Manganese Phthalocyanine-Based Magnetic Core–Shell Composites with Peroxidase Mimetic Activity for Colorimetric Detection of Ascorbic Acid and Glutathione

Abstract

Ascorbic acid (AA) and glutathione (GSH) play a pivotal role in health assessment, drug development, and quality control of nutritional supplements. The development of a new and efficient method for their detection is highly desired. In this work, we fabricated magnetic core–shell nanocomposites (Fe₃O₄@MnPc-NDs) by a one-pot hydrothermal method with citric acid and manganese tetraamino phthalocyanine (MnTAPc) as precursors. Fe₃O₄@MnPc-NDs exhibited enhanced peroxidase activity compared to bare Fe₃O₄ nanoparticles, enabling catalytic oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue ox-TMB in the presence of H₂O₂. Leveraging the antioxidant properties of AA/GSH to reduce ox-TMB, a colorimetric assay achieved a low detection limit of 0.161 μM for AA and 0.188 μM for GSH with broad linear ranges. Moreover, this method displayed high specificity against 12 interfering substances and excellent recyclability (>90% activity after five cycles). Finally, the Fe₃O₄@MnPc-NDs could act as an efficient colorimetric sensor for accurately detecting AA in genuine VC tablets and GSH in whitening serums with high accuracy. Therefore, Fe₃O₄@MnPc-NDs exhibited great potential in bioassay applications, benefiting from their outstanding sensitivity and high recycling rate.

1. Introduction

Ascorbic acid, commonly known as vitamin C (AA), holds a pivotal position as a vital vitamin and potent antioxidant in human nutrition, essential for disease prevention and enhancement of immune defenses [1,2,3]. Similar to AA, glutathione (GSH) plays an important role in providing immunity, enhancing antioxidant function, removing free radicals to delay aging [4,5,6,7], and reducing stress and a broad range of ailments, including Parkinson’s disease, Alzheimer’s disease, HIV, diabetes, and cancer [8,9,10,11,12]. However, it is commonly acknowledged that the inherent concentration of AA/GSH in the human body often falls short of fulfilling the requirements for optimal physiological functions, thereby posing a heightened risk of developing various diseases when their levels are depleted. Humans need to consume AA/GSH from their daily diet or functional foods to maintain health. Therefore, to provide guidance for selecting foods, it is of great significance to develop effective and quantitative methods for the determination of AA and GSH [13,14,15,16].

Based on their antioxidant effects, it is feasible to realize the detection of ascorbic acid and glutathione through specific oxidative reactions [17,18]. Among the diverse measurement techniques, colorimetric approaches harnessing peroxidase-mimicking activity have garnered significant attention due to their swiftness, cost-efficiency, and user-friendliness [19,20,21,22]. Horseradish peroxidase [23] is a natural peroxidase that has some inherent disadvantages, such as being time-consuming to extract, easy to inactivate, and difficult to preserve, thus leading to limited practical applications [24]. Therefore, it was imminent to develop potential peroxidase-mimicking enzymes to realize efficient detection. Fe₃O₄ exhibited catalytic activity that was analogous to that of horseradish peroxidase, demonstrating its potential as a peroxidase mimic for various applications [25,26,27]. Its preparation into nanoparticles endowed them with peroxidase properties as well as unique properties such as magnetism, low toxicity, and high biocompatibility, which were highly promising for applications [28,29]. Due to the distinctive macrocyclic conjugated aromatic structure, porphyrins have garnered extensive attention in numerous fields, including biomimetics, pharmaceutical science, medicinal chemistry, catalysis, materials chemistry, and coordination chemistry [30,31]. Metal porphyrin compounds were widely present in living organisms in nature and played an important role in life activities, among which horseradish peroxidase was composed of iron porphyrin. The structure of phthalocyanine was similar to porphyrin and belonged to porphyrin derivatives with better catalytic activity and stability [32,33]. Therefore, the combination of metal phthalocyanine with Fe₃O₄ might provide exceptional applications in the domains of artificial enzyme mimicry, biosensing platforms, and electrochemical catalysis.

