Yellow fluorescent carbon quantum dots for pathogenic bacteria targeting imaging and preparation method and application thereof

Abstract

The application discloses pathogenic bacteria-targeted yellow fluorescent carbon quantum dots and a preparation method and application thereof, wherein the preparation method provided by the application takes D-threonine and o-phenylenediamine as precursors, and nitrogen-doped yellow fluorescent quantum dots are prepared through a hydrothermal method. The reaction does not need to pretreat the D-threonine and o-phenylenediamine, nor does it need to add other reagents, and has the advantages of simple process and low production cost, and is suitable for large-scale batch production. The prepared yellow fluorescent carbon quantum dots can target pathogenic bacteria cells, enter the inside of the pathogenic bacteria and monitor the internal glutathione.

Description

Technical Field

[0001] This invention belongs to the field of pathogen monitoring technology, specifically relating to a yellow fluorescent carbon quantum dot for pathogen-targeted imaging, its preparation method, and its application. Background Technology

[0002] Pathogen monitoring primarily employs agar culture followed by biochemical probe detection. While this conventional method yields relatively reliable results, the lengthy culture time often misses the critical window for pathogen eradication. Existing pathogen monitoring methods struggle to quickly and effectively address major nosocomial infections and large-scale pathogen surveillance within the hospital environment, easily leading to strain on medical resources. There is a need to develop faster, more accurate, and more portable pathogen monitoring technologies for real-time monitoring of pathogens in the hospital environment. Methods such as polymerase chain reaction (PCR) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) can rapidly monitor pathogens, but the high cost of equipment, the use of toxic and hazardous reagents, the need for specialized facilities and personnel limit their application in real-time pathogen monitoring. Summary of the Invention

[0003] One of the objectives of this invention is to overcome the deficiencies of the prior art and provide a yellow fluorescent carbon quantum dot for targeted imaging of pathogens.

[0004] Another objective of this invention is to provide a method for preparing the above-mentioned yellow fluorescent carbon quantum dots.

[0005] Another object of the present invention is to provide an application of the above-mentioned yellow fluorescent carbon quantum dots.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing yellow fluorescent carbon quantum dots for pathogen-targeted imaging includes the following steps:

[0008] (1) Mix D-threonine and o-phenylenediamine in a mass ratio of 1:1 until homogeneous, and then react at 200 °C for 12 h to obtain a crude solution of carbon quantum dots;

[0009] (2) The crude carbon quantum dot solution synthesized under the above optimal conditions was cooled to room temperature, and then centrifuged and filtered. The filtrate was retained, and the filtrate was dialyzed with deionized water. The pH was then adjusted to 8, and the solution was freeze-dried under vacuum to obtain yellow fluorescent carbon quantum dots.

[0010] The dialysis operation uses 100-500D cellulose dialysis bags, and the filtration operation uses a filter pore size of 0.22 μM.

[0011] In some preferred implementations, the dialysis procedure is as follows: the filtrate is placed in a 100-500D cellulose dialysis bag and then left to stand in deionized water for 24-72 hours, during which the dialysis fluid is replaced every 4 hours.

[0012] In some preferred implementations, the preparation method further includes step (3): dissolving yellow fluorescent carbon quantum dots in deionized water to obtain an aqueous solution of yellow fluorescent carbon quantum dots, and then performing rotary evaporation and vacuum drying operations in sequence to obtain a solid powder of yellow fluorescent carbon quantum dots.

[0013] In some preferred implementations, in step (2), the reagent used to adjust the pH to 8 is a 1M NaOH solution.

[0014] A yellow fluorescent carbon quantum dot was prepared by the above method.

[0015] In some preferred implementations, the average particle size is 2.40 nm.

[0016] Application of yellow fluorescent quantum dots prepared by the above method in pathogen monitoring.

[0017] The application of the yellow fluorescent quantum dots prepared by the above method in the preparation of pathogen monitoring compositions.

[0018] A pathogen monitoring composition, wherein yellow fluorescent quantum dots prepared by the above method are used to monitor pathogens.

[0019] In some preferred implementations, the concentration of yellow fluorescent carbon quantum dots in the pathogen monitoring composition is 2.0 mg / mL.

[0020] The present invention has at least the following beneficial effects:

[0021] (1) The preparation method provided by the present invention uses D-threonine and o-phenylenediamine as precursors to prepare nitrogen-doped yellow fluorescent quantum dots by hydrothermal method. This reaction does not require pretreatment of D-threonine and o-phenylenediamine, nor does it require the addition of other reagents. It has the advantages of simple process and low production cost, and is suitable for large-scale mass production.

[0022] (2) The yellow fluorescent carbon quantum dots prepared by the present invention can target pathogen cells, enter the inside of pathogens and monitor the glutathione inside them, with a detection limit of 7.884 nM. Attached Figure Description

[0023] Figure 1 The image shows an aqueous solution of yellow fluorescent carbon quantum dots under 365 nm ultraviolet light, where yellow fluorescence can be observed.

