Electrochemical nanosensor for detecting intracellular lactate in tumor cells and preparation method and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to an electrochemical nanosensor for detecting intracellular lactic acid in tumor cells and a preparation method and application thereof. A carbon fiber nanoelectrode (CFNE) with a tip diameter of about 150 nm is prepared by using a flame etching technique, and platinum nanoparticles (Pt NPs) are modified on the surface of the carbon fiber nanoelectrode to enhance the electrocatalytic activity of the electrode. Lactate dehydrogenase (LDH) is covalently cross-linked and fixed by using polyethyleneimine to construct an enzyme nanoelectrode (LDH / Pt / CFNE) for electrochemical detection of intracellular lactic acid in single cells. The sensing electrode has the characteristics of high sensitivity, good selectivity and excellent stability, realizes real-time and in-situ quantitative detection of intracellular lactic acid in single tumor cells, and is applied to tumor metabolism research, drug evaluation and mouse tumor model verification.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to an electrochemical nanosensor for detecting lactate in tumor single cells, its preparation method, and its application. Background Technology

[0002] Lactic acid is not only the end product of glycolysis but also a key signaling molecule regulating the tumor immune microenvironment. In the tumor microenvironment, high lactic acid accumulation induces immune escape through mechanisms such as acidifying the microenvironment, inhibiting T cell and NK cell function, and promoting the proliferation of regulatory T cells (Tregs), becoming a significant factor in immunotherapy resistance. Therefore, accurately analyzing the dynamic changes of lactic acid within tumor cells is of great significance for revealing the mechanisms of tumor immune escape and assessing treatment efficacy.

[0003] Existing methods for detecting intracellular lactate, including spectrophotometry, high-performance liquid chromatography (HPLC), and mass spectrometry, while capable of precise quantification, all require cell destruction for sample pretreatment. These methods only provide average data for a population of cells, masking the metabolic heterogeneity of tumor cells and failing to provide spatial distribution and dynamic changes in lactate. Imaging methods (magnetic resonance / optical imaging): While magnetic resonance spectroscopy offers non-invasive detection, its low spatial resolution prevents it from reaching the single-cell level. Existing fluorescent probes or sensors, although possessing high spatiotemporal resolution, often face problems such as phototoxicity, photobleaching, and metabolic interference, making it difficult to achieve long-term stable in-situ monitoring of single cells while maintaining cell viability. Furthermore, methods utilizing LDH immobilized on electrodes to detect oxygen consumption or H2O2 generation during enzyme-catalyzed reactions can indirectly determine lactate content. This method offers rapid response (seconds to minutes), ease of use, and small instrument size, and has wide applications in serum lactate detection and lactate determination in exercise physiology. However, traditional enzyme electrodes are in the range of millimeters to hundreds of micrometers in size, and can only be used for detection at the level of ex vivo samples or cell populations. They cannot meet the requirements for in situ analysis at the single-cell or even subcellular level. Summary of the Invention

[0004] The purpose of this invention is to provide an electrochemical nanosensor for detecting lactate in single tumor cells, its preparation method and application, thereby overcoming the shortcomings of the prior art, realizing real-time, in-situ quantitative detection of lactate in single cells at the live cell level, and applying it to tumor lactate metabolism research, drug evaluation and mouse tumor model detection.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: On one hand, the present invention provides an electrochemical nanosensor (LDH / Pt / CFNE) for detecting lactate in tumor single cells, comprising a working electrode and a platinum nanoparticle layer, a polyethyleneimine layer and an enzyme-modified layer sequentially modified on its surface; The enzyme-modified layer includes lactate dehydrogenase, which can recognize and detect lactate in tumor single cells.

[0006] A high-precision carbon fiber nanoelectrode (CFNE) was fabricated using flame etching technology. Platinum nanoparticles (Pt NPs) were then modified onto its surface to enhance the electrode's electrocatalytic activity. Lactate dehydrogenase (LDH) was immobilized by covalent cross-linking with polyethyleneimine, thus constructing an LDH / Pt / CFNE nanosensor. This nanosensor, with its nanoscale precision, enables non-invasive in-situ detection of single cells, achieving specific recognition and detection of lactate within single cells through the modified LDH.

[0007] In some other embodiments, the enzyme-modified layer is formed by curing a mixed enzyme solution containing lactate dehydrogenase, a cross-linking agent, and serum albumin; The crosslinking agent includes polyethylene glycol diglycidyl ether, in which the epoxy groups of polyethylene glycol diglycidyl ether are covalently linked to the amino groups in the polyethyleneimine layer and the amino groups in lactate dehydrogenase, respectively. Serum albumin includes bovine serum albumin.

[0008] In some other embodiments, the working electrode is a carbon fiber nanoelectrode; Carbon fiber nanoelectrodes have needle-like nano-tip with a diameter of 100-200 nm.

[0009] Secondly, the present invention provides a method for preparing an electrochemical nanosensor for detecting lactate in single tumor cells, comprising the following steps: Using carbon fiber nanoelectrodes as working electrodes, platinum nanoparticles were loaded onto their surfaces by electrochemical deposition to prepare carbon fiber nanoelectrodes modified with platinum nanoparticle layers. The carbon fiber nanoelectrode modified with platinum nanoparticles was immersed in a polyethyleneimine solution, and after being taken out, dried and cleaned, a polyethyleneimine layer was formed on the electrode surface. A mixed enzyme solution containing lactate dehydrogenase, a cross-linking agent, and serum albumin is added dropwise to an electrode with a polyethyleneimine layer, and then dried and cross-linked to obtain the final product.

[0010] In some other embodiments, electrochemical deposition includes CV pulse electrodeposition; CV pulse electrodeposition consists of a three-electrode system: a carbon fiber nanoelectrode as the working electrode, a platinum sheet as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The three-electrode system was placed in a chloroplatinic acid deposition solution with a concentration of 0.5-1.0 mM, and cyclic voltammetric pulse electrodeposition was performed at a scan rate of 0.08 V / s to 0.12 V / s within a voltage window of -0.5 V to 0.0 V. The mass-volume concentration of the polyethyleneimine solution is 0.01-0.2%.

[0011] In some other embodiments, the enzyme activity of lactate dehydrogenase in the mixed enzyme solution is 500-1200 U / mL; The mass ratio of cross-linking agent to serum albumin is (8-12):1; Preferably, the enzyme activity of lactate dehydrogenase in the mixed enzyme solution is 1000 U / mL; The mass ratio of cross-linking agent to serum albumin is 10:1; Crosslinking agents include polyethylene glycol diglycidyl ether; Serum albumin includes bovine serum albumin.

[0012] In some other embodiments, the carbon fiber nanoelectrodes are prepared as follows: Carbon fiber is connected to copper wire, cured, cleaned and dried to obtain carbon fiber lead body; The carbon fiber lead is inserted into the tip capillary, and the length of the carbon fiber protruding from the tip capillary tip is controlled to be 2~5 μm; then epoxy resin is injected into the tail end of the electrode for sealing and fixation. By flame etching the exposed tip of the carbon fiber, a needle-like nano-tip is formed at the front end of the carbon fiber, thus obtaining the carbon fiber nanoelectrode.

[0013] Thirdly, this invention provides the application of an electrochemical nanosensor for detecting lactate in tumor single cells in a model for detecting biomarkers in tumor single cells.

[0014] In some other embodiments, the method for constructing a biomarker model for detecting tumor single cells is as follows: an electrochemical nanosensor for detecting lactate in tumor single cells is inserted into the tumor single cell, and the lactate content is determined by detecting the response electrical signal.

[0015] This electrode exhibits excellent linear detection range (5 ~ 70 mM) for lactate detection, while also demonstrating good reproducibility (RSD = 2.1%), stability, and anti-interference capabilities.

[0016] Fourthly, this invention provides the application of an electrochemical nanosensor for detecting lactate in single tumor cells in the preparation of diagnostic and targeted cancer therapy products, including breast cancer, colorectal cancer, lung cancer, pancreatic cancer, and glioma; the products include drugs and reagent kits.

[0017] Using a constructed LDH / Pt / CFNE nanosensor, in-situ detection of lactate in single cells of different cell types was performed. The results showed a significant difference in lactate concentration between tumor cells and normal cells, indicating that lactate can serve as a tumor marker and effectively distinguish between normal and tumor cells. Furthermore, within the same tumor cell population, there was significant heterogeneity in lactate concentration among different single cells, and the higher the degree of tumor metastasis, the more significant the intracellular lactate heterogeneity.

[0018] The beneficial effects of this invention are: (1) The electrochemical nanosensor (LDH / Pt / CFNE) for detecting lactate in tumor single cells provided by this invention achieves highly selective and sensitive real-time detection of lactate in tumor single cells through a quadruple structural design integrating a needle-like nano-tipped carbon fiber electrode, a platinum nanoparticle catalytic layer, a polyethyleneimine cross-linking layer, and a lactate dehydrogenase recognition layer. The nano-tipped tip endows the sensor with excellent single-cell puncture capability, the platinum nanoparticles significantly enhance electron transfer efficiency and electrochemical signal response, the polyethyleneimine layer combined with a cross-linking agent constructs a stable biocompatible interface, and the lactate dehydrogenase ensures specific recognition of lactate molecules only. This solves the technical problem of traditional sensors being unable to simultaneously achieve minimally invasive single-cell detection, anti-interference capability, and long-term stability. The electrode exhibits excellent linear detection range (5 ~ 70 mM) for lactate detection, while also possessing good reproducibility (RSD = 2.1%), stability, and anti-interference capability.

[0019] (2) This invention achieves the precise construction of a multilayer sensor structure by combining electrochemical deposition, physical adsorption and covalent cross-linking. The method is simple, mild and reproducible. The particle size and density of platinum nanoparticles are precisely controlled by cyclic voltammetric pulse electrodeposition to improve conductivity. With the help of the covalent cross-linking of polyethyleneimine layer and polyethylene glycol diglycidyl ether (PEGDE), not only is the high loading and biological activity of lactate dehydrogenase on the electrode surface maintained, but the microenvironment is also optimized by serum albumin. Thus, a nanosensor with high sensitivity, strong stability and excellent biocompatibility is prepared, which is very suitable for the precise detection of lactate in tumor single cells.

