A method and apparatus for detecting a biological analyte based on a blocking effect

Abstract

The present application relates to a polymer membrane potential type biosensor, in particular to a method and device for detecting biological analyte based on hindering effect. A solid-state polymer membrane ion selective electrode with a layer of recognition molecules on the surface is used as a working electrode, which is incubated with a sample to be detected, then inserted into a background solution to be detected, and an indicating ion capable of mutual recognition with the ion carrier in the polymer membrane of the working electrode is added. The hindering of the diffusion of the indicating ion by the binding of the recognition molecules on the working electrode and the target biological analyte on the membrane surface is used to generate a kinetic potential response change, and then the biological analyte in the sample to be detected is qualitatively and / or quantitatively detected. The present application has strong universality, simple operation, fast detection speed, low cost, and is suitable for detection under complex matrix conditions.

Description

Technical Field

[0001] This invention relates to polymer membrane potential biosensors, specifically a method and apparatus for detecting bioanalytes based on the barrier effect. Background Technology

[0002] Polymer membrane ion-selective electrodes are potentiometric sensors used for ion detection. They offer advantages such as low cost, ease of operation, and miniaturization, and are widely used in environmental monitoring, clinical diagnostics, and industrial analysis. Currently, these sensors have successfully determined a variety of analytes, including pH, electrolyte ions, carbonates, nutrients, and heavy metals. Furthermore, the emergence of novel recognition materials such as aptamers, peptides, and molecularly imprinted polymers has made the detection of bioanalytes possible. However, since the interaction between receptors and biomolecules typically does not generate measurable potential signals, specific and direct potentiometric sensing of biological species without chemical labeling remains a significant challenge.

[0003] Constructing potential sensors based on the principle of surface barrier is a promising emerging sensing strategy. In such sensors, recognition elements capable of selectively binding target molecules are typically introduced onto the surface of the sensing membrane. Surface recognition events can block the diffusion flux of ions to the ion-selective membrane interface, resulting in a potential change. In principle, any recognition mode can be used to design such sensors, including antibody-antigen binding, DNA-DNA hybridization, and artificial bioreceptor-analyte interactions, thus providing a universal method for label-free detection of various biomolecules. To date, due to the rapid and accurate control of the ion extraction process, related achievements mainly involve pulsed constant-current chronopotentiometry. This strategy has been successfully used to measure the non-specific (surfactants and polyelectrolytes) or specific (biotin-avidin and molecularly imprinted polymers) interactions between various bioanalytes and receptors. However, in these systems, macroscopic command control programs must be used to repeatedly switch between constant-potential and constant-current steps, inevitably leading to complex and cumbersome device operation. In contrast, zero-current potentiometry is more advantageous because it does not require external current or voltage. In practice, however, this passive diffusion technique relying on zero current struggles to effectively block indicator ions. Antibody-virus binding can be detected by utilizing the interference of continuous outward ion flux on the membrane surface, but this method suffers from drawbacks such as low sensitivity and long detection time. Furthermore, this non-equilibrium potential assay has poor robustness.

[0004] Therefore, there is an urgent need to develop a novel zero-current potential sensor based on the impedance effect to achieve simpler, faster, and more durable detection of biological substances. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings and provide a solution to achieve the above objective. The technical solution adopted by this invention is as follows:

[0006] A method for detecting bioanalytes based on the barrier effect uses an all-solid polymer membrane ion-selective electrode with a surface-modified recognition molecule layer as the working electrode. The electrode is inserted into the background solution to be tested, and indicator ions that can be selectively recognized by the ion carriers in the polymer membrane of the working electrode are added. The kinetic potential response change generated by the binding of the recognition molecules on the working electrode and the target bioanalyte on the membrane surface hinders the diffusion of the indicator ions, thereby qualitatively and / or quantitatively detecting the bioanalytes in the sample.

[0007] The sample to be tested contains a bioanalyte that is specifically recognized by the recognition molecule of the working electrode. The two combine to form a binding layer, which hinders the diffusion of indicator ions in the background solution to the polymer film in the working electrode, resulting in a change in ion concentration and a change in the kinetic potential response, thereby enabling qualitative and / or quantitative detection of the bioanalyte in the sample to be tested.

[0008] Specifically:

[0009] a. Immerse the working electrode in an activation solution containing indicator ions for 0.5-12 hours, and then incubate it in standard solutions containing different concentrations of the analyte; at the same time, incubate the working electrode activated by the indicator ions in a blank solution without the analyte as a control electrode;

[0010] b. Insert the above-mentioned incubated working electrode and control electrode into the measuring cell containing the background solution, respectively. After the electrodes stabilize, obtain the baseline potential. Then, add indicator ions into the measuring cell and record the potential response of the working electrode and control electrode.

[0011] c. Plot a standard curve for the target concentration based on the difference between the potential response of the working electrode and the control electrode after the addition of indicator ions;

[0012] d. After the working electrode is activated by the indicator ion, it is inserted into the sample solution to be tested and incubated. Then, it is inserted into the measuring cell containing the background solution and the potential response is recorded as the test potential response.

[0013] e. By calculating the difference between the test potential response and the control electrode potential response when plotting the standard curve, the concentration of the analyte can be obtained from the standard working curve.

