Preparation and application of extracellular multi-ion real-time monitoring sensor for detecting cell damage and ion change in recovery process

Abstract

The invention discloses preparation and application of an extracellular multi-ion real-time monitoring sensor capable of being used for detecting cell damage and ion change in a recovery process, and belongs to the technical field of biosensing. The preparation method of the multi-ion real-time monitoring sensor comprises the following steps: carrying out patterning treatment on a polyimide film through a semiconductor diode laser engraving system, and engraving a carbon electrode; connecting the obtained carbon electrodes in pairs by using a conductive copper wire, bonding the carbon electrodes by using conductive silver paste, and then coating the surface of the conductive silver paste with polydimethylsiloxane to serve as a passivation layer; bonding a hollow ring body, wherein the hollow ring body intersects with all the conductive copper wires, and half of the carbon electrode is contained in the hollow inner part of the hollow ring body; and finally, modifying the carbon electrode in the hollow ring body to obtain the multi-ion real-time monitoring sensor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensing, and particularly relates to a preparation and application of an extracellular multi-ion real-time monitoring sensor for detecting cell damage and ion changes in recovery process. BACKGROUND

[0002] Oxidative stress is an important mechanism leading to imbalance of cell ion homeostasis, and is closely related to the occurrence and development of many diseases such as alcoholic liver disease. At present, the detection of ion concentration in and outside cells mainly relies on fluorescent probes or large analytical instruments, which has problems such as inability to monitor in real time for a long time, complex operation, high cost and the like. Electrochemical sensors have the advantages of high sensitivity, good real-time performance and easy miniaturization, but the existing sensors such as patents CN 114323852 A, CN 108828033 A and CN 115389589 A are all for single ion, and it is difficult to realize simultaneous monitoring of multiple ions, and the biocompatibility and stability need to be improved. SUMMARY

[0003] TECHNICAL PROBLEM The present application aims to solve the problem of inability to monitor the dynamic changes of multiple extracellular ions in real time, synchronously and for a long time in the prior art, and provides a multi-ion real-time monitoring sensor with high sensitivity, high selectivity and good biocompatibility.

[0004] TECHNICAL SCHEME The first object of the present application is to provide a preparation method of a multi-ion real-time monitoring sensor (MRMS), comprising the following steps: (1) patterning treatment is performed on a polyimide film by a semiconductor diode laser engraving system, and the treatment conditions are a power of 0.5-0.6 W, and a carbon electrode is engraved on the polyimide film; (2) the carbon electrode obtained in step (1) is connected with a conductive copper wire, and is bonded using conductive silver paste, and then polydimethylsiloxane is coated on the surface of the conductive silver paste as a passivation layer; (3) a hollow ring body is bonded on the polyimide film treated in step (2), the hollow ring body intersects with all the conductive copper wires and contains half of the carbon electrodes in the hollow interior; (4) the carbon electrodes in the hollow ring body are modified, and a multi-ion real-time monitoring sensor is obtained.

[0005] Further, the multi-ion real-time monitoring sensor can realize detection of H + , Ca 2+ , K + , Na + four kinds of ions.

[0006] Further, the thickness of the polyimide film is 2-5 μm.

[0007] Furthermore, the patterning depth on the polyimide film is 0.2~0.5μm.

[0008] Furthermore, the preparation process of the polyimide film is as follows: S1. Spin-coat a polyimide solution onto a glass substrate, and then pre-bake it at 100~120 ℃ for 5~10 min to remove the solvent and pre-cure it into a thin film. S2. Place the film obtained in step S1 at 180~220 ℃ for 10~30 min to cure, and obtain a polyimide film.

[0009] Furthermore, the shape of the carbon electrode is not fixed; selectable shapes include square, circular, rectangular, or trapezoidal.

[0010] Furthermore, the number of carbon electrodes is not fixed and can be any integer greater than 2.

[0011] Specifically, when real-time detection of H is required + Ca 2+ K + Na + When any two of the four ions are present, the number of carbon electrodes must be at least two.

[0012] Specifically, when real-time detection of H is required + Ca 2+ K + Na + When any three of the four ions are present, the number of carbon electrodes must be at least three.

[0013] Specifically, when real-time detection of H is required + Ca 2+ K + and Na + When there are four types of ions, the number of carbon electrodes must be at least four.

[0014] Furthermore, the material of the hollow ring may include glass, quartz, or plastic.

[0015] Furthermore, the shape of the hollow ring is not fixed, and the possible shapes include circles, ellipses, squares, rectangles or regular hexagons.

[0016] Furthermore, if real-time detection of H is required... +In step (4), one or more carbon electrodes are immersed in an aqueous solution containing 0.2-0.5 M aniline monomer and 0.2-0.5 M H2SO4, and a PANI thin film is electrodeposited as a pH sensing layer using cyclic voltammetry. During the deposition process, the potential range is -0.4 V to +0.8 V, the scan rate is 0.01-0.02 V / s, and the number of cycles is 50-60.

[0017] Furthermore, if real-time detection of Ca is required... 2+ Ions, in step (4) one or more of the carbon electrodes are immersed in Ca 2+ Ca is obtained by treating the solution in an ion-selective membrane under vacuum for 1-2 hours. 2+ Ion-selective electrode; the Ca 2 + The ion-selective membrane solution consists of 0.5–2 wt% calcium ion carrier II, 0.3–1 wt% sodium tetrakis(3,5-bis(trifluoromethyl)phenyl)borate, 63–67 wt% dioctyl sebacate, and 30–35 wt% PVC.

[0018] Specifically, the Ca 2+ The ion-selective membrane solution consists of 1 wt% calcium ion carrier II, 0.55 wt% sodium tetrakis(3,5-bis(trifluoromethyl)phenyl)borate, 65.45 wt% dioctyl sebacate and 33 wt% PVC.

