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

<ol>
	<li>Luo, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technology+roadmap+for+flexible+sensors.">Technology roadmap for flexible sensors.</a> Acs Nano. 17 (6), 5211-5295 (2023).</li><li>Din, M. O., Martin, A., Razinkov, I., Csicsery, N., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacing+gene+circuits+with+microelectronics+through+engineered+population+dynamics.">Interfacing gene circuits with microelectronics through engineered population dynamics.</a> Sci Ad. 6 (21), eaaz8344 (2020).</li><li>Markovic, D., Mizrahi, A., Querlioz, D., Grollier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physics+for+neuromorphic+computing.">Physics for neuromorphic computing.</a> Nat Rev Phys. 2 (9), 499-510 (2020).</li><li>Daniel, R., Rubens, J. R., Sarpeshkar, R., Lu, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+analog+computation+in+living+cells.">Synthetic analog computation in living cells.</a> Nature. 497 (7451), 619-623 (2013).</li><li>Ates, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-to-end+design+of+wearable+sensors.">End-to-end design of wearable sensors.</a> Nat Rev Mater. 7 (11), 887-907 (2022).</li><li>Rivnay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+electrochemical+transistors.">Organic electrochemical transistors.</a> Nat Rev Mater. 3 (2), e17086 (2018).</li><li>Rashid, R. B., Ji, X. D., Rivnay, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+electrochemical+transistors+in+bioelectronic+circuits.">Organic electrochemical transistors in bioelectronic circuits.</a> Biosens Bioelectron. 190, 113461 (2021).</li><li>Bernards, D. A., Malliaras, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+and+transient+behavior+of+organic+electrochemical+transistors.">Steady-state and transient behavior of organic electrochemical transistors.</a> Adv Funct Mater. 17 (17), 3538-3544 (2007).</li><li>Lubrano, C., Matrone, G. M., Iaconis, G., Santoro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+frontiers+for+selective+biosensing+with+biomembrane-based+organic+transistors.">New frontiers for selective biosensing with biomembrane-based organic transistors.</a> Acs Nano. 14 (10), 12271-12280 (2020).</li><li>Guo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+single-molecule+detection+of+COVID-19+and+MERS+antigens+via+nanobody-functionalized+organic+electrochemical+transistors.">Rapid single-molecule detection of COVID-19 and MERS antigens via nanobody-functionalized organic electrochemical transistors.</a> Nat Biomed Eng. 5 (7), 666-677 (2021).</li><li>Pappa, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+metabolite+detection+with+an+n-type+accumulation+mode+organic+electrochemical+transistor.">Direct metabolite detection with an n-type accumulation mode organic electrochemical transistor.</a> Sci Adv. 4 (6), eaat0911 (2018).</li><li>Siuti, P., Yazbek, J., Lu, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+circuits+integrating+logic+and+memory+in+living+cells.">Synthetic circuits integrating logic and memory in living cells.</a> Nat Biotechnol. 31 (5), 448-452 (2013).</li><li>Brophy, J. A. N., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+genetic+circuit+design.">Principles of genetic circuit design.</a> Nat Meth. 11 (5), 508-520 (2014).</li><li>Rubens, J. R., Selvaggio, G., Lu, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+mixed-signal+computation+in+living+cells.">Synthetic mixed-signal computation in living cells.</a> Nat Comm. 7, 11658 (2016).</li><li>Rizik, L., Danial, L., Habib, M., Weiss, R., Daniel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+neuromorphic+computing+in+living+cells.">Synthetic neuromorphic computing in living cells.</a> Nat Comm. 13 (1), 5602 (2022).</li><li>Tseng, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution-deposited+and+patternable+conductive+polymer+thin-film+electrodes+for+icmrobial+bioelectronics.">Solution-deposited and patternable conductive polymer thin-film electrodes for icmrobial bioelectronics.</a> Adv Mater. 34 (13), 2109442 (2022).</li><li>Dundas, C. M., Graham, A. J., Romanovicz, D. K., Keitz, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+electron+transfer+by+Shewanella+oneidensis+controls+palladium+nanoparticle+phenotype.">Extracellular electron transfer by Shewanella oneidensis controls palladium nanoparticle phenotype.</a> Acs Synth Biol. 7 (12), 2726-2736 (2018).</li><li>Catania, C., Karbelkar, A. A., Furst, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+interface+between+electroactive+bacteria+and+electrodes.">Engineering the interface between electroactive bacteria and electrodes.</a> Joule. 5 (4), 743-747 (2021).</li><li>Logan, B. E., Rossi, R., Ragab, A., Saikaly, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroactive+microorganisms+in+bioelectrochemical+systems.">Electroactive microorganisms in bioelectrochemical systems.</a> Na Rev Microbiol. 17 (5), 307-319 (2019).</li><li>Graham, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+living+materials+via+extracellular+electron+transfer.">Transcriptional regulation of living materials via extracellular electron transfer.</a> Nat Chem Biol. 20 (10), 1329-1340 (2024).</li><li>Cao, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+plasmid+toolkit+for+Shewanella+oneidensis+MR-1.">A synthetic plasmid toolkit for Shewanella oneidensis MR-1.</a> Front Microbiol. 10, 410 (2019).</li><li>Mehes, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+microbial+electrochemical+transistor+monitoring+extracellular+electron+transfer.">Organic microbial electrochemical transistor monitoring extracellular electron transfer.</a> Adv Sci. 7 (15), 2000641 (2020).</li><li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+transistor+with+transcriptionally+controlled+computation+and+plasticity.">A hybrid transistor with transcriptionally controlled computation and plasticity.