Biomarker content detection method combined with molecularly imprinted polymer (MIP)

Abstract

The invention belongs to the technical field of biomarker detection, and discloses a biomarker content detection method combined with a molecularly imprinted polymer (MIP), and the specific technical scheme is as follows: step 1, preparing specific MIPs of three serum markers of Hcy, hs-CRP and Lpa; 2, combining the specific MIPs of the three serum markers, synthesizing the MIP, and combining the synthesized MIP with the nano composite material; step 3, carrying out surface modification on the screen-printed carbon electrode, and constructing an unmarked electrochemical biosensor system for detecting serum Hcy, hs-CRP and Lpa; and step 4, carrying out independent detection and differential signal marking on the three different markers by using three oxidation-reduction probes, and realizing subsequent current signal change monitoring. The method has the advantages of high detection precision, shortened detection time, simple operation and reusability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarker detection, and specifically relates to a method for jointly detecting HCY, hs-CRP and LPa contents by an electrochemical method combined with a molecularly imprinted polymer (MIP). Background Art

[0002] Homocysteine ​​(Hcy), high-sensitivity C-reactive protein (hs-CRP), and peripheral blood lipoprotein a (Lpa)—these three biomarkers are important intermediates in the body's metabolic processes. Accumulation and elevation of homocysteine ​​in the blood due to various factors can lead to hyperhomocysteinemia, or hyperhomocysteinemia. Elevated hs-CRP levels due to various factors often indicate the presence of an acute inflammatory response. Furthermore, studies have shown that elevated hs-CRP levels are closely associated with the risk of cardiovascular events such as atherosclerosis and coronary heart disease. Therefore, hs-CRP is often used for early screening and risk assessment of cardiovascular disease. Furthermore, abnormal lipoprotein metabolism is closely associated with a variety of diseases, including atherosclerosis, diabetes, obesity, and cancer. Multiple studies have shown that the levels of these three biomarkers are negatively correlated with arterial elasticity and are independent risk factors for atherosclerosis and stroke. Currently, there are various methods used to test them in medical examinations, such as enzyme immunoassay, chromatography, polarization immunoassay, ELISA, immunoturbidimetry, electrophoresis, centrifugation, etc., but all of them have some shortcomings.

[0003] The shortcomings of current detection methods are now described:

[0004] 1. Methods for measuring homocysteine:

[0005] The disadvantages of the amino acid analysis method are: the operation is relatively complicated, it takes a long time, and it is insensitive to sulfur-containing amino acids. This method is no longer used to determine plasma Hcy.

[0006] The disadvantages of paper chromatography are: limited sensitivity, accuracy and quantitative analysis ability, and susceptibility to interference from other components.

[0007] The disadvantages of the isotope method are: the operation is cumbersome and there is radioactive pollution. At the same time, the isotopes are easy to decay and are harmful to the human body, which limits their use and has not been widely used.

[0008] The disadvantages of radioenzyme analysis are: HPLC method is easily interfered by reducing agents, radioenzyme analysis is radioactive and the operation is cumbersome.

[0009] The disadvantages of gas chromatography-mass spectrometry (GC-MS) are: complex operation, time-consuming, and expensive instruments.

[0010] The disadvantages of chromatography are: expensive equipment, very complicated operation, time-consuming, high requirements for instrumentation, and difficulty in promoting its use in routine clinical chemistry laboratories.

[0011] 2. Methods for measuring high-sensitivity C-reactive protein:

[0012] Disadvantages of latex-enhanced immunoturbidimetry: There are problems with precision, correlation, linear regression analysis results and consistency.

[0013] Disadvantages of chemiluminescent immunoassay (CLIA): It requires a dedicated chemiluminescence instrument and is relatively expensive.

[0014] Disadvantages of electrochemiluminescence immunoassay (ECLIA): high cost, complex operation, and high sample processing requirements.

[0015] Disadvantages of enzyme-linked immunosorbent assay (ELISA): many steps, long time consumption, and low degree of automation.

[0016] Disadvantages of mass spectrometry technology (LC-MS / MS): The equipment is expensive and the operation is complicated, and it is mostly used for research or standardized verification.

[0017] 3. Methods for measuring lipoprotein a:

[0018] Disadvantages of immunoturbidimetry: Molecular size polymorphism may affect antibody binding efficiency, leading to biased results.

