A transferrin / flurographene modified electrode and a preparation method and application thereof

Abstract

The application discloses a transferrin / fluorinated graphene modified electrode and a preparation method and application thereof. Fluorinated graphene is ultrasonically dispersed into a solvent to form a suspension, transferrin is added into the suspension to perform a substitution reaction, and a transferrin / fluorinated graphene composite material is obtained; the transferrin / fluorinated graphene composite material is mixed with a liquid binder, and is coated on a working electrode after drying, so that the transferrin / fluorinated graphene modified electrode is obtained. The method has the advantages of small operation difficulty, high efficiency and easy scaling, and the obtained transferrin / fluorinated graphene modified electrode is applied to preparation of a uranyl ion electrochemical sensor, and the sensitivity and selectivity of the uranyl ion electrochemical sensor are improved.

Description

Technical Field

[0001] This invention relates to a sensing electrode, particularly to a transferrin / fluorinated graphene modified electrode, and also to its preparation method and its application in the electrochemical detection of uranyl ions, belonging to the field of electrochemical detection technology. Background Technology

[0002] Uranium is widely found in natural environments such as rocks, soil, and sediments. A certain amount of uranium is also present in the air in uranium mining areas and nuclear power plant regions. In non-uranium mining environments, uranium mainly exists as soluble uranyl ions (UO2). 2+ Uranium exists in water bodies in the form of heavy metal toxicity and radioactive toxicity. Excessive exposure to uranium can cause both heavy metal toxicity and radioactive toxicity to the human body. Therefore, it is essential to detect trace amounts of uranium in water bodies.

[0003] Currently, the detection of uranyl ions mainly relies on instrumental analysis methods (inductively coupled plasma atomic emission spectrometry, ion chromatography, X-ray fluorescence spectrometry, etc.). These methods all require pre-concentration and pre-extraction processes to some extent, placing demands on the detection instruments and failing to meet the requirements for rapid on-site detection. Therefore, researching and developing a simple and rapid on-site trace uranium detection method for the on-site detection of trace uranium in water bodies is of certain significance for analyzing and controlling the level of human exposure to uranium and reducing the dual harm of uranium.

[0004] Electrochemical detection methods offer advantages such as simple instrumentation, low cost, and portability, making them suitable for on-site detection of trace uranium in aquatic environments. Further modification of the working electrode with electrode modification materials can lower the electrochemical detection limit of uranyl ions and improve detection stability and selectivity. Fluorinated graphene (FG), a two-dimensional material with high specific surface area, high electronegativity, and high stability, can increase the active surface area of ​​the electrode and enhance the response of the electrochemical signal when used as an electrode modification material. However, it has detection limitations for extremely low concentrations of uranium, which restricts the sensitivity and accuracy of electrochemical detection based on fluorinated graphene probe electrodes for trace uranium detection in real-world aquatic environments. Therefore, how to modify fluorinated graphene and combine it with electrochemical detection methods to improve the detection of trace uranium in water has become a challenge in existing technologies. Summary of the Invention

[0005] To further reduce the electrochemical detection limit of uranyl ions in existing electrochemical detection methods and to improve the stability and selectivity of detection, the first objective of this invention is to provide a transferrin / fluorinated graphene-modified electrode. This electrode uses a transferrin / fluorinated graphene composite material as a probe to modify the surface of the working electrode, combining the advantages of both fluorinated graphene and transferrin. Its participation in the construction of a uranyl ion electrochemical sensor can improve the sensitivity and selectivity for trace uranyl ions in the aquatic environment.

[0006] The second objective of this invention is to provide a method for preparing a transferrin / fluorinated graphene modified electrode. This method utilizes the fact that fluorinated graphene, while adsorbing transferrin, undergoes nucleophilic substitution with the amino groups in transferrin. The CF half-ionic bonds in the fluorinated graphene break to form new CN covalent bonds, resulting in a composite material of fluorinated graphene and transferrin (Tf / FG). This method has advantages such as ease of operation, high efficiency, and easy scalability.

[0007] The third objective of this invention is to provide an application of a transferrin / fluorinated graphene modified electrode, which can be used as a sensing electrode for the electrochemical detection of uranyl ions. This can improve the sensitivity and selectivity of the uranyl ion electrochemical sensor and is expected to be applied in the rapid detection of trace uranyl ions in aquatic environments with high sensitivity and selectivity.

[0008] To achieve the above-mentioned technical objectives, the present invention provides a method for preparing a transferrin / fluorinated graphene modified electrode. The method involves ultrasonically dispersing fluorinated graphene in a solvent to form a suspension, adding transferrin to the suspension to carry out a substitution reaction, and obtaining a transferrin / fluorinated graphene composite material. The transferrin / fluorinated graphene composite material is then mixed with a liquid binder and coated onto a working electrode, followed by drying to obtain the final product.

