CN109387493A - 一种pH响应型自辅因子DNAzyme ZnO纳米探针及其制备方法与应用 - Google Patents
一种pH响应型自辅因子DNAzyme ZnO纳米探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种pH响应型自辅因子DNAzyme ZnO纳米探针及其制备方法与应用,包括如下步骤:1)将ZnO纳米粒子的Tris溶液与多巴胺Tris溶液混合后,超声处理设定时间;2)离心去除多余的多巴胺溶液,将ZnO纳米粒子分散在DEPC处理的超纯水中,得到混合液A;3)将混合液A与设定浓度的识别发夹DNA混合后,涡旋设定时间,离心去除多余发夹DNA,即得。
Description
技术领域
本发明属于功能纳米材料和生物检测领域,具体涉及一种pH响应型自辅因子DNAzyme ZnO纳米探针及其制备方法与在microRNA的多重检测和活细胞成像中的应用。
背景技术
MicroRNA(miRNA)是一类存在于真核细胞内的非编码内源性RNA分子,由18-24个碱基组成。MiRNA通过降解靶基因mRNA或者抑制其转录后翻译调控基因表达。MiRNA能够调控一系列生理学过程,例如细胞的增殖、分化和凋亡。越来越多的研究表明miRNA的异常表达与癌症的发生和发展密切相关。因此,miRNA已经被作为癌症的诊断标志物和治疗靶点使用。在miRNA的调控网络中,多种miRNA以同时和协同的方式调控生物学过程。因此,细胞内miRNA的多重检测对于癌症的准确诊断和治疗至关重要。
最近,基于一步链置换的方法被提出用于细胞内miRNA的检测。然而,由于细胞内miRNA较低的表达水平,所以需要具有更高灵敏度的方法。基于此,一些基于酶的信号放大方法被提出。然而,将酶递送进细胞需要将细胞固定和脱水。这可能会造成细胞的损伤和死亡,不能满足活细胞内miRNA检测的需求。为解决此问题,一些无酶的信号放大方法被提出,例如催化发夹自组装、杂交链式反应和DNAzyme切割放大反应。其中,DNAzyme切割放大反应由于较低的背景信号和较快的反应速率受到了广泛关注。乐晓春课题组构建了一种DNAzyme驱动的三维DNA行走器用于活细胞内miRNA的检测。然而,细胞内的金属离子不足以驱动DNAzyme切割放大反应,需要额外的辅因子递送过程。因此,急需更加自动化的基于DNAzyme的策略用于活细胞内miRNA的检测。
发明内容
针对上述现有技术中存在的技术问题,本发明的第一个目的是提供一种pH响应型自辅因子DNAzyme ZnO纳米探针。
本发明的第二个目的是提供上述pH响应型自辅因子DNAzyme ZnO纳米探针的制备方法。
本发明的第三个目的是提供上述pH响应型自辅因子DNAzyme ZnO纳米探针在microRNA的多重检测和活细胞成像中的应用。
为了解决以上问题,本发明的技术方案为:
一种pH响应型自辅因子DNAzyme ZnO纳米探针的制备方法,包括如下步骤:
1)将ZnO纳米粒子的Tris溶液与多巴胺Tris溶液混合后,超声处理设定时间;
2)离心去除多余的多巴胺溶液,将ZnO纳米粒子分散在DEPC处理的超纯水中,得到混合液A;
3)将混合液A与设定浓度的识别发夹DNA混合后,涡旋设定时间,离心去除多余发夹DNA,即得。
ZnO纳米粒子具有独特的理化性质,其遇酸溶解的性质使得其具有生物学应用的潜能。本发明中利用ZnO遇酸溶解的性质,制备了一种pH响应型自辅因子DNAzyme ZnO纳米探针,并将其用于miRNA的多重检测和活细胞成像。聚多巴胺在碱性条件下通过自聚反应沉积在ZnO纳米粒子表面。发夹DNA通过π-π相互作用吸附在聚多巴胺夹层上。细胞内核内体和溶酶体的pH值在5.0附近。制备的pH响应型自辅因子DNAzyme ZnO纳米探针具有核壳状结构,包括ZnO纳米粒子核,聚多巴胺夹层和功能发夹DNA外层。
优选的,步骤1)中,ZnO纳米粒子的Tris溶液的pH值为8.5。
优选的,步骤1)中,ZnO纳米粒子与多巴胺的质量浓度比为2:1。
进一步优选的,步骤1)中,ZnO纳米粒子的Tris溶液的浓度为1mg/mL。
