Carbon nano chemical indicator, preparation method thereof and application of carbon nano chemical indicator in bacterial proliferation monitoring

Abstract

The invention discloses a carbon nano chemical indicator, a preparation method thereof and application of the carbon nano chemical indicator in bacterial proliferation monitoring. The method comprises the following steps: S1, preparing nitrogen-sulfur co-doped carbon quantum dots; and S2, carrying out surface modification by adopting 1, 2-naphthoquinone-4-sodium sulfonate, so as to obtain the carbon nano chemical indicator. The intelligent response type chemical indicator based on the carbon quantum dots is innovatively developed, according to the scheme, a specific response system is constructed through surface modification of the carbon quantum dots, and real-time and in-situ visual monitoring of bacterial proliferation is achieved through fluorescence / color dual-mode changes triggered by a bacterial metabolism microenvironment; compared with a traditional method, the method has the outstanding advantages that the response is fast (lt; the method can realize dynamic monitoring, does not need complex pretreatment, supports nondestructive detection, reduces the detection cost by more than 80%, has double functions of naked eye visualization and instrument quantitative detection, and is suitable for universal detection of a plurality of common bacteria.

Description

Technical Field

[0001] This invention relates to the field of biology, and in particular to a carbon nanotube chemical indicator, its preparation method, and its application in bacterial proliferation monitoring. Background Technology

[0002] In the field of microbial detection, bacterial proliferation monitoring techniques mainly include plate counting, turbidimetry (OD600 method), flow cytometry, and ATP bioluminescence assay. Among them, plate counting, as the gold standard for bacterial counting, counts colony-forming units (CFU) through serial dilution and plate culture, offering accurate and reliable results. However, it has significant drawbacks such as long detection cycles (usually 24-48 hours) and cumbersome operation. Turbidimetry indirectly reflects bacterial concentration by measuring absorbance at 600 nm. Although simple to operate and suitable for high-throughput detection, it lacks sensitivity for low-concentration samples (<10^6 CFU / mL) and is easily affected by culture medium turbidity. Flow cytometry, combined with fluorescent labeling, achieves bacterial counting and activity analysis, offering high sensitivity at the single-cell level, but relies on expensive instruments and professional operators. ATP bioluminescence assay reflects the number of viable bacteria by detecting adenosine triphosphate (ATP) content, providing rapid detection (minute-level), but is easily affected by free ATP in the sample and has poor stability.

[0003] The main technical bottlenecks faced by existing technologies are as follows: poor timeliness, traditional methods are difficult to achieve real-time dynamic monitoring, such as plate counting which requires long-term incubation; high operational complexity, most methods require sample pretreatment (such as dilution, staining) or destructive sampling; cost barriers, high-precision detection relies on large instruments, which are difficult to promote at the grassroots level; and limited sensitivity, conventional turbidimetric methods are insufficient for low bacterial count samples. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a carbon nanotube chemical indicator, its preparation method and its application in bacterial proliferation monitoring, in order to address the shortcomings of the prior art.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a method for preparing a carbon nanoparticle chemical indicator, comprising the following steps:

[0006] S1. Preparation of nitrogen-sulfur co-doped carbon quantum dots (N / S-CNDs):

[0007] S1-1. Dissolve o-phenylenediamine and ascorbic acid in ultrapure water, add thiourea, and disperse by ultrasonication to obtain a mixture;

[0008] S1-2. The mixture is transferred to a reaction vessel and subjected to a hydrothermal reaction under heating to prepare nitrogen-sulfur co-doped carbon quantum dots: N / S-CNDs;

[0009] S2. Sodium 1,2-naphthoquinone-4-sulfonate was used to modify the surface of N / S-CNDs to obtain carbon nanotube chemical indicator: N / S-CNDs-NQ.

[0010] Preferably, in step S1-1, the molar ratio of o-phenylenediamine to ascorbic acid is 2:1.

[0011] Preferably, in the mixture of step S1-1, the concentrations of o-phenylenediamine and ascorbic acid are 0.05-0.2M and 0.025-0.1M, respectively, and the mass percentage of thiourea is 5-20%.

[0012] Preferably, the temperature of the hydrothermal reaction in step S1-2 is 160-200℃, and the reaction time is 4-16 hours.

[0013] Preferably, step S2 specifically includes:

[0014] N / S-CNDs were dispersed in PBS buffer, EDC and NHS were added, and the mixture was stirred in the dark. Then, under nitrogen protection, a PBS solution of sodium 1,2-naphthoquinone-4-sulfonate was added, and the reaction was carried out in the dark. After the reaction was completed, the product was dialyzed in PBS buffer using a dialysis bag. The dialysate in the dialysis bag was filtered through a filter membrane, and the filtrate was freeze-dried to obtain the carbon nanotube chemical indicator: N / S-CNDs-NQ.

