Dual-mode optical sensor based on copper-based peroxide nanodots and application of dual-mode optical sensor in gastric cancer Warburg effect

Abstract

The invention develops a dual-mode optical sensor based on CuO2 nanodots. The dual-mode optical sensor is mainly applied to detection and research of a Warburg effect of gastric cancer. The CuO2 nanodots can catalyze the reaction of o-phenylenediamine (OPD) and hydrogen peroxide (H2O2), so that the color of the solution is changed from colorless to yellow along with the increase of absorbance and fluorescence intensity. Based on the reaction, a dual-mode sensor is designed, and the activity of lactic acid and lactate oxidase is synchronously detected through colorimetry and fluorescence. In the process of catalyzing lactic acid to generate H2O2 by lactic acid oxidase, the generated H2O2 promotes the color of a solution to change remarkably, so that the sensitive detection of the activity of lactic acid and oxidase thereof is indirectly realized. As the Warburg effect of the gastric cancer causes rise of the lactic acid level, the sensor can effectively associate the lactic acid level with the metabolic characteristics of the gastric cancer. In addition, in combination with an RGB colorimetric analysis technology, a smart phone is used for capturing color changes and carrying out image quantitative analysis, a portable, low-cost and high-sensitivity detection system is formed, and the application blank of the CuO2 nanodots in the field is filled up.

Description

Technical Field

[0001] This invention belongs to the fields of functional materials and biosensing technology, specifically relating to the preparation method and application of a dual-mode optical sensor based on copper-based peroxide (CuO2) nanodots. This sensor combines colorimetric and fluorescence dual-mode detection, achieving highly sensitive detection of target molecules through color changes and fluorescence signals caused by catalytic reactions. In particular, this invention enables on-site detection via a smartphone, utilizing the reaction between lactate oxidase (LOx) and its substrate lactate (LA) to generate hydrogen peroxide (H2O2), which then catalyzes the reaction of o-phenylenediamine (OPD), enabling the analysis and detection of lactate oxidase and its inhibitors. This method combines optical sensing technology with portable smart devices, providing a new technical means for the on-site detection of Warburg effect-related metabolites in gastric cancer, and has broad application potential and practical value. Background Technology

[0002] Malignant tumors such as gastric cancer often exhibit abnormal metabolic characteristics, most notably the "Warburg effect," where tumor cells, even under aerobic conditions, still rely on glycolysis to produce large amounts of lactate, rather than the normal mitochondrial oxidative phosphorylation pathway. Lactic acid (LA) accumulation is not only a marker of rapid tumor cell metabolism but is also closely related to tumor invasiveness, metastatic potential, and treatment resistance. Therefore, high LA levels in body fluids reflect the degree of abnormal metabolic activity in tumor cells and can serve as a potential biomarker for early diagnosis and disease progression monitoring of gastric cancer. Lactate oxidase (LOx) can be used for in vitro LA detection. It catalyzes the production of pyruvate and hydrogen peroxide (H2O2) from LA, and the LA concentration is rapidly and accurately measured using a colorimetric reaction. This method can be used for non-invasive or minimally invasive screening of gastric cancer, especially during periods of active tumor metabolism. Furthermore, dynamic monitoring of LA levels can help assess patient response to treatment, determine tumor metabolic status, and provide a basis for adjusting treatment plans. While locus oxen (LOx) themselves are not directly used for treatment, detecting leukocyte-lactamase (LA) levels can provide data to support personalized treatment. High LA levels indicate an over-reliance on the glycolytic pathway in tumors, allowing doctors to select metabolic inhibitors or targeted therapies to block the energy supply to tumor cells. Simultaneously, LOx testing can identify patients with high LA accumulation, high invasiveness, or metastatic potential, enabling more precise treatment strategies. Therefore, although LOx is not present in the human body, detecting LA levels is significant for the screening and treatment of gastric cancer. By monitoring LA metabolism, researchers and clinicians can detect gastric cancer earlier and more accurately, and by regulating LA metabolism, provide new treatment ideas, thereby improving patient survival rates and quality of life.

[0003] Copper-based peroxide nanodots (CuO2 nanodots) are novel nanomaterials that have attracted much attention in the fields of nanotechnology and materials science in recent years. Their unique structure endows them with excellent peroxidase-like (POD-like) activity, enabling them to catalyze the generation of reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals, in biological and chemical systems. This enzyme-like activity gives them great potential in fields such as biosensing and environmental monitoring, especially under low pH conditions, where their catalytic activity is significantly enhanced, thus their application in acidic microenvironments such as tumors is also of great interest. In the field of analytical sensors, the main application of copper-based peroxide nanodots lies in sensor development, where their superior catalytic ability makes them ideal catalysts for various analytical methods. Furthermore, copper-based peroxide nanodots show significant promise in cancer detection, particularly in detecting lactic acid or hydrogen peroxide produced by tumor metabolism. By combining optical and electrochemical dual-mode sensor technology, real-time monitoring of the metabolic state of cancer cells can be achieved. The development of such sensors provides a new tool for early cancer detection and metabolic research in the biomedical field. In summary, copper-based peroxide nanodots, with their unique enzyme-like activity and excellent catalytic properties, have become important materials in the field of analytical sensing, especially with broad application prospects in environmental monitoring and biomedical diagnostics.

[0004] This invention relates to a dual-mode optical sensor based on CuO2 nanodots and its application in the Warburg effect of gastric cancer. CuO2 nanodots possess catalytic properties, capable of catalyzing the reaction of o-phenylenediamine (OPD) with H2O2, causing the solution to change from colorless to yellow, accompanied by an increase in absorbance and fluorescence intensity. Based on this catalytic reaction, a dual-mode optical sensor with CuO2 nanodots as its core was constructed, capable of sensitively detecting the activity of LA and LOx through both colorimetric and fluorescence modes. During the process of LOx catalyzing the formation of H2O2 from LA, as H2O2 is gradually produced, the color of OPD gradually changes from colorless to yellow, thus achieving indirect detection of LA and LOx activity. Since the Warburg effect in gastric cancer leads to a large accumulation of LA, this sensor can effectively correlate LA levels with the metabolic state of gastric cancer, providing a new approach for analyzing the relationship between LA, LOx, and the Warburg effect. Furthermore, this invention introduces RGB color space analysis technology, which uses a smartphone to capture images of the color changes in the reaction solution, enabling color-based quantitative analysis. This further expands the research on LA and LOx and their relationship with the Warburg effect in gastric cancer, fills the gap in the application of CuO2 nanodots in this field, and provides a portable solution suitable for clinical testing. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a dual-mode optical sensor based on copper-based peroxide nanodots that is visual, low-cost, highly sensitive, specific, and easy to operate, and its application in the Warburg effect of gastric cancer.

