CN108226249B - Disposable nanopore biosensor and manufacturing method thereof - Google Patents
Disposable nanopore biosensor and manufacturing method thereof Download PDFInfo
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- CN108226249B CN108226249B CN201810019657.0A CN201810019657A CN108226249B CN 108226249 B CN108226249 B CN 108226249B CN 201810019657 A CN201810019657 A CN 201810019657A CN 108226249 B CN108226249 B CN 108226249B
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Abstract
The invention discloses a disposable nanopore biosensor, which comprises a substrate, an electrode layer and a top cover which are sequentially stacked, wherein the top cover is provided with micropores with the diameter of 5-200 mu m, and the micropores are used for injecting nanopores and a phospholipid film. The nanopore biosensor has the advantages of simple structure, convenience and quickness in detection and low cost, can be added at the time of adding a reagent section, ensures the stability of the nanopore and the phospholipid film, is particularly suitable for occasions of disposable use, avoids the mutual interference of detection results among patients caused by the repeated use of the sensor, and improves the accuracy of the detection results. The manufacturing method of the biosensor is also disclosed, the processing method has simple steps, is easy to operate, can realize mass rapid production, greatly reduces the production cost of the nanopore biosensor, and is suitable for mass industrial production and wide popularization and application.
Description
Technical Field
The invention belongs to the technical field of biological detection, relates to a biosensor and a manufacturing method thereof, and particularly relates to a disposable nanopore biosensor and a manufacturing method thereof.
Background
Around the great research and rapid development of high-sensitivity, high-selectivity rapid sensing analysis of biomolecules in recent years, researchers have combined various nanotechnologies to develop a series of electrochemical biosensors using advanced nanomaterials prepared and obtained, in which a nanopore biosensor is one of important bio-detection sensors.
The nanopore generally refers to a pore channel structure with a pore diameter of 1-10nm, the pore diameter of the pore channel structure is equivalent to the size of a single biological macromolecule, usually, the nanopore is arranged in an organic phospholipid film to separate a sample (such as serum) to be analyzed into two parts, when voltage is applied to two sides of the nanopore, charged ions and molecules in the sample can pass through the nanopore to form current, and a generated current signal can be collected together specifically and analyzed. When biological macromolecules pass through the nano-pores, due to different properties such as size, charge and the like, specific current characteristic signals can be generated, so that information such as the structure, the type, the concentration and the like of the biological macromolecules in a sample can be analyzed according to the current signals collected by an instrument.
The nanopore comprises two major types, namely a solid nanopore and a biological nanopore, the nanopore in common application is a biological nanopore, the biological nanopore, an artificial phospholipid film supporting the biological nanopore and a support supporting the phospholipid film are synthesized into the nanopore biosensor, the nanopore biosensor and the nanopore technology are widely applied to qualitative and quantitative analysis of biological macromolecules, such as DNA sequencing, tumor marker detection, environmental heavy metal ion detection and the like, the nanopore technology is a third-generation gene sequencing technology which is popular in the world, and due to the huge application potential in the fields of biology, chemistry, medicine, food, environment and the like, the nanopore has been developed into a new research field crossing multiple subjects, such as biology, chemistry, medicine, fluid, electronics, materials, machinery and the like.
In the field of in vitro diagnosis, microfluidic/nanofluidic and molecular detection, a nanopore product has many advantages such as high sensitivity, simple operation, high detection speed, low equipment cost, and the like, the core of the nanopore product is a nanopore biosensor, but most of the existing nanopore biosensors are reusable products, and the reusable sensors easily cause mutual interference among different patients, which affects the accuracy of detection results.
Disclosure of Invention
Therefore, the technical problems to be solved by the invention are that the traditional nanopore biosensor is repeatedly used, the detection result is not accurate enough, the disposable sensor is complex to operate, high in cost and incapable of being widely applied, and therefore, the disposable nanopore biosensor capable of being quickly detected and low in cost and the manufacturing method thereof are provided.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a disposable nanopore biosensor, which comprises a substrate, an electrode layer and a top cover which are sequentially stacked, wherein the top cover is provided with micropores with the diameter of 5-200 mu m for injecting nanopores and a phospholipid film.
