A diatomic nano-enzyme-based rapid detection method for cephalexin and lead

Abstract

This invention discloses a rapid detection method for cephalexin and lead using a diatomic nanozyme. The method involves synthesizing Fe and Cu coordinated teratophorphene (4-carboxyphenyl)-1, which is then pyrolyzed at high temperature to form a diatomic nanozyme (Cu,Fe-N-C). The coexistence of iron and copper active sites in Cu,Fe-N-C exhibits high peroxidase-mimicking enzyme activity due to its synergistic effect. Cephalexin significantly inhibits the Cu,Fe-N-C + TMB + H₂O₂ system. The complexation of cephalexin with metallic Cu reduces the affinity of Cu,Fe-N-C for the substrate and the number of active sites, leading to a decrease in the absorbance of the system. Meanwhile, Pb... 2+ The addition of Pb restored the activity of the peroxidase nanozyme. 2+ Competitively forming a complex with cephalexin, it releases the iron and copper active sites in the Cu,Fe-N-C nanozyme, leading to increased affinity of Cu,Fe-N-C for the substrate and a corresponding increase in absorbance. This establishes a novel, highly sensitive, and selective rapid detection method for cephalexin and lead, with limits of quantitation of 0.010 and 0.012 mg / kg, respectively, meeting relevant national food safety requirements. This method is characterized by its simplicity, high sensitivity, and rapid operation.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a rapid detection method for cephalexin and lead using a two-atom nanozyme base. Background Technology

[0002] Cephalexin is a β-lactam antibiotic with a broad antibacterial spectrum, no cross-resistance, low residue, and minimal toxicity, making it widely used in clinical medicine and veterinary medicine. Therefore, research on the determination of cephalexin is of great significance. Currently, reported methods for the determination of cephalexin include: microbial detection methods, high-performance liquid chromatography, photochemical fluorescence analysis, and optical rotation. Lead ions (Pb) 2+ Pb is a non-degradable toxic metal ion. The World Health Organization (WHO) recommends a minimum daily intake of Pb in drinking water. 2+ The maximum limit for Pb in food is 10 μg / L. 2+ The maximum limit is 0.02-0.5 mg / kg, for the determination of Pb. 2+ Lead content is a crucial aspect of food safety testing. Currently, methods for lead determination primarily include atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), inductively coupled plasma mass spectrometry (ICP-MS), electrochemical methods, and colorimetric probe methods. Among these, colorimetric probe methods have garnered significant attention due to their simplicity, speed, and lack of complex instrumentation. To achieve Pb... 2+ For ultrasensitivity measurements, signal amplification is usually required to obtain good sensitivity and low detection limits.

[0003] Single-atom nanozymes (SAzymes), with isolated metal atoms on a support, are a novel type of nanozyme with atomically dispersed active sites. They hold significant importance in the development of nanozymes due to their high catalytic activity, maximum utilization efficiency of metal atoms, and simple modeling of active sites. Currently, research on single-atom nanozymes focuses on single metal atoms and related ligands, with limited research on nanozymes using nitrogen-doped carbon (FeCu / NC) with bimetallic atomic sites. Our research group's findings indicate that FeCu / NC exhibits strong enzyme-mimicking activity. This enhanced performance can be attributed to a unique synergistic effect between Cu and Fe single atoms. Specifically, Cu-N4 acts as an electron donor, increasing the electron density of the Fe-N4 active center, thereby promoting O2 activation.