[0014] The working electrode potential response and control potential response for plotting the standard curve, as well as the test potential response for detecting the sample to be tested, are all the differences between the electrode potential and the baseline potential within the same fixed time period before the potential reaches equilibrium after the addition of indicator ions; wherein, the fixed time can be 1-600s.

[0015] The activation solution is a solution containing indicator ions, and the final concentration of indicator ions in the activation solution can be 10. -6 ~10 - 1 M; where the indicator ion is a substance that can selectively recognize the ion carrier in the polymer film of the working electrode.

[0016] The background solution is a solution with or without indicator ions, wherein the concentration of indicator ions is determined based on the sensitivity of the working electrode.

[0017] The incubation time for each step can be 1 to 120 minutes.

[0018] The working electrode is a solid electrode to which a transduction layer material, a polymer film, and a recognition molecule layer are sequentially adhered.

[0019] The polymer membrane is composed of a polymer matrix material, a plasticizer, an ion carrier, and a lipophilic ion exchanger, mixed in a weight ratio of 20-60:20-60:0.1-10:0.1-10. After mixing, the mixture is dissolved in tetrahydrofuran and stirred to form a homogeneous solution. An appropriate amount of the solution is drop-coated or spin-coated onto an electrode carrier coated with a transconducting layer and left at room temperature for 4-12 hours to form a polymer membrane on the electrode carrier.

[0020] The polymer matrix material is polyvinyl chloride, polybutyl acrylate, polybutyl acrylate, polyetherimide, rubber, or sol-gel membrane; the plasticizer is o-nitrophenyl octyl ether (o-NPOE), di-2-ethylhexyl decyl ester, dibutyl sebacate, or dioctyl sebacate; the ion carrier is a cationic carrier or anionic carrier; the lipophilic ion exchanger is a cation exchanger (borate derivative or dinonylnaphthalenesulfonic acid or its salt) or an anion exchanger (quaternary ammonium salt organic compound).

[0021] The indicator ion is a substance that can selectively recognize the above-mentioned ion carriers, such as calcium ions with ETH129, potassium ions with potassium ion carrier II, and butyrylcholine with β-cyclodextrin. At the same time, the function of the above-mentioned indicator ions is only the complexation between them and the ion carriers in the polymer membrane, and they do not participate in the interaction between the recognition molecules and the bioanalytes. Therefore, any pair of indicator ions and ion carriers can be used in various detection samples.

[0022] The solid electrode is a glassy carbon electrode, a gold electrode, or a screen-printed electrode; the transconducting layer material is a conductive polymer, mesoporous carbon, porous graphene, or a graphene composite material doped with metal nanoparticles.

[0023] The transconducting layer is prepared by drop coating or electrodeposition using the above-mentioned transconducting layer material and then adhered to the bottom of the solid electrode. Furthermore, the transconducting layer formed with different transconducting layer materials is suitable for the detection of different bioanalytes. Since it only conducts without affecting the potential response and does not participate in the process of recognizing molecules and bioanalytes, it has universality.

[0024] The recognition molecule is an antibody, polypeptide, nucleic acid aptamer, bacteriophage, bacteriophage receptor-binding protein, or molecular imprint; the bioanalyte is a protein, virus, bacteria, cell, or other biological macromolecule; wherein the recognition molecule is capable of specifically recognizing the bioanalyte.

[0025] The aforementioned recognition molecules are antibodies, and their corresponding bioanalytes can be antigens, including proteins, viruses, bacteria, cells, etc., such as Synagis monoclonal antibody and respiratory syncytial virus, rabbit anti-human HCG antibody and human chorionic gonadotropin (HCG), alpha-fetoprotein antibody and alpha-fetoprotein, Candida albicans antibody and Candida albicans, and anti-human carcinoembryonic antigen antibody and cancer cells.

[0026] The identified molecule is a polypeptide, and the corresponding bioanalytes can be proteins, viruses, bacteria, or cells. The polypeptide sequences of the corresponding analytes can be obtained through screening using phage display technology or through artificial synthesis.

[0027] The recognition molecule is a nucleic acid aptamer, and the corresponding bioanalytes can be proteins, viruses, bacteria, and cells. All of these can be identified by nucleic acid aptamer screening technology, which can yield nucleic acid aptamer sequences with good affinity as recognition molecules.

[0028] The recognition molecule is a bacteriophage, and the corresponding bioanalytes can be its host bacteria, such as Escherichia coli and Escherichia coli bacteriophages MS2, T2, T4 or M13, Salmonella typhimurium and P22 bacteriophage;

[0029] The recognition molecule is a phage receptor-binding protein, and the corresponding bioanalytes can be the corresponding bacteria, such as Salmonella and Salmonella phage LPST144 tail fibrous receptor-binding protein.

[0030] The identified molecule is a molecular imprint, and the corresponding bioanalyte can be the template molecule used in the synthesis of the molecular imprint (overall imprint) or a substance containing the structure of the template molecule (fragment imprint), including but not limited to proteins, viruses, bacteria, and cells.

[0031] Meanwhile, the aforementioned recognition molecules can be formed on the polymer membrane surface through covalent fixation or surface imprinting. For example, carboxyl groups can be modified with dopamine or introduced using carboxylated PVC on the polymer membrane surface, and amino-containing peptides, nucleic acid aptamers, antibodies, or bacteriophages can be covalently fixed to the surface with carboxyl groups through NHS / EDC coupling. Alternatively, amino groups can be modified with dopamine or introduced using amino-containing PVC on the polymer membrane surface, and amino-containing peptides, nucleic acid aptamers, antibodies, or bacteriophages can be covalently fixed to the surface with carboxyl groups through glutaraldehyde coupling. Or, surface imprinting of template molecules can be synthesized on the polymer surface.

