Electrochemical Treatment in KOH Renews and Activates Carbon-Fiber Microelectrode Surfaces

Abstract

Carbon-fiber microelectrodes (CFMEs) are the standard electrodes for fast-scan cyclic voltammetry (FSCV) detection of neurotransmitters. CFMEs are generally used untreated but the surface can be activated with different treatments to improve electrochemical performance. In this work, we explored electrochemical treatments to clean and activate the CFME surface. We used different solution conditions for electrochemical treatment, and found electrochemical pretreatment in KOH outperforms treatment in KCl, H₂O₂ or HCl by accelerating the surface renewal process. The etching rate of carbon with electrochemical treatment in KOH is 37 nm/min, which is 10 times faster than in the other solutions. Electrochemical treatment in KOH for several minutes regenerates a new carbon surface, which introduces more oxygen functional groups beneficial for adsorption and electron transfer. The KOH-treated CFMEs improved the limit of detection (LOD) to 9 ± 2 nM from 14 ± 4 nM for untreated CFMEs, and they successfully detected stimulated dopamine release in rat brain slices, demonstrating that they are stable and sensitive enough to use in biological systems. Electrochemical treatment in KOH completely restores the electrode sensitivity after biofouling. The proposed electrochemical treatment is simple, fast, and can be applied prior to using CFMEs or after use to restore the surface. Thus, the method has potential to be a standard step to clean the carbon surface, or restore the sensitivity of electrodes from biofouling.

Introduction

Carbon fibers are the standard electrode material for in vivo detection of neurotransmitters because of their conductivity, biocompatibility and small size.[1, 2] The tiny carbon-fiber microelectrode (CFME, usually 7–10 µm diameter) is advantageous to approach the release sites of neurotransmitters, and fast-scan cyclic voltammetry (FSCV) allows real-time monitoring of the concentration changes.[3–5] Carbon electrodes can adsorb impurities on the surface which leads to decreased electrochemical activity, so treatments to clean the carbon surface are beneficial to the electrochemical properties.[6] For example, polishing before use is a standard procedure for glassy carbon electrodes to obtain a clean carbon surface.[6] However, CFMEs are tiny and typically fabricated into cylindrical electrodes with glass capillary insulation, so they cannot be easily polished or mechanically cleaned. In most of the biological applications, CFMEs are directly used without any mechanical treatment, which may lead to decreased sensitivity and electron transfer kinetics and differences in surface properties between electrodes.[7, 8] Treatments may activate the surface and lead to a more consistent carbon surface property among electrodes. Also, carbon fiber electrodes are subject to electrode fouling by adsorbing the biomolecules and polymerized products.[9–11] Therefore, a simple and effective treatment method is needed to regenerate the electrode surface.

Various treatments for CFMEs have been employed to improve the adsorption, electron transfer kinetics, and anti-fouling properties.[12–15] Most of the dry etching methods, including heat treatment,[16, 17] laser treatment,[18, 19] flame etching,[20] and plasma treatment[21] clean the electrode surface, and also increase surface roughness or add functional groups.[19, 21] However, these methods require specialized equipment and the surface may not be stable as it adsorbs impurities from air or slowly deactivates in tens of minutes.[18] Electrochemical treatment is in contrast very simple; using a high potential, the surface carbon is oxidized which increases the amount of oxygen functional groups on the surface.[6, 22] The Wightman group demonstrated an extended anodic waveform in buffer causes oxidative etching of the CFME surface, which generates a clean surface and restores sensitivity from electrode fouling.[23] The Mao group demonstrated KOH treatment enhances the electron transfer kinetics for a carbon-nanotube modified CFME.[24, 25] In other work, acid and oxidative reagent were also used with electrochemical treatment of carbon electrodes.[26, 27] While different solution media have been applied to electrochemically activate the carbon surface, there has been no systematic study of how the treatment solution affects electrochemical treatment. In particular, studies that correlate surface properties and electrochemical behavior are needed for understanding the mechanism of electrochemical treatment on carbon electrodes.

