CN110487870B - Method and device for detecting polypeptide and detecting bacteria by taking polypeptide as recognition molecule - Google Patents

Method and device for detecting polypeptide and detecting bacteria by taking polypeptide as recognition molecule Download PDF

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CN110487870B
CN110487870B CN201910782118.7A CN201910782118A CN110487870B CN 110487870 B CN110487870 B CN 110487870B CN 201910782118 A CN201910782118 A CN 201910782118A CN 110487870 B CN110487870 B CN 110487870B
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丁家旺
吕恩广
秦伟
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Yantai Institute of Coastal Zone Research of CAS
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Abstract

The invention relates to a polymer sensitive membrane ion selective electrode, in particular to a method and a device for detecting polypeptide and detecting bacteria by taking the polypeptide as a recognition molecule. The polypeptide and the derivative thereof with positive electricity or negative electricity in the sample to be detected generate potential response on the polymer membrane electrode doped with the ion exchanger, thereby realizing rapid quantitative/qualitative detection of the polypeptide and the derivative thereof in the sample. In the invention, the polypeptide is simultaneously used as an identification element and an indicator ion, so that the application range is wide and the universality is realized; the electrode has quick response, simple operation and good practical application prospect.

Description

Method and device for detecting polypeptide and detecting bacteria by taking polypeptide as recognition molecule
Technical Field
The invention relates to a polymer sensitive membrane ion selective electrode, in particular to a method and a device for detecting polypeptide and detecting bacteria by taking the polypeptide as a recognition molecule.
Background
Polypeptides are compounds of different kinds and numbers of amino acids linked together by peptide bonds. Amino acid side chains are diverse, resulting in different properties for each amino acid, thereby imparting different properties to the polypeptide. Polypeptides carry different amounts of charge in a particular solution and the hydrophobicity of different polypeptides is also different. As an ion, the polypeptide can induce a potential response on the polymer-sensitive membrane electrode. Meanwhile, polypeptides are also often used as indicator molecules to detect ions, molecules, microorganisms, etc. in the environment and in organisms. Thus, the detection of polypeptides has important physiological and environmental implications. Most of the existing polypeptide sensors use optical instruments as detection means, however, the methods are easily interfered by turbidity and chromaticity, and the instruments are complex in structure, high in cost and not suitable for rapid detection. It is still a difficult problem to develop a polypeptide sensor with simple structure, low cost and easy miniaturization. The potential sensor based on the polymer membrane ion selective electrode is simple to manufacture, simple and convenient to operate, high in response speed, free of expensive instruments and particularly suitable for rapid field detection. Conventional polyion-selective electrodes measure the potential response of the electrode at zero current. When low-concentration polyions are measured, the extraction process of the non-equilibrium state is slow, and the response time of the electrode is long. The research generally adopts stirring to improve the mass transfer rate of polyion, thereby shortening the response time and improving the detection sensitivity. Meanwhile, the conventional polyion-selective electrode is difficult to be used for detecting large substances such as bacteria and the like.
Disclosure of Invention
The invention aims to provide a method and a device for detecting polypeptide and detecting bacteria by taking polypeptide as recognition molecule
In order to achieve the purpose, the invention adopts the technical scheme that:
the method for detecting polypeptide is characterized by that the polypeptide and its derivative in the sample to be detected can produce potential response on the polymer membrane electrode doped with ion exchanger so as to implement quick quantitative/qualitative detection of polypeptide and its derivative in the sample.
The detection adopts an open circuit potential mode or multi-current step technology.
The detection can be carried out under zero current or applied current; potentiometry at zero current was performed by inserting a reference electrode and a working electrode together into a measuring cell and recording the potential change using an electrochemical external measuring device. The measurement of potential under applied current is performed by inserting a reference electrode, a working electrode, and an auxiliary electrode into a measuring cell, applying a specific current for a certain period of time to the working electrode using an electrochemical external measuring device, performing time measurement of potential, and then performing open circuit potential measurement.
The polypeptide is a peptide chain formed by connecting the same or different types and numbers of amino acids in a peptide bond mode; the polypeptide derivative is the modified polypeptide, and further a product obtained after the functional group of the polypeptide is modified by some small molecular compounds is the polypeptide derivative, wherein the product modified at the N end becomes the N-end derivative of the polypeptide, such as N-acetylated polypeptide obtained after N-terminal amino group is acetylated, and N-fatty acid acylated polypeptide obtained after fatty acid acylation are common polypeptide N-end derivatives; the product of the modified C-terminal of the polypeptide becomes a C-terminal derivative of the polypeptide, for example, C-terminal carboxyl is converted into amide to obtain polypeptide amide, ester to obtain polypeptide ester, and thioester to obtain polypeptide thioester, which belong to the polypeptide C-terminal derivative; for example, the polypeptide may be labeled with fluorescence (Cy3, FITC) or Biotin (Biotin) at the N-terminus and/or C-terminus, or the polypeptide may be modified with an aminothiol at the C-terminus.
