A detection method based on urine nanovesicle multi-protein marker array

Abstract

The application discloses a detection method based on a urine nanovesicle multi-protein marker array and belongs to the technical field of biomedical detection. Step 1, after differential centrifugation of urine, a podocin magnetic bead is used to capture a subpopulation of nanovesicles derived from podocytes; step 2, after membrane treatment by saponin, biotin-labeled anti-Nephrin, anti-Podocalyxin and anti-Synaptopodin antibodies are added to form an immune complex; step 3, amplification is performed by alternately layering streptavidin-horseradish peroxidase, biotinylated anti-HRP antibody, streptavidin and biotinylated HRP, catalyzing the co-deposition of biotin-tyramide and fluorescein-tyramide, combining quantum dot-655, dual-channel imaging and spectral crosstalk correction, and calculating the signal ratio R=I 655 / I 525 ; step 4, the arithmetic mean and coefficient of variation of the R value of positive events are counted, and a characteristic vector representing the comprehensive state of multi-protein markers is output. The method realizes single-particle ratio self-calibration, provides non-invasive quantitative detection basis for early podocyte function state from two dimensions of overall intensity and population heterogeneity.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a detection method based on a urine nanovesicle multiprotein biomarker array. Background Technology

[0002] Kidney function can undergo a gradual process of change. Before routine biochemical indicators show significant abnormalities, functional changes may occur at the molecular level in renal parenchymal cells. However, the aforementioned routine indicators often fail to effectively capture these early signals.

[0003] Currently, commonly used indicators for assessing kidney function include indirect indicators such as serum creatinine, estimated glomerular filtration rate, and the ratio of urine protein to creatinine. The presence of specific proteins detectable in urine (such as microalbumin) usually indicates significant structural changes in the glomerular filtration barrier. Over a longer period prior to this, molecular events such as renal cell stress, changes in podocyte function, and extracellular matrix remodeling in the mesangial area may have already occurred at the cellular level, but the aforementioned conventional indicators often fail to effectively capture these potential signals still at the cellular level. While histopathological evaluation allows direct observation of morphological changes in kidney tissue, it is invasive, carries risks of bleeding and infection, and is not suitable for repeated use, making it difficult for large-scale screening or dynamic monitoring.

[0004] In recent years, extracellular vesicles in liquid biopsy technology have become a research hotspot. Urinary nanovesicles, especially exosomes with diameters ranging from 30 to 150 nanometers, are released by cells from various segments of the urinary system. The proteins, nucleic acids, and other biomolecules loaded on their lumen and surface can reflect the immediate physiological or functional state of their source cells. Theoretically, by analyzing the protein composition of urinary exosomes, it is possible to inversely deduce changes in cell function in different segments such as the glomeruli and renal tubules, which provides the possibility for the early detection of changes in the functional state of kidney cells. However, existing research and technical solutions face three bottlenecks that restrict their translational application.

[0005] The first problem is the conflict between the singularity of target capture and biological redundancy. Most existing methods focus on detecting a specific protein on exosomes, such as capturing exosomes indiscriminately using CD63 and then detecting the expression level of a single biomarker. This single-target strategy ignores the fact that changes in kidney cell function are a complex process involving multiple cells and pathways, including podocytes, renal tubular epithelial cells, and endothelial cells. The sensitivity and specificity of a single biomarker are highly susceptible to individual differences and biological noise, failing to depict a complete picture of cellular functional states.

[0006] The second problem is exosome capture conformation and signal distortion. Current techniques typically use a single antibody, such as an anti-CD63 antibody, to indiscriminately capture exosomes from all sources. This broad-spectrum capture method results in extremely high background noise, with a large number of exosomes from non-renal tissues or non-target cells being enriched, severely masking target-specific signals. Plasma exosomes from filtered blood also become contaminated, and their protein signals distort the molecular signature of the renal region.

[0007] The third issue is the limitation of detection sensitivity. In the early stages of changes in kidney cell function, the number of nanovesicles released that are associated with functional alterations is extremely small, and the trace multi-target protein signals they carry are far below the detection limits of conventional immunoassay methods. Traditional enzyme-linked immunosorbent assays (ELISA) or chemiluminescence assays, due to their limited signal amplification capabilities, cannot simultaneously and accurately analyze these weak, multiple signals at the single-particle level, leading to a large number of false negative results.

[0008] Therefore, there is an urgent need for a method that can specifically isolate and identify subpopulations of renal podocyte-derived nanovesicles from complex urinary matrix, and simultaneously, with high sensitivity and self-calibrating quantification, detect characteristic multi-protein biomarker arrays derived from podocytes, thereby obtaining multi-dimensional quantitative detection parameters that can characterize the functional state of podocytes. Summary of the Invention

[0009] To achieve the above objectives, the present invention provides a detection method based on a urine nanovesicle multiprotein biomarker array, comprising the following steps:

[0010] Step 1: The urine sample was pretreated by differential centrifugation to remove cells, debris and large particles, and the pretreated urine sample was obtained. Magnetic beads with anti-Podocin antibody on the surface were used to capture and enrich podocyte-derived nanovesicle subpopulations from the pretreated urine sample.

[0011] Step 2: The podocyte-derived nanovesicle subsets captured and enriched in Step 1 are permeabilized with saponin solution, and then biotin-labeled anti-Nephrin antibody, biotin-labeled anti-Podocalyxin antibody and biotin-labeled anti-Synaptopodin antibody are added to form an immune complex carrying the three detection probes.

[0012] Step 3: Streptavidin-labeled horseradish peroxidase is bound to the immune complex formed in Step 2. A multi-level horseradish peroxidase stack is constructed by alternately adding biotin-labeled anti-horseradish peroxidase antibody to streptavidin and biotin-labeled horseradish peroxidase in a stratification cycle. A biotin-tyramine and fluorescein-tyramine mixed substrate and hydrogen peroxide are added to catalytically deposit a co-deposited polymer layer containing a biotin reporter group derived from biotin-tyramine and a fluorescein internal reference group derived from fluorescein-tyramine. Streptavidin-labeled quantum dots-655 are added and bind to the biotin reporter group. Signals from the 525 nm and 655 nm channels are acquired using a single-particle fluorescence microscopy system. After spectral crosstalk correction, the signal ratio of each positive event is calculated. Signal ratio The calculation formula is:

[0013]

[0014] in, Report the signal strength for the corrected quantum dot-655. The corrected signal intensity of the fluorescein internal reference;

[0015] Step 4: Calculate the signal ratio of all positive events obtained in Step 3. Value, calculate signal ratio arithmetic mean and coefficient of variation coefficient of variation The calculation formula is:

[0016]

[0017] in, Signal ratio The standard deviation of the dataset; the arithmetic mean and coefficient of variation Together, they serve as output parameters, constituting a feature vector for characterizing the joint detection response of the multi-protein biomarker array in podocyte-derived nanovesicles.

[0018] Preferably, the concentration of the saponin solution in step 2 is 0.01% to 0.05% by mass volume, and the permeabilization time is 5 to 15 minutes. After permeabilization, the retention rate of the immune signal of the vesicle surface marker Podocin is greater than 90%, and the peak value change of the vesicle particle size distribution measured by nanoparticle tracking analysis is less than 15%, and the change of the full width at half maximum (FWHM) is less than 20%.

[0019] Preferably, in step 2, the molar ratio of biotin to antibody in the biotin-labeled anti-Nephrin antibody, biotin-labeled anti-Podocalyxin antibody, and biotin-labeled anti-Synaptopodin antibody is 4:1 to 6:1.

