阅读说明：本技术 一种基于碳点的双发射比率荧光探针、制备方法及在多巴胺检测中的应用 (Double-emission-ratio fluorescent probe based on carbon dots, preparation method and application in dopamine detection ) 是由 刘玉菲 安佳 陈美珠 胡楠 于 2019-09-30 设计创作，主要内容包括：本发明公开一种基于碳点的双发射比率荧光探针、制备方法及在多巴胺检测中的应用。本发明的荧光探针包括碳点和香豆素,且碳点与香豆素之间通过酰胺键偶联,当用300nm的单一激发光照射该碳点基双发射比率荧光探针时,其在455nm和505nm处分别具有发射峰。本发明的制备方法简单,且得到的探针能够对多巴胺进行精确检测,不仅可以有效消除背景信号的干扰,而且具有灵敏度高、选择性高等优点。(The invention discloses a carbon dot-based dual-emission-ratio fluorescent probe, a preparation method and application in dopamine detection. The fluorescent probe comprises a carbon dot and coumarin, the carbon dot and the coumarin are coupled through an amide bond, and when the carbon dot-based dual-emission-ratio fluorescent probe is irradiated by single excitation light of 300nm, the carbon dot-based dual-emission-ratio fluorescent probe respectively has emission peaks at 455nm and 505 nm. The preparation method is simple, and the obtained probe can accurately detect dopamine, can effectively eliminate the interference of background signals, and has the advantages of high sensitivity, high selectivity and the like.)

1. A carbon dot-based dual emission ratio fluorescent probe, comprising a carbon dot and coumarin coupled to each other through an amide bond, wherein the carbon dot-based dual emission ratio fluorescent probe has emission peaks at 455nm and 505nm when irradiated with a single excitation light of 300 nm.

2. The carbon dot-based dual emission ratio fluorescent probe according to claim 1, wherein the amide bond is obtained by chemically reacting a carbon dot having a carboxyl group with coumarin having an amino group.

3. The carbon dot-based dual emission ratio fluorescent probe according to claim 1, wherein the amino-containing coumarin is 7-amino-4 methylcoumarin.

4. The carbon dot-based dual emission ratio fluorescent probe according to claim 1, wherein the carbon dot is a yellow carbon dot.

5. A preparation method of a carbon dot-based dual-emission-ratio fluorescent probe is characterized by comprising the following steps:

(1) reacting carbon points with thionyl chloride in a first solvent to obtain carbon points of acyl chloride;

(2) and reacting the acyl-chlorinated carbon dot with coumarin with an amino group in a second solvent for 1-24 hours to obtain the carbon dot-based dual-emission ratio fluorescent probe.

6. The method as claimed in claim 5, wherein in step (1), the first solvent is acetonitrile and the amount of acetonitrile is 1.5-3.5 times of volume of thionyl chloride, and the reaction conditions include heating at a reaction temperature of 100-250 ℃ until the carbon dots are completely dispersed, and then continuing heating until the first solvent is evaporated to dryness to obtain the chlorinated carbon dots.

7. The method for preparing the carbon dot-based dual emission ratio fluorescent probe according to claim 5, wherein in the step (2), the second solvent is anhydrous acetonitrile, and the amino group-containing coumarin is added in an amount sufficient to make the carbon dot weight 10-50 times of the amino group-containing coumarin.

8. The method of claim 5, further comprising (3) treating the carbon dot-based dual emission ratiometric fluorescent probe obtained in step (2) with a treating agent, wherein the treating agent comprises 1, 3-propanesultone, dioxane and triethylamine.

9. The application of the carbon dot-based dual-emission-ratio fluorescent probe in dopamine detection is characterized in that the carbon dot-based dual-emission-ratio fluorescent probe is the carbon dot-based dual-emission-ratio fluorescent probe according to any one of claims 1 to 4 or the carbon dot-based dual-emission-ratio fluorescent probe prepared by the method according to any one of claims 5 to 8.

