A method and device for detecting apoptotic bacteria using polypeptides as recognition molecules

Abstract

The present invention relates to a polymer-sensitive membrane ion-selective electrode, specifically a method and apparatus for detecting apoptotic bacteria using polypeptides as recognition molecules. Apoptotic bacteria in a sample are captured using polypeptides or their derivatives as recognition and signaling molecules. The polypeptides alter the potential of the ion-exchanger-doped polymer membrane electrode, enabling qualitative and quantitative detection of apoptotic bacteria. In this method, the polypeptides serve as both recognition elements and indicator ions, resulting in a fast electrode response and simple operation, promising promising practical applications.

Description

Technical Field

[0001] The present invention relates to a polymer sensitive membrane ion selective electrode, in particular to a method and a device for detecting apoptotic bacteria by using polypeptides as recognition molecules. Background Art

[0002] The identification of live and apoptotic bacteria in bacterial cell suspensions is frequently used in bacterial cell proliferation assays, drug sensitivity tests, drug screening, and other aspects. Therefore, it is particularly important to establish an accurate and effective detection method to determine whether the bacteria in the sample are apoptotic or not. Under normal circumstances, phosphatidylserine (PS) is located inside the bacterial cell membrane. When bacteria undergo apoptosis, phosphatidylserine can be reversed from the inside of the cell membrane to the surface of the cell membrane. Annexin, as a phospholipid-binding protein, can specifically recognize and bind to phosphatidylserine. Currently, the most commonly used method for detecting bacterial apoptosis is a fluorescence-based staining technique, in which annexin is labeled with fluorescein to prepare a fluorescent probe to detect apoptosis. Early fluorescence-based microscopy observation technology was easy to operate and was therefore widely used for signal analysis. However, since quantification in the counting chamber is more cumbersome, microscopes usually only provide qualitative results. To address this limitation, researchers have used fluorescence-based microplate readers, fluorometers, and flow cytometers to develop methods for quantitatively detecting apoptotic bacteria. However, the binding of annexin to phosphatidylserine has Ca 2+ The bacterial cell wall partially blocks the binding of annexins to phosphatidylserine on the outer side of the cell membrane, resulting in a low measured bacterial apoptosis rate. Furthermore, the aforementioned instruments and equipment are relatively complex and require high technical skills from the operator. Potentiometric sensors based on polymer-sensitive membrane ion-selective electrodes are simple to manufacture, easy to operate, and have a fast response speed. They do not require expensive instruments and are particularly suitable for rapid on-site testing. However, there are currently no relevant potentiometric biosensors for the direct detection of apoptotic bacteria. Summary of the Invention

[0003] The purpose of the present invention is to provide a method and device for detecting apoptotic bacteria using polypeptides as recognition molecules

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] A method for detecting apoptotic bacteria using a polypeptide as a recognition molecule, wherein a polypeptide or its derivatives are used as recognition molecules and signal transduction molecules to capture apoptotic bacteria in a sample to be detected (the capture process does not require Ca 2+ The presence of ion exchangers causes the polypeptide to change the electrode potential of the polymer membrane doped with the ion exchanger, thereby achieving quantitative detection of apoptotic bacteria in the test solution.

[0006] The polypeptide for recognizing apoptotic bacteria can specifically recognize and bind to phosphatidylserine, and its sequence is NFRLKAGAKIRFG.

[0007] The polypeptide or its derivatives serving as recognition molecules and signal transduction molecules are modified on the surface of magnetic beads to form polypeptide-functionalized magnetic beads, which are used to specifically recognize and bind to the phosphatidylserine on the cell membrane of apoptotic bacteria (the amino acids in the polypeptide or its derivatives can generate electrostatic and hydrophobic interactions with the phosphatidylserine on the surface of the cell membrane of apoptotic bacteria), thereby achieving the capture of apoptotic bacteria.

[0008] The polypeptide and its derivatives are added to a specific buffer solution with a certain amount of positive or negative charges;

[0009] The polypeptide or polypeptide derivative is fixed on the magnetic beads, specifically the polypeptide, modified polypeptide, polypeptide derivative or modified polypeptide derivative is fixed on the magnetic beads;

[0010] For example, a polypeptide with amino and / or carboxyl groups at either end reacts with carboxylated (or amino) magnetic beads, and the polypeptide is fixed on the magnetic beads;

[0011] The peptide modified with aminothiol at the C-terminus reacts with the surface thiol-modified magnetic beads, and the peptide is fixed on the magnetic beads;

[0012] The peptide labeled with biotin at the N-terminus and / or C-terminus reacts with streptavidin-modified magnetic beads, and the peptide is fixed on the magnetic beads;

[0013] If the polypeptide derivatives have amino and / or carboxyl groups at either end, they react with carboxylated (or amino) magnetic beads and are fixed on the magnetic beads;

[0014] If the peptide derivative has aminothiol modified at its C-terminus, it reacts with surface thiol-modified magnetic beads and is fixed on the magnetic beads; etc.

