Carbon quantum dot for targeted detection of petroleum degrading bacteria as well as preparation method and application of carbon quantum dot

Abstract

The invention discloses carbon quantum dots for targeted detection of petroleum degrading bacteria as well as a preparation method and application of the carbon quantum dots, and belongs to the technical field of biochemical detection. The carbon quantum dot is prepared by the following method: mixing L-histidine and diethylenetriamine, adding concentrated hydrochloric acid and water, and carrying out high-temperature reaction; after the reaction is finished, obtaining a brown yellow liquid; filtering, dialyzing and freeze-drying the brownish yellow liquid to obtain a brownish yellow intermediate; mixing the brownish yellow intermediate with acetone, adding EDC and NHS, then adding octadecanoic acid, adjusting the pH to be neutral, and stirring for reaction to obtain a brownish yellow liquid; and then filtering, dialyzing and freeze-drying the brown yellow liquid to obtain the carbon quantum dots. The prepared carbon quantum dots are novel in structure, high in sensitivity and good in cell membrane permeability, and targeted dynamic detection of petroleum degrading bacteria can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of biochemical detection technology, specifically relating to a carbon quantum dot for targeted detection of petroleum-degrading bacteria, its preparation method, and its application. Background Technology

[0002] Oil-contaminated soil is highly toxic, causing birth defects, cancer, and mutations, posing a significant potential threat to human health and ecological security. As the persistence of oil pollution in soil increases significantly, the difficulty of remediation also rises, making the treatment and remediation of oil-contaminated soil an urgent priority. Currently, the primary method for removing petroleum hydrocarbons from soil is microbial degradation. This method involves isolating biodegradable bacterial strains from oil-contaminated soil to degrade and remove the oil from the soil.

[0003] Studies have shown that microorganisms that can grow in petroleum hydrocarbon-contaminated environments typically produce biosurfactants, increasing their degradation rate. Furthermore, the hydrophobicity of bacteria facilitates the hydroxylation of petroleum hydrocarbons, thereby enhancing their biotransformation efficiency. Therefore, during the metabolism of petroleum hydrocarbons, degrading bacteria generate a large amount of hydrophobic agents, resulting in strong cell surface hydrophobicity. This allows them to "miscible" with petroleum hydrocarbons, causing them to target and aggregate on the bacterial surface, where they are then endocytosed, completing the degradation of petroleum hydrocarbons.

[0004] To investigate the degradation relationship between petroleum pollutants and degrading bacteria, a proper method for detecting petroleum-degrading bacteria needs to be established. Currently known detection methods include: radiolabeled membrane-permeable weak acid or alkali methods, etc. 31 Methods such as nuclear magnetic resonance (NMR) and microelectrode methods are commonly used for detecting petroleum pollutants metabolized by degrading bacteria. However, these methods generally suffer from problems such as complex operation, low sensitivity, and low spatiotemporal resolution in detecting petroleum-degrading bacteria. Therefore, developing a low-cost, highly sensitive, moderately hydrophobic, targeted, and fluorescence imaging reagent for detecting petroleum-degrading bacteria is of great importance and significance for the dynamic detection of these bacteria, understanding their colonization dynamics in petroleum-contaminated saline soils, revealing the mechanism of petroleum hydrocarbon metabolism by these bacteria, and elucidating the mechanism by which they remediate petroleum-contaminated saline soils. Summary of the Invention

[0005] This invention provides carbon quantum dots for targeted detection of petroleum-degrading bacteria, wherein the carbon quantum dots are L-Histidine-N,Cl-CDs-C. 18 .

[0006] The above-mentioned carbon quantum dots were prepared by the following method: L-histidine and diethylenetriamine were mixed, and concentrated hydrochloric acid and water were added. The mixture was reacted at high temperature. After the reaction was completed, a brownish-yellow liquid was obtained. The brownish-yellow liquid was filtered, dialyzed, and freeze-dried to obtain a brownish-yellow intermediate. The brownish-yellow intermediate was mixed with acetone, EDC and NHS were added, and then octadecanoic acid was added. The pH was adjusted to neutral, and the mixture was stirred to obtain a brownish-yellow liquid. The brownish-yellow liquid was then filtered, dialyzed, and freeze-dried to obtain carbon quantum dots.

