CN108445062B - Based on NiCo2O4Construction of nano sheet material and trypsin electrochemical sensing platform with peptide chain cleavage effect - Google Patents
Based on NiCo2O4Construction of nano sheet material and trypsin electrochemical sensing platform with peptide chain cleavage effect Download PDFInfo
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Abstract
The invention discloses a method based on NiCo2O4Construction of a nano flaky material and a trypsin electrochemical sensing platform with a peptide chain cleavage effect. Using NiCo2O4The nano flaky material is used as an electroactive substrate material, and the peptide (g-C) is marked by nano graphite phase carbon nitride3N4@ petide) on a substrate material, and an electrochemical sensor is prepared and applied to detection of trypsin. Based on NiCo2O4The stability and excellent conductivity of the nano flaky material can accelerate the electron transfer speed and improve the current signal, and g-C3N4And the introduction of a peptide chain plays a role in space obstruction, and obviously reduces current signals. When the sensing interface is incubated in trypsin solution, the trypsin can catalyze and hydrolyze peptide bonds, and has a cleavage effect on the peptide bonds, so that g-C3N4@ petide is released from the electrode surface, thereby increasing the current signal. The method is used for monitoring the concentration of the trypsin and can be further applied to screening of inhibitors thereof.
Description
Technical Field
The invention belongs to the technical field of novel functional materials and biosensing detection, and particularly relates to a NiCo-based biosensor2O4Construction of a nano flaky material and a trypsin electrochemical sensing platform with a peptide chain cleavage effect.
Background
In recent years, there has been growing interest in medical diagnosis and pathogen identification to achieve simple protein detection using the construction of efficient biosensors. The prediction and diagnosis of cancer to enable early intervention and better disease management, as a diagnostic tool, tumor markers play an important role. Trypsin is an important digestive enzyme produced by the pancreas, which controls the exocrine functions of the pancreas. In addition, a number of diseases are closely associated with varying levels of trypsin concentration, such as cystic fibrosis, pancreatitis, gangrene, and meconium ileus. Therefore, it would be of great therapeutic interest to develop a simple and effective method for the bioassay of trypsin and the monitoring of its inhibitors. At present, enzyme-linked immunosorbent assay, electrochemistry, fluorescence spectrum and the like are mainly used as bioassay methods for detecting trypsin. However, further enhancement of the sensitivity of these methods and the exploration of a sensing platform with low background signal, good reproducibility and wide response range remain urgent requirements for trypsin assays.
Electrochemical detection is a new analytical method established based on electrochemical process and chemical/biological recognition process. The method comprises reacting [ Ru (NH)3)6]3+As a signal source, the current is used as a detection signal, the method has the advantages of high sensitivity, quick response, simple equipment, easiness in miniaturization and the like, and a powerful means is provided for the fields of clinical diagnosis, environmental monitoring, food safety and the like. The electrochemical analysis and detection is an electric signal, and different forms of energy are adopted as an excitation signal and a detection signal, so that the excitation signal and the detection signal are not interfered with each other, the background signal is low, and high sensitivity can be obtained. The type and performance of the electric material are directly and closely related to the realization of the electrochemical process, and the electrochemical property, the preparation method, the composite effect, the shape control, the charge conduction rate and the like of the electric material have important influence on the smooth realization of the electrochemical process.
NiCo2O4The nano material has the characteristics of good electrocatalytic activity, conductivity, biocompatibility, nontoxicity, environmental friendliness and the like, so that the nano material becomes an ideal material of an electrochemical sensor. NiCo2O4The properties of the nanosheets are generally affected by the nanostructure, etc. NiCo2O4The super structure is a nano-layered structure, compared to other NiCo2O4Nanostructured, NiCo2O4The super structure can not only improve the connection efficiency of electrolyte and electrode materials, but also improve the ion and electron transfer rates in the electrode and at the interface of the electrode and electrolyte solution, thereby further improving the NiCo2O4Is used for the electrical conductivity of (1). The invention provides a novel electrochemical detection method for trypsin. The method utilizes NiCo2O4The nano flaky material is used as an electroactive substrate material, and the peptide (g-C) is marked by nano graphite phase carbon nitride3N4@ petide) on a substrate material, and an electrochemical sensor is prepared and applied to detection of trypsin. Based on NiCo2O4The stability and excellent conductivity of the nano flaky material can accelerate the electron transfer speed and improve the current signal, and g-C3N4And the introduction of the peptide chain can obviously reduce current signals, when the sensing interface is incubated in a trypsin solution, the trypsin can catalyze and hydrolyze peptide bonds to play a role in cutting the peptide chain, so that g-C3N4@ petide is released from the electrode surface, thereby increasing the current signal. The current signal and trypsin concentration of the sensing interface are 1 × 10-10mg/mL–10-4Linear in the mg/mL range. The method can be used to monitor the concentration of trypsin in different disease processes and can be further applied to screening of inhibitors thereof.
