U.S. patent application number 11/560966 was filed with the patent office on 2007-08-23 for microarray substrate, method of use, and products comprising the microarray substrate.
This patent application is currently assigned to SAMSUNG ELECTRONICS CO., LTD.. Invention is credited to Jong-myeon PARK, Sang-hyun PEAK, Chang-eun YOO.
Application Number | 20070196836 11/560966 |
Document ID | / |
Family ID | 38428676 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070196836 |
Kind Code |
A1 |
PEAK; Sang-hyun ; et
al. |
August 23, 2007 |
MICROARRAY SUBSTRATE, METHOD OF USE, AND PRODUCTS COMPRISING THE
MICROARRAY SUBSTRATE
Abstract
Provided are a microarray substrate comprising a solid substrate
coated with a chemical having a functional residue represented by
Formula 1 or 2 below, a method of analyzing a biomolecule using the
microarray substrate, and a lab-on-a-chip comprising the microarray
substrate: ##STR1## wherein n, the structure within brackets [ ],
R.sub.1, R.sub.2, R.sub.3, R.sub.10, n and I are as defined in the
specification.
Inventors: |
PEAK; Sang-hyun; (Seoul,
KR) ; PARK; Jong-myeon; (Seoul, KR) ; YOO;
Chang-eun; (Seoul, KR) |
Correspondence
Address: |
CANTOR COLBURN, LLP
55 GRIFFIN ROAD SOUTH
BLOOMFIELD
CT
06002
US
|
Assignee: |
SAMSUNG ELECTRONICS CO.,
LTD.
416, Maetan-dong, Yeongtong-gu
Suwon-si
KR
|
Family ID: |
38428676 |
Appl. No.: |
11/560966 |
Filed: |
November 17, 2006 |
Current U.S.
Class: |
435/6.11 ;
427/2.11; 435/287.2 |
Current CPC
Class: |
B01J 2219/00576
20130101; B01J 2219/00659 20130101; B01J 2219/00722 20130101; B01J
2219/00637 20130101; B01J 2219/00585 20130101; B01J 2219/00626
20130101; B01J 2219/00677 20130101; B01J 2219/00527 20130101; B01J
19/0046 20130101; B01J 2219/00605 20130101; B01J 2219/00612
20130101; B01J 2219/005 20130101; B01J 2219/0061 20130101; B01J
2219/00497 20130101; B01J 2219/00596 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 427/002.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34; B05D 3/00 20060101
B05D003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 3, 2006 |
KR |
10-2006-0000468 |
Claims
1. A microarray substrate comprising a solid substrate coated with
a composition comprising a functional residue represented by
Formula 1: ##STR10## wherein, n is an integer of 1 to 10; the
structure within brackets [ ] represents a moiety formed by
reaction of a tetracarboxylic acid anhydride with an amine to
produce --NH(CH.sub.2).sub.nNH.sub.2 and with an amino group to
produce --NH(CH.sub.2).sub.nNH-- at opposite sides of the
tetracarboxylic acid anhydride; wherein the tetracarboxylic acid
anhydride is selected from the group consisting of pyromellitic
dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride,
2,2',3,3'-biphenyltetracarboxylic dianhydride,
2,3,3',4'-biphenyltetracarboxylic dianhydride,
1,2,4,5-benzenetetracarboxylic dianhydride,
3,3',4,4'-benzophenonetetracarboxylic dianhydride,
2,2',3,3'-benzophenonetetracarboxylic dianhydride,
2,3,3',4'-benzophenonetetracarboxylic dianhydride,
bis(3,4-dicarboxyphenyl)ether dianhydride,
bis(3,4-dicarboxyphenyl)sulfone dianhydride,
1,4,5,8-naphthalenetetracarboxylic dianhydride,
1,2,5,6-naphthalenetetracarboxylic dianhydride,
2,3,6,7-naphthalenetetracarboxylic dianhydride,
2,2-bis(3,4-dicarboxyphenyl)-hexafluoropropane dianhydride,
cyclobutanetetracarboxylic dianhydride,
methylcyclobutanetetracarboxylic dianhydride, and
1,2,3,4-tetracarboxybutane dianhydride; and R.sub.10 is determined
by the selected tetracarboxylic acid anhydride.
2. The microarray substrate of claim 1, wherein the functional
residue is represented by Formula 3: ##STR11## wherein, n is an
integer of 1 to 10.
3. The microarray substrate of claim 2, wherein n is 2.
4. A microarray substrate comprising a solid substrate coated with
a composition comprising a functional residue represented by
Formula 2: ##STR12## wherein, R.sub.1 is --OH or
--NH(CH.sub.2).sub.nNH.sub.2; R.sub.2 is --OH or
--NH(CH.sub.2).sub.nNH.sub.2; n is an integer of 1 to 10; R.sub.3
is C.sub.110 alkyl; and I is an integer of 1 to 30,000.
