Mechanistic and kinetic insights into size-dependent activity in ultra-small Pt/CNTs nanozymes during antibacterial process

2.1 Materials

Raw multi-walled carbon nanotubes (CNTs) with a diameter of 5–15 nm and a length of 0.5–2 μm were purchased from J&K Scientific ltd. (China). Trimethyl(methylcyclopentadienyl)platinum (IV) (MeCpPtMe₃), dimethyl sulfoxide (DMSO), hydrogen peroxide (H₂O₂), 3,3′,5,5′-Tetramethylbenzidine (TMB) and the other chemicals were all obtained from Sigma-Aldrich.

2.2 Synthesis of cPt/CNTs nanozymes

Firstly, the raw CNTs were pretreated with HNO₃ in a 110 °C oil bath for 4 h to remove amorphous carbon and open the CNTs ends, and then washed with deionized water until there was no further change in pH (pH ≈ 7). After that, the samples were further washed with absolute ethanol and then filtered. Finally, the functionalized CNTs were dried overnight in an oven at 100 °C.

Before ALD, the functionalized CNTs were dispersed in ethanol by ultrasonic stirring, dropped onto a quartz wafer, and dried in air. Pt nanoparticles were deposited onto CNTs using a hot-wall, closed-chamber ALD reactor and the prepared nanozymes were named cPt/CNTs (c = 5, 10, 20, 30, 50 and 70). Then, the size of Pt nanoparticles can be precisely controlled by tuning the cycle number (c) of Pt ALD. The deposition temperature for Pt was 270 °C and MeCpPtMe₃ was kept at 60 °C. Nitrogen was used as carrier and purge gas.

2.3 Characterization

The XRD patterns were collected using a Bruker D8 Advance X-ray diffractometer, whose Cu Kα radiation (λ = 1.540 Å) is in the 2θ range from 5^° to 90^°. Pt loading in all nanozymes was determined by an inductively coupled plasma atomic emission spectrometer (ICP-AES, Thermo ICAP 6300). Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were taken with a JEOL-2100F microscope. X-ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo ESCALAB 250 xi, which uses Al-Kα line as the radiation source.

2.4 POD-like activity assay

The POD-like activity of cPt/CNTs nanozymes (2 μmol of Pt) was characterized by oxidation of TMB (1 mM) in the presence of H₂O₂ (20 mM). The experiment was carried out in 0.2 M sodium acetate buffer (NaAc-HAc, pH = 3.6), and the absorbance at 652 nm was measured with a spectrophotometer.

The POD-like activity of cPt/CNTs nanozymes was quantitatively characterized by the standard method established in the literature. (Jiang et al., 2018) In short, using 0.5 mM TMB as a colorimetric substrate, various amounts (5 to 30 µg/mL) of cPt/CNTs nanozymes were added at 37 °C with 40 mM H₂O₂ in 0.2 M NaAc-HAc buffer (pH = 3.6), and the colorimetric response at 652 nm was measured by absorbance every 10 s for up to 600 s. Plot the relationship between absorbance at 652 nm and reaction time to obtain a reaction-time curve. Calculate the specific activity (SA) using the following equation: a_nanozyme = V/(ε × l)×(ΔA/Δt)/[n], where ananozyme is the SA expressed in units per mole (U mol⁻¹) of nanozyme; V is the total volume of reaction system (μL); ε is the molar absorption coefficient of TMB (39000 M⁻¹cm⁻¹); l is the length of light propagation path in the cuvette (cm); A is the absorbance after subtracting the blank value; and ΔA/Δt is the initial change rate of absorbance at 652 nm min⁻¹; [n] is the mole of Pt atom.

2.5 Detection of hydroxyl radical (•OH)

The generation of •OH was evaluated by electron spin resonance (ESR) spectrometer using DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin-trapping adduct. First, 0.1 mL of cPt/CNTs (c = 5, 10, 20, 30, 50, and 70) nanozymes were dispersed in pH 4.5 NaAc buffer (0.2 M). Then, 20 mM of DMPO and 1 ML H₂O₂ were added to the mixture solution, respectively. The mixture was reacted at room temperature and air for 10 min, the solutions were then aspirated into quartz capillaries for ESR analysis. (Meng et al., 2022).

