Intracellular biomass detection device integrating electroporation sampling and nanopore detection

Abstract

This invention discloses an integrated intracellular biomass detection device combining electroporation sampling and nanopore detection, relating to the field of cell detection technology. This invention integrates electroporation sampling and nanopore detection on the same substrate, and introduces a unidirectional main channel with a Tesla valve structure on the substrate to achieve physical connectivity on the fluid side and maintain effective isolation on the electrical side. Transport is completed through a unidirectional path while maintaining high cell viability, avoiding damage to cell activity and eliminating the need for manual transfer of extracted biomolecules. Based on the characteristics of the Tesla valve structure, this invention supports the integration of multiple electroporation sampling methods and enables diverse detection methods.

Description

Technical Field

[0001] This utility model relates to the field of cell detection technology, and more specifically, to an intracellular biomass detection device that integrates electroporation sampling and nanopore detection. Background Technology

[0002] Intracellular and in vivo biomolecules such as nucleic acids, enzymes, proteins, and neurotransmitters are crucial in regulating cellular activity and bodily functions. Accurately obtaining their dynamic expression in living cells and organisms (i.e., quantifying their biomass) is of great significance for the early diagnosis of major diseases, new drug development, and precision medicine.

[0003] Current technologies require two separate devices for biomolecule sampling and biomass detection in cells. This involves inserting an optical probe into the cell for molecule extraction, followed by the injection of labeled materials into the cell for fluorescence resolution if necessary, and then transferring the extracted biomolecules to a detection device for biomass detection. On the one hand, these extraction methods can cause irreversible damage to cells or lead to long-term accumulation of labeled materials within the cells, potentially causing cytotoxicity and interfering with biological functions, making it difficult to meet the requirements for dynamic, label-free, and low-disturbance detection of live cells. On the other hand, the transfer and injection of extracted biomolecules into the detection device is generally done manually by operators, which can easily lead to biomolecule loss and temporal drift. Utility Model Content

[0004] Therefore, it is necessary to provide an intracellular biomass detection device that integrates electroporation sampling and nanopore detection, addressing the problems that existing methods can easily cause cell viability damage, biomolecule loss, and temporal drift.

[0005] This utility model is achieved using the following technical solution: This utility model discloses an intracellular biomass detection device that integrates electroporation sampling and nanopore detection, comprising: at least one detection unit.

[0006] The detection unit includes a substrate and a cover plate stacked together.

[0007] The substrate has a unidirectional main channel with a Tesla valve structure along its width on the side facing the cover plate. Several cell sampling slots are located at one end of the substrate, and a molecular detection slot is located at the other end. When the substrate is erected along its length, the cell sampling slots are located at the top of the substrate, the molecular detection slots are located at the bottom, and the unidirectional main channel is located between them. The bottom of the cell sampling slots is connected to the unidirectional main channel via unidirectional branch channels. The cell sampling slots are equipped with cell sampling components for electroporation sampling and for allowing the sampled biomolecules to flow from the unidirectional branch channels into the unidirectional main channel. A pump is connected to the forward inlet of the unidirectional main channel, and the forward outlet is connected to the molecular detection slot. The molecular detection slot is equipped with molecular detection components for biomass detection via nanopores.

[0008] As a further embodiment of this utility model: the substrate is any one of glass plate, silicon plate, PDMS plate, and COC plate; Or / and, the cover plate is made of transparent sheet material.

[0009] As a further embodiment of this utility model: the cell sampling assembly includes: a sampling top electrode and a sampling bottom electrode; When the substrate is erected along its length, the top sampling electrode is located at the top of the cell sampling groove, and the bottom sampling electrode is located at the bottom of the cell sampling groove. The bottom sampling electrode has a through hole and is connected to the positive inlet of the unidirectional branch channel. A filter membrane is installed in the through hole to prevent cells from flowing through. The top sampling electrode and the bottom sampling electrode are connected to an external pulse signal to realize electroporation sampling of cells in the cell sampling groove.