[0007] In the above method for preparing carbon quantum dots, the components are selected from the following proportions: 100-350 parts L-histidine, 50-1500 parts diethylenetriamine, 0.1-0.5 parts concentrated hydrochloric acid, 10-50 parts water, 10-50 parts acetone, 100-300 parts EDC, 100-300 parts NHS, and 200-1500 parts octadecanoic acid.

[0008] When the component is a solid component, the quantity represents milligrams; when the component is a liquid component, the quantity represents milliliters; in practical applications, the quantity can be increased or decreased proportionally.

[0009] In the above method for preparing carbon quantum dots, the molar ratio of L-histidine to diethylenetriamine is selected from 1:(1~10); preferably 1:1, 1:3, 1:5, 1:8 or 1:10.

[0010] In the above method for preparing carbon quantum dots, the conditions for the high-temperature reaction are: a high-temperature reaction at 150~250℃ for 5~15h.

[0011] In the above method for preparing carbon quantum dots, the stirring reaction conditions are: stirring at 20~30℃ for 5~10h.

[0012] This invention provides the application of the above-mentioned carbon quantum dots in the targeted detection of petroleum-degrading bacteria.

[0013] In the above applications, the petroleum-degrading bacteria are preferably petroleum-degrading bacteria BF04 or petroleum-degrading bacteria L10.

[0014] This invention provides a method for targeted detection of petroleum-degrading bacteria, comprising the following steps: Carbon quantum dots were added to the sample solution of petroleum-degrading bacteria and incubated. After incubation, the fluorescence distribution of carbon quantum dots in the cells of petroleum-degrading bacteria was observed under the excitation wavelength to achieve the detection of petroleum-degrading bacteria.

[0015] In the above detection method, the amount of carbon quantum dots used is 10~100 mg / L; preferably 50 mg / L.

[0016] In the above detection method, the incubation conditions are selected from: incubation at 20~30℃ for 20~240 min.

[0017] In the above detection method, the excitation wavelength is 306 nm.

[0018] The beneficial effects of this invention are as follows: The carbon quantum dots prepared in this invention have a novel structure, high sensitivity, and good cell membrane permeability, enabling targeted dynamic detection of petroleum-degrading bacteria. Using these carbon quantum dots for dynamic detection of petroleum-degrading bacteria allows for understanding the colonization dynamics of these bacteria in petroleum-contaminated saline soils. This is crucial for revealing the mechanisms by which petroleum-degrading bacteria metabolize petroleum hydrocarbons and elucidating their mechanisms for remediating petroleum-contaminated saline soils. The carbon quantum dots of this invention demonstrate significant application value in the field of biochemical detection technology. Attached Figure Description

[0019] Figure 1 The images show TEM images and particle size distribution of carbon quantum dots.

[0020] Figure 2 The image shows the infrared spectrum of carbon quantum dots.

[0021] Figure 3 This is the ultraviolet absorption spectrum of carbon quantum dots.

[0022] Figure 4 This is the fluorescence emission spectrum of carbon quantum dots.

[0023] Figure 5 The antibacterial activity of carbon quantum dots against Staphylococcus aureus (a), Escherichia coli (b), and petroleum-degrading bacteria BF04 (c) was studied.

[0024] Figure 6 The images show fluorescence images of carbon quantum dots in petroleum-degrading bacteria BF04. A, b, and c represent the first group of experiments: a is a bright-field image; b is the fluorescence image after incubation with octadecane for 30 min; c is the fluorescence image after incubation with octadecane for 30 min followed by incubation with carbon quantum dots for 60 min. D, e, and f represent the second group of experiments: d is a bright-field image; e is the fluorescence image after incubation with only carbon quantum dots for 30 min; and f is the fluorescence image after incubation with only carbon quantum dots for 60 min.

