CN108998012B

CN108998012B - Blue fluorescent quantum dot, preparation method thereof and copper ion detection application

Info

Publication number: CN108998012B
Application number: CN201810936227.5A
Authority: CN
Inventors: 黄赛朋; 成杰伟; 薛伟明; 李文帅; 温惠云
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2018-08-16
Filing date: 2018-08-16
Publication date: 2021-07-23
Anticipated expiration: 2038-08-16
Also published as: CN108998012A

Abstract

The invention discloses a blue fluorescent quantum dot and a preparation method and application thereof, and belongs to the technical field of nano materials. The preparation method comprises the following steps: 1) preparing a carbon quantum dot precursor by using citric acid as a carbon source and using a polyethylene diamine dendritic polymer with amino as a nitrogen source through a decocting method; 2) and modifying the carbon quantum dot precursor by using ethylenediamine as a modifying molecule and methyl acrylate as a linking molecule to prepare the blue fluorescent quantum dot. The preparation method has the advantages of reasonable route design, simple operation, good repeatability and low requirement on equipment; the prepared blue fluorescent quantum dot has good water solubility, high stability and excellent fluorescence performance, and can be used for detecting copper ions specifically.

Description

Blue fluorescent quantum dot, preparation method thereof and copper ion detection application

Technical Field

The invention belongs to the technical field of nano materials, and particularly relates to a blue fluorescent quantum dot and a preparation method and application thereof.

Background

The life activities of any living matter in nature cannot be kept away from water. However, with the rapid development of society, human dependence on water resources is increasing, and the problem of water pollution caused by improper treatment of industrial wastes is attracting much attention. Heavy metals are very easy to be enriched in organisms and transferred along with a biological chain, so that great threat is brought to the ecological environment and the life health of human beings. Tin, copper, lead, zinc, mercury, chromium, etc. have significant biological toxicity, and these heavy metals can be converted into substances more toxic to organisms by the action of microorganisms. Therefore, they have been blacklisted for controlling contaminants. Relevant regulations are provided by governments of various countries and united nations of the world and environmental protection organizations and the like, and the heavy metal content in drinking water and food is strictly limited. China also makes a clear regulation on the content of heavy metals in drinking water and food: the maximum limit of chromium in the drinking water is 0.05 mg.L < -1 >, the maximum limit of aluminum is 0.2 mg.L < -1 >, and the maximum limit of copper is 1 mg.L < -1 >. The grain has a copper content of 10 mg/kg-1, a lead content of 0.2 mg/kg-1 and a chromium content of 0.2 mg/kg-1. The content of lead in livestock meat should not exceed 0.2 mg/kg-1, the content of chromium should not exceed 0.1 mg/kg-1, and the content of copper should not exceed 10 mg/kg-1. Meanwhile, according to the regulation of the health and environmental protection organization of the United nations, the content of copper ions in the drinking water must not exceed 10 mu mol.L-1

Copper is the metal that has been found in the earliest days in humans, and is also the metal used most widely in humans, and is widely used in agricultural and industrial products, such as: reagents, pesticides, plasticizers, emulsifiers, coatings, and the like. Meanwhile, copper also plays an important role in regulating physiological activities of organisms. On the one hand, a moderate amount of copper plays no alternative role in normal physiological activities in the organism. In the body, many enzymes involved in physiological activities are involved in copper, such as ascorbate oxidase, superoxide dismutase, ascorbate oxidase, etc., which are inseparable from electron transfer redox reactions in biotransformation. At the same time, the metabolism of many enzymes is also closely related to copper. In the organism, the copper ions play an important role in the absorption and transportation of the iron element. The lack of copper element can inhibit the synthesis of hemoglobin in an organism, so that the content of the hemoglobin is reduced, and functional anemia is caused; meanwhile, copper ions also have certain influence on endocrine system and nervous system. The deficiency of copper has an important influence on the activity of ceruloplasmin, superoxide dismutase, cytochrome oxidase, etc. in nerve tissues, and clinically, symptoms such as Wilson Disease, Alzheimer Disease, Parkinsonism, Central Nervous System Disease, etc. are mainly manifested; meanwhile, the physiological activities of copper bones and cartilages and connective tissues thereof also have a crucial influence, and the deficiency of copper elements can influence the bone growth of children. On the other hand, too high content of copper in the body may also cause certain harm. According to the world health organization study, the safe copper intake of infants, children and adolescents was 80,40 and 30 mug kg-1 per day, respectively. Meanwhile, according to the Chinese Nutrition Society report, the safe intake of copper for adults every day is 210-300 mg; similarly, the National food nutrition Association (National Nutrition Foods Association) recommends an approval of a safe daily intake of 115-310 mg of copper. According to related researches, the excessive copper content in the organism can cause hemolysis. Excessive copper intake for a long time can cause memory decline, nausea, abnormal liver function, enlargement of the liver and the like.

The traditional detection method of copper ions comprises a precipitation method, a color development method, a fluorescence probe method, a metal quantum dot method and the like, but the methods have the problems of low sensitivity, insufficient accuracy and the like, and are difficult to detect trace copper ions in organisms; meanwhile, the biotoxicity brought by the fluorescence probe method and the metal quantum dot method greatly limits the on-line detection and application of the metal quantum dot method. Therefore, development of a highly sensitive and low-biotoxicity method for detecting copper ions has been expected to detect a trace amount of copper ions, and to realize early diagnosis of diseases caused by accumulation of copper ions.

In recent years, with the development of nanomaterials, carbon quantum dots have been receiving attention as a new fluorescent nanomaterial. At present, the quantum yield of the fluorescent carbon quantum dots is improved by mainly using an organic solvent as a passivating agent to fill in the defects on the surfaces of the carbon quantum dots. However, the subsequent treatment of the passivating agent with an organic solvent is complicated. Therefore, researchers are looking for cheaper and simpler methods to improve the fluorescence quantum yield of carbon quantum dots. Because the radiuses of the N atoms and the C atoms are similar, the nitrogen-doped carbon quantum dots (N-CDs) with excellent performance can be obtained by carrying out nitrogen doping treatment on the carbon quantum dots in the process of preparing the carbon quantum dots.

