A method for detecting urine small extracellular vesicle PLA2R1 membrane protein for distinguishing membranous nephropathy

Abstract

The application discloses a urine small extracellular vesicle PLA2R1 membrane protein detection method for distinguishing membranous nephropathy, and belongs to the technical field of biomedical detection. The detection method comprises the following steps: preparing an anti-CD63 antibody surface fixed plasmonic metasurface sensing chip to directly capture small extracellular vesicles in urine; preparing an anti-PLA2R1 antibody modified biofunctionalized gold nanoparticle complex to specifically combine with the PLA2R1 membrane protein on the surface of the small extracellular vesicles; detecting the reflection spectrum of the chip after antibody fixation and gold nanoparticle combination through a spectrometer with an integrated optical fiber probe; calculating the resonance wavelength shift amount; measuring the expression level of the PLA2R1 membrane protein; and realizing accurate distinction of healthy people, membranous nephropathy gray area and confirmed patients. The non-invasive detection method is simple in operation and rapid in detection, the sensitivity and the specificity are improved by means of the gold nanoparticle signal amplification effect, and is suitable for large-scale clinical screening and early diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a method for detecting PLA2R1 membrane protein in urinary microcellular extracellular vesicles to identify membranous nephropathy, for the differentiation and screening of samples related to membranous nephropathy. Background Technology

[0002] Membranous nephropathy (MN) is an organ-specific autoimmune disease and one of the leading causes of adult nephrotic syndrome. Early and accurate identification is crucial for disease intervention and improved prognosis. Currently, the gold standard for diagnosing membranous nephropathy is renal biopsy, but this method is invasive and can cause problems such as hematuria, perirenal hematoma, and arteriovenous fistula. Therefore, non-invasive detection based on biomarkers has become an important next-generation method for detecting membranous nephropathy.

[0003] Clinical studies have shown that phospholipase A2 receptor (PLA2R) is the pathogenic target antigen in patients with primary membranous nephropathy, a disease caused by autoantibodies (aPLA2Rab) produced in the patient's body that attack the PLA2R protein on glomerular podocytes. Currently, traditional non-invasive biosensor methods for detecting membranous nephropathy are mostly based on enzyme-linked immunosorbent assay (ELISA) to detect aPLA2Rab antibodies, which has limited sensitivity. When using indirect immunofluorescence assays to detect membranous nephropathy, there is a certain rate of misdiagnosis, especially for patients in the detection gray area, where the risk of misdiagnosis is significantly increased. In such cases, invasive renal biopsy is still required as an auxiliary detection method, highlighting the limitations of this approach.

[0004] Urinary microvesicles are nanoscale vesicles secreted by cells, carrying cell-derived membrane protein markers. Among them, PLA2R1 (M-type phospholipase A2 receptor) membrane protein is a specific marker of membranous nephropathy (MRN), and its expression level is closely related to the occurrence and development of MRN. Compared to blood samples, urine samples more specifically reflect kidney damage, directly indicating pathological changes and the degree of damage, providing an ideal sample source for non-invasive detection of MRN. It should be noted that although PLA2R protein is present in the urine of MRN patients, direct detection of free PLA2R protein in urine has low sensitivity; it usually needs to be combined with other indicators such as urinary creatinine to improve accuracy. Previous studies have confirmed the presence of PLA2R protein on the surface of urinary microvesicles in MRN patients, and the expression level of this protein is closely related to the progression of MRN, providing important marker support for non-invasive detection of MRN.

[0005] In existing technologies, the detection of small extracellular vesicles in urine typically requires complex pretreatment steps such as ultracentrifugation, ultrafiltration, and enrichment. These procedures are cumbersome, time-consuming, and result in significant sample loss, making it difficult to achieve highly sensitive detection of low-abundance PLA2R1 membrane proteins. Surface plasmon resonance metasurface sensors, due to their speed, simplicity, and portability, have been widely used for the clinical diagnosis of diseases by detecting surface membrane proteins of small extracellular vesicles. Gold nanoparticles, due to their inherent localized plasmon resonance effect, are often used to amplify molecular signals and improve detection sensitivity in the detection of low-abundance biomolecules.

