KR20230114850A - Membrane structure comprising a porous membrane and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a membrane structure for separating biomolecules included in a sample. The membrane structure of the present invention has a porous film including a first porous film and a second porous film. The porous membrane includes fine holes having a diameter of a specific size, so that biomolecules can be more precisely separated. In addition, the porous membrane has increased stability and improved filter performance compared to conventional porous membranes.

Description

Membrane structure comprising a porous membrane and manufacturing method thereof

In general, a biomolecule refers to a material constituting a living body or a material derived from a living body, such as nucleic acids, proteins, and microvesicles. Recently, as it has become known that diseases such as cancer or Alzheimer's can be diagnosed using exosomes, which are signaling substances between cells, interest in and research on technology that can efficiently isolate specific biomolecules such as exosomes is on the rise.

However, as disclosed in U.S. Patent Publication No. 2012-0142001, the existing technology for isolating vesicles in bodily fluids by immobilizing antibodies that stick to vesicle proteins on a microchip requires a centrifugation process as a pretreatment process and fixes the antibodies. Since expensive equipment must be used to do this, there is a problem in that biomolecules are damaged and a lot of time and money are required for the biomolecule separation process.

Furthermore, in the case of a biomolecular filter manufactured using photolithography, there is a limit to manufacturing a biomolecular filter including pores having a diameter less than a certain size due to equipment limitations. In this case, it is difficult to more precisely separate extracellular vesicles such as exosomes.

1. US Patent Publication No. 2012-0142001

The present invention is a membrane including a porous membrane that can prevent damage to biomolecules in the process of separating biomolecules contained in a sample, save time and cost, and more precisely separate extracellular vesicles such as exosomes. It is an object of the present invention to provide a structure and a manufacturing method thereof.

As an example, the present invention provides a membrane structure for filtering biomolecules included in a sample.

In this case, in one embodiment, the structure may include a filtering unit for filtering biomolecules from the sample; and

A support portion extending from the filtering portion to support the filtering portion; includes;

The filtering unit includes a window area in which a plurality of window cells are formed in a matrix form and a blocking area in which the window cells are not formed;

The window cell has micro holes through which biomolecules pass,

The structure may include a substrate having a through hole constituting the window cell; and

It has a laminated structure including; a porous film laminated on the substrate and covering an opening on one side of the through hole,

The porous film may include a first porous film made of a first silicon-based compound; and a second porous film made of a second silicon-based compound.

In one embodiment, the present invention provides a membrane structure in which the microholes have a diameter of 60 nm to 150 nm.

In one embodiment, the present invention provides a membrane structure in which a second porous film is deposited on the first porous film.

In one embodiment, the present invention provides a membrane structure in which the first silicon-based compound and the second silicon-based compound are different silicon-based compounds.

In one embodiment of the present invention, the first silicon-based compound is silicon nitride (Si3N4),

The second silicon-based compound provides a membrane structure of silicon oxide (SiO2).

In one embodiment, the present invention provides a membrane structure in which the substrate includes a silicon substrate.

In one embodiment, the present invention provides a membrane structure wherein the substrate further comprises a silicon oxide (SiO 2 ) layer stacked on the silicon substrate.

As an example, the present invention provides a method for manufacturing a membrane structure for filtering biomolecules included in a sample.

At this time, in one embodiment, the method includes forming a porous film,

The step may include forming a first porous film; and

Forming a second porous film by depositing an additional thin film on the surface of the first porous film; provides a method for manufacturing a membrane structure comprising a.

In one embodiment, the present invention provides a method for manufacturing a membrane structure in which the step of forming the first porous film includes forming a porous film using a lithography process.

In one embodiment, the present invention provides a method for manufacturing a membrane structure in which the lithography process is a photolithography process.

In one embodiment, the present invention provides a method for manufacturing a membrane structure in which the additional thin film deposition uses PECVD.

The membrane structure of the present invention separates biomolecules included in a sample by filtering through a structure having a porous structure, thereby preventing damage to biomolecules and reducing the time and cost required for the biomolecule separation process. .

The membrane structure of the present invention includes a porous membrane having fine holes having a specific diameter, so that biomolecules included in a sample can be more precisely separated.

When the porous film of the membrane structure of the present invention is composed of a silicon nitride (Si3N4) layer and a silicon oxide (SiO2) layer, since the residual stress of each layer is opposite, the stability of the thin film can be increased, and thus the strength of the thin film can be increased. can

When the porous film of the membrane structure of the present invention includes a silicon oxide (SiO2) layer, since the silicon oxide has a large zeta potential, the stability of the thin film can be increased, and thus the strength of the thin film can be increased. . In addition, when a silicon oxide (SiO2) layer is located on the surface of the porous film, the silicon oxide can minimize aggregation with biological materials, thereby improving filter performance. has excellent reactivity, and the surface of the porous film can be easily modified.

