CN112126428A - Orange fluorescent carbon dot and preparation method and application thereof - Google Patents

Abstract

The invention provides an orange fluorescent carbon dot and a preparation method and application thereof, belonging to the field of preparation of fluorescent nano materials. The preparation method comprises the following steps: adding 5-aminosalicylic acid and citric acid into deionized water, and uniformly stirring to obtain a mixed solution; transferring the mixed solution into a hydrothermal reaction kettle for hydrothermal reaction; the obtained product is centrifuged to remove insoluble substances, and dialyzed to remove impurities, so as to obtain an orange fluorescent carbon dot solution. The invention has simple process and low requirement on preparation conditions; and the orange color obtainedThe fluorescent carbon dots have low toxicity and good biocompatibility. The prepared orange fluorescent carbon dots can be used for detecting Cu in a fluorescent/colorimetric dual mode²⁺And pH and cell imaging.

Description

An orange fluorescent carbon dot and its preparation method and application

technical field

The invention relates to fluorescent nanomaterials, in particular to an orange fluorescent carbon dot and a preparation method thereof, and the orange fluorescent carbon dot is used for fluorescence/colorimetric dual-mode Cu ²⁺ and pH sensing and cell imaging.

Background technique

Carbon dots, as a new type of carbon nanomaterials, have received extensive attention since their discovery in 2004. At present, there are many methods for synthesizing carbon dots, which can be classified into two categories: chemical methods and physical methods. Chemical methods include electrochemical synthesis method, combustion/pyrolysis/hydrothermal/acid oxidation method, template synthesis method, microwave/ultrasonic method, other chemical methods, etc. Physical methods include arc discharge, laser ablation/passivation, and plasma treatment, among others.

Carbon dots have the advantages of excellent photoluminescence properties, good photostability, excellent water solubility, and low toxicity. These properties make them promising for applications in imaging, drug delivery, medical diagnostics, fingerprint detection, and fluorescent inks. In particular, carbon dots have been widely used as chemical sensors and biosensors. However, most of these sensors can only measure one target. To further enrich its detection capabilities, many efforts have been made to build carbon dot-based multifunctional sensors. Currently, most of the synthesized multifunctional carbon dots emit blue fluorescence under UV excitation. In biological-related fields, blue fluorescent carbon dots are not attractive as optical probes, because the autofluorescence of organisms is generally blue, which will interfere with detection; in addition, ultraviolet light excitation will cause damage to organisms. Therefore, it is of great significance to develop multifunctional carbon dot-based fluorescent probes with long-wavelength emission.

Colorimetry is also an important detection method, which can determine the content of the component to be tested by comparing or measuring the color depth of the solution of colored substances. Fluorescence/colorimetric dual-mode assays have been extensively studied because they not only provide highly sensitive fluorescence detection methods, but also visualize targets with the naked eye. However, the research on C-dot-based fluorescence and colorimetric dual-mode sensors is still at an early stage and has a lot of research space.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an orange fluorescent carbon dot and a preparation method thereof. The preparation method should be simple in process and low in preparation conditions, and the prepared orange fluorescent carbon dot has excellent photostability and biocompatibility, and can be used for Fluorescence/colorimetric dual-mode Cu ²⁺ and pH sensing and cellular imaging.

The technical scheme provided by the present invention is:

A preparation method of orange fluorescent carbon dots, comprising the following steps:

(1) adding citric acid and 5-aminosalicylic acid to deionized water by mass 1:1～2:70～170 to prepare a mixed solution;

(2) the mixed solution that step (1) obtains is transferred in the hydrothermal reactor, and carries out hydrothermal reaction;

(3) centrifuging the product obtained in step (2) to remove insolubles to obtain a clear brown solution, and dialysis with a dialysis bag to remove impurities to obtain an orange fluorescent carbon dot solution;

(4) freeze-drying the orange fluorescent carbon dot solution obtained in step (3) to obtain the target orange fluorescent carbon dots.

In the step (1), the mass ratio of citric acid, 5-aminosalicylic acid and deionized water is 1:1-1.5:80-170.

In the step (2), the temperature of the hydrothermal reaction is 180-220° C., and the time is 1-5 h.

The dialysis in the step (3) is to use a dialysis bag with a molecular weight cut-off of 500 to 1000 Da for 12 to 24 hours.

