Application of a fluorescent pH-sensitive antibacterial agent based on dendrobium officinale carbon quantum dots

Abstract

The application discloses application of dendrobium officinale-based carbon quantum dots as fluorescent pH-sensitive antibacterial agents, wherein the carbon quantum dots are prepared from dendrobium officinale by a reaction kettle hydrothermal reaction method. In the application, the dendrobium officinale-based carbon quantum dots have excellent pH-sensitive fluorescent performance and high-efficiency broad-spectrum antibacterial activity: the fluorescent intensity is enhanced with the decrease of pH, and the dendrobium officinale-based carbon quantum dots exhibit good bacteriostatic activity to staphylococcus aureus and escherichia coli; and the dendrobium officinale is used as a carbon source, and deionized water is used as a solvent, so that the preparation process is extremely environment-friendly and simple.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological new material preparation, and particularly relates to application of dendrobium officinale-based carbon quantum dots as fluorescent pH-sensitive antibacterial agents. BACKGROUND

[0002] In the development of new antibacterial strategies, real-time monitoring of the pH value of the bacterial infection site is not a simple technical addition, but a key link with core strategic value. Its importance stems from the "information hub" role played by pH in the infection process, which is manifested in the following aspects: achieving early diagnosis and accurate positioning of infection; guiding precise treatment to achieve "on-demand switch" of drug delivery; dynamically evaluating efficacy and predicting prognosis to serve clinical decision-making; and promoting basic research on infection biology and new drug development (Mater. Today Bio. 2023, 19, 100574). In addition, the prevention and treatment of bacterial infections are also facing severe challenges due to the emergence of antibiotic resistance (Ecotoxicol. Environ. Saf. 2023, 254, 114734; Microb. Pathog. 2024, 193, 106741). Therefore, the development of new nanomaterials with pH sensitivity and antibacterial properties has far-reaching significance for the development of new antibacterial strategies.

[0003] Currently, widely used organic fluorescent dyes and traditional quantum dots for in vivo pH detection have problems such as poor light stability and heavy metal toxicity, respectively (Chem. Sci. 2013, 4, 3725-3730). In the field of antibacterial applications, the misuse of antibiotics has led to the emergence of drug-resistant strains, and some inorganic antibacterial nanomaterials (such as silver nanoparticles) have long-term biological safety uncertainties and high costs. Therefore, both biological detection and antibacterial applications have put forward very high requirements for the biocompatibility and safety of materials.

[0004] As a new carbon-based nanomaterial, carbon quantum dots have shown great potential in solving the above problems due to their excellent biocompatibility, low toxicity and easy functionalization (Mater. Today Bio. 2024, 30, 101428; Mater. Today Commun. 2022, 33, 104347). However, the functions of carbon quantum dots in existing research are often single: carbon quantum dots used for pH sensing usually do not have significant antibacterial activity; and carbon quantum dots designed for antibacterial activity are rarely explored for their performance as fluorescent probes. This fragmentation of functions is partly due to the limitations of carbon source selection and preparation process, making it difficult to integrate efficient sensing units and antibacterial units in a single material. Developing a "dual-function" carbon quantum dot that can break this functional boundary and has both pH-sensitive detection capability and high-efficiency antibacterial activity has become a technical bottleneck that needs to be broken through in the field. SUMMARY

[0005] The purpose of the present application is to provide an application of dendrobium officinale-based carbon quantum dots as fluorescent pH-sensitive antibacterial agents. The dendrobium officinale-based carbon quantum dots have excellent pH-sensitive fluorescent properties and high-efficiency broad-spectrum antibacterial activity as antibacterial agents.

[0006] The present application provides the following technical solutions: An application of dendrobium officinale-based carbon quantum dots as fluorescent pH-sensitive antibacterial agents, wherein the carbon quantum dots are prepared by a reaction kettle hydrothermal reaction method using dendrobium officinale as raw material.

[0007] The dendrobium officinale-based carbon quantum dots provided by the present application are a dual-function carbon quantum dot that has both excellent pH-sensitive fluorescent properties and high-efficiency broad-spectrum antibacterial activity.