[0019] Disadvantages of enzyme-linked immunosorbent assay (ELISA): many steps, long time consumption, and low degree of automation.

[0020] Disadvantages of chemiluminescent immunoassay (CLIA): Requires special instruments.

[0021] Disadvantages of immunoturbidimetry (ITA): It is easily interfered by sample turbidity (such as lipemia) and requires calibration and quality control.

[0022] Disadvantages of mass spectrometry technology (LC-MS / MS): The equipment is expensive and the operation is complicated. It is mainly used for research and standardized reference methods.

[0023] Disadvantages of molecular biology methods: Lpa concentration cannot be directly measured and needs to be combined with protein detection.

[0024] 4. Methods for combined detection of three biomarkers:

[0025] Disadvantages of the multi-index automated biochemical analysis platform: high cost, possible influence of lipemia / hemolysis, and different sources of calibrators for different projects may lead to biased results.

[0026] Disadvantages of chemiluminescence immunoassay (CLIA) multiplex testing: strong instrument dependence, need for a dedicated chemiluminescence instrument, high reagent cost, and limited popularization in small and medium-sized hospitals.

[0027] Disadvantages of enzyme-linked immunosorbent assay (ELISA) combined testing: cumbersome operation, limited sensitivity, and difficulty in standardization.

[0028] Disadvantages of LC-MS / MS technology: expensive equipment, high technical threshold, long detection cycle, and not suitable for emergency or routine screening.

[0029] In summary, the shortcomings of current detection methods are mainly concentrated in:

[0030] 1. It takes a long time: This is mainly because the original method of testing the content is to use chemical detection methods to perform chemical reactions combined with color development reactions for measurement, and sometimes chromatographic separation is required, which increases the time of testing and makes it take longer;

[0031] 2. Low measurement sensitivity: This is mainly because some measured indicators in some tests are insensitive to hcy, hs-CRP and Lpa, resulting in low sensitivity;

[0032] 3. Unreliable experimental data: This is mainly because for a rough measurement method such as paper chromatography, a rough color is obtained and then compared with a standard chromatogram. The results measured by this method are not only inaccurate but also difficult to believe.

[0033] 4. Complicated operation: Radioactive contamination is caused by the addition of radioactive elements in some experimental designs. Radioactive elements will decay and may cause radioactive contamination if not handled properly.

[0034] 5. Complex experimental operation: This is mainly because the experimental process requires both substrate conversion and substrate relevance determination. The operation cycle is long and the operation technology is complex, making it difficult to achieve the expected results.

[0035] 6. The instrument is expensive, mainly because it involves the use of some precision instruments, such as gas chromatographs, which increases costs. Summary of the Invention

[0036] To solve the technical problems existing in the prior art, the present invention provides a method for detecting the content of biomarkers in combination with molecularly imprinted polymers (MIPs), which has high detection accuracy, shortened detection time, simple operation, and is reusable.

[0037] To achieve the above objectives, the technical solution adopted by the present invention is: a method for detecting the content of biomarkers in combination with molecularly imprinted polymers (MIPs), the specific steps of which are as follows:

[0038] Step 1: Preparation of specific MIPs for three serum markers: Hcy, hs-CRP, and Lpa;

[0039] Step 2: combining the specific MIPs of the three serum markers to synthesize MIPs, and combining the synthesized MIPs with the nanocomposite material;

[0040] Step 3: Surface modification of the screen-printed carbon electrode was performed to construct a label-free electrochemical biosensor system for detecting serum Hcy, hs-CRP, and Lpa;

[0041] Step 4: Use three redox probes to independently detect three different markers, differentiate signal labels and monitor subsequent current signal changes.

[0042] In step 1, serum Hcy is mixed with the functional monomer methacrylic acid to prepare a monomer mixture, which is then mixed with a cross-linking agent, trimethylolpropane, trimethacrylate, and a free radical-induced polymerization initiator, azobisisobutyronitrile. After the mixture is polymerized, it is dehydrated, and the serum Hcy bound to the MIP is eluted sequentially with methanol, NaOH solution, acetic acid solution, and deionized water to obtain Hcy-MIP.

