定向控制金纳米颗粒生长及其在免疫诊断和核酸诊断中的应用研究

The progress of in vitro diagnosis is extremely significant to rapid diagnosis and screening of diseases. Currently, immunodiagnosis and nucleic acid-based diagnosis are two important diagnostic systems in the clinic. Due to various drawbacks (such as moderate sensitivity and requirement of sophisticated instruments, etc), the two systems are being limited only to a few clinical settings. We here plan to develop a rapid diagnostic platform with high sensitivity and visualization, which will be applied into immunodiagnosis and nucleic acid-based diagnosis. The mechanism of this method is based on the formation of gold nanoparticle dimer induced by detection targets. The dimeric structure will grow orientedly in the presence of gold growth solution, causing the strong coupling of surface plasmon resonance. Consequently, the color of the solutions as well as their corresponding absorption bands changed significantly. Compared to the clinical in vitro diagnostic methods, this platform has several advantages such as no requirement of advanced instruments, high universality and rapidity for use, and the reagents are relatively inexpensive and easy to be kept. Therefore, this system is particularly suitable for in vitro diagnosis. Finally, we plan to test a variety of clinical samples using this platform, and the accuracy of the results will be confirmed by the standard methods clinically used. The feasibility of this platform in clinical applications will be comprehensively evaluated.

体外诊断技术的进步对疾病快速诊断和筛查有着十分重要的现实意义。临床上，免疫诊断和核酸诊断是两类重要的体外诊断技术。目前，由于各种缺陷（如检测灵敏度不够高、依赖大型复杂仪器等）的存在，限制了这两类诊断方法在临床上的广泛使用。在本项目中，我们拟发展一种高灵敏度、可视化的快速检测平台，用于免疫诊断和核酸诊断。该检测方法的原理是：待检测物使得不对称功能化金纳米颗粒形成二聚体结构，再通过控制金纳米颗粒的定向生长，导致颗粒表面等离子共振耦合发生突跃，从而使溶液颜色和相应的吸收峰发生显著变化。与临床上使用的体外诊断平台相比，该诊断系统不依赖昂贵设备，普适性高，检测速度快，所用试剂廉价且易于存放，故特别适合于体外诊断。最后，我们将对大量临床样品进行测试，并与临床标准诊断方法进行对比，验证本诊断系统的准确性，并全方位评估本诊断系统在临床应用方面的可行性。

体外诊断技术的进步对疾病快速诊断和筛查有着十分重要的现实意义。临床上，免疫诊断和核酸诊断是两类重要的体外诊断技术。目前，由于各种缺陷（如检测灵敏度不够高、依赖大型复杂仪器等）的存在，限制了这两类诊断方法在临床上的广泛使用。本项目通过制备一系列功能化金纳米颗粒（尤其是不对称金纳米颗粒）,使之应用于免疫检测和核酸检测，目前已取得的进展简述如下：. 在免疫检测方面，我们发展了几种基于金纳米粒子生长的可视化探针并用于快速免疫检测。一方面，胶体金可以作为ELISA的酶催化基底，通过控制胶体金生长来增大其消光系数，以此提高酶联免疫可视化检测的灵敏度；另一方面，用于免疫检测的抗体可直接修饰到小尺寸金纳米粒子表面，金纳米粒子探针与待测抗原结合，形成三明治夹心结构。再通过金纳米颗粒生长，从而引起胶体金溶液由无色到紫色的变化，来实现蛋白的定性与定量检测。与传统的ELISA相比，该方法免去了多次洗涤与孵化的步骤，具有快速、使用方便、成本低廉等特点。. 在核酸检测方面，我们将金纳米粒子的定向修饰方法与高灵敏度检测技术相结合，发展了一系列高灵敏度检测方法。例如，我们通过合成具有不对称表面修饰的胶体金拉曼增强基底，使其一侧功能化特异性识别配体，另一侧修饰水溶性高分子，使得这种不对称修饰的拉曼增强基底在复杂样品中能保持优越的特异性、灵敏度和单分散性。这种独特的拉曼增强基底与相应的靶标分子识别，导致拉曼热点的形成，实现靶标物质触发的原位拉曼增强效应，从而实现待测核酸在原位的高信背比检测与成像。