A micro-integrated device and method for measuring electrolyte content of urine

Abstract

This invention belongs to the field of urine electrolyte content detection technology, specifically relating to a miniature integrated device and method for measuring urine electrolyte content. The device includes a first electrode on a silicon substrate, an internal reference liquid reservoir and a third microfluidic channel on a third chamber layer, a sample reservoir and a second microfluidic channel in a second chamber layer, a second electrode and a connecting cell on a silicon-based electrode layer, a first microfluidic channel in the first chamber layer, a potential measuring instrument on a cover plate, and a first, second, third, fourth, and fifth injection port on the entire device. Based on silicon micro-etching technology and combined with the principle of ion-selective electrode detection, this invention, through miniaturization of electrodes and electrolyte chambers, provides a low-cost, portable biological electrolyte detection instrument. Furthermore, by increasing the number of internal reference liquid containers, the number of ion types that the product can detect can be further increased.

Description

Technical Field

[0001] This invention belongs to the field of urine electrolyte content detection technology, specifically relating to a miniature integrated device and method for measuring urine electrolyte content. Background Technology

[0002] The detection of electrolytes such as Na+, K+, Ca2+, and Cl- in urine is a common test with significant clinical importance. Abnormal urinary sodium concentration is closely related to various kidney diseases, such as acute adrenal tubular necrosis and adrenal insufficiency. Potassium ion concentration is crucial for maintaining normal myocardial and neuromuscular excitability. Excessive urinary potassium concentration can cause cardiovascular disease, leading to myocardial depression, decreased myocardial tension, and in severe cases, arrhythmias. Conversely, insufficient urinary potassium concentration can also cause severe symptoms such as muscle weakness and arrhythmias. Furthermore, urinary sodium and potassium levels are closely related to blood pressure and arterial stiffness in hypertensive patients. Abnormal urinary calcium concentration may lead to serious conditions such as hypocalcemia and nephrotic syndrome, while abnormal urinary chloride concentration may cause symptoms such as epilepsy, syncope, and convulsions. Therefore, accurate, convenient, and rapid detection of urinary electrolytes is essential for mobile rapid testing scenarios such as emergency rooms, home healthcare, and field rescue.

[0003] Microfluidics is a technology for manipulating fluids at the micrometer scale. Early research focused primarily on the chemical electrophoretic analysis of continuous-phase fluids, hence it is also known as a micro total analysis system or a lab-on-a-chip. One of the key characteristics of microfluidics is the unique fluid properties it exhibits in a microscale environment, such as laminar flow and droplet flow. Leveraging these fluid phenomena, microfluidics can achieve microfabrication and micromanipulation that are difficult to accomplish using conventional methods.

[0004] Currently, the most common electrolyte detection method is electrochemical electrolyte analysis, which uses ion-selective electrode methods with relatively large instruments to determine the concentration of target ions. The principle involves using a galvanic cell to measure the membrane potential between a solution with a known ion concentration and the test solution, and then using this potential to calculate the target ion concentration, based on the Nernst equation. Other methods include microelectrode array chip detection (using highly conductive PMMA hydrogels combined with catalytically active metallographic composites to prepare sensing microelectrodes for detecting trace amounts of potassium ions in human electrolytes) and ion-selective electrode test strips (measuring finger-prick blood electrolytes by fabricating test strip electrodes and integrating a reference solution within the test strip).

[0005] Current urine electrolyte tests often require large instruments, which are not very convenient, are costly, and cannot meet the need for rapid detection of electrolyte concentrations in urine. Furthermore, they are complex to operate and require the assistance of professional medical personnel. Current methods for detecting electrolyte concentrations in body fluids, such as microelectrode array chip detection and ion-selective electrode test strips, offer some convenience, but most are designed for detecting electrolytes in human blood, and their accuracy and stability need improvement. Summary of the Invention

[0006] To overcome the above shortcomings, the present invention provides a miniature integrated device and method for measuring the electrolyte content of urine. Based on silicon micro-etching technology and combined with the principle of ion-selective electrode detection method, it reduces the size while improving performance to meet higher usage requirements. By miniaturizing the electrodes and electrolyte chamber, a low-cost and portable biological electrolyte detection instrument can be obtained. Furthermore, by increasing the number of internal reference liquid containers, the types of ions that the product can detect can be further increased.

[0007] A miniature integrated device for measuring urine electrolyte content includes a silicon substrate 1, a third chamber layer 2, a sensitive membrane layer 3, a second chamber layer 4, a salt bridge 5, a silicon-based electrode layer 6, a first chamber layer 7, and a cover plate 8 connected together from bottom to top.

[0008] Multiple first electrodes 9 are integrated on the silicon substrate 1. Multiple internal reference liquid pools 10 corresponding to each of the first electrodes 9 are provided in the third chamber layer 2. A third microfluidic channel 11 communicating with the front end of the internal reference liquid pool 10 is provided on the third chamber layer 2. The third chamber layer 2 located on the outer side of the internal reference liquid pools 10 on the left and right sides is also provided with a third microfluidic channel 11 communicating with the corresponding internal reference liquid pool 10.

[0009] The second chamber layer 4 is provided with a sample pool 12, and the second chamber layer 4 on both sides of the sample pool 12 is provided with a second microfluidic channel 13 that communicates with the sample pool 12.

[0010] The silicon-based electrode layer 6 is provided with a second electrode 14 and a connecting cell 15;

[0011] The first chamber layer 7 is provided with a reference pool 21 and a first microfluidic channel 22 connected thereto;

[0012] A potential measuring instrument 23 is provided on the top of the cover plate 8;

[0013] The micro-integrated device is provided with a first injection hole 16 penetrating the cover plate 8; a second injection hole 17 sequentially penetrating the cover plate 8, the first chamber layer 7, the silicon-based electrode layer 6, and the salt bridge 5; and a third injection hole 18, a fourth injection hole 19, and a fifth injection hole 20 sequentially penetrating the cover plate 8, the first chamber layer 7, the silicon-based electrode layer 6, the salt bridge 5, the second chamber layer 4, and the sensitive film layer 3.