[0004] This invention synthesizes Fe and Cu coordinated medium-tetra(4-carboxyphenyl)porphyrin, which is then pyrolyzed at high temperature to form a diatomic nanozyme (Cu,Fe-NC). The coexistence of iron and copper active sites in Cu,Fe-NC exhibits high peroxidase-mimicking enzyme activity due to their synergistic effect. Cephalexin significantly inhibits the Cu,Fe-N-C+TMB+H2O2 system. The complexation of cephalexin with metallic Cu reduces the affinity of Cu,Fe-NC for the substrate and the number of active sites, leading to a decrease in the system's absorbance. Meanwhile, Pb... 2+ The addition of Pb restored the activity of the peroxidase nanozyme. 2+ Competitively forming a complex with cephalexin, it releases the iron and copper active sites in the Cu,Fe-NC nanozyme, leading to increased affinity of Cu,Fe-NC for the substrate and a corresponding increase in absorbance. This establishes a novel, highly sensitive, and selective rapid detection method for cephalexin and lead, with limits of quantitation of 0.010 mg / kg and 0.012 mg / kg, respectively. This method meets relevant national food safety requirements and features simple operation, high sensitivity, and rapid detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a rapid detection method for cephalexin and lead using a two-atom nanozyme, utilizing the inhibitory effect of cephalexin on the activity of Cu,Fe-NC nanozymes and the effect of Pb... 2+ The method for rapid "off-on" colorimetric detection of cephalexin and lead was established to restore the activity of the nanozyme inhibition system.

[0006] The method for rapid detection of cephalexin and lead using a dual-atom nanozyme-based approach of the present invention is as follows:

[0007] (1) Using methyl-tetra(4-carboxyphenyl)porphyrin as a ligand (TCPP), Cu,Fe-TCPP complexes were synthesized. Cu,Fe-TCPP was used as a precursor for high-temperature pyrolysis and acid treatment to obtain diatomic Cu,Fe-NC nanozymes.

[0008] (2) Add Cu,Fe-NC nanozyme solution to cefalexin (CEX) standard solution, then add 3,3',5,5'-tetramethylbenzidine (TMB) solution and H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well, generate blue oxTMB oxide, let stand for 5-10 min, centrifuge, take the supernatant and measure the absorbance at 654 nm wavelength, establish the quantitative relationship between absorbance and cefalexin concentration, draw a standard curve, and obtain the regression equation;

[0009] (3) Add Cu,Fe-NC nanozyme solution and Pb to cephalexin solution. 2+The standard solution was prepared, followed by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) solution and H2O2. 0.1 mmol / L pH 4.0 HAc-NaAc buffer was added to a final volume of 3 mL, and the mixture was shaken well. Blue oxTMB oxide was generated. The mixture was allowed to stand for 5-10 min, centrifuged, and the supernatant was measured at 654 nm. An analogy was established between absorbance and Pb. 2+ The quantitative relationship of concentration was determined, a standard curve was plotted, and the regression equation was obtained.

[0010] (4) Add Cu,Fe-NC nanozyme solution to the sample solution containing cephalexin, then add 3,3',5,5'-tetramethylbenzidine (TMB) solution and H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well, let stand for 5-10 min, centrifuge, take the supernatant and measure the absorbance at 654 nm wavelength, substitute into the regression equation, and calculate the concentration of cephalexin in the sample solution.

[0011] In Pb 2+ Cu,Fe-NC nanozyme solution was added to the sample solution to be tested, followed by 3,3',5,5'-tetramethylbenzidine (TMB) solution, H2O2, and cephalexin solution. Then, 0.1 mmol / L pH 4.0 HAc-NaAc buffer was added to a final volume of 3 mL. The mixture was shaken well, allowed to stand for 5-10 min, centrifuged, and the supernatant was measured at 654 nm. The absorbance was then substituted into the regression equation to calculate the Pb concentration in the sample solution. 2+ concentration;

[0012] The Cu,Fe-NC nanozyme was prepared as follows:

[0013] (1) Weigh 2.5-3.0g CuCl2 and dissolve it in 10-15mL methanol, mix it evenly by ultrasonication, and use it as solution A; dissolve 4.5-5.5g of medium-tetra(4-carboxyphenyl)porphyrin (TCPP) and 2.0-2.5g of FeCl3 in 20-25mL methanol, mix them evenly by ultrasonication, and use it as solution B; mix solutions A and B evenly and dry them in an oven at 80-90℃ for 2h to obtain dark green dry powder Cu,Fe-TCPP;

[0014] (2) Weigh 4.5-5.5g of Cu,Fe-TCPP dry powder into a quartz boat, and calcine it in a tube furnace at a heating rate of 5°C / min to 750°C under N2 protection for 2-2.5 h to obtain a black powder. Soak the powder in sulfuric acid solution (H2SO4:H2O=1:9, volume ratio) for 12 h, rinse it repeatedly with water, and dry it to obtain Cu,Fe-NC nanozyme;

[0015] The concentration of the cephalexin standard solution is 0.010~52.76 mg / L, Pb 2+ The concentration of the standard solution was 0.012–45.38 mg / L; the concentration of Cu,Fe-NC nanozyme was 0.2 mg / mL, and the amount added was 100 μL; the concentration of TMB solution was 5 mmol / L, and the amount added was 20–200 μL; the concentration of H2O2 was 50 mmol / L, and the amount used was 20–200 μL.

