Exosome detection method for Alzheimer's disease

Abstract

The invention discloses an exosome detection method for Alzheimer's disease, and relates to exosome separation and extraction. The method comprises the following steps: modifying the surface of a biological sensing chip, and forming an S-Au bond by utilizing MUA and a gold film to construct a self-assembled monomolecular layer rich in carboxyl; activating carboxyl by using an EDC-NHS solution, anchoring an antibody capable of recognizing exosome membrane protein secreted by brain-derived neuronal cells, and closing non-specific sites by using bovine serum albumin; a serum sample and a chip are incubated, exosomes are separated through a glycine buffer solution with the pH value being 2.0, the pH value is adjusted to be neutral, and a lysis solution containing 1% of Triton X-100 is added for lysis; and finally, mounting an integrated optical fiber probe to measure a reflection spectrum and monitor resonance wavelength, and realizing quantitative detection by combining multi-marker conjoint analysis. The exosome can be enriched and purified with high purity and high specificity, the process is simple, the detection sensitivity is high, and the AD detection accuracy is remarkably improved. The process is simple, complex operations such as ultracentrifugation are avoided, and the detection time is shortened.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome separation and extraction, and specifically relates to a method for detecting exosomes in Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a major neurodegenerative disorder facing the world today. Because its pathogenesis remains unclear, effective clinical treatments are still lacking. Therefore, early detection, early detection, and early intervention are crucial strategies for slowing progression.

[0003] Exosomes are small extracellular vesicles ranging in diameter from 30 to 150 nm, with a bilayer lipid membrane. Exosomes are produced by virtually all cell types under both physiological and pathological conditions and are widely distributed in readily accessible bodily fluids such as blood, saliva, breast milk, and urine. For many years, exosomes were considered "junk" waste generated during cellular maturation. However, with the recent isolation of diverse proteins, lipids, and genetic material (such as miRNAs, mRNAs, DNA molecules, and long noncoding RNAs) from different exosome types, their crucial roles in cellular communication and epigenetic regulation have been recognized. Importantly, under both pathological and physiological conditions, the contents of exosomes are meticulously regulated by their parent cells to transmit information from them to other cells to achieve specific functions. Conversely, the functional status of parent cells can be estimated by analyzing their exosome contents, laying the foundation for exosome-based diagnostics, particularly noninvasive liquid biopsies.

[0004] Currently, commonly used diagnostic methods for Alzheimer's disease include clinical and neuropsychological assessments, cerebrospinal fluid testing, PET imaging, and blood testing. However, clinical and neuropsychological assessments are highly subjective, making early diagnosis difficult; cerebrospinal fluid testing is highly invasive and has low patient acceptance; PET imaging is expensive, hindering widespread clinical application; and blood testing has limited sensitivity, and correlation with brain pathology has not yet been established. Exosome isolation and purification methods include ultracentrifugation (UC), polymer precipitation, ultrafiltration, size exclusion chromatography, and immunoaffinity. Ultracentrifugation (UC) is the gold standard for exosome isolation and purification, but it is time-consuming, has low yield, lacks specificity, and relies on expensive precision equipment. Furthermore, repeated ultracentrifugation may damage the structural integrity of Alzheimer's exosomes, hindering their downstream analytical applications. Other isolation techniques based on the physical properties of Alzheimer's exosomes, such as size, density, and solubility, also suffer from low yield, low purity, time-consuming procedures, and lack of specificity. Therefore, there is an urgent need to establish a new method that can achieve high-purity and high-specificity rapid enrichment of Alzheimer's exosomes and content detection and analysis. Summary of the Invention

[0005] The purpose of the present invention is to address the problems of poor specificity, low sensitivity and complex procedures in the existing AD exosome detection technology. This paper provides an Alzheimer's disease exosome detection method based on a functionalized biosensor chip. By targeting and capturing exosomes and quantitatively analyzing the AD-specific markers they carry, rapid and accurate disease diagnosis can be achieved.

[0006] The present invention provides a method for detecting exosomes in Alzheimer's disease, comprising the following steps:

[0007] 1) Modification of the biosensor chip surface: Immersing the biosensor chip in a mercaptoundecanoic acid (MUA) solution allows the thiol groups to form S-Au bonds with the gold film, thereby constructing a carboxyl (-COOH)-rich self-assembled monolayer (SAMs) on the chip surface.

