KR101349332B1 - Protein biosensor comprising pore structure using diffusive flow and fabricating method thereof - Google Patents

Abstract

The present invention relates to: a protein biosensor comprising a pore structure using a diffusive flow which uses the pore structure for checking the existence of antigens and measuring the concentration of the antigens in target fluid using the diffusive flow without the external power supply; and a fabricating method thereof. The protein biosensor using a diffusive flow by the present invention comprises the following: a nanopore formed on the upper side of a substrate in a hollow hole form for supplying target fluid; a first micropore formed on both ends of the nanopore with a step, having a bigger diameter than the nanopore; and a second micropore formed on both ends of the first micropore with a step, having a bigger diameter than the first micropore. Standard fluid is supplied to the lower side of the substrate.

Description

Protein biosensor having a pore structure using a diffusion flow and a method of manufacturing the same {PROTEIN BIOSENSOR COMPRISING PORE STRUCTURE USING DIFFUSIVE FLOW AND FABRICATING METHOD THEREOF}

The present invention relates to a protein biosensor and a method for manufacturing the same, and more particularly, having a pore structure, which can measure the presence and concentration of antigen contained in a measurement fluid using a diffusion flow without external power supply. It relates to a protein biosensor using a diffusion flow and a method of manufacturing the same.

A variety of biosensors for detecting biomolecules in a sample have been developed, and among them, nanopores having a diameter of 100 nm are mainly used.

In relation to an analysis device using a nanopore structure, Korean Patent Laid-Open Publication No. 10-2012-0000520 discloses a conductive layer 511 and first insulating layers on both sides of the conductive layer 511, as shown in FIG. A nanopore film 500 including 512a and 512b and a second insulating layer 513 surrounding the first insulating films 512a and 512b is formed, and the nanopore 520 is formed in the nanopore film 500. By detecting the DNA from the sample passing through the nanopore membrane 520 is disclosed.

However, in the technique related to the conventional nanoflow, it is necessary to apply an external auxiliary unit, for example, an external power source, in order to generate a fluid flow of a test sample such as a sample, that is, to flow ions in the sample. However, this external unit has a problem in that the configuration for controlling the increase of the equipment and the power supply to be applied is complicated.

Technical problem of the present invention devised to improve the above problems, by using a concentration gradient between the nanopore (nanopore) and micropore (micropore) formed on the substrate, through the nanopore without external power supply It is an object of the present invention to provide a nanopore structure of a protein biosensor using a diffusion flow capable of detecting an antigen contained in an incoming measurement fluid and a method of manufacturing the same.

According to another embodiment of the present invention, by forming a nanopore between the measurement fluid and the reference fluid, by increasing the ion concentration of the measurement fluid, the diffusion flow that can move the particles of the measurement fluid through the nanopore without external power supply An object of the present invention is to provide a nanopore structure of the protein biosensor and a method of manufacturing the same.

According to another embodiment of the invention, the antibody is coated on the nanopore wall, the antigen interacting with the antibody is attached to the antibody to adjust the diameter of the nanopore, and detect the amount of current flowing through the nanopore measuring fluid Disclosure of Invention It is an object of the present invention to provide a nanopore structure of a protein biosensor using a diffusion flow capable of detecting an antigen contained therein and a method of manufacturing the same.

However, the objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

Protein biosensor using a diffusion flow according to an embodiment of the present invention to achieve the above object, the nanopores formed in a hollow form on the upper side of the substrate for supplying a measurement fluid; A first micropore formed stepwise from both ends of the nanopore and having a diameter larger than that of the nanopore; And a second micropore formed stepwise from both ends of the first micropore and having a diameter larger than that of the first micropore, wherein the reference fluid is supplied to the lower side of the substrate.

The protein biosensor according to the present invention is characterized in that the reference fluid contains a lower ion concentration than the measurement fluid.

The protein biosensor according to the present invention is characterized in that the substrate is a silicon-on-insulator (SOI) wafer comprising a device layer, a SiO ₂ buried layer, and a P-type handle wafer.

