A composite nanozyme and a kit for simultaneous recognition of multiple exosomes.

Abstract

This invention discloses a composite nanozyme and a kit for simultaneous recognition of multiple exosomes. The kit includes an aptamer / composite nanozyme solution, exosome standards, TMB substrate solution, H2O2 solution, and 96-well plate enzyme labeling strips. This invention innovatively utilizes Ti3C2T... x A nanozyme (MX-MnNF) formed by combining MXene nanosheets and MnO2 nanoflowers was constructed, and a colorimetric sensor array was built by modifying it with three aptamers: EpCAM, MUC1, and HER2. Based on the peroxidase-like activity of the nanozyme, it can catalyze the TMB-H2O2 reaction to produce color changes, enabling the simultaneous recognition of multiple exosomes. This method can complete the detection within 10 minutes, with a detection sensitivity of up to 10 particles / mL and an accuracy of up to 95% for clinical samples. It has the advantages of simple operation, rapid detection, and high sensitivity, providing a new technical means for tumor liquid biopsy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a kit for the simultaneous recognition of multiple exosomes. Background Technology

[0002] Exosomes, as nanoscale vesicles secreted by cells, carry abundant biomolecules (such as proteins, nucleic acids, and lipids) and demonstrate significant value in liquid biopsy fields such as tumor diagnosis and prognostic assessment. Exosome membrane protein expression profiles can reflect disease states, providing key biomarkers for early cancer diagnosis. Therefore, developing efficient and reliable exosome detection technologies is crucial for advancing precision medicine. Currently, common detection methods for exosomal proteins mainly include enzyme-linked immunosorbent assay (ELISA) and Western blotting. These methods rely on the binding of specific antibodies to target proteins. Although they have a certain degree of sensitivity, they have significant limitations: First, antibodies are expensive and have poor stability, easily affected by storage conditions; second, the operation procedures are cumbersome, requiring multiple washing and incubation steps, with detection cycles lasting several hours; third, multiplex detection is difficult to achieve, as a single antibody typically targets only a single target, making it impossible to simultaneously analyze multiple exosomal protein biomarkers, thus limiting their application in the diagnosis of complex diseases. In recent years, biosensor array technology, by integrating multiple sensing elements, can generate response signals to multi-component targets, forming specific "fingerprints" and providing new ideas for the multiplex detection of exosomes. For example, sensor arrays based on fluorescence, colorimetry, or electrochemistry have been used for the detection of extracellular vesicles. However, these methods still face challenges: fluorescent sensors are susceptible to photobleaching and background interference, resulting in insufficient detection stability; electrochemical sensors require complex electrode modification processes, leading to poor reproducibility; and existing sensor arrays mostly rely on a single signal mode, resulting in limited sensitivity for detecting low concentrations of exosomes (typically above 10). 3 (particles / mL), which is insufficient to meet the testing needs of clinical samples. Furthermore, while the application of nanomaterials in biosensing has improved detection performance, the catalytic activity of most nanoenzyme materials (such as noble metal nanoparticles) is significantly affected by pH and temperature, and they are prone to aggregation and inactivation, resulting in insufficient long-term stability. At the same time, the data processing of existing sensor arrays largely relies on simple threshold judgments, lacking intelligent algorithm support, making it difficult to cope with the heterogeneity of clinical samples, and the accuracy of identification needs to be improved.

[0003] The design of nanozymes has long been a technical challenge in this field, especially in the detection of low-concentration biomarkers such as exosomes, which places extremely high demands on the catalytic activity and stability of signal amplification elements. Therefore, it is crucial to develop a novel high-performance nanozyme. Summary of the Invention

[0004] The purpose of this invention is to provide a composite nanozyme and a kit for the simultaneous recognition of multiple exosomes. This composite nanozyme solves the problems of low catalytic activity, poor stability, and limited activity over a wide pH range associated with single nanozyme materials. The kit exhibits high sensitivity and good stability. To achieve the aforementioned objective, the present invention adopts the following technical solution: In a first aspect of the present invention, a composite nanozyme is provided, said composite nanozyme being composed of Ti3C2T x MXene nanosheets were used as a substrate, and MnO2 nanoflowers were formed on them by in-situ growth.

[0005] Furthermore, the Ti3C2T x The mass ratio of MXene to MnO2 is 1:0.5-4.

[0006] Furthermore, the Ti3C2T x The thickness of MXene nanosheets is 1-5 nm, and the particle size of MnO2 nanoflowers is 50-200 nm.

