CN112378971B - 一种CRISPR/Cas13a驱动的催化可再生电化学生物传感器及其应用 - Google Patents
一种CRISPR/Cas13a驱动的催化可再生电化学生物传感器及其应用 Download PDFInfo
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Abstract
本发明公开了一种CRISPR/Cas13a驱动的催化可再生电化学生物传感器，包括CRISPR/Cas13a系统/发夹DNA催化回路两级信号放大体系和可再生丝网印刷电极芯片。体系包括Cas13a蛋白、crRNA、启动分子和DNA发夹；芯片包括丝网印刷电极和装载样本的软体孔板，所述电极包括金工作电极、铂对电极和银/氯化银指示电极，核酸单分子层锚定在金工作电极表面。当检测到血液中的RNA，Cas13a蛋白切割启动分子，引发CHDC生成中间产物，核酸链捕获中间产物给出电信号。本发明传感器具有快速、高灵敏度和特异性、低成本等优点，可用于各种疾病、食品和环境安全等领域的分子检测。
Description
技术领域
本发明涉及一种CRISPR/Cas13a驱动的催化可再生电化学生物传感器，更具体涉及所述CRISPR/Cas13a驱动的催化可再生电化学生物传感器的制备方法及其在疾病检测中的应用。
背景技术
一种疾病越早被诊断，就越有可能被治愈。采用小型化设备原位快速检测核酸分子，特别是小分子RNA(microRNA)和信使RNA(mRNA)，可以实现及时诊断和监测治疗，提高对病人的救治率。鉴于mRNA和microRNA在调节基因表达和其突变体的功能障碍在人类疾病中的重要地位，它们已成为癌症检测的有效生物标志物。传统的RNA检测方法，包括qRT-PCR、微阵列和RNA测序，通常依赖于大型贵重的仪器，复杂的样本准备程序和冗长的检测周期，这些限制了其广泛应用。因此，迫切需要开发快速、便携式、高特异性和灵敏度的RNA检测新技术及设备。
在生物传感器领域中，电化学信号和光学信号是最常用的信号传导模式。基于光学检测的生物传感器操作简单、快速，但是灵敏度较低，并且依赖非一次性的、相当庞大的光密度读数器及控制器用于信号放大/输出，器件集成能力较差，这些因素限制了它们在即时检测(POCT)和现场诊断中的应用。与之相比，电化学生物传感器为即时检测应用提供了强大的功能，包括系统集成化、检测设备小型化、信号放大和多分析物检测。
CRISPR/Cas是一项强大的基因编辑技术，广泛应用于基因功能研究和基因修饰与治疗。CRISPR/Cas13a系统，包含Cas13a蛋白和CRISPR RNA(crRNA)。研究表明在crRNA的指导下，Cas13a可以高特异性和高效率地结合并切割目标RNA特定位点。Cas13a/crRNA特异性识别目标RNA后，一个被激活的Cas13a可在数分钟内非特异性地切割附近数千个信号分子，呈现指数级荧光信号放大。基于Cas13a蛋白的“反式切割”效应，CRISPR-Cas13a系统已被开发成一种快速、低成本且高灵敏的核酸检测工具。发夹核酸链催化回路(CHDC)是一种无酶DNA回路，将碱基互补区域嵌入发夹核酸链的双链茎内，目标核酸链与发夹核酸链的锚点碱基域杂交引发多个杂交催化反应，实现高灵敏检测。同时，CHDC的催化回路特征可实现与CRIPSR/Cas、电化学传感器等组件相结合达到更高的性能。目前尚未见基于CRISPR/Cas13a、CHDC与电化学传感器相结合的技术报道，也未见该技术用于癌症早期检测的报道。
发明内容
针对现有技术中存在的上述不足，本发明所要解决的技术问题之一在于提供一种新的CRISPR/Cas13a驱动的催化可再生电化学生物传感器。本发明中，二茂铁(Fc)修饰的核酸链被锚定在固体基板的表面形成单分子层提供参照信号，其中识别元件包括CRISPR/Cas13a系统/发夹DNA催化回路(Cas-CHDC)两级信号放大体系。与传统的电化学生物传感器相比，本发明中的CRISPR/Cas13a驱动的催化可再生电化学生物传感器具有两级电信号放大功能、两种氧化还原基团构成一对比率型探针提供稳定的信号输出、传感器可再生性能实现重复使用。本发明的CRISPR/Cas13a驱动的催化可再生电化学生物传感器用于检测癌症或其他疾病早期的核酸分子，具有较高的选择性和灵敏性。