[0008] Further, the preparation method of the carbon quantum dots comprises the following steps: 1) adding dendrobium officinale powder into a solvent and ultrasonically mixing to obtain a reaction stock solution; 2) high-temperature reacting the reaction stock solution at 160-240 ℃ to obtain dendrobium officinale-based carbon quantum dots.

[0009] In step 1), the solvent is water, and the use amount ratio of the dendrobium officinale powder to water is 0.1-0.3 g: 5.0-15.0 mL, and the ultrasonic time is 30-90 min.

[0010] The preparation method comprises: 3) extracting the yellow-brown liquid obtained in step 2) with dichloromethane, taking the water phase to obtain dendrobium officinale carbon quantum dot crude product; 4) further dialysis purifying the crude product to obtain dendrobium officinale-based carbon quantum dots.

[0011] In step 3), the ratio of dichloromethane solvent to the yellow-brown liquid used for extraction is 5.0-15.0 mL: 5.0-15.0 mL.

[0012] In step 4), dialysis purification is performed for 3-6 days using a dialysis bag with a molecular weight cutoff of 7000-10000. Dialysis purification is used to remove unreacted reactants and impurities.

[0013] This invention regulates the particle size of Dendrobium officinale-based carbon quantum dots by controlling the amount of Dendrobium officinale powder in the reaction stock solution and the high-temperature reaction temperature.

[0014] Furthermore, the particle size of the Dendrobium officinale-based carbon quantum dots is 3.7 ± 1.1 nm; the lattice spacing of the Dendrobium officinale-based carbon quantum dots is 0.325 nm. Under these size conditions, the Dendrobium officinale-based carbon quantum dots exhibit superior pH-sensitive fluorescence properties and highly efficient broad-spectrum antibacterial activity.

[0015] The fluorescence intensity of the Dendrobium officinale-based carbon quantum dots increased as the pH decreased. Furthermore, the fluorescence intensity of the Dendrobium officinale-based carbon quantum dots exhibited a linear relationship within the pH range of 4-12.

[0016] Furthermore, the Dendrobium officinale-based carbon quantum dots are used to inhibit bacterial growth, and the bacterial mortality rate increases with the increase of Dendrobium officinale-based carbon quantum dot concentration.

[0017] Furthermore, the bacteria are *Escherichia coli* and *Staphylococcus aureus*. The *Dendrobium officinale*-based quantum dots provided by this invention exhibit good antibacterial activity against both *Staphylococcus aureus* and *Escherichia coli*.

[0018] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention utilizes *Dendrobium officinale* as raw material and employs a hydrothermal reaction method in a reaction vessel, using *Dendrobium officinale* as the sole carbon source. Through a one-step hydrothermal process without any post-modification, carbon quantum dots with dual functions of pH detection and antibacterial activity have been successfully prepared, offering advantages such as green raw materials and a simple preparation method. The obtained carbon quantum dots exhibit high sensitivity and good responsiveness to pH changes, enabling real-time, dynamic fluorescence detection and imaging of pH in vivo. Furthermore, these carbon quantum dots demonstrate strong inhibitory effects on both Gram-positive bacteria (such as *Staphylococcus aureus*) and Gram-negative bacteria (such as *Escherichia coli*), and exhibit good biocompatibility, providing a new potential solution to the problem of antibiotic resistance. This dual-functionality makes this material highly promising for constructing an integrated "detection-treatment" platform. Attached Figure Description

[0019] Figure 1 TEM image and crystal lattice (top right corner) of the Dendrobium officinale-based CDs prepared in Example 1. Figure 2 The optimal excitation and emission wavelength spectra of Dendrobium officinale-based CDs prepared in Example 1 are shown. Figure 3 The fluorescence spectra of Dendrobium officinale-based CDs prepared in Example 1 at different times; Figure 4 The UV-Vis absorption spectrum of Dendrobium officinale-based CDs prepared in Example 1 is shown below. Figure 5 XPS image of Dendrobium officinale-based CDs prepared in Example 1; Figure 6 The fluorescence spectra of Dendrobium officinale-based CDs prepared in Example 1 in PBS buffer solutions with different pH values ​​and the linear relationship between fluorescence intensity and pH value are shown. Figure 7 Laser confocal microscopy images of 4T1 cells incubated with Dendrobium officinale-based CDs prepared in Example 1 at different pH values; Figure 8 The inhibition zone diagram of the Dendrobium officinale-based CDs prepared in Example 1 against the bacterial strain; Figure 9 The graph shows the determination of the minimum inhibitory concentration (MIC) of Dendrobium officinale-based CDs prepared in Example 1. Detailed Implementation

