Microfluidic Technologies For Microbial Screening,Immobilization And Self-assembly

The study of microorganism holds an enormous significance in humans’ lives,as the production of food,chemicals,pharmaceuticals,bioenergy,biomaterials and enzymes often involves the use of microorganisms,such as yeast,bacteria and fungi.With the development of systems biology,synthetic biology and medicine,the study of high-throughput screening of specific microbial individuals,efficient immobilization of microorganisms and controllable assembly of bacterial cellulose(BC)fibers have become important parts of current biomedical engineering research.The conventional microbial research methods are mainly based on agarose plate and multi-well plate.However,those techniques are cumbersome,low-throughput,not suitable for precise manipulation of microbial cells.The microfluidic technology has expanded dramatically in recent decades,due to its advantages such as easy fabrication,low consumption,simple operation and high-throughput.Moreover,the microfluidic technology has been recognized as an ideal operating platform for microbial research due to the dimension of the chip microstructures which could be designed freely to match the sizes of the microorganisms perfectly.In this thesis,an agarose-based microwell array chip was designed for the functional microorganisms screening,and polydimethylsiloxane(PDMS)based porous materials were developed to investigate microbial immobilization and the controllable assembly of BC fibrils.(1)An agarose-based microwell array chip was designed for screening functional microorganisms at the monoclonal level.Green fluorescent protein(GFP)labeled E.coli was used to assess the distribution and growth of seeded cells in wells.Lipase-expressing bacillus subtilis was used as a model strain to verify the expression and detection of bacterial products on chips.Our results shown that the bacterial cells could be captured and immobilized by agarose-based microwell quickly,and express enzyme freely.The corresponding positive clones can be detected and screened by fluorescence.(2)A novel PDMS porous material was designed for microbial immobilization.The * This work was supported by the National Natural Science Foundation of China(21775049,31700746)and the Fundamental Research Funds for the Central Universities(HUST: 2018JYCXJJ044).porous material was prepared by polymerizing emulsion,in which the paraffin oil was used as diluent and emulsion water droplets act as porogen.We found that the paraffin oil could promote water emulsified in the PDMS solution and prevent emulsified water droplets to evaporate.After curing,a PDMS porous material with a uniform microstructure was obtained.The results indicated that the as-prepared PDMS porous material possessed great elasticity and porosity,could be used for efficient microbial immobilization.(3)A surface-structured PDMS porous material mold was fabricated by soft lithography technology,and used for bio-fabricating patterned BC.The PDMS porous material mold is introduced at the gas-liquid interface of the bacterial culture.Upon bacterial fermentation,the generated BC nanofibers were assembled at the structures of mold surface.The results shown that the BC with great pattern precision and thickness can be obtained by the patterned porous material mold.(4)Based on the wax-loss process,a hollow structure PDMS porous material mold was designed to control the self-assembly of BC nanofiber for customizable 3D structure BC bio-fabrication.The hollow PDMS porous material mold was introduced at the gas-liquid interface of the bacterial culture.Upon bacterial fermentation,the generated BC nanofibers were assembled at the structures of mold internal surface.The results shown that the BC with 3D structure such as ear or cervical vertebrae can be obtained by PDMS porous material mold.The 3D structure BC could be further used for organ transplantation researches.In summary,we developed an agarose-based microwell chip and PDMS-based porous materials to realize microbial high-throughput screening,efficient immobilization and structured BC fabrication.These attempts will provide more powerful tools for engineering applications of microorganisms.