Paper-based sensor for detecting blood potassium as well as preparation method and application of paper-based sensor

Abstract

The invention relates to the technical field of blood potassium detection, in particular to a blood potassium detection paper-based sensor and a preparation method and application thereof.The sensor is obtained by integrating a substrate, a sensing electrode and a whole blood microfluid channel, and the sensing electrode is located between the substrate and the whole blood microfluid channel; the whole blood microfluid channel comprises a serum diffusion area, a whole blood separation area positioned above the serum diffusion area and a blood potassium detection area positioned below the serum diffusion area; a blood separation membrane is also arranged in the whole blood separation area; the substrate is qualitative filter paper, and the sensing electrode comprises an all-solid-state PBA working electrode and an Ag / AgCl reference electrode; compared with the prior art, calibration is not needed when potassium ions in whole blood are detected, the whole blood sample can be directly used, centrifugal separation is not needed, the detection speed is increased, the hemolysis problem of the whole blood sample in blood potassium detection can be avoided, and the detection sensitivity, accuracy and stability are further improved.

Description

Technical Field

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

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

[0003] Traditional clinical detection methods for blood potassium mainly include ion exchange chromatography, flame photometry, ion selective electrode method, etc. These methods usually rely on complex equipment and professional technicians to achieve detection, which limits their application in on-site first aid, home self-monitoring, and environments with limited resources. With the increasing demand for health monitoring, especially for some patients who need to regularly monitor blood potassium, such as patients with abnormal renal function, there is an urgent need for a method and device that are easy to operate, low-cost, and can quickly detect blood potassium in whole blood on-site.

[0004] In recent years, with the development of point-of-care testing (POCT) technology and microfluidic technology, more and more portable and low-cost blood potassium detection sensors have been developed. However, most of them are only applicable to blood samples that have been centrifuged and separated, and it is difficult to directly apply them to untreated whole blood samples. This is because when the existing detection sensors directly use whole blood samples for blood potassium detection, biological substances such as proteins and cells are easily adsorbed on the sensor surface, resulting in electrode passivation, which reduces the performance of the sensor, shortens the service life of the sensor, and affects the sensitivity and stability of the detection results. Moreover, the composition of whole blood samples is complex, and direct detection will interfere with the detection results, easily resulting in false positives or false negatives, affecting the accuracy of the detection results. At the same time, the sensor requires a certain reaction time to obtain a stable detection signal when detecting blood potassium, the detection speed is slow, and the detection time is too long. And too long detection time will cause hemolysis. The potassium ion concentration in red blood cells is much higher than that outside the cells. Once hemolysis occurs, the potassium ions in red blood cells will be released into the plasma, ultimately leading to a false increase in the measured value of blood potassium and affecting the accuracy of the detection results.

[0005] At present, due to its advantages such as low cost, portability, and simple operation, paper-based sensors have received extensive attention in the field of biological detection. However, current paper-based sensors are rarely used for potassium ion detection in whole blood. This is because when detecting potassium ions in untreated whole blood, it is restricted by the detection speed and hemolysis problems, and there are also deficiencies in aspects such as detection accuracy, sensitivity, and stability. Moreover, most of them require calibration before detection, and the detection process is cumbersome and time-consuming. Therefore, it is of great practical significance to develop a highly efficient, accurate, and convenient paper-based sensor for potassium ion detection in whole blood. Summary of the Invention

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

[0007] A potassium ion detection paper-based sensor includes a substrate and a sensing electrode located on the substrate, and also includes a whole blood microfluidic channel. The whole blood microfluidic channel is an integrated structure, including a serum diffusion area, a whole blood separation area located above the serum diffusion area, and a potassium ion detection area located below the serum diffusion area; a blood separation membrane is also provided in the whole blood separation area; the whole blood microfluidic channel is prepared from qualitative filter paper. Among them, 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 PBAs material.

[0008] Further, 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, where 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, W, and B is at least one of Fe, Mn, Co, Ni, Cu, Zn, La, V, Ti, Mo, W.

[0009] Preferably, the general formula of the PBAs material is K x Fe y Mn z [Fe(CN)6] m ·nH2O, where x = 1.5 - 2.0, y = 0.1 - 0.5, z = 0.5 - 0.9, m = 0.9 - 1.0, n = 0.5 - 1.0.

[0010] Preferably, the general formula of the PBAs material is 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.

[0011] Preferably, the general formula of the PBAs material is 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.

