CN113640515A - 一种利用多种标志物联合检测外泌体的方法及试剂盒 - Google Patents
一种利用多种标志物联合检测外泌体的方法及试剂盒 Download PDFInfo
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Abstract
本发明涉及肿瘤来源的外泌体检测技术领域,具体涉及一种利用多种标志物联合检测外泌体方法及试剂盒,该试剂盒包括用于捕获外泌体的捕获探针、邻近探针P1和P2以及信号探针S,P1包含第一核酸适配体序列以及有荧光素标记的序列,P2包含第二核酸适配体序列以及有荧光素标记的序列,信号探针S由Sa和Sb组成,Sa包含与有荧光素标记的序列互补的序列,Sb包含与Sa互补的序列以及有罗丹明标记的序列。该试剂盒,利用邻近杂交介导荧光共振能量转移实现外泌体表面多种标志物联合检测,以期为提高肿瘤早期诊断的准确度提供检测方法。
Description
技术领域
本发明属于外泌体检测技术领域,具体涉及一种利用多种标志物联合检测外泌体的方法及试剂盒。
背景技术
肺癌是全球范围内的重大公共卫生问题,2020年全球肺癌发病率和死亡率分别为28.3/10万人和23.0/10万人,是死亡率最高的癌症。肺癌早期诊断并规范治疗可将不足20%的五年生存率提高到70%~80%。发展灵敏度高、特异性强的非创伤性诊断方法对肺癌早期诊断具有重要意义。“液体活检”可通过反复采样检测肿瘤生物标志物随时间推移的变化,在肿瘤早期诊断中具有创伤性小、标本易获得等优势,而生物标志物的选择是决定液体活检结果准确度的关键。
外泌体(Exosome,Exo)是由活细胞主动分泌的粒径为30nm~150nm的细胞外囊泡,介导细胞间的通讯,与肺癌的发生发展、血管生成和转移过程密切相关。Exo作为液体活检的检测目标之一,与循环肿瘤细胞和循环肿瘤DNA相比具有以下特点:Exo由肿瘤细胞主动分泌,在一定程度上与肿瘤细胞数量和活性水平相关;Exo表面蛋白具有细胞来源特异性,可反映肿瘤类型及进展情况;作为细胞间通讯的有效介体,其分泌、转运和消除均具有一定的时效性。因此,Exo用于癌症诊断具有灵敏度高、特异性好和时效性强的独特优势,是最具应用潜力的肿瘤早期诊断标志物之一。
常用的Exo传统检测方法包括纳米颗粒追踪分析法(Nanoparticle trackinganalysis,NTA)、免疫印迹法(Western blot,WB)、酶联免疫吸附分析法(Enzyme-linkedimmuno sorbent assay)和流式细胞术(Flow cytometry)等。这些方法检测灵敏度低、所需样本量大、操作过程繁琐,难以满足临床即时检测的需要。为克服上述检测方法的缺陷,多种简便方法已被开发用于Exo的高灵敏检测,例如表面增强拉曼散射、微流控芯片技术、荧光或比色传感检测法等。然而,这些检测方法仍存在一定的局限性:一方面,多数检测方法仅依赖于对Exo普遍表达的跨膜蛋白CD63的特异性识别进行检测,难以区分正常人和肿瘤患者;即使利用CD63与另一种Exo表面肿瘤标志蛋白联合检测,能够在一定程度上改善肿瘤Exo检测的特异性,但目前尚无一种Exo特异性蛋白标志物能够明确诊断肺癌,选择Exo表面多种肿瘤标志物联合检测,可以提高诊断的准确度。另一方面,单强度的传感探针容易受复杂生物样品的干扰,产生假阳性信号。
“邻近连接技术”是指一对分别标记有亲和配体的寡核苷酸探针(称为邻近探针)通过同时识别并结合目标分子,只有当二者由于距离拉近产生邻近效应时,才能给出检测信号,从而保证了检测的高特异性,被成功用于蛋白质的高灵敏和高特异性检测,及细胞表面多种标志物的共定位研究。荧光共振能量转移(FRET)利用激发供体,监测受体荧光发射,能够降低检测背景,减少干扰。因此,结合邻近效应介导的DNA组装及FRET构建Exo表面多种标志物联合检测平台,有望提高肿瘤Exo检测的特异性和抗干扰能力。
发明内容
本发明的目的是提供一种利用多种标志物联合检测外泌体的试剂盒,以提高肿瘤来源外泌体检测的准确性。
本发明的另一个目的是提供一种利用多种标志物联合检测外泌体的方法。
为了实现以上目的,本发明采取的技术方案为:
一种利用多种标志物联合检测外泌体的试剂盒,包括用于捕获外泌体的捕获探针、邻近探针P1和P2以及信号探针S,P1包含第一核酸适配体序列以及有荧光素(FAM)标记的序列,P2包含第二核酸适配体序列以及有荧光素标记的序列,信号探针S由Sa和Sb组成,Sa包含与有荧光素标记的序列互补的序列,Sb包含与Sa互补的序列以及有罗丹明(TAMRA)标记的序列。
进一步地,所述P1的第一核酸适配体序列以及有荧光素标记的序列之间有P1间隔序列,P2的第二核酸适配体序列以及有荧光素标记的序列之间有P2间隔序列。
进一步地,所述P1包含的序列如SEQ ID NO:1所示,P2包含的序列如SEQ ID NO:2所示。
进一步地,所述Sa的两端序列分别与P1的有荧光素标记的序列和P2的有荧光素标记的序列互补。
进一步地,所述Sb与Sa的中间序列互补,Sb的两端标记有罗丹明。
进一步地,所述Sa的序列如SEQ ID NO:3所示,Sb包含的序列如SEQ ID NO:4所示。
进一步地,所述第一核酸适配体可特异性识别非小细胞肺癌来源Exo中表达升高的表皮生长因子受体(EGFR),第二核酸适配体可特异性识别非小细胞肺癌来源Exo中表达升高的上皮细胞粘附分子(EpCAM)。
进一步地,所述捕获探针为生物素化的CD63适配体修饰在链霉亲和素磁珠(MNPs-SA)表面制备得到。
进一步地,还包括与所述捕获探针序列互补的c-DNA序列,用于将捕获的外泌体释放。
上述利用多种标志物联合检测外泌体的方法,包括以下步骤:
1)使用所述捕获探针富集外泌体,之后用c-DNA分离得到外泌体上清液;
2)加入邻近探针P1和P2,反应后加入信号探针S,反应后检测荧光素在522nm的荧光发射强度I522以及罗丹明在580nm处的荧光发射强度I580,I580/I522与外泌体的浓度呈正相关,即可检测出外泌体的浓度。
本发明的有益效果:
本发明的利用多种标志物联合检测外泌体的试剂盒,基于核酸适配体(Aptamer)对外泌体(Exo)表面蛋白标志物的特异性识别,以Exo通用标志物CD63为捕获靶标,选择非小细胞肺癌来源Exo中表达升高的表皮生长因子受体(EGFR)和上皮细胞粘附分子(EpCAM)为联合检测靶标,建立了一种灵敏度高、特异性好、操作简便的Exo表面三种标志物联合检测(即共表达CD63/EGFR/EpCAM Exo)方法。其中,CD63适配体功能化的纳米磁珠用于从待测样品中特异性富集和分离外泌体,避免复杂样品基质对检测的干扰。荧光素标记的一对邻近探针同时与外泌体表面的EGFR和EpCAM结合,方可通过邻近效应介导其与罗丹明标记的信号探针形成稳定的DNA自组装体,进而触发FRET,实现对共表达CD63/EGFR/EpCAM Exo的高特异性检测。本发明利用邻近杂交介导FRET实现Exo表面多种标志物联合检测,以期为提高肿瘤早期诊断的准确度提供检测方法。
附图说明
图1为邻近杂交介导FRET实现Exo表面三种标志物联合检测的原理图;
图2为不同浓度Exo存在下对应的荧光光谱图;
图3为共表达CD63/EGFR/EpCAM Exo检测标准曲线图;
图4为不同稀释倍数无外泌体胎牛血清中外泌体检测结果图;
图5为非小细胞肺癌患者和健康者血浆中共表达CD63/EGFR/EpCAM Exo检测结果图;
图6为邻近杂交介导FRET用于Exo表面三种标志物联合检测的结果对比图;
图7为随机序列DNA代替Aptamer对I580/I522的影响结果图;
图8为相同浓度A549-Exo和BEAS-2B Exo存在下对应的I580/I522图。
具体实施方式
下面将结合本发明实施例以及附图对本发明作进一步说明。
试剂与材料
所有核酸探针均由生工生物工程(上海)股份有限公司合成,并经高效液相色谱(HPLC)纯化,使用前用TE缓冲液配制成100μmol/L的储备液,探针序列见表1。链霉亲和素磁珠(MNPs-SA,300nm)购自于中国无锡百迈格BioMag生物科技有限公司。A549人肺腺癌细胞系、BEAS-2B人正常支气管上皮细胞购买于中国科学院细胞库。胎牛血清购自乌拉圭Lonsera。RPMI 1640培养基、DMEM高糖培养基、胰蛋白酶-EDTA消化液、Tris-HCl(1.0mol/L,pH 7.4)、HEPES(1.0mol/L,pH 7.2-7.4)、TE(0.01mol/L,pH 8.0)和PBS(pH 7.4)缓冲液购自于北京索莱宝生物科技有限公司。