An electrochemical immunosensor and its preparation method and application

Abstract

The present invention belongs to the technical field of electrochemical sensors, and discloses an electrochemical immunosensor, a preparation method thereof and an application. The preparation method includes the following steps: S1. Dissolve the NiCoP / g-C3N4 nanocomposite in nitric acid, and carry out an oil bath reaction at 120-130 °C to obtain a COOH-NiCoP / g-C3N4 nanocomposite; S2. Prepare the COOH-NiCoP / g-C3N4 obtained in S1 into a suspension and drop it onto the surface of the treated electrode for the first incubation; then, after carrying out a condensation reaction on 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and coupling with a monoclonal antibody, drop it onto the electrode surface for the second incubation to obtain an electrochemical immunosensor. The present invention constructs a label-free electrochemical immunosensor for the detection of procalcitonin, which has excellent electrochemical sensing performance and a wider detection range.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical sensors, and in particular relates to an electrochemical immunosensor and a preparation method and application thereof. Background Art

[0002] Procalcitonin (PCT) is an effective biomarker for a variety of diseases caused by bacterial infections and has played an important role in clinical medical practice. Nowadays, a variety of detection methods have been established, including enzyme-linked immunosorbent assay (ELISA), protein microarray, electrochemiluminescence immunosensor, surface plasmon resonance biosensor and electrochemical biosensor, for the detection of PCT. Among them, electrochemical biosensor is a powerful analytical method that has attracted much attention due to its multiple intrinsic advantages such as high sensitivity, fast response, relatively low cost and easy miniaturization. However, choosing the right electrode material remains a challenge to achieve high-performance electrochemical sensors.

[0003] Nanocomposites based on graphene and its derivatives have attracted much attention in the field of sensing in recent years. Graphitic carbon nitride (g-C3N4) is a two-dimensional planar conjugated material that has recently been proposed as a promising candidate for direct solar water splitting, visible light photocatalytic pollutant degradation, optoelectronics, SERS sensing, and bioimaging due to its unique electronic band structure, large specific surface area, good biocompatibility, and excellent electronic and physicochemical properties. Despite these excellent properties, the application of g-C3N4 in the field of electrochemical sensing is still limited due to poor electrical conductivity and large contact resistance. To overcome this shortcoming, some modifications, such as doping or coupling with other nanomaterials, have been adopted to improve the conductivity of g-C3N4 and make it suitable for electrochemical sensor applications.

[0004] Transition metal phosphides such as Ni2P, CoP and Cu3P have been widely used in research fields such as photocatalysis and supercapacitors due to their metalloid properties, good conductivity, stability and high electrocatalytic activity. However, as electrochemical materials, single metal phosphides have high overpotential and large charge transfer impedance, and their electrochemical performance is not ideal. Their application in the field of electrochemical sensing is still limited. Therefore, it is of great significance to improve the performance of immunosensors and use them for the detection of procalcitonin. Summary of the invention

[0005] The purpose of the present invention is to provide an electrochemical immunosensor and its preparation method and application in view of the above existing problems, prepare a NiCoP / g-C3N4 nanocomposite material and use it as a sensing platform, construct a label-free electrochemical immunosensor for procalcitonin analysis, and provide a new method for procalcitonin detection. In addition, the highly sensitive NiCoP / g-C3N4 sensor can be used for procalcitonin detection in human serum samples.

[0006] In order to achieve the above purpose, the technical solution adopted in this application is:

[0007] A method for preparing an electrochemical immunosensor comprises the following steps:

[0008] S1. Carboxylation of NiCoP / g-C3N4 nanocomposite: dissolving the NiCoP / g-C3N4 nanocomposite in nitric acid for oil bath reaction, cooling to room temperature for washing, adjusting the pH to neutral, and drying to obtain COOH-NiCoP / g-C3N4 nanocomposite;

[0009] S2. Preparation of electrochemical immunosensor: the COOH-NiCoP / g-C3N4 suspension obtained in S1 is added dropwise onto the treated electrode surface for the first incubation; after the incubation, the electrode surface is washed, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are condensed with monoclonal antibodies and then added dropwise onto the electrode surface for the second incubation; after the incubation, the unspecified binding sites are covered with bovine serum albumin, and the electrode is rinsed again to obtain the electrochemical immunosensor.