5. The microarray substrate of claim 4, wherein the functional
residue is represented by Formula 4: ##STR13## wherein m is an
integer of 1 to 10.
6. The microarray substrate of claim 5, wherein m is 2.
7. The microarray substrate of claim 5, wherein a ratio of --OH
group to --NH(CH.sub.2).sub.nNH.sub.2 group is about 2 to 1.
8. The microarray substrate of claim 1, wherein the solid substrate
is made of a material selected from the group consisting of
silicone, glass, and a plastic material.
9. The microarray substrate of claim 4, wherein the solid substrate
is made of a material selected from the group consisting of
silicone, glass, and a plastic material.
10. A method for analyzing a target biomolecule, comprising:
obtaining a target biomolecule; and hybridizing a probe
biomolecule, immobilized on the microarray substrate of claim 1 at
a first pH, and the target biomolecule at a second pH.
11. The method of claim 10, wherein the functional residue is
represented by Formula 3 below: ##STR14## wherein n is an integer
of 1 to 10.
12. The method of claim 11, wherein n is 2.
13. The method of claim 10, wherein the first pH is about 2 to
about 3.5, the second pH is about 5 to about 10.
14. The method of claim 10, wherein the solid substrate is made of
a material selected from a group consisting of silicone, glass, and
a plastic material.
15. The method of claim 10, wherein the biomolecule is a nucleic
acid.
16. A method of analyzing a target biomolecule, comprising:
obtaining a target biomolecule; and hybridizing a probe
biomolecule, immobilized on the microarray substrate of claim 4 at
a first pH, and the target biomolecule at the second pH.
17. The method of claim 16 wherein the functional residue is
represented by Formula 4 below: ##STR15## wherein, wherein m is an
integer of 1 to 10.
18. The method of claim 17, wherein m is 2.
19. The method of claim 17, wherein a ratio of --OH group to
--NH(CH.sub.2).sub.nNH.sub.2 group is about 2 to 1.
20. The method of claim 16, wherein the first pH is about 2 to
about 3.5, and the second pH is about 5 to about 10.
21. The method of claim 16, wherein the solid substrate is made of
a material selected from the group consisting of silicone, glass,
and a plastic material.
22. The method of claim 16, wherein the biomolecule is a nucleic
acid.
23. A lab-on-a-chip comprising the microarray substrate of claim
1.
24. A lab-on-a-chip comprising the microarray substrate of claim
4.
25. A method of manufacturing a microarray substrate, comprising
immobilizing a tetracarboxylic acid anhydride on a solid substrate
coated with an amino group: and reacting the tetracarboxylic acid
anhydride with an amine compound represented by
NH.sub.2(CH.sub.2).sub.nNH.sub.2, wherein n is an integer of 1 to
10.
26. A method of manufacturing a microarray substrate, comprising
immobilizing a compound represented by Formula 5 on an amino
group-coated substrate; and reacting the compound with an amine
compound represented by NH.sub.2(CH.sub.2).sub.nNH.sub.2, wherein n
is an integer of 1 to 10; ##STR16## wherein, R.sub.3 is C.sub.1-10
alkyl; and I is an integer of 1 to 30,000.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATION
[0001] This application claims priority to Korean Patent
Application No. 10-2006-000468, filed on Jan. 3, 2006, in the
Korean Intellectual Property Office, and all the benefits therefrom
under 35 U.S.C. .sctn.119, the disclosure of which is incorporated
herein in its entirety by reference.
BACKGROUND
[0002] 1. Field of the Invention
[0003] The present invention relates to a microarray substrate, a
method of use, and products, e.g., a microarray and Lab-On-a-Chip
(LOC) system, comprising the microarray substrate.
[0004] 2. Description of the Related Art
[0005] A microarray refers to a high-density array of molecules
immobilized in predetermined regions of a substrate. One way in
which a microarray can be classified is based on the molecule
immobilized on the substrate, e.g, a polynucleotide microarray, a
protein microarray, etc. The term "polynucleotide microarray"
refers to a high-density array of polynucleotides immobilized in
predetermined regions of a substrate. Such microarrays are well
known in the art. Examples of such microarrays are disclosed in
U.S. Pat. No. 5,445,934 and U.S. Pat. No. 5,744,305.
[0006] Immobilization of polynucleotides on a solid substrate can
be achieved by direct synthesis of polynucleotides on the solid
substrate or by deposition of polynucleotides on predetermined
regions of the solid substrate (spotting method). Illustrative
methods for manufacturing such polynucleotide microarrays are
disclosed in U.S. Pat. No. 5,744,305, U.S. Pat. No. 5,143,854, and
U.S. Pat. No. 5,424,186.
