Bimetal-loaded SOX nanoflower, preparation method thereof and sarcosine detection method

Abstract

The invention provides a bimetallic-loaded SOX nanoflower, a preparation method thereof and a method for detecting sarcosine by using the bimetallic-loaded SOX nanoflower. The preparation method of the bimetallic loaded SOX nanoflower comprises the following steps: mixing a Ce (NO3) 3.6 H2O aqueous solution and a CuSO4. 5H2O aqueous solution, and adding the mixture into a phosphate buffer solution containing sarcosine oxidase and bovine serum albumin; then, reacting the obtained mixture at room temperature; and after the reaction is finished, centrifuging and drying in vacuum to obtain the product. According to the method, firstly, an organic-inorganic hybrid nanoflower with excellent sarcosine oxidase activity and peroxidase-like activity is synthesized, efficient enzyme loading and catalytic capacity is achieved, on the basis, a nano platform based on multi-enzyme cascade reaction is developed, and high-sensitivity and rapid detection of sarcosine is achieved.

Description

Technical Field

[0001] The present invention relates to a bimetal-loaded SOX nanoflower, a preparation method thereof, and a method for detecting sarcosine, belonging to the technical field of analytical chemistry. Background Art

[0002] Prostate cancer is one of the common malignant tumors in men. Early detection and diagnosis of prostate cancer are crucial for improving the survival rate of patients. In recent years, sarcosine has attracted wide attention as a potential biomarker for prostate cancer. Sarcosine is a nitrogen-containing organic acid generated during human metabolism, mainly stored in muscle tissues, and plays an important role in energy metabolism. Research shows that abnormal energy metabolism in prostate cancer cells can lead to significant changes in sarcosine levels. Therefore, detecting changes in sarcosine levels can provide strong support for the early screening and diagnosis of prostate cancer.

[0003] Currently, the commonly used methods for detecting prostate cancer in clinics mainly include serum prostate-specific antigen (PSA) detection, tissue biopsy, imaging examination, etc. Although PSA detection has high sensitivity in early screening, its specificity is insufficient, prone to false positives and false negatives, and other detection means are needed for further confirmation. In addition, although tissue biopsy has high accuracy, it is an invasive examination, and patients may suffer great pain and have certain risks. Therefore, it is of great significance to develop a simple, low-cost, and highly sensitive method for detecting sarcosine to provide a more efficient prostate cancer screening program.

[0004] Currently, the methods for detecting sarcosine mainly include high-performance liquid chromatography (HPLC), mass spectrometry (MS), electrochemical sensors, etc. For example: Chinese patent document CN104438291A discloses a method for rapidly detecting the content of sarcosine in human plasma by high-performance liquid chromatography, which includes the sample pretreatment and chromatographic analysis processes. Using the separation technology of high-performance liquid chromatography and combining with an ultraviolet detector, rapid and quantitative detection of sarcosine is achieved. Chinese patent CN108792173A discloses a method for detecting sarcosine based on an electrochemical sensor. The electrochemical sensor reflects the concentration of sarcosine through the current change of the redox reaction. This method is simple to operate and has low cost, suitable for batch screening. Chinese patent CN109621917A discloses a method for detecting blood sarcosine based on mass spectrometry technology. Using the high resolution of mass spectrometry, trace sarcosine in blood can be detected with high sensitivity, but this method has high cost and high requirements for detection equipment.

[0005] Although the above sarcosine detection methods have certain detection effects in different application scenarios, they also have the following deficiencies: (1) Some methods have high instrument and equipment costs and complex operation processes, and are not suitable for large-scale screening; (2) Certain detection methods have insufficient sensitivity and are easily affected by other interfering substances, resulting in inaccurate results; (3) Some methods require complex sample pretreatment and take a long time.

[0006] Therefore, it is of great significance to develop a convenient, fast and highly sensitive detection method for sarcosine. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, especially the problems of insufficient sensitivity and convenience in detecting sarcosine in the prior art, the present invention provides a bimetal-loaded SOX nanoflower and its preparation method and a method for detecting sarcosine. The present invention first synthesizes an organic-inorganic hybrid nanoflower (Cu3(PO4)2:Ce@SOX) with both excellent sarcosine oxidase (SOX) activity and peroxidase-like activity, realizing efficient enzyme loading and catalytic ability. On this basis, a nano-platform based on multi-enzyme cascade reaction is developed to achieve highly sensitive and rapid detection of sarcosine.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A preparation method of a bimetal-loaded SOX nanoflower (Cu3(PO4)2:Ce@SOX) comprises the following steps:

[0010] Mix an aqueous solution of Ce(NO3)3·6H2O and an aqueous solution of CuSO4·5H2O, and add them to a phosphate buffer solution containing sarcosine oxidase and bovine serum albumin (BSA); then, react the obtained mixture at room temperature; after the reaction is completed, centrifuge and vacuum dry to obtain the bimetal-loaded SOX nanoflower (Cu3(PO4)2:Ce@SOX).

[0011] Preferably according to the present invention, the concentration of the aqueous solution of Ce(NO3)3·6H2O is 200 mmol / L, and the concentration of the aqueous solution of CuSO4·5H2O is 200 mmol / L; the molar ratio of CuSO4·5H2O to Ce(NO3)3·6H2O is 4-6:1.

[0012] Preferably according to the present invention, the concentration of the phosphate buffer solution is 10 mmol / L, the pH value is 7.4, and it is obtained by mixing an aqueous solution of K2HPO4·3H2O with a concentration of 10 mmol / L and an aqueous solution of NaH2PO4·2H2O with a concentration of 10 mmol / L in a volume ratio of 1:4.

