Extracellular vesicle metabolite detection system and tumor metabolism marker detection method

Abstract

The invention discloses an extracellular vesicle metabolite detection system which comprises a vacuum self-driven micro-fluidic chip, the vacuum self-driven micro-fluidic chip is arranged to be of a three-layer structure composed of a PDMS covering layer, a reaction layer embedded with a micro-column array and a glass substrate layer, the PDMS covering layer constructs a vacuum cavity through the porous characteristic of PDMS, and the micro-column array is composed of micro-columns with the perimeter being 600 microns; the detection unit is connected with the vacuum self-driven micro-fluidic chip, is set as a group of three-channel independent detection areas, is preloaded with a biological enzyme-nano enzyme coupled double-enzyme system, and is used for realizing metabolite specific detection by combining a fluorescence signal amplification substrate; the biological enzyme-nano enzyme coupled double-enzyme system is formed by loading lactate oxidase LOX and choline oxidase COD on the surface of O-MoS2 through electrostatic adsorption. According to the invention, the detection signal of the low-abundance metabolite can be effectively amplified, and the detection flux and efficiency can be improved, so that the clinical rapid and accurate detection requirements can be met.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and specifically to a high-sensitivity detection system for low-abundance metabolites (such as lactate and choline) in extracellular vesicles based on dual-enzyme cascade amplification and microfluidic chips, a detection platform integrating bioenzyme-nanozyme coupled dual-enzyme signal amplification and vacuum negative pressure microfluidic chips, and a detection method for its application in the detection of tumor metabolic markers. Background Art

[0002] The occurrence and development of malignant tumors are closely related to cellular metabolic reprogramming, among which abnormalities in sugar metabolism and lipid metabolism are particularly significant. Lactic acid, as the main metabolite of the glycolysis pathway of tumor cells (Warburg effect), and choline, as a key component of cell membrane phospholipid metabolism, are two important small molecule metabolites that characterize the metabolic characteristics of tumor cells.

[0003] In recent years, studies have shown that extracellular vesicles (EVs) secreted by tumor cells not only carry nucleic acid and protein information, but are also enriched with metabolic small molecules from the source cells. Therefore, detecting the lactate and choline content in EVs is expected to be a non-invasive means to reflect the metabolic status of their parent tumor cells in real time, and has important potential application value in the early diagnosis of tumors (especially lung cancer), efficacy evaluation (such as immunotherapy response monitoring), and prognosis.

[0004] However, developing metabolites in EVs (such as lactate and choline) into clinically applicable biomarkers faces major technical challenges. The most important technical bottleneck is that the concentration of metabolites in EVs is extremely low. Existing metabolomics-based detection methods (such as mass spectrometry and nuclear magnetic resonance) usually require expensive equipment, complex sample pretreatment processes and professional operators, and have extremely high detection sensitivity requirements. These factors greatly limit the widespread application and promotion of such detection technologies in clinical settings.

[0005] To this end, this application specifically proposes an extracellular vesicle metabolite detection system and a tumor metabolic marker detection method to solve the above technical problems. Summary of the Invention

[0006] The main purpose of the present invention is to provide an extracellular vesicle metabolite detection system and a tumor metabolic marker detection method, which can effectively amplify the detection signal of low-abundance metabolites and improve the detection flux and efficiency to meet the clinical needs of rapid and accurate detection, thereby solving the technical problems raised in the background technology.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] An extracellular vesicle metabolite detection system, comprising:

[0009] The vacuum self-driven microfluidic chip (ExoMeta chip) is a three-layer structure consisting of a PDMS cover layer, a reaction layer embedded with a microcolumn array, and a glass substrate layer. The PDMS cover layer constructs a vacuum cavity through the porous properties of PDMS, which uses the porous properties of PDMS to form a "vacuum battery" to achieve pump-free negative pressure driven sampling. The microcolumn array uses microcolumns with a circumference of 600 μm (C600 group), which has low flow rate (1.8 mm / s), low shear rate (average 15s -1 ), uniform pressure distribution (pressure difference ~ 3Pa) characteristics to ensure sufficient reaction;

[0010] The detection unit is connected to the vacuum self-driven microfluidic chip and is set up as a set of three independent detection areas (control / lactate / choline group). It is pre-loaded with a bioenzyme-nanozyme coupled dual enzyme system for combining with a fluorescent signal amplification substrate (AmplifluRed) to achieve metabolite-specific detection;

[0011] The bioenzyme-nanozyme coupled dual enzyme system (O-MoS2-LOX / COD) is formed by electrostatically adsorbing lactate oxidase LOX and choline oxidase COD on the O-MoS2 surface.

