Preparation method of Mn-AMP metal organogel nano-enzyme and application of Mn-AMP metal organogel nano-enzyme in colorimetric sensing

Abstract

The invention discloses a preparation method of a Mn-AMP metal organic gel nano-enzyme and application of the Mn-AMP metal organic gel nano-enzyme in colorimetric sensing, and the Mn-AMP metal organic gel with peroxidase-like activity is prepared by taking adenosine-5 'monophosphate (AMP) as a ligand. A colorimetric biosensor used for detecting ascorbic acid (AA) is constructed on the basis, a portable detection method is designed in combination with a smart phone application program 'Color Grab', and a new solution is provided for on-site rapid detection of AA. The preparation method disclosed by the invention is mild in condition and simple in synthesis path, has good water solubility and biocompatibility, is an ideal choice for nano-enzyme materials, and shows a good application prospect in the aspects of biosensing, bacteriostasis and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of nanoenzymes and colorimetric sensing detection, and in particular relates to a preparation method of a metal organic gel nanoenzyme and an application thereof in ascorbic acid detection. Background Art

[0002] Nanozymes are a new type of artificially designed functional nanomaterials that have both the unique properties of nanomaterials and the catalytic activity of simulating biological enzymes. Nanozymes have the advantages of low cost, good stability, adjustable catalytic performance, modifiability, easy storage and large-scale preparation. These characteristics just make up for the defects of biological enzymes, making nanozymes a powerful substitute for biological enzymes. So far, the discovery and development of a series of nanozymes has become an active research field that has attracted much attention, showing broad application prospects in biosensing, disease treatment, antibacterial, anti-oxidation and bioimaging.

[0003] Metal organic gel (MOG) is a coordination polymer, which is a semi-solid material self-assembled by metal ions and organic ligands through non-covalent intermolecular interactions. In recent years, MOG has been widely used in catalysis, adsorption, energy storage, sensing, analytical detection and other fields. Compared with metal organic framework (MOF), MOG has mild synthesis conditions and simple synthesis pathways, and has unique advantages as a new type of nanozyme. The highly interconnected pore structure and abundant redox sites can effectively promote the effective diffusion of substrates and products, giving it superior catalytic activity. It has a broader application prospect in practical detection such as the design of biosensors and the preparation of convenient gel kits. MOG has become an ideal choice as a nanozyme material due to its unique advantages.

[0004] Nucleotides are composed of three functional groups: nitrogenous bases, pentoses, and phosphate groups. These are potential binding sites for forming stable coordination bonds with metal ions. When nucleotides act as ligands with metal ions, they often exhibit a variety of coordination modes, and the resulting products have the characteristics of structural diversity. At the same time, nucleotides have the advantages of good water solubility, high biocompatibility, low toxicity, high stability, and low price, making them an ideal ligand for building materials. Most products with nucleotides as ligands are nanoparticles, and only a few products are gels. Xu et al. found that the products of the reaction of six heavy lanthanides with adenosine 5′-monophosphate (AMP) can be transformed from nanoparticles to gels. Liang et al. reported that Zn 2+Coordination with AMP can form a gel, and it was found that the mechanically broken gel can be re-formed after centrifugation, and the encapsulation effect of Zn-AMP gel on guest molecules was explored. Liang et al. then further explored the encapsulation performance of Zn-AMP gel on glucose oxidase and horseradish peroxidase, and found that compared with the free enzyme, the catalytic efficiency of the composite enzyme after gel encapsulation was improved, and a colorimetric biosensor for glucose detection was constructed through a cascade catalytic system. So far, metal organic gels with nucleotides as ligands are often used as carriers to encapsulate guest molecules such as natural enzymes, but reports of such gel materials with enzyme-like activity are relatively rare. Summary of the invention

[0005] The purpose of the present invention is to provide a kind of AMP as ligand, Mn 2+ The invention discloses a preparation method and application of a Mn-AMP coordination polymer gel nanozyme as a metal ion active center. The gel has peroxidase-like activity and can be used to detect ascorbic acid.

[0006] The implementation process of the present invention is as follows: A method for preparing a Mn-AMP metal organic gel comprises the following steps: (1) preparing adenosine-5′ monophosphate AMP and a soluble manganese salt into an aqueous solution; (2) adding AMP and soluble manganese salt aqueous solution into HEPES buffer and mixing well; (3) A gel is formed upon standing at room temperature, and the Mn-AMP metal organic gel is obtained by washing and drying.

[0007] In the above step (1), the soluble manganese salt is selected from manganese chloride, manganese nitrate, manganese acetate or manganese sulfate.

[0008] In the above step (2), the molar ratio of AMP to soluble manganese salt is 1:(0.9-1.1); In the above step (3), the drying method is preferably freeze-drying.