[0008] 2) Activation of carboxyl groups on the chip surface: Activation of carboxyl groups on the surface of the biosensor chip using EDC-NHS solution;

[0009] 3) Anchoring Antibody: Adding an antibody solution to the activated chip surface to immobilize the antibody on the surface of the biosensor chip, wherein the antibody is an antibody that can recognize exosome membrane proteins secreted by brain-derived neuronal cells;

[0010] 4) Blocking nonspecific sites: Use bovine serum albumin (BSA) solution to block the active sites on the biosensor chip that are not bound to the antibody;

[0011] 5) Capturing exosomes: The serum sample containing exosomes was incubated with the biosensor chip from step 4), and the exosomes were separated using a pH 2.0 glycine buffer. The pH was adjusted to neutral by adding NaOH solution to obtain an exosome extract.

[0012] 6) Lysis of exosomes: Add lysis buffer containing 1% Triton X-100 and protease inhibitor PMSF to lyse and obtain exosome content solution;

[0013] 7) Exosome detection: Add a biofunctionalized biosensor chip to the exosome content solution, install an integrated fiber optic probe, measure the reflection spectrum vertically, and monitor and record the resonance wavelength at this time.

[0014] In step 1), the surface of the biosensor chip is modified with MUA to form an S-Au bond, and a 5-50mM mercaptoundecanoic acid (MUA) solution is used for self-assembly on the surface of the gold film substrate chip. The chip is completely immersed in the MUA solution and placed at 4°C for 3 hours. The thiol (-SH) reacts with the gold film to form a stable S-Au bond, thereby constructing a self-assembled monolayer (SAMs) rich in carboxyl (-COOH).

[0015] In step 2), the EDC-NHS solution is a mixture of 0.4 M EDC (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 0.1 M NHS (N-hydroxysuccinimide). Deionized water is added and stirred until completely dissolved. The total volume is 1 mL. The mixture is left to stand for activation at room temperature for 30 minutes.

[0016] In step 3), the antibodies in the antibody solution are anti-exosome marker antibodies, including anti-NMDAR2A, anti-Aβ40, anti-Aβ42, anti-tau or anti-p-tau181 antibodies that can recognize exosome membrane proteins secreted by brain-derived neuronal cells. The concentration of the antibody solution is 12.5 μg / mL.

[0017] In step 4), the BSA blocks nonspecific sites, and the unreacted active sites can be blocked by a 5% bovine serum albumin (BSA) solution to eliminate the nonspecific adsorption interference of miscellaneous proteins in the serum.

[0018] In step 5), the incubation can be carried out at room temperature for 0.5-2 hours; exosomes are separated using a pH 2.0 glycine buffer. A 0.1-1M glycine buffer with a pH of 2.0 can be used to separate exosomes. This buffer destroys the ionic bonds between the antibody and exosomes, allowing the exosomes to be peeled off the chip surface; the addition of a NaOH solution to adjust the pH to neutral can be performed by preparing a 1M NaOH solution and adding the NaOH solution to the solution after the exosomes are separated to adjust the pH of the solution to 7.0-7.4, thereby stabilizing the exosome membrane structure and obtaining an exosome extract.

[0019] In step 6), the lysis solution is RIPA buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, as well as various inhibitors such as sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin, and 1 nM protease inhibitor (PMSF); the lysis can be performed in a 4°C refrigerator for 10-15 minutes to obtain an exosome content solution.

[0020] In step 7), an integrated fiber optic probe is installed, which has a wavelength detection range of 400-1000nm, an incident angle of 90°, and a resolution of 0.1nm. The vertical reflection spectrum is measured by placing the probe vertically on the chip, vertically measuring the reflection spectrum of the chip surface, and monitoring and recording the resonant wavelength in real time.

[0021] A concentration-resonance wavelength shift calibration curve was established based on different concentrations of standards and the corresponding resonance wavelength shift. Combined with the joint analysis of multiple markers such as anti-NMDAR2A, anti-Aβ40, anti-Aβ42, anti-tau and anti-p-tau181, the diagnostic efficacy was calculated using the ROC curve to achieve quantitative analysis of exosomes and detection of Alzheimer's disease.

