Potassium ion-sensitive microstructural device, preparation method and application thereof

Abstract

The present invention relates to the field of electrochemistry technology, and specifically to a potassium ion-sensitive microstructural device, a preparation method thereof, and an application thereof. The potassium ion-sensitive sensor includes (a) a micro reaction cell and (b) a potassium ion-sensitive membrane. Imitating the principle of the cell membrane transporting potassium ions, a bilayer liquid membrane formed by an amphiphilic compound containing valinomycin is used to capture and transport potassium ions. The patterned electrode at the bottom of the micro reaction cell monitors the electrical signal of the potassium ion-sensitive membrane and leads it out, so as to quantitatively or qualitatively detect potassium ions.

Description

Technical Field

[0001] The present invention relates to the field of electrochemistry technology, and particularly to a potassium ion-sensitive microstructure device, a preparation method thereof and an application thereof. Background Art

[0002] Potassium ions widely exist in human bodies, animals, plants, and many foods, beverages, and medicines, and play important roles therein. For example, potassium ions are one of the most important elements in the human body and are crucial for body functions (such as maintaining extracellular osmotic pressure, enzyme activation, formation of collagen or elastin, blood pressure regulation, and pH balance); potassium ions widely exist in traditional Chinese medicine injections, and the "Compilation of Guiding Principles and Relevant Regulations for the Development of Traditional Chinese Medicine Injections" has limited the potassium ions in traditional Chinese medicine injections, stipulating that the potassium ions should be below 1.0 mg / mL. When the potassium ion concentration is too high, the effectiveness and safety of traditional Chinese medicine injections will be affected; potassium ions are one of the three essential nutrient elements for the growth and development of vegetable crops. Studying the rational fertilization technology of potassium ions is beneficial to improving fertilizer utilization efficiency, reducing production costs, and reducing ecological environmental pollution. It can be seen that there is a need to measure potassium ions in many fields. Therefore, it is important to quickly, reliably, and quantitatively measure the content of potassium ions.

[0003] An ion-sensitive sensor is an electrochemistry sensor that uses an ion-selective electrode to convert the sensed ion amount into an available output signal. Traditional potassium ion-sensitive sensors are based on potassium ion-sensitive electrodes, and a solid-state ion-sensitive membrane is prepared on the electrode. Such sensitive membranes are mostly based on PVC polymers and molecules with specific binding ability to potassium ions, such as crown ethers. However, the method of the solid-state ion-sensitive membrane, when making a microsensor, due to the reduction of the sensitive area, the response signal and sensitivity also decrease accordingly. This is because the PVC polymer binds to the crown ether-like molecules in an embedded form, and its sensitive sites are hidden and cannot selectively permeate potassium ions efficiently, thus hindering the transmission of ion signals.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a potassium ion-sensitive sensor.

[0006] The second object of the present invention is to provide a preparation method of the above potassium ion-sensitive sensor.

[0007] The third object of the present invention is to provide an application of the above potassium ion-sensitive sensor.

[0008] In order to achieve the above objects, the present invention adopts the following solutions:

[0009] A potassium ion-sensitive sensor, the potassium ion-sensitive sensor includes:

[0010] (a) A microreaction cell, the bottom of the microreaction cell having a concave channel array, and a patterned electrode and an electrode lead being disposed on the surface of the concave channel array; and

[0011] (b) A potassium ion-sensitive membrane, the potassium ion-sensitive membrane being disposed on the bottom of the microreaction cell in (a) to form a microreaction cavity with the concave channel, and a buffer solution being filled in the microreaction cavity;

[0012] The potassium ion-sensitive membrane is a liquid membrane containing valinomycin, and the liquid membrane is formed by at least one amphiphilic compound, and the amphiphilic compound forms a bilayer in which valinomycin is incorporated.

[0013] Further, the maximum width of the channels in the concave channel array is not greater than 10 μm, the width ranges from 1 to 10 μm, and the channel spacing ranges from 1 μm to 5 mm.

[0014] Further, the amphiphilic compound is a lipid and its mixture, preferably phospholipid and / or cholesterol.

