Preparation method of PFC-1-UiO-66 heterojunction nano-enzyme and application of PFC-1-UiO-66 heterojunction nano-enzyme in multi-mode detection of catechol

Abstract

The invention discloses a preparation method of PFC-1-UiO-66 heterojunction nano-enzyme and application of the PFC-1-UiO-66 heterojunction nano-enzyme in multi-mode detection of catechol, and relates to the technical field of biochemical analysis and detection. The method comprises the following steps: dissolving cerium salt in a mixed solvent, heating to react, cooling with ice water, adding DMF (Dimethyl Formamide) dissolved with terephthalic acid, and reacting to obtain UiO-66; and dissolving 1, 3, 6, 8-tetra (4-carboxybenzene) pyrene in DMF (Dimethyl Formamide), adding UiO-66, heating to react, cooling after the reaction, adding acetone and stirring, centrifuging, washing and drying to obtain the heterojunction nano-enzyme. A detection reagent constructed on the basis of the heterojunction nano enzyme can react with TMB (tetramethylbenzidine) under an acidic condition to realize 652 nm colorimetric detection, can react with 2, 4-dichlorophenol / 4-aminoantipyrine under a neutral condition to realize 510 nm colorimetric detection, and can generate a photo-thermal signal through 808 nm near-infrared light illumination to realize colorimetric and photo-thermal dual-mode detection. The method has the advantages of high detection sensitivity, strong interference resistance and good stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical analysis testing, and particularly relates to a preparation method of a PFC-1-UiO-66 heterojunction nanozyme and application of the PFC-1-UiO-66 heterojunction nanozyme in catechol multi-mode detection. BACKGROUND

[0002] Catechol is a persistent pollutant in industrial wastewater due to its high water solubility and oxidation activity. It causes metabolic disorders in aquatic organisms by producing reactive oxygen species (ROS) and quinone intermediates, and may cause liver and kidney damage in humans and even carcinogenesis through the food chain. At present, enzyme-linked colorimetric methods based on natural enzymes are widely used due to their high specificity and good sensitivity. However, natural enzymes have the disadvantages of high preparation cost, poor stability, harsh storage conditions and easy inactivation, which limit their large-scale and on-site instant detection (POCT) in practical applications.

[0003] To break through the bottleneck of natural enzymes in practical detection applications, nanozymes, as a kind of functional nanomaterials with enzyme-like catalytic activity, are widely introduced into the fields of biosensing and in vitro diagnosis due to their high stability, low cost and scalable preparation. However, most of the reported nanozymes can only simulate single enzyme activity, which is difficult to support the detection requirements of multiple signal channels and high anti-interference ability. SUMMARY

[0004] In view of the above technical problems, the present application provides a preparation method of a PFC-1-UiO-66 heterojunction nanozyme and application of the PFC-1-UiO-66 heterojunction nanozyme in catechol multi-mode detection.

[0005] The technical solution adopted by the present application is as follows: The preparation method of the PFC-1-UiO-66 heterojunction nanozyme comprises the following steps: (1) Dissolve cerium salt in a mixed solvent, perform a first heating reaction, control the temperature of the first heating reaction to be 60-65 DEG C, and control the reaction time to be 20-30 minutes; after the reaction is completed, cool the obtained reaction solution I with ice water; then add N,N-dimethylformamide solution in which terephthalic acid is dissolved, perform a second heating reaction, control the temperature of the second heating reaction to be 80-85 DEG C, and control the reaction time to be 20-30 minutes; after the reaction is completed, cool the obtained reaction solution II to room temperature, centrifugally collect white precipitate, and then wash and dry the white precipitate to obtain UiO-66 powder; (2) Dissolve 1,3,6,8-tetra(4-carboxyphenyl)pyrene in N,N-dimethylformamide, add the UiO-66 powder prepared in step (1), and carry out a third heating reaction. Control the temperature of the third heating reaction to be 110-120℃ and the reaction time to be 30-35 minutes. After the reaction is completed, cool the obtained reaction solution to room temperature, add acetone and stir, so that 1,3,6,8-tetra(4-carboxyphenyl)pyrene forms a PFC-1 framework structure in situ on the surface of UiO-66. After the stirring reaction is complete, centrifuge, wash and dry to obtain PFC-1-UiO-66 heterojunction nanozyme. In step (1), the molar ratio of the cerium salt to terephthalic acid is 1:0.9-1:1.1; in step (2), the mass ratio of 1,3,6,8-tetra(4-carboxyphenyl)pyrene to UiO-66 powder is 1:1.5-1:2.0.