[0012] Preferably, the general formula of the PBAs material is 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.

[0013] Preferably, the general formula of the PBAs material is 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.

[0014] Furthermore, the sensor is integrally formed 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.

[0015] Furthermore, the whole blood separation area includes a V-shaped separation channel and a whole blood dropping port communicating with both ends of the V-shaped separation channel, and the whole blood dropping port is a circular structure; the blood potassium detection area includes a convex-shaped detection channel and a detection port communicating with both ends of the convex-shaped detection channel.

[0016] Furthermore, the serum diffusion area is a stepped structure formed by the thickness difference generated at the junction of the V-shaped separation channel and the convex-shaped detection channel.

[0017] Furthermore, a conductive layer is provided between the sensing electrode and the substrate. 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 ends are rectangular structures.

[0018] Furthermore, the detection port is a D-shaped structure. Among them, the semi-circular end of the D shape communicates with the two bottom ends of the convex-shaped detection channel, and the straight end of the D shape is separated from the electronic component connection end.

[0019] The present invention also provides a preparation method for the above-mentioned potassium blood detection paper-based sensor, including the following steps: (1) Substrate treatment: Printing the designed pattern on qualitative filter paper and drying it in an oven; (2) Preparation of the sensing electrode: Printing silver ink and carbon ink on qualitative filter paper respectively. After all the printed layers are thoroughly dried, cutting out the electrodes. Dropping the PBAs material solution on the carbon contact points and drying to form a fully solid-state working electrode, and dropping the FeCl3 solution on the silver contact points and drying to form a reference electrode; (3) Preparation of the whole blood microfluidic channel: Overlapping qualitative filter paper and a blood separation membrane on a glass slide, performing μPAD patterning through an iron mold to obtain a whole blood separation area and a potassium blood detection area, and combining the qualitative filter paper and the blood separation membrane using the wax dipping technique. After cooling to room temperature, peeling to obtain an integrated whole blood microfluidic channel; (4) Preparation of the paper-based sensor: Pasting the whole blood microfluidic channel obtained in step (3) and the sensing electrode obtained in step (2) on the substrate processed in step (1) to integrate and obtain the paper-based sensor.

[0020] Furthermore, during 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.

[0021] Furthermore, during the substrate treatment process in step (1), the drying temperature is 52 °C, and the drying time is 5 min; during the preparation process of the sensing electrode in step (2), the drying temperature of the fully 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.

[0022] Furthermore, the potassium blood detection paper-based sensor is used to detect potassium ions in whole blood samples.

[0023] Furthermore, during the process of detecting potassium ions in whole blood samples, whole blood is added dropwise to the whole blood dropping area in batches. The total number of dropwise additions is 6 times, and each time 50 μL is added.

