Preparation and application of electrochemical immune biosensor based on Prussian blue analogue

Abstract

The invention discloses preparation and application of an electrochemical immune biosensor based on a Prussian blue analogue, and belongs to the technical field of nano materials and electrochemical sensing. The electrochemical immune biosensor comprises an electrochemical sensing chip working electrode substrate, a PBAs modification layer and a Protein A modification layer which are arranged in sequence. According to the electrochemical immune biosensor, PBAs is ingeniously introduced into the electrochemical immune biosensor to serve as an electron transfer medium, the medium can specifically interact with a biological recognition element through Protein A, the electron transfer efficiency between biomolecules and an electrode interface is greatly improved, and rapid and efficient electronic communication between the biomolecules and the electrode interface is achieved. The electrochemical immune biosensor provided by the invention is a label-free electrochemical immune sensor combining biological recognition specificity and PBAs electrochemical activity, and shows huge application potential and wide market prospect in the field of biomedical sensing.

Description

Technical Field

[0001] The present invention relates to the technical fields of nanomaterials and electrochemical sensing technologies, and particularly to the preparation and application of an electrochemical immunosensor based on a Prussian blue analogue. Background Art

[0002] Electrochemical biosensing technology has the advantages of simple operation, fast response time, low cost, high detection sensitivity, etc., and is therefore widely used in the clinical diagnosis of various physiological indicators. In an electrochemical biosensor, a biomolecule acts as a receptor, and an electrode acts as a transducer to convert biological information into an electrical signal, thereby allowing the electrical signal to be correlated with the concentration of the analyte. And through electrochemical analysis technology, label-free immunoassay can also be achieved. Compared with common immuno-labeling methods, label-free immunosensors do not require the labeling process between signal molecules and biomolecules, have a shorter analysis time, and are simpler to operate. In order to improve the performance of electrochemical biosensors, optimizing the performance of the electrode and firmly immobilizing the biorecognition element on the electrode surface have become an important aspect indispensable for constructing an efficient biosensor. However, there is currently no generally applicable method that can ensure that biorecognition elements such as enzymes or antibodies can be effectively immobilized on the electrode surface while still maintaining their original catalytic and recognition characteristics unchanged. In some cases, the activity, stability, and selectivity of the biorecognition element even decrease after immobilization. How to solve this problem is the focus of current research by those skilled in the art. Summary of the Invention

[0003] The purpose of the present invention is to provide the preparation and application of an electrochemical immunosensor based on a Prussian blue analogue to solve the problems existing in the above-mentioned prior art.

[0004] To achieve the above purpose, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention: An application of a Prussian blue analogue in the preparation of an electrochemical immunosensor.

[0006] Using the Prussian blue analogue to prepare an electrochemical immunosensor can utilize the Prussian blue analogue to simultaneously achieve signal amplification in electrochemical immunosensing and the immobilization of biorecognition elements such as protein A + antibodies.

[0007] Another technical solution of the present invention: An electrochemical immunosensor based on Prussian blue analogues (PBAs), comprising an electrochemical sensing chip working electrode substrate, a Prussian blue analogue modification layer, and a Protein A modification layer arranged in sequence.

[0008] The electrochemical immunosensor based on Prussian blue analogs realizes the complexation of His-tag mediated Protein A and Prussian blue analogs by utilizing the unique affinity between the metal active sites on Prussian blue analogs and the histidine tag (His-tag) at the end of Protein A. Subsequently, by using the specific binding ability of Protein A to the Fc end of biorecognition elements such as antibodies, Protein A that has been immobilized on the sensing interface is used as a bridge to achieve the directional immobilization of biorecognition elements such as antibodies on the sensing interface. This process completes the construction of the immunosensing interface. This immobilization strategy can be used for the immobilization of various types of biorecognition elements and has the same or similar effects.

[0009] PBAs have excellent electrochemical activity. When introduced into an electrochemical immunosensor as an electron transfer medium, their open framework structure can not only provide rich ion transport channels but also promote the progress of electrochemical reactions. At the same time, PBAs have a high specific surface area, which enables more Protein A to be adsorbed on their surface and thus more biorecognition elements to be adsorbed, thereby enhancing the sensitivity and selectivity of the sensor. In addition, the metal active sites in PBAs can be optimized by adjusting their composition and structure to meet the requirements of different biomolecules.

[0010] Furthermore, the Prussian blue analog is a copper-iron-based Prussian blue analog.

[0011] The third technical solution of the present invention: The preparation method of the above-mentioned electrochemical immunosensor based on Prussian blue analogs includes the following steps:

[0012] Deposit Prussian blue analogs on the surface of the working electrode substrate of the electrochemical sensing chip to form a Prussian blue analog modified layer; incubate Protein A on the surface of the Prussian blue analog modified layer to obtain the electrochemical immunosensor based on Prussian blue analogs.