In this work, we studied electrochemical treatment using acidic, basic, and oxidative solutions, and compared the surface structures and electrochemical properties of carbon electrodes after treatment. X-ray photoelectron spectroscopy (XPS) and Raman spectra were used to characterize the surface functional groups. By systematically studying the electrochemical treatment conditions, we found that electrochemical treatment in KOH outperforms the other treatments with a rapid renewal of the surface. KOH accelerates the etching rate of carbon surface and generates more carbon-oxygen bonds on the surface. Electrochemical treatment in KOH improves the sensitivity, enhances electron transfer rate, and restores the sensitivity of CFMEs after fouling. Electrochemical treatment in KOH is simple and fast, and therefore it is promising to be used as a standard pre-treatment step of CFMEs. In addition, it is an effective cleaning method for reuse and restoration of the carbon electrode surface.

Experimental section

Chemicals

Dopamine was purchased from Acros Organics (Morris Plains, NJ). A 10 mM stock solution of dopamine was prepared in 0.1 M HClO₄. The working solutions were prepared by diluting the stock solution in a phosphate-buffered saline (PBS) (131.2 mM NaCl, 3.0 mM KCl, 10 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.0 mM Na₂SO₄, and 1.2 mM CaCl₂ with pH adjusted to 7.4) to the desired concentration. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). 1 M KCl, H₂O₂, HCl, and KOH were prepared for the electrochemical treatments (H₂O₂ solution was prepared in 1 M KCl to promote the conductivity). A 5 mM solution of K₃Fe(CN)₆ was prepared in 1 M KCl.

Fabrication of carbon fiber microelectrodes

Carbon-fiber microelectrodes were fabricated by pulling a T-650 carbon fiber (7 µm diameter, Cytec Engineering Materials, West Patterson, NJ) into a glass capillary by vacuum aspiration. The capillary was then pulled into two electrodes by an electrode puller (model PE-21, Narishige, Tokyo, Japan). The exposed carbon fiber was cut to an approximate length of 150 µm. After that, each electrode was sealed by dipping in an 80 °C solution of Epon Resin 828 (Danbury, CT) with 14% (w/w) m-phenylenediamine (Acros Organics, Morris Plains, NJ) for 30 seconds. The electrodes were then simply soaked in acetone for 5 seconds. The epoxied electrode was left overnight at room temperature, then cured in an oven at 100 °C for 2 h, and at 150 °C overnight.

Larger carbon fiber electrodes (diameter ~30 µm, World Precision Instruments, Sarasota, FL) were prepared for SEM imaging, because 3 h electrochemical treatment in KOH completely consumed the 7 µm carbon fiber. The larger carbon fiber electrodes were also used for CV and EIS measurements, because larger surface area results in obvious CV peaks instead of plateau. Otherwise, the regular 7 µm carbon fiber were used. The carbon fibers were inserted in glass capillary and sealed with 5-min epoxy (J-B weld, Sulphur Springs, TX).

Cyclic Voltammetry and Electrochemical Impedance

Cyclic voltammetry and electrochemical impedance spectroscopy were performed with Gamry Reference 600 potentiostat (Warminster, PA). A three-electrode system was used, where the working electrode was a 30 µm CFME, the reference electrode was a standard Ag/AgCl electrode, and the counter electrode was a Pt wire. The CV was performed in 5 mM K₃Fe(CN)₆, from – 0.2 V to 0.6 V at a scan rate of 100 mV/s. In electrochemical impedance measurements, the frequency ranged 100 kHz to 0.1 Hz, the biased DC was 0.2 V, and the amplitude of the AC voltage was 10 mV.