[0013] Preferably according to the present invention, in the phosphate buffer solution containing sarcosine oxidase and bovine serum albumin (BSA), the concentration of sarcosine oxidase is 0.1 mg / mL, and the concentration of bovine serum albumin (BSA) is 0.05 mg / mL; the activity of sarcosine oxidase is 0.8 U / mg.

[0014] Preferably according to the present invention, the molar ratio of CuSO4·5H2O in the CuSO4·5H2O aqueous solution to the mass of sarcosine oxidase is 30 - 50 mmol:1 mg.

[0015] Preferably according to the present invention, the reaction time is 50 - 60 h.

[0016] Preferably according to the present invention, the centrifugation speed is 8000 - 10000 r / min, and the centrifugation time is 5 - 15 min; the temperature of vacuum drying is 40 - 45 °C, and the vacuum drying time is 10 - 12 h.

[0017] According to the present invention, the obtained bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) are stored in a dark environment at 4 °C.

[0018] The present invention also provides a kind of bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX), which are prepared by the above preparation method; the obtained bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) are in the shape of multi-layer nanoflowers.

[0019] According to the present invention, the above bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) are used for detecting sarcosine.

[0020] The present invention also provides a method for detecting sarcosine based on bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX), which includes the following steps:

[0021] (1) Mix the aqueous solution of bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) with aqueous solutions of sarcosine at different concentrations, add PBS buffer solution, and incubate at 37 °C to obtain a mixed solution;

[0022] (2) Add 3,3’,5,5’-tetramethylbenzidine (TMB) ethanol solution and NaAc-HAc buffer solution to the mixed solution obtained in step (1) respectively, incubate at room temperature, and obtain sarcosine standard solutions with different concentrations; use an ultraviolet-visible absorption spectrophotometer to measure the absorbance of the sarcosine standard solutions with different concentrations, record the absorbance at a wavelength of 652 nm, and draw a standard curve with the concentration of the sarcosine standard solution as the abscissa and the absorbance at 652 nm as the ordinate;

[0023] (3) Measure the absorbance of the aqueous solution of the sample to be tested at a test wavelength of 652 nm according to the method in steps (1)-(2), and calculate the content of sarcosine in the sample to be tested by comparing the measured absorbance with the standard curve.

[0024] Preferably according to the present invention, in step (1), the concentration of the aqueous solution of the bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) is 1 mg / mL; the volumes of both the aqueous solution of the bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) and the aqueous solution of sarcosine are 30 μL.

[0025] Preferably according to the present invention, in step (1), the concentration of the PBS buffer solution is 0.2 mol / L and the pH is 7.4; the volume of the PBS buffer solution is 100 μL.

[0026] Preferably according to the present invention, in step (1), incubate at 37 °C for 20 - 35 min.

[0027] Preferably according to the present invention, in step (2), the concentration of the 3,3’,5,5’-tetramethylbenzidine (TMB) ethanol solution is 10 mmol / L; the volume of the 3,3’,5,5’-tetramethylbenzidine (TMB) ethanol solution is 30 μL.

[0028] Preferably according to the present invention, in step (2), the concentration of the NaAc-HAc buffer solution is 0.2 mol / L and the pH is 5.0; the volume of the NaAc-HAc buffer solution is 810 μL.

[0029] Preferably according to the present invention, in step (2), the incubation time is 5 - 15 min, preferably 5 - 11 min.

[0030] Preferably according to the present invention, in step (2), the concentration of the sarcosine standard solution is 0.18 - 60 μmol / L.

[0031] The present invention also provides a cotton swab for detecting sarcosine in a colorimetric mode by using bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX), which is prepared according to the following method:

[0032] (i) Mix the aqueous solution of bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX), the 3,3’,5,5’-tetramethylbenzidine (TMB) ethanol solution, the aqueous solution of gelatin, and the NaAc-HAc buffer solution, and ultrasonically mix them evenly to obtain a mixed solution;

[0033] (ii) Insert a medical cotton swab into the mixed solution and let it stand still to allow the mixed solution to fully penetrate the cotton swab. Then, freeze the cotton swab loaded with bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) and perform freeze-drying treatment to obtain a cotton swab for colorimetric detection of sarcosine with bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX).

[0034] Preferably according to the present invention, in step (i), the concentration of the aqueous solution of bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) is 1 mg / mL, the concentration of the ethanol solution of 3,3’,5,5’-tetramethylbenzidine (TMB) is 10 mmol / L, the concentration of the aqueous solution of gelatin is 10 mg / mL, the concentration of the NaAc-HAc buffer solution is 0.2 mol / L, and the pH is 5.0; the volume ratio of the aqueous solution of bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX), the ethanol solution of 3,3’,5,5’-tetramethylbenzidine (TMB), the aqueous solution of gelatin, and the NaAc-HAc buffer solution is 3:3:2:5.

[0035] Preferably according to the present invention, in step (ii), the standing time is 5 - 10 min; the freezing is carried out at -20 °C for 4 hours; the freeze-drying is carried out at -20 °C for 10 - 15 h.

[0036] According to the present invention, the obtained cotton swab for colorimetric detection of sarcosine with bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) is stored in a dark environment at 4 °C for standby.

[0037] According to the present invention, a method for detecting sarcosine using the above-mentioned cotton swab for colorimetric detection of sarcosine with bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) includes the following steps:

[0038] (I) Add 20 μL of sarcosine aqueous solutions with different concentrations into 50 μL of PBS buffer solution to obtain a mixed solution, and then immerse the cotton swab for colorimetric detection of sarcosine with bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) in the mixed solution.

