Electrochemical immunosensor and preparation method and application thereof

Abstract

This invention discloses an electrochemical immunosensor, its preparation method, and its application, belonging to the field of biosensing technology. The invention also proposes a method for preparing the electrochemical immunosensor, comprising: modifying the working region of an electrode with NiHCF nanoparticles; and sealing residual non-specific sites on the electrode surface with bovine serum albumin solution to obtain the electrochemical immunosensor. Furthermore, this invention proposes an application of the electrochemical immunosensor prepared by the above method or the above electrochemical immunosensor in the detection of blood tumor markers. This invention enables direct reagent-free detection of blood samples.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensing technology, specifically to an electrochemical immunosensor, its preparation method, and its application. Background Technology

[0002] Tumor markers are specific biomolecules in the blood that reflect the presence and progression of tumors. Changes in their concentrations are crucial for early screening, auxiliary diagnosis, efficacy evaluation, and prognostic monitoring of malignant tumors. In clinical practice, the specificity of a single tumor marker is limited. Combined detection of multiple blood tumor markers (such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), carbohydrate antigens CA125, CA15-3, CA19-9, CA72-4, cytokeratin 19 fragment CYFRA21-1, neuron-specific enolase (NSE), and human epididymal protein 4 (HE4), among other major markers, can significantly improve the accuracy and subtyping ability of cancer diagnosis. Therefore, developing an electrochemical immunosensing platform with a universal interface construction strategy, capable of highly sensitive, highly specific, and low-cost detection of multiple blood tumor markers, and suitable for primary healthcare and point-of-care testing (POCT) scenarios, has significant clinical value and social implications.

[0003] Currently, clinical detection methods for blood tumor markers mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and electrochemiluminescent immunoassay (ECLIA). ELISA is cumbersome, time-consuming, and has limited sensitivity; while CLIA and ECLIA, although highly sensitive and automated, rely on large automated analytical instruments, which are expensive, bulky, and not portable, severely limiting their widespread application in point-of-care testing (POCT) scenarios such as primary hospitals, community clinics, and home testing.

[0004] Electrochemical immunosensors, with their outstanding advantages such as high sensitivity, fast response speed, portable devices, low cost, and ease of integration and miniaturization, have become an important development direction for point-of-care testing (POCT) of blood tumor markers. However, existing electrochemical immunosensors still have the following key shortcomings in actual clinical blood sample testing: (1) Over-reliance on exogenous redox probes: Most existing electrochemical immunosensors rely on the addition of potassium ferrocyanide / potassium ferrocyanide ([Fe(CN)6]) to the electrolyte. 3- / 4-Using potassium ferricyanide as a redox probe to obtain electrochemical signals not only increases the number of operational steps and reagent costs, but also makes the exogenous probe susceptible to severe interference from reducing substances in blood samples (such as ascorbic acid, uric acid, glutathione, etc.), leading to signal drift and false positive results. In addition, potassium ferricyanide solution is prone to photolysis and auto-oxidation during long-term storage, which further affects the stability and reproducibility of the detection.

[0005] (2) Large steric hindrance at the antibody immobilization interface: Most existing immunosensors use a single thiol carboxylic acid self-assembled monolayer (SAM) to immobilize antibodies. The high carboxyl group density and disordered spatial orientation in the SAM layer make the antibody prone to spatial conflict and conformational distortion during the coupling process, thereby reducing the exposure rate of the antibody's active recognition site and affecting the sensitivity of subsequent antigen capture.

[0006] (3) Existing NiHCF preparation processes are difficult to meet the needs of mass production: Existing NiHCF is mostly prepared on a single electrode by electrochemical deposition, resulting in weak bonding between the deposited layer and the substrate, poor batch stability, and low efficiency; there is no mature solution-based mass production process or standardized ink formulation, and there are no reports of integrating NiHCF with gold nanoparticle immune interface systems for direct detection of blood tumor markers.

[0007] In summary, existing electrochemical immunosensors for hematological tumor markers suffer from limitations in simultaneously addressing issues related to signal probe introduction methods, antibody immobilization interface regulation, and the mass production of NiHCF materials. There is an urgent need to achieve reagent-free direct detection of hematological tumor markers. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the aforementioned technologies and develop a universal immunosensing platform that uses NiHCF nanoparticles prepared by solution method as built-in redox probes, combines MUA / MU mixed self-assembled monolayer to regulate carboxyl group density, and uses EDC / NHS for efficient covalent coupling and fixation of antibodies, thereby enabling reagent-free direct detection of hematological tumor markers. This invention provides an electrochemical immunosensor, its preparation method, and its applications, solving the technical problem of how to achieve reagent-free direct detection of hematological tumor markers in the prior art.

[0009] To achieve the above-mentioned technical objectives, the present invention provides a method for preparing NiHCF nanoparticles, comprising adding a solution A containing nickel acetate and sodium citrate dropwise to a solution B containing potassium ferricyanide under stirring, and then aging to obtain NiHCF nanoparticles.

