A low cross-talk composite electrode layer for adherent cell killing microfluidic chip

Abstract

This invention belongs to the technical field of life science analytical instruments and equipment and clinical testing equipment, specifically relating to a microfluidic chip for adhering cell killing based on discrete photosensitive gating. It includes a flow layer, an upper electrode, and a composite electrode layer arranged sequentially from top to bottom; a parallel counter electrode is formed between the upper electrode and the composite electrode layer; the composite electrode layer includes a protective electrode, a gating electrode, and a lower transparent electrode substrate arranged sequentially from top to bottom. The protective electrode is a patterned microelectrode array, which includes multiple independent electrode units, and a culture chamber is disposed on the upper surface of each electrode unit; the gating electrode and the protective electrode are patterned in the same or similar manner to form multiple independent conductive channels. The gating electrode is a photosensitive electrode. For cases with a large number of cells, this application provides a "photoelectric synergy" reverse precision killing scheme for individual adhering cells.

Description

[0001] Divisional application This application is a divisional application of Chinese invention patent application filed on July 29, 2025 [Application No.: 202511056007X] [Title: A Microfluidic Chip for Adherent Cell Killing Based on Discrete Photosensitive Gating]. Technical Field

[0002] This invention belongs to the technical field of life science analytical instruments and equipment and clinical testing equipment, specifically relating to a low lateral crosstalk composite electrode layer for microfluidic chips used for adhering cell killing. Background Technology

[0003] Microfluidics is a cutting-edge technology that integrates basic operational units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analytical processes onto a micrometer-scale chip, enabling the entire analytical process to be completed automatically. Due to its enormous application potential in biology, chemistry, and medicine, this technology has become a hot topic of interdisciplinary research, integrating biology, chemistry, medicine, fluid mechanics, electronic materials, and mechanical engineering. Microfluidic chip analysis uses a microchip as the operating platform, is based on analytical chemistry, relies on microelectromechanical systems (MEMS) technology to construct microchannel network structures, and primarily focuses on life sciences, representing the core direction of current development in micro total analytical systems.

[0004] As the basic unit of life, the study of cell structure and function is of great significance for revealing the laws of life, disease mechanisms, and treatment strategies. With the increasing demand for single-cell / single-class cell analysis, microfluidic technology has become an important tool for cell sorting due to its advantages of high throughput and precise control. Currently, cell sorting mostly adopts a forward selection strategy (i.e., directly capturing target cells), but this method is extremely sensitive to voltage parameters. If the electric field control is improper, target cells are prone to die during the sorting process due to irreversible electroporation (electric breakdown).

[0005] To this end, the applicant proposed a microfluidic chip device for cell fusion and in-situ sorting in its prior patent publication number CN119432592A, which includes a PDMS microfluidic chip, a PCB circuit board, and a patterned gold electrode microarray layer arranged sequentially from top to bottom; the PDMS microfluidic chip is provided with an inlet, an outlet, and a collection chamber, all of which are connected through an experimental chamber; the experimental chamber is provided with several independent channels and a capture chamber connected to the independent channels, and a dual-chamber capture structure is provided at the entrance of the capture chamber. In this scheme, although the cells in the capture chamber can be independently controlled by precise control, the function is focused on electrofusion and dielectric release, and cannot support the precise killing and sorting of adherent cells.

[0006] Therefore, there is an urgent need for a microfluidic device for high-precision sorting of adherent cells. Summary of the Invention

[0007] The purpose of this invention is to provide a microfluidic chip for killing adherent cells based on discrete photosensitive gating, so as to improve the killing accuracy of cells to be killed.

[0008] To solve the aforementioned technical problems, the present invention specifically adopts the following technical solution: A microfluidic chip for adhering cell killing based on discrete photosensitive gating includes a flow layer, an upper electrode, and a composite electrode layer arranged sequentially from top to bottom; a parallel counter electrode is formed between the upper electrode and the composite electrode layer. The composite electrode layer includes a protective electrode, a gate electrode, and a lower transparent electrode substrate arranged sequentially from top to bottom. The protective electrode is a patterned microelectrode array, which includes multiple independent electrode units. A culture chamber is provided on the upper surface of each electrode unit. The gate electrode and the protective electrode are patterned in the same or similar way to form multiple independent conductive channels. The gate electrode is a photosensitive electrode. When multiple cells are placed in different culture chambers for culture, the cells adhere to and grow on the electrode unit. When the light spot generated by the external optical path system shines upward from the bottom of the composite electrode layer, the light spot covers the killing area of ​​the composite electrode layer. The resistance of the gate electrode located in the killing area decreases, thereby loading the killing electrical signal and destroying the cells to be killed under the action of the killing electrical signal.

[0009] As an improvement, the gate electrode includes multiple independent electrode units, and the electrode units of the protection electrode correspond one-to-one with the electrode units of the gate electrode to form multiple independent conductive channels, with an isolation structure provided between two adjacent conductive channels.

[0010] As an improvement, the isolation structure is disposed on the lower transparent electrode substrate, and the height of the isolation structure is equal to or greater than the thickness of the gate electrode.

[0011] As an improvement, the side length of the electrode unit is 1μm-50μm, and the spacing between two adjacent electrode units is 1μm-30μm.

[0012] As an improvement, the upper electrode and the lower transparent electrode substrate are respectively connected to a kill signal generator, which is used to apply a kill electrical signal to the upper electrode and the lower transparent electrode substrate.

[0013] As an improvement, an insulating layer is also included, which is disposed between the upper electrode and the protective electrode. The insulating layer has a plurality of independent through holes corresponding to the pattern of the protective electrode, and the through holes cooperate with the upper surface of the protective electrode to form the culture chamber.

[0014] As an improvement, the side length of the through hole is smaller than the side length of the electrode unit, and the thickness of the insulating layer is 1μm-5μm.

[0015] As an improvement, the lower surface of the flow layer is provided with a mounting cavity for mounting the upper electrode, the height of the upper electrode being less than the height of the mounting cavity, so that a culture space is formed between the upper electrode and the protective electrode, and the culture space is connected to the culture chamber.

