Ultrasensitive Ti3C2Tx@Pt-Based Immunochromatography with Catalytic Amplification and a Dual Signal for the Detection of Chloramphenicol in Animal-Derived Foods

Herein, a catalytic amplification enhanced dual-signal immunochromatographic assay (ICA) based on Pt nanoparticles (Pt NPs) modified with Ti3C2Tx MXene (Ti3C2Tx@Pt) was first developed for chloramphenicol (CAP) in animal-derived foods. Due to the large specific surface area and abundant active sites of Ti3C2Tx@Pt, they can be loaded with hundreds of Pt NPs to enhance their catalytic activity, resulting in a significant increase in the detection sensitivity; the sensitivity was up to 50-fold more sensitive than the reported ICA for CAP. The LODs of the developed method for milk/chicken/fish were 0.01 μg/kg, the LOQs were 0.03 μg/kg and the recovery rates were 80.5–117.0%, 87.2–118.1% and 92.7–117.9%, with corresponding variations ranging from 3.1 to 9.6%, 6.0 to 12.7% and 6.0 to 13.6%, respectively. The linear range was 0.0125–1.0 μg/kg. The results of the LC-MS/MS confirmation test on 30 real samples had a good correlation with that of our established method (R2 > 0.98), indicating the practical reliability of the established method. The above results indicated that an ICA based on the Ti3C2Tx@Pt nanozyme has excellent potential as a food safety detection tool.


Introduction
Chloramphenicol (CAP) is a highly effective bacteriostatic broad-spectrum antibiotic that has been widely used in the production, processing and manufacturing of livestock, poultry and aquatic products [1].CAP residues in food can cause great harm to human health, including the following: aplastic anemia, bone marrow transplantation, leukemia, gray baby syndrome, etc. [2].At present, the United States, the European Union and China have issued relevant laws and regulations prohibiting the use of CAP in the treatment of animal diseases, and it must not be detected in food [3].However, due to its low cost and good effect, the illegal use of chloramphenicol is still banned repeatedly [4].Therefore, establishing a fast and convenient method for detecting CAP residues in food plays an important role in ensuring food safety.
Currently, the detection methods of CAP in food are mainly instrumental methods, such as LC-MS/MS [5,6], GC-ECD [7,8], etc.While the above instrumental approaches are highly selective and specific, they need costly equipment and skilled operators, and the sample pretreatment process is tedious and not suitable for the quick on-site screening of large-scale samples [9,10].Immunochromatography (ICA) based on the specific binding Foods 2024, 13,1416 2 of 14 of an antigen (Ag) and antibody (Ab) is widely used in on-site rapid detection due to its simple operation, fast reaction speed and instant results [9].The current signal labels used for detecting chloramphenicol (CAP) in ICA are mainly gold nanoparticles (GNPs).However, the low molar extinction coefficient and narrow particle size range limit its detection sensitivity [11].In order to improve sensitivity, many new nanomaterials have been used to replace GNPs, including quantum dots [12], fluorescent microspheres [13], organic dyes such as crystal violet [14], etc.The above labeling materials are all singlesignal outputs, which are increasingly unable to meet the demand for the trace detection of CAP in food.Nanozymes, nanomaterials with enzyme-like characteristics, are excellent signal-reporting molecules that can catalyze the conversion of chromogenic substrates into other visible colors, achieving dual-signal output.Compared with natural enzymes, nanozymes have lower preparation costs, greater stability and higher catalytic efficiency, which makes them ideal carriers for probes in immunoassays [15,16].
In recent years, MXene-based composites as nanozymes have attracted attention [17].The excellent hydrophilicity, large specific surface area and abundant surface groups of Ti 3 C 2 Tx MXene can provide more attachment sites for the anchoring of noble metal particles and improve the catalytic performance of composites [18].At present, MXene-based nanozyme composite materials are mainly used in fields such as biosensing [19] and catalysis [20].For example, when MXene@Pt is under NIR-II laser irradiation, hot electrons can be excited, promoting nano-enzymatic activity.Compared to original nanozymes, MXene-based nanozymes improved tumor suppression [21].The high number of oxygenated groups on the MXene surface can increase the loading capacity of Pt NPs, and the introduction of MXene enhances the catalytic ability of Pt NPs for Hg 2+ , enhancing their adsorption capacity for Hg 2+ [22].The above research results indicated that Ti 3 C 2 Tx MXene was an excellent nanomaterial loading matrix, and the addition of MXene enhanced the catalytic activity of single-atom metal particles.As a signal reporter molecule, it had broad applications in providing stable signal output and improving the stability of biosensors.At present, the application of ICA based on Pt NPs modified with MXene Ti 3 C 2 Tx is relatively limited, with only medical reports on its use for detecting HIV-DNA, and there is still a gap in small molecule detection [23].
Here, we synthesized Ti 3 C 2 Tx@Pt nanocomposites through simple in situ reduction and applied them for the detection of CAP in animal-derived foods.Due to the large specific surface area and abundant active sites of Ti 3 C 2 Tx@Pt, they can be loaded with hundreds of Pt NPs to enhance their catalytic activity, resulting in a significant increase in the detection sensitivity of ICA.The successful establishment of this method has expanded the application of MXene materials in the field of POCT.

