Hydroxytyrosol electrochemical sensor based on ni3fe@lsb, preparation method and application thereof

Abstract

The application discloses a kind of based on Ni3Fe (1:5) @LSB construction hydroxytyrosol electrochemical sensor, and preparation method and application thereof.The preparation of the hydroxytyrosol electrochemical sensor includes the synthesis of LSB, the synthesis of Ni3Fe alloy, the preparation of Ni3Fe@LSB modified electrode and the preparation of electrochemical sensor.The sensor is used for hydroxytyrosol detection, without derivatization pretreatment, can effectively simplify detection process.Its working mechanism is that LSB has abundant pore structure, which provides favorable sites for target enrichment and electron transport, and combined with the excellent electrocatalytic activity of Ni3Fe alloy, the two form synergistic effect, which significantly improves the detection sensitivity of hydroxytyrosol and reduces the adsorption of interfering substances.The electrochemical sensor constructed by the sensor has a wide linear range (0.001 µM~40 µM) for hydroxytyrosol detection, a low detection limit, excellent stability and anti-interference ability, and the detection recovery rate can reach more than 98%, which can be used for rapid and sensitive detection of hydroxytyrosol in actual samples.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensing technology, and relates to a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, its preparation method and application. Background Technology

[0002] Hydroxytyrosol (3,4-Dihydroxyphenylethanol, DOPET) is a polyphenol compound with important physiological activity. Its detection mainly relies on chromatographic methods (such as HPLC-UV, LC-MS / MS) and spectroscopic methods (such as fluorescence spectroscopy). However, these methods have the following technical disadvantages: (1) Pretreatment is complicated and time-consuming. Hydroxytyrosol is highly polar and contains ortho-dihydroxyl groups, making it chemically active. If HPLC analysis is used, its retention on the reversed-phase column is weak. It is necessary to optimize the mobile phase or derivatize to improve the resolution. However, the derivatization step may introduce byproducts and prolong the analysis time. (2) Poor anti-interference ability, such as the poor selectivity of C18 / HLB in conventional solid phase extraction adsorbents, which are easy to co-extract interfering substances; the spectroscopic law is easily affected by background interference, and both are difficult to achieve selective detection of complex samples; (3) High equipment cost: large precision analytical instruments such as liquid chromatography-tandem mass spectrometry systems are expensive and complex to maintain, and must be operated by professional technicians, making them difficult to apply to the needs of rapid on-site detection or resource-limited scenarios; (4) Insufficient detection sensitivity: conventional detectors, such as ultraviolet light for hydroxytyrosol, have high detection limits, while high-sensitivity detectors, such as mass spectrometry, significantly increase the analysis cost.

[0003] Electrochemical sensors have become an important detection method in fields such as food safety, natural product analysis, and environmental monitoring due to their fast detection speed, simple operation, low cost, and ease of field use. With increasing public attention to healthy foods and natural active substances, the rapid, accurate, and sensitive determination of these substances has become a growing demand in related industries and research fields. To improve electrochemical sensing performance, the selection and optimization of electrode materials are crucial. Biomass porous carbon is widely available, green, and renewable, possessing advantages such as large specific surface area, good conductivity, strong chemical stability, and abundant pore structure. As a carrier material, it can effectively load active sites and accelerate mass transfer and electron transfer. Bimetallic alloys, due to their electronic synergistic effect and dual-active-site catalytic activity, exhibit higher catalytic activity, selectivity, and stability compared to single metals, significantly improving the electrocatalytic response to target molecules. Loading bimetallic alloys onto biomass porous carbon can fully leverage the synergistic advantages of the carrier and active components, solving problems such as insufficient catalytic sites in single materials, easy metal aggregation, and low signal sensitivity. Currently, there is a lack of electrochemical sensors for the detection of hydroxytyrosol that combine high sensitivity, high selectivity, low cost, and green stability. Their preparation methods, structural regulation, and sensing performance still need further innovation and improvement.

[0004] Therefore, this invention provides a method for preparing biomass porous carbon supported on a bimetallic alloy and its application in a hydroxytyrosol electrochemical sensor, aiming to achieve controllable material structure, excellent catalytic performance, and stable and reliable detection results, thereby meeting the needs for rapid, sensitive, and accurate detection of hydroxytyrosol in actual samples. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB that is free from derivatization, highly selective, low-cost and portable, and has ultra-high sensitivity, as well as its preparation method and application.

[0006] To address the aforementioned technical problems, the present invention proposes the following technical solutions.

[0007] A hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB includes a working electrode, an auxiliary electrode, and a reference electrode. The working electrode is a Ni3Fe(1:5)@LSB modified electrode, which consists of a glassy carbon electrode and a Ni3Fe(1:5)@LSB composite material modified on the glassy carbon electrode. The Ni3Fe(1:5)@LSB composite material includes an LSB framework and a Ni3Fe alloy loaded on the LSB framework. The LSB framework is made from longan shell powder through activation and sintering, and the Ni3Fe alloy is made from metal salt through impregnation reduction and annealing.

[0008] In the aforementioned hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, preferably, the carbon framework is a porous carbon framework, and the porous carbon framework is co-doped with carbon and oxygen, wherein the carbon framework ensures structural stability and conductivity, and oxygen doping imparts surface activity and polarity.

[0009] In the aforementioned hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, preferably, the auxiliary electrode is a platinum wire electrode and the reference electrode is an Ag / AgCl electrode.

[0010] As a general technical concept, the present invention also provides a method for preparing the above-mentioned hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, comprising the following steps:

[0011] (1) Synthesis of LSB: The washed longan shells were ground into a uniform powder for later use. Potassium citrate was weighed and dissolved in deionized water. The longan shell powder was then transferred to the potassium citrate solution, heated and stirred until the water was evaporated, and a solid mixture was obtained. The obtained solid was heated to 700℃~800℃ under N2 atmosphere for carbonization. The resulting black product was washed successively with dilute hydrochloric acid and deionized water. The porous carbon material obtained after drying was called LSB.

