A wide linear range for the detection of Cu 2+ Ratio-type fluorescent probe and preparation method thereof

Abstract

The present invention discloses a method for detecting Cu in a wide linear range. 2+ Ratio-type fluorescent probe and preparation method thereof, the wide linear range detection Cu 2+ The ratiometric fluorescent probe is obtained by mixing nitrogen-doped graphene quantum dots with dithiothreitol, and the wide linear range detection of Cu 2+ Ratiometric fluorescent probe for Cu 2+ The LOD of the detection is 0.05 μmol / L; the wide linear range of Cu 2+ Ratiometric fluorescent probe for the detection of Cu 2+ When, with Cu 2+ The coordination reaction generates a new complex, which quenches the fluorescence of nitrogen-doped graphene quantum dots emitting 445nm blue light and generates copper nanoparticles emitting 666nm red fluorescence, realizing a ratiometric fluorescence detection of Cu by "down" and "up". 2+ , broadened Cu 2+ The scope of detection.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probe detection technology, and in particular to a method for detecting Cu 2+ Ratiometric fluorescent probe and preparation method thereof. Background Art

[0002] Compared with traditional detection of Cu 2+ Compared with traditional analytical methods such as atomic absorption spectroscopy, ultraviolet spectrophotometry and electrochemical analysis, fluorescence analysis has become a powerful tool for analysis and detection in various fields such as environment, biology, medicine, and food due to its simple operation, high sensitivity and strong selectivity. So far, researchers have developed a large number of single fluorescence signal ("on" or "off" type) or ratiometric detection methods based on a series of materials such as carbon quantum dots, metal nanoclusters, metal organic frameworks, lanthanide metal oxides, vanadates, phosphates, organic polymers, etc. 2+ It is a fluorescent probe that is used in the analysis and detection of environmental samples, physiological samples, and cell imaging.

[0003] Currently reported detection of Cu 2+ The fluorescent probes have the following technical problems that need to be improved:

[0004] (1) The reported Cu 2+ The linear range of the fluorescent probes detected is relatively narrow, which greatly limits the scope of actual sample measurement and cannot simplify the actual operation process.

[0005] (2) Detection of Cu based on a single fluorescence signal value 2+ Most of the reports are about fluorescent probes, which are easily disturbed by the external environment, thus affecting the accuracy of actual detection.

[0006] Therefore, it is very necessary to develop a wide linear range detection method for Cu 2+ ratiometric fluorescent probe. Summary of the Invention

[0007] The present invention provides a method for detecting Cu in a wide linear range. 2+ The purpose of the ratiometric fluorescent probe and the preparation method thereof is to solve the above-mentioned problems existing in the background technology.

[0008] The technical solutions provided by the present invention are as follows:

[0009] A wide linear range for the detection of Cu 2+ Ratiometric fluorescent probe with a wide linear range for detection of Cu 2+ The ratiometric fluorescent probe is obtained by mixing nitrogen-doped graphene quantum dots with dithiothreitol, and the wide linear range detection of Cu 2+ Ratiometric fluorescent probe for Cu2+ The LOD of the assay was 0.05 μmol / L;

[0010] The wide linear range for detection of Cu 2+ Ratiometric fluorescent probe for the detection of Cu 2+ When, with Cu 2+ A coordination reaction occurs to generate a new complex, which quenches the fluorescence of the nitrogen-doped graphene quantum dots that emit 445nm blue light, and generates copper nanoparticles that emit 666nm red fluorescence.

[0011] The present invention also provides a method for detecting Cu in a wide linear range. 2+ The method for synthesizing a ratiometric fluorescent probe comprises the following steps:

[0012] The first mass of citric acid is reacted with the second mass of thiourea, and solid nitrogen-doped graphene quantum dots are obtained after centrifugation, purification, and freeze-drying;

[0013] The nitrogen-doped graphene quantum dots are prepared to obtain a first volume of a first concentration nitrogen-doped graphene quantum dot solution, a second volume of a second concentration of dithiothreitol solution and a third volume of a pH=8.0 Tris-HCl buffer solution are added, and the mixture is uniformly mixed to obtain the wide linear range detection of Cu 2+ Ratiometric fluorescent probes;

[0014] The concentration range of Cu was 0-360 μmol / L. 2+ Fluorescence spectrometry titration experiments were performed to obtain the wide linear range for detecting Cu 2+ Linear range of ratiometric fluorescent probes;

[0015] The wide linear range for Cu 2+ The ratiometric fluorescence probe was used to measure the fluorescence spectrum multiple times, and the Cu 2+ The LOD of the detection is calculated as follows:

[0016] LOD=3σ / k

[0017] Wherein, k is the slope of the linear equation, σ is the wide linear range for detecting Cu 2+ The standard deviation of multiple measurements of ratiometric fluorescent probes.

