Application of CDs and their paper-based sensors in detection of antibiotic rifampicin

Abstract

The application discloses application of coumarin-polyethyleneimine fluorescent carbon dots in detection of antibiotic rifampicin. The fluorescent carbon dots have good selectivity, high sensitivity and strong anti-interference property as a probe. In a rifampicin concentration range of 0-25 muM, the change rate of carbon dot fluorescence has a linear relationship with the rifampicin concentration, and the detection limit is 0.67 muM. The fluorescent carbon dots are applied to detection of rifampicin in environmental water, the recovery rate is 95%-115%, the maximum relative standard deviation is 2.3%, and the fluorescent carbon dots have wide application prospects in the field of environmental monitoring. The fluorescent carbon dots are prepared into a portable paper-based sensor, and in a rifampicin concentration range of 0-90 muM, the RGB total value (R+G+B) captured by a mobile phone has a linear relationship with the rifampicin concentration. The paper-based sensor has low preparation cost, does not need large-scale instruments, and can realize in-situ and rapid visual detection of rifampicin by means of a smart phone software, so that the detection method is simple and efficient.

Description

Technical Field

[0001] This invention belongs to the field of preparation and application technology of fluorescent carbon dot (CDs) materials, specifically relating to a fluorescent probe for the detection and identification of the antibiotic rifampicin, a visualization carbon dot test paper based thereon, a preparation method, and applications. Background Technology

[0002] Rifampin, an important antibiotic for treating serious infectious diseases such as tuberculosis, leprosy, and AIDS, is widely used in medicine. However, long-term or excessive use of rifampin can lead to side effects such as joint pain, indigestion, fever, allergic rashes, and immune disorders, and in severe cases, even liver damage and necrosis. Furthermore, because rifampin cannot be completely absorbed by patients, some of it enters the environment through excretion and persists there, leading to the development of drug-resistant bacteria. Traditional methods for detecting rifampin include high-performance liquid chromatography (HPLC), mass spectrometry (MS), electrochemical methods, and chemiluminescence methods. These methods typically cannot provide rapid on-site detection, require cumbersome experimental procedures, and suffer from drawbacks such as complex operation, long testing cycles, and expensive equipment, resulting in high detection costs. Therefore, developing a rapid, selective, sensitive, and quick method for detecting rifampin is of great significance. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides an application of a novel fluorescent carbon quantum dot fluorescent material in the detection of rifampicin, which is fabricated into a paper-based sensor and integrated with newly developed mobile phone software for the visual and rapid detection of rifampicin.

[0004] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0005] The application of a fluorescent carbon dot synthesized from coumarin and polyethyleneimine in the detection of the antibiotic rifampin, namely the application of coumarin-polyethyleneimine fluorescent carbon dots in the detection of the antibiotic rifampin.

[0006] Carbon dots (CDs) are a new class of carbon-based fluorescent nanomaterials that have emerged in the fields of nanoscience and materials science in recent years. They typically refer to carbon nanoparticles with sizes ranging from 1 to 10 nanometers, possessing abundant surface functional groups and tunable optical properties. Due to their unique fluorescence properties, high chemical stability, good biocompatibility, and resistance to photobleaching, they show great application potential in fields such as environmental monitoring, biomedical diagnostics, and food safety.

[0007] Na+ was added to the fluorescent carbon dot solution synthesized from coumarin and polyethyleneimine. + OH - SO3 2- Ba+ Gd 2+ Hg 2+ Mn 2+ ,ClO - CN - The carbon dots exhibit good selectivity for the recognition of rifampin in aqueous solutions of rifopentine (RFP), rifabutin (RFB), rifoxitin (RFX), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), tetracycline (TC), sulfamethoxazole (SMX), vancomycin hydrochloride (VAN), erythromycin (ERM), chloramphenicol (CM), mitoxantrone (Mi), and azithromycin (AZM).