[0012] Preferably, the vacuum self-driven microfluidic chip is combined with the detection unit to construct an EVs metabolite detection platform. The workflow of the EVs metabolite detection platform includes: EVs cleavage → metabolites enter the microfluidic chip → vacuum drive flows through the microcolumn reaction area → coupled dual-enzyme cascade catalysis → fluorescence signal output (excitation / emission: 570 / 585 nm).

[0013] Preferably, the sample volume of the EVs metabolite detection platform is ≤10 μL, and the detection time is ≤15 minutes.

[0014] Preferably, the bioenzyme-nanozyme coupled dual enzyme system uses bulk MoS2 as a precursor, adopts 15% H2O2 solution for controllable oxidation, and combines ultrasonic exfoliation to prepare ultrathin porous O-MoS2 nanosheets (particle size of about 145 nm).

[0015] Preferably, the construction process of the bioenzyme-nanozyme coupled dual enzyme system includes:

[0016] Take 100 mg of bulk MoS2 powder, add 20 mL of H2O2 solution of specified concentration (30% stock solution dilution), and stir in a 60°C water bath for 6 hours to obtain reaction solution A;

[0017] After the reaction solution A was centrifuged, the supernatant was discarded, and the precipitate was washed three times with deionized water, redispersed in water, and then ultrasonicated in an ice bath for 1 hour to obtain an O-MoS2 dispersion.

[0018] Lactate oxidase LOX and choline oxidase COD were dissolved in PBS buffer, and O-MoS2 dispersion of specified concentration was added. The reaction was shaken at room temperature for 2 hours. After centrifugation to remove unbound enzymes, the precipitate was resuspended to obtain O-MoS2-LOX / COD.

[0019] Preferably, the vacuum self-driven microfluidic chip uses an SU-8 mold to photoetch a microcolumn array (circumference 600 μm), which is bonded to a glass substrate after PDMS casting;

[0020] The vacuum self-driven microfluidic chip is used to connect the reaction channel of the detection unit preloaded with O-MoS2-LOX / COD (0.1 mg / mL) and is vacuum-encapsulated synchronously with the detection unit during the manufacturing process.

[0021] A method for detecting tumor metabolic markers, based on any of the above-mentioned extracellular vesicle metabolite detection systems, includes the following specific operating procedures:

[0022] After obtaining the sample markers, the sample cell supernatant was collected, ultracentrifuged for 1 h, and extracellular vesicles were extracted and treated with lysate to release metabolites;

[0023] 10 μL of sample was added to the inlet of the chip channel. The vacuum drive was started by the vacuum self-driven microfluidic chip. The sample flowed through the microcolumn reaction area within 60 seconds. After 15 minutes of reaction at room temperature in the dark, the Ampliflu Red oxidation product was generated.

[0024] After the sample enters the channel of the detection unit, a three-channel signal is collected in the detection unit through a fluorescence microscope (Ex / Em=570 / 585nm):

[0025] Channel 1: collect background fluorescence as a blank control;

[0026] Channel 2: collects lactate signals and serves as the LOX group;

[0027] Channel 3: collects choline signals as the COD group;

[0028] Tumor metabolic marker detection results can be obtained based on three-channel signals.

[0029] Preferably, the method for obtaining the tumor metabolism marker detection results includes:

[0030] Taking the background fluorescence of the blank control in channel 1 as the benchmark, after deducting the background signal, the fluorescence intensity of the LOX group in channel 2 and the COD group in channel 3 were determined by the specified standards;

[0031] (1) If the fluorescence intensity of the LOX group or COD group is higher than that of the blank control group by a specified multiple, it indicates the presence of the corresponding metabolites in the sample.

[0032] (2) If the metabolite concentration corresponding to the fluorescence signal intensity of the LOX group or COD group is higher than the preset detection limit, it can be judged as a positive result, and the output result is: there may be abnormal tumor metabolic markers; otherwise, it is a negative result, and the output result is: there is no abnormal tumor metabolic marker.

[0033] (3) If the fluorescent signal of lactate or choline detected in the extracellular vesicles is consistent with the expression trend of the corresponding metabolites in the parental cells and is judged to be positive, the output result is: there is a high possibility of abnormal tumor metabolic markers.

