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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 stability^3,4. 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.

Protokół

1. Preparing analytical solutions

1. Preparing 0.1 M EDOT in an organic solution
   1. Weigh out 0.213 g of LiClO₄ and transfer it into a 20 mL volumetric flask.
   2. Use a measuring cylinder to take 20 mL of PC from the bottle.
   3. 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.
   4. 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.
   5. Before the electrochemical test, transfer 1 mL of the prepared LiClO₄ solution (0.1 M) into an electrochemical cell (see the Table of Materials).
   6. Use a micropipette (10-100 µL) to add 10.68 µL of EDOT monomer (density: 1.331 g/mL) to the electrochemical cell containing the prepared LiClO₄ solution.
   7. 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).
   8. 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).
   9. 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.
   10. 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).
2. Preparing 0.01 M EDOT in an aqueous solution
   1. Use a micropipette to take 10.68 µL of EDOT and add to 1 mL of ACN in a glass vial.
   2. Add 9 mL of deionized water (18.2 MΩ/cm at 25 °C) to the vial to prepare 10 mL of 0.01 M EDOT solution.
   3. Add 0.11 g of LiClO₄ powder 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.
   4. 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.
   5. Characterize the surface of this modified electrode by SEM.
3. Preparing 0.1 M sodium perchlorate solution
   1. Weigh out 0.245 g of NaClO₄ and transfer it to a glass vial containing 20 mL of deionized water (18.2 MΩ/cm at 25 °C).
   2. 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).
4. Preparing buffer solution
   1. 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 MΩ/cm at 25 °C).
   2. Place the flask in an ultrasonic bath until the powder dissolves completely in the water, resulting in a 0.2 M solution.
   3. 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.
   4. 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.
5. Preparing target analyte solutions
   1. 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.
   2. 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.
6. Preparing milk samples for analysis
   1. 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.
   2. Use a 5 mL micropipette to take 5 mL of each milk sample from the freshly opened bottles.
   3. 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.
7. Preparing electrode pretreatment solutions
   1. 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.
   2. Use the 0.1 M NaOH solution to remove the residue of PEDOT formed on the microelectrode surface after each run.
   3. 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.
   4. Top up the flask to the line with deionized water to prepare 1 L of a 0.5 M H₂SO₄ solution.
      ​NOTE: Prepare H₂SO₄ solution under a fume hood for safety. Use the H₂SO₄ solution in the final electrochemical cleaning step of the microelectrode.

2. Pretreatment of the gold microelectrode

1. Polish the Au microelectrode (10 µm diameter, 3.5 mm width x 7 cm long) on an alumina polishing pad placed on a glass polishing plate (dimensions: 3" x 3" squares) using an alumina slurry for 30 s with circular and eight-shaped hand motions during polishing.
2. 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.
3. 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.
4. 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.

3. Cyclic voltammetry technique

1. Use a suitable potentiostat to run CV as the electrochemical technique of interest.
2. Turn on the potentiostat and the computer attached to it. Make sure the system is connected.
3. 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.
4. Open the potentiostat software on the computer, and in the Setup menu, choose Technique. From the opening window, choose the cyclic voltammetry (CV). Again, go back to the Setup menu and click on Parameters to enter the experimental parameters for the CV run.
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
5. Prepare three electrode setups in a glass electrochemical cell including a working electrode (Au microelectrode (10 µm diameter)), a reference electrode (e.g., silver/silver chloride (Ag/AgCl) in 3 M sodium chloride (NaCl), and a platinum wire counter electrode.
6. 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.
7. Ensure that there are no bubbles on the electrode surfaces.
   1. 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.
   2. If there are bubbles around the reference electrode, tap the tip gently.
   3. 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.
8. 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.
9. Run all experiments at room temperature. For milk samples, let the temperature of the milk samples reach the ambient temperature before running CV.

4. Data collection and analysis

1. 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.
2. 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.

5. Techniques to characterize PEDOT

1. 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.
2. 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.
3. 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.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Acetonitrile	Baker Analyzed HPLC Ultra Gradient Solvent	75-05-8	HPLC grade
Alumina polishing pad	BASi, USA	MF-1040	tan/velvet color
Belgian chocolate milk	Puhoi Valley dairy company, Auckland, NZ	_	Buy from local supermarket
Caramel/white chocolate milk	Puhoi Valley dairy company, Auckland, NZ	_	Buy from local supermarket
CH instrument	CH instruments, Inc. USA	_	Model CHI660E
Counter electrode	BASi, USA	MW-1032	7.5 cm long platinum wire (0.5 mm diameter) auxiliary/counter electrode, 99.95% purity
Disodium hydrogen phosphate (Na2HPO4, 2H2O)	Scharlau Chemie SA, Barcelona, Spain	10028-24-7	Weigh 17.8 g
DURAN bottle	University of Auckland	_	The glasswares were made locally at the University of Auckland
Electrochemical cell	BASi, USA	MF-1208	5-15 mL volume, glass
Electrode Polishing Alumina Suspension	BASi, USA	CF-1050	7 mL of 0.05 µm particle size alumina polish
Espresso milk	Puhoi Valley dairy company, Auckland, NZ	_	Buy from local supermarket
3,4-Ethylenedioxythiophene (EDOT), 97%	Sigma-Aldrich	126213-50-1	Take 10.68 μL from bottle
FEI ESEM Quanta 200 FEG	USA	_	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.
Gold microelectrode	BASi, USA	MF-2006	Working electrode (10 μm diameter)
Lithium perchlorate, ACS reagent, ≥95%	Sigma-Aldrich	7791-03-9	Make 0.1 M solution
Micropipettes	Eppendorf	_	10-100 μL and 100-1000 volumes
MilliQ water	Thermo Scientific, USA	_	18.2 MΩ/cm at 25°C, Barnstead Nanopure Diamond Water Purification System
Propylene carbonate, Anhydrous, 99.7%	Sigma-Aldrich	108-32-7	Take 20 mL from bottle
Reference electrode	BASi, USA	MF-2052	Silver/silver chloride (Ag/AgCl) electrode to be kept in 3 M sodium chloride
Replacement glass polishing plate	BASi, USA	MF-2128	Glass plate as a stand to attach the polishing pad on it
Sodium dihydrogen phosphate  (NaH2PO4, 1H2O)	Sigma-Aldrich	10049-21-5	Weigh 13.8 g
Sodium hydroxide pearls, AR	ECP-Analytical Reagent	1310-73-2	Make 0.1 M solution
Sodium perchlorate, ACS reagent, ≥98%	Sigma-Aldrich	7601-89-0	Make 0.1 M solution
Sulfuric acid (98%)	Merck	7664-93-9	Make 0.5 M solution
Uric acid	Sigma-Aldrich	69-93-2	Make 1 mM solution
Whole milk	Anchor dairy company, Auckland, NZ		Blue cap milk, buy from local supermarket