CN106970130B - Nanopore detection system based on nanotube and preparation method and application thereof - Google Patents
Nanopore detection system based on nanotube and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano-tube-based nano-hole detection system, a preparation method and application, wherein the system comprises a nano-tube-based nano-hole structure, a micro-channel structure, a cover plate for covering the micro-channel structure and a current detection system for detecting an object to be detected; the nano-tube based nano-pore structure comprises a substrate and a nano-tube, wherein the nano-tube is dispersed or grown on the substrate, the left end and the right end of the nano-tube are opened, and the side wall of the nano-tube is provided with a nano-pore; the micro-channel structure is processed on the substrate and is provided with 3 independent electrolyte solution chambers, the nanotube penetrates through the three chambers, and a left opening, a nanopore and a right opening of the nanotube are respectively communicated with the first chamber, the second chamber and the third chamber; one end of the current detection system is arranged in the second chamber, and the other end of the current detection system is arranged in the first chamber or the third chamber. The invention can greatly improve the resolution and the sensitivity of the biosensor and is expected to realize the idea of direct sequencing of single-molecule DNA.
Description
Technical Field
The invention relates to the technical field of nanopore detection systems, in particular to a nanopore detection system based on a nanotube structure and capable of being used for detecting a long-chain polymer (DNA, RNA or polypeptide) and a preparation method thereof.
Background
Nanopore technology is a powerful tool for analyzing biomolecules, and is specifically applied to the following three fields: first, biophysical studies at the single molecule level, such as folding/unfolding of DNA molecules, melting of double-stranded DNA, single-molecule DNA-protein interactions, and force-spectroscopy measurements of biomolecules; second, a broad sense of "early diagnosis" is performed at the single molecule level. Such as important attribute recognition, specific short segment discrimination and the like of single biomolecules; third, single molecule DNA was directly sequenced.
The biological nanopore is used as a nanopore which is firstly applied to DNA sequencing, has good biological sensitivity and a fixed-size nanochannel, ensures the repeatability and stability of DNA detection, but has the defects of poor stability, short service life, sensitivity to environmental change and incapability of regulating and controlling the pore diameter of the nanopore, and greatly limits the application prospect. The occurrence of the solid-state nanopore makes up the defects of the biological nanopore, but the traditional silicon-based nanopore channel is at least a few nanometers and is far larger than the base stacking distance of 0.34nm on a DNA molecular chain, so a plurality of bases can exist in the solid-state nanopore at the same time, and the obtained ion current signal is the result of the combined action of the bases and is not enough for distinguishing the information of a single base. The appearance of two-dimensional material films such as graphene and h-boron nitride provides possibility for preparing nano-holes with atomic-scale thickness, but the two-dimensional material nano-hole sensor manufactured by the existing technical means has at least the following three problems. First, the mechanical stability of the two-dimensional material film is poor. The two-dimensional material film with the sub-nanometer level thickness is easy to generate mechanical vibration in an electrolyte solution, and the generated vibration noise can submerge a weak signal generated by ion current into the noise and cannot be identified; secondly, defects are generated in the process of transferring the two-dimensional materials such as graphene to the supporting substrate, and leakage current generated by the defects is considered to be one of the reasons for low signal-to-noise ratio of current signals detected by the two-dimensional material nanopore. Thirdly, compared with the traditional silicon-based nanopore, although the atomic-scale pore canal length of the two-dimensional material nanopore can improve the spatial resolution of a current signal, the DNA molecule is enabled to maintain a larger degree of freedom when passing through the pore, so that the molecular configuration is unstable, and the signal-to-noise ratio is reduced.
Most of the existing nanotube biosensors based on the nanopore detection principle integrate nanotubes into silicon-based nanopores (CN103796947B) or form nanopores by using radial slices of the nanotubes (CN 1994864B), but the nanopores formed by the methods still have the problems of low spatial resolution and low signal-to-noise ratio.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention is directed to a nanopore detection system based on nanotubes, which can improve the sensitivity, resolution and signal-to-noise ratio of a nanopore-based biosensor, accurately detect and distinguish biomolecules, and achieve the purpose of direct sequencing of single-molecule DNA.