[0024] The advantages of the present invention are as follows: 1. The present invention provides a potassium blood detection paper-based sensor, which can directly detect whole blood samples, with high sensitivity and portability; when directly detecting potassium ions in whole blood, through a whole blood microfluidic channel with a blood filtration function, the separation of whole blood can be completed within several minutes, avoiding the passivation of electrodes and improving the detection efficiency; compared with the prior art, it can avoid the occurrence of hemolysis problems in the potassium blood detection of whole blood samples, improve the detection sensitivity, accuracy and stability of detection results, has a simple preparation process, low cost, is more portable, and is more suitable for use in other outdoor scenarios such as small and medium-sized hospitals, home use, and travel. 2. The potassium blood detection paper-based sensor of the present invention integrates a filter paper substrate and a all-solid-state PBA working electrode. The PBAs material used in the all-solid-state PBA working electrode can not only be used as a solid contact layer, but also as an ion-selective membrane, reducing the complexity and cost of the electrode structure, lowering the requirements for the process, and reducing the instability of the phase boundary potential of the solid ion-selective electrode without dropping an ion-selective membrane, improving the repeatability between electrodes. At the same time, the dual characteristics of the ion-selective membrane and 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 . When preparing the PBAs material, the redox potential of the material can be controlled through the preparation process to make E 0 it unified. Different electrodes can share a calibration curve, making the paper-based sensor prepared by the present invention available for calibration-free detection. Compared with the prior art, it is more convenient to use and significantly improves the detection speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the integration process of the potassium blood detection paper-based sensor of the present invention ( Figure 1 a is the substrate, Figure 1 b is the sensing electrode, Figure 1 c is the whole blood microfluidic channel, Figure 1 d is the integrated potassium blood detection paper-based sensor); Figure 2 is a diagram of the integration process of the substrate and sensing electrode of the potassium blood detection paper-based sensor of the present invention; Figure 3 is a schematic diagram after the integration of the substrate and sensing electrode of the potassium blood detection paper-based sensor of the present invention; Figure 4 is a schematic diagram of the whole blood microfluidic channel of the potassium blood detection paper-based sensor of the present invention; Figure 5 is a cyclic voltammogram of Example 1 and Comparative Example 2 in Test Example 2 of the present invention; Figure 6It is the electrochemical impedance spectroscopy diagram of Example 1 and Comparative Example 2 in Test Example 2 of the present invention; Figure 7 It is the comparison diagram of whole blood dripping methods in Test Example 1 of the present invention ( Figure 7 a is to drip 125 uL of whole blood at one time, Figure 7 b is to drip 50 μL of whole blood 5 times each time, Figure 7 c is to drip 50 μL of whole blood 6 times each time, Figure 7 d is to drip 50 μL of whole blood 7 times each time); Figure 8 It is the potassium ion response test diagram at different concentrations in Test Example 3 of the present invention; Figure 9 It is the standard curve diagram of the first batch of sensors for the calibration solution in Test Example 4 of the present invention; Figure 10 It is the standard curve diagram of the second batch of sensors for the calibration solution in Test Example 4 of the present invention; Figure 11 It is the standard curve diagram of the third batch of sensors for the calibration solution in Test Example 4 of the present invention; Figure 12 It is the standard curve diagram of the fourth batch of sensors for the quality control serum in Test Example 5 of the present invention; Figure 13 It is the standard curve diagram of the fourth batch of sensors for the standard solution in Test Example 5 of the present invention; Explanation of reference numerals: 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-shaped detection channel; 32. Detection port; 4. Electrode end; 5. Electronic component connection end. Detailed implementation manners

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0027] It should be noted that the installation methods and technical terms mentioned in the present invention are all technical terms that are well-known in the technical field, so no further explanation will be made. In addition, the same reference numerals are used for the same components, but this does not affect and should not constitute an accurate understanding of the technical solution by those skilled in the art.

[0028] Example 1 This example provides a blood potassium detection paper-based sensor, and the structure is as Figure 1 shown in d, and it is composed of a substrate (Figure 1 a), a sensing electrode ( Figure 1 b), and a whole blood microfluidic channel ( Figure 1 c) are integrally formed. The substrate, the sensing electrode, and the whole blood microfluidic channel are all prepared from qualitative filter paper (the qualitative filter paper used in this embodiment is Whatman No.1 filter paper).

[0029] As Figure 2 and Figure 3 shown, a conductive layer (the conductive layer in this embodiment refers to Figure 2 the silver layer and the carbon layer in 1.85 Fe 0.33 Mn 0.67 [Fe(CN)6] 0.98 ·0.77H2O) is further provided between the sensing electrode and the substrate. The conductive layer includes a circular electrode end 4 and rectangular electronic component connection ends 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 PBAs material of the all-solid-state PBA working electrode in this embodiment is K Figure 3 shown.

[0030] As Figure 4 shown, the whole blood microfluidic channel is an integrated structure, which successively includes a whole blood separation area 1, a serum diffusion area 2, and a blood potassium detection area 3 from top to bottom; a blood separation membrane is further provided in the whole blood separation area 1 (the whole blood separation area 1 in this embodiment is formed by combining Whatman No.1 filter paper and a blood separation membrane, and the blood separation membrane in this embodiment is Whatman VF2 lateral flow chromatography whole blood separation membrane). The whole blood separation area 1 includes a circular whole blood dropping port 12 and a V-shaped separation channel 11 communicated with the whole blood dropping port 12. The circular whole blood dropping port 12 is located at the two top ends of the V-shaped separation channel 11; the blood potassium detection area 3 includes a convex-shaped detection channel 31 and a detection port 32 communicated with the two bottom ends of the convex-shaped detection channel 31; the detection port 32 is a D-shaped structure, wherein the semi-circular end of the D shape is communicated with the two bottom ends of the convex-shaped detection channel 31, and the straight end of the D shape is separated from the electronic component connection end 5 (separating the electronic component connection end 5 and the sensing electrode); the serum diffusion area 2 is a stepped structure (formed by the thickness difference generated by the V-shaped separation channel 11 and the convex-shaped detection channel 31).