[0010] As a further embodiment of this invention: several cell sampling slots are arranged side by side and separated by partitions; the two top sampling electrodes of adjacent cell sampling slots are connected together; the two bottom sampling electrodes of adjacent cell sampling slots are separated by partitions.

[0011] As a further embodiment of this utility model: a one-way valve is provided in the one-way branch channel; Alternatively, the unidirectional branch channel may have a Tesla valve structure.

[0012] As a further embodiment of this invention: the molecular detection component includes: a separator membrane, a top detection electrode, and a bottom detection electrode; the separator membrane divides the molecular detection cell into cis-compartment and trans-compartment, the cis-compartment being connected to the forward outlet of the unidirectional main channel; the separator membrane is provided with at least one nanopore to connect the cis-compartment and trans-compartment; the top detection electrode is placed in the cis-compartment, and the bottom detection electrode is placed in the trans-compartment; the top detection electrode and the bottom detection electrode are externally biased to cause the biomolecules flowing into the cis-compartment to undergo electrophoretic movement and interact with the nanopore on the separator membrane to generate a current signal characterizing biomass information.

[0013] As a further embodiment of this invention, the separator is any one of SiN film, SiO2 film, graphene film, and h-BN film.

[0014] As a further embodiment of this utility model: when the base is erected along its length, the oriented compartment is located above the inverted compartment; the top detection electrode is located at the top of the oriented compartment; and the bottom detection electrode is located at the bottom of the inverted compartment.

[0015] As a further embodiment of this utility model, the detection unit also includes: a pulse signal source, a bias voltage source, a signal acquisition device, and a host computer.

[0016] A pulse signal source is electrically connected to the sampling top and bottom electrodes to provide pulse signals. A bias voltage source is electrically connected to the detection top and bottom electrodes to provide bias voltage. A signal acquisition unit is positioned near the nanopores on the septum membrane to acquire current signals characterizing biomass information. A host computer communicates with the signal acquisition unit to obtain the current signals acquired by the acquisition unit and analyzes and processes them to obtain biomass information.

[0017] As a further embodiment of this utility model: the opposite surfaces of the base and the cover plate are provided with matching mating holes and mating posts.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention integrates electroporation sampling and nanopore detection on the same substrate, and introduces a unidirectional main channel with a Tesla valve structure on the substrate to achieve physical connection on the fluid side and maintain effective isolation on the electrical side. The transport is completed through a unidirectional path and high cell viability is maintained, which not only avoids damage to cell activity, but also eliminates the need for manual transfer of extracted biomolecules.

[0019] 2. Based on the characteristics of the Tesla valve structure, this utility model supports the integration of multiple sets of electroporation samples and supports the realization of diversified detection. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this utility model or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A top view of the intracellular biomass detection device integrating electroporation sampling and nanopore detection provided by this utility model; Figure 2A side view of an intracellular biomass detection device that integrates electroporation sampling and nanopore detection; The symbols in the attached diagram have the following meanings: 100. Base; 110. Cover plate; 210. One-way main flow channel; 220. One-way branch flow channel; 230. One-way valve; 240. Pump. 300. Cell sampling groove; 310. Sampling top electrode; 320. Sampling bottom electrode; 410. Detection top electrode; 420. Detection bottom electrode; 430. Cis-compartment; 440. Separating membrane; 450. Reverse-compartment. Detailed Implementation

[0022] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0023] It should be noted that when a component is said to be "installed on" another component, it can be directly on the other component or it may be in a component that is centered on it. When a component is said to be "set on" another component, it can be directly set on the other component or it may also be in a component that is centered on it. When a component is said to be "fixed to" another component, it can be directly fixed to the other component or it may also be in a component that is centered on it.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "or / and" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] Example 1 This embodiment provides an intracellular biomass detection device integrating electroporation sampling and nanopore detection, comprising at least one detection unit. See also... Figure 1 This diagram shows the structure of a single detection unit, which integrates electroporation sampling and nanopore detection on the same substrate. Specifically, electroporation sampling is performed at the electroporation sampling point on one side, and the sample is transmitted to the nanopore detection point on the other side through a unidirectional main channel for nanopore detection.

