Thanks to the funding provided by the New Zealand Ministry of Business, Innovation and Employment (MBIE) within the &#34;High Performance Sensors&#34; program.

1. Preparing analytical solutions
<ol>
	<li>Preparing 0.1 M EDOT in an organic solution
	<ol>
		<li>Weigh out 0.213 g of LiClO4 and transfer it into a 20 mL volumetric flask.</li>
		<li>Use a measuring cylinder to take 20 mL of PC from the bottle.</li>
		<li>Add PC to the 20 mL volumetric flask containing LiClO4. Mix the solution by placing the flask in an ultrasonic bath for 30 min. Transfer the solution to a 20 mL glass vial.</li>
		<li>Cover the vial with aluminum foil and insert a long needle attached to a nitrogen pipe into the solution to degas for 10 min. Then, remove the aluminum foil and cap the vial tightly. 
		NOTE: Prepare LiClO4 fresh on the day of the experiment.</li>
		<li>Before the electrochemical test, transfer 1 mL of the prepared LiClO4 solution (0.1 M) into an electrochemical cell (see the Table of Materials).</li>
		<li>Use a micropipette (10-100 &#181;L) to add 10.68 &#181;L of EDOT monomer (density: 1.331 g/mL) to the electrochemical cell containing the prepared LiClO4 solution.</li>
		<li>Run the CV method (see section 3.4 for CV parameters) to start electropolymerization of EDOT on the bare Au microelectrode surface after inserting all electrode setups in the solution. Use this modified electrode to characterize the surface by scanning electron microscopy (SEM).</li>
		<li>To use this modified electrode for sensing purposes, first acclimatize its surface to an aqueous solution by running CV scans in the sodium perchlorate (NaClO4) solution (see section 3.4 for CV parameters).</li>
		<li>Use this organically PEDOT-modified and acclimatized microelectrode (from 1.1.8) to run CV (see section 3.4 for its CV parameters) of a phosphate buffer solution to be used as a background scan. 
		NOTE: Rinse the electrode after each step.</li>
		<li>Finally, take out the electrode from the buffer solution without rinsing, and immediately insert it into uric acid solutions or milk samples for running CV scans (see section 3.4 for CV parameters).</li>
	</ol>
	</li>
	<li>Preparing 0.01 M EDOT in an aqueous solution
	<ol>
		<li>Use a micropipette to take 10.68 &#181;L of EDOT and add to 1 mL of ACN in a glass vial.</li>
		<li>Add 9 mL of deionized water (18.2 M&#937;/cm at 25 &#176;C) to the vial to prepare 10 mL of 0.01 M EDOT solution.</li>
		<li>Add 0.11 g of LiClO4 powder&#160;to the prepared EDOT solution to obtain 0.1 M LiClO4 solution, and mix gently. 
		NOTE: Prepare the electrolyte solutions freshly on the day of the experiment.</li>
		<li>Transfer the prepared solution to the electrochemical cell and start electropolymerization of 0.01 M EDOT on the electrode surface by the CV method (see section 3.4 for CV parameters) after inserting the electrode in the aqueous/ACN solution.</li>
		<li>Characterize the surface of this modified electrode by SEM.</li>
	</ol>
	</li>
	<li>Preparing 0.1 M sodium perchlorate solution
	<ol>
		<li>Weigh out 0.245 g of NaClO4 and transfer it to a glass vial containing 20 mL of deionized water (18.2 M&#937;/cm at 25 &#176;C).</li>
		<li>Use this solution to acclimatize the surface of the organically made PEDOT-modified Au microelectrode to an aqueous solution and to remove excess EDOT. For this purpose, rinse the electrode and insert it into the NaClO4 solution; then run CV for 10 cycles (see section 3.4 for CV parameters).</li>
	</ol>
	</li>
	<li>Preparing buffer solution
	<ol>
		<li>Weigh out 13.8 g of sodium dihydrogen phosphate (NaH2PO4. 1H2O) in a weighing boat. Transfer it to a 500 mL volumetric flask (i.e., the required final volume) and top it up to the line with deionized water (18.2 M&#937;/cm at 25 &#176;C).</li>
		<li>Place the flask in an ultrasonic bath until the powder dissolves completely in the water, resulting in a 0.2 M solution.</li>
		<li>In a new weighing boat, weigh out 17.8 g of disodium hydrogen phosphate (Na2HPO4. 2H2O) and transfer it to another 500 mL volumetric flask. Top it up with deionized water to obtain a 0.2 M solution. Place the flask in an ultrasonic bath to dissolve properly.</li>
		<li>Mix 62.5 mL of sodium dihydrogen phosphate solution with 37.5 mL of disodium hydrogen phosphate solution in a measuring cylinder and transfer the mixture to a 250 mL glass bottle (see the Table of Materials). Top it up with another 100 mL of deionized water to obtain 200 mL of 0.1 M of phosphate buffer solution, pH 6.6. Refrigerate the phosphate buffer for long-term use. 
		NOTE: Bring the buffer to room temperature before each experiment.</li>
	</ol>
	</li>
	<li>Preparing target analyte solutions
	<ol>
		<li>Weigh out 0.0084 g of uric acid (UA) in a weighing boat, and dissolve it in 50 mL of phosphate buffer (pH 6.6) in a volumetric flask to obtain a 1 mM UA solution.</li>
		<li>Degas the solution by nitrogen purging for 10 min. 
		NOTE: It is advisable to prepare the UA solution fresh on the day of the experiment.</li>
	</ol>
	</li>
	<li>Preparing milk samples for analysis
	<ol>
		<li>Obtain a whole milk sample and some milk samples with different flavors (e.g., Espresso milk, Caramel/white chocolate milk, and Belgian chocolate milk) from a local supermarket for electroanalysis. Do not pretreat or dilute the milk samples.</li>
		<li>Use a 5 mL micropipette to take 5 mL of each milk sample from the freshly opened bottles.</li>