[0015] Preferably, the preparation method of the carbon nanoparticle chemical indicator includes the following steps:

[0016] S1. Preparation of nitrogen-sulfur co-doped carbon quantum dots (N / S-CNDs):

[0017] S1-1. Dissolve o-phenylenediamine and ascorbic acid in ultrapure water, add thiourea, and sonicate for 30 min to obtain a mixture; the concentrations of o-phenylenediamine and ascorbic acid in the mixture are 0.05-0.2M and 0.025-0.1M, respectively, and the mass percentage of thiourea is 5-20%;

[0018] S1-2. The mixture is transferred to a high-pressure reactor lined with polytetrafluoroethylene and reacted at 180°C for 8 hours. After cooling to room temperature, it is centrifuged at 6000-24000 rpm for 10-40 minutes. The supernatant is filtered through a 0.2-0.3 μm filter membrane. The filtrate is dialyzed in ultrapure water for 24-96 hours using a dialysis bag with a molecular weight cutoff of 0.5-2 kDa. The ultrapure water is replaced every 3-12 hours. The dialysate in the dialysis bag is freeze-dried to obtain nitrogen-sulfur co-doped carbon quantum dots: N / S-CNDs.

[0019] S2. Surface modification of N / S-CNDs using sodium 1,2-naphthoquinone-4-sulfonate:

[0020] Disperse 5-20 mg of N / S-CNDs in 5-20 mL of PBS buffer with pH 7-8, add 2.5-10 mg of EDC and 1.5-6 mg of NHS, stir at room temperature in the dark for 15-60 minutes, then add 0.5-2 mL of PBS solution containing 7.5-30 mg of sodium 1,2-naphthoquinone-4-sulfonate under nitrogen protection, and react in the dark for 6-24 hours. After the reaction is complete, dialyze the product in PBS buffer using a dialysis bag with a molecular weight cutoff of 0.5-2 kDa for 12-48 hours. Filter the dialysate in the dialysis bag through a 0.2-0.3 μm filter membrane, freeze-dry the filtrate, and obtain the carbon nanotube chemical indicator: N / S-CNDs-NQ.

[0021] Preferably, the preparation method of the carbon nanoparticle chemical indicator includes the following steps:

[0022] S1. Preparation of nitrogen-sulfur co-doped carbon quantum dots (N / S-CNDs):

[0023] S1-1. Dissolve o-phenylenediamine and ascorbic acid in ultrapure water, add thiourea, and sonicate for 30 min to obtain a mixture; the concentrations of o-phenylenediamine and ascorbic acid in the mixture are 0.1 M and 0.05 M, respectively, and the mass percentage of thiourea is 10%.

[0024] S1-2. The mixture was transferred to a high-pressure reactor lined with polytetrafluoroethylene and reacted at 180°C for 8 hours. After cooling to room temperature, it was centrifuged at 12,000 rpm for 20 minutes. The supernatant was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed in ultrapure water for 48 hours using a dialysis bag with a molecular weight cutoff of 1 kDa. The ultrapure water was replaced every 6 hours. The dialysate in the dialysis bag was freeze-dried to obtain nitrogen-sulfur co-doped carbon quantum dots: N / S-CNDs.

[0025] S2. Surface modification of N / S-CNDs using sodium 1,2-naphthoquinone-4-sulfonate:

[0026] 10 mg of N / S-CNDs was dispersed in 10 mL of PBS buffer at pH 7.4. 5 mg of EDC and 3 mg of NHS were added, and the mixture was stirred at room temperature in the dark for 30 minutes. Then, under nitrogen protection, 1 mL of PBS solution containing 15 mg of sodium 1,2-naphthoquinone-4-sulfonate was added, and the reaction was carried out in the dark for 12 hours. After the reaction was completed, the product was dialyzed in PBS buffer for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa. The dialysate in the dialysis bag was filtered through a 0.22 μm filter membrane, and the filtrate was freeze-dried to obtain the carbon nanotube chemical indicator: N / S-CNDs-NQ.

[0027] A second aspect of the present invention provides a carbon nanotube chemical indicator, which is prepared by the method described above.

[0028] A third aspect of the present invention provides the application of the carbon nanotube chemical indicator as described above in bacterial proliferation monitoring.