Fast-scan Cyclic Voltammetry

FSCV experiments were performed with a ChemClamp potentiostat and head-stage (Dagan, Minneapolis, MN). A two-electrode system was used, where the working electrode was a CFME and reference electrode was Ag/AgCl. 1 M KCl solution was filled into the glass capillary from the opening end to make an electrical connection from the CFME to the headstage. The buffer and neurotransmitter solutions were injected through a flow cell at 2 mL/min by a syringe pump (Harvard Apparatus, Holliston, MA) and modulated by a six-port loop injector with an air actuator (VIVI Valco Instruments, Houston, TX). The electrochemical data were collected and analyzed with HDCV Analysis software (Department of Chemistry, University of North Carolina at Chapel Hill).

Electrochemical Treatments

The FSCV system was used to treat the T-650 CFMEs for dopamine detection. A regular waveform (-0.4 V to 1.3 V, 400 V/s, 10 Hz) was applied to the CFMEs until the background current stabilized in PBS buffer (usually ~15 min). For pretesting, the response to 1 µM dopamine was measured. Then, a constant 1.5 V was applied to the electrode in the treatment solutions. The treatment solutions were 1 M KCl, H₂O₂, HCl, and KOH. After the treatment, the electrodes were stabilized in PBS buffer again (usually ~5 min), and the background and dopamine current were measured. Unless otherwise specified, the electrochemical treatment was a constant 1.5 V for 3 minutes.

The Gamry potentiostat was used to treat 30 µm diameter CFMEs. For SEM imaging, the 30 µm diameter CFMEs were treated with constant 1.5 V for 3 hours. For CV and EIS tests, the CFMEs were also treated with a constant 1.5 V for 3 minutes.

Surface Characterization

Scanning electron microscope (SEM) images were taken on FEI Quanta 650 SEM (Thermo Fisher Scientific, Waltham, MA). Secondary electron detector was used at an accelerating voltage of 2.0 kV.

X-ray photoelectron spectrometer (Physical Electronics, Chanhassen, MN) was used to obtain elemental composition and bonding information. The Al Kα monochromatic X-ray source (1486.6 eV) was used with a pass energy of 224 eV for elemental composition and 55 eV for electronic state information. The XPS spectra were analyzed with MultiPak software, and the C1s peaks were fitted with sp2 C (283.7 eV), sp3 C (285.2 eV), C-O (287.0 eV), C=O (288.9 eV) and π-π* (291.1 eV) bonds.

Raman spectra was performed on Renishaw InVia Confocal Raman microscope (Renishaw, Hoffman Estates, IL) with 1800 lines/mm diffraction grating. A 514 nm laser was focused on the CFME samples through a 50x objective. Laser intensity was 50% and scan range was 100 cm^-1 to 3200 cm^-1.

Brain Slice Experiments

The animal experiments were approved by the Animal Care and Use Committee in University of Virginia. Male C57BL/7 mice from Jackson Labs (6–8 weeks old) were housed and fed in the vivarium. Mice were anesthetized with isoflurane and decapitated immediately. The mouse brain was removed and transfer to artificial cerebral spinal fluid (aCSF) buffer (0–5°C) within 2 minutes. After 2 minutes recovery in the cold buffer, four hundred-micrometer slices of caudate-putamen were obtained by coronal slicing the brain using a vibratome (LeicaVT1000S, Bannockburn, IL, USA). Slices were transferred into oxygenated aCSF buffer (95% oxygen, 5% CO2), and recovered for 30 minutes before the experiment. Oxygenated aCSF (maintained at 35–37 °C) flowed over the brain slices using a perfusion pump (Watson-Marlow 205U, Wilmington, MA, USA) at a rate of 2 mL/min for all experiments. The electrode was placed 50–100 µm away from the stimulation electrode, and the electrode was inserted around 75 µm depth in the brain tissue. Dopamine release was evoked via biphasic stimulation pulses (300 µA, 5 pulses at 60 Hz).