The protein biosensor according to the present invention is characterized in that the ion particles of the measurement fluid are moved to the second micropores through the nanopores.

The protein biosensor according to the present invention is characterized in that the ion particles of the measurement fluid are moved in a diffusion flow through the concentration gradient of the nanopores and the first micropores and the second micropores.

Method for producing a protein biosensor using a diffusion flow according to an embodiment of the present invention, the first step of forming a nanopore (nanopore) of the hollow form on the upper side of the substrate; A second step of forming a hollow microsecond micropore at a point coinciding with the center of the nanopore under the substrate; And a third step of forming a first micropore between the nanopores and the second micropores, wherein the first micropores are formed stepped from both ends of the nanopores to have a larger diameter than the nanopores. The second micropores are formed stepped from both ends of the first micropores, and are larger in diameter than the first micropores.

In the method of manufacturing a protein biosensor according to the present invention, in the first step, the nanopores are formed by a reactive ion etching method.

In the method of manufacturing a protein biosensor according to the present invention, in the second step, the second micropores are formed by deep reactive ion etching (DRIE).

In the method of manufacturing a protein biosensor according to the present invention, in the third step, the first micropores are formed through a thermal oxidation process.

In the method of manufacturing a protein biosensor according to the present invention, a measurement fluid is supplied to an upper side of the substrate, and a reference fluid containing an ion concentration lower than the measurement fluid is supplied to a lower side of the substrate.

In the method of manufacturing a protein biosensor according to the present invention, the ion particles of the measurement fluid are moved to the second micropores through the nanopores, and the movement of the ion particles of the measurement fluid is performed by the nanopores and the first micropores. And a diffusion flow through the concentration gradient of the pore and the second micropores.

Protein biosensor using a diffusion flow according to another embodiment of the present invention, the nanopores formed in a hollow form on the upper side of the substrate for supplying a measurement fluid; A first micropore formed stepwise from both ends of the nanopore and having a diameter larger than that of the nanopore; Second micropores formed stepped from both ends of the first micropores, the second micropores having a larger diameter than the first micropores; And a copolymer coating layer continuously formed on the inner circumferential surface of the second micropores on the inner circumferential surface of the nanopores, wherein the reference fluid is supplied to the lower side of the substrate.

Protein biosensor according to the invention, at least one antibody attached to the copolymer coating layer formed on the nanopore wall; And a blocking layer formed between each of the antibodies and the copolymer coating layer, wherein the blocking layer minimizes a space between the antibodies.

Protein biosensor according to the invention, the antibody attached to the copolymer coating layer is characterized in that the anti-GFP (Green Fluorescent Protein).

According to the protein biosensor of the present invention, by using a concentration gradient between a nanopore and a micropore formed on a substrate, the protein biosensor is included in a measurement fluid flowing through the nanopore without an external power supply. There is an advantage to detect antigen present.

Further, according to the present invention, the nanopores are formed between the measurement fluid and the reference fluid, and the ion concentration of the measurement fluid is increased, so that particles of the measurement fluid can be moved through the nanopores without an external power supply.

In addition, according to the present invention, the antibody is coated on the nanopore wall, the antigen interacting with the antibody is attached to the antibody to control the diameter of the nanopore, and the amount of current flowing through the nanopore is included in the measurement fluid. There is an advantage that can detect the antigen.