[0009] The Mn-AMP metal organic gel prepared by the above method has peroxidase-like activity and can be used to construct a colorimetric biosensor for ascorbic acid detection. In specific applications, the colorimetric biosensor is combined with a smartphone application "Color Grab" to achieve on-site visual portable detection.

[0010] The present invention successfully prepared a Mn-AMP coordination polymer gel with peroxidase-like activity, explored its synthesis conditions and synthesis mechanism, constructed a colorimetric biosensor for the detection of AA based on the Mn-AMP coordination polymer gel, and designed a convenient gel detection method in combination with the smartphone application "Color Grab" to further realize on-site rapid detection.

[0011] Compared with the prior art, the advantages of the present invention are: (1) Under room temperature, Mn 2+ It can be spontaneously formed in about 20 seconds after simple mixing with AMP. Compared with other current gel materials, it does not require the use of additional gelling agents such as agarose and alginate, and the synthesis method is simple and fast.

[0012] (2) The Mn-AMP coordination polymer gel has good peroxidase-like activity. On this basis, a portable gel detection method was designed in combination with the smartphone "Color Grab". The (G + B) / 2R showed a good linear relationship with the concentration of ascorbic acid. The innovation of this detection method lies in its portability and intelligence, which improves the detection efficiency of the colorimetric biosensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 a, b are scanning electron microscope images of Mn-AMP gel. Figure 1 c is the energy dispersive X-ray spectrum of Mn-AMP gel; Figure 2 Mn 2+ Isothermal titration curves with different ligands; Figure 3 The five isomers of Mn-AMP gel and their relative energies at the B3LYP-D3 (BJ) / 6-31G**-SDD theoretical level; Figure 4 a is the reaction phenomenon between different metal ions and AMP, Figure 4 b is Mn 2+ Reaction phenomena with different ligands; Figure 5 a is the absorption spectrum of Mn-AMP gel in TMB / H2O2 system, Figure 5 b is the electron paramagnetic resonance spectrum of Mn-AMP gel nanozyme catalyzing H2O2 to produce •OH; Figure 6 a is the absorption spectrum of the colorimetric biosensor detecting different concentrations of AA. Figure 6 b is Δ A 652 The linear relationship between AA concentration and the concentration range is 20-700 μM. Figure 6 c is the specificity test of colorimetric biosensor, Figure 6 d is stability test; Figure 7 A convenient method for AA detection based on TMB@Mn-AMP composite gel combined with smartphone application “Color Grab” was designed and its detection results. DETAILED DESCRIPTION

[0014] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the present invention is described in detail with reference to the following specific embodiments.

[0015] Example 1: Preparation of Mn-AMP coordination polymer gel material (1) Dissolve AMP and MnCl2·4H2O in water to prepare aqueous solutions of corresponding concentrations; (2) AMP (200 mM, 250 μL) and MnCl2·4H2O (200 mM, 250 μL) were added to HEPES buffer (100 mM, pH 7.4, 500 μL) at a concentration volume ratio of 1:1 and vortexed to mix; (3) Standing at 25°C for about 20 s to obtain a Mn-AMP coordination polymer gel; (4) The gel product was centrifuged at 12,000 rpm for 10 min, washed three times with ultrapure water, and freeze-dried. The obtained dry gel was used for subsequent characterization experiments.

[0016] The prepared Mn-AMP coordination polymer gel was characterized, mainly including scanning electron microscopy test and energy dispersive X-ray spectroscopy test, and the results are as follows: like Figure 1 a and b show scanning electron micrographs of Mn-AMP coordination polymer gels. It can be observed that the Mn-AMP gel exhibits a nanofiber morphology with a diameter of about 100-200 nm. These nanofibers aggregate and cross-link together to form a three-dimensional network structure. Figure 1 As shown in Figure c, it can be seen from the energy dispersive X-ray spectrum that C, N, O, P, and Mn elements are evenly distributed throughout the Mn-AMP gel, proving the successful preparation of the Mn-AMP coordination polymer gel.

[0017] The inventors selected different types of manganese salts, such as manganese nitrate, manganese acetate and manganese sulfate, and obtained similar coordination polymer gels.

[0018] Study on the synthesis mechanism of Mn-AMP coordination polymer gel materials.

[0019] The synthesis mechanism of the prepared Mn-AMP coordination polymer gel was investigated by isothermal titration calorimetry experiments and density functional theory calculations. The results are as follows: Figure 2 Mn 2+ Isothermal titration curves with different ligands. In the reaction between MnCl2·4H2O and four monophosphate nucleotides, the obtained Δ H and Δ S All of them are greater than 0, indicating that the binding of MnCl2·4H2O with the four monophosphate nucleotides is an endothermic and entropy-increasing process, and hydrophobic interaction is the main driving force of the reaction. S | > |Δ H |, so the effect of entropy increase is greater than enthalpy change. According to Δ G = Δ H - TΔ S , determines Δ G <0, indicating that the reaction is spontaneous and is an entropy-driven process. Comparison of the binding constants of the four groups of reactions K a , it was found that the binding constant of AMP titrated by MnCl2·4H2O was the largest, indicating that the binding force between the two was the strongest (Table 1).