Preferably, the top cover has a hollow sample limiting area, an insulating cover is arranged in the sample limiting area, the surface of the insulating cover is lower than the surface of the side wall of the sample limiting area, and the micropores are formed in the insulating cover.
Preferably, the substrate and the top cover are both plastic layers, and the electrode is a silver electrode layer arranged on the surface of the substrate.
Preferably, the silver electrode layer is composed of a pair of silver electrodes, the insulating cover covers one surface of the silver electrode, and the micro-holes on the insulating cover are arranged on the top of the silver electrode, so that the silver electrode is exposed only through the micro-holes.
Preferably, the size of the disposable nanopore biosensor is not less than 5mm by 5mm, and the diameter of the sample confinement region is not less than 1.5 mm.
Preferably, the micropores are round holes, and the aperture of the round holes is 20 μm.
The invention also provides a method for manufacturing the disposable nanopore biosensor, which comprises the following steps:
s1, according to a preset graph, performing injection molding on a medical plastic base material to form a substrate and a top cover, performing injection molding on the top cover to form a sample limiting area, forming and manufacturing an insulating covering piece inside the sample limiting area, and manufacturing micropores on the insulating covering piece, wherein the top surface of the insulating covering piece is lower than the surface of the side wall of the sample limiting area;
s2, preparing an electrode layer on the surface of the substrate;
and S3, covering the top cover on the surface of the substrate with the electrode layer, and welding and fixing the substrate and the top cover.
Preferably, the electrode layer is prepared by a thin film evaporation technique.
Alternatively, the electrode layer is preferably prepared by embedding silver wires into the substrate.
Alternatively, the electrode layer is preferably prepared by covering the surface of the substrate with a silver foil of a specific pattern.
Preferably, the method further comprises the step of injecting the nanopore and the phospholipid membrane into the micropore.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the disposable nanopore biosensor comprises a substrate, an electrode layer and a top cover which are sequentially stacked, wherein the top cover is provided with micropores with the diameter of 5-200 mu m, and the micropores are used for injecting nanopores and a phospholipid film. When the functional biosensor is used, the nanopore and the phospholipid film are injected into the micropore to obtain the functional biosensor, and when biological macromolecules pass through the nanopore, specific current characteristic signals are generated due to different properties such as size, charge and the like, so that information such as the structure, the type, the concentration and the like of the biological macromolecules in a sample can be analyzed according to the collected current signals. The nanopore biosensor has the advantages of simple structure, convenience and quickness in detection and low cost, can be added at the time of adding a reagent section, ensures the stability of the nanopore and the phospholipid film, is particularly suitable for occasions of disposable use, avoids the mutual interference of detection results among patients caused by the repeated use of the sensor, and improves the accuracy of the detection results.
(2) The invention relates to a method for manufacturing a disposable nanopore biosensor, which comprises the steps of firstly manufacturing a substrate and a top cover made of plastic materials through an injection molding process, manufacturing a hollow space at the top of the top cover to form a sample limiting area for limiting a detection sample and an insulating covering piece for containing a dropping reagent and enabling the dropping reagent to cover a micropore and an electrode layer, wherein the insulating covering piece is lower than the side wall surface of the sample limiting area to prevent the reagent from overflowing, preparing the electrode layer on the surface of the substrate according to a designed electrode pattern through evaporation or other modes, and then welding the substrate and the top cover to form a sensor support. The substrate and the top cover are made of plastic materials and are formed by pressing through a traditional injection molding process, mass rapid production can be achieved, the area of a silver coating is reduced on the premise that the requirement is met through structural design before electrode preparation, the production cost of the nanopore biosensor is greatly reduced, and the nanopore biosensor is suitable for mass industrial production and wide popularization and application. The processing method has simple steps and easy operation, and reduces the single use cost of the nano biosensor.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a top view of a disposable nanopore biosensor according to an embodiment of the invention;
fig. 2 is a schematic structural diagram of a disposable nanopore biosensor according to an embodiment of the invention.