1 is a plan view showing a membrane structure having a matrix structure according to an example of the present invention.
2 is an enlarged view showing a window cell portion of the membrane structure shown in FIG. 1;
3 is a vertical cross-sectional view showing a portion AA′ shown in FIG. 2;

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless defined otherwise.

biomolecules

As used herein, the term 'biomolecule' refers to materials constituting living organisms such as nucleic acids, proteins, and microvesicles, and materials derived from living organisms, as well as all materials formed by combining, disassembling, modifying, or mutating these materials. do.

step coverage

The term 'step coverage' used in this specification means step coverage, and means the ratio of the deposition thickness according to the position when a certain material is deposited. For example, when coating a surface with steps (or irregularities), it shows the ratio of the degree of coating on an inclined part to the degree of coating on a flat part, that is, the degree of uniformity of the coating. Specifically, when a certain material is deposited, it may mean an expression expressing the thickness accumulated on the wall and the thickness accumulated on the flat bottom as a ratio. The size of the fine hole diameter of the porous membrane included in the membrane structure of the present invention can be adjusted using step coverage. As a specific example, the porous film of the present invention may be composed of a first porous film and a second porous film, and at this time, in the process of depositing the second porous film on the first porous film, the diameter of the fine holes in the porous film is used by using step coverage. can be adjusted to the desired size.

Hereinafter, the membrane structure of the present invention will be described in detail with reference to the drawings.

I. Membrane structure (130)

The membrane structure 130 of the present invention is a membrane structure configured to filter biomolecules included in a sample.

The membrane structure 130 includes, for example, a filtering unit 132 and a support unit 134 . At this time, the shape and size of the membrane structure 130, the positions of the filtering unit 132 and the support unit 134, etc. may be variously changed depending on the environment in which the membrane structure 130 is applied. As a specific example, when the membrane structure 130 is configured in the form of a rectangular flat plate, the filtering unit 132 is located at the center of the membrane structure 130, and the support unit 134 is located at the edge of the membrane structure 130. can be located However, it is not limited thereto. Details of the configuration of the membrane structure 130 are described below.

1. Configuration of the membrane structure 130

1) Filtering unit 132

The filtering unit 132 is a part where filtering of biomolecules is performed. The filtering unit 132 includes a window area Dw in which the window cells w are formed and a blocking area Db in which the window cells w are not formed. For example, the filtering unit 132 may include a plurality of window areas Dw. In this case, the plurality of window areas Dw may be sequentially spaced apart and disposed side by side with the blocking area Db interposed therebetween.

i) Window area (Dw)

The window area Dw is an area in which a plurality of window cells w are formed along the movement path of the sample, and is formed in a matrix form and is configured to contact the sample. The filtering unit 132 filters biomolecules from the sample moving along the window area Dw. To this end, each window cell (w) formed in the window area (Dw) has a plurality of fine holes (h) through which biomolecules of a certain size or less pass. The fine hole (h) means a fine hole (h) formed in the porous film.

It should be noted in the present invention that the biomolecules to be filtered included in the sample are not separated by the window cells (w) formed in the window area (Dw), but the fine holes (h) formed in each window cell (w). ) are separated by That is, the size and shape of the window cell w are independent of the size of the biomolecule to be filtered.

A plurality of fine holes h are formed in each window cell w formed in the filtering unit 132 of the membrane structure 130 . The size of the window cell w may be, for example, about 1.3*1.3mm to 1.1*1.1mm when the membrane structure 130 has a size of 25*25mm. As a specific example, the size of the window cell w may be about 1.25*1.1.25mm when the membrane structure 130 has a size of 25*25mm. That is, the size and shape of the window cell w are independent of the size of the biomolecule to be filtered, and can be variously changed depending on the strength and thickness of the structure of the window cell w.

The size of the fine hole h included in the window cell w is determined according to the size of the biomolecule to be filtered. For example, the minute hole h formed in the window cell w may have a diameter ranging from 10 nm to 3000 nm. As a specific example, when the biomolecule to be filtered is an exosome, the plurality of fine holes h formed in the window cell w may have a diameter ranging from 10 nm to 300 nm. As a more specific example, 30 nm or more and 300 nm or less, 40 nm or more and 250 nm or less, 50 nm or more and 200 nm or less, 60 nm or more and 150 nm or less, 60 nm or more and 200 nm or less, 60 nm or more and 250 nm or less, or It may be configured to have a diameter corresponding to a range of 60 nm or more and 300 nm or less.

i) Blocking area (Db)

The blocking area Db is an area excluding the window area Dw from the entire area of the filtering unit 132, and is an area in which biomolecule filtering does not occur.