[0019] Furthermore, if real-time detection of K is required... + Ions, in step (4) one or more of the carbon electrodes are immersed in K + K is obtained by treating the solution in an ion-selective membrane under vacuum for 1-2 hours. + Ion-selective electrode; the K + The ion-selective membrane solution consists of 1–3 wt% valine, 0.2–1 wt% sodium tetraphenylborate, 62–66 wt% dioctyl sebacate, and 30–35 wt% PVC.

[0020] Specifically, the K + The ion-selective membrane solution consists of 2 wt% valine, 0.6 wt% sodium tetraphenylborate, 64.7 wt% dioctyl sebacate and 32.7 wt% PVC.

[0021] Furthermore, if real-time detection of Na is required... + In step (4), one or more of the carbon electrodes are immersed in Na. + Na is obtained by treating the solution in an ion-selective membrane under vacuum for 1-2 hours. +ion selective electrode; the Na + The ion selective membrane solution is composed of 0.5-2 wt% sodium ion carrier X, 0.3-1 wt% sodium tetra(3,5-bis(trifluoromethyl)phenyl)borate, 62-67 wt% dioctyl sebacate and 30-35 wt% PVC.

[0022] Specifically, the Na + The ion selective membrane solution is composed of 1 wt% sodium ion carrier X, 0.55 wt% sodium tetra(3,5-bis(trifluoromethyl)phenyl)borate, 65.45 wt% dioctyl sebacate and 33 wt% PVC.

[0023] The application provides a multi-ion real-time monitoring sensor prepared according to the above method.

[0024] The multi-ion real-time monitoring sensor provided by the application is applied to the field of ion detection.

[0025] Further, in the application, the liquid to be detected is poured into the hollow ring body, and then the ion detection is performed by connecting the electrochemical workstation.

[0026] The multi-ion real-time monitoring sensor provided by the application can also realize real-time detection of multiple ions in cell culture.

[0027] The application also provides a method for detecting ion changes in a cell model by using the above multi-ion real-time monitoring sensor, and the method comprises the following steps: S1: sterilizing the multi-ion real-time monitoring sensor; S2: washing the sterilized multi-ion real-time monitoring sensor with PBS; S3: adding a culture medium into the hollow ring body of the washed multi-ion real-time monitoring sensor, and incubating at 35-40 DEG C for 1-5 h; S4: pouring a cell culture solution into the culture medium of the multi-ion real-time monitoring sensor, and continuing to culture for 20-24 h to make the cells grow adherently; S5: subsequently, removing the culture medium, replacing it with 1×D-PBS buffer, and then performing ion detection by connecting the electrochemical workstation.

[0028] Advantages (1) The application realizes the detection of H + , Ca 2+ , K + , Na +Real-time synchronous monitoring of four ions; by optimizing the laser engraving process, electrodes with excellent conductivity and chemical stability are prepared, which can overcome the technical bottlenecks of serious cross-interference and difficult to improve selectivity in multi-ion systems, and thus endow the sensor with excellent detection performance of parallel high sensitivity and high selectivity.

[0029] (2) The present application solves the problems of insufficient stability of traditional electrodes in complex physiological environment and difficulty in supporting long-term cell culture by biocompatibility modification on the multi-sensor interface, realizes the repeatability and long-term stability of the sensor in the cell culture process, and successfully expands its wide application in the fields of alcoholic liver disease model research, high-throughput drug screening and cell metabolism dynamics monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 : MRMS schematic diagram; Figure 2 : MRMS electrode characterization; Figure 3 : Potential response and linear relationship of ion electrodes in MRMS; Figure 4 : Ion selectivity test results of MRMS; Figure 5 : Reversibility, water layer effect and long-term stability test of the sensor; Figure 6 : Performance retention of the sensor after ECM modification; Figure 7 : Ion dynamic monitoring results in the process of alcohol damage and fucoxanthin treatment in the HepG2 cell model; Figure 8 : Ion dynamic monitoring results in the process of alcohol damage and fucoxanthin treatment in the HepG2 cell model; Figure 9 : Sensor performance verification after cell experiment. DETAILED DESCRIPTION

[0031] The present application is not limited to the specific conditions and details described in the following embodiments. Various specific technical features can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, all possible combinations are not described again in the present application. Any person skilled in the art can make simple modifications and substitutions according to their own circumstances within the technical scope described in the present application, and these simple modifications are within the protection scope of the present application. In the practice of the present application, various alternatives of the present application described in the present application can be used. The examples of the present application will be described in detail below with reference to the accompanying drawings, which are intended to explain the present application, to describe the technical solutions clearly and completely, and are not limited to the reagents or instruments used in the present application.

[0032] The application will be further described below by specific examples.

[0033] Raw material sources Polyimide (FPS-1000) was purchased from Xi'an Boyan Micro-nano Information Technology Co., Ltd.; Valinomycin, polyvinyl chloride (PVC) and dioctyl sebacate (DOS) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; polydimethylsiloxane (PDMS) was purchased from Dow Corning Corporation; calcium ionophore II (ETH129), sodium ionophore X, tetrahydrofuran (THF), sodium tetra (3, 5-bis (trifluoromethyl) phenyl) borate (NaTFPB) and sodium tetraphenylborate (NaTPB) were purchased from Shanghai McLean Biochemical Co., Ltd.; high glucose DMEM medium and fetal bovine serum were purchased from Gibco Company; trypsin-EDTA solution and penicillin-streptomycin solution were purchased from Vivacell Company; 1×PBS (for cell culture) and 1×D-PBS buffer (containing calcium and magnesium ions) were provided by Beikong Technology Co., Ltd.; KH2PO4 and K2HPO4 were supplied by Tianjin Kermel Chemical Reagent Co., Ltd.; BCECF AM, Fluo-4 AM and Matrigel matrix (ECM) were purchased from Biyun Tian Biotechnology Co., Ltd. Ultra-micro ATPase detection kit (Na + / K + -ATPase, Ca 2+ / Mg 2+ -ATPase and total ATPase) were purchased from Nanjing Jiancheng Biological Engineering Institute; fucoxanthin (FX) was purchased from Shandong Clean Group Co., Ltd.; all reagents were analytical pure.

