A flexible and bend-resistant working electrode for a flexible electrochemical biosensing device

Abstract

The application discloses a flexible working electrode for a flexible electrochemical bioactive substance detection sensor, which comprises a flexible substrate and a graphene electrode layer on the flexible substrate, a noble metal nanoparticle layer deposited on the graphene electrode layer, a chemical cross-linking oxidase fixing layer covering the noble metal nanoparticle layer, and a mass transfer limiting layer covering the chemical cross-linking oxidase fixing layer; and the oxidase is glucose oxidase or lactic acid oxidase. The application has good detection accuracy, stable performance and good bending resistance.

Description

Technical Field

[0001] This invention relates to the field of biosensor manufacturing technology, and in particular to a flexible electrochemical bioactive substance detection sensor with bend resistance. Background Technology

[0002] Bioactive substances such as glucose and lactic acid play an important role in maintaining optimal human health. By continuously monitoring the blood glucose levels of diabetic patients, the occurrence of related complications can be effectively reduced and the quality of life of patients can be improved.

[0003] Currently, hospitals primarily test blood glucose levels in diabetic patients by drawing venous or finger-prick blood. However, due to the nature of diabetes, patients often need to monitor their blood glucose levels multiple times a day. Repeatedly pricking the finger or drawing venous blood causes significant pain, unnecessary stress and psychological burden, and increases the risk of cross-infection. Commercially available home blood glucose testing devices often use rigid substrates, such as stainless steel needles, which have certain biocompatibility defects. Inflammation and foreign body reactions can lead to the accumulation of biological tissue, reducing the actual performance of the sensor and causing discomfort to the patient, making long-term implantation unsuitable.

[0004] Sensors based on flexible materials have developed rapidly in recent years, employing techniques such as vacuum magnetic sputtering, photolithography, and screen printing. However, these techniques often suffer from drawbacks, including poor adhesion to the substrate leading to easy detachment, complex and cumbersome fabrication processes, and material waste. Recently, LIG ​​(Laser-induced Graphene) technology has offered a new direction. It utilizes the photothermal effect of long-wavelength and relatively long-pulse lasers to provide extremely high local temperatures. This high temperature easily breaks the CO, C=O, and NC bonds on flexible polymer films, causing these atoms to recombine and be released as gases. Aromatic compounds are then rearranged to form a graphite structure. Graphene with a three-dimensional porous structure produced by this technology exhibits a large specific surface area and high conductivity, and the fabrication process does not require high temperatures or solvents. However, this graphene electrode is not resistant to bending and easily detaches from the flexible substrate, leading to open circuits. Summary of the Invention

[0005] The purpose of this invention is to provide a flexible electrochemical bioactive substance detection sensor with good bending resistance, high detection accuracy, stable performance, and good bending resistance.

[0006] The technical solution adopted by this invention to solve its technical problem is: A flexible, bend-resistant electrochemical bioactive substance detection sensor's working electrode includes a flexible substrate and a graphene electrode layer on the flexible substrate. A noble metal nanoparticle layer is deposited on the graphene electrode layer. A chemically cross-linked oxidase immobilization layer is covered on the noble metal nanoparticle layer, and a mass transfer restriction layer is covered on the chemically cross-linked oxidase immobilization layer. The oxidase is glucose oxidase or lactate oxidase. This invention is used for the detection of glucose or lactate.

[0007] Preferably, the flexible substrate is a PI film or a PEI film.

[0008] Preferably, the graphene electrode layer is formed by laser engraving on a flexible substrate. The laser engraving machine parameters are set as follows: engraving mode, intensity 3-9W, and speed 100-500mm / s.

[0009] Preferably, the graphene electrode layer is treated with acetic acid before depositing the noble metal nanoparticle layer. The acetic acid treatment specifically involves: first, washing the graphene electrode layer with deionized water, then immersing it in a 1-2% (v / v) acetic acid solution and allowing it to stand at room temperature for 1-4 hours. Acetic acid treatment can increase the content of C-C bonds and improve the electrode's electrical performance.

[0010] Preferably, the noble metal nanoparticle layer is formed by depositing a graphene electrode layer in a noble metal plating solution using a constant potential method. The constant potential method voltage is -1.5 to -2.5V, and the energizing time is 100-300s.

