Preparation method and application of near-infrared fluorescent carbon dot probe targeting fusobacterium nucleatum

Abstract

The invention discloses a preparation method and application of a near-infrared fluorescent carbon dot probe targeting fusobacterium nucleatum, and the preparation method is characterized by comprising the following steps: taking malic acid, dihydric alcohol and formamide, stirring, ultrasonically and uniformly mixing, transferring the solution to a beaker, and carrying out a microwave method at 500-1000W for 3-8min; after the reaction is finished, cooling to room temperature, adding deionized water, carrying out high-speed centrifugation to remove large-particle impurities, filtering supernate by using a 0.22 mu m filter membrane, transferring the supernate into a dialysis bag, dialyzing, and freeze-drying to obtain black powder, namely the near-infrared fluorescent carbon dot probe targeting fusobacterium nucleatum. Wherein the mixing ratio of the malic acid to the dihydric alcohol to the formamide to the deionized water is 2g: 1mL: 16mL: 30mL, and the fusobacterium nucleatum fluorescence probe has the advantages that specificity, fluorescence signal output and biocompatibility are integrated on a single nano platform, efficiency loss caused by multi-step labeling is avoided, and a brand new solution is provided for rapid and accurate fusobacterium nucleatum detection and living imaging.

Description

Technical Field

[0001] This invention relates to a near-infrared fluorescent carbon dot probe, and more particularly to a method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum and its application. Background Technology

[0002] Fusobacterium nucleatum (Fn) is a Gram-negative anaerobic bacterium that widely colonizes the human oral cavity and intestines, and is one of the key pathogens of oral biofilms. Recent studies have shown that Fusobacterium nucleatum binds specifically to host cells through its surface adhesion factor FadA, playing a significant role not only in local infections such as periodontitis and premature birth, but also in systemic diseases such as colorectal cancer and breast cancer. For example, Fusobacterium nucleatum can bind to host E-cadherin via FadA protein, activating the β-catenin signaling pathway and promoting tumor cell proliferation and metastasis. Therefore, detection methods targeting FadA are of great significance for the early diagnosis, efficacy evaluation, and mechanistic research of Fusobacterium nucleatum-related diseases.

[0003] Current detection technologies for *Fusobacterium nucleatum* have limitations. For example, existing PCR technology requires a cumbersome DNA extraction process and cannot effectively distinguish between live and dead bacteria, leading to a risk of false positives. Immunological methods such as enzyme-linked immunosorbent assay (ELISA) rely on antibody detection, which may suffer from cross-reactivity (similar to epitopes of other *Fusobacterium* species), poor antibody stability, and high cost. Fluorescent labeling has gained widespread attention for bacterial labeling and imaging due to its high sensitivity, visualization, and non-invasiveness. Traditional fluorescent dyes suffer from photobleaching effects and aggregation-induced quenching (ACQ) phenomena, severely limiting their long-term imaging stability. Inorganic luminescent nanomaterials, such as semiconductor quantum dots and rare-earth luminescent materials, while possessing excellent photophysical properties, inevitably suffer from heavy metal toxicity. Fluorescent carbon dots, as an emerging luminescent nanomaterial, have many advantages such as simple preparation, low toxicity, excellent optical properties, and easy functionalization, showing excellent application potential in biochemical sensing, bioimaging, and diagnostics, and have been proven to be a highly competitive class of labeled probe materials. Currently, there are no research reports on near-infrared fluorescent carbon dot probes targeting *Fusobacterium nucleatum*. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a near-infrared fluorescent carbon dot probe for targeting Fusobacterium nucleatum with high specificity and detection sensitivity, and its application.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: a method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum, comprising the following steps: taking malic acid, diol and formamide, stirring and ultrasonically mixing, transferring the solution to a beaker, microwave method 500-1000W, 3-8min; after the reaction is completed, cooling to room temperature, adding deionized water, centrifuging at high speed to remove large particulate impurities, filtering the supernatant with a 0.22μm filter membrane, transferring the supernatant to a dialysis bag, dialysis and freeze-drying to obtain a black powder, which is the near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum.

[0006] Furthermore, the mixing ratio of the malic acid, the diol, the formamide, and the deionized water is 2g:1mL:16mL:30mL.

[0007] Furthermore, the diol is ethylene glycol, propylene glycol, or butanediol.