At present, the reported nitrogen sources mainly include ethylenediamine, amino acids, dendrimers, and the like. However, these nitrogen-containing substances as raw materials are doped on the surface of the carbon quantum dots after the molecules are resolvable again through a violent reaction. The amino group on the surface of the carbon quantum dot can capture copper ions to form a copper ammonia complex and cover the surface of the carbon quantum dot, so that the fluorescence of CDs is quenched. However, most of the amino groups are oxidized, resolved or recombined during the preparation of the carbon quantum dots, so that the number of the prepared amino groups of the carbon quantum dots is reduced, and the sensitivity of the detection of copper ions is reduced.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide the blue fluorescent quantum dot and the preparation method and the application thereof, and the preparation method has the advantages of reasonable route design, simple operation, good repeatability and low requirement on equipment; the prepared blue fluorescent quantum dot has good water solubility, high stability and excellent fluorescence performance, and can be used for detecting copper ions specifically.

The invention is realized by the following technical scheme:

1) preparing a carbon quantum dot precursor by using citric acid as a carbon source and using a polyethylene diamine dendritic polymer with amino as a nitrogen source through a decocting method;

2) and modifying the carbon quantum dot precursor by using ethylenediamine as a modifying molecule and methyl acrylate as a linking molecule to prepare the blue fluorescent quantum dot.

Preferably, the step 1) of preparing the carbon quantum dot precursor by adopting a decoction method comprises the following steps:

1.1) adding citric acid: polyethylene diamine dendrimers with amino groups: water ═ 1 g: 0.5 g: dispersing citric acid and polyethylene diamine dendritic polymer in water at the dosage ratio of 5mL, and decocting at 200 ℃ for 2h to prepare brown solution;

and 1.2) centrifuging the brown solution, collecting supernatant, and sequentially filtering, dialyzing and drying the supernatant through a microporous filter membrane to obtain the carbon quantum dot precursor.

Further preferably, in the step 1.2), the supernatant is filtered by a 0.22 μm syringe filter membrane, and the filtered solution is dialyzed by a 10000Da dialysis bag for 8h to prepare the carbon quantum dot precursor with the average particle size of 4.4 nm.

Preferably, in the step 2), the carbon quantum dot precursor is modified by using ethylenediamine as a modifying molecule and methyl acrylate as a linking molecule, and the specific operations include:

2.1) extracting the carbon quantum dot precursor with methanol for several times, collecting methanol extract, dripping methyl acrylate into the methanol extract, reacting for 24 hours at 30 ℃, performing rotary evaporation on the solution after reaction to remove methanol, and washing with methanol for several times;

2.2) as methanol: ethylenediamine ═ 10: 16, adding methanol into the washed solution, dropwise adding ethylenediamine, and reacting at 30 ℃ for 24 hours; removing methanol from the reacted solution by rotary evaporation, and washing the solution for a plurality of times by using methanol to prepare a yellow solution; and dialyzing the obtained yellow solution, and carrying out freeze drying treatment to obtain the blue fluorescent quantum dots.

Preferably, the specific preparation method of the polyethylene diamine dendritic polymer with amino groups is as follows:

step 1: fully and uniformly stirring ethylenediamine and methanol according to the volume ratio of 4:10, dropwise adding methyl acrylate into the solution, and reacting for 24 hours at 30 ℃; the volume ratio of methyl acrylate to ethylenediamine is 1: 4;

step 2: performing rotary evaporation on the solution reacted in the step 1 to remove methanol, washing the solution for a plurality of times by using methanol, then adding methanol into the washed solution, performing ultrasonic dispersion, dropwise adding ethylenediamine into the dispersed solution, and reacting for 24 hours at the temperature of 30 ℃; the volume ratio of the methanol to the ethylenediamine is 10 (3-10);

and step 3: removing methanol from the solution reacted in the step 2 by rotary evaporation, and washing the solution for a plurality of times by using methanol to obtain yellow oily liquid which is a generation of dendrimer 1G-PAMAM;

and 4, step 4: fully and uniformly stirring the first generation of dendrimer 1G-PAMAM and methanol according to the volume ratio of 4:10, dropwise adding methyl acrylate into the solution, and reacting for 36 hours at 35 ℃; the mass ratio of the methyl acrylate to the first generation dendrimer 1G-PAMAM is 1: 4;

and 5: performing rotary evaporation on the solution reacted in the step 4 to remove methanol, washing the solution for a plurality of times by using methanol, then adding the methanol into the washed solution, performing ultrasonic dispersion, dropwise adding a generation of dendrimer 1G-PAMAM into the dispersed solution, and reacting for 48 hours at 35 ℃; the volume ratio of the methanol to the first generation of dendrimer 1G-PAMAM is 20 (3-10);

step 6: removing methanol from the solution reacted in the step 5 by rotary evaporation, and washing the solution for a plurality of times by using methanol to obtain the second generation dendrimer 2G-PAMAM;

the first generation of dendrimer 1G-PAMAM and the second generation of dendrimer 2G-PAMAM are both polyethylene diamine dendrimers with amino groups which can be used as nitrogen sources.

The invention also discloses the blue fluorescent quantum dot prepared by the method, wherein the average particle size of the blue fluorescent quantum dot is 25.3nm, and the surface charge is +94.4 mV.

The invention also discloses application of the blue fluorescent quantum dot as a copper ion detection fluorescent probe.

