Preparation method and application of high-stability hydrogen sulfide detection card
By immobilizing dithizone-Pb complexes in a sol-gel system prepared under weakly alkaline conditions, the problems of stability and quantitative detection of hydrogen sulfide detection cards were solved, enabling rapid and stable drug sensitivity analysis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2024-10-31
- Publication Date
- 2026-05-01
AI Technical Summary
Existing hydrogen sulfide detection cards have poor stability, cannot be stored for long periods in normal environments, and cannot be used for quantitative detection, thus failing to meet the need for rapid antibiotic screening.
A highly stable hydrogen sulfide detection card was prepared by immobilizing a dithizone-Pb complex in a sol-gel system prepared under weakly alkaline catalytic conditions. The detection was achieved by a chemical colorimetric reaction between the silica gel carrier and hydrogen sulfide.
The prepared hydrogen sulfide detection card exhibits high stability under alkaline conditions and can rapidly and quantitatively detect hydrogen sulfide concentration, meeting the needs for rapid screening of effective drugs and reducing the detection time to within 6 hours.
Smart Images
Figure CN121955281A_ABST
Abstract
Description
A method for preparing a highly stable hydrogen sulfide detection card and its application Technical Field
[0001] This application relates to a method for preparing a highly stable hydrogen sulfide detection card and its application, belonging to the field of rapid detection of hydrogen sulfide gas and bacterial drug sensitivity analysis. Background Technology
[0002] Escherichia coli (E. coli) is a Gram-negative bacterium belonging to the Enterobacteriaceae family and is one of the most common bacteria in the intestines of humans and warm-blooded animals. They are generally harmless, but certain species or serotypes of E. coli are pathogenic, causing illnesses ranging from mild to severe, such as intestinal infections and hemolytic uremic syndrome, requiring antibiotic treatment when necessary. To avoid problems such as insufficient antibiotic dosage leading to prolonged illness or antibiotic resistance due to overuse, it is necessary to screen for sensitive antibiotics for rapid and effective administration. Traditional methods for detecting bacteria and other microorganisms typically require microbial culture or nucleic acid-based polymerase chain reactions, but these methods are very time-consuming and cannot meet the demand for timely and effective disease treatment.
[0003] An investigation into the gaseous composition produced by *Escherichia coli* during its metabolic activities revealed that the bacteria generate a variety of organic and inorganic compounds, with hydrogen sulfide gas being the most abundant, as shown in Table 1. Therefore, we can indirectly determine the inhibitory effect of antibiotics on *Escherichia coli* by testing the concentration of hydrogen sulfide gas, thereby determining the minimum inhibitory concentration (MIE) of the relevant drugs and providing a solution for the rapid screening of sensitive drugs.
[0004] Common hydrogen sulfide test strips are simple to prepare, simply by immersing filter paper in a lead acetate solution and then drying it. However, due to the poor stability of lead acetate, these test strips require storage in dry, low-temperature conditions to prevent decomposition, resulting in stringent storage requirements and a short shelf life. Furthermore, the precipitation of dry lead acetate after the solution dries leads to uneven test results, making quantitative detection impossible. Therefore, this type of test strip is unsuitable for testing the concentration of hydrogen sulfide generated during bacterial culture. To obtain a stable and quantitative hydrogen sulfide test strip that can be used continuously in the aforementioned specific environments, this application proposes a method for preparing a hydrogen sulfide colorimetric test strip by immobilizing an indicator that can chemically react with hydrogen sulfide in a sol-gel system prepared under weakly alkaline catalytic conditions, and realizes its application in the rapid analysis of drug susceptibility in Escherichia coli.
[0005] Table 1. Concentration of gases produced by the metabolic activities of Escherichia coli
[0006] Serial number, detected gas concentration, ppb: 1. Acetaldehyde 2.22, 2. Ethanol 3.20, 3. Pentanol 1.715, 4. Acetone 9.56, 5. Hydrogen sulfide 4.025, 3.66, 6. Methanethiol 1.425, 6.7, 7. Indole 2.765, 8. 2-Aminoacetophenone 4.0, 9. Propylene 1.5 surface Summary of the Invention
[0007] The purpose of this invention is to provide a hydrogen sulfide detection card and apply it to the rapid analysis of drug susceptibility of Escherichia coli. This hydrogen sulfide detection card has good stability, can be quantified, is easy to use, and can quickly provide detection results in bacterial drug susceptibility scenarios, meeting the need for rapid screening of effective drugs in actual situations.
