A nano-channel urease detection device, method and application thereof

Abstract

The application provides a nanochannel urease detection device, method and application thereof. The detection method comprises the following steps: placing a PET film at a communication position of an electrolytic cell, and preparing a tapered nanochannel by one-way etching with a sodium hydroxide solution; and performing electrodeposition of a metal organic framework compound (MOF) of ZIF-8 in the tapered nanochannel, and detecting the activity of bacterial secreted urease by using the rapid and selective transmission effect of the MOF on OH ‑ The application measures the change of the ion conductance of the nanochannel by linear scanning to obtain the kinetic parameters of urea decomposition by urease, and analyzes the urease activity by using the parameters. The detection device is simple in design, low in price and good in electrochemical detection performance, and is successfully applied to the evaluation of bacterial secreted urease activity and the preventive monitoring of dental caries, and provides a new quantifiable detection means for the prevention of dental caries.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, and relates to a nanochannel-based urease detection device, method, and its application. Background Technology

[0002] Existing methods for urease detection include qualitative, semi-quantitative, and quantitative detection. Qualitative detection includes bacterial histological staining, pH indicator method, Nessler's reagent colorimetric method, latex agglutination test, and polymerase chain reaction (PCR) urease gene detection. Bacterial histological staining, pH indicator method, and Nessler's reagent colorimetric method all achieve indirect detection of urease through colorimetric reactions. The latex agglutination test detects antibodies in the sample by binding urease antigen to latex particles or colloidal gold. The specific binding between antigen and antibody results in a visible agglutination reaction, which qualitatively detects urease. PCR determines the presence of urease by designing primers and performing PCR amplification.

[0003] The aforementioned methods for detecting urease can only roughly indicate the intensity of urease activity. To determine the urease content, semi-quantitative detection methods have been developed: radionuclide analysis and immunoblotting. These methods directly or indirectly detect the urease content by detecting changes in the mass of the substrate and products before and after the urease decomposes urea. However, these methods suffer from problems such as insufficient quantitative accuracy, long processing time, complex operation, and the need for specialized technical personnel.

[0004] Furthermore, for the quantitative detection of urease, enzyme activity can be indirectly determined by detecting the ability of urease to catalyze the decomposition of urea through enzyme kinetic experiments. However, current methods for detecting urease activity still have some limitations. One is the rapid detection and evaluation of bacteria that secrete urease, such as Helicobacter pylori. Existing methods rely on radioactive isotopes such as C15. 13 C 14 Labeling methods are not readily available; however, there is no rapid and sensitive analytical method for evaluating the activity of oral streptococci in dental caries risk. We utilized the sub-nanochannels of ZIF-8 to detect OH... - The selective transport effect of ions improves the sensitivity of detecting urease activity secreted by Streptococcus salivarius, and the detection concentration of Streptococcus salivarius has reached 1 CFU. Finally, by comparing urease activity in the oral cavity of healthy individuals and patients with dental caries, we found that oral urease activity can be used as a means of preventing dental caries, thereby advancing the prevention and treatment of dental caries to the pre-disease stage and achieving the goal of preventing disease before it occurs. Summary of the Invention

[0005] The purpose of this invention is to provide a nanochannel-based urease detection device, method, and its application, specifically referring to the use of ZIF-8's own sub-nanochannels to detect OH-- Ion-selective transport characteristics enable rapid detection of urease activity at low voltages (≤1V) and high ionic strengths (100mM).

[0006] This application also provides a detection method based on the nanochannel-based urease detection device, comprising the following steps:

[0007] Step 1: The PET membrane is placed in the passage connecting two electrolytic cells. The PET nanochannel membrane is etched with sodium hydroxide solution. One side of the etched PET nanochannel membrane is irradiated with heavy ions. 1 mol / L KCl solution is added to the two electrolytic cells. Ag / AgCl electrodes are inserted. The curve is recorded using linear sweep voltammetry (LSV). The conductivity is calculated. The pore size of the PET membrane is calculated using formula (1).

[0008]

[0009] Where L represents the thickness of polyethylene terephthalate (PET); K represents the conductivity of the 1M KCl bulk solution;

[0010] D base The fixed diameter of the track after heavy ion irradiation of PET; N represents the average number of nanopores in the etched area.

