Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors

Our research focuses on developing bioelectronics that integrate bacterial extracellular electron transfer or EET to expand biosensing and biocomputing applications. We are seeking answers to how EET interacts with electronic materials, how to regulate EET genetically in order to optimize electrical performance and whether there are new emergent features in these electrochemical systems that contain living cells. Advancements in bacterial HR cell or electron transfer research use synthetic biology for engineering EET pathways, electrochemical system for redox monitoring, for instance, and microscopy to cell activities, conducting atomic force microscopy and macro electrodes for electron flow analysis and advanced spectroscopy like UAVs Raman for material biology interface characterization.

We demonstrate that genetically engineered shown on disease could modulate OECT outputs, enabling biologically-driven electrical responses. Findings include illustrating the direct and indirect EET pathways impacting bioelectronics performance, coupling genetic logic to electrical results and tuning genetic plasticity via EET. Compared to traditional OECT work, living cells offer advantages such as dynamic genetically programmable controls through extracellular electron transfer and ability to leverage cellular metabolisms for real time responses.

Compared to traditional biological circuits that use fluorescence reporters, OECT users provide faster direct electrical results with high sensitivity. Our future work includes expanding genetic circuits to drive EET and developing epigenetics or optogenetics to enable electronics to modulate EET gene expression. We are also interested in creating control loops for applications like neural network training.

Finally, we are exploring diverse OECT channel materials and the inclusion of microfluidics and institute oxygen generation to further control device performance. To begin autoclave the organic electrochemical transistor or OECT slides, polydimethylsiloxane or PDMS sheets and silver, silver chloride reference electrode. Bake the autoclave components at 80 degrees Celsius to dry them.

Transfer the autoclave OECT slides and silver, silver chloride reference electrode into the glove box. Subject the PDMS sheets to a vacuum in the antichamber of the glove box for four hours to desorb the trapped oxygen. Then to assemble the OECT device, place the PDMS sheets over the OECT slides.

Inject 45 microliters of Shewanella basal medium, or SBM into each OECT chamber. Cover the OECT chamber ports with PDMS sheets to prevent medium evaporation and contamination. Begin the initial OECT electrochemical measurement by measuring the channel current IDS for a gate voltage step from zero to 0.2 volts.

Monitor the OECT channel drain course current or IDS under a constant channel drain source voltage or VDS of minus 0.05 volts and gate voltage VGS of 0.2 volts until the current stabilizes. Configure instrument channel one to measure the OECT channel current IDS. Set the technique name to Fast Amperometry, equilibration time to one second, equilibration voltage to minus 0.05 volts, bias voltage to minus 0.05 volts, runtime to 14 seconds and sample interval to 0.5 milliseconds.

Next, configure instrument channel two to control the gate voltage VGS. Set the technique name to Mixed Mode, condition time to one second, condition voltage to zero volts, stage one mode to constant E, stage one bias voltage to zero volts. Stage one runtime to four seconds.

Stage two mode to constant E, stage two bias voltage to 0.2 volts. Stage two runtime to 10 seconds and sample interval to 0.5 milliseconds. Apply a constant channel voltage VDS of minus 0.05 volts and gate voltage VGS of 0.2 volts to all OECT devices.

Centrifuge the Shewanella oneidensis suspension at 1500 3G for four minutes and wash the cell pellet thrice with one milliliter of fresh growth medium. After the last wash, resuspend the cells in 0.5 milliliter or half the original culture volume to obtain a concentrated cell suspension with an optical density of 600 of one to 3.5. Transfer the concentrated cell suspension into the glove box.

Prepare the inoculum by diluting the cells to an intended optical density of 0.1, using the SBM+supplemented with lactate prepared freshly in the glove box with purged media stocks. Stop the potentiostat. Inject five microliters of the prepared inoculum into the OECT chamber containing 45 microliters SBM to achieve a final absorbance of 0.01.

Cover the OECT chamber ports with PDMS sheets to avoid medium evaporation and contamination. For anaerobic cultures, dilute the Shewanella cell culture tenfold using a dilution medium with the same constitution as the growth medium. After stopping the potentiostat, inject five microliters of the anaerobic inoculum into the OECT chamber, containing 45 microliters of SBM medium.

Cover the OECT chamber ports with PDMS sheets. Apply constant bias voltages to the OECT channels with a channel voltage VDS of minus 0.05 volts, and gate voltage VGS of 0.2 volts for 24 hours. Disconnect the potential stat from individual OECT devices only during time point measurements for characterization.

Monitor significant changes in the OECT channel doping state during the initial eight hours. After 24 hours, insert the silver, silver chloride reference electrode into the OECT chamber by gently twisting and pushing it through the fluid exchange port. Measure the transfer curves of the OECT by sweeping the gate voltage from minus 0.1 volts to 0.6 volts while monitoring the channel current IDS with a constant bias voltage VDS of minus 0.05 volts.

Simultaneously, measure the accurate gate and source electrode potentials against the reference electrode. Configure instrument channel one to measure the OECT channel current IDS using the chronoamperometry technique. Set equilibration time to five seconds, equilibration voltage to minus 0.05 volts, bias voltage to minus 0.05 volts, runtime to 35 seconds and sample interval to 0.09915 seconds.

Then configure instrument channel two to control the OECT gate voltage VGS using linear sweep voltammetry. Set the equilibration time to five seconds, equilibration voltage to minus 0.1 volts. Begin voltage to minus 0.1 volts.

End voltage to 0.6 volts. Voltage step to 0.002 volts and scan rate to 0.02 volts per second. Configure instrument channel three to measure the OECT source potential VS against the silver, silver chloride reference electrode using Open Circuit Potentiometry.

Set the runtime to 40 seconds and the sample interval to 0.09915 seconds. Configure instrument channel four to measure the OECT gate potential VG against the reference electrode using Open Circuit Potentiometry. Set the runtime to 40 seconds and sample interval to 0.09915 seconds.

After each measurement, rinse the reference electrode with 70%ethanol and wipe it with a new low lint towel. The fitted rate constants for electron transfer activity showed significant differences between strains with the Delta MtrC and Delta MTR mutants displaying reduced constants compared to wild-type S.oneidensis MR-1. Complementation of mutants with MtrC or MTRC AB restored rates to wild-type levels under inducer conditions.

The OECT channel current demonstrated rapid detection of electron transfer activity with drain source current and normalized drain source current showing statistically significant differences between strains within 1.5 hours post-inoculation. Abiotic controls and uninduced mutants displayed minimal changes reinforcing the sensitivity of the hybrid system.