Conductive nano nickel oxide/hydroxide paper electrochemical sensor for serotonin detection in genetically engineered Drosophila

Abstract

Serotonin is considered an integral part in neuropsychiatric diseases, such as major depressive disorder, schizophrenia, post-traumatic stress disorder, obsessive-compulsive disorder, anxiety disorder, and substance use disorder. Understanding the levels of serotonin under different disease conditions is important. Herein, we explored the development of an efficient electrochemical sensor utilizing sustainable paper electrode integrated with nanocomposites through a simultaneous electrochemical deposition strategy. The as-developed sensor is further investigated with surface and electrochemical studies to understand the robust fabrication of the sensor as well as the electrochemical characteristics to show the improved electron transfer kinetics and detection capabilities even in the presence of common interfering biomolecules. The sensor demonstrated a broad linear range from 0.007 nM to 500 μM, with an impressive limit of detection of 0.024 nM for the low concentration range (0.007–0.48 nM) and 383.7 nM for the high concentration range both falling well within the clinically relevant detection levels of serotonin. To evaluate the practical performance, the developed sensor was tested on brain homogenates obtained from genetically modified Drosophila melanogaster models with different serotonin levels. The sensor effectively detected the in vivo changes in serotonin level, and the results were validated against gold-standard HPLC analysis and immunohistochemical staining experiments. The sensors’ notable stability, selectivity, and sensitivity towards serotonin make them a valuable tool for neurochemical research and clinical applications, particularly in studying serotonin-related neurological conditions and advancing personalized treatments.

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Introduction

Serotonin (5-HT) is a well-established neurotransmitter that plays a crucial role in regulating neural activity, neuropsychological functions, and various physiological processes, including cardiovascular health, bowel motility, ejaculatory latency, and bladder control.^1,2 Recent findings underscore that 5-HT also impacts certain processes, such as platelet aggregation, through receptor-independent mechanisms that involve transglutaminase-dependent covalent bonding to cellular proteins. On the other hand, serotonin syndrome (SS) is a potentially life-threatening reaction that can occur from consuming medications or substances that affect 5-HT levels in the brain.^3,4 Common symptoms of SS include alterations in mental state, autonomic nervous system issues, and heightened neuromuscular activity^3,4 and can lead to significant morbidity and mortality if not addressed promptly. Therefore, it is crucial to recognize and manage these serotonin-modulated issues effectively⁵ by accurately detecting and monitoring 5-HT levels, which is important for human disease pathogenesis, progression, and treatment.

In recent decades, several methods for detecting 5-HT have emerged, including colorimetric detection,^6,7 HPLC (high-performance liquid chromatography),⁸ spectrofluorimetry,⁹ surface-enhanced Raman spectroscopy,¹⁰ liquid chromatography coupled with tandem mass spectrometry (LC-MS),¹¹ near-infrared (nIR) fluorescent nanosensors (NIRSer),¹² and ELISA (enzyme-linked immunosorbent assay).¹³ While these techniques can accurately measure 5-HT levels, they often require expensive, complex equipment and can be time-consuming, particularly liquid chromatography. Developing more straightforward methods for detecting 5-HT is challenging due to the molecule's complexity and the frequent interference from other neurotransmitters with similar structures, such as dopamine (DA), epinephrine (E), and norepinephrine (NE).

Electrochemical detection (ECD) offers high sensitivity and specificity, making it a powerful tool for studying 5-HT in depression and aiding clinical applications.^14,15 The electrochemical sensors’ performance relies on an effective electrode surface to reduce noise and boost sensitivity.¹⁶ Paper-based electrodes, when modified with advanced nanomaterials, serve as a low-cost and efficient platform for sensing applications.^17,18

Polypyrrole (Ppy) is a well-known conducting polymer characterized by its π-electron conjugation. It is recognized for its excellent electrical conductivity, strong environmental stability under ambient conditions, and minimal toxicological concerns. However, Ppy undergoes significant volumetric changes due to the intercalation and deintercalation of electrolyte ions, resulting in shrinkage and swelling, further compromising long-term stability in electrochemical devices.^19–21 One effective solution to improve properties is to combine Ppy with carbonaceous materials, such as graphene and transition metal sulfides.^22–24 Notably, rGO offers high surface area, excellent electron mobility, and functional groups for modification.²⁵ Depositing metal/metal oxide nanoparticles on it enhances surface activity and electron transfer, which is imperative for assessing electrochemical characteristics.²⁶ Previous studies have shown that electrodes modified with noble metals and their alloys can be used for sensing applications. While these noble metal-based sensors offer excellent electrocatalytic activity and high sensitivity, their associated high cost and limited availability significantly hinder their commercial viability.^27–29

In contrast, transition metals are more cost-effective and abundant, yet still exhibit outstanding electrocatalytic properties, making them widely used in non-enzymatic 5-HT sensors. Among these, nickel-based materials – such as nickel (Ni), nickel oxide (NiO), and nickel(II) hydroxide (Ni(OH)₂) have garnered significant interest due to their remarkable electrocatalytic performance.^30–32 NiO shows faster charge transfer and stronger surface activity, especially in physiological pH, leading to better serotonin detection. Nano Ni(OH)₂ with a small crystalline size exhibits a high proton diffusion coefficient, leading to superior electrochemical performance.³³

In terms of redox activity, nickel oxide (NiO) generally exhibits stronger and more reversible redox behavior compared to iron oxides (Fe₂O₃ or Fe₃O₄) which were employed in our previous work, making it a more effective material for electrochemical applications. While iron oxides are also redox-active, the redox transitions involved (e.g., Fe²⁺/Fe³⁺) are typically less reversible and may not significantly contribute to enhanced conductivity. For instance, the oxidative transformation of iron to form rust is a largely irreversible process, limiting its utility in applications that require frequent charge/discharge cycling. Consequently, NiO's superior redox reversibility provides distinct advantages in electrochemical sensing, where high sensitivity and fast electron transfer are critical.^34,35

Hence, we believe integrating Ppy, rGO, and NiO/Ni(OH)₂ into a disposable paper electrode creates a synergistic nanocomposite material that could facilitate enhanced sensitivity, selectivity, and stability for 5-HT detection.

