M.D.Y, S.M.G-S., and L.M.T. acknowledge the Office of Naval Research (Award #N0001415WX01038 and N0001415WX00195), the Naval Research Laboratory, and the Naval Research Laboratory Nanosciences Institute; M.Y.E.-N. is supported by the U.S. Department of Energy Grant DE-FG02-13ER16415.

1. Interdigitated microelectrode array (IDA) preparation
<ol>
	<li>Obtain commercially available IDA electrodes patterned on a nonconductive substrate or synthesize them using standard lithographic methods.28 
	NOTE: IDA dimensions and/or materials can be varied based on desired conditions for different experiments. IDAs used here were obtained commercially and consisted of two interdigitated gold microelectrodes patterned on a glass substrate connected to large electrode pads at opposite ends of the array. The electrodes are exposed while the bus lines connecting the electrodes to the large contact pads are coated with thin insulating material. IDAs used here are comprised of two sets of 10 &#181;m-wide and 2 mm long gold microelectrode bands (source and drain) spaced 5 &#181;m apart patterned on a flat glass surface. IDAs used here had 65 electrode pairs (130 total interdigitated bands). Alternating bands are connected to electrode pads at opposite ends of the array.</li>
	<li>Wire and insulate IDA
	<ol>
		<li>Attach a wire to each large electrode pad using conductive silver epoxy.
		<ol>
			<li>Prepare conductive epoxy according to manufacturer&#39;s instructions for the specific epoxy in use with a mixing rod or pipette tip (instructions may vary by manufacturer).</li>
			<li>Place a wire on each gold pad, secure in place using lab tape. Cover the wire and pad with silver epoxy using a mixing rod or pipette tip.</li>
			<li>Carefully move to an 80 &#176;C oven for 1 h to cure or cure based on manufacturer&#39;s recommendations for the specific conductive silver epoxy used.</li>
			<li>After the epoxy cures, use a multimeter to ensure electrical connectivity between the end of the wire and the pads. The resistance between the wire and the pad should be &#60; 5 &#937;. 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 a scribe to isolate the leads.</li>
		</ol>
		</li>
		<li>Insulate the epoxied connection by coating the connection with a thermal, electrical, and waterproof insulating material. Flame retardant polyurethane resins are often suitable.
		<ol>
			<li>Remove the tip of a 15-mL conical centrifuge tube (at approximately the 2.5 mL mark) as a mold for the insulating material. Make two small holes in the bottom for the wires to protrude through using a 21g needle.</li>
			<li>Insert the IDA into the mold and wires through the holes in the bottom of the mold.</li>
			<li>Prepare the specific insulating material. Follow instructions provided with the specific resin obtained.</li>
			<li>Pipette the insulator into the mold so that the silver epoxy is completely covered (~2.5 mL) and allow to dry according to manufacturer&#39;s specifications.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Electrochemical reactor setup, testing, and inoculation
<ol>
	<li>Set up the electrochemical cell.
	<ol>
		<li>Insert IDA, counter electrode, and reference electrode into the electrochemical cell. 
		NOTE: The electrochemical reactor used can vary widely, as long as all of the electrodes fit inside. One consideration is that the reference electrode should be as close as possible to the working electrode given the practical limits of the reactor. Here, we use a single chamber reactor with the reference electrode ~2-3 cm from the working electrodes. Also, the counter electrode should be larger than the working electrode to ensure that it is not limiting in the system.</li>
		<li>If working with a pure culture, sterilize the reactor with the counter electrode inside. Sterilize the reference electrode by soaking in bleach for 10 min and sterile deionized water for 10 min. Sterilize the IDA by dipping in bleach for 5 s followed by sterile deionized water for 10 s before insertion into the reactor.</li>
		<li>Fill the electrochemical cell with sterile medium suitable for biofilm growth. For G. sulfurreducens17,18, use freshwater medium excluding fumarate. For biocathode MCL,9 use artificial seawater medium.9</li>
		<li>Connect the electrodes to a bipotentiostat. Connect one IDA electrode to the working lead 1, the other IDA electrode to the working lead 2, the reference electrode to the reference lead, and the counter electrode to the counter lead.</li>
	</ol>
	</li>
	<li>Electrochemical testing of IDAs. 
	NOTE: The major goal of this test is to make sure that the two electrodes are electrically isolated. All electrochemical techniques are available in the software used to control the bipotentiostat.
	<ol>
		<li>Perform control conductivity tests (before biofilm growth) to ensure proper IDA function using a bipotentiostat.
		<ol>
			<li>Measure the open circuit potential of each electrode for 1 min. 
			NOTE: On some instruments, open circuit potential must be achieved by using the galvanostatic collection program and setting the current to 0 mA.</li>
			<li>Measure the open circuit potential of electrode 2 while performing cyclic voltammetry on electrode 1 between +0.2 V to +0.6 V (vs. SHE) at 20 mV/s. 
			NOTE: Other bounds and scan rates for CV can be used if desired. However, avoid potentials that will result in hydrogen or oxygen generation.</li>
			<li>Verify that the open circuit potential measured at electrode 2 does not mirror the potential of electrode 1 during the CV using the potentiostat software. 
			NOTE: The open circuit potential of electrode 2 may change, but it should be independent of the potential of electrode 1.</li>
			<li>Repeat steps 2.2.1.1 through 2.2.1.3 except control the potential of electrode 2 and measure the open circuit potential of electrode 1.</li>
		</ol>
		</li>
		<li>Set the potential of working electrodes to the desired growth potential of the relevant electroactive biofilm using the bipotentiostat software. For example, use +0.5 V (vs. SHE) for Geobacter sulfurreducens or +0.31 V (vs. SHE) for biocathode MCL.</li>
	</ol>
	</li>