The reference numbers in the figures denote: 1-a substrate; 2-an electrode layer; 3-a top cover; 31-a sample confinement zone; 32-an insulating cover; 33-microwell.
This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as providing a full and complete disclosure and set forth fully to those skilled in the art, and the scope of the present invention is defined by the claims. In the drawings, the size and relative sizes of various devices may be exaggerated for clarity.
Detailed Description
Examples
The embodiment provides a disposable nanopore biosensor which can be used in various application fields such as DNA sequencing, tumor marker detection, heavy metal ion detection and chemical analysis.
The disposable nanopore biosensor is shown in fig. 1, and comprises a plastic substrate 1, a silver electrode layer 2 and a plastic top cover 3 which are sequentially stacked from bottom to top, wherein the plastic top cover 3 is prepared with a sample limiting area 31 for accommodating and limiting a sample to be detected through injection molding, the sample limiting area 31 is an open hollow position manufactured in the top cover 3, the sample is limited inside the sample limiting area 31 by four walls of the hollow position, the sample is prevented from leaking, an insulating covering part 32 for accommodating a dropped reagent is arranged in the center of the sample limiting area 31, in the implementation, the insulating covering part 32 is a cuboid plastic part, the insulating covering part 32 and the top cover are integrally molded, the top surface of the insulating covering part is lower than the top surface of the side wall of the sample limiting area 31, and the reagent on the insulating covering part 32 is prevented from overflowing. The insulating cover 32 is provided with a micro-hole 33 for injecting a nano-hole and a phospholipid film into the biosensor, the micro-hole may be in the shape of a round hole or a square hole, preferably a round hole in this embodiment, the pore diameter of the micro-hole 33 is 5-200 μm, preferably 20 μm in this embodiment, and the micro-hole 33 is a through hole and is communicated with the electrode layer 2 below.
Further, the silver electrode layer 2 is composed of a pair of silver electrodes, the insulating cover 32 covers one surface of the silver electrode, and the micro-holes 33 are formed on the top of the silver electrode, so that the silver electrode is exposed only through the micro-holes 33 to serve as an insulating cover.
The overall size of the disposable nanopore biosensor is not less than 5mm x 5mm, preferably 2cm x 1cm in this embodiment, the diameter of the sample restriction region 31 is not less than 1.5mm, and in this embodiment, the sample restriction region 31 is rectangular, and the single side size thereof is not less than 2 mm.
The present embodiment also provides a method for manufacturing the disposable nanopore biosensor, comprising the steps of:
s1, manufacturing a substrate 1 and a top cover 3 from a medical plastic base material by an injection molding process according to a preset shape, processing the top cover 3 by the injection molding process, manufacturing a hollowed-out sample limiting area 31 inside the top cover 3 by the injection molding process, enclosing the sample limiting area 31 with four walls outside the sample limiting area 31 to form a dam structure, preventing a sample from overflowing, manufacturing an insulating cover 32 at the central position of the sample limiting area 31 by the injection molding process, making the surface of the insulating cover 32 lower than the surface of the side wall of the sample limiting area 31, preventing a dripped reagent from overflowing, and manufacturing the micropores 33 in the insulating cover 32 by the injection molding process.
S2, preparing the silver electrode layer 2 on the surface of the substrate 1 by a thin film vapor deposition process, or as a switchable embodiment, embedding a silver wire on the plastic substrate 1 as the silver electrode layer, or as a switchable embodiment, covering the surface of the substrate with a silver foil having a specific pattern as the silver electrode layer.
