Bubble-plasma enhanced fluorescence immunoassay detection system, preparation method, application and detection method thereof

Abstract

The application discloses a bubble-plasma enhanced fluorescence immunoassay detection system, a preparation method, application and a detection method, and belongs to the technical field of biomedical detection, liquid biopsy and optical biosensing. In view of the technical problems that the existing tumor-derived extracellular vesicle detection technology has complicated separation and purification steps, low signal amplification efficiency, weak anti-interference ability and cannot realize integration of separation, enrichment and detection, the system comprises an immunofunctionalized hollow glass microbubble and a plasma fluorescent probe. The former takes the hollow glass microbubble with a surface modified target antibody as a capture carrier, realizes target specific capture and buoyancy enrichment, and the latter takes a gold nanorod wrapped by silicon dioxide as a core, and covalently connects a fluorescent dye and a target aptamer on the surface to realize metal enhanced fluorescence signal amplification. The application is mainly used for super-sensitive detection of biomarkers such as tumor-derived extracellular vesicles, and provides an integrated new detection tool for early diagnosis and dynamic monitoring of tumor efficacy.

Description

Technical Field

[0001] This invention relates to the fields of biomedical detection, liquid biopsy, and optical biosensing. More specifically, this invention relates to a bubble-plasma-enhanced fluorescence immunoassay system, its preparation method, applications, and detection methods. Background Technology

[0002] Extracellular vesicles (EVs), a core target in tumor liquid biopsy, are found in low abundance in biological fluids, and their rapid and accurate quantification remains a technical challenge. Enzyme-linked immunosorbent assays (ELISA) and conventional fluorescence immunoassays have limited signal amplification efficiency when detecting tumor-derived EVs, and require cumbersome purification steps such as ultracentrifugation or kit separation, making the process complex and time-consuming. Proteins, lipids, and cell debris in complex biological matrices (such as plasma) can easily cause non-specific interference, affecting the accuracy of detection results. While metal-enhanced fluorescence (MEF) technology can improve fluorescence signal output, traditional MEF systems typically rely on planar substrates or colloidal probes, making it difficult to integrate EV separation and enrichment with signal amplification into a single detection process, thus limiting its translational application in clinical sample testing. Furthermore, existing methods are insufficient in simultaneously achieving high sensitivity, high throughput, and interference resistance, particularly lacking an integrated solution capable of directly separating EVs from complex samples and performing ultrasensitive detection without relying on external force fields (such as centrifugation or magnetic fields). Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a bubble-plasma-enhanced fluorescence immunoassay system that can integrate one-step separation, enrichment, and ultrasensitive fluorescence detection of tumor-derived extracellular vesicles without the need for complex sample pretreatment or external force fields such as centrifugation or magnetic fields. It relies on the dual-mode synergistic amplification of signals through the physical and optical enhancement of hollow glass microbubbles and the metal enhancement of fluorescence by gold nanorods, which greatly improves the detection sensitivity and resistance to matrix interference. At the same time, it is suitable for high-throughput clinical sample detection, providing non-invasive, convenient, and accurate liquid biopsy technology support for early tumor diagnosis and dynamic monitoring of treatment efficacy.

[0005] To achieve these objectives and other advantages of the present invention, a bubble-plasma-enhanced fluorescence immunoassay detection system is provided, comprising immunofunctionalized hollow glass microbubbles and plasma fluorescence probes. The immunofunctionalized hollow glass microbubbles are composed of targeting antibodies covalently linked to the surface of the hollow glass microbubbles, which are used to specifically capture tumor-derived extracellular vesicles and achieve buoyancy separation by relying on their own buoyancy. The plasma fluorescence probe consists of a gold nanorod core, a silica spacer layer wrapped around the surface of the gold nanorod, and a fluorescent dye and a targeting aptamer covalently connected to the surface of the silica spacer layer. It is used to amplify the signal by enhancing fluorescence through metal. Among them, the cavity structure of the immunofunctionalized hollow glass microbubble is used to provide physical optical enhancement, which, together with the metal-enhanced fluorescence provided by the plasma fluorescent probe, constitutes a dual-mode signal amplification of the target. The microbubbles in the hollow glass have a particle size of 10-30 μm, the longitudinal localized surface plasmon resonance peak of the gold nanorods is 600-800 nm, and the thickness of the silica spacer layer is 10-20 nm.

[0006] Preferably, the targeting antibody is one or more of the following: anti-epidermal cell adhesion molecule (EpCAM) antibody, anti-epidermal growth factor receptor (EGFR) antibody, anti-differentiation cluster 24 (CD24) antibody, anti-programmed death ligand 1 (PD-L1) antibody, or anti-differentiation cluster 63 (CD63) antibody; The fluorescent dye is either IRDye 680 near-infrared fluorescent dye (IR680) or IRDye 800CW water-soluble near-infrared fluorescent dye (IR800CW).

[0007] Preferably, the targeting aptamer is a nucleic acid aptamer that specifically recognizes proteins on the surface of extracellular vesicles.

[0008] A method for preparing a bubble-plasma-enhanced fluorescence immunoassay detection system includes the following steps: S1: Hollow glass microbubbles were sequentially activated by piranha solution hydroxylation, modified by 3-mercaptopropyltrimethoxysilane silanization, covalently coupled with targeted antibody, and blocked by bovine serum albumin to obtain immunofunctionalized hollow glass microbubbles. S2: Gold nanorods were prepared by seed growth method. A modified Stuber method was used to coat the surface of the gold nanorods with a silica spacer layer. The coated gold nanorods were then reacted with 3-azidopropyltrimethoxysilane to introduce azide groups. Then, the fluorescent dye and the targeting aptamer were covalently linked to the azide-functionalized silica surface through a copper-free click chemistry reaction to obtain a plasma fluorescent probe. S3: The immunofunctionalized hollow glass microbubbles and the plasma fluorescent probe are used as complementary functional components, and are independently packaged and combined to obtain a bubble-plasma-enhanced fluorescence immunoassay detection system.

[0009] Preferably, in step S1, the hydroxylation activation is performed by immersion in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 7:3 for 0.5-1.5 h. The silanization modification uses an ethanol solution of 3-mercaptopropyltrimethoxysilane, and the reaction is carried out by rotation at room temperature for 1-3 h; the covalent coupling of the targeting antibody is carried out by rotating the silanized hollow glass microbubbles with a concentration of 1-10 μg / mL of the targeting antibody at room temperature for 1-3 h, and then blocking with 1-5 mg / mL of bovine serum albumin for 0.5-1.5 h.

[0010] Preferably, the improved Stobol process is as follows: gold nanorods are dispersed in water, the pH is adjusted to 7.5-8.0, heated to 40-45 °C, and an ethanol solution of tetraethyl orthosilicate is added in three portions, with an interval of 30-40 min between each addition. After the addition is complete, the reaction continues for 12-18 h to obtain gold nanorod@silica (AuNR@SiO2) core-shell particles with a silica spacer layer thickness of 10-20 nm.

