A new quantitative method for polyion-selective electrodes

Abstract

This invention relates to a quantitative method for polyion-selective electrodes, specifically a novel method for rapid, accurate, and sensitive quantification based on the initial potential change rate of a polyion-selective electrode. Using an all-solid-state ultrathin polymer membrane polyion-selective electrode as the working electrode, it is inserted into the sample to be tested, causing the target polyions to accumulate at the membrane interface, generating an initial potential change rate. The concentration of polyions in the sample is then obtained based on the corresponding value of the initial potential change rate of the sample on a standard curve. This novel quantitative method for polyion-selective electrodes developed in this invention has advantages such as simple operation, short detection time, low operating cost, and suitability for on-site detection. It is of great significance for the direct potential determination of polyions and for counterion determination, enzyme assays, and immunoassays using polyion electrodes as signal converters.

Description

Technical Field

[0001] This invention relates to a quantitative method for polyion-selective electrodes, specifically a novel method for rapid, accurate, and sensitive quantification based on the rate of change of the initial potential of a polyion-selective electrode. Background Technology

[0002] Polymer membrane polyion-selective electrodes are potential sensors used for detecting multi-charged ions. They offer advantages such as low cost and simple operation, and include polycation-selective electrodes and polyanion-selective electrodes. In the polycation-selective electrode, the sensitive membrane is doped with a cation exchanger (borate derivative or dinonylnaphthalenesulfonic acid or its salt) as the active site, while the active site of the anion-selective electrode is an anion exchanger (quaternary ammonium salt organic compounds). The working principle of this type of sensor is as follows: polyions in the sample solution can form ion pairs with the ion exchanger in the sensitive membrane, and are thus selectively extracted into the sensitive membrane. Simultaneously, polyions continuously transport from the sample solution bulk phase to the sample solution-sensitive membrane interface, forming an inward ion flow in the aqueous phase; the polyions extracted into the membrane further transport from the interface to the membrane bulk phase, forming an inward ion flow in the membrane phase. When the ion flows of polyions in the aqueous and membrane phases reach kinetic equilibrium, a quasi-steady-state potential response is observed. The difference between this potential response value and the baseline potential is the potential signal, which is positively correlated with the polyion concentration in the aqueous phase.

[0003] Currently, traditional potentiometric analysis methods for polyion detection mainly include direct potentiometry or potentiometric titration, which can detect the potentials of a range of polycations (such as protamine, molecular folds, and dendritic compounds) and polyanions (such as heparin, dextran sulfate, DNA, and chondroitin sulfate). Additionally, antigen-antibody recognition reactions, polyion-cleaving enzymes, and their inhibitors and activators can also be indicated via polyion electrodes. However, direct potentiometry requires reading the potential signal at a quasi-steady state, which often takes a long time for low-concentration polyions, affecting detection efficiency. Potentiometric titration has the advantage of being unaffected by lipophilic small ions in complex samples, but its apparatus is complex, it requires waiting for the titration equilibrium potential to appear, and its accuracy depends on reducing the titration rate, leading to longer detection times and hindering improved detection efficiency.

[0004] Furthermore, further improvements in the sensitivity of polyion-selective electrodes have attracted widespread attention. The potential of polymer membrane polyion-selective electrodes depends on the polyion concentration at the solution-side membrane interface, and its sensitivity is related to the ion current intensity in both the aqueous and membrane phases. Researchers have attempted to suppress diffused ion currents in the membrane phase by reducing the proportion of plasticizers and the content of ion carriers, thereby promoting polyion enrichment at the membrane interface and improving sensitivity. However, due to the presence of unbound ion carriers in the membrane phase, ion diffusion within the membrane phase cannot be completely eliminated. It has been demonstrated that ultrathin films, due to their spatial limitations, can fundamentally prevent this intramembrane diffusion, which is beneficial for improving sensitivity. However, this strategy of ultrathin films has not yet been applied in the field of polyion-selective electrodes.