[0025] Figure 7The images show fluorescence images of carbon quantum dots in petroleum-degrading bacteria L10 and Staphylococcus aureus. Among them, a, b, and c are fluorescence images of Staphylococcus aureus; a is a bright-field image, b is a fluorescence image after 30 min of incubation, and c is a fluorescence image after 60 min of incubation. d, e, and f are fluorescence images of petroleum-degrading bacteria L10; d is a bright-field image, e is a fluorescence image after 30 min of incubation, and f is a fluorescence image after 60 min of incubation. Detailed Implementation

[0026] In this invention, the octadecane used is n-octadecane, i.e., a straight-chain alkane. The EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the NHS is N-hydroxysuccinimide.

[0027] In this invention, the petroleum-degrading bacterium BF04 was isolated from *Gnaphalium affine* growing in the heavily saline-alkali land of the Yellow River Delta, which is currently preserved in our laboratory. Through liquid culture diesel degradation experiments, this strain demonstrated good degradation capabilities for straight-chain alkanes, branched alkanes, and monoaromatic hydrocarbons in diesel fuel. Related experiments can be found in the following literature: Wu Tao, Xu Jie, Xie Wenjun, et al. Screening and degradation characteristics of endophytic hydrocarbon-degrading bacteria in the salt-tolerant plant *Gnaphalium affine* [J]. Journal of Agricultural Environmental Science, 2017, 36(11): 2267-2274.

[0028] In this invention, the petroleum-degrading bacterium L10 is a reed endophytic bacterium isolated from saline soil in the Yellow River Delta, which is widely distributed and capable of consuming petroleum hydrocarbons. It is currently preserved at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 9406. Upon contact with petroleum hydrocarbons, this bacterium generates a large amount of hydrophobic agents, resulting in strong cell surface hydrophobicity, which helps degrade pollutants and promotes reed growth. Related experiments can be found in the following literature: Wu T, Xu J, Xie WJ, et al. Pseudomonas aeruginosa L10: a hydrocarbon-degrading, biosurfactant-producing, and plant-growth-promoting endophytic bacterium isolated from a Reed (Phragmites australis)[J]. Frontiers in Microbiology, 2018, 9(5): 1087.

[0029] In this invention, the structural formula of the carbon quantum dot is as follows: Its preparation process is as follows: In this invention, the MS medium formulation is as follows: The formula contains ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, calcium chloride 440 mg / L, magnesium sulfate 370 mg / L, and potassium dihydrogen phosphate 170 mg / L. Trace elements include potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, and cobalt chloride 0.025 mg / L. Organic components include inositol 100 mg / L, niacin 0.5 mg / L, vitamin B6 0.5 mg / L, vitamin B1 0.1 mg / L, and glycine 2 mg / L. Additionally, 30 g / L sucrose is added as a carbon source, and 6 g / L agar is added as a solidifying agent.

[0030] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0031] Example 1 Preparation of carbon quantum dots (L-Histidine-N,Cl-CDs-C) 18 The steps are as follows: 155 mg of L-histidine and 103 mg of diethylenetriamine were added to a 50 mL beaker, followed by 150 μL of concentrated hydrochloric acid (36%) and 15 mL of deionized water. After the L-histidine was completely dissolved, the mixture was transferred to a 50 mL reaction vessel, which was then placed in an oven at 180 °C for 10 h. After cooling to room temperature, a brownish-yellow liquid was obtained. The brownish-yellow liquid was filtered through a disposable syringe aqueous filter (13*0.22 μm) to remove the residue. The filtrate was placed in a regenerated cellulose membrane dialysis bag (200 Da) and dialyzed for 3 h. After dialysis, the liquid was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 196 mg of a brownish-yellow intermediate (L-Histidine-N, Cl-CDs); the yield was 63.77%.