[0022] Compared with the prior art, the present invention has the following outstanding technical effects and advantages:

[0023] By functionalizing the surface of a biosensor chip and using antibodies to specifically capture exosomes, this method achieves high-purity and highly specific enrichment and purification of Alzheimer's disease exosomes, as well as measurement and analysis of exosome contents. This method effectively differentiates between AD patients and healthy individuals (with a combined diagnostic area under the curve (AUC) of 0.99), compared to the AUC of approximately 0.7 achieved by traditional clinical diagnostics. This method significantly improves upon this and enables precise screening for AD. Antibody anchoring enables specific recognition and capture of exosomes carrying AD-specific markers, specifically targeting specific proteins within exosomes (Aβ40, Aβ42, tau, and p-tau181), reducing nonspecific binding. The biosensor chip enriches brain-derived neuronal exosomes and analyzes the specific binding of AD-related proteins within the exosomes, resulting in more specific and accurate AD detection results. Accurate measurement of the resonant wavelength enables detection of low concentrations of exosomes and their contents, improving detection sensitivity. The present invention has a simple process, avoids complex operations such as ultracentrifugation, shortens detection time, and reduces dependence on expensive instruments and equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of a method for isolating and enriching AD exosomes provided in an embodiment of the present invention;

[0025] Figure 2 This is a scanning electron microscope image of exosomes captured by the metasurface chip provided in an embodiment of the present invention;

[0026] Figure 3 This is a specific experimental curve diagram of the exosome protein Aβ40 in a specific embodiment of the present invention;

[0027] Figure 4 This is a graph showing the detection results of protein Aβ40 in the exosomes of the sample to be tested in a specific embodiment of the present invention.

[0028] Figure 5 This is a ROC curve diagram of the metasurface chip used in a specific embodiment of the present invention to measure the resonance wavelength shift of Aβ40, Aβ42, tau, and p-tau181 for distinguishing AD patients from healthy subjects, verifying the diagnostic efficacy of the method (AUC = 0.997); DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention. It should also be noted that, for ease of description, only portions relevant to the present invention are shown in the accompanying drawings.

[0030] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention. It should be noted that similar reference numerals and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0031] An embodiment of the present invention provides a method for detecting exosomes in Alzheimer's disease, comprising the following steps:

[0032] 1) Preparation of an exosome capture chip: The surface of a biosensor chip is modified with mercaptoundecanoic acid (MUA) to form an S-Au bond. The carboxyl groups on the surface of the biosensor chip are activated with an EDC-NHS solution, and then an antibody solution is added to anchor the antibody on the biosensor chip surface. Nonspecific sites are blocked with bovine serum albumin (BSA) to obtain a functionalized biosensor chip. The surface of the biosensor chip is modified with MUA to form an S-Au bond. A 5-50 mM mercaptoundecanoic acid (MUA) solution is used for self-assembly on the surface of a gold film substrate chip. Stable S-Au bonds are formed between the thiol (-SH) groups and the gold film, thereby constructing a molecular layer rich in carboxyl groups (-COOH). The EDC-NHS solution is a mixture of 0.4 M EDC and 0.1 M NHS, and the activation time is 30 minutes. The antibody in the antibody solution is an anti-exosome marker antibody, including anti-NMDAR2A or anti-Aβ40. Or anti-Aβ42 or anti-tau or anti-p-tau181 antibodies, the concentration of the antibody solution is 12.5ug / mL; the BSA blocks nonspecific sites, and unreacted active sites can be blocked by a 5% bovine serum albumin (BSA) solution to eliminate nonspecific adsorption interference of miscellaneous proteins in the serum.

[0033] 2) Build a detection platform: Install an integrated fiber optic probe, place a functionalized biosensor chip in a deionized water solution, and vertically position the probe on the chip. Measure the chip surface reflection spectrum vertically and record the resonance trough wavelength 1. The integrated fiber optic probe has a wavelength detection range of 400-1000 nm, an incident angle of 90°, and a resolution of 0.1 nm. Monitor and record the resonance wavelength at this time.

