Preparation method and application of LDHB antibody and POx enzyme bifunctionalized AuNPs and SERS detection platform

Abstract

The application provides an LDHB antibody and POx enzyme bifunctional AuNPs and a preparation method and application of a SERS detection platform, and the preparation method of the detection platform comprises the following steps: (1) synthesizing AuNPs; (2) modifying the AuNPs with a MUA and SH-PEG compound reagent to obtain carboxylated AuNPs; (3) coupling LDHB antibodies after activation with DMTMM, blocking, and then coupling POx enzymes to obtain AuNP@LDHB-POx; (4) fixing the AuNP@LDHB-POx on a PDA modified alumina ceramic chip and blocking to obtain a SERS substrate; (5) synthesizing AgNPs, modifying the AgNPs with a MHA and SH-PEG compound reagent, coupling LDHB antibodies after activation with DMTMM, and blocking to obtain a SERS nanolabel. By constructing a double-signal SERS platform, simultaneous and non-interfering quantification of metabolites pyruvic acid and proteins LDHB is realized in the same detection system, which provides a new technical means for early warning and auxiliary diagnosis of ACS-SCD, helps to promote the transformation of multi-omics findings into clinical diagnosis products, and has important clinical application value and transformation prospects.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical detection, nanomaterials and surface-enhanced Raman scattering technology; specifically, it relates to a method for preparing a bifunctional AuNPs and SERS detection platform for LDHB antibody and POx enzyme and its application in the preparation of products for the detection or auxiliary diagnosis of sudden cardiac death caused by acute coronary syndrome. Background Technology

[0002] Sudden cardiac death (SCD) due to acute coronary syndrome (ACS) is one of the most dangerous clinical endpoints in the cardiovascular field. Data shows that among patients admitted to the hospital for ACS and ruled out for myocardial infarction, 3%–5% still have occult myocardial injury; and among out-of-hospital SCD patients, as many as 62%–85% have a history of ACS. However, current clinical risk assessment for ACS-SCD mainly relies on traditional myocardial injury markers such as troponin and creatine kinase isoenzymes. These indicators reflect passive release after myocardial necrosis and lack early warning capabilities for sudden death risk. In other words, the existing diagnostic system cannot answer the crucial question of "which ACS patients are about to develop SCD."

[0003] In recent years, multi-omics studies have provided new perspectives for overcoming this bottleneck. Research shows that during acute myocardial ischemia, mitochondrial oxidative phosphorylation is inhibited, forcing myocardial metabolism to shift towards anaerobic glycolysis. This metabolic reprogramming process leaves captureable molecular traces in circulating extracellular vesicles (EVs). Lactate dehydrogenase B (LDHB), the dominant LDH isoenzyme in myocardial tissue, catalyzes the interconversion between lactate and pyruvate, maintaining redox balance and energy supply in cardiomyocytes under hypoxic conditions. Pyruvate, a key intermediate metabolite in glycolysis, directly reflects the metabolic state of cardiomyocytes under hypoxic stress. A multi-omics study simultaneously detected significant increases in pyruvate and LDHB in the plasma EVs of ACS-SCD patients. The combination of these two enzymes increased the AUC for SCD risk prediction to 0.786, and further combination with creatine kinase isoenzymes reached 0.9. It is noteworthy that the study observed that the elevation of pyruvate and LDHB in EVs was not reflected in the total plasma concentration, indicating that EVs have a unique diagnostic advantage as a "molecular concentrator of metabolic stress".

[0004] However, translating this metabolomics discovery into a usable clinical early warning tool faces a key technological bottleneck: pyruvate (a metabolite) and LDHB (a protein) are two chemically distinct classes of biomolecules with significant differences in abundance and detection mechanisms, making it difficult for existing platforms to simultaneously quantify both in a single assay. While SERS technology possesses high sensitivity and molecular fingerprinting capabilities, most applications remain limited to the detection of single-category biomarkers. To date, no research has constructed a dual-signal SERS platform capable of simultaneously and without interference quantifying pyruvate and LDHB in the same detection system. This gap directly hinders the translation of the aforementioned multi-omics discovery into clinical diagnostic products. Therefore, developing a SERS platform capable of simultaneously detecting pyruvate and LDHB across categories is an urgent technological necessity for achieving early warning of ACS-SCD. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention aims to provide a method for preparing and applying a bifunctional AuNPs and SERS detection platform for LDHB antibody and POx enzyme.