[0010] Preferably, the mass volume ratio of the NiCoP / g-C3N4 nanocomposite material to nitric acid is 1 g:100 mL, and the concentration of the nitric acid is greater than or equal to 12 mol / L.

[0011] Preferably, in S1, the oil bath reaction time is 24 hours; the drying is vacuum drying, the drying temperature is 30-40° C., and the drying time is 10-15.

[0012] Preferably, in S2, the electrode is a glassy carbon electrode, which is processed by polishing the glassy carbon electrode with 0.3 mm alumina slurry, then washing it with water and ethanol for multiple times under ultrasonic treatment, and drying it with nitrogen.

[0013] Preferably, in S2, the COOH-NiCoP / g-C3N4 suspension is prepared by dispersing COOH-NiCoP / g-C3N4 in a solvent and ultrasonically treating the solvent for 30-40 min;

[0014] The volume mass ratio of the COOH-NiCoP / g-C3N4 to the solvent is 1 mg:1-5 mL; the solvent is ethanol.

[0015] Preferably, in S2, the volume ratio of the mixed solution to the monoclonal antibody is 1:1; the concentration of the monoclonal antibody is 14 μg / mL, the concentration of the mixed solution is 2.0 mM, and the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 4:2.3.

[0016] Preferably, in S2, the first incubation time is 3-5 hours, the second incubation time is 2-3 hours; and the mass concentration of the bovine serum albumin solution is 2%.

[0017] In addition, the present invention also provides an electrochemical immunosensor prepared by the above preparation method.

[0018] The present invention also provides an application of the electrochemical immunosensor for detecting procalcitonin in serum, wherein the detection method comprises the following steps:

[0019] (1) A three-electrode system was used for detection, wherein an Ag / AgCl electrode was used as a reference electrode, a platinum plate electrode was used as a counter electrode, and an electrochemical immunosensor was used as a working electrode. The serum sample was centrifuged and filtered to obtain a supernatant, which was diluted with Tris-HCl buffer, and different standard concentrations of procalcitonin were added. In the presence of 5 mM K3Fe(CN)6 / K4Fe(CN)6, its response value was measured by differential pulse voltammetry, and a standard curve was established by comparing the response value with the procalcitonin concentration;

[0020] (2) The serum sample to be tested is prepared into a test solution according to the method of step (1), and its response value is measured by differential pulse voltammetry. The response value is inserted into the standard curve to calculate the concentration of procalcitonin.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention uses NiCoP / g-C3N4 nanocomposite materials to construct an electrochemical immunosensor, providing a new label-free electrochemical immunosensor for procalcitonin detection, which has excellent electrochemical sensing performance and a wider detection range, and can be used for procalcitonin detection in human serum samples, which has important scientific significance and application value.

[0023] (2) The present invention utilizes the larger surface area of ​​g-C3N4 and the higher electron transfer ability of NiCoP / g-C3N4, and the NiCoP / g-C3N4 nanocomposite material as the sensing interface to enhance the sensing performance, thereby improving the detection sensitivity of the electrochemical immunosensor, with a strong response signal, fast response speed, good stability, a wide detection range, and a minimum detection limit of 0.5 ag / mL.