[0034] 3) Exosome Capture: The serum sample containing exosomes is incubated with the capture chip, and the resonance trough wavelength 2 at this time is recorded using the platform in step 2). Elution is then initiated, and the exosomes are separated using a pH 2.0 glycine buffer. NaOH solution is added to adjust the pH to neutral to obtain an exosome extract. Specifically, the serum sample containing exosomes is incubated with the capture chip. Specifically, the functionalized chip is immersed in the serum sample, and the exosomes are captured in a targeted manner through antibody-antigen specific binding (e.g., NMDAR2A protein recognizes exosome membrane structure, and proteins such as Aβ40 recognize exosome contents). The exosomes are separated using a 0.1-1 M glycine buffer (pH 2.0) to disrupt the ionic bond between the antibody and exosomes, thereby stripping the exosomes from the chip surface. The addition of NaOH solution to adjust the pH to neutral can adjust the pH of the stripping solution to 7.0-7.4 using a 1 M NaOH solution to stabilize the exosome membrane structure and prevent structural damage caused by the acidic environment.

[0035] 4) Exosome lysis: Add lysis buffer containing 1% Triton X-100 and 1 nM protease inhibitor PMSF to obtain the exosome content solution. The lysis buffer contains 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and various inhibitors such as sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin, and 1 nM protease inhibitor (PMSF). Lyse the cells in a refrigerator (4°C) for 10-15 minutes.

[0036] 5) Marker Detection: The exosome-containing solution is incubated with the functionalized biosensor chip from step 1). Deionized water is then added to the solution. Using the detection platform constructed in step 2), the resonance trough wavelength (3) is monitored and recorded. This is compared with the resonance wavelength (1) measured in step 2) without the sample. This is the resonance wavelength shift, which enables quantitative marker analysis. The incubation time can be 30 minutes. The resonance wavelength shift (Δλ, resonance trough position 3 minus resonance trough position 1) caused by marker binding can be recorded using surface plasmon resonance (SPR) or metasurface resonance. A concentration-Δλ calibration curve is established using standards. Combined with multi-marker analysis, the diagnostic efficacy (AUC = 0.99) is calculated using a receiver operating characteristic (ROC) curve, enabling early screening and disease assessment for AD.

[0037] The first aspect of the present invention provides a method for enriching Alzheimer's disease (AD) exosomes. Figure 1 A schematic diagram showing the isolation and enrichment method of AD exosomes (EVs) of the present invention is shown. The enrichment method is as follows:

[0038] 1. Prepare serum samples: Serum contains exosomes to be enriched.

[0039] 2. Sample and sensor incubation: MUA is modified on the biosensor chip surface to form S-Au bonds. EDC-NHS solution is used to activate the carboxyl groups on the biosensor chip surface. Antibody solution is then added to anchor the antibody to the biosensor chip surface. BSA blocks nonspecific sites, resulting in a functionalized biosensor chip. Serum, a serum sample containing exosomes, is incubated with a plasmonic metasurface sensor (Metasensor). The functionalized chip is immersed in the serum sample, and the exosomes are captured by membrane protein antibodies. The incubation is carried out at room temperature for 0.5-2 hours. During this process, the functionalized sensor surface recognizes and captures the exosomes through the membrane protein antibodies.

[0040] 3. Stripping of exosomes: After incubation, glycine buffer (pH 2.0) is used to destroy the ionic bonds between the antibody and exosomes, allowing the exosomes to be stripped from the sensor surface.

[0041] 4. Adjust pH and purify: Add sodium hydroxide (NaOH) solution to adjust the pH of the stripping solution to neutral, and then perform purification treatment to finally obtain purified exosomes (EVs).

[0042] This process, based on a plasmonic metasurface sensor, enables the targeted capture, exfoliation, and subsequent processing of exosomes, aiming to enrich them for subsequent analysis and detection. This method can ensure the structural integrity of exosomes, stabilize the exosome membrane structure, and avoid structural damage caused by acidic environments.