[0008] According to one aspect of this application, a method for preparing a highly stable hydrogen sulfide detection card is provided, comprising at least the following steps:
[0009] Step I: Disperse dithizone in ethanol to obtain a dithizone solution;
[0010] Step II: Dissolve lead acetate in water to obtain a lead acetate solution;
[0011] Step III: Under alkaline conditions, the dithizone solution obtained in Step I and the lead acetate solution obtained in Step II are mixed to obtain the dithizone-Pb complex.
[0012] Step IV: Mix a methanol solution containing tetrabutylammonium hydroxide, N,N-dimethylformamide, water, and ethanol, and react in a water bath to obtain an alkaline silica gel solid support;
[0013] Step V: Disperse the dithizone-Pb complex obtained in Step III in the alkaline silica gel solid support obtained in Step IV to obtain the dithizone-Pb complex solid support.
[0014] Step VI: The dithizone-Pb complex solid carrier obtained in step V is drop-coated onto a thin film to obtain a hydrogen sulfide detection card.
[0015] Optionally, in step I, the concentration of dithizone in the dithizone solution is 0.1–0.3 mol / L.
[0016] Optionally, in step II, the concentration of lead acetate in the lead acetate solution is 0.05–0.15 mol / L.
[0017] Optionally, in step III, the pH of the alkaline conditions is 9 to 9.5.
[0018] Optionally, the volume ratio of the dithizone solution to the lead acetate solution is 1:1 to 1:3.
[0019] Optionally, in step IV, the concentration of tetrabutylammonium hydroxide in the methanol solution containing tetrabutylammonium hydroxide is 1 to 1.5 mol / L.
[0020] Optionally, the mass ratio of the mixture containing tetrabutylammonium hydroxide methanol solution, N,N-dimethylformamide, water, and ethanol is 1:(1-1.5):(3-5):(8-10):(10-15).
[0021] Optionally, the conditions for the water bath reaction are as follows:
[0022] The temperature of the water bath reaction is 50–60°C;
[0023] The water bath reaction time is 2 to 3 hours.
[0024] Optionally, in step V, the concentration of the dithizone-Pb complex in the dithizone-Pb complex solid carrier is 0.005–0.01 mol / L.
[0025] Optionally, in step VI, the film is selected from at least one of polyvinylidene fluoride and PET film.
[0026] According to another aspect of this application, a highly stable hydrogen sulfide detection card is provided for the rapid analysis of drug sensitivity in Escherichia coli, characterized in that the hydrogen sulfide detection card is fixed on the lid of a culture dish containing antibiotic drugs and Escherichia coli culture medium, incubated, and the color change of the hydrogen sulfide detection card at different times is observed.
[0027] Optionally, the hydrogen sulfide detection card needs to undergo an aging process.
[0028] Optionally, the aging conditions are as follows:
[0029] The aging process needs to be carried out under light-protected conditions;
[0030] The aging time is 24–36 hours;
[0031] The aging temperature is 20–30°C.
[0032] Optionally, the antibiotic is selected from at least one of gentamicin, ciprofloxacin, cefazolin, meropenem, amikacin, and ampicillin;
[0033] The concentration of Escherichia coli in the culture medium was 5 × 10⁻⁶. 8 ~10×10 8 CFU / mL;
[0034] The pH of the culture medium is 7.2–7.4.
[0035] Optionally, the incubation conditions are as follows:
[0036] The incubation temperature is 30–35°C;
[0037] The incubation time is 5 to 6 hours.
[0038] The beneficial effects that this application can produce include:
[0039] 1) The method for preparing silica gel under alkaline conditions provided in this application can be used to immobilize organic-metal binary complex indicators, and the resulting hydrogen sulfide detection card has high stability and can realize the quantitative detection of hydrogen sulfide gas.
[0040] 2) The hydrogen sulfide detection card prepared in this application can be used for drug susceptibility analysis of Escherichia coli. The entire detection process only takes 6 hours to obtain results, which greatly improves the efficiency of effective drug screening. Attached Figure Description
[0041] Figure 1 shows the color change of the hydrogen sulfide detection card prepared in Example 1 of this application;
[0042] Figure 2 shows the results of sensitivity analysis of Escherichia coli by the Chinese and American ropenem in Example 4 of this application;
[0043] Figure 3 is a quantitative detection diagram of hydrogen sulfide gas by the detection card in Embodiment 5 of this application. Detailed Implementation
[0044] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.
[0045] Unless otherwise specified, the raw materials and reagents used in the embodiments of this application were all purchased commercially.
