Mn-CDs@Pt nanozyme with high oxidase-like activity for smartphone-assisted colorimetric sensing of acrylamide

Abstract

Herein, Mn-doped carbon dots (Mn-CDs) were prepared and used as a reducing agent to reduce PtCl₆²⁻ to platinum nanoparticles (Pt NPs), forming a stable nanozyme of Mn-CDs@Pt NPs. The prepared Mn-CDs@Pt nanozyme exhibited high oxidase-like activity toward the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) with a low Michaelis-Menten constant (K_m) value of 0.0545 mM and a high maximum initial velocity (V_max) value of 3.42 × 10⁻⁷ M s⁻¹. By exploiting the excellent oxidase-like activity of Mn-CDs@Pt nanozyme and combining the thiol-ene Michael addition between glutathione (GSH) and acrylamide (AA), a sensitive colorimetric detection interface for AA was fabricated. Using UV–vis measurement, the colorimetric sensor for AA detection displayed a broad linear detection range from 0.05 to 5000 μM and a detection limit (LOD) of 0.0371 μM. In addition, a smartphone-assisted detection of AA yielded a linear range from 5 to 5000 μM with an LOD of 4.35 μM. The colorimetric sensor was applied for AA detection in real samples, achieving satisfactory recoveries in the range of 94.8–110.4%. Moreover, the detection results of AA in real samples were in good consistency with those detected using the standard method of HPLC, suggesting that the proposed colorimetric method possesses potential for AA detection in food safety and quality monitoring.

1. Introduction

Acrylamide (AA), one of the typical hazardous contaminants that formed during food processing at over 120 °C (Zheng et al., 2026), has been categorized as a Group 2 A carcinogen (probably carcinogenic to humans) by the International Cancer Research Center (ICRC) (Ge et al., 2025; Zhuang et al., 2022). The recommended maximum daily intake of AA is 1 μg kg⁻¹ (Khoshbin et al., 2024). However, AA is commonly found in various kinds of food products, including fried foods, bakery foods, roasted coffee, etc. To urge manufacturers to improve their processing technologies to ensure food safety, the European Union (EU) has set benchmark values for various foods (F. Liu et al., 2023), such as chips (750 μg kg⁻¹), roasted coffee (400 μg kg⁻¹) and infant foods (40 μg kg⁻¹). Therefore, efficient methods for the detection of AA are of great importance in monitoring food safety.

According to the National standard GB/T 44165.6–2024 of China (Determination of key chemicals in consumer products—Part 6: Acrylamide, Pub. L. GB/T 44165.6–2024, 2024), LC-MS/MS and GC–MS methods remain the standard methods due to the high accuracy they offer for the detection of AA in foods. However, these standard methods require highly professional personnel, costly equipment and complex sample preparation, all of which limit their use in routine research labs (Rong et al., 2024). To date, various nanomaterial-based sensors or biosensors (Hoang et al., 2022; Rayappa et al., 2021) have been developed for the rapid, effective and cost-effective detection of AA in foods. These sensing methods include electrochemical (Asnaashari et al., 2019; N. Li et al., 2020; Wu et al., 2019), fluorescence (Cheng et al., 2024; Rong et al., 2024; Tatlı et al., 2026), photoelectrochemical (Zhu et al., 2025) and colorimetric (Zheng et al., 2026; Zhuang et al., 2022) methods. Among these nanomaterial-based assays for AA detection, numerous colorimetric sensors have been fabricated and investigated owing to their advantages of high sensitivity and selectivity, rapidity and low cost. Most importantly, “naked eye” (F. Liu et al., 2023; Zhuang et al., 2022) and smartphone-assisted (Ge et al., 2025; Khoshbin et al., 2024) detection of AA can be realized by these colorimetric sensors.

There are mainly two strategies applied in nanomaterial-based colorimetric sensors for the detection of AA. One is using the aggregation of nanoparticles (Fang Wong & Mei Khor, 2021; Hoang et al., 2022; Hu et al., 2016; Y. Wang et al., 2025), which induces the color change of the detection system. The other is exploiting the enzyme-like activity (Khoshbin et al., 2024; Tajgardoon et al., 2025; Y. Wang et al., 2025) of the used nanomaterials, which catalyzes the substrate reaction, leading to a color change in the detection system. By far, the nanozyme-based strategy has been widely used in the fabrication of colorimetric sensors for AA detection. The enzyme-like activity of these nanozymes can be classified into oxidase-like (Guo et al., 2024; Tajgardoon et al., 2025), peroxidase-like (F. Liu et al., 2023; Y. Wang et al., 2025; Zheng et al., 2026; Zhuang et al., 2022) and nitroreductase-like (Khoshbin et al., 2024) activities. For example, Ge et al. (Ge et al., 2025) reported a Cu-CN single-atom (Cu-CN SANs) nanozyme-based colorimetric sensor for ultrasensitive detection of AA in fried food. The prepared Cu-CN SANs nanozyme showed excellent oxidase-like activity that can catalyze the colorless TMB into blue-colored oxidized TMB (oxTMB). In the presence of glutathione (GSH), the color reaction was inhibited by GSH, leading to the fading of blue color of the system. However, when AA was added, the content of GSH was reduced due to the Michael addition between GSH and AA, which resulted in the recovery of the blue color. Based on the above strategies, the Cu-CN SANs nanozyme-based colorimetric sensor enabled ultrasensitive detection of AA with a detection limit (LOD) of 0.0154 μM.