2.6 Steady-state kinetic assay

The steady-state kinetic assay of cPt/CNTs nanozymes was determined at room temperature. (Jiang et al., 2018) 20 μg/mL of nanozyme and 1 M H₂O₂ were added into 0.2 M NaAc-HAc buffer (pH = 3.6) and mix well, then various concentrations (0.2 to 1.7 mM) of TMB solution were added, and record the initial rate of change of absorbance at 652 nm. The rate of increase in absorbance was converted to the substrate concentration in micromoles per minute (that is, the catalytic reaction rate, ν) and plotted against substrate concentration to generate a Michaelis-Menten curve. Kinetic constants ν_max and K_m were calculated by fitting the reaction rate values and substrate concentrations to the Michaelis-Menten equation as follows: ν=(ν_max × [S])/(K_m + [S]), where ν is the initial reaction rate, νmax is the maximal reaction rate. [S] is the concentration of the substrate and K_m is the Michaelis constant. The catalytic constant (k_cat) was calculated using the following equation: k_cat = ν_max/[E], where k_cat is the rate constant, which defines the maximum number of substrate molecules converted into products per unit time. [E] is the nanozyme concentration (M).

2.7 Antibacterial effect of Pt/CNTs nanozymes

The antibacterial effect of the Pt/CNTs nanozymes was determined via the number of colony forming units (CFU) using the plate counting method. (Wei et al., 2021; Weng et al., 2022) Antibacterial experiments were performed in PBS to evaluate the effect of cPt/CNTs nanozymes on gram-negative bacteria (Escherichia coli; E. coli) and gram-positive bacteria (Staphylococcus aureus, S. aureus). Add cPt/CNTs nanozymes and H₂O₂ (10 mM) to 1 mL bacterial suspension (1 × 10⁶ CFU·mL⁻¹). The final concentration of cPt/CNTs nanozymes and H₂O₂ is 200 μg·mL⁻¹ and 0.2 mM, respectively. Then continue the incubation at 37 °C for 2 h. After dilution of the bacterial solution, take 100 μL and spread over the surface of the Luria-Bertani (LB) medium, after incubating at 37 °C for 24 h, measure the optical density of the suspension at 652 nm using a microplate reader (MQX 200) and finally calculate the antibacterial rate.

The paper-disk diffusion method was further employed to assay the antibacterial activities of cPt/CNTs nanozymes on E. coli and S. aureus. (Xia et al., 2018; Yang et al., 2019) The medium used for growing and maintaining the bacterial liquid cultures was LB medium, and a solid medium was obtained by adding 2 % agar into the liquid medium. 100 μg of as-prepared samples were uniformly dispersed on the filter paper discs (0.5 cm in diameter). The diameters of the inhibition zones were measured after incubation at 37 °C for 24 h.