[0046] The method for extracting the color change value of the hydrogen sulfide detection card in the embodiments of this application is as follows:
[0047] The extraction of color change values from the hydrogen sulfide detection card is achieved by digitizing the card's color recorded by a camera at different times. The RGB values of the card's color at time 0 and time t are then calculated using the following formula:
[0048]
[0049] The result ΔED is the color change value of the detection card.
[0050] According to one embodiment of this application, a method for preparing a hydrogen sulfide detection card is as follows:
[0051] (1) Preparation of indicator: Under alkaline conditions, equal volumes of dithizone and lead acetate solutions are mixed and reacted to form a red complex. Chloroform or carbon tetrachloride is then added to the mixture for liquid-liquid extraction. The reaction product of dithizone and lead acetate enters the chloroform or carbon tetrachloride layer. This layer is then removed and the chemical colorimetric indicator for detecting hydrogen sulfide, namely the dithizone-Pb complex, is obtained by rotary evaporation.
[0052] (2) Preparation of alkaline silica gel immobilization system: Conventional silica gel preparation usually requires the polymerization of silica gel precursor under acidic conditions to form a network structure for embedding indicators. However, the silica gel system prepared by acid catalysis is usually acidic, which is not conducive to immobilizing the indicators used in this application. Therefore, a weakly alkaline condition is selected to catalyze the condensation of silica gel precursor to obtain silica gel nanoparticles, which can be used as indicators of binary complex structure and exhibit high stability.
[0053] Tetraethoxysilane, tetrabutylammonium hydroxide, N,N-dimethylformamide, deionized water, and ethanol were mixed in a mass ratio of 1:(1-1.5):(3-5):(8-10):(10-15) and hydrolyzed in a water bath at 50°C for 2-3 hours.
[0054] (3) Preparation of detection card: The indicator dithizone-Pb complex was dissolved in an alkaline silica gel immobilization system and drop-coated onto a PVDF membrane to prepare a highly stable hydrogen sulfide detection card;
[0055] (4) Detection card aging: The detection card can be used after being stored in the dark for more than 24 hours. During the preparation of the indicator, the alkaline conditions are adjusted by sodium hydroxide, with a pH of 9-9.5; the concentration of the 50% ethanol solution of dithizone is 0.1-0.3 mol / L, and the concentration of the aqueous solution of lead acetate is 0.05-0.15 mol / L; the volume of chloroform or carbon tetrachloride added is 1 / 4 of the sum of the volumes of the dithizone and lead acetate solutions.
[0056] The tetrabutylammonium hydroxide is a 1 mol / L methanol solution;
[0057] The concentration of the indicator dithizone-Pb complex in the alkaline silica gel immobilization system is 0.005-0.01 mol / L.
[0058] According to one embodiment of this application, the application of a hydrogen sulfide detection card in the rapid analysis of drug susceptibility in Escherichia coli:
[0059] Prepare eight sterile culture dishes, numbered #1-#8. Add sterile culture medium to #1 as a negative control. Add equal amounts of culture medium inoculated with Escherichia coli to culture dishes #2-#8, with #2 serving as a positive control. Then, add six different concentrations of antibiotics to #3-#8. Place the lids of the culture dishes with hydrogen sulfide detection cards fixed in the center on top of the culture media #1-#8. Incubate the culture dishes #1-#8 in a 35°C incubator equipped with a camera for 6 hours. Extract the color change values of the hydrogen sulfide detection cards at different times, plot the relationship curve between the color change of the detection cards and time, obtain the minimum inhibitory concentration (MIC) of the drug, and complete the antibiotic sensitivity analysis.
[0060] The culture medium was Mueller-Hinton (MH) broth, pH 7.2-7.4;
[0061] The culture medium was inoculated with Escherichia coli, and the concentration of Escherichia coli was (5-10)×10⁻⁶. 8 CFU / mL.
[0062] The antibiotic mentioned is one of gentamicin, ciprofloxacin, cefazolin, meropenem, amikacin, and ampicillin.
[0063] Example 1
[0064] The preparation method of the hydrogen sulfide detection card is as follows:
[0065] (1) Preparation of indicator: Under pH=9 conditions, 5 mL of 50% ethanol solution of 0.1 mol / L dithizone and 5 mL of aqueous solution of 0.15 mol / L lead acetate were mixed and reacted to form a red complex. Then, 2.5 mL of chloroform or carbon tetrachloride was added to the mixture for liquid-liquid extraction. The reaction product of dithizone and lead acetate entered the chloroform or carbon tetrachloride layer. The layer was removed and rotary evaporated to obtain the chemical colorimetric indicator for detecting hydrogen sulfide, namely the dithizone-Pb complex.