[0008] Furthermore, the preparation steps of the fluorescent carbon dots include:

[0009] S1: Hydrothermal reaction: Polyethyleneimine and coumarin are dissolved in a solvent to obtain a homogeneous solution, which is then transferred to a hydrothermal reactor and reacted at 90℃~240℃ for 3h~24h.

[0010] S2: Centrifugal filtration: After centrifugation, collect the supernatant and filter it through a 0.22μm to 0.35μm aqueous filter membrane to remove large particles;

[0011] S3: Dialysis: Dialyze for 24-48 hours using a dialysis bag with a molecular weight cutoff of 3000-4000 Da;

[0012] S4: Freeze-drying: Fluorescent carbon dots are obtained by freeze-drying.

[0013] Furthermore, the fluorescent carbon dots are fabricated into a paper-based sensor.

[0014] This invention provides a rapid detection method for rifampicin, using the fluorescent carbon dots to prepare a paper-based sensor.

[0015] Furthermore, by using paper as a substrate and combining it with fluorescent carbon dot materials synthesized from coumarin and polyethyleneimine, a paper-based sensor can be constructed, enabling highly sensitive detection of target analytes.

[0016] Furthermore, the preparation method of the paper-based sensor includes: immersion adsorption: immersing filter paper in a fluorescent carbon dot solution synthesized from coumarin and polyethyleneimine, and adsorption is completed; drying: completely drying to obtain the paper-based sensor.

[0017] Furthermore, the immersion adsorption conditions are: immersion time of 10 to 30 minutes; and the concentration of the fluorescent carbon dot solution is 1 to 5 mg / mL.

[0018] Furthermore, the drying conditions are: drying in an oven for 10 to 15 minutes.

[0019] Furthermore, the method for detecting rifampicin includes the following steps:

[0020] A1: Immerse the paper-based sensor in the sample to be tested, and after adsorption is complete, dry it completely;

[0021] A2: Acquire images from the paper-based sensor;

[0022] A3: Analyze the color of the paper-based sensor to obtain the sum of RGB and Y;

[0023] A4: The value of X, i.e., the rifampicin concentration, is obtained by calculating the formula Y = -1.3050X + 642.84167.

[0024] Furthermore, in step A1, the paper-based sensor is immersed in the sample to be tested for 10 minutes.

[0025] Furthermore, the method for obtaining the paper-based sensor image in step A2 is to place the paper base to be tested flat on a black plane under white light and take a picture from about 30 to 40 cm directly above it.

[0026] Furthermore, in step A2, the paper-based sensor image is obtained by taking a picture of the paper-based sensor using a smartphone camera; in step A3, the color of the paper-based sensor is analyzed using a smartphone program to obtain the sum of RGB, Y(R+G+B); in step A4, the concentration of rifampicin in the solution is automatically determined using a smartphone program.

[0027] Paper-based sensors, as a novel miniature sensing platform, offer several advantages over other sensor devices: ① low manufacturing cost, inexpensive and portable, requiring no expensive precision instruments; ② fast response speed, taking only seconds to minutes; ③ ability to achieve in-situ, visualized, real-time detection. Furthermore, they can be integrated with smartphones to form portable, visualized sensing platforms suitable for various applications, bringing new prospects for rapid on-site detection of target objects.

[0028] The present invention achieves the following beneficial technical effects:

[0029] 1. The coumarin-polyethyleneimine fluorescent carbon dots prepared in this invention have a simple and environmentally friendly synthesis method and can selectively identify the antibiotic rifampicin. Within the rifampicin concentration range of 0–25 μM, the fluorescence change rate of the carbon dots is linearly related to the rifampicin concentration, with a detection limit of 0.67 μM. This fluorescent probe exhibits good selectivity, high sensitivity, and strong anti-interference properties. Application of these fluorescent carbon dots to the detection of rifampicin in environmental water bodies shows satisfactory results, with recoveries of 95%–115% and a maximum relative standard deviation of 2.3%. This carbon dot material can be used for the detection of rifampicin in aquatic environments and has broad application prospects in the field of environmental monitoring.