[0007] In a second aspect of the invention, a method for preparing the composite nanozyme is provided, the method comprising: Ti3C2T x The MXene nanosheet dispersion was mixed with a permanganate solution and subjected to a hydrothermal reaction at 100-150℃ for 2-12 h. After the reaction, the mixture was centrifuged, washed, and dried to obtain the composite nanozyme.

[0008] Preferably, the hydrothermal reaction temperature is 110-130°C, and the reaction time is 4-8 hours. This range exhibits optimal nanoflower morphology and catalytic activity in the embodiments.

[0009] Furthermore, the Ti3C2T x The mass ratio of the MXene nanosheet dispersion to the permanganate was 1:0.5 to 1:2 to control the MnO2 and Ti3C2T content in the final composite. x MXene quality ratio.

[0010] Furthermore, the permanganate is potassium permanganate (KMnO4).

[0011] Furthermore, the mixing is carried out under acidic conditions, with the pH of the reaction system not exceeding 5. The acidic environment is a decisive condition for generating specific nanoflower morphologies rather than other MnO2 morphologies. As a specific embodiment, the acidic conditions are provided by adding hydrochloric acid (HCl) to the reaction system.

[0012] Furthermore, the Ti3C2T x Isopropanol was used as a solvent for the MXene nanosheet dispersion.

[0013] Furthermore, after the hydrothermal reaction is completed, the centrifugation speed is 8000-12000 rpm, the washing is performed by washing with ultrapure water and ethanol 3-5 times in sequence, and the drying is performed by freeze drying.

[0014] As one specific implementation method, the preparation method includes: firstly, 20 mg of Ti3C2T x MXene nanosheets were added to 50 mL of isopropanol and sonicated for 30 minutes, then thoroughly mixed with 109 mg of MnCl2·4H2O. The suspension was heated to 85 °C to reach the boiling point of isopropanol. Then, 5 mL of KMnO4 solution (11 mg / mL dissolved in HCl) was rapidly added to the solution. After reflux for half an hour, the collected brownish-black reactant was centrifuged, washed five times with ultrapure water and ethanol, and freeze-dried to generate MnO2@Ti3C2T. x Nanocomposite material (MX-MnNF).

[0015] In a third aspect of the invention, a kit for simultaneous recognition of multiple exosomes is provided, the kit comprising: (1) Aptamer / nanozyme solution: formed by modifying the surface of the composite nanozyme with exosome-targeting aptamers; (2) TMB substrate solution; (3) H2O2 solution; Furthermore, the kit also includes: exosome standards; 96-well plate enzyme strips.

[0016] Furthermore, the exosome targeting aptamer is selected from at least one of the EpCAM aptamer, MUC1 aptamer, and HER2 aptamer.

[0017] Furthermore, the concentration of the TMB (full name 3,3',5,5'-tetramethylbenzidine) solution is 0.1-1 mg / mL, and the buffer solution is sodium acetate buffer with pH 4.0-5.5.

[0018] Furthermore, the concentration of H2O2 is 1-10 mM.

[0019] In a fourth aspect of the invention, a method for identifying exosomes for non-diagnostic purposes is provided, using the aforementioned kit for in vitro detection and analysis of tumor exosomes.

[0020] In a fifth aspect of the invention, Ti3C2T is provided. xApplication of MXene nanosheets and MnO2 nanoflower composite nanozymes in the preparation of exosome detection reagents, wherein the composite nanozymes have peroxidase-like activity.

[0021] Furthermore, the peroxidase-like activity is characterized by its ability to catalyze a colorimetric reaction in the TMB-H2O2 reaction system. In a sixth aspect of the invention, an exosome sensor array is provided, comprising at least three of the described aptamer / nanozyme solutions, capable of simultaneously identifying and detecting multiple exosomes.

[0022] As a specific implementation method, the detection method for simultaneously identifying multiple exosomes using the aforementioned kit includes the following steps: (1) Provide the aforementioned kit; (2) Mix the exosome sample to be tested with the aptamer / nanozyme solution to form a reaction system; (3) Add TMB substrate solution and H2O2 solution to carry out colorimetric reaction; (4) Measure the change in absorbance of the reaction system; (5) Based on the absorbance change value, the synchronous identification and quantitative analysis of multiple exosomes are achieved through machine learning algorithms. Furthermore, the reaction system described in step (2) is carried out in a sodium acetate buffer solution with a pH of 4.0-5.5, at a reaction temperature of 25-37°C, and for a reaction time of 3-30 minutes.

