WO2021163964A1 - 导电水凝胶的制备方法及其细胞阻抗传感检测方法 - Google Patents

导电水凝胶的制备方法及其细胞阻抗传感检测方法 Download PDF

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WO2021163964A1
WO2021163964A1 PCT/CN2020/076048 CN2020076048W WO2021163964A1 WO 2021163964 A1 WO2021163964 A1 WO 2021163964A1 CN 2020076048 W CN2020076048 W CN 2020076048W WO 2021163964 A1 WO2021163964 A1 WO 2021163964A1
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conductive
hydrogel
forming
cell
chip
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French (fr)
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黄建永
姜楠
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R27/00—Arrangements for measuring resistance, reactance, impedance, or electric characteristics derived therefrom
    • G01R27/02—Measuring real or complex resistance, reactance, impedance, or other two-pole characteristics derived therefrom, e.g. time constant

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  • the present disclosure belongs to the technical field of cell impedance sensing and detection, and in particular relates to a method for preparing a micro-pattern conductive hydrogel and a cell impedance sensing and detection method.
  • ECIS Electro Cell Impedance Sensing
  • NFEIS non-Faradic Electric Impedance Sensing
  • FEIS Faradic Electric Impedance Sensing
  • NFEIS does not use any redox probes and is called a probeless cell impedance sensor.
  • FEIS uses redox probes, based on the principle of surface interface probe molecule transfer, to construct highly sensitive sensors.
  • the impedance value is mainly composed of the impedance of the electrode/solution interface and the impedance of the solution itself.
  • the cell adheres to the base electrode, due to the integrity of the cell membrane, the cell can grow and proliferate on the surface of the electrode as a conductor with poor conductivity.
  • the ionic environment of the local solution at the electrode/solution interface changes, which directly leads to an increase in impedance and adhesion.
  • the traditional substrate electrode is mainly a metal electrode, and the metal electrode will cause certain detection limitations, such as the continuous foreign body reaction due to mechanical mismatch and the limitation of electrochemical performance, which hinders the high efficiency and stability of the cell impedance sensing method. Charge transfer.
  • One aspect of the present disclosure provides a method for preparing a conductive hydrogel, including: forming a conductive chip with a microelectrode array; forming a cell culture cavity by the conductive chip; and forming a polymerized hydrogel on the microelectrode array of the cell culture cavity ; And the formation of a conductive hydrogel with micro-patterns by polymerizing the hydrogel.
  • forming a conductive chip with a microelectrode array includes: forming a photoresist layer on the conductive film layer of the conductive substrate; forming a photoresist pattern on the conductive substrate with the photoresist layer; and A conductive chip with a conductive film pattern is formed on a conductive substrate based on a photoresist pattern, wherein the conductive film pattern is a microelectrode array.
  • forming a cell culture cavity by a conductive chip includes: forming a ring structure surrounding a microelectrode array on the conductive chip, wherein the inner surface of the ring structure is formed between the surface of the conductive chip where the microelectrode array is located Cell culture chamber.
  • forming a polymerized hydrogel on the microelectrode array of the cell culture chamber includes: mixing acrylamide (AM), dialdehyde starch (DAS), anionic dopant, and crosslinking agent according to a preset ratio.
  • the linking agent, the initiator and the catalyst are mixed to form a mixed hydrogel; and the mixed hydrogel is swelled to form a polymerized hydrogel.
  • the mass of the acrylamide (AM) solution accounts for 23%-46%; the mass of the dialdehyde starch (DAS) solution accounts for 12%-35%; and the anion
  • the mass of the dopant accounts for 5%-10%; the mass of the crosslinking agent solution accounts for 10%-13%; the mass of the initiator solution accounts for 23%-25%; and the mass of the catalyst accounts for 2% -3%.
  • the concentration of the acrylamide solution is 0.4 g/mL; the concentration of the dialdehyde starch solution is 0.4 g/mL; the concentration of the crosslinking agent solution is 0.02 g/mL; and the initiator solution The concentration is 0.1g/mL.
  • forming a conductive hydrogel with a micro-pattern through a polymerized hydrogel includes: forming a conductive hydrogel on a conductive chip based on the polymerized hydrogel by an electrochemical deposition method, wherein the micro-pattern and the micro-pattern are The pattern of the electrode array is correspondingly matched, and the conductive hydrogel has an integrated interpenetrating network (IPN) structure.
  • IPN integrated interpenetrating network
  • forming a conductive hydrogel based on a polymerized hydrogel on a conductive chip by an electrochemical deposition method includes: swelling the polymerized hydrogel with a 3,4-ethylenedioxythiophene (EDOT) solution; And a conductive chip with a polymerized hydrogel that has been swollen with a 3,4-ethylenedioxythiophene (EDOT) solution is used as a working electrode for electrodeposition to form a conductive hydrogel.
  • EDOT 3,4-ethylenedioxythiophene
  • forming a conductive hydrogel on a conductive chip based on a polymer hydrogel by an electrochemical deposition method includes: the electrochemical deposition method uses an EDOT solution as an electrolyte.
  • Another aspect of the present disclosure provides a cell impedance sensing detection method, which includes: seeding a cell to be tested on a surface with the above-mentioned conductive hydrogel; The cell impedance detection is performed by electrical connection; and the cell impedance information of the cell to be detected is obtained according to the cell impedance detection.
  • performing cell impedance detection includes: performing a frequency sweep measurement on a conductive chip with a conductive hydrogel of the cell to be detected with an output voltage of 5mV-10mV and an output frequency of 400Hz-50MHz to determine the detection frequency value.
  • inoculating the cells to be tested on the surface of the conductive hydrogel includes: performing incubation pretreatment on the surface of the conductive hydrogel; and inoculating the surface of the conductive hydrogel after the incubation pretreatment to be tested. Detect cells.
  • Fig. 1 is a schematic flow chart of a method for preparing a conductive hydrogel according to an embodiment of the present disclosure
  • FIG. 2 is a schematic flow chart of a method for manufacturing a conductive chip with a microelectrode array according to an embodiment of the present disclosure
  • Fig. 3 is a physical diagram of a conductive hydrogel according to an embodiment of the present disclosure.
  • FIG. 4 is a schematic flowchart of a cell impedance sensing detection method according to an embodiment of the present disclosure
  • FIG. 5 is a schematic diagram of the composition of a cell impedance sensing detection system according to an embodiment of the present disclosure
  • Fig. 6 is a frequency sweep measurement impedance diagram of cell impedance sensing detection according to an embodiment of the present disclosure.
  • the present disclosure provides a preparation of conductive hydrogel Method and its cell impedance sensing detection method.
  • hydrogel can absorb a large amount of water while maintaining its dimensional stability, and maintain the 3D structural integrity of the swollen hydrogel through physical or chemical crosslinking.
  • the chemical cross-linked network has permanent knots, while the physical cross-linked network has transient knots caused by polymer chain entanglement or physical interactions (such as ionic interactions, hydrogen bonds, or hydrophobic interactions).
  • Hydrogels containing "sensor” properties can undergo a reversible volume phase change or a gel-sol phase change when the environmental conditions change slightly. These stimulus-responsive types of hydrogels are also called “smart" hydrogels.
  • Various responses of smart hydrogel systems can be induced by applying many physical and chemical stimuli. Physical stimulation includes temperature, electric field, solvent composition, light, pressure, sound and magnetic field; chemical or biochemical stimulation includes pH, ion and specific molecular recognition effects.
  • Conductive Hydrogels have tissue-like flexibility and provide a new way for bioelectronics to design new technological interfaces with the human body, such as wearable and implantable devices, biosensors, Biological brakes, health record electrodes, medical patches, etc.
  • Conductive hydrogels also solve the limitation of the ability to integrate biologically active molecules caused by conducting polymers (CPs). By synthesizing a composite of conductive polymers (CPs) and hydrogels, the degradation of mechanical properties and electrochemical stability can be overcome.