[0024] (3) There is a synergistic effect between the ternary metal phosphides NiCoP used in the present invention. Due to factors such as higher conductivity, lower electronegativity of phosphorus than oxygen, and less negative charge of phosphorus, it has good electronic conductivity and electronic structure, and has better electrochemical properties. Compared with single metal phosphides, it has better electrochemical properties, lower overpotential and smaller charge transfer impedance, making it suitable for the design of biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation process of the electrochemical immunosensor of the present invention;

[0026] Figure 2 This is the X-ray photoelectron spectrum of g-C3N4 prepared in Example 1 of the present invention;

[0027] Figure 3 It is the scanning electron microscope image of Example 1-3 of the present invention;

[0028] Figure 4 X-ray diffraction pattern of the NiCoP / g-C3N4 composite material prepared in Example 2-3 of the present invention;

[0029] Figure 5 is an X-ray photoelectron spectrum of the NiCoP / g-C3N4 composite material of the present invention;

[0030] Figure 6 The electrochemical impedance performance diagram of Examples 1-3 of the present invention;

[0031] Figure 7 IR spectra of COOH-NiCoP / g-C3N4 in Examples 2 and 4 of the present invention;

[0032] Figure 8 The cyclic voltammetry and electrochemical impedance spectroscopy of Example 2 of the present invention;

[0033] Fig. 9 The response of the electrochemical immunosensor of Invention Example 4 to different concentrations of procalcitonin;

[0034] Fig.10 This is a diagram showing the evaluation effect of the electrochemical immunosensor according to Example 4 of the present invention;

[0035] Fig.11 is a standard curve diagram of procalcitonin concentration in human serum samples of the present invention;

[0036] Fig.12 It is the response diagram of the procalcitonin concentration in the human serum samples of the present invention. DETAILED DESCRIPTION

[0037] The following will combine the data in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods. Among them, nickel (II) chloride hexahydrate (NiCl2·6H2O), cobalt nitrate hexahydrate (CoH 12 N2O 12 ), sodium hypophosphite monohydrate (NaH2PO2·H2O) and urea were purchased from Shanghai Titan Technology Co., Ltd.; procalcitonin (PCT), monoclonal antibody (BSA), interleukin 6 (IL-6) and C-reactive protein (CRP) were purchased from Wuhan Huamei Bioengineering Co., Ltd.; Tris-HCl buffer was purchased from Beijing Solebow Technology Co., Ltd.

[0039] Example 1

[0040] The preparation method of g-C3N4 comprises the following steps:

[0041] Urea was placed in a covered crucible and heat treated at 5°C / min to 550°C under ambient pressure in air and kept in an argon environment for 3 h to obtain yellow g-C3N4. Then, g-C3N4 was dissolved in deionized water, stirred with ultrasound overnight, and then centrifuged and dried for later use.

[0042] Example 2

[0043] The preparation method of 50mgNiCoP / g-C3N4 nanocomposite material comprises the following steps:

[0044] 50mgNiCl2·6H2O, 50mgCoH 12 N2O 12and 50 mgNaH2PO2·H2O sodium were dissolved in 10 mL ultrapure water, 300 mg g-C3N4 was added to the above solution and stirred for 6 h after ultrasonic treatment for 1 h; the solution was then completely dried by vacuum drying, and the remaining powder was ground and hydrothermaled at 350 °C in an argon environment for 2 h to obtain a white powder NiCoP / g-C3N4 nanocomposite material, which was washed three times with ultrapure water and ethanol respectively. The resulting response solid was named 50 mgNiCoP / g-C3N4.

[0045] Example 3

[0046] The preparation method of 100mgNiCoP / g-C3N4 nanocomposite material comprises the following steps:

[0047] 100mgNiCl2·6H2O, 100mgCoH 12 N2O 12 and 100 mgNaH2PO2·H2O sodium were dissolved in 10 mL ultrapure water, 300 mg g-C3N4 was added to the above solution and stirred for 6 h after ultrasonic treatment for 1 h; the solution was then completely dried by vacuum drying, and the remaining powder was ground and hydrothermaled at 350 °C in an argon environment for 2 h to obtain a white powder NiCoP / g-C3N4 nanocomposite material, which was washed three times with ultrapure water and ethanol respectively. The resulting response solid was named 100 mgNiCoP / g-C3N4.