[0018] Furthermore, the first mass of citric acid is reacted with the second mass of thiourea, and solid nitrogen-doped graphene quantum dots are obtained after centrifugation, purification, and freeze-drying, specifically:

[0019] 0.84 g of citric acid and 0.72 g of thiourea were added to 20 mL of distilled water. After fully dissolved, the mixture was transferred to a 50 mL Teflon-lined autoclave and reacted at 160 ° C for 4 hours. After natural cooling, the mixture was centrifuged at 8000 r / min for 10 minutes to obtain the supernatant. The supernatant was purified for 12 hours using a dialysis bag with a molecular weight cutoff of 1000 Da. The purified product was collected and freeze-dried to obtain solid nitrogen-doped graphene quantum dots.

[0020] Furthermore, the nitrogen-doped graphene quantum dots are configured to obtain a first volume of a first concentration nitrogen-doped graphene quantum dot solution, a second volume of a second concentration dithiothreitol solution, and a third volume of a pH=8.0 Tris-HCl buffer solution, specifically:

[0021] The nitrogen-doped graphene quantum dots were prepared to obtain 50 μL of a 70 μg / mL nitrogen-doped graphene quantum dot solution, and 200 μL of a 50 mmol / L dithiothreitol solution and 250 μL of a pH=8.0 Tris-HCl buffer solution were added to a 5 mL colorimetric tube.

[0022] Furthermore, the pH value of the fluorescence spectrum titration experiment of the wide linear range detection Cu2+ ratio-type fluorescent probe was 8.0 and the reaction time was 4 minutes.

[0023] Furthermore, the wide linear range detection of Cu 2+ The ratiometric fluorescent probe was used to measure the fluorescence spectrum 11 times and the Cu 2+ The LOD of the assay was 0.05 μmol / L.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The ratiometric detection method of Cu synthesized by the present invention 2+ Ratio-type fluorescent probe (N-GQDs / DTT) is obtained by directly mixing nitrogen-doped graphene quantum dots (N-GQDs) and dithiothreitol (DTT) by physical methods. From the material point of view, the surface of N-GQDs is rich in electron-rich atoms N and O atoms, while the structure of DTT contains electron-rich atoms S and O atoms. After mixing, the two can form a stable composite material through the action of hydrogen bonds. When the substance to be analyzed is added, Cu 2+ When N-GQDs are rich in -NH2 and -COOH, they can react with Cu 2+ The coordination reaction occurs to form a new complex, which leads to the quenching of the fluorescence of N-GQDs emitting blue light (445nm). At the same time, the DTT structure contains a reducing -SH, which reacts with Cu 2+It also showed a strong bonding effect and in situ generated copper nanoparticles (666nm) that could emit red fluorescence, realizing a ratiometric fluorescence detection of Cu 2+ , broadened Cu 2+ The scope of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For the wide linear range detection of Cu in the embodiment of the present invention 2+ Synthesis flow chart of ratiometric fluorescent probes;

[0027] Figure 2 For the wide linear range detection of Cu in the embodiment of the present invention 2+ Schematic diagram of the synthesis principle of ratiometric fluorescent probe;

[0028] Figure 3 For the wide linear range detection of Cu in the embodiment of the present invention 2+ Response graph of ratiometric fluorescent probe to copper ions;

[0029] Figure 4 Figure a in the middle is Cu 2+ Fluorescence titration curve of the probe system when the concentration is; Figure b is Cu 2+ Linear relationship diagram of the detection;

[0030] Figure 5 The interference substances that may exist in the embodiments of the present invention are Cu 2+ Graph showing the selectivity and interference of the assay. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the embodiments described below are part of the embodiments of the present application, not all of the embodiments. Generally, the components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0032] Therefore, the detailed description of the embodiments of the present application provided below in conjunction with the accompanying drawings is intended to represent only selected embodiments of the present application and is not intended to limit the scope of protection claimed in the present application. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present application without making any creative efforts shall fall within the scope of protection claimed in the present application.

