Extracellular vesicle detection reagent based on binary nano-enzyme immunoprobe and application of extracellular vesicle detection reagent

Abstract

The invention belongs to the technical field of biosensing and clinical detection, and particularly relates to an extracellular vesicle detection reagent based on a binary nano-enzyme immunoprobe and application of the extracellular vesicle detection reagent. According to the extracellular vesicle detection reagent based on the binary nano-enzyme immunoprobe provided by the invention, a universal marker CD63 antibody is coupled with gold nanoparticles with glucose oxidase activity, and a pancreatic cancer specific marker GPC1 antibody is coupled with Janus platinum nanoparticles with peroxidase simulation activity; the efficient binary nano-enzyme immune probe is constructed. According to the binary nano-enzyme immune probe disclosed by the invention, an immunoaffinity capture technology and binary nano-enzyme with high catalytic activity are combined, and ultrasensitive, rapid and specific detection of extracellular vesicles is realized by enhancing mass transfer efficiency at a reaction interface, so that a new method can be provided for real-time diagnosis of tumors; meanwhile, a powerful technical support is provided for early diagnosis and intervention treatment of the pancreatic cancer.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and clinical testing technology, specifically relating to an extracellular vesicle detection reagent based on a binary nanozyme immunoprobe and its application. Background Technology

[0002] Extracellular vesicles (EVs) are membrane-derived structures released from living cells and are widely distributed in various body fluids. As important information carriers, EVs carry multiple functional molecules from their source cells and have been considered highly promising biomarkers for "liquid biopsy." EVs have shown unique application value, especially in the diagnosis of pancreatic cancer. Pancreatic cancer has an insidious onset and lacks early specific symptoms, and existing serum biomarkers such as CA19-9 have limitations in sensitivity and specificity. In recent years, studies have found that EVs derived from pancreatic cancer are rich in various specific molecules, such as glycine nucleoprotein-1 (GPC1), KRAS mutant gene transcripts, CEACAM6, and EGFR. Among them, GPC1-positive EVs have been confirmed by multiple studies to effectively distinguish pancreatic cancer patients from healthy individuals or patients with chronic pancreatitis, demonstrating superior diagnostic accuracy compared to CA19-9. Therefore, compared to traditional single-indicator serum testing, EV-based analysis provides a new direction for the accurate diagnosis of pancreatic cancer.

[0003] However, due to the small size, low abundance, and coexistence with high-abundance serum proteins, rapid and highly sensitive detection of EVs remains a significant challenge. Currently, various quantitative methods have been developed based on the biological characteristics of EVs. Nanoparticle tracking technology can quantify EVs through counting and particle size analysis, but it cannot distinguish EV subtypes and has a high detection limit, making it difficult to detect low-concentration samples. Western blotting is also commonly used for the detection of EV-specific protein biomarkers, but it is cumbersome and time-consuming, unsuitable for point-of-care testing. Furthermore, enzyme-linked immunosorbent assay (ELISA) can also specifically detect EVs, but its sensitivity is limited and it typically requires multiple washing steps, failing to meet the speed and simplicity requirements of point-of-care testing. Meanwhile, while digital PCR and digital ELISA offer high sensitivity, they still require complex equipment and specialized operational procedures, resulting in high costs and hindering large-scale adoption.

[0004] In recent years, nanozymes with enzyme-like catalytic activity have provided a powerful tool for constructing novel biosensors. Compared with traditional natural enzymes, nanozymes have advantages such as low cost, high stability, and large-scale preparation. However, existing nanozyme-based EV detection methods still suffer from the key technical problem of low mass transfer efficiency. In traditional homogeneous or heterogeneous reactions, the target and catalyst need to undergo a slow diffusion process to reach the reaction interface and collide effectively. This slow mass transfer process severely limits the reaction rate, leading to prolonged detection time and difficulty in further improving sensitivity.

[0005] Therefore, there is an urgent need to develop a detection reagent and method that can overcome the aforementioned shortcomings. This reagent can actively and efficiently enrich target EVs at the catalytically active site, significantly enhancing the local reaction rate, thereby achieving ultra-fast and ultra-sensitive detection of EVs while ensuring high specificity, to meet the application needs of point-of-care clinical diagnosis of pancreatic cancer. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, the present invention aims to provide an extracellular vesicle detection reagent based on a binary nanozyme immunoprobe and its application. The extracellular vesicle detection reagent provided by the present invention combines immunoaffinity capture technology with a highly catalytically active binary nanozyme. By enhancing mass transfer efficiency at the reaction interface, it achieves ultrasensitive, rapid, and instantaneous detection of extracellular vesicles, thus solving the problems of low mass transfer efficiency and slow reaction rate inherent in existing nanozymes.

[0007] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution:

[0008] An extracellular vesicle detection reagent based on a binary nanozyme immunoprobe, wherein the extracellular vesicle detection reagent comprises a binary nanozyme immunoprobe; wherein the binary nanozyme immunoprobe comprises a gold nanoparticle glucose oxidase mimic probe and a Janus platinum peroxidase mimic probe.