[0016] As an improvement, the spacing between the upper electrode and the protective electrode is 25μm-500μm.

[0017] As an improvement, the protective electrode is a gold layer, the material of the gate electrode layer is selected from zinc oxide, indium tin oxide, phthalocyanine compounds and GaN photosensitive materials, and the material of the lower transparent electrode substrate is selected from indium tin oxide, tin oxide, indium oxide, zinc oxide, titanium oxide and nickel oxide transparent conductive materials.

[0018] The principle and beneficial technical effects of this invention are as follows: For situations with a large number of cells, this application provides a "photoelectric synergy" reverse precision killing scheme for individual adherent cells. This is particularly suitable for photosensitive electrodes with poor biocompatibility, low wear resistance, poor corrosion resistance, and low photoresistivity. Through the synergistic effect of a patterned photosensitive electrode sheet and a protective electrode with multiple independent electrode units, multiple independent conductive channels are formed. That is, through the synchronous patterning design of the gate electrode and the protective electrode, vertically independent conductive channels are constructed, achieving precise spatial positioning of photoelectric signals. When ultraviolet light irradiates a designated area of ​​the photosensitive electrode sheet, the resistance of the first or more conductive channels within that area changes rapidly, thereby reducing the voltage value in that area. This achieves point-to-point precision killing of one or more cells within that area. Throughout the process, the cells remain in situ, meaning there is no need to introduce the cells into the designated channels. This effectively avoids cell damage caused by cell movement and the accidental killing of unstable suspended cells.

[0019] Furthermore, this solution also provides a low crosstalk technical solution to further reduce the accidental killing of cells in adjacent culture chambers. Specifically, the synergistic design of patterned photosensitive electrodes and insulating structures (including isolation structures) can further reduce the lateral crosstalk of photosensitive electrodes, which is especially suitable for high-precision cell sorting. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0021] Figure 1 This is a schematic diagram of the overall structure of the sorting system in Embodiment 1 of the present invention; Figure 2 These are schematic diagrams of the overall structure of the microfluidic chips in embodiments one to four of the present invention; Figure 3 This is an exploded view of the microfluidic chip in Embodiment 2 of the present invention; Figure 4 This is an exploded view of the microfluidic chip in Embodiment 3 of the present invention; Figure 5 This is an exploded view of the microfluidic chip in Embodiment 4 of the present invention; Figure 6 This is an exploded view of another microfluidic chip in Embodiment 4 of the present invention; Figure 7 This is a schematic diagram of the overall structure of another microfluidic chip in Embodiment 4 of the present invention; Figure 8 This is a front view of the composite electrode layer with an isolation structure in an embodiment of the present invention; Figure 9 This is a cross-sectional view of the microfluidic chip in an embodiment of the present invention; Figure 10 This is a schematic diagram of the optical path system in an embodiment of the present invention; Figure 11 This is a schematic diagram of a lower transparent electrode substrate with a microwell structure in an embodiment of the present invention; Figure 12 This is a comparison image of a single cell before and after sorting in an embodiment of the present invention; Figure 13 These are comparison images of multiple cells before and after sorting in an embodiment of the present invention; Figure 14 This is a flowchart of the control method for the optical path system in Embodiment 5 of the present invention; Figure 15 This is a schematic diagram of the modular structure of the optical path system in Embodiment Six of the present invention.

[0022] The diagram is labeled as follows: 1. Microfluidic chip; 2. Sample injector; 3. Optical system; 4. Killer signal generator; 5. Computer control unit; 101. Flow layer; 102. Upper electrode; 103. Composite electrode layer; 31. Insulating layer; 32. Protective electrode; 33. Gate electrode; 34. Lower transparent electrode substrate; 35. Isolation structure; 36. Culture space; 104. Sample inlet; 105. Sample outlet; 106. Insulating substrate; 107. Insulating hole; 108. Mounting cavity; 109. Through hole; 301. Light source; 302. Collimation and homogenization system; 303. Digital micromirror device; 304. Projection imaging system; 41. Dual telecentric lens group; 42. Right-angle mirror; 305. Microfluidic chip platform; 306. Image acquisition device; 307. Host computer. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] In this document, suffixes such as "module," "component," or "unit" used to denote elements are used solely for the purpose of illustrative purposes and have no specific meaning in themselves. Therefore, "module," "component," or "unit" may be used interchangeably. In this document, terms such as "upper," "lower," "inner," "outer," "front," "rear," "one end," and "the other end," indicating orientation or positional relationships based on the orientation or positional relationships shown in the accompanying drawings, are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0025] In this document, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," and "connected," etc., should be interpreted broadly. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. In this document, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0026] Example 1 This embodiment provides a cell sorting system for in-situ reverse killing of adherent cells. See [link to documentation]. Figure 1 and Figure 2 The system includes: a sample injector 2, a microfluidic chip 1, an optical path system 3, a kill signal generator 4, and a computer control unit 5. The microfluidic chip 1 includes a flow layer 101, an upper electrode 102, and a composite electrode layer 103 arranged sequentially from top to bottom. The composite electrode layer 103 includes at least a photosensitive electrode. The kill signal generator 4 is used to apply a kill electrical signal to the upper electrode 102 and the composite electrode layer 103. The microfluidic chip 1 can be any of the microfluidic chips described in Embodiments 2 to 4.

[0027] The computer control unit 5 includes: Data acquisition module: configured to acquire image information of cells in a microfluidic chip; the cells include target cells and cells to be killed; Pattern generation module: configured to process the image information, generate a pattern that matches the site of the cells to be killed, and feed it back to the optical path system 3; The control module is configured to control the operation of the optical path system 3, generate a light spot corresponding to the pattern, and project the light spot onto the killing area where the cells to be killed are located. This significantly reduces the local resistance of the photosensitive electrode in the killing area, thereby loading a killing electrical signal and destroying the cells to be killed under the action of the killing electrical signal. In other words, by changing the local resistance of the photosensitive electrode, the voltage in that area is increased, causing the cells to be killed to undergo irreversible perforation under the action of the voltage.