[0014] The first injection port 16 is connected to the reference pool 21 of the first chamber layer 7 through the first microfluidic channel 22; the second injection port 17 is connected to the sample pool 12 of the second chamber layer 4 through the second microfluidic channel 13; the third injection port 18, the fourth injection port 19, and the fifth injection port 20 are respectively connected to the three internal reference pools 10 of the third chamber layer 2 through the corresponding third microfluidic channel 11.

[0015] The sensitive membrane layer 3 is composed of three sensitive membranes that selectively respond to sodium, potassium and calcium ions respectively, connected by an adhesive, and the three sensitive membranes sequentially cover the corresponding areas of the three internal reference liquid pools 10.

[0016] The micro-integrated device is provided with a first injection hole 16, a second injection hole 17 and a third injection hole 18 on both the left and right sides, a fourth injection hole 19 at both the front and rear ends of the micro-integrated device, and a fifth injection hole 20 at the front end of the micro-integrated device.

[0017] Both the first electrode 9 and the second electrode 14 are Ag / AgCl electrodes, and the second electrode 14 is the common electrode of all the first electrodes 9.

[0018] The salt bridge 5 is a carbon nanotube bundle membrane.

[0019] The sensitive membrane constituting the sensitive membrane layer 3 is prepared by spin-coating a sensitive membrane solution, which is a mixture of PVC matrix, plasticizer, neutral complexing carrier, ionic additive and solvent, onto a wafer substrate.

[0020] The plasticizer is dibutyl phthalate, dioctyl phthalate, dinonyl phthalate, or dibutyl phthalate of sebacic acid, oxalic acid, sebacic acid, or dibutyl phthalate.

[0021] When the sensitive membrane has a selective response to sodium ions, the neutral complexing support is a Na ion support, specifically one or more of the following: double crown ether, 4′-benzo-12-crown-4, tetraphenyl24-crown ether-8, and 1,1,1-tris[1′-(2′-oxa-4′-oxo-5′-aza-5′-methyl)dodecyl]propane;

[0022] When the sensitive membrane has a selective response to potassium ions, the neutral complexing carrier is a potassium ion carrier, specifically one or more of macrocyclic antibiotics, di-tert-butyl or dimethyl-dibenzo-crown-10, 1,1′-binaphthol-20-crown-6;

[0023] When the sensitive membrane has a selective response to calcium ions, the neutral complexing carrier is a calcium ion carrier, specifically one or more of N,N-di[(11-ethyl ester)undecyl]-N,N'-4,5-tetramethyl-3,6-dioxaoctyl diamide and 1,1,1-tris(N-methyl-N-phenylaminocarbonylmethoxymethyl)propane.

[0024] The ionic additive is one or both of sodium tetraphenylborate and potassium tetraphenylborate.

[0025] The solvent is one or both of tetrahydrofuran and cyclohexanone.

[0026] A method for measuring electrolyte content in urine using a miniature integrated device, comprising the following steps:

[0027] Step 1: Connect all electrodes of the device to the potential measuring instrument 23 on the cover plate 8 via wires;

[0028] Step 2: Inject the reference solution into the device through the first injection hole 16. The reference solution enters the reference pool 21 through the first injection hole 16 and the first microfluidic channel 22 in sequence. Stop the injection when the solution is exposed in the first injection hole 16 on the other side of the cover plate 8.

[0029] Step 3: Inject the internal reference solution into the device through the third injection hole 18, the fourth injection hole 19, and the fifth injection hole 20 respectively. The internal reference solution enters the internal reference liquid pool 10 through the third microfluidic channel 11 in sequence. Stop the injection when solution is exposed in the third injection hole 18, the fourth injection hole 19, and the fifth injection hole 20 on the other side of the cover plate 8.

[0030] Step 4: Inject the urine sample into the device through the second injection hole 17. The urine sample enters the sample pool 12 through the second injection hole 17 and the second microfluidic channel 13 in sequence. Stop the injection when solution is exposed in the second injection hole 17 on the other side of the cover plate 8.

[0031] Step 5: After the reading of the potential measuring instrument 23 stabilizes, read the values ​​corresponding to the three first electrodes 9. These three values ​​are the potential values ​​received by the potential measuring instrument 23, which are calculated by the built-in microprocessor and converted into the corresponding concentrations of sodium, potassium and calcium ions.

[0032] The internal reference solution is 10. -2 M NaCl solution, 10 -2 M KCl solution, 10 -2 M is a CaCl2 solution; the reference solution is a saturated KCl solution.

[0033] A method for manufacturing a miniature integrated device for measuring urine electrolyte levels includes the following steps:

[0034] Step 1: Integrate three first electrodes 9 on the silicon substrate 1 to obtain the indicator electrode;

[0035] Step 2: Take a silicon substrate, deposit a layer of SiO2 on the silicon substrate, spin-coat photoresist on the surface of SiO2, and use MEMS photolithography to etch away part of the photoresist to obtain the third chamber layer 2 structure planar mask; the third chamber layer 2 structure planar mask covers the internal reference liquid pool 10 and the third microfluidic channel 11 to be obtained.

[0036] Step 3: The third chamber layer 2 is etched away by HF etching to remove the SiO2 in the part of the third chamber layer 2 structure that is not covered by the planar mask, and the three-dimensional structure of the third chamber layer 2 is initially obtained.

[0037] Step 4: Remove the planar mask of the third chamber layer structure to obtain the final third chamber layer 2;

[0038] Step 5: Fabricate the second chamber layer 4, the silicon-based electrode layer 6, the first chamber layer 7, and the cover plate 8 using the same methods as in steps 2 to 4;

[0039] Step 6: Integrate a second electrode 14 on the silicon-based electrode layer 6 to obtain a reference electrode;

[0040] Step 7: Prepare three sensitive membrane solutions that selectively respond to sodium, potassium and calcium ions respectively, and spin-coat the sensitive membrane solutions onto the wafer substrate to prepare the corresponding sensitive membranes. Then, connect the three sensitive membranes together with adhesive to form sensitive membrane layer 3.

[0041] Step 8: From bottom to top, bond the silicon substrate 1, third chamber layer 2, sensitive film layer 3, second chamber layer 4, salt bridge 5, silicon-based electrode layer 6, first chamber layer 7, and cover plate 8 together with adhesive.