  • conductive polymers can be electrochemically deposited in a pre-formed hydrogel matrix, or the hydrogel network can be doped with conductive polymers (CPs) for chemical polymerization or two components In order to seamlessly form an integrated Interpenetrating Network (IPN) structure of two different polymer components.
  • IPN Interpenetrating Network
  • One aspect of the present disclosure provides a method for preparing a conductive hydrogel, as shown in FIG. 1, including the following operations:
  • S140 Forming a conductive hydrogel with micro-patterns by polymerizing the hydrogel.
  • conductive polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be used for biosensors and artificial nerve electrodes, etc., in contrast to conventional Compared with metals (such as gold and platinum), conductive polymers (CPs) have high charge transfer capabilities and excellent electrical properties.
  • CPs conductive polymers
  • the limited mechanical stability of conductive polymers (CPs) hinders efficient and stable charge transfer.
  • the present disclosure may adopt composite polymer system materials, especially composite materials of conductive polymers (CPs) and hydrogels.
  • Composite conductive polymer (CPs) hydrogels or conductive hydrogels (CHs) can promote the formation of more stable electrode materials, while having the advantages of two polymer components.
  • conductive polymers (CPs) can be deposited into a pre-formed hydrogel matrix by electrochemical deposition methods and other methods to prepare hydrogels (CHs) with an IPN structure, that is, the aforementioned conductive hydrogels.
  • the formation of the IPN structure can give full play to the advantages of both conductive polymers (CPs) and hydrogels.
  • the method for preparing the conductive hydrogel provided by the present disclosure has a micro-pattern structure to facilitate its application in the cell impedance sensing detection method.
  • the present disclosure can prepare micro-patterned conductive hydrogel by electrochemical deposition on the micro-electrode array conductive chip, and can use it as a cell impedance sensor chip in combination with an impedance meter detection system to establish real-time, label-free detection of cell impedance method.
  • acrylamide (Acrylamide, AM for short), Dialdehyde Starch (DAS), sodium styrene sulfonate (Sodium p- Styrenesulfonate, referred to as SSNa) is the AM-co-DAS-co-SSNa hydrogel prepared by in-situ addition polymerization of monomers, and then the hydrogel-coated conductive chip is used as the working electrode in a three-electrode electrolytic cell. Depositing poly(3,4-ethylenedioxythiophene) (PEDOT) to prepare an interpenetrating network (IPN) structure.
  • PEDOT poly(3,4-ethylenedioxythiophene)
  • the preparation method of the conductive hydrogel proposed in the present disclosure is simple in process, easy to operate, does not require expensive preparation equipment, and is easy to popularize.
  • the soft electrode prepared based on the PEDOT/PSS conductive hydrogel can perform real-time, label-free detection of cell impedance , And then evaluate the physiological and pathological state of cells, and overcome the limitations of metal electrode mechanical mismatch and electrochemical performance.
  • the conductive hydrogel can tailor-made covalently fixed doping groups in the hydrogel matrix, the soft hydrogel electrode can lay a foundation for a new field of living cell biomimetic systems.
  • forming a conductive chip with a microelectrode array includes: forming a photoresist layer on the conductive film layer of the conductive substrate; forming a photoresist pattern on the conductive substrate with the photoresist layer; And forming a conductive chip with a conductive film pattern on the conductive substrate based on the photoresist pattern, wherein the conductive film pattern is a microelectrode array.
  • the conductive chip with the microelectrode array can be prepared by using ITO conductive glass as the substrate of the conductive chip. Specifically, the conductive chip can also select other substrates with a conductive film structure. .
  • the formation of a conductive chip with a microelectrode array specifically, as shown in FIG. 2, it can be formed according to the following preparation process:
  • Pre-baking Set the above-mentioned ITO conductive glass with a photoresist layer on a heating plate, and bake it at a temperature of 110° C. for 90 s to preliminarily cure the photoresist layer.
  • Exposure Expose the ITO conductive glass on which the photoresist layer has been cured to ultraviolet light (UV) for 30 seconds through a printed mask using a mask aligner.
  • the electrode used for the mask is an interdigital electrode with a size of 1 cm in length, 30 to 100 ⁇ m in width, and a distance between adjacent electrodes of 30 to 100 ⁇ m;
  • the exposure machine used is: ultraviolet depth lithography machine, and the exposure ultraviolet light intensity is: 15mW/cm 2.
  • the conductive film pattern is a microelectrode array.
  • Degluing Use a photoresist degluing solution to remove the remaining photoresist for 5 minutes, rinse with deionized water and blow dry with nitrogen to prepare a conductive hydrogel for the next step.
  • forming a cell culture cavity by a conductive chip includes: forming a ring structure surrounding a microelectrode array on the conductive chip, wherein the inner surface of the ring structure is formed between the surface of the conductive chip where the microelectrode array is located Cell culture chamber.
  • a quartz glass ring is selected as the annular structure of the present disclosure, and the microelectrode array surrounding the conductive chip is bonded to the conductive chip to form a preliminary cell culture cavity.
  • the size of the quartz glass ring is 27mm in inner diameter, 30mm in outer diameter, 1.5mm in wall thickness, and 10mm in depth.
  • APTES 3-AminopropylTriethoxy Silane
  • forming a polymerized hydrogel on the microelectrode array of the cell culture chamber includes: mixing acrylamide (AM), dialdehyde starch (DAS), anionic dopant, and crosslinking agent according to a preset ratio.
  • the linking agent, the initiator and the catalyst are mixed to form a mixed hydrogel; and the mixed hydrogel is swelled to form a polymerized hydrogel.
  • the so-called polymeric hydrogel may be an acrylamide-graft-dialdehyde starch-graft-sodium styrene sulfonate (AM -co-DAS-co-SSNa) coating hydrogel, which can be specifically formed by in-situ addition polymerization.
  • AM -co-DAS-co-SSNa acrylamide-graft-dialdehyde starch-graft-sodium styrene sulfonate
  • Table 1 The formulation and composition of AM-co-DAS-co-SSNa coated hydrogel
  • the preparation of AM-co-DAS-co-SSNa coated hydrogels with different proportions can be carried out respectively.
  • SSNa styrene sulfonate
  • AM monomer acrylamide
  • dialdehyde starch a concentration of 0.4g/mL dialdehyde starch
  • DAS concentration of 0.02g/mL N,N'-methylenebisacrylamide
  • TEMED tetramethylethylenediamine
  • concentration of 0.1g/mL ammonium persulfate (APS) solution Mix them evenly to get a mixed hydrogel.
  • sodium styrene sulfonate can be used as an anionic dopant to provide anion doping
  • N,N'-methylene bisacrylamide (BIS) can be used as a cross-linking agent to provide cross-linking
  • ammonium persulfate APS
  • TEMED tetramethylethylenediamine
  • the above-mentioned AM-co-DAS-co-SSNa polymerized hydrogel formed by in-situ addition polymerization is calculated as 100% by the sum of the mass percentages, and the mass percentage of each raw material can be: :
  • the mass proportion of the acrylamide (AM) solution can be 23%-46%;
  • the mass proportion of the dialdehyde starch (DAS) solution can be 12%-35%;
  • the mass proportion of the anionic dopant can be 5%-10%, for example, the mass ratio of styrene sulfonate (SSNa) can be 5%-10%;
  • the mass ratio of the crosslinking agent solution can be 10%-13%, for example, N,N'-
  • the mass proportion of methylene bisacrylamide (BIS) solution can be 10%-13%;
  • the mass proportion of initiator solution can be 23%-25%, for example, the mass proportion of ammonium persulfate (APS) solution It can be 23%-25%; and the mass ratio
  • the concentration of the acrylamide solution weighed or prepared may be 0.4 g/mL; the concentration of the dialdehyde starch solution may be 0.4 g/mL;
  • the concentration of the solution of the coupling agent can be 0.02g/mL, the concentration of the N,N'-methylenebisacrylamide (BIS) solution can be 0.02g/mL; and the concentration of the initiator solution can be 0.1g/mL ,
  • the concentration of ammonium persulfate (APS) solution can be 0.1g/mL.