Noble metal nanoparticles (X. Jiang et al., 2021; C. Wang et al., 2022), including gold nanoparticles (Au NPs), silver nanoparticles (Ag NPs), and platinum nanoparticles (Pt NPs), have been recognized as one of an important type of nanozymes. These nanozymes show multiple enzyme-mimic activities (Chen et al., 2021; He et al., 2020; J. Liu et al., 2025; Xu et al., 2021), which have been applied to the construction of various colorimetric sensing interfaces. However, due to their extremely high specific surface free energy, noble metal nanozymes tend to aggregate, resulting in a dramatic reduction in their catalytic activity (L. Sun et al., 2022). To address this challenge, noble metal nanozymes are often stabilized with small molecules (Dolci et al., 2026; Tang et al., 2022) and other nanostructures (X. Jiang et al., 2021; H. Li et al., 2023) to prevent their aggregation and maintain/improve their catalytic activities. For example, Yi et al. (Yi et al., 2026) used Zr metal-organic framework (PCN-224) as a carrier and stabilizer for the loading of Pt NPs, forming PCN-224@Pt nanocomposite. They demonstrated that PCN-224 can effectively lower the energy barrier for electron transfer, which can enhance the catalytic activity of PCN-224@Pt nanozyme for TMB oxidation. The low Michaelis-Menten constant (K_m) of PCN-224@Pt nanozyme for TMB was 0.18 mM, enabling a sensitive colorimetric sensing interface for tetracycline detection with a LOD of 0.04 μM.

Carbon dots (CDs), especially heteroatom-doped carbon dots (Cui et al., 2023; Lu et al., 2023), exhibit good enzyme-like activity. More importantly, CDs show good reducing ability due to their oxygen-containing groups of CDs (Beygisangchin et al., 2025; Ghosh et al., 2023). Inspired by the excellent enzyme-mimic activity of Pt NPs and the reducing ability of CDs, we used Mn-doped carbon dots (Mn-CDs) as the reducing agent for the formation of Pt NPs and also a stabilizer for the resulting Pt NPs to prepare Mn-CDs@Pt NPs. The oxidase-like activity of Mn-CDs@Pt NPs was investigated, and reactive oxygen species (ROS) were determined to explain the activity toward 3,3′,5,5′-tetramethylbenzidine (TMB). By combining the enhanced oxidase-like activity of Mn-CDs@Pt NPs with the inhibition effect of GSH, and using the thiol-ene Michael addition between GSH and AA, a sensitive colorimetric sensing interface for AA detection was successfully constructed.

2. Materials and methods

2.1. Reagents and apparatus

Polyvinylpyrrolidone (PVP), glutathione (GSH), manganese chloride (MnCl₂), and sodium citrate were ordered from National Medicine Group Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Potassium chloroplatinate (K₂PtCl₄), acrylamide (AA, C₃H₅NO), and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All other chemicals were used as received. Deionized water was used throughout the experiment.

UV–vis spectra were recorded on a UV4820 spectrophotometer (Unico Shanghai Instrument Co., Ltd. Shanghai, China). Fluorescence spectra of Mn-CDs and Mn-CDs@Pt NPs were obtained on a F97pro fluorescence instrument (Shanghai Lengguang Tech. Co., Ltd., Shanghai, China). The electrochemical characterizations were performed on a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). The transmission electron microscopy (TEM) was used to characterize the morphology of the prepared nanomaterials, and the images were obtained on a JEM-2100 F (JEOL, Japan) at an acceleration voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) characterizations were conducted on an ESCALAB250XI X-ray photoelectron spectrometer (Thermo Fisher Scientific, USA) using an Al Kα (1486.6 eV) radiation at a power of 225 W. The generated free radicals during TMB oxidation catalyzed by Mn-CDs@Pt nanozyme were confirmed using an electron paramagnetic resonance (EPR) spectrometer (Bruker EMXplus, Bruker (Beijing) Technology Co., Ltd., Beijing, China).

2.2. Preparation of Mn-CDs and Mn-CDs@Pt NPs

Mn-CDs were prepared via a hydrothermal method using sodium citrate as carbon source and MnCl₂ as the Mn²⁺ doping. Typically, 0.7359 g of sodium citrate and 0.1853 g of MnCl₂ were dissolved in 25 mL water and sonicated for 10 min. Then, the mixture was transferred to a 100 mL Teflon autoclave. The hydrothermal reaction was conducted at 200 °C for 6 h. After cooling down to room temperature, the prepared Mn-CDs were filtered using 0.22 μm pore-size membrane, and the obtained light-yellow solution (Fig. S1 A) was stored at 4 °C for later use.

The Mn-CDs@Pt NPs were prepared according to our previous work (Cao et al., 2022) with some modifications. Briefly, 0.0100 g PVP was dissolved in the prepared Mn-CDs (1 mL) as the reducing agent, followed by the addition of 20 mmol L⁻¹ (700 μL). After sonication for 30 min, the mixture was transferred to a 100 mL Teflon autoclave, and the reduction reaction was performed at 120 °C for 12 h. After cooling to room temperature, mixture was centrifuged at 12,000 rpm for 20 min, and the obtained black supernatant (Fig. S1B) was collected and dialyzed (MWCO = 1000 Da) against water for 24 h. Finally, the dialyzed solution was dried at 60 °C for 12 h, and the obtained Mn-CDs@Pt NPs were stored in desiccator for later use (Scheme. 1A).

Scheme 1.

Colorimetric detection of acrylamide via “inhibition-recovery” of Mn-CDs@Pt oxidase-like activity.

