Cerium-based oxidase for acetaminophen detection and application thereof

Abstract

According to the cerium-based oxidase for detecting the acetaminophen and the application of the cerium-based oxidase, a novel oxidase-like material Ce-CS is prepared, a colorimetric detection method for detecting the acetaminophen is established, the cerium-based oxidase can be used for detecting the content of the acetaminophen in oral liquid preparations and sewage, and the detection method is simple and easy to operate and high in detection efficiency. The sensitivity, the accuracy and the precision of the method are high, the used spectrophotometer is cheap and popularized, and the result is visual.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and particularly relates to a cerium-based oxidase-like enzyme for detecting acetaminophen and its application. Background Art

[0002] Acetaminophen (APAP), commonly known as paracetamol (PCT), is the main ingredient in most cold and flu medications. Clinically, it is mainly used for antipyretic and analgesic purposes, and is applicable to joint pain, cold fever, neuralgia, migraine, and postoperative pain, etc. It is one of the most widely used analgesics. The content of APAP in pharmaceutical preparations should comply with the pharmacopoeia standards. If the content of APAP is insufficient, the treatment effect on patients will not be achieved. Therefore, it is crucial to control the quality of the preparations. In addition, APAP has toxic side effects. If patients overdose on APAP, it will cause symptoms such as nausea, vomiting, coma, and stomach pain. In severe cases, it can lead to drug-induced liver injury and acute liver failure. With the large-scale production and widespread use of APAP, the risk of polluting the ecological environment also increases. Once APAP enters the water circulation system, it will inevitably cause harm to the biosphere. Therefore, it is very necessary to monitor the content of APAP in pharmaceutical factories and domestic sewage.

[0003] Common APAP detection methods include electrochemistry, high performance liquid chromatography, titration, fluorescence, high performance thin layer chromatography, etc. These methods involve expensive instrument equipment or complex test steps, thus limiting their practicality. For example, the patent application with the publication number CN101566608A discloses a method for detecting illegally added acetaminophen in a composition. The method includes the following steps: using high performance liquid chromatography-mass spectrometry (HPLC-MS) to confirm the structure of acetaminophen by monitoring the following ions: parent ion 152.1, daughter ion 110.1, and daughter ion 93.1, and using high performance liquid chromatography to determine the content of acetaminophen. In the HPLC-MS method, the aqueous phase of the mobile phase used is an aqueous solution of ammonium acetate at 10 - 50 mmol / L, and the organic phase is methanol; in the high performance liquid chromatography method, the aqueous phase of the mobile phase is an aqueous solution of phosphoric acid with a mass percentage of 0.1% - 0.5%, and the organic phase is methanol.

[0004] The operation of the colorimetric method is relatively simple, and it can quickly identify the color change of the reaction system with the naked eye. Moreover, the spectrophotometer used for detection is relatively inexpensive and popular. For example, the patent application with the publication number CN102016569A discloses a method for determining the concentration of acetaminophen in an aqueous sample. The method includes the following steps: hydrolyzing acetaminophen to form p-aminophenol by contacting acetaminophen with arylamidase; in the presence of a suitable catalyst, oxidatively coupling the p-aminophenol with a xylenol chromophore to form a colored product, where the catalyst is anhydrous or hydrated MnCl2; and determining the amount of the formed colored product, and the amount of the formed colored product is proportional to the amount of the acetaminophen initially present in the aqueous sample. However, this method uses arylamidase and faces various problems such as high preparation cost, poor stability, and easy inactivation. Summary of the Invention

[0005] In view of the above technical problems existing in the prior art, the present invention provides a cerium-based oxidase-like enzyme for the detection of acetaminophen (APAP) and its application. By preparing a novel oxidase-like enzyme material, a colorimetric detection method for detecting acetaminophen is established, which is simple and easy to operate and has high detection result accuracy.

[0006] The cerium-based oxidase-like enzyme for the detection of acetaminophen provided by the present invention is prepared by the following method: mixing L-cysteine (CS, CAS: 56-89-3) and ammonium cerium nitrate (CAN, CAS: 16774-21-3) evenly under alkaline conditions, and carrying out a complexation reaction (the reaction principle is shown in Figure 1 ), to generate a cerium-L-cysteine complex (Ce-CS), thus obtaining the cerium-based oxidase-like enzyme for the detection of acetaminophen.

[0007] Preferably, in the above method, the molar ratio of L-cysteine to ammonium cerium nitrate is 1:0.3-0.7. Further preferably, the molar ratio of L-cysteine to ammonium cerium nitrate is 1:0.5, and the catalytic activity of the cerium-L-cysteine complex prepared under this condition is relatively high.

[0008] Preferably, the alkaline condition is provided by NaOH, and the solvent used for the complexation reaction is water.

