An integrated intracellular biomass detection device

Abstract

This invention relates to an integrated intracellular biomass detection device in the field of biodetection technology. It includes at least one detection unit. The detection unit includes a sampling chamber, at least one microfluidic channel, and a detection chamber. The sampling chamber has an upper sampling electrode at its top and a lower sampling electrode at its bottom; a sampling hole is provided at the bottom of the sampling chamber; the upper and lower sampling electrodes are externally connected to a pulsed current to perform electroporation sampling of cells within the sampling chamber. The top of the microfluidic channel is connected to the sampling hole; a separator membrane is provided inside the detection chamber. This invention, by setting electrodes in the sampling chamber and detection chamber respectively and applying a low-amplitude pulsed voltage, causes a local electric field to accumulate at the cell membrane, forming reversible nanoscale membrane pores. Simultaneously, a controllable transmembrane mass transfer channel is constructed, allowing charged molecules within the cell to migrate to the external microenvironment under the drive of the electric field, and subsequently achieve real-time, label-free single-molecule detection via the detection chamber.

Description

Technical Field

[0001] This utility model relates to the field of biological detection technology, and in particular to an integrated intracellular biomass detection device. Background Technology

[0002] In the regulation of cellular activity and bodily functions, intracellular and in vivo biomolecules such as nucleic acids, proteins, enzymes, and neurotransmitters play crucial roles. Accurate monitoring of their dynamic changes in living cells and the in vivo environment is of great value for the early identification of major diseases, new drug screening, and precision medicine research. However, current detection technologies mainly rely on in vitro or extracellular analysis, often employing cell lysis and molecular extraction methods, which disrupt the original physiological state of cells, making it difficult to achieve dynamic and continuous monitoring of living cells. Even though some methods attempt to achieve in vivo imaging using optical probes or fluorescent labels, the long-term accumulation of labeled materials can easily lead to cytotoxicity and interfere with biological functions, while also presenting problems such as signal drift and limited spatial resolution. These factors collectively make it difficult for current technologies to achieve highly sensitive, label-free, and low-disturbance real-time detection while maintaining cell viability, becoming a major technical bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0003] To address the technical challenge of achieving highly sensitive, label-free, and low-disturbance real-time detection while maintaining cell viability using existing detection technologies, this invention provides an integrated intracellular biomass detection device.

[0004] This utility model is achieved through the following technical solution: An integrated intracellular biomass detection device includes at least one detection unit. The detection unit includes a sampling chamber, at least one microfluidic channel, and a detection chamber. The sampling chamber has an upper sampling electrode at its top and a lower sampling electrode at its bottom; a sampling orifice is formed at the bottom of the sampling chamber; the upper and lower sampling electrodes are externally connected to a pulsed current to perform electroporation sampling of cells within the sampling chamber; the top of the microfluidic channel is connected to the sampling orifice; a separator membrane is disposed inside the detection chamber, dividing the detection chamber into two chambers; nanopores connecting the two chambers are formed on the separator membrane; an upper detection electrode is disposed in one chamber, and this chamber is connected to the bottom of the microfluidic channel; a lower detection electrode is disposed in the other chamber; a bias voltage is externally connected to the upper and lower detection electrodes to cause biomolecules within the chamber to undergo electrophoretic movement and pass through the nanopores, generating a current signal characterizing biomass information.

[0005] As a preferred example, the upper sampling electrode, lower sampling electrode, upper detection electrode, and lower detection electrode are all sheet-like electrodes, which are attached to the inner wall of the corresponding chamber.

[0006] As a preferred example, the number of microfluidic channels is set to one.

[0007] As a preferred example, the number of microfluidic channels is set to five; the top of each of the five microfluidic channels is connected to the sampling chamber, and the bottom of each is connected to one of the chambers.

[0008] As a preferred example, the materials of the upper and lower sampling electrodes are Pt, Au, ITO, or carbon nanofilms; the materials of the separator are SiN, SiO2, graphene, h-BN, or Al2O3.