[0009] In the preparation method of this invention, on the one hand, fluorinated graphene has a large specific surface area and strong electronegativity, thus possessing a strong adsorption capacity. Transferrin can be adsorbed onto the surface of fluorinated graphene using physical action, forming a compound with a large molecular structure. On the other hand, at a certain temperature, transferrin and fluorinated graphene are fully in contact. The amino groups contained in transferrin can undergo nucleophilic substitution reactions with fluorinated graphene, causing the CF half-ionic bonds of fluorinated graphene to be replaced by CN covalent bonds. Under chemical action, the binding between transferrin and fluorinated graphene is further strengthened, improving the yield and stability of the transferrin / fluorinated graphene composite material, resulting in a fully composite transferrin / fluorinated graphene product.

[0010] In this invention, fluorinated graphene is ultrasonically dispersed in a solvent and then reacted with transferrin to form a suspension of transferrin / fluorinated graphene composite material. This suspension is then mixed thoroughly with detergent and poured into centrifuge tubes for centrifugation to remove unreacted substances. The centrifuged and washed transferrin / fluorinated graphene product is then placed in a freeze dryer for drying. The dried agglomerates are then crushed to obtain a dried powder of the transferrin / fluorinated graphene composite material.

[0011] As a preferred embodiment, the detergent includes, but is not limited to, deionized water, methanol / water mixture (concentration not exceeding 50%), ethanol / water mixture (concentration not exceeding 50%), etc. During centrifugal washing, the centrifugation speed is 1000–10000 rpm, the centrifugation time is 5–40 min, and the number of centrifugal washing cycles is 1–4. More preferably, the detergent is an ethanol / water mixture (concentration not exceeding 30%). More preferably, the centrifugation speed is 3000–6000 rpm, the centrifugation time is 10–20 min, and the number of centrifugal washing cycles is 2–3.

[0012] As a preferred embodiment, the freeze-drying process is carried out at a temperature of -100 to 0°C, and the freeze-drying time for the centrifuged precipitate is 4 to 36 hours. The freeze-drying time depends on the amount of centrifuged precipitate, and generally continues until the mixture clumps together, cracks appear, and there is no moisture remaining. More preferably, the freeze-drying temperature is -40 to -10°C, and the freeze-drying time is 6 to 12 hours.

[0013] As a preferred embodiment, the solvent is at least one selected from water, dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, hydroxyethylpiperazine ethanethiol, phosphate buffer, methanol, ethanol, sodium chloride solution, phosphate solution, sulfate solution, and ionic liquid. When the solvent is a mixture of water and methanol, the methanol concentration is 1–40 vol%; when the solvent is a mixture of water and ethanol, the ethanol concentration is 1–40 vol%. More preferably, it is water, a methanol / water mixture (methanol concentration 10–30 vol%), or an ethanol / water mixture (ethanol concentration 10–30 vol%).

[0014] As a preferred embodiment, the ultrasonic dispersion power is 50–300 W, and the time is 0.5–10 min. More preferably, the ultrasonic dispersion power is 100–200 W, and the time is 1–5 min.

[0015] As a preferred embodiment, the mass ratio of fluorinated graphene to transferrin is (0.1–10):1. Appropriate amounts of fluorinated graphene and transferrin are beneficial for their composite composition and their specific recognition ability as probe materials, thereby improving the overall performance of the sensor. If the mass ratio of fluorinated graphene to transferrin is too high, the nucleophilic substitution reaction and composite effect will be insignificant, resulting in too few uranyl ion specific binding sites on the modified electrode and low charge transport efficiency on the electrode surface. Conversely, if the mass ratio of fluorinated graphene to transferrin is too low, transferrin will aggregate, and the steric hindrance effect of the protein will become significant, thus reducing the composite efficiency of fluorinated graphene and transferrin, and simultaneously reducing the mass transfer efficiency of uranyl ions reaching the effective binding reaction sites. All of these will lead to an increase in the detection limit, and a decrease in selectivity and stability of the prepared sensor. A further preferred mass ratio of fluorinated graphene to transferrin is (1–7):1.

[0016] As a preferred embodiment, the concentration of the fluorinated graphene added to the solvent is 0.1–10 g / L; a more preferred concentration is 1–5 g / L.

[0017] As a preferred embodiment, the concentration of transferrin added to the suspension is 1–500 mg / mL; a more preferred concentration is 20–200 mg / L.

[0018] As a preferred embodiment, the transferrin has a purity >90.0 wt.%, a water content <10 wt.%, and an iron-binding capacity of 100–1000 μg / g. More preferably, the transferrin has a purity >95.0 wt.%, a water content <5 wt.%, and an iron-binding capacity of 400–500 μg / g.

