Layered double hydroxide-based electrode sensitive material and electrochemical immunosensor

Abstract

The invention relates to an electrode sensitive material based on layered double hydroxides and an electrochemical immunosensor. A FeNi LDH nano material and copper salt are subjected to a heating reaction in a citrate aqueous solution to prepare a layered double hydroxide Cu-FeNi LDH composite nano material; an electrode sensitive material with excellent electrochemical response characteristics and good selectivity is obtained by loading and modifying AuNPs, a glassy carbon electrode is coated with the electrode sensitive material, a PCT antibody is modified, the electrode sensitive material is sealed with bovine serum albumin, an electrochemical immunosensor is constructed and used for detecting the bacterial infection biomarker PCT, and the lowest detection limit within the concentration range of 1 pg / mL to 100 ng / mL is 30 fg / mL.

Description

Technical Field

[0001] This invention belongs to the field of nanocomposite materials and electrochemical immunosensor technology, and relates to an electrochemical immunosensor that uses gold nanoparticles loaded with iron-nickel-copper metal-based crystalline double hydroxide composite material as a carrier and signal amplification material for the detection of procalcitonin, a biomarker of bacterial infection. Background Technology

[0002] Against the backdrop of accelerated global population mobility and increasing internationalization, bacterial infections have become a major public health problem threatening human health, drawing high attention from the World Health Organization and health departments in various countries. Bacterial infections typically result from abnormal immune regulation triggered by the invasion of pathogenic microorganisms such as bacteria, viruses, or fungi into various parts of the body, leading to systemic inflammatory response syndrome. This can further induce multiple organ dysfunction, posing a serious threat to the patient's health and even endangering their life. Therefore, early detection, accurate diagnosis, and timely treatment are crucial for controlling disease progression and reducing the risk of complications and mortality.

[0003] Procalcitonin (PCT), as the propeptide of calcitonin, has no hormonal activity. It shows a specific and significant increase in systemic inflammatory states such as bacterial infection and sepsis, and has become a key biomarker for clinical diagnosis of infectious diseases, guiding the rational use of antibiotics, and assessing prognosis.

[0004] In healthy individuals, PCT concentrations are extremely low (<0.05 ng / mL), making them difficult to detect using conventional methods. However, during bacterial infections, PCT secreted by thyroid C cells and neuroendocrine cells in the lungs and intestines can rapidly increase to over 2 ng / mL, exhibiting high specificity and excellent stability both in vivo and in vitro. This makes it a specific marker for bacterial infections, distinguishing it from other inflammatory markers. Notably, in inflammatory responses caused by non-bacterial infections such as viral infections or autoimmune inflammation, the increase in PCT is usually significantly lower than in bacterial infections, but its rise may occur earlier than other inflammatory markers, giving it potential value in early diagnosis. Based on these characteristics, developing rapid and accurate PCT detection technologies is of great significance for clinical diagnosis and treatment.

[0005] Traditional PCT detection methods are limited by their complex procedures, long testing cycles, and high costs, making them difficult to widely apply. Immunoassay, with its antigen-antibody specific recognition mechanism, provides an effective approach to achieving highly sensitive and selective PCT detection.

[0006] Electrochemical immunosensors quantitatively detect PCT concentration by immobilizing biorecognition elements (such as antibodies, antigens, or aptamers) on the electrode surface and utilizing changes in electrochemical signals (current, potential, or impedance), offering the advantage of rapid response. With the continuous development of nanotechnology, using nanomaterials as signal amplification elements has significantly improved the performance of electrochemical immunosensors.

[0007] Layered double hydroxides (LDHs) have shown application potential in the field of electrochemical sensing due to their tunable two-dimensional layered structure and efficient biomolecule loading capacity; gold nanoparticles (AuNPs), on the other hand, have become ideal electrode modification materials due to their excellent conductivity, morphology controllability and biocompatibility.

[0008] Liu et al. (Sensitive electrochemical biosensor for Uracil-DNA glycosylase detection based on self-linkable hollow Mn / Ni layered doubled hydroxides asoxidase-like nanozyme for cascade signal amplification [J]. Biosensors and Bioelectronics (2021, 194: 113607.) A sensitive electrochemical immunobiosensor for detecting uracil-DNA glycosylation enzyme (UDG) was developed by preparing hollow Mn / Ni LDHs spheres (h-Mn / NiLDHs) via a one-step hydrothermal method. The detection of UDG was achieved with a detection limit as low as 4.8 × 10⁻⁶. -4 U / mL.

[0009] Furthermore, Zhu et al. (An efficient biosensor using a functionalized microneedle of Cu2O-based CoCu-LDH for glucose detection [J]. RSC advances , 2023, 13(46):32558-32566.)Cauliflower-shaped Cu2O nanoparticle clusters were directly deposited on the tip surface of a stainless steel acupuncture needle electrode (ANE) by electrochemical deposition, and then neatly arranged CoCu-LDH nanosheets were further prepared. The nanosheets were interconnected to form a 3D porous nanohybrid structure, which was successfully applied to the high-sensitivity and selective electrochemical sensing detection of glucose.

[0010] Although LDH-based composite materials have attracted much attention in the sensing field, their application in PCT electrochemical immunoassay remains unexplored. Developing PCT electrochemical immunosensors based on LDH composite sensitive materials, and optimizing material interface properties and signal transduction mechanisms, holds promise for achieving wide linear range and low detection limit analysis of PCT, providing a novel technological platform for the early diagnosis of bacterial infections. Summary of the Invention

[0011] The purpose of this invention is to provide an electrochemical immunosensor for the detection of the bacterial infection biomarker PCT. This is achieved by preparing a layered double hydroxide composite material with AuNPs loaded with iron, nickel and copper metals as a carrier and signal amplification material, thereby constructing an electrochemical immunosensor for the detection of the bacterial infection biomarker PCT.

