Modified Sawhorse Waveform for the Voltammetric Detection of Oxytocin

Abstract

Carbon fiber microelectrodes (CFMEs) have been used to detect neurotransmitters and other biomolecules using fast-scan cyclic voltammetry (FSCV) for the past few decades. This technique measures neurotransmitters such as dopamine and, more recently, physiologically relevant neuropeptides. Oxytocin, a pleiotropic peptide hormone, is physiologically important for adaptation, development, reproduction, and social behavior. This neuropeptide functions as a stress-coping molecule, an anti-inflammatory agent, and serves as an antioxidant with protective effects especially during adversity or trauma. Here, we measure tyrosine using the Modified Sawhorse Waveform (MSW), enabling enhanced electrode sensitivity for the amino acid and oxytocin peptide. Applying the MSW, decreased surface fouling and enabled codetection with other monoamines. As oxytocin contains tyrosine, the MSW was also used to detect oxytocin. The sensitivity of oxytocin detection was found to be 3.99 ± 0.49 nA/μM, (n=5). Additionally, we demonstrate that applying the MSW on CFMEs allows for real time measurements of exogenously applied oxytocin on rat brain slices. These studies may serve as novel assays for oxytocin detection in a fast, sub-second timescale with possible implications for in vivo measurements and further understanding of the physiological role of oxytocin.

Introduction

Small molecule neurotransmitters relay signals across synapses and control a wide array of physiological processes in a fast time scale. These neurotransmitters (NTs) have many important physiological roles in communication and modulation. For example, dopamine is implicated in cognition and emotional regulation¹. Neuropeptides are larger molecules composed of amino acid chains that are formed and secreted by neurons, controlling slow-onset and lasting modulation of synaptic transmission². One such neuropeptide, oxytocin, is involved in forming relationships, stress, depression, and anxiety³. In women, it functions in the progression of labor and lactation^2,4. Furthermore, oxytocin plays a crucial role in the development of mood and anxiety disorders, schizophrenia, and autism where rapid oxytocin monitoring has the potential to lead to treatment breakthroughs^3,5. The development of rapid oxytocin detection is important as it is involved in stress-management, anti-inflammatory effects, and serves as an antioxidant with protective effects during periods of trauma^6,7.

Several assays have been used for the detection of oxytocin. Oxytocin is typically measured by collecting and assaying a blood sample via immunoassays³. This process can be tedious, inaccurate and is unable to capture real time fluctuations⁸. Mass spectrometry⁹ and chromatography^10,11 may also be used to detect neuropeptides like oxytocin. However, these methods are time consuming, have low temporal resolution, and are sample destructive. Additionally, as these existing techniques have been primarily used to detect serum levels, they are indirect measurements and not fully representative of true oxytocin levels in the brain¹². The speed and accuracy of detecting endogenous neuropeptides is also crucial, where the rapid metabolism of compounds in the brain presents further challenges. Various studies on electrochemical techniques^13–16 have shown potential in this area, utilizing the redox active properties of some amino acids to provide faster sample analysis with a higher degree of accuracy. Methods such as conventional “slow scan” cyclic voltammetry (CV) have seen varying degrees of success in this field, but have relatively low temporal resolution and lack of reproducibility¹⁷.

Fast scan cyclic voltammetry (FSCV) with carbon fiber microelectrodes (CFMEs) is an electrochemical technique used to detect neurotransmitters with a subsecond temporal resolution^16,18. Using FSCV along with CFMEs provides for rapid analysis with a high degree of accuracy for representative biomolecule concentration changes in the brain^18–20. This technique was used to detect monoamines in the brain such as dopamine²¹ serotonin^22,23, norepinephrine²⁴, amino acids^25–27, and others²⁸ Recently, it has been adapted for use in purinergic²⁹ signaling³⁰ and the detection of peptide³¹ neurotransmitters³². FSCV has also been used to detect analytes that foul such as melatonin in live mesenteric lymph node slices, further exemplifying the versatility of this technique³¹. This method can rapidly identify a large range of molecules with a high spatiotemporal resolution and provides chemical selectivity not available with other electrochemical methods³³. However, there have been challenges detecting tyrosine-containing compounds⁶. In previous studies, the Sombers group developed a modified sawhorse waveform (MSW) to specifically detect the opioid methionine-enkephalin (M-ENK)⁶ through tyrosine oxidation. This is accomplished by two specific scan rates in the anodic sweep and a short holding period at the switching potential. This enabled high specificity toward tyrosine and the elimination of fouling on the electrode surface. Also, the differentiation of leu-enkephalin and met-enkephalin both in vitro and ex vivo was observed. Additionally, sawhorse waveforms have been previously used to detect and differentiate compounds such as adenosine, ATP, and hydrogen peroxide³². Metals, such as zinc have also been measured with modified sawhorse waveforms³⁴.