Preparation of Ti 3 C 2 Tx
The multilayer Ti 3 C 2 Tx two-dimensional nanosheets were prepared by LiF/HCl in situ etching.First, LiF (3.2 g) was dissolved in 40 mL of HCl (9 M) and stirred evenly.The as-prepared multilayer nanosheets were centrifuged (1300× g, 5 min) several times with ultrapure water until the supernatant reached pH 6.The precipitates containing numerous layers of nanosheets were dissolved in ultrapure water and sonicated for 2 h to prepare a thin layer of Ti 3 C 2 Tx.Finally, the supernatant colloidal dispersion containing several layers of Ti 3 C 2 Tx was collected by centrifugation (21,000× g, 10 min) and stored under nitrogen gas at 4 • C.

Preparation of Ti 3 C 2 Tx@Pt Nanocomposites
The composite material, Ti 3 C 2 Tx@Pt, was prepared using an in situ reduction strategy.Briefly, 500 µL of H 2 PtCl 6 •6H 2 O (19.3 mM) was added to 10 mL of a thin layer of the Ti 3 C 2 Tx solution and stirred vigorously for 30 min.Subsequently, 200 µL of NaBH 4 (0.1 M) was quickly added to the reaction mixture and stirred gently for 3 h.Ti 3 C 2 Tx@Pt was then separated through centrifugation (21,000× g, 10 min) and dried overnight.

Preparation of Ti 3 C2Tx
The multilayer Ti3C2Tx two-dimensional nanosheets were prepared by LiF/HCl in situ etching.First, LiF (3.2 g) was dissolved in 40 mL of HCl (9 M) and stirred evenly.The as-prepared multilayer nanosheets were centrifuged (1300× g, 5 min) several times with ultrapure water until the supernatant reached pH 6.The precipitates containing numerous layers of nanosheets were dissolved in ultrapure water and sonicated for 2 h to prepare a thin layer of Ti3C2Tx.Finally, the supernatant colloidal dispersion containing several layers of Ti3C2Tx was collected by centrifugation (21,000× g, 10 min) and stored under nitrogen gas at 4 °C.

Preparation of Ti 3 C2Tx@Pt Nanocomposites
The composite material, Ti3C2Tx@Pt, was prepared using an in situ reduction strategy.Briefly, 500 µL of H2PtCl6•6H2O (19.3 mM) was added to 10 mL of a thin layer of the Ti3C2Tx solution and stirred vigorously for 30 min.Subsequently, 200 µL of NaBH4 (0.1 M) was quickly added to the reaction mixture and stirred gently for 3 h.Ti3C2Tx@Pt was then separated through centrifugation (21,000× g, 10 min) and dried overnight.

The Peroxidase-like Activity of Ti 3 C 2 Tx@Pt
Steady-state kinetic tests were performed by monitoring changes in the absorbance of TMB oxidation products under 652 nm.The TMB or H 2 O 2 substrate concentration was constant, while the other substrate concentrations were varied.The concentration ranges of TMB and H 2 O 2 were 2-28 mM and 0.2-1.6M, respectively.The two substrates were mixed and dissolved at different concentrations in citrate disodium phosphate buffer (pH 4.5).
Foods 2024, 13, 1416 4 of 14 The kinetic parameters were calculated using the Lineweaver-Burk plots of the double reciprocal of the Michaelis-Menten equation: 1/ν = (K m /V max ) (1/[S] + 1/K m ), where ν is the initial velocity, and V max is the maximum reaction velocity.

