阅读说明：本技术 一种蓝色铁离子碳量子点及制备方法和应用 (Blue iron ion carbon quantum dot and preparation method and application thereof ) 是由 黄赛朋 谢梦瑶 罗庆 周雨霏 杨丹 王文显 温惠云 薛伟明 于 2021-07-26 设计创作，主要内容包括：本发明公开了一种蓝色铁离子碳量子点及制备方法和应用,将L-精氨酸和柠檬酸加入水中,通过微波反应得到固体混合物；将固体混合物和乙醇混合后,进行水热反应,反应结束后,将所得溶液冷却至室温后离心,取上清液并去除乙醇,通过聚苯乙烯凝胶色谱柱分离,在二氯甲烷/乙醇程序洗脱下,收集洗脱比为5：1组分,减压旋转蒸发掉有机溶剂二氯甲烷和乙醇得油状混合物,将油状混合物加入超纯水,然后低温超声,使碳量子点均匀分散在超纯水中,然后用平均孔径为0.22μm的微孔滤膜进行过滤,过滤后用分子量500道尔顿的透析袋在超纯水中透析；然后冷冻干燥,获得蓝色铁离子碳量子点。本发明所制备的碳量子点能实现细胞水平微量铁离子的快速高灵敏检测。(The invention discloses a blue iron ion carbon quantum dot and a preparation method and application thereof, wherein L-arginine and citric acid are added into water, and a solid mixture is obtained through microwave reaction; mixing the solid mixture with ethanol, carrying out hydrothermal reaction, after the reaction is finished, cooling the obtained solution to room temperature, centrifuging, taking supernate, removing ethanol, separating by using a polystyrene gel chromatographic column, eluting by using a dichloromethane/ethanol program, collecting the eluate with the ratio of 5: 1, performing reduced pressure rotary evaporation to remove organic solvents dichloromethane and ethanol to obtain an oily mixture, adding the oily mixture into ultrapure water, performing low-temperature ultrasonic treatment to uniformly disperse carbon quantum dots in the ultrapure water, filtering by using a microporous filter membrane with the average pore diameter of 0.22 mu m, and dialyzing in the ultrapure water by using a dialysis bag with the molecular weight of 500 daltons after filtering; and then freeze-drying to obtain the blue iron ion carbon quantum dots. The carbon quantum dots prepared by the invention can realize rapid and high-sensitivity detection of cell-level trace iron ions.)

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

the method comprises the following steps: adding L-arginine and citric acid into water, and performing microwave reaction to obtain a solid mixture;

step two: mixing the solid mixture obtained in the step one with ethanol, carrying out hydrothermal reaction, and cooling the obtained solution to room temperature after the reaction is finished;

step three: centrifuging the solution cooled in the step two, and taking supernatant;

step four: removing ethanol from the supernatant, separating by polystyrene gel chromatography column, eluting with dichloromethane/ethanol, collecting the eluate at a ratio of 5: 1, performing reduced pressure rotary evaporation to remove organic solvents dichloromethane and ethanol to obtain an oily mixture, adding the oily mixture into ultrapure water, performing low-temperature ultrasonic treatment to uniformly disperse carbon quantum dots in the ultrapure water, filtering by using a microporous filter membrane with the average pore diameter of 0.22 mu m, and dialyzing in the ultrapure water by using a dialysis bag with the molecular weight of 500 daltons after filtering;

step five: and D, freeze-drying the filtrate purified in the step four to obtain the blue iron ion carbon quantum dots.

2. The method for preparing blue iron ion carbon quantum dots according to claim 1, wherein 0.174g L-arginine and 0.21g citric acid per 20mL water are added in the first step.

3. The method for preparing blue iron ion carbon quantum dots according to claim 1, wherein the microwave reaction time in the first step is 4-13 min.

4. The method for preparing the blue iron ion carbon quantum dot according to claim 1, wherein the volume ratio of ethanol to water in the second step is 3: 2.

5. The method for preparing the blue iron ion carbon quantum dot according to claim 1, wherein the temperature of the hydrothermal reaction in the second step is 180 ℃ and the time is 10 hours.

6. The method for preparing the blue iron ion carbon quantum dots according to claim 1, wherein the centrifugation speed in the third step is 10000rpm for 10 minutes.