[0015] An ion exchanger-doped polymer membrane electrode is placed in an electrochemical cell. Under the influence of an external magnetic field, peptide-functionalized magnetic beads are effectively extracted into the polymer-sensitive membrane phase. The electrode potential changes before and after the peptide-functionalized magnetic beads interact with the bacteria being tested are recorded. Based on these changes in electrode potential, this potentiometric sensor can achieve both qualitative and quantitative detection of apoptotic bacteria in the test fluid.

[0016] The polypeptide-functionalized magnetic beads capture apoptotic bacteria in the sample to be tested. Since the isoelectric point of the protein on the bacterial surface is low, the apoptotic bacteria are negatively charged, resulting in changes in the charge and charge density of the polypeptides in the polypeptide-functionalized magnetic beads. Under the action of an external magnetic field, the amount of polypeptides effectively extracted from the polypeptide-functionalized magnetic beads to the polymer sensitive membrane changes, causing a change in the electrode potential, thereby achieving quantitative detection of apoptotic bacteria in the liquid to be tested.

[0017] The bacteria can be Escherichia coli, Candida albicans, Staphylococcus aureus and other microorganisms whose apoptosis causes phosphatidylserine externalization in the cell membrane.

[0018] The ion exchanger-doped polymer membrane electrode is a polymer membrane doped with an ion exchanger adhered to the bottom of the electrode, and the membrane components are, by weight, 20%-80% membrane matrix, 20%-80% plasticizer, and the balance ion exchanger; the ion exchanger is an anion exchanger or a cation exchanger; wherein the anion exchanger is tridecylmethylammonium chloride, a tridecylmethylammonium chloride derivative, tridecylmethylammonium chloride, or a tridecylmethylammonium 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, dinonylnaphthalenesulfonic acid salt or borate, or derivatives of the above compounds;

[0019] The membrane matrix is ​​polyvinyl chloride, polybutyl acrylate, polyetherimide, rubber or sol-gel membrane; the plasticizer is o-nitrophenyl octyl ether, di-2-ethylhexyl decyl ester, dibutyl sebacate or dioctyl sebacate;

[0020] After the potential measurement, the direction of the external magnetic field is changed to achieve separation or exudation of the polypeptide functionalized magnetic beads on the membrane, thereby achieving reversible and reusable use of the electrodes.

[0021] A device for the detection method includes a detection cell, a working electrode, a reference electrode, an electrochemical external measuring device and an external magnetic field; the working electrode is a polymer membrane electrode doped with an ion exchanger, which is placed in the detection cell, and the working electrode, reference electrode and counter electrode are respectively connected to the electrochemical external measuring device.

[0022] The sensor device electrode is single-channel or multi-channel.

[0023] The device includes an external magnetic field, which can be a small flat magnet or an electromagnet.

[0024] The polymer film electrode can be a traditional polymer film electrode with internal liquid filling, a new solid-state electrode or a printed electrode.

[0025] The electrochemical external measuring device is an electrochemical workstation, an ion meter or a potentiometer.

[0026] The reference electrode may be a saturated calomel electrode or a silver-silver chloride electrode; the auxiliary electrode may be a platinum wire.

[0027] The working electrode of the printed electrode can be a carbon electrode covered with a polymer sensitive film, and the reference electrode can be a silver-silver chloride electrode.

[0028] An application of the detection method, and application of the method in evaluating the antibacterial properties of antibacterial materials.

[0029] The polypeptide is used to detect bacteria treated with different antibacterial materials, and the antibacterial performance of the antibacterial material is evaluated based on the change in potential before and after incubation of the polypeptide with the bacteria.

[0030] The above-mentioned polypeptides or their derivatives are used as recognition molecules and signal transduction molecules to interact with apoptotic bacteria. Different antibacterial material treatments lead to different bacterial apoptosis rates, which causes different changes in the electrode potential of the ion exchanger-doped polymer membrane. Based on the different potential changes, the antibacterial properties of the antibacterial materials are evaluated and the bacterial apoptosis mechanism is verified.

[0031] Alternatively, peptides or peptide derivatives can be modified onto magnetic beads to create peptide-functionalized magnetic beads. Peptides act as recognition and signaling molecules, interacting with apoptotic bacteria. Different antimicrobial material treatments lead to different bacterial apoptosis rates, resulting in different changes in electrode potential at the ion exchanger-doped polymer membrane before and after interaction with the peptide-functionalized magnetic beads. This allows for the evaluation of the antimicrobial properties of the antimicrobial material and the verification of the bacterial apoptosis mechanism.

[0032] The antibacterial materials can all cause the externalization of phosphatidylserine on the bacterial cell membrane, and can be tetracycline antibiotics such as mitomycin, oxytetracycline, chlortetracycline, etc.; β-lactam antibiotics such as ampicillin, penicillin, cephalosporin, etc.; quinolone antibiotics such as enrofloxacin, norfloxacin, ciprofloxacin, etc.; aminoglycoside antibiotics such as spectinomycin, gentamicin, kanamycin, streptomycin, etc.; antimicrobial peptides; flavonoid lignan compounds such as silymarin; phenolic compounds such as curcumin; nanomaterials such as nanosilver, nanozinc oxide, etc.