[0009] The present invention also provides a method for detecting acetaminophen, including the following steps:

[0010] (1) Dispersing the above cerium-based oxidase-like enzyme in a solvent to obtain a Ce-CS dispersion;

[0011] (2) Add the Ce-CS dispersion obtained in step (1) and the test solution to the buffer solution in sequence, mix well, and let an oxidation-reduction reaction occur between Ce-CS and paracetamol in the test solution to obtain Solution 1;

[0012] (3) Add 3,3',5,5'-tetramethylbenzidine (TMB, CAS: 54827-17-7) to Solution 1, mix well, and let a catalytic oxidation reaction occur for TMB to obtain Solution 2;

[0013] (4) Measure the absorbance of Solution 2 at 652 nm as A1;

[0014] (5) In step (2), do not add the test solution, repeat steps (1)-(4), and measure the absorbance as A2;

[0015] (6) Compare A2 - A1 with the standard curve to obtain the concentration of paracetamol in the test solution.

[0016] Detection principle: Ce-CS has an activity similar to that of oxidase and can catalytically oxidize colorless TMB into a blue oxide (oxTMB), and oxTMB has an obvious absorption at 652 nm. When paracetamol (APAP) is present, due to the reducibility of APAP, APAP can react with Ce 4+ with peroxidase-like activity. The phenol structure in the APAP structure is oxidized to a quinone-like structure, and Ce in Ce-CS 4+ is reduced to Ce 3+ . Therefore, APAP will inhibit the peroxidase-like activity of Ce-CS, resulting in a decrease in the generation amount of the blue oxide oxTMB, and the absorbance of the reaction system at 652 nm will decrease accordingly (see the detection mechanism in Figure 2 ). The degree of decrease in absorbance is related to the added amount of APAP. Based on this, a peroxidase inhibition colorimetric detection method for APAP can be established.

[0017] Reaction mechanism of Ce-CS catalytic oxidation of TMB: Ce-CS has a multi-channel void structure and a large specific surface area, and can quickly adsorb dissolved oxygen O2 in the solution. The O2 molecules are activated at the active sites on Ce-CS to generate ·O2 - which obtains electrons from the substrate TMB, and TMB is oxidized into the blue oxide oxTMB, resulting in the reaction system showing a blue color. The whole catalytic oxidation process can be simply expressed as follows:

[0018] Ce 4+ +O2→Ce 3+ +·O2 - ;

[0019] TMB+·O2 - →H2O+oxTMB.

[0020] Preferably, the solvent in step (1) is water, ethanol or petroleum ether. Preferably, the pH of the buffer solution in step (2) is 3-5. More preferably, the pH of the buffer solution in step (2) is 4, under which condition the catalytic activity of Ce-CS is relatively high.

[0021] Preferably, the ratio of Ce-CS added in step (2) to TMB added in step (3) is 125-625 g:1 mol. More preferably, the ratio of Ce-CS added in step (2) to TMB added in step (3) is 500 g:1 mol, under which condition the amount of TMB oxidized to oxTMB is relatively large.

[0022] Preferably, in step (3), the reaction temperature is -7-50 °C and the reaction time is 3-30 min. More preferably, in step (3), the reaction temperature is 25 °C and the reaction time is 10 min, under which condition the amount of TMB oxidized to oxTMB is relatively large.

[0023] Preferably, the buffer solution is HAc-NaAc buffer solution.

[0024] Preferably, in step (6), the method for making the standard curve is as follows: Prepare a series of APAP standard solutions with different concentrations, replace the solution to be tested with this series of APAP standard solutions with different concentrations, perform the operations of steps (1)-(5) respectively, and then make the response relationship curve between A2 - A1 and the concentration of the acetaminophen standard solution, thus obtaining the standard curve.

[0025] More preferably, the linear range of the standard curve is 50-2000 μmol·L -1 .

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The colorimetric detection method for APAP based on peroxidase-like Ce-CS provided by the present invention can be used to detect the content of APAP in oral liquid preparations and sewage. This detection method is simple and easy to operate, with relatively high sensitivity, accuracy and precision. The spectrophotometer used is relatively inexpensive and popular, and the results are visualizable. Description of the Drawings

[0028] Figure 1 It is the synthesis principle diagram of Ce-CS of the present invention.

[0029] Figure 2 It is the mechanism diagram for detecting acetaminophen of the present invention.

[0030] Figure 3Morphology diagrams of Ce-CS and its raw materials in Example 1. Among them, Figure 3 A in it is the macroscopic diagram of the peroxidase-like Ce-CS; Figure 3 B, C, and D in it are the 50-μm size diagrams of ammonium cerium nitrate (CAN), L-cysteine (CS), and Ce-CS obtained by scanning electron microscopy (SEM), respectively; Figure 3 E and F in it are the 5-μm and 2-μm size diagrams of Ce-CS obtained by SEM.

[0031] Figure 4 Elemental composition analysis of the Ce-CS prepared in Example 1. Among them, Figure 4 A in it is the EDS point scan of Ce-CS; Figure 4 B, C, D, E, and F in it are the elemental mapping spectra of Ce, N, O, S, and C, respectively.

[0032] Figure 5 Partial characterization results of the Ce-CS prepared in Example 1. Among them, Figure 5 A in it is the pore size distribution of the Ce-CS in Example 1; Figure 5 B in it is the nitrogen adsorption-desorption isotherm curve of Ce-CS; Figure 5 C in it is the XRD spectrum of Ce-CS; Figure 5 D in it is the infrared spectrum of Ce-CS.