[0006] This invention provides a method for preparing a bifunctional AuNPs and SERS detection platform using LDHB antibody and POx enzyme, comprising the following steps: (1) Synthesis of AuNPs; (2) AuNPs were modified with a combination of MUA and SH-PEG to obtain carboxylated AuNPs; (3) After activation with DMTMM, LDHB antibody was coupled and blocked, and then POx enzyme was coupled to obtain AuNP@LDHB-POx; (4) AuNP@LDHB-POx was fixed onto a PDA-modified alumina ceramic chip and sealed to obtain a SERS substrate; (5) Synthesize AgNPs, modify them with a combination of MHA and SH-PEG, activate them with DMTMM, conjugate them with LDHB antibody and block them to obtain SERS nanotags.

[0007] Furthermore, in step (1), the concentration of HAuCl4 solution is (0.008-0.012) wt%, and the volume is (40-60) mL; the trisodium citrate solution is (0.7-0.9) mL of (0.8-1.2) wt% solution; and the average diameter of the obtained AuNPs is (35-45) nm.

[0008] Furthermore, in step (2), the amounts of MUA and SH-PEG used are (8-12) μL of (0.8-1.2) mM MUA and (9-13) μL of (0.08-0.12) mM SH-PEG, MUA:SH-PEG≈(8:1)~(10:1); the amount of AuNP solution is (0.8-1.2) mL; and the centrifugation speed is (7000-9000) rpm.

[0009] Furthermore, in step (3), the amount of DMTMM used is 1.5 to 2.0 molar equivalents relative to the surface carboxyl group; the LDHB antibody is (15-25) μL of (8-12) μg / mL solution; and the BSA blocking solution is (4-6)% BSA.

[0010] Furthermore, in step (3), the POx coupling buffer contains (1.5-2.5) mM MgCl2, (0.15-0.25) mM TPP and (8-12) μM FAD, pH 5.5-6.5, and the POx concentration is (8-12) U / mL; the volume ratio of AuNP@LDHB solution to POx-containing solution is (0.8-1.2) mL: (80-120) μL.

[0011] Furthermore, in step (4), the dopamine solution is (1.5-2.5) mg / mL, pH 8.0-9.0; and the BSA blocking solution is (4-6)%.

[0012] Furthermore, in step (5), AgNO3 is (8-10) mg, dissolved in (40-60) mL of deionized water; trisodium citrate is (0.8-1.2) mL of (0.8-1.2)% solution; and the centrifugation speed is (7500-9000) rpm.

[0013] Furthermore, in step (5), the amount of MHA and SH-PEG used is (16-20) μL of (0.8-1.2) mM MHA and (1.5-2.5) μL of (0.08-0.12) mM SH-PEG, MHA:SH-PEG≈ (8:1)~(10:1); the amount of AgNP suspension is (350-450) μL; the centrifugation speed is (7000-9000) rpm.

[0014] Furthermore, in step (5), the amount of DMTMM used is 1.5 to 2.0 molar equivalents relative to the surface carboxyl group; the LDHB antibody is (8-12) μL of (8-12) μg / mL solution; and the BSA blocking solution is (4-6)%.

[0015] This invention also provides the application of LDHB antibody and POx enzyme bifunctional AuNPs and SERS detection platform in the preparation of detection kits for early warning or auxiliary diagnosis of sudden cardiac death caused by acute coronary syndrome.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention overcomes the limitation that pyruvate and LDHB are difficult to quantify simultaneously in a single assay due to their vastly different chemical properties, large abundance differences, and different detection principles. By constructing a dual-signal SERS platform, simultaneous and interference-free quantification of the metabolite pyruvate and the protein LDHB is achieved in the same detection system, breaking the limitation of existing SERS technologies that are mostly limited to the detection of single-category biomarkers. This platform has high sensitivity, strong specificity, and is easy to operate, providing a new technical means for the early warning and auxiliary diagnosis of ACS-SCD, and helping to promote the transformation of multi-omics discoveries into clinical diagnostic products, which has important clinical application value and translational prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein: Figure 1 To synthesize TEM images of AuNPs; Figure 2 To synthesize TEM images of AgNPs; Figure 3 The particle size distribution of synthesized AuNPs; Figure 4 The particle size distribution of synthesized AgNPs; Figure 5 The results of this invention on the detection of pyruvate levels in exosomes of ACS-SCD patients and healthy controls; Figure 6 The results of this invention on the detection of LDHB levels in exosomes of ACS-SCD patients and healthy controls. Detailed Implementation

[0018] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0019] Example 1: Synthesis of AuNPs.