[0012] To achieve the above-mentioned objectives, the present invention first provides a layered double hydroxide composite material with a uniform hydrangea-like microstructure, which is a Cu-FeNi LDH composite nanomaterial obtained by reacting FeNi LDH nanomaterials with water-soluble copper salts in an aqueous solution containing citrate at 50-100°C.

[0013] The FeNi LDH nanomaterial is prepared by first reacting a water-soluble ferrous salt with triethanolamine to form a complex, then adding a water-soluble nickel salt, and finally conducting a closed hydrothermal reaction at 80–160°C in an aqueous solution containing urea.

[0014] The Cu-FeNi LDH composite nanomaterial of this invention is a dark blue solid powder with a uniform hydrangea-like microstructure and a particle size of about 3 μm.

[0015] Furthermore, the preferred molar ratio of the water-soluble copper salt to the iron element in the FeNi LDH nanomaterial is (2-6):1.

[0016] Furthermore, the water-soluble copper salt includes, but is not limited to, copper chloride, copper acetate, or copper sulfate.

[0017] Furthermore, the preferred reaction time for preparing Cu-FeNi LDH composite nanomaterials is 3 to 24 hours.

[0018] More specifically, the preferred molar ratio of water-soluble ferrous salt to water-soluble nickel salt used in preparing FeNi LDH nanomaterials is 1:(1-6).

[0019] More specifically, the water-soluble ferrous salt includes, but is not limited to, ferrous chloride, ferrous nitrate, or ferrous sulfate; the water-soluble nickel salt includes, but is not limited to, nickel nitrate or nickel sulfate.

[0020] More specifically, the hydrothermal reaction time for preparing FeNi LDH nanomaterials is preferably 4 to 10 hours.

[0021] Secondly, the present invention also provides a specific method for preparing the layered double hydroxide composite material, comprising:

[0022] S1) A deep green Fe is formed by mixing water-soluble ferrous salt with triethanolamine. 2+ -TEA complex precipitation;

[0023] S2) Dissolve water-soluble nickel salt and urea in water to form a solution, add the above complex to precipitate, and perform a closed hydrothermal reaction at 80-160℃ to prepare FeNi LDH nanomaterial solution;

[0024] S3) A mixed solution is formed by dissolving water-soluble copper salt and citrate in water;

[0025] S4) Mix the above mixed solution with the FeNi LDH nanomaterial solution evenly, and heat to 50-100℃ to carry out the reaction;

[0026] S5) Collect the reaction precipitate, wash and dry it to obtain a dark blue Cu-FeNi LDH composite nanomaterial solid powder.

[0027] Further, preferably, the mixed solution is mixed with the FeNi LDH nanomaterial solution, stirred slowly for no less than 1.5 hours, and then heated to carry out the reaction.

[0028] Furthermore, it is preferable to wash the precipitate collected after the reaction with anhydrous ethanol first, and then with deionized water.

[0029] Furthermore, it is preferable to dry the washed precipitate at 60°C for 12–24 hours.

[0030] Third, the present invention also provides an electrode sensitive material, which is obtained by using the layered double hydroxide Cu-FeNi LDH composite nanomaterial with a uniform hydrangea-like morphology as a support, and dispersing and loading a large number of uniform and regular spherical AuNPs between the hydrangea petals of Cu-FeNi LDH.

[0031] Furthermore, the specific preparation method of the electrode sensitive material of the present invention is to dropwise add a 1-5 mol% AuNPs solution onto the surface of Cu-FeNi LDH composite nanomaterial, and allow it to dry at room temperature to obtain an AuNPs@Cu-FeNi LDH composite structure as an electrode sensitive material.

[0032] The AuNPs can be nanomaterials prepared using any conventional method reported in the literature, and the present invention does not limit the specific preparation method thereto.

[0033] The uniform morphology of the Cu-FeNi LDH composite nanomaterial with a uniform hydrangea-like structure is beneficial to the dispersion and distribution of AuNPs, while the loading of AuNPs can greatly increase and improve the surface area of ​​the composite material. Therefore, the AuNPs@Cu-FeNi LDH electrode sensitive material prepared by this invention provides more active sites for electrocatalytic oxidation, enhances the catalytic activity and stability of the electrode material, and can be used to construct electrochemical immunosensors.

[0034] Therefore, the present invention also provides an electrochemical immunosensor for detecting the bacterial infection biomarker PCT constructed using the aforementioned electrode-sensitive material.

[0035] Specifically, the electrochemical immunosensor uses an activated glassy carbon electrode (GCE) as a matrix, coats its surface with an AuNPs@Cu-FeNi LDH electrode sensitive material modification layer, incubates and binds PCT antibody (Ab) to the surface of the electrode sensitive material, and adds bovine serum albumin (BSA) to block the active sites on the surface of the electrode sensitive material that are not occupied by PCT antibody, thus constructing the electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE.

[0036] Furthermore, the specific preparation method of the electrochemical immunosensor of the present invention includes:

[0037] S1) The prepared electrode-sensitive material AuNPs@Cu-FeNi LDH was dispersed in anhydrous ethanol and ultrasonically dispersed to form a stable and uniform suspension. It was then fixed on the activated and pretreated GCE surface and naturally dried to form a uniform nanomaterial modification layer, thus obtaining the AuNPs@Cu-FeNi LDH / GCE electrode.

[0038] S2) An Ab solution was uniformly drop-coated onto the surface of the nanomaterial modified layer and incubated at 4°C to fix the antibody onto the surface of the nanomaterial modified layer through Au-S bonds and physical adsorption, forming an Ab / AuNPs@Cu-FeNi LDH / GCE electrode.

[0039] S3) Add BSA solution dropwise to the electrode surface, let it stand and dry, and seal the active sites on the electrode surface that are not occupied by the antibody to construct the electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE.

[0040] The constructed electrochemical immunosensor needs to be stored at 4°C to avoid inactivation of biomolecules.