[0042] The salt bridge 5 is fabricated using a thermal decoupling CVD method.

[0043] Step 1: Flow a mixture of He at 100 sccm and H2 at 400 sccm in a tubular furnace for 10 minutes while heating the furnace to 775°C.

[0044] Step 2: Insert the wafer into the furnace using a magnetically coupled transfer arm, and then maintain the same gas and flow rate as in Step 1, keeping the furnace at 775°C for 10 minutes.

[0045] Step 3: Change the gas and flow rate to a mixed gas of 100 sccm C2H4, 400 sccm He and 100 sccm H2 for 3 min, and heat it at 775℃. After carbon nanotubes are grown on the wafer, keep the same gas and flow rate, cool the furnace to below 100℃, and finally purge with 1000 sccm He for 5 min to obtain a carbon capillary array.

[0046] Step 4: Deposit a layer of SiO2 on the silicon substrate, spin-coat photoresist on the SiO2 surface, and use MEMS photolithography to etch away part of the photoresist to obtain the salt bridge 5 injection hole planar mask. The planar mask covers the injection holes to be obtained. Etch the SiO2 deposition layer by HF etching, and etch the SiO2 part that is not protected by the photoresist to obtain the three-dimensional structure of each injection hole. Remove the photoresist mask to obtain the SiO2 hard mask of each injection hole.

[0047] Step 5: Cover the carbon capillary array obtained in step 2 with the SiO2 hard mask obtained in step 4, and etch the carbon capillary array with KOH at a mass concentration of 35%. Etch the carbon capillary array portion not protected by the SiO2 hard mask to obtain the three-dimensional structure of each injection hole. Remove the SiO2 hard mask to obtain the salt bridge 5 with each injection hole.

[0048] In step seven, the sensitive membrane is prepared as follows:

[0049] The first step is to prepare 20 ml of selective membrane solutions that selectively respond to sodium, potassium, and calcium ions respectively; the selective membrane solutions for the three ions use one or two of cyclohexanone and tetrahydrofuran as mixed solvents; sodium tetraphenylborate and potassium tetraphenylborate are used as ion additives; polyvinyl chloride is used as the PVC matrix and dioctyl sebacate is used as a plasticizer.

[0050] The second step is to drop the prepared selective film solution onto the wafer substrate and spin-coat it evenly.

[0051] The third step is to allow the mixed solvent to evaporate at room temperature to obtain the sensitive membrane, and then cut the sensitive membrane into an appropriate size shape.

[0052] When preparing the sodium ion selective membrane solution, tetraphenyl 24-crown ether-8 is used as the sodium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:24:74.

[0053] When preparing the potassium ion selective membrane solution, 1,1′-binaphthyl-20-crown-6 is used as the potassium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:33:65.

[0054] When preparing the calcium ion selective membrane solution, 1,1,1-tris(N-methyl-N-phenylaminocarbonylmethoxymethyl)propane is used as the calcium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:32:66.

[0055] The mixed solvent used is a mixture of 20 ml of cyclohexanone and tetrahydrofuran.

[0056] The beneficial effects of this invention are:

[0057] 1. By utilizing MEMS silicon micro-etching technology, the size of the detector is significantly reduced, enabling it to maximize the integration of indicator electrodes and reference electrodes, thereby reducing the cost of bioelectrolyte detection instruments; accurate detection results are obtained while reducing the amount of detection solution used.

[0058] 2. By increasing the internal reference liquid chamber, multiple bioelectrolytes can be detected simultaneously; by changing the sensitive membrane and internal reference solution, specific ion concentrations can be detected.

[0059] 3. MEMS silicon micro-etching technology is now relatively mature. Using MEMS silicon micro-etching technology can greatly improve its portability and integration while ensuring detection performance. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the content of the embodiments of the present invention and these drawings without creative effort.

[0061] Figure 1 This is a schematic diagram of the overall exploded structure of the present invention.

[0062] Figure 2 This is a side view of the structure of the present invention.

[0063] Figure 3 This is a schematic diagram of the silicon layer substrate structure of the present invention.

[0064] Figure 4 This is a schematic diagram of the third chamber layer structure of the present invention.

[0065] Figure 5 This is a schematic diagram of the sensitive film structure of the present invention.

[0066] Figure 6 This is a schematic diagram of the second chamber layer structure of the present invention.

[0067] Figure 7 This is a schematic diagram of the salt bridge structure of the present invention.

[0068] Figure 8 This is a schematic diagram of the silicon-based electrode layer structure of the present invention.

[0069] Figure 9 This is a schematic diagram of the first chamber layer structure of the present invention.

[0070] Figure 10 This is a schematic diagram of the cover plate structure of the present invention.

[0071] The structure includes: silicon substrate 1, third chamber layer 2, sensitive film layer 3, second chamber layer 4, salt bridge 5, silicon-based electrode layer 6, first chamber layer 7, cover plate 8, first electrode 9, internal reference liquid pool 10, third microfluidic channel 11, sample pool 12, second microfluidic channel 13, second electrode 14, connecting pool 15, first injection hole 16, second injection hole 17, third injection hole 18, fourth injection hole 19, fifth injection hole 20, reference pool 21, first microfluidic channel 22, and potential measuring instrument 23. Detailed Implementation

[0072] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the present invention, and not all of the structures.

[0073] Example 1

[0074] like Figure 1-10 As shown, a miniature integrated device for measuring urine electrolyte content includes a silicon substrate 1, a third chamber layer 2, a sensitive membrane layer 3, a second chamber layer 4, a salt bridge 5, a silicon-based electrode layer 6, a first chamber layer 7, and a cover plate 8 connected together from bottom to top.

[0075] Multiple first electrodes 9 are integrated on the silicon substrate 1. Multiple internal reference liquid pools 10 corresponding to each of the first electrodes 9 are provided in the third chamber layer 2. A third microfluidic channel 11 communicating with the front end of the internal reference liquid pool 10 is provided on the third chamber layer 2. The third chamber layer 2 located on the outer side of the internal reference liquid pools 10 on the left and right sides is also provided with a third microfluidic channel 11 communicating with the corresponding internal reference liquid pool 10.