[0019] As a preferred embodiment, the aqueous solution formed by the transferrin should be transparent and turbid, showing a single distinct band in SDS-PAGE, with a pH of 5.5–8.0, and negative for Hiv 1 & 2, HBsAg, Hcv, and Hbc antibodies. A further preferred pH is 6.5–7.8. In this invention, pH has a significant impact on the properties of transferrin. Too low or too high a pH will lead to denaturation and inactivation of transferrin, disrupting its tertiary structure, including the uranyl ion-accommodating cavity (i.e., the specific recognition site), which is detrimental to subsequent material preparation and detection performance.

[0020] As a preferred embodiment, the fluorinated graphene has a purity ≥95 wt.%, a fluorination degree (i.e., the ratio of fluorine atoms to carbon atoms) ≥20 at.%, a thickness of one atomic layer to one hundred atomic layers, and a lateral dimension ≥10 nm. More preferably, the fluorinated graphene powder has a purity ≥99 wt.%, a fluorination degree (i.e., the ratio of fluorine atoms to carbon atoms) ≥50 at.%, a thickness of one atomic layer to twenty atomic layers, and a lateral dimension ≥100 nm. Too low a fluorination degree in the fluorinated graphene will result in too few fluorine atom active sites for nucleophilic substitution reactions with transferrin, leading to low binding density and strength, which is detrimental to its application as a device probe material.

[0021] As a preferred embodiment, the substitution reaction conditions are: temperature 20–70°C and time 4–24 h. Within the temperature range of this invention, the special tertiary structure of transferrin can be preserved while promoting sufficient contact between transferrin and fluorinated graphene. This facilitates the nucleophilic substitution reaction between fluorinated graphene and the amino groups contained in transferrin during the adsorption of transferrin, resulting in the replacement of the CF half-ionic bonds of fluorinated graphene with CN covalent bonds. This improves the yield and stability of the transferrin / fluorinated graphene composite material, yielding a fully composite transferrin / fluorinated graphene product. Excessive temperature will destroy the special tertiary structure of transferrin, while excessively low temperature will prevent the reaction activation energy from being reached, thus hindering the reaction. A further preferred temperature is 30–50°C, and the time is 12–24 h.

[0022] As a preferred embodiment, the liquid binder is at least one of Nafion, polyvinylidene fluoride, polyacrylic acid, polyvinyl alcohol, polytetrafluoroethylene, carboxymethyl cellulose, chitosan, sodium alginate, and β-cyclodextrin, with a content of 1-20 wt.%. More preferably, the liquid binder is at least one of Nafion, polyvinylidene fluoride, polyacrylic acid, and polyvinyl alcohol, with a content of 3-10 wt.%.

[0023] As a preferred embodiment, the solid-liquid ratio of the transferrin / fluorinated graphene composite material to the liquid binder is (0.1–2) mg: 1 mL. By controlling the solid-liquid ratio of the transferrin / fluorinated graphene composite material to the liquid binder, the coating thickness and area of ​​the transferrin / fluorinated graphene composite material on the working electrode can be controlled. If the solid-liquid ratio is too high, it cannot be effectively fixed on the electrode; if the solid-liquid ratio is too low, it will reduce the sensitivity and selectivity of the uranyl ion electrochemical sensor.

[0024] As a preferred embodiment, the ferritin / fluorinated graphene composite material is mixed with the liquid binder by shaking or stirring for 0.1–10 min until a uniform and stable gray-black suspension is produced. More preferably, a vortex mixer is used for stirring for 0.1–1 min.

[0025] As a preferred embodiment, the working electrode is one of glassy carbon, carbon paste, platinum, gold, and graphite; other materials that can be used as electrodes are also applicable to this invention. Electrode shapes include, but are not limited to, disc-shaped, ring-shaped, foil-shaped, filament-shaped, rod-shaped, and mesh-shaped. Glassy carbon or platinum electrodes are further preferred; the shapes are disc-shaped and foil-shaped.

[0026] As a preferred embodiment, the working area of ​​the working electrode is 0.1–10 cm². 2 The working area of ​​the working electrode can affect the sensor's detection limit, stability, and accuracy to a certain extent. For example, if the working area of ​​the working electrode is too small, the sensor's detectable area will be too small, and the detection signal obtained at the same uranyl ion concentration will be weaker, thus increasing the lowest detectable concentration, i.e., increasing the sensor's detection limit. If the working area of ​​the working electrode is too large, it will be difficult to control the uniformity of the film formed on the electrode surface, thereby reducing the sensor's detection stability, selectivity, and accuracy. A further preferred size is 0.25–3 cm. 2 .

[0027] As a preferred embodiment, the working electrode is polished. The polishing method includes polishing with polishing powder, ultrasonic cleaning, and drying. The polishing steps are as follows: the working electrode is polished sequentially for 3 to 10 minutes with a mixture of alumina powder / ultrapure water with particle sizes of 1.0, 0.3, and 0.05 μm dropped onto a polishing table; then it is ultrasonically cleaned in ultrapure water for 0.5 to 5 minutes; and then it is dried in an oven for 30 to 120 minutes at a drying temperature of 60 to 100°C.

