Preparation method and application of low working potential hydrogen sulfide sensor

Abstract

This invention belongs to the field of hydrogen sulfide sensor technology, specifically relating to a method for preparing and applying a low-operating-potential hydrogen sulfide sensor. The method for preparing the low-operating-potential hydrogen sulfide sensor provided by this invention involves adding biomass carbon material Bcn and nano-CuFe2O4 catalyst to ultrapure water for ultrasonic dispersion and composite formation to obtain a CuFe2O4 / Bcn composite material. This composite material is then modified onto the surface of a glassy carbon electrode to form a working electrode. A saturated calomel electrode is used as the reference electrode, and a platinum wire is used as the counter electrode to form a three-electrode unit structure connected to an electrochemical workstation, thus obtaining the electrochemical sensor. The low-operating-potential hydrogen sulfide sensor provided by this invention has a low detection limit, avoids the deposition of oxidation byproduct sulfur on the electrode surface and interference from electroactive substances in the biological environment, and the electrode modification steps are simple and easy to operate. It allows for real-time continuous detection, has a wide linear detection range, and exhibits high stability, selectivity, sensitivity, and reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of hydrogen sulfide sensor technology, specifically relating to a method for preparing and applying a low-operating-potential hydrogen sulfide sensor. Background Technology

[0002] H₂S is an important reactive sulfur species with multiple physiological functions, including vasodilation, antioxidant regulation, and anti-inflammatory effects, playing a crucial role in various physiological processes. Literature reports that the average concentration of endogenous H₂S is in the mM range, but due to the rapid synthesis and metabolism of H₂S within cells, its concentration can vary rapidly, ranging from mM to several nM. Electrochemical methods enable continuous, in-situ detection of H₂S in the biological environment, and electrochemical sensors also possess advantages such as high sensitivity, high selectivity, low detection limit, fast response time, and strong miniaturization capabilities.

[0003] Among the many types of electrochemical sensors, ion-selective electrodes can measure sulfide ions (S₂O₃) with high selectivity and detection limits as low as 100 nM. 2- H₂S concentration is therefore widely used in commercial serum measurements. However, samples must undergo alkaline pretreatment to shift the proton dissociation equilibrium of H₂S to S. 2- The result is the concentration of total sulfides, not the actual H2S content in the sample. Furthermore, Ag2S forms on the electrode surface, requiring daily electrode repair and calibration.

[0004] Enzyme-based electrochemical sensors also have many applications in H2S detection. Iman Shahidi Pour Savizi prepared an amperometric biosensor for sulfide determination by immobilizing *Gnaphalium affine* peroxidase on a screen-printed electrode surface. Ampere measurements were performed at an application potential of 150 mV in the presence of hydroquinone as an electron medium and a 0.1 M phosphate buffer solution at pH 6.5. The sensor exhibited a linear range of 1.09–16.3 μM and a detection limit of 0.3 μM. Zhuang Liu developed a novel microbial sensor using recombinant *E. coli* expressing sulfides. The electrode showed good linear response in the 50 μM–5 mM range for sulfide detection, with a detection limit of 2.55 μM. Martin Dulac et al. covalently grafted hemoglobin onto the electrode surface, enabling reversible and rapid detection of H2S in aqueous solutions at concentrations up to 10 μM, with a detection limit of 0.35 μM. Good analytical performance was also obtained in human plasma without significant interference from the biological matrix. However, the detection limit of enzyme-based electrochemical sensors is at the micromolar level, making them unsuitable for use in biological environments. Furthermore, enzyme activity is highly environment-dependent and cannot be used long-term.

[0005] Direct electrochemical oxidation of H₂S typically occurs at high overpotentials and is susceptible to interference from other electroactive substances. Furthermore, the direct electrochemical oxidation of sulfides is highly irreversible; the deposited elemental sulfur passivates the electrode surface, thus reducing analytical sensitivity. Oxidized H₂S forms polysulfides upon contact with the sulfide layer, increasing the insulating layer and hindering any potential for continuous measurement. Modifying the electrode surface with a redox medium is one solution. In recent years, carbon nanomaterials, Cu, Mn, Zn, and Co-containing catalysts, and Ag and Pd noble metal materials have been primarily used as modified electrode materials for H₂S electrochemical sensing. However, the operating potential of these sensors has decreased, mostly falling between 0 and 0.55 V.

[0006] Carbon nanomaterials, such as carbon nanotubes (CNTs), carbon nanofibers (CNFs), and graphene, possess excellent electrochemical properties and have been widely used in electrode modification materials. However, their preparation heavily relies on fossil fuel-based precursors, involves stringent synthesis conditions, exerts significant environmental pressure, and incurs high production costs. Natural biomass materials, rich in carbon and possessing unique natural microstructures, can be transformed into carbon material frameworks, while minerals can be converted into catalysts, making them high-performance carbon materials for modifying electrodes. Common methods for preparing carbon nanomaterials from biomass include pyrolysis, chemical vapor deposition, cyclic oxidation, mechanical activation, and combustion.

[0007] Therefore, how to effectively utilize biocarbon materials to prepare a high-sensitivity, low-operating-potential hydrogen sulfide sensor has become an urgent technical problem to be solved. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention aims to provide a method for preparing and applying a low-operating-potential hydrogen sulfide sensor. The low-operating-potential hydrogen sulfide sensor provided by the present invention has a significantly reduced potential response, a low detection limit, avoids the deposition of sulfur by-products of oxidation on the electrode surface and interference from electroactive substances in the biological environment, and the electrode modification steps are simple and easy to operate. It can perform real-time continuous detection, and has a wide linear detection range, high stability, selectivity, sensitivity and reproducibility.