[0010] In any embodiment, the concentration of nickel acetate in solution A is 1-10 mM, and the concentration of sodium citrate is 1-15 mM; or, the dropping rate is 10-30 mL / min; or, the concentration of potassium ferricyanide in solution B is 1-8 mM; or, the aging time is 1-36 hours.

[0011] In addition, the present invention also proposes a NiHCF nanoparticle prepared by the above preparation method.

[0012] Furthermore, this invention also proposes a method for preparing an electrochemical immunosensor, comprising the following steps: S1. Modify the above-mentioned NiHCF nanoparticles into the working area of ​​the electrode to construct an embedded redox probe layer; S2. The built-in redox probe layer is immersed in a mixed alcohol solution containing 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol to construct a mixed self-assembled monolayer and obtain a carboxylated electrode. S3. The carboxyl groups on the surface of the carboxylated electrode are activated using the EDC / Sulfo-NHS system; S4. Add the capture antibody solution corresponding to the tumor marker to be detected to the activated electrode working area to achieve covalent fixation of the antibody; S5. The residual non-specific sites on the electrode surface were sealed with bovine serum albumin solution to obtain an electrochemical immunosensor.

[0013] In any embodiment, in step S2, the molar ratio of 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol in the mixed alcohol solution is (1~2):(1~2), and the total concentration of 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol in the mixed solution is 1~10 mM.

[0014] In any embodiment, in step S3, the concentration of EDC in the EDC / Sulfo-NHS system is 2~10mM, and the concentration of Sulfo-NHS is 5~25mM; the activation solvent is 0.1M MES buffer with pH 5.5~6.5; and the activation time is 30 minutes to 2 hours.

[0015] In any embodiment, in step S4, the concentration of the antibody is 1~25 μg / mL; or, in step S1, the electrode is a gold nanoparticle-modified screen-printed carbon electrode.

[0016] In any embodiment, in step S4, the antibody is selected from at least one of anti-AFP, anti-CEA, anti-PSA, anti-CA125, anti-CA15-3, anti-CA19-9, anti-CA72-4, anti-CYFRA21-1, anti-NSE, and anti-HE4; or, in step S5, the concentration of the bovine serum albumin solution is 0.5%~3% (w / v) (i.e., 0.5~3 mg / mL); and the blocking time is 20~60 minutes.

[0017] Furthermore, this invention proposes an electrochemical immunosensor prepared by the above-described method.

[0018] Furthermore, this invention proposes an electrochemical immunosensor prepared by the above-described method or its application in the detection of blood tumor markers.

[0019] Compared with the prior art, the beneficial effects of the present invention include: the NiHCF nanoparticles proposed in the present invention can serve as a built-in redox probe system, completely eliminating dependence on exogenous probes and enabling direct detection of blood samples without reagents.

[0020] A controlled co-precipitation method was used to prepare NiHCF nanoparticle ink in batches. This invention employs sodium citrate as a stabilizer to regulate nucleation kinetics, achieving [Fe(CN)6] nucleation through constant-rate dropwise addition. 3- with Ni 2+ Controllable lattice growth was used to obtain NiHCF nanoparticles with uniform particle size (30~50nm), high crystallinity, and regular cubic phase structure. More than 10 g of NiHCF powder can be prepared in a single batch, which can meet the modification needs of more than 5,000 sensors, providing a material basis for large-scale production.

[0021] This invention employs a controlled co-precipitation method to prepare NiHCF nanoparticles, which are then stably solidified onto the electrode surface as a standardized "ink." The NiHCF lattice contains [Fe(CN)6] fixed within it. 3- / 4- redox couple and hydration K + The channel synergy provides highly reversible electron and ion transport capabilities, with its redox signal originating from the built-in solid-phase probe itself rather than from exogenous solution additives. This materials science design fundamentally eliminates dependence on exogenous potassium ferricyanide, avoiding photodegradation and self-oxidation of exogenous probes, as well as interference from reducing substances in the blood (ascorbic acid, uric acid, glutathione, etc.), thus enabling "on-demand, on-feed, on-test" POCT mode for blood samples.

[0022] The "AuNPs—NiHCF—MUA / MU—Ab—BSA" multilayer immune interface architecture exhibits excellent versatility. The immune interface architecture constructed in this invention, while maintaining structural integrity, allows for the construction of sensors targeting ten mainstream hematologic tumor markers—AFP, CEA, PSA, CA125, CA15-3, CA19-9, CA72-4, CYFRA21-1, NSE, and HE4—simply by changing the capture antibody. This achieves universal adaptability to multiple markers with a single platform design, significantly reducing development and production costs in multi-cancer screening scenarios.

