Hemoglobin-cobalt phosphate-cobalt phosphate iron nanoflower and preparation method and application thereof
By preparing hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflowers, the problems of low catalytic activity of hemoglobin and difficult electron transfer were solved, and efficient hydrogen peroxide electrocatalytic reduction performance was achieved, which is suitable for electrochemical biosensors.
Patent Information
- Application Number
- CN202310633837.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-31
- Publication Date
- 2025-10-17
- Estimated Expiration
- 2043-05-31
AI Technical Summary
In the existing technology, hemoglobin has low catalytic activity and is easily variable, and the electron transfer between the electroactive center and the electrode of organic-inorganic hybrid nanoflowers in electrochemical biosensors is difficult to achieve, which limits its application.
Hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflowers were prepared by a one-pot method. Water-soluble starch was used as a dispersant to grow cobalt iron phosphate nanosheets vertically on the surface of cobalt phosphate nanopetals to form a rose-like structure, expose the electroactive centers, and utilize the synergistic effect of iron and cobalt ions to improve conductivity.
The immobilization stability of hemoglobin and the high exposure of the electroactive center are achieved, which improves the electrochemical performance. As a sensitive material of electrochemical biosensors, it efficiently catalyzes the reduction of hydrogen peroxide and has good electroactivity and conductivity.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of composite materials, and particularly relates to a hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower as well as a preparation method and application thereof. BACKGROUND
[0002] As the most widely studied redox protein, hemoglobin has a low price and an active center iron porphyrin similar to the structure of horseradish peroxidase. Since the iron porphyrin is deeply buried in the globin, it is difficult to directly contact with the catalytic substrate, so the catalytic activity of hemoglobin is much lower than that of horseradish peroxidase. Researchers solve the problems of low catalytic activity and easy variability of hemoglobin by fixing hemoglobin on nanomaterials. Traditional fixing methods include adsorption, embedding, covalent bonding and cross-linking. These methods can improve the catalytic activity, stability and recyclability of hemoglobin to a certain extent, but they do not essentially solve the problem of deep burial of iron porphyrin.
[0003] The organic-inorganic hybrid nanoflower is a three-dimensional hybrid nanoflower formed by growing copper phosphate nanosheets based on biological enzymes. The nanoflower combines the high catalytic activity and specificity of enzymes and the stability of copper phosphate, and is an excellent enzyme immobilization technology. However, the insulation of enzymes and the poor conductivity of copper phosphate make it difficult to realize direct electron transfer between the electroactive center and the electrode, which greatly limits the application of the organic-inorganic hybrid nanoflower in electrochemical biosensors. SUMMARY
[0004] The present application aims to overcome the problems in the prior art and provide a hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower as well as a preparation method and application thereof. The nanoflower has the characteristics of stable hemoglobin immobilization, high exposure of electroactive center and good electrochemical performance, and can be used as a sensitive material for hydrogen peroxide reduction in electrochemical biosensors.
[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0006] A preparation method of a hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower, comprising the following steps:
[0007] In water isolated from oxygen and continuously stirred, water-soluble starch, bovine hemoglobin freeze-dried powder, CoSO4 solution, PBS solution, FeSO4 solution and PBS solution are sequentially added, and the reaction is allowed to stand to obtain the hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower.
[0008] Further, the ratio of the amount of water to the amount of water-soluble starch is 10-20 mL:0.05-0.2 mg.
[0009] Further, the ratio of the amount of water to the amount of bovine hemoglobin freeze-dried powder is 10-20 mL:1-10 mg.
[0010] Further, the volume ratio of water to CoSO4 solution is 10-20 mL:1.25-5 mL, and the concentration of the CoSO4 solution is 10 mmol / L;
[0011] The volume ratio of water to FeSO4 solution is 10-20 mL:1.25-5 mL, and the concentration of the FeSO4 solution is 10 mmol / L.
[0012] Further, the volume ratio of the total amount of the PBS solution to water is 1-2:4.
[0013] Further, when the water-soluble starch, the bovine hemoglobin freeze-dried powder, the CoSO4 solution, the PBS solution, the FeSO4 solution and the PBS solution are added in sequence, the time interval for adding each raw material is 5-10 min.
[0014] Further, the temperature for standing reaction is 10-25 DEG C, and the time is 24-72 h.