[0020] Table 1 Comparison of thermodynamic constants of MnCl2·4H2O binding to different ligands

[0021] Density functional theory calculations were used to investigate the geometry of the Mn-AMP coordination polymer gel and the Mn 2+ The microscopic binding mechanism, including the specific binding site of AMP, will be further explored.

[0022] At the B3LYP-D3 (BJ) / 6-31G**-SDD theoretical level, the structure of AMP was first optimized, and then the Mn 2+ The possible action sites and binding forms of AMP are calculated, and several isomers of coordination polymers are formed after binding, and the configuration with the lowest energy, that is, the most stable configuration, is obtained. Purple represents Mn, orange represents P, red represents O, gray represents C, blue represents N, and white represents H. According to the calculation results, Figure 3As shown in the figure, the structure with the lowest interaction energy is used as the zero point, and the five configurations are arranged in order of energy from low to high, and the relative energy value of each configuration based on the zero point is calculated. Through calculation, type I conformation is the most stable among these conformations. In type I, the Mayer bond orders of Mn-O(23), Mn-N(30) and Mn-O(2) are 0.14, 0.36 and 0.25 respectively. The N3 position on the adenine of AMP (N(30) in the figure) and the O atom of P=O on the phosphate group (O(2) in the figure) show strong interaction with Mn 2+ In these isomers, the amino group on adenine does not coordinate with Mn 2+ This is because of the resonance effect between the amino group and the adenine ring. The rotation of the amino group destroys its p-π conjugated interaction with the adenine ring. The energy increased by this step cannot be absorbed by the chelated Mn 2+ In summary, in the formation process of Mn-AMP coordination polymer gel, Mn 2+ The interaction between O(23) and Mn is weak. N(30) and O(2) are the main 2+ The main coordination site of the interaction.

[0023] Example 2: Preparation of other transition metal coordination polymer gel materials AMP reacts with other transition metal ion salt solutions, such as Figure 4 As shown in a, Mn 2+ and / or Zn 2+ Reacts with AMP to form gel, other metal ions (Fe 3+ , Cu 2+ 、Co 2+ or Ni 2+ ) reacts with AMP to form a precipitate. 2+ No nanozyme activity, subsequent targeting of Mn 2+ The phenomenon of gel formation by reaction with AMP was studied. Pentose, adenine and phosphate groups are the basic components of AMP structure and are also potential binding sites for metal ions. Figure 4 b. MnCl2·4H2O reacts with other monophosphate nucleotides (GMP, CMP, TMP). MnCl2·4H2O reacts with GMP to generate a white precipitate, but no obvious phenomenon occurs when reacting with CMP or TMP, and no gel formation is observed. Therefore, it is preliminarily believed that adenine on AMP plays an important role in the formation of Mn-AMP coordination polymer gel.

[0024] MnCl2·4H2O reacted with ADP, ATP, Adenosine, and Na3PO4 respectively. There was no obvious phenomenon when MnCl2·4H2O reacted with adenine nucleoside, indicating that the phosphate group is indispensable in the formation of gel; but when MnCl2·4H2O reacted with Na3PO4, a white precipitate was produced, indicating that free PO4 3- No gel can be produced; no obvious phenomenon occurs when MnCl2·4H2O reacts with ADP or ATP, indicating that the ligand with a single phosphate group connected to a pentose is the most likely candidate for the MnCl2·4H2O ligand. 2+ Therefore, the adenine and phosphate groups in AMP play a vital role in the formation of Mn-AMP coordination polymer gel. 2+ The key ligand for gel formation.

[0025] Example 3: Study on the peroxidase-like activity of Mn-AMP coordination polymer gel material In this example, TMB is used as a substrate. When H2O2 is present, TMB undergoes an oxidation reaction under the catalysis of a peroxidase-like enzyme to generate a blue oxidation product oxTMB. The peroxidase-like activity of the Mn-AMP coordination polymer gel is tested using a spectrophotometer. The results are as follows: like Figure 5 As shown in a, the TMB alone, TMB + H2O2 and TMB + Mn-AMP gel solutions were almost colorless after the reaction, and there was no obvious absorption peak in the absorption spectrum, indicating that oxTMB was not generated during the reaction. During the reaction of TMB + H2O2 + Mn-AMP gel, the solution gradually turned dark blue, and a strong absorption peak appeared at 652 nm, indicating that oxTMB was generated, proving that the Mn-AMP coordination polymer gel has peroxidase-like activity. Figure 5 As shown in b, the electron paramagnetic resonance experiment proves that the Mn-AMP coordination polymer gel produces •OH in the TMB-H2O2 system, causing TMB to be oxidized to oxTMB, and the solution shows a color reaction from colorless to blue.

