Process for producing array plate for biomolecules having hydrophilic and hydrophobic regions

1. Field of the Invention

The present invention relates to a method for manufacturing an array plate of hydrophilic and hydrophobic regions based on semiconductor manufacturing processes and a method for manufacturing a biochip by immobilizing biomolecules on the array plate.

2. Description of the Related Art

According to a conventional method for manufacturing a nucleic acid array plate including hydrophilic and hydrophobic regions, microdroplets of reactant solution spread by means of a piezoelectric pump can be migrated onto separate binding sites on the array plate due to a difference in surface tension between the hydrophilic and hydrophobic regions.

In particular, as illustrated in FIG. 1 , the conventional method for manufacturing the nucleic acid array plate involves: (a) coating a support surface with a positive or negative photoresist which is subsequently exposed to light and developed to create a patterned region of a first exposed region; (b) reacting the first exposed region with a fluoroalkylsilane to form a stable fluoroalkylsiloxane hydrophobic matrix on the first exposed region; (c) removing the remaining photoresist to form a second exposed region; and (d) reacting the second exposed region with a hydroxy or aminoalkylsilane to form functionalized hydrophilic binding site regions.

Alternatively, as illustrated in FIG. 2 , the nucleic acid array plate may be manufactured by: (a) reacting a support surface with a hydroxy or aminoalkylsilane to form a functionalized hydrophilic support surface; (b) reacting the support surface from step (a) with o-nitrobenzyl carbonyl chloride acting as a temporary photolabile blocking agent to provide a photoblocked support surface; (c) exposing the photoblocked support surface of step (b) to light through a mask to create unblocked areas on the support surface with unblocked hydroxy or aminoalkylsilane; (d) reacting the exposed surface of step (c) with perfluoroalkanoyl halide or perfluoroalkylsulfonyl halide to form a stable hydrophobic (perfluoroacyl or perfluoroalkylsulfonamido)allyl siloxane matrix; and (e) exposing the remaining photoblocked support surface to create patterned regions of the unblocked hydroxy or aminoalkylsilane as functionalized hydrophilic binding site regions.

In practice, however, it is difficult to control the thickness of the molecular layer, which is measured in tens of angstroms, using these methods for manufacturing nucleic acid arrays. Moreover, the strength of the thin molecular layer made using these methods is weak. Additionally, since the molecular layer is thin and transparent, it is difficult to distinguish the hydrophilic and hydrophobic regions of the molecular layer from one another for sample applying through a nozzle. It is also difficult to automate pattern detection by a detector system. In addition, the molecular layer is formed of materials which are unusual in the manufacture of semiconductor devices, and it is difficult to establish an alignment key for the exposure of such a molecular layer according to general semiconductor manufacturing processes.

According to a feature of the present invention, there is provided a method for manufacturing an array plate for biomolecules that includes distinct hydrophilic and hydrophobic regions, based on conventional semiconductor manufacturing processes and in which the thickness of the thin material layers is easily controlled.

According to another feature of the present invention, there is provided a method for manufacturing a biochip using an array plate for biomolecules formed using the above method, with high accuracy and high yield.

According to an aspect of an embodiment of the present invention, there is provided a method for manufacturing an array plate for biomolecules, the method comprising: (a) coating a surface of a substrate with a hydrophobic material to form a hydrophobic layer; (b) etching the hydrophobic layer through an etch mask placed thereon to form a hydrophilic binding site; (c) removing the remaining etch mask; and (d) processing the remaining region of the hydrophobic layer to recover its original hydrophobic properties.

In another aspect, the invention provides a method for manufacturing a biochip, comprising: processing the surface of the hydrophilic binding site of an array plate manufactured using the above method, and applying a solution containing biomolecules to the surface of the hydrophilic binding site.

The above and other features, aspects and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIGS. 1 and 2 illustrate examples of conventional methods for manufacturing nucleotide array plates;

FIG. 3 illustrates the structure of an array plate for biomolecules and a method for manufacturing the same according to an embodiment of the present invention; and

FIG. 4 illustrates the progressive migration of a droplet of biomolecular solution spotted on a hydrophobic region of an array plate to a hydrophilic region due to surface tension.

Korean Patent Application No. 2002-26730, filed on May 15, 2002, and entitled “Process for producing array plate for biomolecules having hydrophilic and hydrophobic regions,” is incorporated herein in its entirety.

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which a preferred embodiment of the invention is shown. Embodiments of methods for manufacturing an array plate for biomolecules and a biochip using the array plate according to the present invention are provided below. The invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. It will also be understood that when a layer is referred to as being “on” another layer or substrate, it can be directly on the other layer or substrate, or intervening layers may also be present.