[0039] (II) Take out the immersed cotton swab, incubate it at 37 °C for 20 - 30 min, then add it into 50 μL of NaAc-HAc buffer solution and incubate it at 37 °C for 5 - 15 min; finally, take a photo of the incubated cotton swab to obtain an image.

[0040] (III) Extract and analyze the R value, G value, and B value of the color of the cotton swab in the image obtained in step (2), and convert them into a gray value according to the following formula: Gray = 0.299R + 0.587G + 0.114B; for each cotton swab image, at least 10 points are taken to calculate the average gray value, and a standard curve is plotted with the logarithm of the sarcosine concentration as the abscissa and the average gray value as the ordinate;

[0041] (IV) For the aqueous solution of the sample to be tested, calculate the average gray value according to the methods of steps (1)-(3), and calculate the content of sarcosine in the aqueous solution of the sample to be tested by comparing the obtained average gray value with the standard curve.

[0042] Preferably according to the present invention, the concentration of sarcosine in the mixed solution in step (I) is 2.44 - 625 μmol / L.

[0043] Preferably according to the present invention, the concentration of the PBS buffer solution in step (I) is 0.2 mol / L, and the pH is 7.4.

[0044] Preferably according to the present invention, the soaking time in step (I) is 5 - 10 min.

[0045] Preferably according to the present invention, the concentration of the NaAc-HAc buffer solution in step (II) is 0.2 mol / L, and the pH is 5.0.

[0046] Preferably according to the present invention, in step (II), the pixel of the device used to take a photo of the incubated cotton swab is greater than or equal to 8 million; the device used to take a photo of the incubated cotton swab only needs to have a photo-taking function, preferably a smart phone; when taking a photo of the incubated cotton swab, the photo-taking environment is under the condition of sufficient light, but direct sunlight of strong light sources and the appearance of reflection should be avoided to obtain a clear photo of the cotton swab, such as taking a photo under the uniform light of an incandescent lamp.

[0047] According to the present invention, in step (III), the method for extracting and analyzing the R value, G value, and B value of the color of the cotton swab in the obtained image is to use existing application programs capable of analyzing the RGB values of the photo color, which can be mobile phone application programs such as ColorGrab, color recognition apps, ImageJ, color cards, etc.; or computer software such as Photoshop, etc. to obtain the R value, G value, and B value of the color of the cotton swab in the photo. The R value, G value, and B value are the R value, G value, and B value in the RGB color (R = Red, G = Green, B = Blue), which can better and more stably reflect the color change of the cotton swab of the present invention.

[0048] According to the present invention, in step (III), to improve accuracy, for each swab image, multiple points are randomly and evenly selected for extracting and analyzing the R value, G value, and B value of the color, and the average gray value is calculated. Preferably, 10 - 15 points are selected.

[0049] According to the present invention, in step (IV), when detecting the concentration of the aqueous solution of the sample to be tested, the photographing conditions of the swab after incubation are the same as those in step (II).

[0050] The technical features and beneficial effects of the present invention are as follows:

[0051] 1. The present invention proposes a synthesis method of organic - inorganic self - assembled nanoflowers for optimizing the existing sarcosine detection technology. By synthesizing an organic - inorganic hybrid nanoflower (Cu3(PO4)2:Ce@SOX) with both excellent sarcosine oxidase (SOX) activity and peroxidase - like activity, efficient enzyme loading and catalytic ability are achieved. On this basis, a nano - platform based on multi - enzyme cascade reaction is developed, and a colorimetric method is used to achieve highly sensitive detection of sarcosine. The prepared Cu3(PO4)2:Ce@SOX composite material has excellent performance, with an encapsulation efficiency as high as 84.5%. Compared with free SOX, its catalytic activity is increased by 1.26 to 1.29 times. The colorimetric detection range of this nano - platform is 0.18 μmol / L to 60 μmol / L, and the detection limit is as low as 0.12 μmol / L. Further, a smartphone - based portable sensor system developed based on the organic - inorganic hybrid nanoflower (Cu3(PO4)2:Ce@SOX) can achieve instant analysis of sarcosine through a swab without the support of additional instruments. This system has good repeatability and stability, and is particularly suitable for detecting sarcosine in human urine samples. The reactants and reagents used in the present invention are all commercially available products, with simple operation and low cost. The proposed nano - platform technology has broad application prospects, and can not only be used for sarcosine detection, but also provides an important reference for the development of portable and efficient biosensors for other analytes.

[0052] 2. The present invention discloses a method for detecting sarcosine in a colorimetric mode based on Cu3(PO4)2:Ce@SOX. By utilizing the specific triggering of SOX activity and the potential influence of by - products on the peroxidase - like activity of Cu3(PO4)2:Ce@SOX, indirect detection of sarcosine is realized; sarcosine is detected through the colorimetric mode Cu3(PO4)2:Ce@SOX + TMB system, with a detection limit as low as 0.12 μmol / L and a detectable range as wide as 0.18 - 60 μmol / L; at the same time, the content of sarcosine in human urine is detected by using a smartphone - based portable sensor system, verifying its great potential in disease diagnosis and treatment.

[0053] In this specification, the term "nanozyme" specifically refers to a nanomaterial with catalytic activity similar to that of natural enzymes. Nanozymes are a class of materials that exhibit the characteristics of natural enzymes at the nanoscale, can catalyze biochemical reactions mediated by natural enzymes under physiological conditions, and show reaction kinetics and catalytic mechanisms similar to those of natural enzymes.