[0020] Example 1 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots specifically includes the following steps: 1) Dry the stems of Dendrobium officinale and grind them into an ultrafine powder; 2) Add 0.2 g of the above-mentioned dried Dendrobium officinale stem ultrafine powder to 10 ml of deionized water solvent, and mix by sonication for 30 min to obtain the reaction stock solution; 3) Transfer the reaction stock solution to a high-pressure reactor, place the reactor in a high-temperature oven, and react at 220 °C for 12 h to obtain a yellow-brown liquid; 4) Take 5 ml of the yellowish-brown liquid obtained in step 3), add 5 ml of dichloromethane, extract, and take the aqueous phase to obtain the crude product of Dendrobium officinale-based carbon quantum dots; 5) The crude product from step 4) was further purified by dialysis for 3 days using a dialysis bag with a molecular weight cutoff of 8000 to remove unreacted reactants and impurities, and to obtain pure Dendrobium officinale-based carbon quantum with antibacterial effects.

[0021] like Figure 1 As shown, the TEM and crystal lattice of the Dendrobium officinale-based CDs prepared in Example 1 are shown. The morphology is spherical with a particle size of 3.7 ± 1.1 nm and a lattice spacing of 0.325 nm.

[0022] 0.1 mg of Dendrobium officinale-based CDs were dispersed in 1.0 ml of PBS solution at pH 7.4, and measurements were performed using a fluorescence spectrophotometer. A semi-micro fluorescence cuvette was used as the sample cell to determine the optimal excitation and emission wavelengths, such as... Figure 2 The image shows the optimal excitation and emission wavelength spectra of the Dendrobium officinale-based CDs prepared in Example 1. It can be seen that the optimal excitation wavelength is 346 nm and the optimal emission wavelength is 436 nm.

[0023] 0.2 mg of Dendrobium officinale-based CDs were dispersed in 1.0 ml of PBS solution at pH 7.4, and measurements were performed using a fluorescence spectrophotometer at 0 h, 12 h, 24 h, 36 h, and 48 h. A semi-micro fluorescence cuvette was used as the sample cell, and the excitation wavelength was 346 nm. All spectra were measured at room temperature. Figure 3 As shown, the fluorescence spectra of Dendrobium officinale-based CDs prepared in Example 1 at different times are shown. It can be seen that the fluorescence intensity does not change significantly with the extension of time.

[0024] 0.1 mg of Dendrobium officinale-based CDs were dispersed in 1.0 ml of PBS solution at pH 7.4, and measurements were performed using a UV spectrophotometer. A UV cuvette was used as the sample cell to measure the UV-Vis absorption spectrum at room temperature. Figure 4 The image shows the UV-Vis absorption spectrum of the Dendrobium officinale-based CDs prepared in Example 1, which shows a distinct UV absorption peak at 280 nm.

[0025] 2.0 mg of Dendrobium officinale-based CDs were dispersed in 1.0 ml of deionized water and measured using X-ray photoelectron spectroscopy (XPS). The Dendrobium officinale-based CDs solution was dropped onto the sample stage and then rapidly frozen into a solid state in liquid nitrogen. The frozen sample was then transferred to the sample chamber of the XPS instrument, forming a dry, solute-containing solid film. This dry solid film was then introduced into the analysis chamber for measurement at room temperature and ultra-high vacuum. Figure 5The figure shows the XPS full spectrum (a) and high-resolution XPS spectra of C 1s (b), N 1s (c), and O 1s (d) of the Dendrobium officinale-based CDs prepared in Example 1. The XPS full spectrum of the Dendrobium officinale-based CDs shows that the carbon quantum dots are mainly composed of C, N, and O elements, with atomic proportions of 67.78%, 4.58%, and 27.64%, respectively. The fine spectrum details the bonding between C, N, and O. The high-resolution C 1s spectrum shows three peaks at 284.8 eV, 286.26 eV, and 288.15 eV, which are attributed to C=C / CC, CO / CN, and C=O / C=N bonds, respectively. In the fine N 1s spectrum, the peak at 399.82 eV is attributed to a C=N bond. In the high-resolution spectrum of O1s, three peaks are observed at 532.27 eV, 531.13 eV, and 535.92 eV, which are attributed to C=O, -OH, and -OO- bonds, respectively. This indicates that C, N, and O elements are all present in the carbon quantum dots of *Dendrobium officinale*, and the possible functional groups include C=N, C=O, CN, and -OO-.

