Photoelectric synchronous detection chip for nanopores

Abstract

The invention discloses a photoelectric synchronous detection chip for nanopores. The photoelectric synchronous detection chip comprises a covering layer, an upper runner layer, a supporting layer, a lower runner layer and a substrate layer, the covering layer is provided with liquid through holes which are symmetrically arranged; the upper runner layer is provided with a separated liquid runner; tiny through holes are formed in the supporting layer; the lower runner layer is provided with a plurality of common liquid runners; liquid inlet and outlet holes are formed in the substrate layer; and the liquid through hole, the separated liquid flow channel, the micro through hole, the multi-path shared liquid flow channel and the liquid inlet and outlet hole form a multi-channel detection micro pool. According to the invention, multi-channel and continuous sample photoelectric synchronous detection can be realized.

Description

Technical Field

[0001] The invention relates to a nanopore photoelectric synchronous detection chip, belonging to the field of nanopore photoelectric synchronous detection chips. Background Art

[0002] Nanopore detection technology uses the specific current signal generated when molecules pass through nanometer-sized channels driven by electric fields to study the structure or dynamics of individual molecules. Due to its advantages such as high sensitivity and high spatiotemporal resolution, it has great potential in the fields of DNA or protein sequencing, drug screening, and analytical detection. However, traditional nanopore detection methods still rely on changes in ionic current to identify molecules, but this method is easily interfered by electromagnetic noise and has limitations in flux and multi-parameter analysis. Optical detection technology (such as confocal microscopy and total internal reflection imaging) can provide richer molecular information through fluorescent labeling or photon scattering characteristics without electromagnetic interference, such as using multi-color fluorescence encoding of molecular beacons to achieve single-base resolution DNA sequencing, or enhancing signals through surface plasmon resonance to achieve single amino acid recognition.

[0003] In order to realize the photoelectric synchronous detection of nanopores, the nanopore chip needs to be able to meet the low-noise current signal measurement and be compatible with general microscopic optical measurement equipment. The utility model patent (name: A nanopore photoelectric detection microcell, application number: 201320623353.8) discloses a nanopore photoelectric detection microcell, which realizes the photoelectric detection of nanopores through a planar double cell design, but the microcell body is thick and cannot meet the requirements of certain optical detection methods (such as total internal reflection imaging). It has only a single channel, its flux is small, and it cannot realize the detection of multiple samples at the same time. Although the MECAopto-inv chip manufactured by Nanion Technologies is designed with multiple channels and the cell body thickness can meet the needs of optical experiments, the liquid in the chip cannot flow, which is not conducive to liquid replacement and continuous sample detection.

[0004] In summary, how to achieve nanopore multi-channel, continuous sample detection and photoelectric synchronous detection is a technical problem that needs to be solved urgently. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a nanopore photoelectric synchronous detection chip which can realize multi-channel, continuous liquid replacement, has a small cell volume, and can meet the requirements of various optical detection methods.

[0006] Technical solution: To solve the above technical problems, the present invention provides a nano-pore optoelectronic synchronous detection chip, which includes a cover layer, an upper flow channel layer, a support layer, a lower flow channel layer, a base layer, and a nano-pore membrane pore system that are closely attached in sequence; a plurality of pairs of liquid through holes are provided on the cover layer; a plurality of separation liquid flow channels are provided on the upper flow channel layer; a plurality of micro through holes are provided on the support layer; a plurality of multi-way shared liquid flow channels are provided on the lower flow channel layer; liquid inlet and outlet through holes are provided on the base layer; and the nano-pore membrane pore system is closely attached above the micro through holes.

[0007] Among them, the liquid through holes are located at both ends of the separation liquid flow channels and within the coverage range of the separation liquid flow channels; the micro through holes are located below the middle position of the separation liquid flow channels; the multi-way shared liquid flow channels are located below the micro through holes and cover all the micro through holes; the liquid inlet and outlet through holes are located at both ends of the multi-way shared liquid flow channels and within the coverage range of the multi-way shared liquid flow channels.

[0008] Among them, the multi-way shared liquid flow channels are perpendicular to the separation liquid flow channels.

[0009] Among them, the number of pairs of liquid through holes is the same as the number of separation liquid flow channels provided on the upper flow channel layer and the number of micro through holes.

[0010] Among them, the width of the liquid flow channels is greater than the diameter of the liquid through holes.

[0011] Among them, the diameter of the liquid inlet and outlet through holes is less than the width of the multi-way shared liquid flow channels, preferably 1 mm.