[0043] In a specific embodiment, the specific antibody anchored to the plasmon metasurface is anti-NMDAR2A. NMDAR2A (N-methyl-D-aspartate receptor 2A), also known as GluN2A, is an important subunit of the NMDA receptor (N-methyl-D-aspartate receptor). This antibody can capture exosomes secreted by brain-derived neuronal cells in the serum, so anti-NMDAR2A can accurately obtain exosomes secreted by brain-derived neurons in AD patients. However, anti-NMDAR2A cannot accurately diagnose AD, and the enriched exosomes need to be tested for their contents. Figure 2 , which is a scanning electron micrograph of exosomes captured by the functionalized chip using this method. It can be seen that the membrane structure is intact and the exosomes enriched by this method are intact, without any damage to the exosome membrane, making them suitable for the next step of content detection and analysis.

[0044] A second aspect of the present invention provides a method for detecting exosome contents in Alzheimer's disease, comprising the following steps:

[0045] Step 1: Incubate S-Au bonds on the metasurface biosensor chip

[0046] The gold surface of the metasurface biosensor chip cannot directly bind to biomolecules, so MUA needs to be introduced as an intermediate layer to connect the gold surface and biomolecules. Then, antibodies are introduced to give the metasurface the ability to specifically capture biomolecules.

[0047] Immerse the gold film substrate chip (such as the ultra-surface gold film chip, with a gold film thickness of 50 nm) in a 10 mM mercaptoundecanoic acid (MUA) ethanol solution and place it in a refrigerator (4°C) for 3 h to form a stable S-Au bond with the gold film through the thiol (-SH) group, thereby constructing a self-assembled monolayer (SAMs) rich in carboxyl (-COOH) groups.

[0048] Step 2: Prepare EDC-NHS solution to activate carboxyl groups

[0049] After the self-assembled layer forms on the gold surface, it needs to be activated to bind biomolecules. Add 76.8 mg of EDC and 11.5 mg of NHS to 1 ml of deionized water and stir until completely dissolved. Soak the biosensor chip in the prepared EDC-NHS solution for 1 hour.

[0050] Step 3: Antibody functionalization

[0051] To achieve specific capture of exosomes, antibodies must be anchored to the self-assembled layer. The activated sensor was immersed in an antibody solution (12.5 μg / ml, anti-Aβ40) for 30 minutes at 37°C to ensure antibody activity. The activated self-assembled layer then attached active anti-Aβ40 antibodies, allowing it to capture the Aβ40 protein after exosome cleavage.

[0052] Step 4: Blocking of groups not anchored to the antibody

[0053] During the previous functionalization step, some free ester groups may not be attached to the antibody. The polymer material of the culture dish also makes the culture environment more complex. Therefore, bovine serum albumin is used to block the active sites of unbound antibodies to prevent interference with subsequent operations.

[0054] Step 5: Install the integrated fiber optic probe, measure the reflection spectrum vertically, and monitor the resonance wavelength in real time; and measure and record the resonance wavelength 1 of step 4;

[0055] Step 6: Lysis of exosomes

[0056] The enriched AD exosome extract according to the first aspect of the present invention is used. Lysis buffer is added to the exosome solution, and the solution is placed in a refrigerator (4°C) and allowed to stand for 15 minutes to obtain an exosome content solution. The solution is then dripped onto the antibody-functionalized metasurface chip in step 3 and incubated at room temperature for 0.5 hours.

[0057] Step 6: Measure the Resonant Wavelength

[0058] Use the monitoring platform in step 5 to record the resonance wavelength 2 at this time and compare it with the resonance wavelength 1 before adding the sample to be tested, so as to obtain the resonance wavelength shift.

[0059] In a preferred embodiment, referring to Figure 3Four plasmonic chips were used. After the plasmonic metasurface formed a self-assembled monolayer and activated it, PBS was used to clean the remaining EDC and NHS from the chip surface and establish a stable baseline spectrum. Anti-Aβ40 was immobilized on the chip surface, and the resonant wavelength shift was observed and recorded to ensure successful antibody immobilization. BSA solution was then injected for 30 minutes to prevent nonspecific adsorption to the metasurface. Only slight wavelength shifts were observed during this process, demonstrating that there were few binding sites for BSA molecules on the metasurface, further confirming the successful immobilization of anti-Aβ40. This is crucial for detecting resonant wavelength shifts caused by antigen-antibody interactions in immunoassays. PBS was then added to remove unbound BSA from the metasurface, and the resonant wavelength at this point was recorded as the first resonant wavelength. PBS, Aβ40 protein solution (60 ng / ml), AFP protein solution (60 ng / ml), and a mixed Aβ40 and AFP protein solution (60 ng / ml) were then added to the four chips, respectively, and allowed to stand for 30 minutes. During this time, the anti-Aβ40 immobilized on the metasurface specifically binds to the Aβ40 protein, while the AFP protein does not, preventing any wavelength shift. Finally, PBS solution is passed through to remove any unbound protein. The resonant wavelength at this point is recorded as the second resonant wavelength. The resonant wavelength shift is equal to the second resonant wavelength minus the first resonant wavelength.

