CN116103374B - 一种基于CRISPR-Cas***的检测外泌体的荧光生物传感器 - Google Patents
一种基于CRISPR-Cas***的检测外泌体的荧光生物传感器 Download PDFInfo
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Abstract
本发明属于生物传感器技术领域,提供了一种基于CRISPR‑Cas***的检测外泌体的荧光生物传感器,一种基于CRISPR‑Cas***的检测外泌体的荧光生物传感器,包括cas12a蛋白,phi29酶,三链体Aptamer,报告探针,核苷酸序列如SEQ ID NO:4‑7所示的发夹H1链、发夹H2链、crRNA和环形模板CT;所述三链体Aptamer由核苷酸序列如SEQ ID NO:1‑3所示PD‑L1Apt和EpCAM Apt、Trigger链杂交形成。该传感器具有检测速度快、操作简单、检测限低、特异性高等优点,可以弥补外泌体现有检测方法的缺陷与不足,实现对肿瘤外泌体的快速、准确的定量检测,适用于肿瘤外泌体的检测和生物传感器产业化的实际应用。
Description
技术领域
本发明属于生物传感器技术领域,涉及一种基于CRISPR-Cas***的检测外泌体的荧光生物传感器。
背景技术
外泌体是由不同细胞分泌的胞外囊泡,直径为30-150 nm之间,包含有蛋白、核酸等物质广泛分布于各种体液中。在肿瘤细胞中,外泌体参与多种胞间活动帮助肿瘤细胞完成侵袭和转移;同时,它们携带多种源代细胞的蛋白质和核酸作为信号分子进一步诱导受体细胞发生病变。这些特征使得外泌体成为癌症早期监测的重要生物标志物。
肿瘤衍生的外泌体含有特定抗原,这些抗原在癌症发展过程中起着重要作用,可以作为液体活检在癌症诊断中的生物标志物,如EpCAM蛋白可以作为肿瘤标志物进行检测。然而,外泌体的表面蛋白并不完全是特异性,这样就会导致肿瘤来源和非肿瘤来源外泌体之间的标记重叠。目前已有的一些方法针对于外泌体上存在的标志物数量,很难确定该标志物来源于肿瘤细胞还是非肿瘤细胞,如何实现基于外泌体的肿瘤诊断以及外泌体作为标志物的生物学功能仍是挑战。
CRISPR/Cas技术作为一种革命性的基因编辑工具,被广泛应用于基因编辑、基因组成像等领域。因其独特的精准识别能力,具有的模块化和可编程的特点,再加上该技术的易用性和抗干扰性,CRISPR/Cas***在生物传感领域也展现出广阔的应用前景。
发明内容
为了解决现有技术检测外泌体的标记重叠的问题,本发明提供了一种特异性和灵敏度高、检测速度快的基于CRISPR/Cas技术检测外泌体的生物传感器。
本发明的另一目的是提供一种上述生物传感器在检测外泌体中的应用与方法。
为实现上述目的,本发明采用如下技术方案。
一种基于CRISPR-Cas***的检测外泌体的荧光生物传感器,包括cas12a蛋白,phi29酶,三链体Aptamer,报告探针,核苷酸序列如SEQ ID NO: 4-7所示的发夹H1链、发夹H2链、crRNA和环形模板CT;
所述三链体Aptamer由核苷酸序列如SEQ ID NO: 1-3所示PD-L1Apt和EpCAM Apt、Trigger链杂交形成;
所述报告探针序列为:TTATT,且5’端修饰FAM,3’端修饰BHQ。
所述三链体Aptamer的制备方法如下:将核苷酸序列如SEQ ID NO: 1-3所示的PD-L1Apt和EpCAM Apt、Trigger链在缓冲液中变性后退火获得。
所述环形模板CT的制备方法如下:将核苷酸序列如SEQ ID NO: 7-8所示的Padlock probe链和Ligation probe链在缓冲液中变性后退火,再加入T4连接酶,在适宜温度下于含有ATP的缓冲液中反应,高温灭活酶并恢复室温后加入核酸外切酶Ⅰ和核酸外切酶Ⅲ,灭活酶后获得环形模板CT。
上述荧光生物传感器可用于制备检测外泌体的试剂盒。
优选的,所述试剂盒还包含缓冲溶液、外泌体样品。