[0028] As a preferred embodiment, the drying conditions are: temperature 0–20°C, time 4–24 hours. In this invention, the working electrode requires low-temperature, long-term drying primarily to prevent irreversible denaturation and coagulation of transferrin due to excessively high temperatures during the drying process. Low-temperature, long-term drying ensures that the transferrin's structure is not damaged during drying, while maintaining its activity, which is beneficial for improving the material's sensing performance. A further preferred method is a drying temperature of 0–10°C and a time of 8–12 hours.

[0029] As a preferred embodiment, the coating is a drop coating. The drop coating method includes, but is not limited to, using a pipette, microsyringe, dropper or other equipment to draw up the suspension and then drop coating it onto the surface of the working electrode. The amount of drop coating is until a thin film of suspension is uniformly coated on the surface of the electrode at each point, while ensuring that the suspension does not overflow the surface of the electrode.

[0030] This invention also provides a transferrin / fluorinated graphene-modified electrode, obtained by the above preparation method. This electrode uses a transferrin / fluorinated graphene composite material as a probe to modify the surface of the working electrode, combining the advantages of both fluorinated graphene and transferrin. Its participation in the construction of a uranyl ion electrochemical sensor can improve the sensitivity and selectivity for trace uranyl ions in the aquatic environment.

[0031] The present invention also provides an application of a transferrin / fluorinated graphene modified electrode, which can be used as a sensing electrode for the electrochemical detection of uranyl ions to improve the sensitivity and selectivity of the uranyl ion electrochemical sensor.

[0032] The mechanism by which the transferrin / fluorinated graphene-modified electrode of this invention improves the sensitivity and selectivity of the uranyl ion electrochemical sensor lies in the following: Fluorinated graphene, a two-dimensional material with advantages such as high specific surface area, high electronegativity, and high stability, increases the active surface area of ​​the electrode and enhances the response of the electrochemical signal when used as an electrode modifier. The transferrin in the composite material possesses a unique crystal structure advantage; it consists of a polypeptide chain folded into two homologous lobes connected by a short polypeptide. Each lobe-like structure contains a metal-binding site. The two tyrosine residues, one aspartic acid group, and one carbonate ion in these two metal-binding sites combine with uranyl ions to form a relatively stable uranyl-transferrin derivative, thereby increasing the specific recognition ability of uranyl ions. When transferrin combines with fluorinated graphene, its multiple active sites and large specific surface area allow for rapid recognition and capture of uranyl ions, thus significantly improving the sensitivity and selectivity of the uranyl ion electrochemical sensor.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1) The transferrin / fluorinated graphene modified electrode provided by the present invention uses the transferrin / fluorinated graphene composite material as a probe to modify the surface of the working electrode, which combines the advantages of fluorinated graphene and transferrin. It can improve the sensitivity and selectivity of trace uranyl ions in the water environment by participating in the construction of uranyl ion electrochemical sensor.

[0035] 2) The preparation method of this invention utilizes fluorinated graphene to undergo nucleophilic substitution with the amino groups in transferrin while adsorbing transferrin. The CF semi-ionic bonds of fluorinated graphene break to form new CN covalent bonds, resulting in a composite material of fluorinated graphene and transferrin (Tf / FG). This method has the advantages of simple operation, high efficiency, and easy scalability.

[0036] 3) This invention constructs a novel electrochemical biosensor by modifying the working electrode surface of an electrochemical sensor with a transferrin / fluorinated graphene composite material as a probe material. By grafting and adsorbing abundant transferrin with specific recognition and adsorption capabilities for uranyl ions onto the surface of fluorinated graphene with a large specific surface area and strong adsorption capacity, trace detection of uranyl ions in low-concentration aquatic environments can be performed, resulting in a highly sensitive, selective, and stable electrochemical sensor modified with a transferrin / fluorinated graphene composite material.

[0037] 4) This invention provides a convenient, sensitive, low-cost, and fast-responding electrochemical sensing detection method for detecting trace uranyl ions in aquatic environments, which combines the advantages of two-dimensional materials and biological materials. It has certain application value in the fields of wastewater treatment and discharge, water environment monitoring and protection. Attached Figure Description

[0038] Figure 1 This is a flowchart of the fabrication process for transferrin / fluorinated graphene modified electrodes and a flowchart of the electrochemical uranyl ion detection process.

[0039] Figure 2 These are atomic force microscopy images of pure fluorinated graphene (FG), pure transferrin (Tf), and the transferrin / fluorinated graphene composite material (Tf / FG) prepared in Example 1.