[0032] 196 mg of the brownish-yellow intermediate product was placed in a 50 mL round-bottom flask and dissolved in 20 mL of acetone. Then, 169 mg of EDC and 165 mg of NHS were added and dissolved separately. Next, 284 mg of octadecanoic acid was added, and the pH was adjusted to 7. The mixture was stirred at room temperature for 6 h to obtain a brownish-yellow liquid. The brownish-yellow liquid was filtered through a disposable needle oil filter (13*0.22 μm) to remove the residue. The filtrate was placed in a polypropylene membrane dialysis bag (300 Da) and dialyzed in acetone for 5 h. After dialysis, the filtrate was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 341 mg of carbon quantum dots (L-Histidine-N, Cl-CDs-C). 18 The yield was 51.11%.

[0033] The size and shape of carbon quantum dots were characterized by transmission electron microscopy (TEM), such as... Figure 1 As shown, carbon quantum dots are spherical in shape and exhibit good dispersibility. Their particle size distribution ranges from 1.49 to 9.12 nm, with an average particle size of 2.89 ± 0.56 nm and a lattice spacing of 0.33 nm.

[0034] The functional groups of carbon quantum dots were characterized using Fourier transform infrared spectroscopy (FTIR), such as... Figure 2 As shown, at 3398 cm -1 and 3102 cm -1 There are two distinct absorption peaks nearby, corresponding to NH and CH respectively, and another peak at 1628 cm⁻¹. -1 1345 cm -1 1105 cm -1 818 cm -1 and 536 cm -1 The nearby absorption peaks correspond to C=O, CO, CC, CN, and C-Cl, respectively, with C-Cl indicating successful doping of chlorine.

[0035] The ultraviolet absorption spectra of carbon quantum dots were measured using a UV-Vis spectrophotometer and a 1.0 cm cuvette. The carbon quantum dots were transferred to a 5.0 mL test tube and diluted to an appropriate concentration with methanol solution, allowing it to stand for 5 minutes to ensure thorough mixing. The mixed solution was then placed in a quartz cuvette, and scanning was performed in the wavelength range of 200–800 nm, using methanol as a reference. The obtained ultraviolet absorption spectra of the carbon quantum dots are shown below. Figure 3 As can be observed from the figure, the optimal absorption peak of carbon quantum dots in the ultraviolet absorption spectrum is located at 306 nm.

[0036] The fluorescence spectra of L-Histidine-N,Cl-CDs-C18 were measured using a fluorescence spectrometer and a 1.0 cm cuvette. The carbon quantum dots were transferred to a 5.0 mL test tube and diluted to an appropriate concentration with methanol, allowing it to stand for 5 minutes to ensure thorough mixing. The mixed solution was then placed in a quartz cuvette, and scanning was performed in the wavelength range of 200–800 nm, using methanol as a reference. Figure 4 It can be observed that the optimal emission peak in the fluorescence spectrum of carbon quantum dots is located at 456 nm.

[0037] Example 2 Preparation of carbon quantum dots (L-Histidine-N,Cl-CDs-C) 18 The steps are as follows: 155 mg L-histidine and 309 mg diethylenetriamine were added to a 50 mL beaker, followed by 200 μL of concentrated hydrochloric acid (36%) and 20 mL of deionized water. After the L-histidine was completely dissolved, the mixture was transferred to a 50 mL reaction vessel, which was then placed in an oven at 200 °C for 12 h. After cooling to room temperature, a brownish-yellow liquid was obtained. The brownish-yellow liquid was filtered through a disposable syringe aqueous filter (13*0.22 μm) to remove the residue. The filtrate was placed in a regenerated cellulose membrane dialysis bag (200 Da) and dialyzed for 4 h. After dialysis, the liquid was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 264 mg of a brownish-yellow intermediate (L-Histidine-N, Cl-CDs); the yield was 85.87%.