[0009] The gold nanoparticle glucose oxidase mimic probe was prepared by modifying gold nanoparticles with CD63 antibody; the Janus platinum peroxidase mimic probe was prepared by modifying Janus Pt nanoparticles with GPC1 antibody.

[0010] Preferably, the preparation method of the gold nanoparticle glucose oxidase mimic probe includes the following steps: preparing a gold nanoparticle solution; then incubating the gold nanoparticle solution with CD63 antibody by shaking, followed by blocking and centrifugation to obtain the gold nanoparticle glucose oxidase mimic probe, denoted as AuNPs-CD63 probe; wherein the gold nanoparticles are prepared by heating a chloroauric acid solution and a citric acid solution. This step modifies the surface of the gold nanoparticles with CD63 antibody through interfacial interaction labeling, forming an immune probe with glucose oxidase mimicry activity.

[0011] More preferably, the concentration of the gold nanoparticle solution is 40~60 μg / mL; for every 0.8~1.2 mL of gold nanoparticle solution, the corresponding amount of CD63 antibody is 4~6 μg.

[0012] More preferably, the average particle size of the gold nanoparticles is 10-20 nm; the concentration of the chloroauric acid solution is 1-3 mM; the concentration of the citric acid solution is 0.5-2 wt%; the volume ratio of the chloroauric acid solution to the citric acid solution is 10-15:1; the temperature of the heating reaction is 90-105 °C, and the heating reaction time is 20-60 min.

[0013] Preferably, the preparation method of the Janus platinum peroxidase mimic probe includes the following steps: preparing a Janus Pt nanoparticle solution; then incubating the Janus Pt nanoparticle solution with a GPC1 antibody by shaking, followed by blocking and centrifugation to obtain the Janus platinum peroxidase mimic probe, denoted as the Janus Pt-GPC1 probe. The Janus Pt nanoparticles are prepared by reacting platinum nanoparticles with tetraethyl silicate by stirring. This step modifies the surface of the silicon component of the Janus platinum nanoparticles with the pancreatic cancer-specific antibody GPC1 through interfacial interaction coupling, forming an immune probe with peroxidase mimicry activity.

[0014] More preferably, the concentration of the Janus Pt nanoparticle solution is 0.8~1.2 mg / mL; each 0.8~1.2 mL of Janus Pt nanoparticle solution corresponds to 4~6 μg of GPC1 antibody.

[0015] More preferably, the average particle size of the platinum nanoparticles is 20-30 nm; the platinum nanoparticles are prepared by heating a chloroplatinic acid solution with a solution containing ascorbic acid and citric acid; the heating reaction temperature is 90-105°C, and the heating reaction time is 20-60 min. The volume ratio of the platinum nanoparticles to tetraethyl silicate is 200-300:1; the stirring reaction time is 8-15 h.

[0016] Preferably, the shaking incubation time is 3-5 hours; the sealing is performed by using a 0.5-3 wt% BSA solution for 0.5-2 hours; the centrifugation speed is 8000-12000 r / min, and the centrifugation time is 5-20 minutes.

[0017] The second aspect of the present invention adopts the following technical solution:

[0018] An extracellular vesicle detection kit based on a binary nanozyme immunoprobe includes the extracellular vesicle detection reagent as described above, and also includes glucose and tetramethylbenzidine.

[0019] Furthermore, glucose and tetramethylbenzidine are catalytic substrates in the assay process of the kit. Glucose is used to trigger the glucose oxidase-mimicking activity of the gold nanoparticles and generate H2O2; tetramethylbenzidine is used to be oxidized by H2O2 and colorimetrically generated under the peroxidase-mimicking activity of the Janus Pt nanoparticles.

[0020] Preferably, when using an extracellular vesicle detection kit to detect extracellular vesicles, the detection method includes the following steps:

[0021] (1) The biological sample to be tested was pretreated to obtain a liquid sample; gold nanoparticle glucose oxidase mimic probe and platinum peroxidase mimic probe were respectively prepared into gold nanoparticle glucose oxidase mimic probe solution and platinum peroxidase mimic probe solution; glucose and tetramethylbenzidine were respectively prepared into glucose solution and tetramethylbenzidine solution.

[0022] (2) Mix the gold nanoparticle glucose oxidase mimic probe solution and the platinum peroxidase mimic probe solution with the liquid sample to obtain a mixture;

[0023] (3) The mixture is first incubated, then glucose solution and tetramethylbenzidine solution are added for catalytic reaction. The absorbance value or catalytic reaction rate is then measured. The absorbance value or catalytic reaction rate is substituted into the standard curve to obtain the content of extracellular vesicles.

[0024] More preferably, the biological sample to be tested is a serum sample; the pretreatment involves coagulating and centrifuging the biological sample to be tested, and the resulting supernatant is the liquid sample.