Preferably, the detection concentration interval of the blue fluorescence quantum dots to copper ions is 0.01 mu mol.L^-1～600μmol·L^-1。

Preferably, the blue fluorescent quantum dots can specifically detect copper ions in cells.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention uses citric acid as a carbon source, modified polyethylene diamine dendritic polymer as a nitrogen source, and unmodified carbon quantum dot precursors (bare-CDs) are obtained by a decoction method. Using ethylenediamine as a modifying molecule and methyl acrylate as a connecting molecule, modifying ethylenediamine on the surface of a carbon quantum dot precursor through an amide reaction to form an aminated carbon dot, and preparing blue fluorescent quantum dot NH₂CDs, blue fluorescent quantum dots NH₂The CDs emit blue fluorescence under a fluorometer or a laser confocal instrument and are quenched by the fluorescence of copper ions. The morphology and the structure of the bara are characterized by a dynamic light scattering particle size analyzer and a transmission electron microscope, the surface functional groups of the bara are characterized by an infrared spectrometer (FT-IR), and the prepared bare-CDs and NH are₂Average particle diameters of CDs of 4.4nm and 25.3nm, respectively, and surface charges of +40.1mV and +94.4mV, respectively, by infrared characterization, NH₂CDs at 1654cm^-1And 1706cm^-1The characteristic peak of amido bond appears, which indicates that the ethylenediamine is successfully grafted on the surface of bare-CDs.

The experiments of over-hydroxylation, carbon carboxyl and EDTA verification show that NH is promoted₂The main reason for quenching of the fluorescence of CDs is the capture of Cu in the solution by the amino groups on the surface²⁺And formation of copper ammonia complex. The optical performance of the material is researched by adopting an ultraviolet-visible spectrophotometer (UV-Vis) and a fluorescence spectrophotometer (PL), and PL and UV-Vis spectra show that Cu2+ can quench NH2-CDs fluorescence and Cu²⁺The detection interval of (a) is 0.01 to 600. mu. mol. L^-1The detection limit is 0.01 mu mol.L^-1. By specific experiments, NH₂CDs can be used as a specific high-efficiency detection reagent for Cu²⁺The fluorescent probe of (1). Meanwhile, the NH is explored by taking MCF-7, HepG-2 and L-929 cells as models₂-cytotoxicity of CDs, and observation of intracellular fluorescence imaging behavior by confocal laser microscopy, the experimental results show that Cu²⁺The presence of (A) can significantly quench NH₂Fluorescence of CDs, quenching of intracellular fluorescence with the addition of copper ions for 16s, indicating NH₂CDs can effect on Cu in cells²⁺The rapid detection of (2).

Drawings

FIG. 1 is a schematic diagram of a process for preparing and detecting copper ion flux of blue fluorescent quantum dots of the present invention;

FIG. 2A is a transmission electron micrograph and a particle size distribution plot of a carbon quantum dot precursor bare-CDs;

FIG. 2B shows blue quantum dots NH₂-transmission electron microscopy and particle size distribution plots of CDs;

FIG. 2C shows bare-CDs and NH₂-surface charge of CDs;

FIG. 2D shows bare-CDs and NH₂-ir spectra of CDs;

FIG. 3A is NH₂-plots of fluorescence intensity of CDs as a function of concentration;

FIG. 3B is NH₂-emission patterns of CDs under excitation at different wavelengths;

FIG. 4 shows 100. mu.g/ml^-1NH₂-ultraviolet-visible absorption spectra, fluorescence excitation and emission profiles of CDsA spectrum;

FIG. 5 shows 100. mu.g/ml^-1NH₂CDs at different concentrations of Cu²⁺A medium fluorescence spectrum;

FIG. 6 shows 100. mu.g/ml^-1NH₂CDs at a concentration of 10. mu. mol. L^-1Fluorescence intensity in different metal ion (A) and amino acid (B) solutions;

FIG. 7 shows the concentration of 100. mu.g/mL^-1The hydroxylated carbon site (A) and the carboxylated carbon site (B) of (A) are 10. mu. mol. multidot.mL^-1Fluorescence intensity in different metal ions;

FIG. 8 shows NH₂-fluorescence intensity of CDs in buffer (a) and NaCl solution (B) at different concentrations;

FIG. 9 shows (A) 100. mu.g/mL^-1NH of (2)₂Detection of Cu by CDs in solutions of different pH²⁺A fluorescence intensity map; (B) NH (NH)₂CDs at a concentration of 0,400. mu. mol. L^-1Cu²⁺And 400. mu. mol. L^-1Cu²⁺And 400. mu. mol. L^-1Fluorescence intensity map in EDTA solution;

FIG. 10 shows NH concentrations₂-effect of CDs on HepG-2, MCF-7 and L-929 cytotoxicity;

FIG. 11 shows HepG-2 and NH₂Co-cultivation with-CDs for 4h and then with Cu²⁺Confocal laser mapping at different time points;

FIG. 12 laser confocal, 100. mu.g/ml NH₂The fluorescent response of CDs to different concentrations of copper ions (250nM, 10. mu.M and 100. mu.M) in MCF-7 and HepG-2 cells.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1 preparation of carbon quantum dots

The amino-containing polyethylene diamine dendritic polymer used as the nitrogen source in the invention can be selected from commercially available products and can also be selected to be prepared by self.

Referring to fig. 1, the blue fluorescent quantum dot NH of the present invention₂-a process for the preparation of CDs comprising the steps of:

step 1: preparation of G-PAMAM

1) To a round bottom flask were added 4mL of ethylenediamine and 10mL of methanol, respectively, and stirred for 5 min. After stirring, 16mL of methyl acrylate is dropwise added into the solution and reacted for 24 hours at the temperature of 30 ℃;

2) after the reaction was completed, methanol was removed at 30 ℃ by a rotary evaporator, and washed three times with methanol to remove a large amount of methyl acrylate. To the solution obtained after the above rotary evaporation, 10mL of methanol was added and uniformly dispersed with sonication. Dropwise adding 16mL of ethylenediamine into the dispersed solution, and continuously reacting for 24 hours at the temperature of 30 ℃;

3) after the reaction, methanol was removed by a rotary evaporator at 30 ℃ and washed three times with methanol to remove a large amount of ethylenediamine, and a yellow oily liquid, i.e., a generation of dendrimer (1G-PAMAM), was obtained after the treatment.

