FeCo Prussian blue derivative as well as preparation method and application thereof

Abstract

The invention provides a FeCo Prussian blue derivative as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing a stabilizer and potassium ferricyanide in an ethanol / water mixed solution, and then adding cobalt acetate in batches for reaction to prepare the FeCo Prussian blue derivative. The FeCo Prussian blue derivative prepared by the method is in a square block shape, is small in particle size and uniform in distribution, has oxidoid enzyme and peroxidase-like activity, and can discolor TMB, ABTS and OPD and generate a characteristic absorption peak; the invention also provides an ascorbic acid concentration detection method which comprises the following steps: mixing the color-changing substrate solution with the FeCo Prussian blue derivative, adding ascorbic acid with gradient concentration, detecting corresponding absorbance, constructing a linear relation between the absorbance and the ascorbic acid concentration, and detecting ascorbic acid with unknown concentration by using the constructed linear relation. The detection method is simple, easy to operate and high in speed.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a FeCo Prussian blue derivative and a preparation method and application thereof. Background Art

[0002] Ascorbic acid, or vitamin C, can act as an antioxidant and free radical scavenger to relieve oxidative stress in a variety of diseases and has important physiological functions. However, the human body cannot synthesize vitamin C directly, and vitamin C is mainly supplemented by consuming fresh fruits, vegetables or medicines. Therefore, determining the content of vitamin C in food and medicine is of scientific significance for evaluating the nutritional value of food, the quality of medicines, and guiding the supplementation of vitamin C through diet. In recent years, methods for detecting ascorbic acid have developed rapidly, mainly including titration analysis, photometry, electrochemical method, chemiluminescence method, flow injection method, liquid chromatography and atomic absorption indirect method. Among them, ultraviolet spectrophotometry has become the main method for laboratory quantitative analysis of ascorbic acid because the required instruments and equipment are relatively mature and have high accuracy.

[0003] Natural enzymes play an important role in biology and chemistry, and can specifically catalyze biochemical reactions and accelerate reaction rates. However, most natural enzymes are composed of proteins, so they have disadvantages such as high cost, poor storage conditions, poor stability, and easy inactivation. Therefore, the development of artificial enzymes that mimic natural enzymes has received widespread attention. Nanoenzyme materials including quantum dots, nanosheets, metal and oxide nanoparticles, metal organic frameworks (MOFs), etc., have been widely used in sensing, environmental protection, disease diagnosis and treatment (such as cancer treatment) due to their strong catalytic activity, excellent stability, and low production and storage costs. Prussian blue derivative (PBA) nanocubes are a well-studied MOF material with a chemical formula of A x T[M(CN)6]·nH2O (A=K, Li, Na; T=Co, Fe, Ni, Cu, etc.; M=Co, Fe, Mn, etc.). In recent years, the enzyme-mimicking properties of PBA have also been gradually developed, but not comprehensive enough. Unlike peroxidase, oxidase can catalyze specific substrates without exogenous H2O2, which can effectively avoid the shortcomings of H2O2 such as instability, high toxicity, volatility and self-decomposition. By utilizing the excellent oxidase-like activity of PBA, an effective detection method for reducing small molecules such as glutathione and ascorbic acid can be established, obtaining a new method for ascorbic acid detection that is simpler, more efficient, more sensitive and less costly. Summary of the invention

[0004] The invention provides a FeCo Prussian blue derivative and a preparation method and application thereof, aiming to provide a FeCo Prussian blue derivative which is small in size, uniform in distribution, and has oxidase-like activity and peroxidase-like activity, and to provide a simpler, more efficient, rapid and sensitive ascorbic acid detection method.

[0005] In order to achieve the above technical effects, the technical solution provided by the present invention includes:

[0006] The invention provides a method for preparing a FeCo Prussian blue derivative, comprising first dissolving and mixing a stabilizer and potassium ferrocyanide in a mixed solution of ethanol / water, then adding cobalt acetate in batches to form a reaction system for reaction, and preparing the FeCo Prussian blue derivative;

[0007] The FeCo Prussian blue derivative has Fe II and Fe III 、Co II and Co III The mixed valence state provides a redox potential, so that the FeCo Prussian blue derivative has oxidase-like activity and peroxidase-like activity.