[0009] The technical solution of this invention is: A method for preparing a low-operating-potential hydrogen sulfide sensor, characterized by comprising the following steps: S1. Pretreatment of glassy carbon electrode: Polish and clean the glassy carbon electrode to obtain the pretreated glassy carbon electrode. S2. Activation of glassy carbon electrode: The glassy carbon electrode after the pretreatment in step S1 is activated by cyclic voltammetry to obtain the activated glassy carbon electrode. S3. Modification of glassy carbon electrode: The electrode is modified by drop coating: CuFe2O4 / Bcn composite material is drop coated onto the surface of the activated glassy carbon electrode in step S2, and then air-dried at room temperature to obtain CuFe2O4 / Bcn composite material modified electrode.

[0010] Furthermore, the preparation method of the CuFe2O4 / Bcn composite material includes the following steps: Nano-CuFe2O4 and biochar material Bcn were weighed into sample tubes and dispersed in ultrapure water to a concentration of 2.0 mg / mL. The tubes were then placed in an ultrasonic cleaner and ultrasonically dispersed for 5-10 min. The dispersed nano-CuFe2O4 and biochar material Bcn dispersions were then mixed at a volume ratio of 1:1 and ultrasonically dispersed for another 30 min to obtain the CuFe2O4 / Bcn composite material.

[0011] Furthermore, the preparation method of the nano-CuFe2O4 is as follows: Copper acetate and ferrous oxalate were ground evenly, and then anhydrous ethanol was added and stirred for 48 h. The mixture was then ground until it became a powder with a particle size of 1-3 μm. The powder was then calcined and ground to obtain nano-CuFe2O4. The obtained nano-CuFe2O4 had a particle size of 20-30 nm.

[0012] Furthermore, in the preparation method of the nano-CuFe2O4, the addition ratio of copper acetate, ferrous oxalate and anhydrous ethanol is 1g:1.8g:3ml; in the preparation method of the nano-CuFe2O4, the heating rate during calcination is 5 ℃ / min, the calcination setting temperature is 500℃, and the calcination time is 6 h.

[0013] Furthermore, the preparation method of the biochar material Bcn is as follows: take lotus leaves, rinse them with distilled water, dry them, then crush and carbonize them, cool them and grind them to obtain the biochar material Bcn.

[0014] Furthermore, in the preparation method of the biocarbon material Bcn, the drying process is as follows: drying at 80°C for 1 h in a drying oven; pulverizing by passing through a 100-mesh sieve, then placing it in a crucible and carbonizing at 800°C for 10 h in a muffle furnace under nitrogen protection; and finally grinding to a particle size of 1~10 μm after cooling.

[0015] Furthermore, the pretreatment process of the glassy carbon electrode is as follows: the glassy carbon electrode is initially polished with metallographic sandpaper, and then Al2O3 powder slurry with particle sizes of 1.0 μm, 0.3 μm and 0.05 μm is used to polish it on chamois leather until it becomes a mirror surface. Then, the glassy carbon electrode is cleaned sequentially with ethanol aqueous solution (volume ratio of anhydrous ethanol to water is 1:1), HNO3 aqueous solution (volume ratio of HNO3 to water is 1:1) and ultrapure water to obtain the pretreated glassy carbon electrode.

[0016] Further, the activation process of the glassy carbon electrode is as follows: the pretreated glassy carbon electrode obtained in step S1 is activated in a 0.5 M H2SO4 electrolyte solution using cyclic voltammetry, with a scan range of -1.0 to 1.0 V, and the scan is repeated until a stable cyclic voltammetric curve is reached. Finally, the electrode performance is tested at 1×10⁻⁶ V. -3 M Fe(CN)6 3- / 4- Cyclic voltammetry scans were performed in the solution, with a potential range of -0.3 to 0.6 V. When symmetrical redox peaks appeared in the curve and the peak potential difference was below 80 mV, the activated glassy carbon electrode was obtained.

[0017] The present invention also provides an application of the low working potential hydrogen sulfide sensor prepared by the above preparation method in the detection of hydrogen sulfide.

[0018] Furthermore, the low operating potential hydrogen sulfide sensor provided by this invention is used for real-time continuous detection of hydrogen sulfide, and the specific steps are as follows: (1) The CuFe2O4 / Bcn composite material modified electrode was used as the working electrode, the saturated calomel electrode was used as the reference electrode, and the platinum wire was used as the counter electrode to form a three-electrode unit structure and connect it to the electrochemical workstation for electrochemical testing. The supporting electrolyte in the electrolytic cell was PBS buffer solution. (2) When measuring hydrogen sulfide, place the three electrodes in PBS solution or wound simulation solution and perform constant potential amperometric experiment. Inject a certain amount of H2S every 50s and record the change of current with time to obtain the time-current step curve. Calculate the concentration of hydrogen sulfide sample using the standard curve method. (3) Plot the standard curve corresponding to hydrogen sulfide with hydrogen sulfide concentration as the abscissa and the average value of the step current as the ordinate, obtain the regression equation, and determine the linear relationship between hydrogen sulfide and peak current; put the sample to be tested into the electrolytic cell, and detect the peak current corresponding to hydrogen sulfide in the sample to be tested by an electrochemical workstation according to the methods of steps (1) and (2), substitute it into the regression equation, and calculate the content of hydrogen sulfide in the sample to be tested.

