| 张李钰 何威 杨颖 孙宏利 宋佳凝 冯帆 武海滨.新型核酸适配体荧光纳米微粒用于人干扰素-γ的测定[J].,2016,16(27):5241-5245 |
| 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 Detectionof Human IFN-γ based on Aptamer |
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| DOI： |
| 中文关键词: 核酸适配体 IFN-γ 磁性纳米颗粒 三明治夹心结构 荧光 |
| 英文关键词: Aptamer IFN-γ Nanomagnetic Beads Sandwich structure Fluorescence |
| 基金项目:西安市科技计划项目（SF1510(4)） |
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| 中文摘要: |
| 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-酌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-酌孵育的磁珠表面荧光强度，绘制IFN-酌检测标准曲线；检测血清对检测特异性的影响。结果：B1-4 和T2 对IFN-酌的结合特
异性高；三明治夹心结构对IFN-酌检测特异性好，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-酌的响应线性浓度为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。 |
| 英文摘要: |
| 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-γand
magnetic separation technology of magnetic nanoparticles.Methods:Flow cytometry was employed to monitor the binding specificities
of aptamer B1-4 and T2 to IFN-酌; Aptamer B1-4 modified with biotin was linked to the magnetic beads which were coated by streptavidin,
then these beads could capture IFN-酌because of the B1-4's binding affinity to IFN-酌. Next, another IFN-酌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-γfor 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-γdetection system; Flow cytometry was utilized to assess the fluorescence intensities under different IFN-γconcentration
to make the linear equation of the IFN-γdetection; The influence caused by serum to detection specificity of nanoparticle systemwas
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-γdetection 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 meanthe
fluorescence of nanoparticlewasstable; Serumhad noobvious effect onthe detectionspecificityof sandwichstructure.Conclusion:Successfully
established a novel nano-particle which is based on fluorescent nanomagnetic beads mediated by aptamer for the detection of human
IFN-γ. |
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