Application of Cobalt Tetraoxide Nanocages as Dual Enzyme Mimics in Biosensors

Abstract

The present invention proposes an application of a cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor. The cobalt tetroxide nanocage of the present invention is both a peroxidase mimetic enzyme and an oxidase mimetic enzyme. The cobalt tetroxide nanocage has both the activity of peroxidase and the activity of oxidase. The cobalt tetroxide nanocage is used to measure the concentrations of hydrogen peroxide, ascorbic acid and acid phosphatase. The cobalt tetroxide nanocage of the present invention is a dual mimetic enzyme of a peroxidase mimetic enzyme and an oxidase mimetic enzyme. When used in a biosensor, it not only realizes the rapid detection of the concentration of hydrogen peroxide, but also realizes the rapid detection of the concentration of ascorbic acid, and also realizes the indirect detection of the concentration of acid phosphatase. It has high sensitivity, good stability and reusability, and is widely used. The cobalt tetroxide nanocage is a hollow cage structure, has a large internal space and a high specific surface area, and effectively increases the contact reaction area with the substrate.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme simulation, in particular to the application of a cobalt tetroxide nanocage as a dual enzyme simulation in a biosensor. Background Art

[0002] Nanozymes are nanomaterials with enzyme-like activity. Compared with natural enzymes, they have the advantages of simple synthesis, controllable cost, stable storage, strong catalytic performance, large specific surface area, easy modification, and integration with biological agents. To date, more and more nanomaterials with different enzyme-like activities have been discovered, and they have been shown to mimic natural enzymes such as peroxidase, oxidase, and superoxide dismutase. These nanozymes can integrate multiple reaction steps together, making the detection process more convenient and efficient, and have great advantages and broad application prospects.

[0003] Some nanomaterials that simulate enzymes have also appeared in the prior art. For example, Cao et al. obtained cerium oxide nanomaterials by calcining the precursor Ce-MOFs. This cerium oxide nanomaterial exhibited good oxidase-like activity (Cao et al., Chem. Mater. 30 (2018) 7831-7839); in addition, Li et al. synthesized cobalt-nitrogen co-doped hierarchical porous carbon (Co, N-HPC) nanoparticles by pyrolysis of dimethylimidazole cobalt (ZIF-67). The nanoparticles have a multilayer structure and a large specific surface area, and have high oxidase-like activity (Li et al., Sens. Actuators B Chem. 264 (2018) 312-319). However, these nanomaterials simulate enzymes only have single enzyme activity, which limits their application. Moreover, this nanomaterial simulates enzyme activity is low, the stability is poor, and the sensitivity is low. Summary of the invention

[0004] The purpose of the present invention is to provide a cobalt oxide nanocage for use as a dual enzyme mimic in a biosensor, aiming to solve the problem that cerium oxide nanomaterials and cobalt-nitrogen co-doped graded porous carbon nanoparticles used as nanomaterial enzyme mimics in the prior art have only a single enzyme activity, which limits their application and has low activity, poor stability and low sensitivity.

[0005] In order to solve the above technical problems, the technical solution of the present invention is achieved as follows:

[0006] The invention discloses an application of a cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor. The cobalt tetroxide nanocage is both a peroxidase mimetic enzyme and an oxidase mimetic enzyme. The cobalt tetroxide nanocage has both peroxidase activity and oxidase activity. The cobalt tetroxide nanocage is used to measure the concentrations of hydrogen peroxide, ascorbic acid and acid phosphatase.

[0007] The cobalt tetroxide nanocage of the present invention is a dual mimetic enzyme of a peroxidase mimetic enzyme and an oxide mimetic enzyme. When used in a biosensor, it not only realizes the rapid detection of the concentration of hydrogen peroxide, but also realizes the rapid detection of the concentration of ascorbic acid, and also realizes the indirect detection of the concentration of acid phosphatase. It has high detection efficiency, high sensitivity, good stability and reusability, and is widely used. The cobalt tetroxide nanocage is a hollow cage-like structure, has a large internal space and a high specific surface area, and effectively increases its contact reaction area with the substrate.