[0033] Figure 6 X-ray photoelectron spectroscopy characterization results of the Ce-CS prepared in Example 1.

[0034] Figure 7 Optimization of the synthesis conditions of Ce-CS and verification of peroxidase-like activity. Among them, Figure 7 A in it is the analysis diagram for selecting the optimal concentration of CAN solution for preparing Ce-CS in Detection Example 1; Figure 7 B in it is the ultraviolet-visible absorption spectrum diagram of different reaction systems in Detection Example 8.

[0035] Figure 8 Curves showing the effects of temperature, buffer pH, Ce-CS concentration, and incubation time on the catalytic activity of Ce-CS in Detection Example 9.

[0036] Figure 9 Study on the catalytic mechanism and catalytic kinetics of Ce-CS. Among them, Figure 9 A in it is the EPR spectrum diagram of DMPO capturing superoxide anions by Ce-CS in methanol dispersion in Detection Example 10;

[0037] Figure 9 B in it is the verification experiment of hydroxyl radicals (·OH) in Detection Example 10; Figure 9C in it is to detect the steady-state kinetic properties of Ce-CS in Detection Example 11; Figure 9 D in it is to detect the Lineweaver-Burk double-reciprocal plot (n = 3) in Detection Example 11.

[0038] Figure 10 It is the detection result of Detection Example 12. Among them, Figure 10 A in it is the ultraviolet absorption spectrum corresponding to different concentrations of APAP in the range of 500 nm to 800 nm; Figure 10 B in it is the linear relationship between ΔA and APAP within the concentration range of 0.05 - 2 mmol·L -1 concentration range.

[0039] Figure 11 It is ΔA of the Ce-CS / TMB system under different interferences in Detection Example 13.

[0040] Figure 12 It is the absorbance value of the reaction system under different solvents in Example 7. Specific implementation manners

[0041] 1. Reagents

[0042] Acetaminophen (APAP), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), glucose (Gs), glycine (Gly), L-histidine (L-His), sodium acetate, L-cystine (CS), salicylic acid (SA), all were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; acetic acid and sodium hydroxide were purchased from Zhejiang Zhongxing Chemical Reagent Co., Ltd.; anhydrous calcium chloride, sucrose, ammonium chloride, potassium dihydrogen phosphate, magnesium chloride and ferrous sulfate heptahydrate were purchased from Sinopharm Chemical Reagent Co., Ltd.; anhydrous sodium carbonate was purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd.; magnesium sulfate and ammonium cerium nitrate (CAN) were purchased from Shanghai Macklin Biochemical Co., Ltd.; acetaminophen oral solution (2.4%, Guangdong Nanguo Pharmaceutical Co., Ltd.); secondary distilled water.

[0043] 2. Instruments

[0044] Cary 60 UV-Vis spectrophotometer (Agilent Technologies Inc.); Apreo S scanning electron microscope (Thermo Fisher Scientific Inc.); specific surface area and pore size analyzer (Beijing Jingwei Gaobo Instrument Co., Ltd.); Spectrum two infrared spectrometer (PerkinElmer, UK); XRD-7000 X-ray single crystal diffractometer (Shimadzu Corporation, Japan); Nexsa G2 X-ray photoelectron spectrometer (Thermo Fisher Scientific Inc.); energy dispersive X-ray spectrometer (Thermo Fisher Scientific Inc.); BRUKE EMXPLUS electron paramagnetic resonance instrument (Bruker, Germany).

[0045] Example 1

[0046] Take 1mL of 50mmol·L -1 CAN solution, 2mL concentration of 50mmol·L -1 CS solution and 260 μL of 1 mol·L -1 The NaOH solution (to promote dissolution) was placed in a centrifuge tube. The solvent of the above solution was double distilled water. Then a yellow flocculent precipitate was produced. After sufficient shaking, the reaction was carried out at room temperature (25°C) for 20 minutes. Then, the solution was centrifuged (5000rpm, 1min) and washed with double distilled water. After repeating the above operation three times, the precipitate was freeze-dried to obtain a light yellow Ce-CS powder ( Figure 3 When using, weigh it accurately and disperse it evenly in a certain volume of double distilled water.

[0047] Preparation of APAP standard solution: accurately prepare 10mmol·L -1 The APAP solution was used as the mother solution, and then different volumes of APAP mother solution were accurately pipetted with a pipette and diluted with secondary water to prepare a series of APAP standard solutions (0 mmol·L -1 , 0.05mmol·L -1 , 0.1mmol·L -1 , 0.25mmol·L -1 , 0.5mmol·L -1 , 1mmol·L -1 , 1.5mmol·L -1 , 2mmol·L -1 ).