		<li>First, run CV of phosphate buffer, pH 6.6, as a background signal. Then, add the 5 mL milk sample into the electrochemical cell, and insert freshly and organically made, PEDOT-modified Au microelectrode and other electrodes into the milk samples and run CV. See section 4 of the protocol for how to analyze the collected data.</li>
	</ol>
	</li>
	<li>Preparing electrode pretreatment solutions
	<ol>
		<li>Weigh out 0.2 g of sodium hydroxide (NaOH) powder and transfer it to a 50 mL volumetric flask to prepare a 0.1 M solution.</li>
		<li>Use the 0.1 M NaOH solution to remove the residue of PEDOT formed on the microelectrode surface after each run.</li>
		<li>Use a glass pipette to withdraw 27.2 mL from a 98% sulfuric acid (H2SO4) bottle. Add it very slowly to a 1 L volumetric flask half-filled with deionized water.</li>
		<li>Top up the flask to the line with deionized water to prepare 1 L of a 0.5 M H2SO4 solution. 
		&#8203;NOTE: Prepare H2SO4 solution under a fume hood for safety. Use the H2SO4 solution in the final electrochemical cleaning step of the microelectrode.</li>
	</ol>
	</li>
</ol>
2. Pretreatment of the gold microelectrode
<ol>
	<li>Polish the Au microelectrode (10 &#181;m diameter, 3.5 mm width x 7 cm long) on an alumina polishing pad placed on a glass polishing plate (dimensions: 3&#34; x 3&#34; squares) using an alumina slurry for 30 s with circular and eight-shaped hand motions during polishing.</li>
	<li>Rinse the Au microelectrode with deionized water, insert it in a glass vial containing 15 mL of absolute ethanol (LR grade), and ultrasonicate for 2 min.</li>
	<li>Rinse the Au microelectrode with ethanol and water and again ultrasonicate it for 4 min in deionized water to remove excess alumina from the electrode surface.</li>
	<li>Finally, remove additional impurities by cycling in 0.5 M H2SO4 for 20 segments between 0.4 and 1.6 V potentials (vs. Ag/AgCl) at a 50 mV/s scan rate. Ensure there are two clear peaks due to the formation and reduction of gold oxide at consistent anodic and cathodic potentials each time the electrode is cleaned in H2SO4.</li>
</ol>
3. Cyclic voltammetry technique
<ol>
	<li>Use a suitable potentiostat to run CV as the electrochemical technique of interest.</li>
	<li>Turn on the potentiostat and the computer attached to it. Make sure the system is connected.</li>
	<li>To test the communication between the computer and the instrument, start the software and switch on the instrument. Use the Hardware Test command under the Setup menu. If a Link Failed error appears, check the connection and port settings.</li>
	<li>Open the potentiostat software on the computer, and in the Setup&#160;menu, choose Technique. From the opening window, choose the cyclic voltammetry (CV). Again, go back to the&#160;Setup menu and&#160;click on Parameters to&#160;enter the experimental parameters for the CV run.
	<ol>
		<li>Use the following CV parameters to run PEDOT electropolymerization in an organic electrolyte on the bare Au microelectrode: initial potential: -0.3 V, final potential: -0.3 V, high potential: 1.2 V, number of segments: 8, scan rates: 100 mV/s, direction: positive.</li>
		<li>Use the following CV parameters to run PEDOT electropolymerization in an aqueous/ACN electrolyte on the bare Au microelectrode: initial potential: -0.3 V, final potential: -0.3 V, high potential: 1.2 V, number of segments: 20, scan rates: 100 mV/s, direction: positive.</li>
		<li>Use the following CV parameters to run the acclimatization step of the organically made PEDOT-modified Au microelectrode: initial potential: -0.2 V, final potential: -0.2 V, high potential: 0.8 V, number of segments: 20, scan rates: 100 mV/s, direction: positive.</li>
		<li>Use the following CV parameters for UA standard solutions and phosphate buffer (pH 6.6) with the bare Au microelectrode: initial potential: 0 V, final potential: 0 V, high potential: 1 V, number of segments: 2, scan rates: 100 mV/s, and direction: positive.</li>
		<li>Use the following CV parameters for UA standard solutions and phosphate buffer (pH 6.6) on the organically made, PEDOT-modified Au microelectrode: initial potential: 0 V, final potential: 0 V, high potential: 0.6 V, number of segments: 2, scan rates: 100 mV/s, and direction: positive.</li>
		<li>Use the following CV parameters for the milk samples and phosphate buffer (pH 6.6) on the organically made, PEDOT-modified Au microelectrode: initial potential: 0 V, final potential: 0 V, high potential: 0.8 V, number of segments: 2, scan rates: 100 mV/s, direction: positive.</li>
	</ol>
	</li>
	<li>Prepare three electrode setups in a glass electrochemical cell including a working electrode (Au microelectrode (10 &#181;m diameter)), a reference electrode (e.g., silver/silver chloride (Ag/AgCl) in 3 M sodium chloride (NaCl), and a platinum wire counter electrode.</li>
	<li>Pass these clean and dried electrodes through the holes of an electrode holder attached to a stand. Then, place the holder above the electrochemical cell to insert the electrodes in the target solution or sample.</li>
	<li>Ensure that there are no bubbles on the electrode surfaces.
	<ol>
		<li>If there are bubbles, remove the electrodes, rinse with deionized water again and pat dry with a tissue. Place the electrodes back into the stand holder and in the solution.</li>