[0008] As a preferred embodiment, the detection limit of the cobalt tetroxide nanocage for determining the concentration of hydrogen peroxide is 0.0046 μM, the detection limit of the cobalt tetroxide nanocage for determining the concentration of ascorbic acid is 0.15 μM, and the detection limit of the cobalt tetroxide nanocage for determining the concentration of acid phosphatase is 0.0068 mU / mL. In the present invention, the cobalt tetroxide (Co3O4) nanocage acts as a peroxidase that catalyzes the redox reaction of the substrate, thereby realizing the rapid detection of the concentration of hydrogen peroxide (H2O2). The cobalt tetroxide (Co3O4) nanocage also acts as an oxidase that catalyzes the redox reaction of the substrate, thereby realizing the rapid detection of the concentration of ascorbic acid (AA), and realizing the indirect detection of the concentration of acid phosphatase (ACP), and is widely used; the detection limits of the hydrogen peroxide concentration, ascorbic acid concentration and acid phosphatase concentration are low, the sensitivity is high, and the detection range is wide.

[0009] As a preferred embodiment, when the cobalt oxide nanocage measures the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, hydrogen peroxide, ascorbic acid and acid phosphatase are the test objects, and the molar ratio of the cobalt oxide nanocage to the test object is 0.3-0.5: 1. In the present invention, the cobalt oxide nanocage is used as a dual mimetic enzyme of peroxide mimetic enzyme and oxide mimetic enzyme, and when measuring the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, its dosage is small, the cost is low, and the utilization value is high.

[0010] As a preferred embodiment, when the cobalt oxide nanocage measures the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, 3,3',5,5'-tetramethylbenzidine is used as a color developer, and the molar ratio of the color developer to the substance to be measured is 0.7-0.9: 1. In the present invention, the cobalt oxide nanocage is used as a dual mimetic enzyme of a peroxidase mimetic enzyme and an oxide mimetic enzyme. When measuring the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, the substances to be measured (hydrogen peroxide, ascorbic acid and acid phosphatase) and the color developer are called substrates, and 3,3',5,5'-tetramethylbenzidine is used as a color developer. The reaction system changes from colorless to blue, and the color change is obvious, which is convenient for observation.

[0011] As a preferred embodiment, when the cobalt oxide nanocage measures the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, a phosphate buffer is used as the buffer solution, the pH value of the buffer solution is 4.0, and the molar ratio of the buffer solution to the test object is 28-32: 1. In the present invention, a phosphate buffer is used as the buffer solution, and the molar concentration of the phosphate buffer is usually 50mmol / L. This phosphate buffer is easy to prepare, has a fast dissolution rate, high solubility, and good use effect.

[0012] As a preferred embodiment, the particle size of the cobalt oxide nanocage is 800-900nm. In the present invention, the cobalt oxide nanocage is a black powder solid with a small particle size. It is a nanoparticle with a hollow cage structure, a large internal space and a high specific surface area, which effectively increases its contact reaction area with the substrate.

[0013] As a preferred embodiment, the preparation method of the cobalt tetroxide nanocage is: 1) taking cobalt nitrate, adding it to methanol, stirring evenly, to obtain a cobalt nitrate methanol solution, the mass concentration of the cobalt nitrate methanol solution is 19-20 mg / mL; 2) taking 2-methylimidazole, adding it to methanol, stirring evenly, to obtain a 2-methylimidazole methanol solution, the mass concentration of the 2-methylimidazole methanol solution is 98-99 mg / mL; 3) mixing the cobalt nitrate methanol solution obtained in step 1) and the 2-methylimidazole methanol solution obtained in step 2), the mass ratio of cobalt nitrate to 2-methylimidazole is 3-5:2, and mixing them in a sealed manner at room temperature for 20-30 hours to obtain a precursor; 4) centrifuging the precursor obtained in step 3), washing, drying, calcining at 350-450° C. for 20-40 minutes under the action of an inert gas, and keeping warm for 20-40 minutes in an air atmosphere to obtain a cobalt tetroxide nanocage.

[0014] The cobalt tetroxide nanocage of the invention is obtained by a coprecipitation and calcination method. In the structure of the cobalt tetroxide nanocage, the molar ratio of the cobalt (Co) element to the oxygen (O) element is 3:4. The preparation process of the cobalt tetroxide nanocage is simple, easy to control, low reaction temperature, low energy consumption, short reaction time, high production efficiency, low cost, and has wide application in biological analysis and drug research.

[0015] As a preferred embodiment, in step 4), washing is performed with methanol for 3-5 times. In the present invention, the precursor is a purple precipitate, which is washed with methanol, centrifuged after each washing, and washed 3-5 times to ensure thorough washing.