[0028] In some embodiments, the microfluidic chip may be one of the chips in Embodiments 2 to 4.

[0029] In some embodiments, the flow layer 101 is provided with an inlet and an outlet. The inlet is connected to the sample injector, and the outlet is connected to the conduit. The inlet continuously introduces complete culture medium into the fluid channel at a certain flow rate, so that adherent cells can grow normally and adhere to the electrode layer.

[0030] In some embodiments, the sample injector 2 may be a fluid manipulation device such as a syringe or a peristaltic pump, which is connected to the injection port through an injection pipe. As for the computer control unit and the kill signal generator (e.g., an electrical signal controller), they are existing technologies and will not be described in detail again.

[0031] In some embodiments, the catheter is connected to a rear-end collection container. During use, the sorting system with the above structure first pumps cells forward into the microfluidic chip via a sample injector 2 (e.g., a syringe), and after culture until adherence, performs a sterilization and sorting operation. After sorting, proteases or other digestive agents are pumped to digest the target cells, and then the digested cells are pumped into the rear-end collection container for collection.

[0032] In some embodiments, see Figure 10 The optical path system includes a light source 301, a digital micromirror device (DMD) 303, a projection imaging system 304, a microfluidic chip platform 305, an image acquisition device 306 (preferably a CCD camera), and a host computer 307. The host computer 307 is used to control the digital micromirror device 303 to generate the light spot according to the instructions of the control module. The host computer 307 may be part of the computer control unit. The light source 301 is an ultraviolet light source, which may be an ultraviolet mercury lamp, an ultraviolet laser, an ultraviolet LED, etc., with a wavelength of 300nm-400nm (preferably 365nm).

[0033] In some embodiments, the optical path system further includes a collimation and homogenization system 302, which includes a collimating lens and a homogenization lens group. After the LED light source is emitted, the collimation is within 10° and the light spot uniformity is above 90% after passing through the collimation and homogenization system.

[0034] In some embodiments, the projection imaging system 304 includes a dual telecentric lens group 41 and a right-angle mirror 42. The dual telecentric lens group 41 is based on a 4f Fourier transform optical path configuration and a dual telecentric optical structure. Its optical characteristics satisfy a strict Fourier conjugate relationship: the image-side focal plane of the former coincides precisely with the object-side focal plane of the latter. An aperture stop is set at this conjugate position as a spatial frequency filter, which can proportionally reduce the light spot reflected by the DMD and illuminate the bottom of the microfluidic chip from bottom to top through the right-angle mirror 42.

[0035] In some embodiments, the microfluidic chip platform 305 consists of a gripper and two three-axis moving platforms, through which the CCD camera and the microfluidic chip can be aligned and matched.

[0036] In summary, this application provides a "photo-electric synergy" single-cell precision reverse killing scheme for adherent cells, which can effectively improve the accuracy of killing cells and reduce damage to target cells and accidental killing.

[0037] In other words, this application uses a dynamic spot positioning method to effectively improve the killing accuracy. Specifically, based on the cell position in the microfluidic chip, a pattern corresponding to the cell to be killed is generated, and the corresponding pattern is projected onto the photosensitive electrode through the spot, causing a change in the resistance of the area (killing area), thereby killing the cells located in the killing area. During the entire killing process, there is no need to move the cells. That is to say, the cells are directly attached to the wall and grow on the microfluidic chip. The cells always remain in place, which can effectively avoid cell damage caused by cell movement as in the prior art (CN115895877B), as well as the situation where suspended cells are not fixed and are accidentally killed.

[0038] Example 2 See Figure 3 and Figure 9 This embodiment provides a microfluidic chip for in situ reverse killing of adherent cells, comprising a flow layer 101, an upper electrode 102, and a composite electrode layer 103 arranged sequentially from top to bottom; the upper electrode 102 and the composite electrode layer 103 form a parallel counter electrode.

[0039] The composite electrode layer 103 includes a protective electrode 32, a gate electrode 33, and a lower transparent electrode substrate 34 arranged sequentially from top to bottom. The protective electrode 32 is patterned to form several independent electrode units, and a culture chamber (not shown in the figure) is provided on the upper surface of the electrode unit. The gate electrode 33 is a photosensitive electrode. The lower transparent electrode substrate 34 is a common electrode.

[0040] When multiple cells are placed in different culture chambers for culture, the cells adhere to the electrode unit and grow. When the light spot generated by the external optical path system 3 shines upward from the bottom of the composite electrode layer 103, the light spot covers the killing area of ​​the composite electrode layer 103. The resistance of the gate electrode 33 located in the killing area decreases, thereby loading the killing electrical signal and destroying the cells to be killed under the action of the killing electrical signal.

[0041] In some embodiments, the gate electrode 33 is a photosensitive electrode sheet, which covers the bottom of the protective electrode. A vertical conductive channel is formed between the overlapping upper electrode 102, the protective electrode 32, the gate electrode 33, and the lower transparent electrode substrate 34, so that the cells to be killed on the protective electrode can be killed under voltage when conductive.

[0042] Microfluidic chips with the above structure are particularly suitable for photosensitive electrodes with poor biocompatibility, poor wear resistance, and poor corrosion resistance. Specifically, through the synergistic effect of the photosensitive electrode sheet and the protective electrode with multiple independent electrode units, when ultraviolet light irradiates the killing area corresponding to the photosensitive electrode sheet (the photosensitive electrode sheet completely covers the electrode units), the resistance of the area changes rapidly, and the voltage value of this part decreases, so as to achieve precise killing of cells in the area. Throughout the process, the cells remain in place, that is, there is no need to introduce the cells into the designated channel. In this way, cell damage caused by moving cells and the accidental killing of unstable suspended cells can be effectively avoided.