		<li>If there are bubbles around the reference electrode, tap the tip gently.</li>
		<li>If there are bubbles around the counter electrode after it starts running, clean the counter electrode. If the CV scan becomes noisy, clean the electrode surface and check the system connections, wires, and clips.</li>
	</ol>
	</li>
	<li>Ensure that all the three wire connections for reference, working, and counter electrodes are correctly connected, and then start the experiment by clicking on Run at the bottom.</li>
	<li>Run all experiments at room temperature. For milk samples, let the temperature of the milk samples reach the ambient temperature before running CV.</li>
</ol>
4. Data collection and analysis
<ol>
	<li>After running CV, save the data in the desired format (CSV or Bin) in a folder, and then use a USB memory stick to collect it. Analyze the data using appropriate software. Convert CSV files to spreadsheets for easier analysis. 
	NOTE: If data are saved in the format of a Binary file, convert it to the format of Text Comma before data collection in a USB memory stick.</li>
	<li>To analyze the CV of milk samples, subtract the CV of milk from the background CV (i.e., CV of phosphate buffer (pH 6.6) taken before running each milk sample) to produce curves due to milk profile oxidation.</li>
</ol>
5. Techniques to characterize PEDOT
<ol>
	<li>Use a specific type of high-performance SEM to characterize the PEDOT layers made in different electrolytes. 
	NOTE: Here, FEI Quanta 200 ESEM FEG was used; it is equipped with a Schottky field emission gun (FEG) for better spatial resolution. This instrument provides different working modes such as high vacuum, low vacuum, and environmental SEM modes and is equipped with a SiLi (Lithium drifted) Super Ultra-Thin Window EDS detector.</li>
	<li>Check the surface morphology of both bare and PEDOT-modified Au (PEDOT-Au) microelectrodes by SEM after PEDOT electropolymerization in organic and aqueous solutions. Perform the PEDOT electropolymerization on bare Au microelectrodes in aqueous/ACN and organic solutions immediately before checking them by SEM.</li>
	<li>Place the freshly prepared electrodes (a bare Au microelectrode and two of the PEDOT-Au microelectrodes) on the SEM stage horizontally, with their head above the stage at a certain angle.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+conformational+stability+and+electronic+structures+of+modified+polymers+based+on+polythiophene.">Understanding the conformational stability and electronic structures of modified polymers based on polythiophene.</a> Macromolecules. 25 (18), 4652-4657 (1992).</li><li>Kundu, K., Giri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+electronic+structure+of+cyclic+polythiophene+upon+bipolaron+doping.">Evolution of the electronic structure of cyclic polythiophene upon bipolaron doping.</a> Journal of Chemical Physics. 105 (24), 11075-11080 (1996).</li><li>Thomas, C. A., Zong, K., Schottland, P., Reynolds, J. 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L., Martin, D., Otto, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(3,4-ethylene+dioxythiophene)+(PEDOT)+as+a+micro-neural+interface+material+for+electrostimulation.">Poly(3,4-ethylene dioxythiophene) (PEDOT) as a micro-neural interface material for electrostimulation.</a> Frontiers in Neuroengineering. 2, 7 (2009).</li><li>Pranti, A. S., Schander, A., B&#246;decker, A., Lang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+stable+PEDOT:PSS+coating+on+gold+microelectrodes+with+improved+charge+injection+capacity+for+chronic+neural+stimulation.">Highly stable PEDOT:PSS coating on gold microelectrodes with improved charge injection capacity for chronic neural stimulation.</a> Proceedings. 1 (4), 492 (2017).</li><li>&#352;tul&#237;k, K., Amatore, C., Holub, K., Marecek, V., Kutner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrodes:+Definitions,+characterization,+and+applications+(Technical+report).">Microelectrodes: Definitions, characterization, and applications (Technical report).</a> Pure and Applied Chemistry. 72 (8), 1483-1492 (2000).</li><li>&#352;tul&#237;k, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+solid+electrodes.">Activation of solid electrodes.</a> Electroanalysis. 4 (9), 829-834 (1992).</li><li>Motshakeri, M., Travas-Sejdic, J., Phillips, A. R. J., Kilmartin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+electroanalysis+of+uric+acid+and+ascorbic+acid+using+a+poly(3,4-ethylenedioxythiophene)-modified+sensor+with+application+to+milk.">Rapid electroanalysis of uric acid and ascorbic acid using a poly(3,4-ethylenedioxythiophene)-modified sensor with application to milk.</a> Electrochimica Acta. 265, 184-193 (2018).</li><li>Motshakeri, M., Phillips, A. R. J., Travas-Sejdic, J., Kilmartin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+study+of+gold+microelectrodes+modified+with+PEDOT+to+quantify+uric+acid+in+milk+samples.">Electrochemical study of gold microelectrodes modified with PEDOT to quantify uric acid in milk samples.</a> Electroanalysis. 32 (9), 2101-2111 (2020).</li><li>Motshakeri, M., Phillips, A. R. J., Kilmartin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+cyclic+voltammetry+to+analyse+uric+acid+and+reducing+agents+in+commercial+milks.">Application of cyclic voltammetry to analyse uric acid and reducing agents in commercial milks.</a> Food Chemistry. 293, 23-31 (2019).</li></ol>