[0008] Furthermore, the addition rate of the cobalt acetate solution is 1-2 mL / min.

[0009] Furthermore, the molar ratio of potassium ferrocyanide to cobalt acetate is 5:4-4:5.

[0010] Furthermore, after adding cobalt acetate, the mixture is stirred at 20-60° C. for 12-48 hours to carry out the reaction.

[0011] Further, when the reaction temperature is 15-25°C, the size of the FeCo Prussian blue derivative is 144.3±33.73 nm; and / or, when the reaction temperature is 35-45°C, the size of the FeCo Prussian blue derivative is 226.3±55.68 nm; and / or, when the reaction temperature is 55-65°C, the size of the FeCo Prussian blue derivative is 285.1±87.91 nm.

[0012] Furthermore, the stabilizer includes any one of polyvinyl pyrrolidone, trisodium citrate, bovine serum albumin, and polyethylene glycol.

[0013] Furthermore, the ethanol / water mixture is prepared by mixing ethanol and water in a volume ratio of 1:3-4:1.

[0014] Furthermore, when the ethanol content in the ethanol / water mixed solution in which the stabilizer and potassium ferrocyanide are dissolved increases, the particle size of the FeCo Prussian blue derivative decreases.

[0015] Furthermore, the amount of the stabilizer added to every 30 mL of the reaction system is 0.1-0.5 g.

[0016] The FeCo Prussian blue derivative is prepared by the preparation method provided by the invention.

[0017] Furthermore, the FeCo Prussian blue derivative has oxidase-like activity and peroxidase-like activity. The FeCo Prussian blue derivative can catalyze H2O2 to produce hydroxyl radicals, so that the absorbance value of 3,3',5,5'-tetramethylbenzidine (TMB) at 652nm increases with the increase of H2O2 concentration.

[0018] Furthermore, the FeCo Prussian blue derivative can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), so that 3,3',5,5'-tetramethylbenzidine (TMB) changes from colorless to blue.

[0019] Furthermore, the FeCo Prussian blue derivative can catalyze the oxidation of 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS), so that 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS) changes from colorless to green.

[0020] Furthermore, the FeCo Prussian blue derivative can catalyze the oxidation of o-phenylenediamine (OPD), so that o-phenylenediamine (OPD) changes from colorless to orange.

[0021] Furthermore, the particle size of the FeCo Prussian blue derivative is 100-600 nm.

[0022] The present invention also provides a method for detecting ascorbic acid, comprising: mixing the FeCo Prussian blue derivative prepared by the preparation method provided by the present invention with a color-changing substrate solution to form a detection system, adding different amounts of ascorbic acid, detecting corresponding absorbances, constructing a linear relationship between absorbance and ascorbic acid concentration, adding ascorbic acid to be detected to the detection system with a known linear relationship, detecting the absorbance of the color-changing substrate at a specific position by ultraviolet light, and detecting the concentration of the ascorbic acid to be detected in combination with the constructed linear relationship.

[0023] Furthermore, the detection method is carried out at 22-26°C.

[0024] Furthermore, before the detection, the concentration of the FeCo Prussian blue derivative in the detection system was 20 μg / mL, and the concentration of the 3,3′,5,5′-tetramethylbenzidine (TMB) was 0.5 mM.

[0025] Furthermore, before the detection, the pH of the system was adjusted to 5 using a citric acid-sodium citrate buffer solution.

[0026] Furthermore, the concentrations of ascorbic acid added to the detection system were 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30 and 35 μM, respectively, and the linear relationship was A=0.0282*C-0.009, with a linear correlation coefficient R 2 is 0.9951, where A is the absorbance of 3,3′,5,5′-tetramethylbenzidine (TMB) detected by ultraviolet light at 652 nm, and C is the concentration of ascorbic acid to be measured.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The FeCo Prussian blue derivative prepared by the preparation method provided by the present invention is small in size, in the shape of nano-scale blocks, and is evenly dispersed.

