Preparation method of pt single-atom nanozyme and application in detection of cardiac troponin i

Abstract

The application relates to a preparation method of a Pt single-atom nanoscale enzyme and application of the Pt single-atom nanoscale enzyme in detection of cardiac troponin I, and the preparation method comprises the following steps: 1) ultrasonic dispersion of cobalt salt, selenium powder, chloroplatinic acid, urea and sodium chloride in an alcohol solvent, heating and stirring until the solvent is completely volatilized, grinding the solid mixture into a powder; 2) performing Joule heat treatment on the powder in a heating device under an air atmosphere, and performing washing and vacuum drying on the reactants to obtain the Pt single-atom nanoscale enzyme. In the one-step chemical reaction process, Se doping is introduced to construct oxygen vacancies, rapid heating and structure shaping are realized by combining the Joule heat treatment, a Pt single-atom catalyst loaded with Se-doped Co3O4 is obtained, the oxygen vacancies and the Se doping sites are used as anchor sites in cooperation, the Pt single atoms are effectively fixed, and the loading capacity and the dispersity of the Pt single atoms are improved. The obtained material shows excellent POD-like enzyme activity, can be used for constructing a high-sensitivity colorimetric immunoassay system, and realizes rapid and accurate detection of cTnI.

Description

Technical Field

[0001] This invention belongs to the field of nanozyme preparation technology, specifically relating to a method for preparing Pt single-atom nanozymes and their application in the detection of cardiac troponin I. Background Technology

[0002] Cardiac troponin I (cTnI) is an important biomarker for cardiovascular diseases such as acute myocardial infarction (AMI), and changes in its level provide crucial information for early diagnosis and prognostic assessment of heart disease. Detecting cTnI levels is essential for timely detection of cardiac damage and guiding clinical treatment. Currently, enzyme-linked immunosorbent assay (ELISA) is widely used clinically for the detection of cardiac troponin I due to its high sensitivity and specificity, and has achieved significant results. Traditional ELISA methods rely on natural enzymes as signal amplification markers; however, natural enzymes are sensitive to environmental factors, easily inactivated, and have high production costs, and their catalytic efficiency and stability are insufficient to meet the requirements of ultra-high sensitivity detection.

[0003] Nanozymes possess high stability, good tunability, and low cost, and can mimic the activity of natural enzymes, replacing traditional natural enzymes in various biochemical reactions to improve detection sensitivity and efficiency. However, existing nanozymes still have shortcomings in catalytic activity, especially in the detection of low-concentration analytes, where their catalytic efficiency is insufficient to meet the demands of high-sensitivity detection.

[0004] Single-atom catalysts significantly improve catalytic activity and selectivity by precisely positioning the catalytic active center as a single metal atom. Loading these single-atom catalysts onto oxide supports to form nanozymes not only further enhances catalytic activity and reaction stability but also effectively overcomes the insufficient catalytic efficiency of traditional nanozymes, thereby significantly improving the efficiency of enzyme-catalyzed reactions and enhancing the sensitivity of ELISA detection. However, traditional synthesis methods typically require the preparation of an oxide support before loading noble metal single atoms. This process is not only cumbersome and time-consuming, but also results in some metal atoms failing to be effectively loaded onto the support during the feeding process, leading to low atomic utilization of the noble metal. For example, Chinese invention patent publication number CN119114100B discloses a method for preparing single-atom peroxidase-like (POD) nanomaterials. This method involves reacting copper salts with amine compounds via a hydrothermal reaction to obtain defective metal oxides, then mixing these oxides with Os salts, stirring, and finally loading Os single atoms onto the oxides via an impregnation method. Although this method can achieve a certain degree of single-atom loading, its synthesis process is relatively complex. Furthermore, during the impregnation process, due to the limited binding efficiency between the metal oxide support and Os, it is difficult to fully utilize the Os atoms, resulting in a waste of precious metals and failing to meet the requirements for industrial applications.

