阅读说明：本技术 用于谷胱甘肽检测的碳点荧光探针及其制备方法 (carbon dot fluorescent probe for glutathione detection and preparation method thereof ) 是由 李力 董文飞 梅茜 岳娟 葛明锋 常智敏 邵昊华 李林 于 2019-08-28 设计创作，主要内容包括：本发明公开了一种用于谷胱甘肽检测的碳点荧光探针及其制备方法,所述碳点荧光探针包括碳点及复合在所述碳点表面的2,4-二硝基苯磺酸酰基。其制备方法包括以下步骤：1)制备碳点；2)在得到的碳点表面修饰2,4-二硝基苯磺酸酰基。本发明基于碳点设计了一种针对谷胱甘肽检测的碳点荧光探针,该碳点荧光探针本身无荧光,随着谷胱甘肽浓度的增加,碳点荧光探针的荧光显著增强；该碳点荧光探针具有灵敏度高、特异性好等特点。(the invention discloses a carbon dot fluorescent probe for detecting glutathione and a preparation method thereof, wherein the carbon dot fluorescent probe comprises a carbon dot and 2, 4-dinitrobenzene sulfonic acid acyl compounded on the surface of the carbon dot. The preparation method comprises the following steps: 1) preparing carbon dots; 2) modifying the surface of the obtained carbon dot with 2, 4-dinitrobenzene sulfonic acid acyl. The carbon dot fluorescent probe for detecting the glutathione is designed based on the carbon dot, the carbon dot fluorescent probe has no fluorescence, and the fluorescence of the carbon dot fluorescent probe is obviously enhanced along with the increase of the concentration of the glutathione; the carbon dot fluorescent probe has the characteristics of high sensitivity, good specificity and the like.)

1. A carbon dot fluorescent probe for detecting glutathione is characterized by comprising a carbon dot and 2, 4-dinitrobenzene sulfonic acid acyl compounded on the surface of the carbon dot.

2. The method for preparing the carbon dot fluorescent probe for detecting glutathione as claimed in claim 1, which is characterized by comprising the following steps:

1) Preparing carbon dots;

2) modifying the surface of the obtained carbon dot with 2, 4-dinitrobenzene sulfonic acid acyl.

3. The method for preparing a carbon dot fluorescent probe for glutathione detection according to claim 2, wherein the step 1) comprises: dissolving o-phenylenediamine in DMF, stirring uniformly to dissolve the o-phenylenediamine completely, transferring the solution into a reaction kettle, heating and reacting; and filtering the reacted solution, dialyzing by using a dialysis bag, taking the internal solution after dialysis, and freeze-drying after rotary evaporation to obtain carbon dots.

4. The method for preparing the carbon dot fluorescent probe for detecting glutathione according to claim 3, wherein the step 1) specifically comprises the following steps: weighing 600mg of o-phenylenediamine, dissolving the o-phenylenediamine in 50mL of DMF (dimethyl formamide), uniformly stirring the o-phenylenediamine to be completely dissolved, transferring the solution into a reaction kettle with a polytetrafluoroethylene substrate of 80mL, and reacting the solution for 8 hours at 180 ℃;

after the reaction kettle is cooled, filtering the solution after reaction, dialyzing the solution in water for 24 hours by using a dialysis bag with the molecular weight cutoff of 500D, and changing the water for 2 times; and (4) taking internal liquid after dialysis, carrying out rotary evaporation, and freeze-drying to finally obtain the carbon dots.

5. The method for preparing the carbon dot fluorescent probe for detecting the glutathione as claimed in claim 2, wherein the step 2) comprises the steps of dissolving the carbon dot powder prepared in the step 1) in anhydrous acetonitrile, adding K ₂ CO ₃, adding the anhydrous acetonitrile solution containing the 2, 4-dinitrobenzenesulfonyl chloride dropwise into the solution, stirring the obtained mixture in an ice bath, removing the solvent in vacuum after the reaction is completed, dissolving the residue in water, dialyzing the mixture in water by using a dialysis bag, taking the internal solution after the dialysis, performing rotary evaporation, and performing freeze drying to obtain the carbon dot fluorescent probe.

6. The method for preparing the carbon dot fluorescent probe for glutathione detection as claimed in claim 5, wherein the step 2) specifically comprises weighing 100mg of carbon dot powder, dissolving the carbon dot powder in 10mL of anhydrous acetonitrile, adding 200mg of K ₂ CO ₃, adding 10.0mL of anhydrous acetonitrile solution containing 288.0mg of 2, 4-dinitrobenzenesulfonyl chloride dropwise into the solution, stirring the obtained mixture in an ice bath for 3 hours, removing the solvent in vacuum after the reaction is completed, dissolving the residue in a small amount of water, dialyzing the obtained mixture in water for 24 hours by using a dialysis bag with molecular weight cutoff of 500D, taking the internal solution after dialysis, carrying out rotary evaporation, and carrying out freeze drying.