3.1 Preparation and characterization

The schematic preparation process of Pt/CNTs nanozymes via ALD is displayed in Fig. 1A. The section diagram of the preparation process is shown in Figure S1. First of all, with CNTs as the support, a certain cycle number (c₁) of Pt is deposited on its surface by ALD and named c₁Pt/CNTs nanozyme. Then the size of Pt nanoparticles can be accurately controlled by tuning the cycle number (c₂) of Pt ALD, that is, c₂Pt/CNTs nanozyme. To characterize the morphology of the as-deposited cPt/CNTs (c = 5, 10, 20, 30, 50, and 70) nanozymes, TEM measurements were conducted, and the results are depicted in Fig. 1B-1G. For the sample of 5Pt/CNTs, the Pt nanoparticles on the surface of the CNTs are so small that they are barely visible (as shown in red circles), which is due to the relatively few cycles of Pt ALD, resulting in a lower load rate of Pt on its surface. As the cycle number of Pt ALD increases, the size of Pt nanoparticles gradually becomes larger and uniformly distribute on the surface of CNTs. The mean particle sizes over cPt/CNTs nanozymes were counted according to the TEM images to more clearly characterize the size of Pt nanoparticles. As shown in Figure S2, the mean particle sizes of the six nanozymes are 0.55 nm, 0.82 nm, 1.42 nm, 1.69 nm, 2.05 nm and 2.81 nm, respectively. The HRTEM image highlights the well-defined lattice spacing of ∼ 0.223 nm for Pt nanoparticles (Figure S3A), corresponding to the Pt(1 1 1) planes. (Zhang et al., 2019; Ramachandran et al., 2016) Moreover, based on ICP-AES measurements, the Pt loadings of cPt/CNTs (c = 5, 10, 20, 30, 50, and 70) nanozymes are 0.19 wt%, 0.34 wt%, 1.27 wt%, 1.61 wt%, 2.75 wt%, and 3.92 wt%, respectively (Table S1). These results demonstrate the superiority of ALD and corroborate that ALD can be used as a novel way to precisely synthesize Pt-based nanozymes with ultrasmall and tunable size.

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Fig. 1

Synthesis and structural characterizations of Pt/CNTs nanozymes. (A) Illustration of the preparation process of Pt/CNTs nanozymes. (B-G) TEM images of 5Pt/CNTs, 10Pt/CNTs, 20Pt/CNTs, 30Pt/CNTs, 50Pt/CNTs and 70Pt/CNTs, respectively. Note: red circles refer to Pt nanoparticles. (H, I) XRD patterns and XPS survey spectra of cPt/CNTs nanozymes.

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The X-ray diffraction (XRD) was further conducted to reveal the chemical structure of nanozymes, and the spectra of cPt/CNTs are presented in Fig. 1H. The XRD patterns of 5Pt/CNTs, 10Pt/CNTs, 20Pt/CNTs, 30Pt/CNTs, 50Pt/CNTs and 70Pt/CNTs exhibit three strong diffraction peaks at around 25.9^°, 42.8^°, and 52.1^°, which can be assigned to the C(0 0 2), C(1 0 1) and C(0 0 4) planes of the graphite structure of CNTs (PDF#75–1621), respectively. (Li et al., 2018; Xing et al., 2022) However, no characteristic peak of Pt could be observed, probably because its high dispersion and/or low content of Pt nanoparticles. Moreover, the XPS measurement was further conducted to investigate the surface elemental composition of cPt/CNTs nanozymes. Fig. 1I shows the presence of carbon and oxygen atoms in the XPS survey spectra of all the cPt/CNTs nanozymes. When the cycle number of Pt ALD is less than 10, no peak of Pt atom is observed in the XPS survey spectra. Still, the peak of Pt atom begins to appear when the cycle number of Pt ALD increases to 20, which may be due to the low content of Pt and/or the relatively high detection limit of XPS instruments, which is consistent with the above XRD results. The above results demonstrate that Pt/CNTs nanozymes are successfully prepared by ALD, and ALD can be used as a novel way to accurately synthesize ultra-small size and highly dispersed nanozymes.