[0012] Among them, the diameter of the micro through holes is less than the width of the separation liquid flow channels.

[0013] Among them, the diameter of the micro through holes is 10 - 100 μm, preferably 50 μm.

[0014] Among them, only the liquid through holes, separation liquid flow channels, micro through holes, multi-way shared liquid flow channels, and liquid inlet and outlet through holes form an interconnected channel system between the layers.

[0015] Among them, the thicknesses of the upper flow channel layer, the support layer, and the lower flow channel layer do not exceed 0.5 mm, and they are made of non-conductive and hydrophobic materials.

[0016] Among them, the cover layer and the base layer are made of transparent materials for observing the liquid flow state.

[0017] Among them, the cover layer and the base layer are made of transparent materials with a thickness not exceeding 1 mm.

[0018] Among them, the width of the separation liquid flow channels is greater than the diameter of the liquid through holes, preferably 1 - 2 mm, and the diameter of the liquid through holes is preferably 1 mm.

[0019] Among them, the multi-channel shared liquid flow path is preferably 2 to 5 mm.

[0020] Among them, the width of the multi-channel shared liquid flow path is 1 to 10 mm.

[0021] Among them, only through the liquid through holes, the separated liquid flow paths, the micro through holes, the liquid inlet and outlet through holes of the multi-channel shared liquid flow path and the nanoporous membrane pore system among the layers, an interconnected channel system is formed.

[0022] Among them, the upper flow channel layer, the support layer and the lower flow channel layer are thin layer materials that are non-conductive, hydrophobic and have no specific adsorption to grease.

[0023] Among them, the nanoporous membrane pore system is a thin film with nanopores, which is used to separate solutions.

[0024] Among them, the nanopores are endogenous or exogenous biological, solid nanopores or microchannels.

[0025] Among them, each pair of independent liquid through holes provided in the covering layer and each independent separated liquid flow path provided in the upper flow channel layer corresponding thereto, each independent micro through hole provided in the support layer, the multi-channel shared liquid flow path layer provided in the lower flow channel layer, and the liquid inlet and outlet through holes provided in the base layer constitute a complete detection microcell, serving as a detection channel.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The present invention can be used for multi-channel continuous acquisition of ion current signals of different samples; A nanopore photoelectric synchronous detection chip of the present invention has a thin layer body and a small volume, and can be applicable to various microscopic imaging techniques (such as confocal microscopes) to collect optical signals (such as fluorescence imaging, fluorescence spectroscopy, Raman spectroscopy). Description of the Drawings

[0027] Figure 1 Is an exploded view of the nanopore photoelectric synchronous detection chip provided by the present invention;

[0028] Figure 2 Is a top perspective view of the nanopore photoelectric synchronous detection chip provided by the present invention;

[0029] Figure 3 Is an actual effect diagram of the nanopore photoelectric synchronous detection chip provided by the present invention;

[0030] Figure 4 Is a schematic diagram of the cross-section of use and the solution flow direction of the nanopore photoelectric synchronous detection chip provided by the present invention;

[0031] Figure 5 Is a partial experimental data diagram of the nanopore photoelectric synchronous detection chip provided by the present invention;

[0032] Figures 1-4 In:

[0033] 1 - Overlay layer; 2 - Upper flow channel layer; 3 - Support layer; 4 - Lower flow channel layer; 5 - Substrate layer; 6 - Solution; 7 - Nanopore membrane pore system; 8 - Common counter electrode; 9 - Working electrode; 10 - Current filtering amplifier; 11 - Photoelectric signal controller; 12 - Optical microscopic imaging device; 101 - Liquid through hole; 201 - Separate liquid flow channel; 301 - Tiny through hole; 401 - Multichannel shared liquid flow channel; 501 - Liquid inlet and outlet through hole. Specific embodiments

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0035] To achieve synchronous detection of multi-channel photoelectric signals, the present invention provides a nanopore photoelectric synchronous detection chip, as Figure 1 shown, which includes: an overlay layer 1 serving as the upper flow channel wall, an upper flow channel layer 2 providing a flow channel for the liquid medium 6, a support layer 3 serving as a support for the nanopore membrane pore system 7, a lower flow channel layer 4 providing a multi-channel shared liquid medium 6 flow channel, and a substrate layer 5 serving as a support for the multi-layer structure. Among them, the overlay layer 1 contains a plurality of symmetrically arranged liquid through holes 101 that completely penetrate the overlay layer 1, and the liquid through holes 101 are used to introduce the liquid medium 6. The upper flow channel layer 2 is provided with a plurality of independent separate liquid flow channels 201 that completely penetrate the upper flow channel layer 2. A plurality of independent tiny through holes 301 are provided on the support layer 3. The lower flow channel layer 4 is provided with a completely penetrating multi-channel shared liquid flow channel 401 parallel to the lower flow channel layer 4. The substrate layer 5 is provided with liquid inlet and outlet through holes 501 for the liquid medium 6 to enter and exit. The nanopore membrane pore system 7 is constructed above the tiny through holes 301.