[0034] As can be seen from the above technical solutions, the present invention provides an extracellular vesicle metabolite detection system and a tumor metabolic marker detection method. Compared with the existing technology, the present invention has the following advantages:

[0035] 1. In the preparation process of O-MoS2 nanozyme, the present invention uses bulk MoS2 as a precursor and adopts H2O2 solution for gradient oxidation, which can regulate the degree of oxidation of the material, retain the original properties of the material and form an ultra-thin sheet structure with a large number of holes, so that O-MoS2 has efficient peroxidase-like activity, thereby improving the catalytic efficiency of the nanozyme and providing a highly sensitive signal amplification basis for measuring metabolite detection.

[0036] 2. The present invention forms a three-layer structure consisting of a PDMS cover layer, a reaction layer embedded with a micropillar array, a glass base layer, and a vacuum chamber in a microfluidic chip. The porous properties of PDMS are utilized to form a "vacuum battery" to achieve pump-free negative pressure drive, and the micropillar array is optimized to enable it to control flow rate, reduce shear rate, and uniformly distribute pressure, thereby ensuring sufficient reaction between the sample and the coupled dual enzyme, and realizing low-sample-volume and high-efficiency rapid detection, thereby significantly improving detection throughput and efficiency to meet the needs of clinical portable detection.

[0037] 3. The present invention loads lactate oxidase LOX and choline oxidase COD on the O-MoS2 surface through electrostatic adsorption to construct a bioenzyme-nanozyme coupled dual-enzyme system. It can utilize the synergistic effect of the dual enzyme proximity to shorten the substrate diffusion path, thereby enhancing the catalytic efficiency and achieving metabolite-specific fluorescence signal amplification, ultimately breaking through the traditional detection sensitivity limit, facilitating the accurate detection of low-abundance lactate and choline in EVs, and providing a new tool for tumor diagnosis.

[0038] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present invention, nor is it intended to limit the scope of the present invention. Other features of the present invention will become easy to understand through the following description. Of course, it is not necessary to achieve all of the above-mentioned advantages simultaneously in order to implement any product of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0040] Figure 1 This is a schematic diagram of the overall process of the ExoMeta chip detection method for detecting EVs metabolites based on efficient nanozymes of the present invention;

[0041] Figure 2 Schematic diagram of the construction and catalytic performance comparison analysis of the coupled dual-enzyme system of the present invention, wherein: (A) is a schematic diagram of the catalytic principle of the coupled dual-enzyme system, (B) is a schematic diagram of the zeta point analysis comparison, and (C) is a schematic diagram of the activity analysis comparison of the coupled dual-enzyme system;

[0042] Figure 3 This is a schematic plan view of the C600 microfluidic chip design of the present invention;

[0043] Figure 4 Schematic diagrams of flow field and velocity simulations for three types of micropillar arrays in the ExoMeta chip of the present invention, including: (A) a schematic diagram of the overall structure of the ExoMeta chip, (B) a schematic diagram of the flow field and flow rate simulation for micropillar arrays of different perimeters, and (C) a schematic diagram of the flow rate comparison for micropillar arrays of different perimeters;

[0044] Figure 5 Schematic diagrams of the fitting simulation of the control mechanism of the geometric parameters of the three micropillar array schemes of the present invention on the flow field characteristics of the ExoMeta chip, wherein: (A) is a simulation diagram of the shear rate of the ExoMeta chip for micropillar arrays with different perimeters, (B) is a comparison diagram of the shear rates of micropillar arrays with different perimeters, (C) is a comparison diagram of the average shear rates of micropillar arrays with different perimeters, (D) is a simulation comparison diagram of the boundary surface pressure of micropillar arrays with different perimeters, (E) is a comparison diagram of the boundary surface pressure of micropillar arrays with different perimeters as they change with length, and (F) is a comparison diagram of the boundary surface pressure of micropillar arrays with different perimeters;

[0045] Figure 6Schematic diagram of the detection areas of the lactate group and the choline group, demonstrating the detection performance of the ExoMeta chip of the present invention, wherein: (A) is a schematic diagram of the change of the ExoMeta chip detection over time, (B) is a schematic diagram of the comparison of the detection results of the ExoMeta chip, and (C) is a schematic diagram of the comparison of the detection intensity of the ExoMeta chip;

