基于近红外光镊技术的金属增强上转换发光纳米传感器及其在血清肿瘤标志物检测中的应用

Based on the principle of optical tweezers trapping microparticles,this project aims to develop a new strategy to sensitively detect tumor markers (TM) in serum. Firstly, microspheres and up-converting luminescent nanoparticles are employed to enrich target molecules by dual-antibody sandwich approach on the surface of the microspheres, and then near-infrared laser beam is focused to optically trap the above microspheres, and simultaneously the up-converting luminescence signals from the microspheres are detected for TM determination. By making use of the principle of noble metal enhanced fluorescence, microspheres that are modified or unmodified with gold island film are manupulated by optical tweezers to control the distance between the surface of the trapped microspheres and the metal island substate on cover glass, and thus single and dual enhancement model for TM sensing are developed, respectively, to significantly increase the detection sensitivity, and more importantly, the mechnism of up-conversion luminescence enhancement will be studied deeply using this apporach. Due to the use of near-infrared laser of 980 nm wavelength, the scattering and autofluorescence inside the serum sample will be avoided. Therefore, this strategy will show the advantages such as high sensitivity, excellent selectivity, speed, small sample volume and no need for sample treatment, etc. Finally, the results not only provide ultrasensitive detection strategy for TM in serum, but also provide a novel detection setup which has potential for wide applications.

本项目基于光镊捕获微米粒子的基本原理，拟构建一种快速、灵敏检测血清中肿瘤标志物(TM)的上转换发光纳米检测新方法。首先利用微球和上转换发光纳米粒子，通过双抗夹心免疫反应，将靶分子富集于微球表面，再通过聚焦近红外激光形成光镊捕获上述微球，同时对源于该微球表面的上转换发光信号进行探测以实现TM的定量分析。利用贵金属增强荧光的原理，通过光镊操纵并精密调控微球（表面修饰或不修饰金岛膜）表面和金岛膜基底盖片的距离，分别构建单重增强和双重增强传感模式，以显著提高检测的灵敏度，并对上转换发光增强机制进行深入研究。由于采用波长980nm的近红外激光为信号激发源，避免了血清中复杂基质的散射光和自发荧光的干扰。因此，本方法具有灵敏度高、选择性好、速度快、样品用量少且无需预处理等优点。本项目结果不仅提供一种血清TM的高灵敏度检测方法，同时提供一种有广泛应用前景的新型检测装置。

本项目基于光镊捕获微米粒子的基本原理，构建了一系列快速、灵敏检测肿瘤标志物(TM)的上转换发光纳米检测新方法。首先利用微球和上转换荧发光纳米粒子，通过双抗夹心免疫反应，将靶分子富集于微球表面，再通过聚焦近红外激光形成光镊捕获上述微球，同时对源于该微球表面的上转换发光信号进行探测以实现TM的定量分析。由于本方法借助了上转换发光极低的背景发光信号，可对MicroRNA-21序列进行“非放大”检测，其检出限达12 fM，并具有很好的选择性，甚至可实现单碱基错配检测。此外，这种分析技术可对低至100个癌细胞中MicroRNA-21序列的含量进行准确定量，其结果与传统的RT-PCR结果吻合。利用全息光镊捕获上述多颗偶联复合微球以形成3×3微球阵列，同时激发出微球表面上转换纳米颗粒的上转换发光信号，实现双通道发光信号的采集和微球成像，对CEA和AFP的检出限分别达到2.5 pg/mL和2.7 pg/mL。采用量子点为纳米探针，构建了双光子荧光检测模式，实现了对患者血清中CEA的检测，在缓冲溶液和人全血清介质中对于CEA的检出限分别达到7.8 pg/mL和8.6 pg/mL。由于采用波长980nm的近红外激光为信号激发源，避免了血清中复杂基质的散射光和自发荧光的干扰。因此，本方法具有灵敏度高、选择性好、速度快、样品用量少且无需预处理等优点。同时，采用RuBpy荧光分子、荧光碳点和金团簇构建了多种金属增强荧光生物传感体系。通过抗体-抗原-抗体作为金岛膜和碳点之间的间隔层，并分别探究了免疫杂交链式反应和金属增强荧光效应对荧光信号的放大和增强的影响。当免疫杂交链式反应和金属增强荧光双重信号放大同时存在时，荧光最大增强倍数达到17.2。对AFP检测的线性范围跨五个数量级(0.0005 - 5 ng/mL)，检出限达94.3 fg/mL。本项目不仅提供了一种血清和癌变组织TM的高灵敏度检测方法，同时提供一种有广泛应用前景的新型检测装置，在生物医学分析和疾病诊断方面具有重要的应用价值。