High catalytic nickel−platinum nanozyme enhancing colorimetric detection of Salmonella Typhimurium in milk

Colorimetric qualitative and sensitive quantitative detection of Salmonella Typhimurium ( S. Typhimurium) holds significant importance for ensuring food safety and preventing foodborne illnesses. In the study, an ultra-high catalytic activity and biocompatible nickel− platinum nanoparticle (NiPt NP) nanozyme is successful synthesized to prepare a NLISA strategy for the detection of S. Typhimurium. The synthesized NiPt NPs exhibit high oxidase-like catalytic efficiency, with a Michaelis constant (Km) of 0.493 mM, similar to that of natural horseradish peroxidase (HRP). The maximal reaction velocity (Vmax) was determined to be 1.97 × 10 −7 M·s −1 exhibiting a 1.97-fold higher than that of the HRP (1.0 × 10 −7 M·s −1 ). Meanwhile, the antibody employed in this NiPt NPs-based NLISA exhibits exceptional capture efficacy, generating a stable immune complex with S. Typhimurium. The NiPt NPs-based NLISA demonstrates sensitivity, specificity, convenience, and cost-efficiency for the detection of S. Typhimurium. Under optimal conditions, this NiPt NPs-based NLISA demonstrates a quantitative range of 10 3 ~10 6 cfu/mL with a detection limit as low as 10 3 cfu/mL. A single-blind experimental testing detects different concentrations of S. Typhimurium spiked skim milk, indicating the application potential of the proposed NLISA in real samples. In all, this research provides novel insights into the synthesis of nanozymes with excellent catalytic activity and their applications in S. Typhimurium biosensing.