[0005] Therefore, there is an urgent need to develop a simple, efficient, and more precious metal-utilizing method for synthesizing metal oxide-supported single-atom catalysts, so that metal oxide single-atom nanozymes can be better applied to cTnI activity detection, improve the detection sensitivity of nanozyme-based ELISA, and promote their widespread application in the diagnostic field. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide a method for preparing Pt single-atom nanozymes and their application in the detection of cardiac troponin I. This invention solves the problems of cumbersome reaction steps, low single-atom loading, easy aggregation and migration, and low catalyst activity and colorimetric immunoassay performance in the preparation of noble metal single-atom catalysts.

[0007] The first aspect of this invention is to provide a method for preparing Pt single-atom nanozymes, the method comprising the following steps:

[0008] 1) Cobalt salt, selenium powder, chloroplatinic acid, urea and sodium chloride are ultrasonically dispersed in an alcohol solvent, and then heated and stirred until the alcohol solvent is completely evaporated to obtain a solid mixture, which is then ground into powder;

[0009] 2) The powder was transferred to a quartz crucible and placed in the graphite sample stage groove of a Joule heating device. Joule heat treatment was carried out in an air atmosphere. The reaction product was centrifuged, washed, and vacuum dried to obtain Pt single-atom nanozyme.

[0010] Furthermore, in step 1), cobalt salt is cobalt dichloride, and alcohol solvent is ethanol.

[0011] Further, in step 1), the ratio of cobalt salt, selenium powder, chloroplatinic acid, urea, sodium chloride and alcohol solvent is: 1 mmol: 7-12 mmol: 0.002-0.007 mmol: 140-180 mmol: 30-50 mmol: 40-60 mL.

[0012] Furthermore, the heating temperature in step 1) is 50-80℃.

[0013] Furthermore, the Joule heat treatment conditions in step 2) are: heat treatment temperature 500-800℃, heating rate 1500-3000℃ / s, and 2-7 Joule heat treatments. One Joule heat treatment consists of heating from room temperature to the target temperature and then naturally cooling to room temperature.

[0014] Furthermore, the washing conditions in step 2) are: centrifugation with deionized water, washing 3 to 5 times, vacuum drying at a temperature of 40-80℃, and drying time of 5 to 10 hours.

[0015] A second aspect of the present invention is to provide a Pt single-atom nanozyme prepared by the above-described preparation method.

[0016] Furthermore, the platinum is dispersed in single-atom form on a selenium-doped Co3O4 support, which has oxygen vacancies and a hierarchical porous structure.

[0017] Furthermore, the specific surface area of ​​the Pt single-atom nanozyme is not less than 60 m² / g.

[0018] A third aspect of the invention is to provide the use of Pt single-atom nanozymes in the preparation of reagents for the detection of cardiac troponin I.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) This invention employs a salt and urea-assisted Joule heat treatment process to construct oxide-supported single atoms. Compared with traditional high-temperature calcination methods, this process requires no complex steps, is simple to operate, and offers high catalyst preparation efficiency and low energy consumption. Simultaneously, this method significantly improves the utilization rate of noble metal precursors, avoids metal waste, and effectively enhances the dispersion and stability of noble metal single atoms on the oxide support, demonstrating promising prospects for industrial application.

[0021] 2) This invention introduces Se doping to construct oxygen vacancies during the one-step synthesis process and combines it with Joule heat treatment to achieve rapid heating and cooling during synthesis, thereby effectively improving the defect concentration of the support and the loading, dispersion, and stability of Pt single atoms. Joule heat treatment has the characteristics of rapid heating and cooling, which can stabilize oxygen vacancies during crystal formation, thus significantly enhancing the bonding strength between Pt single atoms and the support. At the same time, due to the extremely rapid heating and cooling process, Pt elements do not undergo significant agglomeration, thereby achieving efficient single-atom dispersion and overcoming the problem of metal agglomeration under traditional calcination conditions.