The aim of this study is to develop a reusable magnetic core–shell nanocomposite (Fe₃O₄@MnPc-NDs) with enhanced peroxidase-like activity for the dual detection of AA and GSH in complex matrices. By integrating manganese phthalocyanine MnPc with Fe₃O₄ nanoparticles through a one-pot hydrothermal synthesis, we address the limitations of natural enzymes (e.g., instability and high cost) and existing nanozymes (e.g., low catalytic efficiency and poor specificity). Through systematic characterization of structural properties and kinetic analysis, this work elucidates the synergistic mechanism between MnPc and Fe₃O₄, which significantly improves the catalytic oxidation of TMB in the presence of H₂O₂. Furthermore, we establish a colorimetric platform leveraging the antioxidant properties of AA/GSH to reduce ox-TMB, achieving ultra-low detection limits of 0.161 μM for AA and 0.188 μM for GSH, respectively. Finally, the colorimetric method based on Fe₃O₄@MnPc-NDs was used to determine the AA content in vitamin C tablets and the GSH content in whitening serum with high precision and reliability.

2. Experimental Sections

2.1. Synthesis of Manganese Tetranitro Phthalocyanine

As shown in Figure 1, a 6.0 g amount (100 mmol) of urea, 4.0 g (20 mmol) of 4-nitrophthalic anhydride, and 656 mg (5.2 mmol) of manganese chloride were dissolved in 30 mL of nitrobenzene solution. Afterward, 26 mg (0.02 mmol) of ammonium molybdate was added to the reaction mixture, which was then heated at 185 °C for 4 h under a nitrogen atmosphere. The reaction mixture was then cooled to room temperature and diluted with toluene. The suspension was centrifuged to collect the formed sediment, which was washed thoroughly with toluene, water, methanol/ether (1:9), and ethyl acetate/hexane (2:1) successively. Finally, the solid was dried under vacuum to obtain manganese tetranitro phthalocyanine (MnPc-NO₂) in 80% yield.

2.2. Synthesis of Manganese Tetraamino Phthalocyanine

A 7.4 g amount (30.9 mmol) of sodium sulfide nonahydrate and 1.92 g (2.5 mmol) of manganese tetranitro phthalocyanine were dissolved in 50 mL of DMF. Under nitrogen atmosphere, the reaction mixture was heated to 60 °C and agitated for a period of 1.5 h. After cooling to ambient temperature, the reaction solution was diluted with 150 mL of water. The formed precipitate was then separated by centrifugation, followed by washing with methanol/ether mixture (1:9) and ethyl acetate successively. Finally, the solid was dried under vacuum to obtain manganese tetranitro phthalocyanine (MnPc-NH₂) in 75% yield.

2.3. Synthesis of Magnetic Fe₃O₄ Nanoparticles

Magnetic Fe₃O₄ nanoparticles were fabricated using the solvothermal approach. Firstly, 0.75 g of PSSMA was dissolved in 20 mL of ethylene glycol and stirred for 15 min. Then 0.8 g of FeCl₃·6H₂O and 2.25 g of magnesium acetate were added sequentially and reacted for another 15 min. The reaction mixture was then carefully transferred to a hydrothermal reactor and heated at 200 °C for a duration of 10 h. Upon completion of the reaction, the resultant material was thoroughly rinsed three times using deionized water and anhydrous ethanol, to ensure the removal of impurities. Finally, the material was dried under vacuum for 12 h to yield the desired powder.

2.4. Synthesis of Fe₃O₄@MnPc-NDs

Firstly, 50 mg of Fe₃O₄ nanoparticles were dispersed by sonication in 10 mL of ultrapure water for 10 min. Then 15 mg of manganese tetraamino phthalocyanine and 150 mg of citric acid were sequentially added. After sonication for 10 min, the reaction solution was transferred to a hydrothermal reactor. After heated at 200 °C for 4 h, the resulting solid was separated and washed three times with deionized water and anhydrous ethanol, respectively. Finally, the desired composites were dried under vacuum to obtain the magnetic Fe₃O₄@MnPc-NDs with core–shell structure.