[0013] Furthermore, depositing Prussian blue analogs on the surface of the working electrode substrate of the electrochemical sensing chip to form a Prussian blue analog modified layer includes: dispersing Prussian blue analogs in a perfluorosulfonic acid polymer solution to obtain a Prussian blue analog slurry; depositing the Prussian blue analog slurry on the surface of the working electrode substrate of the electrochemical sensing chip, and drying to form a Prussian blue analog modified layer.

[0014] Furthermore, the concentration of the Prussian blue analog slurry is 1 - 50 mg / mL; the perfluorosulfonic acid polymer solution is formed by mixing absolute ethanol and Nafion solution in a volume ratio of 2 - 12:1.

[0015] Further, the concentration of the Nafion solution is 5 wt%.

[0016] Further, the specific operation of dispersing the Prussian blue analogue in the perfluorosulfonic acid polymer solution to obtain the Prussian blue analogue slurry is as follows: adding the Prussian blue analogue into the perfluorosulfonic acid polymer solution and performing ultrasonic treatment for 15 - 45 min to obtain the Prussian blue analogue slurry.

[0017] Further, the preparation steps of the Prussian blue analogue include: mixing a copper salt solution and a hexacyanoferrate solution and aging to obtain the Prussian blue analogue.

[0018] Further, the copper salt solution is an aqueous solution of a copper salt; the copper salt includes copper nitrate, copper acetate or copper chloride; the concentration of the copper salt solution is 0.01 - 0.06 mmol / mL.

[0019] Further, the preparation method of the hexacyanoferrate solution is: dispersing a hexacyanoferrate and sodium citrate in water to obtain the hexacyanoferrate solution; the dosage ratio of the hexacyanoferrate (K 3 [Fe(CN) 6 )), sodium citrate and water is 0.5 mmol: 0.05 - 1 mmol: 50 mL.

[0020] Further, the volume ratio of the copper salt solution to the hexacyanoferrate solution is 1: 0.2 - 1.

[0021] Further, the mixing of the copper salt solution and the hexacyanoferrate solution includes: slowly adding the hexacyanoferrate solution to the copper salt solution under stirring conditions, and continuing to stir for 5 - 60 min after the addition of the hexacyanoferrate solution is completed.

[0022] Further, the temperature of the aging is room temperature and the time is 6 - 72 h.

[0023] Further, after the aging, it also includes the steps of centrifugation, washing and drying.

[0024] Further, the incubation of Protein A on the surface of the Prussian blue analogue modified layer to obtain the Prussian blue analogue-based electrochemical immunosensor includes: soaking the working electrode substrate of the electrochemical sensing chip with the Prussian blue analogue modified layer deposited on its surface in the Protein A solution and incubating for 10 - 14 h, and then rinsing with a phosphate buffer solution to obtain the Prussian blue analogue-based electrochemical immunosensor.

[0025] Further, the concentration of the Protein A solution is 10 μg / mL.

[0026] Furthermore, the incubation temperature is 4°C.

[0027] Technical solution four of the present invention: Application of the above-mentioned electrochemical immunosensor based on Prussian blue analogues in biomolecule detection.

[0028] Furthermore, the steps of the application include: incubating a biorecognition element corresponding to the biomolecule to be detected on the surface of the electrochemical immunosensor based on Prussian blue analogues, then immersing the electrochemical immunosensor based on Prussian blue analogues with the immobilized biorecognition element in the solution of the biomolecule to be detected for incubation, and performing an electrochemical response performance test after incubation.

[0029] Furthermore, incubating the biorecognition element corresponding to the biomolecule to be detected on the surface of the electrochemical immunosensor based on Prussian blue analogues includes: immersing the electrochemical immunosensor based on Prussian blue analogues in the solution of the biorecognition element corresponding to the biomolecule to be detected, incubating at 0 - 8°C for 8 - 18 h, then rinsing with phosphate buffer solution, putting it into a blocking buffer solution after rinsing, incubating at 37°C for 40 - 80 min, and then rinsing with phosphate buffer solution again.

[0030] Furthermore, immersing the electrochemical immunosensor based on Prussian blue analogues with the immobilized biorecognition element in the solution of the biomolecule to be detected for incubation includes: the incubation temperature is 37°C and the time is 40 - 80 min.

[0031] Furthermore, the electrochemical response performance test is carried out by differential pulse voltammetry, and the test conditions are 0 - 0.6 V.

[0032] Furthermore, the pH value of the phosphate buffer solution is 6.8 - 7.4.

[0033] Furthermore, the volume concentration of the blocking buffer solution is 10%.

[0034] Furthermore, the electrochemical immunosensor based on Prussian blue analogues is a current-type label-free electrochemical immunosensor.

[0035] The present invention discloses the following technical effects:

[0036] (1) The present invention discloses an electrochemical immunosensor based on Prussian blue analogs, which includes an electrochemical sensing chip working electrode substrate, a Prussian blue analog modified layer, and a Protein A modified layer arranged in sequence. By utilizing the unique affinity between the metal active sites on the Prussian blue analogs and the histidine tag (His-tag) at the end of Protein A, the electrochemical immunosensor based on Prussian blue analogs first realizes the complexation of Protein A mediated by His-tag with Prussian blue analogs. Subsequently, by using the specific binding ability of Protein A to the Fc end of biorecognition elements such as antibodies, the Protein A already immobilized on the sensing interface is used as a bridge to achieve the directional immobilization of biorecognition elements such as antibodies on the sensing interface. This process completes the construction of the immunosensing interface. This immobilization strategy can be used for the immobilization of various types of biorecognition elements and has the same or similar effects.