[0023] Performance testing has verified that the sensor of this invention has detection limits as low as 0.02 ng / mL to 2 pM (depending on the biomarker) for ten mainstream blood tumor markers, a linear range covering 3 to 4 orders of magnitude, a specificity better than 90%, and a blood sample spike recovery rate of 95% to 105%, which fully demonstrates the reliability and practicality of the process of this invention in clinical POCT applications. Attached Figure Description

[0024] Figure 1 This diagram illustrates the overall fabrication and detection process of the reagent-free electrochemical immunosensor based on nickel hexacyanoferrate (NiHCF) and gold nanoparticle-modified screen-printed carbon electrodes (AuNPs / SPCE) of this invention. Figure a shows the layer-by-layer synthesis and modification process on the working electrode surface; figure b shows the detection principle based on the immobilization of antibodies (Abs) using 11-mercaptoundecanoic acid (MUA) and an EDC / NHS covalent coupling strategy; figure c shows the specific binding of the target analyte (AFP) and the electrochemical signal detection mechanism; and figure d shows the extraction and immediate detection process of actual clinical blood samples.

[0025] Figure 2 The images show the surface morphology and elemental characterization of the various modified nanomaterials prepared in Example 1 of this invention. Image a is a SEM image of the bare screen-printed carbon electrode (SPCE); image b is a TEM image of the nickel hexacyanoferrate (NiHCF) cubic nanoparticles; image c is a SEM image of the surface of the NiHCF / AuNPs composite modified electrode; image d is a HAADF-STEM image of the NiHCF nanoparticles; and images e and f are EDS elemental mappings of nickel (Ni) and iron (Fe) elements in the corresponding NiHCF nanoparticles, respectively.

[0026] Figure 3The graph shows the electrochemical stability test results of the NiHCF / AuNPs / SPCE modified electrode prepared in Example 1 of this invention. Figure a shows a comparison of the curves of the composite modified electrode in the 1st, 25th, and 50th cyclic voltammetry (CV) tests in 0.1 M KCl supporting electrolyte; Figure b shows a comparison of the peak current decay rate of the three modified electrodes made of different materials—Prussian blue, pure NiHCF, and the NiHCF / AuNPs / SPCE of this invention—during 50 consecutive cyclic scans.

[0027] Figure 4 Electrochemical characterization diagrams of the layer-by-layer modified interfaces of the electrochemical immunosensor constructed in Example 2 of this invention are shown. In diagram a, cyclic voltammetry (CV) response curves of the electrode at each modification stage (NiHCF modification, AuNPs / SPCE modification, MUA / MU carboxylation, antibody immobilization, and BSA blocking) are shown; in diagram b, differential pulse voltammetry (DPV) response curves are shown for each layer-by-layer modification stage.

[0028] Figure 5 This is a graph showing the clinical application evaluation and specificity verification of the electrochemical immunosensor prepared in Example 3 of the present invention. Figure a is a box plot comparing the electrochemical signal (current) of the sensor in detecting AFP in real blood samples from healthy and diseased individuals; figure b is a bar chart comparing the current signal changes of the sensor for the target biomarker (AFP) and various interfering tumor biomarkers (CA19-9, CEA, PSA, CA125). Detailed Implementation

[0029] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0030] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0031] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0032] Combination Figure 1 This specific embodiment provides a method for preparing NiHCF nanoparticles, comprising adding a solution A containing nickel acetate and sodium citrate dropwise to a solution B containing potassium ferricyanide under stirring, and then aging for 1 to 36 hours to obtain NiHCF nanoparticles; wherein the concentration of nickel acetate in solution A is 1 to 10 mM, and the concentration of sodium citrate is 1 to 15 mM; the dropping rate is 10 to 30 mL / min; and the concentration of potassium ferricyanide in solution B is 1 to 8 mM.

[0033] This specific embodiment also proposes a NiHCF nanoparticle, which is prepared by the above preparation method.

[0034] Combination Figure 1 Furthermore, this specific embodiment also proposes a method for preparing an electrochemical immunosensor, including the following steps: S1. Modify the working area of ​​the electrode with NiHCF nanoparticles to construct an embedded redox probe layer; the electrode is a screen-printed carbon electrode modified with gold nanoparticles; disperse the NiHCF nanoparticles in water to prepare NiHCF nanoparticle ink, and drop the ink onto the electrode surface using an automated non-contact spotting system, with a spotting volume of 1-5 μL / electrode, and allow it to dry naturally at room temperature for 15-60 minutes. S2. The built-in redox probe layer is immersed in a mixed alcohol solution containing 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol to construct a mixed self-assembled monolayer, thereby obtaining a carboxylated electrode; in the mixed alcohol solution, the molar ratio of 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol is (1~2):(1~2), and the total concentration of 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol in the mixed solution is 1~10 mM; S3. Activate the carboxyl groups on the surface of the carboxylated electrode using an EDC / Sulfo-NHS system; in the EDC / Sulfo-NHS system, the concentration of EDC is 2~10 mM, and the concentration of Sulfo-NHS is 5~25 mM; the activation solvent is 0.1 M MES buffer solution with pH 5.5~6.5; the activation time is 30 minutes to 2 hours. S4. The capture antibody solution corresponding to the tumor marker to be detected is added dropwise to the activated electrode working area to achieve covalent fixation of the antibody; the concentration of the antibody is 1~25 μg / mL; the antibody is selected from at least one of anti-AFP, anti-CEA, anti-PSA, anti-CA125, anti-CA15-3, anti-CA19-9, anti-CA72-4, anti-CYFRA21-1, anti-NSE, and anti-HE4. S5. The residual non-specific sites on the electrode surface are blocked with bovine serum albumin solution (i.e., BSA) to obtain an electrochemical immunosensor; the concentration of the bovine serum albumin solution is 0.5%~3% (w / v); the blocking time is 20~60 minutes.

