阅读说明：本技术 一种高荧光纳米荧光素有机硅点及其制备方法和应用 (High-fluorescence nano fluorescein organic silicon dot and preparation method and application thereof ) 是由 熊维伟 郑芬芬 姚海洋 王敬敬 陈佩雅 孙莎莎 袁爱华 吴王聪 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种高荧光纳米荧光素有机硅点及其制备方法和应用,该高荧光纳米荧光素有机硅点荧光素和硅烷化试剂通过摩尔比为1:2-2:1反应获得。本发明制备合成的高荧光纳米荧光素有机硅点为具有高荧光,发射最高荧光量子产率可达100％,合成产率高,形貌较均一,荧光稳定性好,并且具有良好的生物相容性和稳定的抗光致荧光漂白以及硅纳米荧光光学调控；同时本发明的制备方法简单方便,原料来源广,成本低,适用于工业化生产应用。本发明制备的高荧光纳米荧光素有机硅点发生了很大的红移,展现出良好的激发波长依赖的荧光光谱性质等优点,基于各种优良特性可以用于制备成荧光探针作为荧光标记物应用于细胞成像和多色细胞成像中。(The invention discloses a high-fluorescence nano-fluorescein organic silicon dot and a preparation method and application thereof, wherein the high-fluorescence nano-fluorescein organic silicon dot fluorescein is obtained by reacting with a silanization reagent at a molar ratio of 1:2-2: 1. The high-fluorescence nano fluorescein organic silicon dots prepared and synthesized by the method have high fluorescence, the yield of the highest emitted fluorescence quantum can reach 100%, the synthesis yield is high, the appearance is uniform, the fluorescence stability is good, and the high-fluorescence nano fluorescein organic silicon dots have good biocompatibility, stable anti-photoluminescence bleaching and silicon nano fluorescence optical regulation; meanwhile, the preparation method is simple and convenient, has wide raw material sources and low cost, and is suitable for industrial production and application. The high-fluorescence nano fluorescein organic silicon dots prepared by the method have great red shift, show the advantages of good fluorescence spectrum property depending on the excitation wavelength and the like, and can be used for preparing a fluorescence probe as a fluorescence marker to be applied to cell imaging and multicolor cell imaging based on various excellent characteristics.)

1. The high-fluorescence nano fluorescein organic silicon dot is characterized in that fluorescein and a silanization reagent react at a molar ratio of 1:2-2:1 to obtain the high-fluorescence nano fluorescein organic silicon dot.

2. The highly fluorescent nano-fluorescein organosilicon dot as claimed in claim 1, wherein the silylation agent is diethylenetriaminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, N- (beta-aminoethyl) -gama-aminopropyltrimethoxysilane.

3. The high fluorescence nano-fluorescein silicone dot as claimed in claim 1 or 2, wherein the high fluorescence nano-fluorescein silicone dot is an amorphous nanoparticle.

4. The high fluorescence nano-fluorescein silicone dot as claimed in claim 1 or 2, wherein the high fluorescence nano-fluorescein silicone dot has a diameter of between 2nm and 20 nm.

5. The high-fluorescence nano-fluorescein silicone dot as claimed in claim 1 or 2, wherein the high-fluorescence nano-fluorescein silicone dot has fluorescence property and has a strong fluorescence emission peak near the wavelength of 450nm to 550 nm.

6. The preparation method of the high-fluorescence nano-fluorescein organic silicon dot as claimed in claim 1, which is characterized by comprising the following steps:

(1) adding a silanization reagent and fluorescein into an aqueous solution according to a proportion, fully mixing and dissolving, heating for reaction by a one-step hydrothermal method, stopping heating, cooling, taking out the reaction solution, dialyzing unreacted substances, and finally centrifuging at a low speed to remove precipitates to obtain a high-fluorescence nano fluorescein organic silicon dot aqueous solution;

(2) and (2) freeze-drying the aqueous solution of the high-fluorescence nano-fluorescein organic silicon dots in the step (1) to obtain the solid powder of the product, namely the high-fluorescence nano-fluorescein organic silicon dots.

7. The method as claimed in claim 6, wherein the one-step hydrothermal method in step (1) is carried out at a reaction temperature of 140 ℃ and a reaction temperature of 200 ℃ for 2-12 hours.

8. The method according to claim 6, wherein the withdrawing of the reaction solution in the step (1) is dialyzed against unreacted materials, and the withdrawn reaction solution is transferred to a dialysis membrane for 24 to 36 hours.

9. An application of the high fluorescence nano fluorescein organic silicon dot as claimed in claim 1 in the preparation of biological fluorescent probe.

10. The use of claim 9, wherein the use comprises making a fluorescent probe in the field of bioluminescent imaging, the fluorescent probe having good biocompatibility and being stable against photofluorescent bleaching.

