CN107843631A - Protease detection electrochemical sensor and preparation method and detection method - Google Patents
Protease detection electrochemical sensor and preparation method and detection method Download PDFInfo
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- CN107843631A CN107843631A CN201711421008.5A CN201711421008A CN107843631A CN 107843631 A CN107843631 A CN 107843631A CN 201711421008 A CN201711421008 A CN 201711421008A CN 107843631 A CN107843631 A CN 107843631A
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- polypeptide
- electrochemical sensor
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- detection
- pyrene
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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
- G01N27/28—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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- 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
Abstract
Protease detection electrochemical sensor, described electrochemical sensor use the polypeptide graphene complex that one layer of pyrene fourth mark is modified in glassy carbon electrode surface, and the sequence signature of described polypeptide is：Described polypeptide can be cut by corresponding protease, and three amino acid of the cleavage site toward carbon teminal direction is histidine, and the 4th amino acid is the lysine that pyrene butyl group has been modified on side chain.The detection method of protease detection, comprises the following steps：1：The preparation of electrochemical sensor, including following sub-step：1.1：The preparation of polypeptide graphene complex：1.2：The preparation of electrochemical sensor：2：The detection of protease.This method belongs to the high flux electrochemical sensing method of " signal enhancing ", has the advantages that background current is low, strong antijamming capability.
Description
Technical field
The present invention relates to a kind of electrochemical sensor and preparation method and detection method for protease detection, belonging to
Learn detection technique field.
Background technology
Protease can be in aminosal or polypeptide peptide bond.It digest and assimilate, the side such as Wound healing, function are immune
Face has decisive role.The generation of some major diseases and the content of protease and activity change are closely related, for example, cancer
Disease, Apoptosis, Alzheimer disease, AIDS etc..Therefore, diagnosis and treatment of the protease detection in these major diseases
Research etc. is significant.Method currently used for detecting protease mainly has high performance liquid chromatography, gel chromatography, matter
Spectrum and fluorescence spectrum etc., but high performance liquid chromatography, gel chromatography, mass spectrum need that special instrument, sensitivity be relatively low, analysis cost
It is higher.Fluorescent spectrometry is generally required to enzyme substrate（Typically polypeptide）Both ends are marked, taken time and effort, in addition, polypeptide two
The efficiency of protease catalysis cutting is likely to affect after end is labeled.Between Protein cleavage reaction in research vital movement and disease
Significant role in disease preventing and treating, establish it is a kind of it is inexpensive, simple and quick, suitable for monitoring protease in common laboratory or clinic
The method of content or activity change is one of focus of current research.
Electrochemical sensing technology is according to electrochemical principle, and the change in concentration of test substance is converted into change in electric
A kind of sensing technology.The outstanding feature of the technology is research information on the surface“Focus on”With enhancing, the requirement pole of sample
It is micro-, to isolate and purify simplicity, high sensitivity, selectivity good etc..The main of electrochemical sensor currently used for protease detection is set
Meter theory is：By the one of substrate polypeptide it is terminal modified on can produce the groups of electrochemical signals（Such as ferrocene, methylene blue, enzyme
Label etc.）, for producing electric signal, another terminal modified special amino acid（Typically cysteine）, for by polypeptide
Fixed to electrode surface（Typically gold electrode）.After polypeptide is cut by protease, electric signal group will be from electrode surface
Come off, cause current reduction.But existing subject matter is this kind of electrochemical sensing method in actual applications：First, electricity
Signal group itself easily degraded or rotten, the long-time for being unfavorable for electrode chip preserve；Second, the inspection of this " signal attenuation "
Survey pattern has larger background current, and sensitivity is relatively low, error is larger.Therefore, a kind of simple and sensitive, " signal enhancing " are developed
High flux electrochemical sensing method, for protease detection and some major diseases monitoring it is significant.
The content of the invention
It is an object of the invention to overcome above mentioned problem present in current protease detection, there is provided a kind of protease inspection
Survey electrochemical sensor and preparation method and detection method.