[0022] 3) This invention utilizes urea to form stable chelates with Pt and Co ions, enabling a highly uniform distribution of metal elements at the molecular level. During pyrolysis, urea decomposes, releasing gases and generating carbon-based sacrificial templates, providing reaction sites for the nucleation and growth of Se-doped Co3O4 crystals, and inducing the formation of a hierarchical porous structure within the material. This significantly increases the specific surface area of ​​the resulting support, providing abundant anchoring sites for Pt single atoms, which is beneficial for the fixation and retention of single atoms, while also endowing the catalyst with good structural stability and high activity.

[0023] 4) This invention introduces sodium chloride into the system, creating a polar chemical environment during heat treatment. This not only effectively inhibits the aggregation of Pt atoms but also promotes their single-atom dispersion on the support surface. This polar environment also improves the stability of surface defects on the support, thereby further enhancing the uniformity and stability of Pt single-atom loading and providing a guarantee for constructing high-performance single-atom catalysts.

[0024] 5) The Se-doped Co3O4-supported Pt single-atom nanozyme prepared in this invention possesses an ultra-high specific surface area (65.27 m² / g), abundant oxygen vacancies, and uniformly dispersed Pt single atoms, exhibiting excellent POD catalytic activity. In the colorimetric immunoassay of cardiac troponin I, this nanozyme achieved an extremely low limit of detection (LOD 2.1 pg / mL), indicating that this material not only has outstanding advantages in basic catalytic performance but also demonstrates extremely high sensitivity and application potential in bioassay applications. Attached Figure Description

[0025] Figure 1 The image shows the X-ray powder diffraction (XRD) pattern of the sample from Example 1.

[0026] Figure 2 The images show scanning electron microscope (SEM) and elemental distribution (EDS mapping) images of the sample from Example 1.

[0027] Figure 3 The image shows a scanning transmission electron microscope (STEM) image of the sample from Example 1.

[0028] Figure 4 The image shows the results of a fully automated specific surface area and porosity analyzer (BET) for the sample in Example 1.

[0029] Figure 5 The image shows the electron paramagnetic resonance (EPR) image of the sample from Example 1.

[0030] Figure 6 The graph shows the POD performance test results of the sample in Example 1.

[0031] Figure 7 and Figure 8 This is a colorimetric immunoassay chromatogram of the sample from Example 1. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments in order to better understand the technical solution.

[0033] Example 1

[0034] (1) Disperse 1 mmol CoCl2, 9 mmol selenium powder, 0.006 mmol H2PtCl6, 160 mmol urea and 40 mmol NaCl in 50 mL of ethanol by ultrasonication, and then stir at 60 °C until the ethanol is completely evaporated. Grind the remaining solid into powder to obtain the first solid A.

[0035] (2) The first solid A was transferred to a quartz crucible and placed in the groove of the graphite sample stage of the Joule heating device. It was subjected to four Joule heat treatments in air atmosphere at a temperature of 600℃ and a heating rate of 2300℃ / s. The remaining solid was washed four times by centrifugation with deionized water and then vacuum dried at 60℃ for 8 hours to obtain Se-doped Co3O4-supported Pt single-atom nanozyme.

[0036] The product obtained in Example 1 was characterized as follows:

[0037] XRD diagram as follows Figure 1 As shown in the figure, the characteristic diffraction peaks in the figure are in high agreement with the standard card PDF#00-009-0418, indicating that the sample is composed of the Co3O4 phase.

[0038] SEM image as follows Figure 2 As shown, the results indicate that Se, Co, and O are uniformly distributed in the sample, indicating that Se has been successfully doped into Co3O4.

[0039] STEM image as follows Figure 3 As shown in the figure, the results indicate that Pt single atoms exhibit atomic-level dispersion on the Se-doped Co3O4 support.

[0040] BET diagram as shown Figure 4 As shown, the results indicate that the sample has a significant hysteresis loop, indicating that the material has a rich mesoporous structure and a large specific surface area of ​​65.27 m² / g.

[0041] EPR diagram as follows Figure 5 As shown in the figure, the results show that the sample has a strong EPR signal peak at g=2.003, indicating that there are abundant oxygen defects in the sample.