[0012] (2) Synthesis of Ni3Fe alloy: Ni(CH3COO)2·4H2O and Fe(CH3COO)2 were dissolved in deionized water and stirred until homogeneous. Then, NaBH4 aqueous solution was slowly added and the reaction was continued with stirring. The product was washed several times with ethanol and deionized water. The obtained amorphous nickel-iron alloy was annealed at 500℃~600℃ under N2 atmosphere to obtain Ni3Fe alloy powder;

[0013] (3) Preparation of Ni3Fe(1:5)@LSB: Three ratios of Ni3Fe@LSB materials were prepared (ratios of 1:10, 1:5, and 1:2, respectively). Taking the Ni3Fe(1:5)@LSB with the optimal mass ratio (Ni3Fe to LSB support mass ratio of 1:5) as an example, the synthesis steps are as follows: The LSB support material was dispersed in deionized water and stirred and sonicated to ensure uniform dispersion. A certain proportion of Ni(CH3COO)2·4H2O and Fe(CH3COO)2 were weighed and dissolved in deionized water. The above metal salts were added to the support suspension and stirred to ensure full adsorption of metal ions. Under magnetic stirring, NaBH4 solution was added dropwise to the above mixture to reduce the metal ions in situ to alloy nanoparticles. After the reaction, the product was washed with ethanol and deionized water and dried under vacuum. The dried sample was annealed at 500℃~600℃ under N2 atmosphere. The resulting product is the Ni3Fe(1:5)@LSB composite material;

[0014] (4) Preparation of modified electrode: The glassy carbon electrode is pretreated to obtain a pretreated glassy carbon electrode. The obtained Ni3Fe(1:5)@LSB suspension droplets are accurately transferred by a pipette to 4 µL to 6 µL and dropped onto the surface of the pretreated glassy carbon electrode. The electrode is dried under an infrared lamp to obtain the Ni3Fe(1:5)@LSB modified electrode.

[0015] (5) Preparation of electrochemical sensor: Using the Ni3Fe(1:5)@LSB modified electrode obtained in step (4) as the working electrode, a three-electrode working system was constructed by combining the auxiliary electrode and the reference electrode to obtain a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB.

[0016] In the preferred embodiment of the above-mentioned preparation method of the hydroxytyrosol electrochemical transmitter based on Ni3Fe(1:5)@LSB, in step (1), the mass ratio of potassium citrate to longan shell powder is 1.3 g~1.7 g∶0.3 g~0.7 g, the volume of water is 25 mL~30 mL, the heating temperature is 60℃~80℃, the continuous stirring time is 7 h~9 h, the concentration of hydrochloric acid is 1.116 mol / L~1.402 mol / L, and the carbonization reaction time is 2 h~2.5 h.

[0017] The above-mentioned method for preparing a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB is characterized in that, in step (2), the mass-volume ratio of Ni(CH3COO)2·4H2O, Fe(CH3COO)2 and water is 2 g~2.2 g∶0.48 g~0.5 g∶50 mL, the mass-volume ratio of NaBH4 and water is 0.95 g~0.98 g∶15 mL~20 mL, the dropping rate is one drop every 3 seconds~5 seconds, the heating rate is 3℃ / min~5℃ / min, and the annealing time is 2 h~2.5 h.

[0018] In the preferred embodiment of the above-mentioned method for preparing a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, in step (3), the mass-to-volume ratio of LSB to water is 18 mg~20 mg∶18 mL~22 mL, the mass-to-volume ratio of Ni(CH3COO)2·4H2O to water is 12.6 mg~12.8 mg∶1 mL~1.5 mL, the mass-to-volume ratio of Fe(CH3COO)2 to water is 3.0 mg~3.2 mg∶0.3 mL~0.8 mL, the mass-to-volume ratio of NaBH4 to water is 26 mg~26.2 mg∶6.9 mL, and the annealing time is 2 h~2.5 h.

[0019] In the above-mentioned method for preparing a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, preferably, in step (4), the pretreatment process is as follows: the glassy carbon electrode is polished with Al2O3 powder of 1.0 µm, 0.3 µm and 0.05 µm in succession, and then ultrasonically cleaned with ethanol and deionized water for 5 min to 10 min in succession. The cleaned glassy carbon electrode is then dried at room temperature to obtain the pretreated glassy carbon electrode.

[0020] The above-mentioned method for preparing a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB is characterized in that, in step (4): the mass-to-volume ratio of Ni3Fe(1:5)@LSB to water is 2 mg: 1 mL; 4 µL to 6 µL of the Ni3Fe(1:5)@LSB suspension is dropped onto the pretreated glassy carbon electrode; and the power of the infrared lamp is 250 W to 300 W.

[0021] As a general technical concept, the present invention also provides an application of the above-mentioned electrochemical sensor of hydroxytyrosol based on Ni3Fe(1:5)@LSB or the electrochemical sensor of hydroxytyrosol based on Ni3Fe(1:5)@LSB prepared by the above-mentioned method in the detection of hydroxytyrosol.

[0022] In the above application, preferably, the hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB has a detection sensitivity of 0.001 µM to 40 µM for hydroxytyrosol and a detection limit of 0.56 nM for hydroxytyrosol.

[0023] The main innovation of this invention is as follows:

[0024] 1. Technical Effect Logic

[0025] Improved conductivity: Potassium citrate activates porous biochar based on longan shells to construct a highly conductive three-dimensional carbon framework. The Ni3Fe alloy and biochar interface form an efficient charge transport channel, which greatly accelerates electron transfer efficiency.

[0026] Selective mechanism:

[0027] Size sieving: The porous structure of Ni3Fe(1:5)@LSB biochar can form a steric hindrance effect, repelling large molecular interferences such as humic acid and protein in the sample matrix.

[0028] Catalytic enhancement: The d-band electron regulation effect of Ni3Fe bimetallic alloy optimizes the electron distribution of active sites on the biochar surface, accelerates the oxidation kinetics of hydroxytyrosol, and improves the catalytic reaction efficiency.