[0020] (3) The sensor of the present invention can deeply analyze the heterogeneity of lactate levels in tumor cells and the impact of metabolism-related drugs on tumor development. It provides new research methods and experimental evidence for lactate-related cell research, and strongly promotes the development of single-cell electrochemical analysis technology in basic tumor research and clinical translational applications. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 A schematic diagram illustrating the construction of a lactic acid nanoelectrochemical sensor; Figure 2 The images are scanning electron microscope (SEM) images of a lactic acid nanoelectrochemical sensor, where A represents CFNE, B represents Pt / CFNE, C represents PEI / Pt / CFNE, and D represents LDH / PEI / Pt / CFNE. Figure 3 The elemental characterization diagram of the lactic acid nanosensor is shown below. Figure 4 The graphs show the electrochemical characterization and performance verification of the lactic acid nanosensor. In the graphs, A is the electrochemical response of the electrodes at different modification stages in potassium ferricyanide solution, B is the response curve of cyclic voltammetry for detecting lactic acid, H2O2 and PBS, and C is the repeatability test graph of multiple electrodes (RSD = 2.1%). Figure 5 The diagram shows the optimization of the modification layer conditions for the lactic acid nanosensor. In the diagram, A represents the effect of different concentrations of PEI solution on the electrochemical response of the electrode, and B represents the effect of different concentrations of LDH solution on the sensing signal of the electrode. Figure 6 The graphs show the performance verification of the lactic acid nanosensor. A represents the cyclic voltammetry curves for different concentrations of lactic acid; B represents the chronocurrent response of lactic acid at a constant potential of +0.6 V; C represents the current-concentration linear fitting curve; D represents the sensor's anti-interference capability against lactic acid and interfering substances; and E represents the sensor's stability after 7 days of storage at 4°C. n = 3, where n represents the three sensors used in the experiment. * This indicates that there is a significant difference. *** p <0.001, ns indicates no significant difference; Figure 7 This diagram illustrates the effect of lactate nanosensors on cell viability. Figure A shows the fluorescence changes of the lactate nanosensor before and after cell insertion, and Figure B shows the fluorescence changes of the nanosensor at different puncture depths at 1 mM Ru(NH3)6³. + Electrochemical response curves in solution, C is the photon count of 5 nanosensors puncturing cells, D is the fluorescence photon count of a single cell during a 20 s real-time monitoring period, n = 5, where n represents 5 cells randomly selected in the experiment, and ns indicates no significant difference; Figure 8The diagram shows the current response of lactate electrochemical detection in different cell lines. A represents the single-cell lactate electrochemical response of CT26, B represents the single-cell lactate electrochemical response of RPE1, C represents the single-cell lactate electrochemical response of MCF-7, D represents the single-cell lactate electrochemical response of MDA-MB-231, and E represents the single-cell lactate electrochemical response of MCF-10A. Figure 9 The graph shows the differences in lactate concentration among different cell lines. A represents the lactate content of CT26 single cells, B represents RPE1, C represents the lactate content of MCF-7 single cells, D represents the lactate content of MDA-MB-231 single cells, and E represents the lactate content of MCF-10A single cells. Figure 10 This diagram illustrates the differences in lactate concentration and regulatory proteins among different cell lines. A represents the statistical graph of single-cell electrochemical signals from different cell lines; B represents the statistical graph of intracellular lactate concentration from single cells in different cell lines; C represents the heatmap of lactate concentration distribution; and D represents the expression characteristics of MCTs proteins. n = 30, where n represents the number of cells randomly selected from each cell line in the experiment. * This indicates that there is a significant difference. *** p <0.001; Figure 11 The graph shows the inhibition of cell proliferation by lactate-metabolizing drugs. In the graph, A represents the inhibition of MCF-7 cell proliferation by CHC, B represents the inhibition of MDA-MB-231 cell proliferation by CHC, C represents the inhibition of MCF-7 cell proliferation by GNE-140, and D represents the inhibition of MDA-MB-231 cell proliferation by GNE-140. n = 3, where n represents the 3 independent experiments in the MTT assay. Figure 12 The electrochemical responses of different cell lines after treatment with lactate metabolism drugs are shown in Figure 1. A represents the electrochemical response of MCF-7 single cells before and after drug administration, B represents the electrochemical response of MDA-MB-231 single cells before and after drug administration, and C represents the electrochemical response of MCF-10A single cells before and after drug administration. Figure 13 The graphs show the statistical analysis of single-cell electrochemical signals and the quantitative analysis of intracellular lactate concentration. A represents the statistical analysis of single-cell electrochemical signals in MCF-7 cells after drug intervention; B represents the statistical analysis of single-cell electrochemical signals in MDA-MB-231 cells after drug intervention; C represents the statistical analysis of single-cell electrochemical signals in normal mammary epithelial cells MCF-10A after drug intervention; D represents the quantitative intracellular lactate concentration in MCF-7 cells calculated based on the standard curve; E represents the quantitative intracellular lactate concentration in MDA-MB-231 cells calculated based on the standard curve; and F represents the quantitative intracellular lactate concentration in normal mammary epithelial cells MCF-10A calculated based on the standard curve. n = 15, where n represents the number of cells randomly selected in each experimental group.* This indicates that there is a significant difference. * p <0.05, ** p <0.01, *** p <0.00, ns indicates no significant difference; Figure 14 The graph shows the proliferation activity of MCF-7 cells and the electrochemical analysis of lactate in single cells under exogenous lactate intervention. In the graph, A represents the effect of different concentrations and treatment durations of exogenous lactate on MCF-7 cell proliferation; B represents the electrochemical response of single cells after treatment with lactate at concentrations of 0 mM, C, 40 mM, D, and E (80 mM); and F represents the statistical analysis of electrochemical signals for each group. Data in A are the mean ± standard deviation of three independent experiments. In F, n = 10, where n represents the number of cells randomly selected in each experiment. * This indicates that there is a significant difference. * p <0.05, ** p <0.01, ns indicates no significant difference; Figure 15 The graphs show the effects of GSK2837808A intervention on the proliferation activity of MCF-7 cells and the electrochemical analysis of lactate in single cells. In Figure A, the effect of different concentrations and treatment durations of GSK2837808A on the proliferation of MCF-7 cells is shown. Figure B represents 0 μM, Figure C represents 10 μM, Figure D represents 30 μM, and Figure E represents the electrochemical response of single cells to GSK2837808A at a concentration of 50 μM. Figure F shows the statistical analysis of electrochemical signals for each group. Data in Figure A represent the mean ± standard deviation of three independent experiments. In Figure F, n = 10, where n represents the number of cells randomly selected in each experiment. * This indicates that there is a significant difference. * p <0.05, *** p <0.001, ns indicates no significant difference; Figure 16 The following are heatmaps showing the quantitative analysis and distribution of intracellular lactate concentration in MCF-7 cells from different treatment groups: A represents the quantitative analysis of intracellular lactate concentration in the lactate intervention group; B represents the distribution heatmap of intracellular lactate concentration in the lactate intervention group; C represents the quantitative analysis of intracellular lactate concentration in the GSK2837808A treatment group; and D represents the distribution heatmap of single-cell lactate concentration in the GSK2837808A treatment group. n = 10, where n represents the number of cells randomly selected in each experimental group. * This indicates that there is a significant difference. * p <0.05, **p <0.01, *** p <0.001, ns indicates no significant difference; Figure 17 shows the construction diagram of the mouse tumor model; Figure 18 The images show the therapeutic effects of CHC treatment. A shows a comparison of tumor size between the control and experimental groups of mice; B shows the tumor weight statistics between the two groups; C shows the total body weight statistics of the three groups of mice; and D shows the tumor volume statistics between the control and experimental groups. In B, n = 3, where n represents 3 mouse tumors. * This indicates that there is a significant difference. *** p <0.001; in CD, n = 3, where n represents the mean ± standard deviation of 3 mice; Figure 19 The graph shows the lactate detection and analysis in primary mouse tumor cells. In this graph, A represents the electrochemical signal of lactate in a single primary tumor cell, B represents the statistical analysis of electrochemical signals in primary tumor cells, and C represents the quantitative analysis of lactate concentration in primary tumor cells. In B and C, n = 20, where n represents the number of cells randomly selected in each experimental group. * This indicates that there is a significant difference. ** p <0.01; Figure 20 The images show HE staining patterns in mice after CHC treatment. Image A shows HE staining of the heart, liver, spleen, lungs, and kidneys of mice, while image B shows a comparison of HE staining of tumor tissues in the control and experimental groups. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] Traditional lactate detection methods lack the ability to detect lactate in situ, dynamically, and in real time at the single-cell or even subcellular level. There are significant differences in metabolic levels among tumor cells, and the average results obtained cannot reflect the true metabolic state of individual cells. Furthermore, the spatial and temporal resolution is insufficient to reflect cellular heterogeneity and transient metabolic changes.

[0025] This invention utilizes flame etching technology to fabricate carbon fiber nanoelectrodes (CFNEs) with a tip diameter of approximately 150 nm. Platinum nanoparticles (PtNPs) are then modified onto the CFNE surface to enhance its electrocatalytic activity. Lactate dehydrogenase (LDH) is covalently cross-linked and immobilized using polyethyleneimine to construct an LDH / Pt / CFNE nanosensor. The electrode morphology and elemental composition were characterized using scanning electron microscopy and X-ray energy dispersive spectroscopy. The electrode performance was characterized using cyclic voltammetry and chronoamperometry. This nanosensor, with its nanoscale tip characteristics, enables non-invasive in-situ detection of single cells. The modified LDH allows for the specific recognition and detection of lactate within single cells. This sensor provides a deeper understanding of the heterogeneity of lactate levels in tumor cells and the impact of related metabolic drugs on tumor development, offering new research methods and experimental evidence for lactate-related cell studies and significantly promoting the development of single-cell electrochemical analysis technology in basic tumor research and clinical translational applications.