4) Respectively adding 4mL of first-generation dendrimer 1G-PAMAM and 10mL of methanol into a round-bottom flask, stirring for 5min, dropwise adding 16mL of methyl acrylate into the solution after stirring, and reacting at 30 ℃ for 24 h;

5) after the reaction was completed, methanol was removed at 30 ℃ by a rotary evaporator, and washed three times with methanol to remove a large amount of methyl acrylate. Adding 10mL of methanol into the solution obtained after the rotary evaporation, and uniformly dispersing the methanol by using ultrasonic waves;

6) and dropwise adding 16mL of first-generation dendrimer 1G-PAMAM into the dispersed solution, continuing to react for 24h at 30 ℃, removing methanol at 30 ℃ by using a rotary evaporator after the reaction is finished, preparing second-generation dendrimer 2G-PAMAM, and storing in a refrigerator at 4 ℃.

The first generation of dendrimer 1G-PAMAM and the second generation of dendrimer 2G-PAMAM can be used as the polyethylene diamine dendrimer with amino groups to provide a nitrogen source required by the reaction.

Step 2: preparation of carbon dots by hydrothermal decocting method

1G of citric acid, 0.5G of the 2G-PAMAM prepared in step 1 were weighed accurately into a round bottom flask and dispersed by sonication with 5mL of purified water. And setting the temperature of an electric heating jacket to 200 ℃ to heat the solution for 2h to obtain brown liquid. The resulting brown solution was centrifuged at 10000rpm in a high speed centrifuge to remove substances carbonized during heating. And the supernatant obtained by centrifugation was filtered through a 0.22 μm syringe filter to obtain carbon quantum dot precursors (bare-CDs) having a smaller particle size. The filtered solution was dialyzed for 8h against a dialysis bag of molecular weight 1000Da to remove unreacted citric acid and 2G-PAMAM. The purified solution of bare-CDs was lyophilized to obtain solid bare-CDs and placed in a desiccator.

And step 3: NH (NH)₂Preparation of-CDs

The bare-CDs prepared in step 2 were extracted three times with methanol and the extracts were transferred to a round bottom flask. 16mL of methyl acrylate were measured and added dropwise to the extract to react at 30 ℃ for 24 h. After the reaction was completed, the reacted solution was rotary evaporated at 30 ℃ to remove methanol, and washed three times with methanol to remove unreacted methyl acrylate. To the above washed solution, 10mL of methanol was added and 16mL of ethylenediamine was added dropwise to react at 30 ℃ for 24 hours. After completion of the reaction, methanol was removed by rotary evaporation at 30 ℃ and washed three times with methanol to remove unreacted ethylenediamine. The resulting yellow solution was transferred to a dialysis bag with a molecular weight cut-off of 1000Da and dialyzed for 8h to remove unreacted ethylenediamine and methyl acrylate. The purified NH is removed₂-freeze-drying the CDs solution and placing the resulting dried solid in a desiccator.

Example 2 characterization of carbon quantum dots

1. Average particle diameter and Zeta potential characterization

Determination of bare-CDs and NH by ZEN3600 Malvern dynamic light scattering particle size analyzer₂Average particle size and Zeta potential of two samples of CDs, the test medium being water and the test temperature being 25 ℃.

2. TEM microscopic morphology and structure characterization

Taking small amounts of bare-CDs and NH₂And (3) respectively putting CDs into 5mL centrifugal tubes, adding 2mL anhydrous methanol into the centrifugal tubes, performing ultrasonic dispersion for 5min, uniformly dripping the samples on a 400-mesh copper net, and observing the appearance of the samples by adopting a Tecnai G2F20 field emission transmission electron microscope under 80KV accelerating voltage. Microscopic morphology observation was performed on the prepared bare-CDs and NH2-CDs samples using a Transmission Electron Microscope (TEM), a dynamic light scattering particle sizer (DLS) and a Fourier infrared spectrometer (FT-IR).

As shown in FIG. 2A, it can be seen that the bare-CDs prepared by hydrothermal decocting have regular spherical shape, good monodispersity among particles, and uniform size. The average particle size of bare-CDs was 4.4nm as determined by scanning with a Malvern dynamic light scattering particle sizer. Ethylenediamine is used as a surface modifier, methyl acrylate is grafted on the surfaces of bare-CDs through amido bonds, and bare-CDs are aminated to obtain NH₂-CDs. NH shown in FIG. 2B₂The average particle size of the CDs increased to 25.3 nm. As can be seen from the TEM image, NH₂The CDs have good monodispersity, uniform size and no agglomeration. Meanwhile, as can be seen from FIG. 2C, the Zeta potential of bare-CDs is +40.1mV, NH₂The Zeta potential of the CDs is +94.4 mV. This is because 2G-PAMAM is used as nitrogen source to construct carbon dots by a decocting method, and the amino groups on 2G-PAMAM promote the enrichment of bare-CDs with a large amount of amino groups, thereby enabling bare-CDs to show positive charges. While ethylenediamine is a modifier, the number of amino groups is increased by grafting methyl acrylate on the surface of bare-CDs, thus NH₂The Zeta potential of-CDs was increased by 54.3mV over bare-CDs.