[0041] The electrochemical immunosensor constructed in this invention can specifically bind to PCT antigen through PCT antibody attached to its surface. Therefore, it can be used for qualitative and quantitative detection of PCT, a biomarker of bacterial infection, providing a new method for PCT detection and enabling early diagnosis of bacterial infection.

[0042] The nano-hydrangea-like AuNPs@Cu-FeNi LDH electrode sensitive material prepared in this invention provides abundant active sites, which is beneficial for the immobilization of biomolecules and the conduction of electrochemical reactions. Furthermore, the introduction of Cu further modulates the electronic structure of FeNi LDH, thereby improving the conductivity of the material. The AuNPs attached between the hydrangea petals make the surface of the electrode sensitive material easier to functionalize, thereby achieving specific binding with PCT antibodies and improving the selectivity of the electrochemical immunosensor. The surface plasmon resonance effect and catalytic activity of AuNPs further enhance the electrochemical signal of the prepared electrochemical immunosensor.

[0043] The electrochemical immunosensor prepared according to the present invention is used for the detection of PCT, with a detection limit of 30 fg / mL and a linear range of 1 pg / mL to 100 ng / mL. It also has excellent repeatability, long-term stability and selectivity.

[0044] The electrochemical immunosensor prepared according to this invention was used to detect normal human serum samples. The RSD range was 1.63% to 3.37%, and the recovery rate ranged from 97.00% to 111.20%, which showed good accuracy and precision and could meet the requirements for the detection of PCT in actual serum samples. Attached Figure Description

[0045] Figure 1 These are SEM images of Cu-FeNi LDH (A) and AuNPs@Cu-FeNi LDH (B) prepared in Example 1.

[0046] Figure 2 The images show the XPS spectra of AuNPs@Cu-FeNi LDH (A) and the high-resolution spectra of Ni 2p, Fe 2p, Cu 2p, Au 4f and O 1s (B-F).

[0047] Figure 3 This is a SEM image of Cu-FeNi LDH prepared in Comparative Example 1.

[0048] Figure 4The CV curves (A) and EIS curves (B) for different electrodes are shown, where: a) GCE, b) AuNPs@Cu-FeNi LDH / GCE, c) Ab / AuNPs@Cu-FeNi LDH / GCE, d) BSA / Ab / AuNPs@Cu-FeNi LDH / GCE, e) PCT / BSA / Ab / AuNPs@Cu-FeNi LDH / GCE.

[0049] Figure 5 These are the cyclic voltammetry curves for GCE, FeNi LDH, and Cu-FeNi LDH.

[0050] Figure 6 The electrochemical immunosensor of the present invention shows the SWV response (A) of different PCT concentrations and the linear fitting curve of its peak current response to the logarithmic concentration (B).

[0051] Figure 7 The results show the DPV response of five electrochemical immunosensors from different batches (A), the DPV response of five electrochemical immunosensors from the same batch (B), and the long-term stability (C) and selectivity (D) of the electrochemical immunosensors at 4°C for 7, 14, 21, 28, 35 and 42 days. Implementation

[0052] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can better understand and utilize the present invention, and are not intended to limit the scope of protection of the present invention.

[0053] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0054] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0055] AuNPs were prepared according to conventional literature methods:

[0056] Weigh 0.0588 g (0.2 mmol) of Na3C6H5O7·2H2O and add it to 50 mL of deionized water. Stir until completely dissolved. Separately weigh 0.095 g (2.5 mmol) of NaBH4 and dissolve it in 5 mL of ice water.

[0057] Take 30 mL of Na3C6H5O7·2H2O solution and keep it in a 60℃ water bath for 5 min. Add 600 µL of 0.1 mM HAuCl4·4H2O solution dropwise and stir at 60℃ for 5 min. Immediately add 200 µL of NaBH4 solution and stir vigorously until the solution color gradually changes from colorless to light red, wine red, and dark purple, indicating the formation of AuNPs. Remove the solution and stop the reaction. Example

[0058] Example 1

[0059] 0.0149 g (0.075 mmol) of ferrous chloride tetrahydrate was mixed with 150 µL of triethanolamine (TEA) until a dark green Fe3+ solution was formed. 2+ -TEA complex precipitation.

[0060] Weigh 0.0654 g (0.225 mmol) of nickel nitrate hexahydrate and 0.1013 g (1.688 mmol) of urea, add them to 15 mL of deionized water, stir continuously for 30 min, mix them evenly with the above complex precipitate, transfer them to a 50 mL polytetrafluoroethylene high-pressure reactor, and perform hydrothermal reaction in a 120 °C forced-air drying oven for 6 h to prepare FeNi LDH solution.

[0061] Weigh 0.0748 g (0.375 mmol) of copper acetate monohydrate and 0.1045 g (3 mmol) of sodium citrate pentahydrate, dissolve them in 40 mL of deionized water, stir for 30 min with a magnetic stirrer, then add the above FeNi LDH solution and mix, continue to stir slowly for 1.5 h, and heat in a 60 °C drying oven for 24 h.

[0062] The reaction product was centrifuged at 10,000 rpm for 10 min, and the lower filter cake was collected. It was washed three times with anhydrous ethanol and then three times with deionized water. It was then dried at 60 °C in a forced-air drying oven to constant weight to prepare Cu-FeNi LDH powder, wherein the Fe:Ni:Cu molar ratio was 1:3:5.

[0063] A 5µL aqueous dispersion of 4mol% AuNPs was added dropwise to the surface of Cu-FeNi LDH using a pipette, and the mixture was allowed to dry at room temperature to obtain the electrode-sensitive material AuNPs@Cu-FeNi LDH.