[0048] Example 4

[0049] A method for preparing an electrochemical immunosensor comprises the following steps:

[0050] S1. Carboxylation of NiCoP / g-C3N4 nanocomposite material: 50 mg of NiCoP / g-C3N4 nanocomposite material obtained in Example 2 was dissolved in 100 mL of nitric acid, the mixture was reacted in an oil bath at 125° C. for 24 h, and then cooled to room temperature, solid matter was collected, washed with ultrapure water, pH was adjusted to neutral, and vacuum dried at 35° C. for 12 h to obtain COOH-NiCoP / g-C3N4 nanocomposite material;

[0051] S2. Preparation of electrochemical immunosensor: first polish the glassy carbon electrode with 0.3 mm alumina slurry, the diameter of the glassy carbon electrode is 0.3 cm, then wash it with water and ethanol for multiple times under ultrasonic treatment, and dry it with nitrogen for use; 20 μL of COOH-NiCoP / g-C3N4 suspension obtained in S1 (COOH-NiCoP / g-C3N4 is dispersed in a solvent and ultrasonically treated for 30 min to obtain COOH-NiCoP / g-C3N4 suspension) is added dropwise onto the treated electrode surface and incubated for the first time for 3 h; After the reaction, the electrode surface was washed with ultrapure water, and a total of 20 μL of a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and a monoclonal antibody was added to the electrode surface for a condensation coupling reaction, wherein the mass ratio of EDC to NHS was 4:2.3, the concentration of the mixed solution was 2.0 mM, the concentration of the monoclonal antibody was 14 μg / mL, and the second incubation was performed at room temperature for 2 h; after the incubation, the electrode was rinsed with a pH 7.0 Tris-HCl buffer, and unspecified binding sites were covered with 20 μL BSA (2%, w / v), and then the electrode was rinsed with a pH 7.0 Tris-HCl buffer to obtain an electrochemical immunosensor, and its preparation process is as follows: Figure 1 shown.

[0052] Example 5

[0053] The preparation method is the same as that of Example 4, except that in S1, the NiCoP / g-C3N4 nanocomposite material is 100 mg NiCoP / g-C3N4 prepared in Example 3.

[0054] Figure 2 The X-ray photoelectron spectrum of g-C3N4 prepared in Example 1 of the present invention is as follows: Figure 2 As shown, Figure 2 As shown in A, the peaks of C, N, and O can be seen; in the enlarged view Figure 2 In B, the C1 peak can be decomposed into four peaks, which are derived from graphite (284.3 eV), CN / CO combination (285.2 eV), cyanide / cyanoquinone (287.5 eV) and heptazine-type carbon (292.6 eV); Figure 2 C, for N1s spectrum, heptazine N (397.97 eV), pyrrole N (399.3 eV), graphite N (400.3 eV) and oxidized N (403.6 eV). All characteristic peaks are in good agreement with those of synthetic g-C3N4.

[0055] Figure 3 The scanning electron microscope images of Examples 1-3 of the present invention are shown in Figure 1. Figure 3A) Compared with the nanoparticles dispersed on the surface of g-C3N4, 50mg NiCoP / g-C3N4 prepared in Example 2 ( Figure 3 B) is about 20 nm in size. Example 3 Preparation of 100 mg NiCoP / g-C3N4 ( Figure 3 C) is about 10 nm in size.

[0056] Figure 4 The X-ray diffraction pattern of the NiCoP / g-C3N4 composite material prepared in Example 2-3 of the present invention is as follows: Figure 4 As shown, the g-C3N4 loaded with NiCoP of different mass contents in Examples 2-3 are located at 30.62, 32.02, 35.51, 40.99, 44.89, 47.58, 54.44, 54.74 and 55.33, which can be indexed to (110), (101), (200), (111), (201), (210), (300), (002) and (211), and the planes of hexagonal NiCoP 18, 22, 23 (JCPDS No. 71-2336). The results show that NiCoP nanoparticles are successfully loaded on the surface of g-C3N4.