[0042] In summary, the sample prepared in Example 1 consists of Se-doped Co3O4 and Pt single atoms, exhibiting abundant mesoporous structure and oxygen vacancies. The atomic-level dispersion of Pt and oxygen vacancies enhance the active sites. Simultaneously, the mesoporous structure and larger specific surface area increase the contact area of ​​the reactants, thereby improving the overall catalytic performance of the sample in Example 1 during the POD reaction.

[0043] The POD activity of the sample obtained in Example 1 was tested using the following method:

[0044] Se-doped Co3O4-supported Pt single-atom nanozyme suspensions (1 mg / mL), TMB (1 mM), and H2O2 (80 mM) solutions were prepared using pure water. Subsequently, 50 µL of Se-doped Co3O4-supported Pt single-atom nanozyme, 150 µL of H2O2, and 200 µL of TMB were mixed in 2600 µL of sodium acetate-acetic acid (NaAc-HAc, pH=4.5, 0.2 M) buffer solution and incubated at room temperature for 20 minutes. The absorbance of the reaction solution was measured at 652 nm using a UV-Vis spectrophotometer to evaluate the activity of the Se-doped Co3O4-supported Pt single-atom nanozyme.

[0045] Test results are as follows Figure 6 As shown, Figure 6 The POD enzyme activity of the samples prepared in Examples 2-3 and Comparative Examples 1-5 was also compared. Figure 6 It can be seen that the order of the absorption peak intensity at 652 nm represents the order of POD enzyme activity. The results are: Example 1 > Example 3 > Example 2 > Comparative Example 4 > Comparative Example 5 > Comparative Example 1 > Comparative Example 3 > Comparative Example 2. This shows that the Se-doped Co3O4-loaded Pt single-atom nanozyme (Example 1 sample) prepared under the synthesis conditions of Example 1 has the best POD activity.

[0046] The samples obtained in Example 1 were subjected to colorimetric immunoassay, and the test method is as follows:

[0047] 2 μL of monoclonal antibody mAb1 (12 μg / mL) was mixed with 50 μL of PBS-Tween solution and added to the wells of a 96-well plate, then incubated at 4°C for 12 hours. Subsequently, the plate was washed with 10 mM PBS buffer (containing 0.05% Tween 20, pH 7.4) to remove excess mAb1. Next, unbound nonspecific binding sites were blocked with 1% (w / v) bovine serum albumin (BSA), and the plate was washed again with PBS buffer to remove excess BSA. 50 μL of human serum sample was added to each well and incubated at 37°C for 120 minutes. Then, 50 μL of pAb2-SiO2-ALP (12 μg / mL) was added and incubated at 37°C for 60 minutes. After each incubation, the plate was washed three times with PBS buffer. Next, 50 μL of 1.0 mM ascorbic acid 2-phosphate (AAP) solution was added to each well, and the mixture was incubated at 37°C for 60 minutes. ALP catalyzed the conversion of AAP to ascorbic acid (AA). Finally, the enzymatic product generated from the reaction was transferred to the Example 1 sample-TMB solution, which consisted of 50 μL of Example 1 sample (1 mg / mL), 50 μL of TMB (10 mM), and 200 μL of H2O2 (20 mM). The absorbance of the colorimetric reaction was finally measured at a wavelength of 652 nm.

[0048] Test results are as follows Figure 7 and Figure 8 As shown, by Figure 7 It can be seen that within the low concentration range of cTnI, the absorbance of the colorimetric reaction gradually decreases with increasing cTnI concentration; while within the high concentration range of cTnI, the absorbance of the colorimetric reaction changes less, indicating that high concentrations of cTnI can fully trigger the enzymatic reaction, and the generated product effectively reduces oxidized TMB (ox-TMB). Figure 8 It was found that the response of the target signal was negatively correlated with the absorbance of the TMB solution from Example 1, and exhibited a good linear relationship within a dynamic range of 0.003 to 70 ng / mL. The fitting equation was: Y = -0.28947 × lg C[cTnI] + 0.63631 (R² = 0.99692, n = 12), with a detection limit of 2.1 pg / mL. This indicates that the sample from Example 1 exhibits excellent performance in terms of linear range and LOD in colorimetric immunoassay.