[0046] Figure 7 Schematic diagram of the detection sensitivity, consistency and biological relevance verification of the ExoMeta chip of the present invention, wherein: (A) is a schematic diagram of the comparison of the standardized lactate signal of EVs metabolite detection between the standard kit and other conventional schemes, (B) is a schematic diagram of the comparison of the standardized choline signal of EVs metabolite detection between the standard kit and other conventional schemes, (C) is a schematic diagram of the comparison of the critical flow coefficient FL of lactate and choline under the detection of the standard kit, (D) is a schematic diagram of the comparison of the relative expression signals of lactate and choline in extracellular vesicles EVs and conventional cells, (E) is a schematic diagram of the relationship between the relative expression signal of intracellular lactate and the relative expression signal of lactate in extracellular vesicles EVs, and (F) is a schematic diagram of the relationship between the relative expression signal of intracellular choline and the relative expression signal of choline in extracellular vesicles EVs. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0048] In the embodiment, see Figures 1 to 7 .

[0049] refer to Figure 1 The extracellular vesicle metabolite detection system proposed in the embodiment of the present invention includes:

[0050] The vacuum self-driven microfluidic chip (ExoMeta chip) is a three-layer structure consisting of a PDMS cover layer, a reaction layer embedded with a microcolumn array, and a glass substrate layer. The PDMS cover layer constructs a vacuum cavity through the porous properties of PDMS. It uses the porous properties of PDMS to form a "vacuum battery" to achieve pump-free negative pressure driven sampling. The microcolumn array uses microcolumns with a circumference of 600 μm (C600 group), which has low flow rate (1.8 mm / s), low shear rate (average 15s -1 ), uniform pressure distribution (pressure difference ~ 3Pa) characteristics to ensure sufficient reaction;

[0051] The detection unit is provided on the vacuum self-driven microfluidic chip and connected to the inlet channel of the vacuum self-driven microfluidic chip. It is set as a set of three independent detection areas (control / lactate / choline group) and is pre-loaded with a bioenzyme-nanozyme coupled dual enzyme system for combining with a fluorescent signal amplification substrate (Ampliflu Red) to achieve metabolite-specific detection. In addition, in one embodiment, the detection unit can also be provided independently;

[0052] The bioenzyme-nanozyme coupled dual enzyme system (O-MoS2-LOX / COD) is formed by electrostatic adsorption of lactate oxidase LOX and choline oxidase COD on the O-MoS2 surface.

[0053] It's important to note that O-MoS2 is prepared via a relatively simple one-pot oxidation process, using bulk MoS2 as a precursor and hydrogen peroxide (H2O2) as an oxidant. Compared to other strong oxidants (such as potassium permanganate or nitric acid), H2O2 can effectively and directly adjust its oxidizing properties by adjusting its concentration, thus avoiding over-oxidation or structural damage to the two-dimensional nanomaterial, thereby better preserving the original properties of the material. Therefore, it was chosen as a controllable surface modification oxidant.

[0054] Furthermore, the vacuum self-driven microfluidic chip and the detection unit are combined to construct an EVs metabolite detection platform. The workflow of the EVs metabolite detection platform includes: EVs cleavage → metabolites enter the microfluidic chip → vacuum drive flow through the microcolumn reaction area → coupled dual-enzyme cascade catalysis → fluorescence signal output (excitation / emission: 570 / 585nm).

[0055] What can be explained in detail is that the principle is to use it to oxidize bulk MoS2. During the oxidation process, the Mo-S bonds of MoS2 are partially broken, and the S atoms are replaced by excess O atoms. As the concentration of the H2O2 solution increases, more S atoms in MoS2 are replaced by O atoms, which weakens the van der Waals force between adjacent layers. Therefore, O-MoS2 can be easily obtained through ultrasonic exfoliation treatment.

[0056] Furthermore, in a specific embodiment, the particle size of O-MoS2 can be measured by dynamic light scattering (DLS) to be approximately 145 nm. In summary, under the same conditions of controlled reaction time and ultrasonic exfoliation, there is no significant difference in appearance and size of O-MoS2. The composition changes before and after oxidation were studied by X-ray photoelectron spectroscopy (XPS).

[0057] Further in a specific embodiment, in O-MoS2 with different oxidation degrees (8%, 10%, 15%), the 3d3 / 2 and 3d5 / 2 peaks of Mo4+ of O-MoS2 (8%) are located at 228.8eV and 232.8eV, respectively. As the oxidation degree increases (8% to 15%), the 3d3 / 2 of Mo6+ (236eV) gradually increases, indicating that the formation ratio of Mo-O bonds increases from 3.24% to 15.15%. This phenomenon confirms that the oxidation treatment causes the transformation of Mo4+ to Mo6+, accompanied by the oxidation of part of the surface MoS2 structure.