The authors have nothing to disclose.

The CV method allows for fast and simple measurement of different analytes in foods, wine and beverages, plant extracts, and even biological samples. This technique produces a wide variety of data, including oxidation/reduction peak potentials, peak current values of the target analyte (proportional to concentration), and all other current and potential values after each CV run. Although using CV is relatively easy, the collected data sometimes need to be converted from Binary files to Text Comma format, depending on the potentiostat system used. For instance, in the case of the CH instrument, the data can be saved in Text Comma or CSV formats directly after each run. This makes the data analysis easier in a spreadsheet after converting texts to columns. After the CV scans of the milk or UA standard samples were obtained at the same potential ranges, they were plotted on a single graph for direct comparison. To present the data for publications, graphs can also be plotted in Origin or SigmaPlot and then exported as TIF or the required graphic file types.
Common problems with this method can be artifacts in the CV trace. These can arise from electrical connection errors, likely due to the connection clips (i.e., clips that attach wires to each electrode) that have become rusted or due to gold microelectrodes not being cleaned properly. Using sandpaper to remove rust from the clips or replacing them, and re-cleaning the microelectrode and re-running CV cycles after inserting it in the H2SO4 solution may resolve the issue.
Cleaning the microelectrode is an important step in this experiment, which can otherwise result in a low current signal or noise. Cleaning the microelectrode is also very important as bubbles can form when the microelectrode is not very clean. When the locations of the gold oxidation and reduction peaks and the peak heights obtained are consistent and correct, the electrode is ready to run the electropolymerization. When the potentiostat or electrode connections are faulty, there will be noise in the CV scan, or the output will appear like spreading dots. Before a run, it is important to double-check that all the electrode connections are connected correctly, that there is no gas bubble near the tip of the Ag/AgCl reference electrode, and that the electrodes are not touching in the electrochemical cell. The replacement of the clips and connection wires or tapping the reference electrode tip with a finger can be a useful troubleshooting approach.
During the formation of a PEDOT electrode, as the chosen conducting polymer, the organic electrolyte (LiClO4 in PC) and the aqueous NaClO4 solutions should be degassed before running the electropolymerization. It is imperative to use an EDOT chemical that has not expired or oxidized or been contaminated by other analytical grade chemicals. The fresh PEDOT layers that are formed every time on the electrode surface are different in terms of current growth. If the procedure is kept constant and the electrode is cleaned sufficiently, the CV cycles of electropolymerization would grow by the same current value each time, confirming the accuracy and consistency of the method. It is also worth noting that the amount of the EDOT monomer used in the organic solution was 10 times higher than the EDOT monomer in the aqueous/ACN solution. Although this may seem not comparable, it was considered preferable because our preliminary experiments showed that an aqueous 0.1 M EDOT solution did not form a stable PEDOT layer due to lower solubility in an aqueous electrolyte solution. In contrast, the PEDOT layer formed using 0.01 M EDOT in an organic solution did not have sufficient growth on the electrode surface compared to the aqueous 0.1 M EDOT solution. Hence, those EDOT amounts used for organic and aqueous electropolymerization were selected for this study.
One of the limitations of the CV method when bare electrodes are used is the difficulty to separate peaks when interfering agents exist. However, this problem was resolved when PEDOT was used to modify the electrode surface. For instance, when UA was the target analyte to be detected in milk, it was identified separately from its interfering agent, ascorbic acid, due to the redox mediating role of PEDOT, leading to an earlier and well-separated peak for ascorbic acid. At the same time, even with the PEDOT electrode, when analyzing flavored milk, it can be challenging to separate the UA peak properly from the other ingredients that have close oxidation potentials to UA, leading to a merging of peaks.
To conclude, although troubleshooting may be required intermittently, the use of the CV and PEDOT nanolayers on the electrode surface is advantageous for detecting target analytes such as UA in standard solutions and complex matrix solutions, such as milk samples, without any pretreatments. Compared to the high-performance liquid chromatography technique, this CV method is fast and does not need time-consuming pretreatment steps to remove fat or proteins from milk samples. Further, PEDOT makes the microelectrode highly selective and sensitive, giving a sharp peak for UA analysis.

