CN108007989B - Detection method based on split polypeptide as recognition molecule - Google Patents
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Abstract
The invention relates to a detection method based on 'split type' polypeptide as a recognition molecule, in particular to a method for detecting the content of a target substance by splitting a complete peptide chain capable of recognizing a target substance into two polypeptides as the recognition molecule. Two 'split' short peptides a and b capable of specifically recognizing a target object to be detected are used as recognition molecules, the recognition molecules and the target object to be detected are specifically recognized to form a three-dimensional structure, and a sandwich mode is formed, so that quantitative detection of the target object to be detected is realized. The split polypeptide provided by the invention has good selective recognition performance on a target object, can be used as a main component of various sensors, and has important scientific value and wide market prospect.
Description
Technical Field
The invention relates to a detection method based on 'split type' polypeptide as a recognition molecule, in particular to a method for detecting the content of a target substance by splitting a complete peptide chain capable of recognizing a target substance into two polypeptides as the recognition molecule.
Background
The polypeptide is a compound with a molecular structure between amino acids and proteins, and is formed by combining one or more amino acids through peptide bonds according to a certain arrangement sequence. The polypeptide is used as a high-efficiency recognition molecule for recognizing and detecting a target object. The polypeptide as an excellent artificial receptor mainly shows the following points: 1. the technology is relatively mature; 2, the cost is relatively low; 3, the modification is easy. Like antibodies, aptamers, polypeptides also exhibit specific recognition of ions, molecules, proteins, cells and show good performance.
However, the existing sensor based on polypeptide as recognition molecule mostly adopts competitive bonding, the modification and determination process is complex, the sensor is difficult to be used in the sandwich method reaction, the application range is narrow, two sections of peptide chains can be respectively modified by adopting split polypeptide as recognition molecule, the one-step recognition and detection of the target object are realized, and the application of the polypeptide in the sensor field is greatly expanded.
Disclosure of Invention
The invention aims to provide a detection method based on a split polypeptide as a recognition molecule.
In order to achieve the purpose, the invention adopts the technical scheme that:
a detection method based on 'split type' polypeptide as a recognition molecule takes two 'split type' short peptides a and b capable of specifically recognizing a target object to be detected as the recognition molecules, and the recognition molecules and the target object to be detected are specifically recognized to form a three-dimensional structure, so that a sandwich mode is formed, and further, the quantitative detection of the target object to be detected is realized.
The whole of the two short peptides in the cleavage type is a peptide chain at least comprising 10 amino acids; wherein the "cleavage" site should be in the central third to two thirds of the entire peptide chain.
The two ends of the short peptide a and the short peptide b which can specifically identify the target object to be detected are respectively modified and are applied to the sensor of the sandwich method.
The C end (or N end) of the short peptide a is modified through covalent bonds or affinity reaction; the N end (or C end) of the short peptide b is modified by fluorescent labeling, covalent modification enzyme or covalent bond.
The whole peptide of the two split short peptides is a polypeptide which can be identified by ions, small molecules, polysaccharides, proteins or bacteria; the target substance is an ion, a small molecule, a polysaccharide, a protein, or a bacterium.
The two split-type sequences a and b of the short peptide capable of specifically identifying the target object to be detected are a, GVHRLLANGK and b, NWGEAFSA, a ', FSAGVHRLANGK and b ', biotin-NWGEA or a ', RLANGK respectively; b', NWGEAFSAGVH; thereby specifically recognizing the listeria monocytogenes. As used herein, a "split" polypeptide includes fragments a and b formed by splitting an intact peptide chain at appropriate sites, which specifically bind to a target. The complete peptide chain has the specific recognition capacity for a target object; the proper site is that the polypeptide after the complete peptide chain is split at the site can still maintain the specific recognition capability to the target object; the fragment a and the fragment b do not interact when the target object does not exist, and the fragment a and the fragment b are specifically combined with the target object when the target object exists. The C end (or N end) of the fragment a can be modified on the surface of the electrode through a covalent bond, or modified on magnetic beads or magnetic nanoparticles through an affinity reaction, so that the purpose of immobilization or separation is achieved. The N end (or C end) of the fragment b can be subjected to fluorescence labeling, covalent modification of enzyme and covalent modification of an electrochemical active group, so that the aim of identifying a target object is fulfilled. When the target exists, the two split peptide chains are combined with the target to form a special three-dimensional structure. The active substance on the fragment b can directly or indirectly cause the change of electrochemical signals and fluorescence signals, and the magnitude of the change of the signals is proportional to the concentration of the target substances, so that the concentration of the target substances is measured.
The covalent bond is modified on the surface of the electrode through an amido bond and a gold-sulfur bond.