2.3. Determination of the oxidase-like activity of Mn-CDs@Pt NPs

The oxidase-like activity of Mn-CDs@Pt NPs was investigated using TMB as substrate under air atmosphere at room temperature (Scheme. 1B). The assays were performed in a 500 μL system containing 470 μL acetate buffer (0.2 M, pH 4.5), 10 μL Mn-CDs@Pt NPs suspension (1.5 mg mL⁻¹), and 20 μL various concentrations of TMB (0.5, 0.75, 1.0, 1.5, 2.0 and 2.5 mM). Time-dependent curves were recorded at a fixed wavelength of 652 nm. The initial reaction velocities (v₀) for various TMB concentrations were obtained from these time-dependent curves. The Michaelis-Menten constant (K_m) and maximum reaction velocity (V_max) were obtained using eq. 1 (Eq. 1):

$$v_0=\frac{V_{\mathit{max}}+\left[S\right]}{K_m+\left[S\right]}$$ (1)

where [S] is the substrate concentration of TMB.

Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) and were carried out to explain the underlying kinetic differences in the oxidase-like activity between Mn-CDs and Mn-CDs@Pt NPs. The measurements were performed a CHI660D configuring with a conventional three-electrode system at room temperature. A bare or modified glassy carbon electrode (GCE, Φ = 3 mm) was employed as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl (saturated KCl) electrode as the reference electrode. CV curves were recorded in 0.1 M PBS (pH 7.4) in the absence and presence of TMB (1.0 mM) over a potential window from −0.2 V to 1.0 V at a scan rate of 100 mV s⁻¹. EIS measurements were performed in 0.1 M KCl containing 5.0 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1:1) as the redox probe, with a frequency range of 0.01 Hz to 100 kHz and an AC amplitude of 5 mV. The impedance spectrum was fitted using Zview software.

2.4. Confirmation of free radicals in TMB oxidation catalyzed by Mn-CDs@Pt nanozyme

The effect of dissolved oxygen, the ROS and free radicals produced during the catalytic reaction were investigated to explore the mechanisms of TMB oxidation by Mn-CDs@Pt nanozyme.

The effect of dissolved oxygen was evaluated by UV–vis characterization. Typically, 470 μL of acetate buffer (0.2 M, pH 4.5) and 20 μL of TMB (2.0 M) were mixed under O₂/N₂ flow. After that, 10 μL of Mn-CDs@Pt NPs suspension (1.5 mg mL⁻¹) was added into the above mixture under continuous O₂/N₂ bubbling, and the reaction was allowed to proceed for 10 min. Finally, the UV–vis curve was recorded and compared with the controlled group that performed under air atmosphere.

The ROS that generated during TMB oxidation catalyzed by Mn-CDs@Pt nanozyme were investigated using p-benzoquinone (PBQ), tryptophan (Trp) and isopropyl alcohol (IPA) as ROS scavengers (Yi et al., 2025). Briefly, 450 μL of acetate buffer (0.2 M, pH 4.5), 20 μL of TMB (2.0 M), and 20 μL of various concentrations of ROS scavengers (0, 1 and 10 mM) were mixed thoroughly, and the absorbance at 652 nm was measured after 10 min of reaction. The presence of O₂^•− in the reaction system was further confirmed by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping agent.

2.5. Detection of AA

The detection of AA was performed by following two procedures. First, 20 μL of GSH (0.6 mM, dissolved in 0.1 M PBS, pH 9.0) and 20 μL of AA with various concentrations (dissolved in 0.1 M PBS, pH 9.0) were mixed thoroughly, and the thiol-ene Michael addition was carried out at 50 °C for 1.5 h in the dark. Then, 20 μL of the above mixture was mixed thoroughly with 450 μL of acetate buffer (0.2 M, pH 4.5) and 20 μL of TMB (2 mM), followed by the addition of 10 μL of Mn-CDs@Pt nanozyme (1.5 mg mL⁻¹). The absorbance at 652 nm was measured after 10 min of chromogenic reaction (Scheme. 1C). At the same time, the photos of the above solutions were taken for RGB analysis.

2.6. Detection of AA in food samples

Food samples (bread, fried chips, and cookies) were bought from a local market and treated using the QuEChERS method. Typicaly, each sample (5.0 g) was dried at 90 °C for 10 h. Then, the dried sample was ground into a powder, followed by mixing 20 mL of n-hexane and shaking for 15 min to degrease. After that, 10 mL of water and 10 mL of acetonitrile were added, and the mixture was shaken for another 15 min. Subsequently, 4.0 g of anhydrous magnesium sulfate and 0.5 g of sodium chloride were added. After vigorous shaking for 10 min, the mixture was centrifuged at 10,000 rpm for 10 min to separate the organic and aqueous phases. The upper acetonitrile phase containing AA was collected and filtered through a 0.22 μm membrane. The obtained solution was stored at 4 °C for later use.

The detection of AA in these samples followed the procedures described in section 2.4.

3. Results and discussion

3.1. Characterizations of Mn-CDs and Mn-CDs@Pt NPs

The prepared CDs, Mn-CDs and Mn-CDs@Pt NPs were first characterized by UV–vis and fluorescence spectroscopy. The UV–vis spectra of Mn-CDs (Fig. S2A, red line) and CDs (Fig. S2A, black line) gave two peaks at proximately 270 and 350 nm. The former absorption band can be assigned to the π-π* transition of the aromatic C C and C − C in sp² hybrid domains (Cao et al., 2022; Mandani et al., 2015; Yang et al., 2018), and the latter to the n-π* transition of C O in Mn-CDs and CDs (Arroyave et al., 2021; Seven et al., 2019). A broad spectral continuum spanning from the visible to ultraviolet region was observed in the UV–vis spectrum of Mn-CDs@Pt NPs (Fig. S2 A, blue line). Furthermore, the broad absorption peak of Pt NPs in this range obscures the absorption peaks of Mn-CDs at 270 nm and 350 nm. This suggests that Pt⁴⁺ was completely reduced to Pt⁰ (Dauthal & Mukhopadhyay, 2018; Rashid et al., 2015) by Mn-CDs. The fluorescence spectra of Mn-CDs and Mn-CDs@Pt NPs are shown in Fig. S2B. Mn-CDs (Fig. S2B, black line) showed strong fluorescence emission at 420 nm upon excitation at an optimal wavelength of 360 nm (Fig. S3). However, the emission of Mn-CDs in Mn-CDs@Pt NPs disappeared. The quenched intensity of Mn-CDs indicates the successful preparation of Mn-CDs@Pt NPs.