Adding the polypeptide to be detected and the derivative thereof into a specific buffer solution to enable the polypeptide to be detected and the derivative thereof to have a certain number of positive charges or negative charges; by adjusting the pH, the polypeptide can carry at least one charge and potential detection can be performed at the electrodes. Wherein the buffer is phosphate buffer with pH 7.
After the polypeptide is determined, the current in a specific direction is applied to realize the exudation of the polypeptide in the membrane, so that the reversible and repeated utilization of the electrode is realized.
A method for using polypeptide as recognition molecule to detect bacteria, the polypeptide is used as recognition molecule and signal conduction molecule, and interacts with the bacteria in the liquid to be detected, so that the potential of the polymer membrane electrode doped with ion exchanger changes, and the qualitative/quantitative detection of the bacteria in the liquid to be detected is realized;
or the polypeptide derivative is fixed on the magnetic beads to obtain polypeptide functionalized magnetic beads, and the polypeptide serving as a recognition molecule and a signal conduction molecule interacts with bacteria in the liquid to be detected, so that the potential of the polypeptide functionalized magnetic beads is changed on the polymer membrane electrode doped with the ion exchanger, and qualitative/quantitative detection of the bacteria in the liquid to be detected is realized.
The polypeptide in the polypeptide functionalized magnetic bead interacts with bacteria in a sample to be detected (amino acid in the polypeptide can interact with functional groups on the surface of a bacterial membrane), so that the charge and the charge density of the polypeptide in the polypeptide functionalized magnetic bead are changed, the amount of the polypeptide on the polypeptide functionalized magnetic bead effectively extracted to a polymer sensitive membrane is reduced under the action of an external magnetic field, the potential of an electrode is changed, and the qualitative/quantitative detection of the bacteria in a liquid to be detected is realized.
And (3) placing the polymer membrane electrode in an electrochemical cell, controlling the effective extraction of the polypeptide functionalized magnetic beads before and after the combination with the bacteria to be detected to a polymer sensitive membrane phase by adopting an external magnetic field, and recording the potential change of the electrode. According to the potential change of the electrode before and after the action of the polypeptide and the bacteria, the potential sensor can realize high-sensitivity detection of the bacteria in the aqueous solution.
The polypeptide is a peptide chain formed by connecting the same or different types and numbers of amino acids in a peptide bond mode; the polypeptide derivative is the modified polypeptide.
The polypeptide or the polypeptide derivative is fixed on the magnetic beads, specifically, the polypeptide or the polypeptide derivative is modified and fixed on the magnetic beads;
for example, a polypeptide optionally having an amino group and/or a carboxyl group at both ends is reacted with a carboxylated (or aminated) magnetic bead, and the polypeptide is immobilized on the magnetic bead;
reacting the polypeptide with the amino mercaptan modified at the C terminal with the magnetic beads with sulfhydrylation surfaces, and fixing the polypeptide on the magnetic beads;
reacting polypeptide with an N end and/or a C end marked by Biotin (Biotin) with streptavidin modified magnetic beads, wherein the polypeptide is fixed on the magnetic beads;
if the polypeptide derivative has amino and/or carboxyl at both ends, the polypeptide derivative reacts with carboxylated (or aminated) magnetic beads and is fixed on the magnetic beads;
if the polypeptide of the polypeptide derivative is modified with amino mercaptan at the C terminal, the polypeptide reacts with the magnetic beads with sulfhydrylation surfaces, and the polypeptide is fixed on the magnetic beads; and so on.
The polypeptide is a polypeptide sequence VFQFLGKIIHHVGNFVHGFSHVF-NH for recognizing gram-negative bacteria2The polypeptide sequence WKKQLQLQLQLQLQLKKGGGC for identifying broad-spectrum bacteria, the polypeptide sequence GIGKFLHSAGKFGKAFVGEIMKS for identifying escherichia coli, the polypeptide sequence RVRSAPSSS for identifying staphylococcus aureus, the polypeptide sequence WGEAFSAGVHRLAN for identifying listeria monocytogenes, the polypeptide sequence NRPDSAQFWLHHGGGSC for identifying salmonella, the polypeptide sequence NRPDSAQFWLHHGGGSC for identifying salmonella, the polypeptide sequence KKHRKHRKHRKHGGSGGSKNLRRIIRKGIHIIKKYGC for identifying pseudomonas aeruginosa, the polypeptide sequence WVWPARLGGK for identifying edwardsiella tarda or semi-selective polypeptide KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW or a random sequence with a recognition effect on pathogenic bacteria and microorganisms.