[0011] Preferably, in step S3, the immunofunctionalized hollow glass microbubbles are dispersed in phosphate buffer, bovine serum albumin is added to a final concentration of 1-5 mg / mL, blocked at room temperature for 30-90 min, washed, resuspended, and individually aliquoted; the plasma fluorescent probe is dispersed in phosphate buffer containing 0.05-0.1 volume percentage Tween-20 and individually aliquoted; the two individually aliquoted components together constitute the detection system.

[0012] Application of a bubble-plasma-enhanced fluorescence immunoassay system in the preparation of ultrasensitive detection kits for tumor-derived extracellular vesicles, circulating tumor cells, or proteins.

[0013] Preferably, the test sample is cell culture supernatant or human plasma.

[0014] A method for detecting tumor-derived extracellular vesicles for non-disease diagnostic purposes based on bubble-plasma-enhanced fluorescence, employing the aforementioned bubble-plasma-enhanced fluorescence immunoassay system, includes the following steps: 1) The sample to be tested is mixed with immunofunctionalized hollow glass microbubbles and incubated to allow the immunofunctionalized hollow glass microbubbles to specifically capture tumor-derived extracellular vesicles in the sample to be tested. 2) Add the plasma fluorescent probe to the incubated mixture and continue incubation to allow the plasma fluorescent probe to specifically bind to tumor-derived extracellular vesicles captured on the surface of immunofunctionalized hollow glass microbubbles, forming a recognition complex; 3) The mixture is left to stand. The recognition complex is floated to the surface of the liquid by the buoyancy of the immunofunctionalized hollow glass microbubbles. The lower layer of liquid is removed, and the mixture is washed and resuspended. The signal is collected by fluorescence imaging to achieve centrifugation-free separation and quantitative detection of the target.

[0015] The present invention has at least the following beneficial effects: The bubble-plasma-enhanced fluorescence immunoassay (BP-FLISA) detection system of this invention uses immunofunctionalized hollow glass microbubbles as targets to capture, separate, and enrich carriers, and uses plasma fluorescent probes as specific recognition and signal amplification units to construct a complete immunosensing mechanism integrating "specific capture - buoyancy separation - dual-mode signal amplification - ultrasensitive detection": First, immunofunctionalized hollow glass microbubbles, activated by hydroxylation with piranha solution, modified with 3-mercaptopropyltrimethoxysilane silanization, and covalently coupled with targeting antibodies, can achieve highly specific recognition and efficient capture of tumor-derived extracellular vesicles in samples such as cell culture supernatants or human plasma through high-density fixed anti-EpCAM, anti-EGFR, and other targeting antibodies on their surface. At the same time, relying on the buoyancy self-driving characteristics of their low-density hollow structure with a particle size of 10-30µm, they can quickly and spontaneously float to the liquid surface after capturing the target, without any external force field assistance such as centrifugation, magnetic field, or filtration, to complete the rapid separation of the target and the sample matrix. While completing the target enrichment in one step, it effectively removes non-specific interference from complex matrices such as proteins, lipids, and cell debris in plasma. Subsequently, the longitudinal localized surface plasmon resonance peak is located at 600-800. With a core of 10-20 nm gold nanorods, a 10-20 nm thick silica spacer layer, and a copper-free click-covalently linked near-infrared fluorescent dye such as IR800CW and a plasma fluorescent probe with a targeting aptamer, this complex can specifically bind to surface target proteins of tumor-derived extracellular vesicles captured on the surface of microbubbles, forming a sandwich immune complex of "immunofunctionalized hollow glass microbubbles - tumor-derived extracellular vesicles - plasma fluorescent probe". In this process, the gold nanorods maintain the optimal interaction distance with the fluorescent dye through the precisely regulated silica spacer layer, and their local surface plasmon resonance effect can generate a highly efficient metal-enhanced fluorescence effect, significantly improving the fluorescence signal compared to free fluorophores while avoiding fluorescence quenching. The cavity structure of the hollow glass microbubbles can generate a unique physical optical enhancement effect through light scattering and microcavity resonance, further enhancing the intensity of the local excitation light field. This, together with the metal-enhanced fluorescence effect of the gold nanorods, forms a dual-mode synergistic signal amplification mechanism, significantly improving the detection signal-to-noise ratio. Finally, accurate quantitative detection of tumor-derived extracellular vesicles can be achieved through near-infrared fluorescence imaging.