[0029] 2. The FeCo Prussian blue derivative prepared by the preparation method provided by the present invention has oxidase-like activity and peroxidase-like activity, can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2-azo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS) and o-phenylenediamine (OPD), and cause them to change color, and can catalyze H2O2 to produce a large number of hydroxyl radicals.

[0030] 3. The ascorbic acid detection method provided by the present invention can quickly and accurately detect the ascorbic acid concentration, is simple to operate, and the minimum ascorbic acid concentration detected is 0.3 μM.

[0031] 4. The method for preparing the FeCo Prussian blue derivative provided by the present invention has a simple process and readily available raw materials.

[0032] 5. The method for preparing the FeCo Prussian blue derivative provided by the present invention can control the particle size of the FeCo Prussian blue derivative by the reaction temperature, and control the morphology of the FeCo Prussian blue derivative by controlling the volume ratio of ethanol / water. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 It is a schematic diagram of the enzyme properties of the FeCo Prussian blue derivative prepared in Example 1 of the present invention and the detection of ascorbic acid.

[0035] Figure 2 This is a TEM image of the FeCo Prussian blue derivative prepared in Example 1 of the present invention.

[0036] Figure 3 It is a TEM comparison picture of the FeCo Prussian blue derivatives prepared in Examples 1-4 of the present invention in mixed solutions with ethanol / water volume ratios of 1:3, 1:1, 3:1, and 4:1, respectively.

[0037] Figure 4 TEM images of FeCo Prussian blue derivatives prepared in Example 1 and Examples 5-6 of the present invention at temperatures of 20° C., 40° C. and 60° C.

[0038] Figure 5 It is a comparison chart of X-ray diffraction spectra of the FeCo Prussian blue derivative and Prussian blue nanoparticles prepared in Example 1 of the present invention.

[0039] Figure 6 It is a high-resolution C1s, N1s, Co2p and Fe2p photoelectron spectrum of the FeCo Prussian derivative prepared in Example 1 of the present invention.

[0040] Figure 7 The oxidase-like activity of the FeCo Prussian blue derivative prepared in Example 1 of the present invention at different pH values.

[0041] Figure 8 It is an ultraviolet spectrum diagram of the FeCo Prussian blue derivative prepared in Example 1 of the present invention catalyzing TMB, ABTS and OPD in a buffer solution with a pH of 5 to generate characteristic peaks.

[0042] Fig. 9 The photoelectron spectrum of the FeCo Prussian blue derivative prepared in Example 1 of the present invention catalyzing H2O2 to generate hydroxyl radicals using dimethylpyridine N-oxide electron scavenger (DMPO) as a scavenger for hydroxyl radicals.

[0043] Fig.10 It is an ultraviolet spectrum diagram showing that the FeCo Prussian blue derivative prepared in Example 1 of the present invention exerts peroxidase-like activity and catalyzes different concentrations of H2O2 to generate hydroxyl radicals to change the characteristic absorption peak of the substrate TMB.

[0044] Fig.11A It is a UV absorption spectrum of the FeCo Prussian blue derivative prepared in Example 1 of the present invention for detecting ascorbic acid.

[0045] Fig. 11B It is a standard curve diagram of ascorbic acid detected by the FeCo Prussian blue derivative prepared in Example 1 of the present invention.

[0046] Fig.12 It is a TEM image of the product obtained in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0047] The present invention will be more fully understood through the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are only exemplary of the present invention, which can be embodied in various forms. Therefore, the specific functional details disclosed herein should not be interpreted as limiting, but only as the basis of the claims and as a representative basis for teaching those skilled in the art to adopt the present invention in different ways in virtually any appropriate detailed embodiment.