Results and discussion

Characterization of Electrochemical Treatment in Different Solutions

To explore the effects of different electrochemical treatments on neurotransmitter detection, we performed FSCV and collected the background current and dopamine response before and after the treatment. Four groups of CFMEs were electrochemically treated: 1 M KCl as a control (Figure 1a), 1 M hydrogen peroxide as an oxidizing condition (Figure 1b), 1 M HCl as an acidic condition (Figure 1c), and 1 M KOH as a basic condition (Figure 1d). The potential was held at 1.5 V vs. Ag/AgCl in the solution for 3 minutes. Electrochemical treatments in KCl and H₂O₂ increase both the background and dopamine current approximately 1.2-fold, indicating the activation of the carbon surface, probably due cleaning of the carbon surface, exposing more active surface. For HCl treatment, the background and dopamine current are nearly identical before and after treatment, indicating electrochemical treatment in HCl does not change the properties of the electrode surface. In KOH, electrochemical treatment does not increase the background current, but does increase the dopamine response about 1.5-fold.

Figure 1.

FSCV of dopamine with different treatments. Cyclic voltammograms of background current (top) and detection of dopamine (bottom). Cyclic voltammograms are normalized so the current before treatment is 1. The cyclic voltammograms were collected prior to and after the 3-minute electrochemical treatments in 1 M (a) KCl, (b) H₂O₂, (c) HCl, and (d) KOH solutions. The electrochemical pretreatment was holding at 1.5 V for 3 min. The FSCV waveform was – 0.4 V to 1.3 V at a scan rate of 400 V/s and frequency of 10 Hz.

Surface Characterization after Electrochemical Treatment in Different Conditions

The CFMEs were characterized by SEM to explore the effects of the electrochemical treatments. To amplify the phenomenon of electrochemical etching, we used thicker CFMEs (advertised 30 µm diameter) and prolonged the treatment time to 3 hours. Figure 2 shows the SEM image of the CFMEs treated in different solutions, and Table 1 lists their measured radii and calculated etching rate. After electrochemical etching for 3 hours, the CFMEs radius slightly shrinks in KCl (-0.5 µm), H₂O₂ (-0.4 µm) and HCl (-0.2 µm), indicating electrochemical etching under these conditions is a slow process. In contrast, KOH greatly accelerates the electrochemical etching process and the fiber shrinks by 13.4 µm, which is one magnitude faster than in KCl solution. Also, the treatment in KOH solution results in a rough surface, indicating the treatment increased surface roughness. The electrochemical etching rate of CFMEs in KOH solution is calculated as 37.4 nm/min by simply dividing the radius shrinkage by time. Although the etching rate is dependent on the electrode size, the results imply that several minutes of electrochemical treatment in KOH solution is sufficient to completely renew the carbon surface.

Figure 2.

SEM images of CFMEs (a) without any treatment, (b) treated in 1 M KCl, (c) treated in 1 M H₂O₂, (d) treated in 1 M HCl, and (e) treated in 1 M KOH. The electrodes were treated with a constant potential of 1.5 V for 3 hours.

Table 1.

Radius of the CFMEs before and after the 3 h electrochemical treatment

	No Treatment	KCl	H2O2	HCl	KOH
Radius (n=3)	17.3 ± 0.2	16.8 ± 0.2	16.9 ± 0.1	17.1 ± 0.1	10.6 ± 0.2
Etching rate (nm/min)	-	3.3	2.2	1.5	37.2

Values are mean ± standard deviation.

XPS and Raman spectra were used to explore the chemistry of carbon surface. Figure 3A-E show the C1s spectra of CFME surface after the treatments. The C1s peaks were further deconvoluted into sp² C (283.7 eV), sp³ C (285.2 eV), C-O (287.0 eV), C=O (288.9 eV) and π-π* (291.1 eV) bonds,[28] and their components are listed in Table 2. The C1s peak shape and bond components of KCl group are close to the control group, indicating electrochemical treatment in KCl solution does not affect the surface groups. Treatment in H₂O₂ shifts the peak to smaller binding energy and more sp² component, but the total of sp² and sp³ components are the same to control and KCl groups (Table 2). Treatment in HCl adds more carbon-carbon bonds but decreases carbon-oxygen bonds, which explains why treatment in HCl does not improve the electrochemical response (Figure 1c). In contrast, electrochemical treatment in KOH introduces more C-O bonds, and the oxygen rich surface is beneficial for electron transfer kinetics and adsorption.[6]

Figure 3.