Referring to FIG. 3 , a method for manufacturing an array plate for biomolecules according to an embodiment of the present invention involves coating a surface of a substrate 1 with a hydrophobic material to form a hydrophobic layer 3 (step (a)). The hydrophobic layer 3 is then etched through an etch mask 4 ′ placed thereon to form a hydrophilic binding site 5 (step (b)). After the etch mask 4 ′ is removed (step (c)), the remaining region of the hydrophobic layer, i.e., hydrophobic region 6 , is processed to recover its original hydrophobic properties (step (d)). Details of each of these steps will be described below.

In step (a), suitable materials for the substrate include, but are not limited to, glass, quartz, silicon, ceramic, plastic, metal, etc. The hydrophobic material may be any material capable of being formed as a film and having hydrophobic properties. Examples of such hydrophobic materials include, for example, fluorocarbon polymers such as polytetrafluoroethylene, fluorinated polyimide, fluorinated polyacrylate, fluorinated polyether, and the like; polyhydrocarbons such as polystyrene, polyethylene and derivatives thereof, polynorbornene and derivatives thereof, and the like; polyesters such as polyethylene terephthalate (PET), and the like; diamond-like carbon; and the like.

In step (a), the hydrophobic layer ( 3 ) may be formed using a coating of hydrophobic material through known processes in the related fields, for example, spin coating, plasma enhanced chemical vapor deposition (PECVD), a method of forming a self-assembled monolayer, evaporation, dipping, and the like. The resulting hydrophobic layer 3 may have, but is not limited to, a thickness of from tens of angstroms to a few micrometers. After completion of the formation of the hydrophobic layer 3 , the substrate 1 having the layer of hydrophobic layer 3 is subject to soft baking and hard baking at a temperature between about 100-200° C. for about 1-30 minutes to remove a solvent and for outgassing from the hydrophobic layer. The hydrophobic layer is then subject to activation using argon (Ar) or oxygen (O 2 ) gas in a plasma state for subsequent spin coating with a photoresist or another metallic mask material.

A method for manufacturing an array plate for biomolecules according to the present invention may further include, before step (a) of coating the hydrophobic layer, forming a hydrophilic layer 2 on the surface of the substrate 1 , as shown in FIG. 3 . The hydrophilic layer 2 may be a native oxide layer. Such a hydrophilic layer, including native oxide layers, is formed of; for example, SiO 2 , Al 2 O 3 , SiON, aluminum nitride, polymethylmethacrylate (PMMA), polymethylacrylate (PMA), polyvinyl alcohol (PVA), polyhydroxystyrene, polyacetonitrile, polyacrylic acid, or polytetrahydrofuran. The hydrophilic layer 2 may be formed using, for example, spin coating, thermal wet oxidation, thermal dry oxidation, plasma enhanced chemical vapor deposition (PECVD), or other suitable methods.

According to the present invention, in step (b), the hydrophobic layer 3 formed in step (a) is coated with a mask material and subject to exposure, development, and patterning to form an etch mask 4 ′. The etch mask 4 ′ is then used to expose a region of the hydrophobic layer 3 that will become a hydrophilic region. It is this hydrophilic region that acts as a binding site 5 in a resulting array plate. The etch mask may be formed of, for example, photosensitive polymers containing negative or positive photoresist, metal hard mask materials, SiO 2 , polysilicon, or silicon nitride. Such a hydrophilic region may be formed in a recessed or an elevated configuration with respect to the hydrophobic region depending on the nature and characteristics of a desired binding site.

In step (b), the region of the hydrophobic layer exposed through the etch mask 4 ′ is etched to form the hydrophilic binding site 5 . Etching may be performed using dry etching or wet etching. For wet etching, the substrate 1 with the hydrophobic layer 3 may be immersed in an etchant to dissolve and wash away the exposed region of the hydrophobic layer 3 and expose the hydrophilic binding site 5 . Suitable examples of such an etchant include, but are not limited to, H 2 SO 4 , H 3 PO 4 , H 2 O 2 , HF, HCl, and NH 4 OH. Dry etching may be performed using plasma gas through reactive ion etching (RIE), ashing, and the like until the hydrophilic binding site 5 is exposed. Suitable examples of such etching gas include, but are not limited to, O 2 , Ar, SF 6 , CF 4 , BCl 3 , Cl 2 , and SiCl 2 , and O 2 or Ar is preferred.

In step (c), the etch mask 4 ′ remaining after step (b) of etching is removed to expose a hydrophobic region 6 . During the dry etching such as RIE or the wet etching using chemicals, the photoresist etch mask 4 ′ may not be completely removed. For example, when the photoresist etch mask 4 ′ is too thick or has a low etch rate, some amount of the etch mask may remain after etching is conducted to expose the hydrophobic layer 3 . Accordingly, any remaining photoresist etch mask 4 ′ may be removed using a photoresist stripper, such as H 2 SO 4 , H 2 O 2 , acetone, and the like. Alternatively, any remaining photoresist etch mask 4 ′ may be removed by extending the duration or period of etching to the hydrophobic layer 3 . When a metallic hard mask is used, an etching technique appropriate for removing the hard mask may be applied.

