Ultrathin plasmonic PtCu nanosheets as an efficient peroxidase-like nanozyme for colorimetric detection of H₂O₂

Abstract

Bimetallic PtCu nanosheets were synthesized as a nanozyme with remarkable peroxidase-like catalytic activity for colorimetric detection of H₂O₂ in milk. In an analytical application, they demonstrated a wide linear range (0.001–5 mM) and a low limit of detection (0.58 µM), as well as a good anti-interference performance.

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Hydrogen peroxide (H₂O₂) is a strong oxidant and is used in the food industry as a bleaching agent and disinfectant.¹ It is often utilized as a preservative to prevent spoilage in foods such as milk, cheeses, and fresh-cut fruits.² H₂O₂ as an additive can extend the shelf life of foods by reducing the entry of pathogens and killing harmful microorganisms.³ However, excessive levels of residual H₂O₂ in food can cause an imbalance in the cellular redox environment of human body, which in turn can lead to gastric disorders, cardiovascular diseases, depression, diabetes, Alzheimer's disease, and even cancers.⁴

The food and drug administration (FDA) of the United States stipulates that the maximum residual limit of H₂O₂ in finished packaged food is 0.5 ppm.⁵ The maximum level of H₂O₂ allowed in food according to the Chinese National Standard (GB5009.226-2016) is 3 mg kg⁻¹.⁶ Unregulated uses of H₂O₂ in the food industry always result in substantial food safety problems. Therefore, the development of a cost-effective tool to sensitively, selectively, and rapidly detect H₂O₂ levels in food is urgently needed.

Nanozyme-enabled H₂O₂ biosensors possess the significant advantages of peroxidase-like nanozymes, such as high surface-to-volume ratio, catalytic stability, low production cost, and performance tunability.^7–9 Peroxidase-like nanozymes can catalyze the H₂O₂ oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) into a typical blue product for colorimetric detection of H₂O₂.^10–12 Various nanomaterials have been explored for the construction of H₂O₂ biosensors, such as metal,¹³ metal oxide,¹⁴ metal sulfide,¹⁵ carbon nanomaterial,¹⁶ and metal organic framework.¹⁷

Noble monometallic nanomaterials have been reported as promising alternatives of natural enzymes to fabricate colorimetric biosensors for the detection of H₂O₂.^4,18–20 However, noble monometallic nanomaterials demonstrate relatively low peroxidase-like activity. An alloy strategy can create abundant active sites for catalytic reactions due to the surface and lattice strain in the alloy nanostructures.^13,21–26 Compared with single noble-metal nanostructures, noble-metal alloy nanostructures exhibit enhanced peroxidase-like activity because of their coordination effect, and therefore, they are promising as a peroxidase mimic for constructing highly sensitive nanozyme-enabled H₂O₂ biosensors (Scheme 1).^13,20,21,25

Scheme 1 Solvothermal synthesis of ultrathin PtCu nanosheets as a peroxidase-like nanozyme to construct a colorimetric biosensor for the detection of H₂O₂ in milk.

In this work, two-dimensional PtCu nanosheets were prepared by a solvothermal method with copper(II) acetylacetonate and platinum(II) acetylacetonate as precursors. PtCu nanosheets can rapidly catalyze the H₂O₂ oxidation of TMB with a Michaelis constant (K_m) and maximum reaction rate (V_max) of 0.44 mM and 10.21 µM min⁻¹, respectively. Based on the intrinsic peroxidase-like activity of PtCu nanosheets, a visual colorimetric biosensor for sensitive and accurate detection of H₂O₂ was constructed, which was able to function in the high-performance analysis of H₂O₂ in milk, indicating that PtCu nanosheets can be a promising nanozyme to fabricate a prospective colorimetric biosensor for the detection of H₂O₂ in food.

As shown from the transmission electron microscopy (TEM) images in Fig. 1a and b and their corresponding lateral size distribution in Fig. S1, our prepared PtCu nanosheets display a lateral size distribution of approximately 8 nm, with two-dimensional morphology. The high-resolution TEM (HRTEM) images in Fig. 1c and d show crystal lattices with an interplanar spacing of 0.219 nm, which is ascribed to the (111) plane of the PtCu alloy nanostructure.²⁷ The energy-dispersive X-ray spectroscopy (EDS) elemental mapping images in Fig. 1e–h confirm the compositions of Pt and Cu in the product. The X-ray diffraction (XRD) spectrum in Fig. 2a shows typical diffraction peaks of 40.5 °C and 47.8 °C, which correspond to the (111) and (200) planes of the PtCu alloy nanostructure, respectively.^27,28

Fig. 1 (a and b) TEM, (c and d) HRTEM, and (e–h) corresponding EDS mapping images of PtCu nanosheets.

Fig. 2 (a) XRD and (b) survey XPS spectra of PtCu nanosheets. High-resolution XPS spectra of (c) Pt 4f and (d) Cu 2p in PtCu nanosheets.