To realize the purpose of the present invention, following technical schemes is employed：Protease detection electrochemical sensor, it is described
Electrochemical sensor use polypeptide-graphene complex that one layer of pyrene fourth mark is modified in glassy carbon electrode surface, described is more
The sequence signature of peptide is：Described polypeptide can be cut by corresponding protease, three ammonia of the cleavage site toward carbon teminal direction
Base acid is histidine, and the 4th amino acid is the lysine that pyrene butyl group has been modified on side chain.
The preparation method of protease detection electrochemical sensor, comprises the following steps：
A：The preparation of polypeptide-graphene complex：The polypeptide solution ultrasonic mixing 30 that graphene nanometer sheet marks with pyrene fourth is divided
Clock, the sequence signature of described polypeptide are：Described polypeptide can be cut by corresponding protease, and cleavage site is toward carbon teminal side
To the 3rd amino acid be histidine, the 4th amino acid be modified on side chain pyrene butyl group lysine, centrifuge reject
The remaining polypeptide in upper strata, then with phosphate buffer solution by resulting polypeptide-graphene complex centrifuge washing 3 times, in 4
Saved backup at DEG C；
B：The preparation of electrochemical sensor：Polypeptide-graphene complex of the pyrene fourth being prepared in step A mark is used into phosphorus
The concussion of acid buffering solution is scattered, takes out the glassy carbon electrode surface that 5 microlitres of mixed liquors are added drop-wise to a diameter of 3 mm, naturally dry, obtains
To electrochemical sensor.
The detection method of protease detection, comprises the following steps：
1：The preparation of electrochemical sensor, including following sub-step：
1.1：The preparation of polypeptide-graphene complex：By the polypeptide solution ultrasonic mixing 30 of graphene nanometer sheet and pyrene fourth mark
Minute, the sequence signature of described polypeptide is：Described polypeptide can be cut by corresponding protease, and cleavage site is toward carbon teminal
3rd amino acid in direction is histidine, and the 4th amino acid is the lysine that pyrene butyl group has been modified on side chain, and centrifugation is abandoned
Except the remaining polypeptide in upper strata, then with phosphate buffer solution by resulting polypeptide-graphene complex centrifuge washing 3 times, in 4
Saved backup at DEG C；
1.2：The preparation of electrochemical sensor：Polypeptide-graphene complex that the pyrene fourth being prepared in step A marks is used
Phosphate buffer solution concussion is scattered, takes out the glassy carbon electrode surface that 5 microlitres of mixed liquors are added drop-wise to a diameter of 3 mm, naturally dry,
Obtain electrochemical sensor；
2：The detection of protease：
The electrode that step 1 is obtained is soaked in protein enzyme solution to be detected, is rinsed well after taking-up with secondary water, then is being wrapped
Cyclic voltammetry scan test is carried out in phosphate buffer solution containing 50 μM of copper sulphate, described phosphate buffer solution concentration is 0.2
M, pH value 7.4；
Further, cyclic voltammetry scan uses three-electrode system, and the electrochemical sensor that step 1 obtains is working electrode, satisfies
The Ag/AgCl electrodes of sum are reference electrode, and Pt electrodes are auxiliary electrode.
The advantages of this technology is invented with effect be：(1) preparation process of sensing electrode is simple, cost is relatively low；(2) it is not required to
To enter horizontal electrical signal mark in advance to protease-based bottom, electric signal directly can be produced by the oxidation of electro-catalysis hydrone, have
Have that error is small, advantages of environment protection；(3) polypeptide fragment after cleavage can be complexed with copper ion, and formation can be with catalytic water
The elctro-catalyst of oxidation, there is high sensitivity, advantages of environment protection；（4）This method belongs to the high-flux electric of " signal enhancing "
Chemical sensitisation method, with the increase of protease concentration, electric signal gradually strengthens, and has that background current is low, strong antijamming capability
The advantages that, the Electrochemical Detection for succeeding in developing achievable polytype protease of the technology.
Brief description of the drawings
Fig. 1 is cyclic voltammogram of the sensor when passing through and being soaked without caspase-3 solution.
Fig. 2 is the linearity curve to caspase-3 detections.
Fig. 3 is the selectivity to caspase-3 detections.
Fig. 4 is the linearity curve to trypsase detection.