[0019] Furthermore, the concentration of the PBS buffer solution in step (1) is 0.1 M and the pH is 7.4; in the constant potential amperometry experiment in step (2), a fixed point of -0.3 V is selected for detection.

[0020] Furthermore, the wound simulation solution in step (2) is a deoxyPBS solution of 10% fetal bovine serum.

[0021] This invention prepares biocarbon material (Bcn) from lotus leaves using a pyrolysis method, serving as the framework and excellent conductive agent for electrode-modified composite materials. Nano-CuFe2O4 is prepared from copper acetate and ferrous oxalate using a high-temperature solid-state reaction method, serving as an electrocatalytic material for H2S sensing. Modifying the electrode surface with a stable and dispersed composite material of these two materials allows for sensitive sensing of H2S in biological liquids. Furthermore, this invention utilizes different electrochemical methods to investigate and verify the sensor's sensitivity, selectivity, and stability. Electrochemical experiments demonstrate that the CuFe2O4 / Bcn composite material prepared in this invention exhibits superior electrocatalytic performance for H2S, significantly reducing the oxidation potential of H2S, improving current response, and avoiding the deposition of sulfur, a byproduct of oxidation, on the electrode surface and interference from electroactive substances in the biological environment.

[0022] Experiments have verified that the sensor constructed from the CuFe2O4 / Bcn composite material used in this invention can sensitively and accurately generate a response signal to H2S within a concentration range of 5 nM to 10 μM, with a detection limit as low as 2.1 nM. Furthermore, practical application tests were conducted in a simulated biological environment, and the experiments showed that the low-operating-potential hydrogen sulfide sensor provided by this invention exhibits high sensitivity and retention rate for H2S in simulated wound fluid.

[0023] Compared with existing technologies, the preparation method and application of the low operating potential hydrogen sulfide sensor provided by this invention have the following advantages: (1) The method for preparing a low working potential hydrogen sulfide sensor provided by the present invention uses lotus leaves as raw material to synthesize biocarbon material Bcn by high temperature pyrolysis and prepares nano CuFe2O4 catalyst by high temperature solid phase synthesis. The CuFe2O4 / Bcn composite material obtained by combining the two has excellent conductivity and loading capacity and excellent catalytic performance for H2S. The composite modification of the two on the surface of glassy carbon electrode can be used for H2S sensing in biological liquids.

[0024] (2) The detection limit of the low working potential hydrogen sulfide sensor prepared by the present invention is 2.1 nM and the detection range is 5 nM-10 μM. It can be seen that the detection limit of the present invention is low and the detection range is wide. It can monitor hydrogen sulfide in real time and continuously, and at the same time, it can significantly reduce the potential response.

[0025] (3) The low working potential hydrogen sulfide sensor prepared by the present invention has a simple and easy-to-operate electrode modification step, which reduces the cost of electrode modification and reduces the error caused by the traditional electrode modification process. It has high detection stability, sensitivity and reproducibility. Attached Figure Description

[0026] Figure 1 The XPS spectrum of nano-CuFe2O4 prepared in Example 1; Figure 2 The total reflectance infrared spectra of nano-CuFe2O4 prepared in Example 1 and its synthetic raw materials, iron oxalate and copper acetate; Figure 3 SEM images of the working electrodes for biocarbon material Bcn (a) and CuFe2O4 / Bcn / GCE (b, c, d); Figure 4 The cyclic voltammetric response and differential pulse voltammetric response curves for 0.02 mM H2S with different electrode pairs are shown, where a is the cyclic voltammetric response curve and b is the differential pulse voltammetric response curve. Figure 5 The cyclic voltammetric and differential pulse voltammetric responses of the CuFe2O4 / Bcn / GCE working electrode to 0.02 mM H2S and blank are shown, where a is the cyclic voltammetric response curve and b is the differential pulse voltammetric response curve. Figure 6 The differential pulse voltammetric response (a) and calibration curve (b) of the CuFe2O4 / Bcn / GCE working electrode to different concentrations of H2S in the range of 0~100 μM are shown. Figure 7 The cyclic voltammetric response (a) and calibration curve (b) of the electrochemical sensor pair at different scan rates in the range of 50–150 mV / s under 0.02 mM H2S are shown. Figure 8 Differential pulse voltammetry curves of 0.05 mM H2S for electrochemical sensor pairs at different pH values ​​within the pH range of 5.9 to 7.9; Figure 9 Figure a shows the amperometric time-current response of the CuFe2O4 / Bcn / GCE electrochemical sensor to 10 μM H2S at different potentials in the range of -0.5 V to 0.1 V; figure b shows the amperometric time-current curve of the electrochemical sensor with continuous addition of H2S in 0.1 M pH 7.4 PBS solution from 5 nM to 10 μM; figure c is a magnified view of figure b; and figure d is the calibration curve of the response current against the H2S concentration. Figure 10 Stability (a) and repeatability evaluation (b) of CuFe2O4 / Bcn / GCE electrochemical sensor; Figure 11 In Figure a, the amperometric-time-current response curves of the CuFe2O4 / Bcn / GCE electrochemical sensor in 0.1 M pH 7.4 PBS solution to different interferences and H2S are shown. In Figure b, the differential pulse voltammetric response curve of the CuFe2O4 / Bcn / GCE electrochemical sensor to H2S in wound simulation solution is shown. Detailed Implementation

[0027] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0028] In the following examples and comparative examples, reagents not specifically mentioned are conventional reagents, all of which can be purchased from conventional reagent manufacturers and distributors. The methods used, unless otherwise specified, are existing technologies. Information on some raw material manufacturers is as follows: The copper acetate (Cu(CH3COO)2) and ferrous oxalate (FeC2O4) were purchased from Aladdin Reagent Co., Ltd., and the anhydrous ethanol and sodium sulfide were purchased from Sinopharm Reagent Co., Ltd. The lotus leaves were collected from Zhaowanghe Park in Heze City, washed and dried for later use.