[0029] 2. Key technologies

[0030] (1) Composition and structural design of Ni3Fe(1:5)@LSB composite material: using agricultural waste longan shell as carbon source, porous biochar was prepared by high-temperature activation with potassium citrate. Ni3Fe alloy nanoparticles were uniformly loaded onto the surface / pores of biochar by impregnation stirring and calcination reduction method to construct carbon-oxygen co-doped Ni3Fe(1:5)@LSB composite structure.

[0031] Technical features:

[0032] ① Potassium citrate-activated longan shell biochar has a rich pore structure, providing an ultra-high specific surface area, abundant ion transport channels and a continuous conductive carbon network, enabling efficient pre-enrichment of hydroxytyrosol.

[0033] ②The Ni3Fe alloy nanoparticles form a strong interfacial bond with biochar, and the bimetallic synergistic effect exposes more active sites, which can significantly enhance the electrocatalytic oxidation activity of hydroxytyrosol.

[0034] ③ The composite material combines the structural stability of biochar with the high catalytic activity of Ni3Fe alloy, thereby improving electrochemical sensing performance and cycle life.

[0035] (2) Selective adsorption and catalytic oxidation mechanism of hydroxytyrosol: The π-π stacking effect of longan shell biochar and the hydrogen bonding between oxygen-containing functional groups such as hydroxyl / carboxyl groups and hydroxytyrosol molecules are used to achieve specific enrichment. Combined with the synergistic catalytic effect of Ni3Fe bimetal, the oxidation reaction of hydroxytyrosol is accelerated.

[0036] Technical features:

[0037] ① The size sieving effect of the multi-pore channel reduces matrix interference and improves detection selectivity.

[0038] ②Ni3Fe bimetallic synergistic catalysis promotes the two-electron oxidation reaction of hydroxytyrosol and reduces the oxidation overpotential.

[0039] ③ The specific interaction between oxygen-containing functional groups on the surface of biochar and hydroxytyrosol enables the targeted enrichment of the target analyte, further improving detection sensitivity.

[0040] (3) Electrochemical sensor construction and detection method: Based on the modified electrode of Ni3Fe(1:5)@LSB / GCE, the precise detection of trace hydroxytyrosol in complex matrices is achieved by combining differential pulse voltammetry (DPV).

[0041] Technical features:

[0042] ① Wide linear detection range (0.001 µM~40 µM) and ultra-low detection limit (0.56 nM) to meet the trace detection needs of hydroxytyrosol in different samples.

[0043] ② The modified electrode exhibits high reproducibility and stability, with a signal retention rate >90% after multiple cyclic detections.

[0044] ③ It is suitable for direct detection of complex matrices such as food, plant extracts, and water, without the need for complicated pretreatment.

[0045] This invention achieves triple innovation in materials, interface, and method. At the materials level, high-performance porous biochar is prepared from agricultural waste longan shells via green activation with potassium citrate. This biochar, combined with a Ni3Fe alloy, forms a Ni3Fe(1:5)@LSB porous biochar composite material, exhibiting high conductivity, high specific surface area, high catalytic activity, and structural stability. Simultaneously, it enables the high-value utilization of agricultural waste, aligning with green environmental protection principles. At the detection level, it integrates size sieving, specific adsorption, and synergistic catalysis, achieving rapid, highly selective, and highly sensitive detection of hydroxytyrosol. At the application level, this invention is suitable for trace detection of hydroxytyrosol in complex matrices such as food, plant extracts, and water. Furthermore, the preparation and detection equipment costs are significantly lower than those of chromatography-mass spectrometry (GC-MS), making it economical, portable, and practical.

[0046] Compared with the prior art, the advantages of the present invention are as follows:

[0047] 1. This invention addresses the technical challenges of large matrix interference, high detection cost, complex pretreatment, and insufficient sensitivity in the trace detection of hydroxytyrosol. Using agricultural waste longan shells as a carbon source, porous biochar is prepared by activation with potassium citrate. Ni3Fe alloy is loaded onto the biochar through impregnation stirring and calcination reduction. An innovative electrochemical sensor for hydroxytyrosol constructed from Ni3Fe(1:5)@LSB longan shell-based porous biochar is designed. The hydroxytyrosol electrochemical sensor of this invention can directly realize the electrochemical detection of the target analyte without derivatization treatment, greatly simplifying the detection process and reducing the detection time by more than 50%. Through the pore sieving effect of Ni3Fe(1:5)@LSB, the specific hydrogen bonding and π-π stacking effect between the oxygen-containing functional groups on the biochar surface and hydroxytyrosol are synergistically achieved, enabling selective capture of hydroxytyrosol and effectively avoiding the adsorption of interfering substances on the electrode surface. The actual sample detection recovery rate is >98%. The raw material for the preparation of this sensor is low-cost agricultural waste, reducing equipment costs by more than 90%. It only needs to be used with a miniaturized electrochemical workstation to complete the detection, greatly reducing detection costs and site requirements. Utilizing the interfacial synergistic catalytic effect of Ni3Fe(1:5)@LSB, the electron transfer efficiency and catalytic oxidation activity are significantly enhanced, achieving an ultra-low detection limit for hydroxytyrosol and meeting the needs of trace detection.

[0048] The specific advantages are as follows:

[0049] (1) Rapid detection without derivatization: Relying on the efficient synergistic catalytic oxidation effect of Ni3Fe(1:5)@LSB, hydroxytyrosol can be directly detected electrochemically, avoiding cumbersome sample derivatization and complicated pretreatment steps, and greatly improving detection efficiency.

[0050] (2) Ultra-high selectivity and anti-interference ability: The sensor combines the spatial size sieving effect of longan shell biochar pores with the specific adsorption effect of biochar surface functional groups on hydroxytyrosol, thus enhancing the sensor's anti-matrix interference ability in a dual synergistic manner. It is suitable for the detection of complex samples such as food and plant extracts.