[0033] Detection principle: The present invention uses polypeptides or their derivatives as recognition molecules and signal transduction molecules, and utilizes the specific recognition of phosphatidylserine on the surface of the cell membrane of apoptotic bacteria by polypeptides to achieve the capture of apoptotic bacteria, resulting in changes in the charge and charge density of the polypeptide. Under the action of an external magnetic field, the polypeptide-functionalized magnetic beads are extracted or adsorbed to the surface of the ion exchanger-doped polymer membrane electrode, resulting in changes in the potential response, thereby achieving qualitative / quantitative detection of apoptotic bacteria. In addition, different antibacterial materials cause different apoptosis rates of bacteria. According to the changes in the potential response of the polypeptide-functionalized magnetic beads on the surface of the polymer membrane electrode before and after the action of bacteria treated with different antibacterial materials, the antibacterial properties of different antibacterial materials can be evaluated. The present invention utilizes the specific recognition of apoptotic bacteria by polypeptides, which can achieve apoptotic bacteria detection, antibacterial drug screening, material toxicity testing, apoptotic cell detection and disease screening, etc., so it has universality.

[0034] The advantages of the present invention are:

[0035] 1. Compared with the apoptosis detection method based on annexin, the recognition and capture of apoptotic bacteria by the peptide in the detection process does not require Ca 2+ existence.

[0036] 2. The present invention uses polypeptides as recognition molecules. Compared with membrane-attached proteins, polypeptides have higher stability and smaller molecular weight. They can effectively reduce the obstruction of bacterial cell walls on recognition molecules, increase the binding rate of recognition molecules with phosphatidylserine on the outside of bacterial cell membranes, and have higher sensitivity in detecting apoptotic bacteria.

[0037] 3. The present invention utilizes a polymer membrane ion-selective electrode containing an ion exchanger to achieve real-time potentiometric detection of apoptotic bacteria. An external magnetic field is used to control the extraction or adsorption of polypeptide-functionalized magnetic beads onto the surface of the polymer membrane electrode, resulting in a simple, rapid, and easy-to-operate process. Furthermore, by changing the direction of the external magnetic field, the present invention achieves separation or exudation of the polypeptide-functionalized magnetic beads from the membrane, enabling reversible and reusable electrode utilization. The electrode has a simple structure, low manufacturing cost, high stability, fast analysis speed, and ease of operation.

[0038] 4. The polypeptide-functionalized magnetic beads of the present invention can achieve rapid separation and enrichment of apoptotic bacteria in the sample under the action of an external magnetic field, eliminating the interference of the complex matrix of the background solution on the potential response. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram and actual picture of a sensor device based on a single-channel screen-printed electrode provided by an embodiment of the present invention.

[0040] Figure 2 Schematic diagram and actual picture of a sensor device based on multi-channel screen-printed electrodes provided by an embodiment of the present invention.

[0041] Figure 3 The results of phosphatidylserine detection by magnetic beads modified with selected polypeptides in the examples of the present invention are shown.

[0042] Figure 4 Figure 1 is a schematic diagram of the principle of detecting apoptotic bacteria according to an embodiment of the present invention, wherein Figure A is a schematic diagram of the capture of apoptotic bacteria by polypeptide-functionalized magnetic beads, and Figure B is a potential response diagram at the polymer membrane electrode before and after incubation of polypeptide-functionalized magnetic beads with bacteria.

[0043] Figure 5 Figure 1 shows the detection results of different concentrations of apoptotic bacteria using the selected polypeptide-modified magnetic beads in the examples of the present invention. Figure A shows the potential response of the polypeptide-functionalized magnetic beads after incubation with different concentrations of apoptotic bacteria, and Figure B shows the calibration curve of the potential change of the polypeptide-functionalized magnetic beads versus the bacterial concentration.

[0044] Figure 6These are scanning electron microscopy characterizations of the selected polypeptide-modified magnetic beads capturing apoptotic bacteria in an example of the present invention, wherein Figure A shows the morphology of untreated polypeptide-functionalized magnetic beads, and Figure B shows the morphology of the magnetic bead-bacteria complex formed after incubation of the polypeptide-functionalized magnetic beads with bacteria.

[0045] Figure 7 : This is the potential response of the polypeptide-functionalized magnetic beads incubated with bacteria with different apoptosis rates in the examples of the present invention.

[0046] Figure 8 These are the evaluation results of the antibacterial properties of various antibacterial materials in the examples of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. In the following examples, the experimental methods without specific conditions are selected according to conventional methods and conditions.

[0048] The polypeptide and its derivatives in the present invention can generate a potential response on the polymer membrane electrode doped with an ion exchanger, and the detection of the polypeptide can be achieved based on the potential response.