[0011] Preferably, the precious metal plating solution is composed of the following components by mass percentage: 3% chloroplatinic acid, 0.25% lead acetate, and the remainder hydrochloric acid solution; the concentration of the hydrochloric acid solution is 0.5 mol / L.

[0012] Preferably, the processing method of the chemically cross-linked oxidase immobilization layer is as follows: the chitosan solution of the oxidase is drop-coated onto the noble metal nanoparticle layer, and after drying and curing into a film, it is soaked in genipin solution for 0.5-4 hours.

[0013] Preferably, the chitosan solution containing the oxidase is a mixture of the oxidase and the chitosan solution, wherein the concentration of the oxidase is 1-50 g / L; the chitosan solution is a mixture of chitosan and a 0.5%-10% (v / v) aqueous solution of glacial acetic acid, wherein the concentration of the chitosan is 10-50 g / L. The concentration of the oxidase is preferably 10-20 g / L. The concentration of the chitosan is preferably 10-20 g / L.

[0014] Preferably, the genipin solution is prepared by mixing genipin and citrate-sodium citrate buffer, wherein the concentration of genipin is 10-100 g / L, the concentration of citrate-sodium citrate buffer is 0.1 M, and the pH is 4-5. The concentration of genipin is preferably 10-30 g / L.

[0015] Preferably, the mass transfer restriction layer is formed by depositing a chemically cross-linked oxidase immobilization layer in a Nafion dispersion of carbon nanotubes using a constant potential method. The constant potential method has a voltage of 1V and an energizing time of 90s. The Nafion dispersion of carbon nanotubes is a mixture of 7.5% (w / w) of an aqueous dispersion of carbon nanotubes and 5% (w / w) of a Nafion solution in a volume ratio of 1:4 to 4:1.

[0016] The beneficial effects of this invention are: (1) Using laser-induced graphene technology, the operation is convenient and the steps are simple. Graphene electrodes can be directly prepared in one step, and the mass production of micro electrode patterns can be achieved at a lower cost. (2) The prepared flexible graphene electrode has the characteristics of high electron transfer rate and large specific surface area, and the flexible substrate can effectively reduce the patient's pain during implantation. (3) The deposition of noble metal nanoparticles on graphene electrodes can increase the specific surface area of ​​the electrodes, improve the conductivity of the electrodes, and play a good catalytic role in the oxidation and decomposition of glucose, thereby improving the performance of the sensor. (4) By immobilizing the enzyme on the electrode through chemical cross-linking, the enzyme shedding phenomenon is effectively reduced, the sensor performance is stabilized and the life is extended; at the same time, the drop coating method is used to greatly reduce enzyme waste. (5) A hydrogel layer is formed by cross-linking genipin and chitosan. On the one hand, it serves as a biological component to fix enzymes. Compared with traditional components such as glutaraldehyde, it greatly reduces biotoxicity and provides a good foundation for the long-term stability of implantable sensors. On the other hand, the hydrogel layer plays a good role in fixing graphene to the substrate, solving the problem of easy detachment of laser-induced graphene. (6) By using Nafion and carbon nanotubes to form a mass transfer confinement layer, on the one hand, the diffusion of glucose is reduced, which indirectly increases the proportion of oxygen in the electrode area and broadens the detection range of the sensor; on the other hand, it shields the interference of other interfering substances on glucose oxidation and ensures the accuracy of the sensor response. Attached Figure Description

[0017] Figure 1 This is an image of the sensor formed by the working electrode in Embodiment 1 of the present invention. Figure 2 This is an image of the sensor formed by the working electrode in Embodiment 2 of the present invention. Figure 3 This is an image of the sensor formed by the working electrode in Verification Example 1 of this invention; Figure 4 This is a graph showing the relationship between the number of times the electrode is bent and the electrode resistance under different treatments; Figure 5 yes Figure 4 A magnified view of the first 60 bends; Figure 6 This is a physical image of the sensor formed by the working electrode in Embodiment 2 of the present invention. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0019] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0020] Example 1: Fabrication of graphene electrodes on flexible substrates (1) Preparation of processing materials A 0.125mm thick polyimide film was cleaned with anhydrous ethanol and deionized water, respectively. The polyimide film was then cut into 5cm x 5cm pieces, laid out as flat as possible, and fixed to the substrate of the laser engraving machine with insulating tape to prevent unevenness of the film surface caused by deformation during laser ablation, which could affect the overall performance of the electrode.