[0029] Preferably, the application method is as follows:

[0030] Carbon nanoparticle chemical indicators were added to bacterial suspensions, mixed thoroughly, and images of the mixture were collected at different times. The collected images were then analyzed in RGB, and the bacterial proliferation was determined based on the analysis results.

[0031] The beneficial effects of this invention are:

[0032] This invention innovatively develops a smart responsive chemical indicator based on carbon quantum dots. This scheme constructs a specific response system through carbon quantum dot surface modification and utilizes fluorescence / color dual-mode changes triggered by the bacterial metabolic microenvironment to achieve real-time, in-situ visual monitoring of bacterial proliferation. Compared with traditional methods, the outstanding advantages of this invention are specifically reflected in: rapid response (<3 hours) and dynamic monitoring capability, no need for complex pretreatment and support for non-destructive testing, reduction of detection cost by more than 80%, dual functionality of naked-eye visualization and instrument quantitative detection, and applicability to the universal detection of a variety of common bacteria.

[0033] The chemical indicator of this invention will effectively meet the urgent need in the field of modern microbial detection for rapid, simple and low-cost monitoring methods, and has important application value in rapid screening of food safety, clinical infection diagnosis and environmental microbial monitoring. Attached Figure Description

[0034] Figure 1 TEM images of N / S-CNDs prepared in Example 1;

[0035] Figure 2 The color changes of bacteria at different concentrations under bright field were captured at 1-minute intervals.

[0036] Figure 3 The color changes of bacteria at different concentrations under bright field were captured at 10-minute intervals.

[0037] Figure 4 The image shows a line graph illustrating the RGB analysis of continuous growth of Escherichia coli at four different concentrations. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0039] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0041] Example 1

[0042] A carbon nanotube chemical indicator, the preparation method of which includes the following steps:

[0043] S1. Preparation of nitrogen-sulfur co-doped carbon quantum dots (N / S-CNDs) using a hydrothermal method:

[0044] S1-1. Dissolve o-phenylenediamine (o-PD) and ascorbic acid (AA) in ultrapure water, add thiourea as a sulfur dopant source, and ultrasonically disperse for 30 min until completely dissolved to obtain a mixture; the concentrations of o-phenylenediamine and ascorbic acid in the mixture are 0.1M and 0.05M, respectively, and the mass percentage of thiourea is 10%.

[0045] S1-2. The mixture was transferred to a high-pressure reactor lined with polytetrafluoroethylene and placed in an oven at 180°C for 8 hours (the temperature and time parameters were optimized to ensure sufficient carbon quantum dot formation while avoiding excessive graphitization that could lead to increased particle size). After cooling to room temperature, a brownish-black solution was obtained. The solution was centrifuged at 12,000 rpm for 20 minutes to remove large precipitate particles. The supernatant was filtered through a 0.22 μm polyethersulfone membrane to obtain a clear carbon quantum dot solution (filtrate). To remove unreacted small molecule impurities, the filtrate was dialyzed in ultrapure water for 48 hours using a dialysis bag with a molecular weight cutoff of 1 kDa. The ultrapure water was replaced every 6 hours. The dialysate in the dialysis bag was freeze-dried to obtain nitrogen-sulfur co-doped carbon quantum dot powder: N / S-CNDs. The powder was stored at 4°C in the dark for later use.

[0046] S2. Surface modification of N / S-CNDs with sodium 1,2-naphthoquinone-4-sulfonate was achieved via EDC / NHS activation coupling to impart redox responsive properties. The specific steps are as follows:

[0047] 10 mg of N / S-CNDs was dispersed in 10 mL of PBS buffer at pH 7.4. 5 mg of EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and 3 mg of NHS (N-hydroxysuccinimide) were added. The mixture was stirred at room temperature in the dark for 30 minutes to activate the carboxyl groups on the surface of the carbon quantum dots. Then, under nitrogen protection, 1 mL of PBS solution containing 15 mg of sodium 1,2-naphthoquinone-4-sulfonate was slowly added. The reaction was carried out in the dark for 12 hours. After the reaction was completed, the product was dialyzed in PBS buffer for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa to remove unreacted naphthoquinone derivatives and byproducts. The dialysate in the dialysis bag was filtered through a 0.22 μm filter membrane, and the filtrate was freeze-dried to obtain the carbon nanoparticle chemical indicator: N / S-CNDs-NQ.

[0048] The entire modification process requires strict control of the pH within the range of 7-8, and avoidance of light and oxygen exposure to ensure the stability of the naphthoquinone group.