The morphology of the prepared Mn-CDs and Mn-CDs@Pt NPs was characterized by TEM. The high-resolution TEM (HRTEM) image showed that the prepared Mn-CDs are spherical (Fig. 1A) with an average size of 1.39 ± 0.20 nm (Fig. 1B). The HRTEM image of Mn-CDs exhibited obvious lattices in the carbon cores, and the lattice spacing was measured to be 0.217 nm (Fig. 1A inset), corresponding to the (100) plane of graphitic carbon (Qiu et al., 2023). The HRTEM image of Mn-CDs@Pt NPs exhibited that the prepared nanoparticles possess spherical morphology (Fig. 1C) with an average size of 2.61 ± 0.66 nm (Fig. 1D). Furthermore, well-defined lattice spacing can be observed and measured to be approximately 0.228 nm, corresponding to (111) plane of Pt (W. Jiang et al., 2024) surface. This confirms the reduction of PtCl₆²⁻ by using Mn-CDs as a reducing agent. Moreover, the prepared Mn-CDs@Pt NPs can be well dispersed in water for months, demonstrating that Mn-CDs served as a stabilizer and prevent PtNPs from aggregation.

Fig. 1.

(A) TEM image of Mn-CDs. (B) Particle size distribution of Mn-CDs. (C) TEM image of Mn-CDs@Pt NPs. (D) Particle size distribution of Mn-CDs@Pt NPs.

The XPS characterization was performed to analyze the surface composition and chemical bonding of Mn-CDs and Mn-CDs@Pt NPs. The XPS spectrum of Mn-CDs (Fig. 2A, red line) showed the peaks of Mn 2p (640.86 eV), O 1 s (531.25 eV) and C 1 s (284.79 eV). This indicates that Mn-CDs contain Mn, O and C elements. Apart from the peaks of Mn 2p (642.68 eV), O 1 s (531.12 eV) and C 1 s (284.79 eV) observed for Mn-CDs@Pt NPs, a new peak was found at 71.42 eV corresponding to Pt 4f (Fig. 2A, black line). The deconvoluted Pt 4f spectrum of Mn-CDs@Pt NPs (Fig. 2B) showed four peaks: the two major peaks at 74.15 and 70.90 eV refers to Pt⁰ 4f_5/2 and Pt⁰ 4f_7/2 (Zhang et al., 2019), respectively; another two weak peaks at 76.50 and 72.30 eV can be assigned to Pt²⁺ 4f_5/2 and Pt²⁺ 4f_7/2 (Kappen et al., 2025), respectively. Furthermore, the percentages of Pt⁰ and Pt²⁺ were proximately 93.3% and 6.67%, respectively. These results indicate that PtCl₆²⁺ was reduced to Pt⁰ by Mn-CDs, forming Mn-CDs@Pt NPs. The XPS survey showed that the percentages of C, O and Mn in Mn-CDs were proximately 51.34%, 47.92% and 0.74% respectively, whereas in Mn-CDs@Pt NPs, C, O, Mn, and Pt were 82.45%, 16.79%, 0.25%, and 0.51%, respectively. The increase of C and decline of O in Mn-CDs@PtNPs may result from the oxygen-containing groups in CDs being involved in the reduction of PtCl₆²⁻ (Beygisangchin et al., 2025). The binding energies at 652.66 and 641.32 eV in Mn 2p spectrum for Mn-CDs (Fig. 3C, top line) were assigned to Mn 2p_1/2 and Mn 2p_3/2, and the peak at 646.94 eV was assigned to the satellite of Mn 2p_3/2. The binding energies of Mn 2p_1/2 and Mn 2p_3/2 for Mn-CDs@PtNPs were almost the same as those for Mn-CDs (Fig. 3C, lower line), suggesting that the valence of Mn was the same as that in Mn-CDs. The multiplet splitting (ΔE) between the two peaks in the Mn 3 s spectrum for Mn-CDs (Fig. 3D) was measured to be 5.10 eV. The average oxidation state (AOS) of Mn was calculated to be 3.21 according to the equation AOS = 8.956–1.126 × ΔE (M. Li et al., 2026). This suggests that Mn(III) is the main valence state in both Mn-CDs and Mn-CDs@Pt NPs. In the high-resolution C 1 s spectrum of Mn-CDs (Fig. 3E, top line), four peaks at 284.80, 286.38, 288.22, and 289.14 eV were assigned to C=C/C-C, C—O, C O, and O-C=O, respectively. The atomic percentage of C=C/C-C in Mn-CDs was 55.28% (Table S1), and the total atomic percentage of the oxygen-containing groups in Mn-CDs was 44.72% (Table S1). For the high-resolution C 1 s spectrum of Mn-CDs@PtNPs (Fig. 3E, lower line), only three peaks of C=C/C-C (284.76 eV), C—O (285.89 eV) and C O (287.51 eV) were observed, and the atomic percentages of C=C/C-C and oxygen-containing groups were 68.19% (Table S1) and 31.81% (Table S1), respectively. Compared to Mn-CDs, the increase in the atomic percentage of C=C/C-C and decrease in the atomic percentage of oxygen-containing groups for Mn-CDs@Pt NPs further demonstrate that the oxygen-containing groups in Mn-CDs participated in the reduction of PtCl₆²⁻. The O 1 s spectra (Fig. 3F) of Mn-CDs and Mn-CDs@Pt NPs can reveal which chemical bonds of the oxygen-containing groups were specifically involved in the reduction reaction. Three peaks of C O (531.25 eV), C-OH (532.75 eV) (D. Wang et al., 2026) and O-C=O/Mn-O (535.75 eV) (M. Li et al., 2026) were observed for Mn-CDs (Fig. 3F, top line), and the atomic percentages of the three chemical bonds were 80.00%, 7.78% and 12.22% (Table S1), respectively. Whereas only two peaks of C O (531.25 eV) with an atomic percentage of 97.23% (Table S1) and C-OH (532.75 eV) with an atomic percentage of 2.77% (Table S1) were found for Mn-CDs@Pt NPs. This indicates that hydroxyl oxygen or Mn—O were involved in the reduction of PtCl₆²⁻.

