CN114686354A - A solid-state nanopore gene pool and solid-state nanopore gene sequencing device - Google Patents

Abstract

The invention discloses a solid-state nanopore gene pool, comprising an upper solution pool, a lower solution pool, a gene-attached glass slide and a nanopore chip; the nanopore chip is arranged at the bottom of the upper solution pool, and the upper solution pool has a The lower through-hole is arranged corresponding to the nano-hole of the nano-pore chip; the gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nano-pore chip; the upper surface of the gene-attached glass slide includes A columnar high platform; the columnar high platform is used to fix the gene to be detected. The present invention can effectively shorten the distance between the gene to be detected and the nanopore when facing the face angle of the gene attachment glass slide and the nanopore chip, so that the gene to be detected is easier to be detected by the nanopore Nearby electric field trapping improves detection efficiency while significantly increasing the effective length of genes in the assay. The present invention also provides a solid-state nanopore gene sequencing device with the above beneficial effects.

Description

A solid-state nanopore gene pool and solid-state nanopore gene sequencing device

technical field

The invention relates to the field of gene sequencing, in particular to a solid-state nanopore gene pool and a solid-state nanopore gene sequencing device.

Background technique

As one of the important means for human beings to explore the secrets of life, gene sequencing technology has played a huge role in promoting the technological development in biology, life sciences, medicine and other fields. As an emerging fourth-generation gene sequencing technology, solid-state nanopore sequencing technology has the advantages of low cost, high read length, and easy integration. The current solid-state nanopore gene sequencing technology is to create a nano-scale pore size on a solid-state silicon nitride thin film chip, and make DNA chains pass through the solid-state nanopore to obtain DNA base sequences.

However, the current solid-state nanopore sequencing technology also faces many problems. For example, in the process of gene sequencing, the DNA is first planted on the gene-attached glass slide (hereinafter referred to as the glass slide). During the detection process, due to the glass slide and the nanopore chip ( (hereinafter referred to as the chip) is relatively non-parallel, and there is actually a face angle, so that there is a shortest distance from the glass surface to the chip, and this shortest distance will change with the size of the glass and chip and the size of the face angle, in other words, Due to the surface angle, one end of the silicon wafer is in contact with the glass slide first, but at this time, the gene growth point may not be in contact with the chip, which makes the DNA unable to effectively reach the electric field capture radius near the nanopore. This leads to low detection efficiency and short DNA effective detection length.

To sum up, how to avoid the difficulty of effective contact between the gene to be detected and the nanopore chip caused by the face angle, resulting in low detection efficiency and too short effective detection length of the gene, is an urgent problem to be solved by those skilled in the art.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a solid-state nanopore gene pool and a solid-state nanopore gene sequencing device, so as to solve the problem that the gene to be detected is difficult to contact effectively with the nanopore chip caused by the face angle in the prior art, which in turn leads to low detection efficiency and gene sequencing. Effectively detect problems with too short lengths.

In order to solve the above technical problems, the present invention provides a solid-state nanopore gene pool, including an upper liquid pool, a lower liquid pool, a gene-attached glass slide and a nanopore chip;

The nanopore chip is arranged at the bottom of the upper liquid pool, and the lower through holes of the upper liquid pool are correspondingly arranged with the nanopores of the nanopore chip;

The gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nanopore chip;

The upper surface of the gene-attached glass slide includes a column-shaped high platform; the column-shaped high platform is used to fix the gene to be detected.

Optionally, in the solid-state nanopore gene pool, the solid-state nanopore gene pool further includes a correction plateau;

The correction high platform is arranged around the columnar high platform, and the correction high platform is higher than the columnar high platform;

The ratio of the height of the correction plateau to the shortest distance from the correction plateau to the edge of the gene-attached glass slide is not lower than the contact threshold.

Optionally, in the solid-state nanopore gene pool, the correction plateau is an annular plateau;

The columnar platform is located inside the annular platform.

Optionally, in the solid-state nanopore gene pool, the annular high platform includes an electrolyte exchange port.

Optionally, in the solid-state nanopore gene pool, the height difference between the correction plateau and the columnar plateau ranges from 2 microns to 10 microns, inclusive.

Optionally, in the solid-state nanopore gene pool, the size of the gene-attached glass slide ranges from 1 mm to 10 mm.

Optionally, in the solid-state nanopore gene pool, the height of the correction plateau ranges from 20 microns to 500 microns, inclusive.

Optionally, in the solid-state nanopore gene pool, the gene-attached glass slide is a silicon-based glass slide.

Optionally, in the solid-state nanopore gene pool, the columnar platform is a cylindrical platform.

