The preparation of fluorescent carbon dots as nano-labeling materials is as follows:
In recent years, semiconductor fluorescent quantum dots have been widely used in biology, medicine and photoelectric devices because of their excellent photoelectric properties. However, most of the most mature quantum dots used in biology and medicine are CdTe, CdSe and CdS, which contain heavy metal cadmium, which limits their application in biomedical field. Therefore, reducing and eliminating the toxicity of fluorescent quantum dots has always been a topic that researchers pay close attention to. Until 2006, Sun et al. used laser to ablate carbon targets and experienced a series of acidification and CDs. Carbon point.
As a new type of fluorescent carbon nanomaterial, carbon dots not only has excellent optical properties and small size, but also has good biocompatibility, good water solubility, low price and low cytotoxicity. It is a good choice to replace traditional heavy metal quantum dots. Water-soluble carbon dots are widely concerned because they contain a large number of water-soluble groups such as carboxyl and hydroxyl, and are compatible with many organic, inorganic and biological molecules. These properties determine that carbon dots have greater application prospects in the fields of biological imaging and biological probes. Hong Chu and Wang Shanshan treated the aqueous solution of PEG-200 and saccharides by microwave heating, and obtained carbon dots with different fluorescence properties. Although microwave can synthesize and modify carbon dots in one step, the yield of fluorescence quantum is not significantly improved compared with hydrothermal method. At present, the research work in this field mainly focuses on three aspects: the formation mechanism and properties of carbon dots, especially the photoluminescence mechanism, how to prepare carbon dots with excellent performance simply and quickly, and how to successfully and efficiently apply carbon dots to practice.
In this paper, several factors affecting the synthesis of fluorescent carbon dots were analyzed by single factor method, and the best synthesis conditions of high performance fluorescent carbon dots were found. The advantages and disadvantages of microwave synthesis and hydrothermal synthesis of fluorescent carbon dots are compared, which provides a certain experimental basis and scientific method for preparing high-performance fluorescent nano-labeled materials.
1 experimental part
1. 1 reagents and instruments
Glucose (AR, China Pharmaceutical Group Shanghai Chemical Reagent Company), polyethylene glycol (PEG-200, AR, China Pharmaceutical Group Shanghai Chemical Reagent Company), thioglycolic acid (TGA, AR, Sinopharm Group Chemical Reagent Co., Ltd.), CS (Dalian Xindie), bovine serum albumin (BSA >: 99%, Merck) from Wuhan Ling Fei Biotechnology Company); Hydrochloric acid (HCl, AR, Xinyang Chemical Reagent Factory); Disodium hydrogen phosphate dodecahydrate (Na2HPO4? 12H2O, AR, chemical reagent co., ltd of sinopharm group); Sodium dihydrogen phosphate dihydrate (NaH2PO4? 2H2O, AR, Chemical Reagent Co., Ltd.); Sodium hydroxide (NaOH, AR, Sinopharm Chemical Reagent Co., Ltd.).
Fluorescence spectrophotometer (LS55, Perkin Elmer, USA); Ultraviolet-visible absorption spectrometer (U-30 10, Hitachi, Japan); Pure water instrument (UP type, Shanghai Youpu Industrial Co., Ltd.); Desktop electrothermal constant temperature drying oven (model 202-00A, Tianjin Test Instrument Co., Ltd.); Fourier transform infrared spectrometer (Vertex 70, Brook Company, Germany); Transmission electron microscope (JEM -2 100UHR STEM/EDS, Japan); Microwave reactor (Milestone, Italy); Electronic balance (mettler toledo, mettler toledo Instruments (Shanghai) Co., Ltd.); Electric stirrer (DJIC-40, Jintan Dida Automation Instrument Factory); Intelligent constant temperature electric heating sleeve (ZNHW type, Wuhan Cole Instrument Equipment Co., Ltd.); Digital display constant temperature water bath pot (HH-S2s, Jintan Dida Automation Instrument Factory); Ultraviolet lamp.
All spectral analysis was carried out at room temperature. The water used in the experiment is high pure water with resistivity greater than 18 Mcm. The ultraviolet-visible absorption photometer is set as follows: the gap is 2 nm, the scanning speed is 600 nm/min, and the scanning range is 200 ~ 600 nm. The fluorescence spectrophotometer is set as follows: excitation wavelength 350 nm, scanning range 350 ~ 650 nm, scanning speed 600 nm/min, excitation gap 10 nm, emission gap 15 nm.