[0011] G: Conductivity measured in nanoporous membranes;

[0012] Step 2, MOF modification process: The etched PET film is sputtered with gold, and then the treated PET film is sandwiched in the passage connecting two electrolytic cells. 50 ml of precursor solution is added to each electrolytic cell, and cathodic deposition is performed at a certain current density. After deposition is completed, the surface of the ZIF-8 modified film is rinsed with deionized water and methanol, and then placed in a vacuum drying oven to dry overnight.

[0013] Step 3: Place the ZIF-8 modified sub-nanoporous membrane in the passage connecting two electrolytic cells, and then add 5 U / mL, 25 U / mL and 55 U / mL urease to the two electrolytic cells in sequence, and perform LSV scanning to measure the ionic conductivity of the ZIF-8 modified sub-nanoporous membrane.

[0014] Step 4: Add 3 μM, 5 μM, 7 μM, 9 μM, and 11 μM urea sequentially to an electrolytic cell containing urease solution, performing an LSV scan after each addition. The urea in the electrolytic cell is decomposed by urease, producing OH-. - Diffusion through changes in conductivity of ZIF-8 modified sub-nanoporous membranes;

[0015] Step 5: Input the change in conductivity across the nanochannel after urease decomposes urea into the previously measured Michaelis constant working curve and read the Michaelis constant of urease.

[0016] This application also provides a nanochannel-based urease detection device, the device comprising a ZIF-8 modified PET conical nanochannel membrane, two electrolytic cells and an Ag / AgCl electrode, wherein the two electrolytic cells are interconnected, the ZIF-8 modified PET nanochannel membrane is fixedly clamped in the interconnected pathway of the two electrolytic cells, the two electrolytic cells contain electrolytes, one end of the Ag / AgCl electrode is placed in the electrolytic cell solution, and the other end of the Ag / AgCl electrode is connected to an electrochemical workstation.

[0017] This application also provides the application of the nanochannel-based urease detection device in the detection of bacteria.

[0018] Beneficial effects

[0019] The key advantage of the method of this invention is that it can rapidly detect the enzyme activity of urease in a short time, solving the problem that urease cannot be detected on-site and rapidly.

[0020] 1. Because urease secreted by bacteria can decompose urea to produce NH4. + and OH - Due to the presence of OH groups on both sides of the nanoporous membrane - The concentration gradient causes the generated ions to diffuse through the nanoporous membrane to the side without urea, thereby altering the ionic conductivity of the nanochannel. This invention enables rapid detection of urease in a short time (<5 min) without bacterial isolation, traditional plate culture, or labeling. The sub-nanochannels of ZIF-8 are used to detect OH... - Selective transport characteristics enable rapid detection of urease at low voltages (≤1V) and high ionic strengths (100mM).

[0021] 2. This invention enables rapid detection of Streptococcus salivarius in saliva, with a detection limit of 1 CFU / mL.

[0022] 3. This invention can realize the actual detection of oral saliva, detect the activity of oral urease, evaluate oral health status, and provide preventive suggestions for the occurrence of tooth decay.

[0023] 4. This invention enables rapid on-site detection of urease-producing bacteria and can be used as a new method for detecting urease-producing bacteria.

[0024] 5. The detection device of this invention is simple in design, inexpensive, and possesses excellent electrochemical detection performance. It has been successfully applied to the preventative monitoring of urease activity secreted by *Streptococcus salivarius* and dental caries, overcoming the drawback of traditional bacterial detection methods that require long-term culture. Furthermore, the detection of urease-producing *Streptococcus salivarius* in the oral cavity provides a new method and approach for the early detection of oral health conditions. It also offers a new pathway for the detection of urease-producing bacteria. Attached Figure Description

[0025] Figure 1 Schematic diagram of the urease detection system;

[0026] Figure 2 ZIF-8 PET nanochannels;

[0027] Figure 3 a. Comparison of IV curves before and after ZIF-8 deposition; b. Reaction kinetic parameters showing the relationship between the change in conductivity of 13 μM Urea and 25 U / mL Urease and time; c. C during the reaction of different active Ureases with urea. urea d. Relationship between (μM) and ΔG(μS); d. Michaelis constants for different active Ureases;

[0028] Figure 4 a. Liquid culture of *Streptococcus salivarius*; b. Plate culture of *Streptococcus salivarius*; c. At a constant urea concentration, C s.s Graph showing the relationship between (CFU / mL) and ΔG(μS);

[0029] Figure 5 Bar graph showing the conductivity values ​​of salivary urease in oral cavity of healthy individuals and patients with dental caries. "***" indicates p<0.001.