[0020] Preferably, the specific operations for combining streptavidin-labeled horseradish peroxidase and performing stratification cycling in step 3 are as follows:

[0021] The immune complex formed in step 2 was incubated with streptavidin-labeled horseradish peroxidase, wherein the molar ratio of streptavidin to horseradish peroxidase in the streptavidin-labeled horseradish peroxidase was 1:1, ensuring that each streptavidin molecule retained an average of at least two free biotin-binding sites. The streptavidin-labeled horseradish peroxidase bound to the biotin on the detection probe via streptavidin. After washing, biotin-labeled anti-horseradish peroxidase antibody was added. The biotin-labeled anti-horseradish peroxidase antibody reacted with the already bound horseradish peroxidase. Horseradish peroxidase (HRP) specifically binds to form a first-level bridging layer. After washing, streptavidin is added, and streptavidin binds to biotin on the first-level bridging layer. After washing, biotin-labeled horseradish peroxidase is added, and biotin-labeled horseradish peroxidase binds to streptavidin to form a second-level horseradish peroxidase amplification layer. The steps of adding streptavidin and biotin-labeled horseradish peroxidase are repeated 2 to 5 times, so that the alternating layering of streptavidin and biotin-labeled horseradish peroxidase is performed 3 to 6 times.

[0022] Preferably, in the biotin-tyramine and fluorescein-tyramine mixed substrate in step 3, the molar ratio of biotin-tyramine molecules to fluorescein-tyramine molecules is 20:1 to 100:1; the final concentration of added hydrogen peroxide is 0.01% to 0.05% by volume; the catalytic deposition reaction time is 60 seconds to 120 seconds; and before formal sample detection, the fluorescein internal reference signal intensity is determined by setting at least 5 deposition time gradients in a standard experiment. The working range increases linearly with deposition time, and the validity of individual positive events within this working range is verified. The ratio of the signal intensity to the average background signal intensity is greater than 10, and the catalytic deposition reaction time selected in step 3 falls within the linear operating range.

[0023] Preferably, the method for spectral crosstalk correction in step 3 is as follows:

[0024] A first monochromatic control sample with only fluorescein-tyramine deposition was prepared beforehand. Signals from the 525 nm and 655 nm channels were acquired under the same imaging conditions, and the crosstalk coefficient from fluorescein to the 655 nm channel was calculated. , The value is the average signal intensity of the first monochrome control sample in the 655 nm channel divided by the average signal intensity of the first monochrome control sample in the 525 nm channel;

[0025] A second monochromatic control sample labeled only with quantum dot-655 was prepared beforehand. Signals from the 525 nm and 655 nm channels were acquired under the same imaging conditions, and the crosstalk coefficient from quantum dot-655 to the 525 nm channel was calculated. , The value is the average signal intensity of the second monochrome control sample in the 525 nm channel divided by the average signal intensity of the second monochrome control sample in the 655 nm channel;

[0026] For each positive event, spectral crosstalk correction is performed using the following formula:

[0027]

[0028]

[0029] in, To correct the reported signal strength acquired in the previous 655 nm channel, To correct the intrinsic parameter signal intensity acquired in the first 525 nm channel, Report the signal strength for the corrected quantum dot-655. The signal intensity of the fluorescein internal reference is the corrected value.

[0030] Preferably, the criterion for determining a positive event in step 4 is: the strength of the corrected quantum dot-655 reporting signal. Larger than the background area Three times the standard deviation of the mean, and the corrected fluorescein internal reference signal intensity Larger than the background area The background region is defined as the blank area in the single-particle fluorescence microscopy field of view that is more than 5 micrometers away from the center of any captured first magnetic bead and does not overlap with the fluorescence signal of the magnetic bead-nanovesicle complex. Mean, standard deviation, and background area The mean and standard deviation are obtained by statistically analyzing the correction signal strength of all pixels in the region.

[0031] Preferably, the magnetic beads with anti-Podocin antibodies coupled to their surfaces in step 1 have a particle size of 1 to 5 micrometers, and the anti-Podocin antibodies are covalently linked to the surface of the magnetic beads.

[0032] Preferably, the specific operation of the differential centrifugation pretreatment in step 1 is as follows: under the condition of 4 degrees Celsius, centrifuge at 300 times the acceleration of gravity for 10 minutes, 2000 times the acceleration of gravity for 20 minutes, and 10000 times the acceleration of gravity for 30 minutes in sequence, and take the supernatant after each centrifugation for the next step; the volume of the urine sample is 50 ml to 100 ml.

[0033] The beneficial effects of this invention are:

[0034] 1. This invention achieves targeted capture of podocyte-derived nanovesicle subpopulations by targeting the podocyte characteristic membrane protein Podocin, eliminating background interference from non-target cell-derived vesicles at the source. Based on this, an array of three podocyte characteristic proteins—Nephrin, Podocalyxin, and Synaptopodin—reflecting slit septum integrity, apical membrane charge barrier state, and podocyte actin scaffold remodeling, respectively, is employed. A transient permeabilization treatment with saponins makes the cytoplasmic Synaptopodin epitope accessible, enabling simultaneous co-localization acquisition of multi-dimensional damage signals. The synergistic design of one capture marker and three detection markers fundamentally filters out biological noise from individual markers, and its comprehensive characterization capability far exceeds that of simple linear superposition of individual markers.

[0035] 2. This invention designs an enzyme cascade amplification strategy using streptavidin-labeled horseradish peroxidase as the starting anchor and biotin-labeled anti-horseradish peroxidase antibody as the bridging medium. Through alternating cascades of streptavidin and biotin-labeled horseradish peroxidase, a programmable-density-enhanced stacking structure is constructed at a single nanovesicle recognition site, achieving strong signal amplification for rare targets. Based on this, a biotin-tyramine and fluorescein-tyramine co-deposition at a catalytic molar ratio of 50:1 is used to form a polymer layer carrying both reporter and internal reference signal groups at each recognition site. This is achieved through a signal ratio R = / Achieving ratio self-calibration quantification at the single-particle level completely eliminates batch-to-batch variation introduced by external factors such as probe binding efficiency, differences in the microenvironment of the stacking cycle, and drift of excitation power. The quantitative reproducibility far exceeds that of the conventional single-signal external standard method.

[0036] 3. This invention not only extracts the arithmetic mean reflecting the overall expression level of multiple biomarkers at the output end, but also... Furthermore, it creatively introduced a coefficient of variation that reflects the heterogeneity of damage in the examined vesicle population. In the early stages of changes in podocyte functional state, the damage exhibits a focal segmental distribution, leading to differences in the combination and abundance of protein markers carried by nanovesicles from different podocyte origins. This spatial heterogeneity information is compressed within... In the midst of. By and The combination of two orthogonal dimensional features into a feature vector output can capture early heterogeneous signal patterns that cannot be reflected by simply relying on average signal intensity. This significantly advances the quantitatively characterizable podocyte state window, providing accurate quantitative basis for subsequent physiological state assessment and dynamic monitoring. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0038] Figure 1 This is a flowchart of the steps of the method of the present invention. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should also be noted that, to make the embodiments more comprehensive, the following embodiments are the best and preferred embodiments, and those skilled in the art can use other alternative methods to implement some well-known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0040] Please see Figure 1 This invention provides a detection method based on a multi-protein biomarker array of urinary nanovesicles. The core concept is to specifically separate podocyte-derived nanovesicle subpopulations by targeting the podocyte characteristic membrane protein Podocin, and to achieve joint recognition of multiple biomarkers using a detection probe array containing three podocyte characteristic proteins: Nephrin, Podocalyxin, and Synaptopodin. Furthermore, an enzyme stratification and cyclic amplification strategy is designed, using streptavidin-labeled horseradish peroxidase as the starting anchor and biotin-labeled anti-horseradish peroxidase antibody as the bridging medium. This strategy couples biotin-tyramine and fluorescein-tyramine dual-substrate catalytic co-deposition, simultaneously generating reporter signal groups and internal reference signal groups at a single nanovesicle recognition site. This achieves ratio self-calibrated quantitative detection at the single-particle level, and finally outputs a feature vector composed of the arithmetic mean of the signal ratio and the coefficient of variation, comprehensively reflecting the expression levels of multiple biomarkers and population heterogeneity of podocyte-derived nanovesicles.