[0009] As a preferred example, the microfluidic channel is arranged along the direction of gravity to unidirectionally guide intracellular molecules into the chamber by gravity.

[0010] As a preferred example, the microfluidic channel is configured as a three-dimensional spiral with the following geometry: spiral radius R: channel diameter D = 5~30:1; curvature k = 1 / R ≥ 0.05mm. -1 The number of microfluidic channels should be at least 2.

[0011] As a preferred example, the chamber communicating with the microfluidic channel is configured as a funnel shape, with the bottom of the funnel-shaped chamber corresponding to the nanopore.

[0012] As a preferred example, the detection unit further includes: a pulse voltage source, a driving voltage source, a signal acquisition unit, and a host computer. The pulse voltage source is electrically connected to the upper and lower sampling electrodes and provides a pulse current. The driving voltage source is electrically connected to the upper and lower detection electrodes and provides a bias voltage. The signal acquisition unit is used to acquire current signals characterizing biomass information. The host computer is communicatively connected to the signal acquisition unit to obtain the current signals acquired by the signal acquisition unit and to analyze and process them to obtain biomass information.

[0013] As a preferred example, the detection unit also includes a substrate; both the sampling chamber and the detection chamber are located on the substrate; and the substrate has mounting grooves for mounting microfluidic channels.

[0014] As a preferred example, a through groove is formed on the substrate; transparent cover plates are attached to both sides of the substrate; the cover plates on both sides seal the groove to form a sampling chamber and a testing chamber.

[0015] The beneficial effects of this utility model are as follows: This invention couples electroporation sampling with solid-state nanopore single-molecule detection within the same chip platform. By setting electrodes in the sampling chamber and the detection chamber respectively and applying low-amplitude pulsed voltage, a local electric field is generated at the cell membrane to form reversible nanoscale membrane pores. At the same time, a controllable transmembrane mass transfer channel is constructed, allowing charged molecules in the cell to migrate to the external microenvironment under the drive of the electric field. Subsequently, real-time, label-free single-molecule detection is achieved in the detection chamber. This enables high sensitivity, low perturbation, and high throughput detection performance while maintaining high cell viability, providing a new integrated analysis platform for drug screening, metabolic regulation, and disease model research. Attached Figure Description

[0016] 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.

[0017] Figure 1 This is a schematic diagram of the structure of an integrated intracellular biomass detection device; Figure 2 This is a schematic diagram of the structure of an integrated intracellular biomass detection device in another embodiment.

[0018] In the figure: substrate 1, sampling chamber 11, upper sampling electrode 111, lower sampling electrode 112, mounting groove 12, microfluidic channel 121, upper detection electrode 131, upper chamber 132, septum 133, lower chamber 134, lower detection electrode 135. Detailed Implementation

[0019] The technical solutions in the embodiments of this utility model will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this utility model, and not all embodiments. Based on the embodiments of this utility model, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this utility model.

[0020] 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.

[0021] 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.

[0022] This invention enables low-disturbance sampling, unidirectional transport, and single-molecule resolution detection of charged molecules within cells. Specifically, this embodiment proposes an integrated intracellular biomass detection device, comprising at least one detection unit, with each detection module being independently configured without interconnection. Taking one detection unit as an example, it includes: a sampling chamber 11, at least one microfluidic channel 121, a detection chamber, a pulse voltage source, a driving voltage source, a signal acquisition unit, and a host computer. Please refer to... Figure 1 The sampling chamber 11 and the detection chamber can both be formed on a rectangular substrate 1. The substrate 1 also has mounting grooves 12 for mounting the microfluidic channel 121. The substrate 1 is vertically arranged in the direction of gravity, and from top to bottom, it consists of the sampling chamber 11, the microfluidic channel 121, and the detection chamber, allowing intracellular molecules to be unidirectionally drawn into the chambers by gravity. Specifically, a sheet-like upper sampling electrode 111 is provided at the top of the sampling chamber 11, and a sheet-like lower sampling electrode 112 is provided at the bottom. A sampling hole is also provided at the bottom of the sampling chamber 11. This sampling hole, through its size design or the inclusion of a filter membrane, prevents the cells to be detected from passing through, while allowing intracellular molecules to pass through. A low-amplitude, short-duration electrical pulse can be generated between the upper sampling electrode 111 and the lower sampling electrode 112. This electrical pulse creates nanoscale reversible pores in the cell membrane, allowing charged molecules within the cell to be released into the external microenvironment under the drive of the electric field, maintaining high cell activity and low damage.