[0006] This invention also provides the application of the PFC-1-UiO-66 heterojunction nanozyme prepared by the above method in multimodal detection of catechols, including: (1) Under acidic conditions, the sample to be tested was contacted with the reaction system containing PFC-1-UiO-66 heterojunction nanozyme and TMB substrate, and incubated under light for 1-5 minutes. The absorbance at 652 nm was measured and ΔA was calculated. 652 ; (2) Or / and under neutral conditions, the sample to be tested is contacted with a reaction system containing PFC-1-UiO-66 heterojunction nanozyme and substrates of 2,4-dichlorophenol and 4-aminoantipyrine, and incubated under light for 1-5 minutes. The absorbance at 510 nm is measured and ΔA is calculated. 510 ; (3) or / and under acidic conditions, the sample to be tested is prepared into a reaction solution with the reaction system containing PFC-1-UiO-66 heterojunction nanozyme and TMB substrate, and irradiated with 808 nm near-infrared laser for 1-5 minutes, and the temperature change value ΔT is recorded. The above ΔA 652 ΔA 510 Both ΔT and ΔA show a quantitative relationship with the concentration of catechols in the sample, as determined by ΔA. 652 ΔA 510 Or / and ΔT determines the catechol concentration.

[0007] Compared with the prior art, the present invention has the following beneficial technical effects: (1) The heterojunction structure composed of UiO-66 and PFC-1 is successfully constructed by a step-by-step method, the prepared PFC-1-UiO-66 heterojunction nanometer enzyme has excellent oxidation enzyme and laccase simulation activity, can efficiently catalyze the oxidation of oxygen to develop the substrate without adding H2O2, and can directly catalyze the oxidation of catechol, thereby providing a double signal channel for detection, and improving the flexibility and reliability of detection.

[0008] (2) The unique heterojunction interface of the prepared PFC-1-UiO-66 heterojunction nanometer enzyme promotes electron transmission and substrate adsorption, and produces a significant synergistic effect, so that the oxidation enzyme and laccase simulation activity of the heterojunction nanometer enzyme are both significantly higher than those of single UiO-66 or HOF material.

[0009] (3) The PFC-1-UiO-66 heterojunction nanometer enzyme prepared in the application is used for catechol multi-mode detection, and has high sensitivity and low detection limit for catechol. In addition, the heterojunction nanometer enzyme also has photothermal properties, and can be further used to construct a photothermal detection mode to realize multi-mode signal output, and effectively improve the accuracy and anti-interference ability of detection.

[0010] (4) The application also has the advantages of simple preparation method, easy operation, no need for complex post-treatment operation, rapid detection, and overcoming the disadvantages of natural enzyme such as easy deactivation and high price. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 It is a synthesis schematic diagram of the PFC-1-UiO-66 heterojunction nanometer enzyme in Example 1 of the application; Figure 2 It is a TEM scanning diagram of the PFC-1-UiO-66 heterojunction nanometer enzyme prepared in Example 1 of the application; Figure 3 It is an XRD comparison diagram of the PFC-1-UiO-66 heterojunction nanometer enzyme prepared in Example 1 of the application, and UiO-66 prepared in Comparative Example 1 and PFC-1 prepared in Comparative Example 2; Figure 4a It is a simulation oxidation enzyme catalytic spectrum diagram of the PFC-1-UiO-66 heterojunction nanometer enzyme prepared in Example 1 of the application; wherein, the condition of curve a is PFC-1-UiO-66+TMB+light, the condition of curve b is PFC-1-UiO-66+TMB, the condition of curve c is UiO-66+TMB, curve d is PFC-1-UiO-66, curve e is UiO-66, and curve f is the oxidation enzyme catalytic spectrum diagram of the TMB system; Figure 4bThe PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the catalytic spectrum of laccase; wherein, the condition of curve a is PFC-1-UiO-66+2,4-dichlorophenol (2,4-dp)+4-aminoantipyrine (4-ap)+light, the condition of curve b is PFC-1-UiO-66+2,4-dichlorophenol (2,4-dp)+4-aminoantipyrine (4-ap), the condition of curve c is PFC-1+2,4-dichlorophenol (2,4-dp)+4-aminoantipyrine (4-ap), the condition of curve d is 2,4-dichlorophenol (2,4-dp)+4-aminoantipyrine (4-ap), and curve e is the laccase catalytic spectrum of the 2,4-dichlorophenol (2,4-dp) system; Figure 5a The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the UV absorption spectrum of the colorimetric detection of different catechol concentrations by oxidase; Figure 5b The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the UV absorption spectrum of the colorimetric detection of different catechol concentrations by laccase; Figure 5a The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the UV absorption spectrum of the colorimetric detection of different catechol concentrations by laccase; Figure 6a The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the UV absorption spectrum of the colorimetric detection of different catechol concentrations by laccase; Figure 6b The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the UV absorption spectrum of the colorimetric detection of different catechol concentrations by laccase; Figure 6a The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the UV absorption spectrum of the colorimetric detection of different catechol concentrations by laccase; Figure 7a The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the temperature-time graph of the photothermal detection of different catechol concentrations; Figure 7b The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the temperature-time graph of the photothermal detection of different catechol concentrations; Figure 7a The PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application simulates the temperature-time graph of the photothermal detection of different catechol concentrations. DETAILED DESCRIPTION