[0076] The second chamber layer 4 is provided with a sample pool 12, and the second chamber layer 4 on both sides of the sample pool 12 is provided with a second microfluidic channel 13 that communicates with the sample pool 12.

[0077] The silicon-based electrode layer 6 is provided with a second electrode 14 and a connecting cell 15;

[0078] The first chamber layer 7 is provided with a reference pool 21 and a first microfluidic channel 22 connected thereto;

[0079] A potential measuring instrument 23 is provided on the top of the cover plate 8;

[0080] The micro-integrated device is provided with a first injection hole 16 penetrating the cover plate 8; a second injection hole 17 sequentially penetrating the cover plate 8, the first chamber layer 7, the silicon-based electrode layer 6, and the salt bridge 5; and a third injection hole 18, a fourth injection hole 19, and a fifth injection hole 20 sequentially penetrating the cover plate 8, the first chamber layer 7, the silicon-based electrode layer 6, the salt bridge 5, the second chamber layer 4, and the sensitive film layer 3.

[0081] The first injection port 16 is connected to the reference pool 21 of the first chamber layer 7 through the first microfluidic channel 22; the second injection port 17 is connected to the sample pool 12 of the second chamber layer 4 through the second microfluidic channel 13; the third injection port 18, the fourth injection port 19, and the fifth injection port 20 are respectively connected to the three internal reference pools 10 of the third chamber layer 2 through the corresponding third microfluidic channel 11.

[0082] The sensitive membrane layer 3 is composed of three sensitive membranes that selectively respond to sodium, potassium and calcium ions respectively, connected by an adhesive, and the three sensitive membranes sequentially cover the corresponding areas of the three internal reference liquid pools 10.

[0083] The micro-integrated device is provided with a first injection hole 16, a second injection hole 17 and a third injection hole 18 on both the left and right sides, a fourth injection hole 19 at both the front and rear ends of the micro-integrated device, and a fifth injection hole 20 at the front end of the micro-integrated device.

[0084] Both the first electrode 9 and the second electrode 14 are Ag / AgCl electrodes, and the second electrode 14 is the common electrode of all the first electrodes 9.

[0085] The salt bridge 5 is a carbon nanotube bundle membrane, which can be a porous glass or polymer frit, polymer membrane, or carbon capillary (bundle) (also called carbon nanotube bundle membrane), preferably a carbon nanotube bundle membrane, which is a carbon capillary array with a high aspect ratio.

[0086] The sensitive membrane constituting the sensitive membrane layer 3 is prepared by spin-coating a sensitive membrane solution, which is a mixture of PVC matrix, plasticizer, neutral complexing carrier, ionic additive and solvent, onto a wafer substrate.

[0087] The plasticizer is dibutyl phthalate, dioctyl phthalate, dinonyl phthalate, or dibutyl phthalate of sebacic acid, oxalic acid, sebacic acid, or dibutyl phthalate.

[0088] When the sensitive membrane has a selective response to sodium ions, the neutral complexing support is a Na ion support, specifically one or more of the following: double crown ether, 4′-benzo-12-crown-4, tetraphenyl24-crown ether-8, and 1,1,1-tris[1′-(2′-oxa-4′-oxo-5′-aza-5′-methyl)dodecyl]propane;

[0089] When the sensitive membrane has a selective response to potassium ions, the neutral complexing carrier is a potassium ion carrier, specifically one or more of macrocyclic antibiotics, di-tert-butyl or dimethyl-dibenzo-crown-10, 1,1′-binaphthol-20-crown-6;

[0090] When the sensitive membrane has a selective response to calcium ions, the neutral complexing carrier is a calcium ion carrier, specifically one or more of N,N-di[(11-ethyl ester)undecyl]-N,N'-4,5-tetramethyl-3,6-dioxaoctyl diamide and 1,1,1-tris(N-methyl-N-phenylaminocarbonylmethoxymethyl)propane.

[0091] The ionic additive is one or both of sodium tetraphenylborate and potassium tetraphenylborate.

[0092] The solvent is one or both of tetrahydrofuran and cyclohexanone.

[0093] A method for measuring electrolyte content in urine using a miniature integrated device, comprising the following steps:

[0094] Step 1: Connect all electrodes of the device to the potential measuring instrument 23 on the cover plate 8 via wires;

[0095] Step 2: Inject the reference solution into the device through the first injection hole 16 on the left (or right). The reference solution enters the reference pool 21 through the first injection hole 16 and the first microfluidic channel 22 on the left (or right). Stop the injection when the solution is slightly exposed in the first injection hole 16 on the other side of the cover plate 8.

[0096] Step 3: Inject the internal reference solution into the device through the third injection hole 18, the fourth injection hole 19, and the fifth injection hole 20 on the left (or right) side, respectively. The internal reference solution enters the internal reference liquid pool 10 through the third microfluidic channel 11 on the left (or right) side of the third injection hole 18 (fourth injection hole 19, fifth injection hole 20); stop the injection when the solution is slightly exposed in the third injection hole 18, the fourth injection hole 19, and the fifth injection hole 20 on the other side of the cover plate 8.

[0097] Step 4: Inject the urine sample into the device through the second injection hole 17 on the left (or right). The urine sample enters the sample pool 12 through the second injection hole 17 and the second microfluidic channel 13 on the left (or right) in sequence. Stop the injection when the solution is slightly exposed in the second injection hole 17 on the other side of the cover plate 8.

[0098] Step 5: After the reading of the potential measuring instrument 23 stabilizes, read the values ​​corresponding to the three first electrodes 9. These three values ​​are the concentrations of sodium, potassium and calcium ions in the urine that are calculated by the built-in microprocessor based on the potential values ​​received by the potential measuring instrument 23.

[0099] A method for manufacturing a miniature integrated device for measuring urine electrolyte levels includes the following steps:

[0100] Step 1: Three first electrodes 9 are integrated on the silicon substrate 1 by chemical, electrochemical or screen printing methods to obtain the indicator electrode;

[0101] Step 2: Take a silicon substrate, deposit a layer of SiO2 on the silicon substrate, spin-coat photoresist on the surface of SiO2, and use MEMS photolithography to etch away part of the photoresist to obtain the third chamber layer 2 structure planar mask; the third chamber layer 2 structure planar mask covers the internal reference liquid pool 10 and the third microfluidic channel 11 to be obtained.