In this study, we developed an MSW to detect oxytocin through the oxidation of the tyrosine residue. Oxytocin and tyrosine were adsorption controlled to the surface of the carbon fiber microelectrode and applying the MSW significantly enhanced oxytocin and tyrosine detection with respect to the traditional triangle waveform. Additionally, we co-detected varying concentrations of tyrosine and oxytocin with dopamine and were able to clearly distinguish cyclic voltammogram (CV) peaks using the MSW. We measured exogenously applied oxytocin ex vivo on coronal rat brain slices as a proof of principle experiment for future in vivo studies. This will allow for potential measurement of oxytocin in real time in vivo, and lead to a better understanding of the neuropeptide’s physiological role and significance in the human body.

Experimental

Chemicals

Tyrosine and Dopamine (≥98.0% purity) were purchased from Sigma-Aldrich (St. Louis, MO). Oxytocin was obtained from GenScript Biotech (Piscataway, NJ) (≥95.0% purity). Electrochemical experiments were carried out in phosphate-buffered saline (PBS) (131.5 mM NaCl, 3.25 mM KCl, 1.2 mM CaCl₂, 12.5 mM NaH₂PO₄, 1.2 mM MgC12, and 2.0 mM Na₂SO₄ at pH 7.4) (Sigma-Aldrich, St. Louis, MO). The PBS solution was made by dissolving the salts into deionized water and pH adjusted to 7.4 to match physiological conditions. All aqueous solutions were made with deionized water (Millipore, Billerica, MA).

CFME Fabrication

Carbon fiber-microelectrodes (CFME) were constructed as previously described⁷. T-650 carbon fibers are polyacrylonitrile (PAN) based. Briefly, a 7 μm diameter strand of carbon fiber was aspirated into a glass capillary with an inner diameter of 0.68 mm and outer diameter of 1.2mm (A-M Systems, Sequim, WA) using a vacuum pump (Gast, Model DOA-P704-AA, Benton Harbor, MI). The glass was tapered using a micropipette puller (Narishige, PC-100, Tokyo, Japan). The protruding carbon fiber was trimmed to a length of 100-150 μm from the tapered side of the glass capillary. The glass-carbon fiber interface was sealed with a mixture of EPON 828 and hardener diethylenetriamine (DETA), that was cured for 4 hours at 125°C. Electrodes were backfilled with a 0.1M KCl solution to create an electrical connection.

Fast Scan Cyclic Voltammetry

FSCV recordings were conducted using the WaveNeuro FSCV System with a 5 MΩ headstage (Pine Instruments). High-definition cyclic voltammetry (HDCV) software with PC1e-6363 multifunction I/O device (National Instruments, Austin, TX), was used for data collection and analysis. The traditional triangle waveform was applied with a holding potential of −0.2 V and switching potential of 1.2 V with a 400 V/s scan rate vs. a silver-silver chloride (Ag/AgCl, −0.197V) reference electrode at a frequency of 10 Hz. The MSW was applied at 10 Hz with the holding potential at −0.2 V, then increased to +0.7 V at 100 V/s, and further increased to +1.2 V at 400 V/s. This potential was held for 3 ms before sweeping back down to −0.2 V at 100 V/s. The buffer solution continuously washed over the tip of the electrode at a rate of 1 mL/min (NE-300 Just Infusion Syringe Pump). For each trial, 0.2 mL samples of tyrosine or oxytocin were injected. Each electrode was allowed to equilibrate with the waveform at least 30 min before in vitro measurements in the flow cell (Pine Research, Durham, NC).

Statistical Analysis

All figures and linear regression calculations such as slope and R² values were obtained using GraphPad Prism 9.

Tissue Extraction

Tissue samples were collected from female Sprague Dawley rats in accordance with IACUC and animal facility protocols at American University and Protocol #20-09. Rats were housed in 12-hour light and dark cycles and provided food and water ad libidum. During the day of the experiments, rats were removed from their home cage one at a time and placed in a CO₂ euthanasia chamber. Once reflexes were assessed via toe and tail pinch, euthanasia was confirmed by cervical dislocation. The head was decapitated using surgical scissors and the skull exposed by removing surrounding tissue. Large, surgical rongeurs were used to quickly peel away the skull bones to expose and remove the brain. The excised brain was placed into a vial of artificial cerebrospinal fluid (aCSF) and stored on ice until use. For slice preparation, multiple coronal cuts were made to target either the caudate putamen or the hippocampus. Coordinates and anatomical landmarks were located using the Paxinos Rat Brain Atlas³⁵.

