A robust dual-readout ratiometric assay for dopamine based on NiMn₂O₄-catalyzed DHNP fluorophore conversion

Abstract

Dopamine (DA), a critical biomarker closely associated with the onset and progression of neurological disorders, was sensitively quantified using dual enzyme-mimetic NiMn₂O₄ nanoflowers. The nanozyme simultaneously exhibits peroxidase- and oxidase-like activities, catalyzing a highly selective cyclization reaction between DA and 1, 3-dihydroxynaphthalene (DHNP). As the DA concentration increased, the intrinsic fluorescence of DHNP at 440 nm was progressively quenched, accompanied by the formation of a new optically active fluorophore with emission at 485 nm and a corresponding absorbance at 455 nm. This dual-mode ratiometric fluorescence–colorimetric sensing strategy enabled self-calibrated detection, effectively minimizing signal fluctuation and matrix interference. The platform displayed excellent linearity over the range of 0–250 µM for fluorescence and absorbance responses, allowing accurate, sensitive, and selective DA determination. Owing to its robustness and reliability, the proposed method is well suited for high-throughput dopamine analysis in diverse biological sample matrices.

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1. Introduction

Dysregulation of dopamine (DA) signaling plays a central role in the pathogenesis of Parkinson's disease and has been extensively associated with schizophrenia and other neuropsychiatric disorders.^1–4 Beyond neurological dysfunction, aberrant elevations of DA in biological fluids have been identified as clinically significant biomarkers for neuroendocrine tumors, including pheochromocytomas and paragangliomas, as well as related catecholamine-secreting malignancies.^5,6 Pheochromocytomas and paragangliomas remain challenging to diagnose owing to their nonspecific and overlapping clinical manifestations, which frequently lead to delayed recognition and compromised therapeutic outcomes.^7,8 Aberrations in DA metabolism have also been linked to severe neurological dysfunction and increased risk of premature mortality.⁹ Consequently, accurate and reliable determination of DA levels is of critical importance for timely diagnosis, disease stratification, and therapeutic management, establishing DA as a pivotal biomarker in neurological and neuroendocrine disorders. A wide range of analytical strategies has been developed for DA determination, including LC-MS/MS,¹⁰ capillary electrophoresis,¹¹ and HPLC.¹² Although these approaches offer high analytical accuracy, they often rely on complex sample pretreatment, expensive and bulky instrumentation, and time-consuming operational procedures,¹³ which limit their practicality for routine or point-of-care analysis. Consequently, there is an increasing demand for simplified, rapid, and cost-effective detection platforms.^14,15 Among emerging alternatives, fluorescence-based assays have gained particular attention due to their exceptional sensitivity,¹⁶ high selectivity, operational simplicity, and reliable analytical performance.¹⁷

Natural enzymes, predominantly protein-based or catalytic RNA systems, exhibit outstanding selectivity and sensitivity for biomolecular recognition and detection.^18,19 Despite their indispensable roles in biological processes, their practical deployment is severely constrained by poor operational stability under non-physiological conditions, including elevated temperatures, extreme pH environments, and the presence of organic solvents.^20–22 These inherent drawbacks significantly limit their robustness and reusability, thereby hindering their widespread application in hydrogen peroxide (H₂O₂) sensing for biological and clinical analyses. Owing to their high sensitivity to biochemical environments and costly, labor-intensive production, natural enzymes often suffer from limited practicality in real-world applications. Consequently, artificial enzymes (nanozymes) have attracted growing interest because of their superior catalytic robustness, tunable activity, and long-term stability, particularly in peroxidase-related systems.^23,24 To this end, diverse nanomaterial platforms—including metal and metal oxide nanoparticles, transition metal dichalcogenides, and carbon-based nanostructures—have been extensively investigated as readily synthesized and structurally stable enzyme mimics.

Spinel-type transition metal oxides have emerged as attractive functional materials owing to their inherent biocompatibility, low toxicity, rich redox chemistry, and cost-effective large-scale synthesis.^25–27 In contrast to noble metals, whose limited abundance and high cost restrict widespread use, spinel oxides offer tunable electronic and catalytic properties through controlled composition and morphology engineering, thereby enabling performance optimization for diverse sensing platforms.²⁸ Among spinel oxides, NiMn₂O₄ has been extensively investigated owing to its spinel crystal structure and rich physicochemical characteristics arising from mixed-valence Ni and Mn redox centers.²⁹ This material exhibits high catalytic activity while remaining environmentally benign, earth-abundant, cost-effective, and non-toxic, making it a particularly attractive candidate for sensing and electrocatalytic applications.³⁰ NiMn₂O₄ nanostructure offers high surface area and enhanced charge transfer, improving sensing performance through faster response, higher sensitivity, and selectivity.³¹

