A method for preparing a pepsin sensor electrode

Abstract

The present application belongs to the field of electrochemistry, and relates to a preparation method of a pepsin sensing electrode. The pepsin sensing electrode is prepared by a polyol reduction method. Specifically, polyvinylpyrrolidone (PVP) and silver nitrate are dissolved in a glycol solvent to prepare a reaction solution, and then a hydrothermal synthesis is performed. After centrifugation and cleaning, silver nanowire precipitates are obtained, which are dispersed in an ethanol solution containing 1,4-benzenedithiol, and then incubated. After centrifugation and cleaning, the silver nanowire precipitates are dispersed in a cobalt nitrate aqueous solution, and then ammonia water is added dropwise. After hydrothermal reaction and centrifugation, a cobalt hydroxide / silver nanowire composite material is obtained, which is dispersed in a mixed solution of ultrapure water and chitosan to obtain a composite material mixed solution. The composite material mixed solution is drop-coated on a bare gold working electrode, and then dried at room temperature to obtain the electrode. The electrode is modified by the cobalt hydroxide / silver nanowire composite material with high conductivity and high catalytic activity, and pepsin in bronchoalveolar lavage fluid is used as a detection marker, so that high-sensitivity and high-specificity detection of aspiration pneumonia is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemistry, and relates to a preparation method of a pepsin sensing electrode. BACKGROUND

[0002] Pepsin is a proteolytic enzyme produced in the digestive system, which is converted from pepsinogen in the acidic environment of the stomach through autolysis, and is also one of the main harmful factors of gastric reflux. According to research, diseases such as aspiration pneumonia and throat reflux are closely related to the presence of pepsin, so pepsin detection has become the focus of research on disease markers, disease mechanisms, and drug development. Aspiration pneumonia refers to the inhalation of oral pharyngeal or gastric contents and other irritating substances into the larynx, trachea, bronchus and lower respiratory tract, causing chemical pneumonia, and in severe cases, even respiratory failure. Due to the synergistic effect of the epiglottis and swallowing protective reflex, food and foreign matter are not easy to enter the lower respiratory tract, and a small amount of liquid can also be expelled by coughing. However, when there are esophageal diseases such as esophageal achalasia, upper esophageal cancer, etc., food swallowed cannot all enter the stomach and will reflux into the trachea, especially in the case of increased incidence of cerebrovascular and degenerative nervous system diseases in the elderly, difficulty in swallowing and impaired cough reflex, aspiration can easily lead to aspiration pneumonia. The severity of aspiration pneumonia is related to the concentration of acid inhaled gastric juice, the amount of aspiration, and the distribution in the lungs. Generally, aspiration of gastric acid with pH<2.5 can severely damage lung tissue. After aspiration of gastric contents, gastric acid stimulates the bronchus, causing severe bronchospasm, followed by acute inflammatory response of bronchial epithelium and infiltration of inflammatory cells around the bronchus, and the gastric juice entering the alveoli rapidly spreads to the surrounding lung tissue, the alveolar epithelial cells are damaged and the capillary wall is involved, the vascular permeability increases, the alveolar capillary wall is damaged, and interstitial pulmonary edema is formed. Pulmonary edema and hemorrhage in the alveoli are gradually absorbed and a transparent membrane is formed after a few days, then acute and chronic fibrosis occurs. If not controlled in time, severe cases can lead to respiratory failure and even heart failure. The current diagnostic methods for aspiration pneumonia mainly include general laboratory tests, imaging tests and histological examination of cases. These methods either require expensive and bulky instruments, take a long time to detect, are complex to operate, or have side effects on the human body, and the diagnosis is already obvious. Therefore, how to achieve safe and efficient detection and early prevention and control of aspiration pneumonia is a problem faced by the medical industry. In recent years, electrochemical sensing has received more and more attention in cancer detection. Through the specific recognition between biomolecules, the target molecule is converted into an electrical signal such as capacitance, current, potential, conductivity, etc., thereby realizing rapid and accurate detection of the target analyte. Pepsin does not exist in normal respiratory samples, and when a patient has oropharyngeal aspiration, pepsin is detected in bronchoalveolar lavage fluid, which can assist in the diagnosis of the presence of gastroesophageal reflux disease. However, there is no ideal electrochemical sensor reported for rapid and effective detection of pepsin. SUMMARY