[0015] A hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower prepared according to the method has a rose flower-like appearance, and cobalt phosphate nanoflower petals vertically grow on the surface of cobalt phosphate nanosheets.
[0016] A hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower prepared according to the method is used as sensitive material of an electrochemical biosensor and applied to electrocatalytic reduction of hydrogen peroxide.
[0017] Further, a hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower suspension with a mass concentration of 1-1.5 mg / mL is mixed with a Nafion solution according to a volume ratio of 1-2:1, 5.0-10 muL of the mixture is coated on the surface of a polished glassy carbon electrode to obtain a working electrode, and a three-electrode system is used for electrocatalytic reduction of hydrogen peroxide, a reference electrode is Ag / AgCl, a counter electrode is a platinum wire, and a potential window is-0.7-0.1 V.
[0018] Further, the electrocatalytic reduction of hydrogen peroxide is carried out under a nitrogen atmosphere.
[0019] Compared with the prior art, the present application has the following beneficial effects:
[0020] The preparation method of the hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower of the application adopts hemoglobin as an organic component, cobalt and ferrous ion solution as inorganic components, and water-soluble starch as a dispersant, and the hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower is prepared by a one-pot reaction method, which has the advantages of simple preparation process, easily available raw materials, green environmental protection, no need for special equipment, and batch preparation. The nanoflower is in the shape of a rose flower and is self-assembled by rectangular nanosheets pointing to the center, and small nanosheets grow vertically on the surface of large nanoflower petals, which has a large specific surface area and is beneficial to direct contact of the catalytic substrate. The surface of the nanoflower has rich electroactive centers, one of which is that the quaternary structure of hemoglobin is unfolded to expose the electroactive center iron porphyrin, and the other of which is that cobalt ions and ferrous ions are coordinated with hemoglobin to form variable valence electroactive centers.
[0021] Further, the water-soluble starch is used as a dispersant to inhibit the rapid growth of phosphate crystals, so that the prepared nanoflower has uniform particle size and regular morphology.
[0022] Further, the nitrogen atmosphere is maintained to prevent the oxidation of ferrous ions by oxygen to generate Fe(OH)3 particles.
[0023] Further, the order of adding the bovine hemoglobin lyophilized powder, CoSO4 solution and PBS solution is changed, or the interval time is too long or too short, which cannot obtain the rose flower structure in the application.
[0024] Further, the PBS solution is not added in batches or the dropping speed is too fast, which cannot obtain the structure that small nanosheets grow vertically on the surface of large nanoflower petals.
[0025] The hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower of the application can be used as a sensitive material of an electrochemical biosensor and applied in the electrocatalytic reduction of hydrogen peroxide. The synergistic effect between iron and cobalt ions not only promotes the electron exchange between the electroactive center and the catalytic substrate, but also improves the intrinsic conductivity of the organic-inorganic hybrid nanoflower and accelerates the electron transmission between the electroactive center and the electrode. The sensitive material has excellent electrochemical performance, can efficiently catalyze the reduction of hydrogen peroxide to generate water, is easy to recover, non-toxic and non-polluting, and has application prospects in the field of electrochemical biosensors. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 The XRD pattern of the hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower prepared in Example 3 is shown in the figure;
[0027] Figure 2 The EDS pattern of the hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower prepared in Example 3 is shown in the figure;
[0028] Figure 3SEM image of the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower prepared in Example 3;
[0029] Figure 4 TEM image of the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower prepared in Example 3;
[0030] Figure 5 The relationship curve between the concentration of hydrogen peroxide and the reduction peak current value obtained in Example 6 is tested.
[0031] Figure 6 The relationship curve between the concentration of hydrogen peroxide and the reduction peak current value obtained in Example 6 is tested. DETAILED DESCRIPTION
[0032] The present application is described by specific examples, but the present application is not limited thereto. The hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower provided by the present application and its application in electrocatalytic reduction of hydrogen peroxide will be further described below.