进一步优选的,步骤1)中,多巴胺Tris溶液的pH值为8.5。
优选的,步骤1)中,超声处理的时间为1h,在室温下超声。
优选的,步骤3)中,所述识别发夹DNA为H1和H3。
优选的,步骤3)中,涡旋的时间为1h,室温下涡旋。
优选的,所述制备方法还包括将制备得到的ZnO纳米探针分散在pH值为7.4的HEPES缓冲溶液中的步骤。
上述制备方法制备得到的pH响应型自辅因子DNAzyme ZnO纳米探针。
上述pH响应型自辅因子DNAzyme ZnO纳米探针在microRNA的多重检测和活细胞成像中的应用。
pH响应型自辅因子DNAzyme ZnO纳米探针检测microRNA的方法,具体包括如下步骤:将ZnO纳米探针与pH值为5.0的Tris缓冲溶液混合,孵育设定时间后,加入HEPES缓冲溶液、DEPC处理的超纯水和待测的microRNA样品,孵育后进行荧光测量。
pH响应型自辅因子DNAzyme ZnO纳米探针应用于活细胞成像的方法,具体包括如下步骤:将待测细胞分散在玻璃皿中,在37℃下5%的CO2气氛中孵育24h。然后加入用细胞培养基稀释的ZnO纳米探针,再在37℃下孵育4h。最后,用PBS缓冲溶液将细胞清洗后成像。
本发明的有益效果为:
当纳米探针通过内吞作用进入细胞后,细胞内的酸性环境会使ZnO纳米粒子核分解,释放出功能发夹DNA和Zn2+。释放出的Zn2+可以作为DNAzyme切割放大反应的辅因子,避免了额外的辅因子递送过程。识别发夹DNA(H1和H3)识别目标miRNA后暴露出DNAzyme序列。暴露出的DNAzyme序列在Zn2+的存在下切割报告发夹DNA(H2和H4)的DNAzyme底物部分,使荧光团和猝灭团相互远离,荧光恢复。识别发夹DNA和miRNA的杂交双链继续切割报告发夹DNA,产生增强的荧光信号用于灵敏检测miR-21和miR-373。几种细胞内miR-21的不同表达水平被区分。而且,同一活细胞内两种miRNA的灵敏和同时成像被实现。上述结果表明,该策略在癌症的准确诊断和治疗方面具有较大的应用潜力。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。
图1是基于pH响应型自辅因子DNAzyme ZnO纳米探针的miRNA检测原理示意图;
图2是不同情况下两种miRNA检测的荧光光谱;
图3是不同浓度两种miRNA的荧光发射光谱以及与F-F0的线性关系;
图4是不同miRNA的荧光响应;
图5是不同孵育时间下HepG-2细胞内miR-21的荧光成像;
图6是不同细胞内miR-21的荧光成像;
图7是HepG-2细胞内miR-21和miR-373的荧光成像。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
实验部分
试剂和仪器
所有的DNA和miRNA核酸序列由生工生物工程有限公司(中国,上海)合成。该策略中的DNA和miRNA序列在表格1中列出。ZnO胶体溶液(ZnO纳米粒子,50%)购自麦克林生化科技有限公司(中国,上海)。盐酸多巴胺购自Sigma-Aldrich(中国,上海)。所有的细胞培养试剂购自BI(以色列,Beit-Haemek)。0.1%DEPC(焦碳酸二乙酯)处理的超纯水被用于整个实验过程中。
所有的荧光光谱都通过Hitachi F-7000荧光光谱仪(日立有限公司,日本,东京)测量。MiR-21检测的激发波长为488nm,发射光谱范围为505~620nm。MiR-373检测的激发波长为633nm,发射光谱范围为650~740nm。荧光成像实验通过Axio Observer 3倒置荧光显微镜(卡尔蔡司,德国)执行。
表1.该工作使用到的寡核苷酸序列
ZnO纳米粒子的功能化
首先,ZnO胶体溶液通过离心清洗后分散在pH为8.5的Tris缓冲溶液中,浓度为1mg/mL。然后,1mL ZnO纳米粒子溶液与10mL多巴胺Tris溶液(50μg/mL,pH 8.5)混合,室温超声1h。随后,该混合溶液被离心去除多余的多巴胺溶液,并分散在DEPC超纯水中。然后,1mL上述溶液(0.1mg/mL)与60μL发夹DNA(10μM)混合,在室温下涡旋1h。最后,该混合溶液被离心去除多余的DNA,并分散在pH为7.4的HEPES缓冲溶液中。