[0007] The spotting method has been widely used for covalent
attachment of biomolecules on solid substrates. For example, one
widely used method of immobilizing biomolecules on a solid
substrate includes: activating a surface of the solid substrate by
introducing a nucleophilic functional group (e.g., an amino group)
on the solid substrate, coupling biomolecules (e.g.,
polynucleotides) activated with a good leaving group to the
surface-activated solid substrate, and removing any unreacted
reactants. Drawbacks of this method include low attachment
efficiency of polynucleotides to the coated surface and
non-specific binding of target molecules to the coated surface or
binding of impurities in the target sample to the coated
surface.
[0008] Meanwhile, pH-dependent ion-exchange materials have been
used in the separation of nucleic acids. For example, U.S. Patent
Publication No. 2001/0018513 discloses a method of isolating a
nucleic acid using a material having an ionizable group. The
material is positively charged at a first pH to permit nucleic acid
binding to the material; the bound nucleic acid is released from
the material at a second pH higher than the first pH. Examples of
such a material having an ionizable group include
N-2-acetamido-2-aminoethanesulfonic acid (ACES),
N-2-acetamido-2-imidodiacetic acid (ADA),
N-trihydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), and
trihydroxymethylaminoethane. Additionally, U.S. Pat. No. 6,310,199
discloses a method of isolating a nucleic acid using a pH-dependent
ion-exchange material including a silica magnetic particle and a
plurality of ion-exchange ligands. Each ion exchange ligand
comprises an aromatic hydrocarbon ring; a spacer covalently
attached to the aromatic hydrocarbon ring; and a linker including a
linker alkyl chain attached to the silica magnetic particle at a
first end of the linker alkyl chain and attached to the spacer at a
second end of the linker alkyl chain.
[0009] The present inventors have studied coating materials
applicable to the solid substrate of a microarray substrate and
have discovered that coating a microarray substrate with a
pH-dependent ion-exchange material helps to solve the
aforementioned problems of the prior art.
SUMMARY OF THE INVENTION
[0010] In one aspect, the invention provides a microarray substrate
that enhances the immobilization efficiency of a probe biomolecule
and reduces non-specific binding of impurities, such as proteins,
and of target biomolecules, and furthermore, can simultaneously
perform cell lysis, PCR, and probe-target hybridization.
[0011] In an embodiment, the microarray substrate comprises a solid
substrate coated with a composition comprising a functional residue
represented by Formula 1 or 2 below: ##STR2##
[0012] wherein,
[0013] n is an integer of 1 to 10;
[0014] the structure within brackets [ ] represents a moiety formed
by reaction of a tetracarboxylic acid anhydride with an amine to
produce --NH(CH.sub.2).sub.nNH.sub.2 and with an amino group to
produce --NH(CH.sub.2).sub.nNH-- at opposite sides of the
tetracarboxylic acid anhydride, wherein the tetracarboxylic acid
anhydride is selected from the group consisting of pyromellitic
dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride,
2,2',3,3'-biphenyltetracarboxylic dianhydride,
2,3,3',4'-biphenyltetracarboxylic dianhydride,
1,2,4,5-benzenetetracarboxylic dianhydride,
3,3',4,4'-benzophenonetetracarboxylic dianhydride,
2,2',3,3'-benzophenonetetracarboxylic dianhydride,
2,3,3',4'-benzophenonetetracarboxylic dianhydride,
bis(3,4-dicarboxyphenyl)ether dianhydride,
bis(3,4-dicarboxyphenyl)sulfone dianhydride,
1,4,5,8-naphthalenetetracarboxylic dianhydride,
1,2,5,6-naphthalenetetracarboxylic dianhydride,
2,3,6,7-naphthalenetetracarboxylic dianhydride,
2,2-bis(3,4-dicarboxyphenyl)-hexafluoropropane dianhydride,
cyclobutanetetracarboxylic dianhydride,
methylcyclobutanetetracarboxylic dianhydride, and
1,2,3,4-tetracarboxybutane dianhydride; and R.sub.10 is determined
by the selected tetracarboxylic acid anhydride, and ##STR3##
[0015] wherein,
[0016] R.sub.1 is --OH or --NH(CH.sub.2).sub.nNH.sub.2;
[0017] R.sub.2 is --OH or --NH(CH.sub.2).sub.nNH.sub.2;
[0018] R.sub.3 is C.sub.1-10 alkyl; and
[0019] I is an integer of 1 to 30,000.
