Synergistic Tuning of Active Sites and π-Conjugation in 2D Conductive MOFs Boosts Uric Acid Electrosensing

Abstract

Uric acid (UA) detection is critical for human health monitoring, necessitating the development of electrochemical sensing electrodes suitable for physiological environments. This study evaluated four 2D conductive metal–organic frameworks (2D c-MOFs), namely Cu-HHTP, Ni-HHTP, Cu-HAB, and Ni-HAB, which share identical graphene-like 2D sheet structures but differ in π-conjugation extent and catalytic active centers [MX₄] (M = Cu or Ni; X = O or NH) as electrosensing electrodes. Electrochemical sensing performance was compared by detecting UA in phosphate-buffered saline (PBS). Herein, the Ni-HHTP electrode demonstrated superior sensitivity (6.79 μA·μM⁻¹·cm⁻²), the lowest oxidation potential (0.272 V), and the lowest detection limit (0.44 μM). Langmuir adsorption isotherm analysis revealed that the Ni-HHTP electrode possesses the highest surface coverage (Γ_A) (5061.16 pmol cm⁻²) and the most favorable Gibbs adsorption free energy (ΔG°) (−18.775 kJ mol⁻¹), indicating its strongest UA adsorption capacity and molecular interaction. This enhanced performance is attributed to the optimal synergy between [NiO₄] catalytic centers and extended ligand π-conjugation, facilitating greater analyte adsorption and electron transfer efficiency. This work establishes clear structure–performance relationships for 2D c-MOF electrodes in UA detection, providing key insights for designing advanced electrosensing materials.

1. Introduction

Electrochemical sensing technology has become an important means for detecting in vivo physiological environments due to its high sensitivity, rapid response, and real-time monitoring capabilities [1]. This technology can dynamically track metabolite concentrations, providing critical data for disease diagnosis and health management [2]. Among the key biomarkers in the human body, UA, as the terminal product of purine metabolism, plays essential physiological roles in maintaining redox balance and scavenging free radicals [3]. Recent studies have further associated aberrantly elevated UA levels with metabolic syndrome, cardiovascular disease risk, and neurodegenerative disease progression, establishing UA as a high-value broad-spectrum biomarker [4]. However, inherent limitations of existing electrode materials severely constrain the precise electrochemical detection of UA. Specifically, conductive polymer electrodes suffer from low electrical conductivity due to limited π-electron delocalization capacity, along with poor electrochemical cycling stability caused by polymer swelling/shrinking-induced active site degradation [5]. Carbon nanotube (CNT) electrodes undergo van der Waals-driven agglomeration during modification, causing large interfacial resistance fluctuations that undermine layer reproducibility [6]. Metal oxide electrodes face challenges in morphology control during synthesis due to Ostwald ripening, while their wide bandgap (>3 eV) restricts active facet utilization and charge transfer efficiency [7]. These constraints limit their capability in UA electrochemical sensing, impeding its broader implementation in precision medicine. Therefore, the development of new electrode materials that combine good conductivity, multiple active sites, and stable and well-defined structures with clear structure–activity relationships has become a key path to breaking through the limitations of electrochemical sensing technology for biomarkers.

The rise of 2D conductive metal–organic framework (2D c-MOF) electrodes has provided a revolutionary platform that meets these requirements through their tunable π-conjugated systems and atomic-level structural precision [8,9,10]. 2D c-MOFs are porous crystalline materials formed via the self-assembly of conjugated organic ligands with ortho-substituents (-OH, -SH, or -NH₂) and transition metal ions (such as Zn²⁺, Cu²⁺, and Ni²⁺) in planar square coordination geometries [8,11]. They have significant advantages: dual redox-active centers from synergistic metal–ligand combinations that enhance electrocatalytic activity by providing abundant active sites [12]; efficient charge transfer through extended π-conjugation that yields conductivities orders of magnitude higher than those of conventional MOFs, enabling enhanced sensing sensitivity [13]; and tunable metal–ligand structures and well-defined structure–activity relationships that permit targeted design for specific analytes [12,13]. While the feasibility of 2D c-MOF electrodes for the electrochemical detection of biomarkers (e.g., UA [14,15], dopamine (DA) [15,16], and ascorbic acid (AA) [17,18]) has been preliminarily validated, the correlation between variations in active-center composition and electrosensing performance has not yet been clearly elucidated. A systematic survey of MOF-based electrochemical sensors, particularly those targeting UA detection (Figure S1), reveals a critical knowledge gap: the synergistic regulation of UA sensing selectivity and sensitivity by π-conjugated frameworks and active-site chemistry remains systematically unexamined. This necessitates a fundamental investigation into how these dual structural elements collectively govern detection performance. Through a comparative electrochemical analysis of distinct material systems, this study elucidates the underlying mechanism of this synergy, thereby advancing theoretical frameworks and establishing a robust experimental foundation for high-performance UA sensor design.