[0026] Example 1 I. Experimental Materials and Reagents 1. Materials, reagents and equipment are shown in Table 1 and Table 2.

[0027] Table 1 Materials and Reagents

[0028] Table 2 Instruments and Equipment

[0029]

[0030] 2. Preparation of relevant solutions (1) Preparation of 10 mM potassium ferricyanide (K3[Fe(CN)6]) solution: Weigh 0.1317 g K3[Fe(CN)6] and 0.2982 g KCl, add ultrapure water to make up to 40 mL, stir until completely dissolved, and prepare fresh before use.

[0031] (2) Preparation of 10 mM pH = 7.4 phosphate buffered solution (PBS): Weigh 8.0144 g NaCl, 0.2012 g KCl, 3.6 g Na2HPO4·12H2O, and 0.272 g KH2PO4, and make up to 1 L with ultrapure water. After filtration through a filter membrane, autoclave the solution.

[0032] (3) Saturated KCl solution: Take a clean and dry beaker, add solid KCl and an appropriate amount of distilled water, and stir continuously until excess KCl remains in the beaker to obtain a saturated KCl solution.

[0033] (4) Preparation of 0.5 mM chloroplatinic acid (H2PtCl6) solution: Take 100 μL of 200 mM chloroplatinic acid stock solution, add 0.2 M K2SO4 solution to make up to 20 mL, stir well, use K2SO4 solution as supporting electrolyte, and adjust the pH to 7.4 with NH3·H2O. The solution needs to be stored at 4°C.

[0034] (5) 1 mM hexaammineruthenium (Ru(NH3)6) 3+ Solution preparation: Weigh 4.5 mg of hexaammineruthenium powder, add purified water, mix well, and bring the volume to 15 mL. Prepare fresh before use.

[0035] (6) Preparation of 5% skim milk powder blocking solution: Weigh 0.5 g skim milk powder, add 1 × TBST solution to make up to 10 mL, stir well, and prepare fresh before use.

[0036] (7) Preparation of 5× Running Buffer solution: Weigh 15.1 g Tris, 94 g glycine and 5 g SDS, add purified water and mix well, then make up to 1 L. Dilute to 1× for use in the experiment. The solution can be stored at room temperature.

[0037] (8) Preparation of 5× Transfer Buffer solution: Weigh 15.1 g Tris and 75.1 g glycine, add purified water and mix well, then make up to 1 L. Dilute to 1× for use in experiments. The solution should be stored at 4°C.

[0038] (9) 5 mg / L Methyl thiazolyl tetrazolium (MTT): Weigh 5 mg MTT and dissolve it in 1 mL of sterile water, taking care to avoid light.

[0039] 3. Experimental cell lines, as shown in Table 3.

[0040] Table 3 Experimental cell lines

[0041] II. Experimental Methods 1. Fabrication of carbon fiber nanoelectrodes: The lactic acid nanosensor used was based on CFNE.

[0042] (1) Connection of carbon fiber to conductive lead Take a carbon fiber with a diameter of approximately 7 μm and a length of approximately 2 cm, and use graphite conductive silver paste to bond it together with a copper wire with a diameter of 0.4 mm and a length of 10 cm. The copper wire can be used as an electrode lead. After the conductive silver paste has cured, wash the carbon fiber in anhydrous ethanol and then air dry it.

[0043] (2) Drawing of glass capillaries Borate glass capillaries (with an inner diameter of approximately 0.58 mm) were drawn into glass tubes with a tip diameter of less than 1 μm using a laser drawing instrument (P1000 model) to ensure that the size of the subsequent insulating part is small enough.

[0044] (3) Carbon fiber is inserted into the glass tube The carbon fiber, already connected to the copper wire, is slowly inserted into the drawn glass capillary, leaving approximately 2-5 μm of carbon fiber protruding from the tip. This insertion process must be extremely slow and careful to prevent excessive force from puncturing the tip of the glass tube. The electrode end is sealed with epoxy resin, and the copper wire at the end is then secured.

[0045] (4) Fabrication of nano-tip by flame etching The 50-100 μm portion of the carbon fiber tip is etched over a butane flame to form a needle-like nanoscale tip (approximately 150 nm in diameter).

[0046] 2. Construction of Lactic Acid Nanosensor LDH / Pt / CFNE: Based on CFNE, a lactic acid-specific nanoelectrode sensor was constructed by immobilizing LDH.

[0047] (1) Pt NPs electrodeposition To improve the catalytic efficiency of the electrode for H2O2, Pt NPs were electrochemically coated onto a CFNE. The electrode was immersed in a 0.5 mM H2PtCl6 solution, and a three-electrode system was constructed using an Ag / AgCl electrode as a reference and auxiliary electrode. CV pulse electrodeposition was performed at a scan rate of 0.1 V / s within the range of -0.5 to 0.0 V to obtain uniform Pt NPs on the electrode. After deposition, the electrode was removed from the solution and thoroughly rinsed with ultrapure water. Due to the excellent electrocatalytic performance of Pt NPs, the H2O2 generated in the subsequent enzymatic reaction can be converted into a readable signal, which can then be used to indirectly determine the lactate level in individual cells. Pt NPs provide a sensitive electrochemical detection platform.

[0048] (2) Modification of electrostatic adsorption layer One end of the CFNE electrode loaded with Pt NPs was immersed in a 0.1% (w / v) polyethyleneimine (PEI) solution to coat the electrode tip with a layer of PEI. The electrode was then removed, air-dried at room temperature, and finally rinsed in a deionized water solution to remove excess PEI. PEI is a cationic polymer containing a large number of amino groups. Utilizing its positive charge, it is adsorbed onto the electrode via electrostatic attraction, providing more enzyme linkage sites for subsequent enzyme immobilization and thus improving electrode stability.

[0049] (3) Immobilization of enzyme-modified layer An enzyme solution containing 10 mg / mL polyethylene glycol diglycidyl ether (PEGDE), 1 mg / mL bovine serum albumin (BSA), and LDH (lactate dehydrogenase with an activity of 1000 U / mL) was prepared and then dropped onto the electrode tip and dried overnight at 4°C. LDH specifically catalyzes the oxidation of lactate to pyruvate and H₂O₂. PEGDE, acting as a cross-linking agent, covalently cross-links with the amino groups of PEGDE and LDH, thus firmly immobilizing LDH onto the electrode. BSA, as a stabilizer, protects the protein, helps maintain LDH activity, and reduces enzyme inactivation.

[54] .

[0050] 3. Cell Culture (1) Cell thawing: Preheat the complete culture medium in a 37°C constant temperature water bath; take out the cell cryovials and quickly place them in a preheated 37°C water bath. Thaw within 1-2 minutes. Disinfect the outside of the cryovials with 75% ethanol and then transfer them to a clean bench. Use a pipette to aspirate the thawed cell suspension into a pre-prepared 15 mL centrifuge tube containing 2 mL of complete culture medium and gently pipette to mix. Centrifuge at 1000 rpm / min for 3 minutes, discard the supernatant, resuspend the cells in an appropriate amount of complete culture medium, transfer them to a culture dish, and incubate at 37°C in a 5% CO2 incubator. Observe the cell adhesion and growth status on the second day and replace the culture medium with fresh medium as needed for continued culture.

[0051] (2) Cell passage: When the cell density reaches 80%, begin passage. Discard the old culture medium and wash the cells 2-3 times with PBS to remove any remaining serum. Slowly add 2 mL of trypsin along the wall of the dish and shake well to cover the entire dish. Place the dish in an incubator and digest for 1-3 min (depending on the cell type). When the cells are observed to be round and partially detached under an inverted microscope, add an equal volume of complete culture medium to stop the digestion. Gently pipette the cell layer repeatedly until all cells detach. Transfer the cells to a 15 mL centrifuge tube and centrifuge at 1000 rpm / min for 3 min. Discard the supernatant, add an appropriate amount of fresh complete culture medium, mix well, and resuspend the cells evenly. Passage the cells at a ratio of 1:2 to 1:5. Place the cells in a new culture dish and add an appropriate amount of fresh culture medium. Incubate the dish at 37°C with 5% CO2 for further culture.

[0052] (3) Cell cryopreservation: Cells growing in the logarithmic growth phase were collected, and the culture medium was changed to fresh one day before cryopreservation. Cells were digested and collected according to the conventional passage method, and cell counts were performed. The cells were centrifuged at 1000 rpm / min for 3 min, the supernatant was discarded, and cell cryopreservation solution was added to adjust the cell density to 1 ~ 5 × 10⁶ cells / min. 6 Aliquoting cells / mL, fill cryovials (1-1.5 mL) and label the cryovials with the cell name, passage number, freezing time, and operator's name. Place the cryovials in a controlled-rate cooling box and freeze at -80°C overnight. The next day, transfer them to a liquid nitrogen tank for long-term storage.

[0053] 4. Cell viability assay MCF-7 cells at an appropriate density were seeded in confocal microplates and cultured overnight. After cell adhesion, calcein-AM and propidium iodide (PI) were added to the culture medium at a final concentration of 3 μg / mL for staining. Cells were then incubated at 37°C in a 5% CO2 incubator for 15 min, followed by washing the cells three times with PBS for fluorescence imaging. Using a real-time in situ single-cell analyzer, the prepared lactate nanosensors were inserted into the cells, and the changes in the number of fluorescence photons within 20 seconds were recorded before carefully removing the sensors. Bright-field and fluorescence images were recorded using the in situ single-cell analyzer, and changes in the number of photons within multiple cells were also recorded.

[0054] MCF-7, MDA-MB-231, and RPE1 cells were cultured in DMEM complete medium, CT26 cells were cultured in RPMI-1640 supplemented complete medium, and MCF-10A cells were cultured in MCF-10A specialized culture medium. Cell resuscitation, passage, and cryopreservation procedures were the same as described above.

[0055] 5. Western blot analysis of proteins Western blotting (WB) is used to detect the expression levels of specific proteins in cells or tissues.