The bacteria can be Escherichia coli, Listeria monocytogenes, Listeria ovis, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Edwardsiella tarda, Vibrio vulnificus, Bacillus subtilis, Bacillus anthracis, Vibrio cholerae, Shewanella, Bacillus subtilis, Streptococcus, and Proteus.
The ion exchanger doped polymer membrane electrode is a polymer membrane with an ion exchanger adhered to the bottom of an electrode, and the membrane comprises, by weight, 20% -80% of a membrane matrix, 20% -80% of a plasticizer, and the balance of an ion exchanger; the film substrate is polyvinyl chloride, polybutyl acrylate, polyetherimide, rubber or sol-gel film; the plasticizer is o-nitro benzene octyl ether, di-2-ethylhexyl decyl ester, dibutyl sebacate or dioctyl sebacate; the ion exchanger is an anion exchanger or a cation exchanger; wherein the anion exchanger is dotriacontanyl methyl ammonium chloride, dotriacontanyl methyl ammonium chloride derivative, tetratriacontyl methyl ammonium chloride or tetratriacontyl methyl ammonium chloride derivative; the cation exchanger is potassium tetrakis (4-chlorophenyl) borate, sodium tetrakis (p-tolyl) borate, sodium tetrakis (3, 5-bis (trifluoromethyl) phenyl) borate, dinonylnaphthalenesulfonic acid, dinonylnaphthalenesulfonate or borate or derivatives of the above compounds;
after the potential measurement, the separation or exudation of the polypeptide modified magnetic beads on the membrane is realized by changing the direction of the external magnetic field, and the reversible and repeated utilization of the electrode is realized.
A method for detecting a polypeptide, which is a device for a method for detecting a bacterium using a polypeptide as a recognition molecule, characterized in that: the device comprises a detection cell, a working electrode, a reference electrode, a counter electrode and an electrochemical external measuring device; the working electrode is a polymer membrane electrode doped with an ion exchanger and is arranged in the detection cell, and the working electrode, the reference electrode and the counter electrode are respectively connected with an electrochemical external measuring device.
The device includes an externally applied magnetic field.
The polymer membrane electrode can be a traditional polymer sensitive membrane electrode with internal liquid filling, a novel solid electrode or a printed electrode.
The electrochemical external measuring device is an electrochemical workstation, an ion meter or a potentiometer.
The reference electrode can be a saturated calomel electrode or a silver-silver chloride electrode; the auxiliary electrode may be a platinum wire.
The working electrode of the printed electrode can be a carbon electrode covered by a polymer sensitive film, the reference electrode can be a silver-silver chloride electrode, and the auxiliary electrode can be a platinum sheet electrode.
The detection principle is as follows: the polypeptide is used as an identification molecule and a signal conduction molecule, so that the polypeptide can be rapidly combined with a target object and simultaneously generate signal conduction, and the target object can be rapidly and quantitatively/qualitatively detected; furthermore, most of polypeptides with low charge density and low charge are modified with magnetic beads, and further the potential change caused by extraction or adsorption of the polypeptides to the surface of the polymer membrane electrode doped with the ion exchanger is controlled through an external magnetic field, so that obvious and rapid response is realized. The invention can realize the detection of various substances by utilizing different polypeptides to recognize different target molecules, thereby having universality.
The invention has the advantages that:
1. the invention realizes the real-time potential detection of the polypeptide by using the polymer membrane added with the ion exchanger; meanwhile, after the polymer membrane ionic selection electrode provided by the invention detects polypeptide, the ionic selection electrode can be repeatedly utilized under the condition of specific current; the electrode has simple structure and simple and convenient operation, and can be produced in large scale; the device does not need to depend on complex instruments and equipment, can be carried about, and realizes the on-site rapid detection of the target object.
2. The polypeptide is used as a new recognition molecule in the detection process, so that the selective recognition of various pathogenic bacteria can be realized, and the characteristic of polyions of the polypeptide can be used for signal conduction of potential signals.
3. The invention adopts an external magnetic field to control the magnetic beads with polypeptide functionalization to be directly extracted to the surface of the polymer sensitive membrane electrode, and generates rapid and high-sensitivity potential response. The extraction process is simple and rapid. In addition, under the condition of an external magnetic field, the polypeptide coupled magnetic beads can realize the rapid separation and enrichment of pathogenic bacteria and eliminate the matrix effect of a background solution.