[0058] In order to explore the catalytic performance of O-MoS2 in simulating peroxidase, in a specific embodiment, TMB was selected as a typical oxidation substrate, and its catalytic behavior was analyzed by UV-visible absorption spectroscopy. In the presence of H2O2, O-MoS2 can catalyze the oxidation of TMB to generate an oxidized product with a characteristic absorption peak at 652nm. As the degree of oxidation of O-MoS2 increases (i.e., surface defect modification using 8% to 30% H2O2 solution), the absorption intensity is strongest in the O-MoS2 (15%) group, and then decreases as the degree of oxidation continues to increase. Therefore, O-MoS2 (15%) is preferably selected as the optimal peroxidase.

[0059] also, Furthermore, in a specific embodiment, the catalytic effect of the catalyst on H2O2 can be examined by fluorescence analysis. The results show that the intensity of the fluorescence signal increases with the increase of H2O2 concentration, showing a good linear relationship.

[0060] At this time, the sample volume of the EVs metabolite detection platform is ≤10μL, and the detection time is ≤15 minutes. The detection limit is: H2O2 is 0.391μM (fluorescence method), and the sensitivity of lactate / choline is better than that of commercial kits. Therefore, EVs metabolites are strongly correlated with metabolites in parent cells (in the specific experiment, lactate R 2 =0.9008, choline R 2 =0.9601).

[0061] Furthermore, the bio-enzyme-nanozyme coupled dual enzyme system uses bulk MoS2 as a precursor, adopts 15% H2O2 solution for controllable oxidation, and combines ultrasonic exfoliation to prepare ultrathin porous O-MoS2 nanosheets (particle size of about 145nm).

[0062] In addition, in a specific embodiment, during the preparation experiment of O-MoS2 nanozyme, by using bulk MoS2 as a precursor, H2O2 solution can also be used for gradient oxidation (such as 8%, 10%, and 15% concentration control). Based on experimental adjustments, the degree of oxidation of the material can be precisely controlled, thereby retaining the original properties of the material and forming an ultra-thin sheet structure with a large number of holes, so that O-MoS2 has high-efficiency peroxidase-like activity (H2O2 detection limit reaches 0.391μM), and finally can improve the catalytic efficiency of the nanozyme, providing a highly sensitive signal amplification basis for trace metabolite detection.

[0063] Therefore, the construction process of the bioenzyme-nanozyme coupled dual enzyme system can be:

[0064] In a specific embodiment, first, 100 mg of bulk MoS2 powder was placed in a 50 mL reaction bottle, 50 mL of diluted H2O2 solution (concentration gradient of 8%, 10%, and 15%) was added, and the mixture was magnetically stirred at 80°C for 24 hours; after the reaction, the precipitate was centrifuged and washed, resuspended in deionized water, and subjected to 500 W ultrasonic stripping for 1 hour (pulse mode: on 2 seconds / off 2 seconds), dialyzed and purified for 48 hours, and then freeze-dried to obtain porous O-MoS2 nanosheets.

[0065] Furthermore, in the peroxidase-like performance test, TMB was used as the substrate: the reaction system contained PBS (pH 4.0), 10 mM TMB, 100 mM H2O2 and 50 μg O-MoS2. To improve the catalytic specificity, lactate oxidase (LOX) and choline oxidase (COD) were coupled with O-MoS2 (15%) by electrostatic adsorption to form a dual enzyme system, as shown in Figure 2. Figure 2 As shown, the principle refers to Figure 2 In addition, you can also refer to Figure (A) in Figure 2 Figure (B) in the figure can be combined with Zeta potential to verify the binding mechanism, and its activity in a specific experiment is compared with Figure 2 As shown in Figure (C), using AmplifluRed as the fluorescent probe, the catalytic fluorescence intensity of the coupled group (Coupled) is 2.3 times that of the uncoupled group (Uncoupled), confirming that the proximity effect enhances catalytic efficiency.

[0066] Furthermore, the method is constructed as Figure 3 The integrated microfluidic chip shown in Figure 4 Figure (A) consists of a PDMS cover layer, a reaction layer embedded with a micropillar array, and a glass substrate. The vacuum negative pressure is generated by the PDMS porous layer to drive the pump-free injection.