[0016] Based on the above detection principle, this invention achieves multi-dimensional technological breakthroughs and performance improvements compared to existing extracellular vesicle detection technologies. Its core beneficial effects are reflected in: Firstly, this invention is the first to synergistically couple the buoyancy separation and enrichment of hollow glass microbubbles, physical optical enhancement, and metal-enhanced fluorescence mediated by gold nanorods, realizing the integrated process of separation, enrichment, and detection of tumor-derived extracellular vesicles. It eliminates the need for cumbersome ultracentrifugation or pre-purification steps using commercial reagent kits, greatly simplifying the operation process and shortening the detection cycle. At the same time, the capture efficiency of tumor-derived extracellular vesicles can reach about 70%, which is significantly better than traditional separation methods and effectively improves the enrichment capacity of low-abundance targets in body fluids. Secondly, relying on the dual-mode synergistic signal amplification mechanism, this invention achieves ultra-high detection sensitivity, with a detection limit as low as 187 particles / mL and a linear range spanning 5 orders of magnitude. It can achieve single-molecule-level detection capability without nucleic acid amplification, and can accurately identify tumor-derived extracellular vesicles in extremely low abundance in the body fluids of early-stage tumor patients, providing reliable technical support for early tumor diagnosis and dynamic monitoring of treatment efficacy. Third, this invention has excellent resistance to matrix interference and detection repeatability. Even in complex biological matrices with a high concentration of 10% serum, it can still maintain stable signal output and can be directly used for the detection of clinical human plasma samples. At the same time, it maintains extremely low signal fluctuation when performing 384 high-throughput repeated detections on the same sample, and has excellent batch consistency and method stability, which is fully adapted to the standardized detection needs of large clinical samples and multiple batches. Fourth, the materials used in this invention have excellent versatility and stability. The plasma fluorescent probe can be stably stored for more than one month without significant fluorescence signal decay under light-protected conditions at 4 ℃, solving the pain points of easy degradation and quenching of traditional fluorescent probes. Furthermore, the surface of hollow glass microbubbles can be flexibly coupled with various recognition units such as antibodies, aptamers, and peptides through silanization, click chemistry, etc. It can not only be used for the detection of tumor-derived extracellular vesicles, but can also be further extended to the separation and analysis of various targets such as circulating tumor cells, bacteria, proteins, and miRNAs. With a wide range of applications, it provides a brand-new integrated detection tool for non-invasive liquid biopsy of tumors.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the process for detecting cancer-derived EV surface proteins using high-throughput bubble-plasma enhanced fluorescence immunoassay (BP-FLISA) as described in this invention. Figure 2The images show the fluorescence characterization of the immunofunctionalized hollow glass microbubbles of the present invention; where a is the fluorescence intensity statistics of the control group containing the unmodified antibody and the EpCAM antibody-modified group; b is the fluorescence micrograph of the EpCAM antibody-modified group; and c is the fluorescence micrograph of the control group without the modified antibody. Figure 3 The images show the fluorescence characterization of EVs captured by the immunofunctionalized hollow glass microbubbles of the present invention; where a is the fluorescence intensity statistics of the fluorescence microscopy results of the blank control group containing IgG modification and the EpCAM immunobubble group after capturing DiI-labeled EVs; b is the fluorescence microscopy image of the EpCAM immunobubble group after capturing DiI-labeled EVs; and c is the fluorescence microscopy image of the blank control group containing IgG modification. Figure 4 The images shown are scanning electron microscope (SEM) images of the immune-functionalized hollow glass microbubbles capturing EVs according to the present invention; where a is the SEM image of the control group at a scale bar of 2 μm; b is the SEM image of the control group at a scale bar of 500 nm; c is the SEM image of the EpCAM group at a scale bar of 2 μm; and d is the SEM image of the control group at a scale bar of 500 nm. Figure 5 This is a graph showing the results of using nanoparticle tracking analysis (NTA) to detect EV concentrations before and after immune bubble capture in this invention. Figure 6 This is a schematic diagram of the preparation process of the plasma fluorescent probe of the present invention; Figure 7 The UV-Vis absorption spectrum of the AuNR prepared in this invention; Figure 8 The images show the morphology and size characteristics of the AuNR prepared in this invention; where a is a transmission electron microscope (TEM) morphology image of the AuNR; and b is a histogram of the particle size distribution of the length and width of the AuNR and a Gaussian fitting curve, used to characterize the morphological uniformity and size distribution characteristics of the AuNR. Figure 9 Characterization images of the AuNR@SiO2 core-shell structure prepared in this invention are shown below; where a is a low-magnification transmission electron microscope (TEM) image of AuNR@SiO2; b is a high-magnification TEM image of a single particle; c is a superimposed distribution map of Au and Si elements; d and e are EDS surface scan distribution maps of Au and Si elements, respectively; f is the EDS line scan intensity distribution curve of Au and Si elements along the short axis of AuNR@SiO2. Figure 10 This is a graph showing the zeta potential changes of each intermediate during the synthesis of the plasma fluorescent probe of this invention. Figure 11 This is a comparison of the fluorescence performance of the plasma fluorescent probe of this invention and the free IR800CW; Figure 12 This is a feasibility analysis diagram of the BP-FLISA system for detecting tumor-derived EVs according to the present invention; Figure 13 This is a sensitivity analysis diagram of the BP-FLISA system for detecting tumor-derived EVs according to the present invention; Figure 14 This is a diagram illustrating the anti-interference capability analysis of the BP-FLISA system of this invention. Figure 15 This is a high-throughput detection repeatability analysis diagram of the BP-FLISA system of the present invention; where a is a statistical diagram of fluorescence intensity distribution of 384 repeated detections; and b is a density cloud diagram of the corresponding detection signal. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0021] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0022] This invention, based on a bubble-plasma enhanced fluorescence immunoassay platform, constructs an integrated ultrasensitive detection technology route for tumor-derived extracellular vesicles. For example... Figure 1 As shown, this approach first uses hollow glass microbubbles as a substrate, and through hydroxylation activation, silanization modification, and targeted antibody conjugation, prepares immunofunctionalized hollow glass microbubbles capable of specifically capturing targets. Simultaneously, gold nanorods are synthesized using a seed growth method, encapsulated with a silica spacer layer for azide functionalization, and then coupled with a near-infrared fluorescent dye and a targeting aptamer via copper-free click chemistry to prepare a plasma fluorescence signal amplification probe. Finally, the sample to be tested is incubated with the immunobubbles to achieve target capture, and the probe is added to form a sandwich immune complex. Centrifugation-free separation is achieved using the buoyancy of the microbubbles, and quantitative fluorescence detection of the target is completed based on the dual-mode synergistic effect of bubble physical-optical enhancement and gold nanorod metal-enhanced fluorescence.

[0023] Example 1: Preparation of Immunofunctionalized Hollow Glass Microbubbles 1) Weigh 0.5 g of hollow glass microbubbles (GB) pretreated with piranha solution (H2O2 to H2SO4 volume ratio of 3:7) and add them to a 15 mL centrifuge tube. Add 6 mL of anhydrous ethanol containing 20 µL of 3-mercaptopropyltrimethoxysilane (MPTMS) and mix at room temperature on a rotary mixer for 2 h. After the reaction is complete, centrifuge at 1000 rpm for 1 min, collect the floating microbubbles in the upper layer, and remove the lower layer solution. Wash with ultrapure water, centrifuge again, repeat the washing 5 times, and dry under vacuum at 80 ℃ to obtain silanized hollow glass microbubbles GB@MPTMS. The hollow glass microbubbles are iM30K hollow glass microbubbles manufactured by 3M. 2) Weigh 2 mg of the silanized hollow glass microbubbles from step 1) into an EP tube, add EpCAM antibody (5 µg, 500 µL, concentration 10 µg / mL), mix at room temperature on a rotary mixer for 2 h, centrifuge at 1000 rpm for 1 min after the reaction is complete, remove the lower layer solution; add phosphate Tween buffer (PBST) to wash, centrifuge again, and repeat the washing three times.

[0024] 3) Weigh 5 mg of bovine serum albumin (BSA), dissolve it in 1 mL of phosphate-buffered saline (PBS), add it to step 2), mix at room temperature on a rotary mixer for 1 h, centrifuge at 1000 rpm for 1 min, collect the upper floating microbubbles, remove the lower layer solution; add PBST to wash, centrifuge again, repeat washing three times to obtain immunofunctionalized hollow glass microbubbles (GB@BSA@Tz) with targeted enrichment function, i.e., the immunofunctionalized glass microbubbles, and store at 4 ℃.

[0025] Example 2: Fluorescence characterization of immunofunctionalized hollow glass microbubbles To verify the successful preparation of immunofunctionalized hollow glass microbubbles (immuno-GB), specifically whether the EpCAM antibody was successfully covalently coupled to the MPTMS-modified GB surface, a characterization experiment was performed using fluorescence microscopy. The specific steps are as follows: 1. Sources of experimental materials and reagents Hollow glass microspheres (GB): 3M iM30K, particle size 10-30 µm; Anti-human EpCAM polyclonal antibody: Wuhan Sanying Biotechnology Co., Ltd.; Cy5 fluorescently labeled goat anti-rabbit IgG secondary antibody: Wuhan Sanying Biotechnology Co., Ltd., excitation wavelength 647 nm, emission wavelength 667 nm; PBST washing buffer: 0.01 M PBS (pH 7.4) containing 0.1% Tween-20; Blocking solution: 2% bovine serum albumin (BSA) / PBS.