[0056] (1) Protein sample preparation Collect untreated or drug-treated cells, remove the culture medium from the culture dish, wash the cells 2-3 times with pre-cooled PBS, add an appropriate amount of pre-cooled protein lysis buffer (protein lysis buffer to protease inhibitor volume ratio of 1000:1), and incubate on ice for 15 min to lyse the cells. Then transfer the lysate to a 1.5 mL centrifuge tube, centrifuge at 4°C and 15000 rpm for 10 min, discard the precipitate, transfer the supernatant to a new centrifuge tube, add an appropriate amount of 5× SDS sample buffer, adjust the final concentration of the system to 1×, and store at -20°C for later use.

[0057] (2) Making adhesive The target proteins MCT1 and MCT4 have molecular weights of 40 kDa and 42 kDa, respectively. A 10% stacking gel was prepared based on these molecular weights. The cleaned gel plates were assembled and secured. A leak check was performed using pure water. After the leak was checked, excess pure water on the surface of the gel plates was absorbed with filter paper. An appropriate amount of the pre-prepared 10% stacking gel was added, followed by a small amount of isopropanol to smooth the surface. Once the stacking gel solidified, the isopropanol was removed. Then, the upper separating gel was added, and the comb was gently and smoothly inserted. The mixture was allowed to stand until the separating gel solidified.

[0058] (3) Electrophoresis Place the gel plate into the electrophoresis tank, pour in 1× running buffer, slowly remove the comb, and straighten the sample wells with a syringe. Add appropriate amounts of marker and prepared protein samples to the sample wells. Adjust the electrophoresis voltage to 30 V and start electrophoresis, checking for any rising air bubbles. After about 10 minutes, level the protein front to a horizontal line. Then, switch to a constant voltage of 80 V and electrophoresis for about 30 minutes, until the bromophenol blue front enters the separating gel and the marker bands separate. Change the voltage to a constant voltage of 120 V and electrophoresis for 60-90 minutes, stopping when the bromophenol blue front reaches the bottom of the gel.

[0059] (4) Transfer membrane Before electrophoresis is complete, prepare the PVDF membrane, activate it with pre-cooled methanol, and then equilibrate it in 1× Transfer Buffer. Trim the gel, removing excess edges, and stack the sponge, filter paper, gel, and PVDF membrane in sequence, following the direction of "black side for gel, white side for membrane," gently squeezing to remove air bubbles with a gel cutter. Place this stack into the transfer holder (black side facing black, white side facing red), add crushed ice to the ice box of the transfer tank, and pour in pre-cooled Transfer Buffer. Transfer at a constant current of 260 mA for 60–120 min (adjust the time according to the molecular weight of the target protein). During the transfer process, place the apparatus in an ice-water bath at approximately 4°C to avoid overheating and damaging the protein structure.

[0060] (5) Closed After protein transfer, the PVDF membrane was removed, excess parts were cut off and rinsed 3 times with 1 × TBST. The PVDF membrane was then placed in a blocking solution containing 5% skim milk powder and incubated on a shaker at room temperature for 1 h for blocking. After blocking, it was rinsed 3 times again with TBST.

[0061] (6) Incubation of primary antibody Prepare the primary antibody working solutions for MCT1 and MCT4 using 5% skim milk powder according to the dilution ratio recommended in the instructions. Cut the blocked PVDF membrane into strips according to the molecular weight of the target protein, place them in the primary antibody working solution, and incubate overnight on a rotary shaker at 4°C. Then, recover the primary antibody working solution and wash the membrane strips three times with 1 × TBST for 10 min each time.

[0062] (7) Incubation of secondary antibodies The HRP-conjugated secondary antibody was diluted with a 5% skim milk powder solution according to the specified ratio. The membrane was then immersed in the secondary antibody dilution solution and incubated on a shaker at room temperature for 1 h. Subsequently, the secondary antibody dilution solution was recovered, and the membrane was washed three times with 1 × TBST for 10 min each time.

[0063] (8) Development According to the ECL chemiluminescence kit instructions, prepare the luminescence solution by mixing equal parts of solution A and solution B (in a 1:1 ratio). Drop the prepared luminescence solution onto a PVDF membrane and incubate at room temperature for 1-2 minutes. Take a picture using a chemiluminescence analysis system and save the image.

[0064] 6. MTT Experiment The MTT assay can detect cell proliferation capacity and drug cytotoxicity. The principle is as follows: In living cells, mitochondria contain succinate dehydrogenase, which reduces exogenous MTT to water-insoluble blue formazan crystals that are deposited within the cell. Dead cells lack this function. The crystals are then dissolved in dimethyl sulfoxide (DMSO), and the absorbance at 490 nm is measured using a microplate reader to indirectly represent the number of living cells.

[0065] (1) Cell inoculation Logarithmic growth phase cells were collected, digested with trypsin to obtain a cell suspension, and counted. Cell density was adjusted using complete culture medium (typically 5 × 10⁶ cells / year). 4 (cells / mL) Using a pipette, a suitable amount of cell suspension is transferred into each well of a 96-well culture plate, 100 μL per well (approximately 5 × 10⁻⁶ cells / mL). 3 The plate was divided into cells / wells, with zeroing wells (culture medium + cell-free) and control wells (cells + drug-free intervention). Sterile PBS was added to the outer wells of the cell wells to reduce edge effects, and the plate was incubated overnight in an incubator to allow the cells to adhere.

[0066] (2) Drug treatment After the cells have adhered, the test drug is diluted to the required concentration gradient using complete culture medium. The original culture medium in the 96-well plate is aspirated, and fresh culture medium containing different concentrations of the test drug is added to each well (100 μL per well). Multiple replicates are set for each drug concentration, and the plates are placed in an incubator for incubation according to the preset time.

[0067] (3) MTT test After incubation, remove excess drug from the culture plate, add 20 μL of MTT solution (5 mg / mL) to each well, mix gently, and incubate for 4 h. After removing the culture plate, carefully remove the supernatant from each well, being careful not to aspirate the formazan crystals at the bottom. Then add 100 μL of DMSO solution to each well. Place the sample in a shaker and shake slowly for 10 min until the crystals are completely dissolved. Measure the absorbance at 490 nm using a microplate reader.

[0068] 7. Construction of mouse tumor model (1) Selection of mouse strains Human breast cancer cells MCF-7 were used as the seed cells for the mouse tumor model. Female immunodeficient nude mice aged 4 to 6 weeks were selected as the experimental mouse strain. At this age, the immune system of the mice is not yet fully mature, and the tumor formation rate is relatively high.

[0069] (2) Animal preparation Upon arrival, mice should be housed in an SPF-grade barrier facility for 3-5 days to acclimatize and eliminate transport stress. Feed, bedding, and water must be sterilized. After entering the normal mouse housing, mice should be weighed, their weight recorded, and they should be divided into normal, experimental, and control groups based on their weight.

[0070] (3) Preparation of cell suspension Select cells in the logarithmic growth phase, with a density of 80%–90% and in good growth condition. Perform trypsin digestion on the logarithmic phase cells, add culture medium to stop digestion, collect the cell suspension, centrifuge, discard the supernatant, resuspend in PBS, and wash at least twice (to remove trypsin and serum). Then resuspend in serum-free culture medium until the cell concentration reaches 2 × 10⁻⁶ cells / year. 7 The cell suspension was mixed with the matrix gel at a 1:1 ratio on ice until the final cell density was 1 × 10⁻⁶ cells / mL. 7 cells / mL.

[0071] (4) Subcutaneous inoculation of tumor The right axilla subcutaneous tissue was chosen as the inoculation site, as this area has a rich blood supply, a high tumor formation rate, and is easy to operate in. After fixing the mouse's head and back, the skin at the injection site was disinfected 2-3 times with a 75% ethanol cotton ball. If the mouse was difficult to fix, an anesthetic could be administered intraperitoneally to facilitate tumor inoculation. A syringe containing 1 mL of cell suspension (1 × 10⁻⁶) was used. 7 Approximately 100 μL (containing approximately 1 × 10 cells / mL) 6 After expelling the air from the syringe, insert the needle at a 30° angle to the skin, to a depth of approximately 1 cm. Gently move the needle to ensure it remains subcutaneous. Slowly advance the syringe plunger to inject the cell suspension, pause briefly, and then withdraw the syringe to prevent leakage. Return the mouse to its enclosure and mark it. Monitor the mouse's mental state, activity, and feeding status 1-3 days post-injection. Check the injection site for redness, swelling, or ulceration. Replace any unsuitable mice with replacement tumors promptly.

[0072] (5) Tumor growth monitoring When a noticeable mass appears at the injection site in mice between days 3 and 7 after inoculation, the body weight and tumor volume should be measured and recorded every 2 days. The long diameter (a) and short diameter (b) of the tumor should be measured using calipers, and the volume should be calculated as V = 0.5 × a × b². If the tumor volume exceeds the upper limit stipulated by animal ethics, or if ulceration, necrosis, infection, or other conditions occur, the experiment should be terminated.

[0073] (6) Drug therapy Prepare a CHC solution (18 mg / mL) in advance using sterile physiological saline (NaOH solution as a solubilizer).

[55] In the experimental group, 100 μL of drug (100 mg / kg) was injected using a sterile syringe, while the control group received the same dose of physiological saline solution. Air was expelled from the syringe before injection. Treatment was administered via intraperitoneal injection. The skin on the back of the neck of the mouse was grasped, the head was fixed, and the mouse was turned over with its abdomen facing upwards. The injection site was selected near the midline of the lower abdomen. The skin at the injection site was disinfected with a 75% ethanol cotton ball, and the injection was performed after the ethanol evaporated. The needle was inserted subcutaneously at a 30-45° angle to the skin. After insertion, the needle angle was reduced to 10-20°, and the needle was continued to be inserted parallel to the skin for about 0.5 cm until there was no resistance after penetration. The syringe was gently aspirated to observe for any fluid backflow, indicating whether other organs had been punctured. If no backflow was observed, the drug solution was slowly and evenly injected before the needle was withdrawn. If any fluid was aspirated, the needle was immediately withdrawn, the injection site was changed, and the wound was wiped with a cotton ball soaked in iodine. The control group received the same amount of physiological saline solution using a sterile syringe, following the same procedure. The treatment lasted for 21 consecutive days, with one injection of medication per day.