[0012] In the actual detection of catechol, the nanoszyme system with both oxidase and laccase activities shows significant advantages: the oxidase activity can directly catalyze the color development reaction using oxygen without hydrogen peroxide (H2O2), greatly simplifying the detection process and improving the operation safety; the laccase activity provides another efficient path for directly oxidizing catechol substrates, improving the specificity and reliability of the detection. Therefore, developing a stable and efficient nanoszyme system that can simultaneously exhibit both enzyme activities is of great significance for realizing the dual-channel and high-sensitivity detection of catechol.

[0013] The application provides a preparation method of a PFC-1-UiO-66 heterojunction nanometer enzyme with a tight interface and high stability, and application thereof in catechol multi-mode detection. The method first synthesizes UiO-66 by using cerium salt, and then adds 1,3,6,8-tetra(4-carboxyphenyl)pyrene and other reactions to construct a heterojunction. The prepared PFC-1-UiO-66 heterojunction nanometer enzyme is composed of UiO-66 with cerium (Ce) as a metal center and terephthalic acid as an organic ligand, and PFC-1 with 1,3,6,8-tetra(4-carboxyphenyl)pyrene as a building unit, and the UiO-66 is in situ grown on the surface of the rod-shaped PFC-1 in an octahedral morphology.

[0014] The PFC-1-UiO-66 heterojunction nanometer enzyme has dual simulation activities of oxidase and laccase, can complete the reaction and output colorimetric and photothermal signals under the condition of no additional hydrogen peroxide. The application in catechol multi-mode detection is colorimetric detection based on the oxidase simulation activity or the laccase simulation activity of the heterojunction nanometer enzyme; the application also includes photothermal detection based on the photothermal effect of the heterojunction nanometer enzyme, thereby constructing a multi-mode sensing platform.

[0015] The heterojunction nanometer enzyme has synergistically enhanced dual activities of oxidase and laccase, and realizes high-sensitivity and high-selectivity quantitative detection of catechol through colorimetric and photothermal dual-mode signal output. For example, the detection limit of catechol based on the oxidase channel is not higher than 0.30 μM, the detection limit based on the laccase channel is not higher than 3.1 μM, and the detection limit based on the photothermal channel is not higher than 0.80 μM.

[0016] The application will be further described below in conjunction with the drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the application and not to limit the application.

[0017] The chemical reagents used in the following examples are commercially available unless otherwise specified.

[0018] Example 1: (1) Synthesis of UiO-66; (NH4)2Ce(NO3)6 (1.17 g, 2.135 mmol) and acetic acid (122 μL) were dissolved in 4 mL of deionized water in a reaction bottle, and then the obtained solution was heated to 60 ℃ for 30 minutes. After the reaction was completed, the reaction bottle was quickly transferred into an ice water bath for cooling.

[0019] Terephthalic acid BDC (0.355 g, 2.135 mmol) was dissolved in 18.76 mL of N,N-dimethylformamide (DMF) and added to the above reaction solution cooled in an ice water bath to obtain a mixed solution. The above mixed solution was heated at 80 ℃ for 30 minutes, and then cooled to room temperature.

[0020] The obtained white precipitate was collected by centrifugation (8000 rpm, 5 min) and washed with fresh DMF by centrifugation three times to completely remove the unreacted raw materials and byproducts. Finally, the obtained white solid product was placed in a 60 ℃ vacuum drying oven for drying for 12 hours to obtain a UiO-66 powder.

[0021] The obtained UiO-66 powder product was observed by scanning electron microscopy (SEM) to be a uniform octahedral structure.

[0022] (2) Constructing a heterojunction; 36 mg of the UiO-66 powder prepared in step (1) and 20 mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene (Pyrene-1,3,6,8-tetracarboxylic acid) were weighed and dispersed in 2 mL of DMF, and after complete dispersion, heated at 120 ℃ for 30 minutes.

[0023] After the reaction was completed, the reaction solution was naturally cooled to room temperature. To the cooled solution, 16 mL of acetone was added and stirred at room temperature for 12 hours to induce the complete formation and precipitation of PFC-1.

[0024] After the precipitation was completed, the obtained product was collected by centrifugation (8000 rpm, 5 min) and washed with fresh acetone by centrifugation four times to ensure complete removal of the DMF solvent and unreacted organic molecules. The final product was placed in a 60 ℃ vacuum drying oven for drying overnight to obtain a PFC-1-UiO-66 heterojunction nanoscale enzyme.