[0026] 2. Experimental Procedure S1: Preparation of thiolized GB: GB was activated with piranha solution (H2O2 to H2SO4 volume ratio of 3:7), and then silanized with 2% MPTMS ethanol solution. The reaction was carried out at room temperature by rotation for 2 h, washed three times with ultrapure water, and dried under vacuum at 80 °C to obtain MPTMS-modified GB.

[0027] S2: Weigh 2 mg of GB from step S1, disperse it in 1 mL of PBS, add 10 μg of EpCAM antibody (500 µL, concentration 10 µg / mL), and incubate at room temperature for 2 h to allow the antibody to couple to the GB surface. Control group: Under the same conditions, no EpCAM antibody was added, and all other treatments were exactly the same.

[0028] S3: After washing away unbound antibodies and incubation, centrifuge at 1000 rpm for 1 min, remove the lower layer liquid, and wash 3 times with 200 µL PBST to thoroughly remove unconjugated free antibodies.

[0029] S4: Fluorescent secondary antibody incubation. Add 100 µL of Cy5 fluorescent secondary antibody diluted 1:500 to each of the two GB groups and incubate at room temperature in the dark for 15 min.

[0030] S5: Washing and Imaging: Wash three times with PBST to remove unbound secondary antibody. Add GB to the slide and collect fluorescence signal using a fluorescence inverted microscope under 647 nm excitation.

[0031] The results are as follows Figure 2 As shown, in the experimental group (with EpCAM antibody): GB showed continuous and bright Cy5 red fluorescence at the edge, and the fluorescence signal was uniformly distributed on the surface of the microspheres. Figure 2 b) indicates that the EpCAM antibody has been successfully and stably coupled to the GB surface; control group (no antibody added): no obvious fluorescence signal was observed on the surface of the microspheres, only the bright field outline of the microspheres themselves was observed ( Figure 2 c) indicates that the fluorescent secondary antibody cannot bind non-specifically, resulting in extremely low background in the system. The statistical results of the fluorescence intensity for the two groups are shown below. Figure 2 a.

[0032] Example 3: Fluorescent characterization of immunofunctionalized hollow glass microbubbles capturing extracellular vesicles (EVs) 1. DiI cell membrane fluorescent dye labeling EV Add 100 µL EV (1×10⁻⁶) to the EP tube. 8The sample was reacted with 98 µL of PBS and 2 µL of LDiI (an orange-red fluorescent lipophilic membrane dye, 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole carbonylcyanine iodide) for 20 min, followed by the addition of 40 µL of BSA (10%) and a further 20 min reaction to adsorb excess dye. The mixture was then transferred to a 600 µL ultrafiltration tube and centrifuged at 9000 g for 10 min. This process was repeated three times, and the final volume was adjusted to 100 µL. 20 µL of LExoQuick-TC was then added and incubated overnight at 4 °C. The mixture was then centrifuged at 1500 g for 30 min, resuspended in 100 µL of PBS, and aliquoted for use.

[0033] 2. Immunization against GB-induced DiI-EV Experimental group: 2 mg of EpCAM was used to immunize GB, 100 μL of PBS was added to resuspend the immunized immunization, 100 μL of the above DiI-EV suspension was added, and the immunization was incubated with gentle shaking at room temperature for 40 min.

[0034] Control group: Blank control GB modified with IgG, all other conditions were exactly the same.

[0035] 3. Buoyancy self-separation and washing After incubation, let stand for 3 min to allow the GB-EV complex to float rapidly; remove the lower layer of liquid and wash 3 times with 200 μL PBST to thoroughly remove unbound free EVs and impurities.

[0036] 4. Fluorescence Imaging and Signal Analysis GB was placed on polydimethylsiloxane (PDMS) and observed using a fluorescence microscope at 549 nm excitation and 565 nm emission.

[0037] 5. Results as follows Figure 3 As shown: In the EpCAM-immunized GB group, strong and dense red fluorescence appeared on the surface of the microspheres. Figure 3 (b) indicates that a large number of DiI-EVs were specifically captured and enriched. IgG control group: only very weak background fluorescence was observed, with no obvious EV binding signal ( Figure 3 c), the differences between the two groups were significant. The corresponding fluorescence intensity statistics are shown in [the table below]. Figure 3 a.

[0038] The results showed that EpCAM-modified immunoglobulin (IGG) can specifically and efficiently recognize, bind to and enrich tumor-derived EVs, exhibiting excellent targeted separation and enrichment performance, and can be directly used for the rapid capture of low-abundance EVs in complex biological samples.

[0039] Example 4: Scanning electron microscopy (SEM) characterization of immunofunctionalized hollow glass microbubbles capturing EVs To more clearly demonstrate the successful capture of EVs by the antibody-modified GB, SEM was used to observe the EV capture on the GB surface. First, pre-stained EVs were captured by the EpCAM antibody-modified GB. To maintain the original morphology and structure of the EVs and prevent deformation or degradation in subsequent processing, the EVs were fixed in 2.5% glutaraldehyde (GA) solution overnight at 4 °C. After fixation, to remove moisture and prevent damage or deformation of the sample during electron microscopy, a gradient ethanol dehydration process was performed. The samples were dehydrated sequentially with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol (twice), each time allowing the sample to stand at room temperature for 10 min. After dehydration, the samples were dried in a vacuum drying oven for 12 h to remove residual ethanol. Then, the samples were adhered to conductive adhesive and sputtered with gold to increase conductivity. Finally, the samples were placed on a sample stage for observation.

[0040] The results are as follows Figure 4 As shown, GB modified with IgG antibodies does not have obvious vesicle structures on its surface. Figure 4 a and b). On the other hand, a large number of EVs are uniformly distributed on the EpCAM antibody-modified GB surface ( Figure 4 (c and d) indicate that GB has a good separation and enrichment ability for EV. Further magnification of the images shows that EV exhibits a typical concave morphology due to dehydration and fixation, which is a characteristic structure of EV after dehydration treatment.

[0041] Example 5: Efficiency of separating EVs using microbubbles from immunofunctionalized hollow glass To verify the efficiency of immunofunctionalized hollow glass microbubbles in separating EVs, immunofunctionalized hollow glass microbubbles (50 μL, 2 mg / mL) were mixed with 50 μL of HCT116 cell-derived EVs (concentration 10). 8 Incubate with the nanoparticles / mL solution for 60 min; after the reaction is complete, allow the complex to float to the top of the solution and take 50 μL of the lower layer solution for nanoparticle tracking analysis; finally, calculate the capture efficiency according to the formula: capture efficiency = (EV number before capture - EV number after capture) / EV number before capture × 100%.

[0042] like Figure 5 As shown, the efficiency of separating EVs using microbubbles from immunofunctionalized hollow glass is approximately 70%.

[0043] For EVs derived from other tumors such as liver cancer, lung cancer, and breast cancer, immunofunctionalized hollow glass microbubbles also exhibited similar excellent efficacy to the EV capture assays derived from HCT116 cells described above. Specific experimental details and results will not be elaborated here.