[0048] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative in all aspects and are not intended to limit the present invention, the scope of the present invention is defined only by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0049] Example 1

[0050] This embodiment provides a FeCo Prussian blue derivative, specifically, the preparation method includes:

[0051] 0.3g polyvinylpyrrolidone (PVP) K30 and 0.04mmol potassium ferrocyanide were dissolved in 20mL of ethanol / water mixed solution with a volume ratio of 1:3, and stirred by magnetic force to form a transparent yellow solution; 0.05mmol cobalt acetate was dissolved in 10mL water, and the cobalt acetate solution was added to the potassium ferrocyanide / PVP solution at a drop rate of 1mL / min through a constant pressure dropping funnel, stirred at 20℃ for 24h, centrifuged at 14500rpm for 15min, and washed with distilled water 3 times to obtain FeCo Prussian blue derivative nanocubes. Finally, the FeCo Prussian blue derivative nanocube particles were dispersed in ultrapure water and stored in a refrigerator at 4℃ for use.

[0052] Example 2

[0053] The only difference between this embodiment and embodiment 1 is that the volume ratio of ethanol / water for dissolving PVP and potassium ferrocyanide is 1:1, and the other conditions are consistent with those in embodiment 1.

[0054] Example 3

[0055] The only difference between this embodiment and embodiment 1 is that the volume ratio of ethanol / water for dissolving PVP and potassium ferrocyanide is 3:1, and the other conditions are consistent with those in embodiment 1.

[0056] Example 4

[0057] The only difference between this embodiment and embodiment 1 is that the volume ratio of ethanol / water for dissolving PVP and potassium ferrocyanide is 4:1, and the other conditions are consistent with those in embodiment 1.

[0058] Example 5

[0059] The only difference between this embodiment and embodiment 1 is that the reaction temperature is 40° C., and the other conditions are the same as those in embodiment 1.

[0060] Example 6

[0061] The only difference between this embodiment and embodiment 1 is that the reaction temperature is 60° C., and the other conditions are the same as those in embodiment 1.

[0062] Example 7

[0063] The only difference between this embodiment and embodiment 1 is that, in the reaction system, there are 0.05 mmol of potassium ferrocyanide and 0.04 mmol of cobalt acetate, and the molar ratio of potassium ferrocyanide to cobalt acetate is 5:4. The other conditions are the same as those in embodiment 1.

[0064] Example 8

[0065] The only difference between this embodiment and embodiment 1 is that the reaction system is stirred at 20° C. for 12 h, and the other conditions are the same as those in embodiment 1.

[0066] Example 9

[0067] The only difference between this embodiment and embodiment 1 is that the reaction system is stirred at 20° C. for 48 h, and the other conditions are the same as those in embodiment 1.

[0068] Example 10

[0069] The only difference between this embodiment and embodiment 1 is that 0.1 g of sodium citrate as a stabilizer is added, and the other conditions remain the same as those of embodiment 1.

[0070] Embodiment 11

[0071] The only difference between this embodiment and embodiment 1 is that 0.5 g of bovine serum albumin as a stabilizer is added, and the other conditions remain the same as those in embodiment 1.

[0072] Example 12

[0073] The only difference between this embodiment and embodiment 1 is that the cobalt acetate solution is added dropwise at a dropping rate of 2 mL / min through a constant pressure dropping funnel to carry out the reaction, and the other conditions are the same as those in embodiment 1.

[0074] Comparative Example 1

[0075] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, polyvinyl pyrrolidone (PVP) K30, potassium ferrocyanide and cobalt acetate are added to the ethanol / water mixed solution at the same time, and the other conditions are consistent with those in Example 1.

[0076] See also Figure 2 , is a TEM image of the FeCo Prussian blue derivative prepared in Example 1 of the present invention, Fig.12 This is a TEM image of the product obtained in Comparative Example 1 of the present application. Compared with the sample in which PVP, potassium ferrocyanide and cobalt acetate are directly mixed, the sample added at a rate of 1 mL / min shows better dispersibility and more uniform morphology.