[0037] (2) The present invention ingeniously introduces Prussian blue analogs (PBAs) as an electron transfer medium in the electrochemical immunosensor. This medium can not only specifically interact with biorecognition elements through Protein A, but also greatly promote the electron transfer efficiency between biomolecules and the electrode interface, realizing fast and efficient electronic communication between the two. In addition, the active sites of PBAs can achieve the directional immobilization of biorecognition elements through Protein A as a bridge. This selective binding can reduce non-specific adsorption and interference, improving the detection accuracy and reliability of the sensor.

[0038] (3) The electrochemical immunosensor constructed by the present invention using PBAs can immobilize biorecognition elements such as antibodies directionally and orderly on the surface of the electrochemical immunosensor, maintaining their original biological activity and specific recognition ability. This provides an efficient and stable platform for immunoassay. Due to the highly specific and stable interaction between biorecognition elements such as antibodies and PBAs under the bridging action of Protein A, the electrochemical immunosensor can be used as a general strategy and applied to various immunoassay systems. Whether it is for the rapid screening of specific pathogens or the detection of trace targets in complex biological samples, it can demonstrate its unique advantages and potential.

[0039] (4) By introducing PBAs as an electron transfer medium into the electrochemical immunosensor, the present invention can not only effectively improve the detection performance of the sensor for biomarkers, but also simplify the preparation process of the sensor and reduce the production cost. This label-free electrochemical immunosensor combining biorecognition specificity and the electrochemical activity of PBAs shows great application potential and broad market prospects in the field of biomedical sensing. Brief Description of the Drawings

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0041] Figure 1 XRD and infrared characterization diagrams of three PBAs prepared in Example 1 and Comparative Examples 1-2. Among them, a is the XRD diagram of CoFePBA, b is the XRD diagram of NiFe PBA, c is the XRD diagram of CuFe PBA, and d is the infrared characterization diagram of the three PBAs;

[0042] Figure 2 SEM characterization diagrams of three PBAs prepared in Example 1 and Comparative Examples 1-2. Among them, a is CoFe PBA, b is NiFe PBA, and c is CuFe PBA;

[0043] Figure 3 Physical diagram of a three-channel screen-printed electrode;

[0044] Figure 4 Electrochemical activity test results of three PBAs prepared in Example 1 and Comparative Examples 1-2. Among them, a is the cyclic voltammetry curve (CV) of the three PBAs, and b is the electrochemical impedance spectroscopy test curve (EIS) of the three PBAs;

[0045] Figure 5 Fluorescence detection results after fixing fluorescent goat anti-human IgG and fluorescent human IgG on the surface of the electrochemical immunosensor based on PBAs prepared in Application Example 1;

[0046] Figure 6 Nyquist detection results of the working electrode substrate of the electrochemical sensing chip, the electrochemical sensing chip modified with PBAs (CuFe PBA) prepared in Example 2 (denoted as CuFe), the electrochemical immunosensor based on PBAs prepared in Example 3 (denoted as CuFe / SPA), CuFe / SPA / Ab prepared in Test Example 2, and CuFe / SPA / Ab / Ag prepared in Test Example 2;

[0047] Figure 7Test results of the electrochemical response performance of the PBA-based electrochemical immunosensor prepared in Application Example 1 for human IgG. Among them, a shows the change in DPV signal after the electrochemical immunosensor surface was incubated with different concentrations of goat anti-human IgG antibody solution, b shows the change in DPV signal after the electrochemical immunosensor surface incubated with 10 μg / mL goat anti-human IgG antibody was further incubated with different concentrations of human IgG antigen solution, and c is the standard curve plotted based on b.

[0048] Figure 8 Test results of the electrochemical response performance of the PBA-based electrochemical immunosensor prepared in Application Example 1 for Tau protein. Among them, a shows the change in DPV signal after the electrochemical immunosensor surface incubated with Tau antibody was further incubated with different concentrations of human Tau protein solution, and b is the standard curve plotted based on a. Detailed implementation manners

[0049] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0050] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0051] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0052] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0053] As used herein, terms such as "comprising", "including", "having", "containing", etc. are all open-ended terms, meaning including but not limited to.

[0054] As a first aspect of the present invention, the present invention provides an application of a Prussian blue analogue in the preparation of an electrochemical immunosensor.

[0055] As a second aspect of the present invention, the present invention provides an electrochemical immunosensor based on a Prussian blue analogue, comprising an electrochemical sensing chip working electrode substrate, a Prussian blue analogue modification layer, and a Protein A (abbreviated as SPA) modification layer arranged in sequence.

[0056] As a preferred embodiment of the present invention, the Prussian blue analogue is a copper-iron-based Prussian blue analogue.