[0102] Step 3: The third chamber layer 2 is etched away by HF etching to remove the SiO2 in the part of the third chamber layer 2 structure that is not covered by the planar mask, and the three-dimensional structure of the third chamber layer 2 is initially obtained.

[0103] Step 4: Remove the planar mask of the third chamber layer structure to obtain the final three-dimensional structure of the third chamber layer 2, namely the internal reference liquid pool 10 and the third microfluidic channel 11.

[0104] Step 5: Fabricate the second chamber layer 4, the silicon-based electrode layer 6, the first chamber layer 7, and the cover plate 8 using the same methods as in steps 2 to 4;

[0105] The fabrication method of the second chamber layer 4 is as follows: a layer of SiO2 is deposited on a silicon substrate, photoresist is spin-coated on the surface of SiO2, and part of the photoresist is etched away using MEMS photolithography to obtain a planar mask of the second chamber layer 4 structure; the planar mask covers the sample pool 12, the second microfluidic channel 13 and the corresponding injection hole to be obtained, and the second chamber layer 4 is etched by HF etching to etch the SiO2 part that is not protected by the photoresist to obtain the three-dimensional structure of the sample pool 12, the second microfluidic channel 13 and the corresponding injection hole; the photoresist mask, i.e., the planar mask of the second chamber layer 4 structure, is removed to obtain the sample pool 12, the second microfluidic channel 13 and the corresponding injection hole;

[0106] The method for fabricating the silicon-based electrode layer 6 is as follows: a layer of SiO2 is deposited on a silicon substrate, photoresist is spin-coated onto the surface of the SiO2, and part of the photoresist is etched away using MEMS photolithography to obtain a planar mask of the silicon-based electrode layer 6 structure; the planar mask covers the connected cell 15 and the corresponding injection hole to be obtained, and the silicon-based electrode layer 6 is etched by HF etching to etch the SiO2 portion not protected by the photoresist to obtain the three-dimensional structure of the connected cell 15 and the corresponding injection hole; the photoresist mask is removed to obtain the connected cell 15 and the corresponding injection hole.

[0107] The fabrication method of the first chamber layer 7 is as follows: a layer of SiO2 is deposited on a silicon substrate, photoresist is spin-coated on the surface of SiO2, and part of the photoresist is etched away using MEMS photolithography to obtain a planar mask of the first chamber layer 7 structure; the planar mask covers the reference cell 21, the first microfluidic channel 22 and the corresponding injection hole to be obtained, and the first chamber layer 7 is etched by HF etching to etch the SiO2 part that is not protected by the photoresist to obtain the three-dimensional structure of the reference cell 21, the first microfluidic channel 22 and the corresponding injection hole; the photoresist mask is removed to obtain the reference cell 21, the first microfluidic channel 22 and the corresponding injection hole;

[0108] The fabrication method of the cover plate 8 is as follows: a layer of SiO2 is deposited on a silicon substrate, photoresist is spin-coated on the surface of SiO2, and part of the photoresist is etched away using MEMS photolithography to obtain a planar mask of the cover plate 8 structure. The part covered by the planar mask is the injection hole to be obtained. The cover plate 8 is etched by HF etching to etch the SiO2 part that is not protected by the photoresist to obtain the three-dimensional structure of each injection hole. The photoresist mask is removed to obtain each injection hole.

[0109] Step 6: Integrate a second electrode 14 on the silicon-based electrode layer 6 by chemical, electrochemical or screen printing methods to obtain a reference electrode;

[0110] Step 7: Prepare three sensitive membrane solutions that selectively respond to sodium, potassium, and calcium ions, respectively, and spin-coat the solutions onto a wafer substrate to prepare corresponding sensitive membranes. Then, connect the three sensitive membranes together with adhesive to form sensitive membrane layer 3. The three sensitive membranes are initially separate and are subsequently joined together with adhesive. This larger joined membrane is sensitive membrane layer 3. When the three sensitive membranes are stacked on the third chamber layer 2, they each cover the corresponding internal reference liquid pool 10 on the third chamber layer 2.

[0111] Step 8: From bottom to top, bond the silicon substrate 1, third chamber layer 2, sensitive film layer 3, second chamber layer 4, salt bridge 5, silicon-based electrode layer 6, first chamber layer 7, and cover plate 8 together with adhesive.

[0112] The salt bridge 5 is fabricated using a thermal decoupling CVD method.

[0113] Step 1: Flow a mixture of He at 100 sccm and H2 at 400 sccm in a tubular furnace for 10 minutes while heating the furnace to 775°C.

[0114] Step 2: Insert the wafer into the furnace using a magnetically coupled transfer arm, and then maintain the same gas and flow rate as in Step 1, keeping the furnace at 775°C for 10 minutes.

[0115] Step 3: Change the gas and flow rate to a mixed gas of 100 sccm C2H4, 400 sccm He and 100 sccm H2 for 3 min, and heat it to 775℃. After carbon nanotubes are grown on the wafer, keep the same gas and flow rate, cool the furnace to below 100℃, and finally purge with 1000 sccm He for 5 min to obtain a carbon capillary array.

[0116] Step 4: Deposit a layer of SiO2 on the silicon substrate, spin-coat photoresist on the SiO2 surface, and use MEMS photolithography to etch away part of the photoresist to obtain the salt bridge 5 injection hole planar mask. The planar mask covers the injection holes to be obtained. Etch the SiO2 deposition layer by HF etching, and etch the SiO2 part that is not protected by the photoresist to obtain the three-dimensional structure of each injection hole. Remove the photoresist mask to obtain the SiO2 hard mask of each injection hole.

[0117] Step 5: Cover the carbon capillary array obtained in step 2 with the SiO2 hard mask obtained in step 4, and etch the carbon capillary array with KOH at a mass concentration of 35%. Etch the carbon capillary array portion that is not protected by the SiO2 hard mask to obtain the three-dimensional structure of each injection hole. Remove the SiO2 hard mask to obtain the salt bridge 5 with each injection hole.