[0054] In addition, the term "peroxidase-like enzyme" specifically refers to a material with peroxidase catalytic activity. Specifically, the peroxidase-like enzyme of the present invention uses hydrogen peroxide as an electron acceptor, and generates a colored product by catalytically oxidizing a chromogenic substrate to achieve colorimetric detection, providing core technical support for the detection method of the present invention.

[0055] In this specification, the term "Cu3(PO4)2:Ce@SOX" is the abbreviated name of bimetal-loaded SOX nanoflowers, and the two can be used interchangeably.

[0056] In this specification, the term "TMB" is the abbreviated name of the compound "3,3',5,5'-tetramethylbenzidine", and the two can be used interchangeably.

[0057] In this specification, the term "TMBox" is the abbreviated name of the oxidation product of the compound "3,3',5,5'-tetramethylbenzidine", and the two can be used interchangeably.

[0058] In this specification, the term "SOX" refers to sarcosine oxidase, and the two can be used interchangeably.

[0059] In this specification, the term "Sar" refers to sarcosine, and the two can be used interchangeably. Description of the Drawings

[0060] Figure 1 SEM image of the bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) prepared for Example 1.

[0061] Figure 2 UV-visible absorption spectra of the catalytic oxidation of TMB in different reaction systems in Example 1.

[0062] Figure 3 Effect diagram of the conditional synthesis optimization of the Cu3(PO4)2:Ce@SOX material in Example 2.

[0063] Figure 4 Optimization result diagram of the catalytic process of Cu3(PO4)2:Ce@SOX in Example 3, where a is the pH optimization diagram of the Cu3(PO4)2:Ce@SOX peroxidase-like enzyme, and the inset is a photo of different pH solutions; b is the absorbance of the Cu3(PO4)2:Ce@SOX + TMB system at different reaction times.

[0064] Figure 5 For the UV-visible absorption spectra of reaction systems with different H2O2 concentrations in Example 4 (a) and the fitted straight line showing the relationship between H2O2 concentration and absorbance (b).

[0065] Figure 6 For the UV-visible absorption spectra of the reaction of sarcosine concentration affecting the Cu3(PO4)2:Ce@SOX+TMB system in Example 5 (a) and the fitted straight line showing the relationship between sarcosine concentration and absorbance at 652 nm (b).

[0066] Figure 7 For the effects of different common ions on the detection of sarcosine in Example 6.

[0067] Figure 8 For the detection performance of the smartphone-based recognition system in Example 7, where (a) are the grayscale values of sarcosine solutions with different concentrations and (b) is the fitted straight line showing the relationship between the logarithm of sarcosine concentration and grayscale value. Detailed implementation manners

[0068] To more clearly illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will now be described in more detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the content of the present invention and do not constitute a limitation on the present invention.

[0069] Sarcosine oxidase was purchased from Aladdin Industrial Corporation.

[0070] Example 1

[0071] A preparation method of bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) includes the following steps:

[0072] (1) Prepare phosphate (PBS) buffer solution: Mix an aqueous solution of K2HPO4·3H2O (10 mmol / L, 80 mL) and an aqueous solution of NaH2PO4·2H2O (10 mmol / L, 320 mL) to obtain a phosphate (PBS) buffer solution with a concentration of 10 mmol / L and a pH value of 7.4;

[0073] (2) Mix an aqueous solution of Ce(NO3)3·6H2O (200 mmol / L, 50 μL) and an aqueous solution of CuSO4·5H2O (200 mmol / L, 200 μL), and gradually add the mixture dropwise to a phosphate buffer solution (10 mL) containing SOX (0.1 mg / mL, activity 0.8 U / mg) and bovine serum albumin (BSA, 0.05 mg / mL) to obtain a mixed solution; transfer the obtained mixed solution to a beaker and react at room temperature for 56 h; after the reaction is completed, centrifuge the obtained reaction solution at a rotation speed of 10000 r / min for 15 min, collect the blue-brown precipitate, and dry it in vacuum at 40 °C for 12 h to obtain bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX); to maintain the biological enzyme activity required for Sar catalysis, store the prepared bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) in a dark environment at 4 °C.

[0074] The bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) obtained in Example 1 herein are in the shape of multi-layer nanoflowers, and their SEM images are as Figure 1 shown.

[0075] The application experiment of Cu3(PO4)2:Ce@SOX as a peroxidase-like catalyst for the H2O2 oxidation of TMB reaction has the following steps:

[0076] Prepare a reaction mixture: Take 910 μL of NaAc-HAc buffer solution (pH 5.0, 0.2 mol / L), add 30 μL of an aqueous solution of Cu3(PO4)2:Ce@SOX (1 mg / mL), 30 μL of an aqueous solution of H2O2 (10 mmol / L), and 30 μL of a TMB ethanol solution (10 mmol / L); incubate the obtained mixture at 37 °C for 8 minutes to promote the color development reaction of TMB. The color of the liquid changes from colorless to blue with time. Measure the ultraviolet-visible absorption spectrum of the above mixed solution using an ultraviolet-visible absorption spectrophotometer; at the same time, use a mixed solution containing only TMB (970 μL of NaAc-HAc buffer solution + 30 μL of TMB ethanol solution) and a mixed solution containing only TMB + H2O2 (940 μL of NaAc-HAc buffer solution + 30 μL of an aqueous solution of H2O2 + 30 μL of TMB ethanol solution) as controls. Figure 2 The UV-Vis diagrams for the catalytic oxidation of TMB reactions in different systems are shown in Figure 2It can be seen that only when TMB, H2O2 and Cu3(PO4)2:Ce@SOX coexist, a color reaction will occur; when TMB or TMB+H2O2 exists, no color reaction will occur. This reaction is caused by the H2O2 in the Cu3(PO4)2:Ce@SOX catalytic system producing hydroxyl radicals, and the color developer (TMB) undergoing a redox reaction with the hydroxyl radical ions, proving that the prepared Cu3(PO4)2:Ce@SOX has good peroxidase-like activity.

