基于RCA反应设计的DNA智能水凝胶在生物分析中的应用

DNA smart hydrogel is a portable detection platform with a highly tailored designability, high sensitivity and selectivity for targeted analyte based on the recognition ability of functional nucleic acid. Present DNA smart hydrogel systems are limited to their complex modification and synthesis, or high concentrations of DNA dosage (up to concentration of 500 μM), leading to their cumbersome preparation steps, high cost, and not conducive to practical applications. In this proposal, RCA (Rolling Circle Amplification) isothermal amplification technique is employed to amplify low concentration of DNA (lower to nM scale) to high enough concentration for hydrogel formation, which highly reduces the cost of hydrogel. The method also exploits the property of DNA hydrogel with good tailored designability to incorporate functional nucleic acid with RCA reaction to finally obtain smart hydrogel with target responsiveness. Combined with the visualization method, we obtain DNA smart hydrogel with high sensitivity and selectivity for targeted analyte in bioanalysis. The method enhances the application potential of DNA smart hydrogel and provides a new opportunity for its practical applications by simplifying preparation process of and reducing preparation cost of hydrogel.

DNA智能水凝胶可针对生物分析物中特定目标分子进行定制化设计，利用功能核酸的识别能力，结合可视化检测方法，实现对目标分子高灵敏度、高特异性的便携检测。现有的DNA智能水凝胶体系，局限于复杂的修饰合成过程，高浓度的DNA用量（大于500 μM），其步骤繁琐、成本偏高，不利于实际应用。本项目提出利用滚环扩增反应（Rolling Circle Amplification，RCA）将微量的模板DNA（低至nM浓度）扩增到可形成凝胶的浓度范围，大大降低水凝胶制备成本；并充分利用DNA水凝胶可定制化设计的特点，将功能核酸与RCA反应结合，制备出目标分子响应型的智能水凝胶。结合可视化检测方法，最终实现对生物样品中重要分析物的高灵敏特异性的便携检测。该方法简化水凝胶制备过程，降低水凝胶制备成本，提升了DNA智能水凝胶的应用前景，为DNA智能水凝胶的实际应用提供了新的手段。

DNA智能水凝胶具有良好的稳定性、生物相容性和良好的可设计性。现有的DNA智能水凝胶体系，局限于复杂的修饰合成过程，高浓度的DNA用量（大于500 μM），其步骤繁琐、成本偏高，不利于其实际应用。本项目利用滚环扩增反应（RCA）将微量的模板DNA（低至nM浓度）扩增至可形成凝胶的浓度范围，同时结合功能核酸进行功能设计，研究了初始序列设计、自组装行为对水凝胶的影响，制备了目标分子响应型的智能水凝胶。研究包括：1一步法合成强度、体积、稳定性、pH响应性可调的水凝胶，并揭示了其中的成胶、解胶机理；2结合DNA模板合成银簇的方法，制备高化学稳定性及光学稳定性的银簇，并将其用于细胞内的羟基自由基的定量检测；3在可发生自组装的Y型DNA结构中接入具有聚集诱导荧光性质的芘分子，Y型DNA识别miRNA进而自组装的过程，可被芘的荧光变化记录，从而实现在细胞内对特定miRNA的定量检测。我们发展的方法可通过序列设计及RCA过程控制，方便地对水凝胶性质，如强度、体积、稳定性、响应性等进行调控；同时该方法简化了水凝胶制备过程，降低了水凝胶制备成本，有望提升DNA智能水凝胶的应用前景，为DNA智能水凝胶的实际应用提供了新的思路。