A paper-based sensor for blood potassium detection and its preparation method and application

Abstract

The present invention relates to the technical field of blood potassium detection, and in particular to a paper-based sensor for blood potassium detection, and its preparation method and application. The sensor is integrated with a substrate, a sensing electrode, and a whole blood microfluidic channel, wherein the sensing electrode is located between the substrate and the whole blood microfluidic channel. The whole blood microfluidic channel includes a serum diffusion zone, a whole blood separation zone located above the serum diffusion zone, and a blood potassium detection zone located below the serum diffusion zone. A blood separation membrane is also provided in the whole blood separation zone. The substrate is qualitative filter paper, and the sensing electrode includes an all-solid-state PBA working electrode and an Ag / AgCl reference electrode. Compared with the prior art, the sensor does not require calibration when detecting potassium ions in whole blood, and can directly use whole blood samples without centrifugation, thereby increasing detection speed, avoiding the occurrence of hemolysis of whole blood samples during blood potassium detection, and further improving detection sensitivity, accuracy, and stability.

Description

Technical Field

[0001] The invention relates to the technical field of blood potassium detection, and in particular to a paper-based sensor for blood potassium detection, a preparation method and an application thereof. Background Art

[0002] Potassium ion is one of the important electrolytes for maintaining normal physiological functions of the human body, accounting for 98% of the total amount in cells. It is of great significance in maintaining the osmotic pressure, volume and material metabolism of intracellular fluid, acid-base balance, excitability of neuromuscular cell membranes and maintaining normal myocardial function. Abnormal concentration of potassium ion is associated with many diseases. Clinically, timely and accurate measurement of potassium ion content in the blood plays an important role in maintaining fluid balance.

[0003] Traditional clinical methods for measuring serum potassium include ion exchange chromatography, flame photometry, and ion-selective electrodes. These methods typically rely on complex equipment and specialized technicians, limiting their application in first aid, home self-monitoring, and resource-limited settings. With the increasing demand for health monitoring, especially for patients who require regular monitoring of serum potassium, such as those with renal dysfunction, there is an urgent need for a simple, low-cost method and device for rapid, on-site whole-blood potassium measurement.

[0004] In recent years, with the advancement of point-of-care testing (POCT) and microfluidics, a growing number of portable and low-cost blood potassium sensors have been developed. However, most of these sensors are only suitable for use with centrifuged blood samples and are difficult to apply directly to unprocessed whole blood samples. This is because when using whole blood samples directly for blood potassium testing, existing sensors are susceptible to adsorption of biological substances such as proteins and cells on the sensor surface, leading to electrode passivation, which degrades sensor performance and reduces sensor lifespan, affecting both sensitivity and stability of the test results. Furthermore, the complex composition of whole blood samples can interfere with test results, leading to false positives or false negatives and affecting test accuracy. Furthermore, sensors require a certain reaction time to achieve a stable signal, resulting in slow detection and prolonged testing times. This prolonged testing time can lead to hemolysis, in which the potassium ion concentration inside red blood cells is much higher than outside the cells. Once hemolysis occurs, potassium ions within the red blood cells are released into the plasma, ultimately causing a false increase in the blood potassium value, affecting the accuracy of the test results.

[0005] Paper-based sensors have attracted widespread attention in the field of biological testing due to their low cost, portability, and ease of operation. However, current paper-based sensors are rarely used for whole blood potassium testing. This is because testing of blood potassium in unprocessed whole blood is limited by detection speed and hemolysis issues. There are also deficiencies in detection accuracy, sensitivity, and stability. Moreover, most of them require calibration before testing, making the detection process cumbersome and time-consuming. Therefore, the development of an efficient, accurate, and convenient paper-based sensor for whole blood potassium detection is of great practical significance. Summary of the Invention

[0006] In order to solve the problems in the existing technology, the present invention provides a paper-based sensor for blood potassium detection, its preparation method and application, which can directly detect whole blood samples, has a fast detection speed, avoids the occurrence of hemolysis problems, improves the accuracy and stability of the test results, and has high sensitivity and portability.

[0007] A paper-based sensor for blood potassium detection comprises a substrate and a sensing electrode located on the substrate, and also comprises a whole blood microfluidic channel. The whole blood microfluidic channel is an integrated structure comprising a serum diffusion zone, a whole blood separation zone located above the serum diffusion zone, and a blood potassium detection zone located below the serum diffusion zone. A blood separation membrane is also provided in the whole blood separation zone. The whole blood microfluidic channel is made of qualitative filter paper.

[0008] Wherein, the substrate is qualitative filter paper, the sensing electrode includes an all-solid-state PBA working electrode and an Ag / AgCl reference electrode, and the all-solid-state PBA working electrode is prepared from PBA material.

