Preparation method of pfc-1-uiO-66 heterojunction nano-enzyme and application thereof in catechol multi-mode detection

Abstract

The application discloses a preparation method of a PFC-1-UiO-66 heterojunction nanoscale enzyme and application thereof in catechol multi-mode detection, and relates to the technical field of biochemical analysis and examination. The method comprises the following steps: dissolving cerium salt in a mixed solvent, heating and reacting, adding DMF in which terephthalic acid is dissolved into ice water after cooling, and obtaining UiO-66 through reaction; dissolving 1,3,6,8-tetra(4-carboxyphenyl)pyrene in DMF, adding UiO-66, heating and reacting, cooling after reaction, adding acetone and stirring, and obtaining the heterojunction nanoscale enzyme through centrifugation, washing and drying. The detection reagent constructed based on the heterojunction nanoscale enzyme can react with TMB under acidic conditions to realize 652 nm colorimetric detection, can react with 2,4-dichlorophenol / 4-aminoantipyrine under neutral conditions to realize 510 nm colorimetric detection, and can generate a photothermal signal through 808 nm near-infrared light irradiation to realize dual-mode detection of colorimetry and photothermal effect. The application has the advantages of high detection sensitivity, strong anti-interference and good stability.

Description

TECHNICAL FIELD

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

[0002] Catechol is a stubborn 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 shortcomings 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] In order 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 order to solve the above technical problems, the application provides a preparation method of a PFC-1-UiO-66 heterojunction nanoenzyme and application of the PFC-1-UiO-66 heterojunction nanoenzyme in catechol multi-mode detection.

[0005] The technical solution adopted by the application is as follows:

[0006] The preparation method of the PFC-1-UiO-66 heterojunction nanoenzyme comprises the following steps:

[0007] (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 of the first heating reaction 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 of the second heating reaction 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;

[0008] (2) 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene is dissolved in N,N-dimethylformamide, and the UiO-66 powder prepared in step (1) is added, a third heating reaction is carried out, and the temperature of the third heating reaction is controlled to be 110-120 DEG C, and the reaction time is 30-35 minutes; after the reaction is completed, the obtained reaction liquid is cooled to room temperature, and then acetone is added and stirred, so that 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene forms a PFC-1 framework structure on the surface of UiO-66 in situ, after the stirring reaction is completed, PFC-1-UiO-66 heterojunction nanometer enzyme is obtained after centrifugation, washing and drying;

[0009] 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-tetrakis(4-carboxyphenyl)pyrene to UiO-66 powder is 1:1.5-1:2.0.

[0010] The application further provides the application of the PFC-1-UiO-66 heterojunction nanometer enzyme prepared by the above method in catechol multi-mode detection, which comprises:

[0011] (1) under acidic conditions, the sample to be detected is contacted with a reaction system containing the PFC-1-UiO-66 heterojunction nanometer enzyme and a TMB substrate, incubated under light for 1-5 minutes, the absorbance at 652 nm is measured, and ΔA is calculated 652 ;

[0012] (2) or / and under neutral conditions, the sample to be detected is contacted with a reaction system containing the PFC-1-UiO-66 heterojunction nanometer enzyme and 2,4-dichlorophenol and 4-aminoantipyrine substrates, incubated under light for 1-5 minutes, the absorbance at 510 nm is measured, and ΔA is calculated 510 ;

[0013] (3) or / and under acidic conditions, the sample to be detected is contacted with a reaction system containing the PFC-1-UiO-66 heterojunction nanometer enzyme and a TMB substrate to form a reaction liquid, and the reaction liquid is irradiated with 808 nm near-infrared laser for 1-5 minutes, and the temperature change value ΔT is recorded;

[0014] The above ΔA 652 , ΔA 510 and ΔT are quantitatively related to the catechol concentration in the sample to be detected, and the catechol concentration is determined by ΔA 652 , ΔA 510 or / and ΔT.