Drawings
FIG. 1 is a potential response diagram of cationic polymer membrane electrode detection polypeptide.
FIG. 2 is a potential response diagram of the anionic polymer membrane electrode detecting polypeptide.
Fig. 3 is a schematic diagram and an object diagram of a sensor device based on a printed electrode according to an embodiment of the present invention.
Fig. 4 is a schematic diagram and an actual diagram of a sensor device based on a polymer sensitive membrane electrode with an internal liquid filling according to an embodiment of the present invention.
Fig. 5 is a schematic diagram and an object diagram of a sensor device based on an all-solid-state ion-selective electrode according to an embodiment of the present invention.
FIG. 6 shows the results of detection of Staphylococcus aureus by selected polypeptides in accordance with the present invention and a standard curve.
FIG. 7 shows the results of the potential measurements of eight bacteria by four polypeptides in the examples of the present invention.
FIG. 8 is a graph of the classification of bacteria using linear discriminant analysis in an example 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.
The polypeptide and the derivative thereof can generate ion potential response on the polymer membrane electrode doped with the ion exchanger, so that the potential detection of the polypeptide can be realized.
Furthermore, the polypeptide and the derivatives thereof are used as recognition molecules and signal conduction molecules, after interaction with bacteria, amino acids in the polypeptide and the derivatives thereof can interact with functional groups with charges on the surface of a bacterial membrane, so that the charge quantity and the charge density of the polypeptide are changed, the electrode potential change of the polypeptide on the surface of a sensitive membrane sensor is further caused, and the potential detection of the bacteria can be realized.
In order to improve the detection sensitivity, the printed electrode is arranged on a small flat magnet, and an external magnetic field is adopted to control the polypeptide functionalized magnetic beads to be effectively extracted to a polymer sensitive film phase to generate electrode potential change. In the measurement, the change in potential between the working electrode and the reference electrode on the printed electrode was recorded using an electrochemical system. The device is as follows: the working electrode and the reference electrode on the printed electrode are respectively connected with an electrochemical system. The printed electrode is inserted into a detection cell containing a detection solution. The detection cell is placed on a small flat plate magnet. And the detection of the object to be detected is realized by adopting an open-circuit potential measurement technology. In the invention, the polypeptide is used as a recognition element and an indicator ion at the same time, and the application range is wide.
Example 1
The device comprises a detection cell, an ion selective polymer membrane electrode, a reference electrode, a counter electrode and an electrochemical external measuring device. The ion selective polymer membrane electrode is arranged in a detection cell, and the ion selective polymer membrane electrode, the reference electrode and the counter electrode are respectively connected with an electrochemical external measuring device. Wherein, the reference electrode is a silver-silver chloride electrode, and the counter electrode is a platinum wire electrode.
1. Preparation of ion selective polymer membrane electrode
Weighing 340.06mg of polyvinyl chloride, 340.06mg of o-nitrooctyl ether, 6.94mg of dinonylnaphthalene disulfonic acid and 6.94mg of tetradodecyl ammonium tetrakis (4-chlorophenyl) borate in 6mL of tetrahydrofuran, uniformly stirring, pouring into a glass ring with the inner diameter of 5cm fixed on a glass plate, after the tetrahydrofuran is volatilized, cutting the film into small wafers with the diameter of 3mm by using a puncher, adhering the small wafers to a pipetting gun head with a PVC pipe at the bottom, and using the small wafers as polymer film electrodes for later use.
2. Potentiometric determination of polypeptides
The polypeptide (GVHRLANG) has positive charge in phosphate buffer solution (1mM, pH 7) at pH 7, different concentrations of polypeptide phosphate buffer solution (1mM, pH 7) are prepared, placed in detection cell, and the above electrodes are inserted into different concentrations of polypeptide (5.0 × 10)-7、1.0×10-6、2.5×10-6、5.0×10-6、1.5×10-5、3.0×10-5M) in a phosphoric acid buffer, recording the potential change. The real-time response curve and the calibration curve are shown in FIG. 1.
3. Recycling of electrodes
Transferring the electrode after the polypeptide is measured into the phosphate buffer solution with the pH value of 7, applying external cation current of 5 microamperes to the electrode by utilizing a multi-current step technology for 1 second to promote the polypeptide in the membrane to seep out, and then recycling the electrode.
Example 2
The ion selective polymer membrane electrode treated by the multi-current step technique in example 1 was used as a working electrode, a reference electrode was a silver-silver chloride electrode, and a counter electrode was a platinum wire electrode, which were connected to an electrochemical workstation, respectively, and a buffer solution (pH 7) containing polypeptide (GVHRLANG) was detected by applying a current using the multi-current step technique.
