Integrated sensing device for organ chip barrier integrity and ion in-situ detection

Abstract

The invention discloses an integrated sensing device for organ chip barrier integrity and ion in-situ detection. The integrated sensing device comprises a bottom electrode layer, an organ chip layer and a top electrode layer which are sequentially arranged from bottom to top, the organ chip layer comprises a lower runner layer, a porous membrane and an upper runner layer which are sequentially arranged from bottom to top; the upper runner layer and the lower runner layer are both provided with long grooves which are through up and down; the long grooves in the upper runner layer and the lower runner layer are correspondingly called as upper runners and lower runners; the upper runners and the lower runners are divided into upper chambers and lower chambers by porous membranes; a plurality of electrodes are arranged on the lower surface of the top electrode layer and the upper surface of the bottom electrode layer; one end of each electrode of the top electrode layer is connected with the upper runner, and the other end is connected with an external circuit; one end of each electrode of the bottom electrode layer is connected with the lower runner, and the other end is connected with an external circuit; the upper runner layer and the lower runner layer are insulators; the number of electrodes on the top electrode layer is greater than or equal to 2; the number of electrodes on the top electrode layer is not less than 5. The compact layout that the upper layer and the lower layer are each provided with multiple electrodes is adopted, and the space utilization rate of the organ chip is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of organ-on-a-chip, and particularly to an integrated sensing device for detecting the barrier integrity and in-situ ions of an organ-on-a-chip. Background Art

[0002] The organ-on-a-chip is a new in-vitro experimental platform developed with the cross-integration of microfluidic technology, cell culture technology, and biomaterials science. By constructing cell culture chambers in microscale channels to simulate the human microenvironment, it provides a model closer to the in-vivo real conditions for pathological research, drug development, and disease modeling. Compared with the traditional Transwell static cell culture model or in-vivo animal experiments, the organ-on-a-chip can dynamically regulate the culture conditions, better simulate the human environment, and does not involve ethical issues, thus having broad application prospects.

[0003] Transendothelial electrical resistance (TEER) detection is an important means to evaluate the functional integrity of an organ-on-a-chip. By using the electrochemical impedance spectroscopy (EIS) method to measure the cell layer resistance, the tight junctions of the cell layer can be quantified. Compared with destructive methods such as immunofluorescence staining, TEER detection has the advantages of real-time and non-invasive, and can evaluate the formation and destruction of the organ-on-a-chip on a time scale.

[0004] Sodium ions and calcium ions are important ions that maintain the osmotic pressure balance inside and outside cells, and their concentration changes directly affect cell functions. Detecting the ion concentration in an organ-on-a-chip is crucial for studying cell functions and drug effects. Monitoring the concentration changes of sodium ions and calcium ions in the integrated sensing device for detecting the barrier integrity and in-situ ions of an organ-on-a-chip plays an important role in the research of various central nervous system diseases such as Alzheimer's disease; monitoring the concentration changes of sodium ions and calcium ions in a cardiac organ-on-a-chip is crucial for studying arrhythmia and the role of calcium ions in cardiac functions; monitoring the concentration changes of sodium ions and calcium ions in an intestinal organ-on-a-chip can evaluate the effects of drugs on intestinal functions.

[0005] By integrating the TEER value and ion concentration detection on the same organ-on-a-chip platform, the in-situ measurement of cell barrier construction and ion concentration can be achieved, which is expected to improve the accuracy of the organ-on-a-chip detection results, provide new solutions for drug development, disease modeling, and personalized medicine, and enhance the future application prospects. Summary of the Invention

[0006] The present invention provides an integrated sensing device for detecting the barrier integrity and in-situ ions of an organ-on-a-chip to solve the technical problems existing in the known technology.

[0007] The technical solution adopted by the present invention to solve the technical problems existing in the known art is as follows:

[0008] An integrated sensing device for in-situ detection of organ chip barrier integrity and ions, the device comprising a bottom electrode layer, an organ chip layer, and a top electrode layer sequentially arranged from bottom to top; the organ chip layer comprises a downstream channel layer, a permeable porous membrane, and an upstream channel layer sequentially arranged from bottom to top; both the upstream and downstream channel layers are provided with long grooves penetrating up and down; the long grooves in the upstream and downstream channel layers are correspondingly referred to as the upstream and downstream channels, and the upstream and downstream channels are separated by a porous membrane into a structure of an upper chamber and a lower chamber; the lower surface of the top electrode layer and the upper surface of the bottom electrode layer are both provided with a plurality of electrodes; each electrode of the top electrode layer is connected to the upstream channel at one end and connected to an external circuit at the other end; each electrode of the bottom electrode layer is connected to the downstream channel at one end and connected to an external circuit at the other end; both the upstream and downstream channel layers are insulators; the number of electrodes on the top electrode layer ≥ 2; the number of electrodes on the top electrode layer ≥ 5.