INTRODUCTION
Salmonella, a genus of pathogenic bacteria, poses a significant threat to human health by causing foodborne infectious diseases.Over 2,000 serotypes have been identified (Majowicz et al., 2010).Among the various strains, Salmonella Enterica serovar Typhimurium (S.Typhimurium) is a predominant serotype frequently implicated in human illnesses because of the high virulence (Roumagnac et al., 2006).S. Typhimurium is commonly transmitted through contaminated food via oral and fecal routes, infecting healthy people and causing secondary infections.According to the European Commission (EC) regulations, the detection of Salmonella in foodstuffs such as milk powder, and cheeses should yield a "negative" result.Therefore, there is a significant need for sensitive and specific methods to detect S. Typhimurium to ensure food safety.
Immunological techniques, including enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow strip (LFS), are characterized by its elevated sensitivity and specificity.These methods are the most commonly used for identifying S. Typhimurium (Lee et al., 2015).The CLIA provides an ultrasensitive method, yet the CLISA requires the employment of expensive equipment and does not support rapid colorimetric detection (Xiao andXu, 2020, Feng et al., 2022).The LFS is characterized by its simplicity and rapid execution.However, it is limited to qualitative or semiquantitative assessments and exhibits lower sensitivity compared with ELISA (Ji et al., 2023).As of today, ELISA remains a prevalent immunoassay for both qualitative colorimetric and quantitative analyses of S. Typhimurium.This is attributed to its equipment-free colorimetric detection signal, as well as its high sensitivity and selectivity (Singh et al., 2024).
ELISA is based on the fundamental principle of the specific antigen-antibody binding reaction, resulting in the formation of complexes labeled with enzymes (Cohen and Walt, 2019).The concentration of the detection target is determined by the intensity of the chromogenic reaction, which is catalyzed by the labeled enzyme through the oxidation of substrates.Therefore, the sensitivity of ELISA is dependent on the enzymatic activity of the biocatalysts employed.Natural enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (AP), and p-lactose operon β-galactosidase are commonly employed in assays due to their ability to offer superior sensitivity and selectivity (van Beilen and Li, 2002).However, these natural enzymes are characterized by 3 inadequacies (Bolivar et al., 2022): (i) they are primarily proteins produced from living cells, and their preparation process is complex; (ii) the extraction equipment is expensive, leading to increased prices; (iii) they demonstrate poor stability, necessitating strict storage conditions.Fortunately, numerous nanozymes have been investigated to circumvent the aforementioned limitations and to identify viable substitutes for natural enzymes (Wang et al., 2018, Li et al., 2024).
Since the discovery of ferromagnetic nanoparticles (Fe 3 O 4 NPs) by Yan research group in 2007 (Gao et al., 2007), which exhibit catalytic activity similar to that of horseradish peroxidase (HRP), nanozymes have attracted considerable attention from researchers, thereby paving the way for the exploration of nanomaterials as a new generation of artificial enzymes (Huang et al., 2019, Zhao et al., 2023).Nanozymes, a class of nanomaterials, exhibit catalytic activity similar to that of natural enzymes.They are characterized by several advantages, such as facile purification, cost-effectiveness, high stability, and robust environmental resilience.In the last decade, different types of nanozymes have been applied to immunosorbent assays, leading to the development of the nanozyme-linked immunosorbent assay (NLISA), which has been utilized for biomarker detection (Xi et al., 2021), nucleic acid detection (Broto et al., 2022), and food safety screening (Zhao et al., 2024), and environmental contaminants monitoring.However, most nanozymes were often fabricated using a substantial quantity of costly noble metals, including Pt NPs, Pt-Au NPs, and so on.Alternatively, they may be derived from Fe 3 O 4 with protracted preparation durations and intricate procedures, which constrained their catalytic efficacy and limited their utility in the domain of NLISA.The nickelplatinum nanoparticles (NiPt NPs), composed of costeffective nickel cores and platinum shells, demonstrated significantly superior catalytic efficiency compared with HRP and various other nanozymes (Xi et al., 2021, Wang et al., 2023).
Therefore, as shown in Scheme 1, an ultra-high catalytic activity and biocompatible NiPt NP nanozyme was successful prepared by using a fast and simple one-pot synthesis method (Scheme 1A).Subsequently, thiol-PEG-COOH served as a linker molecule to facilitate the labeling of NiPt NPs with streptavidin (SA).The NiPt NPs-SA probe was successfully immobilized to the 96well plates via a sandwich-type recognition involving the antibody specific to S. Typhimurium and biotin-labeled antibody (Scheme 1B).The NiPt NPs and NiPt NPs-SA probe both exhibited efficient peroxidase-like activity, facilitating the transformation of colorless tetramethylbenzidine (TMB) into blue oxidized products (oxTMB) (Scheme 1C).This oxidation process is accompanied by a transition to blue color and turns yellow after the addition of H 2 SO 4 , which can be leveraged for colorimetric and quantitative detection.Hereto, a NLISA method for the detection of S. Typhimurium was constructed based on high catalytic performance of NiPt NPs.This NiPt NPsbased NLISA was successfully used for high sensitivity and specificity for the detection of S. Typhimurium in skim milk.We believe this NLISA will provide not only a merely stronger stability, intricate instrumentation-free, and economical method for application in resourcelimited environments, but also an excellent method for monitoring foodborne pathogens in laboratory settings.

Materials and Reagents
Pertinent materials and reagents are meticulously documented in the supplementary information.The bacterial name and numbering are detailed in Table S1.

Preparation of NiPt NPs
The synthesis method of NiPt NPs was based on previous studies (Xi et al., 2021, Wang et al., 2023).Platinum acetone, nickel acetate (II) tetrahydrate, polyvinyl pyrrolidone and tetraethylene glycol were mixed in a 100 mL 3-neck flask at 500 rpm by magnetic stirring.Then, the mixture was degassed at a mild N 2 of 100°C for 30 min to form a clear solution.One mL of absolute acetaldehyde was rapidly injected into the mixture under the overlay of N 2 .The mixture solution was heated to 280°C at 10°C/min and held at 280°C for 1 h before cooling to room temperature.Subsequently, the precipitation was used as a product by adding 100 mL acetone to the reaction solution and centrifugating at 6797 × g for 10 min.The product was subsequently washed twice by centrifu-