[0026] Example 4: Preparation of colorimetric biosensor for detecting ascorbic acid based on Mn-AMP coordination polymer gel nanozyme AMP (100 mM, 30 μL) and MnCl2·4H2O (100 mM, 30 μL) were placed in a 1.5 mL centrifuge tube cap and mixed by pipetting to prepare gel. NaAc-HAc buffer (100 mM, pH 4.0, 60 μL), TMB (20 mM, 20 μL) and H2O2 (10 mM, 20 μL) were added to the centrifuge tube and mixed. After adding 10 μL of AA solution, the centrifuge tube cap was closed and inverted to allow the solution in the centrifuge tube to contact the Mn-AMP gel in the cap for 20 min. The solution in the centrifuge tube was quickly transferred to a 96-well plate in turn, and the absorbance of the solution at 652 nm was recorded to calculate the concentration of AA. The experiment was performed in a dark environment throughout.

[0027] Based on the above steps, this colorimetric biosensor was combined with the smartphone application “Color Grab” to design a portable detection method. Firstly, TMB (40 mM, 10 μL), AMP (100 mM, 30 μL) and MnCl2·4H2O (100 mM, 30 μL) were mixed in a centrifuge tube cap by a one-pot method to prepare TMB@Mn-AMP composite gel. NaAc-HAc buffer (100 mM, pH 4.0, 80 μL) and H2O2 (10 mM, 20 μL) were added to the centrifuge tube. Finally, 10 μL of AA solution of different concentrations were added respectively. The centrifuge tube cap was closed and the centrifuge tube was inverted for 20 min to ensure that the solution in the centrifuge tube was in full contact with the TMB@Mn-AMP composite gel in the cap. The centrifuge tube caps containing the color gel were cut off in turn and arranged neatly in order from low to high AA concentration. The APP "Color Grab" was used to identify and generate the RGB value of each gel sample. After data analysis, a linear relationship was obtained with AA concentration as the horizontal axis and (G + B) / 2R as the vertical axis, thus establishing a (G + B) The relationship between / 2R and AA concentration enables convenient detection of AA. The experiment was performed in a dark environment. The results are as follows: like Figure 6 As shown in a and b, when the AA concentration is in the range of 20-700 μM, Δ A 652 and c AA Shows a good linear relationship: Δ A 652 = 9.1336 × 10 -4 c + 0.01522 ( R 2 = 0.9971), LOD = 4.10 μM, the colorimetric biosensor has a wide detection range and a low detection limit. Figure 6 c, d, showing excellent specificity and stability. The recovery rate obtained in the actual sample detection experiment is in line with the reasonable range of spiked recovery rate (95%-105%), with good applicability, and can accurately detect the AA content in two actual samples of tomatoes and lemons.

[0028] A portable detection method designed in conjunction with "Color Grab", such as Figure 7 As shown, the RGB values ​​of the samples are c AA It shows a good linear relationship, and the linear equation is (G + B) / 2R = - 0.0010 c AA +1.7475( R 2 = 0.9972), thus realizing the on-site rapid detection of AA.

[0029] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a Mn-AMP metal organic gel, characterized in that The following steps are involved: (1) preparing adenosine-5′ monophosphate AMP and a soluble manganese salt into an aqueous solution; (2) adding AMP and soluble manganese salt aqueous solution into HEPES buffer and mixing evenly; (3) A gel is formed upon standing at room temperature, and the Mn-AMP metal organic gel is obtained by washing and drying.

2. The method for preparing the Mn-AMP metal organic gel according to claim 1, characterized in that: In step (1), the soluble manganese salt is selected from manganese chloride, manganese nitrate, manganese acetate or manganese sulfate.

3. The method for preparing the Mn-AMP metal organic gel according to claim 1, characterized in that: In step (2), the molar ratio of AMP to soluble manganese salt is 1:(0.9-1.1).

4. The method for preparing the Mn-AMP metal organic gel according to claim 1, characterized in that: In step (3), the drying method is preferably freeze-drying.

5. The Mn-AMP metal organic gel prepared by the method according to any one of claims 1 to 4.

6. The Mn-AMP metal organic gel according to claim 5, characterized in that: The Mn-AMP metal organic gel has peroxidase-like activity.

7. Use of the colorimetric biosensor constructed with the Mn-AMP metal organic gel according to claim 5 in the detection of ascorbic acid.

8. The use according to claim 7, wherein the colorimetric biosensor is combined with a smartphone application "Color Grab" to achieve on-site visual portable detection.

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