The Preparation of the Test Strip
The test strip consists of four components: a nitrocellulose membrane (NC), PVC gasket, absorbent pads and sample pads.The sample pads were immersed in 0.1 M Borax borate buffer (BB, pH 8.0, containing 0.15 M NaCl, 0.3% Polyvinylpyrrolidone (PVP), 0.5% Tween ® -20) and dried at 37 • C overnight.The dried sample pads and absorbent pads were cropped to 1.5 cm × 30 cm and 2.5 cm × 30 cm, respectively.The goat anti-rabbit IgG (0.12 mg/mL) and coating Ag (0.19 mg/mL) were squirted on the NC membrane to form C-line and T-line, respectively.The above prepared materials were assembled as shown in Scheme 1B and cut into 3.05 mm wide test strips.

Sample Pretreatment and Detection Process
Chicken and fish: First, the CAP-free chicken and fish samples were homogenized, and 4 g of the uniform sample was precisely weighed into a 15 mL tube.A total of 2 mL of ultrapure water and 4 mL of ethyl acetate were added and stirred vigorously for 3 min; after 10 min of centrifugation at 6800× g, the upper ethyl acetate layer was moved to another 10 mL tube and dried in the air at 50 • C. The mixture was then redissolved in PB buffer for testing.
Milk: the milk samples were tested directly without pretreatment.

Detection process:
The above sample solution (150 µL) was added to the microwells previously coated with 4.5 µL of Ti 3 C 2 Tx@Pt-Abs probe and incubated 3 min.Placing the test paper upright in the microtiter wells, the reaction took 8 min.The catalytic reaction was then carried out by immersing the test paper in AEC substrate solution (13.3 mM) for 30 s.The color intensity of the test paper was taken with a smartphone, and the data were processed with Image J 1.53K software.
2.8.The Performance Assessment of the Developed Ti 3 C 2 Tx@Pt-ICA 2.8.1.Sensitivity Different concentrations of CAP were added to the samples, and each sample was tested three times to obtain the visual limit of detection (vLOD), limit of detection (LOD), the limit of quantitation (LOQ) and linear detection range.The vLOD was the lowest CAP concentration at which the T-line color intensity was weaker than the C-line color intensity.The Ti 3 C 2 Tx@Pt-ICA is based on the principle that the target and the coating Ag compete to bind limited Ab probes; the color intensity of the T-line is inversely proportional to the concentration of the target (Scheme 1B).The calibration curves were exported by plotting the logarithm of CAP concentration as the X-axis and B/B 0 as the Y-axis (B 0 represents the T/C value of negative samples; B represents the T/C value of positive samples).The LOD and LOQ are defined as the mean of 20 blank samples plus 3 or 10 times the standard deviation (SD).The IC 20 -IC 80 of the calibration curve is the linear detection range.

Accuracy and Precision
Three different concentrations of CAP (0.03/0.05/0.26µg/kg, 0.03/0.05/0.27µg/kg, 0.02/0.04/0.20 µg/kg) were spiked in milk, chicken and fish samples.All samples were Foods 2024, 13, 1416 5 of 14 collected for testing in triplicate within three days.The recovery and coefficient of variation (CV) determined the accuracy and precision of Ti 3 C 2 Tx@Pt-ICA.

Confirmatory Test
A total of 30 samples of chicken, fish and milk were bought at a local market (Guangzhou, China), and the quantity of each sample was confirmed to be 10 without CAP.Different levels of CAP were added to all samples; only the operator knew their actual concentration.Each sample was simultaneously tested in triplicate by Ti 3 C 2 Tx@Pt-ICA and LC-MS/MS.A detailed description can be found in the Supplementary Material for LC-MS/MS.

Statistical Analysis
Three repetitions of each experiment were performed (n = 3).Origin 2021 was used for statistical analysis.The significance level was defined as p less than 0.01 (p < 0.01).The data are shown as the average ± standard deviation (SD).

