Droplet Microfluidics For Isothermal Synthesis Of DNA Origami Nano Structures

DNA origami is a promising nanomaterial that has been widely used in biosensing,cancer therapy,and metamaterials.The traditional method for synthesizing DNA origami is usually to set a specific gradient cooling program by means of a PCR instrument or gradient dialysis by means of an organic denaturant.Both processes have to take several hours or even hundreds of hours.In particular,the construction of the 3D DNA origami structure usually requires,complex gradient cooling procedures and therefore costs long synthesis time.There is still no suitable countermeasure at the moment.The droplet microfluidic can significantly reduce the amount of reagents,shorten the reaction time,and improve the reaction accuracy.Therefore,there has been increasing interest in synthesizing nanomaterials through a droplet microfluidic platform.On the basis of this,the droplet microfluidic platform also holds great potential for synthesizing DNA origami,overcoming the disadvantages of the traditional gradient cooling program setting and the long synthesis time.In this work,we used "+" channels to synthesize DNA Origami with uniform size.In view of the limitations of traditional methods,we mainly carried out the following research:The "+" channels with inner diameters of 10 ?m,30 ?m,and 60 ?m were constructed,respectively.A serpentine path was designed at the far end of the "+" channel,which increased the reaction time of droplets in the channel and facilitated the isothermal synthesis of DNA origami.The platform for isothermal synthesis of DNA origami based on a droplet microfluidic system was constructed.It is mainly composed of the following three modules: The Power Drive,The Reaction Synthesis,and The Real-time Observation and Imaging.According to feedback information from The Real-time Observation and Imaging module,the two-phase sampling parameters of The Power Drive module was adjusted and the PDMS chip of the "+" channel in The Reaction Synthesis module was replaced.The diameters of 10 ?m,30 ?m,60 ?m,100 ?m,130 ?m,and even larger droplets were constructed based on this platform.The hollow equilateral triangle DNA origami was selected as an example to explore the conditions of isothermal synthesis of DNA origami.It was found that when the droplet size was 30 ?m,the concentration ratio of M13/Staple was 1:10,and the melting rate was 5 mins before entering the constant temperature synthesis platform,the yield of DNA origami was also improved.The isothermal synthesis of 2D DNA origami: The method was then extended to other 2D DNA origami,including hollow rectangular DNA origami,cross-shaped DNA origami,boat-shaped DNA origami,and solid rectangular DNA origami.The isothermal synthesis of 3D DNA origami: Using this platform,We also extended the method of isothermal synthesis of DNA origami to 3D DNA origami and successfully synthesized cylindrical 3D DNA origami.In summary,we develpe a new method for synthesizing DNA origami at constant temperature based on droplet microfluidic system,which overcomes the problems of complex gradient cooling program setting and long synthesis time of the traditional methods.This work is expected to expand the toolbox for the efficient synthesis ofDNA origami,especially for 3D DNA origami.