[0006] Currently, there is a lack of detection methods that integrate direct capture of plasmon metasurfaces, broad-spectrum recognition of small extracellular vesicles, specific recognition of PLA2R1, and signal amplification of gold nanoparticles, making it difficult to meet the clinical needs for non-invasive, rapid, highly sensitive, and highly specific sample discrimination of membranous nephropathy. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of traditional methods for detecting membranous nephropathy, such as invasiveness, low sensitivity, poor specificity, and difficulty in early screening. This invention provides a method for detecting PLA2R1 membrane protein in urinary microcellular vesicles to identify membranous nephropathy. This method has the advantages of being non-invasive, fast, accurate, and low-cost, and can be used for scientific research, early screening of membranous nephropathy, and auxiliary assessment.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A method for detecting PLA2R1 membrane protein in urinary microcellular extracellular vesicles to diagnose membranous nephropathy, using urine as the detection target, directly captures urinary microcellular extracellular vesicles based on a plasmonic metasurface sensing chip, specifically recognizes and amplifies the PLA2R1 membrane protein using gold antibody composite nanoparticles, and quantitatively identifies the PLA2R1 membrane protein by measuring the resonance wavelength shift; the specific steps of the method are as follows:

[0010] Step 1: Self-assemble 11-mercaptoundecanoic acid (MUA) monolayer on the surface of the plasmonic metasurface sensor chip;

[0011] Step 2: Activate the MUA monolayer using a mixed solution of ethyl dimethylaminopropyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS);

[0012] Step 3: Reaction with anti-CD63 antibody solution to achieve coupling, and immobilization of anti-CD63 antibody on the activated chip surface;

[0013] Step 4: Use bovine serum albumin (BSA) solution to seal non-specific sites on the chip surface, and detect and record the initial resonance wavelength λ0;

[0014] Step 5: Immerse the sealed chip in the urine sample to be tested for incubation, and directly capture small extracellular vesicles in the urine;

[0015] Step 6: Preparation of gold-labeled antibody-nanoparticles modified with anti-PLA2R1 antibody;

[0016] Step 7: Incubate the gold-labeled antibody-nanoparticle composite with the chip after capturing small extracellular vesicles to allow it to specifically bind to the PLA2R1 membrane protein on the surface of the small extracellular vesicles, and detect and record the final resonance wavelength λ2.

[0017] Step 8: Calculate the resonance wavelength shift to measure the shift caused by PLA2R1 membrane protein: Δλ = λ2-λ0. Based on the magnitude of Δλ, the samples related to membranous nephropathy are distinguished from healthy control samples.

[0018] Furthermore, in step one, the plasmonic metasurface sensing chip comprises a substrate layer, a periodic nanostructured metal layer, and a biofunctionalized layer; the substrate layer is a silicon wafer or a glass wafer, preferably a silicon wafer; the periodic nanostructured metal layer is gold, silver, or aluminum, preferably gold; the periodic nanostructure is a nanopore, nanosphere, or nanopillar, preferably a quadrilateral nanopore; the chip size is 3 mm × 3 mm, the nanopore diameter is 120–250 nm, the pore depth is 150–300 nm, the period is 500 nm, and the metal layer thickness is 100–200 nm; preferably, the nanopore diameter is 130 nm, the pore depth is 275 nm, the period is 500 nm, and the metal layer thickness is 150 nm.

[0019] Furthermore, in step one, the specific steps for the self-assembly of the MUA monolayer on the surface of the sensing chip can be as follows: prepare a 10 mmol / L MUA ethanol solution, place the plasmonic metasurface in the solution and react for 5 hours to form a self-assembled film, then aspirate the solution and thoroughly rinse the metasurface with a phosphate-buffered saline (PBS) solution at pH=7.4 to remove residual solution.