[0005] Therefore, developing a new quantitative method for polyion-selective electrodes to achieve more sensitive and rapid detection of polyions is of great significance for the efficient, accurate, and low-cost determination of polyions. Summary of the Invention

[0006] The purpose of this invention is to provide a new method for rapid, accurate, and sensitive quantification based on the rate of change of the initial potential of a polyion-selective electrode, addressing the aforementioned shortcomings.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A novel quantitative method for polyion-selective electrodes uses an all-solid-state ultrathin polymer film polyion-selective electrode as the working electrode, which is inserted into the sample to be tested. The polyions to be tested accumulate at the membrane interface, generating an initial potential change rate. The concentration of polyions in the sample is obtained by calculating the corresponding value of the initial potential change rate of the sample on a standard curve.

[0009] The standard curve is obtained by adding the working electrode to solutions containing different concentrations of the polyions to be detected, recording the change in the initial potential of the electrode after adding different concentrations of polyions using a potentiometer, calculating the rate of change of the initial potential, and plotting the standard working curve based on the rate of change of the initial potential and the concentration.

[0010] The initial potential change rate is the ratio of the potential signal (the difference between the potential value at that time point and the baseline) to the time before reaching the quasi-steady-state equilibrium potential within a fixed time period; wherein, the fixed time period can be 1-300s.

[0011] When the polyion to be detected has the opposite charge to the polyion selectively responded to by the working electrode, an indicator polyion (i.e., a polyion selectively responded to by the working electrode that can electrostatically complex with the polyion to be detected) is added to different concentrations of solutions or test solutions containing the polyion to be detected to plot the standard curve. The standard curve is plotted by measuring the initial potential change of the remaining indicator polyion after complexing with the polyion to be detected with the polyion of opposite charge at different concentrations, and the concentration of the polyion to be detected in the test solution is detected.

[0012] The amount of indicator polyion added is such that it can completely electrostatically complex the polyions in the test solution, with some remaining (it is generally selected according to the type of indicator polyion, the binding ratio with the counterion, and the linear range of the electrode).

[0013] The all-solid-state ultrathin polymer film polyion-selective electrode consists of a solid electrode with a transduction layer material adhered to the bottom, and an ultrathin polymer film adhered to the surface of the transduction layer material. The thickness of the ultrathin polymer film is 200 nm-20 μm, preferably 5 μm or less.

[0014] The specific testing method is as follows

[0015] a. Immerse the working electrode in a Tris-HCl buffer solution containing 0.12M NaCl for 0.5-3 hours to activate it, and then insert it into a measuring cell containing the buffer solution. After the electrode stabilizes, obtain the baseline potential.

[0016] b. Add the polyions to be detected at different standard concentrations into the measuring cell. The polyions accumulate on the surface of the electrode membrane, causing changes in the membrane interface potential.

[0017] c. Plot a standard curve of polyion concentration based on the rate of change of initial potential after the addition of polyions;

[0018] d. The activated working electrode is directly inserted into the measuring cell containing the sample to be tested to generate a sample potential signal; the concentration of the polyion to be tested is obtained by comparing the initial potential change rate of the sample solution with the standard working curve.

[0019] When the charge of the polyion to be detected is opposite to that of the polyion selectively responded to by the working electrode, after inserting the working electrode in steps b) and d), a polyion that can electrostatically complex with the polyion to be detected is added as an indicator polyion.

[0020] The polyion to be detected is a polycation or a polyanion; the polycation is protamine, polycationic polypeptide, polyethylenediamine dendritic compound, or polypropyleneimine dendritic compound; the polyanion is heparin, sodium pentose polysulfate, nucleic acid, carrageenan, dextran sulfate, chondroitin polysulfate, or persulfated chondroitin polysulfate.

[0021] The carrier in the working 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.

[0022] The polymer membrane is composed of a polymer matrix material, a plasticizer, an ion carrier, and a lipophilic inert salt, mixed in a weight ratio of 20-60:20-60:0.1-10:0.1-10. After mixing, it is dissolved in an excess of tetrahydrofuran and stirred to form a homogeneous solution. Then, it is diluted 10-300 times with tetrahydrofuran. An appropriate amount of the diluted 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 an ultrathin polymer sensitive membrane on the electrode carrier.