试管内miRNA的检测
10μL ZnO纳米粒子探针与2μL pH为5.0的Tris缓冲溶液混合,在37℃下孵育0.5h。随后,加入10μL 5×HEPES缓冲溶液,23μL DEPC超纯水和5μL miRNA,在37℃下孵育2h。
细胞培养
L02细胞在添加有10%牛血清和1%双抗的DMEM培养基中孵育。Hela细胞和HepG-2细胞在添加有10%牛血清和1%双抗的RPMI-1640培养基中孵育。所有的细胞在37℃下5%的CO2气氛中孵育。
荧光显微成像
L02细胞、HepG-2细胞和Hela细胞被分散在20mm的玻璃皿中,在37℃下5%的CO2气氛中孵育24h。然后,加入用细胞培养基稀释的10μg/mL的ZnO纳米探针,再在37℃下孵育4h。最后,用PBS缓冲溶液将细胞清洗三遍,成像。
结果与讨论
原理
基于pH响应型自辅因子DNAzyme ZnO纳米探针的miRNA检测原理如图1所示。首先,聚多巴胺在碱性条件下通过自聚反应沉积在ZnO纳米粒子表面。发夹DNA通过π-π相互作用吸附在聚多巴胺夹层上。识别发夹DNA(H1和H3)包括miRNA的识别序列和被锁住的DNAzyme序列。当miRNA不存在时,DNAzyme序列不能切割报告发夹DNA的底物部分,保证了较低的背景信号。当纳米探针通过内吞作用进入细胞后,细胞内的酸性环境会使ZnO纳米粒子核分解,释放出功能发夹DNA和Zn2+。释放出的Zn2+可以作为DNAzyme切割放大反应的辅因子,避免了额外的辅因子递送过程。当miRNA存在时,识别发夹DNA识别目标miRNA后暴露出DNAzyme序列。暴露出的DNAzyme序列在Zn2+的存在下切割报告发夹DNA,使荧光团和猝灭团相互远离,荧光恢复。识别发夹DNA和miRNA的杂交双链继续切割报告发夹DNA,产生增强的荧光信号用于灵敏检测miR-21和miR-373。几种细胞内miR-21的不同表达水平被区分。而且,同一活细胞内两种miRNA的灵敏和同时成像被实现。上述结果表明,该策略在癌症的准确诊断和治疗方面具有较大的应用潜力。
该传感系统的可行性
我们通过测量不同情况下反应体系的荧光发射光谱验证该策略的可行性,如图2所示。图2中A图是miR-21检测的荧光光谱图。曲线a是只有H2存在时的荧光光谱。当miR-21不存在时,系统表现出很弱的荧光信号(曲线b,中间的曲线)。此结果表明当miRNA不存在时,被封住的DNAzyme序列不能切割报告发夹DNA的底物部分,保证了较低的背景信号。与之相反,当miRNA存在时,系统表现为明显的荧光强度增强(曲线c),这表明miRNA能够与H1结合释放出DNAzyme序列,触发后续的DNAzyme切割放大反应。对于miR-373检测获得了相同的实验现象(图2中B图)。以上实验结果表明,该检测方法是可行的,能够用于同时检测两种miRNA。
灵敏度
该方法的分析性能在试管实验中被验证。如图3中A图所示(miR-21的浓度从a到k分别是0、100pM、500pM、1nM、5nM、10nM、15nM、20nM、25nM、30nM、50nM),随着miR-21浓度从0增加到50nM,荧光强度逐渐增加。如图3中B图所示,净信号F-F0与100pM-30nM浓度范围的miR-21呈线性关系,检测限为54pM。如图3中C图所示(miR-373的浓度从a到j分别是0、100pM、500pM、2nM、4nM、8nM、12nM、16nM、20nM、50nM),随着miR-373浓度从0增加到50nM,荧光强度逐渐增加。如图3中D图所示,净信号F-F0与100pM-20nM浓度范围的miR-373呈线性关系,检测限为38pM。该方法较高的灵敏度主要归因于DNAzyme切割放大反应高的放大效率。
选择性
该方法对于两种miRNA的选择性在试管实验中被验证。如图4中A图和B图所示,干扰miRNA的荧光强度与背景信号相当。与干扰miRNA相比,目标miRNA表现出显著增强的荧光信号。以上结果表明,该方法具有良好的特异性。
活细胞成像实验