[0021] (2) Electrode processing The drawn electrode pattern is imported into a laser engraving machine. The machine is first set to engraving mode with a power of 6W, a speed of 300mm / s, and a wavelength of 450±5nm. A layer of graphene is formed on the surface of the polyimide film through laser ablation. Then, the laser engraving machine is switched to cutting mode, with the current intensity adjusted to 15% and the speed to 100mm / s. The formed graphene electrode area is cut from the polyimide film along its outer edge, resulting in a graphene electrode composited on a flexible substrate.

[0022] Example 2: Working electrode for a flexible electrochemical glucose detection sensor The preparation method includes the following steps: (1) Prepare a 1% acetic acid solution by using a pipette to transfer 10 μL of glacial acetic acid and add it dropwise to 990 μL of deionized water. Shake the solution for 30 seconds to ensure thorough mixing and form a 1% acetic acid solution.

[0023] (2) After rinsing the graphene electrode processed in Example 1 three times with deionized water and drying it, immerse the electrode below the surface of 1% acetic acid solution so that the graphene electrode is fully wetted by acetic acid solution. After standing at room temperature for 2 hours, rinse it three times with deionized water and dry it.

[0024] (3) Prepare platinum plating solution (precious metal plating solution): Weigh appropriate amounts of chloroplatinic acid and lead acetate, dissolve them in 0.5 mol / L hydrochloric acid to form platinum plating solution, wherein the mass fraction of chloroplatinic acid is 3% and the mass fraction of lead acetate is 0.25%.

[0025] (4) Connect the LIG (graphene) electrode treated in step (2) to the Chenhua 660A electrochemical workstation. Use a platinum mesh electrode as the reference electrode and counter electrode. Place the LIG electrode in the platinum plating solution prepared in step (3) so that the electrode is immersed to a depth of about 10 mm below the liquid surface. Deposit nano-platinum particles on the LIG electrode using a voltage of -2.0 V for 300 s.

[0026] (5) After rinsing the platinum-plated LIG electrode with deionized water, immerse it in 0.01 mol / L PBS solution and let it stand for 30 minutes. This step is only for testing purposes and can effectively reduce the electrode's leveling time and lower the baseline current during testing.

[0027] (6) Prepare chitosan solution: Dissolve an appropriate amount of chitosan in 1% glacial acetic acid solution to make the concentration of chitosan solution 10g / L. Add a magnetic stir bar to a centrifuge tube containing chitosan solution and place the chitosan solution on a Guohua HJ-2 magnetic stirrer to stir until the chitosan is fully dissolved and there is no suspension.

[0028] (7) Prepare a chitosan-glucose oxidase mixed solution. Dissolve an appropriate amount of glucose oxidase in the chitosan solution prepared in step (6) so that the concentration of glucose oxidase in the solution is 10 g / L.

[0029] (8) Prepare genipin solution: Take 0.1M citrate-sodium citrate buffer solution with pH 4.5, and dissolve an appropriate amount of genipin in the citrate-sodium citrate buffer solution to make the concentration of genipin 10g / L.

[0030] (9) The gel-like chitosan-glucose oxidase mixed solution prepared in step (7) is drop-coated onto the graphene electrode after treatment in step (5), and placed in an oven to dry at 50°C for half an hour. After a solid film is formed on the electrode surface, it is then immersed in the genipin solution prepared in step (8) for 2 hours.

[0031] (10) Preparation of Nafion dispersion of carbon nanotubes: A 7.5% CNT aqueous dispersion and a 5% Nafion solution were mixed at a volume ratio of 1:4. The electrode was connected to a Chenhua 660A electrochemical workstation, and a platinum mesh electrode was used as the reference electrode and counter electrode. The electrode was immersed in the Nafion dispersion of carbon nanotubes. Using the potentiostatic method, the working voltage was set to 1.0V and the working time was 90s. A mass transfer confinement layer was electrophoretically adsorbed on the electrode to form the working electrode for the flexible electrochemical glucose detection sensor.

