CN117430770B - Light response oxidase material based on COF, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of polymer material synthesis, and specifically relates to a COF-based light-responsive oxidase-like material and its preparation method and application. The present invention uses 1,3,6,8-tetrakis(4-aminophenyl)pyrene (TPy) and pyridine-containing aromatic dialdehydes (including 6-(4-formylphenyl)nicotinic aldehyde (FPY), [2 ,2'-bipyridyl]-5,5'-dicardehyde (BPY) or 2,5-pyridine dialdehyde (PY), etc.) as raw materials, a COF-based photoresponsive oxidation was created through the Schiff base reaction Enzyme material (named TPy COF). The TPy COF photocatalyst prepared by the invention has a narrow bandgap, high carrier mobility and a small electron transfer barrier, which is beneficial to improving light capture ability, accelerating charge separation and transport, and effectively improving photocatalytic activity. The TPy COF prepared by the present invention has significant light-responsive oxidase-like activity, can effectively catalyze the chromogenic substrate of tetramethylbenzidine (TMB), and realize the detection of glutathione (GSH), uric acid (UA) and Highly sensitive and selective detection of L-cysteine (L-Cys).

Description

A COF-based light-responsive oxidase-like material and its preparation method and application

Technical field

The invention belongs to the technical field of polymer material synthesis, and specifically relates to a light-responsive oxidase-like material based on a covalent organic framework (COF) and its preparation method and application.

Background technique

Colorimetric sensors have important application value in environmental pollution, food industry, biochemical analysis, biomedicine and other fields. Specifically, molecular testing in the biomedical field has attracted considerable attention due to its potential applications in disease diagnosis and treatment. Because it does not require expensive instruments, it is suitable for disease diagnosis in resource-limited areas or primary care settings. Since natural enzymes such as glucose oxidase (GOx), oxidase (OXD) and peroxidase (POD) have excellent catalytic activity, high sensitivity and substrate specificity, they have been proven to be useful in designing efficient biosensors. Active ingredient for detecting a variety of biomarkers, including glucose, hydrogen peroxide, xanthine, lactate, L-cysteine (L-Cys), etc. Although natural enzymes have high sensitivity in colorimetric detection, they still have some limitations due to their inherent shortcomings such as high cost, instability, and difficulty in production and storage.

Biocatalysts have the advantages of low cost, good stability, simple synthesis process, and controllable nanomaterial activity, so they have broad application prospects. Due to the POD-like activity of ferrous oxides, a large number of mimetic enzymes with excellent catalytic activity have been developed. Since the surface structure of nanomaterials is difficult to control, the source of biocatalytic sites is unknown, enzyme-like activity is low, and the catalytic mechanism is complex. Therefore, it is necessary to pay attention to the design of ideal biocatalysts with excellent catalytic activity and selectivity. At the same time, it is also very important to construct biocatalysts to make them highly controllable and expand their applications in the field of colorimetric sensors. Recent studies have shown that covalent organic frameworks (COFs) with highly designable structures possess highly photoresponsive catalytic activity. Photoactive and nonmetallic compounds are expected to provide an alternative, environmentally friendly, cheap, and prospective strategy for the development of simulated oxidases.

Contents of the invention

In order to solve the above problems, the present invention uses 1,3,6,8-tetrakis(4-aminophenyl)pyrene (TPy) and pyridine-containing aromatic dialdehydes (including 6-(4-formylphenyl)nicotinic aldehyde ( FPY), [2,2'-bipyridyl]-5,5'-dicarboxaldehyde (BPY) or 2,5-pyridine dialdehyde (PY), etc.) were used as raw materials to create a COF-based light-responsive oxidase. Material.

Specifically, in one aspect, the present invention provides a method for preparing a photoresponsive oxidase-like material based on a covalent organic framework, which includes combining 1,3,6,8-tetrakis(4-aminophenyl)pyrene and The pyridine-containing aromatic dialdehyde undergoes a solvothermal reaction in the reaction solvent under the action of acid, and the reaction product is collected, and after washing and drying, the photoresponsive oxidase-like material based on the covalent organic framework is obtained.