[0032] A detection device for the method is provided, comprising a working electrode and a reference electrode; one end of the working electrode and the reference electrode are respectively connected to a potentiometer via wires, and the other end is inserted into a detection cell; wherein a background solution is added to the detection cell, and the working electrode is an all-solid polymer membrane ion-selective electrode with a surface modified with a recognition molecular layer.

[0033] Add a stirring device, such as a magnetic stir bar, to the above-mentioned detection pool.

[0034] The working electrode is a solid electrode to which a transduction layer material, a polymer film, and a recognition molecule layer are sequentially adhered.

[0035] Detection Principle: The potential of an ion-selective electrode depends on the concentration of the main ion at the solution-side membrane interface. This invention utilizes surface-immobilized recognition molecules to identify and confine the target analyte to the polymer membrane surface, thereby forming a binding layer between the solution and membrane interface. Since bioanalytes are typically large in size, the presence of this binding layer hinders the diffusion of indicator ions from the solution phase to the polymer membrane interface, causing a decrease in the dynamic ion concentration at the membrane interface, and consequently a decrease in the kinetic potential signal. The higher the concentration of the target analyte in the test solution, the more target analyte is bound to the polymer membrane surface, and the stronger the hindering effect, ultimately leading to a larger change in the potential response; that is, the change in the electrode's dynamic potential signal is positively correlated with the concentration of the target analyte. Therefore, the detection of bioanalytes can be achieved using this kinetic potential response signal (i.e., the difference in potential response compared to the control electrode). Simultaneously, since the thermodynamic extraction equilibrium of the main ion at the membrane interface depends on the membrane composition and is not affected by the binding layer, the binding of the target analyte to the sensing membrane surface does not change the equilibrium potential value of the electrode response. This kinetic response signal generated by an electrode conforming to the Nernst potential response is more reliable for continuous monitoring than completely non-equilibrium potential determination methods. Furthermore, this method can quantify using the potential difference at any fixed time before equilibrium is reached, thus enabling faster quantification.

[0036] The advantages of this invention are:

[0037] 1. This invention uses a surface-fixed recognition molecule method, and the selection of recognition molecules and target objects is not limited, thus having universality.

[0038] 2. The relationship between the kinetic response signal and the concentration of the analyte is quantified by utilizing the potential change over any fixed time before equilibrium, which greatly shortens the detection time.

[0039] 3. The working electrode of the present invention conforms to the Nernst potential response, which is more reliable for continuous monitoring than the completely non-equilibrium potential measurement method.

[0040] 4. The separation, enrichment and potential detection of the sample in this invention are performed in steps, which can eliminate the interference of background ions in complex samples. Therefore, this method can be used for the detection of bioanalytes in complex samples.

[0041] 5. The electrode of this invention is simple to prepare, low in cost, and has an all-solid-state design without internal liquid filling, making it easy to store and carry, simple to operate, and suitable for on-site monitoring. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the electrode (working electrode) of the present invention (where 1 is the electrode carrier, 2 is the transduction layer, 3 is the polymer sensitive membrane, and 4 is the recognition molecule modification layer).

[0043] Figure 2 This is a schematic diagram of the testing device for the electrode of the present invention (where 1 is the working electrode, 2 is the target object, 3 is the reference electrode, 4 is the potentiometer, 5 is the detection cell, 6 is the background solution, and 7 is the magnetic stir bar with stirring function).

[0044] Figure 3 This serves as a comparison of the response of the unmodified electrode (a) and the phage-modified all-solid polymer membrane ion-selective electrode (b) to the indicator ion.

[0045] Figure 4 The equilibrium potential of a phage-modified all-solid polymer membrane ion-selective electrode after incubation with different concentrations of host bacteria.

[0046] Figure 5 The phage-modified all-solid-state polymer membrane ion-selective electrode of this invention was used to measure the potential signal response curves of different concentrations of host bacteria, indicating that the ion concentration change was 10. -5 M~3×10 -5 M.

[0047] Figure 6 The phage-modified all-solid-state polymer membrane ion-selective electrode of this invention was used to measure the potential signal response curves of different concentrations of host bacteria, indicating that the change in ion concentration was 3 × 10⁻⁶. -6 M~10 -5 M.

[0048] Figure 7 The phage-modified all-solid-state polymer membrane ion-selective electrode of this invention was used to measure the potential signal response curves of different concentrations of host bacteria, indicating that the change in ion concentration was 3 × 10⁻⁶. -6 M~10 -5 M.

[0049] Figure 8 The standard working curve for measuring different concentrations of host bacteria using the electrode of this invention indicates that the change in indicator ion concentration is 3 × 10⁻⁶. -6 M~10 -5 M.

[0050] Figure 9 This invention relates to the selectivity of the electrode for non-host bacteria.

[0051] Figure 10 This is the electrode repeated detection data of the present invention.

[0052] Figure 11 This data represents the actual concentration of host bacteria in the test solution determined using the method of this invention. Detailed Implementation

[0053] To illustrate the present invention more clearly, the invention will be further described below with reference to the accompanying drawings and embodiments. However, the specific descriptions below are illustrative and not restrictive, and should not be used to limit the scope of protection of the present invention. The scope of protection of the present invention should include all the contents of the claims.