[0064] The morphology of Cu-FeNi LDH powder and the electrode-sensitive material AuNPs@Cu-FeNiLDH was observed using scanning electron microscopy, as follows: Figure 1 As shown in (A), Cu-FeNi LDH exhibits a regular and uniform hydrangea-like nanoflower structure with a diameter of approximately 6 µm; from Figure 1 (B) It can be seen that AuNPs in the electrode sensitive material are largely dispersed between the petals of Cu-FeNi LDH hydrangea, and have high electrochemical reactivity. They can be used as sensitive materials for electrochemical immunosensors to achieve sensitive detection of PCT.

[0065] Figure 2 The elements contained in the electrode-sensitive material AuNPs@Cu-FeNi LDH were determined by X-ray photoelectron spectroscopy. Figure 2 (A) The AuNPs@Cu-FeNi LDH full spectrum shows the coexistence of Fe, Ni, Cu, Au, C, O, and Na elements; the XPS spectrum of Ni2p ( Figure 2 In (B), the two locations at 855.6 and 873.3 eV are Ni2p... 3 / 2 and Ni2p 1 / 2 The two corresponding strong peaks originate from the interaction between Ni and -OOH in FeNi LDH; Figure 2 (C) is the high-resolution spectrum of Fe2p, with binding energies at 711.5 and 725.1 eV corresponding to Fe2p and Fe2p, respectively. 3+ 2p 3 / 2 and 2p 1 / 2 Track; from Figure 2 The high-resolution XPS spectrum of Cu2p in (D) shows that the two main peaks at 934.5 and 953.4 eV represent Cu(II)2p. 3 / 2 and Cu(II)2p 1 / 2 The two binding energies of 932.2 and 952.1 eV correspond to Cu(I)2p. 3 / 2 and Cu(I)2p 1 / 2 Furthermore, the peak at 942.4 eV further confirms the presence of Cu, which may enhance the redox activity of the material; Figure 2 (E) is the high-resolution spectrum of Au4f, where the binding energies of 83.87 and 87.47 eV correspond to Au4f. 7 / 2 and Au4f 5 / 2 This indicates that AuNPs are uniformly loaded in a metallic state on the AuNPs@Cu-FeNi LDH nanoflower structure, without significant aggregation or oxidation; furthermore... Figure 2 (F) is the XPS spectrum of O1s, with two prominent peaks. The peak at 531.03 eV may be the peak of hydroxyl oxygen in LDH, while the peak at 532.31 eV may be the peak of adsorbed water on the material surface.

[0066] Example 2

[0067] Add 150 µL of triethanolamine (TEA) to 0.0114 g (0.075 mmol) of ferrous sulfate and mix thoroughly to form a dark green Fe... 2+ -TEA complex precipitation.

[0068] Weigh 0.0348 g (0.225 mmol) of nickel sulfate and 0.1013 g (1.688 mmol) of urea, add them to 15 mL of deionized water, stir continuously for 30 min, mix them evenly with the above complex precipitate, transfer them to a 50 mL polytetrafluoroethylene high-pressure reactor, and hydrothermally react them in an 80 °C forced-air drying oven for 10 h to prepare FeNi LDH solution.

[0069] Weigh 0.0299 g (0.150 mmol) of copper acetate monohydrate and 0.1045 g (3 mmol) of sodium citrate pentahydrate, dissolve them in 40 mL of deionized water, stir for 30 min with a magnetic stirrer, then add the above FeNi LDH solution and mix, continue to stir slowly for 1.5 h, and react in a 40 °C forced-air drying oven for 24 h.

[0070] The reaction product was centrifuged at 10,000 rpm for 10 min, and the lower filter cake was collected. It was washed three times with anhydrous ethanol and then three times with deionized water. It was then dried at 60°C in a forced-air drying oven to constant weight to prepare Cu-FeNi LDH powder, wherein the Fe:Ni:Cu molar ratio was 1:3:2.

[0071] A 5µL aqueous dispersion of 1mol% AuNPs was added dropwise to the surface of Cu-FeNi LDH using a pipette, and the mixture was allowed to dry at room temperature to obtain the electrode-sensitive material AuNPs@Cu-FeNi LDH.

[0072] Example 3

[0073] Add 150 µL of triethanolamine (TEA) to 0.0114 g (0.075 mmol) of ferrous sulfate and mix thoroughly to form a dark green Fe... 2+ -TEA complex precipitation.

[0074] Weigh 0.0348 g (0.225 mmol) of nickel sulfate and 0.1013 g (1.688 mmol) of urea, add them to 15 mL of deionized water, stir continuously for 30 min, mix them evenly with the above complex precipitate, transfer them to a 50 mL polytetrafluoroethylene high-pressure reactor, and hydrothermally react them in an 80 °C forced-air drying oven for 10 h to prepare FeNi LDH solution.

[0075] Weigh 0.0767 g (0.450 mmol) of copper chloride and 0.1045 g (3 mmol) of sodium citrate pentahydrate, dissolve them in 40 mL of deionized water, stir for 30 min with a magnetic stirrer, then add the above FeNi LDH solution and mix, continue to stir slowly for 1.5 h, and react in a 100 °C forced-air drying oven for 24 h.

[0076] The reaction product was centrifuged at 10,000 rpm for 10 min, and the lower filter cake was collected. It was washed three times with anhydrous ethanol and then three times with deionized water. It was then dried at 60 °C in a forced-air drying oven to constant weight to prepare Cu-FeNi LDH powder, wherein the Fe:Ni:Cu molar ratio was 1:3:6.

[0077] A 5µL aqueous dispersion of 5mol% AuNPs was added dropwise to the surface of Cu-FeNi LDH using a pipette, and the mixture was allowed to dry at room temperature to obtain the electrode-sensitive material AuNPs@Cu-FeNi LDH.

[0078] Comparative Example 1

[0079] 0.0149 g (0.075 mmol) of ferrous chloride tetrahydrate was mixed with 150 µL of triethanolamine (TEA) until a dark green Fe3+ solution was formed. 2+ -TEA complex precipitation.