[0051] (3) Green, low cost and portable: The carbon carrier is prepared by green activation with potassium citrate using agricultural waste longan shells as raw material. The raw materials are readily available and inexpensive, and at the same time, the high-value utilization of agricultural waste is realized. The detection process only requires a small electrochemical workstation to replace the large and expensive chromatography-mass spectrometry equipment, which greatly reduces the equipment investment and maintenance costs and enables rapid on-site detection.

[0052] (4) Ultra-high detection sensitivity: The synergistic catalytic effect of Ni3Fe alloy optimizes the electron distribution of active sites and reduces the oxidation overpotential of hydroxytyrosol. The high specific surface area and specific adsorption of longan shell biochar enable the pre-enrichment of target substances. At the same time, the carbon skeleton ensures efficient charge transfer. The three work together to achieve an ultra-low detection limit of hydroxytyrosol and meet the needs of trace detection.

[0053] (5) Excellent structural and detection stability: The strong interfacial bonding between Ni3Fe alloy and longan shell-based biochar, as well as the high chemical and structural stability of the biochar itself, enable the modified electrode to have excellent electrochemical cycling stability, reproducibility and reliability in actual sample detection, and the detection results are accurate and stable.

[0054] 2. In the preparation method of this invention, the carbon-oxygen co-doped porous LSB material is synthesized through activated pyrolysis of longan shell powder, while the Ni3Fe(1:5)@LSB material is prepared by impregnation, stirring, and calcination reduction, embedding bimetallic Ni3Fe nanoparticles into a porous carbon framework. The synergistic effect of the Ni3Fe alloy optimizes the electronic structure and promotes the adsorption and redox kinetics of hydroxytyrosol molecules. The sensor of this invention has a detection sensitivity of 0.001 µM to 40 µM for hydroxytyrosol, a detection limit of 0.56 nM, and maintains a signal stability of more than 95% even in the presence of 10 times the concentration of interfering substances (such as ferulic acid and glucose). In the detection of actual samples (extract, hand cream, olive oil, olive fruit), the recovery rate is 98.14% to 102.9%, and the relative standard deviation is less than 3%, which is significantly better than traditional chromatographic methods, providing an efficient tool for the detection and control of hydroxytyrosol. Attached Figure Description

[0055] Figure 1 The Ni3Fe(1:5)@LSB of Example 1, the LSB of Comparative Example 1, the Ni3Fe(1:10)@LSB of Comparative Example 2, the Ni3Fe(1:2)@LSB of Comparative Example 3, and the Ni3Fe of Example 4, along with a bare glassy carbon electrode, were used in an environment containing 2.5 × 10⁻⁶ N·m³. -6DPV response plot of the oxidation peak of M-hydroxytyrosol in PBS solution (pH=7).

[0056] Figure 2 The X-ray diffraction patterns are those of Ni3Fe(1:5)@LSB in Example 1, LSB in Comparative Example 1, and Ni3Fe in Example 4 of this invention.

[0057] Figure 3 The X-ray diffraction patterns are of Ni3Fe(1:5)@LSB in Example 1 of the present invention, LSB in Comparative Example 1, Ni3Fe(1:10)@LSB in Comparative Example 2, and Ni3Fe(1:2)@LSB in Comparative Example 3.

[0058] Figure 4 The images show SEM images (A) of LSB in Comparative Example 1 of the present invention; SEM images (B, C) of Ni3Fe(1:5)@LSB composite material in Example 1; and SEM image (D) of Ni3Fe in Comparative Example 4.

[0059] Figure 5 The images show the Raman spectra of Ni3Fe(1:5)@LSB from Example 1 of the present invention, and the LSB from Comparative Example 1, Ni3Fe(1:10)@LSB from Comparative Example 2, and Ni3Fe(1:2)@LSB from Comparative Example 3.

[0060] Figure 6 The Ni3Fe(1:5)@LSB of Example 1 of this invention contains 2.5 × 10⁻⁶ ppm. -6 (A) CV curves of M-hydroxytyrosol in PBS solution (pH=6.5) at different pH values; (B) Relationship between current of oxidation peak and pH.

[0061] Figure 7 The Ni3Fe(1:5)@LSB of Example 1 of this invention contains 2.5 × 10⁻⁶ ppm. -6 DPV response plot of the oxidation peak at enrichment time in PBS solution of M-hydroxytyrosol (pH=6.5); DPV response plot of the oxidation peak at enrichment potential; DPV response plot of the oxidation peak at drop volume.

[0062] Figure 8 The Ni3Fe(1:5)@LSB of Example 1 of this invention contains 2.5 × 10⁻⁶ ppm. -6 (A) CV plots at different scan rates in PBS solution of M-hydroxytyrosol (pH=6.5); (B) Relationship between oxidation peak current and scan rate.

[0063] Figure 9 The DPV plot (A) and linear fitting plot (B) are for the quantitative analysis of hydroxytyrosol in Ni3Fe(1:5)@LSB of Example 1 of the present invention.

[0064] Figure 10 The hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, as described in Example 1 of this invention, is used in a substrate containing 2.5 × 10⁻⁶ N·m³. -6 DPV response plot of hydroxytyrosol in PBS solution after eight repeated measurements (A); DPV response plot of eight independent electrochemical sensors prepared under the same conditions (B); Comparison plot of oxidation peak current of hydroxytyrosol in the presence of different interfering substances (C).

[0065] Figure 11 The UV-Vis spectrum (A) and linear calibration curve (B) of the hydroxytyrosol standard solution in the present invention in the range of 200 nM to 600 nM are shown.

[0066] Figure 12 The following are UV-Vis spectrophotometric images of actual samples from Example 1 of this invention: (A) UV-Vis absorption spectrum of the extract sample; (B) UV-Vis absorption spectrum of the hand cream sample; (C) UV-Vis absorption spectrum of the olive oil sample; (D) UV-Vis absorption spectrum of the olive fruit sample.