In ligand selection, 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) offers a polycyclic aromatic hydrocarbon structure with large π-conjugation, which grants superior electrical conductivity; its hydroxyl functional groups enhance redox activity, significantly boosting electrochemical response sensitivity. Conversely, hexaaminobenzene (HAB) features an ultracompact molecular structure with a high packing density that maximizes redox-active sites per unit volume, while its nitrogen-rich character facilitates efficient electron transport pathways through robust coordination with metal centers. This work employs HHTP and HAB as ligands and catalytically active nickel and copper as metal nodes to synthesize four 2D c-MOFs, namely, Cu-HHTP [14,19], Ni-HHTP [20,21], Cu-HAB, and Ni-HAB [14,22], respectively (Figure 1a). Through comparative electrochemical UA detection performance using these four 2D c-MOF electrodes with different active sites of [MX₄] (M = Cu, Ni; X = O, NH), the effects of metal identity, ligand functional groups, and conjugacy on electrosensing performance are elucidated. Additionally, by calculating Langmuir adsorption isotherms, the ΔG° is further determined to compare the strength of interactions between UA and the four 2D c-MOFs, thereby analyzing the mechanistic basis for observed performance variations.

2. Materials and Methods

2.1. Materials and Synthesis

All reagents and solvents were used as-received without further purification unless otherwise noted. Synthetic protocols followed established literature methods, with subsequent refinement through iterative experimental optimization.

Synthesis of Cu-HHTP MOF: Copper (II) nitrate trihydrate (13.7 mg, 0.057 mmol) and the organic linker HHTP (15 mg, 0.046 mmol) were dissolved in a blend composed of 0.25 mL N,N-dimethylformamide (DMF) and 1.75 mL water contained within a 10 mL glass vessel followed by ultrasonic dispersion for 10 min. The homogeneous mixture underwent crystallization under static hydrothermal conditions at 85 °C for three days. After allowing the reaction to cool to ambient temperature, the precipitated solid was isolated by centrifugation. Subsequent purification involved sequential washing cycles using deionized water and acetone, each repeated three times, to remove impurities. The resulting material was then dehydrated under vacuum at 120 °C for 24 h to yield the purified Cu-HHTP MOF powder.

Synthesis of Ni-HHTP MOF: Nickel (II) acetate tetrahydrate (45.6 mg, 0.183 mmol) and the organic ligand HHTP (20 mg, 0.062 mmol) were dissolved in 2.8 mL of deionized water within a 10 mL reaction vessel, assisted by 10 min of ultrasonication to ensure homogeneity. This aqueous mixture underwent crystallization under static thermal conditions at 85 °C for 24 h. Following natural cooling to room temperature, the formed crystalline precipitate was isolated via centrifugation. Subsequent purification involved multiple rinses with deionized water and acetone, repeated three times for each solvent. The purified material was ultimately dried under vacuum at 120 °C for 24 h to yield the final Ni-HHTP MOF powder.