[0077] The morphologies of the FeCo Prussian blue derivatives prepared in Examples 1-4 at different volume ratios of ethanol / water solution and in Examples 5-6 at different reaction temperatures were observed under a transmission electron microscope. Figure 3 , Figure 4 , Figure 3 Figures AD are TEM images of FeCo Prussian blue derivatives prepared with ethanol / water volume ratios of 1:3, 1:1, 3:1, and 4:1, respectively. It can be seen in the figure that the particle size of the FeCo Prussian blue derivative is 100-600nm. The ethanol in the mixed solvent affects the conformational change of the PVP molecule by controlling the polymer-polymer interaction, polymer-water interaction, and polymer-ethanol interaction. This conformational change further affects the structural morphology and size of the FeCo Prussian blue derivative. When the ethanol content in the ethanol / water mixture increases, the particle size of the FeCo Prussian blue derivative decreases.

[0078] Depend on Figure 3 It can be seen that the FeCo Prussian blue derivative prepared in the ethanol / water mixed solution with a volume ratio of 1:3 has the most obvious block structure, more uniform morphology and particle size distribution of the Prussian blue derivative. Preferably, the particle size of the FeCo Prussian blue derivative does not exceed 150 nm.

[0079] See also Figure 4 , which are TEM images of FeCo Prussian blue derivatives prepared in Example 1 (a: 20°C), Example 5 (b: 40°C) and Example 6 (c: 60°C) of the present invention. Different reaction temperatures have an effect on the morphology of FeCo Prussian blue derivatives. Figure 4 It can be seen that with the increase of reaction temperature, the size of the obtained FeCo Prussian blue derivative gradually increases, while still maintaining good morphology and dispersibility. The size ranges of FeCo prepared at 20, 40 and 60 °C measured by dynamic light scattering are 144.3±33.73nm, 226.3±55.68nm and 285.1±87.91nm, respectively.

[0080] See also Figure 5 , is a comparison diagram of the X-ray diffraction spectra of the FeCo Prussian blue derivative and Prussian blue nanoparticles prepared in Example 1 of the present invention. Compared with the XRD spectrum of the classic Prussian blue nanoparticles, the characteristic peak of FeCo shifts to a small angle, which means that the doping of heteroatoms larger than the main body (Fe), Co atoms, increases the unit cell parameters and the interplanar spacing.

[0081] See also Figure 6 , which is a high-resolution C1s, N1s, Co2p and Fe2p spectrum of the photoelectron spectrum of the FeCo Prussian blue derivative prepared in Example 1 of the present invention. In the Fe2p spectrum, Fe 2+ and Fe 3+ The Co2P spectrum also shows the coexistence of the +II and +III oxidation states of Co, and a single peak at 396.7 eV belonging to CN is observed in the N1s spectrum. It can be inferred that the mixed valence state of Fe and Co in FeCo provides a variable redox potential, with the chemical formula K 0.18 Co 1.55 [Fe 1.31 (CN)6], the atomic ratio of Fe and Co is about 1:1.2, which makes FeCo likely to have multiple types of enzyme activities.

[0082] Performance Characterization

[0083] Detection of oxidase activity of FeCo Prussian blue derivatives

[0084] 10.2 mg of 3,3'5,5'-tetramethylbenzidine was dissolved in 5 mL of DMSO and ultrasonicated for 3 minutes to obtain a 10.0 mM TMB solution.

[0085] Weigh 2.1013 g of citric acid (C6H8O7·H2O), dissolve it in ultrapure water and make up to 100 mL. Then weigh 2.9421 g of sodium citrate (Na3C6H5O7·2H2O), dissolve it in ultrapure water and make up to 100 mL to obtain 0.1 M citric acid and 0.1 M sodium citrate solutions, respectively. Citric acid-sodium citrate buffer solutions with pH values ​​of 3, 4, 5, and 6 were prepared, respectively.