[0040] Figure 3 These are Raman spectra of pure fluorinated graphene (FG), pure transferrin (Tf), and the transferrin / fluorinated graphene composite material (Tf / FG) obtained in Example 1. Figure 3 This indicates that the present invention has successfully prepared a transferrin / fluorinated graphene composite material.

[0041] Figure 4 It is the polished glassy carbon electrode obtained in Example 1. Figure 4 (a) Macro chart and Figure 4 (c) Microscopic images, and the polished electrode after modification with transferrin / fluorinated graphene composite material. Figure 4 (b) Macro chart and Figure 4 (d) Microscopic image.

[0042] Figure 5The graph shows a comparison of peak currents of uranyl ions and other metal interfering ions detected by the DPV method using a glassy carbon electrode modified with the transferrin / fluorinated graphene composite material prepared in Example 1 of this invention.

[0043] Figure 6 The graph shows the relationship between the DPV peak current signal of uranyl ions detected by the glassy carbon electrode modified with the transferrin / fluorinated graphene composite material prepared in this invention and the exposure time of the electrode in air.

[0044] Figure 7 The graph shows the relationship between the DPV peak current signal of uranyl ions and the enrichment time of the electrode in the uranyl ion solution when the glassy carbon electrode is modified with the transferrin / fluorinated graphene composite material prepared in this invention is used. Detailed Implementation

[0045] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described below are only a part of the embodiments, and all other embodiments obtained by those skilled in the art without creative effort are still within the protection scope of this invention.

[0046] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0047] Example 1

[0048] 1) Weigh 50 mg of fluorinated graphene powder (purity 99.5 wt.%, thickness 1–8 atomic layers, fluorination degree 60 at.%, lateral dimension ≥50 nm) and add it to 100 mL of 20% ethanol / water mixed solution. Disperse the solution ultrasonically at 150 W for 3 min to ensure uniform mixing of the fluorinated graphene and solvent, forming a grayish-white suspension. Then, weigh 50 mg of transferrin powder (protein purity >98.0 wt.%, water content <2 wt.%, transferrin iron-binding capacity 400–500 μg / g, and the pH of the aqueous solution formed by transferrin is 6.7–7.5) and add it to the fluorinated graphene suspension. Vortex mix for 3 min to obtain a uniform mixed suspension. Then heat and stir at 40°C for 12 h, causing the suspension to change from grayish-white to grayish-black. This indicates that the fluorinated graphene has fully adsorbed the transferrin and undergone an amino nucleophilic substitution reaction with it. The sample was then washed three times by centrifugation with a 20% ethanol / water mixture at a speed of 5000 rpm for 15 min. The centrifuged precipitate was then placed in a freeze dryer with the cold trap temperature lowered to -40℃ for 12 h to obtain transferrin / fluorinated graphene powder.

[0049] 2) The glassy carbon electrode was polished sequentially for 10 min with an ultrapure water mixture containing alumina powder with particle sizes of 1.0, 0.5, and 0.03 μm, which was dropped onto the polishing table. Then it was ultrasonically cleaned in ultrapure water for 1 min and then dried in an oven for 120 min at a drying temperature of 60℃.

[0050] 3) Weigh 5 mg of transferrin / fluorinated graphene composite powder and dissolve it in 5 mL of 20% ethanol / water mixture. Add 40 μl of Naifion and mix well to obtain a 1 mg / mL transferrin / fluorinated graphene solution. Take 10 μL of the 1 mg / mL transferrin / fluorinated graphene solution and uniformly drop-coat it onto the surface of a glassy carbon electrode. Dry it at 5 °C for 12 h to obtain a glassy carbon working electrode modified with transferrin / fluorinated graphene.

[0051] 4) A three-electrode system was constructed by combining a transferrin / fluorinated graphene-modified glassy carbon working electrode (disc-shaped, 3 mm in diameter), a saturated calomel reference electrode, and a platinum wire auxiliary electrode to create an electrochemical sensor for uranyl detection. The electrolyte was an ultrapure aqueous solution with a uranyl ion concentration of 0.1 ppm (approximately 0.04 μM) and contained 0.1 M KCl as a supporting electrolyte. Other interfering metal ions of similar or equal concentration were also added. The electrode system was placed in the electrolyte, and ion concentration was detected at room temperature (25 ± 2 °C) using the DPV method. The AC voltage amplitude was 5 mV, the applied potential was 200 mV, and the voltage frequency ranged from 0.01 Hz to 100 kHz.

[0052] 5) Environmental stability testing was performed on the electrochemical sensor for uranyl detection. The test method involved exposing the transferrin / fluorinated graphene-modified glassy carbon electrode to air and measuring the DPV signal of uranyl ions at different time periods, under the same conditions as in step 4).