[0016] As a preferred embodiment, in step 4), the centrifugal speed is 5000-6000 r / min and the centrifugal time is 4-6 min. The precursor of the present invention is separated into precipitates by centrifugation. By controlling the centrifugal speed and centrifugal time, effective separation is achieved and the operation is convenient.

[0017] As a preferred embodiment, in step 4), the drying is oven drying, the drying temperature is 50-70°C, and the drying time is 10-14h. The precursor of the present invention is dried in an oven after washing, and the solvents such as methanol contained therein are removed by low-temperature drying; this drying method is convenient and easy to control.

[0018] Compared with the prior art, the invention has the following beneficial effects: the cobalt tetroxide nanocage of the invention is a dual mimetic enzyme of a peroxidase mimetic enzyme and an oxide mimetic enzyme, and when used in a biosensor, not only the rapid detection of the concentration of hydrogen peroxide is realized, but also the rapid detection of the concentration of ascorbic acid is realized, and the indirect detection of the concentration of acid phosphatase is realized, and the detection efficiency is high, the sensitivity is high, the stability and reusability are good, and the application is wide; the cobalt tetroxide nanocage is a hollow cage structure, has a large internal space and a high specific surface area, and effectively increases the contact reaction area with the substrate; moreover, the preparation process is simple, easy to control, the reaction temperature is low, the energy consumption is small, the reaction time is short, the production efficiency is high, the cost is low, and the application is wide in biological analysis and drug research. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the XRD pattern of the cobalt tetroxide nanocage obtained in Example 1 of the present invention;

[0020] Figure 2 This is a TEM image of the cobalt oxide nanocage obtained in Example 1 of the present invention;

[0021] Figure 3 The wavelength and absorbance curve of the reaction system obtained in Example 2 of the present invention;

[0022] Figure 4 The reaction kinetics curve of the reaction system obtained in Example 3 of the present invention;

[0023] Figure 5 The concentration and absorbance curve of the reaction system obtained in Example 3 of the present invention;

[0024] Figure 6 The reaction kinetics curve of the reaction system obtained in Example 4 of the present invention;

[0025] Figure 7 The concentration and absorbance curve of the reaction system obtained in Example 4 of the present invention is shown;

[0026] Figure 8 This is a reaction kinetics curve diagram of the reaction system obtained in Example 5 of the present invention;

[0027] Fig. 9 The concentration and absorbance curve of the reaction system obtained in Example 5 of the present invention;

[0028] Fig.10 This is a stability bar graph of the reaction system obtained in Example 6 of the present invention;

[0029] Fig.11 This is a stability curve diagram of the reaction system obtained in Example 6 of the present invention;

[0030] Fig.12 This is a selectivity diagram of the hydrogen peroxide series reaction system obtained in Example 6 of the present invention;

[0031] Fig.13 This is a selectivity diagram of the ACP series reaction system obtained in Example 6 of the present invention. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The invention discloses an application of a cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor. The cobalt tetroxide nanocage is both a peroxidase mimetic enzyme and an oxidase mimetic enzyme. The cobalt tetroxide nanocage has both peroxidase activity and oxidase activity. The cobalt tetroxide nanocage is used to measure the concentrations of hydrogen peroxide, ascorbic acid and acid phosphatase.

[0034] Preferably, the detection limit of the cobalt tetroxide nanocage for determining the concentration of hydrogen peroxide is 0.0046 μM, the detection limit of the cobalt tetroxide nanocage for determining the concentration of ascorbic acid is 0.15 μM, and the detection limit of the cobalt tetroxide nanocage for determining the concentration of acid phosphatase is 0.0068 mU / mL.

[0035] Preferably, when the cobalt oxide nanocage measures the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, hydrogen peroxide, ascorbic acid and acid phosphatase are the analytes, and the molar ratio of the cobalt oxide nanocage to the analyte is 0.3-0.5:1.

[0036] Preferably, when the cobalt tetroxide nanocage is used to measure the concentrations of hydrogen peroxide, ascorbic acid and acid phosphatase, 3,3',5,5'-tetramethylbenzidine is used as a color developer, and the molar ratio of the color developer to the analyte is 0.7-0.9:1.

[0037] Preferably, when the cobalt tetroxide nanocage is used to measure the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, a phosphate buffer is used as the buffer solution, the pH value of the buffer solution is 4.0, and the molar ratio of the buffer solution to the analyte is 28-32:1.