[0009] Further, both the bottom electrode layer and the top electrode layer comprise a glass substrate, and the electrodes are fabricated by the following method: First, deposit 50 nm of titanium tungsten on the glass substrate as an adhesion layer, then deposit 200 - 250 nm of gold, and finally obtain gold electrodes with the desired shape by etching or lift-off method.

[0010] Further, both the upstream and downstream channels are 0.5 - 2 cm in length, 1.5 - 2.5 mm in width, and 1 - 2 mm in height; each electrode of the top electrode layer is perpendicular to the upstream channel and the length of the part connected to the upstream channel ≥ the width of the upstream channel; each electrode of the bottom electrode layer is perpendicular to the downstream channel and the length of the part connected to the downstream channel ≥ the width of the downstream channel.

[0011] Further, two of the electrodes on the top electrode layer are used to measure TEER, and these two electrodes are respectively referred to as electrode E1 and electrode E2; two of the electrodes on the bottom electrode layer are used to measure TEER, and these two electrodes are respectively referred to as electrode E3 and electrode E4; the TEER measurement method adopts the electrochemical impedance spectroscopy EIS method.

[0012] Further, form a TEER measurement circuit with E1 to E4, and measure the impedance value between two electrodes in the frequency range of 10 Hz - 10 kHz; Let: the resistance of the porous membrane and the cell layer be R m ; the resistance between electrode E1 and E2 be R 12 , the resistance between electrode E1 and E3 be R 13 , the resistance between electrode E1 and E4 be R 14 , the resistance between electrode E2 and E3 be R 23 , the resistance between electrode E2 and E4 be R 24 , the resistance between electrode E3 and E4 be R 34 , R m The calculation formula of is as follows:

[0013]

[0014] The overlapping area of the upstream and downstream channels is the cell layer culture area; let the cell layer culture area be A cult , and the calculation formula of the TEER value is as follows:

[0015] TEER = A cult ·R m .

[0016] Furthermore, for the three electrodes used to measure the concentrations of sodium ions and calcium ions, one serves as a reference electrode, one is used to measure the sodium ion concentration, and one is used to measure the calcium ion concentration; a solution containing sodium and calcium ions is injected into the downstream channel; first, measure the open-circuit potential of the electrode in standard sodium ion solutions and standard calcium ion solutions at different concentrations, establish the linear relationship between the open-circuit potential and the ion concentration according to the open-circuit potential at different ion concentrations, and then measure the open-circuit potential of the electrode in the solution to be measured. From the linear relationship between the open-circuit potential and the ion concentration, obtain the ion concentrations of sodium ions and calcium ions in the solution to be measured.

[0017] Furthermore, the reference electrode is modified with a reference electrode protective film, the electrode for measuring the sodium ion concentration is modified with a sodium ion selective membrane; the electrode for measuring the calcium ion concentration is modified with a calcium ion selective membrane.

[0018] The advantages and positive effects of the present invention are as follows: The electrodes are integrated on a transparent glass substrate through photolithography technology and combined with an organ chip manufactured using microfluidic technology, which can realize in-situ detection of measuring the TEER value and ion concentration, improving the consistency and reliability of data; by adjusting the electrode surface modification material, the detection of sodium ions and calcium ions can also be extended to the detection of other types of ions or molecules, having strong application prospects; the electrode layout with multiple gold electrodes on each of the upper and lower layers is compact, improving the space utilization rate of the miniaturized organ chip. Description of the Drawings

[0019] Figure 1 It is a schematic structural diagram of an integrated sensing device for organ chip barrier integrity and ion in-situ detection of the present invention.

[0020] Figure 2 It is a schematic structural diagram of the connection between the upstream channel layer and the top electrode layer in an integrated sensing device for organ chip barrier integrity and ion in-situ detection of the present invention.