Drosophila is widely used as a model system due to its genetic similarity to mammals and the ease of performing genetic manipulations.³⁶ The nervous system of Drosophila utilizes 5-HT as one of its neurotransmitters, displaying conserved functional homology with that of humans.^37–39 Hence, analysing 5-HT levels in the brains of Drosophila melanogaster can provide valuable insights into the fundamental mechanisms of 5-HT regulation and its impact on behavioral and neurological disorders. We employed the Gal-UAS transgenic system⁴⁰ to genetically modify the expression of the key enzyme tryptophan hydroxylase (TRH),⁴¹ which is involved in the synthesis of 5-HT. In order to alter 5-HT production, we crossed the TRH-Gal4 line, which drives the expression of the gene responsible for producing tryptophan hydroxylase, with UAS-TeTxLC and UAS-rpr lines. The TeTxLC is a microbial toxin that affects the release of neurotransmitters by acting on the synaptic vesicle-associated protein neuronal-synaptobrevin.⁴² The pro-apoptotic gene reaper (rpr) inhibits proteins that deactivate activated caspases by competitively binding to them.⁴³ Herein, TeTxLC and rpr are used to reduce 5-HT production, and observed changes are detected using the developed serotonin sensor, thus validating the efficacy of the sensor. We further validated the results through high-performance liquid chromatography (HPLC) analysis. Our findings confirm the sensor's reliability and accuracy, demonstrating its potential for precise 5-HT detection in complex biological systems and establishing a solid foundation for future applications in neurochemical research and clinical diagnostics.

Experimental section

Materials

All chemicals and reagents were used as received without any further purification. Pyrrole (>99%), graphite, nickel chloride hexahydrate, ascorbic acid (99.9%), sodium nitrate, sodium acetate, potassium permanganate, potassium ferricyanide (K₃Fe(CN)₆) (99%), potassium ferrocyanide (K₄Fe(CN)₆), hexamine ruthenium(III) chloride (Ru(NH₃)₆Cl₃) (99.9%), serotonin, dopamine, uric acid, epinephrine, and norepinephrine were purchased from Merck-Sigma-Aldrich. The paper electrodes were made using SU-8 photoresist material from Gersteltec in Pully, Switzerland, Whatman CHR paper from GE Healthcare in the UK, and conductive ink (both carbon ink and Ag/AgCl ink) from Kayaku Advanced Materials, Inc. in Massachusetts, USA. All experimental protocols were conducted using a 0.1 M phosphate buffer solution (PB) with a pH of 7.4. This buffer was prepared by precisely mixing the necessary amounts of sodium phosphate monobasic (NaH₂PO₄) and sodium phosphate dibasic (Na₂HPO₄). Before use, all the solutions were purged with nitrogen to eliminate dissolved gases or impurities. Deionized (DI) water (purified to a resistance of 18 MΩ using the Milli-Q Reagent Water System, Millipore Corp, Billerica, MA) was used in all experiments and solution preparations.

Instrumentation and characterizations

Fourier-transform infrared (FT-IR) spectra were recorded using a Shimadzu IR Spirit FT-IR spectrometer. Morphological analysis was performed with a Carl Zeiss Sigma field emission scanning electron microscope (FE-SEM) and a Flex-Axiom atomic force microscope (AFM) from Nanosurf, Switzerland. Electrochemical experiments were conducted using a CH 708E electrochemical workstation and a Zensor ECAS 100 potentiostat. X-ray photoelectron spectroscopy (XPS) experiments were carried out with a K-Alpha spectrometer from Thermo Scientific. The XPS data were analyzed and curve-fitted using the XPS PEAK 41 software.

Fabrication of paper-based electrodes (PPE)

The fabrication of disposable paper-based paper electrodes (PPEs) was accomplished using in-house photolithography and punching techniques, as previously described by our research group.^18,19 In summary, the photolithography process involved creating electrode patterns on the paper substrate by selectively forming hydrophobic and hydrophilic regions with an SU-8 photoresist and an appropriate photomask. The SU-8-treated paper, along with the photomask, was exposed to UV light for 12 minutes to define the hydrophilic regions and establish hydrophobic barriers. After exposure, the treated paper was thoroughly cleaned with isopropyl alcohol and acetone and then dried at ambient temperature. Next, the electrode patterns were etched into the hydrophobic regions using a simple crown punching technique. The working and counter electrodes were made from conductive carbon ink, while the reference electrode was composed of silver/silver chloride (Ag/AgCl) ink functioning as pseudo-reference electrode. Finally, the fabricated 3-in-1 PPEs were dried in an oven at 90 °C for one hour. The geometric calculations determined the area of the working electrode to be 0.07 cm².

Electrochemical modification of PPE with Ppy–rGO–NiO/Ni(OH)₂

At first, GO was synthesized via a modified Hummers’ method,⁴⁴ and 2 μL of GO (2 mg mL⁻¹) was drop-coated onto the working electrode of PPE. Subsequently, after drying at 45 °C, the PPE is treated with an aqueous electrolyte solution comprising monomer pyrrole (Py, 0.01 M) dissolved in phosphate buffer (PB) at pH 7.4. Next, the in situ electrochemical reduction and polymerization were carried out by cycling between the potential ranges of −1.2 V to 1.2 V, with a scan rate of 0.05 V s⁻¹ for 20 cycles at room temperature. After the electrochemical process, the modified electrodes were cleaned with Milli-Q water and dried at room temperature before proceeding with the electrochemical deposition of NiO/Ni(OH)₂ nanocomposites. An acetate buffer (pH 5.2) containing 0.04 M NiCl₂·7H₂O was used for the electrodeposition of NiO/Ni(OH)₂. The electrochemically reduced and polymerized PPE–Ppy–rGO is scanned for 14 cycles between the potential range of −1.2 V to 0.5 V, at a scan rate of 0.05 V s⁻¹. The method is developed by following the previously reported procedure⁴⁵ for the realization of PPE–Ppy–rGO–NiO/Ni(OH)₂. The detailed fabrication procedure for the development of Ppy–rGO–NiO/Ni(OH)₂ nanocomposites onto the PPE is exhibited in Scheme 1. After fabrication, all electrodes were thoroughly washed with Milli-Q water and dried under ambient conditions.