[0031] The preparation method of the blood potassium detection paper-based sensor in this embodiment includes the following steps: (1) Substrate treatment: The pattern designed by CAD is 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 penetrates the paper. (2) Preparation of sensing electrodes: The prepared silver ink and carbon ink are respectively printed on Whatman No.1 filter paper by screen printing. After all the printed layers are thoroughly dried, the electrodes are cut out. After dropping 5 μL of the PBAs material solution on the carbon contact points, it is dried in an oven at 60 °C for 15 min to make the PBAs material adhere to the carbon contact points, thus fabricating a all-solid-state PBA working electrode. Then, after dropping 2 μL of 0.1 M FeCl3 solution on the silver contact points, it is dried in an oven at 60 °C for 15 min to make a reference electrode, and a reference membrane is covered on the reference electrode. (3) Preparation of whole blood microfluidic channels: Cut out Whatman No.1 filter paper with a size of 1.5×2.5 cm and Whatman VF2 blood separation membrane with a size of 1.7×2.5 cm. Overlap the Whatman No.1 filter paper and Whatman VF2 blood separation membrane on the glass slide by about 1 mm. Through the μPAD manufacturing process, define the iron molds for the whole blood separation area and the potassium blood detection area, and use the permanent magnet on the back of the glass slide to fix the iron molds on the paper. Then, melt the wax on a hot plate at 125 °C, and immerse the assembly composed of Whatman No.1 filter paper and Whatman VF2 blood separation membrane into the melted wax for 1 second and then take it out. 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. (4) Preparation of paper-based sensor: The whole blood microfluidic channel obtained in step (3) and the sensing electrodes obtained in step (2) are pasted on the substrate treated in step (1) using 3M waterproof glue to integrate and obtain a paper-based sensor.

[0032] The preparation process of the PBAs material solution is as follows: ① Dissolve K4Fe(CN)6·3H2O in 100 mL of saturated KCl solution to obtain a mixed solution A with a concentration of 2 mM. ② Dissolve FeSO4·7H2O and MnSO4·H2O in 80 mL of saturated KCl solution respectively to obtain a mixed solution B (there is 0.8 mM of FeSO4 and 1.6 mM of MnSO4 in the mixed solution B). ③ Under magnetic stirring at 60 °C, slowly drop the mixed solution B obtained in step ② into the mixed solution A obtained in step ①. After stirring for 12 hours, centrifuge the formed precipitate, thoroughly wash it with deionized water, and dry it in air at 80 °C for 12 hours to obtain the PBAs material. ④ Add 4 mg of the PBAs material obtained in step ③, 1 mg of PVDF and 200 μL of NMP, mix them and ultrasonicate for 30 minutes to obtain a PBAs material solution.

[0033] The preparation process of the reference membrane is as follows: Pour 79.1 mg of PVB and 50 mg of KCl into 1 mL of methanol, ultrasonicate for 30 minutes until dissolved uniformly, and obtain the reference membrane after drying.

[0034] The potassium ion detection paper-based sensor in this example is used to detect potassium ions in whole blood samples. The detection process is as follows: Drop whole blood at the whole blood dropping port 12 of the whole blood separation area 1. After filtering through the Whatman VF2 blood separation membrane in the lower layer, the separated serum flows along the V-shaped separation channel 11 to the serum diffusion area 2, gradually diffuses through the stepped flow channel of the serum diffusion area 2 to the potassium ion detection area 3, flows into the detection port 32 through the convex-shaped detection channel 31 in the potassium ion detection area 3, and the concentration of potassium ions in the whole blood is obtained by detecting the potential through the sensing electrode under the detection port 32.

[0035] Example 2 The preparation process of the potassium ion detection paper-based sensor provided in this example is the same as that in Example 1. The difference is that in the process of preparing the all-solid-state PBA working electrode in this example, the PBAs material is K 1.6 Co 0.5 Ni 0.5 [Fe(CN)6] 0.7 ·0.6H2O, and the dropping amount of the PBAs material solution is 3 μL.

[0036] Example 3 The preparation process of the potassium ion detection paper-based sensor provided in this example is the same as that in Example 1. The difference is that in the process of preparing the all-solid-state PBA working electrode in this example, the PBAs material is K 1.8 Cu 0.3 Zn 0.7 [Fe(CN)6] 0.9 ·1.2H2O, and the dropping amount of the PBAs material solution is 4 μL.