[0054] This invention is based on an all-solid-state polymer membrane ion-selective electrode. It utilizes the binding of the recognition molecule and target analyte on the membrane surface to hinder the diffusion of indicator ions, generating a kinetic potential response change. The degree of this potential response change is positively correlated with the sample concentration. Therefore, this kinetic potential response signal (i.e., the difference in potential response compared to the control electrode) can be used to quantify bioanalytes. The working electrode of this invention conforms to the Nernst potential response, and quantification is performed using its kinetic response signal and the relationship between the analyte concentration, offering advantages of reliability and speed. The surface-immobilized recognition molecule method is suitable for various combinations of recognition molecules and target analytes. Sample separation and enrichment and potential detection are performed stepwise, eliminating interference from background ions in complex samples, which is beneficial for the detection of bioanalytes in complex samples. Simultaneously, the all-solid-state electrode structure makes it easy to store and carry. In summary, this novel bioanalyte potential detection mechanism developed in this invention has advantages such as simple operation, short detection time, low operating cost, and suitability for on-site detection, and is of great significance for the potential measurement of bioanalytes in environmental, industrial, and clinical fields.

[0055] Example 1

[0056] The potential response of a phage-modified all-solid-state polymer membrane ion-selective electrode to an indicator ion (butyrylcholine).

[0057] Fabrication of the working electrode (Au / PGR-Pt) coated with a transconducting layer:

[0058] The electrode was prepared by one-step electrochemical co-reduction of graphene oxide (GO) aqueous dispersion and H2PtCl4 on the surface of an Au electrode using a three-electrode system via cyclic voltammetry. Specifically, it was prepared using an aqueous GO solution (1.0 mg / mL) containing 0.1 M LiClO4 and 2.5 mM H2PtCl4. -1 The electrolyte solution was gold, the working electrode was a platinum sheet (10 mm × 10 mm), and the reference electrode was a saturated calomel electrode (SCE). Cyclic voltammetry electrodeposition was performed with a potential window of -1.5 V to 0 V and a scan rate of 100 mV·s. -1 The scanning circle count was 20 revolutions. After electrodeposition reduction, the electrode surface was first gently rinsed with deionized water, and then soaked in deionized water for at least 1 hour to remove GO adsorbed on the electrode surface. Finally, the electrode was soaked in deionized water again to remove LiClO4, yielding the Au / PGR-Pt electrode.

[0059] Preparation of an all-solid-state polymer membrane ion-selective electrode using butyrylcholine as the indicator ion: A mixture of 100 mg of β-cyclodextrin, sodium tetrakis(3,5-di(trifluoromethyl)phenyl)borate, polyvinyl chloride, and o-nitrophenyl octyl ether was dissolved in 1 mL of tetrahydrofuran solution at a mass percentage ratio of 1%:1%:49%:49%. The solution was stirred to form a homogeneous solution. 80 μL of the membrane solution was dropped onto an Au / PGR-Pt electrode coated with a transconductance layer and left at room temperature. After about 12 h, the tetrahydrofuran evaporated completely, yielding the all-solid-state polymer membrane ion-selective electrode.

[0060] Preparation of phage-modified all-solid-state polymer membrane ion-selective electrode:

[0061] First, the all-solid polymer membrane ion-selective electrode prepared above was immersed in a solution containing 0.5 mg·mL⁻¹ -1A thin polydopamine (PDA) layer was formed by immersing the electrode in Tris-HCl buffer (10 M, pH 8.5) for 1 hour. The electrode was then washed with deionized water and immersed in a 2 mg / mL glycine solution (10 M Tris-HCl buffer, pH 8.5) for 3 hours. In this way, glycine was covalently grafted onto the PDA coating surface via amino groups. The carboxyl groups of glycine were then chemically activated using EDC / NHS. Specifically, the functionalized electrode was immersed in 2-(N-morpholine)ethanesulfonic acid (MES) buffer (0.1 M, pH 5.5) containing 0.4 M 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and 0.1 M N-hydroxysulfonylimide (NHS) for 1 hour and washed with MES buffer to remove unreacted EDC / NHS molecules.

[0062] Subsequently, the activation electrode was mixed with phage MS2 (ATCC 15597-B1) solution (10 10 Incubate with PFU / mL solution for 3 h, then rinse with 0.02 M pH 7.4 sodium phosphate buffer (PBS) to remove unbound phages. Next, treat the electrode with 1 mM ethanolamine solution to inactivate unreacted -NHS groups, then incubate with 0.1% BSA solution for 30 min to block nonspecific adsorption sites. Finally, wash the surface with PBS and air dry. See [link to electrode structure] for details. Figure 1 .

[0063] Working electrode activation: The electrode prepared above is placed in a solution containing 10 -2 Activate in mol / L butyrylcholine solution (0.02M sodium phosphate buffer, pH 7.4) for 12 h.

[0064] Detection apparatus: The all-solid-state polymer membrane ion-selective electrode and the phage-modified all-solid-state polymer membrane ion-selective electrode obtained above are used as working electrodes (positive electrodes), and the Ag / AgCl (3M KCl) electrode is used as the reference electrode (negative electrode). The positive and negative electrodes are connected to the CHI 660E electrochemical workstation via wires (see [link to relevant documentation]). Figure 2 Select the "open circuit potential-time" technique to measure the potential value. A magnetic stirrer is provided at the bottom of the detection tank to rotate the magnetic stir bar.

