Biomass electrochemical sensing probe, electrochemical sensor, preparation method and application

Abstract

The present invention relates to a biomass electrochemical sensing probe, an electrochemical sensor, a preparation method and an application. The biomass electrochemical sensing probe comprises a substrate and an active component combined by electrostatic action, and the mass ratio of the substrate to the active component is (8-12):1; the substrate comprises polydopamine nanospheres, and the active component comprises Prussian blue nanoparticles. The electrochemical sensor is a biomass electrochemical sensing probe modified on the working electrode surface of a flexible screen-printed electrode by a probe adhesive. The biomass electrochemical sensing probe and the electrochemical sensor of the present invention have the advantages of safety, non-toxicity and good stability. The probe material is biomimetic to green biological materials. When used to detect vitamin C, it has a low detection limit, high sensitivity and a large linear detection range.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical sensors, and in particular to a biomass electrochemical sensing probe, an electrochemical sensor, and a preparation method and application thereof. Background Art

[0002] With the advent of the "big health" era, people are paying more and more attention to their own health, especially for some diseases caused by vitamin deficiency. For example, problems such as oral ulcers are caused by vitamin C deficiency in the human body. What's more frightening is that as the deficiency of vitamin C worsens, the patient's connective tissue will be affected, leading to poor wound healing and spontaneous bleeding. It is worth noting that an appropriate amount of vitamin C can enhance the function of the human immune system, while excessive supplementation, that is, abuse of vitamin C, can cause nausea, diarrhea, and rash. Therefore, maintaining endogenous vitamin C levels within the normal range is of extraordinary significance to human health. It is worth noting that the vitamin C content in different human body fluids varies greatly. For example, the standard range of vitamin C in human blood is 10 - 90 μM, and the value of vitamin C in urine needs to be less than 0.6 mg L -1 This means that real-time monitoring of vitamin C content in the human body requires tools with multiple detection channels, and also puts higher requirements on the detection sensitivity of vitamin C.

[0003] Currently, a variety of detection methods such as chromatography, mass spectrometry, spectroscopy, and immunoassay are used for the quantitative determination of vitamin C. However, these traditional methods still suffer from cumbersome sample processing, complex test operations, and difficulties in operating in bulky instruments. Therefore, we urgently need to develop an efficient and convenient diagnostic tool for real-time detection of vitamin C in human body fluids. Fortunately, electrochemical sensors have achieved remarkable results in recent years due to their high sensitivity and low cost, and have broad application prospects in real-time home disease management. In particular, vitamin C, as a recognized reducing agent, is very suitable for electrochemical catalysis to produce obvious current or impedance signals. Therefore, it is a reasonable prospect to manufacture a portable electrochemical sensor to monitor the level of vitamin C in the human body.

[0004] In addition, there have been some literature reports on the use of electrochemical methods to detect vitamin C. However, considering the safety of home testing and human application, the green and harmless standards of electrochemical sensing probes also need to be achieved. Therefore, the determination of vitamin C in human body fluids using green and non-toxic biomaterials or biomimetic materials has become a major research goal. However, there have been no reports on the research of such electrochemical vitamin C sensors, especially ensuring the detection sensitivity and stability of the probe, which is a research difficulty of this work. Therefore, it is of great practical value to develop a portable electrochemical sensor for detecting vitamin C to fill the research gap. Summary of the invention

[0005] The purpose of the present invention is to overcome the above technical deficiencies, provide a biomass electrochemical sensing probe, an electrochemical sensor, and a preparation method and application, to solve the technical problems of insufficient detection sensitivity and stability of green electrochemical sensors in the prior art.

[0006] In order to achieve the above technical purpose, the technical solution provided by the present invention is:

[0007] In a first aspect, the present invention provides a biomass electrochemical sensing probe, comprising a substrate and an active component combined by electrostatic action, wherein the mass ratio of the substrate to the active component is (8-12):1; the substrate comprises polydopamine nanospheres, and the active component comprises Prussian blue nanoparticles.

[0008] In a second aspect, the present invention provides a method for preparing a biomass electrochemical sensing probe, comprising the following steps: stirring and mixing polydopamine nanospheres and Prussian blue nanoparticles in a mass ratio of (8-12):1 in deionized water, centrifuging and drying to obtain a biomass electrochemical sensing probe.

[0009] In a third aspect, the present invention provides an electrochemical sensor for detecting vitamin C, comprising a flexible screen-printed electrode (SPCE), and a biomass electrochemical sensing probe modified on the working electrode surface of the flexible screen-printed electrode by a probe adhesive; the probe adhesive is obtained by dissolving chitosan in an acetic acid solution.

[0010] In a fourth aspect, the present invention provides a method for preparing an electrochemical sensor for detecting vitamin C, comprising the following steps: dispersing a biomass electrochemical sensing probe in a probe adhesive to obtain a probe modification liquid; dropping the probe modification liquid on the working electrode surface of a flexible screen-printed electrode, and obtaining the electrochemical sensor after drying.

[0011] In a fifth aspect, the present invention provides an application of an electrochemical sensor in detecting vitamin C.

