AVS 72 Session BI-MoM: Biofilms, Biofouling, and Biomolecules at Interfaces

Nature utilizes various materials such as peptides, nucleotides, and lipids to create order within complex environments such as cell interiors. Imitating nature, we combine DNA nanotechnology with peptide nanotechnology, specifically peptide based liquid-liquid phase separations, to access advanced functionalities. Peptide-based liquid-liquid phase separated domains, also known as coacervates, are neither pure homogenous liquid phase nor a heterogenous aggregate, displaying varying degrees of order. Coacervates are of keen interest in cell biology, where they function as membraneless organelles, dynamically colocalizing enzymes and other biomolecules (e.g. RNA) to enhance translation and other operations. We recently demonstrated that sequestration of DNA biosensors within coacervates allows for i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. We have also found that coacervates protect DNA from nuclease degradation while subsequently allowing for release from the coacervate upon proper stimuli, which could have implications for nucleotide delivery applications. We hypothesize that the strong peptide-DNA interaction within the coacervates inhibits degradation and have preliminary evidence that this can be applied to other biological moieties, e.g. enzymes. In a third demonstration, enzymatic cascades are sequestered into coacervates improving product flux through the system by exploiting enzyme stabilization and intermediate channeling.

Here we demonstrate that both graphene oxide (GO)-coated and GO-impregnated bioreactors result in faster growth and more robust biofilm formation than uncoated bioreactors when inoculated with Marinobacter atlanticus. GO is a nanomaterial typically used as either an irreversible binding substrate to trap microbial cells or as an antimicrobial coating, often dependent on GO concentration. Here, GO was incorporated either prior to the final resin cure or as a coating to 3D-printed bioreactors in order to increase cellular adhesion, enhance biofilm formation, and ultimately, increase bioproduction capabilities. Initial assays showed dense biofilm formation on bioreactor segments coated 10x with 0.1 wt% GO-in-water, compared to other solutions. In contrast, crystal violet biofilm assays demonstrated maximum biofilm formation on segments coated only 1x, indicating that high GO concentrations transition from biofilm-promoting to antimicrobial. In bioreactor experiments, effluent oxygen concentrations and final protein concentrations demonstrated that M. atlanticus biofilms colonized and grew faster in GO-coated or GO-impregnated systems than in uncoated bioreactors. Recovery, defined as the highest effluent oxygen concentration measured immediately following inoculation, reached only 40.5±13.3% oxygen and 55.5±5.7% oxygen in GO-impregnated and GO-coated bioreactors, respectively, compared to uncoated bioreactors (89.6± 4.1% oxygen). Similarly, biofilms within GO-impregnated and GO-coated bioreactors grew to maximum biomass (here, measured as effluent oxygen concentration < 2%) in less time (3.5±0.8 h and 10.5±2.3 h, respectively) than biofilms within uncoated bioreactors (15.2±3.9 h). Additionally, the same total biomass (as measured by protein concentration) was achieved in half the time with GO-impregnated bioreactors than with uncoated bioreactors (50 h vs. 100 h). When M. atlanticus was inoculated into bioreactors with medium that did not contain trace minerals, biofilm growth was fastest in GO-coated bioreactors than in GO-impregnated or uncoated bioreactors, with maximum biomass achieved within 13.9±2.5 h. These results suggest that GO can promote microbial growth, potentially through electron transfer, as previously seen with GO-modified electrodes, although more research is necessary. These data indicate a complex relationship between GO concentration and microbial growth, which requires further investigation to improve and optimize GO coatings for enhanced microbial production.

Despite recent advances in medical device technology, seamless integration in biological environments remains a challenge. When interfacing with biological environments, devices are prone to the accumulation of fouling proteins, microorganisms, and biofilms that lead to inflammation, infections, foreign body response, and failure or rejection. Thus, there is an urgent need for long-lasting, highly-stable solutions to prevent biofouling to the wide range of materials, designs, and length scales that comprise medical devices. In this talk, we will present the design of zwitterionic polymer coatings as a bioinspired surface engineering approach to avoid the attachment of proteins, bacteria, and other microorganisms to soft devices. We have recently developed surface functionalization methods compatible with medical-grade silicones (which are typically inert and difficult to functionalize), as well as zwitterionic photoresin formulations compatible with 3D-printing technology, that we have implemented in soft anti-biofouling devices. We will demonstrate their application in two case studies: (i) zwitterionic polymer coatings in urinary catheters that reduce >99% bacterial attachment and the formation of biofilms, outperforming state-of-the-art commercial antimicrobial catheter devices, and (ii) 3D-printed microrobots that avoid immune recognition and clearance by macrophages, significantly prolonging the functional lifetime of medical microrobots.

Collagen is the most abundant protein in mammals and plays a critical role in tissue formation, stability, and mechanics, closely linked to its unique triple-helix structure. We used atomic force microscopy (AFM) and the surface forces apparatus (SFA) to study fibril-forming type I and III human collagen absorbed on atomically smooth mica surfaces. The two-dimensional contours of individual collagen molecules showed non-uniform curvature for both types, which persisted after drying the surface and dehydrating the molecules. This finding suggests that collagen either has an intrinsic three-dimensional curvature in solution, or it acquires a two-dimensional curvature due to a particularly strong interaction with the surface. The former case has direct relevance to the self-assembly and elasticity of collagen fibrils, whereas the latter has potential implications for biomaterial design and tissue engineering.

SFA measurements showed strong adhesion between a collagen-coated and an uncoated mica surface, and repulsion between equally coated surfaces, suggesting that collagen-mica interaction is mainly electrostatic. Moreover, AFM force measurements showed a large adhesive force for a single collagen molecule being peeled by one end from the mica surface. Interestingly, a collagen molecule could be pulled and stretched without failing to a contour length of up to 900 nm—nearly three times its native length—over thousands of stretching cycles until the triple-helix started unravelling. During this process, the alpha-chains slipped progressively, irreversibly, and almost entirely past each other, before being caught by strong physical interactions between overlapping chain ends. These findings indicate that a controlled slippage mechanism at the molecular scale underpins the exceptional toughness of collagen fibrils and collagen-rich tissues such as tendons and skin.