[0032] (11) Connect the working electrode obtained in step (10) to the electrochemical workstation, using the Ag / AgCl electrode as the reference electrode and the platinum mesh electrode as the counter electrode. Figure 6 The chronoamperometry method was used in an electrochemical workstation. The initial potential was set to 0.55 V, and 400 mol / L glucose was added dropwise to a 0.01 mol / L PBS solution every 300 s. The resulting t-image is shown below. Figure 1 As shown, the prepared sensor exhibits a good linear response to glucose, with a sensitivity of 3.922 uA / mmol (R0) in the concentration range of 0-20 mmol / L. 2 =0.99).

[0033] Example 3: Working electrode for a flexible electrochemical lactic acid detection sensor The preparation method includes the following steps: (1) Prepare a 1% glacial acetic acid solution. Use a pipette to transfer 10 μL of glacial acetic acid and add it dropwise to 990 μL of deionized water. Use a test tube shaker to shake for 30 seconds to mix the solution thoroughly, forming a 1% glacial acetic acid solution by volume.

[0034] (2) After rinsing the graphene electrode processed in Example 1 three times with deionized water and drying it, immerse the electrode below the surface of the 1% glacial acetic acid solution prepared in step 1 so that the LIG electrode is fully wetted by the glacial acetic acid solution. After standing for 2 hours, rinse it three times with deionized water and dry it.

[0035] (3) Prepare platinum plating solution (precious metal plating solution): Weigh appropriate amounts of chloroplatinic acid and lead acetate, dissolve them in 0.5 mol / L hydrochloric acid to form platinum plating solution, wherein the mass fraction of chloroplatinic acid is 3% and the mass fraction of lead acetate is 0.25%.

[0036] (4) Connect the LIG (graphene) electrode treated in step (2) to the Chenhua 660A electrochemical workstation. Use a platinum mesh electrode as the reference electrode and counter electrode. Place the LIG electrode in the platinum plating solution prepared in step (3) so that the electrode is immersed to a depth of about 10 mm below the liquid surface. Deposit nano-platinum particles on the LIG electrode using a voltage of -2.0 V for 300 s.

[0037] (5) After rinsing the platinum-plated LIG electrode with deionized water, immerse the platinum-plated portion in 0.01 mol / L PBS solution and let it stand for 30 minutes. This step is only for testing purposes and can effectively reduce the electrode's leveling time and lower the baseline current during testing.

[0038] (6) Prepare chitosan solution: Dissolve an appropriate amount of chitosan in 1% glacial acetic acid solution to make the concentration of chitosan solution 10g / L. Add a magnetic stir bar to a centrifuge tube containing chitosan solution and place the chitosan solution on a Guohua HJ-2 magnetic stirrer to stir until the chitosan is fully dissolved and there is no suspension.

[0039] (7) Prepare a chitosan-lactic acid oxidase mixed solution: Dissolve an appropriate amount of lactic acid oxidase in the chitosan solution prepared in step (6) so that the concentration of lactic acid oxidase in the solution is 10 g / L.

[0040] (8) Prepare genipin solution: Take 0.1M citrate-sodium citrate buffer solution with pH 4.5, and dissolve an appropriate amount of genipin in the citrate-sodium citrate buffer solution to make the concentration of genipin 10g / L.

[0041] (9) The gel-like chitosan-lactic acid oxidase mixed solution prepared in step (7) is drop-coated onto the graphene electrode after treatment in step (5), and placed in an oven to dry at 50°C for half an hour. After a solid film is formed on the electrode surface, it is then immersed in the genipin solution prepared in step (8) for 2 hours.

[0042] (10) Preparation of Nafion dispersion of carbon nanotubes: A 7.5% CNT aqueous dispersion and a 5% Nafion solution were mixed at a volume ratio of 1:4. The electrode was connected to a Chenhua 660A electrochemical workstation, and a platinum mesh electrode was used as the reference electrode and counter electrode. The electrode was immersed in the Nafion dispersion of carbon nanotubes. Using the potentiostatic method, the working voltage was set to 1.0V and the working time was 90s. A mass transfer confinement layer was electrophoretically adsorbed on the electrode to form the working electrode for the flexible electrochemical lactic acid detection sensor.

