Application of CDs and paper-based sensor thereof in detection of antibiotic rifampicin

Abstract

The invention discloses an application of coumarin-polyethyleneimine fluorescent carbon dots in detection of antibiotic rifampicin. The fluorescent carbon dots as the probe have the advantages of good selectivity, high sensitivity and strong anti-interference performance. In a rifampicin concentration range of 0-25 [mu] M, the change rate of carbon dot fluorescence and the rifampicin concentration form a linear relationship, and the detection limit is 0.67 [mu] M. 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 [mu] M, an RGB sum value (R + G + B) captured by a mobile phone has a linear relationship with the rifampicin concentration. The paper-based sensor is low in preparation cost, large instruments are not needed, in-situ and rapid rifampicin visual detection can be achieved by means of intelligent mobile phone software, and the detection method is simple and efficient.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation and application of fluorescent carbon dots (CDs) materials, and specifically relates to a fluorescent probe for detecting and identifying the antibiotic rifampicin and a visualized carbon dot test paper based thereon, as well as a preparation method and application thereof. Background Art

[0002] Rifampicin is an important antibiotic drug for the treatment of serious infectious diseases such as tuberculosis, leprosy and AIDS, and is widely used in medical treatment. However, long-term or excessive use of rifampicin can cause side effects such as joint pain, indigestion, fever, allergic rash and immune disorders, and in severe cases, it can even cause liver damage and liver necrosis. At the same time, since rifampicin cannot be completely absorbed by the patient, some rifampicin will enter the environment through excretion and exist in the environment for a long time, leading to the production of drug-resistant bacteria. Traditional rifampicin detection methods include high-performance liquid chromatography (HPLC), mass spectrometry (MS), electrochemical method, chemiluminescence method, etc. These methods are usually unable to detect quickly on site, require cumbersome experimental steps, have the disadvantages of complex operation, long test cycle, expensive instruments, etc., resulting in high detection costs. Therefore, it is of great significance to develop a rapid, selective, sensitive and quick-operation rifampicin detection method. Summary of the invention

[0003] To solve the above problems, the present invention provides a novel fluorescent carbon quantum dot fluorescent material for use in rifampicin detection, and prepares it into a paper-based sensor, integrates a newly developed mobile phone software, and applies it to the visual rapid detection of rifampicin.

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

[0005] The invention discloses an application of fluorescent carbon dots synthesized with coumarin and polyethyleneimine as raw materials in detecting the antibiotic rifampicin, namely, an application of coumarin-polyethyleneimine fluorescent carbon dots in detecting the antibiotic rifampicin.

[0006] Carbon dots (CDs) are a new type of carbon-based fluorescent nanomaterials that have emerged in the fields of nanoscience and materials science in recent years. They usually refer to carbon nanoparticles with a size range of 1-10 nanometers, with rich surface functional groups and adjustable optical properties. Due to their unique fluorescence properties, high chemical stability, good biocompatibility and resistance to photobleaching, they have shown great application potential in environmental monitoring, biomedical diagnosis and food safety.

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

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

[0009] S1: Hydrothermal reaction: dissolve polyethyleneimine and coumarin in a solvent to obtain a homogeneous solution, transfer it to a hydrothermal reactor, and react at 90°C to 240°C for 3h to 24h;

[0010] S2: Centrifugal filtration: After centrifugation, the supernatant was collected and filtered through a 0.22 μm to 0.35 μm water filter membrane to remove large particles;

[0011] S3: Dialysis: Use a dialysis bag with a molecular weight cutoff of 3000Da to 4000Da for 24h to 48h;

[0012] S4: Freeze-drying: freeze-dried fluorescent carbon dots.

[0013] Furthermore, the fluorescent carbon dots are prepared into paper-based sensors.

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

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

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

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

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

[0019] Furthermore, the detection method of rifampicin comprises the following steps:

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

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

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

[0023] A4: The formula Y=-1.3050X+642.84167 is used to calculate the X value, i.e., the rifampicin concentration.

[0024] Furthermore, in the 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-based substrate to be tested flat on a black plane under white light, and take a photo and capture it at about 30 to 40 cm above it.