In order to achieve the above purpose, the invention specifically provides the following scheme:
1. a nanopore detection system based on nanotubes comprises a nanopore structure based on nanotubes, a micro-channel structure, a cover plate for covering the micro-channel structure, and a current detection system for detecting an object to be detected;
the nano-tube based nano-pore structure comprises a substrate and a nano-tube, wherein the nano-tube is dispersed or grown on the substrate, the left end and the right end of the nano-tube are opened, and the side wall of the nano-tube is provided with a nano-pore;
the micro-channel structure is processed on the substrate and is provided with 3 independent electrolyte solution chambers, namely a first chamber, a second chamber and a third chamber, the nanotube penetrates through the three chambers, and the left opening, the nanopore and the right opening of the nanotube are respectively communicated with the first chamber, the second chamber and the third chamber;
the cover plate is provided with liquid inlet holes which are respectively matched with the first chamber, the second chamber and the third chamber;
one end of the current detection system is arranged in the second chamber, the other end of the current detection system is arranged in the first chamber or the third chamber, and the current detection system is communicated with the electrolyte solution in the electrolyte solution chamber to form a circuit for detecting the object to be detected.
Further preferably, the nanopore is located on the sidewall of the middle section of the nanotube.
Further preferably, the current detection system comprises a power supply, an electrode I, an electrode II and an ammeter;
the electrode I is arranged in the first or third chamber, and the electrode II is arranged in the second chamber;
the power supply, the electrode I, the electrode II and the ammeter are connected in series to form a circuit for detecting the object to be detected.
Preferably, the substrate is silicon oxide, silicon nitride, quartz, glass or PMMA; the nanotube is a nanotube-shaped structural material which is not chemically modified or is chemically modified.
Further preferably, the nanotube is a single-walled carbon nanotube, a multi-walled carbon nanotube, a boron nitride nanotube, an aluminum oxide nanotube, a zinc oxide nanotube or a polymer nanotube.
Further preferably, the diameter of the nanotube is 1-500 nm, and the length of the nanotube is 1-1000 μm; the height of the micro-channel structure is 0.5-500 mu m, the width of the micro-channel structure is 0.5-500 mu m, the diameters of the left opening and the right opening of the nano tube are consistent with those of the nano tube, and the diameter of the nano hole is 0.1-100 nm.
2. The preparation method of the nano-tube based nanopore detection system comprises the following steps:
(1) dispersing or growing nanotubes on the surface of the substrate;
(2) processing a micro-channel structure on the substrate of the selected nanotube region by using a photoetching method; 3 independent electrolyte solution chambers, namely a first chamber, a second chamber and a third chamber, are arranged, and the nanotube penetrates through the three chambers;
(3) forming a nano hole on the side wall of the middle section of the nano tube after packaging the micro-channel structure by using a cover plate or forming a hole and then packaging, wherein the left opening, the nano hole and the right opening of the nano tube are respectively communicated with the first chamber, the second chamber and the third chamber;
(4) and arranging an electrode I of the current detection system in the first or third chamber, and arranging an electrode II in the second chamber to form a loop for detecting the object to be detected.
Further, the method for forming the nano-holes on the side wall of the middle section of the nano-tube comprises the following steps:
scheme A: forming a nano hole on the side wall of the middle section of the nano tube after packaging the micro-channel structure:
(1) performing opening end treatment on two ends of the nanotube by a micro-nano processing method;
(2) packaging the formed micro-channel structure by using a cover plate;
(3) respectively introducing electrolyte solution into the first chamber, the second chamber and the third chamber and filling the micro-channels with the electrolyte solution;
(4) inserting an electrode I and an electrode II into the first chamber and the third chamber respectively, and applying voltage to fill the nanotube with electrolyte solution;
(5) respectively inserting an electrode I and an electrode II into the first chamber and the second chamber, and applying voltage to perform electric breakdown operation on the side wall of the middle section of the nanotube exposed in the electrolyte solution to form a nanopore;
scheme B: forming nano holes on the side wall of the middle section of the nano tube to package the micro channel structure:
(1) performing opening processing on the nanotube by using a micro-nano processing method, and processing the side wall of the middle section of the nanotube to form a nanopore;
(2) packaging the formed micro-channel structure with a cover sheet
(3) Electrolyte solution is introduced into the first chamber, the second chamber and the third chamber and is filled in the micro-flow channels;
(4) an electrode I and an electrode II are respectively inserted into the first chamber and the third chamber, and voltage is applied to fill the nanotube with electrolyte solution.
Further preferably, the micro-nano processing method is a focused ion beam or transmission electron microscope processing method.
3. The application of the nano-tube based nanopore detection system in the detection of DNA, RNA or polypeptide.
The invention has the beneficial effects that: the invention utilizes point breakdown or focused ion beam or transmission electron microscope to form nano-hole on the side wall of the nano-tube, and the nano-hole and the nano-tube are in an integral structure and have good mechanical stability. Meanwhile, the nanotube is not easy to form defects on the tube wall when dispersed or grown on the substrate, and the problem that the defects are easy to generate when the two-dimensional material film is transferred is effectively avoided. In addition, before passing through the nanopore, biomolecules such as DNA pass through a nanotube channel with a diameter of several nanometers to several hundred nanometers, so that the configuration of the biomolecules such as DNA before passing through the nanopore is stabilized. Therefore, the invention can greatly improve the resolution and the sensitivity of the biosensor and is expected to realize the idea of direct sequencing of single-molecule DNA.