XPS spectra in C1s region and Raman spectra of the treated CMFEs. The XPS C1s peaks were deconvoluted into sp² C (283.7 eV), sp³ C (285.2 eV), C-O (287.0 eV), C=O (288.9 eV) and π-π* (291.1 eV) bonds.

Table 2.

Binding energy (eV) and percentage contributions of XPS C 1s peaks for the untreated and electrochemically treated CFMEs.

	Binding Energy (eV)	Control (%)	KCl (%)	H2O2 (%)	HCl (%)	KOH (%)
sp2 C	283.7	36.3	37.3	50.1	38.9	36.3
sp3 C	285.2	41.1	41.3	28.2	41.6	37.5
Sum of sp2 C and sp3 C		77.4	78.6	78.3	80.5	73.8
C-O	287.0	11.1	10.8	11.9	9.8	15.6
C=O	288.9	9.3	8.2	7.9	8.5	8.4
π-π*	291.1	2.1	2.4	1.3	1.2	2.2

Figure 3F shows the Raman spectra of CFMEs after electrochemical treatments in different solutions. The spectra contain two characteristic peaks for carbon, a D band (~1360 cm^-1) due to defects and a G band (~1580 cm^-1) due to graphitic carbon.[29] The D/G ratio, calculated by the area under the two peaks, is used to evaluate the disorder level of carbon. The D/G ratios after treatment in HCl, H₂O₂, or KOH are slightly less than control, indicating a more graphitic structure. The KOH group exhibits a higher D/G ratio than the other treatments, more similar to control, indicating more edge-plane carbon sites, which are beneficial for neurotransmitter detection.[30, 31] Overall, the Raman spectra are not extremely different after treatment. This may be due to the Raman sampling tens of nanometers to micrometer depths into the surface,[32] while the electrochemical treatments only renews the surface without changing the structure of bulk carbon. In a comparison, XPS characterizes only 1–10 nm depth, which is more indicative of surface structures.[33]

The SEM images and surface characterizations explain the two factors that contribute to the increased Faradaic current of dopamine. The treatment in KOH induces more carbon-oxygen bonds resulting in larger current responses;[34] but it also etches the surface carbon, which slightly reduces the surface area. Therefore, the background current does not increase because the two factors counteract each other. As for dopamine detection, the current response is improved because the renewed surface promotes adsorption.[30]

Optimizing electrochemical pretreatment parameters.

To better optimize the treatment and understand the mechanism, we changed the conditions for electrochemical pretreatment in KOH. First, we compared chemical treatment (without a voltage applied) to electrochemical treatment. When CFMEs are treated with KOH without any potential applied, neither the background current nor the dopamine response changes (Fig. S1). Thus, carbon electrodes are electrochemically etched but not chemically etched by KOH, and effect of KOH solution is to accelerate the electrochemical process.