[0043] (11) Connect the lactic acid sensor obtained in step (10) to an electrochemical workstation using the working electrode, with the Ag / AgCl electrode as the reference electrode and the platinum mesh electrode as the counter electrode, and use the chronoamperometry method in the electrochemical workstation. Set the initial potential to 0.55V, and add 40mol / L lactic acid dropwise to 0.01mol / L PBS solution every 300s. Obtain the following image: Figure 2 As shown, the prepared sensor exhibits a good linear response to lactic acid, with a sensitivity of 2.431 uA / mmol in the concentration range of 0-1.2 mmol / L (R0). 2 =0.98).

[0044] Verification Example 1: Hydrogen Peroxide Sensor The preparation method includes the following steps: (1) Prepare a 1% glacial acetic acid solution. Use a pipette to transfer 10 μL of glacial acetic acid and add it dropwise to 990 μL of deionized water. Use a test tube shaker to shake for 30 seconds to mix the solution thoroughly, forming a 1% glacial acetic acid solution by volume.

[0045] (2) After rinsing the graphene electrode processed in Example 1 three times with deionized water and drying it, immerse the electrode below the surface of the 1% glacial acetic acid solution prepared in step 1 so that the LIG electrode is fully wetted by the glacial acetic acid solution. After standing for 2 hours, rinse it three times with deionized water and dry it.

[0046] (3) Prepare platinum plating solution (precious metal plating solution): Weigh appropriate amounts of chloroplatinic acid and lead acetate, dissolve them in 0.5 mol / L hydrochloric acid to form platinum plating solution, wherein the mass fraction of chloroplatinic acid is 3% and the mass fraction of lead acetate is 0.25%.

[0047] (4) Connect the LIG (graphene) electrode treated in step (2) to the Chenhua 660A electrochemical workstation. Use a platinum mesh electrode as the reference electrode and counter electrode. Place the LIG electrode in the platinum plating solution prepared in step (3) so that the electrode is immersed to a depth of about 10 mm below the liquid surface. Deposit nano-platinum particles on the LIG electrode using a voltage of -2.0 V for 300 s.

[0048] (5) After rinsing the platinum-plated LIG electrode with deionized water, immerse the platinum-plated part in 0.01 mol / L PBS solution and let it stand for 30 minutes. After taking it out and drying it, the working electrode for the hydrogen peroxide sensor can be obtained.

[0049] (6) Connect the hydrogen peroxide sensor obtained in step (5) to the electrochemical workstation using the working electrode, with the Ag / AgCl electrode as the reference electrode and the platinum mesh electrode as the counter electrode, and use the chronoamperometry method in the electrochemical workstation. Set the initial potential to 0.55V, and add 400mol / L hydrogen peroxide dropwise to 0.01mol / L PBS solution every 300s. The obtained image is shown below. Figure 3 As shown, the prepared sensor exhibits a good linear response to hydrogen peroxide, with a sensitivity of 3.492 mA / mmol (R0) in the concentration range of 0-8 mmol / L. 2 =0.99), with a sensitivity of 1.673 mA / mmol in the concentration range of 8-20 mmol / L (R = 0.99). 2 =0.97). This verification example demonstrates that the above-described working electrode scheme of the present invention is feasible.

[0050] Comparative example This example provides a control group where LIG electrode detachment was significantly improved after chitosan crosslinking: Four graphene electrodes processed in Example 1 were taken, rinsed three times with deionized water and dried. The electrodes were then immersed below the surface of a 1% glacial acetic acid solution to ensure that the LIG electrodes were fully wetted by the glacial acetic acid solution. After standing for 2 hours, they were rinsed three times with deionized water and dried. They were labeled as No. 1, 2, 3, and 4, respectively.

[0051] Electrode 1 was left untouched. Electrodes 2, 3, and 4 were platinum-plated using the method described in Example 2. Electrode 4, after platinum plating, underwent crosslinking using chitosan-genipin as described in Example 2. Electrode 3 was crosslinked using the method described in Example 2, except that the chitosan+genipin crosslinking was replaced with a 5wt% glutaraldehyde solution.

[0052] After the above operations are completed, use a multimeter to measure the resistance between the four electrodes. It can be seen that the resistance values ​​of the four resistors are not much different, ranging from 400 to 600 Ω.

