Preparation method and application of a pH sensor based on nanocapillaries

Abstract

The present application provides a preparation method and application of a pH sensor based on nanocapillary, wherein the preparation method heats and stirs a chitosan solution and a polyvinyl pyrrolidone solution, adds a glutaraldehyde solution and continues to heat and stir to prepare a chitosan-PVP sol. The chitosan-PVP sol is then filled into the capillary and heated on a hot plate to form a gel in the capillary. The difference in the hydrophilic and hydrophobic properties of the CS / PVP gel under different pH conditions is utilized to induce the nanocapillary ion current to change, and a gel nanocapillary with pH sensitivity is prepared. The pH sensor provided by the present application can sense changes in pH by observing changes in current.

Description

Technical Field

[0001] The present application belongs to the technical field of material preparation and electrochemical measurement, and specifically relates to a preparation method of a pH sensor based on nanocapillaries and its application. Background Art

[0002] Currently, the current standards for clinically used antibiotic susceptibility testing (AST) are broth dilution and disk diffusion methods. By observing changes in bacterial optical density and the extent of the inhibition zone on agar, the minimum inhibitory concentration (MIC) is determined, thereby predicting the therapeutic efficacy of different antibiotics and providing a basis for clinical antibiotic dosage. However, these methods are time-consuming, typically requiring one to two days to obtain reliable results.

[0003] To address the time-consuming problem, researchers have developed various rapid AST methods. Genotypic AST typically uses common genetic tools (e.g., sequence-specific amplification by polymerase chain reaction (PCR), padlock probe-mediated rolling circle amplification, or whole genome sequencing) to detect specific genetic markers (plasmids, genes, or mutations) associated with the resistance phenotype. Although this method can achieve rapid AST detection in a short period of time, due to the current lack of markers for drug-resistance genes, it is difficult to reliably predict the sensitivity of specific antibiotics or resistance genes, and it is also impossible to monitor bacterial growth during the action of antibiotics.

[0004] Using optical methods to monitor the growth rate of individual bacterial cells can measure MIC within tens of minutes, but this method limits the scale of the assay, and the cost of its optical components is also a huge challenge. Monitoring bacterial metabolism can also achieve rapid AST. Although oxygen nanoprobes can be used to monitor bacterial metabolic levels, rapid bacterial AST detection can be achieved within 2 to 3 hours, their stability is poor and detection errors are prone to occur. Although various rapid AST detection methods have been developed, rapid AST detection still has shortcomings such as poor stability, high cost, and the inability to perform sensitivity tests for specific antibiotics. Summary of the Invention

[0005] In response to the above problems, the present invention aims to provide a method for constructing a nanocapillary-based pH sensor, which specifically includes: a method for preparing a nanocapillary-based pH sensor, wherein the nanocapillary-based pH sensor includes a nanocapillary, a chitosan-PVP graft copolymer hydrogel, and an electrode; the preparation method includes the following steps:

[0006] Preparation of chitosan-PVP sol: Chitosan solution and polyvinyl pyrrolidone solution are heated and stirred, glutaraldehyde solution is added and continued to heat and stir to prepare chitosan-PVP sol;

[0007] Preparation of gel in nanocapillaries: Chitosan-PVP sol is then filled into the capillaries and heated on a hot plate to form a gel in the capillaries;

[0008] Connecting and constructing a nanocapillary-based pH sensor: connecting the nanocapillary, chitosan-PVP graft copolymer hydrogel and electrodes to construct a nanocapillary pH sensor, connecting the nanocapillary pH sensor to an electrochemical workstation, and detecting the current response signal at a fixed voltage to obtain pH changes.

[0009] The nanocapillary-based pH sensor utilizes the different hydrophilic and hydrophobic properties of CS / PVP gel under different pH conditions to induce changes in the nanocapillary ion current, thereby preparing a pH-sensitive gel nanocapillary.

[0010] The present application also provides a nanocapillary-based pH sensor prepared by the preparation method.

[0011] The present application also provides the use of the nanocapillary-based pH sensor in pH monitoring during bacterial growth.

[0012] Beneficial effects

[0013] The nanocapillary-based pH sensor provided in this application utilizes the different hydrophilic and hydrophobic properties of CS / PVP gel under different pH conditions to induce changes in the nanocapillary ion current, thereby preparing a pH-sensitive gel nanocapillary that can achieve current response in the pH range of 7.3 to 4.3, and is used to measure pH changes during bacterial and plant growth and metabolism.