[0049] Furthermore, peptides and their derivatives are used as recognition molecules and signal transduction molecules. Peptides and their derivatives capture apoptotic bacteria by recognizing phosphatidylserine on the bacterial cell membrane. Since the isoelectric point of the protein on the bacterial surface is low, the apoptotic bacteria are negatively charged, resulting in changes in the charge and charge density of the peptide, which in turn causes changes in the potential of the peptide and its derivatives on the surface of the sensitive membrane electrode. Qualitative / quantitative detection of apoptotic bacteria is achieved based on the potential change.

[0050] To improve the sensitivity of peptide and apoptotic bacterial detection, peptides are modified on the surface of magnetic beads through biotin-streptavidin, and an external magnetic field is used to extract / adsorb the peptide-functionalized magnetic beads to the surface of a polymer membrane electrode doped with an ion exchanger, thereby generating a potential response. After the peptide-functionalized magnetic beads capture apoptotic bacteria, the negatively charged bacteria cause the charge and charge density on the surface of the peptide-functionalized magnetic beads to change, resulting in a change in the potential response. Based on the potential change, qualitative / quantitative detection of apoptotic bacteria is achieved. During the potential measurement, an electrochemical system is used to record the potential change between the working electrode and the reference electrode on the screen-printed electrode. The device is as follows: the working electrode and the reference electrode of the screen-printed electrode are respectively connected to the electrochemical external measurement device and inserted into a detection cell containing a background solution. The detection cell is placed on a small flat magnet.

[0051] Example 1

[0052] Devices such as Figure 1As shown, the device includes an electrochemical external measurement device, a detection cell, a screen-printed electrode, and an external magnetic field. The screen-printed electrode is placed in the detection cell, and the working electrode and reference electrode of the printed electrode are respectively connected to the electrochemical external measurement device.

[0053] 1. Preparation of Ion-Selective Polymer Membrane Electrodes

[0054] Weigh 122.5 mg of polyvinyl chloride, 122.5 mg of o-nitrophenyl octyl ether, 2.5 mg of dinonylnaphthalene disulfonic acid, and 2.5 mg of tetra(dodecyl)ammonium tetra(4-chlorophenyl)borate in 2 mL of tetrahydrofuran and stir until evenly combined. Add 10 μL of the membrane solution dropwise onto the carbon electrode of the screen-printed electrode and allow to dry before use as the working electrode.

[0055] 2. Quantitative Detection of Phosphatidylserine

[0056] A peptide (NFRLKAGAKIRFG) with selective recognition for phosphatidylserine was selected and biotin-labeled according to existing technology, namely biotin-NFRLKAGAKIRFG. Streptavidin-modified magnetic beads (commercially available) were washed five times with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride and resuspended to the original concentration. 30 μL of the resuspended streptavidin magnetic beads were mixed with 270 μL of 10 -5 After incubation for 20-30 min, the beads were washed twice with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride and resuspended to 30 μL to prepare peptide-functionalized magnetic beads for detection of apoptotic bacteria:

[0057] During the measurement, the open circuit potential measurement technique was used, 5 μL of polypeptide functionalized magnetic beads were dropped onto the working electrode, and the open circuit potential E1 was measured.

[0058] Phosphatidylserine (10 -7 -10 -5 M);

[0059] 5 μL of the peptide-functionalized magnetic beads were removed by magnetic separation, and then mixed with the phosphatidylserine solution obtained above for one hour. After incubation, the peptide-functionalized magnetic beads were washed twice with 1 mM phosphate buffer (pH 7.4) containing 1 mM sodium chloride and resuspended to 5 μL. 5 μL of the peptide-functionalized magnetic beads incubated with the phosphatidylserine solution was added dropwise to the working electrode of the screen-printed electrode, and the open circuit potential (E2) was measured.

[0060] Calculate the potential response change (potential difference between E1 and E2) caused by the peptide functionalized magnetic beads on the electrode before and after incubation with phosphatidylserine solution. Figure 3 As shown in the figure, due to the presence of phosphate groups, phosphatidylserine is negatively charged at pH 7.4. Therefore, as the concentration of phosphatidylserine increases, the charge and charge density of the polypeptide in the polypeptide functionalized magnetic beads change more, and thus the potential response of the polypeptide functionalized magnetic beads decreases (corresponding to Figure 3 The potential response curve ea) shows that the detection range of this electrode for phosphatidylserine is 10 -7 -10 -5 M; and the electrode response stabilization time after sample addition is 25-30s, and the electrode response is fast.

[0061] Example 2

[0062] Devices such as Figure 1 As shown, the screen-printed electrode is placed in a detection cell, a flat magnet is placed at the bottom of the detection cell to provide an external magnetic field, and the working electrode and reference electrode of the printed electrode are respectively connected to an electrochemical external measurement device.

[0063] 1. Preparation of Ion-Selective Polymer Membrane Electrodes

[0064] The electrode was obtained according to the preparation method described in Example 1 above.