[0041] The technical content of this invention that may cause misunderstandings or unclear issues will be described in detail below.

[0042] Regarding the capture strategy for podocyte-derived nanovesicle subsets:

[0043] Step 1 of this invention uses magnetic beads with anti-Podocin antibodies conjugated to their surfaces to capture a subset of podocyte-derived nanovesicles from pretreated urine samples. Podocin is a specific membrane protein on the slit diaphragm of glomerular podocytes and is not expressed in other urinary system cells. Therefore, using Podocin as a capture target enables the targeted enrichment of podocyte-derived nanovesicles, fundamentally eliminating background interference from vesicles originating from non-target cells such as renal tubular epithelial cells, bladder epithelial cells, and reproductive tract cells. This is fundamentally different from existing techniques that use general exosome markers such as CD63 and CD9 for broad-spectrum capture. The latter captures vesicle populations from complex sources, including a large number of vesicles not derived from the kidneys or even from hemofiltration, severely masking local molecular signature signals in the kidneys.

[0044] Pre-treated urine samples were prepared using differential centrifugation, sequentially centrifuged at 300x, 2000x, and 10000x gravity to remove cells, large debris, apoptotic bodies, micron-sized vesicles, and larger vesicles and protein aggregates with diameters greater than 200 nanometers, respectively. It is important to note that centrifugation at 10000x gravity is insufficient to precipitate target nanovesicles with diameters ranging from 30 to 150 nanometers; the target nanovesicles remained intact in the supernatant. Therefore, the purpose of this pre-treatment step was to remove interfering substances rather than recover exosome particles, and the subsequent immunocapture step was performed directly in the pre-treated supernatant.

[0045] Regarding membrane permeation treatment and membrane integrity verification:

[0046] In step 2, aescin solution was used to permeabilize the captured podocyte-derived nanovesicles. The saponin referred to in this invention is specifically aescin, a triterpenoid saponin compound extracted from the seeds of plants in the *Aesculus* genus. Aescin can specifically bind to cholesterol molecules in the lipid bilayer of the nanovesicles, forming transiently reversible perforations on the membrane. The perforation pore size is approximately 6 to 8 nanometers (the perforation size of the aescin-cholesterol complex has been reported in relevant literature, for example, Mellor et al., *Biophysical Journal*, 1998, 74(6):3154-3160), sufficient to allow immunoglobulin G antibody molecules with a molecular weight of approximately 150 kilodaltons to enter the vesicle cavity without causing irreversible disintegration of the vesicle membrane. After washing and diluting with phosphate buffer to remove aescin, the lipid bilayer can automatically close and recover its integrity.

[0047] The selection of a concentration range of 0.01% to 0.05% (w / v) and a treatment time range of 5 to 15 minutes is based on the following: Below 0.01%, perforation efficiency is insufficient, making it difficult for 150 kDalton antibody molecules to effectively enter the vesicle cavity; above 0.05%, the perforation density is too high, potentially leading to irreversible disintegration of the lipid bilayer. Treatment times shorter than 5 minutes result in insufficient antibody entry, while times longer than 15 minutes, even within a safe concentration range, may cause some vesicles to rupture due to cumulative effects. The aforementioned concentration and time ranges represent the optimized window determined through orthogonal experiments, balancing antibody accessibility and vesicle integrity within this window.

[0048] Verification of membrane integrity employed a dual quality control index. The first quality control index was the retention rate of the podocin immune signal on the vesicle surface before and after permeabilization treatment. Podocin is a transmembrane protein, and its extracellular domain is always recognized by antibodies on the vesicle membrane surface. If permeabilization treatment leads to membrane rupture, the podocin protein will disperse in the solution after vesicle rupture, significantly reducing the surface immune signal. Therefore, a podocin immune signal retention rate greater than 90% after permeabilization treatment was used as the criterion for intact membrane integrity. The specific method for determining the immune signal retention rate was as follows: the first aliquot of the sample was taken from the podocyte-derived nanovesicle subpopulation before permeabilization treatment, and the surface was stained with a fluorescently labeled anti-podocin antibody. The average fluorescence intensity of the podocin-positive vesicles was measured by flow cytometry or single-particle fluorescence microscopy, denoted as . After permeabilization, a second aliquot of the sample was taken from the same batch of treatment system and stained with the same fluorescently labeled anti-Podocin antibody. The average fluorescence intensity of the Podocin-positive vesicles was measured and recorded as _____. The formula for calculating the retention rate is:

[0049]

[0050] The second quality control indicator was the change in vesicle particle size distribution before and after permeabilization treatment. Nanoparticle tracking analysis was used to determine the vesicle particle size distribution before and after permeabilization treatment, and the peak particle size was recorded. and and half-peak full width and If permeabilization treatment leads to vesicle disintegration, the particle size distribution will change significantly, manifested as a large shift in peak particle size or a sharp broadening of the full width at half maximum (FWHM). Therefore, a change in peak particle size of less than 15% and a change in FWHM of less than 20% are used as the second criterion for ensuring that the membrane integrity has not been compromised.

[0051] Regarding the selection and coordination logic of the three detection probes:

[0052] The three detection probes used in step 2 are biotin-labeled anti-Nephrin antibody, biotin-labeled anti-Podocalyxin antibody, and biotin-labeled anti-Synaptopodin antibody. These three protein markers are all derived from podocytes, but they reflect different functional dimensions of podocytes.

[0053] Nephrin is a transmembrane protein on the slit septum of podocytes and a core structural component of the glomerular filtration barrier. The extracellular domain of nephrin is naturally accessible on the surface of nanovesicle membranes, and changes in its expression level reflect the integrity of the slit septum.

[0054] Podocalyxin is a transmembrane glycoprotein on the apical membrane of podocytes. It is anchored to the cell membrane via glycosylphosphatidylinositol and is a key molecule in maintaining the negative charge barrier of the apical membrane of podocytes. The extracellular domain of Podocalyxin is naturally accessible on the surface of nanovesicle membranes, and changes in its expression level reflect the state of the charge barrier of the apical membrane of podocytes.

[0055] Synaptopodin is a podocyte-specific actin-binding protein located on the cytoplasmic side of the foot process and is closely related to the polymerization and remodeling of the foot process actin cytoplasm. Because Synaptopodin is located on the cytoplasmic side rather than the membrane surface, anti-Synaptopodin antibodies cannot access its antigenic epitopes on intact nanovesicles without permeabilization treatment. This invention utilizes the transient reversible perforation formed by the aescin permeabilization treatment in step 2 to allow anti-Synaptopodin antibodies to enter the vesicle lumen, recognize, and bind to the Synaptopodin protein. After washing to remove the aescin, the lipid bilayer closes spontaneously, and the bound antibody is retained within the vesicle lumen, enabling effective detection of intraluminal protein markers.

