Electrochemical Detection of Serotonin

• The paper demonstrates that engineered electrode materials with tailored surface chemistries enable effective pre-concentration and selective redox reactions of serotonin.
• FSCV and DPV methodologies yield distinct voltammetric signatures with peaks at 0.27 V and 0.68 V, achieving low detection limits down to 1.2 nM under physiological conditions.
• Advanced sensor platforms show high reproducibility, rapid temporal resolution, and robust interference resistance, validating their performance in real biological sample analysis.

Electrochemical detection of serotonin (5-hydroxytryptamine, 5-HT) encompasses the identification and quantification of this monoaminergic neurotransmitter using electrode-based transduction of redox reactions. Accurate, sensitive, and selective detection of serotonin is essential for investigations of neurochemical processes, diagnosis and monitoring of neurological disorders, and pharmacodynamic studies. Principal methodologies draw on engineered electrode materials with tailored surface chemistries, advanced waveform control, and system-level integration that together optimize selectivity amid interfering species and achieve detection limits aligned with physiological concentrations.

1. Electrode Architectures and Material Functionalization

Two principal approaches have been recently validated for serotonin electrochemical detection: (i) glassy carbon (GC) microelectrode arrays fabricated via a carbon MEMS (C-MEMS) pattern transfer protocol (Castagnola et al., 2020), and (ii) glassy carbon electrode (GCE) modification through copper sulfide-hydroxypropyl-β-cyclodextrin-reduced graphene oxide (Cu₂S/Hβcd-rGO) nanocomposites (Santhan et al., 6 Nov 2025).

Glassy carbon microelectrode arrays exploit spin-coated SU-8 negative resist, UV patterning (1.5 µm thickness, arrays of 1 × 500 µm² detection pads, 220 µm inter-pad spacing), and N₂ pyrolysis at 1000 °C to yield defect-rich GC, which is subsequently transferred and insulated on flexible polyimide substrates. Transmission Electron Microscopy and Raman spectroscopy (G-peak ≈ 1605 cm⁻¹, I_D/I_G ≈ 1.1) confirm the formation of crumpled graphene stacks, high defect density, and edge plane termination with carboxyl, carbonyl, and hydroxyl groups. At physiological pH (7.4), these surface oxygenated moieties confer a net negative charge and dipole moment, facilitating strong electrostatic adsorption of protonated serotonin via its primary amine (pK_a ≈ 9.8), thus pre-concentrating the analyte.

Cu₂S/Hβcd-rGO/GCE sensors emerge from hydrothermal synthesis of nanocrystalline Cu₂S (D ≈ 32.4 nm), chemical reduction of graphene oxide (rGO), and supramolecular functionalization with hydroxypropyl-β-cyclodextrin (Hβcd), followed by dispersion and drop-casting of the composite ink onto alumina-polished GCEs. Structural analysis (XRD, Raman, FT-IR, XPS, FE-SEM, TEM) establish phase purity, defect density enhancement (ID/IG = 1.01), and uniform Cu₂S nanoparticle decoration on rGO sheets. Hβcd confers host–guest inclusion capabilities (binding constant >10³), acting as a molecular anchor for serotonin at the electrode interface via π–π stacking, van der Waals interactions, and electrostatic attraction, collectively increasing surface enrichment and facilitating selective sensing.

2. Fast-Scan Cyclic Voltammetry and Waveform Optimization

Glassy carbon arrays are coupled with fast-scan cyclic voltammetry (FSCV), wherein waveform parameters—holding potential (E_H = –0.4 V vs Ag/AgCl), switching potential (E_S = +1.0 V), scan rate (ν = 400 V/s, frequency 10 Hz)—are optimized for co-detection of serotonin and dopamine. The rationale for peak resolution is that moderate scan rate and voltage excursion (ΔE = 1.4 V) afford sufficient time for multi-electron, multi-step serotonin oxidation to yield two discernible anodic peaks (E_p,ox1 = 0.27 ± 0.04 V, E_p,ox2 = 0.68 ± 0.03 V), contrasting dopamine's mono-peak oxidation (E_p,ox ≈ 0.65 V). This waveform design prevents overlap, limits surface fouling, and enhances kinetic selectivity via cationic pre-concentration at E_H. The mathematical description follows:

• Forward scan: $$E(t) = E_H + \nu t,\quad 0 \leq t \leq (E_S-E_H)/\nu$$
• Reverse scan: $$E(t) = E_S - \nu [t-(E_S-E_H)/\nu],\quad (E_S-E_H)/\nu \leq t \leq 2(E_S-E_H)/\nu$$

The multi-peak cyclic voltammetric signature demarcates two redox stages: (i) carbocation formation, and (ii) subsequent conversion to quinone–imine.

In Cu₂S/Hβcd-rGO/GCE platforms (Santhan et al., 6 Nov 2025), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) are employed, with DPV optimizing signal-to-noise ratios for ultrasensitive detection (pulse amplitude 50 mV, width 50 ms, step 4 mV, scan 20 mV/s). Scan-rate studies indicate that serotonin oxidation is predominantly adsorption-controlled, with the peak current linearly proportional to the scan rate ($$I_{pa} = 0.0476 (\mu{\rm A}/\text{mV\,s}^{-1})∙ν + 3.602$$, $R^2 = 0.9922$), and pH-dependence consistent with a 2H⁺/2e⁻ mechanism.

3. Analytical Performance Metrics and Calibration Protocols

Quantitative evaluation of electrochemical sensitivity, linear range, limit of detection (LOD), selectivity, and stability is fundamental for sensor characterization. Calibration curves for GC MEA/FSCV in PBS (pH 7.4) in the 10–200 nM 5-HT range yield:

• Main peak: $i_p (nA) = 0.028 [C_{5-HT}] (nM) + 0.45$, $R^2 > 0.99$
• Blank-noise: $\sigma_{blank} ≈ 0.10$ nA
• $LOD = 3\sigma_{blank}/m ≈ 10.7$ nM

Cu₂S/Hβcd-rGO/GCE provides dual linear detection ranges in DPV:

• Low: $0.019$–$0.299$ μM $$I_{pa} (\mu{\rm A}) = 1.1159 [C_{5-HT}] (\mu{\rm M}) + 0.8918$$, $R^2 = 0.9966$
• High: $4.28$–$403.14$ μM $$I_{pa} (\mu{\rm A}) = 0.0031 [C_{5-HT}] (\mu{\rm M}) + 1.3143$$, $R^2 = 0.9934$
• LOD: $1.2$ nM ($0.0012$ μM), Sensitivity: $15.9$ μA μM⁻¹ cm⁻² (normalized to 0.07 cm² geometric area)
• Charge-transfer resistance $R_{ct}$: $61$ Ω (vs $497$ Ω for bare GCE)

Stability is affirmed by <5 % drift over 15 days, and reproducibility with RSD <3 %.

4. Selectivity, Interference, and Application to Biological Samples

Selectivity against key interferents (dopamine, epinephrine, hydroquinone, 4-aminophenol, melatonin, Cl⁻, etc.) is requisite, given the closely spaced voltammetric peaks of neurotransmitters. GC MEA/FSCV achieves peak separation of ΔE ≈ 380 mV between serotonin and dopamine, allowing for discrimination in mixed environments (Castagnola et al., 2020). In Cu₂S/Hβcd-rGO/GCE sensors, DPV signal change of the serotonin peak remains <5 % even with 50 μM interferents (Santhan et al., 6 Nov 2025), attributed to both kinetic differentiation (distinct oxidation potentials) and the host–guest inclusion capabilities of Hβcd that enrich serotonin over competing species.

Real sample validation applies these sensors to human blood serum (Sigma-Aldrich, diluted 1:10 in PBS, pH 7.0, spiked with 4–25 μM serotonin), yielding recoveries from 98.7–99.8 % with minimal drift and high reproducibility.