10. The use according to claim 9, wherein said use comprises contacting said carbon dot-based dual emission ratio fluorescent probe with dopamine, followed by excitation with excitation light at 300nm, followed by measurement of the spectrum of emission wavelengths between 320 and 580 nm.

Technical Field

The invention relates to the field of dopamine detection, in particular to a carbon dot-based dual-emission-ratio fluorescent probe, a preparation method and application thereof in dopamine detection.

Background

Dopamine, an important neurotransmitter, is involved in many biological processes in the central nervous system, hormones and cardiovascular system. Many studies have shown that dysregulation of dopamine levels can lead to a variety of disorders in the nervous system, such as huntington's disease, parkinson's disease, alzheimer's disease and schizophrenia. Therefore, accurate detection of dopamine is of great significance for disease diagnosis. The existing methods for detecting dopamine comprise a chromatography method, an ultraviolet spectrophotometry method, an electrochemical method, a chemiluminescence method, an enzymatic method and the like. However, these methods have some drawbacks to a greater or lesser extent. For example, the requirement for testing personnel is high, the detection is time-consuming, the accuracy is poor, and the like. Therefore, it is urgently required to develop a method which can be operated simply and quickly and accurately.

Carbon dots, which is a material that has attracted attention in recent years, has the advantages of inexpensive and readily available raw materials, good water solubility and biocompatibility, low toxicity, and the like. The abundant oxygen-containing structure on the surface of the carbon dots not only enables the carbon dots to be well dispersed in an aqueous solution, but also has the potential of further modification and application. Many methods for preparing carbon dots are currently developed, including hydrothermal methods, chemical oxidation methods, electrochemical oxidation methods, laser ablation methods, and the like. The application of carbon dots mainly focuses on detection, biological imaging, photocatalysis, photoelectric devices and other fields.

Recently, many researchers have used carbon spots to detect dopamine. For example, CN105675559A discloses a method for detecting dopamine by using carbon dots as fluorescent probes. The method utilizes the phenomenon that the specificity of dopamine to a fluorescent carbon dot is enhanced, realizes the double functionalization of functional groups containing nitrogen and boron on the surface of the carbon dot by preparing a specific fluorescent probe, realizes the detection of trace dopamine, improves the detection sensitivity, and further realizes the detection of the dopamine with the concentration as low as 0.1 pM.

For another example, Qingdao university discloses a preparation method of a carbon dot/copper nanocluster complex ratiometric fluorescent dopamine probe in CN 106970061A. Firstly, preparing carbon dots for blue fluorescence emission, modifying the surfaces of the carbon dots with aminobenzene boric acid, then preparing bovine serum albumin stable copper nanoclusters for red fluorescence emission, mixing the carbon dots and the copper nanoclusters for reaction to prepare a double-fluorescence-emission carbon dot/copper nanocluster compound, then adding dopamine into a water dispersion of the carbon dot/copper nanocluster compound, measuring a fluorescence emission spectrum by using a fluorescence spectrometer, fitting a linear relation between the intensity of a ratio fluorescence peak and the coexistence concentration of the dopamine, and further constructing a ratio fluorescence dopamine probe based on the carbon dot/copper nanocluster compound. The probe has the advantages of simple preparation process, low preparation cost and high sensitivity of products.

For example, Hoviager et al [ ratio metric fluorescence and visual imaging detection of nanoparticles on carbons/coppers nanocapsules [ J ]. Talanta,2018,178:109-115 ] use probes prepared from carbon dots and gold nanoclusters for dopamine detection.

In summary, although many carbon-based probes have been developed to detect dopamine, these probes are mainly focused on sensitivity and still need to be further improved with respect to selectivity.