[0074] (7) Sampling of mouse tumors After 21 days of treatment, a significant difference in tumor size was observed between the experimental and control groups. Mice were removed from the SPF-grade barrier facility, and feeding was stopped one day in advance. Dissection instruments such as trays, ophthalmic scissors, and ophthalmic forceps were disinfected with alcohol. Mice were euthanized by cervical dislocation. The subcutaneous skin of the right axilla was wiped with 75% ethanol to prevent contamination. The skin was cut along the edge of the tumor with ophthalmic scissors to separate the tumor from the surrounding connective tissue. The tumor was completely dissected, and adhering fat, necrotic tissue, and blood vessels were removed. The tumor tissue was washed with physiological saline, weighed, and then subjected to subsequent primary culture.

[0075] (8) Sampling of major organs of mice After removing the tumor tissue, the skin and peritoneum were incised along the midline of the mouse abdomen, followed by incision of the diaphragm and splitting of the sternum to fully expose the thoracic and abdominal cavities. The liver, spleen, kidneys, lungs, and heart were removed in sequence. Fat and blood vessels on the surface of each organ were removed using ophthalmic scissors. After being thoroughly rinsed with physiological saline, the organs were stored in 4% paraformaldehyde (PFA) fixative for hematoxylin and eosin (HE) staining analysis.

[0076] 8. Primary culture of mouse tumor cells This method is used to isolate single cells from mouse tumor tissue for subsequent experiments.

[0077] (1) Material selection and cleaning Fresh tumor fragments were detached from mice and washed several times (usually 2-3 times) in PBS buffer containing penicillin and streptomycin to remove blood, cell debris, and collagen from the surface. For larger tumor fragments, large areas of necrotic tissue and the capsule should be removed.

[0078] (2) Tissue block trimming The cleaned tumor tissue was placed in a sterile culture dish and infiltrated with a small amount of culture medium containing serum. The tumor tissue was then processed using surgical scissors or ophthalmic scissors to remove the necrotic parts. The healthy tumor tissue was then cut into small pieces of 1-2 mm³. During the resection process, care was taken to keep the tumor pieces moist to prevent dehydration.

[0079] (3) Tissue block inoculation Using a bent pipette or sterile forceps, evenly place the trimmed tissue blocks at the bottom of the culture dish, maintaining a distance of approximately 0.5 cm between each block to ensure sufficient space for subsequent cell growth. After inoculation, aspirate excess liquid to ensure close contact between the tissue blocks and the culture surface.

[0080] (4) Let it stand still against the wall Place the petri dishes in an incubator at 37°C with 5% CO2 and incubate statically for 2-4 hours. The purpose of static incubation is to allow the tissue blocks to fully adhere to the surface of the petri dish, preventing the tissue blocks from floating when culture medium is added later.

[0081] (5) Add culture medium Once the tissue block has firmly adhered to the bottom of the dish, add an appropriate volume of complete culture medium containing 10% to 20% FBS. When adding the medium, pour it slowly along the inner wall of the dish to avoid liquid impact that could cause the tissue block to detach. Then place the dish in an incubator for further culture.

[0082] (6) Observation and fluid replacement During the initial stage of culture, the tissue should be kept as still as possible to avoid movement that could affect cell attachment. After 24 to 48 hours of culture, use an inverted microscope to examine the edges of the tissue pieces for signs of cell migration. Cells can usually be observed to emerge from the periphery of the tissue piece within 3 to 7 days. Based on changes in the color of the culture medium and cell growth, replace half of the culture medium every 2 to 3 days.

[0083] (7) Cell passage When the primary cells have grown to 50% to 80% of the bottom area of ​​the dish, perform the first passage, discard the original culture medium, add trypsin solution to remove the cells from the culture dish, collect the cell suspension and dilute it with an appropriate multiple, and then inoculate it into new culture dishes.

[0084] III. Results and Discussion 1. Morphological characterization of lactic acid nanosensors To achieve in-situ, real-time detection of lactate in single living cells while avoiding fatal cell damage, this study employed flame etching to prepare CFNE (carbon fiber nanoparticles). The electrode tip diameter was controlled within 100–200 nm, exhibiting excellent single-cell puncture capability and biocompatibility. The construction strategy of the lactate nanosensor is shown in Figure 1. First, Pt NPs were modified onto the CFNE surface via electrochemical deposition, increasing the specific surface area and thus enhancing electrocatalytic activity, significantly improving the electrode's detection sensitivity. Subsequently, LDH was covalently immobilized on the electrode surface using PEI as a crosslinking agent with the assistance of PEGDE. Based on the specific catalytic reaction between the enzyme and the substrate, this sensor exhibits highly selective recognition of lactate. Thanks to its nanoscale tip size, the constructed lactate nanosensor enables minimally invasive puncture of single living cells, ultimately achieving real-time, in-situ, and quantitative electrochemical monitoring of intracellular lactate molecules.

[0085] This study used scanning electron microscopy (SEM) to characterize the morphology of four progressively modified CFNEs: bare CFNE, platinum-deposited CFNE, PEI-modified CFNE, and LDH-fixed CFNE. The results showed that the surface of bare CFNE was smooth and flat. Figure 2 In section A), the electrode tip diameter is approximately 100-200 nm, meeting the requirements for single-cell detection. After electrochemical deposition of Pt NPs, the CFNE surface is uniformly covered by dense, continuous nanoparticles, forming a catalytically active layer with a high specific surface area. Figure 2 (B) After PEI modification, a uniform organic film is visible on the electrode surface, indicating that PEI has successfully adhered to the electrode surface. Figure 2 After immobilizing LDH (C in the figure), a thick and uniform biofilm layer appeared on the electrode surface, confirming that LDH had been effectively coated and immobilized. Figure 2(D in the middle).

[0086] Furthermore, energy dispersive x-ray spectroscopy (EDX) analysis also verified the uniform dispersion of each element on the lactic acid nanosensor. Figure 3 This confirms that the modified layer is uniformly loaded.

[0087] To further verify the lactic acid detection performance of the constructed lactic acid nanosensor, the electrodes at different modification stages were characterized using electrochemical methods. CFNEs modified with different functional layers were placed in K3[Fe(CN)6] solution for detection. The results showed that the electrochemical signal gradually decreased with the stepwise assembly of the sensor, confirming the successful construction of the lactic acid nanosensor. Figure 4 (A) The response behavior of the electrode in 5 mM lactic acid, 5 mM H2O2 and PBS solutions was investigated using cyclic voltammetry (CV). The results are as follows: Figure 4 As shown in Figure B, at a working potential of +0.6 V, the sensor exhibits a significant current response to lactic acid solution but almost no current response to H₂O₂. Furthermore, no obvious signal is observed in the PBS blank solution, indicating good specificity for lactic acid recognition. Parallel tests were performed on four independently prepared lactic acid nanosensors under the same lactic acid concentration conditions, and the electrochemical response signals showed high consistency. Figure 4 The relative standard deviation (RSD) was calculated to be 2.1%, indicating that the prepared lactic acid nanosensor has good reproducibility. These results demonstrate that the lactic acid nanosensor constructed in this study possesses excellent electrochemical response, sensitivity, and repeatability.

[0088] To further improve the electrode sensing performance, this study systematically optimized the preparation conditions of the surface modification layer for the nanosensor. First, the effect of PEI concentration on the modification effect was analyzed. Under the condition that other factors remained the same, 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), and 0.2% (w / v) PEI solutions were used to modify CFNEs after Pt NPs deposition. The results are shown in Figure 5A. The electrode modified with 0.1% (w / v) PEI exhibited the best electrochemical response. Excessive PEI concentration easily leads to the formation of an overly thick and uneven film, increasing the electrode charge transfer resistance and thus weakening the current response. Conversely, excessively low concentration results in a thin film, exposing the substrate and providing insufficient amino binding sites, thereby reducing the efficiency of subsequent enzyme loading. Based on this, the LDH fixation concentration was further optimized, with enzyme solutions of 500 U / mL, 800 U / mL, 1000 U / mL, and 1200 U / mL used for modification. Figure 5 As shown in B, when modified with 1000 U / mL enzyme solution, the lactate nanosensor can obtain the optimal electrochemical signal. Further increasing the enzyme concentration will not increase the current response. This is because excessive non-conductive enzyme membrane will hinder the electron transfer between the electrode and the electrolyte, resulting in a decrease in sensing performance.

[0089] 2. Performance verification of lactic acid nanosensors This study further investigated the quantitative detection performance of lactic acid nanosensors. First, lactic acid standard solutions with concentrations of 0, 5, 10, 20, 30, 40, 60, and 80 mM were prepared, and electrochemical detection was performed using cyclic voltammetry. Figure 6 (A) The results showed that as the concentration of lactic acid solution gradually increased, the electrochemical response signal of the lactic acid nanosensor showed a gradient upward trend; at the same time, the oxidation peak potential remained stable at around +0.6 V without significant shift, confirming that the sensing electrode has excellent detection sensitivity and stable electrochemical response characteristics.

[0090] Under a constant potential of +0.6 V, quantitative detection of lactic acid solutions at gradients of 5, 10, 20, 35, 50, and 70 mM was performed using chronoamperometry (it). Figure 6 (B in the original text). Different concentrations of lactic acid elicited significant and regular current responses from the nanosensor, further validating its high sensitivity for lactic acid detection. A current-concentration standard calibration curve was constructed by linearly fitting the detected current response values ​​to the corresponding lactic acid concentrations. Figure 6 (C in the equation), the two show a good linear correlation, and R is calculated to be... 2 = 0.99, which meets the needs of accurate quantitative detection of lactic acid.

[0091] Furthermore, this study systematically evaluated the specificity and storage stability of the lactate nanosensor. Common intracellular endogenous interfering agents, including glucose (Glc), reduced glutathione (GSH), lysine (Lys), L-arginine (L-Arg), ascorbic acid (AA), and hydrogen peroxide (H2O2), were selected for anti-interference verification experiments. Figure 6 As shown in D, the electrochemical response signal of the sensor to lactic acid is significantly stronger than that of various interfering substances, indicating that the sensor has excellent specific recognition ability for lactic acid and good anti-interference performance.