7. The method for preparing the blue iron ion carbon quantum dots according to claim 1, wherein the supernatant is subjected to rotary evaporation under reduced pressure at a temperature of 50 ℃ in the fourth step to remove ethanol, and the low-temperature ultrasonic treatment time in the fourth step is 5-15 min; and in the fourth step, the ultrapure water is dialyzed for 24 hours in the ultrapure water by using a dialysis bag with the molecular weight of 500 daltons, and the ultrapure water is replaced every 6 hours.

8. The method for preparing the blue iron ion carbon quantum dot according to claim 1, wherein the freeze-drying conditions in the fifth step are as follows: freezing the purified filtrate into ice, and freeze-drying for 6-18h in vacuum.

9. A blue iron ion carbon quantum dot, which is characterized by being prepared by the preparation method of the blue iron ion carbon quantum dot according to any one of claims 1 to 8.

10. The method for preparing the blue iron ion carbon quantum dot in Fe according to claim 9³⁺Application to detection.

Technical Field

The invention relates to the field of carbon quantum dot preparation, in particular to a blue iron ion carbon quantum dot and a preparation method and application thereof.

Background

Iron is the most abundant trace element in the human body and is of great importance in many physiological processes. Abnormal iron concentrations can lead to anemia, hemochromatosis, liver damage, and the like. Recent studies have shown that excess ferric ions have a serious detrimental effect on the environment and on the human body. Excess iron in the blood and cells leads to an increase in reactive oxygen species generated by the Fenton reaction, which react with biomolecules and can lead to cell damage and even death. If a large amount of ferrous iron is oxidized to ferric iron in the body, methemoglobinemia is developed, which is a relatively rare metabolic disease. Chronic toxic symptoms may also appear: the liver and spleen are heavily iron-deposited and may be manifested by liver cirrhosis, osteoporosis, calcification of cartilage, dark brown or gray skin, and decreased insulin secretion leading to diabetes, and Fe³⁺As one of the main sources of industrial process contaminants, pollution of the ecological environment occurs. The burning of mineral materials leads the iron content in the atmosphere to be seriously over-standard and causes serious pollution to the atmosphere; iron-containing wastewater discharged by a factory influences the color, bromine and taste of a water body; rock weathering is an enrichment process of iron in soil, further polluting farmlands. Therefore, there is an urgent need to establish a method with high efficiency, rapidity, high selectivity, high sensitivity and short response timeDetecting Fe in biological systems and environments³⁺And (4) content.

Traditional methods for detecting ferric ions include inductively coupled plasma mass spectrometry, spectrophotometry, atomic absorption spectrometry, and stripping voltammetry, but their use is limited by the time-consuming pretreatment of the sample. Fluorescent sensors are receiving more and more attention due to their advantages of simple operation, high cost performance, high sensitivity, intuitive and fast response, etc. Among them, the fluorescence detection method constructed based on the carbon quantum dots has received much attention because of its advantages such as good selectivity, high sensitivity, fast response, and simple operation.

Carbon quantum dots are widely used in fluorescence analysis and detection as a novel fluorescent material. Carbon quantum dots (CDs) are approximately spherical Carbon nanomaterials with size less than 10nm, and are generally made of amorphous structures or sp²The carbon core of the hybrid nanocrystal structure is composed of functional groups (such as carboxyl, hydroxyl, aldehyde, amino, etc.) rich on the surface. In recent years, carbon quantum dots have received much attention in chemical and biological sensing, photocatalysis, bio-imaging, light emitting diodes and solar cells, and have good stability, low cytotoxicity and high biocompatibility as compared with organic fluorophores and semiconductor quantum dots.

As known from the research results in the literature, carbon dots based on fluorescent probes are used for detecting Fe³⁺Ions are still rare. Therefore, the development of a green, low-cost and high-sensitivity carbon point silver ion detection probe is urgent. In addition, CDs have excellent quantum yield, and can be used for fluorescence detection of heavy metal ions without further chemical modification and surface functionalization. An effective enhancement of fluorescence emission in freshly prepared CDs was observed, i.e. addition of Fe at lower concentrations³⁺The CDs can be significantly enhanced. Therefore, a selective and sensitive fluorescent platform can be established for Fe in an aqueous medium³⁺Detection of (3).