Embodiment
In order to more fully explain the implementation of the present invention, there is provided embodiment of the invention.These embodiments are only
Elaboration to the technique, is not limited the scope of the invention, and is illustrated in the present invention with following examples, but be not limited to following implementations
Example, any change are included in the technical scope of the present invention.
The sequence signature of polypeptide used in the present invention is：Described polypeptide can be cut by corresponding protease, and
Three amino acid of the cleavage site toward carbon teminal direction is histidine, and the 4th amino acid is that pyrene butyl group has been modified on side chain
Lysine, corresponding protease are the protease to be detected.In Fig. 1, curve a is polypeptide（Ac-GDEVDSGHK-Pyrene fourth）- stone
The circulation volt curve that the electrode of black alkene compound modification obtains when being soaked not in caspase-3 solution, curve b is the modification
After electrode soaks certain time in caspase-3 solution, then the circulation scanned in the phosphate buffer solution comprising copper ion
Volt-ampere curve, caspase-3 concentration is 50 ng/mL.Fig. 2 is the oxidation current value and caspase-3 concentration at 0.8 V
Linearity curve, caspase-3 concentration is 0.05,0.5,5,10,20 ng/mL successively.Fig. 3 is the selection of sensor
Property.What is be corresponding in turn to from 1 to 7 is：Blank control is tested, 20 ng/mL caspase-3,20 ng/mL bovine serum albumins, and 20
Ng/mL fibrin ferments, 20 ng/mL trypsase, 20 ng/mL beta-secretases, 10% serum.Fig. 4 is polypeptide（Ac-GKGGHK-
Pyrene fourth）The linearity curve that-graphene complex modified electrode detects to trypsase.Polypeptide（Ac-GDEVDSGHK-Pyrene fourth）And
Polypeptide A c-GKGGHK-Pyrene fourth produces for Shanghai Qiang Yao biotechnologies company.
Embodiment 1：
1：The preparation of electrochemical sensor：
1.1：The preparation of polypeptide-graphene complex：1 mL is added into 0.5 mg graphene nanometer sheets and includes 0.1 mM polypeptides
（Ac-GDEVDSGHK-Pyrene fourth）Phosphate buffer solution（10 mM, pH 7.4）, ultrasonic mixing 30 minutes, centrifugation reject upper strata
Remaining polypeptide, then with phosphate buffer solution by resulting polypeptide-graphene complex centrifuge washing 3 times, at 4 DEG C
Save backup；
The preparation of 1.2 electrochemical sensors：The polypeptide being prepared in step 1.1-graphene complex phosphoric acid buffer is molten
Liquid concussion is scattered, takes out 5 microlitres of mixed liquors, is added drop-wise to a diameter of 3 mm glassy carbon electrode surface, naturally dry, is obtained at room temperature
To polypeptide（Ac-GDEVDSGHK-Pyrene fourth）The electrode of-graphene complex modification, i.e. electrochemical sensor of the invention；
2：Caspase-3 detection
The polypeptide that will be obtained in 1（Ac-GDEVDSGHK-Pyrene fourth）The electrode of-graphene complex modification is respectively comprising different dense
Spend in caspase-3 phosphate buffer solution and soak 20 minutes, gently rinse electrode surface with secondary water, then electrode is soaked in
Include the phosphate buffer solution of 50 μM of copper sulphate（0.2 M, pH 7.4）Middle carry out cyclic voltammetry scan, sweep speed 50
MV/s, scanning range are 0.3 ~ 1.0V.It will be seen from figure 1 that polypeptide（Ac-GDEVDSGHK-Pyrene fourth）- graphene complex
The electrode of modification does not have redox peaks in electrolyte solution（Curve a）, show that the modified electrode can not be catalyzed hydrone
, still, after the electrode soaks certain time in caspase-3 solution, there is an obvious oxidation peak in oxidation（Curve
b）, after showing that the polypeptide of graphenic surface is cut, remain in the polypeptide fragment of electrode surface（SGHK- pyrene fourths）Can with copper from
Son forms complex compound, so as to be catalyzed the electroxidation of hydrone.Fig. 2 is linear between oxidation current value and caspase-3 concentration
Relation, it can be seen that current value increases with the increase of caspase-3 concentration.Therefore, the modified electrode can be used
In caspase-3 detection, detection is limited to 0.01 ng/mL, and the range of linearity is 0.05 ~ 20 ng/mL.