[0035] This specific embodiment also proposes an electrochemical immunosensor, which is prepared by the above-described preparation method.

[0036] Furthermore, this specific embodiment also proposes an electrochemical immunosensor prepared by the above preparation method or the application of the above electrochemical immunosensor in the detection of blood tumor markers.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0039] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0040] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0041] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0042] In the following embodiments, the AuNPs / SPCE electrode substrate is prepared by the following steps: (1) Chemical activation pretreatment of electrode surface Commercial screen-printed carbon electrodes (model C301) were chemically activated by immersing them in a 0.1 mol / L sulfuric acid solution for 30 minutes, rinsed with ultrapure water, and dried for later use.

[0043] (2) Construction of molecular bridging layer The electrode was immersed in a 10 mM cysteine ​​hydrochloride aqueous solution and allowed to stand at room temperature for 30 minutes. The amino terminus of the cysteine ​​binds to the functional groups on the carbon surface, while the thiol terminus is exposed outwards, forming a molecular bridging layer, such as... Figure 1 As shown in a. Remove the electrode, rinse it, and allow it to air dry at room temperature for later use.

[0044] (3) Formulation of weak ligand-type gold nanoparticle ink Gold nanoparticles were synthesized using the sodium borohydride-chloroauric acid reduction method, a technique already known in the art. The synthesized gold nanoparticles had a particle size of 5-15 nm. They were then prepared into an aqueous dispersion with a concentration of 1 mg / mL, and 0.5 wt% sodium citrate was added as a weak ligand dispersant.

[0045] (4) Automated precise spotting and in-situ chemical bonding Automated spotting equipment was used to precisely spot ink onto the electrode working area, with a single spot volume of 0.5 μL. After deposition, the thiol groups in the bridging layer replaced the sodium citrate on the surface of the gold particles, forming Au-S bonds through ligand exchange.

[0046] (5) Heat treatment curing and aging stabilization The spotted electrode was placed in an oven and heat-treated at 60°C for 60 minutes to obtain the metal nanoparticle-modified electrode. This process achieves solvent evaporation, stress relief, and deep stabilization of chemical bonding, forming a dense modified layer.

[0047] Example 1: Solution-based batch preparation of NiHCF nanoparticle ink and construction of an embedded redox probe layer (1) Step 1: Preparation of AuNPs / SPCE electrode substrate. The obtained gold nanoparticle modified screen-printed carbon electrode (AuNPs / SPCE) is used as the sensor substrate.

[0048] (2) Step 2: Solution-based batch preparation of NiHCF nanoparticle ink ① Preparation of Solution A: Weigh 29.9 mg of nickel acetate tetrahydrate (Ni(CH3COO)2·4H2O) and 52.9 mg of sodium citrate dihydrate (Na3C6H5O7·2H2O), dissolve them in 20 mL of deionized water, and stir magnetically until completely dissolved to obtain a mixed aqueous solution containing 6 mM nickel acetate and 9 mM sodium citrate (sodium citrate / nickel acetate molar ratio of 1.5:1). Sodium citrate acts as a key stabilizer, reacting with Ni through its carboxyl group. 2+ The weak coordination effect regulates the nucleation rate and avoids the aggregation of NiHCF particles.

[0049] ② Preparation of solution B: Weigh 26.3 mg of potassium ferricyanide (K3[Fe(CN)6]) and dissolve it in 20 mL of deionized water to obtain an iron source aqueous solution with a concentration of 4 mM.

[0050] ③ Controlled coprecipitation reaction: Place solution B in a 100 mL beaker and stir magnetically at 500 rpm; use a syringe pump to slowly add solution A dropwise to solution B at a constant rate of 20 mL / min. The controlled dropping rate is crucial to ensuring the regular growth of the NiHCF crystal lattice and obtaining a uniform cubic phase structure. After the addition is complete, the solution is a clear, light yellow-green color.

[0051] ④ Reaction aging: The reaction was carried out with continuous stirring at room temperature (approximately 22°C) for 24 hours. After the reaction was completed, the solution gradually turned into a cloudy olive green.

[0052] ⑤ Separation and washing: Centrifuge the reaction solution at 10,000 rpm for 10 minutes and discard the supernatant; resuspend in deionized water and centrifuge again, repeat washing twice to remove unreacted precursors and free sodium citrate.