2.5. Peroxidase Like Catalytic Activity of Fe₃O₄@MnPc-NDs

To assess the peroxidase-like properties of Fe₃O₄@MnPc-NDs, the catalyzed oxidation of TMB by H₂O₂ was determined. In brief, TMB (5 mM, 50 μL) and Fe₃O₄@MnPc-NDs (1 mg/mL, 20 μL) were added into HAc-NaAc buffer (pH = 4.0, 2.8 μL). The combined solution was maintained at 25 °C for 7 min. Fe₃O₄@MnPc-NDs were then separated from the solution using an external magnet, followed by the measurement of the maximum absorbance at 652 nm.

2.6. Kinetic Measurements

The kinetic analysis of Fe₃O₄@MnPc-NDs was evaluated by Michaelis–Menten model [18]. In brief, different concentrations of H₂O₂ or TMB were added into a HAc-NaAc buffer solution (pH 3.8) containing Fe₃O₄@MnPc-NDs (1 µg/mL, 20 μL). The intensity of absorption at 652 nm was monitored at room temperature over time. ${\mathrm{k}}_{\mathrm{m}}$ is the Michaelis constant, ${\mathrm{v}}_0$ and ${\mathrm{v}}_{\mathrm{m}}$ are the initial and maximum reaction velocities of the reaction, respectively, and $\left[\mathrm{s}\right]$ is the concentration of the substrate. The computation of this value is carried out using the Michaelis–Menten equation (Equation (1)).

$${\displaystyle \frac{1}{{\mathrm{v}}_0}}={\displaystyle \frac{{\mathrm{k}}_{\mathrm{m}}}{{\mathrm{v}}_{\mathrm{m}}\left[\mathrm{s}\right]}}+{\displaystyle \frac{1}{{\mathrm{v}}_{\mathrm{m}}}}$$

(1)

where v₀ and v_m represent the initial rate and maximum rate, respectively. k_m stands for the Michaelis–Menten constant and [s] stands for substrate (H₂O₂ or TMB) concentration.

2.7. Detection of H₂O₂ by the Colorimetric Method

A colorimetric detection method for H₂O₂ was established based on the relationship between the concentration of H₂O₂ and the absorbance of system at 652 nm. Specifically, 50 μL of different concentrations of H₂O₂, 20 μL of Fe₃O₄@MnPc-NDs suspension (1.0 mg/mL), and 50 μL of TMB solution (5 mM) were added to 2800 μL of HAc-NaAc buffer solution (pH 3.8), which was stirring at 25 °C for 7 min. Fe₃O₄@MnPc-NDs were then isolated from the solution by magnet, followed by the measurement of the absorbance at 652 nm. To further investigate the specificity of this method, interfering substances such as ascorbic acid (AA), citric acid (CA), dopamine (DA), Na⁺, K⁺, Ca²⁺, and glucose were selected as control samples.

2.8. Determination of AA and GSH by the Colorimetric Method

A colorimetric detection platform was devised based on the antioxidant activity of AA/GSH. Specifically, 20 μL of suspension containing Fe₃O₄@MnPc-NDs (1.0 mg/mL), 50 μL of H₂O₂ (0.1 M), 50 μL of TMB solution (5 mM), and 50 μL of AA (or GSH) of different concentrations were sequentially added to 2830 μL of HAc-NaAc buffer solution (pH 3.8). After stirring at 25 °C for 7 min, the absorbance at 652 nm was recorded by UV–vis absorption spectrum. The AA or GSH content was measured by observing the variation in absorbance at 652 nm. The selectivity of AA/GSH was determined by comparing the variations in absorbance at 652 nm between AA/GSH and twelve distinct interfering substances (Arg (arginine), His (histidine), Lys (lysine), Gly (glycine), Ala (alanine), Phe (phenylalanine), Val (valine), Mg²⁺, Zn²⁺, Na⁺, K⁺, and Ca²⁺). The concentration of AA/GSH was 60 μM, while the concentrations of the interfering substances were set at a level tenfold higher than that of AA/GSH.