[0043] hs-CRP was mixed with the functional monomer acrylamide to prepare a monomer mixture, which was then mixed with the cross-linker N,N'-methylenebisacrylamide, the free radical-induced polymerization initiator ammonium persulfate, and tetramethylethylenediamine. After the polymerization of the mixture was completed, the hs-CRP bound to MIP was eluted with sodium dodecyl sulfate, glycine-HCl buffer, Tris-HCl buffer containing NaCl, phosphate-buffered saline, and deionized water in sequence to obtain hs-CRP-MIP.

[0044] Lpa was mixed with functional monomers methacrylic acid and vinyl imidazole to prepare a monomer mixture, which was then mixed with a cross-linker polyethylene glycol dimethacrylate, a free radical-induced polymerization initiator ammonium persulfate, and tetramethylethylenediamine. After the mixture was polymerized, it was dehydrated and the Lpa bound to the MIP was eluted with sodium dodecyl sulfate, Tris-HCl buffer containing NaCl, ethylenediaminetetraacetic acid, and deionized water to obtain Lpa-MIP.

[0045] In step 2, serum Hcy-MIP and carbon nanotube dispersion are mixed in proportion, hs-CRP-MIP and chitosan are mixed in proportion, and LPa-MIP and ionic liquid are mixed in proportion.

[0046] In step 4, Hcy standard solutions of different concentrations were applied to completely interact with the active sites on the surface of the Hcy-MIP modified electrode. The electrode was cleaned with deionized water to remove nonspecific binding substances. The electrode potential was scanned, and the peak current of K4[Fe(CN)6] was observed using DPV to determine the linear relationship between the DPV peak current drop value and the Hcy concentration.

[0047] hs-CRP standard solutions of different concentrations were added dropwise to the surface of the hs-CRP-MIP modified electrode and incubated at room temperature. The electrode was rinsed with deionized water to remove unbound hs-CRP and nonspecific adsorbates. The electrode potential was scanned, and the peak current of MB was observed using CV. The logarithmic relationship between the CV peak current difference and the hs-CRP concentration was determined.

[0048] LPa standard solutions of different concentrations were added dropwise to the surface of the LPa-MIP modified electrode and incubated at room temperature. The electrode was gently rinsed with deionized water to remove unbound LPa and nonspecific adsorbates. The electrode potential was scanned to observe the positive shift of the oxidation peak potential, and an S-shaped curve of ΔE and LPa concentration was fitted.

[0049] The blood was divided into three equal parts and interacted with the Hcy-MIP biosensor, hs-CRP-MIP biosensor, and LPa-MIP biosensor respectively. The electrodes were cleaned with deionized water to remove non-specific binding substances. A scanning electrode potential was applied, and the current signal changes of K4[Fe(CN)6]3- / 4-, methylene blue, and ferrocene were recorded by CV and DPV, respectively.

[0050] Among them, the nanocomposite material is composed of carbon nanotubes, chitosan and ionic liquid.

[0051] The present invention utilizes molecularly imprinted polymer (MIP) combined with differential pulse voltammetry (DPV) to detect homocysteine ​​(HCY), high-sensitivity C-reactive protein (hs-CRP) and lipoprotein a (LPa). The specific beneficial effects are embodied in:

[0052] 1. High Specificity: MIP achieves specific binding by creating recognition sites within the polymer that are complementary in shape, size, and functional groups to the target molecule (HCY, hs-CRP, or LP(a)). DPV uses pulsed potential to measure peak current changes, further amplifying the signal difference caused by target molecule binding. The MIP recognition site only allows the embedding of specific target molecules. Compared to traditional methods, such as chemiluminescent immunoassay (CMIA), MIP-DPV avoids the problem of antibody cross-reactivity through molecular imprinting technology, significantly improving detection specificity, and performing particularly well in complex biological matrices such as serum.

[0053] 2. High Sensitivity and Low Detection Limit: DPV applies small pulses and measures the current difference, reducing charge current interference and improving the signal-to-noise ratio. The high binding capacity of the MIP further enhances the electrochemical signal changes caused by the target molecule. The DPV pulse design optimizes electrochemical signal acquisition, and the MIP's nanopore structure increases the capture efficiency of the target molecule. The detection limit of HCY-MIP-DPV is 1.2 µM, far below the threshold required for clinical diagnosis (5–15 µM). The detection range of lipoprotein a MIP (such as LP(a)-MIP) is 4–400 mg / dL, covering clinically relevant concentrations. The high binding capacity of CRP-MIP (1.04 μg / cm²) supports low-concentration detection, significantly improving sensitivity compared to traditional HPLC, ELISA (which typically have detection limits in the µM range or higher), or MIP-DPV.