[0049] Example 2

[0050] (1) Disperse 1 mmol CoCl2, 7 mmol selenium powder, 0.002 mmol H2PtCl6, 140 mmol urea and 30 mmol NaCl in 40 mL of ethanol by ultrasonication, and then stir at 50 °C until the ethanol is completely evaporated. Grind the remaining solid into powder to obtain the first solid A.

[0051] (2) The first solid A was transferred to a quartz crucible and placed in the groove of the graphite sample stage of the Joule heating device. It was subjected to two Joule heat treatments in air atmosphere at a temperature of 800℃ and a heating rate of 1500℃ / s. The remaining solid was centrifuged with deionized water, washed three times, and vacuum dried at 40℃ for 10h to obtain Se-doped Co3O4-supported Pt single-atom nanozyme.

[0052] The samples prepared in Example 2 were subjected to XRD and BET tests, and the test results are shown in Table 1.

[0053] As shown in Table 1, the XRD test results of the sample in Example 2 indicate that the sample is composed of the Co3O4 phase, which is consistent with the phase composition of the sample in Example 1. Furthermore, the BET test results of the sample in Example 2 show that it also possesses abundant mesoporous structure and a large specific surface area of ​​60.82 m² / g.

[0054] The samples prepared in Example 2 were subjected to POD activity testing and colorimetric immunoassay. The test results are shown in [Figure 1]. Figure 6 See Table 1.

[0055] Depend on Figure 6 As shown in Table 1, the POD enzyme activity exhibited by the sample in Example 2 was superior to that of the comparative sample, with a LOD of 4.7 pg / mL, significantly lower than that of the comparative sample. This indicates that the Se-doped Co3O4-supported Pt single-atom nanozyme synthesized under the conditions of Example 2 possesses excellent POD activity.

[0056] Example 3

[0057] (1) Disperse 1 mmol CoCl2, 12 mmol selenium powder, 0.007 mmol H2PtCl6, 180 mmol urea and 50 mmol NaCl in 60 mL of ethanol by ultrasonication, and then stir at 80 °C until the ethanol is completely evaporated. Grind the remaining solid into powder to obtain the first solid A.

[0058] (2) The first solid A was transferred to a quartz crucible and placed in the groove of the graphite sample stage of the Joule heating device. It was subjected to 7 Joule heat treatments in air atmosphere at a temperature of 500℃ and a heating rate of 3000℃ / s. The remaining solid was centrifuged with deionized water, washed 5 times, and vacuum dried at 80℃ for 5h to obtain Se-doped Co3O4-supported Pt monoprim nanozyme.

[0059] The samples prepared in Example 3 were subjected to XRD and BET tests, and the test results are shown in Table 1.

[0060] As shown in Table 1, the XRD test results of the sample in Example 3 indicate that the sample is composed of the Co3O4 phase, which is consistent with the phase composition of the sample in Example 1. Furthermore, the BET test results of the sample in Example 3 show that it also possesses a rich mesoporous structure and a large specific surface area, which is 67.41 m² / g.

[0061] The samples prepared in Example 3 were subjected to POD activity testing and colorimetric immunoassay. The test results are shown in [Figure 1]. Figure 6 See Table 1.

[0062] Depend on Figure 6 As shown in Table 1, the absorbance of the sample in Example 3 at 652 nm was 0.2486, indicating superior peroxidase-like activity compared to the comparative sample. Its LOD (Level of Displacement) was 3.4 pg / mL, significantly lower than that of the comparative sample. This demonstrates that the Se-doped Co3O4-supported Pt single-atom nanozyme synthesized under the conditions of Example 3 also exhibits good POD activity.

[0063] Comparative Example 1

[0064] The difference between Comparative Example 1 and Example 1 is that no selenium powder was added in step (1), while everything else remained the same.

[0065] The sample prepared in Comparative Example 1 was subjected to XRD, BET, and EPR tests. The test results are shown in Table 1 and 2. Figure 5 (EPR).