[0020] Also disclosed herein is a method of analyzing a
biomolecule, comprising: obtaining a target biomolecule; and
hybridizing a probe biomolecule, immobilized on the microarray
substrate coated with a composition comprising a functional residue
represented by Formula 1 or 2 at a first pH; and the target
biomolecule at the second pH:
[0021] Also disclosed is a microarray comprising the microarray
substrate and a Lab-on-a-Chip (LOC) comprising the microarray
substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a schematic drawing of DNA binding to or releasing
from a microarray substrate according to the present invention with
respect to pH change;
[0023] FIG. 2 is a schematic drawing of a binding interaction
between a probe DNA and a target DNA on a substrate before and
after PCR on a chip;
[0024] FIGS. 3 and 4 are fluorescence intensity data obtained after
target DNAs hybridized with probe DNAs immobilized on a control
microarray and test microarrays (CRS 1 and 2) according to the
present invention;
[0025] FIG. 5A shows fluorescence images obtained after target DNAs
are hybridized with probe DNAs immobilized on a control microarray
and a test microarray (CRS 2) according to the present invention
that have been immersed in a pH 3 solution, and FIG. 5B show
fluorescence images obtained after target DNAs are hybridized with
probe DNAs immobilized on a control microarray and a test
microarray (CRS 2) according to the present invention that have not
been immersed in a pH 3 solution; and
[0026] FIG. 6 shows PCR yields after solution-phase PCR (positive
control) and after PCR using CRS beads according to the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The invention now will be described more fully hereinafter
with reference to the accompanying drawings, in which embodiments
of the invention are shown. This invention may, however, be
embodied in many 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.
[0028] In one embodiment, the invention provides a microarray
substrate comprising a solid substrate coated with a composition
comprising a functional residue represented by Formula 1:
##STR4##
[0029] wherein,
[0030] n is an integer of 1 to 10;
[0031] the structure within brackets [ ] represents a moiety formed
by reaction of a tetracarboxylic acid anhydride with an amine to
produce --NH(CH.sub.2).sub.nNH.sub.2 and with an amino group to
produce --NH(CH.sub.2).sub.nNH-- at opposite sides of the
tetracarboxylic acid anhydride; wherein the tetracarboxylic acid is
selected from the group consisting of pyromellitic dianhydride,
3,3',4,4'-biphenyltetracarboxylic dianhydride,
2,2',3,3'-biphenyltetracarboxylic dianhydride,
2,3,3',4'-biphenyltetracarboxylic dianhydride,
1,2,4,5-benzenetetracarboxylic dianhydride,
3,3',4,4'-benzophenonetetracarboxylic dianhydride,
2,2',3,3'-benzophenonetetracarboxylic dianhydride,
2,3,3',4'-benzophenonetetracarboxylic dianhydride,
bis(3,4-dicarboxyphenyl)ether dianhydride,
bis(3,4-dicarboxyphenyl)sulfone dianhydride,
1,4,5,8-naphthalenetetracarboxylic dianhydride,
1,2,5,6-naphthalenetetracarboxylic dianhydride,
2,3,6,7-naphthalenetetracarboxylic dianhydride,
2,2-bis(3,4-dicarboxyphenyl)-hexafluoropropane dianhydride,
cyclobutanetetracarboxylic dianhydride,
methylcyclobutanetetracarboxylic dianhydride, and
1,2,3,4-tetracarboxybutane dianhydride; and R.sub.10 is determined
by the selected tetracarboxylic acid anhydride.
[0032] In an embodiment, the invention provides a microarray
substrate comprising a solid substrate coated with a composition
comprising a functional residue represented by Formula 2:
##STR5##
[0033] wherein,
[0034] R.sub.1 is --OH or --NH(CH.sub.2).sub.nNH.sub.2; R.sub.2 is
--OH or --NH(CH.sub.2).sub.nNH.sub.2; n is an integer of 1 to 10;
R.sub.3 is C.sub.1-10 alkyl; and I is an integer of 1 to
30,000.
[0035] In the microarray substrate, the functional residues of
Formulae 1 and 2 are amino- and carboxyl-containing materials,
i.e., bifunctional materials that are positively charged at a first
pH and negatively charged at a second pH.
[0036] The value of the first pH can be lower than the pKa value of
the carboxyl groups of the functional residues of Formulae 1 and 2.
Specifically, the first pH can be about 2 to about 3.5 but the
present invention is not limited thereto. The value of the second
pH is not limited to a particular range provided that it is higher
than the value of the first pH. Specifically, the second pH can be
about 5 to about 10.
[0037] The solid substrate can be a plastic substrate made of
polyethylene, polypropylene, polystyrene, or polyurethane; a glass
substrate; a silicone wafer; or a modified substrate thereof, but
the present invention is not limited to the illustrated
examples.