[0037] Example 4 The preparation process of the potassium ion detection paper-based sensor provided in this example is the same as that in Example 1. The difference is that in the process of preparing the all-solid-state PBA working electrode in this example, the PBAs material is K 1.7 Mo 0.5 W 0.5 [Fe(CN)6] 0.7 ·0.9H2O, and the dropping amount of the PBAs material solution is 6 μL.

[0038] Comparative Example 1 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 of this comparative example.

[0039] Comparative Example 2 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 the process of preparing the all-solid-state PBA working electrode in this example, the amount of PBAs material solution added is 0 μL.

[0040] Test Example 1 Cyclic voltammetry and electrochemical impedance tests were performed on Examples 1-4 and Comparative Example 2 to test the electrochemical performance of the all-solid-state PBA working electrode obtained by dropping different types and volumes of PBA material solutions. The results showed that compared with Comparative Example 2, the all-solid-state PBA working electrode 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 FIG. Figure 5 As shown, it can be seen that the curve of Example 1 has a large area, a large capacitance, and a high redox peak, indicating that compared with Comparative Example 2, the addition of 5 μL of PBAs material in Example 1 has better redox performance; the electrochemical AC impedance diagrams of Example 1 and Comparative Example 2 are shown in FIG. Figure 6 As shown, 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 smaller 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.

[0041] Test Example 2 This test example characterizes the dripping method of whole blood. When the potassium ion detection paper-based sensor prepared in Example 1 is used to detect potassium ions in whole blood samples, whole blood is dripped once and in batches to determine the best dripping method and dripping amount. The corresponding results are shown in FIG. Figure 7 As shown, from Figure 7 As can be seen from a, 125 μL of whole blood is dripped at each of the two whole blood dripping ports at one time. After dripping, congestion will occur, and the separated serum cannot be filled due to the congestion of blood volume; Figure 7 b It can be seen that when whole blood is added 5 times (50 μL each time) at each of the two whole blood drop ports, the separated serum cannot fill the detection area completely due to the small amount of blood. Figure 7It can be seen that whole blood (50 μL each time) was added dropwise 6 times at each of the two whole blood addition ports, and the detection area was completely filled with the separated serum; from Figure 7 It can be seen that whole blood (50 μL each time) was added dropwise 7 times at each of the two whole blood addition ports. Since the amount of blood exceeded the capacity of the separation area, the whole blood overflowed from the separation area and effective separation could not be carried out. From the test results, it can be concluded that when the potassium ion detection paper-based sensor prepared in Example 1 is used to detect potassium ions in whole blood samples, the best way of adding dropwise is to add whole blood to the whole blood addition area in batches, the best number of adding dropwise is 6 times, and the best amount of adding dropwise is 50 μL each time.

[0042] Test Example 3 The potassium ion concentration of the potassium ion detection paper-based sensor obtained from the optimal Example 1 was tested to analyze the response of the prepared potassium ion detection paper-based sensor to potassium ions at different concentrations. 7 paper-based sensors were prepared according to the method of Example 1 to detect the response of potassium ions in potassium chloride aqueous solutions from 10 -6 M to 1 M, and the measured potentials are shown in Table 1: Table 1 Potential changes of potassium ions at different concentrations

[0043] From the change results of the potential with the concentration in Table 1, based on the Nernst equation, the Nernst response can be obtained as 50.32 mV / decade, approaching the theoretical value, and there is a good linear relationship in the potassium ion aqueous solutions from 10 -5 M to 1 M (as Figure 8 shown), indicating that the potassium ion detection paper-based sensor of Example 1 has a high sensitivity, and the lowest concentration of potassium ion response is 10 -5 M.

[0044] Test Example 4 The potassium ion detection paper-based sensor obtained from the optimal Example 1 was used for 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 potassium ion detection paper-based sensor is used for potassium ion detection in whole blood.

[0045] 18 potassium ion detection paper-based sensors were prepared according to the method of Example 1 and divided into three batches for detection. 6 identical paper-based sensors were used in each batch and evenly divided into three groups (A, B, C) for detection.