[0023] The polymer matrix material is polyvinyl chloride, polybutyl acrylate, polybutyl acrylate, polyetherimide, rubber, or sol-gel film; the plasticizer is o-nitrophenyl octyl ether (o-NPOE), di-2-ethylhexyl decyl ester, dibutyl sebacate, or dioctyl sebacate; the ion carrier is tri-dodecylmethyl ammonium chloride, tri-tetradecylmethyl ammonium chloride, dinonylnaphthalene sulfonic acid, dinonylnaphthalene sulfonate, or borate derivative; the lipophilic inert salt is tetra(dodecyl)-tetra(4-chlorophenyl)borate ammonium (ETH500).

[0024] Detection Principle: The potential of a polyion-selective electrode depends on the polyion concentration at the solution-side membrane interface. Its sensitivity is related to the ion current intensity in the aqueous and membrane phases. This invention utilizes the spatial limitation of the ultrathin polymer sensitive membrane to prevent the polyions extracted into the membrane from diffusing further into the membrane phase from the interface, thus suppressing the ion current within the membrane phase. This accelerates the enrichment of the target polyions at the membrane interface, causing a significant enhancement of the potential signal, i.e., an increased rate of initial potential change. This makes it easier for low-concentration polyions to accumulate on the membrane surface, thereby improving the electrode's sensitivity. Since the ion current intensity in the aqueous phase is positively correlated with the polyion concentration, the real-time potential signal of the thin-film electrode is limited by the ion current intensity in the aqueous phase and is positively correlated with the target polyion concentration. Therefore, polyion detection can be achieved using this real-time non-equilibrium potential response signal (i.e., the rate of initial potential change before potential equilibrium). Furthermore, thanks to the improved electrode response sensitivity of this invention, sensitive detection of low-concentration polyions can be achieved, avoiding the drawback of traditional polyion-selective electrodes that require a long waiting time for low-concentration polyions to reach a quasi-steady state before reading the potential signal for quantification.

[0025] Simultaneously, since polycations and polyanions form potential-free complexes, this can be used for the detection of polyions with opposite charges. Taking the detection of polyanions using a polycation-selective electrode as an example, a fixed amount of polycation is added to the test solution. The electrostatic complexation of the polyanions in the test solution reduces the concentration of the added free polycation, causing a decrease in the potential signal. Therefore, further utilizing the quantitative method based on the initial potential rate described above, using a polycation-selective electrode as an example, the polycation-selective electrode acts as a signal converter. By adding polycations as indicator ions to the test solution and detecting the concentration of the indicator ions remaining after the polyanion to be detected complexes, direct detection of polyanions with opposite charges can be achieved. Similarly, the detection of polycations can be achieved using a polyanion-selective electrode.

[0026] The advantages of this invention are:

[0027] 1. This invention uses the relationship between the initial potential change rate and the concentration of the analyte for quantification. It utilizes the potential change within any fixed time before quasi-steady-state equilibrium, eliminating the need to wait for the quasi-steady-state response as with traditional polyion electrodes, thus greatly shortening the detection time for polyions, especially low-concentration polyions.

[0028] 2. The electrode of this invention has high sensitivity and can achieve direct detection of low-concentration polyions.

[0029] 3. The electrode of this invention can realize direct potential detection of counterions by utilizing the polyion complexation phenomenon, without the need for the complex equipment and cumbersome steps of traditional titration, and can also eliminate the interference of background ions in complex samples. Therefore, this method can be directly used for the detection of polyions in complex samples.

[0030] 4. 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

[0031] Figure 1 This is a schematic diagram of the electrode of the present invention (where 1 is the electrode carrier, 2 is the transconducting layer, and 3 is the polymer sensitive membrane).

[0032] Figure 2 This is a schematic diagram of the testing device for the electrode of the present invention (where 1 is the electrode carrier, 2 is the transconducting layer, 3 is the polymer sensitive membrane, 4 is the external reference electrode, 5 is the potentiometer, 6 is the detection cell, 7 is the detection liquid, and 8 is the magnetic stir bar with stirring function).

[0033] Figure 3 This serves as a comparison of the protamine response of the electrode of this invention and a conventional polymer membrane protamine-selective electrode of varying thicknesses.