[0020] Furthermore, in step two, the specific steps for activating the MUA monolayer can be as follows: prepare a mixed solution of 400 mmol / L EDC and 100 mmol / L NHS, soak the metasurface in this solution for more than 40 minutes to activate the MUA, and then rinse it again with PBS solution to remove residual solution.

[0021] Furthermore, in step three, the specific steps for the conjugated antibody can be as follows: the supersurface from step two is immersed in a 25 μg / mL anti-CD63 solution for 1 h, so that the activated MUA of the plasmon supersurface binds to anti-PLA2R1.

[0022] Furthermore, in step four, the specific steps for blocking the unbound antibody sites can be as follows: add a BSA solution with a concentration of 50 μg / mL to the plasmonic metasurface in step three and react for 30 min to block the unbound antibody MUA sites, measure its reflectance spectrum and record the resonance trough of the spectrum.

[0023] Furthermore, in step five, the small extracellular vesicles in the urine sample do not require ultracentrifugation, ultrafiltration, or enrichment and purification treatment and can be directly used for capture.

[0024] Furthermore, in step five, the chip is incubated in the urine sample for 30–50 minutes.

[0025] Furthermore, in step six, the pH of the gold nanoparticle solution is adjusted by potassium carbonate (K2CO3) before being coupled with the anti-PLA2R1 antibody.

[0026] Further, in step seven, the gold-antibody composite nanoparticles are biofunctionalized as follows: potassium carbonate (K₂CO₃) is added to 1 mL of gold nanoparticle solution to adjust the pH to 7.5–8.5, then 50 μg / mL of anti-PLA2R1 antibody is added. The solution is allowed to stand for 25–30 min to allow the anti-PLA2R1 antibody to fully bind with the gold nanoparticles, forming the gold-antibody composite nanoparticle solution. The gold nanoparticles can have a particle size of 30–50 nm, and the gold nanoparticle solution is prepared from chloroauric acid at a concentration of 0.04%.

[0027] Furthermore, in step seven, the contact incubation time is 30–90 min, preferably 40–60 min; the final resonance wavelength is detected and recorded by detecting the reflection spectrum of the sensor chip after antibody fixation in step S1 and after binding with gold nanoparticles in step S4, and recording its resonance trough wavelength.

[0028] Furthermore, the resonant wavelength offset Δλ is positively correlated with the expression level of PLA2R1 membrane protein on the surface of small extracellular vesicles, which can distinguish between healthy samples, borderline gray zone samples, and samples related to membranous nephropathy. Specifically, Δλ≤3.52 nm indicates healthy individuals, 3.52 nm<Δλ<6.09 nm indicates the gray zone of membranous nephropathy, and Δλ≥6.09 nm indicates samples related to membranous nephropathy.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] This invention provides a method for detecting PLA2R1 membrane protein in urinary microcellular extracellular vesicles to differentiate membranous nephropathy. The method utilizes a plasmonic metasurface to capture microcellular extracellular vesicles in urine, and then employs gold antibody-based composite nanoparticles to measure the specific PLA2R1 membrane protein on the surface of these vesicles. This achieves the separation and purification of microcellular extracellular vesicles in urine and can non-destructively and with high sensitivity distinguish between patients with membranous nephropathy and healthy controls. A two-sample t-test was used to compare the results among healthy individuals, the gray zone of disease, and the disease group, with P < 0.0001. This method has broad application prospects in practical applications such as disease screening. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the plasmonic metasurface sensing chip of the present invention;

[0032] Figure 2 This is a SEM image of the plasmonic metasurface chip of the present invention;

[0033] Figure 3 This is a TEM image of small extracellular vesicles in membranous nephropathy according to the present invention;

[0034] Figure 4 This is a particle size distribution diagram of small extracellular vesicles in membranous nephropathy according to the present invention;

[0035] Figure 5 This is a schematic diagram of the process for measuring PLA2R1 membrane protein on the surface of extracellular vesicles in urine for the detection of membranous nephropathy according to the present invention.

