Design And Research Of Microfluidic Cell Counting System Based On Impedance Method

Cell counting has a wide range of applications in clinical diagnosis,biomolecular detection,environmental testing and climate change.Currently,the most used cell counting methods are microscopy,image counting and fluorescence-based flow cytometry,but most of these counting devices are large,expensive,and require long pre-processing time and labeling of the target sample,which greatly consumes counting time and increases counting cost.Microfluidic-based impedance method flow cytometer has a promising future in replacing conventional cell counting due to its portability,non-invasive and label-free features,but its further development is also greatly limited by the location-dependent problem of impedance signal of cells in the sensing region.In this paper,an impedance-based microfluidic cell counting chip and related signal detection and processing system are designed and developed by using the principle of impedance-based cell counting combined with the concept of liquid electrode.Firstly,the cell counting chip structure is determined according to the design requirements,and the counting and focusing module is analyzed theoretically.For the dielectrophoretic focusing module,this paper proposes a new dielectrophoretic focusing structure based on the existing dielectrophoretic focusing research status,which based on sheet electrodes.In addition,COMSOL simulation software is used to analyze the distribution of flow field and electric field within the dielectrophoretic focusing structure formed by liquid electrode pairs,and the design rules and structure dimensions of the liquid electrode structure are determined by combining relevant literature:the metal electrode position at the bottom of the closed flow channel is 30μm with the same height of the micro-channel,and the width of the liquid electrode,the width of the main channel and the width of the insulating block between the liquid electrodes are 50μm.Then,the dielectrophoretic focusing chip structure and size is designed and determined.Simulation model is established according to the dielectrophoretic focusing structure,and the particle focusing situation is simulated and analyzed under the conditions of the model.The parameters of particle focusing effect were determined from the particle focusing trajectory as the particle focusing width d at the focusing exit and the dimensionless parameterΦ,whereΦis the ratio between the focusing width d and the third-to-last pair of particle focusing width d₀ at the exit of liquid electrode.The finite element simulation software was used to simulate and analyze the influence of three factors,namely the number of liquid electrode,the voltage amplitude and the sample flow rate,on the focusing effect of dielectrophoresis.The simulation results showed that:liquid electrode pairs and voltage amplitude has a positive effect on the dielectrophoretic focusing,with the increase of the value the particle focusing width gradually decreases and the focusing effect keeps improving;while the sample flow rate has the opposite effect on the dielectrophoretic focusing,with the increase of the sample flow rate the particle focusing width increases and the focusing effect decreases.Next,in this paper,focusing experiments of dielectrophoresis were designed and studied based on the simulation results.The trajectory of particles at the exit of the focusing area was captured by high-speed camera,which was superimposed by Image J software to determine the focusing width of particles and calculate the measurement parameters of the focusing effect of particles.The results of single factor experiment show that the particle focusing trend is consistent with the simulation results,which proves the accuracy of the simulation model and the practicability of the focusing chip structure designed in this paper.In order to further determine the importance of each experimental factor on the focusing effect,an orthogonal experiment was designed for each factor.The orthogonal experiment results showed that the main and secondary factors affecting the focusing effect were the number of the liquid electrode pairs,the voltage amplitude and the sample velocity.The optimal focusing parameters were determined as follows:the number of the liquid electrode pair was 100 pairs,the voltage amplitude was 20 Vpp,and the sample flow rate was 6μL/min.Finally,the impedance counting chip structure was determined,and the impedance signal simulation model was built to simulate and analyze the changes of electrical signals caused by cells or particles passing through the detection area under the condition of the electrode structure.The simulation results are used to select relevant signal detection electronic components and design a signal detection and processing system,which is used to conduct counting experiments.The counting experiment was carried out with the standard sample solution to determine the signal detection SNR of the counting system,and the linear relationship curve between the number of particles passing through the detection area and the concentration of the standard sample solution in the detection time was obtained（correlation coefficient is over 0.99）.In addition,the yeast cell solution was used for counting experiments,and the results were compared with those of the blood cell counting plate,and the relative deviation of the counting results was tested to be 3.56%,indicating the validity of the counting results of this counting system.