[0029] All electrochemical measurements were performed at room temperature using a CHI660E electrochemical workstation.

[0030] Example 1: Preparation of Nano CuFe2O4 The preparation method of the nano-CuFe2O4 includes the following steps: Nano-CuFe2O4 was prepared by high-temperature solid-state reaction: First, take 1.0 g of copper acetate and 1.8 g of ferrous oxalate, grind them evenly in an agate mortar, add 3 mL of ethanol and stir for 48 h. After the ethanol evaporates, continue grinding until the particle size is 1~3 μm. Transfer the mixture to a crucible and place it in a muffle furnace (box-type muffle furnace SX2-5-12TP, Jinan Precision Scientific Instruments Co., Ltd.) for calcination. The heating rate is 5 ℃ / min, the calcination temperature is set at 500℃, and the calcination time is 6 h. After calcination, remove the product from the muffle furnace and allow it to cool naturally at room temperature. Weigh the product and grind it again in an agate mortar until the particle size is 20~30 nm, which is the nano-CuFe2O4 particles.

[0031] The reaction equation is 2Cu(CH3COO)2·H2O + 4FeC2O4·2H2O + O2→2CuFe2O4+ 4CH3COOH +4CO2+ 4CO + 8H2O.

[0032] Example 2 Preparation of biocarbon material Bcn The lotus leaves were rinsed three times with tap water and three times with distilled water, and then dried in a drying oven at 80°C for 1 hour. After being crushed and passed through a 100-mesh sieve, 5.0 g of the crushed leaves were placed in a crucible and carbonized in a muffle furnace at 800°C for 10 hours under nitrogen protection. After cooling at room temperature, the leaves were ground in a mortar until the particle size was 1-10 μm to obtain the biocarbon material Bcn.

[0033] Example 3: Preparation of CuFe2O4 / Bcn composite material The preparation method of the CuFe2O4 / Bcn composite material dispersion includes the following steps: Take the nano-CuFe2O4 prepared in Example 1 and the biochar material Bcn prepared in Example 2 into sample tubes respectively, add ultrapure water to disperse them, and make the concentration of both 2.0 mg / mL. Then place them in an ultrasonic cleaner and ultrasonically disperse for 10 min. Then take 2 mL of each dispersion and mix them, and ultrasonically disperse for 30 min to obtain CuFe2O4 / Bcn composite material. Store it in a refrigerator at 4℃ for later use. Before each use, it is necessary to ultrasonically disperse it well.

[0034] Example 4: A method for fabricating and applying a low-operating-potential hydrogen sulfide sensor. The method for preparing the low operating potential hydrogen sulfide sensor includes the following steps: S1. Pretreatment of glassy carbon electrode: The glassy carbon electrode is initially polished with metallographic sandpaper, and then polished on chamois leather with Al2O3 slurry with particle sizes of 1.0 μm, 0.3 μm and 0.05 μm until it becomes mirror-like. Then, the glassy carbon electrode is cleaned sequentially with ethanol aqueous solution (volume ratio of anhydrous ethanol to water is 1:1), HNO3 aqueous solution (volume ratio of HNO3 to water is 1:1) and ultrapure water to obtain the pretreated glassy carbon electrode. S2. Activation of the glassy carbon electrode: The pretreated glassy carbon electrode obtained in step S1 was activated in a 0.5 M H2SO4 electrolyte solution using cyclic voltammetry. The scan range was -1.0 to 1.0 V, and the scans were repeated until a stable cyclic voltammetric curve was obtained. Finally, the electrode performance was tested at 1×10⁻⁶ V. -3 M Fe(CN)6 3- / 4- Cyclic voltammetry scans were performed in the solution, with a potential range of -0.3 to 0.6 V. If symmetrical redox peaks appeared in the curve and the peak potential difference was below 80 mV, the glassy carbon electrode could be used, i.e., the activated glassy carbon electrode was obtained. S3. Modification of glassy carbon electrode: Take 5 μL of CuFe2O4 / Bcn composite material prepared in Example 3 and drop it onto the surface of the activated glassy carbon electrode in step S2. After air drying at room temperature, the CuFe2O4 / Bcn composite material modified electrode is obtained, which can be used for electrochemical experiments.