[0054] 3. Low cost and high stability: MIP does not rely on expensive biological antibodies, has high chemical / thermal stability, and is reusable. DPV is based on simple electrochemical equipment with low operating costs. MIP uses inexpensive monomers (such as methyl methacrylate (MAA)) and cross-linkers (such as TRIM), and the preparation process is simple, such as precipitation polymerization. The cost of DPV equipment is much lower than that of HPLC or mass spectrometers. The repeatability coefficient of variation of HCY-MIP sensors is 2.27–3.50%, and the reproducibility is 3.42–4.22%, indicating high stability. MIP can be reused multiple times without significantly reducing performance. Compared with CMIA, which requires expensive antibodies and reagents, the cost of MIP-DPV is reduced by about 50–70%.

[0055] 4. Application of specific MIP: Molecular imprinting polymers (MIPs) are designed for three key markers of myocardial infarction, Hcy, hs-CRP and LP(a), to achieve highly selective detection of multiple targets. Compared with single marker detection, this integration improves the comprehensiveness and efficiency of diagnosis.

[0056] 5. Sample splitting testing strategy: Dividing the blood into three equal parts for separate testing avoids cross-interference in multi-marker testing and improves detection specificity, which is particularly suitable for scenarios with limited resources.

[0057] 6. Combination innovation of nanocomposite materials: The ternary composite system of carbon nanotubes (high conductivity), chitosan (biocompatibility) and ionic liquid (stability) optimizes the electron transfer ability, anti-pollution property and stability of the modified layer of the electrode, significantly enhancing the sensitivity of the sensor.

[0058] 7. Low-cost electrode modification: The low-cost characteristics of screen-printed carbon electrodes (SPCEs) combined with nanocomposites provide an economically feasible technical path for the development of portable detection devices.

[0059] 8. Differentiated signal labeling: Three redox probes, K4[Fe(CN)6], methylene blue, and ferrocene, are used to independently detect current changes corresponding to different markers through CV and DPV, reducing the risk of signal overlap and improving the accuracy of multi-index detection.

[0060] 9. Remove the cleaning step of non-specific adsorption and introduce a deionized water cleaning step to further reduce background interference and enhance detection reliability.

[0061] 10. Dynamic quantitative risk scoring: Converting marker concentrations into graded risk scores (low, medium, and high risk) to construct a comprehensive scoring system, which simplifies the clinical decision-making process and has greater predictive value than a single indicator.

[0062] 11. Data-driven grading standards: Setting thresholds based on clinical research data enhances the scientific nature and practicality of the model and provides support for personalized medicine.

[0063] 12. Platform-based design thinking: Modular integration of MIP synthesis, nanomaterial modification, electrochemical detection, and data analysis leaves room for subsequent addition of more biomarkers (such as troponin).

[0064] 13. Potential for rapid testing: Combined with the portability of SPCE, this solution is expected to be developed into a point-of-care (POCT) tool to promote the popularization of early screening for myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Flowchart of the present invention. DETAILED DESCRIPTION

[0066] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0067] like Figure 1 As shown, a method for detecting the content of biomarkers combined with molecular imprinting polymers (MIPs) is described. The MIP selectively absorbs the target analyte and then generates a characteristic electrochemical signal to determine the content of the target analyte. The specific steps are as follows: preparing specific MIPs for three serum markers: homocysteine ​​(Hcy), high-sensitivity C-reactive protein (hs-CRP), and lipoprotein (LP). The synthesized MIPs are then combined with nanocomposites (carbon nanotubes / chitosan / ionic liquids) respectively, and the screen-printed carbon electrode (SPCE) is surface-modified to create a label-free electrochemical biosensor system for detecting serum Hcy, hs-CRP, and LP.