[0077] Example 2

[0078] Optimization of Cu3(PO4)2:Ce@SOX enzyme activity

[0079] (1) Synthesis of different Cu 2+ / Ce 3+ The specific preparation method is as described in Example 1, except that the volumes of the CuSO4·5H2O aqueous solution and the Ce(NO3)3·6H2O aqueous solution in step (2) are 250 μL and 0 μL, respectively (Cu 2+ / Ce 3+ molar ratio of 1:0), 214 μL and 36 μL (Cu 2+ / Ce 3+ molar ratio of 6:1), 167 μL and 83 μL (Cu 2+ / Ce 3+ molar ratio of 2:1), 125 μL and 125 μL (Cu 2+ / Ce 3+ molar ratio of 1:1), 83 μL and 167 μL (Cu 2+ / Ce 3+ molar ratio of 1:2), 50 μL and 200 μL (Cu 2+ / Ce 3+ molar ratio of 1:4), 36 μL and 214 μL (Cu 2+ / Ce 3+ molar ratio of 1:6), 0 μL and 250 μL (Cu 2+ / Ce 3+ The molar ratio is 0:1).

[0080] (2) Take 920 μL of NaAc-HAc buffer (0.2 mol / L, pH 5.0), add 50 μL of 10 mmol / L TMB ethanol solution and 30 μL of 1 mg / mL Cu3(PO4)2:Ce@SOX aqueous dispersion with different metal ratios, mix well, and react at room temperature for 5 minutes. During the reaction, the color of the liquid gradually changes from colorless to blue. Comparing the absorbance, it is found that when Cu2+ / Ce 3+ When the ratio is 4:1, the reaction activity is the best ( Figure 3 ), so all subsequent experiments used Cu3(PO4)2:Ce@SOX which was prepared according to the method described in Example 1.

[0081] Example 3

[0082] 1. Take 890 μL of NaAc-HAc buffer (0.2 mol / L) with pH values ​​of 2, 3, 4, 5, 6, and 7, respectively, and add 50 μL of 10 mmol / L TMB ethanol solution, 30 μL of H2O2 aqueous solution (10 mmol / L), and 30 μL of 1 mg / mL Cu3(PO4)2:Ce@SOX aqueous solution, mix well, and react at room temperature for 5 minutes. During the reaction, the liquid color gradually changes from colorless to blue;

[0083] 2. Use a UV-Vis absorption spectrophotometer to measure the UV-Vis absorption spectrum of the mixed solution. The results are as follows: Figure 4 As shown in a, with the increase of pH value, the absorbance value first increases and then decreases, and the absorbance value reaches a peak value at pH 5.0, showing the best catalytic activity.

[0084] 3. Take 890μL of 0.2mol / L pH5.0 NaAc-HAc buffer, add 50μL of 10mmol / L TMB ethanol solution, 30μL of H2O2 aqueous solution (10mmol / L) and 30μL of 1mg / mL Cu3(PO4)2:Ce@SOX aqueous solution in sequence, mix well, and use a UV-visible absorption spectrophotometer to measure the UV-visible absorption spectrum at different reaction times. As the reaction time increases, the absorbance value gradually increases. Figure 4 As shown in b, the absorbance reaches 0.8 after 5 minutes of reaction, which is sufficient to meet the detection requirements.

[0085] Example 4

[0086] UV-Vis absorption spectrum of Cu3(PO4)2:Ce@SOX+H2O2+TMB reaction system

[0087] 1. Prepare a reaction solution for detecting H2O2: Take 30 μL of Cu3(PO4)2:Ce@SOX aqueous solution (1 mg / mL) and 30 μL of H2O2 aqueous solutions with different concentrations, add 910 μL of NaAc-HAc buffer solution (pH 5.0, 0.2 mol / L). After the reaction ends in 5 min, transfer the mixed solution to a quartz cuvette, and add 30 μL of TMB ethanol solution (10 mmol / L) to the above system and react for 5 min; the final concentrations of H2O2 in the system are 0.075 μmol / L, 0.3 μmol / L, 2.4 μmol / L, 6.0 μmol / L, 14 μmol / L, 18 μmol / L, 25 μmol / L, 36 μmol / L, 60 μmol / L, 90 μmol / L respectively;

[0088] 2. Record the ultraviolet-visible spectrum of this system for further analysis of the reaction situation. The results are as Figure 5 shown in a. The liquid color of the Cu3(PO4)2:Ce@SOX + H2O2 + TMB system has a strong ultraviolet absorption signal at 652 nm with the increase of H2O2 concentration; taking the H2O2 concentration as the abscissa and the absorbance at 652 nm as the ordinate, draw a curve, and the regression equation is A 652 = 0.00713[H2O2] + 0.108 (R 2 = 0.993) ( Figure 5 b), where A 652 is the absorbance at 652 nm, [H2O2] is the H2O2 concentration, and the detectable range of this method for H2O2 concentration is 0.12 - 36.0 μmol / L (linear detection range), and it has excellent detection effect.

