Double-emission carbon dot probe with dynamic adjustable range and preparation method and application thereof

Abstract

The invention provides a dual-emission carbon dot probe with a dynamic adjustable range for glutathione as well as a preparation method and application of the dual-emission carbon dot probe. The carbon dot probe is synthesized from alizarin red, carbon powder and ethylenediamine through a hydrothermal method, and the carbon dot probe shows concentration-dependent nonlinear fluorescence response in the presence of glutathione. By adjusting the concentration of the phosphate buffer solution, the detection parameters of the probe can be continuously and adjustably changed, and the wide-range detection of glutathione from 0.46 mu M to 3.2 mM is realized. The carbon dot probe has the advantages of high sensitivity, high selectivity, simplicity and convenience in operation, low cost and the like, and can be widely applied to the fields of environmental monitoring, food safety, biomedical diagnosis and the like.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry and biosensor technology, and in particular to a dual-emission carbon dot probe with a dynamically adjustable range for glutathione (GSH), a preparation method thereof, and applications thereof. Background Art

[0002] GSH is one of the most abundant intracellular non-protein thiols and is widely involved in maintaining intracellular redox homeostasis, signal transduction, and gene regulation, crucially impacting cell growth and function. Furthermore, as an antioxidant, GSH protects against free radical damage in intracellular compartments such as the cytoplasm, mitochondria, and nucleus. Changes in intracellular GSH levels are closely associated with a variety of diseases, including liver damage, neurological disorders, Alzheimer's disease, AIDS, and cancer. Therefore, accurate monitoring of GSH is of great significance in biochemical research, clinical diagnosis, and environmental monitoring.

[0003] Currently, methods for detecting GSH include electrochemical methods, high-performance liquid chromatography, and fluorescence methods. Although these methods have high selectivity and high sensitivity, their application in daily environmental and food safety monitoring is limited by the complex equipment, cumbersome operation, and high cost. In contrast, colorimetric sensors have become an ideal detection platform due to their advantages such as speed, convenience, and the absence of complex instruments and professional personnel. However, traditional colorimetric sensors face challenges in achieving quantitative detection of analytes within a wide dynamic range, and it is difficult to meet the detection requirements of biomarker concentrations in actual samples that span multiple orders of magnitude. Therefore, the development of a sensor with an adjustable detection range that can simultaneously detect targets of different concentrations has important scientific research and application value. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a dual-emission carbon dot probe with an adjustable dynamic range for GSH, as well as its preparation method and application, to achieve high-sensitivity and high-selectivity detection of GSH, and the detection range is adjustable, which can be adapted to the detection needs of different GSH concentrations.

[0005] To achieve this objective, the present invention provides a dual-emission ratiometric carbon dot (DRCD) probe that exhibits a concentration-dependent, nonlinear fluorescence response to GSH, demonstrating a characteristic threshold-response fluorescence transition. By simply adjusting the phosphate buffer (PBS) concentration, the probe's detection parameters can be continuously and tunably varied, enabling accurate detection of GSH from low to high concentrations.

[0006] Preparation method of DRCDs: The DRCDs preparation method comprises the following steps: (1) Dissolve alizarin red, carbon powder, and ethylenediamine in ultrapure water and mix well; (2) The mixed solution was transferred to a high-pressure reactor and reacted at 160°C for 2 hours; (3) After the reaction is completed, the mixture is cooled to room temperature, centrifuged, filtered, and dialyzed to obtain a dual-emission carbon dot probe.

[0007] Preparation method: DRCDs were synthesized via a hydrothermal method using alizarin red, carbon powder, and ethylenediamine as raw materials. The steps are as follows: 0.02 g of alizarin red, 0.01 g of carbon powder, and 10 μL of ethylenediamine were dissolved in 10 mL of ultrapure water and stirred thoroughly. The solution was transferred to a 50 mL Teflon-lined stainless steel autoclave and heated at 160°C for 2 hours. After the reaction, the mixture was cooled to room temperature, centrifuged to remove large particles, and filtered to remove impurities. The resulting supernatant was dialyzed (molecular weight cutoff 1000 Da) to remove small molecule impurities, yielding a purified DRCDs solution. The dialyzed solution was lyophilized to yield black solid DRCDs.