Characterization of Ti 3 C 2 Tx@Pt
The dense layered structure of Ti 3 AlC 2 was shown by scanning electron microscopy (SEM) (Figure 1A), and the sparse layered structure of Ti 3 C 2 Tx after etching (Figure 1B) was also observed, whose large surface area can load more Pt NPs.SEM (Figure 1C) and transmission electron microscopy (TEM, Figure 1E) showed that Pt NPs were highly dispersed among the layer and on the surface of Ti 3 C 2 Tx.X-ray energy spectroscopy (EDS) (Figure 1D) showed that the Ti 3 C 2 Tx@Pt nanocomposites contained Ti, O, C and Pt elements, all of which were evenly spread on the Ti 3 C 2 Tx surface, further confirming the large amount of Pt NPs loaded on Ti 3 C 2 Tx.
X-ray photoelectron spectroscopy (XPS) spectra of Ti 3 C 2 Tx and Ti 3 C 2 Tx@Pt showed the presence of elements such as Pt, C, F and O (Figure 1F).Two new peaks of Ti 3 C 2 Tx@Pt appeared in the XPS spectrum, Pt 4d and Pt 4f.As shown in the high-resolution Pt 4f spectrum of Figure 1G, two distinct peaks at the −74.3 eV and −70.9 eV binding energies correspond to 4f 5/2 and 4f 7/2 .The peaks can be divided further into two groups, corresponding to Pt 2+ and Pt 0 .These showed that a robust relationship exists within Ti 3 C 2 Tx with Pt NPs, which positively affects the adsorption properties of Pt NPs on the Ti 3 C 2 Tx surface.Moreover, by comparing the XPS spectra of Ti 3 C 2 Tx and Ti 3 C 2 Tx@Pt, it was found that there were three clear peaks in the O 1s spectrum of Ti 3 C 2 Tx in Figure 1H, including Ti-O and Ti-OH bonds, as well as the adsorbed oxygen in the surface sample (O ads); the binding energies were approximately 529.75 eV, 531.2 eV and 532.8 eV.Meanwhile, Ti 3 C 2 Tx@Pt can also detect the Pt-O peak with a binding energy of approximately 530.4 eV.These results further confirmed that Pt NPs were successfully loaded onto Ti 3 C 2 Tx and distributed uniformly.The X-ray diffraction results (XRD, Figure 1I) showed a peak of the highest Ti 3 AlC 2 (Figure S1) with JCPDS card no.52-0875 as an indicator that completely disappears at about 39.5 • , and the diffraction peak of the Ti 3 C 2 Tx reflection surface (002) was observed at about 6.2 • , indicating that the Al layer in Ti 3 AlC 2 was basically completely etched.At 39.76 • , 46.24 • and 67.45 • , the peaks of diffraction belong to the (111), ( 200) and (220) crystal faces of the Pt NPs, respectively (JCPDS certificate no.040802).In addition, as shown in Figure 1J, the potential of Ti 3 C 2 Tx loaded with negatively charged Pt NPs changed from −30.7 mV to −22.57mV, indicating that the Ti 3 C 2 Tx@Pt was successfully prepared.In particular, after coupling Abs with Ti 3 C 2 Tx@Pt, the potential changed from −22.57mV to −25.9 mV.These results showed that the Ti 3 C 2 Tx@Pt-Abs probe had been successfully prepared.
changed from −30.7 mV to −22.57mV, indicating that the Ti 3 C 2 Tx@Pt was successfully prepared.In particular, after coupling Abs with Ti 3 C 2 Tx@Pt, the potential changed from −22.57mV to −25.9 mV.These results showed that the Ti 3 C 2 Tx@Pt-Abs probe had been successfully prepared.XRD spectra of Ti3C2Tx and Ti3C2Tx@Pt nanocomposites.(J) Zeta potentials of Ti3C2Tx, Ti3C2Tx @Pt and Ti3C2Tx @Pt-Abs.(K) UV-vis absorption spectra of various solutions.

Evaluation of Ti 3 C 2 Tx@Pt Peroxidase-like Enzyme Activity
The catalytic oxidation of the chromogenic substrate TMB in the presence of H 2 O 2 produces a blue product with a characteristic adsorption peak at 652 nm to assess the peroxidase-like activity of Ti 3 C 2 Tx@Pt.Figure 1K shows that when TMB + H 2 O 2 + Ti 3 C 2 Tx@Pt was present simultaneously, a blue-colored product with a highly distinctive absorption peak at 652 nm can be generated, while the peaks for the other groups are barely visible.It was particularly emphasized that Ti 3 C 2 Tx itself does not exhibit peroxidase-like activity (blue line).However, due to their large specific surface area and active binding sites, the loading of Pt NPs results in their strong peroxidase-like activity.