Scheme 1 Schematic representation of the electrochemical fabrication process involved for the development of PPE–Ppy–rGO–NiO/Ni(OH)₂ and the detection of 5-HT.

Drosophila melanogaster brain sample extraction

Drosophila melanogaster flies [TRH-Gal4(control), TRH-Gal4/UAS-TeTxLC, and TRH-Gal4/UAS-rpr] were reared under controlled environmental conditions. 5–6 days old F1 generation flies were collected for the experiment. The flies were anesthetized with carbon dioxide, and approximately 100 females were gathered from each group. The flies underwent decapitation, and their heads were homogenized using a pellet pestle in 500 μL of 0.1 M phosphate buffer, with the process repeated for three cycles. The homogenate was then centrifuged at 12 000 rpm for 10 minutes to yield a clear supernatant, which was carefully collected for subsequent neurotransmitter quantification using the sensor (Scheme 2). In parallel to analyse the serotonin level in the brain samples, a histochemical approach was employed. For this, the brains were dissected from the F1 generation flies and fixed in 1× paraformaldehyde (PFA) for 1 hour at room temperature, followed by washing with 1× PBT for 3–4 times with 20-minute intervals. The brain samples were incubated with the antibody against serotonin (catalog no: S5545, Sigma Aldrich, dilution 1 : 600) for visualizing serotonin and the antibody against Dlg (catalog no: 4F3, DSHB, dilution 1 : 200) for labelling morphological landmarks in the brain. The primary antibodies are incubated overnight with constant shaking using a rocker. Unbound primary antibodies are removed with washing using 1× PBT for 3–4 times with 20-minute intervals. Brain samples are further incubated with secondary antibodies – goat anti-mouse Alexa 568 (catalog no: A-11004, Invitrogen, dilution 1 : 200) and goat anti-rabbit 488 (catalog no: A-11008, Invitrogen, dilution 1 : 200) overnight. Unbound secondary antibodies were washed using 1× PBT. The stained whole brain samples are imaged using a Zeiss LSM 800 confocal microscope (DST-PURSE central facility, Mangalore University). The individual confocal stacks were analyzed in Image J (NIH). The size, contrast, and brightness of the resulting images were adjusted in Photoshop CS (Adobe Systems, San Jose, CA).

Scheme 2 Schematic illustration of the extraction of a genetically modified Drosophila melanogaster brain sample.

Results and discussion

Electrochemical fabrication of PPE–Ppy–rGO–NiO/Ni(OH)₂

One-step simultaneous electro-polymerization of Ppy and electrochemical reduction of GO has been shown in Fig. 1A. The observed anodic peak corresponds to the electropolymerization of the pyrrole monomer, and the oxidation of the monomer begins when the potential reaches 0.6 V, on the surface of the GO-coated paper electrode. A significant increase in current at 0.6 V and the observance of a pronounced anodic peak at 0.9 V indicates the initiation of the polymerization process⁴⁶ and the charging currents gradually increases with subsequent scanning cycles indicates that the process starts at a lower potential, optimal conditions for maximum polymer formation occur at a higher voltage due to increased monomer activation and reaction rates at 0.9 V, which supports the electropolymerization and oxidation of the Py monomer on the PPE surface. On the other hand, electrochemical reduction of GO offers a non-toxic alternative to chemical reduction methods.⁴⁷

Fig. 1 Cyclic voltammograms (CV) for (A) simultaneous electroreduction of GO to rGO and electropolymerization of Py to Ppy (B) electrodeposition of NiO/Ni(OH)₂.

As can be seen from Fig. 1A, a profound reduction peak at −0.59 V, indicating the strong reduction of GO during the first cycle of CVs, and in the subsequent cycles, the cathodic peak response starts decreasing due to the less availability of surface oxygen functional groups on GO. The observed results are akin to the previous reports where these peaks are associated with oxygen-containing functionalities on the graphene surface reduced using the cyclic voltammetry technique.^48,49 Furthermore, it is understandable from Fig. 1B that the cathodic peak observed at −0.19 V corresponds to the oxidation of nickel from the nickel chloride solution in acetate buffer. The highest current density was measured within the lowest potential range (−1.0 to −1.2 V), where water splitting can be observed. The effects of proton concentration and pH can explain the shift of the anodic peak to more negative values during the electrodeposition of nickel hydroxide using nickel chloride hexahydrate in acidic conditions. The high concentration of protons (H⁺) in acidic solutions affects the electrochemical reactions. For reactions involving protons, the Nernst equation (eqn (1)) indicates that an increase in proton concentration (lower pH) shifts the electrode potential.

The mechanism of the electrodeposition of nickel involves two consecutive one-electron charge transfers, and the participation of an anion with the formation of an adsorbed complex, which depends primarily upon the pH of the system. Based on this hypothesis, the possible mechanism for nickel deposition from acetate solutions at pH 5.2 is represented below.^50,51

Ni(CH₃COO)₂ → (NiCH₃COO)⁺ + CH₃COO⁻

Ni(CH₃COO)⁺ → Ni²⁺ + CH₃COO⁻

NiCl₂ → Ni²⁺ + 2Cl⁻

Ni²⁺ + H₂O → Ni(OH)_(aq)⁺ + H⁺

The predominant step for the initiation of deposition at pH 5.2 is as follows.