我们考察了不同孵育时间下HepG-2细胞内miR-21的成像情况。如图5所示,随着孵育时间的增加,HepG-2细胞内的荧光强度逐渐增加,在4h时达到饱和。荧光强度不会随着时间的增加再增加。该结果表明,该策略可以用于活细胞内miRNA的成像。
不同细胞内的成像实验
为了证明该方法能够区分miRNA的不同表达水平,进行了L02细胞、HepG-2细胞和Hela细胞内miR-21的成像实验。如图6所示,HepG-2细胞和Hela细胞内表现为较强的荧光强度,表示HepG-2细胞和Hela细胞内相对较高的miR-21表达水平。相反的,L02细胞内表现为较弱的荧光强度,表示L02细胞内相对较低的miR-21表达水平。以上结果表明,该方法能够区分活细胞内miRNA不同的表达水平。
活细胞内miRNA的多重成像实验
进行了活细胞内miR-21和miR-373的同时成像实验。如图7所示,FAM和Cy5荧光分别代表miR-21和miR-373的表达。该结果清晰地表明了HepG-2细胞内miR-21和miR-373的不同表达水平和空间分布。以上结果表明,该方法能够用于活细胞内两种miRNA的同时成像。
总结
总体来说,一种pH响应型自辅因子DNAzyme ZnO纳米探针被构建用于miRNA的多重检测和活细胞成像。细胞内酸性环境降解ZnO纳米粒子产生Zn2+可以作为DNAzyme切割放大反应的辅因子,避免了额外的辅因子递送过程。DNAzyme切割放大反应保证了较高的灵敏度。几种细胞内miR-21的不同表达水平被区分。而且,同一活细胞内两种miRNA的灵敏和同时成像被实现。上述结果表明,该策略在癌症的准确诊断和治疗方面具有较大的应用潜力。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
Claims (10)
1.一种pH响应型自辅因子DNAzyme ZnO纳米探针的制备方法,其特征在于:包括如下步骤:
1)将ZnO纳米粒子的Tris溶液与多巴胺Tris溶液混合后,超声处理设定时间;
2)离心去除多余的多巴胺溶液,将ZnO纳米粒子分散在DEPC处理的超纯水中,得到混合液A;
3)将混合液A与设定浓度的识别发夹DNA混合后,涡旋设定时间,离心去除多余发夹DNA,即得。
2.根据权利要求1所述的制备方法,其特征在于:步骤1)中,ZnO纳米粒子的Tris溶液的pH值为8.5;
优选的,步骤1)中,ZnO纳米粒子与多巴胺的质量浓度比为2:1;
进一步优选的,步骤1)中,ZnO纳米粒子的Tris溶液的浓度为1mg/mL;
进一步优选的,步骤1)中,多巴胺Tris溶液的pH值为8.5。
3.根据权利要求1所述的制备方法,其特征在于:步骤1)中,超声处理的时间为1h。
4.根据权利要求1所述的制备方法,其特征在于:步骤3)中,所述识别发夹DNA为H1和H3。
5.根据权利要求1所述的制备方法,其特征在于:步骤3)中,涡旋的时间为1h。
6.根据权利要求1所述的制备方法,其特征在于:所述制备方法还包括将制备得到的ZnO纳米探针分散在pH值为7.4的HEPES缓冲溶液中的步骤。
7.权利要求1-6任一所述制备方法制备得到的pH响应型自辅因子DNAzyme ZnO纳米探针。
8.权利要求6所述pH响应型自辅因子DNAzyme ZnO纳米探针在microRNA的多重检测和活细胞成像中的应用。
9.pH响应型自辅因子DNAzyme ZnO纳米探针检测microRNA的方法,其特征在于:具体包括如下步骤:将ZnO纳米探针与pH值为5.0的Tris缓冲溶液混合,孵育设定时间后,加入HEPES缓冲溶液、DEPC处理的超纯水和microRNA样品,孵育后进行荧光测量。
10.pH响应型自辅因子DNAzyme ZnO纳米探针应用于活细胞成像的方法,其特征在于:具体包括如下步骤:将待测细胞分散在玻璃皿中,在37℃下5%的CO2气氛中孵育24h。然后加入用细胞培养基稀释的ZnO纳米探针,再在37℃下孵育4h。最后,用PBS缓冲溶液将细胞清洗后成像。
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