CN115290732B - An electrochemical biosensor for detecting trypsin and preparation method thereof - Google Patents
An electrochemical biosensor for detecting trypsin and preparation method thereof Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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Abstract
本发明提供一种检测胰蛋白酶的电化学生物传感器及其制备方法，将多肽(HGC‑Fc)通过金硫自组装固定到金电极表面，用MCH封闭后，得到MCH/HGC‑Fc/GE修饰电极，即得到检测胰蛋白酶的电化学生物传感器。本发明基于多肽(HGC‑Fc)胰蛋白酶之间的特异性识别并切割构建了一种检测胰蛋白酶的电化学生物传感器，该传感器具有灵敏度高、特异性强、成本低和分析速度快等特点。
The present invention provides an electrochemical biosensor for detecting trypsin and a preparation method thereof. A polypeptide (HGC-Fc) is fixed to the surface of a gold electrode by gold-sulfur self-assembly, and after being sealed with MCH, an MCH/HGC-Fc/GE modified electrode is obtained, i.e., an electrochemical biosensor for detecting trypsin is obtained. The present invention constructs an electrochemical biosensor for detecting trypsin based on the specific recognition and cleavage between the polypeptide (HGC-Fc) and trypsin, and the sensor has the characteristics of high sensitivity, strong specificity, low cost, and fast analysis speed.
Description
Technical Field
The invention belongs to the field of medical instruments, and relates to an electrochemical biosensor for detecting trypsin and a preparation method thereof.
Background
Trypsin is an important serine protease produced by the pancreas that promotes conversion of pancreatic proenzymes into an active form and controls exocrine pancreatic function. The lack of trypsin can lead to pancreatic diseases such as meconium ileus and hereditary pancreatitis. Trypsin thus acts as a specific biomarker and plays a vital role in diagnosing important biological diseases such as pancreatitis, pancreatic cancer, cystic fibrosis, biliary cirrhosis, etc. Various methods are available for detecting trypsin, such as colorimetry (Colorimetry), liquid-mass chromatography (Liquid chromatography-mass spectrometry), raman spectroscopy (Colorimetry), and the like. However, these methods are complicated in operation procedures, are long in time consumption and high in detection cost, and are inconvenient to apply to conventional diagnosis. Thus, there is a need to establish a rapid, sensitive and simple assay for the detection of trypsin.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an electrochemical biosensor for detecting trypsin and a preparation method thereof, which can detect trypsin in time, and has high sensitivity, strong specificity, simplicity and low cost.
The invention provides a preparation method of an electrochemical biosensor for detecting trypsin, which is characterized by comprising the following steps:
step 1, preparation of trypsin-specifically cleaved polypeptide
Step 1.1, dissolving a polypeptide (HGC-Fc) in water-acetonitrile (V: V=4:1) to obtain a probe solution for trypsin specific cleavage, wherein the amino acid sequence of the polypeptide is HWRGWVC- (CH 2)6 -Cys;
step 2, assembly of electrochemical biosensor
Step 2.1, adding polypeptide chain (HGC-Fc) solution dropwise to the surface of a gold electrode for self-assembly modification, and then flushing with Tris-HCl buffer solution to obtain an HGC-Fc/GE electrode;
and 2.2, dropwise adding MCH solution to the electrode surface obtained in the step 2.1 to seal blank sites, which are not occupied by HGC-Fc, on the electrode surface, and flushing the electrode surface with Tris-HCl buffer solution after sealing to obtain an MCH/HGC-Fc/GE electrode, namely the electrochemical biosensor for detecting trypsin.
Preferably, step 1 comprises the steps of:
Preferably, in step 1.1, the concentration of HGC-Fc in the trypsin-specifically recognizing probe solution is 30 to 60. Mu.M.
Preferably, before the polypeptide chain solution is dripped on the surface of the gold electrode in the step 2.1, polishing and cleaning the gold electrode, taking the cleaned gold electrode as a working electrode, taking a platinum wire electrode as a counter electrode and Ag/AgCl as a reference electrode to form a three-electrode system, and carrying out cyclic voltammetry scanning in an electrochemical solution until the cyclic voltammogram is stable.
Further, the scanning potential range is-0.3V to +1.5V, and the scanning speed is 0.1 V.s -1.