As used herein, pyridine-containing aromatic dialdehyde means that at least one phenyl group in the aromatic dialdehyde contains one nitrogen heteroatom.

Further, the pyridine-containing aromatic dialdehyde is selected from 6-(4-formylphenyl)nicotinic aldehyde, [2,2'-bipyridyl]-5,5'-dicarboxaldehyde or 2,5-pyridine dialdehyde. Preferably, the pyridine-containing aromatic dialdehyde is selected from 6-(4-formylphenyl)nicotinic aldehyde or 2,5-pyridine dialdehyde. More preferably, the pyridine-containing aromatic dialdehyde is 6-(4-formylphenyl)nicotinic aldehyde.

Further, in the preparation method of the present invention, the molar ratio of 1,3,6,8-tetrakis(4-aminophenyl)pyrene and the pyridine-containing aromatic dialdehyde is 1:2.

Further, the reaction solvent is 1,4-dioxane, mesitylene, N,N-dimethylformamide, N,N-dimethylacetamide, o-dichlorobenzene, n-butanol, One of benzyl alcohol, methanol, ethanol, dimethyl sulfoxide, acetonitrile, cyclohexane or a mixed solvent of several of them.

Further, the reaction solvent is a mixed solvent of 1,4-dioxane and mesitylene. Further, the volume ratio of 1,4-dioxane and mesitylene in the mixed solvent is 1:1.

Further, the acid is 3-6 mol/L acetic acid. Further, the volume ratio of the reaction solvent to the acid is 6:1. In the case where the reaction solvent is a mixed solvent of 1,4-dioxane and mesitylene, the volume ratio of 1,4-dioxane, mesitylene and the acid is 3:3:1 .

Further, the solvothermal reaction is carried out under sealed and degassed conditions. Further, the degassing includes rapid freezing in a liquid nitrogen bath and degassing through three freezing pump-thawing cycles.

Further, the solvothermal reaction includes reaction at 120-140°C for 3-7 days. Further, the solvothermal reaction included reaction at 120°C for 5 days.

Further, the washing includes washing with acetone and tetrahydrofuran.

Further, the drying includes vacuum drying. Further, the drying includes vacuum drying at 120°C.

In other aspects, the present invention provides a photoresponsive oxidase-like material based on a covalent organic framework prepared according to the methods described herein.

Further, the photoresponsive oxidase-like material based on a covalent organic framework has the following structural units: or or .

Preferably, the photoresponsive oxidase-like material based on a covalent organic framework has the following structural units: or .

More preferably, the photoresponsive oxidase-like material based on a covalent organic framework has the following structural units: .

In other aspects, the present invention provides the use of a covalent organic framework-based light-responsive oxidase-like material as described herein for constructing a colorimetric sensor.

Further, the use is based on the light-responsive oxidase-like materials based on covalent organic frameworks as described herein, which can be used to treat glutathione (GSH), uric acid (UA) and L-cysteine (L-Cys). ) for highly sensitive and selective detection. Therefore, the present invention provides photoresponsive oxidase-like materials based on covalent organic frameworks as described herein for use in constructing colorimetric sensors to detect glutathione (GSH), uric acid (UA) and L-cysteine (L-Cys) one or more uses.

Beneficial effects of the invention

The present invention uses 1,3,6,8-tetrakis(4-aminophenyl)pyrene (TPy) and pyridine-containing aromatic dialdehydes (including 6-(4-formylphenyl)nicotine aldehyde (FPY), [ A COF-based light-responsive oxidase-like material (named TPy COF, the TPy COF corresponding to the above three raw materials are named TPy-FPY, TPy-BPY and TPy-PY respectively). The TPy COF (especially TPy-FPY) photocatalyst prepared by the invention has a narrow bandgap, high carrier mobility and a small electron transfer barrier, which is beneficial to improving light capture ability, accelerating charge separation and transport, and effectively improving Photocatalytic activity. TPy COF (especially TPy-FPY) has significant photoresponsive oxidase-like activity and can effectively catalyze the chromogenic substrate of tetramethylbenzidine (TMB). Taking advantage of these advantages, a highly sensitive and selective detection of glutathione (GSH), uric acid (UA), and L-cysteine (L-Cys) was performed using the photoactivated COF photoresponsive oxidase-like activity, and Its application prospects in complex biological samples were further explored. We believe that this efficient nanoreactor with oxide-like oxidation activity can provide some experience for the subsequent construction of colorimetric sensors with wide applications.

