An electrochemical biosensor for detecting trypsin and preparation method thereof

Abstract

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

[0001] The invention belongs to the field of medical devices and relates to an electrochemical biosensor for detecting trypsin and a preparation method thereof. Background Art

[0002] Trypsin is an important serine protease produced by the pancreas, which can promote the conversion of pancreatic zymogen into active form and control the exocrine function of the pancreas. The lack of trypsin can lead to pancreatic diseases such as meconium ileus and hereditary pancreatitis. Therefore, trypsin, as a special biomarker, plays a vital role in the diagnosis of some important biological diseases, such as pancreatitis, pancreatic cancer, cystic fibrosis and biliary cirrhosis. At present, there are many methods for detecting trypsin, such as colorimetry, liquid chromatography-mass spectrometry, Raman spectroscopy, etc. However, these methods have complicated operating procedures, long time consumption and high detection costs, which are not convenient for routine diagnosis. Therefore, it is necessary to establish a rapid, sensitive and simple analytical method for detecting trypsin. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention provides an electrochemical biosensor for detecting trypsin and a preparation method thereof, which can detect trypsin in real time, has high sensitivity, strong specificity, and is simple and cheap.

[0004] The present invention provides a method for preparing an electrochemical biosensor for detecting trypsin, which is characterized by comprising the following steps:

[0005] Step 1: Preparation of trypsin-specific peptides

[0006] Step 1.1, dissolving the polypeptide (HGC-Fc) in water-acetonitrile (V:V=4:1) to obtain a probe solution for specific trypsin cleavage, wherein the amino acid sequence of the polypeptide is: HWRGWVC-(CH2)6-Cys;

[0007] Step 2, Assembly of electrochemical biosensor

[0008] Step 2.1, adding a polypeptide chain (HGC-Fc) solution to the surface of the gold electrode for self-assembly modification, and then washing with a Tris-HCl buffer solution to obtain a HGC-Fc / GE electrode;

[0009] Step 2.2, adding MCH solution to the electrode surface obtained in step 2.1 to block the blank sites on the electrode surface not occupied by HGC-Fc, and after blocking, rinsing the electrode surface with Tris-HCl buffer solution to obtain MCH / HGC-Fc / GE electrode, which is an electrochemical biosensor for detecting trypsin.

[0010] Preferably, step 1 comprises the following steps:

[0011] Preferably, in step 1.1, the concentration of HGC-Fc in the probe solution specifically recognized by trypsin is 30-60 μM.

[0012] Preferably, before the polypeptide chain solution is added to the surface of the gold electrode in step 2.1, the following treatment is performed: the gold electrode is polished and cleaned, and the cleaned gold electrode is used as the working electrode, the platinum wire electrode is used as the counter electrode, and Ag / AgCl is used as the reference electrode to form a three-electrode system, and a cyclic voltammetry scan is performed in the electrochemical solution until the cyclic voltammetry spectrum is stable.

[0013] Furthermore, the scanning potential range was -0.3 V to +1.5 V, and the scanning rate was 0.1 V·s -1 .

[0014] Furthermore, the gold electrode polishing is specifically polishing with aluminum oxide on a polishing cloth.

[0015] Further, cleaning was performed by ultrasonic cleaning in ethanol and deionized water for 2-5 min, respectively.

[0016] Preferably, in step 2.1, the time for self-assembly modification of the polypeptide chain (HGC-Fc) is 10-15 hours.

[0017] Preferably, in step 2.2, the concentration of the MCH solution is 0.1-10 μM.

[0018] Preferably, in step 2.2, the MCH blocking time is 10-30 min.

[0019] Compared with the prior art, the present invention has the following beneficial technical effects:

[0020] Trypsin is an important serine protease that can catalyze the hydrolysis of peptide bonds and ester bonds containing lysine and arginine residues at the C-terminus. Compared with various other proteolytic enzymes, trypsin has a high degree of specificity and the ability to recognize a few specific sequences. Therefore, HGC can be used as a probe that can be specifically recognized and cut to detect trypsin. The present invention uses a peptide chain HGC-Fc with lysine and arginine residues as a substance that can be specifically recognized and cut by trypsin, marks Fc on HGC as a signal molecule, and simultaneously marks "(CH2)6-Cys" to functionally modify the peptide chain to obtain a polypeptide chain HGC-Fc. HGC-Fc is used as a molecular recognition substance. Because the "(CH2)6-Cys" marked on HGC-Fc carries a thiol group, HGC-Fc can be fixed on the surface of a gold electrode through Au-S self-assembly, and an electrochemical biosensor for detecting trypsin can be obtained after being sealed with MCH. In the absence of trypsin, Fc is oxidized to produce a differential pulse voltammetry (DPV) signal; when trypsin specifically cuts HGC-Fc, Fc falls off the electrode surface, causing the DPV signal to weaken, thereby achieving highly selective and sensitive detection of trypsin. The schematic diagram of the electrochemical biosensor for detecting trypsin is shown in Figure 2. Figure 1 As shown. The advantages of the present invention are: (1) High sensitivity. The differential pulse voltammetry technology used in the present invention is used as a signal output method. The technology itself has extremely high sensitivity. The electrochemical biosensor in the present invention can quantitatively detect 0.075pg / ml trypsin; (2) Good selectivity. The present invention detects trypsin based on the principle of specific recognition and cleavage of HGC-Fc by trypsin, so that the differential pulse voltammetry signal is weakened. Common interfering proteins: MMP-2, BSA, Thrombin, CEA, lysozyme, and MUC1 have no interference with the detection. (3) The steps are simple and the operation is convenient. After the electrochemical biosensor is assembled, it can be detected after combining with the trypsin solution to be tested for 30 minutes; (4) The instrument is inexpensive and the amount of test material used is small.

[0021] The electrochemical biosensor for detecting trypsin obtained by the present invention, when the sensor interacts with trypsin, trypsin specifically recognizes and cuts HGC-Fc, causing the signal molecule Fc to fall off the electrode surface, thereby reducing the DPV signal, based on which high selectivity and high sensitivity detection of trypsin is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The schematic diagram of the electrochemical biosensor for detecting trypsin of the present invention is used for detecting trypsin.

[0023] Figure 2 The differential pulse voltammetry (DPV) spectra of trypsin at different concentrations.

[0024] Figure 3 This is the linear relationship between DPV signal value and trypsin concentration.

[0025] Figure 4 Figure 2 shows the selectivity of the electrochemical biosensor for the detection of trypsin. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific embodiments, which are intended to explain the present invention rather than to limit it.

[0027] A method for preparing an electrochemical biosensor for detecting trypsin, the specific preparation steps are as follows:

[0028] (1) Pretreatment of gold electrodes

[0029] The gold electrode was polished to a mirror surface on a soft polishing cloth with 1.0μM, 0.3μM and 0.05μM aluminum oxide, and the electrode surface was rinsed with deionized water after polishing. The gold electrode was then ultrasonically cleaned in ethanol and deionized water for 2-5 minutes. A three-electrode system was composed of the cleaned gold electrode as the working electrode, the platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode (saturated KCl) in a 0.1-0.5M H2SO4 solution for cyclic voltammetry scanning until the cyclic voltammetry spectrum was stable. The scanning potential range was -0.3V to +1.5V, and the scanning rate was 0.1V·s -1 .

[0030] (2) Configuring a specific recognition probe for detecting trypsin

[0031] a. Prepare 30-60 μM HGC-Fc with water-acetonitrile (V:V=4:1) to obtain a probe solution that can be specifically recognized and cleaved by trypsin.

[0032] (3) Assembly of electrochemical biosensors

[0033] a. 10 μL of peptide (HGC-Fc) solution was added to the surface of the pretreated gold electrode (φ=2.0 mm) for modification for 10-15 h, and then rinsed with Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances to obtain HGC-Fc / GE. The amino acid sequence of the peptide (HGC-Fc) is: HWRGWVC-(CH2)6-Cys.

[0034] b. 10-30 μL of 0.1-10 μM MCH solution was added dropwise to the surface of HGC-Fc / GE, and after blocking for 10-30 min, the electrode surface was rinsed with 10 mM Tris-HCl (pH 7.4) buffer solution to obtain MCH / HGC-Fc / GE, which is an electrochemical biosensor capable of detecting trypsin. The concentration of the Tris-HCl buffer solution can be any value within the range of 0.01-0.1 M, and the pH value can be any value within the range of 7-8.

[0035] The method for using the electrochemical biosensor for detecting trypsin specifically comprises the following steps:

[0036] (1) The electrochemical biosensor for detecting trypsin prepared as above was used as the working electrode, the Ag / AgCl electrode (saturated KCl) was used as the reference electrode, and the platinum wire electrode was used as the counter electrode to construct a three-electrode system;

[0037] (2) 10 μL of a known concentration of trypsin solution prepared with 10 mM Tris-HCl (pH 7.4) was added to the surface of the working electrode. After 30 minutes, any adsorbed substances were removed by washing with 10 mM Tris-HCl (pH 7.4). The DPV signal value (μA) was tested in a 10 mM Tris-HCl-0.1 M NaClO4 (pH 7.4) test solution. The electrochemical method used was differential pulse voltammetry; the scanning range was 0.65-0.2 V, the pulse width was 0.06 s, the amplitude was 0.05 V, the sampling width was 0.02 s, and the scanning rate was 100 mV·s -1 Repeat this step to obtain multiple groups of DPV signal values ​​corresponding to different trypsin concentrations; the trypsin concentration range is 0.1 pg / mL-1.0 pg / mL.

