CN106629659A - Preparation method of fluorescent carbon quantum dots employing algae as carbon sources and application - Google Patents

Abstract

The invention discloses a method for preparing fluorescent carbon quantum dots employing algae as raw materials. The method comprises the following steps of (1) cleaning the alga raw materials to remove impurities, and crushing the algae and water to obtain alga slurry; (2) putting a proper amount of alga slurry into a sealing system for high-temperature hydrothermal reaction, and carbonizing the alga slurry; (3) carrying out post-treatment on the carbonized product in the step (2) to remove insoluble substances and large-size components, thereby obtaining a fluorescent carbon quantum dot solution; and (4) carrying out freeze drying on the fluorescent carbon quantum dot solution in the step (3) to obtain fluorescent carbon quantum dot powder. Cheap algae, especially enteromorpha prolifera, are effectively converted into the carbon quantum dots with fluorescence characteristics, and high additional values and harmless conversion of the enteromorpha prolifera are achieved; and meanwhile, the preparation method is simple in process, high in operability and low in cost, massive preparation of the carbon quantum dots can be achieved, and other chemicals are not needed, so that the method is environment-friendly; and the yield of a product is high in the preparation process.

Description

A kind of preparation method and application of fluorescent carbon quantum dots using seaweed as carbon source

technical field

The invention belongs to the technical field of functional nanometer materials, and in particular relates to a method for preparing carbon quantum dots with fluorescence characteristics, in particular to a method for preparing carbon quantum dot cell labeling probes with fluorescence characteristics by using seaweed as a carbon source.

Background technique

Functional nanomaterials are a technical field that has developed very rapidly in recent years. Scientists have successfully prepared organic polymer beads, liposomes, semiconductor quantum dots, silicon quantum dots, upconversion nanoprobes, and noble metal nanoparticles. , magnetic nanospheres, etc., and use their various excellent properties to develop a variety of technical means, which are widely used in medical testing, disease diagnosis, energy batteries, environmental assessment, pollution control and other fields. for the application.

Carbon quantum dots are a new research hotspot in the field of nanomaterials. It refers to microspheres of carbon atoms with a diameter in the range of 2-40 nanometers. While retaining various excellent physical and chemical properties of carbon materials, carbon quantum dots exhibit remarkable fluorescence characteristics and good aqueous phase dispersibility. Experiments show that carbon quantum dots can be directly used as a safe and effective fluorescent probe, which can be internalized by various cells to fluorescently label these cells, and surface-modified carbon quantum dots can specifically label tumor cells. In short, carbon quantum dots have important application value in the fields of biological detection and biological analysis.

There are two main methods for the preparation of carbon quantum dots. One is to use high-purity graphite, carbon black and carbon nanotubes as raw materials, and use electron beam photolithography, electrochemical oxidation, chemical exfoliation, microwave and ultrasonic assistance, oxygen plasma Treatment and other methods destroy the interaction force between graphite sheets, and at the same time form a large number of oxidized groups in the sheets, and cut the graphene sheets to obtain small-sized carbon quantum dots; another type is to use citric acid and Small molecular compounds such as ethylenediamine undergo condensation reaction at a certain temperature to generate carbon quantum dots with higher molecular weight. The raw materials of these two preparation methods are high-purity compounds, and the cost is relatively high. Moreover, concentrated nitric acid and concentrated sulfuric acid are often used in the synthesis process, and the harmless treatment of the reaction by-products is relatively complicated. In recent years, scientists have discovered a variety of methods for preparing carbon quantum dots from natural organic substances. For example, Chinese patent CN2014108419973 discloses a method for carbon quantum dot fluorescent labeling materials using egg white as a carbon source, and Chinese patent CN2012102644112 discloses a method for photoluminescence The preparation method of carbon quantum dots discloses a method for preparing carbon quantum dots using soybean milk residue as a raw material. Chinese patent 2013102199000 discloses a method for preparing carbon quantum dots using plant leaves as raw materials. However, low product yields are common in the preparation process of these natural organic substances , if the yield of soybean milk residue is 5-7.3%, and the use of chemicals such as sulfuric acid and sodium hydroxide is involved in the process, it does not belong to the green production process. In addition, these raw materials still have the disadvantages of relatively high prices and limited sources.

