| Microfluidic technology is a technology that accurately manipulates fluids in a micro-scale space;droplet microfluidics is the most attractive microfluidic technology in application currently.The droplet microfluidic technology means chemical reagents are compartmented with the solvent at micrometer scales in sizes.Since each droplet is surrounded by immiscible liquids,the potential interference between the droplets is reduced greatly.Every microdroplet can be regarded as an independent reactor,and the result of the droplet is regarded as an independent sample in statistics.Compared with conventional approaches of chemistry,the same amount of reagents can produce a larger sample space,which has the advatanges of reagent saving,high efficiency,high sensitivity,high throughput,automation,and low pollution.These characteristics make the droplet microfluidic technology a great prospect in the field of biomedicine.In order for the microfluidics technology come into practice,we have carried out integration of droplet microfluidic technology with the cytometer.This article mainly improves microfluidic technology,in a hope that the above functions can be realized under the standard working conditions of cytometer.Here,we propose to produce droplets using functional materials,hoping the existing equipment can detect and screen them in the mode of calibration by particles.First,we study on the feasibility of light-curing materials which is the most popular.Based on the principle of droplet generation,we compared and analyzed the respective advantages of one-step and two-step droplet generation devices and chose one-step method to produce double emulsion for its simplicity.At the same time,the influence of surfactants on the stability of double emulsion were considered.Compared with the surfactant Span80,the droplets are more stable within EM90.After photopolymerization,droplets successfully encapsulated the liquid flame retardant material DMMP,for the purpose of efficient use of flame-retardant materials.Because the ultraviolet light irradiation during the droplet solidification is harmful to DNA,we have to apply for agarose.Second,in the case of agarose,we first obtained the characteristics of the droplet generators,including the two-phase flow rates,droplet generation sizes,generation frequency and pump pressure.The result shows the polydispersity of droplet is 3.58%in size,while the droplet generation frequency is up to 5000Hz.Then,the sol-gel transition of agarose is found by spectral absorbance experiment to occur at temperature ranging from 15? to 27?,while the gel-sol transition of agarose is from 50? to 70?.Finally,we choose 35? as the working temperature where it can produce agarose droplets at liquid state,and the produced agarose beads in the way we discussed above are uniform in size ranging from 30?m to 250?m in diameter.Third,in order to collect single molecules,we have to know experimentally the influence of agarose concentration on the PCR reaction as well as on molecular fluorescence detection.The experiment results show:(1)Adding agarose has little influence for PCR amplification,and the observed fluorescence intensity is the same as usual.(2)The fluorescence intensity of the hybridization of single aptamer(058)with target(AFB1)was enhanced upon adding agarose in the dPBS buffer.Last,in order to verify the feasibility of sorting agarose beads(1.5wt%)by flow cytometer,we test two kinds of beads:(1)agarose beads containing fluorescent dyes.(2)agarose beads containing PCRmix which would be fluorescent after the PCR amplification.The sorting result proved that it is good enough to enrich the target molecule by using agarose bead for the collection of single molecules.Owing to the adjustment of equipment,the final part of the single molecule collection experiment based on agarose beads cannot be completed,it suggested to continue the experiment after condition available.In addition,it is worth making further research on the interaction between agarose and fluorescence. |
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