[0057] The composition and chemical state of the NiCoP / g-C3N4 composite material were further tested by XPS. Figure 5 is the X-ray photoelectron spectrum (XPS) of the NiCoP / g-C3N4 composite material of the present invention, such as Figure 5 As shown in A, the complete XPS survey spectrum shows the presence of C, N, Ni, Co, P and O (O may come from air contact); the high-resolution P2p spectrum ( Figure 5 B) shows that the peak binding energy at 127.7 eV is close to that of P 2p3 / 2, indicating the presence of elemental P24,25, while the peak at 131.9 eV can be attributed to the oxidized phosphorus species in contact with air26; for Co 2p ( Figure 5 C) region, the binding energies of 777.25 eV and 792.2 eV are attributed to Co2p3 / 2 and Co 2p1 / 2 of metallic Co due to the formation of Co-P27,28; similarly, for Ni 2p( Figure 5 In region D, the strong binding energy of 852.25 eV is very close to that of metallic nickel (852.6 eV), which means that there are partially charged Ni species, further proving that NiCoP has been successfully loaded on the g-C3N4 surface.

[0058] Figure 6 The electrochemical impedance performance of Examples 1-3 of the present invention is as follows: Figure 6As shown, compared with g-C3N4, the interfacial electron transfer resistance (Ret) of NiCoP / g-C3N4 after direct deposition on the GCE surface is significantly reduced, indicating that the conductivity of g-C3N4 is higher when NiCoP is loaded on the surface; in addition, compared with Example 1 (g-C3N4) and Example 3 (100mgNiCoP / g-C3N4), 50mg NiCoP / g-C3N4 (Example 2) exhibits higher electron transfer capacity, and these results can be attributed to the agglomeration of excess NiCoP, which may prevent or reduce the activity of certain reaction sites. According to the above results, 50mg NiCoP / g-C3N4 nanocomposite materials were used for subsequent studies.

[0059] Figure 7 The infrared spectra of NiCoP / g-C3N4 prepared in Example 2 of the present invention and NiCoP / g-C3N4 after carboxylation in Example 4 are as follows: Figure 7 As shown, the spectral bands with different wave numbers correspond to the specific vibrations of the molecular functional groups, 806cm -1 The band at 1240-1643 cm -1 The bands in the range can be attributed to the CN stretching of g-C3N4, 3165cm -1 The broad band at 1330 cm-1 may correspond to the primary and secondary amines, their intermolecular hydrogen bonds and the OH stretching vibration of water molecules; in addition, -1 and 1558cm -1 The peak at corresponds to the -COO- bending band, and these results indicate that the carboxyl groups have been successfully functionalized on NiCoP / g-C3N4.

[0060] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to demonstrate the modification steps of the electrode surface, such as Figure 8 The cyclic voltammetry and electrochemical impedance spectroscopy of Example 2 of the present invention show that in terms of EIS, [Fe(CN)6] 3- / [Fe(CN)6] 4- As a redox probe, the semicircle diameter is equivalent to the electron transfer resistance; in 5mM [Fe(CN)6] 3- / 4- In the figure, the bare electrode is almost a straight line ( Figure 8 A curve a), which is the characteristic of the mass diffusion-limited step of the electron transfer process; when 50 mg COOH-NiCoP / g-C3N4 prepared in Example 2 is self-assembled onto the bare drain electrode by π-π stacking, Ret increases ( Figure 8 A curve b), which can be attributed to the fact that the immobilized 50 mg COOH-NiCoP / g-C3N4 prevented the [Fe(CN)6] 3- / 4-The diffusion of the redox probe and increased the interfacial electron transfer resistance, after which the antibody (Ab) was cross-linked on the 50 mg COOH-NiCoP / g-C3N4 modified electrode by zero-length amine-reactive crosslinkers EDC and NHS, Ret increased significantly because the large size of curve b hindered the diffusion of the redox probe to the electrode surface, increasing the electron transfer resistance. These results are consistent with those obtained from CV measurements ( Figure 8 B). Both results indicate that the sensing interface has been successfully prepared.