[0034] Figure 4The effect of the thickness of the protamine selective electrode polymer film on the protamine response sensitivity of the present invention.

[0035] Figure 5 The potential signal response curves of different concentrations of protamine sulfate were measured using the electrode of this invention.

[0036] Figure 6 This invention provides a standard working curve for measuring different concentrations of protamine sulfate using the electrode.

[0037] Figure 7 The graph shows the concentration curve of protamine in the test solution when the method of the present invention was actually used. Detailed Implementation

[0038] 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.

[0039] This invention is based on an ultrathin polyion-sensitive membrane. It utilizes the principle that the thin film accelerates the accumulation of analyte polyions at the membrane interface, enhancing the potential signal and improving electrode sensitivity. It exhibits a non-equilibrium potential response that increases over time and is positively correlated with sample concentration. Therefore, unlike traditional direct potential detection methods that require reading the potential signal at a quasi-steady state for quantification, this invention, with its improved electrode sensitivity, can utilize this non-equilibrium potential response signal to detect polyions. Specifically, the electrode potential changes after adding different concentrations of polyions are recorded using a potentiostat to obtain the initial potential change rate at different concentrations. A standard working curve is plotted using the initial potential change rate against the concentration, and the polyion concentration in the unknown sample is obtained by comparing it with the standard working curve. Furthermore, since polycations and polyanions form potential-free complexes, this polyion-selective electrode can also act as a signal converter to directly detect polyions with opposite charges, eliminating the need for complex potentiometric titration devices. Additionally, the all-solid-state electrode structure makes it easy to store and carry. In summary, the novel quantitative method for polyion-selective electrodes developed in this invention has advantages such as simple operation, short detection time, low operating cost, and suitability for on-site detection. It is of great significance for the direct potential determination of polyions and the determination of counterions, enzymes, and immunoassays using polyion electrodes as signal converters.

[0040] Example 1

[0041] Sensitivity determination of highly sensitive polycationic selective electrode.

[0042] Preparation of the working electrode coated with a transconductance layer: The Au / PGR-Pt electrode was prepared by one-step electrochemical co-reduction of graphene oxide (GO) aqueous dispersion and H2PtCl4 on the surface of the 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.

[0043] Preparation of the polycationic selective working electrode: A mixture of 100 mg of dinonylnaphthalenesulfonic acid, tetra(dodecyl)-tetra(4-chlorophenyl)borate ammonium, polyvinyl chloride, and o-nitrobenzeneoctyl 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. After being placed at room temperature for about 12 hours, the tetrahydrofuran evaporated completely, and a polycationic selective electrode with a conventional thickness (150 μm) was obtained. For the working electrode, 50 μL, 200 μL, and 400 μL of membrane solution were diluted to 1 mL with tetrahydrofuran. 40 μL of each diluted membrane solution was then dropped onto the Au / PGR-Pt electrode coated with the transduction layer. After approximately 4 hours at room temperature, the tetrahydrofuran evaporated, forming uniform polymer sensitive films of different thicknesses on the aforementioned working electrode (Au / PGR-Pt electrode). The polymer sensitive film thicknesses were approximately 5 μm, 20 μm, and 40 μm, thus obtaining thin-film working electrodes with different sensitive film thicknesses. For specific electrode structures, please refer to [link to specific electrode structure description]. Figure 1 .

[0044] Working electrode activation: The above-mentioned thin-film working electrode was activated for 1 h in a Tris-HCl buffer solution (pH 7.4) containing 0.12 mol / L NaCl, while the conventional thickness working electrode was activated for 12 h.

[0045] Detection apparatus: The polycation-selective electrodes of the polymer sensitive films of different thicknesses 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 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.

[0046] Electrode sensitivity enhancement test: Using the above-mentioned different working electrodes (5μm, 150μm), under magnetic stirring at 3000rpm, with 15mL of Tris-HCl buffer solution (pH 7.4) containing 0.12mol / L NaCl as the background electrolyte solution, after the baseline potentials stabilized, different concentrations of protamine (1μg / mL or 2μg / mL) were added. Potential signals were generated after sample addition, and the sensitivity of each electrode was measured (see [reference]). Figure 3 ).

