A photoelectrochemical biosensor for detecting urease and its preparation method

Abstract

The present invention relates to a photoelectrochemical biosensor for detecting urease and a preparation method thereof. The photoelectrochemical biosensor adopts a three-electrode system, with an ITO electrode modified with a Y6-PM6 composite material as a working electrode, an Ag / AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode. The photoelectrochemical biosensor for detecting urease provided by the present invention uses ITO / Y6-PM6 as an electrode material, and the concentration of urease is within 10-3000mU / mL. The photocurrent of the sensor shows a good linear relationship with the concentration of urease, and the optical properties such as the specificity and stability of the sensor also reach a high level with good reproducibility. In addition, the ITO / Y6-PM6-based PEC sensor has a fast response, simple equipment, and is easy to miniaturize. It is suitable for detecting urease activity in various occasions and has good application prospects in the fields of clinical diagnosis, food science, and environmental analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of testing or analyzing materials by means of measuring the chemical or material properties of the materials, and particularly relates to a photoelectric chemical biosensor for detecting urease and a preparation method thereof. Background Art

[0002] Urease is a nickel-containing oligoenzyme that specifically and efficiently catalyzes the hydrolysis of urea to produce ammonia and carbon dioxide. In medicine, bacterial urease is a significant pathogenic factor, contributing to numerous diseases such as gastritis, pyelonephritis, ammonia encephalopathy, hepatic coma, peptic ulcers, urinary catheter obstruction, and infected urinary stones. In agriculture, excessive soil urease activity rapidly decomposes urea in fertilizers into ammonia, which is released into the atmosphere, causing economic losses and environmental pollution. Furthermore, soy products such as soy milk and milk replacer containing urease can cause diarrhea and other symptoms after consumption. Therefore, urease detection is of great significance in fields such as medicine, agronomy, and food production.

[0003] Currently, a variety of technologies have been developed for the quantitative or qualitative determination of urease, including volumetric methods, near-infrared spectroscopy, electrochemical methods, and spectrophotometry. These methods often suffer from drawbacks such as expensive instrumentation, complex sample pretreatment, a narrow range of applications, significant background noise interference, low sensitivity, and time-consuming assays. These methods present limitations and cannot rapidly and accurately detect the analyte. Therefore, the development of new, convenient, sensitive, accurate, and safe detection technologies for the sensitive detection of urease is urgently needed. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a photoelectrochemical biosensor for detecting urease and a preparation method thereof, so as to achieve rapid and highly sensitive detection of urease.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] Provided is a photoelectrochemical biosensor for detecting urease. The photoelectrochemical biosensor adopts a three-electrode system, with an ITO electrode modified with a Y6-PM6 composite material as a working electrode, an Ag / AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode.

[0007] The present invention also includes a method for preparing the above-mentioned photoelectrochemical biosensor for detecting urease, wherein the steps of preparing the working electrode are as follows:

[0008] 1) adding acetone (as an auxiliary stabilizer) dropwise to chloroform and mixing uniformly to obtain a composite solvent;

[0009] 2) dissolving Y6 in the composite solvent obtained in part of step 1) to obtain a Y6 solution, dissolving PM6 in the mixed solvent obtained in part of step 1) to obtain a PM6 solution, then mixing the Y6 solution with the PM6 solution to obtain a Y6-PM6 mixed solution, spin-coating the Y6-PM6 mixed solution on a clean ITO glass, and drying to obtain an ITO electrode modified with a Y6-PM6 composite material (ITO / Y6-PM6), further preparing a photoelectrochemical biosensor for detecting urease.

[0010] According to the above scheme, the chloroform and acetone in step 1) are both analytically pure, and the volume percentage of acetone in the composite solvent is 1-3%.

[0011] According to the above scheme, the Y6 concentration in the Y6 solution in step 2) is 2-5 mg / mL.

[0012] According to the above scheme, the PM6 concentration in the PM6 solution in step 2) is 2-5 mg / mL.

[0013] According to the above scheme, the mass ratio of Y6 to PM6 in the Y6-PM6 mixed solution in step 2) is 0.8-1.2:1.

[0014] According to the above scheme, in step 2), the Y6-PM6 mixed solution is spin-coated on the ITO glass at a spin-coating speed of 800-1500 rpm.

[0015] The present invention also includes the use of the aforementioned photoelectrochemical biosensor for detecting urease. Urea is exogenously added to the system being detected, and the photoelectrochemical biosensor detects the photocurrent signal in the system. When urease is present in the system being detected, the urease hydrolyzes the urea to produce ammonia. The alkaline environment destroys the Y6 molecular structure, causing a change in the detection signal. The change in the current signal can then be used to monitor the urease concentration. Because the change in the detection signal is generated by the unique biocatalytic reaction of urease, the detection of urease is highly sensitive and selective.