[0020] 50 mL of 0.01 wt% HAuCl4 solution was heated to boiling and refluxed for 10 min. Then, 0.8 mL of 1 wt% trisodium citrate solution was rapidly added with vigorous stirring. The reaction mixture was boiled for another 30 min to ensure complete reduction of HAuCl4. The resulting AuNPs were characterized by SEM and TEM, showing an average diameter of 40 nm and a narrow size distribution.

[0021] Example 2: Preparation of LDHB antibody and POx enzyme bifunctionalized AuNPs.

[0022] To co-functionalize AuNPs with LDHB antibody and POx, the surface of citrate-stabilized AuNPs (1 mL) was first modified with 11-mercaptoundecanoic acid (MUA) to introduce carboxyl groups. Specifically, 10 μL of 1 mM MUA ethanol solution and 11 μL of 0.1 mM SH-PEG solution were mixed thoroughly to make a MUA:SH-PEG ratio of approximately 9:1. The mixture was then added dropwise to 1 mL of citrate-stabilized AuNP solution under gentle stirring at room temperature in the dark. The mixture was stirred gently overnight at room temperature in the dark. After the reaction was complete, the mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of PBS. The centrifugation and washing process was repeated 1–2 times to remove excess MUA and SH-PEG. Finally, the precipitate was resuspended in 1 mL of PBS and stored at 4 °C in the dark.

[0023] Carboxylated AuNPs, which were resuspended in 1 mL PBS (pH 7.2–7.5) in the previous step, were activated by adding DMTMM (1.5–2.0 molar equivalents relative to the surface carboxyl groups, which can be dissolved in a small amount of PBS first) and gently stirring at room temperature for 30 min. Then, 20 μL of LDHB antibody (10 μg / mL) was added, and the mixture was incubated at 4 °C with shaking in the dark for 4–12 h. The solution was centrifuged at 8000 rpm for 10 min to remove unbound antibody. The precipitate was resuspended in 50 μL of 5% BSA and incubated at room temperature for 30 min to block non-specific binding. After centrifugation again (10000 rpm, 10 min), the precipitate was resuspended in 1.2 mL PBS.

[0024] Prepare a PBS buffer (pH 6.0) containing 2 mM MgCl2, 0.2 mM TPP, and 10 μM FAD to achieve a final POx concentration of 10 U / mL. Slowly add 1 mL of AuNP@LDHB solution to 100 μL of the POx-containing solution, gently vortex, and incubate at 4 °C for 24 h. Wash the resulting AuNP@LDHB-POx conjugate with PBS, centrifuge at 10,000 rpm for 10 min, and resuspend in PBS for later use.

[0025] Example 3: Preparation of functionalized SERS substrate.

[0026] Alumina ceramic chips were coated with a 2 mg / mL dopamine solution in Tris buffer (pH 8.5). The chips were immersed in the dopamine solution and incubated at room temperature for 2 h to allow spontaneous polymerization and uniform PDA deposition. After coating, the PDA-modified ceramic substrate was incubated overnight in AuNP@LDHB-POx solution at 4 °C to achieve robust immobilization through PDA interactions. The chips were then thoroughly rinsed with PBS to remove unbound nanoparticles and incubated in 5% BSA solution at room temperature for 30 min to block nonspecific sites. After washing, the resulting “Gemini” SERS substrate was air-dried and stored in a light-protected environment at 4 °C, avoiding moisture and light exposure.

[0027] Example 4: Preparation of SERS nanotags.

[0028] Dissolve 9 mg AgNO3 in 50 mL of deionized water and heat to boiling. Then add 1 mL of 1% trisodium citrate solution and reflux for 1 h. After cooling, collect AgNPs by centrifugation at 8400 rpm for 5 min and resuspend in 5 mL of deionized water.

[0029] 6-Mercaptohexanoic acid (MHA, 1 mM, 18 μL) and SH-PEG (0.1 mM, 20 μL) were mixed thoroughly to achieve an MHA:SH-PEG ratio of approximately 9:1. This mixture was then added to 400 μL of AgNP suspension and stirred gently in the dark for 2 h. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min and washed twice with PBS to remove unbound MHA and SH-PEG.

[0030] DMTMM (1.5–2.0 molar equivalents relative to the surface carboxyl groups, which can be dissolved in a small amount of PBS first) was added to AgNPs modified with complex ligands and activated by gentle stirring at room temperature for 30 min. Then, 10 μL of LDHB antibody solution (10 μg / mL in PBS) was added, and the mixture was incubated overnight at 4 °C with gentle shaking in the dark. After antibody conjugation, the mixture was centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in 30 μL of 5% BSA and incubated at room temperature for 1 h to block non-specific sites. Finally, the mixture was centrifuged (10000 rpm, 10 min), and the SERS nanotags were resuspended in PBS and stored at 4 °C for later use.