[0066] XRD results of Comparative Example 1 showed that the sample consisted of Co3O4 and Pt phases, indicating that the Pt in Comparative Example 1 existed in the form of nanoparticles rather than single Pt atoms. Furthermore, the specific surface area of ​​Comparative Example 1 was 59.75 m² / g, significantly lower than that of Example 1. The EPR signal peak intensity exhibited by Comparative Example 1 at g=2.003 was also weaker than that of the Example 1 sample. Figure 5 This indicates that the oxygen defect content of the Comparative Example 1 sample is much lower than that of the Example sample. The main reason is that the Se doping in the Example can partially replace the O element positions in Co3O4, thereby forming oxygen vacancies.

[0067] The POD activity test and colorimetric immunoassay were performed on the sample of Comparative Example 1. The POD performance test method was the same as that for the sample of Example 1. The test results are shown in [Figure 1]. Figure 6 See Table 1.

[0068] from Figure 6 As shown in Table 1, Comparative Example 1 had an absorbance of 0.1346 at 652 nm, indicating significantly lower POD-like activity compared to the Example samples. Simultaneously, its LOD was 77.5 pg / mL, far exceeding that of the Example samples. These results demonstrate that the lack of Se doping leads to a reduction in the number of oxygen vacancies, preventing Pt from being dispersed atomically and instead forming Pt nanoparticles. This significantly reduces the number of Pt active sites, ultimately resulting in weakened enzyme activity.

[0069] Comparative Example 2

[0070] The difference between Comparative Example 2 and Example 1 is that chloroplatinic acid was not added in step (1), while everything else remained the same.

[0071] XRD and BET tests were performed on the comparative sample 2, and the test results are shown in Table 1.

[0072] As shown in Table 1, the sample prepared in Comparative Example 2 is composed of the Co3O4 phase. Furthermore, similar to the samples in the examples, the specific surface area of ​​the sample in Comparative Example 2 is 58.98 m² / g, indicating that the sample in Comparative Example 2 has a high specific surface area.

[0073] The samples prepared in Comparative Example 2 were subjected to POD activity testing and colorimetric immunoassay. The POD performance testing method was the same as that used for the samples in Example 1. The test results are shown in [Figure 1]. Figure 6 See Table 1.

[0074] Depend on Figure 6 As shown in Table 1, Comparative Example 2 had an absorbance of 0.1788 at 652 nm, indicating that its peroxidase-like activity was lower than that of the Example sample. Furthermore, its LOD was 110.3 pg / mL, significantly higher than that of the Example sample. This suggests that Pt single atoms were the main POD nanozyme active sites in the examples.

[0075] Comparative Example 3

[0076] The difference between Comparative Example 3 and Example 1 is that no urea was added in step (1), while everything else remained the same.

[0077] The samples prepared from the three comparative examples were subjected to XRD and BET tests, and the test results are shown in Table 1.

[0078] As shown in Table 1, the XRD results of the sample prepared in Comparative Example 3 indicate that the sample consists of Co3O4 and Pt phases, suggesting that the Pt in Comparative Example 3 exists in the form of nanoparticles rather than single Pt atoms. Furthermore, the specific surface area of ​​the sample in Comparative Example 3 is 33.36 m² / g, which is significantly lower than the test results of the sample in Example 1. In the absence of urea, the dispersion of Pt and Co ions is poor, and the lack of gas generation and carbon-based template results in insufficient pore structure and specific surface area of ​​the Se-doped Co3O4, thus causing Pt to exist in the form of nanoparticles rather than single Pt atoms.

[0079] The sample prepared in Comparative Example 3 was subjected to POD activity testing and colorimetric immunoassay. The POD performance testing method was the same as that used for the sample in Example 1. The test results are shown in [Figure 1]. Figure 6 See Table 1.

