Data-driven statistical modeling of the emergent behavior of biohybrid microrobots.

Multi-agent biohybrid microrobotic systems, owing to their small size and distributed nature, offer powerful solutions to challenges in biomedicine, bioremediation, and biosensing. Synthetic biology enables programmed emergent behaviors in the biotic component of biohybrid machines, expounding vast potential benefits for building biohybrid swarms with sophisticated control schemes. The design of synthetic genetic circuits tailored toward specific performance characteristics is an iterative process that relies on experimental characterization of spatially homogeneous engineered cell suspensions. However, biohybrid systems often distribute heterogeneously in complex environments, which will alter circuit performance. Thus, there is a critically unmet need for simple predictive models that describe emergent behaviors of biohybrid systems to inform synthetic gene circuit design. Here, we report a data-driven statistical model for computationally efficient recapitulation of the motility dynamics of two types of Escherichia coli bacteria-based biohybrid swarms-NanoBEADS and BacteriaBots. The statistical model was coupled with a computational model of cooperative gene expression, known as quorum sensing (QS). We determined differences in timescales for programmed emergent behavior in BacteriaBots and NanoBEADS swarms, using bacteria as a comparative baseline. We show that agent localization and genetic circuit sensitivity strongly influence the timeframe and the robustness of the emergent behavior in both systems. Finally, we use our model to design a QS-based decentralized control scheme wherein agents make independent decisions based on their interaction with other agents and the local environment. We show that synergistic integration of synthetic biology and predictive modeling is requisite for the efficient development of biohybrid systems with robust emergent behaviors.

Construction of Bacteria-Based Cargo Carriers for Targeted Cancer Therapy.

Despite significant recent progress in nanomedicine, drug delivery to solid tumors remains a formidable challenge often associated with low delivery efficiency and limited penetration of the drug in poorly vascularized regions of solid tumors. Attenuated strains of facultative anaerobes have been demonstrated to have exceptionally high selectivity to primary tumors and metastatic cancer, a good safety profile, and superior intratumoral penetration performance. However, bacteria have rarely been able to completely inhibit tumor growth in immunocompetent hosts solely by their presence in the tumor. We have developed a Nanoscale Bacteria-Enabled Autonomous Drug Delivery System (NanoBEADS) in which the functional capabilities of tumor-targeting bacteria are interfaced with chemotherapeutic-loaded nanoparticles, an approach that would amplify the therapeutic potential of both modalities. Here, we describe two biomanufacturing techniques to construct NanoBEADS by linking different bacterial species with polymeric theranostic vehicles. NanoBEADS are envisioned to significantly impact current practices in cancer theranostics through improved targeting and intratumoral transport properties.

Development of an Optical Nanosensor Incorporating a pH-Sensitive Quencher Dye for Potassium Imaging.

One of the key challenges in the design of a sensor for measuring extracellular changes in potassium concentration is selectivity against the competing cation, sodium. Here, we present an optode-based nanosensor selective to potassium ions, owing to the addition of a pH-sensitive quencher molecule paired with a static fluorophore. The nanosensor was fabricated using emulsification and characterized in solution by absorbance and fluorescence spectroscopy. The resulting nanosensor detected potassium with nearly 1 order of magnitude higher selectivity compared to our chromoionophore-based optode nanosensors. In addition to the improved selectivity, the nanosensor has the following properties required for measurements in a biological environment: (1) a physiologically relevant dynamic range, (2) response to potassium ions at a physiological ionic strength, and (3) response to serum potassium in the presence of fouling biological components. The potassium nanosensor described in this study is envisioned to have application in cellular imaging and drug screening.

3D Insulator-based dielectrophoresis using DC-biased, AC electric fields for selective bacterial trapping.

Insulator-based dielectrophoresis (iDEP) is a well-known technique that harnesses electric fields for separating, moving, and trapping biological particle samples. Recent work has shown that utilizing DC-biased AC electric fields can enhance the performance of iDEP devices. In this study, an iDEP device with 3D varying insulating structures analyzed in combination with DC biased AC fields is presented for the first time. Using our unique reactive ion etch lag, the mold for the 3D microfluidic chip is created with a photolithographic mask. The 3D iDEP devices, whose largest dimensions are 1 cm long, 0.18 cm wide, and 90 µm deep are then rapidly fabricated by curing a PDMS polymer in the glass mold. The 3D nature of the insulating microstructures allows for high trapping efficiency at potentials as low as 200 Vpp. In this work, separation of Escherichia coli from 1 µm beads and selective trapping of live Staphylococcus aureus cells from dead S. aureus cells is demonstrated. This is the first reported use of DC-biased AC fields to selectively trap bacteria in 3D iDEP microfluidic device and to efficiently separate particles where selectivity of DC iDEP is limited.