[0021] Figure 3 It is a schematic structural diagram of the connection between the downstream channel layer and the bottom electrode layer in an integrated sensing device for organ chip barrier integrity and ion in-situ detection of the present invention.

[0022] Figure 4The graph showing the relationship between the impedance value measured by the impedance detection method of the integrated sensing device for organ chip barrier integrity and in-situ ion detection of the present invention and the frequency in the frequency range of 10 kHz - 10 Hz.

[0023] Figure 5 The graph showing the relationship between the TEER value calculated by the impedance detection method of the integrated sensing device for organ chip barrier integrity and in-situ ion detection of the present invention and the corresponding frequency.

[0024] Figure 6 The electrical schematic diagram of the TEER measurement circuit composed of E1 to E4 of the present invention.

[0025] Figure 7 The surface morphology of the sodium ion selective electrode described in the present invention.

[0026] Figure 8 The graph showing the sodium ion measurement result of the present invention.

[0027] In the figure:

[0028] 1. Upstream channel input section; 2. Upstream channel detection section; 3. Upstream channel output section; 4. Downstream channel input section; 5. Downstream channel detection section; 6. Downstream channel output section; 7. Top electrode layer; 8. Upstream channel layer; 9. Porous membrane; 10. Downstream channel layer; 11. Bottom electrode layer.

[0029] A represents the through hole on the top electrode layer that communicates with the input end of the upstream channel.

[0030] B represents the through hole on the top electrode layer that communicates with the output end of the upstream channel.

[0031] C represents the through hole on the top electrode layer that communicates with the input end of the downstream channel.

[0032] D represents the through hole on the top electrode layer that communicates with the output end of the downstream channel.

[0033] G represents the through hole on the porous membrane that communicates with the input end of the downstream channel.

[0034] H represents the through hole on the porous membrane that communicates with the output end of the downstream channel.

[0035] E1, E2 represent the two electrodes on the top electrode layer for measuring TEER.

[0036] E3, E4 represent the two electrodes on the bottom electrode layer for measuring TEER.

[0037] E6 represents the reference electrode.

[0038] E5, E7 represent the working electrodes.

[0039] R1 is the resistance between the external connection port of electrode E1 and the porous membrane and the cell layer.

[0040] R2 is the resistance between the external connection port of electrode E2 and the porous membrane and the cell layer.

[0041] R3 is the resistance between the external connection port of electrode E3 and the porous membrane and the cell layer.

[0042] R4 is the resistance between the external connection port of electrode E4 and the porous membrane and the cell layer. Detailed implementation manners

[0043] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0044] In the description of the present invention, the orientation or positional relationship indicated by the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", etc. is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing the present invention rather than requiring the present invention to be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of the present invention. The terms "connected" and "connected" used in the present invention should be understood in a broad sense. For example, it can be a fixed connection or a detachable connection; it can be directly connected or indirectly connected through an intermediate component; it can also be an electrical connection or a signal transmission; for those of ordinary skill in the art, the specific meanings of the above terms can be understood according to specific circumstances.

[0045] Please refer to Figures 1 to 8 , an integrated sensing device for in-situ detection of organ chip barrier integrity and ions. The device includes a bottom electrode layer 11, an organ chip layer, and a top electrode layer 7 arranged in sequence from bottom to top; the organ chip layer includes a downstream channel layer 10, a permeable porous membrane 9, and an upstream channel layer 8 arranged in sequence from bottom to top; both the upstream channel layer 8 and the downstream channel layer 10 are provided with long grooves penetrating up and down; the long groove in the upstream channel layer 8 is called the upstream channel, and the long groove in the downstream channel layer 10 is called the downstream channel. The upstream and downstream channels are separated by the porous membrane 9 into a structure of an upper chamber and a lower chamber; both the lower surface of the top electrode layer 7 and the upper surface of the bottom electrode layer 11 are provided with a plurality of electrodes; each electrode of the top electrode layer 7 is connected to the upstream channel at one end and to an external circuit at the other end; each electrode of the bottom electrode layer 11 is connected to the downstream channel at one end and to an external circuit at the other end; both the upstream channel layer 8 and the downstream channel layer 10 are insulators; the number of electrodes on the top electrode layer 7 ≥ 2; the number of electrodes on the top electrode layer 7 ≥ 5.