[0025] More preferably, the volume ratio of the gold nanoparticle glucose oxidase mimic probe solution, platinum peroxidase mimic probe solution, liquid sample, glucose solution, and tetramethylbenzidine solution is (1.8~2.2):(1.8~2.2):(0.8~1.2):(0.8~1.2):(3.8~4.2), and more preferably 2:2:1:1:4. The incubation treatment temperature is 35~38℃, and the time is 20~40 min; the absorbance value is the absorbance value at a certain time point at a wavelength of 650 nm; the catalytic reaction rate is the rate of change of absorbance within a catalytic reaction time of 0~30 min. In this step, on the one hand, the content of extracellular vesicles in the sample can be obtained by measuring the absorbance value at a certain time point and substituting it into a pre-established standard curve of absorbance value versus extracellular vesicle concentration. On the other hand, the content of extracellular vesicles in the sample can also be obtained by measuring the rate of change of absorbance during the catalytic reaction time of 0-30 min and substituting it into a pre-established standard curve of catalytic reaction rate and extracellular vesicle concentration.

[0026] In the above detection method, if extracellular vesicles (EVs) originating from the target tumor are present, the CD63 and GPC1 proteins on their surfaces will bind to the gold nanoparticle glucose oxidase mimic probe (AuNPs-CD63) and the platinum peroxidase mimic probe (Janus Pt-GPC1), respectively, forming an EVs / AuNPs-CD63-Janus Pt-GPC1 sandwich immune complex. This complex formation tightly pulls the two nanozymes closer together. The brought-up gold nanoparticles (Au NPs) and Janus Pt nanoparticles (Janus Pt) constitute a highly efficient microreactor. Au NPs catalyze the oxidation of glucose to gluconic acid and H2O2. The newly generated H2O2 can be efficiently captured by the neighboring Janus Pt without long-distance diffusion, and immediately catalyzes the oxidation of colorless tetramethylbenzidine (TMB) to blue TMB oxide. Due to the extremely short mass transfer distance, this cascade reaction is extremely fast, resulting in a rapid accumulation of the blue product in a short time. Semi-quantitative analysis can be achieved by visually observing the intensity of the blue color in the solution, or by using a UV-Vis spectrophotometer to monitor the rate of change in absorbance at a wavelength of 650 nm (i.e., the slope of the reaction kinetic curve) to achieve quantitative detection of the signal. The magnitude of the reaction rate directly reflects the mass transfer efficiency, and is thus proportional to the concentration of the target EVs.

[0027] The third aspect of the present invention adopts the following technical solution:

[0028] The application of the extracellular vesicle detection reagent or the extracellular vesicle detection kit described above in the preparation of diagnostic reagents for pancreatic cancer.

[0029] Preferably, in diagnosing pancreatic cancer, serum samples from both pancreatic cancer patients and healthy individuals are used as biological samples for testing. The aforementioned extracellular vesicle detection method is employed to obtain absorbance values ​​or catalytic reaction rates. By comparing the absorbance values ​​or catalytic reaction rates of serum samples from pancreatic cancer patients and healthy individuals, the likelihood of the patient having pancreatic cancer can be determined, thereby achieving early diagnosis of pancreatic cancer.

[0030] More preferably, for ease of determination, the catalytic reaction rate or absorbance value at a specific time point of serum samples from pancreatic cancer patients and healthy individuals can be recorded. By comparing the statistical differences in signal intensity of the catalytic reaction rate (or absorbance value) between pancreatic cancer patients and healthy individuals, a diagnostic threshold is determined. If the signal intensity of the sample to be tested is higher than this threshold, it is judged as positive; if the signal intensity of the sample to be tested is lower than this threshold, it is judged as negative. This enables immediate diagnosis of pancreatic cancer patients, thus providing a new direction for the early diagnosis of pancreatic cancer.

[0031] The technical solution provided by this invention has the following advantages and beneficial effects:

[0032] The present invention provides an extracellular vesicle detection reagent and kit based on binary nanozyme immunoprobes. It utilizes gold nanoparticles with glucose oxidase activity and Janus platinum nanozymes with peroxidase-mimicking activity to construct a highly efficient binary nanozyme catalytic system. Two specific recognition probes are constructed by conjugating the universal biomarker CD63 antibody to gold nanoparticles and the pancreatic cancer-specific biomarker GPC1 antibody to Janus platinum nanozymes.

[0033] When extracellular vesicles (EVs) derived from pancreatic cancer are present, two nanozyme probes simultaneously bind to the EVs via sandwich immunoaffinity, rapidly bringing the two nanozymes closer together in space to form a "nanozyme pair." This dramatic reduction in spatial configuration significantly shortens the diffusion distance of hydrogen peroxide, an intermediate product generated in the cascade catalytic reaction, thereby significantly enhancing local mass transfer efficiency and overall reaction rate, achieving effective signal amplification. Furthermore, the detection process of this invention's reagent is not only simple and rapid, requiring no complex instruments or multiple washing processes, but also enables highly sensitive and specific detection of disease-specific EVs in the serum of pancreatic cancer patients within a short time by monitoring changes in the catalytic reaction rate. Moreover, by analyzing changes in the catalytic reaction rate in serum samples, it is possible to effectively distinguish between healthy individuals and pancreatic cancer patients.

