Research On Key Technologies Of The Droplet Generation System For Digital Nucleic Acid Amplification Based On Vibration-induced Interfacial Emulsification

The outbreak and pandemic of the novel coronavirus(COVID-19)from 2019 to the present has caused serious threats to the safety of global human life and huge social and economic losses.The detection and analysis of pathogenic microorganisms is extremely important for epidemic prevention and control.Nucleic acid amplification molecular diagnostic technology has become the gold standard for pathogen detection and has been widely used in the detection of viruses and microorganisms.Among them,the digital nucleic acid amplification technology based on droplets has developed rapidly,with its high sensitivity and strong resistance to inhibition.So it shows unique advantages in the early prevention and control of infectious diseases and oncology research.The preparation of water-in-oil droplets is the core technology for digital nucleic acid amplification.However,the current methods such as microfluidic chips and microchamber chips for preparing droplets are difficult to design and process.The high cost limits the promotion and application of digital nucleic acid quantitative technology.Vibration-induced interfacial emulsification is a new droplet preparation method proposed by domestic scholars.It has the advantages of simple operation,easy control,and low cost of consumables,which is expected to overcome the above-mentioned limiting factors.However,the technology still needs to be improved and strengthened in terms of stability and automation control.To this end,a systematic study on the droplet preparation process based on the principle of vibration-induced interfacial emulsification is carried out by this paper.And theoretical reference and key technical support for building an automated droplet preparation system is provided by this paper,which has important scientific significance and application value.The specific work and achievements of this paper are summarized as follows:1.A new method of simulating the vibration-induced interfacial emulsification process is proposed based on the traditional co-flow model coupled with the interface shear force,which proves that the interface shear force is the key driver of droplet generation.2.According to the theoretical analysis,comparative experiments are designed to select and optimize experimental materials and methods of vibration-induced interfacial emulsification,which improves the thermal stability of the droplet array.Meanwhile,an injection method driven by oil is designed,which not only avoids the pollution of injection pump,but also realizes the accurate control of sample velocity in the process of droplet generation.3.Oil level detection module based on machine vision and automatic sample injection module based on three-way valve structure are designed,which realizes the automatic control of the droplet generation.The system can automatically generate the droplets array with good homogeneity according to the given volume.4.Using ?DNA as the verification nucleic acid material,through the implementation of complete digital LAMP(Loop-mediated Isothermal Amplification)quantitative detection and analysis,effective detection of samples with a concentration as low as 1.5 copies per microliter is realized.It verifies that the droplet array prepared by this system meets the ultra-sensitive digital nucleic acid quantification testing needs.