Portable 3D Printing Droplet PCR Instrument And Its Application In Cancer MiRNA Detection

miRNA,a class of non-coding small RNA,is abnormally expressed in cancer cells and often used as markers of cancer.The common detection methods for miRNAs are microarray method,miRNA sequencing method and PCR method.It's worth noting that PCR is an extremely important tool for molecular biology research.After many years of research,PCR has evolved from qualitative analysis to quantitative analysis.To suit the complex experimental scenarios,one development direction of the PCR instrument is portability.The key to the portability of the PCR instrument is the PCR microfluidic technology.The PCR microfluidic technology is divided into spatial domain PCR and time domain PCR.The spatial domain PCR need short reaction time and easily combination modules that attract the interest of researchers.The traditional manufacture technology of PCR microfluidic chip is mainly based on soft lithography which is complicated in operation,requires large instruments during preparation and has high technical requirements.The disadvantage of traditional manufacture technology has restricted the PCR microfluidic chip's further development.In recent years,3D printing technology,a new microfluidic technology,has been applied to the manufacture of microfluidic chips.Its manufacture process basically requires no manual operation,simple operation and low cost.However,the use of 3D printing technology to manufacture PCR microfluidic chips is relatively small.Based on the above research status,the droplet PCR microfluidic chip was manufacture by 3D printing technology in this paper,and manufacture portable droplet PCR instrument to detect miRNA in cancer cells.Firstly,four style spatial domain PCR chips are designed in this paper to explore the shape and size design of microfluidic channels.According to the preliminary experiments,the electrothermal snake PCR chip has the advantages of fast heating,easy shape processing,and multiple cycles.So,the electrothermal snake PCR chip was selected for subsequent research.The flow rate of the microfluidic chip channel during the PCR experiment was further calculated.What's more,the generation performance of the droplets under different flow rates of the continuous phase and the dispersed phase was investigated to realize small droplet formation.In order to complete the PCR experiment in a short time,the flow rates of the continuous and dispersed phases were 23 ?L/min and 1 ?L/min,respectively.Then,this paper constructs PI heating membrane temperature control system,heating ceramic temperature control system,and cartridge heater temperature control system.we design experiments to explore the temperature distribution of the chip under three temperature control systems on 3D printing PCR microfluidic chip and the flow rate is same.The infrared image processing is carried out on the temperature distribution of the chip under the three temperature control systems.After the temperature value analysis,we find the cartridge heater has fast heating speed,good uniformity and stability which are beneficial to PCR experiment.Therefore,the cartridge heater temperature control system was selected for the next droplet PCR experiment.Finally,PCR experiments were performed on a portable droplet PCR machine prepared by 3D printing.Amplification was performed on the 100 bp fragment of the bacterial Lacz gene,and further experiments showed that the sensitivity of the portable droplet PCR instrument was 1 ng/?L.The miRNA-21 of MCF-7 breast cancer cells was amplified to verify the amplification of miRNA-21 on a portable droplet PCR instrument.When the temperature of the high temperature region of the cartridge heater was set to 105 ° C,and the temperature of the low temperature region was set to 60 ° C,the result of experiment is better.The expression of miRNA-21 from MCF-10 A normal cells and MDA-MB-231 breast cancer cells is different,and the amplified products were analyzed by gel electrophoresis.MDA-MB-231 breast cancer cells were detected by different expression levels of miRNA-21.In summary,the portable droplet PCR instrument manufactured by 3D printing technology successfully implements droplet PCR and detects miRNAs in cancer cells.It not only proves the feasibility of manufacture PCR microfluidic chip by 3D printing technology,but also realizes miniaturization of PCR instrument.The portable droplet PCR instrument is manufactured by 3D printing technology,which also embodies the advantages of personalized production and integral molding of 3D printing technology.The application scenario of the PCR instrument will be expanded due to its great advantages.It is believed portable droplet PCR instruments manufactured by 3D printing technology will be widely used in the field of biomedicine.