JP2021195434A - Manufacturing method of carbon-based nanoparticle luminescent material and fluorescent labeling method of cell and animal and plant using the same - Google Patents

Abstract

To introduce a water soluble substituent for improving excellently dissolubility and dispersibility to a culture solution and a culture medium so that a carbon quantum dot luminescent material can be used for fluorescent labeling in a living body such as bioimaging, in view of the fact that the carbon quantum dot luminescent material of a narrow band width-luminescence triangular shape, derived from solvothermal reaction of resorcinol having high biocompatibility without containing a heavy metal, a sharp luminescence peak, a high luminous efficiency, and light resistance is studied for application to a luminescent material for a light-emission diode.SOLUTION: A manufacturing method of a carbon-based nanoparticle luminescent material includes subjecting to dehydration condensation reaction by adding 3,5-dihydroxyphenyl-β-D-glucopyranoside to a solution of a compound refined in the middle of or after dehydration condensation reaction of a solution of phloroglucinol and resorcinol, in a water-solubility improvement method of the carbon-based nanoparticle luminescent material by dehydration condensation reaction of phloroglucinol and resorcinol.SELECTED DRAWING: None

Description

The present invention relates to a light emitting material for a fluorescent label having improved water solubility.

For bioimaging applications such as fluorescent labeling of cells, semiconductor quantum dots, which have higher light resistance than general fluorescent dyes, can change the emission color depending on the particle size, and have a sharp emission spectrum suitable for simultaneous multicolor observation, are being studied. .. The semiconductor quantum dots are covered with CdSe, CdTe, etc. as the light emitting core, ZnS, etc., which has a larger energy gap than the core, as the shell, and the surface is coated with carboxylated polyethylene glycol, acrylic acid polymer, etc. to make them water-soluble. It has been studied, and recently quantum dots consisting of lead perovskite have also been studied.

However, if the semiconductor quantum dot material contains cadmium or lead, which are highly cytotoxic heavy metals, there is a concern that they will dissolve out and cause damage in the body or cells. Therefore, InP-based quantum dots that do not use heavy metals have also been developed (Non-Patent Document 1). However, the carcinogenicity of InP has also begun to be pointed out (Non-Patent Document 2).

Therefore, as a safer light-emitting material, carbon-based nanoparticle light-emitting materials made from natural products that do not contain heavy metals and have high biocompatibility (hereinafter referred to as carbon dot light-emitting materials, or those that show particle size dependence of absorption or light emission are carbon. It is expected to be a quantum dot light emitting material), and its production by various synthetic methods is being studied (Non-Patent Document 3).

The main methods for synthesizing carbon dot luminescent materials include a hydrothermal synthesis method in which heating is performed in water and a solvothermal method in which heating is performed in an organic solvent. A raw material, a solvent, and a catalyst as needed are added to an autoclave consisting of a closed stainless steel container with a Teflon (registered trademark) liner inside, and the reaction is carried out for several hours to several tens of hours under high temperature and high pressure of 100 to 200 and several tens of degrees Celsius. Is widely used. Also, a method of heating in a microwave oven for several minutes or more is being studied.

Carbon dot luminescent materials are often studied for fluorescent labeling of cells, especially for imaging cancer cells. Membrane proteins such as glucose transporters, amino acid transporters, and folic acid receptors are overexpressed on the membrane surface of many cancer cells (Non-Patent Document 4). Therefore, if a carbon dot luminescent material hydrothermally synthesized using folic acid as a raw material is used, it is used for imaging because it binds to the folic acid receptor overexpressed on the surface of cancer cells by the folic acid residue on the surface and emits fluorescence (non-patented). Document 5).

In addition, carbon dot luminescent materials having those residues on the surface from a mixture of glucose and amino acids can be sent into brain tumor cells through the blood-brain barrier because they are nanomaterials, and bind to the surface of cancer cells to cause cancer. It is also possible to obtain a fluorescence imaging image of a tissue (Non-Patent Document 6).

In addition, research is being conducted to use carbon dot luminescent materials not only for cancer imaging but also for treatment. For example, a drug delivery system in which a luminescent material in which an anticancer agent Doxorubicin is bound to folic acid-modified carbon dots adsorbed on bovine serum albumin is transported to cancer cells has also been studied (Non-Patent Document 7).

Research is also being conducted on the use of carbon dot luminescent materials for the treatment of RNA interference. For example, by adsorbing siRNA (anionic) that inhibits mRNA of vascular growth factor on carbon dots citrate cation-modified with polyethyleneimine, it is difficult to be degraded in cells, and RNA interference suppresses vascular growth to cancer cells. There is also research to make it (Non-Patent Document 8).