[0003] The present application aims at the problems existing in the traditional inhalation pneumonia detection method, and proposes a pepsin detection method based on cobalt hydroxide / silver nanowire composite material. The purpose of the present application is to prepare a pepsin sensing electrode for the detection of inhalation pneumonia markers.

[0004] In order to achieve the above purpose, the present application is realized by using the following technical scheme:

[0005] A preparation method of a pepsin sensing electrode, the steps are as follows:

[0006] (1) Preparation of silver nanowire

[0007] A polyol reduction method is used, with ethylene glycol as a solvent and a reducing agent, and polyvinylpyrrolidone (PVP) as a dispersant. The polyvinylpyrrolidone is dissolved in ethylene glycol, and after the solution is clear, sodium chloride ethylene glycol solution is added to obtain solution A. Silver nitrate is dissolved in ethylene glycol to obtain solution B. Then solution B is added to solution A in batches, and stirring is continuously carried out to obtain a reaction liquid. The reaction liquid is transferred into a hydrothermal kettle for hydrothermal synthesis reaction to obtain a silver nanowire solution. Centrifugation and washing are carried out to obtain a silver nanowire precipitate.

[0008] (2) Preparation of cobalt hydroxide / silver nanowire composite material

[0009] The silver nanowire precipitate is dispersed in ethanol to obtain dispersion liquid C. 1,4-benzenedithiol ethanol solution is added to dispersion liquid C, and a 1,4-benzenedithiol rigid self-assembled film is obtained by standing at room temperature. After centrifugation and washing, the film is dispersed in deionized water to obtain dispersion liquid D. Cobalt nitrate solution is added to dispersion liquid D to obtain mixture E. After stirring uniformly, ammonia water is added dropwise, and the mixture is transferred into a hydrothermal kettle for hydrothermal synthesis reaction to obtain a cobalt hydroxide / silver nanowire composite material solution. Centrifugation and washing are carried out to obtain a composite material precipitate.

[0010] (3) Preparation of sensing electrode

[0011] The composite material precipitate is added to a chitosan suspension liquid, and mixed uniformly to obtain a mixture. The bare gold working electrode is washed with ultrapure water, and the mixture is added dropwise to the surface of the working electrode. After drying at room temperature, the working electrode is washed with ultrapure water to obtain a sensing electrode.

[0012] As preferred, the volume ratio of ethylene glycol to silver nitrate in step (1) is 4:1, the concentration of polyvinylpyrrolidone in solution A is 6-20 g / L, the concentration of silver nitrate in solution B is 24-80 g / L, the concentration of sodium chloride ethylene glycol solution is 0.2-0.6 M, the volume ratio of sodium chloride ethylene glycol solution to solution A is 1:(35-160), the hydrothermal synthesis temperature of the reaction solution is 160-200 DEG C, and the hydrothermal synthesis time is 1-3 h.

[0013] As preferred, the silver nanowire solution in step (1) is centrifuged once with isopropanol, once with ethanol, and twice with deionized water, and the centrifugal conditions are all at a speed of 8000 r / min for 6 min, and the silver nanowire precipitate is obtained by pouring off the supernatant after centrifugation.

[0014] As preferred, the concentration of 1,4-benzenedithiol ethanolic solution in step (2) is 1-5 mM, the standing time at room temperature is 6-24 h, the concentration of cobalt nitrate solution is 1-5 mM, the volume ratio of dispersion C to ethanol in 1,4-benzenedithiol ethanolic solution is 1:1, the volume fraction of ammonia is 25%, the volume ratio of deionized water: cobalt nitrate solution: ammonia in dispersion D is 1:1:0.006, the hydrothermal synthesis reaction temperature is 120-200 DEG C, and the hydrothermal synthesis time is 1-9 h, and then the product is washed with deionized water at a centrifugal speed of 8000 r / min for 6 min.

