Blue fluorescent carbon dots and preparation method and application thereof

Abstract

The application discloses blue fluorescent carbon dots and a preparation method and application thereof. The preparation method of the blue fluorescent carbon dots comprises the following steps: taking folic acid, cysteine and citric acid, mixing them uniformly in ultrapure water, reacting by using a one-pot hydrothermal method, filtering the obtained reaction product, collecting the filtrate, and freeze-drying to obtain the blue fluorescent carbon dots. The blue fluorescent carbon dots can emit single blue light, have high luminance, and have good application potential in bacterial imaging detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of fluorescent nanomaterials, and particularly relates to a blue fluorescent carbon dot and a preparation method and application thereof. BACKGROUND

[0002] In the field of clinical imaging of microorganisms, a detection method based on fluorescence microscopy is commonly used; the method has high usability and high resolution, and is considered as one of the preferred methods for gram bacteria detection. At present, the small molecule fluorescent probes widely used in the detection method based on fluorescence microscopy have problems of poor stability, high toxicity and high cost, which further limits the further application of the method.

[0003] Carbon dots are a new type of fluorescent nanomaterials, and have advantages of good water solubility, simple preparation, high biocompatibility and the like. In recent years, carbon dots are widely applied in the field of biomedicine, and the excellent optical performance of the carbon dots provides a new direction for real-time monitoring and diagnosis of clinical bacteria. However, the research on the imaging function of carbon dots is still in the initial stage, and still faces great challenges.

[0004] Traditional semiconductor quantum dots need to be doped with heavy metals in the preparation process, so that the synthesized quantum dots have toxicity; compared with semiconductor quantum dots, carbon quantum dots (carbon dots) as a new type of carbon nanomaterial greatly reduce the toxicity. Generally, people use toxic amine-based benzene organic matter as a carbon source to prepare fluorescent carbon dots to realize the targeted imaging of bacteria, or use strong acid treatment or metal surface modification to cooperatively prepare; such a preparation method increases the toxicity of the carbon dot precursor on the one hand, and is difficult to popularize and apply; on the other hand, the cost of the preparation process of the carbon dots is also increased. At the same time, the prepared fluorescent carbon dot material must be quickly and simply adsorbed with the detected microorganism, so as to shorten the detection time, reduce the operation time of the tester, and reduce the operation error of the personnel. Therefore, it has become a research focus in the field to prepare carbon dots with excellent optical performance by using cheap and easily available, natural and non-toxic raw materials in a simple and effective way. SUMMARY

[0005] To solve the above problems, the application provides a blue fluorescent carbon dot and a preparation method and application thereof; the carbon dot can emit single blue light, and has high luminance, and has good application potential in bacterial imaging detection.

[0006] The application is implemented by the following technical solutions:

[0007] The application first provides a preparation method of a blue fluorescent carbon dot, which comprises the following steps: taking folic acid, cysteine and citric acid, mixing them uniformly in ultrapure water, performing reaction by using a hydrothermal method, then filtering the obtained product, collecting the filtrate, and freeze-drying to obtain the blue fluorescent carbon dot.

[0008] Preferably, the amounts of the raw materials in the present application are as follows: folic acid 0.2-0.3 g, cysteine 0.04-0.08 g, citric acid 0.25-0.3 g, and ultrapure water 20 mL.

[0009] Preferably, the reaction temperature is 120-220°C, more preferably 160°C.

[0010] Preferably, the reaction time is 6-12 hours, more preferably 8-10 hours.

[0011] Specifically, the mixed raw material solution is placed in a sealed reaction container and then heated and reacted in a heating device.

[0012] Preferably, the reaction container is a hydrothermal reaction kettle or a microwave reaction kettle, and the heating container is an oil bath pot, an electric heating constant-temperature air drying oven, or a microwave digestion and extraction instrument.

[0013] The present application also provides blue fluorescent carbon dots prepared by the above method.

[0014] The present application also provides the use of the blue fluorescent carbon dots in bacterial imaging detection.

[0015] The blue fluorescent carbon dots are preferably used in rapid imaging detection of bacteria.

[0016] Specifically, the bacteria include but are not limited to gram bacteria.

[0017] Preferably, in the above use, the blue fluorescent carbon dots are prepared into a solution with a concentration of 0.5-2 mg / mL using ultrapure water as the solvent, added to the bacteria to be detected for culture, and observed under a confocal fluorescence microscope; the culture time is 5 min.