[0038] (3) In the concentration range of 0.1 pg / mL-1.0 pg / mL, the DPV signal value decreases with the increase of trypsin concentration (DPV signal value spectrum corresponding to different concentrations of trypsin, such as Figure 2 According to the DPV signal values ​​corresponding to the multiple groups of different trypsin concentrations obtained in step (2), a fitting curve between the trypsin concentration and the DPV signal value is simulated.

[0039] (4) After the test solution is added to the surface of the working electrode and allowed to bind for a period of time, the electrode after binding is used as the working electrode. The DPV signal value is tested in 10 mM Tris-HCl-0.1 M NaClO4 (pH 7.4). The concentration of trypsin in the test solution is obtained based on the fitting curve between the trypsin concentration and the DPV signal value, in units of pg / mL.

[0040] The electrochemical biosensor used in the present invention can be used to detect trypsin, and the testing steps are simple. After the electrochemical biosensor is assembled, it can be detected in one step after combining with the trypsin solution to be tested for 30 minutes, which is beneficial to the diagnosis and treatment of various diseases.

[0041] The present invention is further described in detail below with reference to the accompanying drawings. Specific embodiment 1

[0043] A method for preparing an electrochemical biosensor for detecting trypsin, the specific preparation steps are as follows:

[0044] (1) Pretreatment of gold electrodes

[0045] a. The gold electrode was polished to a mirror surface on a soft polishing cloth with 1.0μM, 0.3μM and 0.05μM aluminum oxide, respectively. After polishing, the electrode surface was rinsed with deionized water. The gold electrode was then ultrasonically cleaned in ethanol and deionized water for 2 minutes. A three-electrode system was composed of the cleaned gold electrode as the working electrode, the platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode (saturated KCl) in a 0.5M H2SO4 solution for cyclic voltammetry scanning until the cyclic voltammetry spectrum was stable. The scanning potential range was -0.3V to +1.5V, and the scanning rate was 0.1 V·s -1 .

[0046] (2) Preparation of specific recognition probe for detection of trypsin

[0047] 30 μM HGC-Fc was prepared with water-acetonitrile (V:V=4:1) to obtain a probe solution that can be specifically recognized and cleaved by trypsin.

[0048] (3) Assembly of electrochemical biosensors

[0049] a. Gold electrode after pretreatment 10 μL of 30 μM peptide (HGC-Fc) solution was added to the surface for modification for 10 h, and then the electrode surface was fully rinsed with 10 mM Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances, thereby obtaining HGC-Fc / GE. The amino acid sequence of the peptide (HGC-Fc) is: HWRGWVC-(CH2)6-Cys.

[0050] b. 30 μL of 0.1 μM MCH solution was added dropwise to the surface of the HGC-Fc / GE electrode and blocked for 10 min to obtain MCH / HGC-Fc / GE, which is the electrochemical biosensor for detecting trypsin. Specific embodiment 2

[0052] A method for preparing an electrochemical biosensor for detecting trypsin, the specific preparation steps are as follows:

[0053] (1) Pretreatment of gold electrodes

[0054] a. The gold electrode was polished to a mirror surface on a soft polishing cloth with 1.0μM, 0.3μM and 0.05μM aluminum oxide, respectively. After polishing, the electrode surface was rinsed with deionized water. The gold electrode was then ultrasonically cleaned in ethanol and deionized water for 3 minutes. A three-electrode system was composed of the cleaned gold electrode as the working electrode, the platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode (saturated KCl). Cyclic voltammetry was performed in 0.5M H2SO4 solution until the cyclic voltammetry spectrum was stable. The scanning potential range was -0.3V to +1.5V, and the scanning rate was 0.1 V·s -1 .

[0055] (2) Preparation of specific recognition probe for detection of trypsin

[0056] 45 μM HGC-Fc was prepared with water-acetonitrile (V:V=4:1) to obtain a probe solution that can be specifically recognized and cleaved by trypsin.