[0036] Among them, the number of pairs of liquid through holes 101 provided on the overlay layer 1, the number of separate liquid flow channels 201 provided on the upper flow channel layer 2, and the number of tiny through holes 301 provided on the support layer 3 are kept consistent. The present invention preferably has 5 pairs of liquid through holes 101, 5 separate liquid flow channels 201, and 5 tiny through holes 301.

[0037] The overlay layer 1, the upper flow channel layer 2, the support layer 3, the lower flow channel layer 4, and the substrate layer 5 are closely attached to each other in sequence. The close attachment method can be pasting or clamping with a fixture, and preferably pasting.

[0038] To facilitate observing the liquid flow state, the overlay layer 1 can be made of any transparent material, including but not limited to glass and transparent plastics, etc., and the thickness is less than 1 mm, preferably a glass slide with a thickness of 0.2 mm.

[0039] To satisfy the acquisition of optical signals such as fluorescence spectra and Raman spectra by the optical microscopy imaging device 12 (such as a confocal microscope) and the construction of the nanopore membrane pore system 7, the upper flow channel layer 2, the support layer 3, and the lower flow channel layer 4 are made of a non-conductive and hydrophobic material with a thickness not exceeding 0.5 mm, preferably a PTFE film with a thickness of 0.1 mm.

[0040] Among them, the width of the separation liquid flow channel 201 is greater than the diameter of the liquid through hole 101, preferably 1 - 2 mm; the diameter of the liquid through hole 101 is preferably 1 mm; the diameter of the micro through hole 301 is less than the width of the separation liquid flow channel 201, preferably 50 μm; the multi-channel shared liquid flow channel 401 is preferably 2 - 5 mm; the diameter of the liquid inlet and outlet through hole 501 is less than the width of the multi-channel shared liquid flow channel 401, preferably 1 mm. As Figure 2 shown, the covering layer 1 is located above the upper flow channel layer 2, and the liquid through holes 101 on the covering layer 1 are respectively arranged at both ends of the separation liquid flow channel 201 and within the coverage range of the separation liquid flow channel 201; the micro through holes 301 on the support layer 3 are respectively located below the middle position of the separation liquid flow channel 201; the multi-channel shared liquid flow channel 401 on the lower flow channel layer 4 is located below the micro through holes 301 and covers all the micro through holes 301; the liquid inlet and outlet through holes 501 on the base layer 5 are located at both ends below the multi-channel shared liquid flow channel 401 and within the coverage range of the multi-channel shared liquid flow channel 401; the upper surfaces of the liquid through holes 101, the separation liquid flow channel 201, and the micro through holes 301 form the upper pool body of the detection microcell; the lower surfaces of the liquid inlet and outlet through holes 501, the multi-channel shared liquid flow channel 401, and the micro through holes 301 form the lower pool body of the detection microcell; each pair of liquid through holes 101 and the corresponding separation liquid flow channel 201, each micro through hole 301, multi-channel shared liquid flow channel 401, and liquid inlet and outlet hole 501 form a complete detection microcell, serving as a detection channel. The actual effect diagram of the present invention for the nanopore optoelectronic synchronous detection chip is as Figure 3 shown.

[0041] As Figure 4As shown, in the upper cell body, solution 6 enters through the liquid inlet hole 101. Solution 6 flows into the corresponding separation liquid flow channel 201 below through each pair of liquid inlet holes 101. Part of solution 6 flows out through the liquid inlet hole 101 on the other side, and part of solution 6 flows into the lower cell body through the nanopores of the nanopore membrane pore system 7. In the lower cell body, solution 6 flows in through the liquid inlet and outlet through hole 501 on one side. Part of solution 6 flows into the liquid inlet and outlet through hole 501 on the other side through the multi-channel shared liquid flow channel 401 and then flows out, and part of solution 6 flows into the upper cell body through the micro through hole 301 and the nanopores of the nanopore membrane pore system 7. The nanopore optoelectronic synchronous detection chip of the present invention uses the nanopore membrane pore system 7 to connect the upper and lower cell bodies. Solution 6 can pass through the nanopores to electrically conduct the entire detection microcell. The multi-channel working electrodes 9 and the common counter electrodes 8 are respectively connected to the upper and lower cell bodies of the chip and connected to the current filtering amplifier 10. The current filtering amplifier 10 is connected to the optoelectronic signal controller 11 to collect the ionic current signals during the detection process of the nanopore membrane pore system 7 in multiple channels. At the same time, the chip can be placed in the optical microscopy imaging device 12 to realize the synchronous acquisition of nanopore optoelectronic signals, and the solution 6 in the upper and lower cell bodies can be continuously replaced during the detection process to realize the continuous detection of different samples.

