Name: Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors (Video) | JoVE Journal
Uploaded: 2025-01-31T15:00:00.000+00:00
Duration: 10 min 44 s
Description: Learn how organic electrochemical transistors translate extracellular electron transfer activity in Shewanella oneidensis into measurable electrical signals for bioelectronics research.

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Base plasmids for the NAND circuit were generously provided by the Voigt Lab via Addgene (#49375, #49376, #49377). This research was financially supported by the Welch Foundation (Grant F-1929, B.K.K.), the National Institutes of Health under award number R35GM133640 (B.K.K.), an NSF CAREER award (1944334, B.K.K.), and the Air Force Office of Scientific Research under award number FA9550-20-1-0088 (B.K.K.). A.J.G. was supported through a National Science Foundation Graduate Research Fellowships (Program Award No. DGE-1610403). The authors acknowledge the use of shared research facilities supported in part by the Texas Materials Institute, the Center for Dynamics and Control of Materials: an NSF MRSEC (DMR-1720595), and the NSF National Nanotechnology Coordinated Infrastructure (ECCS-1542159). We gratefully acknowledge the use of facilities within the core microscopy lab of the Institute for Cellular and Molecular Biology, University of Texas at Austin.

Measuring Electron Transfer Activity in Shewanella oneidensis Using OECTs

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<li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hybrid+transistor+with+transcriptionally+controlled+computation+and+plasticity%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">A hybrid transistor with transcriptionally controlled computation and plasticity.</a> Nat Commun. 15 (1), 1598(2024).</li>
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The authors declare no competing interests.

Electrochemical Cell (EC) Comparison 
One major advantage of fitting the rate constant from the OECT channel current is that it minimizes device variation by focusing on the underlying dynamics of IDS changes rather than the raw output. Combined with the inherent signal amplification capability of OECTs, this approach enhances the robustness of hybrid OECT systems compared to traditional electrochemical cells (EC). For example, at the end of each 24-hour OECT experiment, transfer curves a...

Devices that can transduce and amplify biological and chemical activities into electrical signals are crucial across various fields, such as sensing1,2, neuromorphic computing3,4, and wearable electronics5. Among these, organic electrochemical transistors (OECTs) have emerged as exceptional interfaces between biological systems and electronic readouts due to their compatibility with aqueous environments and low operating voltages6,7. OECTs differ from conventional electronics by utilizing ions in an electrolyte to modulate the conductivity of an organic channel, which couples ionic and electronic transport to achieve exceptional transconductance8. These characteristics make OECTs ideal for interfacing biological systems with electronics, as they can amplify weak biological signals and translate them into electrical readouts.
OECTs operate by altering the doping state of a mixed ionic-electronic conducting channel through ionic diffusion, typically controlled by applying a voltage at the gate electrode. However, biological or redox reactions can also change the channel&#39;s conductivity, enabling OECTs to respond to a variety of chemical and biological stimuli. Functionalizing OECTs with lipid bilayers, ion channels, or biomolecules allows them to detect specific analytes, making them useful for sensing applications9,10. For instance, OECTs have been integrated with redox-active enzymes like glucose oxidase to directly transfer electrons to the channel, tuning its conductivity in response to glucose concentration11. While such configurations are effective for biosensing, they are limited in their computational capacity due to the relatively simple behavior of individual enzymes or proteins.
In contrast, living cells, particularly bacteria, offer a versatile platform capable of performing complex and robust computations4,12,13,14,15. Electroactive bacteria such as Shewanella oneidensis and Geobacter sulfurreducens have the unique ability to transfer electrons across their cell membranes in a process known as extracellular electron transfer (EET). Under anaerobic conditions, these bacteria couple their metabolic processes to the reduction or oxidation of external electron acceptors, including metals, metal oxides, and synthetic materials like conducting polymers16 and nanoparticles17 (Figure 1A). This capability has been exploited in microbial fuel cells for power generation and offers the potential for more advanced bioelectronic applications18,19. Additionally, advancements in synthetic biology have enabled precise genetic manipulation of electroactive bacteria to control EET pathways. By engineering genetic circuits that regulate the expression of EET-related genes, researchers can modulate electron flux in response to specific environmental cues or computational logic operations20,21. This genetic control over EET opens avenues for creating bio-hybrid systems where bacterial computations are directly interfaced with electronic devices like OECTs. For example, bacterial genetic circuits could be designed to respond to combinations of chemical inputs, turning on or off EET pathways and thereby modulating the conductivity of an OECT channel. This would allow for direct electrical readouts of bacterial computations, bypassing the need for fluorescent or other traditional biological reporters.
Recent advances have demonstrated the potential of coupling OECTs with electroactive bacteria. For example, M&#233;hes et al. used S. oneidensis to monitor real-time EET activity with a p-type OECT, illustrating how bacterial metabolism could be tracked electrically22. While this work highlights the possibility of using OECTs to detect bacterial activity, the potential for the hybrid system for biosensing and biocomputing remains underexplored. To address this, we recently developed hybrid transistors incorporating genetically engineered S. oneidensis into p-type OECTs23. The results demonstrated that the OECT channel could be de-doped through bacterial EET activities (Figure 1B). To further enhance biosensing capabilities and gain mechanistic insights into the de-doping process, we engineered S. oneidensis strains with genetic circuits that regulate EET flux. This results in predictable OECT output changes in response to environmental cues such as inducer molecules. Moreover, by integrating Boolean logic into these genetic circuits, we enabled direct electrical readouts of complex bacterial computations.
Here, we present the comprehensive protocol for the hybrid transistor operation, covering the device fabrication, cell culture preparation, measurement procedures, and data analysis. Additionally, we address key considerations such as device cleaning, reusability, and the potential for automation in high-throughput testing.

