Development And Application Of The Microfluidic Single-cell Impedance Detection Systems

Single-cell biophysical characterization has been widely employed for demonstrating the structure,function and pathological status of individual cells,or revealing the heterogeneity among various populations.Thus,it plays an important role in the identification of cell types as well as the early diagnosis of cancer and other diseases.Microfluidic device,which consists of microchannel networks with dimensions comparable to cell diameters,has a unique advantage in the micro-environment control,movement regulation and property characterization of biological cells.In recent years,microfluidics has developed into a frontier technology integrating with mechanical engineering,electronic engineering,material science,fluid mechanics,biology and medicine.Microfluidics-based single-cell biophysical characterization is expected to be a potential breakthrough for the next generation devices of POCT(Point-of-Care Testing).In this dissertation,the recent advances and existing problems in the microfluidics for characterizing single-cell biophysical properties are comprehensively reviewed.A portable electrical impedance measurement system and three kinds of single-cell impedance analyzers with different functions have been developed,which can be potentially applied for the identification,counting and status monitoring of CTCs(Circulating Tumor Cells)from whole blood.The innovative achievements are mainly described as follows.(1)Cell dielectric properties and portable electrical impedance measurement system.The Maxwell’s mixture theory and the equivalent circuit model describing the dielectric properties of a cell dispersed in a suspending medium are established,then the dielectric properties of cell mixture system are also analysed in time domain.After that,a new method to obtain broadband single-cell impedance spectroscopy is proposed,based on the pseudorandom m-sequence correlation identification.The spectrum lies between 1.953 KHz and 1 MHz with a total of 512 frequencies can be measured in a very short period of time(0.5115 ms).The excitation of m-sequence signals and the acquisition of response signals are executed by using a current amplifier and a data acquisition card with a LabVIEW synchronization sampling program.The broadband impedance spectroscopy is obtained by sequentially applying a fast M transform and a fast Fourier transform to the measured response signals with a MATLAB data processing program.The reliability of m-sequence based broadband impedance measurement technique is verified by measuring an RC test circuit with known electrical parameters.Since the excitation signal generation,the response signal acquisition and the impedance signal processing are all achieved through software programming,the developed electrical impedance measurement system has a good portability,an d th e DC or AC impedance detection of individual cells can be easily conducted by calling the corresponding program.(2)DC impedance-based microfluidic Coulter counter for tumor cell detection.A microfluidic Coulter counter integrated with inertial focusing and liquid electrode is developed to achieve high-throughput cell counting,cell size characterization and cell population discrimination.An asymmetrically curved microchannel is used to inertially focus cells into a determinate train to reduce the possibility of cell adhesions and ensure that only one cell passes through detection region at a time,which improves the stability and accuracy of downstream DC impedance measurements.The liquid electrodes are constructed by inserting Ag/AgCl wires into the electrode chambers filled with flowing highly conductive electrolyte solutions,which reduces the length of detection region,and effectively improves the sensitivity of the measured DC impedance peak to the cell size.The main sample flow rate and the feed flow rate in the electrode chambers are first optimized by measuring the standard polystyrene particles.On the basis of the optimized system,an exactly linear relationship between the amplitude of impedance peaks and the volume of size-calibrated particles is established,and a high detection throughput of～5000 cells/s is achieved.Using the calibrated microfluidic Coulter counter,the human breast tumor cells(MCF-7 cells)and leukocytes(WBCs)are measured,and the average diameters of MCF-7 cells and WBCs are respectively calculated to be 14.28 μm and 9.01 μm.Finally,a detection threshold is set according to the statistical impedance peak distributions of two pure cell populations,and the MCF-7 cells are successfully distinguished from the mixed cells based on the threshold.(3)AC impedance-based microcytometer for tumor cell detection.A microfluidic impedance flow cytometer integrated with viscoelastic focusing and broadband electrical impedance measurement is developed to conduct the AC impedance detection for continuously flowing cells.The cells suspended in PEO solution are well-ordered at the center of the straight microchannel with square cross-section,due to the inertial and viscoelastic coupling effects of the PEO solution.The focused cells are then detected by using the developed broadband impedance spectroscopy,through the underneath ITO coplanar electrodes.The necessity of viscoelastic focusing unit is verified by comparing the measured particle impedance signals with PEO solution(Newtonian fluid)and PBS solution(viscoelastic fluid).The effects of sample flow rates on the viscoelastic focusing behaviors of MCF-7 cells are also systematically studied.By analyzing the impedance changes caused by particle translocations at different frequencies,the optimal frequency for the AC impedance detection is determined.After that,the influences of microchannel sizes and sample flow rates on the electrical impedance measurement are explored.Finally,the proposed microfluidic impedance flow cytometer is used to measure the MCF-7 cells spiked into leukocyte suspension,and the MCF-7 cells are successfully identified from the mixed population of cells based on an impedance peak threshold.(4)Microelectrical impedance spectroscopy for tumor cell monitoring.A microelectrical impedance spectroscopy integrated with hydrodynamic cell trapping and broadband electrical impedance measurement is developed to realize the automatic capture and status monitoring of tumor cells.Using the least flow resistance path principle,the hydrodynamic cell trapping in serpentine microchannel arrays can be carried out in a deterministic and automatic manner without the assistance of any external fields.The trapped cells are then monitored by using the developed broadband impedance spectroscopy,through the ITO electrode arrays which are exactly corresponding to the cell trap arrays.Three microfluidic devices with different serpentine microchannels are designed and fabricated to systematically investigate the cell trapping behaviors.The experimental results show that the cell trap rate of higher than 95%can be easily achieved in all cell trapping microdevices.Subsequently,the optimal frequency range for monitoring tumor cells is determined by analyzing the measured full-frequency impedance spectrum of MCF-7 cells under different capture states.Finally,the developed microelectrical impedance spectroscopy is used to monitor the trapping process of single or multiple MCF-7 cells.During the trapping process,the position,state,speed,direction and number of MCF-7 cells are successfully probed according to the changes of the measured electrical impedance signals.