[0046] Preferably, the top electrode layer 7 may be provided with through holes A and B corresponding to and communicating with both ends of the upstream channel, and through holes C and D corresponding to and communicating with both ends of the downstream channel. The upstream channel layer 8 may be provided with through holes G and H corresponding to and communicating with both ends of the downstream channel, and the through holes G and H communicate with the through holes C and D correspondingly; the through holes A, B, C, and D do not communicate with each other; the solution can enter from the through holes A and C and flow out from the through holes B and D.

[0047] Preferably, both the upstream and downstream channels can be divided into the following three segments: an input segment, a detection segment, and an output segment; that is, the upstream channel can be divided into the following three segments: an upstream channel input segment 1, an upstream channel detection segment 2, and an upstream channel output segment 3; the downstream channel can be divided into the following three segments: a downstream channel input segment 4, a downstream channel detection segment 5, and a downstream channel output segment 6.

[0048] The detection segments of both the upstream and downstream channels are vertically aligned; that is: the upstream channel detection segment 2 and the downstream channel detection segment 5 are vertically aligned.

[0049] The input segments of both the upstream and downstream channels are in a V shape; that is: the upstream channel input segment 1 and the downstream channel input segment 4 form a V shape when projected downward.

[0050] The output segments of both the upstream and downstream channels are in a V shape; that is: the upstream channel output segment 3 and the downstream channel output segment 6 form a V shape when projected downward.

[0051] The electrodes of the top electrode layer 7 and the bottom electrode layer 11 are correspondingly connected to the detection segments of the upstream and downstream channels, that is, the electrode of the top electrode layer 7 is connected to the upstream channel detection segment 2, and the electrode of the bottom electrode layer 11 is connected to the downstream channel detection segment 5.

[0052] Preferably, both the bottom electrode layer 11 and the top electrode layer 7 can include a glass substrate, and the electrodes can be fabricated by the following method: First, deposit 50 nm of titanium tungsten on the glass substrate as an adhesion layer, then deposit 200 - 250 nm of gold, and finally obtain the gold electrodes of the desired shape through etching or lift-off methods.

[0053] Preferably, both the upstream and downstream channels can be 0.5 - 2 cm in length, 1.5 - 2.5 mm in width, and 1 - 2 mm in height; each electrode of the top electrode layer 7 can be perpendicular to the upstream channel and the length of the part connected to the upstream channel ≥ the width of the upstream channel; each electrode of the bottom electrode layer 11 can be perpendicular to the downstream channel and the length of the part connected to the downstream channel ≥ the width of the downstream channel.

[0054] Preferably, both the bottom electrode layer 11 and the top electrode layer 7 can further include a cover layer fabricated by a PDMS molding method; the cover layer of the top electrode layer 7 is located on the upper surface of the glass substrate, and the cover layer of the bottom electrode layer 11 is located on the lower surface of the glass substrate.

[0055] Preferably, two of the electrodes in the top electrode layer 7 can be used to measure TEER, and these two electrodes are respectively called electrode E1 and electrode E2; two of the electrodes in the bottom electrode layer 11 can be used to measure TEER, and these two electrodes are respectively called electrode E3 and electrode E4; the TEER measurement method can adopt the electrochemical impedance spectroscopy (EIS) method.

[0056] Preferably, E1 to E4 can form a TEER measurement circuit to measure the impedance value between two electrodes in the frequency range of 10 Hz to 10 kHz; it can be set that the resistance of the porous membrane 9 and the cell layer is R m ; the resistance between electrode E1 and E2 is R 12 ; the resistance between electrode E1 and E3 is R 13 ; the resistance between electrode E1 and E4 is R 14 ; the resistance between electrode E2 and E3 is R 23 ; the resistance between electrode E2 and E4 is R 24 ; the resistance between electrode E3 and E4 is R 34 ; R m The calculation formula of is as follows:

[0057]

[0058] The overlapping area of the upstream and downstream channels is the cell layer culture area; it can be set that the cell layer culture area is A cult ; the calculation formula of the TEER value is as follows:

[0059] TEER = A cult ·R m .