[0118] The internal reference solution is 10. -2 M NaCl solution, 10 -2 M KCl solution, 10 -2 M is a CaCl2 solution.

[0119] The reference solution is a saturated KCl solution.

[0120] In step seven, the sensitive membrane is prepared as follows:

[0121] The first step is to prepare 20 ml of selective membrane solutions that selectively respond to sodium, potassium, and calcium ions respectively; the selective membrane solutions for the three ions use one or two of cyclohexanone and tetrahydrofuran as mixed solvents; sodium tetraphenylborate and potassium tetraphenylborate are used as ion additives; polyvinyl chloride is used as the PVC matrix and dioctyl sebacate is used as a plasticizer.

[0122] The second step is to drop the prepared selective film solution onto the wafer substrate and spin-coat it evenly.

[0123] The third step is to allow the mixed solvent to evaporate at room temperature to obtain the sensitive membrane, and then cut the sensitive membrane into an appropriate size shape.

[0124] When preparing the sodium ion selective membrane solution, tetraphenyl 24-crown ether-8 is used as the sodium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:24:74.

[0125] When preparing the potassium ion selective membrane solution, 1,1′-binaphthyl-20-crown-6 is used as the potassium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:33:65.

[0126] When preparing the calcium ion selective membrane solution, 1,1,1-tris(N-methyl-N-phenylaminocarbonylmethoxymethyl)propane is used as the calcium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:32:66.

[0127] The mixed solvent used is a mixture of 20 ml of cyclohexanone and tetrahydrofuran.

[0128] Example 2

[0129] A miniature integrated device for measuring urine electrolyte levels includes a silicon substrate 1 positioned below, on which three Ag / AgCl first electrodes 9 are integrated; an internal reference solution pool 10 in a third chamber layer 2 is used to hold internal reference solution, which enters the internal reference solution pool 10 through a fourth injection port 19, and then enters a third microfluidic channel 11, filling the internal reference solution pool 10 under injection pressure; three sensitive membranes are bonded to the third chamber layer 2, which selectively respond to sodium, potassium, and calcium ions respectively; a second chamber layer 4 is connected to the sensitive membrane layer 3, where a sample pool 12 holds the sample obtained through a second injection port 19. The sample solution enters through the injection hole 16 and microfluidic channel 13; then it is connected to the salt bridge 5, which is a carbon nanotube bundle membrane with low impedance and is not easily contaminated by the sample solution; above the salt bridge 5 is a silicon-based electrode layer 6, which integrates a second electrode 14 and a connecting cell 15 on a silicon substrate. The connecting cell 15 allows ion exchange between the reference solution in the first chamber layer 7 and the sample solution in the sample cell 12, thereby conducting the circuit; the first chamber layer 7 is connected to the silicon-based electrode layer 6, and the reference cell 21 in it is used to hold the reference solution entering through the second injection hole 16 and microfluidic channel 22; the top layer is sealed with a cover plate 8 etched on the silicon substrate. A potentiometer 23 is integrated on the cover plate 8 for measuring the ion activity of the sample solution.

[0130] A miniature integrated device for measuring electrolyte levels in urine. (See structural diagram below.) Figure 1 The system includes a silicon substrate 1 located below, on which three Ag / AgCl first electrodes 9 are integrated; an internal reference liquid pool 10 in the attached third chamber layer 2 is used to hold the internal reference liquid, which enters the third microfluidic channel 11 through the injection hole 20 and fills the internal reference liquid pool 10 under injection pressure; three sensitive membranes are bonded to the chamber layer 2, which are selectively responsive to sodium, potassium, and calcium ions, respectively; a second chamber layer 4 is connected to the sensitive membrane layer 3, in which a sample pool 12 holds the sample solution entering through the second injection hole 17 and the second microfluidic channel 13; and then connected to... A salt bridge 5, made of carbon nanotube bundle membrane, has low impedance and is not easily contaminated with the sample solution. Above the salt bridge 5 is a silicon-based electrode layer 6, which integrates an Ag / AgCl second electrode 14 and a connecting cell 15 on a silicon substrate. The connecting cell 15 allows ion exchange between the reference solution in the first chamber layer 7 and the sample solution in the sample cell 12, thereby conducting the circuit. The silicon-based electrode layer 6 is connected to the first chamber layer 7, where the reference cell 21 is used to hold the reference solution entering through the first injection hole 16 and the first microfluidic channel 22. The top layer is sealed with a cover plate 8 etched on a silicon substrate. A potentiometer 23 is integrated on the cover plate 8 for measuring the ion activity of the sample solution. The entire structure constitutes a battery system, which can be represented as Ag|AgCl(s), KCl (saturated)|0.1mol / L LiAc||sample solution||sensitive membrane|0.01mol / L Cl - AgCl(s)|Ag. The silicon substrate 1, the chamber, and the sensitive film layer 3 constitute the indicator electrode, while the cover plate 8, the chamber, the silicon-based electrode layer 6, and the salt bridge 5 constitute the reference electrode. The overall structure dimensions are 2cm*8cm*1cm.

[0131] Example 1: A method for patterned etching of the third chamber layer.

[0132] 1. A 1 mm thick SiO2 layer is grown on the cleaned silicon surface using PECVD;

[0133] 2. Apply photoresist to the sample surface using spin coating. Specifically, the silicon wafer is vacuum-adsorbed and rotated at high speed on a centrifugal spin coater to evenly coat the photoresist droplets onto the wafer surface.

[0134] 3. Heat and evaporate part of the solvent in the photoresist to allow the photoresist layer to initially solidify.

[0135] 4. Align the photomask with the silicon wafer by marking, expose the photoresist to cause structural changes in some areas, and transfer the pattern of the third chamber layer structure.

[0136] 5. Dissolve and remove the light-exposed portion of the silicon wafer in the developer solution to obtain a planar mask with a third chamber layer structure.