Disclosure of Invention

In order to solve at least some of the technical problems of the prior art, the present inventors have conducted intensive studies and found that by coupling a carbon dot (particularly a yellow carbon dot) and a blue coumarin, peaks emitted at 455nm and 505nm are observed upon excitation with 300nm excitation light, and further that dopamine simultaneously quenches the two types of fluorescence of the carbon dot and coumarin to different degrees, and based on this, dopamine can be quantitatively detected by the ratio of the fluorescence intensities of the carbon dot and coumarin. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.

In a first aspect of the invention, a carbon dot-based dual emission ratiometric fluorescent probe is provided. The carbon dot-based dual-emission ratiometric fluorescent probe comprises a carbon dot and coumarin, and the carbon dot and the coumarin are coupled through an amide bond. The carbon dot-based dual emission ratio fluorescent probe has two emission peaks at 455nm and 505nm when irradiated with a single excitation light of 300 nm.

In certain exemplary embodiments, the amide bond is obtained by chemically reacting a carbon site having a carboxyl group with a coumarin having an amino group.

In certain exemplary embodiments, the amino-bearing coumarin is 7-amino-4 methylcoumarin.

In certain exemplary embodiments, the carbon dot is a carbon dot with yellow fluorescence and the coumarin is a coumarin with blue fluorescence.

In a second aspect of the present invention, there is provided a method for preparing a carbon dot-based dual emission ratio fluorescent probe, comprising the steps of:

(1) reacting carbon points with thionyl chloride in a first solvent to obtain carbon points of acyl chloride;

(2) and reacting the acyl-chlorinated carbon dot with coumarin with an amino group in a second solvent for 1-24 hours to obtain the carbon dot-based dual-emission ratio fluorescent probe.

In certain exemplary embodiments, the first solvent in step (1) is acetonitrile and is used in an amount of 1.5 to 3.5 times the volume of thionyl chloride, and the reaction conditions include heating at a reaction temperature of 100 ℃ and 250 ℃ until the carbon sites are completely dispersed, and then continuing heating until the first solvent is evaporated to dryness to obtain the carbon sites of the acyl chloride.

In certain exemplary embodiments, the second solvent in step (2) is anhydrous acetonitrile and the amount of amino-bearing coumarin added is sufficient to provide a weight of the carbon dots that is 10 to 50 times the weight of the amino-bearing coumarin.

In certain exemplary embodiments, the method for preparing the carbon dot-based dual emission ratiometric fluorescent probe further comprises (3) a step of treating the carbon dot-based dual emission ratiometric fluorescent probe with a treating agent, wherein the treating agent comprises 1, 3-propanesultone, dioxane, and triethylamine.

In a third aspect of the present invention, there is provided a use of the carbon dot-based dual emission ratio fluorescent probe according to the first aspect of the present invention or the carbon dot-based dual emission ratio fluorescent probe obtained by the method according to the second aspect of the present invention in dopamine detection.

In certain exemplary embodiments, the application of the present invention comprises contacting a carbon dot-based dual emission ratio fluorescent probe with a dopamine solution, followed by excitation with excitation light at 300nm, followed by measurement of the spectrum of emission wavelengths between 320 and 580 nm.

The fluorescent probe of the present invention is a ratiometric fluorescent probe, which is an important probe in a fluorescence detection method. The ratio type fluorescent probe quantitatively detects a target substance by using the ratio of two fluorescent substances as a detection signal. The ratio of the fluorescence intensity of the carbon dots and the coumarin in the ratiometric fluorescent probe shows a good linear relation in the range of 0-33.6 mu M, so that the dopamine can be accurately detected, the interference of background signals can be effectively eliminated, and the ratiometric fluorescent probe has the advantages of high sensitivity and accuracy, good selectivity or specificity and the like. In addition, the preparation method of the ratio type fluorescent probe is simple.

Drawings

FIG. 1 is a schematic diagram of an exemplary carbon dot-based dual emission ratio fluorescent probe preparation process.

FIG. 2 is a fluorescence spectrum of the carbon dot-based dual emission ratio fluorescent probe of the present invention.