Our country has limited arable land resources, but has a vast ocean area, and the output of seaweed is huge, which can be used as a low-cost and high-volume biomass resource. Enteromorpha is a kind of large natural wild seaweed, which has the characteristics of strong reproductive ability and wide environmental adaptability. In recent years, with changes in a series of environmental factors such as global warming and eutrophication of water bodies, green tides of Enteromorpha marine have continued to break out. Enteromorpha appeared in the Yellow Sea of China as early as 2007, and the green tide disaster of Enteromorpha has occurred for nine consecutive years so far. For example, in June 2015, there was a large area of Enteromorpha floating in the coastal waters of Shandong Province. The green tide covered an area of about 430 square kilometers, and the distribution area was about 35,200 square kilometers (Xinhua News). A large number of Enteromorpha floated and gathered on the sea surface, blocking the passage of ships. At the same time, the massive growth and proliferation of Enteromorpha and the process of decay and decomposition require a large amount of oxygen, which poses a serious threat to the survival of other aquatic organisms in the sea area where it grows. Enteromorpha that is not removed in time emits a stench when it rots, which affects the development of coastal tourism. How to treat Enteromorpha harmlessly and turn it into treasure is a severe challenge we are facing. At present, the main way of harmless treatment of Enteromorpha is to make it into feed, or process it for consumption.

Based on the above discussion, designing an effective way to transform Enteromorpha into functional fluorescent carbon quantum dot materials has important practical significance for the high value-added harmless transformation of Enteromorpha.

Contents of the invention

The purpose of the present invention is to address the deficiencies in the existing carbon quantum dot synthesis technology, to provide a method for preparing and producing fluorescent carbon quantum dots using seaweed as a raw material and applying them as cell labeling probes, which solves the problem of seaweed, especially Enteromorpha high The problem of harmless treatment of added value.

In order to realize the above-mentioned technical scheme, the preparation method of fluorescent carbon quantum dots using seaweed as the carbon source that the present invention relates to specifically comprises the following steps:

(1) cleaning the seaweed raw material to remove impurities, then pulverizing the seaweed and water together to obtain a seaweed slurry;

(2) Putting an appropriate amount of seaweed slurry into a sealed system for high-temperature hydrothermal reaction and carbonizing the seaweed slurry;

(3) Carry out post-treatment to the carbonized product in step (2) to remove insoluble matter and large-sized components, obtain fluorescent carbon quantum dot solution;

(4) Freeze-drying the fluorescent carbon quantum dot solution in step (3) to obtain fluorescent carbon quantum dot powder.

The seaweed involved in the present invention is one or more of enteromorpha, kelp, wakame and seaweed, but not limited to these, and the water is preferably pure water.

The Enteromorpha involved in the present invention is one or more of Enteromorpha barbadensis, Enteromorpha marginatus, Enteromorpha shovel and Flat Enteromorpha.

In the step (1) involved in the present invention, seaweed and water with a mass ratio of (1.5-2.5): 1 are put together into a tissue grinder and pulverized for 3-10 minutes to obtain a seaweed slurry preferably with a mass ratio of seaweed and water of 2: 1. The crushing time is 5 minutes.

The sealing system in the step (2) involved in the present invention is a closed reaction kettle, and the seaweed slurry in the step (2) accounts for one-third of the volume of the closed reaction kettle. The high-temperature hydrothermal reaction and carbonization process are specifically: in the heating device The closed reaction kettle is heated to 180-200°C and maintained for 60-90 minutes. In order to ensure the yield and quality of fluorescent carbon quantum dots, the temperature fluctuation of the heating device is controlled within ±5°C.

The heating device involved in the present invention is one of microwave reactor, oven or muffle furnace.

The post-treatment process in the step (3) involved in the present invention is one of centrifugation, filtration, and dialysis, but is not limited to these types.

The centrifugation of the present invention is specifically: put the carbonized product in step (2) into a centrifuge, and centrifuge for 10-20 minutes under the condition of a rotating speed of 5000-12000 rpm, and the centrifuged supernatant is fluorescent carbon quantum dots Solution, the preferred rotating speed is 10000 rpm, and the centrifugation time is 15 minutes.