本发明的检测原理如图1所示,用到9条序列:
PD-L1 Apt(Anti-exosome surface protein 1 Apt)
5’-TACAGGTTCTGGGGGGTGGGTGGGGAACCTGTTACTGCC-3’;
EpCAM Apt(Anti-exosome surface protein 2 Apt)
5’-GTCTCACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3’;
Trigger链
5’-CTGTAGTGAGACGGCAGTAACAGGTTC-3’;
发夹H1链
5’-GCCGTCTCACTACAGGTAGTGCGAGCTTTTCTGTAGTGTTTACT-3’;
发夹H2链
5’-AGCTCGCACTACCTGTAGTGAGACGGCTTTAGGTAGTGTTTACT-3’;
Padlock probe链
5’-AGTAAACACTACCTATTTCGACCGGCTCGGAGAAGAGATTTTTTACTATTTCGACCGGCTCGGAGAAGAGATAAGTAGA-3’;
Ligation probe链
5’-TAGTGTTTACTTCTACTTATCTC-3’;
crRNA
5’-TAAGTAGAAGTAAACACTAC-3’;
报告探针(FQ)
5’-FAM-TTATT-BHQ-3’;
如图1A所示:PD-L1 Apt和EpCAM Apt中双下划线部分与Trigger链分别互补配对形成三链体Aptamer。当目标物存在时,PD-L1 Apt和EpCAM Apt分别与目标物特异性结合,释放Trigger链,用于触发HCR反应(DNA的杂交链式反应);PD-L1蛋白作为免疫治疗中一个重要诊断指标,但其本身并不具有肿瘤特异性,EpCAM蛋白作为肿瘤来源外泌体的特异蛋白,可通过其在外泌体中的表达用于癌症的检测,通过同时检测PD-L1和EpCAM蛋白可定位至肿瘤外泌体上的PD-L1蛋白。
如图1B所示:释放的Trigger链与H1链部分互补配对打开H1链的发卡结构,从而暴露出能够与H2链互补的部分,进而与H2链互补打开H2链的发卡结构;Padlock probe链和Ligation probe链在T4酶的作用下形成环形模板CT,而H1和H2暴露的部分会与环形模板CT杂交,在phi29酶的作用下,进行滚环扩增反应;滚环扩增一方面会将Trigger链置换下来重新进行循环,另一方面得到的核酸链中包含CRISPR-Cas12a***的目标链,在向导RNA(crRNA)的引导下,Cas12a-crRNA复合体识别并结合目标链,激活Cas12a的反式切割活性,切割报告探针,产生荧光信号,通过荧光变化检测外泌体的量。
本发明具有以下优点:
本发明提供的生物传感器检测限低,利用核酸适配体与外泌体的“双靶标识别”实现了对目标物的高特异性检测;当目标物存在时,两个捕获探针会同时结合在外泌体表面PD-L1和EpCAM蛋白,从而释放Trigger,而Trigger引发HCR反应,反应后Trigger又会被被置换下来重新进入循环,极大提高了其检测的灵敏度;利用Cas12a的“附属切割”活性切割体系中的荧光报告基团,产生可定量的信号。
该传感器构建简单,具有操作简单、反应速度快、性能稳定等优势;检测原理的主要过程均是在均相中实现的,提高了反应速度,降低了操作的复杂程度,实现了目标物的快速,简单,灵敏的检测。该传感器具有检测速度快、操作简单、检测限低、特异性高等优点,可以弥补外泌体现有检测方法的缺陷与不足,实现对肿瘤外泌体的快速、准确的定量检测,适用于肿瘤外泌体的检测和生物传感器产业化的实际应用。
附图说明
图1为本发明生物传感器的原理图;
图2为捕获探针与Trigger链的浓度摩尔比优化检测结果图;
图3为温度优化检测结果图;
图4为生物传感器对不同浓度外泌体的荧光扫描。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 荧光生物传感器的制备
