Coordination-Driven Electrodeposition of Chitosan-Cu²⁺/SWCNT Hydrogel for Enhanced Non-Enzymatic Glucose Sensing

Materials

Chitosan from crab shells (highly viscous), copper (II) sulfate penta-hydrate (CuSO₄ · 5H₂O, 98%), carbon nanotube-single walled (≥95% as carbon nanotubes), potassium sulfate (K₂SO₄), sodium hydroxide (NaOH, ≥97.0%), acetic acid (CH₃COOH, ≥99.7%), D-glucose, L-ascorbic acid (99%) and dopamine hydrochloride (≥98%) were purchased from Sigma-Aldrich. All chemicals and reagents used in this work were of reagent grade. Chitosan hydrogel was synthesized on a screen-printed gold electrode (SPGE, Metrohm DRP-250AT) with a 4 mm diameter gold working electrode, a platinum counter electrode, and a silver quasi reference electrode for comparative study and glucose sensor fabrication. The SPGE was electrochemically cleaned via cyclic voltammetry (CV) in the potential range of –1.0~1.0 V (vs. Ag/AgCl QRE) in 0.1 M H₂SO₄ solution for 30 cycles to remove organic contaminants. Ultrapure water (>18 MΩ·cm) was obtained from a Millipore Milli-Q purification system.

Electrodeposition of copper-coordinated chitosan hydrogel

The chitosan solutions with varying concentrations (0.5~2.0 wt%) were prepared by dissolving chitosan powder in acetic acid (2% v/v) under continuous magnetic stirring overnight. The pH of the chitosan solution was then adjusted to 5.5 by the dropwise addition of 1 M NaOH, resulting in a viscous, cream-like formulation. To synthesize copper-coordinated chitosan hydrogel (CSCu) with carbon nanotubes (CSCuCNT), single walled carbon nanotubes (SWCNTs) were added to the chitosan solution at a concentration of 1 mg / mL.

For the coordination-driven deposition of chitosan hydrogel, a two-step electrodeposition method was employed on a gold electrode. First, copper solution containing 50 mM CuSO₄, 0.5 M H₂SO₄, and 0.1 M K₂SO₄ was introduced to the pre-cleaned SPGE. A reduction potential of –0.6 V (vs. Ag/AgCl QRE) was applied for 10~120 s, resulting in the electrodeposition of copper onto the gold surface. Subsequently, the chitosan solution was introduced onto the copper coated gold electrode, and an oxidation potential of 0.6 V was applied for twice the deposition time to fully oxidize the copper, generating Cu²⁺ ions. The formation of bluish chitosan hydrogel on the gold electrode indicated the successful synthesis of copper-coordinated chitosan hydrogel. For CSCuCNT, the same procedure was performed using a chitosan solution containing SWCNTs.

For comparison, chitosan hydrogel without Cu²⁺ ions was prepared by cathodic electrodeposition based on pH-induced precipitation. The chitosan solution (pH 5.5) was introduced onto the gold electrode, and a cathodic reduction potential of –1.5 V was applied for 120 s. Under this strong reduction potential, hydrogen evolution increased the local pH near the electrode surface beyond pH 5.5. This local pH increase, induced cathodic precipitation of the chitosan hydrogel, which was subsequently used for further experiments.

Characterization of chitosan hydrogel

The topography and roughness factor of chitosan hydrogel was investigated using the non-contact mode of an NX-10 AFM (Park Systems, Republic of Korea) equipped with a AC160TS (Olympus, Japan) probe which has resonance frequency of 300 kHz and tip radius of ~7 nm. FTIR spectra of chitosan hydrogel was obtained with ALPHA II (Bruker, Germany) spectrophotometer with ATR mode. The surface morphology of SPGE with and without copper electrodeposition and after synthesizing the chitosan hydrogel was confirmed by using FE-SEM (TESCAN, MIRA III) equipped with a secondary electron detector. To confirm the copper was well deposited and fully oxidized, the energy-dispersive spectrometer (EDS, Oxford Instrument, UK) was used. A digital microscope (AM4113, Dino-Lite) was used to measure the height of chitosan hydrogel.