	<li>Grow relevant electroactive biofilm
	<ol>
		<li>Inoculate the electrochemical reactor from a stock culture/enrichment of the desired electrochemically active microorganisms using standard aseptic microbiological techniques. For standard tests, inoculate in a 1:20 ratio (inoculum to reactor volume) of an OD600 = 0.5 culture.</li>
		<li>Set the stirring in the reactor to the desired level (~200 rpm) and the incubator/ water bath to the desired temperature based on the growth conditions of the biofilm of interest. For G. sulfurreducens, use 30 &#176;C for optimal growth.</li>
		<li>Incubate the system based on specific requirements of the microorganism(s) of interest until the biofilm bridges the gap separating the two electrodes. For stationary G. sulfurreducens biofilm, incubate for ~7-10 days. For biocathode MCL, incubate ~7 days. In each case, control the temperature at 30 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Electrochemical gating experiments
<ol>
	<li>Select the experimental parameters that will be used to determine the current-potential dependence for the gating measurements.
	<ol>
		<li>Determine the range of gate potentials that will be applied to the IDA to obtain the conducted current (ISD) versus gate potential (EG) curve for the system. 
		NOTE: The range of gate potentials examined should cover all potentials with possible redox activity. If no information about the system of interest is available, a wide potential range should be used (-0.55 to +0.6 V vs. SHE). A trial and error approach can be used to fine tune the range based on the system under study. Gate potential is defined as: 
		<img alt="Equation 1" src="/files/ftp_upload/54671/54671eq1.jpg" /> 
		where ED is the potential of the drain electrode and ES is the potential of the source electrode. The range of gate potentials used is constrained by the requirements and limitations of the specific system of interest.18 
		NOTE: Source and drain potentials that will result in oxygen and hydrogen evolution should be avoided as these processes can damage the biofilm.</li>
		<li>Determine the source-drain voltage (VSD) that will be used as the driving force for electron transport through the film from the source to the drain. Source-drain voltage is defined as: 
		<img alt="Equation 2" src="/files/ftp_upload/54671/54671eq2.jpg" /> 
		NOTE: The source-drain voltage should be sufficiently small so that the ISD scales linearly with VSD.17</li>
		<li>Choose a scan rate (v) at which EG is changed linearly with time that is independent of ISD so that the system can be approximated to be at steady state for each gate potential. 
		NOTE: A scan rate of less than 0.002 V/s is often used for biological systems.29,30</li>
		<li>Set up the bipotentiostat software to perform the gating measurements (i.e. sweep the gate potential) over the selected range, at the selected source-drain voltage, and at the desired scan rate based on the above considerations. 
		NOTE: For previous gating measurements using G. sulfurreducens,17,19 EG = -0.55 V to 0.6 V (vs. SHE), VSD = 0.01 V or 0.1 V, v = 0.001 V/s. For Biocathode MCL, EG = 0.25 V to 0.7 V (vs. SHE), VSD = 0.002 V, v = 0.0002 V/s.
		<ol>
			<li>Additionally, perform a baseline measurement with VSD = 0.000 V (i.e., a CV at each individual electrode taken simultaneously) at the same v chosen in Step 3.1.3.</li>
		</ol>
		</li>
		<li>Perform gating measurements using the conditions in 3.1.4 under both turnover (with soluble electron donor or acceptor present) and non-turnover (without soluble electron donor or acceptor) conditions. 
		NOTE: Non-turnover conditions are advantageous because measurements are not obscured by the oxidation or reduction of soluble compounds for cellular metabolism, although similar results should be obtained regardless of which condition is used after subtracting background currents (detailed in 3.1.8).
		<ol>
			<li>Achieve turnover conditions by using the same reactor medium as used for bacterial growth on the electrode. This medium contains a soluble electron donor, such as acetate for G. sulfurreducens,17 or acceptor, such as oxygen for Biocathode MCL.</li>
			<li>Achieve non-turnover conditions by making the same reactor medium used for bacterial growth on the electrode, except omit the soluble electron donor or acceptor. After ensuring that the potentiostat is off, remove the medium aseptically and add in fresh medium without the electron donor for anodic systems or acceptor for cathodic systems. Alternatively, a continuous flow system can be set up to slowly replace the medium with the desired medium for nonturnover conditions. 
			NOTE: If oxygen is the soluble electron acceptor (as for biocathode MCL), sparge the system with a mixture of ~15% CO2 and 85% N2 (or a gas mixture that will maintain the correct pH in the medium).</li>
		</ol>
		</li>
		<li>After completion of the gating measurements, use the potentiostat software to set the potential of each electrode back to the growth potential to allow the system to re-stabilize (using the same values as in 2.2.2).</li>
		<li>If all the conditions described above are met (ISD is independent of v and scales linearly with VSD), convert ISD values to conductivity using the following equations as previously described31 
		<img alt="Equation 3" src="/files/ftp_upload/54671/54671eq3.jpg" /> 
		<img alt="Equation 4" src="/files/ftp_upload/54671/54671eq4.jpg" /></li>
		<li>where G is the conductance and S is a scaling factor that is system dependent, and factors in variables such as electrode size, gap size, and biofilm height. For certain systems, S can be determined from predetermined equations.31 Alternatively, S can be calculated numerically using a modeling software, as detailed previously.17</li>
		<li>Subtract the background currents to identify the shape and magnitude of the conducted current. Either subtract the current generated at VSD = 0.00 V from the current generated at VSD = 0.01 V or subtract the drain current from the source current generated with a VSD = 0.01 V. Either method removes background currents, leaving only the conducted current.</li>
	</ol>
	</li>