[0056] The molar ratio of biotin to antibody in all three detection probes was controlled within the range of 4:1 to 6:1. This range was chosen because: when the molar ratio is below 4:1, the number of biotin groups attached to each antibody molecule is insufficient, resulting in inadequate anchoring efficiency of the subsequent streptavidin-labeled horseradish peroxidase and a decrease in signal amplification; when the molar ratio is above 6:1, over-modification may affect the antigen-binding activity of the antibody and increase the risk of non-specific aggregation between detection probes via biotin-streptavidin bridging.

[0057] Detailed explanation of the enzyme stratification cycle amplification strategy:

[0058] The signal amplification strategy in step 3 is one of the core inventive aspects of this invention. Its design logic is as follows: using the biotin group on the detection probe as the starting anchor, horseradish peroxidase (HRP) is first directionally immobilized at the recognition site through the specific binding of streptavidin-labeled HRP; then, biotin-labeled anti-HRP antibody is used to immunorecognize the bound HRP, forming a first-level bridging layer; next, streptavidin is added to bind to the biotin on the first-level bridging layer; finally, biotin-labeled HRP is added to form a second-level HRP amplification layer. By repeating the alternating addition of streptavidin and biotin-labeled HRP, the enzyme is stacked in a stratified manner.

[0059] In streptavidin-labeled horseradish peroxidase, the molar ratio of streptavidin to horseradish peroxidase was controlled at 1:1, and the conjugation was performed using the sodium periodate oxidation method. Specifically, horseradish peroxidase was dissolved in carbonate buffer, and sodium periodate was added for oxidation, oxidizing the glycosyl groups on the horseradish peroxidase to aldehyde groups. After removing excess sodium periodate by dialysis, the activated horseradish peroxidase was mixed with streptavidin at a 1:1 molar ratio and reacted under alkaline conditions to form a Schiff base. Sodium borohydride was added for reduction to form a stable covalent conjugate. The conjugate was purified by gel filtration chromatography, and the streptavidin-labeled horseradish peroxidase fraction was collected. The number of free biotin binding sites in the conjugated product was determined using the HABA (2-(4′-hydroxyazobenzene)benzoic acid) colorimetric method. Horseradish peroxidase labeled with a known concentration of streptavidin was reacted with an excess of biotin standard solution, followed by the addition of HABA reagent. The absorbance at 500 nm was measured, and the number of unoccupied binding sites per molecule of streptavidin-labeled horseradish peroxidase was calculated based on the standard curve. Verification showed that each streptavidin molecule retained an average of 2.3 free biotin binding sites. The purpose of this design was to provide sufficient binding capacity for streptavidin and biotin-labeled horseradish peroxidase in subsequent alternating stratification cycles. If the conjugation molar ratio is too high, resulting in insufficient free biotin binding sites, the increment in each round of stratification will decrease progressively, failing to achieve effective enzyme density enhancement.

[0060] The rationale for performing alternating layering 3 to 6 times is as follows: With fewer than 3 layers, the enzyme packing density is insufficient, resulting in limited signal amplification and the inability to generate detectable signals for extremely rare target nanovesicles. Parallel experiments verified that the number of positive events detected and the signal-to-noise ratio under the conditions of 3, 4, 5, and 6 layers were significantly better than those in Comparative Example 1, which did not undergo layering cycles. Performing 3 layers already met the detection sensitivity requirements and offered the best operational efficiency; therefore, 3 layers were the preferred choice. When the number of layers exceeds 6, due to the steric hindrance effect and the nonlinearity of enzyme activity superposition, the marginal contribution of further increasing the number of layers to signal amplification decreases significantly, while the background signal begins to increase noticeably. Therefore, the upper limit was set at 6 layers.

[0061] After each cycle, the sample is washed with phosphate buffer. This step removes unbound reagents and reaction byproducts, maintains a clean local enzyme reaction environment, and prevents non-specific bridging of free components between cycles. If washing is not performed promptly during cycling, residual free streptavidin from the previous cycle may bind with biotin-labeled horseradish peroxidase added in the next cycle, forming non-specific solution-phase aggregates. These aggregates may non-specifically deposit on magnetic beads or tube walls, causing an increase in background signal.

[0062] Detailed explanation of dual-substrate catalytic co-deposition and ratio self-calibration:

[0063] After lamination amplification, a mixed substrate solution consisting of biotin-tyramine and fluorescein-tyramine was added for catalytic deposition. In the presence of hydrogen peroxide, the multilayered horseradish peroxidase catalyzed the free radical polymerization of the tyramine derivative, forming a co-deposited polymer layer in its local microenvironment. Because biotin-tyramine and fluorescein-tyramine molecules coexist in the reaction system at a fixed molar concentration ratio, they are covalently integrated into the same polymer layer, resulting in each recognition site carrying both a reporter signal group and an internal reference signal group.

[0064] The molar ratio of biotin-tyramine to fluorescein-tyramine was set to 20:1 to 100:1. This range was chosen because: the biotin-tyramine ratio needs to be sufficiently high to ensure adequate biotin reporter groups in the co-deposited polymer layer, providing abundant sites for subsequent binding of quantum dots-655 and generating a sufficiently strong reporter signal; the fluorescein-tyramine ratio needs to be sufficiently low to avoid self-quenching of fluorescein at high local concentrations, while ensuring that the generated internal reference signal intensity has a statistically acceptable Poisson noise level. Refer to the deposition time gradient experiment in step 3 of the following examples; when the molar ratio is in the range of 20:1 to 100:1, the fluorescein internal reference signal intensity of a single positive event... The ratio of the signal intensity to the average background signal intensity is greater than 10, which meets the signal-to-noise ratio requirements for single-event ratio measurement.

[0065] The catalytic deposition reaction time was set to 60 to 120 seconds, a range determined through prior standard experiments. The specific method for the standard experiments is described in step 3 of the following example. The basic procedure is as follows: using the same batch of standard samples, five deposition time gradients were set: 30 seconds, 60 seconds, 90 seconds, 120 seconds, and 150 seconds. Under each time condition, the fluorescein internal reference signal intensity of a single positive event was measured. Plotting deposition time on the x-axis, and representing all positive events under that time condition. Using the median as the ordinate, plot the curve of deposition time versus signal intensity to determine... The working range increases linearly with deposition time. The time within this linear working range is selected as the deposition time for formal detection to ensure the fluorescein internal reference signal. Linear response characteristics as a quantitative reference.

[0066] Streptavidin-labeled quantum dots-655 were added to a co-deposited polymer layer, enabling them to specifically bind to the biotin reporter group in the polymer layer. The streptavidin-labeled quantum dots-655 are commercially available products with a CdSe / ZnS core-shell structure and a carboxylated polymer coating layer. They have a particle size of approximately 15-20 nm, an emission peak center wavelength of 655 nm, and a high absorption cross-section at an excitation wavelength of 488 nm. Streptavidin was covalently linked to the carboxyl groups on the quantum dot surface via coupling 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride with N-hydroxysuccinimide. These quantum dots exhibit excellent photostability, and the sufficient wavelength separation between their 655 nm emission peak and the 525 nm emission peak of fluorescein facilitates spectral separation using a dichroic mirror and bandpass filter. Streptavidin provides high-affinity biotin recognition capabilities, ensuring that quantum dot-655 binds only to the co-deposited polymer layer regions containing biotin reporter groups.