In this study, NiMn₂O₄ nanoflowers are introduced for the first time as a bifunctional nanozyme system exhibiting both peroxidase- and oxidase-like activities for DA sensing. The hierarchical NiMn₂O₄ architecture efficiently promotes a selective oxidative cyclization reaction between 1, 3-dihydroxynaphthalene (DHNP) and DA under aerobic conditions in the presence of H₂O₂, yielding a stable fluorescent product. With increasing DA concentration, the native emission of DHN at 440 nm is gradually suppressed, while a concurrent enhancement in fluorescence at 485 nm and absorbance at 455 nm is observed, reflecting the formation of the cyclized fluorophore (Scheme 1). This synchronized signal evolution enables a dual-mode ratiometric fluorescence–colorimetric readout, which provides intrinsic self-referencing capability and effectively mitigates signal fluctuations arising from photobleaching, probe instability, and complex matrix effects. Owing to the synergistic catalytic activity of the mixed-metal oxide and the robustness of ratiometric detection, the proposed sensing platform achieves high sensitivity, improved accuracy, and reliable quantification of DA in clinically relevant samples, highlighting its potential for practical bioanalytical applications.

Scheme 1 Schematic diagram for nanozyme-catalyzed cyclization reaction for DA detection.

2. Experimental

2.1. Materials and reagents

Dopamine hydrochloride (≥99.7%), tetramethylbenzidine (TMB, 98.8%), 1,3-dihydroxynaphthalene (DHNP, 98.8%), uric acid (≥98.3%), ascorbic acid (≥99.8%), glutathione (≥98.8%), glucose (≥96.8%), glycine (≥97.8%), cysteine (≥98.3%), tryptophan (≥98.8%), alanine (≥97.9%), adenosine triphosphate sodium (98.8%), methionine (99.5%), bovine serum albumin (≥98.9%), epinephrine (≥97.9%), norepinephrine (≥98.6%), and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were purchased from Sigma-Aldrich (Germany). Potassium permanganate (KMnO₄), nickel (II) chloride hexahydrate (NiCl₂·6H₂O), hydrochloric acid (HCl), sodium hydroxide (NaOH), acetic acid, sodium acetate, acetonitrile, hydrogen peroxide (H₂O₂), dipotassium hydrogen phosphate (K₂HPO₄), and potassium dihydrogen phosphate (KH₂PO₄) were obtained from Merck (Germany).

2.2. Instrumentation

Fluorescence measurements were performed using a Shimadzu spectrofluorometer (Tokyo, Japan) equipped with a xenon flash lamp and a 1 cm quartz cuvette. Spectra were recorded at a photomultiplier tube voltage of 900 V, a scan rate of 500 nm min⁻¹, and excitation/emission slit widths set to 5 nm. UV–visible absorption spectra were obtained using a Shimadzu UV-1600 double-beam spectrophotometer (Tokyo, Japan). The morphology of the NiMn₂O₄ was investigated using scanning electron microscopy (SEM) on a JEOL JEM-2100 microscope (Tokyo, Japan). Fourier-transform infrared (FTIR) spectra were recorded with a Bruker ALPHA II spectrometer (Germany). Zeta potential measurements were conducted using a Malvern Zetasizer (Malvern Panalytical, UK). Elemental analysis was conducted via EDX on a SEM fitted with an Oxford X-Max 20 detector. XPS measurements were carried out using a Thermo Fisher K-ALPHA system (USA) with Al Kα radiation (10–1350 eV), a 400 µm spot, and 10⁻⁹ mbar pressure. Spectral scans included full-range (200 eV) and high-resolution (50 eV) modes. X-ray diffraction (XRD) analysis was conducted with a Tongda TD-3700 diffractometer employing Cu Kα radiation (λ = 1.5406 Å). A 6420 Triple Quadrupole LC-MS/MS system (Agilent Technologies, USA), equipped with a Q-TOF mass spectrometer and an electrospray ionization (ESI) source, was employed for compound analysis. Data were acquired using MassHunter Workstation software. The ESI source operated in positive ion mode with a fragmentor voltage of 150 V, capillary voltage of 3500 V, and skimmer voltage of 60 V. Nitrogen was used as the drying gas (320 °C, 10 L min⁻¹), nebulizing gas (45 psi), and as the collision gas for CID experiments.

2.3. Synthesis of NiMn₂O₄ nanoflowers

NiMn₂O₄ was synthesized via a base-free hydrothermal method. Briefly, 22 mL of an aqueous solution of NiCl₂·6H₂O (1.24 mM) and 30 mL of KMnO₄ (3.72 mM) were mixed and diluted to a total volume of 100 mL with ultrapure water, followed by magnetic stirring for 45 min to ensure complete homogenization. The resulting precursor solution was transferred into a Teflon-lined stainless-steel autoclave, sealed, and heated at 140 °C for 5 h. After natural cooling to room temperature, the obtained precipitate was separated by centrifugation at 4000 rpm for 20 min, washed repeatedly with ultrapure water and ethanol to remove residual ions and by-products, and then dried in an oven at 70 °C for 24 h. The dried solid was finally ground into a fine dark-brown powder, yielding the NiMn₂O₄ product.