Disclosure of Invention
One of the objects of the invention is based on NiCo2O4Construction of a nano flaky material and a trypsin electrochemical sensing platform with a peptide chain cleavage effect.
The other purpose of the invention is to apply the electrochemical sensor to the high-sensitivity detection of the trypsin.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
(1) pretreatment of GCE: firstly, mechanically grinding and polishing a chamois leather paved with alumina powder by GCE, washing residual powder on the surface by secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally completely washing the chamois leather by ethanol, dilute acid and water in sequence;
(2)NiCo2O4/PAMAM/peptide@g-C3N4preparing a modified electrode: 4 mul of 5mg/mLNiCo is added dropwise2O4And 0.3 percent of PAMAM suspension is put on the surface of a clean glassy carbon electrode, dried under an infrared lamp and cooled to room temperature; immersing the electrode in g-C3N4The marked peptide is dried in the room temperature for 60min, namely the marked peptide is modified on the surface of the electrode; immersing the modified electrode into 5 mu L BSA with the concentration of 1.0wt.% for 50min, sealing the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water and naturally airing at room temperature to prepare NiCo2O4/PAMAM/peptide@g-C3N4Modifying the electrode;
(3) detection of trypsin: the measurement is carried out using a three-electrode system with NiCo2O4/PAMAM/peptide@g-C3N4The modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is a counter electrode, an electrochemical workstation is used for detection, and the electrochemical workstation is used for detection in PBS buffer solution with the pH value of 7.4 to obtain 1 multiplied by 10-10mg/mL–10-4A series of trypsin standard solutions with different solubilities are obtained in a mg/mL mode, and a working curve is drawn by recording different current signals generated before and after the trypsin solution is added; the sample solution to be detected replaces a trypsin standard solution for detection, and the detection result can be obtained through checking a working curve.
The biological peptide used in the invention is arginine-containing peptide (CAGRAAADAD), the amino acid sequence list of which is Cys-Ala-Gly-Arg-Ala-Ala-Ala-Asp-Ala-Asp, and is purchased from Karelay Biochemical company.
The above NiCo2O4Preparing a nano sheet material:
1mmol of Ni (NO)3)2·6H2O, 2mmol of Co (NO)3)2·6H2O and 4.5mmol of hexamethylenetetramine are dissolved in 20mL of ethanol and 40mL of deionized water, the solution is transferred into a 100mL of a polytetraethylene reaction kettle, and the solution is stirred for 24 hours at 90 ℃ by a magnetic stirrer, howeverThen cooling to room temperature, centrifuging to obtain precipitate, washing with deionized water and ethanol for several times, drying the product at 60 deg.C for 12 hr, and calcining at 350 deg.C for 2 hr to remove residual organic matter to obtain NiCo2O4A nano-platelet material.
G to C above3N4Preparation of a @ petide solution: 1) graphite phase carbon nitride (g-C)3N4) The preparation of (1): heating dicyandiamide in static air at 550 ℃ for 4h at a ramp rate of 2.3 ℃/min; cooling at 1 deg.C/min to form yellow agglomerates, grinding into powder in a mortar, placing the powder in an open ceramic container and heating at 550 deg.C with a ramp rate of 5 deg.C/min for 2h to obtain g-C3N4A nanolayer; 2) g-C3N4Preparation of a @ petide solution: 10mg of 3,4,9, 10-benzenetetracarboxylic acid (PTCA) was added to 1mL of distilled water, stirred, and mixed with 1mg/mLg-C3N4Mixing, standing overnight at 4 deg.C, centrifuging and ultrafiltering for several times and redispersing the obtained precipitate in ultrapure water; 20mM EDC and NHS were added to g-C3N4In solution with functionalized PTCA; subsequently, 1mg/mL peptide chain dissolved in 10mL absolute ethanol was added to the mixed solution and left overnight at 4 ℃. In this process, the graphite-phase carbon nitride is coupled with the 3,4,9, 10-benzenetetracarboxylic acid pi-pi conjugate, and the amine group (-NH) of the 3,4,9, 10-benzenetetracarboxylic acid2) And then carrying out classical EDC and NHS coupling reaction with carboxyl (-COOH) of the biological peptide to obtain the graphite-phase carbon nitride labeled peptide. Finally, the prepared graphite-phase carbon nitride labeled peptide is centrifugally washed for a plurality of times and then is dispersed into 100 mu L of phosphate buffer solution with pH7.4 again.