[0016] The centrifugation was performed at 8000-10000 r / min for 5-10 min;

[0017] Advantages and technical effects of the present invention:

[0018] 1. This invention introduces bimetallic atoms (Fe, Cu) into a dual-site nanozyme with methyl-tetra(4-carboxyphenyl)porphyrin as a ligand to prepare a biatomic embedded nitrogen-doped carbon nanozyme (Cu,Fe-NC). Due to the unique synergistic effect between Cu and Fe single atoms, Cu-N4 acts as an electron donor, increasing the electron density of the Fe-N4 active center, giving Cu,Fe-NC ultra-high peroxidase-like activity. In the presence of H2O2, TMB is oxidized to blue oxTMB, while the addition of cephalexin inhibits the peroxidase activity of Cu,Fe-NC. Due to the reduction of the active sites in Cu,Fe-NC, when Pb... 2+ When present, it can interact with cephalexin, thereby releasing Cu,Fe-NC active sites, restoring Cu,Fe-NC activity, and resulting in increased absorbance, thus establishing a new colorimetric detection method for cephalexin and lead;

[0019] 2. The colorimetric detection method for cephalexin and lead established in this invention has high detection sensitivity, with limits of quantitation for cephalexin and lead reaching 0.010 and 0.012 mg / L, respectively. Other coexisting metal ions, other β-lactam antibiotics, and coexisting substances do not interfere with the determination, and the method has good selectivity.

[0020] 3. The method of the present invention can be used to detect cephalexin and lead residues in various foods. It has a high recovery rate, accurate detection of standard substances, and features high sensitivity, strong specificity, simple operation, and speed. Attached Figure Description

[0021] Figure 1 TEM image of Cu,Fe-NC prepared in Example 1 of this invention;

[0022] Figure 2The solutions in Example 1 of this invention are (Cu,Fe-N-C+TMB +H2O2), (Cu,Fe-N-C+TMB +H2O2+CEX), and (Cu,Fe-N-C+TMB +H2O2+CEX + Pb). 2+ The ultraviolet absorption spectrum of )

[0023] Figure 3 This is a diagram showing the verification results of the Cu-N4 and Fe-N4 active sites of the Cu,Fe-NC nanozyme in Example 1 of this invention.

[0024] Figure 4 The Michaelis-Menten kinetic curves for the oxidation of H2O2+TMB by Cu,Fe-NC in Example 1 are shown.

[0025] Figure 5 The Michaelis-Menten kinetic curves for the oxidation of H2O2+TMB by Cu,Fe-N-C+CEX in Example 1 are shown.

[0026] Figure 6 shows the Cu,Fe-N-C+CEX+ Pb example 1. 2+ Michaelis kinetics of H2O2+TMB oxidation;

[0027] Figure 7 The CEX linear UV-Vis absorption spectrum (left) and regression equation (right) of the Cu,Fe-N-C+TMB+H2O2 system in Example 1 are shown.

[0028] Figure 8 For the detection of Pb in the Cu,Fe-N-C+TMB+H2O2+ CEX system in Example 1 2+ Linear UV-Vis absorption spectrum (left) and regression equation (right);

[0029] Figure 9 The results of the influence of coexisting substances on CEX in Example 1;

[0030] Figure 10 In Example 1, the coexisting metal ion pair is Pb 2+ The impact of the results. Implementation

[0031] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto;