[0057] As a third aspect of the present invention, the present invention provides a preparation method of the above-mentioned electrochemical immunosensor based on a Prussian blue analogue, comprising the following steps:

[0058] Deposit a Prussian blue analogue on the surface of the electrochemical sensing chip working electrode substrate to form a Prussian blue analogue modification layer; incubate Protein A on the surface of the Prussian blue analogue modification layer to obtain the electrochemical immunosensor based on the Prussian blue analogue.

[0059] As a preferred embodiment of the present invention, depositing a Prussian blue analogue on the surface of the electrochemical sensing chip working electrode substrate to form a Prussian blue analogue modification layer includes: dispersing the Prussian blue analogue in a perfluorosulfonic acid polymer solution to obtain a Prussian blue analogue slurry; depositing the Prussian blue analogue slurry on the surface of the electrochemical sensing chip working electrode substrate, and drying to form a Prussian blue analogue modification layer.

[0060] As a preferred embodiment of the present invention, the preparation steps of the Prussian blue analogue include: mixing a copper salt solution and a hexacyanoferrate solution, and aging to obtain the Prussian blue analogue.

[0061] As a preferred embodiment of the present invention, incubating Protein A on the surface of the Prussian blue analogue modification layer to obtain the electrochemical immunosensor based on the Prussian blue analogue includes: incubating the electrochemical sensing chip working electrode substrate with a surface-deposited Prussian blue analogue modification layer and a Protein A solution for 10-14 h, and then rinsing with a phosphate buffer solution to obtain the electrochemical immunosensor based on the Prussian blue analogue.

[0062] As a preferred embodiment of the present invention, the preparation method of the electrochemical immunosensor based on the Prussian blue analogue is more specifically as follows:

[0063] (1) Preparation of Prussian blue analog: Disperse a copper salt (copper nitrate, copper acetate or copper chloride) in water to obtain a copper salt solution with a concentration of 0.01 - 0.06 mmol / mL; disperse hexacyanoferrate and sodium citrate in water (the dosage ratio of hexacyanoferrate, sodium citrate and water is 0.5 mmol: 0.05 - 1 mmol: 50 mL) to obtain a hexacyanoferrate solution; slowly add the hexacyanoferrate solution to the copper salt solution under stirring conditions (the volume ratio of the copper salt solution to the hexacyanoferrate solution is 1: 0.2 - 1). After the addition of the hexacyanoferrate solution is completed, continue stirring for 5 - 60 min, and then age at room temperature for 6 - 72 h (a precipitate is formed during the aging process). After the aging is completed, centrifuge, wash, and vacuum dry at 60 °C to obtain the Prussian blue analog;

[0064] (2) Preparation of an electrochemical immunosensor: Add the Prussian blue analog to a perfluorosulfonic acid polymer solution (a mixed solution of absolute ethanol and Nafion solution, the concentration of the Nafion solution is 5 wt%, by volume ratio, V 乙醇 : V Nafion is 2 - 12: 1), and perform ultrasonic treatment for 15 - 45 min to obtain a uniformly dispersed Prussian blue analog slurry (the concentration of the Prussian blue analog is 1 - 50 mg / mL). Deposit the Prussian blue analog slurry on the surface of the working electrode substrate of the electrochemical sensing chip and dry it at room temperature to form a Prussian blue analog modified layer; incubate the working electrode substrate of the electrochemical sensing chip with a surface-deposited Prussian blue analog modified layer in a Protein A solution at 4 °C for 10 - 14 h (to complex Protein A with metal ions in the Prussian blue analog). After the incubation is completed, rinse with a phosphate buffer solution to remove the residual Protein A to obtain the electrochemical immunosensor based on the Prussian blue analog.

[0065] As an embodiment of the present invention, the working electrode substrate of the electrochemical sensing chip can be a working electrode of an electrochemical sensing chip with any structure.

[0066] As a fourth aspect of the present invention, the present invention provides the application of the above-mentioned electrochemical immunosensor based on Prussian blue analog in biomolecule detection.

[0067] As a preferred embodiment of the present invention, the steps of the application include: incubating a biorecognition element corresponding to a biomolecule to be detected on the surface of the electrochemical immunosensor based on Prussian blue analog, and then immersing the electrochemical immunosensor based on Prussian blue analog with the immobilized biorecognition element in a solution of the biomolecule to be detected for incubation, and performing an electrochemical response performance test after the incubation.

[0068] As a preferred embodiment of the present invention, the incubation of the biorecognition element corresponding to the biomolecule to be detected on the surface of the electrochemical immunosensor based on Prussian blue analogs includes: soaking the electrochemical immunosensor based on Prussian blue analogs in the solution of the biorecognition element corresponding to the biomolecule to be detected, incubating at 0 - 8 °C for 8 - 18 h, then rinsing with phosphate buffer solution, and after rinsing, putting it into the blocking buffer solution and incubating at 37 °C for 40 - 80 min, and then rinsing with phosphate buffer solution again.