[0038] Preferably, the particle size of the cobalt oxide nanocage is 800-900 nm.

[0039] Preferably, the preparation method of the cobalt tetroxide nanocage is: 1) taking cobalt nitrate, adding it to methanol, stirring evenly, to obtain a cobalt nitrate methanol solution, the mass concentration of the cobalt nitrate methanol solution is 19-20 mg / mL; 2) taking 2-methylimidazole, adding it to methanol, stirring evenly, to obtain a 2-methylimidazole methanol solution, the mass concentration of the 2-methylimidazole methanol solution is 98-99 mg / mL; 3) mixing the cobalt nitrate methanol solution obtained in step 1) and the 2-methylimidazole methanol solution obtained in step 2), the mass ratio of cobalt nitrate to 2-methylimidazole is 3-5:2, and mixing them in a sealed manner at room temperature for 20-30 hours to obtain a precursor; 4) centrifuging the precursor obtained in step 3), washing, drying, calcining at 350-450° C. for 20-40 minutes under the action of an inert gas, and keeping warm for 20-40 minutes in an air atmosphere to obtain a cobalt tetroxide nanocage.

[0040] Preferably, in step 4), washing is performed with methanol, and the number of washing times is 3-5 times.

[0041] Preferably, in step 4), the rotation speed during centrifugation is 5000-6000 r / min, and the centrifugation time is 4-6 min.

[0042] Preferably, in step 4), the drying is oven drying, the drying temperature is 50-70° C., and the drying time is 10-14 h.

[0043] Embodiment 1

[0044] Preparation of Cobalt Tetraoxide Nanocages

[0045] 1) Take 582 mg of cobalt nitrate hexahydrate, add it to 30 mL of methanol, stir evenly, and obtain cobalt nitrate methanol solution;

[0046] 2) Take 984 mg of 2-methylimidazole, add it to 10 mL of methanol, stir evenly to obtain a 2-methylimidazole methanol solution;

[0047] 3) Mix 30 mL of the cobalt nitrate methanol solution obtained in step 1) and 10 mL of the 2-methylimidazole methanol solution obtained in step 2), and seal and mix at room temperature for 24 hours to obtain the precursor ZIF-67;

[0048] 4) The precursor obtained in step 3) was centrifuged at 5000 r / min for 5 min, washed three times with methanol, dried in an oven at 60° C. for 12 h, placed in a tubular furnace, and calcined at 400° C. for 30 min in a nitrogen atmosphere. The nitrogen was turned off and the mixture was kept warm for 30 min in an air atmosphere to obtain cobalt tetroxide nanocages.

[0049] The obtained cobalt oxide nanocages were placed on a Rigaku Ultima IV X-ray diffractometer produced by Rigaku Corporation of Japan for measurement. Figure 1 It can be seen that the positions of most of the diffraction peaks of the cobalt oxide nanocage obtained in the present invention are consistent with the diffraction peak positions of standard cobalt oxide Co3O4 (JCPDS NO 74-2120), and no impurity phase appears; therefore, the sample obtained in the present invention is a Co3O4 nanocrystalline phase structure. Figure 1 It can also be seen that the diffraction peak intensity of the sample obtained in the present invention is relatively large and the diffraction peak is relatively sharp, which indicates that the Co3O4 obtained in the present invention has good crystallinity.

[0050] The cobalt oxide nanocages obtained above were placed on a JEOL JEM-2100 transmission electron microscope produced by Japan Electron Technology Co., Ltd. for measurement. Figure 2 It can be seen that the Co3O4 obtained in the present invention is a nanoparticle, the overall appearance of which is a hollow cage-like structure, and the average size is 800-900nm.

[0051] Embodiment 2

[0052] Application of Cobalt Tetraoxide Nanocages as Dual Enzyme Mimics in Biosensors

[0053] 1) Prepare phosphate buffered saline (PBS) with a pH of 4.0 and a concentration of 50 mmol / L, TMB ethanol solution with a concentration of 8 mmol / L, and H2O2 solution with a concentration of 5 mmol / L for later use;

[0054] 2) adding water to the cobalt tetroxide nanocage obtained in Example 1 to prepare a cobalt tetroxide nanocage dispersion with a concentration of 1 mg / mL for later use;

[0055] 3) Add water to the precursor ZIF-67 obtained in Example 1 to prepare a ZIF-67 dispersion with a concentration of 1 mg / mL for later use;