[0015] As preferred, the mass fraction of chitosan in the chitosan suspension in step (3) is 1-5%, the mass concentration of the composite material precipitate in the mixed solution is 6-15 mg / L, the drop coating amount is 2-5 μL, and the room temperature drying time is 6-24 h.

[0016] The application provides application of the sensing electrode prepared by the above method in a pepsin detection instrument and a detection method.

[0017] The application provides a pepsin detection sensor based on a cobalt hydroxide / silver nanowire composite material, which comprises a pepsin sensing electrode and a detection marker, the pepsin electrode takes a gold electrode as a working electrode, a platinum wire electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode, wherein the surface of the working electrode is covered with a cobalt hydroxide / silver nanowire composite material, and the pepsin marker is a pepsin (PP) ovalbumin conjugate (which can be commercially purchased).

[0018] The cobalt hydroxide / silver nanowire composite material of the present application needs to strictly control the hydrothermal synthesis time in the synthesis process, the PVP molecules will cover the crystal surface of the seed, and only a part of the crystal surface is covered, so that the crystal surface is easy to adsorb the reduced silver to slowly grow into a silver nanowire, and the hydrothermal synthesis condition will affect the morphology such as thickness and length of the silver nanowire, and then affect the binding site of the subsequent cobalt hydroxide growth. Controlling the hydrothermal synthesis time can regulate the thickness and length of the silver nanowire and the growth morphology of the cobalt hydroxide crystal. The appropriate hydrothermal synthesis time can make the cobalt hydroxide crystal grown on the surface of the nanowire more uniform, and avoid the occurrence of agglomeration phenomenon. In the sensing modification material, the silver nanowire has high conductivity, so that the sensor has small electron transfer resistance in the detection process, shortens the response time, and the cobalt hydroxide can make the signal peak of the material in the detection process more obvious, which is more conducive to the presentation of detection performance. The high length-diameter ratio of the silver nanowire is beneficial to form more Ag-S bonds to fix the 1,4-benzenedithiol molecules, and the 1,4-benzenedithiol further provides a guiding ability for the growth of cobalt hydroxide, so that it grows uniformly and directionally on the nanowire; the cobalt hydroxide has good catalytic activity and can better realize the biosensing detection. The combination of the two materials solves the problems of low sensitivity and poor conductivity existing in electrochemical sensing. The antigen-antibody specific binding mode of the sensor prepared by the present application can produce a sensing signal for a very small marker, realizing trace detection.

[0019] Compared with the prior art, the present application has the advantages and positive effects that:

[0020] The present application uses the cobalt hydroxide / silver nanowire composite material with high conductivity and high catalytic activity as the electrode modification material, and uses pepsin in bronchoalveolar lavage fluid as the detection marker, to realize high sensitivity and high specificity detection of aspiration pneumonia. In addition, the detection object of the sensor is the marker extracted from the bronchoalveolar lavage fluid, the detection effect is good, the harm to the human body during detection is small, the detection meets the requirements of clinical pepsin detection, and is suitable for large-scale clinical detection application and promotion, providing a new idea for early screening of aspiration pneumonia in the medical field. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The scanning electron microscope image of the silver nanowire synthesized by the hydrothermal method in Example 1.

[0022] Figure 2 The scanning electron microscope image of the cobalt hydroxide / silver nanowire composite material synthesized by the hydrothermal method in Example 1.

[0023] Figure 3 The TEM scanning image of the cobalt hydroxide / silver nanowire composite material synthesized by the hydrothermal method in Example 1.

[0024] Figure 4DPV scan chart of the pepsin sensor prepared in Example 1 for different concentrations of the marker. DETAILED DESCRIPTION

[0025] In order to enable a more complete understanding of the above-mentioned objects, features and advantages of the present application, the application will be described in further detail below with reference made to specific embodiments. It is to be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict, if necessary.