$\begin{array}{cccc} \hline \text{Sample} & \text{Added}\ [\mu\mathrm{M}] & \text{Found}\ [\mu\mathrm{M}] & \text{Recovery\ (\%)} \ \hline \text{Serum\,1} & 4.94 & 4.88\pm0.12 & 98.8 \ & 9.87 & 9.76\pm0.18 & 98.7 \ & 14.90& 14.80\pm0.27& 99.3 \ & 19.91& 19.82\pm0.30& 99.5 \ & 24.95& 24.85\pm0.35& 99.8 \ \hline \text{Serum\,2} & 4.96 & 4.92\pm0.10 & 99.2 \ & 9.90 & 9.80\pm0.16 & 99.0 \ & 14.93&14.86\pm0.24 & 99.5 \ & 19.94&19.84\pm0.29 & 99.7 \ & 24.92&24.81\pm0.33 & 99.6 \ \hline \end{array}$

Temporal resolution of 100 ms in vitro and maintained sensor function for >8 h continuous scanning in flow cell configurations further substantiate real-time neuroscience applications.

5. Mechanistic Interpretation of Serotonin Redox Processes

Electrochemical oxidation of serotonin mechanistically proceeds as a two-stage transformation: first, the generation of a carbocation intermediate, then subsequent electron transfer and water activation convert serotonin into serotonin-quinoneimine.

$\ce{5\!-\!HT\ +\ 2H2O\ ->\ quinoneimine\ +\ 2H^+\ +\ 2e^-}$

At the Cu₂S/Hβcd-rGO interface, rapid electron transfer is facilitated by Cu₂S high-activity sites and rGO's conductivity; Hβcd confers inclusion enrichment, and the layered composite architecture supports efficient preconcentration and kinetic resolution of serotonin analytes. Similar preconcentration and oxygenation effects are achieved on GC MEAs through defect- and oxygen-rich basal planes.

A plausible implication is that further tuning of interfacial charge, defects, and host–guest properties might improve selectivity and functionality for poly-analyte detection or point-of-care adaptation.

6. Comparative Evaluation and Limitations

GC MEA/FSCV provides robust performance for simultaneous detection of dopamine and serotonin, albeit with a lower sensitivity for serotonin ($m_{5-HT} = 0.028$ nA/nM) and LOD of $\sim$10.7 nM, but benefits from clear voltage peak separation and stable sub-second temporal resolution. Dopamine exhibits higher sensitivity ($m_{DA} = 0.058$ nA/nM, LOD $\sim$5.2 nM).

The Cu₂S/Hβcd-rGO/GCE configuration achieves ultra-low LOD (1.2 nM), high sensitivity ($15.9\;\mu{\rm A}\,\mu{\rm M}^{-1}\,\text{cm}^{-2}$), exceptional selectivity, and operational stability; limitations primarily relate to potential electrode fouling by oxidation products and the necessity for precise control of composite ratios for optimal performance. Integration with microfluidic or screen-printed approaches is identified as a potential future direction, as is the exploration of alternative cyclodextrin derivatives and metal-sulfide phases.

7. Future Directions and Outlook

Advancement in electrochemical sensing of serotonin is progressing toward higher spatial resolution (multichannel MEAs), lower detection limits, multiplexed neurotransmitter identification, long-term stability, and facile integration with point-of-care systems. Potential areas for improvement include surface regeneration protocols to counteract fouling, further optimization of material ratios, and systematic assessment in complex biological matrices and in vivo microenvironments. The chemical versatility of hybrid architectures such as Cu₂S/Hβcd-rGO combined with the microfabrication advantages of GC MEAs is suggestive of a trajectory leading to platform technologies for neurochemical analytics.

Research in this domain is aligned with the increasing requirements of neuroscience, clinical diagnostics, and real-time neuropharmacological monitoring, substantiated by rigorous materials engineering, waveform optimization, and robust analytical validation (Castagnola et al., 2020, Santhan et al., 6 Nov 2025).