[0032] Example 1: CEX in milk samples and Pb in edible fungi 2+ Measurement

[0033] 1. Preparation of Cu,Fe-NC nanozymes

[0034] (1) Weigh 2.5g CuCl2 and dissolve it in 10 mL of methanol, mix it evenly by ultrasonication, and use it as solution A; dissolve 4.5g of medium-tetra(4-carboxyphenyl)porphyrin (TCPP) and 2.0g of FeCl3 in 20 mL of methanol, mix them evenly by ultrasonication, and use it as solution B; mix solutions A and B evenly and dry them in an oven at 80℃ for 2 hours to obtain dark green dry powder Cu,Fe-TCPP;

[0035] (2) Weigh 4.5 g of dried Cu,Fe-TCPP powder into a quartz boat and calcine it in a tube furnace at a heating rate of 5°C / min to 750°C for 2 h under N2 protection to obtain a black powder. Soak the powder in sulfuric acid solution (H2SO4:H2O=1:9, volume ratio) for 12 h, rinse it repeatedly with water, and dry it to obtain Cu,Fe-NC nanozyme. Figure 1 TEM images of Cu,Fe-TCPP were prepared. As expected, TEM characterization confirmed the nanosheet structure of the nanozyme, and no aggregated Cu and Fe species were observed after post-treatment.

[0036] 2. Evaluation of Cu,Fe-NC nanozyme peroxidase activity: Take 100 μL of 5 mmol / L TMB, add 100 μL of 0.2 mg / mL Cu,Fe-NC and 100 μL of 50 mmol / L H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer solution to 3 mL, shake well, let stand for 10 min, centrifuge at 10000 r / min for 5 min, and measure the absorbance of the supernatant at 654 nm. Simultaneously, take 100 μL of 0.2 mg / mL Cu,Fe-NC and 100 μL of 10 mg / L CEX aqueous solution, add 100 μL of 5 mmol / L TMB and 100 μL of 50 mmol / L H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer solution to 3 mL, shake well, let stand for 5-10 min, centrifuge at 10000 r / min for 5 min. After min, the supernatant was taken and its absorbance was measured at 654 nm. 100 μL of 0.2 mg / mL Cu,Fe-NC and 100 μL of 10 mg / L CEX aqueous solution were added, along with 100 μL of 10 mg / L Pb. 2+Then add 100 μL of 5 mmol / L TMB and 100 μL of 50 mmol / L H2O2, and add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL. Shake well, let stand for 5-10 min, centrifuge at 10000 r / min for 5 min, and measure the absorbance of the supernatant at 654 nm. The results are as follows. Figure 2 Cu,Fe-NC oxidation of TMB exhibits strong peroxidase activity under acidic conditions. The absorbance of the system is significantly inhibited upon the addition of CEX, while the addition of Pb... 2+ Subsequently, the absorbance of the system recovered and increased. Simultaneously, referring to the literature (Anal. Chem. 2020, 92, 3373−3379), KSCN was used to block Cu and Fe sites to confirm the active sites, and the results were as follows. Figure 3 When KSCN was added, the absorbance of the Cu,Fe-N-C+TMB+H2O2 system decreased significantly, which proves that Cu-N4 and Fe-N4 are active sites.

[0037] The experiment also included the determination of Michaelis catalytic kinetic parameters. Figure 4 , Figure 5 , Figure 6 (and Table 1), before and after adding CEX and after adding Pb 2+ The subsequent Michaelis constant K m The reaction rate constants were 3.25 × 10⁻⁶ mM, 1.150 mM, and 0.534 mM, respectively. -8 M∙s -1 1.57×10 -8 M∙s -1 and 2.80×10 -8 M∙s -1 This indicates that the addition of CEX reduced the affinity of Cu,Fe-NC nanozymes for the substrate and the reaction rate, while Pb... 2+ The addition of Cu,Fe-NC nanozymes improved the affinity and reaction rate between the substrate and the substrate;

[0038] Table 1 Michaelis catalytic kinetic parameters

[0039] Enzymes Substrate <![CDATA[ K m (mM)]]> <![CDATA[ V max (10 -8 M∙s -1 )]]> Cu,Fe-NC <![CDATA[H2O2]]> 0.237 3.25 Cu,Fe-NC +CEX <![CDATA[H2O2]]> 1.150 1.57 <![CDATA[Cu,Fe-N-C+CEX+ Pb 2+ ]]> <![CDATA[H2O2]]> 0.534 2.80