[0025] Figure 1 Schematic diagram for the synthesis of a PFC-1-UiO-66 heterojunction nanoscale enzyme. Figure 2 TEM scanning diagram of the prepared PFC-1-UiO-66 heterojunction nanoscale enzyme. Through transmission electron microscopy (TEM) observation, it can be clearly observed that the octahedral UiO-66 is successfully grown on the surface of the rod-shaped PFC-1, and the two form a closely combined heterojunction structure.

[0026] Example 2: (1) Synthesis of UiO-66; (NH4)2Ce(NO3)6(1.17 g, 2.135 mmol) and acetic acid (122 μL) were dissolved in 4 mL of deionized water in a reaction flask, and the resulting solution was heated to 60 °C for 30 minutes. After the reaction was completed, the reaction flask was quickly transferred into an ice water bath for cooling.

[0027] Terephthalic acid BDC (0.355 g, 2.135 mmol) was dissolved in 18.76 mL of N,N-dimethylformamide (DMF) and added to the above reaction solution cooled in the ice water bath to obtain a mixed solution. After the above mixed solution was heated at 80 °C for 30 minutes, it was cooled to room temperature.

[0028] The resulting white precipitate was collected by centrifugation (8000 rpm, 5 min) and washed with fresh DMF by centrifugation three times to completely remove unreacted raw materials and byproducts. Finally, the obtained white solid product was placed in a 60 °C vacuum drying oven for drying for 12 hours to obtain a UiO-66 powder.

[0029] The obtained UiO-66 powder product was observed by scanning electron microscopy (SEM) to be a uniform octahedral structure.

[0030] (2) Constructing a heterojunction; 24 mg of the UiO-66 powder prepared in step (1) and 20 mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene (Pyrene-1,3,6,8-tetracarboxylic acid) were weighed and dispersed in 2 mL of DMF, and after complete dispersion, heated at 120 °C for 30 minutes.

[0031] After the reaction was completed, the reaction solution was naturally cooled to room temperature. To the cooled solution, 16 mL of acetone was added and stirred at room temperature for 12 hours to induce complete formation and precipitation of PFC-1.

[0032] After the precipitation was completed, the obtained product was collected by centrifugation (8000 rpm, 5 min) and washed with fresh acetone by centrifugation four times to ensure complete removal of the DMF solvent and unreacted organic molecules. The final product was placed in a 60 °C vacuum drying oven for drying overnight to obtain a PFC-1-UiO-66 heterojunction nanoszyme.

[0033] Example 3: (1) Synthesis of UiO-66; (NH4)2Ce(NO3)6(1.17 g, 2.135 mmol) and acetic acid (122 μL) were dissolved in 4 mL of deionized water in a reaction flask, and the resulting solution was heated to 60 °C for 30 minutes. After the reaction was completed, the reaction flask was quickly transferred into an ice water bath for cooling.

[0034] Terephthalic acid BDC (0.355 g, 2.135 mmol) was dissolved in 18.76 mL of N,N-dimethylformamide (DMF) and added to the above reaction solution cooled in the ice water bath to obtain a mixed solution. After the above mixed solution was heated at 80 °C for 30 minutes, it was cooled to room temperature.

[0035] The resulting white precipitate was collected by centrifugation (8000 rpm, 5 min) and washed with fresh DMF by centrifugation three times to completely remove unreacted raw materials and byproducts. Finally, the obtained white solid product was placed in a 60 °C vacuum drying oven for drying for 12 hours to obtain a UiO-66 powder.

[0036] The obtained UiO-66 powder product was observed by scanning electron microscopy (SEM) to be a uniform octahedral structure.

[0037] (2) Constructing a heterojunction; 12 mg of the UiO-66 powder prepared in step (1) and 20 mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene (Pyrene-1,3,6,8-tetracarboxylic acid) were weighed and dispersed in 2 mL of DMF, and after complete dispersion, heated at 120 °C for 30 minutes.

[0038] After the reaction was completed, the reaction solution was naturally cooled to room temperature. To the cooled solution, 16 mL of acetone was added and stirred at room temperature for 12 hours to induce complete formation and precipitation of PFC-1.

[0039] After the precipitation was completed, the obtained product was collected by centrifugation (8000 rpm, 5 min) and washed with fresh acetone by centrifugation four times to ensure complete removal of the DMF solvent and unreacted organic molecules. The final product was placed in a 60 °C vacuum drying oven for drying overnight to obtain a PFC-1-UiO-66 heterojunction nanoszyme.

