Fe single atom and single atom cluster nanozyme, and preparation method and application thereof

Abstract

The application discloses a kind of based on Fe single atom and single atom cluster nano enzyme (Fe AC / Fe SA ‑NC) and preparation method and application thereof, Fe (II) o-phenanthroline complex solution is mixed with ZIF-8 dispersion, and the precipitate is dried, and calcination is obtained Fe AC / Fe SA ‑NC single atom nano enzyme by heating to 900-1000 DEG C under protective atmosphere.After calcination, iron element and nitrogen element enter carbon skeleton, form the composite material of graphite carbon, FeN5 / Fe3 cluster, with large specific surface area and abundant active site, improve the catalytic property of carbon-based nano enzyme;Fe AC / Fe SA ‑NC multifunctional nano enzyme of the application has oxidase mimic enzyme property, can catalyze oxidation Aplex Red to produce strong red fluorescence, can be used for detecting acetylcholinesterase, low cost, wide application range.

Description

Technical Field

[0001] This invention belongs to the field of biosensing nanozymes, specifically relating to a method based on Fe single atoms and single-atom clusters (Fe). AC / Fe SA -NC) nanozymes, their preparation methods, and applications. Background Technology

[0002] In recent decades, nanozymes, as a substitute for natural enzymes, have attracted widespread attention from experts in various fields due to their advantages such as low cost, high activity, and good stability. Metal-organic frameworks (MOFs), with their large specific surface area and ordered arrangement of metal sites and ligands, are considered ideal precursors for synthesizing porous carbon materials. ZIF-8, in particular, contains nitrogen-rich organic ligands. After pyrolysis, zinc evaporates from the MOF, allowing iron to be incorporated into the zinc-zeolite imidazole framework, which contains a special cavity structure. Further pyrolysis yields porous carbon materials with controllable morphology and abundant FeN5 / Fe3 cluster sites.

[0003] Acetylcholinesterase is a key enzyme in biological neurotransmission. At cholinergic synapses, this enzyme degrades acetylcholine, terminating the excitatory effect of neurotransmitters on the postsynaptic membrane and ensuring the normal transmission of nerve signals within the body. Aplex Red luciferin itself is non-fluorescent, but upon oxidation, it generates oxidized Aplex Red, producing strong red fluorescence. With the advancement of nanozymes, nanozyme-based biosensors offer new avenues for the detection of acetylcholinesterase. Summary of the Invention

[0004] This invention aims to provide an oxidase-like enzyme based Fe single atom and single atom cluster (Fe AC / Fe SA -NC) nanozymes, their preparation methods, and applications.

[0005] Another object of the present invention is to provide a method for using Fe single atoms and single-atom clusters (Fe AC / Fe SA A method for detecting acetylcholinesterase using NC nanozymes.

[0006] The technical solution of this invention is as follows:

[0007] A Fe single atom and single atom cluster (Fe AC / Fe SA The preparation method of Fe(II) o-phenanthroline nanozyme includes the following steps: mixing Fe(II) o-phenanthroline complex solution with ZIF-8 dispersion, drying the precipitate, and calcining it at 900-1000℃ under a protective atmosphere.

[0008] Preferably, the calcination conditions are: heating to 900-1000℃ at a heating rate of 5-6℃ / s and holding at that temperature for 2-6 hours, preferably 3-4 hours.

[0009] The molar ratio of Fe(II) o-phenanthroline complex to ZIF-8, based on Fe and Zn, is 1:35-60, preferably 1:40-50, and more preferably 44-45.

[0010] The preparation method of the Fe(II) o-phenanthroline complex is as follows: ferrous salt is mixed with 1,10-phenanthroline and water, and the molar ratio of ferrous ions to 1,10-phenanthroline is 1:3-5.

[0011] Preferably, the ferrous salt is ferrous sulfite.

[0012] The ZIF-8 has a particle size of 150-300 nm, preferably 180-240 nm; in a preferred embodiment of the present invention, the particle size is approximately 200 nm. The ZIF-8 is prepared by dissolving zinc salt and 2-methylimidazole separately in a mixture of methanol and DMF, then mixing the two solutions and reacting for 1-10 hours to collect the precipitate. The molar ratio of zinc salt to 2-methylimidazole, based on elemental zinc, is 1:6-10, preferably 1:8.