[0067] The microcolumn design is optimized by COMSOL fluid simulation. Figure 4Figure (A) shows the ExoMeta chip structure and channel design. Figure 4 Figures (B) and (C) and Figure 5 These are all simulation results, including the simulation results of various indicators such as shear rate, arc, pressure, etc., specifically including: Figure 4 (B) is the flow field simulation result of micropillar arrays with different perimeters. Figure 4 (C) is the flow rate comparison result of micropillar arrays with different perimeters. Figure 5 (A) is the simulation result of the shear rate of ExoMeta chip with micropillar arrays of different perimeters. Figure 5 (B) is the shear rate comparison simulation result of micropillar arrays with different perimeters. Figure 5 (C) is the simulation result of the average shear rate comparison of micropillar arrays with different perimeters. Figure 5 (D) is the simulation comparison result of boundary surface pressure of micropillar arrays with different perimeters. Figure 5 (E) is the simulation result of the boundary surface pressure comparison of micropillar arrays with different perimeters varying with length. Figure 5 (F) is the simulation result of boundary surface pressure comparison of micropillar arrays with different perimeters.

[0068] Comparing C600 / C1200 / C1800 μm circumference microcolumns, the C600 protocol (flow rate 1.8 mm / s, shear force 15 s) was selected. -1 The chip is fabricated using SU-8 molds photolithographically to form PDMS, with the reaction channel preloaded with O-MoS2-LOX / COD coupled enzymes. During operation, the chip is vacuum-sealed and 10 μL EVS lysis solution is added dropwise, completing the test within 15 minutes. The process is as follows: Figure 6 As shown in Figure (A), the results can be referred to Figure 6 As shown in Figures (B) and (C).

[0069] The three-channel design includes: channel 1 (no enzyme control), channel 2 (O-MoS2-LOX for lactic acid measurement), channel 3 (O-MoS2-COD for choline measurement); according to Figure 6 The detection results showed that the lactate / choline channel signal was significantly enhanced by fluorescence imaging.

[0070] Performance verification: Verification results such as Figure 7 As shown, according to Figure 7 Comparing the test results of Figures (A), (B), and (C), the sensitivity of the ExoMeta chip in detecting lactate / choline is better than that of the commercial kit and is highly consistent with the results of the kit (R 2 >0.98); according to Figure 7 Comparison of the detection results in Figures (D), (E), and (F) shows that EVs metabolites are strongly correlated with parental cell expression (lactate R2 =0.9008, choline R 2 =0.9601), thus the detection reliability can be confirmed.

[0071] In another specific embodiment, the construction process of the bioenzyme-nanozyme coupled dual enzyme system may also include:

[0072] To 100 mg of bulk MoS2 powder, add 20 mL of a 15% H2O2 solution (diluted from a 30% stock solution) and stir in a 60°C water bath for 6 hours. The reaction mixture is centrifuged (8000 rpm, 10 minutes) and the supernatant is discarded. The precipitate is washed three times with deionized water, redispersed in water, and sonicated with a probe (300 W, ice bath) for 1 hour to obtain an O-MoS2 dispersion.

[0073] In this example, O-MoS2 (50 μg / mL) reacted with TMB (0.2 mM) and H2O2 (10 mM) in acetate buffer (pH 4.0). LOX (5 U / mL) and COD (5 U / mL) were dissolved in PBS (pH 7.4), and a 15% O-MoS2 dispersion (0.1 mg / mL) was added. The reaction was shaken at room temperature for 2 hours. Unbound enzyme was removed by centrifugation (12,000 rpm, 15 minutes), and the precipitate was resuspended to yield O-MoS2-LOX / COD.

[0074] At this point, the chip preparation process includes:

[0075] A micropillar array (600 μm circumference) was photolithographically fabricated using a SU-8 mold and bonded to a glass substrate using PDMS casting. The reaction channel was preloaded with O-MoS2-LOX / COD (0.1 mg / mL) and vacuum-sealed for later use.