[0015] Further, the shape of the patterned electrode can be square, circular, annular, triangular, rhombic, polygonal and combinations thereof;

[0016] Preferably, the thicknesses of the patterned electrode and the electrode lead are each independently 1 to 1000 nm.

[0017] Further, the microreaction cell is made of PDMS, PET, PMMA, glass, quartz or silicon wafer;

[0018] Preferably, the height of the microreaction cell is 1 to 10 mm.

[0019] The preparation method of the above potassium ion-sensitive sensor is to lay a potassium ion-sensitive membrane on the bottom of the microreaction cell to obtain the potassium ion-sensitive sensor.

[0020] Further, the laying process of the patterned electrode and the electrode lead at the bottom of the microreaction cell is sequential photolithography, patterned mask evaporation / sputtering of metal; or

[0021] The laying method of the patterned electrode and the electrode lead at the bottom of the microreaction cell is patterned printing.

[0022] Further, the method of laying the potassium ion-sensitive membrane is to form a film of a solution containing valinomycin and an amphiphilic compound on the bottom of the microreaction cell;

[0023] Preferably, a solution containing valinomycin and an amphiphilic compound is added to the bottom of the microreaction cell to self-assemble into a film; or

[0024] First, a film is formed at the bottom of the micro-reaction cell with a solution containing an amphiphilic compound, and then valinomycin is added to allow valinomycin to spontaneously insert into the bilayer;

[0025] Preferably, a solution containing an amphiphilic compound is added to the bottom of the micro-reaction cell, and then valinomycin is added to self-assemble into a film, allowing valinomycin to spontaneously insert into the bilayer.

[0026] The preparation method of the potassium ion sensitive sensor includes:

[0027] S1: Construct a concave channel array on the surface of the substrate by means of laser engraving or casting;

[0028] S2: Lay a patterned electrode and an electrode lead on the surface of the concave channel array in S1;

[0029] S3: Construct the side wall of the micro-reaction cell on the surface of the concave channel array in S2 by means of 3D printing or casting to obtain a micro-reaction cell; wherein, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for electrical signal output;

[0030] S4: Lay a potassium ion sensitive membrane in the micro-reaction cell in S3.

[0031] The present invention also relates to the application of the above potassium ion sensitive sensor in the detection of potassium ion content.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. The present invention uses an amphiphilic compound to form a bilayer liquid membrane, and valinomycin is immobilized thereon to simulate the action mechanism of the functional cell membrane, which can transfer potassium ions on the membrane to under the membrane with high sensitivity and high selectivity. Compared with the traditional method of preparing ion-selective electrodes by polymer embedding, the surface sites of this potassium ion sensitive sensor will not fail due to being embedded, and it has higher selectivity for potassium ions.

[0034] 2. The concave channel array cooperating with the potassium ion sensitive membrane serves as a support layer for the potassium ion sensitive membrane. Its advantage lies in that it forms a quantitative microcavity with the potassium ion sensitive membrane above it, making the volume and shape of the target solution for electrical signal extraction certain, and realizing stable and accurate reading of electrical signals. Another function of the quantitative microcavity is that it forms a microspace under the membrane, enabling valinomycin to play a more efficient role in potassium ion transport, and the micron-sized channel size can provide stable support for the potassium ion sensitive membrane.

[0035] 3. The functions of the micro-reaction cell are: 1) making the reaction solution quantitative and confining it within a certain geometric shape to ensure the consistency of detection; 2) there is a tension between the inner wall of the micro-reaction cell and the bilayer liquid membrane, which helps the bilayer liquid membrane to spread out flat, enabling the sensitive sites on the surface of the sensor to be well exposed.