  • forming a conductive hydrogel with a micro-pattern through a polymerized hydrogel includes: forming a conductive hydrogel on a conductive chip based on the polymerized hydrogel by an electrochemical deposition method, wherein the micro-pattern and the micro-pattern are formed on a conductive chip.
  • the pattern of the electrode array is correspondingly matched, and the conductive hydrogel has an interpenetrating network structure (IPN structure).
  • IPN structure interpenetrating network structure
  • the above-mentioned polymerized hydrogel can be electrochemically deposited based on an electrochemical deposition method to seamlessly integrate them to form PEDOT and AM-co-DAS-co -Interpenetrating network (IPN) structure of SSNa polymeric hydrogel.
  • IPN Interpenetrating network
  • forming a conductive hydrogel based on a polymerized hydrogel on a conductive chip by an electrochemical deposition method includes: swelling the polymerized hydrogel with a 3,4-ethylenedioxythiophene (EDOT) solution; And a conductive chip with a polymerized hydrogel that has been swollen with a 3,4-ethylenedioxythiophene (EDOT) solution is used as a working electrode for electrodeposition to form a conductive hydrogel.
  • EDOT 3,4-ethylenedioxythiophene
  • the AM-co-DAS-co-SSNa polymeric hydrogel coating sample was immersed in a 3,4-ethylenedioxythiophene (EDOT) solution (EDOT ⁇ 97%) for about 1 hour, In order to swell the polymerized hydrogel and make the EDOT monomer reach equilibrium in the entire polymerized hydrogel.
  • the independent Ag/AgCl electrode is used as the reference electrode
  • the platinum electrode is used as the counter electrode
  • the AM-co-DAS-co-SSNa polymer hydrogel coating conductive chip is used as the working electrode to construct a three-electrode system.
  • a conductive hydrogel with micropatterns is formed on a conductive chip (ITO conductive glass).
  • forming a conductive hydrogel on a conductive chip based on a polymer hydrogel by an electrochemical deposition method includes: the electrochemical deposition method uses an EDOT solution as an electrolyte.
  • EDOT solution poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (Poly(2,3-dihydrothieno-1,4-dioxin)-poly(styrenesulfonate), referred to as PEDOT/PSS)
  • PEDOT/PSS poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid)
  • PEDOT/PSS Poly(2,3-dihydrothieno-1,4-dioxin)-poly(styrenesulfonate
  • PEDOT/PSS poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid)
  • PEDOT/PSS Poly(2,3-d
  • the selection parameters of the above-mentioned constituent materials can make it possible to fully deposit EDOT in the polymerized hydrogel and avoid excessive oxidation of the material. Since the counter ion PSS is provided by the hydrogel, no additional supporting electrolyte is required.
  • the completed sample is washed in a phosphate (Dulbecco's Phosphate-Buffered Saline, DPBS) buffer solution, and unreacted EDOT is extracted, that is, PEDOT and AM-co-DAS-co-SSNa with micro-patterns can be obtained on the conductive chip
  • DPBS Dulbecco's Phosphate-Buffered Saline
  • the above-mentioned AM-co-DAS-co-SSNa conductive hydrogel with the micropattern of the IPN structure was prepared according to the material component ratio of the ratio described in Table 1-a.
  • Figure 3-A is a real photo of the micro-patterned conductive hydrogel coated ITO glass chip
  • Figure 3-B is a partial magnification of the micro-patterned conductive hydrogel under a fluorescent inverted microscope IX71 picture.
  • the light-colored band is AM-co-DAS-co-SSNa polymerized hydrogel
  • the black band is the conductive polymer PEDOT and AM-co-DAS-co-SSNa polymerized hydrogel.
  • the IPN structure of the interpenetrating network Therefore, we can see the successful electrodeposition of PEDOT in the conductive hydrogel IPN network.
  • the conductive chip with the micro-patterned IPN structure conductive hydrogel prepared by the electrochemical deposition process can be applied to bioelectrodes in the field of cell impedance sensing and detection.
  • Bioelectrodes are an important part of many neuroprosthetic devices to transfer electric charges and record cell activities.
  • Traditional electrodes are usually metal electrodes (such as platinum, platinum alloy or gold).
  • the limitations of metal electrodes include mechanical mismatch leading to continuous foreign body reactions and limitations in electrochemical performance that hinder efficient and stable charge transfer.
  • conductive polymers (CPs) have better impedance characteristics.
  • the present disclosure prepares an IPN network of conductive polymers (CPs) and polymerized hydrogels by electrodeposition, and provides a softer mechanical interface through the surface of the hydrogels.
  • the conductive hydrogel in the IPN structure not only plays a role in regulating and improving the mechanical properties of the IPN structure, but also provides a low-polluting surface and long-term storage for water-soluble bioactive agents.
  • the present disclosure provides the preparation method of the conductive hydrogel, and the preparation method of the conductive hydrogel.
  • the present disclosure prepares a microstructure with an integrated interpenetrating network structure (IPN structure) on a conductive chip of a microelectrode array by an electrochemical deposition method.
  • IPN structure integrated interpenetrating network structure
  • Patterned soft conductive hydrogel which can effectively replace metal electrodes in the field of cell impedance sensing technology, combined with the impedance meter online detection system, effectively realizes efficient and stable charge in the process of cell impedance sensing and detection Transfer, making cell impedance detection achieve the technical effect of label-free, full-process dynamic, and real-time analysis.
  • FIG. 4 Another aspect of the present disclosure provides a cell impedance sensing detection method, as shown in FIG. 4, including:
  • S220 Electrically connect the conductive chip of the conductive hydrogel inoculated with the cells to be detected with the impedance meter to perform cell impedance detection;
  • a cell impedance sensor detection system is constructed to conduct electricity on the conductive chip.
  • Cells seeded on the hydrogel can be detected in real time and without labels.
  • the cells to be tested are seeded on the conductive hydrogel with the micro-pattern design and the IPN structure of the conductive chip, and the cell culture chamber formed by the conductive chip is placed in an incubator at 37° C. and 5% CO 2 for culture.
  • the impedance analyzer is connected to the monitoring electrode (ie, the cell impedance sensor chip) where the cell to be detected is located through a wire through a metal clip, to measure the impedance value of the cell to be detected.
  • the monitoring electrode ie, the cell impedance sensor chip
  • an impedance meter 520 is electrically connected to a lead electrode of the monitoring electrode.
  • a current amplifier 530 electrically connected to the other lead electrode of the monitoring electrode.
  • the current amplifier 530 is also electrically connected to the impedance meter 520 to construct a cell impedance sensing detection system, and the impedance meter 520 of the system is connected to the computer system 540 , To realize the online combined analysis of the impedance information data of the cells to be tested.
  • the micro-patterned conductive hydrogel of the present disclosure can complete its application in cell impedance sensing.
  • the micro-patterned conductive hydrogel-coated ITO conductive glass chip and the impedance meter are used to construct an online combined system, which can detect various adherent cells, including cell proliferation detection, cell toxicity detection of various compounds, and cell Co-culture detection, cell adhesion and extension detection, receptor-mediated signal pathway detection, etc.
  • the dynamic monitoring of the combined system ensures the instantaneous response of cells and the acquisition of long-term effects, and can be extended to the research fields of cell therapy killing quality control, environmental toxicology, food toxicology evaluation, etc.
  • performing cell impedance detection includes: performing a frequency sweep measurement on a conductive chip with a conductive hydrogel of the cell to be detected with an output voltage of 5mV-10mV and an output frequency of 400Hz-50MHz to determine the detection frequency value.