[0048] Preparation of standard curve: Pipette 1.75 mL of HAC-NaAC buffer (50 mmol / L -1, pH = 4) eight portions, each portion was added with 50 μL of Ce-CS dispersion (1 mg / mL), and then 100 μL of the above-prepared APAP standard solution was added successively. After shaking well and standing for 1 h, 100 μL of substrate TMB (1 mmol·L -1 ) was added to a total volume of 2 mL. After shaking well and standing for 10 min at room temperature of 25 °C, the absorbance of each reaction system at 652 nm was measured. The absorbance differences ΔA between the reaction system of the blank solution (without APAP) and the reaction systems of APAP standard solutions with different concentrations were calculated respectively. Then, a standard curve was plotted with ΔA against the concentration of the APAP standard solution.

[0049] Sample preparation and detection: (1) Preparation and detection of APAP oral liquid samples: Randomly select paracetamol oral liquid (APAP oral liquid) sold on the market. After diluting it 200 times with secondary water, referring to the detection method of the above standard curve, the sample solution was taken instead of the standard solution for detection, and it was detected 5 times in parallel, and the average value was calculated. Then, a standard addition recovery experiment was carried out, that is, 1 mL of the above sample solution diluted 200 times was taken in three portions, and 1 mL of APAP standard solutions with different concentrations (0.4 mmol·L -1 , 0.8 mmol·L -1 , 1 mmol·L -1 ) were added respectively. After mixing evenly, it was detected in the same way, and the standard addition recovery rate and relative standard deviation (RSD) were calculated to evaluate the precision and accuracy of the peroxidase-like inhibition colorimetric method for detecting APAP oral liquid; (2) Preparation and detection of APAP simulated sewage samples: Take the river water on the campus of Jiaxing University. After filtration treatment, no APAP was detected. Then, 1 mL of river water was taken in three portions, and 1 mL of APAP standard solutions with different concentrations (0.4 mmol·L -1 , 0.8 mmol·L -1 , 2 mmol·L -1 ) were added respectively. After mixing evenly, three kinds of APAP simulated sewage samples with different concentrations were prepared. Referring to the detection method of the above standard curve, the sample solution was taken instead of the standard solution for detection, and it was detected 5 times in parallel, and the average value and RSD were calculated, and the blank standard addition recovery rates of the three concentrations were calculated to evaluate the precision and accuracy of the peroxidase-like inhibition colorimetric method for detecting APAP simulated sewage.

[0050] The results are shown in Table 1 - Table 3. The recovery rate in the spiked samples was 97.90% - 101.75%, and the RSD was 1.804% - 4.571%. It can be seen that this method is reliable and practical, and can provide accurate and rapid data support for detecting the content of APAP.

[0051] Table 1 Absorbance data in the detection of APAP concentration in oral liquid and simulated sewage

[0052] Absorbance 1 Absorbance 2 Absorbance 3 Absorbance 4 Absorbance 5 Blank control 0.666 0.662 0.677 0.654 0.664 Oral liquid detection 0.373 0.344 0.380 0.353 0.359 Simulated sewage detection 0.651 0.632 0.678 0.643 0.634

[0053] Table 2 Absorbance data in the recovery experiments of oral liquid and simulated sewage spiked with standard substances

[0054]

[0055] Table 3 Detection results of APAP concentrations and spiked recoveries of oral liquid and simulated sewage

[0056]

[0057] Relative standard deviation (RSD) represents precision; Recovery = experimental test result / theoretical value × 100%.

[0058] Example 2

[0059] Differing from Example 1, CAN solution with a concentration of 30 mmol·L -1 was used to prepare Ce-CS.

[0060] Example 3

[0061] Differing from Example 1, CAN solution with a concentration of 40 mmol·L -1 was used to prepare Ce-CS.

[0062] Example 4

[0063] Differing from Example 1, CAN solution with a concentration of 50 mmol·L -1 was used to prepare Ce-CS.

[0064] Example 5

[0065] Differing from Example 1, CAN solution with a concentration of 60 mmol·L -1 was used to prepare Ce-CS.

[0066] Example 6

[0067] Differing from Example 1, CAN solution with a concentration of 70 mmol·L -1 was used to prepare Ce-CS.

[0068] Example 7

[0069] The Ce-CS prepared in Example 1 was respectively dispersed in absolute ethanol, 50% ethanol, petroleum ether, and water to prepare 1 mg / mL dispersion liquids. 50 μL of different dispersion liquids were respectively taken and placed into 1.85 mL of HAC-NaAC buffer solution (50 mmol·L -1, in a solution with pH = 4), then add 100 μL of TMB solution (1 mM). After reacting at room temperature of 25 °C for 10 min, use a UV-Vis spectrophotometer to detect the absorbance of the reaction system at 652 nm. Figure 12 is the corresponding absorbance value. It was found that Ce-CS can be dispersed in ethanol, petroleum ether or water, and has the best dispersion effect in water.

[0070] Detection Example 1

[0071] Determine the peroxidase-like activity of the Ce-CS prepared in Examples 1-6. A commonly used chromogenic substrate for investigating peroxidase-like activity is 3,3',5,5'-tetramethylbenzidine (TMB). TMB can be catalytically oxidized by peroxidase-like enzymes to form a blue oxide oxTMB, and its maximum absorption wavelength is 652 nm.