[0049] The modification efficiency of sodium 1,2-naphthoquinone-4-sulfonate can be verified by detecting the change of the characteristic absorption peak of naphthoquinone at 320 nm using ultraviolet-visible spectroscopy, combined with X-ray photoelectron spectroscopy (XPS) analysis of the binding energy shifts of nitrogen and sulfur elements.

[0050] Example 2 uses the carbon nanotube chemical indicator prepared in Example 1 to monitor bacterial proliferation.

[0051] The bacterial proliferation monitoring experiment used *Escherichia coli* (ATCC 25922) as the model strain. The specific procedures were as follows: Single colonies of *E. coli* preserved on LB solid medium were transferred to LB liquid medium and cultured at 37°C with shaking for 12 hours until the logarithmic growth phase. The concentration of the bacterial suspension was determined using an ultra-micro UV-Vis spectrophotometer, with initial concentration gradients of 2.8 × 10⁵, 2.8 × 10⁶, 2.8 × 10⁷, and 2.8 × 10⁸ CFU / mL. After the system stabilized for 1 hour, N / S-CNDs-NQ prepared in Example 1 was added to a final concentration of 10–40 μg / mL. After thorough mixing, the mixture was placed in a 37°C constant temperature environment, and images were acquired every minute using a timed imaging system. Using the same analysis window, the RGB color parameters of the acquired images were quantitatively analyzed using image analysis software. A colorimetric change graph and trend line graph were plotted with time as the abscissa, ultimately achieving dynamic monitoring of the bacterial proliferation process.

[0052] Test results are as follows Figures 2-4 As shown, Figure 2 The image shows the color changes of bacteria at different concentrations under bright field, captured at 1-minute intervals. Figure 3The image shows the color changes of bacteria at different concentrations under bright field, captured at 10-minute intervals; all images are arranged in chronological order. In the image, bacterial concentrations A through D are 2.8 × 10⁻⁶. 5 2.8×10 6 2.8×10 7 and 2.8×10 8 .

[0053] Figure 4 The graph shows the RGB analysis of continuous growth of E. coli at four different concentrations (where the coordinates are chromaticity values, represented by the H values ​​in the HSV color model using Python). The bacterial concentrations from curve A to curve D are 2.8 × 10⁻⁶. 5 2.8×10 6 2.8×10 7 and 2.8×10 8 ,and Figure 2 and Figure 3 The results are consistent. It can be seen that the slope change of the curve conforms to the "S"-shaped growth curve of bacteria, indicating that the RGB results can be used to determine bacterial proliferation. After setting the environment and the shooting interval, no further intervention is required throughout the experiment; dynamic monitoring of bacterial proliferation can be achieved based on the RGB image analysis results.

[0054] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for preparing a carbon nanoparticle chemical indicator, characterized in that, Includes the following steps: S1. Preparation of nitrogen-sulfur co-doped carbon quantum dots (N / S-CNDs): S1-1. Dissolve o-phenylenediamine and ascorbic acid in ultrapure water, add thiourea, and disperse by ultrasonication to obtain a mixture; S1-2. The mixture is transferred to a reaction vessel and subjected to a hydrothermal reaction under heating to prepare nitrogen-sulfur co-doped carbon quantum dots: N / S-CNDs; S2. Sodium 1,2-naphthoquinone-4-sulfonate was used to modify the surface of N / S-CNDs to obtain carbon nanotube chemical indicator: N / S-CNDs-NQ.

2. The method for preparing the carbon nanotube chemical indicator according to claim 1, characterized in that, In step S1-1, the molar ratio of o-phenylenediamine to ascorbic acid is 2:

1.

3. The method for preparing the carbon nanoparticle chemical indicator according to claim 2, characterized in that, In the mixture of step S1-1, the concentrations of o-phenylenediamine and ascorbic acid are 0.05-0.2M and 0.025-0.1M, respectively, and the mass percentage of thiourea is 5-20%.

4. The method for preparing the carbon nanoparticle chemical indicator according to claim 1, characterized in that, The temperature of the hydrothermal reaction in step S1-2 is 160-200℃, and the reaction time is 4-16 hours.

5. The method for preparing the carbon nanotube chemical indicator according to claim 1, characterized in that, Step S2 is as follows: N / S-CNDs were dispersed in PBS buffer, EDC and NHS were added, and the mixture was stirred in the dark. Then, under nitrogen protection, a PBS solution of sodium 1,2-naphthoquinone-4-sulfonate was added, and the reaction was carried out in the dark. After the reaction was completed, the product was dialyzed in PBS buffer using a dialysis bag. The dialysate in the dialysis bag was filtered through a filter membrane, and the filtrate was freeze-dried to obtain the carbon nanotube chemical indicator: N / S-CNDs-NQ.