In addition, a method of destroying and treating cancer cells by irradiating a carbon dot luminescent material accumulated in cancer tissue with laser light is also being studied. For example, carbon dot red luminescent material synthesized from a polythiophene polymer is accumulated in cancer tissue for fluorescence imaging, and the cancer tissue in which carbon dots are accumulated is irradiated with an infrared laser to generate active oxygen and kill cancer cells. Photodynamic therapy to cause is being studied (Non-Patent Document 9).

Further, since cancer cells are sensitive to heat, photothermal excision therapy and the like in which cancer cells accumulating carbon dots are irradiated with a laser to generate heat and kill the cancer cells are also being studied (Non-Patent Document 10).

In addition, the carbon dot luminescent material, which does not contain heavy metals and has low cytotoxicity, can be fluorescently stained while animals and plants are alive when used as a food or medium component for animals and plants. For example, a flower having blue fluorescence is obtained by absorbing a carbon dot light emitting material having blue fluorescence in an ornamental plant (Non-Patent Document 11). Further, a luminescent nematode that emits red, blue, and green light depending on the excitation wavelength is obtained by mixing a carbon dot light emitting material made from a fruit with the food of the nematode (Non-Patent Document 12). Further, fluorescent silk is obtained by mixing with silk moth food (Non-Patent Document 13).

Research has also been conducted to cause energy transfer from a luminescent substrate of a luminescent organism to a carbon dot luminescent material (Non-Patent Document 14). In the future, it may be absorbed by luminescent organisms such as luminescent mushrooms, luminescent fish, and luminescent insects, and the original yellow or green emission may be changed to a longer wavelength emission color to emit light.

Most of the carbon dot light emitting materials have a blue emission color, and until around 2018, there were few reports of red emission carbon dots having high luminous efficiency (Non-Patent Document 15).

In addition, the conventional carbon dot light emitting material has a broad emission spectrum because the band end cannot emit light due to irregular particle size and functional group state, molecular structure defects, etc., and a narrow band width light emitting material having a narrow emission half width is available. There wasn't. However, in June 2018, a group of Beijing Normal University and others used the solvothermal method of heating at 200 ° C in ethanol and autoclave for several hours to 24 hours using fluoroluminol as a raw material to produce a sharp emission spectrum from blue to red. A narrow band width light emitting triangular carbon quantum dot light emitting material (corresponding to an armchair type triangular graphene nanodisk) was synthesized. Conventionally, synthesis of triangular carbon dots having a similar molecular skeleton required several steps of synthesis and purification as in Non-Patent Document 16, for example, but the yield is unknown by a simple solvothermal method, but synthesis is easy. I can now do it.

The growth of molecular particle size is controlled by the synthesis time and the addition of an acid catalyst, and blue, green, and red luminescent materials are produced separately by purification by column chromatography, and the half width of their emission spectra is extremely as about 30 nm. A narrow luminescent material is obtained. For example, it has been reported that the red luminescent material has a luminescence peak of 598 nm, a luminescence quantum yield of 54%, and a half width of 29 nm in the luminescence spectrum in an ethanol solution. Utilization of these light-emitting materials as light-emitting materials for organic EL devices was examined by utilizing their narrow emission spectra. However, in order to use it for a wide color gamut 4K8K display, an emission peak wavelength close to 630 nm is required for the primary color red, and it is also necessary to improve the emission quantum yield (Non-Patent Document 17, Patent Document 1).

In addition, if the light emission with a narrow half-value width can be made more water-soluble, it can be expected to be applicable to simultaneous multicolor observation in fluorescence maging.

After that, in 2019, a group such as Beijing Normal University synthesized a triangular carbon quantum dot light emitting material with improved luminous efficiency and peak wavelength using resorcinol, which is more reactive than phloroglucinol. It was possible to produce green and red luminescent materials separately in a shorter reaction time. By increasing the reactivity of the raw material, the heating reaction time can be shortened to about 1/5, the particle size of the nanoparticles can be increased as compared with the case of using fluoroglucolcinol, and the emission peak is 610 nm in the ethanol solution. A high-performance red carbon quantum dot light emitting material having a quantum yield of 72% and a half-value width of 33 nm in the emission spectrum was obtained (Non-Patent Document 18).