[0012] Compared with the prior art, the beneficial effects of the present invention include:

[0013] The present invention provides a biomass electrochemical sensing probe and an electrochemical sensor for detecting vitamin C, which have the advantages of safety, non-toxicity and good stability. The probe material is biomimetic to green biological materials, safe and edible, and can be biodegraded in the natural environment, and has good application prospects in vitamin C detection. The preparation method of the biomass electrochemical sensing probe and the electrochemical sensor of the present invention has the characteristics of simple and fast process and green environmental protection, and has higher production capacity and is suitable for industrial-scale production. When used for detecting vitamin C, the detection limit is low, the sensitivity is high, and the linear detection range is large. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Field emission scanning electron microscope (FESEM) images of PDA, PB and PDA-PB in Example 1, wherein (a) is PDA, (b) is PB, and (c) is PDA-PB;

[0015] Figure 2 : is the energy spectrum element distribution diagram of PDA-PB in Example 1, wherein (a) is carbon, (b) is oxygen, (c) is nitrogen, and (d) is iron;

[0016] Figure 3 is the particle size distribution diagram of PDA and PB in Example 1;

[0017] Figure 4 is the Zeta potential distribution diagram of PDA and PB in Example 1;

[0018] Figure 5 The Fourier transform infrared (FTIR) spectra of PDA, PB and PDA-PB in Example 1, wherein (a) is PDA, (b) is PB, and (c) is PDA-PB;

[0019] Figure 6 The powder X-ray diffraction (XRD) patterns of PDA, PB and PDA-PB in Example 1, wherein (a) is PDA, (b) is PB, and (c) is PDA-PB;

[0020] Figure 7 is the energy level diagram of all elements in the X-ray photoelectron spectroscopy (XPS) of PDA-PB in Example 1;

[0021] Figure 8 The XPS peak spectra of each element in PDA-PB in Example 1, wherein (a) is carbon, (b) is oxygen, (c) is nitrogen, and (d) is iron;

[0022] Fig. 9 Cyclic voltammetry (CV) curves of the bare SPCE and the electrochemical sensor PDA-PB-SPCE in Example 6;

[0023] Fig.10 The electrochemical impedance spectroscopy (EIS) curves of the bare SPCE and the electrochemical sensor PDA-PB-SPCE in Example 6;

[0024] Fig.11 CV curve of the electrochemical sensor PDA-PB-SPCE in Example 6 after 10 cycles;

[0025] Fig.12 CV curves of the bare SPCE at different bending angles in Example 6;

[0026] Fig.13 CV curves of the electrochemical sensor PDA-PB-SPCE in Application Example 1 for standard solutions of vitamin C with different concentrations, wherein (a) is a current density diagram after CV reaction between PDA-PB-SPCE and vitamin C with different concentrations, (b) is a vitamin C concentration-current density fitting curve (high linear detection range), and (c) is a vitamin C concentration-current density fitting curve (low linear detection range);

[0027] Fig.14 The electrochemical sensor PDA-PB-SPCE in Application Example 2 detects vitamin C in actual human body fluids, where (a) is saliva detection and (b) is urine detection;

[0028] Fig.15 The X-ray powder diffraction (XRD) pattern of PB obtained in Comparative Example 2;

[0029] Fig.16 is the powder X-ray diffraction (XRD) pattern of PB obtained in Comparative Example 3;

[0030] Fig.17 Schematic diagram of the electrochemical sensing mechanism of vitamin C and the electrochemical sensor PDA-PB-SPCE, including the reaction route, proton-electron transfer and energy loss. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] In a first aspect, the present invention provides a biomass electrochemical sensing probe, comprising a substrate and an active component combined by electrostatic action, wherein the mass ratio of the substrate to the active component is (8-12):1; the substrate comprises polydopamine (PDA) nanospheres, and the active component comprises Prussian blue nanoparticles.

[0033] The present invention uses safe and non-toxic PDA as the probe substrate and human-friendly PB as the electrochemically active component. The two are combined through electrostatic action to obtain a biomass electrochemical sensing probe PDA-PB, which is stable in nature and helps to improve the overall electrochemical activity. The technical difficulty overcome by the present invention lies in the design and preparation of green and non-toxic material sensing probes, wherein PDA is consistent with the main component of cuttlefish juice, is a natural melanin, can be eaten, and has almost no harm to the human body; PB is a common antidote for thallium poisoning and can also be eaten safely. The combination of the two PDA-PB ensures safety during human sample detection.

[0034] In a second aspect, the present invention provides a method for preparing a biomass electrochemical sensing probe, comprising the following steps:

[0035] Polydopamine nanospheres and Prussian blue nanoparticles were stirred and mixed evenly in deionized water at a mass ratio of (8-12):1, and then centrifuged and dried to obtain a biomass electrochemical sensing probe.

[0036] The preparation method of the biomass electrochemical sensing probe of the present invention is simple, rapid, green and environmentally friendly.

[0037] Preferably, the preparation steps of polydopamine nanospheres include: adding dopamine hydrochloride solution to an alkaline solution to obtain a first mixed solution, subjecting the first mixed solution to a self-polymerization reaction to obtain a PDA suspension, and subjecting the mixture to centrifugation and drying to obtain polydopamine nanospheres.

[0038] Further preferably, the dopamine hydrochloride solution is obtained by mixing dopamine hydrochloride and deionized water, and the alkaline solution is obtained by mixing 25-30wt% ammonia water, anhydrous ethanol and deionized water; in the first mixed solution, the mass concentration of dopamine hydrochloride is 2-5 mg / mL, and the volume concentration of ammonia water is 0.3-1% (here, the ammonia water concentration refers to the ratio of the volume of ammonia water to the volume of the first mixed solution).