Compilation of 1 2 carbon points
There are many factors affecting the fluorescence properties of carbon dots, mainly the molar ratio of reactants, reaction temperature and reaction time. In order to better control the experimental conditions and improve the performance of carbon dots, an orthogonal experimental method with three factors and three levels was adopted. This method completes the optimal selection under multiple conditions with less experiments. The carbon source is glucose, the surface modifier is PEG, and the temperature is 65438 050℃ respectively. At 160℃ and 180℃, the time is 1. Five minutes, two. Five minutes and three seconds. The molar ratio of PEG to glucose is 4, 5 and 6, respectively. In addition, the experimental conditions, toxicity and biocompatibility of the product should be considered in addition to the fluorescence intensity of carbon dots when determining the optimal conditions. Weigh 2g of glucose, dissolve it in 3ml of water, and mix it with different volumes of PEG-200 to obtain a clear solution, then put it in a microwave reactor or an electrothermal constant-temperature water bath pot, set a certain temperature and reaction time, and microwave radiation or water bath heating to obtain different brown-red solutions, that is, carbon point stock solution; Then the carbon point stock solution was separated and purified by centrifugation at different rotational speeds, and its optical properties were measured and compared. Finally, centrifugal separation and purification with the rotating speed of 6000 r /min were selected, and the supernatant was diluted for different time to characterize.
The characterization of 1 Three carbon points
The obtained carbon dots were diluted by different times, and their photoluminescence properties were tested by U-30 10 UV-Vis absorption spectrometer and LS55 fluorescence spectrophotometer respectively.
Determination of UV-Vis absorption spectrum: The prepared carbon dots are diluted several times (the absorption value at the excitation wavelength is 0. 1), and its absorption peak position was determined by ultraviolet scanning. By taking the ultraviolet absorption peak wavelength of carbon point as the excitation wavelength, the excitation wavelength is determined to be 3500nm, and the excitation and emission slits are both 5. The PMT voltage is set to 700 V, and the excitation wavelength is 290 ~ 435 nm.
Fluorescence spectrum measurement: take about 2. 5 mL of carbon point solution to be detected was placed in a fluorescent cuvette, and its fluorescence was detected by LS55 fluorescence spectrometer at room temperature. The excitation wavelength is 350 nm, the width of excitation and emission slits is 5 nm, the scanning wavelength range is 300 ~ 650 nm, and the scanning speed is 65438 0200 nm/min.
The microstructure and size of carbon point samples were observed by transmission electron microscope (accelerating voltage 200 kV). The obtained carbon point stock solution was mixed with the same volume of anhydrous ethanol, then dripped on KBr tablets, dried to dryness in a desktop electrothermal constant temperature drying oven, and then put into a Fourier transform spectrometer to obtain infrared spectra.
2 Results and discussion
2. 1 factor analysis of microwave synthesis of carbon dots.
In this experiment, three influencing factors (molar ratio of reactants (n), reaction temperature (t) and reaction time (t)) were selected, and three different levels were selected for each factor, that is, three factors and three levels orthogonal experiment method was used to arrange the experiment. The factors affecting the fluorescence intensity of carbon dots prepared by microwave method were discussed, and the best synthesis conditions were found. According to the conditions of three factors and three levels, orthogonal table 34 is selected.
In the synthesis of carbon dots, the trends of different influencing factors change at different levels. Under the same factor, with the change of level, the experimental indexes also change. According to the trend in the figure, the optimum conditions for microwave synthesis of carbon dots are: the molar ratio of PEG to glucose is 6, the reaction temperature is 180℃, and the reaction time is 2. Five minutes. The fluorescence intensity of carbon quantum synthesized under these conditions is the best. It can also be seen from the trend chart that the longer the microwave-assisted reaction time, the better. However, when the reaction time is less than 3. With the decrease of reaction time, the fluorescence intensity of carbon point tends to increase.
Through the intuitive analysis of the above orthogonal experiments, the optimized conditions were obtained, and the fluorescent carbon dots were synthesized by microwave under these conditions. The fluorescence emission spectra of the carbon dots prepared in the experimental group under the optimized conditions and the carbon dots prepared under the optimal experimental conditions 9 were obtained. Under the same other conditions, the fluorescence intensity of the optimized carbon point is 234, which is much higher than that of the experimental group No.9 carbon point (153. 17).
The experimental results were analyzed by changing the pH value of the precursor solution (3, 7 and 9 respectively). With the increase of pH value of the solution, the fluorescence intensity of carbon dots first decreases and then increases. Under alkaline condition (pH = 9), the fluorescence intensity of carbon point is the highest, followed by acidic condition (pH = 3) and neutral condition (pH = 7). The reason may be that in the glucose -PEG system, the surface of carbon dots prepared is rich in hydroxyl and carboxyl functional groups (as shown in Figure 8). Under acidic conditions, because a large number of hydroxyl groups on the surface of carbon dots form a large number of hydrogen bonds with H+, the system is relatively stable and the carbon dots can be well dispersed, so it emits good fluorescence. Under alkaline conditions, carboxyl groups interact with OH- on the surface of carbon dots, which makes the system more stable and the carbon dots can be well dispersed. However, under neutral conditions, the generated carbon dots are aggregated due to their high surface energy, which leads to the increase of particle size and the broadening of particle size distribution.
2.2 Comparison of Microwave Method and Hydrothermal Method
Under the same optimization conditions, carbon dots were synthesized by microwave method and hydrothermal method respectively, and their optical properties were preliminarily compared.