[0069] As a preferred embodiment of the present invention, the soaking of the electrochemical immunosensor based on Prussian blue analogs with the immobilized biorecognition element in the solution of the biomolecule to be detected for incubation includes: the incubation temperature is 37 °C and the time is 40 - 80 min.

[0070] As a preferred embodiment of the present invention, the electrochemical response performance test is carried out by differential pulse voltammetry, and the test conditions are 0 - 0.6 V.

[0071] The electrochemical immunosensor based on Prussian blue analogs of the present invention can be commonly used for the detection of various biomolecules such as human IgG antigen, Alzheimer's disease biomarker protein Tau, β-amyloid protein (Aβ), etc., and all have good detection effects. Among them, the detection effects for human IgG antigen and Alzheimer's disease biomarker protein Tau are better, and the detection sensitivity and accuracy for the two are high.

[0072] The preparation method and application of the electrochemical immunosensor based on Prussian blue analogs of the present invention will be further described below in conjunction with specific examples.

[0073] In the specific embodiments of the present invention, the room temperature specifically refers to 20 - 30 °C.

[0074] All raw materials used in the following examples and application examples are ordinary commercially available products.

[0075] The pH value of the phosphate buffer solution used in the following application examples is 7.4.

[0076] Example 1

[0077] A Prussian blue analogs (PBAs) electrode material is prepared according to the following steps:

[0078] (1) Prepare solution A and solution B at room temperature: Disperse 1 mmol of copper nitrate in 50 mL of deionized water and stir evenly to obtain a copper nitrate solution, denoted as solution A; Disperse 0.5 mmol of K 3 [Fe(CN) 6Disperse 1 mmol of [substance] and 1 mmol of sodium citrate in 50 mL of deionized water, stir evenly to obtain a hexacyanoferrate solution, denoted as Solution B.

[0079] (2) Slowly add Solution B to Solution A under stirring conditions. After adding Solution B completely, continue stirring for 10 min, then age the mixed solution at room temperature for 24 h. Centrifuge the aged solution at 8000 rpm at high speed, collect the precipitate, wash it alternately 3 times with absolute ethanol and deionized water. Finally, dry it in vacuum at 60 °C for 12 h to obtain a Prussian blue analogue, denoted as CuFe PBA.

[0080] Comparative Example 1

[0081] A Prussian blue analogue electrode material is prepared as follows:

[0082] (1) Prepare Solution A and Solution B at room temperature: Disperse 1 mmol of cobalt nitrate in 50 mL of deionized water, stir evenly to obtain a cobalt nitrate solution, denoted as Solution A; Disperse 0.5 mmol of K 3 [Fe(CN) 6 and 1 mmol of sodium citrate in 50 mL of deionized water, stir evenly to obtain a hexacyanoferrate solution, denoted as Solution B.

[0083] (2) Slowly add Solution B to Solution A under stirring conditions. After adding Solution B completely, continue stirring for 10 min, then age the mixed solution at room temperature for 24 h. Centrifuge the aged solution at 8000 rpm at high speed, collect the precipitate, wash it alternately 3 times with absolute ethanol and deionized water. Finally, dry it in vacuum at 60 °C for 12 h to obtain a Prussian blue analogue, denoted as CoFe PBA.

[0084] Comparative Example 2

[0085] A Prussian blue analogue electrode material is prepared as follows:

[0086] (1) Prepare Solution A and Solution B at room temperature: Disperse 1 mmol of nickel nitrate in 50 mL of deionized water, stir evenly to obtain a nickel nitrate solution, denoted as Solution A; Disperse 0.5 mmol of K 3 [Fe(CN) 6 and 1 mmol of sodium citrate in 50 mL of deionized water, stir evenly to obtain a hexacyanoferrate solution, denoted as Solution B.

[0087] (2) Slowly add Solution B to Solution A under stirring conditions. After the addition of Solution B is complete, continue stirring for 10 min, then age the mixed solution at room temperature for 24 h. Centrifuge the aged solution at 8000 rpm at high speed, collect the precipitate, wash it alternately 3 times with absolute ethanol and deionized water. Finally, dry it in vacuum at 60 °C for 12 h to obtain a Prussian blue analogue, denoted as NiFe PBA.

[0088] Example 2

[0089] Preparation of an electrochemical sensing chip modified with PBAs is as follows:

[0090] Add 4 mg of PBAs (CuFe PBA prepared in Example 1, or CoFe PBA prepared in Comparative Example 1, or NiFe PBA prepared in Comparative Example 2) to a mixed solution of 100 μL of absolute ethanol and 25 μL of 5 wt% Nafion solution, and ultrasonically treat it for 30 min to obtain a uniformly dispersed Prussian blue analogue slurry with a concentration of 32 mg / mL. Take 0.5 μL of the Prussian blue analogue slurry and drop it on the working electrode substrate of an electrochemical sensing chip with a diameter of 3.6 mm (the working electrode substrate of the electrochemical sensing chip is specifically a three-channel screen-printed carbon electrode, and its physical diagram is as Figure 3 shown) on the surface, and dry it at room temperature to obtain three different electrochemical sensing chips modified with PBAs.