[0060] Preferably, for the three electrodes used to measure the concentrations of sodium ions and calcium ions, one can be used as a reference electrode, one can be used to measure the sodium ion concentration, and one can be used to measure the calcium ion concentration; inject a solution containing sodium and calcium ions into the downstream channel; first measure the open-circuit potential of the electrode in standard sodium ion solutions and standard calcium ion solutions at different concentrations, and a linear relationship between the open-circuit potential and the ion concentration can be established according to the open-circuit potential at different ion concentrations. Then measure the open-circuit potential of the electrode in the solution to be measured, and the ion concentrations of sodium ions and calcium ions in the solution to be measured can be obtained from the linear relationship between the open-circuit potential and the ion concentration.

[0061] Preferably, the reference electrode can be modified with a reference electrode protective film, the electrode for measuring the sodium ion concentration can be modified with a sodium ion selective membrane, called a sodium ion selective electrode; the electrode for measuring the calcium ion concentration can be modified with a calcium ion selective membrane, called a calcium ion selective electrode.

[0062] The present invention also provides a method for manufacturing the above-mentioned integrated sensing device for in-situ detection of organ-on-a-chip barrier integrity and ions. The upper and lower flow channel layers 10 are both manufactured according to the following method: A mold is made by means of a photolithography mask or 3D printing for casting. PDMS and a curing agent are mixed in a ratio of 10:1, stirred evenly, evacuated, and then poured into the mold and baked. After curing, it is taken out.

[0063] The bottom and top electrode layers 7 can be manufactured according to the following method: Glass is used as the substrate material. After the substrate is cleaned, HMDS is used for adhesion enhancement treatment; the photoresist is modified, and then soft baking, exposure, post-baking, and development are carried out to expose the electrode pattern; a metal sputtering deposition method is used on the substrate, the adhesion layer is titanium tungsten, and the electrode layer is gold with a thickness of 200 - 250 nm; the remaining photoresist is removed, and etching is carried out according to the designed electrode size; a dicing knife can be used to cut the glass substrate into the required electrode size.

[0064] The cleaned bottom electrode layer 11, lower flow channel layer 10, porous membrane 9, upper flow channel layer 8, and top electrode layer 7 are integrally bonded together from bottom to top through plasma bonding; put into an oven for baking to make the bonding tighter.

[0065] Cell suspensions are respectively injected into the upper and lower flow channels. As the cells in the upper and lower flow channels adhere to the wall, a cell layer is formed on the porous membrane 9, thus forming an integrated sensing device for in-situ detection of organ-on-a-chip barrier integrity and ions.

[0066] For the working electrode, a conductive layer is deposited using a three-electrode system of an electrochemical workstation. The counter electrode uses a platinum wire, and the reference electrode uses silver / silver chloride; 0.01 M EDOT monomer and 0.05% by mass carboxylated single-walled carbon nanotubes are added to ultrapure water and mixed to prepare a PEDOT / single-walled carbon nanotube electrolyte; cyclic voltammetry is used, and 5 cycles are scanned between voltages of -1 V and 1.3 V to deposit PEDOT / single-walled carbon nanotubes on the electrode.

[0067] The sodium ion-selective membrane solution, calcium ion-selective membrane solution, and reference electrode protective membrane solution can be respectively prepared according to the following methods:

[0068] The preparation method of the sodium ion-selective membrane solution is as follows: 64.8 mg of PVC, 131.4 mg of DOS, 1.8 mg of NaTFPB, and 2 mg of sodium ionophore X are dissolved in 1500 μL of tetrahydrofuran, and after ultrasonic treatment for 15 minutes, they are mixed evenly.

[0069] The preparation method of the calcium ion-selective membrane solution is as follows: 33 mg of PVC, 665.45 mg of DOS, 0.55 mg of NaTFPB, and 1 mg of calcium ionophore II are dissolved in 660 μL of tetrahydrofuran, and after ultrasonic treatment for 15 minutes, they are mixed evenly.

[0070] The reference electrode protective film solution was prepared by dissolving 79.1 mg of PVB, 50 mg of NaCl in 1 mL of methanol, and adding 2 mg of F127 and 0.2 mg of multi-walled carbon nanotubes to reduce potential drift.

[0071] The silver / silver chloride paste was coated on the gold electrode. After drying, the reference electrode protective film solution was dropped on the surface of the reference electrode and dried for 10 - 24 hours to form a silver / silver chloride reference electrode.