[0039] Further preferably, the mass volume ratio of dopamine hydrochloride to deionized water in the dopamine hydrochloride solution is (0.4-0.6) g:10 mL; the volume ratio of ammonia water, anhydrous ethanol and deionized water in the alkaline solution is (0.5-0.8):(35-45):(80-100).

[0040] The present invention provides alkalinity through ammonia water; the alkaline environment, i.e., the volume concentration of ammonia water in the first mixed solution, directly affects the synthesis effect of PDA. If the concentration is lower than the above range, dopamine hydrochloride cannot undergo self-polymerization to obtain PDA; if the concentration is higher than the above range, more PDA with intact morphology cannot be generated, which wastes costs. By adding ethanol to provide a mild reaction environment, it is beneficial to promote the increase of PDA yield, and the generated PDA has a more uniform morphology and better dispersibility.

[0041] More preferably, the temperature of the self-polymerization reaction is room temperature, the self-polymerization time is 24 to 72 hours, and the drying method is freeze-drying.

[0042] The synthesis of PDA is directly related to the polymerization time. If the time is lower than the above range, the ideal spherical PDA cannot be synthesized, and the PDA particle size is too small to be used as a probe substrate. The drying method will also affect the resulting PDA. For example, when high-temperature vacuum drying is used, the obtained PDA is in block form and needs to be ground to obtain a powdered sample, which increases the cost.

[0043] Preferably, the preparation steps of Prussian blue nanoparticles include: mixing potassium ferrocyanide solution, polyethyleneimine (PEI) solution and ferric chloride solution in an acidic environment to obtain a second mixed solution; refluxing the second mixed solution at high temperature to obtain a PB solution, and then centrifuging and drying to obtain Prussian blue nanoparticles (PB).

[0044] Further preferably, the molar concentrations of the potassium ferrocyanide solution and the ferric chloride solution are both 4-6 mmol / L; the mass concentration of the polyethyleneimine solution is 2-5 wt %, and the number average molecular weight of the polyethyleneimine is 300-500.

[0045] Further preferably, in the second mixed solution, the molar ratio of potassium ferrocyanide to ferric chloride is 1:1; and the ratio of ferric chloride to polyethyleneimine is (4-6) mmol:3g.

[0046] Further preferably, the second mixed solution is obtained by mixing potassium ferricyanide solution and polyethyleneimine (PEI) solution for 3 to 7 minutes and then mixing with ferric chloride solution; wherein the volume ratio of potassium ferricyanide solution, ferric chloride solution and polyethyleneimine solution is (9 to 11): (9 to 11): 1; the pH value of potassium ferricyanide solution and ferric chloride solution is 0.5 to 1.5. By controlling the mixing order of the solutions, the acid anion of potassium ferricyanide first reacts with PEI to produce a reaction precursor, and then reacts quickly with FeCl3 to ensure the yield.

[0047] Further preferably, the reaction environment for PB preparation is regulated by inorganic hydrochloric acid, sulfuric acid or nitric acid. Since the volume of PEI solution is much smaller than that of the other two liquids, it has little effect on the pH of the mixed solution. Therefore, the pH value is determined by the pH value of potassium ferrocyanide solution and ferric chloride solution; more preferably, hydrochloric acid is selected to control the reaction pH to about 1.

[0048] Since PB is a strict crystal, no matter it is higher or lower than the above range, crystalline PB will not be obtained. At the same time, PEI is the key to the two trivalent iron salts to generate divalent iron and obtain the final PB (ferric ferrocyanide). If the concentration is higher or lower than the above range, strictly crystalline PB cannot be obtained; if the PEI molecular weight is higher or lower than the range of 300-500, it will affect the reaction synthesis rate and thus affect the morphology of PB. In addition, when the pH of the reaction system is too high, the XRD diffraction pattern of the product is a typical amorphous state, and when the pH of the reaction system is too low, some amorphous impurities will be produced.

[0049] More preferably, the high temperature reflux temperature is 100-105° C., the reflux time is 1-3 h; and the drying method is freeze drying.

[0050] In the present invention, if the reaction is not refluxed, the blue precipitate is dark blue and the product is not PB. Only when the reaction is refluxed sufficiently can bright blue PB be obtained.

[0051] Preferably, the mass ratio of polydopamine nanospheres to deionized water is (1-5):1000; the mixing temperature of PDA and PB in deionized water is room temperature, and the mixing time of polydopamine nanospheres and Prussian blue nanoparticles in deionized water is 2-4 hours.

[0052] In a third aspect, the present invention provides a portable electrochemical sensor for detecting vitamin C, comprising a flexible screen-printed electrode (SPCE), and a biomass electrochemical sensing probe modified on the working electrode surface of the flexible screen-printed electrode by a probe adhesive; the probe adhesive is obtained by dissolving chitosan in an acetic acid solution.

[0053] In the packaging process of the electrochemical sensing probe PDA-PB, the present invention also uses biomass chitosan as the main component of the probe adhesive, thereby avoiding the toxicity of the traditional adhesive perfluorosulfonic acid resin and being suitable for human body fluid detection; thereby obtaining a safe, non-toxic, and stable portable electrochemical sensor for detecting vitamin C.

[0054] Preferably, the substrate of the SPCE is a polyethylene terephthalate (PET) film, and the electrodes are printed with graphite ink and silver paste ink.