[0040] Comparative Example 1: A UiO-66 nanoszyme was obtained by the preparation method comprising the following steps: Dissolve (NH4)2Ce(N03)6(1.17 g, 2.135 mmol) and acetic acid (122 μL) in 4 mL of deionized water. Heat the above solution to 60 °C for 30 minutes. After the reaction is completed, quickly transfer the reaction flask into an ice-water bath to cool down, obtaining a cerium solution.

[0041] Dissolve BDC (0.355 g, 2.135 mmol) in 18.76 mL of N,N-dimethylformamide (DMF) and add the above cerium solution. Heat the resulting mixed solution at 80 °C for 30 minutes and then cool to room temperature.

[0042] The obtained white precipitate is collected by centrifugation (8000 rpm, 5 min) and washed with fresh DMF by centrifugation three times to completely remove unreacted starting materials and byproducts. Finally, the obtained white solid product is placed in a 60 °C vacuum drying oven for 12 hours to obtain UiO-66 powder.

[0043] Comparative Example 2: This comparative example obtained a single-component PFC-1 nanoszyme, and the preparation method comprises the following steps: Weigh 20 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene, disperse it in 2 mL of DMF, and ultrasonic until completely dissolved. Heat to 120 °C for 30 minutes.

[0044] After the reaction is completed, the reaction solution is naturally cooled to room temperature. Add 16 mL of acetone to the cooled solution and stir at room temperature for 12 hours to induce the complete formation and precipitation of PFC-1.

[0045] After the precipitation is completed, the obtained product is collected by centrifugation (8000 rpm, 5 min) and washed with fresh acetone by centrifugation four times to ensure complete removal of DMF solvent and unreacted organic molecules. The final product is placed in a 60 °C vacuum drying oven overnight to obtain the PFC-1 nanoszyme.

[0046] Figure 3The XRD comparison chart of PFC-1-UiO-66 heterojunction nanoszyme prepared in Example 1 of the present application, UiO-66 prepared in Comparative Example 1 and PFC-1 prepared in Comparative Example 2. It can be seen from the figure that UiO-66 shows characteristic diffraction peaks at 7.1° and 8.2°, corresponding to (111) and (200) crystal planes, respectively. PFC-1 shows multiple characteristic diffraction peaks in the range of 4.3° to 13.8°. In the PFC-1-UiO-66 heterojunction structure, the intensity of the specific diffraction peak corresponding to PFC-1 significantly decreases. This phenomenon can be attributed to the dispersion growth of small octahedral UiO-66 on the surface of rod-like PFC-1, forming surface coverage, which produces absorption and shielding effects on the X-ray of PFC-1, thereby significantly weakening the diffraction peak intensity of the PFC-1 phase. Secondly, the strong interaction formed by Ce-O-C covalent bonding at the heterojunction interface of PFC-1-UiO-66 may introduce local lattice strain or defects in the PFC-1 lattice, causing the diffraction peak intensity to weaken.

[0047] Example 4: This example tests the oxidase and laccase mimetic activities of Example 1 (PFC-1-UiO-66 heterojunction nanoszyme), Comparative Example 1 (UiO-66 nanoszyme) and Comparative Example 2 (PFC-1 nanoszyme) to demonstrate the synergistic enhancement effect of the heterojunction.

[0048] (1) Oxidase mimetic activity test; A sodium acetate-acetic acid buffer solution with pH = 4.0 is configured. The nanoszyme sample is configured into a dispersion of 0.3 mg / mL with ultrapure water. 1600 μL of buffer, 200 μL of nanoszyme dispersion and 200 μL of 1.0 mM TMB solution are sequentially added to prepare a standard reaction system of 2 mL. After three minutes of irradiation, the change curve of absorbance with wavelength is monitored using a UV-visible spectrophotometer.

[0049] Test results: The absorbance of Example 1 (PFC-1-UiO-66 heterojunction nanoszyme) is the highest at 652 nm (see Figure 4a , and the oxidase activity is the highest.

[0050] Figure 4a The curves b-f in the figure are controls, b is PFC-1-UiO-66 + TMB, i.e. keeping other conditions the same as above, the difference is that the light irradiation condition is omitted. c is UiO-66 + TMB, i.e. keeping other conditions the same as above, the difference is that the UiO-66 nanoszyme is used instead of the PFC-1-UiO-66 heterojunction nanoszyme, etc.

[0051] (2) Laccase mimetic activity test; A sodium phosphate buffer solution with pH = 7.0 was prepared. The nanoscale enzyme sample was configured into a dispersion of 0.3 mg / mL with ultrapure water. In turn, 1600 μL of buffer, 200 μL of nanoscale enzyme dispersion, 100 μL of 2,4-dp solution (2 mg / mL), and 100 μL of 4-ap solution (2 mg / mL) were added to prepare 2 mL of a standard reaction system. After three minutes of irradiation, the absorbance curve was monitored using a UV-visible spectrophotometer.

