基于光微流激光阵列的快速准确DNA分析与筛查

Distinguishing the target DNA from the single-base mismatched counterpart provides critical information for disease diagnosis and basic biochemical research. High resolution melting (HRM) is a newly developed post-PCR (polymerase chain reaction) analysis method for identifying genetic variation. It employs fluorescence from intercalating dyes to differentiate the melting curve of two DNA sequences. As compared to other DNA analysis methods, HRM is simple and fast. However, with the increase of DNA length, the resulting fluorescence difference between the target and the single-base mismatched DNA becomes very small, which can not be easily distinguished. How to sensitively discern the small intrinsic thermal dynamic difference becomes crucial when we analyze two DNA sequences of thousands of bases long but that differ by only a single base. We propose to develop a paradigm-changing intra-cavity DNA melting analysis with an optofluidic DNA laser that is capable of differentiating single-base mismatched DNAs of thousands of bases long in nano-liter sized solution. In this scheme, DNA samples, probes, and fluorophores are flowed inside a laser cavity, thus becoming part of the DNA laser gain medium. As compared to conventional HRM, the proposed intra-cavity analysis has many distinct advantages. (1) It employs stimulated laser emission, rather than fluorescence as the sensing signal. Due to the strong optical feedback provided by the laser cavity, the small signal difference between the target and the single-base mismatched DNA will be significantly amplified for a few orders of magnitudes even with DNA of thousands of bases long. (2) When temperature increases, the laser undergoes a sharp phase transition from stimulated emission and spontaneous emission, which allows us to precisely determine the melting temperature difference of two DNA sequences; (3) In addition to temperature ramping, the high differential signal allows us to scan the excitation with different pumping intensities at a fixed temperature to distinguish two DNA sequences, which provides another means for rapid, high-throughput DNA analysis; (4) The optofluidic DNA laser is highly compatible with on-chip PCR and requires only nano-liter sized sample volumes. The realization of this project can promote the application in biomedic and other related fields.

实现完全配对与单碱基错配DNA检测对疾病诊断和生化基础研究具有非常重要的意义。高分辨熔解曲线分析(HRM)是近年来兴起的一种简便、可实现DNA突变快速扫描的后聚合酶链反应技术。然而随着序列长度的增加，不同DNA所产生荧光强度差异越来越小，将难以分辨。 本项目提出一种基于光微流激光的腔内高灵敏度HRM检测方法，可用来分辨具有上千碱基对的DNA序列，所需样品低至纳升量级。DNA样品及荧光素被用做激光器增益介质，与传统HRM相比：1) 激光取代荧光被用做传感信号，利用共振腔极强光反馈，有望将传统HRM中的传感信号差提高数千至数万倍；2)利用激光器由受激辐射到自发辐射锐利相变过程，可精确标定不同DNA熔解温度；3)提供了一种保持温度恒定，检测信号强度随激发光强变化这一快速、高通量的新型检测方法；4)与PCR及微流控技术高度兼容，可实现芯片上检测。本项目的实现可推动生物医药应用和相关研究领域的发展。

实现完全配对DNA与单碱基错配DNA的精确、快速检验对疾病诊断与基础生化研究等的发展具有重要意义。本项目提出一种基于稳定平面凹面(p-c) FP微腔的光流控激光阵列，该微腔有亚pl模体积。该FP腔采用激光烧蚀技术在熔融石英玻璃表面上制备微凹面结构，然后在其表面上镀上多层分布布拉格反射(DBR)介质层封装制成。达到了高的品质因数为5.6×10^5，超过通常所用的FP腔100倍。以1mM 溶于酒精的R6G染料作为增益介质，得到90nj/mm²的超低阈值。高的精细度和低的激光阈值使得我们的FP腔的性能接近光流控环形谐振腔激光器的性能。光流控环形谐振腔激光器具有创纪录的高精细度(和q因子)和低激光阈值。因为生物疾病检测普遍存在着检测灵敏度低、所需样品量大的现象，本项目中的FP激光谐振腔满足生物疾病检测所需要的高灵敏度、使用样品量少以及容易集成等要求。.本项目提出一种新型的基于FP腔的光微流控激光器的DNA高分辨率熔融（DNA-HRM）分析方法。与传统的HRM相比，基于激光的HRM具有信噪比高、温度分辨率高以及容易集成等优点。另外，还可以通过调Q技术和降低泵浦强度等手段降低熔解温度。本项目主要分析了多种长度以及多种类型的DNA。在单碱基替换型DNA中分析了长度由25个碱基对至两种GC含量的130个碱基对DNA。对单碱基变异DNA（SNP）也进行了实验分析。SNP是人类基因可遗传变异中最常见的一类疾病，对SNP的快速准确检验对人类疾病检测具有重要意义。.对此，我们提出了一种在固定温度下扫描DNA的方法，这种方法是传统的HRM所不能使用的。实验结果显示错配DNA与正配DNA或者SNP变异后DNA与未变异DNA之间的斜率是不同的。而后我们也对G-四链体（一种DNA的高级结构）进行分析研究。G-四链体与人类癌症细胞有重要关联，合理利用G四链体具有治疗癌症的潜能。.本项目的开展进一步推动了微流控激光芯片检测技术的应用，同时推动了生物医药应用和相关领域的发展。

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