[0030] Figure 6 .10 6 CFU / mL Bacillus subtilis urease detection, C urea The graph shows the relationship between (μM) and ΔG(nS), with a Michaelis constant of 10.4μM. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0032] One embodiment of this application provides a method for detecting urease, comprising the following steps:

[0033] Step 1: The PET membrane is placed in a passage connecting two electrolytic cells. One side of the PET membrane is etched with sodium hydroxide solution. The etched side of the PET nanochannel membrane is then irradiated with heavy ions. A 1 mol / L KCl solution is added to both electrolytic cells, and an Ag / AgCl electrode is inserted. The curve is recorded using linear sweep voltammetry (LSV), and the conductivity is calculated. The pore size D of the PET membrane is calculated using formula (1). tip ;

[0034]

[0035] L: Thickness of polyethylene terephthalate (PET); K: Conductivity of 1M KCl bulk solution; D base : Fixed diameter of track after heavy ion irradiation of PET; N: Average number of nanopores per area of ​​etched region;

[0036] G: Conductivity measured in nanoporous membranes;

[0037] Step 2, MOF modification process: The etched PET film is sputtered with gold, and then the treated PET film is sandwiched in the passage connecting two electrolytic cells. 50 ml of precursor solution is added to each electrolytic cell, and cathodic deposition is performed at a certain current density. After deposition is completed, the surface of the ZIF-8 modified film is rinsed with deionized water and methanol, and then placed in a vacuum drying oven to dry overnight.

[0038] Step 3: Place the ZIF-8 modified sub-nanoporous membrane in the passage connecting two electrolytic cells, and then add 5 U / mL, 25 U / mL and 55 U / mL urease to the two electrolytic cells in sequence, and perform LSV scanning to measure the ionic conductivity of the ZIF-8 modified sub-nanoporous membrane.

[0039] Step 4: Add 3 μM, 5 μM, 7 μM, 9 μM, and 11 μM urea sequentially to an electrolytic cell containing urease solution, performing an LSV scan after each addition. The urea in the electrolytic cell is decomposed by urease, producing OH-. - Diffusion through changes in conductivity of ZIF-8-modified subnanoporous membranes; see [link to relevant documentation]. Figure 3 b

[0040] Step 5: Input the change in conductivity across the nanochannel after urease decomposes urea into the pre-determined Michaelis constant working curve and read the Michaelis constant of urease.

[0041] In one embodiment, the precursor solution is a mixture of 0.85 mol / L 2-MIM (2-methylimidazole) and 0.014 mol / L Zn(Ac)₂. In one embodiment, the specified current density is 0.13 mA / cm². 2 .

[0042] In one embodiment, the cathode deposition time is 1 hour to 5 hours.

[0043] In one embodiment, the temperature for overnight drying in a vacuum drying oven is 60°C to 100°C.

[0044] In one embodiment, the formula for calculating the Michaelis constant is shown in formula (2).

[0045]

[0046] Where v represents the product formation rate; V represents the maximum reaction rate of the enzyme; K m [A] represents the Michaelis constant of the enzyme; [A] represents the concentration of the substrate.

[0047] Based on the Michaelis equation, the relationship between the change in conductivity ΔG and the Michaelis constant and maximum reaction rate of the enzyme was calculated, as shown in formula (3).

[0048]

[0049] Where 1 / ΔG represents the reciprocal of the ionic conductivity on both sides of the conical nanochannel;

[0050] c represents the concentration of urea;

[0051] The apparent Michaelis constant of urease is represented by 1 / c; the experimental data are plotted with 1 / c on the x-axis and 1 / ΔG on the y-axis, which simplifies formula (3) to formula (4).

[0052]

[0053] Plot the relationship between 1 / c and 1 / ΔG. The ratio of the slope to the intercept of the curve is the apparent Michaelis constant for the enzyme-catalyzed reaction.

[0054] Among them, the larger the Michaelis constant value, the weaker the affinity between the enzyme and the substrate, and the smaller the value, the stronger the affinity between the enzyme and the substrate.

[0055] In one embodiment, in step 1, the PET nanochannel membrane is etched with sodium hydroxide solution. One side of the PET nanochannel membrane, which has been irradiated with heavy ions, is filled with 15 mL of 6M NaOH solution as an etching solution, and the other side of the PET conical nanochannel membrane is filled with 15 mL of a mixed solution of 1M HCOOH and 1M KCl as a stop solution. The outer side is connected to an electrochemical workstation. A Pt electrode is inserted into the solutions on both sides and a transmembrane potential of 1V is applied for etching monitoring (it). The size of the etched membrane pores is controlled by monitoring the current value. Then, the membrane is rinsed with deionized water to remove the etching solution and stop solution remaining on the surface of the polyethylene terephthalate membrane and in the ion channels.