[0080] Weigh 0.0654 g (0.225 mmol) of nickel nitrate hexahydrate and 0.1013 g (1.688 mmol) of urea, add them to 15 mL of deionized water, stir continuously for 30 min, mix them evenly with the above complex precipitate, transfer them to a 50 mL polytetrafluoroethylene high-pressure reactor, and perform hydrothermal reaction in a 120 °C forced-air drying oven for 6 h to prepare FeNi LDH solution.

[0081] Weigh 0.0748 g (0.375 mmol) of copper acetate monohydrate and 0.1045 g (3 mmol) of sodium citrate pentahydrate, dissolve them in 40 mL of deionized water, stir for 30 min with a magnetic stirrer, then add the above FeNi LDH solution and mix, continue to stir slowly for 1.5 h, and place in a 60 °C forced-air drying oven to heat and react for 30 h.

[0082] The reaction product was centrifuged at 10,000 rpm for 10 min, and the lower filter cake was collected. It was washed three times with anhydrous ethanol and then three times with deionized water. It was then dried at 60 °C in a forced-air drying oven to constant weight to prepare Cu-FeNi LDH powder, wherein the Fe:Ni:Cu molar ratio was 1:3:5.

[0083] Figure 3The morphology of the Cu-FeNi LDH powder prepared in the above comparative example is shown. Compared with the product prepared by heating and reacting for 24 hours in Example 1, the Cu-FeNi LDH nanosheet structure was significantly destroyed after the heating and reaction time was extended to 30 hours, and irregular impurities appeared on the surface. It lost the original regularly distributed nanostructure, which is not conducive to the electrochemical catalytic reaction.

[0084] Example 4

[0085] Alumina with particle sizes of 1.0 μm and 0.05 μm was used as grinding powder to thoroughly polish the GCE. Then, its surface was thoroughly cleaned with anhydrous ethanol and deionized water to remove the surface oxide layer, resulting in a smooth and flat electrode surface visible to the naked eye.

[0086] The polished GCE electrode was placed in a three-electrode system with sulfuric acid as the electrolyte and chemically activated using cyclic voltammetry (CV). The scan rate was set to 50 mV / s, and the voltage range was -1 to 1 V. After several scans, the electrode surface was rinsed with deionized water and ethanol and dried to obtain the activated GCE electrode.

[0087] Take 5 mg of the electrode-sensitive material AuNPs@Cu-FeNi LDH prepared in Example 1, disperse it in 1 mL of anhydrous ethanol, and sonicate it for 30 min until a stable and uniform suspension is formed. Use a pipette to take 5 µL of the suspension and drop it onto the surface of the activated GCE electrode. Allow it to stand and dry at room temperature to form a uniform nanomaterial modification layer, and obtain the AuNPs@Cu-FeNi LDH / GCE electrode.

[0088] Continue to drop-coat the surface of the AuNPs@Cu-FeNi LDH / GCE electrode with an Ab solution of 10 µg / mL and incubate at 4°C for 12 h to fix the antibody on the AuNPs@Cu-FeNi LDH surface. Wash with PBS (pH=7.4) to remove the Ab that has not been incubated on the electrode surface and allow it to dry naturally at room temperature to prepare the Ab / AuNPs@Cu-FeNi LDH / GCE electrode.

[0089] 1 wt% BSA was added dropwise to the surface of the Ab / AuNPs@Cu-FeNi LDH / GCE electrode, and allowed to stand at room temperature for 1 h. After air drying, the active sites on the electrode material surface not occupied by the antibody were sealed to prevent non-specific adsorption interference in subsequent detection. The electrode was then washed with PBS (pH=7.4) and allowed to air dry at room temperature to obtain the electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE.

[0090] The prepared electrochemical immunosensor was stored in a refrigerated environment at 4°C to prevent the inactivation of biomolecules.

[0091] Example 5

[0092] 30 μL of 10 ng / mL PCT antigen was dropped onto the surface of the electrochemical immunosensor prepared in Example 4, and incubated at a constant temperature for 6 h to allow the antibody and antigen to bind fully and specifically, thus obtaining the PCT detection sensor PCT / BSA / Ab / AuNPs@Cu-FeNiLDH / GCE.

[0093] Using GCE electrode, AuNPs@Cu-FeNi LDH / GCE electrode, Ab / AuNPs@Cu-FeNi LDH / GCE electrode, BSA / Ab / AuNPs@Cu-FeNi LDH / GCE electrochemical immunosensor and the PCT detection sensor PCT / BSA / Ab / AuNPs@Cu-FeNi LDH / GCE prepared above as working electrodes, the relevant electrochemical performance tests, including CV curves and electrochemical impedance (EIS) curves, were completed within 24 hours.

[0094] Both tests were performed in a three-electrode system, using different working electrodes. A silver / silver chloride (Ag / AgCl) electrode saturated with KCl solution was used as the reference electrode, and a 1×1.5 cm platinum mesh (Pt) was used as the counter electrode. The electrolyte solution was 10.0 mM [Fe(CN)6]. 3- / 4- A 1:1 mixture with 0.2M phosphate buffer (PBS, pH=7.4).

[0095] Figure 4 In the figure, (A) is the CV curve, with a test scan rate of 50mV / s and a voltage range of -1 to 1V; (B) is the EIS curve, with a test frequency of 100mHz to 100kHz.

[0096] In the figure: a) GCE, b) AuNPs@Cu-FeNi LDH / GCE, c) Ab / AuNPs@Cu-FeNi LDH / GCE, d) BSA / Ab / AuNPs@Cu-FeNi LDH / GCE, e) PCT / BSA / Ab / AuNPs@Cu-FeNi LDH / GCE.