[0092] Three lactic acid nanosensors with uniform performance were prepared and stored in a sealed container at 4°C. Their electrochemical response to lactic acid was monitored for 7 consecutive days. Figure 6 (E in the figure). The results show that the lactic acid detection performance of the sensor did not significantly decrease after 7 days of low-temperature storage, confirming that the lactic acid nanoelectrochemical sensor has good storage stability and application reliability.

[0093] 3. Verification of the biocompatibility of lactic acid nanosensors with cells Given the excellent sensing performance of the prepared lactic acid nanosensor, this study further verified its biocompatibility. The lactic acid nanosensor was inserted into cells and then slowly withdrawn. Using a real-time in-situ single-cell analysis system, cells stained with both Calcium-AM and PI fluorescence were observed, and dynamic changes in the number of fluorescent photons in single cells were simultaneously acquired. The differences in cell activity before and after electrode insertion were compared by statistically analyzing the fluorescence signals. Calcium-AM contains a lipophilic methyl acetate group, allowing it to penetrate intact cell membranes and enter living cells. After hydrolysis by intracellular esterases, it emits green fluorescence. PI is a nuclear-specific fluorescent dye that binds to nuclear DNA to produce red fluorescence, but it can only penetrate damaged cell membranes and cannot enter normal living cells. Therefore, it is often used in combination with Calcium-AM to achieve dual-color qualitative assessment of cell activity.

[0094] Depend on Figure 7 As shown in section A, the cells consistently exhibited green fluorescence before and after insertion of the lactate nanosensor, and no red fluorescence was observed throughout the process, confirming that the intracellular puncture operation of the lactate nanosensor did not significantly affect cell viability. Subsequently, the feasibility of using this nanosensor for single-cell electrochemical analysis was systematically evaluated. Ru(NH3)6 3+ As a hydrophilic probe molecule, it cannot penetrate intact living cell membranes; therefore, 1 mM Ru(NH3)6 was selected for the experiment. 3+The solution was used as a cell bath fluid, and the intracellular implantation status of the lactic acid nanosensor was determined by detecting the electrochemical response of the nanosensor to this solution. Figure 7 As shown in B, curve 1 represents the sensor in Ru(NH3)6 before electrode insertion into the cell. 3+ The baseline current response in solution, curves 2 and 3 correspond to the electrochemical signals when the electrode is inserted into the cell at approximately 50% and 80% depths, respectively. It can be seen that Ru(NH3)6... 3+ The reduction current gradually decreases with increasing cell penetration depth, and when the electrode is completely inside the cell, the reduction current almost disappears completely (curve 4). These results indicate that the lactic acid nanosensor possesses excellent electrochemical and mechanical properties, and can stably and accurately perform intracellular detection of single living cells.

[0095] Utilizing an in-situ single-cell analysis platform, micro / nano fiber optic probes are positioned in the near-field of single cells. Laser light is transmitted via fiber to the probe tip to capture single-cell fluorescence signals. A portion of the fluorescence signal is backcoupled back into the fiber and transmitted to a high-sensitivity photodetector for photon counting and signal analysis. In the fluorescence photon counting results, five cells were randomly selected for independent puncture; no significant attenuation was observed in the total fluorescence photon count across all groups. Figure 7 (C in the text); and the fluorescence photon signal of a single cell remained stable without significant decrease during a 20-second real-time monitoring period (C in the text). Figure 7 (D in the original text). In summary, this confirms that the lactate nanosensor does not cause significant cell damage during puncture and in-situ detection. Given the excellent performance of the lactate nanosensor, it will be applied to single-cell intracellular electrochemical detection in the next step. 4. Electrochemical detection of lactate in single cells of different cell lines Compared to normal cells, tumor cells preferentially convert glucose into lactate even in aerobic environments, exhibiting a Warburg effect that produces large amounts of lactate. This results in significantly higher lactate concentrations within tumor cells compared to normal cells. Five cell lines were selected for experiments: three human breast cancer cells (MCF-7 and MDA-MB-231), mouse colon cancer cells (CT26), and two normal human epithelial cells (MCF-10A and RPE1). Real-time dynamic monitoring of intracellular lactate levels in single cells was performed using an immunoassay (IT) method. This method is simple to operate and reacts rapidly, indirectly reflecting differences in intracellular lactate content among different cells through differences in the detected electrical signals.

[0096] The study randomly selected 30 single cells from each cell line for electrochemical lactate detection and analysis. Figure 8 It can be seen that, specifically, the normal cell line is human normal epithelial cell MCF-10A ( Figure 8 E in the middle), RPE1 ( Figure 8Compared to B in the text, the tumor cell line specifically refers to human breast cancer cells MCF-7 ( Figure 8 C in the middle), MDA-MB-231 ( Figure 8 D), mouse colon cancer cells CT26 ( Figure 8 The electrochemical signal responses measured in the A-cell line were generally higher, indicating a significant difference in lactate metabolism levels between tumor cell lines and normal cell lines.

[0097] By combining the concentration fitting curve, the detected electrochemical signal can be further converted into intracellular lactate concentration, thus intuitively reflecting the differences in single-cell lactate content in different cell lines. The results show that the normal cell line specifically refers to human normal epithelial cells MCF-10A (… Figure 9 E in the middle), RPE1 ( Figure 9 B) and tumor cell lines specifically human breast cancer cells MCF-7 ( Figure 9 C in the middle), MDA-MB-231 ( Figure 9 D), mouse colon cancer cells CT26 ( Figure 9 There were significant differences in lactate concentration among cells in group A). ​​Furthermore, compared to normal cells, the differences in lactate concentration among individual cells within the tumor cell population were even more pronounced.

[0098] Statistical analysis of single-cell electrochemical detection signals ( Figure 10 (A in the standard curve), and convert the electrical signal into the corresponding intracellular lactate concentration based on the standard curve (A in the standard curve). Figure 10 (B in the original text). The results showed that the intracellular lactate concentration in tumor cells was mainly concentrated between 10 and 30 mM, while the intracellular lactate concentration in normal cells was below 5 mM. This confirmed that the lactate accumulation level in tumor cells could reach 2 to 10 times that of normal cells, fully demonstrating that tumor cells produce and accumulate large amounts of lactate through glycolysis via the unique Warburg effect, while normal cells generate only trace amounts of lactate through OXPHOS energy supply. Lactic acid concentration distribution heatmap results ( Figure 10 As shown in C), tumor cells not only have a higher overall lactate content, but also exhibit more significant fluctuations in concentration among individual cells, suggesting that tumor cells possess unique lactate metabolism characteristics and exhibit cellular heterogeneity. This heterogeneous phenotype is speculated to be closely related not only to differences in cellular glycolytic metabolic activity but may also be regulated by multiple intrinsic factors, including cellular genetic background.

[0099] In addition, the expression of MCT1 and MCT4, key lactate transport proteins, in five cell lines was detected by Western blotting. Figure 10In normal cells, the protein expression levels of MCT1 and MCT4 are relatively low, while tumor cells generally upregulate the expression of MCT proteins to adapt to the metabolic demands of high-intensity glycolysis. MCF-7 cells express both MCT1 and MCT4, possessing both lactate uptake and efflux capabilities. They can both take up lactate for OXPHOS and mediate the efflux of lactate produced by glycolysis to avoid cellular acidosis. In contrast, MDA-MB-231 cells show predominantly high expression of MCT4 and almost no expression of MCT1, indicating that MDA-MB-231 cells are highly glycolytic cells. They rely on MCT4 to efflux large amounts of lactate produced by glycolysis, thus avoiding the toxic damage caused by high concentrations of lactate accumulation and remodeling the tumor microenvironment (TME) through lactate secretion, promoting tumor proliferation and invasion. This may also explain the high invasiveness of MDA-MB-231 cells.

[0100] 5. Investigate the effects of lactate metabolism drugs on lactate metabolism in single cells. This study first investigated the effects of two lactate metabolism inhibitors, CHC (MCTs inhibitor) and GNE-140 (LDH inhibitor), on the proliferation of MCF-7 and MDA-MB-231 tumor cells. The MTT assay was used to verify the inhibitory effects of the two drugs. Cells were cultured in 96-well plates. CHC concentrations of 0, 1, 2, 10, and 20 mM were applied to MCF-7 and MDA-MB-231 cells, respectively, for incubation times of 6, 12, 24, and 48 h. After incubation, the absorbance (OD value) of each well was measured at 490 nm using a microplate reader. After subtracting the background OD value from blank wells, the data were normalized. The results showed that low concentrations or short incubation times did not significantly inhibit tumor cell proliferation. However, after treatment with 10 mM CHC for 24 h, the inhibitory effect on MCF-7 cells (MCF-7 and MDA-MB-231) was significantly reduced. Figure 11 The activity of A in MDA-MB-231 cells decreased to about 50%. Figure 11 The activity of B) in the drug decreased to about 40%, so this drug concentration and incubation time were selected as the conditions for subsequent experiments.

[0101] Simultaneously, the inhibitory effect of GNE-140 was assessed using the same MTT assay, with drug concentrations set at 0, 10, 30, 50, and 100 μM, and incubation times at 6, 12, 24, and 48 h. The results showed that, compared to CHC, GNE-140 did not cause a significant decrease in tumor cell viability, but still significantly inhibited cell proliferation; after treatment with 50 μM GNE-140 for 24 h, MCF-7 (… Figure 11 C in the middle) and MDA-MB-231 ( Figure 11 The viability of D) cells decreased to about 70%, exhibiting a mild and stable inhibitory effect on proliferation.

[0102] After determining the drug concentration and duration of action, we further conducted single-cell electrochemical detection of lactate in cells treated with CHC and GNE-140 to explore the regulation of intracellular lactate content changes in tumor cells and normal cells by lactate metabolism-related inhibitors. In this experiment, we selected the following preliminary screening conditions: 10 mM CHC treatment for 24 h and 50 μM GNE-140 treatment for 24 h. Breast cancer cells MCF-7 and MDA-MB-231, and normal breast epithelial cells MCF-10A were used as research subjects. Single-cell electrochemical signal detection was performed after drug incubation.