[0034] The preparation process of Gal-SEVs is as follows: S1, inoculate Lactobacillus plantarum Lp90 in MRS broth medium and culture at 37 ℃ for 36 h. Centrifuge at 3000 g, 4 ℃ for 15 min, collect the lower precipitate and resuspend in sterile PBS; S2, take the resuspension obtained in step S1 and add 10 mg / mL lysozyme, react at 37 ℃, 500 rpm for 6 h, then centrifuge at 3500 g, 4 ℃ for 15 min for the second time, collect the precipitate and centrifuge with sterile PBS according to the above parameters for 2 times, and finally resuspend in sterile PBS; S3, the liquid obtained in step S2 was placed under an ultrasonic cell disruptor, the power was set to 200 W, and the time was 30 min (intermittent ultrasonic, working for 2 s and stopping for 2 s) for ice bath ultrasonic treatment. After ultrasonic treatment, the third centrifugation was performed at 4 ℃ and 8000 g, and the supernatant was collected and filtered using a 0.45 μm filter membrane; S4, the filtrate obtained in step S3 was treated by a tangential flow ultrafiltration system, the treatment time was 500 mL / h, and the filter membrane of the ultrafiltration system was selected to have a size of 100 KDa.

[0035] S5, the ultrafiltration concentrated liquid obtained in step S4 was placed under an ultrasonic cell disruptor, the power was set to 200 W, and the time was 30 min (intermittent ultrasonic, working for 2 s and stopping for 2 s) for ice bath ultrasonic treatment. Subsequently, it was transferred to ice for incubation for 1 h, and Lactobacillus plantarum Lp90 engineered nanovesicles (SEVs) were obtained. S6, Distearylphosphatidylethanolamine-polyethylene glycol 2000-galactose was prepared. D- (+) -galactosamine (500 mg, 60 μmol) was completely dissolved in distilled water, distearylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG2000-COOH) was dissolved in a dimethylformamide solution, and then the carboxyl group was activated with N-hydroxysuccinimide (50.0 μmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (60.0 μmol). After stirring at room temperature for 12 hours, the aqueous galactosamine solution was added (the mass ratio of galactosamine to distearylphosphatidylethanolamine-polyethylene glycol 2000-carboxyl was 15:1), and stirring was performed at 4 ℃ for 24 hours. The obtained viscous solution was dialyzed (MWCO 3500) with deionized water for 48 hours, and freeze-drying was performed to obtain distearylphosphatidylethanolamine-polyethylene glycol 2000-galactose. 5 mg of distearylphosphatidylethanolamine-polyethylene glycol 2000-galactose was dissolved in heavy water.

[0036] S7, the Lactobacillus plantarum Lp90 engineered nanovesicles in step S5 were mixed with the distearylphosphatidylethanolamine-polyethylene glycol 2000-galactose obtained in step S6 according to a mass ratio of 15:1, stirring was performed at 4 ℃ and 200 rpm / h for 12 h, and galactosyl-modified Lactobacillus plantarum Lp90 engineered nanovesicles (Gal-SEVs) were obtained.

[0037] The preparation process of FX-SEVs is as follows: S1, the prepared SEVs were mixed with a 5 mg / mL fucoxanthin ethanol solution, and the mass ratio was 50:1.

[0038] S2: The mixed solution in S2 was placed in an ultrasonic cell disruptor and ice-bath ultrasonic treatment was performed for 30 min (intermittent ultrasonic, working for 2 s and stopping for 2 s), with a power of 200 W. After the treatment, the solution was incubated on ice for 1 h.

[0039] S3: The ethanol solvent in S2 was removed by rotary evaporation treatment at a water bath temperature of 37 °C, thereby obtaining the fucoxanthin-loaded Lactobacillus plantarum Lp90 engineered nanovesicle delivery system (FX-SEVs).

[0040] The preparation process of Gal-FX-SEVs was as follows: (1) the obtained Gal-SEVs were mixed with a 5 mg / mL fucoxanthin ethanol solution at a mass ratio of 50:1; (2) the mixed solution was placed in an ultrasonic cell disruptor and ice-bath ultrasonic treatment was performed for 30 min, with a power of 200 W. After the treatment, the solution was incubated on ice for 1 h; (3) the incubated mixed solution was subjected to rotary evaporation treatment at a water bath temperature of 37 °C to remove the ethanol solvent, thereby obtaining Gal-FX-SEVs.

[0041] Example 1: Preparation and characterization of a multi-ion real-time monitoring sensor (1) A polyimide solution was spin-coated on a circular glass substrate at a rotation speed of 2000 rpm.

[0042] (2) The obtained product in step (1) was subjected to a pre-baking treatment at 120 °C for 10 min to remove the solvent and preliminarily solidify into a thin film with a thickness of 3 μm.

[0043] (3) The thin film obtained in step (2) was placed on a hot stage at 200 °C for solidification for 30 min to ensure that the polyimide (PI) thin film was completely solidified and good mechanical stability was obtained. After solidification, the PI thin film had a thickness of 2 μm.

[0044] (4) The solidified PI thin film obtained in step (3) was subjected to patterning treatment by a computer-controlled semiconductor diode laser engraving microprocessing system (model Diatools K6, purchased from Shanghai Diatools Industrial Co., Ltd., with a laser head power of 3 W). The treatment conditions were a power of 20% and a depth of 15%. The PI film was patterned into a carbon electrode, and the carbon electrode was obtained.

