A ratio fluorescent sensor for high spatial resolution detection of reduced glutathione and preparation method and application thereof

Abstract

This invention relates to the field of ratiometric fluorescence sensor fabrication technology. More specifically, it relates to a ratiometric fluorescence sensor for high spatial resolution detection of reduced glutathione, its fabrication method, and its application. The ratiometric fluorescence sensor comprises: silicon nanowires modified with an azide-based silane reagent to improve spatial resolution and provide one-dimensional axial resolution; a response unit A for responding to the content of reduced glutathione; and a reference unit B; wherein the structure of the response unit A is shown in Formula I; and the structure of the reference unit B is shown in Formula II; both the response unit A and the reference unit B are covalently modified on the surface of the silicon nanowires. This ratiometric fluorescence sensor can achieve a high spatial resolution response to reduced glutathione in the environment, while also exhibiting excellent selectivity and good photostability, and can be applied to single-cell detection without interference from the protein crown phenomenon. Ⅰ; Ⅱ

Description

Technical Field

[0001] This invention relates to the field of ratiometric fluorescence sensor fabrication technology. More specifically, it relates to a ratiometric fluorescence sensor for high spatial resolution detection of reduced glutathione, its fabrication method, and its applications. Background Technology

[0002] Reduced glutathione (GSH) is a short-chain tripeptide composed of L-glutamic acid (Glu), cysteine ​​(Cys), and glycine (Gly). It is mainly found in the cytoplasm and mitochondrial matrix, participating in the regulation of several key physiological activities. In the cytoplasm, one of the most important functions of GSH is maintaining cellular redox homeostasis, effectively scavenging free radicals and eliminating reactive oxygen species (ROS) and their toxic byproducts. The main metabolite of GSH is oxidized glutathione (GSSG). The GSH / GSSG redox pair is the most widely distributed and abundant redox pair in cells, representing the intracellular redox state and acting as a signal to regulate cellular state. By altering the GSH / GSSG redox potential, a series of molecular switches can be activated within the cell, enabling the cell to transition to different stages of growth and development. Furthermore, GSH plays important roles in regulating gene expression, maintaining normal protein function, and resisting ferroptosis. Abnormal changes in GSH levels are associated with various diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. In Alzheimer's disease neurons, the absence of GSH significantly leads to intracellular redox imbalance, which promotes neuronal degeneration and death. Furthermore, GSH exhibits uneven distribution across different regions of Alzheimer's cells, which is closely related to the pathogenesis and progression of Alzheimer's disease. Therefore, detecting GSH levels at different sites within cells is of great significance, and the development of related probes and sensors has gradually attracted considerable attention from researchers.

[0003] Due to the complexity of the cellular biological environment, fluorescent probes face challenges such as background fluorescence and interference from other biomolecules when applied. Therefore, using a single fluorescence channel signal as a detection indicator has limitations; differences between individual cells and fluctuations in experimental conditions can introduce significant errors into the detection results. For example, in 2020, Chen et al. developed a fluorescent probe characterized by coumarin as a luminescent group (Chen S., Luo Y., Wang N., et al. Cascade reaction-based trinal-site probe for sensing and imaging of cysteine ​​and glutathione [J]. Talanta, 2020, 208: 119934.), which was successfully applied to fluorescence imaging to monitor changes in endogenous and exogenous GSH in cells. This probe exhibits excellent selectivity and sensitivity; however, its single-wavelength fluorescence intensity response makes it susceptible to fluctuations in experimental conditions and interference from the cellular environment. Furthermore, the drift characteristics of the probe molecule limit its application in detecting GSH at different cellular sites. To address this issue, Niu et al. reported a fluorescent probe characterized by chlorocoumarin in 2020 (Niu T., Yin G., Yu T., et al. A novel fluorescent probe for detection of Glutathione dynamics during ROS-induced redox imbalance[J]. Analytica Chimica Acta, 2020, 1115: 52-60.). This probe exhibits a change in the position of its fluorescence emission peak before and after the response. Using the ratio of fluorescence intensity at the peak positions before and after the response as a detection indicator can effectively reduce interference from the cellular environment and significantly increase the reliability of the detection results. However, this invention still fails to solve the problem of low spatial resolution caused by the drift characteristics of the probe molecule, which limits its further application in cell detection. Summary of the Invention

[0004] To address the aforementioned problems, the first objective of this invention is to provide a ratiometric fluorescence sensor for high spatial resolution detection of reduced glutathione.