[0060] In another preferred embodiment, referring to Figure 4 After the plasmonic metasurface formed a self-assembled monolayer and activated it, PBS was used to clean the remaining EDC and NHS from the chip surface and establish a stable baseline spectrum. Anti-Aβ40 was immobilized on the chip surface, and the resonant wavelength shift was observed and recorded to ensure successful antibody immobilization. BSA solution was then added for 30 minutes to prevent nonspecific adsorption to the metasurface. Only slight wavelength shifts were observed during this process, demonstrating that there were few binding sites for BSA molecules on the metasurface, further confirming the successful immobilization of anti-Aβ40. This is crucial for detecting resonant wavelength shifts caused by antigen-antibody interactions in immunoassays. PBS was then added to remove unbound BSA from the metasurface, and the resonant wavelength at this point was recorded as the first resonant wavelength. Two sets of test samples were prepared: one set (sample 1) consisted of exosomes from an AD patient, and the other (sample 2) consisted of exosomes from a healthy individual. The contents of the lysed AD exosomes were then added for another 30 minutes. During this time, the immobilized antibody specifically bound to the Aβ40 in the contents, demonstrating that the plasmonic metasurface biosensor successfully achieved real-time detection in immunoassays. Finally, PBS solution was passed through to remove unbound contents and the resonance wavelength shift was observed. Figure 4The test results of the sample to be tested are shown, and the vertical axis is the resonance wavelength offset, where It indicates that when the significance level is 0.0001, the mean values ​​of the resonance wavelength shifts are significantly different.

[0061] Reference Figure 4 A two-sample t-test was performed on the resonance wavelengths obtained for samples ① and ②. The results showed that at a significance level of 0.0001, the mean values ​​of the resonance wavelength shifts were significantly different, indicating that the anti-Aβ40-injected metasurface biodetection chip could clearly distinguish between samples ① and ②, verifying the accuracy of the metasurface biodetection chip in detecting AD.

[0062] Furthermore, to compare the effects of combined anti-Aβ40, anti-Aβ42, anti-tau and anti-p-tau181 detection with the effects of using the four antibodies alone, Figure 5 The ROC curves for the resonance wavelength shifts of Aβ40, Aβ42, tau, and p-tau181 in exosomes measured by the plasmon metasurface chip are shown. The areas under the ROC curves (AUCs) for distinguishing between sample ① and sample ② using exosome Aβ40, Aβ42, tau, and p-tau181 were 0.915, 0.890, 0.891, and 0.888, respectively, while the AUC for combined diagnosis reached 0.997. Therefore, combined detection of exosome Aβ40, Aβ42, tau, and p-tau181 facilitates the identification of different samples and improves detection specificity.

[0063] This invention achieves highly specific capture of exosomes using a functionalized biosensor chip and, combined with metasurface resonance technology, enables label-free quantitative analysis of biomarkers. This approach overcomes the low specificity and complex workflows of existing methods, providing a highly effective solution for the early in vitro diagnosis of AD. Experimental data demonstrate that this method significantly outperforms conventional technologies in terms of capture efficiency, structural preservation, and detection sensitivity, demonstrating promising clinical application prospects.

[0064] The above describes the specific embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

[0065] In the description of this application, it should be understood that the terms "upper", "lower", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limiting this application. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "one" or "an" preceding an element does not exclude the presence of multiple such elements. The simple fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used for improvement. Any reference signs in the claims should not be interpreted as limiting the scope.