[0035] The low-operating-potential hydrogen sulfide sensor described above is used for real-time continuous detection of hydrogen sulfide, and specifically includes the following steps: (1) A three-electrode unit structure was formed by using CuFe2O4 / Bcn composite modified electrode as working electrode, saturated calomel electrode as reference electrode, and platinum wire as counter electrode. The structure was connected to an electrochemical workstation for electrochemical testing. The electrolyte in the electrolytic cell was 0.1 M PBS buffer solution with pH 7.4. (2) When measuring hydrogen sulfide, the three electrodes are placed in PBS solution, and a fixed point of -0.3 V is selected for the constant potential amperometric experiment. A certain amount of H2S is injected every 50s, and the current changes with time is recorded to obtain the time-current step curve. The concentration of hydrogen sulfide sample is calculated using the standard curve method. (3) Plot the standard curve corresponding to hydrogen sulfide with hydrogen sulfide concentration as the abscissa and the average value of the step current as the ordinate, obtain the regression equation, and determine the linear relationship between hydrogen sulfide and peak current; put the sample to be tested into the electrolytic cell, and detect the peak current corresponding to hydrogen sulfide in the sample to be tested by an electrochemical workstation according to the methods of steps (1) and (2), substitute it into the regression equation, and calculate the content of hydrogen sulfide in the sample to be tested.

[0036] In step (2), within the hydrogen sulfide concentration range of 5 nM to 10 μM, the current response is linearly related to the hydrogen sulfide concentration, and the linear equation is I(μA) = -1.2752 + 1.5348E -3 ×C(nM), R=0.9977, the lowest real-time detection limit can reach 2.1 nM, see Experimental Example 4 for details. Example 1: Characterization of nano-CuFe2O4 and biocarbon material Bcn (1) The composition and morphology of the nano-CuFe2O4 prepared in Example 1 were characterized. First, the elemental composition of the nano-CuFe2O4 prepared in Example 1 was analyzed using X-ray photoelectron spectroscopy (XPS, ThermoFisher ESCALAB Xi+). Figure 1The XPS spectrum of nano-CuFe2O4 shows the presence of four elements: copper, iron, oxygen, and carbon. Elemental abundance analysis (as shown in Table 1) revealed a ratio of copper, iron, and oxygen atoms of 1.0:2.2:6.3, which is consistent with the elemental composition of CuFe2O4, indicating successful synthesis of nano-CuFe2O4. The presence of extra C and O atoms may be due to excess unreacted raw materials in the product. Figure 2 The total reflectance IR absorption spectrum of the prepared nano-CuFe2O4 (the synthesized material was analyzed using a Thermo Fisher Scientific iS50 FTIR spectrometer), with a wavenumber range of 400–4000 cm⁻¹. -1 .from Figure 2 As can be seen from the data, compared with the infrared spectrum of the raw material, the prepared nano-CuFe2O4 only shows a value at 520 cm⁻¹. -1 and 400 cm -1 There are two sharp infrared vibrational absorption peaks nearby. These can be attributed to Fe. 3+ -O 2- The stretching vibration peak of oxygen anions at the tetrahedral positions formed and Cu 2+ -O 2- The stretching vibration peak of the oxygen anion at the octahedral position formed. The infrared absorption spectrum of the product shows no other obvious absorption peaks, indicating that the material prepared by this invention has high purity.

[0037] Table 1. Relative abundance of elements in the XPS spectra of CuFe2O4 O1s 530.13 62.23 Fe2p 711.06 22.16 Cu2p3 934.2 9.9 C1s 285.09 5.7 (2) Morphological characterization of biochar material Bcn and CuFe2O4 / Bcn composite material The surface morphology of the biochar material Bcn prepared in Example 2 and the CuFe2O4 / Bcn composite material prepared in Example 3 was characterized using SEM (Zeiss Gemini Sigma 300 / VP ultra-high resolution field emission scanning electron microscope). Figure 3 . Figure 3Figure a shows the surface morphology of Bcn dispersed on the conductive adhesive. It was found that the biochar particles were uneven in size and irregular in shape, with most particles in the micrometer range and many small fragments. This structure is unfavorable for adsorption on the electrode surface. Therefore, Bcn was ground in an agate mortar and then ultrasonically dispersed for a long time. The upper dispersion was then mixed with CuFe2O4 to prepare a composite material, which was observed on the electrode surface under an electron microscope. Figures b, c, and d show the morphology of the CuFe2O4 / Bcn composite material at different magnifications. It was found that the treated CuFe2O4 / Bcn composite material had a uniform particle size of about 1-2 μm and was uniformly dispersed on the electrode surface (Figure b). At higher magnifications (Figures c and d), particles of approximately tens of nanometers in size were observed uniformly dispersed on the Bcn surface; these are precisely the nano-CuFe2O4 adsorbed on the surface.

[0038] Experimental Example 2: Electrocatalytic Response of CuFe2O4 / Bcn Nanomaterials to H2S The electrochemical catalytic performance of CuFe2O4 / Bcn composite material for H2S was characterized by constructing an electrochemical sensor using a three-electrode system. A three-electrode unit structure was formed, consisting of a CuFe2O4 / Bcn composite modified electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the counter electrode. This structure was connected to an electrochemical workstation for electrochemical testing. The electrolyte in the electrolytic cell was a 0.1 M PBS buffer solution at pH 7.4. The electrochemical catalytic performance of the material was verified by cyclic voltammetry and differential pulse voltammetry.

[0039] Figure 4 and 5 The cyclic voltammetric response and differential pulse response of the sensor in the voltage range of -0.5 to 0 V are shown under different working electrodes. Figure 4 Figure 'a' shows the cyclic voltammetric response curves of different electrodes to 0.02 mM H2S. It can be seen that within the applied voltage range, the current change of the bare electrode is small, indicating that sulfur ions do not undergo redox reactions on the bare electrode. When the working electrode surface is modified with Bcn, the electrode current response is significantly enhanced, and the area of ​​the CV curve increases considerably, indicating that Bcn has good electron transport capabilities, which facilitates the rapid diffusion of sulfur ions at the electrolyte / electrode interface, providing a larger electrochemically active surface area for the electrochemical reaction. For the CuFe2O4 / Bcn composite film modified electrode, a significant oxidation peak appears in the CV curve around -0.2 V, suggesting that the peak current is due to the oxidation of sulfur ions or the redox reaction of CuFe2O4 itself. Figure 4 Figure b shows the differential pulse curves for 0.02 mM H2S under different electrodes, which further verifies the existence of redox reactions under the composite film modified electrode, while no redox reaction occurs on the bare electrode surface within the applied voltage range.