[0005] A second objective of this invention is to provide a method for preparing the ratiometric fluorescence sensor as described above.

[0006] A third objective of this invention is to provide an application of the ratiometric fluorescence sensor described above in the detection of reduced glutathione for purposes other than disease diagnosis and treatment.

[0007] A fourth objective of this invention is to provide an application of the ratiometric fluorescence sensor described above in the preparation of a product for the simultaneous detection of cell body and synaptic reduced glutathione in a single living cell.

[0008] The fifth objective of this invention is to provide a detection device for simultaneously detecting reduced glutathione in the cell body and synapse of a single living cell.

[0009] To achieve the first objective mentioned above, the present invention adopts the following technical solution: This invention discloses a ratiometric fluorescence sensor for high spatial resolution detection of reduced glutathione, comprising: Azide-based silane-modified silicon nanowires for improving spatial resolution and providing one-dimensional axial resolution for ratiometric fluorescence sensors; Response unit A for responding to reduced glutathione levels; and Reference Unit B; The structure of the response unit A is shown in Equation I and is named CyQ-al: Ⅰ; The structure of the reference unit B is shown in Formula II and is named Nap-OH; II; The response unit A emits red fluorescence, and its fluorescence intensity increases with the increase of reduced glutathione content; the reference unit B emits green fluorescence, and its fluorescence intensity remains essentially unchanged in the cellular environment. Both the response unit A and the reference unit B are covalently bonded to the surface of the silicon nanowire.

[0010] This ratiometric fluorescence sensor can achieve a high spatial resolution response to reduced glutathione in the environment, while also exhibiting excellent selectivity and good photostability. It can be applied to single-cell detection without interference from the protein crown phenomenon, enabling simultaneous detection of reduced glutathione at different sites in a single live cell.

[0011] It should be noted that response unit A can be replaced with other compounds according to actual application needs, provided that at least the following two requirements are met: 1) there is a certain mathematical relationship with the change in the content of reduced glutathione; 2) the structure contains an alkynyl group.

[0012] Similarly, reference unit B can be replaced with other compounds according to the actual application needs, provided that at least the following two requirements are met: 1) the fluorescence intensity remains stable in the cellular environment; 2) the structure contains an alkynyl group.

[0013] Furthermore, the molar ratio of the response unit A and the reference unit B used during modification is 1-10:1-10. For example, the molar ratio of the response unit A and the reference unit B can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.

[0014] Furthermore, the silicon nanowires have a length of 10-200 micrometers and a diameter of 100-300 nanometers.

[0015] To achieve the second objective mentioned above, the present invention adopts the following technical solution: This invention discloses a method for preparing the ratiometric fluorescence sensor as described above, comprising the following steps: S1. The surface-hydroxylated silicon nanowire array was immersed in toluene solution, and the silane reagent 3-(azidopropyl)triethoxysilane was added under nitrogen atmosphere. The mixture was heated to reflux and reacted. After the reaction was completed, the array was washed and dried to obtain the silicon nanowire array modified with azidosilane reagent. The above example uses 3-(azidopropyl)triethoxysilane as a modifying agent to modify hydroxylated silicon nanowires. It can be replaced with other organosilane reagents containing azido groups and capable of hydrolysis, depending on the actual application requirements. S2. Prepare an organic solution containing response unit A and an organic solution containing reference unit B in advance. Separate the silicon nanowires modified with azide-silane reagent from the silicon nanowire array and disperse them in deionized water. Add the organic solution containing response unit A and reference unit B, and then add a catalyst solution containing copper sulfate and sodium ascorbate. Stir in the dark and filter under reduced pressure to obtain the ratiometric fluorescence sensor precursor. S3. Disperse the ratiometric fluorescence sensor precursor in a polyethylene glycol aqueous solution, stir in the dark, and filter under reduced pressure to obtain the ratiometric fluorescence sensor.