[0076] In summary, by designing a three-layer structure (PDMS cover layer, reaction layer embedded with micropillar array, glass substrate layer) and a vacuum cavity in the microfluidic chip, the porous properties of PDMS are used to form a "vacuum battery" to achieve pump-free negative pressure drive, and the micropillar array is optimized (such as the C600 group with a circumference of 600 μm), which can control the flow rate (1.8 mm / s), reduce the shear rate (average 15s -1), uniform pressure distribution (pressure difference ~ 3Pa), thereby ensuring sufficient reaction between the sample and the coupled dual enzyme, realizing rapid detection, and significantly improving detection throughput and efficiency to meet the needs of clinical portable detection. In addition, by loading lactate oxidase (LOX) and choline oxidase (COD) on the O-MoS2 surface through electrostatic adsorption, a bioenzyme-nanozyme coupled dual enzyme system is constructed, which can also play a role in shortening the substrate diffusion path by utilizing the synergistic effect of the dual enzyme proximity, thereby enhancing the catalytic efficiency (the fluorescence signal is increased by about 2.3 times compared with the uncoupled system), realizing metabolite-specific fluorescence signal amplification, and finally breaking through the sensitivity limit of traditional detection, accurately detecting low-abundance lactate and choline in EVs, and providing a new tool for tumor diagnosis.

[0077] On the other hand, Figure 1 As shown, the present invention also discloses a method for detecting tumor metabolic markers, which is based on any of the above-mentioned extracellular vesicle metabolite detection systems and includes the following specific operating procedures:

[0078] After obtaining the sample markers, the sample cell supernatant was collected, ultracentrifuged for 1 h, and extracellular vesicles were extracted and treated with lysate to release metabolites;

[0079] Add 10 μL of sample to the inlet of the chip channel, tear off the sealing film and start vacuum driving. The vacuum drive is activated by the vacuum self-driving microfluidic chip, and the sample flows through the microcolumn reaction area within 60 seconds. After reacting in the dark at room temperature for 15 minutes, the AmplifluRed oxidation product is generated.

[0080] After the sample enters the channel of the detection unit, a three-channel signal is collected in the detection unit through a fluorescence microscope (Ex / Em=570 / 585nm):

[0081] Channel 1: collect background fluorescence as a blank control;

[0082] Channel 2: collects lactate signals and serves as the LOX group;

[0083] Channel 3: collects choline signals as the COD group;

[0084] The tumor metabolism marker detection results can be obtained based on the three-channel signal. In addition, based on the specific chip constructed in the above embodiment (using 20mL of 15% H2O2 solution), during the detection process, after subtracting the background, the fluorescence intensity of the lactate group was 8.7 times that of the blank group.

[0085] After collecting three-channel signals through a fluorescence microscope in the detection unit, the detection results of tumor metabolism markers (lactate, choline) can be determined according to the following judgment criteria:

[0086] Background subtraction and channel comparison: Using the background fluorescence of channel 1 (blank control) as a benchmark, after subtracting the background signal, analyze the fluorescence intensity of channel 2 (lactate group) and channel 3 (choline group). If the fluorescence intensity of the lactate group or choline group is significantly higher than that of the blank control (e.g., more than 8 times that of the blank group, refer to Example 1 where the fluorescence intensity of the lactate group is 8.7 times that of the blank group), it indicates the presence of the corresponding metabolite in the sample.

[0087] Correlation between fluorescence signal intensity and detection limit: Because this detection system has superior sensitivity for lactate and choline compared to commercial kits, and a detection limit of 0.391 μM for H2O2, a positive result is considered if the metabolite concentration corresponding to the fluorescence signal intensity in the lactate or choline group is above the detection limit, indicating the possible presence of abnormal tumor metabolic markers.

[0088] Correlation verification with parent cell metabolites: EVs metabolites have a strong correlation with parent cell metabolites (lactate R 2 =0.9008, choline R 2 =0.9601). If the fluorescent signals of lactate or choline detected in EVs are consistent with the expression trends of the corresponding metabolites in parental cells, it further supports the detection results of tumor metabolic markers.

[0089] Comparison with standard curve or kit: By comparing with the standard curve, lactate and choline can be quantitatively analyzed. If the quantitative results are outside the normal range, combined with clinical data, it can be judged as a positive tumor metabolism marker. In addition, the test results of this system are highly correlated with those of the standard kit (lactate R 2 =0.9488, choline R 2 =0.9887), and the results can also be compared with those of the standard test kit to assist in judgment.

