基于核酸分子门控染料释放的多色荧光信号同时获取传感器阵列用于肿瘤标志物检测

Rapid, highly sensitive and high-throughput detection of multiplex biomarkers plays an important role in cancer diagnostics. The project has proposed a multicolor fluorescent sensor array for detection of cancer biomarkers in sera and cells. We plan to use a series of oligonucleotides, which have different binding affinities with biomarkers, to seal mesoporous silica nanoparticles(MSN) containing fluorophores with different excitation and emission waves. As fluorescent quenchers are covalently immobilized on the inner walls of mesopores, the fluorescence is quenched when MSNs are sealed. In the presence of biomarkers, the oligonucleotides attached on the surface of MSNs with electrostatic adsorption can be moved away and release fluorophores into solutions as a result of the competitive displacement. Since the binding affinities between oligonucleotides and biomarkers are different, the release rates of different fluorophores are different so that distinct multicolor fluorescence recovery patterns are obtained, which can be discriminated as “fingerprints”. The most attractive advantages of oligonucleotide-gated dyes release are signal enhancement and multidimensional signal obtaining, which can significantly improve the sensitivity and differentiability of a sensor array. Using this strategy, we hope array-based sensing of multiplex cancer biomarkers and sera diagnostics can be demonstrated for the first time.

快速、多组分、高灵敏、高通量检测肿瘤标志物在肿瘤诊断和治疗中具有重要的意义。本项目提出一种多色荧光信号同时获取的传感器阵列，用于血清以及细胞内的肿瘤标志物检测，拟利用一系列与各肿瘤标志物亲和力不一的核酸分子作为识别单元，以静电吸附作用包裹填充有不同荧光染料的多孔硅球，染料在硅球孔道中处于荧光淬灭状态。肿瘤标志物加入后，因其将各核酸分子从硅球表面竞争下来的能力具有特征性差异，致使各色荧光染料释放速度快慢不一，各色荧光的恢复情况也就有特征性差异。通过同时检测各色荧光信号随时间变化的曲线，得到各肿瘤标志物的“指纹图谱”，从而实现区分检测。本项目通过核酸门控荧光染料释放来实现信号放大和多维信息同时获取，大大提高传感器阵列的灵敏度和分辨能力，以期首次实现传感器阵列对多种肿瘤标志物的高灵敏度检测以及实际血清样品的肿瘤诊断和分期。

本项目针对肿瘤标志物的便捷检测和肿瘤筛查，利用新型纳米材料和阵列传感思路，构建了一系列光谱传感器阵列。首先，利用磁分离和等离子体荧光增强设计了非标记荧光纳米簇传感器对两种标志物在两个波长通道进行同时检测。然后，利用肿瘤标志物与金属纳米颗粒特征性的相互作用构建了显色-荧光双信号通路传感器阵列用于肿瘤标志物的识别和正常人与肿瘤病人血清样品的区分。最后，基于核酸分子门控介孔硅球的染料释放实现了多色荧光恢复对三种肿瘤标志物的同时检测。这些方法与传统免疫检测方法相比，无需使用抗体或标记，检测方便快速，成本低廉，并在肿瘤筛查和细胞成像中有较大的应用前景。