[0065] 2. Apoptotic Bacteria Detection

[0066] A peptide (NFRLKAGAKIRFG) with selective recognition for apoptotic bacteria was selected and biotin-labeled according to existing techniques, namely biotin-NFRLKAGAKIRFG. Streptavidin-modified magnetic beads (commercially available) were washed five times with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride and resuspended to the original concentration. 30 μL of the resuspended streptavidin magnetic beads were mixed with 270 μL of 10 -5 M peptide (biotin-NFRLKAGAKIRFG) was mixed and incubated for 20-30 min. After that, it was washed twice with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride and resuspended to 30 μL to prepare peptide functionalized magnetic beads. Then, apoptotic bacteria were detected. The principle is as follows Figure 4 As shown, Figure A is a schematic diagram of the capture of apoptotic bacteria by peptide-functionalized magnetic beads, and Figure B is a potential response diagram at the polymer membrane electrode before and after incubation of peptide-functionalized magnetic beads with bacteria:

[0067] During the measurement, the open circuit potential measurement technique was used, 5 μL of peptide functionalized magnetic beads were added dropwise to the working electrode, and the open circuit potential E1 was measured.

[0068] Escherichia coli was cultured in a conventional manner, washed with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride, and then resuspended to 10 8 CFU / mL, the E. coli suspension was treated with UV to a certain apoptosis rate, and then the treated E. coli was diluted to 10 7 , 10 6 , 10 5 , 10 4 CFU / mL;

[0069] 5 μL of the peptide-functionalized magnetic beads obtained above were removed by magnetic separation, and then mixed with the apoptotic E. coli suspension obtained above at different concentrations and incubated for 1 hour. Following incubation, the peptide-functionalized magnetic beads were washed twice with 1 mM phosphate buffer (pH 7.4) containing 1 mM sodium chloride and resuspended to 5 μL. 5 μL of the peptide-functionalized magnetic beads, after incubation with bacteria, was dripped onto the working electrode of the screen-printed electrode, and the open-circuit potential (E2) was measured.

[0070] The change in potential response (the potential difference between E1 and E2) caused by the polypeptide-functionalized magnetic beads on the electrode before and after incubation with apoptotic E. coli was calculated, and the logarithm of the apoptotic E. coli concentration was plotted to draw a standard curve.

[0071] Test results such as Figure 5 As shown in Figure A, the potential response diagram of peptide functionalized magnetic beads after incubation with different concentrations of apoptotic bacteria, and Figure B is the calibration curve of the potential change of peptide functionalized magnetic beads versus bacterial concentration. The detection range of this electrode for Escherichia coli with an apoptosis rate of 50% is 10 4 -10 7 CFU / mL; As the concentration of E. coli increases, the number of apoptotic bacteria captured by the peptide functionalized magnetic beads increases, and then the potential response of the peptide functionalized magnetic beads decreases (corresponding to the potential curve fa in Figure A). The potential change value has a good linear relationship with the concentration of E. coli, and the electrode responds quickly. The morphology of the peptide functionalized magnetic beads before and after incubation with bacteria was observed using a scanning electron microscope. The results are as follows Figure 6 As shown, Figure A shows the morphology of untreated polypeptide-functionalized magnetic beads, and Figure B shows the morphology of the magnetic bead-bacteria complex formed after incubation of polypeptide-functionalized magnetic beads with bacteria. It can be seen that the polypeptide-functionalized magnetic beads successfully captured apoptotic bacteria after incubation with bacteria.

[0072] Example 3

[0073] The difference from Example 2 is that the detection device includes an electrochemical external measuring device, a detection cell, a screen-printed electrode and an external magnetic field. The screen-printed electrode is a multi-channel screen-printed electrode, including a reference electrode and multiple working electrodes. Figure 2As shown, the multi-channel screen-printed electrode is placed in a detection cell, and each working electrode and reference electrode is connected to an electrochemical external measurement device respectively.

[0074] Example 4

[0075] The difference from Example 2 is that the treatment method for Escherichia coli is as follows:

[0076] Escherichia coli was cultured in a conventional manner, washed with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride, and then resuspended to 10 7 CFU / mL, E. coli suspensions with different apoptosis rates were obtained after UV treatment for different times;

[0077] Take 5 μL of the polypeptide functionalized magnetic beads obtained above, remove the suspension by magnetic separation, and then mix them with the Escherichia coli suspensions with different apoptosis rates obtained above and incubate them for 1 hour. After incubation, wash the polypeptide functionalized magnetic beads twice with phosphate buffer solution containing 1 mM sodium chloride (1 mM, pH 7.4) and resuspend them to 5 μL and add them dropwise to the working electrode of the screen-printed electrode to measure the open circuit potential E2.

[0078] The results are as follows Figure 7 As shown in Figures a to f, the time of UV treatment of the E. coli suspension is shortened successively, and the apoptosis rate of E. coli is reduced successively. Therefore, the number of apoptotic E. coli captured by the polypeptide functionalized magnetic beads is reduced, and the potential response is increased successively.