Currently, the synthesis methods of carbon nanoparticles can be divided into two categories: top-down and bottom-up. The top-down method refers to a physical method for preparing carbon nano particles by peeling from a larger carbon structure, and mainly comprises an arc discharge method, a laser etching method and an electrochemical oxidation method; chemical methods for preparing carbon nanoparticles from molecular precursors by the bottom-up method mainly include combustion methods, organic matter carbonization methods, support synthesis methods, microwave methods, ultrasonic methods, and hydrothermal synthesis methods. Compared with other synthesis methods, the hydrothermal synthesis method has the advantages of simple synthesis steps, easily controlled reaction conditions, low energy consumption, sustainable large-scale production and high fluorescence quantum yield of the product, and is considered to be an economical and effective method. The quantum dot overcomes the defects of the traditional metal quantum dot, has the advantages of no toxicity, high fluorescence quantum yield, good water solubility, good biocompatibility, good light stability and the like, and leads CDs to be widely explored in the fields of metal ion detection, photocatalysis, biomedical imaging and the like.

Disclosure of Invention

The invention aims to provide a blue iron ion carbon quantum dot, a preparation method and application thereof, and aims to overcome the problems in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of blue iron ion carbon quantum dots comprises the following steps:

the method comprises the following steps: adding L-arginine and citric acid into water, and performing microwave reaction to obtain a solid mixture;

step two: mixing the solid mixture obtained in the step one with ethanol, carrying out hydrothermal reaction, and cooling the obtained solution to room temperature after the reaction is finished;

step three: centrifuging the solution cooled in the step two, and taking supernatant;

step four: removing ethanol from the supernatant, separating by polystyrene gel chromatography column, eluting with dichloromethane/ethanol, collecting the eluate at a ratio of 5: 1, performing reduced pressure rotary evaporation to remove organic solvents dichloromethane and ethanol to obtain an oily mixture, adding the oily mixture into ultrapure water, performing low-temperature ultrasonic treatment to uniformly disperse carbon quantum dots in the ultrapure water, filtering by using a microporous filter membrane with the average pore diameter of 0.22 mu m, and dialyzing in the ultrapure water by using a dialysis bag with the molecular weight of 500 daltons after filtering;

step five: and D, freeze-drying the filtrate purified in the step four to obtain the blue iron ion carbon quantum dots.

Further, in step one, 0.174g L-arginine and 0.21g citric acid per 20mL of water were added.

Further, the microwave reaction time in the step one is 4-13 min.

Further, the volume ratio of ethanol to water in the step two is 3: 2.

Further, the temperature of the hydrothermal reaction in the second step is 180 ℃ and the time is 10 hours.

Further, in the third step, the centrifugal speed was 10000rpm, and the time was 10 minutes.

Further, performing rotary evaporation of the supernatant at 50 deg.C under reduced pressure to remove ethanol, and performing low temperature ultrasonic treatment for 5-15 min; and in the fourth step, the ultrapure water is dialyzed for 24 hours in the ultrapure water by using a dialysis bag with the molecular weight of 500 daltons, and the ultrapure water is replaced every 6 hours.

Further, the freeze-drying conditions in step five are: freezing the purified filtrate into ice, and freeze-drying for 6-18h in vacuum.

A blue iron ion carbon quantum dot is prepared by the preparation method of the blue iron ion carbon quantum dot.

Blue iron ion carbon quantum dot in Fe³⁺Application to detection.

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

according to the invention, L-arginine (L-Arg) and Citric Acid (CA) are used as raw materials, CDs are synthesized by a one-step hydrothermal method, transmission electron microscopy, ultraviolet visible spectrum, infrared spectrum and fluorescence spectrum characterization are carried out on the CDs, the selectivity and anti-interference capability of a carbon quantum dot are simultaneously considered, and the constructed carbon quantum can selectively identify iron ions, and the fluorescence intensity of the carbon quantum dot is in a linear relation with the concentration of the iron ions within a certain range. Biological experiments prove that the carbon quantum dots constructed by the invention can realize rapid and high-sensitivity detection of cell-level trace iron ions.

Drawings

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a transmission electron microscope image of a fluorescent carbon quantum dot prepared according to the present invention;

FIG. 2 is an infrared spectrum of a fluorescent carbon quantum dot prepared according to the present invention;

FIG. 3 shows the UV-VIS absorption spectrum of the fluorescent carbon quantum dots prepared according to the present invention;

FIG. 4 is a fluorescence emission spectrum of a fluorescent carbon quantum dot prepared according to the present invention;

FIG. 5 is a fluorescence quenching graph of different metal ions for fluorescent carbon quantum dots;

FIG. 6 shows fluorescence intensity as a function of Fe³⁺A trend plot of increase in concentration;

FIG. 7 shows fluorescence intensity and Fe³⁺A linear relationship of concentration;

FIG. 8 is a graph showing the results of co-culture of carbon quantum dots with cells;

FIG. 9 carbon quantum dots to monitor Fe at cellular level³⁺Applicability and feasibility.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.