Compared with the prior art, the present invention uses substances used as chemical catalysis in the prior art as light-responsive oxidases. Although COF is also used as a photocatalytic enzyme material in the prior art, there is no prior art. It is pointed out that the COF synthesized with pyridine dialdehyde is used as a photocatalytic material in the present invention, and the present invention reveals this and proves that it can be used as a colorimetric sensor; while using dialdehyde without pyridine such as 4,4' -COF prepared from biphenyl dicarboxaldehyde (BPD) and terephthalaldehyde (PD) has very poor effect when used as a photocatalytic oxidase material, as shown in Figures 16b and 16c of the description; in addition, as shown in Figure 13, TPy-BPD and TPy-PD are not suitable as oxidase materials due to their wide band gaps. In addition, the catalysts in the prior art produce hydrogen peroxide through photocatalysis, while the catalyst of the present invention can produce active oxygen under photocatalysis.

Description of drawings

Figure 1 shows the synthesis process and chemical structure of TPy-1P.

Figure 2 shows the synthesis process and chemical structure of TPy-2P.

Figure 3 shows SEM images of TPy-PD at different magnifications, (a) magnification of 10 μm, (b) magnification of 5 μm, and (c) magnification of 2 μm.

Figure 4 shows SEM images of TPy-PY at different magnifications, (a) magnification of 10 μm, (b) magnification of 5 μm, and (c) magnification of 2 μm.

Figure 5 shows SEM images of TPy-BPD at different magnifications, (a) magnification is 10 μm, (b) magnification is 5 μm, (c) magnification is 2 μm.

Figure 6 shows SEM images of TPy-FPY at different magnifications, (a) magnification of 10 μm, (b) magnification of 5 μm, and (c) magnification of 2 μm.

Figure 7 shows SEM images of TPy-BPY at different magnifications, (a) magnification of 10 μm, (b) magnification of 5 μm, and (c) magnification of 2 μm.

Figure 8 shows PXRD patterns presenting experimental data and simulation patterns of (a) TPy-BPD, (b) TPy-FPY, (c) TPy-BPY, (d) TPy-PD and (e) TPy-PY. .

Figure 9 shows the FT-IR spectra of (a) TPy-BPD, (b) TPy-FPY, (c) TPy-BPY, (d) TPy-PD and (e) TPy-PY.

Figure 10 shows the XPS spectral analysis of (a) TPy-BPD, TPy-FPY and TPy-BPY and (b) TPy-PD and TPy-PY.

Figure 11 shows UV-DRS analysis of (a) TPy-2P and (b) TPy-1P.

Figure 12 shows the Mott-Schottky plots of (a) TPy-BPD, (b) TPy-FPY, (c) TPy-BPY, (d) TPy-PD and (e) TPy-PY.

Figure 13 shows the band gap distribution of (a) TPy-BPD, TPy-FPY and TPy-BPY and (b) TPy-PD and TPy-PY.

Figure 14 shows the steady-state photoluminescence measurements of (a) TPy-BPD, TPy-FPY and TPy-BPY and (b) TPy-PD and TPy-PY.

Figure 15 shows the transient photocurrent response of (a) TPy-BPD, TPy-FPY and TPy-BPY and (b) TPy-PD and TPy-PY and (c) TPy-BPD, TPy-FPY and Tpy-BPY. and (d) Nyquist plots of TPy-PD and TPy-PY.

Figure 16 shows the schematic diagram of (a) TMB oxidation and the time-dependent oxidase-like activities before and after light exposure of (b) TPy-BPD, TPy-FPY and Tpy-BPY and (c) TPy-PD and TPy-PY, where In each figure of (b) and (c), the left half of the figure shows the time-dependent oxidase-like activity before illumination, and the right half of the figure shows the time-dependent oxidase-like activity after illumination.

Figure 17 shows the light-controlled oxidase-like activities of TPy-FPY (left panel) and TPy-PY (right panel).