总外泌体提取试剂(细胞培养液)购自美国ThermoFisher。兔抗人CD63抗体、羊抗兔HRP-IgG抗体购自于英国Abcam。鼠抗人HSP70抗体、鼠抗人TSG101抗体和兔抗鼠IgG-HRP抗体购自于成都正能生物技术有限责任公司。5×蛋白上样缓冲液、TBST缓冲液、SDS-PAGE凝胶配制试剂盒购自于雷根生物技术有限公司。聚偏二氟乙烯(PVDF)膜、Ultra超滤管(100KD)和无菌针头式过滤器(0.22μm)购买于德国Millipore。所用其他试剂均为分析纯,实验用水Milli-Q超纯水(电阻率大于18.2MΩ·cm)。实验所用缓冲液如下:(1)buffer A:含有1.0mol/L NaCl和0.05%(v/v)Tween-20的TE缓冲液;(2)反应缓冲液:含20mmol/L NaCl和5mmol/L MgCl2的Tris-HCl缓冲液(50mmol/L,pH 7.4)。
实施例1
本实施例的利用多种标志物联合检测外泌体的试剂盒,包括的探针序列如表1所示。
表1.Exo表面多种标志物联合检测所用探针序列
由图1可见,邻近探针P1和P2均由三个区域组成:I区为识别部分,P1和P2的I区分别为EGFR适配体(AptEGFR)和EpCAM适配体(AptEpCAM);II区是一段间隔序列,即P1间隔序列和P2间隔序列,可减少与信号探针结合时的位阻效应;III区为标记有FAM的尾部序列。P1的I区包含的序列为tgccgtttcttctctttcgctttttttgcttttgagcat,II区包含的序列为tttatgtcatgatct,III区包含的序列为tttttttttt。P2的I区包含的序列为cactacagaggttgcgtctgtcccacgttgtcatggggggttggcctg,II区包含的序列为tctagtactcatttt,III区包含的序列为tttttttttt。信号探针S由两条部分互补的单链DNA构成,分别是Sa和Sb,Sa的两端序列与P1和P2中III区互补,Sb与Sa的中间序列互补配对,且两端标记有TAMRA。
基于“邻近杂交介导FRET”实现外泌体(Exo)表面多种标志物联合检测的原理如图1所示。AptCD63为生物素化的CD63适配体,MNPs@AptCD63为将生物素化的AptCD63修饰在MNPs-SA表面制备得到。AptCD63的核苷酸序列如SEQ ID NO:5所示。MNPs@AptCD63识别Exo表面的CD63来特异性捕获和分离待测样品中的Exo,然后利用与AptCD63序列互补的单链核酸(c-DNA),将捕获的Exo释放。c-DNA的序列如SEQ ID NO:6所示。随后,Exo中加入邻近杂交探针体系:包括FAM标记的邻近探针P1、P2,及TAMRA标记的信号探针S。当Exo表面仅存在标志蛋白CD63/EGFR或CD63/EpCAM时,P1和P2上不能自发与S的两端杂交,FAM与TAMRA因距离较远而不能发生FRET,TAMRA荧光不能被监测到;当Exo表面同时存在标志蛋白CD63、EGFR和EpCAM时,P1和P2通过识别EGFR和EpCAM同时结合到Exo表面,有效地拉近P1和P2距离,从而产生“邻近效应”,促进其与S的杂交,导致P1和P2上标记的FAM与S上标记的TAMRA因距离拉近而产生高效的FRET,伴随着TAMRA在580nm荧光强度(I580)的显著增强及FAM在522nm荧光强度(I522)的降低。I580/I522与目标Exo浓度呈正相关,即可实现对共表达CD63/EGFR/EpCAMExo的比率型检测。
本实施例的利用多种标志物联合检测外泌体的试剂盒的检测方法,包括以下步骤:
1、细胞培养及外泌体提取
在A549细胞和BEAS-2B细胞中加入含有10%(v/v)胎牛血清的RPMI 1640培养基,置于37℃,5%CO2恒温培养箱中进行培养。当细胞生长至60%~70%融合度时,将细胞培养液置换为不含胎牛血清的培养基,继续培养48h。收集细胞培养上清液,并离心10min(4℃,2000g)去除残留的细胞和细胞碎片;随后,使用0.22μm滤膜过滤上清液,并将滤液转移至超滤管(100KD)中,超滤离心30min(4℃,4000g),将超滤管中的沉淀重悬于PBS中,再次超滤离心30min(4℃,4000g);收集截留产物,并加入0.5体积的总外泌体提取试剂,涡旋混匀,4℃孵育过夜后,离心1h(4℃,10000g),弃去上清液,将获得的Exo重悬于PBS中,置于4℃或-80℃保存。将上述所提取的A549-Exo通过鉴定和定量后作为标准品应用于后续方法构建及评价。