The following detailed description is illustrative of the embodiments and is intended to provide further details of the invention. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention.

Example 1

A preparation method of blue iron ion carbon quantum dots is characterized in that L-arginine (L-Arg) and Citric Acid (CA) are used as raw materials, and CDs are synthesized by a one-step hydrothermal method. A mixture of 0.174g L-Arg, 0.21g CA and 20mL water was reacted with microwave for 8min to obtain a solid mixture, and then 30mL ethanol was added thereto and mixed, and the mixture was put into a 50 mL-lined autoclave and then reacted at 180 ℃ for 10 hours. After the reaction was completed, the resulting solution was cooled to room temperature. Centrifugation was carried out at 10000rpm for 10 minutes, and the supernatant was collected. The supernatant was then rotary evaporated at 50 ℃ under reduced pressure for 8min to remove the ethanol, separated by polystyrene gel chromatography, eluting with a dichloromethane/ethanol program, and the fractions collected were separated at an elution ratio of 5: 1, performing reduced pressure rotary evaporation to remove organic solvents dichloromethane and ethanol to obtain an oily mixture, adding 50ml of ultrapure water, performing low-temperature ultrasonic treatment for 10min to uniformly disperse carbon quantum dots in the ultrapure water, filtering by using a microporous filter membrane with the average pore diameter of 0.22 mu m, dialyzing for 24h in the ultrapure water by using a dialysis bag with the molecular weight of 500 daltons after filtering, and replacing the ultrapure water once every 6 h. And finally, freezing the purified filtrate into ice, and carrying out vacuum freeze drying for 12h to obtain dark brown solid powder, namely the blue iron ion carbon quantum dots.

The transmission electron micrograph (figure 1) shows that the synthesized carbon quantum dots are approximately spherical in shape, about 5nm in size and good in dispersibility.

FIG. 2 is an infrared spectrum of fluorescent carbon quantum dots, wherein 3392cm^-1The absorption peaks at (a) can be attributed to the tensile vibration of O-H and N-H. 2982 and 1437cm^-1The absorption band at (a) is assigned to the stretching and bending vibrations of C-H. At 1130 and 1077cm^-1The characteristic peak at (a) reflects the stretching of the C-O bond. 1707cm^-1The peak at (b) is due to tensile vibration of the C ═ O bond, whereas 1286 and 1024cm^-1The spectral band of (b) is assigned to the C-N vibration. Meanwhile, the surface of the carbon quantum dot contains abundant hydrophilic functional groups such as hydroxyl, carboxyl, carbonyl and the like, so that the carbon quantum dot has good water solubility and excellent fluorescence property.

The present invention measures the ultraviolet-visible absorption spectrum and the fluorescence emission spectrum. As shown in FIG. 3, the UV-VIS absorption spectrum of CDs shows two peaks at 209 and 374 nm. The characteristic absorption peak of the carbon dot at 374nm is attributed to n → pi transition absorption of C ═ O double bond in the carboxyl group on the surface of the carbon quantum dot. Subsequently, the fluorescence property of CDs was measured, and as shown in fig. 4, at the optimal excitation wavelength (368nm), a distinct fluorescence emission peak was present at 449nm of the nitrogen-doped carbon quantum dots, and the peak position and intensity of the emission peak were significantly changed with the excitation wavelength. When the excitation wavelength is increased from 320nm to 370nm, the fluorescence emission peak position is red-shifted, the fluorescence intensity is obviously increased, and then the fluorescence intensity is reduced along with the continuous increase of the emission wavelength, and the characteristic of the dependence of the excitation wavelength is considered to be caused by the size effect or the difference of surface luminescence sites of the carbon quantum dots.

From FIG. 5, Fe can be seen³⁺Has obvious fluorescence quenching effect on fluorescent carbon quantum dots, and other metal ions (Fe)^{2 +}，Mn²⁺，Ba²⁺，Ca²⁺，K⁺，Zn²⁺，Mg²⁺，Na⁺Etc.) has little influence on quenching of fluorescence intensity of carbon quantum dots, and thus, the prepared carbon quantum dots have little influence on Fe³⁺Has stronger selectivity for detecting Fe and different metal ions³⁺Has little interference of fluorescence quenching.