[0047] Depend on Figure 3 The sensitivity comparison shows that within 150 seconds, the conventionally thick polymer-sensitive membrane working electrode did not exhibit a significant potential response to protamine at concentrations of 1 μg / mL or even 2 μg / mL, while the 5 μm ultrathin membrane working electrode showed a potential response of 24 mV to a 1 μg / mL protamine sample and a high response of 80 mV to a 2 μg / mL protamine sample. This demonstrates that the ultrathin polymer-sensitive membrane electrode of this invention significantly improves sensitivity, enabling quantitative detection of protamine based on initial potential changes or their rates.

[0048] Simultaneously, the sensitivity was further tested using working electrodes with polymer sensitive membranes of different thicknesses. Using the aforementioned working electrodes (5μm, 20μm, 40μm), under magnetic stirring at 3000rpm, with 15mL of Tris-HCl buffer solution (pH 7.4) containing 0.12mol / L NaCl as the background electrolyte solution, after the baseline potentials stabilized, different concentrations of protamine sulfate (0.4μg / mL or 0.8μg / mL) were added. Potential signals were generated after sample addition, and the sensitivity of each electrode was measured (see [reference]). Figure 4 ).

[0049] Depend on Figure 4 Sensitivity is evident; as the thickness of the electrode sensitive film increases, the potential signal decreases significantly. Beyond 20 μm, the sensitivity cannot be significantly improved, confirming that ultrathin films can indeed improve the sensitivity of polyion-selective electrodes, and the thinner the sensitive film, the higher the sensitivity.

[0050] Example 2

[0051] The highly sensitive polycation-selective electrode (polymer sensitive membrane thickness of 5 μm) prepared in Example 1 was used as the working electrode to determine protamine. The determination steps are as follows:

[0052] Working electrode activation: The above electrode was activated in a Tris-HCl buffer solution (pH 7.4) containing 0.12 mol / L NaCl for 1 h to obtain a highly sensitive polycation-selective electrode.

[0053] 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.

[0054] Under magnetic stirring at 3000 rpm, using 15 mL of Tris-HCl buffer solution (pH 7.4) containing 0.12 mol / L NaCl as the background electrolyte solution, after the baseline potential stabilized, different amounts of protamine (0-1.4 μg / mL) were added. Different standard signals were generated after each addition, and the potential changes within 150 s were recorded to obtain potential signal response curves for different concentrations (see [reference]). Figure 5 By reading the potential change values ​​over 150 seconds at different concentrations, calculating the ratio of this value to time to obtain the initial potential change rate, and plotting this initial potential change rate against the corresponding concentration, the standard working curve of protamine can be obtained (see...). Figure 6 ).

[0055] To determine the concentration of protamine sulfate of unknown concentration, under the same magnetic stirring conditions, a selective electrode was inserted into the protamine sulfate solution of unknown concentration, and the potential change value was read within 150 seconds (see [reference]). Figure 7 Calculate the initial potential change rate and compare it with the standard working curve (e.g., Figure 6 Calculate the corresponding concentration.

[0056] Depend on Figure 7 The initial potential change rate was calculated to be 0.116, which is consistent with the standard curve ( Figure 6 According to the comparison, the concentration of the unknown protamine was 0.78 μg.

[0057] Example 3

[0058] The highly sensitive polycation-selective electrode (polymer sensitive film thickness of 5 μm) prepared in Example 1 was used as the working electrode to determine heparin in sheep blood. The determination steps are as follows:

[0059] Working electrode activation: The above electrode was activated in a Tris-HCl buffer solution (pH 7.4) containing 0.12 mol / L NaCl for 1 h to obtain a highly sensitive polycation-selective electrode.

[0060] Detection apparatus: A polycation-selective electrode serves as the working electrode (positive electrode), and an Ag / AgCl (3M KCl) electrode serves as the reference electrode (negative electrode). The positive and negative electrodes are connected to a CHI 660E electrochemical workstation via wires (see [link]). 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.