[0026] See Figure 2Taking a single detection unit as an example, it includes: a substrate and a cover plate stacked together; it may also include: a pulse signal source, a bias voltage source, a signal acquisition device, and a host computer.

[0027] The substrate and cover plate can be integrated using bonding methods such as plasma bonding, hot pressing, or adhesive bonding. Of course, for ease of processing, matching mating holes and posts can be pre-set on the opposing surfaces of the substrate and cover plate for positioning. As the base plate of the entire detection unit, the substrate needs to provide a certain degree of structural support. Generally, it is recommended to use any one of the following substrates: glass plate, silicon plate, PDMS plate, or COC plate. For easy observation, a transparent material—such as a glass plate—is recommended for the cover plate.

[0028] See Figure 1 The substrate, facing the cover plate, has a unidirectional main channel with a Tesla valve structure along its width. Generally, the unidirectional main channel is fabricated on the substrate using methods such as etching, milling, or injection molding. Figure 1 In the central direction, the unidirectional main channel is set along the left and right directions, with the positive inlet located at the left end and the positive outlet at the right end. Several cell sampling slots are set at one end of the substrate, and molecular detection slots are set at the other end. Similarly, the cell sampling slots and molecular detection slots are fabricated on the substrate using methods such as etching, milling, and injection molding.

[0029] The cell sampling cell is equipped with a cell sampling assembly for electroporation sampling of cells, allowing the sampled biomolecules to flow from a unidirectional branch channel into a unidirectional main channel. The unidirectional main channel serves as the transport channel from electroporation sampling to nanopore detection. To ensure smooth flow, a pump is connected to the forward inlet of the unidirectional main channel, and the forward outlet is connected to the molecular detection cell. The molecular detection cell is equipped with a molecular detection assembly to achieve biomass detection via nanopores.

[0030] like Figure 1 As shown, when the substrate is erected along its length, the cell sampling groove is located at the top of the substrate, the molecular detection groove is located at the bottom of the substrate, and the unidirectional main channel is located between the two. The bottom of the cell sampling groove is connected to the unidirectional main channel through a unidirectional branch channel.

[0031] In addition, the substrate or cover plate should have necessary through holes (controlled by plugs) for the cell sampling and molecular detection cells to facilitate media injection before detection (e.g., injecting an electrolyte solution containing cells into the cell sampling cell, injecting an appropriate amount of electrolyte solution into the molecular detection cell), and injection and drainage of rinsing solution after detection for cleaning. It is important to note that during sampling and detection in the detection unit, the pump provides inlet pressure to the unidirectional main channel; during cleaning of the detection unit, the pump can introduce rinsing solution to flush the unidirectional main channel.

[0032] ① For the electroporation sampling side: Specifically, an electrolyte solution containing cells is injected into the cell sampling tank. For a single cell sampling assembly, it includes a top sampling electrode and a bottom sampling electrode. When the substrate is erected along its length, the top sampling electrode is located at the top of the cell sampling tank, and the bottom sampling electrode is located at the bottom. The bottom sampling electrode has a through-hole connected to the forward inlet of a one-way branch channel; a filter membrane is installed within the through-hole to prevent cells from flowing through; the top and bottom sampling electrodes are externally connected to a pulse signal to achieve electroporation sampling of cells in the cell sampling tank—the pulse signal causes the cell membrane to form instantaneous reversible pores, allowing biomolecules to flow from the cell into the electrolyte solution within the cell sampling tank for sampling. The cell membrane can subsequently recover, avoiding damage to cell viability. The pulse signal can be input via an external signal line—for example, by setting up a pulse signal source and electrically connecting it to the top and bottom sampling electrodes to provide the pulse signal.

[0033] like Figure 1 As shown, in this embodiment, several cell sampling slots are arranged side by side and separated by partitions; the two sampling top electrodes of adjacent cell sampling slots are connected together; the two sampling bottom electrodes of adjacent cell sampling slots are separated by partitions. This can save the number of wiring connections for applying signals to the sampling top electrodes.

