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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 design and fabricate a new type of fluorescent nano-particles based on the magnetic separation technology of sandwich structure and magnetic nanoparticles constructed by high affinity of two nucleic aptamers and Interferon-γ (IFN-γ) Particles, to establish a novel aptamer-based fluorescent nanoparticles for the detection of human IFN-γ. Methods: Flow cytometry was used to detect the binding specificity of IFN-γ aptamers B1-4 and T2 to IFN-γ. Biotin-modified B1-4 was immobilized on streptavidin-coated magnetic nanoparticles , The magnetic beads connected with B1-4 can capture IFN-γ by the affinity of B1-4 and IFN-γ, and another B1-modified nucleic acid aptamer T2 can be used to form (B1-4) - ( IFN-γ) - (T2) sandwiched sandwich structure to construct a novel aptamer fluorescent nanoparticle. IFN-γ and aptamer fluorescent nanoparticles were incubated at different times to explore the IFN-γ and magnetic beads The optimal incubation time was determined by flow cytometry. Fluorescence changes of magnetic beads were detected by flow cytometry to explore the influence of time on the detection system. Flow cytometry was used to detect the fluorescence intensity of magnetic beads incubated with different concentrations of IFN- Standard curve; detect the effect of serum on test specificity. Results: The binding specificity of B1-4 and T2 to IFN-γ was high. Sandwich sandwich structure was specific to IFN-γ detection. Fluctuation of the fluorescence signal on the surface of the magnetic beads was significantly reduced by any factor change. The sandwich sandwich structure failed to be constructed. The linear response of IFN-γ to IFN-γ was 0-50 ng / L with a linear equation of y = 3.512x + 1.060 with a sensitivity of 1 ng / L. The fluorescence signal measured within 12 h was stable with no significant attenuation Serum had no obvious effect on the specificity of the system. Conclusion: The aptamer fluorescent nanoparticles were successfully constructed for the determination of human IFN-γ.