2.4. Nanoenzyme activity

The enzyme-like catalytic activity of NiMn₂O₄ was evaluated by monitoring the oxidation of TMB in the presence of H₂O₂. In a typical assay, acetate buffer (550 µL, pH 5.5) was mixed with NiMn₂O₄ (200 µL, 20 µg mL⁻¹), TMB (100 µL, 5.0 mM), and H₂O₂ (150 µL, 0.6–8.0 mM). The reaction mixture was incubated at 37 °C under continuous shaking (2000 rpm), and the absorbance of the oxidized TMB product was recorded at 655 nm after 5 min of reaction.

For kinetic studies, the concentration of TMB was varied from 0.6 to 20.0 mM while maintaining a constant H₂O₂ concentration of 5.0 mM under identical experimental conditions. The apparent kinetic parameters, including the maximum reaction velocity (V_max) and the Michaelis constant (K_m), were determined by fitting the experimental data to the Michaelis–Menten model and further analyzed using Lineweaver–Burk double-reciprocal plots.^32,33

2.5. Stability under different pH and temperature

The catalytic activity of NiMn₂O₄ was evaluated over a pH range of 3.0–10.0. In a typical assay, 550 µL of buffer solution was mixed with NiMn₂O₄ (200 µL, 20 µg mL⁻¹), TMB (100 µL, 5.0 mM), and H₂O₂ (150 µL, 0.6–8.0 mM). The reaction mixture was incubated at 37 °C for 15 min under constant shaking (2000 rpm), and the absorbance of the oxidized TMB product was recorded at 655 nm. Thermal stability was investigated by conducting the reaction at temperatures ranging from 20 to 55 °C under otherwise identical conditions, using acetate buffer (pH 5.5). After 5 min of incubation, the absorbance at 655 nm was measured to assess temperature-dependent catalytic performance.

2.6. Steps for DA detection

Aliquots of dopamine (DA, 100 µL) at different concentrations were mixed with 550 µL of acetate buffer (pH 5.5). Subsequently, 1,3-dihydroxynaphthalene (DHN, 100 µL, 10.0 mM) was added, followed by NiMn₂O₄ nanozyme (200 µL, 20 µg mL⁻¹) and H₂O₂ (150 µL, 4.5 mM). The resulting reaction mixture was incubated at 37 °C for 20 min under constant shaking at 900 rpm. Fluorescence emission spectra were recorded at 440 and 485 nm using an excitation wavelength of 340 nm, while the corresponding absorbance was measured at 455 nm.

2.7. Procedures for samples

For DA analysis in injectable formulations, aliquots of the sample were used directly without any pretreatment, diluted appropriately with acetate buffer (pH 5.5), and then analyzed using the proposed dual-mode sensing strategy. For DA quantification in human serum, 200 µL of serum was mixed with 1.0 mL of acetonitrile to precipitate proteins, followed by centrifugation at 5000 rpm for 10 min. The resulting supernatant was collected and analyzed using the dual-mode fluorescence–colorimetric platform both before and after spiking with known concentrations of DA for recovery evaluation. The detection of DA was carried out using standard addition method. For urine sample, the urine from healthy volunteer was spiked with DA, centrifuged at 4000 rpm, and filtered. Then, 200 µL of the urine was measured using dual-mode detection methods. The detection of DA was carried out using standard addition method. In compliance with Egyptian regulations, the study received ethical clearance from Assiut University and included only participants who had provided informed consent.

3. Results and discussions

3.1. Characterization

SEM analysis confirmed the formation of well-defined flower-like NiMn₂O₄ nanozyme composed of radially oriented nanosheets (Fig. S1A). This hierarchical architecture affords a large exposed surface area and a high density of accessible catalytic sites. Particle size analysis revealed a narrow distribution between 285 and 315 nm, with an average diameter of 297.7 ± 3.21 nm (Fig. S1B), indicating good morphological uniformity. Such a structurally ordered and homogeneous nanoarchitecture is favorable for rapid electron transport and efficient mass diffusion of reactants, which collectively contributes to the pronounced peroxidase–mimetic activity observed for the NiMn₂O₄ nanozyme. Fig. S1C presents the EDX spectrum of the as-synthesized material, confirming the presence of Ni, Mn, and O as the principal constituent elements.