[0043] In some embodiments, the photosensitive electrode sheet is configured with an insulating structure through doping. Specifically, the photosensitive electrode sheet is fabricated as follows: first, a grid-like insulating structure is sputtered onto the lower transparent electrode substrate 34; then, photosensitive material is sputtered into the blank areas of the grid-like insulating structure, ultimately making the insulating structure and the photosensitive material coplanar to jointly form the photosensitive electrode sheet. The material of the insulating structure can refer to the material of the isolation structure in Embodiment 3, which is also referred to as an insulating substrate in Embodiment 4. By regionally dividing the photosensitive area of ​​the photosensitive electrode sheet, lateral crosstalk on the photosensitive electrode sheet can be effectively reduced, improving the elimination accuracy.

[0044] In some embodiments, the protective electrode 32 is a patterned microelectrode array, which includes multiple independent electrode units. The side length of each electrode unit is 1 μm-50 μm, and the spacing between two adjacent electrode units is 1 μm-30 μm. This solution limits the size of the electrode units, effectively providing a regionalized elimination method. Specifically, one cell typically corresponds to one electrode unit. Of course, there may also be cases where one cell spans two electrode units. Regardless of the situation, this application achieves "point-to-point" precise elimination by conducting to a single electrode in a regionalized manner, thereby effectively improving the accuracy of elimination.

[0045] In some embodiments, the patterned microelectrode array is square, but it can also be rectangular or other shapes.

[0046] In some embodiments, the lower surface of the flow layer 101 is provided with a mounting cavity 108 for mounting the upper electrode 102, and the height of the upper electrode 102 is less than the height of the mounting cavity 108, so that a culture space 36 is formed between the upper electrode 102 and the protective electrode 32.

[0047] In some embodiments, the composite electrode layer further includes an insulating layer 31 disposed between the upper electrode 102 and the protective electrode 32. The insulating layer 31 has a plurality of independent through holes 109 corresponding to the pattern of the protective electrode 32. The through holes 109 cooperate with the upper surface of the protective electrode 32 to form the culture chamber (not shown in the figure). By providing the insulating layer, the upper electrode and the lower transparent electrode substrate in areas without the protective electrode are separated, avoiding electrical conduction in such areas and thus preventing crosstalk and reduced sterilization accuracy.

[0048] The through-hole 109 has a shape and size that are basically the same as the electrode unit of the protective electrode 32, but is slightly smaller than the electrode unit. This ensures that the lower transparent electrode substrate 34, which serves as the common electrode, will not be exposed and form an ineffective conductive channel with the upper electrode 102. In other words, it avoids uncontrollable conduction between the lower transparent electrode substrate 34 and the upper electrode 102, which could lead to uncontrollable cell killing. Preferably, the side length of the through-hole 109 is 1 μm to 10 μm smaller than the side length of the electrode unit, and the thickness of the insulating layer is 1 μm to 5 μm.

[0049] In some embodiments, the culture chamber and the culture space are connected.

[0050] In some embodiments, the flow layer 101 is provided with an inlet and an outlet, which are respectively connected to the culture chamber so that the culture medium can be continuously introduced into the culture chamber at a certain flow rate. In other embodiments, the inlet 104 and the outlet 105 are respectively connected to the culture chamber and the culture space so that the culture medium can be continuously introduced into the culture chamber and the culture space at a certain flow rate, thereby enabling adherent cells to grow normally and adhere to the electrode layer.

[0051] In some embodiments, the distance between the upper electrode 102 and the protective electrode 32 is 25 μm-500 μm. That is, the distance between the upper electrode 102 and the protective electrode 32, or the height of the culture space, is 25 μm-500 μm.

[0052] The protective electrode 32 is used to improve biocompatibility and protect the gate electrode. The gate electrode 33 is a semiconductor material that acts as a "optical switch". Under 365nm wavelength ultraviolet light irradiation, its resistance will drop significantly. The lower transparent electrode substrate 34 serves as a common electrode and is used to supply power to the protective electrode 32 and the gate electrode 33. In some embodiments, the flow layer may be one of polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA), the protective electrode 32 may be a gold layer, the material of the gate electrode 33 may be one of photosensitive materials such as zinc oxide (ZnO), indium tin oxide (ITO), phthalocyanine compounds and gallium nitride (GaN), and the material of the lower transparent electrode substrate 34 may be one of transparent conductive materials such as indium tin oxide (ITO), tin oxide, indium oxide, zinc oxide, titanium oxide, and nickel oxide. The above materials can be sputtered onto glass to form a thin film, so as to achieve the function of "transparent conductivity" under the conditions of nanoscale thin film preparation and doping control. Of course, those skilled in the art can also select other photosensitive materials as gate electrodes according to actual needs. As for the protective electrode and the lower transparent electrode substrate, they can also be replaced as needed. All of these should be within the protection scope of this application.

[0053] In some embodiments, the number of electrode units is 100-1×10 8 Of course, those skilled in the art can also make adjustments according to actual needs.

[0054] In some embodiments, the upper electrode 102 and the lower transparent electrode substrate 34 are respectively connected to a kill signal generator 4. The kill signal generator 4 is used to apply a kill electrical signal to the upper electrode 102 and the lower transparent electrode substrate 34 to form a plurality of conductive channels between the upper and lower electrodes. The kill electrical signal can be a pulse signal, a direct current signal, or an alternating current signal. When the kill electrical signal is a pulse signal, a bipolar pulse signal can be selected, with an amplitude of 0 V-100 V, a pulse width of 1 μs-1000 μs, a pulse interval of 0.1 s-1 s, and a pulse count of 10-100 times.

[0055] In some embodiments, see Figure 11The upper surface of the lower transparent electrode substrate 34 is provided with a plurality of micro-well openings 37 corresponding to the electrode units, so as to reduce the light reflection or refraction generated when ultraviolet light passes through the lower transparent electrode substrate 34. By achieving a balance between the requirements of light spot positioning accuracy and conductivity loss, the overall elimination accuracy is improved. Specifically, the micro-well opening 37 is a micro-recessed structure formed by processing on the upper surface of the lower transparent electrode substrate 34. Each micro-well corresponds to an electrode unit (i.e., the smallest functional unit of the photosensitive electrode), forming a periodic or arrayed arrangement. The opening 37 of the micro-well is located on the surface of the electrode substrate and can be circular, square or polygonal (circular is preferred to reduce light scattering).