A solid-state nanopore gene sequencing device, the solid-state nanopore gene sequencing device comprising the solid-state nanopore gene pool according to any one of the above.

The solid-state nanopore gene pool provided by the present invention includes an upper solution pool, a lower solution pool, a gene-attached glass slide and a nanopore chip; the nanopore chip is arranged at the bottom of the upper solution pool, below the upper solution pool The through-holes are arranged corresponding to the nano-holes of the nano-pore chip; the gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nano-pore chip; the upper surface of the gene-attached glass slide comprises a columnar High platform; the columnar high platform is used to fix the gene to be detected.

In the present invention, the columnar high platform is arranged on the gene attachment glass slide, so that the attachment position of the gene to be detected is higher than the plane of the gene attachment glass slide, and when facing the gene attachment glass slide and the nanopore When the face-to-face angle of the chip is used, the distance from the gene to be detected to the nanopore can be effectively shortened, so that the gene to be detected is more easily captured by the electric field near the nanopore, the detection efficiency is improved, and the number of genes under detection is significantly increased. Effective length. The present invention also provides a solid-state nanopore gene sequencing device with the above beneficial effects.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention or the prior art more clearly, the following will briefly introduce the accompanying drawings used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only For some embodiments of the present invention, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic partial structure diagram of a specific embodiment of a solid-state nanopore gene pool provided by the present invention;

Figure 2 is a schematic partial structure diagram of another specific embodiment of the solid-state nanopore gene pool provided by the present invention;

FIG. 3 is a top view of the partial structure of another specific embodiment of the solid-state nanopore gene pool provided by the present invention;

FIG. 4 is a top view of the partial structure of another specific embodiment of the solid-state nanopore gene pool provided by the present invention.

Detailed ways

In order to make those skilled in the art better understand the solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The core of the present invention is to provide a solid-state nanopore gene pool. The schematic structural diagram of a specific embodiment of the gene pool is shown in FIG. 1, which is called specific embodiment 1, including an upper liquid pool, a lower liquid pool, and a gene attachment glass slide. and nanopore chips;

The nanopore chip is arranged at the bottom of the upper liquid pool, and the lower through holes of the upper liquid pool are correspondingly arranged with the nanopores of the nanopore chip;

The gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nanopore chip;

The upper surface of the gene-attached glass slide includes a columnar high platform 10; the columnar high platform 10 is used to fix the gene to be detected.

In addition, the gene-attached glass slide is a silicon-based glass slide, which can be a silicon glass slide or a silica glass slide.

As a summary of the preferred embodiments, the columnar platform 10 is a columnar platform, which is convenient for production and processing, and can greatly improve the production efficiency and production yield of the solid-state nanopore gene pool.

Of course, in order to facilitate the alignment of the nanopore with the columnar platform 10 and to ensure that the columnar platform 10 has the same effect when dealing with face angles in all directions, the columnar platform 10 is preferably arranged on the gene attachment glass. the central area of the slice.

It should be noted that, FIGS. 1 to 4 in the accompanying drawings of the present invention are schematic diagrams of the local structure of the solid-state nanopore gene pool, and more specifically, are schematic diagrams of the structure of the gene-attached glass slide.

The upper surface of the gene-attached glass slide refers to the surface close to the nanopore chip.

The solid-state nanopore gene pool provided by the present invention includes an upper solution pool, a lower solution pool, a gene-attached glass slide and a nanopore chip; the nanopore chip is arranged at the bottom of the upper solution pool, below the upper solution pool The through-holes are arranged corresponding to the nano-holes of the nano-pore chip; the gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nano-pore chip; the upper surface of the gene-attached glass slide comprises a columnar The high platform 10; the columnar high platform 10 is used to fix the gene to be detected. In the present invention, the columnar high platform 10 is arranged on the gene attachment glass slide, so that the attachment position of the gene to be detected is higher than the plane of the gene attachment glass slide, and when facing the gene attachment glass slide and the nanometer When the surface angle of the hole chip is high, the distance from the gene to be detected to the nanopore can be effectively shortened, so that the gene to be detected is more easily captured by the electric field near the nanopore, the detection efficiency is improved, and the detection efficiency is significantly increased. Gene effective length.