Ni(OH)_(aq)⁺ + e⁻ → Ni(OH)_(ads)⁺

Direct discharge of the hydroxylated complex

Ni(OH)_(ads)⁺ + Ni²⁺ + 2e⁻ → Ni(OH)_(ads) + Ni

On drying at 40 °C the residual Ni(OH)_(ads) converts to nickel oxide⁵¹

Ni(OH)_(ads) → NiO + H₂O

Surface morphological characterization of PPE–Ppy–rGO–NiO/Ni(OH)₂

To investigate the robust formation of modified electrode materials, we have studied the morphological features involved during each step of the electrode fabrication and were revealed through SEM imaging (GEMINI 300, Carl Zeiss, and Germany), EDX, AFM, FT-IR, and XPS studies.

The SEM images represented in Fig. 2A of bare PPE illustrate the presence of fluffy conductive carbon flakes, similar to what we observed during our previous studies for PPE.^18,19 Interestingly after the simultaneous electorpolymerisation and electrochemical reduction procedure, the modified PPE shows an interconnected nanocomposite network consists of Ppy and rGO, displaying a wrinkled sheet-like structure (Fig. 2B). The electrochemically deposited NiO/Ni(OH)₂ nanocomposite exhibits a flower-like morphology akin to previous reports.⁵² The analysis revealed a well-developed flower structure composed of distinct sheets or petals (inset of Fig. 2C), which are more densely packed towards the center and less compact at the outer edges, highlighting the variations in sheet density within the same porous structure, as previously reported.⁵³ The energy-dispersive spectroscopy (EDS) spectra shown in Fig. 2D–F indicate the elemental distribution of corresponding the modified electrodes, predominantly showing presence of carbon, and oxygen functional groups, with trace amounts of nitrogen, and nickel in accordance with the steps of modifications.

Fig. 2 SEM images of (A) bare PPE, (B) PPE–Ppy–rGO, (C) PPE–Ppy–rGO–NiO/Ni(OH)₂, and the corresponding EDS mapping for (D)–(F). AFM images of (G) PPE, (H) PPE–Ppy–rGO (I) PPE–Ppy–rGONiO/Ni(OH)₂.

Furthermore, atomic force microscopy (AFM) studies (Fig. 2(G)–(I)) supported the SEM findings, revealing significant morphological changes after each modification step. The modified PPE–Ppy–rGO surface displayed mountain-like ridges, while further deposition of NiO/Ni(OH)₂ led to a more pronounced surface texture.⁵⁴ These features suggest an increase in surface area and electroactive sites, favourable for electrochemical applications. On the other hand, the surface thickness also varied according to each modification step, resulting in a thickness of 0.94 nm, 0.2 μm, and 0.82 μm, respectively confirms the sequential layer-by-layer deposition of the nanocomposites. The roughness and thickness parameters are calculated using Gwyddion software, are detailed in Table S1 (ESI†).

Further, the PPE, PPE–Ppy–rGO, and PPE–Ppy–rGO–NiO/Ni(OH)₂ nanocomposites were examined using FT-IR analysis (Fig. S1, ESI†). In the FT-IR spectrum of bare PPE, peaks observed at 1058, 1327, 1558, and 1721 cm⁻¹ correspond to C–O alkoxy, C–O–H deformation vibration, C–O epoxy stretching, and C=O carbonyl stretching vibrations, respectively. After the simultaneous electrodeposition and electroreduction step, a shift in the FT-IR bands can be observed. The peaks at 1440 cm⁻¹ and 1558 cm⁻¹ are attributed to the C–N and N–H stretching vibrations of the Py ring. Additionally, peaks centred at 1963 cm⁻¹ and 2159 cm⁻¹ represent the asymmetric and symmetric stretching vibrations of CH₂ in Ppy. In the FT-IR spectra of Ppy–rGO–NiO/Ni(OH)₂, a significant shift in the characteristic peaks of Ppy and rGO occurs due to the presence of metallic nickel on Ppy–rGO. Therefore, the FT-IR analysis also confirms the successful formation of the Ppy–rGO–NiO/Ni(OH)₂ nanocomposite.^55,56

X-ray photoelectron spectroscopy (XPS) analysis was performed to evaluate the valence states of the elements on the modified electrode surface. The XPS analysis provides valuable insights into the elemental composition and surface chemistry modifications occurring at each step of the layer-by-layer assembly process. Changes observed in the contents of carbon, oxygen, nitrogen, and nickel confirmed the successful integration of each component and supported the formation of a multifunctional nanocomposite material. The investigation included a wide scan (Fig. S2, ESI†) and deconvoluted XPS spectra of C 1s, O 1s, N 1s, and Ni 2p for the PPE–Ppy–rGO–NiO/Ni(OH)₂ composite (Fig. 3). The bare PPE exhibited a predominant composition of carbon (82.99%) and oxygen (17.01%), with no detectable nitrogen or nickel (Table S2, ESI†), indicating that the PPE initially consists of a carbon-rich material with oxygen-containing functional groups. Upon the simultaneous electrochemical reduction and deposition experimental steps on PPE, there was a decrease in carbon content (71.71%) and an increase in oxygen content (26.91%) observed (Fig. S2 and Table S2, ESI†), suggests the successful deposition of rGO, which introduces additional oxygen-containing functional groups. On the other hand, the appearance of nitrogen (1.38%) indicates the incorporation of Ppy molecules.

Fig. 3 XPS deconvoluted spectra of PPE–Ppy–rGO–NiO/Ni(OH)₂. (A) C 1s spectrum (B), O 1s spectrum, (C) N 1s spectrum, and (D) Ni 2p spectrum.