[0056] When using a microfluidic chip with the above structure, a cell suspension containing adherent cells is first introduced into the culture space and / or culture chamber through the inlet, so that the cells are randomly distributed on the electrode unit. Then, the chip is placed in an incubator with suitable conditions (e.g., 37°C, 5% CO2 concentration) and perfused until the cells adhere and grow on the exposed electrodes.

[0057] Then, the microfluidic chip is connected to the sorting system described in Example 1 for sterilization. Specifically, during sterilization, the ultraviolet light spot generated by the optical path system irradiates the bottom of the microfluidic chip from bottom to top. At this time, the resistance of the gate electrode in the part irradiated by ultraviolet light drops significantly, thereby increasing the voltage on the surface of the gold electrode, enhancing the electric field focusing effect, and forming a conductive channel. This causes irreversible electroporation of the cells to be sterilized in this part (sterilization area), thereby achieving "point-to-point" precise sterilization of the cells to be sterilized. Throughout the process, the cells remain in situ, thus effectively avoiding cell damage caused by cell movement and the accidental killing of unstable suspended cells. In other words, this solution does not require introducing cells into a designated channel and has at least the following advantages: 1. The entire sterilization process does not require digestion, the target cells have high activity, and morphological information in the adherent state can be obtained; 2. It supports fluorescence + cell image composite analysis; 3. High throughput; 4. High accuracy; 5. It can obtain dynamic long-term cell information in culture, etc.

[0058] In summary, this embodiment provides a low-cost, "photoelectric synergy" reverse precision killing scheme for individual adherent cells, which is especially suitable for situations with a small total number of cells (when there are few cells, some electrode units may not have cells at their corresponding positions, meaning that there may be at least one electrode unit between two adjacent cells. In this case, even if a small amount of crosstalk occurs, it is difficult to affect adjacent cells).

[0059] Example 3 This embodiment is basically as follows: Figure 4 and Figure 8 As shown: This embodiment provides a microfluidic chip for adhering cell killing based on discrete photosensitive gating. Unlike the microfluidic chip in Embodiment 2, the gating electrode 33 and the protection electrode 32 of the microfluidic chip in this embodiment adopt the same patterned design. Specifically, the gating electrode 33 can also be composed of multiple independent electrode units. The electrode units in the gating electrode 33 and the protection electrode 32 correspond one-to-one to form multiple independent conductive channels.

[0060] In some embodiments, an isolation structure 35 is provided between two adjacent conductive channels. The isolation structure 35 may be at least one of non-conductive materials such as photoresist, aluminum nitride, titanium dioxide, polytetrafluoroethylene, silicon nitride, and fiberglass. Multiple isolation structures 35 together form a grid-like structure to isolate multiple electrode units of the gate electrode 33.

[0061] In some embodiments, the isolation structure 35 is disposed on the lower transparent electrode substrate 34, and the height of the isolation structure 35 is equal to the height of the gate electrode 33, such that two adjacent electrode units in the gate electrode 33 are completely isolated laterally, thereby reducing crosstalk. In other embodiments, the height of the isolation structure 35 is greater than the height of the gate electrode 33, such that the entire gate electrode 33 and at least a portion of the protection electrode 32 are isolated, thereby further reducing crosstalk between adjacent electrode units.

[0062] Microfluidic chips with the above structure form multiple independent conductive channels through the synergistic effect of patterned photosensitive electrodes and protective electrodes with multiple independent electrode units. In other words, through the synchronous patterning design of the gating electrode and the protective electrode, vertically independent conductive channels are constructed to achieve precise spatial positioning of photoelectric signals. This is especially suitable for sorting systems with a large number of cells or higher requirements for sorting accuracy. In other words, even when the photoresistivity of the gating electrode is relatively low, it can still ensure low crosstalk and high-precision killing.

[0063] Example 4 This embodiment is basically as follows: Figures 5-8 As shown: This embodiment provides a simplified microfluidic chip for adhering cell killing based on photosensitive gating. Unlike the microfluidic chip in Embodiment 2, the composite electrode layer in this embodiment adopts a "double-layer structure" and does not require a protective electrode. The composite electrode layer 103 includes a lower transparent electrode substrate 34 and a gating electrode 33 disposed on the upper surface of the lower transparent electrode substrate 34. The gating electrode 33 is a photosensitive electrode, and a culture chamber is disposed on the upper surface of the gating electrode 33. That is to say, the composite electrode layer 103 in this embodiment only has a lower transparent electrode substrate 34 and a gating electrode 33, which is especially suitable for gating electrodes 33 with good performance (e.g., biocompatibility, corrosion resistance, wear resistance, etc.).

[0064] In use, a cell suspension containing adherent cells is first introduced into the culture space and / or culture chamber through the inlet. The cells grow directly on the upper surface of the gate electrode. Then, the cells are placed in an incubator under suitable conditions (e.g., 37°C, 5% CO2 concentration) and perfused until they adhere to the exposed electrode. Next, the microfluidic chip is connected to the sorting system described in Example 1 for cell elimination. Specifically, during elimination, an ultraviolet light spot generated by the optical path system irradiates the bottom of the microfluidic chip from bottom to top. At this time, the resistance of the gate electrode in the ultraviolet-irradiated part decreases significantly, the voltage in this part increases, the electric field focusing effect is enhanced, and a conductive channel is formed. This causes irreversible electroporation of the cells to be eliminated in this part (elimination area), thereby achieving precise "point-to-point" elimination of the cells.