[0036] 4. The concave channel array is directly designed with electrodes to lead out ion signals through electrical signals. The electrodes are directly under the potassium ion-sensitive membrane. When potassium ions penetrate the membrane, it will immediately cause a change in the electrical signal, greatly improving the response speed of the device. Description of the Drawings

[0037] The following further describes each technical feature of the present invention and the relationships between them with reference to the drawings. The drawings are exemplary. Some technical features are not shown in actual proportion, and in some drawings, technical features that are customary in the technical field to which the present invention belongs and are not essential for understanding and implementing the present invention may be omitted, or technical features that are not essential for understanding and implementing the present invention may be additionally shown. That is, the combination of the technical features shown in the drawings is not used to limit the present invention. In addition, throughout the present invention, the content referred to by the same reference numerals is the same. The specific description of the drawings is as follows:

[0038] Figure 1 is a front cross-sectional view of the potassium ion sensor of the present invention without the potassium ion-sensitive membrane laid;

[0039] Figure 2 is a top view of the potassium ion sensor of the present invention without the potassium ion-sensitive membrane laid;

[0040] Figure 3 is a front cross-sectional view of a potassium ion sensor of the present invention;

[0041] Figure 4 is a substrate with a concave channel array of the present invention;

[0042] Figure 5 is a concave channel array substrate of the present invention with patterned electrodes and electrode leads laid;

[0043] Figure 6 is a detection result diagram of Example 1 and Comparative Example 1 of the response signal in the experimental example of the present invention;

[0044] Figure 7 is a detection result diagram of Example 1 and Comparative Example 2 of the sensitivity in the experimental example of the present invention;

[0045] Among them, 1 - side wall of the micro-reaction cell; 2 - patterned electrode; 3 - concave channel array at the bottom of the micro-reaction cell; 4 - electrode lead; 5 - potassium ion-sensitive membrane; 6 - micro-reaction cavity. Detailed Embodiments

[0046] Unless otherwise defined, all technical and scientific terms used throughout this invention shall have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of any inconsistency, the meaning stated in the full text of this invention or the meaning derived from the content recorded in the full text of this invention shall prevail. Additionally, the terms used in this specification are only for the purpose of describing the embodiments of this invention and are not intended to limit this invention.

[0047] It can be understood that those skilled in the art can combine the features mentioned in one or more of the embodiments mentioned throughout this invention with the features in other embodiments in any appropriate manner to implement this invention.

[0048] Next, the specific embodiments of this invention will be described in detail with reference to the accompanying drawings.

[0049] In this invention, the "potassium ion sensitive membrane" also refers to "(a liquid membrane containing valinomycin)", "(a bilayer containing valinomycin)", "(a bilayer liquid membrane containing valinomycin)", "(a bilayer liquid membrane containing valinomycin)", "(a liquid membrane bilayer thin film)".

[0050] The potassium ion sensitive sensor of this invention includes (a) a micro reaction cell and (b) a potassium ion sensitive membrane.

[0051] (a) The micro reaction cell has a concave channel array at its bottom, and a patterned electrode and an electrode lead are laid on the surface of the concave channel array. As shown in the front view sectional drawing of the micro reaction cell in Figure 1 , the micro reaction cell is a micro container with an open upper end, and it has a concave channel array 3 at its bottom. The channel can be linear (as shown in the top view of the micro reaction cell in Figure 2 ), or it can be in a serpentine bending shape. After the amphiphilic compound forms a film in the micro reaction cell, it can be sealed with the concave part of the channel to form a quantitative micro reaction cavity. The height of the side wall 1 of the micro reaction cell is such that it can realize the formation of a film of the amphiphilic compound on its bottom surface while constraining the shape of the liquid membrane. The shape of the micro reaction cell can be Figure 2 circular as shown in Figure 2 , or it can be of other shapes to meet specific requirements. As shown in Figure 2 , a patterned electrode 2 and an electrode lead 4 are laid on the surface of the concave channel array 3. The patterned electrode is used to detect and lead out the change in the electrical signal of the sensitive membrane, and it is led out of the micro reaction cell through the electrode lead and tested by the subsequent measurement circuit. It can be understood that the patterned electrode and the electrode lead can achieve this function. The patterned electrode can be laid on all or part of the bottom of the micro reaction cell (such as

[0052] (b) The potassium ion sensitive membrane, and this potassium ion sensitive membrane 5 is laid on the bottom of (a) the micro reaction cell (as shown in Figure 3The front elevation sectional view of the potassium ion sensitive sensor), forms a micro reaction cavity 6 with the concave channel, and a buffer solution is filled in the micro reaction cavity.