The electrode is a glassy carbon electrode, a graphite electrode, a metal electrode, an ITO conductive glass electrode, a screen printing electrode or a paper chip electrode.
The affinity reaction is that biotin and avidin are respectively modified on a peptide chain and a magnetic bead (or magnetic nano-particle), the biotin and the avidin generate the affinity reaction, and the peptide chain is connected on the magnetic bead (or the magnetic nano-particle).
The fluorescent label is used for labeling FITC, FAM, rhodamine, AFC, AMC, Rox, Sulforhodamine101, 5-TAMRA and EDANS Texas Red on a peptide chain or labeling the peptide chain with quantum dots and gold nano-labels; the covalent modification enzyme is to modify catalase and phosphatase on a peptide chain; covalently modifying the electrochemically active group is to modify ferrocene onto a peptide chain.
The electrochemical signal refers to a current signal generated by a redox substance on the surface of an electrode or a potential response generated by an electroactive substance on an ion selective membrane; the fluorescence signal is the change in signal of the fluorescent labeling substance on the fluorescence detector.
The detection principle of the invention is as follows: the fragment a or the fragment b can not identify the target object independently, the target object can be identified and folded into a spiral structure only when the fragment a and the fragment b exist simultaneously, and different markers can be decorated at the two ends of the fragment a and the fragment b to expand the applicability of the method. The main modes of application of the detection principle according to the method of the present invention include a "sandwich" reaction mode, a competitive bonding reaction mode.
The invention has the advantages that:
1. the split polypeptide provided by the invention is easy to synthesize, and the selective recognition performance on a target object is reserved.
2. The invention can selectively identify the target object by utilizing the split polypeptide, the two split peptide chains can be respectively used as a capture and signal probe, and the two probes simultaneously act on the target object only when the target object exists, thereby achieving the purposes of screening and detecting.
3. The present invention can be applied to various detection methods. By means of the characteristic that the polypeptide is easy to mark, the polypeptide can be fixed and modified, the recognition performance of the polypeptide cannot be influenced, the polypeptide can be used for various detection devices, and the application of the polypeptide in the field of sensors is greatly expanded.
Drawings
FIG. 1 is a schematic diagram of the principle of detecting Listeria monocytogenes by a potentiometric sensor using cleaved polypeptide as a recognition molecule according to an embodiment of the present invention.
FIG. 2 is a standard graph of the response of an electrode to catalase catalyzed TMB product in example 1 of the present invention.
FIG. 3 is a standard curve diagram of the polypeptide for identifying Listeria with FITC labeling for fluorescence detection in example 2 of the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The following examples were conducted in accordance with conventional methods and conditions, and experimental methods without specifying specific conditions were used.
Specifically, the split polypeptides are fixed or marked, when a target substance exists, two split polypeptides interact with the target substance to form a compound, photoelectric signals are directly or indirectly obtained through fluorescent substances and enzymes on the polypeptides, and different electric signals caused by the target substances with different concentrations are different, so that the detection of the target substance is realized. The split polypeptide provided by the invention has good selective recognition performance on a target object, can be used as a main component of various sensors, and has important scientific value and wide market prospect.
Example 1
And detecting the listeria monocytogenes by using a potentiometric sensor based on the cleaved polypeptide as the recognition molecule. The determination steps are as follows:
1) the peptide chain GEAFSAGVHRLANG is used as a complete peptide chain for identifying the listeria monocytogenes, and because the identification process can form a three-dimensional structure, after the peptide chain is split into two peptide chains of a and b, the identification process can still form a three-dimensional spiral structure to interact with a target. Two peptide chains are respectively synthesized, and different amino acids are added at two ends in order to increase the hydrophilicity of the peptide chains and easily modify the peptide chains. Biotin was then modified for both peptide chains. The sequence is as follows: a, GVHRLANGK-biotin; b, biotin-NWGEAFSA. In order to verify the influence of different sites on identification, four peptide chains a', FSAGVHRLANGK-biotin which are split by different sites are respectively synthesized; b', biotin-NWGEA; a', RLANGK-biotin; b', biotin-NWGEAFSAGVH.
2) As shown in FIG. 1, the peptide chain a, a 'was attached to the magnetic beads by affinity reaction, and the peptide chain b, b' was attached to horseradish peroxidase (HRP) by affinity reaction. The concentration of the peptide chains a, a ', b, b' is 10-3M, 100 microliters of magnetic beads and 1U/mL of horseradish peroxidase concentration.