[0040] To verify the assignment of the oxidation peak, the same modified electrode was used to perform scans in blank and 5 mM Na2S PBS solutions for comparison. Figure 5 As shown, Figure 5 Figures a and b show the cyclic voltammetric and differential pulse voltammetric responses of the CuFe₂O₄ / Bcn / GCE working electrode to 0.02 mM H₂S and blank, respectively. Figure 5 As shown in Figure a, the same oxidation peak only appears in the cyclic voltammogram of the PBS solution electrode containing sulfur, confirming that the oxidation peak belongs to the electrocatalytic reaction of CuFe₂O₄ with sulfur ions. Figure 5 The results of the differential pulse method in b are consistent, except that the oxidation peak potential of the differential pulse voltammetry is slightly off at -0.3 V. This is due to the different measurement methods. The potential waveform of the differential pulse voltammetry method is the superposition of a linearly increasing voltage and a rectangular pulse with a constant amplitude, which reduces the interference of capacitive current in the background current and has higher detection sensitivity and lower detection limit.

[0041] Experiments show that the use of CuFe2O4 / Bcn composite materials not only increases the response current but also significantly reduces the oxidation potential. This is related to the excellent charge conduction properties of Bcn and the electrocatalytic performance of nano-CuFe2O4. The fine nano-CuFe2O4 adheres to the Bcn surface, increasing the active sites on the electrode surface, reducing ion diffusion and shuttle, and accelerating the interfacial reaction kinetics.

[0042] The presence of oxides or hydroxides readily leads to the electro-oxidation of sulfides, and the Cu(III) component plays a greater role in electron transport mediation than Cu(I) and Cu(II). Therefore, the electro-oxidation of sulfides on Cu-based hybrid material modified electrodes may occur through the reaction with CuOOH to generate Cu(OH)2. This application proposes a possible mechanism for H2S sensing on CuFe2O4 / Bcn / GCE as follows: CuO + OH − → CuOOH + e − 2CuOOH + H2S → 2Cu(OH)2 + S CuO + HS − → CuS + OH − CuS + OH−→ CuSOH + e − Based on the electrochemical mechanism of H2S oxidation catalyzed by CuFe2O4 / Bcn, the performance of the prepared electrochemical sensor was investigated using differential pulse voltammetry. A series of different Na2S concentrations were measured in 0.1M PBS at pH 7.4, and the results are as follows: Figure 6 As shown in Figure a, with the increase of Na₂S concentration (1~100 μM), the oxidation peak current gradually increases, while the oxidation potential remains basically unchanged. A graph is plotted between the peak current values ​​and the corresponding Na₂S concentrations, and linear correction is performed. Figure 6 (b) A linear relationship was found between the two. The regression equation was I(μA) = 7.6791 + 0.4952×C(μM), R = 0.9968. This further verifies that the CuFe2O4 / Bcn composite material prepared in this invention has good electrocatalytic performance for H2S.

[0043] Experimental Example 3: Optimization of Experimental Conditions for H2S Detection by the Sensor Prepared in This Invention Since electrochemical voltammetry is affected by electrochemical parameters, electrode interfaces, and electrochemical media, this invention further investigated the effects of the scan rate at the CuFe₂O₄ / Bcn / GCE interface and the pH of the electrolyte solution on H₂S oxidation, thereby optimizing the electrochemical reaction conditions. A 0.1 M PBS buffer solution supporting an electrolyte pH of 7.4 was used during the reaction, and the scan rate ranged from 50 mV / s to 150 mV / s. Figure 7 , Figure 7 The cyclic voltammetric response and calibration curves of the electrochemical sensor pair at different scan rates in the range of 50–150 mV / s for 0.02 mM H₂S are presented. Figure 7 As can be seen, the oxidation potential of H2S at the working electrode increases slightly with increasing scan rate, and the oxidation current increases accordingly. The cyclic voltammetry curve shows only one oxidation peak, indicating an irreversible electrode process. Based on data analysis and fitting results, the calibration curve (…) is obtained. Figure 7 (b) The peak current is linearly related to the scan rate, but the fit to the square root of the scan rate deviates from a straight line and bends upwards. This indicates that the electrode process is adsorption-controlled, primarily governed by the kinetic reaction rate. The linear fitting equation is: i pa (µA) = 1.5271 + 0.0335 v (mV / s), R=0.9984.