Electrically conducting polymers are organic materials widely used in bioelectronic devices to improve interfaces. Similar to conventional polymers, conducting polymers are easy to synthesize and are flexible during processing1. Conducting polymers can be synthesized using chemical and electrochemical methods; however, electrochemical synthesis approaches are particularly favorable. This is mainly due to their ability to form thin films, allow simultaneous doping, capture molecules in the conducting polymer, and most importantly, the simplicity of the synthesis process1. In addition, conducting polymers form uniform, fibrous, and bumpy nanostructures, firmly adherent to the electrode surface, which increases the active surface area of the electrode2.
In the 1980s, certain polyheterocycles, such as polypyrrole, polyaniline, polythiophene, and PEDOT, were developed that showed good conductivity, ease of synthesis, and stability3,4. Although polypyrrole is better understood than other polymers (e.g., polythiophene derivatives), it is prone to irreversible oxidation5. Thus, PEDOT has certain advantages over the rest as it has a much more stable oxidative state and retains 89% of its conductivity compared to polypyrrole under similar conditions6. In addition, PEDOT is known for high electroconductivity (~500 S/cm) and a moderate band gap (i.e., band gaps or energy gaps are regions with no charge and refer to the energy difference between the top of a valence band and the bottom of a conduction band)7.
Furthermore, PEDOT has electrochemical properties, needs lower potentials to be oxidized, and is more stable over time than polypyrrole after being synthesized7. It also has good optical transparency, which means its optical absorption coefficient, especially in the form of PEDOT-polystyrene sulfonate (PEDOT-PSS), is in the visible region of the electromagnetic spectrum at 400-700 nm7. In the formation of PEDOT electrochemically, EDOT monomers oxidize at the working electrode to form radical cations, which react with other radical cations or monomers to create PEDOT chains that deposit on the electrode surface1.
Different controlling factors are involved in the electrochemical formation of PEDOT films, such as electrolyte, electrolyte type, electrode setup, deposition time, dopant type, and solvent temperature1 PEDOT can be generated electrochemically by passing current through an appropriate electrolyte solution. Different electrolytes such as aqueous (e.g., PEDOT-PSS), organic (e.g., PC, acetonitrile), and ionic liquids (e.g., 1-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF4)) can be used8.
One of the advantages of PEDOT coatings is that it can significantly decrease the impedance of a Au electrode in the 1 kHz frequency range by two or three orders of magnitude, which makes it helpful to increase the sensitivity of direct electrochemical detection of neural activity9. Moreover, the charge storage capacity of the PEDOT-modified electrodes increases and results in faster and lower potential responses when stimulation charge is transferred through PEDOT10. In addition, when polystyrene sulfonate (PSS) is used as a dopant for PEDOT formation on Au microelectrode arrays, it creates a rough, porous surface with a high active surface area, lower interface impedance, and higher charge injection capacity11. For the electropolymerization step, EDOT-PSS usually makes a dispersion in an aqueous electrolyte.
However, EDOT is soluble in chloroform, acetone, ACN, and other organic solvents such as PC. Therefore, in this study, a mixture of water was used with a small volume of ACN in a 10:1 ratio to make a soluble EDOT solution before electropolymerization starts. The purpose of using this aqueous electrolyte is to omit the acclimatization step in the preparation of PEDOT-modified microelectrode and shorten the steps. The other organic electrolyte used to compare with the aqueous/ACN electrolyte is PC. Both electrolytes contain LiClO4 as a dopant to help in oxidizing the EDOT monomer and forming the PEDOT polymer.
Microelectrodes are voltammetric working electrodes with smaller diameters than macroelectrodes, approximately tens of micrometers or less in dimension. Their advantages over macroelectrodes include enhanced mass transport from the solution toward the electrode surface, generating a steady-state signal, a lower ohmic potential drop, a lower double-layer capacitance, and an increased signal-to-noise ratio12. Similar to all solid electrodes, microelectrodes need to be conditioned before analysis. The appropriate pretreatment or activation technique is mechanical polishing to obtain a smooth surface, followed by an electrochemical or chemical conditioning step, such as potential cycling over a particular range in a suitable electrolyte13.
CV is very commonly used in the electrochemical polymerization of PEDOT by inserting electrodes in a monomer solution that involves a suitable solvent and dopant electrolyte. This electrochemical technique is beneficial in providing direction information such as the reversibility of conducting polymer doping processes and the number of transferred electrons, diffusion coefficients of analytes, and the formation of reaction products. This paper describes how two different electrolytes used for the electropolymerization of PEDOT can generate thin nanostructure films with a potential sensing application that depends on the morphology and other intrinsic properties.