Technical Field

The invention belongs to the field of chemical nano materials, and particularly relates to a high-fluorescence nano fluorescein organic silicon dot and a preparation method and application thereof.

Background

Silicon quantum dots are a zero-dimensional semiconductor fluorescent nano material, and are receiving more and more attention due to the unique optical characteristics of the silicon quantum dots. Especially, the silicon quantum dots have good biocompatibility and stable photofluorescence bleaching resistance. Making it an ideal candidate material for substituting fluorescent dyes for biological determination and fluorescence imaging. However, the hydrophobic silicon quantum dots prepared by the traditional "top-down" method have low fluorescence quantum yield, and the fluorescence quantum yield is further reduced after hydrophilic modification, so that the application of biological imaging is limited. In 2013, professor He Yao et al used sodium citrate to reduce 3-aminopropyl-trimethoxy silane, and synthesized silicon quantum dots in a water phase by using a bottom-up method, wherein the fluorescence quantum yield is 20-25%. In 2017, Wu et al adopt rose bengal and three silanization reagents to react to generate organic fluorescent silicon dots, and the highest fluorescence quantum yield of the synthesized organic silicon dots can reach 100%. The achievement of the research result develops a good prospect for further development and practical application of the silicon dots.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the high-fluorescence nano-fluorescein organic silicon dot which is prepared and synthesized by the invention, has high fluorescence, the highest fluorescence quantum yield can reach 100%, the synthesis yield is high, the appearance is uniform, the fluorescence stability is good, and the phenomenon that the fluorescence intensity of a fluorescent dye is obviously reduced due to illumination is solved.

The invention also provides a preparation method and application of the high-fluorescence nano fluorescein organic silicon dots.

The technical scheme is as follows: in order to achieve the purpose, the high-fluorescence nano fluorescein organic silicon dot is obtained by reacting fluorescein and a silanization reagent in a molar ratio of 1:2-2: 1.

Wherein the silanization reagent is diethylenetriaminopropyltrimethoxysilane (AEEA); 3-Aminopropyltriethoxysilane (AMEO); 3-Aminopropyltrimethoxysilane (APTMS); n- (beta-aminoethyl) -gama-aminopropyltrimethoxysilane (DAMO).

Preferably, the method comprises the following steps: the molar ratio of fluorescein to the four silanization reagents is 1:1, and the high-fluorescence nano fluorescein organosilicon points obtained by reaction are respectively marked as C5, C6, C7 and C8.

Wherein the high-fluorescence nano fluorescein organic silicon dots are amorphous nano particles.

Wherein the diameter of the high-fluorescence nano fluorescein organic silicon dots is between 2nm and 20nm, and the diameter is increased along with the prolonging of the reaction time.

Wherein, the high fluorescence nanometer fluorescein organic silicon point has fluorescence property and has strong fluorescence emission peak near the wavelength of 450 nm-550 nm. The four fluorescein organosilicon points have high fluorescence performance and have the strongest fluorescence generation peak at 530 nm.

The preparation method of the high-fluorescence nano fluorescein organic silicon dot comprises the following steps:

(1) adding a silanization reagent and fluorescein into a certain amount of aqueous solution according to a certain proportion, fully mixing and dissolving, heating for reaction by a one-step hydrothermal method (heating can be carried out by a hydrothermal method or can be carried out by loading into a pressure-resistant pipe for heating), then stopping heating, cooling, taking out the reaction solution, dialyzing unreacted substances, and finally centrifuging at a low speed to remove precipitates to obtain the high-fluorescence nano fluorescein organosilicon point aqueous solution;

(2) and (2) freeze-drying the aqueous solution of the high-fluorescence nano-fluorescein organic silicon dots in the step (1) to obtain the solid powder of the product, namely the high-fluorescence nano-fluorescein organic silicon dots.

Preferably, the one-step hydrothermal method in the step (1) is carried out at a reaction temperature of 140 ℃ and 200 ℃ for 2-12 hours. Most preferably, the reaction temperature is 160 ℃ and the reaction time is 4 hours.

Wherein, the reaction solution is taken out for dialysis to remove unreacted substances in the step (1), and the reaction solution is taken out and transferred to a 500ka dialysis membrane for dialysis for 24 to 36 hours.

The invention relates to application of a high-fluorescence nano fluorescein organic silicon dot in preparation of a biological fluorescent probe.

The application comprises the preparation of a fluorescent probe in the field of biological fluorescence imaging, wherein the fluorescent probe has good biocompatibility and stable photofluorescence bleaching resistance.

The starting materials in the present invention are commercially available, fluorescein CAS no: 518-44-5.