[0014] The nanocapillary-based pH sensor provided in the present application can achieve stability in the ion response current to pH within 10 seconds.

[0015] The nanocapillary-based pH sensor provided in this application can realize pH monitoring during bacterial growth, providing technical conditions for real-time monitoring of bacterial growth process.

[0016] The nanocapillary-based pH sensor provided in this application can realize antibiotic sensitivity detection and provide a basis for the determination of bacterial MIC.

[0017] The nanocapillary-based pH sensor provided in this application can determine the MIC of antibiotics against bacteria within 1 hour.

[0018] The detection device of the present invention comprises nanocapillaries, a chitosan-PVP graft copolymer hydrogel, and electrodes. The pore size of the gel replaces that of the capillaries. Under acidic conditions, protons bind to -NH2 groups on the chitosan, increasing the hydrophilicity of the gel and making it easier for solutions to flow into and out of the gel nanocapillaries. This, in turn, increases the ion response current of the gel nanocapillaries. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the nanocapillary pH sensor structure

[0020] Figure 2 Current response of the nanocapillary pH sensor to phosphate buffers of different pH values, including (a) ion response current of the gel nanocapillary to different pH values, and (b) the linear relationship between the ion response current and the relative pH value;

[0021] Figure 3 The current response of the nanocapillary pH sensor to Escherichia coli supernatant at different culture time periods, including (a) the ion response current of the gel nanocapillary to different pH values, and (b) the growth curves drawn by the standard method and the OD600 method;

[0022] Figure 4 Effects of different concentrations of tetracycline (a) and norfloxacin (c) on the growth of Escherichia coli, where (b) and (d) are the relevant inhibitory fractions. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] One embodiment of the present application provides a method for preparing a pH sensor based on nanocapillaries (see Figure 1 ), the nanocapillary-based pH sensor comprises a nanocapillary, a chitosan-PVP graft copolymer hydrogel and an electrode; the preparation method comprises the following steps:

[0027] Preparation of chitosan-PVP sol: Chitosan solution and polyvinyl pyrrolidone solution are heated and stirred, glutaraldehyde solution is added thereto and continued heating and stirring to prepare chitosan-PVP sol;

[0028] Preparation of gel in nanocapillaries: Chitosan-PVP sol is then filled into the capillaries and heated on a hot plate to form a gel in the capillaries;

[0029] Connecting and constructing a pH sensor based on nanocapillaries: constructing a nanocapillary pH sensor with the nanocapillary and chitosan-PVP grafted copolymer hydrogel, and connecting the nanocapillary and the electrode to an electrochemical workstation. Under a fixed voltage, the current response signal is detected to obtain the pH change.

[0030] Among them, the pH sensor based on nanocapillaries uses the different hydrophilic and hydrophobic properties of CS / PVP gel under different pH conditions to induce changes in the nanocapillary ion current, thereby preparing a pH-sensitive gel nanocapillary.

[0031] In one embodiment, the nanocapillary comprises quartz and glass nanocapillary.

[0032] In one embodiment, the concentration of the chitosan solution is 0.5% to 3%.

[0033] In one embodiment, the concentration of the polyvinyl pyrrolidone solution is 3% to 5%.

[0034] In one embodiment, the ratio of the chitosan solution to the polyvinyl pyrrolidone solution is 1:2 to 1:8.

[0035] In one embodiment, the chitosan solution and the polyvinyl pyrrolidone solution are heated and stirred at 60° C. for 30 minutes.

[0036] In one embodiment, the concentration of the glutaraldehyde solution is 0.1-0.5%.

[0037] In one embodiment, the glutaraldehyde solution is added and the heating and stirring are continued for 40 minutes.

[0038] In one embodiment, the nanocapillary filled with chitosan-PVP sol was heated on a heating stirrer at 60°C for 5 h to convert the chitosan-PVP sol into a gel inside the nanocapillary.

[0039] In one embodiment, the electrode is made of Ag / AgCl.

[0040] In one embodiment, the fixed voltage is 0.1V to 5V.

[0041] An embodiment of the present application provides a nanocapillary-based pH sensor prepared by the preparation method.

[0042] One embodiment of the present application provides the application of the nanocapillary-based pH sensor in pH monitoring during bacterial growth.