The orange fluorescent carbon dots prepared by the method of the invention can be used for fluorescence/colorimetric dual-mode Cu ²⁺ and pH detection and cell imaging.

Compared with the prior art, the advantages of the present invention are:

(1) The prepared orange fluorescent carbon dots have good orange luminescence properties, and can be used for biological labeling and cell imaging to avoid the interference of biological autofluorescence.

(2) The prepared orange fluorescent carbon dots have strong stability, less toxic and side effects, and good water solubility.

(3) The prepared orange fluorescent carbon dots can be used for fluorescence/colorimetric dual-mode Cu ²⁺ and pH detection and cell imaging.

Description of drawings

Fig. 1 is the photo of the orange fluorescent carbon dot solution prepared in Example 1 under the excitation of fluorescent lamp and wavelength of 560 nm, respectively

Fig. 2 is the transmission electron microscope image and size distribution diagram of the orange fluorescent carbon dots prepared in Example 1

Fig. 3 is the infrared spectrogram of the orange fluorescent carbon dots prepared in Example 1

Fig. 4 is the X-ray photoelectron spectrogram of the orange fluorescent carbon dots prepared in Example 1

Fig. 5 is the ultraviolet absorption spectrum and fluorescence emission spectrum of the orange fluorescent carbon dots prepared in Example 1

Fig. 6 is the fluorescence emission spectra of orange fluorescent carbon dots prepared in Example 1 under different excitation wavelengths

FIG. 7 is a graph of the selectivity of the orange fluorescent carbon dots prepared in Example 1 to Cu ²⁺ (fluorescence method)

Figure 8 is a graph of the selectivity of the orange fluorescent carbon dots prepared in Example 1 to Cu ²⁺ (colorimetric method)

Fig. 9 is the fluorescence emission spectrum of the orange fluorescent carbon dot solution prepared in Example 1 with the change of Cu ²⁺ concentration

Figure 10 is the photo of the orange fluorescent carbon dot solution prepared in Example 1 under fluorescent lamp with the change of Cu ²⁺ concentration

Figure 11 is the fluorescence emission spectrum of the orange fluorescent carbon dot solution prepared in Example 1 as a function of pH

Figure 12 is a photo of the orange fluorescent carbon dot solution prepared in Example 1 under fluorescent lamp with pH changes

Figure 13 is the laser confocal image of the orange fluorescent carbon dot-labeled HeLa cells prepared in Example 1 before and after adding Cu ²⁺

Figure 14 is a laser confocal image of the orange fluorescent carbon dot-labeled HeLa cells prepared in Example 1 as a function of pH

Detailed ways

The following examples further illustrate the content of the present invention, but the present invention is not limited to these examples.

Example 1

Preparation of orange fluorescent carbon dots:

(1) 0.24g of citric acid and 0.30g of 5-aminosalicylic acid were added to 20mL of deionized water to obtain a mixed solution;

(2) The mixed solution obtained in (1) was transferred to a hydrothermal reaction kettle, and hydrothermally reacted at 200° C. for 3 h;

(3) centrifuge the product obtained in (2) at 4000r/min for 20min to remove insolubles to obtain a clear brown solution, and dialyze the product with a dialysis bag with a molecular weight cut-off of 500-1000Da for 24h to obtain an orange fluorescent carbon dot solution;

(4) After freeze-drying the solution of orange fluorescent carbon dots obtained in (3), orange fluorescent carbon dots are obtained, and the fluorescence quantum yield (based on Rhodamine 6G) is 14.0%.

The photos of the prepared orange fluorescent carbon dots solution under the excitation of fluorescent lamp and the wavelength of 560nm are shown in Figure 1. The left picture is the picture under the excitation of the wavelength of 560nm, the color is orange, and the right picture is the orange fluorescent carbon dot solution under the illumination of the fluorescent lamp. picture, the color is brown.

The TEM image and size distribution of the prepared orange fluorescent carbon dots are shown in Figure 2.

The infrared spectrum of the prepared orange fluorescent carbon dots is shown in Figure 3.

The X-ray photoelectron spectrum of the prepared orange fluorescent carbon dots is shown in Figure 4.

The UV absorption spectrum and fluorescence emission spectrum of the prepared orange fluorescent carbon dots are shown in Figure 5.