On the basis of the first embodiment, a correction high platform 20 is further added for the gene attachment glass slide, and the second embodiment is obtained. and nanopore chips;

The nanopore chip is arranged at the bottom of the upper liquid pool, and the lower through holes of the upper liquid pool are correspondingly arranged with the nanopores of the nanopore chip;

The gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nanopore chip;

The upper surface of the gene attachment glass slide includes a columnar high platform 10; the columnar high platform 10 is used to fix the gene to be detected;

The solid-state nanopore gene pool also includes a correction platform 20;

Described correction high platform 20 is arranged around described columnar high platform 10, and described correction high platform 20 is higher than described columnar high platform 10;

The ratio of the height of the correction plateau 20 to the shortest distance from the correction plateau 20 to the edge of the gene-attached glass slide is not lower than the contact threshold; it should be noted that the contact threshold is used to ensure that the nanopore The chip is firstly contacted with the correction platform 20 , and the contact threshold can be selected in the range of 0.03 to 0.06. Of course, it can be adjusted according to actual needs.

In this specific embodiment, the correction plateau 20 is added to the solid-state nanopore gene pool, and the correction plateau 20 is arranged around the columnar plateau 10 and is higher than the columnar plateau 10 and defines the The ratio range between the height of the correction high platform 20 and the shortest value of the correction high platform 20 to the edge of the glass slide is to ensure that the nanopore chip is in contact with the correction high platform 20 in the process of pressing down. The distance of the columnar platform 10 that grows the described gene to be detected from the correction platform 20 is far less than the distance between the contact point knife of the nanopore chip and the gene attachment chip without the correction platform 20 and the distance of the columnar platform 10 Therefore, compared with the situation without the correction high platform 20, the specific embodiment adds the correction high platform 20, so that under the same face angle, the distance from the gene to be detected to the nanopore is significantly shortened , to further improve the detection efficiency and the effective gene length in detection.

In addition, after adding the correction platform 20, a certain space is reserved between the nanopore chip and the gene-attached glass slide to avoid the occurrence of the nanopore chip and the gene-attached glass slide in the prior art. The influence of the dust particles between them further expands the face angle. In addition, by using the correction platform 20, the contact area between the nanopore chip and the gene-attached glass slide is significantly increased, and the difference between the nanopore chip and the gene-attached glass slide due to surface angle differences is avoided. There is a situation in which simple line contact occurs during the pressing process, resulting in excessive pressure and fragmentation, which reduces the accident rate in detection.

As a preferred embodiment, the correction high platform 20 is an annular high platform;

The columnar high platform 10 is located inside the annular high platform; the correction high platform 20 is an annular high platform, which can effectively deal with the face angles in all directions, so that no matter which direction the nanopore chip is oriented, it can be corrected first. The elevated platform 20 is in contact, and preferably, the columnar elevated platform 10 is located at the center of the annular elevated platform. Of course, the correction high platform 20 may not be a ring, but can be a rectangular frame-shaped high platform, or a scattered point-shaped high platform, etc. FIG. 3 is the embodiment corresponding to FIG. 2 when the correction high platform 20 is a ring high platform. A plan view, FIG. 4 is a plan view of a distribution mode in which the correction platform 20 is a scattered point-shaped platform, and the specific pattern can be selected according to actual needs.

Further, the annular high platform includes an electrolyte exchange port 21, and the electrolyte exchange port 21 is used for the circulation and exchange of electrolytes on the inside and outside of the annular shape. Referring to FIG. 2, the annular high platform in FIG. 2 is divided into Four sections, each gap is the above-mentioned electrolyte exchange port 21. Of course, the electrolyte exchange port 21 may not destroy the continuity of the annular high platform, but only open holes on the side wall of the annular high platform. Of course, It can be adjusted according to actual needs.

Further, the range of the height difference between the correction high platform 20 and the columnar high platform 10 is 2 microns to 10 microns, including the endpoint value, such as any one of 2.0 microns, 6.5 microns or 10.0 microns, of course, the specific situation can be based on Actual demand changes.

Still further, the height range of the described correction high platform 20 is 20 micrometers to 500 micrometers, including the endpoint value, such as any one of 20.0 micrometers, 120.3 micrometers or 500.0 micrometers. The ratio between the height and the shortest distance between the correction high platform 20 and the edge of the gene-attached glass slide is not lower than the contact threshold, and the height of the correction high platform 20 is at least 20 microns, then the correction high platform 20 is to the gene The maximum value of the shortest distance to the edge of the attached slide can be 500 microns.

In addition, the size of the gene attachment slides ranges from 1 mm to 10 mm, inclusive, such as any of 1.0 mm, 5.2 mm, or 10.0 mm. Since the gene-attached slides are mostly square slides, the range of the size refers to the range of the square slides.