	<li>Temperature dependent electrochemical gating measurements
	<ol>
		<li>Determine the range of temperatures of interest. This range is highly dependent on the system under study, however physiologically relevant temperatures should be used. 
		NOTE: Previous studies have used a temperature range of 10 &#176;C to 30 &#176;C to study electron transport under physiologically relevant conditions for mesophilic microorganisms.17</li>
		<li>Obtain a recirculating water bath or incubator to regulate reactor temperature and a control reactor to ensure that the set point and actual temperature of the medium is the same.
		<ol>
			<li>Place a thermometer or thermocouple in a control reactor where the IDA would be.</li>
		</ol>
		</li>
		<li>Make ISD measurements (see 3.1.4) over the range of temperatures selected under turnover and nonturnover (described in Step 3.1.5) conditions using the bipotentiostat following one of the two procedures described below.
		<ol>
			<li>Generate ISD vs. EG curves, as described above (see 3.1), for each temperature over the temperature range of interest. For materials that exhibit redox conductivity, such as G. sulfurreducens and Biocathode MCL, the peak current from each temperature is used to determine the ISD vs. T dependence. 
			NOTE: This method is used to generate a full ISD vs. EG curve for each temperature, but requires more time than the second option.</li>
			<li>Alternately, set and hold the IDA at the gate potential that yields maximum conducted current using the bipotentiostat (obtained from the ISD vs EG curve, Step 3.1.4) and 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. 
			NOTE: With the electrode/reactor geometry used here, the ISD and T are allowed to stabilize for at least 20 min and an average stable ISD is used for each temperature. More or less stabilization time may be required based on the specific system. This method is faster than the first and causes less stress to the biofilm. However, full gating curves are not generated.</li>
			<li>Cycle the temperature from one set point to the other and back again using the onboard controls of the incubator or the water bath to determine the reversibility of the reaction to ensure that the temperature cycling does not harm the biofilm.</li>
		</ol>
		</li>
		<li>Reset the temperature back to the normal growth temperature using the onboard controls of the incubator or water bath and allow the system to stabilize. 
		NOTE: For a redox conductor, the ISD versus 1/T data can be fit with the Arrhenius rate expression, which allows the calculation of activation energy as follows: 
		<img alt="Equation 5" src="/files/ftp_upload/54671/54671eq5.jpg" /> 
		<img alt="Equation 6" src="/files/ftp_upload/54671/54671eq6.jpg" /> 
		where Ea is the activation energy for electron transfer between adjacent redox cofactors and k is the Boltzmann constant.</li>
	</ol>
	</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geobacter+sulfurreducens+pili+support+ohmic+electronic+conduction+in+aqueous+solution.">Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution.</a> Phys Chem Chem Phys. 19, 21791-21799 (2017).</li><li>Fricke, K., Harnisch, F., Schr&#246;der, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+cyclic+voltammetry+for+the+study+of+anodic+electron+transfer+in+microbial+fuel+cells.">On the use of cyclic voltammetry for the study of anodic electron transfer in microbial fuel cells.</a> Energ Environ Sci. 1, 144-147 (2008).</li><li>Marsili, E., Rollefson, J. B., Baron, D. B., Hozalski, R. M., Bond, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilm+voltammetry:+direct+electrochemical+characterization+of+catalytic+electrode-attached+biofilms.">Microbial biofilm voltammetry: direct electrochemical characterization of catalytic electrode-attached biofilms.</a> Appl Environ Microbiol. 74, 7329-7337 (2008).</li><li>Kankare, J., Kupila, E. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+conductance+measurement+during+electropolymerization.">In-situ conductance measurement during electropolymerization.</a> J Electroanal Chem. 322, 167-181 (1992).</li><li>Byun, H. S., Pirbadian, S., Nakano, A., Shi, L., El-Naggar, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Monte+Carlo+Simulations+and+Molecular+Conductance+Measurements+of+the+Bacterial+Decaheme+Cytochrome+MtrF.">Kinetic Monte Carlo Simulations and Molecular Conductance Measurements of the Bacterial Decaheme Cytochrome MtrF.</a> Chem Electro Chem. 1, 1932-1939 (2014).</li><li>El Kasmi, A., Wallace, J. M., Bowden, E. F., Binet, S. M., Linderman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+interfacial+electron-transfer+kinetics+of+cytochrome+c+with+mixed+self-assembled+monolayers.">Controlling interfacial electron-transfer kinetics of cytochrome c with mixed self-assembled monolayers.</a> J Am Chem Soc. 120, 225-226 (1998).</li><li>Bortolotti, C. 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=The+Reorganization+Energy+in+Cytochrome+c+is+Controlled+by+the+Accessibility+of+the+Heme+to+the+Solvent.">The Reorganization Energy in Cytochrome c is Controlled by the Accessibility of the Heme to the Solvent.</a> J Phys Chem Lett. 2, 1761-1765 (2011).</li><li>Gallaway, J. W., Calabrese Barton, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+Redox+Polymer-Mediated+Enzyme+Electrodes.">Kinetics of Redox Polymer-Mediated Enzyme Electrodes.</a> J Am Chem Soc. 130, 8527-8536 (2008).</li><li>Thackeray, J. W., White, H. S., Wrighton, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(3-methylthiophene)-coated+electrodes:+optical+and+electrical+properties+as+a+function+of+redox+potential+and+amplification+of+electrical+and+chemical+signals+using+poly(3-methylthiophene)-based+microelectrochemical+transistors.">Poly(3-methylthiophene)-coated electrodes: optical and electrical properties as a function of redox potential and amplification of electrical and chemical signals using poly(3-methylthiophene)-based microelectrochemical transistors.</a> J Phys Chem-US. 89, 5133-5140 (1985).</li><li>Jugnet, Y., Tourillon, G., Duc, T. 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The authors have nothing to disclose.