[0033] It should be understood that, in the description of the embodiments of the present invention, the terms "first," "second," etc. are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features indicated. Therefore, features specified as "first," "second," etc. may explicitly or implicitly include one or more of the described features.

[0034] In the description of the embodiments of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "disposed," "installed," "connected," and "connected" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections, electrical connections, or mutual communication; they can refer to direct connections or indirect connections through an intermediate medium; they can refer to internal communication between two elements or an interaction between two elements. Those skilled in the art can understand the specific meanings of the above terms in the embodiments of the present invention based on specific circumstances.

[0035] See Figure 1 The present invention provides a wide linear range detection method for Cu 2+ The method for synthesizing a ratiometric fluorescent probe comprises the following steps:

[0036] Step S1: reacting a first mass of citric acid with a second mass of thiourea, and obtaining solid nitrogen-doped graphene quantum dots after centrifugation, purification, and freeze-drying.

[0037] Step S2: The nitrogen-doped graphene quantum dots are configured to obtain a first volume of a first concentration nitrogen-doped graphene quantum dot solution, a second volume of a second concentration dithiothreitol solution, and a third volume of a pH = 8.0 Tris-HCl buffer solution, and the mixture is uniformly mixed to obtain the wide linear range detection Cu 2+ Ratiometric fluorescent probe.

[0038] Step S3: Cu with a concentration range of 0-360 μmol / L 2+ Fluorescence spectrometry titration experiments were performed to obtain the wide linear range for detecting Cu 2+ Linear range of ratiometric fluorescent probes.

[0039] Step S4: Detecting Cu in the wide linear range 2+ The ratiometric fluorescence probe was used to measure the fluorescence spectrum multiple times, and the Cu 2+ The LOD of the detection is calculated as follows:

[0040] LOD=3σ / k

[0041] Wherein, k is the slope of the linear equation, σ is the wide linear range for detecting Cu2+ The standard deviation of multiple measurements of ratiometric fluorescent probes.

[0042] The present invention directly mixes nitrogen-doped graphene quantum dots (N-GQDs) and dithiothreitol (DTT) by physical methods to construct a wide linear range detection device for Cu 2+ Ratio-type fluorescent probe (N-GQDs / DTT). Cu 2+ When DTT reacts with Cu 2+ In situ reaction occurs to generate copper nanoparticles (DTT-CuNPs) with red fluorescence (666nm); at the same time, N-GQDs also react with Cu 2+ The combination quenches the blue fluorescence (445 nm) of N-GQDs, achieving a wide linear range ratiometric detection of Cu 2+ goals, such as Figure 2 shown.

[0043] Optionally, in step S1, a first mass of citric acid is reacted with a second mass of thiourea, and solid nitrogen-doped graphene quantum dots are obtained after centrifugation, purification, and freeze-drying, specifically:

[0044] 0.84 g of citric acid and 0.72 g of thiourea were added to 20 mL of distilled water. After fully dissolved, the mixture was transferred to a 50 mL Teflon-lined autoclave and reacted at 160 ° C for 4 hours. After natural cooling, the mixture was centrifuged at 8000 r / min for 10 minutes to obtain the supernatant. The supernatant was purified for 12 hours using a dialysis bag with a molecular weight cutoff of 1000 Da. The purified product was collected and freeze-dried to obtain solid nitrogen-doped graphene quantum dots.

[0045] Optionally, in step S2, the nitrogen-doped graphene quantum dots are configured to obtain a first volume of a first concentration nitrogen-doped graphene quantum dot solution, a second volume of a second concentration dithiothreitol solution, and a third volume of a pH=8.0 Tris-HCl buffer solution are added, specifically:

[0046] The nitrogen-doped graphene quantum dots were prepared to obtain 50 μL of a 70 μg / mL nitrogen-doped graphene quantum dot solution, and 200 μL of a 50 mmol / L dithiothreitol solution and 250 μL of a pH=8.0 Tris-HCl buffer solution were added to a 5 mL colorimetric tube.