[0030] 2. The carbon dot material provided by this invention can be used to prepare a portable paper-based sensor. In the rifampicin concentration range of 0 to 90 μM, the sum of RGB values ​​(R+G+B) captured by the paper-based sensor using a mobile phone is linearly related to the rifampicin concentration.

[0031] 3. This invention presents a mobile application for detecting rifampicin. This paper-based sensor has low manufacturing costs, requires no large instruments, and, with the help of smartphone software, enables in-situ, rapid, and visual detection of rifampicin. The detection method is simple and efficient. Attached Figure Description

[0032] Figure 1 This is a bar chart showing the selective analysis of the response of fluorescent carbon dots to different ions and antibiotics in this invention.

[0033] Figure 2 A bar chart illustrating the anti-interference ability of rifampicin in the detection of fluorescent carbon dots in this invention.

[0034] Figure 3 Emission spectra of the fluorescent carbon dot solution of this invention with different concentrations (0-30 μM) of rifampicin added.

[0035] Figure 4 This is a linear fitting graph showing the change rate of fluorescence intensity of the fluorescent carbon dots in this invention versus the concentration of rifampicin.

[0036] Figure 5 This is a color change diagram of the paper-based sensor of the present invention at rifampicin concentrations of 0–90 μM.

[0037] Figure 6 Linear fitting graph of the sum of RGB values ​​of the paper-based sensor of this invention and rifampicin concentration.

[0038] Figure 7 The application diagram of the rapid detection of rifampicin in this invention: (A) is the interface of the mobile application applet, and (B) and (C) respectively show the results of measuring the concentration of rifampicin in the solution at 30 μM and 90 μM using the applet. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments shown in the accompanying drawings, but this does not constitute any limitation on the present invention.

[0040] Example 1

[0041] A method for preparing multifunctional coumarin-polyethyleneimine fluorescent carbon dots includes the following steps:

[0042] S1: Hydrothermal reaction: 1 part by mass of polyethyleneimine with a molecular weight of 10000 Da and 3 parts by mass of coumarin were dissolved in 20 mL of methanol to obtain a homogeneous solution, which was then transferred to a hydrothermal reactor and reacted at 90 °C for 9 h.

[0043] S2: Centrifugal filtration: After centrifugation, collect the supernatant and filter it through a 0.22μm aqueous filter membrane to remove large particles;

[0044] S3: Dialysis: Dialyze for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da;

[0045] S4: Freeze-drying: Fluorescent carbon dots are obtained by freeze-drying.

[0046] Example 2

[0047] Experiment 1: Rifampin Selectivity

[0048] (1) Take 0.1 mL of carbon dot solution (1 mg / mL) in a cuvette, dilute it to 3 mL with ultrapure water, and use it as a blank control group. Perform the test at an excitation wavelength of 280 nm, and measure the maximum fluorescence emission intensity as F0.

[0049] (2) The recognition ability of carbon dots for different types of ions and antibiotics (including rifamycin antibiotics and anti-tuberculosis synergists) was studied. 0.1 mL of Na+ was added to 0.1 mL of carbon dot solution (1 mg / mL). + OH - SO3 2- Ba + Gd 2+ Hg 2+ Mn 2+ ,ClO - CN - The following drugs were prepared: rifopentine (RFP), rifabutin (RFB), rifoxitin (RFX), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), tetracycline (TC), sulfamethoxazole (SMX), vancomycin hydrochloride (VAN), erythromycin (ERM), chloramphenicol (CM), mitoxantrone (Mi), and azithromycin (AZM). An aqueous solution (1 mM) was diluted to 3 mL with ultrapure water. The tests were performed at an excitation wavelength of 280 nm, and the maximum fluorescence emission intensity was measured as F.