3.2 Size-dependent POD-like activities of Pt/CNTs nanozymes

Pt nanoparticles with intrinsic POD-like catalytic activity have attracted significant interest due to their ability to replace specific POD enzymes in POD-based applications. (Liu et al., 2018; Ge et al., 2018; Chen et al., 2021) We evaluated the POD-like activities of the cPt/CNTs nanozymes with different sizes of Pt through the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB, a typical POD substrate (Jiang et al., 2018) by H₂O₂ as a model catalytic reaction. As shown in Fig. 2A, the cPt/CNTs nanozymes rapidly catalyzed the oxidation of TMB in the presence of H₂O₂, producing a blue-colored oxidation product with an absorbance maximum at 652 nm. In contrast, bare CNTs, without Pt nanoparticles, alone did not lead to a color change. Moreover, the 30Pt/CNTs nanozyme has the highest catalytic activity of TMB oxidation by H₂O₂ in all cPt/CNTs nanozymes. Then, we combined the absorbance values of the cPt/CNTs nanozyme at 652 nm with the size of the Pt nanoparticles, as shown in Fig. 2B, it is found that the absorbance value at 652 nm of Pt nanozymes exhibits a volcanic relationship with the size of Pt nanoparticles. The Pt particles with a size of 1.69 nm (30Pt/CNTs nanozyme) present the highest absorbance value. The nanozyme activity standardization method was introduced to quantify the catalytic activities. The SA is defined as activity units (one nanozyme activity unit (U) is defined as the amount of nanozyme that catalytically produces 1 μmol of product per minute.) of per mole nanozyme. (Jiang et al., 2018) The reaction-time curves were obtained by plotting the absorbance at 652 nm against the reaction time (Figure S3B). As depicted in the inset of Figure S3B, for initial 60 s of the reaction, the nanozyme produces product at an initial rate that is approximately linear to the reaction time. The SA values of 5Pt/CNTs, 10Pt/CNTs, 20Pt/CNTs, 30Pt/CNTs, 50Pt/CNTs, and 70Pt/CNTs were calculated as 0.23 U μmol⁻¹, 1.03 U μmol⁻¹, 4.95 U μmol⁻¹, 5.58 U μmol⁻¹, 3.64 U μmol⁻¹, and 1.20 U μmol⁻¹, respectively, using the nanozyme activity standardization method (Figure S4 and Table S2). Then, plotting the SA values of the cPt/CNTs nanozymes against the size of the Pt nanoparticles, as shown in Fig. 2C, it is found that the SA of cPt/CNTs nanozymes also exhibits a volcanic relationship with the size of Pt nanoparticles, and the Pt particles with a size of 1.69 nm (30Pt/CNTs nanozyme) present the highest specific catalytic activity. To further verify the POD-like activity of cPt/CNTs nanozymes, the generated •OH was detected by ESR spectroscopy (Fig. 2D). (Meng et al., 2022) The ESR signal intensity of •OH in cPt/CNTs nanozymes also exhibits a volcanic relationship with the size of Pt nanoparticles, and the 30Pt/CNTs nanozyme present the highest intensity, confirming the presence of •OH, which demonstrates that the Pt/CNTs nanozyme is an efficient POD-like catalyst. The above experimental results indicate that cPt/CNTs nanozymes fabricated by ALD have a size-dependent activity in POD-like catalytic reaction, and the POD-like activity can be optimized by regulating the particle size of Pt by ALD.

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Fig. 2

POD-like property of the cPt/CNTs nanozymes. (A) UV–vis spectroscopy of cPt/CNTs (c = 0, 5, 10, 20, 30, 50, and 70) nanozymes (the same molar amount of Pt). Inserted images (tubes) represent the visual color changes of TMB. (B) The absorption value of TMB at 652 nm compared to different sizes of Pt nanoparticles. (C) The SA of cPt/CNTs nanozymes at 652 nm compared to different sizes of Pt nanoparticles. (D) DMPO spin-trapping ESR spectra of the ultrasmall TA-Ag nanozyme for H₂O₂.

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3.3 Mechanistic and kinetic insights into size-dependent POD-like activity in Pt/CNTs nanozymes