[0038] The microarray substrate coated with a composition
comprising the functional residue of Formula 1 and the microarray
substrate coated with a composition comprising the functional
residue of Formula 2 have similar effects in terms of increasing
the immobilization efficiency of a probe biomolecule, reducing
non-specific binding of a target biomolecule, or the non-specific
binding of impurities, for example, proteins, that may be contained
in the target sample, and furthermore, can simultaneously perform
cell lysis, PCR, and probe-target hybridization.
[0039] In the microarray substrate of the present invention, the
functional residue of Formula 1 can be a functional residue
represented by Formula 3 below and the functional residue of
Formula 2 can be a functional residue represented by Formula 4
below: ##STR6##
[0040] wherein,
[0041] n is an integer of 1 to 10, and ##STR7##
[0042] wherein,
[0043] R.sub.1 can be --OH or --NH(CH.sub.2).sub.nNH.sub.2; and
R.sub.1 can be --OH or --NH(CH.sub.2).sub.nNH.sub.2; where n is an
integer of 1 to 10; m is an integer of 1 to 10; and I is an integer
of 1 to 30,0000.
[0044] Each of the functional residues of Formulae 1 and 2
comprises an amino group and a carboxyl group.
[0045] For the functional residue of Formula 1, a ratio of positive
charge to negative charge at the first pH can be about 2 to 1. A
positive charge of plus 1 (+1) is preferred. More specifically, the
functional residue of Formula 1 can be the functional residue of
Formula 3 where n is 2.
[0046] For the functional residue of Formula 2, the ratio of the
--OH group to the --NH(CH.sub.2).sub.nNH.sub.2 group represented by
R.sub.1 and/or R.sub.2 can be about 2 to 1. More specifically, the
functional residue of Formula 2 can be the functional residue of
Formula 4 where m is 2 and I is an integer of 1 to 30,000.
[0047] The preparation of the microarray substrate can be performed
using any method known in the art. For example, the microarray
substrate coated with a composition comprising the functional
residue of Formula 1 can be prepared by immobilizing a
tetracarboxylic acid anhydride, for example
1,2,4,5-benzenetetracarboxylic anhydride, on a solid substrate
(e.g., a silicone wafer, a glass substrate, or a plastic substrate)
coated with an amino group (e.g.,
--NH.sub.2(CH.sub.2).sub.nNH.sub.2) followed by reaction with a
compound (e.g., ethylenediamine) represented by
NH.sub.2(CH.sub.2).sub.nNH.sub.2 where n is an integer of 1 to 10.
The microarray substrate coated with a composition comprising the
functional residue of Formula 2 can be prepared by immobilizing a
compound, for example the compound represented by Formula 5 below,
on an amino group-coated substrate followed by reaction with a
compound (e.g., ethylenediamine) represented by
NH.sub.2(CH.sub.2).sub.nNH.sub.2 where n is an integer of 1 to 10:
##STR8##
[0048] wherein,
[0049] R.sub.3 is C.sub.1-10 alkyl; and
[0050] I is an integer of 1 to 30,000.
[0051] The compound of Formula 5 can be a compound represented by
Formula 6 below: ##STR9##
[0052] wherein,
[0053] m is an integer of 1 to 10; and
[0054] I is an integer of 1 to 30,000.
[0055] The compound of Formula 5 is a polyanhydride polymer. The
polyanhydride polymer can be easily separated from a solution using
a separation method (e.g., centrifugation).
[0056] The present invention also provides a method of analyzing a
biomolecule using the microarray substrate. The method comprises
obtaining a target biomolecule; and hybridizing a probe
biomolecule, immobilized on the microarray substrate coated with a
composition comprising a functional residue represented by Formula
1 or 2 at a first pH; and the target biomolecule at a second
pH.
[0057] A probe biomolecule is immobilized on the microarray
substrate at a first pH to prepare a microarray. The probe
biomolecule can be, but is not limited to, a nucleic acid. When a
probe biomolecule is a nucleic acid, the first pH may be lower than
the pKa values of the carboxyl groups of the functional residues of
Formulae 1 or 2. Specifically, the first pH may be about 2 to about
3.5. However, the first pH is not limited thereto.
[0058] Common problems associated with the prior art are
non-specific binding of target biomolecules, e.g. nucleic acid, to
a microarray substrate and non-specific binding of impurities, e.g.
proteins that may be contained in a target sample. Therefore, it
has been common to treat the microarray substrate with chemicals
after immobilization of a probe nucleic acid to reduce the
aforementioned non-specific binding. For example, in order to block
non-specific binding of a target DNA with an unreacted amino group
on a substrate, the substrate may be treated with anhydrous
succinic acid after immobilization of a probe DNA on the
substrate.
[0059] For the microarray substrate according to the invention,
however, the above mentioned non-specific binding can be
significantly reduced without any post-immobilization treatment.
Further, non-specific binding of impurities such as proteins is
also reduced using the disclosed microarray substrate.