The current is applied by utilizing a multi-current step technique for 1 second and 5 muA, so that the target polypeptide can be quickly transferred to an electrode sensitive membrane phase to cause the potential change of the electrode, the potential change before and after the current is applied is recorded, and the polypeptide in the sample can be quickly and sensitively detected.
Example 3
The difference from the embodiment 1 is that:
1. the preparation of the middle polymer membrane of the ion selective polymer membrane electrode is as follows
The polymer membrane component is 178.2mg of polyvinyl chloride, 178.2mg of o-nitrophenyl octyl ether and 3.6mg of thirty-dialkyl methyl ammonium chloride, the components are weighed and dissolved in 3.6mL of tetrahydrofuran, the mixture is poured into a glass ring with the diameter of 3.6cm after being uniformly stirred, and the elastic polymer sensitive membrane is obtained after the tetrahydrofuran is volatilized.
The polypeptide (NWGEAFSA) has negative charge in phosphate buffer solution (1mM, pH 7) at pH 7, phosphate buffer solutions (1mM, pH 7) with different concentrations of polypeptide are prepared, placed in detection cells, and the electrodes are inserted into the phosphate buffer solutions (5.0 × 10) with different concentrations of polypeptide-7、1.0×10-6、2.5×10-6、5.0×10-6、1.5×10-5、3.0×10-5M) and recording the potential change, the real-time response curve and the calibration curve are shown in figure 2.
Example 4
The difference from example 1 was that the middle polymer membrane of the ion-selective polymer membrane electrode was prepared as follows
The ion selective electrode membrane comprises 178.2mg of polyvinyl chloride, 178.2mg of o-nitrophenyl octyl ether and 3.6mg of sodium tetra (p-tolyl) borate, the components are weighed and dissolved in 3.6mL of tetrahydrofuran, the mixture is poured into a glass ring with the diameter of 3.6cm after being uniformly stirred, and the elastic polymer sensitive membrane is obtained after the tetrahydrofuran is volatilized.
The polymer film obtained in the above way is attached to an electrode substrate to obtain an electrode, and the measurement of the polypeptide with positive charge in the buffer solution to be measured can be carried out.
Example 5
The difference from example 1 is that an ion selective polymer membrane electrode was prepared as follows:
the components of the ion selective electrode membrane are 122.5mg of polyvinyl chloride, 122.5mg of o-nitrooctyl ether, 2.5mg of dinonylnaphthalene disulfonic acid and 2.5mg of tetradodecyl ammonium tetrakis (4-chlorophenyl) borate which are dissolved in 2mL of tetrahydrofuran and stirred uniformly. And (3) dropwise adding 10 mu L of the membrane solution onto a carbon electrode of a printed electrode, and airing.
The electrodes obtained as described above can be used for the determination of positively charged polypeptides in the particular buffer solution to be tested.
Example 6
The device comprises a detection cell, a printed electrode, an electrochemical external measuring device and an external magnetic field as shown in figure 3; the printed electrode is arranged in the detection cell, a magnetic field is applied, and the working electrode and the reference electrode of the printed electrode are respectively connected with an electrochemical external measuring device. The working electrode-printed electrode was obtained in the preparation manner described in example 5 above.
Selecting polypeptide GGGRVRSAPSSSRRR with selective recognition effect on staphylococcus aureus, and labeling the polypeptide GGGRVRSAPSSSRRR with biotin according to the prior art to obtain biotin-GGGRVRSAPSSSRRR; and further detecting staphylococcus aureus:
during the measurement, an open circuit potential measurement technology is adopted. 80 μ L of 10-5M polypeptide biotin-GGGRVRSAPSSSRRR was mixed with 20. mu.L of streptavidin (streptavidin) -modified magnetic beads (commercially available) and incubated for 30After min, the beads were washed 2 times with 1mM phosphate buffer (containing 1mM NaCl) pH 7.4 to prepare polypeptide-modified magnetic beads. 10 mu L of polypeptide modified magnetic beads are dripped on the electrode, and the open circuit potential E1 is measured.
Culturing Staphylococcus aureus by conventional method, and preparing culture solution (0, 10, 100, 10) containing bacteria with different concentrations3、104、105、106CFU/mL)；
And mixing the obtained 10 mu L of polypeptide modified magnetic beads with the obtained bacterial culture solution with different concentrations, respectively incubating for one hour, washing for 2 times after incubation, and diluting to 10 mu L. 10 μ L of the diluted solution containing different bacteria culture solutions was dropped onto the electrode for potential measurement, and the potential change of bacteria at different concentrations was obtained as E2.