[0036] Figure 6 This invention provides spectral shifts of different functionalization processes of PLA2R1 membrane protein on the surface of extracellular vesicles in urine for the detection of membranous nephropathy.

[0037] Figure 7 This is an experimental comparison of urine samples from patients with membranous nephropathy and healthy individuals, used in the detection of membranous nephropathy by measuring the PLA2R1 membrane protein on the surface of extracellular vesicles in urine according to the present invention.

[0038] Figure 8 A comparative experimental image showing the direct detection of anti-CD63 in the urine of patients with membranous nephropathy and healthy individuals;

[0039] Figure 9 A comparative experimental image showing the direct detection of anti-PLA2R1 in the urine of patients with membranous nephropathy and healthy individuals;

[0040] Figure 10 This is a comparative experimental image showing the detection of urine samples from patients with membranous nephropathy and healthy individuals using gold antibody composite nanoparticles coated with anti-CD63 and anti-PLA2R1. Detailed Implementation

[0041] To more clearly illustrate the embodiments of the present invention, a detailed and complete description will be provided below with reference to the accompanying drawings used in the embodiments. The described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] This invention discloses a method for detecting PLA2R1 membrane protein in urinary microcellular vesicles to identify membranous nephropathy, which solves the problems of traditional methods for detecting membranous nephropathy being invasive, having low sensitivity, and poor specificity, and enables early screening of membranous nephropathy in clinical practice.

[0043] The biological reagents used in this invention include: phosphate buffer (PBS), 11-mercaptoundecanoic acid solution (MUA), N-hydroxysuccinimide (NHS), ethyldimethylaminopropylcarbodiimide (EDC), bovine serum albumin (BSA), PLA2R1 antibody (anti-PLA2R1), and CD63 antibody (anti-CD63).

[0044] Reference Figure 1 The detection system of this invention includes a plasmon metasurface sensing chip 1, a capture antibody 2, small extracellular vesicles 3, PLA2R1 membrane protein 4, gold nanoparticles 5, and an anti-PLA2R1 antibody 6. The plasmon metasurface sensing chip 1 includes a substrate layer, a periodic nanostructured metal layer, and a biofunctionalized layer, forming a stable sensing substrate. (Refer to...) Figure 2 Specifically, the image shows a SEM image of the plasmonic metasurface chip of this invention. The substrate layer of the plasmonic metasurface sensing chip is preferably a silicon wafer (purity ≥99.9%), and the periodic nanostructured metal layer is preferably a gold nanostructure. The structure of the periodic nanostructured gold layer is preferably quadrilateral nanopores, which can generate a strong localized surface plasmon effect and improve detection sensitivity. The size of the plasmonic metasurface sensing chip is preferably 3 mm × 3 mm, the nanopore diameter is preferably 130 nm, the pore depth is preferably 275 nm, the period is 500 nm, and the metal layer thickness is preferably about 150 nm. The capture antibody 2 can be any one of carbohydrate antigen 9 (anti-CD9), anti-carbohydrate antigen 63 (anti-CD63), and anti-carbohydrate antigen 81 (anti-CD81), all of which are antibodies specific to the surface of small extracellular vesicles, with anti-CD63 being preferred.