Toward development of an autonomous network of bacteria-based delivery systems (BacteriaBots): spatiotemporally high-throughput characterization of bacterial quorum-sensing response.

Characterization of bacterial innate and engineered cooperative behavior, regulated through chemical signaling in a process known as quorum sensing, is critical to development of a myriad of bacteria-enabled systems including biohybrid drug delivery systems and biohybrid mobile sensor networks. Here, we demonstrate, for the first time, that microfluidic diffusive mixers can be used for spatiotemporally high-throughput characterization of bacterial quorum-sensing response. Using this batch characterization method, the quorum-sensing response in Escherichia coli MG1655, transformed with a truncated lux operon from Vibrio fischeri, in the presence of 1-100 nM exogenous acyl-homoserine lactone molecules has been quantified. This method provides a rapid and facile tool for high-throughput characterization of the quorum-sensing response of genetically modified bacteria in the presence of a wide concentration range of signaling molecules with a precision of ±0.5 nM. Furthermore, the quorum-sensing response of BacteriaBots has been characterized to determine if the results obtained from a large bacterial population can serve as a robust predictive tool for the small bacterial population attached to each BacteriaBot.

Directed transport of bacteria-based drug delivery vehicles: bacterial chemotaxis dominates particle shape.

Several attenuated and non-pathogenic bacterial species have been demonstrated to actively target diseased sites and successfully deliver plasmid DNA, proteins and other therapeutic agents into mammalian cells. These disease-targeting bacteria can be employed for targeted delivery of therapeutic and imaging cargos in the form of a bio-hybrid system. The bio-hybrid drug delivery system constructed here is comprised of motile Escherichia coli MG1655 bacteria and elliptical disk-shaped polymeric microparticles. The transport direction for these vehicles can be controlled through biased random walk of the attached bacteria in presence of chemoattractant gradients in a process known as chemotaxis. In this work, we utilize a diffusion-based microfluidic platform to establish steady linear concentration gradients of a chemoattractant and investigate the roles of chemotaxis and geometry in transport of bio-hybrid drug delivery vehicles. Our experimental results demonstrate for the first time that bacterial chemotactic response dominates the effect of body shape in extravascular transport; thus, the non-spherical system could be more favorable for drug delivery applications owing to the known benefits of using non-spherical particles for vascular transport (e.g. relatively long circulation time).

Antimicrobial surfaces using covalently bound polyallylamine.

We investigated the antimicrobial properties of the cationic polymer polyallylamine (PA) when covalently bonded to glass. The objective was to obtain a robust attachment, yet still allow extension of the polymer chain into solution to enable interaction with the bacteria. The PA film displayed strong antimicrobial activity against Staphylococcus epidermidis , Staphylococcus aureus , and Pseudomonas aeruginosa , which includes both Gram-positive and Gram-negative bacteria. Glass surfaces were prepared by a straightforward two-step procedure of first functionalizing with epoxide groups using 3-glycidoxypropyltrimethoxy silane (GOPTS) and then exposing to PA so that the PA could bind via reaction of a fraction of its amine groups. The surfaces were characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy to verify the presence of the polymer on the surface, zeta potential measurements to estimate the surface charge of the films, and atomic force microscopy to determine the extension of the polymer chains into solution. Antimicrobial properties of these coatings were evaluated by spraying aqueous suspensions of bacteria on the functionalized glass slides, incubating them under agar, and counting the number of surviving cell colonies.

Embedded passivated-electrode insulator-based dielectrophoresis (EπDEP).

Here, we introduce a new technique called embedded passivated-electrode insulator-based dielectrophoresis (EπDEP) for preconcentration, separation, or enrichment of bioparticles, including living cells. This new method combines traditional electrode-based DEP and insulator-based DEP with the objective of enhancing the electric field strength and capture efficiency within the microfluidic channel while alleviating direct contact between the electrode and the fluid. The EπDEP chip contains embedded electrodes within the microfluidic channel covered by a thin passivation layer of only 4 µm. The channel was designed with two nonaligned vertical columns of insulated microposts (200 µm diameter, 50 µm spacing) located between the electrodes (600 µm wide, 600 µm horizontal spacing) to generate nonuniform electric field lines to concentrate cells while maintaining steady flow in the channel. The performance of the chip was demonstrated using Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial pathogens in aqueous media. Trapping efficiencies of 100% were obtained for both pathogens at an applied AC voltage of 50 V peak-to-peak and flow rates as high as 10 µl/min.