The potential changes (E1 and E2) caused by the polypeptide-modified magnetic beads on the electrodes before and after incubation with the bacteria were calculated and plotted against the logarithm of the bacterial concentration, and a standard curve was drawn.
The detection result is shown in FIG. 6, the detection range of the electrode to Staphylococcus aureus is 100-106CFU/mL; and the electrode response is fast.
Example 7
The device comprises a detection cell, a traditional polymer membrane electrode with inner liquid filling, an electrochemical external measuring device and an external magnetic field as shown in figure 4; the method mainly comprises the following steps:
1. preparation of ion selective polymer membrane electrode
Weighing 340.06mg of polyvinyl chloride, 340.06mg of o-nitrooctyl ether, 6.94mg of dinonylnaphthalene disulfonic acid and 6.94mg of tetradodecyl ammonium tetrakis (4-chlorophenyl) borate in 6mL of tetrahydrofuran, uniformly stirring, pouring into a glass ring with the inner diameter of 5cm fixed on a glass plate, after the tetrahydrofuran is volatilized, cutting the film into small wafers with the diameter of 3mm by using a puncher, and adhering the small wafers to a pipetting gun head with a PVC pipe at the bottom to prepare a polymer membrane electrode for later use.
2. Bacterial detection
Selecting polypeptide GGGRVRSAPSSSRRR with selective recognition effect on staphylococcus aureus, and labeling the polypeptide GGGRVRSAPSSSRRR with biotin according to the prior art to obtain biotin-GGGRVRSAPSSSRRR; and further detecting staphylococcus aureus:
during the measurement, an open circuit potential measurement technology is adopted. 80 μ L of 10-5M polypeptide biotin-GGGRVRSAPSSSRRR was mixed with 20. mu.L of streptavidin-modified magnetic beads, incubated for 30min, and washed 2 times with 1mM phosphate buffer (containing 1mM sodium chloride) pH 7.4 to prepare polypeptide-modified magnetic beads. 10 mu L of polypeptide modified magnetic beads are dripped on the electrode, and the open circuit potential E1 is measured.
Culturing Staphylococcus aureus by conventional method, and preparing culture solution (0, 10, 100, 10) containing bacteria with different concentrations3、104、105、106CFU/mL)；
And mixing the obtained 10 mu L of polypeptide modified magnetic beads with the obtained bacterial culture solution with different concentrations, respectively incubating for one hour, washing for 2 times after incubation, and diluting to 10 mu L. 10 μ L of the diluted solution containing different bacteria culture solutions was dropped onto the electrode for potential measurement, and the potential change of bacteria at different concentrations was obtained as E2.
The potential changes (E1 and E2) caused by the polypeptide-modified magnetic beads on the electrodes before and after incubation with the bacteria were calculated and plotted against the logarithm of the bacterial concentration, and a standard curve was drawn.
Example 8
The device comprises a detection cell, a novel all-solid-state ion selective polymer membrane electrode, an electrochemical external measuring device and an external magnetic field as shown in figure 5; the method mainly comprises the following steps:
preparing a novel all-solid-state ion selective polymer membrane electrode: the membrane component was 122.5mg of polyvinyl chloride, 122.5mg of o-nitrocetophenone, 2.5mg of dinonylnaphthalene disulfonic acid, and 2.5mg of tetradodecyl ammonium tetrakis (4-chlorophenyl) borate dissolved in 2mL of tetrahydrofuran, and the mixture was stirred uniformly. And (3) dropwise adding 10 mu L of membrane solution onto the solid electrode, and airing.
Selecting polypeptide GGGRVRSAPSSSRRR with selective recognition effect on staphylococcus aureus, and labeling the polypeptide GGGRVRSAPSSSRRR with biotin according to the prior art to obtain biotin-GGGRVRSAPSSSRRR; and further detecting staphylococcus aureus:
during the measurement, an open circuit potential measurement technology is adopted. 80 μ L of 10-5M polypeptide biotin-GGGRVRSAPSSSRRR was mixed with 20. mu.L of streptavidin-modified magnetic beads, incubated for 30min, and washed 2 times with 1mM phosphate buffer (containing 1mM sodium chloride) pH 7.4 to prepare polypeptide-modified magnetic beads. 10 mu L of polypeptide modified magnetic beads are dripped on the electrode, and the open circuit potential E1 is measured.