Technical Field

the invention relates to the field of analytical chemistry and nano materials, in particular to a carbon dot fluorescent probe for detecting glutathione and a preparation method thereof.

Background

Glutathione (GSH) is a non-protein sulfhydryl compound with high content in cells, is a small molecular peptide consisting of cysteine, glutamic acid and glycine, is used as an important free radical scavenger and antioxidant in vivo, can combine harmful substances such as heavy metal, free radicals and the like, and discharges the harmful substances out of the body, and plays a very important role in human bodies. Thus, changes in glutathione content can be associated with a number of diseases, such as neurodegeneration, cancer, psoriasis, aids, diabetes, and the like. The research on the recognition and the sensing of the glutathione is an important subject in the fields of biochemistry and pharmaceutical chemistry, can provide a premise for detecting the pathogenicity of a plurality of diseases, further can more fully understand the characteristics of the occurrence, the development and the like of the diseases, and provides powerful help for the targeted treatment of the diseases.

Compared with traditional detection methods such as spectrophotometry, chromatography, capillary electrophoresis, electrochemistry and the like, the fluorescence analysis method based on the fluorescent probe has the advantages of simple test, high selectivity, short response time and the like. More importantly, the fluorescent probe can also be applied to real-time monitoring and biological imaging research in a living body, so that the fluorescent probe is widely applied to the fields of biomedicine, analytical chemistry, chemical biology and the like.

carbon dots are a novel carbon nano material, have good water solubility and biocompatibility, and have the characteristics of adjustable fluorescence emission wavelength, high fluorescence quantum yield, stable structure, strong photobleaching resistance and the like, so that the carbon dots are used as fluorescent probes and applied to biochemical detection, which are more and more concerned by people, but the carbon dots are lack of the fluorescent probes at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing a carbon dot fluorescent probe for detecting glutathione and a preparation method thereof aiming at the defects in the prior art.

In order to solve the technical problems, the invention adopts the technical scheme that: a carbon dot fluorescent probe for detecting glutathione comprises a carbon dot and 2, 4-dinitrobenzene sulfonic acid acyl compounded on the surface of the carbon dot.

the preparation method comprises the following steps:

1) Preparing carbon dots;

2) Modifying the surface of the obtained carbon dot with 2, 4-dinitrobenzene sulfonic acid acyl.

Preferably, the step 1) includes: dissolving o-phenylenediamine in DMF, stirring uniformly to dissolve the o-phenylenediamine completely, transferring the solution into a reaction kettle, heating and reacting; and filtering the reacted solution, dialyzing by using a dialysis bag, taking the internal solution after dialysis, and freeze-drying after rotary evaporation to obtain carbon dots.

Preferably, the step 1) specifically includes: weighing 600mg of o-phenylenediamine, dissolving the o-phenylenediamine in 50mL of DMF (dimethyl formamide), uniformly stirring the o-phenylenediamine to be completely dissolved, transferring the solution into a reaction kettle with a polytetrafluoroethylene substrate of 80mL, and reacting the solution for 8 hours at 180 ℃;

After the reaction kettle is cooled, filtering the solution after reaction, dialyzing the solution in water for 24 hours by using a dialysis bag with the molecular weight cutoff of 500D, and changing the water for 2 times; and (4) taking internal liquid after dialysis, carrying out rotary evaporation, and freeze-drying to finally obtain the carbon dots.

Preferably, the step 2) comprises the steps of dissolving the carbon dot powder prepared in the step 1) in anhydrous acetonitrile, adding K ₂ CO ₃, dropwise adding an anhydrous acetonitrile solution containing 2, 4-dinitrobenzenesulfonyl chloride into the solution, stirring the obtained mixture in an ice bath, removing the solvent in vacuum after the reaction is finished, dissolving the residue in water, dialyzing the residue in water by using a dialysis bag, taking the inner liquid after the dialysis, and performing rotary evaporation and freeze drying to obtain the carbon dot fluorescent probe.

Preferably, the step 2) specifically comprises the steps of weighing 100mg of carbon dot powder, dissolving the carbon dot powder in 10mL of anhydrous acetonitrile, adding 200mg of K ₂ CO ₃, dropwise adding 10.0mL of anhydrous acetonitrile solution containing 288.0mg of 2, 4-dinitrobenzenesulfonyl chloride into the solution, stirring the obtained mixture in an ice bath for 3 hours, removing the solvent in vacuum after the reaction is finished, dissolving the residue in a small amount of water, dialyzing the residue in water for 24 hours by using a dialysis bag with the molecular weight cutoff of 500D, taking the internal solution after dialysis, and freeze-drying the internal solution after rotary evaporation.

the invention has the beneficial effects that: the carbon dot fluorescent probe for detecting the glutathione is designed based on the carbon dot, the carbon dot fluorescent probe has no fluorescence, and the fluorescence of the carbon dot fluorescent probe is obviously enhanced along with the increase of the concentration of the glutathione; the carbon dot fluorescent probe has the characteristics of high sensitivity, good specificity and the like.

