An electrochemical biosensor for the detection of C-reactive protein in plasma and whole blood

Abstract

The present invention discloses an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood. The electrode interface of the electrochemical biosensor is constructed based on electrochemical polymerization and electrochemical deposition. It is simple and rapid to prepare, requires minimal reagents, has low human toxicity, and is environmentally friendly and pollution-free. The sensor exhibits a good linear relationship with the electrical signal detected by the sensor within a C-reactive protein concentration range of 0.1 to 25 μg / mL, with a detection limit of 0.096 μg / mL. The sensor exhibits high sensitivity, simple operation, high selectivity, environmental friendliness, and high biosafety. It facilitates the rapid diagnosis of sepsis and the immediate monitoring of disease progression. It also provides technical support for the development of biosensors for biomarker detection in complex media.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology and relates to an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood. The electrochemical biosensor can be used in the fields of rapid diagnosis of sepsis and guidance of individualized medication. Background Art

[0002] Sepsis is a clinical syndrome characterized by life-threatening organ dysfunction caused by a dysregulated systemic response to infection. It is characterized by rapid progression, high mortality, and numerous sequelae. Currently, the diagnosis of sepsis relies primarily on the Sequential Organ Failure Assessment (SOFA) and rapid Sequential Organ Failure Score (SOS) 3.0 diagnostic criteria, as well as bacterial cell culture. However, bacterial cells are present in very low abundance in the blood of septic patients, requiring at least 12 hours for diagnosis and a high incidence of false positives. These diagnostic criteria are derived from complex retrospective studies, potentially leading to delays in diagnosis and treatment. Furthermore, early diagnosis of sepsis is challenging due to its overlap with other medical conditions and the lack of reliable adjunctive sepsis testing tools. Furthermore, antimicrobial therapy for sepsis lacks robust diagnostic protocols and supporting evidence, leading to prolonged antibiotic therapy, multidrug resistance, and adverse events such as renal impairment. Therefore, there is an urgent need for clinical indicators and methods that can rapidly and immediately diagnose sepsis to guide antibiotic use and prognosis. Biomarkers are naturally occurring molecules, genes, or proteins that can be used to diagnose diseases and determine the physiological or pathological processes of diseases. Sepsis biomarkers refer to detectable indicators expressed in various aspects such as physiology, immunity, and biochemistry after pathogens invade the human body, which can quantitatively evaluate the patient's pathophysiological process. Monitoring the levels of sepsis-related biomarkers is of great significance in guiding personalized drug treatment for sepsis. By measuring the changes in the content of sepsis-related markers, it is helpful to quickly and immediately diagnose sepsis, monitor the course of the disease in real time, and provide patients with tailored treatment plans based on their characteristics. Therefore, it is necessary to establish a simple, economical, and immediate biomarker detection method for the rapid diagnosis of sepsis, and to evaluate the course of the disease in real time based on the changes in the content of sepsis markers, so as to provide patients with more immediate and effective treatment plans.

[0003] C-reactive protein (CRP), synthesized by the liver, is an acute-phase protein that activates complement, enhances phagocytosis by phagocytes, and clears invading pathogens and damaged, necrotic, and apoptotic tissue cells. It is not only an important protective factor in the immune system but also a sensitive marker of inflammation. Plasma CRP levels are low in healthy adults (CRP <3 mg / L). They begin to rise 6 to 8 hours after the onset of an inflammatory response and remain elevated for 36 to 50 hours. The magnitude of the increase is positively correlated with the severity of the infection or inflammation. A CRP concentration of 50 mg / L is the optimal threshold for diagnosing sepsis. An 80% decrease in CRP levels in patients is an indicator of antibiotic discontinuation. Changes in plasma CRP levels are instructive for the diagnosis and prognosis of infection. Because of its short half-life, dynamic monitoring of CRP levels in patients can aid clinical diagnosis and treatment, providing immediate monitoring of infection, inflammatory responses, and the effectiveness of antibiotic therapy.

[0004] Electrochemical biosensors are a new type of analytical device that can convert chemical changes in target analytes into detectable electrical signals through specific bioaffinity or biocatalytic behavior. Electrodes are used as signal converters to output and amplify current, potential or impedance signals for highly sensitive and selective analysis of target objects. Currently, electrochemical biosensors can be divided into label-free electrochemical sensors and enzyme-labeled electrochemical sensors. Traditional enzyme-labeled electrochemical sensors require the coupling of redox-active enzymes to target proteins on the electrode surface, which usually requires multi-layer electrode assembly and the participation of substances that promote electrode transfer. Label-free electrochemical sensors have the advantages of low cost, rapidity, and simplicity in terms of electrode assembly and enzyme-labeled substances. The use of label-free electrochemical biosensors as detection markers can reduce operational difficulty and save costs. Conductive polymer composites are widely used in the field of electrochemical biosensors due to their advantages such as high conductivity and good electrochemical activity. Polyaniline (PANI) is a classic conductive material with redox activity. When detecting current signals, it does not require additional solutions to provide electron transfer and can also improve the current signal. It can be used as an excellent conductive material in the construction of label-free electrochemical sensors.