[0053] The transferrin / fluorinated graphene composite material prepared in step 1) of this embodiment was characterized by atomic force microscopy, and the results are as follows: Figure 2 As shown in the figure, FG, Tf, and Tf / FG represent atomic force microscopy images of the initial fluorinated graphene, the initial transferrin, and the transferrin / fluorinated graphene composite, respectively. Comparing the three images, it can be seen that after the substitution reaction between fluorinated graphene and transferrin, the transferrin is uniformly distributed on the fluorinated graphene sheets.

[0054] The transferrin / fluorinated graphene composite material prepared in step 1) of this embodiment was characterized by Raman spectroscopy, and the results are as follows: Figure 3As shown, FG, Tf, and Tf / FG represent the Raman spectra of the initial fluorinated graphene, the initial transferrin, and the transferrin / fluorinated graphene composite, respectively. Comparing the three spectra reveals that fluorinated graphene and transferrin each have corresponding characteristic peaks in their Raman spectra. The Raman spectrum of the composite material shows the presence of both characteristic peaks, indicating a reaction between transferrin and fluorinated graphene. Furthermore, the tertiary structure of the transferrin protein in the transferrin / fluorinated graphene composite material, which adsorbs uranyl ions, remains intact, confirming that transferrin and fluorinated graphene combine to form a new transferrin / fluorinated graphene complex.

[0055] The macroscopic and microscopic images of the surface of the glassy carbon working electrode modified with transferrin / fluorinated graphene prepared in step 3) of this embodiment are shown below. Figure 4 As shown, the surface of the glassy carbon electrode modified with transferrin / fluorinated graphene material is no longer a mirror surface, but is uniformly covered with a thin film. The changes on the electrode surface under a metallographic microscope further indicate that the transferrin / fluorinated graphene material is uniformly and firmly distributed on the surface of the glassy carbon electrode.

[0056] The anti-metal ion interference results of the electrochemical sensor for uranyl detection constructed in step 4) of this embodiment are as follows: Figure 5 As shown, a distinct DPV current signal peak of uranyl ion reduction (reduction peak position: -0.22V, intensity: 15.59μA) can be detected at a concentration of 0.1ppm, while the current change intensity of interfering ions is significantly weaker than that of uranyl ions.

[0057] The environmental stability test results of the electrochemical sensor for uranyl detection constructed in step 4) of this embodiment are as follows: Figure 6 As shown, the transferrin / fluorinated graphene-modified electrode, after being exposed to air for 28 days, still showed a significant uranyl ion detection signal, and the signal intensity began to decrease gradually, indicating that the prepared transferrin / fluorinated graphene-modified electrode has good environmental stability.

[0058] The effect of the uranyl ion enrichment time on the detection results of the electrochemical sensor for uranyl detection constructed in step 4) of this embodiment, such as... Figure 7 As shown (test conditions are the same as in step 3), when the enrichment time is 8 minutes, the change in the peak current signal of the DPV test of uranyl ions begins to enter the plateau period. The required enrichment time is relatively short, which meets the conditions for rapid detection and is suitable for simple and rapid on-site detection of trace uranium.

[0059] Example 2

[0060] The preparation steps and process conditions are the same as in Example 1, except that the amount of fluorinated graphene powder is changed to 100 mg and the amount of transferrin powder is changed to 40 mg.

[0061] The obtained transferrin / fluorinated graphene composite material was tested by atomic force microscopy and Raman spectroscopy. The test results were similar to those of Example 1, confirming that transferrin and fluorinated graphene formed a transferrin / fluorinated graphene complex through a substitution reaction. The transferrin was uniformly distributed on the fluorinated graphene sheets, and the distribution density was reduced, which was attributed to the decrease in transferrin content.

[0062] Optical microscopy revealed that the surface of the glassy carbon working electrode modified with transferrin / fluorinated graphene was similar to that in Example 1, except that the flatness of the covering film was slightly reduced in the microscopic images, which was attributed to the increased content of fluorinated graphene sheets. This confirms that a transferrin / fluorinated graphene modified electrode can be successfully prepared using this ratio of fluorinated graphene to transferrin powder.

[0063] The constructed uranyl ion electrochemical sensor was subjected to DPV testing to resist interference from metal ions. A significant DPV current signal peak for uranyl ion reduction was detected at a concentration of 0.1 ppm (reduction peak position: -0.22 V, intensity: 12.42 μA). The decrease in current intensity was attributed to the reduction of transferrin on the electrode surface and its uranyl ion-specific binding sites, as well as the reduced charge transport efficiency at the reduction site. The current change intensity of interfering ions was significantly weaker than that of uranyl ions.

[0064] The constructed uranyl ion electrochemical sensor was subjected to environmental stability tests. After being exposed to air for 25 days, the transferrin / fluorinated graphene modified electrode still showed a significant uranyl ion detection signal, and the signal intensity decline trend began to level off, indicating that the prepared transferrin / fluorinated graphene modified electrode has good environmental stability.

