High Resolution Melting Curve Analysis Technology Based On Fabry-Perot Microcavity Laser

In this work,we studied a high-resolution melting?HRM?analysis method based on laser signal in the high quality Fabry-Perot?FP?optical microcavity we fabricated.We utilize the FP optical microcavity as the micro-laser cavity,intercalating saturation dyes as the gain media and emitted laser s ignal as the detection signal to achieve high-resolution detection and screening of target and mismatched DNA by temperature scanning.We investigate on the melting curves of target and mismatched DNA with 25bases,50bases,99bases,130bases respectively.Theoretical and experimental results indicate that the HRM detection method based on laser signal has lower melting temperature and higher signal-to-noise ratio.The specific work is as follows:1.We set up a CO₂ laser processing platform,and a micro-concave structure was fabricated on the fused silica glass substrate by laser micromachining method.The lateral dimension of the curved structure was controlled in 40-110?m with depth between 3 and10?m.At the same time,a planar structure was fabricated on a single crystal silicon wafer,and a highly reflective dielectric film was plated onto two prepared substrates.Finally,a microfabricated mirror with a single crystal silicon wafer-silica substrate and a silicone encapsulation process were used to fabricate a closed Fabry-Perot optical microcavity and an optical microfluidic system to form an optical microfluidic system.The chip realizes the integration of light microfluidic laser devices.2.Firstly,the fluorescent and laser-based HRM curves of DNA molecules with different base pairs were theoretically simulated,and the results were analyzed.Subsequently,experimentally,a 488 nm pulsed laser was used as a pumping source,and a 250?M concentration of DNA and SYTO13 dye mixture was used as a gain medium.In combination with the high-resolution melting curve technique,a fluorescence-based and laser-based HRM technique was studied.The results show that the laser-based HRM technology has a lower melting temperature and a higher signal-to-noise ratio.3.On the basis of laser HRM detection,we proposed a novel DNA detection method that can not be achieved by conventional HRM technology is proposed,that is,keeping a constant temperature,we detection signal intensity changes with the excitation light intens ity,and providing another rapid,high-throughput DNA detection method.