在本发明的第一方面，提供了一种CRISPR/Cas13a驱动的催化可再生电化学生物传感器，所述CRISPR/Cas13a驱动的催化可再生电化学生物传感器包括CRISPR/Cas13a系统/发夹DNA催化回路两级信号放大体系和可再生丝网印刷电极芯片，CRISPR/Cas13a系统/发夹DNA催化回路特异性识别目标核酸链，并生成两级信号放大后的中间产物，锚定在可再生丝网印刷电极芯片上的核酸链捕获信号放大后的产物给出电信号。
优选的，所述可再生丝网印刷电极芯片包括丝网印刷电极和装载液体样本的软体孔板。所述丝网印刷电极的固体基板为玻璃，硅晶片，聚甲基丙烯酸甲酯、陶瓷或其它固体材料。所述装载液体样本的软体孔板为聚二甲基硅氧烷(PDMS)、聚苯醚(PPO)或其它工程塑料；孔的尺寸为5到15毫米。
优选的，所述CRISPR/Cas13a系统包括Cas13a蛋白、crRNA和启动分子。所述启动分子为RNA修饰发夹DNA核酸链，其特征在于在5’或3’末端自身形成一个12个碱基对左右的发夹结构并且内部环状结构有2到5个核糖核苷酸标记的寡核苷酸。其中标记的核糖核苷酸包括腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或其中几种。
所述CRISPR/Cas13a系统用于检测核酸分子，如mRNA、microRNA等。
优选的，所述发夹DNA催化回路(CHDC)由以下组分组成：1)两个可潜在结合成双链的发夹DNA(C-H1和C-H2)；2)其中一个发夹DNA的5’或3’末端标记氧化还原基团，并且5’或3’末端含有3-7个尿嘧啶碱基。
优选的，所述的被锚定在金工作电极表面的核酸链单分子层包括硫醇化DNA，氨基修饰的DNA，或RNA。核酸链单分子层的长度在5-50个核苷酸之间。
优选的，所述氧化还原基团包括但不限于二茂铁、亚甲基蓝、蒽醌等。
所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器用于定量样本中目标核酸链包括以下步骤：
(1)将软体孔板通过微接触粘贴技术固定到丝网印刷电极上制成芯片。
(2)加入核酸链/竞争链溶液于孔板中，在金电极表面形成自组装单分子层；
(3)将目标核酸链与CRISPR/Cas13a系统和发夹DNA催化回路混合；
(4)将(3)中的混合液滴加到装载液体样本的软体孔板中，通过分析电信号来定量目标核酸链，电信号与目标核酸链的浓度成正比。
所述竞争链包含与核酸链互补的碱基序列。
为了防止非特异性的核酸分子吸附，我们选择用末端硫醇化的β-巯基乙醇(βME)保护芯片中的非阵列区，核酸单分子层形成微阵列区。
为了丝网印刷电极芯片的可再生，我们选择用尿嘧啶DNA糖基化酶和核酸内切酶定点剪切发夹DNA(C-H1或C-H2)末端的尿嘧啶碱基，使得中间产物与锚定在金电极表面的核酸单分子层脱离。
相比于常规的电化学生物传感器，本发明中的CRISPR/Cas13a驱动的催化可再生电化学生物传感器用于核酸的检测具有以下优点：
1)CRISPR/Cas13a驱动的催化可再生电化学生物传感器具有Cas-CHDC两级信号放大体系，能够检测到癌症早期少量的目标RNA，具有较高的选择性和灵敏性，准确率也较高；
2)芯片具有可再生功能，可多次循环使用；
3)可连续检测不同生物标志物，实现多元检测；
4)检测时间短，少于6分钟。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是本发明生物传感器制备的示意图；
图2是电极表面核酸单分子层的可再生情况原子力电镜图；
图3是第1、20、45次测量中，SWV读取的亚甲基蓝(MB)和二茂铁(Fc)的典型氧化峰值图；
图4是比率校正(IMB/IFc)随次数增多的变化关系图；
图5是在不同浓度的miR-17(0.5fM–5nM)和PBS作为背景信号的情况下，在6分钟的时间点检测MB和Fc的电信号图；
图6是IMB/IFC与miR-17浓度(0.5fM–5nM)的变化关系图；
图7是以miR-17为目标链的MB氧化峰图；
图8是以miR-155为目标链的MB氧化峰图；
图9是以TTF-1mRNA为目标链的MB氧化峰图；
图10是传感器芯片将miR-17与同一家族的非靶RNA区分开的结果图；
图11是同一患者样品中miR-17，miR-155和TTF-1mRNA串扰风险评估图；
图12是miR-17在四个不同患者样品中的串扰风险评估图。
具体实施方式
下面结合具体实施例对本发明做进一步的解释和说明，但本发明并不受其限制。
【实施例1】
CRISPR/Cas13a驱动的催化可再生电化学生物传感器的制备及理化性能表征：