1 is a perspective view showing a conventional nano-pore membrane structure.
2 is a cross-sectional view showing a pore structure having nanopores according to an embodiment of the present invention.
Figure 3 shows an optical micrograph of the pore structure according to an embodiment of the present invention from the micropore side.
4 is an exemplary view of a cross section of a pore structure according to an embodiment of the present invention with a scanning electron microscope.
5 to 7 are cross-sectional views showing pore structures having nanopores in another embodiment of the present invention.
8 is a graph showing the current-time characteristics of anti-GFP attached to the polymer layer in the nanopores according to the present invention.
9 is a graph showing current-time characteristics for the interaction of anti-GFP / GFP.
10 is a graph showing the current value according to the experimental process.
FIG. 11 is a graph showing current values for BSA flow instead of anti-GFP. FIG.
12 is a graph showing current-time characteristics of 1.5 nM (40 ng / mL) of PBS based GFP solution.
FIG. 13 is a graph showing current-time characteristics of 0.15 nM (4 ng / mL) of PBS based GFP solution.
14 is a graph showing changed current versus GFP concentration.
15 is a graph showing the time to reach a steady state according to the GFP concentration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a detailed description of preferred embodiments of the present invention will be given with reference to the accompanying drawings. In the following description of the present invention, detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

Embodiments in accordance with the concepts of the present invention can make various changes and have various forms, so that specific embodiments are illustrated in the drawings and described in detail in this specification or application. It is to be understood, however, that it is not intended to limit the embodiments according to the concepts of the present invention to the particular forms of disclosure, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

It is to be understood that when an element is referred to as being "connected" or "connected" to another element, it may be directly connected or connected to the other element, . On the other hand, when an element is referred to as being "directly connected" or "directly connected" to another element, it should be understood that there are no other elements in between. Other expressions that describe the relationship between components, such as "between" and "between" or "neighboring to" and "directly adjacent to" should be interpreted as well.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms "comprises ",or" having ", or the like, specify that there is a stated feature, number, step, operation, , Steps, operations, components, parts, or combinations thereof, as a matter of principle.

≪ Example 1 >

2 is a cross-sectional view showing a pore structure having a nano-pore according to an embodiment of the present invention.

As shown, the pore structure 10 in the protein biosensor 1 using the diffusion flow has both ends of the nanopores 20 and 20 formed in a hollow shape on the upper side of the substrate for supplying the measurement fluid. It is formed stepped from, and comprises a first micropores 30 having a larger diameter than the nanopores (20).

In addition, it may include a second micropore 40 formed stepped from both ends of the first micropore 30, the diameter larger than the first micropore 30, the reference fluid is supplied to the lower side of the substrate Can be.

In the present invention, it is preferable that the diameter of the nanopore 20 is 175 nm, the diameter of the first micropore 30 is 2.0 mu m, and the diameter of the second micropore 40 is 100 mu m. It is not. In addition, the nanopore 20 may be formed in various shapes such as a cone (funnel) shape, a round shape, or a square shape depending on the shape.

In addition, the reference fluid supplied to the lower side of the substrate may contain lower ion concentrations than the measurement fluid supplied to the upper side of the substrate. Thus, the ion particles of the measurement fluid may be moved to the second micropores 40 through the nanopores 20, in particular, the ion particles of the measurement fluid may be nanopores 20 and the first micropores 30 and The concentration of the second micropores 40 is transferred to a diffusive flow through a concentration gradient.

Although not shown, the substrate may be an SOI (Silicon-On-Insulator) wafer comprising an element layer, an SiO ₂ buried layer, and a P-type handle wafer, and a plurality of pore structures including nanopores 20 may be formed on one substrate .

Also, though not shown, the nanopore 20 may be formed in the device layer, the first micropore 30 may be formed in the SiO2 buried layer, and the second micropore 40 may be formed in the P-type handle wafer. In particular, a substrate made of an N-type handle wafer may be used instead of the P-type handle wafer.

A nanopore according to the present invention is fabricated on a silicon-on-insulator (SOI) wafer using two lithography steps and three etching steps.

1) samples use polished 100 mm diameter silicon-on-insulator (SOI) wafers with a 340 nm device layer, a 1 탆 thick SiO ₂ buried layer and a 450 탆 thick p-type handle wafer, both sides of which can be used .

2) A 60 nm SiO ₂ layer is thermally grown in a dry oxidation atmosphere at 1000 캜 for 60 minutes to etch the device layer to function as a hard mask.

3) The device layer was spin-coated with 3% poly (methyl methacrylate) in methoxybenzene at 5,000rpm to obtain a film thickness of 100nm. PPMA is soft baked at 175 ° C. for 15 minutes on a hot plate.