[0053] ⑥ Preparation of NiHCF ink: Disperse the washed NiHCF nanoparticles in 5 mL of deionized water, control the concentration at 5 mg / mL, and ultrasonically disperse for 10 minutes to obtain uniform and stable NiHCF nanoparticle ink. Store at 4℃ in the dark for later use.

[0054] (3) Step 3: Modification of the NiHCF built-in redox probe layer An automated non-contact spotting system was used to precisely drop the NiHCF ink at a volume of 2 μL / electrode onto the working area (approximately 3 mm in diameter) of AuNPs / SPCE. The electrode was then allowed to dry naturally at room temperature for 30 minutes to obtain the NiHCF / AuNPs / SPCE composite electrode.

[0055] (4) Performance evaluation ① Microscopic morphology and elemental characterization: such as Figure 2 As shown, the SEM images reveal that the bare SPCE surface exhibits a typical porous carbon sheet structure. Figure 2 a); TEM images show that the prepared NiHCF nanoparticles exhibit a uniform cubic phase morphology, with a particle size distribution concentrated in the range of 30–50 nm, and good dispersibility. Figure 2 b); The surface of the NiHCF / AuNPs composite modified electrode shows that cubic NiHCF particles are uniformly distributed on the AuNPs modification layer. Figure 2 c); HAADF-STEM images further reveal that the cubic lattice of the NiHCF nanoparticles is clearly discernible. Figure 2 d); EDS elemental mapping confirms the presence of Ni element ( Figure 2 e) and Fe element ( Figure 2 f) Spatial overlap was achieved within the nanoparticle range, with a molar ratio close to 1:1, confirming the successful construction of the NiHCF lattice.

[0056] ② Electrochemical stability characterization: To verify the stability advantages of the NiHCF built-in probe system of this invention, the following two sets of comparative electrodes were set up. Comparative group 1 (traditional commercial Prussian blue modified electrode): Prepared using the classic electrodeposition method widely reported in the literature. A mixed electroplating solution containing 2 mM FeCl3, 2 mM K3[Fe(CN)6], 0.1 M KCl and 0.01 M HCl was prepared. Using bare SPCE as the working electrode, electrodeposition was performed in a potential range of -0.2 to +0.8 V (vs. Ag / AgCl) at a scan rate of 50 mV / s for 15 cycles. Subsequently, it was heat-treated in a 50℃ oven for 1 hour to promote the lattice stabilization of Prussian blue, thus obtaining the Prussian blue / SPCE comparative electrode.

[0057] Comparative Group 2 (Pure NiHCF Comparative Electrode): The AuNPs underlayer modification step was omitted. The NiHCF ink prepared in Example 1 was directly dropped onto the bare SPCE working area with the same spotting volume (2 μL) and dried at room temperature for 30 minutes to obtain the NiHCF / SPCE comparative electrode.

[0058] like Figure 3As shown, NiHCF / AuNPs / SPCE exhibits a reversible redox peak at approximately +0.15 V (vs. Ag / AgCl) in a 0.1 M KCl supporting electrolyte. The 1st, 25th, and 50th CV curves highly overlap. Figure 3 a) Peak current retention rate >96%; comparative experiments show that ( Figure 3 (b) The conventional commercial Prussian blue modified electrode (comparative group 1) showed that the peak current decayed to less than 25% of the initial value after 50 cycles (attenuation rate of about 75%), which is consistent with the phenomenon reported in the literature that the PB system is prone to structural dissolution and poor stability in neutral / weakly alkaline electrolytes; the pure NiHCF system (comparative group 2) had a peak current retention rate of about 88% after 50 cycles; while the NiHCF / AuNPs / SPCE system of this invention had a peak current retention rate of >94% after 50 cycles, which fully confirms the stabilizing synergistic effect of the AuNPs bottom layer on the NiHCF built-in probe.

[0059] ③ Optimization of NiHCF ink concentration and spotting volume: Based on the above scheme in this embodiment, the effects of different NiHCF ink concentrations (3, 5, 7, 10 mg / mL) and spotting volumes (1, 2, 3, 5 μL) on the electrochemical response of the composite electrode were further investigated. The results showed that when the ink concentration was 5 mg / mL and the spotting volume was 2 μL, the NiHCF built-in probe layer exhibited the largest and most stable DPV peak current response. Too low an ink concentration (3 mg / mL) would lead to insufficient NiHCF loading and a weak signal, while too high a concentration (≥7 mg / mL) or too large a volume (≥3 μL) would lead to an excessively thick modified layer, increased interfacial resistance, and decreased subsequent antibody immobilization efficiency.