[0050] Test results are as follows Figure 1 As shown, the fluorescence intensity change rate (F0-F) / F0 changed significantly after the addition of rifampin (RIF), with a change rate of 83%. However, after the addition of other substances, the fluorescence intensity change rate (F0-F) / F0 was relatively stable, with a maximum change rate of only 37%, indicating that the carbon dot has good selectivity for the recognition of rifampin.

[0051] Experiment 2: Anti-interference capability

[0052] Add 0.1 mL of carbon dots (1 mg / mL), 0.1 mL of rifampicin solution (1 mM), and 0.1 mL of different interfering solutions (1 mM) to a cuvette, and dilute to 3 mL with ultrapure water. Perform the test at 280 nm excitation, and measure the maximum fluorescence emission intensity as F. Use the solution containing only carbon dots and rifampicin as a blank sample.

[0053] Test results are as follows Figure 2 As shown, the fluorescence intensity of the probe in the presence of interfering ions was not significantly different from that of rifampicin alone, indicating that the probe has strong anti-interference ability. Experiment 3: Linear relationship with rifampicin concentration

[0054] Different concentrations of RIF solution were added to 0.1 mL of carbon dot solution (1 mg / mL) and finally diluted to 3 mL. The test was performed at an excitation wavelength of 280 nm. The maximum fluorescence emission intensity was recorded as F, and the solution with only carbon dots was used as a blank sample and recorded as F0.

[0055] The fluorescence intensity of carbon dots changes as the rifampicin concentration increases, as shown in the following trend. Figure 3 As shown in the figure. Within the concentration range of 0–30 μM, the fluorescence intensity of the mixed system continuously decreased with increasing rifampicin concentration. A linear analysis was performed on the fluorescence intensity change rate (F0-F) / F0 of the mixed system versus the rifampicin concentration, and the results are shown in the figure. Figure 4 As shown, within the rifampicin concentration range of 0–25 μM, the fluorescence intensity change rate (F0-F) / F0 showed a good linear relationship with the rifampicin concentration, with the linear equation being: Y = 0.0091X + 0.1234, and the correlation coefficient R. 2 =0.9911, and the lowest detectable concentration was calculated to be 0.67 μM using LOD = 3σ / S.

[0056] Experiment 4: Ability to identify rifampin in real-world water environments

[0057] Different doses (final concentrations of 5, 10, and 15 μM) of rifampicin solution (1 mM) were added to 0.1 mL of carbon dot solution (1 mg / mL). The solution was then diluted to 3 mL with lake water, tap water, or plateau water. The samples were tested under 280 nm excitation, and the maximum fluorescence emission intensity was recorded as F. A blank sample containing only lake water, tap water, or plateau water was used, and its fluorescence intensity was recorded as F0. The concentration of rifampicin in the environmental water samples was determined by calculating the fluorescence intensity change rate (F0-F) / F0. The results are shown in Table 1. The prepared fluorescent carbon dots can effectively identify rifampicin in natural environmental water bodies, with recoveries ranging from 94% to 115% and a maximum relative standard deviation of 7.7%.

[0058] Table 1 shows the spiked recoveries of rifampin in different environmental water bodies detected by fluorescent carbon dots.

[0059]

[0060] Example 3: Coumarin-Polyethyleneimine Fluorescent Carbon Dot Paper-Based Sensor

[0061] By using paper as a substrate and combining it with fluorescent carbon dot materials synthesized from coumarin and polyethyleneimine, a paper-based sensor can be constructed, enabling highly sensitive detection of target analytes.

[0062] Furthermore, the preparation method of the paper-based sensor includes: immersion adsorption: immersing filter paper in a fluorescent carbon dot solution synthesized from coumarin and polyethyleneimine, and adsorption is completed; drying: completely drying to obtain the paper-based sensor.

[0063] Filter paper discs cut to a diameter of 1 cm are immersed in a 1-5 mg / mL carbon dot solution for 10-20 minutes. After that, the carbon dot-coated filter paper discs are removed and placed in an oven to dry for 10 minutes to completely dry them, thus obtaining the paper-based sensor.