[0005] Although electrochemical biosensors have high sensitivity, they are susceptible to various contaminants (such as proteins, cells, and polysaccharides) adhering to the sensing interface in complex biological fluids, resulting in decreased sensitivity and specificity. To alleviate the problem of biofouling at the sensor interface, antifouling materials are selected and integrated into the sensing interface of the biosensor to effectively reduce nonspecific adsorption. Amyloid bovine serum albumin (AL-BSA) is based on the phase transition of bovine serum albumin (BSA). The natural α-helical structure of BSA is rapidly converted to β-sheet stacking after tris(2-carboxyethyl)phosphine (TCEP) reduces the disulfide bonds in the BSA molecule, resulting in the amyloid aggregation of unfolded BSA chains, forming AL-BSA. AL-BSA oligomers aggregate at solid / liquid and gas / liquid interfaces to form macroscopic two-dimensional films. Due to their uniform distribution of positive and negative charges, they have excellent antifouling properties against proteins, carbohydrates, lipids, and microorganisms, effectively preventing the formation of biofilms. Choosing AL-BSA as the antifouling material for constructing antifouling biosensors has the advantages of stability, economy and environmental protection. It not only resists nonspecific adsorption, but also avoids the occurrence of false positives, which helps to improve the accuracy of diagnosis.

[0006] Therefore, the present invention proposes an electrochemical biosensor for the detection of C-reactive protein in plasma and whole blood. The conductive polymer PANI is used as the electrode interface conductive material of the label-free sensor, and is bonded with an AL-BSA anti-fouling coating to construct a label-free and anti-fouling electrochemical biosensor for the detection of C-reactive protein in plasma or blood. It has potential application value in the rapid diagnosis of sepsis and real-time monitoring of the disease. Summary of the Invention

[0007] 1. The purpose of the present invention is to provide an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood.

[0008] 2. The electrochemical biosensor for detecting C-reactive protein in plasma and whole blood described in the present invention uses the conductive polymer polyaniline as a signal conversion element, amyloid bovine serum albumin (AL-BSA) as an anti-fouling coating, and a specific C-reactive protein antibody as a target recognition component, and can achieve direct detection of C-reactive protein in human plasma or blood samples.

[0009] 3. The electrochemical biosensor for detecting C-reactive protein in plasma and whole blood described in the present invention utilizes the changes in electrical signals generated by the blockade of polyaniline electron transport before and after target recognition to establish a direct quantitative relationship between C-reactive protein (CRP) concentration and the current signal, thereby achieving label-free electrochemical quantitative detection of CRP.

[0010] 4. The electrochemical biosensor for detecting C-reactive protein in plasma and whole blood described in the present invention uses tris(2-carboxyethyl)phosphine (TCEP) to disrupt the disulfide bonds in bovine serum albumin (BSA), forming a denser anti-fouling structure, which can detect CRP concentrations in complex samples.

[0011] 5. A method for detecting C-reactive protein concentration in plasma and whole blood using an electrochemical biosensor, characterized in that it comprises the following steps: (1) generating an endogenous electrical signal through the intrinsic conductive state of polyaniline, and using the antibody probe on the electrode surface to bind to the target protein to be detected to affect the polyaniline electron transfer efficiency on the electrode surface, thereby correlating it with the target content to be detected; (2) preparing gold nanoparticles on the electrode surface by electrochemical deposition, and modifying the electrode surface with amyloid bovine serum albumin through gold-sulfur bonds to form an anti-fouling coating with good biocompatibility to resist non-specific adsorption caused by serum proteins, cells, plasma, etc.; (3) assembling specific C-reactive protein antibodies onto the amyloid bovine serum albumin-modified electrode surface by chemical cross-linking using a carbodiimide condensation method; and (4) detecting the current change before and after incubation with the plasma or blood sample by differential pulse voltammetry to calculate the C-reactive protein (CRP) concentration in the plasma or whole blood sample.