  • the voltage applied during the cell impedance measurement can output range: 5-10mV; use the sweep frequency measurement in the range of 400Hz-50MHz, according to Select the appropriate detection frequency value for the measurement result.
  • the real-time impedance information of the cells to be detected collected from the corresponding detection frequency values is further analyzed and processed by MATLAB.
  • impedance sweep frequency measurement was performed on the conductive chip seeded with 40 ⁇ 10 4 cells/mL MDCK cells in a 37°C 5% CO 2 cell incubator for 24 hours.
  • the frequency is swept in the range of 10-40MHz, and the applied voltage output is 5mV.
  • Figure 6-A is the real part of the cell impedance
  • Figure 6-B is the imaginary part of the cell impedance. It can be seen from Figure 6-A and Figure 6-B that when the applied voltage is constant, the impedance of the cell is different at different frequencies. It is necessary to select a certain frequency range for sweep frequency measurement, and select the appropriate detection frequency value according to the measurement result.
  • inoculating the cells to be tested on the surface of the conductive hydrogel includes: performing incubation pretreatment on the surface of the conductive hydrogel; and inoculating the surface of the conductive hydrogel after the incubation pretreatment to be tested.
  • the chip culture chamber needs to be sterilized by UV irradiation for 30min-1h before cell inoculation.
  • the micro-patterned conductive hydrogel in the cell culture chamber where the conductive chip is located needs to be soaked in DPBS buffer, and the soaking time is up to one week or more, and During this period, the DPBS buffer needs to be replaced every day to fully remove substances that are toxic to cell culture in the conductive hydrogel.
  • the hydrogel component of the conductive hydrogel of the present disclosure provides a matrix that can be combined with brittle conductive polymers.
  • conductive hydrogels have been shown to have improved mechanical properties and biocompatibility, and have improved Electric activity.
  • the increase in conductivity of conductive polymers is attributed to the three-dimensional structure (3D), which enables charge transfer to occur over a larger area.
  • 3D three-dimensional structure
  • ITO conductive glass chip as the conductive chip can make it have good cell compatibility when used for real-time sensing and detection of cell impedance.
  • the present disclosure provides a conductive hydrogel conductive chip with micropatterns and a cell impedance sensing detection method thereof. Specifically, the present disclosure prepares the AM-co-DAS-co-SSNa polymer hydrogel coating by in-situ addition reaction, and further uses the conductive chip of the polymer hydrogel coating microelectrode array as the working electrode, The electrochemical deposition process is used to prepare PEDOT into the above-mentioned polymerized hydrogel to form a conductive hydrogel with an integrated interpenetrating network (IPN) structure, and the conductive chip with the above-mentioned conductive hydrogel and an impedance meter are used to construct a cell impedance sensing detection system Combined with the online analysis system, a new method for real-time, label-free, and dynamic detection of cell impedance information data is realized.
  • IPN integrated interpenetrating network