[0045] Reference Figure 3This is a TEM image of extracellular vesicles in membranous nephropathy according to the present invention. The extracellular vesicle 3 was directly captured from urine without additional separation processes such as ultracentrifugation, thus preserving the integrity of the small extracellular vesicles to the greatest extent possible. Its particle size is approximately 100 nm. (See reference...) Figure 4 The image shows the particle size distribution of extracellular vesicles in membranous nephropathy cells, as described in this invention. Dynamic light scattering was used for detection, and the results indicate that the concentration of extracellular vesicles in urine from membranous nephropathy cells is approximately 1 × 10⁻⁶. 10 The concentration is measured in particles per milliliter. PLA2R1 membrane protein 4 is an extracellular vesicle membrane surface protein and a specific protein of membranous nephropathy. Its expression level is positively correlated with the severity of membranous nephropathy. The expression level of PLA2R1 membrane protein on the surface of small extracellular vesicles in the urine of healthy individuals is extremely low, while the expression level is significantly increased in patients with membranous nephropathy (including early-stage gray zone patients), making it a core target for distinguishing membranous nephropathy from healthy individuals. The gold nanoparticles 5 and the anti-PLA2R1 antibody 6 are combined through electrostatic interactions to form gold-antibody composite nanoparticles. These composite nanoparticles can specifically recognize and bind to the PLA2R1 membrane protein on the surface of extracellular vesicles, used to measure the content of small extracellular vesicle membrane surface proteins. Simultaneously, utilizing the signal amplification effect of localized surface plasmon resonance of gold nanoparticles, the detection sensitivity is significantly improved, achieving accurate measurement of PLA2R1 membrane protein.

[0046] Reference Figure 5 This is a schematic diagram of the process for measuring the PLA2R1 membrane protein on the surface of small extracellular vesicles in urine for the detection of membranous nephropathy according to the present invention. It mainly includes two steps: capturing small extracellular vesicles in the urine of patients with membranous nephropathy using a plasmonic metasurface chip, and coupling gold antibody composite particles with the specific PLA2R1 membrane protein on the surface of small extracellular vesicles. The specific steps include the following (all operations are performed at room temperature and under sterile conditions):

[0047] Step 1: Self-assembly of MUA monolayer on sensor chip surface: Prepare a 10 mmol / L MUA ethanol solution (using anhydrous ethanol as solvent), place the plasmonic metasurface in this solution and react for 5 h, so that MUA molecules form stable Au-S covalent bonds with the gold nanostructure surface through thiol groups (-SH), forming a self-assembled MUA film. After the MUA ethanol solution is removed, the metasurface is repeatedly rinsed 3 times with PBS solution (pH=7.4), each time for 1 min, to thoroughly remove residual MUA solution and unbound MUA molecules from the chip surface. After drying with nitrogen, it is ready for use.

[0048] Step 2: Activation of MUA monolayer: Prepare a mixed solution of 400 mmol / L EDC and 100 mmol / L NHS. Soak the metasurface chip treated in Step 1 in this mixed solution for 40 min to activate the carboxyl groups (-COOH) at the ends of the MUA molecules, forming active ester groups (-CO-NHS). After incubation, rinse the chip three times with PBS solution for 1 min each time to remove residual EDC-NHS mixed solution, and then dry it with nitrogen.

[0049] Step 3: Antibody conjugation: The metasurface chip activated in Step 2 is immersed in a 25 μg / mL anti-CD63 solution for 1 hour to allow the activated MUA molecules of the plasmon metasurface to bind with the anti-CD63 antibody, thereby achieving stable conjugation of the anti-CD63 antibody on the chip surface.

[0050] Step 4: Blocking sites where no antibody binds: Add a 50 μg / mL BSA solution to the plasmon metasurface chip treated in Step 3 and react for 30 min. Use BSA molecules to block the MUA active sites on the chip surface where no anti-CD63 antibody is attached. Use a spectrometer with an integrated fiber optic probe to measure the reflectance spectrum and record the wavelength of the resonance trough of the spectrum (denoted as λ0).

[0051] Step 5: Capturing small extracellular vesicles in urine: Collect fresh urine samples from patients with membranous nephropathy and healthy individuals and store them rapidly at -80°C. o The C-type refrigerator prevents protein degradation. During the experiment, the chips were rapidly thawed at room temperature without centrifugation. The metasurface chips processed in step four were directly immersed in urine samples from patients and healthy individuals for 30 min. The anti-CD63 antibody coupled to the chip surface specifically binds to the CD63 membrane protein on the surface of small extracellular vesicles in the urine, capturing the small extracellular vesicles on the metasurface chip. The chip was washed three times with PBS buffer to remove unbound urine impurities and free substances, dried with nitrogen, and its reflectance spectrum was measured and the wavelength of the resonance trough of the spectrum (denoted as λ1) was recorded.