[0023] In this embodiment, five microfluidic channels 121 are configured, with their tops all connected to the sampling chamber 11 and their bottoms all connected to the detection chamber, enabling multi-channel high-throughput detection and improving molecular detection efficiency. Taking one of the microfluidic channels 121 as an example, the microfluidic channel 121 is configured as a three-dimensional spiral, with the following geometry: spiral radius R: channel diameter D = 5~30:1. The curvature k is: k = 1 / R ≥ 0.05mm. -1The microfluidic channel 121 has at least two turns. Through its unique geometric path, the microfluidic channel 121 generates a secondary flow and inertial rectification effect, enabling natural unidirectional flow of charged molecules even without a pump or under low-pressure external pump conditions. This suppresses backflow and turbulence, and significantly reduces fluid pressure drop and external interference. The curved structure of the spiral channel further forms a hydraulic impedance buffer within the detection device, effectively blocking the transient electromagnetic disturbances caused by electrical pulses in the perforated unit on the current baseline in the downstream nanopore unit, improving the stability and repeatability of the detection signal. Simultaneously, the microfluidic channel 121 employs a dead-volume-free structural design, utilizing continuous curves and a branch-free dead-angle structure to effectively reduce molecular retention, cross-contamination, and time drift. This microfluidic channel 121 significantly improves the stability, sensitivity, and integration of the detection device.

[0024] A separator membrane 133 is installed inside the detection chamber. The separator membrane 133 divides the detection chamber into two chambers: an upper chamber 132 located above and a lower chamber 134 located below. Nanopores are formed on the separator membrane 133 connecting the two chambers 132 and 134. The upper chamber 132 is funnel-shaped, with its upper opening facing the microfluidic channel 121 and its bottom aligned with the nanopores to prevent molecules from remaining in the upper chamber 132. A sheet-like upper detection electrode 131 is disposed within the upper chamber 132. A sheet-like lower detection electrode 135 is disposed within the lower chamber 134. The upper detection electrode 131 and the lower detection electrode 135 generate a current through an external bias voltage. When charged molecules pass through the nanopores, a current blocking phenomenon occurs, thereby perturbing the current signal passing through the pores. This perturbed current signal is acquired and analyzed in real time to detect intracellular biomass.

[0025] The pulse voltage source, drive voltage source, signal acquisition unit, and host computer can all be mounted on one side of the substrate 1, and do not need to be directly connected to the substrate 1. The pulse voltage source is electrically connected to the upper sampling electrode 111 and the lower sampling electrode 112, providing a pulse voltage. The drive voltage source is electrically connected to the upper detection electrode 131 and the lower detection electrode 135, providing a bias voltage. The signal acquisition unit is connected to the septum membrane 133 to collect current signals characterizing biomass information. The host computer is communicatively connected to the signal acquisition unit to obtain the current signals collected by the signal acquisition unit and analyze and process them to obtain biomass information.

[0026] In another embodiment, the difference from the biomass detection device in the above embodiment lies in the number of microfluidic channels 121. Please refer to... Figure 2In this biomass detection device, one perforated unit is connected to a microfluidic channel 121, which greatly simplifies the complexity and manufacturing difficulty of the device while providing molecular transfer functionality. To facilitate observation of the internal conditions of the sampling chamber 11 and the detection chamber, a through groove (such as the sampling chamber 11 and the detection chamber) can be first opened on the substrate 1, and then transparent cover plates, such as glass, can be attached to both sides of the substrate 1, thereby sealing the groove to form the sampling chamber 11 and the detection chamber while allowing observation of the internal conditions through the cover plates.