3) As shown in figure 1, the peptide chains a, b, the peptide chains a ', b' or the peptide chains a ", b" with the above concentrations and Listeria monocytogenes with different concentrations are respectively added into a beaker at the same time, two peptide chains are specifically combined with Listeria monocytogenes, and the beaker is kept still for 1 hour to ensure that the reaction can be completed. Separating the magnetic beads by using a magnet, connecting the separated magnetic beads with Listeria monocytogenes through a peptide chain, connecting HRP enzyme on the peptide chain b, and removing the solution after reaction by using a pipette gun.
4) And (5) manufacturing an electrode. The electrode film comprises the following components: 171mg of polyvinyl chloride, 171mg of o-nitrophenyloctyl ether and 10mM/kg of sodium tetraphenylborate. Weighing the electrode membrane components, dissolving the electrode membrane components in 3mL of tetrahydrofuran solution, standing the solution in a constant temperature and humidity box for 12 hours, and forming an elastic electrode membrane after the tetrahydrofuran is volatilized. The electrode film was cut into a circle and adhered to a pvc pipe to prepare a simple working electrode. The electrode was activated prior to use in a 15mM sodium chloride solution, 50mM test solution, pH 7.4 phosphate buffered saline. The Ag/AgCl electrode is used as a reference electrode to form a two-electrode system for subsequent determination.
5) And (4) electrode measurement. 2mL of phosphate buffer solution was added to the cell, followed by 0.1mM TMB, 0.5mMH2O2And finally adding the compound of the polypeptide and the bacteria separated out previously. Catalase catalysis modified on the complex can catalyze TMB in solution, and the intermediate product can generate potential change on the electrode manufactured in the front.
6) Different cleavage sites. The two peptide chains a ', b' and a ", b" were used separately in the listeria assays as described above. The experimental result shows that the two peptide chains a and b have the best recognition effect on the listeria monocytogenes, so that the peptide chains a and b are adopted for subsequent determination.
7) And drawing a standard curve. The standard curve was generated by using two peptide chains a and b to measure different concentrations of listeria, and different concentrations of HRP enzyme were linked, resulting in different potential changes (see fig. 2). Example 2
Fluorescence detection of listeria monocytogenes based on split polypeptide as recognition molecule
The difference from the embodiment 1 is that:
1) the peptide chain b is used for marking a fluorescent marker FITC through a covalent bond, and the sequence of the peptide chain b is FITC-NWGEAFSA.
2) Will 10-3After the M peptide chains a and b interact with Listeria monocytogenes with different concentrations, the compound is separated by utilizing the magnetism of magnetic beads, and redundant fluorescence-labeled peptide chain b is removed.
3) The fluorescence intensity of the complex was detected on a fluorescence detector and a standard curve was generated (see FIG. 3). Example 3
Electrochemical detection of listeria monocytogenes based on split polypeptide as recognition molecule
The difference from the embodiment 1 is that:
1) the peptide chain a is modified with sulfydryl by adding cysteine and is fixed on an electrode by virtue of a gold-sulfur bond, and the peptide chain b is modified with ferrocene by virtue of a covalent bond.
2) The electrodes were inserted into solutions containing peptide chain b and different concentrations of listeria, and two peptide chains specifically bound to listeria.
3) And (4) electrode measurement. And (3) carrying out differential pulse voltammetry detection, wherein the ferrocene connected with the listeria produces electrochemical signals on the electrode, only the ferrocene on the peptide chain b connected with the listeria reacts on the electrode, and different electrochemical signals are produced according to the listeria with different concentrations to make a standard curve.
Example 4
Nanogold colorimetric method for detecting listeria monocytogenes based on split polypeptide as recognition molecule
The difference from the embodiment 1 is that:
1) the peptide chain a and the peptide chain b modify sulfydryl by adding cysteine, and modify the gold nanoparticles through gold-sulfur bonds.
2) When no listeria exists, the nano-gold exists in a dispersed state; when listeria is added, the peptide chain on the nanogold is recognized and connected to the listeria, so that the nanogold is enriched, and color change is caused.
3) The listeria at different depths causes different enrichment degrees of the nanogold and different color changes, thereby achieving the purpose of detecting the listeria.
Example 5
Quantum dot-labeled split polypeptide-detected Listeria monocytogenes
The difference from the embodiment 1 is that:
1) and modifying the CdS quantum dots at the N end of the peptide chain b through amido bonds.
2) When the peptide chain a, b is present together with ListeriaIdentifying Listeria to form a compound by the split polypeptide, separating the compound by magnetic beads, and catalyzing CdS quantum dots by using hydrogen peroxide to obtain Cd2+. The concentrations of listeria monocytogenes-linked peptide chains b were different, and therefore the amount of CdS quantum dots separated was different.
3) Using chromium ion-selective electrode pairs for Cd2+The concentration of the listeria monocytogenes is measured by potential to indirectly obtain the concentration of the listeria monocytogenes, and a standard curve is drawn.