2、捕获探针(MNPs@AptCD63)的制备
将生物素化的CD63适配体AptCD63修饰在MNPs-SA表面制备捕获探针MNPs@AptCD63,用以从待测样品中特异性富集和分离Exo。将10.0mg/mL的MNPs-SA置于涡旋混匀仪上20s混匀,取100μL于离心管中,磁分离,弃上清,加入buffer A清洗3次;随后,加入494μL的bufferA和6.0μL的100μmol/L的生物素标记的AptCD63,室温避光,涡旋反应30min;反应结束后磁分离,用buffer A清洗3次除去未反应的AptCD63,重悬于1mL buffer A中,得到捕获探针MNPs@AptCD63(浓度为1.0mg/mL,以MNPs计)。
3、信号探针(S)的制备
信号探针S由两条部分互补的单链DNA(Sa和Sb)杂交形成,将10μL 100μmol/L的Sa、10μL 100μmol/L的Sb和30μL的反应缓冲液混合,室温涡旋反应60min,即得信号探针S(浓度为20μmol/L),置于4℃短时间保存(1天)。
4、方法构建
用反应缓冲液将MNPs@AptCD63稀释后加入96孔板,每孔100μL,磁分离后弃上清;加入一系列反应缓冲液稀释的不同浓度的Exo,每孔100μL,室温避光反应40min后,磁分离弃上清,加入反应缓冲液清洗3次;随后,加入500nmol/L c-DNA,室温避光反应40min,磁分离,将上清液转移至新96孔板中;加入邻近探针P1和P2各100nmol/L(终浓度),室温避光反应40min后,加入500nmol/L信号探针S(终浓度),室温避光反应40min后,利用SpectraMax i3x多功能酶标仪(Molecular Devices,美国)监测FAM在522nm、TAMRA在580nm的荧光发射强度,设定激发波长为480nm。将所构建的邻近效应介导FRET方法用于共表达CD63/EGFR/EpCAM Exo的检测。不同浓度Exo存在下对应的荧光光谱如图2所示,随Exo浓度的增加,P1、P2和S可形成的稳定自组装体的量逐渐增多,FAM与TAMRA产生有效的FRET,可观察到580nm处TAMRA的荧光强度(I580)增加,同时伴随着522nm处FAM的荧光强度(I522)降低。以Exo浓度为横坐标,以荧光强度比值(I580/I522)为纵坐标,绘制标准曲线如图3所示,I580/I522与1×103~4×106particles/μL范围内的Exo浓度具有良好的线性关系,R2为0.9912。另外,通过实验测定方法的检测限(LOD)。测定10组空白样品获得其中,和SD分别为10组Exo空白样品检测获得的I580/I522的平均值和标准差。以对应的Exo浓度作为方法的LOD。因此,实验测得邻近效应介导FRET方法用于共表达CD63/EGFR/EpCAM Exo的检测的LOD为400particles/μL。
实施例2
复杂生物样品中外泌体的检测
胎牛血清超速离心2.5h(4℃,100,000g)除去Exo。将已知浓度的A549细胞来源的Exo分别添加到10%、20%和50%的无外泌体胎牛血清中,并按实施例1中步骤4构建的方法进行检测。结果如图4所示。由图4可见,通过独立样本t检验分析,在Exo在10%,20%和50%UC-FBS中产生的I580/I522与缓冲液中的结果均无显著差异(P>0.05)。表明该方法通过采用比率型信号进行自校准,可避免复杂生物样品基质对检测结果的影响,具有良好的抗干扰能力。
实施例3
临床适用性检测
将该方法用于非小细胞肺癌患者(15例)和健康志愿者(15例)血浆中Exo的检测。非小细胞肺癌患者(15例)和健康志愿者(15例)的血浆样本取自郑州大学第一附属医院,均为临床检测剩余样品。该实验已通过郑州大学生命科学伦理审查委员会审批,并按照相关规定进行。收集的血浆样品3000rpm离心5min,上清液经0.22μm的滤膜过滤,并用反应缓冲液稀释5倍后,采用实施例1中的步骤4所构建的方法进行检测。结果如图5所示。对检测结果进行独立样本t检验,发现非小细胞肺癌患者血浆中共表达CD63/EGFR/EpCAM Exo的量显著高于健康人(P<0.001)。因此,该方法在复杂生物样品中的检测具有良好的稳定性,并可在一定程度上区分非小细胞肺癌患者和健康人,具有很大的临床应用潜力。