[0018] 1. The method of the present application has the advantages of simple operation process, good repeatability, and convenience for industrialized production; the raw materials used have less harm to the human body and can be produced on a large scale.

[0019] 2. The carbon dots prepared by the present application can emit single blue light, have high luminance (the fluorescence intensity is maximum under 380 nm excitation light), good imaging performance, and low biological toxicity, and thus have strong application potential in the fields of image probes and fluorescent tracing, especially in bacterial imaging detection.

[0020] 3. The carbon dots of the present application have stable fluorescence properties, and the fluorescence peak position does not change substantially (about 460 nm) under the condition of 360-400 nm excitation light.

[0021] 4. The blue fluorescent carbon dots of the present application can achieve the purpose of targeted fluorescent staining imaging in a very short culture time (5 minutes) with gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli), so the blue fluorescent carbon dots of the present application can be applied to clinical rapid detection of bacteria and the like; and since the culture time can be reduced, the bacterial imaging detection time is shortened, the operation time of the tester is reduced, and the operation error of the tester is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a fluorescence spectrum of the blue fluorescent carbon dot solution of Examples 1 to 3;

[0023] Figure 2 is a confocal fluorescence imaging diagram of the effect of the blue fluorescent carbon dots of Example 1 on Staphylococcus aureus;

[0024] Figure 3 is a confocal fluorescence imaging diagram of the effect of the blue fluorescent carbon dots of Example 1 on Escherichia coli. DETAILED DESCRIPTION

[0025] The following examples are only used to illustrate the present application, and the protection scope of the present application is not limited to the following examples only. The person skilled in the art can achieve the purpose of the present application according to the above disclosure and the range of each parameter.

[0026] Example 1

[0027] (1) 0.22 g of folic acid, 0.06 g of cysteine and 0.276 g of citric acid were uniformly mixed in 20 mL of ultrapure water to prepare a mixed solution;

[0028] (2) The mixed solution obtained in step (1) was transferred to a hydrothermal reaction kettle, and hydrothermal reaction was carried out at 160°C for 8 h;

[0029] (3) The product obtained in step (2) was filtered through a 0.22 μm filter membrane to obtain a carbon dot solution;

[0030] (4) The carbon dot solution obtained in step (3) was freeze-dried to obtain blue fluorescent carbon dots.

[0031] The blue fluorescent carbon dots prepared were prepared into a solution with a concentration of 2 mg / mL with ultrapure water as the solvent, and the fluorescence spectrum was measured under the condition of 380 nm excitation light.

[0032] Example 2

[0033] (1) 0.22 g of folic acid, 0.06 g of cysteine and 0.276 g of citric acid were uniformly mixed in 20 mL of ultrapure water to prepare a mixed solution;

[0034] (2) The mixed solution obtained in step (1) was transferred to a hydrothermal reactor, and hydrothermal reaction was carried out at 190 DEG C for 8h;

[0035] (3) The product obtained in step (2) was filtered through a 0.22 μm filter membrane to obtain a carbon dot solution;

[0036] (4) After freeze-drying of the carbon dot solution obtained in step (3), blue fluorescent carbon dots were obtained.

[0037] The blue fluorescent carbon dots prepared were prepared into a solution with a concentration of 2 mg / mL using ultrapure water as a solvent, and the fluorescence spectrum was measured under the condition of 380 nm excitation light.

[0038] Example 3

[0039] (1) 0.22g folic acid, 0.06g cysteine, 0.276g citric acid were uniformly mixed in 20mL ultrapure water to prepare a mixed solution;

[0040] (2) The mixed solution obtained in step (1) was transferred to a hydrothermal reactor, and hydrothermal reaction was carried out at 210 DEG C for 8h;

[0041] (3) The product obtained in step (2) was filtered through a 0.22 μm filter membrane to obtain a carbon dot solution;

[0042] (4) After freeze-drying of the carbon dot solution obtained in step (3), blue fluorescent carbon dots were obtained.

[0043] The blue fluorescent carbon dots prepared were prepared into a solution with a concentration of 2 mg / mL using ultrapure water as a solvent, and the fluorescence spectrum was measured under the condition of 380 nm excitation light.

[0044] Example 4

[0045] (1) 0.28g folic acid, 0.04g cysteine, 0.29g citric acid were uniformly mixed in 20mL ultrapure water to prepare a mixed solution;

[0046] (2) The mixed solution obtained in step (1) was transferred to a hydrothermal reactor, and hydrothermal reaction was carried out at 160 DEG C for 8h;

[0047] (3) The product obtained in step (2) was filtered through a 0.22 μm filter membrane to obtain a carbon dot solution;

[0048] (4) After freeze-drying of the carbon dot solution obtained in step (3), blue fluorescent carbon dots were obtained.