[0016] The present invention prepares an electrode modified with Y6 and PM6 as optically active materials. Y6 has a narrow band gap, which is conducive to electron transition and promotes the effective separation and transport of charges. This plays an important role in increasing the current of the photoelectrochemical sensor and improving its sensitivity. The combination of Y6 and PM6 can promote the transfer of photoelectrons to each other. In addition, as an organic semiconductor with excellent performance, the terminal group of Y6 will destroy the molecular conjugated structure after combining with hydroxide, thereby causing the signal of its photoelectrochemical sensor to weaken. Based on this, the present invention uses Y6-PM6 as a signal converter between the concentration of the detected substance urease and the photocurrent for the first time to achieve sensitive detection of urease. The ammonia produced by the hydrolysis of urea by urease is electrolyzed to produce hydroxide, which reacts with Y6 loaded on indium tin oxide, thereby affecting its electron transmission and realizing the change of the photoelectric signal. The photoelectrochemical sensor based on Y6-PM6 of the present invention shows good detection accuracy and significant stability for urease.

[0017] The beneficial effects of the present invention are: 1. The photoelectrochemical biosensor for detecting urease provided by the present invention uses ITO / Y6-PM6 as the electrode material, and the concentration of urease is within the range of 10-3000mU / mL. The photocurrent of the sensor shows a good linear relationship with the concentration of urease, and the optical properties of the sensor, such as specificity and stability, also reach a high level with good reproducibility. In addition, the ITO / Y6-PM6-based PEC sensor has a fast response, simple equipment and easy miniaturization, and is suitable for detecting urease activity in various occasions, and has good application prospects in the fields of clinical diagnosis, food science and environmental analysis; 2. The preparation method of the present invention has simple steps, mild reaction conditions, low cost, and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The mass spectra of the Y6 solution before and after the reaction with hydroxide in Example 1 of the present invention are shown;

[0019] Figure 2 Absorption spectra of the ITO / Y6-PM6 electrode and ITO / Y6-PM6-OH prepared in Example 1;

[0020] Figure 3 This is a photocurrent test graph of the photoelectrochemical biosensor prepared in Example 1 to different concentrations of urease;

[0021] Figure 4 is a linear relationship diagram between the photocurrent intensity and the urease concentration in Example 1;

[0022] Figure 5 This is a selectivity test diagram of the photoelectrochemical biosensor prepared in Example 1 for detecting urease. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings.

[0024] Example 1

[0025] A photoelectrochemical biosensor for detecting urease, wherein the photoelectrochemical biosensor adopts a three-electrode system, with an ITO electrode modified with a Y6-PM6 composite material as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The steps for preparing the working electrode are as follows:

[0026] 1) adding acetone (analytical grade) dropwise to chloroform (analytical grade) to obtain a composite solvent, wherein the volume percentage of acetone in the composite solvent is 1%;

[0027] 2) Weigh 5 mg of Y6 and 5 mg of PM6, dissolve each in 1 mL of the complex solvent prepared in step 1), stir thoroughly in the dark for 24 h to obtain a Y6 solution and a PM6 solution, and then mix the obtained Y6 solution with the PM6 solution to obtain a Y6-PM6 mixed solution;

[0028] 3) An ITO glass (1 cm × 2 cm) was cleaned with ultrapure water, ethanol, and acetone in sequence under ultrasonication and dried at 60°C overnight. Then, 10 μL of the Y6-PM6 mixed solution obtained in step 2) was spin-coated on the ITO glass at a spin coating speed of 1000 rpm. The actual coating area was 0.071 cm 2 , dried at room temperature, and the obtained photoelectrode was named ITO / Y6-PM6.

[0029] The above-mentioned ITO / Y6-PM6 was used as a working electrode, an Ag / AgCl electrode was used as a reference electrode, and a platinum wire electrode was used as a counter electrode to obtain a photoelectrochemical biosensor.

[0030] 5 mg of Y6 was weighed and dissolved in 1 mL of the complex solvent prepared in step 1) of this example. After sufficient stirring for 24 h in the dark, a Y6 solution was obtained. A base (100 μL of a 0.1 mol / L NaOH solution) was added to the obtained Y6 solution. The mass spectra of the Y6 solution before and after the reaction with hydroxide were measured (see Figure 1 ),from Figure 1 It can be clearly seen that the relative atomic mass difference between Y6 and Y6-OH is 36, which is exactly the sum of the relative atomic masses of the two hydroxyl groups and the two hydrogen atoms on the other side of the carbon-carbon double bond, confirming that Y6 can undergo the theoretically predicted addition reaction under alkaline conditions.