[0067] Figure 13 This is a comparison chart of the detection results of the electrochemical detection method and the ultraviolet spectrophotometric method in Example 1 of the present invention. Detailed Implementation

[0068] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. The reagents and instruments used in the following embodiments are all commercially available. The experimental reagents are shown in Table 1, and the experimental instruments are shown in Table 2. In the following embodiments, M refers to mol / L.

[0069] Table 1. Experimental reagents for Example 1

[0070] Table 2. List of experimental instruments used in the process operation in Example 1

[0071] Example 1

[0072] A hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB includes a working electrode, an auxiliary electrode, and a reference electrode. The working electrode is a Ni3Fe(1:5)@LSB modified electrode, which consists of a glassy carbon electrode and a Ni3Fe(1:5)@LSB composite material modified on the glassy carbon electrode. The Ni3Fe(1:5)@LSB composite material includes an LSB framework and a Ni3Fe alloy loaded on the LSB framework. The LSB framework is made by activating and sintering longan shell powder, and the Ni3Fe alloy is made by impregnation, reduction, and annealing of metal salt.

[0073] In this embodiment, the carbon framework is a porous carbon framework, and the porous carbon framework is co-doped with carbon and oxygen. The carbon framework ensures structural stability and conductivity, while oxygen doping imparts surface activity and polarity.

[0074] In this embodiment, the auxiliary electrode is a platinum wire electrode, and the reference electrode is an Ag / AgCl electrode.

[0075] A method for fabricating a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB according to this embodiment includes the following steps:

[0076] (1) Synthesis of LSB: Longan shells were repeatedly washed with ethanol and deionized water to remove surface impurities, dried, and ground into a uniform powder for later use. 1.5 g of potassium citrate was dissolved in 30 mL of deionized water, and 0.5 g of the above longan shell powder was transferred to the potassium citrate solution. The mixture was heated and stirred until the water was evaporated to obtain a solid mixture. The obtained solid was heated to 800 °C at a heating rate of 5 °C / min under a high-purity nitrogen atmosphere and pyrolyzed for 2 h. The black product obtained was washed with dilute hydrochloric acid and deionized water, and the dried material was called LSB.

[0077] (2) Synthesis of Ni3Fe alloy: 2.12 g Ni(CH3COO)2·4H2O and 0.494 g Fe(CH3COO)2 were placed in 50 mL of deionized water and stirred at 800 rpm for 15 min. Then, 20 mL of 1.28 M NaBH4 aqueous solution was slowly added, and stirring was continued for 10 min. The product was washed several times with ethanol and deionized water. The prepared amorphous nickel-iron alloy catalyst was annealed at 600 °C for 2 h under N2 atmosphere.

[0078] (3) Preparation of Ni3Fe(1:5)@LSB: Three ratios of Ni3Fe(1:5)@LSB material were prepared (ratios of 1:10, 1:5, and 1:2, respectively). Taking the Ni3Fe(1:5)@LSB with the optimal mass ratio (Ni3Fe to LSB support mass ratio of 1:5) as an example, the synthesis steps are as follows: Accurately weigh 20 mg of LSB support material and disperse it in 20 mL of deionized water. Under N2 atmosphere, stir and sonicate for 30 min to ensure uniform dispersion. Weigh 12.72 mg of Ni(CH3COO)2·4H2O and 3.12 mg of Fe(CH3COO)2 and dissolve them in 2 mL of water and 1 mL of water, respectively. Add the above metal salt solution to the support suspension and stir for 2 h to allow the metal ions to be fully adsorbed. Weigh 26.1 mg of NaBH4 as a reducing agent and dissolve it in 6.9 mL of deionized water. Under magnetic stirring, freshly prepared NaBH4 solution was added dropwise to the above mixture, and the reaction was continued for 30 min to reduce metal ions in situ to alloy nanoparticles. After the reaction, the product was collected by centrifugation and washed multiple times with ethanol and deionized water to remove residual impurity ions. The washed product was dried under vacuum. The dried sample was then heated to 600℃ at a heating rate of 5℃ / min under N2 atmosphere and annealed for 2 h. The resulting product is the Ni3Fe(1:5)@LSB composite material.

[0079] (3) Preparation of modified electrode: The glassy carbon electrode was polished successively with Al2O3 powder of 1.0 µm, 0.3 µm, and 0.05 µm, and then ultrasonically cleaned with ethanol and deionized water for 5 min each. The cleaned glassy carbon electrode was dried at room temperature to obtain a pretreated glassy carbon electrode. A suspension was prepared with Ni3Fe(1:5)@LSB and water at a mass-to-volume ratio of 2 mg:1 mL. 6 μL of the Ni3Fe(1:5)@LSB suspension was dropped onto the pretreated glassy carbon electrode. The infrared lamp had a power of 250 W.

[0080] (4) Preparation of electrochemical sensor: Using the Ni3Fe(1:5)@LSB modified electrode obtained in step (3) as the working electrode, the platinum wire electrode as the auxiliary electrode, and the Ag / AgCl electrode as the reference electrode, a three-electrode working system was constructed to obtain a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, which is abbreviated as Ni3Fe(1:5)@LSB / GCE.

[0081] An application of the hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB prepared in this embodiment in the detection of hydroxytyrosol shows that the detection sensitivity of hydroxytyrosol is 0.001 µM to 40 µM and the detection limit of hydroxytyrosol is 0.56 nM.

[0082] Comparative Example 1

[0083] An electrochemical sensor is basically the same as the electrochemical sensor in Example 1, except that the modified electrode is an LSB modified electrode.

[0084] Comparative Example 2

[0085] An electrochemical sensor is basically the same as the electrochemical sensor in Example 1, except that the modified electrode is a Ni3Fe(1:10)@LSB modified electrode.

[0086] Comparative Example 3

[0087] An electrochemical sensor is basically the same as the electrochemical sensor in Example 1, except that the modified electrode is a Ni3Fe(1:2)@LSB modified electrode.