Evaluation of Ti 3 C 2 Tx@Pt Peroxidase-like Enzyme Activity
The catalytic oxidation of the chromogenic substrate TMB in the presence of H 2 O 2 produces a blue product with a characteristic adsorption peak at 652 nm to assess the peroxidase-like activity of Ti 3 C 2 Tx@Pt.Figure 1K shows that when TMB + H 2 O 2 + Ti 3 C 2 Tx@Pt was present simultaneously, a blue-colored product with a highly distinctive absorption peak at 652 nm can be generated, while the peaks for the other groups are barely visible.It was particularly emphasized that Ti 3 C 2 Tx itself does not exhibit peroxidase-like activity (blue line).However, due to their large specific surface area and active binding sites, the loading of Pt NPs results in their strong peroxidase-like activity.
The steady-state kinetics were used to further confirm Ti 3 C 2 Tx@Pt peroxidase-like activity (Figure S3).The lower K m value showed the strength of the enzyme's affinity for the substrate [24].Table S1 [25][26][27][28][29][30][31][32][33][34][35][36] summarized the activity of nanozymes based on Pt.The results showed that when using TMB as the substrate, the K m of Ti 3 C 2 Tx@Pt (0.17 mM) was the lowest, which means that the peroxidase activity of Ti 3 C 2 Tx@Pt was the strongest.

Optimizing the Synthesis of Ti
The amount of H 2 PtCl 6 •6H 2 O had a significant impact on the loading and signal enhancement in Pt NPs on Ti 3 C 2 Tx [37].The T-line color intensity became stronger with an increasing amount of H 2 PtCl 6 •6H 2 O, but the inhibition ratio deteriorated, as shown in Figure S2A.The color intensity and inhibition ratio reached the desired level when the amount of H 2 PtCl 6 •6H 2 O was 25 µL.Therefore, 25 µL of H 2 PtCl 6 •6H 2 O was chosen as the optimal amount of H 2 PtCl 6 •6H 2 O.

Optimization of Synthesis Time
As the synthesis time increased, more Pt NPs were loaded onto Ti 3 C 2 Tx.As shown in Figure S2B, the loading amount of Pt NPs on Ti 3 C 2 Tx tended to reach saturation when the reaction time was 9 h.Meanwhile, the inhibition ratio and color intensity reached the ideal state.Therefore, the optimal synthesis time was 9 h.

The Optimization of the Reductant Amount
The more reductant was used, the smaller the particles.The smaller the amount of reductant, the larger the particles, which can increase the molar absorption coefficient and enhance color development [38].The color intensity and inhibition ratio were optimal at 60 µL of NaBH 4 , as shown in Figure S2C, so the optimal reductant amount was 60 µL.

Optimization of Ti 3 C 2 Tx@Pt-ICA
To achieve the best detection performance of Ti 3 C 2 Tx@Pt-ICA, we optimized various critical technical parameters, which included the coupling pH, the amount of Ab, blocking agent, catalytic reaction time, catalytic substrate and catalytic methods.

Optimization of Coupling pH
A suitable coupling pH is beneficial for the strong binding of Ab to Ti 3 C 2 Tx@Pt, improving the ability to recognize Ag [39]. Figure 2A shows that the T-line color intensity decreased with increasing pH, but the inhibition effect became better.The desired inhibition ratio and color intensity effect were achieved at a coupling pH of 8.4.Therefore, the optimal coupling pH was 8.4.

Optimization of Ab Amount
Too little Ab was not enough to achieve the desired color intensity on a competitive ICA.Excessive amounts of Ab will result in excess Ab binding to the coating Ag, thus reducing sensitivity [40].Figure 2B shows the T-line color intensity increased with increasing Ab content, but the inhibition became weaker.When the Ab amount was 40 µg, the ideal color intensity and best inhibition effects were obtained.Therefore, the optimal amount of Ab was chosen to be 40 µg.

Optimization of Blocking Agent
A suitable blocking agent can prevent the non-specific adsorption of nanoprobes and increase the sensitivity of the approach [41].Figure 2C shows four commonly used blocking agents (10% glycine, 10% PEG-2000, 10% BSA, 10% skim milk powder); using 10% BSA as the blocking agent resulted in a higher inhibition ratio and better color intensity.Therefore, the optimal blocking agent was chosen to be 10% BSA.