[0016] Furthermore, in step S1, the ratio of the amount of hydroxylated silicon nanowire array to silane reagent is 200-300 μL of silane reagent added per 1 cm × 2 cm hydroxylated silicon nanowire array.

[0017] Furthermore, in the organic solution of step S2, the concentration of the response unit A is 1-10 mM, the concentration of the reference unit B is 1-10 mM, and the mass-volume ratio of the silicon nanowire modified with the azide-silane reagent to the organic solution of the response unit A is 0.2-5 mg:200 μL.

[0018] Furthermore, in the catalyst solution of step S2, the molar ratio of copper sulfate to sodium ascorbate is 1-2:2-4, the concentration of copper sulfate is 8-24 mM, and the mass-volume ratio of silicon nanowires modified with azidosilane reagent to catalyst solution is 0.2-5 mg:300 μL.

[0019] Furthermore, in step S3, the average molecular weight of the polyethylene glycol used is 2000-20000, the concentration of the polyethylene glycol solution is 1-5 mg / mL, and the mass-to-volume ratio of the ratiometric fluorescent sensor precursor to the polyethylene glycol solution is 0.2-5 mg:5 mL.

[0020] Furthermore, the reflux reaction time in step S1 is 24-48 hours, the reaction time in step S2 with stirring in the dark is 24-72 hours, and the stirring time in step S3 with stirring in the dark is 10-120 minutes.

[0021] To achieve the third objective mentioned above, the present invention adopts the following technical solution: This invention discloses the application of a ratiometric fluorescence sensor prepared using the ratiometric fluorescence sensor described above or the preparation method described above in the detection of reduced glutathione for purposes other than disease diagnosis and treatment.

[0022] To achieve the fourth objective mentioned above, the present invention adopts the following technical solution: This invention discloses the application of a ratiometric fluorescence sensor prepared using the ratiometric fluorescence sensor described above or the preparation method described above in the preparation of a product for the simultaneous detection of cell body and synaptic reduced glutathione in a single live cell.

[0023] To achieve the fifth objective mentioned above, the present invention adopts the following technical solution: This invention discloses a detection device for simultaneously detecting reduced glutathione in the cell body and synapse of a single living cell, comprising a micromanipulator, a laser scanning confocal microscope, and a ratiometric fluorescence sensor as described above.

[0024] By mounting a ratiometric fluorescence sensor onto a micromanipulator, the micromanipulator is controlled to precisely implant the sensor into a single living cell. A laser scanning confocal microscope is then used to perform fluorescence ratio imaging of the sensor, thereby enabling the simultaneous detection of reduced glutathione pairs in the cell body and synapse of a single living cell.

[0025] The beneficial effects of this invention are as follows: The ratiometric fluorescence sensor provided by this invention is based on a single silicon nanowire and its surface-modified responsive unit CyQ-al that responds to the content of reduced glutathione, and a reference unit Nap-al that is luminescent and stable in the cellular environment. Compared with the prior art, it has the following advantages: 1) The ratiometric fluorescence sensor provided by the present invention uses one-dimensional silicon nanowires as the load substrate material. Based on the characteristics of this structure, the ratiometric fluorescence sensor can effectively overcome the spatial drift problem of sensors such as small molecules and nanoparticles, significantly improve the spatial resolution of intracellular imaging, and realize fluorescence imaging of different regions of the cell.

[0026] 2) The ratiometric fluorescence sensor provided by the present invention uses the fluorescence ratio of the response unit to the reference unit as the detection index, which can effectively reduce the interference from other substances in the cell and reduce the influence of background fluorescence on the results.

[0027] 3) The ratiometric fluorescence sensor provided by this invention has the characteristics of high sensitivity and excellent photostability, and is effective against Na+. + K + Ca 2+ Mg 2+ Cu 2+ Fe 3+ Competing substances such as H2O2, GSH, Cys and Hcy have strong anti-interference properties and exhibit excellent selectivity for the detection of reduced glutathione.

[0028] 4) The ratiometric fluorescence sensor provided by the present invention has a polyethylene glycol protective layer coated on its surface, which can effectively reduce the impact of protein crowning in the biological environment on the test and improve the reliability of the test results.