[0103] Depend on Figure 12 The results showed that, compared with the blank control group, after CHC intervention, MCF-7 ( Figure 12 A in the middle) and MDA-MB-231 ( Figure 12 In both types of tumor cells (B) and GNE-140, intracellular electrochemical signals were significantly upregulated, while GNE-140 treatment significantly reduced intracellular electrochemical response signals in tumor cells. These results confirm that both lactate metabolism inhibitors can effectively intervene in the lactate metabolism level of tumor cells. Conversely, normal mammary epithelial cells MCF-10A (… Figure 12 In the case of C), after intervention with the two drugs, no significant difference was observed in intracellular electrochemical signals, indicating that CHC and GNE-140 have almost no regulatory effect on lactic acid metabolism in normal breast cells and have good specificity for tumor cell intervention.

[0104] Further statistical analysis was performed on the single-cell electrochemical detection signals, and the electrochemical response signals were converted into corresponding intracellular lactate concentrations using a standard curve. The results showed that after CHC intervention, the intracellular lactate concentrations of both tumor cell types were significantly upregulated, and compared to MCF-7 cells (… Figure 13 D in MDA-MB-231 Figure 13 The changes in intracellular lactate content in single cells of type E were more pronounced, and this difference is presumably related to the difference in protein expression levels of MCTs in the two cell types. Meanwhile, GNE-140 treatment significantly reduced intracellular lactate concentration in both types of tumor cells, confirming that this LDH inhibitor can effectively block lactate production in tumor cells, thereby interfering with the metabolism and proliferation of tumor cells.

[0105] For normal mammary epithelial cells MCF-10A ( Figure 13 C in the middle), compared to MCF-7 cells ( Figure 13 (A) and MDA-MB-231 ( Figure 13 In the B section, there was no significant difference in electrochemical signals before and after drug intervention, indicating that the two inhibitors have no effect on normal cellular lactate metabolism ( Figure 13The F in the sample has almost no effect, a characteristic that may be related to the physiological characteristic that normal cells hardly synthesize lactate through the Warburg effect.

[0106] 6. Investigate the effect of changes in lactate concentration on lactate metabolism in tumor cells. To further investigate the effects of changes in intracellular lactate concentration on lactate metabolism in tumor cells, this study treated MCF-7 breast cancer cells with exogenous lactate and the LDHA-specific inhibitor GSK2837808A, respectively. Exogenous lactate and GSK2837808A can more directly affect the intracellular lactate concentration. By artificially upregulating or downregulating intracellular lactate content, we explored the regulation of lactate metabolism in tumor cells by changes in lactate concentration gradient.

[0107] This study first focused on the effects of exogenous lactic acid, using the MTT assay to verify the influence of different concentrations of lactic acid solution on the proliferation of MCF-7 cells. Given that the stock lactic acid solution is acidic, excessively low pH values ​​can cause non-specific cytotoxicity and interfere with the reliability of experimental results. Therefore, during reagent preparation, the pH of each gradient lactic acid solution was pre-adjusted to neutral using NaOH solution to match the physiological environment of cell culture. After adjustment, the solutions were filtered through a sterile membrane to ensure the sterility and stability of the drug delivery system and to avoid experimental errors caused by impurities and microbial contamination. The experiment set lactic acid concentration gradients of 0, 10, 40, 60, 80, and 100 mM, and MCF-7 cells were subjected to gradient intervention for 6, 12, 24, and 48 h, respectively. Figure 14 The results in section A show that as the concentration of exogenous lactic acid increases and the incubation time is prolonged, the proliferation capacity of MCF-7 cells shows a continuous downward trend, confirming that high concentrations of exogenous lactic acid can inhibit the proliferative activity of tumor cells.

[0108] Based on the above proliferation screening results, this study ultimately selected three gradient lactate concentrations of 40, 60, and 80 mM to uniformly treat MCF-7 cells for 24 h. Electrochemical analysis was then performed on single cells after drug incubation to analyze changes in intracellular lactate levels. From 0 mM ( Figure 14 B in), 40 mM ( Figure 14 (C), 60 mM ( Figure 14 D in the middle) and 80 mM ( Figure 14 The electrochemical response of single-cell lactate after treatment with E concentration lactate showed that, compared with the blank control group, the electrochemical response signal of the lactate intervention group was significantly enhanced, and the signal intensity increased stepwise with the increase of exogenous lactate concentration, which directly confirms that exogenous lactate can be effectively taken up by tumor cells, thereby increasing the intracellular lactate enrichment level.

[0109] Statistical analysis was performed on the electrochemical detection signals of each group. Figure 14The results further clarified that exogenous lactate intervention can directly affect lactate metabolism in MCF-7 cells, and the higher the concentration of exogenous lactate, the more significant the regulatory effect on cellular lactate metabolism.

[0110] To further investigate the effect of single-cell lactate concentration on tumor lactate metabolism, intracellular lactate concentration was reduced using an LDHA inhibitor. The inhibitory effect of the LDHA inhibitor GSK2837808A on MCF-7 cell proliferation was verified using an MTT assay. GSK2837808A concentration gradients of 0, 5, 15, 30, and 60 μM were used to treat MCF-7 cells for 6, 12, 24, and 48 h, respectively. Figure 15 As shown in A, with increasing drug concentration and prolonged intervention time, the proliferation activity of MCF-7 cells showed a gradient decreasing trend, and tumor growth was significantly inhibited, but no large-scale cell death occurred, indicating that the drug can gradually inhibit tumor cell proliferation.

[0111] Based on the above proliferation screening results, this study ultimately determined 10, 30, and 50 μM as the subsequent intervention concentrations. MCF-7 cells were uniformly cultured at a constant temperature for 24 h after drug administration. Subsequently, a lactate nanosensor was used to perform in-situ lactate detection in single cells, analyzing the changes in intracellular lactate levels before and after drug intervention. From 0 mM ( Figure 15 B in), 10 mM ( Figure 15 (C), 30 mM ( Figure 15 D in the middle) and 50 mM ( Figure 15 The electrochemical response of single-cell lactate after treatment with GSK2837808A at concentration E (E) showed that, compared with the blank control group, the electrochemical response signal of single cells in the GSK2837808A-treated group was significantly decreased, and the inhibitory effect was significantly concentration-dependent, with the electrochemical signal response continuously decreasing with increasing concentration. Statistical analysis was performed on the electrochemical detection signals of cells in each group (…). Figure 15 The results showed that GSK2837808A can directly regulate the lactate metabolism pathway in MCF-7 cells, and the higher the drug concentration, the more significant the inhibitory effect on cellular lactate production.

[0112] Based on the previously established standard fitting curve for lactate concentration, the response signal obtained from single-cell electrochemical detection was converted into a quantitative intracellular lactate concentration. Quantitative analysis results showed that the intracellular lactate concentration in MCF-7 single cells of the exogenous lactate intervention group increased significantly in a concentration-dependent manner, confirming that exogenous lactate can effectively cross the membrane into the cell, disrupting intracellular lactate homeostasis and promoting its accumulation. Figure 16 A in the middle). Based on the heatmap results of single-cell lactate concentration detection ( Figure 16Figure B in the figure further reveals the heterogeneity and distribution patterns of lactate metabolism at the single-cell level in different treatment groups. Among them, the lactate concentration distribution in single cells of the lactate intervention group showed a significant gradient difference; the higher the added lactate concentration, the more dispersed the color in the heatmap, indicating that the addition of exogenous lactate not only increased the overall lactate level of the cells but also exacerbated the heterogeneity of lactate metabolism among single cells. It is speculated that this may be related to differences in lactate transport capacity and metabolic enzyme activity among different single cells, leading to a discrete distribution of lactate concentration at the single-cell level.

[0113] Conversely, the intracellular lactate concentration in single cells of the GSK2837808A-treated group was significantly lower than that in the blank control group. Figure 16 The presence of C in the figure further confirms that GSK2837808A has a significant inhibitory effect on lactate production in MCF-7 cells. The thermogram of the GSK2837808A-treated group showed significantly lower overall color brightness than the blank control group, and the color of the thermogram gradually became more uniform with increasing drug concentration, indicating that GSK2837808A, while inhibiting intracellular lactate production, can also reduce the heterogeneity of lactate metabolism among single cells. Figure 16 (D in the text). This may be because inhibiting the activity of enzymes related to lactate synthesis synchronously suppresses the lactate production levels of different single cells, reducing fluctuations in lactate concentration caused by differences in metabolism within single cells, thereby making the lactate distribution of the entire cell population more uniform.

[0114] In summary, the quantitative concentration data and heatmap visualization results fully demonstrate the regulatory effect of exogenous lactate on intracellular lactate concentration and the effective inhibitory effect of GSK2837808A on lactate production and metabolic heterogeneity in MCF-7 cells, providing a solid experimental basis for further research on the role of lactate metabolism regulation in tumor treatment.

[0115] 7. Application of lactic acid nanosensors in mouse tumor models To further verify the feasibility of the constructed lactic acid nanosensor in actual sample detection, this study constructed a mouse tumor model and detected the lactic acid content in tumor tissue to evaluate the tumor treatment effect of the MCTs inhibitor CHC.

[0116] This experiment strictly adhered to relevant ethical guidelines and was approved by the institutional review committee and the institution's animal care and use committee. Four- to six-week-old female immunodeficient nude mice were used in the experiment and housed in a specific pathogen-free (SPF) environment. The mice had free access to food and water, and the ambient temperature was controlled at 25 ± 2°C. The mouse tumor model construction process is as follows: Figure 17 As shown, the experimental mice were divided into a control group and an experimental group, and each group of mice was subcutaneously inoculated with 1×10⁻⁶ mg / L. 6MCF-7 cells were collected, and when the tumor volume reached 50-100 mm³, the experimental group mice were injected with CHC and treated continuously for 21 days, while the control group mice were injected with an equal volume of physiological saline as a blank control. Three additional mice were selected as the blank control group.

