A Microchip For Cell Survival Rate Determination Based On Hypertonic Stimulation

Cell survival rate(CSR)is a very important parameter in biological and medical fields.Today,the main methods for determining this parameter are dyeing/staining and flow cytometry.The dyeing method is simple,but the dyeing process is time-consuming and easy to be affected by human operation.The flow cytometry has high accuracy and sensitivity,but the device is expensive.In addition,both methods require the tested cells to be labeled,causing the labeled cells usually unsuitable to be reused in later experiments.With the development of Lap-on-a-chip(LOC)technology,cell manipulations on chip for fundamental researches have become research focuses and hotspots,making it necessary to determine the CSR in a fast,label-free and on-line mode;however,the conventional methods mentioned above are difficult to be integrated onto the chip.Here,a hypertonic stimulus-based impedance flow cytometry(HSIFC)was developed for determining the CSR.On the chip,a hypertonic solution was used to stimulate the cell sample to cause the volume difference between living and dead cells,then the volume difference was detected using the impedance analysis method,and finally the CSR was calculated according to the amplitude distribution of impedance signals.The main contents of this thesis are as follows:(1)HSIFC was designed and fabricated.In the HSIFC,the staggered herringbone mixer design was adopted to realize the fast mixing of cell suspension and hypertonic solution and to complete on-chip hypertonic stimulus of cells.The size of cells passing through the chip is measured using contactless impedance measuring electrodes,and the CSR was calculated according to the statistical results.In the fabrication process,the polydimethylsiloxane(PDMS)layer with fluid flow passages was formed using the template fabricated by lithography technology,indium tin oxide electrodes were fabricated on glass using laser etching technology,and the detection chip is finally formed by bonding the PDMS layer and the glass layer through PDMS film.(2)The subfunctions of the HSIFC were confirmed experimentally.Firstly,the mixing performance of the staggered herringbone mixer was confirmed using fluorescence experiments.The accuracy of impedance signal recognition was analyzed using microbead experiments and cell experiments,and the relationship between microbead/cell size and impedance amplitude was studied.The results showed that the staggered herringbone mixer could rapidly mix hypertonic solution and cell suspension;the electrodes could accurately capture the impedance pulse signals of cells and microbeads when they passed through the detection pore;the diameter of microbeads was in proportion to the amplitude of signals;and the size of cells was positively related to the amplitude of signals.(3)The performance of the HSIFC was studied experimentally in terms of determining the CSR.The CSRs of the mixed cell samples under different mixing ratios,death modes and cell types were determined using HSIFC and then the results were compared with the expected values and the values determined using flow cytometry.The results showed that the HSIFC could accurately predict the CSRs,and the results were in agreement with the theoretical values and in consistence with the values from flow cytometry.The determination coefficient(R2)of the two methods was 98.4%.Here,a new method for on-chip,label-free,and on-line determination of the CSR was developed and thus this work could contribute to the development of LOC technology.