对比例
为验证邻近杂交介导FRET检测共表达多种标志物Exo(CD63/EGFR/EpCAM)的技术效果,在Exo中加入P1/S或P2/S探针组合,其他条件以及步骤与实施例1相同,分别模拟CD63/EGFR或CD63/EpCAM Exo;同时,以Exo空白的P1/P2/S探针组合为对照。结果如图6所示,当Exo中仅存在P1/S或P2/S探针组合时(相当于Exo表面仅存在标志蛋白CD63/EGFR或CD63/EpCAM时),可观察到522nm处FAM强烈的荧光,而580nm处TAMRA荧光强度较弱,FRET效率极低,证明单独存在的P1或P2均无法与S形成稳定的二级结构,FAM与TAMRA距离较远而无法产生有效FRET。当在Exo中加入P1/P2/S探针组合时,TAMRA荧光强度显著升高,并伴随着FAM荧光强度降低,表明P1和P2同时与外泌体的结合,可使P1与P2之间的距离拉近而产生邻近效应,促进二者与S之间的组装,并介导近距离的FAM与TAMRA间产生高效的FRET。同时,对照试验表明不存在Exo时,TAMRA荧光强度较弱,即P1/P2/S探针组合不能自发形成稳定的自组装体。上述结果表明,目标Exo的存在,可促使探针体系通过邻近杂交形成稳定的自组装体,介导FAM与TAMRA间高效FRET的发生,实现对Exo表面多种标志物的联合检测。
试验例
1、特异性
选取3种不同的随机序列DNA:r-DNA1,r-DNA2和r-DNA3,r-DNA1包含的序列如SEQID No:7所示,r-DNA2包含的序列如SEQ ID NO:8所示,r-DNA3包含的序列如SEQ ID NO:9所示。r-DNA1,r-DNA2和r-DNA3如表2所示。
表2.r-DNA1,r-DNA2和r-DNA3探针序列
r-DNA1代替AptCD63,r-DNA2代替AptEGFR,r-DNA3代替AptEpCAM,用来评价方法的特异性,其他条件与实施例1相同,结果如图7所示。由图7可见,当随机序列DNA替代任一种Aptamer时,所测得的I580/I522与Exo空白时无明显差异;仅当三种Aptamer和Exo同时存在时,可观察到I580/I522显著升高,即体系中FAM与TAMRA发生有效FRET。这一结果表明,AptCD63,AptEGFR和AptEpCAM能够分别与Exo表面的蛋白标志物CD63、EGFR和EpCAM特异性结合,从而保证共表达CD63/EGFR/EpCAM Exo检测的选择性。
以人正常支气管上皮细胞(BEAS-2B)来源的Exo为对照,其他条件与实施例1相同,评价该方法用于肺癌细胞来源Exo检测的特异性,结果如图8所示。在相同浓度Exo存在下,A549-Exo产生的I580/I522显著高于BESA-2B Exo(P<0.001),提示A549-Exo中共表达CD63/EGFR/EpCAM Exo的量显著高于BESA-2B Exo。
综上,本发明基于邻近杂交介导FRET,提出了一种用于Exo表面多种标志物联合检测的新策略。该策略具有以下优势:首先,选择Aptamer作为Exo表面蛋白的特异性识别分子,可利用DNA组装的可程序化设计性能,方便设计邻近杂交探针体系;其次,通过Exo通用标志物CD63富集和分离Exo,及一对邻近探针同时与Exo表面两种肿瘤标志物的特异性结合,促进邻近杂交并介导有效FRET的产生,实现共表达多种肿瘤标志物Exo的检测,可提高肿瘤来源Exo检测的选择性;再者,利用FRET产生的比率型检测信号可降低检测背景,提高方法的抗干扰能力。该方法用于A549-Exo中共表达CD63/EGFR/EpCAM Exo检测的LOD为400particles/μL,在50%UC-FBS中的加标回收率和RSD分别为83.8%~107.0%和5.7%~7.3%。重要的是,血清检测结果显示非小细胞肺癌患者血浆(n=15)中共表达CD63/EGFR/EpCAM Exo的量显著高于健康人(n=15)(P<0.001),表明该方法在肺癌液体活检方面具有潜在的应用价值。另外,通过简单地改变用于标志物识别的Aptamer,该方法可方便地扩展到其他肿瘤来源Exo表面多种标志物的联合检测,这将为提高肿瘤早期诊断准确率提供潜在的检测方法。