Electrochemical measurement of chitosan hydrogel and glucose oxidation properties of CSCu-hydrogel

All the electrochemical measurements were performed using a conventional three-electrode system with a CHI 6011E potentiostat (CH Instruments, USA). For investigating the mass transport properties of Cu²⁺ ions in chitosan hydrogel, the CSCu modified SPGE was used. The CV was conducted in the range of –0.5~0.5 V (vs. Ag/AgCl QRE) with various scan rate (10, 20, 50, 70, 100 mV/s) in the supporting electrolyte containing 0.1 M K₂SO₄. The glucose oxidation properties were examined using CV on CSCu- and CSCuCNT- modified SPGEs in the potential range of 0~0.9 V (vs. Ag/AgCl QRE) in 0.1 M NaOH solution, both with and without glucose, at a scan rate of 50 mV/s. Electrochemical impedance spectroscopy (EIS) measurements were performed using a Gamry 1010E potentiostat (Gamry Instruments, USA) equipped with a frequency response analyzer. Experiments were conducted at an applied potential of 0.7 V (vs. Ag/AgCl QRE) in 0.1 M NaOH solution containing glucose. The frequency range was scanned from 100 kHz to 0.1 Hz with an AC perturbation amplitude of 10 mV. To determine the detection limit of glucose sensing, chronocoulometry was performed by applying potential of 0.7 V for 5 seconds.

Comparative analysis of cathodic and Cu²⁺-coordinated electrodeposition mechanisms of chitosan hydrogels

In order to confirm the difference between the two electrodeposition mechanisms, Au substrate was used as the cathodic electrode, and related cathodic neutralization process is depicted in Scheme 1a. Photographs of the chitosan hydrogel obtained by cathodic electrodeposition showed extremely rough and porous surface due to the evolution of H₂ bubbles during deposition process (Fig. S1a). Also, as-prepared chitosan hydrogel was expanded by the H₂ gas, resulting in a thick and irregular film on the substrate (Fig. S1b). From the viewpoint of fabricating biopolymer, these results indicate that H₂ bubbles significantly affect the surface smoothness and introduces defects on the deposited film, which may potentially impede the subsequent application of the deposited material. Meanwhile, Cu-modified Au substrate was used as the anodic electrode for the coordinated electrodeposition of chitosan (depicted in Scheme 1b, c). Photographs of the as-prepared chitosan hydrogel by this method revealed completely different surface condition compared to cathodic process. The deposited hydrogel exhibited a transparent and uniform surface with a consistent thickness across the entire area without any bubbles (Fig. S1c, d).

Scheme 1.

Schematic illustration of chitosan electrodeposition methods. (a) Cathodic electrodeposition of chitosan hydrogel, (b) Cu²⁺-coordinated electrodeposition of chitosan hydrogel, and (c) stepwise process of Cu²⁺-coordinated electrodeposition.

To evaluate whether the Cu on the SPGE remained after Cu²⁺-coordinated hydrogel formation and could affect subsequent electrochemical applications, the surface morphology and elemental composition of the SPGE were examined. After hydrogel formation, the chitosan layer was removed from the electrode, and the exposed Au surface was analyzed using FE-SEM equipped with EDS (Fig. S2). Prior to hydrogel formation, SEM images revealed the presence of aggregated Cu nanoparticles, typically several tens of nanometers in diameter, distributed on the Au substrate (Fig. S2a). These Cu nanostructures were clearly distinguishable from the underlying Au crystalline surface, and the corresponding EDS spectra confirmed strong Cu signals in addition to the dominant Au peaks (Fig. S2c). In contrast, after oxidation of Cu to Cu²⁺ followed by coordination with chitosan, the SEM images no longer showed Cu nanoparticles, exposing only the bare Au crystalline structure beneath the hydrogel layer (Fig. S2b). Consistently, the EDS spectra indicated that the Cu content was reduced to a negligible level, demonstrating that Cu was almost completely consumed during hydrogel formation (Fig. S2d). Minor Si and Ti peaks observed in the spectra originated from the supporting substrate.

Additionally, AFM analysis was performed to examine the microscopic structures of each generated hydrogel network. The 2D and 3D AFM images of the cathodic and anodic electrodeposited hydrogels are presented in Fig. 1. As shown in Fig. 1a and 1c, cathodically generated chitosan hydrogels exhibited a rough surface with repeated canyon-like features among bright and dark domains, reflecting its high surface roughness. The RMS roughness value (R_q) obtained over a 2×2 μm² area was 10.242 nm. In contrast, the Cu-coordinated hydrogel (Fig. 1b and 1d) showed a more regular and finely pore structure, with a significantly lower R_q value of 1.923 nm. These results suggest that coordinated electrodeposition enables the formation of biopolymer hydrogels with homogeneous surfaces and uniform thickness, which are beneficial for further applications.