Optimization of Catalytic Reaction Time
The catalytic reaction time determined the effect of the signal amplification and detection sensitivity [42].The test strip was soaked in an AEC/H 2 O 2 solution and recorded the T-line color intensity from 10 to 40 s, respectively.As shown in Figure 2D, as the soaking time increased, the color intensity in the T-line became progressively stronger, but the inhibition effect was the best at 30 s.Thus, the optimal catalytic reaction time was chosen to be 30 s.

Optimization of Ab Amount
Too little Ab was not enough to achieve the desired color intensity on a competitive ICA.Excessive amounts of Ab will result in excess Ab binding to the coating Ag, thus reducing sensitivity [40].Figure 2B shows the T-line color intensity increased with increasing Ab content, but the inhibition became weaker.When the Ab amount was 40 µg, the ideal color intensity and best inhibition effects were obtained.Therefore, the optimal amount of Ab was chosen to be 40 µg.

Optimization of Blocking Agent
A suitable blocking agent can prevent the non-specific adsorption of nanoprobes and increase the sensitivity of the approach [41].Figure 2C shows four commonly used blocking agents (10% glycine, 10% PEG-2000, 10% BSA, 10% skim milk powder); using 10% BSA as the blocking agent resulted in a higher inhibition ratio and better color intensity.Therefore, the optimal blocking agent was chosen to be 10% BSA.

Optimization of Catalytic Substrate
The amplification effect of catalytic signals varies with different catalytic substrates.The catalytic effects of TMB and AEC are shown in Figure 2E; there was a phenomenon of the dissolution and diffusion of TMB after oxidation, which was consistent with previous reports [43].On the contrary, AEC precipitated on the T-line, producing a bright and clear red signal.

Optimization of Catalytic Methods
The results of two different catalytic methods are shown in Figure 2F.The soaking method was more effective than the spot sampling method in enhancing the color intensity of the strips, improving the smoothness of sample running and reducing background interference.Therefore, soaking was chosen as the optimal catalytic method.

Selectivity
Table S2 shows the selectivity results for Ti 3 C 2 Tx@Pt-ICA.CAP Ab showed a high CR on CAPSS which was 160.8%.This may be due to the similarity in structure between CAPSS and CAP [44].There was almost no CR for FF, FFA and TAP, and the CR value was below 0.1%.The results indicated that Ti 3 C 2 Tx@Pt-ICA can simultaneously detect

Selectivity
Table S2 shows the selectivity results for Ti 3 C 2 Tx@Pt-ICA.CAP Ab showed a high CR on CAPSS which was 160.8%.This may be due to the similarity in structure between CAPSS and CAP [44].There was almost no CR for FF, FFA and TAP, and the CR value was below 0.1%.The results indicated that Ti 3 C 2 Tx@Pt-ICA can simultaneously detect CAPSS and CAP.

Confirmatory Test
Table 1 shows that the developed method was in good agreement with LC-MS/MS, with a significant correlation rate of 0.98 (Figure S4).The data showed that the method we developed had excellent reliability for rapid testing CAP in animal tissues.

Comparison of ICA for CAP Detection
Table 2 summarizes the ICAs used to detect CAP.Firstly, on the ICA platform, the most commonly used labeling material was GNPs.This study prepared a Ti 3 C 2 Tx@Pt nanocomposite with peroxidase-like activity as the signal amplification label.The loading of noble Foods 2024, 13, 1416 11 of 14 metal Pt NPs enhanced the enzyme activity of Ti 3 C 2 Tx@Pt, giving it significant advantages in expanding the detection range.The linear range was doubled.Secondly, our test can detect milk, chicken and fish at the same time, while most of the approaches described previously can only detect one or two of them.Thus, the range of samples monitored was substantially increased by our method.Finally, the Ti 3 C 2 Tx@Pt nanocomposite had a large specific surface area and could carry more Ab, which increased the detection sensitivity up to 50-fold compared to the reported GNPs-ICA.Therefore, this method had significant advantages in terms of the linear detection range, sample adaptability and sensitivity.However, the material we used is still in the preliminary research stage compared to the traditional GNPs, and there is still a long way to go before commercialization.Therefore, strengthening the research of this material in the field of food safety testing, leveraging its advantages in sensitivity, stability, etc. and accelerating its commercialization process are the research directions we will strive for in the future.