[0056] In one embodiment, controlling the size of the etched film aperture by monitoring the current value is achieved when the current value reaches a preset range of 1.2 × 10⁻⁶. -7 —3.0×10 -6 A. Immediately add 15 mL of stop solution to each of the two electrolytic cells to stop the etching.

[0057] One embodiment of this application also provides a nanochannel-based urease detection device. The device includes a ZIF-8 modified PET conical nanochannel membrane, two electrolytic cells, and an Ag / AgCl electrode. The two electrolytic cells are interconnected, and the PET conical nanochannel membrane is sandwiched in the interconnecting pathway of the two electrolytic cells. The two electrolytic cells contain electrolytes. One end of the Ag / AgCl electrode is placed in the electrolyte solution, and the other end of the Ag / AgCl electrode is connected to an electrochemical workstation.

[0058] In one embodiment, the PET conical nanochannel membrane is clamped in the passage connecting the two electrolytic cells and fixed with a U-shaped clamp.

[0059] In one embodiment, a tapered nanochannel is etched on the PET nanochannel membrane, the narrow end of the tapered nanochannel having a pore size of 20-200 nm, and ZIF-8 MOF material is deposited on the inner wall of the PET nanochannel and the outer surface of the membrane by electrodeposition, further reducing the channel size to the sub-nanometer scale.

[0060] In one embodiment, the ZIF-8 modified nanochannel utilizes the sub-nanochannels of ZIF-8 itself, with a channel size of 0.386 nm. The nanochannel current after ZIF-8 modification is significantly lower than that of the unmodified PET nanochannel. Figure 3 a)

[0061] In one embodiment, the electrolyte in both electrolytic cells is 50 mL of KCl with a concentration of 100 mM / L to 1 mol / L.

[0062] One embodiment of this application provides the application of the nanochannel-based urease detection device in the detection of bacteria.

[0063] In one embodiment, the bacteria include Streptococcus salivarius, oral saliva mixed bacteria, and Bacillus subtilis.

[0064] Etching of cone-shaped nanochannel membranes

[0065] The apparatus for preparing a cone-shaped nanochannel membrane based on a PET membrane comprises: an unetched PET nanochannel membrane, a U-shaped electrolytic cell, a Pt electrode, and an electrochemical workstation CHI660, wherein the PET cone-shaped nanochannel membrane is sandwiched in the middle of the U-shaped electrolytic cell and fixed with a U-shaped clamp, as shown below. Figure 1 As shown, one side irradiated with heavy ions was filled with 15 mL of etching solution (6M NaOH solution), and the other side was filled with 15 mL of stop solution (a mixed solution of 1M HCOOH and 1M KCl). The outer side was connected to an electrochemical workstation. A Pt electrode was inserted into both solutions and a transmembrane potential of 1V was applied for etching monitoring (it). The size of the etched film pores was controlled by monitoring the current value. Once the current reached the preset value (1.2 × 10⁻⁶), the etching was stopped. -7 —3.0×10 -6 A) Immediately add 15 mL of stop solution to the double-sided electrolytic cell to prevent further enlargement of the membrane pores. Then rinse thoroughly with deionized water to remove residual etching solution and stop solution from the surface of the polyethylene terephthalate (PET) membrane and within the ion channels. Next, replace the double-sided electrolytic cell with a 1 mol / L KCl solution, insert an Ag / AgCl electrode, record the curve using linear sweep voltammetry (LSV), calculate the conductivity, and substitute it into Equation 1 to calculate the pore size etched into the PET tapered nanochannel membrane.