[0086] Measure 16.6 mL of 85% phosphoric acid and add water to make up to 1000 mL as liquid A, weigh 71.63 g of disodium hydrogen phosphate, add water to dissolve it to 1000 mL as liquid B; take 14.5 mL of liquid A and mix it with 5.5 mL of liquid B to prepare a phosphoric acid-sodium dihydrogen phosphate buffer solution with a pH of 2.

[0087] Weigh 27.6 g of NaH2PO4·H2O, dissolve it in water and make up to 1000 mL as liquid C, weigh 53.6 g of Na2HPO4·7H2O, dissolve it in water and make up to 1000 mL as liquid D; mix 7.8 mL of liquid C with 12.2 mL of liquid D to prepare a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7; mix 1.06 mL of liquid C with 18.94 mL of liquid D to prepare a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 8.

[0088] At 25°C, 100uL of 10mM TMB and a certain amount of FeCo Prussian blue derivative aqueous dispersion prepared in Example 1 were added to 900μL of different buffer solutions of pH=2, pH=3, pH=4, pH=5, pH=6, pH=7 and pH=8, respectively, so that the concentration of TMB in each sample was 1mM and the concentration of FeCo was 20μg / mL. After mixing for 2 minutes, the absorbance of the blue mixed solution at 652nm was measured by UV-visible spectrophotometer to obtain the optimal pH value for the oxidase activity of FeCo Prussian blue derivative. Figure 7 When TMB was used as substrate, the FeCo Prussian blue derivative showed the best oxidase-like activity in a buffer solution of pH 5.

[0089] Prepare 10mM stock solutions of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS) and o-phenylenediamine (OPD). Dilute TMB, ABTS and OPD to 1mM with a pH 5 citric acid-sodium citrate buffer solution, add 40μg / mL FeCo Prussian blue derivative aqueous dispersion, and measure the UV absorption spectra of TMB, ABTS and OPD in the range of 400-800nm ​​before and after the addition of FeCo using a UV-visible spectrophotometer. Figure 8 As shown, FeCo Prussian blue derivatives can catalyze the oxidation of TMB, ABTS and OPD, and the colors of the colors are changed from colorless to blue, green and orange, respectively, and characteristic absorption peaks are generated.

[0090] Detection of Peroxidase-like Activity of FeCo Prussian Blue Derivatives

[0091] FeCo Prussian blue derivative can catalyze H2O2 to oxidize substrate to produce color reaction. At 25°C, the FeCo Prussian blue derivative prepared in Example 1, TMB and H2O2 of different concentrations of 5, 25 and 50 mM were added to a citric acid-sodium citrate buffer solution with a pH of 5, mixed for 30 minutes, and then the ultraviolet absorption spectrum of TMB in the range of 400-800 nm before and after the addition of FeCo Prussian blue derivative and H2O2 was measured by a UV-visible spectrophotometer. Fig. 9As shown in the figure, when DMPO is used as the scavenger of hydroxyl radicals, the FeCo Prussian blue derivative can catalyze H2O2 to produce hydroxyl radicals of different intensities under light irradiation of 1, 2 and 3 min, proving the peroxidase-like activity of the FeCo Prussian blue derivative. Fig.10 As shown, when TMB is used as a substrate, the hydroxyl radicals generated by H2O2 catalyzed by the FeCo Prussian blue derivative can cause TMB to further change its color. As the concentration of H2O2 increases, the characteristic absorption peak of TMB at 652nm also increases.

[0092] The products obtained in the other embodiments all showed nanoscale block-like morphology and were evenly dispersed. The enzyme-like activities thereof were detected by the same detection method, and all had oxidase-like activity and peroxidase-like activity, and were capable of catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS) and o-phenylenediamine (OPD), and causing them to change color, and were capable of catalyzing H2O2 to produce a large number of hydroxyl radicals.

