新型核酸适配体荧光纳米微粒用于人干扰素-γ的测定

Establishment of a Novel Fluorescent Nanomagnetic Beads for the Detection of Human IFN-γ based on Aptamer

目的：利用由两条核酸适配体与人干扰素-γ（Interferon-γ,IFN-γ）的高亲和力构建的三明治结构和磁性纳米颗粒的磁性分离技术,设计并制作了一种新型荧光纳米微粒,建立基于核酸适配体的新型荧光纳米微粒用于人IFN-γ的检测。方法：流式细胞术检测IFN-γ核酸适配体B1-4和T2对IFN-γ的结合特异性;将生物素修饰的B1-4固定于链霉亲和素包被的纳米磁珠上,连接B1-4的纳米磁珠可通过B1-4与IFN-γ的亲和性捕获IFN-γ,再利用另一条FAM修饰的IFN-γ核酸适配体T2形成（B1-4）-（IFN-γ）-（T2）三明治夹心结构,构建新型核酸适配体荧光纳米微粒;IFN-γ分别与核酸适配体荧光纳米微粒孵育不同时间以探索该检测系统中IFN-γ与磁珠的最佳孵育时间;流式细胞术检测磁珠12 h内荧光变化,探索时间对检测体系的影响;流式细胞术检测与不同浓度IFN-γ孵育的磁珠表面荧光强度,绘制IFN-γ检测标准曲线;检测血清对检测特异性的影响。结果：B1-4和T2对IFN-γ的结合特异性高;三明治夹心结构对IFN-γ检测特异性好,任意改变其中一因素则磁珠表面荧光信号显著下降,三明治夹心结构构建失败;此法对IFN-γ的响应线性浓度为0~50 ng/L,其线性方程为y=3.512x＋1.060,敏感度为1 ng/L;12 h内测得的荧光信号稳定,并无明显衰减;血清对体系检测特异性无明显影响。结论：成功构建核酸适配体荧光纳米微粒用于人IFN-γ的测定。

Objective： To establish and produce a novel nanoparticle which was based on fluorescent nanomagnetic beads mediated by aptamers for the detection of human IFN-γ, we constructed a sandwich structure utilized two aptamers’ binding abilities to IFN-γ and magnetic separation technology of magnetic nanoparticles. Methods： Flow cytometry was employed to monitor the binding specificities of aptamer B1-4 and T2 to IFN-γ; Aptamer B1-4 modified with biotin was linked to the magnetic beads which were coated by streptavidin, then these beads could capture IFN-γ because of the B1-4’s binding affinity to IFN-γ. Next, another IFN-γ aptamer T2 modified with FAM was added to form sandwich structure（B1-4）-（IFN-γ）-（T2）, then the aptamer mediated fluorescent nanomagnetic beads were constructed successfully; Fluorescence intensities of nanomagnetic beads incubated with IFN-γ for different time were detected to study the optimum IFN-γ incubation time; Flow cytometry was employed to detect the fluorescence change of beads within 12 h to study the influence of time to IFN-γ detection system; Flow cytometry was utilized to assess the fluorescence intensities under different IFN-γ concentration to make the linear equation of the IFN-γ detection; The influence caused by serum to detection specificity of nanoparticle system was investigated. Results： B1-4 and T2 showed high binding specificity to IFN-γ; The sandwich structure showed high binding specificity to IFN-γ, and the fluorescence intensities decreased significantly when changed anyone of the three elements in sandwich structure,which mean the construction failure of sandwich structure; The response linear range of IFN-γ detection is 0~50 ng/L, and the linear equation is y=3.512x＋1.060, the sensitivity is 1 ng/L; The fluorescence intensities showed no obvious decline within 12 h which mean the fluorescence of nanoparticle was stable; Serum had no obvious effect on the detection specificity of sandwich structure. Conclusions： Successfully established a novel nano-particle which is based on fluorescent nanomagnetic beads mediated by aptamer for the detection of human IFN-γ.