2.9. Identification of AA and GSH Concentrations in Real Samples

To explore the practicality of the colorimetric method, a certain weight of vitamin C tablets was dissolved in water and further adjusted to the required volume to determine the amount of AA in vitamin C tablets. For accuracy verification, a standard AA solution was spiked into the diluted vitamin C tablet sample, and the absorbance variation at 652 nm upon vitamin C tablet addition was documented, respectively. Meanwhile, whitening serum was obtained from a nearby beauty shop and the spiked recovery method was employed to quantify the glutathione level in the sample.

3. Results and Discussion

3.1. Characterization of Fe₃O₄@MnPc-NDs

The size and morphology of Fe₃O₄ nanospheres and Fe₃O₄@MnPc-NDs were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As indicated in Figure 2A, the size of Fe₃O₄ nanoparticles was about 257 nm. After functionalization with manganese phthalocyanine, the composites with core–shell structures were formed along with the increase in particle size to 285 nm (Figure 2B). The thickness of the shell layer in Fe₃O₄@MnPc-NDs was approximated to be 15 nm. FESEM (Figure 2C,D) demonstrated that the surface of Fe₃O₄@MnPc-NDs was rougher than that of bare Fe₃O₄ nanoparticles, further indicating the successful modification of phthalocyanine to the Fe₃O₄ surface.

The elemental composition of Fe₃O₄@MnPc-NDs was confirmed by XPS analysis. As indicated in Figure 3A, the distinct peaks at 285, 400, 531, 643, and 726 eV were observed, attributable to C 1s, N 1s, O 1s, Mn 2p, and Fe 2p, respectively. The XPS analysis at high resolution for C 1s orbital (Figure 3B) unveiled four discrete peaks positioned at 284.2, 285.1, 285.9, and 288.0 eV, each corresponding to C-C/C=C, C-N, C-O, and C=O/C=N bonds, respectively. The high-resolution XPS spectrum of N 1s (Figure 3C) showed C=N (398.1 eV) attributed to nitrogen of phthalocyanine ring, C-N (399.4 eV) attributed to the coordination of manganese and nitrogen, and O=C-N (401.1 eV) attributed to amide on the of nitrogen. The XPS analysis at high resolution for O 1s (Figure 3D) showed two peaks at 533.69 and 533.08 eV, attributed to C=O and C-O, respectively. The XPS spectra of Mn 2p could be fitted into two peaks at 652.98 and 643.68 eV, attributable to Mn 2p_1/2 and Mn 2p_3/2, respectively, indicating the presence of trivalent manganese ions in Fe₃O₄@MnPc-NDs (Figure 3E). Four peaks were observed in the high-resolution XPS spectra of Fe 2p (Figure 3F), matching Fe²⁺ 2p_3/2 (713.58 eV), Fe²⁺ 2p_1/2 (725.28 eV), Fe³⁺ 2p_3/2 (711.78 eV), and Fe³⁺ 2p_1/2 (726.68 eV) of Fe₃O₄ nanoparticles, respectively. Thus, the XPS spectrum clearly confirmed the presence of Fe and Mn elements in Fe₃O₄@MnPc-NDs in addition to C, N, and O.