<table><tbody><tr><td>Acetonitrile</td><td>Baker Analyzed HPLC Ultra Gradient Solvent</td><td>75-05-8</td><td>HPLC grade</td></tr><tr><td>Alumina polishing pad</td><td>BASi, USA</td><td>MF-1040</td><td>tan/velvet color</td></tr><tr><td>Belgian chocolate milk</td><td>Puhoi Valley dairy company, Auckland, NZ</td><td>_</td><td>Buy from local supermarket</td></tr><tr><td>Caramel/white chocolate milk</td><td>Puhoi Valley dairy company, Auckland, NZ</td><td>_</td><td>Buy from local supermarket</td></tr><tr><td>CH instrument</td><td>CH instruments, Inc. USA</td><td>_</td><td>Model CHI660E</td></tr><tr><td>Counter electrode</td><td>BASi, USA</td><td>MW-1032</td><td>7.5 cm long platinum wire (0.5 mm diameter) auxiliary/counter electrode, 99.95% purity</td></tr><tr><td>Disodium hydrogen phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Scharlau Chemie SA, Barcelona, Spain</td><td>10028-24-7</td><td>Weigh 17.8 g</td></tr><tr><td>DURAN bottle</td><td>University of Auckland</td><td>_</td><td>The glasswares were made locally at the University of Auckland</td></tr><tr><td>Electrochemical cell</td><td>BASi, USA</td><td>MF-1208</td><td>&amp;nbsp;5-15 mL volume, glass</td></tr><tr><td>Electrode Polishing Alumina Suspension</td><td>BASi, USA</td><td>CF-1050</td><td>7 mL of 0.05 &amp;micro;m particle size alumina polish</td></tr><tr><td>Espresso milk</td><td>Puhoi Valley dairy company, Auckland, NZ</td><td>_</td><td>Buy from local supermarket</td></tr><tr><td>3,4-Ethylenedioxythiophene (EDOT), 97%</td><td>Sigma-Aldrich</td><td>126213-50-1</td><td>Take 10.68 &amp;mu;L from bottle</td></tr><tr><td>FEI ESEM Quanta 200 FEG</td><td>USA</td><td>_</td><td>SEM equipped with a Schottky field emission gun (FEG) for optimal spatial resolution. The instrument can be used in high vacuum mode (HV), low-vacuum mode (LV) and the so called ESEM (Environmental SEM) mode.&amp;nbsp;</td></tr><tr><td>Gold microelectrode</td><td>BASi, USA</td><td>MF-2006</td><td>Working electrode (10 &amp;mu;m diameter)</td></tr><tr><td>Lithium perchlorate, ACS reagent, &amp;ge;95%</td><td>Sigma-Aldrich</td><td>7791-03-9</td><td>Make 0.1 M solution</td></tr><tr><td>Micropipettes</td><td>Eppendorf</td><td>_</td><td>10-100 &amp;mu;L and 100-1000 volumes</td></tr><tr><td>MilliQ water</td><td>Thermo Scientific, USA</td><td>_</td><td>18.2 M&amp;Omega;/cm at 25&amp;deg;C, Barnstead Nanopure Diamond Water Purification System</td></tr><tr><td>Propylene carbonate, Anhydrous, 99.7%</td><td>Sigma-Aldrich</td><td>108-32-7</td><td>Take 20 mL from bottle</td></tr><tr><td>Reference electrode</td><td>BASi, USA</td><td>MF-2052</td><td>Silver/silver chloride (Ag/AgCl) electrode to be kept in 3 M sodium chloride</td></tr><tr><td>Replacement glass polishing plate</td><td>BASi, USA</td><td>MF-2128</td><td>Glass plate as a stand to attach the polishing pad on it</td></tr><tr><td>Sodium dihydrogen phosphate&amp;nbsp; (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 1H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>10049-21-5</td><td>Weigh 13.8 g</td></tr><tr><td>Sodium hydroxide pearls, AR</td><td>ECP-Analytical Reagent</td><td>1310-73-2</td><td>Make 0.1 M solution</td></tr><tr><td>Sodium perchlorate, ACS reagent, &amp;ge;98%</td><td>Sigma-Aldrich</td><td>7601-89-0</td><td>Make 0.1 M solution</td></tr><tr><td>Sulfuric acid (98%)</td><td>Merck</td><td>7664-93-9</td><td>Make 0.5 M solution</td></tr><tr><td>Uric acid</td><td>Sigma-Aldrich</td><td>69-93-2</td><td>Make 1 mM solution</td></tr><tr><td>Whole milk</td><td>Anchor dairy company, Auckland, NZ</td><td></td><td>Blue cap milk, buy from local supermarket</td></tr></tbody></table>

electrochemical preparation of poly(3,4-ethylenedioxythiophene) layers on gold microelectrodes for uric acid-sensing applications

Two different methods for the synthesis of poly(3,4-ethylenedioxythiophene) (PEDOT) on gold electrodes are described, using electropolymerization of 3,4-ethylenedioxythiophene (EDOT) monomer in an aqueous and an organic solution. Cyclic voltammetry (CV) was used in the synthesis of PEDOT thin layers. Lithium perchlorate (LiClO4) was used as a dopant in both aqueous (aqueous/acetonitrile (ACN)) and organic (propylene carbonate (PC)) solvent systems. After the PEDOT layer was created in the organic system, the electrode surface was acclimatized by successive cycling in an aqueous solution for use as a sensor for aqueous samples. 
The use of an aqueous-based electropolymerization method has the potential benefit of removing the acclimatization step to have a shorter sensor preparation time. Although the aqueous method is more economical and environmentally friendly than the organic solvent method, superior PEDOT formation is obtained in the organic solution. The resulting PEDOT electrode surfaces were characterized by scanning electron microscopy (SEM), which showed the constant growth of PEDOT during electropolymerization from the organic PC solution, with rapid fractal-type growth on gold (Au) microelectrodes.