[0027] In other embodiments, the sampling upper electrode 111 and sampling lower electrode 112 are made of Pt, Au, ITO, or carbon nanofilm. The separator 133 is made of SiN, SiO2, graphene, h-BN, or Al2O3. The multi-turn channels of the three-dimensional helical microfluidic channel 121 can be configured with variable curvature, or the helical radius of the microfluidic channel 121 can be varied stepwise, or the pitch of the microfluidic channel 121 can be varied in segments, etc., to enhance the fluid inertial bias and rectification capabilities. In addition to using gravity to unidirectionally extract molecules, the microfluidic channel 121 can also utilize any one or two of the following methods for unidirectional molecular extraction: pump (recommended pump pressure range is 1-5 kPa), capillary drive, micro-pressure regulation, or electroosmotic kinetic energy.

[0028] 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.

[0029] 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. An integrated intracellular biomass detection device, characterized in that, It includes: At least one detection unit; the detection unit includes: a sampling chamber, at least one microfluidic channel, and a detection chamber; The sampling chamber has an upper sampling electrode at the top and a lower sampling electrode at the bottom; a sampling hole is located at the bottom of the sampling chamber; the upper and lower sampling electrodes are connected to an external pulsed current to perform electroporation sampling of cells within the sampling chamber; the top of the microfluidic channel is connected to the sampling hole; a separator membrane is installed inside the detection chamber, dividing the detection chamber into two chambers; nanopores connecting the two chambers are formed on the separator membrane; an upper detection electrode is installed in one chamber, and this chamber is connected to the bottom of the microfluidic channel; a lower detection electrode is installed in the other chamber; a bias voltage is applied to the upper and lower detection electrodes to cause biomolecules within the chamber to undergo electrophoretic movement and pass through the nanopores, generating a current signal characterizing biomass information.

2. The integrated intracellular biomass detection device according to claim 1, characterized in that, The upper sampling electrode, lower sampling electrode, upper detection electrode, and lower detection electrode are all sheet-like electrodes, which are attached to the inner wall of the corresponding chamber.

3. The integrated intracellular biomass detection device according to claim 1, characterized in that, The number of microchannels is set to one; Alternatively, the number of microfluidic channels can be set to five; the top of each of the five microfluidic channels is connected to the sampling chamber, and the bottom of each is connected to one of the chambers.

4. The integrated intracellular biomass detection device according to claim 1, characterized in that, The upper and lower sampling electrodes are made of Pt, Au, ITO, or carbon nanofilm; the separator is made of SiN, SiO2, graphene, h-BN, or Al2O3.

5. The integrated intracellular biomass detection device according to claim 1, characterized in that, The microfluidic channels are positioned along the direction of gravity to allow intracellular molecules to be unidirectionally exported into the chamber by gravity.

6. The integrated intracellular biomass detection device according to claim 1, characterized in that, The microfluidic channel is designed as a three-dimensional spiral with the following geometry: spiral radius R : channel diameter D = 5~30 : 1; curvature k = 1 / R ≥ 0.05mm -1 The microfluidic channel should have at least two loops.

7. The integrated intracellular biomass detection device according to claim 1, characterized in that, The chamber connected to the microfluidic channel is designed in a funnel shape, with the bottom of the funnel-shaped chamber corresponding to the nanopore.

8. The integrated intracellular biomass detection device according to claim 1, characterized in that, The detection unit also includes: A pulse voltage source is electrically connected to the upper sampling electrode and the lower sampling electrode and provides pulse voltage. A driving voltage source is electrically connected to the upper and lower probe electrodes and provides a bias voltage. A signal acquisition device, used to acquire 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.

9. The integrated intracellular biomass detection device according to claim 1, characterized in that, The detection unit also includes a substrate; the sampling chamber and the detection chamber are both located on the substrate; the substrate has mounting slots for installing microfluidic channels.

10. The integrated intracellular biomass detection device according to claim 9, characterized in that, A through groove is formed on the substrate; transparent cover plates are attached to both sides of the substrate; the cover plates on both sides seal the groove to form a sampling chamber and a testing chamber.

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