[0006] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a dual-mode optical sensor based on copper-based peroxide nanodots and its application in the Warburg effect of gastric cancer. The specific steps are as follows:

[0007] 1. Preparation of CuO2 Nanodots

[0008] Dissolve 0.5 g of polyvinylpyrrolidone (PVP) in 5 mL of deionized water containing CuCl2·2H2O (0.01 M). Stir thoroughly to allow copper ions to fully combine with PVP and form a stable complex. Add 5 mL of 0.02 M NaOH solution dropwise to the mixture, ensuring the pH of the solution remains stable between 8 and 9. This helps prevent the rapid formation of copper hydroxide precipitate while ensuring that copper ions in the solution fully participate in the reaction. Slowly add 100 μL of 30% H2O2 solution while stirring. It is recommended to use a titrator or syringe pump to control the H2O2 addition rate at 5-10 μL per minute to ensure a gentle reaction and avoid the formation of byproducts. Stir for 1 hour instead of 30 minutes; extending the reaction time helps to promote the uniform formation of CuO2 nanoparticles and improves their stability. Collect the PVP-coated CuO2 nanoparticles by ultrafiltration or centrifugation at 10,000 rpm for 20 minutes to ensure complete separation of the nanoparticles. Wash repeatedly with deionized water 3-5 times to remove unreacted reagents and byproducts, ensuring that pure CuO2 nanodots are obtained (estimated concentration 100 μg / mL).

[0009] 2. Fabrication of Dual-Mode Optical Sensor and Smartphone Color Picking Sensor

[0010] (1) Constructing a dual-mode optical sensor based on CuO2 Nanodots

[0011] To 70 μL of 0.1M pH 7.4 PBS buffer, add 2.5 μL of 10mM OPD (final concentration 250 μM), 1 μL of CuO2 Nanodots, 5 μL of 1mM H2O2, and 21.5 μL of deionized water, and premix at 37°C for 30 minutes. Transfer the above solution to a fluorescence spectrophotometer for analysis, measuring the fluorescence intensity in the wavelength range of 460–660 nm. Simultaneously, transfer the above solution to a microplate reader and measure the absorbance in the wavelength range of 370–500 nm. Finally, use a high-definition camera to capture the colors for smartphone analysis.

[0012] (2) Construction of mobile phone sensors

[0013] To achieve on-site detection of target analytes, this study designed a portable detection system based on a smartphone, utilizing the high-performance camera of the iPhone 11 and a self-made darkroom device. This system achieves precise analysis of target analytes by capturing the fluorescence intensity and brightness variations of oxOPD. The detection system mainly consists of a specially designed darkroom, a blue light excitation device, and white background paper. The top of the darkroom has a small hole matching the iPhone 11 camera to stabilize the phone and ensure accurate camera alignment with the sample. The blue light excitation device effectively excites the fluorescence signal in the sample. The white background paper inside the darkroom enhances the color contrast of the image, improving detection accuracy. The operation procedure is as follows: the prepared fluorescence sensor is placed in the darkroom, the blue light excitation device is activated, and an image of the sample is captured using the iPhone 11. The image is then imported into free colorimetric software on the phone. By analyzing the RGB (red-green-blue) color values, especially the R / B ratio, and combining this with a pre-established standard curve, the concentration of lactate oxidase (LOx) in the sample is accurately calculated. This design not only demonstrates the potential of smartphones in modern analytical techniques but also provides a rapid, economical, and user-friendly detection method. Its portability and ease of use make it ideal for rapid on-site testing outside the laboratory, especially in resource-constrained environments. This innovative method significantly improves the efficiency and accuracy of LOx concentration change detection, providing important support for early liver cancer screening and the analysis of other biomarkers.

[0014] (3) Sensors are used for the analysis of LA, LOx and their inhibitors.

[0015] A, LA detection

[0016] To 70 μL of 0.1M pH 7.4 PBS buffer, add 1.2 μL of 5000 U / L LOx (final concentration 6 U / L), 1 μL of CuO2 Nanodots, 5 μL of 1mM lactate (final concentration 50 μM), 2.5 μL of 10mM OPD (final concentration 250 μM), and 20.3 μL of deionized water, and premix at 37°C for 30 minutes. Transfer the above solution to a fluorescence spectrophotometer for analysis, measuring the fluorescence intensity in the wavelength range of 460–660 nm. Simultaneously, transfer the above solution to a microplate reader and measure the absorbance in the wavelength range of 370–500 nm. Finally, use a high-definition camera to capture the colors for smartphone analysis.

[0017] B. Detection of lactate oxidase

[0018] To 70 μL of 0.1M pH 7.4 PBS buffer, add 1.2 μL of 5000 U / L LOx (final concentration 6 U / L), 1 μL of CuO2 Nanodots, 5 μL of 1 mM LA (final concentration 50 μM), 2.5 μL of 10 mM OPD (final concentration 250 μM), and 20.3 μL of deionized water, and premix at 37°C for 30 minutes. Transfer the above solution to a fluorescence spectrophotometer for analysis, measuring the fluorescence intensity in the wavelength range of 460–660 nm. Simultaneously, transfer the above solution to a microplate reader and measure the absorbance in the wavelength range of 370–500 nm. Finally, use a high-definition camera to capture the colors for analysis via smartphone.

[0019] C. Detection of inhibitors

[0020] First, incubate the inhibitor with LOx for 5 minutes. Then, add the other reagents for the enzyme reaction. In 70 μL of 0.1M pH 7.4 PBS buffer, add 1.2 μL of 5000 U / L LOx (final concentration 6 U / L), 1 μL of CuO2 Nanodots, 5 μL of 1mM LA (final concentration 50 μM), 2.5 μL of 10mM OPD (final concentration 250 μM), 2 μL of 0.1 μM FeCl3 (final concentration 0.01 μM), and 18.3 μL of deionized water, and premix at 37°C for 30 minutes. Transfer the above solution to a fluorescence spectrophotometer for analysis, measuring the fluorescence intensity in the wavelength range of 460–660 nm. Simultaneously, transfer the above solution to a microplate reader and measure the absorbance in the wavelength range of 370–500 nm. Finally, use a high-definition camera to capture the colors for smartphone analysis.