The crystal structures of Fe₃O₄ nanoparticles and Fe₃O₄@MnPc-NDs were characterized through an X-ray diffraction (XRD) curve. As shown in Figure 4A, the XRD spectrum of Fe₃O₄ nanoparticles exhibited distinct diffraction peaks positioned at angles of 30.1°, 35.5°, 43.2°, 53.5°, 57.3°, and 62.9°, attributed to the characteristic lattice planes of (220), (311), (400), (422), (511), and (440), respectively, which were consistent with the reported data [34] (JCPDS No. 16-692). The XRD spectra of Fe₃O₄@MnPc-NDs were similar to that of Fe₃O₄ nanoparticles, indicating the functionalization of phthalocyanine did not affect the crystal structures of Fe₃O₄ nanoparticles. The functional groups on the surface were further characterized via infrared spectroscopy. As shown in Figure 4B, the characteristic peak at 579 cm⁻¹ was ascribed to the Fe-O-Fe vibration in the Fe₃O₄ nanoparticles. The peaks at 3437, 1713, and 1613 cm⁻¹ were assigned to the stretching vibrations of the O-H, C=O, and C=C, respectively. In addition, the peaks at 1393 and 1194 cm⁻¹ were ascribed to the stretching vibrations of the C-N and C-O bonds, respectively. The magnetic properties of Fe₃O₄ nanoparticles and Fe₃O₄@MnPc-NDs were investigated using hysteresis loops. As indicated in Figure 4C, both Fe₃O₄ nanoparticles and Fe₃O₄@MnPc-NDs displayed obvious magnetic properties with saturation magnetization strengths of 49.46 and 36.37 emu/g, respectively. In addition, with the help of an external magnetic field, Fe₃O₄@MnPc-NDs dispersed in aqueous solution can be collected in a short time, indicating their excellent magnetism and ability for rapid magnetic separation.

3.2. Peroxidase-Mimicking Activity of Fe₃O₄@MnPc-NDs

The peroxidase-mimicking activity of Fe₃O₄@MnPc-NDs was evaluated with TMB and H₂O₂ as substrates. The absorption spectrum of the system was recorded through UV–vis absorption spectroscopy. As depicted in Figure 5A, only the “TMB + H₂O₂ + Fe₃O₄@MnPc-NDs” system exhibited an obvious absorption peak at 652 nm by comparing with the control groups, along with the appearance of a distinct blue color in solution. Moreover, the structural advantages of Fe₃O₄@MnPc-NDs were illustrated by comparing the catalytic activities of raw materials and composites. As shown in Figure 5B, the UV–visible spectrum of Fe₃O₄@MnPc-NDs exhibited a significant absorption peak at 652 nm. Moreover, the absorption intensity at 652 nm of Fe₃O₄@MnPc-NDs was higher than that of other control samples (Fe₃O₄ nanoparticles and MnPc-NDs), indicating the higher catalytic activity after functionalization.

3.3. Optimization of the Catalyzed Conditions

Similar to other peroxidase-like artificial enzymes, the catalytic activity of Fe₃O₄@MnPc-NDs might also depend on the H₂O₂ concentration, pH values, and temperature. Accordingly, the investigations were conducted to examine the catalytic activity of Fe₃O₄ nanoparticles and Fe₃O₄@MnPc-NDs under varying experimental conditions, specifically encompassing H₂O₂ concentrations ranging from 0.8 to 13 mM, pH values spanning from 1.7 to 11, and temperatures ranging from 20 to 70 °C. As depicted in Figure 6A,D, the catalytic activity of two materials started to increase and then leveled off with the increase in H₂O₂ concentration. The best catalytic activity was achieved at an H₂O₂ concentration of 10 mM, lower than that of bare Fe₃O₄ nanoparticles (255 mM). As shown in Figure 6E, Fe₃O₄@MnPc-NDs exhibited the highest activity at pH 3.8, which was similar to that of Fe₃O₄ nanoparticles (Figure 6B). Moreover, a temperature of 25 °C was found to be the most favorable for the catalytic performance of Fe₃O₄@MnPc-NDs (Figure 6F), while a higher temperature (55 °C) was required for Fe₃O₄ nanoparticles to realize the maximum catalytic activity (Figure 6C). Therefore, Fe₃O₄@MnPc-NDs exhibited the maximum catalytic activity under the milder conditions compared with that of bare Fe₃O₄ nanoparticles, and H₂O₂ concentration of 10 mM, pH 3.8, and temperature 25 °C were chosen as the most suitable conditions for studying the catalytic activity of Fe₃O₄@MnPc-NDs.