Drawings
In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:
FIG. 1 is a cross-sectional view of a nanopore detection system based on nanotubes and a schematic diagram of the principle of molecular detection.
Detailed Description
The preferred embodiment of the present invention will be described in detail below with reference to the accompanying drawing 1:
FIG. 1 is a cross-sectional view of a nanopore detection system based on nanotubes and a schematic diagram of the principle of molecular detection. The method comprises the following steps of 1-power supply, 2-amperemeter, 3-electrode I, 4-electrode II, 5-substrate, 6-micro-channel structure, 7-cover plate, 8-first liquid inlet hole, 9-second liquid inlet hole, 10-third liquid inlet hole, 11-first chamber, 12-second chamber, 13-third chamber, 14-nanotube, 15-nanopore and 16-molecular sample.
Example 1
The embodiment provides a nanopore detection system based on single-walled carbon nanotubes. The specific embodiment is as follows:
growing a nanotube 14 with the diameter of 1-10nm and the length of 500-1000 μm on the surface of a substrate 5 with a silicon dioxide film, spin-coating a negative photoresist on the substrate 5 with the nanotube 14, and processing a negative photoresist micro-channel structure 6 with the thickness of 20 μm and the width of 7 μm on the nanotube 14 with the selected diameter of 2nm and the length of 500 μm by a photoetching method to form a first chamber 11, a second chamber 12 and a third chamber 13.
The first chamber 11 is not communicated with the second chamber 12, the second chamber 12 is not communicated with the third chamber 13, and the first chamber 11 is communicated with the third chamber 13 only by the nanotube 14. The two ends of the nanotube 14 are treated by the focused ion beam to ensure that the two ends of the nanotube 14 are not blocked. The first, second and third wells 8, 9 and 10 are formed on the cover plate 7, and the micro flow channel structure 6 is encapsulated in alignment with the first, second and third chambers 11, 12 and 13, respectively, by methods known in the art.
The current detection system comprises a power supply 1, an electrode I3, an electrode II4 and an ammeter 2, electrolyte solutions are respectively injected into a first chamber 11, a second chamber 12 and a third chamber 13, an electrode I3 and an electrode II4 are respectively inserted into the first chamber 11 and the third chamber 13, and the power supply 1 is turned on to enable the electrolyte solutions to be filled in the single-walled carbon nanotubes 14. Removing the electrode II4 in the third chamber 13, inserting the electrode I3 and the electrode II4 in the first chamber 11 and the second chamber 12 respectively, and turning on the power supply 1 to perform an electric breakdown operation on the side wall of the middle section of the carbon nanotube 14 exposed in the electrolyte solution to form a nanopore 15, wherein the diameter of the nanopore 15 is 1 nm.
A single-stranded DNA molecule sample 16 is added to the first chamber 11 and a bias voltage is applied to drive the single-stranded DNA molecule sample 16 through a nanopore 15 formed in the sidewall of the nanotube 14 and into the second chamber 12. Analyzing the current signal generated by the single-stranded DNA molecule 16 passing through the nanopore 15 ammeter 2 to obtain the sequence of the DNA molecule to be detected.
In this embodiment, the substrate 5 is a silicon wafer, the nanotubes 14 are single-walled carbon nanotubes, and the cover 7 is a PDMS cover.
Example 2:
the embodiment provides a nanopore detection system based on a boron nitride nanotube. The specific embodiment is as follows:
dispersing nanotubes 14 with the diameter of 300-500nm and the length of 10-90 μm on the surface of a substrate 5, spin-coating a positive photoresist on the substrate with the nanotubes, and processing a positive photoresist micro-channel structure 6 with the thickness of 15 μm and the width of 8 μm on the selected nanotubes 14 with the diameter of 400nm and the length of 20 μm by a photoetching method to form a first chamber 11, a second chamber 12 and a third chamber 13.
The first chamber 11 is not communicated with the second chamber 12, the second chamber 12 is not communicated with the third chamber 13, and the first chamber 11 is communicated with the third chamber 13 only by the selected nanotubes 14. The two ends of the nanotube 14 are treated by the focused ion beam to ensure that the two ends of the nanotube 14 are not blocked. The portion of the nanotube 14 exposed in the second chamber 12 is processed with a focused ion beam to form a nanopore 15, the nanopore 15 having a diameter of 100 nm. The first, second and third wells 8, 9 and 10 are formed on the cover plate 7, and the micro flow channel structure 6 is encapsulated in alignment with the first, second and third chambers 11, 12 and 13, respectively, by methods known in the art.