[0044] Example 6: Preparation of Plasma Fluorescence (PF) Probe 1. Preparation of gold nanorods (AuNR) 1) Preparation of gold seed solution Add 0.6 mL of ice-cold sodium borohydride (NaBH4, 10 mM) solution to a mixed solution containing 25 μL of chloroauric acid (HAuCl4·3H2O, 100 mM) solution and 9.75 mL of hexadecyltrimethylammonium bromide (CTAB, 0.1 M), and then stir vigorously. The solution color will change from yellow to brown, indicating that the preparation of the gold seed solution is complete.

[0045] 2) Preparation of AuNR growth solution and synthesis reaction 2 mL of chloroauric acid (0.01 M) aqueous solution, 48 mL of CTAB (0.1 M), 450 μL of silver nitrate (AgNO3, 0.01 M), 0.8 mL of hydrochloric acid (HCl, 1 M), and 280 μL of ascorbic acid (AA, 0.1 M) were added sequentially to a clean container. After each addition of a reagent, the mixture was gently mixed to prepare the AuNR growth solution. Then, 20 μL of the prepared gold seed solution was added to the growth solution. After gently inverting and mixing, the container was placed in a dark environment and left to stand at room temperature overnight to complete the growth and synthesis of AuNR.

[0046] 3) Purification and preservation of AuNR The synthesized AuNR solution was centrifuged at 5000 g for 20 min at 25 °C to remove the supernatant and remove unreacted CTAB, silver ions and other impurities from the solution. The AuNR precipitate obtained by centrifugation was redispersed with ultrapure water, and the above centrifugation and washing steps were repeated twice. Finally, the AuNR was redispersed in ultrapure water, and the absorbance of the solution at a wavelength of 780 nm was adjusted to about 1.5 (corresponding to a gold nanorod concentration of 2 nM). It was then stored at 4 °C in the dark for later use. Figure 6 This is a schematic diagram of the preparation process of the plasma fluorescent probe of the present invention.

[0047] Transmission electron microscopy characterization revealed that the prepared AuNR exhibited a uniform and regular rod-shaped morphology, with no obvious agglomeration or impurities. The average size was 79±4 nm in length and 20±1 nm in width, with a stable and controllable aspect ratio. The longitudinal local surface plasmon resonance peak was located at 780 nm, which meets the design requirement of 600-800 nm, providing a core material basis for the subsequent preparation of plasma fluorescent probes.

[0048] 2. Coating with a silicon dioxide layer to obtain AuNR@SiO2 First, take 10 mL of AuNR solution and add 125 μL of sodium hydroxide (NaOH, 0.1 M) to adjust the pH to 7.6. Equilibrate at 40 °C for 30 min. Then, add a mixture of tetraethyl orthosilicate (TEOS) and ethanol (10%) in three portions, 45 μL each time, every 30 min. React overnight at 40 °C. Centrifuge the synthesized AuNR@SiO2 at 25 °C with a centrifugal force of 5000 g for 20 min, remove the supernatant, and disperse the precipitate in ethanol for later use.

[0049] 3. Silanization introduces azide groups. 1 mL of 2% (v / v) 3-azidopropyltrimethoxysilane ethanol solution was added to the AuNR@SiO2 ethanol dispersion prepared above. After mixing, the mixture was heated at 80 °C for 10 h to ensure complete azide functionalization of the AuNR@SiO2 surface. After the reaction was completed, the mixture was purified by centrifugation at 7000 g for 20 min. The supernatant was discarded, and the precipitate was redispersed with ethanol. The mixture was centrifuged and washed three times to completely remove excess unreacted azides, and the azide-functionalized AuNR@SiO2@N3 was obtained and dispersed in ethanol for later use.

[0050] 4. Click to chemically couple IR800CW with aptamer Take 1 mL of 2 nM AuNR@SiO2@N3 dispersion, add 50 µL of 10 µM DBCO-modified nucleic acid aptamers (Apt-DBCO) targeting EpCAM, EGFR, PD-L1, and other proteins on the EV surface, and mix at room temperature for 30 min. Then add 50 µL of 10 µM DBCO-modified near-infrared fluorescent dye IR800CW (IR800-DBCO) to the reaction system, mix at room temperature in the dark, and complete the covalent coupling of the fluorescent dye and aptamer through a copper-free click chemistry reaction. After the reaction, centrifuge at 7000 g for 20 min, discard the supernatant, and resuspend the precipitate in 10 mL of PBS solution; then centrifuge a second time at 15000 g for 20 min to completely remove unbound free IR800-DBCO and aptamer, finally obtaining the plasma fluorescent (PF) probe, which is stored at 4 ℃ in the dark for later use.

[0051] Example 7: UV-Vis spectroscopic and fluorescence spectral characterization of AuNR The prepared AuNR colloidal solution was subjected to full-spectrum absorption spectroscopy scanning using a UV-Vis spectrophotometer, with a scanning range of 400-900 nm. Simultaneously, the excitation and emission spectra of the near-infrared fluorescent dye IR800CW were measured using a fluorescence spectrophotometer. The longitudinal localized surface plasmon resonance (LSPR) absorption peak of AuNR was compared with the excitation and emission peaks of IR800CW to evaluate the resonance matching degree between the two.

[0052] The results are as follows Figure 7 As shown, the LSPR absorption band of AuNR highly overlaps with the excitation band of IR800CW, forming an excellent spectral resonance match, which can maximize the metal-enhanced fluorescence effect.

[0053] Example 8: Transmission Electron Microscopy (TEM) Characterization of AuNR To verify the morphology, size uniformity, and dispersion of the AuNRs prepared in this invention, TEM was used to characterize the morphology and perform statistical analysis on the size of the AuNRs. The specific experimental steps and results are as follows: S1: Sample preparation Take an appropriate amount of AuNR colloidal solution and dilute it with ultrapure water to a suitable concentration; take 10 µL of the diluted sample and drop it onto the copper grid of the carbon support film, let it dry naturally at room temperature, and prepare the TEM test sample.

[0054] S2: TEM morphology imaging uses transmission electron microscopy to image the AuNR sample, acquiring the overall morphology of AuNR and high-resolution images of individual particles. Figure 8 a) Observe the morphological characteristics and dispersion state of AuNR.

[0055] S3: Size Statistical Analysis. Randomly select no fewer than 200 AuNR particles from the field of view, and use ImageJ software to measure and statistically analyze their length and width, and plot the size distribution histogram and Gaussian fitting curve. Figure 8 b) Calculate the average size and standard deviation of AuNR.

[0056] The prepared AuNR exhibits a uniform and regular rod-shaped morphology, without obvious agglomeration, deformation, or impurity particles. Figure 8 a) The particles are well dispersed with no obvious aggregation, providing a structural basis for subsequent SiO2 coating, functionalization modification and plasma fluorescence effect; the size distribution shows typical normal distribution characteristics, proving that the AuNR prepared by this invention has excellent size uniformity and batch-to-batch consistency.