[0047] Optionally, the pH value of the fluorescence spectrum titration experiment for the wide linear range detection of Cu2+ ratio-type fluorescent probe is 8.0 and the reaction time is 4 minutes.

[0048] Optionally, the wide linear range detection of Cu 2+The ratiometric fluorescent probe was used to measure the fluorescence spectrum 11 times and the Cu 2+ The LOD of the assay was 0.05 μmol / L.

[0049] Specifically, the present invention provides a method for detecting Cu in a wide linear range. 2+ The method for synthesizing a ratiometric fluorescent probe comprises the following steps:

[0050] (1) Preparation of probe material: 0.84 g of citric acid and 0.72 g of thiourea were added to 20 mL of distilled water, respectively. After sufficient dissolution, the mixture was transferred to a 50 mL Teflon-lined autoclave and reacted at 160°C for 4 h. After natural cooling, the mixture was centrifuged at 8000 rpm for 10 min. After large particles were removed, the supernatant was purified using a dialysis bag with a molecular weight cutoff of 1000 Da for 12 h. The purified product was collected and freeze-dried to obtain solid N-GQDs.

[0051] (2) Wide linear range detection of Cu 2+ Synthesis of ratiometric fluorescent probe: The solid N-GQDs obtained in step (1) was prepared into 50 μL of 70 μg / mL N-GQDs solution. 200 μL of 50 mmol / L (dithiothreitol) DTT solution, 50 μL of 70 μg / mL N-GQDs solution and 250 μL of pH = 8.0 Tris-HCl buffer solution were added to a 5 mL colorimetric tube in sequence and mixed to obtain a wide linear range for detection of Cu. 2+ Ratiometric fluorescent probes, such as Figure 3 shown.

[0052] (3)Cu 2+ Determination: Under the optimized detection conditions (pH=8.0, Cu 2+ Detect Cu with wide linear range 2+ The ratiometric fluorescent probe has a reaction time of t = 4 min and is sensitive to Cu in the concentration range of 0-360 μmol / L. 2+ Fluorescence spectroscopic titration experiments were carried out and its linear range was obtained, such as Figure 4 shown.

[0053] (4) Determination of limit of detection (LOD): Detection of Cu in a wide linear range against blank 2+ The fluorescence spectrum of the ratiometric fluorescent probe was measured 11 times in parallel, and the LOD was calculated according to the formula: LOD = 3σ / k (k is the slope of the linear equation, σ is the standard deviation of the 11 empty probe detections). 2+ The LOD of the detection was 0.05 μmol / L.

[0054] (5)Cu 2+Selectivity and interference experiments: 19 substances (Al 3 + 、Zn 2+ 、Cd 2+ , K + 、Ba 2+ Mg 2+ , Ca 2+ 、Co 2+ 、Ni 2+ 、Na + NH4 + 、F - 、NO3 - Br - 、CO3 2- 、Cl - 、SO3 2- , I - 、SO4 2- ) were tested for selectivity and anti-interference, in which Cu 2+ The concentration is 200 μmol / L, except Co 2+ and Ni 2+ Except for the one with a concentration of 20 μmol / L, the concentrations of other interfering substances were all 400 μmol / L.

[0055] The ratiometric detection method of Cu synthesized by the present invention 2+ Ratio-type fluorescent probe (N-GQDs / DTT) is obtained by directly mixing nitrogen-doped graphene quantum dots (N-GQDs) and dithiothreitol (DTT) by physical methods. From the material point of view, the surface of N-GQDs is rich in electron-rich atoms N and O atoms, while the structure of DTT contains electron-rich atoms S and O atoms. After mixing, the two can form a stable composite material through the action of hydrogen bonds. When the substance to be analyzed is added, Cu 2+ When N-GQDs are rich in -NH2 and -COOH, they can react with Cu 2+ The coordination reaction occurs to form a new complex, which leads to the quenching of the fluorescence of N-GQDs emitting blue light (445nm). At the same time, the DTT structure contains a reducing -SH, which reacts with Cu 2+ It also showed a strong bonding effect and in situ generated copper nanoparticles (666nm) that could emit red fluorescence, realizing a ratiometric fluorescence detection of Cu 2+ , broadened Cu 2+ The scope of detection.

