Research And Application Of Surface Acoustic Wave Biochip In Rare Cells Sample Pretreatment

Surface acoustic wave(SAW)is one of the acoustic waves that exist in nature,and it was first discovered in the study of seismic waves.With the deepening of people's understanding of SAW principles and the improvement of the preparation process of electronic devices,people have successfully constructed SAW electronic devices using piezoelectric crystals in the last century.After the birth of SAW devices,it quickly occupied an important position in the field of precision electronic sensor devices.With the further development of micro-nano processing technology,SAW has become widely used.In recent years,it has been applied to the field of biomedical materials and has shown important application prospects.The use of acoustic field structure design and acoustic wave intensity adjustment methods can be used to precisely control micro-nano materials.Therefore,the "acoustic tweezers" technology corresponding to the"optical tweezers" technology was born.As the name suggests,SAW "acoustic tweezers" technology can realize a series of functions such as cells drive,transport,enrichment,manipulation and even lysis at the micro-nano scale,and has great application significance in the processing of biological samples.Before performing sensing tests on target biochemical molecules in specific cells in a sample of multicellular biological tissues,the liquefied biological sample often needs to go through three necessary and related steps for processing,that is,the separation and purification of the cells in the sample,Cell enrichment and cell lysis.Accurate separation of target cells from biological samples with two or more types of different cells with a size difference of only a few microns often uses a centrifugal method,and the separation efficiency and sample capture effect are poor.In addition,when the sample concentration in the liquid sample is too low to meet the minimum requirement for detection,the method of high-speed centrifugation is often used to solve the problem.This not only requires higher equipment performance,but also needs a large number of samples to be enriched multiple times to meet the requirements.Therefore,it is urgent to develop an enrichment method for micro-nanoparticles or cells in micro-liquid samples.For cell samples that have been separated,purified and enriched,lysis is necessary to obtain key compounds such as intracellular proteins and nucleic acids for subsequent detection.Commonly used lysis equipment always requires a considerable amount of sample,while the cell lysis reagent has a certain degree of harm to the operator due to its strong toxicity.Therefore,there is an urgent need for a fast and harmless lysis method for small samples and low-concentration cell samples.Based on the above problems,this paper will propose to study the new SAW microfluidic technology to achieve precise manipulation on the micro-nano scale,to achieve the pre-processing of cell liquid samples.Providing new technologies and new SAW devices to achieve the inspection of cell contents and provide effective and reliable Cell manipulation and the release of intracellular biochemical molecules.This study combines the design of the acoustic field with the acoustic properties of biological cell materials to understand the propagation characteristics of SAW at the solid-liquid interface and explore the influence of SAW on the behavior of cells and particles in liquid.At the same time,on the basis of the design and preparation of the corresponding functional devices,the acoustic interference effect and the acoustic streaming effect that occurred in the SAW microfluidic system were realized and observed.The separation of animal and plant cells and the efficient lysis of cells were achieved.Besides,the self-focusing enrichment effect is point out theoretically.The research content of this paper mainly includes the following four aspects:(1)Excitation and propagation of SAW,functional acoustic field design,design and preparation SAW functional deviceThrough the finite element simulation,the excitation and propagation of SAW are realized in the surface of lithium niobate crystal(128°YX-LiNbO3).Besides,the characteristic mode and transient propagation are observed,which matches to the theory.Furthermore,the control of the position distribution of the center sample flow in the laminar flow structure was realized.Combined with the simulation of the standing SAW field,the rapid separation of polystyrene(PS)particles was obtained.Through MATLAB modeling and calculation,the influence of input power,channel flow rate and inclination angle on the separation distance of particles was discussed when the standing SAW and the microfluidic channel have a relative inclination angle.The acoustic coupling on the lithium niobate substrate-microfluidic channel wall and the total reflection phenomenon occurring at the channel wall-liquid domain interface are discussed.Furthermore,it was discovered that the diffraction wave at the intersection of the lithium niobate substrate-the wall of the microfluidic channel-the fluid domain has an important effect on the transmission of acoustic waves in the fluid domain.After plane wave approximation,it is proposed that the diffraction wave interferes with the leakage wave coupled into the fluid domain by the lithium niobate substrate,and further pointed out that this discovery is expected to realize the enrichment of micro-nano particles.In addition,by using the acoustic streaming effect of SAW on micro-droplets,a method of adding particles in cell size to microdroplets is proposed to achieve an efficient cell lysis mechanism.Based on the above theory and actual manufacturing conditions,the corresponding functional devices were designed and prepared,and the drive test system was built.