[0072] After the conductive layer was deposited, the prepared ion-selective membrane was dropped on the surface of the conductive layer of the working electrode and left standing overnight; after drying, 0.1 M sodium chloride solution was dropped on the surface of the sodium ion-selective electrode, and 0.1 M calcium chloride solution was dropped on the surface of the calcium ion-selective electrode and left standing for 10 - 24 hours. The surface solution was washed off before use.

[0073] The following further illustrates the structure and working principle of the present invention with several preferred embodiments of the present invention:

[0074] An integrated sensing device for in-situ detection of organ chip barrier integrity and ions, the device includes a bottom electrode layer 11, an organ chip layer, and a top electrode layer 7 arranged in sequence from bottom to top; the organ chip layer includes a downstream channel layer 10, a permeable porous membrane 9, and an upstream channel layer 8 arranged in sequence from bottom to top; both the upstream channel layer 8 and the downstream channel layer 10 are provided with long grooves that penetrate up and down; the long groove in the upstream channel layer 8 is called the upstream channel, and the long groove in the downstream channel layer 10 is called the downstream channel. The upstream and downstream channels are separated by the porous membrane 9 into a structure of an upper chamber and a lower chamber; both the lower surface of the top electrode layer 7 and the upper surface of the bottom electrode layer 11 are provided with a plurality of electrodes; each electrode of the top electrode layer 7 is connected to the upstream channel at one end and to an external circuit at the other end; each electrode of the bottom electrode layer 11 is connected to the downstream channel at one end and to an external circuit at the other end; both the upstream channel layer 8 and the downstream channel layer 10 are insulators.

[0075] The top electrode layer 7 is provided with through holes A, B corresponding to and communicating with both ends of the upstream channel, and through holes C, D corresponding to and communicating with both ends of the downstream channel. The upstream channel layer 8 is provided with through holes G, H corresponding to and communicating with both ends of the downstream channel. The through holes G, H communicate with the through holes C, D correspondingly; the through holes A, B, C, D are not connected to each other; the solution enters from the through holes A and C and flows out from the through holes B and D.

[0076] Both the upstream and downstream channel layers 10 are made of PDMS, and the material of the porous membrane 9 is PETE.

[0077] The top and bottom electrode layers 11 further include a covering layer manufactured by the PDMS molding method; the covering layer of the top electrode layer 7 is located on the upper surface of the glass substrate, and the covering layer of the bottom electrode layer 11 is located on the lower surface of the glass substrate to completely wrap the glass substrate.

[0078] Both the upstream and downstream channels are 0.5 - 2 cm in length, 1.5 - 2.5 mm in width, and 1 - 2 mm in height; each electrode of the top electrode layer 7 is perpendicular to the upstream channel and the length of the part connected to the upstream channel is ≥ the width of the upstream channel; each electrode of the bottom electrode layer 11 is perpendicular to the downstream channel and the length of the part connected to the downstream channel is ≥ the width of the downstream channel. The electrodes are rectangular and cover the channels, with a length of 2 mm and a width of 1 - 2 mm.

[0079] The electrodes are connected to the outside of the channels through leads with a width of 0.5 - 2 mm. A gold electrode with a length of 2 mm and a width of 1 - 2 mm is left outside the channels and connected to an electrochemical workstation.

[0080] The electrodes are fabricated by photolithography. First, a 50 - nm - thick titanium tungsten is deposited as an adhesion layer, and then a 200 - 250 - nm - thick gold is deposited.

[0081] There are two gold electrodes in the top electrode layer 7 for measuring TEER; there are five gold electrodes in the bottom electrode layer 11. Among them, two are used to measure TEER simultaneously with the gold electrodes in the top electrode layer 7, and the remaining three are used for measuring the ion concentrations of sodium ions and calcium ions. Among the three electrodes for measuring ion concentrations, the middle electrode is modified with silver / silver chloride as a reference electrode, and the two electrodes on both sides are respectively modified with a sodium - ion - selective membrane and a calcium - ion - selective membrane to detect the ion concentrations of sodium ions and calcium ions.

[0082] In the present invention, the TEER measurement adopts the electrochemical impedance spectroscopy (EIS) method; the ion concentration is detected in - situ by an electrochemical method, and the ion concentration measurement adopts the open - circuit potential method.

