New Bioanalytical Strategies Based On Microfluidic Flow Control

With advantages of low sample consumption,high integration,miniaturization,controllable flow,rapid analysis and satisfied biocompability,microfluidic technoogy has developed enormously both in research and industry,and has been applied in fields of biomedical diagnosis,environmental analysis and food safety monitoring.Flow control,which is the core of microfluidic,enables integrated analysis including sample input,reaction,separation and analysis in a single device.Traditional microfluidic-based flow control methods are based on optics,electricity,sound,magnetics,pressure,centrifugal force,gravity and capillarity,etc.However,traditional methods rely on external instruments to achieve precise flow control,which requires severe environments and professional users,thus limiting the extensive usage of microfluidic-based devices in real life.To this end,this thesis aims to develop simple and user-friendly microfluidic-based flow control methods and its applications in biomedical analysis.The main parts of this thesis are as follows:1.Capillarity controlled target responsive hydrogel combined with microfluidic paper-based analytical devices(μPADs)A capillarity controlled integrated target responsive hydrogel-μPAD was developed,where hydrogels trapped with enzymes were used for molecular recognition,and ptPADs enabled signal transduction and amplification.A capillary device was designed for integration of hydrogels and μPADs,and visualized assays were performed based on colorimetry.Moreover,μPADs with parallel detection zones were designed,allowing multiplex sample-in-answer-out analysis.In consideration of cost-effectiveness,user-friendliness and portability,the hydrogel-μPAD system shows potential in fields of telemedicine,health monitoring and environmental analysis.2.Gravity and capillarity controlled distance-based origami μPADsGravity and capillarity controlled dstance-based origami μPADs were developed for detection of small molecules and proteins.Functionalized beads were used for target recognition,signal transduction and amplification was based on "one-step" enzymatic cascaded reactions.Moreover,distance-based signal output was performed,and integrated analysis was achieved simply by folding and flipping of μPADs.In view of small molecules analysis,a probe separation strategy based on size difference was developed.Upon target introduction,it preferentially bound with the aptamer,releasing the probes immobilized on functionalized beads.Subsequently,released probes were transferred to detection zones to initiate integrated analysis,whereas functionalized beads with larger sizes than cellulose pores were excluded.And the method only requires small amounts of samples(20 μL)to achieve visualized analysis within 30 min In view of protein analysis,ELISA was used for signal amplification,and feasibility was demonstrated by establishing the relationship between the target concentration and distance signals.Such method requires no software assistance and eliminates the influence of user interpretation variance.Morevoer,no off-chip performance is required,endowing sample-in-answer-out detection.Overall,the developed system shows combined merits of μPADs and distance-based methods,providing new platforms for rapid and visualized analysis.3.Differential fluid resistance-based microparticle dispenserA differential fluid resistance-based microparticle dispenser was developed for rmcroparticle isolation.Instead of acquiring driven forces from external devices,the developed dispenser was initiated by handheld injectors to achieve rapid and simple microparticle isolation based on differential fluid resistance.Taking beads and cells as models,the dispenser was demonstrated with advantages of high capture efficiency,high stability,user-friendliness,small batch effects and long recycled times.Morevoer,dispensers with multiple channels were also developed for parallel isolation of microparticles.Overall,the developed dispenser is simple and stable,thus holding practical potentials in single-cell analysis.4.Single-cell RNA sequencing platform based on the microparticle dispenserA single-cell RNA sequencing platform based on the dispenser was developed for efficient and simplified cell barcoding and sample preparation.96-well plates with isolated barcode beads were prepared by the dispenser,followed by single cell isolation via FACS,where rapid single cell barcoding was achieved.The platform requires no external instruments but a simple dispenser for cell barcoding,thus dramatically decreasing the total cost of single barcode bead isolation.Moreover,the combination of FACS simplifies the overall procedure of sample collection,transferration and pretreatment,thus largely preserved the intrinsic information of cells.Aside from using FACS for cell isolation,the dispenser also enables single cell transferration from trace amounts of samples especially for rare cells.Overal,the developed platform enables single-cell RNA sequencing with low technological thresholds,low costs and high flexibility.