[0038] 264 mg of the brownish-yellow intermediate product was placed in a 50 mL round-bottom flask and dissolved in 20 mL of acetone. Then, 169 mg of EDC and 165 mg of NHS were added and dissolved separately. Next, 852 mg of octadecanoic acid was added, and the pH was adjusted to 7. The mixture was stirred at room temperature for 6 h to obtain a brownish-yellow liquid. The brownish-yellow liquid was filtered through a disposable needle oil filter (13*0.22 μm) to remove the residue. The filtrate was placed in a polypropylene membrane dialysis bag (300 Da) and dialyzed in acetone for 5 h. After dialysis, the filtrate was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 630 mg of carbon quantum dots (L-Histidine-N, Cl-CDs-C). 18 The yield was 70.01%.

[0039] Example 3 Preparation of carbon quantum dots (L-Histidine-N,Cl-CDs-C) 18 The steps are as follows: 155 mg L-histidine and 1030 mg diethylenetriamine were added to a 50 mL beaker, followed by 150 μL of concentrated hydrochloric acid (36%) and 20 mL of deionized water. After the L-histidine was completely dissolved, the mixture was transferred to a 50 mL reaction vessel, which was then placed in an oven at 220 °C for 15 h. After cooling to room temperature, a brownish-yellow liquid was obtained. The brownish-yellow liquid was filtered through a disposable syringe aqueous filter (13*0.22 μm) to remove the residue. The filtrate was placed in a regenerated cellulose membrane dialysis bag (200 Da) and dialyzed for 5 h. After dialysis, the liquid was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 250 mg of a brownish-yellow intermediate (L-Histidine-N, Cl-CDs); the yield was 81.39%.

[0040] 250 mg of the brownish-yellow intermediate product was placed in a 50 mL round-bottom flask and dissolved in 20 mL of acetone. Then, 169 mg of EDC and 165 mg of NHS were added and dissolved separately. 1420 mg of octadecanoic acid was added to adjust the pH to 7, and the mixture was stirred at room temperature for 6 h to obtain a brownish-yellow liquid. The brownish-yellow liquid was filtered through a disposable needle oil filter (13*0.22 μm) to remove the residue. The filtrate was placed in a polypropylene membrane dialysis bag (300 Da) and dialyzed in acetone for 5 h. After dialysis, the filtrate was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 583 mg of carbon quantum dots (L-Histidine-N, Cl-CDs-C). 18 The yield was 68.31%.

[0041] Example 4 Preparation of carbon quantum dots (L-Histidine-N,Cl-CDs-C) 18 The steps are as follows: 310 mg L-histidine and 515 mg diethylenetriamine were added to a 50 mL beaker, followed by 150 μL of concentrated hydrochloric acid (36%) and 25 mL of deionized water. After the L-histidine was completely dissolved, the mixture was transferred to a 50 mL reaction vessel, which was then placed in an oven at 190 °C for 12 h. After cooling to room temperature, a brownish-yellow liquid was obtained. The brownish-yellow liquid was filtered through a disposable syringe aqueous filter (13*0.22 μm) to remove the residue. The filtrate was placed in a regenerated cellulose membrane dialysis bag (200 Da) and dialyzed for 4 h. After dialysis, the liquid was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 512 mg of a brownish-yellow intermediate (L-Histidine-N, Cl-CDs); the yield was 83.43%.

[0042] 512 mg of the brownish-yellow intermediate product was placed in a 50 mL round-bottom flask and dissolved in 20 mL of acetone. Then, 169 mg of EDC and 165 mg of NHS were added and dissolved separately. 1136 mg of octadecanoic acid was added, and the pH was adjusted to 7. The mixture was stirred at room temperature for 6 h to obtain a brownish-yellow liquid. The brownish-yellow liquid was filtered through a disposable needle oil filter (13*0.22 μm) to remove the residue. The filtrate was placed in a polypropylene membrane dialysis bag (300 Da) and dialyzed in acetone for 5 h. After dialysis, the sample was transferred to a beaker, sealed with a sealing film, and frozen. The frozen sample was dried using a refrigerated dryer to obtain 1175 mg of carbon quantum dot probe (L-Histidine-N, Cl-CDs-C). 18 The yield was 67.22%.