The survey X-ray photoelectron spectroscopy (XPS) spectrum in Fig. 2b presents binding energy peaks of Pt 4f and Cu 2p, confirming the elemental compositions of the formed alloy nanostructure. The high-resolution XPS spectra of Pt 4f (Fig. 2c) and Cu 2p (Fig. 2d) show the binding energies of Pt 4f_7/2 (70.7 eV) and Pt 4f_5/2 (74.1 eV) of zero-valent Pt, and Cu 2p_3/2 (931.8 eV) and Cu 2p_1/2 (951.3 eV) of zero-valent Cu, further proving the formation of PtCu alloy nanosheets.²⁹

To investigate the intrinsic peroxidase-like activity of the PtCu nanosheets, experiments were performed through the catalytic oxidation reaction of TMB in the presence of H₂O₂ and PtCu nanosheets. As shown in Fig. 3a, a characteristic absorption peak ascribed to oxidized TMB appears at 652 nm in the reaction system containing TMB, H₂O₂, and PtCu nanosheets. The blue color of the reaction system was quickly observed after PtCu nanosheets were added to a solution containing TMB and H₂O₂. However, the reaction systems of PtCu nanosheets, PtCu nanosheets + TMB, and TMB + H₂O₂ showed no evident absorption at 652 nm with color changes. This indicates that the PtCu nanosheets can catalyze the oxidation of TMB in the presence of H₂O₂, confirming the peroxidase-like catalytic activity of PtCu nanosheets.

Fig. 3 (a) UV-Vis-NIR absorption spectra of oxidized TMB derived from catalytic oxidation of H₂O₂ in the presence of PtCu nanosheets. The influences of (b) pH values, (c) concentrations of PtCu nanosheets, and (d) reaction temperatures on the catalytic H₂O₂ oxidation of TMB.

The influences of pH values, concentrations of PtCu nanosheets, and reaction temperatures on the peroxidase-like catalytic reaction of PtCu nanosheets were further explored. As shown in Fig. 3b–d, the catalytic activity of PtCu nanosheets increases with the rise of pH values from 3 to 4, and decreases after the pH value is greater than 4.0. The catalytic reaction rate increases with increasing concentration of PtCu nanosheets, and reaches the maximum after the concentration of PtCu nanosheets is greater than 10 µg mL⁻¹. The catalytic activity of PtCu nanosheets increases as the temperature rises from 20 to 30 °C and is inhibited when the incubation temperature exceeds 30 °C. The experimental results in Fig. 3b–d confirm that the optimum conditions of the catalytic reaction are the pH value of 4.0, 10 µg mL⁻¹ PtCu nanosheets, and a reaction temperature of 30 °C.

The steady-state kinetics of the peroxidase-like catalytic reaction were studied by changing the concentration of H₂O₂ or TMB while keeping the other constant. The experimental results were measured by a UV-Vis spectrophotometer at selected time intervals to obtain the typical Michaelis–Menten curves for H₂O₂ or TMB. Fig. 4 shows that the peroxidase-like catalytic reaction of PtCu nanosheets towards H₂O₂ and TMB follows the Michaelis–Menten mechanism.

Fig. 4 (a) The velocity (ν) versus H₂O₂ concentrations. (b) Double reciprocal plots of ν versus H₂O₂ concentrations. (c) The ν versus TMB concentrations. (d) Double reciprocal plots of ν versus TMB concentrations.

As shown in Fig. 4a and b, the K_m and V_max for H₂O₂ substrate were calculated to be 26.94 mM and 10.18 µM min⁻¹, respectively, according to the Lineweaver–Burk plot. Fig. 4c and d demonstrate that the K_m and V_max for TMB substrate are 0.44 mM and 10.21 µM min⁻¹, respectively, which are comparable to those of natural horseradish peroxidase (HRP). Moreover, PtCu nanosheets present a lower K_m value and higher V_max value than other reported nanozymes in Table S1,^{20,21,23,26,30–33} suggesting the increased affinity and peroxidase-like catalytic activity of PtCu nanosheets. It is indicated that PtCu nanosheets with excellent peroxidase-like activity can serve as a promising alternative to HRP in the biosensing field.

A colorimetric biosensor based on the peroxidase-like activity of PtCu nanosheets was constructed to detect H₂O₂. Fig. 5a and b show that the absorbance of oxidized TMB at 652 nm is linear with H₂O₂ concentrations from 0.001 to 5 mM. The limit of detection (LOD) of the PtCu nanosheet-enabled colorimetric biosensor was calculated to be 0.58 µM according to the formula of LOD = 3s/k, where s and k denote the relative standard deviation of parallel controlled measurements and the slope of the linear calibration plots, respectively. In this formula, s = 0.34 × 10⁻⁴, k = 1.8601 × 10⁻⁴, and LOD = 3s/k = 0.58 µM. Compared with the biosensors based on other nanozymes such as Fe₃O₄-Ag,³⁴ Pt-Pd/MoS₂,²⁵ F-MoS₂-FePt NCs,²⁴ NiV₂O₆,³³ PAnFc/Pt foil,³⁵ PAA(DS)SPCE,¹⁴ and H@M,³⁶ there is a wider linear range and lower LOD for the PtCu nanosheet-enabled colorimetric biosensor (Table S2).