The XRD patterns confirm the well-crystallized nature of the NiMn₂O₄ nanozyme (Fig. S1D). The diffraction profile displays a series of sharp and well-defined reflections at 2θ values of 19.6°, 28.9°, 34.2°, 43.8°, 56.5°, and 63.4°, which can be indexed to the (111), (220), (311), (400), (511), and (440) crystal planes, respectively. These diffraction features are in excellent agreement with the standard spinel NiMn₂O₄ phase (JCPDS No. 71-0852),³⁴ confirming the successful formation of a pure and highly crystalline mixed-metal oxide structure.

The FTIR spectrum further verifies the successful formation of the NiMn₂O₄ nanozyme by revealing characteristic vibrational features of the nanocatalyst (Fig. S1E). The broad absorption bands centered at 3380 cm⁻¹ and 1640 cm⁻¹ are attributed to O–H stretching and bending modes of surface-adsorbed water molecules, respectively.³⁵ In the fingerprint region, distinct bands appearing at 780 cm⁻¹ and 670 cm⁻¹ are assigned to Mn–O vibrations in octahedral sites and Ni–O vibrations in tetrahedral sites, respectively. These metal–oxygen stretching modes are in good agreement with the typical spinel lattice of NiMn₂O₄, confirming the successful establishment of the mixed-metal oxide framework.³⁴

The Raman spectrum of the NiMn₂O₄ nanozyme exhibits distinct vibrational features characteristic of a spinel lattice (Fig. S1F). The intense A₁g mode observed at 648 cm⁻¹ is attributed to symmetric Mn–O stretching vibrations within MnO₆ octahedra, while the band centered at 545 cm⁻¹ corresponds to the F₂g mode associated with Ni–O stretching. These Raman signatures further corroborate the formation of the spinel NiMn₂O₄ framework and are consistent with the phase identification obtained from XRD analysis.³⁵

XPS analysis was performed to elucidate the surface elemental composition and oxidation states of the NiMn₂O₄ nanozyme (Fig. 1). The survey and high-resolution spectra confirm the presence of O 1s, Mn 2p, and Ni 2p signals, verifying the successful incorporation of all constituent elements (Fig. 1A). The high-resolution O 1s spectrum (Fig. 1B) can be deconvoluted into two components located at binding energies of 542.6 and 545.4 eV, which are assigned to H–O–H species associated with surface-adsorbed water and metal–oxygen (M–O) bonds within the oxide lattice, respectively.³⁶ These features reflect the coexistence of lattice oxygen and surface oxygen species, which are commonly observed in transition-metal oxide nanozymes. Fig. 1C shows Ni 2p peaks at 856.1 eV (2p₃/₂) and 873.3 eV (2p₁/₂), with a spin-energy separation of ∼18 eV, consistent with Ni²⁺ species. Satellite peaks at 858.7 and 868.9 eV confirm this assignment.³⁷ In Fig. 1D, Mn 2p peaks at 643.3 and 654.3 eV correspond to Mn 2p_3/2 and Mn p_1/2, respectively.³⁸

Fig. 1 (A) Full XPS survey of NiMn₂O₄ nanozyme while (B–D) are deconvoluted spectra of O 1s, Ni 2p, and Mn 2p, respectively.

3.2. Kinetic analysis

To quantitatively evaluate the peroxidase-like catalytic activity of the NiMn₂O₄ nanozyme, enzyme-mimetic kinetics were systematically investigated using classical Michaelis–Menten analysis. Kinetic experiments were conducted by maintaining one substrate at a constant concentration (either TMB or H₂O₂) while varying the concentration of the other. The resulting reaction rate profiles exhibited typical Michaelis–Menten behavior, confirming that the catalytic process follows enzyme-like saturation kinetics (Fig. 2A and B). To extract the kinetic parameters, the experimental data were further analyzed using Lineweaver–Burk double-reciprocal plots (Fig. 2C and D). The apparent Michaelis constant (K_m) and maximum reaction velocity (V_max) for H₂O₂ were determined to be 1.671 mM and 21.750 × 10⁻⁸ M min⁻¹, respectively, while the corresponding values for TMB were 0.119 mM and 19.621 × 10⁻⁸ M min⁻¹. The relatively low Km value toward H₂O₂ indicates a strong substrate affinity, suggesting efficient adsorption and activation of peroxide species on the NiMn₂O₄ surface (Table S1). This enhanced catalytic performance can be attributed to the synergistic redox coupling between Ni²⁺/Ni³⁺ and Mn³⁺/Mn⁴⁺ centers, which facilitate rapid electron transfer and promotes the generation of reactive oxygen intermediates. Moreover, the hierarchical nanoarchitecture of NiMn₂O₄ provides abundant accessible active sites, thereby accelerating substrate diffusion and turnover. Collectively, these features endow the NiMn₂O₄ nanozyme with high catalytic efficiency, enabling effective DHNP cyclization and highlighting its potential as a robust artificial enzyme for oxidative sensing applications.