FIG. 3 is a fluorescence spectrum of the carbon dot-based dual emission ratio fluorescence probe of the present invention with different concentrations of dopamine added.

FIG. 4 is a graph of fluorescence intensity ratio versus dopamine concentration for an exemplary carbon-dot based dual emission ratio fluorescent probe of the present invention.

FIG. 5 shows the effect of interferents on dopamine assay in the dopamine assay of the present invention. In the figure, every two columns form a group, the left column in each group of columns is probe + interference, and the right side is probe + interference + DA.

FIG. 6 shows the results of selective detection of carbon dot-based dual emission ratio fluorescent probe according to the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

In the present invention, the term "carbon-spot-based dual emission ratio fluorescent probe", which may also be referred to as "carbon-spot-coumarin fluorescent probe", or "carbon-spot-based dual emission ratio fluorescent probe", refers to a complex having two detectable signals. Unlike the existing complex, the carbon-dot-based dual emission ratiometric fluorescent probe herein has not only a carbon dot as an inorganic moiety but also coumarin as an organic moiety.

In the present invention, the term "coumarin having an amino group" refers to a compound in which any atom or group on the coumarin is substituted with an amino group. The amino group may be directly bonded to an atom in the cyclic structure of the following formula (I), or may be directly bonded to an atom in a substituent at the 3-8 position in the structure of the following formula (I), and is not particularly limited herein. In certain embodiments, the amino group is located anywhere from 3-8 positions of the cyclic structure of formula (I) by substitution with a hydrogen atom. The number of amino groups in one molecule of coumarin is not limited, and may be one amino group or two or more amino groups.

[ carbon dot-based double emission ratio fluorescent probe ]

In a first aspect of the invention, a carbon dot-based dual emission ratiometric fluorescent probe is provided, which comprises a carbon dot and coumarin, wherein the carbon dot and the coumarin are coupled through an amide bond. The carbon dot-based dual emission ratio fluorescent probe of the present invention is preferably a water-soluble probe, and for this reason, it may be preferable to increase its water solubility by water-soluble treatment. When the carbon dot-based dual emission ratiometric fluorescent probe was irradiated with 300nm single excitation light, it had emission peaks at 455nm and 505nm, respectively.

In the invention, the carbon dots refer to fluorescent carbon nano-materials with particle size less than 10nm and good biocompatibility, and the structure of the fluorescent carbon nano-materials includes but is not limited to a crystal lattice or a non-crystal lattice structure. The carbon dots can be prepared by known methods using known products. The specific synthesis method is not particularly limited, and may be a top-down synthesis or a bottom-up synthesis. Wherein from top to bottom means that a zero-dimensional carbon dot is formed by cutting and cracking a three-dimensional or two-dimensional carbon material. Namely, the carbon quantum dots with small size are stripped from the carbon source with large size by a physical or chemical method. The bottom-up method is mainly to obtain carbon dots by condensation and carbonization of small molecules or polymers. Such as chemical oxidation, combustion, hydrothermal/solvothermal, microwave synthesis, or templating.

In certain embodiments, the carbon dots used in the present invention are yellow carbon dots. The invention finds that the yellow carbon dots can avoid the interference of other components in the sample, particularly components derived from organisms, and further improves the detection specificity and sensitivity of dopamine. Preferably, the yellow carbon dot of the present invention has a single emission peak of 505nm when irradiated with a single laser of 300 nm. The yellow carbon dots of the present invention can be synthesized by chemical oxidation. In an exemplary synthesis method, the synthesis of the yellow carbon dot comprises the steps of: adding 0.1-1g of carbon fiber powder into 30 ml of mixed acid of concentrated sulfuric acid and concentrated nitric acid, keeping the solution boiling, and refluxing for 0.5-6 h; neutralizing the solution with sodium bicarbonate to pH7 after the reaction, filtering with a filter (e.g., a 0.22 μm filter) twice, and dialyzing the obtained filtrate for 1 to 10 days; the samples were freeze-dried and stored in a desiccator to obtain the carbon dots of the present invention.