Culturing Staphylococcus aureus by conventional method, and preparing culture solution (0, 10, 100, 10) containing bacteria with different concentrations3、104、105、106CFU/mL)；
And mixing the obtained 10 mu L of polypeptide modified magnetic beads with the obtained bacterial culture solution with different concentrations, respectively incubating for one hour, washing for 2 times after incubation, and diluting to 10 mu L. 10 μ L of the diluted solution containing different bacteria culture solutions was dropped onto the electrode for potential measurement, and the potential change of bacteria at different concentrations was obtained as E2.
The potential changes (E1 and E2) caused by the polypeptide-modified magnetic beads on the electrodes before and after incubation with the bacteria were calculated and plotted against the logarithm of the bacterial concentration, and a standard curve was drawn.
According to the methods of the embodiments, when other targets (i.e., other gram-negative or gram-positive bacteria) are detected, the recognition molecule and the signal transduction marker polypeptide are replaced by the polypeptide capable of specifically recognizing the target, and then the ion exchanger in the ion-selective polymer membrane electrode is replaced correspondingly according to the method described above, so that the detection of any target can be realized, and further, the method has certain universality.
Example 9
According to the detection apparatus of example 6 above, eight kinds of bacteria were subjected to potential detection using four kinds of polypeptides:
modifying four polypeptides through the mutual action of biotin-streptavidin, incubating the modified magnetic beads at the same time to enable the polypeptides to be used as recognition molecules and signal conduction molecules, carrying out potential detection on eight bacteria by using the four polypeptides, calculating the leading-out of the polypeptide-modified magnetic beads on an electrode before and after incubation of the bacteriaA change in potential of the electrode; wherein, four polypeptides: peptide 01, GIGKFLHSAGKFGKAFVGEIMKS; peptide 02: WVWPARLGGK; peptide 03: WKKQLQLQLQLQLQLKKGGG; peptide 04: VFQFLGKIIHHVGNFVHGFSHVF-NH2. The eight species of bacteria are Staphylococcus Aureus (SA), Escherichia coli O157: H7(E. coli), Listeria Monocytogenes (LM), Listeria Iduanii (LI), Salmonella Typhimurium (ST), Pseudomonas Aeruginosa (PA), Edwards iella tarda (EA), and Vibrio Vulgaris (VV), respectively.
Taking polypeptide Peptide 01 as an example, the polypeptide Peptide comprises the following specific components: 80 μ L of 10-5M polypeptide biotin-GIGKFLHSAGKFGKAFVGEIMKS was mixed with 20. mu.L of streptavidin-modified magnetic beads, incubated for 30min, and washed 2 times with 1mM phosphate buffer (containing 1mM sodium chloride) pH 7.4 to prepare polypeptide-modified magnetic beads. 5 mu L of polypeptide modified magnetic beads are dripped on the electrode, and the open circuit potential E1 is measured.
Taking 5 μ L of polypeptide-modified magnetic beads and different bacteria (10)4CFU/mL) for one hour, washed 2 times and diluted to 5 μ L; dripping 5 mu L of the solution on the surface of an electrode in the solution for potential measurement to obtain the potential change E2 of different bacteria, and calculating the potential change (namely the potential difference between E1 and E2 of different bacteria) caused by polypeptide modified magnetic beads on the electrode before and after incubation with the bacteria; the potential detection results are shown in FIG. 7.
The detection in FIG. 7 shows that the electrode potential changes differently after different bacteria interact with the polypeptide modified magnetic beads; classifying and identifying the eight bacteria by a linear discriminant analysis method; the results of the analysis are shown in FIG. 8, where the 8 bacteria above are clearly distinguished. Thus, the four polypeptides used above allow for the differential identification of the eight bacteria selected.

Claims (7)

1. A method for detecting a polypeptide, comprising: the polypeptide and the derivative thereof with positive electricity or negative electricity in the sample to be detected generate potential response on the polymer membrane electrode doped with the ion exchanger, so that the rapid quantitative/qualitative detection of the polypeptide and the derivative thereof in the sample is realized;
the detection adopts an open circuit potential mode or multi-current step technology;
the ion exchanger doped polymer membrane electrode is a polymer membrane with an ion exchanger adhered to the bottom of an electrode, and the membrane comprises, by weight, 20% -80% of a membrane matrix, 20% -80% of a plasticizer, and the balance of an ion exchanger; the film substrate is polyvinyl chloride, polybutyl acrylate, polyetherimide, rubber or sol-gel film; the plasticizer is o-nitro benzene octyl ether, di-2-ethylhexyl decyl ester, dibutyl sebacate or dioctyl sebacate; the ion exchanger is an anion exchanger or a cation exchanger; wherein the anion exchanger is dotriacontanyl methyl ammonium chloride, dotriacontanyl methyl ammonium chloride derivative, tetratriacontyl methyl ammonium chloride or tetratriacontyl methyl ammonium chloride derivative; the cation exchanger is potassium tetrakis (4-chlorophenyl) borate, sodium tetrakis (p-tolyl) borate, sodium tetrakis (3, 5-bis (trifluoromethyl) phenyl) borate, dinonylnaphthalenesulfonic acid, dinonylnaphthalenesulfonate or borate or a derivative of each of the above compounds.