3. FT-IR characterization

Taking bare-CDs and NH₂Lyophilized samples of CDs, potassium bromide pellets. Measuring an infrared absorption spectrum by adopting a Tensor 27 type infrared spectrometer, wherein the scanning range is 500-4000 cm^-1. The results are shown in FIG. 2D, which is an infrared spectrum of bare-CDs, 3332cm^-1And 1560cm^-1Respectively has an amino N-H stretching vibration peak and a bending vibration peak on the surface of bare-CDs, which are 1148cm^-1A bending vibration peak of 1328cm at C-N^-1Bending vibration peak at-OH, 1483cm^-1The peak is the bending vibration peak of-OH on carboxyl. After grafting ethylenediamine onto the surface of bare-CDs via methyl acrylate via amide linkage. At 1654cm^-1Shows a bending vibration peak of amide bond N-H at 1706cm^-1And the C-N bending vibration peak of the amido bond is shown. In addition, 3075cm^-1Is represented by-NH₂The broader peak of stretching vibration in (2) indicates that the degree of amination at the carbon site is higher after modification with ethylenediamine. And 1560cm^-1At a position of 1406cm^-1N in each case being a surface amino group-H bending vibration peak and-OH bending vibration peak.

4. Fluorescent properties

Preparing certain concentration of bare-CDs, NH₂And (3) measuring a fluorescence spectrum of the-CDs, 2G-PAMAM solution by using a fluorescence spectrophotometer, wherein the scanning range is 200-700 nm. The results are shown in FIG. 3A, where it can be seen that NH₂The fluorescence intensity of CDs is directly related to its concentration. When NH is present₂Concentration of-CDs less than 18. mu.g.mL^-1When is NH₂The intensity of the fluorescence of the CDs increases sharply with increasing concentration. However, when the concentration is more than 18. mu.g.mL^-1When it comes with NH₂The increase in the concentration of CDs slows down the increase in fluorescence intensity. Due to NH in the process of detecting copper ions₂The concentration of CDs has a significant effect on the sensitivity of the detection. When NH is present₂Errors due to instrument signal-to-noise ratio dominate at lower concentrations of CDs. When NH is present₂At higher concentrations of CDs, the effect of the signal-to-noise ratio of the instrument can be eliminated, but at higher concentrations, the sensitivity of the detection is reduced. Thus, NH was determined from repeated experiments₂The detection concentration of-CDs was 100. mu.g.mL^-1。

FIG. 3B is NH₂The fluorescence emission spectra of CDs under excitation at different wavelengths. As can be seen, NH is generated under different wavelength excitation₂There is no major change in the emission wavelength of the CDs. However, the emission wavelength of the fluorescent light is increased and then decreased with the excitation wavelength. This is because NH modified with ethylenediamine₂-CDs increase in particle size to 25nm relative to bare-CDs. When the particle size of the carbon quantum dot is larger than 10nm, the quantum effect is weakened, and the emission wavelength of the carbon quantum dot does not have a dependence on the excitation wavelength. Thus, NH synthesized₂CDs have relatively stable spectroscopic properties.

Example 3 detection of copper ions by carbon dots

1. Copper ion response test

Due to NH₂The amino group on the surface of CDs can rapidly capture Cu in the solution²⁺Forming copper ammonia complex and covering the surface. According to the effect of fluorescence internal rate, the formed copper ammonia complex is covered on NH₂-the surface of CDs.At this time, NH₂The copper ammonia complex formed on the surface of the CDs prevents the latter from receiving external ultraviolet stimuli, or prevents the emission of its fluorescence when it receives external stimuli. Thus, when NH₂Capture of Cu by CDs²⁺The copper ammonia complex then formed on the surface changes its spectral behavior.

Results referring to fig. 4, a c e in fig. 4 respectively represent carbon quantum dots NH₂-ultraviolet absorption, excitation and emission spectra of CDs; b d f each represents NH₂-uv absorption, excitation and emission spectra after binding of CDs with copper ions; g and h represent carbon quantum dots NH under fluorescent lamp₂Fluorescence before and after the binding of CDs to copper ions. As can be seen from FIG. 4, NH₂The ultraviolet characteristic absorption peak of CDs is 232.2 nm. When Cu is added²⁺Then, NH₂Blue-shift of the UV characteristic peak of CDs to 206.4 nm. At the same time, a new peak appeared at 365 nm. This is due to the amino group being bound to Cu²⁺Resulting from the formation of copper ammonia complexes. From NH₂The excitation and emission profiles of the CDs can be seen. In the absence of copper ions, NH₂The excitation and emission wavelengths of the CDs are 349nm and 454nm, respectively, with corresponding intensities of 563.6 and 444.2, respectively; NH after addition of copper ions₂The excitation and emission wavelengths of the CDs do not change, the corresponding intensities decrease to 100.1 and 85.4, respectively. Due to Cu²⁺And NH₂-CDs surface-NH₂Complexing to form a copper ammonia complex covering the NH₂-the surface of CDs. According to the effect of fluorescence inner filtering, the formed complex is covered on NH₂The CDs surface will block NH₂CDs are excited by UV and block the emission spectrum of the carbon dots, resulting in NH₂CDs trapping Cu²⁺Which is then attenuated to some extent. As can be seen, NH is produced₂CDs show blue light under UV 365nm excitation, when Cu is added²⁺Then, the fluorescence intensity is obviously reduced under 365nm excitation.

2、Cu²⁺Linear relationship detection

Accurately weighing NH prepared by the method₂-CDs and dissolved in PBS (pH 7.4) buffer and diluted stepwise to a concentration of 100 μ g/mL^-1NH of (2)₂-a CDs mother liquor; sequentially preparing the solution with concentration of 1 mu mol.L by a stepwise dilution method by using a PBS (pH 7.4) buffer solution as a solvent^-1，1mmol·L^-1Cu of (2)²⁺And (4) mother liquor.

With the above-mentioned Cu²⁺The solution was a mother solution, and PBS (pH 7.4) buffer was a solvent, and the concentrations of the solutions were 0, 0.01,0.05,0.25,0.5,0.75,10,50,100,250,400,600 μmol · L^-1Cu of (2)²⁺And (6) liquid to be detected. Adding NH prepared by i into the to-be-detected₂-CDs mother liquor, NH in the solution to be tested₂Concentration of-CDs 10. mu.g.mL^-1. Detecting NH in the prepared solution in a fluorescence spectrophotometer at 365nm₂-fluorescence intensity of CDs.