[0034] Therefore, this invention can provide a new method for the point-of-care diagnosis of tumors, provide strong technical support for the early diagnosis and intervention of pancreatic cancer, and also provide new directions for the development of extracellular vesicle detection reagents and pancreatic cancer diagnostic reagents, and has broad application prospects in the point-of-care diagnosis of pancreatic cancer tumors. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings involved in the present invention are briefly described below;

[0036] Figure 1 Transmission electron microscope (TEM) images of the gold nanoparticles and Janus platinum nanoparticles used in this invention.

[0037] Figure 2 The potential, particle size, and circular dichroism chromatographic results of GPC1 antibody, Janus Pt nanoparticles, Janus Pt-GPC1 probe, CD63 antibody, gold nanoparticles, and AuNPs-CD63 probe in this invention are shown.

[0038] Figure 3 The time-absorbance curves of extracellular vesicle (EV) standards at different concentrations in this invention, as well as the linear relationship curves between EV concentration and corresponding reaction rate;

[0039] Figure 4 The results show the specificity of the binary nanozyme immunoprobe based on the present invention for detecting different samples.

[0040] Figure 5 The results of the diagnosis of pancreatic cancer patients and healthy controls using a single nanozyme immunoprobe based on AuNPs-CD63 in this invention are presented.

[0041] Figure 6 The results of the single nanozyme immunoprobe based on Janus Pt-GPC1 used in this invention to diagnose pancreatic cancer patients and healthy controls are presented.

[0042] Figure 7 This presents the results of the binary nanozyme immunoprobe constructed based on AuNPs-CD63 probe and Janus Pt-GPC1 probe in this invention for diagnosing pancreatic cancer patients and healthy controls. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and test examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other implementation schemes obtained by those skilled in the art without creative effort, such as implementation schemes obtained by modification, variation or simple substitution, should fall within the scope of protection of the present invention.

[0044] In the following embodiments, the technical terms and abbreviations used have their conventional meanings in the art. The room temperature is 20-30°C. The extracellular vesicle-associated membrane protein 3 antibody (CD63 antibody) and phosphatidylinositol proteoglycan 1 antibody (GPC1 antibody) used in the following embodiments were purchased from Abcam. The phosphate-buffered saline (PBS) concentration was 0.01M, pH=7.4.

[0045] The gold nanoparticles (Au) used in the following embodiments were prepared by a method including the following steps: 50 mL of 2 mM chloroauric acid solution was mixed with 4 mL of 1 wt% citric acid solution and boiled for 30 min to obtain gold nanoparticles (average particle size 15 nm).

[0046] The Janus Pt nanoparticles used in the following embodiments were prepared using a method comprising the following steps: First, 0.6 mL of a 20 mM chloroplatinic acid solution was added to 30 mL of deionized water containing 0.41 wt% ascorbic acid and 0.33 wt% citric acid and boiled for 30 min to obtain platinum nanoparticles (average particle size 25 nm). Subsequently, 5 mL of the obtained platinum nanoparticles were mixed with 0.02 mL of tetraethyl orthosilicate and reacted at room temperature with stirring for 10 h. After centrifugation at 8000 rpm for 10 min, the resulting precipitate was the Janus Pt nanoparticles. Other raw materials and reagents not specified were commercially available.

[0047] Example 1

[0048] This embodiment provides a reagent for detecting extracellular vesicles based on binary nanozyme immunoprobes, including a gold nanoparticle glucose oxidase mimic probe (AuNPs-CD63 probe) and a Janus platinum peroxidase mimic probe (JanusPt-GPC1 probe).

[0049] The preparation method of the AuNPs-CD63 probe includes the following steps: 50 μg of gold nanoparticles are dispersed in 1 mL of phosphate-buffered saline (PBS) buffer to obtain a gold nanoparticle solution with a concentration of 50 μg / mL. 1 mL of the 50 μg / mL gold nanoparticle solution is incubated with 5 μg of CD63 antibody at room temperature with shaking for 4 h. Then, 0.1 mL of 1 wt% BSA solution is added for blocking treatment for 1 h to block non-specific sites. Subsequently, the resulting liquid is centrifuged at 10000 r / min for 10 min to remove unbound antibody. The precipitate obtained after centrifugation is the AuNPs-CD63 probe. The probe is resuspended in 1 mL of PBS buffer to obtain the AuNPs-CD63 probe solution, which is stored at 4℃ for later use.