[0042] Among them, for nanopore detection, Aerolysin nanopore membrane pore system, α-Hemolysin nanopore membrane pore system, MspA membrane pore system, solid nanopore system, etc. can be selected. For example, Figure 4 As shown, in this experiment, the K238Q Aerolysin nanopore and DPhPC bilayer membrane system were taken as examples. Under the condition of solution 6 containing 1M Tris-KCl, 1mM EDTA, pH 7.4 and a single-stranded DNA molecule with 6 adenine deoxyribonucleotide lengths modified at the end with tetraphenylethylene (poly(dA)6-TPE molecule), a nanopore membrane pore system 7 composed of DPhPC bilayer membrane and mutant K238Q Aerolysin nanopore protein was constructed above the micro through hole 301, and the poly(dA)6-TPE molecule was detected under the applied potential of +100 mV. Figure 5 shows the ionic current signal segment caused by the poly(dA)6-TPE molecule passing through the K238Q Aerolysin nanopore in a single detection channel.

Claims

1. A nanopore optoelectronic synchronous detection chip, characterized in that, It includes a covering layer (1), an upper flow channel layer (2), a support layer (3), a lower flow channel layer (4), a base layer (5) and a nanoporous membrane pore system (7) that are closely attached in sequence; several pairs of liquid through holes (101) are provided on the covering layer (1); several separation liquid flow channels (201) are provided on the upper flow channel layer (2); several micro through holes (301) are provided on the support layer (3); multiple shared liquid flow channels (401) are provided on the lower flow channel layer (4); liquid inlet and outlet through holes (501) are provided on the base layer (5); and the nanoporous membrane pore system (7) is closely attached above the micro through holes (301).

2. The nanopore optoelectronic synchronous detection chip according to claim 1, wherein The liquid through holes (101) are located at both ends of the separation liquid flow channels (201) and within the coverage range of the separation liquid flow channels (201); the micro through holes (301) are located below the middle position of the separation liquid flow channels (201); the multiple shared liquid flow channels (401) are located below the micro through holes (301) and cover all the micro through holes (301); the liquid inlet and outlet through holes (501) are located at both ends of the multiple shared liquid flow channels (401) and within the coverage range of the multiple shared liquid flow channels (401).

3. The nanopore optoelectronic synchronous detection chip according to claim 1, wherein The multiple shared liquid flow channels (401) are perpendicular to the separation liquid flow channels (201).

4. The nanopore optoelectronic synchronous detection chip according to claim 1, characterized in that, The width of the separation liquid flow channels (201) is greater than the diameter of the liquid through holes (101).

5. The nanopore optoelectronic synchronous detection chip according to claim 1, characterized in that, The diameter of the liquid inlet and outlet through holes (501) is less than the width of the multiple shared liquid flow channels (401).

6. The nanopore optoelectronic synchronous detection chip according to claim 1, wherein The diameter of the micro through holes (301) is less than the width of the liquid flow channels (401).

7. The nanopore optoelectronic synchronous detection chip according to claim 1, wherein Only the liquid through holes (101), separation liquid flow channels (201), micro through holes (301), multiple shared liquid flow channels (401), liquid inlet and outlet through holes (501) and the nanoporous membrane pore system (7) form an interconnected channel system between the layers.

8. The nanopore optoelectronic synchronous detection chip according to claim 1, wherein The covering layer (1) and the base layer (5) are made of transparent materials.

9. The nanopore optoelectronic synchronous detection chip according to claim 1, wherein The upper flow channel layer (2), the support layer (3) and the lower flow channel layer (4) are thin layer materials that are non-conductive, hydrophobic and have no specific adsorption to grease.

10. The nanopore optoelectronic synchronous detection chip according to claim 1, characterized in that, The nanoporous membrane pore system (7) is a thin film with nanopores.

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