[0057] This invention successfully prepared gold nanorods with regular morphology, uniform size, and excellent dispersibility, with an average size of 79±4 nm (length) and 20±1 nm (width). The aspect ratio is stable and controllable, providing a core material basis for the subsequent construction of AuNR@SiO2 core-shell structures, plasma fluorescence probes, and BP-FLISA detection systems. This invention also verifies the rationality and reliability of the AuNR preparation process.

[0058] Example 9: TEM characterization of AuNR@SiO2 To verify the successful construction and morphological uniformity of the AuNR@SiO2 core-shell structure of the plasma fluorescence (PF) probe of this invention, morphology and elemental distribution characterization were carried out using transmission electron microscopy (TEM) combined with energy dispersive spectroscopy (EDS). The specific experimental steps and results are analyzed as follows: S1: Sample preparation Take an appropriate amount of AuNR@SiO2 colloidal solution and dilute it with ultrapure water to a suitable concentration; take 10 μL of the diluted sample and drop it onto the carbon support film copper grid, let it dry naturally at room temperature, and prepare the TEM test sample.

[0059] S2: TEM morphology characterization was performed using transmission electron microscopy at an accelerating voltage of 200 kV to image the AuNR@SiO2 sample, acquiring high-resolution TEM images of individual nanoparticles to observe the core-shell structure morphology and coating thickness.

[0060] S3: EDS elemental distribution characterization. Energy dispersive X-ray spectroscopy (EDS) surface scan analysis was performed on AuNR@SiO2 particles in the TEM field of view to collect distribution maps of gold (Au) and silicon (Si) elements, and to verify the correspondence between elemental distribution and core-shell structure.

[0061] S4: Line scan element analysis along Figure 9 The scanning path marked by the yellow arrow in the middle is used for EDS line scan analysis, and the intensity distribution curves of Au and Si elements along the scanning path are collected. Figure 9 f), quantitatively verifying the elemental distribution trend of the core-shell structure.

[0062] The results are as follows Figure 9As shown, a) is a low-magnification transmission electron microscope (TEM) image of AuNR@SiO2; b) is a high-magnification TEM image of a single particle, where the gold nanorod core presents a high-contrast dark area and the silica coating layer is a low-contrast bright area, with clear interfaces and uniform coating; c) is a superimposed distribution map of Au and Si elements, with yellow arrows indicating the line scan path; d and e are EDS surface scan distribution maps of Au and Si elements, respectively. Au elements are concentrated in the rod-shaped core region, while Si elements are uniformly distributed in the outer coating region. Their positions correspond one-to-one with the core-shell structure in the TEM image, further verifying the elemental distribution characteristics of the core-shell structure. The above results fully demonstrate that the present invention successfully prepared a well-formed, uniformly coated, and structurally complete AuNR@SiO2 core-shell nanostructure. The gold nanorod core is completely wrapped by a uniformly thick SiO2 spacer layer, with clear elemental distribution and distinct interfaces. This provides a solid structural foundation for the subsequent realization of azidofunctionalization, fluorescent dye coupling, and metal-enhanced fluorescence effects, verifying the rationality and reliability of the PF probe core structure.

[0063] Example 10: Zeta potential characterization of the PF probe To verify the surface charge changes and reaction progress of each synthesis step of the plasma fluorescence (PF) probe of this invention, the key intermediates in the PF probe preparation process were characterized using Zeta potential (dynamic light scattering, DLS) technology. The specific experimental steps and results analysis are as follows: The original zeta potential of AuNR was measured by diluting an appropriate amount of AuNR colloidal solution with ultrapure water to a suitable concentration, transferring it to a zeta potential sample cell, and measuring the zeta potential value under constant temperature conditions of 25 °C. The same method was used for AuNR@SiO2, azide-functionalized (AuNR@SiO2@N3), and plasma fluorescence (PF).

[0064] The results are as follows Figure 10 As shown, the positive zeta potential of AuNR decreased from 37.1 mV to -3.1 mV due to the presence of silanol groups after coating the SiO2 shell. After azide functionalization, the surface charge of the nanorods approached zero due to the presence of neutral azides. Finally, after loading with IR800CW, the potential decreased to approximately -6.2 mV, indicating that IR800CW-DBCO affected the negatively charged surface when loaded onto the SiO2 shell.

[0065] Example 11: Fluorescence performance test of PF To verify the fluorescence enhancement efficiency and optical performance of the PF constructed in this invention compared to a single free fluorescent dye, a fluorescence performance comparison experiment was conducted using the near-infrared fluorescent dye IR800CW as a reference. The specific steps are as follows: S1: Sample preparation: Prepare PF suspension and free IR800CW dye solution with the same molar concentration. The concentration of each solution is adjusted to the detectable linear range. Three replicates are set for each group.

[0066] S2: Fluorescence signal acquisition. The two types of samples were added to a 96-well plate, and imaging was performed using a near-infrared fluorescence imaging system at an excitation wavelength of 780 nm and an emission wavelength of 800 nm. The fluorescence intensity values ​​of each sample were recorded.

[0067] S3: Data comparison and analysis: Extract the average fluorescence intensity data of the PF probe group and the free IR800CW group, calculate the fluorescence enhancement factor, and evaluate the signal amplification efficiency of the PF probe.

[0068] The results are as follows Figure 11 As shown, the bar chart and fluorescence imaging clearly demonstrate the comparison of fluorescence signals between the two systems: The PF group (PF) detected extremely high intensity near-infrared fluorescence signals, which were about 1,000 times higher than those of the free IR800CW group.

[0069] Example 12: BP-FLISA detection of EV performance 1. Add immunoglobulin GB (50 µL, 2 mg / mL), different concentrations of EV samples (10 µL), and PBS buffer (40 µL) sequentially to each well of a 96-well plate, for a total reaction volume of 100 µL / well. Incubate the 96-well plate at room temperature on a microplate shaker. After the reaction, wash each well three times with PBST buffer (200 µL) containing 0.1% (v / v) Tween-20 to remove unbound free components.

[0070] 2. Add 10 µL of plasma-fluorescent probe (2 pM) to each well and incubate the reaction at room temperature in the dark on a shaker. After the reaction is complete, wash three times with 200 µL of PBST buffer containing 0.1% (v / v) Tween-20.

[0071] 3. Add 100 µL of PBS buffer (pH 7.4) to each well, and use an eight-channel pipette to aspirate 10 µL of the post-reaction sample and spot it onto the PDMS cover plate. Use a near-infrared imaging system to perform scanning imaging.

[0072] 4. Establish a standard curve with a linear range of 10. 2 -10 6 particles / mL, LOD=187 particles / mL.