[0033] The preparation method of the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower comprises the following steps:
[0034] Preparation by one-pot method: nitrogen is introduced into 10-20 mL ultrapure water to exclude oxygen and maintain a nitrogen atmosphere. Under continuous magnetic stirring, 0.05-0.2 mg water-soluble starch is added after nitrogen is introduced for 20-30 min. 1-10 mg of lyophilized bovine hemoglobin powder is added after 5-10 min. 1.25-5 mL of 10 mmol / L CoSO4 solution is quickly poured in after 5-10 min. 1.25-5 mL of PBS solution with a concentration of 20 mmol / L and pH=7.4 is slowly dripped using a syringe after 5-10 min. 1.25-5 mL of 10 mmol / L FeSO4 solution is quickly poured in after 5-10 min. 1.25-5 mL of PBS solution is slowly dripped again using a syringe after 5-10 min. The stirring and nitrogen introduction are stopped after 5-10 min, and the reaction container is sealed and incubated at 10-25℃ for 24-72 h to obtain the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower.
[0035] The water-soluble starch is a dispersant that inhibits the rapid growth of phosphate crystals, and the prepared nanoflower has uniform particle size and regular morphology.
[0036] The nitrogen atmosphere is maintained to prevent the oxidation of ferrous ions by oxygen to form Fe(OH)3 particles.
[0037] The order of adding the lyophilized bovine hemoglobin powder, CoSO4 solution and PBS solution is changed, or the interval time is too long or too short, which cannot obtain the rose-like structure in the present application.
[0038] The PBS solution cannot be added in batches or at too high a dropwise speed to obtain the large nanoflower petal surface vertical growth small nanosheet structure in the application.
[0039] The hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower prepared in the application has a rose flower shape, utilizes biological mineralization, and grows cobalt phosphate crystals based on hemoglobin molecules to vertically grow small iron cobalt phosphate nanosheets on the surface of large cobalt phosphate nanoflower petals.
[0040] (2) Application of hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower to electrocatalytic reduction of hydrogen peroxide
[0041] 4-6 mg of hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower freeze-dried powder is weighed and dispersed in 4 mL of a PBS solution to obtain a suspension; the suspension is mixed with Nafion (perfluorosulfonic acid, 5 wt%) at a volume ratio of 1-2:1 to obtain an electrode modification solution; 5.0-10 μL of the electrode modification solution is coated on the surface of a polished glassy carbon electrode to obtain a Nafion / hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower / glassy carbon electrode.
[0042] Specifically, the pretreatment of the glassy carbon electrode: the surface of the polishing cloth is polished with 1.0, 0.3 and 0.05 μm aluminum oxide powder in sequence, and the electrode surface is blown dry with a nitrogen gas flow after being ultrasonically cleaned in ethanol and ultrapure water for 6 times.
[0043] An electrochemical test is performed using a three-electrode system, the reference electrode is Ag / AgCl (3M KCl), the counter electrode is a platinum wire, and the Nafion / hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflower / glassy carbon electrode is the working electrode, and the potential window is -0.7-0.1 V.
[0044] Nitrogen is bubbled in the PBS electrolyte for 20-30 min before the electrochemical test, and a nitrogen atmosphere is maintained above the electrolyte surface during the test to prevent the current peak generated by oxygen from interfering with the hydrogen peroxide reduction signal.
[0045] The preparation process of the application is simple, the raw materials are easy to obtain, it is green and environmentally friendly, no special equipment is needed, and it can be prepared in batches. The nanoflower prepared has a rose flower shape, has a large specific surface area and rich electroactive centers. The synergistic effect between iron and cobalt ions not only promotes the exchange of electrons between the electroactive centers and the catalytic substrate, but also improves the intrinsic conductivity of the organic-inorganic hybrid nanoflower, accelerates the electron transfer between the electroactive centers and the electrode, and can efficiently catalyze the reduction of hydrogen peroxide.
[0046] The application will be described below in conjunction with specific examples.
[0047] Example 1
[0048] Preparation of hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower.
[0049] Step 1: Measure 10 mL of ultrapure water, pass nitrogen and start magnetic stirring, exhaust oxygen;
[0050] Step 2: After passing nitrogen for 20 min, add 0.05 mg of water-soluble starch;
[0051] Step 3: After 5 min, add 1 mg of bovine hemoglobin freeze-dried powder;
[0052] Step 4: After 5 min, quickly pour 1.25 mL of 10 mmol / L CoSO4 solution;
[0053] Step 5: After 5 min, slowly drop 1.25 mL of PBS solution with a concentration of 20 mmol / L pH=7.4 using a syringe;
[0054] Step 6: After 5 min, quickly pour 1.25 mL of 10 mmol / L FeSO4 solution;
[0055] Step 7: After 5 min, slowly drop 1.25 mL of PBS solution using a syringe again;
[0056] Step 8: After 5 min, stop stirring and pass nitrogen, seal and incubate at 10°C for 24 h to obtain hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower.