[0053] The potassium ion sensitive membrane is a liquid membrane containing valinomycin, and the liquid membrane is formed by at least one amphiphilic compound, and the amphiphilic compound forms a bilayer in which valinomycin is incorporated.

[0054] Most functions of biological membranes are achieved through proteins. One type of protein is selective for the permeation of ions and controls the transport of substances inside and outside the membrane. For example, valinomycin can selectively bind to K + ions to form a lipophilic complex, making K + easily pass through the lipid bilayer of the membrane to achieve transmembrane transport.

[0055] The potassium ion sensitive sensor of the present invention mimics the principle of the cell membrane transporting potassium ions, prepares a liquid membrane bilayer thin film on the micro reaction cell of the micro device, and valinomycin is uniformly carried on the film as a sensitive site. The advantage of this product is that by means of a bionic method, the liquid membrane bilayer thin film has better biocompatibility, and valinomycin can be naturally embedded outside the lipid polymer liquid membrane bilayer thin film, so that the sensitive sites are evenly exposed outside the sensitive membrane, making full use of the high selectivity of valinomycin for transporting potassium ions to achieve highly sensitive detection of potassium ions.

[0056] At the same time, the potassium ion sensitive membrane is arranged in a special micro reaction cell, and the micro reaction cell is used to provide good support for the sensitive membrane and facilitate the extraction of the sensor signal. The potassium ion sensitive membrane is prepared on the concave channel array at the bottom of the micro reaction cell, so that the sensitive membrane and the channel below it are sealed to form a quantitative micro reaction cavity. The surface of the channel forming the micro reaction cavity with the sensitive membrane can be paved with a patterned electrode and an electrode lead to facilitate the detection and extraction of the change in the electrical signal of the sensitive membrane, which is tested by the backend measurement circuit. The geometric size of the potassium ion sensitive membrane is restricted by the micro reaction cell.

[0057] In order to give sufficient support to the potassium ion sensitive membrane and meet the efficient transport of potassium ions, the maximum width of the channels in the concave channel array is not greater than 10 μm, the width range is 1 - 10 μm, and the channel spacing range is 1 μm - 5 mm.

[0058] It is understandable that the widths between channels and different regions within a channel can be the same or different. However, in order not to affect the supporting effect on the potassium ion-sensitive membrane, the maximum width of the channel is not greater than 10 μm. If the channel width is too wide, the potassium ion-sensitive membrane will collapse accordingly. If there are no channels at the bottom of the microreaction cell but just a flat surface, it will not be possible to seal with the sensitive membrane to form a quantitative microcavity, affecting the accurate reading of electrical signals. At the same time, the mechanism of valinomycin transport is to carry potassium ions on the membrane and release them under the membrane. If there is not enough space under the membrane, this transport efficiency will be greatly reduced. The width of the channel can be, but is not limited to, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm or 10 μm. The spacing between channels can be, but is not limited to, 1 μm, 10 μm, 50 μm, 100 μm, 300 μm, 500 μm, 1 mm, 2 mm, 3 mm, 4 mm or 5 mm.

[0059] In the potassium ion sensor of the present invention, for the capture and transport of potassium ions in the sample to be measured, the potassium ion-sensitive membrane utilizes the biomimetic principle of biological membranes. Therefore, the potassium ion-sensitive membrane can be prepared with an amphiphilic compound forming a bilayer to form a liquid membrane, and valinomycin is inserted therein to achieve the capture and transport of potassium ions. Therefore, the amphiphilic compound can form a bilayer incorporating valinomycin, preferably amphiphilic lipids and their mixtures. The advantage of amphiphilic lipids is that they can self-assemble into membranes, such as phospholipids, phosphoglycerides, sphingolipids and cardiolipin, and their mixtures, such as phospholipids, such as 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or mixtures of phospholipids; it can also be cholesterol, etc.

[0060] In a preferred embodiment, the shape of the patterned electrode in the present invention can be square, circular, annular, triangular, rhombic, polygonal and their combinations. The thicknesses of the patterned electrode and the electrode lead are both independently 1 - 1000 nm.

[0061] In some embodiments, the materials of the bottom and side walls of the microreaction cell can be the same or different, and can specifically be PDMS, PET, PMMA, glass, quartz or silicon wafers, etc. The materials of the patterned electrode and the electrode lead can be common electrode materials such as gold, copper, platinum, aluminum and their combinations.