However, in the case of PPE–Ppy–rGO–NiO/Ni(OH)₂, a significant change in carbon content (73.61%) and oxygen content (16.7%), along with the appearance of nickel (0.81%), confirms the integration of nickel compounds into the nanocomposite. Notably, the calculated atomic ratios of O/C, N/C, and Ni/C provide insights into the composition of the Ppy–rGO and NiO/Ni(OH)₂ composite. The O1/C1 ratio for bare PPE and PPE–P(py)–rGO–NiO/Ni(OH)₂ was found to be 0.2049 and 0.227, respectively, indicating a notable presence of oxygen within the composite. The abundance of oxygen is attributed to the oxygen-containing functional groups present in the rGO and NiO/Ni(OH)₂ components. The improved oxygen content can enhance the interactions between Ppy, rGO, and NiO/Ni(OH)₂, leading to better stability and performance in applications such as sensing and catalysis. Although nitrogen is present in smaller quantities, it signifies the inclusion of the Ppy component in the composite. The N1/C1 ratios for PPE–Ppy–rGO and PPE–Ppy–rGO–NiO/Ni(OH)₂ are 0.019 and 0.026, respectively. The increase in nitrogen content in the final composite suggests an ongoing presence and further interaction of Ppy with the new components. Here, Ppy contributes nitrogen atoms, which can enhance electrical conductivity and provide active sites for electrochemical reactions. The NiO/Ni(OH)₂ component, with a Ni/C1 ratio of 0.011, can significantly influence the composite's catalytic and electrochemical properties, even at low concentrations. Nickel oxide is known for its catalytic activity, particularly in oxidation reactions, and can enhance the overall performance of the composite in applications such as supercapacitors, batteries, and sensors.

The C 1s spectrum (Fig. 3A) of the PPE–Ppy–rGO–NiO/Ni(OH)₂ hybrid structure shows three distinct binding energies. The peak at 284.8 eV corresponds to the C–C bond, while the peak at 286.7 eV indicates the C–N bond, confirming the successful complexation of reduced graphene oxide (rGO) and pyrrole. The peak at 288.5 eV is associated with the C=O bond energy generated by the rGO network. In the deconvoluted O 1s spectrum (Fig. 3B), we observe bonds between metal and hydroxides at 530.1 eV and the absorption of oxygen at 531.2 eV.^57,58 The N 1s spectrum (Fig. 3C) displays a major peak that can be broken down into three main components: –N= at 398.5 eV, –NH– at 399.9 eV, and –N⁺– at 401.1 eV.⁵⁹ The Ni 2p spectrum (Fig. 3D) reveals four peaks. The broad peaks at 861.3 eV and 879.6 eV correspond to shake-up satellites. The two main peaks at 873.8 eV and 856 eV are attributed to Ni 2p_1/2 and Ni 2p_3/2, respectively. The spin energy separation of 17.8 eV between these main peaks indicates that nickel in the composite is in the +2 valence state.^57,60 The XPS data confirms the successful layer-by-layer modification of the PPE surface with Ppy, rGO, and NiO/Ni(OH)₂.

Electrochemical behavior of 5-HT at PPE–Ppy–rGO–NiO/Ni(OH)₂

Cyclic voltammetry (CV) measurements were conducted using a three-electrode system with a 0.1 M KOH solution as the electrolyte. Fig. S3 (ESI†) displays the CV curves for the PPE–Ppy–rGO–NiO/Ni(OH)₂ nanocomposite electrodes, recorded over a potential range of −0.1 to 0.6 V at a scan rate of 0.05 V s⁻¹ over 8 cycles. The CV curve shows a pair of redox peaks corresponding to the Ni(OH)₂/NiOOH surface redox couple, associated with the faradaic reactions occurring at the electrode surface. Two well-defined peaks are evident, an anodic peak at 0.44 V, which represents the oxidation of Ni²⁺ to Ni³⁺ during the forward scan according to the reaction Ni(OH)₂ + OH⁻ → NiOOH + H₂O + e⁻, and a cathodic peak at 0.214 V, which corresponds to the reduction of Ni³⁺ to Ni²⁺ during the reverse scan, represented by the reaction NiOOH + H₂O + e⁻ → Ni(OH)₂ + OH⁻. This process involves the reversible insertion and extraction of OH⁻ ions. The symmetric nature of the redox peaks suggests that the Ni(OH)₂ nanosheets exhibit high reversibility. Furthermore, the increasing current density observed with successive sweep segments indicates that the ultrathin Ni(OH)₂ nanoparticles facilitate faster redox reactions.⁶¹

The electrochemical performance of the modified electrodes was evaluated through voltammetric experiments using a commonly employed cationic redox probe, Ru(NH₃)₆Cl₃. The probe displayed a prominent redox peak with a high current, as illustrated in Fig. S4 (ESI†). The voltammograms indicated an increase in the oxidation peak potential with each modification, accompanied by a slight anodic shift. This shift in peak current suggests that the electrode modifications have successfully enhanced its electrochemical properties. The addition of Ppy and rGO improves the overall conductivity of the electrode. Here, Ppy provides a conductive polymer matrix, while the oxidation of Py molecules results in the formation of polarons, which combine to form bipolarons. This process enhances charge transport within the polymer matrix, leading to improved electrochemical performance. Additionally, rGO and NiO/Ni(OH)₂ nanoparticles provide excellent electron transport properties due to their high conductivity and large surface area, which offers more active sites for redox reactions. Furthermore, NiO/Ni(OH)₂ acts as a catalyst, accelerating the rate of electrochemical reactions. This catalytic effect reduces the energy required for the redox processes, thus enhancing the peak currents. The modified electrode exhibited a ΔE_p greater than 60 mV and an I_pa/I_pc ratio exceeding unity, indicating a quasi-reversible process at the electrode surface.⁶² The electroactive surface area of the PPE–Ppy–rGO–NiO/Ni(OH)₂ was evaluated using the Randles–Sevcik equation,^62–64 as follows:

where D is the diffusion coefficient of Ru(NH₃)₆Cl₃ (9.1 × 10⁻⁶ cm² s⁻¹), “I_p” is the forward peak current, C is the concentration of the Ru(NH₃)₆Cl₃ (mol cm⁻³), A is the electroactive area (cm²), “n” is the number of transferred electrons and “v” is the applied scan rate (V s⁻¹), “R” is the universal gas constant and “T” is temperature. The estimated active surface area values are 0.37, 0.46, and 0.99 cm² for bare PPE, PPE–Ppy–rGO, and PPE–Ppy–rGO–NiO/Ni(OH)₂, respectively. The electroactive area of the PPE–Ppy–rGO composite increased by 1.2 times compared to that of bare PPE, and the enhancement is attributed to the functional groups on the modified surface of the PPE–Ppy–rGO composite, which in turn improve the electrochemical properties (Table S3, ESI†). Additionally, after the deposition of NiO/Ni(OH)₂, a further increase of 2.7 times in electroactive area was observed due to the enhanced conductivity and catalytic activity provided by NiO/Ni(OH)₂.

Further investigation of the electrocatalytic oxidation of 1 mM 5-HT was conducted using cyclic voltammetry with a PPE–Ppy–rGO–NiO/Ni(OH)₂ nanocomposite-modified electrode at a scan rate of 0.05 V s⁻¹ in the potential range of −0.1 to 0.45 V with 0.1 M PBS as electrolyte. The electrocatalytic activity of the PPE–Ppy–rGO–NiO/Ni(OH)₂, PPE–Ppy–rGO, and bare PPE electrodes for the oxidation of serotonin (5-HT) was compared, as shown in Fig. 4A. The bare PPE electrode exhibited an ill-defined voltammogram, whilst a significant oxidation peak was observed with PPE–Ppy–rGO electrode. Interestingly, an enhanced oxidation peak was recorded with the PPE–Ppy–rGO–NiO/Ni(OH)₂, indicating superior electrocatalytic activity towards 5-HT oxidation. For better visualization and comparison of the CV responses in the presence and absence of 5-HT, corresponding individual scan for modified electrode is provided in the ESI† (Fig. S5). The enhanced electrocatalytic current is mainly attributed to the increased conductivity, electron transfer, electroactive surface area and roughness surface characteristics resulted during the modification of PPE with Ppy–rGO–NiO/Ni(OH)₂. Overall, the PPE–Ppy–rGO–NiO/Ni(OH)₂ nanocomposite demonstrated excellent performance in detecting 5-HT. The oxidation process generally follows a two-step mechanism, which is characterized by the transfer of two electrons and two protons. In this mechanism, 5-HT is initially oxidized to generate a carbocation intermediate, followed by a second oxidation step that leads to the formation of a quinone imine,⁶⁵ as shown in eqn (3)

5-HT → 5-HT^+· → 5-HT⁺ → quinone imine (3)

Fig. 4 (A) CV profiles of PPE, PPE-Ppy, PPE–Ppy–rGO, and PPE–Ppy–rGO–NiO/Ni(OH)₂ in 1 mM 5-HT in 0.1 M PB solution (pH 7.4), recorded at a scan rate of 0.05 V s⁻¹. (B) CV analysis of PPE–P(py)–rGO–NiO/Ni(OH)₂ at different scan rates from 0.01 to 0.17 V s⁻¹ in 0.1 M PB solution (pH 7.4) containing 500 μM 5-HT and the corresponding (C) linear relationship between the peak current (I_p) and scan rate (ν) (D) calibration plot for log of peak current (I_p) and log of scan rate.

The effect of scan rates on the electrochemical behaviour of 5-HT at the PPE–Ppy–rGO–NiO/Ni(OH)₂ electrode was investigated using the CV studies. Fig. 4B displays the CVs of the PPE–Ppy–rGO–NiO/Ni(OH)₂ electrode at various scan rates (ν) ranging from 0.01 to 0.17 mV s⁻¹ in phosphate-buffered saline (PBS, pH 7.4) containing 500 μM of 5-HT. Both the peak potential (E_p) and peak current (I_p) were influenced by the scan rate. As the scan rate increased, the oxidation peak current (I_pa) also increased (Fig. 4C), while the peak potential shifted positively. To determine whether the process was controlled by diffusion or adsorption, the relationship between the logarithmic vales of peak current (log I_p) and scan rate (log ν) was analysed (Fig. 4D). For the 500 μM concentration of 5-HT in pH 7.4 PBS, the log I_pversus log ν showed a linear relationship, resulted a linear equation with a slope of 0.86 indicating that the oxidation process is controlled by adsorption, suggesting that the electrochemical process is predominantly surface-controlled.

Next, electrochemical impedance spectroscopy (EIS) was employed to investigate the surface properties of the modified electrode in a 0.1 M KCl solution containing 5 mM [Fe(CN)₆]^3−/4−, measured at open circuit potential (OCP) over a frequency range of 100 mHz to 1 MHz (Fig. 5). The PPE–Ppy–rGO–NiO/Ni(OH)₂ composite exhibited the lowest electron transfer resistance, characterized by the smallest semicircle diameter compared to the other electrodes. In contrast, the significantly larger semicircle diameter observed for the bare PPE (Fig. 5, pink) indicates that the electron transfer between the redox probe and the electrode was hindered. The analysis utilized the Randles equivalent circuit, which consists of four main components: solution resistance (R_s), double-layer capacitance (C_dl), charge-transfer resistance (R_ct), and Warburg impedance (Z_W). In the Nyquist plot, the initial point of the semicircle corresponds to the solution resistance (R_s), while the diameter of the semicircle represents the R_ct. The bare electrode displayed a high charge-transfer resistance of 5394.74 Ω, whereas the PPE–Ppy–rGO–NiO/Ni(OH)₂ composite demonstrated a significantly lower R_ct of 129.73 Ω, which highlights its superior conductivity and enhanced electron transfer efficiency.