[0057] (3) Assembly of electrochemical biosensors

[0058] a. Gold electrode after pretreatment 10 μL of 45 μM peptide (HGC-Fc) solution was added to the surface for modification for 15 h, and then the electrode surface was fully rinsed with 10 mM Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances, thereby obtaining HGC-Fc / GE. The amino acid sequence of the peptide (HGC-Fc) is: HWRGWVC-(CH2)6-Cys.

[0059] b. 20 μL of 10 μM MCH solution was added dropwise to the surface of the HGC-Fc / GE electrode and blocked for 20 min to obtain MCH / HGC-Fc / GE, which is the electrochemical biosensor for detecting trypsin. Specific embodiment 3

[0061] A method for preparing an electrochemical biosensor for detecting trypsin, the specific preparation steps are as follows:

[0062] (1) Pretreatment of gold electrodes

[0063] a. The gold electrode was polished to a mirror surface on a soft polishing cloth with 1.0μM, 0.3μM and 0.05μM aluminum oxide, respectively. After polishing, the electrode surface was rinsed with deionized water. The gold electrode was then ultrasonically cleaned in ethanol and deionized water for 5 minutes. A three-electrode system was composed of the cleaned gold electrode as the working electrode, the platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode (saturated KCl) in a 0.5M H2SO4 solution for cyclic voltammetry scanning until the cyclic voltammetry spectrum was stable. The scanning potential range was -0.3V to +1.5V, and the scanning rate was 0.1 V·s -1 .

[0064] (2) Preparation of specific recognition probe for detection of trypsin

[0065] 60 μM HGC-Fc was prepared with water-acetonitrile (V:V=4:1) to obtain a probe solution that can be specifically recognized and cleaved by trypsin.

[0066] (3) Assembly of electrochemical biosensors

[0067] a. Add 10μL of 60μM peptide (HGC-Fc) solution to the surface of the pretreated gold electrode (φ=2.0mm) for 12h, then rinse the electrode surface with 10mM Tris-HCl (pH 7.4) buffer solution to remove any adsorbed substances, and obtain HGC-Fc / GE. The amino acid sequence of the peptide (HGC-Fc) is: HWRGWVC-(CH2)6-Cys.

[0068] b. 10 μL of 1 μM MCH solution was added dropwise to the surface of the HGC-Fc / GE electrode and blocked for 30 min to obtain MCH / HGC-Fc / GE, which is the electrochemical biosensor for detecting trypsin.

[0069] Application Examples

[0070] Example 1 Detection of trypsin

[0071] A method for using an electrochemical biosensor for detecting trypsin comprises the following steps:

[0072] (1) The electrochemical biosensor for detecting trypsin prepared in the above-mentioned Preparation Example 3 was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire electrode was used as the counter electrode to construct a three-electrode system;

[0073] (2) Add 10 μL of 10 mM Tris-HCl (pH 7.4) to the working electrode surface. -1 , 0.3pg·mL -1 , 0.5pg·mL-1 , 0.7pg·mL -1 、 0.9pg·mL -1 , 1pg·mL -1 The trypsin test solution was combined for 30 minutes and then rinsed with 10mM Tris-HCl (pH 7.4) to remove any adsorbed substances. The DPV signal value (μA) was tested in 10mM Tris-HCl-0.1MNaClO4 (pH 7.4) test solution. The electrochemical method used was differential pulse voltammetry; the scanning range was 0.65-0.2V, the pulse width was 0.06s, the amplitude was 0.05V, the sampling width was 0.02s, and the scanning rate was 100mV·s -1 .

[0074] (3) According to the test results, the DPV signal value I is 0.1 pg mL -1 -1.0pg·mL -1 The linear relationship is I=-0.0967C+0.1588(C unit: pg·mL -1 , R 2 =0.9905), the DPV signal values ​​corresponding to different concentrations of trypsin are shown in FIG2 , and the linear relationship between the DPV signal value and the trypsin concentration is shown in FIG2 . Figure 3 shown.

[0075] (4) 10 μL of the trypsin test solution was added to the working electrode surface. After binding for 30 min, the DPV signal value was obtained by testing in a 10 mM Tris-HCl-0.1 M NaClO4 (pH 7.4) test solution. The concentration of trypsin in the test solution was obtained based on the fitting curve between the trypsin concentration and the DPV signal value. The unit is pg mL -1 .

[0076] It can be seen from Example 1 that the DPV signal value is linearly related to the trypsin concentration in the concentration range of 0.1 pg / mL-1.0 pg / mL. Therefore, according to the detection limit calculation formula 3σ / S, it is calculated that the detection limit of the electrochemical biosensor for detecting trypsin prepared in Example 1 can reach 0.075 pg / mL, which shows that the electrochemical biosensor of the present invention has a high sensitivity.