[0066] (2) Preparation of alkaline silica gel immobilization system: 1g tetraethoxysilane, 1.5g methanol solution of 1mol / L tetrabutylammonium hydroxide, 3g N,N-dimethylformamide, 10g deionized water and 10g ethanol were mixed and hydrolyzed in a water bath at 50℃ for 3 hours.
[0067] (3) Preparation of detection card: The indicator dithizone-Pb complex obtained in step (1) was dissolved in the silica-based gel immobilization system obtained in step (2) to a concentration of 0.01 mol / L, and then dropped onto the PVDF membrane to prepare a highly stable hydrogen sulfide detection card.
[0068] (4) Test card aging: After storing the test card at 25°C in the dark for 24 hours, it can be used for subsequent applications.
[0069] Example 2.
[0070] The preparation method of the hydrogen sulfide detection card is as follows:
[0071] (1) Preparation of indicator: Under pH=9.5 conditions, 5 mL of 50% ethanol solution of 0.3 mol / L dithizone and 15 mL of aqueous solution of 0.05 mol / L lead acetate were mixed. After the reaction to form a red complex, 5 mL of chloroform or carbon tetrachloride was added for liquid-liquid extraction. The reaction product of dithizone and lead acetate entered the chloroform or carbon tetrachloride layer. The layer was removed and rotary evaporated to obtain the chemical colorimetric indicator for detecting hydrogen sulfide, namely the dithizone-Pb complex.
[0072] (2) Preparation of alkaline silica gel immobilization system: 1g tetraethoxysilane, 1g methanol solution of 1mol / L tetrabutylammonium hydroxide, 5g N,N-dimethylformamide, 10g deionized water and 15g ethanol were mixed and hydrolyzed in a water bath at 50℃ for 2.5 hours.
[0073] (3) Preparation of detection card: The indicator dithizone-Pb complex obtained in step (1) was dissolved in the alkaline silica gel immobilization system obtained in step (2) to a concentration of 0.008 mol / L, and then dropped onto the PVDF membrane to prepare a highly stable hydrogen sulfide detection card.
[0074] (4) Test card aging: After storing the test card at 30℃ in the dark for 24 hours, it can be used for subsequent applications.
[0075] Example 3.
[0076] The preparation method of the hydrogen sulfide detection card is as follows:
[0077] (1) Preparation of indicator: Under pH=9.2 conditions, 5 mL of 50% ethanol solution of 0.2 mol / L dithizone and 10 mL of aqueous solution of 0.1 mol / L lead acetate were mixed. After the reaction to form a red complex, 3.75 mL of chloroform or carbon tetrachloride was added for liquid-liquid extraction. The reaction product of dithizone and lead acetate entered the chloroform or carbon tetrachloride layer. The layer was removed and rotary evaporated to obtain the chemical colorimetric indicator for detecting hydrogen sulfide, namely the dithizone-Pb complex.
[0078] (2) Preparation of alkaline silica gel immobilization system: 1g tetraethoxysilane, 1.2g methanol solution of 1mol / L tetrabutylammonium hydroxide, 4g N,N-dimethylformamide, 9g deionized water and 12g ethanol were mixed and hydrolyzed in a water bath at 50℃ for 2 hours.
[0079] (3) Preparation of detection card: The indicator dithizone-Pb complex obtained in step (1) was dissolved in the alkaline silica gel immobilization system obtained in step (2) to a concentration of 0.005 mol / L, and then dropped onto the PVDF membrane to prepare a highly stable hydrogen sulfide detection card.
[0080] (4) Detection card aging: After storing the detection card at 20℃ in the dark for 24 hours, it can be used for subsequent applications.
[0081] Example 4.
[0082] The susceptibility of meropenem to Escherichia coli was analyzed using the hydrogen sulfide detection card prepared in Example 1.
[0083] Prepare 8 sterile petri dishes and Mueller-Hinton (MH) broth medium (pH 7.2), numbered #1-#8. Sterile medium was added to #1 as a negative control. Equal volumes of culture medium inoculated with *Escherichia coli* were added to petri dishes #2-#8 at an inoculation concentration of 5 × 10⁻⁶. 8 CFU / mL, #2 served as the positive control group. Subsequently, six different concentrations of meropenem were added to culture dishes #3-#8, at concentrations of 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 μg / mL, respectively. The lids of the culture dishes with the hydrogen sulfide detection card fixed in the center were then placed on top of the culture media #1-#8. Culture dishes #1-#8 were placed in an incubator equipped with a camera at 35℃ for 6 hours. Color change values of the hydrogen sulfide detection cards at different time points were extracted, and the relationship between color change and time was plotted, as shown in Figure 2. The figure shows that the minimum inhibitory concentration of meropenem against Escherichia coli is 4 μg / mL.