[0012] The method for preparing an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood according to the present invention comprises the following steps in sequence:

[0013] (1) pre-treating the screen-printed electrode, including washing with dilute sulfuric acid, washing with water, and drying with nitrogen, to obtain a pre-treated screen-printed electrode SPCE;

[0014] (2) constructing a polyaniline (PANI) coating on the surface of the pretreated screen-printed electrode (SPCE) obtained in step (1) by an electropolymerization method to prepare a polyaniline coating / screen-printed electrode (PANI) / SPCE;

[0015] (3) depositing gold nanoparticles on the surface of the polyaniline coating / screen-printed electrode PANI / SPCE prepared in step (2) by an electrodeposition method to prepare gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE;

[0016] (4) first reducing bovine serum albumin with tri(2-carboxyethyl)phosphine to prepare amyloid bovine serum albumin AL-BSA, and then assembling AL-BSA onto the surface of the gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE prepared in step (3) through gold-sulfur bonds to obtain amyloid bovine serum albumin / nanogold / polyaniline coating / screen-printed electrode AL-BSA / AuNPs / PANI / SPCE;

[0017] (5) using the carbodiimide condensation method, C-reactive protein capture monoclonal antibody (CRP Ab) was assembled onto the surface of the amyloid bovine serum albumin / nano-gold / polyaniline coating / screen-printed electrode AL-BSA / AuNPs / PANI / SPCE prepared in step (4) by chemical cross-linking to obtain CRP Ab modified amyloid bovine serum albumin / nano-gold / polyaniline coating / screen-printed electrode CRP Ab / AL-BSA / AuNPs / PANI / SPCE;

[0018] (6) adding a sample containing CRP to the surface of the CRP Ab modified amyloid bovine serum albumin / nano-gold / polyaniline coating / screen-printed electrode CRP Ab / AL-BSA / AuNPs / PANI / SPCE prepared in step (5) and incubating the surface to obtain an incubated electrode;

[0019] (7) After incubation, the electrode surface is cleaned and then placed in a phosphate buffer solution. Differential pulse voltammetry is used for electrochemical detection. The CRP concentration in the sample is calculated based on the change in the electrical signal.

[0020] Furthermore, the method for preparing an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood is characterized by comprising the following specific steps:

[0021] (1) Pretreatment of screen-printed electrode (SPCE): 100 μL of 0.5 mol / L H2SO4 was added to the surface of the SPCE and scanned under cyclic voltammetry with a scanning range of 0 to 1.5 V, a scanning speed of 1 V / s, and 30 scanning cycles. The electrode was then rinsed with ultrapure water and dried with N2 to obtain the pretreated screen-printed electrode SPCE for use.

[0022] (2) Polyaniline (PANI) deposition: Prepare a mixture containing 0.5 mol / L A mixed solution of H2SO4 and 0.02mol / L aniline was prepared, and the screen-printed electrode SPCE pretreated in step (1) was subjected to cyclic voltammetry scanning for 20 cycles at a scanning rate of 0.05V / s in the potential range of -0.2 to 0.9V to obtain a polyaniline PANI film modified electrode; the polyaniline PANI film modified electrode was taken out, washed with deionized water, and then placed in a 0.5mol / LH2SO4 solution, electrolyzed at +0.90V as the electrolysis potential, and electrolyzed at a constant potential for 60s, and then electrolyzed at -0.2V as the electrolysis potential, and electrolyzed at a constant potential for 60s, and then scanned by cyclic voltammetry at a scanning rate of 0.1V / s in the potential range of -0.2V to 0.9V to obtain a modified electrode, and the aniline that was not firmly adsorbed or polymerized on the surface of the modified electrode was cleaned until the cyclic voltammetry curve was stable, and then washed with ultrapure water and dried with N2 to prepare a polyaniline coating / screen-printed electrode PANI / SPCE for standby use;

[0023] (3) Deposition of gold nanoparticles (AuNPs): 2.8 mmol / L chloroauric acid solution was added dropwise onto the surface of the polyaniline coating / screen-printed electrode PANI / SPCE, and AuNPs were electrodeposited using the time-current method. The electrodeposition potential was -0.2 V, and the deposition time was 100 s. The AuNPs were then washed with ultrapure water and dried with nitrogen to obtain the gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE, which was then used for later use.

[0024] (4) Assembly of AL-BSA: 7 μL of AL-BSA solution pre-reacted for 2 h was added dropwise to the surface of the gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE. After incubation for 1 h, the solution was washed with 10 mmol / L pH 7.4 phosphate buffer (PBS) and dried with N2 to obtain AL-BSA / AuNPs / PANI / SPCE for later use. The AL-BSA solution was prepared by 2 mg / mL bovine serum albumin and 50 mmol / L tris(2-carboxyethyl)phosphine in a volume ratio of 1:1.

[0025] (5) Activation by carbodiimide condensation (EDC / NHS) method: 7 μL of EDC / NHS solution prepared with 0.4 mol / L EDC and 0.1 mol / L NHS was added dropwise to the surface of the AL-BSA / AuNPs / PANI / SPCE electrode prepared in step (4). After incubation at room temperature for 1 h, the electrode was washed with 10 mmol / L pH 7.4 PBS and dried with N2 to obtain EDC-NHS / AL-BSA / AuNPs / PANI / SPCE for later use.