Figure 18 shows a schematic diagram of absorbance under different reaction conditions.

Figure 19 shows the steady-state kinetic measurements (a) and the corresponding double reciprocal plot (b) of TPy-FPY (left panel) and TPy-PY (right panel) as catalyst using TMB as substrate under visible light irradiation.

Figure 20 shows the effect of free radical scavengers on the light-responsive oxidase-like activity of TPy-FPY (left panel) and TPy-PY (right panel).

Figure 21 shows the EPR spectra of TPy-FPY (left picture) and TPy-PY (right picture) for ·O ₂ ^- and ¹ O ₂ signals before and after illumination.

Figure 22 shows a schematic diagram of TMB oxidation by active oxygen.

Figure 23 shows (a) Schematic diagram of selective colorimetric detection of glutathione, uric acid and cysteine based on the oxidase-like activity of TPy-FPY; (b) TMB in the presence of different concentrations of glutathione UV-visible absorption spectrum of +TPy-FPY mixture; (c) Linear calibration plot of ΔA (ΔA=A ₀ -A, A ₀ is the initial absorbance, A is the absorbance detected after adding GSH) and GSH concentration; (d ) UV-visible absorption spectra of TMB+TPy-FPY mixture in the presence of different concentrations of UA; (e) ΔA (ΔA=A ₀ -A, A ₀ is the initial absorbance, A is the absorbance detected after adding UA) and Linear calibration chart of UA concentration; (f) UV-visible absorption spectrum of TMB+TPy-FPY mixture in the presence of different concentrations of Cys; (g) ΔA (ΔA=A ₀ -A, A0 is the initial absorbance, A is after adding Cys Linear calibration plot of detected absorbance) versus Cys concentration.

Figure 24 shows the ΔA of TPy-PY (ΔA=A ₀ -A, A ₀ is the initial absorbance, A is the absorbance detected after adding GSH/UA/Cys) versus (a) GSH, (b) UA and (c )Linear calibration plot of Cys concentration.

Figure 25 shows the steady-state kinetics determination of TPy-BPY as catalyst using TMB as substrate under visible light irradiation (left panel) and the corresponding double reciprocal plot (right panel).

Figure 26 shows the ΔA of TPy-BPY (ΔA=A ₀ -A, A ₀ is the initial absorbance, A is the absorbance detected after adding GSH/UA/Cys) versus (a) GSH, (b) UA and (c )Linear calibration plot of Cys concentration.

Detailed ways

The present invention will be further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Example 1: Synthesis of light-responsive oxidase-like materials based on covalent organic frameworks

We use the 1,3,6,8-tetrakis(4-aminophenyl)pyrene (TPy) building block with a series of linear dialdehydes including phenyl (1P) and twisted biphenyl (2P) derived building blocks. Imine-linked COFs are generated in the solvothermal synthesis (Figures 1 and 2 show the synthesis schematics of TPy-1P COF and TPy-2P COF, respectively). Regarding the selection of linear dialdehyde building blocks, we selected 1,4-o-phthalaldehyde (PD), 2,5-pyridine dialdehyde (PY), 4,4'-biphenyl dialdehyde (BPD), 6- (4-formylphenyl)nicotinic aldehyde (FPY) and [2,2'-bipyridyl]5,5'-dicarboxaldehyde (BPY) were used to construct our COF to explore the possible effects of introducing the pyridine module on the material properties. Impact.

Specifically, the process of synthesizing COF using the 1,3,6,8-tetrakis(4-aminophenyl)pyrene (TPy) building block and the above-mentioned linear dialdehyde building block as raw materials is as follows.

Synthesis of TPy-PD COF. In a typical synthesis process, 1,3,6,8-tetrakis(4-aminophenyl)pyrene (85 mg, 0.15 mmol), 1,4-o-phthalaldehyde (32 mg, 0.3 mmol), 1,4 - Dioxane (1.5 mL) and mesitylene (1.5 mL) were added to a 15 mL Pyrex tube. After ultrasonic treatment for 10 minutes, add 0.5 mL of 3M acetic acid. After this, the heat-resistant glass tubes were sealed, quickly frozen in a liquid nitrogen bath, degassed through three freeze pump-thawing cycles, and then heated at 120°C for 5 days. After cooling to room temperature, the precipitate was collected by filtration and washed with acetone and tetrahydrofuran. The further purified COF was dried under vacuum at 120°C.