[0045] (5) The electrode obtained in step (4) was connected with a conductive copper wire, and the conductive copper wire was bonded with conductive silver paste. The other end of the conductive copper wire was connected with a common electrode. Then, PDMS was used to coat the surface of the conductive silver paste as a passivation layer to prevent electric leakage.

[0046] (6) The obtained product in step (5) was bonded with a glass circular ring with a height of 5 cm and a diameter of 2 cm at the center position of the microelectrode array using PDMS. The carbon electrode was completely included in the interior of the glass circular ring.

[0047] (7) One of the carbon electrodes inside the glass ring was immersed in a 0.50 M H2SO4 solution containing 0.25 M aniline monomer, and a PANI film was electrodeposited as a pH sensing layer by cyclic voltammetry. The chemical deposition process was carried out at a potential range of -0.4 V ~ +0.8 V, a scan rate of 0.01 V / s, and 60 cycles.

[0048] (8) The Ca 2+ , K + , and Na + ion selective membrane (ISM) solutions were prepared according to the proportions in Table 1.

[0049] Table 1

[0050] (9) 1 µL of each of the Ca 2+ , K + , and Na + ISM solutions prepared in step (8) was dropped onto the surface of the other carbon electrodes inside the glass ring, and the electrodes were placed in a vacuum environment for 1 h to obtain Ca 2+ , K + , and Na + ion selective electrodes. The sensors containing both the modified PANI film and the Ca 2+ , K + , and Na + ion selective membrane are MRMS (as shown in Figure 1 , where the colored ones are the modified carbon electrodes, and the black ones are ordinary electrodes for connecting to the electrochemical workstation).

[0051] (10) The microstructure of the carbon electrodes in (4) and the pH-sensitive electrode in the multi-ion electrochemical sensor in (7) was characterized by field emission scanning electron microscopy (NOVA NanoSEM 450), and the Raman spectra of the carbon electrodes and the pH-sensitive electrode were recorded using a Raman spectrometer (DXR SmartRaman, Thermo Fisher) with a 325 nm laser excitation.

[0052] (11) The microstructure of the modified ion selective membrane electrode in (8) was characterized using the field emission scanning electron microscope in step (10).

[0053] Comparative Example 1 The preparation process described in Reference Example 1 was followed, except that in step (4) the patterning treatment was adjusted to a power of 15% and a depth of 15%, and the PI film was patterned into carbon electrodes, and the subsequent operations and steps remained unchanged.

[0054] Comparative Example 2 Reference is made to the preparation process described in Example 1, wherein only in step (4) the patterning treatment is adjusted to power 25%, depth 15%, and the PI film is patterned into a carbon electrode, and the subsequent operations and steps remain unchanged.

[0055] Example 2: Performance test of a multi-ion real-time monitoring sensor The electrochemical measurement results were recorded by an electrochemical workstation (CHI 1440, Shanghai Chenhua Instruments). A self-made ion selective electrode was used as the working electrode, a commercial Ag / AgCl electrode was used as the reference electrode, and the open circuit potential method was used for testing. The test solution was poured into the glass ring at the center of the MRMS.

[0056] S1: The MRMS obtained in step (9) of Example 1 was tested for open circuit potential responsiveness of the MRMS by changing the ion concentration in the test solution at a temperature of 25°C (298.15 K) under atmospheric conditions, i.e., the electrochemical response test of the pH electrode in the MRMS was performed in a PBS buffer solution (containing 10 mM NaH2PO4 and 10 mM Na2HPO4) with a pH of 7.44. The pH was adjusted every 50 s of testing, and the pH gradient was 7.00, 6.49, 6.13, and 5.79. The electrochemical response test of the Ca 2+ electrode was performed in a HEPES buffer solution with a pH of 7.4, and the initial buffer solution had a Ca 2+ concentration of 0.1 mM. The Ca 2+ concentration was adjusted every 100 s of testing, and the concentration gradient was 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, and 3.2 mM. The electrochemical response test of the K + electrode was performed in a PBS buffer solution (containing 10 mM NaH2PO4 and 10 mM Na2HPO4) with a pH of 7.44, and the initial buffer solution had a K + concentration of 1 mM. The K + concentration was adjusted every 50 s of testing, and the concentration gradient was 2 mM, 4 mM, 8 mM, 16 mM, and 32 mM. The electrochemical response test of the Na + electrode was performed in a PBS buffer solution (containing 10 mM NaH2PO4 and 10 mM Na2HPO4) with a pH of 7.44, and the initial buffer solution had a Na + concentration of 10 mM. The Na + concentration was adjusted every 100 s of testing, and the concentration gradient was 20 mM, 40 mM, 80 mM, 160 mM, and 320 mM.

[0057] The response linear correlation curve of all electrodes in MRMS Nernst calculation equation is as follows:

[0058] In the formula, E ISE is the electrode potential, E 0 is the standard electrode potential (constant term), R is the molar constant of ideal gas, the value is 8.314 J·mol -1 ·K -1 ; T is the thermodynamic temperature; n is the charge number carried by the ion to be measured; F is the Faraday constant, the value is 96485 C / mol; α i is the activity of the ion to be measured.

[0059] S2: The ion selectivity of the sensor obtained in step (9) in Example 1 was detected. When it was a pH sensitive electrode, 150 mM Na + , 10 mM K + , 2 mM Ca 2+ , 2 mM Mg 2+ and pH 3.53 buffer solution were added in sequence in a HEPES buffer solution with pH of 7.4. When it was a Ca 2+ electrode, 1 mM Ca 2+ , 150 mM Na + , 10 mM K + , 2 mM Mg 2+ and 5 mM Ca 2+ were added in sequence in a test solution containing 0.1 mM Ca 2+ . When it was a K⁺ electrode, 4 mM K + , 150 mM Na + , 2 mM Ca 2+ , 2 mM Mg 2+ and 10 mM K + were added in sequence in a solution containing 1 mM K + . When it was a Na + electrode, 40 mM Na + , 10 mM K + , 2 mM Ca 2+ , 2 mM Mg 2+ and 140 mM Na + were added in sequence in a solution containing 4 mM Na + .