[0040] 3. Preparation of CEX working curve: Add 50 μL of 0.5 mg / mL Cu,Fe-NC and CEX standard solution with a concentration ranging from 0.010 to 52.76 mg / L to a 5 mL stoppered colorimetric tube. Add 100 μL of 5 mmol / L TMB and 100 μL of 50 mmol / L H2O2. Add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to a final volume of 3 mL. Shake well, let stand for 5-10 min, centrifuge at 10000 r / min for 5 min, and measure the absorbance of the supernatant at 654 nm. Plot the standard curve with CEX concentration on the x-axis and absorbance A on the y-axis to obtain the regression equation. See [link to relevant documentation]. Figure 7 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2.

[0041] 4. Pb 2+ Preparation of working curve: Add 50 μL of 0.5 mg / mL Cu,Fe-NC and 100 μL of 40 mg / L CEX standard solution to a 5 mL stoppered colorimetric tube, and add Pb at a concentration ranging from 0.012 to 45.38 mg / L. 2+ The standard solution was prepared by adding 100 μL of 5 mmol / L TMB and 100 μL of 50 mmol / L H2O2, then adding 0.1 mmol / L pH 4.0 HAc-NaAc buffer to a final volume of 3 mL. The solution was shaken well, allowed to stand for 5–10 min, centrifuged at 10000 r / min for 5 min, and the supernatant was used to measure the absorbance at 654 nm. A standard curve was plotted with CEX concentration on the x-axis and absorbance A on the y-axis, yielding the regression equation (see [reference needed]). Figure 8 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2.

[0042] Table 2. Linear equation, correlation coefficient, relative standard deviation, and linear range

[0043] target Working curve <![CDATA[Coefficient of correlation (R 2 ).]]> Linear range mg / L RSD%(n=3) LOQ mg / kg CEX y = 0.029x + 1.551 0.995 0.010~52.76 2.85 0.010 <![CDATA[Pb 2+ ]]> y = 0.024x + 0.082 0.996 0.012~45.38 3.10 0.012

[0044] 5. Method specificity study: CEX was mixed with other coexisting substances, and the effect of the coexisting substances on CEX in the above detection system was detected. The concentration of CEX was 5 mg / kg, and the concentration of the interfering substances was 50 mg / kg. Figure 9The results show the effects of coexisting substances (glucose, glutamic acid, tyrosine, serine, arginine, leucine, proline, ampicillin, clindamycin, aminophylline, cefixime, cefuroxime, DL-tryptophan, and theophylline) on CEX. The figures indicate that the Cu,Fe-NC detection system has good selectivity and specificity, with CEX showing significant inhibition of oxidation reactions, while other substances show almost no effect. The method for determining CEX exhibits good selectivity and specificity. The determination of Pb was also investigated. 2+ The selectivity of Pb 2+ Mixing with other potentially coexisting substances to detect the effect of metal ions on the detection system, Pb 2+ The concentration was 5 mg / kg, and the concentration of other interfering substances was 50 mg / kg. Figure 10 It is a coexisting metal ion (Na) + K + Ca 2+ Mg 2+ Cu 2+ Zn 2+ Fe 2+ Ni 2+ Hg 2+ (etc.) on Pb 2+ The effects are shown in the figure, with only Pb showing an impact. 2+ It significantly enhances the catalytic activity of nanozymes, while having almost no effect on other substances, and the method has good selectivity and specificity.

[0045] 6. Determination of CEX in milk samples

[0046] (1) Sample preparation: Weigh 4 mL of milk into a 10 mL centrifuge tube and centrifuge at 5000 rpm for 10 min. Remove the upper lipid layer; carefully pipette 500 mL of the intermediate layer into a clean test tube, add 2 mL of 0.1 mmol / L pH 4.0 HAc-NaAc buffer, and vortex to mix. Dilute the treated sample 5 times.