The labeling of the NiPt NPs
The method for labeling the protein to the NiPt NPs was based on a prior study by Prof. Xia's group (Xi et al., 2021).First, the NiPt NPs surface was functionalized with thiol-PEG-COOH.Under magnetic stirring, the 300 μL of thiol-PEG-COOH aqueous solution (0.5 mM) and 300 μL of NiPt NPs solution (8.5 × 10 −8 M) were mixed for 3 h.Then, the unbound thiol-PEG-COOH was removed by centrifugation at 20817 × g for 10 min.Precipitation product (NiPt NPs-S-PEG (COOH) ) was washed twice with DI water and redispersed in 300 μL of DI water.
Second, the NPs-S-PEG (COOH) was conjugated to the streptavidin (SA) by EDC / NHS cross-linking.50 μL of the prepared NP-S-PEG (COOH) solution was added to 450 μL of DI water.Five μL of EDC (25 mM) and 5 μL of NHS (50 mM) aqueous solution were then added to the solution under magnetic stirring.After 30 min, the precipitation was collected by centrifugation at 20817 × g for 10 min and redispersed by 50 μL of 1 × PBS buffer (10 mM, pH = 7.4).Subsequently, 50 μL of SA (2.5 mg/mL in PBS) was added to the solution and mixed by the shaker.After 1 h of incubation at room temperature, the reaction solution was stored in the refrigerator at 4°C overnight.Then, 100 μL of blocking solution was added to the solution (5% BSA).After 1 h, the final product (NPs-SA) was collected by centrifugation at 20817 × g for 10 min and washed twice by 1 × PBS.Finally, the precipitation was redispersed in 100 μL 1 × PBS containing 1% BSA for future use.

Sensitivity analysis of NiPt NPs-based NLISA for the detection of S. Typhimurium
S. Typhimurium was detected in PBS and skim milk (10-fold dilution) and diluted into a series of concentration gradients (0, 10 3 cfu/mL, 3 × 10 3 cfu/mL, 10 4 cfu/ mL, 3 × 10 4 cfu/mL, 10 5 cfu/mL, 3 × 10 5 cfu/mL, 10 6 cfu/ mL).First, 100 μL of monoclonal antibody (6H7H6G4) at a concentration of 10 μg/ml was added to 96-well microplate plate at 37°C for 1 h.Subsequently, 200 μL of 5% BSA was added per well to block for 1 h.After washing steps, the diluted S. Typhimurium spiked solution was incubated at 37°C for 40 min and absorbed by the antibody labeling at a 96-well microplate plate.After washing steps, 100 μL of biotin-antibody (1 μg/ mL) was added to the 96-well microplate plate for 30 min.After washing procedures, 100 μL of NiPt NPs-SA (2.5 mg/mL) was added to each well for 30 min.Then 50 μL of TMB was added for 10 min after washing steps.The color development was observed.Finally, 50 μL of H 2 SO 4 (2 mol/L) stop solution was added to each well.The 96-well microplate plate was sent to the microplate reader for the determination of OD450.

Exploration of the specificity of NiPt NPs-based NLISA for S. Typhimurium detection and its detection performance for real milk samples
Specificity exploration.Under the same detection conditions, the other common pathogenic bacteria (The different pathogenic bacteria were listed in Table.S1) were detected at the same concentration of 1 × 10 6 cfu/mL and followed according to the detection step .
Detection performance in real samples.The skim milk sample was obtained from a local supermarket to assess the practicality of our NiPt NPs-based NLISA.The absence of S. Typhimurium in the skim milk was confirmed using traditional culture-based assays before inoculation.The absence of S. Typhimurium was reaffirmed at the beginning of each experiment in all skim milk samples.Single-blind experiments were conducted for the detection of S. Typhimurium in spiked skim milk.First, different concentrations of S. Typhimurium with ranging from 1.0 × 10 4 cfu/mL to 5.0 × 10 6 cfu/mL were added to the 24 skim milk samples.-Then, the 24 spiked samples were detected by our NiPt NPs-based NLISA.The detection recovery rate (P) was calculated by formula (P = (C1/C2) × 100% (Zhou et al., 2019, Jing et al., 2023), C1 represents the detection concentration of S. Typhimurium by the NiPt NPs-based NLISA, and C2 is the concentration of S. Typhimurium in the spiked milk samples.)