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

[0027] As a new type of micro-sensing platform, paper-based sensors have the following advantages compared with other sensors: ① low preparation cost, cheap and portable, and do not require expensive precision instruments; ② fast response speed, only a few seconds to a few minutes; ③ can achieve in-situ visual real-time detection. In addition, it can also be integrated with a smartphone to form a portable visual sensing platform, which is suitable for a variety of application fields and brings new prospects for rapid on-site detection of targets.

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

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

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

[0031] 3. The present invention designs a mobile phone application applet that can be used to detect rifampicin. The paper-based sensor has low preparation cost and does not require large instruments. With the help of smartphone software, it can quickly and in situ realize visual detection of rifampicin. The detection method is simple and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a bar graph showing the selectivity of the fluorescent carbon dots of the present invention in response to different ions and antibiotics.

[0033] Figure 2 This is a bar graph showing the anti-interference ability of the fluorescent carbon dots used in the present invention in detecting rifampicin.

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

[0035] Figure 4 The linear fitting diagram of the fluorescence intensity change rate of the fluorescent carbon dots of the present invention and the rifampicin concentration is

[0036] Figure 5 This is a color change diagram of the paper-based sensor of the present invention when the concentration of rifampicin is 0 to 90 μM

[0037] Figure 6 Linear fitting diagram of the RGB sum value of the paper-based sensor of the present invention and the concentration of rifampicin

[0038] Figure 7 The application diagram of the present invention for rapid detection of rifampicin: (A) is the interface of the mobile phone application applet, and (B) and (C) respectively show the results of using the applet to determine the concentration of rifampicin in the solution as 30 μM and 90 μM. DETAILED DESCRIPTION

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

[0040] Example 1

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

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

[0043] S2: Centrifugal filtration: After centrifugation, the supernatant was collected and filtered through a 0.22 μm water filter membrane to remove large particles;

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

[0045] S4: Freeze-drying: freeze-dried fluorescent carbon dots.

[0046] Example 2

[0047] Experiment 1: Rifampicin selectivity

[0048] (1) Take 0.1 mL of carbon dot solution (1 mg / mL) in a cuvette and dilute it to 3 mL with ultrapure water to serve as a blank control group. Test at an excitation wavelength of 280 nm, and count 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 synergistic drugs) was studied. 0.1 mL of Na + OH - 、SO3 2- , Ba + , Gd 2+ , Hg 2+ , Mn 2+ , ClO - , CN - , rifapentine (RFP), rifabutin (RFB), rifaximin (RFX), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), tetracycline (TC), sulfamethoxazole (SMX), vancomycin hydrochloride (VAN), erythromycin (ERM), chloramphenicol (CM), mitoxantrone (Mi), azithromycin (AZM) aqueous solution (1mM), diluted to 3mL with ultrapure water. Tested at an excitation wavelength of 280nm, the maximum fluorescence emission intensity was counted as F.

[0050] The test results are as follows Figure 1 As shown in the figure, when rifampicin (RIF) was added, the fluorescence intensity change rate (F0-F) / F0 changed significantly, with a change rate of 83%. After adding 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 dots have good selectivity for the recognition of rifampicin.

[0051] Experiment 2: Anti-interference ability

[0052] 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) were added to the cuvette and diluted to 3 mL with ultrapure water. The test was performed under 280 nm excitation, and the maximum fluorescence emission intensity was calculated as F. The solution with only carbon dots and rifampicin was used as the blank sample.

[0053] The test results are as follows Figure 2 As shown in the figure, in the presence of interfering ions, the fluorescence intensity of the probe is not significantly different from that of rifampicin alone, indicating that the probe has a 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 solution. The test was carried out 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] As the concentration of rifampicin increases, the fluorescence intensity of carbon dots changes as shown in Figure 3 As shown. In the concentration range of 0 to 30 μM, as the concentration of rifampicin increases, the fluorescence intensity of the mixed system continues to weaken. A linear analysis of the change rate of the fluorescence intensity of the mixed system (F0-F) / F0 and the concentration of rifampicin was performed. The results are shown in Figure 4 As shown. In the range of rifampicin concentration from 0 to 25 μM, the fluorescence intensity change rate (F0-F) / F0 showed a good linear relationship with the rifampicin concentration. The linear equation was: Y = 0.0091X + 0.1234, and the correlation coefficient R 2 =0.9911, and the minimum detection concentration was calculated to be 0.67 μM using LOD=3σ / S.