[0023] Furthermore, the exosome sample described in step (2) is first enriched by immunomagnetic bead method, specifically including: using CD63 antibody-modified magnetic beads to capture exosomes from plasma samples, magnetically separating them and then resuspending them in PBS buffer.

[0024] Further, in step (3), the concentration of the TMB substrate solution is 0.1-1 mg / mL, and the concentration of the H2O2 solution is 1-10 mM.

[0025] Furthermore, the absorbance measurement in step (4) is performed at a wavelength of 650 nm, with a detection sensitivity of 10⁻¹⁰. 9 particles / mL.

[0026] Furthermore, the machine learning algorithm described in step (5) is linear discriminant analysis or principal component analysis, specifically including the following sub-steps: (5.1) Collect the absorbance response values ​​of exosomes from three aptamer sensors and form a response matrix; (5.2) Project the multidimensional data into a low-dimensional space through linear discriminant analysis; (5.3) Calculate the Euclidean distance between the sample point and the reference point, and establish a concentration-distance standard curve; (5.4) Quantitative identification of exosomes based on standard curves.

[0027] Furthermore, the linear range of the concentration-distance standard curve is 10. 2 -10 9 particles / mL, goodness of fit R²≥0.99. Furthermore, in step (5), data analysis is performed using R language v4.3.1 or RStudio v2023.06.2, including data preprocessing, feature extraction and pattern recognition.

[0028] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention achieves breakthroughs in sensitivity, speed, multiplexing, and accuracy through an innovative combination of nanozyme-aptamer sensor arrays and machine learning, providing an efficient and reliable solution for the diagnosis of exosome-related diseases. Specifically: (1) Ultra-high detection sensitivity This invention enables the detection of extremely low concentrations of exosomes, with a detection limit of 10 particles / mL, far exceeding the detection limit of traditional methods (such as ELISA, which typically has a detection limit of 10). 3 -10 4 This is mainly attributed to the synergistic catalytic effect of Ti3C2TxMXene and MnO2 nanoflower composite nanozyme (MX-MnNF), whose peroxidase-like activity significantly enhances signal amplification, thereby enabling precise capture and signal conversion of trace exosomes. (2) Rapid detection efficiency The entire detection process, including exosome enrichment, colorimetric reaction, and signal reading, can be completed within 10 minutes, significantly reducing detection time (traditional methods require several hours). This is thanks to the rapid catalytic properties of nanozymes and optimized reaction system design, such as reagent premixing and short incubation. Multiple batches of samples can be processed within 30 minutes, improving the feasibility of high-throughput detection and making it suitable for large-scale clinical screening. (3) Multiple synchronous recognition capability By constructing a 3×n colorimetric sensor array and utilizing nanozymes modified with three aptamers (EpCAM, MUC1, and HER2), simultaneous recognition of multiple exosomal membrane protein markers was achieved. This "one-to-many" detection mode overcomes the limitations of traditional one-to-one methods, reduces inter-group variability, and improves detection efficiency. (4) High accuracy and intelligent analysis By combining machine learning algorithms (such as linear discriminant analysis (LDA)) to perform pattern recognition on multi-channel absorbance data, visual classification and quantitative analysis of exosomes were achieved. Clinical validation showed that the detection accuracy in plasma samples from cancer patients reached up to 95%, significantly improving diagnostic reliability. (5) Easy to operate and widely available equipment The assay requires only a standard microplate reader, without the need for complex instruments or specialized technical skills. The kit uses standardized components (such as pre-coated 96-well plates and ready-to-use reagents), simplifying the process and making it suitable for widespread application in primary healthcare institutions. Examples show that absorbance signals can be read within 3 minutes of a single sample addition, significantly reducing human error. (6) Good stability and repeatability The MX-MnNF nanozyme exhibited high and stable catalytic activity under optimized reaction conditions (pH 4.0-5.5, temperature 25-37℃), and aptamer modification further improved its binding specificity. Batch-to-batch experiments showed a coefficient of variation (CV) of less than 5%, ensuring excellent reproducibility of the detection method. (7) High clinical application value This method provides a novel tool for non-invasive liquid biopsy, which can be used for early tumor screening, treatment monitoring, and prognostic assessment. Exosome membrane proteomic analysis holds promise for cancer subtyping and personalized treatment. In the examples, the analysis of 156 clinical samples showed good discriminative ability (healthy group vs. cancer group), demonstrating its clinical translational potential. Attached Figure Description