Other experiment procedures
For details of the experimental procedures, please refer to the Supplementary Information [Section S1, Page S1-S3].

Characterization of the NiPt NPs
As can be seen from Figure 1A-C, the Transmission Electron Microscopy (TEM) image revealed that NiPt NPs were scattered and exhibited nearly spherical particles with a particle size of about 15.8 ± 1.9 nm.The surface morphology and microstructure of NiPt NPs were observed by Scanning Electron Microscopy (SEM) in Fig. S1.As depicted in Figure 1D-G, energy dispersion x-ray spectroscopy (EDS) was used to analyze the element distribution in NiPt NPs (Figure 1D-F), and the STEM-HAADF (high-angle annular dark field) mode image was performed to display the atomic number comparison (Figure 1G).These images demonstrated that Ni elements were uniformly distributed in the central region, while Pt elements encompass Ni elements.Moreover, as shown in Figure 1H, the characteristic element signal of NiPt NPs can be easily observed in the EDX spectrum, which was consistent with the results of the elemental mapping.The x-ray diffraction (XRD) analysis of NiPt NPs was shown in Fig. S2, the results indicated that the diffraction peak (111) was divided into 2 separate peaks possibly caused by Pt-rich shell (40.76°) and Ni-rich core (42.89°).All these results demonstrated the successful synthesized of NiPt NPs.Meanwhile, the hydrated particle size of NiPt NPs was determined by the Malvern particle size analyzer, which revealed a distribution ranging from 12 to 18 nm with excellent dispersion (Figure 1I).

Exploring the catalytic activity of NiPt NPs
The peroxidase activity of NiPt NPs was evaluated by analyzing the absorption spectra derived from the oxidation of TMB, catalyzed by NiPt NPs.As shown in Fig. S3, the color of the solution transitions from a light blue to a dark blue as the concentration of NiPt NPs escalates.And the maximum peak of absorption spectra at 652 nm gradually increased with the increase in NPs concentration.Before exploring the catalytic activity of NiPt NPs nanozymes, the key parameters used for the assay such as concentration, temperature, pH, and time were optimized for the assay.As can be seen from Fig. S4A, the optimal concentration of NiPt NPs for detection was 500 μg/mL.The optimal detection temperature was 55°C (Fig. S4B).The optimal detection pH was 4 (Fig. S4C).As shown in Fig. S4D, the maximum change of OD652 values were achieved in less than 10 min.As shown in Fig. S5, the NiPt NPs retained approximately 91.8% of their initial peroxidase activity after 5 mo of storage at 4°C.Furthermore, the NiPt NPs maintained 77.3% of their initial peroxidase activity following 9 mo of stor-Zhu et al.: High catalytic nickel−platinum… age at 4°C, suggesting that the NiPt NPs exhibit commendable storage stability.The catalytic activity of NiPt NPs was investigated by collecting the OD652 values at 37°C.The peroxidase-like activity of NiPt NPs can be quantitatively evaluated by steady-state kinetic analysis.The kinetic constants were calculated according to the following (Ocsoy et al., 2015, Ji et al., 2021): The initial reaction rate (V) was calculated using the formula SlopeInitial/ (εTMB -653nm × L).In this equation, SlopeInitial is derived from the first derivative of each absorbance versus time plot.The εTMB -652nm is the molar extinction coefficient of TMB at 652 nm, which is equal to 3.9 × 10 4 M −1 cm −1 .Vmax is the maximum reaction velocity, [S] is the substrate concentration, and Km is the Michaelis-Menten constant obtained from the Lineweaver-Burk plot.As shown in Figure 2A-B, the kinetic analysis of NiPt NPs was conducted using H 2 O 2 as the substrate for the peroxidase reaction.The Km derived from the analysis was 0.047 mM, and the Vmax was determined to be 23.635 × 10 −8 M•s −1 .The Km value of NiPt NPs was smaller than that of the HRP (3.7 mM) (Gao et al., 2007), indicating that NiPt NPs have a higher affinity for the H 2 O 2 substrate and exhibit better peroxidase-like catalytic activity in comparison to HRP.As shown in Figure 2C-D, the kinetic analysis of NiPt NPs was conducted using TMB as the substrate for the peroxidase reaction.The Km derived from the analysis was 0.493 mM, and the Vmax was determined to be 19.662× 10 −8 M•s −1 .The affinity of NiPt NPs to the substrate TMB was similar to that of HPR (0.434 mM).Meanwhile, the Vmax was much higher than that of the HRP.Overall, the above results substantiated that our NiPt NPs possess superior peroxidase-like catalytic activity.