[0052] Test results: At 510 nm, the absorbance of Example 1 (PFC-1-UiO-66 heterojunction nanoscale enzyme) was the highest (see Figure 4b ), and the laccase activity was the highest.

[0053] Figure 4b The curves b-e are controls, b is PFC-1-UiO-66+2,4-dp+4-ap, that is, the other conditions remain the same as above, and the difference is that the light irradiation condition is omitted. c is PFC-1+2,4-dp+4-ap, that is, the other conditions remain the same as above, and the difference is that PFC-1 nanoscale enzyme is used instead of PFC-1-UiO-66 heterojunction nanoscale enzyme, and so on.

[0054] Example 5: This example demonstrates the use of the PFC-1-UiO-66 heterojunction nanoscale enzyme prepared in Example 1 of the present application to perform high-sensitivity dual-mode colorimetric detection of 3,5-di-tert-butylcatechol based on the oxidative enzyme simulation activity and laccase simulation activity of the PFC-1-UiO-66 heterojunction nanoscale enzyme.

[0055] (1) Detection based on oxidative enzyme activity; Under acidic conditions, the heterojunction nanoscale enzyme of the present application can catalyze the oxidation of the enzyme substrate TMB (3,3',5,5'-tetramethylbenzidine) to generate blue oxTMB, which has a characteristic absorption peak at 652 nm. When the target 3,5-DTBC is present, it will bind to the active site of the nanoscale enzyme or compete with TMB, inhibiting the oxidative enzyme activity of the nanoscale enzyme, resulting in a decrease in the generation of blue product and a decrease in absorbance at 652 nm. The concentration of 3,5-DTBC is directly proportional to the degree of inhibition of absorbance.

[0056] Detection steps: A series of 3,5-DTBC standard solutions with different concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM) were prepared in ethanol.

[0057] In turn, the following were added in sequence: A standard reaction system of 2 mL was prepared by mixing 1400 μL sodium acetate-acetic acid buffer (pH = 4.0), 200 μL heterojunction nanozyme dispersion prepared in Example 1 (0.3 mg / mL), 200 μL 3,5-DTBC solution of a specific concentration, and 200 μL TMB solution (1.0 mM).

[0058] The reaction was carried out under light irradiation for three minutes, and the absorbance value of the reaction system at a wavelength of 652 nm was measured (A). 652 ).

[0059] Results and calculations: The absorbance of the system without 3,5-DTBC (concentration of 0) is used as the blank control value (A0), and the absorbance of the systems containing different concentrations of 3,5-DTBC is used as the measured value (A). x ), ΔA = A0 - A x .

[0060] like Figure 5a , 5b As shown, the concentration of 3,5-DTBC exhibits a good linear relationship in the range of 1 to 10 μM. The linear regression equation is y = 0.02932x + 0.0067 (R² = 0.99), where y is ΔA and x is the concentration of 3,5-DTBC (μM). Based on a signal-to-noise ratio of 3 (S / N=3), the limit of detection (LOD) for 3,5-DTBC based on oxidase activity is 0.2582 μM.

[0061] (2) Detection based on laccase activity; Under neutral conditions, the heterojunction nanozyme of this invention can mimic laccase activity, catalyzing the coupled oxidation reaction of the substrate 2,4-dichlorophenol (2,4-dp) with 4-aminoantipyrine (4-ap) to generate a red quinone imine dye with a characteristic absorption peak at 510 nm. In the presence of 3,5-DTBC, it competes for the active site of the nanozyme, inhibiting its laccase activity, leading to a reduction in the formation of the red product and a decrease in absorbance at 510 nm. The concentration of 3,5-DTBC is directly proportional to the degree of absorbance inhibition.

[0062] Testing steps: A series of 3,5-DTBC standard solutions of different concentrations (10, 25, 35, 45, 55, 60, 70, 80 μM) were prepared in ethanol.

[0063] Add them in order: A standard reaction system of 2 mL was prepared by mixing 1400 μL phosphate buffer (pH=7.0), 100 μL 4-ap solution (2 mg / mL), 100 μL 2,4-dp solution (2 mg / mL), 200 μL 3,5-DTBC solution of a specific concentration, and 200 μL heterojunction nanozyme dispersion prepared in Example 1.

[0064] The reaction was carried out under light irradiation for three minutes, and the absorbance value of the reaction system at a wavelength of 510 nm was measured (A). 510 ).

[0065] Results and calculations: The absorbance of the system without 3,5-DTBC (concentration of 0) is used as the blank control value (A0), and the absorbance of the systems containing different concentrations of 3,5-DTBC is used as the measured value (A). x ), ΔA = A0 - A x .