[0083] As Figure 4 shown, R1 is the resistance between the external connection port of electrode E1 and the porous membrane 9 and the cell layer, R2 is the resistance between the external connection port of electrode E2 and the porous membrane 9 and the cell layer, R3 is the resistance between the external connection port of electrode E3 and the porous membrane 9 and the cell layer, and R4 is the resistance between the external connection port of electrode E4 and the porous membrane 9 and the cell layer. R1 to R4 are the resistances of other regions of the upstream and downstream channels except the porous membrane 9 and the cell layer, and R m is the resistance of the porous membrane 9 and the cell layer. The impedances between E1E2, E1E3, E1E4, E2E3, E2E4, and E3E4 are measured respectively, and R1 to R4 are eliminated using the Gaussian elimination method, and only R m is obtained, that is, the common impedance value of the porous membrane 9 and the cell layer.

[0084] The TEER value is obtained by measuring the impedance value between two electrodes in the frequency range of 10 kHz - 10 Hz. Six groups of data are measured by combining the four electrodes in pairs. By using the Gaussian elimination method, the influence of other factors such as solution resistance, electrode resistance, and double - layer capacitance can be subtracted, and only the accurate TEER value of the cell barrier layer is obtained.

[0085] By selecting the impedance value at a certain frequency within a relatively stable frequency range and with little change over time as the measured TEER value.

[0086] The ion in-situ detection method is used to detect the concentrations of sodium ions and calcium ions. Three of the electrodes in the bottom electrode layer 11 are used to measure the concentrations of sodium ions and calcium ions. Among these three electrodes, E6 represents the reference electrode, and E5 and E7 represent the working electrodes. E5 serves as the sodium ion-selective electrode for measuring the sodium ion concentration, and E7 serves as the calcium ion-selective electrode for measuring the calcium ion concentration. A solution containing sodium and calcium ions is injected into the downstream channel; first, the open-circuit potential of the electrodes is measured in standard sodium ion solutions and standard calcium ion solutions at different concentrations. Based on the open-circuit potential at different ion concentrations, a linear relationship between the open-circuit potential and the ion concentration is established. Then, the open-circuit potential of the electrodes is measured in the solution to be tested, and from the linear relationship between the open-circuit potential and the ion concentration, the ion concentrations of sodium ions and calcium ions in the solution to be tested are obtained.

[0087] The working principle of the present invention:

[0088] In biological detection, the calculation of TEER is based on the relationship between voltage and current in Ohm's law. However, direct current will damage cells and electrodes. Therefore, the most widely used and commercially available TEER measurement system is the EVOM system developed by WPI Corporation, which uses "chopstick electrodes" with a measurement range of 1–9999Ω and a resolution of 1Ω. However, due to limitations in the position of the electrodes, the current density generated by the electrodes is inconsistent, affecting the accuracy of TEER measurement. Moreover, its frequency is fixed at 12.5Hz, and its functions are limited.

[0089] Another one is impedance spectroscopy. This method applies a variable-frequency alternating voltage to the system to be measured and obtains the impedance amplitude and current phase response of the system at different frequencies. As Figure 4 What is shown is the schematic diagram of measuring TEER by the impedance spectroscopy EIS method. R1 to R4 are the resistances of other regions in the upstream and downstream channels except for the porous membrane 9 and the cell layer, and R m is the resistance of the porous membrane 9 and the cell layer. The impedance between E1E2, E1E3, E1E4, E2E3, E2E4, and E3E4 is measured through four electrodes, and R1 to R4 are eliminated using the Gaussian elimination method, and only the common impedance value of the porous membrane 9 and the cell layer is obtained. In the intermediate frequency range, the impedance value is mainly affected by the cells, the imaginary part of the impedance approaches zero, and the real part corresponds to a pure resistance. At this time, the impedance value can represent the TEER value. The value at the frequency with the most obvious change in the TEER value over time is taken as the final TEER value.

[0090] When measuring the TEER value by impedance spectroscopy, information related to the capacitance of the cell layer can also be obtained. Through the equivalent circuit analysis of the measured impedance spectrum, electrical parameters can be obtained to characterize the mechanical properties of the cell barrier, etc.

[0091] The embodiments described above are only used to illustrate the technical idea and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The patent scope of the present invention cannot be limited only by these embodiments. That is, any equivalent changes or modifications made based on the spirit disclosed by the present invention still fall within the patent scope of the present invention.