For tissue sample recording, the set-up procedure was adapted from Papouin and Haydon where a 600 mL plastic beaker was used as the holding container³⁶. A petri dish with the bottom removed had nylon hosiery super glued using cyanoacrylate glue. The petri dish was also superglued to the empty beaker. The sliced brain tissue was placed on top of the nylon and saturated with the cold aCSF. A steady flow of the buffer was provided to maintain saturation and to emulate a flow cell. ACSF used for the duration of the experiment was oxygenated by bubbling in carbogen (95% O₂, 5% CO₂, Roberts Oxygen) using an air stone (Tetra, Blacksburg, VA) and airline tubing (Tetra).

The CFMEs were lowered until they penetrated the brain tissue and allowed to equilibrate for 30 min applying the MSW. Afterwards, oxytocin (20 mL of a 10-50 μM solution) was exogenously applied using a micropipette. The flowing aCSF ensured the applied oxytocin was washed away from the slice. Trials were repeated with a 10-min wait period in between runs.

Results and Discussion

The redox active moieties in oxytocin include a tyrosine residue and a disulfide bridge. In strictly controlled environments (protein electron transfer chains and studies of model compounds), tyrosine oxidation involves a proton-coupled electron transfer event that leads to the formation of a tyrosyl radical³⁷ (Scheme 1). For these reactions in proteins, the proton is shuttled to either water or a structurally proximal acceptor while the electron is passed along to a catalytically-active reaction center³⁸. In less controlled systems, the full mechanism for tyrosine oxidation becomes muddled. The formed tyrosyl radical can become involved in cross-linking between amino acids³⁹. This radical can also lead to cleavage of peptide bonds^40,41. The initial steps of tyrosine oxidation, as expected for our system, are shown in Scheme 1. While the ultimate fate of this oxidized tyrosine is unknown, there is likely a heterogeneous set of products. For the purpose of this report, however, it is only the initial oxidation at the surface of the electrode that is important for detection. The scan rates were chosen, in part, to ensure that the oxidized product would diffuse away from the electrode surface before any fouling might occur^6,7

Scheme 1.

Proposed electrochemical oxidation schemes for tyrosine (A) and oxytocin (B) on CFMEs with FSCV at a physiological pH of 7.4.

The observed oxidation of tyrosine at the surface of CFMEs allows for the indirect detection of oxytocin. Dimerization of two cysteine residues to create cystine facilitates the formation of a tightly bonded disulfide bridge, unlike similar residues like methionine which does not contain a disulfide bridge. The high bond dissociation energy (60 kcal/mol)⁴² of the disulfide bridge suggests that the cleavage and subsequent oxidation of the cystine residue is unlikely when scanning up to 1.2 V/sec. Tyrosine readily donates a proton, which enables its facile oxidation. Due to the negligible contribution from the cystine disulfide bridge, we hypothesized that the oxidation of the tyrosine residue enables the measurement of oxytocin at physiological pH.

Tyrosine was first detected using a triangle waveform, which scans from a −0.2 V holding potential, to a switching potential of 1.2 V, and back down to −0.2 V at 400 V/s (Fig. 1A). The adapted MSW has a −0.2 V holding potential, and scans at a rate of 100 V/s to a transition potential of 0.7 V. The scan rate is subsequently swept to 400 V/s and held at the switching potential of 1.2 V for 3 ms, before scanning down to −0.2 V at 100 V/sec (Fig. 1C). Since the MSW is held for 3 ms at 1.2 V, we propose that this allows for increased adsorption and facilitates electron transfer kinetics at the CFME’s surface. Holding the potential at 1.2 V enables tyrosine oxidation and produces faster electron transfers between the analyte and the CFME surface. This is evident by the defined cyclic voltammogram peak shape (Fig. 1D). The broader cyclic voltammogram peak shape, lower peak oxidative current, and increase in noise produced from the triangle waveform further indicate the enhanced sensitivity and selectivity of the MSW in comparison to the triangle waveform (Fig. 1B). Figure 1E shows the CV of oxytocin oxidation using the MSW. The Sombers group have previously noted the challenge in measuring tyrosine-containing peptides using the triangle waveform⁶. They also have applied the MSW onto CFMEs to detect neuropeptides such as leu-enkephalin and met-enkephalin. Applying the MSW clearly enables the detection of oxytocin as it cannot be detected using the aforementioned triangle waveform⁷.