Synthesis of Cu-HAB MOF: Cu(NO₃)₂·3H₂O (33 mg, 0.138 mmol) and the HAB ligand (15 mg, 0.545 mmol) were separately aliquoted into individual 20 mL vials. Each solid was dissolved in 2.5 mL deionized water using ultrasonication. Subsequently, 0.2 mL ammonium hydroxide (NH₄OH, 30 wt%) was introduced into the HAB solution before combining it with the copper solution. The resulting mixture was allowed to react under ambient aerobic conditions with continuous stirring at room temperature for 2 h. The precipitated solid product was then isolated by centrifugation, subjected to three sequential washing cycles with deionized water and acetone to remove soluble residues, and finally vacuum-dried at room temperature for 12 h to obtain the Cu-HAB MOF powder.

Synthesis of Ni-HAB MOF: Nickel (II) nitrate hexahydrate (15.95 mg, 0.055 mmol) and the organic ligand HAB (6.05 mg, 0.036 mmol) were individually weighed and transferred to separate 20 mL vials. Each compound was dissolved in 5 mL deionized water assisted by 10 min of ultrasonication. Then, 0.4 mL ammonium hydroxide (NH₄OH, 30 wt%) was introduced into the HAB solution prior to combining the two components. The resulting mixture underwent brief sonication for homogenization before undergoing crystallization under static thermal conditions at 65 °C for 4 h. After cooling, the precipitated solid was isolated via centrifugation, sequentially rinsed with deionized water and acetone to remove reaction residues, and vacuum-dried overnight at ambient temperature to afford the Ni-HAB MOF powder.

2.2. Preparation and Testing of Electrodes

ITO glass was sonicated in acetone, ethanol, and water separately for 15 min and rinsed with distilled water. It was then treated in a 1:1:5 (v/v/v) mixed solution of H₂O₂ (30 wt%), NH₄OH (30 wt%), and H₂O at 85 °C for 30 min, rinsed with distilled water, and dried under nitrogen.

Based on our previous experience in preparing electrodes, 2 mg of the prepared 2D c-MOF powder was combined with 1 mg of carbon black and 20 μL of Nafion. The mixture was sonicated in 200 μL of ethanol for 30 min then drop-cast onto ITO (1 × 2 cm²) and vacuum-dried to prepare MOF-based electrodes. All electrochemical measurements employed a three-electrode configuration using the MOF-based electrode as the working electrode, platinum wire as the counter electrode, and Ag/AgCl as the reference electrode. Physiological conditions were simulated in a 0.1 M phosphate buffered saline (PBS) (pH = 7.4) buffer solution. All electrochemical tests were conducted using a CHI760 electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., Shanghai, China).

2.3. Calculation of Langmuir Adsorption Isotherms

The Langmuir adsorption isotherm [23] is based on the isotherm model of Irving Langmuir and can be used to determine the Γ_A and adsorption strength of liquid-phase analytes on electrode surfaces. By using the Langmuir adsorption isotherm, the interaction between MOFs and analytes can be quantified, thereby further quantifying the adsorption capacity of four MOFs for UA.

The Γ_A (in pmol cm⁻²) of UA on the electrode can be calculated from its cyclic voltammetry (CV) oxidation peak current ($i_p$, in A) using Equation (1). The equation incorporates the number of electrons transferred (n), the Faraday constant (F, in C mol⁻¹), the scan rate (v, in V s⁻¹), the electrode area (A, in m²), the ideal gas constant (R, in J mol⁻¹ K⁻¹), and the temperature (T, in K).

$$i_p={\displaystyle \frac{n^2{F}^{2}vA{\Gamma }_A}{4RT}}$$

(1)

The correlation between analyte concentration [A] (μmol·L⁻¹) and Γ_A is fitted to the Langmuir adsorption Equation (2). By optimizing the weighted least-squares curve, the saturated surface coverage (Γ_S, pmol·cm⁻²) and thermodynamic equilibrium constant (β, cm³·pmol⁻¹) are derived.

$${\Gamma }_A={\displaystyle \frac{{\Gamma }_s\beta \left[A\right]}{1+\beta \left[A\right]}}$$

(2)

The ΔG° (at 298 K) was calculated after obtaining β (Equation (3)):

$$∆G°=-RT\ln \left(\beta \right)$$

(3)