序列表
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Claims (10)
1.一种利用多种标志物联合检测外泌体的试剂盒,其特征在于,包括用于捕获外泌体的捕获探针、邻近探针P1和P2以及信号探针S,P1包含第一核酸适配体序列以及有荧光素标记的序列,P2包含第二核酸适配体序列以及有荧光素标记的序列,信号探针S由Sa和Sb组成,Sa包含与有荧光素标记的序列互补的序列,Sb包含与Sa互补的序列以及有罗丹明标记的序列。
2.根据权利要求1所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,所述P1的第一核酸适配体序列以及有荧光素标记的序列之间有P1间隔序列,P2的第二核酸适配体序列以及有荧光素标记的序列之间有P2间隔序列。
3.根据权利要求2所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,所述P1包含的序列如SEQ ID NO:1所示,P2包含的序列如SEQ ID NO:2所示。
4.根据权利要求1-3任一项所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,所述Sa的两端序列分别与P1的有荧光素标记的序列和P2的有荧光素标记的序列互补。
5.根据权利要求4所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,所述Sb与Sa的中间序列互补,Sb的两端标记有罗丹明。
6.根据权利要求5所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,所述Sa的序列如SEQ ID NO:3所示,Sb包含的序列如SEQ ID NO:4所示。
7.根据权利要求1所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,所述第一核酸适配体可特异性识别表皮生长因子受体,第二核酸适配体可特异性识别上皮细胞粘附分子。
8.根据权利要求1所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,所述捕获探针为生物素化的CD63适配体修饰在链霉亲和素磁表面制备得到。
9.根据权利要求1所述的利用多种标志物联合检测外泌体的试剂盒,其特征在于,还包括与所述捕获探针序列互补的c-DNA序列,用于将捕获的外泌体释放。
10.如权利要求9所述的利用多种标志物联合检测外泌体的方法,其特征在于,包括以下步骤:
1)使用所述捕获探针富集外泌体,之后用c-DNA分离得到外泌体上清液;
2)加入邻近探针P1和P2,反应后加入信号探针S,反应后检测荧光素在522nm的荧光发射强度I522以及罗丹明在580nm处的荧光发射强度I580,I580/I522与外泌体的浓度呈正相关,即可检测出外泌体的浓度。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114280016A (zh) * | 2021-12-07 | 2022-04-05 | 广州兆瑞医学生物科技有限公司 | 一种外泌体检测方法 |
CN114634973A (zh) * | 2022-03-11 | 2022-06-17 | 郑州大学 | 一种核酸适配体识别的肿瘤外泌体检测方法 |
CN114657184A (zh) * | 2022-02-10 | 2022-06-24 | 南京邮电大学 | 一种多价适体功能化dna纳米结构探针及其制备方法和应用 |
CN115772525A (zh) * | 2022-11-29 | 2023-03-10 | 中国科学院基础医学与肿瘤研究所(筹) | 一种检测早期卵巢癌的核酸适体组合及其应用 |
Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1570140A (zh) * | 2003-07-25 | 2005-01-26 | 宋克 | 双探针基因芯片信号放大方法 |