[0089] Example 5

[0090] A method for detecting sarcosine based on bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) includes the following steps:

[0091] 1. Take 30 μL of Cu3(PO4)2:Ce@SOX aqueous solution (1 mg / mL) and 30 μL of sarcosine aqueous solutions with different concentrations and mix them, add 100 μL of PBS buffer solution (pH 7.4, 0.2 mol / L), and incubate at 37 °C for 30 min to obtain a mixed solution;

[0092] 2. Then, 30 μL of TMB ethanol solution (10 mmol / L) and 810 μL of NaAc-HAc buffer solution (pH 5.0, 0.2 mol / L) were successively added to the mixed solution, and incubated at room temperature for 5 min to obtain sarcosine standard solutions with different concentrations; the concentrations of the sarcosine standard solutions were 0.18 μmol / L, 0.5 μmol / L, 4 μmol / L, 10 μmol / L, 25 μmol / L, 30 μmol / L, 45 μmol / L, 60 μmol / L, 100 μmol / L, 150 μmol / L, and 300 μmol / L respectively;

[0093] 3. The absorbance of the above standard solutions at 652 nm was measured by an ultraviolet-visible absorption spectrophotometer, and a standard working curve was plotted with the concentration of the sarcosine standard solution as the abscissa and the absorbance at 652 nm as the ordinate.

[0094] The results of detecting the sarcosine concentration by this method are as Figure 6 shown. Among them, Figure 6 a shows that as the concentration of sarcosine increases, the absorbance of the solution gradually increases. Figure 6 b is a fitting straight line with the concentration of the sarcosine standard solution as the abscissa and the absorbance at 652 nm as the ordinate. The regression equation is A 652 = 0.00395[Sar] + 0.0463 (R 2 = 0.991) indicates that the detectable range of the sarcosine concentration by this method is 0.18 - 60 μmol / L (linear detection range), the detection limit is 0.12 μmol / L, and it has excellent detection effects.

[0095] Example 6

[0096] Detecting the selectivity of sarcosine using the reaction system of Example 5

[0097] 1. To evaluate the anti-interference ability of the sarcosine analysis platform, the colorimetric response of the target was observed under the addition of different common ions and small molecules; 50 μL of Cu3(PO4)2:Ce@SOX aqueous solution at 1 mg / mL and 20 μL of sarcosine aqueous solution at 10 μmol / L were added to a 1.5 mL centrifuge tube respectively, and reacted at 37 °C for 30 min; then 900 μL of NaAc-HAc solution (0.2 mol / L, pH 5.0) was added to the mixed solution and mixed evenly, and then 30 μL of TMB ethanol solution at 10 mmol / L was added to the above mixed solution, and reacted at room temperature for 5 min. The absorbance of the above mixed solution at 652 nm was measured by an ultraviolet-visible absorption spectrophotometer;

[0098] 2. Add 10 μL of common metal ions (Fe 3 + , Ca 2+ , and Na + ) with different concentrations of 10 mmol / L, anions (PO4 3 -, SO4 2 -, and CO3 2 -), and small molecules (glucose (Glu), adenosine triphosphate (ATP), glycine (Gly), arginine (Arg), alanine (Ala), and urea (Urea)) to the detection system containing Cu3(PO4)2:Ce@SOX respectively, observe the changes in fluorescence emission and TMB color reaction, and repeat the experiment;

[0099] The results are as Figure 7 shown. Except for Fe 3+ , the effects of these common ions and small molecules on the fluorescence emission and TMB color reaction catalyzed by Cu3(PO4)2:Ce@SOX can be ignored; due to the peroxidase-like activity of Fe 3+ accelerating the color reaction and affecting the accurate detection of sarcosine, adding ethylenediaminetetraacetic acid (EDTA, 10 mmol / L) as a chelating agent can effectively eliminate the interference caused by Fe 3+ .

[0100] Example 7

[0101] A method for detecting sarcosine with a cotton swab based on the colorimetric mode of bimetal-loaded SOX nanoflowers (Cu3(PO4)2:Ce@SOX) for detecting sarcosine, comprising the following steps:

[0102] 1. Prepare a colorimetric test cotton swab: Add 30 μL of Cu3(PO4)2:Ce@SOX aqueous solution (1 mg / mL), 30 μL of TMB ethanol solution (10 mmol / L), 20 μL of gelatin aqueous solution (10 mg / mL), and 50 μL of NaAc-HAc buffer solution (pH 5.0, 0.2 mol / L) to a test tube, and mix well by ultrasound. Insert a medical cotton swab into the test tube and let it stand for 5 minutes to allow the mixed solution to fully penetrate the cotton swab. The prepared cotton swab is frozen at -20 °C for 4 hours, and then freeze-dried (temperature is -20 °C, time is 12 h) to obtain a cotton swab; the dried cotton swab is stored in a 4 °C dark environment for later use.

[0103] 2. Smartphone-assisted sarcosine measurement: Add 20 μL of aqueous sarcosine solutions with different concentrations into 50 μL of PBS buffer (pH 7.4, 0.2 mol / L) to obtain a mixed solution. The concentrations of sarcosine in the mixed solution are 2.44, 4.88, 9.76, 19.53, 39.06, 78.12, 156.25, 312.50, 625.0, 1250, 2500, 5000 μmol / L respectively. Then immerse a cotton swab into the mixed solution, take out the cotton swab after soaking for 5 min, and incubate it at 37 °C for 25 minutes. Next, add it into 50 μL of NaAc-HAc buffer (pH 5.0, 0.2 mol / L), and then incubate it at 37 °C for 10 minutes. Observe the color change of the cotton swab with the naked eye, take a photo of the incubated cotton swab using a smartphone, extract the color R value, G value, and B value of the cotton swab in the analyzed image through the color card application program of the smartphone, and calculate and convert it into a gray value according to the following formula: Gray = 0.299R + 0.587G + 0.114B. Randomly take 12 points evenly from each cotton swab photo for extraction and analysis. The specific gray values are as Figure 8 shown in a. In the figure, 1-13 are the gray values of concentrations of 5000, 2500, 1250, 625.0, 312.50, 156.25, 78.12, 39.06, 19.53, 9.76, 4.88, 2.44 μmol / L and the blank cotton swab respectively.