The specific process of filtration in the present invention is as follows: the carbonized product in step (2) is treated through a 0.10-0.45 μm filter membrane, and the filtrate is a fluorescent carbon quantum dot solution, preferably with a filter membrane pore size of 0.22 μm.

The specific process of filtration in the present invention is as follows: the carbonized product in step (2) is processed through a dialysis membrane with a molecular weight cut-off of 500 Da to remove small molecular impurities, and the intercepted solution inside the dialysis bag is a carbon quantum dot solution.

The fluorescent carbon quantum dots with seaweed as the carbon source involved in the present invention are used in various directions such as biomarker, heavy metal analysis, electrode composite material preparation, antibody coupling and composite photocatalyst preparation.

Compared with the prior art, the present invention has the following advantages: (1) effectively transform cheap seaweed, especially Enteromorpha, into nanometer-sized carbon quantum dots with fluorescent properties, and realize the high added value and harmless of Enteromorpha Transformation; (2) The preparation method is simple in process, strong in operability, low in cost, and can realize large-scale preparation of carbon quantum dots without using other chemicals, and is environmentally friendly; (3) The prepared fluorescent carbon quantum dots are used for biomarkers , heavy metal analysis, antibody labeling and cell staining; (4) The product yield is high during the preparation process; (5) The crushing process is conducive to the dispersion of the components in the seaweed in the aqueous solution to improve the efficiency of the subsequent hydrothermal reaction and carbonization process.

Description of drawings:

Fig. 1 is the transmission electron microscope characterization figure of embodiment 1 fluorescent carbon quantum dots involved in the present invention;

Fig. 2 is the ultraviolet-visible light absorption spectrogram of embodiment 1 fluorescent carbon quantum dots involved in the present invention;

Fig. 3 is the fluorescence emission spectrogram of embodiment 1 fluorescent carbon quantum dots involved in the present invention;

Fig. 4 is the influence of embodiment 1 fluorescent carbon quantum dots involved in the present invention on cell growth and reproduction;

Fig. 5 is the confocal microscope imaging result after co-cultivating cells with fluorescent carbon quantum dots at a concentration of 500 mg/L in Example 1 of the present invention for 5 hours.

detailed description

The present invention will be further described below in conjunction with specific embodiment:

Example 1:

(1) wash enteromorpha with tap water, remove impurities such as surface silt, then rinse with pure water;

(2) Weigh 200 grams of Enteromorpha raw material (about 10 grams in dry weight) after step (1), mix Enteromorpha and pure water according to the mass ratio of Enteromorpha and pure water, and put Tissue the raw material container of the mashing machine and pulverize for 5 minutes to obtain the enteromorpha slurry;

(3) Take by weighing 10 grams of Enteromorpha slurry that step (2) obtains, put into 30mL polytetrafluorohydrothermal reaction kettle and add a cover and put into the outsourcing sleeve to carry out airtight;

(4) Put the reaction kettle with Enteromorpha slurry into the microwave reactor, set the temperature to 200°C, and the pressure to 20atm, react for 60 minutes, then stop heating;

(5) When the temperature of the reactor is lowered to below 40°C, the lid of the reactor is opened, and the reaction product is a reddish-brown solution containing a little black insoluble matter;

(6) Collect the reaction product mixture obtained in step (5) into a centrifuge tube, set the rotation speed at 10000 rpm, centrifuge for 20 minutes, transfer the supernatant of the centrifuge tube to a clean test tube, and discard the insoluble precipitate directly;

(7) Freeze-dry the fluorescent carbon quantum dot solution obtained in step (6) to obtain fluorescent carbon quantum dot powder. The calculated fluorescence quantum yield is 4.7%, and the product yield is 9.6%.