[0066] like Figure 6a , 6b As shown, the concentration of 3,5-DTBC exhibits a good linear relationship in the range of 0 to 70 μM. The linear regression equation is y = 0.00248x + 0.01501 (R² = 0.99), where y is ΔA and x is the concentration of 3,5-DTBC (μM). Based on a signal-to-noise ratio of 3 (S / N=3), the limit of detection (LOD) for 3,5-DTBC based on laccase activity is 3.036 μM.

[0067] Example 6: This embodiment demonstrates the photothermal effect of the PFC-1-UiO-66 heterojunction nanozyme system prepared based on Example 1 of the present invention, and provides highly sensitive photothermal detection of 3,5-di-tert-butylcatechol.

[0068] This invention relates to a heterojunction nanozyme that catalyzes the oxidation of the enzyme substrate TMB under acidic conditions, generating blue oxidized TMB (oxTMB). oxTMB is an excellent photothermal reagent, exhibiting a significant photothermal effect under 808 nm near-infrared laser irradiation, causing a rapid rise in solution temperature. The presence of the target compound 3,5-DTBC inhibits the catalytic activity of the nanozyme, leading to a reduction in the amount of blue oxTMB generated, thereby decreasing the photothermal conversion efficiency of the solution. Therefore, the higher the concentration of 3,5-DTBC, the lower the temperature rise of the reaction system after laser irradiation. Quantitative detection of 3,5-DTBC can be achieved by measuring the temperature change.

[0069] Testing steps: A series of 3,5-DTBC standard solutions (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM) with different concentrations were prepared in ethanol.

[0070] In order, add: 1400 μL sodium acetate-acetic acid buffer (pH = 4.0), 200 μL of the prepared heterogeneous nano-enzyme dispersion solution (0.3 mg / mL) of Example 1, 200 μL of 3,5-DTBC solution with a specific concentration, 200 μL of TMB solution (1.0 mM) to prepare a 2 mL standard reaction system.

[0071] After the reaction, all the solutions were transferred to a clean 2 mL centrifuge tube. The center of the solution surface was vertically irradiated using an 808 nm near-infrared laser (power density of 1.5 W / cm 2 ), and the irradiation distance was fixed at 15 cm. At the same time, the highest temperature (T) of the solution after 3 minutes of irradiation was monitored and recorded in real time using an infrared thermal imager.

[0072] The system without 3,5-DTBC (concentration of 0) was used as a blank control, and the highest temperature after irradiation was recorded as T0. The temperature change value in the presence of different concentrations of 3,5-DTBC was calculated.

[0073] Results and calculations: The system without 3,5-DTBC (concentration of 0) was used as a blank control, and the highest temperature after irradiation was recorded as T0. The temperature change value in the presence of different concentrations of 3,5-DTBC was calculated. x x .

[0074] As shown in Figure 7a , 7b , the concentration of 3,5-DTBC showed a good linear relationship in the range of 1 to 10 μM. The linear regression equation was y = 0.02932x + 0.0067 (R 2 = 0.99), where y was ΔA and x was the concentration of 3,5-DTBC (μM). According to the calculation of 3 times the signal-to-noise ratio (S / N = 3), the detection limit (LOD) of 3,5-DTBC based on oxidase activity was 0.2582 μM.

[0075] The concentration of 3,5-DTBC showed a good linear relationship with the ΔT value in the range of 1 to 20 μM. The linear regression equation was y = 1.04026x - 0.31285 (R 2 ​= 0.99), wherein y is the value of AT (℃), and x is the concentration of 3,5-DTBC (μM). The limit of detection (LOD) of 3,5-DTBC based on the photothermal effect is 0.7257 μM.

[0076] The present application successfully combines the enzyme-catalyzed color reaction with the photothermal analysis for the detection of catechol in multiple modes. The method realizes the detection by measuring the temperature, an intuitive physical signal, and has potential advantages such as strong anti-background interference ability, simple equipment, and can be used for on-site detection of complex samples. Combined with the colorimetric method in Example 3, a dual-mode signal output can be formed, which verifies each other, greatly improving the reliability of the detection results.