(1)三链体Aptamer的制备
合成序列如SEQ ID NO: 1-3所示的捕获探针PD-L1 Apt和捕获探针EpCAM Apt、Trigger链;
将DEPC水、NEB buffer 2.1、PD-L1 Apt和EpCAM Apt和Trigger链加入到预先准备好的灭菌的EP管中,震荡离心,95℃下退火5 min,缓慢冷却至室温杂交为探针,储存于4℃备用。
(2)环形模板CT的制备
合成核苷酸序列如SEQ ID NO: 7和8所示的Padlock probe链(PP)和Ligationprobe链(LP),加水稀释成浓度为100μM;
在1.5 mL EP管中,加入17.5 μL RNase-free水,3 μL T4 DNA连接酶Buffer(10×),3 μL PP溶液(100 μM),3 μL LP溶液,95℃退火5 min,自然冷却至室温;
加入1.5 μL T4 DNA连接酶,16℃过夜(8 h),65℃保温10 min灭活酶,冷却至室温;
加入1 μL核酸外切酶I和1 μL核酸外切酶III,37℃下放置8 h,85℃保温20 min灭活酶,获得浓度为10μM的环形模板CT,于4℃下保存备用。
(3)合成序列如SEQ ID NO: 4-6所示的发夹H1链、发夹H2链和crRNA,合成报告探针。
实施例2 荧光生物传感器检测外泌体的条件优化
(1)外泌体的制备
黑色素瘤细胞(A375 cell)培养在37℃包含5% CO2潮湿空气中,培养基为MEM、10%的小牛血清、1%抗生素,每两天传代一次:用移液器吸出培养基,加入新培养基,吹打成单细胞悬液,分瓶即可;
将A375细胞培养三天,吸出培养基,将细胞培养液在4℃条件下,5000×g离心10min,获取上清,然后利用外泌体提取试剂盒(exoEasy Maxi Kit,QIAGEN)按照说明书进行提取,提取过程在超净操作台中进行,尽可能减少RNA酶的污染;提取得到的外泌体保存在-80℃的冰箱中备用。
(2)三链体Aptamer与Trigger链的浓度摩尔比
按照不同比例的PD-L1 Apt、EpCAM Apt和Trigger链(0:0:1、1:1:1、1.5:1.5:2、2:2:1)制备三链体Aptamer,储存于4℃备用;
在1.5 mL的暗管中分别加入3 μLA375细胞的外泌体,3 μL三链体Aptamer溶液(100nM)、3 μL H1溶液(100 μM)、3 μL H2溶液(100 μM)、3 μL环状模板CT溶液、3 μL phi29酶buffer、2 μL dNTP溶液、1 μL phi29酶液(10000U/mL),混合物在37℃下反应1 h。反应完成后,继续向管中加入2 μLRNA酶抑制剂、2 μL buffer 2.1、3 μLCas蛋白溶液(1 μM)、3 μLcrRNA溶液(1 μM)以及3 μL报告探针溶液(10 μM),在37℃下反应1 h,获得检测液;
将上述检测液与70 μL的超纯水混合后在荧光仪下检测荧光强度,激发波长为485nm,检测波长为525 nm,荧光光谱的激发和发射狭缝宽度均为10 nm。结果见图2,从图中可以看出,随着捕获探针与Trigger链的摩尔比增加,荧光信号逐渐降低。当捕获探针与Trigger链的摩尔比浓度达到2:2:1时,荧光信号最低。因此选取捕获探针与Trigger链的摩尔比浓度为2:2:1。
(3)孵育温度
按照PD-L1 Apt、EpCAM Apt和Trigger链摩尔比2:2:1制备三链体Aptamer,储存于4℃备用;