During the setup of the IDA, it is critical to test that the source and the drain are not shorted together prior to electrochemical gating measurements, as this will alter the ISD vs. EG curve and could lead to erroneous results and interpretations. It is also critical to select VSD and v such that the current is linearly dependent on VSD and independent of v. If this is not the case, then the equations described above cannot be utilized to calculate conductivity.
<p class=...

Extracellular electron transport (EET) is a process that enables certain microorganisms to transport electrons between intracellular metabolic processes and insoluble electron acceptors or donors that reside outside the cell, ranging from natural minerals to electrodes. In some cases, EET enables microorganisms to form electrically conductive multi-cell thick biofilms on electrode surfaces, in which cells not in direct contact with the electrode can still utilize it as a metabolic electron acceptor or donor. There is considerable interest in such biofilms as electrode catalysts for various applications, such as microbial electrosynthesis, contaminant sensing/removal, and remote energy generation and storage,7,8,9,10,11,12,13,14 due to the diversity of metabolic processes performed by microorganisms and the durability of microbial biofilms compared to enzyme-based bioelectrodes.15,16 In addition, EET pathways may potentially be utilized to electrically control or signal changes in naturally occurring or genetically engineered microbial metabolic processes involved, for example, in production of a desired product or detection of a target analyte or stimulus. The electrical conductivity of electrocatalytic biofilms, which sets them apart from other biological materials, is a central aspect of their electrocatalytic properties, yet little is understood about the underlying EET process in the electrode environment, and that which is known is highly contested.17,18,19,20,21,22,23,24
Described here is a 2-electrode method to measure conductivity through living, electrode-grown biofilms using interdigitated electrode arrays (IDAs). IDAs consist of parallel rectangular electrodes patterned on flat glass surface such that every other band is connected at opposite sides of the array resulting in 2 electrodes (the source and drain). Careful examination of an IDA (see for example, Figure 6.12b of ref #1) reveals that that the gaps separating adjacent bands are also connected in such a way as to form a single gap that weaves back and forth across the array separating the two electrodes. The result is a long and narrow gap separating the source and drain electrodes, yielding very high source-drain currents when a conductive material is formed, cast, polymerized, or grown (in the case of the type of biofilms considered here) over the array. In addition, the small size of the electrodes results in small background current due to capacitance charging and to change in oxidation state of the conductive material with change in gate potential, since the amount of material needed to make conductivity measurements using IDAs is so small. The technique of IDA-based electrochemical gating described here, developed to characterize thin film conductive polymers,2,3,4,25 has only recently been applied to living systems.18 Another technique used to measure conductivity of living biofilms utilized a large format split source and drain electrodes and source meters to set the gate potential.26,27 However, concerns over these methods have been detailed previously.18
The protocol below encapsulates our experience with making conductivity measurements of living Geobacter sulfurreducens and biocathode MCL biofilms. G. sulfurreducens is a model electrode reducing organism able to use insoluble materials, including electrodes, as the sole metabolic electron acceptor. Additionally, it forms thick biofilms that are able to transport electrons over multiple cell lengths, making it an ideal model organism to study anodic long-distance extracellular electron transfer. We also include details for the study of biocathode MCL, an aerobic, autotrophic mixed community biofilm isolated from the cathode of a benthic microbial fuel cell. Biocathode MCL (named for the three primary constituents &#8211; Marinobacter, Chromatiaceaea and Labrenzia) is capable of oxidizing an electrode as its sole electron donor and transporting electrons over multiple cell lengths, making it an interesting cathodic system to study. Additionally, biocathode MCL has the highest reported conductivity for a living system to date using these methods. The inclusion of these diverse electroactive biofilms in this protocol is meant to highlight that this technique is applicable to measure the transport of electrons through any living biofilm able to electrically interact with electrodes.