[0067] Detailed explanation of spectral crosstalk correction:

[0068] In single-particle fluorescence microscopy systems, spectral crosstalk is unavoidable between the 525 nm and 655 nm channels. There are two main sources of this crosstalk: first, the emission spectrum of fluorescein exhibits a long-wavelength tail, causing some of its emission light to leak into the 655 nm channel; second, the absorption spectrum of quantum dot-655 covers the 488 nm excitation wavelength, causing some of the emission light generated by quantum dot-655 under 488 nm excitation to leak into the 525 nm channel due to its broad emission spectrum. Without correction, this spectral crosstalk can lead to inaccurate measurements. The value is influenced by the contribution of fluorescein. The value incorporates the contribution of quantum dot-655, thus affecting the signal ratio. Deviation from the true value impairs the accuracy of ratio self-calibration.

[0069] This invention employs a dual monochromatic control method to determine the spectral crosstalk coefficient. First, a first monochromatic control sample is prepared by depositing only fluorescein-tyramine without adding quantum dots-655. Signals from the 525 nm and 655 nm channels are acquired under the same imaging conditions. Since quantum dots-655 are absent in this sample, any signal detected in the 655 nm channel originates from spectral leakage of fluorescein. Therefore, the crosstalk coefficient from fluorescein to the 655 nm channel is calculated. Then, a second monochromatic control sample was prepared by labeling only quantum dots-655 without depositing fluorescein-tyramine. Two-channel signals were acquired under the same imaging conditions, and the crosstalk coefficient from quantum dots-655 to the 525 nm channel was calculated. In formal sample detection, a correction formula is applied to each positive event to subtract crosstalk contributions from the original signal, obtaining the corrected true signal strength.

[0070] Detailed explanation of positive event determination and background area definition:

[0071] A positive event is determined using a dual threshold standard: the corrected quantum dot-655 reporting signal strength. Larger than the background area Three times the standard deviation of the mean, and the corrected fluorescein internal reference signal intensity Larger than the background area Three times the standard deviation of the mean. The basis for using three times the standard deviation as the threshold is that, assuming the background noise follows a normal distribution, three times the standard deviation corresponds to a confidence level greater than 99.7%, which can effectively distinguish between real signals and random background fluctuations.

[0072] The background region is defined as a blank area within the field of view of single-particle fluorescence microscopy that is more than 5 micrometers away from the center of any captured first magnetic bead and does not overlap with the fluorescence signal of the magnetic bead-nanovesicle complex. The 5-micrometer distance threshold is based on the fact that the particle size of the first magnetic bead is 1 to 5 micrometers, the diameter of the nanovesicle is 30 to 150 nanometers, and the fluorescence signal of the magnetic bead-nanovesicle complex is mainly concentrated on the surface of the magnetic bead and its adjacent area. Experiments have verified that the region beyond 5 micrometers from the center of the magnetic bead is not affected by diffraction or scattering of the complex fluorescence signal and can accurately reflect the background noise level of the detection system. average value, Standard deviation Average and The standard deviation is obtained by statistically analyzing the corresponding corrected signal strength of all pixels in the region.

[0073] Explanation regarding the output feature vector:

[0074] Step 4 output is the arithmetic mean and coefficient of variation The eigenvectors formed. Arithmetic mean. This reflects the overall expression level of the multi-protein biomarker array on the captured podocyte-derived nanovesicle population; a higher value indicates a higher combined abundance of the three biomarkers on a single vesicle. Coefficient of variation This reflects the degree of difference in the expression levels of multiple protein markers among different nanovesicles. A higher value indicates a greater difference in protein expression patterns among different individuals within the vesicle population. In the early stages of population heterogeneity in the multi-protein marker characteristics of podocyte-derived nanovesicles, individuals within the vesicle population may exhibit significant differences in marker combinations and abundance. This heterogeneity leads to differences in the combinations and abundance of protein markers carried by nanovesicles from different podocytes, directly manifested as… The increase. Therefore, and The combination into a feature vector can comprehensively characterize the multi-protein biomarker features of podocyte-derived nanovesicles from two orthogonal dimensions: overall strength and population heterogeneity.

[0075] Example

[0076] In this embodiment, the test subject is a midstream sample of morning urine with a volume of 80 ml.

[0077] Step 1: Specific isolation and enrichment of podocyte-derived urinary nanovesicles

[0078] Urine samples were aliquoted into two 50 mL centrifuge tubes, 40 mL each, and centrifuged at 300x gravity for 10 minutes in a benchtop centrifuge pre-cooled to 4°C. The supernatant was carefully transferred to a new 50 mL centrifuge tube, and the precipitate was discarded. The supernatant was then centrifuged at 2000x gravity for 20 minutes at 4°C, and the precipitate was carefully transferred to a new centrifuge tube. The supernatant was then centrifuged at 10000x gravity for 30 minutes at 4°C, and the precipitate was carefully transferred to a new centrifuge tube, yielding approximately 78 mL of pretreated urine sample. This process ensured that target nanovesicles with diameters ranging from 30 nm to 150 nm were completely preserved in the supernatant.

[0079] 200 μL of the first immunomagnetic bead suspension was added to the pretreated urine sample. The first immunomagnetic beads were magnetic beads covalently coupled with anti-Podocin antibody via amide bonds. The anti-Podocin antibody was a commercially available rabbit anti-human Podocin (NPHS2) polyclonal antibody, which can be purchased from Abcam (the immunogen is the synthetic peptide from the C-terminal intracellular domain of human Podocin, approximately amino acid position 350 to the C-terminus). The bead size was 2.8 μm, and the anti-Podocin antibody to magnetic bead conjugation mass ratio was 1:30. The mixture was incubated at 4°C for 2 hours by rotation. After incubation, the centrifuge tubes were placed on a magnetic separator and allowed to stand for 3 minutes. The supernatant was discarded, retaining the magnetically adsorbed bead-vesicle complex. 1 mL of pre-chilled phosphate buffer was added, and the mixture was gently resuspended by pipetting. The supernatant was discarded again by magnetic separation. This washing procedure was repeated 3 times. Approximately 200 μL of podocyte-derived urine nanovesicle subset suspension was obtained.

[0080] Step 2: Joint recognition of multi-protein biomarker arrays

[0081] The podocyte-derived urine nanovesicle subset suspension obtained in step 1 was resuspended in 500 μL of reaction buffer containing aescin, with a mass-volume percentage concentration of 0.03% and a purity of not less than 95%. The reaction tube was gently shaken and incubated for 10 minutes at room temperature.

[0082] Before permeabilization, a 20 μL aliquot of the first sample was taken from the suspension and surface-stained with an Alexa Fluor 647 fluorescently labeled anti-Podocin antibody. This anti-Podocin antibody was from the same clone as the one used in step 1. The average fluorescence intensity of the Podocin-positive vesicles was measured using a single-particle fluorescence microscopy system and recorded. After permeabilization, a second aliquot of 20 μL was taken from the same batch of treatment system and surface-stained with the same Alexa Fluor 647 fluorescently labeled anti-Podocin antibody. The average fluorescence intensity was measured and recorded. Calculate the retention rate:

[0083]

[0084] The retention rate is greater than 90%, which meets the first quality control indicator for membrane integrity.