2.2. Ultraviolet-visible absorption spectrum of1carbon point
The ultraviolet-visible absorption spectra of carbon dots obtained by the two methods show that the absorption peak positions of the two methods are around 280 nm, and the absorption peak positions do not change with the change of heating methods, indicating that the mechanism of forming carbon dots by the two heating methods may be the same. In addition, under the same synthesis conditions, the ultraviolet-visible absorption spectrum intensity of carbon dots prepared by microwave method is less than that by hydrothermal method.
2.2.2 Fluorescence emission spectrum of carbon point
After diluting a group of carbon dots synthesized by microwave optimization, the excitation wavelength was increased in turn, and the change of fluorescence emission wavelength was observed. The fluorescence emission spectra of carbon dots synthesized by microwave at different excitation wavelengths (340 ~ 450 nm) show that with the increase of excitation wavelength, the fluorescence emission peak shifts red, and the fluorescence intensity first increases and then decreases. Among them, when the excitation wavelength is 350 nm, the fluorescence emission intensity of carbon point is the largest. Therefore, 350 nm is chosen as this experiment.
2.2.3 Discussion on the Mechanism of Carbon Point Fluorescence
The fluorescence properties of carbon dots mainly come from two different types of emission, one is its surface energy trap emission, and the other is its internal state emission, that is, the emission produced by the combination of electrons and holes, which is also known as the TEM image of carbon dots caused by the quantum size effect of quantum dots. On the one hand, the surface energy trap emission of carbon points produced by high temperature pyrolysis of glucose produces fluorescence; On the other hand, PEG can be used as a surface passivator for carbon dots. In this study, the precursor is a mixture of glucose and PEG. Therefore, PEG acts as a stabilizer on the one hand and a surface modifier on the other. PEG contains a large number of hydroxyl groups and other functional groups. Under alkaline conditions, functional groups such as hydroxyl groups are introduced into the surface of carbon dots, which inhibits the emission of defects in carbon dots and makes the radiation combination of electrons and holes that can generate fluorescence more convenient.
2.2. 4 Transmission Electron Microscope in Carbon Point
It can be seen that carbon dots are similar to semiconductor quantum dots, with spherical appearance, good dispersibility and uniform size distribution, and the average particle size is about 5 ~ 8 nm. It shows that polyethylene glycol plays a good role as dispersant and surface modifier in the process of preparing carbon dots from glucose pyrolysis, which can effectively prevent the agglomeration of carbon dots.
2.2.5 Infrared spectrum of carbon point
Infrared spectra of carbon dots prepared by different methods (a. microwave method; B. hydrothermal method) under the same optimization conditions, microwave method and hydrothermal method.
The peak position and shape of infrared spectra of carbon dots obtained by the two methods are basically the same, but the absorption peak intensity is slightly different, which may be related to the concentration of carbon dots.
The stretching vibration spectrum of hydroxyl group appears in 3 700 ~ 3 100 cm ~ 1. In most hydroxyl-containing compounds, due to strong intermolecular hydrogen bonds, a very strong and wide band appears in the region of 3 500 ~ 3 100 cm- 1. Around 3 370cm- 1, the carbon dots prepared by the two methods have a broadened absorption peak, which is the characteristic peak of the stretching vibration of O-H bond. At the same time, there are strong absorption peaks in the fingerprint regions of110/cm-1and 1 247cm- 1, which belong to the symmetric contraction and asymmetric expansion oscillation of C-O-C, respectively, which proves the existence of hydroxyl groups. At the same time, the absorption peak of both of them is observed at 1 643 cm- 1, which is the stretching vibration of C = O, which proves the existence of carboxyl groups. Judging from this, the surface of carbon dots has hydroxyl and carboxyl functional groups, which not only enhances the water solubility and biocompatibility of quantum dots, but also provides useful guidance for the subsequent modification of such carbon dots.
3 Conclusion
Through orthogonal experiments, the suitable experimental conditions for preparing nano-fluorescent carbon dots by microwave method are determined as follows: reaction time is 2. The reaction temperature was 65438 080℃, the molar ratio of PEG to glucose was 6, and the pH was 9. The order of influencing factors in synthesis is: reaction time >; Molar ratio >; Reaction temperature. At the same time, it is found that the range of R blank > R temperature indicates that there are other important factors to be discussed in the experiment, and the most easily overlooked factor is stirring.
Under the same optimization conditions, the optical properties of carbon dots synthesized by hydrothermal method are slightly better than those synthesized by microwave. Besides the use of the stirring device mentioned in this paper, the reason may also be related to the growth rate, surface modification degree and state of carbon dots in the synthesis process. The comprehensive effect of these factors leads to the failure to control the lattice defects of fluorescent carbon dots well. However, surface defects and edge effects will lead to the generation of trapped electrons or hole pairs, which will further affect the luminescent properties of quantum dots, which needs further experimental verification. In a word, the fluorescent carbon dots prepared by the two heating methods have good optical properties and are expected to be used in the field of fluorescent labeling.