Key Technology Of Microfluidic Chip For The Measurement Of [Ca²⁺]_I And Experimental Study

Microfluidic chip can complete sample preparation, reaction, separation, detection and otherfunctions which involved in the chemical and biological domains. Microfluidic chip have a lot ofadvantages including that the sample consumption is small, the processing time is short and thecost is low.Therefore, microfluidic chip have been widely applied in chemistry, biology andmedicine and other aspects, to replace the conventional chemical or biological laboratoryfunction. Calcium ions act as very important intracellular second messengers, widely involved insignal transduction process. Calcium ions play an important role in many biological activitiessuch as muscle contraction, nerve transfer, enzyme and hormone secretion.In recent years, many studies show that TRP channels are the way of Ca2+.TRP channelsmediate feeling signal transduction, regulate of cell calcium balance and affect the developmentand other functions. TRPV4are non-selective cation channels and they have always permeabilityon the calcium ion. They involved in the maintenance of homeostasis, have important sense oncompleting many physiological functions. New ideas are provided to study the clinicaldysfunction such as a pathological pain, touch pressure sensation disorders by studying ofTRPV4channels action mechanism on regulation of intracellular Ca²⁺homeostasis andtransmission of extracellular signals.This thesis was supported by Tianjin Natural Science Foundation Project The research ofmicrofluidic chip and detection system used in intracellular free calcium.The major content includes: 1. The basic theory of fluid dynamics was described. The continuity equation, eulerequation and navier-stokes equations were discussed. CFD which solving complex fluid flowproblem, the continuity equation momentum equations and micro channel diffusion equationsof microfluidics were studied. The microfluidic chip mixture model was established and thecomputer numerical simulation of micro channel flow was introduced.2. This part is mainly related to numerical study on the diffusion and minxing in themicro-channel flow. The numerical simulation of the diffusion-based mixing of the species in themicro-channel and some experiments were carried out on the basis of different parameters ofliquid mixing. A lot of detailed research work show that there are many factors which affect themixing of microfluidic, such as the inlet velocity, diffusion coefficient, and so on. Based on it,the difference of mixing flow in the microfluidic chip is discussed.3. On the basis of studies of fluorescence detection calcium ion concentration, the methodof detecting the concentration of calcium ion in the cells using microfluidic chips was presentedand the microfluidic fluorescent detection system was built. We have designed and manufacturedsome microfluidic chips based on PDMS for the detection of calcium concentration.4. In this study, a set of experiments were completed by microfluidic fluorescent detectionsystem.Through the analysis of measurement results of fluorescent ratio change, a lot of detailedresearch work were completed that TRPV4has influence on transport mechanism of calcium ionInnovative works in the thesis include as follows:1. The numerical simulation of the diffusion-based mixing of the species in themicro-channel and some experiments were carried out on the basis of different parameters ofliquid mixing. Based on it, designing of the microfluidic was discussed. The research work hasimportant meaning and reference value for microfluidic chip sample of rapid response andtesting.2. Microfluidic fluorescent detection system was used to detect the change of calciumconcentration and distribution in multiple sets of experiments. In different temperature, lowosmotic pressure, shear stress, phorbol ester derivatives and other physical and chemicalstimulus characteristics, the detailed study of the intracellular concentration of calcium ion wascompleted. And further produce mechanism of the change was discussed.3. In order to learn the mechanism of TRPV4which have the mechanical sensitivity and temperature sensitivity, a set of experiments were completed which through the analysis ofmeasurement results of fluorescent ratio change. Contrast experiments show that the changes in[Ca²⁺])ilevel induced by those stimuli in synoviocytes are similar to the function of TRPV4.4. A lot of detailed research work include that the TRPV4activation has influence ontransport mechanism of calcium ion. Variety of reagent have been used in contrast experiments,and that heat and hyposmotic stimulation increase [Ca²⁺])iby activation of TRPV4were found. Inthis study, we proved that the level of [Ca²⁺])iwas increased via Ca²⁺influx in the synoviocytes.