基于CaCO3晶体原位生长的QCM检测新方法的研究

Quartz Crystal Microbalance is widely applied in multiple areas like clinical diagnose, genetic analysis and environmental pollution and so on. However its relative high LOD limited its further development. In order to overcome this bottleneck, mass amplifiers like PCR, metal nanoparticle, enzyme based deposition were applied to enhance the sensitivity of QCM. While, their disadvantages such as expensive, rely on enzyme, introduce error restricted their practicability. In this work, we take the advantage of the CaCO3 crystallization and take the CaCO3 crystals as mass amplifier to enhance the QCM signal. In order to prevent the random amplification, -N(CH3)3 was utilized to passive the sensor surface to prevent the unspecific amplification. Using the CaCO3 crystallization strategy to construct a real time biosensor with high sensitivity and specificity. Compare with the traditional amplification way, the CaCO3 crystallization strategy has the advantage of low-cost, no need enzyme, no need PCR and so on. The –COOH is utilized to enhance the CaCO3 crystallization at specific sites, meanwhile the –N(CH3)3 is utilized to reduce the unspecific crystallization. This method overcomes the drawback of low signal-to-noise ratio, multistep amplification of traditional methods. Through the modification of modified oligo, the novel QCM method based on in-situ CaCO3 crystallization can be applied in the detection of nucleic acid, protein, cell and so on.

石英晶体微天平（QCM）被广泛应用于生物分析、食品安全、环境污染监测等诸多领域，相比于其他传感器有原理简单、在线检测、免标记等优点。但随着对痕量分子检测要求的提高，其检测限也亟需提高。常用的信号放大方法有：基因扩增、金属纳米颗粒偶联、酶反应沉淀等，但是存在造价高、依赖生物酶、需多步放大等缺陷。所以有待开发新的信号放大方法，本项目创造性地将原位生长的CaCO3作为质量增强原件，放大由目标引发的检测信号，并且同时钝化晶片表面背景，有效防止非特异性放大，从而建立新颖、灵敏、特异的QCM分析方法。与传统信号放大方法相比，有操作简单、造价低 、不依赖生物酶等优势。晶片表面被-N(CH3)3钝化后，控制CaCO3只在目标位点结晶生长，有效排除假阳性，克服了传统方法的信噪比低的缺点。通过对修饰序列的简单更改，基于CaCO3原位晶体生长的QCM检测方法可实现核酸、蛋白、细胞水平的多种检测。

本项目以石英晶体微天平为检测平台进行了生化分析研究，探究了官能团对碳酸钙定量生长的调控作用。实验通过调节两种不同功能基团实现晶体生长的定量控制证明该方法可以作为一个有效的质量扩增机制用于石英晶体微天平的信号放大，以此提高石英晶体微天平的分析灵敏度。研究了基于碳酸钙原位选择性生长的石英晶体微天平检测技术对信号放大的作用，并将该方法应用于DNA分子的检测中。实验证明该方法在DNA检测中可以获得良好的检测效果，并且具有很高的选择性。在引入适配体后，将该方法应用于Ramos细胞的检测中。实验证明该方法在细胞检测中可以获得良好的检测效果。同时，我们将该方法与其他基于质量信号放大的石英晶体微天平检测方法进行了对比，通过对比证明了我们的实验具有很好的灵敏度。本项目利用选择性结晶技术，开发了基于石英晶体微天平的生物分子灵敏检测平台，基于该方法，可建立多种生物活性分子的通用检测平台。在本项目的资助下，发表了5篇学术论文，其中TOP论文一篇，发表在美国分析化学《Analytical Chemistry》期刊上，出版学术专著一章，申报专利3项，获奖一项。培养硕士研究生6名。