Claims

1. A method for detecting exosomes in Alzheimer's disease, characterized in that The following steps are involved: 1) Modifying the biosensor chip surface: Immersing the biosensor chip in a mercaptoundecanoic acid solution allows the thiol groups to form S-Au bonds with the gold film, thereby constructing a carboxyl-rich self-assembled monolayer on the chip surface. 2) Activation of carboxyl groups on the chip surface: Activation of carboxyl groups on the surface of the biosensor chip using EDC-NHS solution; 3) Anchoring Antibody: Adding an antibody solution to the activated chip surface to immobilize the antibody on the surface of the biosensor chip, wherein the antibody is an antibody that can recognize exosome membrane proteins secreted by brain-derived neuronal cells; 4) Blocking nonspecific sites: Use bovine serum albumin solution to block the active sites on the surface of the biosensor chip that are not bound to the antibody; 5) Capturing exosomes: The serum sample containing exosomes was incubated with the biosensor chip from step 4), and the exosomes were separated using a pH 2.0 glycine buffer. The pH was adjusted to neutral by adding NaOH solution to obtain an exosome extract. 6) Lysis of exosomes: Add lysis buffer containing 1% Triton X-100 and protease inhibitor PMSF to lyse and obtain exosome content solution; 7) Exosome detection: Add a biofunctionalized biosensor chip to the exosome content solution, install an integrated fiber optic probe, measure the reflection spectrum vertically, and monitor and record the resonance wavelength at this time.

2. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 1), the surface of the biosensor chip is modified by using a 5-50mM mercaptoundecanoic acid solution to self-assemble on the surface of the gold film substrate chip. The chip is completely immersed in the mercaptoundecanoic acid solution and placed at 4°C for 3 hours. The mercapto groups react with the gold film to form a stable S-Au bond, thereby constructing a carboxyl-rich self-assembled monolayer.

3. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 2), the EDC-NHS solution is a mixture of 0.4 M EDC and 0.1 M NHS, deionized water is added and stirred until completely dissolved, the total volume is 1 mL, and the mixture is left to stand for activation for 30 minutes at room temperature.

4. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 3), the antibodies in the antibody solution are anti-exosomal marker antibodies, including anti-NMDAR2A, anti-Aβ40, anti-Aβ42, anti-tau or anti-p-tau181, and the concentration of the antibody solution is 12.5 μg / mL.

5. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 4), the non-specific sites are blocked by blocking unreacted active sites with 5% bovine serum albumin (BSA) solution to eliminate the non-specific adsorption interference of miscellaneous proteins in the serum.

6. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 5), the incubation is carried out at room temperature for 0.5-2 hours; exosomes are separated using a pH 2.0 glycine buffer, and the exosomes are separated using a 0.1-1M, pH 2.0 glycine buffer. This buffer destroys the ionic bonds between the antibody and exosomes, causing the exosomes to be peeled off the chip surface; the NaOH solution is added to adjust the pH to neutral, and a 1M NaOH solution is prepared. The NaOH solution is added to the solution after the exosomes are separated, and the pH of the solution is adjusted to 7.0-7.4 to stabilize the exosome membrane structure and obtain an exosome extract.

7. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 6), the lysis solution is RIPA buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and various inhibitors such as sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin, and 1 nM protease inhibitor (PMSF) is added; the lysis is carried out in a 4°C refrigerator for 10-15 minutes to obtain an exosome content solution.

8. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 7), the probe has a wavelength detection range of 400-1000 nm, an incident angle of 90°, and a resolution of 0.1 nm.

9. The method for detecting exosomes in Alzheimer's disease according to claim 1, wherein In step 7), the reflectance spectrum is measured vertically by placing the probe vertically on the chip, and the reflectance spectrum of the chip surface is measured vertically. The resonance wavelength at this time is monitored and recorded in real time; the exosome samples of AD patients and healthy people are distinguished based on the difference in the mean value of the resonance wavelength offset.

10. The method for detecting exosomes in Alzheimer's disease according to claim 9, characterized in that A concentration-resonance wavelength shift calibration curve was established based on standards of different concentrations and the corresponding resonance wavelength shifts. The diagnostic efficacy was calculated using the ROC curve to achieve quantitative analysis of exosomes and detection of Alzheimer's disease.

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