Synthesis of TPy-PY COF. In a typical synthesis process, 1,3,6,8-tetrakis(4-aminophenyl)pyrene (85 mg, 0.15 mmol), 2,5-pyridine dialdehyde (32 mg, 0.3 mmol), 1,4- Dioxane (1.5 mL) and mesitylene (1.5 mL) were added to a 15 mL Pyrex tube. After ultrasonic treatment for 10 minutes, add 0.5 mL of 3M acetic acid. After this, the heat-resistant glass tubes were sealed, quickly frozen in a liquid nitrogen bath, degassed through three freeze pump-thawing cycles, and then heated at 120°C for 5 days. After cooling to room temperature, the precipitate was collected by filtration and washed with acetone and tetrahydrofuran. The further purified COF was dried under vacuum at 120°C.

Synthesis of TPy-BPD COF. In a typical synthesis process, 1,3,6,8-tetrakis(4-aminophenyl)pyrene (85 mg, 0.15 mmol), 4,4'-biphenyldicarboxaldehyde (64 mg, 0.3 mmol), 1, 4-Dioxane (1.5 mL) and mesitylene (1.5 mL) were added to a 15 mL Pyrex tube. After ultrasonic treatment for 10 minutes, add 0.5 mL of 3M acetic acid. After this, the heat-resistant glass tubes were sealed, quickly frozen in a liquid nitrogen bath, degassed through three freeze pump-thawing cycles, and then heated at 120°C for 5 days. After cooling to room temperature, the precipitate was collected by filtration and washed with acetone and tetrahydrofuran. The further purified COF was dried under vacuum at 120°C.

Synthesis of Tpy-FPY COF. In a typical synthesis process, 1,3,6,8-tetrakis(4-aminophenyl)pyrene (85 mg, 0.15 mmol), 6-(4-formylphenyl)nicotinic aldehyde (64 mg, 0.3 mmol) ), 1,4-dioxane (1.5 mL) and mesitylene (1.5 mL) were added to a 15 mL Pyrex tube. After ultrasonic treatment for 10 minutes, add 0.5 mL of 3M acetic acid. After this, the heat-resistant glass tubes were sealed, quickly frozen in a liquid nitrogen bath, degassed through three freeze pump-thawing cycles, and then heated at 120°C for 5 days. After cooling to room temperature, the precipitate was collected by filtration and washed with acetone and tetrahydrofuran. The further purified COF was dried under vacuum at 120°C.

Synthesis of Tpy-BPY COF. In a typical synthesis, 1,3,6,8-tetrakis(4-aminophenyl)pyrene (85 mg, 0.15 mmol), [2,2'-bipyridyl]-5,5'-dicarboxaldehyde ( 64 mg, 0.3 mmol), 1,4-dioxane (1.5 mL) and mesitylene (1.5 mL) were added to a 15 mL Pyrex tube. After ultrasonic treatment for 10 minutes, add 0.5 mL of 3M acetic acid. After this, the heat-resistant glass tubes were sealed, quickly frozen in a liquid nitrogen bath, degassed through three freeze pump-thawing cycles, and then heated at 120°C for 5 days. After cooling to room temperature, the precipitate was collected by filtration and washed with acetone and tetrahydrofuran. The further purified COF was dried under vacuum at 120°C.

Example 2: Structural characterization of light-responsive oxidase-like materials based on covalent organic frameworks

The scanning electron microscope (SEM) images of TPy-PD COF, TPy-PY COF, TPy-BPD COF, TPy-FPY COF and TPy-BPY COF are shown in Figure 3-7 respectively.