[0052] Step Six: Biofunctionalization of Gold-Antibody Composite Nanoparticles: Add an appropriate amount of potassium carbonate (K2CO3) to 1 mL of gold nanoparticle solution to adjust the pH of the solution to 7.5-8.5. Add 50 μg / mL of anti-PLA2R1 antibody solution to the gold nanoparticle solution and let it stand for 30 min. This allows the anti-PLA2R1 antibody to fully bind to the surface of the gold nanoparticles through electrostatic interaction, forming a gold-antibody composite nanoparticle solution.

[0053] Step 7: Coupling of the gold antibody-antibody composite nanoparticles to the PLA2R1 membrane protein: Add 100 μL of the gold antibody-antibody composite nanoparticle solution prepared in Step 6 to the metasurface chip treated in Step 5 and react for 40 min to fully couple the anti-PLA2R1 antibody in the gold antibody-antibody composite nanoparticles with the PLA2R1 membrane protein. Rinse the chip three times with PBS buffer to remove unbound gold antibody-antibody composite nanoparticles, dry it with nitrogen, measure its reflectance spectrum and record the wavelength of the resonance trough of the spectrum (denoted as λ2).

[0054] Step 8: Calculate the resonance wavelength shift to measure the PLA2R1 membrane protein: Subtract the resonance trough wavelength λ2 of the spectrum in Step 7 from the resonance trough wavelength λ0 of the spectrum in Step 4. This is the resonance wavelength shift caused by the PLA2R1 membrane protein (Δλ=λ2-λ0). The magnitude of the shift is used to distinguish between patients with membranous nephropathy, patients in the disease gray zone, and healthy individuals. Among them, healthy individuals have Δλ≤3.52 nm, patients in the disease gray zone have 3.52 nm<Δλ<6.09 nm, and patients with membranous nephropathy have Δλ≥6.09 nm, thus achieving accurate differentiation among the three groups.

[0055] Reference Figure 6 This is a spectral shift diagram showing the different functionalization processes of PLA2R1 membrane protein on the surface of urinary microcellular extracellular vesicles for the detection of membranous nephropathy, according to the present invention. The present invention is based on... Figure 5 The detection steps shown involved measuring the reflectance spectral resonance wavelengths of different functionalization processes on the chip (blank chip, anti-CD63 conjugation, capture of small extracellular vesicles, and conjugation of gold antibody composite nanoparticles). Figure 6 As can be seen, the blank plasmonic metasurface chip resonates at a wavelength of 731.5 nm. When the anti-CD63 antibody is attached to the metasurface, the adsorption of antibody molecules causes a change in the refractive index of the chip surface, resulting in a redshift of the resonance wavelength to 736.4 nm. When small extracellular vesicles in urine are captured onto the chip surface, the adsorption of these vesicles further alters the refractive index of the chip surface, causing the resonance wavelength to redshift to 740.4 nm. Furthermore, after coupling with gold antibody-associated nanoparticles, the signal amplification effect of the gold nanoparticles further shifts the resonance wavelength to 743.64 nm. These results demonstrate that the plasmonic metasurface exhibits high detection sensitivity, and every minute change in environmental refractive index caused by biomolecule linkages is effectively addressed through a resonance shift response. This also indicates the feasibility of the biosensing method of this invention for screening membranous nephropathy.