[0091] Test Example 1

[0092] Performance characterization of Prussian blue analogue electrode materials

[0093] Figure 1 XRD and infrared spectra of the three PBAs prepared in Example 1 and Comparative Examples 1-2 are shown. Among them, a is the XRD pattern of CoFePBA, b is the XRD pattern of NiFe PBA, c is the XRD pattern of CuFe PBA, and d is the infrared spectrum of the three PBAs. It can be Figure 1 seen that the XRD diffraction patterns of the three PBAs are all in line with the standard XRD characteristic peaks, proving that the synthesized Prussian blue analogue conforms to the standard face-centered cubic structure and belongs to the Fm3m space group. The main diffraction peaks of the three PBAs materials are strong and sharp, which proves that the material purity is relatively high. The infrared spectrum shows that the three PBAs materials have similar structural compositions. Among them, the relatively strong absorption peak near 2100 cm -1 corresponds to the stretching vibration of the C≡N group; the absorption peak located near 1610 cm -1 proves the existence of the hydroxyl functional group (-OH), indicating that the PBAs materials contain a certain amount of coordinated water. Figure 2SEM characterization diagrams of three kinds of PBAs prepared in Example 1 and Comparative Examples 1-2. Among them, a is CoFe PBA (CoFe in the figure represents CoFe PBA), b is NiFe PBA (NiFe in the figure represents NiFe PBA), and c is CuFe PBA (CuFe in the figure represents CuFe PBA). Figure 2 The microstructure shown in Figure 2 is a PBA cube. It can be seen from

[0094] that the main particle size of PBA particles is distributed between 50-200 nm. Among them, CuFe PBA has a relatively clear edge and regular cubic structure, while the other two show obvious agglomeration phenomena. This may be because the reaction rate is too fast to form regular and uniform-sized particles, and the too-fast reaction rate leads to adhesion between particles. Such a morphology is not conducive to the performance of the material. Figure 4 As shown in Figure 4 , where a is the cyclic voltammogram (CV) of three kinds of PBAs, and b is the electrochemical impedance spectroscopy test curve (EIS) of three kinds of PBAs. It can be observed from

[0095] that CuFe PBA exhibits a higher oxidation-reduction peak current. Further, through impedance spectroscopy analysis, it is found that in the potassium ferricyanide solution, the impedance value of CuFe PBA is the lowest. Based on these results, CuFe PBA is selected as the electrode material for the subsequent preparation of an electrochemical immunosensor.

[0095] Example 3

[0096] Preparation of an electrochemical immunosensor based on PBAs, the steps are as follows:

[0097] Add 4 mg of CuFe PBA prepared in Example 1 to a mixed solution of 100 μL of absolute ethanol and 25 μL of 5 wt% Nafion solution, and ultrasonically treat for 30 min to obtain a uniformly dispersed Prussian blue analogue slurry with a concentration of 32 mg / mL. Deposit the Prussian blue analogue slurry on the surface of the working electrode substrate of the electrochemical sensing chip and dry it at room temperature. The working electrode substrate of the electrochemical sensing chip specifically uses a three-channel screen-printed carbon electrode, and its physical diagram is as Figure 3A Prussian blue analogue modification layer is formed on the surface as shown; the working electrode substrate of the electrochemical sensing chip with a Prussian blue analogue modification layer deposited on its surface (i.e., the PBAs-modified electrochemical sensing chip) is immersed in a Protein A solution (10 μg / mL) and incubated at 4 °C for 12 h to allow Protein A to complex with metal ions in PBAs. After incubation, it is rinsed with phosphate buffer solution to remove the residual Protein A, and an electrochemical immunosensor based on PBAs is obtained.

[0098] Test Example 2

[0099] Immunosensing Feasibility Test

[0100] (1) Fluorescence Detection

[0101] The electrochemical immunosensor based on PBAs prepared in Example 3 is immersed in a solution of fluorescent goat anti-human IgG antibody (goat anti-human IgG-RBITC) (concentration 10 μg / mL, solvent is phosphate buffer solution) and incubated at 4 °C for 12 h. The taken-out electrochemical immunosensor is rinsed with phosphate buffer solution and then put into a blocking buffer (Blocking Buffer, solvent is bovine serum albumin (BSA)) with a volume fraction of 10% and incubated at 37 °C for 1 h. After taking out the electrochemical immunosensor, it is rinsed with phosphate buffer solution to remove the residual blocking buffer.

[0102] Subsequently, the electrochemical immunosensor immobilized with goat anti-human IgG antibody is put into a solution of fluorescent human IgG antigen (human IgG-FITC) (concentration 5 μg / mL, solvent is phosphate buffer solution) and incubated at 37 °C for 1 h. Red fluorescence emitted by RBITC goat anti-human IgG is observed under a confocal microscope, proving the successful immobilization of the antibody. At the same time, green fluorescence emitted by FITC human IgG antigen is also observed, proving the successful recognition of the antigen, as Figure 5 shown.