[0060] Target molecules can be obtained according to various
methods known to those skilled in the art. When the target molecule
is a nucleic acid, preparation methods include, but are not limited
to, organic extraction and precipitation, PCR amplification,
ion-exchange chromatography, reversed phase chromatography,
affinity chromatography and a combination of the above methods.
[0061] After immobilizing the probe biomolecule, a target
biomolecule can be attached to a probe-free region of the
microarray substrate at the first pH. Since the functional residue
of Formula 1 or 2 immobilized on the microarray substrate can bind
with the target biomolecule, like the probe biomolecule, at the
first pH, the target biomolecule can be attached to the probe-free
region of the microarray substrate, permitting processes such as
cell lysis and purification of a target biomolecule to be performed
on the microarray substrate.
[0062] For example, if using a nucleic acid from a cell as the
target biomolecule, a sample containing such cells is applied to a
probe-free region of the microarray substrate. Then, the cells are
lysed at the first pH, and the target biomolecule released from the
lysed cells is attached to a probe-free region of a microarray
substrate via the functional residue of Formula 1 or 2. After
attaching the target biomolecule to the microarray substrate, the
microarray substrate may be washed to remove components of the cell
lysate other than the bound target biomolecule.
[0063] The purified target biomolecule is then eluted from the
microarray substrate at a second pH and amplified. When the target
molecule is a nucleic acid, a crude PCR product can be directly
used as a target nucleic acid sample with no purification after PCR
amplification since non-specific binding of proteins on the
microarray substrate is significantly reduced at a second pH.
Therefore, the use of the microarray substrate of the present
invention enables simultaneous execution of cell lysis, PCR, and
probe-target hybridization.
[0064] After amplifying the target biomolecule, the probe
biomolecule immobilized on the microarray is hybridized with the
target biomolecules at a second pH.
[0065] The value of the second pH is not limited to a particular
range provided that it is higher than the value of the first pH.
Specifically, the second pH may be about 5 to about 10. A
probe-target hybrid can be washed with a washing buffer (e.g.
SSPET, SSC buffer).
[0066] After performing hybridization between the probe biomolecule
and the target biomolecule, the target biomolecule is analyzed, for
example by using fluorescence intensity, etc.
[0067] In another aspect, the invention provides a microarray
comprising the microarray substrate with a probe biomolecule
immobilized thereon and a Lab-On-a-Chip (LOC) comprising the
microarray substrate.
[0068] Hereinafter, the present invention will be described more
specifically with reference to the following working examples,
which are not intended to limit the scope of the invention.
EXAMPLES
Example 1
Preparation of Microarray Substrates According to the Present
Invention
[0069] (1) Manufacturing of Microarray Substrates According to the
Present Invention
[0070] Microarray substrates coated with a composition comprising a
functional residue of Formula 3 where n was 2 were
manufactured.
[0071] Gamma-aminopropyltriethoxysilane (GAPS)-coated silicone
wafers (LG Siltron, Korea) were immersed in a solution of 100 mM
1,2,4,5-benzenetetracarboxylic anhydride in N-methyl-2-pyrrolidone
(NMP) for one hour, washed with acetone, and dried in vacuum. The
resultant 1,2,4,5-benzenetetracarboxylic anhydride-covalently bound
silicone wafers were immersed in a solution of 100 mM
ethylenediamine in NMP for one hour, washed with ethanol, and
dried. The resulting microarray substrates according to the present
invention are hereinafter, designated as "test substrates".
[0072] (2) Preparation of Control Substrates
[0073] In the following working examples, GAPS-coated silicone
wafers were used as control substrates.
Example 2
Manufacturing of Polynucleotide Microarrays
[0074] (1) Manufacturing of Polynucleotide Microarrays (Including
Post-Treatment after Probe Immobilization)
[0075] Polynucleotide microarrays in which 5'-end functionalized
DNAs were arranged in two or more groups of spots on the test
substrates prepared in (1) of Example 1 were manufactured as
follows.
[0076] A spotting solution was prepared. The composition of the
spotting solution was as follows: 50% formamide, 25% a solution of
polyethyleneglycol (PEG) (MW: 10,000) in phosphate buffer (100 mM,
pH 3), and 25% a solution containing a probe DNA (SEQ ID NO: 1)
having an amino (NH.sub.2) group at the 5'-end. The final
concentration of DNA in the spotting solution was 20 .mu.M.
[0077] The spotting solution thus prepared was spotted on the test
substrates using a Pix5500 spotter (Cartesian) (500 pl per spot).