Fig. 5 (a) Absorbance of oxidized TMB at 652 nm by catalytic reaction of PtCu nanosheets with different concentrations of H₂O₂ and (b) its corresponding calibration curve. (c) The selectivity and (d) reproducibility of H₂O₂ detection by the PtCu nanosheet-enabled colorimetric biosensor.

To investigate the selectivity of the PtCu nanosheet-enabled colorimetric biosensor for H₂O₂ detection, arginine, cysteine, Mg²⁺, Zn²⁺, K⁺, Na⁺, SO₄²⁻, glucose, sucrose, lactose, and citric acid were used as controls for H₂O₂ determination experiments. As shown in Fig. 5c, the impact level of all interfering agents against H₂O₂ detection is less than 14%, suggesting that there is high selectivity of the PtCu nanosheet-enabled colorimetric biosensor for H₂O₂ detection.

The stability of our constructed colorimetric biosensor was measured by detecting its peroxidase-like catalytic activity after different storage times (5, 10, 20, 30, 40, 50, and 60 days). The experimental results in Fig. 5d show that the peroxidase-like catalytic activity of the PtCu nanosheet-enabled colorimetric biosensor remains at 95% over 60 days. All the above analytical results demonstrate that PtCu nanosheets with remarkable peroxidase-like catalytic activity can offer a sensitive, selective, and convenient colorimetric sensing strategy for H₂O₂ detection.

It is well known that H₂O₂ is often used as a stabilizer or preservative agent in commercial milk. Because residual H₂O₂ in milk may be harmful to human health, it is of significance to accurately detect H₂O₂ in milk. Encouraged by the satisfactory analytical performance of the PtCu nanosheet-enabled colorimetric biosensor, we used our proposed biosensor to determine the content of H₂O₂ in sour milk. Experiments were carried out by adding different concentrations of H₂O₂ to the pretreated milk sample, and then, the mixed sample was added to the reaction system containing the PtCu nanosheets and TMB.

As shown in Table 1, the content of H₂O₂ in 20-fold diluted sour milk is determined to be 17.128 µM according to the linear equation, and the concentration of H₂O₂ in the sour milk is calculated to be 342.56 µM, which is nearly that of the measured result (340.98 µM) by the iodometric standard method,³⁷ indicating the accuracy of the PtCu nanosheet-enabled colorimetric biosensor. The recovery for detecting H₂O₂ in three milk samples ranges from 96.8% to 103.0%, with a relative standard deviation (RSD) of 3.25%, which indicates that our constructed biosensor is promising for detecting H₂O₂ in real samples.

Table 1 Detection of H₂O₂ in sour milk by the PtCu nanosheet-enabled colorimetric biosensor (n = 3)

Added H2O2 (µM)	Found H2O2 (µM)	RSD (%)	Recovery (%)
0	<1	0	0
5	4.838	5.6	96.8
10	10.304	6.5	103.0
20	19.641	3.1	98.2
Sour milk diluted 20-fold	17.128	—	—

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In summary, PtCu nanosheets are prepared as a peroxidase-like mimic to construct a sensitive and convenient colorimetric biosensor for the detection of H₂O₂. The experimental results show that PtCu nanosheets exhibit remarkable peroxidase-like catalytic activity and can efficiently catalyze the oxidation reaction of TMB in the presence of H₂O₂. The PtCu nanosheet-enabled colorimetric biosensor demonstrates a satisfactory analytical performance with a wide linear range (0.001–5 mM) and a low limit of detection (0.58 µM), as well as a good anti-interference performance, which can be applied to accurately detect H₂O₂ in milk samples. Our work facilitates the practical applications of PtCu nanosheets in food additive detection.

Author contributions

Z. Cao, F. Saleem, Z. Luo, and Y. Zhang conceived the project, designed the studies, and analyzed the results. Z. Cao, F. Mu, Q. Shang, Y. Chen, P. Zhang, F. Chen, and C. Shen carried out the experiments. Z. Cao, Z. Luo, and Y. Zhang wrote the manuscript. F. Saleem, Z. Luo, and Y. Zhang supervised and revised the project.

Conflicts of interest

There are no conflicts to declare.

Data availability

All relevant data are available from the corresponding author upon reasonable request.

Supplementary information (SI): materials, methods, characterizations. See DOI: https://doi.org/10.1039/d5ay01909e.

Acknowledgements

We acknowledge the National Natural Science Foundation of China (22275096, 62288102, 52002396, 22473060), the Natural Science Foundation of Jiangsu Province-Major Project (BK20212012), the Natural Science Key Fund for Universities in Jiangsu Province (22KJA430007), the Key Research and Development Program of Jiangsu (BE2018732), the China Postdoctoral Science Foundation (2022M711691), and the General Project of Natural Science Research in Colleges and Universities of Jiangsu Province (22KJB350001).

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