[0077] The above detailed description does not constitute a limitation on the protection scope of the present application. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a PFC-1-UiO-66 heterojunction nanoszyme, characterized in that Comprising the following steps: (1) Dissolve cerium salt in mixed solvent, carry out the first heating reaction, control the temperature of the first heating reaction at 60-65℃, the reaction time is 20-30 minutes; after the reaction is completed, the obtained reaction solution is cooled with ice water; then add N,N-dimethylformamide solution dissolved with terephthalic acid, carry out the second heating reaction, control the temperature of the second heating reaction at 80-85℃, the reaction time is 20-30 minutes; after the reaction is completed, the obtained reaction solution is cooled to room temperature, centrifugal collection white precipitate, then wash, dry to obtain UiO-66 powder; (2) Dissolve 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene in N,N-dimethylformamide, add the UiO-66 powder prepared in step (1), carry out the third heating reaction, control the temperature of the third heating reaction at 110-120℃, the reaction time is 30-35 minutes; after the reaction is completed, the obtained reaction solution is cooled to room temperature, then add acetone and stir, so that 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene forms PFC-1 framework structure in situ on the surface of UiO-66, after the stirring reaction is completed, centrifugal, wash, dry to obtain PFC-1-UiO-66 heterogeneous nanoenzyme; In step (1), the molar ratio of cerium salt to terephthalic acid is 1:0.9-1:1.1; in step (2), the mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to UiO-66 powder is 1:1.5-1:2.

0.

2. The method for preparing a PFC-1-UiO-66 heterojunction nanoszyme according to claim 1, characterized in that, In step (1): the cerium salt is cerium ammonium nitrate; the mixed solvent is obtained by mixing deionized water and acetic acid, wherein the volume ratio of deionized water to acetic acid is 30:1-35:

1.

3. The method for preparing a PFC-1-UiO-66 heterojunction nanoszyme according to claim 2, characterized in that, In step (1): the mass-volume ratio of the cerium salt to the mixed solvent is 0.25-0.35 g / mL; the mass-volume ratio of the terephthalic acid to N,N-dimethylformamide is 0.015-0.025 g / mL.

4. The method according to claim 1, wherein, In step (1): when centrifugal collection white precipitate, control the centrifugal speed at 7000-8000 rpm, centrifugal time is 5-7 minutes; when washing, use N,N-dimethylformamide, centrifugal washing 3-5 times; dry in a vacuum drying oven, control the drying temperature at 60-70℃, drying time is 10-12 hours.

5. The method for preparing a PFC-1-UiO-66 heterojunction nanoszyme according to claim 1, characterized in that, In step (2): the mass-volume ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to N,N-dimethylformamide is 8-12 mg / mL.

6. The method for preparing a PFC-1-UiO-66 heterojunction nanoszyme according to claim 1, characterized in that, In step (2): after adding acetone, control stirring at room temperature for 10-12 hours.

7. The method according to claim 1, wherein, In step (2): control the centrifugal speed at 8000-8500 rpm, centrifugal time is 5-7 minutes; when washing, use acetone, centrifugal washing 3-5 times; dry in a vacuum drying oven, control the drying temperature at 70-80℃, drying time is 10-15 hours.

8. Use of the PFC-1-UiO-66 heterojunction nanoszyme prepared according to any one of claims 1-7 in catechol multi-modal detection, characterized in that, Comprising: (1) Under acidic conditions, the sample to be tested, PFC-1-UiO-66 heterojunction nanometer enzyme dispersion and TMB substrate were mixed to prepare the reaction system, incubated under light for 1-5 minutes, the absorbance at 652 nm was measured and ΔA was calculated 652 ; (2) or / and under neutral conditions, the sample to be tested is mixed with PFC-1-UiO-66 heterojunction nanometer enzyme dispersion liquid and 2,4-dichlorophenol and 4-aminoantipyrine substrate, a reaction system is prepared, incubated under light for 1-5 minutes, the absorbance at 510 nm is measured and ΔA is calculated 510 ; (3) or / and under acidic conditions, the sample to be tested is mixed with PFC-1-UiO-66 heterojunction nanoscale enzyme dispersion and TMB substrate, the reaction system is prepared, 808 nm near-infrared laser irradiation is carried out for 1-5 minutes, and the temperature change value ΔT is recorded; The above ΔA 652 , ΔA 510 and ΔT are all quantitatively related to the catechol concentration in the sample to be measured, and the catechol concentration is determined by ΔA 652 , ΔA 510 or / and ΔT.

9. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 8 in catechol multimodal detection, characterized in that, The PFC-1-UiO-66 heterojunction nanoscale enzyme dispersion is prepared by adding PFC-1-UiO-66 heterojunction nanoscale enzyme into ultrapure water, and the concentration of the PFC-1-UiO-66 heterojunction nanoscale enzyme dispersion is 0.1-1.0 mg / mL; In steps (1) and (3), the acidic condition is achieved by adding sodium acetate-acetic acid buffer solution into the reaction system to control the pH to be 3.5-5.0; In step (2), the neutral condition is achieved by adding sodium phosphate buffer solution into the reaction system to control the pH to be 6.5-7.

5.

10. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 8 in catechol multimodal detection, characterized in that, The application is suitable for quantitative detection of catechol and its homologous phenolic compounds in environmental water, food samples or biological samples.

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