The following provides a method for making a gene-attached glass slide including the correction platform 20, including (parameters in the steps can be changed according to actual conditions):

(1) Cleaning the slides;

(2) Magnetron sputtering deposition of aluminum film 500nm;

(3) Apply photoresist 1.7um;

(4) photolithography, development, and preparation of process patterns;

(5) Etch the aluminum film and transfer the lithography pattern;

(6) etching glass slide, depth 2um, diameter 10-50um, obtains the preposition of described columnar high platform;

(7) Remove the glue and peel off the metal aluminum film;

(8) Magnetron sputtering deposition of aluminum film 3um;

(9) Apply photoresist 1.7um;

(10) photolithography, development, and preparation of process patterns;

(11) Etch the aluminum film and transfer the lithography pattern;

(12) etching the glass slide, the depth is 20-100um, forming the columnar high platform and the correction high platform, and the thickness of the annular correction high platform is 25um;

(13) Remove glue and peel off the metal aluminum film;

(14) Sectioning, the gene attachment glass slide of 2*2mm can be obtained.

A solid-state nanopore gene sequencing device, the solid-state nanopore gene sequencing device comprising the solid-state nanopore gene pool according to any one of the above. The solid-state nanopore gene pool provided by the present invention includes an upper solution pool, a lower solution pool, a gene-attached glass slide and a nanopore chip; the nanopore chip is arranged at the bottom of the upper solution pool, below the upper solution pool The through-holes are arranged corresponding to the nano-holes of the nano-pore chip; the gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nano-pore chip; the upper surface of the gene-attached glass slide comprises a columnar The high platform 10; the columnar high platform 10 is used to fix the gene to be detected. In the present invention, the columnar high platform 10 is arranged on the gene attachment glass slide, so that the attachment position of the gene to be detected is higher than the plane of the gene attachment glass slide, and when facing the gene attachment glass slide and the nanometer When the surface angle of the hole chip is high, the distance from the gene to be detected to the nanopore can be effectively shortened, so that the gene to be detected is more easily captured by the electric field near the nanopore, the detection efficiency is improved, and the detection efficiency is significantly increased. Gene effective length.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the various embodiments may be referred to each other. As for the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant part can be referred to the description of the method.

It should be noted that in this specification, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply these entities or operations There is no such actual relationship or order between them. Moreover, the terms "comprising", "comprising" or any other variation thereof are intended to encompass non-exclusive inclusion such that a process, method, article or device comprising a list of elements includes not only those elements, but also includes not explicitly listed or other elements inherent to such a process, method, article or apparatus. Without further limitation, an element qualified by the phrase "comprising a..." does not preclude the presence of additional identical elements in a process, method, article or apparatus that includes the element.

The solid-state nanopore gene pool and the solid-state nanopore gene sequencing device provided by the present invention are described in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (10)

1. a solid-state nanopore gene pool, is characterized in that, comprises upper liquid pool, lower liquid pool, gene attachment glass slide and nanopore chip; The nanopore chip is arranged at the bottom of the upper liquid pool, and the lower through holes of the upper liquid pool are correspondingly arranged with the nanopores of the nanopore chip; The gene-attached glass slide is located on the upper surface of the center of the lower liquid pool, and is arranged corresponding to the nanopore chip; The upper surface of the gene-attached glass slide includes a column-shaped high platform; the column-shaped high platform is used to fix the gene to be detected. 2. The solid-state nanopore gene pool of claim 1, wherein the solid-state nanopore gene pool further comprises a correction plateau; The correction high platform is arranged around the columnar high platform, and the correction high platform is higher than the columnar high platform; The ratio of the height of the correction plateau to the shortest distance from the correction plateau to the edge of the gene-attached glass slide is not lower than the contact threshold. 3. The solid-state nanopore gene pool as claimed in claim 2, wherein the correction plateau is an annular plateau; The columnar platform is located inside the annular platform. 4 . The solid-state nanopore gene pool of claim 3 , wherein the annular high platform comprises an electrolyte exchange port. 5 . 5 . The solid-state nanopore gene pool according to claim 2 , wherein the height difference between the correction plateau and the columnar plateau is in a range of 2 μm to 10 μm, inclusive. 6 . 6. The solid-state nanopore gene pool of claim 2, wherein the size of the gene-attached slide is in the range of 1 mm to 10 mm. 7 . The solid-state nanopore gene pool of claim 6 , wherein the height of the correction plateau ranges from 20 μm to 500 μm, inclusive. 8 . 8 . The solid-state nanopore gene pool of claim 1 , wherein the gene-attached glass slide is a silicon-based glass slide. 9 . 9 . The solid-state nanopore gene pool according to claim 1 , wherein the columnar plateau is a cylindrical plateau. 10 . 10. A solid-state nanopore gene sequencing device, wherein the solid-state nanopore gene sequencing device comprises the solid-state nanopore gene pool according to any one of claims 1 to 9.

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