[0015] Compared with the prior art, the application has the following beneficial technical effects:

[0016] (1) The heterojunction structure composed of UiO-66 and PFC-1 is successfully constructed by a step-by-step method, and 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.

[0017] (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.

[0018] (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, thereby effectively improving the accuracy and anti-interference ability of detection.

[0019] (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

[0020] Figure 1 It is a synthesis schematic diagram of the PFC-1-UiO-66 heterojunction nanometer enzyme in Example 1 of the application;

[0021] Figure 2 It is a TEM scanning diagram of the PFC-1-UiO-66 heterojunction nanometer enzyme prepared in Example 1 of the application;

[0022] 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;

[0023] 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;

[0024] Figure 4b The images show the catalytic spectra of laccase-like nanozymes prepared by PFC-1-UiO-66 in Example 1 of this invention. Curve a is given under the following conditions: PFC-1-UiO-66 + 2,4-dichlorophenol (2,4-dp) + 4-aminoantipyrine (4-ap) + light irradiation; curve b is given under the following conditions: PFC-1-UiO-66 + 2,4-dichlorophenol (2,4-dp) + 4-aminoantipyrine (4-ap); curve c is given under the following conditions: PFC-1 + 2,4-dichlorophenol (2,4-dp) + 4-aminoantipyrine (4-ap); curve d is given under the following conditions: 2,4-dichlorophenol (2,4-dp) + 4-aminoantipyrine (4-ap); and curve e is the catalytic spectrum of laccase in the 2,4-dichlorophenol (2,4-dp) system.

[0025] Figure 5a The UV absorption spectra of the PFC-1-UiO-66 heterojunction nanoenzyme mimicking oxidase prepared in Example 1 of this invention for colorimetric detection of different catechol concentrations.

[0026] Figure 5b To be Figure 5a The graph showing the relationship between the UV absorbance and catechol concentration after the signal is converted according to Lambert-Beer's law;

[0027] Figure 6a The image shows the ultraviolet absorption spectrum of the PFC-1-UiO-66 heterojunction nanozyme simulated laccase prepared in Example 1 of this invention for colorimetric detection of different catechol concentrations.

[0028] Figure 6b To be Figure 6a The graph showing the relationship between the UV absorbance and catechol concentration after the signal is converted according to Lambert-Beer's law;

[0029] Figure 7a This is a temperature-time graph of the PFC-1-UiO-66 heterojunction nanozyme prepared in Example 1 of the present invention for photothermal detection of different catechol concentrations.

[0030] Figure 7b To be Figure 7a The graph shows the relationship between the temperature change and catechol concentration after the signal is converted according to the Lambert-Beer law. Detailed Implementation

[0031] In the actual detection of catechol, the nanoscale enzyme system with both oxidase and laccase activities shows significant advantages: the oxidase activity can directly catalyze the color 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 nanoscale enzyme system capable of simultaneously exerting the two enzyme activities is of great significance to realize the dual-channel and high-sensitivity detection of catechol.

[0032] The application provides a preparation method of a PFC-1-UiO-66 heterojunction nanoscale enzyme with a tight interface and high stability, and application of the PFC-1-UiO-66 heterojunction nanoscale enzyme in catechol multi-mode detection. The method first synthesizes UiO-66 by using cerium salt, and then adds 1,3,6,8-tetrakis (4-carboxyphenyl) pyrene to perform a reaction for constructing a heterojunction. The prepared PFC-1-UiO-66 heterojunction nanoscale 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-tetrakis (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.

[0033] The PFC-1-UiO-66 heterojunction nanoscale enzyme has both oxidase and laccase dual simulation activities, can complete the reaction without additional hydrogen peroxide and output colorimetric and photothermal signals. The application in catechol multi-mode detection is colorimetric detection based on the oxidase simulation activity or the laccase simulation activity of the heterojunction nanoscale enzyme; the application also includes photothermal detection based on the photothermal effect of the heterojunction nanoscale enzyme, thereby constructing a multi-mode sensing platform.

