Study Of Three-Dimensional Dynamic Adhesion Behavior Of Bacteria On Polymeric Surfaces

Polymeric interfacial materials are everywhere in our daily life and industries.Indwelling venous catheters,reverse osmosis membranes in water treatments plants,and paints on ship hulls and other marine facilities are such examples.Generally,these materials suffer from the colonization of bacteria and other microorganisms in service,leading to low efficacy,increased maintenance cost,and even destruction of the engineering or life threatening in extreme cases.Study of bacterial adhesion on polymeric materials can help to understand the mechanisms behind the formation of biofouling and clinical infection,and to develop approaches to inhibit them.Bacterial adhesion on polymeric surface is largely determined by how they move and disperse close to the surface in three dimensions.To comprehensively characterize bacterial adhesion,we set up digital holographic microscopy(DHM)capable of tracking the three-dimensional(3-D)motion of bacteria in real time.Through extracting the location and profile of massive numbers of motile bacteria in the near-surface volume with a vertical depth of tens of micrometers from a single planar interference pattern,DHM allows non-invasive,real-time,and high-throughput observation of the 3-D dynamic behaviors and adhesion of bacteria,thus making up for the drawback of 2-D,static-state-only adhesion measurements by conventional microscopy.We designed and prepared a series of polymeric interfacial materials,whose surface properties are tunable in a de-coupled manner,aiming to set up linkages between surface properties and the 3-D behaviors and adhesion of bacteria.The main contents of this dissertation are as follows.(1)DHM set-up and calibration for the observation of the 3-D dynamic behaviors of microparticles and bacteria.We built the in-line transmission light path of DHM based on a cage system,and recorded the holograms of particles and bacteria.A numerical reconstruction algorithm compiled based on the convolution theory and the Rayleigh-Sommerfeld propagation function was used to reconstruct the scattering light field and to refocus on the sample.DHM was calibrated,and its computation parameters were optimized for the tracking of certain bacterial targets.(2)DHM study on bacterial adhesion on polymeric surfaces.We prepared polymer brushes with varied surface charge property and hydrophobicity via surface-initiated atom transfer radical polymerization(SI-ATRP).By using DHM,we investigated the effect of surface properties on the spatial distribution,3-D velocity,tumbling frequency,and surface collision of Escherichia coli.Atomic force microscopy(AFM)was used to measure the adhesion force between bacteria and the surfaces.It reveals that the bacterial adhesion rate is positively correlated to the adhesion force.Accordingly,hydrophobic interactions reduce the swimming speed of E.coli upon colliding with the surface,and promote bacterial adhesion.(3)Anti-biofouling mechanism of dynamics surfaces.We fabricated dynamics surfaces consisting of poly(e-caprolatone)(PCL)based homo-and copolymers with varied degradation rates.The 3-D motion of E.coli and marine Pseudomonas sp.upon the polymeric surfaces were monitored by DHM,and the effects of degradation rate on the spatial distribution,motion orientation,and tumbling frequency were analyzed.AFM was used to measure the microscale interaction forces between bacteria and the surfaces.It reveals that the dynamic surface with a higher degradation can reduce the bacterial adhesion force,and the low molecular weight compounds generated by the degradation cause E.coli to present chemotactic repellence and escape from the surface.Thus,triggering the active responsive behavior in bacteria is probably a novel strategy towards reducing bacterial adhesion and biofouling.(4)Adhesion of bacteria on micropatterned polymeric surfaces.We used soft lithography together with surface chemistry modification to fabricate micropatterned polyacrylamide(PAAm)brushes and poly(ethylene glycol)diacrylate(PEGDA)hydrogel microwell arrays.The materials exhibited tuned surface hydrophobicity in the microscale,rendering 2-D or 3-D adhesion-promoting microenvironments.The micropatterned surfaces were used to achieve controlled adhesion of Pseudomonas fluorescens,where the 3-D microwell array was further used for the segregated incubation of bacterial biofilms.The micropatterned surfaces can be used to study chemical communication between bacterial microcolonies or biofilms.By using DHM with unique 3-D real-time imaging capability,we restore the near-surface motion and consequent adhesion of bacteria as a unified process,shedding light on a blind area left by conventional 2-D static state adhesion assessments.Our studies demonstrate that the physico-chemical properties of surfaces profoundly influence bacterial adhesion through shortrange surface collisions,and the effect of active response of bacteria cannot be neglected,which markedly alters bacterial adhesion via affecting their near-surface behaviors.3-D dynamic monitoring of bacterial adhesion makes significance for the design and fabrication of antibacterial and anti-biofouling materials.