[0053] Fix one end of each of the four resistors and bend them outward at about 90°. Measure and record the resistance every five bends until the electrode breaks off at a certain point, making the resistance too high to measure.

[0054] Plotting the obtained data shows that ( Figure 4-5Chitosan-crosslinked electrodes significantly improved the inherent detachment tendency of LIG electrodes. Untreated LIG electrodes exhibited large-scale detachment and open circuits after only 20 bends. Other treated electrodes showed slight improvement, only experiencing open circuits after 50 bends. While the resistance of chitosan-crosslinked electrodes increased significantly after more than 150 bends, they maintained a conductive path without complete breakage. Furthermore, the increase in resistance was relatively small before 140 bends. Within 50 bends, the resistance of chitosan-crosslinked electrodes remained significantly lower than that of glutaraldehyde-crosslinked electrodes for the same number of bends. Therefore, it can be concluded that chitosan crosslinking significantly improves the detachment tendency of LIG electrodes.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A flexible, bend-resistant working electrode for a flexible electrochemical biosensing sensor, comprising: a flexible, bend-resistant substrate; and a working electrode disposed on the substrate, wherein the working electrode comprises a conductive material and a binder, and wherein the working electrode is configured to be used in a sensor for detecting a biological analyte. The invention includes a flexible substrate and a graphene electrode layer on the flexible substrate, a noble metal nanoparticle layer deposited on the graphene electrode layer, a chemically cross-linked oxidase immobilization layer covered on the noble metal nanoparticle layer, and a mass transfer restriction layer covered on the chemically cross-linked oxidase immobilization layer; the oxidase is glucose oxidase or lactate oxidase. The processing method of the chemically cross-linked oxidase immobilization layer is as follows: the chitosan solution of the oxidase is drop-coated onto the noble metal nanoparticle layer, and after drying and curing into a film, it is soaked in genipin solution for 0.5-4 hours.

2. The working electrode of claim 1, wherein, The flexible substrate is a PI film or a PEI film.

3. The working electrode of claim 1, wherein, The graphene electrode layer is formed by laser engraving on a flexible substrate. The laser engraving machine parameters are set as follows: engraving mode, intensity 3-9W, and speed 100-500mm / s.

4. The working electrode according to claim 1, characterized in that, The graphene electrode layer was treated with acetic acid before depositing a noble metal nanoparticle layer. The acetic acid treatment was performed by first cleaning the graphene electrode layer with deionized water, then immersing it in an acetic acid solution with a volume concentration of 1-2%, and letting it stand at room temperature for 1-4 hours.

5. The working electrode according to claim 1, characterized in that, The noble metal nanoparticle layer is formed by depositing a graphene electrode layer in a noble metal plating solution using a constant potential method. The constant potential method voltage is -1.5 to -2.5V, and the energizing time is 100-300s.

6. The working electrode according to claim 5, characterized in that, The precious metal plating solution is composed of the following components by mass percentage: 3% chloroplatinic acid, 0.25% lead acetate, and the remainder hydrochloric acid solution; the concentration of the hydrochloric acid solution is 0.5 mol / L.

7. The working electrode according to claim 1, characterized in that, The chitosan solution of the oxidase is composed of a mixture of oxidase and chitosan solution, wherein the concentration of oxidase is 1-50 g / L; the chitosan solution is composed of a mixture of chitosan and a 0.5%-10% (v / v) aqueous solution of glacial acetic acid, wherein the concentration of chitosan is 10-50 g / L.

8. The working electrode according to claim 1, characterized in that, The genipin solution is prepared by mixing genipin and citrate-sodium citrate buffer, wherein the concentration of genipin is 10-100 g / L, the concentration of citrate-sodium citrate buffer is 0.1 M, and the pH is 4-5.

9. The working electrode according to claim 1, characterized in that, The mass transfer confinement layer is formed by depositing a chemically cross-linked oxidase immobilization layer in a Nafion dispersion of carbon nanotubes using a constant potential method. The constant potential method has a voltage of 1V and an energizing time of 90s. The Nafion dispersion of carbon nanotubes is a mixture of 7.5% (w / w) of an aqueous dispersion of carbon nanotubes and 5% (w / w) of a Nafion solution in a volume ratio of 1:4 to 4:1.

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