[0021] A. Detection of H2O2

[0022] (1) Based on step 2-(1) above, by changing the H2O2 concentration in step 2-(1) (final concentration: 0, 0.001, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200)

[0023] With the other steps unchanged, the fluorescence spectrum in the range of 460-660 nm was measured using a fluorescence spectrophotometer, and a linear relationship graph of different concentrations of H2O2 was established based on the maximum fluorescence intensity at 553 nm, thereby realizing the fluorescence analysis and detection of different concentrations of H2O2; in addition, the visualization detection of different concentrations of H2O2 can be realized through colorimetric images.

[0024] (2) Based on step 2-(1) above, by changing the H2O2 concentration in step 2-(1) (final concentration: 0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200 μM), while keeping other steps unchanged, the ultraviolet spectrum in the range of 370-500 nm is measured using an enzyme-linked immunosorbent assay (ELISA) reader, and a linear relationship graph of different concentrations of H2O2 is established based on the maximum absorbance at 420 nm, thereby realizing the ultraviolet analysis and detection of different concentrations of H2O2; in addition, the visualization detection of different concentrations of H2O2 can be realized through colorimetric images.

[0025] B. Lactic acid detection

[0026] (1) Based on step 2-(3)-A above, by changing the LA concentration in step 2-(3)-A (final concentration: 0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200 μM), while keeping other steps unchanged, the fluorescence spectrum in the range of 460-660 nm is measured with a fluorescence spectrophotometer, and a linear relationship graph of different LA concentrations is established with the maximum fluorescence intensity at 553 nm, thereby realizing the fluorescence analysis and detection of different LA concentrations; in addition, the visualization detection of different LA concentrations can be realized through colorimetric images.

[0027] (2) Based on step 2-(3)-A above, by changing the LA concentration in step 2-(3)-A (final concentration: 0, 0.1, 0.3, 0.5, 0.8, 1, 3, 5, 10, 20, 30, 50, 80, 100, 150, 200 μM), while keeping other steps unchanged, the ultraviolet spectrum in the range of 370-500 nm is measured with an enzyme-linked immunosorbent assay (ELISA) reader, and a linear relationship graph of different LA concentrations is established with the maximum absorbance at 420 nm, thereby realizing the LA analysis and detection of different concentrations of lactic acid; in addition, the visualization detection of different concentrations of LA can be realized through colorimetric images.

[0028] C, LOx detection

[0029] (1) Based on step 2-(3)-B above, by changing the LOx concentration in step 2-(3)-B (final concentration: 0, 0.001, 0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 1, 1.6, 2, 4, 6, 10, 20, 40 U / L), while keeping other steps unchanged, the fluorescence spectrum in the range of 460-660 nm is measured with a fluorescence spectrophotometer, and a linear relationship graph of different concentrations of LOx is established with the maximum fluorescence intensity at 553 nm, thereby realizing the fluorescence analysis and detection of different concentrations of LOx; in addition, the visualization detection of different concentrations of LOx can be realized through colorimetric images.

[0030] (2) Based on step 2-(3)-B above, by changing the LOx concentration in step 2-(3)-B (final concentration: 0, 0.001, 0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 1, 1.6, 2, 4, 6, 10, 20, 40 U / L), while keeping other steps unchanged, the ultraviolet spectrum in the range of 370-500 nm is measured using an enzyme-linked immunosorbent assay (ELISA) reader, and a linear relationship graph of different concentrations of lactate oxidase is established based on the maximum absorbance at 420 nm, thereby realizing the ultraviolet analysis and detection of different concentrations of lactate oxidase; in addition, the visualization detection of different concentrations of lactate oxidase can be realized through colorimetric images.

[0031] D. Detection of inhibitors

[0032] (1) Based on step 2-(3)-C above, by changing the FeCl3 concentration in step 2-(3)-C (final concentration: 0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200 μM), while keeping other steps unchanged, the fluorescence spectrum in the range of 460–660 nm was measured using a fluorescence spectrophotometer. The maximum fluorescence intensity at 553 nm was used to establish the different concentrations of FeCl3. 3+ A linear relationship graph was generated to achieve the analysis of Fe at different concentrations. 3+ Fluorescence analysis was used to detect and calculate Fe. 3+ The half-inhibition concentration of LOx; in addition, different concentrations of Fe can be obtained through colorimetric imaging. 3+ Visual detection.

[0033] (2) Based on step 2-(3)-C above, by changing the FeCl3 concentration in step 2-(3)-C (final concentration: 0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200 μM), while keeping other steps unchanged, the ultraviolet spectrum in the range of 370-500 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The maximum absorbance at 420 nm was used to establish different concentrations of FeCl3. 3+ A linear relationship graph was generated to achieve the analysis of Fe at different concentrations. 3+ Ultraviolet analysis was used to detect and calculate Fe. 3+ The half-inhibition concentration of LOx; in addition, different concentrations of Fe can be obtained through colorimetric imaging. 3+ Visual detection.

[0034] Invention Principle: This invention constructs a dual-mode optical sensor based on CuO2 nanodots and applies it to the study of the Warburg effect in gastric cancer. CuO2 nanodots catalyze the reaction of OPD with H2O2, causing the solution color to change from colorless to yellow, while simultaneously enhancing absorbance and fluorescence intensity. This sensor sensitively detects LA and LOx activities through colorimetric and fluorescence modes. During the process of LOx catalyzing the formation of H2O2 from LA, the color of OPD gradually changes with the production of H2O2, thus achieving indirect detection of LA and LOx activities. Due to lactic acid accumulation induced by the Warburg effect, this sensor can correlate lactic acid levels with the metabolic state of gastric cancer. Furthermore, by utilizing smartphones and RGB color space analysis technology, this method achieves quantitative analysis of color changes, providing a portable solution suitable for clinical testing and filling a gap in the application of CuO2 nanodots in this field.

[0035] Its advantages are:

[0036] (1) Novel Target Material. This is the first time that CuO2 nanodots have been used to construct a LOx fluorescence-UV dual-mode sensor and innovatively applied to early gastric cancer screening. This novel detection method breaks through the limitations of traditional detection methods and provides a new analytical tool for the detection of LOx activity. Currently, there are few reports on LOx activity detection. The dual-mode sensor designed in this invention provides new opportunities and challenges for a deeper understanding of the role of LOx in physiological and pathological processes and for developing treatment strategies for related diseases. In particular, in the Warburg effect of gastric cancer, LOx is closely related to LA levels, which can provide a new approach for assessing the metabolic status of cancer.