Fig. 2 Enzyme-like kinetics study of NiMn₂O₄ nanozyme and conditions optimization. Enzymatic reaction kinetics curves of NiMn₂O₄ nanozyme when (A) TMB and (B) H₂O₂ as substrate, respectively. Lineweaver–Burk inverse equation curve of NiMn₂O₄ nanozyme when (C) TMB and (D) H₂O₂ as substrate, respectively. Peroxidase-like activity of NiMn₂O₄ nanozyme at (E) different pH and (F) temperature.

As illustrated in Fig. 2E, the NiMn₂O₄ nanozyme maintained pronounced peroxidase-like activity over a wide pH window ranging from 3.0 to 8.0, markedly broader than that reported for many conventional nanozymes. This wide pH tolerance is particularly advantageous for practical and biological applications, as it enables stable catalytic performance under both acidic reaction conditions and near-physiological environments. The robust pH adaptability may be attributed to the structural stability of the mixed-valence Ni–Mn oxide framework, which preserves redox-active sites and prevents proton-induced deactivation. In addition, the NiMn₂O₄ nanozyme exhibited outstanding thermal stability, retaining high catalytic activity over the temperature range of 20–55 °C (Fig. 2F). Such thermal robustness reflects the intrinsic inorganic nature of the nanozyme and the strong metal–oxygen bonding within the spinel structure, which effectively suppresses thermal denaturation commonly observed in natural enzymes. Together, the broad pH tolerance and excellent thermal stability underscore the suitability of NiMn₂O₄ nanozyme for reliable operation in complex analytical and bio-related environments.

3.3. Reaction conditions optimization

Like most catalytic nanomaterials, the enzyme-mimetic activity of the NiMn₂O₄ nanozyme is inherently sensitive to surrounding reaction conditions. Therefore, to achieve reliable and highly sensitive DA detection, the key experimental parameters governing the catalytic reaction were systematically optimized. As depicted in Fig. 3A, the nanozyme activity exhibited a pronounced dependence on solution pH, which significantly influenced both the fluorescence intensity and the emission spectral profile of the reaction system. An optimal pH of 5.5 was identified, a condition that is typically unfavorable for many reported nanozymes, thereby underscoring the exceptional pH adaptability of the NiMn₂O₄ catalyst. The sensing reaction was carried out in acetate buffer at pH 5.5 because this condition afforded the highest fluorescence response and best signal reproducibility. Although biological samples are typically near neutral pH, they were diluted or pretreated in acetate buffer before analysis to provide a unified reaction medium and minimize matrix-dependent pH variation. Importantly, mildly acidic conditions help suppress the spontaneous autoxidation of DA, thereby reducing background interference and allowing the NiMn₂O₄/H₂O₂ system to dominate the oxidative cyclization process. Time-dependent kinetic studies (Fig. 3B) demonstrated a gradual increase in product formation followed by a plateau at approximately 20 min, indicating reaction saturation and completion. Accordingly, a reaction time of 20 min was selected to balance analytical sensitivity and throughput. Temperature was also found to play a critical role in catalytic efficiency, with the maximum fluorescence response observed at 37 °C (Fig. 3C), which is particularly advantageous for biological and clinical applications. The concentration of H₂O₂ proved to be a crucial parameter for accurate signal generation. While low peroxide levels limited catalytic turnover, excessive H₂O₂ induced non-catalytic oxidation of DHNP, leading to signal distortion and reduced selectivity. An optimal H₂O₂ concentration of 4.5 mM was therefore established (Fig. 3D). In addition, owing to the inner filter effect (IFE) arising from spectral overlap between NiMn₂O₄ and DHNP, the nanozyme dosage was carefully optimized. Fluorescence measurements revealed that a NiMn₂O₄ concentration of 20 µg mL⁻¹ provided the highest signal response without compromising spectral integrity (Fig. 3E).

Fig. 3 Fluorescence spectra recorded under varying experimental conditions: (A) pH values, (B) reaction time, (C) temperature, (D) H₂O₂ concentrations, and (E) NiMn₂O₄ catalyst concentrations.