[0009] Furthermore, the PBAs material is a multi-metal co-doped Prussian blue analogue, and its general formula is K x A y B z [Fe(CN)6] m nH2O, wherein x=1.0-2.2, y=0.1-0.7, z=0.3-0.9, m=0.7-1.0, n=0.5-1.5, A is at least one of Fe, Mn, Co, Ni, Cu, Zn, La, V, Ti, Mo, and W, and B is at least one of Fe, Mn, Co, Ni, Cu, Zn, La, V, Ti, Mo, and W.

[0010] Preferably, the PBAs material has the general formula K x Fe y Mn z [Fe(CN)6] mnH2O, where x=1.5-2.0, y=0.1-0.5, z=0.5-0.9, m=0.9-1.0, and n=0.5-1.0.

[0011] Preferably, the PBAs material has the general formula K x Co y Ni z [Fe(CN)6] m nH2O, where x=1.2-1.8, y=0.3-0.7, z=0.3-0.7, m=0.7-1.0, and n=0.5-1.2.

[0012] Preferably, the PBAs material has the general formula K x Cu y Zn z [Fe(CN)6] m nH2O, where x=1.5-2.2, y=0.1-0.4, z=0.6-0.9, m=0.7-1.0, and n=0.8-1.5.

[0013] Preferably, the PBAs material has the general formula K x V y Ti z [Fe(CN)6] m nH2O, where x=1.0-1.8, y=0.2-0.5, z=0.5-0.8, m=0.7-1.0, and n=0.8-1.5.

[0014] Preferably, the PBAs material has the general formula K x Mo y W z [Fe(CN)6] m nH2O, where x=1.0-1.8, y=0.4-0.6, z=0.4-0.6, m=0.7-1.0, and n=0.7-1.2.

[0015] Furthermore, the sensor is integrated by a substrate, a sensing electrode and a whole blood microfluidic channel, and the sensing electrode is located between the substrate and the whole blood microfluidic channel.

[0016] Furthermore, the whole blood separation area includes a V-shaped separation channel and a whole blood dripping port connected to both ends of the V-shaped separation channel, and the whole blood dripping port is a circular structure; the blood potassium detection area includes a convex-shaped detection channel and a detection port connected to both ends of the convex-shaped detection channel.

[0017] Furthermore, the serum diffusion zone has a step-shaped structure, and the step-shaped structure is formed by the thickness difference generated at the junction of the V-shaped separation channel and the convex-shaped detection channel.

[0018] Furthermore, a conductive layer is provided between the sensing electrode and the substrate, and the conductive layer includes an electrode end and electronic component connection ends located on both sides of the electrode end. The electrode end matches the sensing electrode, the electrode end is a circular structure, and the electronic component connection end is a rectangular structure.

[0019] Furthermore, the detection port is a D-shaped structure, wherein the semicircular end of the D-shape is connected to the two bottom ends of the convex detection channel, and the straight end of the D-shape is separated from the electronic component connection end.

[0020] The present invention also provides a method for preparing the above-mentioned paper-based sensor for detecting blood potassium, comprising the following steps:

[0021] (1) Substrate treatment: Print the designed pattern on qualitative filter paper and dry it in an oven;

[0022] (2) Preparation of sensing electrodes: Silver ink and carbon ink were printed on qualitative filter paper respectively. After all printed layers were completely dried, the electrodes were cut out. PBAs material solution was dripped on the carbon contact points and dried to make a fully solid-state working electrode. FeCl3 solution was dripped on the silver contact points and dried to make a reference electrode.

[0023] (3) Preparation of whole-blood microfluidic channel: Qualitative filter paper and blood separation membrane were overlapped on a glass slide, and μPAD patterning was performed using an iron mold to obtain a whole-blood separation area and a blood potassium detection area. The qualitative filter paper and blood separation membrane were combined using wax dipping technology, and after cooling to room temperature, they were peeled off to obtain an integrated whole-blood microfluidic channel;

[0024] (4) Preparation of paper-based sensor: The whole blood microfluidic channel obtained in step (3) and the sensing electrode obtained in step (2) are pasted on the substrate treated in step (1) to form a paper-based sensor.

[0025] Furthermore, in the preparation process of the whole blood microfluidic channel in step (3), the overlapping thickness of the qualitative filter paper and the blood separation membrane is 1 mm; the temperature of the wax dipping technique is 125° C., and the time is 1 s.

[0026] Furthermore, in the substrate treatment process of step (1), the drying temperature is 52° C. and the drying time is 5 min; in the preparation process of the sensing electrode of step (2), the drying temperature of the all-solid-state working electrode and the reference electrode is 60° C. and the drying time is 15 min; the printing method in steps (1) and (2) is screen printing.

[0027] Furthermore, the blood potassium detection paper-based sensor is used to detect potassium ions in a whole blood sample.