[0037] (2) Convenience: This invention integrates the colorimetric device of a smartphone, greatly improving the convenience of testing. Through the use of the smartphone camera and dedicated software, users can easily achieve rapid and portable on-site testing without the need for complex experimental equipment or professional operators. This design not only significantly simplifies the testing process but also provides a convenient and practical solution for non-professional users, making it particularly suitable for environments with limited resources or where conditions do not permit the use of laboratory equipment.

[0038] (3) High sensitivity and high selectivity: The sensor utilizes the linear relationship between fluorescence intensity and UV absorption and LOx concentration to achieve accurate detection of low concentrations of LOx with extremely high sensitivity. The linear correlation between fluorescence intensity and the logarithm of LOx concentration ensures accurate concentration differentiation. The correlation equation between UV absorption and fluorescence signals indicates that the detection results possess high accuracy and reliability. Furthermore, the sensor exhibits high selectivity; other interfering substances such as alkaline phosphatase and glucose oxidase do not significantly interfere with the detection system, ensuring specificity of the detection.

[0039] (4) Low cost: Compared with traditional detection methods, the preparation process of this invention is simple and does not require expensive reagents and complex instruments. The synthesis cost of CuO2 nanodots is low and the materials are readily available, making them suitable for large-scale application. Especially in resource-limited environments, it provides an economical and efficient detection solution that meets the needs of extensive clinical testing and large-scale screening.

[0040] (5) Broad Application Prospects: This sensor is particularly suitable for detecting LOx in circulating tumor cells of gastric cancer, and has broad clinical application value. Accurate monitoring of LOx levels is expected to enable early screening and precise diagnosis of gastric cancer, helping to improve patient survival rates and quality of life. Furthermore, this detection technology can be extended to the detection of other metabolic-related diseases, possessing extremely high potential for biomedical research and application, and can drive the development of future disease biomarker detection.

[0041] In summary, this invention constructs a sensor based on copper peroxide nanodots and its application in early gastric cancer screening. It has the advantages of rapid analysis, simple operation, high sensitivity, good selectivity, and low cost, and has good application prospects. Attached Figure Description

[0042] Figure 1 This is a feasibility experiment diagram of the dual-mode colorimetric sensor of the present invention;

[0043] Figure 2 This is a graph showing the linear relationship between different concentrations of H2O2 and RGB values ​​for the dual-mode sensor of this invention;

[0044] Figure 3 This is a linear relationship graph of different LA concentrations and RGB values ​​for the dual-mode sensor of this invention;

[0045] Figure 4 This is a graph showing the linear relationship between different concentrations of LOx and RGB values ​​for the dual-mode sensor of this invention;

[0046] Figure 5 The dual-mode sensor of this invention is for detecting different concentrations of Fe. 3+ Linear relationship graph with RGB values;

[0047] Figure 6 This is an experimental diagram showing the selectivity of the dual-mode sensor of the present invention for LOx;

[0048] Figure 7 This is an experimental diagram showing the analysis and detection of bacteria using the dual-mode sensor of this invention;

[0049] Figure 8 This is an experimental diagram illustrating the analysis and detection of bacteria with the addition of inhibitors using the dual-mode sensor of this invention.

[0050] Figure 9This is an experimental diagram showing the analysis and detection of the dual-mode sensor of the present invention in a blood sample;

[0051] Figure 10 This is an experimental diagram of the analysis and detection of the present invention's dual-mode sensor in simulated gastric fluid samples. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0053] Example 1: Preparation of CuO2 Nanodots

[0054] Dissolve 0.5 g of polyvinylpyrrolidone (PVP) in 5 mL of deionized water containing CuCl2·2H2O (0.01 M). Stir thoroughly to allow copper ions to fully combine with PVP and form a stable complex. Add 5 mL of 0.02 M NaOH solution dropwise to the mixture, ensuring the pH of the solution remains stable between 8 and 9. This helps prevent the rapid formation of copper hydroxide (Cu(OH)2) precipitate while ensuring that copper ions in the solution fully participate in the reaction. Slowly add 100 μL of 30% H2O2 solution while stirring. It is recommended to use a titrator or syringe pump to control the H2O2 addition rate at a rate of 5-10 μL per minute to ensure a gentle reaction and avoid the formation of byproducts. Stir for 1 hour instead of 30 minutes; extending the reaction time helps to promote the uniform formation of CuO2 nanoparticles and improves their stability. Collect the PVP-coated CuO2 nanoparticles by ultrafiltration or centrifugation at 10,000 rpm for 20 minutes to ensure complete separation of the nanoparticles. Wash repeatedly with deionized water 3-5 times to remove unreacted reagents and byproducts, ensuring that pure CuO2 nanodots are obtained.

[0055] (2) Constructing a dual-mode optical sensor based on CuO2 Nanodots

[0056] To 70 μL of 0.1M pH 7.4 PBS buffer, add 2.5 μL of 10mM OPD (final concentration 250 μM), 1 μL of CuO2 Nanodots, 5 μL of 1mM H2O2, and 21.5 μL of deionized water, and premix at 37°C for 30 minutes. Transfer the above solution to a fluorescence spectrophotometer for analysis, measuring the fluorescence intensity in the wavelength range of 460–660 nm. Simultaneously, transfer the above solution to a microplate reader and measure the absorbance in the wavelength range of 370–500 nm. Finally, use a high-definition camera to capture the colors for smartphone analysis.

[0057] The fluorescence intensity of the sensor was detected in the wavelength range of 460–660 nm, such as Figure 1As shown in Figure A, CP nanodots can effectively catalyze the reaction between H2O2 and OPD, and a significant fluorescence emission peak was observed at 553 nm. Figure 1 A, Curve 5, purple). In contrast, almost no fluorescence signal was detected in the CP nanodots and OPD system without added H2O2 ( Figure 1 A, curve 4, green). In addition... Figure 1 B shows a distinct ultraviolet absorption peak at 420 nm. Figure 1 B, curve 5, gray-blue), while no significant absorbance signal was detected in the system without added H2O2 (). Figure 1 B, Curve 4, green). These results indicate that the reaction of the constructed copper peroxide nanodot system depends on the presence of H2O2, laying the foundation for subsequent analysis of lactate oxidase-catalyzed reactions.