[0097] As can be seen from the CV curves, within the voltage range of -0.4 to 0.8 V, the bare GCE electrode surface exhibits a distinct redox curve, with peak anodic and cathodic currents corresponding to [Fe(CN)6]. 3- / 4-Electron transfer was observed. After modifying GCE with AuNPs@Cu-FeNiLDH nanocomposite as the electrode sensing material, the peak current of the electrode was significantly enhanced, which is attributed to the excellent electronic conduction and catalytic activity of AuNPs@Cu-FeNiLDH. Subsequently, when Ab was introduced further onto the electrode surface, the current response decreased significantly. This was due to the hindering effect of biomolecules on electron transport, resulting in a reduction in the peak current. After effective encapsulation with BSA, the peak current decreased again, indicating that it, as a biological protein, again hindered the electron transport channel. Finally, the electrochemical response of the PCT detection sensor modified with PCT decreased to 87.56 μA.

[0098] The peak current in the figure first increases and then decreases, indicating that the electrode modified with the electrode-sensitive material has high activity. In order to make the electrode specific for PCT, although the activity is reduced after modification with Ab and protein, it still has good electrochemical responsiveness.

[0099] To gain a more comprehensive understanding of the surface modification process of the electrodes, the resistance characteristics of different working electrodes were studied using EIS technology. The high-frequency region of the EIS plot is mainly composed of semicircles, representing the charge transfer process; while the low-frequency region is linear, corresponding to the diffusion process. The diameter of the semicircle can be quantified as the charge transfer resistance of different electrodes. This resistance value reflects key information about the surface modification effect of the electrodes and the interfacial reaction kinetics.

[0100] The EIS curves show that the GCE has a larger semicircle radius, indicating a higher electron transfer resistance. Modifying the electrode sensing material AuNPs@Cu-FeNi LDH reduces the corresponding semicircle radius in the EIS curve, suggesting that the introduction of the active material promotes electron transfer between the electrolyte and the electrode surface. The sequential loading of Ab, BSA, and PCT antigens leads to an increase in the semicircle radius, indicating that the specific binding of the immune complexes increases the electron transfer resistance, hindering electron transfer and ion diffusion at the sensor interface. This result is consistent with the CV curve results, both demonstrating the successful construction of the BSA / Ab / AuNPs@Cu-FeNi LDH / GCE electrochemical immunosensor, which can be used for PCT detection.

[0101] Example 6

[0102] Take 5 mg of the electrode-sensitive material AuNPs@Cu-FeNi LDH prepared in Example 3, disperse it in 1 mL of anhydrous ethanol, and sonicate it for 30 min until a stable and uniform suspension is formed. Use a pipette to take 5 μL and fix it on the activated GCE electrode. Let it stand at room temperature and dry naturally to form a uniform nanomaterial modification layer.

[0103] Then, a 10 µg / mL Ab solution was continuously and evenly dripped onto the electrode surface, and incubated at 4°C for 12 h to fix the antibody on the AuNPs@Cu-FeNi LDH surface. The unincubated Ab on the electrode surface was removed by washing with PBS (pH=7.4), and the electrode was allowed to dry naturally at room temperature.

[0104] Then, 1 wt% BSA was added to the electrode surface, allowed to stand at room temperature for 1 hour, and allowed to air dry naturally to block the active sites on the electrode material surface that were not occupied by the antibody. The electrode was then washed again with PBS (pH=7.4) and allowed to air dry naturally at room temperature to prepare the electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE, which was stored in a refrigerated environment at 4℃.

[0105] Comparative Example 2

[0106] 0.0149 g (0.075 mmol) of ferrous chloride tetrahydrate was mixed with 150 µL of triethanolamine (TEA) until a dark green Fe3+ solution was formed. 2+ -TEA complex precipitation.

[0107] Weigh 0.0654 g (0.225 mmol) of nickel nitrate hexahydrate and 0.1013 g (1.688 mmol) of urea, add them to 15 mL of deionized water, stir continuously for 30 min, mix them evenly with the above complex precipitate, transfer them to a 50 mL polytetrafluoroethylene high-pressure reactor, and perform hydrothermal reaction in a 120 °C forced-air drying oven for 6 h to prepare FeNi LDH solution.

[0108] The reaction product was separated by centrifugation, the filter cake was collected, washed three times with anhydrous ethanol, then washed three times with deionized water, and dried at 60°C in a forced-air drying oven to constant weight to obtain FeNi LDH solid powder.

[0109] Take 5 mg of the FeNi LDH prepared above and the Cu-FeNi LDH solid powder prepared in Example 1, respectively, disperse them in 1 mL of anhydrous ethanol, and sonicate for 30 min until a stable and uniform suspension is formed. Use a pipette to take 5 μL of each and fix it on the activated GCE electrode. Allow it to stand at room temperature and dry naturally to form a uniform nanomaterial modification layer. Construct the electrode materials FeNiLDH / GCE and Cu-FeNi LDH / GCE as working electrodes, and perform electrochemical performance tests according to the three-electrode system provided in Example 5.

[0110] Test results are as follows Figure 5As shown, the oxidation current of bare GCE is relatively low, around 56.39 μA. After growing FeNi LDH on the GCE substrate, the peak current increases significantly. This phenomenon is attributed to the high specific surface area and abundant active sites of FeNi LDH, which promotes the oxidation of [Fe(CN)6]. 3- / 4- Electron transfer; and the peak current of the electrode modified Cu-FeNi LDH composite nanomaterial is further improved to 96.73 μA, exhibiting strong redox reaction.

[0111] The above results indicate that Cu doping not only enhances the conductivity of the composite nanomaterials but may also further accelerate interfacial electron transfer through synergistic catalysis. Figure 4 The CV curves of AuNPs@Cu-FeNi LDH / GCE further demonstrate that loading AuNPs increases the sensitivity of the electrochemical immunosensor.

[0112] Application Example 1

[0113] Using the electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE constructed in Example 4 as the detection target, its electrochemical detection performance for PCT was quantitatively characterized by square wave voltammetry (SWV), and key performance parameters such as its linear range and limit of detection for PCT were investigated.