[0050] The preparation method of the Janus Pt-GPC1 probe includes the following steps: 1 mg of Janus Pt nanoparticles are dispersed in 1 mL of PBS buffer to obtain a Janus Pt nanoparticle solution with a concentration of 1 mg / mL. 5 μg of GPC1 antibody is added to the 1 mL Janus Pt nanoparticle solution, and the mixture is incubated with shaking at room temperature for 4 h. Then, 0.1 mL of 1 wt% BSA solution is added for blocking for 1 h. The resulting liquid after blocking is then centrifuged at 10000 r / min for 10 min. The precipitate obtained after centrifugation is the Janus Pt-GPC1 probe. The probe is resuspended in 1 mL of PBS buffer to obtain the Janus Pt-GPC1 probe solution, which is stored at 4 °C for later use.

[0051] The method for detecting extracellular vesicles using the above reagents includes the following steps:

[0052] (1) Sample preparation: The blood sample to be tested was allowed to stand and coagulate, and then centrifuged at 3000 r / min for 15 min to collect the serum sample; then the serum sample was centrifuged at 10000 g for 30 min to remove impurities in the serum, and the supernatant was taken as the pretreated serum sample for subsequent detection.

[0053] (2) Sandwich immunoassay and catalytic reaction: 50 μL of the prepared AuNPs-CD63 probe solution, 50 μL of Janus Pt-GPC1 probe solution, and 25 μL of pretreated serum sample were added to a 96-well clear microplate to obtain a mixture. A blank control group was set up, in which 25 μL of PBS buffer was used instead of the pretreated serum sample, while other conditions remained unchanged.

[0054] The mixture was incubated at 37°C for 30 minutes to allow the immune sandwich structure to fully form. After incubation, 25 μL of glucose solution (final glucose concentration of 6 mM) and 100 μL of tetramethylbenzidine substrate solution (TMB solution, final TMB concentration of 10 mM) were added to initiate the catalytic reaction.

[0055] (3) Signal detection and data analysis: The reaction solution after adding TMB solution in step (2) is immediately transferred to a cuvette and then placed in a UV-Vis spectrophotometer. The absorbance value is recorded at a wavelength of 650 nm. The absorbance value is substituted into the standard curve to obtain the content of extracellular vesicles.

[0056] The technical effects of the present invention will be illustrated below through detailed experimental examples.

[0057] Experimental Example 1: Morphology Analysis of Nanoparticles

[0058] The prepared gold nanoparticles and Janus Pt nanoparticles were analyzed using transmission electron microscopy (TEM). For testing, gold nanoparticles and Janus Pt nanoparticles were prepared into 0.5 mg / mL solutions, and then 5 μL of each solution was loaded onto a conventional carbon film. Imaging was performed at an accelerating voltage of 100 kV. The obtained TEM images are shown below. Figure 1 As shown. Figure 1 In the image, Figure A is a transmission electron microscope (TEM) image of gold nanoparticles, and Figure B is a TEM image of Janus Pt nanoparticles.

[0059] like Figure 1 As shown, the obtained gold nanoparticles exhibit a monodisperse spherical structure (A), and the Janus Pt nanoparticles have a clearly defined silica component and Pt nanoparticle composition.

[0060] Experimental Example 2: Characterization of Janus Pt-GPC1 and AuNPs-CD63 probes

[0061] GPC1 antibody (GPC1), Janus Pt nanoparticles (Pt Janus NRs), and the JanusPt-GPC1 probe (Pt Janus NRs-GCP1) prepared in Example 1 were each prepared into 1 mg / mL solutions. Then, 2 mL of each of the 1 mg / mL solutions were added to cuvettes for potential measurement, particle size analysis, and circular dichroism spectroscopy identification.

[0062] Meanwhile, CD63 antibody (CD63), gold nanoparticles (Au NPs), and the AuNPs-CD63 probe (AuNPs-CD63) prepared in Example 1 were each prepared into a 1 mg / mL solution. Subsequently, 2 mL of each of the 1 mg / mL solutions were added to cuvettes for potential measurement, particle size analysis, and circular dichroism chromatographic identification.

[0063] The potential, particle size, and circular dichroism analysis results of antibodies, nanoparticles, and corresponding probes are as follows: Figure 2 As shown. Figure 2 Figure A shows the potential measurement results of GPC1 antibody, Janus Pt nanoparticles, and Janus Pt-GPC1 probe; Figure B shows the particle size measurement results of Janus Pt nanoparticles and Janus Pt-GPC1 probe; Figure C shows the circular dichroism chromatographic analysis results of GPC1 antibody, Janus Pt nanoparticles, and Janus Pt-GPC1 probe; Figure D shows the potential measurement results of CD63 antibody, gold nanoparticles, and AuNPs-CD63 probe; Figure E shows the particle size measurement results of gold nanoparticles and AuNPs-CD63 probe; Figure F shows the circular dichroism chromatographic analysis results of CD63 antibody, gold nanoparticles, and AuNPs-CD63 probe.