[0056] Reference Figure 7This image shows a comparative experimental study of urine samples from patients with membranous nephropathy and healthy individuals, using the measurement of PLA2R1 membrane protein on the surface of urinary microvesicles for the detection of membranous nephropathy. Five patients diagnosed with membranous nephropathy (disease duration 1-3 years) and five healthy volunteers (without kidney disease or autoimmune diseases) were selected for the experiment. The experiments were conducted according to the aforementioned detection steps, and the resonance spectral shifts of the two groups were compared. The results showed that when anti-CD63 was used to directly capture microvesicles in the urine of both patients and healthy individuals, the shift caused by healthy individuals was approximately 1 nm, while the shift caused by patients was approximately 3 nm. Although the shift caused by patients was higher than that of healthy individuals, the difference was small and difficult to distinguish accurately. When gold antibody-associated nanoparticles were used to connect to the PLA2R1 membrane protein on the surface of microvesicles, the shift caused by healthy individuals was approximately 2 nm, while the shift caused by patients increased to approximately 8 nm. The amplification effect of the composite nanoparticles significantly increased the distinguishability of the spectral shifts between patients and healthy individuals, indicating that this invention has higher sensitivity in diagnosis.

[0057] Reference Figure 8 To compare the results of direct detection of urine samples from patients with membranous nephropathy and healthy individuals using anti-CD63, a two-sample t-test was used to analyze the resonance shift between the patient and healthy groups. The results showed that when using a plasmonic metasurface coupled with anti-CD63 to directly detect small extracellular vesicles in urine, the probability of detecting the resonance wavelength shift between healthy individuals and patients was P < 0.0001. (P < 0.0001) can clearly distinguish patients. However, for patients in the gray zone, the offset is small compared to healthy individuals, and they cannot be effectively distinguished, indicating that the detection sensitivity of the single anti-CD63 antibody capture method is low.

[0058] Reference Figure 9 This image shows a comparative experiment comparing the direct detection of small extracellular vesicles in urine from patients with membranous nephropathy and healthy individuals using anti-PLA2R1. The experiment employed a plasmonic metasurface coupled with anti-PLA2R1 to directly detect small extracellular vesicles in urine. A two-sample t-test was used to compare the resonance wavelength shift between the healthy and patient groups. The results showed that the probability of a significant difference in resonance wavelength shift between healthy and patient groups was P < 0.0001. Even with a p-value < 0.0001, patients can be clearly distinguished. Furthermore, for patients in the gray zone, effective differentiation is achieved due to the specific recognition of PLA2R1 membrane proteins by anti-PLA2R1, indicating that anti-PLA2R1 is specific for PLA2R1 membrane proteins on the surface of extracellular vesicles. However, in the single PLA2R1 capture method, the differentiation between the gray zone and patients is low, and further improvements in detection sensitivity are needed to enhance its detection performance.

[0059] Reference Figure 10 This image shows a comparative study of urine samples from patients with membranous nephropathy and healthy individuals using anti-CD63 and anti-PLA2R1-coated gold antibody composite nanoparticles. This detection method combines the high-efficiency capture capability of anti-CD63 with the connection between anti-PLA2R1-coated gold antibody composite nanoparticles and the PLA2R1 membrane protein on the surface of small extracellular vesicles. Utilizing the signal amplification effect of the gold nanoparticles, experimental results show that the resonance shift in healthy individuals is approximately 2 nm, the shift in patients in the gray zone of disease is approximately 4.5 nm, and the resonance shift in patients increases to 8 nm. The significant difference in shift among the three groups allows for accurate differentiation. This method enhances the signal induced by the disease-specific membrane protein PLA2R1 while avoiding other background interference. Two-sample t-test results show statistically significant differences between the healthy group and the gray zone group, and between the gray zone group and the disease group (P<0.0001), effectively increasing detection sensitivity and achieving effective differentiation between healthy individuals, the gray zone of disease, and the disease group.

[0060] This invention utilizes plasmonic metasurfaces to capture extracellular vesicles in urine, and then employs gold antibody-based composite nanoparticles to measure the specific PLA2R1 membrane protein on the surface of these small extracellular vesicles. This achieves the separation and purification of small extracellular vesicles in urine, and can non-destructively and with high sensitivity distinguish between patients with membranous nephropathy and healthy controls. The detection method is highly accurate and simple to operate. A two-sample t-test was used to compare experiments between healthy individuals, the gray zone of disease, and the disease group, with P < 0.0001, demonstrating good repeatability. It requires no large-scale detection equipment and has broad application prospects in practical applications such as disease screening for membranous nephropathy.