[0043] I. Antibacterial Experiment Take 3 mg of carbon quantum dots from Example 1, dissolve ice in deionized water and bring the volume to 10 mL. Sonicate for 10 min until completely dissolved to obtain a 0.3 mg / mL stock solution. Using a 10-fold serial dilution method, take 1 mL of the stock solution and add it to a sterile centrifuge tube. Add deionized water to bring the volume to 10 mL, mix thoroughly, and repeat the process to prepare four concentration gradients, ranging from 0.3 to 3 × 10⁻⁶ mg / mL. -4 Four sterile centrifuge tubes were stored at -4℃. Staphylococcus aureus, Escherichia coli, and petroleum-degrading bacteria BF04 in the logarithmic growth phase were spread onto agar medium using a sterile spreader. Filter paper discs with a diameter of 2.5 cm were immersed in carbon quantum dot solutions of different concentrations for 30 min. Excess solution was removed from the sterile filter paper discs, and the discs containing different concentrations of carbon quantum dot solutions were then flattened onto the surface of the culture medium using sterile forceps. Filter paper discs immersed in sterile water served as a control group. The center-to-center distance between each filter paper disc was at least 24 mm. After incubation at 35℃ for 18 h, the diameter of the inhibition zone around each filter paper disc was measured and recorded using calipers. The antibacterial test for each concentration of carbon quantum dot solution was performed independently three times, and the data were taken as mean ± standard deviation.

[0044] The diameter of the inhibition zone = the total inhibition zone diameter - the filter paper diameter.

[0045] Antibacterial effect such as Figure 5 As shown: At four concentration gradients, no inhibition zones were formed around the filter paper in any of the three culture dishes. The three types of bacteria (Staphylococcus aureus, Escherichia coli, and petroleum-degrading bacteria BF04) grew outward from the filter paper as the center. The colony density around the filter paper was consistent with that in the distant region. This indicates that carbon quantum dots do not have a significant antibacterial effect on the three types of bacteria without external energy stimulation. This non-antibacterial property makes carbon quantum dots a faster, more effective, and more precise positioning tool.

[0046] II. Fluorescence Imaging of Targeted Petroleum-Degrading Bacteria BF04 Using octadecane as a representative petroleum pollutant, petroleum-degrading bacteria were cultured in MS medium. Then, carbon quantum dots (Example 1) were used to detect the petroleum-degrading bacteria BF04, verifying the targeted fluorescence imaging activity of the carbon quantum dots.

[0047] Petroleum-degrading bacteria BF04 suspension (10 8 ~10 9 The CFU / mL solution was inoculated into MS medium and then incubated at 25°C.

[0048] Set up the following two groups: Group 1: After inoculating with petroleum-degrading bacteria BF04, 50 mg / L octadecane was added to MS medium and incubated for 30 min; then 50 mg / L carbon quantum dots were added and incubated for 60 min.

[0049] Group 2: After inoculating with petroleum-degrading bacteria BF04, only 50 mg / L of carbon quantum dots were added for incubation.

[0050] Samples were then taken at different time points (after 30 min and 60 min of incubation). Before fluorescence imaging, the cells were rinsed three times with PBS buffer (0.1 M, pH 7.4) to remove any residual extracellular carbon quantum dots. The fluorescence distribution of carbon quantum dots in petroleum-degrading bacteria BF04 cells was observed under an upright fluorescence microscope (40x objective) with an excitation wavelength of 306 nm (fluorescence imaging). A bright-field image after 30 min of incubation was used as a control.

[0051] The test results are as follows Figure 6 As shown: like Figure 6 As shown, figures a, b, and c represent the results of the first group of experiments. Figure a is a bright-field image, figure b is an upright fluorescence microscope image after incubation with octadecane for 30 min, and figure c is a fluorescence image after incubation with carbon quantum dots for 60 min. Figures D, e, and f represent the results of the second group of experiments. Figure d is a bright-field image, and figures e and f are fluorescence images after incubation with only carbon quantum dots for 30 min and 60 min, respectively.