Drawings

FIG. 1 is a schematic illustration of carbon dot synthesis in one embodiment of the present invention;

FIG. 2 is a schematic illustration of a carbon dot surface modification in an embodiment of the present disclosure;

FIG. 3 is a transmission electron micrograph of a carbon dot prepared in one embodiment of the present invention;

FIG. 4 is an excitation and emission spectrum of a fabricated carbon dot in an embodiment of the present invention;

FIG. 5 is a diagram illustrating the detection result of a carbon dot fluorescent probe in an embodiment of the present invention;

FIG. 6 is a schematic diagram of the detection mechanism of the carbon dot fluorescent probe in an embodiment of the present invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The carbon dot fluorescent probe for glutathione detection is characterized by comprising a carbon dot and 2, 4-dinitrobenzenesulfonyl acyl compounded on the surface of the carbon dot.

the preparation method of the carbon dot fluorescent probe comprises the following steps:

1) Preparing carbon dots;

2) Modifying the surface of the obtained carbon dot with 2, 4-dinitrobenzene sulfonic acid acyl.

In this embodiment, the preparation method of the carbon dot fluorescent probe specifically comprises:

1) Preparing a carbon dot: weighing 600mg of o-phenylenediamine, dissolving the o-phenylenediamine in 50mL of DMF (dimethyl formamide), uniformly stirring the o-phenylenediamine to be completely dissolved, transferring the solution into a reaction kettle with a polytetrafluoroethylene substrate of 80mL (namely the reaction kettle with the volume of 80 mL), and reacting the solution for 8 hours at 180 ℃;

After the reaction kettle is cooled, filtering the solution after reaction, dialyzing the solution in water for 24 hours by using a dialysis bag with the molecular weight cutoff of 500D, and changing the water for 2 times; and (4) taking the internal liquid after dialysis, carrying out rotary evaporation, and freeze-drying to finally obtain brown powder, namely the prepared carbon dots. Referring to FIG. 1, a schematic diagram of carbon dot synthesis is shown.

2) Carbon dot surface modification, namely weighing 100mg of carbon dot powder, dissolving the carbon dot powder in 10mL of anhydrous acetonitrile, adding 200mg of K ₂ CO ₃, dropwise adding 10.0mL of anhydrous acetonitrile solution containing 288.0mg of 2, 4-dinitrobenzenesulfonyl chloride into the solution, stirring the obtained mixture in an ice bath for 3 hours, removing the solvent in vacuum after the reaction is finished, dissolving the residue in a small amount of water, dialyzing the residue in water for 24 hours by using a dialysis bag with the molecular weight cutoff of 500D, taking the internal liquid after the dialysis, performing rotary evaporation, and performing freeze drying, wherein the schematic diagram of the carbon dot surface modification is shown in figure 2.

Referring to fig. 3, a Transmission Electron Microscope (TEM) photograph of the carbon dot prepared in step 1). As can be seen from the figure, the size of each carbon dot is about 5-6nm, and the uniformity of the carbon dot is good.

Referring to fig. 4, the excitation spectrum and the emission spectrum of the carbon dot (without surface modification) prepared in step 1) are shown, and it can be seen from the figure that the excitation spectrum of the carbon dot is 440nm, the emission spectrum is 550nm, and the green light can be seen with naked eyes.

The detection capability of the carbon dot fluorescent probe prepared in the step 2) is tested. First, probe solutions with a certain concentration are prepared, glutathione aqueous solutions (0, 10,20,30,40,50,60,70 and 100 μ M) with different concentrations are respectively added, and the peak positions in fig. 5 are 0, 10,20,30,40,50,60,70 and 100 μ M from bottom to top in sequence through arrows. Then, the fluorescence intensity was measured by irradiating the sample with 440nm excitation light. As can be seen from FIG. 5, the fluorescence intensity of the carbon dot fluorescent probe at 550nm significantly increased with increasing GSH concentration. The probe is proved to be capable of effectively detecting the concentration of GSH in the solution.

The carbon dot fluorescent probe disclosed by the invention has no fluorescence, and the fluorescence of the probe is obviously enhanced along with the increase of the concentration of glutathione. The detection mechanism of the carbon dot fluorescent probe is as follows: the carbon dot fluorescent probe is formed by compounding a carbon dot and 2, 4-dinitrobenzenesulfonyl on the surface, and a Photoinduced Electron Transfer (PET) process exists between the carbon dot and the 2, 4-dinitrobenzenesulfonyl, so that the fluorescence emission of the carbon dot is inhibited. As shown in FIG. 6, in the presence of GSH, the 2, 4-dinitrobenzenesulfonyl group on the surface of the carbon spot was reacted and detached, and the PET mechanism was interrupted, thereby recovering the fluorescence of the carbon spot.

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.