4) Electron beam lithography (EBL) is used to pattern the circles and then the pattern is grown.

5) The opening in the PPMA is transferred to the SiO ₂ hard mask by reactive ion etching (RIE).

6) By utilizing a SiO ₂ hard mask in the ICP (inductively coupled plasma, ICP) RIE with 500W Coil Power Using general etching equipment, 50W platen power and 2 10sccm of Cl ₂ for a minute, the upper silicon device layer Is etched.

Then, the back side process of the wafer is performed.

7) The backside of the wafer was spin coated at 2500 rpm to AZ4620 and soft baked on a hot plate at 90 ° C for 90 seconds.

8) Back-side photolithography is completed by utilizing patterned features through the EBL on the front side as reference alignment markers, so as to form a 100 [mu] m circle pattern perpendicular to the element side circle.

9) The handle wafer is etched in DRIE (deep reactive ion etcher) with SF ₆ and C ₄ F ₈ using the Bosch process.

10) The back side is then spin-coated with AZ4620 and soft baked to fill the 100 μm aperture.

11) The wafer is immersed in a relaxed oxide etchant (20: 1) to etch the buried SiO ₂ through the upper side aperture.

12) The wafer is then placed in a thermal oxidation furnace to grow a layer of SiO ₂ . The thermal oxidation process is conformal; The film thus grows on the sidewalls of the pore and controllably reduces the diameter of the pore.

3 is an optical micrograph of the pore structure according to the embodiment of the present invention seen from the micropore side, in particular an optical micrograph of the back side micropores having an area in which a diameter of 100 μm and a diameter of 2 μm or less are connected to each other Indicates. In addition, Figure 4 shows an SEM image of the cross-section of the 40nm nanopore as an illustration of the cross-sectional view of the pore structure according to an embodiment of the present invention by a scanning electron microscope. The large region below the nanopore represents a connected region of 2 탆 or less, that is, a region connected to the micropores.

As described above, by using the diffusion flow due to the concentration gradient between the nanopores 20 and the micropores 30 and 40, the ion currents changed by moving the ion particles contained in the measurement fluid toward the reference fluid with low ion concentration. It is possible to measure the component, the concentration and the like of the measurement fluid through the measured ion current. Therefore, in the present invention, there is a feature that can induce the flow of the ion particles without applying additional energy, that is, an external power source.

5 to 7 are cross-sectional views showing pore structures having nanopores in another embodiment of the present invention.

As shown, the pore structure in the protein biosensor using the diffusion flow includes a nanopore 20, a first micropore 30 and a second micropore 40, one embodiment described above It has the same form and function as the pore structure 10 in. Therefore, detailed description corresponding to one embodiment will be omitted.

In this other embodiment, the copolymer coating layer 50 is formed continuously from the inner circumferential surface of the nanopore 20 to the inner circumferential surface of the second micropore 40. In addition, a plurality of antibodies 60, for example, anti-GFP (Green fluorescent Protein) is attached to the copolymer coating layer 50 formed on the wall of the nanopores 20. In general, the silicon-based device has a property that proteins do not adhere well, while a protein-type antibody (copolymer) is coated on the wall surface of the nanopores 20 with the copolymer coating layer 50. 60) is more easily attached.

In addition, a blocking layer 70 is formed between each antibody 60 and the copolymer coating layer 50 formed on the inner circumferential surface of the nanopores 20. Thus, the blocking layer 70 minimizes the space between each antibody 60, and in particular, can prevent the antigens to be attached to each antibody 60, which will be described later, to adhere to the copolymer coating layer 50.

Therefore, according to the present invention, the measurement fluid is moved through the nanopores 20, in which case the antigen 80 included in the measurement fluid is also moved. At this time, the antigen 80 is attached to each antibody 60 attached to the wall of the nanopores 20, and the diameter of the nanopores 20 is reduced by the antigens 80 attached thereto.