Off-chip passivated-electrode, insulator-based dielectrophoresis (OπDEP).

In this study, we report the first off-chip passivated-electrode, insulator-based dielectrophoresis microchip (OπDEP). This technique combines the sensitivity of electrode-based dielectrophoresis (eDEP) with the high-throughput and inexpensive device characteristics of insulator-based dielectrophoresis (iDEP). The device is composed of a permanent, reusable set of electrodes and a disposable, polymer microfluidic chip with microposts embedded in the microchannel. The device operates by capacitively coupling the electric fields into the microchannel; thus, no physical connections are made between the electrodes and the microfluidic device. During operation, the polydimethylsiloxan (PDMS) microfluidic chip fits onto the electrode substrate as a disposable cartridge. OπDEP uses insulting structures within the channel as well as parallel electrodes to create DEP forces by the same working principle that iDEP devices use. The resulting devices create DEP forces which are larger by two orders of magnitude for the same applied voltage when compared to off-chip eDEP designs from literature, which rely on parallel electrodes alone to produce the DEP forces. The larger DEP forces allow the OπDEP device to operate at high flow rates exceeding 1 mL/h. In order to demonstrate this technology, Escherichia coli (E. coli), a known waterborne pathogen, was trapped from water samples. Trapping efficiencies of 100% were obtained at flow rates as high as 400 µL/h and 60% at flow rates as high as 1200 µL/h. Additionally, bacteria were selectively concentrated from a suspension of polystyrene beads.

Effect of body shape on the motile behavior of bacteria-powered swimming microrobots (BacteriaBots).

Swimming microrobots are envisioned to impact minimally invasive diagnosis, localized treatment of diseases, and environmental monitoring. Dynamics of micro-scale swimming robots falls in the realm of low Reynolds number, where viscous forces exerted on the robots are dominant over inertia. Viscous forces developed at the interface of the swimming microrobots and the surrounding fluid are a strong function of the body geometry. In this work, a collection of bacteria-powered micro-robots (BacteriaBots) with prolate spheroid, barrel, and bullet-shaped bodies is fabricated and the influence of body shape on the dynamics of the BacteriaBots is investigated. We have experimentally demonstrated that using non-spherical geometries increases the mean directionality of the motion of the BacteriaBots but does not significantly affect their average speed compared with their spherical counterparts. We have also demonstrated that directionality of non-spherical BacteriaBots depends on the aspect ratio of the body and for the case of prolate spheroid, a higher aspect ratio of two led to a larger directionality compared to their low aspect ratio counterparts.

Selective E. coli trapping with 3D insulator-based dielectrophoresis using DC-biased, AC electric fields.

We present the development of a batch trapping, insulator-based dielectrophoretic (iDEP) device with three-dimensional design. The microfluidic devices use DC-biased, AC electric fields to selectively manipulate biological particles based on their electric properties. The mold for the polymer microdevices is fabricated using an RIE-lag technique which creates microchannels with varying depths using a single etch process. The resulting three-dimensional insulating constrictions permit operation at low applied voltages. By varying both the applied frequency and the ratio of AC to DC electric fields, the iDEP device can trap and separate polystyrene beads and E. coli cells.

Enhanced directionality of bio-hybrid mobile microrobots using non-spherical body geometries.

Mobile microrobots are envisioned to be employed for several applications including drug delivery, diagnostic imaging and environmental monitoring. In the bio-hybrid microrobot that is presented here, microparticles are used as the body of the microrobot and bacterial cells are utilized to realize on-board actuation. In this work, the importance of body shape on the dynamics of bacteria-propelled swimming microrobots (BacteriaBots) is investigated. We have shown that, with the use of non-spherical microparticles, average directionality of the BacteriaBots is enhanced compared with the spherical BacteriaBots.

Computational and experimental study of chemotaxis of an ensemble of bacteria attached to a microbead.

Micro-objects propelled by whole cell actuators, such as flagellated bacteria, are being increasingly studied and considered for a wide variety of applications. In this work we present theoretical and experimental investigations of chemotactic motility of a 10 µm diameter microbead propelled by an ensemble of attached flagellated bacteria. The stochastic model presented here encompasses the behavior of each individual bacterium attached to the microbead in a spatiotemporally varying chemoattractant field. The computational model shows that in a chemotactic environment, the ensemble of bacteria, although constrained, propel the bead in a chemotactic manner with a 67% enhancement in displacement to distance ratio (defined as directionality) compared to nonchemotactic propulsion. The simulation results are validated experimentally. Close agreement between theory and experiments demonstrates the possibility of using the presented model as a predictive tool for other similar biohybrid systems.