[0057] Example 2
[0058] Preparation of hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower.
[0059] Step 1: Measure 10 mL of ultrapure water, pass nitrogen and start magnetic stirring, exhaust oxygen;
[0060] Step 2: After passing nitrogen for 30 min, add 0.05 mg of water-soluble starch;
[0061] Step 3: After 10 min, add 1 mg of bovine hemoglobin freeze-dried powder;
[0062] Step 4: After 10 min, quickly pour 1.25 mL of 10 mmol / L CoSO4 solution;
[0063] Step 5: After 10 min, slowly drop 1.25 mL of PBS solution with a concentration of 20 mmol / L pH=7.4 using a syringe;
[0064] Step 6: After 10 min, quickly pour 1.25 mL of 10 mmol / L FeSO4 solution;
[0065] Step 7: After 10 min, slowly drop 1.25 mL of PBS solution using a syringe again;
[0066] Step 8: After 10 min, stop stirring and pass nitrogen, seal and incubate at 25°C for 72 h to obtain hemoglobin-cobalt phosphate-cobalt ferric phosphate hybrid nanoflowers.
[0067] Example 3
[0068] Preparation of hemoglobin-cobalt phosphate-cobalt ferric phosphate hybrid nanoflowers.
[0069] Step 1: Measure 20 mL of ultrapure water, pass nitrogen and start magnetic stirring, and vent oxygen;
[0070] Step 2: After passing nitrogen for 30 min, add 0.2 mg of water-soluble starch;
[0071] Step 3: After 10 min, add 10 mg of bovine hemoglobin freeze-dried powder;
[0072] Step 4: After 10 min, quickly pour in 5 mL of 10 mmol / L CoSO4 solution;
[0073] Step 5: After 10 min, slowly drop 5 mL of PBS solution with a concentration of 20 mmol / L pH = 7.4 using a syringe;
[0074] Step 6: After 10 min, quickly pour in 5 mL of 10 mmol / L FeSO4 solution;
[0075] Step 7: After 10 min, slowly drop 5 mL of PBS solution using a syringe again;
[0076] Step 8: After 10 min, stop stirring and pass nitrogen, seal and incubate at 25°C for 72 h to obtain hemoglobin-cobalt phosphate-cobalt ferric phosphate hybrid nanoflowers.
[0077] Example 4
[0078] Preparation of hemoglobin-cobalt phosphate-cobalt ferric phosphate hybrid nanoflowers.
[0079] Step 1: Measure 15 mL of ultrapure water, pass nitrogen and start magnetic stirring, and vent oxygen;
[0080] Step 2: After passing nitrogen for 30 min, add 0.1 mg of water-soluble starch;
[0081] Step 3: After 7 min, add 4 mg of bovine hemoglobin freeze-dried powder;
[0082] Step 4: After 7 min, quickly pour in 2 mL of 10 mmol / L CoSO4 solution;
[0083] Step 5: 7 min later, slowly drop 2.5 mL of PBS solution with a concentration of 20 mmol / L, pH = 7.4 using a syringe;
[0084] Step 6: 7 min later, quickly pour in 2 mL of 10 mmol / L FeSO4 solution;
[0085] Step 7: 7 min later, again slowly drop 2.5 mL of PBS solution using a syringe;
[0086] Step 8: 7 min later, stop stirring and nitrogen flow, seal and incubate at 25°C for 72 h to obtain hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers.
[0087] Example 5
[0088] Preparation of hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers.
[0089] Step 1: Measure 12 mL of ultrapure water, introduce nitrogen and start magnetic stirring, and vent oxygen;
[0090] Step 2: After 30 min of nitrogen flow, add 0.15 mg of water-soluble starch;
[0091] Step 3: 6 min later, add 6 mg of bovine hemoglobin lyophilized powder;
[0092] Step 4: 6 min later, quickly pour in 3 mL of 10 mmol / L CoSO4 solution;
[0093] Step 5: 6 min later, slowly drop 1.5 mL of PBS solution with a concentration of 20 mmol / L, pH = 7.4 using a syringe;
[0094] Step 6: 6 min later, quickly pour in 5 mL of 10 mmol / L FeSO4 solution;
[0095] Step 7: 6 min later, again slowly drop 1.5 mL of PBS solution using a syringe;
[0096] Step 8: 10 min later, stop stirring and nitrogen flow, seal and incubate at 25°C for 72 h to obtain hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers.