[0026] 2.0 mg of Dendrobium officinale-based CDs were dispersed in 1.0 ml of PBS at different pH values, and their concentrations were measured using a fluorescence spectrophotometer. Figure 6 The image shows the fluorescence spectra of the Dendrobium officinale-based CDs prepared in Example 1 in PBS buffer solutions at different pH values, and the linear relationship between fluorescence intensity and pH value. It can be seen that within the pH range of 4-12, the fluorescence intensity increases with decreasing pH, exhibiting a very good linear relationship.

[0027] Using 4T1 cells as a representative biological sample, Dendrobium officinale-based CDs were co-incubated with 4T1 cells for 2 h, followed by treatment with PBS buffer solutions at different pH values ​​(4.0, 6.0, and 8.0) for 15 min, and then fluorescence imaging was performed. Figure 7 The image shows laser confocal microscopy images of 4T1 cells incubated with Dendrobium officinale-based CDs prepared in Example 1 at different pH values. It can be seen that the intracellular fluorescence intensity increases as the cell pH decreases.

[0028] The antibacterial activity of *Dendrobium officinale*-based CDs was studied using the paper disc method. *Dendrobium officinale*-based CDs prepared at the same temperature and different reaction times (6 h, 8 h, and 12 h) were dissolved in sterile water to a concentration of 2 mg / ml; sterile water served as a negative control. The paper discs prepared by a perforator were immersed in the above solution for 10 min. In a clean bench, appropriate bacterial suspensions (Staphylococcus aureus or Escherichia coli) were evenly spread on solid culture media using sterile cotton swabs. Then, the soaked CDs were gently placed on the surface of the culture medium with the bacterial suspension using sterile forceps. The media were then inverted and incubated at 37°C for 24 h. The results were recorded by photographing, and each experiment was repeated three times. Figure 8 As shown, the inhibition zones of the Dendrobium officinale-based CDs prepared in Example 1 against Staphylococcus aureus and Escherichia coli strains are shown. It can be seen that under the same reaction temperature conditions and a reaction time of 12 h, the CDs prepared have good antibacterial activity against Staphylococcus aureus and Escherichia coli. The diameter of the inhibition zone against Staphylococcus aureus can reach 29.0±1.0 mm, and the diameter of the inhibition zone against Escherichia coli can reach 13.0±1.0 mm.

[0029] The minimum inhibitory concentration (MIC) was determined in 96-well plates using a serial dilution method. 0.5 mg / ml ampicillin sodium solution served as a positive control, and sterile water as a negative control. Different concentrations of *Dendrobium officinale*-based CDs were added to each well of the 96-well plate, with six wells replicated at the same concentration. Next, 100 μl of *Escherichia coli* or *Staphylococcus aureus* suspension was added to each well. The 96-well plates were then incubated at 37 ℃ for 18 h. After incubation, 20 μl of TTC (2,3,5-triphenyltetrazolium chloride) solution was added to each well. Finally, the plates were incubated at 37 ℃ for another 2 h before the results were observed. Figure 9 The figure shows the MIC determination of Dendrobium officinale-based CDs prepared in Example 1 against Staphylococcus aureus and Escherichia coli strains, respectively. It can be seen that the MIC of Dendrobium officinale-based CDs against Staphylococcus aureus (a) is 0.0125 mg / ml, and the MIC against Escherichia coli (b) is 0.25 mg / ml.

[0030] Example 2 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: in step 2), 0.1 g of the above-mentioned Dendrobium officinale dried stem ultrafine powder is added to 5 ml of deionized water solvent and ultrasonically mixed for 30 min to obtain the reaction stock solution.