3.4. Steady-State Kinetic Determination of Fe₃O₄@MnPc-NDs

To further understand the catalytic mechanism of Fe₃O₄@MnPc-NDs, a series of kinetic assessments were performed by incrementally adjusting the concentration of one substrate while maintaining a constant level of the other substrate throughout the entire series of measurements. As displayed in Figure 7A,C, the typical Michaelis–Menten curves for TMB and H₂O₂ were measured within their appropriate concentration ranges. The corresponding relationships between the reciprocals of initial velocity and substrate concentration were obtained (Figure 7B,D). The oxidation reaction of Fe₃O₄@MnPc-NDs upon TMB and H₂O₂ conformed to the typical Michaelis–Mente equation. To assess the key kinetic attributes of the enzyme, including the Michaelis–Menten constant (K_m) and the maximum initial reaction rate (V_max), the Lineweaver–Burk plot was utilized (

Table 1. Comparison of apparent steady-state kinetic parameters between Fe₃O₄@MnPc-NDs and other artificial enzyme mimics.

Material	Catalytic Substrate	Km (mM)	Vmax (10−8 M S−1)	Reference
Fe3O4@MnPc-NDs	TMB	0.037	3.78	This work
	H2O2	0.47	4.82
Fe3O4	TMB	0.098	3.44	[35]
	H2O2	154	9.78
HRP	TMB	0.434	10	[35]
	H2O2	3.7	8.71
Cu-Ag/rGO	TMB	8.61	7.02	[36]
	H2O2	0.634	4.26

). K_m serves as an indicator of enzyme affinity for substrates. As the K_m value decreases, the affinity of the enzyme toward its substrate increases. Compared with HRP and Fe₃O₄ nanoparticles, Fe₃O₄@MnPc-NDs exhibited lower K_m values for H₂O₂, indicating that H₂O₂ can be detected at lower concentrations.

3.5. Detection of H₂O₂

Based on the peroxidase-mimicking properties of Fe₃O₄@MnPc-NDs, a simple colorimetric approach for the quantification of H₂O₂ was devised. As depicted in Figure 8A,C, a distinct enhancement in the absorbance of the mixture of Fe₃O₄@MnPc-NDs and TMB at 652 nm was observed, with the H₂O₂ concentration ranging from 0.2 to 15 mM. Based on the variation of absorbance and color change in solution, a linear calibration curve was obtained with the H₂O₂ concentration ranging from 20 to 250 μM (Figure 8B). According to the obtained linear equation, the detection limit (LOD) of H₂O₂ was calculated to be 4.7 μM, which was lower than the values previously reported in the literature (

Table 2. Comparison of detection limit and linear range for detecting H₂O₂ using different peroxidase mimetic enzymes.

Material	Linear Range (μM)	Detection Limit (μM)	Reference
Fe3O4@MnPc-NDs	20–250	4.7	This work
Fe3O4	5–100	3	[37]
Copper	10–1000	10	[38]
Pt-MnO2/GOP	2–13,330	5	[39]
Au@Ag	10–10,000	6	[40]

). In order to evaluate the influence of interfering substances, a range of potential interferences, including dopamine (DA), citric acid (CA), ascorbic acid (AA), glucose, as well as ions like Na⁺, K⁺, and Ca²⁺ were chosen. As depicted in Figure 8D, the solution color of Fe₃O₄@MnPc-NDs and TMB did not show any significant change after adding the interfering substances, even though their concentration was five times higher than that of H₂O₂, indicating the high selectivity and specificity for H₂O₂ detection.