3.4. Detection mechanism

To validate the catalytic contribution of NiMn₂O₄ in DA sensing, a series of control experiments were conducted using TMB as a chromogenic probe. As shown in Fig. 4A, systems lacking the NiMn₂O₄ catalyst exhibited no discernible absorbance at 655 nm, confirming the absence of spontaneous TMB oxidation. In contrast, the introduction of NiMn₂O₄ induced a pronounced absorption band accompanied by an intense blue coloration, irrespective of external H₂O₂ addition. This behavior indicates the efficient in situ generation of reactive oxygen species (ROS), particularly superoxide (O₂˙⁻) and hydroxyl radicals (˙OH), arising from the intrinsic redox cycling between Ni and Mn active centers. The ability of NiMn₂O₄ to sustain radical formation under mild, near-physiological conditions highlights its robust peroxidase/oxidase-like activity and underpins its suitability as a catalytic platform for highly sensitive DA detection. To elucidate the reactive intermediates responsible for the DA–DHNP cyclization, electron spin resonance (ESR) spectroscopy was employed using DMPO as a spin-trapping agent. As illustrated in Fig. 4B, the characteristic quartet and sextet ESR signatures corresponding to DMPO–OH˙ and DMPO–O₂˙⁻ adducts were clearly observed, providing direct evidence for the concurrent generation of ˙OH and O₂˙⁻ radicals. When the reaction was conducted under a nitrogen atmosphere, a pronounced decrease in product formation was observed (Fig. 4C), underscoring the indispensable role of dissolved molecular oxygen in sustaining the catalytic cycle. Furthermore, the stepwise attenuation of the fluorescence signal upon the addition of superoxide dismutase and isopropanol—specific scavengers for O₂˙⁻ and ˙OH, respectively—further corroborated the cooperative involvement of both radical species in driving the cyclization process (Fig. 4D). Collectively, these results establish a dual-radical-mediated mechanism underlying the NiMn₂O₄-catalyzed DA–DHNP reaction.

Fig. 4 (A) Absorbance spectra of NiMn₂O₄ + H₂O₂, TMB + H₂O₂, NiMn₂O₄ + TMB, NiMn₂O₄ + H₂O₂ + TMB, NiMn₂O₄; (B) radical trapping assays demonstrated that NiMn₂O₄ generates O₂˙⁻ and ˙OH; (C) catalytic performance of NiMn₂O₄ was assessed under ambient and nitrogen atmospheres to investigate oxygen dependence; (D) ROS involvement was confirmed through radical scavenging assays using superoxide dismutase and isopropanol.

3.5. Quantitative data

Using a validated analytical protocol, DA was quantitatively determined with excellent linear responses in both fluorescence and absorbance modes. As shown in Fig. 5A and B, the ratiometric fluorescence signal (F₄₈₅/F₄₄₀) increased linearly with DA concentration according to the equation (F₄₈₅/F₄₄₀) = 0.0312 [DA] + 0.5315, exhibiting an outstanding correlation coefficient (R² = 0.9977) over the range of 0–250 µM. In parallel, the colorimetric response (ΔA) displayed a similarly robust linear relationship (ΔA = 0.0032 [DA] + 0.1387, R² = 0.9972; Fig. 5C and D). Based on a signal-to-noise ratio of 3 : 1, the limits of detection were estimated to be 3 nM and 17 nM for the fluorometric and colorimetric assays, respectively. The superior sensitivity achieved in the fluorescence mode can be attributed to the intrinsic ratiometric readout, which effectively suppresses background fluctuations and instrumental drift, thereby enhancing analytical reliability for trace-level DA quantification. The analytical performance of the proposed sensing platform was systematically benchmarked against previously reported colorimetric and fluorometric methods for DA determination, as summarized in Table 1. Key quantitative parameters—including linear dynamic range, limit of detection, and detection modality—were compared to highlight the relative sensitivity and reliability of the present approach. Notably, the proposed dual-mode strategy demonstrates competitive, and in several cases superior, analytical performance, particularly in terms of detection limit and operational simplicity, underscoring its potential advantages for practical DA analysis. The ratiometric fluorescence assay exhibited excellent accuracy and precision across different matrices, achieving recoveries of 95.3–104.9% in buffer solutions (RSDs: 2.15–3.87%, Table S2), 95.0–107.0% in human serum (RSDs: 3.10–4.46%, Table S3), and 95.6–105.2% in urine samples (RSDs: 2.76–3.87%, Table S4). Consistently strong performance was also observed for the colorimetric method, with recoveries ranging from 97.7–102.7% in buffer (RSDs: 1.76–3.19%, Table S5), 97.4–106.9% in serum (RSDs: 2.87–4.34%, Table S6), and 97.4–104.1% in urine (RSDs: 1.89–3.10%, Table S7). The narrow recovery ranges and low relative standard deviations collectively confirm minimal matrix effects and high analytical robustness in complex biological media.

Fig. 5 (A) Fluorescence readings of NiMn₂O₄ + DHNP after addition of different amounts of DA (0–250 µM); (B) plot a relationship between (F₄₈₅/F₄₄₀) against concentration of DA; (C) absorbance readings of NiMn₂O₄ + DHNP after addition of different amounts of DA (0–250 µM); (D) plot a relationship between ΔA against concentration of DA. Number of replicates is four.