[0080] Depend on Figure 6 As shown in Table 1, the absorbance of Comparative Example 3 sample at 652 nm was 0.13, indicating that its POD activity was lower than that of the Example sample. Furthermore, its LOD was 92.6 pg / mL, significantly higher than that of the Example sample. This suggests that urea effectively disperses Pt and Co ions by chelating with metal ions. During high-temperature calcination, urea decomposes to generate gas and forms a carbon-based template, promoting the uniform dispersion of Se-doped Co3O4, increasing the pore structure and specific surface area, and simultaneously contributing to the uniform distribution of Pt single atoms. These changes not only optimize the material structure but also significantly enhance the POD activity.

[0081] Comparative Example 4

[0082] The difference between Comparative Example 4 and Example 1 is that sodium chloride was not added in step (1), while everything else remained the same.

[0083] The samples prepared in Comparative Example 4 were subjected to XRD, BET, and EPR tests, respectively. The test results are shown in Table 1 and 2. Figure 5 (EPR).

[0084] As shown in Table 1, the XRD results of Comparative Example 4 indicate that the sample consists of Co3O4 and Pt phases, suggesting that the Pt in Comparative Example 4 exists in the form of nanoparticles rather than single Pt atoms. Furthermore, the specific surface area of ​​Comparative Example 4 is 50.23 m² / g, lower than that of Example 1. Compared to Example 1, the EPR signal peak of Comparative Example 4 at g=2.003 is weaker (…). Figure 5The results indicate that the oxygen defect concentration in Comparative Example 4 was lower than that in Example 1. This is mainly because the addition of sodium chloride created a polar chemical environment during the heat treatment process, which not only effectively suppressed the aggregation of Pt atoms but also promoted their single-atom dispersion on the support surface. This polar environment also improved the stability of defects on the support surface, thereby further improving the uniformity and stability of Pt single-atom loading and providing a guarantee for the construction of high-performance single-atom catalysts.

[0085] The sample prepared in Comparative Example 4 was subjected to POD activity testing and colorimetric immunoassay. The POD performance testing method was the same as that used for the sample in Example 1. The test results are shown in [Figure 1]. Figure 6 See Table 1.

[0086] from Figure 6 As shown in Table 1, the absorbance of Comparative Example 4 sample at 652 nm was 0.12, indicating that its peroxidase-like activity was lower than that of the Example sample. Simultaneously, its LOD was 40.2 pg / mL, significantly higher than that of the Example sample. These results suggest that the absence of sodium chloride resulted in Pt existing in nanoparticle form rather than as single Pt atoms, which simultaneously reduced the oxygen vacancy concentration in the carrier. This significantly reduced the number of active sites in Comparative Example 4 sample, ultimately leading to weakened enzyme activity.

[0087] Comparative Example 5

[0088] The difference between Comparative Example 5 and Example 1 lies in the heating device used in step (1). The Joule heating device is replaced with a muffle furnace, while the other steps remain unchanged. The specific operation is as follows:

[0089] The first solid A was transferred to a quartz crucible and placed in a muffle furnace for calcination in air atmosphere at a temperature of 600℃ and a heating rate of 5℃ / min. The reaction was held at this temperature for 1 hour. The remaining solid was centrifuged with deionized water, washed four times, and vacuum dried at 60℃ for 8 hours to obtain the comparative sample 5.

[0090] XRD, BET, and EPR tests were performed on the five comparative samples, and the results are shown in Table 1 and 2. Figure 5 (EPR).

[0091] As shown in Table 1, the XRD results of Comparative Example 5 indicate that the sample consists of Co3O4 and Pt phases, suggesting that the Pt in Comparative Example 5 exists in the form of nanoparticles rather than single Pt atoms. Furthermore, the specific surface area of ​​Comparative Example 5 is 45.30 m² / g, significantly lower than that of Example 1. The EPR signal peak intensity of Comparative Example 5 at g=2.003 is weaker than that of Example 1. Figure 5This is mainly because Joule heat treatment has the characteristics of rapid heating and cooling, which can avoid the sintering of Se-doped Co3O4 caused by prolonged high-temperature calcination. At the same time, it stabilizes oxygen vacancies during the formation of Se-doped Co3O4, thereby significantly enhancing the bonding strength between Pt single atoms and the Se-doped Co3O4 support. Furthermore, due to the extremely rapid heating and cooling process, Pt elements do not undergo significant agglomeration, thus achieving highly efficient single-atom dispersion.