如图1所示，实施例1描述了一个简单的方法制备CRISPR/Cas13a驱动的催化可再生电化学生物传感器。将PDMS模板通过微接触粘贴技术固定到丝网印刷电极上制成芯片。加入硫醇化DNA溶液于孔板中，在金电极表面形成自组装单分子层。Cas13a/crRNA复合体通过碱基序列互补特异性识别目标核酸链，并激活Cas13a蛋白的反式切割活性，剪切触发链(Trigger)，从而引发发夹DNA催化回路(H1、H2和Reporter)，产生大量的C-H1和C-H2的复合物C-I2。金电极表面锚定的核酸单分子层特异性识别C-I2。通过电化学工作站检测电信号来定量目标核酸链。图2描述了电极表面核酸单分子层的可再生情况。原子力电镜图所示：金电极表面(A)、硫醇化DNA修饰后金电极表面(B)、与互补核酸链杂交后电极表面(C)和经酶切反应后金电极表面(D)。
【实施例2】
CRISPR/Cas13a驱动的催化可再生电化学生物传感器的可再生性能和灵敏度测试：
在本实施例中，为了评估传感器的可再生性能，在单个芯片上进行了45次重复的目标核酸链检测实验。图3显示了在第1、20、45次测量中，SWV读取的亚甲基蓝(MB)和二茂铁(Fc)的典型氧化峰。对于单独的MB，类似于仅具有一个报告分子的电化学生物传感器，其氧化峰随着测量次数的增加而逐渐降低，从而导致34.8％的广泛差异。但是，比率校正(IMB/IFc)的应用显示出0.61的平均响应，并将该变异性大幅降低至2.2％(如图4所示)。我们观察到，在单个传感器芯片上至少可以进行37次连续且稳定的测量(如图2B中灰色突出显示的区域)。
【实施例3】
CRISPR/Cas13a驱动的催化可再生电化学生物传感器的灵敏度测试：
在本实施例中，在不同浓度的miR-17(0.5fM–5nM)和PBS作为背景信号的情况下，在6分钟的时间点检测MB和Fc的电信号(如图5所示)。图5中显示MB氧化峰与目标核酸链(miR-17)浓度成正比，而Fc的信号响应在不同miR-17浓度下未显示明显变化。使用室温下的直接电化学读数，传感器芯片能够检测miR-17低至0.5fM的水平，动态范围扩展至5nM。校准曲线表明，IMB/IFC与miR-17浓度(0.5fM–5nM)成比例增加(如图6所示)。通过将测得的数据点拟合为四参数的S形曲线，可以确定miR-17的检出限(LOD)为50aM，同时每个反应消耗的样品/试剂体积小于10μL，检测时间不超过6分钟。
【实施例4】
CRISPR/Cas13a驱动的催化可再生电化学生物传感器的特异性测试：
在本实施例中，将目标RNA与具有一到几个碱基错配的其他RNA进行区分。与5pMmiR-17的MB氧化峰相比，相同的浓度miR-155或TTF-1mRNA几乎没有出峰。miR-17的IMB/IFc分别比miR-155和TTF-1mRNA高7.8和6.8倍(如图7所示)。在miR-155和TTF-1mRNA分别作为目标链时也获得了相似的结果(如图8和图9)。并且传感器芯片能够将miR-17与同一家族的非靶RNA区分开：miR-106a(1-碱基错配)，miR-20a和miR-20b(2-碱基)不匹配)(p<0.0001)(如图10所示)。
【实施例5】
CRISPR/Cas13a驱动的催化可再生电化学生物传感器用于NSCLC病人血清中多目标RNA检测：
对非小细胞肺癌(NSCLC)患者的血清样品，miR-17，miR-155和TTF-1mRNA(在同一样品中，如图11所示)和miR-17(在四个不同样品中，如图12所示)进行串扰风险评估，结果表明血清样品之间的测量没有串扰。
以上实施例仅为本发明的示例性实施例，不用于限制本发明，本发明的保护范围由权利要求书限定。本领域技术人员可以在本发明的实质和保护范围内，对本发明做出各种修改或等同替换，这种修改或等同替换也应视为落在本发明的保护范围内。
Claims (10)
1.一种CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，所述CRISPR/Cas13a驱动的催化可再生电化学生物传感器包括：
(a)可再生丝网印刷电极芯片，包括丝网印刷电极和装载液体样本的软体孔板，其中丝网印刷电极包括金工作电极、铂对电极和银/氯化银指示电极；
(b)氧化还原基团修饰的核酸链单分子层被锚定在金工作电极表面；
(c)CRISPR/Cas13a系统，包括Cas13a蛋白、crRNA和启动分子；