[0068] Preparation of specific MIP: 10 μmol Hcy was mixed with 200 μmol functional monomer methacrylic acid (MAA) to prepare a monomer mixture, which was then mixed with 50 μmol cross-linking agents trimethylolpropane, trimethacrylate (TRIM) and 3 μmol free radical-induced polymerization initiator azobisisobutyronitrile (AIBN). After the mixture was polymerized, it was dehydrated and the Hcy bound to the MIP was eluted with methanol, 10% NaOH solution, 1% acetic acid solution and deionized water in sequence to obtain 30 mg of MIP that selectively bound Hcy.

[0069] A monomer mixture was prepared by mixing 0.125 mg of hs-CRP with 5.0 mg of the functional monomer acrylamide (AAM), which was then mixed with 25 mg of the cross-linker N,N'-methylenebisacrylamide (MBA), 0.5 mg of the free radical-induced polymerization initiator ammonium persulfate (APS) and 1 μL of tetramethylethylenediamine (TEMED). After polymerization, the mixture was dehydrated and the hs-CRP bound to the MIP was eluted with 0.5% sodium dodecyl sulfate (SDS), glycine-HCl buffer at pH 2, Tris-HCl buffer containing NaCl (150 mmol NaCl, 50 mmol Tris-HCl at pH 7.4), phosphate-buffered saline (PBS) at a concentration of 0.1 mmol pH 7, and deionized water, respectively, to obtain 30 mg of MIP selectively bound to hs-CRP.

[0070] 0.15 mg of LPa was mixed with 4 mg of functional monomers methacrylic acid (MAA) and vinylimidazole (VIM) to prepare a monomer mixture, which was then mixed with 24 mg of cross-linker polyethylene glycol dimethacrylate (PEGDMA, MW 550) and 0.6 mg of free radical-induced polymerization initiators ammonium persulfate (APS) + tetramethylethylenediamine (TEMED). After the polymerization was completed, the mixture was dehydrated and the LPa bound to MIP was eluted with 1% sodium dodecyl sulfate (SDS), Tris-HCl buffer containing NaCI (NaCl concentration of 150 mmol, Tris-HCl concentration of 50 mmol, pH = 7.4), 1 mmol ethylenediaminetetraacetic acid (EDTA), and deionized water to obtain 30 mg of MIP selectively bound to LPa.

[0071] Serum Hcy-MIP (30 mg / 10 ml, 1.5 mg / mL) was mixed with a carbon nanotube dispersion (100 mg / 10 ml, 5 mg / mL) at a 1:1 volume ratio; hs-CRP-MIP (30 mg / 12 ml, 2.5 mg / mL) was mixed with chitosan (120 mg / 8 ml, 15 mg / mL) at a 1:1.5 volume ratio; and LP(a)-MIP (30 mg / 2 ml, 136.4 mg / mL) was mixed with an ionic liquid (685 mg / 0.5 ml, 1.37 g / mL) at a 1:2 volume ratio. These three complexes were then used to modify the surface of screen-printed carbon electrodes, forming three label-free electrochemical biosensor systems that specifically bind to serum markers.

[0072] After modification, three redox probes, K4[Fe(CN)6]3- / 4-, methylene blue, and ferrocene, were used, all at 5 mol / L, to combine with the specific MIPs of the three modules on the electrode to monitor subsequent current signal changes.

[0073] 150 μL of Hcy standard solution with different concentrations was applied to fully interact with the active sites on the surface of the Hcy-MIP modified electrode for 10 minutes. The electrode was then cleaned with deionized water to remove any potential nonspecific binding substances. The electrode potential was scanned from -0.5 V to 0.8 V, and the peak current of (K4[Fe(CN)6]) was observed using DPV. A linear relationship between the DPV peak current decrease value (ΔI) and the Hcy concentration was established (ΔI = k1·[Hcy] + b1).

[0074] 150 μL of hs-CRP standard solution of different concentrations was added dropwise to the surface of the hs-CRP-MIP modified electrode and incubated at room temperature for 15 min. The electrode was gently rinsed with deionized water to remove unbound hs-CRP and nonspecific adsorbates. The electrode potential was scanned from -0.4 V to 0 V. The peak current of MB was observed using CV, and the logarithmic relationship between the CV peak current difference (ΔI) and the hs-CRP concentration was established (ΔI = k2·log[hs-CRP] + b2).