[0047] (2) Determination of CEX in milk samples: Add 50 μL of 0.5 mg / mL Cu,Fe-NC and 1 mL of the above test solution to a 5 mL stoppered colorimetric tube, add 100 μL of 5 mmol / L TMB and 100 μL of 50 mmol / L H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well, let stand for 5-10 min, centrifuge at 10000 r / min for 5 min, take the supernatant and measure the absorbance at 654 nm wavelength, substitute into the regression equation, CEX was not detected;

[0048] (3) Recovery and precision experiments: Three different concentrations of CEX standard solution were added to the milk sample; each concentration was measured in parallel three times, the spiked recovery rate was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 3. The spiked recovery rate of CEX was found to be 96.1% to 102.7%, and the RSD was 2.31% to 3.86%. This method has good accuracy and precision.

[0049] Table 3. Spike recoveries and RSDs of samples (n = 3)

[0050] 7. Pb in rice quality control samples 2+ Measurement

[0051] (1) Sample pretreatment: Rice quality control sample RMS-A025 was selected as the sample to be tested (Zhongyuan Standard Material Center); 0.3000 g of dried rice flour sample was weighed, placed in a glass digestion tube, 5 mL of nitric acid was added, the cap was tightened, and the sample was soaked overnight. The sample tube was then transferred to the reaction vessel, the vessel was completely sealed, nitrogen was added, and the pressure was increased to 4000 kPa. The external temperature was set to below 40 ℃. The digestion temperature increase program is shown in Table 4. After the digestion was completed and the pressure was completely released, the sample was taken out and transferred to a 10 mL volumetric flask, mixed evenly, and tested. A blank test was also performed.

[0052] Table 4 Digestion Heating Procedure

[0053] step Temperature (°C) Hold time / min Power / W Heating time / min 1 120 10 1600 7 2 150 5 1600 5 3 180 15 1600 10

[0054] (2) Sample determination: Add 50 μL of 0.5 mg / mL Cu,Fe-NC and 1 mL of the above test solution and 100 μL of 40 mg / L CEX standard solution to a 5 mL stoppered colorimetric tube, add 100 μL of 5 mmol / L TMB and 100 μL of 50 mmol / L H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well, let stand for 5-10 min, centrifuge at 10000 r / min for 5 min, take the supernatant and measure the absorbance at 654 nm wavelength, substitute into the regression equation, calculate the Pb concentration as 1.15 mg / kg, and the standard value as 1.17 ± 0.50 mg / kg;

[0055] Example 2: CEX in pork samples and Pb in tea samples 2+ Measurement

[0056] 1. Preparation of Cu,Fe-NC nanozymes

[0057] (1) Weigh 2.7 g CuCl2 and dissolve it in 12 mL of methanol, mix it evenly by ultrasonication, and use it as solution A; dissolve 4.7 g of medium-tetra(4-carboxyphenyl)porphyrin (TCPP) and 2.3 g FeCl3 in 23 mL of methanol, mix them evenly by ultrasonication, and use it as solution B; mix solutions A and B evenly and dry them in an oven at 85 °C for 2 h to obtain dark green dry powder Cu,Fe-TCPP;

[0058] (2) Weigh 5.0 g of Cu,Fe-TCPP dry powder into a quartz boat, and calcine it in a tube furnace at a heating rate of 5°C / min to 750°C for 2.5 h under N2 protection to obtain a black powder. Soak the powder in sulfuric acid solution (H2SO4:H2O=1:9, volume ratio) for 12 h, rinse it repeatedly with water, and dry it to obtain Cu,Fe-NC nanozyme;

[0059] 2. Evaluation of Cu,Fe-NC nanozyme peroxidase activity: Same as in Example 1;

[0060] 3. CEX working curve creation: Same as in Example 1;

[0061] 4. Pb 2+ Creating the working curve: Same as in Example 1;

[0062] 5. Determination of CEX content in pork samples

[0063] (1) Sample pretreatment method: Weigh 4.00 g of homogenized sample into a 50 mL centrifuge tube, add 12 mL of acetonitrile aqueous solution (15:2, volume ratio), homogenize for 30 s, centrifuge at 4℃ 4000 r / min for 5 min, and transfer the supernatant to a 50 mL centrifuge tube; take another centrifuge tube, add 8 mL of acetonitrile aqueous solution, and wash the homogenizer blade; crush the precipitate in the centrifuge tube with a glass rod, add the solution used to wash the homogenizer blade, shake on a vortex mixer for 1 min, centrifuge at 4000 r / min for 5 min, combine the supernatant into a 50 mL centrifuge tube, wash the blade with 8 mL of acetonitrile aqueous solution and extract once, combine into a 50 mL centrifuge tube, make up to 50 mL, and take 20 mL into a 100 mL volumetric flask;