Verified by powder X-ray diffraction (PXRD) patterns (Fig. 8a-e), TPy-2P (i.e., TPy-BPD COF, Tpy-FPYCOF and Tpy-BPY COF) has two positions at 2θ≈3.2° and ≈6.4° respectively. Sharp diffraction peaks, TPy-1P (i.e. TPy-PDCOF, TPy-PY COF) has two sharp diffraction peaks at 2θ≈3.7° and ≈7.5° respectively, indicating that they have high AA superposition crystallinity. The same position and similar intensity of the diffraction peaks indicate that the introduced pyridine unit has little effect on the crystal structure of COF.

We collected Fourier transform infrared (FT-IR) spectra to study the chemical structures of TPy-2P COF and TPy-1P COF. As shown in Figure 9, after the reaction, the characteristic adsorption of FT-IR spectra in the precursors including -NH (≈3344cm ^-1 ) in TPy and -C=O (≈1695cm ^-1 ) in 1P and 2P The peaks drop sharply with the -C=N- bond (≈1621 cm ^-1 ) appearing in five COFs, indicating the successful formation of imine bonds through the Schiff base reaction.

In addition to this, XPS analysis (Figure 10) also shows that with the introduction of the pyridine block, the transfer of N1s binding energy shifts to a high value, which is confirmed by 2P-COF and 1P-COF(SI).

These results fully demonstrate that two different series of target compounds with the same structure, similar skeleton, and different positions were successfully synthesized through Schiff base reaction.

Example 3: Characterization of photophysical properties of light-responsive oxidase-like materials based on covalent organic frameworks

The photophysical properties of TPy-2P and TPy-1P were systematically studied to demonstrate their potential as simulated light-responsive oxidases. The energy levels of the valence band maximum (VB) and conduction band minimum (CB) of TPy-2P and TPy-1P were studied using Tauc diagrams and Mott-Schottky (MS) diagrams. The band gaps of TPy-BPD, TPy-FPY and TPy-BPY are estimated to be 2.49, 2.39 and 2.31 eV respectively, and the band gaps of TPy-PD and TPy-pY are estimated to be 2.38 and 2.20 eV respectively (Fig. 11). This makes us believe that the introduction of pyridine groups can effectively adjust the band gap of TPy-COF, including TPy-2P and TPy-1P. The positive slopes of the MS patterns of TPy-2P and TPy-1P indicate their n-type semiconductor behavior. By calculation, the flat band potentials of TPy-BPD, TPy-FPY and TPy-BPY are -1.29, -0.98 and -0.43V respectively relative to the general hydrogen electrode (NHE) (Figure 12a-c). Therefore, CB is estimated to be -1.49, -1.18 and -0.63V (V vs. NHE). For TPy-BPD, TPy-FPY and TPy-BPY, the calculated VB values based on band gap and CB are 1.00, 1.21 and 1.68V respectively. Data related to TPy-1P are given in Figure 12d–e. For comparison, the band data and the standard reduction potential ^of O2 _/ · _O2- were collected in Figure 13. Obviously, the CB values of TPy-2P and TPy-1P are more negative than the standard reduction potential of _O2 /· _O2- , ^indicating their ability to achieve · _O2 ^- generation. The properties of TPy-2P and TPy-1P under photoexcitation were studied using steady-state photoluminescence spectroscopy. The data of TPy-2P and TPy-1P show that with the introduction of pyridine blocks, the fluorescence intensity of the materials decreases, which means that electrons in the pyridine-free COF excited state tend to combine with holes and release energy in the form of photons (Fig. 14). The results show that the introduction of pyridine blocks can promote charge separation. In addition, photocurrent response curves and electrochemical impedance analyzes were performed on these materials. With the introduction of pyridine blocks, the material exhibits better electron hole generation and separation capabilities as well as carrier transport capabilities, indicating that pyridine-containing COFs have great potential to construct overall light-responsive oxidase simulation properties (Fig. 15a-b and cd). However, we surprisingly found that TPy-BPY has lower photocurrent density and higher charge transfer resistance compared with TPy-FPY, which may result in the final performance of TPy-BPY being inferior to Tpy-FPY.