[0024] Figure 2The UV-Vis absorption spectrum of yellow fluorescent carbon quantum dots;

[0025] Figure 3 The fluorescence excitation and emission spectrum of yellow fluorescent carbon quantum dots;

[0026] Figure 4 Transmission electron microscopy image of yellow fluorescent carbon quantum dots;

[0027] Figure 5 X-ray photoelectron spectrum of yellow fluorescent carbon quantum dots;

[0028] Figure 6 The infrared spectrum of yellow fluorescent carbon quantum dots;

[0029] Figure 7 The graph shows the fluorescence quantum yield of yellow fluorescent carbon quantum dots.

[0030] Figure 8 Feasibility study of detecting glutathione using yellow fluorescent carbon quantum dots;

[0031] Figure 9 This is a result of imaging glutathione in bacteria using yellow fluorescent carbon quantum dots. Detailed Implementation

[0032] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0033] In the following embodiments, the water used may be one or more of distilled water, purified water, and drinking water; unless otherwise specified, the detection methods in the following embodiments are conventional detection methods; unless otherwise specified, the reagents in the following embodiments are all purchased from commercial channels. In the following embodiments, the dialysate is deionized water.

[0034] Example 1: Yellow fluorescent quantum dots (h-DCQD) S Preparation of )

[0035] (1) Accurately weigh 1.0000 g of D-threonine and 1.0000 g of o-phenylenediamine solid, place them in a 10 mL beaker, mix well, transfer to a polytetrafluoroethylene reaction vessel, heat to 200 °C in a forced-air drying oven, and react for 12 h to obtain a crude carbon quantum dot liquid;

[0036] (2) Cool the above carbon quantum dot crude solution to room temperature, centrifuge and pass it through a 0.22 μM filter membrane, retain the filtrate, and then dialyze it in deionized water for 24 hours using a 100-500D cellulose dialysis bag, changing the deionized water every 4 hours during the process. Then adjust the pH to 8.0 with 1M NaOH to obtain yellow fluorescent carbon quantum dots.

[0037] (3) The above yellow fluorescent carbon quantum dots were freeze-dried under vacuum and then dissolved in ultrapure water to obtain an aqueous solution of yellow fluorescent carbon quantum dots with a concentration of 2.0 mg / mL.

[0038] Example 2 Structural characterization of yellow fluorescent quantum dots

[0039] (1) When the aqueous solution of the yellow fluorescent carbon quantum dots prepared in Example 1 is irradiated under a 365 nm ultraviolet lamp, yellow fluorescence can be observed (see Figure 1 ).

[0040] (2) A small amount of the aqueous solution of the yellow fluorescent carbon quantum dots prepared in Example 1 was diluted, and its ultraviolet-visible absorption spectrum was scanned. The characteristic absorption peak of the yellow fluorescent carbon quantum dot solution targeting bacteria was located at 430 nm (see Figure 2 ).

[0041] (3) Take a small amount of the aqueous solution of the yellow fluorescent carbon quantum dots prepared in Example 1 and dilute it. Scan its excitation and emission spectra. From the spectra, it can be seen that the optimal excitation wavelength and the optimal emission wavelength of the carbon quantum dot solution are 285 nm / 430 nm and 558 nm, respectively (see Figure 3 ).

[0042] (4) The aqueous solution of the yellow fluorescent carbon quantum dots prepared in Example 1 was coated on a copper grid and examined by transmission electron microscopy. The yellow fluorescent carbon quantum dots targeting bacteria were regular monodisperse spheres with an average particle size of 2.40 nm (see Figure 4 ).

[0043] (5) The aqueous solution of yellow fluorescent carbon quantum dots prepared in Example 1 was subjected to rotary evaporation and vacuum drying to obtain a solid powder of yellow fluorescent carbon quantum dots targeting bacteria. X-ray photoelectron spectroscopy (XPS) was performed on the obtained powder. The spectrum showed that the surface of the carbon quantum dots contained a large amount of C, O, and N elements (see [link to XPS]). Figure 5 ).

[0044] (6) Fourier transform infrared (FTIR) spectroscopy was performed on the solid powder of yellow fluorescent carbon quantum dots, which further confirmed the presence of oxygen-containing groups (CO, -COOH, C=O) and -NH2 as well as CC (see Figure 6 ).

[0045] (7) Using Rhodamine 6 G dissolved in ethanol as a reference (420 nm as the excitation wavelength, absorbance at 420 nm, QY=0.95), the emission spectra of different concentrations of h-DCQDs and Rhodamine 6 G under excitation at 420 nm and the corresponding concentrations of ultraviolet absorption spectra were measured. A graph was then plotted with the absorbance at 420 nm in the corresponding absorption spectrum at the same concentration as the X-axis and the area integral of the emission spectrum as the Y-axis. The slope of the curve was calculated, and then the equation was used:

[0046]

[0047] The relative fluorescence quantum yield was calculated, where “Φ” represents the fluorescence quantum yield, “m” represents the slope of the curve obtained by plotting the area integral of the emission spectrum against the absorbance value, “η” is the refractive index of the solvent (where the refractive index of Rhodamine 6 G dissolved in ethanol is 1.36, and the refractive index of carbon quantum dots dissolved in ultrapure water is 1.33), and “ST” and “X” represent the standard and sample solutions, respectively. The results are as follows: Figure 7 As shown, the relative fluorescence quantum yield of h-DCQDs was calculated to be 19.23%.