The results are shown in FIG. 5, where the excitation and emission channels are both 5nm, at 100. mu.g.mL^-1NH₂The CDs fluorescence intensity is 468.9. When the concentration of copper ions in the solution is continuously increased, NH₂The fluorescence intensity of the-CDs also decreases when Cu²⁺The concentration was increased to 600. mu. mol. mL^-1The fluorescence intensity was reduced to 81.9, which was 0.17 times the fluorescence intensity without copper ions. NH can be seen by fitting a curve₂The copper ion detection interval of CDs is 0.01-600 mu mol.L^-1The fitting equation is that Y is 3.4281+0.34718X, R²0.94289, has a wide detection range. The probe concentration of 100. mu.g.mL can be seen by fitting a curve^-1The limit of the detection of copper ions is 0.01 mu mol.L^-1。

3、NH₂Selective detection of CDs

1) Metal impurity ion interference experiment

The concentration of the solution was adjusted to 1 mmol. L by stepwise dilution using PBS (pH 7.4) buffer as a solvent^-1Hetero ion mother liquor (M) of⁺:Co²⁺,Mn²⁺,Ag⁺,Cs²⁺,Zn²⁺,Hg²⁺,Na⁺,Fe²⁺,K⁺,Mg²⁺,Ba²⁺,Ni²⁺,Ca²⁺,Fe³⁺). Diluting the mixed ion mother liquor to obtain the mixed ion mother liquor with the concentration of 10 mu mol.L^-1The solution to be tested contains the hetero-ions. Adding into the above solution to be testedNH prepared by the method₂-CDs mother liquor, NH in the solution to be tested₂Concentration of-CDs 10. mu.g.mL^-1. Detecting NH in the prepared solution in a fluorescence spectrophotometer at 365nm₂-fluorescence intensity of CDs.

2) Amino acid interference experiments

The concentration of the solution was adjusted to 1 mmol. L by stepwise dilution using PBS (pH 7.4) buffer as a solvent^-1The amino acid mother liquor (L-Pro, L-ALa, L-Lys, L-Met, L-Ser, L-GLy, L-GLu, L-Leu, L-Hyp, L-His, L-Cys). Diluting the prepared amino acid mother liquor to obtain the amino acid mother liquor with the concentration of 10 mu mol.L^-1The amino acid solution to be tested. Adding NH prepared by the method into the liquid to be detected₂-CDs mother liquor, NH in the solution to be tested₂Concentration of-CDs 10. mu.g.mL^-1. Detecting NH in the prepared solution in a fluorescence spectrophotometer at 365nm₂-fluorescence intensity of CDs.

To detect the NH produced₂CDs can only be substituted by Cu²⁺Quenching, we selected different kinds of metal ions and amino acids to verify. As shown in FIG. 6 (A), the concentration was 100. mu.g/mL^-1NH₂Fluorescence intensity of CDs in different metal ion solutions under 365nm UV excitation. Detected 15 metal ions only Cu²⁺Ion energy of NH₂Quenching of the CDs fluorescence. Especially with Cu in the periodic Table²⁺Adjacent Ni²⁺And Zn²⁺None of the ions quenches the fluorescence. Illustrating the NH synthesized₂CDs have good specificity, and can specifically detect Cu in solution²⁺. FIG. 6(B) shows 100. mu.g/mL^- ¹NH₂Fluorescence intensity profiles of CDs in different amino acid solutions, it being possible to see the addition of amino acids versus NH₂None of the fluorescence intensities of the CDs affected. Lateral demonstration of the prepared NH₂Application of CDs to Cu in vivo²⁺And (6) detecting.

3) Stability test

Respectively preparing NH by using distilled water as solvent by adopting a stepwise dilution method₂Concentration of-CDs 10. mu.g.mL^-1And the concentration of NaCl is 0.1,0.5,0.75,1,1.25,1.5,1.75,2mol ·L^-1And detecting NH in the prepared solution in a fluorescence spectrophotometer at 365nm₂-fluorescence intensity of CDs.

Respectively preparing the concentration of 1 mu mol.L by using distilled water as a solvent by adopting a stepwise dilution method^-1With respect to PBS (pH 7.4), Tris-HCl, Tris-HAc, Tris-NaAc, CA-SC, Tris-EDTA and HePes-Tris buffer, NH was prepared in each buffer as a mother solution₂Concentration of-CDs 10. mu.g.mL^-1And the concentration of each buffer solution is 100 nmol.L^-1Containing NH of₂-CDs buffer and detecting NH at 365nm in a fluorospectrophotometer with the prepared solution₂-fluorescence intensity of CDs.

To verify NH₂CDs fluorescence quenching is the capture of Cu by amino groups on its surface²⁺Caused by the formation of a layer of complex on the surface. And detecting the metal impurity ions by adopting hydroxylation and carboxylation carbon points. As a result, as shown in FIG. 7, it can be seen that the fluorescence intensities of hydroxylated and carboxylated CDs are 412 and 550, respectively, at the same channel width. With the prepared NH₂The reason for the slight difference in CDs is mainly that the functional group on the surface of CDs is the donor of electrons of the CDs fluorophore, and the surface functional group transfers electrons to the fluorescent luminophore under UV excitation. Thus, differences in the surface functionality of CDs can result in different fluorescence intensities of CDs. On the other hand, Cu²⁺The fluorescence intensity of the two CDs cannot be changed, because the hydroxyl and carboxyl can not capture free Cu in the solution²⁺So that a layer of complex can not be formed on the surface of the CDs, and the CDs can still receive the stimulation of external ultraviolet to emit fluorescence. As the carbon source of the prepared CDs is citric acid, the error of the citric acid as the carbon source on the experimental result is eliminated under the condition that the carbon source is the same, and the experimental result fully explains that the addition of Cu²⁺After leading to NH₂The main reason for quenching of the fluorescence of CDs is the capture of Cu in the solution by the amino groups on the surface²⁺And a layer of complex is generated on the surface of the fluorescent material so that the fluorescence is quenched.