Example 4: Light-driven oxidase-like activity of light-responsive oxidase-like materials based on covalent organic frameworks

The excellent photocatalytic properties endow the oxidase-like potential. Using TMB as the chromogenic substrate, the photosensitive oxidase mimicking ability of COF was studied. As shown in Figure 16a and bc, the small molecule substrate is oxidized by TPy-COF under light and produces the dark blue product oxTMB, among which TPy-FPY and TPy-PY have the best catalytic performance among TPy-2P and TPy-1P respectively. Good material, which is consistent with the above photophysical property test results. In view of the excellent properties of TPy-FPY and TPy-PY, they were selected for further research. The light-driven oxidase-like activity of TPy-FPY and TPy-PY exhibits a step-like behavior by turning light on or off (Fig. 17). Possible influencing factors in the catalytic system were further studied. As shown in Figure 18, with the pH value (pH=3.5-7.5) and catalyst dosage (10-40mg/L), oxygen emissions (argon gas injected into the system for 0-10min) and TMB content (0.015-0.24mg/L ) increases, the absorbance of the system decreases. Then, the light-driven oxidase-like activities of TPy-FPY and TPy-PY were studied using the steady-state kinetics method. As shown in Figure 19a and b, the K _m and V _max of TPy-FPY are 0.45mM and 0.79μMs ^-1 respectively, while those of TPy-PY are 0.88mM and 0.73μMs ^-1 respectively. Compared with the catalytic abilities of reported nanozymes, TPy-FPY and TPy-PY showed high affinity and catalytic activity towards TMB.

Example 5: Catalytic mechanism of light-responsive oxidase-like materials based on covalent organic frameworks

In addition, the catalytic mechanisms of TPy-FPY and TPy-PY were also studied. First, the effect of _O2 on this experiment was determined to explore the formation of ROS. As mentioned previously, there is a positive correlation between absorbance and oxygen concentration, and the absorbance signal decreases after the solution is bubbled with argon for 10 min (SI). Therefore, _O2 is related to TPy-FPY or TPy-PY catalyzed TMB oxidation. Secondly, p-benzoquinone (p-BQ), sodium azide (NaN ₃ ) and oxalic acid ((NH ₄ ) ₂ C ₂ O ₄ ) were selected as superoxide anion (·O ₂ ^- ), singlet oxygen ( ¹ Scavenger for O ₂ ) and photopores (h ⁺ ). As shown in Figure 20, in the presence of p-BQ, the absorption peak of oxTMB is obviously suppressed, and the absorption peak decreases slightly after adding NaN ₃ and (NH ₄ ) ₂ C ₂ O ₄ . In addition, electron spin resonance (EPR) experiments were performed to verify ·O ₂ ^- and ¹ O ₂ . After 5 minutes of irradiation, the mixed system greatly confirmed the presence of ·O ₂ ^- and ¹ O ₂ (Fig. 21). However, ·OH radicals are not observed in the figure. Therefore, as shown in Figure 22, ·O ₂ ^- , ¹ O ₂ and h ⁺ are the main reaction intermediates catalyzed by the oxidase simulation of TPy-FPY and TPy-PY.

Example 6: Application of light-responsive oxidase-like materials based on covalent organic frameworks in colorimetric biosensors

Since TPy-FPY has excellent oxidase-like activity, it is expected to develop multifunctional colorimetric biosensors based on different nanozyme activities. Glutathione is an important intracellular antioxidant in organisms and plays an important role in physiological and pathological processes. Sensitive and selective detection of glutathione is of great significance for the early diagnosis and prevention of many diseases. To this end, we established a simple colorimetric sensing platform for GSH target detection. As shown in Figure 23a, the colorimetric assay of GSH was established based on the oxidase-like activity of TPy-FPY. Under light conditions, TPy-FPY catalyzes the blue oxidation of colorless TMB to generate oxTMB. As a typical reducing substance, GSH can effectively reduce TMB to oxTMB, resulting in blue fading and absorbance decrease. Figure 23b shows the absorption spectra of the TMB+TPy-FPY system in the presence of different concentrations of glutathione. In the range of 0~120μM, the absorbance decreases proportionally with the glutathione concentration. As shown in Figure 23c, there is a good linear relationship between the absorbance at 652 nm and GSH concentration in the range of 0 to 60 μM, with a correlation coefficient ( ^R2 ) of 0.9935 and a limit of detection (LOD) estimated to be 0.63 μM, which is consistent with the previous Colorimetric methods for GSH detection based on biocatalysts are comparable or slightly higher. This ultra-thin TPy-FPY nanosheet has a large surface area, which allows the Tpy-FPY nanosheet to effectively contact GSH and promotes the interaction on the surface of the Tpy-FPY nanosheet, thus improving the sensitivity of the Tpy-FPY nanosheet. It has good sensitivity and wide detection range, making the Tpy-FPY method an ideal method for determining glutathione.