[0093] Method for detecting ascorbic acid using FeCo Prussian blue derivatives

[0094] TMB, FeCo Prussian blue derivative aqueous dispersion and different volumes of ascorbic acid solution were added to 12 groups of citric acid-sodium citrate buffer solutions with a pH of 5 at 25°C, so that the total volume of each sample was 1 mL, in which the final concentration of TMB was 0.5 mM, the concentration of FeCo was 20 μg / mL, and the concentrations of ascorbic acid were 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30 and 35 μM, respectively. Due to the oxidase-like activity of the FeCo Prussian blue derivative, TMB was oxidized to generate blue TMB imide salt, and the mixed system turned blue. When different amounts of ascorbic acid were added to the mixed system, the color of the solution gradually faded. After mixing for 5 minutes, the UV absorption spectrum of each sample in the range of 400-800nm ​​was measured by UV-visible spectrophotometer; and the absorbance value of each sample at 652nm was used to construct a linear relationship between absorbance and the final concentration of ascorbic acid. The constructed linear relationship was used to detect unknown concentrations of ascorbic acid.

[0095] After adding ascorbic acid solution, the blue color of FeCo / TMB system gradually becomes lighter, and the degree of lightening is positively correlated with the amount of ascorbic acid added. Fig.11A and Fig. 11B, within a certain concentration range of ascorbic acid, the absorbance of the solution is linearly related to the concentration of ascorbic acid added, and within the concentration range of 0.5-35μM, the ascorbic acid concentration is linearly related to the absorbance of the mixed solution. The linear equation A = 0.0282*C-0.009, where A is the absorbance, C is the concentration of ascorbic acid added, and the linear correlation coefficient R 2 The detection limit of ascorbic acid is as low as 0.3 μM.

[0096] The present invention provides a method for detecting ascorbic acid by utilizing the enzyme activity of the FeCo Prussian blue derivative prepared by the present invention. The principle of the method mainly includes: because the FeCo Prussian blue derivative has oxidase-like activity, it can catalyze the solution oxygen to oxidize TMB, ABTS and OPD to produce color changes, and produce characteristic absorption peaks at 652nm, 734nm and 450nm. When TMB is used as a substrate, when the reducing biological molecule ascorbic acid is introduced into TMB, due to the reducing effect of ascorbic acid, the blue TMB imide salt (TMBox) generated by FeCo oxidation in the system undergoes a reduction reaction to generate colorless TMB, see Figure 1 Therefore, as the concentration of biological reducing molecules such as ascorbic acid increases, the blue color in the system gradually becomes lighter. Within a certain concentration range, the degree of lightening of the blue color in the system is proportional to the concentration of ascorbic acid. From this, a linear equation can be established to obtain a method for detecting ascorbic acid.

[0097] This detection method is simple to operate and can quickly obtain the ascorbic acid concentration in the sample, with a detection limit as low as 0.3 μM.

[0098] See Table 1 for a comparison of the linear range and detection limit of different nanozymes:

[0099] Table 1 Comparison of linear range and gun detection limit of different nanozyme guns

[0100]

[0101] Compared with most other oxidase mimetics, the detection method based on the FeCo Prussian blue derivative-like oxidase activity provided by the present invention has a comparable or even lower detection limit for ascorbic acid.

Claims

1. A method for preparing a FeCo Prussian blue derivative, characterized in that: The method comprises first dissolving and mixing a stabilizer and potassium ferrocyanide in a mixed solution of ethanol / water, and then adding cobalt acetate in batches to form a reaction system for reaction to obtain the FeCo Prussian blue derivative; The FeCo Prussian blue derivative has Fe II and Fe III 、Co II and Co III The mixed valence state provides a redox potential, so that the FeCo Prussian blue derivative has oxidase-like activity and peroxidase-like activity.

2. The method for preparing the FeCo Prussian blue derivative according to claim 1, characterized in that include: Firstly, the stabilizer and potassium ferrocyanide are dissolved and mixed in a mixture of ethanol / water, and then the cobalt acetate solution is added in batches at an addition rate of 1-2 mL / min, and the mixture is mixed to form a reaction system for reaction to obtain the FeCo Prussian blue derivative.