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			<td height="21" style="height:21px;width:259px;">IDAs</td>
			<td style="width:192px;">CH Instruments</td>
			<td style="width:219px;">012125</td>
			<td style="width:337px;">Manufactured by ALS-Japan; sold by CH Instruments</td>
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			<td height="21" style="height:21px;width:259px;">Wire</td>
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			<td height="43" style="height:43px;width:259px;">Conductive silver epoxy</td>
			<td style="width:192px;">Electron microscopy sciences</td>
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			<td>Scotchast flame retardant compound</td>
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			<td height="21" style="height:21px;width:259px;">5 mL pipette tips</td>
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			<td height="21" style="height:21px;width:259px;">5 mL pipettor</td>
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			<td>89079-976</td>
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			<td height="43" style="height:43px;width:259px;">Freshwater medium components</td>
			<td style="width:192px;">Sigma Aldrich</td>
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			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Ammonium chloride</td>
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			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium phosphate monobasic</td>
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			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium bicarbonate</td>
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			<td height="43" style="height:43px;width:259px;">Artificial seawater medium components</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:219px;">All standard laboratory chemicals</td>
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			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium chloride</td>
			<td style="width:192px;"></td>
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			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Magnesium chloride hexahydrate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Magnesium sulfate heptahydrate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Potassium chloride</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Sodium bicarbonate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Calcium chloride dihydrate</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Ammonium chloride</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">&#160;&#160;&#160; Potassium phosphate dibasic</td>
			<td style="width:192px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Ag/AgCl reference electrode</td>
			<td style="width:192px;">Basi</td>
			<td style="width:219px;">MF-2079</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:259px;">Graphite rod counter electrode</td>
			<td style="width:192px;">Electron microscopy sciences</td>
			<td>70230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Recirculating water bath</td>
			<td style="width:192px;">Thermo Scientific</td>
			<td>152-5256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Bipotentiostat</td>
			<td style="width:192px;">Pine Instruments</td>
			<td style="width:219px;">WD-20</td>
			<td>http://www.voltammetry.net/pine/aftermath/user</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Stir bars</td>
			<td style="width:192px;">VWR</td>
			<td>58947-114</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">G. sulfurreducens culture</td>
			<td style="width:192px;">ATCC</td>
			<td style="width:219px;">51573</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Jacketed reactor</td>
			<td style="width:192px;">Pine Instruments</td>
			<td>RRPG085</td>
			<td></td>
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characterizing electron transport through living biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