US20080124735A1 (en) * | 2006-10-16 | 2008-05-29 | Matthias Schuster | Method for detection of one or more CpG positions |
CN105392895A (zh) * | 2013-03-13 | 2016-03-09 | 中尺度技术有限责任公司 | 改进的测定方法 |
US20170275696A1 (en) * | 2015-12-10 | 2017-09-28 | Surexam Bio-Tech Co., Ltd. | Circulating tumour cell typing and identification kit |
CN107860765A (zh) * | 2017-11-06 | 2018-03-30 | 首都医科大学 | 一种金属离子检测用探针、试剂盒、制备方法、应用 |
CN107893101A (zh) * | 2017-12-22 | 2018-04-10 | 郑州大学 | 一种用于肿瘤疾病早期诊断的试剂盒、方法及应用 |
CN108872438A (zh) * | 2018-08-06 | 2018-11-23 | 杭州迪相实业有限公司 | 一种外泌体中肺癌标志物gk5快速检测试剂盒 |
CN109504775A (zh) * | 2018-12-27 | 2019-03-22 | 杭州迪相实业有限公司 | 一种外泌体肺部肿瘤标志物lsm2的快速检测试剂盒 |
CN109507416A (zh) * | 2018-12-27 | 2019-03-22 | 杭州迪相实业有限公司 | 一种外泌体肿瘤标志物pdl1的快速检测试剂盒 |
CN110468190A (zh) * | 2019-08-23 | 2019-11-19 | 郑州大学 | 一种基于构型变化的自组装体探针及其用于外泌体的无标记检测方法 |
CN111394470A (zh) * | 2020-05-27 | 2020-07-10 | 郑州大学第一附属医院 | 一组早期食管鳞状细胞癌筛查标志物及其在探针试剂盒中的应用 |
CN112391448A (zh) * | 2020-04-29 | 2021-02-23 | 湖北中医药大学 | 一种用于外泌体及表面蛋白分析的dna纳米分子机器及应用 |
KR20210093266A (ko) * | 2018-12-27 | 2021-07-27 | 항저우 디시앙 컴퍼니 리미티드 | 엑소좀 막단백질과 mRNA를 동시에 검출하는 방법 |
-
2021
- 2021-08-09 CN CN202110907130.3A patent/CN113640515B/zh active Active
Patent Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1570140A (zh) * | 2003-07-25 | 2005-01-26 | 宋克 | 双探针基因芯片信号放大方法 |
US20080124735A1 (en) * | 2006-10-16 | 2008-05-29 | Matthias Schuster | Method for detection of one or more CpG positions |
CN105392895A (zh) * | 2013-03-13 | 2016-03-09 | 中尺度技术有限责任公司 | 改进的测定方法 |
US20170275696A1 (en) * | 2015-12-10 | 2017-09-28 | Surexam Bio-Tech Co., Ltd. | Circulating tumour cell typing and identification kit |
CN107860765A (zh) * | 2017-11-06 | 2018-03-30 | 首都医科大学 | 一种金属离子检测用探针、试剂盒、制备方法、应用 |
CN107893101A (zh) * | 2017-12-22 | 2018-04-10 | 郑州大学 | 一种用于肿瘤疾病早期诊断的试剂盒、方法及应用 |