[0065] Electrode Nernst potential response test: Using the above different working electrodes under magnetic stirring at 600 rpm, with 10 mL of 0.02 M pH 7.4 sodium phosphate buffer solution as the background electrolyte solution, after the baseline potentials stabilized, butyrylcholine was added sequentially to make the butyrylcholine concentration in the solution change continuously in a gradient of (1) 10 -7 (2) 3×10 -7 (3) 10 -6(4) 3×10 -6 (5) 10 -5 (6) 3×10 -5 (7) 10 -4 (8) 3×10 -4 (9)10 -3 (10) 3×10 -3 (11) 10 -2 M. A potential signal is generated after sample addition (see...). Figure 3 ).

[0066] Depend on Figure 3 The potential comparison shows that at 3×10 -6 M to 10 -2 Within the range of M, the potential response of the unmodified electrode is linear, with a Nernst response slope of 61.5 mV / decade and a detection limit of 1.7 × 10⁻⁶. -7 M. Within the same range, the modified electrode exhibited a Nernst response slope (60.7 mV / decade) and a detection limit (1.8 × 10⁻⁶). -7 M) is comparable to that before modification. Therefore, there is no significant difference in the performance of the modified phage electrode in terms of linear range, response slope, and detection limit. These data indicate that surface modification does not affect the thermodynamic extraction equilibrium of the butyrylcholine selective electrode, and the potential response of the phage-modified electrode still conforms to the conventional Nernst equation.

[0067] Example 2

[0068] The phage-modified all-solid-state polymer membrane ion-selective electrode prepared in Example 1 was used as the working electrode to determine the host bacteria (Escherichia coli, purchased from the American Culture Collection Center, accession number ATCC 15597) and record its dynamic potential response and equilibrium potential characteristics. The determination steps are as follows:

[0069] Working electrode activation: The phage-modified all-solid-state polymer membrane ion-selective electrode prepared above is activated in a solution containing 10... -2 The phage-modified all-solid polymer membrane ion-selective electrode was obtained by activating it in mol / L butyrylcholine solution (0.02M sodium phosphate buffer, pH 7.4) for 12 h.

[0070] Sample incubation: The activated phage-modified all-solid polymer membrane ion-selective electrode was inserted into PBS blank solution and solutions containing different concentrations of host bacteria (300 CFU / mL), respectively. -1 7000 CFU·mL -1 Incubate in PBS solution for 15 min.

[0071] Detection apparatus: Working electrode (positive electrode), Ag / AgCl (3M KCl) electrode as reference electrode (negative electrode), the positive and negative electrodes are connected to the CHI 660E electrochemical workstation via wires (see...). Figure 2 Select the "open circuit potential-time" technique to measure the potential value. A magnetic stirrer is installed at the bottom of the detection tank to rotate the magnetic stir bar.

[0072] Under magnetic stirring at 600 rpm, 10 mL of 3 × 10 -6 M butyrylcholine PBS solution was used as the background electrolyte solution. After the baseline potential stabilized, 7 μL of 10-method PBS solution was added. -2 M-butyrylcholine indicator ions increase the butyrylcholine concentration in the background electrolyte solution to 10. -5 M, recording different concentrations of host bacteria (300 CFU·mL) -1 7000 CFU·mL -1 The potential signal response curve after incubation (see) Figure 4 ).

[0073] Depend on Figure 4 It can be seen that, compared with the electrode after incubation in the blank solution, the equilibrium potential of the electrode after incubation in the bacterial solution did not change. However, the response time of the electrode increased with the increase of the host bacteria concentration in the sample. That is, the kinetic potential response of the electrode, rather than the thermodynamic potential response, changed, which provides experimental support for quantification using the kinetic potential response.

[0074] Example 3

[0075] The phage-modified all-solid-state polymer membrane ion-selective electrode prepared in Example 1 was used as the working electrode to determine different concentrations of host bacteria (Escherichia coli purchased from the American Culture Collection Center, accession number ATCC 15597; different concentrations were 10... 3 CFU·mL -1 5×10 3 CFU·mL -1 10 4 CFU·mL -1 The effect of indicator ion concentration on the potential response signal is determined by the following steps:

[0076] Working electrode activation: The phage-modified all-solid-state polymer membrane ion-selective electrode prepared above is activated in a solution containing 10... -2 The phage-modified all-solid polymer membrane ion-selective electrode was obtained by activating it in mol / L butyrylcholine solution (0.02M sodium phosphate buffer, pH 7.4) for 12 h.

[0077] Sample incubation: The activated phage-modified all-solid polymer membrane ion-selective electrode was inserted into PBS blank solution (control condition) and solutions containing different concentrations of host bacteria (10). 3 CFU·mL -1 5×10 3 CFU·mL -1 10 4 CFU·mL -1 Incubate in PBS solution for 15 min each.

[0078] Detection apparatus: Working electrode (positive electrode), Ag / AgCl (3M KCl) electrode as reference electrode (negative electrode), the positive and negative electrodes are connected to the CHI 660E electrochemical workstation via wires (see...). Figure 2 Select the "open circuit potential-time" technique to measure the potential value. A magnetic stirrer is installed at the bottom of the detection tank to rotate the magnetic stir bar.