[0043] One embodiment of the present application provides a method for preparing a pH sensor based on nanocapillaries. 0.5% chitosan solution and 3% polyvinyl pyrrolidone (PVP) solution are heated and stirred at 60°C for 30 minutes at a ratio of 1:2. 1 ml of 0.1% glutaraldehyde solution is added thereto and heated and stirred for 50 minutes to prepare a chitosan-PVP sol. The chitosan-PVP sol is then filled into the capillary and heated at 60°C on a hot plate for one and a half hours to form a gel in the capillary. The electrode uses Ag / AgCl, and the above steps are used to construct a pH sensor based on nanocapillaries.

[0044] One embodiment of the present application provides a method for preparing a pH sensor based on nanocapillaries. A 3% chitosan solution and a 5% polyvinyl pyrrolidone (PVP) solution are heated and stirred at 60°C for 30 minutes at a ratio of 1:8. 1 ml of a 0.5% glutaraldehyde solution is added thereto and heated and stirred for 50 minutes to prepare a chitosan-PVP sol. The chitosan-PVP sol is then filled into a capillary and heated at 60°C on a hot plate for one and a half hours to form a gel in the capillary. The electrode uses Ag / AgCl, and the above steps are used to construct a pH sensor based on nanocapillaries.

[0045] One embodiment of the present application provides a method for preparing a pH sensor based on nanocapillaries, wherein 2% chitosan solution and 4% polyvinyl pyrrolidone (PVP) solution are heated and stirred at 60°C for 30 minutes at a ratio of 1:4, 1 ml of 0.4% glutaraldehyde solution is added thereto, and the mixture is heated and stirred for 50 minutes to prepare a chitosan-PVP sol. The chitosan-PVP sol is then filled into the capillary and heated on a heating plate at 60°C for one and a half hours to allow the chitosan-PVP sol to form a gel in the capillary. The electrode uses Ag / AgCl, and the above steps are used to construct a pH sensor based on nanocapillaries. At a voltage of 0.5V, the pH of the buffer solution is changed to observe the change in current within 60s, and different current response signals are obtained.

[0046] Example 1 Detection of Current Changes in Phosphate Buffers of Different pH Values

[0047] A 2% chitosan solution and a 4% polyvinylpyrrolidone (PVP) solution were heated and stirred at 60°C for 30 minutes. To this solution, 1 ml of a 0.4% glutaraldehyde solution was added and heated and stirred for another 50 minutes to prepare a chitosan-PVP sol. The chitosan-PVP sol was then filled into a capillary tube and heated on a hot plate at 60°C for 1.5 hours to form a gel within the capillary tube. Using Ag / AgCl electrodes, the above steps were used to construct a nanocapillary pH sensor. At a voltage of 0.5 V, the pH of the buffer solution was varied and the current was observed over 60 seconds, yielding different current response signals.

[0048] To measure the current response of the pH sensor to buffers of different pH values, the gel nanocapillary was placed in 0.2M phosphate buffer at different pH values, a voltage of 0.5V was applied, and the current change was observed within 60 seconds. After measuring the ion current, the gel nanocapillary was rinsed with 1M KCl for 60 seconds. The current change was examined in the pH range of 7.3 to 4.3. The results showed that the ion response current of the gel nanocapillary to different pH values ​​can reach stability within 10 seconds ( Figure 2 In a), there is a good linear relationship between the ion response current and the relative pH value. The sensitivity of the gel nanocapillary for pH detection is 3.06nA / pH through linear fitting, and the linear fitting degree is 0.99 ( Figure 2 b)

[0049] Example 2 Detection of pH changes during the growth of Escherichia coli

[0050] 1 ml of frozen E. coli culture was added to 50 ml of glucose broth and cultured overnight at 37 °C. The overnight culture was diluted in glucose broth to ensure that the initial concentration of bacteria was maintained at 1 × 10 5 CFU / ml, continue to culture. Collect the E. coli culture every hour and centrifuge it at 5000rpm for 5min in a desktop high-speed centrifuge, and wait for the supernatant of E. coli in different culture time periods. Use gel nanocapillary to measure pH changes. At a wavelength of 600nm, use an ultraviolet spectrophotometer to measure the optical density of E. coli in different culture time periods. That is OD600. The Boltzmann equation is used to fit the ion current and OD value of E. coli and draw a growth curve. The results show that with the increase of culture time, the current is increasing and the pH is decreasing ( Figure 3 (a) The changes are consistent with the growth trend of E. coli. By comparing with the standard method OD600, it was found that the trends of the growth curves drawn by the two methods are roughly similar ( Figure 3 b), which shows that the method of the present application can provide technical conditions for real-time monitoring of the growth of Escherichia coli and detection of bacterial counts.