[0092] POD activity was tested on Comparative Example 5. The POD performance testing method was the same as that used for Sample 1 in Example 1. The test results are shown in [Figure 1]. Figure 6 See Table 1.

[0093] from Figure 6 As shown in Table 1, the absorbance of Comparative Example 5 sample at 652 nm was 0.1134, indicating that its peroxidase-like activity was lower than that of Examples 1-3. Meanwhile, its LOD was 68.7 pg / mL, significantly higher than that of Example 1 sample. This is mainly because, compared to Comparative Example 5, the Example samples have a higher specific surface area and more abundant oxygen vacancies, both of which are beneficial for the adsorption of reactants and the desorption of products during the enzyme-catalyzed reaction. Furthermore, the Pt single atoms in the Example samples expose more active sites, thus enabling the Example samples to exhibit superior POD catalytic activity and a lower detection limit for cardiac troponin I.

[0094] Table 1

[0095] Name XRD BET(m 2 / g) Limit of detection (pg / mL) Example 1 Co304, Pt monatomic 65.27 2.1 Example 2 Co304, Pt monatomic 60.82 4.7 Example 3 Co304, Pt monatomic 67.41 3.4 Comparative Example 1 Co304, Pt nanoparticles 59.75 77.5 Comparative Example 2 Co304 58.98 110.3 Comparative Example 3 Co304, Pt nanoparticles 33.36 92.6 Comparative Example 4 Co304, Pt nanoparticles 50.23 40.2 Comparative Example 5 Co304, Pt nanoparticles 45.30 68.7 .

Claims

1. A method for preparing a Pt single-atom nanozyme, characterized in that, The preparation method includes the following steps: 1) Cobalt salt, selenium powder, chloroplatinic acid, urea, and sodium chloride are ultrasonically dispersed in an alcohol solvent, and then heated and stirred until the alcohol solvent is completely evaporated to obtain a solid mixture, which is then ground into powder; the ratio of the amount of cobalt salt, selenium powder, chloroplatinic acid, urea, sodium chloride, and alcohol solvent is: 1 mmol: 7-12 mmol: 0.002-0.007 mmol: 140-180 mmol: 30-50 mmol: 40-60 mL; 2) Transfer the powder to a quartz crucible and place it in the graphite sample stage groove of the Joule heating device. Perform Joule heat treatment in an air atmosphere. After centrifugation, washing, and vacuum drying, Pt single-atom nanozyme is obtained. The Joule heat treatment conditions are: heat treatment temperature 500-800℃, heating rate 1500-3000℃ / s, and 2-7 Joule heat treatments. One Joule heat treatment is performed by heating from room temperature to the target temperature and then naturally cooling to room temperature.

2. The method for preparing a Pt single-atom nanozyme as described in claim 1, characterized in that, In step 1), cobalt salt is cobalt chloride, and alcohol solvent is ethanol.

3. The method for preparing a Pt single-atom nanozyme as described in claim 1, characterized in that, The heating temperature in step 1) is 50-80℃.

4. The method for preparing a Pt single-atom nanozyme as described in claim 1, characterized in that, The washing conditions in step 2) are: centrifugation with deionized water, washing 3 to 5 times, vacuum drying temperature of 40-80℃, and drying time of 5 to 10 hours.

5. Pt single-atom nanozymes prepared by any of the preparation methods of claims 1-4.

6. The Pt single-atom nanozyme as described in claim 5, characterized in that, The platinum is dispersed in single-atom form on a selenium-doped Co3O4 support, which has oxygen vacancies and a hierarchical porous structure.

7. The Pt single-atom nanozyme as described in claim 6, characterized in that, The specific surface area of ​​the Pt single-atom nanozyme is not less than 60 m² / g.

8. The use of the Pt single-atom nanozyme as described in claim 5 in the preparation of reagents for detecting cardiac troponin I.

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