[0058] Example 2: Detection of H2O2

[0059] (1) Based on Examples 1-(2), by changing the final concentration of H2O2 (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.3, 1, 3, 5, 10, 30, 50, 100, 150, 200 μM) while keeping other steps unchanged, the fluorescence spectrum in the range of 460–660 nm was measured. Based on the maximum fluorescence intensity at 553 nm, a linear relationship graph of the Log values ​​of different concentrations of H2O2 was established, thereby realizing the fluorescence analysis and detection of H2O2. At the same time, the visualization results of different concentrations of H2O2 were visually displayed through colorimetric images. Figure 2 A shows the standard curve for H2O2 detection, where the Δfluorescence intensity exhibits a good linear relationship with the Log value of H2O2 concentration. As the H2O2 concentration increases, the fluorescence intensity of OPD increases, and the solution color gradually changes from colorless to deep yellow. The linear equation for Δfluorescence intensity versus the Log value of H2O2 concentration is: y = 242.376x + 959.045, R0 2 =0.998, detection limit is 0.42 nM (3σ / slope), linear range is 0 to 100 μM. Figure 2 B illustrates the linear relationship between the B / R ratio and the logarithmic value of the H₂O₂ concentration. As the H₂O₂ concentration increases, the solution color gradually deepens, and the B / R ratio also increases. The linear equation is: y = 0.0233x + 0.116, R 2 =0.992.

[0060] (2) Based on Examples 1-(2), by changing the final concentration of H2O2 (0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200 μM) while keeping other steps unchanged, the ultraviolet spectrum in the range of 370–500 nm was measured. A linear relationship graph of the Log values ​​of different concentrations of H2O2 was established based on the maximum absorbance at 420 nm, thereby achieving ultraviolet analysis and detection of H2O2. The visualization effect of different concentrations of H2O2 can also be intuitively displayed through colorimetric images. Figure 2 C shows the standard curve for H2O2 detection, where Δabsorbance exhibits a good linear relationship with H2O2 concentration. As the H2O2 concentration increases, the absorbance of OPD increases, and the solution color gradually changes from colorless to deep yellow. The linear equation for Δabsorbance versus the logarithmic value of H2O2 concentration is: y = 0.0738x + 0.174, R0 2 =0.994, detection limit is 3.2 nM (3σ / slope), linear range is 0 to 100 μM. Figure 2 D shows the linear relationship between the R / B value and the Log value of the H2O2 concentration. As the H2O2 concentration increases, the solution color gradually deepens, and the R / B value increases accordingly. The linear equation is: y = 0.538x + 2.211, R 2 =0.987. These results indicate that the sensor can sensitively detect different concentrations of H2O2.

[0061] Example 3: Sensor used for the analysis of lactic acid, lactate oxidase and its inhibitors

[0062] A. Lactic acid detection

[0063] To 70 μL of 0.1M pH 7.4 PBS buffer, add 1.2 μL of 5000 U / L LOx (final concentration 6 U / L), 1 μL of CuO2 Nanodots, 5 μL of 1 mM LA (final concentration 50 μM), 2.5 μL of 10 mM OPD (final concentration 250 μM), and 20.3 μL of deionized water, and premix at 37°C for 30 minutes. Transfer the solution to a microplate reader, fluorescence spectrophotometer, and cuvettes for analysis and testing, and take photographs using a high-resolution camera.

[0064] (1) Based on the above steps, by adjusting the final concentration of LA (0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200 μM) while keeping other experimental steps unchanged, the fluorescence spectrum in the range of 460–660 nm was measured. A linear relationship graph of different LA concentrations was established at the maximum fluorescence intensity at 553 nm, thereby enabling fluorescence analysis and detection of LA. Furthermore, the results of different LA concentrations can be visually displayed through colorimetric images. Figure 3 A shows the standard curve for LA detection, demonstrating a good linear relationship between Δfluorescence intensity and the Log value of LA concentration. As the LA concentration increases, the fluorescence intensity of OPD gradually increases, and the solution color changes from colorless to deep yellow. The linear equation between Δfluorescence intensity and the Log value of LA concentration is: y = 228.490x + 517.200, R0 2 =0.983, detection limit is 5.3 nM (3σ / slope), linear range is 0 to 100 μM. Figure 3 B shows the linear relationship between the B / R value and the logarithmic value of the LA concentration. As the LA concentration increases, the solution color deepens, and the B / R value gradually increases. The linear equation is: y = 0.0305x + 0.133, R 2 =0.991, indicating that the sensor has high sensitivity to LA detection.

[0065] (2) Based on the above experimental steps, by adjusting the final concentration of LA (0, 0.1, 0.3, 0.5, 0.8, 1, 3, 5, 10, 20, 30, 50, 80, 100, 150, 200 μM) while keeping other steps unchanged, the ultraviolet spectrum in the range of 370–500 nm was measured. A linear relationship graph of different LA concentrations was established at the maximum absorbance at 420 nm, thereby achieving ultraviolet analysis and detection of LA. Simultaneously, visual detection of LA was achieved through colorimetric imaging. Figure 3 C shows the standard curve for LA detection, where Δabsorbance exhibits a good linear relationship with LA concentration. As LA concentration increases, the absorbance of OPD gradually increases, and the linear equation between Δabsorbance and the Log value of LA concentration is: y = 0.103x + 0.140, R0 2 =0.998, detection limit is 36 nM (3σ / slope), linear range is 0 to 100 μM. Figure 3 D shows the linear relationship between the R / B value and the logarithmic value of the LA concentration. As the LA concentration increases, the solution color deepens, and the R / B value gradually increases. The linear equation is: y = 0.778x + 1.842, R 2 =0.994. These results indicate that the sensor can sensitively detect different concentrations of LA.

[0066] B. Detection of lactate oxidase

[0067] To 70 μL of 0.1M pH 7.4 PBS buffer, add 1.2 μL of 5000 U / L LOx (final concentration 6 U / L), 1 μL of CuO2 Nanodots, 5 μL of 1 mM LA (final concentration 50 μM), 2.5 μL of 10 mM OPD (final concentration 250 μM), and 20.3 μL of deionized water, and premix at 37°C for 30 minutes. Transfer the solution to a microplate reader, fluorescence spectrophotometer, and cuvettes for analysis and testing, and take photographs using a high-resolution camera.