Conclusions
In this study, Ti 3 C 2 Tx@Pt composites with excellent peroxidase-like activity were successfully synthesized, and AEC-based Ti 3 C 2 Tx@Pt-ICA was prepared for ultrasensitive CAP detection in milk, chicken and fish.The excellent enzyme-like activity of Ti 3 C 2 Tx@Pt was attributed to the abundance of oxygenated groups on the Ti 3 C 2 Tx MXene surface and the large specific surface area, which enabled it to load hundreds of Pt NPs.Ti 3 C 2 Tx@Pt can catalyze the color development of the chromogenic substrate AEC and achieve the catalytic amplification of the ICA signal.The developed Ti 3 C 2 Tx@Pt-ICA showed LODs and LOQs of 0.01 µg/kg and 0.03 µg/kg for milk, fish and chicken, respectively.Ti 3 C 2 Tx@Pt-ICA was 50-fold more sensitive than the reported GNPs-ICA.Finally, the Ti 3 C 2 Tx@Pt-ICA sensing platform was successfully applied to real sample detection.The results of the LC-MS/MS confirmation test on 30 real samples had a good correlation with that of our established method (R 2 > 0.98).This result tells us that in practical applications, we can first use our established method for rapid screening, and the suspected positive samples screened out can be confirmed by confirmation methods such as LC-MS/MS, which can greatly improve detection efficiency and reduce detection costs.Our research has pioneered the application of MXene (Ti 3 C 2 Tx@Pt) in the field of food safety detection, providing a new signal label for the establishment of high-performance ICA.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/foods13091416/s1, Figure S1: XRD spectra of Ti 3 AlC 2 .Figure S2: The optimization of Ti 3 C 2 Tx@Pt synthesis: (A) the amount of H 2 PtCl 6 6H 2 O, (B) the synthesis time of Ti 3 C 2 Tx@Pt and (C) the amount of reductant.Figure S3: A steady-state kinetic assay of Ti 3 C 2 Tx@Pt: (A) the double reciprocal plots between reaction velocity and TMB concentration and (B) the TMB concentration dependence of the initial reaction velocity (v). Figure S4: Correlation analysis between Ti 3 C 2 Tx@Pt-ICA and LC-MS/MS.Table S1: Comparison of the K m and V max of different enzymes; Table S2: The IC 50 and CR values of icELISA and Ti 3 C 2 Tx@Pt-ICA (n = 3); Table S3: The recovery of Ti 3 C 2 Tx@Pt-ICA for the detection of CAP in milk, chicken and fish samples (n = 3).

Figure 2 .
Figure 2. The synthesis optimization of Ti 3 C 2 Tx@Pt-ICA: including (A) the coupling pH value, (B) the amount of Ab, (C) the type of blocking agent, (D) the catalytic reaction time, (E) the type of catalytic substrate and (F) the type of catalytic methods.The inhibition ratio was calculated as indicated: inhibition ratio (%) = [(T/C negative − T/C positive )/(T/C negative )] × 100.

Foods 2024 , 15 Figure 3 .
Figure 3.A performance evaluation of Ti3C2Tx@Pt-ICA.The calibration curves of Ti3C2Tx@Pt-ICA for the detection of CAP in (A) milk, (B) chicken, (C) fish, respectively.The calibration curves of catalytic signals for the detection of CAP in (D) milk, (E) chicken, (F) fish, respectively.

Figure 3 .
Figure 3.A performance evaluation of Ti 3 C 2 Tx@Pt-ICA.The calibration curves of Ti 3 C 2 Tx@Pt-ICA for the detection of CAP in (A) milk, (B) chicken, (C) fish, respectively.The calibration curves of catalytic signals for the detection of CAP in (D) milk, (E) chicken, (F) fish, respectively.

Author Contributions:
Writing-original draft, methodology, software, formal analysis, M.L. and Z.G.; software, data curation, resources, Z.Q., Y.D. and Y.C.; resources, supervision, writingreview and editing, Y.W. and X.L.All authors have read and agreed to the published version of the manuscript.Funding: This work was funded by the National Key Research and Development Program of China (2023YFF1105004), the National Natural Science Foundation of China (32272404).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Determination of CAP in milk, chicken and fish by LC-MS/MS and Ti 3 C 2 Tx@Pt-ICA (n = 3).

Table 2 .
Comparison of ICAs for detecting CAP in animal-derived foods.