[0064] Depend on Figure 2 Based on the potential, particle size, and circular dichroism chromatographic identification results, it can be seen that the present invention successfully prepared AuNPs-CD63 probe and Janus Pt-GPC1 probe.

[0065] Experimental Example 3: Determination of Standard Curve and Analysis of Detection Limit

[0066] Extracellular vesicles (EVs) derived from pancreatic cancer were purified and prepared into standards with a concentration of 5 × 10⁻⁶. 2 1×10 3 5×10 3 1×10 4 5×10 4 1×10 5 2.5×10 5 5×10 5 1×10 6 2.5×10 6 5×10 7A solution with particles / μL was prepared. In a 96-well clear microplate, 50 μL of AuNPs-CD63 probe solution, 50 μL of Janus Pt-GPC1 probe solution, and 25 μL of extracellular vesicle (EV) solutions of different concentrations were added to obtain a mixture. The mixture was incubated at 37°C for 30 min to allow for the full formation of the immune sandwich structure. After incubation, 25 μL of glucose solution (final glucose concentration 6 mM) and 100 μL of TMB solution (final TMB concentration 10 mM) were added to initiate the catalytic reaction. The reaction solution after adding TMB solution was immediately transferred to a cuvette and placed in a UV-Vis spectrophotometer. The absorbance changes were continuously monitored from 0 to 30 min at a wavelength of 650 nm, and the reaction time-absorbance curve was recorded.

[0067] Furthermore, based on the obtained reaction time-absorbance kinetic curves, the slope (ΔA / min) of each curve in the initial linear phase was calculated. This slope is the catalytic reaction rate (V), which directly reflects the mass transfer efficiency of the binary nanozyme system. Linear relationship curves between EV concentration and corresponding reaction rates were established using different catalytic reaction rate values.

[0068] Time-absorbance curves of extracellular vesicle (EV) standards at different concentrations and linear relationship curves between EV concentration and corresponding reaction rates are shown below. Figure 3 As shown. Figure 3 In Figure A, the time-absorbance curves are shown from 0 to 30 min; Figure B is a linear graph showing the relationship between EV concentration and the corresponding catalytic reaction rate. In Figure A, the absorbance curves from bottom to top represent the absorbance results of EV concentrations from low to high.

[0069] Depend on Figure 3 It can be seen that within 30 minutes, the absorbance values ​​of different EV-containing systems increase with increasing reaction time and EV concentration (Figure A). Furthermore, the reaction rate V increases with increasing EV concentration, exhibiting a good linear relationship (Figure B). In addition, further monitoring revealed that the absorbance intensity increases further with prolonged time (beyond 30 minutes), but this becomes a second-order reaction, and the rate is difficult to define. Therefore, the optimal testing time was selected as 30 minutes.

[0070] Furthermore, based on the catalytic reaction rates corresponding to different concentrations of EVs standards, a standard curve was established for the catalytic reaction rate (V) as a function of EVs concentration ([EVs]). The obtained standard curve is V = 0.4Lg[EVs]⁻¹.3, R 2 =0.97, therefore the detection linear range of EVs is determined to be 5×10. 3 ~5×10 6Particles / μL, detection limit is 28 particles / μL.

[0071] Experimental Example 4: Specificity Analysis

[0072] 25 μL of bovine serum albumin (BSA) solution, alpha-fetoprotein (AFP) solution, human serum immunoglobulin (IgG) solution, and extracellular vesicle standard (EVs) solution, as well as 25 μL of a mixture of BSA and EVs solutions of the above concentrations (BSA+EVs), a mixture of AFP and EVs solutions (AFP+EVs), and a mixture of IgG and EVs solutions (IgG+EVs), were added to 96-well clear microplates. Then, 50 μL of AuNPs-CD63 probe solution and 50 μL of Janus Pt-GPC1 probe solution were added to obtain a mixture. The mixture was incubated at 37°C for 30 min. After incubation, 25 μL of glucose solution (final glucose concentration 6 mM) and 100 μL of TMB solution (final TMB concentration 10 mM) were added to initiate the catalytic reaction. The reaction solution after adding TMB was transferred to a cuvette and then placed in a UV-Vis spectrophotometer. The absorbance change was continuously monitored from 0 to 30 minutes at a wavelength of 650 nm, and the reaction time-absorbance curve was recorded. Based on the obtained reaction time-absorbance kinetic curves, the slope (ΔA / min) of each curve in the initial linear phase was calculated. This slope is the catalytic reaction rate (V). The detection specificity was determined based on the catalytic reaction rate. The specificity detection results for each sample are as follows: Figure 4 As shown.

[0073] Depend on Figure 4 It can be seen that when BSA, AFP, IgG, EVs, BSA+EVs, AFP+EVs, and IgG+EVs are added to the reaction system, the catalytic rate changes significantly only when EVs are present in the system, indicating that the detection probe and detection method of the present invention have good specificity.