However, when resorcinol with two hydroxyl groups is used as the raw material, the number of hydroxyl groups on the side surface of the carbon quantum dot molecule becomes only three, which is significantly smaller than when fluoroglycylol having three hydroxyl groups is used as the raw material. When used in an electronic device such as a light emitting dopant of an EL element, it is considered that deterioration due to moisture absorption may be reduced, but when used in bioimaging, it is considered that a higher water solubility is conversely desirable.

In 2019, a group of the Indian Scientific Cultivation Association obtained a water-soluble carbon dot luminescent material by heating resorcinol with phosphoric acid at 190 ° C. for 6 hours while oxygen-oxidizing it in the air. However, although the emission peak was 600 nm, the emission quantum yield in water was as low as 25% (Non-Patent Document 19).

Currently, development of carbon dot light emitting materials having high biocompatibility, high water solubility, narrow bandwidth emission (small half price width), and high luminous efficiency that do not contain heavy metals is expected.

International Publication No. 2019/134068 (WO, A1)

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Semiconductor quantum dots having a sharp emission spectrum used for fluorescent labels and displays that can be multicolored contain Cd and have a problem of being harmful to humans and the environment. In recent years, a narrow band width emission triangular carbon quantum dot derived from the sorbothermal reaction of resorcinol, which does not contain heavy metals and has high biocompatibility, sharp emission peak, high emission efficiency, and light resistance, has been developed and used as a light emitting material for light emitting diodes. Has been studied. A method for producing a luminescent material for improving good solubility and dispersibility in a culture solution or a medium so that these carbon quantum dot luminescent materials can be used for fluorescent labeling in a living body such as bioimaging. The issue was to provide.

The present invention is a method for producing a carbon-based nanoparticles luminescent material by a dehydration condensation reaction of phloroglucinol and resorcinol. This is a method for producing a carbon-based nanoparticles luminescent material by adding phenyl-β-D-glucopyranoside and causing a dehydration condensation reaction.

Further, in the method for producing a carbon-based nanoparticle light-emitting material by a dehydration-condensation reaction of fluoroglucolcinol and resorcinol, a carbon-based nanoparticles material purified after the dehydration-condensation reaction of a solution of fluoroglucolcinol and resorcinol and 3,5-dihydroxy. This is a method for producing a carbon-based nanoparticle luminescent material by subjecting a solution containing phenyl-β-D-glucopyranoside to a dehydration condensation reaction. Further, it is a fluorescent labeling method for cells and animals and plants by dissolving the carbon-based nanoparticles luminescent material in an aqueous solution and allowing the living body and cells to absorb the material.

Narrow band width emission triangular carbon quantum dot that can obtain a sharp emission spectrum with high emission efficiency By introducing a group derived from β-D-glucopyranoside group into the side chain of the emission material, water solubility is increased, and cell culture medium and animals and plants are improved. It will be easier to add to the medium and food, and it will be expected to be used for fluorescent labeling and bioimaging by suppressing aggregation and concentration quenching of carbon quantum dots in an aqueous solution, and to be taken up by a glucose transporter of cancer cells.

Next, an embodiment of the present invention will be described with reference to the drawings.

<Narrow band width light emitting triangular carbon quantum dot synthesis method using PG as a raw material according to Non-Patent Document 17>
The production of carbon-based nanoparticles luminescent material by dehydration condensation reaction of phloroglucinol (hereinafter also referred to as PG) and resorcinol is carried out according to the synthetic method of narrow band width luminescent triangular carbon quantum dots of Patent Document 1, Non-Patent Documents 17 and 18. Can be synthesized.

<Synthesis of blue and green luminescent materials>
Dissolve 500 mg (3.96 mmol) of PG in 10 ml of ethanol, sonicate for 10 minutes and then transfer to an autoclave with a 25 ml Teflon® inner container at 200 ° C., 9 hours (blue), 24 hours (blue). Green) Heat and react. After the reaction, return to room temperature by water cooling or natural cooling.

<Synthesis of yellow and red luminescent materials>
500 mg (3.96 mmol) of PG is dissolved in 10 ml of ethanol and 2 ml of concentrated hydrochloric acid or concentrated sulfuric acid is added as a catalyst. The obtained transparent solution is transferred to an autoclave with an inner container of 25 ml of Teflon (registered trademark) and heated at 200 ° C. for 2 hours (yellow) and 5 hours (red) to react. After the reaction, the solution is returned to room temperature by water cooling or natural cooling, and the solution is directly heated for 30 minutes to remove hydrochloric acid, or neutralized with sodium hydroxide and the supernatant is collected by a centrifuge.