Fig. 5 Electrochemical impedance spectroscopy of bare PPE (pink), PPE–Ppy (black), PPE–Ppy–rGO (red), PPE–Ppy–rGO–NiO/Ni(OH)₂ (blue) in 0.1 M KCl solution containing 5 mM [Fe(CN)₆]^3−/4−.

The charge-transfer resistance (R_ct) is related to several factors including the gas constant (R), absolute temperature (T), the standard rate constant for heterogeneous electron transfer (K₀), the number of electrons transferred (n), Faraday's constant (F), the surface area of the electrode (A), and the concentration of the redox species (C) in solution, as described by eqn (4).⁶⁶

R_ct = RT/K₀n²F²AC (4)

The calculated K₀ values for PPE, PPE–P(py)–rGO and PPE–Ppy–rGO–NiO/Ni(OH)₂ were 0.099 × 10⁻⁷ and 2.9 × 10⁻⁷, and 4.1 × 10⁻⁷, respectively. The combination of a low R_ct and a high K₀ for the nanocomposite material can be attributed to a synergistic effect, significantly enhancing the sensor's performance, making it well-suited for detecting 5-HT.

To further test the detection of 5-HT, we have performed SWV analysis of the fabricated sensor under optimal conditions. As shown in Fig. 6A, the oxidation peak current significantly increases with rising concentrations of 5-HT, ranging from 500 μM to 0.007 nM. This suggests that the PPE–Ppy–rGO–NiO/Ni(OH)₂ sensor has potential for the quantitative detection of 5-HT. While the overall calibration curve shows a linear and non-linear relationship between current and 5-HT concentrations, a closer examination reveals two linear ranges, largely influenced by the higher concentration region (0.48 nM to 500 μM), and the lower concentration region (0.007 nM to 0.48 nM). The presence of two linear ranges are due to concentration gradient limitations, where reduced mass transport and limited analyte–electrode interactions affect the electrochemical response as illustrated in Fig. 6B. The limit of detection (LOD) defines the minimum amount of analyte that can be detected by the sensor. It is calculated using the formula: LOD = 3.3 × S/b, where “S” represents the standard deviation of the lowest concentration of 5-HT and “b” is the slope of the calibration curve obtained from the SWV. The resulting LOD values were 0.024 nM for the low concentration range and 383.7 nM for the high range, which is lower than values reported in the literature, as detailed in Table S4 (ESI†). In summary, the PPE–Ppy–rGO–NiO/Ni(OH)₂ nanocomposite exhibits excellent sensing performance, a strong linear relationship, high sensitivity, and a low LOD.

Fig. 6 (A) Square wave voltammetry (SWV) profile for the different concentrations of 5-HT from 0.007 nM to 500 μM (inset shows the SWV for lower concentrations of 5-HT from 0.007 nM to 60 nM) (B) the corresponding calibration plot for the higher concentration region (0.48 nM to 500 μM), and the lower concentration region (0.007 nM to 0.48 nM).

The ability to selectively detect specific molecules is crucial for the effective use of electrochemical sensors and biosensors. In the case of sensors targeting 5-HT, the presence of other electroactive substances such as ascorbic acid (AA), uric acid (UA), dopamine (DA), adrenaline (E), and noradrenaline (NE) in real samples can significantly impact sensor performance. These compounds are often oxidized at a potential close to that of serotonin on conventional electrodes, leading to interference in the electrochemical detection of 5-HT.^13,58,59 To assess the ability of the PPE–Ppy–rGO–NiO/Ni(OH)₂ electrode to avoid interference and confirm its selectivity towards 5-HT sensing, the SWV response was recorded. As illustrated in Fig. S6(A)–(F) (ESI†), the sensor displayed minimal interference from common electroactive species, including E, NE, DA, UA, and AA (all at 100 μM each). However, the CV also shows there is a substantial decrease in current meant to be for 5-HT in the presence of AA (Fig. S6F, ESI†) and satisfactory results in the case of NE, E and UA. As outlined in earlier studies, DA and other interferents such as ascorbic acid (AA) and uric acid (UA) are known to oxidize within close potential windows to 5-HT, especially on carbon-based electrodes. Their presence can result in overlapping or slightly shifted peaks due to changes in the double-layer composition, competitive oxidation, or co-adsorption effects. Interestingly, as can be observed from the Fig. S6B (ESI†), in the presence of DA, there is second peak meant for DA oxidation at 0 mV obtained in the presence of 5-HT. It should be noted that, the increased current response for 5-HT upon the addition of DA can be attributed to the enhanced conductivity of the electrode or to the synergistic effects between the analytes. DA appears to improve electron transfer at the electrode, indirectly increasing the oxidation current of 5-HT.⁶⁷ The differing oxidation potentials for DA and 5-HT allows the opportunity for selective detection, which is a hallmark of a well-designed sensor, that can quantify both neurotransmitters independently in mixed samples. The stability of the developed 5-HT biosensor was also studied (Fig. S7, ESI†). After being stored at room temperature for 90 days, the biosensor maintained 95% of its initial CV response for 100 μM 5-HT for the first 45 days. However, a decline in activity was observed beyond this period, likely due to degradation of the electroactive surface. This may involve moisture absorption or interaction with atmospheric CO₂ affecting the polypyrrole matrix, as well as surface passivation or leaching of the NiO/Ni(OH)₂ component, ultimately impairing conductivity and redox performance.