[0013] Preferably, the zinc salt is zinc nitrate. Preferably, the volume ratio of methanol to DMF is 3-6:1, more preferably 4:1.

[0014] A Fe single atom and a single atom cluster (Fe) were obtained through the above preparation method. AC / Fe SA -NC), having a FeN5 / Fe3 cluster structure, with a particle size of 150-300 nm, preferably 180-240 nm; in a preferred embodiment of the present invention, Fe single atoms and single-atom clusters (Fe AC / Fe SA -NC) nanozymes have a particle size of approximately 200 nm.

[0015] Fe single atoms and single-atom clusters (Fe AC / Fe SA In the -NC) nanozyme, the Fe content is 1wt%-3wt%, preferably 1.5wt%-2wt%, and more preferably 1.6wt%-1.8wt%.

[0016] The Fe single atom and single atom cluster (Fe) AC / Fe SA -NC) nanozymes have peroxidase or oxidase activity and can be used to prepare peroxidase or oxidase.

[0017] The Fe single atom and single atom cluster (Fe) AC / FeSA -NC nanozymes can also be used to prepare biosensors or detection reagents for detecting acetylcholinesterase.

[0018] A method for detecting acetylcholinesterase includes the following steps: adding acetylcholine to the sample to be tested and incubating at pH 6-7 for 15-30 min; then adding the aforementioned Fe single atoms and single-atom clusters (Fe... AC / Fe SA Incubate the NC nanozyme for 15-30 min, add Aplex Red, and detect the fluorescence intensity after reacting for 5-15 min.

[0019] This invention involves coating a Fe(II)-phenanthroline complex onto ZIF-8, removing zinc ions through calcination, and utilizing ZIF-8 to form a dodecahedral carbon framework structure. Fe coordinates with N to form a FeN5 / Fe3 structure, thus preparing Fe single atoms and single-atom clusters (Fe...). AC / Fe SA -NC) nanozymes. These single-atom nanozymes possess oxidase activity, catalyzing the oxidation of Aplex Red to produce strong red fluorescence. When the reaction products of acetylcholinesterase and thioacetylcholine are added, the nanozyme activity is inhibited, resulting in a decrease in the fluorescence of Aplex Red, thereby enabling the detection of acetylcholinesterase.

[0020] The beneficial effects of this invention are as follows: after calcination, iron and nitrogen elements are introduced into the carbon skeleton of the ZIF-8@Fe-Phen composite material to form a graphite carbon and FeN5 / Fe3 composite material, which has a large specific surface area and abundant active sites, effectively improving the catalytic properties of carbon-based nanozymes.

[0021] The Fe single atom and single atom cluster (Fe) of the present invention AC / Fe SA -NC) Multifunctional nanozymes possess oxidase-mimicking enzyme properties and can catalyze the oxidation of Aplex Red to produce strong red fluorescence. When the reaction products of acetylcholinesterase and thioacetylcholine are added, the nanozyme activity is inhibited, resulting in a decrease in the fluorescence of Aplex Red. The biosensor established in this way is inexpensive and has a wide range of applications. Attached Figure Description

[0022] Figure 1 These are scanning electron microscope (SEM) images (Figure A) and transmission electron microscope (TEM) images (Figure B) of ZIF-8 prepared in Example 1.

[0023] Figure 2 These are scanning electron microscope (SEM) images (Figure A) and transmission electron microscope (TEM) images (Figure B) of ZIF-8@Fe-Phen prepared in Example 1.

[0024] Figure 3The Fe single atom and single atom cluster (Fe) prepared in Example 1 AC / Fe SA Scanning electron microscope (SEM) image (Fig. A), transmission electron microscope (SEM) image (Fig. B), and high-angle annular dark-field scanning transmission electron microscope (CD) image of the NC nanozyme.