3. Results and Discussion

3.1. Synthesis and Characteristics of 2D c-MOF Electrodes with Different [MX₄] Active Sites

2D c-MOFs were employed as modular electrodes in our experimental design. This subclass of MOFs typically exhibits high electrical conductivity, permanent porosity, and regularly distributed redox-active sites, enabling their direct application as working electrodes in analytical devices. These characteristics are considered essential for achieving enhanced current intensities required to boost electrochemical sensor sensitivity. Crucially, the structural tunability and compositional modularity of 2D c-MOFs facilitate the integration of required electrocatalytic components within their architectures. In this study, we synthesized four distinct 2D c-MOFs, Cu-HHTP, Ni-HHTP, Cu-HAB, and Ni-HAB, through the strategic design of conjugated ligand sizes, coordination functional groups, and metal node selection (Figure 1a). These analogues feature different catalytic active centers [MX₄] (M = Cu, Ni; X = O, NH) and exhibit variations in layer stacking modes, resulting in significantly different pore sizes (~0.8 to 1.6 nm) that potentially influence analyte adsorption capacity and catalytic efficiency. Precursor molecular design is thus directly correlated with resultant electrode structure–property relationships through the systematic comparison of these structurally related 2D c-MOF electrodes.

The four 2D c-MOFs were prepared via solvothermal/hydrothermal synthesis. Powder X-ray diffraction (PXRD) analysis confirmed their structural integrity and crystallinity (Figure 1b–e), while scanning electron microscopy (SEM) revealed surface morphologies (Figure 1f–i). PXRD patterns demonstrate good phase purity for all MOFs, with characteristic peaks matching simulated patterns and literature data. Specifically, Cu-HHTP exhibits diffraction peaks at 4.75°, 9.56°, 12.82°, and 27.77°, corresponding to (100), (200), (210), and (002) planes, respectively (Figure 1b) [24]. Ni-HHTP displays peaks at 4.53°, 13.77°, 16.03°, and 26.75°, corresponding to (100), (300), (220), and (004) planes (Figure 1c) [25]. The sharp characteristic peaks of M-HHTP MOFs indicate excellent crystallinity. Conversely, Cu-HAB exhibits characteristic peaks at 7.74°, 15.45°, and 27.67° (Figure 1d), while Ni-HAB has peaks at 7.74°, 15.46°, and 27.89° (Figure 1e), corresponding to the (100), (200), and (001) crystal planes, respectively [26]. These broader, lower-intensity peaks indicate their limited long-range order. Consistent with SEM analysis, both HAB-based MOFs exhibit irregular nanoparticulate morphologies (Figure 1h,i), suggesting substantial defect formation during synthesis that limited crystal growth, in line with their poorer PXRD crystallinity. In contrast, M-HHTP analogues form well-defined nanorod morphologies (Figure 1f,g), indicating superior structural order and integrity resulting from the enhanced π-conjugation capability of HHTP ligands, which facilitates out-of-plane ordered stacking.

3.2. Assessing the Electrochemical Response of 2D c-MOF Electrodes with Different [MX₄] Active Sites Toward UA

Within 2D c-MOFs, organic linkers engage analytes through intermolecular interactions (e.g., electrostatic forces or hydrogen bonding), while catalytically active metal nodes (Ni or Cu) drive redox transformations of electroactive analytes. Atomic-level control over the chemical composition of 2D c-MOF electrodes enables the precise tuning of MOF–analyte interactions, thereby achieving differential sensitivity and resolution in detecting biologically crucial redox molecules. To evaluate UA detection capabilities in physiological environments, uniform MOF slurries were drop-cast onto indium tin oxide (ITO) substrates to fabricate working electrode films. All electrochemical measurements were conducted under simulated physiological conditions of the 0.1 M PBS buffer (pH = 7.4).