[0137] 6. The SiO2 layer was wet-etched using a 40% HF solution to obtain the three-dimensional structure of the third chamber layer;

[0138] The development method of the micro-integrated bioelectrolyte detector based on MEMS silicon micro-etching of this invention is based on the ion-selective electrode method in potentiometric analysis, which is commonly used to detect the concentration of specific ions in a solution. Combining MEMS silicon micro-etching technology improves the integration of the detection device and achieves ultra-miniaturization. This invention allows for the selective addition or removal of internal reference cells and corresponding sensitive membranes according to specific medical bioelectrolyte detection scenarios to detect specific bioelectrolytes. First, the chamber layers are etched using MEMS silicon micro-etching technology, then the electrode layers, sensitive membranes, and salt bridges are bonded together, ultimately achieving the fabrication of the ion-selective electrode detector at the micrometer scale.

[0139] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the scope of protection of the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, any person skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention within the scope of the technology disclosed in the present invention. These simple modifications are all within the scope of protection of the present invention.

[0140] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0141] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A miniature integrated device for measuring the electrolyte content of urine, characterized in that, It includes a silicon substrate (1), a third chamber layer (2), a sensitive film layer (3), a second chamber layer (4), a salt bridge (5), a silicon-based electrode layer (6), a first chamber layer (7), and a cover plate (8) connected together from bottom to top. Multiple first electrodes (9) are integrated on the silicon substrate (1). Multiple internal reference liquid pools (10) corresponding to each of the first electrodes (9) are provided in the third chamber layer (2). A third microfluidic channel (11) communicating with the internal reference liquid pool (10) is provided on the third chamber layer (2) at the front end of the internal reference liquid pool (10). The third chamber layer (2) located on the outer side of the internal reference liquid pools (10) on the left and right sides is also provided with a third microfluidic channel (11) communicating with the corresponding internal reference liquid pool (10). The second chamber layer (4) is provided with a sample pool (12), and the second chamber layer (4) on both sides of the sample pool (12) is provided with a second microfluidic channel (13) that communicates with the sample pool (12). The silicon-based electrode layer (6) is provided with a second electrode (14) and a connecting cell (15). The first chamber layer (7) is provided with a reference pool (21) and a first microfluidic channel (22) connected thereto. A potential measuring instrument (23) is provided on the top of the cover plate (8); The micro-integrated device is provided with a first injection hole (16) penetrating the cover plate (8); a second injection hole (17) penetrating the cover plate (8), the first chamber layer (7), the silicon-based electrode layer (6), and the salt bridge (5) in sequence; and a third injection hole (18), a fourth injection hole (19), and a fifth injection hole (20) penetrating the cover plate (8), the first chamber layer (7), the silicon-based electrode layer (6), the salt bridge (5), the second chamber layer (4), and the sensitive film layer (3) in sequence. The first injection hole (16) is connected to the reference pool (21) of the first chamber layer (7) through the first microfluidic channel (22); the second injection hole (17) is connected to the sample pool (12) of the second chamber layer (4) through the second microfluidic channel (13); the third injection hole (18), the fourth injection hole (19), and the fifth injection hole (20) are respectively connected to the three internal reference pools (10) of the third chamber layer (2) through the corresponding third microfluidic channel (11); The sensitive membrane layer (3) is formed by bonding three kinds of sensitive membranes that have selective responses to sodium, potassium and calcium ions respectively with adhesive, and the three kinds of sensitive membranes are sequentially covered in the corresponding areas of the three internal reference liquid pools (10). The salt bridge (5) is a carbon nanotube bundle membrane.

2. The miniature integrated device for measuring urine electrolyte content according to claim 1, characterized in that, The micro-integrated device is provided with a first injection hole (16), a second injection hole (17) and a third injection hole (18) on both the left and right sides, a fourth injection hole (19) at both the front and rear ends of the micro-integrated device, and a fifth injection hole (20) at the front end of the micro-integrated device.

3. The miniature integrated device for measuring urine electrolyte content according to claim 1, characterized in that, The sensitive membrane constituting the sensitive membrane layer (3) is prepared by spin-coating a sensitive membrane solution, which is a mixture of PVC matrix, plasticizer, neutral complexing carrier, ionic additive and solvent, onto a wafer substrate; The plasticizer is dibutyl phthalate, dioctyl phthalate, dinonyl phthalate, or dibutyl phthalate of sebacic acid, oxalic acid, sebacic acid, or dibutyl phthalate. When the sensitive membrane has a selective response to sodium ions, the neutral complexing support is a Na ion support, specifically one or more of the following: double crown ether, 4′-benzo-12-crown-4, tetraphenyl24-crown ether-8, and 1,1,1-tris[1′-(2′-oxa-4′-oxo-5′-aza-5′-methyl)dodecyl]propane; When the sensitive membrane has a selective response to potassium ions, the neutral complexing carrier is a potassium ion carrier, specifically one or more of macrocyclic antibiotics, di-tert-butyl or dimethyl-dibenzo-crown-10, 1,1′-binaphthol-20-crown-6; When the sensitive membrane has a selective response to calcium ions, the neutral complexing carrier is a calcium ion carrier, specifically one or more of N,N-di[(11-ethyl ester)undecyl]-N,N'-4,5-tetramethyl-3,6-dioxaoctyl diamide and 1,1,1-tris(N-methyl-N-phenylaminocarbonylmethoxymethyl)propane. The ionic additive is one or both of sodium tetraphenylborate and potassium tetraphenylborate. The solvent is one or both of tetrahydrofuran and cyclohexanone.

4. The miniature integrated device for measuring urine electrolyte content according to claim 1, characterized in that, The first electrode (9) and the second electrode (14) are both Ag / AgCl electrodes, and the second electrode (14) is the common electrode of all the first electrodes (9).