[0085] Simultaneously, 10 μL samples were taken from the suspension before and after membrane permeation treatment, and the vesicle size distribution was determined using a nanoparticle tracking analyzer. The peak particle size before membrane permeation treatment was... 98 nanometers, full width at half maximum (FWHM) The peak particle size is 42 nanometers. After membrane treatment, the peak particle size is... 103 nanometers, full width at half maximum (FWHM) The particle size is 46 nanometers. The peak particle size variation is 5.1%, and the full width at half maximum (FWHM) variation is 9.5%, both of which meet the second quality control index for membrane integrity.

[0086] After the permeabilization process, three detection probes were added directly to the reaction system. The first detection probe was a biotin-labeled commercially available rabbit anti-human Nephrin polyclonal antibody (Thermo Fisher Scientific), with a biotin to antibody molar ratio of 5:1. The second detection probe was a biotin-labeled commercially available goat anti-human Podocalyxin polyclonal antibody (R&D Systems), with a biotin to antibody molar ratio of 5:1. The third detection probe was a biotin-labeled commercially available rabbit anti-human Synaptopodin polyclonal antibody (Novus Biologicals), with a biotin to antibody molar ratio of 5:1. All three polyclonal antibodies underwent cross-adsorption treatment to immunogenic peptides associated with human podocytes. Specific staining of human podocyte-derived nanovesicles was verified by an irrelevant IgG substitution control. The final concentration of each detection probe was 2 μg / mL. The system was incubated with gentle shaking at 37°C for 1 hour.

[0087] After incubation, place the reaction tube on a magnetic separator and let it stand for 3 minutes, then discard the supernatant. Add 1 mL of phosphate buffer and wash, then magnetically separate and discard the supernatant. Repeat the washing process 3 times. During the washing process, aescin is gradually diluted and removed, and the lipid bilayer of the nanovesicles closes and recovers its integrity, while the bound detection probe is retained.

[0088] Step 3: Enzyme stratification cycle amplification and ratio self-calibration quantitative detection

[0089] The capture-detection immune complex washed in step 2 was resuspended in 500 μL of buffer containing streptavidin-labeled horseradish peroxidase (HRP), with a final concentration of 5 μg / mL. The streptavidin-labeled HRP was prepared using the sodium periodate oxidation method, with a streptavidin to HRP molar ratio of 1:1. HABA colorimetric assay showed that each streptavidin molecule retained an average of 2.3 free biotin-binding sites. The mixture was incubated at 37°C for 30 minutes. After incubation, magnetic separation was performed, followed by washing once with phosphate buffer to remove unbound streptavidin-labeled HRP.

[0090] The complex was resuspended in 500 μL of buffer containing biotin-labeled anti-horseradish peroxidase antibody to a final concentration of 5 μg / mL, and incubated at 37°C for 30 minutes to allow the biotin-labeled anti-horseradish peroxidase antibody to specifically bind to the bound horseradish peroxidase, forming the first-level bridging layer. Magnetic separation was performed, followed by washing once with phosphate buffer.

[0091] The complex was resuspended in 500 μL of streptavidin-containing buffer to a final concentration of 5 μg / mL and incubated at 37°C for 30 min, allowing streptavidin to bind to the biotin groups on the first-stage bridging layer. Magnetic separation was then performed, followed by washing once with phosphate-buffered saline.

[0092] The complex was resuspended in 500 μL of buffer containing biotin-labeled horseradish peroxidase to a final concentration of 5 μg / mL, and incubated at 37°C for 30 min. The biotin-labeled horseradish peroxidase bound to streptavidin, forming a secondary horseradish peroxidase amplification layer. The layer was then magnetically separated and washed once with phosphate buffer.

[0093] The streptavidin incubation step and the biotin-labeled horseradish peroxidase incubation step were repeated twice each, so that the alternating layering of streptavidin and biotin-labeled horseradish peroxidase was performed a total of 3 times.

[0094] The amplified complex obtained through the above-described chromatographic process was resuspended in 100 μL of a mixed substrate solution containing biotin-tyramine and fluorescein-tyramine. The concentration of biotin-tyramine in the mixed substrate solution was 250 μmol / L, and the concentration of fluorescein-tyramine was 5 μmol / L, i.e., a molar ratio of 50:1. Hydrogen peroxide was added to a final concentration of 0.03% (v / v), and the catalytic deposition reaction was initiated, with the reaction time precisely controlled at 90 seconds. The 90-second reaction time was verified in pre-experimental studies to fall within the fluorescein internal control signal range. Within its linear operating range.

[0095] The specific method for the standard sample experiment is as follows: using the same batch of standard sample, five deposition time gradients were set: 30 seconds, 60 seconds, 90 seconds, 120 seconds, and 150 seconds. Under each deposition time condition, the fluorescein internal reference signal intensity of a single positive event was measured. Plotting deposition time on the x-axis, and representing all positive events under that time condition. The median value was used as the ordinate to plot the relationship between deposition time and signal intensity. It was determined that... The working range that linearly increases with deposition time is 30 to 120 seconds, and a 90-second reaction time falls within this linear working range. Under a 90-second deposition time condition, the single positive event... The ratio of the signal strength to the average background signal strength is 18.5, which is much greater than 10, thus meeting the signal-to-noise ratio requirements for single-event ratio measurement.

[0096] After the deposition reaction was complete, 1 mL of pre-cooled phosphate buffer was immediately added to dilute and terminate the reaction, followed by one magnetic separation and washing. The complex was resuspended in 200 μL of buffer containing streptavidin-labeled quantum dots-655, with a final concentration of 2 nanomoles per liter. The streptavidin-labeled quantum dots-655 have a CdSe / ZnS core-shell structure, a carboxylated polymer coating layer, a particle size of 15-20 nm, and an emission peak center wavelength of 655 nm. Incubation at 37°C for 30 min allowed for specific binding of quantum dots-655 to the biotin reporter group in the co-deposited polymer layer. After incubation, magnetic separation was performed, followed by two washes with phosphate buffer.

[0097] The final complex was resuspended in 100 μL of phosphate buffer, and 10 μL was dropped onto a glass-bottomed culture dish and placed on the stage of a single-particle fluorescence microscopy system. The imaging system was configured as follows: the excitation source was a 488 nm single-wavelength laser with a laser power density of 0.5 kW / cm²; the emission light route used a dichroic mirror to guide the signal into the 525 nm and 655 nm channels. The 525 nm channel was equipped with a bandpass filter with a center wavelength of 525 nm and a bandwidth of 20 nm, and the 655 nm channel was equipped with a bandpass filter with a center wavelength of 655 nm and a bandwidth of 20 nm; the detector was an electron multiplication charge-coupled device (ECD) camera with an exposure time of 100 ms and an electron multiplication gain of 300x.