[0034] The heterojunction nanoscale enzyme has synergistically enhanced oxidase and laccase dual activities, 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.

[0035] 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.

[0036] The chemical reagents used in the following examples are commercially available unless otherwise specified.

[0037] Example 1:

[0038] (1) Synthesis of UiO-66;

[0039] (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.

[0040] 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.

[0041] 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.

[0042] The obtained UiO-66 powder product was observed to have a uniform octahedral structure by scanning electron microscopy (SEM).

[0043] (2) Constructing a heterojunction;

[0044] 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, heating was performed at 120 °C for 30 minutes.

[0045] 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.

[0046] After the precipitation was completed, the resulting 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.

[0047] Figure 1 A schematic diagram for the synthesis of a PFC-1-UiO-66 heterojunction nanoszyme. Figure 2TEM scanning image of the prepared PFC-1-UiO-66 heterojunction nanoszyme. By transmission electron microscopy (TEM) observation, it can be clearly observed that the octahedral UiO-66 is successfully grown on the surface of the rod-like PFC-1, and the two form a closely combined heterojunction structure.

[0048] Example 2:

[0049] (1) Synthesis of UiO-66;

[0050] (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 resulting 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.

[0051] 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 ℃ for 30 minutes, it was cooled to room temperature.

[0052] The obtained white precipitate was collected by centrifugation (8000 rpm, 5 min) and washed with fresh DMF three times by centrifugation to completely remove 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 UiO-66 powder.

[0053] By scanning electron microscopy (SEM) observation, the obtained UiO-66 powder product was a uniform octahedral structure.

[0054] (2) Construction of heterojunction;

[0055] 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, they were heated at 120 ℃ for 30 minutes.

[0056] 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.

[0057] After the completion of the precipitation, the obtained product was collected by centrifugation (8000 rpm, 5 min) and washed with fresh acetone by centrifugation four times to ensure the complete removal of DMF solvent and unreacted organic molecules. The final product was dried in a vacuum oven at 60 °C overnight to obtain PFC-1-UiO-66 nanoszyme.

[0058] Example 3:

[0059] (1) Synthesis of UiO-66;

[0060] (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 °C for 30 minutes. After the reaction was completed, the reaction bottle was quickly transferred into an ice water bath for cooling.

[0061] 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.

[0062] The obtained 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 vacuum drying oven at 60 °C for 12 hours to obtain UiO-66 powder.

[0063] The obtained UiO-66 powder product was observed by scanning electron microscopy (SEM) to be a uniform octahedral structure.

[0064] (2) Construction of heterojunction;

[0065] 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.

[0066] After the completion of the reaction, 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.

[0067] 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 the complete removal of DMF solvent and unreacted organic molecules. The final product was placed in a 60 °C vacuum drying oven to dry overnight, obtaining the PFC-1-UiO-66 heterojunction nanozyme.

[0068] Comparative Example 1:

[0069] The comparative example prepared obtained a UiO-66 nanozyme, the preparation method comprising the following steps:

[0070] Dissolve (NH4)2Ce(NO3)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 bottle into an ice water bath to cool, obtaining a cerium solution.

[0071] Dissolve BDC (0.355 g, 2.135 mmol) in 18.76 mL of N,N-dimethylformamide (DMF) and add the above cerium solution. After the obtained mixed solution is heated at 80 °C for 30 minutes, cool to room temperature.

[0072] 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 raw materials and byproducts. Finally, the obtained white solid product is placed in a 60 °C vacuum drying oven to dry for 12 hours, obtaining a UiO-66 powder.

[0073] Comparative Example 2:

[0074] The comparative example prepared obtained a single-component PFC-1 nanozyme, the preparation method comprising the following steps:

[0075] Weigh 20 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene, disperse it in 2 ml of DMF, and ultrasonic to completely dissolve. Heat to 120 °C for 30 minutes.