It is presumed that the synthesized blue-green carbon quantum dots include carbon quantum dots having the structures represented by the formulas 1 to 2 as the dehydration condensation of PG progresses. Further, since addition, transition, and desorption of hydroxyl groups may occur during hydrothermal synthesis, Equation 2 is included in Equation 3.

Further, it is considered that the structure of red emission includes a structure in which condensation proceeds further and can be represented by the formula 4.

In addition to ethanol, the solvent can be replaced with a general solvent such as formamide, N, N-dimethylformamide, and water by optimizing the reaction conditions. In addition, various general solvents such as ethanol, formaldehyde, and N, N-dimethylformamide can also synthesize narrow band width emission triangular carbon quantum dots having different emission wavelengths by optimum conditions.

For example, red narrow band width luminescent triangular carbon quantum dots can also be made by reflux of PG in concentrated hydrochloric acid or concentrated sulfuric acid and ethanol. Green and yellow narrow luminescent triangular carbon quantum dots can also be produced by reflux of PG in a small amount of concentrated hydrochloric acid or concentrated sulfuric acid, N, N-dimethylformamide and formamide as a catalyst.

<Purification method according to Non-Patent Document 17>
Narrowbandwidth luminescent triangular carbon quantum dots are purified by silica column chromatography. Dichloromethane and methanol are used as developing solvents, and the ratio of dichloromethane to methanol is dynamically changed on the way to ensure the efficiency of separation and purification. Specifically, during the separation and purification process, the volume ratio was 6: 1 to 2: 1 for blue, 10: 1 to 4: 1 for green, 16: 1 to 8: 1 for yellow, and red. Case change from 25: 1 to 10: 1. Silica column chromatography is repeated several times to obtain pure narrowband width emission triangular carbon quantum dots.

<Synthesis and purification method from resorcinol raw material according to Non-Patent Document 18>
Dissolve 50 mg (454 μmol) of resorcinol in 10 ml of ethanol, transfer to an autoclave with an inner container of 25 ml of Teflon (registered trademark), heat at 200 ° C. for 7 hours (red) and 4 hours (green), and then cool to room temperature by natural cooling. .. The obtained solution is purified by silica column chromatography using a mixture of petroleum ether and ethyl acetate as a developing solvent.

The structure represented by the formula 5 is described as a molecular structure synthesized and purified in Non-Patent Document 18. Considering that the substance represented by the formula 6 shown in Non-Patent Document 16 is green fluorescence in chloroform, it is considered that the luminescent substance having the structure of the formula 5 contains a green luminescent component.

Further, since the addition, transition, and desorption of hydroxyl groups may occur during hydrothermal synthesis, it is considered that the compound represented by the formula 3 is produced.

Further, when dehydration condensation progresses to become a red light emitting material, it is considered that a compound having a structure represented by the formula 4 is generated and contained as a red light emitting component.

In the present invention, as shown in Non-Patent Documents 17 and 18, the step of synthesizing triangular carbon dots by the step of condensing phloroglucinol and resorcinol is performed halfway as the first step. At that time, during the reaction in the autoclave, it is desirable to use a degassing solvent in order to prevent the carboxylation of the raw materials and the reactants by carbon dioxide, and to have an inert atmosphere such as helium and argon.

Next, as a second step, fluoroline (3,5-dihydroxyphenyl-β-D-glucopyranoside) is added, and the condensation reaction with the lateral phenolic group of the carbon dots obtained in the first step is continued. Although the second step can be carried out continuously to the first step, it is desirable to carry out the second step by adding florin after purifying the compound synthesized in the first step in order to obtain a highly pure material. .. At that time, in order to prevent the condensation oligomerization reaction due to caramelization of glucopyranoside groups, it is desirable to carry out the reaction at a glucose caramelization temperature of less than 160 ° C.

When the reaction is carried out at a low temperature, the outside of the reaction vessel can be cooled to a constant temperature of less than 100 ° C. from room temperature through a jacket for flowing water or oil or a pipe for flowing water or oil inside. Furthermore, in order to efficiently heat the reactants and promote the condensation reaction, microwave irradiation is performed using a solvent having a small dielectric loss and difficult to absorb microwaves or a mixed solvent containing them, and the reaction is promoted by a non-heating effect. You can also do it. The dielectric loss of the solvent is 22.87 for ethanol, but water (9.9), butyl alcohol (9.76), dimethylformamide (6.07), dichloromethane (0.38), tetrahydrofuran (0.35). By irradiating microwaves with a solvent that has a smaller dielectric loss than ethanol such as toluene (0.1) and is difficult to absorb microwaves or a mixed solvent containing them, the reaction is carried out at a lower temperature than when ethanol is used. You can also do it.