<table><tbody><tr><td>3-oxohexanoyl-homoserine lactone&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>anhydrotetracycline hydrochloride&amp;nbsp;</td><td>VWR</td><td></td><td></td></tr><tr><td>casamino acids&amp;nbsp;</td><td>VWR</td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Ethylene glycol&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td>anhydrous 99.8%,&amp;nbsp;</td></tr><tr><td>HEPES buffer solution&amp;nbsp;</td><td>VWR</td><td></td><td>1 M in water, pH = 7.3</td></tr><tr><td>isopropyl &amp;szlig;-D-1-thiogalactopyranoside&amp;nbsp;</td><td>Teknova</td><td></td><td></td></tr><tr><td>kanamycin sulfate&amp;nbsp;</td><td>Growcells</td><td></td><td></td></tr><tr><td>Magnesium(II) sulfate heptahydrate&amp;nbsp;</td><td>VWR</td><td></td><td></td></tr><tr><td>PEDOT:PSS aqueous suspension&amp;nbsp;</td><td>Heraeus Epurio LLC</td><td>Clevios PH1000</td><td></td></tr><tr><td>Potassium phosphate dibasic&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Potentiostat&amp;nbsp;</td><td>PalmSens BV</td><td>MultiPalmSens4</td><td></td></tr><tr><td>Quartz microscopic slides&amp;nbsp;</td><td>AdValue&amp;nbsp;</td><td>FQ-S-003</td><td></td></tr><tr><td>Quartz microscopic slides&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>VWR</td><td></td><td></td></tr><tr><td>Sodium DL-lactate&amp;nbsp;</td><td>TCI</td><td></td><td>60% in water</td></tr><tr><td>Sodium fumarate&amp;nbsp;</td><td>VWR</td><td></td><td>98%</td></tr><tr><td>Sulfuric acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td>95.0%-98.0%</td></tr><tr><td>Two-part silicone elastomer&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>Sylgard184</td><td></td></tr><tr><td>Wolfe&#039;s Trace Mineral Mix&amp;nbsp;</td><td>ATCC</td><td></td><td></td></tr></tbody></table>

translating extracellular electron transfer activities with organic electrochemical transistors

Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors

Extracellular electron transfer (EET) is a process through which certain microorganisms can transfer electrons across their cell membranes to external electron acceptors, linking cellular metabolism to their environment. While Geobacter and Shewanella have been the primary models for EET research, emerging studies reveal that EET-active species are also associated with fermentation and the human gut microbiome. Leveraging the ability of EET to bridge biological and electronic systems, we present a protocol for using organic electrochemical transistors (OECTs) to translate microbial EET activity into easily detectable electrical signals. This system enables the use of cellular responses to external stimuli for biosensing and biocomputing applications. Specifically, we demonstrated the de-doping of the p-type poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT: PSS) channel in the OECT is driven by cellular EET from Shewanella oneidensis. By transcriptionally controlling EET flux by genetic circuits, we establish the biosensing capability of this hybrid OECT system to detect chemical stimuli, such as inducer molecules. Furthermore, we introduce plasmid-based Boolean logic gates within the cells, allowing them to process environmental signals and drive current changes in the OECTs, further demonstrating the biocomputing potential of these devices. This method provides a novel interface between biological systems and electronics, enabling future high-throughput screening, biosensing, and biocomputing applications.

Fitted rate constant k 
The fitted rate constant k of the OECT channel current IDS serves as a reliable metric for assessing the EET activity of the sample. While the constant gate bias voltages would affect the rate constants, we choose 0.2 V to ensure positive bias to encourage bacterial electron transfer while avoiding fast de-doping at higher gate voltages and providing and minimizing electrochemical stress on the bacteria cells23...

Here, we present a protocol for using organic electrochemical transistors (OECTs) to translate extracellular electron transfer (EET) activity in Shewanella oneidensis into electrical signals. The hybrid OECT system provides enhanced robustness, sensitivity, and potential for rapid, high-throughput testing, making it an effective tool for EET measurements.

Extracellular electron transfer (EET) is a process through which certain microorganisms can transfer electrons across their cell membranes to external ...

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Bioengineering

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Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

Characterizing Electron Transport through Living Biofilms

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Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.

Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

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Spray-Coated Melanin/PEDOT:PSS Films for Sustainable Organic Electrochemical Transistors

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes.  Here we present a general procedure for the fabrication and use of such sensors.

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

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We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Learn how organic electrochemical transistors translate extracellular electron transfer activity in Shewanella oneidensis into measurable electrical signals for bioelectronics research.

Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors

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