Fig. 2.

(A) XPS survey spectra of Mn-CDs and Mn-CDs@Pt NPs; (B) High-resolution XPS spectrum of Pt 4f for Mn-CDs@Pt NPs; (C, E, F) High-resolution XPS spectra of Mn 2p, C 1 s, and O 1 s for Mn-CDs and Mn-CDs@Pt NPs; (D) High-resolution XPS spectrum of Mn 3 s for Mn-CDs@Pt NPs.

Fig. 3.

(A) Time-dependent curve at the wavelength of 652 nm for various concentrations of TMB. (B) Michaelis-Menten equation curve for TMB catalyzed by Mn-CDs@Pt NPs. (C) The Lineweaver-Burk plot for TMB. (D) The UV–vis curves obtained in the atmosphere of O₂ (blue), air (black) and N₂ (red). (E) Effects of varying concentrations (0.5, 1.0, and 5.0 mM) of ROS scavengers (PBQ, IPA, and Trp) on the absorbance at 652 nm of the TMB-Mn-CDs@Pt system. Error bars represent the standard deviation of three independent measurements. (F) EPR spectrum for O₂^•− in Mn-CDs@Pt NPs using DMPO as the spin-trapping agent. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. Oxidase-like catalytic activity and mechanism of Mn-CDs@Pt NPs

The oxidase-mimic activity of Mn-CDs@Pt NPs was investigated using TMB as the chromogenic substrate. Fig. S4A shows the UV–vis curves obtained for different reaction solutions. When TMB was not catalyzed by nanozymes, no distinct absorption peak at 652 nm was observed. However, the absorption peak at 652 nm was observed for the reaction system of Mn-CDs@Pt NPs + TMB. This indicates that Mn-CDs@Pt NPs shows oxidase-like activity that can catalyzed TMB into oxTMB. Moreover, the absorption peak at 652 nm for different reaction systems was found to follow the order of Mn-CDs@Pt NPs + TMB > Mn-CDs + TMB > CDs + TMB. This demonstrates that Mn-CDs@Pt NPs shows the strongest oxidase-mimicking activity, followed by Mn-CDs, and CDs the weakest. It is worth noting that the improved activity of Mn-CDs over CDs can be attributed to Mn(III) doping (M. Li et al., 2026), which endows Mn-CDs with more active sites on their surface than CDs possess. The time-dependent curves at 652 nm were also recorded for different reaction systems, and the plot is shown in Fig. S4B. The reaction system of Mn-CDs@Pt NPs + TMB reached its maximum absorbance at 10 min, and V₀ was calculated to be 1.84 × 10⁻⁷ M s⁻¹. For the Mn-CDs + TMB and CDs + TMB systems, the absorbances at 652 nm at 10 min were far lower than that of the Mn-CDs@Pt NPs + TMB system, and the V₀ values were calculated to be 3.11× 10⁻⁸ and 3.21× 10⁻⁹ M s⁻¹, respectively. The V₀ value for Mn-CDs@Pt NPs + TMB system was 5.92 times higher than that of the Mn-CDs + TMB system, and 57.3 times than that of the CDs + TMB system. This further demonstrates that Mn-CDs@Pt NPs exhibits the highest oxidase-like activity among these nanomaterials. The excellent oxidase-like activity of Mn-CDs@Pt NPs can be attributed to two reasons. For one reason, Pt NPs possess outstanding enzyme activity, especially when supported by other nanomaterials (W. Jiang et al., 2024; Zeng et al., 2025). In our present work, Pt NPs were stabilized with Mn-CDs, which prevents their aggregation and thus maintains the high catalytic activity. For another, Mn-CDs themselves also exhibit good oxidase-like activity.

The EIS and CV measurements were performed to further elucidate the intrinsic electron transfer pathway underlying the synergistic effect between Mn-CDs@Pt NPs and Mn-CDs. As shown in Fig. S5A, only a weak electrocatalytic current toward TMB oxidation was observed on bare GCE (purple line). However, on the Mn-CDs/GCE (red dashed line), the oxidation of TMB (10 μM) started at a higher potential of 0.385 V and yielded a lower peak current of 1.24 μA at 0.539 V. This shows that Mn-CDs exhibit good catalytic activity toward TMB oxidation. Furthermore, the oxidation started at 0.281 V on Mn-CDs@Pt/GCE (Fig. S5A, green line), with a maximum peak current of 2.32 μA at 0.453 V. The lower onset potential, lower oxidation peak potential, and higher peak current observed on the Mn-CDs@Pt/GCE demonstrate that Mn-CDs@Pt NPs possess superior oxidation activity toward TMB oxidation compared to Mn-CDs.