[0060] S3: The reversibility of the sensor obtained in step (9) of Example 1 was detected. In the detection of the reversibility of the pH electrode, the pH of the test solution was decreased from alkaline (7.41) to acidic (5.59) and then increased to alkaline (7.56), and the electrochemical value change was measured. Similarly, the Ca 2+ The reversibility of the electrode was detected by adjusting the Ca 2+ concentration from 0.1 mM to 1.6 mM and then to 0.1 mM. The K + The reversibility of the electrode was detected by adjusting the K + concentration from 1 mM to 16 mM and then to 1 mM. The Na + The reversibility of the electrode was detected by adjusting the Na + concentration from 10 mM to 320 mM and then to 10 mM.

[0061] S4: The water layer test and potential drift test of the sensor obtained in step (9) of Example 1 were performed. That is, the water layer test of the ion selective electrode was detected by using the electrochemical workstation to detect the electrical signal of the ion selective electrode in CaCl2 solution and NaCl solution, wherein the pH electrode was tested alternately between PBS and NaCl solution, and other electrodes were tested alternately between KCl and NaCl solution, and after each change of solution, a two-minute pause was made, followed by continuing the potential measurement. The stability test of the ion selective electrode in the MRMS was performed by detecting the electrical signal of the ion selective electrode in its fixed corresponding ion solution for 24 h.

[0062] Example 3: Application of a multi-ion real-time monitoring sensor in detecting ion changes in the process of alcohol-induced HepG2 cell oxidative stress generation and relief S1: The MRMS obtained in step (9) of Example 1 was sterilized by using 75% alcohol and ultraviolet light irradiation for 60 min.

[0063] S2: The sterilized MRMS in S1 was washed with PBS for 3 times, and the PBS was discarded after the last washing.

[0064] S3: 10 μg / mL of ECM was added to the MRMS washed in S2, and incubated at 37°C for 2 h. The incubated MRMS was used for cell culture.

[0065] S4: The cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody (37°C, 5% CO2). When the cells grew to 80%, they were washed with PBS and digested with trypsin for 2 min, and then the digestion was terminated by adding culture medium. The cells were collected by centrifugation at 1000 rpm for 4 min. Subsequently, the cells were dispersed in DMEM medium at a concentration of 1×10 5Cells were seeded into MRMS obtained from S3 at a density of cells / mL and cultured in an incubator for 24 h to allow the cells to adhere and grow.

[0066] S5: Three groups were set up: Control group, Ethanol group, and sample intervention groups 1, 2, and 3. The Ethanol group culture medium consisted of DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-antibody solution, with 150 mM anhydrous ethanol added. The sample intervention groups cultured Ethanol medium with the addition of free FX (sample intervention group 1), FX-SEVs (sample intervention group 2), and Gal-FX-SEVs (sample intervention group 3), respectively. The final concentration of FX in the medium was 2.5 μg / mL. FX-SEVs were used to improve the water solubility of FX in the delivery system, while Gal-FX-SEVs were used to improve the water solubility and hepatocyte targeting of FX. Cells were cultured in MRMS for 24 h. Subsequently, the culture medium was removed, replaced with 1×D-PBS (containing calcium and magnesium) buffer, and the MRMS was used to test changes in extracellular ions for 1 h.

[0067] S6: HepG2 cells were seeded in 12-well plates and cultured for 24 h to promote adherent growth. Oxidative damage and sample intervention were performed on the cells as described in S5. Cells were incubated at 37 °C for 30 min using BCECF AM and Fluo-4 AM fluorescent probes, and intracellular pH and Ca2+ were measured. 2+ The cell nuclei were labeled with DAPI to determine their concentration. After incubation, the staining solution was discarded, and the cells were washed three times with PBS. Finally, the cells were observed under a fluorescence microscope. Fluorescence intensity was analyzed using ImageJ software.

[0068] Experimental results (1) MRMS characterization results By optimizing laser parameters (power 20%, etching depth 15%), graphite materials (LIG) with a highly ordered porous structure were successfully prepared on the surface of polyimide. Figure 2 As shown in figure a, scanning electron microscopy (SEM) reveals that this structure possesses three-dimensional interconnected pores, significantly increasing the specific surface area and providing an ideal interface for ion-electron transduction. Figure 2 As shown in b, the Raman spectrum is at 1357 cm⁻¹. -1 (D belt), 1590 cm -1 (G-band) and 2804 cm -1 Characteristic peaks were observed at the (2D band), confirming that laser etching achieved complete carbonization and graphitization of polyimide, resulting in materials with excellent conductivity and chemical stability. Secondly, polyaniline (PANI) was successfully polymerized on the LIG electrode surface using cyclic voltammetry. Raman spectra were observed at 1167 cm⁻¹. -1(C-H bending vibration) and 1499 cm -1 (C=N stretching vibration). Figure 2 The SEM of c shows that a dense and continuous PANI coating layer is formed, which ensures efficient proton conduction and ion transmission path. Figure 2 d shows that the ion-selective membrane (ISM) is uniformly modified on the LIG porous structure by vacuum-assisted deposition technology, forming a deep penetration interface configuration. The MRMS carbon electrode patterns obtained in Comparative Example 1 and Comparative Example 2 are not complete, and the conductivity is poor, and the subsequent ion detection results are not ideal, and there is a phenomenon of mutual interference of ions.