[0029] Figure 1 This study investigated the peroxidase-like activities of MX-MnNF composite nanozymes before and after modification with three aptamers. Figure A shows a schematic diagram of the preparation of the MX-MnNF composite nanozyme and the aptamer modification process of MX-MnNF. Figure B shows the Ti3C2T... x Peroxidase-like activities of MXene nanosheets, MnO2 nanoflowers and MX-MnNF composite nanozymes. Figure C shows the absorbance at 650 nm of peroxidase activities of MXene nanosheets and MX-MnNF composites before and after modification with aptamers. Figure 2To optimize the catalytic reaction conditions of the MX-MnNF composite nanozyme, the following data are presented: A) Changes in the UV absorption curves of MXene and MX-MnNF within the pH range of 3.0 to 7.0; B) Analysis of the difference in absorbance of the MXene and MX-MnNF reaction systems at 650 nm within the pH range of 3.0 to 7.0; C) Changes in the UV absorption curves of MXene and MX-MnNF within the temperature range of 15°C to 55°C; D) Analysis of the difference in absorbance of the MXene and MX-MnNF reaction systems at 650 nm within the temperature range of 15°C to 55°C; E) Changes in the UV absorption curves of the system at different reaction times (0-30 minutes); and F) Analysis of the difference in absorbance of the system at 650 nm within different reaction times (0-30 minutes). Figure 3 The effect of different concentrations of exosomes on the activity of MX-MnNF composite nanozymes as peroxidases is shown in Figure A, where A represents the change in absorption spectrum after adding different concentrations of exosomes to the reaction system, and B represents the linear relationship between exosome concentration and system absorbance. Figure 4 This study demonstrates the ability of an aptamer-mediated nanozyme sensor array to simultaneously identify and quantify exosomes from various tumor cell sources. Figure 4 A: The specific response "fingerprint" generated when the sensor array detects exosomes from five different sources (A549, 4T1, HeLa, MDA-MB-231, SK-OV-3). Figure 4 B: The two-dimensional scatter plot obtained after processing the above fingerprint data by linear discriminant analysis (LDA) shows that the five exosomes form distinct clusters. Figure 4 C: Scatter plot distribution of different concentrations of A549-derived exosomes (A549-Exo) after LDA analysis in a simulated clinical environment containing 0.5% serum. Figure 4 D: The linear standard curve between exosome concentration and the calculated Euclidean distance (ED) demonstrates the quantitative reliability of this method.

[0030] Figure 5 Figure 1 shows the detection performance of the aptamer-mediated nanozyme sensor array in clinical sample testing trials. In the figure, A is the LDA scatter plot, B is the significance between the healthy group and the cancer group assessed by one-way ANOVA (****p<0.0001, unpaired t test), C is the confusion matrix showing that the accuracy of the LDA diagnostic model between the healthy group and the cancer group is 95.0%, and D is the ROC analysis of all samples (n = 156) from healthy and cancer patients. Detailed Implementation

[0031] The following detailed description of the embodiments and examples will illustrate the present invention in more detail, thereby making the advantages and various effects of the embodiments more clearly apparent. Those skilled in the art should understand that these detailed embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0032] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. In the event of any conflict, this specification shall prevail.

[0033] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained by purchasing them on the market or by existing methods.

[0034] The overall concept of this invention is as follows: The core of this invention lies in constructing a novel colorimetric sensor array system through the integration of multidisciplinary technologies, enabling rapid and highly sensitive simultaneous identification of various exosomes. The overall approach is based on synergistic innovation across materials, methods, and algorithms, with specific innovations as follows: 1. Innovative design of nanoenzyme materials: Ti3C2T x Composite structure of MXene and MnO2 nanoflowers Innovation: The first application of two-dimensional material Ti3C2T x MXene was combined with MnO2 nanoflowers to construct a nanozyme (MX-MnNF) with high peroxidase-like activity. The layered structure of MXene provides a large specific surface area and abundant active sites, while the MnO2 nanoflowers enhance catalytic stability and signal amplification ability through in-situ growth. Technical benefits: This composite nanozyme overcomes the problem of insufficient catalytic activity of single materials, maintains high activity over a wide pH range, and provides a stable signal basis for colorimetric detection. Figure 2 The preparation process of the composite nanozyme and the comparison of enzyme activities were demonstrated.