[0066] Urease test

[0067] like Figure 1 As shown, the device consists of a ZIF-8 modified nanoporous membrane (see Figure 1). Figure 2Two electrolytic cells and an Ag / AgCl electrode were used, with the two cells interconnected. A ZIF-8 modified nanoporous membrane was fixedly clamped in the connecting passage between the two electrolytic cells. Each electrolytic cell contained 50 mL of 100 mM KCl electrolyte. One end of the Ag / AgCl electrode was placed in the electrolyte solution, and the other end was connected to an electrochemical workstation. First, urease solutions of 1 U / mL, 10 U / mL, 25 U / mL, and 55 U / mL were prepared, respectively, and dispensed into 1 mL centrifuge tubes and stored at 4°C for easy access. The solutions on both sides of the electrolytic cells were 50 mL of 100 mM KCl. To ensure equal concentration differences between the solutions on both sides of the electrolytic cells, 1 mL of the prepared 1 U / mL, 10 U / mL, 25 U / mL, and 55 U / mL urease solutions were added to each electrolytic cell, respectively. A ZIF-8 modified nanoporous membrane was used as the working electrode (Ag / AgCl) and as the reference electrode (Ag / AgCl) and counter electrode (Pt) on the other side. A transmembrane voltage ranging from -1V to 1V was applied, and the intra-phase (IV) curve was measured to determine its ionic conductivity. Urea at concentrations of 1μM, 3μM, 5μM, 7μM, 9μM, 11μM, and 13μM was added to an electrolytic cell containing urease, and its IV curve was measured to determine the ionic conductivity. The Michaelis constants for different enzyme activities were then determined based on the relationship between conductivity and urea concentration (see [reference]). Figure 3 c and d.

[0068] Example 1: Standard Product Testing

[0069] like Figure 4 As shown in a and 4b, *Streptococcus salivarius* (BNCC 337521) was cultured in BHI broth at 37°C with shaking for 12 h, and counted on BHI plates. After centrifugation, the supernatant was collected and stored at 4°C for later use (no more than 2 weeks). *Streptococcus salivarius* standards at concentrations of 1, 5, 10, and 20 CFU / mL were extracted, and their urease production activity could be rapidly detected using the urease detection method described above. (See [reference]). Figure 4 c. At a constant urea concentration, as the concentration of *Streptococcus salivarius* increases, the urease content increases, urease activity increases, and the amount of urease binding with the substrate urea increases, resulting in the production of OH-. - As the amount of OH increases, the concentration difference across the membrane widens. - As the diffusion rate increases, the change in electrical conductivity also increases.

[0070] Example 2: Saliva Sample Detection

[0071] In healthy individuals, saliva and the gingival sulcus continuously secrete urea. The oral cavity contains *Streptococcus salivati*, which secretes urease. Urea is rapidly hydrolyzed in the oral cavity, and the resulting ammonia increases the pH of the oral environment, thereby altering the dental plaque biofilm and reducing the incidence of tooth decay. The activity of urease produced by *Streptococcus salivati* in patients with dental caries is significantly lower than that in healthy individuals. We collected 0.1 mL of saliva from both healthy individuals and patients with dental caries for testing. Due to the lower urease content in the oral cavity of patients with dental caries, the conductivity change of the hydrolysis reaction between urease and urea was also significantly lower in the caries-affected population compared to the healthy population (P < 0.001). See [link to relevant documentation]. Figure 5 .

[0072] Example 3: Detection of Bacillus subtilis

[0073] like Figure 6 As shown, Bacillus subtilis (BNCC337524) was cultured in nutrient broth at 37°C for 12 h and counted on nutrient agar plates. Then, 10 [units of measurement were extracted]. 6 Using the experimental method described above for constructing a urease model, the Michaelis constant of Bacillus subtilis with a concentration of CFU / mL can be rapidly detected within 5 minutes, and the Michaelis constant is 10.4 μM.

[0074] Control experiment

[0075] The oral cavity of healthy individuals contains *Streptococcus salivans* that secrete urease. We collected 0.1 mL of saliva from healthy individuals for actual sample testing. The results showed that the conductivity change caused by the reaction of urease with urea in healthy individuals was significantly higher than that in patients with dental caries, indicating that their urease activity was much higher. (See [link to relevant documentation]). Figure 5 .

