Microfluids And Size Differences Based On Sorting And Fish Detection For White Blood Cells

With the development of society and national economy,people’s living standard has been greatly improved,but with it comes a series of diseases.Among them,the number of patients with leukemia increased with an alarming growth rate year by year.Different pathologic classification of leukemia requires different clinical diagnosis and treatment,so the early classification of leukemia has important clinical research value.The key of medical detection based on molecular biology and cell biology lies in the precise and efficient separation of target cells with pathologic significance.In recent years,with the development of microprocessing technology,microfluidic technology has a wide application prospect in specific target cell sorting and other fields.Conventional cell sorting methods include antibody-antigen binding sorting,electroosmosis,magnetic sorting and so on.But this kind of method usually separation purity is not enough,interference cell is many,detection effect is poor;The microfluidic chip with complex structure has better separation effect,but its preparation process is complicated,the production cost is high,the cycle is long,which is not conducive to popularization and application.Fluorescence In Situ Hybridization technique(Fluorescence-In-Situ Hybridization,FISH)in detection and pathological classification of leukemia achievements In their application,and is widely applied In the accurate diagnosis of various diseases,but still face detection reagent consumption big,time is long,high test cost,rely heavily on manual operation,such problems as lack of automation equipment,greatly limits the FISH detection technology popularization and application In the field of molecular detection.In this paper,a new method of cell separation based on microfluidic flow field and size difference is proposed.This method can continuously and efficiently separate target cells in microfluidic channels,and then use microdam microfluidic chip to enrich the above-mentioned isolated cells twice before FISH detection.This method combining separation and detection makes cell detection faster and cheaper,and is more suitable for clinical diagnosis.This method has the advantages of easy manufacture and operation,low cost and fast detection speed.The research contents of this paper are as follows:(1)To understand the development status and trend of microfluidic technology,cell sorting technology and FISH detection technology,and summarize the development status,existing problems and challenges of FISH detection technology based on microfluidic chip;(2)Design and manufacture microfluidic chips based on flow field and size difference,including the design and optimization of chip structure,so as to achieve the optimal separation effect;(3)Cell sorting experiments based on flow field and size differences,including the construction of sorting experimental equipment,fluid parameter optimization and experiments,analysis and summary;(4)Design and manufacture white blood cell capture and FISH staining chips based on microdam array,including design and optimization of chip structure,production of mask,chip characterization and chip testing;(5)WBC capture and FISH staining experiments based on microdam array,including optimization of different cell pretreatment methods,influence of key parameters(gap height and sample injection speed)on WBC enrichment efficiency,FISH staining experiment,and analysis,summary and prospect of experimental results.The new method of cell separation based on microfluidic flow field and size difference proposed in this paper has the following innovations:(1)FISH detection based on microfluidic chip has low detection cost and low reagent consumption,especially the consumption of expensive probe(2-3μl),which can be reduced to 1/4 of traditional FISH probe consumption(about 10μl);(2)The microfluidic chip based on microdam array and the dyeing system built can realize by myself the automatic FISH dyeing process,and the detection time(about 4h)is shortened to one sixth of the traditional FISH detection time(24 hours);(3)The target cells were pre-selected by the microfluidic flow field and size difference,and then enriched by the microdam array chip,which could minimize the interference of blood impurities,improve the experimental effect of FISH staining,and increase the success rate and repeatability of FISH staining.