[0062] In order to enable the electrode lead to lead out and detect the electrical signal from the microreaction cell, a concave channel array can be constructed on the plane of the substrate by means of laser engraving or casting (as Figure 4 shown), and then a patterned electrode and an electrode lead are formed on the surface of the concave channel array by means of photolithography → evaporation → patterning of metal or patterned printing, etc. (as Figure 5As shown in the figure, the micro-reaction cell is then fabricated by 3D printing or casting to enclose the patterned electrode, where a part of the electrode lead is led out outside the micro-reaction cell to realize the electrical signal output (as Figure 2 shown).

[0063] In a preferred embodiment, the side wall height of the micro-reaction cell is 1-10 mm. The side wall of the micro-reaction cell is used to realize the quantification of the reaction solution and confine it within a certain geometric shape to ensure the consistency of detection. At the same time, it helps the bimolecular liquid film to spread out flatly, so that the sensitive sites on the sensor surface are well exposed.

[0064] The present invention also provides a preparation method of a potassium ion sensitive sensor. A potassium ion sensitive film is laid on the bottom of the micro-reaction cell to obtain the potassium ion sensitive sensor.

[0065] In a preferred embodiment, the fabrication process of the patterned electrode and the electrode lead at the bottom of the micro-reaction cell is lithography, patterned mask evaporation / sputtering of metal in sequence; or, the method of laying the patterned electrode and the electrode lead is patterned printing. Other common means in the art can also be used for preparation.

[0066] In a preferred embodiment, the method of laying the potassium ion sensitive film is to form a film with a solution containing valinomycin and amphiphilic compounds at the bottom of the micro-reaction cell. Further preferably, a solution containing valinomycin and amphiphilic compounds is added to the bottom of the micro-reaction cell for self-assembly into a film; or

[0067] First, a film is formed with a solution containing amphiphilic compounds at the bottom of the micro-reaction cell, and then valinomycin is added to allow valinomycin to spontaneously insert into the bilayer. Preferably, a solution containing amphiphilic compounds is added to the bottom of the micro-reaction cell for self-assembly into a film, and then valinomycin is added to allow valinomycin to spontaneously insert into the bilayer.

[0068] After the amphiphilic compound solution is added to the bottom of the micro-reaction cell, the amphiphilic compound spontaneously forms a bilayer liquid film on the surface. The buffer solution fills the micro-reaction cavity formed by the liquid film and the channel. When valinomycin on the liquid film captures potassium ions, it releases them through the liquid film into the buffer solution in the channel under the film. The patterned electrode on the surface of the channel will immediately detect and form an electrical change signal, and the electrical signal is transmitted out through the electrode lead for detection.

[0069] In a preferred embodiment, the preparation method can be prepared by the following steps:

[0070] S1: Construct a concave channel array on the surface of the substrate by laser engraving or casting;

[0071] S2: Lay a patterned electrode and an electrode lead on the surface of the concave channel array in S1;

[0072] S3: On the surface of the concave channel array in S2, construct the side walls of the micro-reaction cell by means of 3D printing or casting to obtain the micro-reaction cell; wherein, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for electrical signal output;

[0073] S4: Lay a potassium ion-sensitive membrane in the micro-reaction cell in S3.

[0074] The potassium ion sensor provided by the present invention can accurately detect the potassium ion content. The specific usage method is to add the sample to be tested into the micro-reaction cell of the potassium ion sensor. The potassium ions in the sample to be tested will bind to valinomycin in the potassium ion-sensitive membrane. Valinomycin carries potassium ions through the bilayer composed of amphiphilic compounds, and releases potassium ions into the buffer solution in the micro-reaction cavity formed by the potassium ion-sensitive membrane and the channel. The patterned electrode on the surface of the channel monitors the change of the electrical signal and transmits the electrical signal to obtain the potassium ion content in the sample to be tested.

[0075] The electrical signal can be a potential signal converted from the selective adsorption effect of valinomycin, which changes the charge density on the upper surface of the electrode; or it can be an impedance signal converted from the selective adsorption effect of valinomycin, which changes the equivalent impedance of the sensor.