[0029] 5) The ratiometric fluorescence sensor provided by this invention can be combined with a micromanipulator and a laser scanning confocal microscope to be precisely implanted inside a single cell and located in different cellular regions, enabling simultaneous detection of the content of reduced glutathione in the cell body and synapse of a single living cell.

[0030] 6) The method for preparing the ratiometric fluorescence sensor provided by this invention is simple and can be mass-produced. Attached Figure Description

[0031] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0032] Figure 1 A schematic diagram of the synthesis steps for a ratiometric fluorescence sensor is shown.

[0033] Figure 2 The graph shows the fluorescence ratio of the ratiometric fluorescence sensor of Example 1 as a function of the concentration of reduced glutathione.

[0034] Figure 3 The diagram shows the selectivity test of reduced glutathione by the ratiometric fluorescence sensor of Example 2.

[0035] Figure 4The photostability test diagram of the ratiometric fluorescence sensor of Example 3 is shown.

[0036] Figure 5 This illustrates the testing process of the ratiometric fluorescence sensor of Example 1 for high spatial resolution simultaneous detection of cell body and synaptic reduced glutathione within a single living cell, wherein... Figure 5 Image A shows a bright-field image of a ratiometric fluorescence sensor precisely implanted inside a single cell, while image B shows a multi-channel fluorescence image corresponding to the bright-field image. Detailed Implementation

[0037] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. It should be understood that the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0038] Example 1 This embodiment designs and synthesizes a ratiometric fluorescence sensor with high spatial resolution for detecting reduced glutathione. The specific synthesis steps are as follows, and the flowchart is shown below. Figure 1 : S1. Preparation of silicon nanowires modified with silane reagents: A 1 cm × 2 cm silicon nanowire array rich in hydroxyl groups was taken, and 50 mL of anhydrous toluene was added. The system was placed under nitrogen protection. The mixture was heated to reflux with toluene, and residual water was removed from the system using a water separator. Then, 200 μL of 3-(azidopropyl)triethoxysilane was added, and the reaction was continued under reflux for 24 hours. After the reaction was completed, the silicon nanowire array was washed and dried to obtain a silicon nanowire array modified with an azidosilane reagent.

[0039] S2. Preparation of the ratiometric fluorescence sensor precursor: The silicon nanowires modified with azide-silane reagent were separated from the silicon nanowire array and dispersed in deionized water at a concentration of 0.2 mg / mL. Prepare DMSO solutions containing CyQ-al and Nap-al, respectively, with a concentration of 1 mM. Named CyQ-al; Named Nap-OH; Compounds CyQ-al and Nap-al can be synthesized in-house or purchased from external sources, referring to publicly available technologies. Dissolve 8 mg of copper sulfate and 20 mg of sodium ascorbate in 6 mL of deionized water to obtain a catalyst solution; Take 5 mL of an aqueous solution of silicon nanowires dispersed with a azidosilane-modified reagent, add 100 μL of DMSO solution containing CyQ-al, 200 μL of DMSO solution containing Nap-al, and 300 μL of catalyst solution, and stir the reaction system at room temperature in the dark for 36 hours. After the reaction is complete, remove the solvent by vacuum filtration to obtain the ratiometric fluorescence sensor precursor; S3. Fabrication of a ratiometric fluorescence sensor: 30 mg of PEG-10000 was dispersed in 10 mL of deionized water to obtain a modified solution; The ratiometric fluorescent sensor precursor was dispersed in the modified solution at a concentration of 0.2 mg / mL, stirred in the dark for 60 minutes, and filtered under reduced pressure to obtain the ratiometric fluorescent sensor.

[0040] Example 2 This embodiment designs and synthesizes a ratiometric fluorescence sensor with high spatial resolution for detecting reduced glutathione. The specific synthesis steps are as follows: S1. Preparation of silicon nanowires modified with silane reagents: A 1 cm × 2 cm silicon nanowire array rich in hydroxyl groups was taken, and 50 mL of anhydrous toluene was added. The system was placed under nitrogen protection. The mixture was heated to reflux with toluene, and residual water was removed from the system using a water separator. Then, 200 μL of 3-(azidopropyl)triethoxysilane was added, and the reaction was continued under reflux for 24 hours. After the reaction was completed, the silicon nanowire array was washed and dried to obtain a silicon nanowire array modified with an azidosilane reagent.