[0064] (2) Determination of CEX in samples: Same as in Example 1, CEX was not detected in pork samples;

[0065] 6. Pb in tea 2+ Measurement

[0066] (1) Sample pretreatment: Accurately weigh 0.5000 g of tea powder sample into a polytetrafluoroethylene dissolving cup, add 5 mL of HNO3 and 2 mL of H2O2 respectively, mix well and let stand overnight; put into an outer container, place in a microwave digester, and digest according to 4 steps (0.5 MPa 2 min 400 W, 1.0 MPa 2 min 600 W, 1.5 MPa 2 min 800 W, 2.0 MPa 2 min 1000 W); after digestion, cool and open the container, wash the digestion liquid into a volumetric flask, make up to 25 mL with high-purity water, and store for later use; at the same time, prepare a reagent blank;

[0067] (2) Sample determination: Same as in Example 1, Pb in tea 2+ The content is 0.5 mg / kg;

[0068] Example 3: CEX in chicken samples and Pb in vegetables 2+ Measurement

[0069] 1. Preparation of Cu,Fe-NC nanozymes

[0070] (1) Weigh 3.0g CuCl2 and dissolve it in 15 mL of methanol, mix it evenly by ultrasonication, and use it as solution A; dissolve 5.5g of medium-tetra(4-carboxyphenyl)porphyrin (TCPP) and 2.5g of FeCl3 in 25 mL of methanol, mix them evenly by ultrasonication, and use it as solution B; mix solutions A and B evenly and dry them in an oven at 90℃ for 2 hours to obtain dark green dry powder Cu,Fe-TCPP;

[0071] (2) Weigh 5.5g of Cu,Fe-TCPP dry powder into a quartz boat, and calcine it in a tube furnace at a heating rate of 5°C / min to 750°C for 2.5 h under N2 protection to obtain a black powder. Soak the powder in sulfuric acid solution (H2SO4:H2O=1:9, volume ratio) for 12 h, rinse it repeatedly with water, and dry it to obtain Cu,Fe-NC nanozyme;

[0072] 2. Evaluation of Cu,Fe-NC nanozyme peroxidase activity: Same as in Example 1;

[0073] 3. CEX working curve creation: Same as in Example 1;

[0074] 4. Pb 2+ Creating the working curve: Same as in Example 1;

[0075] 5. Determination of CEX content in chicken samples

[0076] (1) Sample pretreatment method: same as in Example 1;

[0077] (2) CEX determination in samples: Same as in Example 1, CEX was not detected in chicken samples;

[0078] 6. Pb in tea 2+ Measurement

[0079] (1) Sample pretreatment: Celery standard material GBW10048 was selected as the sample to be tested (Institute of Geophysical and Geochemical Exploration); 1.000 g of vegetable sample was accurately weighed and placed in a glass digestion tube. 5 mL of nitric acid and 1 mL of hydrogen peroxide were added in sequence. The tube was capped and the sample tube was transferred to the reaction vessel. The vessel was completely sealed, nitrogen gas was added, and the pressure was increased to 4000 kPa. The external temperature was set below 40 ℃. The digestion temperature program is shown in Table 5. After digestion was completed and the pressure was completely released, the sample was taken out and transferred to a 25 mL volumetric flask. The mixture was stirred evenly and ready for testing. A blank test was also performed.

[0080] (2) Sample determination: Same as in Example 1, Pb in celery 2+ The concentration was 61.0 µg / kg, the standard value was 62.3±1.9 µg / kg, and the measurement result was within the error range.

[0081] Table 5 Digestion Heating Procedure

[0082] step Temperature (°C) Hold time / min Power / W Heating time / min 1 110 15 1500 5 2 150 10 1500 5 3 190 10 1500 5

[0083] The CEX and Pb established in this invention 2+ The assay method has advantages in practical testing because it involves fewer processing steps, shorter processing time, lower processing costs, simpler operation, and does not require large instruments or equipment.