Claims

1. An integrated sensing device for in-situ detection of barrier integrity and ions in an organ chip, characterized in that: The device comprises a bottom electrode layer, an organ chip layer and a top electrode layer which are arranged in sequence from bottom to top; the organ chip layer comprises a lower flow channel layer, a permeable porous membrane and an upper flow channel layer which are arranged in sequence from bottom to top; the upper and lower flow channel layers are both provided with long grooves which pass through from top to bottom; the long grooves in the upper and lower flow channel layers are correspondingly referred to as upper and lower flow channels, and the upper and lower flow channels are separated by a porous membrane into an upper chamber and a lower chamber structure; a plurality of electrodes are provided on the lower surface of the top electrode layer and the upper surface of the bottom electrode layer; each electrode of the top electrode layer is connected to the upper flow channel at one end and to an external circuit at the other end; each electrode of the bottom electrode layer is connected to the lower flow channel at one end and to an external circuit at the other end; the upper and lower flow channel layers are both insulators; the number of electrodes on the top electrode layer is ≥2; the number of electrodes on the top electrode layer is ≥5.

2. The integrated sensing device for in-situ detection of barrier integrity and ions in an organ chip according to claim 1, characterized in that: The bottom electrode layer and the top electrode layer both include a glass substrate, and the electrodes are made by the following method: first, a 50nm thick titanium tungsten is deposited on the glass substrate as an adhesion layer, then a 200-250nm thick gold is deposited, and finally a gold electrode of the desired shape is obtained by etching or stripping.

3. The integrated sensing device for in-situ detection of barrier integrity and ions in an organ chip according to claim 1, characterized in that: The upper and lower flow channels are 0.5-2 cm long, 1.5-2.5 mm wide and 1-2 mm high; each electrode of the top electrode layer is perpendicular to the upper flow channel and the length of the part connected to the upper flow channel is ≥ the width of the upper flow channel; each electrode of the bottom electrode layer is perpendicular to the lower flow channel and the length of the part connected to the lower flow channel is ≥ the width of the lower flow channel.

4. The integrated sensing device for in-situ detection of barrier integrity and ions in an organ chip according to claim 1, characterized in that: Two electrodes of the top electrode layer are used to measure TEER, and the two electrodes are called electrode E1 and electrode E2 respectively; two electrodes of the bottom electrode layer are used to measure TEER, and the two electrodes are called electrode E3 and electrode E4 respectively; the TEER measurement method adopts the electrochemical impedance spectroscopy EIS method.

5. The integrated sensing device for in-situ detection of barrier integrity and ions in an organ chip according to claim 4, characterized in that: E1 to E4 form a TEER measurement circuit to measure the impedance value between two electrodes in the frequency range of 10Hz to 10kHz; Assume that the resistance of the porous membrane and the cell layer is R m ; The resistance between electrodes E1 and E2 is R 12 , the resistance between electrodes E1 and E3 is R 13 , the resistance between electrodes E1 and E4 is R 14 , the resistance between electrodes E2 and E3 is R 23 , the resistance between electrodes E2 and E4 is R 24 , the resistance between electrodes E3 and E4 is R 34 , R m The calculation formula is as follows: The overlapping area of ​​the upper and lower channels is the cell layer culture area; let the cell layer culture area be A cul , the calculation formula of TEER value is as follows: TEER=A cult ·R m 。 6. The integrated sensing device for in-situ detection of barrier integrity and ions in an organ chip according to claim 1, characterized in that: Three electrodes are used to measure the concentration of sodium ions and calcium ions, one is used as a reference electrode, one is used to measure the concentration of sodium ions, and one is used to measure the concentration of calcium ions; a solution containing sodium and calcium ions is injected into the downstream channel; the open circuit potential of the electrode is first measured in standard sodium ion solutions and standard calcium ion solutions at different concentrations, and a linear relationship between the open circuit potential and the ion concentration is established based on the open circuit potential at different ion concentrations; the open circuit potential of the electrode is then measured in the solution to be tested, and the ion concentrations of sodium ions and calcium ions in the solution to be tested are obtained from the linear relationship between the open circuit potential and the ion concentration.

7. The integrated sensing device for in-situ detection of barrier integrity and ions in an organ chip according to claim 6, characterized in that: The reference electrode is modified with a reference electrode protective membrane, the electrode for measuring sodium ion concentration is modified with a sodium ion selective membrane; and the electrode for measuring calcium ion concentration is modified with a calcium ion selective membrane.

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