[0041] S2. Preparation of the ratiometric fluorescence sensor precursor: The silicon nanowires modified with azide-silane reagent were separated from the silicon nanowire array and dispersed in deionized water at a concentration of 0.2 mg / mL. Prepare DMSO solutions containing CyQ-al and Nap-al, respectively, with a concentration of 1 mM. Dissolve 10 mg of copper sulfate and 20 mg of sodium ascorbate in 6 mL of deionized water to obtain a catalyst solution; Take 3.75 mL of an aqueous solution of silicon nanowires dispersed with a azidosilane-modified reagent, add 30 μL of DMSO solution containing CyQ-al, 300 μL of DMSO solution containing Nap-al, and 45 μL of catalyst solution. Stir the reaction system at room temperature in the dark for 24 hours. After the reaction is complete, remove the solvent by vacuum filtration to obtain the ratiometric fluorescent sensor precursor. S3. Fabrication of a ratiometric fluorescence sensor: 10 mg of PEG-2000 was dispersed in 10 mL of deionized water to obtain a modified solution; The ratiometric fluorescent sensor precursor was dispersed in the modified solution at a concentration of 0.04 mg / mL, stirred in the dark for 10 minutes, and filtered under reduced pressure to obtain the ratiometric fluorescent sensor.

[0042] Example 3 This embodiment designs and synthesizes a ratiometric fluorescence sensor with high sensitivity for detecting reduced glutathione. The specific synthesis steps are as follows: S1. Preparation of silicon nanowires modified with silane reagents: A 1 cm × 2 cm silicon nanowire array rich in hydroxyl groups was taken, and 50 mL of anhydrous toluene was added. The system was placed under nitrogen protection. The mixture was heated to reflux with toluene, and residual water was removed from the system using a water separator. Then, 300 μL of 3-(azidopropyl)triethoxysilane was added, and the reaction was continued under reflux for 48 hours. After the reaction was completed, the silicon nanowire array was washed and dried to obtain a silicon nanowire array modified with an azidosilane reagent.

[0043] S2. Preparation of the ratiometric fluorescence sensor precursor: The silicon nanowires modified with azide-silane reagent were separated from the array and dispersed in deionized water at a concentration of 0.2 mg / mL. Prepare DMSO solutions containing CyQ-al and Nap-al, respectively, with a concentration of 1 mM. Dissolve 16 mg of copper sulfate and 20 mg of sodium ascorbate in 6 mL of deionized water to obtain a catalyst solution; Take 25 mL of an aqueous solution of silicon nanowires dispersed with a azidosilane-modified reagent, add 200 μL of DMSO solution containing CyQ-al, 20 μL of DMSO solution containing Nap-al, and 300 μL of catalyst solution, and stir the reaction system at room temperature in the dark for 72 hours. After the reaction is complete, remove the solvent by vacuum filtration to obtain the ratiometric fluorescence sensor precursor; S3. Fabrication of a ratiometric fluorescence sensor: 50 mg of PEG-20000 was dispersed in 10 mL of deionized water to obtain a modified solution; The ratiometric fluorescent sensor precursor was dispersed in the modified solution at a concentration of 1 mg / mL, stirred in the dark for 120 minutes, and filtered under reduced pressure to obtain the ratiometric fluorescent sensor.

[0044] Reduced glutathione response test An appropriate amount of the ethanol solution of the ratiometric fluorescence sensor prepared in Example 1 was dropped onto a glass-bottomed culture dish. After the solution evaporated, PBS buffer solution was added. A silicon nanowire was selected in the field of view using a laser scanning confocal microscope, and its fluorescence intensity ratio was measured. Subsequently, keeping the position of this silicon nanowire unchanged, a pre-prepared reduced glutathione solution (PBS buffer solution) was added to adjust the concentration of reduced glutathione in the solution, and the mixture was stirred evenly using a pipette. The concentration of reduced glutathione in the solution in the culture dish at this time can be calculated based on the amount of reduced glutathione solution added, and the fluorescence intensity ratio data at this concentration is recorded. The above steps were repeated to obtain the fluorescence intensity ratio data of the silicon nanowire at different reduced glutathione concentrations. The results are as follows. Figure 2 As shown, with increasing concentration of reduced glutathione, the orange fluorescence F of CyQ-al increases. orange The intensity gradually increases, while the green fluorescence F of Nap-al... green The intensity remains basically unchanged, and the ratio of their fluorescence intensities, F... orange / F green The fluorescence intensity continued to rise. As the concentration of reduced glutathione increased from 0 mM to 5 mM, the fluorescence intensity ratio changed by 6-7 times, and the calculated detection limit was 70 μM, demonstrating the sensor's high sensitivity to reduced glutathione concentration.