[0049] Example 5

[0050] The imaging effect of the blue fluorescent carbon dots of Example 1 on Staphylococcus aureus (a representative of gram-positive bacteria) was verified, and the specific operation was as follows:

[0051] Staphylococcus aureus cells were cultured in LB medium at 37℃ for 12h, then 1mL of the culture suspension was centrifuged at 1800rpm for 5min, then the precipitate was suspended in 1mL PBS buffer after washing twice with phosphate buffer solution, then 1mL of the blue fluorescent carbon dots solution (2mg / mL) of Example 1 was added to the PBS solution containing Staphylococcus aureus, and incubated for 5min. Centrifugation was performed at 1800rpm, the supernatant was removed, 50μL of PBS was added for resuspension, 10μL was dropped onto a cover glass, and observed under a confocal fluorescence microscope.

[0052] Example 6

[0053] The imaging effect of the blue fluorescent carbon dots of Example 1 on Escherichia coli (a representative of gram-negative bacteria) was verified, and the specific operation was as follows:

[0054] Escherichia coli cells were cultured in LB medium at 37℃ for 12h, then 1mL of the culture suspension was centrifuged at 1800rpm for 5min, then the precipitate was suspended in 1mL PBS buffer after washing twice with phosphate buffer solution, then 1mL of the blue fluorescent carbon dots solution (2mg / mL) of Example 1 was added to the PBS solution containing Escherichia coli, and incubated for 5min. Centrifugation was performed at 1800rpm, the supernatant was removed, 50μL of PBS was added for resuspension, 10μL was dropped onto a cover glass, and observed under a confocal fluorescence microscope.

[0055] Figure 1 The fluorescence spectrum of the carbon dot solution prepared in Example 1, Example 2 and Example 3 under the condition of 380nm excitation light. As can be seen from the spectrum, the fluorescence intensity of the carbon dots decreases first and then increases with the increase of the preparation temperature, and the fluorescence intensity is the largest when the carbonization reaction is at 160℃. And the carbon dots obtained in the examples all show stable fluorescence properties, and the fluorescence peak position does not change basically (about 460nm) under the condition of 380nm excitation light.

[0056] Figure 2 The imaging picture of the fluorescent carbon dots prepared in Example 1 on Staphylococcus aureus. Staphylococcus aureus can emit strong blue fluorescence in a very short time after being cultured with the carbon dots, and the imaging contrast with the black background is clear. The blue fluorescent carbon dots prepared in the application can be successfully used for gram-positive bacteria targeted fluorescent labeling.

[0057] Figure 3 The imaging picture of the fluorescent carbon dots prepared in Example 1 on Escherichia coli. Escherichia coli can emit strong blue fluorescence in a very short time after being cultured with the carbon dots, and the imaging contrast with the black background is clear. The blue fluorescent carbon dots prepared in the application can be successfully used for gram-negative bacteria targeted fluorescent labeling.

[0058] The present application can be summarized in other specific forms without departing from the spirit or main features of the present application; for example, the amount of raw materials can be expressed in terms of mass ratio or molar ratio, i.e. the use of folic acid, cysteine, citric acid and water in the same mass ratio or molar ratio as the present application also falls within the scope of the technical solutions of the present application. The above embodiments of the present application can only be considered as a description of the present application rather than a limitation. Therefore, any minor modification, equivalent change and modification made to the above embodiments in accordance with the essence of the present application shall fall within the scope of the technical solutions of the present application.

Claims

1. A method for preparing blue fluorescent carbon dots, characterized in that, The method comprises the following steps: taking folic acid, cysteine and citric acid, mixing them uniformly in ultrapure water, reacting by using a hydrothermal method, filtering the product obtained in the reaction, collecting the filtrate, and freeze-drying to obtain blue fluorescent carbon dots. The amount of each raw material is as follows: folic acid 0.2-0.3 g, cysteine 0.04-0.08 g, citric acid 0.25-0.3 g, and ultrapure water 20 mL; the reaction temperature is 120-220 DEG C, and the reaction time is 6-12 hours. 2.The blue fluorescent carbon dots prepared by the preparation method of claim 1. 3.The blue fluorescent carbon dots of claim 2 are applied to bacterial imaging detection.

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