6. The method for preparing the carbon nanoparticle chemical indicator according to claim 5, characterized in that, Includes the following steps: S1. Preparation of nitrogen-sulfur co-doped carbon quantum dots (N / S-CNDs): S1-1. Dissolve o-phenylenediamine and ascorbic acid in ultrapure water, add thiourea, and sonicate for 30 min to obtain a mixture; the concentrations of o-phenylenediamine and ascorbic acid in the mixture are 0.05-0.2M and 0.025-0.1M, respectively, and the mass percentage of thiourea is 5-20%; S1-2. The mixture is transferred to a high-pressure reactor lined with polytetrafluoroethylene and reacted at 180°C for 8 hours. After cooling to room temperature, it is centrifuged at 6000-24000 rpm for 10-40 minutes. The supernatant is filtered through a 0.2-0.3 μm filter membrane. The filtrate is dialyzed in ultrapure water for 24-96 hours using a dialysis bag with a molecular weight cutoff of 0.5-2 kDa. The ultrapure water is replaced every 3-12 hours. The dialysate in the dialysis bag is freeze-dried to obtain nitrogen-sulfur co-doped carbon quantum dots: N / S-CNDs. S2. Surface modification of N / S-CNDs using sodium 1,2-naphthoquinone-4-sulfonate: Disperse 5-20 mg of N / S-CNDs in 5-20 mL of PBS buffer with pH 7-8, add 2.5-10 mg of EDC and 1.5-6 mg of NHS, stir at room temperature in the dark for 15-60 minutes, then add 0.5-2 mL of PBS solution containing 7.5-30 mg of sodium 1,2-naphthoquinone-4-sulfonate under nitrogen protection, and react in the dark for 6-24 hours. After the reaction is complete, dialyze the product in PBS buffer using a dialysis bag with a molecular weight cutoff of 0.5-2 kDa for 12-48 hours. Filter the dialysate in the dialysis bag through a 0.2-0.3 μm filter membrane, freeze-dry the filtrate, and obtain the carbon nanotube chemical indicator: N / S-CNDs-NQ.

7. The method for preparing the carbon nanotube chemical indicator according to claim 6, characterized in that, Includes the following steps: S1. Preparation of nitrogen-sulfur co-doped carbon quantum dots (N / S-CNDs): S1-1. Dissolve o-phenylenediamine and ascorbic acid in ultrapure water, add thiourea, and sonicate for 30 min to obtain a mixture; the concentrations of o-phenylenediamine and ascorbic acid in the mixture are 0.1 M and 0.05 M, respectively, and the mass percentage of thiourea is 10%. S1-2. The mixture was transferred to a high-pressure reactor lined with polytetrafluoroethylene and reacted at 180°C for 8 hours. After cooling to room temperature, it was centrifuged at 12,000 rpm for 20 minutes. The supernatant was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed in ultrapure water for 48 hours using a dialysis bag with a molecular weight cutoff of 1 kDa. The ultrapure water was replaced every 6 hours. The dialysate in the dialysis bag was freeze-dried to obtain nitrogen-sulfur co-doped carbon quantum dots: N / S-CNDs. S2. Surface modification of N / S-CNDs using sodium 1,2-naphthoquinone-4-sulfonate: 10 mg of N / S-CNDs was dispersed in 10 mL of PBS buffer at pH 7.

4. 5 mg of EDC and 3 mg of NHS were added, and the mixture was stirred at room temperature in the dark for 30 minutes. Then, under nitrogen protection, 1 mL of PBS solution containing 15 mg of sodium 1,2-naphthoquinone-4-sulfonate was added, and the reaction was carried out in the dark for 12 hours. After the reaction was completed, the product was dialyzed in PBS buffer for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa. The dialysate in the dialysis bag was filtered through a 0.22 μm filter membrane, and the filtrate was freeze-dried to obtain the carbon nanotube chemical indicator: N / S-CNDs-NQ.

8. A carbon nanoparticle chemical indicator, characterized in that, It is prepared by the method described in any one of claims 1-7.

9. The application of the carbon nanoparticle chemical indicator as described in claim 8 in bacterial proliferation monitoring.

10. The application according to claim 9, characterized in that, The application method is as follows: Carbon nanoparticle chemical indicators were added to bacterial suspensions, mixed thoroughly, and images of the mixture were collected at different times. The collected images were then analyzed in RGB, and the bacterial proliferation was determined based on the analysis results.

Images