品中的应用。
5.根据权利要求4所述的一种基于EXPAR的无酶指数扩增生物传感器在制备检测乳腺癌生物标志物的产品中的应用,其特征在于:所述乳腺癌生物标志物为microRNA-1246。
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