[0088] Comparative Example 4

[0089] An electrochemical sensor is basically the same as the electrochemical sensor in Example 1, except that the modified electrode is a Ni3Fe modified electrode.

[0090] like Figure 2 , Figure 3 As shown, the crystal structures of LSB, Ni3Fe alloy, and Ni3Fe@LSB composites in three proportions were characterized by X-ray diffraction (XRD). Figure 2 It can be seen that the LSB sample obtained by pyrolysis at 800℃ exhibits two broad diffraction peaks near 25° and 44°, corresponding to the (002) and (101) crystal planes of amorphous carbon, respectively, confirming its typical biomass-derived porous carbon structure. The diffraction peaks of the Ni3Fe alloy perfectly match those of the standard card PDF#38-0419, indicating its high crystallinity and phase purity. The XRD pattern of the optimal ratio Ni3Fe(1:5)@LSB retains both the broad peaks of the LSB carbon matrix and the characteristic sharp peaks of the Ni3Fe alloy, and the main peaks of the Ni3Fe alloy all correspond to those of the standard card, confirming that the crystalline Ni3Fe alloy has been successfully loaded into the LSB framework without obvious impurities. Figure 3 The figures show Ni3Fe@LSB composites with different Ni / Fe ratios. As can be seen from the figures, all three composite samples retain the characteristic broad peak of LSB at approximately 25°, indicating that the amorphous carbon structure is completely preserved. At the same time, sharp diffraction peaks corresponding to the Ni3Fe alloy (PDF#38-0419) can be clearly observed in all spectra, indicating that the Ni3Fe alloy crystalline phase was successfully formed under different ratios.

[0091] To intuitively investigate the microstructure and structural characteristics of the Ni3Fe(1:5)@LSB composite material, a systematic characterization was performed using scanning electron microscopy (SEM). The results are as follows: Figure 4 As shown. Figure 4 As shown in Figure A, the LSB carbon material derived from longan shells exhibits a typical three-dimensional porous network framework structure. Figure 4 Images B and C show the morphology of Ni3Fe(1:5)@LSB. The Ni3Fe alloy loaded on the LSB surface exhibits a uniformly dispersed nanoparticle morphology without obvious agglomeration. This indicates that the porous carbon framework of the LSB effectively restricts the growth and agglomeration of Ni3Fe alloy particles, achieving uniform dispersion of bimetallic nanoparticles. In contrast, as... Figure 4 As shown in Figure D, the morphology of the Ni3Fe alloy is characterized by agglomerated nanoparticles.

[0092] like Figure 5 Raman spectroscopy revealed typical D and G peaks at approximately 1350 cm⁻¹ and 1580 cm⁻¹ for all samples, corresponding to the defect / disorder structure of carbon materials and the graphitization vibrations of sp² hybridized carbon, respectively. These exhibit typical porous carbon structural characteristics. Quantitative analysis of the characteristic peak intensities yielded the Ig values ​​for each sample. D / I G The ratios are as follows: I of LSB D / I G The ratio is 0.99, while the I of Ni3Fe(1:10)@LSB, Ni3Fe(1:5)@LSB and Ni3Fe(1:2)@LSB is... D / I G The ratios were 0.98, 1.01, and 0.97, respectively. The Ig of all samples... D / I G The ratios are all close to 1, indicating that the material has good structural stability and a moderate degree of disorder, which ensures both electron transport efficiency and provides sufficient reactive sites. Ni3Fe(1:5)@LSB exhibits the highest Ig ratio. D / I G The ratio (1.01) indicates that it has the highest defect density, the most abundant active sites, and the best structural advantages.

[0093] Electrochemical detection:

[0094] Electrochemical detection was performed on a CHI660 electrochemical workstation using a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB prepared in this embodiment. The electrolyte solution used for electrochemical detection was 0.1 M PBS (phosphate buffer), and the detection was conducted under a nitrogen atmosphere throughout. The potential range for cyclic voltammetry (CV) was set to -0.2 V to 0 V, and the scan rate was set to 100 mV·s. -1 The potential range of the linear sweep voltammetry (LSV) was also set to -0.3 V to 0.5 V, and the electrochemical impedance spectroscopy (EIS) experiment was carried out in a 5 mM potassium ferricyanide / potassium ferrocyanide mixed solution containing 0.1 M potassium chloride.

[0095] (1) Electrochemical response of hydroxytyrosol on different modified electrodes

[0096] In containing 2.5×10⁻ 6 In a 0.1 M PBS (pH=7) system containing hydroxytyrosol, the electrochemical response behavior of different modified electrodes to hydroxytyrosol was investigated using differential pulse voltammetry (DPV). The results showed that the oxidation peak currents of LSB / GCE, Ni3Fe / GCE, Ni3Fe(1:10)@LSB / GCE, Ni3Fe(1:5)@LSB / GCE, and Ni3Fe(1:2)@LSBNi3Fe / GCE were 12.02 µA, 2.22 µA, 17.83 µA, 26.5 µA, and 29.93 µA, respectively. Among them, the Ni3Fe(1:5)@LSB modified electrode exhibited the strongest electrochemical response, with an oxidation peak current as high as 29.93 µA, significantly better than other control samples. This indicates that the composite material effectively improves the electron transfer efficiency at the electrode interface and the electrocatalytic oxidation activity of hydroxytyrosol.