[0025] Figure 4 The Fe single atom and single atom cluster (Fe) prepared in Example 1 AC / Fe SA Fluorescence activity diagram of oxidase mimicry enzymes of (NC) nanozymes.

[0026] Figure 5 The Fe single atom and single atom cluster (Fe) prepared in Example 1 AC / Fe SA Feasibility diagram for detecting acetylcholinesterase using NC nanozymes.

[0027] Figure 6 The Fe single atom and single atom cluster (Fe) prepared in Example 1 AC / Fe SA Fluorescence spectra of acetylcholinesterase at different concentrations detected by NC nanozymes. Detailed Implementation

[0028] To facilitate a clear understanding of the technical means, creative features, objectives, and effects of this invention, the invention will be further described below in conjunction with specific embodiments.

[0029] Example 1

[0030] 4 mmol of zinc nitrate hexahydrate was dissolved in a mixed solution of methanol and DMF (V methanol:V DMF = 4:1) to make a total volume of 15 mL, forming a homogeneous solution A. Then, 32 mmol of 2-methylimidazole was dissolved in a mixed solution of methanol and DMF (V methanol:V DMF = 4:1) to form a colorless and transparent solution B with a total volume of 10 mL.

[0031] Finally, pour solution A into solution B, stir magnetically for 2 minutes, and let stand for 2 hours after mixing thoroughly.

[0032] The precipitate was used to obtain a white solid ZIF-8, which was washed three times by centrifugation with methanol and then dispersed in 40 mL of methanol to form solution C.

[0033] 0.05 g of Fe(SO)₄·7H₂O (0.18 mmol) and 0.1 g of 1,10-o-phenanthroline (0.5 mmol) were dissolved in 20 mL of water and ultrasonically dispersed to obtain the Fe(II)o-phenanthroline complex. 10 mL of the Fe(II)o-phenanthroline complex was added to the above solution C, stirred at room temperature for 4 hours, washed with methanol, centrifuged three times, and dried under vacuum to obtain the light yellow precursor ZIF-8@Fe-Phen.

[0034] The light yellow powder ZIF-8@Fe-Phen obtained above was transferred to a ceramic boat and heated from room temperature to 900°C at a rate of 5°C / min under a flowing nitrogen atmosphere, and held for 3 hours. This yielded Fe single atoms and single-atom clusters (Fe...). AC / Fe SA -NC) nanozymes.

[0035] The obtained ZIF-8, ZIF-8@Fe-Phen, Fe AC / Fe SA -NC was analyzed by scanning electron microscopy, transmission electron microscopy, and high-angle annular dark-field scanning transmission electron microscopy. Results are shown below. Figures 1 to 3 .

[0036] Depend on Figure 1 It can be seen that the obtained ZIF-8 has a dodecahedral structure and good dispersibility, with a diameter of about 200 nm.

[0037] Depend on Figure 2 It can be seen that the obtained composite material ZIF-8@Fe-Phen has Fe-Phen complex particles on ZIF-8, indicating that Fe-Phen was successfully coated onto ZIF-8 and the dispersion was still well maintained, with the diameter remaining at about 200 nm.

[0038] Depend on Figure 3 As can be seen, the obtained material has a deformed dodecahedral carbon framework structure, with no Fe nanoparticles generated and no Fe-Phen complex particles appearing, indicating that Fe successfully coordinated with N to form a FeN5 / Fe3 cluster structure with good dispersion and a diameter still around 200 nm. High-angle annular dark-field scanning transmission electron microscopy showed the coexistence of Fe single atoms (red circles) and Fe single-atom clusters (blue circles), indicating that Fe AC / Fe SA Successful preparation of -NC. The Fe content was 1.73 wt%.

[0039] Example 2: Fe single atoms and single-atom clusters (Fe AC / Fe SA Verification of oxidase mimicry activity of NC)

[0040] 0.5 mg / mL Fe AC / Fe SA 15 μL of NC nanozyme aqueous solution was added to PB buffer (20 mM, pH 6.0) containing Aplex Red, bringing the total reaction volume to 200 μL. In the reaction system, Fe... AC / Fe SA -NC nanozyme content 37.5 μg / mL, Aplex Red content 2 μM. After mixing thoroughly, react at 25℃ for 10 minutes, excite at 550 nm using a microplate reader, and scan the fluorescence spectrum of the reaction system from 575 nm to 700 nm.