[0065] The enrichment time of the constructed uranyl ion electrochemical sensor was tested. When the enrichment time was 7.8 min, the change in the peak current signal of the DPV test of uranyl ions began to plateau, differing from the enrichment time in Example 1 by only 0.2 min. This indicates that the change in transferrin content has little impact on the enrichment time, and also shows that the binding sites of transferrin can uniformly and rapidly recognize and coordinate uranyl ions. The required enrichment time is short, providing conditions for rapid detection, and is suitable for simple and rapid on-site detection of trace uranium.

[0066] Example 3

[0067] The preparation steps and process conditions are the same as in Example 1, except that the amount of fluorinated graphene powder is changed to 175 mg and the amount of transferrin powder is changed to 25 mg.

[0068] The obtained transferrin / fluorinated graphene composite material was tested by atomic force microscopy and Raman spectroscopy. The test results were similar to those of Example 1, confirming that transferrin and fluorinated graphene formed a transferrin / fluorinated graphene complex through a substitution reaction. Furthermore, the distribution of transferrin on the surface of the fluorinated graphene sheets became sparser, and the uniformity became difficult to control, which was attributed to the further reduction in transferrin content.

[0069] Optical microscopy revealed that the surface of the glassy carbon working electrode modified with transferrin / fluorinated graphene was similar to that in Example 1. However, the smoothness of the covering film was somewhat affected in the microscopic images, becoming somewhat rougher. Occasionally, uncomplexed fluorinated graphene sheets were observed exposed on the film surface, attributed to the further increase in the content of fluorinated graphene sheets. Nevertheless, the integrity and overall quality of the film remained relatively good. This confirms that a transferrin / fluorinated graphene modified electrode can be successfully prepared using the specified ratio of fluorinated graphene to transferrin powder.

[0070] The constructed uranyl ion electrochemical sensor was subjected to DPV testing to resist interference from metal ions. A significant DPV current signal peak for uranyl ion reduction was detected at a concentration of 0.1 ppm (reduction peak position: -0.22 V, intensity: 8.18 μA). The further decrease in current intensity was attributed to the further reduction of transferrin and its uranyl ion-specific binding sites, and the further decrease in charge transport efficiency at the reduction site. However, the current change intensity of interfering ions was still significantly weaker than that of uranyl ions.

[0071] The constructed uranyl ion electrochemical sensor was subjected to environmental stability tests. After being exposed to air for 18 days, the transferrin / fluorinated graphene modified electrode still detected a significant uranyl ion detection signal, and the signal intensity decline trend began to level off, indicating that the prepared transferrin / fluorinated graphene modified electrode still has good environmental stability.

[0072] The enrichment time of the constructed uranyl ion electrochemical sensor was tested. Because the DPV peak current signal of the uranyl ion binding site began to plateau after 7.6 min of enrichment, it was not significantly different from the enrichment time described in Example 1. The required enrichment time is short, providing conditions for rapid detection, and is suitable for simple and rapid on-site detection of trace uranium.

[0073] Comparative Example 1

[0074] The preparation steps and process conditions are the same as in Example 1, except that the amount of fluorinated graphene powder is changed to 200 mg and the amount of transferrin powder is changed to 10 mg.

[0075] Atomic force microscopy (AFM) analysis of the prepared material revealed only the morphology and thickness characteristics of the fluorinated graphene sheets; no significant transferrin particles were detected on the sheet surface. Raman spectroscopy analysis showed only strong Raman spectral peaks for fluorinated graphene, while no characteristic peaks for transferrin were detected. Optical microscopy observation of the glassy carbon working electrode surface modified with the prepared material revealed only the arrangement of fluorinated graphene sheets covered by the thin film. This indicates that although a nucleophilic substitution reaction can occur upon contact between fluorinated graphene and transferrin, the low transferrin content results in insufficient contact between transferrin and fluorinated graphene, making it difficult to confirm the effective preparation of the transferrin / fluorinated graphene composite material and its modified electrode.

[0076] The constructed uranyl ion electrochemical sensor was subjected to DPV testing to resist interference from metal ions. At a concentration of 0.1 ppm, no obvious DPV current signal of uranyl ions or interfering ions was detected. This was also attributed to the absence of transferrin and its uranyl ion-specific binding sites, while fluorinated graphene itself does not have specific binding sites.

[0077] As shown in Comparative Example 1, an excessively high mass ratio of added fluorinated graphite to transferrin will cause the composite material, modified electrode, and sensor to fail, thus failing to meet the requirements of practical applications.

[0078] Comparative Example 2

[0079] The preparation steps and process conditions are the same as in Example 1, except that the amount of fluorinated graphene powder is changed to 10 mg and the amount of transferrin powder is changed to 200 mg.