[0045] Using the same testing process, it was confirmed that the ratiometric fluorescence sensors prepared in Examples 2 and 3 also have the above-mentioned similar characteristics.

[0046] Selective testing An appropriate amount of the ethanol solution of the ratiometric fluorescence sensor prepared in Example 2 was added dropwise to a glass-bottomed culture dish. After the solution evaporated to dryness, PBS buffer solution was added. Multiple silicon nanowires were selected in the field of view using a laser scanning confocal microscope, and their fluorescence intensity ratios were tested. Subsequently, common competitive substances found in cells were added in parallel to the culture dish, and the changes in the fluorescence intensity ratios of the silicon nanowires were tested one by one. Figure 3 As shown, these competing substances include: No. 1, blank control; No. 2, Na. + 15 mM; Serial number 3, K + 145 mM; Serial number 4, Mg 2+ 1 mM; Serial number 5, Ca 2+ 10 μM; Serial number 6, Cu 2+ 400 μM; Serial number 7, Fe 3+ 100 μM; 8. NADH, 300 μM; 9. Cys, 100 μM; 10. Hcy, 100 μM; 11. AA, 300 μM; 12. SO32- 13, H2S, 100 μM; 14, H2O2, 500 μM; 15, reduced glutathione, 1 mM. This ratiometric fluorescence sensor exhibits excellent selectivity.

[0047] Using the same testing procedure, it was confirmed that the ratiometric fluorescence sensors prepared in Examples 1 and 3 also have the above-mentioned similar characteristics.

[0048] Light stability test An appropriate amount of the ethanol solution of the ratiometric fluorescence sensor prepared in Example 3 was dropped onto a glass-bottomed culture dish. After the solution evaporated to dryness, PBS buffer solution was added. A silicon nanowire was selected in the field of view using a laser scanning confocal microscope, and it was continuously photographed. The change in fluorescence intensity ratio was measured after 10 images. The laser power used was 10 μW. The results are as follows: Figure 4 As shown, the ratiometric fluorescence sensor remained stable after 10 consecutive images under 10 μW laser irradiation, demonstrating good photostability.

[0049] Using the same testing procedure, it was confirmed that the ratiometric fluorescence sensors prepared in Examples 1 and 2 also have the above-mentioned similar characteristics.

[0050] High spatial resolution detection of reduced glutathione in cell bodies and synapses First, capillary tubes were fabricated into capillary microneedles with extremely fine tips using a needle puller. Under a microscope, a portion of the tip of the fabricated capillary microneedles was then trimmed off, creating a small opening. Using a micropipette connected to a syringe, the ratiometric fluorescence sensor prepared in Example 1 was precisely aspirated through the small opening of the microneedle, ensuring that at least half of its length was exposed at the tip. Two ratiometric fluorescence sensors mounted on microneedles were fabricated using this method.

[0051] Subsequently, two ratiometric fluorescence sensors mounted on microneedles were connected to two microcontrollers. Further, using a laser scanning confocal microscope, the two ratiometric fluorescence sensors were precisely implanted into the same cell. Bright-field imaging was then used to confirm the sensors' positions within the cell, specifically at the cell body and synapse. Figure 5 As can be seen, the two ratiometric fluorescence sensors are located in the cell body and cell synapse, respectively. Data analysis shows that the concentration of reduced glutathione in the cell body region is significantly higher than that in the synaptic region. This indicates that the ratiometric fluorescence sensor can achieve simultaneous detection of reduced glutathione in both the cell body and synaptic regions.