[0046] (1) First, the three groups of potassium ion detection paper-based sensors in the first batch were calibrated, and calibration solutions with potassium ion concentrations of 4 mM and 8 mM (the pH value and other ions contained in the calibration solution were similar to those in real serum) were detected and standard curves were drawn. The response results are shown in Table 2, and the standard curves are as Figure 9As shown, after obtaining the standard curves of the potassium ion aqueous solution and the calibration solution, the response of the potassium ion in the blood potassium test paper-based sensor to the real fetal bovine serum was detected, and the response results are shown in Table 3: Table 2 Potassium ion response of blood potassium test paper-based sensors of different batches to the calibration solution

[0047] Table 3 Potassium ion response of the first batch of blood potassium test paper-based sensors to fetal bovine serum

[0048] It was found through detection that the potential obtained when detecting 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 for detecting serum was 340.7 mV. Substituting it into the blood potassium test paper-based sensor, the fetal bovine serum measured was 4.66 mM, which was close to the potassium ion concentration of 4.42 mM obtained using the existing commercially available blood potassium ion detection kit.

[0049] (2) Then, the calibration solutions of 4 mM and 8 mM of the first batch were used to calibrate and detect three groups of blood potassium test paper-based sensors of the second batch respectively. The response results are shown in Table 2, and the standard curves were drawn (corresponding to Figure 10 and Figure 11 ) respectively. After obtaining the standard curves of the potassium ion aqueous solution and the calibration solution, the responses of the second batch and the third batch of blood potassium test paper-based sensors to potassium ions in different concentrations of quality control serum (the potassium blood range measured by ISE for low-concentration quality control serum was 3.68 - 4.32 mM, with an average value of 4.00 mM, and the potassium blood range measured by ISE for high-concentration quality control serum was 5.45 - 6.39 mM, with an average value of 5.9 mM) were detected respectively. The response results are shown in Table 4: Table 4 Potassium ion response of blood potassium test paper-based sensors to quality control serum

[0050] It can be found from the test results that the second batch and the third batch of electrodes made at different times using the same method and PBAs material solution had good repeatability in the Nernst response and standard potential. They had good responses to potassium ions in the serum when detecting different concentrations of quality control serum, and the potential differences in the three detections were not significant, indicating that the accuracy and repeat stability of the detection results were relatively high, which could be used for calibration-free detection. Moreover, the potassium ion concentrations measured when detecting the quality control serum were within the range of the quality control serum and were close to each other, and the detection sensitivity was relatively high, which could be used for the detection of blood potassium in actual whole blood.

[0051] Test Example 5 First, prepare the fourth batch of potassium blood detection paper-based sensors according to the method of Example 1, and use standard solutions and quality control sera for detection respectively. The potassium ion responses in standard solutions with different concentrations and quality control sera with different concentrations are shown in Table 5, and standard curves are plotted (as shown in Figure 12 and Figure 13 respectively).

[0052] Table 5 Potassium ion responses of the fourth batch of potassium blood detection paper-based sensors to calibration solutions and quality control sera

[0053] Secondly, prepare potassium blood detection paper-based sensors without a blood separation membrane according to the method of Comparative Example 1. Take whole blood samples and use the potassium blood detection paper-based sensors of Example 1 and the potassium blood detection paper-based sensors of Comparative Example 1 for detection respectively. The responses of the fourth batch of potassium blood detection paper-based sensors of Example 1 and the potassium blood detection paper-based sensors of Comparative Example 1 to potassium ions in whole blood and the corresponding potassium ion detection concentrations under different standard curves are obtained, as shown in Tables 6 and 7. At the same time, after using the prior art to centrifuge and separate whole blood to obtain serum, use a commercially available potassium ion detection kit for ELISA detection as a control group. Analyze the differences in the responses of the potassium blood detection paper-based sensors with a blood separation membrane obtained by the method of Example 1 and the potassium blood detection paper-based sensors without a blood separation membrane obtained by the method of Comparative Example 1 to potassium ions and the potassium ion concentration in the finally obtained whole blood sample: Table 6 Responses of the fourth batch of potassium blood detection paper-based sensors of Example 1 to potassium ions in whole blood

[0054] Table 7 Responses of the potassium blood detection paper-based sensors of Comparative Example 1 to potassium ions in whole blood

[0055] From the results of Tables 6 and 7, it can be seen that the detection results of the fourth batch of potassium blood detection paper-based sensors prepared by the method of the best embodiment 1 of the present invention are still stable, and the response speed to potassium ions is relatively fast, which is close to the results of the control group (the detection result after the whole blood is processed and detected by the prior art in the control group is 4.42 mM). In Comparative Example 1, no blood separation membrane is set, and the result is quite different from that of the control group. The detection result of the finally obtained potassium blood detection paper-based sensor is on the low side because a part of the potassium ions in the blood exist in red blood cells. When the blood coagulates to form serum, a small amount of potassium ions in the red blood cells will be released into the serum, resulting in a low detection result. Thus, it can be seen that directly detecting whole blood samples with the paper-based sensors without setting a blood separation membrane will affect the detection results of potassium ions and the accuracy is poor, while the paper-based sensors with a blood separation membrane can directly detect whole blood samples, and the detection accuracy is relatively high and the stability is good.