[0117] Experimental results are as follows Figure 18 Comparison of mouse tumor samples in A and Figure 18 The tumor weight statistics of mice in group B show that after 21 days of CHC intervention, the tumor volume of mice in the experimental group was significantly smaller than that in the control group, indicating that the MCTs inhibitor CHC can effectively inhibit the growth of MCF-7 tumors. To evaluate the biosafety of CHC, this study continuously monitored and statistically analyzed the body weight of mice in the three groups. Figure 18 As shown in Figure C, the weight change trends of mice in the experimental group and the control group were basically the same as those in the normal group, with no significant statistical difference. Furthermore, no abnormal conditions such as significant weight loss or lethargy were observed, indicating that CHC drugs have good biocompatibility and safety and will not cause significant toxic damage to mice, providing safety support for their potential clinical application.

[0118] Meanwhile, after MCF-7 cells were subcutaneously inoculated to form tumors, the tumor volume of mice in the experimental and control groups was dynamically recorded and statistically analyzed regularly. Figure 18 (D in the text). The results showed that the tumor volume in the control group mice continued to increase over time, while the tumor growth rate in the experimental group mice was significantly inhibited after treatment with CHC, and the growth curve tended to flatten. At the end of 21 days of treatment, the tumor volume in the experimental group mice was much lower than that in the control group. These results further corroborate that the MCTs inhibitor CHC can effectively inhibit tumor cell proliferation by regulating the lactate metabolism pathway, thus exerting a reliable tumor treatment effect, which is consistent with the experimental conclusions at the cellular level mentioned above.

[0119] After the treatment cycle, the experimental mice were dissected, and tumor tissues, as well as major organs such as the heart, liver, spleen, lungs, and kidneys, were collected from each group for subsequent safety assessment and primary cell culture. For the tumor tissues, primary cells were isolated and cultured from the tumor tissues of both the control and experimental groups using the tissue block culture method. The lactate nanosensor constructed in this study was used to perform in-situ electrochemical detection of lactate content within primary tumor single cells, verifying the sensor's feasibility in detecting lactate in real biological samples.

[0120] Electrochemical detection results, such as the electrochemical signal of lactate in single-cell primary tumor cells ( Figure 19 In A) and the statistical analysis of electrochemical signals in primary tumor cells ( Figure 19As shown in B), compared with the control group mouse primary tumor cells, the experimental group mouse primary tumor cells treated with CHC showed a significantly increased electrochemical response signal for lactate detection. Based on the previously established standard fitting curve for lactate concentration, the detected electrochemical signal was converted into the corresponding intracellular lactate concentration (CLC concentration). Figure 19 The results showed that the intracellular lactate concentration in the primary tumor cells of the experimental group was significantly higher than that in the control group, confirming that CHC, as an inhibitor of MCTs, can effectively block the efflux of lactate in tumor cells, leading to abnormal accumulation of lactate in the cells, which in turn causes intracellular acidosis, disrupts the metabolic homeostasis and proliferation capacity of tumor cells, and ultimately inhibits tumor cell proliferation and induces their death. This result is consistent with the experimental results at the cellular level mentioned above.

[0121] Finally, HE staining was used to further verify the tumor-killing effect and biosafety of CHC. HE staining was performed on major organs of mice, including the heart, liver, spleen, lungs, and kidneys. The staining results showed that no obvious pathological damage or abnormal changes were found in the experimental group mice. Figure 20 (A) Meanwhile, HE staining of mouse tumors revealed significant cell death in the tumor tissues of mice treated with CHC, while no significant changes were observed in the tumor tissues of the control group. Figure 20 (B in the image) visually demonstrates the killing effect of CHC on tumor cells.

[0122] In summary, this study validated the tumor therapeutic efficacy and biosafety of CHC using a mouse tumor model. Furthermore, the constructed lactate nanosensor successfully enabled precise detection of lactate concentration in primary mouse tumor cells, with stable and reliable results. This demonstrates that the lactate nanosensor can be used for lactate detection in real biological samples, providing an efficient and accurate detection tool for tumor lactate metabolism research and targeted therapy efficacy evaluation.

[0123] This study addresses the lack of single-cell lactate detection technology in tumor metabolism research by constructing an enzyme nanoelectrode (LDH / Pt / CFNE) for electrochemical detection of lactate in single cells. This sensing electrode features high sensitivity, good selectivity, and excellent stability, enabling real-time, in-situ quantitative detection of lactate in single tumor cells. It has been applied to tumor metabolism research, drug evaluation, and validation of mouse tumor models.

[0124] A constructed LDH / Pt / CFNE sensor was used to quantitatively detect lactate at the single-cell level in various normal and tumor cell types. Results showed that the intracellular lactate concentration in tumor cells was significantly higher than that in normal cells. Further analysis revealed significant heterogeneity in single-cell lactate levels within the tumor cell population. Regarding drug evaluation, the dynamic effects of LDH inhibitors, MCT inhibitors, and exogenous lactate on single-cell lactate levels were investigated. Results showed that LDH inhibitors reduced intracellular lactate in a concentration-dependent manner, while MCT inhibitors inhibited lactate efflux, leading to lactate accumulation. These results provide single-cell-level evidence for precision treatment targeting lactate metabolism. Finally, the constructed LDH / Pt / CFNE sensor was used for actual sample detection and application. In mouse tumor model validation, it was able to distinguish between the normal and experimental groups, verifying the feasibility of the sensor's application.

[0125] In summary, this study successfully constructed a nanoelectrochemical sensor for detecting lactate in single cells, enabling real-time, in-situ quantitative analysis of intracellular lactate. This sensor effectively distinguishes metabolic differences between normal and tumor cells, assesses the efficacy of drugs targeting lactate metabolism, and validates its in vivo application potential in a mouse tumor model. This research provides a new analytical tool for tumor metabolism research, drug screening, and precision treatment evaluation.

[0126] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An electrochemical nanosensor for detecting lactate in single tumor cells, characterized in that, It includes a working electrode and a platinum nanoparticle layer, a polyethyleneimine layer, and an enzyme-modified layer sequentially modified on its surface; The enzyme-modified layer includes lactate dehydrogenase, and the lactate dehydrogenase can recognize and detect lactate in tumor single cells.

2. The electrochemical nanosensor for detecting lactate in single tumor cells as described in claim 1, characterized in that, The enzyme-modified layer is formed by solidifying a mixed enzyme solution containing lactate dehydrogenase, a cross-linking agent, and serum albumin; The crosslinking agent includes polyethylene glycol diglycidyl ether, wherein the epoxy groups in the polyethylene glycol diglycidyl ether are covalently bonded to the amino groups in the polyethyleneimine layer and the amino groups in lactate dehydrogenase, respectively. The serum albumin includes bovine serum albumin.

3. The electrochemical nanosensor for detecting lactate in tumor single cells as described in claim 1, characterized in that, The working electrode is a carbon fiber nanoelectrode; The carbon fiber nanoelectrode has needle-shaped nano-tip with a diameter of 100-200 nm.

4. A method for preparing an electrochemical nanosensor for detecting lactate in tumor single cells according to any one of claims 1-3, characterized in that, Includes the following steps: Using carbon fiber nanoelectrodes as working electrodes, platinum nanoparticles were loaded onto their surfaces by electrochemical deposition to prepare carbon fiber nanoelectrodes modified with platinum nanoparticle layers. The carbon fiber nanoelectrode modified with platinum nanoparticles was immersed in a polyethyleneimine solution, and after being taken out, dried and cleaned, a polyethyleneimine layer was formed on the electrode surface. A mixed enzyme solution containing lactate dehydrogenase, a cross-linking agent, and serum albumin is added dropwise to an electrode with a polyethyleneimine layer, and then dried and cross-linked to obtain the final product.

5. The method for preparing the electrochemical nanosensor for detecting lactate in single tumor cells as described in claim 4, characterized in that, The electrochemical deposition includes CV pulse electrodeposition; The CV pulse electrodeposition is a three-electrode system consisting of a carbon fiber nanoelectrode as the working electrode, a platinum sheet as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The three-electrode system was placed in a chloroplatinic acid deposition solution with a concentration of 0.5-1.0 mM, and cyclic voltammetric pulse electrodeposition was performed at a scan rate of 0.08 V / s to 0.12 V / s within a voltage window of -0.5 V to 0.0 V. The mass-volume concentration of the polyethyleneimine solution is 0.01-0.2%.

6. The method for preparing the electrochemical nanosensor for detecting lactate in tumor single cells as described in claim 4, characterized in that, The enzyme activity of lactate dehydrogenase in the mixed enzyme solution is 500-1200 U / mL; The mass ratio of the cross-linking agent to serum albumin is (8-12):1; The crosslinking agent includes polyethylene glycol diglycidyl ether; The serum albumin includes bovine serum albumin.

7. The method for preparing the electrochemical nanosensor for detecting lactate in single tumor cells as described in claim 4, characterized in that, The method for preparing the carbon fiber nanoelectrode is as follows: Carbon fiber is connected to copper wire, cured, cleaned and dried to obtain carbon fiber lead body; The carbon fiber lead is inserted into the tip capillary, and the length of the carbon fiber protruding from the tip capillary tip is controlled to be 2~5 μm; then epoxy resin is injected into the tail end of the electrode for sealing and fixation. By flame etching the exposed tip of the carbon fiber, a needle-like nanoscale tip is formed at the front end of the carbon fiber, thus obtaining the carbon fiber nanoelectrode.

8. The application of an electrochemical nanosensor for detecting lactate in tumor single cells as described in any one of claims 1-3 in constructing a model for detecting biomarkers in tumor single cells.

9. The application as described in claim 8, characterized in that, The biomarker is lactic acid; the method for constructing a biomarker model for detecting lactic acid in tumor single cells is as follows: an electrochemical nanosensor for detecting lactic acid in tumor single cells is inserted into the tumor single cells, and the lactic acid content is determined by detecting the response electrical signal.

10. The application of an electrochemical nanosensor for detecting lactate in tumor single cells as described in any one of claims 1-3 in the preparation of diagnostic and targeted tumor therapy products, characterized in that, Oncology includes breast cancer, colorectal cancer, lung cancer, pancreatic cancer, and glioma; products include drugs and test kits.

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