[0069] (2) Test results of electrode electrochemical response in MRMS The pH electrode in MRMS shows linear characteristics close to ideal Nernst response in the entire test pH range, with a sensitivity of 59.5 mV / pH and a linear correlation coefficient of 0.999 (R2). Figure 3 a, 3b). The carbon electrode modified by PANI also shows high sensitivity and good linear response to pH changes. In the Ca 2+ detection aspect, the Ca 2+ electrode in MRMS shows excellent performance, with a sensitivity of 30.1 mV / dec and a linear correlation coefficient of 0.999 (R2). Figure 3 c, 3d), which is close to the theoretical limit, indicating that the electrode has excellent detection performance. For K + detection, the K + electrode in MRMS shows excellent performance, with a sensitivity of 52.6 mV / dec and a linear correlation coefficient of 0.999 (R2). Figure 3 e, 3f), showing good linear response ability in the K + detection range. In the Na + detection aspect, the Na + electrode in MRMS has a sensitivity of 58.7 mV / dec and a linear correlation coefficient of 0.999 (R2). Figure 3 g, 3h), indicating that it has excellent performance in Na + concentration change detection, providing a potential reliable tool for accurate measurement of sodium ions in extracellular fluid.

[0070] (3) Test results of electrode selectivity in MRMS As shown in Figure 4 a, in the HEPES buffer solution with pH 7.4, 150 mM Na + , 10 mM K + , 2 mM Ca 2+ and 2 mM Mg 2+Experimental results show that even in the presence of high concentrations of interfering ions, the potential response of the pH electrode in MRMS remains essentially unchanged, with a significant potential increase only occurring when the solution pH changes. This indicates that the electrode possesses excellent anti-interference capabilities and is suitable for pH detection in complex extracellular environments. In Ca... 2+ In electrode testing, 150 mM Na was added. + Although it causes a reverse fluctuation in potential, its amplitude is significantly lower than that of physiological concentrations of Ca. 2+ The resulting potential change; and K + With Mg 2+ The introduction of this did not lead to significant potential fluctuations. Figure 4 b), indicating that Ca 2+ The electrode exhibits excellent anti-interference performance, enabling the control of Ca in an extracellular fluid environment. 2+ Effective detection. Similarly, K in MRMS + electrode( Figure 4 c) with Na + electrode( Figure 4 d) No significant potential changes were observed in the presence of multiple interfering ions, indicating that both exhibit good ion selectivity and can effectively eliminate interference from common ions in extracellular fluid.

[0071] (4) Results of electrode reversibility test in MRMS like Figure 5 As shown in Figure a, during the continuous change of solution pH from alkaline (7.41) to acidic (5.59) and then back to alkaline (7.56), the pH electrode exhibited excellent reversibility, indicating that it could stably output a signal corresponding to pH changes in an alternating acid-base environment without significant hysteresis or irreversible response deviation. In Ca... 2+ In electrode testing, the sensor measures Ca 2+ The response signals were highly consistent, and no significant signal drift or attenuation was observed during the test. Figure 5 b), indicating its effect on Ca 2+ The detection exhibits good repeatability and accuracy. + The electrode experiences K + After cyclic changes in concentration, the deviation between the initial and final potential values ​​was only about 1.4 mV. Figure 5 c), exhibiting excellent reversibility, ensures that the sensor can maintain K during continuous monitoring. + Accurate and reliable measurement of Na. + After continuous testing, the electrode's potential value deviated from its initial state by only 0.3 mV. Figure 5 d) indicates that the electrode possesses rapid recovery capability and high stability, and can maintain its resistance to Na during continuous monitoring. + Accurate detection.

[0072] (5) Results of electrode water layer test and potential drift test in MRMS To evaluate the resistance to water layer effect and long-term stability of each electrode in MRMS, water layer effect testing and potential drift monitoring were conducted. Figure 5 e–5h shows the pH and Ca in MRMS. 2+ K + with Na + Results of the water layer effect test on the electrodes. Experiments show that each electrode can rapidly respond and recover to its initial potential value during solution replacement, indicating that the PANI layer densely deposited on the carbon electrode surface by cyclic voltammetry effectively blocks the interference of the water layer on electrode performance; simultaneously, the vacuum treatment process also achieves Ca… 2+ K + with Na + The tight bonding between the ion-selective membrane (ISM) and the carbon substrate effectively inhibits the formation of water layers.

[0073] like Figure 5 As shown in i–5l, during a long-term monitoring period of 24 hours, the potential drift rates of each electrode in the MRMS were as follows: pH electrode 0.15 mV / h, Ca 2+ Electrode 0.25 mV / h, K + Electrode 0.33 mV / h, Na + The electrode voltage is 0.41 mV / h. These data indicate that MRMS exhibits high signal stability during long-term operation, making it suitable for practical applications requiring continuous and reliable monitoring.

[0074] (6) Performance test results of MRMS after ECM modification To verify whether the sensor's detection performance was maintained after ECM modification, a system test was conducted on the sensor that had been incubated with ECM for 2 hours. Figure 6 As shown in a–6h, all ion-selective electrodes maintained excellent detection performance after modification: the pH electrode sensitivity was 57.3 mV / pH (R0). 2 = 0.999), Ca 2+ The electrode sensitivity is 28.9 mV / dec (R 2 = 0.998), K + The electrode sensitivity is 51.2 mV / dec (R 2 = 0.999), Na + The electrode sensitivity is 54.5 mV / dec (R 2 = 0.999).

[0075] The above results indicate that ECM surface modification improves the biocompatibility of MRMS without sacrificing its core detection performance. Each electrode still possesses high sensitivity and excellent linear response characteristics, providing a reliable guarantee for accurate ion concentration detection in the cell culture environment.

