Electroaddressing Functionalized Polysaccharides as Model Biofilms for Interrogating Cell Signaling

Abstract

Bacteria often reside at surfaces as complex biofilms in which an exopolysaccharide matrix entraps the population while allowing access to its chemical environment. There is a growing awareness that the biofilm structure and activity are integral to a wide array of properties important to health (the microbiome), disease (drug resistance) and technology (fouling). Despite the importance of bacterial biofilms, few experimental platforms and systems are available to assemble complex populations and monitor their activities. Here, a functionalized alginate composite material for creating in vitro model biofilms suitable for cell-cell signaling studies by entrapping bacterial cells in situ is reported. Biofilm assembly is achieved using device-imposed electrical signals to electrodeposit the stimuli-responsive polysaccharide alginate. This electrodeposition mechanism is versatile in that it allows control of the bacterial population density and distribution. For instance, it is demonstrated that a mixed population can be homogeneously distributed throughout the biofilm or can be assembled as spatially segregated populations within a stratified biofilm. The "electroaddressable" biofilms are visualized using both a planar 2D chip with patterned electrodes and a microfluidic bioMEMS device with sidewall electrodes. Specifically, it is observed that bacteria entrapped within the model biofilm recognize and respond to chemical stimuli imposed from the fluidic environment. Finally, reporter cells are used to demonstrate that bacteria entrapped within this model biofilm engage in intercellular quorum sensing. This work demonstrates the functionality of the stimuli-responsive polysaccharide by biofabricating pseudo-3D cell-gel biocomposites, mimicking the formation of biofilms, for interrogating phenotypes of E. coli bacterial populations. In addition to controlling assembly, the microfluidic device allows the biofilm to be monitored through the fluorescence methods commonly used in biological research. This platform technology should be able to be exploited for monitoring biofilm development, as well as for extending the understanding of the interactions between various bacterial species arranged in controlled patterns.

Chitosan to Connect Biology to Electronics: Fabricating the Bio-Device Interface and Communicating Across This Interface

“…We use chitosan primarily for the assembly of proteins to impart either selective binding properties [140] or enzymatic catalytic activities [141]. In contrast, when our goal is to assemble cells at electrode addresses, we generally favor alternative stimuli-responsive biopolymers such as alginate [78,[142][143][144][145][146], agarose [147] and gelatin [148]. Chitosan possesses several properties that facilitate protein assembly in/on an electrodeposited film through entrapment within the hydrogel network, covalent grafting to the polysaccharide backbone, or attachment through non-covalent associations.…”

Bridging the Bio-Electronic Interface with Biofabrication

“…"senders" and "receivers") at distinct addresses, and showed that they interacted across adjacent electrodes to deliver AI-2 and generate a fluorescence response. This concept has also been demonstrated by Cheng et al in a microfluidic chip 14 . We also mimicked the interaction, but instead used an enzyme to synthesize AI-2 for delivery.…”

Using a Redox Modality to Connect Synthetic Biology to Electronics: Hydrogel‐Based Chemo‐Electro Signal Transduction for Molecular Communication

“…First is the physical integration of these labile biological components into a functional system. Traditionally, integrated circuits are fabricated using thin film technologies and recent efforts are extending such technologies to the programmable assembly of biologically compatible hydrogels . Here, we electro‐assemble a dual hydrogel film system at an electrode surface.…”

“…After completing these electrofabrication steps, the dual film coating was stabilized by incubation in 1% CaCl 2 solution for 15 min (i.e., this step hardens the Ca 2+ ‐alginate hydrogel). Importantly, these electrofabrication steps are performed under sufficiently mild conditions to retain viability of the entrapped synbio components (e.g., E. coli cells) in the alginate hydrogel (see Figure S4, Supporting Information).…”