  • this soft conductive hydrogel electrode not only It solves the limitations of metal electrodes, such as the continuous foreign body reaction caused by mechanical mismatch and the limitations of electrochemical performance, which hinder efficient and stable charge transfer, and evaluate the cell by measuring the real-time impedance dynamic change between the cell and the monitoring electrode Physiological and pathological conditions.
  • the microelectrode array conductive chip with micro-patterned conductive hydrogel coating disclosed in the present disclosure not only has important significance for laboratory and clinical drug toxicity detection, but also has important commercial promotion value, which is expected to be used in new drug development and drug development. Safety evaluation and secondary development play a specific role to produce good social and economic value.

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Abstract

本公开提供了一种导电水凝胶的制备方法及其细胞阻抗传感检测方法，该导电水凝胶的制备方法包括：形成具有微电极阵列的导电芯片；通过导电芯片形成细胞培养腔；在细胞培养腔的微电极阵列上形成聚合水凝胶；以及通过聚合水凝胶形成具有微图案的导电水凝胶。本公开通过电化学沉积法在微电极阵列导电芯片上制备了具有集成互穿网络结构(IPN结构)的微图案的软质导电水凝胶，该导电水凝胶可以有效的替代细胞阻抗传感技术领域中的金属电极，结合阻抗仪在线检测系统，有效实现了细胞阻抗传感检测过程中高效稳定的电荷转移，使得细胞阻抗检测达到了无标记、全程动态、实时分析的技术效果。

Description

导电水凝胶的制备方法及其细胞阻抗传感检测方法 技术领域
本公开属于细胞阻抗传感检测技术领域，具体涉及一种微图案导电水凝胶的制备方法及其细胞阻抗传感检测方法。
背景技术
细胞阻抗传感(Electric Cell Impedance Sensing，ECIS)是一种基于交流阻抗技术的细胞分析方法，其原理是通过测量细胞与基底电极之间的阻抗值变化来评估细胞的生理和病理状态，能够实时监测细胞的生长、增殖、毒性、粘附及形态变化等动态生物学过程。作为一种无标记、全程动态、实时的分析方法，ECIS在细胞研究中得到了广泛的应用。在该领域中，用于细胞分析的交流阻抗方法大致可分为两类：非法拉第阻抗谱法(non-Faradic Electric Impedance Sensing，NFEIS)和法拉第阻抗谱法(Faradic Electric Impedance Sensing，FEIS)。NFEIS没有使用任何氧化还原探针，称为无探针细胞阻抗传感器。FEIS则运用氧化还原探针，基于表界面探针分子转移的原理，以用于构筑灵敏度高的传感器。当没有细胞粘附于基底电极时，阻抗值主要由电极/溶液界面的阻抗和溶液本身的阻抗两部分组成。当细胞粘附于基底电极时，由于细胞膜的完整性，细胞可作为导电性差的导体在电极表面生长和增殖，电极/溶液界面局部溶液的离子环境发生改变，直接导致阻抗值升高，粘附的细胞越多，阻抗值越大；细胞发生多种生理和病理状态变化均可通过阻抗值的变化来反应。但是传统的基底电极主要为金属电极，而金属电极会造成一定的检测局限性，例如因机械失配导致持续的异物反应和电化学性能的局限性，因此阻碍了细胞阻抗传感方法中高效稳定的电荷转移。
发明内容
本公开的一个方面提供了一种导电水凝胶的制备方法，包括：形成具有微电极阵列的导电芯片；通过导电芯片形成细胞培养腔；在细胞培养腔的微电极阵列上形成聚合水凝胶；以及通过聚合水凝胶形成具有微图案的 导电水凝胶。
根据本公开的实施例，形成具有微电极阵列的导电芯片包括：在导电衬底的导电膜层上形成光刻胶层；在具有光刻胶层的导电衬底上形成光刻胶图案；以及基于光刻胶图案在导电衬底上形成具有导电膜图案的导电芯片，其中，导电膜图案为微电极阵列。
根据本公开的实施例，通过导电芯片形成细胞培养腔，包括：在导电芯片上形成围设微电极阵列的环形结构，其中，环形结构的内表面和微电极阵列所在的导电芯片表面之间形成细胞培养腔。
根据本公开的实施例，在细胞培养腔的微电极阵列上形成聚合水凝胶，包括：依据预设配比，将丙烯酰胺(AM)、双醛淀粉(DAS)、阴离子掺杂剂、交联剂、引发剂以及催化剂进行混合形成混合水凝胶；以及对混合水凝胶进行溶胀形成聚合水凝胶。
根据本公开的实施例，在聚合水凝胶中，丙烯酰胺(AM)的溶液的质量占比23％-46％；双醛淀粉(DAS)的溶液的质量占比12％-35％；阴离子掺杂剂的质量占比5％-10％；交联剂的溶液的质量占比10％-13％；引发剂的溶液的质量占比23％-25％；以及催化剂的质量占比2％-3％。
根据本公开的实施例，丙烯酰胺的溶液的浓度为0.4g/mL；双醛淀粉的溶液的浓度为0.4g/mL；交联剂的溶液的浓度为0.02g/mL；以及引发剂的溶液的浓度为0.1g/mL。
根据本公开的实施例，通过聚合水凝胶形成具有微图案的导电水凝胶，包括：通过电化学沉积法基于聚合水凝胶在导电芯片上形成导电水凝胶，其中，微图案与微电极阵列的图形对应匹配，导电水凝胶具备集成互穿网络(Interpenetrating Network，简称IPN)结构。
根据本公开的实施例，通过电化学沉积法基于聚合水凝胶在导电芯片上形成导电水凝胶，包括：对聚合水凝胶进行3,4-乙烯二氧噻吩(EDOT)溶液的溶胀；以及将具有已进行溶胀3,4-乙烯二氧噻吩(EDOT)溶液的聚合水凝胶的导电芯片作为工作电极进行电沉积以形成导电水凝胶。
根据本公开的实施例，通过电化学沉积法基于聚合水凝胶在导电芯片上形成导电水凝胶，包括：电化学沉积法以EDOT溶液作为电解质。
本公开的另一个方面提供了一种细胞阻抗传感检测方法，包括：在具 有上述的导电水凝胶的表面接种待检测细胞；将接种待检测细胞的导电水凝胶的导电芯片与阻抗仪电连接进行细胞阻抗检测；以及根据细胞阻抗检测获取待检测细胞的细胞阻抗信息。
根据本公开的实施例，进行细胞阻抗检测，包括：以输出电压为5mV-10mV、输出频率为400Hz～50MHz对具有待检测细胞的导电水凝胶的导电芯片进行扫频测量，以确定检测频率值。
根据本公开的实施例，在导电水凝胶的表面接种待检测细胞，包括：在导电水凝胶的表面进行孵育预处理；以及在进行了孵育预处理后的导电水凝胶的表面接种待检测细胞。
附图说明
图1为根据本公开实施例中导电水凝胶的制备方法的流程示意图；
图2为根据本公开实施例中具有微电极阵列的导电芯片的制备方法的流程示意图；
图3为根据本公开实施例中导电水凝胶的实物图；
图4为根据本公开实施例中细胞阻抗传感检测方法的流程示意图；
图5为根据本公开实施例中细胞阻抗传感检测系统的组成示意图；
图6为根据本公开实施例中细胞阻抗传感检测的扫频测量阻抗图。
具体实施方式
为使本公开的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本公开进一步详细说明。
为解决细胞阻抗传感检测技术领域中，金属电极因检测局限性造成检测过程中电荷转移不能高效稳定，进而影响细胞阻抗传感检测的技术问题，本公开提供了一种导电水凝胶的制备方法及其细胞阻抗传感检测方法。