[0055] Specifically, SPCE is a three-electrode system, namely, a working electrode WE, a counter electrode CE, and a reference electrode RE. The working electrode WE is mainly used to load the probe PDA-PB to detect vitamin C and generate electrochemical signals in the subsequent test process. Two arc-shaped counter electrodes CE and reference electrodes RE are arranged at intervals outside the working electrode WE. The counter electrode CE can ensure that the current of the working electrode is smooth, so that the reaction under study occurs on the working electrode. The reference electrode RE is used to control the potential difference between the electrodes. The diameter of the working electrode WE is 4 to 6 mm, and more preferably 5 mm. The sample amount required in the actual sensing area is as low as 200 μL, and the entire usable area is less than 6 cm 2 , very easy to carry.

[0056] In a fourth aspect, the present invention provides a method for preparing a portable electrochemical sensor for detecting vitamin C, comprising the following steps:

[0057] dispersing the biomass electrochemical sensing probe in a probe adhesive to obtain a probe modification solution;

[0058] 5 to 10 μL of the probe modification solution is dropped onto the working electrode surface of the flexible screen-printed electrode, and the electrochemical sensor is obtained after drying.

[0059] Preferably, the probe adhesive is obtained by dissolving chitosan in an acetic acid aqueous solution with a concentration of 1.5 to 2.5 mol / L.

[0060] Preferably, the concentration of chitosan in the probe adhesive is 1 to 10 mg / mL; the concentration of the biomass electrochemical sensing probe in the probe modification solution is 1 to 5 mg / mL.

[0061] Chitosan is insoluble in water, so an organic solvent is needed to dissolve it. Acetic acid is an organic solvent with low toxicity and good volatility, which can fully dissolve chitosan to obtain a uniform probe adhesive. If the concentration of the probe adhesive is lower than the above range, the bonding effect is not good, and the sensing probe may fall off during the test; if it is higher than the above range, there will be more solid residues after the adhesive dries, which affects the sensing performance of the sensor. If the concentration of the biomass electrochemical sensing probe is lower than the above range, the sensor sensitivity is low; if it is higher than the above range, the sensing probes will be stacked, and the sensitivity of the sensor cannot be effectively improved, which also wastes costs.

[0062] Preferably, after the probe modification solution is added dropwise, the drying method is vacuum drying at 30-40°C.

[0063] In a fifth aspect, the present invention provides an application of a portable electrochemical sensor in detecting vitamin C.

[0064] The present invention is further described in detail below through specific examples.

[0065] Example 1

[0066] The preparation method of the biomass electrochemical sensing probe for detecting vitamin C comprises the following steps:

[0067] (1) Preparation of biomass PDA:

[0068] Under magnetic stirring, 0.6 mL of ammonia water (25 wt%) was fully mixed with 40 mL of anhydrous ethanol and 90 mL of deionized water to obtain liquid a; 0.5 g of dopamine hydrochloride was dissolved in 10 mL of deionized water and shaken to obtain liquid b. Liquid b was added dropwise to liquid a to obtain a first mixed solution, which was subjected to self-polymerization reaction for 24 h. The obtained black suspension was fully washed, centrifuged and freeze-dried to obtain PDA.

[0069] (2) Preparation of Prussian blue nanoparticles PB:

[0070] Dissolve 5 mmol potassium ferrocyanide in 1000 mL deionized water, and adjust the pH to 1 with hydrochloric acid solution to obtain liquid c; dissolve 5 mmol anhydrous ferric chloride (FeCl3) in 1000 mL deionized water, and adjust the pH to 1 with hydrochloric acid solution to obtain liquid d; dissolve 3 g PEI with a number average molecular weight of 423 in 100 mL deionized water to obtain liquid e; mix liquid c with liquid e for 5 min and then mix with liquid d to obtain a second mixed solution; reflux the second mixed solution at 105°C for 3 h, and wash the blue suspension thoroughly, centrifuge it, and freeze-dry it to obtain PB.

[0071] (3) Preparation of biomass electrochemical sensing probe PDA-PB:

[0072] 1 g of PDA was dispersed in 1000 mL of deionized water, and 0.1 g of prefabricated PB was added. The mixture was magnetically stirred for 2 h. The black suspension was thoroughly washed, centrifuged and freeze-dried to obtain the biomass electrochemical sensing probe PDA-PB.

[0073] The PDA, PB and PDA-PB obtained in Example 1 were tested respectively, and the results are as follows: Figure 1-Figure 8 shown.

[0074] Figure 1 Figure 2 is the FESEM images of PDA, PB and PDA-PB. (a) shows that PDA is a regular microsphere with a diameter of about 500 nm and a slightly concave-convex structure on the surface. (b) shows that PB is a nanocube morphology, whose size is quite different from that of PDA. After compounding with PDA, it forms a composite morphology as shown in (c).

[0075] Figure 2 (a)-(d) correspond to the distribution of four elements: carbon, oxygen, nitrogen, and iron. Carbon and nitrogen come from PDA and PB, oxygen comes from PDA, and iron comes from PB.

[0076] Figure 3 Figure 2 is the particle size distribution diagram of PDA and PB, where the average particle size of PDA is 531 nm and the average particle size of PB is 21 nm. The results are consistent with FESEM.

[0077] Figure 4 This is the Zeta potential distribution diagram of PDA and PB, where the surface of PDA is negatively charged and the surface of PB is positively charged, indicating that the binding mode between PDA and PB in PDA-PB is electrostatic interaction.