In step (d) of the present invention, the initial hydrophobic properties of the hydrophobic layer 3 may be recovered. In general, hydrophobic layers formed of, for example, fluorocarbon polymers, polyhydrocarbons, etc., lose their hydrophobic properties at least to some extent while undergoing Ar activation, etching, and etch mask removal processes. For example, the water contact angle of such a hydrophobic layer may be reduced to about 50 degrees. In step (d), the initial hydrophobic properties of the hydrophobic layer 3 that have been lost to some extent are recovered, for example, through thermal treatment. This thermal treatment may be performed at a temperature equal to or above the temperature of baking performed after the coating of the hydrophobic layer 3 , for example, between about 100-200° C. for about 1-30 minutes. As a result, the hydrophobic properties of the hydrophobic layer 3 are recovered, for example, with a water contact angle of 105 degrees. It is preferable that the surface of the final hydrophobic region 6 has a water contact angle of 105 degrees or more and a surface energy of about 20 dynes/cm or less. In addition, it is preferable that the surface of the final hydrophilic region, i.e. hydrophilic binding site 5 , have a water contact angle of less than about 105 degrees.

A method for manufacturing a biochip according to the present invention comprises: manufacturing an array plate using the above-described method according to the present invention; processing the surface of a hydrophilic binding site of the array plate; and applying a solution containing biological molecules to the surface of the hydrophilic binding site.

The surface of the hydrophilic binding site is processed using a compound capable of increasing the affinity of biomolecules to the hydrophilic binding site. For example, the surface of the hydrophilic binding site may be processed using aminopropyltriethoxysilane to attach an amine group on the surface of the hydrophilic binding site.

Any kind of biomolecules, including nucleic acids, proteins, a substrate of an enzyme, cofactor, ligands, and polysaccharides, etc., may be used for a solution to be applied to the hydrophilic binding site. Such biomolecules may be used alone or in conjugation with another compound and may be used after being dissolved in an appropriate solvent.

A gel matrix-DNA conjugate solution is preferred as a solution for the hydrophilic binding site. The gel matrix-DNA conjugate can be prepared by, for example, covalently bonding oligonucleotides to a gel matrix obtained through the coupling reaction between a polyethylene glycol (PEG) derivative having epoxy groups and a cross-linking agent such as polyethylene glycol diamine having a molecular weight of approximately 10,000 or less. Preferably, the gel matrix used herein is a three dimensional (3D) gel matrix prepared by cross-linking a PEG derivative having polyethylene glycol branches in 2 or more directions with a cross-linking agent.

The PEG derivative includes a PEG derivative which may be formed by reacting a pentaerythritol ethoxylate with epichlorohydrin, as shown in Example 3. A solution containing biomolecules may be applied to the hydrophilic binding site in microdroplets through jetting or spotting using, for example, a piezoelectric pump. Alternatively, a solution containing biomolecules may be applied to the hydrophilic binding site of an array plate by dipping the array plate into the solution. The biomolecules in the solution applied to the hydrophilic binding site may be covalently or non-covalently bound to the hydrophilic binding site of the array plate. Referring to FIG. 4 , in a case where the biomolecules 7 are hydrophilic, even though the biomolecules 7 may be spotted on a hydrophobic region 6 , the hydrophilic biomolecules migrate to a hydrophilic binding site 5 due to surface tension.

In the manufacture of a biochip according to the present invention, a solution containing biomolecules may be applied to a substrate with a plurality of arrays or to individual arrays after dicing. In dicing, the top surface of a substrate with a plurality of arrays may be covered with a polymer film or a UV tape to protect the hydrophilic binding site from dust contamination during dicing and then uncovered.

A method for manufacturing a biochip according to the present invention may further include, before the application of a solution containing biomolecules to a hydrophilic binding site, coating the surface of the array plate opposite to the hydrophilic and hydrophobic regions with a magnetic material in order to accurately apply the solution containing biomolecules to a desired hydrophilic binding site. Alternatively, this step of coating a magnetic material may be performed after step (a) of forming the hydrophobic layer 3 on the substrate 1 in the manufacture of an array plate according to the present invention. Such a magnetic material may be coated on a substrate with a plurality of arrays or on individual arrays after dicing, before the application of a biomolecule-containing solution as described above. Providing a coating of such magnetic material provides an alignment key for biomolecules to be accurately immobilized on desired binding sites.

Although the binding site of biomolecules in an array plate according to the present invention is described above as being hydrophilic, in an alternative arrangement, an array plate having a hydrophobic binding site and a hydrophilic matrix may be manufactured using the above-described processes in accordance with the present invention. Examples of hydrophobic biomolecules include cell membrane proteins, lipoproteins, etc.

Preferred embodiments of the present invention will now be further described in greater detail by way of the following examples. These examples are provided for illustrative purposes and are not intended to limit the scope of the invention.