[0097] Example 6
[0098] Preparation of hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers.
[0099] Step 1: Measure 16 mL of ultrapure water, introduce nitrogen and start magnetic stirring, and vent oxygen;
[0100] Step 2: After 30 min of nitrogen bubbling, 0.1 mg of water-soluble starch was added;
[0101] Step 3: After 8 min, 8 mg of lyophilized bovine hemoglobin powder was added;
[0102] Step 4: After 8 min, 4 mL of 10 mmol / L CoSO4 solution was quickly poured in;
[0103] Step 5: After 8 min, 4 mL of PBS solution with a concentration of 20 mmol / L and pH = 7.4 was slowly dripped in using a syringe;
[0104] Step 6: After 8 min, 3 mL of 10 mmol / L FeSO4 solution was quickly poured in;
[0105] Step 7: After 8 min, 4 mL of PBS solution was again slowly dripped in using a syringe;
[0106] Step 8: After 10 min, stirring and nitrogen bubbling were stopped, and the mixture was incubated at 25°C for 72 h in a sealed state to obtain hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers.
[0107] Figure 1 and Figure 2 XRD and EDS patterns of the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers prepared in Example 3. From Figure 1 It can be seen that the diffraction peaks of the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower composite material correspond to the standard diffraction peaks of Co3(PO4)2·8H2O (JCPDS 41-0375) and Co3Fe4(PO4)6 (JCPDS 49-1083), and there are no other impurity peaks, and the peak type is sharp, indicating that the hemoglobin-cobalt phosphate-iron phosphate cobalt composite material is successfully prepared. Figure 2 It is shown that the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers are composed of Co, Fe, P, O, C and N elements, and the C and N elements are derived from hemoglobin.
[0108] Figure 3 and Figure 4 SEM and TEM patterns of the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers prepared in Example 3. From Figure 3 and Figure 4 It can be seen that the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflowers have a rose flower-like morphology, which is self-assembled from rectangular nanosheets pointing to the center, and small nanosheets are vertically grown on the surface of large nanoflower petals, and the flower ball particle size is 3-5 μm.
[0109] Example 7
[0110] Preparation of Nafion / hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower / boron-doped diamond electrode.
[0111] Step 1: 6 mg of hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower lyophilized powder prepared in Example 3 was weighed and dispersed in 4 mL of PBS solution to obtain a suspension;
[0112] Step 2: The suspension was mixed with Nafion (perfluorosulfonic acid, 5 wt%) at a volume ratio of 2:1 to obtain an electrode modification solution;
[0113] Step 3: The boron-doped diamond electrode was polished on the surface of a polishing cloth using 1.0, 0.3 and 0.05 μm aluminum oxide powder in sequence, and was ultrasonically cleaned in ethanol and ultrapure water alternately for 6 times, and the electrode surface was blown dry with nitrogen gas flow;
[0114] Step 4: 10 μL of the electrode modification solution was coated on the polished boron-doped diamond electrode surface to obtain a Nafion / hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower / boron-doped diamond electrode, which was stored in a 4°C refrigerator.
[0115] Example 8
[0116] Preparation of Nafion / hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower / boron-doped diamond electrode.
[0117] Step 1: 4 mg of hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower lyophilized powder prepared in Example 3 was weighed and dispersed in 4 mL of PBS solution to obtain a suspension;
[0118] Step 2: The suspension was mixed with Nafion (perfluorosulfonic acid, 5 wt%) at a volume ratio of 2:1 to obtain an electrode modification solution;
[0119] Step 3: The boron-doped diamond electrode was polished on the surface of a polishing cloth using 1.0, 0.3 and 0.05 μm aluminum oxide powder in sequence, and was ultrasonically cleaned in ethanol and ultrapure water alternately for 6 times, and the electrode surface was blown dry with nitrogen gas flow;
[0120] Step 4: 10 μL of the electrode modification solution was coated on the polished boron-doped diamond electrode surface to obtain a Nafion / hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower / boron-doped diamond electrode, which was stored in a 4°C refrigerator.