According to the invention, fluorescein and 4 different silanization reagents are used for carrying out high-temperature reaction in a hydrothermal reaction kettle (or a pressure-resistant tube) to prepare 4 different organic silicon dots (high-fluorescence nano fluorescein organic silicon dots), the fluorescence quantum yield of the synthesized organic silicon dots is greatly improved compared with that of fluorescein, and the fluorescence quantum yield can reach 100% at most. Meanwhile, the synthesized organic silicon dots have good photofluorescence bleaching resistance. MTT experiments and biological imaging show that the synthesized organic silicon dots have good biocompatibility and can be applied to the field of biological imaging, particularly the field of cell imaging.

The fluorescein and the silanization reagent are polymerized into the nano small particles through an amide reaction at high temperature, and the formed nano particles have better water solubility compared with the fluorescein, have better biocompatibility due to the silicon element, and have better light stability under the illumination compared with fluorescent dye due to the newly generated organic silicon dots.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the high-fluorescence nano fluorescein organic silicon dot prepared by the one-step hydrothermal method is a brand new nano fluorescent material, the prepared high-fluorescence nano fluorescein organic silicon dot shows stronger fluorescence and good water dispersibility, and the fluorescence intensity of the organic silicon dot is obviously enhanced compared with that of fluorescein stock solution by the fluorescence spectrum. The fluorescent material shows good fluorescent spectrum performance, has good biocompatibility, stable photofluorescent bleaching resistance and silicon nano fluorescence optical regulation and control; meanwhile, the preparation method is simple and convenient, has wide raw material sources and low cost, and is suitable for industrial production and application; the high-fluorescence nano fluorescein organic silicon dot is an ideal candidate material for biological determination and fluorescence imaging due to the unique optical property of the high-fluorescence nano fluorescein organic silicon dot.

The high-fluorescence nano fluorescein organic silicon dots prepared by the invention have great red shift, show the advantages of good fluorescence spectrum property depending on the excitation wavelength and the like, are used for preparing fluorescent probes based on various excellent characteristics, and can be used as a fluorescent marker to be applied to cell imaging and multicolor cell imaging.

Drawings

FIG. 1 is a photograph of four fluorescein organosilicon points C5, C6, C7 and C8 prepared by the present invention before and after synthesis;

FIG. 2 is a comparison graph of fluorescence obtained by the fluorescein organosilicon points C5, C6, C7 and C8 prepared by the invention and a fluorescein aqueous solution under sunlight and an ultraviolet lamp (from left to right, C0, C5, C6, C7 and C8 respectively);

FIG. 3 is an ultraviolet absorption spectrum of fluorescein organosilicon points C5, C6, C7 and C8 prepared by reacting for 4 hours at 160 ℃;

FIG. 4 is a fluorescence emission spectrum of fluorescein organosilicon points C5, C6, C7 and C8 prepared by reacting for 4 hours at 160 ℃;

FIG. 5 is an XRD pattern of fluorescein organosilicon points C5, C6, C7 and C8 prepared by reacting for 4h at 160 ℃;

FIG. 6 is an HR-TEM spectrum of a fluorescein organosilicon point C5 prepared by the reaction of the invention for 4h at 160 ℃;

FIG. 7 is an EDS spectrum of fluorescein organosilicon points C5, C6, C7 and C8 prepared by reacting for 4 hours at 160 ℃;

FIG. 8 is an infrared spectrum of fluorescein organosilicon points C5, C6, C7 and C8 prepared by the reaction for 4 hours at 160 ℃;

FIG. 9 is an XPS spectrum of fluorescein organosilicon points C5, C6, C7 and C8 prepared by the reaction at 160 ℃ for 4 hours in the invention;

FIG. 10 is a graph of the fluorescence image of cells using fluorescein organosilicon dot C6 prepared by the present invention;

FIG. 11 shows fluorescence emission spectra of fluorescein organosilicon sites C5, C6, C7 and C8 prepared by the present invention under 180 ℃ reaction conditions;

FIG. 12 shows fluorescence emission spectra of fluorescein organosilicon sites C5, C6, C7 and C8 prepared by the method under the reaction condition of 200 ℃.

FIG. 13 is an HR-TEM spectrum of organosilicon point C5 prepared by reaction of the present invention at 160 ℃ for 2 h;

FIG. 14 is an HR-TEM spectrum of organosilicon point C5 prepared by reaction of the present invention at 160 ℃ for 12 h;

FIG. 15 is a fluorescence stability test of the organic silicon dots C5 prepared by the reaction of the invention at 160 ℃ for 4 h.

FIG. 16 is a cytotoxicity test of organosilicon site C5 prepared by reacting for 4h at 160 ℃.

Detailed Description

The invention is further illustrated by the following figures and examples.