Research On Immunofluorescence Labeling Technology Of Ku70 Protein Based On Microfluidic Chip

With the continuous development of deep space exploration program and the increasing duration of astronauts’in-orbit missions,space radiation risk assessment is becoming more and more important.Conducting space radiation biology research in orbit is an effective way to supplement the deficiencies of current space radiation damage assessment methods and improve the accuracy of assessment results.By monitoring radiation damage biomarkers,the physical health indicators of astronauts can be indirectly reflected,and on this basis,space radiation damage can be warned,radiation damage mechanism can be detected and radiation protection can be studied.In view of the screening and in-orbit monitoring requirements of radiation damage markers,this study investigated the effectiveness of Ku70 protein as a marker of radiation damage,and proposed a microfluidic automated immunofluorescence labeling technology that can be used in microgravity environment,and designed a prototype of its principle,and preliminarily verified its performance.The main research contents include:1)The radiation dose-dependent effect of Ku70 protein under UV-C irradiation was determined for the first time.Human CD4~+T lymphocytes were irradiated with UV-C at different doses,and immunofluorescence was detected by flow cytometry to investigate the linear relationship between Ku70 expression and irradiation dose under different cell damage repair times,and to verify the feasibility of Ku70 as a marker of radiation damage.To provide evidence for the effectiveness of space radiation biology research based on Ku70.The experimental results showed that there was an obvious dependence between Ku70 expression and radiation dose in the irradiation dose range of 0～768 J/m~2 and after 36 h repair time of damaged cells.2)To meet the requirements of in-orbit monitoring of biomarkers for radiation damage,a portable automated immunofluorescent labeling microfluidic technology suitable for microgravity environment and its principle prototype were constructed by combining microfluidic technology,immunomagnetic bead technology and immunofluorescence technology.The main research work includes:(1)Fluorescent immunolabeling fluid module design and processing:liquid storage bag and pinch valve,microfluidic chip and injection pump were used to construct fluorescent immunolabeling flow path based on microfluidic chip,and the chip flow path module was designed and processed;(2)Optimization of three-dimensional magnetic field mixing module:Adjust the distance between vertical and horizontal magnets of three-dimensional magnetic field module(vertical:7 mm,horizontal:5 mm)to make it easier to integrate with other components of the microfluidic device;(3)Establishment of automatic immunofluorescence labeling device and functional verification and testing:Various functional modules were designed,processed and integrated,and a microfluidic automatic immunofluorescence labeling device with automatic liquid exchange and three-dimensional mixing of magnetic beads was built.The device was used for fluorescence immunolabeling of irradiated CD4~+cells,and the experimental results of radiation dose-dependent effect of Ku70protein expression in the range of 0～768 J/m~2 were realized.The automatic immunofluorescence labeling microfluidic technology and principle prototype developed in this subject can realize the functions of reagent storage and fluid automatic control,three-dimensional mixing of immunomagnetic beads,immunofluorescence labeling of cell samples,etc.,providing a solid theoretical and technical basis for the follow-up research and application of in-orbit monitoring of radiation damage markers.