[0056] Experiment 4: Ability to Identify Rifampicin in Real Environmental Water

[0057] To 0.1mL of carbon dot solution (1mg / mL), add different doses (final concentrations of 5, 10, 15μM) of rifampicin solution (1mM), and finally dilute the solution to 3mL with lake water / tap water / plateau water. Test under 280nm excitation, and record the maximum fluorescence emission intensity as F. Use the carbon dot solution with only lake water / tap water / plateau water as a blank sample and record the fluorescence intensity as F0. The concentration of rifampicin in the environmental water sample was obtained 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 a recovery rate of 94% to 115% and a maximum relative standard deviation of 7.7%.

[0058] Table 1 shows the spiked recovery of rifampicin 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 material as a substrate and combining it with fluorescent carbon dot materials synthesized with coumarin and polyethyleneimine as raw materials, a paper-based sensor is constructed, which can achieve highly sensitive detection of target analytes.

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

[0063] A filter paper disc cut into a diameter of 1 cm was immersed in a 1-5 mg / mL carbon dot solution. After 10-20 minutes, the filter paper disc with carbon dots attached was taken out and placed in an oven for 10 minutes to dry it completely, thereby obtaining the paper-based sensor.

[0064] Example 4 Rapid Detection Method for Rifampicin

[0065] (1) Cut a filter paper disc with a diameter of 1 cm and soak it in a 1-5 mg / mL carbon dot solution. After 20 minutes, take out the filter paper disc with carbon dots attached and put it in an oven to dry for 10 minutes to completely dry it, thereby obtaining the paper-based sensor. (The results of the paper-based sensor soaking for 10 to 30 minutes are not much different)

[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 for drying for 10 minutes to completely dry it.

[0067] (3) Under white light, the paper-based sensor was laid flat on a black plane, and a smartphone was placed approximately 30 cm above it. The smartphone camera was used to capture the color changes of the paper-based sensor and extract the RGB values ​​of the paper-based sensor. Linear analysis was performed with the RGB sum (R+G+B) as the Y-axis and the rifampicin concentration as the X-axis.

[0068] The color development results of the paper-based sensor are as follows Figure 5 As shown, within the concentration range of 0 to 90 μM rifampicin solution, as the concentration of rifampicin increases, the color on the paper-based sensor continues to deepen, gradually deepening from light yellow to orange-red.

[0069] The linear fitting results are as follows Figure 6As shown, in the range of 0 to 90 μM rifampicin solution concentration, the linear equation is: Y = -1.3050X + 842.84167, the correlation coefficient R 2 =0.9960. There is a good linear relationship between the sum of RGB and the concentration of rifampicin. The paper-based sensor has the ability to detect the concentration of rifampicin in water. The detection limit is the lowest visual color concentration, so the detection limit LOD = 10μM.

[0070] Example 5 Ability to Identify Rifampicin in Real Environmental Water

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

[0072] The obtained RGB sum (R+G+B) was substituted into the linear equation as the Y value: 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 recovery rate of rifampicin in different water bodies detected by paper-based sensors

[0074]

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

[0076] Example 6

[0077] Based on the above, a WeChat applet was designed to build a visual rapid detection platform for rifampicin. The applet uses the linear equation "Y = -1.3050X + 642.84167" obtained in the above study as the theoretical basis. By directly capturing the color changes of the paper-based sensor in the applet and analyzing them, the obtained RGB sum value is substituted into the linear equation as the Y value: Y = -1.3050X + 842.84167, and the final calculated rifampicin concentration is directly displayed in the applet, realizing the rapid visual quantitative detection of rifampicin. The specific steps are as follows:

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

[0079] (2) Under white light, place the paper substrate to be tested flat on a black surface and place it about 30 to 40 degrees above the black surface.

[0080] Place your phone at a distance of 100 cm.