(2)Particle migration and sorting of cells based on standing SAWBy adjusting the rates of the inlet sheath flow and the sample flow,the position of the sample flow and the particles in the microfluidic channel can be controlled.The phenomenon of crystallization turbidity in the phosphate buffer caused by the solubility change at the laminar flow interface was discovered,and this phenomenon was used to realize the visual observation of the standing SAW.The rapid separation of human breast cancer cells(MDA-MB-231)and human leukemia cells(Jurkat)cells under the influence of the acoustic field was observed in the microfluidic channel parallel to the acoustic field,achieving the rapid separation of the two kinds of cell mixed samples.The lateral migration of polyethylene particles corresponding to the inclination angle and their sorting at the outlet were observed in the microfluidic channel with an inclination angle to the acoustic field.In view of the different acoustic properties of different materials,the separation of polystyrene particles,animal cells,and plant cells of different sizes and the same kind of particles in the microfluidic channel with a relative inclination angle of 15°are obtained.Besides,the separation of two particles of similar sizes and different densities are achieved.For the first time,the sorting of pollen as well as the sorting of intact pollen and germ cells have been realized in the SAW microfluidic system.(3)Enrichment of micro-nanoparticles induced by self-focusing SAW interference fieldThe vibration field at the junction of the microfluidic channel wall-lithium niobate substrate-fluid domain interface was studied,and the formation mechanism of the"diffraction wave" generated by the vibration of the piezoelectric substrate is discussed.After plane wave approximation,the interference phenomenon of the "diffracted wave"and the leakage wave in the fluid coupled from the surface of the lithium niobate substrate was studied.The characteristics of the interference acoustic field when there is only a single time-domain averaged pressure minima in a channel of a specific size is studied.Through volume force and acoustic flow field analysis,the trend of particle enrichment in the fluid is determined.This thsis also discussed the interference acoustic field in different flow channel wall materials and liquid sound velocity,and analyzed its influence on the interference acoustic field distribution.Through the analysis of the particle flow trajectory in the acoustic field,the rapid enrichment of particles with diameters of 10 ?m and 5 ?m was achieved.Analyzing the distribution of the drag force and the acoustic radiation force in the channel with different channel size ratios and the distribution of the force ratio.This thsis puts forward the microfluidic channel size ratio with stronger enrichment ability,and realizes the rapid enrichment of sub-micron particles with diameters of 800 nm and 500 nm.In addition,this method is expected to achieve rapid enrichment of nanoparticles.(4)Efficient cell lysis causes by acoustic streaming induced cell-particle collision in the dropletThe acoustic streaming effect was used to realize the high-speed movement of the fluid inside the droplet on the SAW propagation path.The high-speed movement of the fluid in the droplet can drive the cells and other particles in the droplet to move quickly.Because the speed and accelation of the living cells and particles with different densities and sizes are different,high-frequency collisions occur between the cells and the particles,which results the cell lysis.The principle and process of cell lysis were systematically studied,and the changes of the residual force before and after the cell lysis are analysed.Trypan blue staining were introduced to quickly judge the cell lysis effect before and after lysis,and the influence of particle presence,input power,lysis time,droplet volume and particle size on the efficiency of cell lysis were studied.The integrity of membrane function before and after cell lysis was determined by staining with live cell staining reagent Calcium AM.In addition,the changes of cell membrane structure and function before and after cell lysis were determined by the combined action of cell membrane staining reagent DiO and red fluorescent protein.Combining the scanning electron microscope images,a cell-particle collision lysis model is proposed.The theoretical feasibility of the model is supplemented by the estimation of the dynamics parameters of selected particle materials and the cells.Moreover,this article has achieved high-efficiency lysis of a variety of cells,and provided guidance for the application of low-concentration,micro-cell lysis and cytoplasmic release.In summary,this paper proposes a SAW-based microfluidic biological sample pretreatment method,which realizes the application of SAW standing wave field,SAW interference field and acoustic microfluidic effect in cell sorting,enrichment and lysis.Different application devices are designed and constructed through simulation theory,and the standing wave SAW device is used to realize the migration of particles in the acoustic standing wave field,which further realizes the sorting of animal and plant cells and the sorting of pollen and germ cells for the first time.The SAW interference field is studied by perturbation theory,and rapid micron-sized particle enrichment is achieved.The sub-micron-sized particle enrichment is achieved by adjusting the structure parameters of the microfluidic channel,and it provides a method for nanoparticle enrichment.In addition,the new effect of sono-induced microfluidics drives the high-speed movement and collision of cells and particles in the droplet,realizing the rapid and efficient lysis of a variety of cells.The research work in this article has opened up new application areas for SAW,and the results will provide a variety of "acoustic tweezers" operating platforms for the cells separation and enrichment,as well as the realease of cells intracellular biochemical molecules.