In order to further explore the NH produced₂Optical stability of CDs. The experiment is carried out by designing different types of buffer solutions and different concentrationsThe optical stability of the probe was investigated with NaCl solution. As shown in FIG. 8, FIG. 8 (A) is a graph showing the fluorescence intensity of the probe in PBS, Tris-HCl, Tris-HAc, Tris-NaAC, CA-SC, Tris-EDTA, HePes-Tris buffers, and it can be seen that the fluorescence intensity of the probe in 7 buffers is not greatly varied, indicating that NH was prepared₂The application range of CDs is wide, and the CDs can be applied to the detection of copper ions in various buffers. In FIG. 8, (B) shows the fluorescence intensity of the probe in NaCl solutions of different concentrations, and it can be seen that the concentration is 0.1-2 mol. L^-1NH in NaCl solution₂The fluorescence intensity of the CDs does not vary much, in particular at 0.1 mol. multidot.L^-1The fluorescence intensity in the NaCl solution was equal to that in the PBS buffer. Description of NH prepared according to the invention₂Application of CDs to Cu in physiological saline²⁺Detection of (3).

4、NH₂Study of the mechanism of detection of CDs

The concentration of the solution was adjusted to 1 mmol. L by stepwise dilution using PBS (pH 7.4) buffer as a solvent^-1The mother liquor of ethylenediaminetetraacetic acid (EDTA). Adding 200 μ L of 1 mmol/L^-1Cu of (2)²⁺Mother liquor, 200 mul EDTA mother liquor, 50 mul NH prepared by the method₂-CDs mother liquor and 50. mu.L of ultrapure water. Detecting NH in the prepared solution in a fluorescence spectrophotometer at 365nm₂-fluorescence intensity of CDs. The results are shown in FIG. 9, in which (A) is NH under different pH conditions₂-CDs vs Cu²⁺Detected fluorescence intensity profile. As can be seen from the figure, Cu is not added²⁺NH (g) is₂The fluorescence intensity of CDs differs in different pH environments. At pH < 7, the fluorescence intensity decreases with decreasing pH. At pH > 7, the fluorescence intensity did not fluctuate significantly. Mainly due to the NH produced₂The surface of CDs contains a large number of amino groups, which are protonated in an acidic environment to block electron transition. In neutral and alkaline solution, NH₂-NH of the surface of CDs₂Not protonated, -NH₂As an electron donor, electrons can be supplied to the fluorophore under excitation of ultraviolet light. Meanwhile, when the pH value is between 6 and 8, NH₂CDs are sensitive to copper ion response. NH when pH < 6 and pH > 8₂-CDs vs Cu²⁺The responsivity of (a) is drastically reduced. This is because NH is present in an acidic system environment₂Protonation of amino groups on the surface of CDs prevents the amino groups from trapping Cu²⁺So that its fluorescence cannot be quenched; in alkaline environment, although the amino group is exposed in NH₂CDs surface, but Cu²⁺Combine with hydroxide in solution to produce Cu (OH)₂Precipitating and then NH₂CDs cannot cope with Cu in alkaline solution²⁺And (6) detecting. Due to the NH produced₂The detection pH range of the CDs is 6-8. Therefore, the prepared probe can be applied to normal cells and tissues Cu in organisms²⁺Detection of (3).

In FIG. 9, (B) shows EDTA fluorescence recovery assay. It can be seen that Cu is added to the solution in which fluorescence quenching has occurred²⁺EDTA reagent, NH, in equal concentrations₂The fluorescence intensity of the CDs is restored. In the quenched NH₂EDTA addition to CDs, since EDTA reacts with Cu²⁺Has stronger complexing ability and can react NH₂Surface of-CDs by-NH₂Trapped Cu²⁺Trapped out, thereby destroying NH₂A complex layer on the surface of CDs to convert NH₂-fluorescence recovery of CDs. The results show that the prepared probe can be reused for detecting Cu²⁺。

5. Cell experiments

1) Cytotoxicity test

L-929 is selected as a normal cell model, HepG-2 and MCF-7 are selected as tumor cell models to react with NH₂The cytotoxicity of CDs was investigated. Cells in the logarithmic growth phase were digested with 0.25% trypsin and centrifuged at 2000rpm for 5min to collect cells under sterile conditions. Suspending and counting cells by using fresh DMEM culture solution, and preparing cell suspension to make cell concentration be 5X 10³each.mL^-1. And 5000 cells were seeded into a 96-well plate and 100. mu.L of the medium was added per well and cultured in an incubator at 37 ℃ for 24 hours. The concentrations of the components were 0,2,4,8,10,20,40,60,80, 100. mu.g/mL^-1NH of (2)₂CDs medium solution, and 5 duplicate wells per concentration were designed. All plates were provided with blank wells, i.e. wells contained medium only. Replacing original culture medium in 96-well plate with solution containing medicine, and culturingAnd (5) cultivating for 24 hours. Then using a concentration of 5 mg. mL^-1The MTT solution was incubated for 4 hours in a 96-well plate, the original medium was removed, 100. mu.L of DMSO was pipetted into each well to dissolve formazan, and the absorbance was measured at 490nm using a microplate reader.

Cell survival rate ═ OD_treated/OD_control)×100％

Wherein, OD_controlIs the absorbance, OD, of the wells without drug carrier_treatedIs the absorbance of the drug-containing carrier pores.