[0026] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in a variety of ways beyond the specific embodiments described and should not be limited to the specific embodiments disclosed in the following description.

[0027] Unless otherwise specified, the preparation process of the pepsin electrode in the following examples is as follows: the prepared composite material mixed solution is drop-coated on a bare gold working electrode, and dried at room temperature. Then the electrode is immersed in the activated pepsin monoclonal antibody solution, so that the antibody is grafted onto the material through an amide bond, and then placed in a refrigerator for incubation at 4°C for 6h. Then the unbound antibody is washed away, and the working electrode is sealed with a 1% BSA solution and stored in a refrigerator at 4°C. Figure 4 The DPV characterization method is as follows: first, prepare a 0.1 M KCl, 2 mM [Fe(CN)6] 3− / 4− solution, immerse the three electrodes in the electrolytic cell filled with the solution, connect the working electrode, the reference electrode and the counter electrode sealed with a 1% BSA solution to the interface of the electrochemical workstation, change the amount of the marker by adding different concentrations of the marker into the electrolytic cell, finally set the potential window to 0-0.4V, and perform DPV scanning under different concentrations of the marker. The current signal generated by the hybrid reaction is received by the electrochemical workstation.

[0028] The pepsin monoclonal antibody is purchased from Wuhan Yunklon Technology Co., Ltd. The pepsin (PP) ovalbumin conjugate purchased as a set is used to prepare solutions of various concentrations. The concentration range of the marker detected in the following examples is 10 -12 -10 -7 M, and the standard addition method is used to detect from low concentration to high concentration.

[0029] Example 1

[0030] This embodiment provides a preparation method of a pepsin sensor based on a cobalt hydroxide / silver nanowire composite material, and the steps are as follows.

[0031] (1) Take 0.35 g PVP dissolved in 40 mL ethylene glycol, after the solution is clear, add 600 μL of 0.2 M concentration of NaCl-EG solution to it, get solution A, dissolve 0.45 g silver nitrate in 10 mL ethylene glycol to get solution B, then add solution B to solution A for more than 5 times, each time no more than 2 mL, and constantly stirring, get the reaction liquid, transfer into the hydrothermal kettle 190 ℃ hydrothermal synthesis reaction 1 h, after the reaction, centrifugal, pour off the upper liquid, leave the solid, then use isopropyl alcohol centrifugal washing once, use ethanol centrifugal washing once, and use deionized water centrifugal washing twice, the centrifugal conditions are all 8000 r / min, centrifugal time 6 min, after centrifugal, pour off the upper liquid to get silver nanowire precipitate. The silver nanowire precipitate microstructure is shown in the TEM picture as shown in Figure 1 The silver nanowire precipitate microstructure is shown in the TEM picture as shown in Figure 1 It can be seen that the synthesized silver nanowire presents uniform linear structure.

[0032] (2) Disperse all the silver nanowire precipitate obtained in step (1) in 10 mL ethanol to get the dispersion liquid, take 0.02 g of 1,4-benzenedithiol dissolved in 10 mL ethanol solution, after dissolving, add to the dispersion liquid, incubate at room temperature for 6 h, use deionized water to centrifugal twice to clean the unbound 1,4-benzenedithiol molecules. Pour off the upper liquid, disperse the obtained silver nanowire precipitate with self-assembled film on the surface in 10 mL deionized water to get the dispersion liquid, add 10 mL of 1 mM concentration of cobalt nitrate solution to the dispersion liquid, after stirring uniformly, add 60 μL of 25% ammonia water, the dropwise adding time is controlled within half a minute, then transfer into the hydrothermal kettle, hydrothermal reaction at 120 ℃ for 7 h, after the reaction, centrifugal, remove the supernatant, add deionized water to the precipitate and centrifugal wash 3 times. The centrifugal speed of this step is all 8000 r / min, the centrifugal time is all 6 min, after centrifugal, pour off the upper liquid to get the cobalt hydroxide / silver nanowire composite material precipitate. The cobalt hydroxide / silver nanowire composite material precipitate microstructure is shown in the TEM picture as shown in Figure 2 and Figure 3 The cobalt hydroxide / silver nanowire composite material precipitate microstructure is shown in the TEM picture as shown in Figure 2 It can be seen that the cobalt hydroxide nanoparticles are attached to the surface of the nanowire; the TEM result of Figure 3 shows that the cobalt hydroxide is evenly distributed on the surface of the silver nanowire.