IDAs were wired, insulated and tested to ensure that the two electrodes were electrically isolated from each other (Figure 1). Reactors were assembled, inoculated with G. sulfurreducens, and incubated until a biofilm bridged the gap between the electrodes. The G. sulfurreducens biofilm can be visually seen to be covering the array. Other biofilms may require the researcher to do an electrochemical gating measurements to see if the two electr...

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Characterizing Electron Transport through Living Biofilms

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

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

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8.3K Views.  Naval Research Laboratory. A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

Video: Characterizing Electron Transport through Living Biofilms

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties

We demonstrate a novel synthesis strategy for the preparation of Pr-doped SrTiO3 ceramics via a combination of solid state reaction and spark plasma sintering techniques. Polycrystalline ceramics possessing a unique morphology can be achieved by optimizing the process parameters, particularly spark plasma sintering heating rate. The phase and morphology of the synthesized ceramics were investigated in detail using X-ray diffraction, scanning electron microcopy and energy-dispersive X-ray spectroscopy. It was observed that the grains of these bulk Pr-doped SrTiO3 ceramics were enhanced with Pr-rich grain boundaries. Electronic and thermal transport properties were also investigated as a function of temperature and doping concentration. Such a microstructure was found to give rise to improved thermoelectric properties. Specifically, it resulted in a significant improvement in carrier mobility and the thermoelectric power factor. Simultaneously, it also led to a marked reduction in the thermal conductivity. As a result, a significant improvement (> 30%) in the thermoelectric figure of merit was achieved for the whole temperature range over all previously reported maximum values for SrTiO3-based ceramics. This synthesis demonstrates the steps for the preparation of bulk polycrystalline ceramics of non-uniformly Pr-doped SrTiO3.

Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties

A protocol for the synthesis and processing of polycrystalline SrTiO3 ceramics doped non-uniformly with Pr is presented along with the investigation of their thermoelectric properties.

We demonstrate a novel synthesis strategy for the preparation of Pr-doped SrTiO3 ceramics via a combination of solid state reaction and spark plasma ...