[0072] Disperse the Ce-CS prepared in Examples 1-6 evenly into secondary distilled water to obtain a Ce-CS dispersion with a concentration of 0.5 mg·mL -1 . Perform the following operations respectively: Pipette 50 μL of the Ce-CS dispersion and add it to an HAc-NaAc buffer solution with pH = 4.0 (1.850 mL, 50 mmol·L -1 ), then add a colorless substrate TMB solution (100 μL, 1 mmol·L -1 ) until the reaction volume is 2 mL. Shake well, and after standing for 10 min, use a UV-Vis spectrophotometer to detect the absorbance of the reaction system at 652 nm, and use the magnitude of the absorbance to evaluate the peroxidase-like activity of Ce-CS.

[0073] Results ( Figure 7 A in) show that as the concentration of Ce 4+ increases, the catalytic activity of Ce-CS first increases and then decreases. When the concentration of Ce 4+ is 50 mmol·L -1 , the catalytic activity of the synthesized Ce-CS reaches the maximum value. Analyzing the reason, when the concentration is too high, cerium ions accumulate on the surface of Ce-CS, hindering the binding of the substrate to Ce-CS and weakening its activity.

[0074] Detection Example 2

[0075] The macroscopic image of the Ce-CS powder prepared in Example 1 is shown in Figure 3 A. After sputtering gold on the Ce-CS powder and raw materials prepared in Example 1, use a scanning electron microscope (SEM) for microscopic morphology characterization. The 50-μm size microscopic morphologies of the raw materials CAN, CS and Ce-CS can be observed ( Figure 3Among B, C, and D), the surface of CAN is uneven, and there are uneven lumps on the surface of CS. The new material Ce-CS presents a regular rose-like shape, which is quite different from the microscopic morphologies of the raw materials CAN and CS, indicating that CAN and CS are not simply physically stacked, but a new material Ce-CS with a microscopic regular shape is formed by orientation through the interaction force between them. Further micro-magnification of Ce-CS ( Figure 3 in E and F) shows a pore structure. This structure enables Ce-CS to expose more active sites and has a larger specific surface area for loading substrates. The substrate is more likely to contact the active sites, making Ce-CS exhibit more excellent peroxidase-like activity.

[0076] Detection Example 3

[0077] Elemental composition analysis was carried out on the Ce-CS prepared in Example 1. The test results of the X-ray energy spectrometer (EDS) ( Figure 4 in A) show that characteristic elements such as Ce, N, O, S, and C exist in the synthesized Ce-CS. Among them, the elements N, O, S, and C are derived from the ligand L-cysteine, and the Ce element is derived from ammonium cerium nitrate (CAN). In addition, the EDS surface scan results ( Figure 4 in B-F) show that N, O, S, C, and Ce are highly dispersed and evenly distributed in the synthesized Ce-CS, which is beneficial for each unit in Ce-CS to have the same peroxidase-like activity.

[0078] Detection Example 4

[0079] The nitrogen adsorption-desorption experiment (BET) was carried out on the Ce-CS prepared in Example 1 ( Figure 5 in A and B). According to the nitrogen adsorption-desorption isotherm, the pore structure parameters of Ce-CS were obtained. The pore size distribution was calculated by the Barrett-Joyner-Halenda (BJH) pore size analysis method, and the specific surface area was calculated by the Brunauer-Emmett-Teller (BET) method. The results show that the specific surface area of Ce-CS is 57.085 m 2 ·g -1 , the average pore size is 16.305 nm, and the proportion of the pore size range of 20-50 nm is the largest, reaching 38.6%. The pore size results measured by BET are consistent with those observed by SEM, both less than 1 μm.

[0080] Detection Example 5

[0081] The Ce-CS prepared in Example 1 was characterized by using an X-ray diffractometer (XRD) ( Figure 5In C), it can be seen that diffraction peaks of Ce-CS appear at 18.85°, 28.50°, 33.03°, 34.38°, and 46.43°, which are very consistent with the peak angles of the standard card of its ligand CS. That is, there are characteristic peaks of ligand CS in the XRD pattern of Ce-CS.

[0082] Detection Example 6

[0083] The Ce-CS prepared in Example 1 was characterized by infrared spectroscopy (IR) ( Figure 5 in D), and two peaks at 1565 cm -1 and 1392 cm -1 correspond to the symmetric and asymmetric stretching vibrations of -COO- respectively, and the peak at 535 cm -1 is the Ce-O stretching vibration, indicating that bonding occurs between Ce 4+ and -COO - . Combining the characterization results of XRD can further prove that CAN reacts with CS, and Ce-CS is formed by bonding between Ce 4+ and -COO - .