The steady-state kinetic of oxidation of TMB with H₂O₂ was conducted for cPt/CNTs (c = 5, 10, 20, 30, 50, and 70) to determine the enzyme kinetic parameters to understand the catalytic mechanism of size-dependent POD-like activity. (Jiang et al., 2018) Clearly, within the suitable range of TMB and H₂O₂, typical Michaelis-Menten curves are received for Pt nanoparticles with the sizes of 0.55 nm, 0.82 nm, 1.42 nm, 1.69 nm, 2.05 nm, and 2.81 nm, respectively (Figs. 3 and 4). The data were fitted based on the Michaelis-Menten equation, and the enzymatic parameters were determined with the typical double-reciprocal Lineweaver-Burk plots (Inserted images in Figs. 3 and 4). (Lineweaver and Burk, 1934) The calculated enzyme kinetic parameters are listed in Table S3 and Table S4, which show that 5Pt/CNTs nanozyme displays a smaller K_m value for TMB and H₂O₂, suggesting a higher affinity to substrates than other nanozyme. However, in a catalytic reaction, the reaction substrate is strongly adsorbed by the catalyst and will make it difficult to desorption later, which in turn will lead to a decrease in reactivity. (Chen et al., 2014) It can be seen from the TEM characterization that the size of Pt in 5Pt/CNTs is about 0.55 nm, which indicates that Pt is composed of small clusters of several Pt atoms. In general, this small size cluster has richer surface charge, which are beneficial to adsorb positively charged TMB, resulting in a high affinity. For the surface charge of 5Pt/CNTs, we can determine it by zeta potential. As shown in Figure S5, both CNTs and cPt/CNTs have negative surface zeta potentials, which is not only beneficial to the solubility and dispersibility of Pt/CNTs in water, but also beneficial to adsorb positively charged TMB. It is also found that 5Pt/CNTs has the largest negative surface zeta potential, indicating that it had the strongest adsorption effect on the reaction substrate, which is consistent with kinetic analysis. While K_m values for TMB and H₂O₂ display different trends of change with increasing the size from 0.55 nm to 2.81 nm. In contrast, ν_max and k_cat values for TMB and H₂O₂ show a volcanic trend and 30Pt/CNTs (1.69 nm) nanozyme has the largest ν_max and k_cat, indicating that 30Pt/CNTs exhibits the highest catalytic efficiency among these cPt/CNTs nanozymes. For cPt/CNTs (c = 10, 20, 30, 50 and 70), whose surface contains a small negative potential, has a proper adsorption/desorption effect on the reaction substrate, so they have better catalytic performance, of which 30Pt/CNTs have the best reactivity. Therefore, a proper adsorption of nanozymes with appropriate size on the substrates is more conducive to the catalytic activity. Moreover, it is obvious that this value is higher than that of reported Pt-based nanozymes (Table S3 and S4), demonstrating the excellent performance of 30Pt/CNTs fabricated by ALD.

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Fig. 3

Steady-state kinetics of the cPt/CNTs nanozymes for TMB kinetic assay in the presence of H₂O₂. (A-F) Michaelis-Menten curves for cPt/CNTs nanozymes with varied TMB concentrations in the presence of H₂O₂. Inserted images represent the Lineweaver-Burk plots of the double reciprocal of the Michaelis-Menten equation. Error bars shown represent standard errors derived from three independent experiments.

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Fig. 4

Steady-state kinetics of the cPt/CNTs nanozymes for H₂O₂ kinetic assay in the presence of TMB. (A-F) Michaelis-Menten curves for cPt/CNTs nanozymes with varied H₂O₂ concentrations in the presence of TMB. Inserted images represent the Lineweaver-Burk plots of the double reciprocal of the Michaelis-Menten equation. Error bars shown represent standard errors derived from three independent experiments.