[0076] (7) Results of dynamic ion monitoring during alcohol injury and fucoxanthin treatment in the HepG2 cell model like Figure 7 As shown in Figure a, alcohol-induced damage significantly decreased extracellular pH, indicating increased cellular acid excretion; however, after sample intervention, the decrease in extracellular pH was significantly reduced, indicating that the treatment effectively alleviated the acid load induced by alcohol. Quantitative analysis showed ( Figure 7 (b) The pH change in the Ethanol group within 1 hour was approximately -0.11, about 1.89 times that of the Control group; while the pH change in sample intervention group 3 was only about -0.07, close to the level of the Control group (approximately -0.01). Enzyme activity assay results showed ( Figure 7 c), Gal-FX-SEVs can... + -K + -ATPase activity was increased to 0.064 U / mgprot, significantly restoring its function. These results indicate that FX and its derivatives can alleviate oxidative stress and increase H2O levels. + -K + -ATPase activity effectively maintains extracellular pH homeostasis.

[0077] To verify the reliability of extracellular pH changes measured by MRMS, intracellular pH was further detected using the BCECF AM fluorescent probe. Figure 7 As shown in d, the intensity of green fluorescence in the cells of the Ethanol group decreased, indicating a decrease in intracellular pH; while the fluorescence intensity of all intervention groups rebounded, with the relative fluorescence intensity of intervention group 3 recovering to 43.74%, which was 2.85 times that of the Ethanol group and closest to the level of the Control group. Figure 7 e). This result is consistent with the trend of extracellular pH changes detected by MRMS, jointly demonstrating that alcohol can cause simultaneous acidification inside and outside the cell, and that Gal-FX-SEVs can effectively alleviate this acid-base imbalance, while also verifying the accuracy of MRMS in the detection of the extracellular environment.

[0078] Mitochondrial function and Ca 2+ Closely related to steady state, its decrease in membrane potential will weaken Ca 2+ Buffer capacity leads to cytoplasmic calcium 2 + The concentration increased. To investigate the effect of alcohol on Ca... 2+ The effect of homeostasis, using MRMS to monitor extracellular Ca 2+Concentration changes. For example... Figure 7 As shown in f-7g, extracellular Ca in the Ethanol group 2+ The concentration decreased by 0.10 mM, which was greater than that in the control group (0.04 mM), suggesting the possible presence of Ca. 2+ Influx or intracellular release; while after Gal-FX-SEVs intervention, Ca 2+ The concentration decreased by only 0.05 mM. Further analysis of Ca... 2+ / Mg 2+ -ATPase activity was found to be 85.7% lower in the Ethanol group compared to the Control group, while FX, FX-SEVs, and Gal-FX-SEVs mitigated the decrease to 65.9%, 33.1%, and 26.7%, respectively. Figure 7 h), indicating that Gal-FX-SEVs can significantly maintain Ca. 2+ Steady state, reducing alcohol-induced calcium 2+ overload.

[0079] To eliminate measurement interference and verify Ca 2+ Changes were observed using the Fluo-4 probe to detect intracellular Ca2+. 2+ Fluorescence imaging. For example... Figure 7 As shown in i, intracellular fluorescence was significantly enhanced after ethanol treatment, indicating that Ca 2+ The concentration increased; however, the fluorescence gradually decreased in each intervention group as the drug performance improved, with the fluorescence intensity of the Gal-FX-SEVs group being only 1.67 times that of the Control group. Figure 8 (j) indicates that it has good calcium homeostasis restoration ability. This result is consistent with the MRMS detection results, further confirming that MRMS can still accurately capture ion dynamics in complex cellular environments.

[0080] extracellular K + with Na + The concentration dynamics of ions are a key indicator for assessing cell function and damage status. To investigate the effect of alcohol on hepatocyte ion homeostasis, MRMS was used to measure extracellular potassium (K+). + with Na + The concentration was monitored in real time.

[0081] In K + In terms of testing, such as Figure 8 As shown in a, alcohol stimulation can induce significant K... + Outflow, leading to extracellular K + The concentration increased; however, after sample intervention, this efflux phenomenon was effectively alleviated, indicating that the above-mentioned substances help maintain cell membrane stability and ion balance. Quantitative analysis showed ( Figure 8 b) Within 1 hour, extracellular K+ in the Ethanol group +The concentration increased by 0.3 ± 0.059 mM, which was 2.6 times the change in the control group. Each intervention group recovered K... + The gradient effect was observed in terms of homeostasis, further validating the effectiveness of MRMS in monitoring extracellular K+. + It exhibits good accuracy and reliability in dynamic aspects.

[0082] In Na + In terms of testing, Figure 8 The results showed that alcohol stimulation could cause extracellular Na+. + The decrease in concentration suggests that Na + Inflow or Na + / K + The pump function was impaired. With the intervention of samples (especially sample 3), Na... + The downward trend has been significantly curbed. Quantitative data show that ( Figure 8 d), Sample intervention in 3 groups of extracellular Na + The concentration was only 0.85 mM lower than that of the control group, demonstrating the best ability to maintain ion stability.

[0083] To verify the above Na + The reliability of the changing trend was further tested by examining Na. + / K + -ATPase activity. For example... Figure 9 As shown in Figure e, enzyme activity in the Ethanol group decreased to 0.42 U / mgprot, while Gal-FX-SEV intervention significantly increased its activity to 0.88 U / mgprot, closest to the control group level. This change in enzyme activity reflects the "extracellular Na+" level. + Accumulation and K + The "loss" trend is consistent with the extracellular Na+ observed by MRMS. + Decline and K + The elevated results have clear physiological consistency, thus mutually confirming the role of MRMS in the complex cellular environment of Na+. + K + The ability to accurately monitor ion dynamics.

[0084] (8) Sensor performance test results after cell experiments To verify the reliability of the multichannel sensor after cell experiments, the sensor underwent systematic cleaning and performance retesting. First, cells were digested with trypsin, and then the sensor surface was thoroughly cleaned sequentially with PBS, ethanol, and deionized water to remove biological residues.