[0033] (3) The composite material prepared in step (2) was dispersed in a chitosan suspension with a mass fraction of 1% to prepare a dispersion with a composite material mass fraction of 6 mg / L. 2 μL was dropped on the gold electrode and dried at room temperature for 12 h to obtain a sensing electrode. 0.03 g of EDC powder was dissolved in 0.5 mL of deionized water and 0.0514 g of NHS powder was dissolved in 10 mL of deionized water. 10 μL of each was taken and mixed with 5 μL of 1 mg / mL gastric egg yolk paste. The pepsin (PP) monoclonal antibody solution was mixed and activated for 1 hour. 2 μL of the mixture was added dropwise to the sensing electrode to graft the PP monoclonal antibody onto the material. The mixture was incubated at 5-8°C for 24 hours. The unbound antibody was then rinsed off with PBS solution (pH = 7.4). 2 μL of 1% bovine serum albumin solution was then added dropwise. The mixture was incubated for 2 hours and then rinsed with PBS solution (pH = 7.4) to prepare a pepsin sensing electrode, which was stored at 5-8°C.

[0034] (4) The gold electrode after the above treatment was used as the working electrode, the platinum wire electrode as the counter electrode, and the Ag / AgCl electrode as the reference electrode. Differential pulse voltammetry (DPV) was used to detect markers of different concentrations. A fixed concentration was selected for hybridization time investigation. The test was performed every five minutes until the DPV curve stabilized, which was the response time of the sensor to the marker. According to the test results, the detection response time of the aptamer sensor to the marker was 25 minutes. The detection linear range of the marker was investigated, and the results showed that the detection limit reached 1*10 -12 This indicates that the sensor has a faster response time and a lower detection limit.

[0035] Depend on Figure 1 The SEM characterization shows that the silver nanowires synthesized in step (1) have a good linear structure and the thickness of the nanowires is relatively uniform, which is convenient for the subsequent growth of cobalt hydroxide.

[0036] Depend on Figure 2 The SEM characterization showed that in the cobalt hydroxide / silver nanowire composite material synthesized in step (2), cobalt hydroxide successfully grew on the surface of the nanowires.

[0037] Depend on Figure 3 TEM characterization shows that in the composite material synthesized in step (2), cobalt hydroxide is uniformly attached to the surface of the nanowires, which is beneficial to improving the redox ability of the material.

[0038] Depend on Figure 4 It can be seen from the DPV characterization that the prepared pepsin sensor has a wide detection range and a low detection limit, which is beneficial to the trace detection of pepsin.

[0039] The standard addition method was used to prepare three concentrations of marker samples in the bronchoalveolar lavage fluid (provided by Zhongda Hospital Affiliated to Southeast University) for real sample detection. The results of real sample detection are shown in Table 1.

[0040] Table 1. Results of real sample detection

[0041]

[0042] As can be seen from the results of real sample detection in Table 1, the sensor has high consistency between the detection results of real samples and the theoretical calculation results by using the standard addition method to prepare three concentrations of marker samples in the bronchoalveolar lavage fluid, indicating that the pepsin sensor has extremely high accuracy in the detection of aspiration pneumonia.

[0043] Example 2

[0044] This embodiment provides a pepsin detection method based on a cobalt hydroxide / silver nanowire composite material. The embodiment not specifically explained herein is consistent with Example 1. The steps are as follows.