[0068] (1) Based on the above experimental steps, by adjusting the LOx concentration (final concentration: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.3, 0.5, 1, 1.6, 2, 4, 6, 10, 20, 40 U / L), while keeping other steps unchanged, the fluorescence spectrum in the range of 460–660 nm was measured, and a linear relationship graph of different LOx concentrations was established using the maximum fluorescence intensity at 553 nm, thereby realizing the fluorescence analysis and detection of different LOx concentrations; in addition, the visual detection of different LOx concentrations can be achieved through colorimetric images. Figure 4 A represents the standard curve for LOx detection. The fluorescence intensity (Δ) shows a good linear relationship with the Log value of LOx concentration. As the LOx concentration increases, the fluorescence intensity of OPD also gradually increases. The linear correlation equation between Δ fluorescence intensity and LOx concentration is: y = 304.938x + 878.282, R0 2 =0.994, detection limit is 0.44 mU / L (3σ / slope), linear range is 0~10 U / L; Figure 4 B is a standard curve established to show the relationship between the R / B value and the Log value of LOx concentration. As the LOx concentration increases, the solution color changes from colorless to deep yellow, and the B / R value gradually increases. The linear correlation equation between the B / R value and the Log value of LOx concentration is: y = 0.0322x + 0.122, R 2 =0.987, indicating that the sensor achieves sensitive detection of LOx.

[0069] (2) Based on the above experimental steps, by adjusting the LOx concentration (final concentrations: 0, 0.01, 0.03, 0.05, 0.07, 0.1, 0.3, 0.5, 0.7, 1, 2, 4, 6, 10, 20, 40 U / L), while keeping other steps unchanged, the ultraviolet spectrum in the range of 370–500 nm was measured, and a linear relationship graph of different LOx concentrations was established using the maximum absorbance at 420 nm, thereby realizing the ultraviolet analysis and detection of different LOx concentrations; in addition, the visual detection of different LOx concentrations can be achieved through colorimetric images. Figure 4C represents the standard curve for LOx detection. The absorbance Δ value shows a good linear relationship with the Log value of LOx concentration. The linear correlation equation between absorbance Δ value and LOx concentration is: y = 0.0655x + 0.191, R0 2 =0.989, detection limit is 3.3 mU / L (3σ / slope), linear range is 0~10 U / L; Figure 4 D represents a standard curve establishing the relationship between the R / B ratio and LOx concentration. As the LOx concentration increases, the solution color changes from colorless to deep yellow, and the R / B ratio gradually increases. The linear correlation equation between the R / B ratio and LOx concentration is: y = 0.751x + 2.530, R... 2 =0.990, indicating that the sensor achieves sensitive detection of LOx.

[0070] C. Detection of inhibitors

[0071] First, incubate the inhibitor with lactate oxidase for 5–10 minutes. Then, add the other reagents for the enzyme reaction. In 70 μL of 0.1 M pH 7.4 PBS buffer, add 1.2 μL of 5000 U / L LOx (final concentration 6 U / L), 5 μL of 1 mM LA (final concentration 50 μM), 2.5 μL of 10 mM OPD (final concentration 250 μM), 1 μL of CuO2 Nanodots, 2 μL of 0.1 μM FeCl3 (final concentration 0.01 μM), and 18.3 μL of deionized water, and premix at 37 °C for 30 minutes. Transfer the above solution to a microplate reader, fluorescence spectrophotometer, and cuvettes for analysis and testing, and take photographs using a high-resolution camera.

[0072] (1) Based on the above experimental steps, by adjusting the FeCl3 concentration (final concentration: 0, 0.01, 0.03, 0.05, 0.1, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200, 400 μM), while keeping other steps unchanged, the fluorescence spectrum in the range of 460–660 nm was measured using an ELISA reader. The maximum fluorescence intensity at 553 nm was used to establish the concentrations of FeCl3 at different concentrations. 3+ A linear relationship graph of the Log value is used to achieve the analysis of Fe at different concentrations. 3+ Fluorescence analysis was used to detect and calculate Fe. 3+ The half-inhibition concentration of LOx; in addition, different concentrations of Fe can be obtained through colorimetric imaging. 3+ Visual detection, Figure 5 A represents the standard curve for inhibitor detection. As the inhibitor concentration increases, the fluorescence signal decreases. Figure 5B represents a standard curve establishing the relationship between the R / B value and the Log value of the inhibitor concentration. As the inhibitor concentration increases, the solution color gradually fades, changing from deep yellow to light yellow, and the R / B value gradually decreases. The half-maximal inhibitory concentration (IC50) was obtained using fluorescence sensing and a smartphone method. 50 The concentrations were 4.0 μM and 2.5 μM, respectively, which is within an acceptable range.

[0073] (2) Based on the above experimental steps, by adjusting the FeCl3 concentration (final concentrations: 0, 0.01, 0.03, 0.05, 0.1, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 200, 400 μM), while keeping other steps unchanged, the ultraviolet spectra in the range of 370–500 nm were measured using an enzyme-linked immunosorbent assay (ELISA) reader. The maximum absorbance at 420 nm was used to establish the concentrations of FeCl3 at different concentrations. 3+ A linear relationship graph of the Log value is used to achieve the analysis of Fe at different concentrations. 3+ Ultraviolet analysis was used to detect and calculate Fe. 3+ The half-inhibition concentration of LOx; in addition, different concentrations of Fe can be obtained through colorimetric imaging. 3+ Visual detection, Figure 5 C is an inhibitor of Fe 3+ Detection standard curve, Δ absorbance and inhibitor Fe 3+ The concentration showed a good linear relationship; as the inhibitor concentration increased, the absorbance of the system gradually decreased. Figure 5 D represents the R / B ratio and the inhibitor Fe. 3+ A standard curve was established based on the relationship between concentration and logarithmic values, with the inhibitor Fe... 3+ As the concentration increases, the solution color changes from dark yellow to light yellow, and the R / B value gradually decreases. The half-maximum inhibitory concentration (IC50) was obtained using ultraviolet sensing and smartphone methods. 50 The concentrations were 3.9 μM and 2.7 μM, respectively, which is within an acceptable range.

[0074] Example 4, Specificity Detection

[0075] To verify the selectivity of the sensor, other control substances such as alkaline phosphatase (ALP), glucose oxidase (HRP), terminal deoxynucleotidyl transferase (TdT), acetylcholinesterase (AChE), and lysozyme (LZM) were substituted in the LOx detection. These interfering substances did not interfere with the system. The results are as follows: Figure 6 As shown in A and 6B, it can be seen that the dual-mode colorimetric sensor has a good response to the target LOx and good selectivity.