Evaluation and Optimization of the Properties of the NLISA-based NiPt NPs
To confirm the successful preparation of the NiPt NPs-SA probe, the UV-vis absorption spectra of NiPt NPs, SA and NiPt NPs-SA were analyzed.As shown in Figure 3A, the black curve of NiPt NPs did not exhibit any absorption peaks.Concurrently, the red curve of NiPt NPs-SA showed one absorption peak, which were the characteristic peaks of SA (280 nm), proving the successful conjugation of SA with NiPt NPs.The zeta potential can be used to characterize the positive and negative charges on the surface of the NPs in the solution dispersion system.As depicted in Figure 3B, the measured zeta potential of NiPt NPs, SA, NiPt NPs-PEG and NiPt NPs-SA were −9.0 mV, −16.3 mV, −5.3 mV, and −10.2 mV, respectively.These changes of zeta potential values could prove that SA was successfully labeled to the surface of NiPt NPs. Figure 3C showed that colorless TMB, used as a chromogenic substrate, was significantly catalyzed to generate blue oxTMB with a characteristic absorption peak of 652 nm in the presence of NiPt NPs.In contrast, TMB alone was without an absorption peak.Furthermore, TMB was also significantly catalyzed to generate blue ox-TMB in the presence of NiPt NPs-SA, indicating that the labeled of SA did not affect the catalytic performance of NiPt NPs.
To improve the catalytic performance of NiPt NPsbased NLISA for the detection of S. Typhimurium, optimal parameters including the concentration of biotinylated antibody, the concentration of NiPt NP-SA, and the incubation time of S. Typhimurium were identified, respectively.As shown in Figure 3D, the positive OD450 values showed a plateau intensity when the biotinantibody concentration was at 2 μg/mL.For the negative control samples, the negative OD450 values were stable in the range of 0 μg/mL to 1 μg/mL of biotin-antibody.Nevertheless, when the concentration of biotin-antibody was greater than 1 μg/mL, which led to the interference of false positive.Therefore, the concentration of biotinantibody was determined to be 1 μg/mL.Using the same analytical mechanism, the optimal concentration of NiPt NPs-SA for the detection of S. Typhimurium was explored (Figure 3E).The optimal concentration of NiPt NPs-SA was determined to be 2.5 mg/mL.Finally, as shown in Figure 3F, the optimal incubation time was selected to be 40 min based on the data analysis of the positive signal display over time.
The performance of this NLISA for detecting S. Typhimurium was evaluated under the condition of optimizing the key parameters.The sensitivity of the NLISA was examined by detecting the of S. Typhimurium of gradient concentrations (10 3 ~10 6 cfu/mL).For the PBS matrix groups (Figure 4A), the blued color signals were visually observed with 10 −4 ~10 −6 cfu/mL of S. Typhimurium.In contrast, the blued color signal for detection of 10 −4 cfu/ mL of S. Typhimurium in spiked skim milk matrix was weaker but still observable (Figure 4D).The result validated the food matrix exerted a discernible impact on the NLISA, yet it did not substantially affect the colorimetric qualitative analysis of S. The OD450 values showed a good linear relationship with S. Typhimurium concentrations in the range of 10 4 to 10 6 cfu/mL (Figure 4C and Figure 4F).The linear equations were Y = −2.975+0.911LogC(R 2 = 0.978) and Y = −3.724+1.015LogC(R 2 = 0.963), respectively.The limits of detection (LOD) were 2.31 × 10 3 cfu/mL (PBS) and 5.77 × 10 3 cfu/mL (milk), respectively.(LOD = Mean blank + 3SD, n = 10).To compare the sensitivity results between NLISA and ELISA for the detection of S. Typhimurium, various concentrations of S. Typhimurium in spiked milk samples were tested by ELISA based on the same bispecific sandwich antibodies (Fig. S6).It was obvious that the blued color signals began to change at the concentration of 3.0 × 10 4 cfu/mL (Fig. S6A).As shown in Fig. S6B-C, the OD450 values increased along with the increase of S. Typhimurium concentration.The LOD of ELISA was 18.95 × 10 3 cfu/mL in spiked milk samples, according to LOD = Mean (blank) + 3SD (n = Zhu et al.: High catalytic nickel−platinum… 10).These results indicated that the developed bispecific antibody sandwich NLISA exhibited the better LOD than that of ELISA.Interferences from food matrices may be present.Consequently, it is imperative to re-establish a standard linear calibration curve for the detection of S. Typhimurium in various food matrices.