[0061] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for detecting PLA2R1 membrane protein in urinary microcellular extracellular vesicles to diagnose membranous nephropathy, characterized in that, Includes the following steps: S1. A plasmonic metasurface sensing chip is provided, comprising a silicon substrate, a gold periodic nanopore array metal layer on the substrate, and a biofunctionalized layer on the metal layer; an anti-CD63 antibody is used as the first antibody and is immobilized on the biofunctionalized layer by a carboxyl-amino coupling method for specifically capturing small extracellular vesicles in urine samples. S2. Incubate the untreated urine sample with the sensor chip for 20-60 min to allow small extracellular vesicles in the urine to be specifically captured on the chip surface by the first antibody. S3. A biofunctionalized gold nanoparticle complex is prepared by incubating and conjugating an anti-PLA2R1 antibody with a gold nanoparticle solution after adjusting the pH to 7.5-8.

5. The anti-PLA2R1 antibody, as a second antibody, can specifically bind to the phospholipase A2 receptor 1 (PLA2R1) membrane protein on the surface of small extracellular vesicles. S4. The gold nanoparticle complex is incubated with a sensing chip containing small extracellular vesicles for 30-90 minutes, so that the gold nanoparticle complex binds to the PLA2R1 membrane protein on the surface of the small extracellular vesicles through the anti-PLA2R1 antibody. S5. Detect the reflection spectra of the sensor chip after antibody fixation in step S1 and after binding with gold nanoparticles in step S4, and record the resonance trough wavelengths of the two detections as λ0 and λ2, respectively. S6. Calculate the difference in resonance trough wavelength between step S4 and step S1, Δλ = λ2 - λ0. Based on the redshift of this wavelength, measure the level of PLA2R1 membrane protein on the surface of extracellular vesicles in urine to diagnose membranous nephropathy. Where: Δλ ≤ 3.52 nm is healthy, 3.52 nm < Δλ < 6.09 nm is the gray zone of membranous nephropathy, and Δλ ≥ 6.09 nm is a patient with membranous nephropathy.

2. The method according to claim 1, characterized in that, The periodic nanopore array is a quadrilateral array with nanopore diameters of 120–250 nm, pore depths of 150–300 nm, a period of 500 nm, and a metal layer thickness of 100–200 nm.

3. The method according to claim 1, characterized in that, In step S1, the carboxyl-amine coupling method specifically includes: first, self-assembling an 11-mercaptoundecanoic acid monolayer on the surface of the sensor chip; then, activating the carboxyl groups with a solution of ethyldimethylaminopropylcarbodiimide and N-hydroxysuccinimide; then, reacting with the anti-CD63 antibody solution at a concentration of 10–50 μg / mL to achieve coupling; and finally, blocking unbound sites with bovine serum albumin solution.

4. The method according to claim 1, characterized in that, In step S2, the contact incubation time is 30 to 50 minutes.

5. The method according to claim 1, characterized in that, In step S3, the pH value of the gold nanoparticle solution is adjusted using potassium carbonate solution; the gold nanoparticle solution is prepared from chloroauric acid with a concentration of 0.04%, and the final concentration of the anti-PLA2R1 antibody is 50 μg / mL.

6. The method according to claim 1, characterized in that, In step S4, the contact incubation time is 40 to 60 minutes.

7. A test kit for implementing the method according to any one of claims 1-6, characterized in that, Include: (a) A plasmonic metasurface sensing chip prefabricated by the method according to claim 1, with anti-CD63 antibody immobilized on its surface; (b) A gold nanoparticle complex suspension prepared by the method according to claim 1, with its surface modified with anti-PLA2R1 antibody; (c) Auxiliary reagents: phosphate buffer at pH 7.4, 50 μg / mL bovine serum albumin blocking solution.

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