[0035] 2. Aptamer-modified sensor array: "One-to-many" detection mode Innovation: Nanozymes are modified with three exosome-targeting aptamers (EpCAM, MUC1, and HER2) to form a 3×n colorimetric sensor array. Each aptamer specifically binds to different exosome membrane proteins, generating a unique "fingerprint" through absorbance changes, enabling multiple simultaneous detection. Technical benefits: It breaks through the limitations of traditional one-to-one testing, significantly reduces inter-group differences, and improves testing throughput and accuracy. Figure 1A schematic diagram of the three sensing units in the sensor array assembly is shown. 3. Optimization of detection methods: Integration of rapid colorimetric reaction with machine learning Innovation: Combining nanozyme catalysis (TMB-H2O2 system) with machine learning algorithms (such as linear discriminant analysis LDA). The reaction is completed within 10 minutes, and the absorbance data is intelligently analyzed to achieve automatic classification and quantification of exosomes. Technical benefits: Detection time is reduced from hours to minutes, sensitivity reaches 10 particles / mL, and human error is reduced through algorithms, achieving an accuracy of 95%. Figure 4 and Figure 5 The array's discriminative capabilities and clinical validation results were presented respectively.

[0036] 4. Integrated design of the reagent kit: ease of operation and clinical application orientation Innovation: The kit contains pre-optimized components (aptamer / nanozyme solution, standards, chromogenic reagents, etc.), supporting a one-stop "sample addition-detection-analysis" workflow. The 96-well plate format is compatible with conventional microplate readers, eliminating the need for complex equipment. Technical Benefits: Significantly lowers the operational threshold, making it suitable for primary healthcare institutions and promoting the widespread adoption of liquid biopsy for tumors. 5. Breakthrough advantages brought about by technological collaboration High sensitivity and specificity: Nanozymes amplify signals through catalysis, and aptamers ensure targeted recognition, resulting in a detection limit that is 100 times lower than that of traditional ELISA. Multiple detection capabilities: Simultaneously identify multiple exosome biomarkers, providing a new tool for cancer subtyping and treatment monitoring. Robustness and reproducibility: Nanozymes exhibit high stability in complex environments such as serum, with a batch-to-batch coefficient of variation of <5%. Figure 3 The linear relationship between concentration and signal was demonstrated, supporting the reliability of the quantitative analysis.

[0037] In summary, the overall approach of this invention is to overcome the technical bottlenecks in exosome detection, such as low sensitivity, cumbersome operation, and difficulty in multiplex detection, through a three-layer innovation of "material composite - array design - algorithm integration". This solution not only improves detection performance but also promotes the development of intelligent and portable biosensors, possessing significant scientific research and clinical value.

[0038] The following will provide a detailed description of a kit for simultaneous recognition of multiple exosomes, based on embodiments and experimental data.

[0039] Example 1: Ti3C2T x Preparation and characterization of MXene-MnO2 composite nanozymes (MX-MnNF) 1. Experimental Objective Preparation of Ti3C2T with high peroxidase-like activityx MXene-MnO2 composite nanomaterials were studied, and their morphology, structure, and catalytic performance were systematically characterized. 2. Experimental Procedure (1) Ti3C2T x Synthesis of MXene-MnO2 composite nanozymes: according to Figure 1 As shown in procedure A, 3.2 g of LiF was mixed with 40 mL of 10.5 M HCl solution, and 0.8 g of LiF was added. The mixture was stirred at 45 °C for 24 hours. The precipitate was collected by centrifugation, washed with deionized water until pH=6, and then ultrasonically exfoliated for 2 hours under argon protection to obtain a monolayer of Ti3C2T. x MXene nanosheet suspension (1 mg / mL). Add 20 mL of Ti3C2T... x MXene nanosheet suspension (1 mg / mL) was sonicated for 30 minutes and thoroughly mixed with 109 mg MnCl2·4H2O, then heated to 85 °C to reach the boiling point of isopropanol. Subsequently, 5 mL of KMnO4 solution (11 mg / mL dissolved in HCl) was rapidly added to the solution. After reflux for half an hour, the collected brownish-black reactant was centrifuged, washed five times with ultrapure water and ethanol, and freeze-dried to generate MnO2@Ti3C2T. x Nanocomposite material (MX-MnNF) (2) Aptamer modification: EpCAM, MUC1, and HER2 aptamers were covalently coupled to the surface of MX-MnNF using the EDC / NHS method to obtain three different sensing elements. Specifically, MX-MnNF was dispersed in PBS buffer (pH 7.4) and divided into three portions. EpCAM, MUC1, and HER2 aptamers (molar ratio 1:5) were added to each portion, along with EDC / NHS activator, and the mixture was reacted at room temperature for 12 hours. The aptamer-modified MX-MnNF complex (MX-MnNF / Apt) was obtained by ultrafiltration and centrifugation purification.