[0098] Before performing spectral crosstalk correction, the following calibration procedures were completed. Prepare the first monochromatic control sample: Take an equal amount of the capture-detection immune complex formed in step 2, and proceed with the above-described layering amplification and catalytic deposition steps, but the mixed substrate solution contains only fluorescein-tyramine and not biotin-tyramine. After the deposition reaction, do not add streptavidin-labeled quantum dots-655, only add an equal volume of buffer solution. Under the same imaging conditions as the official sample, acquire signals from the 525 nm and 655 nm channels. The average signal intensity of the 525 nm channel was measured to be 12560 arbitrary units, and the average signal intensity of the 655 nm channel was measured to be 382 arbitrary units. Calculate the crosstalk coefficient of fluorescein to the 655 nm channel. :

[0099]

[0100] Preparation of a second monochromatic control sample: An equal amount of the capture-detection immune complex formed in step 2 was taken, and the layering amplification and catalytic deposition steps described above were followed. However, the mixed substrate solution contained only biotin-tyramine and not fluorescein-tyramine. After the deposition reaction, only streptavidin-labeled quantum dots-655 were added. Under the same imaging conditions, signals were acquired from the 525 nm and 655 nm channels. The average signal intensity of the 525 nm channel was measured to be 265 arbitrary units, and the average signal intensity of the 655 nm channel was 22100 arbitrary units. The crosstalk coefficient from quantum dot-655 to the 525 nm channel was calculated. :

[0101]

[0102] For each positive event in the official sample image, the raw signal is acquired. and Spectral crosstalk correction is performed using the following formula:

[0103]

[0104]

[0105] After correction, the signal ratio for each positive event was calculated:

[0106]

[0107] Step 4: Feature Parameter Extraction and Feature Vector Output

[0108] Positive results were determined for all candidate events in the imaging field of view. First, a blank region within the field of view, greater than 5 micrometers from the center of any captured first magnetic bead and not overlapping with the fluorescence signal of the magnetic bead-nanovesicle complex, was selected as the background region. The corrected signal intensity of all pixels within this region was statistically analyzed. The background region was measured. The mean is 87 arbitrary units, and the standard deviation is 24 arbitrary units; background area The mean is 112 arbitrary units, and the standard deviation is 31 arbitrary units. Calculate the positive threshold: The threshold is 87 plus 3 multiplied by 24, which equals 159 arbitrary units. The threshold is 112 plus 3 multiplied by 31, which equals 205 arbitrary units.

[0109] For each magnetic bead-nanovesicle complex in the field of view, its corrected value is extracted. Value and value. The correction value is greater than 159 units and Events with a correction value greater than any unit of 205 were considered valid positive events. In this example, a total of 127 valid positive events were detected.

[0110] The signal ratio of the 127 valid positive events The values ​​are statistically analyzed, and the arithmetic mean and coefficient of variation are calculated. The measured signal ratio... arithmetic mean of values The standard deviation is 4.73. The coefficient of variation is 1.28. The calculation is as follows:

[0111]

[0112] The final output is the arithmetic mean. and coefficient of variation The feature vector is constructed to characterize the joint detection response of the three podocyte characteristic proteins described in this embodiment on podocyte-derived nanovesicles.

[0113] Comparative Example 1

[0114] To verify the technical effectiveness of the enzyme chromatographic amplification strategy of the present invention, Comparative Example 1 was set up. Comparative Example 1 adopted the exact same operating steps as the Example, except that the enzyme chromatographic amplification step was omitted in step 3. The specific operation was as follows: After the capture-detection immune complex formed in step 2 was bound to streptavidin-labeled horseradish peroxidase and washed, it was not subjected to subsequent incubation with biotin-labeled anti-horseradish peroxidase antibody, streptavidin incubation, and biotin-labeled horseradish peroxidase incubation. That is, the alternating chromatographic cycle was not performed, and it directly entered the catalytic deposition reaction step of the biotin-tyramine and fluorescein-tyramine mixed substrate solution. The other steps of Comparative Example 1 and all reagent concentrations, reaction times, temperatures, and other parameters were exactly the same as those in the Example. In this comparative example, each nanovesicle recognition site before the catalytic deposition reaction was only bound to a single layer of horseradish peroxidase provided by streptavidin-labeled horseradish peroxidase. The biotin-labeled anti-horseradish peroxidase antibody bridging layer and the subsequent alternating lamination steps of streptavidin and biotin-labeled horseradish peroxidase were simultaneously omitted. Therefore, the absolute copy number of horseradish peroxidase and the number of streptavidin-free biotin binding sites were significantly lower than in the example.

[0115] Comparative Example 2

[0116] To verify the technical effectiveness of the ratio self-calibration strategy of this invention, Comparative Example 2 was set up. Comparative Example 2 adopted the exact same operating steps as the example, the only difference being that in step 3, the catalytic deposition reaction used a single substrate solution containing only biotin-tyramine, without fluorescein-tyramine. The specific operation was as follows: After the deposition cycle was completed, the complex was resuspended in 100 μL of a substrate solution containing only biotin-tyramine, with a biotin-tyramine molecule concentration of 250 μmol / L. Hydrogen peroxide was added to a final concentration of 0.03% by volume, and the reaction time was 90 seconds. After the deposition reaction, streptavidin-labeled quantum dots-655 were added, and the signal of only the 655 nm channel was acquired using a single-particle fluorescence microscopy system. Since there was no fluorescein internal reference signal channel, Comparative Example 2 only used... The absolute value is used as the detection signal, and the signal ratio is... Unable to calculate the arithmetic mean and coefficient of variation None of them could be calculated. All other steps and parameters, including reagent concentrations, reaction times, and temperatures, were exactly the same as in the Example.

[0117] Comparative Example 1 and Comparative Example 2 were each repeated 3 times. The results of the 3 repeated experiments of Example 1 are summarized in the table below.

[0118] Number of positive events detected 127±9 24±5 119±11 Arithmetic mean of signal ratios bar{R} 4.73±0.35 2.18±0.62 — Report the average signal value (any unit). 1980±280 — 3850±520 Coefficient of variation 27.1±3.2 41.5±8.7 — Batch repeatability (coefficient of variation of signal ratios bar{R} between batches, %) 7.4 13.2 — Batch repeatability coefficient of variation of absolute values, % 14.1 — 28.6

[0119] Note: Examples The average reported signal is the result of correction for all positive events. The arithmetic mean of the values ​​is expressed as 1980 arbitrary units ± 280 arbitrary units. In Comparative Example 2, the catalytic deposition reaction used a single substrate solution containing only biotin-tyramine; the co-deposited polymer layer did not contain a fluorescein internal reference group, therefore, a fluorescein internal reference signal could not be collected. Therefore, it is impossible to calculate the signal ratio. Arithmetic mean and coefficient of variation The table indicates this with a "—". The batch repeatability of Comparative Example 2 can only be... The absolute value is represented by the inter-batch coefficient of variation. (Example) The absolute value of the inter-batch coefficient of variation was 14.1%, significantly lower than that of Comparative Example 2 (28.6%), further validating the contribution of the ratio self-calibration strategy to reducing inter-batch variation.

[0120] As can be seen from the results in the table above, the number of positive events detected in the example was 127, approximately 5.3 times that of Comparative Example 1. This indicates that the enzyme stratification cycle amplification strategy significantly improved the detection sensitivity, enabling the detection of more low-abundance target events. (Arithmetic mean of signal ratio in the examples) The value was 4.73, significantly higher than the 2.18 of Comparative Example 1. This is because the multilayered enzyme stacking provides a stronger catalytic signal amplification capability. The batch-to-batch reproducibility of the examples is expressed as a signal-to-weight ratio. The inter-batch coefficient of variation was only 7.4%, significantly better than the 13.2% of Comparative Example 1, and far better than Comparative Example 2. The absolute value of 28.6% demonstrates that the ratio self-calibration strategy can effectively correct batch-to-batch variability introduced by external factors such as fluctuations in incubation conditions and drift in excitation power. (Example) The detection rate was 27.1% in Example 1 and 41.5% in Comparative Example 2, indicating that the detection of positive events in the examples was more comprehensive, including low-abundance target events, thus preserving the heterogeneity information of the vesicle population more completely. This invention encompasses any substitutions, modifications, equivalent methods, and schemes made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments, but those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary confusion regarding the essence of this invention, well-known methods, processes, procedures, elements, and circuits are not described in detail.