[0076] 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.

[0077] 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 the complete removal of DMF solvent and unreacted organic molecules. The final product is placed in a 60 °C vacuum drying oven to dry overnight, obtaining the PFC-1 nanozyme.

[0078] Figure 3 The 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. From the figure, it can be seen 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.

[0079] Example 4:

[0080] 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.

[0081] (1) Oxidase mimetic activity test;

[0082] 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 reaction under light irradiation, the change curve of absorbance with wavelength is monitored using a UV-visible spectrophotometer.

[0083] Test results:

[0084] At 652 nm, the absorbance of Example 1 (PFC-1-UiO-66 heterojunction nanoszyme) is the highest (see Figure 4a , and the oxidase activity is the highest.

[0085] Figure 4aThe b-f curves in the middle are controls, such as b is PFC-1-UiO-66+TMB, that is, the other conditions remain the same as above, and the difference is that the light condition is omitted. c is UiO-66+TMB, that is, the other conditions remain the same as above, and the difference is that the UiO-66 nanoscale enzyme is used instead of the PFC-1-UiO-66 heterojunction nanoscale enzyme, and so on.

[0086] (2) Laccase mimetic activity test;

[0087] A sodium phosphate buffer solution with pH = 7.0 is configured. The nanoscale enzyme sample is configured into a dispersion of 0.3 mg / mL with ultrapure water. 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) are sequentially added to prepare 2 mL of a standard reaction system. After irradiation under light for three minutes, the change curve of absorbance with wavelength is monitored using a UV-visible spectrophotometer.

[0088] Test results:

[0089] The absorbance of Example 1 (PFC-1-UiO-66 heterojunction nanoscale enzyme) is the highest at 510 nm (see Figure 4b ), and the laccase activity is the highest.

[0090] Figure 4b The b-e curves in the middle are controls, such as 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 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 the PFC-1 nanoscale enzyme is used instead of the PFC-1-UiO-66 heterojunction nanoscale enzyme, and so on.

[0091] Example 5:

[0092] This example demonstrates that the PFC-1-UiO-66 heterojunction nanoscale enzyme prepared by Example 1 of the present application is used to perform high-sensitivity dual-mode colorimetric detection on 3,5-di-tert-butylcatechol based on the oxidase mimetic activity and the laccase mimetic activity.

[0093] (1) Detection based on oxidase activity;

[0094] Under acidic conditions, the heterojunction nanozyme of this invention catalyzes the oxidation of the enzyme substrate TMB (3,3',5,5'-tetramethylbenzidine) to produce blue oxTMB, generating a characteristic absorption peak at 652 nm. When the target compound 3,5-DTBC is present, it binds to the active site of the nanozyme or competes with TMB, inhibiting the oxidase activity of the nanozyme, leading to a reduction in the formation of the blue product and a decrease in absorbance at 652 nm. The concentration of 3,5-DTBC is directly proportional to the degree of absorbance inhibition.

[0095] Testing steps:

[0096] A series of 3,5-DTBC standard solutions of different concentrations (1,2,3,4,5,6,7,8,9,10 μM) were prepared in ethanol.

[0097] Add them in order:

[0098] 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).

[0099] 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 ).

[0100] Results and calculations:

[0101] 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 .

[0102] 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.

[0103] (2) Detection based on laccase activity;

[0104] Under neutral conditions, the heterojunction nanoszyme of the application can simulate the activity of laccase, catalyze the coupling oxidation reaction of the substrate 2,4-dp and 4-ap to generate red quinone imine dye, and have a characteristic absorption peak at 510 nm. When 3,5-DTBC is present, it will compete for the active site of the nanoszyme and inhibit its laccase activity, resulting in a decrease in the generation of red product and a decrease in absorbance at 510 nm. The concentration of 3,5-DTBC is proportional to the degree of inhibition of absorbance.