[0045] (1) 0.45 g of PVP was dissolved in 40 mL of ethylene glycol, and after the solution was clear, 600 μL of 0.2M NaCl-EG solution was added to obtain solution A. 0.45 g of silver nitrate was dissolved in 10 mL of ethylene glycol to obtain solution B. Then, solution B was added to solution A in batches, and continuously stirred to obtain a reaction solution. The reaction solution was transferred into a hydrothermal kettle for hydrothermal synthesis reaction at 180°C for 2 h. After the reaction was completed, the upper liquid was poured out, and the solid was left. Then, the silver nanowire precipitate was centrifuged once with isopropyl alcohol, once with ethanol, and twice with water. The centrifugation conditions were all at a speed of 8000 r / min for 6 min. After the centrifugation was completed, the upper liquid was poured out to obtain the silver nanowire precipitate.

[0046] (2) The silver nanowire precipitate obtained in step (1) was dispersed in 10 mL of ethanol to obtain a dispersion liquid. 0.02 g of 1,4-benzenedithiol was dissolved in 10 mL of ethanol solution, and then added to the former. After incubation at room temperature for 6 h, the unbound 1,4-benzenedithiol molecules were washed away by centrifugation with water twice. The upper liquid was poured out, and the silver nanowire precipitate with a self-assembled film on the surface was dispersed in 10 mL of water to obtain a dispersion liquid. 10 mL of 1 mM cobalt nitrate solution was added to the dispersion liquid, and then 60 μL of 25% ammonia water was added after stirring uniformly. The mixture was transferred into a hydrothermal kettle for hydrothermal reaction at 140°C for 5 h. After the reaction was completed, the supernatant was removed, and the precipitate was washed by centrifugation with ionized water for 3 times. The centrifugation speed was 8000 r / min, and the centrifugation time was 6 min. After the centrifugation was completed, the upper liquid was poured out to obtain the cobalt hydroxide / silver nanowire composite material precipitate.

[0047] (3) The composite material prepared in step (2) is dispersed in a 2% chitosan suspension to prepare a dispersion liquid with a concentration of 6 mg / mL, 2 μL of which is dropped on a gold electrode and dried at room temperature for 12 h; 1 mg / mL of pepsin (PP) monoclonal antibody is activated in EDC and NHS for 1 h, the volume ratio of PP:EDC:NHS being 1:2:2, 2 μL of which is dropped on the sensing electrode after activation, so that the PP monoclonal antibody is grafted on the material, and the sensing electrode is incubated at 5-8°C for 12 h, then the unbound antibody is washed away with a PBS solution (pH=7.4), and 2 μL of 1% bovine serum albumin solution is dropped thereon, and the sensing electrode is incubated for 2 h and then washed with the PBS solution (pH=7.4) to prepare a pepsin sensing electrode, which is stored at 5-8°C.

[0048] (4) The gold electrode treated as above is used as a working electrode, a platinum wire electrode is used as a counter electrode, and an Ag / AgCl electrode is used as a reference electrode, and differential pulse voltammetry (DPV) is used to detect different concentrations of the marker. A fixed concentration is selected to investigate the hybridization time, and detection is performed every five minutes until the DPV curve is stable, which is the response time of the sensor to the marker. According to the detection results, the detection response time of the aptamer sensor to the marker is 20 min, and the linear range of the marker is investigated, and the results show that the detection lower limit reaches 1*10 -12 M. This indicates that the sensor has a fast response time and a low detection lower limit.

[0049] Example 3

[0050] This example provides a pepsin detection method based on a cobalt hydroxide / silver nanowire composite material. The places not specially explained in this example are consistent with Example 1. The steps are as follows.