The fluorescence emission spectra of the prepared orange fluorescent carbon dots under different excitation wavelengths are shown in Figure 6, where 1 to 8 are the fluorescence spectra under excitation at wavelengths of 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm and 570 nm, respectively.

Example 2

Preparation of orange fluorescent carbon dots:

(1) 0.24g of citric acid and 0.30g of 5-aminosalicylic acid were added to 25mL of deionized water to obtain a mixed solution;

(2) The mixed solution obtained in (1) was transferred to a hydrothermal reaction kettle, and hydrothermally reacted at 180° C. for 3 h;

(3) centrifuge the product obtained in (2) at 4000r/min for 20min to remove insolubles to obtain a clear brown solution, and dialyze the product with a dialysis bag with a molecular weight cut-off of 500-1000Da for 24h to obtain an orange fluorescent carbon dot solution;

Example 3

Preparation of orange fluorescent carbon dots:

(1) 0.24g of citric acid and 0.30g of 5-aminosalicylic acid were added to 30mL of deionized water to obtain a mixed solution;

(2) transfer the mixed solution obtained in (1) into a hydrothermal reaction kettle, and perform a hydrothermal reaction at 220° C. for 3 h;

(3) The product obtained in (2) was centrifuged at 4000r/min for 20min to remove insoluble matter to obtain a clear brown solution, and dialyzed with a dialysis bag with a molecular weight cut-off of 500-1000Da for 24h to obtain an orange fluorescent carbon dot solution.

Example 4

Preparation of orange fluorescent carbon dots:

(1) 0.24g of citric acid and 0.30g of 5-aminosalicylic acid were added to 35mL of deionized water to obtain a mixed solution;

(2) transfer the mixed solution obtained in (1) into a hydrothermal reaction kettle, and perform a hydrothermal reaction at 200° C. for 2 h;

(3) The product obtained in (2) was centrifuged at 4000r/min for 20min to remove insoluble matter to obtain a clear brown solution, and dialyzed with a dialysis bag with a molecular weight cut-off of 500-1000Da for 24h to obtain an orange fluorescent carbon dot solution.

Example 5

Preparation of orange fluorescent carbon dots:

(1) 0.24g of citric acid and 0.30g of 5-aminosalicylic acid were added to 40mL of deionized water to obtain a mixed solution;

(2) The mixed solution obtained in (1) was transferred to a hydrothermal reaction kettle, and hydrothermally reacted at 200° C. for 4 h;

(3) The product obtained in (2) was centrifuged at 4000r/min for 20min to remove insoluble matter to obtain a clear brown solution, and dialyzed with a dialysis bag with a molecular weight cut-off of 500-1000Da for 24h to obtain an orange fluorescent carbon dot solution.

Example 6

The selectivity experiment of the orange fluorescent carbon dots prepared in Example 1 to Cu ²⁺ :

With pH=7.2 0.01mol·L ^-1 Tris-HCl buffer and CuCl ₂ , BiCl ₃ , CaCl ₂ , CdCl ₂ , FeCl ₃ , HgCl ₂ , KCl, AlCl ₃ , BaCl ₂ , MgCl ₂ , MnCl ₂ , NaCl, PbCl ₂ , and ZnCl ₂ were used to prepare solutions with a metal ion concentration of 262 μmol·L ^-1 , respectively, and 0.44 mg of the orange fluorescent carbon dots prepared in Example 1 were dissolved in 1 mL of the above solutions containing different metal ions. The fixed excitation wavelength was 550nm, at 20°C for fluorescence spectrum detection, according to (F/F ₀ ), to achieve the detection of Cu ²⁺ selectivity. According to the color of the solution under sunlight, the colorimetric detection of Cu ²⁺ selectivity can be achieved.

The graph of the selectivity (fluorescence method) of orange fluorescent carbon dots to Cu ²⁺ is shown in Fig. 7: The orange fluorescent carbon dots have the greatest response to Cu ²⁺ .

The graph of the selectivity (colorimetry) of orange fluorescent carbon dots to Cu ²⁺ is shown in Figure 8: when Cu ²⁺ is added to the solution of carbon dots, it is dark brown, and other ions are added to light yellow or light brown. The orange fluorescent carbon dots are selective for Cu ²⁺ .