Further, the gold electrode is polished specifically by polishing with aluminum oxide on a polishing cloth.
Further, the cleaning is ultrasonic cleaning in ethanol and deionized water for 2-5min respectively.
Preferably, in step 2.1, the time for self-assembly modification of the polypeptide chain (HGC-Fc) is 10-15h.
Preferably, in step 2.2, the concentration of the MCH solution is 0.1-10. Mu.M.
Preferably, in step 2.2, the MCH blocking time is 10-30min.
Compared with the prior art, the invention has the following beneficial technical effects:
Trypsin is an important serine protease that catalyzes the hydrolysis of peptide and ester bonds containing lysine and arginine residues at the C-terminus. Trypsin has a high degree of specificity and the ability to recognize a small number of specific sequences compared to other various proteolytic enzymes. Thus, HGC can be used as a probe capable of being specifically recognized and cleaved to detect trypsin. The invention uses peptide chain HGC-Fc with lysine and arginine residues as substances capable of being specifically identified and cut by trypsin, marks Fc on HGC as signal molecules, marks "(CH 2)6 -Cys" to carry out functional modification on the peptide chain to obtain polypeptide chain HGC-Fc. by taking HGC-Fc as molecular identification substances, and the invention has the advantages that (1) the sensitivity is high, the invention utilizes differential pulse current and voltage as signal output mode, the invention can fix HGC-Fc on the surface of a gold electrode through Au-S self-assembly, and can obtain an electrochemical biosensor for detecting trypsin after being blocked by MCH, under the condition of no trypsin, the Fc is oxidized to generate differential pulse volt-ampere (DPV) signals, when trypsin specifically cuts HGC-Fc, the Fc drops from the surface of an electrode, so that DPV signals are weakened, and the high-selectivity and high-sensitivity detection of trypsin are realized based on the basis, the principle of detecting trypsin by the electrochemical biosensor is shown in figure 1. The invention has the advantages that (1) the differential pulse current and voltage are high, the invention has high-sensitivity, and the differential pulse current and voltage-channel can normally interfere with the detection principle of 35.37 ml, thus the invention can detect trypsin in the condition of 35 to complete the specific trypsin, and the detection of 35.37 ml, and the invention has very high-sensitivity, and (4) the kit can be combined with trypsin solution to be detected for 30min, and the instrument has low cost and small detection object consumption.
According to the electrochemical biosensor for detecting trypsin, which is disclosed by the invention, after the sensor interacts with trypsin, the trypsin specifically recognizes and cleaves HGC-Fc, so that a signal molecule Fc drops off from the surface of an electrode, DPV signals are reduced, and the high-selectivity and high-sensitivity detection of trypsin is realized based on the reduced DPV signals.
Drawings
FIG. 1 is a schematic diagram of an electrochemical biosensor for detecting trypsin according to the present invention.
FIG. 2 is a graph of Differential Pulse Voltammetry (DPV) corresponding to different concentrations of trypsin.
FIG. 3 is a graph showing the relationship between DPV signal value and trypsin concentration.
FIG. 4 is a graph showing the selective results of trypsin detection by an electrochemical biosensor.
Detailed Description
The invention will now be described in further detail with reference to specific examples, which are intended to illustrate, but not to limit, the invention.
The preparation method of the electrochemical biosensor for detecting trypsin comprises the following specific preparation steps:
(1) Pretreatment of gold electrodes
Gold electrodes were polished to mirror surfaces with 1.0. Mu.M, 0.3. Mu.M and 0.05. Mu.M aluminum oxide, respectively, on a soft polishing cloth, and the electrode surfaces were rinsed with deionized water after polishing. And then the gold electrode is respectively ultrasonically cleaned in ethanol and deionized water for 2-5min. The cleaned gold electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, and the Ag/AgCl is used as a reference electrode (saturated KCl) to form a three-electrode system, and cyclic voltammetry scanning is carried out in 0.1-0.5M H 2SO4 solution until the cyclic voltammogram is stable. The scanning potential range is-0.3V to +1.5V, and the scanning speed is 0.1V.s -1.
(2) Configuring specific recognition probe for detecting trypsin
A. 30-60 mu M HGC-Fc was prepared with water-acetonitrile (V: V=4:1), to obtain a probe solution specifically recognized and cleaved by trypsin.