[0065] In some embodiments, the gate electrode 33 is a one-piece gate electrode sheet, which is a seamless electrode sheet (see [reference]). Figure 5 Preferably, the gate electrode sheet covers the upper surface of the upper transparent electrode substrate 34. In the absence of ultraviolet light irradiation, the gate electrode sheet has a high resistance, which blocks or limits the current between the upper electrode 102 and the lower transparent electrode substrate 34 to a very small value. When ultraviolet light irradiates a portion of the gate electrode, the resistance of that portion decreases significantly, forming a conductive channel between the upper electrode 102 and the lower transparent electrode substrate 34. This method is particularly suitable for gate electrode sheets with high photoresistivity and dark resistance. The composite electrode layer 103 with the above structure can reduce the structural complexity and fabrication difficulty of the microfluidic chip while ensuring the killing accuracy (reducing lateral crosstalk). In other words, this solution provides a low-cost, simple, high-precision "photoelectric combination" in-situ reverse cell killing solution.

[0066] In other embodiments, the gate electrode is a patterned gate electrode (see [link to documentation]). Figure 6For example, the patterned gate electrode can be composed of multiple independent electrode units, with an isolation structure 35 disposed between adjacent electrode units. The isolation structure 35 can be at least one of non-conductive materials such as photoresist, aluminum nitride, titanium dioxide, polytetrafluoroethylene, silicon nitride, and fiberglass. Multiple isolation structures 35 together form a grid-like structure to isolate the multiple electrode units of the gate electrode 33.

[0067] In some embodiments, the isolation structure 35 is disposed on the lower transparent electrode substrate 34. Preferably, the height of the isolation structure 35 is equal to the height of the gate electrode 33, so that two adjacent electrode units in the gate electrode 33 are completely isolated in the lateral direction, thereby reducing crosstalk.

[0068] In other embodiments, the gate electrode 33 is a patterned gate electrode. An insulating substrate 106 is disposed on the lower transparent electrode substrate, and the insulating substrate 106 has a plurality of insulating holes 107. The patterned gate electrode 33 is disposed within the insulating holes 107, so as to be isolated by the insulating substrate 106 to form multiple independent portions. In some specific embodiments, the thickness of the insulating substrate 106 is the same as the thickness of the gate electrode 33. Specifically, when processing the composite electrode layer, a grid-like insulating substrate 106 can be sputtered onto the lower transparent electrode substrate 34 first, and then photosensitive material can be sputtered into the insulating holes 107 portions of the grid-like insulating substrate 106 to form the gate electrode 33. Finally, the insulating substrate 106 and the photosensitive material are on the same plane, that is, the insulating substrate 106 and the photosensitive material are coplanar. The composite electrode layer with the above structure can further prevent the gate electrode from generating lateral crosstalk, and is especially suitable for sorting systems with higher precision requirements for extermination.

[0069] Example 5 See Figure 14 Based on the sorting system in Embodiment 1, this embodiment provides a control method for a cell reverse killing optical path system based on DMD light patterns. The optical path system includes a dual-mode light source, a digital micromirror device, a projection imaging system, a microfluidic chip platform, an image acquisition device, and a host computer. The dual-mode light source includes a white light mode and an ultraviolet light mode. The control method includes the following steps: S10, an initial image of the microfluidic chip on the microfluidic chip platform is acquired by the image acquisition device; at least two sets of positioning points for calibration are pre-set on the microfluidic chip; and the location of at least one cell to be killed and the location of the positioning points are marked on the initial image; S20, the host computer generates an initial pattern based on the position of the cells to be killed and the position of the positioning point in the initial image; S30, the initial pattern is input into the digital micromirror device, and the dual-mode light source is controlled to illuminate the digital micromirror device in white light mode, and output a light spot corresponding to the initial image; the light spot includes a first light spot corresponding to each cell to be killed, and a second light spot corresponding to the positioning point; S40, Alignment is performed based on the positioning point and the position of the second light spot; S50, the current image of the microfluidic chip superimposed with the first light spot is acquired again; all images can be acquired by a projection imaging system.

[0070] S60, obtain the distance difference between the first light spot and the corresponding cell to be killed in the current image, and determine whether the distance difference is greater than a first preset threshold. If so, mark the first light spot as an incorrect light spot; otherwise, mark it as a correct light spot. The distance difference refers to the straight-line distance between the center point of the first light spot and the center point of the cell to be killed. The first preset threshold can be preset by the user or automatically generated based on the previous killing accuracy of the sorting system.

[0071] S70: Obtain a first ratio of the number of erroneous light spots to the number of correct light spots, and determine whether the first ratio is greater than a first preset ratio. If yes, execute S80; otherwise, execute S90. The first preset ratio can be preset by the user. The larger the first preset ratio, the lower the user's requirement for the killing accuracy. That is, the larger the first preset ratio, the larger the proportion of erroneous light spots, and the greater the possibility of the target cells being mistakenly killed.

[0072] S80: Obtain the offset direction between the erroneous light spot and the corresponding cell to be killed, and determine whether the multiple offset directions are consistent. If so, readjust the relative position of the light spot and the microfluidic chip according to the offset direction; otherwise, correct the multiple erroneous light spots and repeat S40-S70. The criterion for determining whether the offset directions are consistent is as follows: Obtain the angle between the offset direction and the horizontal plane (i.e., the offset angle); calculate the average offset angle of all erroneous light spots; calculate the difference between the offset angle of each erroneous light spot and the average offset angle; if the difference is less than a preset difference (which can be preset by the user), then the offset directions are consistent.

[0073] S90, the white light mode is switched to the ultraviolet light mode to obtain multiple first light spots in the ultraviolet light mode, and these spots are projected onto the microfluidic chip to change the resistance of the photosensitive electrode in the microfluidic chip.