[0076] Example 5: Extraction and purification of lactate oxidase

[0077] (1) Culturing Lactobacillus casei strain

[0078] First, MRS (Man, Rogosa, and Sharpe) medium was prepared using 20 g / L glucose, 10 g / L beef extract, 5 g / L yeast extract, 10 g / L peptone, 5 g / L polyvalent salt, 2 g / L disodium citrate, 0.1 g / L magnesium sulfate, and 0.05 g / L manganese sulfate. The pH of the medium was adjusted to 6.2–6.5 for the growth culture of *Lactobacillus casei*. *Lactobacillus casei* was inoculated into fresh MRS liquid medium and then cultured at 37°C in a static or shake-flask environment (closed). The culture was continued for 24 hours until the logarithmic growth phase. The strain cultured when the OD600 reached 0.8–1.0 was identified as *Lactobacillus casei*.

[0079] (2) Bacterial lysis

[0080] After incubation, centrifuge at approximately 5000g for 10 minutes to separate the bacterial pellet. Discard the upper culture medium, retaining only the bacterial pellet at the bottom. Resuspend the bacterial pellet in sterile PBS, gently agitate or pipette to mix, and then centrifuge again to wash away any remaining culture medium components. Repeat this step 1 to 2 times to ensure the bacteria are clean. Resuspend the washed bacterial pellet in an appropriate amount of cold PBS or Tris-HCl buffer, adding a protease inhibitor to prevent protein degradation. Disrupt the bacterial pellet using an ultrasonic cell disruptor. Set the sonication conditions to: sonicate in an ice bath for 3 seconds, with a 3-second interval, repeating 30 times or longer until the bacteria are completely disrupted (avoid overheating and maintain an ice bath). After disruption, centrifuge (10000g, 20 minutes, 4°C) to remove any undisrupted cell residue and collect the supernatant (which now contains lactate oxidase). Gradually add ammonium sulfate (gradual saturation, typically 40%-60% saturation) to the supernatant, stirring slowly in an ice bath to precipitate the lactate oxidase. Centrifuge the solution at 4°C (10000g, 20 minutes), collect the precipitate, and resuspend it in an appropriate amount of PBS. Place the resuspended protein solution into a dialysis membrane and dialyze to remove residual ammonium sulfate or other impurities, using appropriate PBS. The dialysis time is typically 12 hours, with the dialysis buffer changed multiple times during this period. Through these detailed steps, high-purity lactate oxidase can be obtained for further biosensing applications.

[0081] like Figure 7 As shown in Figure A, the fluorescence signal gradually increases with the increase of the number of bacteria (1000, 2000, 3000, 5000 bacteria / L), indicating that the bacteria contain a certain amount of LOx. Figure 7B illustrates the relationship between the B / R ratio and the number of bacteria in bacterial experiments. The results show that as the number of bacteria increases, the B / R ratio also increases. This is consistent with... Figure 7 The results from A are consistent, further demonstrating that the LOx content increases with the increase in bacterial count. These results indicate that the smartphone detection method developed in this patent has good consistency with the detection results from large-scale instruments. Similarly, the ultraviolet signal also increases with the increase in bacterial count, further indicating the presence of LOx in the bacteria. Figure 7 The change in the B / R value of B further confirms this, highlighting the reliability and consistency of this method.

[0082] To verify that the captured signal was indeed caused by LOx, the experiment introduced the LOx inhibitor Fe. 3+ .like Figure 8 As shown in Figure A, the fluorescence signal significantly weakens with increasing LOx inhibitor concentration, indicating that Fe... 3+ It has a strong inhibitory effect on LOx in bacterial extracts, which is consistent with the results in Example 3 above. Figure 8 B further validated the inhibitor's effect using a smartphone method, showing the same trend. Similarly, as... Figure 8 As shown in Figure C, the UV signal gradually decreases with increasing LOx inhibitor concentration, further indicating that Fe 3+ The inhibitory effect on LOx. Figure 8 D further validated the inhibitor's effect using smartphone detection results. The cross-validation of the two methods demonstrates that this dual-mode colorimetric sensor can effectively detect LOx activity in samples, possessing potential clinical application value.

[0083] Example 6: Application of sensors in the Warburg effect of gastric cancer

[0084] For gastric cancer patients, LOx-based in vitro diagnostic experiments can be used to monitor LA levels in patient body fluids, providing crucial information about tumor metabolic activity. One of the metabolic characteristics of solid tumors such as gastric cancer is excessive glycolysis, known as the "Warburg effect," which leads to a large accumulation of lactic acid. Therefore, measuring LA concentration can reflect the metabolic status and progression of the tumor. This patent utilizes a developed sensor to explore this effect.

[0085] ① Blood Samples: Blood samples from healthy individuals and gastric cancer patients were prepared. The LA in Example 3 was replaced with the dual-mode optical sensor of this patent to directly detect the LA level in the blood samples. The LOx concentration was adjusted to 0, 1, and 6 U / L, and the LA concentration in the sample was measured under each condition. The sensor detected H2O2 generated from lactate oxidase in the solution, and CuO2 nanodots were used to catalyze the reaction between H2O2 and OPD, recording colorimetric and fluorescence signals. Images were captured using a smartphone camera, and the LA concentration was calculated using RGB analysis technology. The differences between healthy individuals and gastric cancer patients were compared, indirectly achieving the analysis and detection of LOx activity.

[0086] The results are as follows Figure 9 As shown in A and 9C:

[0087] In normal human blood samples, LA levels are low, and the enhancement of fluorescence and UV absorbance signals is relatively slow with increasing LOx concentration (0, 1, 6 U / L). Because normal individuals have normal metabolism, LA accumulation is relatively low; therefore, the reaction between CuO2 nanodots and OPD generates less H2O2, and the changes in fluorescence and absorbance signals are not significant.

[0088] Blood samples from gastric cancer patients showed significantly higher LA levels than normal individuals. Fluorescence and UV absorbance signals increased significantly with increasing LOx concentration (0, 1, 6 U / L). This is because the Warburg effect in gastric cancer leads to a large accumulation of LA, which, under the catalysis of LOx, generates more H2O2, thereby enhancing the reaction catalyzed by CuO2 nanodots.