Claims

1. A rapid detection method for cephalexin and lead using a two-atom nanozyme, characterized in that, Includes the following steps: (1) Using neu-tetra(4-carboxyphenyl)porphyrin as ligand TCPP, Cu,Fe-TCPP complex was synthesized. Cu,Fe-TCPP was used as a precursor for high-temperature pyrolysis and acid treatment to obtain diatomic Cu,Fe-NC nanozyme. (2) Add Cu,Fe-NC nanozyme solution to the cephalexin standard solution, then add 3,3',5,5'-tetramethylbenzidine solution and H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well, generate blue oxTMB oxide, let stand for 5-10 min, centrifuge, take the supernatant and measure the absorbance at 654 nm wavelength, establish the quantitative relationship between absorbance and cephalexin concentration, draw a standard curve, and obtain the regression equation; (3) Add Cu,Fe-NC nanozyme solution and Pb to cephalexin solution. 2+ The standard solution was prepared, followed by the addition of 3,3',5,5'-tetramethylbenzidine solution and H2O2. Then, 0.1 mmol / L pH 4.0 HAc-NaAc buffer was added to a final volume of 3 mL. The solution was shaken well, and a blue oxTMB oxide was generated. The mixture was allowed to stand for 5-10 min, centrifuged, and the supernatant was measured at 654 nm. A correlation between absorbance and Pb was established. 2+ The quantitative relationship of concentration was determined, a standard curve was plotted, and the regression equation was obtained. (4) Add Cu,Fe-NC nanozyme solution to the sample solution containing cephalexin, then add 3,3',5,5'-tetramethylbenzidine solution and H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well, let stand for 5-10 min, centrifuge, take the supernatant and measure the absorbance at 654 nm wavelength, substitute into the regression equation, and calculate the concentration of cephalexin in the sample solution. In Pb 2+ Cu,Fe-NC nanozyme solution was added to the sample solution to be tested, followed by 3,3',5,5'-tetramethylbenzidine solution, H2O2, and cephalexin solution. Then, 0.1 mmol / L pH 4.0 HAc-NaAc buffer was added to a final volume of 3 mL. The mixture was shaken well, allowed to stand for 5-10 min, centrifuged, and the supernatant was measured at 654 nm. The absorbance was then substituted into the regression equation to calculate the Pb concentration in the sample solution. 2+ concentration.

2. The method according to claim 1, characterized in that, Cu,Fe-NC nanozymes were prepared as follows: (1) Weigh 2.5-3.0 g CuCl2 and dissolve it in 10-15 mL methanol, mix it evenly by ultrasonication, and use it as solution A; dissolve 4.5-5.5 g of medium-tetra(4-carboxyphenyl)porphyrin and 2.0-2.5 g FeCl3 in 20-25 mL methanol, mix them evenly by ultrasonication, and use it as solution B; mix solutions A and B evenly and dry them in an oven at 80-90℃ for 2 hours to obtain a dark green dry powder Cu,Fe-TCPP; (2) Weigh 4.5-5.5 g of Cu,Fe-TCPP dry powder into a quartz boat, heat it in a tube furnace at a rate of 5°C / min to 750°C, and calcine it under N2 protection for 2-2.5 h to obtain black powder; soak the powder in a solution with a volume ratio of H2SO4:H2O=1:9 for 12 h, rinse it repeatedly with water, and dry it to obtain Cu,Fe-NC nanozyme.

3. The method according to claim 1, characterized in that: The concentration of cephalexin standard solution ranges from 0.010 to 52.76 mg / L, Pb 2+ The concentration of the standard solution was 0.012–45.38 mg / L; the concentration of Cu,Fe-NC nanozyme was 0.2 mg / mL, and the amount added was 100 μL; the concentration of TMB solution was 5 mmol / L, and the amount added was 20–200 μL; the concentration of H2O2 was 50 mmol / L, and the amount used was 20–200 μL.

4. The method according to claim 1, characterized in that: Centrifugation is performed at 8000-10000 r / min for 5-10 min.

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