Measurement of 5-HT in genetically engineered Drosophila melanogaster brain sample

For a newly developed sensor it is essential to check the practical application in complex biological environments and further validation with gold standard methods. Herein, we used genetically engineered Drosophila melanogaster brain samples to evaluate the performance of a novel sensor designed to detect 5-HT using SWV (Fig. 7A–C), and validated the results by comparing them with gold standard method (Table 1). The data presented in Table S5 (ESI†) includes the analysis of accuracy percentages (%) for three spiked samples under three experimental conditions: TRH-Gal4 as the control (Fig. 7A), TRH-Gal4/UAS-TeTxLC (Fig. 7B), and TRH-Gal4/UAS-rpr (Fig. 7C).

Fig. 7 SWV response genetically engineered Drosophila melanogaster brain samples (A) TRH-Gal4 (B) TRH-Gal4/UAS-TeTxLC (C) TRH-Gal4/UAS-rpr and the corresponding 5-HT immunostaining of Drosophila brain samples (in green): (D) TRH-Gal4 (E) TRH-Gal4/UAS-TeTxLC (F) TRH-Gal4/UAS-rpr Immunostaining against Dlg is used to anatomically identify the regions in the brain (in magenta) and the scale bar for the confocal images are 100 μM.

Table 1 Data obtained real sample analysis and comparison with gold standard (HPLC)

Sample	Present method	HPLC	% of accuracy
TRH-Gal4	27.6	26.6	103.7
TRH-Gal4/UAS-TeTxLC	11.9	12.5	95.2
TRH-Gal4/UAS-rpr	7.59	7.44	102

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In the spike sample analysis, we assessed the added amounts, found amounts, recovery percentages for each sample to evaluate the accuracy of the method (Table S5, ESI†). For the control TRH-Gal4, the recoveries were consistently close to 100%, ranging from 98.6% to 97.7%, which indicates high accuracy (Fig. 7A). The combination of high recovery and low variability underscores the reliability and repeatability of the method for this experimental group, making it well-suited for accurate quantification.

The antibody staining against 5-HT in the Drosophila brain samples depicted in Fig. 7(D)–(F) corresponds to the different experimental conditions and will also be considered for evaluation in genetically manipulated conditions. In the case of genetically manipulated conditions where 5-HT-producing cells are suppressed using the expression of tetanus toxins (TRH-Gal4/UAS-TeTxLC), the recoveries remained similarly close to 100%, with values between 96.8% and 100.5%, signifying very high accuracy (Fig. 7B). In the TRH-Gal4/UAS-rpr group (Fig. 7C), where serotoninergic cells were induced to undergo apoptosis, the recovery percentages ranged from 98.1% to 100.4%, reflecting high accuracy. However, slightly lower recoveries were observed in the first and second spikes compared to the other groups. These results suggest that the method is both accurate and precise for this sample group. The overall recovery rates were consistently high, ranging from 96.8% to 100.5%, which confirms the method's excellent accuracy under various conditions. Validation using high-performance liquid chromatography (HPLC) (Fig. S8A–C, ESI†) further corroborated the sensor's accuracy and sensitivity. The agreement between the electrochemical results and the HPLC data highlights the robustness of the sensor for neurochemical analysis.

Conclusions

In this study, we developed a highly sensitive and selective electrochemical sensor based on PPE–Ppy–rGO–NiO/Ni(OH)₂ for the detection of 5-HT. The sensor exhibited excellent performance in terms of sensitivity, with a broad range from 0.007 nM to 500 μM, and an impressive limit of detection (LOD) of 0.024 nM for the low concentration range (0.007–0.48 nM) and 383.7 nM for the high concentration range (0.48 nM–500 μM). The EIS studies confirmed enhanced electron transfer kinetics at the electrode, with calculated K₀ of 4.1 × 10⁻⁷ cm s⁻¹ and reduced R_ct value of 129.73 Ω, further supports improved conductivity and electrochemical activity. The anti-interference capability, validated against common interfering species such as dopamine, epinephrine, norepinephrine, ascorbic acid, and uric acid, exhibited satisfactory outcome. Considering the importance of 5-HT in neurobiology, the brain homogenates from Drosophila melanogaster models with genetically altered serotonin levels validated the sensor's practical applicability. Spike sample analysis demonstrated high accuracy, with recovery values ranging from 98.1% to 100.5% across all genotypes tested, including TRH-Gal4 controls, TRH-Gal4/UAS-TeTxLC, and TRH-Gal4/UAS-rpr lines. The observed results were consistent with immunohistochemical staining and were further corroborated by HPLC analysis, affirming the robustness, precision, and reliability of the sensor platform for real-time neurochemical monitoring. The sensor showed remarkable stability over a period of 45 days when stored at room temperature, making it as a promising candidate for long-term use in neurochemical sensing. These findings suggest that the developed sensor holds significant potential for applications in clinical diagnostics and neurobiological research, particularly in monitoring neurotransmitter levels in real biological systems. On the other hand, the nanocomposites materials could be used in the future as an electrode material for fast-scan cyclic voltammetry and fast-scan controlled adsorption voltammetry probes for real-time measurement of 5-HT.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

All the data's are presented in the manuscript and it is available from the authors up on request. The authors have nothing to declare.

Acknowledgements

SP is grateful to the Department of Science and Technology (DST) for the Women Scientist-A Fellowship (ref. no. DST/WOS-A/CS-53/2021). KSP is thankful for the financial support received from the BIRAC-ETA grant (YTI/ETA/SL/PR009). The authors also appreciate the support provided by the Yenepoya Technology Incubator MedTech rapid prototyping facility, which is backed by the National Biopharma Mission (NBM) and DBT-BIRAC. Additionally, the authors would like to acknowledge the Central Research Facility (CRF) at the National Institute of Technology Karnataka (NITK) for granting access to the Flex-Axiom Atomic Force Microscope (AFM) from M/s Nanosurf, Switzerland.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb01216c

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