Figure 1.

Triangle and MSWs with corresponding cyclic voltammograms (CVs). (A) Triangle waveform shown as voltage potentials plotted vs. time, with scan rates overlaid. (B) CV of tyrosine (10 μM) with the triangle waveform. (C) Modified Sawhorse Waveform. (D) CV of tyrosine (10 μM) detection with the MSW. (E) CV of oxytocin (10 μM) with the MSW.

We observed a significantly higher sensitivity for oxytocin and tyrosine detection when CFMEs were applied with the MSW vs. the triangle waveform. We hypothesize that this is due to more facile oxidation and adsorption of tyrosine to the electrode surface when holding at 1.2 V. Previous studies have shown that scanning to higher potentials renews the electrode surface, breaks carbon-carbon bonds, increases the electroactive surface area/roughness, and functionalizes the electrode with negatively charged oxide groups, which enhances biomolecule adsorption and detection⁴³. Averages from three electrodes measuring 10 μM tyrosine indicate a 12-fold increase in the peak oxidative current on the MSW (50.24 ± 1.57 nA) when compared to the triangle waveform (3.89 ± 0.86 nA) (Fig. 2A). Tyrosine detection is also markedly more sensitive than oxytocin when applying the MSW on CFMEs. The peak oxidative currents of CVs measuring 10 μM tyrosine (48.63 ± 1.56 nA) and oxytocin (9.83 ± 1.09 nA) indicated a nearly 5-fold increase in current for tyrosine detection. We hypothesize that tyrosine adsorption is more facile onto the CFME surface due to its relatively smaller size and free rotation of chemical bonds. On the other hand, the neuropeptide oxytocin is larger, bulker, and probably more difficult to adsorb on the surface of the CFME. Since only the tyrosine residue is electroactive, the remaining peptide hinders the adsorption and oxidation of oxytocin, which explains the lower sensitivity with respect to tyrosine.

Figure 2.

Comparison of tyrosine and oxytocin detection using the MSW and Triangle Waveforms. (A) Direct comparison of tyrosine (10 μM) peak oxidative current on MSW and triangle waveforms. p < 0.0001, (n = 3). Applying the MSW yields a significantly higher peak oxidative current with respect to applying the triangle waveform. (B) Comparison of peak oxidative currents between oxytocin (10 μM) and tyrosine (10 μM) on the MSW. Measuring the same concentration of tyrosine yields a higher peak oxidative current than oxytocin. p < 0.0001, (n = 3).

Concentration experiments indicate a linear relationship between peak oxidative current and tyrosine concentration up to 10 μM and an asymptotic curve at higher concentrations (Fig. 3A). At higher concentrations, the CFME surface becomes saturated as sites for adsorption are occupied, which explains the deviation from linearity at higher concentrations. The linear concentration range of tyrosine detection spans from 0.5 μM - 10 μM when applying the MSW. With the triangle waveform, the linear range spanned from 1 μM - 10 μM, as concentrations below 0.5 μM could not be accurately measured. The application of both waveforms on CFMEs results in an asymptotic curve at 25 μM and beyond, upon which the analytes become saturated at the surface of the electrode. Figure 3B indicates a linear relationship between increases in concentration and peak oxidative current (R²_MSW = 0.9849, R²_Triangle = 0.8933). The MSW provides a significant enhancement for tyrosine detection with respect to the triangle waveform. The MSW also yielded stable peak oxidative currents over a 4-hour period upon detecting 10 μM tyrosine. As shown in Figure 3C, the electrode applied with the MSW was stable for a period of up to four hours, which is the typical timeframe for in vivo experiments. A stable response will ensure the accuracy of pre- and post-calibrations for ex vivo and in vivo experiments.

Figure 3.

Adsorption-Control studies of tyrosine on CFMEs (A) Plot of peak oxidative current vs. concentration. Peak oxidative currents display an asymptotic relationship with respect to concentrations of tyrosine (500 nM to 100 μM). (n = 5). (B) Lower tyrosine concentrations (500 nM to 10 μM) display a linear relationship when plotted against the respective peak oxidative current. (R²_MSW = 0.984, R²_Triangle = 0.893, n = 5). (C) Peak oxidative current for CV of tyrosine detection is stable over a period of 4 h. Measurements of tyrosine (10 μM) are taken each hour (n = 5). (D) There is a linear relationship between scan rate and current between 50 – 400 V/s. (R² = 0.900, n = 3).