[0076] Example 1

[0077] S1: On the surface of the PMMA substrate, construct a concave channel array by means of laser engraving or casting. The channel width is 2 μm, the channels are parallel to each other, and the distance between channels is 10 μm;

[0078] S2: Lay a circular electrode and a strip-shaped electrode lead on the surface of the concave channel array in S1, and the materials are both 50 nm thick gold on top of 10 nm thick Cr;

[0079] S3: On the surface of the concave channel array in S2, construct the side walls of the micro-reaction cell made of PMMA by means of 3D printing. Among them, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for potential signal output;

[0080] S4: Lay a potassium ion-sensitive membrane in the micro-reaction cell in S3. The specific method is to fully mix 1 mg / mL DOPC phospholipid solution and 1 mg / mL valinomycin, then gently blow dry with nitrogen, and dissolve again with 10 mM HEPES buffer solution to form a vesicle suspension, and drop it on the bottom of the micro-reaction cell to self-assemble into a lipid-sensitive membrane with valinomycin.

[0081] Example 2

[0082] S1: On the surface of the PDMS substrate, construct a concave channel array by means of casting. The channel width is 5 μm, the channels are parallel to each other, and the distance between channels is 100 μm;

[0083] S2: Lay a square electrode and a strip-shaped electrode lead on the surface of the concave channel array in S1. Both are made of 5-nm-thick Ti with 100-nm-thick platinum on top.

[0084] S3: Use the replica molding method to construct the side walls of a micro-reaction cell made of PDMS on the surface of the concave channel array in S2. Among them, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for electrical impedance signal output.

[0085] S4: Lay a potassium ion-sensitive membrane in the micro-reaction cell in S3. The specific method is to drop a 1 mg / mL DPPC and cholesterol mixed phospholipid solution onto the bottom of the micro-reaction cell to self-assemble into a lipid membrane. Then drop a 1 mg / mL valinomycin solution. Valinomycin will spontaneously insert into the lipid membrane to form specific sites on the sensor surface.

[0086] Example 3

[0087] S1: Use the laser engraving method to construct a concave channel array on the surface of a PET substrate. The channel width is 10 μm, the channels are parallel to each other, and the spacing between channels is 4 mm.

[0088] S2: Lay a diamond-shaped electrode and a strip-shaped electrode lead on the surface of the concave channel array in S1. Both are made of 100-nm-thick Cr with 500-nm-thick gold on top.

[0089] S3: Use the replica molding method to construct the side walls of a micro-reaction cell made of PET on the surface of the concave channel array in S2. Among them, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for potential signal output.

[0090] S4: Lay a potassium ion-sensitive membrane in the micro-reaction cell in S3. The specific method is to fully mix a 1 mg / mL cholesterol phospholipid solution and 1 mg / mL valinomycin, then gently blow dry with nitrogen, dissolve again with 10 mM HEPES buffer to form a vesicle suspension, and drop it onto the bottom of the micro-reaction cell to self-assemble into a lipid-sensitive membrane with valinomycin.

[0091] Example 4

[0092] S1: Use the laser engraving method to construct a concave channel array on the surface of a glass substrate. The channel width is 8 μm, the channels are parallel to each other, and the spacing between channels is 5 μm.

[0093] S2: Lay a triangular electrode and a strip-shaped electrode lead on the surface of the concave channel array in S1. Both are made of 1-nm-thick Ti with 1-nm-thick platinum on top.

[0094] S3: On the surface of the concave channel array in S2, construct the side walls of the micro-reaction cell made of PMMA by 3D printing. Among them, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for potential signal output;

[0095] S4: Lay a potassium ion-sensitive membrane in the micro-reaction cell in S3. The specific method is to fully mix 1 mg / mL DPPC phospholipid solution and 1 mg / mL valinomycin, then gently dry it with nitrogen, and dissolve it again with 10 mM HEPES buffer to form a vesicle suspension, which is dropped onto the bottom of the micro-reaction cell and self-assembled into a lipid-sensitive membrane with valinomycin.