[0008] 2. Detection method of GSH by DRCDs: Dissolve DRCDs in PBS buffer at different concentrations (1-50 mM) and adjust the solution pH to 7.0. Add the GSH solution at the desired concentration to the DRCDs solution, mix well, and incubate at room temperature for 2 minutes.

[0009] The fluorescence spectra of DRCDs were measured at an excitation wavelength of 365 nm, and the fluorescence intensities at 464 nm and 560 nm were recorded. The fluorescence ratio I was calculated. 560 / I 464 , draw a standard curve of fluorescence ratio and GSH concentration.

[0010] By adjusting the PBS concentration, the detection range and sensitivity of GSH can be controlled.

[0011] 3. Application of DRCDs: It can be used to detect GSH concentration in biological and food samples, such as pig liver, tomato, cucumber, etc. It can be used to distinguish normal cells from tumor cells and has potential clinical diagnostic application value.

[0012] The DRCDs probe of the present invention has the following advantages: Adjustable detection range: By simply adjusting the concentration of PBS buffer, the detection parameters of DRCDs for GSH can be continuously adjusted to achieve accurate detection from low to high concentration ranges to meet the detection needs of different samples.

[0013] High sensitivity and selectivity: DRCDs exhibited a good fluorescence ratio response to GSH, with a detection limit as low as 0.46 μM, and had good anti-interference ability to other interfering substances.

[0014] Easy to operate: The preparation method is simple, the cost is low, and the detection process does not require complicated instruments and equipment, making it suitable for rapid on-site detection.

[0015] Visual detection: The addition of GSH can cause the color of the DRCDs solution to change from red to yellow, which can be observed with the naked eye under natural light conditions, facilitating rapid judgment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Transmission electron microscopy image and size distribution diagram of DRCDs prepared in Example 1 Figure 2 Infrared spectrum of DRCDs prepared in Example 1 Figure 3 X-ray photoelectron spectrum of DRCDs prepared in Example 1 Figure 4 Fluorescence spectrum and ultraviolet absorption spectrum of DRCDs prepared in Example 1 Figure 5 Fluorescence emission spectrum of the DRCDs solution prepared in Example 1 as the GSH concentration changes Figure 6 The titration plot of DRCDs prepared in Example 1 at different GSH and PBS concentrations, where A is the relationship between the fluorescence intensity ratio and the GSH concentration, B is the Boltzmann fit of the fluorescence intensity ratio and the GSH concentration, C is the relationship between the PBS concentration and the detection threshold, and D is the relationship between the PBS concentration and the detection limit. Figure 7 Visual detection of GSH by the DRCDs solution prepared in Example 1, where: A is a photo at different GSH concentrations and PBS concentrations, B is the relationship between the G / B value of the photo and the GSH concentration, C is the specific fitting function relationship, and D is the relationship between the PBS concentration and the detection threshold Figure 8 The following are photos of four cells labeled with DRCDs prepared in Example 1, where ABCD are HepG2, HeLa, BEAS-2B, and HL-7702 cells, respectively, and E is the identification of the four cells. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below in conjunction with the embodiments and the accompanying drawings, but the protection scope of the present invention is not limited thereto.

[0018] 1. Raw material preparation: Alizarin red (purity ≥98%): 0.02 g Carbon powder (particle size ≤ 100 nm): 0.01 g Ethylenediamine (analytical grade): 10 μl Ultrapure water: 10 ml 2. Synthesis steps: Solution preparation: Add Alizarin Red, carbon powder and ethylenediamine to 10 ml of ultrapure water and stir with a magnetic stirrer for 30 minutes until a uniform suspension is formed.

[0019] Hydrothermal reaction: The suspension was transferred to a 50 ml stainless steel autoclave lined with polytetrafluoroethylene, sealed, and placed in an oven with a set temperature of 160°C and a reaction time of 2 hours.

[0020] Cooling and Centrifugation: After the reaction is complete, cool naturally to room temperature. Centrifuge the reaction solution at 4000 rpm for 30 minutes to remove unreacted particles and macromolecules, and collect the supernatant.

[0021] Filtration and dialysis: Filter the supernatant through a 0.22 μm filter membrane. Place the filtrate in a dialysis bag with a molecular weight cutoff of 500-1000 Da and dialyze against ultrapure water at room temperature for 24 hours. Change the dialysate every 4 hours.