(3) Assembly of electrochemical biosensors
A. After pretreatment, 10. Mu.L of polypeptide (HGC-Fc) solution was added dropwise to the surface of the gold electrode (phi=2.0 mm) for modification for 10-15 hours, followed by rinsing with Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances, to obtain HGC-Fc/GE. Wherein the amino acid sequence of the polypeptide (HGC-Fc) is HWRGWVC- (CH 2)6 -Cys).
B. And (3) dropwise adding 10-30 mu L of 0.1-10 mu M MCH solution to the surface of the HGC-Fc/GE, sealing for 10-30min, and then flushing the surface of the electrode with 10mM Tris-HCl (pH 7.4) buffer solution to obtain the MCH/HGC-Fc/GE, namely the electrochemical biosensor capable of detecting trypsin. The concentration of Tris-HCl buffer solution may be any value within 0.01-0.1M and the pH may be any value within 7-8.
The application method of the electrochemical biosensor for detecting trypsin specifically comprises the following steps:
(1) The electrochemical biosensor for detecting trypsin prepared by the method is used as a working electrode, an Ag/AgCl electrode (saturated KCl) is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and a three-electrode system is constructed;
(2) To the surface of the working electrode, 10. Mu.L of trypsin test solution of known concentration prepared with 10mM Tris-HCl (pH 7.4) was added dropwise, and after 30min, any adsorbed substances were removed by washing with 10mM Tris-HCl (pH 7.4). DPV signal value (. Mu.A) was measured in 10mM Tris-HCl-0.1M NaClO 4 (pH 7.4) using an electrochemical method of differential pulse voltammetry, a scan range of 0.65-0.2V, a pulse width of 0.06s, an amplitude of 0.05V, a sample width of 0.02s, and a scan rate of 100 mV.s -1. The step is repeated to obtain a plurality of groups of DPV signal values corresponding to different trypsin concentrations, wherein the trypsin concentration range is 0.1pg/mL-1.0pg/mL.
(3) Within the concentration range of 0.1pg/mL-1.0pg/mL, the DPV signal values decreased with increasing trypsin concentration (DPV signal value profile for different concentrations of trypsin, as shown in FIG. 2). And (3) simulating the DPV signal values corresponding to the plurality of groups of different trypsin concentrations obtained in the step (2) to obtain a fitting curve between the trypsin concentration and the DPV signal value.
(4) After the solution to be tested is dripped on the surface of the working electrode for a period of time for combination, the combined electrode is taken as the working electrode, the DPV signal value is tested in 10mM Tris-HCl-0.1M NaClO 4 (pH 7.4), and the concentration of trypsin in the solution to be tested is obtained according to a fitting curve between the concentration of trypsin and the DPV signal value, wherein the unit is pg/mL.
The electrochemical biosensor can be used for detecting trypsin, has simple steps during testing, can be combined with trypsin solution to be detected for 30min after the electrochemical biosensor is used for detecting trypsin, and is beneficial to diagnosis and treatment of various diseases.
The present invention will be described in further detail with reference to the following examples of embodiments of the drawings.
Example 1
The preparation method of the electrochemical biosensor for detecting trypsin comprises the following specific preparation steps:
(1) Pretreatment of gold electrodes
A. The gold electrode was polished to a mirror surface with 1.0. Mu.M, 0.3. Mu.M and 0.05. Mu.M aluminum oxide, respectively, on a soft polishing cloth, and the electrode surface was rinsed with deionized water after polishing. The gold electrodes were then ultrasonically cleaned in ethanol and deionized water, respectively, for 2min. The cleaned gold electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, and the Ag/AgCl is used as a reference electrode (saturated KCl) to form a three-electrode system, and cyclic voltammetry scanning is carried out in 0.5M H 2SO4 solution until the cyclic voltammogram is stable. The scanning potential range is-0.3V to +1.5V, and the scanning speed is 0.1V.s -1.
(2) Preparation of specific recognition probe for detecting trypsin
30. Mu.M HGC-Fc was prepared with water-acetonitrile (V: V=4:1), to give a probe solution specifically recognized and cleaved by trypsin.