[0061] Sodium citrate was added to fresh sheep blood to prevent coagulation. Heparin samples of different known concentrations were prepared using this blood as the background electrolyte. Under magnetic stirring, a polyion electrode was inserted into a measuring cell containing standard sheep blood samples with different concentrations of heparin. After the electrode stabilized, a baseline potential was obtained. A fixed amount of standard protamine was added to the measuring cell to a concentration of 1.1 μg / mL, resulting in a heparin-to-protamine binding ratio of 10:1. The remaining uncomplexed protamine accumulated on the electrode membrane surface, causing a potential increase. Due to the different concentrations of heparin in the samples, different standard signals were generated after the addition of protamine. The potential changes over 150 seconds were recorded to obtain potential signal response curves in the presence of different heparin concentrations. The potential change value over 150 seconds was read, and the ratio of this value to time (150 seconds) was calculated to obtain the initial potential change rate. Plotting this initial potential change rate against the corresponding heparin concentration yielded the standard working curve for heparin.

[0062] To determine the concentration of heparin of unknown concentration, under the same magnetic stirring conditions, a selective electrode is inserted into a heparin solution of unknown concentration. A fixed amount of standard protamine is added to the measuring cell to make its concentration 1.1 μg / mL, generating a potential signal. The potential change value within 150 s is read to calculate the initial potential change rate, and the corresponding concentration is calculated by referring to the standard working curve.

[0063] If the protamine concentration measured in the test does not cause a significant change in electrode potential, it may be because the heparin concentration in the sample exceeds 0.1 U / mL, resulting in no remaining protamine in the sample or the concentration being below the detection limit. In this case, the sample can be diluted and the above test steps can be repeated.

[0064] Example 4

[0065] The electrode of this invention was used to determine heparin in human blood: using fresh blood as the background electrolyte, heparin samples of different known concentrations were prepared, and the corresponding concentrations were calculated by referring to the standard working curve under magnetic stirring conditions, as per Example 3.

[0066] Example 5

[0067] Highly sensitive polyanion-selective electrode for determining heparin in buffer solution.

[0068] Preparation of the working electrode coated with a transconductance layer: The Au / PGR-Pt electrode was prepared by one-step electrochemical co-reduction of graphene oxide (GO) aqueous dispersion and H2PtCl4 on the surface of the 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.

[0069] Preparation of the working electrode with the polymer-sensitive membrane: A mixture of 100 mg of tri-dodecylmethylammonium chloride, tetra(dodecyl)-tetra(4-chlorophenyl)borate, polyvinyl chloride, and dioctyl sebacate 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, then diluted 20 times with tetrahydrofuran. 40 μL of the diluted membrane solution was dropped onto an Au / PGR-Pt electrode coated with a transconductance layer and left at room temperature. After about 4 hours, the tetrahydrofuran evaporated completely, forming a uniform ultrathin polymer-sensitive membrane on the electrode. For the specific electrode structure, see [link to electrode details]. Figure 1 .

[0070] Detection apparatus: The polyanion-selective electrode is the working electrode (positive electrode), and the Ag / AgCl (3M KCl) electrode is the reference electrode (negative electrode). The positive and negative electrodes are connected to the CHI 660E electrochemical workstation via wires (see [link]). 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.

[0071] The electrode was activated for 1 hour in a Tris-HCl buffer solution (pH 7.4) containing 0.12 mol / L NaCl. This yielded a highly sensitive polyanion-selective electrode after activation.

[0072] Under magnetic stirring at 3000 rpm, using 15 mL of Tris-HCl buffer solution (pH 7.4) containing 0.12 mol / L NaCl as the background electrolyte solution, after the baseline potential stabilized, different amounts of heparin were added to achieve a series of concentrations (0.1-2 U / mL). The potential changes within 30 seconds were recorded to obtain potential signal response curves for different concentrations. The potential change values ​​within 30 seconds at different concentrations were read, and the ratio of this value to time was calculated to obtain the initial potential change rate. The standard working curve of heparin can be obtained by plotting the initial potential change rate against the corresponding concentration.