[0008] The present invention also provides the application of the near-infrared fluorescent carbon dot probe for targeting Fusobacterium nucleatum prepared by the above method in the preparation of Fusobacterium nucleatum detection reagents.

[0009] Compared with the prior art, the advantages of the present invention are as follows: 1. One-step synthesis: Using malic acid, formamide and ethylene glycol as precursors, high quantum yield near-infrared carbon dots are synthesized in one step by microwave method. Their emission wavelength effectively avoids the autofluorescence of biological tissues and improves the imaging penetration depth.

[0010] 2. Targeted Recognition Mechanism: Electrostatic Attraction: Positively charged outer membrane proteins on the surface of *Fusobacterium nucleatum* (e.g., 40-kDa OMP, isoelectric point ≈ 10) are initially anchored to the negatively charged carbon dots through electrostatic interactions. π-π Stacking: Aromatic amino acids (Trp, Phe) in bacterial transmembrane proteins specifically bind to the conjugated structure of carbon dots, enhancing binding strength and selectivity. Structural Specificity: The rigid β-barrel framework and flexible ring conformation of the conserved surface exposed loops (e.g., L1 loops) of bacteria work synergistically to achieve highly specific recognition of *Fusobacterium nucleatum*.

[0011] 3. Multifunctional Integrated Probe: This probe integrates specificity, fluorescence signal output, and biocompatibility into a single nanoplatform, avoiding efficiency losses caused by multi-step labeling. It provides a novel solution for rapid and accurate detection and in vivo imaging of *Fusobacterium nucleatum*. This invention not only fills the gap in the field of highly specific near-infrared bacterial probes but also provides a highly efficient tool for the diagnosis and treatment monitoring of infectious diseases. Attached Figure Description

[0012] Figure 1 Here is a high-resolution electron microscope image of the near-infrared fluorescent carbon dots prepared in Example 1; Figure 2Size distribution diagram of near-infrared fluorescent carbon dots prepared in Example 1; Figure 3 The ultraviolet absorption spectrum of the near-infrared fluorescent carbon dots prepared in Example 1; Figure 4 The near-infrared fluorescent carbon dot fluorescence spectrum prepared in Example 1; Figure 5 The image shows the FTIR spectrum of the near-infrared fluorescent carbon dots prepared in Example 1. Figure 6 The ultraviolet absorption spectrum of the near-infrared fluorescent carbon dots prepared in Example 2; Figure 7 The ultraviolet absorption spectrum of the near-infrared fluorescent carbon dots prepared in Example 3; Figure 8 This is a fluorescence confocal microscopy image of near-infrared fluorescent carbon dot-targeted nucleated Fusobacterium prepared in Example 1. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0014] Specific Example 1: Preparation Method of Near-Infrared Fluorescent Carbon Dot Probe Targeting Fusobacterium nucleatum Example 1: A method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum, comprising the following steps: Weigh 2.0 g of malic acid, 1 mL of ethylene glycol, and 16 mL of formamide. Stir and sonicate to mix thoroughly. Transfer the solution to a beaker and microwave at 700 W for 5 min. After the reaction, cool to room temperature, add 30 mL of deionized water, and centrifuge at high speed to remove large particles. Filter the supernatant through a 0.22 μm filter membrane. Transfer the supernatant to a dialysis bag and dialyze for 48 h. After lyophilization, obtain a black powder, which is the near-infrared fluorescent carbon dot probe targeting *Fusobacterium nucleatum*, abbreviated as NIR-CDs. As shown in Figure 1, NIR-CDs exhibit a uniform morphology in transmission electron microscopy (TEM) images. As shown in Figure 2, the particle size of NIR-CDs is approximately 3 nm. The ultraviolet absorption spectrum of NIR-CDs is as follows: Figure 3 As shown, distinct absorption peaks can be observed near 589 nm and 903 nm. Figure 4 As shown, NIR-I emission can be observed at around 650 nm under different excitations in the PL spectra of NIR-CDs, with the optimal excitation wavelength around 550 nm and the optimal emission wavelength around 650 nm. Each group was characterized by Fourier transform infrared (FTIR) spectroscopy, as shown below. Figure 5 As shown, the C––C bond is at 1600 cm⁻¹ -1The vibration at this location indicates the typical aromatic structure of CDs. A 1710 cm⁻¹ region was also observed in the sample. -1 C––O (electron acceptor group) bond at 1017 cm -1 The C–N bond at 3200 cm⁻¹, the –NH₂ (electron donor group) at 3200 cm⁻¹, and the C–N bond at 1630 cm⁻¹. -1 -1690 cm -1 The vibration of CO-NH (electron donor group) at the point is observed. These characterizations preliminarily demonstrate the successful synthesis of near-infrared carbon dots.