[0060] (5) Integrated detection based on portable detectors and smartphone terminals To evaluate the feasibility of this sensor chip in point-of-care testing (POCT), this embodiment constructs an integrated mobile detection system consisting of an electrochemical sensor chip, a portable wireless card reader, and a smartphone app. The specific detection and result determination process is as follows (e.g., ...). Figure 1 (as shown in d): ① Sample pretreatment and sample addition reaction: Collect clinical whole blood samples and rapidly separate them using a microcentrifuge to obtain supernatant serum samples. Use a pipette to aspirate 10 μL of serum sample and add it to the working electrode area of ​​the sensor chip. Incubate at room temperature for 15 minutes to allow the target AFP molecules in the serum to specifically bind to the capture antibody on the electrode surface, forming a steric hindrance.

[0061] ② Signal Acquisition and Wireless Transmission: After incubation, the electrode surface is rinsed with deionized water to remove unbound impurities. The sensor chip is then inserted into a customized portable handheld electrochemical detector. Upon starting the detector, the system automatically performs differential pulse voltammetry (DPV) scanning to acquire the characteristic peak current of the NiHCF built-in probe after signal attenuation due to antigen-antibody binding.

[0062] ③ Algorithm Interpretation and Mobile Result Display: The electrochemical signal data collected by the detector is wirelessly transmitted to a smartphone terminal in real time via Bluetooth Low Energy (BLE). The dedicated mobile app has a built-in preset AFP standard working curve algorithm. The app automatically calculates the current change value (ΔI) and intelligently classifies the data according to the preset clinical diagnostic positive threshold (Cut-off value): if the detected concentration is higher than the threshold, the mobile phone screen immediately and intuitively displays the positive judgment result ("AFP level: +"), and if it is lower than the threshold, it displays the negative result ("AFP level: -"). This realizes a closed-loop intelligent mobile health detection of "sample input - result output (Sample-in, Answer-out)".

[0063] Example 2: Construction of an AFP immunosensor based on a MUA / MU hybrid SAM and evaluation of its AFP detection performance. This embodiment aims to verify the feasibility of the strategy of regulating carboxyl group density in a MUA / MU hybrid self-assembled monolayer and covalently coupling it with EDC / Sulfo-NHS, and to systematically evaluate the detection performance of the constructed AFP immunosensor. The implementation steps are as follows: (1) Steps one to three: Same as in Example 1, to obtain NiHCF / AuNPs / SPCE composite electrode.

[0064] (2) Step 4: Construction of MUA / MU hybrid self-assembled monolayer The NiHCF / AuNPs / SPCE electrode was immersed in a mixed solution of anhydrous ethanol containing 1 mM MUA and 1 mM MU (molar ratio 1:1, total concentration 2 mM) and reacted at room temperature in the dark for 12 hours. MU, acting as a diluent molecule, effectively reduced the lateral packing density between MUA molecules, forming a carboxylated interface with moderate density and good spatial orientation. After the reaction, the electrode was rinsed three times each with anhydrous ethanol and deionized water, and then dried under nitrogen.

[0065] (3) Step 5: EDC / Sulfo-NHS activation of carboxyl groups Prepare a 0.1 M MES buffer (containing 0.5 M NaCl, pH 6.0) containing 2 mM EDC and 5 mM Sulfo-NHS. Immerse the carboxylated electrode in the activation solution and react at room temperature in the dark for 1 hour. During the reaction, the -COOH group at the MUA terminal is activated into a highly reactive Sulfo-NHS ester. After activation, gently wash three times with 0.01 M PBS (pH 7.4) and proceed immediately to the next step.

[0066] (4) Step Six: Covalent fixation of anti-AFP antibody The anti-AFP monoclonal antibody was diluted to 10 μg / mL with 0.01 M PBS, and 5 μL was precisely added to the activated electrode working area using an automated spotting system. The reaction was carried out at room temperature for 1 hour. The free amino group of the antibody underwent a nucleophilic substitution reaction with the active ester of Sulfo-NHS to form a stable amide bond. After the reaction, the sample was washed three times with PBS to remove any uncoupled free antibody.

[0067] (5) Step 7: BSA blocks nonspecific sites The electrode was immersed in a PBS solution containing 1% (w / v) BSA and sealed at room temperature for 30 minutes. The electrode was then rinsed three times with PBS to obtain the AFP immunosensor, which was then stored at 4°C for later use.

[0068] (6) Step 8: "Reagent-free" electrochemical detection of AFP antigen Different concentrations of AFP antigen standard solutions (0.1, 1, 10, 50, 100, 200, 500, 1000, 2000, and 5000 ng / mL) were added 50 μL to the sensor's working area and incubated at room temperature for 1 hour. After rinsing with PBS, the solution was placed in 10 mL of 0.1 M KCl supporting electrolyte (containing no exogenous redox probes). The NiHCF built-in probe [Fe(CN)6] was recorded using the DPV method. 3- / 4- Redox peak current of the redox couple. DPV test parameters: potential range -0.2~+0.6 V (vs. Ag / AgCl), pulse amplitude 50mV, pulse width 50 ms, step potential 4 mV.