[0052] As shown in Figures b and c, the petroleum-degrading bacterium BF04 shows almost no fluorescent signal. Figures e and f show strong cellular fluorescence intensity. The carbon quantum dot-labeled petroleum-degrading bacterium BF04 exhibits a smooth-edged, mononuclear morphology. This degrading bacterium has a slow cell division and proliferation rate, and weak cell viability.

[0053] The fluorescence imaging experiments described above demonstrated that carbon quantum dots possess fluorescence imaging activity. When octadecane was first added to the petroleum-degrading bacteria BF04 culture system, no fluorescence signal was observed in the cells; after 30 minutes, the addition of carbon quantum dot probes still resulted in no fluorescence signal. However, under the same conditions, direct addition of carbon quantum dots to the petroleum-degrading bacteria BF04 culture system resulted in strong fluorescence signals in the bacteria after both 30 and 60 minutes.

[0054] The above experimental results demonstrate that octadecane enters the cells of the petroleum-degrading bacterium BF04 and can prevent carbon quantum dots from entering the cells. In another set of experiments without octadecane, carbon quantum dots entered the cells and generated a fluorescent signal. This fully illustrates that, on the one hand, carbon quantum dots possess fluorescent imaging properties and can enter the cells of the petroleum-degrading bacterium BF04, thereby enabling the fluorescence imaging and detection of BF04; on the other hand, octadecane (a straight-chain alkane) competes with carbon quantum dots, competing for and preventing carbon quantum dots from entering the cells of the petroleum-degrading bacterium BF04.

[0055] In practical applications, this characteristic can be used to dynamically detect the degradation of straight-chain alkanes by the petroleum-degrading bacterium BF04. For example, to understand the dynamic degradation process of straight-chain alkanes in petroleum pollutants, the petroleum-degrading bacterium BF04 can be pre-imagined with carbon quantum dots. When the petroleum-degrading bacterium BF04 is mixed with petroleum pollutants, the straight-chain alkanes will competitively enter the petroleum-degrading bacterium, thereby causing the carbon quantum dots to be expelled and the fluorescence to be quenched. When fluorescence reappears in the petroleum-degrading bacterium BF04, it indicates that the straight-chain alkanes in the petroleum pollutants have been completely degraded.

[0056] III. Targeted Petroleum-Degrading Bacteria L10 Fluorescence Imaging Petroleum-degrading bacteria were cultured in MS medium. Then, carbon quantum dots (Example 1) were used to detect the targeted fluorescence imaging activity of the petroleum-degrading bacteria L10. Staphylococcus aureus was used as a control.

[0057] Petroleum-degrading bacteria L10 suspension (10 8 ~10 9 The sample was inoculated into MS medium containing CFU / mL, 50 mg / L carbon quantum dots were added, and then incubated at 25°C. Staphylococcus aureus was used as a control.

[0058] Samples were then taken at different time points (after 30 min and 60 min of incubation). Before fluorescence imaging, the samples were rinsed three times with PBS buffer solution (0.1 M, pH 7.4) to remove any residual extracellular carbon quantum dots. The fluorescence distribution of carbon quantum dots in bacterial cells was observed under an upright fluorescence microscope (40x objective) with an excitation wavelength of 306 nm.

[0059] The test results are as follows Figure 7 As shown: like Figure 7 As shown, a, b, and c are fluorescence imaging images of Staphylococcus aureus; where a is a bright-field image after 30 min of incubation, b is a fluorescence image after 30 min of incubation, and c is a fluorescence image after 60 min of incubation; d, e, and f are fluorescence imaging images of petroleum-degrading bacteria L10; d is a bright-field image after 30 min of incubation, e is a fluorescence image after 30 min of incubation, and f is a fluorescence image after 60 min of incubation.

[0060] As shown in Figures b and c, Staphylococcus aureus showed no fluorescent signal. Figures e and f show that the cells exhibited strong fluorescence intensity, indicating that the L10 petroleum-degrading bacteria labeled with carbon quantum dots had a slow cell division and proliferation rate, and weak cell viability.