[0026] (6) Assembly of capture antibody probe: 7 μL of 100 μg / mL CRP Ab was incubated on the surface of the EDC-NHS / AL-BSA / AuNPs / PANI / SPCE electrode prepared in step (5) at room temperature for 3 h, washed with 10 mmol / L pH 7.4 PBS, and dried with N2 to obtain CRP Ab / AL-BSA / AuNPs / PANI / SPCE for later use;

[0027] (7) CRP detection: In the reagent analysis mode that requires a buffer system, 7 μL of a test solution containing CRP at a concentration of 10 μg / mL was added to the surface of the CRP Ab / AL-BSA / AuNPs / PANI / SPCE electrode, incubated at room temperature for 1 h, washed with 10 mmol / L pH 7.4 PBS, blown dry with N2, and electrochemical detection was performed using differential pulse voltammetry in a 0.2 mol / L pH 5.7 phosphate buffer solution system. In the reagent-free analysis mode that does not require detection in a buffer system, 1.68 μL of a 5 mol / L sodium dihydrogen phosphate solution was added to the surface of the CRP Ab / AL-BSA / AuNPs / PANI / SPCE electrode and dried at 37°C. Then, 100 μL of a test solution containing CRP at a concentration of 10 μg / mL was added to the surface of the electrode and incubated at room temperature for 1 h. No electrode cleaning was required, and differential pulse voltammetry electrochemical detection was performed directly. Record the changes in electrical signals and calculate the CRP concentration in the sample. The scanning voltage is -0.6V to 0.6V.

[0028] Furthermore, the above-mentioned preparation method of the present invention produces an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood.

[0029] Advantages of the present invention: The electrochemical biosensor for detecting C-reactive protein in plasma and whole blood employs green, simple, and economical electrochemical synthesis and detection methods, significantly reducing testing costs. The assembly design of a signal transduction element and an antifouling coating enables reagent-free detection of C-reactive protein in plasma or blood samples. This sensor exhibits strong specificity, high anti-interference capabilities, and simple operation. Compared to classic beacon detection methods or enzyme-linked immunosorbent assays, it significantly reduces detection time and reagent costs. It is expected to be used for accurate, efficient, and rapid bedside CRP testing, aiding in the immediate diagnosis of sepsis and ongoing treatment assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the electrochemical biosensor for C-reactive protein of the present invention.

[0031] Figure 2 This is the infrared characterization diagram of AL-BSA of the present invention.

[0032] Figure 3 This is a scanning electron microscope (SEM) characterization image of the AL-BSA / Au / PANI / SPCE prepared in the present invention. Figure 3 A, B, C to D are SEM images of SPCE, PANI / SPCE, AuNPs / PANI / SPCE, and AL-BSA / AuNPs / PANI / SPCE, respectively.

[0033] Figure 4This is an electrochemical impedance spectroscopy (EIS) characterization diagram of the electrode assembly of the present invention.

[0034] Figure 5 This is a diagram showing the feasibility of CRP detection results of the present invention.

[0035] Figure 6 This is the fluorescence staining diagram of the sensor against cell adsorption of the present invention. Figure 6 Figures A, B, and C are the fluorescence staining results of PANI / SPCE, AL-BSA / Au / PANI / SPCE, and CRP Ab / AL-BSA / Au / PANI / SPCE after cell incubation for 48 hours.

[0036] Figure 7 This is a diagram showing the results of the sensor's anti-BSA interference test.

[0037] Figure 8 This is a graph showing the results of the anti-plasma interference test of the sensor of the present invention.

[0038] Figure 9 This is a diagram showing the results of specific analysis of the sensor detection of the present invention.

[0039] Figure 10 The figure shows the results of quantitative analysis of CRP in the present invention. The concentrations of CRP standards are 0.1, 0.5, 1, 5, 10, and 25 μg / mL.

[0040] Figure 11 The results of the quantitative detection and analysis of plasma CRP in the present invention are shown in FIG. The concentrations of the CRP standards are 0.1, 1, 5, 10, and 25 μg / mL, respectively.

[0041] Figure 12 This is a graph showing the results of the quantitative analysis of blood CRP according to the present invention. The concentrations of the CRP standards are 0.1, 1, 5, 10, and 25 μg / mL, respectively.

[0042] Figure 13 This is a graph showing the results of the intra-group reproducibility analysis of the CRP detection of the present invention.

[0043] Figure 14 This is a graph showing the reproducibility analysis results of the CRP detection groups of the present invention. DETAILED DESCRIPTION

[0044] In order to make the technical problems, technical solutions and effects to be solved by the present invention clearer, the present invention is further described in detail below with reference to the embodiments and drawings.