[0040] (3) Study on the activity of nanozymes as peroxidases according to Figure 1 B and C: Sample preparation: Setting up the experimental group (MX-MnNF complex) and the control group: MX-MnNF composite: The experimental group directly used composite MX-MnNF nanomaterials.

[0041] Single MXene: Use the prepared MXene dispersion directly; Single MnO2 nanoflowers (MnNF): Pure MnO2 nanoflowers were prepared using the same procedure as above without the addition of MXene. MXene & MnNF physical mixing group: The prepared single MXene and single MnO2 were physically mixed at a mass ratio of 1:1.

[0042] Reference Figure 1 Method B: Take 100 μg / mL nanozyme solution, add TMB and H2O2 under NaAc-HAc buffer reaction conditions, react at 37℃ for 10 minutes, and then measure the UV absorption spectrum at 400-800 nm.

[0043] according to Figure 1 Option C: Compare the changes in enzyme activity before and after aptamer modification.

[0044] 3. Results and Discussion The embodiments of the present invention compare Ti3C2T X The peroxidase-like activities of MXene nanosheets, MnNF, and MX-MnNF composite nanozymes are as follows: Figure 1 As shown in B, and listed below. Table 1

[0045] As shown above, compared with the highly active single component (MnNF, 1.43), the activity of the MX-MnNF composite nanozyme was increased by approximately 69%; compared with single MXene (0.84), the peroxidase-like activity of the MX-MnNF composite nanozyme was increased by as much as approximately 188% (1.8 times). Through a specific in-situ growth composite process, a strong synergistic catalytic effect was generated between MXene and MnO2, rather than a simple addition of activities. This synergistic effect is the core innovation of this invention and a key piece of evidence for its inventiveness. Figure 1 As shown in C, the activity of the single MXene enzyme decreased slightly after aptamer modification, while the enzyme-like activity of the MX-MnNF composite nanozyme was basically unaffected, which meets the expected design goal.

[0046] like Figure 1 C. A successfully constructed sensor array system was demonstrated, with all three aptamer-modified nanozymes exhibiting excellent absorbance responses at 650 nm. This array enables multiplex recognition of exosomes, laying the foundation for subsequent detection applications.

[0047] Example 2: Optimization of Detection Conditions 1. Experimental Objective This embodiment aims to systematically optimize the reaction conditions (including pH, temperature, and reaction time) of the colorimetric detection system based on MX-MnNF composite nanozymes, and comprehensively evaluate the analytical performance of the optimized system, including sensitivity, linear range, selectivity, anti-interference ability, and actual detection capability in complex biological samples, and finally establish a standardized operating procedure.

[0048] 2. Experimental Procedures and Results Analysis 2.1 System optimization of detection conditions To determine the optimal reaction conditions, we systematically optimized pH, temperature, and reaction time using absorbance as an indicator. All optimization experiments used PBS as the buffer system, and the concentrations of MX-MnNF nanozyme, TMB, and H2O2 were kept constant.

[0049] pH optimization (see...) Figure 2 A, B): The response of the detection system was tested within the pH range of 3.0 to 7.0. Results are as follows... Figure 2 As shown in Figures A and B, the system exhibits the highest absorbance value at pH 4.0. The absorbance value decreases significantly when the pH is above or below 4.0. This indicates that an acidic environment is most favorable for the MX-MnNF nanozyme-catalyzed TMB-H2O2 reaction. Therefore, pH 4.0 was chosen as the optimal acidity condition for the reaction.

[0050] Optimization of reaction temperature (see) Figure 2 C, D): Tests were conducted within a temperature range of 15°C to 55°C. Results are as follows: Figure 2 Figures C and D show that the reaction system maintained high and stable catalytic efficiency within the temperature range of 25°C to 37°C. Considering the convenience and stability for practical applications, 37°C was selected as the standard reaction temperature for subsequent experiments.