[0056] Meanwhile, 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 does not need to wait for processes such as blood coagulation and centrifugation, can directly perform detection, and has a relatively fast response speed to potassium ions, so that the detection speed of potassium ions is relatively fast and the time consumption is short, significantly improving the detection efficiency of potassium ions.

[0057] The detection principle of the blood potassium detection paper-based sensor of the present invention is as follows: The present invention uses a Prussian blue analogue to construct membrane-free K⁺-SC-ISEs (solid contact potassium ion selective electrodes), and its mechanism of action is as follows: the crystal lattice of the PBAs material realizes the ion recognition function through reversible K⁺ insertion / extraction reactions, and at the same time, Fe 2+ / Fe 3 + and Mn 2+ / Mn 3+ These two redox pairs act as electron transfer bridges to convert the K⁺ concentration signal into a detectable potential response.

[0058] Potassium ion concentration calculation principle: According to the Nernst equation, for a potassium ion selective electrode, its electrode potential has a linear relationship with 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, is the activity of potassium ions, and the potassium ion concentration can be calculated by measuring the electrode potential.

[0059] The calculation formula for the electrode potential E is: E = E s - E r , where, E s is the working electrode potential, E r is the reference electrode potential.

[0060] For those skilled in the art, the present inventive concept is not limited to the details of the above-described exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present inventive concept. Therefore, from any perspective, the embodiments should be regarded as exemplary and non-limiting. The scope of the present inventive concept is defined by the appended claims rather than the above description. Thus, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present inventive concept. Any reference signs in the claims should not be construed as limiting the claims involved.

[0061] The above are only the preferred embodiments of the present inventive concept, and are not intended to limit the present inventive concept. Any minor modifications, equivalent substitutions, and improvements made to the above embodiments based on the technical essence of the present inventive concept should be included within the protection scope of the technical solution of the present inventive concept.

Claims

1. A paper-based sensor for detecting blood potassium, comprising a substrate and a sensing electrode located on the substrate, characterized in that: It also comprises a whole blood microfluidic channel, which 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 also provided in the whole blood separation zone (1); the whole blood microfluidic channel is prepared from qualitative filter paper; Wherein, the substrate is a qualitative filter paper, the sensing electrode comprises 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 sensor is integrated by 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.

3. The paper-based sensor for detecting blood potassium according to claim 1 or 2, 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), and 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); and the serum diffusion area (2) is a step-shaped structure.

4. The paper-based sensor for detecting blood potassium according to claim 3, 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) matches the sensing electrode, the electrode end (4) is a circular structure, and the electronic component connection end (5) is a rectangular structure.

5. The paper-based sensor for detecting blood potassium according to claim 4, 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).

6. 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 are printed on qualitative filter paper respectively. After all printed layers are completely dried, the electrodes are cut out, and the PBAs material solution is dripped on the carbon contact point and dried to form a fully solid-state working electrode. The FeCl3 solution is dripped on the silver contact point and dried to form a reference electrode. (3) Preparation of whole blood microfluidic channel: The qualitative filter paper and the 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 the blood separation membrane were combined using wax dipping technology, and then peeled off after cooling to room temperature 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 obtain a paper-based sensor.

7. The method for preparing a paper-based sensor for detecting blood potassium according to claim 6, characterized in that: In the step (3) of preparing the whole blood microfluidic channel, 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.

8. The method for preparing a paper-based sensor for detecting blood potassium according to claim 6, characterized in that: In the step (1), the drying temperature of the substrate treatment process is 52° C. and the drying time is 5 min. In the step (2), the drying temperature of the all-solid-state working electrode and the reference electrode in the preparation process of the sensing electrode is 60° C. and the drying time is 15 min. The printing method in the steps (1) and (2) is screen printing.

9. 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.

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

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