[0104] Draw a standard curve with the logarithm of the sarcosine concentration in the mixed solution as the abscissa and the average gray value as the ordinate. The regression equation is Y = 202.157 - 21.664X, R 2 = 0.989, where Y is the average gray value and X is the logarithm of the sarcosine concentration in the mixed solution, so as to realize the quantitative detection of sarcosine, as Figure 8 shown in b. With the increase of the added amount of sarcosine, under the action of Cu3(PO4)2:Ce@SOX, sarcosine oxidase catalyzes the oxidation of sarcosine to H2O2, and the gray value gradually decreases. The result of detecting the sarcosine concentration by using the smartphone color picking method shows that this method has an excellent detection effect and a good linear relationship for the sarcosine concentration within the range of 2.44 μmol / L - 625 μmol / L.

[0105] Example 8

[0106] Detect the concentration of sarcosine in actual urine by using the method of Example 5

[0107] Approximately 10 mL of early morning urine was collected from a healthy adult male volunteer (without eating before collection). The sample was centrifuged at 5000 rpm for 10 minutes, the supernatant was collected, and stored at -10 °C until analysis. The urine sample was diluted 20-fold and different concentrations of sarcosine were added to prepare spiked samples for subsequent detection; before the experiment, the volunteer signed an informed consent form, and the experimental process strictly followed the ethical guidelines of the Declaration of Helsinki in 1964 and its subsequent amendments or similar ethical guidelines to ensure that the experiment complied with relevant ethical standards. The specific experiment is as follows:

[0108] 1. Take 30 μL of Cu3(PO4)2:Ce@SOX aqueous solution (1 mg / mL) and 30 μL of the urine sample of sarcosine respectively, add 100 μL of PBS buffer (pH 7.4, 0.2 mol / L), and incubate at 37 °C for 30 min to obtain a mixed solution;

[0109] 2. Then add 30 μL of TMB ethanol solution (10 mmol / L) and 810 μL of NaAc-HAc buffer (pH 5.0, 0.2 mol / L) to the mixed solution in sequence to control the total volume to 1 mL, so that the sarcosine concentrations are 5 μmol / L and 10 μmol / L respectively, and react at room temperature for 5 min to obtain a mixed solution;

[0110] 3. Measure the absorbance of the above mixed solution at 652 nm with an ultraviolet-visible absorption spectrophotometer, and draw a standard working curve with the sarcosine concentration as the abscissa and the absorbance at 652 nm as the ordinate.

[0111] The results are shown in Table 1:

[0112] Table 1 Comparison of the detection results of the actual samples by this method and the national standard method

[0113]

[0114] As can be seen from the above table, the reaction system is sensitive to the change of sarcosine content in the actual sample, and the results are more accurate compared with the national standard method.

[0115] As described above, it is only the preferred specific implementation manner of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the description of the present invention, or directly or indirectly applied to other related technical fields, is similarly included in the patent protection scope of the present invention.

Claims

1. A preparation method of bimetal-loaded SOX nanoflowers, comprising the following steps: Mix an aqueous solution of Ce(NO3)3·6H2O and an aqueous solution of CuSO4·5H2O, and add them to a phosphate buffer solution containing sarcosine oxidase and bovine serum albumin; then, react the obtained mixture at room temperature; after the reaction is completed, centrifuge and vacuum dry to obtain bimetal-loaded SOX nanoflowers.

2. The preparation method of the bimetal-loaded SOX nanoflower according to claim 1, characterized in that, The concentration of the aqueous solution of Ce(NO3)3·6H2O is 200 mmol / L, and the concentration of the aqueous solution of CuSO4·5H2O is 200 mmol / L; the molar ratio of CuSO4·5H2O to Ce(NO3)3·6H2O is 4 - 6:1; The concentration of the phosphate buffer solution is 10 mmol / L, and the pH value is 7.

4. It is obtained by mixing an aqueous solution of K2HPO4·3H2O with a concentration of 10 mmol / L and an aqueous solution of NaH2PO4·2H2O with a concentration of 10 mmol / L in a volume ratio of 1:4; In the phosphate buffer solution containing sarcosine oxidase and bovine serum albumin, the concentration of sarcosine oxidase is 0.1 mg / mL, and the concentration of bovine serum albumin is 0.05 mg / mL; the activity of sarcosine oxidase is 0.8 U / mg; The molar ratio of CuSO4·5H2O in the aqueous solution of CuSO4·5H2O to the mass of sarcosine oxidase is 30 - 50 mmol:1 mg; the reaction time is 50 - 60 h; the centrifugation speed is 8000 - 10000 r / min, and the centrifugation time is 5 - 15 min; the temperature of vacuum drying is 40 - 45 °C, and the time of vacuum drying is 10 - 12 h.

