Research And Application Of A New Method For Rapid And Highly Sensitive Blood Cell Function Detection Based On Microfluidic Chips

Microcirculation disorders are related to human aging,the occurrence of tumors,hypertension,diabetes,sepsis and many cardiovascular and cerebrovascular diseases.Therefore,normal microcirculation is very important for human health.Blood rheology is closely related to microcirculation and is one of the main factors causing microcirculation disorders.Red blood cell(RBC)and platelet in blood are the key factors to determine the rheological properties of blood,and their functions directly determine the rheological properties of blood.Red cell deformability(RCD)and platelet function are the main intravascular factors that cause microcirculation disturbance.Microfluidic chips have the characteristics of single cell,high throughput,large scale,parallelism,small sample consumption,fast speed and intuitive data analysis.Polydimethylsiloxane(PDMS)microfluidic chips have the advantages of good light transmission,processability,easy integration,good biocompatibility and approximate vascular elasticity,and can be used to build a microminiature human capillary model close to physiological function in vitro.The main research contents of this thesis are as follows:(1)We apply a gravity-driven deformability cytometry platform(GD-DCP)to profile the drug response of the RCD at the single-cell level using pentoxifylline(PTX)as a model drug,the effect of different concentrations of PTX(0,2,20,200 μg/mL)on RCD in patients with cardiovascular disease was explored.Based on the GD-DCP,about 56%of coronary heart disease patients respond positively to PTX,indicating that PTX has a strong patient dependency on RCD.Moreover,RCD is observed to be significantly inversely correlated with the activation of membrane protein kinase C(PKC)as well as the concentration of Ca2+(both P<0.001).The results of animal experiments show that the protective effects of PTX on myocardial ischemia rats have substantial individual variation,too.It is noted that the effect of PTX on RCD in vitro,which is a potential indicator for individualized therapy and a clinical drug prescreening/dosing indicator,has a high predictive accuracy for in vivo outcomes.(2)On the basis of the second chapter,the complex operation of RCD detection by the integrated GD-DCP imaging system developed in the second chapter is improved.For example,RBC suspension is obtained after repeated centrifugation of the sample,and its deformability can be analyzed only after staining of the RBC suspension.Based on this,we introduce a deterministic flow cytometry(DFC)assay for simple,rapid,sensitive and label-free measurements of the biophysical parameters(apparent size,distribution and RCD)of RBC.The assay can be completed within 2 min using only 10 μL diluted whole blood.Our results show that the unique C-shaped DFC is a sensitive and easy-to-use tool,which can detect not only the biphasic effect of ethanol and oxidative damage of erythrocytes but also significant individualdependent variability and dose dependence under the phenazine methosulfate(PMS)stimuli.In addition,different biophysical characteristics of RBC in a septic rat model are also determined,and the practicability of this method is verified by a septic microcirculation impaired model.The results show that the new method can achieve rapid,highly sensitive and accurate detection of RCD.(3)A new S-shaped pillar DLD chip is designed for rapid assessment of platelet Dapp and distribution in whole blood to determine whether platelets are in an activated state.This rapid,unlabeled platelet Dapp assay requires only 10 μL of whole blood.The whole process takes only two minutes.First,we demonstrated by flow cytometry that the S-shaped DLD chip does not activate platelets by collecting platelets flowing through the S-shaped DLD chip,and that exposure to the activator is the only critical factor affecting platelet activation.Subsequently,we measured the size and distribution of platelets exposed to thrombin by S-shaped DLD chip,and the results showed a significant increase in platelet Dapp.Finally,by establishing a rat model of myocardial ischemia,the changes of platelet Dapp in rats were effectively measured.Compared with healthy rats,the platelet Dapp in myocardial ischemia rats was significantly increased.The results show that our new method can detect platelet function quickly,sensitively and accurately,which is expected to provide a new method for clinical detection of platelet function.