水凝胶作为一种三维(3D)材料，能够在保持其尺寸稳定性的同时吸收大量的水，通过物理或化学交联维持溶胀状态的水凝胶的3D结构完整性。化学交联网络具有永久性结，而物理交联网络具有由聚合物链缠结或物理相互作用(例如离子相互作用，氢键或疏水相互作用)引起的瞬时结。含有“传感器”特性的水凝胶可以在环境条件微小变化时经历可逆体积相 变或凝胶-溶胶相变。这些具有刺激响应性类型的水凝胶也被称为“智能”水凝胶。智能水凝胶系统的各种响应可以应用许多物理和化学刺激来诱导。物理刺激包括温度、电场、溶剂成分、光、压力、声音和磁场；化学或生化刺激包括pH，离子和特定的分子识别效应。
导电水凝胶(Conductive Hydrogels，简称CHs)具有类似组织的柔软性，为设计与人体之间的新技术接口的生物电子学提供了一条新途径，例如可穿戴和可植入设备、生物传感器、生物制动器、健康记录电极和医用贴片等。导电水凝胶(CHs)还解决了由导电聚合物(Conducting Polymers，简称CPs)带来的生物活性分子整合能力的局限性。通过合成导电聚合物(CPs)和水凝胶的复合材料，可以克服机械性能和电化学稳定性的下降。
制备导电水凝胶可以将导电聚合物(CPs)电化学沉积在预先形成的水凝胶基质中，还可以在水凝胶网络中掺杂导电聚合物(CPs)进行化学聚合或两种组分的同时聚合，以无缝形成两个不同聚合物组件的集成互穿网络(Interpenetrating Network，简称IPN)结构。IPN结构的形成对于充分实现由导电聚合物(CPs)和水凝胶组成的复合材料的好处至关重要。
本公开的一个方面提供了一种导电水凝胶的制备方法，如图1所示，包括如下操作：
S110：形成具有微电极阵列的导电芯片；
S120：通过导电芯片形成细胞培养腔；
S130：在细胞培养腔的微电极阵列上形成聚合水凝胶；以及
S140：通过聚合水凝胶形成具有微图案的导电水凝胶。
作为本公开的实施例，导电聚合物(CPs)例如聚3,4-乙烯二氧噻吩(Poly(3,4-ethylenedioxythiophene)，简称PEDOT)可以被用于生物传感器和人工神经电极等，与常规金属(例如金和铂)相比，导电聚合物(CPs)具有高电荷转移能力，具有优异的电性能。但是，由于导电聚合物(CPs)有限的机械稳定性，阻碍了高效稳定的电荷转移。为了解决导电聚合物(CPs)的该局限性，本公开可以采用复合聚合物体系材料，尤其是导电聚合物(CPs)和水凝胶的复合材料。复合导电聚合物(CPs)水凝胶或导电水凝胶(CHs)可以促进更稳定的电极材料的形成，同时具有两种聚合物成分的优势。具体地，本公开可以采用电化学沉积法等方式将导电聚合物 (CPs)沉积到预先形成的水凝胶基质中制备具备IPN结构的水凝胶(CHs)，即上述的导电水凝胶。IPN结构的形成可以充分发挥导电聚合物(CPs)和水凝胶两者的优势。
本公开提供的导电水凝胶的制备方法，其制备得到的导电水凝胶具有微图案结构，以助于其细胞阻抗传感检测方法中的应用。本公开可以在微电极阵列导电芯片上以电化学沉积方式制备微图案导电水凝胶，并可以将其作为细胞阻抗传感芯片与阻抗仪检测系统在线联用，建立细胞阻抗实时、无标记检测方法。在本公开的实施例中，在细胞阻抗传感芯片制备过程中，首先可以以丙烯酰胺(Acrylamide，简称AM)、双醛淀粉(Dialdehyde Starch，简称DAS)、苯乙烯磺酸钠(Sodium p-styrenesulfonate，简称SSNa)为单体通过原位加成聚合反应制备得AM-co-DAS-co-SSNa水凝胶，再在三电极电解池中以水凝胶涂层导电芯片作为工作电极电化学沉积聚3,4-乙烯二氧噻吩(Poly(3,4-ethylenedioxythiophene)，简称PEDOT)制备互穿网络(IPN)结构。本公开所提出的导电水凝胶的制备方法工艺简单、易于操作、无需昂贵制备仪器，易于推广，基于PEDOT/PSS导电水凝胶所制备的软质电极可以进行细胞阻抗的实时、无标记检测，进而评估细胞的生理和病理状态，克服了金属电极机械失配和电化学性能的局限性。另外，由于该导电水凝胶可以水凝胶基质内量身定制共价固定的掺杂基团，因此，软质水凝胶电极可以为活细胞仿生系统的新领域奠定了基础。
根据本公开的实施例，形成具有微电极阵列的导电芯片，包括：在导电衬底的导电膜层上形成光刻胶层；在具有光刻胶层的导电衬底上形成光刻胶图案；以及基于光刻胶图案在导电衬底上形成具有导电膜图案的导电芯片，其中，导电膜图案为微电极阵列。
作为本公开的实施例，进一步地，关于上述具有微电极阵列的导电芯片可以采用ITO导电玻璃作为该导电芯片的衬底进行制备，具体地，该导电芯片还选择其他具有导电薄膜结构的衬底。另外，关于形成具有微电极阵列的导电芯片，具体地，如图2所示，可以依据如下制备过程形成：
S101、旋涂：选取尺寸为40mm×40mm×0.4mm、导电膜层厚度约为185nm的ITO导电玻璃作为导电芯片的衬底，以低速300rpm 20s、高速1000rpm 30s将紫外负性光刻胶旋涂在ITO导电玻璃的导电膜层上， 即在导电衬底(ITO导电玻璃)的导电膜层上形成光刻胶层，以为下一步形成光刻胶图案做准备。
S102、前烘：将上述具有光刻胶层的ITO导电玻璃设置在加热板上，以温度110℃对其烘烤90s，以将光刻胶层进行初步固化。
S103、曝光：使用掩膜对准器通过印刷的掩膜将进行固化了光刻胶层的ITO导电玻璃暴露于紫外光(UV)下30s。其中，掩膜的使用电极为叉指电极，尺寸为长1cm，宽30～100μm，相邻电极间距30～100μm；所用曝光机为：紫外深度光刻机，曝光紫外光强为：15mW/cm 2，即在具有光刻胶层的导电衬底上形成光刻胶图案。
S104、中烘：将上述进行了曝光操作的具有光刻胶层的ITO导电玻璃设置在加热板上，以温度145℃将曝光后的ITO导电玻璃烘烤60s，以将光刻胶层作进一步固化。
S105、显影：将上述中烘后的ITO导电玻璃浸入光刻胶显影液中显影60s，以除去上述未被曝光的光刻胶层的部分，以露出未被光刻胶层图案保护的ITO导电膜层。
S106、刻蚀：以比例HCl:DDI Water:FeCl 3·6H 2O＝3L:1L:25g配制刻蚀液，其中FeCl 3·6H 2O可以为分析纯，含量≥99.0％。使用上述刻蚀液除去未被光刻胶层保护的ITO导电膜层，用去离子水清洗后氮气吹干，即基于光刻胶图案在导电衬底上形成具有导电膜图案的导电芯片，其中，导电膜图案为微电极阵列。
S107、去胶：使用光刻胶去胶液去除残留的光刻胶5min，用去离子水清洗后氮气吹干以备下一步形成导电水凝胶。
根据本公开的实施例，通过导电芯片形成细胞培养腔，包括：在导电芯片上形成围设微电极阵列的环形结构，其中，环形结构的内表面和微电极阵列所在的导电芯片表面之间形成细胞培养腔。
作为本公开的实施例，进一步地，为更好的在具有微电极阵列的导电芯片表面制备导电水凝胶，同时为形成细胞阻抗传感检测过程中细胞的接种和培养条件，需要在导电芯片的导电面上围绕微电极阵列形成一细胞培养腔。具体地，在制得微电极阵列ITO导电玻璃芯片之后，选择石英玻璃圆环作为本公开的环形结构，围设导电芯片上的微电极阵列粘结在导电芯 片上，形成初步的细胞培养腔。其中，粘结剂选择主剂：硬化剂＝20:1的PDMS粘接剂，石英玻璃圆环的尺寸为内径27mm、外径30mm、壁厚1.5mm、深10mm。
将粘结完毕的细胞培养腔结构放入65℃烘箱烘干，形成最终的细胞培养腔。之后，采用无水甲醇清洗培养腔2次，每次3min。以3-氨基丙基三乙氧基硅烷(3-AminopropylTriethoxySilane，简称APTES)为硅烷偶联剂，配制APTES：丙酮＝3:25溶液对培养腔进行硅烷化处理。具体地，可以在27mm内径的玻璃皿加入360μL上述溶液，不加盖置于通风橱中等待30min彻底晾干。然后，用去离子水清洗培养腔2次，每次3min。再配制0.5％戊二醛PBS溶液对该细胞培养腔进行醛基修饰。最后用去离子水清洗培养腔2次，每次3min，在通风橱中晾干，用于后续聚合水凝胶的原位合成。
根据本公开的实施例，在细胞培养腔的微电极阵列上形成聚合水凝胶，包括：依据预设配比，将丙烯酰胺(AM)、双醛淀粉(DAS)、阴离子掺杂剂、交联剂、引发剂以及催化剂进行混合形成混合水凝胶；以及对混合水凝胶进行溶胀形成聚合水凝胶。
在本公开的实施例中，所谓聚合水凝胶可以是经原位加成聚合反应形成的具有交联网络结构的丙烯酰胺-接枝-双醛淀粉-接枝-苯乙烯磺酸钠(AM-co-DAS-co-SSNa)涂层水凝胶，具体可以通过原位加成聚合形成。
