Study Of Cell Separation And In-situ Detection Based On Microfluidic Cell Chip With Multi-modes

Cell detection plays an important role in the prevention,diagnosis and evaluation of therapeutic effects of major diseases.Today,cancer and brain nervous system diseases are two important diseases that affect human health.The detection of tumor cells in human peripheral blood and the detection of leukocytes in cerebrospinal fluid are clinically deficient.The microfluidic chip has the advantages of high throughput,low sample requirements,high integration of flexible and flexible microfluidic channels,and a highly-compatible detection unit.Combining microfluidic chip technology with cell separation detection will help realize the analysis and detection of important cells and provide new methods for clinical cell detection.The main research work and results are as follows:?1?A multifunctional microfluidic chip integrated with multi-orifice flow fractionation?MOFF?and magnetic capture technology was developed to specifically separate and capture the HepG2 cells in artificial samples.The chip contains a glass substrate and a PDMS micro-channel cover plate.The PDMS cover plate consisted of 3long 10mm injection channels,MOFF separation zone and hexagonal cavity cell enrichment detection zone.The total length of MOFF separation zone was 20 mm consisting of 80 semi-rhombic shrinkage and expansion units with a length of 0.18 mm,a depth of 50?m,a shrinkage area width of 0.06 mm,and an expansion area of 0.20mm.The angle between each group of shrinkage and expansion units was 103.0°.HepG2-blood cell suspension was used as the sample.Based on the principle that the magnetic beads surface modified c-Met antibody could specifically bind to HepG2 cells,an immune-magnetic beads?Anti-MNCs?suspension at a concentration of 50?g·mL^-1was prepared by surface carboxylated beads,EDC(1 mg·mL^-1),NHS(1 mg·mL^-1)and c-Met antibody.Under the optimized flow rate of 50?L·min^-1,HepG2 in suspension samples were efficiently captured at the detection zone of chip via a magnetic field.The carbon quantum dots were prepared by microwave heating with citric acid and thiourea to label HepG2 cells which made it possible that the captured HepG2 could be visualized in-situ.The captured cells in the chip detection area were counted by microscope.It was shown that the capture rate of HepG2 cells was 88.5%±6.7%?500?L volume contained 10⁶ blood cells and 10 HepG2 cells?.The results demonstrated the developed multifunctional microfluidic chip may serve as a promising tool for separation and capture of tumour cells.?2?A DEP microfluidic chip for the separation and enrichment of WBC in artificial cerebrospinal fluid?CSF?was designed and manufactured.The H-configured DEP microfluidic chip with four arrays of interdigitated electrodes and one set of parabolic electrodes was independently designed.The 50 pairs of parallel pairs of interdigitated gold electrodes have a length of m=900?m,a width of 20?m,a spacing between each pair of electrodes of n=15?m,a radius of a parabolic electrode of r=750?m,and a spacing between quarter circles of t=55?m.An in situ Raman/in-situ fluorescence detection microsystem for the separation and enrichment of leukocytes from artificial CSF was established.The separation and enrichment of leukocyte in artificial CSF samples theoretically demonstrated,and the performance of the self-designed DEP microfluidic chip for separation and enrichment was tested.Under the condition of the optimized flow rate of 50?L·min^-1,excitation voltage of 5Vp-p and excitation frequency of 4MHz,it was autonomous.The designed DEP microfluidic electrode interdigital electrodes and parabolic electrodes successfully separated and enriched the white blood cells in the artificial CSF sample,capture rate was 78.7%?n=3?.?3?The separation and enrichment of WBC in artificial cerebrospinal fluid based on the self-designed DEP microfluidic chip combined with Raman spectroscopy,the silver colloid synthesized by microwaves was used to amplify the Raman signals of white blood cells,and the interaction time of leukocyte-silver colloid was determined 6h,optimized Raman detection parameters:integration time 10s,detection power 70.2mW,separation and enrichment of WBC from artificial CSF based on the DEP microfluidic chip-in situ Raman detection,two characteristic peaks 477 cm^-1,511 cm^-1 were successfully obtained,captured white blood cells were qualitatively tested.?4?Fluorescent labeling technology was used to combine the self-designed DEP microfluidic chip with a fluorescent chemiluminescent microplate reader by labeling the leukocyte in the artificial cerebrospinal fluid with the fluorescent reagent DTOC6?3?.The relationship between the number of white blood cells and the fluorescence intensity was successfully obtained.A standard curve was established:y=0.0164x-0.0991,R²=0.9928,and the maximum detection amount at the parabola electrode of the independently designed microfluidic chip was detected 200 WBC.Artificial white blood cell-CSF samples were tested using a standard curve established experimentally.The fluorescence intensity value obtained by subtracting the blank group was substituted into the experimentally established standard curve.The number of cells was calculated to be 44.42,95.55,and 141.17 respectively.The number of cells contained in the sample was 50,100,and 150.The accuracy was 88.84%,95.55%,and 94.11%,respectively.Compared with the traditional clinical cerebrospinal fluid test,it has the advantages of rapid,high-efficiency and multi-functionality,and provides a new method for clinical cerebrospinal fluid cell detection.