Analytical performances of the NiPt NPs-based NLISA for S. Typhimurium detection
Four foodborne pathogens at concentrations of 1 × 10 6 cfu/mL, Escherichia coli (E.coil), Staphylococcus aureus (S. aureus), Shigella flexneri (S. flexneri), and Salmonella Enteritidis (S.Enteritidis) were selected to evaluate the selectivity of the proposed NiPt NPs-based NLISA for S. Typhimurium detection.As shown in Figure 5A, the colorimetric detection signal of NLISA for target S. Typhimurium (10 4 cfu/mL) was clearly observed, whereas no colorimetric signals were observed for other foodborne pathogens, even at the highest concentration of 10 6 cfu/mL.The difference in OD450 values of NLISA was employed as a metric to demonstrate the disparity in response between the positive sample and the negative control (Figure 5B).The corresponding OD450 values were increased with the increase of concentration of S. Typhimurium, whereas negligible OD450 values were obtained for other foodborne pathogenic bacteria.The specificity results confirm that the proposed Ni-Pt NPs- based NLISA exhibits good specificity for S. Typhimurium in the presence of other pathogenic bacteria.
Furthermore, to evaluate the performance of this NiPt NPs-based NLISA, a single-blind experimental testing was conducted to detect different concentrations of S. Typhimurium in 24 spiked skim milk samples (1.0 × 10 4 cfu/mL to 5.0 × 10 6 cfu/mL).As shown in Figure 5C, all S. Typhimurium spiked skim milk samples showed the results.The deeper colorimetric results corresponded with the higher concentration of S. Typhimurium.The corresponding OD450 values were depicted in Figure 5D.The OD450 values of 10 4 cfu/mL was 0.3937 (a2) which is slightly higher than the threshold value (0.0946. Figure 4E).To accurately detect the concentration of S. Typhimurium within these 24 single-blind samples, the OD450 values derived from this NLISA were quantified carefully.As shown in Table S1, the concentrations of S. Typhimurium in spiked skim milk samples were quantified within the linear concentration range.This quantification was based on the established linear correlation between the OD450 values and the concentration of S. Typhimurium in skim milk (Figure 4F).The recovery rates ranged from 82.4 to 118.0%, demonstrating the method's excellent applicability and feasibility for analyzing S. Typhimurium in real food samples.