The carbon quantum dot powder prepared in Example 1 was tested, and the results are shown in Figures 1-5. From the transmission electron microscope characterization diagram, it can be seen that the prepared fluorescent carbon quantum dot particles are uniformly dispersed, and the particle size distribution range is relatively narrow. Basically in the range of 2-6nm, the average particle diameter is 3.5nm. It can be seen from the ultraviolet-visible light absorption spectrum that the fluorescent carbon quantum dots have no obvious absorption peaks. From the fluorescence emission spectrum, it can be concluded that when the carbon quantum dots are irradiated with 360nm excitation light, the fluorescent carbon quantum dots have a strong fluorescence emission in the range of 400-600nm, and the emission peak is near 440nm. From the confocal microscope imaging results after co-cultivating with cells for 5 hours, it can be concluded that Enteromorpha carbon quantum dots at a concentration of 500ug/mL have no significant effect on cell growth and proliferation, and can be enriched in the cytoplasm for fluorescent labeling.

Example 2:

(1) wash enteromorpha with tap water, remove impurities such as surface silt, then rinse with pure water;

(2) Weigh 200 grams of Enteromorpha raw material (about 10 grams in dry weight) after step (1), mix Enteromorpha and pure water according to the mass ratio of Enteromorpha and pure water, and put Pound the cup and pulverize for 5 minutes with a tissue masher to obtain Enteromorpha slurry;

(3) Take by weighing 30 grams of Enteromorpha slurry that step (2) obtains, put into 100mL polytetrafluorohydrothermal reaction kettle and add a cover and put into the outsourcing metal casing and carry out airtight;

(4) Put the reactor containing Enteromorpha slurry into an oven, set the temperature to 180°C, and react for 90 minutes. During the reaction, the temperature fluctuation is controlled at ±5°C, and then stop heating;

(5) When the temperature of the reactor is lowered to below 40°C, the lid of the reactor is opened, and the reaction product is a reddish-brown solution containing a little black insoluble matter;

(6) Filter the reaction product mixture obtained in step (5) with a suction filtration device equipped with a 0.22 μm filter membrane, collect the clarified filtrate, transfer it to a clean test tube, and discard the insoluble matter collected by the filter membrane ;

(7) Freeze-drying the fluorescent carbon quantum dot solution obtained in step (6) to obtain fluorescent carbon quantum dot powder. The calculated fluorescence quantum yield is 4.9%, and the product yield is 7.8%.

Example 3:

(1) wash enteromorpha with tap water, remove impurities such as surface silt, then rinse with pure water;

(2) Weigh 200 grams of Enteromorpha raw material (about 10 grams in dry weight) after step (1), mix Enteromorpha and pure water according to the mass ratio of Enteromorpha and pure water, and put Pound the cup and pulverize for 5 minutes with a tissue masher to obtain Enteromorpha slurry;

(3) Take by weighing 30 grams of Enteromorpha slurry that step (2) obtains, put into 100mL polytetrafluorohydrothermal reaction kettle and add a cover and put into the outsourcing metal casing and carry out airtight;

(4) Put the reaction kettle with Enteromorpha slurry into the muffle furnace, set the temperature to 200°C, and react for 60 minutes. During the reaction, the temperature fluctuation is controlled at ±5°C, and then stop heating;

(5) When the temperature of the reactor is lowered to below 40°C, the lid of the reactor is opened, and the reaction product is a reddish-brown solution containing a little black insoluble matter;

(6) Collect and distribute the reaction product mixture obtained in step (5) into centrifuge tubes, set the rotation speed at 10000 rpm, centrifuge for 20 minutes, transfer the supernatant of the centrifuge tube to a clean test tube, and discard the insoluble precipitate directly;

(7) Freeze-drying the fluorescent carbon quantum dot solution obtained in step (6) to obtain fluorescent carbon quantum dot powder. The calculated fluorescence quantum yield is 3.8%, and the product yield is 8.9%.

Example 4:

In Example 4 and Example 1, except that the raw material selected in step (1) is Undaria pinnatifida, the mass ratio of Undaria pinnatifida and pure water in step (2) is 2.5:1, and the rotating speed is 5000rpm in step (6), centrifuged for 10 minutes , the other steps are the same, and the fluorescent carbon quantum dot powder is prepared.

Example 5:

Present embodiment 5 is the same as embodiment 2 except that other steps are all the same, except that the raw material selected in step (1) is laver, the mass ratio of laver and pure water in step (2) is 1.5:1, and step (6) suction filtration device A 0.45 μm filter membrane was used in the method, and the other steps were the same to prepare fluorescent carbon quantum dot powder.