在1.5 mL的暗管中分别加入3 μLA375细胞的外泌体,3 μL三链体Aptamer溶液(100nM)、3 μL H1溶液(100 μM)、3 μL H2溶液(100 μM)、3 μL环状模板CT溶液、3 μL phi29酶buffer、2 μL dNTP溶液、1 μL phi29酶液(10000U/mL),混合物在25℃、31℃、37℃下反应1 h。反应完成后,继续向管中加入2 μLRNA酶抑制剂、2 μL buffer 2.1、3 μLCas蛋白溶液(1 μM)、3 μL crRNA溶液(1 μM)以及3 μL报告探针溶液(10 μM),分别在25℃、31℃、37℃下反应1 h,获得检测液;
将上述检测液与70 μL的超纯水混合后在荧光仪下检测荧光强度,激发波长为485nm,检测波长为525 nm,荧光光谱的激发和发射狭缝宽度均为10 nm。结果见图3,从图中可以看出,检测到的化学发光强度峰值随着温度的增加而增加。在浓度为37℃时,化学发光强度最高,所以选取37℃为最佳实验温度。
应用例1 荧光生物传感器对外泌体的检测
三链体Aptamer的制备:将DEPC水(8μL)、NEB buffer 2.1(3μL)、不同比例的PD-L1Apt、EpCAM Apt和Trigger链(2:2:1)加入到预先准备好的灭菌的EP管中,震荡离心,95℃下退火5 min,缓慢冷却至室温杂交为探针,储存于4℃备用。
在1.5 mL的暗管中分别加入3 μL不同浓度的外泌体悬液(102-106particles/μL)或水(control),3 μL三链体Aptamer溶液(100nM)、3 μL H1溶液(100 μM)、3 μL H2溶液(100 μM)、3 μL环状模板CT溶液、3 μL phi29酶buffer、2 μL dNTP溶液、1 μL phi29酶液(10000U/mL),混合物在37℃下反应1 h。反应完成后,继续向管中加入2 μLRNA酶抑制剂、2μL buffer2.1、3 μLCas蛋白溶液(1 μM)、3 μL crRNA溶液(1 μM)以及3 μL报告探针溶液(10 μM),37℃下反应1 h,获得检测液;
将上述检测液与70 μL的超纯水混合后在荧光仪下检测荧光强度,激发波长为485nm,检测波长为525 nm,荧光光谱的激发和发射狭缝宽度均为10 nm
结果如图4所示,从图中可以看出,随着外泌体浓度的增加,在525 nm处的荧光值逐渐增加。经计算,该生物传感器的检测限为:7.59×102particles/mL。
Claims (3)
1.一种基于CRISPR-Cas***的检测外泌体的荧光生物传感器,其特征在于,包括cas12a蛋白,phi29酶,三链体Aptamer,报告探针,环形模板CT,核苷酸序列如SEQ ID NO:4-6所示的发夹H1链、发夹H2链和crRNA;
所述三链体Aptamer由核苷酸序列如SEQ ID NO: 1-3所示PD-L1 Apt和EpCAM Apt、Trigger链在NEB buffer 2.1缓冲液中变性后退火获得;
所述环形模板CT的制备方法如下:将核苷酸序列如SEQ ID NO: 7-8所示的Padlockprobe链和Ligation probe链在T4 DNA连接酶缓冲液中变性后退火,再加入T4连接酶,在16℃下于T4 DNA连接酶缓冲液中反应,高温灭活酶并恢复室温后加入核酸外切酶Ⅰ和核酸外切酶Ⅲ,灭活酶后获得环形模板CT;
所述报告探针的核苷酸序列为:TTATT,且5’端修饰FAM,3’端修饰BHQ。
2.一种包含如权利要求1所述荧光生物传感器的检测外泌体的试剂盒。
3.根据权利要求2所述的试剂盒,其特征在于,试剂盒还包含缓冲溶液、外泌体样品。
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基于竞争触发滚环扩增的荧光适配体传感器高灵敏检测凝血酶;张松柏;郑丽英;胡霞;沈广宇;刘学文;沈国励;俞汝勤;;分析化学;20151115(11);全文 * |
生物传感器检测白细胞介素-2与其受体的相互作用;林青等;传感器与微***;20091231;第28卷(第12期);全文 * |
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