Embodiment 2：To the selectivity of caspase-3 detections：
By embodiment:Caspase-3 in 1 changes material to be tested into, and the condition of other steps does not change, and each work is prepared
Tested as electrode.Experimental result as shown in figure 3,1 to 7 be corresponding in turn to be：Blank control is tested, 20 ng/mL
Caspase-3,20 ng/mL bovine serum albumins, 20 ng/mL fibrin ferments, 20 ng/mL trypsase, 20 ng/mL beta-secretases
Enzyme, 10% serum.It is it can be seen that basic using electric signal caused by 3 ~ 7 protein and the electric signal of blank control
Equally, illustrate that the method for the present invention can be with selective enumeration method caspase-3.
Embodiment 3：The detection of trypsase
By polypeptide in embodiment 1（Ac-GDEVDSGHK-Pyrene fourth）It is replaced by polypeptide (Ac-GKGGHK-Pyrene fourth), obtain polypeptide (Ac-
GKGGHK-Pyrene fourth)-graphene modified electrode, then respectively in the phosphate buffer solution comprising various concentrations trypsase
Immersion 20 minutes.Other steps and experiment condition are consistent with the detection of caspase-3 in embodiment 1.Fig. 4 is current value and pancreas egg
The linear relationship of white enzyme concentration.It can be seen that current value increases with the increase of trypsinase concentration, the range of linearity
It is 1 ~ 500 ng/mL, detection is limited to 0.5 ng/mL.When using the caspase-3 in embodiment 3, bovine serum albumin, blood coagulation
Enzyme, beta-secretase, when 10% serum does control experiment, the current value of the electric signal of gained and blank control experiment is essentially the same,
Illustrate that detection of the modified electrode to trypsase has selectivity well.Therefore, if the polypeptide of graphenic surface changed
The polypeptide fragment that paired a certain protease specifically responds, this method can be used for the Electrochemical Detection of corresponding protease.
Sequence table
<110>Anyang Teachers College
<120>Protease detection electrochemical sensor and preparation method and detection method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Gly Asp Glu Val Asp Ser Gly His Lys
1 5
<210> 2
<211> 6
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
Gly Lys Gly Gly His Lys
1 5
Claims (4)
1. protease detection electrochemical sensor, described electrochemical sensor uses modifies one layer of pyrene in glassy carbon electrode surface
Polypeptide-graphene complex of fourth mark, it is characterised in that：The sequence signature of described polypeptide is：Described polypeptide can be by
Corresponding protease cutting, and three amino acid of the cleavage site toward carbon teminal direction is histidine, the 4th amino acid is side
The lysine of pyrene butyl group has been modified on chain.
2. the preparation method of protease detection electrochemical sensor, it is characterised in that comprise the following steps：
A：The preparation of polypeptide-graphene complex：The polypeptide solution ultrasonic mixing 30 that graphene nanometer sheet marks with pyrene fourth is divided
Clock, the sequence signature of described polypeptide are：Described polypeptide can be cut by corresponding protease, and cleavage site is toward carbon teminal direction
The 3rd amino acid be histidine, the 4th amino acid be modified on side chain pyrene butyl group lysine, centrifuge reject on
The remaining polypeptide of layer, then with phosphate buffer solution by resulting polypeptide-graphene complex centrifuge washing 3 times, in 4 DEG C
Under save backup；
B：The preparation of electrochemical sensor：Polypeptide-graphene complex of the pyrene fourth being prepared in step A mark is used into phosphorus
The concussion of acid buffering solution is scattered, takes out the glassy carbon electrode surface that 5 microlitres of mixed liquors are added drop-wise to a diameter of 3 mm, naturally dry, obtains
To electrochemical sensor.