[0015] Example 2: A method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum, comprising the following steps: Weigh 2.0 g of malic acid, 1 mL of propylene glycol, and 16 mL of formamide. Stir and sonicate to mix thoroughly. Transfer the solution to a beaker and microwave at 700 W for 5 min. After the reaction is complete, cool to room temperature, add 30 mL of deionized water, and centrifuge at high speed to remove large particulate impurities. Filter the supernatant through a 0.22 μm filter membrane, transfer the supernatant to a dialysis bag, dialyze for 48 h, and then freeze-dry to obtain a black powder. Figure 6 Ultraviolet absorption spectroscopy shows that there are ultraviolet absorption peaks at 610 nm and 700 nm. The positions of the ultraviolet absorption peaks correspond to the absorption of photons with specific energies, which enables molecules to obtain the energy required for excitation.

[0016] Example 3: A method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum, comprising the following steps: Weigh 2.0 g of malic acid, 1 mL of butanediol, and 16 mL of formamide. Stir and sonicate to mix thoroughly. Transfer the solution to a beaker and microwave at 700 W for 5 min. After the reaction is complete, cool to room temperature, add 30 mL of deionized water, and centrifuge at high speed to remove large particulate impurities. Filter the supernatant through a 0.22 μm filter membrane, transfer the supernatant to a dialysis bag, dialyze for 48 h, and then freeze-dry to obtain a black powder. Figure 7 Ultraviolet spectroscopy shows ultraviolet absorption peaks at 510 nm and 610 nm. The positions of these ultraviolet absorption peaks correspond to the absorption of photons with specific energies, providing the molecules with the energy required for excitation.

[0017] Sensitivity analysis of the near-infrared fluorescent carbon dot probes prepared in Specific Example 2 and Specific Example 1.

[0018] Strain culture and preparation: Fusobacterium nucleatum (… Fusobacterium nucleatum, Fn (ATCC 25586) was cultured in an anaerobic incubator (80% N2, 10% H2, 10% CO2) on brain heart infusion agar (BHI) at 37°C for 48 hours. The bacterial cells were collected, washed three times with sterile PBS (pH 7.4), and analyzed by optical density method (OD). 600Adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / mL, serially diluted to 1×10⁻⁶ 3 ~1×10 8 CFU / mL was used as the standard bacterial solution.

[0019] Preparation of fluorescent probe: The NIR-CDs probe prepared in Example 1 was dissolved in PBS buffer to prepare a working solution with a concentration of 100 μg / mL.

[0020] Detection Procedure: Take a 96-well black ELISA plate and add 100 μL of different concentrations of *Fusobacterium nucleatum* bacterial culture to each well (three replicates per group). A blank control (PBS buffer only) should be set up. Add 50 μL of NIR-CDs probe working solution to each well and incubate at 37°C in the dark for 2 hours. After incubation, wash three times with PBS buffer to remove unbound probes. Add 200 μL of PBS buffer to each well and detect fluorescence intensity using a multi-mode microplate reader (excitation wavelength 550 nm, emission wavelength 670 nm).

[0021] Standard curve construction: Using the fluorescence intensity of the blank group as the baseline, after background subtraction, linear regression analysis (R²) was performed. 2 Construct a standard curve of fluorescence intensity (ΔF) versus bacterial concentration (log CFU / mL) ≥0.99), with the equation ΔF / mL. F =110×log(CFU / mL)−280, R 2 =0.992, the limit of detection (LOD) is 1×10 3 CFU / mL.

[0022] Specific analysis of the near-infrared fluorescent carbon dot probe prepared in Specific Example 1, Specific Example 3.