[0114] PCT antigen solutions with different concentration gradients ranging from 1 pg / mL to 100 ng / mL were prepared and drop-coated onto the surface of the electrochemical immunosensor BSA / Ab / AuNPs@Cu-NiFe LDH / GCE. After drying, they were placed in a 10.0 mM [Fe(CN)6] atmosphere. 3- / 4- SWV was detected in an electrolyte solution containing 0.2M PBS (pH=7.4) using a three-electrode system. The test voltage window was -0.5 to 1.0V, the scan rate was 50mV / s, and the peak SWV current signal was recorded.

[0115] like Figure 6 From the SWV curve in (A), it can be observed that within the range of 1 pg / mL to 100 ng / mL, the anodic peak current ( I pa The trend of decreasing PCT concentration is observed as PCT concentration increases. (This is in contrast to the relationship between logarithmic concentration and...) I pa By performing a linear fit, the linear regression equation shown in (B) is obtained. y =–20.828 x +123.747 ( R 2 =0.9995), and the limit of detection of the electrochemical immunosensor can be calculated to be 30 fg / mL based on the linear fitting line.

[0116] Application Example 2

[0117] The detection repeatability of the prepared electrochemical immunosensor was tested using differential pulse voltammetry (DPV).

[0118] Using the same synthesis and assembly methods, but with electrode-sensitive materials synthesized in different batches, five electrochemical immunosensors were prepared and their electrochemical performance was tested; five electrochemical immunosensors were also prepared using electrode-sensitive materials synthesized in the same batch and their electrochemical performance was tested; the detection repeatability of materials from the same batch and different batches was verified respectively.

[0119] like Figure 7 As shown in (A), the detection results of electrochemical immunosensors prepared from five different batches of electrode sensitive materials for 100 ng / mL PCT were evaluated by DPV technology. The average current response was 174.39 µA and the relative deviation was 1.99%.

[0120] Figure 7 (B) The DPV scan test results of the five identical electrochemical immunosensors from the same batch also showed a relatively stable peak current response, with an average value of 170.32 μA and a relative deviation of 2.09%.

[0121] The above test results show that the electrochemical immunosensor prepared in this invention has good detection reproducibility.

[0122] Application Example 3

[0123] To evaluate the long-term stability of the prepared BSA / Ab / AuNPs@Cu-FeNi LDH / GCE electrochemical immunosensor, stability tests were conducted on PCT at mass concentrations of 100 fg / mL and 1 ng / mL using the same batch of sensors for 42 days.

[0124] The sensor was stored in a 4°C refrigerator to simulate storage conditions in real-world applications. Every 7 days, the sensor was removed for PCT DPV testing. Specific test results are as follows: Figure 7 As shown in (C).

[0125] During the test, the peak current response of the fabricated sensor did not fluctuate significantly, with average values ​​of 184.37 and 147.66 μA, and relative deviations of 2.16% and 1.89%, respectively, indicating that the sensor has good stability.

[0126] Application Example 4

[0127] Five common substances from human serum, including carcinoembryonic antigen (CEA), amino acid (AA), prostate-specific antigen (PSA), alpha-fetoprotein (AFP), and hepatitis B virus (HBs), were used as interferants to verify the selectivity of the prepared electrochemical immunosensor.

[0128] Five different antiserum interfering agents were modified with 100 pg / mL onto the electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE, and their SWV current response values ​​were tested.

[0129] The above-mentioned electrochemical immunosensor was used to incubate 100 pg / mL of PCT antigen alone, and a mixture of 100 pg / mL of the above 5 interferants and 100 pg / mL of PCT antigen, and the SWV current response value was tested for comparison.

[0130] like Figure 7 As shown in (D), when the electrochemical immunosensor was modified with five different interfering agents, the measured SWV current response values ​​were all around 230 µA. However, after incubating the PCT antigen alone on the electrochemical immunosensor, the peak current response decreased significantly to 152.8 µA. In comparison, the peak current response of the mixed solution of the interfering agent and PCT did not show a significant change. This result indicates that only PCT can specifically bind to the PCT antibody, and the resistance of the sensor increases after binding, thereby reducing the current. Therefore, the electrochemical immunosensor prepared in this invention has excellent anti-interference and specificity in detecting PCT.

[0131] Application Example 5

[0132] Table 1 below compares in detail the detection performance of the electrochemical immunosensor used for PCT detection with the electrode sensitive material AuNPs@Cu-FeNi LDH prepared in this invention and various electrode sensitive materials reported in the literature, including the limit of detection (LOD) and detection range.

[0133]

[0134] Multi-dimensional data comparison shows that, compared with various electrode-sensitive materials reported in the literature, the electrochemical immunosensor based on the AuNPs@Cu-FeNi LDH electrode-sensitive material of this invention achieves a wide linear range (1 pg / mL to 100 ng / mL) and a low limit of detection (LOD) (30 fg / mL) through material design and interface regulation. It has a wide detection range and high sensitivity, and has important application potential in the fields of early diagnosis of bacterial infection and monitoring of antibiotic efficacy.

[0135] The detection performance of each electrode sensing material and its electrochemical immunosensor in Table 1 is derived from the following literature reports:

[0136] [1] Miao J, Du K, Li X, et al. Ratiometric electrochemicalimmunosensor for the detection of procalcitonin based on the ratios of SiO2-Fc-COOH-Au and UiO-66-TB complexes [J]. Biosens Bioelectron , 2021, 171:112713.

[0137] [2] Xu X, Li X, Miao J, et al. A dual-mode label-free electrochemicalimmunosensor for ultrasensitive detection of procalcitonin based on g-C3N4-NiCo2S4-CNTs-AgNPs [J]. Analyst , 2021, 146(10): 3169–3176.