[0084] Example 5.
[0085] The hydrogen sulfide detection card prepared in Example 1 was used for quantitative analysis of hydrogen sulfide gas. Hydrogen sulfide gas was diluted with nitrogen to concentrations of 0, 50, 100, 200, 400, and 600 ppm. Each concentration of hydrogen sulfide gas was then introduced into the container containing the hydrogen sulfide colorimetric detection card prepared in Example 1 and maintained for 5 minutes. The RGB color values of the detection card before and after the reaction were recorded, and the color change value ΔED was calculated. A standard curve was plotted between the color change value ΔED and the hydrogen sulfide gas concentration, as shown in Figure 3. The figure shows that when the hydrogen sulfide concentration is between 0 and 600 ppm, its concentration is linearly related to the color change value of the detection card. 2 A value greater than 0.9 indicates that the prepared test card can quantitatively determine hydrogen sulfide gas in the range of 0-600 ppm.
[0086] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.
Claims
1. A method for preparing a highly stable hydrogen sulfide detection card, characterized in that, At least the following steps are included: Step I: Disperse dithizone in ethanol to obtain a dithizone solution; Step II: Dissolve lead acetate in water to obtain a lead acetate solution; Step III: Under alkaline conditions, mix the dithizone solution obtained in Step I and the lead acetate solution obtained in Step II to obtain a dithizone-Pb complex; Step IV: Mix a methanol solution containing tetrabutylammonium hydroxide, N,N-dimethylformamide, water, and ethanol, and react in a water bath to obtain an alkaline silica gel solid support; Step V: Disperse the dithizone-Pb complex obtained in Step III in the alkaline silica gel solid support obtained in Step IV to obtain a dithizone-Pb complex solid support; Step VI: Drop-coat the dithizone-Pb complex solid support obtained in Step V onto a thin film to obtain a hydrogen sulfide detection card.
2. The preparation method according to claim 1, characterized in that, In step I, the concentration of dithizone in the dithizone solution is 0.1–0.3 mol / L.
3. The preparation method according to claim 1, characterized in that, In step II, the lead acetate solution has a lead acetate concentration of 0.05–0.15 mol / L.
4. The preparation method according to claim 1, characterized in that, In step III, the pH of the alkaline conditions is 9 to 9.5; preferably, the volume ratio of the dithizone solution to the lead acetate solution is 1:1 to 1:
3.
5. The preparation method according to claim 1, characterized in that, In step IV, the concentration of tetrabutylammonium hydroxide in the methanol solution containing tetrabutylammonium hydroxide is 1–1.5 mol / L; preferably, the mass ratio of the methanol solution containing tetrabutylammonium hydroxide, N,N-dimethylformamide, water, and ethanol is 1:(1–1.5):(3–5):(8–10):(10–15); preferably, the conditions for the water bath reaction are as follows: the temperature of the water bath reaction is 50–60°C; the time of the water bath reaction is 2–3 hours.
6. The preparation method according to claim 1, characterized in that, In step V, the concentration of the dithizone-Pb complex in the dithizone-Pb complex solid carrier is 0.005–0.01 mol / L.
7. The preparation method according to claim 1, characterized in that, In step VI, the film is selected from at least one of polyvinylidene fluoride and PET film.
8. The application of a highly stable hydrogen sulfide detection card in the rapid analysis of drug susceptibility in Escherichia coli, characterized in that, A hydrogen sulfide detection card was fixed to the lid of a culture dish containing antibiotics and Escherichia coli culture medium, incubated, and the color change of the hydrogen sulfide detection card was observed at different times; the hydrogen sulfide detection card was prepared by any one of the preparation methods in claims 1 to 7.
9. The application according to claim 8, characterized in that, The hydrogen sulfide detection card needs to undergo an aging process; preferably, the aging conditions are as follows: the aging needs to be carried out under light-protected conditions; the aging time is 24 to 36 hours; and the aging temperature is 20 to 30°C.
10. The application according to claim 8, characterized in that, The antibiotic is selected from at least one of gentamicin, ciprofloxacin, cefazolin, meropenem, amikacin, and ampicillin; the concentration of Escherichia coli in the culture medium is 5 × 10⁻⁶. 8 ~10×10 8 CFU / mL; pH of the culture medium 7.2–7.4; preferably, the incubation conditions are as follows: the incubation temperature is 30–35°C; the incubation time is 5–6 hours.