Further, it is also possible to protect the hydroxyl group of the glucopyranoside group of Florin with a benzyl group, an acetyl group, a phenylboronic acid ester group or the like and then react them to finally carry out a deprotection reaction.

The luminescent material of the present invention into which a glucopyranoside group and its residues have been introduced can enhance water solubility, can be used for fluorescent staining of animal and plant cells, and can easily cross the blood-brain barrier by the action of a glucose transporter. It is easily taken up by the cancer cells inside, and can be expected to be used as a fluorescent label.

<Example 1>
12.61 mg (100 μmol) of phloroglucinol containing a glucopyranoside group and 66.06 mg (600 μmol) of resorcinol were dissolved in 12 ml of ethanol, transferred to an autoclave with an inner container of 25 ml of Teflon (registered trademark), substituted with argon, and then replaced with argon at 200 ° C. for 3 hours. After heating, the temperature was lowered to 130 ° C. or lower, the internal pressure of the autoclave was lowered to 1 MPa or lower, and 84.68 mg (300 μmol) of phloroglucinol dissolved in about 12 ml of ethanol was dropped and mixed in the autoclave with a high-pressure inert gas, and the mixture was further stirred for 2 hours to room temperature. Cooling.

The molar ratio of the raw material charged is phloroglucinol: resorcinol: florin molar ratio 1: 6: 3. Then purify by HPLC. The product contains at least a water-soluble luminescent substance having a higher water solubility than the compound of the formula 5 containing a green luminescent component having the structure of the formula 7 and the structure of the formula 8, and a water-soluble luminescence having a structure considered to be a dehydration condensate oligomer of the glucopyranoside group thereof. It is thought to contain materials.

<Example 2>
Dissolve 3.8 mg (30 μmol) of phloroglucinol in about 10 ml of ethanol, transfer to an autoclave with an inner container of 25 ml of Teflon (registered trademark), heat at 200 ° C. for 5 hours, cool to room temperature, and divide the compound of formula 9 by HPLC. Take.

Next, 3.24 mg (10 μmol) of the compound of the formula 9 and 33.9 mg (120 μmol) of fluoroline (3,5-dihydroxyphenyl-β-D-glucopyranoside) were dissolved in about 20 ml of dimethylformamide, placed in a pressure-resistant flask with a water-cooled jacket, and replaced with argon. After that, it is irradiated with microwaves for 15 minutes to react. Then purify by HPLC. Since the molar ratio of the final raw material to be charged is fluoroglucolcinol: florin, the molar ratio is 1:12. , It is considered that a water-soluble luminescent material composed of a luminescent material having a higher water solubility than the compound of the formula 5 containing the red luminescent component represented by the formula 11 and a dehydrated condensate oligomer of the glucopyranoside group thereof is contained.

The luminescent material obtained in Examples 1 and 2 can be used for fluorescent staining and labeling of animal and plant cells.

<Example 3>
An aqueous solution containing a hyponica liquid fertilizer containing a luminescent material containing a green luminescent component obtained in Example 1 and a luminescent material containing a red fluorescent component obtained in Example 2 (10 mg / L each) was placed in a bucket of radish. When the main root is attached and the solution is inhaled for one week, the cells of the radish are stained and an ornamental radish showing emission including green and red fluorescent components is obtained by ultraviolet excitation.

Claims (3)

In the method for producing a carbon-based nanoparticle luminescent material by a dehydration condensation reaction of phloroglucinol and resorcinol, 3,5-dihydroxyphenyl-β-glucopyranoside is added during a heat condensation reaction of a solution of phloroglucinol and resorcinol to cause a dehydration condensation reaction. A method for producing a carbon-based nanoparticle light emitting material. In the method for producing a carbon-based nanoparticle luminescent material by a dehydration condensation reaction of fluoroglucolcinol and resorcinol, a carbon-based nanoparticles material purified after a dehydration-condensation reaction of a solution of fluoroglucolcinol and resorcinol and 3,5-dihydroxyphenyl- A method for producing a carbon-based nanoparticle luminescent material, which comprises subjecting a solution to which β-glucopyranoside is added to a dehydration condensation reaction. A method for fluorescent labeling of cells and animals and plants by dissolving the carbon-based nanoparticles luminescent material according to claim 1 or 2 in an aqueous solution and allowing the living body and cells to absorb the material.