[0084] Detection Example 7

[0085] The Ce-CS prepared in Example 1 was characterized by X-ray photoelectron spectroscopy (XPS) ( Figure 6 ), and it can be seen from the full-scan XPS spectrum ( Figure 6 in A) that all the constituent elements in Ce-CS were detected, including the metal element Ce, which is consistent with the test results of EDS and XRD. In addition, it can be seen from the high-resolution spectrum of Ce element ( Figure 6 in B) that characteristic peaks of Ce 4+ appear at 916.1 eV, 906.9 eV, 899.3 eV, 888.9 eV, and 881.4 eV, while characteristic peaks of Ce 3+ appear at 903.5 eV and 884.9 eV, proving that both Ce 3+ and Ce 4+ exist in the synthesized Ce-CS, that is, there are empty orbitals. It can be seen from the high-resolution spectrum of O element ( Figure 6 in C) that the binding energy of C-O bond exists at 533.9 eV, the binding energy of C=O bond exists at 531.8 eV, and the binding energy of Ce-O bond exists at 530.4 eV, proving that bonding occurs between Ce 4+ and -COO - , which is consistent with the characterization results of Detection Example 6. Ce 4+ , as a Lewis acid, provides empty orbitals, and -COO -Provide lone pair electrons as a Lewis base, and the two form a coordination bond to combine.

[0086] Comprehensively integrating the characterization results of Test Examples 3 - 7 can fully prove that CAN and CS pass through Ce 4+ and -COO - to form a coordination bond, and successfully synthesize Ce-CS( Figure 1 ).

[0087] Test Example 8

[0088] By adding different reagents to the HAC-NaAC buffer solution with pH = 4, five groups of experiments were designed to investigate the peroxidase-like activity of Ce-CS. These five groups of experiments are respectively (1) Ce-CS; (2) TMB + Ce-CS; (3) H2O2 + Ce-CS; (4) H2O2 + TMB; (5) TMB + H2O2 + Ce-CS. Among them, Ce-CS was prepared according to Example 4, and the concentrations and volumes of Ce-CS and TMB are the same as those in Test Example 1, and the concentration and volume of H2O2 are the same as those of TMB in Test Example 1. All three are selected secondary distilled water as the solvent. The results are as Figure 7 shown in B. For the (1) and (3) experimental groups without the substrate TMB, there is no obvious absorption at 652 nm; the (4) group has a very weak absorption peak, indicating that TMB can be oxidized by H2O2, but the reaction rate is very slow; the (2) experimental group with both Ce-CS and the substrate TMB produces a strong absorption at 652 nm, indicating that Ce-CS has a high peroxidase-like activity; the absorption of the (5) experimental group with Ce-CS, the substrate TMB, and H2O2 coexisting is significantly weakened at 652 nm, indicating that H2O2 has an inhibitory effect on the peroxidase-like activity of Ce-CS.

[0089] Test Example 9

[0090] According to the method of Test Example 1, investigate the effects of conditions such as temperature (-7 °C, 4 °C, 25 °C, 35 °C, 50 °C), pH (3.0, 3.5, 4.0, 4.5, 5.0), the addition amount of Ce-CS (0.25 mg·mL -1 , 0.5 mg·mL -1 , 0.75 mg·mL -1 , 1 mg·mL -1 , 1.25 mg·mL -1 ), reaction time (0 - 30 min), etc. on the peroxidase-like activity of Ce-CS. The results are as Figure 8 shown. As shown in A of Figure 8 , with the increase of temperature, the catalytic activity of Ce-CS first increases and then decreases, and the activity is the strongest at room temperature of 25 °C. As shown in Figure 8B in [reference] found that as the pH of the reaction system increased, the catalytic activity of Ce-CS first increased and then decreased, and the activity was the strongest at pH = 4. From Figure 8 C in [reference] showed that as the amount of Ce-CS added increased, the absorbance also increased, indicating that the amount of TMB oxidized to oxTMB increased accordingly. When the concentration reached 1 mg·mL -1 after, the absorbance no longer continued to increase. From Figure 8 it can be seen from D in [reference] that as the catalytic reaction time increased, the absorbance of the reaction system also increased, and finally the reaction reached the maximum value after 10 min.

[0091] Detection Example 10

[0092] Generally speaking, the catalytic activity of peroxidase mimics is related to O2 and electron transfer. In order to clarify whether oxygen is involved in the process of Ce-CS catalyzing the oxidation of TMB, electron paramagnetic resonance (EPR) technology was used to detect whether superoxide anion (·O2 - ) was generated. Methanol was used as the solvent and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as the scavenger. The results can be seen from Figure 9 A in [reference], which conforms to the characteristic peaks of ·O2 - free radicals, that is, there are 6 peaks. From left to right, four large and two small (the peak intensities at positions 1, 2, 4, and 6 are almost the same height, and the peak intensities at positions 3 and 5 are lower). It can be inferred that the catalytic activity of Ce-CS stems from activating O2 to generate ·O2 - , thereby oxidizing the substrate TMB.

[0093] In order to study whether hydroxyl radicals (·OH) exist in the catalytic process, salicylic acid (SA) was introduced as a scavenger for ·OH. The principle is that ·OH attacks SA to generate two adducts: 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. Specific operation: In the reaction system of Detection Example 1 method, 100 μL of SA solution with a concentration of 10 mmol·L -1 was added, and the volume of the buffer solution was reduced, and the total volume was 2 mL. The experimental results ( Figure 9 B in [reference]) showed that compared with the control group without the scavenger, the absorbance of the SA group hardly changed, and it can be inferred that no ·OH was generated during the reaction process.