[0097] (2) Study on the linear range of the sensor

[0098] Under the selected optimal conditions (pH=6.5, enrichment time 300 s, enrichment potential 0 V, modifier drop volume 4 µL, scan rate 100 mV・s⁻¹), the quantitative detection performance of the Ni3Fe(1:5)@LSB sensor for hydroxytyrosol was evaluated using differential pulse voltammetry. Figure 9 As shown in Figure A, as the concentration of hydroxytyrosol gradually increased from 0.001 µM to 40 µM, its oxidation peak current showed a continuous increasing trend, and the peak shape remained clear and stable, demonstrating the high response sensitivity of the sensor to the target analyte. Logarithmic fitting of the peak current and concentration revealed two segments of good linear relationship (…). Figure 9 B):

[0099] The linear equation for the low concentration range (0.001 µM ≤ x ≤ 0.01 µM) is:

[0100] y1 = 6.249logx + 1.252, R 2 =0.999 Formula (I)

[0101] In formula (I), y1 is the anodic oxidation peak current of hydroxytyrosol in µA, and x is the concentration of hydroxytyrosol in µM (i.e. µmol / L), where 0.001 µM≤x≤0.01 µM.

[0102] The linear equation for the high concentration range (0.01 µM ≤ x ≤ 40 µM) is:

[0103] y2 = 45.048logx - 79.679, R 2 =0.997 Equation (II)

[0104] The high correlation coefficients across the two linear intervals demonstrate that the sensor exhibits excellent quantitative accuracy across a wide range from 0.001 µM to 40 µM. Specifically, the sensor's limit of detection (LOD) for hydroxytyrosol is as low as 0.56 nM, significantly lower than the typical residual concentration of hydroxytyrosol in existing samples, showcasing excellent potential for trace detection. These results indicate that the Ni3Fe(1:5)@LSB sensor not only possesses an ultra-wide linear range (0.001 µM–40 µM) but also achieves a synergy between low detection limit and high sensitivity, providing a reliable electrochemical sensing platform for the efficient and accurate monitoring of hydroxytyrosol in real-world samples.

[0105] (3) Study on sensor repeatability, reproducibility and anti-interference

[0106] The accuracy, reproducibility, and robustness of the performance analysis are key performance indicators that need to be considered. To evaluate the repeatability and reproducibility of Ni3Fe(1:5)@LSB / GCE of this invention, a solution containing 2.5 × 10⁻⁶ g / LBS was analyzed. -6 DPV was used in a 0.1 M PBS (pH=6.5) solution of M-hydroxytyrosol. For example... Figure 10 As shown in Figure A, after eight repeated measurements, the relative standard deviation (RSD) of the current response was observed to be 1.01%, indicating that the sensor of the present invention exhibits satisfactory stability. Figure 10 As shown in B, reproducibility was tested using eight repeatedly prepared Ni3Fe(1:5)@LSB modified electrodes, and all electrodes showed a stable current response with an RSD of 1.05%.

[0107] The ability of electrochemical sensors to detect target substances in the presence of interfering substances is crucial for their practical application in the detection field. For example... Figure 10 As shown in C, select Al. 3+ Ca 2+ Fe 3+ Mg 2+ Cu 2+ The anti-interference ability of Ni3Fe(1:5)@LSB / GCE in the detection of hydroxytyrosol was studied using oryzanol, L-tyrosine, glucose, tyrosol, ascorbic acid, ferulic acid, palmitic acid, p-hydroxybenzoic acid, vanillic acid, and myristic acid as interfering substances. Multiple groups containing 2.5 × 10⁻⁶ oryzanol were included. -6 In a PBS buffer solution of M-hydroxytyrosol, 50 times the amount of interfering ions, 10 times the amount of biomacromolecules, 5 times the amount of similar molecules, and 2 times the amount of tyrosol were added respectively. It was observed that there was almost no effect on the DPV response and oxidation peak current of hydroxytyrosol, which indicates that the Ni3Fe(1:5)@LSB / GCE of the present invention has excellent anti-interference performance.

[0108] (4) Comparison of Ni3Fe(1:5)@LSB / GCE detection results and UV spectra in actual samples

[0109] Hydroxytyrosol extract, hand cream, olive oil, and olive fruit purchased online were used to investigate the performance of the hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB in detecting hydroxytyrosol in complex real-world samples. To prepare the solution required for the UV spectroscopy experiment, crushed olive fruit was added to ethanol as a solvent and refluxed at 60°C for 2 hours. The supernatant was obtained as the analyte. The hydroxytyrosol content in the samples was determined using the standard addition method, and the spiked recovery rate was calculated.

[0110] Figure 11 UV-Vis spectroscopy results showed that hydroxytyrosol exhibited a characteristic absorption peak in the range of 10 µM to 100 µM, and the peak intensity increased gradient with increasing concentration. At the characteristic absorption wavelength, the absorbance showed a significant linear correlation with the concentration of hydroxytyrosol, with the fitted equation being Abs = 0.00286C. DOPET +0.00913, R 2 =0.998 (see) Figure 11 B). The high sensitivity response in the low concentration range enables accurate detection of trace amounts of hydroxytyrosol, while maintaining stable linearity in the high concentration range, confirming that this method has reliable quantitative analysis capabilities over a wide concentration range and providing an optical basis for the efficient detection of hydroxytyrosol.

[0111] Table 3. Detection results of hydroxytyrosol in actual samples (s / n=3)

[0112] Finally, the oxidation peak current of each actual sample on Ni3Fe(1:5)@LSB / GCE was obtained using DPV, and the concentration of hydroxytyrosol was calculated, as shown in Table 3. The hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB prepared in this invention showed satisfactory results in detecting various natural samples, with recoveries ranging from 98.14% to 102.9% and RSD less than 3% (n=3). This indicates that the hydroxytyrosol electrochemical sensor of this invention can be used for the identification of actual samples.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB, characterized in that, The device includes a working electrode, an auxiliary electrode, and a reference electrode. The working electrode is a Ni3Fe@LSB modified electrode. The Ni3Fe(1:5)@LSB modified electrode consists of a glassy carbon electrode and a Ni3Fe(1:5)@LSB composite material modified on the glassy carbon electrode. The Ni3Fe(1:5)@LSB composite material includes an LSB framework and a Ni3Fe alloy loaded on the LSB framework. The LSB framework is made from longan shell powder through activation and sintering, and the Ni3Fe alloy is made from metal salt through impregnation reduction and annealing.