[0085] The test results of each ion-selective electrode after cleaning are as follows: ​ As shown in ah, its sensitivity and linear response are as follows: pH electrode 53.9 mV / pH (R2 = 0.999), Ca 2+ Electrode 26.2 mV / dec (R 2 = 0.998), K + Electrode 49.4 mV / dec (R 2 = 0.999), Na + Electrode 53.6 mV / pH (R 2 = 0.999).

[0086] The experimental results show that the sensor still maintains high sensitivity and excellent linearity close to the initial level after experiencing cell culture and detection in each channel, which fully verifies the reliability of the test results in complex biological environment, and also shows good reusable potential.

[0087] The above results prove that: the multi-ion real-time monitoring system (MRMS) constructed by the application still maintains high sensitivity and excellent linear response close to the initial state in each ion channel after experiencing complex cell environment detection and strict cleaning process, which shows that the system has excellent stability and reusable capacity, and can realize long-term, accurate and reliable monitoring of various ions (H⁺, Ca 2+ , K + , Na + ) in the extracellular environment, and is suitable for biomedical research fields such as cell metabolism analysis, drug evaluation and toxicity detection.

[0088] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description manner of the specification is only for the sake of clarity, and the person skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be combined appropriately to form other embodiments that can be understood by the person skilled in the art.

Claims

1. A method for preparing a multi-ion real-time monitoring sensor, characterized by, The preparation method comprises the following steps: (1) patterning treatment on the polyimide film by a semiconductor diode laser engraving system, the treatment condition being that the power is 0.5-0.6 W, and a carbon electrode is engraved on the polyimide film; (2) connecting the carbon electrode obtained in step (1) with a conductive copper wire, and using conductive silver paste for bonding, and then using polydimethylsiloxane to coat the surface of the conductive silver paste as a passivation layer; (3) bonding a hollow ring body on the polyimide film treated in step (2), the hollow ring body intersecting all the conductive copper wires and containing half of the carbon electrodes in the hollow interior; (4) modifying the carbon electrodes in the hollow ring body, and obtaining a multi-ion real-time monitoring sensor; If real-time detection H + ions, the carbon electrode is immersed in an aqueous solution containing 0.2-0.5 M aniline monomer and 0.2-0.5 M H2SO4, and a PANI film is electrodeposited by cyclic voltammetry as a pH sensing layer; the potential range during deposition is -0.4 V to +0.8 V, the scanning rate is 0.01-0.02 V / s, and the number of cycles is 50-60. If real-time detection of Ca 2+ ions, the carbon electrode is immersed in a Ca 2+ ion-selective membrane solution and placed in a vacuum environment for 1-2 h to obtain a Ca 2+ ion-selective electrode; the Ca 2+ ion-selective membrane solution is composed of 0.5-2 wt% of a calcium ion carrier II, 0.3-1 wt% of NaTFPB, 63-67 wt% of DOS, and 30-35 wt% of PVC; If real-time detection of K+ + ions, the carbon electrode is immersed in a K+ + ion-selective membrane solution and placed in a vacuum environment for 1-2 h to obtain a K+ + ion-selective electrode; the K+ + ion-selective membrane solution is composed of 1-3 wt% valinomycin, 0.2-1 wt% NaTPB, 62-66 wt% DOS, and 30-35 wt% PVC; If Na + ions are detected in real time, the carbon electrode is immersed in a Na + ion-selective membrane solution and placed in a vacuum environment for 1-2 h to obtain a Na + ion-selective electrode; the Na + ion-selective membrane solution is composed of 0.5-2 wt% of a sodium ion carrier X, 0.3-1 wt% of NaTFPB, 62-67 wt% of DOS, and 30-35 wt% of PVC.

2. The preparation method according to claim 1, characterized in that, The thickness of the polyimide film is 2-5 μm; and the preparation process of the polyimide film is as follows: S1, spin-coating a polyimide solution on a glass substrate, and then performing a pre-baking treatment at 100-120 ℃ for 5-10 min to remove the solvent and preliminarily solidify into a film; S2, curing the film obtained in step S1 at 180-220 ℃ for 10-30 min to obtain a polyimide film.

3. The preparation method according to claim 1, characterized in that, The treatment depth of the patterning treatment in step (1) on the polyimide film is 0.2-0.5 μm.

4. The preparation method according to claim 1, characterized in that, The shape of the carbon electrode includes a square, a circle, a rectangle or a trapezoid.

5. The preparation method according to claim 1, characterized in that, The material of the hollow ring body includes glass, quartz or plastic; and the shape of the hollow ring body includes a circle, an ellipse, a square, a rectangle or a regular hexagon.

6. The production method as claimed in claim 1, characterized in that, The Ca 2+ The ion selective membrane solution consisted of 1 wt% calcium ionophore II, 0.55 wt% sodium tetra(3,5-bis(trifluoromethyl)phenyl)borate, 65.45 wt% dioctyl sebacate, and 33 wt% PVC.

7. The production method as claimed in claim 1, characterized in that, The K + The ion-selective membrane solution consisted of 2 wt% valinomycin, 0.6 wt% sodium tetraphenylborate, 64.7 wt% dioctyl sebacate, and 32.7 wt% PVC.

8. The production method as claimed in claim 1, characterized in that, The Na + The ion-selective membrane solution consisted of 1 wt% of the sodium ionophore X, 0.55 wt% of sodium tetra(3,5-bis(trifluoromethyl)phenyl)borate, 65.45 wt% of dioctyl sebacate, and 33 wt% of PVC.

9. A multi-ion real-time monitoring sensor, characterized by, The multi-ion real-time monitoring sensor is prepared according to the preparation method in any one of claims 1-8.

10. Use of the multi-ion real-time monitoring sensor as defined in claim 9 in the field of ion detection, characterized in that, In the application, the liquid to be detected is poured into the hollow ring body, and then an external electrochemical workstation is used for ion detection.

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