Fig. 1.

2D and 3D AFM images of chitosan hydrogel on Au electrode by (a, c) cathodic electrodeposition and (b, d) Cu²⁺-coordinated electrodeposition from a 1.0 wt% chitosan solution.

Effect of chitosan concentration on hydrogel porosity and structural evolution

To investigate the effect of chitosan concentration on hydrogel porosity, AFM analyses were conducted on hydrogels prepared with different chitosan concentration (Fig. 2). The AFM topography images and corresponding pore size distributions revealed distinct morphological changes as a function of concentration. At the lowest concentration (0.5 wt% CSCu), the hydrogel surface exhibited a highly porous and nonuniform structure, with an average pore size of 177.6 nm and a relatively large standard deviation of 6.4 nm. In the Cu²⁺- coordinated deposition system, the interaction between Cu²⁺ ions and chitosan chains play a crucial role in hydrogel network formation. However, at 0.5 wt%, the chitosan concentration was insufficient to establish a coherent gel matrix, leading to poor structural integrity and irregular network formation. Increasing the concentration to 1.0 wt% markedly reduced the average pore size to 149.1 nm, with a narrower distribution and an increased density of uniformly sized pore domains (Fig. 2c, d). This indicates the development of a more interconnected and homogeneous hydrogel network, likely facilitated by the greater availability of chitosan chains for coordination and cross-linking with Cu²⁺ ions. Further increases in concentration to 1.5 wt% and 2.0 wt% produced progressively denser hydrogel structures, with average pore sizes decreasing to 136.7 nm and 103.8 nm, respectively (Fig. 2e–h). The morphological features became increasingly compact and uniform, indicating tighter hydrogel packing and a more integrated network matrix. In general, the increase in the concentration of chitosan solution for gelation accompanied a monotonic decrease of pore size (Fig. S3). This structural evolution critically regulates electrolyte accessibility, ion diffusion, and interfacial charge transfer, which are key parameters in further electrochemical applications.

Fig. 2.

AFM topographies of Cu²⁺-coordinated chitosan hydrogel prepared different concentration of chitosan: (a) 0.5 wt%, (c) 1.0 wt%, (e) 1.5 wt%, (g) 2.0 wt%. Corresponding gaussian distributions of pore sizes are shown in (b), (d), (f), and (h), respectively.

Effect of chitosan concentration on hydrogel thickness and Cu²⁺ coordination behavior

To elucidate the influence of chitosan concentration on hydrogel growth, we examined the thickness of electrodeposited chitosan hydrogels prepared on SPGE with identical geometric areas. Cross-sectional microscopy revealed that hydrogel thickness varied with chitosan content (Fig. 3a), reflecting differences in hydrogel network formation and Cu²⁺ incorporation. The macroscopic gelation behavior was strongly governed by the overall availability of chitosan chains and their functional groups (–NH₂, –OH) that serve as ligands for Cu²⁺ coordination. At 0.5 wt%, the limited chitosan content was insufficient to form a coherent network, resulting in incomplete gelation and low cross-linking density. The 1.0 wt% CSCu hydrogel exhibited the maximum thickness of 1.10 mm, representing an optimal balance between the availability of chitosan chain and Cu²⁺ ions for coordination. In contrast, at higher concentrations (1.5 and 2.0 wt%), the significantly increased viscosity of the chitosan solution hindered the diffusion of Cu²⁺ ions from the electrode surface into bulk chitosan solution, leading to localized cross-linking near the electrode surface. This restricted ion diffusion leads to the formation of a compact and dense structure at the electrode surface, while the upper region exhibits a loose morphology, resulting in thinner hydrogel than optimized conditions.

Fig. 3.

(a) Cross-sectional microscopic images of electrodeposited chitosan hydrogels with varying chitosan concentrations (0.5–2.0 wt%), showing the hydrogel thickness (L) measured by digital microscopy. (b) FT-IR spectra of cathodically deposited chitosan hydrogel (green), and Cu²⁺-coordinated chitosan hydrogel (CSCu) with increasing concentrations: 0.5 wt% (black), 1.0 wt% (red), 1.5 wt% (blue), and 2.0 wt% (orange).