[0079] Under magnetic stirring at 600 rpm, 10 mL of 3 × 10 -6 M butyrylcholine PBS solution was used as the background electrolyte solution. After the baseline potential stabilized, 7 μL of 10-method PBS solution was added. -2 M-butyrylcholine indicator ions increase the butyrylcholine concentration in the background electrolyte solution to 10. -5 M, recording different concentrations of host bacteria (10 3 CFU·mL -1 5×10 3 CFU·mL -1 10 4 CFU·mL -1 The potential signal response curve after incubation (see) Figure 5 ).

[0080] Under magnetic stirring at 600 rpm, 10 mL of 10 -5 M butyrylcholine PBS solution was used as the background electrolyte solution. After the baseline potential stabilized, 20 μL of 10 M butyrylcholine solution was added. -2 The M-butyrylcholine indicator ion increases the butyrylcholine concentration in the background electrolyte solution by 3 × 10⁻⁶. -5 M, recording different concentrations of host bacteria (10 3 CFU·mL -1 5×10 3 CFU·mL -1 10 4 CFU·mL -1 The potential signal response curve after incubation (see) Figure 6 ).

[0081] Depend on Figure 5 and Figure 6 In comparison, when using a lower concentration of indicator ions, the changes in the potential response of the sensor exposed to different concentrations of bacterial solutions were very significant. However, when using a higher concentration of indicator ions, the difference in potential response between the sensor and the sensor after incubation with different concentrations of bacterial solutions was not very significant. Therefore, the choice of indicator ion concentration can affect the sensitivity of the working electrode and needs to be optimized in practical applications.

[0082] Example 4

[0083] The phage-modified all-solid-state polymer membrane ion-selective electrode prepared in Example 1 was used as the working electrode to determine different concentrations of host bacteria (Escherichia coli purchased from the American Culture Collection Center, accession number ATCC 15597; different concentrations were 3.0 × 10⁻⁶). 2 1.0×10 3 3.0×10 3 5.0×10 3 7.0×10 3 1.0×10 4 1.3×10 4 2.0×10 4 CFU·mL -1 The determination steps are as follows:

[0084] Working electrode activation: The phage-modified all-solid-state polymer membrane ion-selective electrode prepared above is activated in a solution containing 10... -2 The phage-modified all-solid polymer membrane ion-selective electrode was obtained by activating it in mol / L butyrylcholine solution (0.02M sodium phosphate buffer, pH 7.4) for 12 h.

[0085] Sample incubation: The activated phage-modified all-solid polymer membrane ion-selective electrode was inserted into PBS blank solution (control condition) and PBS solution containing different concentrations of bacteria and incubated for 15 min respectively.

[0086] Detection apparatus: Working electrode (positive electrode), Ag / AgCl (3M KCl) electrode as reference electrode (negative electrode), the positive and negative electrodes are connected to the CHI 660E electrochemical workstation via wires (see...). Figure 2 Select the "open circuit potential-time" technique to measure the potential value. A magnetic stirrer is installed at the bottom of the detection tank to rotate the magnetic stir bar.

[0087] Under magnetic stirring at 600 rpm, 10 mL of 3 × 10 -6 M butyrylcholine PBS solution was used as the background electrolyte solution. After the baseline potential stabilized, 7 μL of 10-method PBS solution was added. -2M-butyrylcholine indicator ions increase the butyrylcholine concentration in the background electrolyte solution to 10. -5 M, recording different concentrations of host bacteria (b. 3.0 × 10⁻⁶). 2 c.1.0×10 3 ,d.3.0×10 3 e.5.0×10 3 f.7.0×10 3 g.1.0×10 4 h.1.3×10 4 i.2.0×10 4 CFU·mL -1 The potential signal response curve after incubation (see) Figure 7 Read the potential response over 60 seconds and calculate the potential values ​​of the electrodes after incubation with different concentrations of bacteria, comparing them with those of the electrodes incubated under control conditions (see [reference]). Figure 7 The difference between the median curve (a) and the corresponding bacterial concentration can be used to plot the standard working curve (see [reference]). Figure 8 The detection limit of this electrode was calculated to be 100 CFU·mL using the 3σ method (n=7). -1 The detection limit is comparable to that of previously reported chronopotential sensors based on the barrier effect (Liang, R.; Ding, J.; Gao, S.; Qin, W. Angew. Chem. Int. Ed. Engl. 2017, 56(24), 6833-6837) (50 CFU·mL). -1 Our proposed method does not require the application of additional voltage and current, making it superior in terms of universality and simplicity. Compared with other sensors with zero current impedance (Ozdemir, MS; Marczak, M.; Bohets, H.; Bonroy, K.; Royalans, D.; Stuyver, L.; Vanhoutte, K.; Pawlak, M.; Bakker, E. Anal. Chem. 2013, 85(9), 4770-6) (which require 20 minutes to obtain a potential signal of about 2mV), the electrode of this invention saves more time (only 60s in this embodiment).

[0088] To determine the concentration of bacteria of unknown concentration, under the same magnetic stirring conditions, a selective electrode was inserted into a bacterial solution of unknown concentration for incubation, followed by the addition of 7 μL of 10... -2 M-butyrylcholine indicator ions were added to the measuring cell to adjust its concentration from 3 × 10⁻⁶. -6 M is changed to 10 -5M generates a potential signal, reads the potential change value within 60 seconds and calculates the difference with the control condition, and calculates the corresponding concentration according to the standard working curve. This process is simulated using the standard addition method, and the recovery rate is between 92% and 96%, indicating that the electrode of this invention can accurately determine bacterial concentration (see...). Figure 11 ).