1) Support 134

The support part 134 is configured to extend or extend from the filtering part 132 to support the filtering part 132 . That is, the support part 134 is a part corresponding to a frame that is gripped by a user when the membrane structure 130 is transported or adhered to or inserted into another structure when the membrane structure 130 is installed to support the filtering unit 132. .

2. Structure and detailed configuration of the membrane structure 130

The membrane structure 130 of the present invention may have a laminated structure including a substrate and a porous film.

1) Substrate 130a

The substrate 130a corresponds to a basic skeleton of the membrane structure 130 and may include a silicon substrate L1 forming a core layer. In addition, the substrate 130a may further include a silicon oxide layer L2 stacked on the silicon substrate L1 to prevent deformation of the substrate or generation of cracks due to residual stress. The silicon oxide layer L2 may be stacked on both the upper and lower surfaces of the silicon substrate L1, or may be stacked on only one of the upper and lower surfaces. For example, the silicon oxide layer L2 may be deposited on the silicon substrate L1 through a deposition process of semiconductor manufacturing technology.

A through hole Hw for constituting the window cell w is formed in the substrate 130a. In this case, the through hole Hw may be formed through a lithography process of semiconductor manufacturing technology.

2) Porous membrane (130d)

The porous film 130d corresponds to the porous structure of the window cell w, and has a plurality of fine holes h through which biomolecules of a certain size or less pass through, and is laminated and supported on one side of the substrate 130a, It covers the opening on one side of the through hole Hw.

For example, the porous film 130d may include a first porous film 130b made of a first silicon-based compound and a second porous film 130c made of a second silicon-based compound. As a specific example, the porous film 130d may have a form in which a second porous film is deposited on the first porous film.

In this case, as an example, each of the first silicon-based compound and the second silicon-based compound may be selected from the group consisting of silicon nitride (Si3N4), silicon oxide (SiO2), and silicon carbide (SiC). As a specific example, the first silicon-based compound and the second silicon-based compound may be different silicon-based compounds. As a more specific example, the first silicon-based compound may be silicon nitride (Si3N4), and the second silicon-based compound may be silicon oxide (SiO2). In this case, the porous film 130d may have a form in which a second porous film made of silicon oxide is deposited on a first porous film made of silicon nitride.

Silicon nitride is a material mainly used as a passivation film to prevent alkali ions from diffusing to the semiconductor surface during semiconductor manufacturing. In the present invention, considering the characteristics of silicon nitride, that is, high strength (normal temperature bending strength of 100 to 140 kg/mm2), low thermal expansion coefficient (thermal expansion coefficient of 3 × 10 -6 / ° C), and excellent thermal shock resistance, etc. Thus, silicon nitride is adopted as a material for the porous film 130d.

In addition, when a silicon oxide layer is deposited on a silicon nitride layer, stability of the porous film 130d may be increased because the residual stress between the layers is opposite. Specifically, since the silicon nitride layer has tensile stress and the silicon oxide layer has compressive stress, stability of the porous film 130d may be increased. Additionally, as the absolute value of the zeta potential increases, the repulsive force between particles becomes stronger, resulting in higher dispersion and dispersion stability. Since silicon oxide has a higher zeta potential value, the stability of the porous film 130d increases when the silicon oxide layer is present on the surface. can do. Accordingly, the strength of the thin film can be increased by increasing the stability of the porous film 130d.

In addition, since silicon oxide can minimize aggregation with biomaterials, it can have an effect of improving filter performance, and since it has excellent reactivity with silane compounds, various functional groups are attached to the porous film 130d to provide additional biochemical effects. activity can be added.

The size of the fine holes h formed in the porous film 130d is determined according to the size of the biomolecule to be filtered. That is, the plurality of fine holes (h) formed in the porous film (130d) may be configured to have a diameter corresponding to a range of 10 nm or more and 3000 nm or less. For example, when the biomolecule to be filtered is an exosome, the plurality of fine holes h formed in the porous film 130d may have diameters ranging from 10 nm to 300 nm. As a specific example, the fine hole (h) corresponds to a range of 30 nm to 300 nm, 40 nm to 250 nm, 50 nm to 200 nm, 60 nm to 150 nm, 60 nm to 200 nm, 60 nm to 250 nm, or 60 nm to 300 nm. It can be configured to have a diameter. At this time, the minimum value of the diameter of the fine hole (h) may be determined according to the size of the biomolecule to be filtered. The maximum value of the diameter of the fine hole (h) is the fine hole (h) that can be manufactured only by a lithography process without using a deposition process h) It can have its meaning as the minimum limit value of the size. For example, the diameter of the fine hole (h) that can be obtained when manufactured only through a photolithography process is at least 150 nm, and an additional deposition process is required to have a diameter smaller than that. As described above, the membrane structure of the present invention includes a porous film having fine holes having a diameter in a specific range, so that biomolecules included in a sample can be more precisely separated.

