(2ea) Autonomous Bioelectronic Systems for Multiplex Functions

Research Interests: My future research will be focused on the rational design of autonomous bioelectronics. Both biology and electronics use electrons as information and energy carriers. The disparate capabilities of biological and microelectronics made the combination an extremely attractive technology. Combining biology and microelectrodes as sense-and-respond systems, the sensing information generated by microbes can be encoded into electrical signals received by microelectronics. The integration of synthetic engineered electron transfer pathways into non-native electroactive microbes empowers them to function as bioelectronics. However, one of the challenges encountered in current bioelectronic systems is the dominance of planktonic cells, which results in low biomass concentration and inefficient electron transfer processes. To address the issue, encapsulating bacteria with electrodes were utilized to improve the electron transfer efficiency by increasing biomass density. However, the use of hydrogel for encapsulation forms a thick layer, in order of mili-meters, which limits the mass transfer. Additionally, the current encapsulation techniques mainly focus on simple sealing without incorporating structure design, resulting in a lack of manipulation variables and over-reliance on biological functional design. As a result, the current status of bioelectronics exhibits inefficiency, simplistic structure and limited functionality. Thus, there is a need to have a new phenotype of microorganisms as concentrated biomass for efficient electron transfer and development of diverse structures with more functions. My goal is to create a novel self-assembled electroactive biofilm as a bioelectronics platform, incorporating geometric patterning and offering the capability for multiplex sensing and therapeutic functions.

PREVIOUS ACCOMPLISHMENTS

ASSESSING EXTRACELLULAR ELECTRON TRANSFER ACROSS BIOFILM. Electroactive bacteria ‘catalyze’ bioelectrochemical reactions and can form electronically conductive biofilms on polarized electrodes. Central to their catalytic properties is the extracellular electron transport across the EAB to deliver electrons to the polarized electrode surface, but the underlying bio-molecular mechanisms are still largely under debate. To better understand, quantify and optimize the electron transfer process, I developed a double potential steps chronoamperometry to rapidly monitor and quantify charge transport across Geobacter-dominated anodic EABs. The values of charge transport parameters were comparable with those of pure Geobacter biofilms or abiotic redox polymers. This electrochemical approach opens the possible in vivo assessment of the electron transfer process across variable electron transfer capable biofilms.

PERIODIC POLARIZATION BOOST ELECTRO- ACTIVE BIOFILM ACTIVITY. Low current densities have been a longstanding challenge in the microbial fuel cell or biosensors field. Most research to increase energy production focuses on electrode materials modification or genetic engineering microbes. I develop a novel periodic polarization to significantly improve the energy production of the electroactive biofilms (EAB). The EAB enhancement was reversible in only a few days after polarization modes were shifted. Periodic polarizations also impacted biofilm adhesion and current production under substrate-limiting conditions. We investigated the polarization signal to optimize the EAB electroactivity and the rate of charge production. This electrochemical engineering of EAB opens new routes for enhancing the performance of microbial electrochemical systems.

MULTICHANNEL BIOELECTRONIC SENSOR. By engineering extracellular electron transfer to be dependent on an analyte, researchers have developed whole cell bioelectronic sensors that sense hazards to human and environmental health. However, these sensors regulate a single electron transfer pathway as an electrochemical channel, limiting the sensing information to a single analyte. To increase information content, I developed a multichannel bioelectronic sensor through which different chemicals regulate distinct extracellular electron transfer pathways in Escherichia coli. Then we demonstrate heavy metal responsive promoters to control the two EET processes, respectively. We developed a redox-potential-dependent, which allows us to distinguish variable input signals of each analyte mediated by two EET pathways in vivo. This approach enables a 2-bit signal readout for real-time tracking throughout the entire sensing duration. The multichannel bioelectronic sensors allow for simultaneously detect different chemicals and expand information transmission helping safeguard human and environmental health.

Future research overview. The goal of my future research is to create a novel autonomous bioelectronics, incorporating geometric patterning and offering the capability for multiplex sensing and therapeutic functions. To accomplish this goal, my plan involves the development of synthetic adhesive electroactive bacteria and manipulation of their structures from single-dimensional to multiple-dimensional configurations (Aim1). I will explore novel biofilm lithography techniques to form geometric patterns on conductive surfaces, effectively creating a bio-integrate-circuit (Aim2). I will initially target probiotic Escherichia coli and Bacillus subtilis, promising chassises for applications in the human body, plants and foods. This work will develop a comprehensive platform that enables the intelligent assembly of various bioelectronic components, facilitating the creation of tailored functionalities to meet diverse requirements.