[0045] The electrochemical biosensor for detecting C-reactive protein in plasma and whole blood described in the present invention is used for detecting plasma CRP. The specific operating steps are as follows:

[0046] (1) Preparation of testing instruments, reagents and solutions

[0047] The instruments and equipment used in this invention were purchased from the following companies:

[0048]

[0049] The reagents used in this invention are from the following companies:

[0050]

[0051] (2) Pretreatment of screen-printed electrodes (SPCE)

[0052] 100 μL of 0.5 mol / L H2SO4 was added to the SPCE surface and scanned under cyclic voltammetry (CV) with a scanning range of 0 to 1.5 V, a scanning speed of 1 V / s, and 30 scanning cycles. After that, the electrode was washed with ultrapure water and dried with N2 to obtain the pretreated screen-printed electrode SPCE for use.

[0053] (3) Polyaniline (PANI) deposition

[0054] A mixed solution containing 0.5 mol / L H2SO4 and 0.02 mol / L aniline was prepared, and the pretreated screen-printed electrode SPCE was cyclically scanned for 20 cycles in the potential range of -0.2 to 0.9 V at a scanning rate of 0.05 V / s to obtain a polyaniline (PANI) thin film modified electrode; the modified electrode was taken out, washed with deionized water, and then placed in a 0.5 mol / L H2SO4 solution, electrolyzed at +0.90 V for 60 s, then electrolyzed at -0.2 V for 60 s, and then cyclic voltammetry was scanned in the potential range of -0.2 V to 0.9 V at a scanning rate of 0.1 V / s to clean the aniline that was not firmly adsorbed or polymerized on the surface of the modified electrode until the cyclic voltammetry curve was stable, and then washed with ultrapure water and blown dry with N2 to prepare a polyaniline coating / screen-printed electrode PANI / SPCE electrode for use.

[0055] (4) Deposition of gold nanoparticles (AuNPs)

[0056] 2.8 mmol / L chloroauric acid solution was added to the surface of the polyaniline coating / screen-printed electrode PANI / SPCE, and AuNPs were electrodeposited using the time-current method. The electrodeposition potential was -0.2 V, the time was 100 s, and the electrode was washed with ultrapure water and dried with N2 to obtain a nanogold / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE electrode for later use.

[0057] (5) Assembly of AL-BSA

[0058] 7 μL of AL-BSA solution pre-reacted for 2 h was added dropwise to the surface of the electrode modified with AuNPs / PANI / SPCE. After incubation at room temperature for 1 h, the electrode was washed with 10 mmol / L pH 7.4 phosphate buffer (PBS) and dried with N2 to obtain an AL-BSA / AuNPs / PANI / SPCE electrode for later use. The AL-BSA solution was prepared from 2 mg / mL BSA and 50 mmol / L TCEP in a volume ratio of 1:1.

[0059] (6) Activation by carbodiimide condensation (EDC / NHS)

[0060] 7 μL of EDC / NHS solution prepared with 0.4 mol / L EDC and 0.1 mol / L NHS was dropped onto the surface of the prepared AL-BSA / AuNPs / PANI / SPCE electrode. After incubation at room temperature for 1 h, the electrode was washed with 10 mmol / L pH 7.4 PBS and dried with N2 to obtain the EDC-NHS / AL-BSA / AuNPs / PANI / SPCE electrode for later use.

[0061] (7) Assembly of capture antibody probe

[0062] 7 μL of 100 μg / mL CRP Ab was incubated on the prepared EDC-NHS / AL-BSA / AuNPs / PANI / SPCE electrode surface at room temperature for 3 h, then washed with 10 mmol / L pH 7.4 PBS and dried with N2 to obtain a CRP Ab / AL-BSA / AuNPs / PANI / SPCE electrode for later use.

[0063] (8) CRP assembly (i.e., sample CRP detection process)

[0064] In the reagent analysis mode requiring a buffer system (i.e., analysis requires a buffer system, referred to as the reagent analysis mode), 7 μL of a test solution containing CRP at a concentration of 10 μg / mL was dropped onto the surface of the CRP Ab / AL-BSA / AuNPs / PANI / SPCE electrode, incubated at room temperature for 1 h, washed with 10 mmol / L pH 7.4 PBS, and blown dry with N2. Electrochemical detection was performed using differential pulse voltammetry (DPV) in a 0.2 mol / L pH 5.7 phosphate buffer solution (PBS) system, with a scanning voltage of -0.6 V to 0.6 V. In the reagent-free analysis mode that does not require a buffer system (i.e., analysis does not require a buffer system, referred to as the reagent-free analysis mode), 1.68 μL of 5 mol / L sodium dihydrogen phosphate solution was dripped onto the electrode surface and dried at 37°C. Then, 100 μL of a test solution containing CRP at a concentration of 10 μg / mL was dripped onto the electrode surface and incubated at room temperature for 1 hour. DPV detection can be performed directly without washing, and the scanning voltage is -0.6V to 0.6V.

[0065] Figure 1 The figure is a schematic diagram of an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood according to the present invention. The electrochemical biosensor prepared through the above steps was used for relevant experiments.