[0073] Example 13: Feasibility Study of BP-FLISA To verify the detection feasibility and component synergistic effect of the bubble-plasma enhanced fluorescence immunoassay (BP-FLISA) system constructed in this invention, control systems with different component deficiencies were set up for fluorescence signal comparison analysis. The specific experimental steps are as follows: 1. The system was configured with four parallel control groups, each with three replicates: 1) Complete system group: immune GB + extracellular vesicles derived from human colorectal cancer HCT116 cells (HCT116 EV) + PF probe (all components present); 2) Antibody-deficient group: Blank GB+HCT116 EV+PF probe without modified antibody; 3) Group lacking EV: Immunized with GB + PBS without EV + PF probe; 4) Adaptorless probe group: Immunoglobulin GB + HCT116 EV + PF blank probe without aptor (Apt).

[0074] 2. Experimental Procedure S1: Add immunoglobulin GB (50 µL, 2 mg / mL) to each well of a 96-well plate, followed by 40 µL of PBS; S2: Add 10 μL of HCT116 EV suspension (1×10⁻⁶) to the corresponding group. 7 (particles / mL), the EV-free group was treated with an equal volume of PBS; S3: Incubate at room temperature with shaking for 40 min, let stand for 2 min to separate by buoyancy, and wash 3 times with PBST; S4: Add 10 μL of PF probe to the corresponding group, and add an equal amount of aptamerless PF probe to the blank probe group; S5: Incubate at room temperature in the dark for 30 min, then wash again to remove unbound probes; S6: Transfer each group of GB to a PDMS substrate, acquire signals using a near-infrared fluorescence imaging system, and record fluorescence intensity.

[0075] 3. Results are as follows Figure 12 As shown: Complete system group: exhibits significantly enhanced fluorescence signal, with the highest fluorescence intensity; Antibody-deficient group: unable to effectively capture EVs, fluorescence signal significantly reduced; EV-deficient group: No target is present, probe cannot bind, and only a weak background signal appears; The probe group lacking aptamer: could not specifically recognize EV surface antigens, and the fluorescence signal was greatly weakened.

[0076] Statistical analysis showed that the absence of any key component would cause a significant decrease in fluorescence signal, and only when the immunogens GB, EV, and PF probes were present together could a strong and stable fluorescence signal be generated.

[0077] The above results confirm that the BP-FLISA sensing system constructed in this invention has a clear function, synergistic components, strong specificity, and clear mechanism: hollow glass microspheres (GB) provide a support platform for target capture and optical enhancement, gold nanorods (AuNR) realize metal-enhanced fluorescence signal amplification, and the target aptamer (Apt) ensures recognition specificity. The three work together to achieve highly sensitive and specific detection of EVs.

[0078] Example 14: Sensitivity test of BP-FLISA To verify the detection sensitivity of the bubble-plasma-enhanced fluorescence immunoassay (BP-FLISA) system constructed in this invention, tumor-derived extracellular vesicles (EVs) from human colorectal cancer HCT116 cells were used as the detection target. A gradient concentration EV detection experiment was conducted under optimal detection conditions, and the specific steps are as follows: S1: Add immunoglobulin GB (50 µL, 2 mg / mL) to each well of a 96-well plate, followed by sequentially adding gradient concentrations of HCT116EV suspension (1.87 × 10⁻⁶). 2 1.87×10 3 1.87×10 4 1.87×10 5 1.87×10 6 (particles / mL), with 4 replicate wells in each group, and an equal volume of sterile PBS added to the blank group; S2: Incubate at room temperature with shaking for 40 min, then use the buoyancy self-floating property of GB for rapid separation, wash 3 times with PBST to remove unbound free EVs; S3: Add an equal amount of plasma fluorescent probe, incubate at room temperature in the dark for 30 min, and wash again to remove unbound probe; S4: Transfer the reaction products to a PDMS substrate, acquire signals using a near-infrared fluorescence imaging system, and record the fluorescence intensity of each group; S5: Plot a standard curve with the logarithm of EV concentration on the x-axis and the corresponding signal value on the y-axis, and calculate the detection linear range and the limit of detection.

[0079] Under optimal detection conditions, the detection results of the BP-FLISA system are as follows: Figure 13 As shown: With HCT116 EV concentration at 1.87 × 10⁻⁶ 2 ~1.87×10 6As the concentration of particles / mL gradually increases, the fluorescence signal of the BP-FLISA system shows a good concentration-dependent increasing trend. Clear and recognizable effective signals can be presented even in the low concentration range, and the signal intensity is positively correlated with the EV concentration. Figure 13 The fluorescence response results of this system at different concentrations are shown.

[0080] The BP-FLISA sensing system constructed in this invention has ultra-high detection sensitivity, reaching 1.87 × 10⁻⁶. 2 Effective detection of EVs is achieved at extremely low concentration levels of particles / mL, with a linear range spanning five orders of magnitude.

[0081] Example 15: Anti-interference test of BP-FLISA To verify the anti-interference ability and detection stability of the bubble-plasma-enhanced fluorescence immunoassay (BP-FLISA) system constructed in this invention in complex biological matrices, a comparative detection experiment was conducted using EpCAM, a characteristic protein on the surface of tumor-derived extracellular vesicles, as the detection target under matrix interference conditions with different concentrations of fetal bovine serum. The specific steps are as follows: 1. Experimental Grouping 1) PBS blank matrix group (no serum interference); 2) 1% EV-free fetal bovine serum matrix group; 3) 5% EV-free fetal bovine serum matrix group; 4) 10% EV-depleted fetal bovine serum matrix group.

[0082] 2. Experimental Procedure S1: Add equal amounts of HCT116 EV to the above four matrix systems respectively, mix thoroughly, and prepare test samples containing interfering matrices; S2: Add an equal amount of immunoglobulin GB to each system and incubate at room temperature with shaking for 40 min; S3: Separate by static setting using GB self-floating property, wash 3 times with PBST to remove unbound impurities and free components; S4: Add an equal amount of plasma fluorescent probe and incubate at room temperature in the dark for 30 min; S5: Perform fluorescence imaging after washing, and collect and record the fluorescence signal intensity of each group; S6: Statistically analyze and compare the differences in fluorescence signals among different matrix groups to evaluate the anti-interference performance.

[0083] 3. Experimental Results like Figure 14As shown, the fluorescence signal intensity detected by the BP-FLISA system showed no significant difference in four biological matrices with different levels of complexity: PBS, 1%, 5%, and 10% de-EV fetal bovine serum. The signal output was stable with minimal fluctuations. Even in a 10% high-concentration serum matrix environment, the sensor could still specifically identify and bind to the target EV without significant signal inhibition or increased non-specific adsorption.

[0084] Example 16: Repeatability test of BP-FLISA To verify the detection repeatability, method stability, and result reliability of the bubble-plasma-enhanced fluorescence immunoassay (BP-FLISA) system constructed in this invention, a high-throughput repeatable detection experiment was conducted using tumor-derived extracellular vesicles (EVs) from HCT116 cells at the same concentration as the target. The specific steps are as follows: S1: The concentration is 1×10 2 HCT116EV standard solution with particles / mL was used as a repeatability test sample; S2: Using four 96-well plates, the same sample was tested 384 times using the BP-FLISA system. S3: Add immunoglobulin GB, EV sample and plasma fluorescent probe to each well in sequence, and complete incubation, buoyancy self-separation, washing and fluorescence imaging according to the optimal detection conditions; S4: Collect fluorescence signal values ​​from 384 reaction wells and plot fluorescence signal distribution map and density cloud map; S5: Calculate the within-group signal difference and relative standard deviation (RSD) to evaluate the repeatability and stability of the method.