CN108872438A (zh) * | 2018-08-06 | 2018-11-23 | 杭州迪相实业有限公司 | 一种外泌体中肺癌标志物gk5快速检测试剂盒 |
CN109504775A (zh) * | 2018-12-27 | 2019-03-22 | 杭州迪相实业有限公司 | 一种外泌体肺部肿瘤标志物lsm2的快速检测试剂盒 |
CN109507416A (zh) * | 2018-12-27 | 2019-03-22 | 杭州迪相实业有限公司 | 一种外泌体肿瘤标志物pdl1的快速检测试剂盒 |
KR20210093266A (ko) * | 2018-12-27 | 2021-07-27 | 항저우 디시앙 컴퍼니 리미티드 | 엑소좀 막단백질과 mRNA를 동시에 검출하는 방법 |
CN110468190A (zh) * | 2019-08-23 | 2019-11-19 | 郑州大学 | 一种基于构型变化的自组装体探针及其用于外泌体的无标记检测方法 |
CN112391448A (zh) * | 2020-04-29 | 2021-02-23 | 湖北中医药大学 | 一种用于外泌体及表面蛋白分析的dna纳米分子机器及应用 |
CN111394470A (zh) * | 2020-05-27 | 2020-07-10 | 郑州大学第一附属医院 | 一组早期食管鳞状细胞癌筛查标志物及其在探针试剂盒中的应用 |
Non-Patent Citations (5)
Title |
---|
LIN B, ET AL.: "Tracing Tumor-Derived Exosomal PD-L1 by Dual-Aptamer Activated Proximity-Induced Droplet Digital PCR", ANGEW CHEM INT ED ENGL., vol. 60, no. 14 * |
YAMIN XIONG, ET AL.: "Dual signal amplification strategy for high-sensitivity detection of copper species in bio-samples with a tunable dynamic range", CHEM COMMUN (CAMB), vol. 54, no. 20 * |
ZHAO X, ET AL.: "Aptamer-Cholesterol-Mediated Proximity Ligation Assay for Accurate Identification of Exosomes", ANAL CHEM., vol. 92, no. 7, XP055805951, DOI: 10.1021/acs.analchem.0c00141 * |
张松柏;郑丽英;胡霞;沈广宇;刘学文;沈国励;俞汝勤;: "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", 分析化学, no. 11 * |
熊亚敏;黄沛力: "可用于血清游离铜快速检测的特异性识别元件", 生态毒理学报, vol. 12, no. 005 * |
Cited By (5)
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---|---|---|---|---|
CN114280016A (zh) * | 2021-12-07 | 2022-04-05 | 广州兆瑞医学生物科技有限公司 | 一种外泌体检测方法 |
CN114657184A (zh) * | 2022-02-10 | 2022-06-24 | 南京邮电大学 | 一种多价适体功能化dna纳米结构探针及其制备方法和应用 |
CN114657184B (zh) * | 2022-02-10 | 2023-09-12 | 南京邮电大学 | 一种多价适体功能化dna纳米结构探针及其制备方法和应用 |
CN114634973A (zh) * | 2022-03-11 | 2022-06-17 | 郑州大学 | 一种核酸适配体识别的肿瘤外泌体检测方法 |
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