表1 AM-co-DAS-co-SSNa涂层水凝胶的制备配方组成
Figure PCTCN2020076048-appb-000001
依据上述表1，可以分别进行不同配比的AM-co-DAS-co-SSNa涂层水凝胶的制备。具体地，可以按照上述表1中不同序号对应的配比称取苯 乙烯磺酸钠(SSNa)、浓度为0.4g/mL单体丙烯酰胺(AM)溶液、浓度为0.4g/mL双醛淀粉(DAS)溶液、浓度为0.02g/mL N,N’-亚甲基双丙烯酰胺(BIS)溶液、四甲基乙二胺(TEMED)、浓度为0.1g/mL过硫酸铵(APS)溶液，将其均匀混合，即可得到混合水凝胶。其中，苯乙烯磺酸钠(SSNa)可以作为阴离子掺杂剂，提供阴离子掺杂；N,N’-亚甲基双丙烯酰胺(BIS)可以作为交联剂，提供交联作用；过硫酸铵(APS)可以作为该原位加成聚合反应的引发剂；四甲基乙二胺(TEMED)可以作为催化剂，对原位加成聚合反应进行催化。将上述称取的各组成材料或溶液进行混合之后，快速超声振荡30s，用移液枪吸取126μL混合物快速滴于经上述醛基修饰处理的具有微电极阵列的导电芯片上，用直径为20mm的圆形盖玻片压平，在室温下静置，等待成胶10min，即可获得上述混合水凝胶。在成胶后揭掉上述盖玻片，用DPBS溶液对上述混合水凝胶进行充分溶胀即可获得AM-co-DAS-co-SSNa聚合水凝胶。
根据本公开的实施例，上述经原位加成聚合形成的AM-co-DAS-co-SSNa聚合水凝胶，按质量百分数之和为100％计，各原料在其中所占质量百分数可以为：丙烯酰胺(AM)的溶液的质量占比可以为23％-46％；双醛淀粉(DAS)的溶液的质量占比可以为12％-35％；阴离子掺杂剂的质量占比可以为5％-10％，例如苯乙烯磺酸钠(SSNa)的质量占比可以为5％-10％；交联剂的溶液的质量占比可以为10％-13％，例如N,N’-亚甲基双丙烯酰胺(BIS)溶液的质量占比可以为10％-13％；引发剂的溶液的质量占比可以为23％-25％，例如过硫酸铵(APS)溶液的质量占比可以为23％-25％；以及催化剂的质量占比可以为2％-3％，例如四甲基乙二胺(TEMED)的质量占比可以为2％-3％。
根据本公开的实施例，在上述原位加成聚合反应前，称取或配制的丙烯酰胺的溶液的浓度可以为0.4g/mL；双醛淀粉的溶液的浓度可以为0.4g/mL；交联剂的溶液的浓度可以为0.02g/mL，N,N’-亚甲基双丙烯酰胺(BIS)溶液的浓度可以为0.02g/mL；以及引发剂的溶液的浓度可以为0.1g/mL，过硫酸铵(APS)溶液的浓度可以为0.1g/mL。
根据本公开的实施例，通过聚合水凝胶形成具有微图案的导电水凝胶，包括：通过电化学沉积法基于聚合水凝胶在导电芯片上形成导电水凝胶， 其中，微图案与微电极阵列的图形对应匹配，导电水凝胶具备互穿网络结构(IPN结构)。在本公开的实施例中，在制备得到上述聚合水凝胶之后，可以基于电化学沉积法对上述聚合水凝胶进行电化学沉积，使之无缝集成形成PEDOT与AM-co-DAS-co-SSNa聚合水凝胶的互穿网络(IPN)结构。
根据本公开的实施例，通过电化学沉积法基于聚合水凝胶在导电芯片上形成导电水凝胶，包括：对聚合水凝胶进行3,4-乙烯二氧噻吩(EDOT)溶液的溶胀；以及将具有已进行溶胀3,4-乙烯二氧噻吩(EDOT)溶液的聚合水凝胶的导电芯片作为工作电极进行电沉积以形成导电水凝胶。具体地，在电沉积之前，AM-co-DAS-co-SSNa聚合水凝胶涂层样品设置于3,4-乙烯二氧噻吩(EDOT)溶液(EDOT＜97％)中浸泡约1小时，以使聚合水凝胶溶胀并使EDOT单体在整个聚合水凝胶中达到平衡。基于电化学沉积工艺，以独立的Ag/AgCl电极作为参比电极，铂电极作为对电极以及AM-co-DAS-co-SSNa聚合水凝胶涂层导电芯片作为工作电极构建三电极体系，以基于上述的微电极阵列在导电芯片(ITO导电玻璃)形成具有微图案的导电水凝胶。
根据本公开的实施例，通过电化学沉积法基于聚合水凝胶在导电芯片上形成导电水凝胶，包括：电化学沉积法以EDOT溶液作为电解质。具体地，聚(3,4-亚乙二氧基噻吩)-聚(苯乙烯磺酸)(Poly(2,3-dihydrothieno-1,4-dioxin)-poly(styrenesulfonate)，简称PEDOT/PSS)是通过上述聚合水凝胶网络结构在含有0.01M EDOT的水溶液中以电流密度0.125mA/cm 2和电荷截止量为0.17mC/cm 2对聚合水凝胶进行电流沉积获得的，其中0.01M EDOT的水溶液可以充当电解质作用。上述组成材料的选择参数可以使得既可以充分在聚合水凝胶中沉积EDOT，又可以避免材料的过度氧化。由于平衡离子PSS由水凝胶提供，因此不需要额外的支撑电解质。完成的样品在磷酸盐(Dulbecco's Phosphate-Buffered Saline，简称DPBS)缓冲溶液中洗涤，提取未反应的EDOT，即可以在该导电芯片上得到具有微图案的PEDOT与AM-co-DAS-co-SSNa的IPN结构导电水凝胶。
在本公开的实施例中，按照表1-a所述比例的材料组分比例制备上述的具有IPN结构的微图案的AM-co-DAS-co-SSNa导电水凝胶。其中，结 果如图3所示，其中图3-A微图案导电水凝胶涂层的ITO玻璃芯片的实物照片，图3-B为微图案导电水凝胶在荧光倒置显微镜IX71下的局部放大图。在局部放大图3-B中，浅色条带是AM-co-DAS-co-SSNa聚合水凝胶，黑色条带是导电聚合物PEDOT与AM-co-DAS-co-SSNa聚合水凝胶的互穿网络IPN结构。因此，可见PEDOT在导电水凝胶IPN网络中的成功电沉积。
在本公开的实施例中，上述经电化学沉积工艺制备得到的具有微图案的IPN结构导电水凝胶的导电芯片，可以应用于细胞阻抗传感检测领域中的生物电极。生物电极是许多神经假体装置中传递电荷和记录细胞活动的重要组成部分。传统电极通常为金属电极(例如铂、铂合金或金组成)，金属电极的局限性包括机械失配导致持续的异物反应以及电化学性能的局限性阻碍了高效稳定的电荷转移。与传统的金属电极相比，导电聚合物(CPs)具有更好的阻抗特性。本公开通过电沉积制备导电聚合物(CPs)和聚合水凝胶的IPN网络，通过水凝胶的表面提供了更柔软的机械界面。IPN结构中的导电水凝胶不仅起到了调节和改善IPN结构机械性能的作用，并为水溶性生物活性剂提供低污染的表面和长效贮藏库。
本公开提供的这种导电水凝胶的制备方法，该导电水凝胶的制备方法本公开通过电化学沉积法在微电极阵列导电芯片上制备了具有集成互穿网络结构(IPN结构)的微图案的软质导电水凝胶，该导电水凝胶可以有效的替代细胞阻抗传感技术领域中的金属电极，结合阻抗仪在线检测系统，有效实现了细胞阻抗传感检测过程中高效稳定的电荷转移，使得细胞阻抗检测达到了无标记、全程动态、实时分析的技术效果。
本公开的另一个方面提供了一种细胞阻抗传感检测方法，如图4所示，包括：
S210：在具有上述的导电水凝胶的表面接种待检测细胞；
S220：将接种待检测细胞的导电水凝胶的导电芯片与阻抗仪电连接进行细胞阻抗检测；
S230：根据细胞阻抗检测获取待检测细胞的细胞阻抗信息。
在本公开实施例中，基于上述公开的具有IPN结构和微图案设计的导 电水凝胶的导电芯片作为监测电极，即细胞阻抗传感芯片，构建一细胞阻抗传感检测系统对导电芯片上导电水凝胶上接种的细胞进行实时、无标记的高效检测。具体地，在导电芯片的具有微图案设计和IPN结构的导电水凝胶上接种待检测细胞，将导电芯片所形成的细胞培养腔置于37℃，5％CO 2的培养箱中培养。然后通过阻抗分析仪(阻抗仪或阻抗谱仪)通过导线经金属夹片与待检测细胞所在的监测电极(即细胞阻抗传感芯片)相连，以测量待检测细胞的阻抗值。如图5所示，以表面进行了type I collagen孵育预处理的微图案导电水凝胶涂层的ITO玻璃芯片510作为监测电极，与该监测电极的一引出电极电连接的阻抗仪520，同时与该监测电极的另一引出电极电连接的电流放大器530，该电流放大器530同时也与阻抗仪520电连接，以构建细胞阻抗传感检测系统，并且该系统的阻抗仪520与计算机系统540连接，实现对待检测细胞的阻抗信息数据的在线联用分析。