AIM 1: SYNTHETIC PROGRAM ARTIFICIAL ELECTROACTIVE BIOFILMS.

Motivation. Electroactive biofilm, which contains concentrated biomass, offers effective extracellular electron transfer at the interface between EAB and conductive materials. However, non-native exoelectrogens, such as E.coli and B.subtilis, predominantly exist as planktonic cells, resulting in low biomass density and inefficient electron transfer. To address this challenge, adhesive protein expression can enhance cell attachment to a solid surface, thereby increasing biomass density and decreasing the electron transfer distance. Integrating the adhesive protein into engineered EET capable cells, forming an artificial electroactive biofilm, could enhance the efficiency of the electron transfer process, to enable the future ‘biofilm lithography’ techniques. Strategy. To construct a self-assembled artificial biofilm, I will initially target on introducing several reported adhesive proteins into variable bacteria to construct self-assembled artificial EABs. To integrate the physical adhesive properties with the biological electron transfer process, I will construct an individual and combined-genetic circuit control for gene expression. To facilitate the biofilm formation, I will apply different electrochemical programs to the conductive surface to form variable biofilm morphology. Microscopy will be used to visualize the biofilm structures. Electrochemical analysis will be used to assess the electron transfer process across artificial biofilm matrices, which will also be associated with different EET mechanisms. Impact. This aim will facilitate the identification of suitable aggregation proteins for the assembled artificial biofilms for various microbes and microbiota. The fundamental knowledge on how cell phenotype influences synthetic electron transfer process, will open new opportunities to utilize engineered EET capable bacteria into bioelectronic applications not only stick to energy production. With more knowledge on the integration between variable microbes and conductive surfaces, this aim will benefit on potentially exploring microbiota EET processes.

AIM 2: GEOMETRIC PATTERNING BIO-INTEGRATE-CIRCUIT

Motivation: Planktonic cells perform as dimension less, while artificial biofilm form single/multi-layer biofilm as single-dimensional structure. The single-dimensional structure carries limited information capacities. Analogy to integrate circuits, with geometric mapping of individual electrical components can be integrated into a complex system. To achieve elaborate functional bioelectronics, geometric patterning the EAB into different structures opens the possibility and complexity of using each EAB with different functions. Strategy: The previous strategy to attach cells from biofilm in simple structures without considering materials aspect application, could severely limit the functions of those artificial EABs. I will apply a parallel approach from artificial EAB formation, based on conductive surface modifications with genetic modified adhesive proteins. For this purpose, I will develop different electrode surface modification for cell attaching enhancement or decrease. And also I will integrate the adhesive protein circuit with several different regulation systems, which allows the formation of the biofilm to appear in different geometric patterns, and characterize the electrical signal outputs via different structures. Then I will optimize the programmable biofilm based on specific needs. Due to the small size of microbes, the spatial resolution of EAB could be really small, which might make pattern-EAB working as electrical devices, IDA or OECT (geometric application). Additionally, artificial biofilm as living bioelectronic materials, I will characterize the mechanical properties of different structures with rheology analysis. Impact: Physically diversifying the EAB could create living-electrical devices, which offers numerous advantages in biomaterial properties, such as non-toxic, reversible and genetically programmable.

TEACHING INTEREST: As an international student who has studied in Europe and being a postdoc here in the US, I have had diverse experiences in both learning and teaching. This has made me a keen observer of my teachers and students. I observe their approaches, think about their teaching methods, and assess which methods enhance my own learning. Through my experience teaching microbiology lab classes, I made a conscious effort to take notes on questions and the common challenges they faced. This allowed me to accumulate a valuable collection of effective teaching techniques through careful analysis. A well-organized content helps students quickly grasp the overall structure and better understand the connections between different pieces of information. Using relatable examples, such as those from everyday life or the human body, can make learning more engaging and facilitate concept comprehension. I have also found interpretation is a powerful tool for memorizing knowledge. Rather than simply repeating information, interpreting and expressing unfamiliar concepts in our own words enhances understanding. Lastly, I highly value the encouragement and support I received from my previous teachers as a student. A supportive environment helps students build confidence in their learning journey, as getting started can be challenging.