In the present invention, coumarin is a generic name for a compound having a cyclic structure represented by the above formula (I). Specifically, the coumarin of the invention can be a compound shown in the formula (I) or a compound with a substituent at the 3-8 position of the structure of the formula (I), wherein the substituent is not particularly limited, and is preferably a straight-chain substituent, more preferably a C1-C8 alkyl substituent, such as a C1-C4 alkyl substituent or a C1-C2 alkyl substituent. Preferably, coumarin has an emission peak at 455nm when it is irradiated with a single laser at 300 nm.

In the carbon dot-based dual emission ratio fluorescent probe of the present invention, the molar ratio of the carbon dot to the coumarin is not particularly limited, and may be, for example, 1:1 to 1:10, preferably 1:1 to 1:8, and more preferably 1:1 to 1: 5. In certain embodiments, the molar ratio of carbon to coumarin is 1: 4.

In the present invention, an amide bond means a group formed by a chemical reaction between a carboxyl group and an amino group. The coumarin compound can be formed by chemical reaction of the coumarin with the carboxyl functional group and the carbon point with the amino functional group, or can be formed by chemical reaction of the carbon point with the carboxyl functional group and the coumarin with the amino functional group. In the present invention, an amide bond obtained by a chemical reaction between a carbon point having a carboxyl group and coumarin having an amino group is preferably used.

In the present invention, the number of amide bonds in one molecular probe is not particularly limited. As mentioned above, the number of amide bonds varies with the number of amino groups in the coumarin and the molar ratio of carbon to coumarin. Those skilled in the art can freely set them as needed.

[ production method ]

In a second aspect of the present invention, a method for preparing a carbon dot-based dual emission ratio fluorescent probe is provided, which at least comprises the following two steps:

(1) reacting carbon points with thionyl chloride in a first solvent to obtain carbon points of acyl chloride;

(2) and reacting the acyl-chlorinated carbon dot with coumarin with an amino group in a second solvent for 1-24 hours to obtain the carbon dot-based dual-emission ratio fluorescent probe.

Step (1) of the present invention is a step of obtaining an acylchlorocarbon site. In this step, the first solvent may be tetrahydrofuran, dichloromethane, or acetonitrile. Preferably, acetonitrile is used as solvent, generally in an amount of 1.5 to 3.5 times, preferably 2 to 3 times, the volume of thionyl chloride. In a particular embodiment, 2 to 10ml of acetonitrile may be used as solvent.

The reaction conditions of the step (1) comprise heating at the reaction temperature of 100-250 ℃ until the carbon points are completely dispersed, and continuing heating until the first solvent is evaporated to dryness. Preferably, the reaction temperature is 120-200 deg.C, more preferably 135-160 deg.C.

The step (2) is a step of obtaining the carbon dot-based dual-emission-ratio fluorescent probe. Wherein the second solvent is a pure solvent, preferably anhydrous acetonitrile. The weight ratio of the amount of the coumarin having an amino group added in this step to the carbon dots in step (1) is preferably 1:10 to 50, preferably 1:15 to 40, more preferably 1:20 to 30. In a specific embodiment, the amount of amino group-bearing coumarin added in this step is from 0.01 to 5 mg.

The reaction conditions of the step (2) include carrying out the reaction at ordinary temperature, for example, for 1 to 24 hours, preferably for 1 to 20 hours, more preferably for 2 to 10 hours. The amount of the anhydrous acetonitrile is not particularly limited as long as it can dissolve the carbon dots prepared in the step (1) and uniformly disperse them in the solvent.

In addition to the above steps (1) and (2), the preparation process of the carbon dot-based ratiometric fluorescent probe of the present invention preferably further comprises a step of treating the fluorescent probe sample obtained in step (2) with a treating agent. The treating agents of the present invention include, but are not limited to, 1, 3-propanesultone, dioxane, and triethylamine. Preferably, the treating agent of the present invention consists of 1, 3-propane sultone, dioxane and triethylamine. In general, the ratio of the three components is 0.05-1g of 1, 3-propanesulfonic lactone, 10mL of dioxane and 100-.