[0074] In some embodiments, step S80, the step of correcting the plurality of erroneous light spots, includes: S81, the current image is divided into at least two sub-regions; S82, obtain the first number of cells to be killed in the current sub-region; S83, determine whether the first quantity is greater than a preset quantity. If so, merge multiple light spots in the current sub-region into a fused light spot that can cover the current sub-region; otherwise, independently adjust the position of the current erroneous light spot according to the distance difference and offset direction to adjust the erroneous light spot into a correct light spot. The preset quantity can be pre-set by the user. The method for independently adjusting the position of the current erroneous light spot according to the distance difference and offset direction is as follows: based on the distance difference and offset direction between the erroneous light spot and the cells to be killed, shift the position of the erroneous light spot, and input the shift direction (i.e., the opposite direction of the offset direction) and the shift distance (i.e., the distance difference) to the host computer. The host computer controls the dual-mode light source to irradiate the digital micromirror device in white light mode to generate a corrected light spot. In some embodiments, before step S83, the method further includes: S84, obtain the number of target cells in the current sub-region; wherein, the number of target cells and cells to be killed can be obtained by analyzing the acquired image information (different cells have different morphologies and can be distinguished by manual or machine analysis).

[0075] S85, calculate the actual ratio of the number of cells to be killed to the number of target cells, and determine whether the actual ratio is greater than a preset ratio; if so, execute S83; otherwise, independently adjust the position of the current erroneous spot according to the distance difference to adjust the erroneous spot into a correct spot. In other words, this solution provides a dual judgment mechanism. By imposing dual restrictions on the adjustment of the spot (i.e., firstly, a preliminary judgment is made on the ratio of the erroneous spot to the correct spot; when the ratio of the correct spot is large, the erroneous spot is adjusted separately; further, when the ratio of the erroneous spot is large, the total number of erroneous spots is judged, and cases with a large number of erroneous spots are merged), the killing efficiency is improved while the false kill rate of target cells is made more controllable, thereby further ensuring the killing quality.

[0076] In some embodiments, the step of readjusting the relative position of the light spot and the microfluidic chip according to the offset direction includes: S86, calculate the average distance difference based on the distance difference between the erroneous light spot and the cell to be killed; S87, adjust the angle of the digital micromirror device and / or adjust the position of the microfluidic chip according to the average distance difference and the offset direction.

[0077] In some specific embodiments, the microfluidic chip platform includes at least one three-axis moving platform and a gripper disposed on the three-axis moving platform. The microfluidic chip is clamped on the three-axis moving platform by the gripper and can move synchronously with the three-axis moving platform.

[0078] In step S80, the step of readjusting the relative position of the light spot and the microfluidic chip according to the offset direction includes: S86, calculate the average distance difference based on the distance difference between all the erroneous light spots and the cells to be killed; S87, adjust the angle of the digital micromirror device and / or adjust the position of the microfluidic chip using the three-axis moving platform according to the average distance difference and the offset direction. Specifically, when adjusting the angle of the digital micromirror device, the light spot is moved a specified distance (i.e., the average distance difference) along the offset direction by rotating the angle of the digital micromirror device; when adjusting the position of the microfluidic chip, the microfluidic chip is moved a specified distance (i.e., the average distance difference) in the opposite direction of the offset direction.

[0079] In some embodiments, step S87 specifically includes: If the average distance difference is less than a second preset threshold, the angle of the digital micromirror device is adjusted according to the average distance difference; otherwise, the position of the microfluidic chip is adjusted via the three-axis moving platform according to the average distance difference. The second preset threshold can be preset by the user or automatically generated based on the previous elimination accuracy of the sorting system. In other words, for cases where the overall position of the light spot shifts (the relative position of the light spot and the microfluidic chip shifts), this application also provides a hierarchical control method. Specifically, for cases with large deviations, a lower-precision microfluidic chip platform is used for adjustment, while for cases with small deviations, a higher-precision digital micromirror device is used for adjustment. This reduces the operational difficulty of the equipment and avoids wear and tear caused by frequent adjustments to the high-precision instrument (digital micromirror device), as well as compensating for the limited rotation angle of the digital micromirror device.

[0080] In summary, for optical path systems used for reverse killing of in situ cells, this application provides a real-time image feedback pre-alignment mechanism and a dual-pathway control mechanism to improve the accuracy of the correspondence between the microfluidic chip and the light spot, thereby improving the accuracy of killing adherent cells.

[0081] First, this application uses a white light mode for pre-alignment. By comparing the position of the light spot with the position of the cells to be killed in the microfluidic chip, and when the positional deviation between the two is large, the position of the light spot or the microfluidic chip is specifically adjusted to avoid the "false killing" problem caused by the positional deviation between the two.

[0082] Furthermore, this application distinguishes the deviation between the light spot and the position of the cells to be killed in the microfluidic chip. On the one hand, for cases with large deviations, a microfluidic chip platform with lower precision is used for adjustment, while for cases with small deviations, a digital micromirror device with higher precision is used for adjustment. On the other hand, this reduces the difficulty of operating the equipment and avoids the wear and tear caused by frequent adjustments to the high-precision instrument (digital micromirror device), as well as compensating for the limited rotation angle of the digital micromirror device.

[0083] Furthermore, this application proposes a zoned elimination scheme, which targets areas where cells to be eliminated are concentrated for concentrated elimination, thereby reducing the pressure on light spot regulation and improving elimination efficiency while ensuring the survival rate of target cells.