[0061] Fluorescence imaging experiments demonstrated that carbon quantum dots possess targeted fluorescence imaging activity in petroleum-degrading bacteria L10, but lack targeted fluorescence imaging activity in Staphylococcus aureus.

[0062] The above experiments confirmed that carbon quantum dots possess targeted fluorescence imaging activity in petroleum-degrading bacteria BF04 and L10 cells, but exhibit no targeting activity or fluorescence imaging activity in non-petroleum-degrading bacteria. Therefore, the carbon quantum dots prepared in this invention are a low-cost, highly sensitive, moderately hydrophobic, targeted, and fluorescence imaging reagent for detecting petroleum-polluted degrading bacteria. Utilizing these carbon quantum dots for dynamic detection of petroleum-degrading bacteria allows for understanding the colonization dynamics of these bacteria in petroleum-polluted saline soils, playing a crucial role in revealing the mechanisms by which these bacteria metabolize petroleum hydrocarbons and elucidating their mechanisms for remediating petroleum-polluted saline soils.

[0063] In this invention, petroleum-degrading bacteria possess the ability to consume octadecane, a capability not shared by other bacteria. Since carbon quantum dots contain an octadecane chain, the carbon quantum dots prepared in this invention can be used for targeted detection of petroleum-degrading bacteria, but are not targeted at other bacteria and cannot enter their cells.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A carbon quantum dot for targeted detection of petroleum-degrading bacteria, characterized in that, The carbon quantum dots are L-Histidine-N,Cl-CDs-C 18 .

2. The carbon quantum dot according to claim 1, characterized in that, The carbon quantum dots are prepared by the following method: L-histidine and diethylenetriamine were mixed, concentrated hydrochloric acid and water were added, and the mixture was reacted at high temperature. After the reaction was completed, a brownish-yellow liquid was obtained. The brownish-yellow liquid was filtered, dialyzed, and freeze-dried to obtain a brownish-yellow intermediate. The brownish-yellow intermediate was mixed with acetone, EDC and NHS were added, and then octadecanoic acid was added. The pH was adjusted to neutral, and the mixture was stirred to obtain a brownish-yellow liquid. The brownish-yellow liquid was then filtered, dialyzed, and freeze-dried to obtain carbon quantum dots.

3. The carbon quantum dot according to claim 2, characterized in that, The ingredients are selected from the following quantities: 100-350 parts L-histidine, 50-1500 parts diethylenetriamine, 0.1-0.5 parts concentrated hydrochloric acid, 10-50 parts water, 10-50 parts acetone, 100-300 parts EDC, 100-300 parts NHS, and 200-1500 parts octadecanoic acid.

4. The carbon quantum dot according to claim 2, characterized in that, The molar ratio of L-histidine to diethylenetriamine is selected from 1:(1~10).

5. The carbon quantum dot according to claim 2, characterized in that, The conditions for the high-temperature reaction are: reacting at 150~250℃ for 5~15 hours; the conditions for the stirring reaction are: stirring at 20~30℃ for 5~10 hours.

6. The application of the carbon quantum dots as described in claim 1 in the targeted detection of petroleum-degrading bacteria.

7. The application according to claim 6, characterized in that, The preferred petroleum-degrading bacteria are petroleum-degrading bacteria BF04 or petroleum-degrading bacteria L10.

8. A method for targeted detection of petroleum-degrading bacteria, characterized in that, Includes the following steps: The carbon quantum dots described in claim 1 are added to the sample solution of petroleum-degrading bacteria and incubated. After incubation, the fluorescence distribution of the carbon quantum dots in the petroleum-degrading bacteria cells is observed under the excitation wavelength to achieve the detection of petroleum-degrading bacteria.

9. The method according to claim 8, characterized in that, The amount of carbon quantum dots used is 10~100 mg / L.

10. The method according to claim 8, characterized in that, The excitation wavelength is 306 nm.

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