[0022] Drying and storage: The dialyzed solution was placed in a freeze dryer and freeze-dried to obtain black solid DRCDs, which were collected and placed in a sealed container and stored in a refrigerator at 4°C until use.

[0023] 3. Product characteristics: Transmission electron microscopy (TEM) showed that DRCDs were spherical particles with a particle size of approximately 2.06 ± 0.06 nm and a uniform distribution ( Figure 1 ).

[0024] The infrared spectra of the prepared DRCDs are shown in Figure 2 .

[0025] The X-ray photoelectron spectrum of the prepared DRCDs is shown in Figure 3 .

[0026] The prepared DRCDs exhibited dual emission peaks at 464 nm and 614 nm under an excitation wavelength of 365 nm. The fluorescence spectrum and UV absorption spectrum were shown in Figure 2. Figure 4 .

[0027] 1. Detection principle: In the presence of GSH, the fluorescence intensity of DRCDs at 560 nm increased significantly, while the fluorescence intensity at 464 nm did not change much. 560 / I 464It increases with the increase of GSH concentration and can be used to quantitatively detect GSH ( Figure 5 ).

[0028] 2. Reagents and Instruments: DRCDs solution: Dissolve the freeze-dried DRCDs in ultrapure water to prepare a stock solution of a certain concentration.

[0029] GSH standard solution: Prepare GSH standard solutions of different concentrations using ultrapure water.

[0030] PBS buffer (pH 7.0): Prepare PBS solutions with concentrations of 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM.

[0031] Fluorescence spectrophotometer: used to measure fluorescence spectra.

[0032] 3. Testing steps: Solution preparation: dilute the DRCDs stock solution to the required concentration according to a certain ratio, add different concentrations of PBS buffer, and mix well.

[0033] Sample preparation: Add different concentrations of GSH standard solution to 0.55 ml of DRCDs solution, mix well, and react at room temperature for 2 minutes.

[0034] Fluorescence measurement: Under an excitation wavelength of 365 nm, the fluorescence spectrum of the sample was measured, and the fluorescence intensity at 464 nm and 560 nm was recorded.

[0035] Data processing: Calculation of fluorescence ratio I 560 / I 464 , draw a standard curve of fluorescence ratio and GSH concentration.

[0036] 4. Results and Analysis Detection range: By adjusting the concentration of PBS buffer, the detection range of DRCDs for GSH can be adjusted from 0.46 μM to 3.2 mM ( Figure 6 ).

[0037] Detection limit: In 1 mM PBS, the detection limit can reach 0.46 μM; in 50 mM PBS, the detection limit is 6.64 μM.

[0038] Linear relationship: Under different PBS concentrations, the fluorescence ratio I 560 / I 464 There was a good linear relationship between the concentration of GSH and the

[0039] 5. Optional Experiments: Other possible interfering substances (such as cysteine, glutamic acid, lysine, glucose, vitamin C, etc.) were detected under the same conditions as GSH. The results showed that these substances had little effect on the fluorescence ratio of DRCDs, proving that the probe has good selectivity for GSH.

[0040] 1. Principle Overview: This example utilizes the color change of DRCDs solutions in the presence of varying glutathione concentrations to enable visual identification of GSH. As the concentration of GSH increases, the color of the DRCDs solution gradually changes from red to yellow. This change can be observed with the naked eye under natural light, without the need for complex instrumentation.

[0041] 2. Reagents and Instruments: DRCDs solution: prepared according to the method of Example 1.

[0042] GSH standard solution: prepare GSH solutions of different concentrations.

[0043] PBS buffer (pH 7.0): prepare 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM PBS solutions respectively.

[0044] Transparent cuvette or test tube.

[0045] White background or paper (to observe color changes).

[0046] 3. Testing steps: Solution preparation: Add DRCDs solution into PBS buffer of different concentrations in a certain proportion and mix well.

[0047] Sample preparation: Add different concentrations of GSH standard solution to the PBS solution containing DRCDs, adjust the total volume to the same, mix well, and let it stand for 2 minutes.

[0048] Color observation: Place the solution on a white background and observe the color change of the solution under natural light conditions. Record the color change from red to yellow at different GSH concentrations.