(4) The invention relates to a NiCo-based material2O4The trypsin electrochemical sensing platform comprises a working electrode, a platinum wire electrode as a counter electrode and Ag/AgCl as a reference electrode, and is characterized in that the working electrode adopts NiCo2O4/PAMAM/peptide@g-C3N4A modified electrode prepared by the method of 1) polishing a glassy carbon electrode: the glassy carbon electrode is firstly mechanically polished on chamois coated with alumina powder, the residual powder on the surface is washed away by secondary water, and then the chamois is transferred into an ultrasonic deviceWashing in water bath until the water is completely washed, and finally thoroughly washing with ethanol, dilute acid and water in sequence; 2) NiCo2O4/PAMAM/peptide@g-C3N4Preparing a modified electrode: 4 mul of 5mg/mLNiCo is added dropwise2O4And 0.3 percent of PAMAM suspension is put on the surface of a clean glassy carbon electrode, dried under an infrared lamp and cooled to room temperature; immersing the electrode in g-C3N4The marked peptide is dried in the room temperature for 60min, namely the marked peptide is modified on the surface of the electrode; immersing the modified electrode into 5 mu L BSA with the concentration of 1.0wt.% for 50min, sealing the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water and naturally airing at room temperature to prepare NiCo2O4/PAMAM/peptide@g-C3N4Modifying the electrode;
(5) the invention relates to a NiCo-based material2O4The method for detecting the trypsin by using the trypsin electrochemical sensing platform with the nano sheet material and the peptide chain cleavage effect is characterized by comprising the following steps of: 1) the measurement is carried out using a three-electrode system with NiCo2O4/PAMAM/peptide@g-C3N4The modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is a counter electrode, an electrochemical workstation is used for detection, and the electrochemical workstation is used for detection in PBS buffer solution with the pH value of 7.4 to obtain 1 multiplied by 10-10mg/mL–10-4A series of trypsin standard solutions with different solubilities are obtained in a mg/mL mode, and a working curve is drawn by recording different current signals generated before and after the trypsin solution is added; the sample solution to be detected replaces a trypsin standard solution for detection, and the detection result can be obtained through checking a working curve.
The invention has the following remarkable advantages:
(1) with NiCo2O4The nano sheet material is an electroactive substrate material, NiCo2O4The stability and the excellent conductivity of the nano flaky material can accelerate the transfer speed of electrons and improve current signals.
(2) The graphite-phase carbon nitride is used for marking the peptide biological probe, so that the specificity of the detection method is improved, the signal amplification effect is realized on the substrate material, and the sensitivity of the sensor is further improved.
(3) The peptide chain containing arginine can be hydrolyzed by trypsin and induces the re-promotion reaction of electric signals, and the prepared sensing platform realizes the ultra-sensitive detection of the trypsin.
(4) The invention utilizes NiCo2O4The nano flaky material is used as an electroactive substrate material, and the peptide (g-C) is marked by nano graphite phase carbon nitride3N4@ petide) on a substrate material, and an electrochemical sensor is prepared and applied to detection of trypsin. Based on NiCo2O4The stability and excellent conductivity of the nano flaky material can accelerate the electron transfer speed and improve the current signal, and g-C3N4And the introduction of a peptide chain plays a role in space obstruction, and obviously reduces current signals. When the sensing interface is incubated in trypsin solution, the trypsin can catalyze and hydrolyze peptide bonds, and has a cleavage effect on the peptide bonds, so that g-C3N4@ petide is released from the electrode surface, thereby increasing the current signal. The current signal and trypsin concentration of the sensing interface are 1 × 10-10mg/mL–10-4Linear in the mg/mL range. The method is used for monitoring the concentration of the trypsin and can be further applied to screening of inhibitors thereof.