[0075] 150 μL of LP(a) standard solution of varying concentrations was added dropwise to the surface of the LP(a)-MIP-modified electrode. The solution was incubated at room temperature for 20 minutes. The electrode was then gently rinsed with deionized water to remove unbound LP(a) and nonspecific adsorbents. The electrode potential was scanned from 0 V to 0.6 V at a scan rate of 50 mV / s, and a positive shift in the oxidation peak potential (E_p) was observed. A sigmoid curve (logistic model) was fitted to the relationship between ΔE and LP(a) concentration: ΔE = E_max / (1 + (K_d / [LP(a)])^n) (E_max: maximum displacement, K_d: apparent dissociation constant, n: Hill coefficient)

[0076] 10ml of blood was divided into three equal parts and interacted with the Hcy-MIP biosensor, hs-CRP-MIP biosensor, and LP(a)-MIP biosensor, respectively. The electrodes were carefully cleaned with deionized water to remove any potential nonspecific binding substances. A sweeping electrode potential was applied using an electrochemical workstation, and the current signal changes of K4[Fe(CN)6]3- / 4-, methylene blue, and ferrocene were recorded by CV and DPV, respectively.

[0077] Since the interconversion of the redox reactions of the three electroactive probe molecules unfolds as a surface-controlled process, the imprinted sites are blocked by serum markers and the response current decreases, which also reduces the effective surface area. Therefore, the reduction in the pore availability of the electrode surface is caused by the increase in the concentration of serum markers Hcy, hs-CRP, and LP(a).

[0078] The ΔI of the test sample was substituted into the standard curve to calculate the concentrations of Hcy, hs-CRP and LP(a) in serum.

[0079] Based on clinical study data, biomarkers were assigned risk scores (low risk = 1 point, intermediate risk = 2 points, high risk = 3 points) according to the measured concentrations of the three serum markers.

[0080]

[0081] Assign weight coefficients according to the independent prediction weights of the markers:

[0082] Hcy: weight 1.2 (independent oxidative stress risk);

[0083] hs-CRP: weight 1.5 (inflammation is directly related to plaque instability);

[0084] LP(a): weight 1.3 (strong risk factor for hereditary atherosclerosis);

[0085] Myocardial infarction risk levels are divided according to the comprehensive risk score:

[0086] Comprehensive risk score = (Hcy risk score × 1.2) + (hs-CRP risk score × 1.5) + (LP(a) risk score × 1.3)

[0087] Risk score allocation: low risk = 1 point, medium risk = 2 points, high risk = 3 points.

[0088]

[0089] Overall, the present invention first prepares three specific MIPs, then combines them with nanocomposites. The composites then modify screen-printed carbon electrodes, and finally, redox probes are incorporated into the specific MIPs. The electrode preparation and modular modification involve dividing the screen-printed carbon electrode (SPCE) into three independent detection modules, each modified with molecularly imprinted polymers (MIPs) for HCY, hs-CRP, and LP(a). The three markers are then modified with carbon nanotubes, chitosan, and ionic liquid nanocomposites, respectively, to enhance conductivity. Each module is isolated by a PDMS microfluidic channel to prevent cross-interference, enabling the construction of a substrate for simultaneous detection of three markers. Next, electrochemical detection was performed. After sample injection into the instrument, quantitative analysis was performed using differential pulse voltammetry and cyclic voltammetry (CV). The redox probes ferricyanide (K4[Fe(CN)6]), methylene blue (MB), and ferrocene (Fc) corresponded to HCY, hs-CRP, and LPa, respectively. The peak current change (ΔI) at the electrodes was inversely proportional to the concentrations of HCY (5–150 μM), hs-CRP (0.1–50 μg / mL), and LPa (2–250 mg / dL). The detection limits reached 1.2 μM, 0.05 μg / mL, and 0.8 mg / dL, respectively. Finally, data integration and result output were performed. A multi-channel electrochemical workstation simultaneously acquired signals from three modules. Concentration values ​​were automatically fitted using precalibrated curves. The accuracy of the results was ensured by combining spike recovery (90–105%) and clinical sample validation (R² > 0.98 agreement with ELISA). The final concentration is processed by software and displayed in real time on a portable terminal. The entire process from sample addition to report output is completed within 10 minutes, providing an efficient and low-cost POCT solution for rapid screening of cardiovascular diseases.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims

1. A method for detecting biomarker content in combination with molecularly imprinted polymer (MIP), characterized in that: The specific steps are as follows: Step 1: Preparation of specific MIPs for three serum markers: Hcy, hs-CRP, and Lpa; Step 2: combining the specific MIPs of the three serum markers to synthesize MIPs, and combining the synthesized MIPs with the nanocomposite material; Step 3: Surface modification of the screen-printed carbon electrode was performed to construct a label-free electrochemical biosensor system for detecting serum Hcy, hs-CRP, and Lpa; Step 4: Use three redox probes to independently detect three different markers, differentially label the signals, and monitor the changes in the current signal.

2. The method for detecting the content of a biomarker combined with a molecularly imprinted polymer (MIP) according to claim 1, characterized in that: In step 1, serum Hcy is mixed with methacrylic acid to prepare a monomer mixture, which is then mixed with trimethylolpropane, trimethacrylate, and azobisisobutyronitrile. After the mixture is polymerized, it is dehydrated and serum Hcy bound to MIP is eluted with methanol, NaOH solution, acetic acid solution, and deionized water in sequence to obtain Hcy-MIP. hs-CRP is mixed with acrylamine to prepare a monomer mixture, which is then mixed with N,N'-methylenebisacrylamide, ammonium persulfate, and tetramethylethylenediamine. After the mixture is polymerized, it is dehydrated and the hs-CRP bound to the MIP is eluted sequentially with sodium dodecyl sulfate, glycine-HCl buffer, Tris-HCl buffer containing NaCl, phosphate-buffered saline, and deionized water to obtain hs-CRP-MIP. Lpa is mixed with methacrylic acid and vinylimidazole to prepare a monomer mixture, which is then mixed with polyethylene glycol dimethacrylate, ammonium persulfate, and tetramethylethylenediamine. After the mixture is polymerized, it is dehydrated and the Lpa bound to the MIP is eluted with sodium dodecyl sulfate, Tris-HCl buffer containing NaCl, ethylenediaminetetraacetic acid, and deionized water in sequence to obtain Lpa-MIP.

3. The method for detecting the content of biomarkers combined with molecularly imprinted polymers (MIPs) according to claim 2, characterized in that: In step 2, serum Hcy-MIP and carbon nanotube dispersion are mixed in proportion, hs-CRP-MIP and chitosan are mixed in proportion, and LPa-MIP and ionic liquid are mixed in proportion.

4. The method for detecting the content of a biomarker combined with a molecularly imprinted polymer (MIP) according to claim 3, characterized in that: In step 4, Hcy standard solutions of varying concentrations were applied to completely interact with the active sites on the surface of the Hcy-MIP-modified electrode. The electrode was cleaned with deionized water to remove nonspecific binding substances. The electrode potential was scanned, and the peak current of K4[Fe(CN)6] was observed using DPV. The linear relationship between the DPV peak current drop and Hcy concentration was determined. hs-CRP standard solutions of different concentrations were added dropwise to the surface of the hs-CRP-MIP modified electrode and incubated at room temperature. The electrode was rinsed with deionized water to remove unbound hs-CRP and nonspecific adsorbates. The electrode potential was scanned and the peak current of MB was observed using CV. The logarithmic relationship between the CV peak current difference and the hs-CRP concentration was determined. LPa standard solutions of different concentrations were added dropwise to the surface of the LPa-MIP modified electrode and incubated at room temperature. The electrode was gently rinsed with deionized water to remove unbound LPa and nonspecific adsorbates. The electrode potential was scanned to observe the positive shift of the oxidation peak potential and to fit an S-shaped curve of ΔE and LPa concentration. The blood was divided into three equal parts and interacted with the Hcy-MIP biosensor, hs-CRP-MIP biosensor, and LPa-MIP biosensor respectively. The electrodes were cleaned with deionized water to remove non-specific binding substances. A scanning electrode potential was applied, and the current signal changes of K4[Fe(CN)6]3- / 4-, methylene blue, and ferrocene were recorded by CV and DPV, respectively.

5. The method for detecting the content of biomarkers combined with molecularly imprinted polymers (MIPs) according to claim 4, characterized in that: The nano composite material is composited from carbon nano tubes, chitosan and ionic liquid.

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