[0069] (7) Performance evaluation ① Electrochemical characterization of layer-by-layer modification: such as Figure 4 As shown, the CV and DPV methods were used to characterize each stage of the immune sensor construction. Figure 4 The results show that as NiHCF modification, AuNPs involvement, MUA / MU carboxylation, antibody immobilization, and BSA blocking progress, the CV redox peak current decreases sequentially; the corresponding DPV curves ( Figure 4b) also exhibited the same signal change trend, verifying the successful layer-by-layer construction of the multilayer immune recognition interface. Specific data are as follows: the DPV peak current of naked NiHCF / SPCE was approximately 35 μA; the DPV peak current of NiHCF / AuNPs / SPCE increased to approximately 62 μA, indicating that the AuNPs bottom layer significantly enhanced interfacial electron transport; after MUA / MU carboxylation, the DPV peak current decreased to approximately 44 μA, with the SAM layer moderately hindering electron transport; after anti-AFP antibody immobilization, the DPV peak current further decreased to approximately 38 μA; and after BSA blocking, the DPV peak current was approximately 33 μA.

[0070] ②AFP concentration-response curve: The results showed that the change in DPV peak current (ΔI = I0 - I) and the logarithm of AFP concentration (lg C) exhibited a good linear relationship in the range of 0.1 ng / mL to 5000 ng / mL. The linear regression equation was: ΔI (μA) = 18.5 lg C (ng / mL) + 12.3, R 2 = 0.996. Based on a signal-to-noise ratio of 3 (S / N = 3), the limit of detection (LOD) is as low as 0.05 ng / mL.

[0071] ③ Optimization of MUA / MU molar ratio: The effect of different MUA / MU molar ratios (2:1, 1:1, 1:2) on the response performance of the AFP sensor was further investigated. The results showed that the sensor had the largest ΔI (approximately 32.5 μA) for 100 ng / mL AFP when MUA:MU = 1:1; the ΔI decreased to approximately 28.5 μA when MUA:MU = 2:1 (due to excessive carboxyl density leading to steric hindrance); and the ΔI decreased to approximately 24.8 μA when MUA:MU = 1:2 (due to insufficient carboxyl density). This result confirms the precise regulatory effect of MU dilution molecules on the lateral packing density of MUA.

[0072] ④ Ascorbic acid interference resistance comparison: Under the same conditions, a control AFP sensor was constructed using a traditional exogenous potassium ferricyanide probe system for detection. The results showed that after adding 5 mM ascorbic acid interference, the peak current of the exogenous probe system was severely interfered with (signal change >80%), while the signal of the NiHCF built-in probe system of this invention remained stable (signal change <3%), fully verifying the immunity of the built-in probe system to interference from reducing substances in blood samples.

[0073] Example 3: Validation of the universality of multiple biomarkers and evaluation of clinical blood samples This embodiment aims to verify the universality, specificity, and direct detection capability of the interface architecture of the present invention for ten mainstream hematologic tumor markers, as well as its ability to detect clinical blood samples directly.

[0074] (1) Construction of a multi-marker universal sensor Following the interface construction process described in Example 2, only the antibodies in step six were replaced with anti-CEA, anti-PSA, anti-CA125, anti-CA15-3, anti-CA19-9, anti-CA72-4, anti-CYFRA21-1, anti-NSE, and anti-HE4 monoclonal antibodies (all at a concentration of 10 μg / mL). The remaining steps and process parameters were completely consistent, and the corresponding immunosensors were constructed accordingly. This modular design strategy of "changing only the antibody, not the interface" fully demonstrates the universality advantage of the interface architecture of this invention.

[0075] (2) Detection performance of ten mainstream tumor markers The present invention conducted concentration gradient tests and electrochemical response evaluations on the above-constructed channel sensing electrodes.

[0076] For the representative biomarker alpha-fetoprotein (AFP): AFP standards with concentration gradients ranging from 0.1 to 5000 ng / mL were used in the experiment. The results showed that the DPV peak current signal of the sensor exhibited a regular decrease with increasing AFP concentration. The change in peak current (ΔIΔ) was used as the statistical expression. I The logarithm of AFP concentration ( The sample was fitted and showed excellent linear response within the above concentration range. The calculated limit of detection (LOD) was as low as 0.05 ng / mL (signal-to-noise ratio S / N=3).

[0077] For other mainstream tumor markers (CEA, PSA, CA125, CA15-3, CA19-9, CA72-4, CYFRA21-1, NSE, HE4): the corresponding target antigens were tested within their commonly used clinical concentration ranges. Experimental results showed that after specific immune binding to the corresponding antigens, the steric hindrance formed on the electrode surface hindered interfacial electron transport, thus causing a regular attenuation of the characteristic DPV peak current of the built-in redox probe. Within the characteristic detection range corresponding to each marker (covering a range of 3-4 orders of magnitude), the signal attenuation showed a good linear response trend to the logarithm of the antigen concentration, and the limit of detection for each channel was below its respective clinical diagnostic threshold (cut-off value), meeting the requirements for clinical screening and actual quantitative detection.