[0079] Example 5

[0080] According to the detection device of Example 2 above, different antibacterial materials are used to treat E. coli, and the treated E. coli are subjected to potential detection. The main steps include:

[0081] A peptide (NFRLKAGAKIRFG) with selective recognition for apoptotic bacteria was selected and biotin-labeled according to existing techniques, namely biotin-NFRLKAGAKIRFG. Streptavidin-modified magnetic beads (commercially available) were washed five times with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride and resuspended to the original concentration. 30 μL of the resuspended streptavidin magnetic beads were mixed with 270 μL of 10 -5 M peptide (biotin-NFRLKAGAKIRFG), incubated for 20-30 minutes, and then washed twice with phosphate buffer solution containing 1mM sodium chloride (1mM, pH 7.4) to prepare peptide-functionalized magnetic beads, which were then used to detect apoptotic bacteria:

[0082] The open circuit potential measurement technique was used. 5 μL of peptide-functionalized magnetic beads was added dropwise to the working electrode to measure the open circuit potential E1.

[0083] Escherichia coli was cultured in a conventional manner, the culture medium was washed off and then resuspended in 1 mM sodium chloride solution to 10 7 CFU / mL. Subsequently, mitomycin C (MMC, 5μg / mL), norfloxacin (Norf, 750ng / mL), ampicillin (Amp, 5μg / mL), nanosilver (Nano-Ag, 12-15nm, 0.1mg / mL), and antimicrobial peptide (Anti-pep, 0.8μM) were incubated with E. coli for 1.5 hours. The selected concentrations were the minimum inhibitory concentrations of the antimicrobial materials.

[0084] Take 5 μL of the polypeptide functionalized magnetic beads obtained above, remove the suspension by magnetic separation, and mix them with the Escherichia coli suspension treated with the antibacterial material obtained above and incubate them for 1 hour. After incubation, wash the polypeptide functionalized magnetic beads twice with phosphate buffer solution containing 1 mM sodium chloride (1 mM, pH 7.4) and resuspend them to 5 μL and add them dropwise to the working electrode of the screen-printed electrode to measure the open circuit potential E2.

[0085] The change in electrode potential response (the potential difference between E1 and E2) caused by peptide-functionalized magnetic beads before and after incubation with apoptotic E. coli was calculated. The electrode potential change varied depending on the E. coli treated with different antimicrobial materials and the peptide-functionalized magnetic beads. The greater the change in electrode potential, the higher the apoptosis rate of the E. coli, indicating the better the antimicrobial performance of the antimicrobial material. This potential difference can be used to evaluate the antimicrobial performance of different antimicrobial materials.

[0086] Depend on Figure 8 The test results show that mitomycin and nanosilver have better antibacterial effects on Escherichia coli, followed by ampicillin and norfloxacin. Compared with other materials, the antimicrobial peptides have poor antibacterial effects.

[0087] Example 6

[0088] The device includes a detection cell, a conventional polymer membrane electrode with internal liquid filling, an electrochemical external measurement device, and an external magnetic field; the main steps include:

[0089] 1. Preparation of Ion-Selective Polymer Membrane Electrodes

[0090] Weigh 340.06 mg of polyvinyl chloride, 340.06 mg of o-nitrophenyl octyl ether, 6.94 mg of dinonylnaphthalene disulfonic acid, and 6.94 mg of tetra(dodecyl)ammonium tetra(4-chlorophenyl)borate, dissolve in 6 mL of tetrahydrofuran, stir evenly, and pour into a glass ring with an inner diameter of 5 cm fixed on a glass plate. After the tetrahydrofuran evaporates, an ion-selective electrode membrane is prepared. Use a hole punch to cut the membrane into small discs with a diameter of 3 mm and use tetrahydrofuran to stick them to the pipette tip with a PVC tube at the bottom to make an ion-selective polymer membrane electrode for use.

[0091] 2. Apoptotic Bacteria Detection

[0092] A peptide (NFRLKAGAKIRFG) with selective recognition for apoptotic bacteria was selected and biotin-labeled according to existing techniques, namely biotin-NFRLKAGAKIRFG. Streptavidin-modified magnetic beads (commercially available) were washed five times with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride and resuspended to the original concentration. 30 μL of the resuspended streptavidin magnetic beads were mixed with 270 μL of 10 -5 M peptide (biotin-NFRLKAGAKIRFG), incubated for 20-30 minutes, and then washed twice with phosphate buffer solution (1mM, pH 7.4) containing 1mM sodium chloride to prepare peptide-functionalized magnetic beads.

[0093] The open circuit potential measurement technique was used for the measurement. 5 μL of peptide-modified magnetic beads was added dropwise to the electrode to measure the open circuit potential E1.