[0056] 4) Take 7 centrifuge tubes with a volume of 1.5 mL and mark them as a, b, c, d, e, f and g respectively. In centrifuge tube a, add 600 μL of the above phosphate buffer, 100 μL of the above TMB ethanol solution and 200 μL of the above H2O2 solution; in centrifuge tube b, add 600 μL of the above phosphate buffer, 200 μL of the above H2O2 solution and 100 μL of the above ZIF-67 dispersion; in centrifuge tube c, add 600 μL of the above phosphate buffer, 200 μL of the above H2O2 solution and 100 μL of the above cobalt oxide nanocage dispersion; in centrifuge tube d, add 600 μL of the above phosphate buffer, 100 μL of the above TMB ethanol solution and 100 μL of the above ZIF-67 dispersion; in centrifuge tube e, add 100 μL of the above phosphate buffer, 100 μL of the above TMB ethanol solution and 100 μL of the above ZIF-67 dispersion. , add 600 μL of the above phosphate buffer, 100 μL of the above TMB ethanol solution, 200 μL of the above H2O2 solution and 100 μL of the above ZIF-67 dispersion; in a No. f centrifuge tube, add 600 μL of the above phosphate buffer, 100 μL of the above TMB ethanol solution and 100 μL of the above cobalt oxide nanocage dispersion; in a No. g centrifuge tube, add 600 μL of the above phosphate buffer, 100 μL of the above TMB ethanol solution, 200 μL of the above H2O2 solution and 100 μL of the above cobalt oxide nanocage dispersion; each centrifuge tube was reacted for 7 minutes, the solution changes were observed, and the solution was placed on a U-3900H UV-visible absorption spectrometer produced by Hitachi High-Tech Corporation to measure the absorbance at 400-800 nm.

[0057] By the attached Figure 3 It can be seen that the wavelength of 652nm is the characteristic absorption peak of oxidized TMB; centrifuge tubes a (TMB + H2O2), b (H2O2 + ZIF-67), c (H2O2 + Co3O4) and d (TMB + ZIF-67) have almost no absorption peaks, centrifuge tube e (TMB + H2O2 + ZIF-67) has only a weak absorption peak, and centrifuge tubes f (TMB + Co3O4) and g (TMB + H2O2 + Co3O4) have obvious absorption peaks at 652nm, and the peak shape is better.

[0058] In addition, experimental observations show that the colors of the solutions in centrifuge tubes a (TMB + H2O2), b (H2O2 + ZIF-67), c (H2O2 + Co3O4) and d (TMB + ZIF-67) are all colorless and transparent, with no significant changes; however, the solutions in centrifuge tubes f (TMB + Co3O4) and g (TMB + H2O2 + Co3O4) are obviously blue. This indicates that Co3O4 can catalyze the oxidation of TMB to generate blue oxTMB in the presence or absence of H2O2, thus proving that Co3O4 has excellent peroxidase and oxidase activity.

[0059] Therefore, the cobalt oxide nanocage obtained by the present invention has good catalytic activity of simulating peroxidase and oxidase, and is a dual enzyme-mimicking material; in the presence or absence of H2O2, it can catalyze the oxidation of TMB to generate blue oxides, and this color development reaction can achieve rapid detection of H2O2.

[0060] Embodiment 3

[0061] Application of Cobalt Tetraoxide Nanocages as Dual Enzyme Mimics in Biosensors

[0062] 1) Prepare phosphate buffered saline (PBS) with a pH of 4.0 and a concentration of 50 mmol / L and TMB ethanol solution with a concentration of 8 mmol / L for later use;

[0063] 2) adding water to the cobalt tetroxide nanocage obtained in Example 1 to prepare a cobalt tetroxide nanocage dispersion with a concentration of 100 μg / mL for later use;

[0064] 3) taking hydrogen peroxide (H2O2) and adding water to prepare hydrogen peroxide solutions with concentrations of 0, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 25, 50, 75, and 100 μmol / L;

[0065] 4) Take 12 centrifuge tubes with a volume of 1.5 mL and mark them as ①, ②, ③, ④, ⑤, ⑥, ⑦, ⑧, ⑨, ⑩, No., 700 μL of the above-mentioned phosphate buffer solution (PBS), 100 μL of the above-mentioned TMB ethanol solution, 100 μL of the above-mentioned cobalt tetroxide nanocage dispersion and 100 μL of the above-mentioned hydrogen peroxide solution of different concentrations were added thereto respectively, and the spectrometer was placed on a U-3900H UV-visible absorption spectrometer produced by Hitachi High-Tech Corporation, and the absorbance-time curve was measured at 652 nm.