Next, we studied the effects of treatment time and potential on FSCV background and dopamine currents. Electrochemical treatment time was varied from 0 to 3 minutes and treatment potential was varied from 1.3 V to 1.6 V in KCl (control) and KOH solution. Figures 4a-b show the current responses with different treatment time at a potential of 1.5 V. The background current increases with treatment time in KCl but remains the same in KOH, because KOH etches the surface faster, but also activates the surface. Figure 4b shows the one-minute electrochemical treatment significantly enhances dopamine detection, but there is only a slight increase if the CFMEs are treated for a longer time. Figure 4c-d show the current responses with different treatment potentials for 3 min of electrochemical treatment. When a higher potential is applied in KCl solution, both the background and dopamine current increases due to surface activation.[23] However, in KOH, a very high potential may quickly etch the electrode surface, which decreases the background current slightly (Figure 4c). With a short treatment time (<3 min), dopamine detection is enhanced with larger treatment potential, because higher potential renews the surface faster. We also studied electrochemical treatment with cyclic voltammetry (scanning from-0.4 V to 1.5 V at 400 V/s and 100 Hz). Dopamine current enhancement after cyclic voltammetry treatment in KOH is similar to amperometry treatment (compare Fig. S2 and Figures 4a-b). Thus, short electrochemical treatments in KOH activate the electrode surface, but the CFMEs may lose sensitivity if they were treated for too long or at too high potential due to surface etching. For the rest of the work, we used the 1.5 V, 3 minute condition for further characterization.

Figure 4.

The effects of treatment time and potential. (a) The effect of treatment time on background current (n = 6) with a potential of 1.5 V, (b) the effect of treatment time on dopamine detection (n = 6) with a potential of 1.5 V, (c) the effect of treatment potential on background current (n = 6) with a time of 3 min, and (d) the effect of treatment potential on dopamine detection (n = 6) with a time of 3 min.

Electrochemical characterization CFMEs electrochemically treated in KOH.

Cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were used to characterize the electrochemical properties of CFMEs electrochemically treated in KOH; the same CFME was tested for CV and EIS before and after the treatment. Larger CFMEs were used because larger electrode surface area results in obvious CV peaks. Figure 5a shows the CVs collected in K₃Fe(CN)₆, which is an inner-sphere redox sensitive to surface properties.[6] After the electrochemical treatment in KOH for 3 minutes, the CV exhibits a larger current density and smaller peak separation, indicating a higher sensitivity and faster electron transfer rate. Figure 5b shows the EIS Nyquist plot collected at a range of frequencies, where real part of the impedance is plotted on the x-axis and the imaginary part on the y-axis. The size of semicircle indicates electron transfer resistance, and the linear part shows diffusion.[35] The smaller semicircle after the treatment indicates the faster electron transfer rate. These experiments demonstrate the electrochemical treatment in KOH effectively activates the carbon surface, and can be used as a pre-treatment for CFMEs prior to any electrochemical measurements.

Figure 5.

Electrochemical characterization of CFMEs before and after the treatment at 1.5 V for 3 minutes. (a) CV of 5 mM K₃Fe(CN)₆. The scan rate was 100 mV/s. (b) EIS Nyquist plot.

The KOH-treated CMFEs were characterized for FSCV detection of dopamine. Figure 6a-b shows electrochemical treatment enhances the sensitivity for dopamine detection, and the dopamine anodic current is linear with concentration up to 10 μM. At concentrations above 10 μM, the electrode surface will be saturated with adsorbed dopamine, so the kinetics are controlled by diffusion.[3] Figure 6c displays an example CV for detection of 50 nM dopamine. The limit of detection (LOD) was calculated by three times of signal to noise ratio, and the values are 14 ± 4 nM for untreated CFMEs , and 9 ± 2 nM for CFMEs electrochemically treated in KOH. This improvement of LOD is due to the increase for dopamine signal, while the noise does not increase because the electrode surface area did not change.[36] The etching of glass sheath by KOH might be a concern as a source of noise,[37] but the influence can be ignored using dilute KOH for only 3 minutes.[38] Figure 6d shows that dopamine current increases linearly with scan rate, indicating the kinetics are controlled by adsorption.[3] To test the stability of the treated electrodes, 1 μM dopamine was detected every half an hour over a 5 h time period with an FSCV waveform continually applied (Figure 6e). The anodic peak current drops only about 10–15% after using for 5 h, which is comparable with untreated carbon fiber and other carbon-based electrodes,[39–41] so electrodes are stable for the length of typical biological experiments.

