Research On Label Free Sorting And Analysis Of Tumor Cells Based On Microfluidic Chips

Circulating tumor cells(CTCs)have emerged as liquid biopsy markers for diagnosis,post-healing,and testing of cancer patients,and their efficient,pure,and highly active screening is key in driving the laboratory detection and clinical application of CTCs.Compared to microfluidic separation methods based on biological characteristics,label free microfluidic separation methods based on physical characteristics have low cost,are simple to perform,and have high cell viability after separation,thus have greater potential for applications.Since very small numbers of CTCS are occult in a bulk blood cell background and there are large differences but partially identical in their physical properties(density,size,stiffness,electromagnetic properties,etc.)and blood cells,it is difficult to achieve complete separation of CTCs relying on a single difference in physical properties.This paper summarized the progress of label free micro fluidic sorting technology,and aimed at high-efficiency,high-purity,and high-throughput label free sorting of CTCs.Based on micro fluidic technology and the size difference and stiffness difference of CTCs and blood cells,for the optimization of chip structure,precise screening theory,chip design method,chip preparation process,cell culture method,configuration of cell solution,chip performance test,construction of sorting analysis system,theoretical exploration and experimental studies were carried out,and carried out preliminary explorations for single-cell encapsulation techniques.In this paper,droplet-shaped microcolumns have been developed for the deterministic lateral displacement technique(DLD)to improve the sorting performance;Developed a novel two-stage arrayed DLD chip to achieve high-performance sorting of CTCs;Proposed a label free precisely sorting theory by controlling fluid flow rates;A cone channel chip was developed based on this theory to realize precise screening of CTCs;Based on the developed microfluidic device,a CTCs sorting and analysis system has been built in combination with a Raman microspectrometer system already available in the laboratory;Developed a single-cell encapsulation chip to preliminarily achieve micro droplet fusion.The research works in this paper are as follows:1.The microcolumn morphology of deterministic lateral displacement technique(DLD)was optimized and droplet-shaped microcolumns were obtained,which greatly improved the sorting performance.A Bessel curve was used to fit the conventional circle,and the parameter optimization model was constructed and the computational equation of the width of the first flow beam was derived.The analysis results showed that critical size of droplet-shaped micropillars decreased by 21.25±1.91%compared with that of circular micropillars,and throughput increased by 28%,and was smoother than triangular micropillar to guide fluid,and avoided fluidic reflux and were cell friendly.A fluid-solid coupling simulation considering the role of cell coupling with microfluidics was implemented to accurately predict the trajectory of cells within micropillar arrays.2.A high-performance two-stage DLD sorting chip was designed using dropletshaped microcolumn.Experiments showed that the chip exhibited sorting purity of 98.20±0.22%and 99.74±0.47%,sorting efficiency of 92.02±0.54%and 99.53±0.45%for polystyrene particles of 12μm and 20μm,respectively;Two-stage critical size accuracies were 90%and 94.85%,respectively;In the context of blood cells,the sorting purity was 98.25±2.48%and the recovery efficiency was 96.30±2.10%at a throughput of 2mL/min.The chip was highly reproducible and simple in structure,which enables label free sorting with high efficiency,purity and high throughput.3.A label free CTCs sorting method that precisely controls the flow rate of fluid within the microflow channel was proposed and the underlying theory was constructed.Incorporating cell stiffness into the classification basis,3D solid mechanical contact simulation and fluid mechanical simulation were implemented;The mechanical properties of cells through slit and cone channels were analyzed;The effect of cell stiffness on the separation process was investigated to analyze the relationship between critical inlet flow rate and cell size,cell stiffness.4.A single slit chip was designed to verify the theory,and a cone channel chip was designed to accurately separate leukocytes and CTCs in close size.Experiments showed that cancer cells and leukocytes of close size were separated by a cone channel chip at a flux of 35μL/min,and the cancer cell purity was improved from 9.3%to 16.67%,with a 1.8-fold improvement and 75%recovery efficiency.5.Prostate cancer DU145 cells were cultured and various experimental solutions were prepared;The Young’s modulus of DU 145 cells and leukocytes were measured and calculated;Microfluidic chips of polydimethylsiloxane(PDMS)and glass were fabricated as described.Aiming at the practical application scenarios of the Raman microspectrometer system,glass-holed PDMS-glass chip was prepared,which avoid the influence of PDMS on the Raman signal.6.Based on the developed microfluidic device combined with Raman microspectrometer system,a label free,high-throughput,high-purity and high-efficiency CTCs sorting and high-precision,label free,and damage free CTCs analysis system was constructed,which achieves the all microfluidic label free and high-performance complete CTCs sorting and cell analysis.Aiming at the potential application prospects of this system with the goal of encapsulating cells with sequencing primers,a single-cell encapsulation chip was developed based on droplet microfluidics technology and microdroplet fusion was preliminarily achieved.