[0028] Furthermore, during the process of detecting potassium ions in the whole blood sample, whole blood was dripped into the whole blood dripping area in batches, with a total of 6 dripping times, and 50 μL was dripped each time.

[0029] The advantages of the present invention are:

[0030] 1. The present invention provides a paper-based sensor for blood potassium detection that can directly detect whole blood samples and possesses high sensitivity and portability. When performing direct detection of potassium ions in whole blood, the sensor uses a whole blood microfluidic channel with a blood filtration function to separate the whole blood within minutes, avoiding electrode passivation and improving detection efficiency. Compared with existing technologies, this sensor can avoid the problem of hemolysis in whole blood potassium detection, improve detection sensitivity, and enhance the accuracy and stability of test results. It also features a simple preparation process, low cost, and increased portability, making it more suitable for use in small and medium-sized hospitals, homes, travel, and other outdoor settings.

[0031] 2. The present invention integrates a filter paper substrate and an all-solid-state PBA working electrode. The PBAs material used in the all-solid-state PBA working electrode can serve not only as a solid contact layer but also as an ion-selective membrane, thereby reducing the complexity and cost of the electrode structure and lowering the process requirements. Moreover, without adding an ion-selective membrane, the instability of the phase boundary potential of the solid ion-selective electrode is reduced, and the repeatability between electrodes is improved. At the same time, the dual characteristics of the ion-selective membrane and the transduction layer of the PBAs material simplify the structure of the ion-selective electrode and reduce the influence of the phase boundary potential on the standard electrode potential. E 0 The influence of the redox potential of the material can be controlled during the preparation process when preparing PBAs. E 0 Maintaining uniformity, different electrodes can share a calibration curve, so that the paper-based sensor prepared by the present invention can be used for calibration-free detection. Compared with the existing technology, it is more convenient to use and significantly improves the detection speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the integration process of the paper-based sensor for blood potassium detection of the present invention ( Figure 1 a is the base, Figure 1 b is the sensing electrode, Figure 1 c is the whole blood microfluidic channel, Figure 1 d is an integrated paper-based sensor for blood potassium detection);

[0033] Figure 2 This is a diagram of the integration process of the substrate and sensing electrodes of the paper-based sensor for blood potassium detection of the present invention;

[0034] Figure 3This is a schematic diagram of the substrate and sensing electrodes of the paper-based sensor for blood potassium detection of the present invention after integration;

[0035] Figure 4 Schematic diagram of the whole blood microfluidic channel of the paper-based sensor for blood potassium detection of the present invention;

[0036] Figure 5 1 is a cyclic voltammetry curve of Example 1 and Comparative Example 2 in Test Example 2 of the present invention;

[0037] Figure 6 1 is the electrochemical impedance spectroscopy of Example 1 and Comparative Example 2 in Test Example 2 of the present invention;

[0038] Figure 7 This is a comparison diagram of the whole blood dripping method in Test Example 1 of the present invention ( Figure 7 a is to add 125uL whole blood at one time, Figure 7 b: add 50 μL of whole blood 5 times each time. Figure 7 c is adding 50 μL of whole blood 6 times, Figure 7 d: 50 μL of whole blood was added 7 times);

[0039] Figure 8 This is a test graph of potassium ion response at different concentrations in Test Example 3 of the present invention;

[0040] Figure 9 This is a standard curve diagram of the first batch of sensors versus calibration solution in Test Example 4 of the present invention;

[0041] Figure 10 This is a standard curve diagram of the second batch of sensors versus calibration solution in Test Example 4 of the present invention;

[0042] Figure 11 This is a standard curve diagram of the third batch of sensors versus calibration solution in Test Example 4 of the present invention;

[0043] Figure 12 This is the standard curve of the fourth batch of sensors for quality control serum in Test Example 5 of the present invention;

[0044] Figure 13 This is a standard curve diagram of the fourth batch of sensors versus standard solution in Test Example 5 of the present invention;

[0045] Description of reference numerals:

[0046] 1. Whole blood separation area; 11. V-shaped separation channel; 12. Whole blood dripping port; 2. Serum diffusion area; 3. Blood potassium detection area; 31. Convex detection channel; 32. Detection port; 4. Electrode end; 5. Electronic component connection end. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0048] It should be noted that the various installation methods and technical terms mentioned in the present invention are technical terms that have long been clearly known in the relevant technical field and therefore will not be further explained. In addition, the same reference numerals are used for the same components, but this does not affect nor constitute an accurate understanding of the technical solution by those skilled in the art.

[0049] Example 1 This example provides a paper-based sensor for detecting blood potassium, the structure of which is as follows: Figure 1 d, by the base ( Figure 1 a) Sensing electrodes ( Figure 1 b) and whole blood microfluidic channels ( Figure 1 c) integrated, the substrate, sensing electrodes and whole blood microfluidic channel are all made of qualitative filter paper (the qualitative filter paper used in this example is Whatman No. 1 filter paper).