[0031] The ITO / Y6-PM6 electrode and ITO / Y6-PM6-OH (prepared by adding 0.1 mol / L NaOH solution to the surface of the ITO / Y6-PM6 electrode and allowing it to react) prepared in this example were subjected to absorption tests under the same conditions. The absorption spectra are shown in FIG. Figure 2 As shown, it can be found that ITO / Y6-PM6 has obvious absorption peaks at 420nm, 620nm, and 840nm. The absorption peaks of ITO / Y6-PM6-OH at 420nm and 620nm are retained, which are absorption peaks belonging to PM6 and remain stable in an alkaline environment. The absorption peak at 840nm disappears significantly, which is an absorption peak belonging to Y6 and disappears after the reaction, indicating that hydroxide has a significant impact on its performance. This is also consistent with Figure 3 Therefore, Y6-PM6 was selected to construct a photoelectrochemical sensor to measure the urease concentration, which is conducive to the construction of a photoelectrochemical biosensor with signal amplification and high sensitivity.

[0032] The photoelectric chemical biosensor prepared in this example was tested for the photocurrent of different concentrations of urease (10-3000 mU / mL). Urease hydrolysates (urease is obtained by reacting in the presence of urea to produce hydroxide) at different concentrations were directly dropped onto the surface of the working electrode for reaction. The photocurrent test graph is shown in FIG. Figure 3 As shown in the figure, the results show that as the urease concentration increases, the photocurrent signal gradually decreases. Therefore, a good linear relationship can be established between the photocurrent intensity and the urease concentration. The linear relationship diagram between the photocurrent intensity and the urease concentration is obtained by fitting. Figure 4 As shown, the linear regression equation is y = -0.228C urease +901.194, correlation coefficient R 2 =0.99, and the detection limit was 8.6 mU / mL (S / N=3).

[0033] In actual detection, the photocurrent may react to other substances and change, so the selectivity of the photoelectrochemical biosensor prepared in this example to urease was tested. First, the photoelectrochemical biosensor was tested for urease solution (Urease, 100mUmL -1 ) photocurrent, and then with common small molecules lactic acid (LAC, 100 μM), uric acid (UA, 100 μM), glucose (Glucose, 100 μM), lactose (Lactose, 100 μM), histidine (HIS, 100 μM), phenylalanine (Phe, 100 μM), valine (L-serine, 100 μM), and glucose oxidase (Gox, 100 mU mL -1 ) and other interfering substances to replace urease and test the changes in the photocurrent of the solution. Figure 5As shown: Compared with urease, the photocurrent inhibition of several interferences is negligible, indicating that the photoelectrochemical biosensor based on ITO / Y6-PM6 electrode has good selectivity for urease.

[0034] In summary, the photoelectrochemical biosensor with ITO electrode modified with Y6-PM6 composite material as the working electrode has the characteristics of wide linear range, low detection limit, high sensitivity and good selectivity in the detection of urease, and has good application prospects.

Claims

1. Application of a photoelectrochemical biosensor in detecting urease, characterized in that: The photoelectrochemical biosensor adopts a three-electrode system, with an ITO electrode modified with a Y6-PM6 composite material as a working electrode, an Ag / AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode.

2. Use of the photoelectrochemical biosensor according to claim 1 for detecting urease, characterized in that: The steps of preparing the working electrode are as follows: 1) Acetone is added dropwise to chloroform and mixed evenly to obtain a composite solvent; 2) dissolving Y6 in the composite solvent obtained in part of step 1) to obtain a Y6 solution, dissolving PM6 in the mixed solvent obtained in part of step 1) to obtain a PM6 solution, then mixing the Y6 solution and the PM6 solution to obtain a Y6-PM6 mixed solution, spin-coating the Y6-PM6 mixed solution on a clean ITO glass, and drying to obtain an ITO electrode modified with the Y6-PM6 composite material, and further preparing a photoelectrochemical biosensor for detecting urease.

3. Use of the photoelectrochemical biosensor according to claim 2 for detecting urease, characterized in that: In step 1), the chloroform and acetone are both analytically pure, and the volume percentage of acetone in the composite solvent is 1-3%.

4. Use of the photoelectrochemical biosensor according to claim 2 for detecting urease, characterized in that: Step 2) The Y6 concentration in the Y6 solution is 2-5 mg / mL.

5. Use of the photoelectrochemical biosensor according to claim 2 for detecting urease, characterized in that: Step 2) The PM6 concentration in the PM6 solution is 2-5 mg / mL.

6. Use of the photoelectrochemical biosensor according to claim 2 for detecting urease, characterized in that: Step 2) The mass ratio of Y6 to PM6 in the Y6-PM6 mixed solution is 0.8-1.2:

1.

7. Use of the photoelectrochemical biosensor according to claim 2 for detecting urease, characterized in that: Step 2) Spin-coat the Y6-PM6 mixed solution on the ITO glass at a spin-coating speed of 800-1500 rpm.

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