The overall goal of this experimental procedure is to determine the amount of current that can be conducted through a living material in situ as well as the mechanism employed by the living material to transport the current. This method could help answer key questions in the field of microbial electrochemistry such as, How can certain biofilms move electrons over hundreds of microns to support cellular metabolism? The main advantage of this technique is that the interdigitated electrodes give a very high signal-to-noise ratio and can be scaled to fit into many different reactor configurations.

Those method can provide insight into multi-cell length extracellular electron transport in biofilms. It can also be applied to other systems, such as electrically conductive polymer films. Researchers new to this method will struggle because it requires a bipotentiostat to set the potentials of both electrodes separately and measure current at each electrode separately.

To begin, obtain commercially available IDA electrodes patterned on a non-conductive substrate or synthesize them using standard lithographic methods. Prepare conductive silver epoxy according to the manufacturer's instructions with a mixing rod or pipette tip. Then, place a wire on each gold pad and secure them in place using lab tape.

Cover the wire and pad with silver epoxy using a mixing rod or pipette tip. Carefully move the apparatus to an 80 degree Celsius oven for one hour to cure. After the epoxy cures, use a multimeter to ensure electrical conductivity between the end of the wire and the pads.

The resistance between the wire and the pads should be less than five ohms. Also, use the multimeter to verify that no conductive epoxy is connecting multiple electrode pads, as this can short the array. If conductive epoxy is connecting multiple leads, use the scribe to isolate the leads.

Next, remove the tip of a 15 milliliter conical centrifuge tube to use as a mold for the insulating material. Use a 21 gauge needle to make two small holes in the bottom of the mold for the wires to protrude through. Insert the IDA into the mold and insert the wires through the holes in the bottom of the mold.

Next, prepare a thermal electrical and waterproof insulating material. Flame retardant polyurethane resins are often suitable. Pipette the insulator into the mold so that the silver epoxy is completely covered and allow the insulator to dry according to manufacturer's specifications.

To set up the electrochemical cell, insert the IDA, counter electrode, and reference electrode into the electrochemical cell. Fill the electrochemical cell with sterile medium suitable for biofilm growth. For Geobacter sulfurreducens, use freshwater medium excluding fumarate.

Next, connect the electrodes to a bipotentiostat. Connect one IDA electrode to the working lead one, the other IDA electrode to the working lead two, the reference electrode to the reference lead, and the counter electrode to the counter lead. Perform a control experiment by performing cyclic voltammetry on electrode one and holding electrode two at open circuit to ensure that the electrodes are not connected.

To grow the relevant electroactive biofilm, inoculate the electrochemical reactor from a stock culture or enrichment of the desired electrochemically active microorganisms using standard aseptic microbiological techniques. For standard tests, inoculate in a 1 to 20 ratio of inoculum-to-reactor volume. Set the stirring in the reactor to the desired level and set the incubator or water bath to the desired temperature based on the growth conditions of the biofilm of interest.

Incubate the system based on specific requirements of the microorganism of interest until the biofilm bridges the gap separating the two electrodes. For stationary Geobacter sulfurreducens biofilm, incubate for seven to ten days at 30 degrees Celsius. Begin setup of the experimental parameters that will be used to determine the current potential dependence for the gating measurements as described in the text protocol.

Set up the bipotentiostat software to perform the gating measurements over the selected range at the selected source drain voltage and at the desired scan rate. Additionally, perform a baseline measurement as described in the text protocol. Perform gating measurements using the same conditions under turnover with soluble electron donor or acceptor present in non-turnover without soluble electron donor or acceptor conditions.

Obtain a control reactor to ensure that the set point and actual temperature of the medium is the same. Place a thermometer or a thermocouple in a control reactor where the IDA would be. Then, make source strain current measurements over the range of temperatures selected under turnover and non-turnover conditions using the bipotentiostat.

To do so, set and hold the IDA at the gate potential that yields maximum conducted current using the bipotentiostat. Record the maximum conducted current using the bipotentiostat while the temperature is cycled between the range of temperatures selected using the onboard controls of the water bath or incubator. Cycle the temperature from one set point to the other and back against using the onboard controls of the incubator or the water bath.

This determines the reversibility of the reaction to ensure that the temperature cycling does not harm the biofilm. Finally, reset the temperature back to the normal growth temperature and allow the system to stabilize. Shown here are representative results of an IDA that is functioning properly in freshwater medium.

Versus one where the electrodes have been shorted. Representative electrochemical gating measurements performed on a Geobacter sulfurreducens biofilm under non-turnover conditions with a source strain voltage of 10 millivolts exhibit the peak shaped conducted current versus gate potential curve characteristic of redox conductivity. Here, representative raw data shows the dependence of the conducted current on the temperature of the reactor medium.

The decrease in conducted current with decreasing temperature is another characteristic of redox conductors. Once mastered, this technique can be done in approximately two days if it is performed properly. While attempting this procedure it is important to remember to make sure that the IDA bands are not shorted prior to inoculating the reactor.

The implications of this technique extend towards therapy when applied to study how to disrupt cellular metabolism of biofilm forming pathogens. This procedure can be applied to any living material able to transport electrons in order to address additional questions, such as the correlation between biofilm refology and conductivity.