S3, covering the top cover 3 prepared in step S1 on the surface of the substrate 1 provided with the electrode layer 2, covering the insulating cover 32 of the top cover 3 on the surface of a silver electrode, placing the micro-holes 33 on the top of the silver electrode, and welding and fixing the substrate 1 and the top cover 2 by thermal welding to form a sensor holder, wherein as an alternative embodiment, the substrate 1 and the top cover 2 can be welded and fixed by other welding means. When the functional nanopore biosensor is used for detection, nanopores and a phospholipid film are injected from the micropores 33 to form the functional nanopore biosensor.
The disposable nanopore biosensor can be used for rapidly manufacturing the substrate 1 and the top cover 3 in a large batch through an injection molding process, and by designing the structure of the electrode layer 2, the silver plating area can be reduced to the maximum extent on the premise of meeting requirements, so that the production cost of the biosensor is greatly reduced, the biological detection cost is further reduced, the disposable nanopore biosensor is particularly suitable for occasions with disposable use, the single use cost is low, the mutual interference between detection results of different patients caused by repeated use of the sensor is avoided, and the detection accuracy of the sensor is improved.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (5)
1. A disposable nanopore biosensor, comprising a substrate, an electrode layer and a top cap which are sequentially stacked, wherein the top cap is provided with micropores with the diameter of 5-200 μm for injecting nanopores and a phospholipid membrane; the top cover is provided with a hollow sample limiting area, an insulating covering part is arranged in the sample limiting area, the surface of the insulating covering part is lower than the surface of the side wall of the sample limiting area, and the micropores are formed in the insulating covering part; the substrate and the top cover are made of plastic layers, and the substrate and the top cover are formed by pressing through a traditional injection molding process; the electrode layer is a silver electrode layer arranged on the surface of the substrate, and is prepared by a thin film evaporation technology or is prepared by embedding silver wires into the substrate or is prepared by covering a silver foil with a specific pattern on the surface of the substrate; the silver electrode layer consists of a pair of silver electrodes, the insulating covering piece covers the surface of one silver electrode, and the micropores in the insulating covering piece are arranged at the top of the silver electrode, so that the silver electrode is only exposed through the micropores;
the shape of the sample limiting area is rectangular, and the unilateral size of the sample limiting area is not less than 2 mm.
2. The disposable nanopore biosensor of claim 1, wherein said disposable nanopore biosensor has a size of no less than 5mm by 5mm, and said sample confinement region has a diameter of no less than 1.5 mm.
3. The disposable nanopore biosensor according to claim 2, wherein said micropores are round holes with a pore size of 20 μm.
4. A method of making a disposable nanopore biosensor according to any of claims 1-3, comprising the steps of:
s1, according to a preset graph, performing injection molding on a medical plastic base material to form a substrate and a top cover, performing injection molding on the top cover to form a sample limiting area, forming and manufacturing an insulating covering piece inside the sample limiting area, and manufacturing micropores on the insulating covering piece, wherein the top surface of the insulating covering piece is lower than the surface of the side wall of the sample limiting area;
s2, embedding silver wire into the substrate or on the surface of the substrate by using a thin film evaporation technology
Preparing an electrode layer by covering a silver foil with a specific pattern;
and S3, covering the top cover on the surface of the substrate with the electrode layer, and welding and fixing the substrate and the top cover.
5. The method of claim 4, further comprising the step of injecting the nanopore and the phospholipid membrane into the pore.
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| CN111085280B (en) * | 2018-10-23 | 2021-04-30 | 京东方科技集团股份有限公司 | Biological detection device, preparation method thereof, chip and detection method of biological molecules |
| CN111499705B (en) * | 2019-01-30 | 2023-08-08 | 深圳市梅丽纳米孔科技有限公司 | Mutant NfpAB nanopore, testing system, manufacturing method and application |
| CN111413383B (en) * | 2020-04-20 | 2021-03-02 | 深圳市梅丽纳米孔科技有限公司 | Method for constructing double-recognition-site nanopore |
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