Example 7

Sensitivity experiment of the orange fluorescent carbon dots prepared in Example 1 as ^Cu probes:

The Cu ²⁺ concentration was 0.05μmol·L ^-1 , 0.1μmol·L ^-1 , 0.25μmol·L ^-1 , 0.5μmol using 0.01mol·L ^-1 Tris-HCl buffer at pH=7.2 and CuCl ₂ , respectively. ·L ^-1 , 0.75μmol·L ^-1 , 1μmol·L ^-1 , 2.5μmol·L ^-1 , 5μmol·L ^-1 , 7.5μmol·L ^-1 , 10μmol·L ^-1 , 25μmol·L ^-1 , 50μmol·L ^-1 , 75μmol·L ^-1 , 100μmol·L ^-1 , 125μmol·L ^-1 , 150μmol·L ^-1 , 175μmol·L ^-1 , 200μmol·L ^-1 , 225μmol·L ^-1 , 250μmol· L ^-1 , 275 μmol · L ^-1 , 300 μmol · L ^-1 , 325 μmol · L ^-1 , 350 μmol · L ^-1 , 375 μmol · L ^-1 and 400 μmol · L ^-1 aqueous solutions, respectively, 0.44 mg of Example 1 was prepared The orange fluorescent carbon dots were dissolved in 1 mL of the above aqueous solutions containing different concentrations of Cu ²⁺ , and the excitation wavelength was fixed at 550 nm, and fluorescence spectrum detection was performed at 20 °C.

The fluorescence emission spectra of the orange fluorescent carbon dots solution with the concentration of Cu ²⁺ are shown in Figure 9, where 1 to 27 are the Cu ^{2 +} concentrations of 0μmol·L ^-1 , 0.05μmol·L ^{-1 , and 0.1μmol·L -1 ,} respectively. , 0.25μmol·L ^-1 , 0.5μmol·L ^-1 , 0.75μmol·L ^-1 , 1μmol·L ^-1 , 2.5μmol·L ^-1 , 5μmol·L ^-1 , 7.5μmol·L ^-1 , 10μmol· L ^-1 , 25μmol·L ^-1 , 50μmol·L ^-1 , 75μmol·L ^-1 , 100μmol·L ^-1 , 125μmol·L ^-1 , 150μmol·L ^-1 , 175μmol·L ^-1 , ^200μmol·L -1 ¹ , 225μmol·L ^-1 , 250μmol·L ^-1 , 275μmol·L ^-1 , 300μmol·L ^-1 , 325μmol·L ^-1 , 350μmol·L ^-1 , 375μmol·L ^-1 and 400μmol·L ^-1 The fluorescence emission spectrum of Tris-HCl buffer solution with orange fluorescent carbon dots dissolved; it can be seen from the figure that with the increase of Cu ²⁺ concentration, the fluorescence peak intensity at 595nm gradually decreases.

Figure 10 shows the photo of the orange fluorescent carbon dot solution under fluorescent lamp with the change of Cu ²⁺ concentration. From left to right in Figure 10, the Cu ²⁺ concentration is 0μmol·L ^-1 , 0.05μmol·L ^-1 , 0.1μmol·L ^{- . 1} , 0.25μmol·L ^-1 , 0.5μmol·L ^-1 , 0.75μmol·L ^-1 , 1μmol·L ^-1 , 2.5μmol·L ^-1 , 5μmol·L ^-1 , 7.5μmol·L ^-1 , 10μmol ·L ^-1 , 25μmol·L ^-1 , 50μmol·L ^-1 , 75μmol·L ^-1 , 100μmol·L ^-1 , 125μmol·L ^-1 , 150μmol·L ^-1 , 175μmol·L ^-1 , 200μmol·L -1 ^-1 , 225μmol·L ^-1 , 250μmol·L ^-1 , 275μmol·L ^-1 , 300μmol·L ^-1 , 325μmol·L ^-1 , 350μmol·L ^-1 , 375μmol·L ^-1 and 400μmol·L ^-1 The photo of Tris-HCl buffer solution dissolved with orange fluorescent carbon dots under fluorescent lamp; it can be seen from the figure that with the increase of Cu ²⁺ concentration, the color of the solution gradually changes from light yellow to dark brown.