To further verify the coordination of Cu²⁺ with chitosan at different concentrations, FT-IR spectroscopy was performed (Fig. 3b). All spectra exhibited characteristic absorption bands of chitosan [37], including the broad –OH / –NH stretching vibration around 3400 cm^-1, the C-H stretching at 2920 cm^-1 and 2850 cm^-1, the amide I band near 1633 cm^-1 (C=O stretching of residual N-acetyl groups), the amide II vibration at 1561 cm^-1 (NH bending coupled with C-N stretching), and the polysaccharide C-O-C stretching in the 1150-1020 cm^-1 region. Notably, the intensity of the amide II band at 1561 cm^-1 increased with higher chitosan concentrations, confirming progressive coordination between Cu²⁺ ions and the amino groups of chitosan [38,39]. Subtle variations in the broad –OH/ –NH stretching band suggest that the incorporation of Cu²⁺ perturbs the hydrogen-bonding network of the hydrogel. In contrast, the characteristic C-O-C band of the polysaccharide backbone remained unchanged, indicating that the glycosidic backbone of chitosan was preserved during coordination [40]. Collectively, these spectral features demonstrate that Cu²⁺-amine coordination is a dominant factor governing hydrogel stabilization, consistent with previous reports on metal-chitosan interactions [41,42].

Electrochemical analysis of Cu²⁺ coordination behavior within chitosan hydrogels

To further investigate the effect of chitosan concentration on electrochemical behavior of Cu²⁺ ions and their coordination environment within the hydrogel matrix, cyclic voltammetry (CV) was performed (Fig. 4a). Chitosan hydrogel, prepared by cathodic electrodeposition (dotted line) showed no redox peaks within the examined potential window, while Cu²⁺-coordinated chitosan exhibited well-defined Cu related redox peaks, indicating the electrochemical response arises from coordinated Cu²⁺ species. Furthermore, increasing the chitosan content from 0.5 to 2.0 wt% led to a negative shift in the half-wave potential (E_1/2) of both Cu²⁺/Cu⁺ and Cu⁺/Cu⁰ redox couples compared to the redox behavior of free Cu²⁺ in solution (Fig. S4). The negative shift of the half-wave potential with increasing chitosan concentration indicates stabilization of Cu²⁺ in the coordinated state, making its electrochemical reduction more difficult compared with free Cu²⁺. It is noteworthy that at chitosan concentrations above 1.5 wt%, Cu²⁺ forms stable complexes that favor the direct two-electron reduction pathway (Cu²⁺→Cu), thereby suppressing the intermediate Cu⁺ state and eliminating the Cu²⁺/Cu⁺ peak within the potential window [43]. This potential shift reflects both the strength of the interaction and the effective number of ligation sites involved, and can be quantitatively described by the Lingane relation [44,45]:

Fig. 4.

(a) Cyclic voltammograms of a Cu²⁺ free CS hydrogel (dotted line) and Cu²⁺-coordinated CSCu hydrogels with varying chitosan concentrations (0.5 wt% – 2.0 wt%), (b) plot of log [L] vs half-wave potential shift at a scan rate of 50 mV/s.

Where ΔE_1/2 is the change of half-wave potential relative to that of free Cu²⁺ ions in solution, C_L is the ligand concentration, R is the universal gas constant, T is the temperature, n is the number of electrons involved in the transfer, F is the Faraday constant, K_s is the stability constant, and j represents the effective coordination number. The plot of ΔE_1/2 versus log [chitosan] exhibited a linear relationship (Fig. 4b), from which quantitative analysis yielded an average coordination number of j = 0.67 and a stability constant of K_s = 7.04 × 10⁴ (the detailed calculation process is provided in the Supporting Information).

The fractional coordination number (j<1) indicates that in the heterogeneous polymer-metal system, not all Cu²⁺ ions can fully coordinate with every available donor site on the chitosan monomer [45,46]. This result can be attributed to steric hindrance from the semi-rigid chitosan backbone, partial accessibility of donor groups, and the coexistence of various coordination mode [47,48]. Thus, j = 0.67 represents a thermodynamic average, indicating that the coordination sphere of Cu²⁺ is partially unsaturated and contains open and accessible coordination sites at the metal center, which are highly desirable for catalytic activity [49–51]. These available sites are essential for the binding of substrate molecules (i.e., glucose), which is a prerequisite for an efficient electrocatalytic oxidation process.