[0080] Atomic force microscopy (AFM) analysis of the prepared material revealed only the morphology and thickness characteristics of dispersed transferrin aggregates, with no fluorinated graphene sheets detected. Raman spectroscopy analysis showed only strong Raman peaks characteristic of transferrin, while no characteristic peaks of fluorinated graphene were detected. Optical microscopy observation of the glassy carbon working electrode surface modified with the prepared material revealed only dispersed transferrin aggregates on the coated film. This indicates that although nucleophilic substitution reactions may occur upon contact between fluorinated graphene and transferrin, the low fluorinated graphene content results in insufficient contact between transferrin and fluorinated graphene, making it difficult to confirm the effective preparation of the transferrin / fluorinated graphene composite material and its modified electrode.

[0081] The constructed uranyl ion electrochemical sensor was subjected to DPV testing to resist interference from metal ions. At a concentration of 0.1 ppm, no obvious DPV current signal of uranyl ions or interfering ions was detected. This was attributed to the fact that the low content of fluorinated graphene prevented transferrin from being effectively loaded onto the working electrode surface as a signal probe for detecting ions, resulting in a low level of charge transport signal from the electrochemical reduction reaction of the detected ions. At the same time, the high content of transferrin made its steric hindrance effect very significant, hindering the mass transfer process of the detected ions reaching the electrode surface to undergo electrochemical reduction reaction.

[0082] Comparative Example 2 shows that an excessively low mass ratio of added fluorinated graphite to transferrin can also lead to performance failure of the composite material, modified electrode, and sensor, thus failing to meet practical application requirements.

[0083] Comparative Example 3

[0084] The preparation steps and process conditions are the same as in Example 1, except that transferrin is added to the fluorinated graphene suspension and shaken with a vortex mixer for 3 minutes to obtain a uniform mixed suspension, and then heated and stirred at 80°C for 12 hours.

[0085] Atomic force microscopy revealed no transferrin loading on the graphene surface in the prepared material. This was attributed to the fact that the transferrin had denatured and solidified at 80°C and lost its activity. No effective nucleophilic substitution reaction occurred between the transferrin and fluorinated graphene, and no transferrin / fluorinated graphene composite material was obtained.

Claims

1. An application of a transferrin / fluorinated graphene modified electrode, characterized in that: It is used as a sensing electrode for the electrochemical detection of uranyl ions; The preparation process of the transferrin / fluorinated graphene modified electrode is as follows: fluorinated graphene is ultrasonically dispersed in a solvent to form a suspension, transferrin is added to the suspension to carry out a substitution reaction, and a transferrin / fluorinated graphene composite material is obtained; the transferrin / fluorinated graphene composite material is mixed with a liquid binder and coated on the working electrode, and then dried to obtain the final product.

2. The application of the transferrin / fluorinated graphene modified electrode according to claim 1, characterized in that: The solvent is at least one of water, dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, hydroxyethylpiperazine ethanethiolic acid, phosphate buffer salt, methanol, ethanol, sodium chloride solution, phosphate solution, sulfate solution, and ionic liquid.

3. The application of the transferrin / fluorinated graphene modified electrode according to claim 1, characterized in that: The mass ratio of fluorinated graphene to transferrin is (0.1~10):

1.

4. The application of the transferrin / fluorinated graphene modified electrode according to any one of claims 1 to 3, characterized in that: The transferrin has a purity >90.0 wt.%, a water content of less than 10 wt.%, and an iron-binding capacity of 100~1000 μg / g; The aqueous solution formed by the transferrin should be turbid and transparent, showing a single distinct band in SDS-polyacrylamide gel electrophoresis, with a pH of 5.5-8.0, and negative for Hiv 1 & 2, HBSAg, Hcv and Hbc antibodies; The fluorinated graphene has a purity of ≥95wt.%, a fluorination degree of ≥20at.%, a thickness of 1 atom layer to 100 atom layers, and a lateral dimension of ≥10nm.

5. The application of the transferrin / fluorinated graphene modified electrode according to claim 4, characterized in that: The conditions for the substitution reaction are: temperature 20~70℃, time 4~24h.

6. The application of the transferrin / fluorinated graphene modified electrode according to claim 1 or 2, characterized in that: The liquid binder is at least one of Nafion, polyvinylidene fluoride, polyacrylic acid, polyvinyl alcohol, polytetrafluoroethylene, carboxymethyl cellulose, chitosan, sodium alginate and β-cyclodextrin, with a content of 1~20 wt.%. The solid-liquid ratio of the transferrin / fluorinated graphene composite material to the liquid binder is (0.1~2) mg:1 mL.

7. The application of the transferrin / fluorinated graphene modified electrode according to claim 1, characterized in that: The working electrode is one of glassy carbon, carbon paste, platinum, gold, and graphite; The working area of ​​the working electrode is 0.1~10cm². 2 .

8. The application of the transferrin / fluorinated graphene modified electrode according to claim 1, characterized in that: The drying conditions are: temperature 0~20℃, time 4~24h.

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