Figure 4 shows the adsorption control of oxytocin on CFMEs when applying the MSW. At concentrations higher than 10 μM, oxytocin becomes saturated at the surface of the electrode. In figure 4B, we observe a linear range of 0.5 μM to 10 μM for oxytocin detection using the MSW. Applying the MSW can enable oxytocin detection reliably even at relatively lower concentrations. We hypothesize that this will enable the detection of oxytocin in vivo at physiological levels. The stability experiment shown in Figure 4C indicates that oxytocin detection with MSW is steady and does not fluctuate over a long period of time.

Figure 4.

Measurement of adsorption of oxytocin on CFMEs using the MSW. (A) Peak oxidative currents display an asymptotic relationship at higher concentrations of oxytocin (>10 μM). (n = 5). (B) The linear range of oxytocin detection is from 500 nM to 10 μM. (R²_MSW = 0.950). (C) Peak oxidative current is stable over a detection period of 4 h, for measurements of oxytocin (10 μM) taken each hour. (n=5).

Fouling occurs when an analyte polymerizes and coats the electrode surface in non-conductive polymer, hence blocking sites for further adsorption. It is a particularly important consideration for in vivo experiments involving analytes such as serotonin, which easily fouls the electrode⁴⁴, as a decrease in peak oxidative current due to fouling impedes detection⁴⁵. As shown in Figure 5A, 10 repeated injections of 1 μM tyrosine produced a significantly decreased peak oxidative current between the 1^st and 10^th injections using the triangle waveform. On the other hand, there was no significant decrease in peak oxidative current between the 1^st and 10^th injection of tyrosine using the MSW. Moreover, the same fouling experiment was performed with 1 μM oxytocin and the MSW. We hypothesize that applying the Modified Sawhorse Waveform prevents fouling at the electrode surface. By holding the potential at 1.2 V for approximately 3 ms, we hypothesize that the MSW may be etching the microelectrode surface. As shown in the literature, scanning to higher potentials past 1.0 V etches the electrode surface to break carbon-carbon bonds, increase surface roughness and the electroactive surface area, and functionalize the electrode surface with negatively charged oxide groups^43,46. Renewing the electrode surface through electrochemical etching will prevent analyte fouling at the surface of the electrode by not enabling the formation of non-conductive polymers to coat the electrode surface as it is continually being regenerated.

Figure 5.

Fouling] experiments show differences in peak oxidative current upon repeated injections over 300 seconds or five minutes. (A) Injections of 1 μM tyrosine were compared using the MSW and triangle waveforms. Applying the triangle waveform yielded a significant decrease in peak oxidative current after 10 injections. Applying the MSW yielded no significant decrease in current. p < 0.0001, (n = 5). (B) 10 repeated injections of oxytocin (1 μM) using the MSW did not produce a significant decrease in peak oxidative current. (n = 3).

We then co-detected dopamine with tyrosine and oxytocin using MSW and triangle waveforms. Upon applying the triangle waveform, dopamine and tyrosine oxidize at approximately +0.7 V and +1.15 V, respectively (Fig. 6A–B). Applying the MSW, oxytocin and tyrosine oxidize at approximately + 1.1V, while dopamine oxidizes at + 0.4V , respectively (Fig. 6C–D). This could be explained by the differing rates of heterogeneous electron transfer for dopamine and tyrosine at CFMEs. The heterogeneous electron transfer rates (k₀) for dopamine (1.0 · 10⁻³ cm/s) and tyrosine (1.1 · 10⁻² cm/s) were obtained by fitting the triangle waveform voltammograms using the Bio-Logic EC-Lab software. The results were reproducible across several scan rates and comparable to what have been previously outlined in the literature^46–48. We hypothesize that the rate is greater for tyrosine oxidation because the formation of a free radical is relatively fast and unstable (Scheme 1). We hypothesize that the reason for the separation of the dopamine peak from that of tyrosine/oxytocin, is that the scan rate is 100 V/s when using MSW at the potential window that DA oxidizes and 400 V/s for tyrosine/oxytocin. Since the electron transfer rate for DA is smaller, its oxidation peak shifts more strongly with the scan rate. When the scan rate is reduced, the oxidation peak shifts to more negative potentials. For tyrosine/oxytocin, the peak position remains the same since the scan rate is the same.

