Patent Application
20100052207
This application claims priority from Korean Patent Application No. 10-2008-0084044, filed on Aug. 27, 2008, in the Korean Intellectual Property Office, the contents of which are herein incorporated by reference in their entirety.
1. Field
The present disclosure is directed to a method of preparing a microarray using an array mold comprising a concave portion including a fluidic channel having a fluid inlet and a fluid outlet.
2. Description of the Related Art
Methods of preparing microarrays are well known and widely used. Two such methods include using photolithography and a spotting method. The former is a method of gradually synthesizing a probe material on a surface of a substrate to immobilize the probe material thereon, and the latter is a method of immobilizing a previously synthesized probe material on an activated surface of a substrate.
With regards to the method of immobilizing the previously synthesized probe on the surface of the substrate, a known method of manufacturing a DNA chip includes providing a substrate on which a solidifying agent is coated on a front surface of a slide glass, mounting one by one on the surface of the substrate by spotting a droplet that includes a previously prepared probe DNA, and drying the droplet.
However, in the first method of gradually synthesizing the probe material on the surface of the substrate to immobilize the probe material thereon, a large amount of the photomask, which is expensive, is used, and the used photomask cannot be reused. In addition, the process of immobilizing a biomaterial on a surface of a substrate is generally performed in a liquid phase of the biomaterial. Thus, if a stepper is used in the process, a large amount of the biomaterial is needed, and it is challenging to perform the process in a liquid phase. Therefore, microarray manufacturing costs are high, processes of manufacturing microarrays are complicated, and the yield is low. In addition, in the second method of immobilizing the previously synthesized probe on the surface of the substrate, reaction efficiency is low, and activation may not be uniformly conducted.
Exemplary embodiments of the invention include a method of efficiently preparing a microarray using an array mold including a concave portion.
Exemplary embodiments of the invention may include a method of preparing a microarray, the method comprising: preparing an array mold with a concave portion arranged thereon, the concave portion comprising a fluidic channel having a fluid inlet and a fluid outlet, wherein the array mold comprises an optically transparent material; adhering a substrate to a surface on which the concave portion of the array mold is arranged to form an adhered body of the array mold and the substrate, the adhered body comprising a chamber defined by a space between the concave portion and the substrate; and introducing a biomaterial into the chamber via the fluid inlet of the adhered body to immobilize the biomaterial on a surface of the substrate in the chamber.
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description.
A method according to an embodiment of the invention of preparing a microarray includes preparing an array mold comprising a concave portion including a fluidic channel having a fluid inlet and a fluid outlet, wherein the array mold comprises an optically transparent material.
The fluid inlet includes an inlet through which a material needed for immobilization of a probe of a microarray or a probe is introduced to fill the concave portion of the array mold.
The fluid outlet includes an outlet through which a left over material is exhausted.
The fluidic channel includes the fluid inlet and the fluid outlet, and is connected to the concave portion of the array mold. [0019] The concave portion is a portion that is adhered onto a surface of a substrate and faces the substrate and that includes the fluidic channel including the fluid inlet and fluid outlet in the array mold. There may be a plurality of concave portions in a variety of sizes according to a spot pattern of a microarray.
The array mold comprises an optically transparent material. The array mold may comprise any material that facilitates the preparation of an internal structure such as the concave portion including a fluidic channel having a fluid inlet and a fluid outlet, is optically transparent, and has a strong adhesion with respect to a surface of a microarray substrate. The “optically transparent material” may be indium tin oxide (ITO), glass, optically transparent plastic, or optically transparent silicon. The array mold may comprise polydimethylsiloxane (PDMS).
The array mold may be prepared using, for example, a photolithographic process using a negative photoresist, which is well known to one of ordinary skill in the art.
A method according to an embodiment of the invention of preparing a microarray includes adhering a substrate to a surface of a portion in which the concave portion of the array mold is arranged to form an integrated adhered structure including a chamber defined by a space between the concave portion and the substrate.