3. The method for preparing the FeCo Prussian blue derivative according to claim 1, characterized in that: The molar ratio of potassium ferrocyanide to cobalt acetate is 5:4-4:5; And / or, the stabilizer includes any one of polyvinyl pyrrolidone, trisodium citrate, bovine serum albumin, and polyethylene glycol; and / or, adding cobalt acetate in batches to a stabilizer and potassium ferrocyanide in a mixture of ethanol / water to react, including stirring at 20-60° C. for 12-48 hours to react after adding cobalt acetate; And / or, the ethanol / water mixed solution in which the stabilizer and potassium ferrocyanide are dissolved is prepared by mixing ethanol and water in a volume ratio of 1:3-4:

1.

4. The method for preparing the FeCo Prussian blue derivative according to claim 3, characterized in that: When the reaction temperature is 20-25°C, the size of the FeCo Prussian blue derivative is 144.3±33.73nm; and / or, when the reaction temperature is 35-45° C., the size of the FeCo Prussian blue derivative is 226.3±55.68 nm; And / or, when the reaction temperature is 55-60° C., the size of the FeCo Prussian blue derivative is 285.1±87.91 nm.

5. The method for preparing the FeCo Prussian blue derivative according to claim 3, characterized in that: When the ethanol content in the ethanol / water mixture containing the dissolved stabilizer and potassium ferrocyanide increases, the particle size of the FeCo Prussian blue derivative decreases; And / or, the amount of the stabilizer added to every 30 mL of the reaction system is 0.1-0.5 g.

6. A FeCo Prussian blue derivative, characterized in that: The method is prepared according to any one of claims 1 to 5.

7. The FeCo Prussian blue derivative according to claim 6, characterized in that: The FeCo Prussian blue derivative has oxidase-like activity, and can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine and / or 2,2-azo-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt and / or o-phenylenediamine to produce color change. The FeCo Prussian blue derivative has peroxidase-like activity, and can catalyze H2O2 to produce hydroxyl radicals. Preferably, the FeCo Prussian blue derivative can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine, so that 3,3′,5,5′-tetramethylbenzidine changes from colorless to blue, and produces a characteristic absorption peak at 652 nm; And / or, the FeCo Prussian blue derivative can catalyze the oxidation of 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt, so that the 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt changes from colorless to green, and produces a characteristic absorption peak at 734 nm; And / or, the FeCo Prussian blue derivative can catalyze the oxidation of o-phenylenediamine, causing o-phenylenediamine to change from colorless to orange, and generate a characteristic absorption peak at 450 nm.

8. Use of the FeCo Prussian blue derivative as claimed in claim 6 or 7 in the detection of ascorbic acid.

9. A method for detecting ascorbic acid, characterized in that: The method comprises mixing the FeCo Prussian blue derivative according to claim 6 or 7 with a color-changing substrate solution to form a detection system, adding ascorbic acid to the detection system, detecting the corresponding absorbance, constructing a linear relationship between absorbance and ascorbic acid concentration, adding ascorbic acid to be tested to the detection system with a known linear relationship, and detecting the absorbance of the color-changing substrate solution in combination with the constructed linear relationship to detect the concentration of the ascorbic acid to be tested; Wherein, the color-changing substrate solution includes any one of 3,3′,5,5′-tetramethylbenzidine solution, 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt solution, and o-phenylenediamine solution.

10. The method for detecting ascorbic acid according to claim 9, characterized in that: The detection method is carried out at 22-26°C and pH 3-7; Utilizing 3,3′,5,5′-tetramethylbenzidine / or 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt / or o-phenylenediamine to produce color change, adding gradient concentrations of ascorbic acid solutions to the detection system, respectively, and detecting the absorbance of 3,3′,5,5′-tetramethylbenzidine at 652 nm, or the absorbance of 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diamine salt at 734 nm, or the absorbance of o-phenylenediamine at 450 nm by ultraviolet light to establish a linear relationship, and finally obtaining the concentration of ascorbic acid to be measured; And / or, the concentration of ascorbic acid is at least 0.3 μM.

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