[0049] Result analysis: By comparing the changes in solution color, the concentration range of GSH can be intuitively determined. As the PBS concentration increases, the color change (color change point) corresponding to the GSH concentration also increases accordingly, thus achieving visual detection of GSH in different concentration ranges ( Figure 7 ).

[0050] 4. Results and Analysis In low concentrations of PBS (e.g., 1 mM), the GSH concentration threshold for the solution color to change from red to yellow is low, approximately 0.4 mM.

[0051] As the PBS concentration increases (e.g., to 50 mM), the GSH concentration threshold for the solution color change increases to approximately 2.6 mM.

[0052] This phenomenon indicates that the response range of DRCDs to the color change of GSH can be adjusted by adjusting the concentration of PBS buffer, thus achieving adjustable visual detection.

[0053] In addition, using DRCDs with actual samples (such as food samples) can also achieve visual detection of GSH in samples, which is convenient and fast.

[0054] 1. Sample processing: Pork liver sample: Wash fresh pork liver with tap water and ultrapure water and air dry. Accurately weigh a certain amount of pork liver, add an appropriate amount of ultrapure water, and homogenize using a homogenizer. Centrifuge the homogenized sample at 3000 rpm for 15 minutes and collect the supernatant. Filter the supernatant through a 0.22 μm filter membrane to obtain a clear sample solution. As required for testing, dilute the sample solution with ultrapure water so that its GSH concentration falls within the standard curve range.

[0055] Tomato and cucumber samples: Refer to the above-mentioned processing method for the pig liver sample and process the tomatoes and cucumbers.

[0056] 2. Testing steps: The GSH content in the actual sample was determined according to the detection method in Example 2. The standard addition method was used to investigate the recovery and accuracy of the method.

[0057] 3. Results and Analysis The results for GSH analysis in pig liver samples showed recoveries ranging from 97.71% to 102.97%, with relative standard deviations (RSDs) less than 4.43%. Results for tomato and cucumber samples also demonstrated good accuracy and repeatability (Table 1), demonstrating that this method can be used to accurately detect GSH in real-world samples.

[0058] Table 1

[0059] 1. Cell samples: Normal cells: normal human lung epithelial cells (BEAS-2B), normal human hepatocytes (HL-7702).

[0060] Tumor cells: human hepatocellular carcinoma cells (HepG2), human cervical cancer cells (HeLa).

[0061] 2. Testing steps: The cells were cultured and a cell suspension was prepared according to standard cell culture and treatment methods. The cells were lysed and intracellular GSH was extracted. The GSH content in the cell sample was determined according to the method in Example 2.

[0062] 3. Results and Analysis The test results showed that the GSH content in tumor cells (HepG2 and HeLa) was significantly higher than that in normal cells (BEAS-2B and HL-7702). 560 / I 464 Significantly increased in tumor cells ( Figure 8 ). This indicates that DRCDs probes can be used to distinguish normal cells from tumor cells and have potential clinical diagnostic application value.

Claims

1. A dual-emission carbon dot probe with a dynamically adjustable range for glutathione, characterized in that: The carbon dot probe is prepared from alizarin red, carbon powder and ethylenediamine by a hydrothermal method.

2. The method for preparing the dual-emission carbon dot probe according to claim 1, characterized in that: The following steps are involved: (1) Components in percentage by mass: 0.1% to 0.5% of alizarin red, 0.05% to 0.2% of carbon powder, 0.05% to 0.5% of ethylenediamine, and the balance of ultrapure water; mix well; (2) The mixed solution was transferred to a high-pressure reactor and reacted at 160°C for 2 hours; (3) After the reaction is completed, the mixture is cooled to room temperature, centrifuged, filtered, and dialyzed to obtain a dual-emission carbon dot probe.

3. Use of the dual-emission carbon dot probe according to claim 1 in detecting glutathione.

4. Use of the dual-emission carbon dot probe in detecting glutathione as claimed in claim 3, characterized in that: By adjusting the concentration of phosphate buffer, the detection range and sensitivity of the carbon dot probe for glutathione can be controlled.

5. Use of the dual-emission carbon dot probe according to claim 1 for detecting glutathione in actual samples, wherein the actual samples are biological samples or food samples.

6. Use of the dual-emission carbon dot probe according to claim 1 in tumor cell identification, characterized in that: By detecting the difference in intracellular glutathione content, normal cells and tumor cells can be distinguished.

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