[0034] Of course, to achieve control over electroporation sampling at various locations, an independent pulse signal should be applied to each cell sampling component. For example, the pulse signal source can be set to match the number of cell sampling components and correspond one-to-one with multiple cell sampling components, thus achieving independent control. Alternatively, only one pulse signal source can be set—multiple cell sampling components are connected in parallel and then connected to the same pulse signal source through multiple switches, thereby controlling the operation of the required cell sampling components by controlling the switches.

[0035] ② Regarding the transmission process: For a unidirectional main channel, the Tesla valve structure it adopts has the characteristic of unidirectional flow - that is, it can flow smoothly from the positive inlet to the positive outlet, while there is huge resistance in the reverse direction, which can effectively suppress backflow and cross-contamination.

[0036] Of course, similar to the main unidirectional channel, the unidirectional branch channel needs to avoid backflow. Therefore, a one-way valve can be installed in the unidirectional branch channel, or a unidirectional branch channel with a Tesla valve structure can be used.

[0037] ③ For the nanopore detection side: Specifically, an appropriate amount of electrolyte solution is pre-filled into the molecular detection cell. The molecular detection assembly includes: a septum membrane, a top detection electrode, and a bottom detection electrode.

[0038] A separator membrane divides the molecular detection cell into cis and trans compartments, with the cis compartment connected to the forward outlet of the unidirectional main channel. The separator membrane can be any of SiN, SiO2, graphene, or h-BN films. The separator membrane has at least one nanopore (which can be fabricated through dielectric breakdown, FIB or EB opening, template transfer, etc., and its wetting and selectivity can be controlled by ALD or chemical modification if necessary) to connect the cis and trans compartments. The top detection electrode is placed in the cis compartment, and the bottom detection electrode is placed in the trans compartment. An external bias voltage is applied to the top and bottom detection electrodes to cause biomolecules flowing into the cis compartment to undergo electrophoretic movement and interact with the nanopores on the separator membrane, generating a current signal characterizing biomass information—biomolecules undergo electrophoretic movement under the bias voltage: if they successfully pass through the nanopores, a steady-state current signal is formed; if they block the nanopores, a blocking current signal is formed. The bias voltage can be input via an external signal line—for example, by setting a bias voltage source and electrically connecting it to the top and bottom detection electrodes to provide the bias voltage.

[0039] like Figure 1 As shown, in this embodiment, when the substrate is erected along its length, the compliant compartment is located above the inverse compartment; the top detection electrode is located at the top of the compliant compartment; and the bottom detection electrode is located at the bottom of the inverse compartment.

[0040] To quantify biomass information, a signal acquisition device can be placed near the nanopores on the separator to collect current signals that characterize biomass information. A host computer can be set up to communicate with the signal acquisition device to obtain the current signals collected by the signal acquisition device and analyze and process them to obtain biomass information.

[0041] Of course, if multiple detection units are set up, they work independently and do not interfere with each other, but can share the same host computer. In other words, the above detection units support multi-channel arraying; and combined with the above description, the above detection units are compatible with mainstream microfabrication processes, which is convenient for mass production.

[0042] In summary, due to the inclusion of multiple electroporation sampling points, diverse detection methods can be provided in practical applications: I. Perform single-cell detection: A single cell is placed in a cell sampling slot; Cell electroporation is performed using a cell sampling component, and the sampled biomolecules flow from a unidirectional branch channel into a unidirectional main channel, which then transports the sampled biomolecules to the molecular detection cell. Biomass detection is achieved through nanopore detection using molecular detection components.

[0043] II. Perform multi-cell reuse detection: Multiple cells are placed one-to-one into multiple cell sampling slots; Each cell was processed according to the single-cell detection method described in section I; after processing each cell, the device was rinsed clean before processing the next cell.

[0044] III. Perform pooled detection of multiple cells: Multiple cells are placed one-to-one into multiple cell sampling slots; Each cell is sampled by electroporation using a cell sampling component, and the sampled biomolecules flow from a unidirectional branch channel into a unidirectional main channel. The sampled biomolecules are then transported to the molecular detection cell through the unidirectional main channel to achieve mixing. Biomass detection is achieved through nanopore detection using molecular detection components.