[0044] In sulfide sensing, the pH of the solution significantly affects the electrocatalytic oxidation tendency of the prepared material. Furthermore, H₂S is a diprotic weak acid, and its decomposition equilibrium depends on pH. A phosphate buffer solution at pH 7.4 contains approximately 20% H₂S and 80% HS₂. - A mixture of H2S and S, and under strong acid pH and strong base pH, H2S and S 2-These are the main forms of sulfides. To investigate the effect of pH on the CuFe2O4 / Bcn / GCE sensor, differential pulse voltammetry experiments were performed on solutions containing 1 mM sulfides at different pH values, with 0.1 M PBS used as the supporting electrolyte. The pH range was from 5.9 to 7.9. Figure 8 , Figure 8 The differential pulse voltammetry curves of the electrochemical sensor pair at different pH values ​​within the pH range of 5.9–7.9 are obtained from... Figure 8 It is evident that the electrocatalytic oxidation capacity of the CuFe2O4 / Bcn / GCE modified electrode in H2S determination exhibits a dynamic effect. The oxidation peak current increases within the pH range of 5.9–7.4, reaching its maximum at pH 7.4. When using PBS with a pH higher than 7.4, the peak current for H2S oxidation gradually decreases. Therefore, pH 7.4 was selected as the optimal pH for H2S detection.

[0045] In addition, to use the sensing platform in a biological environment, we used PBS under physiological conditions to ensure that H2S was fully converted into electrochemically detectable HS. - This also avoids the possibility that extreme acidic or alkaline conditions might lead to an overestimation of free H2S in real tissues.

[0046] Example 4: The Ampere-Time Current Response of the Sensor Prepared by the Present Invention to H2S The potentiostatic amperometry method, i.e., time-current curves, is simpler and has higher time resolution than the voltammetric method. The sensitivity of the synthesized CuFe2O4 / Bcn hybrid nanostructure to H2S can also be evaluated using amperometric measurements. First, a suitable operating potential was determined to assess the practicality of surface treatment for a continuous electrochemical H2S sensor. The results from the previous cyclic voltammetry experiments show that H2S oxidation by the modified electrode occurs at approximately -0.3 V. Measurements were taken at applied voltages of 0.1, -0.1, -0.2, -0.3, -0.4, and -0.5 V, and the resulting time-current curves are shown below. Figure 9 As shown in Figure a, the larger the applied voltage, the larger the current response. When the voltage is less than -0.3 V, the current response decreases significantly. When the applied potential is between -0.3 and 0.1 V, the larger the potential, the longer it takes for the electrode polarization to reach a steady state. On the other hand, to avoid more interference with the oxidation of substances at high potentials, a fixed potential of -0.3 V is chosen for the potentiostatic amperometric experiment.

[0047] Furthermore, the sensor prepared using the low working potential hydrogen sulfide sensor preparation method in Example 4 was subjected to a constant potential amperometric experiment. Figure 9Figures b and c depict the steady-state current-time (it) response of the CuFe₂O₄ / Bcn modified electrode when a certain concentration of H₂S is continuously added to a stirred PBS solution (pH=7.4). With the continuous addition of H₂S, a rapid increase in the oxidation current signal feedback occurs, causing the current signal to change from a linear steady-state state to a regular stepwise increase. After fitting the experimental data... Figure 9 d) It was found that within the concentration range of 5 nM to 10 μM, the current response was linearly related to the concentration of hydrogen sulfide, with the linear equation being I(μA) = -1.2752 + 1.5348E -3 ×C(nM), R=0.9977, the minimum real-time detection limit can reach 2.1 nM.

[0048] The abundance of CuFe2O4 active sites on the Bcn framework facilitates sulfide diffusion and activation, thereby improving the charge transfer kinetics on the electrode surface. Compared with redox media detection methods for H2S reported in recent years, the electrochemical sensor constructed using this method exhibits a significantly reduced potential response and a lower detection limit, making it a more advantageous detection method.

[0049] Example 5: Evaluation of the stability, repeatability, selectivity, and compatibility of the H2S sensor prepared according to this invention. This invention also employed the DPV method to study the long-term stability and repeatability of the CuFe2O4 / Bcn / GCE working electrode stored at room temperature. The experimental results are as follows: Figure 10 As shown, by Figure 10 As can be seen from this, the CuFe2O4 / Bcn / GCE electrode prepared in Example 4, when stored at room temperature, maintained more than 92.3% of its original current value after 3, 7, 15, and 30 days of testing. Figure 10 As can be seen from b, the CuFe2O4 / Bcn / GCE electrode prepared in Example 4 was used continuously for 9 times, and no significant change in the 1 mM sulfide oxidation current was observed. The relative standard deviation (RSD) was less than 4.6%.

[0050] The anti-interference capability of the constructed CuFe2O4 / Bcn / GCE electrochemical sensor was evaluated using the amperometric method. Electrochemical experiments were conducted using highly abundant electroactive substances found in biological fluids, including dopamine hydrochloride (DA), ascorbic acid (AA), uric acid (UA), NaNO2, and cysteine ​​(CYS), as the main interfering agents. The electrolyte solution was 0.1 M PBS solution at pH 7.4. An applied potential of -0.3 V was used, and several interfering agents at concentrations of 5 mM or higher were added with continuous stirring. Changes in current were recorded. Figure 11As shown in Figure a, the current change caused by the interfering substance is very small and can be ignored. However, under the same experimental conditions, the Ampere current response of 0.5 mM H₂S is significant. This confirms that the developed electrochemical sensor exhibits good selectivity for H₂S detection and has superior anti-interference capabilities.