[0078] Figure 5 FTIR spectra of PDA, PB and PDA-PB, where 3500 cm -1The characteristic peak near the hydroxyl (-OH) stretching vibration is 2900 cm -1 The characteristic peaks near this are the stretching vibrations of carbon and hydrogen (-CH), 1700 cm -1 The characteristic peaks near 2050 cm -1 The characteristic peak nearby is cyano (-CN).

[0079] Figure 6 Figure 2 shows the XRD patterns of PDA, PB and PDA-PB. PDA is a typical non-crystalline macromolecular polymer with an obvious bulge structure at about 20°. PB is a strict crystal with obvious crystal characteristic peaks in the diffraction pattern. Its lattice corresponds to JCPDS card no. 73-0687.

[0080] Figure 7 Energy level diagram of all elements in XPS of PDA-PB; Figure 8 The XPS peak spectra of each element in PDA-PB, (a)-(d) correspond to the chemical states of carbon, oxygen, nitrogen, and iron respectively; Figure 7 and Figure 8 It can be seen that the two characteristic peaks of carbon element can be fitted as C=O and CC; the oxygen element can be fitted into two characteristic peaks corresponding to O=C and OC respectively; the two characteristic peaks of nitrogen element can be fitted as NH2-C and NC-; the characteristic peaks of iron element can be fitted as 2p 1 / 2 and 2p 3 / 2 and its satellite peaks.

[0081] Example 2

[0082] The preparation method of the biomass electrochemical sensing probe for detecting vitamin C is different from that of Example 1 only in that: in step (1), liquid b is added dropwise to liquid a, and the self-polymerization reaction of the obtained first mixed solution is continued for 48 hours. The other steps and conditions are the same as those of Example 1.

[0083] The results show that the average particle size of the obtained PDA is about 520 nm, which is basically the same as that in Example 1. This shows that the obtained PDA has been shaped when the reaction time is 24 hours, and the reaction tends to a plateau. Therefore, in the process of preparing PDA of the present invention, the self-polymerization reaction time is preferably 24 hours, and the production capacity is more efficient.

[0084] Example 3

[0085] The preparation method of the biomass electrochemical sensing probe for detecting vitamin C is different from that in Example 1 only in that 5 mmol of anhydrous ferric chloride (FeCl3) in step (2) is replaced by 5 mmol of hexahydrate ferric chloride (FeCl3·6H2O), and the other steps and conditions are the same as those in Example 1.

[0086] Example 4

[0087] The preparation method of the biomass electrochemical sensing probe for detecting vitamin C is different from that of Example 1 only in that the amount of ammonia water in step (1) is adjusted to 0.4 mL, and the other steps and conditions are the same as those of Example 1. The average particle size of the obtained PDA is 780 nm.

[0088] Example 5

[0089] The preparation method of the biomass electrochemical sensing probe for detecting vitamin C is different from that of Example 1 only in that the amount of ammonia water in step (1) is adjusted to 0.75 mL, and the other steps and conditions are the same as those of Example 1. The average particle size of the obtained PDA is 380 nm.

[0090] The performance of the biomass electrochemical sensing probes obtained in the above Examples 2-5 is close to that of Example 1, and both meet the requirements of the biomass electrochemical sensing probes.

[0091] Example 6

[0092] The preparation method of a portable electrochemical sensor for detecting vitamin C comprises the following steps:

[0093] (1) Preparation of screen-printed electrodes (SPCE):

[0094] The 0.1 mm thick PET was cleaned with anhydrous ethanol and dried, and then the working electrode (diameter 5 mm) and the counter electrode of the three electrodes were printed by screen printing using graphite ink according to the pre-ordered template and dried; the reference electrode was printed by screen printing using silver paste ink according to the pre-ordered template and dried to obtain SPCE.

[0095] The graphite pattern and silver paste ink were purchased from Maisicheng (Beijing) Technology Co., Ltd.

[0096] (2) Preparation of electrochemical sensor PDA-PB-SPCE:

[0097] 0.02 g of chitosan was dissolved in 2 mL of 2 M acetic acid aqueous solution as a probe adhesive, and 2 mg of the biomass electrochemical sensing probe PDA-PB prepared in Example 1 was dispersed in the above probe adhesive to obtain a probe modification solution; 5 μL of the probe modification solution was added dropwise to the working electrode surface of SPCE and dried to obtain the electrochemical sensor PDA-PB-SPCE.

[0098] The electrochemical performance of the SPCE obtained in step (1) of Example 6 and the electrochemical sensor PDA-PB-SPCE obtained in step (2) were tested. The results are as follows: Figure 9-12 shown.

[0099] Fig. 9 CV curves of bare SPCE and electrochemical sensor PDA-PB-SPCE. The oxidation peak and reduction peak of SPCE slightly increased and shifted after being modified with the sensing probe PDA-PB. Fig.10 Figure 2 shows the EIS curves of bare SPCE and electrochemical sensor PDA-PB-SPCE. The charge transfer resistance of SPCE decreased slightly after being modified with the sensing probe PDA-PB, which proves the successful modification of the probe.

[0100] Fig.11 The CV curve of the electrochemical sensor PDA-PB-SPCE after 10 cycles is presented in the form of a current-time curve. The changes in the oxidation peak and reduction peak signals in each cycle are small and within the allowable error range, proving the stability of the sensor.

[0101] Fig.12 The CV curves of bare SPCE at different bending angles. The electrochemical signal of SPCE changes slightly when bent at 0°, 45°, and 90°, which is within the allowable error range, proving the portable application potential of flexible SPCE.