[0064] Example 4: Rapid Detection Method for Rifampin

[0065] (1) Filter paper discs cut to a diameter of 1 cm were immersed in a 1–5 mg / mL carbon dot solution for 20 minutes. After 20 minutes, the carbon-dot-coated filter paper discs were removed and placed in an oven to dry for 10 minutes until completely dry, thus obtaining the paper-based sensor. (The results of immersing the paper-based sensor within the range of 10–30 minutes showed little difference.)

[0066] (2) The prepared paper-based sensor was immersed in rifampicin solutions of different concentrations (0-90 μM) for 10 minutes, and then taken out and placed in an oven to dry for 10 minutes to completely dry it.

[0067] (3) Under white light, the paper-based sensor is laid flat on a black plane, and a smartphone is placed about 30cm above it. The smartphone camera is used to capture the color change of the paper-based sensor, and the RGB values ​​of the paper-based sensor are extracted. The sum of RGB (R+G+B) is used as the Y-axis and the rifampicin concentration is used as the X-axis for linear analysis.

[0068] The color development results of the paper-based sensor are as follows Figure 5 As shown, within the concentration range of 0–90 μM rifampicin solution, the color on the paper-based sensor deepens continuously with the increase of rifampicin concentration, gradually deepening from pale yellow to orange-red.

[0069] The linear fitting results are as follows Figure 6As shown, within the concentration range of 0–90 μM rifampicin solution, the linear equation obtained is: Y = -1.3050X + 842.84167, with a correlation coefficient R0. 2 =0.9960. A good linear relationship exists between the RGB sum and the rifampicin concentration, indicating that the paper-based sensor is capable of detecting rifampicin concentration in water. The detection limit is set at the lowest visually apparent colorimetric concentration, therefore the detection limit LOD = 10 μM.

[0070] Example 5: Ability to identify rifampin in real-world water environments

[0071] The paper-based sensor was immersed in rifampicin solutions of different concentrations (25, 50, 75 μM) prepared in different water bodies (lake water, tap water). After 10 minutes, it was removed and placed in an oven to dry for 10 minutes until completely dry. The resulting paper-based sensor was placed on a black platform, and the color change of the paper-based sensor was captured using a smartphone camera, followed by RGB analysis.

[0072] The obtained RGB sum (R+G+B) was substituted into the linear equation Y = -1.3050X + 842.84167 to obtain the rifampicin content in the environmental water detected by the paper-based sensor. The results are shown in Table 2.

[0073] Table 2 shows the spiked recoveries of rifampin in different water bodies detected by paper-based sensors.

[0074]

[0075] The prepared paper-based sensor can effectively identify rifampicin in natural water bodies with a recovery rate of 89%–112% and a maximum relative standard deviation of 8.6%.

[0076] Example 6

[0077] Based on the above, a WeChat mini-program was designed to build a visual and rapid detection platform for rifampicin. The mini-program uses the linear equation "Y = -1.3050X + 642.84167" obtained in the above research as its theoretical basis. It directly captures and analyzes the color changes of a paper-based sensor within the mini-program, substitutes the obtained RGB sum value as the Y value into the linear equation: Y = -1.3050X + 842.84167, and directly displays the final calculated rifampicin concentration within the mini-program, achieving rapid, visual, and quantitative detection of rifampicin. The specific steps are as follows:

[0078] (1) Immerse the paper-based sensor in the sample to be tested for 10 minutes, then remove it and dry it completely.

[0079] (2) Under white light, place the paper base to be tested flat on a black plane, and position it approximately 30-40 degrees above it.

[0080] Place the phone at a distance of cm.

[0081] (3) Click “Click to upload image” in the mini program and choose to take a picture directly or select a picture from the album. The change in color of the paper-based sensor captured by the smartphone camera is uploaded to the mini program, and the concentration of rifampicin in the solution to be tested can be read directly.