Therefore, when the diameter of the nanopores 20 is reduced, the number of ions flowing through the nanopores 20 decreases, thereby decreasing the amount of ion current detected. If the antigen is not included in the measurement fluid, the amount of ion current becomes constant, and as the amount of antigen increases, the amount of current detected also decreases.

<Copolymer Preparation>

Copolymers are prepared to attach anti-GFP to the silicon nanopore walls. Monomer [20.17 mmol DMA, 0.42 mmol N, N-acryloyloxysuccinimide (NAS), 0.21 mmol [3- (methacryloyl-oxy) propyl] trimethoxysilane (MAPS)] was dried in a 50 mL round-bottom flask. Soluble in dried tetrahydrofuran.

The solution is degassed by alternating nitrogen purge over a vacuum connection over 30 minutes. Azoisobutyronitrile (AIBN) is added to the solution and heated to 50 ° C. and maintained at this temperature under some positive nitrogen pressure for 24 hours. After the polymerization is completed, the solution is promoted by the addition of petroleum ether.

Remove the supernatant and repeat the whole process twice. The copolymer is dried under vacuum for 4 hours at room temperature. Each monomer confers specific characteristics on the copolymer.

NAS is a reactive group capable of binding amino modified DNA, primary amines of lysines, and arginine in proteins. DMA, which forms a polymer backbone, allows polymer adsorption on the glass surface, and MAPS reacts with free silanol in covalent bonds to stabilize the coating.

The nanopores are pretreated with NaOH 1M for 30 minutes and HCL 1M for 1 hour and then washed with deionized water (DI water, DI water). A DMA.NAS.MAPS copolymer solution (1 w / v% of aqueous ammonium sulfate solution at 20% saturation level) is prepared. GFP (Millipore) contains 1M phosphate buffered saline at concentrations of 15 nM (400 ng / mL), 4 nM (107 ng / mL), 1.5 nM (40 ng / mL), 0.4 (10.7 ng / mL) and 0.15 (4 ng / mL) (phosphate buffered saline, PBS) and mouse anti-GFP (Invitrogen) is dissolved in 1M PBS buffer at a concentration of 400 ng / mL. PBS containing 1w / v% bovine serum albumin (BSA) is prepared as a block, ie barrier layer.

<Experimental Example>

First, the rear side of the pore (micropore side) with the copolymer solution 100 and the front side (nanopore) side with the DI water 200 are filled.

As a result, diffusion molecular flow due to different ion concentrations between copolymer solution (or PBS, anti-GFP and GFP solution) and DI water to produce a test sample flow through the nanopores without using an applied potential. This happens.

In this step, the characteristics of the present invention, as shown in Figure 5, is to coat the nanopores with a copolymer to form a copolymer coating layer (50). In the next step, as shown in FIG. 6, anti-GFP is bound to the copolymer as antibody 60 by filling the back side with DI water 200 and the front side with anti-GFP solution 300. The background is then blocked by filling the nanopore wall, which is not coated with anti-GFP, with the back side with DI water 200 and the front side with BSA, ie, forming a barrier layer 70.

Finally, as shown in FIG. 7, the GFP solution 400 is injected into the front side and DI water 200 is injected into the rear side. Between each step, the substrate is washed with DI water. Thereafter, the ion current passing through the nanopores is measured without applying a voltage.

<Result>

FIG. 8 is a graph showing the current-time characteristics of anti-GFP attached to the polymer layer in the nanopores according to the present invention. FIG. 8 shows the current-time (It) characteristics when anti-GFP is injected and copolymerized in the nanopores. Indicates. The current decreases from 8 mA to a steady state value of approximately 1.5 mA after 15 minutes. 9 is a graph showing the current-time characteristics for the interaction of anti-GFP / GFP. When GFP is injected, GFP binds to the anti-GFP in the nanopores, resulting in a current of 1.2 mA to 1 mA after 6.5 minutes. Falls into.