As can be seen from FIG. 6, with Fe³⁺The fluorescence intensity shows a tendency to gradually decrease with increasing concentration. As can be seen from FIG. 7, when Fe³⁺The concentration is in the range of 0-80 mu mol/L, the two are in good linear relation, the correlation coefficient is 0.993, and y is 0.0222x + 1.1205.

In order to study the time for the carbon quantum dots to enter the cells, the carbon quantum dots and the cells were co-cultured for different time intervals, and it is found from fig. 8 that the carbon quantum dots can enter the cells within 15 minutes and show brighter blue light, and the blue light is more obvious after 1 hour of incubation.

To further investigate CDs to monitor Fe at the cellular level³⁺Applicability and feasibility of using exogenous Fe³⁺Introduced into cells pretreated with CDs. As shown in FIG. 9, the Fe-containing material is used³⁺Only a rather weak blue fluorescence is seen by the cells incubated with the medium, which is attributed to Fe³⁺Quenching of the fluorescence properties of CDs. Therefore, the phenomenon shows that the nano material CDs can be used for intracellular Fe³⁺Detection of (3).

Example 2

A preparation method of blue iron ion carbon quantum dots is characterized in that L-arginine (L-Arg) and Citric Acid (CA) are used as raw materials, and CDs are synthesized by a one-step hydrothermal method. A mixture of 0.174g L-Arg, 0.21g CA and 20mL water was reacted with microwave for 4min to obtain a solid mixture, and then 30mL ethanol was added thereto and mixed, and the mixture was put into a 50 mL-lined autoclave and then reacted at 180 ℃ for 10 hours. After the reaction was completed, the resulting solution was cooled to room temperature. Centrifugation was carried out at 10000rpm for 10 minutes, and the supernatant was collected. The supernatant was then rotary evaporated at 50 ℃ under reduced pressure for 8min to remove the ethanol, separated by polystyrene gel chromatography, eluting with a dichloromethane/ethanol program, and the fractions collected were separated at an elution ratio of 5: 1, performing reduced pressure rotary evaporation to remove organic solvents dichloromethane and ethanol to obtain an oily mixture, adding 50ml of ultrapure water, performing low-temperature ultrasonic treatment for 5min to uniformly disperse carbon quantum dots in the ultrapure water, filtering by using a microporous filter membrane with the average pore diameter of 0.22 mu m, dialyzing in the ultrapure water for 24h by using a dialysis bag with the molecular weight of 500 daltons after filtering, and replacing the ultrapure water once every 6 h. And finally, freezing the purified filtrate into ice, and carrying out vacuum freeze drying for 6h to obtain dark brown solid powder, namely the blue iron ion carbon quantum dots.

Example 3

A preparation method of blue iron ion carbon quantum dots is characterized in that L-arginine (L-Arg) and Citric Acid (CA) are used as raw materials, and CDs are synthesized by a one-step hydrothermal method. A mixture of 0.174g L-Arg, 0.21g CA and 20mL water was reacted with microwave for 13min to obtain a solid mixture, and then 30mL ethanol was added thereto and mixed, and the mixture was put into a 50 mL-lined autoclave and then reacted at 180 ℃ for 10 hours. After the reaction was completed, the resulting solution was cooled to room temperature. Centrifugation was carried out at 10000rpm for 10 minutes, and the supernatant was collected. The supernatant was then rotary evaporated at 50 ℃ under reduced pressure for 8min to remove the ethanol, separated by polystyrene gel chromatography, eluting with a dichloromethane/ethanol program, and the fractions collected were separated at an elution ratio of 5: 1, performing reduced pressure rotary evaporation to remove organic solvents of dichloromethane and ethanol to obtain an oily mixture, adding 50ml of ultrapure water, performing low-temperature ultrasonic treatment for 15min to uniformly disperse carbon quantum dots in the ultrapure water, filtering by using a microporous filter membrane with the average pore diameter of 0.22 mu m, dialyzing in the ultrapure water for 24h by using a dialysis bag with the molecular weight of 500 daltons after filtering, and replacing the ultrapure water once every 6 h. And finally, freezing the purified filtrate into ice, and carrying out vacuum freeze drying for 18h to obtain dark brown solid powder, namely the blue iron ion carbon quantum dots.

The embodiments described above are merely preferred embodiments of the present invention, and should not be considered as limitations of the present invention, and features in the embodiments and examples in the present application may be arbitrarily combined with each other without conflict. The protection scope of the present invention is defined by the claims, and includes equivalents of technical features of the claims. I.e., equivalent alterations and modifications within the scope hereof, are also intended to be within the scope of the invention.