The large stability constant (K_s = 7.04 × 10⁴) corresponds to a free energy of complex formation of approximately –27.7 kJ/mol at 298 K, evidencing spontaneous and thermodynamically favorable complexation [52–55]. The high K_s value accounts for the pronounced potential shift observed with increasing chitosan concentration, showing a steep rise from 0.5 to 1.0 wt% and a saturation behavior above 1.5 wt%, where ligand accessibility becomes limited due to network compaction and steric hindrance. Taken together, these findings demonstrate that Cu²⁺ is strongly stabilized through coordination with chitosan functional groups, while the sub-unity coordination number highlights the heterogeneous and partially accessible nature of the polymer–metal binding environment. This combination of high stability and incomplete site saturation provides fundamental insight into the structural role of chitosan in immobilizing Cu²⁺ within the hydrogel matrix.

Electrochemical evaluation of mass transfer characteristics of Cu²⁺ in the hydrogel matrix

To investigate the mass transport properties of Cu²⁺ within the hydrogel matrix, CV were performed at scan rates ranging from 10 to 100 mV/s for chitosan hydrogels of varying concentrations (Fig. 5). All samples exhibited well-defined Cu²⁺/Cu⁺ and Cu⁺/Cu⁰ redox peaks, confirming the electrochemical activity of coordinated Cu species within the hydrogel network, except that the Cu²⁺/Cu⁺ peak disappeared at concentrations ≥1.5 wt% due to the aforementioned complexation effects.

Fig. 5.

Cyclic voltammograms of Cu²⁺ within chitosan hydrogels at various scan rate (v), recorded for different chitosan concentrations: (a) 0.5 wt%, (c) 1.0 wt%, (e) 1.5 wt%, and (g) 2.0 wt%. The corresponding plots of anodic (Ipa) and cathodic (I_pc) peak currents versus the square root of the scan rate for the Cu / Cu⁺ redox couple are shown in (b), (d), (f), and (h), respectively.

The CV measurements revealed a non-linear dependence of the cathodic peak current (I_pc) on chitosan concentration (0.5–2.0 wt%). As shown in Fig. 5a, c, e, and g, the lowest I_pc was observed at 0.5 wt%, while the maximum value appeared at 1.0 wt%. Further increase in concentration to 1.5 and 2.0 wt% yielded intermediate I_pc values. This behavior can be attributed to the balance between the effective Cu²⁺ concentration at the electrode surface and the structural characteristics of the hydrogel network. At 0.5 wt%, the limited number of –NH₂ and –OH groups provide insufficient coordination sites, leading to a reduced preconcentration of Cu²⁺ at the electrode surface and resulting in a small I_pc. At 1.0 wt%, the density of coordination sites was sufficient to maximize the local accumulation of Cu²⁺ near the electrode while maintaining a porous and conductive hydrogel network, thereby producing the highest I_pc. In contrast, at higher concentrations (≥1.5 wt%), excessive gel formation increased network density and reduced porosity, which hinders ion diffusion and lowers the effective conductivity of the hydrogel matrix. Despite the increased number of coordination sites, these mass-transport and electron-transfer limitations suppressed the current response [56–58]. These results demonstrate that the 1.0 wt% chitosan hydrogel achieves an optimal balance between the effective Cu²⁺ concentration at the electrode surface and the structural integrity of the gel network. Collectively, the results indicate that the 1.0 wt% chitosan hydrogel provides an optimal balance between effective Cu²⁺ concentration at the electrode surface and the structural integrity of the hydrogel network, thereby representing the most promising composition for future application in electrochemical sensing platforms.

In addition to the concentration-dependent variation in I_pc , the corresponding plots of anodic and cathodic peak currents (I_p) versus the square root of the scan rate (v^1/2) (Fig. 5b, d, f, and h) exhibited linear relationships across all hydrogel concentrations. Compared with the peak current versus scan rate (v), a stronger linear correlation was observed for I_p versus v^1/2 (Fig. S5). This behavior is consistent with the Randles-Sevcik equation, indicating that the redox process is predominantly diffusion-controlled [59].