The EIS results provide quantitative evidence for this enhanced electron transfer (Fig. S5B and Table S2). The charge transfer resistance (R_ct) of the bare GCE (Fig. S5B, red line) was measured to be 229.2 Ω. Upon modification with Mn-CDs (Fig. S5B, blue line), the R_ct significantly increased to 618.2 Ω, indicating substantial surface blockage. This increase is likely due to the abundant oxygen-containing functional groups on Mn-CDs (as evidenced by XPS), which may impede interfacial electron transfer. Remarkably, the incorporation of Pt NPs (Fig. S5B, black line) onto Mn-CDs significantly reduced the R_ct to 523.3 Ω, alongside a decrease in Warburg impedance (WR) from 2921 to 2441. This partial recovery in R_ct suggests that Mn-CDs@Pt NPs can facilitate electron transfer more efficiently than that of Mn-CDs. As previously mentioned, the oxygen-containing groups in Mn-CDs participate in the reduction of PtCl₆²⁻. The subsequent formation of metallic Pt NPs on Mn-CDs, along with the significant decrease in oxygen content, may synergistically contribute to the improved electron transfer. Consequently, this enhanced electron transfer is likely responsible for the improved oxidation activity of Mn-CDs@Pt NPs toward TMB.

Steady-state kinetic experiment using TMB as the substrate was performed under optimized condition (Fig. S6) to evaluate the oxidase-like activity of Mn-CDs@Pt NPs. Fig. 3A exhibits the time-dependent curves for various TMB concentrations recorded at the fixed wavelength of 652 nm. All the curves show that the absorbance at 652 nm increased over time until reaching a plateau. Furthermore, v₀ of the catalytic reaction increased with the concentration of TMB (C_TMB). The K_m and V_max values were calculated by analyzing the relationship between v₀ and C_TMB. The well fitted Michaelis-Menten equation curve (Fig. 3B) gave the K_m value of 0.0545 mM and V_max value of 3.42 × 10⁻⁷ M s⁻¹. In addition, the corresponding relationship between 1/v₀ and 1/C_TMB from the Lineweaver-Burk plot (Fig. 3C) can be written as $\frac{1}{v_0}=1.597\times {10}^{-2}\frac{1}{C_{\mathit{TMB}}}+2.932\times {10}^{-1}$ with a R² of 0.995, demonstrating a well linear relationship between 1/v₀ and 1/C_TMB. The K_m and V_max values for TMB by Mn-CDs@PtNPs were compared with other reported oxidase-like nanozymes, and the results are listed in Table 1. Results show that the prepared Mn-CDs@Pt nanozyme has lower K_m and higher V_max values than those of Pt NPs/C (X. Sun et al., 2025), Pt NPs/HOFs (Huang et al., 2025), and Pt—Ni NPs (W. Jiang et al., 2024), demonstrating its superior affinity toward TMB. Furthermore, the affinity of the prepared Mn-CDs@Pt NPs for TMB is comparable to that of Pt NPs/HCNs (W. Jiang et al., 2024) and Pt NCs (Jin et al., 2025), as its K_m and V_max values for TMB are close to those of Pt NPs/HCNs and Pt NCs.

Table 1.

Comparison of steady-state kinetic parameters of Mn-CDs@Pt and other Pt-based nanozymes.

Nanozyme	Km (mM)	Vmax (×10−8, M s−1)	Ref.
Pt-Ni NPs	0.629	20.2	(Jiang et al., 2024)
Pt NPs/HCNs	0.0570	25.2	(Zeng et al., 2025)
Pt NPs/C	0.303	4.18	(Sun et al., 2025)
Pt NPs/HOFs	0.126	1.19	(Huang et al., 2025)
Pt NCs	0.0518	26.0	(Jin et al., 2025)
Mn-CDs@Pt NPs	0.0545	34.2	This work

Note: HOFs, hydrogen-bonded organic frameworks; HCNs, hollow carbon nanospheres; NCs, nanoclusters.

The mechanism of the catalytic reaction by Mn-CDs@Pt nanozyme was then investigated. The role of dissolved O₂ was first investigated by recording the UV–vis curves under air, N₂ and O₂ atmosphere. As shown in Fig. 3D, the absorbance at 652 nm was significantly decreased after bubbling N₂ for 5 min. Compared to the absorbance obtained under the condition of air atmosphere, it decreased by 61.7%. Conversely, under an O₂-saturated atmosphere, the absorbance at 652 nm was markedly increased by 40.7% compared to that under air. The dramatic enhancements under O₂-saturated and significant decrease under N₂-saturated atmosphere clearly indicate that dissolved O₂ plays a critical role in the catalytic oxidation of TMB by Mn-CDs@Pt NPs, presumably as the electron acceptor.

To further quantitatively assess the possible ROS generated during the nanozyme-catalyzed reaction, various concentrations (0.5, 1.0, and 5.0 mM) of PBQ, IPA and Trp were used as ROS scavengers (Yi et al., 2025) for capturing superoxide anion (O₂^•−), hydroxyl radicals (·OH) and singlet oxygen (¹O₂), respectively. As displayed in Fig. 3E, the absorbance at 652 nm decreased in a concentration-dependent manner, with reductions of 45.50%, 47.46%, and 64.11% upon the addition of 0.5, 1.0, and 5.0 mM PBQ, respectively, compared with the control value. In contrast, the addition of IPA and Trp (even at the high concentration of 5.0 mM) resulted in only a negligible decrease in absorbance at 652 nm compared with the control value. These results demonstrate that O₂^•− is the predominant reactive oxygen intermediate involved in the oxidase catalysis process, with negligible contributions from ∙OH and ¹O₂.