[0050] like Figure 2 and Figure 3 As shown, a conductive layer is further provided between the sensing electrode and the substrate (the conductive layer in this embodiment refers to Figure 2 The conductive layer includes a circular electrode end 4 and rectangular electronic component connection terminals 5 located on both sides of the electrode end 4. The shape of the sensing electrode matches the shape of the electrode end 4. The sensing electrode includes an all-solid-state PBA working electrode and an Ag / AgCl reference electrode (the PBA material of the all-solid-state PBA working electrode in this embodiment is K 1.85 Fe 0.33 Mn 0.67 [Fe(CN)6] 0.98 0.77H2O). During the integration of the sensing electrode and the substrate, the carbon layer and the silver layer are located on the printed substrate, the all-solid-state PBA working electrode is located on the carbon layer, the Ag / AgCl reference electrode is located on the silver layer, and there is also a reference film on the Ag / AgCl reference electrode. The schematic diagram after integration is shown in FIG. Figure 3 shown.

[0051] like Figure 4As shown, the whole blood microfluidic channel is an integrated structure, which comprises a whole blood separation zone 1, a serum diffusion zone 2 and a blood potassium detection zone 3 from top to bottom. A blood separation membrane is also provided in the whole blood separation zone 1 (in this embodiment, the whole blood separation zone 1 is formed by combining Whatman No. 1 filter paper and a blood separation membrane. The blood separation membrane in this embodiment is Whatman VF2 lateral flow chromatography whole blood separation membrane), the whole blood separation zone 1 includes a circular whole blood dripping port 12 and a V-shaped separation channel 11 connected to the whole blood dripping port 12, with the circular whole blood dripping port 12 located at the two top ends of the V-shaped separation channel 11; the blood potassium detection zone 3 includes a convex detection channel 31 and a detection port 32 connected to the two bottom ends of the convex detection channel 31; the detection port 32 has a D-shaped structure, wherein the semicircular end of the D is connected to the two bottom ends of the convex detection channel 31, and the straight end of the D is separated from the electronic component connection terminal 5 (separating the electronic component connection terminal 5 from the sensing electrode); the serum diffusion zone 2 has a stepped structure (formed by the thickness difference between the V-shaped separation channel 11 and the convex detection channel 31).

[0052] The preparation method of the paper-based sensor for detecting blood potassium in this embodiment comprises the following steps:

[0053] (1) Substrate treatment: The pattern designed by CAD was printed on Whatman No. 1 filter paper by screen printing on a hot plate and dried in an oven at 52 °C for 5 minutes until the wax completely penetrated the paper;

[0054] (2) Preparation of sensing electrodes: The prepared silver ink and carbon ink were respectively printed on Whatman No. 1 filter paper by screen printing. After all the printed layers were completely dried, the electrodes were cut out and 5 μL of PBA material solution was dropped on the carbon contact points. The solution was then dried in an oven at 60°C for 15 min to allow the PBA material to adhere to the carbon contact points, thereby forming a fully solid-state PBA working electrode. 2 μL of 0.1 M FeCl3 solution was then added to the silver contact points. The solution was then dried in an oven at 60°C for 15 min to form a reference electrode. A reference film was then covered on the reference electrode.

[0055] (3) Preparation of whole-blood microfluidic channel: Cut out Whatman No.1 filter paper of 1.5×2.5 cm size and Whatman VF2 blood separation membrane of 1.7×2.5 cm size, overlap the Whatman No.1 filter paper and Whatman VF2 blood separation membrane on the glass slide by about 1 mm, define the iron mold of the whole-blood separation area and the blood potassium detection area through the μPAD manufacturing process, and use the permanent magnet on the back of the glass slide to fix the iron mold on the paper, then melt the wax on a hot plate at 125°C, and use the wax dipping technique to dip the Whatman No.1 filter paper and Whatman VF2 blood separation membrane into the melted wax for 1 second and then remove it. When the wax cools to room temperature, peel the paper from the glass slide and separate it from the iron mold to obtain an integrated whole-blood microfluidic channel;

[0056] (4) Preparation of paper-based sensor: The whole blood microfluidic channel obtained in step (3) and the sensing electrode obtained in step (2) were adhered to the substrate treated in step (1) using 3M waterproof adhesive to integrate the paper-based sensor.