To further quantify the diffusion characteristics of Cu²⁺ within the chitosan hydrogels, the amount of incorporated Cu²⁺ was estimated by calibration curve obtained from cyclic voltammetry. The Cu²⁺ concentration was found to vary with chitosan content, and the corresponding diffusion coefficient (D) were subsequently calculated (Fig. S6). The obtained D values ranged from 2.0 × 10^-7 cm²/s (0.5 wt%) to 1.65 × 10^-7 cm²/s (2.0 wt%) which are significantly lower than the reported D of free Cu²⁺ ions in aqueous solution at 25°C (7.2 × 10^-6 cm²/s), indicating that ion transport is hindered within the chitosan hydrogel matrix. Notably, the 1.0 wt% hydrogel showed the highest Cu²⁺ incorporation while maintaining a moderate diffusion coefficient (1.88 × 10^-7 cm²/s), suggesting an optimal balance between ion loading and mobility. This behavior can be attributed to dynamic Cu²⁺ coordination with chitosan, in which the ions are strongly associated with the chitosan matrix but not fully immobilized, thereby remaining electrochemically accessible for redox reactions.

Based on the presence of Cu²⁺ diffusion within the hydrogel matrix, additional experiments were carried out to assess the reversibility of the hydrogel network and the Cu²⁺ reduction. Electrochemical reduction at a sufficiently negative potential (–0.6 V. vs Ag/AgCl) did not induce macroscopic dissolution or collapse of the hydrogel, whereas acid treatment applied after gel formation resulted in a loss of structural integrity and reflow of the chitosan gel on the electrode surface (Fig. S7a). In contrast to protonation under acidic conditions, electrochemical reduction does not disrupt the hydrogel network, as coordination interactions between chitosan amino groups and reduced Cu species are maintained even after reduction.

To further evaluate the Cu²⁺ reduction under comparable conditions, electrochemical reduction experiments were performed using CSCu hydrogel and 2.6 mM Cu²⁺ aqueous solution (Fig. S7b). Cu²⁺ reduction occurred in both systems; however, the fraction of reduced Cu generated from the hydrogel was markedly lower (2.88%, Fig. S7c) than that obtained from the freely dissolved Cu²⁺ solution (Fig. S7d). These results indicate that Cu²⁺-chitosan complexes remain sufficiently dynamic to allow diffusion and redox activity, consistent with the reduced diffusion coefficients relative to the bulk solution derived from Randles-Sevick equation. At the same time, the large stability constant obtained from the Lingane equation indicates strong coordination, which effectively suppresses extensive Cu²⁺ reduction and preserves the integrity of the hydrogel network.

Non-enzymatic glucose sensing based on Cu²⁺/Cu³⁺ redox electrocatalysis within CSCu/SWCNT hydrogel

On the basis of the above results, the CSCu hydrogel prepared with 1.0 wt% chitosan was selected as the platform for non-enzymatic glucose sensing. To elucidate the electrocatalytic oxidation characteristics of Cu²⁺, we first examined its behavior in 0.1 M NaOH solution (pH 13) [30]. Fig. 6b shows the CVs of Cu²⁺ to Cu³⁺ oxidation with and without glucose (Fig. S8). In the absence of glucose (Fig. 6b ii), the oxidation current of Cu²⁺ began to increase sharply at ~0.7 V [Reaction (1)], whereas in the presence of glucose (Fig. 6b iii), the onset potential shifted markedly to a lower potential (~0.35 V), indicating a glucose-assisted redox pathway involving Cu²⁺/Cu³⁺. This distinct negative shift in the oxidation potential reflects an electrocatalytic EC′ mechanism, wherein Cu²⁺ is first electrochemically oxidized to Cu³⁺ [Reaction (2)] and subsequently reduced back to Cu²⁺ via chemical oxidation of glucose to gluconolactone [Reaction (3)]. The rapid consumption of Cu³⁺ continuously drives the redox equilibrium forward, thereby lowering the overpotential required for Cu²⁺ oxidation [30,60–62]. In essence, the presence of glucose promotes catalytic turnover, enabling oxidation to proceed at a much lower potential than that thermodynamically required for the direct, non-catalytic oxidation of Cu²⁺.

Fig. 6.