[0078] The above results fully demonstrate that the NiHCF / AuNPs universal sensing interface constructed in this invention does not depend on specific antibodies, and it has excellent compatibility and universal adaptability to a variety of tumor markers with different molecular weights and different isoelectric points, and can be used as a universal technology platform for parallel detection of multiple indicators. (3) Specificity verification: such as Figure 5As shown in b, taking the AFP immunosensor as an example, the current signal changes of 100 ng / mL AFP (the target analyte) and common interfering substances (including CA19-9, CEA, PSA, and CA125) at equal concentrations were tested. The results showed that the DPV peak current change caused by the target analyte AFP was 21±2 μA, while the signal changes caused by CA19-9, CEA, PSA, and CA125 were only 11±3, 12±3, 5±2, and 13±5 μA, respectively, all significantly lower than the AFP response (p<0.01), confirming that the sensor has excellent specific recognition ability for the target biomarker.

[0079] (4) Evaluation of clinical blood sample testing: such as Figure 5 As shown in Figure a, serum samples were collected from 15 healthy volunteers and 15 patients clinically diagnosed with liver cancer. 50 μL of each sample was directly dropped onto the surface of the AFP sensor, incubated at room temperature for 1 hour, rinsed with PBS, and then placed in 0.1 M KCl for DPV detection. The results showed that the median current signal in the healthy group was approximately 30 μA (range approximately 20–42 μA), while the median current signal in the diseased group was approximately 11 μA (range approximately 6–18 μA). The difference between the two groups was highly significant (p < 0.0001). At a threshold current of approximately 20 μA, the diagnostic sensitivity was approximately 93%, and the specificity was approximately 90%. The correlation coefficient (r) with the results obtained by the commercially available ECLIA method was > 0.95.

[0080] (5) Blood sample spike recovery rate: Artificial serum samples containing known concentrations of AFP (spiking concentrations of 50, 200, and 500 ng / mL) were prepared and directly detected without reagents using the sensor of this invention. The spike recovery rates were 98.2%, 103.5%, and 95.8%, respectively, and the relative standard deviations (RSDs) were 4.2%, 3.8%, and 5.1% (n=3), respectively. This further verifies the accurate quantitative detection capability of the sensor of this invention for tumor markers in complex blood matrix.

[0081] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor, characterized in that, Includes the following steps: S1. Modify the working area of ​​the electrode with NiHCF nanoparticles to construct an embedded redox probe layer; S2. The built-in redox probe layer is immersed in a mixed alcohol solution containing 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol to construct a mixed self-assembled monolayer and obtain a carboxylated electrode. S3. The carboxyl groups on the surface of the carboxylated electrode are activated using the EDC / Sulfo-NHS system; S4. Add the capture antibody solution corresponding to the tumor marker to be detected to the activated electrode working area to achieve covalent fixation of the antibody; S5. The residual non-specific sites on the electrode surface were blocked using bovine serum albumin solution to obtain an electrochemical immunosensor; A solution A containing nickel acetate and sodium citrate is added dropwise to a solution B containing potassium ferricyanide under stirring, followed by aging to obtain NiHCF nanoparticles; the concentration of nickel acetate in solution A is 1~10 mM, and the concentration of sodium citrate is 1~15 mM; the dropwise addition rate is 10~30 mL / min; the concentration of potassium ferricyanide in solution B is 1~8 mM; or, the aging time is 1~36 hours.

2. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that, In step S2, the molar ratio of 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol in the mixed alcohol solution is (1~2):(1~2), and the total concentration of 11-mercaptoundecanoic acid and 11-mercaptoundecyl alcohol in the mixed solution is 1~10 mM.

3. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that, In step S3, the concentration of EDC in the EDC / Sulfo-NHS system is 2~10 mM, and the concentration of Sulfo-NHS is 5~25 mM; the activation solvent is MES buffer with pH 5.5~6.5; and the activation time is 30 minutes to 2 hours.

4. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that, In step S4, the concentration of the antibody is 1~25 μg / mL; or, in step S1, the electrode is a gold nanoparticle-modified screen-printed carbon electrode.

5. The method for preparing the electrochemical immunosensor according to claim 1, characterized in that, In step S4, the antibody is selected from at least one of anti-AFP, anti-CEA, anti-PSA, anti-CA125, anti-CA15-3, anti-CA19-9, anti-CA72-4, anti-CYFRA21-1, anti-NSE, and anti-HE4; or, in step S5, the concentration of the bovine serum albumin solution is 0.5~3 mg / mL; and the blocking time is 20~60 minutes.

6. An electrochemical immunosensor, characterized in that, It is prepared by the preparation method according to any one of claims 1-5.

7. The application of an electrochemical immunosensor prepared by the preparation method according to any one of claims 1-5 or the electrochemical immunosensor according to claim 6 in the detection of blood tumor markers.

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