5. The miniature integrated device for measuring urine electrolyte content according to any one of claims 1-4, characterized in that, The salt bridge (5) was fabricated using thermal decoupling CVD, as detailed below: Step 1: Flow a mixture of He at 100 sccm and H2 at 400 sccm in a tubular furnace for 10 minutes while heating the furnace to 775°C. Step 2: Insert the wafer into the furnace using a magnetically coupled transfer arm, and then maintain the same gas and flow rate as in Step 1, keeping the furnace at 775°C for 10 minutes. Step 3: Change the gas and flow rate to a mixed gas of 100 sccm C2H4, 400 sccm He and 100 sccm H2 for 3 min, and heat it at 775℃. After carbon nanotubes are grown on the wafer, keep the same gas and flow rate, cool the furnace to below 100℃, and finally purge with 1000 sccm He for 5 min to obtain a carbon capillary array. Step 4: Deposit a layer of SiO2 on the silicon substrate, spin-coat photoresist on the SiO2 surface, and use MEMS photolithography to etch away part of the photoresist to obtain a salt bridge (5) injection hole planar mask. The planar mask covers the injection holes to be obtained. Etch the SiO2 deposition layer by HF, etch the SiO2 part that is not protected by the photoresist, and obtain the three-dimensional structure of each injection hole. Remove the photoresist mask to obtain the SiO2 hard mask of each injection hole. Step 5: Cover the carbon capillary array obtained in step 2 with the SiO2 hard mask obtained in step 4, and etch the carbon capillary array with KOH of mass concentration of 35%. Etch the part of the carbon capillary array that is not protected by the SiO2 hard mask to obtain the three-dimensional structure of each injection hole. Remove the SiO2 hard mask to obtain the salt bridge with each injection hole (5).

6. The miniature integrated device for measuring urine electrolyte content according to any one of claims 1-4, characterized in that, The preparation method of the sensitive membrane is as follows: The first step is to prepare 20 ml of selective membrane solutions that selectively respond to sodium, potassium, and calcium ions respectively; the selective membrane solutions for the three ions use one or two of cyclohexanone and tetrahydrofuran as mixed solvents; sodium tetraphenylborate and potassium tetraphenylborate are used as ion additives; polyvinyl chloride is used as the PVC matrix and dioctyl sebacate is used as a plasticizer. The second step is to drop the prepared selective film solution onto the wafer substrate and spin-coat it evenly. The third step is to allow the mixed solvent to evaporate at room temperature to obtain the sensitive membrane, and then cut the sensitive membrane into an appropriate size and shape. When preparing the sodium ion selective membrane solution, tetraphenyl-24-crown ether-8 is used as the sodium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:24:

74. When preparing the potassium ion selective membrane solution, 1,1′-binaphthyl-20-crown-6 is used as the potassium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:33:

65. When preparing the calcium ion selective membrane solution, 1,1,1-tris(N-methyl-N-phenylaminocarbonylmethoxymethyl)propane is used as the calcium ion carrier, and the ion additive, ion carrier, polyvinyl chloride and dioctyl sebacate are mixed in a mass ratio of 1.8:0.2:32:

66. The mixed solvent used is a mixture of 20 ml of cyclohexanone and tetrahydrofuran.

7. A method for measuring electrolyte content in urine using a miniature integrated device for measuring electrolyte content in urine as described in any one of claims 1-4, characterized in that, Includes the following: Step 1: Connect all electrodes of the device to the potential measuring instrument (23) on the cover plate (8) via wires; Step 2: Inject the reference solution into the device through the first injection hole (16). The reference solution enters the reference pool (21) through the first injection hole (16) and the first microfluidic channel (22) in sequence. Stop the injection when the solution is exposed in the first injection hole (16) on the other side of the cover plate (8). Step 3: Inject the internal reference solution into the device through the third injection hole (18), the fourth injection hole (19), and the fifth injection hole (20), respectively. The internal reference solution enters the internal reference liquid pool (10) through the third microfluidic channel (11) in sequence. Stop the injection when the solution is exposed in the third injection hole (18), the fourth injection hole (19), and the fifth injection hole (20) on the other side of the cover plate (8). Step 4: Inject the urine sample into the device through the second injection hole (17). The urine sample enters the sample pool (12) through the second injection hole (17) and the second microfluidic channel (13) in sequence. Stop the injection when the solution is exposed in the second injection hole (17) on the other side of the cover plate (8). Step 5: After the reading of the potential measuring instrument (23) stabilizes, read the values ​​corresponding to the three first electrodes (9). These three values ​​are the potential values ​​received by the potential measuring instrument (23) and calculated by the built-in microprocessor and converted into the corresponding concentrations of sodium, potassium and calcium ions.

8. The method for measuring electrolyte content in urine according to claim 7, characterized in that, The internal reference solution is 10. -2 M NaCl solution, 10 -2 M KCl solution, 10 -2 M is a CaCl2 solution; the reference solution is a saturated KCl solution.

9. A method for manufacturing a miniature integrated device for measuring urine electrolyte content as described in any one of claims 1-4, characterized in that, Specifically, the following steps are included: Step 1: Integrate three first electrodes (9) on the silicon substrate (1) to obtain the indicator electrode; Step 2: Take a silicon substrate, deposit a layer of SiO2 on the silicon substrate, spin coat photoresist on the surface of SiO2, and use MEMS photolithography to etch away part of the photoresist to obtain the third chamber layer (2) structure planar mask; the third chamber layer (2) structure planar mask covers the internal reference liquid pool (10) and the third microfluidic channel (11) to be obtained. Step 3: The SiO2 in the part of the third chamber layer (2) not covered by the planar mask is etched away by HF etching of the third chamber layer (2) to obtain the three-dimensional structure of the third chamber layer (2) in the preliminary stage. Step 4: Remove the planar mask of the third chamber layer structure to obtain the final third chamber layer (2). Step 5: The second chamber layer (4), silicon-based electrode layer (6), first chamber layer (7), and cover plate (8) are fabricated using the same methods as in steps 2 to 4. Step 6: Integrate a second electrode (14) on the silicon-based electrode layer (6) to obtain a reference electrode; Step 7: Prepare three sensitive membrane solutions that selectively respond to sodium, potassium and calcium ions respectively, and spin-coat the sensitive membrane solutions onto the wafer substrate to prepare the corresponding sensitive membranes. Then, connect the three sensitive membranes together with adhesive to form a sensitive membrane layer (3). Step 8: From bottom to top, the silicon substrate (1), third chamber layer (2), sensitive film layer (3), second chamber layer (4), salt bridge (5), silicon electrode layer (6), first chamber layer (7) and cover plate (8) are stacked and bonded with adhesive.

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