[0023] Targeted and quantitative detection of Fusobacterium nucleatum in complex microbial environments: This example uses a mixture of Escherichia coli, Staphylococcus aureus, and Fusobacterium nucleatum to simulate a complex microbial environment, as detailed below: Strain selection and culture conditions: Fusobacterium nucleatum ( Fusobacterium nucleatum , Fn (ATCC 25586) was cultured in an anaerobic incubator (80% N2, 10% H2, 10% CO2) on Brain Heart Infusion Agar (BHI) medium at 37°C for 48 hours. Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Klebsiella pneumoniae were cultured in TSB medium at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD). 600≈0.6), Enterococcus faecalis, Candida albicans, Pseudomonas aeruginosa, Morganella morganii, and maltobacterium were cultured in LB medium at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD). 600 ≈0.6).

[0024] Bacterial culture standardization: Each bacterial cell was washed three times with sterile PBS buffer (pH 7.4), and the culture was analyzed by optical density method (OD). 600 Adjust the concentration of the single bacterial culture to 1×10⁻⁶. 8 CFU / mL.

[0025] Preparation of mixed bacterial culture: via OD 600 Adjust the concentration of Fusobacterium nucleatum to 1×10⁻⁶. 6 CFU / mL, Escherichia coli and Staphylococcus aureus up to 1×10⁻⁶ 7 CFU / mL. Mixing ratio: Mix the three components in a volume ratio of 1:1:1.

[0026] Fluorescence detection: Follow the detection procedure in Example 2, with 3 parallel wells set up for each group. Detection parameters: excitation wavelength 550 nm, emission wavelength 670 nm. Using the fluorescence intensity (F0) of the blank group as the baseline, calculate ΔF (ΔF = Fsample - F0) for the experimental group and the control group. Specificity verification was performed according to the standard curve equation established in Example 2: there was no significant difference in fluorescence intensity between the mixed group and the positive group (p>0.05).

[0027] Figure 8 This is a fluorescence confocal microscopy image of near-infrared fluorescent carbon dot-targeted nucleated Fusobacterium. (See image.) Figure 8 As shown, *Fusobacterium nucleatum* exhibits a very distinct and dense red fluorescent signal. This indicates that NIR-CDs can bind well to *Fusobacterium nucleatum*, thus producing strong fluorescence during imaging. Compared with other bacteria, *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Streptococcus pneumoniae*, and *Enterococcus faecium* also show fluorescence signals in the NIR-CDs channel, but the intensity and density of the fluorescence signal are significantly lower than that of *Fusobacterium nucleatum*. This indicates that the binding efficiency of NIR-CDs to these cocci is relatively lower than that of *Fusobacterium nucleatum*. *Candida albicans*, as a fungus, shows a weak fluorescence signal in the NIR-CDs channel, which contrasts sharply with the strong fluorescence signal of *Fusobacterium nucleatum*, showing that the targeting of NIR-CDs to *Fusobacterium nucleatum* is significantly better than that of *Candida albicans*. *Pseudomonas aeruginosa*, *Morganella moschata*, *Klebsiella pneumoniae*, and *Stemonas maltella* also show weaker fluorescence signal intensity and distribution in the NIR-CDs channel than *Fusobacterium nucleatum*, directly demonstrating that the targeting of NIR-CDs to *Fusobacterium nucleatum* is superior. Figure 8 Other bacteria shown in the image.

[0028] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum, characterized in that... The process includes the following steps: Malic acid, diol, and formamide are stirred and ultrasonically mixed. The solution is then transferred to a beaker and microwaved at 500-1000W for 3-8 minutes. After the reaction is complete, the mixture is cooled to room temperature, deionized water is added, and high-speed centrifugation is used to remove large particulate impurities. The supernatant is filtered through a 0.22μm filter membrane and transferred to a dialysis bag. After dialysis, the supernatant is freeze-dried to obtain a black powder, which is the near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum.

2. The method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum according to claim 1, characterized in that: The mixing ratio of the malic acid, the diol, the formamide, and the deionized water is 2g:1mL:16mL:30mL.

3. The method for preparing a near-infrared fluorescent carbon dot probe targeting Fusobacterium nucleatum according to claim 1, characterized in that: The diol mentioned is ethylene glycol, propylene glycol, or butanediol.

4. The use of a near-infrared fluorescent carbon dot probe targeting *Fusobacterium nucleatum* prepared by the method of any one of claims 1-3 in the preparation of a *Fusobacterium nucleatum* detection reagent.

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