FIG. 10 is NH respectively₂Cytotoxicity profiles of CDs against HepG-2, MCF-7 and L-929 cells. From the cytotoxicity profiles of the three cells, it can be seen that 2. mu.g.mL was tested^-1～100μg·mL^-1The survival rate of the three cells is kept above 95% within the concentration range, which indicates that NH is contained_2-The CDs have no toxic effect on three cells within the detection concentration range, and have better biocompatibility.

2) Cell imaging experiments

MCF-7 and HepG-2 are selected as cell models to carry out cell uptake behavior and intracellular copper ion detection research. First, a medium containing 10% Fetal Bovine Serum (FBS) and 1% diabody (penicillin/streptomycin) was prepared. Will be 4X 10⁵The individual cells were dispersed in 8mL of medium and individually placed in 4 laser confocal dishes and incubated in an incubator at 37 ℃ for 24 h. Observing cell morphology under an inverted microscope, and adding 2mL NH into 4 laser confocal dishes when cell status is good₂Concentration of-CDs 10. mu.g.mL^-1And (3) after culturing for 4 hours, removing the material liquid, washing for 2-3 times by using a PBS (phosphate buffer solution), and adding the culture medium solution containing copper ions with different concentrations into three laser confocal dishes. And (3) after soaking for 5min, discarding the culture medium containing copper ions, washing for 2-3 times by using a PBS (phosphate buffer solution), soaking the cells in the PBS, and observing the fluorescence condition in the cells by laser confocal observation.

In order to explore the fluorescent dynamic quenching process of the NH2-CDs fluorescent probe for intracellular detection of copper ions. We co-cultured NH2-CDs with HepG-2 and MCF-7 cells for 4h, then added a certain concentration of copper ions to the culture dish and scanned under a laser confocal microscope. As shown in FIG. 11, in order to dynamically scan 23s HepG-2 cells, the carbon quantum dots NH2-CDs are combined with copper ions under laser confocal, and the fluorescence is gradually quenched as the combination time is prolonged. It can be seen that at 0s, the moment of copper ion addition, the cell still showed clear blue fluorescence. The intracellular blue fluorescence gradually decreased after reaching 6s until the intracellular blue fluorescence was quenched to stabilize for 20 s. This phenomenon indicates that the prepared NH2-CDs can be applied to rapid real-time monitoring of copper ions in cells. Cannot pass through the nuclear membrane of the cell nucleus and enter the cell nucleus.

To evaluate the NH prepared₂Prospect of application of CDs in fluorescence detection in biological systems, FIG. 12 shows the application of prepared NH₂Direct application of CDs to Cu in HepG-2 and MCF-7 cells²⁺The fluorescence detection of (3). Under laser confocal, 100 mu g/ml NH₂The fluorescent response of CDs to different concentrations of copper ions (250nM, 10. mu.M and 100. mu.M) in MCF-7 and HepG-2 cells. It can be observed from the figure that with NH₂CDs incubated HepG-2 and MCF-7 cells exhibit clear blue fluorescence; when copper ions of different concentrations are added, Cu²⁺Freely diffuse into the cell and are trapped by intracellular NH₂CDs capture leads to quenching of intracellular fluorescence. Due to osmotic pressure of fluorescent probe inside and outside cell membrane, and NH₂The biological toxicity of the CDs fluorescent probe is low, and the intracellular Cu can be accurately realized only if the uptake of the CDs fluorescent probe by cells is enough²⁺Fluorescence imaging of the sensitive detection.

Claims

1. A preparation method of blue fluorescent quantum dots is characterized by comprising the following steps:

the preparation method comprises the following steps of:

1.1) adding citric acid: polyethylene diamine dendrimers with amino groups: water =1 g: 0.5 g: dispersing citric acid and polyethylene diamine dendritic polymer in water at the dosage ratio of 5mL, and decocting at 200 ℃ for 2h to prepare brown solution;

1.2) centrifuging the brown solution, collecting supernatant, filtering the supernatant by using a 0.22 mu m syringe filter membrane, dialyzing the filtered solution for 8 hours by using a 10000Da dialysis bag, and drying to prepare a carbon quantum dot precursor with the average particle size of 4.4 nm;

2) modifying a carbon quantum dot precursor by using ethylenediamine as a modifying molecule and methyl acrylate as a linking molecule to prepare a blue fluorescent quantum dot;

wherein, the step 2) specifically comprises the following operations:

2.1) extracting a carbon quantum dot precursor with the average particle size of 4.4nm with methanol for a plurality of times, collecting methanol extract, dropwise adding methyl acrylate into the methanol extract, reacting for 24 hours at 30 ℃, performing rotary evaporation on the reacted solution to remove methanol, and washing with methanol for a plurality of times;

2.2) as methanol: ethylenediamine = 10: 16, adding methanol into the washed solution, dropwise adding ethylenediamine, and reacting at 30 ℃ for 24 hours; removing methanol from the reacted solution by rotary evaporation, and washing the solution for a plurality of times by using methanol to prepare a yellow solution; and dialyzing the obtained yellow solution, and carrying out freeze drying treatment to obtain the blue fluorescent quantum dots, wherein the average particle size of the blue fluorescent quantum dots is 25.3 nm.

2. The method for preparing blue fluorescent quantum dots according to claim 1, wherein the method for preparing the polyethylene diamine dendrimer with amino groups comprises the following steps:

step 6: removing methanol from the solution reacted in the step 5 by rotary evaporation, and washing the solution for a plurality of times by using methanol to obtain the second-generation dendrimer 2G-PAMAM;

3. The blue fluorescent quantum dot prepared by the method of claim 1 or 2, wherein the surface charge of the blue fluorescent quantum dot is +94.4 mV.

4. The use of the blue fluorescent quantum dot as a fluorescent probe for detecting copper ions according to claim 3, wherein the detection concentration interval of the blue fluorescent quantum dot to the copper ions is 0.01 μmol-L^-1~600 μmol·L^-1。

5. The use according to claim 4, wherein the blue fluorescent quantum dots are capable of specifically detecting intracellular copper ions.