Example 8

Experiment of the orange fluorescent carbon dots prepared in Example 1 to detect pH:

Dissolve 0.44 mg of the orange fluorescent carbon dots prepared in Example 1 into 1 mL of Tris-HCl buffers with different pH values, fix the excitation wavelength at 550 nm, and perform fluorescence spectrum detection at 20 °C. Detection of pH value.

The fluorescence emission spectrum of orange fluorescent carbon dots with pH changes is shown in Figure 11, where: 1 to 17 are pH values of 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, and 9.4, respectively. , 9.6, 9.8, 10.0, 10.2 fluorescence emission spectra. It can be seen from the figure that the fluorescence intensity decreases with the increase of pH value.

The photo of orange fluorescent carbon dots with pH changes under fluorescent light is shown in Figure 12. Figure 12 shows pH values of 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2 from left to right. , 9.4, 9.6, 9.8, 10.0, 10.2 photos under fluorescent light, it can be seen from the figure that with the increase of pH, the color of the solution gradually changes from light yellow to dark blue.

Example 9

Experiment of Cu ²⁺ sensing in living cells by orange fluorescent carbon dots prepared in Example 1:

The orange fluorescent carbon dots prepared in Example 1 were added to Tris-HCl buffer at pH 7.2 (the concentration of carbon dots was 1.32 mg/mL) for incubating HeLa cells for 30 min.

The laser confocal images of HeLa cells labeled with orange fluorescent carbon dots before and after adding Cu ²⁺ are shown in Figure 13. In the figure: a is the laser confocal image of HeLa cells labeled with carbon dots (showing orange fluorescence); b is carbon Confocal image (orange fluorescence quenching) of dot-labeled HeLa cells with added Cu ²⁺ (768 μmol·L ^-1 ).

Example 10

Experiment of pH sensing in living cells by orange fluorescent carbon dots prepared in Example 1:

The orange fluorescent carbon dots prepared in Example 1 were respectively added to Tris-HCl buffers with different pH values (the concentration of carbon dots was 1.32 mg/mL) for incubating HeLa cells for 30 min.

The laser confocal images of HeLa cells labeled with orange fluorescent carbon dots as a function of pH are shown in Figure 14. In the figure: a is the laser confocal image of HeLa cells labeled with carbon dots at pH 7.2; b is the laser confocal image of HeLa cells labeled with carbon dots at pH 8.2; c is the laser confocal image of HeLa cells labeled with carbon dots at pH 9.2 Laser confocal image. It can be seen from the figure that with the increase of pH value, the fluorescence intensity of cells gradually weakened.

Claims (9)

1. a preparation method of orange fluorescent carbon dots, is characterized in that, comprises the steps: (1) adding citric acid and 5-aminosalicylic acid to deionized water in a mass ratio of 1:1～2:70～170 to prepare a mixed solution; (2) the mixed solution that step (1) obtains is transferred in the hydrothermal reactor, and carries out hydrothermal reaction; (3) centrifuging the product obtained in step (2) to remove insolubles to obtain a clear brown solution, and dialysis with a dialysis bag to remove impurities to obtain an orange fluorescent carbon dot solution; (4) freeze-drying the orange fluorescent carbon dot solution obtained in step (3) to obtain the target orange fluorescent carbon dots. 2. the preparation method of a kind of orange fluorescent carbon dots as claimed in claim 1, is characterized in that, in described step (1), citric acid, 5-aminosalicylic acid and deionized water are 1:1 by mass ratio ~1.5:80~170. 3 . The method for preparing orange fluorescent carbon dots according to claim 1 , wherein the temperature of the hydrothermal reaction in the step (2) is 180-220° C., and the time is 1-5 h. 4 . 4 . The method for preparing orange fluorescent carbon dots according to claim 1 , wherein the dialysis in the step (3) is dialysis using a dialysis bag with a molecular weight cut-off of 500 to 1000 Da for 12 to 24 hours. 5 . 5. The orange fluorescent carbon dots prepared by the method of any one of claims 1-4. 6 . The application of the orange fluorescent carbon dots as claimed in claim 5 as fluorescent probes in the detection of Cu ²⁺ . 7. The application of the orange fluorescent carbon dots according to claim 5 in the colorimetric detection of Cu ²⁺ . 8. The application of the orange fluorescent carbon dots as claimed in claim 5 as a fluorescent probe in detecting pH. 9. The application of the orange fluorescent carbon dots according to claim 5 in colorimetric detection of pH.

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