[0094] Escherichia coli was cultured in a conventional manner, the culture medium was washed off and then resuspended in 1 mM sodium chloride solution to 10 7 CFU / mL, E. coli suspension was treated with UV to different apoptosis rates;

[0095] Take 5 μL of the polypeptide functionalized magnetic beads obtained above, remove the suspension by magnetic separation, and then mix them with the Escherichia coli suspensions with different apoptosis rates obtained above and incubate them for 1 hour. After incubation, wash the polypeptide functionalized magnetic beads twice with phosphate buffer solution containing 1 mM sodium chloride (1 mM, pH 7.4) and resuspend them to 5 μL and add them dropwise to the working electrode of the screen-printed electrode to measure the open circuit potential E2.

[0096] The potential response change (the potential difference between E1 and E2) caused by the polypeptide-functionalized magnetic beads on the electrode before and after incubation with E. coli was calculated, and the apoptosis rate of bacteria was plotted to draw a standard curve.

[0097] Example 7

[0098] The device includes a detection cell, a novel all-solid-state ion-selective polymer membrane electrode, an electrochemical external measurement device, and an external magnetic field; the main steps include:

[0099] 1. Preparation of Ion-Selective Polymer Membrane Electrodes

[0100] Preparation of a novel all-solid-state ion-selective polymer membrane electrode: Dissolve 122.5 mg of polyvinyl chloride, 122.5 mg of octyl o-nitrophenyl ether, 2.5 mg of dinonylnaphthalene disulfonic acid, and 2.5 mg of tetra(dodecyl)ammonium tetrakis(4-chlorophenyl)borate in 2 mL of tetrahydrofuran and stir thoroughly. Add 10 μL of the membrane solution dropwise to the solid-state electrode and allow to dry.

[0101] 2. Apoptotic Bacteria Detection

[0102] A peptide (NFRLKAGAKIRFG) with selective recognition for apoptotic bacteria was selected and biotin-labeled according to existing techniques, namely biotin-NFRLKAGAKIRFG. Streptavidin-modified magnetic beads (commercially available) were washed five times with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride and resuspended to the original concentration. 30 μL of the resuspended streptavidin magnetic beads were mixed with 270 μL of 10 -5 M peptide (biotin-NFRLKAGAKIRFG), incubated for 20-30 minutes, and then washed twice with phosphate buffer solution (1mM, pH 7.4) containing 1mM sodium chloride to prepare peptide-functionalized magnetic beads.

[0103] The open circuit potential measurement technique was used for the measurement. 5 μL of peptide-modified magnetic beads was added dropwise to the electrode to measure the open circuit potential E1.

[0104] Escherichia coli was cultured in a conventional manner, washed with phosphate buffer solution (1 mM, pH 7.4) containing 1 mM sodium chloride, and then resuspended to 10 7 CFU / mL, E. coli suspension was treated with UV to different apoptosis rates;

[0105] Take 5 μL of the polypeptide functionalized magnetic beads obtained above, remove the suspension by magnetic separation, and then mix them with the Escherichia coli suspensions with different apoptosis rates obtained above and incubate them for 1 hour. After incubation, wash the polypeptide functionalized magnetic beads twice and resuspend them to 5 μL and add them dropwise to the working electrode of the screen-printed electrode to measure the open circuit potential E2.

[0106] The potential response change (the potential difference between E1 and E2) caused by the polypeptide-functionalized magnetic beads on the electrode before and after incubation with E. coli was calculated, and the apoptosis rate of E. coli was plotted to draw a standard curve.

[0107] If the above embodiments can be used to detect other apoptotic bacteria (ie, bacteria whose apoptosis leads to externalization of phosphatidylserine on the cell membrane), it will further prove that the method of the present invention has a certain degree of universality.

[0108] Example 8

[0109] The difference from Example 2 is that the toxicity test of the unknown toxic substance is carried out, and the E. coli treatment method is as follows:

[0110] E. coli are treated with an unknown toxic substance and subjected to potentiometric testing. The change in potential response (the potential difference between E1 and E2) caused by the peptide-functionalized magnetic beads before and after incubation with E. coli is calculated. A larger potential difference indicates a higher apoptosis rate in E. coli. This potential difference can be used to assess the toxicity of the substance to E. coli.

[0111] Example 9

[0112] The difference from Example 2 is that the highly effective antibacterial drug is screened and the E. coli treatment method is as follows:

[0113] E. coli was treated with various drugs and subjected to potentiometric testing. The change in potential response (the potential difference between E1 and E2) caused by the peptide-functionalized magnetic beads before and after incubation with E. coli was calculated. A larger potential difference indicates a higher apoptosis rate in E. coli. This potential difference can be used to screen for highly effective antimicrobial drugs.

[0114] Example 10

[0115] Devices such as Figure 1 As shown, the screen-printed electrode is placed in a detection cell, a flat magnet is placed at the bottom of the detection cell to provide an external magnetic field, and the working electrode and reference electrode of the printed electrode are respectively connected to an external electrochemical measurement device. The working electrode printed electrode is obtained according to the preparation method described in Example 1 above.