[0076] The above description is merely an exemplary embodiment of the present invention and does not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for detecting urease, characterized in that, Includes the following steps: Step 1: The PET membrane is placed in the passage connecting two electrolytic cells. One side of the PET membrane is etched with sodium hydroxide solution. The etched side of the PET nanochannel membrane is then irradiated with heavy ions. A 1 mol / L KCl solution is added to both electrolytic cells, and an Ag / AgCl electrode is inserted. The linear sweep voltammetry (LSV) curve is recorded, and the conductivity is calculated. The pore size of the PET membrane is calculated using formula (1). D tip ; （1） in, L Represents PET thickness; K This represents the conductivity of a 1 M KCl bulk solution. This represents the fixed diameter of the track after heavy ion irradiation of PET; N The average number of nanopores representing the etched area; G The conductivity value represents that measured for nanoporous membranes; Step 2, MOF modification process: The etched PET film is sputtered with gold, and then the treated PET film is sandwiched in the passage connecting two electrolytic cells. 50 ml of precursor solution is added to each electrolytic cell, and cathodic deposition is performed at a certain current density. After deposition is completed, the surface of the ZIF-8 modified film is rinsed with deionized water and methanol, and the ZIF-8 modified film is placed in a vacuum drying oven to dry overnight to obtain a ZIF-8 modified sub-nano porous film. Step 3: Place the ZIF-8 modified sub-nanoporous membrane in the passage connecting two electrolytic cells, and then add 5 U / mL, 25 U / mL and 55 U / mL urease to the two electrolytic cells respectively in a quantitative manner. After each addition, perform LSV scanning to measure the ionic conductivity of the ZIF-8 modified sub-nanoporous membrane. Step 4: Add 3 μM, 5 μM, 7 μM, 9 μM, and 11 μM urea sequentially to an electrolytic cell containing urease solution, performing an LSV scan after each addition. The urea in the electrolytic cell is decomposed by urease, producing OH-. - Diffusion through changes in conductivity of ZIF-8 modified sub-nanoporous membranes; Step 5: Input the change in conductivity across the nanochannel after urease decomposes urea into the pre-determined Michaelis constant working curve and read the Michaelis constant of urease.

2. The detection method according to claim 1, characterized in that, The precursor solution is a mixture of 0.85 mol / L 2-MIM and 0.014 mol / L Zn(Ac)2.

3. The detection method according to claim 1, characterized in that, The formula for calculating the Michaelis constant is shown in the Michaelis equation of formula (2). （2） Where v represents the product formation rate; V represents the maximum reaction rate of the enzyme; K m Michaelis constant, representing the enzyme; [A] represents the concentration of the substrate; Based on the Michaelis equation, the relationship between the change in conductivity ΔG and the Michaelis constant and maximum reaction rate of the enzyme was calculated, as shown in formula (3). （3） in, c Indicates the concentration of urea; The apparent Michaelis constant of urease is expressed; experimental data are presented in... The x-axis is... Using the vertical axis, formula (3) is simplified to formula (4). （4） do and The relationship curve between the two, where the slope of the curve is equal to the intercept, represents the apparent Michaelis constant of the enzyme-catalyzed reaction. .

4. The detection method according to claim 1, characterized in that, In step 1, the PET nanochannel membrane is etched with sodium hydroxide solution. One side of the PET nanochannel membrane, which has been irradiated with heavy ions, is filled with 15 mL of 6 M NaOH solution as an etching solution, and the other side of the PET conical nanochannel membrane is filled with 15 mL of a mixed solution of 1 M HCOOH and 1 M KCl as a stop solution. The outer side is connected to an electrochemical workstation. A Pt electrode is inserted into the solutions on both sides and a transmembrane potential of 1 V is applied for etching monitoring. The size of the etched membrane pores is controlled by monitoring the current value. Then, the membrane is rinsed with deionized water to remove the residual etching solution and stop solution on the surface of the polyethylene terephthalate membrane and inside the ion channels.

5. The detection method according to claim 4, characterized in that, The method of controlling the size of the etched film apertures by monitoring the current value is defined as follows: when the current value reaches a preset range of 1.2 × 10⁻⁶... -7 —3.0×10 -6 A. Immediately add 15 mL of stop solution to each of the two electrolytic cells to stop the etching process.

6. A urease detection device for implementing the detection method as described in any one of claims 1-5, characterized in that, The device includes a ZIF-8 modified PET nanochannel membrane, two electrolytic cells, and an Ag / AgCl electrode. The two electrolytic cells are interconnected, and the ZIF-8 modified PET nanochannel membrane is fixedly clamped in the connecting passage between the two electrolytic cells. The two electrolytic cells contain electrolytes. One end of the Ag / AgCl electrode is placed in the electrolyte solution, and the other end of the Ag / AgCl electrode is connected to an electrochemical workstation.

7. The detection device according to claim 6, characterized in that, The PET nanochannel membrane is etched with tapered nanochannels, the narrow end of which has a diameter of 0.1 to 0.9 nm.

8. The detection device according to claim 6, characterized in that, The electrolyte in both electrolytic cells is KCl.

Images