[0051] Optimization of reaction time (see) Figure 2 E, F): Monitor the change in absorbance of the reaction system at 650 nm over time (0-30 minutes). For example... Figure 2 As shown in E and F, the absorbance values ​​increase rapidly within 25 minutes after the start of the reaction and then plateau. To ensure complete reaction while maintaining detection efficiency, 25 minutes was determined to be the optimal reaction time.

[0052] Example 3: Sensitivity and Linearity Range Validation of Exosome Concentration Detection 1. Experimental Objective like Figure 3 The relationship between exosome concentration and absorbance is shown, and a standard curve for quantitative detection of exosomes is established.

[0053] 2. Experimental Procedure (1) According to Figure 3 Method A: Prepare a concentration gradient of 10 2 10 10 MDA-MB-231 cell-derived exosome standards with particles / mL. (2) Detection procedure: Take 20 μL of exosomes of various concentrations and mix them with aptamer / MX-MnNF solution, add TMB / H2O2 substrate, and after reacting for 25 minutes, measure the UV absorption spectrum of the reaction solution (400-800 nm). (3) Data processing: According to Figure 3 Method B establishes a linear relationship curve between absorbance at 650 nm and exosome concentration. 3. Results and Discussion In 10 7 10 10 Within the particle / mL concentration range, absorbance showed a good linear relationship with exosome concentration (R²=0.992). Example 4: Simultaneous identification and differential analysis of multiple exosomes 1. Experimental Objective Verify the sensor array's ability to identify exosomes from various tumor cells and establish a fingerprint database. 2. Experimental Procedure (1) Sample preparation: according to Figure 4 Protocol A involves preparing exosomes derived from five tumor cell lines: A549, 4T1, HeLa, MDA-MB-231, and SK-OV-3. (2) Array detection: Each exosome reacts with a sensor modified with three aptamers to obtain a 3×5 response matrix. (3) Data analysis: using Figure 4 The linear discriminant analysis (LDA) method shown in B processes the data to obtain a discriminant scatter plot. (4) Quantitative verification: The quantitative detection and analysis of exosomes in a serum detection environment includes the following steps: Step 1: Taking A549 cell-derived exosomes as an example, the exosomes were mixed with 0.5% serum solution to obtain a series of exosome dilutions with concentration gradients (10-fold dilution) (2.1 × 10⁻⁶). 9 2.1 × 10 8 2.1 × 10 7 2.1 × 10 6 2.1 × 10 5 2.1 × 10 4 2.1 × 10 3 2.1 × 10 2 (particles / mL).

[0054] Step 2: Add 20 μL of MX-MnNF / Apt solution and 10 μL of the above series of concentration gradient (10-fold dilution) exosome mixtures to the detection plate in sequence. Perform 6 technical replicates at each concentration and incubate at room temperature for 20 minutes.

[0055] Step 3: Add 20 μL H2O2 (5 mM), 25 μL TMB (10 mM) and sodium acetate buffer to the above well plate in sequence, and incubate in the dark for 10 minutes.

[0056] Step 4: Measure the absorbance at 650 nm using an ELISA reader and analyze the relationship between the absorbance change and the exosome concentration.

[0057] Step 5: Process the absorbance change response data using LDA, and simultaneously calculate the Euclidean distance using cluster analysis to establish a standard curve between "concentration" and "ED" (see details of the standard curve). Figure 4 D).

[0058] 3. Results and Discussion like Figure 4 As shown in A and 4B, the sensor array successfully distinguished exosomes from five different sources, forming clearly separated clusters in LDA analysis (accuracy > 95%). The Euclidean distance showed a good linear relationship with exosome concentration. Figure 4 (D) This demonstrates the quantitative reliability of the method.

[0059] Example 5: Performance evaluation of aptamer-mediated nanozyme sensor array for clinical sample detection 1. Experimental Objective This embodiment aims to evaluate the detection performance of the aptamer-mediated nanozyme sensor array in real clinical samples, including its identification accuracy, sensitivity, specificity, and feasibility for practical application, providing experimental evidence for clinical translation.

[0060] 2. Experimental Materials and Methods 2.1 Clinical Sample Collection and Preparation Sample source: Peripheral blood samples were collected from 96 cancer patients and 60 healthy volunteers (a total of 156 samples) with the approval of the ethics committee and informed consent from the patients.

[0061] Sample processing: Collect whole blood using EDTA anticoagulant tubes, centrifuge at 3000 rpm for 15 minutes to separate plasma, and store at -80℃ for later use.