[0081] (3) In the mini program, click “Click to upload picture” and choose to take a photo directly or select a picture from the photo album. Use the smartphone camera to capture the color change of the paper-based sensor and upload it to the mini program. The concentration of rifampicin in the test solution can be directly read.

[0082] like Figure 7 As shown in A, the designed applet is simple and clear, consisting of only two parts: "select photo" and "display result", and is easy to operate. It only requires using the smartphone camera to take a picture of the paper-based sensor. The applet can capture the color changes of the paper-based sensor and analyze the RGB sum, and automatically calculate the rifampicin concentration in the test solution. Figure 7 B and Figure 7 C shows the results of the paper-based sensor measured by the applet with different rifampicin concentration solutions (30μM, 90μM). At different concentrations, the color of the paper-based sensor has obvious changes, which can be clearly captured by the applet, and RGB analysis can be automatically performed, with the rifampicin concentration results listed directly below the picture. Figure 7 B and Figure 7 C It can be seen that the results analyzed by the applet are 29μM and 93.37μM, respectively, with recovery rates of 97% and 104%. The detailed results are listed in Table 3.

[0083] Table 3 shows the results of the mobile phone application applet using the paper-based sensor to measure the concentration of rifampicin

[0084]

[0085] The recovery rate of rifampicin detected by the mini program is relatively good, ranging from 92% to 113%, with a maximum relative standard deviation of 11.4%. It can be applied to the detection of rifampicin in actual water bodies.

[0086] The above is only a preferred implementation case of the present invention, and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred implementation case as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent implementation cases with equivalent changes by using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above implementation cases based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Application of fluorescent carbon dots synthesized from coumarin and polyethyleneimine in the detection of the antibiotic rifampicin.

2. The use according to claim 1, characterized in that: The preparation steps of the fluorescent carbon dots include: S1: Hydrothermal reaction: dissolve polyethyleneimine and coumarin in a solvent to obtain a homogeneous solution, transfer it to a hydrothermal reactor, and react at 90°C to 240°C for 3h to 24h; S2: Centrifugal filtration: After centrifugation, the supernatant was collected and filtered through a 0.22 μm to 0.35 μm water filter membrane to remove large particles; S3: Dialysis: Use a dialysis bag with a molecular weight cutoff of 3000Da to 4000Da for 24h to 48h; S4: Freeze-drying: freeze-dried fluorescent carbon dots.

3. The use according to claim 1, characterized in that: The fluorescent carbon dots are prepared into paper-based sensors.

4. The use according to claim 3, characterized in that: The preparation method of the paper-based sensor comprises: soaking and adsorption: soaking the paper material in a fluorescent carbon dot solution synthesized with coumarin and polyethyleneimine as raw materials to complete the adsorption; and drying: completely drying to obtain the paper-based sensor.

5. The use according to claim 4, characterized in that: The soaking and adsorption conditions are as follows: soaking time is 10 to 30 minutes; the concentration of the fluorescent carbon dot solution is 1 to 5 mg / mL.

6. The use according to claim 4, characterized in that: The drying condition is: drying in an oven for 10 to 15 minutes.

7. The use according to claim 3, characterized in that: The paper-based sensor is used for rapid detection of rifampicin, comprising the following steps: A1: Immerse the paper-based sensor in the sample to be tested, and dry it completely after adsorption is completed; A2: Acquire the paper-based sensor image; A3: Analyze the color of the paper-based sensor and obtain the RGB sum Y; A4: The formula Y=-1.3050X+642.84167 is used to calculate the X value, i.e., the rifampicin concentration.

8. The use according to claim 7, characterized in that: In the step A1, the paper-based sensor is immersed in the sample to be tested for 10 minutes.

9. The use according to claim 7, characterized in that: The method for obtaining the paper-based sensor image in step A2 is to place the paper-based sensor to be tested flat on a black plane under white light, and take a photo and capture the image at about 30 to 40 cm above the paper-based sensor.

10. The use according to claim 7, characterized in that: In the step A2, the image of the paper-based sensor is obtained by photographing the paper-based sensor using a smartphone camera; in the step A3, the color of the paper-based sensor is analyzed by using a smartphone program to obtain the RGB sum Y; In step A4, the concentration of rifampicin in the solution is automatically calculated using a smartphone program.

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