The generation of O₂^•− was further proved by using electron spin resonance (ESR) spectroscopy. As shown in Fig. 3F, no peaks were observed for the control, while 6 characteristic peaks corresponding DMPO-OOH (Shi et al., 2025) were observed in presence of Mn-CDs@PtNPs, showing a characteristic signal of 1:1:1:1 (Yi et al., 2026; Yue et al., 2025). This confirms that O₂^•− was generated in the catalytic reaction. Considering the above results, it is reasonable to hypothesize that the prepared Mn-CDs@Pt NFs can effectively catalyze the dissolved oxygen into O₂^•− species (Eq. 2), which subsequently enhance the oxidation of the colorless TMB into the blue-colored oxTMB (Eq. 3 and 4). These proposed equations represent our current mechanistic interpretation based on the observed data.

$${\mathrm{Pt}}^0+{\mathrm{O}}_2\to {\mathrm{Pt}}^{2+}+{{\mathrm{O}}_2}^{•-}$$ (2)

$${{\mathrm{O}}_2}^{•-}+{\mathrm{H}}^{+}+\mathrm{TMB}\to \text{oxTMB}+{\mathrm{H}}_2\mathrm{O}$$ (3)

$${\mathrm{Pt}}^{2+}+\mathrm{TMB}\to \text{oxTMB}+{\mathrm{Pt}}^0$$ (4)

3.3. Detection mechanism of acrylamide

The detection of AA was based on the excellent oxidase-like activity of the prepared Mn-CDs@Pt NPs. The feasibility of sensing AA was investigated, and the related UV–vis curves are shown in Fig. S7A. When TMB was incubated with Mn-CDs@Pt NPs, the colorless TMB can be catalyzed into blue-colored oxTMB. Upon addition of GSH to the Mn-CDs@Pt NPs + TMB system, the oxTMB is reduced by GSH (Eq. 5) due to the strong reducing ability of GSH, leading to a decrease in the color intensity of the solution. However, after AA reacting with GSH through thiol-ene Michael addition (Eq. 6), the intensity at 652 nm of the sensing system can be recovered, and the color of the solution turns blue again. This confirms that the thiol-ene Michael addition between GSH and AA reduces the amount of free GSH in the reaction solution, thus increasing the amount of the blue-colored oxTMB in the chromogenic reaction system.

$$\mathrm{GSH}+\text{oxTMB}\to \mathrm{TMB}+\text{2GSSG}+{\text{2H}}^{+}$$ (5)

$${\mathrm{C}}_3{\mathrm{H}}_5\mathrm{NO}+{\mathrm{C}}_{10}{\mathrm{H}}_{17}{\mathrm{N}}_3{\mathrm{O}}_6\mathrm{S}\stackrel{\mathrm{pH}9.0,50°\mathrm{C},1.5\mathrm{h}}{\to }{\mathrm{C}}_3{\mathrm{H}}_{23}{\mathrm{N}}_3{\mathrm{O}}_7$$ (6)

3.4. Colorimetric detection of acrylamide

The parameters were optimized first for the colorimetric detection of AA, and the results are displayed in Fig. S7 B—D. Fig. S7 B shows the effect of color reaction time on the absorbance at 652 nm. The absorbance increased with the increase of reaction time, until it reached a maximum value at 10 min of reaction. However, the relative standard deviation (RSD) of the absorbance at 7 and 10 min was calculated to be 2.8%, showing only a slight difference between these two absorbance values. So, the optimal reaction time was set at 7 min. Fig. S7 C shows the effect of GSH concentration on the absorbance at 652 nm. The absorbance decreased dramatically with the increase of GSH concentration. Compared to the initial (without addition of GSH), the absorbance at 652 nm decreased by 72.8% when the added concentration of GSH was 0.3 mM. However, when the concentration of GSH increased from 0.3 mM to 1.0 mM, the absorbance at 652 nm only decreased slightly with RSD of 15.8%. The quantitative detection strategy for AA was based on the decrease in GSH concentration in the colorimetric system caused by the thiol-ene Michael addition between GSH and AA. A slight decrease in GSH concentration should lead to an increase in oxTMB (color restoration) in the colorimetric system. Therefore, according to the significance of the color change in the chromogenic system, the optimal GSH concentration was determined to be 0.3 mM. Fig. S7 D shows the effect of thiol-ene Michael addition time on the absorbance at 652 nm. The absorbance increased with the increase of reaction time, until it reached a maximum value at 2 h min of reaction. However, the relative standard deviation (RSD) of the absorbance at 1.5 and 2 h was calculated to be 4.2%, showing only a slight difference between these two absorbance values. Hence, the optimal thiol-ene Michael addition time was 1.5 h.

The colorimetric detection of AA was then conducted under optimized conditions. The absorbance at 652 nm increased as expected with the increasing concentrations of AA in the range from 0.05 to 5000 μM (Fig. 4A). The relationship between the absorbance at 652 nm and AA concentration is shown in Fig. 4B. As shown in the figure, a good linear relationship can be obtained in range of 0.05–5000 μM with a linear equation of Abs = 0.0849lgC_AA (μM) + 0.330 (R² = 0.992). The LOD of AA sensing was calculated to be 0.0371 μM (2.64 μg kg⁻¹, S/N = 3). The Mn-CDs@Pt-based colorimetric sensing system was compared with other nanozyme-based colorimetric sensors, and the results are listed in Table 2. It is evident that our sensor shows a broad linear range than other nanozyme-based sensors (Ge et al., 2025; Zheng et al., 2026; Zhuang et al., 2022). Furthermore, it showed a lower LOD than the Fe-PHS (0.27 μM) (F. Liu et al., 2023) AuNPs (0.16 μM) (Zhuang et al., 2022) and PC@Fe (Zheng et al., 2026)-based sensors. In addition, our sensor demonstrates comparable performance to the Cu-CN SANs (Ge et al., 2025) detection system in terms of LOD. Overall, these results confirm that the Mn-CDs@Pt-based colorimetric sensor performs excellent performances for AA detection.

Fig. 4.