[0051] Example 3 Antibiotic Sensitivity Detection

[0052] Before AST determination, the E. coli stock solution was thawed at room temperature and diluted with glucose broth to prepare different concentrations of tetracycline (0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml) and norfloxacin (0.016 μg / ml, 0.03 μg / ml, 0.0625 μg / ml, 0.125 μg / ml) detection solutions. The inoculum size was 5 × 10 5 Overnight cultures of Escherichia coli with CFU / ml were added to different concentrations of antibiotic test solutions, and glucose broth without antibiotics was used as a control group. All test samples were incubated at 37°C for 3 hours. During the AST process of E. coli, we collected the supernatant from the bacterial culture every hour to detect changes in its pH value. Two gel nanocapillaries were used to detect changes in ion currents of tetracycline and norfloxacin at different concentrations. Subsequently, in order to evaluate the effectiveness of antibiotics in inhibiting bacterial activity, the bacterial inhibition fraction (f) of the antibiotics was introduced:

[0053]

[0054] Among them, ΔI control represents the difference between the ion current in E. coli culture medium and the initial state without antibiotics; ΔI antibiotics The difference between the ionic current of E. coli culture during the growth process under the action of antibiotics and the initial state without antibiotics. We define the minimum inhibitory concentration (MIC) as the inhibition fraction ≥ 80%.

[0055] The results showed that as the antibiotic concentration increased, the ion response current of the gel nanocapillary showed a downward trend during the growth of E. coli. At the same time, the growth rate of E. coli also decreased accordingly. Figure 4 (a, c) The bacterial inhibition fraction increased with increasing tetracycline and norfloxacin concentrations, reaching 90% within an hour when the tetracycline and norfloxacin concentrations were increased to 2 μg / ml and 0.125 μg / ml, respectively. This result indicates that the growth of E. coli was completely inhibited in both cases ( Figure 4 (b, d) Thus, within one hour, the MICs of tetracycline and norfloxacin against E. coli were determined to be 2 μg / ml and 0.125 μg / ml, respectively. Therefore, this study enables rapid antibiotic susceptibility testing and provides a basis for MIC determination.

[0056] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a pH sensor based on nanocapillaries, characterized in that: The nanocapillary-based pH sensor comprises a nanocapillary, a chitosan-PVP graft copolymer hydrogel, and an electrode; and the preparation method comprises the following steps: Preparation of chitosan-PVP sol: Chitosan solution and polyvinyl pyrrolidone solution are heated and stirred, glutaraldehyde solution is added and continued to heat and stir to prepare chitosan-PVP sol; Preparing a gel in a nanocapillary: filling the chitosan-PVP sol into the capillary and heating it on a heating plate to form a gel in the capillary; Connecting and constructing a nanocapillary-based pH sensor: connecting the nanocapillary, chitosan-PVP graft copolymer hydrogel and electrodes to construct a nanocapillary pH sensor, connecting it to an electrochemical workstation, and detecting the current response signal at a fixed voltage to obtain the pH change.

2. The preparation method according to claim 1, characterized in that The concentration of the chitosan solution is 0.5% to 3%.

3. The preparation method according to claim 1, characterized in that The concentration of the polyvinyl pyrrolidone solution is 3% to 5%.

4. The preparation method according to claim 1, characterized in that The ratio of the chitosan solution to the polyvinyl pyrrolidone solution is 1:2 to 1:

8.

5. The preparation method according to claim 1, characterized in that The chitosan solution and polyvinyl pyrrolidone solution were heated and stirred at 60° C. for 30 min.

6. The preparation method according to claim 1, characterized in that The concentration of glutaraldehyde solution is 0.1~0.5%.

7. The preparation method according to claim 1, characterized in that Add glutaraldehyde solution and continue heating and stirring for 50 minutes.

8. The preparation method according to claim 1, characterized in that The electrode is an Ag / AgCl electrode.

9. The pH sensor based on nanocapillaries prepared by the preparation method according to claim 1.

10. Use of the nanocapillary-based pH sensor according to claim 9 in pH monitoring during bacterial growth.

Images