[0051] (1) 0.5 g of PVP is dissolved in 40 mL of ethylene glycol, and 600 μL of 0.2M NaCl-EG solution is added to the solution after the solution is clear to obtain solution A, 0.45 g of silver nitrate is dissolved in 10 mL of ethylene glycol to obtain solution B, then solution B is added to solution A in batches and continuously stirred to obtain a reaction liquid, which is transferred into a hydrothermal kettle for hydrothermal synthesis reaction at 170°C for 3 h, after the reaction is completed, the upper liquid is poured off, and the solid is left, then isopropanol is used for centrifugation (washing) once, ethanol is used for centrifugation (washing) once, and water is used for centrifugation (washing) twice, the centrifugation conditions are all at a rotation speed of 8000 r / min and a centrifugation time of 6 min, and after the centrifugation is completed, the upper liquid is poured off to obtain a silver nanowire precipitate.

[0052] (2) The silver nanowire precipitate obtained in step (1) is dispersed in 10 mL of ethanol to obtain a dispersion liquid, 0.02 g of 1,4-benzenedithiol is dissolved in 10 mL of ethanol solution, and then the former is added after dissolution, and incubated at room temperature for 6 h, and then centrifuged twice with water to wash away the unbound 1,4-benzenedithiol molecules. Pour off the upper liquid, and disperse the silver nanowire precipitate with a self-assembled film on the surface in 10 mL of water to obtain a dispersion liquid, add 10 mL of 1 mM cobalt nitrate solution to the dispersion liquid, and then add 60 μL of 25% ammonia water after stirring uniformly, and then transfer into a hydrothermal kettle for hydrothermal reaction at 160°C for 3 h, and then centrifuge after the reaction is completed, and then pour off the supernatant, and then add ionized water and centrifuge for washing 3 times. The centrifugal speed in this step is 8000 r / min, the centrifugal time is 6 min, and then pour off the upper liquid after the centrifugation is completed to obtain a cobalt hydroxide / silver nanowire composite material precipitate.

[0053] (3) The composite material prepared in step (2) is dispersed in a chitosan suspension with a mass fraction of 5% to prepare a dispersion liquid with a concentration of 6 mg / mL, 2 μL of which is dropped on a gold electrode and dried at room temperature for 12 h; 1 mg / mL of pepsin (PP) monoclonal antibody is activated in EDC and NHS for 1 h, and the volume ratio of PP:EDC:NHS is 1:2:2, 2 μL of which is added to the sensing electrode after activation, so that the PP monoclonal antibody is grafted to the material, and then incubated at 5-8°C for 6 h, and then washed with PBS solution (pH=7.4) to remove the unbound antibody, and then 2 μL of 1% bovine serum albumin solution is added, and then incubated for 2 h, and then washed with PBS solution (pH=7.4) to prepare a pepsin sensing electrode, which is stored at 5-8°C.

[0054] (4) The gold electrode treated by the above method is used as a working electrode, a platinum wire electrode is used as a counter electrode, and an Ag / AgCl electrode is used as a reference electrode, and differential pulse voltammetry (DPV) is used to detect different concentrations of the marker. A fixed concentration is selected to investigate the hybridization time, and detection is performed every five minutes until the DPV curve is stable, which is the response time of the sensor to the marker. According to the detection results, the detection response time of the aptamer sensor to the marker is 20 min; and the linear range of the marker is investigated, and the results show that the detection lower limit reaches 1*10 -12 M. This indicates that the sensor has a fast response time and a low detection lower limit.

[0055] The above merely describes preferred embodiments of the present application, but is not intended to limit the present application to other forms, any person skilled in the art can make changes or modifications to the above disclosed technical contents into equivalent embodiments with equivalent changes, and apply to other fields, but any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application without departing from the technical solution content of the present application still belongs to the protection scope of the present application technical solution.