因此，本公开的微图案导电水凝胶能完成其在细胞阻抗传感中的应用。将微图案导电水凝胶涂层的ITO导电玻璃芯片与阻抗仪构建在线联用系统，可实现对各种贴壁细胞的检测，主要包括细胞增殖检测、各种化合物对细胞的毒性检测、细胞间共培养检测、细胞粘附和伸展检测、受体介导的信号通路检测等。该联用系统的动态监测保证了细胞的瞬时响应及长时效应的获取，并可扩展应用于细胞治疗杀伤质控、环境毒理、食品毒理评价等研究领域。
根据本公开的实施例，进行细胞阻抗检测，包括：以输出电压为5mV-10mV、输出频率为400Hz～50MHz对具有待检测细胞的导电水凝胶的导电芯片进行扫频测量，以确定检测频率值。在构建完成的细胞阻抗传感检测系统后，需要对细胞阻抗信息进行检测，其中，细胞阻抗测量时施加的电压可输出范围为：5～10mV；使用400Hz～50MHz范围内进行扫频测量，根据测量结果选择合适的检测频率值。对相应检测频率值采集到的实时待检测细胞阻抗信息进一步用MATLAB分析处理。如图6所示，对接种了40×10 4cells/mL MDCK细胞在37℃5％CO 2细胞培养箱培养了24h后的导电芯片进行阻抗扫频测量。使用10～40MHz范围内进行扫频，施加的电压输出为5mV。其中，图6-A为细胞阻抗的实部，图6-B为细胞阻抗的虚部。从图6-A、图6-B可见，当施加的电压一定时，细胞在不同 频率下阻抗是不同的，需要选择一定的频率范围进行扫频测量，根据测量结果选择合适的检测频率值。
根据本公开的实施例，在导电水凝胶的表面接种待检测细胞，包括：在导电水凝胶的表面进行孵育预处理；以及在进行了孵育预处理后的导电水凝胶的表面接种待检测细胞。具体地，在细胞培养腔接种细胞前，吸取1mL 1mM Sulfo-SANPAH覆盖水凝胶表面，UV活化15min，用50mM HEPES缓冲液(pH＝8.5)清洗2次，每次3min。加入0.2mg/mL type I collagen 4℃孵育过夜；芯片培养腔在接种细胞使用前需UV照射30min-1h灭菌处理。其中，在对导电水凝胶的表面进行孵育预处理之前，还需要对导电芯片所在的细胞培养腔中的微图案导电水凝胶进行DPBS缓冲液的浸泡，浸泡时间长达一周或以上，并且期间每天需要更换DPBS缓冲液，以充分去除导电水凝胶中对细胞培养具有毒性的物质。本公开的导电水凝胶的水凝胶成分提供了可与脆性导电聚合物结合的基质，这些导电水凝胶已显示出具有改善的机械性能和生物相容性，并且提高了导电聚合物的电活性。导电聚合物电导率的提高归因于三维结构(3D)，它使电荷转移能够在更大面积上发生。本公开通过在微图案导电水凝胶上孵育type I collagen，使其具有很好的细胞附着能力。另一方面，采用ITO导电玻璃芯片作为导电芯片，可以使得其在用于细胞阻抗实时传感检测时具有良好的细胞相容性。
本公开提供了一种具有微图案的导电水凝胶导电芯片及其细胞阻抗传感检测方法。具体而言，本公开以原位加成反应制备了AM-co-DAS-co-SSNa聚合水凝胶涂层，进一步以该聚合水凝胶涂层的微电极阵列的导电芯片为工作电极，利用电化学沉积工艺将PEDOT制备到上述聚合水凝胶中形成集成互穿网络(IPN)结构的导电水凝胶，将具有上述导电水凝胶的导电芯片与阻抗仪构建细胞阻抗传感检测系统和在线联用分析系统，实现了细胞阻抗信息数据的实时、无标记、动态检测新方法。由于导电聚合物PEDOT电导率高、环境稳定性好和能隙小等特点，以及PEDOT和水凝胶无缝形成的集成互穿网络(IPN)结构，该软质特性的导电水凝胶电极不仅解决了金属电极的局限性，如机械失配导致持续的异物反应和电化学性能的局限性，阻碍了高效稳定的电荷转移，还通过测 量细胞与监测电极之间的实时阻抗动态变化来评估细胞的生理和病理状态。本公开所公开的具有微图案的导电水凝胶涂层的微电极阵列导电芯片，不仅对于实验室和临床药物毒性检测具有重要的意义，而且拥有重要的商业推广价值，有望在新药开发、药物安全性评价和二次开发等方面发挥特定作用，产生良好的社会和经济价值。
以上的具体实施例，对本公开的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本公开的具体实施例而已，并不用于限制本公开，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (12)

  1. 一种导电水凝胶的制备方法，其中，包括：
    形成具有微电极阵列的导电芯片；
    通过所述导电芯片形成细胞培养腔；
    在所述细胞培养腔的微电极阵列上形成聚合水凝胶；以及
    通过所述聚合水凝胶形成具有微图案的导电水凝胶。
  2. 根据权利要求1所述的导电水凝胶的制备方法，其中，所述形成具有微电极阵列的导电芯片包括：
    在导电衬底的导电膜层上形成光刻胶层；
    在所述具有光刻胶层的导电衬底上形成光刻胶图案；以及
    基于所述光刻胶图案在所述导电衬底上形成具有导电膜图案的导电芯片，
    其中，所述导电膜图案为所述微电极阵列。
  3. 根据权利要求1所述的导电水凝胶的制备方法，其中，所述通过所述导电芯片形成细胞培养腔，包括：
    在所述导电芯片上形成围设所述微电极阵列的环形结构，
    其中，所述环形结构的内表面和所述微电极阵列所在的导电芯片表面之间形成所述细胞培养腔。
  4. 根据权利要求1所述的导电水凝胶的制备方法，其中，所述在所述细胞培养腔的微电极阵列上形成聚合水凝胶，包括：
    依据预设配比，将丙烯酰胺(AM)、双醛淀粉(DAS)、阴离子掺杂剂、交联剂、引发剂以及催化剂进行混合形成混合水凝胶；以及
    对所述混合水凝胶进行溶胀形成所述聚合水凝胶。
  5. 根据权利要求4所述的导电水凝胶的制备方法，其中，在所述聚合水凝胶中，
    所述丙烯酰胺(AM)的溶液的质量占比23％-46％；
    所述双醛淀粉(DAS)的溶液的质量占比12％-35％；
    所述阴离子掺杂剂的质量占比5％-10％；
    所述交联剂的溶液的质量占比10％-13％；
    所述引发剂的溶液的质量占比23％-25％；以及
    所述催化剂的质量占比2％-3％。
  6. 根据权利要求4所述的导电水凝胶的制备方法，其中，
    所述丙烯酰胺的溶液的浓度为0.4g/mL；
    所述双醛淀粉的溶液的浓度为0.4g/mL；
    所述交联剂的溶液的浓度为0.02g/mL；以及
    所述引发剂的溶液的浓度为0.1g/mL。
  7. 根据权利要求1所述的导电水凝胶的制备方法，其中，所述通过所述聚合水凝胶形成具有微图案的导电水凝胶，包括：
    通过电化学沉积法基于所述聚合水凝胶在导电芯片上形成所述导电水凝胶，
    其中，所述微图案与所述微电极阵列的图形对应匹配，所述导电水凝胶具备集成互穿网络(IPN)结构。
  8. 根据权利要求7所述的导电水凝胶的制备方法，其中，所述通过电化学沉积法基于所述聚合水凝胶在导电芯片上形成所述导电水凝胶，包括：
    对所述聚合水凝胶进行3,4-乙烯二氧噻吩(EDOT)溶液的溶胀；以及
    将具有所述已进行溶胀3,4-乙烯二氧噻吩(EDOT)溶液的所述聚合水凝胶的导电芯片作为工作电极进行电沉积以形成所述导电水凝胶。
  9. 根据权利要求7所述的导电水凝胶的制备方法，其中，所述通过电化学沉积法基于所述聚合水凝胶在导电芯片上形成所述导电水凝胶，包括：
    所述电化学沉积法以EDOT溶液作为电解质。
  10. 一种细胞阻抗传感检测方法，其中，包括：
    在权利要求1-9中任一项所述的导电水凝胶的表面接种待检测细胞；
    将接种所述待检测细胞的导电水凝胶的导电芯片与阻抗仪电连接进行细胞阻抗检测，以及
    根据所述细胞阻抗检测获取所述待检测细胞的细胞阻抗信息。
  11. 根据权利要求10所述的细胞阻抗传感检测方法，其中，所述进 行细胞阻抗检测，包括：
    以输出电压为5mV-10mV、输出频率为400Hz～50MH对所述具有所述待检测细胞的导电水凝胶的导电芯片进行扫频测量，以确定检测频率值。
  12. 根据权利要求10所述的细胞阻抗传感检测方法，其中，在所述导电水凝胶的表面接种待检测细胞，包括：
    在所述导电水凝胶的表面进行孵育预处理；以及
    在进行了孵育预处理后的所述导电水凝胶的表面接种待检测细胞。
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