The chamber includes an inner space of the adhered structure, which is isolated from the outside by the adhesion between the array mold and the substrate and in which the immobilization of the probe of the microarray is performed. The adhesion occurs between a portion along side the concave portion of the array mold and a portion along side a spot region immobilized on the surface of the substrate. The adhesion provides the chamber into which the biomaterial introduced through the fluid inlet is immobilized on the surface of the substrate, and prevents contamination between the biomaterials by preventing the biomaterial introduced into the chamber from leaking out to another concave portion. The adhesion may be performed using an adhesive material, or using adhesive properties of the substrate and array mold themselves. The adhered structure includes a structural body obtained by the adhesion between the array mold and the substrate.
A method according to an embodiment of the invention of preparing a microarray includes introducing a biomaterial into the chamber via the fluid inlet of the adhered structure to immobilize the biomaterial on a surface of the substrate in the chamber.
The biomaterial includes a probe or any material comprising a probe in the manufacturing of the microarray. For example, the biomaterial may be one selected from the group comprising DNA, RNA, LNA, PNA, peptide, a virus, and a cell, or a monomer, such as nucleotide, nucleoside, or amino acid. The biomaterial may be derived from a living organism, or synthesized or semi-synthesized.
Immobilization may include depositing previously synthesized probe molecules on a surface of an activated substrate. The immobilization of the biomaterial on the surface of the microarray substrate is well known to those of ordinary skill in the art. A method according to an embodiment of the invention of activating a predefined region of a substrate, and then contacting the predefined region of the substrate with a previously selected monomer solution is provided. The predefined region belongs to the portion of the surface of the substrate of the chamber of the adhered structure, and may be activated by a light source. The remaining portion of the substrate is blocked from the light source, thus remains inert. According to an embodiment of the invention, the predefined region, is either exposed to light to be active, or exposed to a reactive material. The remaining portion of the substrate is a portion adhered to the array mold, and is physically blocked from contact with a reactant. In addition, the surface of the substrate may be treated with a material having a functional group that can bind the biomaterial. The functional group may be, but is not limited to, an amine group, a carboxyl group, an epoxy group, or a sulfur group. The material having the functional group that can bind the biomaterial may be, but is not limited to, γ-aminopropyltriethoxysilane (GAPS). As described above, in the process of immobilizing the biomaterial on the surface of the substrate, adsorption, a chemical reaction, or physical interaction between the substrate and probe may occur. Such reaction may support, accelerate or catalyze the production of the microarray.
The immobilizing of the biomaterial on the surface of the substrate includes: exposing the substrate to light via a top portion of the chamber of the adhered structure to expose an active group screened by a photoremovable protecting group on the surface of the substrate; and contacting the exposed active group with an activated monomer protected by the photoremovable protecting group to couple the active group on the surface of the substrate with the monomer.
Exposing to light includes irradiation of gamma-rays, X-rays, infrared rays, visible rays or ultraviolet rays to the surface of the substrate of the microarray and any irradiation needed in a process according to an embodiment of the invention of preparing the microarray, such as measurement of stabilization and optical analysis of patterned probe, or a UV cross link. The type of light may be appropriately selected according to the type of the photoremovable protecting group.
The top portion of the chamber is a part of the array mold comprising the optically transparent material, and thus the light exposure can be performed where the chamber is filled with the biomaterial without separating the adhered structure into the array mold and the substrate. When a general photolithographic method is used, the light exposure should be performed on the surface of the substrate through a patterned photomask to patternize a spot on the surface of the substrate. However, in preparing the microarray according to an embodiment of the invention, a pattern is formed on the surface of the substrate by the array mold including the concave portion, and thus a separate photomask is not required.
The protecting group is a group, such as an activator, that is chemically bound to a monomer unit and can be removed by a selective exposure to an electromagnetic wave. The photoremovable protecting group is a protecting group that can be removed by light. Photoremovable protecting groups are well known to those of ordinary skill in the art. Examples of the photoremovable protecting group may include, but are not limited to, 6-nitroveratryloxycarbonyl (NVOC), 6-nitropiperonyl (NP), 6-nitropiperonyl oxycarbonyl (NPOC), 6-nitroveratryl (NV), methyl-6-nitroveratryl (MeNV), methyl-6-nitroveratryloxycarbonyl (MeNVOC), methyl-6-nitropiperonyl (MeNP), and methyl-6-nitropiperonyloxycarbonyl (MeNPOC).