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To further reveal the catalytic mechanism of size-dependent POD-like activity, high-resolution Pt 4f XPS characterization was employed to probe the surface composition of Pt nanoparticles and the difference in the electronic properties of the cPt/CNTs nanozymes. As shown in Fig. 5, the Pt 4f region exhibits doublets from the spin–orbit splitting of the 4f7/2 and 4f5/2 states, both of which can be deconvoluted into three peaks, indicating that all the cPt/CNTs nanozymes consist of metallic Pt⁰, Pt²⁺, and Pt⁴⁺. (Gan et al., 2020) The existence of platinum oxide phases can be ascribed to the ultra-size of Pt nanoparticles, which are susceptible to oxidation by O₃ exposure during the ALD process or in the ambient environment. (Chen et al., 2014; Zhang et al., 2017) Meanwhile, the strength of the Pt 4f peak increase with Pt ALD cycle number (Figure S6), suggesting that the Pt content is gradually increasing, which is consistent with the above-mentioned ICP-AES and XPS surveys. According to literature, a high ratio of Pt⁰/Pt²⁺ in the surface composition of Pt nanoparticles is beneficial to improving the POD-like activity of Pt nanoparticles. (Chen et al., 2020) We calculated the ratios of Pt⁰/Pt²⁺; Pt⁰/Pt⁴⁺ and Pt²⁺/Pt⁴⁺ on the surface of cPt/CNTs nanozymes. As shown in Table S5, the ratios of Pt⁰/Pt²⁺ and Pt⁰/Pt⁴⁺ exhibit a volcanic curve with the cycle number of Pt ALD increasing from 5 cycles (0.55 nm) to 70 cycles (2.81 nm), and the 30Pt/CNTs nanozyme with a size of 1.69 nm shows the maximum Pt⁰/Pt²⁺ and Pt⁰/Pt⁴⁺ values, which is consistent with the results of size-dependent POD-like activity. However, the ratio of Pt²⁺/Pt⁴⁺ tends to increase with the cycle number of Pt ALD. The above results demonstrate that Pt⁰ and Pt²⁺ species are the main catalytic reaction species, and their content has a great influence on the catalytic activity. And Pt⁴⁺ species do have a certain impact on the activity of nanozymes, which is consistent with the result of literature. (Fu et al., 2014).

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Fig. 5

High-resolution Pt 4f XPS of 5Pt/CNTs (A), 10Pt/CNTs (B), 20Pt/CNTs (C), 30Pt/CNTs (D), 50Pt/CNTs (E), and 70Pt/CNTs (F) nanozymes.

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As reported in the literature, the POD-like reaction follows the Eley-Rideal mechanism: H₂O₂ molecules are initially adsorbed on the surface of Pt nanoparticles, and the Pt active site rapidly breaks the oxygen–oxygen bond of H₂O₂ to generate two surfaces •OH species (H₂O₂ → 2•OH), which are stabilized on the nanoparticles’ surface via partial electron exchange interactions between the unpaired electrons of the adsorbed radicals and the conduction-band electrons of the metallic particles. Then, the surface •OH species can further oxidize the adsorbed TMB on the surface of Pt nanoparticles to form a blue color product. (Ge et al., 2018; Ma et al., n.d.; Ma et al., 2011) However, the adsorption of TMB and H₂O₂ should not be too strong, otherwise they will be hardly desorbed from the surface of Pt nanoparticles, which probably leads to nanozyme poisoning and inactivation. (Chen et al., 2021) To more clearly study the catalytic mechanism of size-dependent POD-like activity, based on the above results, we have proposed a correlation between the size of Pt nanoparticles and the ratio of Pt⁰/Pt²⁺ or POD-like activities (specific activity, k_cat of TMB and H₂O₂). As shown in Fig. 6, the ratio of Pt⁰/Pt²⁺ and specific activity, as well as k_cat of substrates, exhibit a volcanic relationship with the size of Pt nanoparticles, in which the 30Pt/CNTs nanozyme with a size of 1.69 nm gives rise to a higher catalytic efficiency owing to appropriate adsorption of the nanozyme on substrates.

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Fig. 6

Correlation between the size of Pt nanoparticles and ratio of Pt⁰/Pt²⁺ or POD-like activities (specific activity, k_cat of TMB and H₂O₂).