[0073] To determine the concentration of heparin of unknown concentration, under the same magnetic stirring conditions, a selective electrode is inserted into a heparin solution of unknown concentration, and the potential change value within 30 seconds is read to calculate the initial potential change rate. The corresponding concentration is then calculated by comparing with a standard working curve.

[0074] 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 quantitative method for a polyion-selective electrode, characterized in that: Using an all-solid-state ultrathin polymer membrane polyion-selective electrode as the working electrode, it is inserted into the sample to be tested, so that the polyions to be tested are enriched at the membrane interface to generate an initial potential change rate. The concentration of polyions in the sample to be tested is obtained by the corresponding value of the initial potential change rate of the sample to be tested on the standard curve. The all-solid-state ultrathin polymer film polyion-selective electrode consists of a solid electrode with a transduction layer material adhered to the bottom, and an ultrathin polymer film adhered to the surface of the transduction layer material. The thickness of the ultrathin polymer film is 5 μm or less. The ultrathin polymer film is composed of a polymer matrix material, a plasticizer, an ion carrier, and a lipophilic inert salt, mixed in a weight ratio of 20-60:20-60:0.1-10:0.1-10. After mixing, it is dissolved in an excess of tetrahydrofuran and stirred to form a homogeneous solution. Then, it is diluted 10-300 times with tetrahydrofuran. An appropriate amount of the diluted 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 an ultrathin polymer sensitive film on the electrode carrier.

2. The quantitative method for the polyion-selective electrode according to claim 1, characterized in that: The standard curve is obtained by adding the working electrode to a solution containing different concentrations of the polyions to be detected, recording the change in the initial potential of the electrode after adding different concentrations of polyions using a potentiometer, calculating the rate of change of the initial potential, and plotting the standard working curve based on the rate of change of the initial potential and the concentration.

3. The quantitative method for the polyion-selective electrode according to claim 1, characterized in that: The initial potential change rate is the ratio of the potential signal to time within a fixed time period before reaching the quasi-steady-state equilibrium potential; wherein the fixed time period is 1-300 s.

4. The quantitative method for the polyion-selective electrode according to claim 1, characterized in that: When the charge of the polyion to be detected is opposite to that of the polyion selectively responded to by the working electrode, an indicator polyion is added to solutions or test solutions containing different concentrations of the polyion to be detected to plot the standard curve. The standard curve is plotted by measuring the rate of change of the initial potential of the remaining indicator polyion after complexation with the polyion to be detected at different concentrations, and the concentration of the polyion to be detected in the test solution is detected.

5. The quantitative method for the polyion-selective electrode according to any one of claims 1-4, characterized in that: a. Immerse the working electrode in a Tris-HCl buffer solution containing 0.12 M NaCl for 0.5-3 h to activate it, and then insert it into a measuring cell containing the buffer solution. After the electrode stabilizes, obtain the baseline potential. b. Add the polyions to be detected at different standard concentrations into the measuring cell. The polyions accumulate on the surface of the electrode membrane, causing changes in the membrane interface potential. c. Plot a standard curve of polyion concentration based on the rate of change of initial potential after the addition of polyions; d. The activated working electrode is directly inserted into the measuring cell containing the sample to be tested to generate a sample potential signal; the concentration of the polyion to be tested is obtained by comparing the initial potential change rate of the sample solution with the standard working curve.

6. The quantitative method for the polyion-selective electrode according to claim 5, characterized in that: When the charge of the polyion to be detected is opposite to that of the polyion selectively responded to by the working electrode, after inserting the working electrode in steps b and d, a polyion that can electrostatically complex with the polyion to be detected is added as an indicator polyion.

7. The quantitative method for the polyion-selective electrode according to claim 1, characterized in that: The polyion to be detected is a polycation or a polyanion; the polycation is protamine, polycationic polypeptide, polyethylenediamine dendritic compound or polypropyleneimine dendritic compound; the polyanion is heparin, sodium pentose polysulfate, nucleic acid, carrageenan, dextran sulfate, chondroitin polysulfate or persulfated chondroitin polysulfate.

8. The quantitative method for the polyion-selective electrode according to claim 1, characterized in that: The carrier in the working 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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