Cyclic voltammetry is an easy technique to form a thin PEDOT layer on a Au microelectrode surface to increase the electrode conductivity and sensitivity during electrochemical sensing of target analytes. This protocol demonstrates the method of electropolymerization of 0.1 M EDOT from an organic solution compared to 0.01 M EDOT from an aqueous electrolyte solution. Running 10 cycles in aqueous/ACN solution results in a moderate growth of PEDOT comparable to that observed with the 4 cycles in LiClO4/PC solution. Figure 1 shows a distinct difference between EDOT electropolymerized in aqueous/ACN and organic solutions, with the subsequent PEDOT layers formed by applying CV. It is evident that upon cycling from -0.3 to +1.2 V (vs. Ag/AgCl in 3 M NaCl) at a 100 mV/s scan rate, the polymer started to oxidize at 0.9 V in both electrolyte solutions (Figure 1A and Figure 1C), with an oxidation peak seen at 1 V in the aqueous/ACN solution.
Upon closer inspection, the PEDOT layers made in the organic solution after 4 cycles display higher current values (~2.9 &#181;A) at 1.2 V compared with the current value (0.23 &#181;A) seen for PEDOT layers formed at this potential in the aqueous/ACN solution. When the number of electropolymerization cycles increases during CV runs, the new layers of PEDOT are made gradually on the electrode surface to increase the thickness of layers. This could be due to the redox reactions occurring in the internal PEDOT between the potential range of 0 to 0.7 V (Figure 1B and Figure 1D).&#160;Figure 1B and Figure 1D depict a narrower potential rangeto show the PEDOT growth correctly. The current density values on the right side of each graph were calculated by dividing the current values on the left side of the graph by the geometric surface area of the unmodified Au microelectrode (78.5 &#215; 10-8 cm2, r = 5 &#215; 10-4 cm).
SEM analysis was performed to confirm the efficiency of PEDOT layer formation by electropolymerization in the two electrolyte solutions (Figure 2A-F). The images taken by SEM were chosen at different magnifications (4000x, 30000x, and 60000x). The geometric surface area of bare and PEDOT-Au microelectrodes can be established using these images. Figure 2A confirms a diameter of ~10 &#181;m for the bare gold microelectrode; hence, the surface area is calculated to be ~78.5 &#215; 10-8 cm2. The diameter of the PEDOT nanostructure formed in the organic solution after 4 cycles at the surface of the Au microelectrode was ~40 &#181;m (Figure 2C,D). By contrast, the PEDOT growth on the electrode surface was lower after 10 cycles of electropolymerization. It is seen as mountainous polymeric features on the electrode circumference with a depression in the center (Figure 2E,F).
The SEM images provide evidence for the superiority of the PEDOT growth in the organic solution compared to the aqueous/ACN system and the creation of a very porous nanostructure extending out from the microelectrode in a cauliflower-like shape. This PEDOT microelectrode prepared in an organic solution was used for sensing applications, particularly for UA detection in standard solutions and milk samples. Figure 3 shows the CV for the detection of UA in a standard solution at a bare Au microelectrode and the PEDOT sensor. The performance of the bare Au microelectrode for UA detection is characterized by steady-state currents obtained at potentials higher than 0.8 V due to radial diffusion of UA to the electrode surface (Figure 3A). A linear calibration curve was plotted based on the average currents at 0.8 V for the UA concentration range of 62.5 to 1000 &#181;M after three replicate CV runs (Figure 3B).
By comparing the slope of the calibration curve equations, the PEDOT microelectrode was found to have 100 times higher sensitivity than the bare microelectrode. Interestingly, the detected UA range using the PEDOT sensor made in an organic solution was lower, from 6.25 to 200 &#181;M, calculated by measuring the current value at the tip of the sharp anodic peak (Figure 3C,D). The calibration curve data for the PEDOT electrode were used to measure the limit of detection (LOD) and limit of quantification (LOQ) of the UA for the modified electrode. The slope of the calibration curve equation (b) and the evaluated standard error of the intercept (s) were used to measure the LOD and LOQ values (95% confidence level)-7 &#181;M and 24 &#181;M14, respectively-by using equations (1) and (2).
LOD= 3s/b&#160;(1)
LOQ= 10s/b&#160;(2)
The sensitivity of the organically made PEDOT-modified sensor is an important factor. This is calculated by dividing the calibration curve slope by the geometric surface area of the working electrode, which is 397 &#181;A &#181;M-1 cm-2.
Another application of the PEDOT sensor synthesized in the organic solution was to analyze UA content in real samples, e.g., regular fresh milk and selected flavored milk samples (Figure 4). The advantage of this technique is that UA levels in milk samples can be measured without any pretreatment or dilution. The performance of this PEDOT-Au microelectrode sensor was compared to the PEDOT-modified glassy carbon macroelectrode (PEDOT-GC) prepared by the same method in the organic solution15. The anodic peak current for UA in regular milk at 0.35 V (vs. Ag/AgCl) using the PEDOT microelectrode was ~28.4 nA, which is equivalent to 82.7 &#181;M using the equation of the calibration curve in Figure 3D (y = 0.3x + 2.6, R2 = 0.993). This value was ~83.4 &#181;M for UA in the regular milk determined using the PEDOT-GC15. The second large oxidation peak in the CV scan of regular milk at 0.65 V (Figure 4A) is related to oxidizable compounds, including electroactive amino acids such as cysteine, tryptophan, and tyrosine15,16. The current density of this peak from the regular milk is over 200 times larger than that obtained using a previously reported PEDOT-GC15. This shows a more sensitive response of the microelectrode covered by PEDOT layers compared to the PEDOT-modified macroelectrode.
The CV scans obtained for caramel and white chocolate milk samples can be seen in Figure 4A. It shows a clear peak at 0.36 V for UA, along with an additional peak current of ~42 nA at 0.56 V that is merged with the peak at 0.66 V. This additional peak at 0.56 V can be related to the presence of vanillic acid, one of the ingredients of flavored milk. The CV of the Belgian chocolate milk sample indicates a new set of anodic peaks at 0.26 V, 0.36 V, and 0.66 V and a cathodic peak at 0.22 V. The chocolate profile resembles the catechin redox profile along with the other polyphenolic antioxidants present in chocolate or cocoa15. Thus, the catechin oxidation and reduction peaks appear at 0.26 V and 0.22 V, respectively. The 0.36 V peak current, which appears as a sharp peak at the tail of the catechin peak, is due to UA oxidation. Figure 4B shows a CV of Colombian espresso milk sample, which exhibits broad anodic and cathodic peak currents at 0.35 V and 0.23 V, respectively, at the PEDOT-Au, which are due to the major phenolic antioxidants in coffee, namely, chlorogenic and caffeic acids. Because the geometric surface area of the PEDOT microelectrode is higher than that of the PEDOT macroelectrode, the current densities of the UA peaks in these milk samples are ~150 to 500 times larger at the PEDOT-Au15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62707/62707fig01.jpg" /> 
Figure 1:&#160;Electropolymerization of PEDOT on a gold microelectrode. PEDOT prepared by (A, B) 10 CV scans in an aqueous solution (0.01 M EDOT in 1 mL ACN + 9 mL deionized water + 0.1 M LiClO4); and (C, D) using 4 CV scans in an organic electrolyte solution (0.1 M EDOT in 1 mL of 0.1 M LiClO4/PC). B and D are expanded versions of A and C to visualize the PEDOT currents clearly. Scan rate = 100 mV/s. This figure has been modified from15. Abbreviations: PEDOT = poly(3,4-ethylenedioxythiophene); CV = cyclic voltammetry; EDOT = 3,4-ethylenedioxythiophene; ACN = acetonitrile; LiClO4 = lithium perchlorate; Ag = silver; AgCl = silver chloride. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62707/62707fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62707/62707fig02.jpg" /> 
Figure 2: SEM images. (A and B) Bare gold microelectrode (Au). PEDOT-modified gold microelectrodes prepared in (C and D) organic solution after 4 cycles of electropolymerization and (E and F) aqueous solution after 10 cycles of electropolymerization at different magnifications. This figure has been modified from15. Abbreviations: SEM = scanning electron microscopy; PEDOT = poly(3,4-ethylenedioxythiophene). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62707/62707fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62707/62707fig03.jpg" /> 
Figure 3: Cyclic voltammograms for different concentrations of UA in phosphate buffer, pH 6.6. (A) Bare gold microelectrode (background subtracted) and (C) PEDOT-modified gold microelectrode (background subtracted), measurements taken immediately after inserting the electrode into the solution at a scan rate of 100 mV/s. (B) Plot of limiting current at 0.8 V versus UA concentration on the bare gold microelectrode. (D) Plot of anodic peak current (Ip.a/&#181;A) versus UA concentration on the PEDOT-modified gold microelectrode. (n=3). This figure has been modified from15. Abbreviations: UA = uric acid; PEDOT = poly(3,4-ethylenedioxythiophene). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62707/62707fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62707/62707fig04.jpg" /> 
Figure 4: Cyclic voltammograms (background subtracted). (A) Regular milk, Belgian chocolate milk, caramel, and white chocolate milk, and (B) regular milk and Colombian espresso milk on a PEDOT-modified gold microelectrode (10 &#181;m diameter) at 100 mV/s. This figure has been modified from15. Abbreviation: PEDOT = poly(3,4-ethylenedioxythiophene). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62707/62707fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Electrochemical Preparation of Poly3,4-Ethylenedioxythiophene Layers on Gold Microelectrodes for Uric Acid-Sensing Applications at JoVE.com