[0057] The preparation process of the PBAs material solution is as follows:

[0058] ① Dissolve K4Fe(CN)6·3H2O in 100 mL of saturated KCl solution to prepare a 2 mM solution A;

[0059] ② Dissolve FeSO4·7H2O and MnSO4·H2O in 80 mL of saturated KCl solution to obtain mixed solution B (mixed solution B contains 0.8 mM FeSO4 and 1.6 mM MnSO4);

[0060] ③ Under magnetic stirring at 60°C, the mixed solution B obtained in step ② was slowly added dropwise to the mixed solution A obtained in step ①. After stirring for 12 hours, the formed precipitate was centrifuged, thoroughly washed with deionized water, and dried in air at 80°C for 12 hours to obtain the PBA material;

[0061] ④ Take 4 mg of the PBAs material obtained in step ③, add 1 mg of PVDF and 200 μL of NMP, mix and ultrasonicate for 30 minutes to obtain a PBAs material solution.

[0062] The preparation process of the reference film is as follows:

[0063] 79.1 mg of PVB and 50 mg of KCl were poured into 1 mL of methanol, ultrasonicated for 30 minutes until uniformly dissolved, and dried to obtain a reference film.

[0064] The blood potassium detection paper-based sensor in this embodiment is used to detect potassium ions in a whole blood sample. The detection process is as follows:

[0065] Whole blood is added to the whole blood addition port 12 of the whole blood separation zone 1. After being filtered through the Whatman VF2 blood separation membrane below, the separated serum flows along the V-shaped separation channel 11 to the serum diffusion zone 2. It then gradually diffuses into the blood potassium detection zone 3 through the stepped flow path of the serum diffusion zone 2. The serum then flows through the convex detection channel 31 of the blood potassium detection zone 3 into the detection port 32. The potential is detected by the sensing electrode below the detection port 32 to obtain the potassium ion concentration in the whole blood.

[0066] Example 2 The preparation process of the paper-based sensor for potassium detection provided in this example is the same as that in Example 1, except that in this example, the PBA material is K during the preparation of the all-solid-state PBA working electrode. 1.6 Co 0.5 Ni 0.5 [Fe(CN)6] 0.7 0.6H2O, the amount of PBAs material solution added is 3μL.

[0067] Example 3 The preparation process of the paper-based sensor for blood potassium detection provided in this example is the same as that in Example 1, except that in this example, the PBA material is K during the preparation of the all-solid-state PBA working electrode. 1.8 Cu 0.3 Zn 0.7 [Fe(CN)6] 0.9 ·1.2H2O, the amount of PBAs material solution added is 4μL.

[0068] Example 4 The preparation process of the paper-based sensor for blood potassium detection provided in this example is the same as that in Example 1, except that in this example, the PBA material is K during the preparation of the all-solid-state PBA working electrode. 1.7 Mo 0.5 W 0.5 [Fe(CN)6] 0.7 0.9H2O, the amount of PBAs material solution added is 6μL.

[0069] Comparative Example 1

[0070] The structure and preparation process of the paper-based sensor for blood potassium detection provided in this comparative example are different from those in Example 1 in that no blood separation membrane is provided in the whole blood separation area of ​​the whole blood microfluidic channel in this comparative example.

[0071] Comparative Example 2

[0072] The preparation process of the paper-based sensor for blood potassium detection provided in this comparative example is the same as that of Example 1, except that, in this example, the amount of the PBA material solution added during the preparation of the all-solid-state PBA working electrode is 0 μL.

[0073] Test Example 1

[0074] Cyclic voltammetry and electrochemical impedance spectroscopy were performed on Examples 1-4 and Comparative Example 2 to test the electrochemical performance of the all-solid-state PBA working electrodes obtained by adding different types and volumes of PBA material solutions. The results showed that compared with Comparative Example 2, the all-solid-state PBA working electrodes obtained by adding PBA materials in Examples 1-4 had significantly improved electrochemical performance and good signal response. The cyclic voltammetry curves of Example 1 and Comparative Example 2 are shown in Figure 2. Figure 5 As shown, it can be seen that the area of ​​the curve of Example 1 is large, the capacitance is large, and the redox peak is high, indicating that compared with Comparative Example 2, the addition of 5 μL of PBAs material in Example 1 has better redox performance; the electrochemical impedance spectroscopy diagrams of Example 1 and Comparative Example 2 are shown in FIG. Figure 6 As shown, it is concluded that the addition of PBAs can improve the electrochemical performance of the electrode, and the addition of 5 μL of PBAs in Example 1 makes the electrode have a lower resistance, a faster electron transfer rate, and a faster signal response to potassium ions. Figure 5 and Figure 6 The results show that the all-solid-state PBA working electrode prepared by adding 5 μL of PBAs material solution in Example 1 has the best electrochemical performance and is the optimal embodiment of the present invention.