[0103] From Figure 5 the fluorescence detection results, it can be seen that the electrochemical immunosensor based on PBAs can effectively achieve the immobilization of goat anti-human IgG and human IgG, laying a foundation for subsequent immune reactions. It ensures that the electrochemical immunosensor based on PBAs can be applied in specific biological detections or diagnoses, accurately capture and recognize target molecules, and thus achieve efficient and sensitive immune reactions.

[0104] (2) Nyquist Detection

[0105] The electrochemical immunosensor based on PBAs prepared in Example 3 (denoted as CuFe / SPA) was immersed in a Tau antibody (Ab) solution (concentration: 10 μg / mL, solvent: phosphate buffer solution) and incubated at 4 °C for 12 h. After the taken-out electrochemical immunosensor was rinsed with phosphate buffer solution, it was put into a blocking buffer solution with a volume fraction of 10% (Blocking Buffer, solvent: bovine serum albumin (BSA)) and incubated at a constant temperature of 37 °C for 1 h. After the electrochemical immunosensor was taken out, it was rinsed with phosphate buffer solution to remove the residual blocking buffer solution, and the electrochemical immunosensor immobilized with the antibody was obtained, denoted as CuFe / SPA / Ab.

[0106] Then CuFe / SPA / Ab was put into a solution of the biomarker protein tau of Alzheimer's disease (concentration: 5 μg / mL, solvent: phosphate buffer solution) and incubated at a constant temperature of 37 °C for 1 h, and the electrochemical immunosensor immobilized with protein tau was obtained, denoted as CuFe / SPA / Ab / Ag.

[0107] Nyquist tests were performed on the working electrode substrate of the electrochemical sensing chip (denoted as Blank), the electrochemical sensing chip modified with PBAs (CuFe PBA) prepared in Example 2 (denoted as CuFe), the electrochemical immunosensor based on PBAs prepared in Example 3 (denoted as CuFe / SPA), CuFe / SPA / Ab prepared in this test example, and CuFe / SPA / Ab / Ag prepared in this test example. The results are as Figure 6 shown. The semicircular part in the high-frequency region of the Nyquist plot corresponds to the electron transfer process. The diameter of the semicircle is related to the surface resistance of the electrode, and the low-frequency region corresponds to the diffusion-limited process. As can be seen from Figure 6 it, the blank electrode presented an obvious semicircular curve. After the PBA material was modified, the radius of the semicircular part increased, indicating an increase in the surface resistance. After Protein A (SPA) and the antibody were incubated, the increase in the resistance indicated the successful immobilization of the antibody. Finally, after the antigen was incubated, the specific binding between the antigen and the antibody led to a further increase in the interfacial resistance, indicating the successful immobilization of the biomolecules.

[0108] Application Example 1

[0109] Detection of IgG protein by the electrochemical immunosensor based on PBAs

[0110] The electrochemical immunosensors based on PBAs prepared in Example 3 were respectively immersed in solutions of goat anti-human IgG antibodies with different concentrations (0, 1, 10, 20, 50 μg / mL, and the solvent was phosphate buffer solution), and incubated at 4 °C for 12 h. After the taken-out electrochemical immunosensors were rinsed with phosphate buffer solution, they were put into a blocking buffer solution (Blocking Buffer, bovine serum albumin (BSA)) with a volume fraction of 10%, and incubated at a constant temperature of 37 °C for 1 h. After taking out the electrochemical immunosensors, they were rinsed with phosphate buffer solution to remove the residual blocking buffer solution, and electrochemical immunosensors immobilized with goat anti-human IgG antibodies at different concentrations were obtained. The differential pulse voltammetry was used to test the current response signals of the electrochemical immunosensors immobilized with goat anti-human IgG antibodies at different concentrations in the potential range of 0 - 0.6 V. The results are as Figure 7 shown in Figure 7 a (where background represents that the concentration of goat anti-human IgG antibody solution is 0). As can be seen from

[0111] a, after immobilizing 10 μg / mL of goat anti-human IgG, the differential pulse (DPV) signal decreased significantly, indicating that the number of specifically bound complexes on the electrode surface was the largest. Figure 7 Then, the electrochemical immunosensors immobilized with 10 μg / mL of goat anti-human IgG antibodies were respectively put into solutions of the human IgG antigen to be detected with different concentrations (1, 2, 5, 10 μg / mL, and the solvent was phosphate buffer solution) and incubated at a constant temperature of 37 °C for 1 h. The differential pulse voltammetry was used to test the electrochemical response performance of the electrochemical immunosensors to human IgG at different concentrations in the potential range of 0 - 0.6 V. The results are as shown in Figure 7 b. From Figure 7 b, as the concentration of human IgG increased, the peak current of DPV became lower and lower, indicating that this electrochemical immunosensor could effectively immobilize IgG-type antibodies and detect human IgG antigens at different concentrations. According to Figure 7 b, with the concentration of the human IgG antigen solution as the abscissa and the peak current of DPV as the ordinate, a standard curve was plotted, as shown in Figure 7 c. As can be seen from Figure 7 c, the standard curve equation was: y = a + b * x (a = 90.99668 ± 1.21253, b = -35.46577 ± 1.93098), and the detection limit of the electrochemical immunosensors based on PBAs prepared in Example 3 for the human IgG antigen was 1 μg / mL.