CONCLUSION
In summary, NiPt NPs were synthesized that consist of low-cost nickel cores and platinum shells showed a significantly higher catalytic efficiency than HRP and other types of nanozymes.Meanwhile, the distinctive architecture and composite elements of the NiPt NPs conferred them protracted shelf stability.Then, a NLISA utilizing the ultra-high catalytic activity and biocompatible NiPt NPs, termed NiPt NPs-based NLISA, has been successfully developed to achieve sensitive, specific, and stable detection of S. Typhimurium.This excellent NLISA demonstrated several advantages, including a lower LOD (10 3 cfu/mL), an extended linear range from 10 3 cfu/mL to 10 6 cfu/mL, and detection adaptability in real food matrices.In addition, this NiPt NPs-based NLISA facilitated the naked-eye detection of S. Typhimurium and provided excellent differentiation from other prevalent pathogens, thus ensuring on-site diagnostic capabilities.Compari- son to other S. Typhimurium detection methods such as immunological methods and molecular methods (Table S3), our NiPt NPs-based NLISA performed nicely in S. Typhimurium detection with a low LOD and short detection time (within 2 h) in complex biological samples.
The enhancements are likely to facilitate the sustainable application of this NiPt NPs nanozyme-based detection method, potentially enabling its transition from laboratory settings to field use for detecting other foodborne pathogens.
Zhu et al.: High catalytic nickel−platinum… gation with DI water (20817 × g, 10 min).The products were redispersed in DI water for further use.
Scheme 1. Schematic diagram of the detection of S. Typhimurium by NiPt NPs-based NLISA.(A) Synthesis of NiPt NPs; (B) doubleantibody sandwich NiPt NPs-based NLISA for the detection of S. Typhimurium; (C) TMB chromogenic reaction.

Figure 1 .
Figure 1.Characterization of the NiPt NPs.(A-C) the TEM images of NiPt NPs; the elemental mapping of single NiPt NPs for (D) Pt elemental (E) Ni elemental and (F) Pt and Ni elemental; (G) the representative HAAD F-STEM image; (H) EDS mapping image of NiPt NPs; (I) size distribution of the NiPt NPs.
Typhimurium.The intensity of the colorimetric signals demonstrated a positive correlation with the concentration of S. Typhimurium.By recording the absorbance 450 (OD450) of the solution, as illustrated in Figure 4B-C and Figure 4D-F, it was observed that the OD450 values increased along with the increase of S. Typhimurium concentration.
Figure 2. Enzyme activity assay of NiPt NPs.(A) steady-state kinetics of NiPt NPs for substrate H 2 O 2 at different H 2 O 2 concentrations; (B) the Lineweaver−Burk curve corresponding to H 2 O 2 ; (C) steady-state kinetics of NiPt NPs for TMB substrate at different TMB concentrations; (D) the Lineweaver−Burk curve corresponding to TMB. n = 3; error bars depict the standard deviation.

Figure 4 .
Figure 4. NiPt NPs-based NLISA detects S. Typhimurium down to 10 4 cfu/mL concentration.(A) photograph for detecting different concentrations of S. Typhimurium in PBS buffer before (upside) and after (downside) terminating the reaction; (B) the correlation between OD450 values and various concentrations of S. Typhimurium in PBS buffer; (C) linear relationship between the OD450 values and the concentration of S. Typhimurium in PBS buffer; (D) photograph for detecting different concentrations of S. Typhimurium in spiked skim milk before (upside) and after (downside) terminating the reaction; (E) the correlation between OD450 values and various concentrations of S. Typhimurium in spiked skim milk; (F) linear relationship between the OD450 values and the concentration of S. Typhimurium in spiked skim milk.n = 3; error bars depict the standard deviation.