[0084] Example 6 See Figure 15 This embodiment provides an optical path system for reverse killing of in situ cells, including a dual-mode light source, a digital micromirror device, a projection imaging system, a microfluidic chip platform, an image acquisition device, and a host computer. The dual-mode light source includes a white light mode and an ultraviolet light mode, and further includes: Data acquisition module: configured to acquire an initial image of the microfluidic chip on the microfluidic chip platform via the image acquisition device; the microfluidic chip has at least two sets of positioning points pre-set for calibration; and to mark the location of at least one cell to be killed and the location of the positioning points on the initial image; Pattern generation module: configured to generate an initial pattern via the host computer based on the position of the cells to be killed and the position of the positioning point in the initial image; A light spot generation module is configured to input the initial pattern into the digital micromirror device, control the dual-mode light source to illuminate the digital micromirror device in white light mode, and output a light spot corresponding to the initial image; the light spot includes a first light spot corresponding to each cell to be killed, and a second light spot corresponding to the positioning point; Position calibration module: Aligns the first light spot with the position of the positioning point and the second light spot; obtains the distance difference between the first light spot and the corresponding cell to be killed in the current image, and determines whether the distance difference is greater than a first preset threshold. If so, the first light spot is marked as an incorrect light spot; otherwise, it is marked as a correct light spot. Obtains a first ratio of the number of incorrect light spots to the number of correct light spots, and determines whether the first ratio is greater than a first preset ratio. If so, the position of the light spot is calibrated. Specifically, when calibrating the position of the light spot, the position calibration module is configured to acquire the offset direction between the erroneous light spot and the corresponding cell to be killed, and determine whether the multiple offset directions are consistent. If so, the relative position of the light spot and the microfluidic chip is readjusted according to the offset direction; otherwise, the multiple erroneous light spots are corrected. Mode switching module: configured to switch the white light mode to the ultraviolet light mode, obtain multiple first light spots in the ultraviolet light mode, and project them onto the microfluidic chip to change the resistance of the photosensitive electrode in the microfluidic chip.

[0085] In some embodiments, the optical path system further includes: Correction module: configured to divide the current image into at least two sub-regions; obtain a first number of cells to be killed in the current sub-region; determine whether the first number is greater than a preset number; if so, merge multiple light spots in the current sub-region into a fused light spot that can cover the current sub-region; otherwise, independently adjust the position of the current erroneous light spot according to the distance difference and the offset direction to adjust the erroneous light spot into a correct light spot.

[0086] In some embodiments, the correction module is further configured to: obtain the number of target cells in the current sub-region; calculate the actual ratio of the number of cells to be killed to the number of target cells, and determine whether the actual ratio is greater than a preset ratio; if so, execute S83; otherwise, independently adjust the position of the current erroneous spot according to the distance difference to adjust the erroneous spot into a correct spot.

[0087] In some embodiments, when readjusting the relative position of the light spot and the microfluidic chip according to the offset direction, the position calibration module is specifically configured to: The average distance difference is calculated based on the distance difference between the erroneous light spot and the cells to be killed; the angle of the digital micromirror device and / or the position of the microfluidic chip are adjusted based on the average distance difference and the offset direction.

[0088] In some embodiments, when adjusting the angle of the digital micromirror device and / or adjusting the position of the microfluidic chip based on the average distance difference and the offset direction, the position calibration module is specifically configured to: If the average distance difference is less than a second preset threshold, the angle of the digital micromirror device is adjusted according to the average distance difference; otherwise, the position of the microfluidic chip is adjusted by the three-axis moving platform according to the average distance difference.

[0089] The optical path system with the above structure is particularly suitable for in-situ reverse killing of adherent cells. It provides a real-time image feedback pre-alignment mechanism and a dual-pathway control mechanism to improve the accuracy of the correspondence between the microfluidic chip and the light spot, thereby improving the accuracy of killing adherent cells.

[0090] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0091] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A low lateral crosstalk composite electrode layer for a microfluidic chip used for adhering cell killing, characterized in that, The composite electrode layer includes a protective electrode, a gate electrode, and a lower transparent electrode substrate arranged sequentially from top to bottom. The protective electrode is a patterned microelectrode array, which includes multiple independent electrode units. A culture chamber is provided on the upper surface of each electrode unit. The gate electrode and the protective electrode are patterned in the same or similar way to form multiple independent conductive channels. The gate electrode is a photosensitive electrode. The gate electrode includes multiple independent electrode units, and the electrode units of the protective electrode and the electrode units of the gate electrode correspond one-to-one to form multiple independent conductive channels. An isolation structure is provided between two adjacent conductive channels. The isolation structure is disposed on the lower transparent electrode substrate, and the height of the isolation structure is equal to or greater than the thickness of the gate electrode.

2. The low lateral crosstalk composite electrode layer for adherent cell killing microfluidic chip according to claim 1, characterized in that, The side length of the electrode unit is 1μm-50μm, and the spacing between two adjacent electrode units is 1μm-30μm.

3. The low lateral crosstalk composite electrode layer for microfluidic chips used for adhering cell killing according to claim 1, characterized in that, The protective electrode is a gold layer, the material of the gate electrode layer is selected from zinc oxide, indium tin oxide, phthalocyanine compounds and GaN photosensitive materials, and the material of the lower transparent electrode substrate is selected from indium tin oxide, tin oxide, indium oxide, zinc oxide, titanium oxide and nickel oxide transparent conductive materials.

4. The low lateral crosstalk composite electrode layer for adherent cell killing microfluidic chip according to claim 1, characterized in that, The lower transparent electrode substrate is a common electrode.

5. The low lateral crosstalk composite electrode layer for microfluidic chips used for adhering cell killing according to claim 1, characterized in that, The patterned microelectrode array is square or rectangular.

6. The low lateral crosstalk composite electrode layer for adherent cell killing microfluidic chip according to claim 1, characterized in that, The composite electrode layer further includes an insulating layer, which has a plurality of independent through holes corresponding to the pattern of the protective electrode. The through holes cooperate with the upper surface of the protective electrode to form the culture chamber.

7. The low lateral crosstalk composite electrode layer for adherent cell killing microfluidic chip according to claim 6, characterized in that, The side length of the through hole is smaller than the side length of the electrode unit, and the thickness of the insulating layer is 1μm-5μm.

8. The low lateral crosstalk composite electrode layer for adherent cell killing microfluidic chip according to claim 1, characterized in that, The upper surface of the lower transparent electrode substrate is provided with a plurality of microwell openings corresponding to the electrode units.

9. The low lateral crosstalk composite electrode layer for adherent cell killing microfluidic chip according to claim 8, characterized in that, The microwell opening is a micro-recessed structure on the upper surface of the lower transparent electrode substrate, and each microwell corresponds to an electrode unit.

10. The low lateral crosstalk composite electrode layer for adherent cell killing microfluidic chip according to claim 1, characterized in that, The number of electrode units is 100-1×10 8 indivual.

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