[0074] Experimental Example 5: Comparison of Detection Efficacy between Binary Nanozyme Immunoprobes and Single Nanozyme Immunoprobes

[0075] Blood samples were collected from 20 clinically diagnosed pancreatic cancer patients (10 males and 10 females, mean age 65 years) and 20 healthy volunteers (10 males and 10 females, mean age 65 years). Serum samples were collected by centrifugation at 3000 rpm for 15 minutes. The serum samples were then centrifuged at 10000 g for 30 minutes to remove impurities, and the supernatant was used as the pretreated serum sample for subsequent testing.

[0076] The testing process was set up with the following comparison group:

[0077] ① Add 50 μL of AuNPs-CD63 probe solution and 25 μL of pretreated serum sample to a 96-well clear microplate to obtain a mixture;

[0078] ② Add 50 μL of Janus Pt-GPC1 probe solution and 25 μL of pretreated serum sample to a 96-well clear microplate to obtain a mixture;

[0079] ③ Add 50 μL of AuNPs-CD63 probe solution, 50 μL of Janus Pt-GPC1 probe solution and 25 μL of pretreated serum sample to a 96-well clear microplate to obtain a mixture;

[0080] The mixtures were incubated at 37°C for 30 min. After incubation, 25 μL of glucose solution (final glucose concentration 6 mM) and 100 μL of TMB solution (final TMB concentration 10 mM) were added to initiate the catalytic reaction. The reaction solution after adding TMB solution was transferred to a cuvette and placed in a UV-Vis spectrophotometer. The absorbance was continuously monitored from 0 to 30 min at 650 nm, and the reaction time-absorbance curve was recorded. Based on the obtained reaction time-absorbance kinetic curves, the slope (ΔA / min) of each curve in the initial linear phase was calculated. This slope is the catalytic reaction rate (V). Based on the catalytic reaction rate, the differences in the detection effect of different detection systems on biological samples were determined. Data were statistically analyzed using GraphPad, with a confidence interval of 95%. All data points are the average of three independent measurements, and a two-tailed t-test was used to assess statistical significance. P < 0.05 indicates a significant difference between the experimental groups, and P < 0.0001 indicates a highly significant difference between the experimental groups.

[0081] Among them, the results of serum detection of pancreatic cancer patients and healthy controls using a single nanozyme immunoprobe based on AuNPs-CD63 are as follows: Figure 5 As shown. The results of serum detection of pancreatic cancer patients and healthy controls using a single nanozyme immunoprobe based on Janus Pt-GPC1 are shown below. Figure 6 As shown in the figure. The results of serum detection of pancreatic cancer patients and healthy controls using a binary nanozyme immunoprobe constructed based on AuNPs-CD63 and Janus Pt-GPC1 probes are shown in the figure. Figure 7 As shown. Figures 5-7 In this study, samples from healthy volunteers were denoted as Healthy controls, and samples from pancreatic cancer patients were denoted as PCa. Figures 5-7In the figure, Figure A shows the measurement results of the catalytic reaction rate, and Figure B shows the statistical analysis results of the catalytic reaction rate.

[0082] Depend on Figure 5 It can be seen that, based on the single probe detection of AuNPs-CD63, the P=0.08>0.05 between pancreatic cancer patients and healthy controls indicates no significant difference, thus failing to achieve immediate diagnosis of pancreatic cancer. Figure 6 It can be seen that, based on the Janus Pt-GPC1 single probe detection, the P-value between pancreatic cancer patients and healthy controls was 0.2193 > 0.05, indicating no significant difference, and thus failing to achieve real-time diagnosis of pancreatic cancer. Figure 7 It can be seen that when using AuNPs-CD63 probe and Janus Pt-GPC1 probe for detection, there is a highly significant difference (P < 0.0001) between pancreatic cancer patients and healthy controls, which can effectively achieve immediate diagnosis of pancreatic cancer.

[0083] The above test results demonstrate that the binary nanozyme immunoprobe provided by this invention can achieve immediate diagnosis of pancreatic cancer patients through the detection of extracellular vesicles. Furthermore, by comparing the statistical differences in signal intensity between the patient group and the healthy control group, a diagnostic threshold (V=0.56×10⁻⁶) can be determined. -7 Ms -1 If the signal intensity of the catalytic reaction rate of the sample being tested is higher than this threshold, it is considered positive, meaning the patient is diagnosed with pancreatic cancer at an early stage. If the signal intensity is lower than this threshold, it is considered negative or the detection threshold has not been reached, and further diagnosis can be made by combining other tumor detection methods.

[0084] In summary, the binary nanozyme immunoprobe provided by this invention can achieve highly sensitive and specific detection of EVs in the serum of pancreatic cancer patients, and has broad application prospects in point-of-care tumor diagnosis.