Since the anti-GFP is easily caught by the copolymer, the nanopore diameter is reduced by the anti-GFP coated on the copolymer coating layer. As shown in FIG. 8, a gradual decrease in current is shown due to the attachment of anti-GFP to the copolymer. However, because some of the anti-GFP antigens that bind to the site are in contact with the polymer, the anti-GFPs do not attach uniformly to the copolymer as a result of non-uniform anti-GFP / GFP interactions (FIG. 9). As such, the nanopore currents show fluctuations and sudden decreases. As shown in the figure inserted in FIG. 9, the sudden current decrease is due to the interaction of a large amount of anti-GFP 50 / GFP 70 at the same time.

10 shows current values for the steps of the experimental process. The current decreases until GFP flows, which means that good coating of the copolymer on the nanopore, adhesion of anti-GFP to the copolymer coating layer, and BSA blocking reduce the nanopore diameter, resulting in a current through the nanopore. Decreases. After the BSA block is used, the anti-GFP no longer attaches to the nanopores, as shown in the sixth step of FIG. 10 (eg, the second anti-GFP flow represented by aGFP2).

However, GFP flow reduces current due to anti-GFP / GFP interactions (FIGS. 9 and 10). As shown in the fifth step (eg, the second PBS flow represented by PBS2), it can be seen that the anti-GFP and PBS attached to the copolymer are very stable because there is no current change. In addition, experiments were performed using BSA instead of anti-GFP to confirm that the current change is due to the specific binding of GFP to anti-GFP.

As shown in FIG. 11, the current value of the BSA flow is about 1.15 mA, which is almost the same as that of the anti-GFP. Thus, the current change in the seventh step is due to the specific binding of GFP to anti-GFP.

FIG. 12 shows current-time (It) characteristics of GFP concentrations of 1.5 and 0.15 nM, and FIG. 13 shows current-time characteristics of 0.15 nM (4 ng / mL) of the PBS based GFP solution. At a GFP concentration of 1.5 nM, the current through the nanopores drops some suddenly, and the changed current appears smaller than the current change at a GFP concentration of 15 nM. In the case of the present invention, it can be seen that there is no sudden current drop (for example, a GFP concentration of 0.15 nM). The changed currents for the 1.5 and 0.15 nM concentrations were 0.48 and 0.71 mA, respectively, and the time to reach a stable current for the 1.5 and 0.15 nM concentrations was about 26 and 78 minutes, respectively.

14 shows the current (Δcurrent) changed during the experiment. Δcurrent is inversely proportional to the concentration of GFP, meaning that the change in current at a low GFP concentration is greater than at a high GFP concentration. The starting (starting) current values differ for each of the various experimental cases (eg, the current is inversely proportional to the concentration of GFP), but the stable current is slightly different and the current is inversely proportional to the concentration of GFP.

This result can be seen as a result of the fact that the ion current through the nanopores can be influenced by the GFP molecules. If there are many GFP molecules, the ionic current decreases due to the relative lack of PBS ions. The time to reach a stable current is also inversely proportional to the GFP concentration (FIG. 15). If there are many GFP molecules (eg, high GFP concentrations), the interaction of anti-GFP / GFP ends in a short time, but at low GFP concentrations, due to the lack of GFP, the interaction of anti-GFP / GFP is completed. It can be seen that a lot of time is required.

As described above, according to the present invention, nanopores having a diameter of 175 nm in succession to two micropores having a diameter of 2 μm and 100 μm were produced using an SOI wafer. Since GFP and anti-GFP were used as test samples, and diffusive flow can be obtained through the use of a concentration gradient between the nanopore and micropore sides, the biosensor according to the present invention is There is no need to use an external unit, i.e. an external power source, to create a flow (flow) of test samples.