[0066] By the attached Figure 4 and attached Figure 5 It can be seen that in the range of 0.25-1 μM and 7.5-75 μM, the absorbance and concentration show a good linear relationship, and the detection limit is 0.0046 μM (S / N=3). The linear regression equation is A652nm=116.9326C+0.2669(R 2 =0.9841) and A652 nm = 3.4144C + 0.4126 (R 2 =0.9976), where C is the concentration of hydrogen peroxide (expressed in μM). Therefore, the cobalt tetroxide nanocage as a dual mimetic enzyme has a very low detection limit and ultra-high sensitivity in determining the concentration of hydrogen peroxide.

[0067] Embodiment 4

[0068] Application of Cobalt Tetraoxide Nanocages as Dual Enzyme Mimics in Biosensors

[0069] 1) Prepare phosphate buffered saline (PBS) with a pH of 4.0 and a concentration of 50 mmol / L and TMB ethanol solution with a concentration of 8 mmol / L for later use;

[0070] 2) adding water to the cobalt tetroxide nanocage obtained in Example 1 to prepare a cobalt tetroxide nanocage dispersion with a concentration of 100 μg / mL for later use;

[0071] 3) taking ascorbic acid (AA), adding water to prepare ascorbic acid solutions with concentrations of 0, 0.01, 0.05, 0.10, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, and 5.00 μmol / L;

[0072] 4) Take 12 centrifuge tubes with a volume of 1.5 mL, mark them as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, add 700 μL of the above-mentioned phosphate buffered saline (PBS), 100 μL of the above-mentioned TMB ethanol solution, 100 μL of the above-mentioned cobalt tetroxide nanocage dispersion and 100 μL of the above-mentioned ascorbic acid solution of different concentrations, respectively, place them on a U-3900H UV-visible absorption spectrometer produced by Hitachi High-Tech Corporation, and measure the absorbance-time curve at 652 nm.

[0073] By the attached Figure 6 It can be seen that with the increase of ascorbic acid solution concentration, the absorbance of the system shows a significant downward trend, and the color of the system gradually changes from blue to colorless. Figure 7 It can be seen that within the range of 0.10-4.00 μM, the detection limit is 0.15 μM (S / N=3), and the absorbance and concentration show a good linear relationship. The linear regression equation is A652 nm=-0.0992C+0.5023(R 2 =0.9972), where C is the concentration of AA in mM. This shows that the cobalt oxide nanocage obtained by the present invention has high sensitivity and greater ascorbic acid (AA) detection potential. The present invention uses the oxidase-like activity of Co3O4 and the reducibility of AA to establish an AA colorimetric sensing response platform with reduced absorbance at 652nm. Under the optimal experimental conditions, different concentrations of AA were measured based on the Co3O4 sensing platform.

[0074] Embodiment 5

[0075] Application of Cobalt Tetraoxide Nanocages as Dual Enzyme Mimics in Biosensors

[0076] 1) Prepare phosphate buffered saline (PBS) with a pH of 4.0 and a concentration of 50 mmol / L and TMB ethanol solution with a concentration of 8 mmol / L for later use;

[0077] 2) adding water to the cobalt tetroxide nanocage obtained in Example 1 to prepare a cobalt tetroxide nanocage dispersion with a concentration of 100 μg / mL for later use;

[0078] 3) Take acid phosphatase (AAP), add water to prepare an acid phosphatase solution with a concentration of 50 mmol / L, and set aside;

[0079] 4) Take ascorbic acid phosphate (ACP), add water to prepare ascorbic acid solutions with concentrations of 0, 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.025, 0.05, 0.075, 0.1, 0.5, and 1, respectively, and add 50 μL of the above acid phosphatase solution thereto, mix well, react for 10 minutes, and obtain the test solution;

[0080] 5) Take 12 centrifuge tubes with a volume of 1.5 mL, mark them as (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), and (12), add 700 μL of the above-mentioned phosphate buffered saline (PBS), 100 μL of the above-mentioned TMB ethanol solution, 100 μL of the above-mentioned cobalt tetroxide nanocage dispersion and 100 μL of the above-mentioned test solution, respectively, place them on a U-3900H UV-visible absorption spectrometer produced by Hitachi High-Technologies Corporation, and measure the absorbance-time curve at 652 nm.

