基于核酸适配体的比率荧光探针定量检测血浆外泌体

A novel ratiometric fluorescent probe method for the quantitative detection of exosomes in the plasma based on aptamer

目的:建立一种新型比率荧光探针法用于定量检测血浆中外泌体。方法:将Cy5荧光探针标记的CD63核酸适配体固定到链霉亲和素磁珠(MNPs)上,得到MNPs-Apt,加入SYBR GreenⅠ(SGⅠ)核酸染料构建比率荧光免疫磁珠(MNPs-Apt-SGⅠ)。当外泌体存在时,CD63核酸适配体捕获外泌体而发生结构改变,SGⅠ探针从双链中释放,免疫磁珠上的SGⅠ荧光强度大幅减弱,而Cy5荧光探针的荧光恒定,根据两种荧光探针被激发的荧光信号比值变化[Δ(F_(C)/F_(S))]对外泌体进行定量检测。结果:外泌体浓度在8.0×10^(4)~1.0×10^(7)个/μL范围内,Δ(F_(C)/F_(S))与外泌体浓度呈良好的线性关系。该方法灵敏度高,选择性好,检出限为4.0×10^(4)个/μL。此外,所建立的方法成功地应用于血浆外泌体的检测。结论:构建的比率荧光探针法具有快速、灵敏、准确度高、特异性强等优点,可用于血浆样品外泌体的定量检测。

Aim:To establish a novel ratiometric fluorescent probe method for the quantitative detection of exosomes in the plasma.Methods:The CD63 aptamer modified with Cy5 fluorescent probe(Cy5-Apt) was immobilized to streptavidin magnetic nanoparticles(MNPs) and obatined MNPs-Apt.Secondly,SYBR Green Ⅰ(SGⅠ) fluorescent probe was added to construct ratiometric fluorescent immunomagnetic beads(MNPs-Apt-SGⅠ).When exosomes were present,the aptamer captured exosomes with structural changes,SGⅠ fluorescent probe was released from the double chain,and the fluorescence intensity of the immunomagnetic beads was greatly weakened,while the fluorescence of Cy5 fluorescent probe remained the same.Exosomes were quantitatively detected according to the changes in the ratio of excited fluorescence signals of two fluorescent probes[Δ(F_(C)/F_(S))].Results:In the range of 8.0×10^(4)-1.0×10^(7)/μL,Δ(F_(C)/F_(S)) had a good linear relationship with the concentration of exosomes,which could be used for quantitative detection of exosomes.The method had high sensitivity and selectivity,with the detection limit of 4.0×10^(4)/μL.Besides,the established method had been successfully applied to the detection of exosomes in the plasma.Conclusion:The novel method of quantitative detection of exosomes has been developed by using ratiometric fluorescent probe.It takes on the advantages of being speedy,sensitive,accurate and specific,and could be used for the detection of exosomes in actual plasma samples.