The monomer may be one selected from the group comprising a nucleotide, a nucleoside, and an amino acid. Thus, the nucleotide, the nucleoside, or the amino acid that fills the chamber through the fluid inlet may be prepared as a monomer that comprises the probe of the microarray.
The array mold may include at least two concave portions, each of which includes a fluidic channel having a fluid inlet and a fluid outlet. The concave portions may be configured where a fluid outlet of a first concave portion is connected to a fluid inlet of a second concave portion to form a fluidic channel. Thus, an array mold including a plurality of concave portions can be prepared, thereby preparing a microarray with a variety of probe patterns.
The concave portions may be connected to a single fluid inlet. Thus, the array mold may include at least one fluid inlet according to a probe pattern of a microarray. That is, when the same materials fill the chambers, an array mold with concave portions including a single fluid inlet is configured, and when the materials that fill the chambers are different from each other, an array mold with concave portions including at least two fluid inlets is configured.
The array mold may comprise any material that facilitates the preparation of an internal structure such as the concave portion including a fluidic channel having a fluid inlet and a fluid outlet, is optically transparent, and can strongly adhere to a surface of a microarray substrate. For example, the array mold may comprise PDMS.
The substrate may be one selected from the group comprising silicon, glass, metal, plastic, and ceramic. In particular, the substrate may be one selected from the group comprising silicon, glass, gold, silver, copper, platinum, polystyrene, polymethylacrylate, polycarbonate, and ceramic.
The substrate may be treated with SiO2 as an oxide film-forming material before adhering the substrate and the array mold, when the array mold comprises, for example, silicon. The treatment of the substrate with SiO2 can increase an adhesion between the substrate and the array mold or the biomaterial that fills the chamber through the fluid inlet. The treatment of the substrate with SiO2 may be performed before or after adhering the array mold and the substrate.
The substrate may be treated with a material that can bind the biomaterial on the surface of the substrate. A material for binding the biomaterial may be any material that can treat the surface of the substrate of the microarray to immobilize a probe thereon. For example, the material may be, but is not limited to, a linker, GAP, an amine group, a carboxyl group, an epoxy group, a sulfur group, an aldehyde group, activated ester, maleimide, or a carbohydrate. The treatment of the substrate with the material may be performed before or after adhering the array mold and the substrate.
A material for binding the biomaterial on the surface of the substrate may be one selected from the group comprising DNA, RNA, LNA, PNA, peptide, a virus, and a cell. Thus, DNA, RNA, LNA, PNA, peptide, a virus or a cell, which fills the chamber through the fluid inlet, may be prepared as the probe of the microarray.
The monomer may be one selected from the group comprising a nucleotide, a nucleoside, and an amino acid.
Referring to
Referring to
A process according to an embodiment of the invention of filling each chamber with the biomaterial by using the array mold is as illustrated in
A method according to an embodiment of the invention of preparing a microarray includes exposing the biomaterial that fills the chamber to light in the state in which the array mold and the substrate are adhered to each other. The biomaterial is deposited on the surface of the substrate in an isolated state in the concave portion of the array mold, and the array mold comprises an optically transparent material. Thus, the light exposure may be performed when the array mold and the substrate are adhered to each other.
To prevent contamination between biomaterials that are filled in each chamber of the array mold or the biomaterial from leaking out, the adhesion between the array mold and the microarray substrate should be strong. Thus, the array mold may comprise PDMS, but is not limited thereto. PDMS can strongly adhere to silicon, glass, plastic, or ceramic, each of which may be used as a microarray substrate material. In addition, if the microarray substrate comprises silicon, the microarray substrate may be surface-treated with SiO2 to form an oxide film layer to strengthen the adhesion between the array mold and the microarray substrate.
It should be understood that the exemplary embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2008-0084044 | Aug 2008 | KR | national |