2. The hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB as described in claim 1, characterized in that, The carbon framework is a porous carbon framework, and the porous carbon framework is co-doped with carbon and oxygen. The carbon framework ensures structural stability and conductivity, while oxygen doping imparts surface activity and polarity.

3. The hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB as described in claim 1 or 2, characterized in that, The auxiliary electrode is a platinum wire electrode, and the reference electrode is an Ag / AgCl electrode.

4. A method for preparing a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Synthesis of LSB: The washed longan shells are ground into uniform powder for later use. Potassium citrate is weighed and dissolved in deionized water. The longan shell powder is then transferred to the potassium citrate solution, heated and stirred to evaporate the water. The resulting solid is heated to 700℃~800℃ under N2 atmosphere for carbonization. The resulting black product is washed with dilute hydrochloric acid and deionized water in sequence. The porous carbon material obtained after drying is called LSB. (2) Synthesis of Ni3Fe alloy: Ni(CH3COO)2·4H2O and Fe(CH3COO)2 were dissolved in deionized water and stirred evenly. Then, NaBH4 aqueous solution was slowly added and the reaction was continued with stirring. The product was washed multiple times with ethanol and deionized water. The obtained amorphous nickel-iron alloy was annealed at 500℃~600℃ under N2 atmosphere to obtain Ni3Fe alloy powder. (3) Preparation of Ni3Fe@LSB: Three ratios of Ni3Fe@LSB materials were prepared (ratios of 1:10, 1:5, and 1:2 respectively). Taking the Ni3Fe(1:5)@LSB with the best mass ratio (Ni3Fe to LSB carrier mass ratio of 1:5) as an example, the synthesis steps are as follows: The LSB carrier material is dispersed in deionized water and stirred and sonicated to make it uniformly dispersed; a certain proportion of Ni(CH3COO)2·4H2O and Fe(CH3COO)2 are weighed and dissolved in deionized water. The above metal salts are added to the carrier suspension and stirred to make the metal ions fully adsorbed. Under magnetic stirring, NaBH4 solution is added dropwise to the above mixed system to reduce the metal ions in situ to alloy nanoparticles; after the reaction, the product is washed with ethanol and deionized water and dried under vacuum. The dried sample is annealed at 500℃~600℃ under N2 atmosphere. The obtained product is Ni3Fe(1:5)@LSB composite material. (4) Preparation of modified electrode: The glassy carbon electrode is pretreated to obtain a pretreated glassy carbon electrode. The obtained Ni3Fe(1:5)@LSB suspension droplets are accurately transferred by a pipette to 4 µL to 6 µL and dropped onto the surface of the pretreated glassy carbon electrode. The electrode is dried under an infrared lamp to obtain the Ni3Fe(1:5)@LSB modified electrode. (5) Preparation of electrochemical sensor: Using the Ni3Fe(1:5)@LSB modified electrode obtained in step (4) as the working electrode, a three-electrode working system was constructed by combining the auxiliary electrode and the reference electrode to obtain a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB.

5. The method for preparing the hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB according to claim 4, characterized in that, In step (1), the mass ratio of potassium citrate to longan shell powder is 1.3 g to 1.7 g: 0.3 g to 0.7 g, the volume of water is 25 mL to 30 mL, the heating temperature is 60℃ to 80℃, the continuous stirring time is 7 h to 9 h, the concentration of hydrochloric acid is 1.116 mol / L to 1.402 mol / L, and the carbonization reaction time is 2 h to 2.5 h.

6. The method for preparing the hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB according to claim 4, characterized in that, In step (2), the mass-volume ratio of Ni(CH3COO)2·4H2O, Fe(CH3COO)2 and water is 2 g~2.2 g∶0.48 g~0.5 g∶50 mL, the mass-volume ratio of NaBH4 and water is 0.95 g~0.98 g∶15 mL~20 mL, the dropping rate is one drop every 3 seconds~5 seconds, the heating rate is 3℃ / min~5℃ / min, and the annealing time is 2 h~2.5 h.

7. The method for preparing the hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB according to claim 4, characterized in that, In step (3), the mass-to-volume ratio of LSB to water is 18 mg to 20 mg: 18 mL to 22 mL, the mass-to-volume ratio of Ni(CH3COO)2·4H2O to water is 12.6 mg to 12.8 mg: 1 mL to 1.5 mL, the mass-to-volume ratio of Fe(CH3COO)2 to water is 3.0 mg to 3.2 mg: 0.3 mL to 0.8 mL, the mass-to-volume ratio of NaBH4 to water is 26 mg to 26.2 mg: 6.9 mL, and the annealing time is 2 h to 2.5 h.

8. The method for preparing the hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB according to any one of claims 4 to 7, characterized in that, In step (4), the pretreatment process is as follows: the glassy carbon electrode is polished with Al2O3 powder of 1.0 µm, 0.3 µm and 0.05 µm in succession, and then ultrasonically cleaned with ethanol and deionized water for 5 min to 10 min in succession. The cleaned glassy carbon electrode is dried at room temperature to obtain the pretreated glassy carbon electrode.

9. The method for preparing a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB according to any one of claims 4 to 7, characterized in that, In step (4): the mass-to-volume ratio of Ni3Fe(1:5)@LSB to water is 2 mg:1 mL; 4 µL to 6 µL of the Ni3Fe(1:5)@LSB suspension is dropped onto the pretreated glassy carbon electrode, and the power of the infrared lamp is 250 W to 300 W.

10. The application of a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB constructed according to any one of claims 1 to 3, or a hydroxytyrosol electrochemical sensor based on Ni3Fe(1:5)@LSB constructed according to the preparation method of any one of claims 4 to 9, in the detection of hydroxytyrosol.

11. The application according to claim 10, characterized in that, The electrochemical sensor for hydroxytyrosol based on Ni3Fe(1:5)@LSB has a detection sensitivity of 0.001 µM to 40 µM and a detection limit of 0.56 nM for hydroxytyrosol.

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