[0031] Example 5: SERS-based detection of LDHB and pyruvate.

[0032] Peripheral blood samples were collected from 36 patients diagnosed with sudden cardiac death due to acute coronary syndrome and 36 healthy controls at Sir Run Run Hospital, Nanjing Medical University. Demographic and clinical data, including age, sex, and BMI, were systematically recorded. The study protocol was approved by the Ethics Committee of Sir Run Run Hospital (Ethics Approval No.: 2021-SR-003), and all participants signed written informed consent forms. Whole blood was collected in anticoagulated vacuum blood collection tubes and centrifuged at 2000 rpm for 10 min at 4 ℃ to remove blood cells. The supernatant was then centrifuged at 3000 rpm for 20 min to obtain plasma. To separate exosomes, the plasma was first centrifuged at 10,000 g for 1 h to remove large vesicles and debris. Then, 1 mL of the supernatant was diluted with 9 mL of PBS and ultracentrifuged at 100,000 g for 2 h. Exosome precipitate (transparent, round deposits visible at the bottom of the tube) is resuspended in PBS according to the precipitate volume and stored at -80 °C for later use.

[0033] For LDHB detection, a series of dilutions of the LDHB standard solution were prepared, and 10 μL of each concentration was added to each well of the functionalized SERS chip. The chip was incubated at 37 °C for 30 min to allow LDHB to specifically bind to the AuNP functionalized surface. After incubation, the chip was thoroughly washed with TBST buffer and deionized water to remove unbound proteins. Then, 40 μL of SERS nanotags were added to each well, and the chip was incubated at 37 °C for another 30 min. Excess nanotags were removed by washing with PBS. Raman spectra of each well were acquired, and quantitative analysis was performed using a standard calibration curve derived from a known LDHB concentration.

[0034] For pyruvate detection, 10 μL of pyruvate standard solution was mixed with 40 μL of SERS nanotag and incubated with a functionalized SERS chip at 37 °C for 30 min. Raman spectra were then acquired, and the signal intensity was quantified using a standard curve that correlates SERS intensity with pyruvate concentration.

[0035] Example 6: SERS-based detection of plasma exosomes.

[0036] For exosome detection, 10 μL of exosome solution, after sonication lysis and resuspending, was added to a functionalized SERS chip and incubated at 37 °C for 30 min. After thorough washing with TBST buffer and deionized water, 40 μL of SERS nanotags were added, and the chip was incubated at 37 °C for another 30 min. The chip was then rinsed to remove excess tags, and SERS spectra were acquired. Each sample was measured three times using the same technique, and the spectral data were quantitatively analyzed using an established calibration model.

[0037] The concentrations of LDHB and pyruvate in exosome samples were quantified using an ELISA kit. Standard wells, sample wells, and blank controls were set up according to the kit instructions. A total of 72 exosome samples were analyzed to ensure accuracy and reproducibility.

[0038] For LDHB quantification, exosome lysis buffer was diluted with sample diluent, and 100 μL was added to each well. After incubation at 37 °C for 2 h, the plate was washed, and 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added. The plate was incubated at 37 °C for 1 h. After washing again, 100 μL of LMB substrate solution was added, and the plate was incubated at 37 °C in the dark for 15–20 min. The reaction was terminated by adding 100 μL of stop solution, and the optical density (OD) was measured at 450 nm. A standard curve was constructed to calculate the actual concentration of LDHB.

[0039] For pyruvate quantification, add 10 μL of exosome lysis buffer and 40 μL of sample diluent to each well, followed by 100 μL of HRP-conjugated detection antibody. Incubate at 37 °C for 1 h and wash. Then add 50 μL of substrate A and 50 μL of substrate B, and incubate at 37 °C in the dark for 10 min. After adding 50 μL of stop solution, measure the OD at 450 nm and determine the concentration according to the corresponding standard curve.