Thus, there is a feature that the present invention can be applied to a simple, portable lab-on-chip device that does not require an external unit to generate a flow of test samples.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. will be. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

1: protein biosensor 10: pore structure
20: nanopore 30: first micropore
40: second micropore 50: copolymer coating layer
60 antibody 70 barrier layer
80: antigen

Claims (17)

In protein biosensor using diffusion flow,
Nanopores formed in a hollow form on the upper side of the substrate for supplying a measurement fluid;
A first micropore formed stepwise from both ends of the nanopore and having a diameter larger than that of the nanopore; And
And a second micropore formed stepped from both ends of the first micropores, the second micropores having a larger diameter than the first micropores.
Protein biosensor, characterized in that for supplying a reference fluid to the lower side of the substrate.
The method of claim 1,
And said reference fluid contains a lower ion concentration than said measurement fluid.
The method of claim 1,
The substrate is a protein biosensor, characterized in that the silicon-on-insulator (SOI) wafer consisting of a device layer, a SiO ₂ buried layer and a P-type handle wafer.
The method of claim 1,
The protein biosensor of the measuring fluid is moved to the second micropores through the nanopores.
5. The method of claim 4,
Ion particles of the measurement fluid is characterized in that the protein biosensor is moved to the diffusion flow through the concentration gradient (concentration gradient) of the nanopores and the first and second micropores.
In the method of manufacturing a protein biosensor using a diffusion flow,
A first step of forming a hollow nanopore (nanopore) on the upper side of the substrate;
A second step of forming a hollow microsecond micropore at a point coinciding with the center of the nanopore under the substrate; And
And a third step of forming a first micropore between the nanopores and the second micropores.
The first micropores are formed stepped from both ends of the nanopores to have a larger diameter than the nanopores, and the second micropores are formed stepped from both ends of the first micropores to have a diameter greater than the first micropores. Method for producing a protein biosensor, characterized in that large.
The method according to claim 6,
In the first step, the nanopore is a method of manufacturing a protein biosensor, characterized in that formed by reactive ion etching (Reactive Ion Etching) method.
The method according to claim 6,
In the second step, the second micropore is a manufacturing method of the protein biosensor, characterized in that formed by Deep Reactive Ion Etching (DRIE).
The method according to claim 6,
In the third step, the first micropore is a method of manufacturing a protein biosensor, characterized in that formed through a thermal oxidation process.
The method according to claim 6,
A measurement fluid is supplied to the upper side of the substrate, and a reference fluid containing an ion concentration lower than the measurement fluid is supplied to the lower side of the substrate.
11. The method of claim 10,
Ion particles of the measurement fluid are moved to the second micropores through the nanopores,
The movement of the ion particles of the measuring fluid is a method of manufacturing a protein biosensor, characterized in that the diffusion flow through the concentration gradient (concentration gradient) of the nanopores and the first micropores and the second micropores.
In protein biosensor using diffusion flow,
Nanopores formed in a hollow form on the upper side of the substrate for supplying a measurement fluid containing an antigen;
A first micropore formed stepwise from both ends of the nanopore and having a diameter larger than that of the nanopore;
Second micropores formed stepped from both ends of the first micropores, the second micropores having a larger diameter than the first micropores; And
And a copolymer coating layer continuously formed on the inner circumferential surface of the second micropores on the inner circumferential surface of the nanopores.
Protein biosensor, characterized in that for supplying a reference fluid to the lower side of the substrate.
13. The method of claim 12,
At least one antibody attached to the copolymer coating layer formed on the nanopore wall; And
And a blocking layer formed between each of the antibodies and the copolymer coating layer.
The blocking layer is a protein biosensor, characterized in that to minimize the space between each antibody.
The method of claim 13,
The antibody attached to the copolymer coating layer is a protein biosensor, characterized in that the anti-GFP (Green Fluorescent Protein).
13. The method of claim 12,
And said reference fluid contains a lower ion concentration than said measurement fluid.
13. The method of claim 12,
The substrate is a protein biosensor, characterized in that the silicon-on-insulator (SOI) wafer consisting of a device layer, a SiO ₂ buried layer and a P-type handle wafer.
13. The method of claim 12,
Ion particles of the measurement fluid is characterized in that the protein biosensor is moved to the diffusion flow through the concentration gradient (concentration gradient) of the nanopores and the first and second micropores.

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