[0077] Example 2 Selectivity Test:

[0078] The electrochemical biosensor for detecting trypsin prepared in the above specific example 3 was used as the working electrode. The experimental conditions were the same as those in the above specific example 1. 10 pg / mL trypsin and 1 ng / mL common interfering proteins: MMP-2, BSA, Thrombin, CEA, lysozyme, MUC1 were detected. The results are as follows: Figure 4 shown.

[0079] The results show that, except for the target trypsin, the DPV signal value changes are very small after the other interfering proteins interact with the electrochemical biosensor. This shows that 100 times the common interfering proteins do not affect the detection, and the biosensor of the present invention has good selectivity. This is mainly because after the biosensor interacts with trypsin, trypsin specifically recognizes and cuts HGC-Fc, causing the signal molecule Fc to fall off the electrode surface, thereby reducing the DPV signal, based on which high selectivity and high sensitivity detection of trypsin is achieved.

[0080] Based on the specific recognition and cleavage of HGC-Fc by trypsin, the signal molecule Fc falls off the electrode surface, thereby reducing the DPV signal. An electrochemical biosensor with high sensitivity and specificity for detecting trypsin was constructed. First, the polypeptide (HGC-Fc) was self-assembled on the gold electrode through Au-S bonds, and the blank sites on the electrode surface not occupied by HGC-Fc were blocked with 6-mercapto-1-hexanol (MCH) to complete the assembly of the biosensor. Trypsin can specifically recognize and cleave HGC-Fc, so the MCH / HGC-Fc / GE modified biosensor can be used to detect trypsin, and Fc is used as a probe to detect the change in the DPV signal value on the sensor interface after the interaction between the sensor and the trypsin test solution. 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 present invention proposes a novel method for clinical diagnosis of certain biological diseases by studying the interaction between polypeptide (HGC-Fc) and trypsin, and provides a new research platform for screening drugs for treating these diseases.

[0081] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention, and the protection scope of the present invention shall be subject to the claims.

Claims

1. A method for preparing an electrochemical biosensor for detecting trypsin, characterized in that: The steps include: Step 1: Preparation of trypsin-specific peptides Step 1.1, dissolving the polypeptide HGC-Fc in water-acetonitrile V:V=4:1 to obtain a probe solution for specific trypsin cleavage, wherein the amino acid sequence of the polypeptide chain is: HWRGWVC-(CH2)6-Cys; Step 2, Assembly of electrochemical biosensor Step 2.1, adding a polypeptide HGC-Fc solution to the surface of the gold electrode for self-assembly modification, and then washing with a Tris-HCl buffer solution to obtain a HGC-Fc / GE electrode; Step 2.2, add MCH solution to the electrode surface obtained in step 2.1 to block the blank sites on the electrode surface not occupied by HGC-Fc, and rinse the electrode surface with Tris-HCl buffer solution after blocking to obtain MCH / HGC-Fc / GE electrode, which is an electrochemical biosensor for detecting trypsin.

2. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 1, characterized in that: In step 2.1, the concentration of the polypeptide chain HGC-Fc is 30-60 μM.

3. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 1, characterized in that: Before adding the polypeptide chain HGC-Fc to the surface of the gold electrode in step 2.1, the following pretreatment is performed: the gold electrode is polished and cleaned, and the cleaned gold electrode is used as the working electrode, the platinum wire electrode is used as the counter electrode, and Ag / AgCl is used as the reference electrode to form a three-electrode system, and a cyclic voltammetry scan is performed in the electrochemical solution until the cyclic voltammetry spectrum is stable.

4. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 3, characterized in that: In the pretreatment of the gold electrode, the potential range of the cyclic voltammetry scan is -0.3V to +1.5V, and the scan rate is 0.1V·s-1.

5. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 3, characterized in that: In the pretreatment of the gold electrode, the gold electrode is polished by using aluminum oxide on a polishing cloth.

6. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 3, characterized in that: In the pretreatment of the gold electrode, the cleaning is ultrasonic cleaning in ethanol and deionized water for 2-5 minutes respectively.

7. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 1, characterized in that: In step 2.1, the time for self-assembly modification of the polypeptide chain HGC-Fc on the gold electrode surface is 10-15 hours.

8. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 1, characterized in that: In step 2.2, the concentration of the MCH solution is 0.1-10 μM.

9. The method for preparing the electrochemical biosensor for detecting trypsin according to claim 1, characterized in that: In step 2.2, the time for MCH to block the HGC-Fc / GE electrode is 10-30 min.

10. An electrochemical biosensor for detecting trypsin prepared by the preparation method according to any one of claims 1 to 9.

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