[0085] like Figure 15 As shown, the BP-FLISA system exhibits uniform fluorescence signal distribution in 384 consecutive repeated detections of EV samples at the same concentration, with small inter-well signal differences, low fluctuations, and no obvious outliers or deviations; the signal density cloud map shows a concentrated and stable distribution. The BP-FLISA sensing system constructed in this invention possesses excellent detection repeatability, high-throughput stability, and method reliability. It can achieve standardized detection with high throughput, high precision, and low fluctuations, meeting the clinical needs for large-sample, multi-batch, and high-stability detection. This lays a solid and reliable methodological foundation for subsequent high-throughput detection and translational applications of clinical samples.

[0086] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A bubble-plasma-enhanced fluorescence immunoassay system, characterized in that, Including immunofunctionalized hollow glass microbubbles and plasma fluorescent probes; The immunofunctionalized hollow glass microbubbles are composed of targeting antibodies covalently linked to the surface of the hollow glass microbubbles, which are used to specifically capture tumor-derived extracellular vesicles and achieve buoyancy separation by relying on their own buoyancy. The plasma fluorescence probe consists of a gold nanorod core, a silica spacer layer wrapped around the surface of the gold nanorod, and a fluorescent dye and a targeting aptamer covalently connected to the surface of the silica spacer layer. It is used to amplify the signal by enhancing fluorescence through metal. Among them, the cavity structure of the immunofunctionalized hollow glass microbubble is used to provide physical optical enhancement, which, together with the metal-enhanced fluorescence provided by the plasma fluorescent probe, constitutes a dual-mode signal amplification of the target. The microbubbles in the hollow glass have a particle size of 10-30 μm, the longitudinal localized surface plasmon resonance peak of the gold nanorods is 600-800 nm, and the thickness of the silica spacer layer is 10-20 nm.

2. The bubble-plasma enhanced fluorescence immunoassay system according to claim 1, characterized in that, The targeting antibody is one or more of the following: anti-EpCAM antibody, anti-EGFR antibody, anti-CD24 antibody, anti-PD-L1 antibody, or anti-CD63 antibody; The fluorescent dye is IR680 or IR800CW.

3. The bubble-plasma enhanced fluorescence immunoassay system according to claim 1, characterized in that, The targeted aptamer is a nucleic acid aptamer that specifically recognizes proteins on the surface of extracellular vesicles.

4. The preparation method of the bubble-plasma enhanced fluorescence immunoassay detection system as described in claim 1, characterized in that, Includes the following steps: S1: Hollow glass microbubbles were sequentially activated by piranha solution hydroxylation, modified by 3-mercaptopropyltrimethoxysilane silanization, covalently coupled with targeted antibody, and blocked by bovine serum albumin to obtain immunofunctionalized hollow glass microbubbles. S2: Gold nanorods were prepared by seed growth method. A modified Stuber method was used to coat the surface of the gold nanorods with a silica spacer layer. The coated gold nanorods were then reacted with 3-azidopropyltrimethoxysilane to introduce azide groups. Then, the fluorescent dye and the targeting aptamer were covalently linked to the azide-functionalized silica surface through a copper-free click chemistry reaction to obtain a plasma fluorescent probe. S3: The immunofunctionalized hollow glass microbubbles and the plasma fluorescent probe are used as complementary functional components, and are independently packaged and combined to obtain a bubble-plasma-enhanced fluorescence immunoassay detection system.

5. The preparation method of the bubble-plasma enhanced fluorescence immunoassay detection system according to claim 4, characterized in that, In step S1, the hydroxylation activation is performed by soaking the fish in a piranha solution with a volume ratio of concentrated sulfuric acid to hydrogen peroxide of 7:3 for 0.5-1.5 h. The silanization modification uses an ethanol solution of 3-mercaptopropyltrimethoxysilane, and the reaction is carried out by rotation at room temperature for 1-3 h; the covalent coupling of the targeting antibody is carried out by rotating the silanized hollow glass microbubbles with a concentration of 1-10 μg / mL of the targeting antibody at room temperature for 1-3 h, and then blocking with 1-5 mg / mL of bovine serum albumin for 0.5-1.5 h.

6. The preparation method of the bubble-plasma enhanced fluorescence immunoassay detection system according to claim 4, characterized in that, The improved Stobol process is as follows: gold nanorods are dispersed in water, the pH is adjusted to 7.5-8.0, the mixture is heated to 40-45°C, and an ethanol solution of tetraethyl orthosilicate is added in three portions, with an interval of 30-40 min between each addition. After the addition is complete, the reaction continues for 12-18 h to obtain gold nanorods@silica core-shell particles with a silica spacer layer thickness of 10-20 nm.

7. The preparation method of the bubble-plasma enhanced fluorescence immunoassay detection system according to claim 4, characterized in that, In step S3, immunofunctionalized hollow glass microbubbles are dispersed in phosphate buffer, bovine serum albumin is added to a final concentration of 1-5 mg / mL, and the mixture is blocked at room temperature for 30-90 min. After washing, the mixture is resuspended and individually aliquoted. The plasma fluorescent probe is dispersed in phosphate buffer containing 0.05-0.1% Tween-20 by volume and individually aliquoted. The two individually aliquoted components together constitute the detection system.

8. The use of the bubble-plasma-enhanced fluorescence immunoassay system according to any one of claims 1 to 3 in the preparation of an ultrasensitive detection kit for tumor-derived extracellular vesicles, circulating tumor cells or proteins.

9. The application according to claim 8, characterized in that, The test samples are cell culture supernatant or human plasma.

10. A method for detecting tumor-derived extracellular vesicles for non-disease diagnostic purposes based on bubble-plasma-enhanced fluorescence, characterized in that, The bubble-plasma enhanced fluorescence immunoassay detection system according to any one of claims 1 to 3 includes the following steps: 1) The sample to be tested is mixed with immunofunctionalized hollow glass microbubbles and incubated to allow the immunofunctionalized hollow glass microbubbles to specifically capture tumor-derived extracellular vesicles in the sample to be tested. 2) Add the plasma fluorescent probe to the incubated mixture and continue incubation to allow the plasma fluorescent probe to specifically bind to tumor-derived extracellular vesicles captured on the surface of immunofunctionalized hollow glass microbubbles, forming a recognition complex; 3) The mixture is left to stand. The recognition complex is floated to the surface of the liquid by the buoyancy of the immunofunctionalized hollow glass microbubbles. The lower layer of liquid is removed, and the mixture is washed and resuspended. The signal is collected by fluorescence imaging to achieve centrifugation-free separation and quantitative detection of the target.

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