[0040] This invention uses a "Gemini" SERS sensor to detect the levels of pyruvate and LDHB in exosomes of ACS-SCD patients and healthy controls. In the presence of POx enzyme, pyruvate is catalytically oxidized to H2O2. H2O2 acts on the Raman-responsive molecule 3-mercaptophenylboronic acid (3-MPBA) on the surface of the SERS nanotag, oxidizing it to 3-hydroxythiophenol (3-HTP). The characteristic Raman peak of 3-HTP and the internal standard peak of 3-MPBA remain relatively stable. The ratio of these two peaks is used as the response signal. This ratio shows a linear relationship with the pyruvate concentration in the range of 7.5–100 μM, thus quantifying the pyruvate concentration in the sample. LDHB is a protein and is quantified using a sandwich immunoassay. The quantitative analysis results of pyruvate and LDHB based on the standard curve are shown in the table below:

[0041] Note: Pyruvate was tested using a two-tailed Student's t-test; LDHB was tested using a two-tailed Mann-Whitney U test.

[0042] This invention constructs a dual-signal SERS platform, achieving simultaneous and interference-free quantification of the metabolite pyruvate and the protein LDHB in the same detection system. This overcomes the limitations of existing SERS technologies, which are mostly limited to the detection of single-category biomarkers. The platform is highly sensitive, specific, and easy to operate, providing a new technical means for the early warning and auxiliary diagnosis of ACS-SCD. It also helps to promote the translation of multi-omics discoveries into clinical diagnostic products, possessing significant clinical application value and translational prospects.

[0043] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a bifunctional AuNPs and SERS detection platform using LDHB antibody and POx enzyme, characterized in that, Includes the following steps: (1) Synthesis of AuNPs; (2) AuNPs were modified with a combination of MUA and SH-PEG to obtain carboxylated AuNPs; (3) After activation with DMTMM, LDHB antibody was coupled and blocked, and then POx enzyme was coupled to obtain AuNP@LDHB-POx; (4) AuNP@LDHB-POx was fixed onto a PDA-modified alumina ceramic chip and sealed to obtain a SERS substrate; (5) Synthesize AgNPs, modify them with a combination of MHA and SH-PEG, activate them with DMTMM, conjugate them with LDHB antibody and block them to obtain SERS nanotags.

2. The method according to claim 1, characterized in that, In step (1), the concentration of HAuCl4 solution is (0.008-0.012) wt%, and the volume is (40-60) mL; the trisodium citrate solution is (0.7-0.9) mL of (0.8-1.2) wt% solution; the average diameter of the obtained AuNPs is (35-45) nm.

3. The method according to claim 1, characterized in that, In step (2), the amounts of MUA and SH-PEG used are (8-12) μL of (0.8-1.2) mM MUA and (9-13) μL of (0.08-0.12) mM SH-PEG, MUA:SH-PEG≈(8:1)~(10:1); the amount of AuNP solution is (0.8-1.2) mL; the centrifugation speed is (7000-9000) rpm.

4. The method according to claim 1, characterized in that, In step (3), the amount of DMTMM used is 1.5 to 2.0 molar equivalents relative to the surface carboxyl group; the LDHB antibody is (15-25) μL of (8-12) μg / mL solution; and the BSA blocking solution is (4-6)% BSA.

5. The method according to claim 1, characterized in that, In step (3), the POx coupling buffer contains (1.5-2.5) mM MgCl2, (0.15-0.25) mM TPP and (8-12) μM FAD, pH 5.5-6.5, and the POx concentration is (8-12) U / mL; the volume ratio of AuNP@LDHB solution to POx-containing solution is (0.8-1.2) mL: (80-120) μL.

6. The method according to claim 1, characterized in that, In step (4), the dopamine solution is (1.5-2.5) mg / mL with pH 8.0-9.0; the BSA blocking solution is (4-6)%.

7. The method according to claim 1, characterized in that, In step (5), AgNO3 is (8-10) mg, dissolved in (40-60) mL of deionized water; trisodium citrate is (0.8-1.2) mL of (0.8-1.2)% solution; centrifugation speed is (7500-9000) rpm.

8. The method according to claim 1, characterized in that, In step (5), the amount of MHA and SH-PEG used is (16-20) μL of (0.8-1.2) mM MHA and (1.5-2.5) μL of (0.08-0.12) mM SH-PEG, MHA:SH-PEG≈ (8:1)~(10:1); the amount of AgNP suspension is (350-450) μL; the centrifugation speed is (7000-9000) rpm.

9. The method according to claim 1, characterized in that, In step (5), the amount of DMTMM used is 1.5 to 2.0 molar equivalents relative to the surface carboxyl group; the LDHB antibody is (8-12) μL of (8-12) μg / mL solution; and the BSA blocking solution is (4-6)%.

10. Application of LDHB antibody and POx enzyme bifunctional AuNPs and SERS detection platform in the preparation of detection kits for early warning or auxiliary diagnosis of sudden cardiac death caused by acute coronary syndrome.

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