[0066] Figure 2 The dark curve in the middle is the Fourier transform infrared spectrum result of AL-BSA reduced by TCEP, and the light curve is the Fourier transform infrared spectrum result of BSA. Due to the reduction of disulfide bonds in BSA by TCEP, BSA undergoes phase transition, and the secondary structure changes from α-helix to β-fold, forming an amyloid-like structure. Compared with BSA, AL-BSA has a larger peak at the amide I band (1625cm -1 ) is enhanced, and due to the exposure of a large number of hydroxyl functional groups, the β-folding peak at 3500 cm -1 The (hydroxyl peak) was also enhanced, which showed that the preparation of AL-BSA was successful.

[0067] Figure 3 This is the SEM result of the electrode. Figure 3 A, B, C to D are SEM images of SPCE, PANI / SPCE, AuNPs / PANI / SPCE, and AL-BSA / AuNPs / PANI / SPCE, respectively. Figure 3The SEM characterization results show that the SPCE surface presents a granular rough interface, the PANI / SPCE surface presents a bifurcated three-dimensional porous interface, and the AuNPs / PANI / SPCE surface grows flower-like gold nanoparticles on the basis of the PANI / SPCE surface. Due to the modification of AL-BSA on the surface of the flower-like gold nanoparticles, the fine structure of the nanoparticle surface is passivated, and a membrane structure is formed on the surface of AL-BSA / AuNPs / PANI / SPCE. The results show that the assembly of AL-BSA / AuNPs / PANI / SPCE is successful.

[0068] Figure 4 The EIS characterization diagram of the electrochemical sensor assembly process. The larger the arc in the EIS characterization diagram, the greater the resistance. When PANI is electro-deposited on the bare electrode, PANI has good conductivity and a small resistance. Subsequently, gold nanoparticles (AuNPs) are deposited. Due to the good conductivity of AuNPs, the resistance is further reduced. After AL-BSA is modified on the surface of the gold nanoparticles, the resistance of the AL-BSA / PANI modified electrode increases due to the blocking effect of AL-BSA and poor conductivity. When CRP Ab is immobilized, the charge transfer is further blocked, causing the resistance to continue to increase. Finally, after capturing CRP, the charge transfer is further blocked due to the steric hindrance effect, and the resistance reaches a maximum. Therefore, the EIS characterization results of these different modified electrodes prove that the electrochemical biosensor is successfully assembled.

[0069] Figure 5 When the CRP concentration was 10 μg / mL, the difference between the experimental group and the control group was △I = 20.1 μA. The results showed that the CRP / CRP Ab / AL-BSA / Au / PANI / SPCE of the present invention, that is, the electrochemical sensor of the present invention, is feasible for detecting CRP.

[0070] Figure 6 Figures A, B, and C are the fluorescence staining results of PANI / SPCE, AL-BSA / Au / PANI / SPCE, and CRP Ab / AL-BSA / Au / PANI / SPCE after 48 hours of cell incubation, respectively. The cell line selected is MCF-7 breast cancer cells, and the incubation conditions are 37°C and 5% CO2. After staining with calcein-AM stain, the cells show fluorescence under a fluorescence microscope. The more fluorophores, the more cell adhesion. Compared with Figure A, the cell adhesion at the interfaces treated with AL-BSA in Figures B and C is very small, indicating that the electrochemical biosensor for detecting C-reactive protein in plasma and whole blood of the present invention has good anti-cell adhesion and contamination performance.

[0071] Figure 7PANI / SPCE and CRP / CRP Ab / AL-BSA / AuNPs / PANI / SPCE were immersed in solutions with BSA concentrations of 0%, 0.5%, 1.0%, 2.0%, 5.0%, and 10.0% for 0.5 h. The results showed that in the PANI / SPCE group, as BSA concentration increased, nonspecific adsorption of BSA on the electrode surface blocked PANI electron transfer, resulting in a larger difference in DPV signals. At a BSA concentration of 100 mg / mL (10%), the current signal change rate of the PANI / SPCE decreased by 15.24%. In the CRP / CRP Ab / AL-BSA / AuNPs / PANI / SPCE group, the current signal change rate of the AL-BSA-modified electrode decreased by 6.13% at a BSA concentration of 100 mg / mL, demonstrating that the electrochemical biosensor for detecting C-reactive protein in plasma and whole blood has excellent anti-fouling properties.

[0072] Figure 8 PANI / SPCE and CRP / CRP Ab / AL-BSA / AuNPs / PANI / SPCE were immersed in solutions with plasma concentrations of 0%, 1%, 10%, 25%, 50%, and 100% for 0.5 hours. The results showed that in the PANI / SPCE group, as plasma concentration increased, nonspecific adsorption of plasma on the electrode surface blocked PANI electron transfer, resulting in a larger difference in the DPV signal. At a plasma concentration of 100%, the current signal change rate of the PANI-modified electrode decreased by 19.56%. In the CRP / CRP Ab / AL-BSA / AuNPs / PANI / SPCE group, the current signal change rate of the AL-BSA-modified electrode decreased by 5.84% at a plasma concentration of 100%, demonstrating that the electrochemical biosensor for detecting C-reactive protein in plasma and whole blood has excellent anti-fouling properties.