[0089] Example 5

[0090] The electrode of this invention is used to determine the selectivity for other bacteria during the measurement process. The measurement steps are as follows:

[0091] The phage-modified all-solid-state polymer membrane ion-selective electrode prepared in Example 1 was used as the working electrode to determine bacteria (Escherichia coli purchased from the U.S. Culture Collection, accession number ATCC 15597, labeled E. coli H) and five other non-host bacteria: Escherichia coli O157:H7 (labeled E. coli O), Staphylococcus aureus (labeled SA), Pseudomonas aeruginosa (labeled PA), Bacillus subtilis (labeled BS), and Vibrio parahaemolyticus (labeled VP). All the above strains were purchased from the U.S. Culture Collection, with accession numbers ATCC43889, ATCC29213, ATCC27853, ATCC 6051, and ATCC 33847, respectively. The determination procedure is as follows:

[0092] Working electrode activation: The phage-modified all-solid-state polymer membrane ion-selective electrode prepared above is activated in a solution containing 10... -2 The phage-modified all-solid polymer membrane ion-selective electrode was obtained by activating it in mol / L butyrylcholine solution (0.02M sodium phosphate buffer, pH 7.4) for 12 h.

[0093] Sample incubation: The activated phage-modified all-solid polymer membrane ion-selective electrode was inserted into a PBS blank solution (control conditions) containing 10... 4 CFU·mL -1 The host bacteria and 10 5 CFU·mL -1 Incubate the other five non-host bacteria in PBS solution for 15 min.

[0094] Detection apparatus: Working electrode (positive electrode), Ag / AgCl (3M KCl) electrode as reference electrode (negative electrode), the positive and negative electrodes are connected to the CHI 660E electrochemical workstation via wires (see...). Figure 2 Select the "open circuit potential-time" technique to measure the potential value. A magnetic stirrer is installed at the bottom of the detection tank to rotate the magnetic stir bar.

[0095] Under magnetic stirring at 600 rpm, 10 mL of 3 × 10 -6 M butyrylcholine PBS solution was used as the background electrolyte solution. After the baseline potential stabilized, 7 μL of 10-method PBS solution was added. -2 M-butyrylcholine indicator ions increased the butyrylcholine concentration in the solution to 10. -5 M, record the potential signal response curve. Read the potential response over 60 seconds and calculate the difference between the potential response of the electrode incubated with different bacteria and the electrode incubated under control conditions (see [reference]). Figure 9 ).

[0096] Depend on Figure 9 As can be seen, although the concentration of non-host bacteria is 10 times higher than that of the host strain, the change in electrode potential response is almost negligible compared to that of the host strain. Therefore, the electrode of the present invention has good selectivity for host bacteria.

[0097] Example 6

[0098] The reusability was measured using the electrode of this invention. The measurement steps are as follows:

[0099] Using the same phage-modified all-solid-state polymer membrane ion-selective electrode prepared in Example 1 as the working electrode, the 500 CFU·mL was repeatedly measured. -1 The host bacteria (Escherichia coli, purchased from the American Culture Collection Center, accession number ATCC 15597) were used. The assay procedure is as follows:

[0100] Working electrode activation: The phage-modified all-solid-state polymer membrane ion-selective electrode prepared above is activated in a solution containing 10... -2 The phage-modified all-solid polymer membrane ion-selective electrode was activated in mol / L butyrylcholine solution (0.02M sodium phosphate buffer, pH 7.4) for 12 h to obtain the activated phage-modified all-solid polymer membrane ion-selective electrode. Control incubation conditions: The activated phage-modified all-solid polymer membrane ion-selective electrode was incubated in PBS blank solution for 15 min.

[0101] Detection apparatus: Working electrode (positive electrode), Ag / AgCl (3M KCl) electrode as reference electrode (negative electrode), the positive and negative electrodes are connected to the CHI 660E electrochemical workstation via wires (see...). Figure 2Select the "open circuit potential-time" technique to measure the potential value. A magnetic stirrer is installed at the bottom of the detection tank to rotate the magnetic stir bar.

[0102] Control potential detection: Under magnetic stirring at 600 rpm, 10 mL of 3 × 10⁻⁶ mol / L was used. -6 M butyrylcholine PBS solution was used as the background electrolyte solution. After the baseline potential stabilized, 7 μL of 10-method PBS solution was added. -2 M-butyrylcholine indicator ions increased the butyrylcholine concentration in the solution to 10. -5 M, record the potential signal response curve of the working electrode under the control condition, and read the potential response within 60s of the potential signal response curve.