[0121] Example 9
[0122] Application of Nafion / hemoglobin-cobalt phosphate-cobalt phosphate iron hybrid nanoflower / boron-doped diamond electrode to electrocatalytic reduction of hydrogen peroxide.
[0123] Step 1: prepare 100 mL PBS solution with concentration of 0.1 mol / L and pH=7.4, use the solution as electrolyte, pass nitrogen for 30 min, and discharge oxygen in the solution;
[0124] Step 2: use the Nafion / hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower / borosilicate electrode prepared in Example 8 as working electrode, Ag / AgCl (3 mol / L KCl solution) as reference electrode, and platinum wire as counter electrode, test PBS solution containing different concentrations (0-500 μmol / L, see Figure 6 ) of hydrogen peroxide by cyclic voltammetry, and the potential window is-0.7-0.1 V.
[0125] Figure 5 The cyclic voltammograms of the Nafion / hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower / borosilicate electrode tested in PBS solution containing different concentrations of hydrogen peroxide are shown, and the reduction peak current value gradually increases with the increase of the concentration of hydrogen peroxide, which indicates that the hemoglobin-cobalt phosphate-iron phosphate cobalt hybrid nanoflower exhibits excellent performance in the electrocatalytic reduction of hydrogen peroxide.
[0126] Figure 6 The relationship curve between the concentration of hydrogen peroxide and the reduction peak current value is shown, and the linear relationship is good in the concentration range of 2-350 μmol / L.
[0127] The hybrid material of the application can be used as sensitive material of electrochemical biosensor, and the electrochemical biosensor is constructed for hydrogen peroxide detection.
[0128] The above is only the preferred embodiment in the embodiment of the application, therefore, cannot be used to limit the scope of the application, equivalent changes made according to the claims of the application, still belong to the scope covered by the application.
Claims
1. A method for preparing hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflowers, characterized in that: The following steps are involved: In oxygen-isolated and continuously stirred water, water-soluble starch, bovine hemoglobin freeze-dried powder, CoSO4 solution, PBS solution, FeSO4 solution, and PBS solution were added in sequence and allowed to react to obtain hemoglobin-cobalt phosphate-iron cobalt phosphate hybrid nanoflowers, wherein the PBS solution was slowly added dropwise; When adding water-soluble starch, bovine hemoglobin freeze-dried powder, CoSO4 solution, PBS solution, FeSO4 solution and PBS solution in sequence, the interval between adding each raw material is 5 to 10 minutes, and the PBS solution is added slowly dropwise; The temperature of the static reaction is 10~25℃ and the time is 24~72h; The usage ratio of water to bovine hemoglobin lyophilized powder is 10~20mL:1~10mg.
2. The method for preparing hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflowers according to claim 1, characterized in that: The usage ratio of water to water-soluble starch is 10~20mL:0.05~0.2mg.
3. The method for preparing hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflowers according to claim 1, characterized in that: The ratio of water to CoSO4 solution is 10~20mL:1.25~5mL, and the concentration of CoSO4 solution is 10mmol / L; The ratio of water to FeSO4 solution is 10~20mL:1.25~5mL, and the concentration of FeSO4 solution is 10mmol / L.
4. The method for preparing hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflowers according to claim 1, characterized in that: The volume ratio of the total amount of PBS solution to water is 1 to 2:
4.
5. A hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflower prepared according to the method of any one of claims 1 to 4, characterized in that: The morphology is rose-shaped, with cobalt iron phosphate nanosheets growing vertically on the surface of cobalt phosphate nanopetals.
6. An application of hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflowers prepared according to the method of any one of claims 1 to 4 as a sensitive material for electrochemical biosensors in the electrocatalytic reduction of hydrogen peroxide; A hemoglobin-cobalt phosphate-cobalt iron phosphate hybrid nanoflower suspension with a mass concentration of 1-1.5 mg / mL was mixed with a Nafion solution at a volume ratio of 1-2:1, and 5.0-10 μL of the mixture was coated on the surface of a polished glassy carbon electrode to obtain a working electrode; A three-electrode system was used for the electrocatalytic reduction of hydrogen peroxide, with Ag / AgCl as the reference electrode and platinum wire as the counter electrode, and the potential window was -0.7~0.1V.