Drawings
FIG. 1 shows a NiCo-based composition according to the invention2O4The construction process of the nano flaky material and the trypsin electrochemical sensing platform with the peptide chain cleavage effect is schematically shown.
FIG. 2A is NiCo2O4Electron emission Scanning Electron Microscope (SEM) images of (a).
FIG. 2B shows NiCo2O4The nano-sheet materials are connected with each other to form a TEM image of a porous nano-layered structure.
Fig. 2C is a transmission electron microscope (HRTEM) image of OAM.
Fig. 2D is an X-ray diffraction (XRD) pattern of OAM.
The inset in FIGS. 3A and 3A is 1 × 10 at different concentrations-10mg/mL–10-4Sensing electrode under mg/mL trypsin standard solutionCurrent response graph and trypsin solution concentration of 10-9Reproducibility of electrode current response at mg/mL.
FIG. 3B is a linear plot of current response at the sensing electrode versus concentration of trypsin standard solution.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.
Example 1
Based on NiCo2O4The construction method of the trypsin electrochemical sensing platform of the nano flaky material and the peptide chain cleavage effect (as shown in figure 1) comprises the following steps:
(1) pretreating a glassy carbon electrode: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence;
(2) 4 mul of 5mg/mLNiCo is added dropwise2O4And 0.3wt% PAMAM suspension is put on the surface of a clean glassy carbon electrode, dried under an infrared lamp and cooled to room temperature;
(3) immersing the electrode in g-C3N4@ peltide solution is dried for 60min at room temperature;
(4) immersing the modified electrode obtained in the step (3) into 5 μ L of BSA1h with the concentration of 1.0wt.%, and blocking the nonspecific active sites on the electrode surface. Rinsing the electrode surface with ultrapure water and naturally airing at room temperature to obtain NiCo2O4/PAMAM/peptide@g-C3N4An electrochemical sensing platform.
Example 2
NiCo used in example 12O4Preparing a nano sheet material:
1mmol of Ni (NO)3)2·6H2O, 2mmol of Co (NO)3)2·6H2O and 4.5mmol of hexamethylenetetramine were dissolved in 20mL of ethanol and 40mL of deionized water, and then transferred to a 100mL of a polytetraethylene reactor at 90 deg.CStirring with magnetic stirrer for 24 hr, cooling to room temperature, centrifuging to obtain precipitate, washing with deionized water and ethanol several times, drying at 60 deg.C for 12 hr, and calcining at 350 deg.C for 2 hr to remove residual organic substances to obtain NiCo2O4A nano-platelet material. NiCo2O4Electron emission Scanning Electron Microscope (SEM) picture of (g), NiCo shown in FIG. 2A2O4The nano-sheet materials are connected with each other to form a porous nano-layered structure, and a TEM image (2B) of the structure also proves that NiCo2O4The nano-sheet material is composed of nano-secondary structural units. Transmission Electron Microscopy (HRTEM) and X-ray diffraction (XRD) patterns of OAM, as shown in FIGS. 2C and 2D, indicate NiCo2O4And (4) forming a nano sheet structure.
Example 3
G to C used in example 13N4Preparation of a @ petide solution:
(1)g-C3N4the preparation of (1): heating dicyandiamide in static air at 550 ℃ for 4h at a ramp rate of 2.3 ℃/min; cooling at 1 deg.C/min to form yellow agglomerates, grinding into powder in a mortar, placing the powder in an open ceramic container and heating at 550 deg.C with a ramp rate of 5 deg.C/min for 2h to obtain g-C3N4A nanolayer.
G to C used in example 13N4Preparation of a @ petide solution: 10mg of 3,4,9, 10-benzenetetracarboxylic acid (PTCA) was added to 1mL of distilled water, stirred, and mixed with 1mg/mLg-C3N4Mixing, standing overnight at 4 deg.C, centrifuging and ultrafiltering for several times and redispersing the obtained precipitate in ultrapure water; 20mM EDC and NHS were added to g-C3N4In solution with functionalized PTCA; subsequently, 1mg/mL peptide chain dissolved in 10mL absolute ethanol was added to the mixed solution and left overnight at 4 ℃. The above-mentioned biological peptide is an arginine-containing peptide (CAGRAAADAD), and its amino acid sequence list is Cys-Ala-Gly-Arg-Ala-Ala-Ala-Asp-Ala-Asp, and it is purchased from Karelay Biochemical company. In this process, the graphite-phase carbon nitride is coupled with the 3,4,9, 10-benzenetetracarboxylic acid pi-pi conjugate, and the amine group (-NH) of the 3,4,9, 10-benzenetetracarboxylic acid2) And with the carboxyl (-COOH) group of the biological peptideEDC and NHS coupling reaction to obtain graphite phase carbon nitride labeled peptide. Finally, the prepared graphite-phase carbon nitride labeled peptide was redispersed in 100. mu.L of phosphate buffer solution at pH 7.4.