3. the detection method of protease detection, it is characterised in that comprise the following steps：
1：The preparation of electrochemical sensor, including following sub-step：
1.1：The preparation of polypeptide-graphene complex：By the polypeptide solution ultrasonic mixing 30 of graphene nanometer sheet and pyrene fourth mark
Minute, the sequence signature of described polypeptide is：Described polypeptide can be cut by corresponding protease, and cleavage site is toward carbon teminal side
To the 3rd amino acid be histidine, the 4th amino acid be modified on side chain pyrene butyl group lysine, centrifuge reject
The remaining polypeptide in upper strata, then with phosphate buffer solution by resulting polypeptide-graphene complex centrifuge washing 3 times, in 4
Saved backup at DEG C；
1.2：The preparation of electrochemical sensor：Polypeptide-graphene complex that the pyrene fourth being prepared in step A marks is used
Phosphate buffer solution concussion is scattered, takes out the glassy carbon electrode surface that 5 microlitres of mixed liquors are added drop-wise to a diameter of 3 mm, naturally dry,
Obtain electrochemical sensor；
2：The detection of protease：
The electrode that step 1 is obtained is soaked in protein enzyme solution to be detected, is rinsed well after taking-up with secondary water, then is being wrapped
Cyclic voltammetry scan test is carried out in phosphate buffer solution containing 50 μM of copper sulphate, described phosphate buffer solution concentration is 0.2
M, pH value 7.4.
4. the detection method of protease detection according to claim 3, it is characterised in that：Cyclic voltammetry scan is using three electricity
Polar body system, the electrochemical sensor that step 1 obtains are working electrode, and the Ag/AgCl electrodes of saturation are reference electrode, and Pt electrodes are
Auxiliary electrode.
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| CN201711421008.5A CN107843631B (en) | 2017-12-25 | 2017-12-25 | Protease detection electrochemical sensor and preparation method and detection method |
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| CN201711421008.5A CN107843631B (en) | 2017-12-25 | 2017-12-25 | Protease detection electrochemical sensor and preparation method and detection method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109470757A (en) * | 2019-01-04 | 2019-03-15 | 安阳师范学院 | Electrochemical immunosensor electric signal marker and detection method for prostate specific antigen detection |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149185A (en) * | 2013-02-05 | 2013-06-12 | 苏州大学 | Novel high-efficiency protease activity detecting method |
| CN104049007A (en) * | 2014-04-15 | 2014-09-17 | 南昌大学 | Trypsin-chymotrypsin electrochemical synchronous detection method based on enzyme digestion |
| CN105274000A (en) * | 2014-07-15 | 2016-01-27 | 中国科学院大连化学物理研究所 | Immobilized enzyme reactor and preparation method and application |
| CN106520913A (en) * | 2016-09-22 | 2017-03-22 | 江苏大学 | Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection |
-
2017
- 2017-12-25 CN CN201711421008.5A patent/CN107843631B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149185A (en) * | 2013-02-05 | 2013-06-12 | 苏州大学 | Novel high-efficiency protease activity detecting method |
| CN104049007A (en) * | 2014-04-15 | 2014-09-17 | 南昌大学 | Trypsin-chymotrypsin electrochemical synchronous detection method based on enzyme digestion |
| CN105274000A (en) * | 2014-07-15 | 2016-01-27 | 中国科学院大连化学物理研究所 | Immobilized enzyme reactor and preparation method and application |
| CN106520913A (en) * | 2016-09-22 | 2017-03-22 | 江苏大学 | Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection |
Non-Patent Citations (4)
| Title |
|---|
| HAIBO WANG等: "Graphene Oxide–Peptide Conjugate as an Intracellular Protease Sensor for Caspase-3 Activation Imaging in Live Cells", 《ANGEWANDTE CHEMIE INTERNATIONAL EDITION》 * |
| JUAN LI: "A graphene oxide platform for energy transfer-based detection of protease activity", 《BIOSENSORS AND BIOELECTRONICS》 * |
| NING XIA等: "A signal-on electrochemical strategy for protease detection based onthe formation of ATCUN-Cu(II)", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
| PABLO GARRIDO-BARROS等: "Electronic π‑Delocalization Boosts Catalytic Water Oxidation by Cu(II) Molecular Catalysts Heterogenized on Graphene Sheets", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109470757A (en) * | 2019-01-04 | 2019-03-15 | 安阳师范学院 | Electrochemical immunosensor electric signal marker and detection method for prostate specific antigen detection |
| CN109470757B (en) * | 2019-01-04 | 2020-04-03 | 安阳师范学院 | Electrochemical detection method for prostate specific antigen detection |
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