[0048] Example 3: Detection of glutathione using yellow fluorescent quantum dots

[0049] (1) TEM results of the morphological changes of yellow fluorescent carbon quantum dots induced by the product TNB after the reaction of the aqueous solution of yellow fluorescent carbon quantum dots prepared in Example 1 with DTNB / GSH (see Figure 8 A).

[0050] (2) A small amount of the product from the reaction of yellow fluorescent carbon quantum dots in Example 1 with DTNB / GSH and TNB aqueous solution was diluted, and its UV-Vis absorption spectrum was scanned. The characteristic absorption peak of TNB was located at 430 nm, which coincided with the emission peak of the yellow fluorescent carbon quantum dot solution, indicating that the product TNB can induce fluorescence quenching of TOCDs (see Figure 8 B). Under the same 430nm excitation light, the yellow fluorescent carbon quantum dots produced strong fluorescence emission, while TNB showed almost no fluorescence emission (B). Figure 8 (as shown in C).

[0051] (3) A small amount of the yellow fluorescent carbon quantum dots prepared in Example 1 was diluted with an aqueous solution to verify the feasibility of TNB quenching TOCDs fluorescence. First, 6×10⁶ ppm of PBS buffer was prepared. -6 Glutathione (GSH) at mol / mL and 5×10 -65,5'-Dithiobis(2-nitrobenzic acid) (DTNB) at mol / mL was added. Then, 50 μL of DTNB, 0-150 μL of GSH, and 50 μL of TOCDs were mixed, followed by the addition of PBS buffer to a total volume of 2 mL. The reaction was allowed to proceed for 0.5 h. Figure 8 As can be clearly seen in illustration D, the solution color gradually deepens with increasing glutathione addition, while the fluorescence intensity gradually decreases. Figure 8 As shown in Figure D), the feasibility of TNB quenching TOCDs fluorescence was verified. After sufficient reaction, the emission spectra of each solution were measured under 420 nm excitation light, and the peak value of the emission spectrum (fluorescence intensity at 560 nm) was taken as the fluorescence intensity of the solution. The results are shown in Figure D. Figure 8 As shown in Figure E, the fluorescence intensity decreased drastically as the glutathione concentration increased from 0 to 40 nM. However, when the glutathione concentration exceeded 40 nM, the decrease in fluorescence intensity slowed down (or remained almost constant). Figure 8 The inset in E shows a linear relationship between fluorescence intensity and the concentration of glutathione (0-40 nM), with the fitted linear equation being: Y = -6.47X + 946.48, R0 2 The value is 0.96793. At a confidence interval of 95%, the detection limit for glutathione by this fluorescence sensing system is 7.884 nM.

[0052] Example 4: Imaging of glutathione in bacteria using yellow fluorescent quantum dots

[0053] (1) The aqueous solution of yellow fluorescent carbon quantum dots prepared in Example 1 was incubated with six kinds of bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Serratia marcescens, and Candida albicans. The fluorescence imaging effect of the bacteria stained with TOCDs was significant (see Figure 9 A).

[0054] (2) The aqueous solution of yellow fluorescent carbon quantum dots prepared in Example 1 was incubated with six kinds of bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Serratia marcescens, and Candida albicans. After the addition of GSH, the fluorescence intensity of the bacteria stained with TOCDs decreased significantly (see Figure 9 B).

[0055] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. The application of yellow fluorescent carbon quantum dots in the detection of glutathione, characterized in that, The preparation method of the yellow fluorescent carbon quantum dots includes the following steps: (1) D-threonine and o-phenylenediamine were mixed evenly in a mass ratio of 1:1 and then reacted at 200 °C for 12 h to obtain a crude solution of carbon quantum dots; (2) Cool the above carbon quantum dot crude solution to room temperature, centrifuge and pass it through a 0.22 μM filter membrane, retain the filtrate, and then dialyze it in deionized water for 24 hours using a 100-500 Da cellulose dialysis bag, changing the deionized water every 4 hours during the process. Then adjust the pH to 8.0 with 1M NaOH to obtain yellow fluorescent carbon quantum dots. (3) Dissolve the yellow fluorescent carbon quantum dots in deionized water to obtain an aqueous solution of the yellow fluorescent carbon quantum dots, and then perform rotary evaporation and vacuum drying operations in sequence to obtain a solid powder of the yellow fluorescent carbon quantum dots. 2.The application of the yellow fluorescent carbon quantum dots in detecting glutathione according to claim 1, wherein, The average particle size of the yellow fluorescent carbon quantum dots is 2.40 nm.

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