[0112] Application Example 2

[0113] Detection of the Alzheimer's disease biomarker protein Tau by the electrochemical immunosensor based on PBAs

[0114] The electrochemical immunosensor based on PBAs prepared in Example 3 was immersed in a Tau antibody solution (concentration: 10 μg / mL, solvent: phosphate buffer solution) and incubated at 4 °C for 12 h. The taken-out electrochemical immunosensor was rinsed with phosphate buffered saline and then placed in a blocking buffer with a volume fraction of 10% (Blocking Buffer, solvent: bovine serum albumin (BSA)) and incubated at a constant temperature of 37 °C for 1 h. After taking out the electrochemical immunosensor, it was rinsed with phosphate buffer solution to remove the residual blocking buffer, and an electrochemical immunosensor immobilized with Tau antibody was obtained. Then, the electrochemical immunosensor immobilized with Tau antibody was placed in tau protein solutions with different concentrations (0, 100, 200, 500 ng / mL, solvent: phosphate buffer solution) and incubated at a constant temperature of 37 °C for 1 h. Differential pulse voltammetry was used to test the electrochemical response performance of the electrochemical immunosensor to Tau with different concentrations in the potential range of 0 - 0.6 V. The results are as Figure 8 shown in Figure 8 a. According to Figure 8 a, a standard curve was plotted with the concentration of the Tau antigen solution as the abscissa and the DPV peak current as the ordinate. As shown in Figure 8 b, the standard curve equation is: y = a + b * x (a = 92.80974 ± 0.92872, b = -8.53277 ± 0.48904). It can be

[0115] seen that as the concentration of Tau protein increases, the DPV current signal decreases, and the detection sensitivity can reach 5 pg / mL, indicating that the electrochemical immunosensor prepared in Example 3 can be used for highly sensitive and specific detection of Tau protein.

[0115] The above-described embodiments are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a Prussian blue analogue in the preparation of an electrochemical immunoassay biosensor.

2. An electrochemical immunoassay biosensor based on a Prussian blue analogue, characterized in that: The invention comprises an electrochemical sensor chip working electrode substrate, a Prussian blue analogue modification layer and a Protein A modification layer which are arranged in sequence.

3. The electrochemical immunoassay biosensor based on Prussian blue analogues according to claim 2, characterized in that: The Prussian blue analog is a copper-iron-based Prussian blue analog.

4. The method for preparing an electrochemical immunoassay biosensor based on a Prussian blue analogue according to any one of claims 2 to 3, characterized in that: The following steps are involved: A Prussian blue analog is deposited on the surface of the working electrode substrate of the electrochemical sensor chip to form a Prussian blue analog modification layer; Protein A is incubated on the surface of the Prussian blue analog modification layer to obtain the electrochemical immunobiosensor based on the Prussian blue analog.

5. The preparation method according to claim 4, characterized in that: The method of depositing a Prussian blue analog on the surface of the working electrode substrate of the electrochemical sensor chip to form a Prussian blue analog modification layer comprises: dispersing the Prussian blue analog in a perfluorosulfonic acid polymer solution to obtain a Prussian blue analog slurry; depositing the Prussian blue analog slurry on the surface of the working electrode substrate of the electrochemical sensor chip, and drying to form a Prussian blue analog modification layer.

6. The preparation method according to claim 5, characterized in that: The concentration of the Prussian blue analog slurry is 1-50 mg / mL; the perfluorosulfonic acid polymer solution is prepared by mixing anhydrous ethanol and Nafion solution in a volume ratio of 2-12:

1.

7. The preparation method according to claim 4, characterized in that: The preparation steps of the Prussian blue analogue include: mixing a copper salt solution and a hexacyanoferrate solution, aging, and obtaining the Prussian blue analogue; the volume ratio of the copper salt solution to the hexacyanoferrate solution is 1:0.2-1.

8. The preparation method according to claim 4, characterized in that: The method of incubating Protein A on the surface of the Prussian blue analog modified layer to obtain the electrochemical immunoassay biosensor based on the Prussian blue analog comprises: immersing the working electrode substrate of the electrochemical sensor chip with the Prussian blue analog modified layer deposited on the surface in a Protein A solution for incubation for 10-14 hours, and then rinsing with a phosphate buffer solution to obtain the electrochemical immunoassay biosensor based on the Prussian blue analog.

9. Use of the electrochemical immunobiosensor based on Prussian blue analogues according to any one of claims 2 to 3 in biomolecule detection.

10. The use according to claim 9, characterized in that The application steps include: incubating a biorecognition element corresponding to the biomolecule to be detected on the surface of the electrochemical immunoassay biosensor based on Prussian blue analogue, then immersing the electrochemical immunoassay biosensor based on Prussian blue analogue with the biorecognition element fixed thereon in a solution of the biomolecule to be detected for incubation, and performing an electrochemical response performance test after incubation.

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