[0138] [3] Ding H, Yang L, Jia H, et al. Label-free electrochemicalimmunosensor with palladium nanoparticles functionalized MoS2 / NiCoheterostructures for sensitive procalcitonin detection [J]. Sensors and Actuators B: Chemical , 2020, 312: 127980.

[0139] [4] Li Y, Liu L, Liu X, et al. A dual-mode PCT electrochemicalimmunosensor with CuCo2S4 bimetallic sulfides as enhancer [J]. Biosensors and Bioelectronics , 2020, 163: 112280.

[0140] [5] Wang XY, Feng YG, Wang AJ, et al. Facile construction of ratiometric electrochemical immunosensor using hierarchical PtCoIr nanowires and porous SiO2@Ag nanoparticles for accurate detection of septicemia biomarker [J]. Bioelectrochemistry , 2021, 140: 107802.

[0141] Application Example 6

[0142] To investigate the reliability and authenticity of the electrochemical immunosensor in detecting PCT in real samples, the standard spiking method was used to detect normal human serum samples and evaluate its recovery performance in real human serum samples.

[0143] PCT antigen at concentrations of 0.1, 1, 5, and 20 ng / mL was added to serum samples, and its SWV current response was detected using the electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE of this invention. The detection amount was then calculated.

[0144] As can be seen from the results in Table 2, the relative standard deviation (RSD) of the detection ranged from 1.63 to 3.37%, and the recovery rate ranged from 97.00 to 111.20%.

[0145]

[0146] The above test results show that the electrochemical immunosensor constructed based on AuNPs@Cu-FeNi LDH electrode sensing material has good accuracy and precision, can meet the detection requirements of PCT in actual serum samples, and has broad application prospects.

[0147] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A layered double hydroxide composite material with uniform ballflower-like micro-morphology, which is a Cu-FeNi LDH composite nanomaterial obtained by heating a water-soluble copper salt and the FeNi LDH nanomaterial in a water solution containing citrate to 50-100℃. wherein The FeNi LDH nanomaterial is prepared by first reacting a water-soluble ferrous salt with triethanolamine to form a complex, then adding a water-soluble nickel salt, and carrying out a sealed hydrothermal reaction in a water solution containing urea at 80-160℃.

2. The layered double hydroxide composite material according to claim 1, characterized by The molar ratio of the water-soluble copper salt to the iron element in the FeNi LDH nanomaterial is (2-6):1, and the molar ratio of the water-soluble ferrous salt to the water-soluble nickel salt is 1:(1-6).

3. The layered double hydroxide composite material of claim 1, characterized by The water-soluble copper salt is copper chloride, copper acetate or copper sulfate; the water-soluble ferrous salt is ferrous chloride, ferrous nitrate or ferrous sulfate; and the water-soluble nickel salt is nickel nitrate or nickel sulfate.

4. The layered double hydroxide composite material of claim 1, characterized by The hydrothermal reaction time for preparing the FeNi LDH nanomaterial is 4-10h, and the reaction time for preparing the Cu-FeNi LDH composite nanomaterial is 3-24h.

5. A method for preparing the layered double hydroxide composite material of claim 1, comprising: Fe is formed by mixing water-soluble ferrous salts with triethanolamine to form a dark green Fe 2+ - TEA complex precipitation; adding a water-soluble nickel salt and urea to water to form a solution, adding the above-mentioned complex to precipitate, and carrying out a sealed hydrothermal reaction at 80-160℃ to prepare a FeNi LDH nanomaterial solution; dissolving a water-soluble copper salt and citrate in water to form a mixed solution; mixing the above-mentioned mixed solution with the FeNi LDH nanomaterial solution uniformly, and heating to 50-100℃ to react; collecting the reaction precipitate, washing and drying to obtain a dark blue Cu-FeNi LDH composite nanomaterial solid powder.

6. An electrode sensitive material, which is obtained by dispersing and loading a large number of uniform and regular spherical AuNPs between the ballflower petals of the Cu-FeNi LDH composite nanomaterial with uniform ballflower-like micro-morphology of claim 1 as a support.

7. A method for preparing the electrode sensitive material of claim 6, which is obtained by adding a 1-5mol% AuNPs solution to the surface of the Cu-FeNi LDH composite nanomaterial, and standing and drying at room temperature to obtain an AuNPs@Cu-FeNi LDH composite structure as the electrode sensitive material.

8. An electrochemical immunosensor, which is constructed by using an activated glassy carbon electrode as a substrate, coating the electrode sensitive material of claim 6 as a modification layer on the surface of the substrate, incubating PCT antibodies on the surface of the electrode sensitive material, and adding bovine serum albumin to block the active sites on the surface of the electrode sensitive material not occupied by the PCT antibodies, to obtain an electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE.

9. A method for preparing the electrochemical immunosensor of claim 8, comprising: The electrode sensitive material AuNPs@Cu-FeNi LDH is dispersed in anhydrous ethanol, ultrasonic dispersion is performed to form a stable and uniform suspension, is fixed on the surface of an activated and pretreated glassy carbon electrode, is naturally dried to form a nanomaterial modified layer, and an AuNPs@Cu-FeNi LDH / GCE electrode is obtained; A PCT antibody solution is uniformly dropped on the surface of the nanomaterial modified layer, and is incubated at 4 DEG C, so that the antibody is fixed on the surface of the nanomaterial modified layer through Au-S bond and physical adsorption, and an Ab / AuNPs@Cu-FeNi LDH / GCE electrode is formed; A bovine serum albumin solution is dropped on the surface of the electrode, is left to dry, and the active sites on the surface of the electrode not occupied by the antibody are blocked, and an electrochemical immunosensor BSA / Ab / AuNPs@Cu-FeNi LDH / GCE is constructed.

10. Application of the electrochemical immunosensor of claim 8 as a sensor for detecting a bacterial infection biomarker PCT, wherein the detection of PCT is performed by specific binding of PCT antigens in a sample to be detected to PCT antibodies on the electrochemical immunosensor.

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