[0075] Test Example 2

[0076] This test example characterizes the dripping method of whole blood. When the potassium ion in the whole blood sample is detected using the blood potassium detection paper-based sensor prepared in Example 1, the whole blood is dripped once and in batches to determine the optimal dripping method and dripping amount. The corresponding results are shown in Figure 2. Figure 7 As shown, from Figure 7 As can be seen from a, when 125 μL of whole blood is dripped into each of the two whole blood dripping ports, congestion will occur after the dripping is completed. Due to the congestion of blood volume, the separated serum cannot be filled; Figure 7 As can be seen from b, when adding 5 drops of whole blood (50 μL each time) at each of the two whole blood dripping ports, the separated serum cannot fill the entire detection area due to the small amount of blood. Figure 7 c As can be seen, six drops of whole blood (50 μL each time) were added to each of the two whole blood dripping ports, and the separated serum completely filled the detection area; Figure 7 d As can be seen, after seven drops of whole blood (50 μL each) were added to each of the two whole blood droplet inlets, the blood volume exceeded the separation zone's capacity, causing it to overflow and preventing effective separation. The test results indicate that the optimal dripping method for the potassium-sensing paper-based sensor prepared in Example 1 for detecting potassium ions in whole blood samples is to drip whole blood into the blood droplet in batches, with the optimal number of drops being six and the optimal dripping volume being 50 μL each.

[0077] Test Example 3

[0078] The potassium ion concentration of the paper-based sensor for blood potassium detection obtained in the best embodiment 1 was tested to analyze the response of the prepared paper-based sensor for blood potassium detection to different concentrations of potassium ions. According to the method of embodiment 1, 7 paper-based sensors were prepared for detection of 10 -6 The response of potassium ions in aqueous potassium chloride solutions ranging from 1M to 1M, and the measured potentials are shown in Table 1:

[0079] Table 1 Potential changes of different concentrations of potassium ions

[0080]

[0081] From the results of the potential change with concentration in Table 1, based on the Nernst equation, the Nernst response can be obtained to be 50.32mV / decade, which is close to the theoretical value and -5 There is a good linear relationship between the potassium ion aqueous solution from M to 1M (such as Figure 8 As shown), it shows that the sensitivity of the paper-based sensor for blood potassium detection in Example 1 is high, and the lowest concentration of potassium ion response is 10 -5 M.

[0082] Test Example 4

[0083] The blood potassium detection paper-based sensor obtained in the optimal embodiment 1 was subjected to whole blood detection in different batches to determine the stability and accuracy of the detection results and the reusability of the sensor when the prepared blood potassium detection paper-based sensor was used to detect potassium ions in whole blood.

[0084] Eighteen paper-based sensors for blood potassium detection were prepared according to the method of Example 1 and divided into three batches for testing. Each batch used six identical paper-based sensors, which were evenly divided into three groups (A, B, and C) for testing.

[0085] (1) First, the first three groups of potassium detection paper-based sensors were calibrated, and the potassium ion concentrations of the calibration solutions were 4mM and 8mM respectively (the pH value and other ions contained in the calibration solutions were similar to those of real serum) and the standard curves were drawn. The response results are shown in Table 2. The standard curves are shown in Table 2. Figure 9 As shown, after obtaining the standard curves of the potassium ion aqueous solution and the calibration solution, the response of the blood potassium detection paper-based sensor to potassium ions in real fetal bovine serum was tested. The response results are shown in Table 3:

[0086] Table 2 Potassium ion response of different batches of potassium detection paper-based sensors to the calibration solution

[0087]

[0088] Table 3 Response of the first batch of potassium detection paper-based sensors to potassium ions in fetal bovine serum

[0089]

[0090] Through testing, it was found that the potential obtained when testing the actual sample was not within the standard curve of the potassium ion aqueous solution, but within the standard curve of the calibration solution. The average potential of the tested serum was 340.7mV, and the fetal bovine serum measured by the blood potassium detection paper-based sensor was 4.66mM, which is close to the potassium ion concentration of 4.42mM obtained using the existing commercially available blood potassium ion detection kit.

[0091] (2) Then, the first batch of 4mM and 8mM calibration solutions were used to calibrate the three groups of potassium detection paper-based sensors in the second batch. The response results are shown in Table 2, and the standard curves were drawn (corresponding to Figure 10 and Figure 11 After obtaining the standard curves of potassium ion aqueous solution and calibration solution, the responses of the second and third batches of blood potassium detection paper-based sensors to potassium ions in quality control serum of different concentrations (the blood potassium range measured by ISE of low-concentration quality control serum was 3.68~4.32mM, with an average of 4.00mM, and the blood potassium range measured by ISE of high-concentration quality control serum was 5.45~6.39mM, with an average of 5.9mM) were tested respectively. The response results are shown in Table 4:

[0092] Table 4 Potassium ion response of the paper-based sensor for blood potassium detection to the quality control serum

[0093]

[0094] The test results show that the second and third batches of electrodes made at different times using the same method and PBAs material solution have good repeatability in Nernst response and standard potential. They all have good responses to potassium ions in serum when detecting quality control serum of different concentrations, and the potentials of the three tests are not much different, indicating that the test results are accurate and repeatable, and can be used for calibration-free testing. In addition, the potassium ion concentrations measured when detecting quality control serum are within the range of quality control serum and are similar, with high detection sensitivity, and can be used for actual whole blood potassium detection.