[0096] Comparative Example 1

[0097] S1: Lay a circular electrode and a strip-shaped electrode lead on the PMMA surface, both made of 10 nm thick Cr with 50 nm thick gold on top;

[0098] S2: On the PMMA surface in S1, construct the side walls of the micro-reaction cell made of PMMA by 3D printing. Among them, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for potential electrical signal output;

[0099] S3: Lay a potassium ion-sensitive membrane in the micro-reaction cell in S2. The specific method is to fully mix 1 mg / mL DOPC phospholipid solution and 1 mg / mL valinomycin, then gently dry it with nitrogen, and dissolve it again with 10 mM HEPES buffer to form a vesicle suspension, which is dropped onto the bottom of the micro-reaction cell and self-assembled into a lipid-sensitive membrane with valinomycin.

[0100] When the solution to be measured is dropped into the micro-reaction cell, valinomycin plays a role in selective adsorption, thereby changing the charge density on the upper surface of the electrode and converting it into a potential signal for reading.

[0101] The difference between this comparative example and Example 1 is that there is no concave channel array constructed on the PMMA substrate surface.

[0102] Comparative Example 2

[0103] S1: On the PMMA substrate surface, construct a concave channel array by laser engraving or casting. The channel width is 2 μm, the channels are parallel to each other, and the spacing between channels is 10 μm;

[0104] S2: On the surface of the concave channel array in S1, lay a circular electrode and a strip-shaped electrode lead, both made of 10 nm thick Cr with 50 nm thick gold on top;

[0105] S3: On the surface of the concave channel array in S2, construct the side walls of the micro-reaction cell made of PMMA by means of 3D printing. Among them, the patterned electrode is located at the bottom of the micro-reaction cell, and the electrode lead is used for potential signal output;

[0106] S4: Lay a potassium ion selective permeable membrane in the micro-reaction cell in S3. The specific method is to fully mix 1 mg / ml PVC solution with 1 mg / mL valinomycin, drop it on the bottom of the micro-reaction cell, and calcine it at 50 °C for 3 min to form a potassium ion selective permeable membrane.

[0107] When the solution to be measured is dropped into the micro-reaction cell, valinomycin plays a role in selective adsorption, thereby changing the charge density on the upper surface of the electrode and converting it into a potential signal for reading.

[0108] The difference between this comparative example and Example 1 is that PVC is used to construct a potassium ion selective permeable membrane at the bottom of the micro-reaction cell.

[0109] Experimental Example

[0110] I. Qualitative Detection

[0111] Three potassium ion sensitive sensors of Example 1 were respectively used, and potassium ion standard solution, sodium ion standard solution, and calcium ion standard solution were added respectively. Detection was carried out by means of testing potential signals or impedance signals. As a result, the sensors added with potassium ion standard solution detected electrical signals respectively, while other sensors did not detect electrical signals. Examples 2-4 were also tested respectively, and the same results were obtained.

[0112] II. Response Signal Size

[0113] The sensors in Example 1 and Comparative Example 1 were respectively operated as follows:

[0114] When the solution to be measured is dropped into the micro-reaction cell, valinomycin plays a role in selective adsorption, thereby changing the charge density on the upper surface of the electrode and converting it into a potential signal for reading. The voltage changes detected by the two sensors over time were respectively detected. The results are as Figure 6 shown.

[0115] It can be seen that the introduction of the concave channel array results in a quantitative microcavity above the electrode lead under the membrane, enabling the valinomycin transporter to have more sufficient space for movement, improving the transport efficiency, and thus improving the response signal of the sensor.

[0116] III. Sensitivity

[0117] The sensors in Example 1 and Comparative Example 2 were respectively operated as follows:

[0118] When the concentration of the potassium ion solution to be measured is successively added dropwise to the micro reaction cell from 0.1 mM to 1 mM, valinomycin plays a role in selective adsorption, thereby changing the equivalent impedance of the sensor, which is converted into an impedance signal and read out by the bottom lead. The current changes detected by the two sensors with the increase of concentration are respectively detected, and the results are as Figure 7 shown.