[0082] like Figure 7 As shown in Figure A, the designed mini-program is simple and clear, consisting of only two parts: "selecting a photo" and "displaying the results". It is easy to operate. Simply use a smartphone camera to take a picture of the paper-based sensor, and the mini-program can capture the color changes of the paper-based sensor, analyze the sum of RGB values, and automatically calculate the concentration of rifampicin in the solution to be tested. Figure 7 B and Figure 7 C shows the results of a paper-based sensor measured with a mini-program at different rifampicin concentrations (30μM, 90μM). The paper-based sensor exhibits significant color changes at different concentrations, which are clearly captured by the mini-program, which automatically performs RGB analysis and lists the rifampicin concentration results directly below the image. Figure 7 B and Figure 7 As shown in Table C, the results analyzed by the mini-program were 29 μM and 93.37 μM, with recovery rates of 97% and 104%, respectively. Detailed results are listed in Table 3.

[0083] Table 3 shows the results of rifampin concentration determination using a paper-based sensor via a mobile application.

[0084]

[0085] The rifampicin detection method using the mini-program showed a relatively good recovery rate of 92%–113%, with a maximum relative standard deviation of 11.4%, making it applicable to the detection of rifampicin in actual water bodies.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations 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 scope of the present invention.

Claims

1. The application of fluorescent carbon dots synthesized from coumarin and polyethyleneimine in the detection of antibiotic rifampicin, characterized in that: The preparation steps of the fluorescent carbon dots include: ​ S1: hydrothermal reaction: polyethyleneimine and coumarin are dissolved in a solvent to obtain a homogeneous solution, which is transferred to a hydrothermal kettle and reacted at 90-240 DEG C for 3-24 h; S2: centrifugal filtration: after centrifugation, the supernatant is collected and filtered through a water-based filter membrane with a pore size of 0.22-0.35 μm to remove large particles; S3: dialysis: dialysis is performed for 24-48 h using a dialysis bag with a molecular weight cutoff of 3000-4000 Da; S4: freeze-drying: freeze-drying of the fluorescent carbon dots.

2. Use according to claim 1, characterized in that: The fluorescent carbon dots are prepared into a paper-based sensor.

3. Use according to claim 2, wherein: The preparation method of the paper-based sensor includes: soaking adsorption: soaking the paper material in the fluorescent carbon dot solution synthesized by using coumarin and polyethyleneimine as raw materials, and adsorbing completely; drying: completely drying to obtain the paper-based sensor.

4. Use according to claim 3, wherein: The soaking adsorption conditions are: soaking time of 10-30 minutes; the concentration of the fluorescent carbon dot solution is 1-5 mg / mL.

5. The use according to claim 3, wherein: The drying conditions are: drying in an oven for 10-15 minutes.

6. The use according to claim 2, characterized in that: The paper-based sensor is used for rapid detection of rifampicin, which includes the following steps: A1: immersing the paper-based sensor in the sample to be tested, and completely drying after adsorption; A2: obtaining the image of the paper-based sensor; A3: analyzing the color of the paper-based sensor to obtain the RGB sum Y; A4: using the formula Y=-1.3050X+642.84167 to calculate the X value, i.e. the concentration of rifampicin.

7. Use according to claim 6, wherein: In the A1 step, the paper-based sensor is immersed in the sample to be tested for 10 minutes.

8. Use according to claim 6, wherein: In the A2 step, the method for obtaining the image of the paper-based sensor is to place the paper-based sensor to be tested on a black plane under white light, and take a photo at a distance of 30-40 cm above it.

9. The use according to claim 6, characterized in that: In the A2 step, the image of the paper-based sensor is captured by using the camera of a smart phone; in the A3 step, the color of the paper-based sensor is analyzed by using the color analysis program of a smart phone to obtain the RGB sum Y; In the A4 step, the concentration of rifampicin in the solution is automatically obtained by using the program of a smart phone.

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