[0095] Test Example 5

[0096] First, the fourth batch of paper-based sensors for blood potassium detection was prepared according to the method of Example 1, and the standard solution and quality control serum were used for detection, and the potassium ion responses in the standard solution and quality control serum with different concentrations were obtained as shown in Table 5, and the standard curves were drawn (respectively as shown in Table 5). Figure 12 and Figure 13 shown).

[0097] Table 5 Potassium ion response of the fourth batch of paper-based sensors for blood potassium detection to calibration solutions and quality control serum

[0098]

[0099] Secondly, according to the method of Comparative Example 1, a blood potassium detection paper-based sensor without a blood separation membrane was prepared, and a whole blood sample was taken. The blood potassium detection paper-based sensor of Example 1 and the blood potassium detection paper-based sensor of Comparative Example 1 were used for detection, respectively. The responses of the fourth batch of blood potassium detection paper-based sensors of Example 1 and the blood potassium detection paper-based sensors of Comparative Example 1 to potassium ions in whole blood under different standard curves and the corresponding potassium ion detection concentrations were obtained, as shown in Tables 6 and 7. At the same time, after the whole blood was centrifuged to obtain serum using the existing technology, an existing commercially available potassium ion detection kit was used for microplate reader detection as a control group. The differences in the responses of the blood potassium detection paper-based sensor with a blood separation membrane obtained by the method of Example 1 and the blood potassium detection paper-based sensor without a blood separation membrane obtained by the method of Comparative Example 1 to potassium ions and the potassium ion concentrations in the whole blood samples finally obtained were analyzed:

[0100] Table 6 Response of the fourth batch of potassium detection paper-based sensors in Example 1 to potassium ions in whole blood

[0101]

[0102] Table 7 Response of the blood potassium detection paper-based sensor of Comparative Example 1 to potassium ions in whole blood

[0103]

[0104] The results in Tables 6 and 7 indicate that the fourth batch of potassium-detecting paper sensors prepared using the method of Best Example 1 of the present invention maintained stable test results and exhibited a rapid response to potassium ions, approaching those of the control group (where the control group achieved a test result of 4.42 mM after whole blood treated and tested using the prior art). However, the results of Comparative Example 1, which lacked a blood separation membrane, differed significantly from those of the control group. The resulting low test results from the potassium-detecting paper sensors are due to the fact that some potassium ions in the blood reside within red blood cells. When the blood coagulates to form serum, a small amount of potassium ions from the red blood cells are released into the serum, resulting in low test results. This indicates that direct potassium ion detection using a paper-based sensor without a blood separation membrane can affect the potassium ion detection results and result in poor accuracy. However, the paper-based sensor with a blood separation membrane can directly detect whole blood samples, achieving higher accuracy and better stability.

[0105] At the same time, compared with the control group of the prior art, the paper-based sensor with a blood separation membrane prepared by the method of the embodiment of the present invention can directly perform detection without waiting for processes such as blood coagulation and centrifugation, and has a faster response speed to potassium ions, making the detection speed of potassium ions relatively fast and time-saving, significantly improving the detection efficiency of potassium ions.

[0106] The detection principle of the paper-based sensor for blood potassium detection of the present invention is as follows:

[0107] The present invention uses Prussian blue analogs to construct membraneless K⁺-SC-ISEs (solid contact potassium ion selective electrodes). Its mechanism of action is as follows: the PBAs material lattice realizes ion recognition function through reversible K⁺ insertion / extraction reaction, while the Fe 2+ / Fe 3 + and Mn 2+ / Mn 3+ These two redox pairs act as electron transfer bridges, converting K⁺ concentration signals into detectable potential responses.

[0108] Principle of potassium ion concentration calculation: According to the Nernst equation, for a potassium ion selective electrode, its electrode potential is linearly related to the logarithm of the potassium ion activity in the solution, and the expression is: , where E is the electrode potential, E 0 is the standard electrode potential, R is the gas constant, T is the absolute temperature, n is the number of electrons transferred in the reaction, F is the Faraday constant, It is the activity of potassium ions. The potassium ion concentration can be calculated by measuring the electrode potential.

[0109] Electrode potential E The calculation formula is: E = E s - E r , where E s is the working electrode potential, E r is the reference electrode potential.

[0110] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention may be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description. It is intended that all variations that fall within the meaning and range of equivalents of the claims be embraced within the present invention. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any minor modifications, equivalent replacements, and improvements made to the above embodiments based on the technical essence of the present invention shall be included in the scope of protection of the technical solution of the present invention.