[0061] A three-electrode system was used for detection, in which the Ag / AgCl electrode was used as the reference electrode, the platinum plate electrode was used as the counter electrode, and the electrochemical immunosensor was used as the working electrode. Different concentrations of procalcitonin were added to pH 7.0 Tris-HCl in the presence of 5 mM K3Fe(CN)6 / K4Fe(CN)6, and its response value was measured by differential pulse voltammetry (DPV) at room temperature. Fig. 9 The response of the electrochemical immunosensor of Example 2 and Example 4 of the present invention to different concentrations of procalcitonin is shown in FIG. Fig. 9 As shown in FIG. 1 , DPV is used to display the response of the sensor to different concentrations of PCT. As the PCT concentration increases from 1 ag / mL to 10 ng / mL, the electrochemical oxidation peak current gradually decreases, and a linear relationship is established between the DPV response value and the procalcitonin concentration. The linear equation of PCT is described as I(A)=88.5-7.82c(R 2 =0.97), and the detection limit was estimated to be 0.5 ag / mL (S / N = 3), which is competitive with other electrochemical methods, dynamic light scattering (DLS), SPR biosensor, and enzyme-free immunosensor methods listed in Table 1.

[0062] Table 1 compares the proposed method with previous reports on PCT detection

[0063]

[0064] The sensitivity of the immunosensor prepared in Example 4 was evaluated by several interfering biomarkers, including CRP and IL-6. CRP is a biomarker of tissue damage and inflammation, and IL-6 is associated with a variety of diseases, such as diabetic osteoarthritis, asthma, and inflammatory bowel disease. The PCT concentration was 1 pg / mL, and the concentration of each interfering biomarker was 10 pg / mL. Fig.10 This is a diagram showing the evaluation effect of the electrochemical immunosensor of Example 4 of the present invention, showing the Rec values ​​of the samples to which each interfering biomarker, PCT, and the mixture with or without PCT added are added. Fig.10As shown, the Rct value of each interfering biomarker was not significant and was similar to that of the control sample, but the Ret value response of the sensor to PCT and a mixture of each interfering biomarker was the same as that of PCT, indicating that the interfering biomarkers had no effect on the detection of PCT. It can be inferred that the electrochemical sensor has a high specificity and selectivity.

[0065] Actual sample analysis application

[0066] In order to evaluate the practical application of the NiCoP / g-C3N4 electrochemical immunosensor prepared in Example 4 in blood samples, the concentration of PCT in human serum samples was analyzed. The serum sample was centrifuged at 5000rpm for 20min, then filtered with a 0.22μm filter membrane to obtain a supernatant, diluted 100 times with Tris-HCl buffer, and detected using a three-electrode system, in which the Ag / AgCl electrode was used as the reference electrode, the platinum plate electrode was used as the counter electrode, and the electrochemical immunosensor was used as the working electrode. Procalcitonin with a final concentration of 0, 0.5, 1, 10, 50, 100, and 150pg / mL was added in sequence, and its response value was measured in the presence of 5mM K3Fe(CN)6 / K4Fe(CN)6, and a linear relationship was established based on the response value and the concentration of procalcitonin in the serum sample. Fig.11 As shown, the standard curve obtained is y = -5.19C PCT +63.29, the serum sample to be tested was centrifuged at 5000 rpm for 20 min, then filtered with a 0.22 μm filter membrane to obtain the supernatant, and diluted 100 times with Tris-HCl buffer to prepare the test solution, and its response value was measured by differential pulse voltammetry (DPV) at room temperature, and the response value was substituted into the standard curve y=-2.17143C+65.17 to calculate the concentration of procalcitonin.