Initial characterization of the electrode redox properties revealed no discernible oxidation peaks using CV testing in the blank electrolyte (Figure 2a–d), indicating the absence of faradaic processes. Variations in baseline voltammetric responses were attributed to differences in electrode architecture, electrolyte composition, ionic strength, and potential additives. Subsequent UA detection (100 μM) demonstrated distinct oxidation peaks across all 2D c-MOF electrodes (Figure 2e–h and Figure S2). The oxidation potentials were recorded as 0.300 V (Cu-HHTP, the lowest), 0.315 V (Ni-HHTP), 0.485 V (Cu-HAB), and 0.490 V (Ni-HAB, the highest). Comparative analysis showed that the Cu-HHTP and Ni-HHTP electrodes generated the highest oxidation currents, while the Cu-HAB and Ni-HAB exhibited the weakest responses. The electrochemical oxidation of UA involves a two-electron/two-proton transfer, typically generating unstable diazomethane intermediates that subsequently convert to iminoalcohols, ultimately forming 4,5-dihydroxyuric acid. These results confirm that all four 2D c-MOFs function as electrocatalysts for UA redox reactions.

Differential pulse voltammetry (DPV) measurements further quantified sensing performance in physiologically relevant conditions with the UA range of 0–100 μM. As shown in Figure 3a–d, all electrodes exhibited concentration-dependent responses with good linear correlations (R² > 0.96, Figure 3e and Figure S3, Table S1). The Cu-HHTP and Ni-HHTP electrodes demonstrated higher currents at lower potentials of 0.268 V and 0.272 V, respectively, compared to HAB-based analogues (0.296 V for Cu-HAB and 0.336 V for Ni-HAB). Ni-HHTP achieved optimal sensitivity (6.79 μA·μM⁻¹·cm⁻²), followed by Cu-HHTP (5.71 μA·μM⁻¹·cm⁻²). HAB-based electrodes displayed inferior sensitivity and linearity. Detection limits were determined as 2.62 μM (Cu-HHTP), 15.94 μM (Cu-HAB), 0.44 μM (Ni-HHTP), and 0.29 μM (Ni-HAB), respectively (Figure 3f and Figure S3). These performance variations stem from distinct chemical compositions of the [MX₄] catalytic centers and the extended π-conjugated systems/larger pore sizes in HHTP-based MOFs, which enhance UA adsorption and catalytic efficiency.

The selectivity of the four 2D c-MOF-based UA sensors was rigorously evaluated in complex environments simulating physiological conditions. PBS buffer solutions were sequentially spiked with common biological interferents, 50 μmol/L AA, 20 μmol/L DA, and 5 mmol/L glucose (Glu), while simultaneously introducing UA across the clinically relevant concentration range from 0 to 100 μmol/L. All sensors maintained excellent UA detection performance without significant interference from these coexisting substances, confirming robust selectivity (Figures S4–S7).

Comparative analysis confirmed Ni-HHTP’s superior detection performance. Long-term stability of the Ni-HHTP MOF electrode was quantified through continuous 72 h monitoring of current response to 100 μM UA, demonstrating excellent signal retention (RSD = 1.5%, Figure S8). Batch reproducibility was further validated across five independently fabricated electrodes tested under identical conditions (100 μM UA), yielding highly consistent responses (RSD = 3.1%, Figure S9). These results collectively establish excellent temporal stability and fabrication reproducibility, confirming both the sensing platform’s reliability and manufacturing process control.

To elucidate the electrochemical reaction kinetics governing UA detection for the 2D c-MOF-modified electrodes, systematic scan-rate-dependent voltammetry was performed across 10–100 mV/s. The linear proportionality (R² > 0.98) between the oxidation peak current (Iₚ) and the square root of the scan rate (v^1/2) establishes diffusion-controlled processes (Figure S10), confirming that the electron transfer kinetics are limited by the mass transport of UA molecules to the electrode interface rather than surface adsorption phenomena.