3. A bimetal-loaded SOX nanoflower, characterized in that, Prepared by the preparation method described in claim 1.

4. Application of the bimetal-loaded SOX nanoflowers described in claim 3 in detecting sarcosine.

5. A method for detecting sarcosine based on the bimetal-loaded SOX nanoflowers described in claim 3, comprising the following steps: (1) Mix an aqueous solution of bimetal-loaded SOX nanoflowers with aqueous solutions of sarcosine at different concentrations, add PBS buffer solution, and incubate at 37 °C to obtain a mixed solution; (2) Add 3,3’,5,5’-tetramethylbenzidine ethanol solution and NaAc-HAc buffer solution to the mixed solution obtained in step (1) respectively, and incubate at room temperature to obtain sarcosine standard solutions with different concentrations; measure the absorbance of the sarcosine standard solutions with different concentrations using an ultraviolet-visible absorption spectrophotometer, record the absorbance at a wavelength of 652 nm, and plot a standard curve with the concentration of the sarcosine standard solution as the abscissa and the absorbance at 652 nm as the ordinate; (3) For the aqueous solution of the sample to be tested, measure the absorbance at a wavelength of 652 nm according to the method of steps (1) - (2), and calculate the content of sarcosine in the sample to be tested by comparing the measured absorbance with the standard curve.

6. The method for detecting sarcosine according to claim 5, wherein, In step (1), the concentration of the aqueous solution of bimetal-loaded SOX nanoflowers is 1 mg / mL; the volumes of both the aqueous solution of bimetal-loaded SOX nanoflowers and the aqueous solution of sarcosine are 30 μL; the concentration of the PBS buffer solution is 0.2 mol / L and the pH is 7.4; the volume of the PBS buffer solution is 100 μL; in step (1), incubation is carried out at 37 °C for 20 - 35 min; In step (2), the concentration of the 3,3’,5,5’-tetramethylbenzidine ethanol solution is 10 mmol / L; the volume of the 3,3’,5,5’-tetramethylbenzidine ethanol solution is 30 μL; the concentration of the NaAc-HAc buffer solution is 0.2 mol / L and the pH is 5.0; the volume of the NaAc-HAc buffer solution is 810 μL; In step (2), the incubation time is 5 - 15 min, preferably 5 - 11 min; The concentration of the sarcosine standard solution is 0.18 - 60 μmol / L.

7. A cotton swab for detecting sarcosine in a colorimetric mode with a bimetal-loaded SOX nanoflower, characterized in that, It is prepared by the following method: (i) Mix the aqueous solution of bimetal-loaded SOX nanoflowers, the 3,3’,5,5’-tetramethylbenzidine ethanol solution, the aqueous solution of gelatin, and the NaAc-HAc buffer solution, and ultrasonically mix them evenly to obtain a mixed solution; (ii) Insert a medical cotton swab into the mixed solution and let it stand still to allow the mixed solution to fully penetrate the cotton swab; then freeze the cotton swab loaded with bimetal-loaded SOX nanoflowers and perform freeze-drying treatment to obtain a cotton swab for detecting sarcosine by the colorimetric mode of bimetal-loaded SOX nanoflowers.

8. The cotton swab for detecting sarcosine by the colorimetric mode of the bimetal-loaded SOX nanoflower according to claim 7, wherein, In step (i), the concentration of the aqueous solution of bimetal-loaded SOX nanoflowers is 1 mg / mL, the concentration of the 3,3’,5,5’-tetramethylbenzidine ethanol solution is 10 mmol / L, the concentration of the aqueous solution of gelatin is 10 mg / mL, the concentration of the NaAc-HAc buffer solution is 0.2 mol / L and the pH is 5.0; the volume ratio of the aqueous solution of bimetal-loaded SOX nanoflowers, the 3,3’,5,5’-tetramethylbenzidine ethanol solution, the aqueous solution of gelatin, and the NaAc-HAc buffer solution is 3:3:2:5; In step (ii), the standing time is 5 - 10 min; the freezing is carried out at -20 °C for 4 hours; the freeze-drying is carried out at -20 °C for 10 - 15 h.

9. A method for detecting sarcosine with a cotton swab for detecting sarcosine by the colorimetric mode of bimetal-loaded SOX nanoflowers according to claim 7, comprising the following steps: (I) Add 20 μL of aqueous solutions of sarcosine with different concentrations to 50 μL of PBS buffer solution to obtain a mixed solution, and then immerse the cotton swab for detecting sarcosine by the colorimetric mode of bimetal-loaded SOX nanoflowers in the mixed solution; (II) Take out the immersed cotton swab, incubate it at 37 °C for 20 - 30 min, then add it to 50 μL of NaAc-HAc buffer solution, and incubate it at 37 °C for 5 - 15 min; finally, take a photo of the incubated cotton swab to obtain an image; (III) Extract and analyze the R value, G value, and B value of the color of the cotton swab in the image obtained in step (2), and convert them into a gray value Gray = 0.299R + 0.587G + 0.114B according to the following formula; for each cotton swab image, at least 10 points are taken to calculate the average gray value, and a standard curve is plotted with the logarithm of the sarcosine concentration as the abscissa and the average gray value as the ordinate; (IV) For the aqueous solution of the sample to be tested, calculate the average gray value according to the methods in steps (1)-(3), and calculate the content of sarcosine in the aqueous solution of the sample to be tested by comparing the obtained average gray value with the standard curve.

10. The method for detecting sarcosine according to claim 9, characterized in that, In step (I), the concentration of the sarcosine aqueous solution is 2.44 - 625 μmol / L; the concentration of the PBS buffer solution is 0.2 mol / L, and the pH is 7.4; the soaking time is 5 - 10 min; In step (II), the concentration of the NaAc-HAc buffer solution is 0.2 mol / L, and the pH is 5.0.

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