The test substrates having the spots were incubated in a constant
temperature and humidity chamber (70.degree. C., 40% humidity) for
one hour so that the probe DNAs were immobilized on the test
substrates. After the reaction was terminated, the test substrates
were washed with distilled water, treated with anhydrous succinic
acid (blocking agent) to block unreacted amino groups, washed with
ethanol, and spin-dried to obtain polynucleotide microarrays with
immobilized probe DNAs (hereinafter, designated as "CRS 1
microarrays"). Each spot group of each CRS 1 microarray consisted
of nine spots. The spacing between the spots was 300 .mu.m.
[0078] Meanwhile, "control microarrays" were manufactured in the
same manner as described above using the GAPS-coated silicone
wafers prepared in (2) of Example 1.
[0079] (2) Manufacturing of Polynucleotide Microarrays (excluding
Post-Treatment after Probe Immobilization)
[0080] Polynucleotide microarrays according to the present
invention (hereinafter, designated as "CRS 2 microarrays") were
manufactured in the same manner as in (1) using the test substrates
of the invention prepared in (1) of Example I except that no
post-treatment after probe immobilization was performed (i.e.,
without the steps of treating the test substrates with immobilized
probe DNAs with anhydrous succinic acid and the subsequent
washes).
Example 3
Evaluation of Immobilization Efficiency of Probe
Polynucleotides
[0081] Hybridization of the probe DNAs immobilized on the
polynucleotide microarrays manufactured in Example 2 with target
DNAs was performed and the hybridization results were detected to
thereby determine the immobilization efficiency of the probe DNAs
on the polynucleotide microarrays.
[0082] Oligonucleotides (SEQ ID NO: 2) having --NH.sub.2-Cy3 at the
5'-end were used as target DNAs. A volume of 15 .mu.l of the target
DNAs (100 pM) was placed in a 1.5 ml tube, vortexed for 10 seconds,
and centrifuged.
[0083] The target DNAs were denatured in a 94.degree. C. heating
block for 5 minutes and placed on ice. In this state, 16 .mu.l of a
hybridization buffer (a diluted solution of
NaH.sub.2PO.sub.4H.sub.2O 138 g, NaCl 876 g, 0.5M EDTA 200 ml, and
10N NaOH 100ml) was added to the target DNAs to reach a total
volume of 31 .mu.l. The hybridization solution was vortexed for 10
seconds, centrifuged for 10 seconds, and applied to the CRS 1 and
CRS 2 microarrays of the invention and to the control microarrays.
The CRS 1 and CRS 2 microarrays and the control microarrays were
incubated at 42.degree. C. for one hour, washed with washing
solution I (3.times.SSPET) and washing solution II (1.times.SSPET)
for 5 minutes (for each), and dried. Fluorescence images were
acquired using a GenePix 4000B scanner (Axon Instruments) and
analyzed using GenePix Pro software (Axon Instruments, Union City,
Calif.). The fluorescence image data and the fluorescence intensity
data are presented in Table 1 below and FIG. 3. The fluorescence
intensity was observed at 532 nm (PMT560 or PMT500Z).
TABLE-US-00001 TABLE 1 Control microarray CRS 1 CRS 2 Section (PMT
560) PMT560 PMT500 PMT560 PMT500 Intensity 31345 65500 43100 65500
54290 Background 57 279 54 281 53
[0084] As shown in Table 1 and FIG. 3, the immobilization
efficiency of the probe DNAs of the microarrays of the present
invention were comparable with or without the post-treatment after
probe immobilization. Both showed superior immobilization
efficiency as compared to that of the control microarrays.
Example 4
Analysis of Non-Specific Binding of Target DNAs on Microarray
Substrates with or without Post-Treatment after Probe
Immobilization
[0085] As in Example 3, hybridization of the probe DNAs immobilized
on the polynucleotide microarrays manufactured in Example 2 with
target DNAs was performed, and background image data and
fluorescence intensities of the microarrays were detected. Images
were obtained using the same machine and data were analyzed using
the same software. The background image data and fluorescence
intensities are presented in Table 2 below and FIG. 4. The
fluorescence intensities were obtained at 532 nm (PMT560).
TABLE-US-00002 TABLE 2 Control microarray CRS1 CRS2 1 2 Average 1 2
Average 1 2 Average Intensity 230 241 235 254 274 264 266 301
283
[0086] As shown in Table 2 and FIG. 4, after probe-target
hybridization, background fluorescence intensities obtained from
the CRS 2 microarrays of the invention (excluding post-treatment
after probe immobilization) were comparable to those from the CRS 1
microarrays of the invention (including post-treatment after probe
immobilization). These results show that the use of a microarray
substrate of the present invention reduces non-specific binding of
a target DNA under probe-target hybridization conditions.
Example 5
Determination of Damage to Probes at pH 3
[0087] Damage to a probe DNA immobilized on a substrate in a pH 3
environment was investigated by measuring the degree of
hybridization between the probe DNA and a target DNA.