Figure 6.

FSCV study of CFMEs electrochemically treated in KOH. The treatment was a constant 1.5 V for 3 minutes. (a) Detection of dopamine when concentration is varied from 1 μM to 100 μM (n = 3). (b) Dopamine current is linear with a concentration up to 10 μM (R² = 0.98 for both untreated and treated CFMEs). (c) Example CV of 50 nM dopamine measurement. (d) Dopamine current is linear with scan rate (R² = 0.99 for untreated CFME and R² = 0.95 for treated CFMEs, n = 3). (e) Stability test. 1 μM dopamine was detected every 0.5 h for 5 h (n = 3).

Brain Slice Measurements and Biofouling reduction with CFMEs electrochemically-treated in KOH

To prove the treated CFMEs can be applied for biological systems, we used the KOH-treated electrodes to monitor stimulated dopamine release in mice brain slices (Figure 7a). The treated electrode has a good time response for dopamine in the brain tissue and the signal is stable for multiple measurements. The inset in Figure 7a, the background subtracted CV proves that the signal after stimulation is dopamine release. Thus, these KOH-treated electrodes can be used in tissue.

Figure 7.

KOH-treated electrodes in brain slices (a) Brain slice experiment. Electrochemical stimulus was applied in caudate–putamen using biphasic stimulation pulses (300 mA, 5 pulses at 60 Hz). The i–t curve of the electrical stimulated dopamine release was collected in caudate–putamen region. (b) The background current and (c) dopamine response before fouling, after fouling and after treated in KOH. (d) The anodic peak current intensity for dopamine.

Once the carbon electrodes are used in biological tissues, the surface may adsorb biomolecules or polymerized electrochemical byproduct , which is known as electrode biofouling.[11] Biofouling decreases the sensitivity for neurotransmitter detection. To test the ability of electrochemical treatment in KOH to restore electrodes after biofouling, we continuously applied the dopamine waveform to an untreated CFME(-0.4 V to 1.3 V, 400 V/s, 10Hz) in a brain slice for 1 h to produce biofouling. After removing the electrode from tissue, we tested its response to dopamine, performed electrochemical treatment in KOH for 3 minutes, and tested its response again. Figure 7b shows the background signals before biofouling, after biofouling, and after KOH treatment. The background currents are largely the same, because the electrode surface area does not change. Figure 7c-d shows that implantation in a brain slice for 1 h decreases the signal for dopamine dramatically, to about 30% of the original signal due to the adsorption of biomolecules. Treatment in KOH for 3 minutes fully restores the sensitivity for dopamine after biofouling, and the slight increase in sensitivity may due to surface activation, but no significant difference is observed (Paired t-test, p > 0.1). The CV slightly shifts right compared with untreated CFMEs, likely due to the changes in surface chemistry. These results demonstrate that treatment in KOH can clean electrodes after biofouling by renewing the surface. Compared with the results by the Wightman group, where the electrodes were treated in buffer for 15 min and the sensitivity was not fully restored,[23] KOH greatly accelerate the surface renewal process.

Conclusions

In summary, we have explored different solution conditions to electrochemically activate the carbon fiber electrodes and found treatment in KOH solution outperforms treatment in KCl, H₂O₂ and HCl solutions. Using a high potential, the surface of the CFME is etched much faster in KOH than in other conditions. Surface and electrochemical characterizations reveal more oxygen groups on the renewed CFME surface treated with KOH, which is beneficial for electron transfer kinetics and adsorption. The treatment improves the sensitivity for dopamine detection and resulting in a smaller limit of detection. In addition, the KOH-treated CFMEs successfully measured stimulated dopamine release in mouse brain slice and treatment removes biofouling after use. The electrochemical treatment using KOH is simple, fast, and effective. Therefore, electrochemical treatment in KOH is useful for pre-treatment to activate the electrodes, restore the electrodes from biofouling, or clean the electrode surface for further modification.