[0073] Figure 9 In this study, we investigated whether the presence of 50 μg / mL SAA, 5 ng / mL PCT, 0.5 ng / mL IL-6, 1 ng / mL LPS, 1 ng / mL FN and 10% BSA would affect the specificity of the constructed electrochemical biosensor. Figure 9 As shown, the current signals of SAA, PCT, IL-6, LPS, FN and 10% BSA are all close to the background value with small differences, and are significantly different from the current signal of CRP, indicating that the electrochemical biosensor for detecting C-reactive protein in plasma and whole blood of the present invention has good specificity.

[0074] Figures 10 to 12The relationship between the CRP concentration in PBS solution, plasma and blood and the detected electrical signal value was investigated. The results showed that within the range of 0.1μg / mL to 25μg / mL, the current difference △I and lg C CRP There is a good linear relationship with a detection limit of 0.096 μg / mL. The linear range and detection limit in plasma and blood are basically consistent with the detection results of PBS solution.

[0075] Figure 13 and Figure 14 Five screen-printed electrodes from the same batch but different batches, as well as five screen-printed electrodes from different batches, were used to detect a 10.0 μg / mL CRP sample. The results showed an intra-group RSD of 0.20% and an inter-group RSD of 1.32%, demonstrating the excellent stability of the electrochemical biosensor for detecting C-reactive protein in plasma and whole blood.

Claims

1. A method for detecting C-reactive protein concentration using an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood, characterized in that: The method comprises the following steps: (1) constructing a polyaniline coating on the electrode surface by an electropolymerization method, generating an endogenous electrical signal through the intrinsic conductive state of polyaniline, and using the antibody probe on the electrode surface to bind to the target protein to be measured to affect the polyaniline electron transfer efficiency on the electrode surface, thereby correlating it with the target content to be measured; (2) reducing bovine serum albumin with tri(2-carboxyethyl)phosphine to prepare amyloid bovine serum albumin, preparing gold nanoparticles on the electrode surface by electrochemical deposition, and modifying the amyloid bovine serum albumin on the electrode surface by gold-sulfur bonds to form an anti-fouling coating with good biocompatibility, which resists non-specific adsorption caused by serum proteins, cells, and plasma; (3) assembling specific C-reactive protein antibodies onto the amyloid bovine serum albumin-modified electrode surface by chemical cross-linking using a carbodiimide condensation method; and (4) detecting the current change before and after incubation with plasma or blood samples by differential pulse voltammetry, and calculating the C-reactive protein (CRP) concentration in the plasma or whole blood samples.

2. A method for preparing an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood, characterized in that: The steps include: (1) Pre-treating the screen-printed electrode, including washing with dilute sulfuric acid, washing with water, and drying with nitrogen, to obtain a pre-treated screen-printed electrode SPCE; (2) constructing a polyaniline (PANI) coating on the surface of the pretreated screen-printed electrode (SPCE) obtained in step (1) by an electropolymerization method to prepare a polyaniline coating / screen-printed electrode (PANI) / SPCE; (3) depositing gold nanoparticles on the surface of the polyaniline coating / screen-printed electrode PANI / SPCE prepared in step (2) by an electrodeposition method to prepare gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE; (4) first reducing bovine serum albumin with tri(2-carboxyethyl)phosphine to prepare amyloid bovine serum albumin AL-BSA, and then assembling AL-BSA onto the surface of the gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE prepared in step (3) through gold-sulfur bonds to obtain amyloid bovine serum albumin / nano-gold / polyaniline coating / screen-printed electrode AL-BSA / AuNPs / PANI / SPCE; (5) Using the carbodiimide condensation method, the C-reactive protein capture monoclonal antibody CRP Ab was assembled onto the surface of the amyloid bovine serum albumin / nano-gold / polyaniline coating / screen-printed electrode AL-BSA / AuNPs / PANI / SPCE prepared in step (4) by chemical cross-linking to obtain CRP Ab modified amyloid bovine serum albumin / nano-gold / polyaniline coating / screen-printed electrode CRP Ab / AL-BSA / AuNPs / PANI / SPCE; (6) dropping a sample containing CRP onto the surface of the CRP Ab-modified amyloid bovine serum albumin / nano-gold / polyaniline coating / screen-printed electrode CRP Ab / AL-BSA / AuNPs / PANI / SPCE prepared in step (5) and incubating the surface to obtain an incubated electrode; (7) After incubation, the electrode surface is cleaned and then placed in a phosphate buffer solution. Differential pulse voltammetry is used for electrochemical detection. The CRP concentration in the sample is calculated based on the change in the electrical signal.