Example 4
Based on NiCo2O4The method for detecting the trypsin by using the trypsin electrochemical sensing platform with the nano flaky material and the peptide chain cleavage effect comprises the following steps:
(1) the assay was carried out using a three-electrode system, with NiCo prepared in example 12O4/PAMAM/peptide@g-C3N4The modified electrode is a working electrode, the Ag/AgCl is a reference electrode, the platinum wire electrode is a counter electrode, and an electrochemical workstation is used for detection.
(2) Detection was carried out by electrochemical workstation in PBS buffer solution at pH7.4 at 1X 10-10mg/mL–10- 4A series of trypsin standard solutions with different solubilities are prepared in mg/mL, and a working curve is drawn by recording different current signals generated before and after adding the trypsin solution. The inset in FIGS. 3A and 3A is 1 × 10 at different concentrations-10mg/mL–10-4Current response diagram of sensing electrode under mg/mL trypsin standard solution and concentration of trypsin solution is 10-9Reproducibility of electrode current response at mg/mL. FIG. 3B is a linear plot of current response at the sensing electrode versus concentration of trypsin standard solution.
(3) And (3) replacing the trypsin standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
Claims (4)
1. Based on NiCo2O4The construction method of the nano flaky material and the trypsin electrochemical sensing platform with the peptide chain cleavage effect is characterized by comprising the following steps of:
(1) pretreatment of the glassy carbon electrode GCE: firstly, mechanically grinding and polishing a chamois leather paved with alumina powder by GCE, washing residual powder on the surface by secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally completely washing the chamois leather by ethanol, dilute acid and water in sequence;
（2）NiCo2O4/PAMAM/peptide@g-C3N4preparing a modified electrode: 4 mul of 5mg/mLNiCo is added dropwise2O4And 0.3wt% PAMAM suspension is put on the surface of a clean glassy carbon electrode, dried under an infrared lamp and cooled to room temperature; immersing the electrode in peptide @ g-C3N4The solution is dried for 60min at room temperature, namely peptide @ g-C3N4Modifying the surface of the electrode; immersing the modified electrode in 5 μ L BSA solution with concentration of 1.0wt.% for 50min, blocking the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water, and naturally drying at room temperature to obtain NiCo2O4/PAMAM/peptide@g-C3N4Modifying the electrode;
(3) detection of trypsin: the measurement is carried out using a three-electrode system with NiCo2O4/PAMAM/peptide@g-C3N4The modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is a counter electrode, an electrochemical workstation is used for detection, and the electrochemical workstation is used for detection in PBS buffer solution with the pH value of 7.4 to obtain 1 multiplied by 10-10mg/mL–10-4A series of trypsin standard solutions with different solubilities are obtained in a mg/mL mode, and a working curve is drawn by recording different current signals generated before and after the trypsin solution is added; the sample solution to be detected replaces a trypsin standard solution for detection, and the detection result can be obtained by checking a working curve;
the peptide @ g-C3N4The solution was prepared by the following method:
(1) graphite phase carbon nitride g-C3N4The preparation of (1): heating dicyandiamide in static air at 550 ℃ for 4h at a ramp rate of 2.3 ℃/min; cooling at 1 deg.C/min to form yellow agglomerates, grinding into powder in a mortar, placing the powder in an open ceramic container and heating at 550 deg.C with a ramp rate of 5 deg.C/min for 2h to obtain g-C3N4A nanolayer;
(2) graphite-phase carbon nitride labeled peptide @ g-C3N4Preparation of the solution: 10mg of 3,4,9, 10-benzenetetracarboxylic acid PTCA was added to 1mL of distilled water, stirred and mixed with 1mg/mLg-C3N4Mixing, standing overnight at 4 deg.C, centrifuging and ultrafiltering for several times and redispersing the obtained precipitate in ultrapure water; 20mM EDC and NHS were added to g-C3N4In solution with functionalized PTCA; subsequently, 1mg/mL peptide chain, which is arginine-containing peptide CAGRAAADAD with the amino acid sequence Cys-Ala-Gly-Arg-Ala-Ala-Ala-Asp-Ala-Asp, dissolved in 10mL absolute ethanol was added to the mixed solution and left overnight at 4 ℃; in this process, the graphite-phase carbon nitride is coupled with the 3,4,9, 10-benzenetetracarboxylic acid pi-pi conjugate, and the amine group-NH of 3,4,9, 10-benzenetetracarboxylic acid2And performing classical EDC and NHS coupling reaction with carboxyl-COOH of the biological peptide to obtain graphite-phase carbon nitride labeled peptide, and finally, performing centrifugal washing on the prepared graphite-phase carbon nitride labeled peptide for several times and then re-dispersing the graphite-phase carbon nitride labeled peptide into a phosphoric acid buffer solution with 100 mu L pH7.4.