[0119] It can be seen that compared with the PVC embedding method where some sensitive sites are easily covered, this method uses a liquid membrane self-assembled by amphiphilic molecules, and valinomycin is naturally embedded, which will increase the density of sensitive sites on the sensor surface and thus improve the sensitivity of the sensor.

[0120] Note that the above is only the preferred embodiment of the present invention and the technical principles applied. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described here, and various obvious changes, re-adjustments and substitutions can be made by those skilled in the art without departing from the protection scope of the present invention. Therefore, although the present invention has been described in more detail through the above embodiments, the present invention is not limited to the above embodiments. Without departing from the technical concept of the present invention, more other equivalent embodiments can also be included, all of which belong to the protection scope of the present invention.

Claims

1. A potassium ion-sensitive sensor, characterized in that, The potassium ion sensitive sensor includes: (a) A micro reaction cell, the bottom of the micro reaction cell having a concave channel array, and a patterned electrode and an electrode lead being disposed on the surface of the concave channel array; and (b) A potassium ion sensitive membrane, the potassium ion sensitive membrane being laid on the bottom of the (a) micro reaction cell to form a micro reaction cavity with the concave channel, and a buffer solution being filled in the micro reaction cavity; The width of the channels in the concave channel array ranges from 1 to 10 μm, and the spacing between the channels ranges from 1 μm to 5 mm; The potassium ion sensitive membrane is a liquid membrane containing valinomycin, the liquid membrane being formed by at least one amphiphilic compound, the amphiphilic compound forming a bilayer in which valinomycin is incorporated, and the amphiphilic compound being a lipid and its mixture.

2. The potassium ion-sensitive sensor according to claim 1, characterized in that, The amphiphilic compound is phospholipid and / or cholesterol.

3. The potassium ion-sensitive sensor according to claim 1, characterized in that, The shape of the patterned electrode is square, circular, ring-shaped, triangular, rhombic, polygonal and combinations thereof; The thicknesses of the patterned electrode and the electrode lead are both independently 1 to 1000 nm.

4. The potassium ion-sensitive sensor according to any one of claims 1-3, characterized in that, The micro reaction cell is made of PDMS, PET, PMMA, glass, quartz or silicon wafer; The height of the micro reaction cell is 1 to 10 mm.

5. A method for preparing the potassium ion-sensitive sensor according to any one of claims 1-4, characterized in that, Laying a potassium ion sensitive membrane on the bottom of the micro reaction cell to obtain the potassium ion sensitive sensor.

6. The preparation method according to claim 5, characterized in that, The laying process of the patterned electrode and the electrode lead on the bottom of the micro reaction cell is lithography and patterned mask evaporation / sputtering of metal in sequence; or The laying method of the patterned electrode and the electrode lead on the bottom of the micro reaction cell is patterned printing.

7. The preparation method according to claim 5, characterized in that, The method of laying the potassium ion sensitive membrane is to form a film of a solution containing valinomycin and an amphiphilic compound on the bottom of the micro reaction cell; or First, form a film of a solution containing an amphiphilic compound on the bottom of the micro reaction cell, and then add valinomycin to enable valinomycin to autonomously insert into the bilayer.

8. The preparation method according to claim 7, characterized in that, Adding a solution containing valinomycin and an amphiphilic compound to the bottom of the micro reaction cell to self-assemble into a film.

9. The preparation method according to claim 7, characterized in that, Adding a solution containing an amphiphilic compound to the bottom of the micro reaction cell to self-assemble into a film, and then adding valinomycin to enable valinomycin to autonomously insert into the bilayer.

10. The preparation method according to any one of claims 5-9, characterized in that, The preparation method includes: S1: Constructing a concave channel array on the surface of a substrate by means of laser engraving or casting; S2: Laying a patterned electrode and an electrode lead on the surface of the concave channel array in S1; S3: Constructing the side wall of the micro reaction cell on the surface of the concave channel array in S2 by means of 3D printing or casting to obtain a micro reaction cell; wherein, the patterned electrode is located at the bottom of the micro reaction cell, and the electrode lead is used for electrical signal output; S4: Laying a potassium ion sensitive membrane in the micro reaction cell in S3.

11. Application of the potassium ion-sensitive sensor according to any one of claims 1-4 in the detection of potassium ion content.

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