[0081] By the attached Figure 8 It can be seen that as the ACP concentration increases, the absorbance of the system at 652nm gradually decreases. When the ACP concentration increases from low to high, the color of the corresponding system gradually changes from dark blue to light blue, which is basically consistent with the UV-visible absorption spectrum. Fig. 9 It can be seen that within the low concentration range of ACP, there is a linear growth trend. The linear equation is A652nm=-1.8138C(mU / mL)+0.3680, and the linear range is 7.5-75mU / mL(R 2=0.9910), and the detection limit of ACP is 0.0068mU / mL (S / N=3). This shows that the cobalt tetroxide nanocage has a wide linear range and a low detection limit. Therefore, the cobalt tetroxide nanocage sensor has outstanding application potential in the indirect determination of ACP. Orthophosphate monoester (AAP) is the hydrolysis substrate of ascorbic acid phosphate (ACP). Under weak acid conditions, ACP can catalyze the generation of a certain amount of AA and inorganic phosphate. Therefore, based on the sensitive response of cobalt tetroxide nanocages to AA, a sensitive colorimetric platform for the indirect determination of ACP was constructed.

[0082] Embodiment 6

[0083] Stability of Cobalt Tetraoxide Nanocages as Dual Enzyme Mimics in Biosensors

[0084] (I) Cyclic catalytic experiment

[0085] According to the method of the centrifuge tube No. f in Example 2, 10 portions of the reaction system were prepared and placed on a U-3900H UV-visible absorption spectrometer produced by Hitachi High-Technologies Corporation to measure the absorbance at 625 nm. Fig.10 and 11 It can be seen that the absorbance and blue signal of all reaction systems at 652 nm did not change significantly, which indicates that the obtained reaction system has good stability.

[0086] (II) Selective Experiment

[0087] According to the method of the centrifuge tube No. g in Example 2, the substrates were prepared to contain hydrogen peroxide, Ca 2+ 、Na + Br - 、NO 3- , Glu, Fru, Trp, Lys and diluted AL reaction system, wherein the concentration of hydrogen peroxide in the reaction system is 0.5mM, Ca 2+ 、Na + Br - 、NO 3- The concentration of Glu, Fru, Trp, Lys and diluted AL in the reaction system is 20.0 mM, and 10 parts of the reaction system are obtained. 2+ 、Na + Br - 、NO 3-, Glu, Fru, Trp, Lys and diluted AL were used as interfering substances of hydrogen peroxide, so that hydrogen peroxide was replaced by different ions and biomacromolecules, while keeping all other conditions unchanged; the 10 solutions obtained were placed on the U-3900H UV-visible absorption spectrometer produced by Hitachi High-Technologies Corporation, and the absorbance at 625nm was measured. Fig.12 It can be seen that under the same conditions, except for the diluted AL (containing 0.5 mM hydrogen peroxide) and hydrogen peroxide, the absorbance of the solutions in the other reaction systems did not increase significantly. This shows that the cobalt tetroxide nanocage colorimetric sensor of the present invention has good specificity.

[0088] According to the method of the centrifuge tube No. g in Example 2, the substrates were prepared to contain ascorbic acid phosphate (ACP), GOx, BSA, Na + , Ca 2+ 、NO 3- , Glu, Fru, Trp and Lys reaction system, wherein the concentration of ascorbic acid phosphate in the reaction system is 0.5 mM, GOx, BSA, Na + , Ca 2+ 、NO 3- The concentration of Glu, Fru, Trp and Lys in the reaction system was 20.0 mM, and 10 reaction systems were obtained. + , Ca 2+ 、NO 3- , Glu, Fru, Trp and Lys were used as interfering substances of ascorbic acid phosphate, so that ascorbic acid phosphate was replaced by different ions and biomacromolecules, while keeping all other conditions unchanged; the 10 solutions obtained were placed on a U-3900H UV-visible absorption spectrometer produced by Hitachi High-Technologies Corporation, and the absorbance at 625nm was measured. Fig.13 It can be seen that under the same conditions, except for the absorbance of the solution in the reaction system containing ascorbic acid phosphate, the absorbance of the solution in other reaction systems at 652nm has no significant difference. This shows that the cobalt tetraoxide nanocage colorimetric sensor is not only reproducible, but also has high detection selectivity.