[0094] In summary, the mechanism of the entire catalytic process can be inferred: Ce-CS has a multi-channel void structure and a large specific surface area, which can quickly adsorb dissolved oxygen O2 in the solution. The O2 molecules are activated at the active sites on Ce-CS to generate ·O2 - obtains electrons from the substrate TMB, and TMB is oxidized to the blue oxide oxTMB, resulting in the reaction system showing blue. The entire catalytic oxidation process can be simply expressed as follows:

[0095] Ce 4+ +O2 → Ce 3+ +·O2 - ;

[0096] TMB + ·O2 - → H2O + oxTMB。

[0097] Detection Example 11

[0098] By changing the concentration of the substrate TMB in the reaction system, the kinetic parameters of the peroxidase-like Ce-CS catalyzing the oxidation of TMB were further evaluated.

[0099] The initial reaction rates of the substrate TMB with the peroxidase-like Ce-CS at different concentrations (0.4 mmol·L -1 , 0.6 mmol·L -1 , 0.8 mmol·L -1 , 1.0 mmol·L -1 , 1.2 mmol·L -1 ) were measured respectively, and then the kinetic parameters (K m , V max ) were calculated using the Lineweaver-Burk plot of the Michaelis-Menten equation:

[0100]

[0101] where: V - apparent initial reaction rate, K m - apparent Michaelis-Menten constant, V max - maximum reaction rate, S - substrate concentration.

[0102] The relationship between the initial reaction rate and the TMB concentration was fitted to obtain the Michaelis-Menten curve (C in Figure 9 ), and at the same time, the Lineweaver-Burk curve (D in Figure 9 ) was plotted. The kinetic constants K m and V max were measured to be 0.0429 mmol·L -1 and 7.51×10 -8 Ms -1 . K m is an important index for evaluating enzyme performance, and K mThe lower the value, the stronger the affinity between the enzyme and the substrate, and vice versa. As shown in Table 4, compared with other types of oxidases (References: [1] FEKE K, TADELE ALULAM. Colorimetric detection of chromium(VI) via its instigation of oxidase-mimic activity of CuO[J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2023, 294: 122539. [2] YUAN C, YIN Y, RONG Y, et al. Intrinsic oxidase-mimicking activity of nitrite upon visible light illumination and its colorimetric detection in saliva[J]. Talanta, 2024, 279: 126649. [3] CHEN Y, XIA Y, LIU Y, et al. Colorimetric and electrochemical detection platforms for tetracycline based on surface molecularly imprinted polyionic liquid on Mn3O4 nanozyme[J]. Biosensors and Bioelectronics, 2022, 216: 114650. [4] WU Y, JIAO L, LUO X, et al. Oxidase-Like Fe-N-C Single-Atom Nanozymes for the Detection of Acetylcholinesterase Activity[J]. Small, 2019, 15(43): 1903108.), the K m value of Ce-CS is lower, indicating that Ce-CS has a higher affinity for the substrate TMB, and its V max is also relatively large.

[0103] Table 4 Comparison of steady-state kinetic parameters of different types of oxidases

[0104] Serial number Catalyst <![CDATA[K m / mmol·L -1 > <![CDATA[V max / Ms -1 > 1 <![CDATA[Copper oxide [1] > 1.097 <![CDATA[3.315×10 -8 > 2 <![CDATA[Nitrite [2] > 5.548 <![CDATA[9.91×10 -8 > 3 <![CDATA[DSMIP@Mn3O4 [3] > 5.1 <![CDATA[9.2×10 -9 > 4 <![CDATA[Fe-N-C SAzymes [4] > 1.81 <![CDATA[6.01×10 -8 > 5 Ce-CS 0.0429 <![CDATA[7.51×10 -8 >

[0105] Detection Example 12

[0106] Prepare the standard curve according to the method in Example 1. Figure 10 Among them, A is the ultraviolet absorption spectrum from 500 nm to 800 nm corresponding to different concentrations of APAP. The obtained standard curve is y = 0.20215X + 0.14338, as Figure 10 shown in B of 2 which the correlation coefficient R -1 = 0.99627, and the linear range is 50 μmol·L -1 to 2 mmol·L -1(S / N = 3). The results showed that the colorimetric detection of APAP based on peroxidase-like Ce-CS had high sensitivity. As shown in Table 5, compared with the performance of detecting APAP using other peroxidase-like enzymes (References: [5] NGO Q T T, PHAM T H, TUFAL T, et al. Rapid and sensitive detection of paracetamol in environmental aqueous samples using MnO2 nanosheet-based colorimetric sensing platform[J]. Colloid and Polymer Science, 2024, 302: 1415-1422. [6] LY C T, PHAN C T, VU C N, et al. Electrodeposition of PEDOT-rGO film in aqueous solution for detection of acetaminophen in traditional medicaments[J]. Advances in Natural Sciences: Nanoscience and Nanotechnology, 2019, 10(1): 015013. [7] GAO Y, CHU T, ZHOU C, et al. Fe, Cu Co-Doped Carbon Nanosheets for Electrochemical-Colorimetric Detection of Acetaminophen[J]. ACS Applied Nano Materials, 2024, 7(12): 14321-14330. [8] ALLWIN RICHARD Y, ANIU LINCY S, SARAVANAKUMAR R, et al. Sensitive detection of acetaminophen in body fluids, pharmaceuticals and herbal medicines at un-doped mesoporous carbon nitride film electrode[J]. Microchemical Journal, 2023, 184: 108175.), its detection range was larger and the detection limit was lower.