An interval between the fine holes h may be, for example, 1 μm to 300 nm. The fine holes h may be positioned in a square pattern or a hexagonal pattern. At this time, the square pattern may mean that the fine holes h are located at the east, west, south, and north positions of one fine hole h, and the hexagonal pattern may mean that the south and north sides of one fine hole h , it may mean that the fine holes (h) are located at southeast, southwest, northeast, and northwest positions. However, the pattern in which the fine holes h are located is not limited thereto.

On the other hand, the porous film (130d), for example, may be configured to have a thickness corresponding to the range of 1μm or less. As a specific example, it may be configured to have a nano-sized thickness, that is, a thickness corresponding to a range of 50 nm or more and 500 nm or less. If the thickness of the porous film 130d is less than 50 nm, the strength of the porous film 130d is low and is easily damaged during manufacturing or use. On the other hand, if the thickness of the porous film 130d exceeds 500 nm, the biomolecule separation time may be prolonged and the separation efficiency may decrease. As a more specific example, in order to simultaneously satisfy membrane strength and biomolecule separation efficiency, the porous membrane 130d may be configured to have a thickness corresponding to a range of 50 nm or more and 300 nm or less.

II. Membrane structure 130 manufacturing method

One example of the present invention provides a method of manufacturing the membrane structure 130 . The membrane structure 130 is, for example, I. It may refer to the structure described in the membrane structure 130 . However, it is not limited thereto.

1. Forming a Porous Film (130d)

The manufacturing method includes forming a porous film. The step may include forming a first porous film 130b; and depositing an additional thin film on the surface of the first porous film 130b to form a second porous film 130c.

1) Forming the first porous film 130b

The manufacturing method may include forming the first porous film 130b. For example, forming the first porous film 130b may include forming the porous film using a lithography process. As a specific example, the lithography process may include photo lithography, electron beam lithography, or X-ray lithography. As a more specific example, the lithography process may be a photolithography process.

2) Forming a second porous film 130c by depositing an additional thin film on the surface of the first porous film 130b

The manufacturing method may include forming a second porous film 130c by depositing an additional thin film on the surface of the first porous film 130b.

In this step, as an additional thin film is deposited on the surface of the first porous film 130b, the diameter of the fine holes h may be reduced. In this case, in the thin film deposition process, the fine holes h may be manufactured to have a desired diameter by using step coverage.

At this time, the thin film deposition includes, for example, physical vapor deposition (PVD) and chemical vapor deposition (CVD) methods, and the chemical vapor deposition (CVD) method may include APCVD, LPCVD, PECVD, HDPCVD, and ALD methods there is. In addition, when an additional silicon layer is deposited by the PECVD method, the temperature can be controlled, so that the size of the diameter of the fine holes (h) can be adjusted without damaging the porous film.

2. Specific examples of manufacturing methods

As a specific example, the manufacturing method includes forming a first porous film 130b made of silicon nitride using a photo lithography process and oxidizing the surface of the first porous film 130b using PECVD It may be a method including forming the second porous film 130c by additionally depositing a thin film made of silicon.

In this case, the membrane structure 130 having a porous film including minute holes h having a diameter smaller than the limit size of photolithography equipment may be manufactured. As a more specific example, the manufacturing method makes it possible to manufacture a membrane structure 130 having a porous film including fine holes h having a diameter of 150 nm to 60 nm.

However, the manufacturing method is not limited to the above specific example, and the second porous film 130c is formed by forming the first porous film 130b and depositing an additional thin film on the surface of the first porous film 130b. It may include any manufacturing method including the step of forming.

III. Characteristics of the membrane structure 130

1. Manufacturing using deposition process

The membrane structure 130 of the present invention includes, for example, fine holes h having a diameter ranging from 60 nm to 150 nm. A diameter in the above range cannot be obtained with photolithography equipment, and may be formed through the deposition process of the present invention. In the membrane structure 130 of the present invention, the size of the diameter of the fine holes h is adjusted using step coverage through a deposition process. Here, step coverage is also referred to as stepped coating, and when coating a surface with steps (or unevenness), the ratio of the degree of coating on the inclined part to the degree of coating on the flat part, that is, the degree of uniformity of the coating is shown. The diameter of the fine hole h of the present invention can be adjusted to a desired size using step coverage.