(d)具有氧化还原基团和尿嘧啶DNA修饰的发夹DNA催化回路；
启动分子为RNA修饰发夹DNA核酸链，在5’或3’末端自身形成一个12个碱基对左右的发夹结构并且内部环状结构有2到5个核糖核苷酸标记的寡核苷酸；
RNA修饰发夹DNA核酸链，RNA修饰包括腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或其中几种；
RNA修饰发夹DNA核酸链，RNA修饰位点会被激活后的Cas13a蛋白切割，从而断开并解链；
(a)两个可潜在结合成双链的发夹DNA，即C-H1和C-H2；
(b)其中一个发夹DNA的5’或3’末端标记氧化还原基团，并且5’或3’末端含有3-7个尿嘧啶碱基；
在目标核酸链存在的情况下，C-H1与C-H2会发生链式杂交反应，生成C-I2；C-I2与金电极表面的核酸单分子层结合生成复合物，C-I2与金电极表面的核酸单分子层结合生成的复合物用来做电信号检测，之后使用溶剂洗去，金电极表面的核酸单分子层是可再生的。
2.如权利要求1所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，
(a)所述丝网印刷电极包括丝网印刷电极的固体基板，所述固体基板为玻璃，陶瓷，硅晶片或聚甲基丙烯酸甲酯；
(b)装载液体样本的软体孔板，所述软体孔板为聚二甲基硅氧烷(PDMS)或聚苯醚(PPO)。
3.如权利要求1所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，被锚定在金工作电极表面的核酸链单分子层包括硫醇化DNA，氨基修饰的DNA，或RNA。
4.如权利要求1所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，氧化还原基团包括二茂铁、亚甲基蓝或蒽醌。
5.如权利要求3所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，核酸链单分子层的长度在5-50个核苷酸之间。
6.如权利要求1所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，CRISPR/Cas13a系统会特异性识别目标核酸链。
7.如权利要求6所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，目标核酸链包括小分子RNA(miRNA)和信使RNA(mRNA)。
8.如权利要求1-7任一所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器在定量样本中目标核酸链的应用，其特征在于，所述应用包括以下步骤：
(1)将软体孔板通过微接触粘贴技术固定到丝网印刷电极上制成芯片；
(2)加入核酸链/竞争链溶液于孔板中，在金电极表面形成自组装单分子层；
(3)将目标核酸链与CRISPR/Cas13a系统和发夹DNA催化回路混合；
(4)将(3)中的混合液滴加到装载液体样本的软体孔板中，通过分析输出的电信号来定量目标核酸链，电信号与目标核酸链的浓度成正比。
9.如权利要求8所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器在定量样本中目标核酸链的应用，其特征在于，竞争链包含与核酸链互补的碱基序列。
10.如权利要求1所述的CRISPR/Cas13a驱动的催化可再生电化学生物传感器，其特征在于，检测的体液样本包括血液、血清、血浆、尿液、唾液。
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| CN113777141B (zh) * | 2021-08-03 | 2024-04-19 | 南京师范大学 | 电化学生物传感器及其制备方法和检测新型冠状病毒的方法 |
| CN114689473A (zh) * | 2022-03-28 | 2022-07-01 | 潍柴动力股份有限公司 | 颗粒物传感器测试分析系统及方法 |
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| CN112567030A (zh) * | 2018-05-08 | 2021-03-26 | 勒芬天主教大学 | 生物传感器 |
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