3. The method for preparing an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood according to claim 2, wherein: The specific steps include: (1) Pretreatment of screen-printed electrode SPCE: 100 μL of 0.5 mol / L H2SO4 was added to the surface of SPCE and scanned under cyclic voltammetry with a scanning range of 0–1.5 V, a scanning speed of 1 V / s, and 30 scanning cycles. The electrode was then washed with ultrapure water and dried with N2 to obtain the pretreated screen-printed electrode SPCE for use. (2) Polyaniline PANI deposition: a mixed solution containing 0.5 mol / L H2SO4 and 0.02 mol / L aniline was prepared, and the screen-printed electrode SPCE pretreated in step (1) was subjected to cyclic voltammetry for 20 cycles at a scan rate of 0.05 V / s in the potential range of -0.2~0.9 V to obtain a polyaniline PANI film modified electrode; the polyaniline PANI film modified electrode was taken out, washed with deionized water, and then placed in a 0.5 mol / L H2SO4 solution, and electrolyzed at +0.90 V for 60 s, then at -0.2 V for 60 s, and then at 0.1 V in the potential range of -0.2 V~0.9 V. V / s scanning rate cyclic voltammetry scan to obtain the modified electrode, clean the aniline adsorbed or polymerized loosely on the surface of the modified electrode until the cyclic voltammetry curve is stable, then rinse with ultrapure water and blow dry with nitrogen to prepare the polyaniline coating / screen-printed electrode PANI / SPCE for use; (3) Deposition of gold nanoparticles AuNPs: 2.8 mmol / L chloroauric acid solution was added to the surface of the polyaniline coating / screen-printed electrode PANI / SPCE, and AuNPs were electrodeposited using the time-current method. The electrodeposition potential was -0.2 V, and the time was 100 s. The surface was washed with ultrapure water and dried with N2 to obtain the gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE, which was then used for standby use. (4) Assembly of AL-BSA: 7 μL of AL-BSA solution pre-reacted for 2 h was added dropwise to the surface of the gold nanoparticles / polyaniline coating / screen-printed electrode AuNPs / PANI / SPCE. After incubation for 1 h, the surface was washed with 10 mmol / L pH 7.4 phosphate buffer (PBS) and dried with N2 to obtain AL-BSA / AuNPs / PANI / SPCE for later use. The AL-BSA solution was prepared by mixing 2 mg / mL bovine serum albumin and 50 mmol / L tris(2-carboxyethyl)phosphine in a volume ratio of 1:

1. (5) Activation by carbodiimide condensation method: 7 μL of EDC / NHS solution prepared with 0.4 mol / L EDC and 0.1 mol / L NHS was added dropwise to the surface of the AL-BSA / AuNPs / PANI / SPCE electrode prepared in step (4). After incubation at room temperature for 1 h, the electrode was washed with 10 mmol / L pH 7.4 PBS and dried with N2 to obtain EDC-NHS / AL-BSA / AuNPs / PANI / SPCE for later use. (6) Assembly of capture antibody probe: 7 μL of 100 μg / mL CRP Ab was incubated on the surface of the EDC-NHS / AL-BSA / AuNPs / PANI / SPCE electrode prepared in step (5) at room temperature for 3 h, washed with 10 mmol / L pH 7.4 PBS, and blown dry with N2 to obtain CRP Ab / AL-BSA / AuNPs / PANI / SPCE for later use; (7) CRP detection: In the reagent analysis mode requiring a buffer system, 7 μL of a test solution containing CRP at a concentration of 10 μg / mL was added to the surface of the CRP Ab / AL-BSA / AuNPs / PANI / SPCE electrode, incubated at room temperature for 1 h, washed with 10 mmol / L pH 7.4 PBS, blown dry with N2, and electrochemical detection was performed using differential pulse voltammetry in a 0.2 mol / L pH 5.7 phosphate buffer solution system; In the reagent-free analysis mode that does not require detection in a buffer system, 1.68 μL of 5 mol / L sodium dihydrogen phosphate solution was dripped onto the surface of the CRP Ab / AL-BSA / AuNPs / PANI / SPCE electrode and dried at 37°C. Then, 100 μL of the test solution containing CRP at a concentration of 10 μg / mL was dripped onto the electrode surface and incubated at room temperature for 1 h. No electrode cleaning was required, and differential pulse voltammetry electrochemical detection was performed directly. The changes in the electrical signal were recorded, and the CRP concentration in the sample was calculated. The scanning voltage was -0.6 V to 0.6 V.

4. The preparation method according to claim 2 or 3 obtains an electrochemical biosensor for detecting C-reactive protein in plasma and whole blood.

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