[0107] Table 5 Comparison of the detection of acetaminophen using different peroxidase-like enzymes

[0108] Serial number Catalyst Detection method <![CDATA[Linear range / mol·L -1 > <![CDATA[Detection limit / mol·L -1 > 1 <![CDATA[MnO2 [5] > Colorimetry <![CDATA[5×10 -4 ~10 -2 > <![CDATA[5.24×10 -7 > 2 <![CDATA[PEDOT-rGO [6] > Electrochemistry <![CDATA[1×10 -5 ~6×10 -5 > <![CDATA[5.74×10 -6 > 3 <![CDATA[FeCu / CNS [7] > Colorimetry <![CDATA[5×10 -7 ~1.625×10 -3 > <![CDATA[3×10 -6 > 4 <![CDATA[GCE [8] > Electrochemistry <![CDATA[1×10 -7 ~1.4×10 -3 > <![CDATA[9.322×10 -8 > 5 Ce-CS Colorimetry <![CDATA[5×10 -5 ~2×10 -3 > <![CDATA[1×10 -7 >

[0109] Detection Example 13

[0110] Examine various possible interfering substances, including metal ions (Na + , Mg 2+ , K + , Ca 2+ ), glucose (Gs), sucrose, glycine (Gly), and L-histidine (L-His). According to the method in Detection Example 10, replace the SA solution with the interfering substance solution. The results are as Figure 11 shown. Even when the concentration of the interfering substance is 10 times that of the APAP concentration, only when APAP is added to the reaction system will the absorbance decrease significantly. ΔA is only significantly correlated with the presence of APAP, indicating that the reaction of APAP with the peroxidase-like Ce-CS is specific. This proves that the colorimetric method for detecting APAP based on the peroxidase-like Ce-CS has high selectivity.

Claims

1. A cerium-based oxidase for paracetamol detection, characterized in that, It is prepared by the following method: L-cysteine and ammonium cerium nitrate are mixed evenly under alkaline conditions to undergo a complexation reaction to form a cerium-L-cysteine complex, and the cerium-based oxidase for paracetamol detection is obtained.

2. The cerium-based oxidase for paracetamol detection according to claim 1, wherein The molar ratio of the L-cysteine to the ammonium cerium nitrate is 1∶0.3-0.

7.

3. The cerium-based oxidase for paracetamol detection according to claim 2, characterized in that, The molar ratio of the L-cysteine to the ammonium cerium nitrate is 1∶0.

5.

4. The cerium-based oxidase for paracetamol detection according to claim 1, characterized in that, The alkaline condition is provided by NaOH, and the solvent used in the complexation reaction is water.

5. A method for detecting paracetamol, characterized in that, Using the cerium-based oxidase for paracetamol detection according to any one of claims 1 to 4, the method comprises the following steps: (1) Dispersing the cerium-based oxidase in a solvent to obtain a Ce-CS dispersion; (2) Sequentially adding the Ce-CS dispersion obtained in step (1) and the test solution into a buffer solution, mixing evenly, and enabling the cerium-based oxidase and paracetamol in the test solution to undergo an oxidation-reduction reaction to obtain solution 1; (3) Adding 3,3',5,5'-tetramethylbenzidine to solution 1, mixing evenly, and enabling 3,3',5,5'-tetramethylbenzidine to undergo a catalytic oxidation reaction to obtain solution 2; (4) Measuring the absorbance of solution 2 at 652 nm as A1; (5) Without adding the test solution in step (2), repeating steps (1)-(4), and measuring the absorbance as A2; (6) Comparing A2−A1 with the standard curve to obtain the concentration of paracetamol in the test solution.

6. The method for detecting paracetamol according to claim 5, characterized in that, In step (1), the solvent is water, ethanol or petroleum ether, and in step (2), the pH of the buffer solution is 3-5.

7. The method for detecting paracetamol according to claim 5, characterized in that, The ratio of the cerium-based oxidase added in step (2) to 3,3',5,5'-tetramethylbenzidine added in step (3) is 125-625 g∶1 mol.

8. The method for detecting paracetamol according to claim 5, characterized in that, In step (3), the reaction temperature is -7-50 °C, and the reaction time is 3-30 min.

9. The method for detecting paracetamol according to claim 5, characterized in that, In step (6), the method for making the standard curve: Prepare a series of paracetamol standard solutions with different concentrations, replace the test solution with this series of paracetamol standard solutions with different concentrations, respectively perform the operations of steps (1)-(5), and then make a response relationship curve of A2−A1 and the concentration of the paracetamol standard solution, and the standard curve is obtained.

10. The method for detecting paracetamol according to claim 9, wherein The linear range of the standard curve is 50 - 2000 μmol·L -1 .

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