[0102] Example 7

[0103] The preparation method of the portable electrochemical sensor for detecting vitamin C is different from that of Example 6 only in that the amount of the biomass electrochemical sensing probe PDA-PB prepared in Example 1 is adjusted to 10 mg, and the other steps and conditions are the same as those of Example 6.

[0104] Application Example 1

[0105] This application example uses the CV method in the electrochemical method as the testing means to study the electrochemical detection effect of the portable electrochemical sensor PDA-PB-SPCE prepared in Example 6 and Example 7 on vitamin C. First, the electrochemical CV detection scheme for vitamin C standard solutions of different concentrations (200 μM, 100 μM, 50 μM, 20 μM, 10 μM, 5 μM, 2 μM, 1 μM, 0.5 μM, 0.2 μM, 0.1 μM) at room temperature and pressure was established and tested. Among them, the total volume of the experimental solution was maintained at 50 mL, the electrolyte was a universal phosphate buffer (PBS), the concentration was 0.01 M, and the pH value was 7.0; after 200 μL of the test solution was added to the three-electrode sensing part of the portable electrochemical sensor PDA-PB-SPCE, the scanning rate was 50 mV s -1, the scanning range is -0.2 to 0.4 V, the current density value is recorded, and the linear detection range and the minimum detection limit of the electrochemical sensor of the present invention for vitamin C are calculated using a linear regression model.

[0106] Fig.13 In the figure, (a) curve represents the current density diagram after PDA-PB-SPCE reacts with different concentrations of vitamin C through CV reaction. The vitamin C concentrations represented by the curve from top to bottom are: 200 μM, 100 μM, 50 μM, 20 μM, 10 μM, 5 μM, 2μM, 1 μM, 0.5 μM, 0.2 μM, 0.1 μM, and the oxidation potential is about 0.1 V. Fig.13 In (b), the horizontal axis is the vitamin C concentration, and the vertical axis is the current density. The vitamin C concentration and current density were fitted and calculated using a linear regression model, and the high linear detection range of PDA-PB-SPCE for vitamin C was 10 - 200 μM, and the linear correlation coefficient was 0.9868; Fig.13 In (c), the horizontal axis is the vitamin C concentration, and the vertical axis is the current density. The linear regression model is used to fit the vitamin C concentration and current density, and the low linear detection range of PDA-PB-SPCE for vitamin C is 0.2 - 10μM, and the linear correlation coefficient is 0.9936. It can be calculated that the linear detection range of the prefabricated sensor PDA-PB-SPCE for vitamin C is 0.2 - 200 μM, the detection limit is 0.03 μM, the quantitative limit is 0.1 μM, and the sensitivity is 0.33 μA μM -1 cm -2 .

[0107] Application Example 2

[0108] This application example uses the chronoamperometry (CA) method in the electrochemical method as a test method to study the electrochemical detection effect of the portable electrochemical sensor PDA-PB-SPCE prepared in Example 6 and Example 7 on vitamin C in human body fluids. First, the electrochemical CA detection scheme for standard solutions of vitamin C in different human body fluids such as saliva and urine at room temperature and pressure was established and tested. Saliva and urine were diluted 1 times with PBS with a concentration of 0.01 M and a pH of 7.0, and then 200 μL of the test solution was added to the three-electrode sensing part of the portable electrochemical sensor PDA-PB-SPCE. The polarization (oxidation) potential was 0.1 V, the test time was 120 s, and the current density value was recorded. The vitamin C concentration in human saliva and human urine was calculated by combining the linear regression equation in Application Example 1.

[0109] Fig.14In the figure, (a) is the electrochemical test result of human saliva sample on PDA-PB-SPCE, and the calculated vitamin C concentration is 62.9 μM; Fig.14 In the figure, (b) is the electrochemical test result of human urine sample on PDA-PB-SPCE, and the calculated vitamin C concentration is 81.2 μM.

[0110] Comparative Example 1

[0111] The preparation of biomass PDA differs from that of Example 1 only in that the type of alkali solution used in the preparation of biomass PDA in step (1) is changed, specifically:

[0112] 64 mL of ethanol and 144 mL of Tris-HCl buffer (pH = 8.8) were fully mixed under magnetic stirring; 0.08 g of dopamine hydrochloride was dissolved in 2 mL of deionized water, and then the above solution was added dropwise. The polymerization reaction was carried out at room temperature for 30 h, and the black suspension was fully washed, centrifuged and freeze-dried to obtain PDA.

[0113] This comparative example studies the self-polymerization effect of dopamine hydrochloride under different alkaline conditions. The PDA particle size obtained by using Tris-HCl buffer as the reaction system is small, with an average particle size of 200-300 nm, and the polymerization reaction time is long and the yield is lower, which is relatively cost-effective and not suitable for large-scale production of biomass electrochemical sensing probes.

[0114] Comparative Example 2

[0115] The preparation of Prussian blue nanoparticles PB differs from that of Example 1 only in that the pH of the reaction system during the PB synthesis process in step (2) is changed, specifically:

[0116] Dissolve 5 mmol potassium ferrocyanide in 1000 mL deionized water to obtain liquid c; dissolve 5 mmol anhydrous ferric chloride (FeCl3) in 1000 mL deionized water to obtain liquid d; dissolve 3 g PEI with a number average molecular weight of 423 in 100 mL deionized water to obtain liquid e; mix liquid c with liquid e for 5 min and then mix with liquid d, reflux the mixture at 105°C for 3 h, and wash the blue suspension thoroughly, centrifuge it, and freeze-dry it to obtain PB.