The current detection system comprises a power supply 1, an electrode I3, an electrode II4 and an ammeter 2, electrolyte solutions are respectively injected into a first chamber 11, a second chamber 12 and a third chamber 13, an electrode I3 and an electrode II4 are respectively inserted into the first chamber 11 and the third chamber 13, and the power supply 1 is turned on to enable the electrolyte solutions to be filled in the nanotubes 14. Electrode II4 in third chamber 13 was removed and electrode I3 and electrode II4 were inserted in first chamber 11 and second chamber 12, respectively.
A sample of RNA molecules is added to the first chamber 11 and a bias voltage is applied to drive the RNA molecules through a nanopore 15 formed in the sidewall of the nanotube 14 and into the second chamber 12. And analyzing the current signal generated by the RNA molecule through the nanopore 15 amperemeter 2 to obtain the information of the detected RNA molecule.
In this example the substrate 5 is quartz, the nanotubes 14 are boron nitride and the cover 7 is PMMA.
Example 3:
the embodiment provides a nanopore detection system based on multi-walled carbon nanotubes. The specific embodiment is as follows:
the method comprises the steps of growing nanotubes with the diameter of 10-90nm and the length of 100-500 mu m on the surface of a substrate 5, spin-coating a negative photoresist on the substrate 5 with the nanotubes, and processing a negative photoresist micro-channel structure 6 with the thickness of 25 mu m and the width of 5 mu m on a selected nanotube 14 by using a photoetching method to form a first chamber 11, a second chamber 12 and a third chamber 13.
The first chamber 11 is not communicated with the second chamber 12, the second chamber 12 is not communicated with the third chamber 13, and the first chamber 11 is communicated with the third chamber 13 only by the nanotube 14. The two ends of the nanotube 14 are treated by the focused ion beam to ensure that the two ends of the nanotube 14 are not blocked. The portion of the nanotube 14 exposed in the second chamber 12 is processed using a transmission electron microscope to form a nanopore 15, the nanopore 15 having a diameter of 50 nm. A first liquid inlet hole 8, a second liquid inlet hole 9 and a third liquid inlet hole 10 are formed on the quartz cover plate 7, and the micro flow channel structure 6 is encapsulated in alignment with a first chamber 11, a second chamber 12 and a third chamber 13, respectively, and the encapsulation method is known in the art.
The current detection system comprises a power supply 1, an electrode I3, an electrode II4 and an ammeter 2, electrolyte solutions are respectively injected into a first chamber 11, a second chamber 12 and a third chamber 13, an electrode I3 and an electrode II4 are respectively inserted into the first chamber 11 and the third chamber 13, and the power supply 1 is turned on to enable the electrolyte solutions to be filled with carbon nano tubes 14. Electrode II4 in third chamber 13 was removed and electrode I3 and electrode II4 were inserted in first chamber 11 and second chamber 12, respectively.
A sample of polypeptide molecules is added to the first chamber 11, and a bias voltage is applied to drive the polypeptide molecules into the second chamber 12 through a nanopore 15 formed in the sidewall of the carbon nanotube 14. The information of the polypeptide molecule 16 to be detected can be obtained by analyzing the current signal generated by the amperemeter 2 when the polypeptide molecule passes through the nanopore 15.
In this embodiment, the substrate 5 is PMMA, the nanotubes 14 are multi-walled carbon nanotubes, and the cover 7 is quartz.
In summary, the present invention utilizes an electric breakdown or focused ion beam or transmission electron microscope to form nanopores in the nanotube sidewalls. The nano-hole and the nano-tube are of an integral structure and have good mechanical stability. Meanwhile, the nanotube is not easy to form defects on the tube wall when dispersed or grown on the substrate, and the problem that the defects are easy to generate when the two-dimensional material film is transferred is effectively avoided. In addition, before passing through the nanopore, biomolecules such as DNA pass through a nanotube channel with a diameter of several nanometers to several hundred nanometers, so that the configuration of the biomolecules such as DNA before passing through the nanopore is stabilized. Thus, the present invention effectively overcomes the disadvantages of the prior art and is of high commercial value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A nanopore detection system based on nanotubes is characterized by comprising a nanopore structure based on nanotubes, a micro-channel structure, a cover plate for covering the micro-channel structure and a current detection system for detecting an object to be detected;
the nano-tube based nano-pore structure comprises a substrate and a nano-tube, wherein the nano-tube is dispersed or grown on the substrate, the left end and the right end of the nano-tube are opened, and the side wall of the nano-tube is provided with a nano-pore;
the micro-channel structure is processed on the substrate and is provided with 3 independent electrolyte solution chambers, namely a first chamber, a second chamber and a third chamber, the nanotube penetrates through the three chambers, and the left opening, the nanopore and the right opening of the nanotube are respectively communicated with the first chamber, the second chamber and the third chamber;
the cover plate is provided with liquid inlet holes which are respectively matched with the first chamber, the second chamber and the third chamber;
one end of the current detection system is arranged in the second chamber, the other end of the current detection system is arranged in the first chamber or the third chamber, and the current detection system is communicated with the electrolyte solution in the electrolyte solution chamber to form a circuit for detecting the object to be detected.