Table 1 Quantitative parameters of the proposed and other reported methods for determination of DA

Technique	Probe/system	Linear range (µM)	LOD (µM)	Response time (min)	Reference
Colorimetry	NAC@AuNPs	7.45–24.40	8.4	120	39
	3D-MoS2/Graphene/TMB	1–400	0.21	10	40
	Cu-MOFs/TMB	10–100	10	10	41
	h-CuS NC/TMB	2–150	1.67	30	42
	Graphene nanoribbons/TMB	0.1–50	0.035	10	43
	HP-Co3O4@C NBs/TMB	0–100	0.24	3	44
	Pt/CoFe2O4/TMB	20–80	0.42	3	45
	β-CD/N@GQDs/TMB	0.12–7.5	0.04	5	46
	NiMn2O4 + DHNP	0–250	0.017	20	This work
Fluorometry	β-CD/N@GQDs/TMB	0.028–1.5	0.009	20	46
	Carbon dots	0.1–15	0.037	90	47
	FNP (OS0.2ANS0.8-LEuH)	1–200	0.29	600	48
	N-CQDs	0–100	0.0972	10	49
	Arg-CDs	10–100	0.28	15	50
	NiMn2O4 + DHNP	0–250	0.003	20	This work

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3.6. Selectivity and stability

To rigorously assess the selectivity of the proposed sensing platform—an essential requirement for reliable biomarker analysis—the response toward DA was evaluated in the presence of a broad panel of potentially interfering species commonly encountered in biological matrices. These included inorganic ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺, Cl⁻), small biomolecules (uric acid, ascorbic acid, glutathione, glucose), amino acids (glycine, cysteine, tryptophan, alanine, methionine), nucleotides (adenosine triphosphate sodium), proteins (bovine serum albumin), and structurally related catecholamines (epinephrine and norepinephrine). All interferents were introduced at concentrations tenfold higher than that of DA as an initial anti-interference test under controlled assay conditions. Although this design is useful for benchmarking selectivity, it does not fully reproduce the endogenous concentration ratios encountered in physiological fluids, where some interferents may be present at much higher excess relative to DA. As illustrated in Fig. 6, only DA induced a pronounced and simultaneous change in both fluorescence and absorbance signals, whereas all other species produced negligible responses. This exceptional selectivity is attributed to the specific DA-driven cyclization reaction and its unique redox behavior within the sensing system, effectively discriminating DA from closely related compounds. These results demonstrate good anti-interference performance under the tested experimental conditions and support the selective response of the platform toward DA. However, further validation under more physiologically representative analyte/interferent ratios would be valuable to extend its applicability to endogenous trace-level DA analysis.

Fig. 6 Selectivity of NiMn₂O₄ + DHNP towards DA detection (30 µM) and 300 µM interfering species using fluorescence method (A, B) and colorimetric method (C, D) where 1. Blank, 2. Dopamine, 3. Na⁺, 4. K⁺, 5. Ca²⁺, 6. Mg²⁺, 7. Zn²⁺, 8. Cl⁻, 9. Uric acid, 10. Ascorbic acid, 11. Glutathione, 12. Glucose, 13. Glycine, 14. Cysteine, 15. Tryptophan, 16. Alanine, 17. Methionine, 18. Adenosine triphosphate sodium, 19. Bovine serum albumin, 20. Epinephrine, and 21. Norepinephrine.

The storage stability of the synthesized NiMn₂O₄ nanozyme was assessed over 25 days (Fig. S2). The response showed no appreciable variation throughout the test period, indicating excellent long-term stability. The nanozyme was stored in a tightly closed vial under dry conditions at 4 °C and shielded from light until use.

3.7. Reaction mechanism

The reaction mechanism involves the oxidation of DA by reactive oxygen species (ROS) generated via the catalytic activity of NiMn₂O₄ in the presence of H₂O₂ and O₂. This oxidation produces a DA-quinone intermediate, which is highly electrophilic. DHNP then acts as a nucleophile, attacking the quinone via a Michael addition, followed by intramolecular cyclization and aromatization, leading to the formation of a tetrahydroisoquinoline derivative. This product is stable and exhibits strong chromophoric and fluorescent properties, allowing its detection and quantification via UV-Vis and fluorescence spectroscopy (Scheme 2). LC-MS/MS confirms the product's molecular weight, and control experiments with scavengers and alternate analytes confirm the selectivity and ROS-dependence of the reaction (Fig. S3).

Scheme 2 Proposed reaction pathway for interaction between DA and DHNP.

3.8. Analysis of real samples

To evaluate the practical performance of the developed nanozyme-based sensor, real human serum samples were analyzed using the standard addition method. Detection results from the dual-mode fluorometric/colorimetric sensor were compared with those obtained via high-performance liquid chromatography (HPLC).⁵¹ As shown in Tables 2 and 3, both detection modes showed strong agreement with HPLC results. The method demonstrated satisfactory recoveries ranging from 95.6% to 106.4% (fluorometric) and 95.6% to 104.8% (colorimetric), confirming its accuracy and reliability for DA detection in real samples. These findings highlight the platform's potential for practical applications in early disease diagnosis.