[0121] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A detection method based on a urine nanovesicle multiprotein biomarker array, characterized in that, Includes the following steps: Step 1: The urine sample was pretreated by differential centrifugation to remove cells, debris and large particles, and the pretreated urine sample was obtained. Magnetic beads with anti-Podocin antibody on the surface were used to capture and enrich podocyte-derived nanovesicle subpopulations from the pretreated urine sample. Step 2: The podocyte-derived nanovesicle subsets captured and enriched in Step 1 are permeabilized with saponin solution, and then biotin-labeled anti-Nephrin antibody, biotin-labeled anti-Podocalyxin antibody and biotin-labeled anti-Synaptopodin antibody are added to form an immune complex carrying the three detection probes. Step 3: Streptavidin-labeled horseradish peroxidase is bound to the immune complex formed in Step 2. A multi-level horseradish peroxidase stack is constructed by alternately adding biotin-labeled anti-horseradish peroxidase antibody to streptavidin and biotin-labeled horseradish peroxidase in a stratification cycle. A biotin-tyramine and fluorescein-tyramine mixed substrate and hydrogen peroxide are added to catalytically deposit a co-deposited polymer layer containing a biotin reporter group derived from biotin-tyramine and a fluorescein internal reference group derived from fluorescein-tyramine. Streptavidin-labeled quantum dots-655 are added and bind to the biotin reporter group. Signals from the 525 nm and 655 nm channels are acquired using a single-particle fluorescence microscopy system. After spectral crosstalk correction, the signal ratio of each positive event is calculated. Signal ratio The calculation formula is: in, Report the signal strength for the corrected quantum dot-655. The corrected signal intensity of the fluorescein internal reference; Step 4: Calculate the signal ratio of all positive events obtained in Step 3. Value, calculate signal ratio arithmetic mean and coefficient of variation coefficient of variation The calculation formula is: in, Signal ratio The standard deviation of the dataset; the arithmetic mean and coefficient of variation Together, they serve as output parameters, constituting a feature vector for characterizing the joint detection response of the multi-protein biomarker array in podocyte-derived nanovesicles.

2. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, The concentration of the saponin solution in step 2 is 0.01% to 0.05% by mass and volume, and the permeabilization time is 5 to 15 minutes. After permeabilization, the retention rate of the immune signal of the vesicle surface marker Podocin is greater than 90%, and the peak value of the vesicle particle size distribution measured by nanoparticle tracking analysis is less than 15% and the full width at half maximum (FWHM) value is less than 20%.

3. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, In step 2, the biotin-labeled anti-Nephrin antibody, biotin-labeled anti-Podocalyxin antibody, and biotin-labeled anti-Synaptopodin antibody all have a biotin to antibody molar ratio of 4:1 to 6:

1.

4. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, The specific steps for combining streptavidin-labeled horseradish peroxidase and performing stratification cycling in step 3 are as follows: The immune complex formed in step 2 was incubated with streptavidin-labeled horseradish peroxidase, wherein the molar ratio of streptavidin to horseradish peroxidase in the streptavidin-labeled horseradish peroxidase was 1:1, ensuring that each streptavidin molecule retained an average of at least two free biotin-binding sites. The streptavidin-labeled horseradish peroxidase bound to the biotin on the detection probe via streptavidin. After washing, biotin-labeled anti-horseradish peroxidase antibody was added. The biotin-labeled anti-horseradish peroxidase antibody reacted with the already bound horseradish peroxidase. Horseradish peroxidase (HRP) specifically binds to form a first-level bridging layer. After washing, streptavidin is added, and streptavidin binds to biotin on the first-level bridging layer. After washing, biotin-labeled horseradish peroxidase is added, and biotin-labeled horseradish peroxidase binds to streptavidin to form a second-level horseradish peroxidase amplification layer. The steps of adding streptavidin and biotin-labeled horseradish peroxidase are repeated 2 to 5 times, so that the alternating layering of streptavidin and biotin-labeled horseradish peroxidase is performed 3 to 6 times.

5. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, In step 3, the molar ratio of biotin-tyramine molecules to fluorescein-tyramine molecules in the biotin-tyramine and fluorescein-tyramine mixed substrate is 20:1 to 100:1; the final concentration of added hydrogen peroxide is 0.01% to 0.05% by volume; the catalytic deposition reaction time is 60 seconds to 120 seconds; before formal sample detection, the fluorescein internal reference signal intensity is determined by setting at least 5 deposition time gradients in standard experiments. The working range increases linearly with deposition time, and the validity of individual positive events within this working range is verified. The ratio of the signal intensity to the average background signal intensity is greater than 10, and the catalytic deposition reaction time selected in step 3 falls within the linear operating range.

6. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, The method for spectral crosstalk correction in step 3 is as follows: A first monochromatic control sample with only fluorescein-tyramine deposition was prepared beforehand. Signals from the 525 nm and 655 nm channels were acquired under the same imaging conditions, and the crosstalk coefficient from fluorescein to the 655 nm channel was calculated. , The value is the average signal intensity of the first monochrome control sample in the 655 nm channel divided by the average signal intensity of the first monochrome control sample in the 525 nm channel; A second monochromatic control sample labeled only with quantum dot-655 was prepared beforehand. Signals from the 525 nm and 655 nm channels were acquired under the same imaging conditions, and the crosstalk coefficient from quantum dot-655 to the 525 nm channel was calculated. , The value is the average signal intensity of the second monochrome control sample in the 525 nm channel divided by the average signal intensity of the second monochrome control sample in the 655 nm channel; For each positive event, spectral crosstalk correction is performed using the following formula: in, To correct the reported signal strength acquired in the previous 655 nm channel, To correct the intrinsic parameter signal intensity acquired in the first 525 nm channel, Report the signal strength for the corrected quantum dot-655. The signal intensity of the fluorescein internal reference is the corrected value.

7. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, The criterion for determining a positive event in step 4 is: the strength of the corrected quantum dot-655 reporting signal. Larger than the background area Three times the standard deviation of the mean, and the corrected fluorescein internal reference signal intensity Larger than the background area The background region is defined as the blank area in the single-particle fluorescence microscopy field of view that is more than 5 micrometers away from the center of any captured first magnetic bead and does not overlap with the fluorescence signal of the magnetic bead-nanovesicle complex. Mean, standard deviation, and background area The mean and standard deviation are obtained by statistically analyzing the correction signal strength of all pixels in the region.

8. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, The magnetic beads with anti-Podocin antibodies coupled to their surfaces in step 1 have a particle size of 1 to 5 micrometers, and the anti-Podocin antibodies are covalently linked to the surface of the magnetic beads.

9. The detection method based on a urine nanovesicle multiprotein biomarker array according to claim 1, characterized in that, The specific operation of differential centrifugation pretreatment in step 1 is as follows: under the condition of 4 degrees Celsius, centrifuge at 300 times the acceleration of gravity for 10 minutes, 2000 times the acceleration of gravity for 20 minutes, and 10000 times the acceleration of gravity for 30 minutes in sequence. After each centrifugation, take the supernatant for the next step; the volume of the urine sample is 50 ml to 100 ml.

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