Claims

1. A paper-based sensor for detecting blood potassium, comprising a substrate and a sensing electrode located on the substrate, characterized in that: The whole blood microfluidic channel is an integrated structure, comprising a serum diffusion zone (2), a whole blood separation zone (1) located above the serum diffusion zone (2), and a blood potassium detection zone (3) located below the serum diffusion zone (2); a blood separation membrane is further provided in the whole blood separation zone (1); the whole blood microfluidic channel is prepared from qualitative filter paper; the serum diffusion zone (2) is a step-shaped structure; the sensor is integrated by a substrate, a sensing electrode, and a whole blood microfluidic channel, and the sensing electrode is located between the substrate and the whole blood microfluidic channel; Wherein, the substrate is a qualitative filter paper, the sensing electrode includes an all-solid-state PBA working electrode and an Ag / AgCl reference electrode, and the all-solid-state PBA working electrode is prepared from PBAs material; The PBAs material is a multi-metal co-doped Prussian blue analogue, and its general formula is K x A y B z [Fe(CN)6] m nH2O, wherein x=1.0-2.2, y=0.1-0.7, z=0.3-0.9, m=0.7-1.0, n=0.5-1.5, A is at least one of Fe, Mn, Co, Ni, Cu, Zn, La, V, Ti, Mo, and W, and B is at least one of Fe, Mn, Co, Ni, Cu, Zn, La, V, Ti, Mo, and W.

2. The paper-based sensor for detecting blood potassium according to claim 1, characterized in that: The whole blood separation area (1) comprises a V-shaped separation channel (11) and a whole blood dripping port (12) connected to both ends of the V-shaped separation channel (11), wherein the whole blood dripping port (12) is a circular structure; the blood potassium detection area (3) comprises a convex-shaped detection channel (31) and a detection port (32) connected to both ends of the convex-shaped detection channel (31).

3. The paper-based sensor for detecting blood potassium according to claim 2, characterized in that: A conductive layer is further provided between the sensing electrode and the substrate, the conductive layer comprising an electrode end (4) and electronic component connection ends (5) located on both sides of the electrode end (4), the electrode end (4) matching the sensing electrode, the electrode end (4) being a circular structure, and the electronic component connection ends (5) being a rectangular structure.

4. The paper-based sensor for detecting blood potassium according to claim 3, characterized in that: The detection port (32) is a D-shaped structure, wherein the semicircular end of the D-shape is connected to the two bottom ends of the convex detection channel (31), and the straight end of the D-shape is separated from the electronic component connection end (5).

5. A method for preparing a paper-based sensor for detecting blood potassium according to claim 1, characterized in that: The following steps are involved: (1) Substrate treatment: Print the designed pattern on qualitative filter paper and dry it in an oven; (2) Preparation of sensing electrodes: Silver ink and carbon ink were printed on qualitative filter paper respectively. After all printed layers were completely dried, the electrodes were cut out. PBAs material solution was dripped on the carbon contact points and dried to make a fully solid-state working electrode. FeCl3 solution was dripped on the silver contact points and dried to make a reference electrode. (3) Preparation of whole-blood microfluidic channel: Qualitative filter paper and blood separation membrane were overlapped on a glass slide, and μPAD patterning was performed using an iron mold to obtain a whole-blood separation area and a blood potassium detection area. The qualitative filter paper and blood separation membrane were combined using wax dipping technology, and after cooling to room temperature, they were peeled off to obtain an integrated whole-blood microfluidic channel; (4) Preparation of paper-based sensor: The whole blood microfluidic channel obtained in step (3) and the sensing electrode obtained in step (2) are pasted on the substrate treated in step (1) to form a paper-based sensor.

6. The method for preparing a paper-based sensor for detecting blood potassium according to claim 5, wherein: In the preparation process of the whole blood microfluidic channel in step (3), the overlapping thickness of the qualitative filter paper and the blood separation membrane is 1 mm; the temperature of the wax dipping technique is 125° C., and the time is 1 s.

7. The method for preparing a paper-based sensor for detecting blood potassium according to claim 5, wherein: The drying temperature during substrate treatment in step (1) is 52° C., and the drying time is 5 minutes. The drying temperature of the all-solid-state working electrode and the reference electrode during preparation of the sensing electrode in step (2) is 60° C., and the drying time is 15 minutes. The printing method in steps (1) and (2) is screen printing.

8. An application of the paper-based sensor for detecting blood potassium according to claim 1, characterized in that: Used to detect potassium ions in whole blood samples.

9. The use of the paper-based sensor for detecting blood potassium according to claim 8, characterized in that: During the test, whole blood was added to the whole blood adding area in batches, with a total of 6 additions, and 50 μL was added each time.

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