(A) UV–vis spectra for various AA concentrations (0, 0.05, 0.5, 5, 50, 500, and 5000 μM). (B) Linear relationship between the absorbance at 652 nm and the AA concentration. (C) Selectivity for the detection of AA using the colorimetric sensing system. (D) Storage stability of the prepared Mn-CDs@Pt NPs. (E) Photos of the colorimetric reaction system containing various AA concentrations. (F) Linear relationship between B/G ratio and AA concentration.

Table 2.

Comparison in analytical performance of various nanozyme-based sensors for colorimetric detection of AA using GSH as inhibitor.

Nanozyme	Linear range (μM)	LOD (μM)	Ref.
PC@Fe	0.074–185	0.0740	(Zheng et al., 2026)
Cu-CN SANs	0.05–15	0.0154 0.146 (smartphone)	(Ge et al., 2025)
Au NPs	0.5–175	0.160	(Zhuang et al., 2022)
Fe-PHS	0.75–36	0.270	(F. Liu et al., 2023)
Mn-CDs@Pt NPs	0.05–5000 5–5000	0.0371 4.35 (smartphone)	This work

Note: PHS, polydopamine coordination hedgehog-like superstructure; Cu-CN SANs, Cu-based single-atom nanozyme; PC, phytate/carnosine.

The selectivity of the AA sensor was investigated using molecules with similar structures to AA, such as succinic acid (SA), L-asparagine (Asn), maleic acid (MaA), methacrylamide (MA), and caffeic acid (CA), as well as other common food components including glucose (Glu) and L-cysteine (Cys), as the interferents. As shown in Fig. 4C, the sensing system showed significant absorbance at 652 nm only for AA (500 μM). However, the absorbance for the interferents (each at 5 mM) were much lower than that of AA and were close to the absorbance of blank signal. This excellent selectivity is attributed to the thiol-ene Michael addition between GSH and AA, which prevents GSH from reducing oxTMB reduction to TMB. These results demonstrate that the Mn-CDs@Pt-based colorimetric sensor shows excellent selectivity toward AA.

The storage stability of Mn-CDs@Pt nanozyme was investigated by measuring the oxidase-like activity. As shown in Fig. 4D, after 25 days of storage, the absorbance at 652 nm barely changed, with a relative standard deviation (RSD) of 0.94% for the measured intensities. This demonstrates excellent storage stability of the prepared Mn-CDs@Pt nanozyme.

3.5. Smartphone-assisted detection of acrylamide

Finally, we used a smartphone to take pictures of the colorimetric reaction system containing various AA concentrations (Fig. 4E). The color of the reaction system was almost colorless when the concentration of AA was 0, and it became deeper as the AA concentration increased from 0.005 mM to 5 mM. Furthermore, the color of the reaction system barely changed when the AA concentration increased from 5 mM to 25 mM. After converting the color intensity into RGB (red, green, blue) values, a linear relationship between B/G ratio and the AA concentration was obtained in the range of 0.005–5 mM (Fig. 4F). The linear equation was B/G = 0.533C_AA (mM) + 1.280 (R² = 0.964), and the LOD was calculated to be 4.35 μM (S/N = 3). Although the LOD for the visual method was much higher than that for the UV–vis method, the smartphone-assisted visual detection of AA can realize on-site detection of AA at concentration as low as 5 μM.

3.6. Analysis of real samples

Thermally processed foods such as bread, chips and biscuits bought from the local market were selected as the real samples. The concentrations of AA in these samples were determined using our presented method, and the results were compared with those determined using the standard method of HPLC (Fig. S8). The results obtained are listed in Table S3. The AA concentrations in these samples were consistent with those measured by the standard method of HPLC. The credibility of the colorimetric sensor was also verified using standard addition method. As shown in the table, the recovery rates were in the range of 94.8–110.4%, demonstrating the reliability of the colorimetric sensor. These results indicate the promising potential for AA detection in real samples.

Despite these advantages, the proposed method has inherent limitations, such as its dependence on light conditions for RGB analysis, the critical time-window of the GSH-AA reaction, and its limitation to screening-level sensitivity compared to quantitative capability of sophisticated lab instruments (such as HPLC or LC-MS). Therefore, future work will focus on integrating separation modules and standardizing the detection environment to further enhance its robustness and practical applicability in complex food matrices.

4. Conclusions

In conclusion, Mn-CDs@Pt NPs were prepared by using Mn-CDs as the reducing agent. The prepared Mn-CDs@Pt NPs showed good stability (remaining stable for 25 days) and excellent oxidase-like activity toward the oxidation of TMB (K_m: 0.0545 mM, V_max: 3.42 × 10⁻⁷ M s⁻¹), making them suitable for the fabrication of a colorimetric sensing interface for AA. The colorimetric assay showed high sensitivity (LODs: 0.0371 μM for UV–vis and 4.35 μM for smartphone-assisted visual detection) and acceptable rapidity (within 1.5 h). Furthermore, it demonstrated excellent reliability, as the results for AA detection were consistent with those obtained by HPLC, enabling its application to the detection of AA in real samples.

CRediT authorship contribution statement

Jiaxuan Tang: Writing – original draft, Investigation, Formal analysis. Wenru Wang: Writing – original draft, Investigation. Weina Yu: Investigation, Data curation. Qiuran Wang: Investigation, Funding acquisition. Yunxuan Li: Investigation. Conglin Feng: Investigation. Jianhang Yang: Investigation. Yifei Pan: Investigation, Funding acquisition. Xiaodong Cao: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Shudong He: Resources, Data curation. Yuhan Sun: Resources, Funding acquisition. Yongkang Ye: Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Funding acquisition. Haisong Zheng: Writing – review & editing, Resources, Methodology, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material.

Data availability

Data will be made available on request.