Single-cell Microorganism@Nanomaterials Composite Microspheres For Liquid Biochips

Liquid biochip-based flow cytometry is an innovative analysis technique by using microspheres as reaction carriers,which can achieve highly sensitive and quantitative detection of one to hundreds of biomolecules.It has extensive application prospects in basic and applied research fields such as biology,medicine and the environment.Thus,it is important to develop a large number of eco-friendly,simple,and low-cost functional microspheres with excellent performance and uniformity for liquid biochips analysis in basic research and practical applications.Based on the above insights,this thesis proposed two methods of functional microspheres synthesis by using single-cell microorganisms.The microorganisms@nanomaterials composites were successfully used for the detection of biomolecules:First,a simple,rapid,inexpensive,eco-friendly,and high-throughput biological strategy for the preparation of functional microspheres on a yeast-cell platform was introduced.Microspheres prepared through the treatment of yeast cells with formaldehyde and decoating buffer exhibited excellent characteristics,such as superior mechanical strength,high sulfhydryl group content,and mesoporous structure.Colloidal gold nanoparticles?Au NPs?rapidly self-assembled onto the surfaces of the yeast-based microspheres?YMs?within 5 min to form rigid yeast@Au microspheres with high monodispersity and uniformity.The yeast@Au microspheres were characterized by TEM,SEM,EDX,HR-TEM,AFM,fluorescence microscope,nitrogen adsorption-desorption,potentiometric titration,XRD,FTIR,Raman spectroscopy and XPS,indicating that the rapid formation of yeast@Au microspheres is mainly due to enhanced sulfhydryl groups and mesoporosity.The yeast@Au microspheres were successfully used in a flow cytometry fluorescent immunoassay to detect pseudorabies viral infection events.The signal-to-noise ratio was enhanced by approximately 49.4-fold.The presence of Au NPs on the YMs also improved sensitivity by decreasing noise through reducing nonspecific adsorption,thus enhancing the fluorescence signal caused by the surface plasmon resonance effect,and increasing the coupling efficiency of the capture protein.The presented method was used to analyze81 clinical swine serum specimens and the results obtained were compared with those of commercial diagnostic kits.The sensitivity,specificity,and efficiency of the developed method were 92.31%,88.24%,and 88.89%,respectively.The excellent characteristics of the yeast@Au microspheres illustrate its great potential for high-throughput immunoassay applications in the fields of disease diagnosis,food safety,and environmental analysis.Second,Here,a novel class of microorganism@metal-organic framework composites was synthesized using a simple and efficient self-assembly approach on the surface of microorganisms?yeast,PCC 6803,and E.coli?with different shapes and sizes.We have successfully prepared a variety of high monodisperse and uniform microorganism@MOF composites.Microorganisms-based particles and microorganism@MOF composites were characterized by various methods?such as XRD,FE-SEM,EDX,nitrogen adsorption-desorption,zeta potential measurement,FTIR,and XPS?.Compared with the microorganisms-based particles,the specific surface area and pore size of the microorganism@MOF composites is significantly increased,and functional groups?such as carboxyl,amino,hydroxyl,and sulfhydryl groups?on the surface of the microorganisms may participate in the formation of composites.Both results of FTIR and XPS revealed that the formation of the composites was a chemical self-assembly process.The yeast@Ui O-66-NH₂,PCC6803@Ui O-66-NH₂ and E.coli@Ui O-66-NH₂ with different sizes could be clearly detected by flow cytometer due to the correlation between the forward scatter signals and as-prepared composites sizes.Then we developed a method for the detection of multiple micro RNAs?mi RNAs?based on these as-prepared composites.The proposed method allowed simultaneous detection of mi RNA-21,mi RNA-182,and mi RNA-17 in the ranges 0.75–1500 pmol L^–1,0.3–500 pmol L^–1,and 0.25–550 pmol L^–1,respectively.The limit of the detection for mi RNA-21,mi RNA-182,and mi RNA-17was measured to be 0.75 pmol L^–1,0.30 pmol L^–1,and 0.25 pmol L^–1,respectively.These results demonstrated high sensitivity,specificity and repeatability in the detection and analysis of colon cancer-related mi RNAs,indicating that the proposed method has potential application prospects in the biomedical field.In summary,the conclusions of advantages in our work are as follows:1)The single cell microorganisms are good carriers for the preparation of functional microspheres,and their advantages include easy operation,low cost,and eco-friendly.2)The microorganisms-based functional microspheres have good performance and are successfully used in the analysis and determination of liquid biochips,which has good research value and application prospects in the field of basic and applied research.