[0105] Detection step:

[0106] A series of 3,5-DTBC standard solutions with different concentrations (10, 25, 35, 45, 55, 60, 70, 80 μM) were prepared in ethanol.

[0107] In order, add:

[0108] 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 with a specific concentration, 200 μL heterojunction nanoszyme dispersion prepared in Example 1, to prepare 2 mL of a standard reaction system.

[0109] Under light irradiation, react for three minutes, and measure the absorbance value of the reaction system at 510 nm wavelength (A 510 ).

[0110] Results and calculations:

[0111] The absorbance value of the system without 3,5-DTBC (concentration of 0) was taken as the blank control value (A0), and the absorbance value of the system containing different concentrations of 3,5-DTBC was taken as the measured value (A x ), ΔA = A0- A x .

[0112] As shown in Figure 6a , 6b , the concentration of 3,5-DTBC showed a good linear relationship in the range of 0 to 70 μM. The linear regression equation was y = 0.00248x + 0.01501 (R² = 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 laccase activity was 3.036 μM.

[0113] Example 6:

[0114] The embodiment demonstrates the photothermal effect of the PFC-1-UiO-66 heterojunction nanoenzyme system prepared in the embodiment 1 of the application, and performs high-sensitivity photothermal detection on 3,5-di-tert-butyl catechol.

[0115] The heterojunction nanoenzyme can catalyze oxidation of the enzyme substrate TMB under acidic conditions to generate blue oxidized TMB (oxTMB). The oxTMB is an excellent photothermal reagent, and can generate significant photothermal effect under 808 nm near-infrared laser irradiation, so that the solution temperature rapidly rises. When the target substance 3,5-DTBC exists, it will inhibit the catalytic activity of the nanoenzyme, resulting in a decrease in the generation amount of blue oxTMB, thereby reducing the photothermal conversion efficiency of the solution. Therefore, the higher the concentration of 3,5-DTBC is, the lower the temperature rise value of the reaction system after laser irradiation is. The quantitative detection of 3,5-DTBC can be realized by measuring the temperature change value.

[0116] Detection step:

[0117] A series of 3,5-DTBC standard solutions (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM) with different concentrations were configured in ethanol.

[0118] In order, add:

[0119] 1400 μL sodium acetate-acetic acid buffer (pH = 4.0), 200 μL heterojunction nanoenzyme dispersion liquid (0.3 mg / mL) prepared in the embodiment 1, 200 μL 3,5-DTBC solution with a specific concentration, 200 μL TMB solution (1.0 mM) to prepare 2 mL standard reaction system.

[0120] After the reaction, all the solutions were transferred to a clean 2 mL centrifuge tube. An 808 nm near-infrared laser (power density of 1.5 W / cm 2 ) was vertically irradiated to the center of the solution liquid surface, and the irradiation distance was fixed at 15 cm. At the same time, the infrared thermal imager was used to monitor and record the highest temperature (T) of the solution at 3 minutes of irradiation.

[0121] 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.

[0122] Results and calculations:

[0123] 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. xCalculate the temperature change under different concentrations of 3,5-DTBC: ΔT = T0 - T x .

[0124] like Figure 7a , 7b 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). 2 = 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.

[0125] 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 (R²). 2 = 0.99), where y is the ΔT value (°C) and x is the 3,5-DTBC concentration (μM). The limit of detection (LOD) for 3,5-DTBC based on the photothermal effect is 0.7257 μM.

[0126] This invention successfully combines enzyme-catalyzed colorimetric reactions with photothermal analysis for multi-mode detection of catechols. This method achieves detection by measuring the intuitive physical signal of temperature, offering potential advantages such as strong resistance to background interference, simple equipment, and applicability to on-site detection of complex samples. Combined with the colorimetric method in Example 3, it enables dual-mode signal output for mutual verification, significantly improving the reliability of the detection results.