[0052] Using the same testing process, it was confirmed that the ratiometric fluorescence sensors prepared in Examples 2 and 3 also have the above-mentioned similar characteristics.

[0053] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A ratiometric fluorescence sensor for high spatial resolution detection of reduced glutathione, characterized in that, The ratiometric fluorescence sensor includes: Azide-based silane reagent-modified silicon nanowires for improving spatial resolution and providing one-dimensional axial resolution; Response unit A for responding to reduced glutathione levels; and Reference Unit B; The structure of the response unit A is shown in Equation I and is named CyQ-al: Ⅰ； The structure of the reference unit B is shown in Formula II and is named Nap-OH; Ⅱ； Both the response unit A and the reference unit B are covalently bonded to the surface of the silicon nanowire.

2. The ratiometric fluorescence sensor according to claim 1, characterized in that, The molar ratio of the amount of the response unit A and the reference unit B used during modification is 1-10:1-10.

3. The ratiometric fluorescence sensor according to claim 1, characterized in that, The silicon nanowires have a length of 10-200 micrometers and a diameter of 100-300 nanometers.

4. The method for fabricating a ratiometric fluorescence sensor as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. The surface-hydroxylated silicon nanowire array was immersed in toluene solution, and the silane reagent 3-(azidopropyl)triethoxysilane was added under nitrogen atmosphere. The mixture was heated to reflux and reacted. After the reaction was completed, the array was washed and dried to obtain the silicon nanowire array modified with azidosilane reagent. S2. The silicon nanowires modified with azide-based silane reagent are separated from the silicon nanowire array, dispersed in deionized water, and an organic solution containing response unit A and reference unit B is added. Then, a catalyst solution containing copper sulfate and sodium ascorbate is added. The mixture is stirred in the dark and filtered under reduced pressure to obtain a ratiometric fluorescence sensor precursor. S3. Disperse the ratiometric fluorescence sensor precursor in a polyethylene glycol aqueous solution, stir in the dark, and filter under reduced pressure to obtain the ratiometric fluorescence sensor.

5. The preparation method according to claim 4, characterized in that, In step S1, the ratio of the amount of hydroxylated silicon nanowire array to silane reagent is 200-300 μL of silane reagent per 1 cm × 2 cm hydroxylated silicon nanowire array.

6. The preparation method according to claim 4, characterized in that, In the organic solution of step S2, the concentration of the response unit A is 1-10 mM, the concentration of the reference unit B is 1-10 mM, and the mass-volume ratio of the silicon nanowire modified with the azide-silane reagent to the organic solution containing the response unit A is 0.2-5 mg:200 μL. Preferably, in the catalyst solution of step S2, the molar ratio of copper sulfate to sodium ascorbate is 1-2:2-4, the concentration of copper sulfate is 8-24 mM, and the mass-volume ratio of silicon nanowires modified with azide-silane reagent to the catalyst solution is 0.2-5 mg:300 μL. Preferably, the polyethylene glycol used in step S3 has an average molecular weight of 2000-20000, the concentration of the polyethylene glycol solution is 1-5 mg / mL, and the mass-to-volume ratio of the ratiometric fluorescent sensor precursor to the polyethylene glycol solution is 0.2-5 mg:5 mL.

7. The preparation method according to claim 4, characterized in that, The reflux reaction time in step S1 is 24-48 hours, the reaction time in step S2 is 24-72 hours with stirring in the dark, and the reaction time in step S3 is 10-120 minutes with stirring in the dark.

8. The use of the ratiometric fluorescence sensor as described in any one of claims 1-3 or the ratiometric fluorescence sensor prepared by the preparation method as described in any one of claims 4-7 in the detection of reduced glutathione for purposes other than disease diagnosis and treatment.

9. The application of the ratiometric fluorescence sensor as described in any one of claims 1-3 or the ratiometric fluorescence sensor prepared by the preparation method as described in any one of claims 4-7 in the preparation of a product for the simultaneous detection of cell body and synaptic reduced glutathione in a single living cell.

10. A detection device for simultaneously detecting reduced glutathione in the cell body and synapse of a single living cell, characterized in that, It includes a micromanipulator, a laser scanning confocal microscope, and a ratiometric fluorescence sensor as described in any one of claims 1-3.

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