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3.4 Size-dependent antibacterial activity in Pt/CNTs nanozymes

POD-like nanozymes have been widely used in antibacterial application. It decomposes H₂O₂ into highly toxic free radicals (•OH, Fig. 2D), thereby destroying bacterial membrane and induce apoptosis. (Chen et al., 2021; Sun et al., 2020) Based on the size-dependent POD-like activity of cPt/CNTs nanozymes, we evaluated the broad-spectrum antimicrobial capability of cPt/CNTs nanozymes against Gram-negative E. coli and Gram-positive S. aureus. As shown in Fig. 7A and 7C, cPt/CNTs nanozymes in the absence of H₂O₂ showed negligible killing effect against E. coli and S. aureus. In the presence of H₂O₂ (0.2 mM), the viability of both E. coli and S. aureus decreases effectively, indicating the antibacterial activity of cPt/CNTs nanozymes employ their POD-like catalytic activity to decompose H₂O₂ into highly toxic •OH that disrupt the bacterial membrane and induce bacterial apoptosis. (Mei et al., 2021) In comparison, the 30Pt/CNTs nanozyme with a size of 1.69 nm exhibits a much higher antibacterial activity than other cPt/CNTs nanozymes under the same experimental conditions. The plate counting method was employed to quantify the antibacterial activity of cPt/CNTs nanozymes. As shown in Fig. 7B and 7D, the antibacterial activity of cPt/CNTs nanozymes exhibits a volcanic relationship with Pt ALD cycle number, in which 30Pt/CNTs nanozymes presented the highest antibacterial activity against E. coli (∼81 %) and S. aureus (∼54 %). In order to fully characterize the antibacterial properties of cPt/CNTs nanozymes, we carried out the inhibition zone experiment. (Xia et al., 2018; Yang et al., 2019) The antibacterial performance of all the samples was displayed in Figure S7. Notably, after incubation for about 24 h, the as-prepared cPt/CNTs nanozymes presents the robust antibacterial activity, comparing with that of bare CNTs samples. And the antibacterial activity of cPt/CNTs nanozymes exhibits a volcanic relationship with Pt ALD cycle number, in which 30Pt/CNTs nanozymes presented the highest antibacterial activity against E. coli (3.8 cm) and S. aureus (3.17 cm). It is also noticeable that E. coli was more sensitive toward antibacterial treatments as reported in the literature, (Yin et al., 2016; Liu et al., 2021; Xue et al., 2022) which could be ascribed to the difference in their membrane structure. (Gupta et al., 2019) And, we compared with the antibacterial properties of relevant nanozymes in the literature and found that the antibacterial properties of Pt/CNTs nanozymes are very close to or even higher than those of Pt-based nanozymes, demonstrating the superiority of ALD technique. (Ge et al., 2018; Chen et al., 2021) The above experimental results indicate that the cPt/CNTs nanozymes have a size-dependent antibacterial activity, consistent with the POD-like activity of the cPt/CNTs nanozymes. Meanwhile, the optimization of the antibacterial activity can be realized by controlling Pt nanoparticle size by ALD.

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Fig. 7

Antibacterial properties of the cPt/CNTs nanozymes. Photographs of bacterial colonies formed by (A) E. coli and (C) S. aureus treated with cPt/CNTs nanozymes with or without H₂O₂. The plate counting method determines the corresponding bacterial viabilities of (B) E. coli and (D) S. aureus after treatment with cPt/CNTs nanozymes in different groups. Error bars shown represent standard errors derived from three independent experiments.

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H₂O₂ is a common fungicide in clinic, which can destroy bacterial cell membrane, protein and nucleic acid. (Kalyanaraman, 2013) However, in the clinic, due to the high concentration of H₂O₂ (2 M) required and poor antibacterial properties, it is easy to develop drug resistance. (Djorić and Christopher, n.d.) The results unveil that a low content of 30Pt/CNTs nanozyme (0.2 mg/mL) could kill ≥ 80 % of E. coli in the presence of extremely low concentrations of H₂O₂ (0.2 mM), while the bactericidal efficiency of bare CNTs was negligible. (Fig. 7A and 7C) Therefore, the development of nanozyme-based high-performance fungicides provides a new strategy to address bacterial infection and drug resistance.