Multiple Displacement Amplification Based On Microfluidics

Droplet microfluidics has been widely used in various fields as a reaction platform or a technical tool since microchips made of polydimethylsiloxane(PDMS)are widespread.The tiny volume of separated droplets contributed to their applications in bio-molecular research.Being encapsulated into large amounts of small droplets,reactions could show new characters.Multiple displacement amplification(MDA)is a common non-specific DNA amplifying technique.With reagents dispersing in many small droplets,droplet MDA has a high amplifying uniformity and has been utilized to amplify genomes of single cells or multi-genomes samples.However,the progress of droplet MDA and traditional MDA are similar,which take long time to reach the efficient amplification period after the reactions begin.This thesis focused on developing a whole microfluidic platform and MDA method,which are settled to amplify DNA rapidly by maintaining the concentration of DNA in a proper range.The main contents of this thesis are as follows:1.Building the operating and analyzing platform for droplet microfluidicsPDMS microfluidic chips were fabricated with soft-photolithography progress.Micro-electrodes were generated by flowing melted low-melting-point metal in to specific channels.Besides,pumps were controlled by external signals to generated different levels of flow fluctuation.Droplet controlling using fluorinated oil as the continuous phase went fluently in the chips.The progress of microfluidics was taken by microscopes and analyzed using ImageJ scripts automatically.The whole process of droplet microfluidics was stable and repeatable.2.Optimizing the traditional droplet merging method and proposing a new droplet reagent loading methodAs a widely used content adding method in droplet microfluidics,traditional droplet merging strategy shows poor robustness.The“self-adjusting”droplet merging strategy was proposed.The success rate of this new method maintained~70%under fluctuation while the traditional method got only~30%under the same condition.The output of the self-adjusting method exhibited deviation near the expected results in volume and brightness instead of the binary results in the traditional method.In addition,the self-adjusting method was confirmed to have the ability of gather the to-be-merging droplets while enlarge the distances between those should not be merged by the size differences among droplets.The“mixing generation”method was proposed as another reagent adding method.Within this method,loading completed in flow focusing channels,which made the process more robust and reproducible.The success rate of this method was slightly higher than self-adjusting method and the CV value of multiple experiments reduced to~5%from~10%.The deviation of every output was stable,making this method suitable for multiple loading,which is the main droplet manipulation method in this thesis.3.Increasing MDA efficiency by sequential dilutionThe efficiency of MDA was increased utilizing the sequential diluting method,which is mainly focused on the controlling of DNA concentration.The initial concentration of DNA was enlarged to shorten the slow amplification period in the first few hours in MDA and after the reaction entering the high speed period,the concentration of DNA was diluted several times to maintain the amplifying speed.The fluorescence signal of the reaction was obtained and the increasing rate of signal was calculated accordingly.The increasing rate exhibited the statuses of reactions and could be used in optimizing the time parameter.The diluting MDA reaction ended 4 times quicker than the traditional MDA when the dilution ratio was set to 10~4.The dilution ratio of droplet diluting MDA was set to 10~3 after testing the performance of droplet MDA with different initial DNA amount.The diluting MDA within droplet completed within~2/5 times of normal droplet MDA.Both aqueous and droplet-encapsulated diluting MDA were validated to be efficient.