2. The method of detecting a polypeptide according to claim 1, wherein: the polypeptide is a peptide chain formed by connecting the same or different types and numbers of amino acids in a peptide bond mode; the polypeptide derivative is the modified polypeptide;
after the polypeptide is measured, the polypeptide is exuded in the membrane by applying current in a specific direction, so that the reversible and repeated utilization of the electrode is realized.
3. A method for detecting bacteria by using polypeptide as a recognition molecule is characterized in that:
the polypeptide or the polypeptide derivative is fixed on the magnetic beads to obtain polypeptide functionalized magnetic beads, the polypeptide is used as a recognition molecule and a signal conduction molecule,
the magnetic field interacts with bacteria in a sample to be detected, so that the polypeptide charge and the charge density in the polypeptide functionalized magnetic beads are changed, the amount of the polypeptide on the polypeptide functionalized magnetic beads effectively extracted to a polymer sensitive film is reduced under the action of an external magnetic field, the potential of an electrode is changed, and the qualitative/quantitative detection of the bacteria in a liquid to be detected is realized;
the detection adopts an open circuit potential mode or multi-current step technology;
the polymer sensitive membrane comprises, by weight, 20-80% of a membrane matrix, 20-80% of a plasticizer and the balance of an ion exchanger; the film substrate is polyvinyl chloride, polybutyl acrylate, polyetherimide, rubber or sol-gel film; the plasticizer is o-nitro benzene octyl ether, di-2-ethylhexyl decyl ester, dibutyl sebacate or dioctyl sebacate; the ion exchanger is an anion exchanger or a cation exchanger; wherein the anion exchanger is dotriacontanyl methyl ammonium chloride, dotriacontanyl methyl ammonium chloride derivative, tetratriacontyl methyl ammonium chloride or tetratriacontyl methyl ammonium chloride derivative; the cation exchanger is potassium tetrakis (4-chlorophenyl) borate, sodium tetrakis (p-tolyl) borate, sodium tetrakis (3, 5-bis (trifluoromethyl) phenyl) borate, dinonylnaphthalenesulfonic acid, dinonylnaphthalenesulfonate or borate or a derivative of each of the above compounds.
4. A method for detecting bacteria using a polypeptide as a recognition molecule according to claim 3, wherein: the polypeptide is a peptide chain formed by connecting the same or different types and numbers of amino acids in a peptide bond mode; the polypeptide derivative is the modified polypeptide.
5. The method for detecting bacteria using a polypeptide as a recognition molecule according to claim 4, wherein: the polypeptide is a polypeptide sequence VFQFLGKIIHHVGNFVHGFSHVF-NH for recognizing gram-negative bacteria2The polypeptide sequence WKKQLQLQLQLQLQLKKGGGC for identifying broad-spectrum bacteria, the polypeptide sequence GIGKFLHSAGKFGKAFVGEIMKS for identifying escherichia coli, the polypeptide sequence RVRSAPSSS for identifying staphylococcus aureus, the polypeptide sequence WGEAFSAGVHRLAN for identifying listeria monocytogenes, the polypeptide sequence NRPDSAQFWLHHGGGSC for identifying salmonella, the polypeptide sequence KKHRKHRKHRKHGGSGGSKNLRRIIRKGIHIIKKYGC for identifying pseudomonas aeruginosa, the polypeptide sequence WVWPARLGGK for identifying edwardsiella tarda or the semi-selective polypeptide KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW or a random sequence with an identification effect on pathogenic bacteria and microorganisms.
6. A method for detecting bacteria using a polypeptide as a recognition molecule according to claim 3, wherein: after the potential measurement, the separation or exudation of the polypeptide modified magnetic beads on the membrane is realized by changing the direction of the external magnetic field, so that the reversible and repeated utilization of the electrode is realized.
7. An apparatus for detecting a signal according to claim 1 or 2, wherein: the device comprises a detection cell, a working electrode, a reference electrode, a counter electrode and an electrochemical external measuring device; the working electrode is a polymer membrane electrode doped with an ion exchanger and is arranged in the detection cell, and the working electrode, the reference electrode and the counter electrode are respectively connected with an electrochemical external measuring device; the device includes an externally applied magnetic field.
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