In the present invention, when the fluorescent probe sample obtained in step (2) is treated with the treating agent, it is preferably carried out in an aqueous environment (preferably, a pure water environment). Specifically, it is necessary to react the fluorescent probe obtained in step (2) with a treating agent in water. For this purpose, it is necessary to first subject the fluorescent probe obtained in step (2) to a solvent removal step, for example, by subjecting the obtained sample to rotary evaporation to remove the solvent. The fluorescent probe after removal of the solvent is then dispersed in an aqueous environment, such as pure water.

In the present invention, the amount of the treating agent to be added is not particularly limited, but generally, when a fluorescent probe obtained with 0.5 to 50mg of carbon dots is dissolved in 1mL of pure water, the amount of the treating agent to be added is 10 to 25 mL.

In the step (3), the reaction conditions for treating the treating agent comprise 35-45 ℃ water bath for 6-48 h. The fluorescent probe treated by the treating agent has improved water solubility. Preferably, the reaction conditions are water baths 38-42 deg.C, more preferably 39-41 deg.C. The reaction time is preferably 10 to 30 hours. Exemplary reaction conditions include a water bath temperature of 40 ℃ and a reaction time of 6 to 48 hours.

In the present invention, after the treatment with the treating agent, an evaporation process may be further included for the fluorescent probe sample in order to remove the solvent. The evaporation process of the present invention may be carried out by any known method, and in particular embodiments, by removing the solvent by rotary evaporation.

Optionally, after the treatment with the treating agent and the removal of the solvent by rotary evaporation, the method further comprises the steps of dispersing the obtained sample in a pure water environment again, removing free coumarin by using an extraction solvent, removing the solvent by rotary evaporation of the sample again, and finally performing dialysis by using a semipermeable membrane. The extraction and semi-permeable membrane treatment processes adopt a common method. Wherein the extractant is neutral organic compound, such as carboxylic ester, etc., and in specific embodiment, ethyl acetate is used as extractant. The semipermeable membrane used is a dialysis bag with molecular weight cut-off of 3500Dalton, and in a specific embodiment, dialysis conditions of 7 days and 4-5 water changes per day are used.

[ detection of dopamine ]

The carbon dot-based dual-emission ratiometric fluorescent probe can be used for detecting dopamine. When the carbon dot-based dual-emission-ratio fluorescent probe prepared by the method is used for detecting dopamine, the fluorescence of the carbon dots and coumarin is quenched to different degrees, and the dopamine can be accurately and quantitatively detected by calculating the intensity ratio of the two characteristic peaks.

In certain embodiments, the assays of the invention (or uses of the invention) comprise: dispersing a carbon dot-based ratio type fluorescent probe sample in phosphate buffer solution, adding the dispersed sample solution into dopamine solutions with different concentrations respectively, incubating for 1-10min, and adding into a cuvette for detection. Preferably, the slit width of excitation and emission is set to 10nm, and the spectrum of emission wavelength 320-580nm is measured at 300nm for the excitation light. In an exemplary embodiment, the buffer used in the assay is Phosphate Buffered Saline (PBS) at pH7.2, 0.01M, and the concentration of dopamine solution is 0-2400. mu.M.

The selectivity of fluorescent probes is a key and essential parameter for evaluating the feasibility of a sensing system or probe. The specificity of the carbon dot-based dual-emission fluorescent probe is researched by comparing the carbon dot-based dual-emission fluorescent probe with substances commonly found in human blood. Specifically, the selective experiment was carried out by replacing the dopamine solution with uric acid, glycine, L-cysteine, ascorbic acid, alanine, glutathione, glucose, urea, sodium chloride and potassium chloride solutions at concentrations of 0.1M, preferably.