Figure 6.

Co-detection of Dopamine (DA) with tyrosine using the MSW and Triangle waveforms, with starting ratios of 1 μM to 1 μM and increasing concentrations of Tyr/OT. (A) CVs of dopamine and tyrosine (Tyr) mixtures using the triangle waveform. (B) CVs of mixtures of dopamine and oxytocin (OT) using the triangle waveform where oxytocin was not detected. (C) CVs of mixtures of various concentrations of dopamine and tyrosine using the MSW. (D) CVs of mixtures of dopamine and oxytocin with the MSW.

Upon applying the MSW, the shape and position of the CV change as faster scan rates outrun electron transfer. Using the triangle waveform, we only observe a broad shoulder for tyrosine detection (Fig. 6A), while oxytocin was not clearly measured. However, upon increasing the concentration of oxytocin, the peak oxidative current shifted to more positive potentials due to the overlap of the tyrosine and dopamine peaks (Fig. 6B). Applying the MSW, we observe a clear separation of oxidation peaks when co-detecting dopamine and oxytocin/tyrosine (Fig. 6C). At lower concentrations of oxytocin, noise is produced as the concentrations approach the limit of detection (Fig. 6D). Dopamine detection is more facile than oxytocin due to its smaller size and faster electron transfer kinetics. Dopamine is a catechol that contains two hydroxyl groups, which are easily oxidized to a quinone. At a physiological pH, the amine of dopamine is protonated, which produces a net positive charge. This causes dopamine and other cationic monoamines to strongly adsorb to the surface of the negatively charged CFMEs through an electrostatic attraction of opposite charges. Tyrosine, the main redox active amino acid of oxytocin, has a net neutral charge because it is zwitterionic and does not contain a charged sidechain. Therefore, dopamine is more sensitive to the electrode surface than tyrosine and has faster electron transfer kinetics.

In addition to co-detection with dopamine, interference studies were completed using the following neurochemicals: norepinephrine (NE), DOPAC (3,4-dihydroxyphenylacetic acid), serotonin (5-HT), and adenosine (AD) (Fig. S1A–E). All the observed interferants were differentiated from oxytocin with the MSW. AD is oxidized at a potential of approximately 1.4 V and the MSW has a maximum voltage potential of 1.2 V, which suggests why there is not a discernable oxidation peak for adenosine (Fig. S1D). Moreover, we also performed a pH study (Fig. S2) where we determined that the detection of oxytocin was pH dependent. At lower pH values, the amine of tyrosine is more likely to be protonated and positively charged, which would explain enhanced sensitivity at the surface of a negatively charged electrode. At higher pH values, carboxyl groups are more likely to be deprotonated to create an overall negative charge, which decreases sensitivity.

Lastly, we performed a proof-of-concept experiment to detect oxytocin exogenously applied in rat brain tissue (Fig. 7). We extracted coronal brain slices from rats and placed them into a custom-made flow cell. The CFME was then slowly inserted in the rat brain tissue. Various concentrations of oxytocin (10 – 50 μM) were then exogenously applied onto the brain slice. We clearly detected oxytocin as the CVs indicated a strong, distinct peak that resembled the in vitro data. We observed approximately 15% of the in vitro signal with the same electrode as only a certain percentage of the analyte diffuses into the brain. This work illustrates that immersion of the electrode into biological tissue such as brain slices does not prevent the detection of neuropeptides such as oxytocin. Therefore, we expect this assay to be useful for the detection of oxytocin in vivo in animal models.

Figure 7.

Exogenous application of oxytocin onto brain slices. (A). CV of oxytocin (10 μM) exogenously applied onto rat brain slice. (B). Current vs. time (I vs. T) trace of exogenously applied oxytocin. (C). Color plot of oxytocin detection.

Conclusions

We utilized the MSW to measure oxytocin through the oxidation of the tyrosine residue. Applying the MSW enhanced the detection of both tyrosine and oxytocin by utilizing a holding period at 1.2 V, allowing for greater electron transfer. Additionally, we observed that the holding potential actively renews the electrode surface to a greater degree than the triangle waveform, which enhances sensitivity and co-detection with several monoamines. Applying MSW onto CFMEs produced greater selectivity for oxytocin and tyrosine, and differentiation from other catecholamines such as dopamine. The detection of exogenously applied oxytocin in brain slices in ex vivo experiments shows the applicability for possible in vivo measurements. Ultimately, the enhanced detection of neuropeptides such as oxytocin will potentially further elucidate their physiological importance.