[0067] Fig.12 is the response of procalcitonin concentration in human serum samples, such as Fig.12 As shown, the DPV curves showed that as the PCT concentration increased from 1 pg / mL to 100 pg / mL, the electrochemical oxidation peak current decreased, and the recovery rates of PCT (1 pg / mL, 10 pg / mL, 100 pg / mL) ranged from 94% to 101.3% (see Table 2), indicating that the NiCoP / g-C3N4-based immunosensor has great potential for the analysis of PCT in actual clinical samples.

[0068] Table 2 Determination of PCT in human serum samples

[0069] sample Add (pg / mL) Found (pg / mL) Discovery rate (%) RSD(%) 1 1 0.94 94 2.56 2 10 9.76 97.6 1.75 3 100 101.3 101.3 2.05

[0070] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0071] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an electrochemical immunosensor, characterized in that: The steps include: S1. Carboxylation of NiCoP / g-C3N4 nanocomposite: dissolving NiCoP / g-C3N4 nanocomposite in nitric acid, reacting in an oil bath at 120-130°C, cooling to room temperature, washing, adjusting the pH to neutral, and drying to obtain COOH-NiCoP / g-C3N4 nanocomposite; S2. Preparation of electrochemical immunosensor: prepare the COOH-NiCoP / g-C3N4 obtained in S1 into a suspension and add it dropwise onto the treated electrode surface for the first incubation; after the incubation, wash the electrode surface, then form a mixed solution with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, then mix the mixed solution with a monoclonal antibody and add it dropwise onto the electrode surface for the second incubation; after the incubation, rinse, then cover the unspecified binding sites with bovine serum albumin, and rinse again to obtain the electrochemical immunosensor.

2. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that: In S1, the mass volume ratio of the NiCoP / g-C3N4 nanocomposite material to nitric acid is 1 g:100 mL, and the concentration of the nitric acid is greater than or equal to 12 mol / L.

3. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that: In S1, the oil bath reaction time is 24 hours; the drying is vacuum drying, the drying temperature is 30-40° C., and the drying time is 10-15 hours.

4. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that: In S2, the electrode is a glassy carbon electrode, and the treatment method is to polish the glassy carbon electrode with 0.3 mm alumina slurry, then wash it with water and ethanol for multiple times under ultrasonic treatment, and dry it with nitrogen.

5. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that: In S2, the COOH-NiCoP / g-C3N4 suspension is prepared by dispersing COOH-NiCoP / g-C3N4 in a solvent and subjecting it to ultrasonic treatment for 30-40 min; The volume mass ratio of the COOH-NiCoP / g-C3N4 to the solvent is 1 mg:1-5 mL; and the solvent is ethanol.

6. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that: In S2, the volume ratio of the mixed solution to the monoclonal antibody is 1:1; the concentration of the monoclonal antibody is 14 μg / mL, the concentration of the mixed solution is 2.0 mM, and the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 4:2.

3.

7. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that: In S2, the first incubation time is 3-5 hours, and the second incubation time is 2-3 hours; the mass concentration of the bovine serum albumin solution is 2%.

8. An electrochemical immunosensor prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the electrochemical immunosensor according to claim 8, characterized in that: Used for the detection of procalcitonin in serum, the detection method comprises the following steps: (1) A three-electrode system was used for detection, wherein an Ag / AgCl electrode was used as a reference electrode, a platinum plate electrode was used as a counter electrode, and an electrochemical immunosensor was used as a working electrode. The serum sample was centrifuged and filtered to obtain a supernatant, which was diluted with Tris-HCl buffer, and different standard concentrations of procalcitonin were added. In the presence of 5 mM K3Fe(CN)6 / K4Fe(CN)6, its response value was measured by differential pulse voltammetry, and a standard curve was established by comparing the response value with the procalcitonin concentration; (2) The serum sample to be tested is prepared into a test solution according to the method of step (1), and its response value is measured by differential pulse voltammetry. The response value is inserted into the standard curve to calculate the concentration of procalcitonin.

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