[0045] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0046] The embodiments described above are merely illustrative of several implementations of this utility model, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the utility model patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this utility model, and these all fall within the protection scope of this utility model. Therefore, the protection scope of this utility model patent should be determined by the appended claims.

Claims

1. A device for detecting intracellular biomass integrating electroporation sampling and nanopore detection, characterized in that, It includes: At least one detection unit; wherein the detection unit includes: a substrate and a cover plate stacked together; The substrate has a unidirectional main channel with a Tesla valve structure along its width on the side facing the cover plate. Several cell sampling slots are located at one end of the substrate, and a molecular detection slot is located at the other end. A pump is connected to the forward inlet of the unidirectional main channel, and the forward outlet is connected to the molecular detection slot. When the substrate is erected along its length, the cell sampling slots are located at the top of the substrate, the molecular detection slots are located at the bottom of the substrate, and the unidirectional main channel is located between them. The bottom of the cell sampling slots is connected to the unidirectional main channel via unidirectional branch channels. The cell sampling slots are equipped with cell sampling components for electroporation sampling and for allowing the sampled biomolecules to flow from the unidirectional branch channels into the unidirectional main channel. The molecular detection slots are equipped with molecular detection components for biomass detection via nanopores.

2. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 1, characterized in that, The substrate can be any one of glass plate, silicon plate, PDMS plate, or COC plate; Or / and, the cover plate is made of transparent sheet material.

3. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 1, characterized in that, The cell sampling assembly includes: a top sampling electrode and a bottom sampling electrode; When the substrate is erected along its length, the top sampling electrode is located at the top of the cell sampling groove, and the bottom sampling electrode is located at the bottom of the cell sampling groove. The bottom sampling electrode has a through hole and is connected to the positive inlet of the unidirectional branch channel. A filter membrane is installed in the through hole to prevent cells from flowing through. The top sampling electrode and the bottom sampling electrode are connected to an external pulse signal to realize electroporation sampling of cells in the cell sampling groove.

4. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 3, characterized in that, Several cell sampling cells are arranged side by side and separated by partitions; the two top sampling electrodes of adjacent cell sampling cells are connected together; the two bottom sampling electrodes of adjacent cell sampling cells are separated by partitions.

5. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 1, characterized in that, A one-way valve is installed in the one-way branch channel; Alternatively, the unidirectional branch channel may have a Tesla valve structure.

6. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 1, characterized in that, The molecular detection assembly includes: a separator membrane, a top detection electrode, and a bottom detection electrode; the separator membrane divides the molecular detection cell into cis-compartment and trans-compartment, with the cis-compartment connected to the forward outlet of the unidirectional main channel; the separator membrane has at least one nanopore to connect the cis-compartment and trans-compartment; the top detection electrode is placed in the cis-compartment, and the bottom detection electrode is placed in the trans-compartment; the top detection electrode and the bottom detection electrode are externally biased to cause biomolecules flowing into the cis-compartment to undergo electrophoretic movement and interact with the nanopore on the separator membrane to generate a current signal characterizing biomass information.

7. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 1, characterized in that, The separator is any one of SiN film, SiO2 film, graphene film, or h-BN film.

8. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 1, characterized in that, When the substrate is erected along its length, the cis-type compartment is located above the inverse-type compartment; the top detection electrode is located at the top of the cis-type compartment; and the bottom detection electrode is located at the bottom of the inverse-type compartment.

9. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 1, characterized in that, The detection unit also includes: A pulse signal source, which is electrically connected to the top sampling electrode and the bottom sampling electrode to provide a pulse signal; A bias voltage source, which is electrically connected to the top and bottom detection electrodes to provide a bias voltage; A signal acquisition device, positioned near nanopores on a septal membrane, acquires current signals characterizing biomass information; and The host computer communicates with the signal acquisition unit to obtain the current signal collected by the signal acquisition unit and analyzes and processes it to obtain biomass information.

10. The intracellular biomass detection device integrating electroporation sampling and nanopore detection according to claim 9, characterized in that, The base and the cover plate have matching mating holes and mating posts on their opposite sides.

Images