2. The nanotube-based nanopore detection system of claim 1, wherein said nanopore is located on a medial sidewall of said nanotube.
3. The nanotube based nanopore detection system of claim 1, wherein said amperometric detection system comprises a power source, electrode i, electrode ii, and an ammeter;
the electrode I is arranged in the first or third chamber, and the electrode II is arranged in the second chamber;
the power supply, the electrode I, the electrode II and the ammeter are connected in series to form a circuit for detecting the object to be detected.
4. The nanotube based nanopore detection system of claim 1, wherein said substrate is silicon oxide, silicon nitride, quartz, glass, or PMMA; the nanotube is a nanotube-shaped structural material which is not chemically modified or is chemically modified.
5. The nanotube-based nanopore detection system of claim 4, wherein the nanotube is a single-walled carbon nanotube, a multi-walled carbon nanotube, a boron nitride nanotube, an aluminum oxide nanotube, a zinc oxide nanotube, or a polymer nanotube.
6. The nanotube-based nanopore detection system of claim 1, wherein said nanotube has a diameter of 1-500 nm and a length of 1-1000 μm; the height of the micro-channel structure is 0.5-500 mu m, the width of the micro-channel structure is 0.5-500 mu m, the diameters of the left opening and the right opening of the nano tube are consistent with those of the nano tube, and the diameter of the nano hole is 0.1-100 nm.
7. The method of any one of claims 1 to 6, wherein the method comprises the steps of:
(1) dispersing or growing nanotubes on the surface of the substrate;
(2) processing a micro-channel structure on the substrate of the selected nanotube region by using a photoetching method; 3 independent electrolyte solution chambers, namely a first chamber, a second chamber and a third chamber, are arranged, and the nanotube penetrates through the three chambers;
(3) forming a nano hole on the side wall of the middle section of the nano tube after packaging the micro-channel structure by using a cover plate or forming a hole and then packaging, wherein the left opening, the nano hole and the right opening of the nano tube are respectively communicated with the first chamber, the second chamber and the third chamber;
(4) and arranging an electrode I of the current detection system in the first or third chamber, and arranging an electrode II in the second chamber to form a loop for detecting the object to be detected.
8. The method of claim 7, wherein the nanopore is formed in a sidewall of the nanotube intermediate section by a method comprising:
scheme A: forming a nano hole on the side wall of the middle section of the nano tube after packaging the micro-channel structure:
(1) performing opening end treatment on two ends of the nanotube by a micro-nano processing method;
(2) packaging the formed micro-channel structure by using a cover plate;
(3) respectively introducing electrolyte solution into the first chamber, the second chamber and the third chamber and filling the micro-channels with the electrolyte solution;
(4) inserting an electrode I and an electrode II into the first chamber and the third chamber respectively, and applying voltage to fill the nanotube with electrolyte solution;
(5) respectively inserting an electrode I and an electrode II into the first chamber and the second chamber, and applying voltage to perform electric breakdown operation on the side wall of the middle section of the nanotube exposed in the electrolyte solution to form a nanopore;
scheme B: forming nano holes on the side wall of the middle section of the nano tube to package the micro channel structure:
(1) performing opening processing on the nanotube by using a micro-nano processing method, and processing the side wall of the middle section of the nanotube to form a nanopore;
(2) packaging the formed micro-channel structure with a cover sheet
(3) Electrolyte solution is introduced into the first chamber, the second chamber and the third chamber and is filled in the micro-flow channels;
(4) an electrode I and an electrode II are respectively inserted into the first chamber and the third chamber, and voltage is applied to fill the nanotube with electrolyte solution.
9. The method of claim 8, wherein the micro-nano processing method is a focused ion beam or transmission electron microscopy processing method.
10. Use of the nanotube based nanopore detection system of any one of claims 1 to 6 for detecting DNA, RNA or a polypeptide.
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