| With the development of space projects,such as China's Moon landing,Mars landing and China Space Station,it will face new challenges for researches on radiation damage mechanisms,risk assessment and early warning for astronauts in long-term on-orbit and deep space exploration,and there is an increasing need for on-orbit radiation biology research.The model organism Caenorhabditis elegans(C.elegans)has been used for many times in research on space radiation biology,but previous nematode-based space biology experiments,such as studying the influence of microgravity on the growth and development of nematodes,the effect of space flight on the mutation rate of nematodes,and the changes in the expression of some nematode proteins after short-term flight,have many problems that need to be solved,including the failures to realize automated cultivation and control,single nematode-based research and on-orbit monitoring of nematode movement and changes in body length and width.Aiming to the requirement of on-orbit detection of nematodes for research in space radiation biology,this project carried out research on the key technologies of microfluidic-based automated culture and detection of single nematode.By overcoming technical problems of nematode loading and separation,automatic control and detection of single nematode,it will enable parallel separation,reversible capture and release,overnight cultivation and array detection of single nematodes on microfluidic chip.The experimental methods and main results of this research are as follows:(1)A method of centrifugal microfluidic-based separation and analysis of nematodes was proposed.A disc-like microfluidic chip containing multiple nematode analysis units is designed,and the chip containing a single analysis unit is fabricated and tested,which includes 16 microchannels for single nematode analysis.Conditions of centrifugal-based nematode loading and separation were also determined.The results show that the method is feasible and preliminary experimental data are obtained;Based on the centrifugal chip containing a single nematode analysis unit,a method of manual sampling and direct injection of single nematodes were preliminarily investigated,and the feasibility was initially verified.(2)Pressure-driven microfluidic chips for nematode separation were developed,which were based on protocols of one-step loading and multi-step loading,respectively.Preliminary results show that the multi-step protocol is significantly better than the one-step method,and the sample of nematodes cultured at low density(about 300 worms/mL)favors the sample in the high-density group(about 700 worms/mL),as for the results of nematode separation.After separation of the two samples on multi-step loading chips,the proportion of culture chambers containing nematodes was 83.3%and 54.2%,respectively.By using a multi-step loading chip with eight microchannels,this work also realized parallel,reversible immobilization and release of single nematodes,as well as replacement of culture medium.(3)To detect single nematode array on a chip,a conventional optical microscope and a CMOS-based lensless microimaging module were utilized.Preliminary results verified the feasibility of one-by-one detection method based on microscopic imaging module and one-time imaging protocol based on lens-free module.It was demonstrated that the microfluidic chips for nematode analysis and methods of nematode control and detection proposed in this work can realize chip-based nematode loading,parallel separation and control of single nematodes,and video recording and microscopic imaging of an array of single nematodes.The results of this project will provide a technical basis for the construction of an microfluidic system for automated culture and detection of single nematodes. |
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