[0056] The above is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions within the technical scope disclosed in this application shall be included in the scope of protection of this application. Therefore, the scope of protection of this application shall be based on the scope of protection of the claims.

Claims

1. A wide linear range for detecting Cu 2+ Ratiometric fluorescent probe, characterized by: The wide linear range for detection of Cu 2+ The ratiometric fluorescent probe is obtained by mixing nitrogen-doped graphene quantum dots with dithiothreitol, and the wide linear range detection of Cu 2+ Ratiometric fluorescent probe for Cu 2+ The LOD of the assay was 0.05 μmol / L; The wide linear range for detection of Cu 2+ Ratiometric fluorescent probe for the detection of Cu 2+ When, with Cu 2+ The coordination reaction generates a new complex, which quenches the fluorescence of the nitrogen-doped graphene quantum dots that emit 445 nm blue light and generates copper nanoparticles that emit 666 nm red fluorescence. The wide linear range for detection of Cu 2+ The method for synthesizing a ratiometric fluorescent probe comprises the following steps: The first mass of citric acid is reacted with the second mass of thiourea, and solid nitrogen-doped graphene quantum dots are obtained after centrifugation, purification, and freeze-drying; The nitrogen-doped graphene quantum dots are configured to obtain a first volume of a first concentration nitrogen-doped graphene quantum dot solution, a second volume of a second concentration of dithiothreitol solution and a third volume of a pH=8.0 Tris-HCl buffer solution are added, and the mixture is uniformly mixed to obtain the wide linear range detection of Cu 2+ Ratiometric fluorescent probes; The concentration range of Cu was 0-360 μmol / L. 2+ Fluorescence spectrometry titration experiments were performed to obtain the wide linear range for detecting Cu 2+ Linear range of ratiometric fluorescent probes; The wide linear range for Cu 2+ The ratiometric fluorescence probe was used to measure the fluorescence spectrum multiple times, and the Cu 2+ The LOD of the detection is calculated as follows: LOD=3σ / k Wherein, k is the slope of the linear equation, σ is the wide linear range for detecting Cu 2+ The standard deviation of multiple measurements of ratiometric fluorescent probes.

2. The wide linear range detection of Cu according to claim 1 2+ A ratiometric fluorescent probe, characterized in that The first mass of citric acid is reacted with the second mass of thiourea, and solid nitrogen-doped graphene quantum dots are obtained after centrifugation, purification, and freeze-drying. Specifically, 0.84 g of citric acid and 0.72 g of thiourea were added to 20 mL of distilled water. After fully dissolved, the mixture was transferred to a 50 mL Teflon-lined autoclave and reacted at 160°C for 4 h. After natural cooling, the mixture was centrifuged at 8000 r / min for 10 min to obtain the supernatant. The supernatant was purified for 12 h using a dialysis bag with a molecular weight cutoff of 1000 Da. The purified product was collected and freeze-dried to obtain solid nitrogen-doped graphene quantum dots.

3. The wide linear range detection of Cu according to claim 1 2+ A ratiometric fluorescent probe, characterized in that The nitrogen-doped graphene quantum dots are configured to obtain a first volume of a nitrogen-doped graphene quantum dot solution of a first concentration, a second volume of a dithiothreitol solution of a second concentration, and a third volume of a Tris-HCl buffer solution of pH=8.0, specifically: The nitrogen-doped graphene quantum dots were prepared to obtain 50 μL of a 70 μg / mL nitrogen-doped graphene quantum dot solution, and 200 μL of a 50 mmol / L dithiothreitol solution and 250 μL of a pH=8.0 Tris-HCl buffer solution were added to a 5 mL colorimetric tube.

4. The wide linear range detection of Cu according to claim 1 2+ Ratiometric fluorescent probe, characterized by: The pH value of the fluorescence spectrum titration experiment for the wide linear range detection of Cu2+ ratiometric fluorescent probe was 8.0 and the reaction time was 4 min.

5. according to the wide linear range detection of Cu in any one of claims 1 to 4 2+ Ratiometric fluorescent probe, characterized by: The wide linear range for Cu 2+ The ratiometric fluorescent probe was used to measure the fluorescence spectrum 11 times and the Cu 2+ The LOD of the assay was 0.05 μmol / L.

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