[0062] Exosome enrichment: Take 100 μL of plasma, add CD63 antibody-modified immunomagnetic beads, incubate at 4°C for 2 hours, and after magnetic separation, resuspend the exosomes in PBS.

[0063] 2.2 Sensor Array Detection Detection procedure: Following the method in Example 2, the enriched exosomes were reacted with the MX-MnNF sensor modified with three aptamers, TMB / H2O2 substrate was added, the reaction was carried out at 37°C for 25 minutes, and the absorbance at 650 nm was measured.

[0064] Data acquisition: Three absorbance values ​​were obtained for each group of samples (corresponding to EpCAM, MUC1, and HER2 aptamers), forming a feature response matrix.

[0065] 2.3 Data Analysis and Model Building Machine learning analysis: Linear discriminant analysis (LDA) was performed using R (v4.3.1) to project the absorbance data of 156 samples into a two-dimensional space and calculate the Euclidean distance between the sample points and the center of the healthy group.

[0066] Model validation: The leave-one-out cross-validation method was used to construct the diagnostic model and evaluate its accuracy, sensitivity, and specificity.

[0067] Statistical tests: The significance of differences between groups was assessed by one-way ANOVA and unpaired t-test (p<0.05 was considered significant).

[0068] 3. Experimental Results The test results for 156 clinical samples (96 cancer patients and 60 healthy volunteers) are as follows: Figure 5 As shown.

[0069] LDA clustering analysis ( Figure 5 A): The sample points of cancer patients and healthy volunteers are clearly separated in two-dimensional space.

[0070] Statistical significance ( Figure 5 B): There was a highly significant difference in the Euclidean distance between the response signals of the groups (****p<0.0001).

[0071] Diagnostic accuracy ( Figure 5 C): The overall classification accuracy of the LDA diagnostic model is as high as 95.0%.

[0072] ROC analysis ( Figure 5 D): The area under the curve (AUC) reaches 0.994, indicating that the method has extremely high discrimination accuracy.

[0073] 4. Experimental Conclusions This embodiment demonstrates that the sensor array has extremely high accuracy in identifying clinical samples, providing a reliable tool for tumor liquid biopsy.

[0074] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0075] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0076] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. A composite nanozyme, characterized in that, The composite nanozyme is composed of Ti3C2T x MXene nanosheets were used as a substrate, and MnO2 nanoflowers were formed on them by in-situ growth.

2. The composite nanozyme according to claim 1, characterized in that, The Ti3C2T x The mass ratio of MXene to MnO2 is 1:0.5-4.

3. The composite nanozyme according to claim 1, characterized in that, The Ti3C2T x The thickness of MXene nanosheets is 1-5 nm, and the particle size of MnO2 nanoflowers is 50-200 nm.

4. A method for preparing the composite nanozyme according to any one of claims 1-3, characterized in that, The method includes: Ti3C2T x The MXene nanosheet dispersion was mixed with a permanganate solution and subjected to a hydrothermal reaction at 100-150℃ for 2-12 hours. After the reaction, the mixture was centrifuged, washed, and dried to obtain the composite nanozyme.

5. The preparation method according to claim 4, characterized in that, The Ti3C2T x The mass ratio of the MXene nanosheet dispersion to the permanganate is 1:0.5 to 1:

2.

6. A kit for simultaneous recognition of multiple exosomes, characterized in that, The kit includes: (1) Aptamer / composite nanozyme solution: formed by surface modification of exosome-targeting aptamers of the composite nanozyme according to any one of claims 1-3; (2) TMB substrate solution; (3) H2O2 solution.

7. The kit for simultaneous recognition of multiple exosomes according to claim 6, characterized in that, The exosome targeting aptamer is selected from at least one of the EpCAM aptamer, MUC1 aptamer, and HER2 aptamer.

8. The kit for simultaneous recognition of multiple exosomes according to claim 6, characterized in that, The concentration of TMB in the TMB substrate solution is 0.1-1 mg / mL, and the buffer solution is sodium acetate buffer with pH 4.0-5.

5.

9. The kit for simultaneous recognition of multiple exosomes according to claim 6, characterized in that, The concentration of H2O2 in the H2O2 solution is 1-10 mM.

10. A Ti3C2T x The application of MXene nanosheets and MnO2 nanoflower composite nanozymes in the preparation of exosome detection reagents is characterized by, The composite nanozyme exhibits peroxidase-like activity.

Images