基于稀土上转换纳米荧光探针的艾滋病早期标志物体外检测研究

Currently, in vitro diagnosis of AIDS is confronted with the long window period problem. The early and accurate identification of AIDS early marker is very important for early diagnosis of HIV. However, due to the shortcomings such as complex operation, time-consuming and expensive, the methods currently used for detecting early markers of AIDS are difficult to popularize in China. Rare-earth upconverting fluorescencent probes are expected to play an important role in the early diagnosis of HIV for the reason that it can improve the detection sensitivity and specificity through effectively avoiding the interference from the biological autofluorescence. In this project we propose a new method based on the upconversion fluorescence for the detection of AIDS early serum marker. We are devoted to the investigation of novel nano-bioprobes based on RE-doped UCNPs for in vitro biodetection of human HIV early markers, propose reliable and universal bioconjugate techniques for specific binding of HIV early markers, and develop the general protocols for both heterogeneous upconversion luminescence (UCL) and homogeneous upconversion fluorescence resonance energy transfer (UC-FRET) assays for sensitive and specific detection of HIV early marker such as p24 antigen. Particularly, it is expected that the detection limit of p24 antigen finally reaches as low as 10 pM, and the window period reduces to 2-3 weeks. We anticipate that this study could provide methodological basis for the early diagnosis and disease surveillance of AIDS in China.

艾滋病的血清抗体体外诊断面临窗口期较长的问题，及早、准确地对艾滋病的早期标志物进行识别是艾滋病早期确诊的关键。目前艾滋病早期标志物的检测方法如病毒RNA检测由于操作复杂、耗时长且花费昂贵而难以在我国基层广泛使用。稀土上转换荧光探针可有效避免生物自荧光干扰而显著提高疾病标志物的检测灵敏度，有望在艾滋病早期诊断等领域发挥重要作用。本项目针对艾滋病早期标志物如HIV-1型p24抗原提出一种艾滋病窗口期上转换荧光检测新方法，致力于研发可用于其体外检测的具有高发光效率的稀土上转换纳米荧光探针，发展成熟可靠的生物偶联技术，建立基于稀土纳米荧光探针的异相上转换发光和均相上转换荧光共振能量传递检测方法，实现对艾滋病早期标志物的高灵敏特异性检测，其中对p24抗原的检测限低于10 pM，窗口期缩短至2-3周，为我国艾滋病的早期诊断和疾病监控提供方法学依据及相关基础。

艾滋病的血清抗体体外诊断面临窗口期较长的问题，及早、准确地对艾滋病的早期标志物进行识别是艾滋病早期确诊的关键。目前艾滋病早期标志物的检测方法如病毒RNA检测由于操作复杂、耗时长且花费昂贵而难以在我国基层广泛使用。稀土纳米荧光探针可有效避免生物自荧光干扰而显著提高疾病标志物的检测灵敏度，有望在艾滋病早期诊断等领域发挥重要作用。为解决这一问题，我们发展了基于稀土氧化物和稀土氧化物复合物纳米探针用于艾滋病早期标志物HIV-1 p24抗原超灵敏检测的方法, 建立了成熟可靠的表面修饰和生物偶联技术，成功地实现了对艾滋病早期标志物HIV-1 p24抗原的超灵敏时间分辨检测，检测限分别低至0.37 pg/mL和25 fg/mL，比第四代ELISA试剂盒降低了约500倍，对应检测窗口期缩短至3周以下。进一步地，我们利用基于该纳米探针的检测方法实测了福州市传染病提供的87例艾滋病患者血清中HIV-1 p24抗原含量，结果与临床商用试剂盒结果一致，并通过血清样品抗干扰实验、变异系数及回收率测定等验证了该检测方法的特异性、精确度和可靠性，为我国艾滋病的早期诊断和疾病监控提供方法学依据及相关基础。在项目执行期间，本项目组已在Coord. Chem. Rev., Adv. Sci., Nano Res., Nanoscale, Int. J. Nanomed., J Mater. Chem. B, Food Chem., Colloid Surface B.等国际期刊发表SCI论文11篇，其中I区论文8篇（影响因子大于10的1篇，大于7的5篇），II区论文3篇；受理中国发明专利2项，圆满完成了预定的研究目标。