Original Experimental Approach for Assessing Transport Fuel Stability

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several technological changes to fulfill environmental and economic requirements. These developments have resulted in increasingly severe operating conditions whose suitability for conventional and alternative fuels needs to be addressed. For example, fatty acid methyl esters (FAMEs) introduced as biodiesel are more prone to oxidation and may lead to deposit formation. Although several methods exist to evaluate fuel stability (induction period, peroxides, acids, and insolubles), no technique allows one to monitor the real-time oxidation mechanism and to measure the formation of oxidation intermediates that may lead to deposit formation. In this article, we developed an advanced oxidation procedure (AOP) based on two existing reactors. This procedure allows the simulation of different oxidation conditions and the monitoring of the oxidation progress by the means of macroscopic parameters, such as total acid number (TAN) and advanced analytical methods like gas chromatography coupled to mass spectrometry (GC-MS) and Fourier Transform Infrared - Attenuated Total Reflection (FTIR-ATR). We successfully applied AOP to gain an in-depth understanding of the oxidation kinetics of a model molecule (methyl oleate) and commercial diesel and biodiesel fuels. These developments represent a key strategy for fuel quality monitoring during logistics and on-board utilization.

Oxidation stability of transport fuels has become a concern for future fuel development. This work presents an original methodology developed by IFP Energies Nouvelles for assessing fuel stability using two different reactors. This methodology was successfully applied to gain an in-depth understanding of the oxidation kinetics and pathways of model molecules and commercial fuels.

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several ...

1,3,5-Triphenylbenzene and Corannulene as Electron Receptors for Lithium Solvated Electron Solutions

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic hydrocarbons (PAH), namely 1,3,5-triphenylbenzene and corannulene, as electron receptors. The solid PAHs were first dissolved in tetrahydrofuran (THF) to form a solution. Metallic lithium was then dissolved into these PAH/THF solutions to yield either blue or greenish blue solutions, colors which are indicative of the presence of solvated electrons. Conductivity measurements at ambient temperature carried out on 1,3,5-triphenylbenzene-based LiSES, denoted by LixTPB(THF)24.7 (x = 1, 2, 3, 4), showed an increase of conductivity with increase of Li:PAH ratio from x = 1 to 2. However, the conductivity gradually decreased upon further increasing the ratio. Indeed the conductivity of LixTPB(THF)24.7 for x = 4 is even lower than for x = 1. Such behavior is similar to that of the previously reported LiSES prepared from biphenyl and naphthalene. Conductivity versus temperature measurements on corannulene-based LiSES, denoted by LixCor(THF)247 (x = 1, 2, 3, 4, 5), showed linear relationships with negative slopes, indicating a metallic behavior similar to biphenyl and naphthalene-based LiSES.

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using 1,3,5-triphenylbenzene (TPB) and corannulene as electron receptors.

The authors report on conductivity studies carried out on lithium solvated electron solutions (LiSES) prepared using two types of polyaromatic ...

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

The Effect of Charging and Discharging Lithium Iron Phosphate-graphite Cells at Different Temperatures on Degradation

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. The degradation of the cells is assessed by using 10 charging and discharging temperature permutations ranging from -20 &#176;C to 30 &#176;C. This allows an analysis of the effect of charge and discharge temperatures on aging, and their associations. A total of 100 charge/discharge cycles were carried out. Every 25 cycles a reference cycle was performed to assess the reversible and irreversible capacity degradation. A multi-factor analysis of variance was used, and the experimental results were fitted showing: i) a quadratic relationship between the rate of degradation and the temperature of charge, ii) a linear relationship with the temperature of discharge, and iii) a correlation between the temperature of charge and discharge. It was found that the temperature combination for charging at +30 &#176;C and discharging at -5 &#176;C led to the highest rate of degradation. On the other hand, the cycling in a temperature range from -20 &#176;C to 15 &#176;C (with various combinations of temperatures of charge and discharge), led to a much lower degradation. Additionally, when the temperature of charge is 15 &#176;C, it was found that the degradation rate is nondependent on the temperature of discharge.

This article describes the effect of dissimilar charging/discharging temperatures on the degradation of lithium iron phosphate-graphite pouch cells, aiming at simulating close to real case scenarios. In total, 10 temperature combinations are investigated in the range -20 to 30 &#176;C in order to analyze the impact of temperature on degradation.

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. ...

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...