[0090] In summary, this method solves the problems of low nanozyme activity, poor detection sensitivity, complex and time-consuming operation in traditional clinical detection of key metabolites in EVs (such as lactate and choline), breaking through the sensitivity limit and realizing minute-level POCT. It involves three technological innovations: materials (gradient oxidation), devices (pumpless chip), and methods (dual-enzyme proximity effect) that work together to overcome the problem of portable detection of trace metabolites and provide new tools for tumor diagnosis.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0092] In addition, it should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0093] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such features. In addition, the meaning of "and / or" appearing throughout the text includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or schemes in which A and B are satisfied at the same time. In addition, in the embodiments of the present invention, "multiple" refers to more than two. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

Claims

1. A system for detecting extracellular vesicle metabolites, characterized in that: include: The vacuum self-driven microfluidic chip is configured as a three-layer structure consisting of a PDMS cover layer, a reaction layer embedded with a micropillar array, and a glass substrate layer. The PDMS cover layer uses the porous properties of PDMS to form a vacuum cavity, and the micropillar array is composed of micropillars with a circumference of 600 μm. The detection unit is connected to the vacuum self-driven microfluidic chip and is set up as a set of three-channel independent detection areas. It is pre-loaded with a bioenzyme-nanozyme coupled dual enzyme system for combining fluorescent signal amplification substrate to achieve metabolite-specific detection; The bioenzyme-nanozyme coupled dual enzyme system is formed by electrostatically adsorbing lactate oxidase LOX and choline oxidase COD on the O-MoS2 surface.

2. The extracellular vesicle metabolite detection system according to claim 1, characterized in that The bioenzyme-nanozyme coupled dual enzyme system uses bulk MoS2 as a precursor, adopts 15% H2O2 solution for controllable oxidation, and combines ultrasonic exfoliation to prepare ultrathin porous O-MoS2 nanosheets.

3. The extracellular vesicle metabolite detection system according to claim 2, wherein: The construction process of the bioenzyme-nanozyme coupled dual enzyme system includes: Take 100 mg of bulk MoS2 powder, add 20 mL of H2O2 solution of specified concentration, and stir in a 60 °C water bath for 6 hours to obtain reaction solution A; After the reaction solution A was centrifuged, the supernatant was discarded, and the precipitate was washed three times with deionized water, redispersed in water, and then ultrasonicated in an ice bath for 1 hour to obtain an O-MoS2 dispersion. LOX and COD were dissolved in PBS buffer, and the specified concentration of O-MoS2 dispersion was added. The reaction was shaken at room temperature for 2 hours. After centrifugation to remove unbound enzymes, the precipitate was resuspended to obtain O-MoS2-LOX / COD.

4. The extracellular vesicle metabolite detection system according to claim 1, wherein The vacuum self-driven microfluidic chip adopts SU-8 mold to photolithographically form microcolumn arrays, and is bonded to a glass substrate after PDMS casting; The vacuum self-driven microfluidic chip is used to connect to the reaction channel of the detection unit preloaded with O-MoS2-LOX / COD, and is vacuum-encapsulated synchronously with the detection unit during the manufacturing process.

5. A method for detecting tumor metabolic markers, based on the extracellular vesicle metabolite detection system according to any one of claims 1 to 4, characterized in that: The specific operation process includes the following: After obtaining the sample markers, the sample cell supernatant was taken, ultracentrifuged to extract extracellular vesicles, and then treated with lysate to release metabolites; 10 μL of sample was added to the inlet of the chip channel, and the vacuum drive was started by the vacuum self-driven microfluidic chip, and the sample flowed through the microcolumn reaction area. After reacting in the dark at room temperature for 15 minutes, the Ampliflu Red oxidation product was generated. After the sample enters the channel of the detection unit, a three-channel signal is collected by a fluorescence microscope inside the detection unit: Channel 1: collect background fluorescence as a blank control; Channel 2: collects lactate signals and serves as the LOX group; Channel 3: collects choline signals as the COD group; Tumor metabolic marker detection results can be obtained based on three-channel signals.

6. The extracellular vesicle metabolite detection system according to claim 5, characterized in that: The method for obtaining tumor metabolism marker detection results includes: Taking the background fluorescence of the blank control in channel 1 as the benchmark, after deducting the background signal, the fluorescence intensity of the LOX group in channel 2 and the COD group in channel 3 were determined by the specified standards; (1) If the fluorescence intensity of the LOX group or COD group is higher than that of the blank control group by a specified multiple, it indicates the presence of the corresponding metabolites in the sample. (2) If the metabolite concentration corresponding to the fluorescence signal intensity of the LOX group or COD group is higher than the preset detection limit, it can be judged as a positive result, and the output result is: there may be abnormal tumor metabolic markers; otherwise, it is a negative result, and the output result is: there is no abnormal tumor metabolic marker. (3) If the fluorescent signal of lactate or choline detected in the extracellular vesicles is consistent with the expression trend of the corresponding metabolites in the parental cells and is judged to be positive, the output result is: there is a high possibility of abnormal tumor metabolic markers.

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