Example 6
Listeria determination based on high-throughput ELISA reaction
The difference from the embodiment 1 is that:
1) peptide chain a was immobilized in a 96-well plate by chemisorption.
2) Adding Listeria and peptide chain b modified with catalase into a microporous plate at the same time, and reacting. 1 hour, the reaction is completed, and the mixture is eluted by using clear water, and TMB, H with the concentration mentioned above is added2O2A color reaction occurs.
3) The color change degree caused by the listeria with different concentrations is different, and the concentration of the listeria is obtained through colorimetric reaction.
4) The method can realize high-throughput analysis of the listeria monocytogenes.
Example 7
A potentiometric sensor based on a cleaved polypeptide as a recognition molecule detects lipopolysaccharide. The determination steps are as follows:
the difference from the embodiment 1 is that:
1) the target to be assayed is the detection of lipopolysaccharides (bacterial endotoxins).
2) The complete peptide chain is KKNYSSSISSIHC, in order to conveniently modify the peptide chain, an amino acid is added at one end of the peptide chain a, and the synthesized peptide chain is a, ISSIHCK-biotin respectively; b, biotin-KKNYSSS.
3) Lipopolysaccharide was detected using the method in example 1. When the concentration of the lipopolysaccharide is increased, the potential signal change is increased, and a standard curve is drawn according to different potential changes caused by different concentrations of the lipopolysaccharide, so that the quantitative analysis of the lipopolysaccharide is completed.
4) The standard curve shows that the two split short peptides can effectively quantitatively detect lipopolysaccharide, and the potential detection of the lipopolysaccharide is completed.
SEQUENCE LISTING
<110> institute of tobacco pipe coastal zone of Chinese academy of sciences
<120> detection method based on cleaved polypeptide as recognition molecule
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Claims (7)
1. A detection method based on 'split type' polypeptide as a recognition molecule is characterized in that: the method comprises the following steps of taking two 'split' short peptides a and b capable of specifically identifying a target object to be detected as identification molecules, specifically identifying the identification molecules and the target object to be detected to form a three-dimensional structure to form a sandwich mode, directly or indirectly causing the change of photoelectric signals through fluorescent substances and enzymes on the identification molecules, and further realizing the quantitative detection of the target object to be detected through detecting the change of the photoelectric signals;
"cleaved" polypeptides include fragments a and b, which are formed by cleaving an intact peptide chain at appropriate sites, and which specifically bind to the target.
2. The method of claim 1 for detecting a "split-type" polypeptide-based recognition molecule, wherein: the whole of the two short peptides in the cleavage type is a peptide chain at least comprising 10 amino acids; wherein the "cleavage" site should be in the central third to two thirds of the entire peptide chain.
3. The method of claim 1 for detecting a "split-type" polypeptide-based recognition molecule, wherein: the two ends of the short peptide a and the short peptide b which can specifically identify the target object to be detected are respectively modified and are applied to the sensor of the sandwich method.
4. A method for detecting a "split-type" polypeptide-based recognition molecule according to claim 3, wherein: the C end or the N end of the short peptide a is modified through covalent bonds or affinity reaction; and the N end or the C end of the short peptide b is modified by fluorescent labeling, covalent modification enzyme or covalent bond.
5. A method for detecting a "split" polypeptide-based recognition molecule according to any one of claims 1 to 3, wherein: the whole peptide of the two split short peptides is a polypeptide which can be identified by ions, small molecules, polysaccharides, proteins or bacteria; the target substance is an ion, a small molecule, a polysaccharide, a protein, or a bacterium.
6. A method for detecting a "split" polypeptide-based recognition molecule according to any one of claims 1 to 3, wherein: the two 'splitting' short peptides a and b capable of specifically identifying the target object to be detected have sequences of GVHRLANGK and NWGEAFSA, or FSAGVHRLANGK and biotin-NWGEA, or RLANGK and NWGEAFSAGVH respectively; thereby specifically recognizing the listeria monocytogenes.
7. A method for detecting a "split" polypeptide-based recognition molecule according to any one of claims 1 to 3, wherein: the two split-type short peptides a and b capable of specifically recognizing the target object to be detected have sequences a and ISSIHCK respectively; and b, KKNYSSS, and the target to be detected is Listeria monocytogenes.
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| CN104109197A (en) * | 2013-11-13 | 2014-10-22 | 叶森 | Preparation method and application for dual recognition antibody fragment |
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| CN104931710A (en) * | 2015-06-17 | 2015-09-23 | 深圳大学 | Double antibody sandwich method for quantitatively detecting cow milk beta-lactoglobulin |
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