[0041] Figure 4 Fe was shown AC / Fe SA The solution fluorescence spectrum of the direct catalytic oxidation of Aplex Red by -NC in the absence of H2O2 showed a significant absorption peak at 585 nm, proving the presence of Fe single atoms and single-atom clusters (Fe). AC / Fe SA -NC) nanozymes possess oxidase-mimicking enzyme activity.

[0042] Example 3 is based on Fe single atoms and single-atom clusters (Fe AC / Fe SA -NC nanozymes are used as biosensors for detecting acetylcholinesterase.

[0043] Different concentrations of acetylcholinesterase (AChE) were mixed with acetylcholine (ATCh) and PBS buffer (20 mM pH 6.0) to make a total volume of 100 μL. The concentrations of acetylcholinesterase in the mixture were 0, 2.5, 5, 10, 15, 25, 50, 75, and 100 mU / mL, and the concentration of acetylcholine was 1 mM. The mixture was incubated at 37°C for 20 minutes, and then 0.5 mg / mL of Fe was added. AC / Fe SA - Add 15 μL of NC buffer and PB buffer (20 mM pH 6.0), incubate for 20 minutes at room temperature, then add Aplex Red (AR) to ensure a final volume of 200 μL, with a final AR concentration of 2 μM and Fe... AC / Fe SA -NC 37.5μg / mL; and reacted for 10 minutes. The enzyme was excited at 550nm using a microplate reader, and the fluorescence spectrum of the reaction system from 575nm to 700nm was scanned. The results are as follows: Figure 5 and 6 .

[0044] Figure 5 It shows Fe single atoms and single-atom clusters (Fe AC / FeSA The feasibility of using NC nanozymes as a biosensor for detecting acetylcholinesterase.

[0045] Figure 6 The fluorescence spectra of the sensor detecting different concentrations of acetylcholinesterase are shown.

[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and various changes and modifications can be made without departing from the spirit and scope of the invention, all of which fall within the scope of the claims. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A Fe single-atom and cluster nanozyme, characterized in that, The Fe single-atom and cluster nanozyme has a particle size of 150-300 nm and has a FeN5 / Fe3 cluster structure. The preparation method includes the following steps: mixing Fe(II) o-phenanthroline complex solution with ZIF-8 dispersion, drying the precipitate, and calcining it under a protective atmosphere; the calcination conditions are: heating to 900-1000℃ at a heating rate of 5-6 ℃ / s and holding at that temperature for 3-4 h; the molar ratio of Fe(II) o-phenanthroline complex to ZIF-8 is 1:35-60 based on Fe and Zn. The Fe(II) o-phenanthroline complex is prepared by mixing ferrous salt with 1,10-phenanthroline and water, and then ultrasonically dispersing the mixture to obtain the Fe(II) o-phenanthroline complex; the molar ratio of ferrous ions to 1,10-phenanthroline is 1:3-5. The ZIF-8 has a particle size of 150-300 nm. It is prepared by dissolving zinc salt and 2-methylimidazole in a mixture of methanol and DMF, respectively, then mixing the two solutions and reacting for 1-10 hours to collect the precipitate.

2. The Fe single-atom and cluster nanozyme according to claim 1, characterized in that, The Fe single-atom and cluster nanozymes possess oxidase-mimicking enzyme properties.

3. The application of the Fe single-atom and cluster nanozymes according to claim 1 in the preparation of biosensors or detection reagents for detecting acetylcholinesterase.

4. A method for detecting acetylcholinesterase, characterized in that, Thioacetylcholine was added to the sample to be tested and incubated at pH 6-7 for 15-30 min. The Fe single-atom and cluster nanozyme described in claim 1 was added and incubated for 15-30 min. Amplex Red was added, and fluorescence was detected after reacting for 5-15 min.

Images