</a> Nat Commun. 15 (1), 1598 (2024).</li><li>Yoo, B., Dodabalapur, A., Lee, D. C., Hanrath, T., Korgel, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germanium+nanowire+transistors+with+ethylene+glycol+treated+poly(3,4-ethylenedioxythiophene):+poly(styrene+sulfonate)+contacts.">Germanium nanowire transistors with ethylene glycol treated poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) contacts.</a> Appl Phys Lett. 90 (7), 072106 (2007).</li><li>Tahernia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+96-well+high-throughput,+rapid-screening+platform+of+extracellular+electron+transfer+in+microbial+fuel+cells.">A 96-well high-throughput, rapid-screening platform of extracellular electron transfer in microbial fuel cells.</a> Biosens Bioelectron. 162, 112259 (2020).</li><li>Ainla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-source+potentiostat+for+wireless+electrochemical+detection+with+smartphones.">Open-source potentiostat for wireless electrochemical detection with smartphones.</a> Anal Chem. 90 (10), 6240-6246 (2018).</li><li>Rowe, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CheapStat:+an+open-source,+&amp;#34;do-it-yourself&amp;#34;+for+analytical+and+educational+applications.">CheapStat: an open-source, &amp;#34;do-it-yourself&amp;#34; for analytical and educational applications.</a> PLoS One. 6 (9), e23783 (2011).</li><li>Salyk, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+electrochemical+transistor+microplate+for+real-time+cell+culture+monitoring.">Organic electrochemical transistor microplate for real-time cell culture monitoring.</a> Appl Scil. 7 (10), 998 (2017).</li><li>Zhao, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+patterning+of+electroactive+bacterial+biofilms.">Light-induced patterning of electroactive bacterial biofilms.</a> Acs Synth Biol. 11 (7), 2327-2338 (2022).</li><li>Tan, S. T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operation+mechanism+of+organic+electrochemical+transistors+as+redox+chemical+transducers.">Operation mechanism of organic electrochemical transistors as redox chemical transducers.</a> J Mater Chem. 9 (36), 12148-12158 (2021).</li><li>White, S. P., Dorfman, K. D., Frisbie, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operating+and+sensing+mechanism+of+electrolyte-gated+transistors+with+floating+gates:+Building+a+platform+for+amplified+biodetection.">Operating and sensing mechanism of electrolyte-gated transistors with floating gates: Building a platform for amplified biodetection.</a> J Phys Chem. 120 (1), 108-117 (2016).</li><li>Huang, B., Gao, S., Xu, Z., He, H., Pan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+mechanisms+and+application+of+electron+shuttles+in+extracellular+electron+transfer.">The functional mechanisms and application of electron shuttles in extracellular electron transfer.</a> Curr Microbiol. 75 (1), 99-106 (2018).</li><li>Light, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flavin-based+extracellular+electron+transfer+mechanism+in+diverse+Gram-positive+bacteria.">A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria.</a> Nature. 562 (7725), 140-144 (2018).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-oxohexanoyl-homoserine lactone&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anhydrotetracycline hydrochloride&#160;</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>casamino acids&#160;</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>anhydrous 99.8%,&#160;</td>
		</tr>
		<tr>
			<td>HEPES buffer solution&#160;</td>
			<td>VWR</td>
			<td></td>
			<td>1 M in water, pH = 7.3</td>
		</tr>
		<tr>
			<td>isopropyl &#223;-D-1-thiogalactopyranoside&#160;</td>
			<td>Teknova</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin sulfate&#160;</td>
			<td>Growcells</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium(II) sulfate heptahydrate&#160;</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEDOT:PSS aqueous suspension&#160;</td>
			<td>Heraeus Epurio LLC</td>
			<td>Clevios PH1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic&#160;</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&#160;</td>
			<td>PalmSens BV</td>
			<td>MultiPalmSens4</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz microscopic slides&#160;</td>
			<td>AdValue&#160;</td>
			<td>FQ-S-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz microscopic slides&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium DL-lactate&#160;</td>
			<td>TCI</td>
			<td></td>
			<td>60% in water</td>
		</tr>
		<tr>
			<td>Sodium fumarate&#160;</td>
			<td>VWR</td>
			<td></td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Sulfuric acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>95.0%-98.0%</td>
		</tr>
		<tr>
			<td>Two-part silicone elastomer&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>Sylgard184</td>
			<td></td>
		</tr>
		<tr>
			<td>Wolfe&#39;s Trace Mineral Mix&#160;</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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</su...

Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors

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 ...

translating-extracellular-electron-transfer-activities-with-organic

Research

JoVE Journal

Bioengineering

364 Views.  University of Texas at Austin. 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.

Video: Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors

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

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"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.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

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.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

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.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...