[0089] Therefore, compared with the prior art, the beneficial effects of the present invention are as follows: the cobalt tetroxide nanocage of the present invention is a dual mimetic enzyme of a peroxidase mimetic enzyme and an oxide mimetic enzyme, and when used in a biosensor, not only the rapid detection of the concentration of hydrogen peroxide is realized, but also the rapid detection of the concentration of ascorbic acid is realized, and the indirect detection of the concentration of acid phosphatase is realized, and the detection efficiency is high, the sensitivity is high, the stability and reusability are good, and the application is wide; the cobalt tetroxide nanocage is a hollow cage structure, has a large internal space and a high specific surface area, and effectively increases the contact reaction area with the substrate, and the preparation process is simple, easy to control, the reaction temperature is low, the energy consumption is small, the reaction time is short, the production efficiency is high, the cost is low, and the application is wide in biological analysis and drug research.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of cobalt trioxide nanocage as a dual enzyme mimic in a biosensor, characterized in that: The cobalt oxide nanocage is both a peroxidase mimic and an oxidase mimic, and has both peroxidase activity and oxidase activity. The cobalt oxide nanocage is used to measure the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase. The preparation method of the cobalt tetroxide nanocage is: 1) Take cobalt nitrate, add it to methanol, stir evenly, and obtain cobalt nitrate methanol solution. The mass concentration of cobalt nitrate methanol solution is 19-20 mg / mL; 2) Take 2-methylimidazole, add it to methanol, stir evenly, and obtain 2-methylimidazole methanol solution. The mass concentration of 2-methylimidazole methanol solution is 98-99 mg / mL; 3) mixing the cobalt nitrate methanol solution obtained in step 1) and the 2-methylimidazole methanol solution obtained in step 2), wherein the mass ratio of cobalt nitrate to 2-methylimidazole is 3-5:2, and mixing them in a sealed state at room temperature for 20-30 hours to obtain a precursor; 4) The precursor obtained in step 3) is centrifuged, washed, dried, calcined at 350-450° C. for 20-40 min under the action of an inert gas, and kept warm for 20-40 min in an air atmosphere to obtain cobalt tetroxide nanocages.

2. The use of the cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor according to claim 1, characterized in that: The detection limit of the cobalt tetroxide nanocage for determining the concentration of hydrogen peroxide is 0.0046 μM, the detection limit of the cobalt tetroxide nanocage for determining the concentration of ascorbic acid is 0.15 μM, and the detection limit of the cobalt tetroxide nanocage for determining the concentration of acid phosphatase is 0.0068 mU / mL.

3. The use of the cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor according to claim 1, characterized in that: When the cobalt tetroxide nanocage measures the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, hydrogen peroxide, ascorbic acid and acid phosphatase are the test objects, and the molar ratio of the cobalt tetroxide nanocage to the test object is 0.3-0.5:

1.

4. The use of the cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor according to claim 3, characterized in that: When the cobalt tetroxide nanocage is used to measure the concentrations of hydrogen peroxide, ascorbic acid and acid phosphatase, 3,3',5,5'-tetramethylbenzidine is used as a color developer, and the molar ratio of the color developer to the object to be tested is 0.7-0.9:

1.

5. The use of the cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor according to claim 4, characterized in that: When the cobalt tetroxide nanocage is used to measure the concentration of hydrogen peroxide, ascorbic acid and acid phosphatase, a phosphate buffer is used as the buffer solution, the pH value of the buffer solution is 4.0, and the molar ratio of the buffer solution to the test object is 28-32:

1.

6. The use of the cobalt oxide nanocage according to any one of claims 1 to 5 as a dual mimetic enzyme in a biosensor, characterized in that: The particle size of the cobalt tetroxide nanocage is 800-900nm.

7. The use of the cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor according to claim 1, characterized in that: In the step 4), washing is performed with methanol, and the number of washing times is 3-5 times.

8. The use of the cobalt oxide nanocage as a dual enzyme mimic in a biosensor according to claim 1, characterized in that: In the step 4), the rotation speed during centrifugation is 5000-6000 r / min, and the centrifugation time is 4-6 min.

9. The use of the cobalt tetroxide nanocage as a dual mimetic enzyme in a biosensor according to claim 1, characterized in that: In the step 4), the drying is performed by oven drying at a drying temperature of 50-70° C. and a drying time of 10-14 hours.

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