3.6. Detection of AA and GSH

Based on the antioxidant effects of AA and GSH, a colorimetric approach for detection was established using the system of Fe₃O₄@MnPc-NDs and TMB. As shown in Figure 9A, the absorbance of the sensing platform at 652 nm exhibited a gradual decline upon the addition of ascorbic acid and GSH (Figure 9A,B), along with the fading of the blue solution. Based on the variation curve of absorbance at 652 nm upon the AA (Figure 9C) or GSH (Figure 9D) concentration, a linear equation was obtained for AA (ΔA = 0.0139 [AA] + 0.0668 (R² = 0.996)) and GSH (ΔA = 0.0156 [GSH] + 0.0999 (R² = 0.991)) (Figure 9E,F). According to these equations, the detection limit was calculated to be 0.161 μM for AA, and 0.188 μM for GSH, which were comparable to those reported by colorimetric methods (

Table 3. Comparison of linear range and detection limit for detecting AA and GSH using different peroxidase mimetics.

Catalyst	Detection	Linear Range (µM)	Detection Limit (μM)	Reference
N-CQDs	AA	5–40	1.77	[41]
N, S-CDs	AA	10–200	4.69	[42]
MIL-88	AA	2.57–10.10	1.03	[43]
Fe3O4@MnPc-NDs	AA	5–45	0.161	This work
QDs	GSH	5–250	0.6	[44]
Cd-Se/ZnS QDs	GSH	10–180	1.5	[45]
Co, N-HPC	GSH	0.05–30	0.036	[46]
Fe3O4@MnPc-NDs	GSH	5–30	0.188	This work

). Using Arg (arginine), Ala (alanine), His (histidine), Lys (lysine), Gly (glycine), Phe (phenylalanine), Val (valine), Mg²⁺, Zn²⁺, Na⁺, K⁺, and Ca²⁺ as interfering substances, the selectivity study for AA/GSH determination was conducted based on Fe₃O₄@MnPc-NDs. As shown in Figure 10, even though the concentrations of numerous interfering substances escalated to tenfold that of AA/GSH, the solution color of Fe₃O₄@MnPc-NDs/TMB/H₂O₂ system did not exhibit any significant change, along with negligible alterations in absorbance at 652 nm.

3.7. Detection of Ascorbic Acid and Glutathione in Actual Samples

To evaluate the practical application of this colorimetric method, the ascorbic acid content in vitamin C was tested. Specifically, vitamin C was diluted to a certain concentration and then added to the Fe₃O₄@MnPc-NDs/TMB/H₂O₂ system. To further corroborate the precision of this method, a known concentration of AA was introduced into the diluted sample of vitamin C tablets. As evident from Table S1, the recovered quantities of AA in these samples were extremely close to the expected values, with recovery rates higher than 98% and the relative standard deviation (RSD) less than 5%. The colorimetric method was also used to detect the content of glutathione in the purchased whitening serum. As shown in Table S2, the sample recoveries were higher than 99%, with an RSD of less than 5%, indicating the high accuracy of this method for the analysis of real samples.

3.8. Recyclability Testing

To evaluate the recyclability of Fe₃O₄@MnPc-NDs, the recovered Fe₃O₄@MnPc-NDs were added into the “TMB + H₂O₂” system, and their catalytic activity was tested. As shown in Figure 11, after five catalytic cycles, Fe₃O₄@MnPc-NDs still possessed high peroxidase activity with catalytic activity around 90%, indicating the good reusability of Fe₃O₄@MnPc-NDs.

4. Conclusions

In summary, the Fe₃O₄@MnPc-NDs composites were effectively synthesized via the hydrothermal method. Fe₃O₄@MnPc-NDs demonstrated enhanced peroxidase-like activity compared with the bare Fe₃O₄ nanoparticles, which could oxidize the colorless TMB to blue ox-TMB in the presence of H₂O₂. Based on the inhibitory effect of AA/GSH on this catalytic process, a highly sensitive and rapid colorimetric sensor was developed, capable of determining AA and GSH with detection limits of 0.161 and 0.188 μM, respectively. This developed sensor exhibited high selectivity and exceptional reproducibility, enabling its application in determining AA levels in vitamin C tablets and GSH concentrations in whitening serum, indicating the practical potential of Fe₃O₄@MnPc-NDs in colorimetric assays.