3.3. Probing the Response Mechanisms of Different [MX₄] Active Sites in 2D c-MOF Electrodes to UA Oxidation

To elucidate the performance differences in UA detection among the four 2D c-MOF electrodes with different [MX₄] active sites, CV responses were measured across a UA concentration region of 10–700 μM. As shown in Figure 4a–d, the UA oxidation process exhibited essential irreversibility. Since electrochemical UA oxidation requires analyte adsorption at electrode surfaces, variations in adsorption characteristics across different 2D c-MOF electrodes and their influence on analytical sensitivity were further investigated. Langmuir adsorption isotherms were constructed and comparatively analyzed using the maximum oxidation peak currents from CV measurements. Th electrode Γ_A at UA concentrations of 10–700 μM was calculated through Equation (1), with subsequent Γ_A versus concentration plots fitted to the Langmuir isotherm model (Equation (2)). This enabled the determination of Γ_S and β, the latter being used to compute ΔG° via Equation (3).

The adsorption isotherms presented in Figure 4e–h revealed significantly higher Γ_S and β values for Cu-HHTP and Ni-HHTP electrodes compared to their HAB-based counterparts. Ni-HHTP exhibited the highest Γ_S value (5061.16 pmol cm⁻²), with Ni-HHTP being greater than Cu-HHTP (1689.76 pmol cm⁻²), which in turn was greater than Ni-HAB (1284.72 pmol cm⁻²), and finally Cu-HAB (826.93 pmol cm⁻²) (Figure 5a). Additionally, the adsorption equilibrium constants decreased sequentially across the series: Cu-HHTP (2.88 cm³ nmol⁻¹) > Ni-HHTP (1.95 cm³ nmol⁻¹) > Cu-HAB (1.01 cm³ nmol⁻¹) > Ni-HAB (0.44 cm³ nmol⁻¹) (Figure 5b). This trend corresponded directly to progressively less favorable ΔG° values (Figure 5c). Comparative analysis with CV measurements established that UA detection performance correlates fundamentally with both Γ_S, β, and ΔG° (Figure 5d). Larger β values yielded more thermodynamically favorable ΔG values (increasingly negative), aligning perfectly with DPV response trends. Furthermore, enhanced Γ_A and Γ_S values directly increased peak currents in the voltammetric analysis, explaining Ni-HHTP’s superior sensitivity. These findings demonstrate that [NiX₄] catalytic centers promote higher UA saturation coverage, while the superior conjugation capability of HHTP-based MOFs strengthens analyte binding. The Langmuir adsorption analysis thus elucidates the origin of electrochemical performance variations among 2D c-MOF electrodes and establishes the enhancement mechanism through [MX₄] node composition regulation, providing fundamental insights for the rational design of 2D c-MOF electrosensing electrodes.

4. Conclusions

In summary, four 2D c-MOFs (Cu-HHTP, Ni-HHTP, Cu-HAB, and Ni-HAB) were synthesized via solvothermal methods. These materials feature similar graphene-like 2D porous architectures but exhibit distinct π-conjugation extents and catalytic active centers [MX₄] (M = Cu or Ni, X = O or NH). Fabricated as working electrodes, their UA detection capabilities were comparatively evaluated in physiologically simulated PBS buffer using CV and DPV methods. The Ni-HHTP electrode demonstrated optimal performance metrics, establishing it as the superior sensing electrode material: highest sensitivity, lowest oxidation potential, and low detection limit. Langmuir adsorption isotherm analysis revealed the mechanistic origin of these performance variations: Ni-HHTP achieves a maximal Γ_S while maintaining favorable adsorption thermodynamics (most negative ΔG°), enabling optimal response performance at equivalent UA concentrations. Through comparative analysis with existing UA detection methods, our approach demonstrates significant advancements over conventional electrodes, such as nanocomposites, uricase biosensors, MXenes, and MOF composites, particularly in terms of wider linear ranges, lower detection limits, and superior detection sensitivity (Table S2). This study demonstrates the systematic modulation of electroactive biomolecule detection through precision design of 2D c-MOF electrodes. The Langmuir isotherm analysis quantitatively correlates structural variations with analyte affinity and adsorption capacity differences, thereby elucidating structure–property relationships governing electrochemical sensing capabilities. These fundamental insights establish design principles for synthesizing high-performance 2D c-MOF electrode materials.