环形激光光解复合牺牲层诱导单细胞无损分离新方法及其机理研究

The laser-induced forward transfer is a potentially promising single cell isolation process for its high efficiency and no nozzles. However, it is limited by two main issues: cell damage and low separation accuracy. In this project, we propose to use a ring-shaped laser to photon-decomposition a hybrid sacrificed layer combining of an azene with small molecule and polydimethylsiloxane (PDMS). With the introduction of a new sacrificed layer, the laser-induced thermal effect is replaced by laser-induced photon-decomposition and no particles generated since the process is constrained by PDMS. The ring-shaped laser is used. Consequently, a ring-shape cavitation is induced. The shock produced by the cavitation will conquer the surface tension, electro-statics, and viscous force, and then block the cells from each other, resulting in sing-cell accuracy. At the end of the process, the shock waves on the inner side will be focused and propel the cell away. To reveal the mechanism of laser-induced hybrid material decomposition, rheology coupling infrared spectra, gas chromatography, mass spectrometry and photon reaction pathway are used to analyze conversion process from laser photon energy to excitation energy, then to cavitation potential energy and to explore the cavitation process, Lattice-Boltzmann method, high-speed camera, and hydrophone are used. The mathematical relation between convergence effect and laser and the sacrificial layer is established to the model of laser-induced two-phase material transferring. This project is profound meaningful from both theoretical and practical aspects.

激光诱导前向转移具有高效、无喷嘴的优点，是非常具有潜力的单细胞分离方法，但存在细胞损伤及分离精度低等问题。本项目提出一种环形激光光解复合牺牲层诱导单细胞无损分离的新方法。该方法引入小分子三氮烯-聚二甲基硅氧烷（PDMS）两相复合牺牲层，利用小分子三氮烯的光解效应取代激光热作用诱导空化；通过交联PDMS的粘弹特性约束分解产物，抑制再生颗粒的产生，避免细胞污染；修正激光光斑为环形光斑，利用环形空化生长的几何对称性隔断细胞间能量传递，利用其内侧汇聚效应驱动细胞，实现单细胞无损分离。项目将通过红外-流变耦合、气相质谱配合光反应途径研究激光光子能量-激发态能量-空化势能的转换过程，揭示激光光解两相复合材料诱导空化成核的机制；利用高速摄影-水听器结合格子波兹曼模型，探索空化隔离细胞间联系的条件，内侧汇聚效应和激光、牺牲层的数理联系，揭示空化作用下细胞转移的机制，具有重要的理论和现实意义。

单细胞分离是单细胞生物的关键步骤，是获得可靠研究样本的重要手段。本项目通过锥透镜对激光光斑进行修正，利用环形激光取代圆形激光，诱导形成环形空化，充分发挥环形空化的对中聚焦和外阻隔断效应，提高激光诱导前向转移单细胞的精度。同时为了保证细胞活性，本项目采用小分子三氮烯的光解效应取代激光热作用诱导空化，结合交联PDMS的粘弹特性约束分解产物。本项目建立了小分子三氮烯的合成工艺，并将吸收峰提高到355nm。通过数字建模与高速摄影发现环形空化受到内外径比的影响严重，只有在0.3-0.7间环形空化的对中聚焦及隔断作用才能凸显。通过本项目的实施，单细胞精度提升到80%以上，细胞没有损伤。