[0085] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A binary nanoszyme immunoprobe-based extracellular vesicle detection reagent, characterized in that, The extracellular vesicle detection reagent comprises a binary nanozyme immunoprobe; the binary nanozyme immunoprobe comprises a gold nanoparticle glucose oxidase mimetic probe and a Janus platinum peroxidase mimetic probe; The gold nanoparticle glucose oxidase mimetic probe is prepared by using a CD63 antibody to modify gold nanoparticles; and the Janus platinum peroxidase mimetic probe is prepared by using a GPC1 antibody to modify Janus Pt nanoparticles.

2. The extracellular vesicle detection reagent based on binary nanoszyme immunoprobes according to claim 1, characterized in that, The preparation method of the gold nanoparticle glucose oxidase mimetic probe comprises the following steps: gold nanoparticles are prepared into a gold nanoparticle solution; then the gold nanoparticle solution is oscillation-incubated with a CD63 antibody, followed by blocking and centrifugation, to obtain the gold nanoparticle glucose oxidase mimetic probe, denoted as AuNPs-CD63 probe; wherein the gold nanoparticles are prepared by heating reaction of chloroauric acid solution and citric acid solution.

3. The extracellular vesicle detection reagent based on binary nanoszyme immunoprobes according to claim 2, characterized in that, The concentration of the gold nanoparticle solution is 40-60 μg / mL; and the amount of CD63 antibody corresponding to 0.8-1.2 mL of the gold nanoparticle solution is 4-6 μg.

4. The binary nanoszyme immunoprobe-based extracellular vesicle detection reagent of claim 1, wherein, The preparation method of the Janus platinum peroxidase mimetic probe comprises the following steps: Janus Pt nanoparticles are prepared into a Janus Pt nanoparticle solution; then the Janus Pt nanoparticle solution is oscillation-incubated with a GPC1 antibody, followed by blocking and centrifugation, to obtain the Janus platinum peroxidase mimetic probe, denoted as Janus Pt-GPC1 probe; wherein the Janus Pt nanoparticles are prepared by stirring reaction of platinum nanoparticles and tetraethyl orthosilicate.

5. The extracellular vesicle detection reagent based on binary nanoszyme immunoprobes according to claim 4, characterized in that, The concentration of the Janus Pt nanoparticle solution is 0.8-1.2 mg / mL; and the amount of GPC1 antibody corresponding to 0.8-1.2 mL of the Janus Pt nanoparticle solution is 4-6 μg.

6. The extracellular vesicle detection reagent based on binary nanoszyme immunoprobes according to claim 2 or 4, characterized in that, The oscillation-incubation time is 3-5 h; the blocking is BSA solution blocking treatment for 0.5-2 h, with a concentration of 0.5-3 wt%; and the centrifugation speed is 8000-12000 r / min, and the centrifugation time is 5-20 min.

7. A binary nanoszyme immunoprobe-based extracellular vesicle detection kit, characterized in that, The extracellular vesicle detection reagent comprises a binary nanozyme immunoprobe; the binary nanozyme immunoprobe comprises a gold nanoparticle glucose oxidase mimetic probe and a Janus platinum peroxidase mimetic probe; 8. The extracellular vesicle detection kit based on binary nanoszyme immunoprobe according to claim 7, characterized in that, When the extracellular vesicle detection kit is used for extracellular vesicle detection, the detection method comprises the following steps: (1) pretreating a biological sample to be detected to obtain a liquid sample; separately preparing a gold nanoparticle glucose oxidase mimetic probe solution and a platinum peroxidase mimetic probe solution from a gold nanoparticle glucose oxidase mimetic probe and a platinum peroxidase mimetic probe; and separately preparing a glucose solution and a tetramethylbenzidine solution from glucose and tetramethylbenzidine; (2) mixing the gold nanoparticle glucose oxidase mimetic probe solution and the platinum peroxidase mimetic probe solution with the liquid sample to obtain a mixture; (3) the mixture is first incubated, then glucose solution and tetramethyl benzidine solution are added for catalytic reaction, and then the absorbance value or catalytic reaction rate is determined, and the absorbance value or catalytic reaction rate is substituted into a standard curve to obtain the content of extracellular vesicles.

9. The extracellular vesicle detection kit based on binary nanoszyme immunoprobe according to claim 8, characterized in that, The volume ratio of the gold nanoparticle glucose oxidase mimic probe solution, the platinum peroxidase mimic probe solution, the liquid sample, the glucose solution, and the tetramethyl benzidine solution is (1.8-2.2):(1.8-2.2):(0.8-1.2):(0.8-1.2):(3.8-4.2); the temperature of the incubation treatment is 35-38 DEG C, and the time is 20-40 min; the absorbance value is an absorbance value at a certain time point at a wavelength of 650 nm; and the catalytic reaction rate is an absorbance change rate within a catalytic reaction time of 0-30 min.

10. Use of the extracellular vesicle detection reagent according to claim 1 or the extracellular vesicle detection kit according to claim 7, characterized in that, Use in preparation of a diagnostic reagent for pancreatic cancer.

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