Tests show that the performance of the fluorescent carbon quantum dot powder prepared in Examples 2-5 is close to that of the fluorescent carbon quantum dot powder prepared in Example 1.

Comparative example 1:

This comparative example 1 and embodiment 1 except that step (4) reaction temperature is 150 ℃, when the reaction time is 30min, other steps are all the same, prepare fluorescent carbon quantum dot powder, calculate that the fluorescence quantum yield is 1.2%, product yield The rate is 2.1%.

Claims (10)

1. a kind of preparation method taking seaweed as the fluorescent carbon quantum dot of carbon source is characterized in that, specifically comprises the following steps: (1) cleaning the seaweed raw material to remove impurities, then pulverizing the seaweed and water together to obtain a seaweed slurry; (2) Putting an appropriate amount of seaweed slurry into a sealed system for high-temperature hydrothermal reaction and carbonizing the seaweed slurry; (3) Carry out post-treatment to the carbonized product in step (2) to remove insoluble matter and large-sized components, obtain fluorescent carbon quantum dot solution; (4) Freeze-drying the fluorescent carbon quantum dot solution in step (3) to obtain fluorescent carbon quantum dot powder. 2. the preparation method of the fluorescent carbon quantum dot with seaweed as carbon source according to claim 1, is characterized in that, seaweed is one or more in enteromorpha, kelp, wakame and seaweed. 3. the preparation method of fluorescent carbon quantum dots taking seaweed as carbon source according to claim 2, is characterized in that, Enteromorpha is the one in Enteromorpha bar, Enteromorpha margina, Enteromorpha shovel and Flat Enteromorpha or more. 4. the preparation method of the fluorescent carbon quantum dot taking seaweed as carbon source according to claim 3 is characterized in that, in step (1), seaweed and water with mass ratio (1.5-2.5): 1 are put into tissue together Pulverize in a masher for 3-10 minutes to obtain seaweed slurry. 5. The method for preparing fluorescent carbon quantum dots using seaweed as a carbon source according to claim 4, wherein the mass ratio of seaweed to water is 2:1, and the crushing time is 5 minutes. 6. according to claim 4 or 5 described taking seaweed as the preparation method of the fluorescent carbon quantum dot of carbon source, it is characterized in that, in step (2), sealing system is airtight reactor, and the seaweed slurry in step (2) Accounting for one-third of the volume of the closed reactor, the high-temperature hydrothermal reaction and carbonization process are as follows: raise the temperature of the closed reactor to 180-200°C in the heating device and maintain it for 60-90 minutes, and control the temperature fluctuation of the heating device at ±5°C. 7. the preparation method of the fluorescent carbon quantum dot taking seaweed as carbon source according to claim 6, is characterized in that, heating device is a kind of in microwave reactor, oven or muffle furnace, and water is pure water, step The post-treatment process in (3) is one of centrifugation, filtration, and dialysis, but it is not limited to these types. 8. the preparation method of the fluorescent carbon quantum dot that takes seaweed as carbon source according to claim 7, is characterized in that, centrifugation is specifically: the carbonized product in step (2) is put into the centrifuge, at rotating speed 5000-12000 Under the condition of rev/min, centrifuge for 10-20 minutes, and the centrifuged supernatant is fluorescent carbon quantum dot solution. The specific process of filtration is as follows: process the carbonized product in step (2) through a 0.10-0.45 μm filter membrane, filter The output liquid is a fluorescent carbon quantum dot solution, and the specific process of filtration is as follows: the carbonized product in step (2) is processed through a dialysis membrane with a molecular weight cut-off of 500Da to remove small molecular impurities, and the intercepted solution inside the dialysis bag is a carbon quantum dot solution. 9. The preparation method of fluorescent carbon quantum dots with seaweed as carbon source according to claim 8, characterized in that, the centrifugal speed is 10000 rpm, the centrifugation time is 15 minutes, and the filter membrane pore size is 0.22 μm. 10. according to the preparation method of the fluorescent carbon quantum dot described in any item of claim 1-9 with seaweed as carbon source, it is characterized in that, fluorescent carbon quantum dot is used for biomarker, heavy metal analysis, preparation of electrode composite material, antibody conjugate Combined and composite photocatalyst preparation.