长余辉发光微流控纸芯片的构建及其应用研究

Microfluidic paper-based analytical devices (μPADs) are one of the most potential point-of-care testing (POCT) methods for tumor marker in body fluid. Among them, the μPADs based on fluorescence detection have shown great application prospect, due to their excellent sensitivity. However, the assay sensitivity of fluorescence μPADs in biological sample will be limited, which is mainly attributed to the autofluofescence, scattering light and light absorption arise from biological matrix and paper substrate. To overcome these problems, this project intends to develop novel luminescence μPADs based on persistent luminescence nanoparticles (PLNPs). Energy transfer process with inorganic nanoquencher (carbon nanoparticles, gold nanoparticles) as the acceptor will be constructed to adjust persistent luminescence intensity. By using persistence luminescence of PLNPs as the detection signal, the optical interference in fluorescence μPADs can be completelly eliminated, and thus, sensitivity of tumor marker detection are enhanced obviously. More importantly, complicated body fluid sample (blood, urine) can be detected directly without any separation steps. Combined with the unique optical properties of PLNPs, the excellent quenching ability of nanoquencher and the advantages of μPADs, this project is expected to provide a new technology and a new method for the simple, rapid and sensitive analysis of clinically complex samples.

微流控纸芯片（μPADs）是目前最有潜力实现体液样本肿瘤标志物现场快速检测（POCT）的方法之一。荧光分析技术是提高μPADs检测灵敏度的一个重要策略。然而，由于生物基质和纸张基底存在本底荧光，散射光和光吸收干扰，使得荧光微流控纸芯片应用于实际样本分析时的灵敏度受到限制。为了解决上述问题，本项目拟设计基于长余辉发光纳米材料（PLNPs）的新型发光微流控纸芯片，构建无机纳米材料（碳纳米颗粒、金纳米颗粒）为受体的能量转移过程，实现长余辉发光强度的有效调控。以长余辉发光作为检测信号，消除荧光微流控纸芯片面临的光学干扰问题，显著提高检测灵敏度，实现复杂体液样本（血液、尿液）的无分离直接检测。本项目结合了PLNPs独特的光学性质，纳米受体优异的猝灭能力和μPADs简单便捷的优势，有望为临床复杂样本的简便、快速和高灵敏分析提供一种新技术和新方法。

本项目旨在通过在微流控纸芯片中引入荧光纳米材料构建新型纸基荧光共振能量转移体系，降低光学干扰，发展高灵敏的纸基分析方法，实现复杂样本中疾病标志物的无分离直接检测。围绕这一目标，本项目制备了长余辉发光纳米材料（PLNPs）、上转换纳米材料（UCNPs）及荧光碳点（Cdots）等性能优良的荧光纳米材料作为能量供体，并对其进行表面修饰和生物偶联；制备了氧化碳纳米颗粒（OCNPs）、二氧化锰纳米片及金纳米颗粒（AuNPs）等无机纳米材料作为能量受体，系统地优化了制备方法，并对其作为受体的应用性能进行了研究；构建了以不同材料为供受体对的纸基能量转移体系，探索了能量供体与纸张基底的组装方法、能量供体与受体的组装方法，并对纸基能量转移效率进行了研究；构建了基于核酸适配体、免疫反应等识别机理的分析方法，实现了体液样本中疾病标志物的检测。本项目所取得的研究成果拓展了荧光微流控纸芯片的应用范围，有望为临床复杂样本中疾病标志物的分析提供新方法。