(3) Assembly of electrochemical biosensors
A. Gold electrode after pretreatment10. Mu.L of 30. Mu.M polypeptide (HGC-Fc) solution was added dropwise to the surface for modification for 10 hours, and then the electrode surface was rinsed thoroughly with 10mM Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances, to obtain HGC-Fc/GE. Wherein the amino acid sequence of the polypeptide (HGC-Fc) is HWRGWVC- (CH 2)6 -Cys).
B. And (3) dropwise adding 30 mu L of 0.1 mu M MCH solution to the surface of the HGC-Fc/GE electrode, and sealing for 10min to obtain the MCH/HGC-Fc/GE, namely the electrochemical biosensor for detecting trypsin.
Example 2
The preparation method of the electrochemical biosensor for detecting trypsin comprises the following specific preparation steps:
(1) Pretreatment of gold electrodes
A. The gold electrode was polished to a mirror surface with 1.0. Mu.M, 0.3. Mu.M and 0.05. Mu.M aluminum oxide, respectively, on a soft polishing cloth, and the electrode surface was rinsed with deionized water after polishing. The gold electrodes were then ultrasonically cleaned in ethanol and deionized water, respectively, for 3min. The cleaned gold electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, and the Ag/AgCl is used as a reference electrode (saturated KCl) to form a three-electrode system, and cyclic voltammetry scanning is carried out in 0.5M H 2SO4 solution until the cyclic voltammogram is stable. The scanning potential range is-0.3V to +1.5V, and the scanning speed is 0.1V.s -1.
(2) Preparation of specific recognition probe for detecting trypsin
45. Mu.M HGC-Fc was prepared with water-acetonitrile (V: V=4:1), to give a probe solution specifically recognized and cleaved by trypsin.
(3) Assembly of electrochemical biosensors
A. Gold electrode after pretreatment10. Mu.L of 45. Mu.M polypeptide (HGC-Fc) solution was added dropwise to the surface for modification for 15 hours, and then the electrode surface was rinsed thoroughly with 10mM Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances, to obtain HGC-Fc/GE. Wherein the amino acid sequence of the polypeptide (HGC-Fc) is HWRGWVC- (CH 2)6 -Cys).
B. And (3) dropwise adding 20 mu L of 10 mu M MCH solution to the surface of the HGC-Fc/GE electrode, and closing for 20min to obtain the MCH/HGC-Fc/GE, namely the electrochemical biosensor for detecting trypsin.
Example 3
The preparation method of the electrochemical biosensor for detecting trypsin comprises the following specific preparation steps:
(1) Pretreatment of gold electrodes
A. The gold electrode was polished to a mirror surface with 1.0. Mu.M, 0.3. Mu.M and 0.05. Mu.M aluminum oxide, respectively, on a soft polishing cloth, and the electrode surface was rinsed with deionized water after polishing. The gold electrodes were then ultrasonically cleaned in ethanol and deionized water, respectively, for 5min. The cleaned gold electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, and the Ag/AgCl is used as a reference electrode (saturated KCl) to form a three-electrode system, and cyclic voltammetry scanning is carried out in 0.5M H 2SO4 solution until the cyclic voltammogram is stable. The scanning potential range is-0.3V to +1.5V, and the scanning speed is 0.1V.s -1.
(2) Preparation of specific recognition probe for detecting trypsin
60. Mu.M HGC-Fc was prepared with water-acetonitrile (V: V=4:1), to give a probe solution specifically recognized and cleaved by trypsin.
(3) Assembly of electrochemical biosensors
A. After pretreatment, 10. Mu.L of 60. Mu.M polypeptide (HGC-Fc) solution was added dropwise to the surface of the gold electrode (phi=2.0 mM) for modification for 12 hours, and then the electrode surface was rinsed thoroughly with 10mM Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances, to obtain HGC-Fc/GE. Wherein the amino acid sequence of the polypeptide (HGC-Fc) is HWRGWVC- (CH 2)6 -Cys).
B. and (3) dropwise adding 10 mu L of 1 mu M MCH solution to the surface of the HGC-Fc/GE electrode, and closing for 30min to obtain the MCH/HGC-Fc/GE, namely the electrochemical biosensor for detecting trypsin.