[0117] This comparative example studies the effect of a higher pH value in the reaction system on the strict crystallinity of PB during the PB synthesis process. Figure 6 The crystal characteristics in (b) have proved the successful preparation of PB in Examples 1 to 5; however, if the hydrochloric acid solution is not used for pH adjustment before the solution is mixed, the obtained suspension is dark blue instead of the classic bright blue, and Fig.15The corresponding crystal characteristic peaks could not be analyzed in the XRD characterization shown, and PB could not be synthesized.

[0118] Comparative Example 3

[0119] The preparation of Prussian blue nanoparticles PB differs from that of Example 1 only in that the pH of the reaction system during the PB synthesis process in step (2) is changed, specifically:

[0120] Dissolve 5 mmol potassium ferrocyanide in 1000 mL deionized water, and use hydrochloric acid solution to adjust the pH to much less than 1 to obtain liquid c; dissolve 5 mmol anhydrous ferric chloride (FeCl3) in 1000 mL deionized water, and use hydrochloric acid solution to adjust the pH to much less than 1 to obtain liquid d; dissolve 3 g PEI with a number average molecular weight of 423 in 100 mL deionized water to obtain liquid e; mix liquid c with liquid e for 5 min and then mix with liquid d, reflux the mixture at 105°C for 3 h, and wash the blue suspension thoroughly, centrifuge it, and freeze-dry it to obtain PB.

[0121] This comparative example studies the effect of a lower pH value in the reaction system on the strict crystallinity of PB during the PB synthesis process. Figure 6 The crystal characteristics in (b) have proved the successful preparation of PB in Examples 1 to 5; however, if an excess of hydrochloric acid solution is used to adjust the pH value of the reaction system to be much less than 1 before the solution is mixed, the obtained suspension is bright blue, but Fig.16 The XRD characterization shown shows residual amorphous impurities, and the characteristic peak has a bulge of amorphous impurities at about 20°, indicating that the synthesized PB is impure.

[0122] It can be seen from Example 1, Comparative Example 2 and Comparative Example 3 that during the PB synthesis process, when the pH value of the reaction system is too high, that is, direct reaction without adding hydrochloric acid, the obtained blue precipitate does not have a clear cubic configuration and is amorphous; if the pH value is too low, some amorphous impurities will be produced.

[0123] Comparative Example 4

[0124] The preparation method of the portable electrochemical sensor for detecting vitamin C is different from that of Example 6 only in that the composition of the probe adhesive in the preparation process of the electrochemical sensor PDA-PB-SPCE in step (2) is changed, and the other steps and conditions are the same as those in Example 6, and the specific differences are:

[0125] Preparation of electrochemical sensor PDA-PB-SPCE: Mix 1 mL of anhydrous ethanol and 1 mL of 5 wt.% perfluorosulfonic acid resin (Nafion) as a probe adhesive, and then disperse 2 mg of biomass electrochemical sensing probe PDA-PB in the above probe adhesive to obtain a probe modification solution; pipette 5 μL of the probe modification solution and drop it on the working electrode surface of SPCE and dry it to obtain the electrochemical sensor PDA-PB-SPCE.

[0126] This comparative example studies the effects of different adhesives on the modification of SPCE with biomass electrochemical sensing probes. Among them, although perfluorosulfonic acid resin (Nafion) can also fix the probe on the surface of the working electrode, and Nafion, as a conductive polymer, will not have a significant effect on the performance of the electrochemical sensor, the toxicity of Nafion still restricts its detection in real human samples and is not suitable for home medical treatment.

[0127] In summary, the present invention evaluates the feasibility of PDA-PB as a biomass electrochemical sensing probe, and further evaluates the feasibility, safety and stability of PDA-PB as a vitamin C electrochemical sensing probe, and studies the preparation methods of PDA, PB, and PDA-PB respectively. Subsequently, the present invention evaluates the feasibility and safety of PDA-PB modified SPCE with different adhesives, prepares a portable electrochemical sensor PDA-PB-SPCE for quantitative detection of vitamin C, and verifies the stability and portability of the sensor. Subsequently, the present invention verifies the feasibility of the portable electrochemical sensor PDA-PB-SPCE for vitamin C detection in real human body fluid samples. This biomass electrochemical sensing probe is biomimetic to green and environmentally friendly biomaterials, is friendly to human health, has low process cost and is simple and convenient. Subsequently, the probe is fixed on the flexible SPCE by the biomass adhesive chitosan. The method is simple and has high repeatability. The portable electrochemical sensor for detecting vitamin C is obtained by complete drying.

[0128] Compared with the commercial medical enzyme method for detecting vitamin C content, the portability of this electrochemical sensor method for detecting vitamins is greatly improved. Fig.17The mechanism diagram of the electrochemical reaction between the electrochemical sensor PDA-PB-SPCE of the present invention and vitamin C is shown. Vitamin C is an organic acid (VC), which preferentially ionizes a hydrogen ion in aqueous solution to obtain VC2. After DFT calculation (calculation software GaussView) of the energy required for the conversion of the intermediate, the negative ion will not preferentially lose electrons to obtain the free radical intermediate VC3-1, but will continue to ionize a hydrogen ion to obtain the intermediate VC3-2 with two negative charges, and then lose two electrons in turn, and obtain the oxidation product o-VC through intramolecular rearrangement reaction; the current density of the entire detection process changes linearly, the detection amount is 200 μL, and the detection time is within 2 min, which is convenient and efficient. Compared with other electrochemical vitamin C sensors, the sensing probe of this portable sensor is non-toxic and environmentally friendly, and all materials can be biodegraded under natural conditions, achieving carbon neutrality. At the same time, in the present invention, the SPCE based on the flexible PET substrate greatly enhances the practicality of the sensor, and realizes portable and efficient selective detection of vitamin C in the solution.