Claims

1. A method for preparing a pepsin sensing electrode, characterized in that: Here are the steps: (1) Preparation of silver nanowires Dissolving polyvinyl pyrrolidone in ethylene glycol, adding sodium chloride ethylene glycol solution after the solution is clarified to obtain solution A, dissolving silver nitrate in ethylene glycol to obtain solution B, and then adding solution B to solution A in batches while continuously stirring to obtain a reaction solution; transferring the reaction solution into a hydrothermal reactor for a hydrothermal synthesis reaction to obtain a silver nanowire solution, which is then centrifuged and washed to obtain a silver nanowire precipitate; (2) Preparation of cobalt hydroxide / silver nanowire composite materials The silver nanowire precipitate is dispersed in ethanol to obtain a dispersion C, a 1,4-benzenedithiol ethanol solution is added to the dispersion C, and the mixture is allowed to stand at room temperature to obtain a 1,4-benzenedithiol rigid self-assembled film. The mixture is centrifuged, washed, and then dispersed in deionized water to obtain a dispersion D. A cobalt nitrate solution is added to the dispersion D to obtain a mixed solution, and ammonia water is added dropwise after stirring. The mixture is transferred to a hydrothermal reactor for a hydrothermal synthesis reaction to obtain a cobalt hydroxide / silver nanowire composite material solution. The mixture is centrifuged and washed to obtain a composite material precipitate. (3) Preparation of sensing electrodes The composite material precipitate is added to the chitosan suspension and mixed evenly to obtain a mixed solution. The bare gold working electrode is washed with ultrapure water, and the mixed solution is added dropwise to the surface of the working electrode, dried at room temperature, and then washed with ultrapure water to obtain a sensing electrode. An aqueous dispersion of EDC powder and an aqueous dispersion of NHS powder were mixed with a pepsin monoclonal antibody solution. After activation for 1 hour, 2 μL was added dropwise to the sensing electrode and incubated for 24 hours. Unbound antibodies were then rinsed off with a PBS solution, and a bovine serum albumin solution was added dropwise. After incubation for 2 hours, the solution was rinsed with a PBS solution to prepare a pepsin sensing electrode, which was stored at 5-8°C.

2. The method for preparing the pepsin sensing electrode according to claim 1, wherein: In step (1), the volume ratio of polyvinyl pyrrolidone to silver nitrate in ethylene glycol is 4:1, the concentration of polyvinyl pyrrolidone in solution A is 6-20 g / L, the concentration of silver nitrate in solution B is 24-80 g / L, the concentration of sodium chloride ethylene glycol solution is 0.2-0.6 M, the volume ratio of sodium chloride ethylene glycol solution to solution A is 1:(35-160), the hydrothermal synthesis temperature of the reaction solution is 160-200 ° C, and the hydrothermal synthesis time is 1-3 h.

3. The method for preparing the pepsin sensing electrode according to claim 1, wherein: In step (1), the silver nanowire solution is centrifugally washed once with isopropanol, once with ethanol, and twice with deionized water. The centrifugation condition is centrifugation at a speed of 8000 r / min for 6 min. After the centrifugation, the upper liquid is discarded to obtain the silver nanowire precipitate.

4. The method for preparing the pepsin sensing electrode according to claim 1, wherein: In step (2), the concentration of the 1,4-benzenedithiol ethanol solution is 1-5 mM, the volume ratio of ethanol in dispersion C to ethanol in the 1,4-benzenedithiol ethanol solution is 1:1; the standing time at room temperature is 6-24 h, the concentration of the cobalt nitrate solution is 1-5 mM, the volume fraction of ammonia water is 25%, the volume ratio of deionized water: cobalt nitrate solution: ammonia water is: 1:1:0.006, the hydrothermal synthesis reaction temperature is 120-200 ° C, and the hydrothermal synthesis time is 1-9 h.

5. The method for preparing the pepsin sensing electrode according to claim 1, characterized in that: In step (3), the mass fraction of chitosan in the chitosan suspension is 1-5%, the mass concentration of the composite material precipitate in the mixed solution is 6-15 mg / L, the drop coating amount is 2-5 μL, and the drying time at room temperature is 6-24 h.

6. Use of the sensing electrode prepared by the method according to any one of claims 1 to 5 in a pepsin detection instrument.

7. A method for detecting pepsin for non-diagnostic and / or therapeutic purposes, characterized in that: Detection is performed using the sensing electrode prepared by the method according to any one of claims 1 to 5.

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