Application instance
EXAMPLE 1 Trypsin detection
A method of using an electrochemical biosensor for detecting trypsin, comprising the steps of:
(1) The electrochemical biosensor for detecting trypsin prepared in the preparation example 3 is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and a three-electrode system is constructed;
(2) On the surface of the working electrode, 10. Mu.L of trypsin test solution with concentration of 0.1pg·mL-1、0.3pg·mL-1、0.5pg·mL-1、0.7pg·mL-1、 0.9pg·mL-1、1pg·mL-1 prepared by 10mM Tris-HCl (pH 7.4) is respectively dripped, after the solution is combined for 30min, any adsorbed substances are removed by washing by 10mM Tris-HCl (pH 7.4), and DPV signal value (mu A) is tested in 10mM Tris-HCl-0.1MNaClO 4 (pH 7.4) test solution by adopting an electrochemical method, wherein the differential pulse voltammetry method is adopted, the scanning range is 0.65-0.2V, the pulse width is 0.06s, the amplitude is 0.05V, the sampling width is 0.02s, and the scanning speed is 100 mV.s -1.
(3) According to the test results, the DPV signal value I is found to be in a linear relation I= -0.0967C+0.1588 (C unit: pg.mL -1,R2 = 0.9905) within the range of 0.1 pg-mL -1-1.0pg·mL-1 of trypsin concentration, DPV signal values corresponding to different concentrations of trypsin are shown in figure 2, and a linear relation diagram between the DPV signal values and the trypsin concentration is shown in figure 3.
(4) And (3) dropwise adding 10 mu L of trypsin to be detected on the surface of the working electrode, combining for 30min, and then testing in 10mM Tris-HCl-0.1M NaClO4 (pH 7.4) test solution to obtain a DPV signal value, wherein the unit of the concentration of trypsin in the solution to be detected is pg.mL -1 according to a fitting curve between the concentration of trypsin and the DPV signal value.
From this example 1, it was found that the DPV signal value and the trypsin concentration were in a linear relationship in the concentration range of 0.1pg/mL-1.0 pg/mL, and therefore, according to the detection limit calculation formula 3σ/S, the detection limit of trypsin detected by the electrochemical biosensor for detecting trypsin prepared in the calculation example 1 can reach 0.075pg/mL, which indicates that the sensitivity of the electrochemical biosensor of the invention is very high.
Example 2 selectivity test:
The electrochemical biosensor for detecting trypsin prepared in the above embodiment 3 is used as a working electrode, experimental conditions are the same as those in the above embodiment 1, and 10pg/mL trypsin and 1ng/mL common interfering proteins, namely MMP-2, BSA, thrombin, CEA, lysozyme and MUC1, are detected, and the results are shown in FIG. 4.
The result shows that the DPV signal value changes little after the other interfering proteins interact with the electrochemical biosensor except the target trypsin. This shows that 100 times of common interfering proteins do not affect detection, and the biosensor of the invention has better selectivity. This is mainly because, after the interaction of the biosensor and the trypsin, the trypsin specifically recognizes and cleaves the HGC-Fc, so that the signal molecule Fc is detached from the electrode surface, thereby reducing the DPV signal, and based on this, the detection of the trypsin with high selectivity and high sensitivity is realized.
The specific recognition and cleavage are carried out on the HGC-Fc based on trypsin, so that the signal molecule Fc falls off from the surface of the electrode, and the DPV signal is reduced, thus constructing the electrochemical biosensor for detecting trypsin with high sensitivity and strong specificity. The polypeptide (HGC-Fc) was first self-assembled on the gold electrode via Au-S bond, and the empty sites on the electrode surface not occupied by HGC-Fc were blocked with 6-mercapto-1-hexanol (MCH) and then the biosensor was completed. The trypsin can specifically recognize and cleave HGC-Fc, so that the MCH/HGC-Fc/GE modified biosensor can be used for detecting trypsin, and the Fc is used as a probe to detect the change of DPV signal value on the interface of the sensor after interaction of the sensor and the trypsin liquid to be detected. The trypsin concentration showed a good linear relationship with the DPV signal value in the range of 0.1pg/mL-1.0pg/mL, and the detection limit was 0.075pg/mL. The invention provides a novel method for clinically diagnosing certain biological diseases by researching the interaction between polypeptide (HGC-Fc) and trypsin, and provides a novel research platform for screening medicaments for treating the diseases.
The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the inventive concept, which fall within the scope of protection of the present invention, which is set forth in the claims.
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