[0129] Different from the prior art, the present invention provides a biomass electrochemical sensing probe, an electrochemical sensor, a preparation method and an application. The structure of the electrochemical sensor includes a flexible screen-printed electrode and a biomass electrochemical sensing probe modified on its surface; the raw materials of the biomass electrochemical sensing probe include polydopamine and Prussian blue, wherein polydopamine is the main component of cuttlefish juice and Prussian blue is a commonly used thallium antidote, which is safe to use. The portable electrochemical sensor disclosed in the present invention has the advantages of safety, non-toxicity and good stability. It can undergo electrochemical oxidation-reduction reactions with vitamin C solutions of different concentrations to determine the vitamin C content, and can evaluate the vitamin C content in real human body fluid samples, and has good application prospects. The present invention also has the characteristics of simple process, green environmental protection, and high production capacity, and is suitable for industrial-scale production.

[0130] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a biomass electrochemical sensing probe for detecting vitamin C, characterized in that: The steps include: Adding a dopamine hydrochloride solution to an alkaline solution to obtain a first mixed solution, subjecting the first mixed solution to a self-polymerization reaction at room temperature for 24 hours to obtain a PDA suspension, and subjecting the first mixed solution to centrifugation and drying to obtain polydopamine nanospheres; the dopamine hydrochloride solution is obtained by mixing dopamine hydrochloride with deionized water, and the alkaline solution is obtained by mixing 28-30% ammonia water, anhydrous ethanol and deionized water; in the first mixed solution, the mass concentration of dopamine hydrochloride is 2-5 mg / mL, and the volume concentration of ammonia water is 0.3-1%; Polydopamine nanospheres and Prussian blue nanoparticles are mixed uniformly in deionized water at a mass ratio of (8-12):1, and then centrifuged and dried to obtain a biomass electrochemical sensing probe; The mass ratio of the polydopamine nanospheres to deionized water is (1-5):1000; the mixing time of the polydopamine nanospheres and the Prussian blue nanoparticles in the deionized water is 2-4 hours.

2. The method for preparing a biomass electrochemical sensing probe for detecting vitamin C according to claim 1, characterized in that: The polydopamine nanospheres are dried by freeze drying.

3. The method for preparing a biomass electrochemical sensing probe for detecting vitamin C according to claim 1, characterized in that: The preparation steps of the Prussian blue nanoparticles include: mixing potassium ferrocyanide solution, polyethyleneimine solution and ferric chloride solution in an acidic environment to obtain a second mixed solution; refluxing the second mixed solution to obtain a PB solution, and then centrifuging and drying to obtain Prussian blue nanoparticles.

4. The method for preparing the biomass electrochemical sensing probe for detecting vitamin C according to claim 3, characterized in that: The molar concentrations of the potassium ferrocyanide solution and the ferric chloride solution are both 4-6 mmol / L; the mass concentration of the polyethyleneimine solution is 2-5%, and the number average molecular weight of the polyethyleneimine is 300-500; In the second mixed solution, the molar ratio of potassium ferrocyanide to ferric chloride is 1:1; the ratio of ferric chloride to polyethyleneimine is (4-6) mmol:3g.

5. The method for preparing a biomass electrochemical sensing probe for detecting vitamin C according to claim 3, characterized in that: The second mixed solution is obtained by mixing a potassium ferrocyanide solution and a polyethyleneimine solution for 3 to 7 minutes and then mixing with a ferric chloride solution; the volume ratio of the potassium ferrocyanide solution, the ferric chloride solution and the polyethyleneimine solution is (9 to 11): (9 to 11): 1; the pH values ​​of the potassium ferrocyanide solution and the ferric chloride solution are between 0.5 and 1.5; The reflux temperature is 100-105°C, and the reflux time is 1-3 h; The Prussian blue nanoparticles are dried by freeze drying.

6. A biomass electrochemical sensing probe for detecting vitamin C prepared according to the preparation method of any one of claims 1 to 5.

7. An electrochemical sensor for detecting vitamin C, characterized in that: It includes a flexible screen-printed electrode, and a biomass electrochemical sensing probe modified on the working electrode surface of the flexible screen-printed electrode by a probe adhesive; The biomass electrochemical sensing probe is a biomass electrochemical sensing probe prepared by the preparation method according to any one of claims 1 to 5 or a biomass electrochemical sensing probe according to claim 6; The probe adhesive is obtained by dissolving chitosan in acetic acid solution.

8. The method for preparing an electrochemical sensor for detecting vitamin C according to claim 7, characterized in that: The following steps are involved: dispersing the biomass electrochemical sensing probe in a probe adhesive to obtain a probe modification solution; The probe modification liquid is dropped onto the working electrode surface of the flexible screen-printed electrode, and the electrochemical sensor is obtained after drying.

9. Use of the biomass electrochemical sensing probe according to claim 6 or the electrochemical sensor according to claim 7 in detecting vitamin C.

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