Table 2 Analysis of real samples using fluorometric method (n = 3)

Sample	Spiked (µM)	NiMn2O4 + DHNP			HPLC/UV^51
		Found (µM)	Recovery %	RSD %	Found (µM)	Recovery %	RSD %
Injection	0.0	9.78 ± 0.23	—	—	9.56 ± 0.45	—	—
	5.0	14.76 ± 0.14	99.6	2.67	14.89 ± 0.34	106.4	3.78
	10.0	20.34 ± 0.26	104.5	2.78	19.96 ± 0.67	104.0	4.19
	20.0	30.45 ± 0.47	103.4	3.21	31.21 ± 0.76	108.3	4.65
Serum	0.0	ND	—	—	ND	—	—
	5.0	4.78 ± 0.38	95.6	2.65	5.23 ± 0.23	104.6	3.78
	10.0	9.97 ± 0.54	99.7	3.19	10.34 ± 0.44	103.4	4.62
	20.0	21.28 ± 0.67	106.4	3.00	19.67 ± 0.64	98.4	3.89
Urine	0.0	ND	—	—	ND	—	—
	5.0	5.32 ± 0.32	106.4	2.89	4.93 ± 0.67	98.6	3.78
	10.0	10.52 ± 0.56	105.2	3.20	10.75 ± 0.65	107.5	4.28
	20.0	19.57 ± 0.55	97.9	3.67	22.16 ± 0.66	110.8	4.67

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Table 3 Analysis of real samples using colorimetric method (n = 3)

Sample	Spiked (µM)	NiMn2O4 + DHNP			HPLC/UV^51
		Found (µM)	Recovery %	RSD %	Found (µM)	Recovery %	RSD %
Injection	0.0	9.63 ± 0.43	—	—	9.55 ± 0.76	—	—
	5.0	14.42 ± 0.44	95.8	1.76	14.09 ± 0.48	90.8	3.38
	10.0	20.04 ± 0.26	104.1	1.88	19.76 ± 0.67	102.1	4.19
	20.0	30.19 ± 0.47	102.8	2.49	31.71 ± 0.66	110.8	4.89
Serum	0.0	ND	—	—	ND	—	—
	5.0	5.24 ± 0.23	104.8	2.65	4.86 ± 0.57	97.2	4.34
	10.0	10.32 ± 0.57	103.2	3.00	9.87 ± 0.37	98.7	3.98
	20.0	19.76 ± 0.54	98.8	2.67	22.27 ± 0.59	111.4	5.03
Urine	0.0	ND	—	—	ND	—	—
	5.0	4.78 ± 0.52	95.6	3.54	5.34 ± 0.67	106.8	3.65
	10.0	9.65 ± 0.66	96.5	2.87	10.60 ± 0.55	106.0	3.98
	20.0	20.89 ± 0.35	104.5	3.77	18.87 ± 0.54	94.4	5.00

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4. Conclusions

In this study, a NiMn₂O₄ nanozyme with intrinsic dual peroxidase- and oxidase-like activities was rationally developed as a multifunctional catalytic platform for dopamine (DA) sensing. Unlike conventional nanozyme-based assays that rely on nonspecific oxidation or single-signal transduction, the proposed system uniquely exploits a reactive oxygen species–driven cyclization reaction between DA and 1,3-dihydroxynaphthalene (DHNP), leading to the in situ formation of a new optically active product. This selective chemical transformation constitutes the core novelty of the present work, as it couples nanozyme catalysis with reaction-specific molecular recognition rather than simple signal amplification. The resulting ratiometric fluorescence–colorimetric dual-mode sensing strategy provides intrinsic self-calibration, significantly improving signal fidelity and robustness against probe instability, photobleaching, and matrix effects. Importantly, the high selectivity originates from the exclusive reactivity of DA toward DHNP under nanozyme-mediated conditions, effectively suppressing interference from structurally similar biomolecules commonly present in biological fluids. Owing to the synergistic integration of selective reaction chemistry, dual-mode readout, and nanozyme catalysis, the method achieves high sensitivity, a wide linear dynamic range, and excellent analytical reliability in complex biological samples. Overall, this work introduces a conceptually new sensing paradigm in which nanozyme-triggered, analyte-specific cyclization reactions are harnessed to construct self-validated ratiometric platforms, thereby extending the functional scope of nanozymes beyond conventional enzyme mimetics. The proposed strategy offers a robust and generalizable approach for accurate DA quantification, with strong potential for early diagnosis of DA-related disorders and therapeutic drug monitoring.

Ethical statement

This study received approval from the Institutional Ethics Committee at the University of Assiut. Written informed consent was obtained from all participants prior to sample collection. All procedures were conducted in compliance with the Declaration of Helsinki and applicable national regulations.

Conflicts of interest

The authors declare no competing interests.

Data availability

Data will be available upon request from the corresponding authors. Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra00996d.

Acknowledgements

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2601).

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