UA and L-Cys exhibit similar reducing properties to GSH. They can also reduce oxTMB to TMB to obtain absorbance changes, which means that colorimetric methods based on Tpy-FPY-like oxidase activity may be equally effective for them. Based on the above principles, we performed similar performance tests in the presence of UA and Cys. In the range of 0~100 μM, the absorbance decreased proportionally with the increase in UA concentration (Fig. 23d). As shown in Figure 23e, there is a good linear relationship between the absorbance at 652 nm and the GSH concentration in the range of 5 to 80 μM, with a correlation coefficient (R ² ) of 0.9912 and an estimated detection limit of 0.72 μM. On the other hand, based on the oxidase-like activity of TPy-FPY, a colorimetric sensor for measuring Cys content was established. The absorbance at 652 nm decreases linearly with increasing Cys concentration in the range of 2 μM to 60 μM (R ² =0.9925). The corresponding detection limit was determined to be 0.42 μM (Fig. 23f-g). Of course, we also tested the colorimetric detection ability of TPy-PY (Figure 24), which has the best oxidase-like activity in 1P-COF. The colorimetric detection capabilities of TPy-BPY are shown in Figures 25 and 26. It can be seen from the test results that among the three, TPy-FPY has the strongest oxidase-like activity and its detection range is relatively wide, which also shows that the catalytic activity is proportional to the detection range to a certain extent.

It should be noted that the preferred embodiments of the present invention are given in the description and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations to the content of the present invention, and are provided for the purpose of making the disclosure of the present invention more thorough and comprehensive. Furthermore, the above technical features can be continuously combined with each other to form various embodiments not listed above, which are all deemed to be within the scope of the description of the present invention; further, for those of ordinary skill in the art, they can be improved or transformed according to the above description. , and all these improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims (9)

1. A preparation method of a photoresponse oxidase-like material based on a covalent organic framework is characterized by comprising the steps of carrying out solvothermal reaction on 1,3,6, 8-tetra (4-aminophenyl) pyrene and pyridine-containing aromatic dialdehyde in a reaction solvent under the action of acid, collecting reaction products, washing and drying to obtain the photoresponse oxidase-like material based on the covalent organic framework,

wherein the pyridine-containing aromatic dialdehyde is selected from 6- (4-formylphenyl) nicotinaldehyde, [2,2 '-bipyridine ] -5,5' -dicarboxaldehyde or 2, 5-pyridinedialdehyde.

2. The method according to claim 1, wherein the molar ratio of 1,3,6, 8-tetrakis (4-aminophenyl) pyrene to pyridine-containing aromatic dialdehyde is 1:2.

3. The preparation method according to claim 1, wherein the reaction solvent is one or a mixed solvent of 1, 4-dioxane, mesitylene, N-dimethylformamide, N-dimethylacetamide, o-dichlorobenzene, N-butanol, benzyl alcohol, methanol, ethanol, dimethyl sulfoxide, acetonitrile and cyclohexane.

4. The method according to claim 3, wherein the reaction solvent is a mixed solvent of 1, 4-dioxane and mesitylene.

5. The method of claim 1, wherein the acid is 3-6mol/L acetic acid.

6. The method of claim 1, wherein the solvothermal reaction comprises reacting at 120-140 ℃ for 3-7 days.

7. A covalent organic framework based photoresponsive oxidase-like material, characterized in that it is prepared by a method according to any one of claims 1-6.

8. Use of the covalent organic framework based photoresponsive oxidase-like material of claim 7 for the construction of a colorimetric sensor.

9. The use according to claim 8, wherein the colorimetric sensor is used for detecting one or several of glutathione, uric acid and L-cysteine.