[0116] A peptide that selectively recognizes phosphatidylserine on the surface of apoptotic cells was selected and biotin-labeled according to existing techniques. Streptavidin-modified magnetic beads (commercially available) were washed five times with 1 mM sodium chloride-containing phosphate buffer (1 mM, pH 7.4) and resuspended to the original concentration. 30 μL of the resuspended streptavidin magnetic beads were mixed with 270 μL of 10 -5 After incubation for 20-30 minutes, the beads were washed twice with phosphate buffer solution (1mM, pH 7.4) containing 1mM sodium chloride to obtain peptide-functionalized magnetic beads.

[0117] The open circuit potential measurement technique was used. 5 μL of peptide-functionalized magnetic beads was added dropwise to the working electrode to measure the open circuit potential E1.

[0118] Take 5 μL of the polypeptide-functionalized magnetic beads obtained above, remove the suspension by magnetic separation, and then mix with the apoptotic cell suspension and incubate for 1 hour. After incubation, wash the polypeptide-functionalized magnetic beads twice with phosphate buffer solution containing 1 mM sodium chloride (1 mM, pH 7.4) and resuspend them to 5 μL and drop them onto the working electrode of the screen-printed electrode to measure the open circuit potential E2.

[0119] The change in the potential response (the potential difference between E1 and E2) induced by the peptide-functionalized magnetic beads before and after incubation with apoptotic cells was calculated and plotted against the logarithm of the number of apoptotic cells to create a standard curve. Besides apoptotic cells, phosphatidylserine can be exposed to the exterior of the cell membrane in various disease states, making this method suitable for screening for diseases such as microbial and viral infections, antiphospholipid syndrome, malaria, and neoplasia.

[0120] In summary, the positively charged peptides of the present invention can generate a potential response on the surface of a polymer-sensitive membrane ion-selective electrode, which can recognize the externalized phosphatidylserine on the cell membrane of apoptotic bacteria, thereby specifically capturing the apoptotic bacteria. The negative charge of apoptotic bacteria causes changes in the peptide's charge and charge density, altering the potential response and thus enabling the detection of apoptotic bacteria. Furthermore, the magnitude of the change in the peptide's potential response can be used to evaluate the antibacterial properties of different antibacterial materials.

Claims

1. A method for detecting apoptotic bacteria using a polypeptide as a recognition molecule, characterized in that: Using polypeptides or their derivatives as recognition molecules and signal transduction molecules to capture apoptotic bacteria in the sample to be tested, the polypeptide changes the electrode potential of the polymer membrane doped with the ion exchanger, thereby achieving qualitative / quantitative detection of apoptotic bacteria; The polypeptide that recognizes apoptotic bacteria can specifically recognize and bind to phosphatidylserine, and the sequence is NFRLKAGAKIRFG; The polypeptide or its derivatives serving as recognition molecules and signal transduction molecules are modified on the surface of magnetic beads to form polypeptide-functionalized magnetic beads, which can capture apoptotic bacteria by specifically recognizing and binding to the outward-turned phosphatidylserine on the cell membrane of apoptotic bacteria.

2. The method for detecting apoptotic bacteria using a polypeptide as a recognition molecule according to claim 1, characterized in that: The polypeptide-functionalized magnetic beads capture apoptotic bacteria in the sample to be tested, resulting in changes in the charge and charge density of the polypeptides in the polypeptide-functionalized magnetic beads. Under the action of an external magnetic field, the amount of polypeptides effectively extracted from the polypeptide-functionalized magnetic beads to the polymer sensitive membrane changes, causing a change in electrode potential, thereby achieving quantitative detection of apoptotic bacteria in the liquid to be tested.

3. The method for detecting apoptotic bacteria using a polypeptide as a recognition molecule according to any one of claims 1 to 2, characterized in that: The ion exchanger-doped polymer membrane electrode is a polymer membrane doped with an ion exchanger adhered to the bottom of the electrode. The membrane components, by weight, are 20%-80% membrane matrix, 20%-80% plasticizer, and the balance is ion exchanger. The ion exchanger is an anion exchanger or a cation exchanger. The anion exchanger is tridecylmethylammonium chloride, a tridecylmethylammonium chloride derivative, tridecylmethylammonium chloride, or a tridecylmethylammonium 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, dinonylnaphthalenesulfonic acid salt or borate, or derivatives of the above compounds.

4. The method for detecting apoptotic bacteria using a polypeptide as a recognition molecule according to claim 3, characterized in that: After the potential measurement, the direction of the external magnetic field is changed to achieve separation or exudation of the polypeptide functionalized magnetic beads on the membrane, thereby achieving reversible and repeated use of the electrodes.

5. An application of the detection method according to claim 1, characterized in that: Use of the method of claim 1 in evaluating the antibacterial properties of antibacterial materials.

6. The use according to claim 5, characterized in that: The polypeptide according to claim 1 is used to detect bacteria treated with different antibacterial materials, and the antibacterial performance of the antibacterial material is evaluated based on the change in potential before and after incubation of the polypeptide with the bacteria.

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