[0103] Sample testing: Insert the working electrode, which has undergone the above potential detection, into a container containing 500 CFU / mL. -1 The host bacteria were incubated in PBS solution for 15 min. Potentiometric detection was performed using the same apparatus and procedure as the working electrode after incubation under control conditions, and the result was recorded at 500 CFU / mL. -1 The potential signal response curve of the working electrode after incubation with host bacteria was recorded. The potential response within 60 seconds was read, and the difference in working electrode potential response (the difference between the potential response after incubation in the bacterial solution and the potential response after incubation under control conditions) was calculated. Working electrode regeneration and testing: After each host bacteria measurement, the working electrode was regenerated by immersing it in 10 mg / mL lysozyme solution (10 mmol Tris-HCl, pH 8.0) for 20 min. The control condition potential detection and sample detection procedures were repeated. The potential signal response curves of the regenerated working electrode after incubation under control conditions and sample conditions were recorded. The control potential test and the sample test after regeneration were counted as one test cycle. The potential response within 60 seconds was read, and the difference in potential response for each test cycle of the regenerated electrode (the difference between the potential response after incubation in the bacterial solution and the potential response after incubation under control conditions) was calculated.

[0104] The potential response signal curve obtained from the above process (see) Figure 10It can be seen that the working electrode can be regenerated and repeated. Calculations show that in four reversible detections of a 500 CFU / mL bacterial sample, the standard deviation (RSD%) of the electrode potential response difference is 1.2%, indicating that the electrode of the present invention has good reproducibility and stability in bacterial detection. Compared with other zero-current methods (Ozdemir, MS; Marczak, M.; Bohets, H.; Bonroy, K.; Roymans, D.; Stuyver, L.; Vanhoutte, K.; Pawlak, M.; Bakker, E. Anal. Chem. 2013, 85(9), 4770-6), the updated electrode needs to re-establish a steady-state diffusion state. The electrode of the present invention is fully activated, and there is no ion diffusion process due to the absence of an indicator ion concentration gradient inside the polymer film, which conforms to the Nernst response of thermodynamic equilibrium. It does not need to re-establish a diffusion steady state, thus exhibiting better stability.

[0105] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for detecting bioanalytes based on barrier effects, characterized in that: Using an all-solid-state polymer membrane ion-selective electrode with a surface-modified recognition molecular layer as the working electrode, the electrode is incubated with the sample to be tested and then inserted into the background solution to be tested. Indicator ions that can recognize each other with the ion carriers in the polymer membrane of the working electrode are added. The kinetic potential response change generated by the binding of the recognition molecules on the working electrode and the target bioanalyte on the membrane surface hinders the diffusion of the indicator ions, thereby qualitatively and / or quantitatively detecting the bioanalyte in the sample to be tested. The sample to be tested contains a bioanalyte that is specifically recognized by the working electrode recognition molecule. The two combine to form a binding layer, which hinders the diffusion of indicator ions in the background solution to the polymer film in the working electrode, resulting in a change in the ion concentration at the polymer film interface and a change in the kinetic potential response, thereby enabling qualitative and / or quantitative detection of the bioanalyte in the sample to be tested. Specifically: a. Immerse the working electrode in an activation solution containing indicator ions for 0.5-12 h, and then incubate it in standard solutions containing different concentrations of the analyte; at the same time, incubate the working electrode activated by the indicator ions in a blank solution without the analyte as a control electrode; b. Insert the above-mentioned incubated working electrode and control electrode into the measuring cell containing the background solution, respectively. After the electrodes stabilize, obtain the baseline potential. Then, add indicator ions into the measuring cell and record the potential response of the working electrode and control electrode. c. Plot a standard curve for the target concentration based on the difference between the potential response of the working electrode and the control electrode after the addition of indicator ions; d. After the working electrode is activated by the indicator ion, it is inserted into the sample solution to be tested and incubated. Then, it is inserted into the measuring cell containing the background solution and the potential response is recorded as the test potential response. e. By calculating the difference between the test potential response and the control electrode potential response when plotting the standard curve, the concentration of the analyte can be obtained from the standard working curve. The activation solution is a solution containing indicator ions, and the final concentration of indicator ions in the activation solution can be 10. -6 ~ 10 -1 M; wherein, the indicator ion is capable of recognizing the ion carrier in the polymer film of the working electrode; the background solution is a solution containing or not containing the indicator ion; The recognition molecule is an antibody, polypeptide, nucleic acid aptamer, bacteriophage, or bacteriophage receptor-binding protein; the bioanalyte is a protein, virus, bacteria, cell, or other biological macromolecule; wherein the recognition molecule is capable of specifically recognizing the bioanalyte.

2. The method for detecting bioanalytes based on the barrier effect according to claim 1, characterized in that: The incubation time for each step can be 1 to 120 minutes.

3. The method for detecting bioanalytes based on the barrier effect according to claim 1, characterized in that: The working electrode is a solid electrode to which a transduction layer material, a polymer film, and a recognition molecule layer are sequentially adhered.

4. The method for detecting bioanalytes based on the barrier effect according to claim 3, characterized in that: The polymer membrane is composed of a polymer matrix material, a plasticizer, an ion carrier, and a lipophilic ion exchanger, mixed in a weight ratio of 20-60:20-60:0.1-10:0.1-10. After mixing, the mixture is dissolved in tetrahydrofuran and stirred to form a homogeneous solution. An appropriate amount of the solution is drop-coated or spin-coated onto an electrode carrier coated with a transconducting layer and left at room temperature for 4-12 hours to form a polymer membrane on the electrode carrier.

5. The method for detecting bioanalytes based on the barrier effect according to claim 3, characterized in that: The solid electrode is a glassy carbon electrode, a gold electrode, or a screen-printed electrode; the transconducting layer material is a conductive polymer, mesoporous carbon, porous graphene, or a graphene composite material doped with metal nanoparticles.

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