[0051] In addition to electrochemically active substances, biofluids may also contain proteins that can contaminate electrode surfaces, hindering electron transfer and affecting electrochemical reactions. Therefore, detection of real samples is an essential part of evaluating the performance of electrochemical sensors. This invention uses wound septic fluid (SWF) as a realistic biological environment, adding different amounts of standard H2S solution, and then performing DPV detection. The concentrations were compared with those obtained from the working curves of the standard buffer solution to evaluate the sensor's performance. Calculations showed that the recovery rate was between 89.9% and 93.2%, indicating that the influence of the real environment was within an acceptable range, making it suitable for the detection of H2S in SWF liquids. Figure 11 b represents the differential pulse voltammetric response of the CuFe2O4 / Bcn / GCE electrochemical sensor to H2S in a wound simulation solution.

[0052] In summary, this invention synthesizes biocarbon material Bcn from lotus leaves via high-temperature pyrolysis and prepares nano-CuFe2O4 catalyst via high-temperature solid-state synthesis. Utilizing the excellent conductivity and loading capacity of Bcn and the catalytic performance of CuFe2O4 for H2S, the two are composite-modified onto the surface of a glassy carbon electrode for H2S sensing in bio-liquids. Electrochemical experiments verify that the composite material exhibits superior electrocatalytic performance for H2S, significantly reducing the oxidation potential of H2S, improving current response, and avoiding the deposition of sulfur, a byproduct of oxidation, on the electrode surface and interference from electroactive substances in the biological environment. The CuFe2O4 / Bcn sensor can sensitively and accurately generate response signals to H2S within a concentration range of 5 nM to 10 μM. It also shows high sensitivity and retention rate for H2S in simulated wound fluid.

[0053] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a low-operating-potential hydrogen sulfide sensor, characterized in that, Includes the following steps: S1. Pretreatment of glassy carbon electrode: Polish and clean the glassy carbon electrode to obtain the pretreated glassy carbon electrode. S2. Activation of glassy carbon electrode: The glassy carbon electrode after the pretreatment in step S1 is activated by cyclic voltammetry to obtain the activated glassy carbon electrode. S3. Modification of glassy carbon electrode: The electrode is modified by drop coating: CuFe2O4 / Bcn composite material is drop coated on the surface of the glassy carbon electrode activated in step S2, and after air drying at room temperature, CuFe2O4 / Bcn composite material modified electrode is obtained. The preparation method of the CuFe2O4 / Bcn composite material includes the following steps: Nano-CuFe2O4 and biochar material Bcn were weighed separately and dispersed in ultrapure water to a concentration of 2.0 mg / mL. Then, they were placed in an ultrasonic cleaner for ultrasonic dispersion. The dispersed nano-CuFe2O4 dispersion and biochar material Bcn dispersion were mixed at a volume ratio of 1:1 and ultrasonically dispersed again to obtain CuFe2O4 / Bcn composite material. The preparation method of the nano-CuFe2O4 is as follows: After grinding copper acetate and ferrous oxalate evenly, anhydrous ethanol was added and stirred for 48 h. The mixture was then ground until it became powder with a particle size of 1~3 μm. After calcination and grinding, nano CuFe2O4 was obtained. The preparation method of the biochar material Bcn is as follows: take lotus leaves, rinse them with distilled water, dry them, then crush and carbonize them, cool them and grind them to obtain the biochar material Bcn. The particle size of the nano-CuFe2O4 is 20~30 nm; In the preparation method of nano-CuFe2O4, the addition ratio of copper acetate, ferrous oxalate and anhydrous ethanol is 1g:1.8g:3ml; in the preparation method of nano-CuFe2O4, the heating rate during calcination is 5 ℃ / min, the calcination set temperature is 500℃, and the calcination time is 6 h.

2. The method for preparing a low-operating-potential hydrogen sulfide sensor according to claim 1, characterized in that, The drying process involves drying at 80°C for 1 hour in a drying oven; pulverizing by passing the material through a 100-mesh sieve, then placing it in a crucible and carbonizing it at 800°C for 10 hours in a muffle furnace under nitrogen protection; and finally grinding it to a particle size of 1~10μm after cooling.

3. The application of a low-operating-potential hydrogen sulfide sensor prepared by the preparation method as described in claim 1 or 2 in the detection of hydrogen sulfide.

4. The application according to claim 3, characterized in that, A low-operating-potential hydrogen sulfide sensor is used for real-time continuous detection of hydrogen sulfide. The specific steps are as follows: (1) The CuFe2O4 / Bcn composite material modified electrode was used as the working electrode, the saturated calomel electrode was used as the reference electrode, and the platinum wire was used as the counter electrode to form a three-electrode unit structure and connect it to the electrochemical workstation for electrochemical testing. The supporting electrolyte in the electrolytic cell was PBS buffer solution. (2) When measuring hydrogen sulfide, place the three electrodes in PBS solution or wound simulation solution and perform constant potential amperometric experiment. Inject a certain amount of H2S every 50s and record the change of current with time to obtain the time-current step curve. Calculate the concentration of hydrogen sulfide sample using the standard curve method. (3) Plot the standard curve corresponding to hydrogen sulfide with hydrogen sulfide concentration as the abscissa and the average value of the step current as the ordinate, obtain the regression equation, and determine the linear relationship between hydrogen sulfide and peak current; put the sample to be tested into the electrolytic cell, and detect the peak current corresponding to hydrogen sulfide in the sample to be tested by an electrochemical workstation according to the methods of steps (1) and (2), substitute it into the regression equation, and calculate the content of hydrogen sulfide in the sample to be tested.

5. The application according to claim 4, characterized in that, The concentration of the PBS buffer solution in step (1) is 0.1 M and the pH is 7.4; in the constant potential amperometry experiment in step (2), a fixed potential of -0.3 V is selected for detection.

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