[0089] Furthermore, such as Figure 9 B. Figure 9 D. The color changes (RGB values) captured by the smartphone will also be more pronounced, reflecting a higher LA concentration. Furthermore, the signal detected in the blood sample is significantly higher than that at LOx (6 U / L), indicating that excessively high LA levels have a stimulating effect on the response, resulting in a significant signal increase. This verifies the metabolic characteristics of the Warburg effect in gastric cancer patients.

[0090] Therefore, this detection system can be used for early gastric cancer screening because an increase in LA concentration is one of the hallmarks of the Warburg effect. By detecting LOx catalytic reactions, the sensor can rapidly reflect changes in LA levels, providing data support for early diagnosis and treatment, and improving the survival rate of patients with early-stage gastric cancer.

[0091] ② Homemade simulated gastric fluid sample: Prepare simulated gastric fluid samples and add 10 μM and 50 μM LA. Replace the LA in Example 3 with the homemade simulated gastric fluid and use a sensor to detect the LA level in the gastric fluid. Similarly, adjust the LOx concentration (0, 1, 6 U / L) and record the LA detection results at each concentration. Analyze the changes in LA level using colorimetric and fluorescence signals from a smartphone via CuO2 nanodot catalysis. Explore the effect of LOx concentration on sensor detection sensitivity and the response of LA content in simulated gastric fluid to the detection signal, indirectly realizing the analysis and detection of LOx activity.

[0092] The results are as follows Figure 10 As shown in A and 10C: Overall, the signal in the 50 μM LA sample is higher than that in the 10 μM LA sample. Figure 10 As shown in B and 10D, the color variations captured by the smartphone are significant.

[0093] Simulated gastric fluid (without LOx): Under 0 U / L LOx conditions, the fluorescence and UV absorbance signals will remain at low levels, indicating that the reaction between CuO2 nanodots and OPD is not significantly enhanced by H2O2, because no LOx participates in the catalytic generation of H2O2.

[0094] Simulated gastric fluid (1 U / L and 6 U / L LOx): As the LOx concentration increased from 1 U / L to 6 U / L, the fluorescence and UV absorbance signals gradually increased. At a LOx concentration of 1 U / L, less H2O2 was generated, resulting in a relatively mild increase in fluorescence and absorbance. However, at a LOx concentration of 6 U / L, a large amount of H2O2 was generated, significantly enhancing the catalytic effect of CuO2 nanodots on the OPD reaction, leading to a significant increase in fluorescence and absorbance signals.

[0095] For both blood and gastric juice samples, the signal was found to be significantly higher in blood samples than in gastric juice samples. This is likely due to the higher LA content in blood samples, further confirming the occurrence of the Warburg effect. Therefore, this sensor can rapidly detect changes in LA levels related to gastric cancer metabolism in body fluid samples, especially in gastric cancer patients, by monitoring LA concentration in gastric juice, further expanding its application value in gastric cancer disease progression tracking and metabolic research. Simulated gastric juice experiments demonstrated the sensitivity and potential for field application of this method in clinical sample analysis, providing a tool for real-time monitoring and treatment progress assessment for gastric cancer patients. This sensor has broad application potential by accurately detecting changes in lactate concentration. Its innovative dual-mode detection provides a rapid and portable tool for gastric cancer metabolic analysis, particularly suitable for research and clinical screening related to the Warburg effect.

[0096] It should also be noted that the specific embodiments described above are not intended to limit the present invention, nor is the present invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the claims of this invention.

Claims

1. A dual-mode optical sensor based on copper-based peroxide nanodots and its application in the Warburg effect of gastric cancer. The mechanism is as follows: CuO2 nanodots are used as the core catalyst to detect lactate oxidase (LOx) activity via fluorescence and ultraviolet dual-mode detection. The sensor works by catalyzing the production of hydrogen peroxide (H2O2) from lactic acid using LOx. CuO2 nanodots accelerate the reaction between H2O2 and o-phenylenediamine (OPD), significantly enhancing the fluorescence emission intensity at 553 nm and the ultraviolet absorbance at 420 nm. Color changes after the reaction are captured using a smartphone camera, and RGB colorimetric values ​​are analyzed using dedicated software to establish a linear relationship between LOx concentration and color change, thereby achieving quantitative analysis of LOx activity and concentration. Furthermore, the sensor incorporates Fe... 3+ As an inhibitor, it is used for LOx inhibitor screening, further expanding its application in biomedicine. This invention is the first to use CuO2 nanodots for LOx activity detection, and achieves rapid, portable and sensitive LOx detection through a combination of fluorescence and ultraviolet dual-mode visualization analysis using a smartphone. It has important applications, especially in early gastric cancer screening and metabolic studies related to the Warburg effect.

2. The dual-mode optical sensor based on copper-based peroxide nanodots according to claim 1 and its application in the Warburg effect of gastric cancer, characterized in that: This invention is the first to develop a fluorescence-UV dual-mode sensor for LOx detection using CuO2 nanodots and apply it to the study of the Warburg effect in gastric cancer. This sensor, combined with a smartphone application and image processing technology, enables rapid on-site detection of LOx, providing an innovative and convenient solution for early screening of gastric cancer and accurate assessment of metabolic status. It fills a gap in the application of CuO2 nanodots in LOx detection and gastric cancer metabolic research.

3. The dual-mode optical sensor based on copper-based peroxide nanodots according to claims 1-2 and its application in the Warburg effect of gastric cancer, characterized in that: The constructed optical sensing method can be used for the analysis and detection of LOx at different concentrations, with detection limits of 0.47 mU / L (fluorescence) and 3.3 mU / L (UV), and a half-inhibition concentration (IC50) of [missing value]. 50 The fluorescence and ultraviolet (UV) values ​​were 4.0 μM and 3.9 μM, respectively, and the results from smartphone detection were not significantly different from those from optical sensing methods, falling within an acceptable range.

4. The dual-mode optical sensor based on copper-based peroxide nanodots according to claims 1-3 and its application in the Warburg effect of gastric cancer, characterized in that: Experimental results showed that the LA level in blood samples from gastric cancer patients was significantly higher than that in normal individuals. With the increase of LOx concentration, the fluorescence and ultraviolet absorbance signals were significantly enhanced, confirming the phenomenon of LA accumulation under the Warburg effect. In contrast, the signal in the blood sample was much higher than that in the simulated gastric juice sample, further indicating a higher LA content in the blood, thus verifying the metabolic characteristics of the Warburg effect in gastric cancer patients.

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