[0127] The specific embodiments described above do not constitute a limitation on the scope of protection of this 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 substitutions, and improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. Use of a PFC-1-UiO-66 heterojunction nanoszyme in catechol multimodal 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, mix the sample to be tested with the PFC-1-UiO-66 heterojunction nanoscale enzyme dispersion and TMB substrate, prepare the reaction system, irradiate with 808 nm near-infrared laser for 1-5 minutes, and record the temperature change value ΔT; The above ΔA 652 , ΔA 510 and ΔT are 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; The preparation method of the PFC-1-UiO-66 heterojunction nanoscale enzyme comprises the following steps: a. 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 the reaction time to be 20-30 minutes; after the reaction is completed, cool the obtained reaction solution one 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 the reaction time to be 20-30 minutes; after the reaction is completed, cool the obtained reaction solution two to room temperature, centrifugally collect white precipitate, and then wash and dry to obtain UiO-66 powder; b. Dissolve 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene in N,N-dimethylformamide, add the UiO-66 powder prepared in step a, perform a third heating reaction, control the temperature of the third heating reaction to be 110-120 DEG C, and the reaction time to be 30-35 minutes; after the reaction is completed, cool the obtained reaction solution three to room temperature, then add acetone and stir, so that 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene forms a PFC-1 framework structure in situ on the surface of UiO-66, after the stirring reaction is completed, centrifugally wash and dry to obtain the PFC-1-UiO-66 heterojunction nanoscale enzyme; In step a, the molar ratio of the cerium salt to terephthalic acid is 1:0.9-1:1.1; in step b, the mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to UiO-66 powder is 1:1.5-1:2.

0.

2. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 1 in catechol multimodal detection, characterized in that, The PFC-1-UiO-66 heterojunction nanoscale enzyme dispersion is prepared by adding the 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, and the pH is controlled to be 3.5-5.0; In step (2), the neutral condition is achieved by adding sodium phosphate buffer solution into the reaction system, and the pH is controlled to be 6.5-7.

5.

3. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 1 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.

4. Use of PFC-1-UiO-66 heterojunction nanoszyme in catechol multi-modal detection according to claim 1, characterized in that, In step a, the cerium salt is cerium ammonium nitrate; the mixed solvent is obtained by mixing deionized water and acetic acid, and the volume ratio of deionized water to acetic acid is 30:1-35:

1.

5. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 1 in catechol multimodal detection, characterized in that, In step a, the mass-volume ratio of the cerium salt to the mixed solvent is 0.25-0.35 g / mL; and the mass-volume ratio of the terephthalic acid to N,N-dimethylformamide is 0.015-0.025 g / mL.

6. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 1 in catechol multimodal detection, characterized in that, In step a, when the white precipitate is collected by centrifugation, the centrifugal speed is controlled to be 7000-8000 rpm, and the centrifugal time is 5-7 minutes; when washing, N,N-dimethylformamide is used, and the centrifugal washing is performed for 3-5 times; and the drying is performed in a vacuum drying box, the drying temperature is controlled to be 60-70℃, and the drying time is 10-12 hours.

7. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 1 in catechol multimodal detection, characterized in that, In step b, the mass-volume ratio of 1,3,6,8-tetra(4-carboxyphenyl)pyrene to N,N-dimethylformamide is 8-12 mg / mL.

8. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 1 in catechol multimodal detection, characterized in that, In step b, after the addition of acetone, the stirring is controlled at room temperature for 10-12 hours.

9. Use of the PFC-1-UiO-66 heterojunction nanoszyme according to claim 1 in catechol multimodal detection, characterized in that, In step b, the centrifugal speed is controlled to be 8000-8500 rpm, and the centrifugal time is 5-7 minutes; when washing, acetone is used, and the centrifugal washing is performed for 3-5 times; and the drying is performed in a vacuum drying box, the drying temperature is controlled to be 70-80℃, and the drying time is 10-15 hours.

Images