(a) Schematic illustration of the fabrication and working principle of CSCuCNT electrodes for glucose sensing. (b) Cyclic voltammograms in 0.1 M NaOH (pH 13) for (i) cathodically electrodeposited chitosan hydrogel (CS) modified SPGE, (ii) a CSCu modified SPGE, (iii) CSCu SPGE with glucose (iV) CSCuCNT SPGE with 5 mM glucose (Scan rate : 50 mV/s). (c) Nyquist plots of (i) CSCu and (ii) CSCuCNT electrodes measured at 0.7 V (vs. Ag/AgCl) in 0.1 M NaOH containing 5mM glucose. (d) Chronocoulometric response at 0.7 V (vs. Ag/AgCl) in 0.1 M NaOH (pH 13) with various glucose concentrations. (e) Calibration plot of the charge values at 5 s in (d).

Although the Cu²⁺-coordinated chitosan hydrogel provided stable electrocatalytic centers for glucose oxidation, its intrinsically low conductivity hindered efficient electron transfer. To address this limitation, SWCNTs were incorporated into the hydrogel, where the −NH₂ and −OH groups of chitosan facilitated their uniform dispersion, preventing aggregation and yielding a homogeneous CSCuCNT composite (see illustration in Fig. 6a, Fig. S9). As shown in Fig. 6b iV, incorporation of SWCNTs caused no noticeable shift in the onset potential of Cu²⁺ oxidation but led to a substantial enhancement of the anodic peak current, attributable to improved electron transport within the hydrogel matrix. Consistent with this observation, Electrochemical Impedance Spectroscopy (EIS) analysis revealed a marked decrease in charge-transfer resistance (R_ct) from 1.38 kΩ (i) to 0.74 kΩ (ii) upon SWCNT incorporation (Fig. 6c), confirming the significant facilitation of interfacial electron-transfer kinetics. These results further demonstrate that the chitosan matrix effectively disperses SWCNTs, thereby enhancing overall conductivity and concurrently improving both interfacial charge transfer and mass transport efficiency during glucose sensing [63,64].

To evaluate the analytical performance of the optimized CSCuCNT system for glucose detection, Chronocoulometry (CC) were performed (Fig. 6d). This technique uses time-integrated current over a fixed period as the sensing signal, is well suited for quantifying total electrocatalytic charge and shows higher reproducibility than potential sweep methods such as CV because long-window integration suppresses capacitive charging currents [57,65]. A series of chronocoulometric curves were recorded for increasing concentrations of glucose, ranging from 31.5 to 1000 μM in 0.1 M NaOH. The results demonstrated a clear and systematic increase in the accumulated charge (Q) over the 5.0 s measurement period according to the glucose concentration. This behavior is a direct consequence of the electrocatalytic mechanism. At a higher glucose concentration, the chemical regeneration of the Cu²⁺ catalyst from the Cu³⁺ intermediate occurs at a faster rate. This accelerated catalytic turnover leads to a greater number of electron transfer events at the electrode surface, resulting in a larger integrated charge. Fig. 6e shows the calibration plot for the charge data obtained at 5.0 s presented in Fig. 6d. The limit of detection (LOD) was estimated as ca. 11.9 μM using Equation (2):

Where S_b is the standard deviation (SD) of the blank and m is the slope of the corresponding calibration curve. The background charge recorded in the absence of glucose was 3.39 μC, with a SD of 1.0 μC. To further confirm the selectivity of the sensor system, cyclic voltammetry and chronoamperometric measurements were performed with successive additions of glucose and common interfering species, including dopamine, ascorbic acid, urea, and hydrogen peroxide (Fig. S10) [60,61,66]. The CSCuCNT electrode exhibited a distinct current increase upon the addition of glucose (4 and 2 mM), whereas negligible current variations were observed for all interfering species at comparable concentrations (2 mM). This excellent performance is attributed to the synergistic combination of the components: the inherent catalytic activity of the Cu²⁺/Cu³⁺ redox couple, which is efficiently regenerated by glucose, and the SWCNT network, which provides a high electroactive surface area and rapid electron transport pathways, thereby amplifying the catalytic signal. Overall, the chitosan-Cu²⁺/SWCNT composite hydrogel serves as a highly effective and sensitive platform for non-enzymatic glucose sensing, demonstrating excellent sensitivity, selectivity, and practical applicability.