2. The method of claim 1, wherein said NiCo is2O4The nano flaky material is prepared by the following method: 1mmol of Ni (NO)3)2·6H2O, 2mmol of Co (NO)3)2·6H2Dissolving O and 4.5mmol of hexamethylenetetramine in 40mL of deionized water containing 20mL of ethanol, transferring the solution into a 100mL of a polytetraethylene reaction kettle, stirring the solution at 90 ℃ for 24h by using a magnetic stirrer, cooling the solution to room temperature, centrifuging the solution to obtain a precipitate, washing the precipitate by using the deionized water and the ethanol for a plurality of times, drying the product at 60 ℃ for 12 h, and calcining the product at 350 ℃ for 2h to remove residual organic matters to obtain NiCo2O4A nano-platelet material.
3. Based on NiCo2O4A trypsin electrochemical sensing platform based on nano sheet material and peptide chain cleavage effect comprises a working electrode, a platinum wire electrode as a counter electrode and Ag/AgCl as a reference electrode, and is characterized in that the working electrode adopts NiCo prepared by the method of any one of claims 1-22O4/PAMAM/peptide@g-C3N4And modifying the electrode.
4. A NiCo-based material prepared by the method of any of claims 1-22O4The method for detecting the trypsin by using the trypsin electrochemical sensing platform with the nano sheet material and the peptide chain cleavage effect is characterized by comprising the following steps of: 1) the measurement is carried out by using a three-electrode system and using GEC/NiCo2O4/PAMAM-peptide@C3N4The modified electrode is a working electrode, the Ag/AgCl is a reference electrode, the platinum wire electrode is a counter electrode, and detection is carried out by utilizing an electrochemical workstation; 2) detection was carried out by electrochemical workstation in PBS buffer solution at pH7.4 at 1X 10-10mg/mL–10-4A series of trypsin standard solutions with different solubilities are obtained in a mg/mL mode, and a working curve is drawn by recording different current signals generated before and after the trypsin solution is added; 3) and (3) replacing the trypsin standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106248656A (en) * | 2016-08-22 | 2016-12-21 | 福建师范大学 | A kind of based on Ni nanoparticle Co2o4the preparation method and application of the electrochemiluminescence immunosensor of@AD bifunctional catalyst |
| CN106290528A (en) * | 2016-07-27 | 2017-01-04 | 福建师范大学 | A kind of based on TiO2it is situated between and sees the trypsin Optical Electro-Chemistry detection method of crystal |
| CN107543850A (en) * | 2017-07-28 | 2018-01-05 | 苏州健雄职业技术学院 | Detect biology sensor of trypsase and its preparation method and application |
-
2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290528A (en) * | 2016-07-27 | 2017-01-04 | 福建师范大学 | A kind of based on TiO2it is situated between and sees the trypsin Optical Electro-Chemistry detection method of crystal |
| CN106248656A (en) * | 2016-08-22 | 2016-12-21 | 福建师范大学 | A kind of based on Ni nanoparticle Co2o4the preparation method and application of the electrochemiluminescence immunosensor of@AD bifunctional catalyst |
| CN107543850A (en) * | 2017-07-28 | 2018-01-05 | 苏州健雄职业技术学院 | Detect biology sensor of trypsase and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| Photoelectrochemical biosensor constructed using TiO2 mesocrystals based multipurpose matrix for trypsin detection;Hong Dai et al.;《Biosensors and Bioelectronics》;20171231;第92卷;全文 * |
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