[0031] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0032] Example 3 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: in step 2), 0.3 g of the above-mentioned Dendrobium officinale dried stem ultrafine powder is added to 15 ml of deionized water solvent and ultrasonically mixed for 30 min to obtain the reaction stock solution.

[0033] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0034] Example 4 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 2) ultrasonic mixing for 40 min to obtain the reaction stock solution.

[0035] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0036] Example 5 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 2) ultrasonic mixing for 50 min to obtain the reaction stock solution.

[0037] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0038] Example 6 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 2) ultrasonic mixing for 60 min to obtain the reaction stock solution.

[0039] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0040] Example 7 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 2) ultrasonic mixing for 70 min to obtain the reaction stock solution.

[0041] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0042] Example 8 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 2) ultrasonic mixing for 80 min to obtain the reaction stock solution.

[0043] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0044] Example 9 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 2) ultrasonic mixing for 90 min to obtain the reaction stock solution.

[0045] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0046] Example 10 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 3) is reacted at a high temperature of 160 ℃ for 12 h.

[0047] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0048] Example 11 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 3) is reacted at a high temperature of 180 ℃ for 12 h.

[0049] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0050] Example 12 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 4) is reacted at a high temperature of 200 ℃ for 12 h.

[0051] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0052] Example 13 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: step 3) is reacted at a high temperature of 240 ℃ for 12 h.

[0053] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0054] Example 14 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: in step 5), dialysis is performed for 6 days using a dialysis bag with a molecular weight cutoff of 7000.

[0055] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0056] Example 15 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: in step 5), dialysis is performed for 5 days using a dialysis bag with a molecular weight cutoff of 9000.

[0057] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0058] Example 16 A method for preparing Dendrobium officinale-based antibacterial carbon quantum dots, the synthesis steps are basically the same as those in Example 1, except that: in step 5), dialysis is performed for 4 days using a dialysis bag with a molecular weight cutoff of 10,000.

[0059] The final prepared Dendrobium officinale-based antibacterial carbon quantum dots are similar to those in Example 1.

[0060] The above embodiments are only used to explain the inventive concept of the present invention, and are not intended to limit the protection of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical and methodological essence of the present invention shall still fall within the scope of the technical and methodological solutions of the present invention.

Claims

1. The application of Dendrobium officinale-based carbon quantum dots as a fluorescent pH-sensitive antibacterial agent, wherein the carbon quantum dots are prepared from Dendrobium officinale by a hydrothermal reaction in a reaction vessel.

2. The application according to claim 1, characterized in that, The method for preparing the carbon quantum dots includes the following steps: 1) Add Dendrobium officinale powder to a solvent and mix using ultrasound to obtain the reaction stock solution; 2) The reaction solution was reacted at a high temperature of 160-240 ℃ to obtain Dendrobium officinale-based carbon quantum dots.

3. The application according to claim 2, characterized in that, In step 1), the solvent is water, and the ratio of Dendrobium officinale powder to water is 0.1-0.3 g: 5.0-15.0 mL, with an ultrasonic time of 30-90 min.

4. The application according to claim 2, characterized in that, The preparation method includes: 3) Extract the yellowish-brown liquid obtained in step 2) with dichloromethane, take the aqueous phase, and obtain the crude product of Dendrobium officinale carbon quantum dots; 4) The crude product was further purified by dialysis to obtain Dendrobium officinale-based carbon quantum dots.

5. The application according to claim 4, characterized in that, In step 3), the ratio of dichloromethane, the solvent used for extraction, to the yellow-brown liquid is 5.0-15.0 mL: 5.0-15.0 mL.

6. The application according to claim 4, characterized in that, In step 4), dialysis purification is performed for 3-6 days using a dialysis bag with a molecular weight cutoff of 7000-10000.

7. The application according to claim 1, characterized in that, The particle size of the Dendrobium officinale-based carbon quantum dots is 3.7 ± 1.1 nm.

8. The application according to claim 1, characterized in that, The fluorescence intensity of the Dendrobium officinale-based carbon quantum dots increased as the pH decreased.

9. The application according to claim 8, characterized in that, The fluorescence intensity of the Dendrobium officinale-based carbon quantum particles exhibits a linear relationship within the pH range of 4-12.

10. The application according to claim 1, characterized in that, The bacteria are Escherichia coli and Staphylococcus aureus.

Images