2. Increased stability of the porous membrane 130d

The membrane structure 130 of the present invention includes, for example, a porous film 130d in which a silicon oxide layer is additionally deposited on a silicon nitride layer. In this case, since the residual stresses of silicon nitride and silicon oxide are opposite, stability of the film may be increased. In addition, since silicon oxide has a high zeta potential value, the stability of the film can be increased. In this way, it has the characteristic of increasing the strength of the porous membrane through the increase in stability of the membrane.

3. Improved biomolecular filter ability

Since the membrane structure 130 of the present invention can precisely separate biomolecules through the fine holes h having a diameter of a specific size or less, the filtering ability is improved. Specifically, it may have a feature capable of more precisely size-separating extracellular vesicles such as exosomes. In addition, the porous film 130d including the silicon oxide layer may minimize aggregation with the biological material, thereby improving filter performance.

4. Possible to add biochemical activity

The membrane structure 130 of the present invention includes, for example, a porous film 130d in which a silicon oxide layer is additionally deposited on a silicon nitride layer. In this case, silicon oxide has excellent reactivity with the silane compound and has a characteristic of easily modifying the surface of the porous film 130d. Accordingly, by attaching various functional groups to the surface of the porous film 130d, it has a feature that desired biochemical activity can be added.

IV. Biomolecular filter including membrane structure 130

The present invention provides, for example, a biomolecular filter including the membrane structure 130 as one component. At this time, as a specific example, in addition to the membrane structure 130, the biomolecule filter collects and discharges the biomolecules filtered through the first structure receiving the sample including the biomolecule to be filtered and the membrane structure 130. 2 structures may be additionally included.

The first structure may be configured to be located on one surface of the membrane structure 130, and may function to receive a sample including a biomolecule to be filtered and move it along the window area Dw of the membrane structure 130. there is. To this end, the first structure may include an inlet through which the sample is introduced, a first outlet through which residual sample from which biomolecules are separated is discharged, and a first flow path connecting the inlet and the first outlet.

The second structure may be configured to be positioned on the other side of the membrane structure 130, and may function to collect and discharge biomolecules coming out through the other side of the membrane structure 130. To this end, the second structure may include a second channel for moving biomolecules coming out through the fine holes of each window cell (w) on the other surface of the membrane structure 130 and a second outlet for discharging the biomolecules. there is.

At this time, the configuration of the biomolecule filter including the membrane structure 130 as one component is not limited to the configuration described above, and may include additional components required to perform the function of filtering biomolecules.

130: membrane structure
132: filtering unit
134: support

Claims (11)

A membrane structure for filtering biomolecules contained in a sample,
The structure may include a filtering unit filtering biomolecules from the sample; and
A support portion extending from the filtering portion to support the filtering portion; includes;
The filtering unit includes a window area in which a plurality of window cells are formed in a matrix form and a blocking area in which the window cells are not formed;
The window cell has micro holes through which biomolecules pass,
The structure may include a substrate having a through hole constituting the window cell; and
It has a laminated structure including; a porous film laminated on the substrate and covering an opening on one side of the through hole,
The porous film may include a first porous film made of a first silicon-based compound; and a second porous film made of a second silicon-based compound.
According to claim 1,
The micro-hole is a membrane structure having a diameter size of 60 nm to 150 nm.
According to claim 1,
The porous film is a membrane structure in the form of depositing a second porous film on the first porous film.
According to claim 1,
The first silicon-based compound and the second silicon-based compound are different silicon-based compounds membrane structure.
According to claim 4,
The first silicon-based compound is silicon nitride (Si3N4),
The second silicon-based compound is a membrane structure of silicon oxide (SiO2).
According to claim 1,
The substrate is a membrane structure comprising a silicon substrate.
According to claim 6,
The substrate further comprises a silicon oxide (SiO 2 ) layer stacked on the silicon substrate.
A method for manufacturing a membrane structure for filtering biomolecules contained in a sample,
The method includes forming a porous film,
The step may include forming a first porous film; and
Forming a second porous film by depositing an additional thin film on the surface of the first porous film; Membrane structure manufacturing method comprising a.
According to claim 8,
Wherein the forming of the first porous film comprises forming a porous film using a lithography process.
According to claim 9,
The lithography process is a method for manufacturing a membrane structure that is a photolithography process.
According to claim 8,
The additional thin film deposition is a membrane structure method using PECVD.