Electrochemical Preparation of Poly3,4-Ethylenedioxythiophene Layers on Gold Microelectrodes for Uric Acid-Sensing Applications

We describe aqueous and organic solvent systems for the electropolymerization of poly(3,4-ethylenedioxythiophene) to create thin layers on the surface of gold microelectrodes, which are used for sensing low molecular weight analytes.

Electrochemical Preparation of Poly(3,4-Ethylenedioxythiophene) Layers on Gold Microelectrodes for Uric Acid-Sensing Applications

Two different methods for the synthesis of poly(3,4-ethylenedioxythiophene) (PEDOT) on gold electrodes are described, using electropolymerization of ...

electrochemical-preparation-poly-34-ethylenedioxythiophene-layers-on

Nanyang Technological University

Research

JoVE Journal

Chemistry

3.7K Views.  The University of Auckland. We describe aqueous and organic solvent systems for the electropolymerization of poly(3,4-ethylenedioxythiophene) to create thin layers on the surface of gold microelectrodes, which are used for sensing low molecular weight analytes.

Video: Electrochemical Preparation of Poly3,4-Ethylenedioxythiophene Layers on Gold Microelectrodes for Uric Acid-Sensing Applications

Reductive Electropolymerization of a Vinyl-containing Poly-pyridyl Complex on Glassy Carbon and Fluorine-doped Tin Oxide Electrodes

Controllable electrode surface modification is important in a number of fields, especially those with solar fuels applications. Electropolymerization is one surface modification technique that electrodeposits a polymeric film at the surface of an electrode by utilizing an applied potential to initiate the polymerization of substrates in the Helmholtz layer. This useful technique was first established by a Murray-Meyer collaboration at the University of North Carolina at Chapel Hill in the early 1980s and utilized to study numerous physical phenomena of films containing inorganic complexes as the monomeric substrate. Here, we highlight a procedure for coating electrodes with an inorganic complex by performing reductive electropolymerization of the vinyl-containing poly-pyridyl complex onto glassy carbon and fluorine doped tin oxide coated electrodes. Recommendations on electrochemical cell configurations and troubleshooting procedures are included. Although not explicitly described here, oxidative electropolymerization of pyrrole-containing compounds follows similar procedures to vinyl-based reductive electropolymerization but are far less sensitive to oxygen and water.

A procedure for performing reductive electropolymerization of vinyl-containing compounds onto glassy carbon and fluorine doped tin-oxide coated electrodes is presented. Recommendations on electrochemical cell configurations and troubleshooting procedures are included. Although not explicitly described here, oxidative electropolymerization of pyrrole-containing compounds follows similar procedures to vinyl-based reductive electropolymerization.

Controllable electrode surface modification is important in a number of fields, especially those with solar fuels applications. Electropolymerization is ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Bioengineering

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

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 ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

Immunology and Infection

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

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

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

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

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

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...

Measuring Electron Transfer Activity in Shewanella oneidensis Using OECTs

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.

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 ...

Electrochemical Preparation of Poly(3,4-Ethylenedioxythiophene) Layers on Gold Microelectrodes for Uric Acid-Sensing Applications

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Bridging the Bio-Electronic Interface with Biofabrication

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

Bioinspired Soft Robot with Incorporated Microelectrodes

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

Translating Extracellular Electron Transfer Activities with Organic Electrochemical Transistors