Construction Of Microdroplet Generation Platform Based On Tip Capillary Vibration And Its Application In Bacteria Research

Droplet-based analytical assays have been widely used in various fields due to the advantages of high throughput,low consumption,and no cross-contamination.The existing droplet generation methods are mainly based on microfluidic chips,which form droplets by exploiting the fluid properties in the channel.However,there are some drawbacks of this method,such as the complexity of chip fabrication,the need for precision instruments for liquid flow rate control,and the difficulty of real-time adjustment of droplet size.Especially,the overall device is large and complicated to operate,and these defects limit the application of droplet technology in field inspection.In this thesis,a portable microdroplet generation platform based on tip capillary vibration is constructed and validated for its application in bacteria research as follows:(1)Construction of a portable droplet generation platform based on tip capillary vibrationIn this study,a portable droplet generation platform based on tip capillary vibration was developed,which consists of only a piezoelectric diaphragm-controlled glass capillary,a portable signal generator,and a droplet reservoir.In the experiment,the tip capillary containing the water phase is fixed on the piezoelectric diaphragm,and the signal generator generates an electrical signal to drive the tip capillary to vibrate in the oil phase to generate centrifugal force to push the water phase of the capillary to pump out from the tip,and the pumped liquid forms a"necking flow"under the action of acoustic flow;When the acoustic signal stops being input,the tip vibration ceased,and the liquid was cut off to form droplets.The droplet generation platform is simple,easy and inexpensive to operate,does not require special manufacturing procedures or external pressure sources,is driven by a battery-powered low-cost signal generator,can be performed on demand,and can be adjusted by the output waveform to continuously regulate droplet size in real time over a wide range,with a large droplet volume range(0.016 n L to 26.40 n L)and a throughput of up to 5000droplets per second,and has good monodispersibility.(2)Bacterial digital counting based on portable microdroplet platformBased on the portable micro-droplet generation platform established in experiment 1.The bacteria activity indicator Alamar Blue and bacterial suspensions were combined as the water phase to encapsulate different concentrations of bacterial suspensions in droplets of varying volumes.Bacterial growth within the droplets resulted in the indicator reacting and producing a red fluorescent signal.After incubation,droplets containing viable bacteria exhibited a fluorescent signal,while those without bacteria or containing dead bacteria showed no significant fluorescence.Combined with our developed digital image processing program,fluorescence imaging allowed for rapid identification and counting of positive and negative droplets.Based on the Poisson distribution,the concentration of viable bacteria in the sample can be accurately calculated.Our method enables rapid and accurate counting of viable bacteria within the range of 5×10~2CFU/m L to 1×10~7 CFU/m L,and the use of multiple volume droplet analysis significantly improves the dynamic range and accuracy of counting compared to single volume droplet analysis.(3)Bacterial antibiotic sensitivity test based on portable microdroplet platformBased on the portable microdroplet generation platform established in experiment 1,a 3D printing rotary sampling switching device is integrated.Through the rotation of the central switching valve,flexible switching of different samples and droplet generation can be realized,meeting the requirements of antibiotic treatment and detection of different concentrations.Bacteria,antibiotics and the active indicator of Alamar Blue were encapsulated in droplets.By calculating the survival rate of bacteria in the samples treated with different concentrations of antibiotics,the relationship curve between antibiotic concentration and bacterial survival rate was fitted,and the minimum inhibitory concentration of antibiotics could be accurately evaluated.The method can shorten the antibiotic sensitivity test time from 20 hours to 5 hours,and the bacterial concentration in a single droplet can be accurately set through the flexible adjustment of the microdroplet volume,and the influence of the initial bacterial inoculation concentration on the antibiotic sensitivity test results can be accurately evaluated.It is expected to quickly and accurately evaluate antimicrobial susceptibility in resource-limited Settings.(4)Array droplet printing based on tip capillary vibration of microdroplet platformAn array droplet printing platform was constructed based on the microdroplet generation device of tip capillary vibration established in Experiment 1 and the XY displacement platform controlled by the Python program.In this device,fill the tip capillary and the polyethylene tube at its tail with the aqueous phase.The tip of the capillary was immersed in an oil phase containing lipids,A customized Python program moves the tip capillary accordingly through the oil phase and controls the signal generator to generate a signal and print the droplets.The phospholipids in the oil phase form a phospholipid monomolecular layer on the droplet surface,and as the droplet descends,the droplet forms tight junctions through the attractive forces between the monomolecular layers.The experimental results show that this device can realize arrayed droplet printing according to the established procedure.In summary,the constructed microdroplet generation platform based on tip capillary vibration is stable,portable,and flexible in terms of liquid volume,which can achieve rapid and accurate bacterial enumeration and antibiotic susceptibility testing in resource-constrained environments,and is expected to be used for droplet array printing-based bacteria community ecology studies.