[0088] (1) The CRS 2 microarrays of the invention and the control
microarrays prepared in Example 2 were immersed in a pH 3 binding
buffer (100 mM phosphate buffer) for 5 minutes. Then, target DNAs
(500 pM, oligonucleotides (SEQ ID NO: 2) having --NH.sub.2-Cy3 at
the 5'-end) as described in Example 3 and a hybridization buffer
(6.times. SSPE-0.1% Triton X-100) were mixed in equal volume. The
reaction solution was applied to the CRS 2 microarrays and the
control microarrays. The CRS 2 microarrays and the control
microarrays were incubated at 42.degree. C. for one hour, washed
with washing buffer I (3.times. SSPE-Triton X-100 0.005%) for 5
minutes and then with washing buffer II (1.times. SSPE-Triton X-100
0.005%) for 5 minutes, and then dried at room temperature for 15
minutes. Fluorescence images were acquired at 532 nm (PMT 560)
using a GenePix 4000B scanner (Axon Instruments) and analyzed using
GenePix Pro software (Axon Instruments, Union City, Calif.). The
results are presented in Table 3 below (see FIG. 5A).
[0089] (2) As a control, hybridizations and post hybridization
washes were carried out under the same conditions for CRS2 and
control microarrays as in (1), except without immersing those
arrays in the pH3 solution. Fluorescence images were obtained and
analyzed using the same machine and software. The results are
presented in Table 3 and FIG. 5B. TABLE-US-00003 TABLE 3 Immersion
in No immersion in pH 3 buffer pH 3 buffer Microarray Intensity
Background Intensity Background Control microarray 31345 60 42653
57 (PMT 560) CRS 2 (PMT 560) 65500 281 65500 279 CRS 2 (PMT 500)
53107 53 54290 54
[0090] As shown in Table 3 and FIGS. 5A and 5B, the degree of
probe-target hybridization for the CRS 2 microarrays of the
invention exposed to the pH 3 environment was similar to that of
the CRS 2 microarrays of the invention unexposed to pH3
environment. These results show that a probe immobilized on a
microarray of the present invention is not damaged under an acidic
condition such as pH 3.
Example 6
Determination of PCR Efficiency
[0091] CRS beads were prepared in the same manner as described in
(1) of Example 1 except that silica beads were used instead of the
silicone wafers.
[0092] Probe DNAs consisting of a nucleotide sequence of SEQ ID NO:
3 were immobilized on the CRS beads in the same manner as described
in Example 2. No post-treatment was performed after probe
immobilization.
[0093] Hybridization between target DNAs (genomic DNAs (gDNAs)
derived from Staphylococcus aureus) and the probe DNAs immobilized
on the CRS beads was performed. Bound target DNAs were eluted from
the CRS beads by incubating the beads in a PCR buffer at 95.degree.
C. for one minute. Then, PCR was performed using the eluted
solution and the PCR efficiency (DNA yield) was measured.
[0094] As a negative control, hybridization between the target DNAs
and the probe DNAs immobilized on the CRS beads was performed, and
the target DNAs were eluted from the CRS beads by incubating the
beads in a PCR buffer at room temperature for one minute. Then, PCR
was performed using the eluted solution and the PCR efficiency was
measured.
[0095] As a positive control, a conventional solution-phase PCR was
performed and the PCR efficiency was measured.
[0096] The PCR was performed for 25 cycles using a PCR GENEAMP.RTM.
thermocycler (Applied Biosystems). The composition of the PCR
solution was as follows: 10 .mu.l 10.times. PCR buffer (Solgent), 5
.mu.l of the eluted solution (template), 0.68 .mu.M primers
(forward primers: oligonucleotides consisting of SEQ ID NO: 4,
reverse primers: oligonucleotides consisting of SEQ ID NO: 5), 0.25
mM dNTPs, and 0.5 unit Taq polymerase (total volume: 50 .mu.l).
[0097] The PCR was performed according to the following program:
denaturation of 95.degree. C. for one minute, 25 cycles of
95.degree. C. for 5 seconds, 62.degree. C. for 13 seconds, and
72.degree. C. for 15 seconds, and extension of 72.degree. C. for
one minute.
[0098] After the PCR reaction, DNA yield was measured using a Lab
chip (Agilent). The yields of the reactions were: positive control:
4.1 ng/ul, negative control: 1.4 ng/.mu.l, and the invention: 4.7
ng/.mu.l (see FIG. 6). The PCR yield of the invention was greater
than that obtained by the positive control. These results show that
cell lysis, nucleic acid purification, and PCR can be performed on
a chip comprising a microarray substrate of the present
invention.
[0099] As described above, microarray substrates of the invention
provide several advantages: enhancing the immobilization efficiency
of a probe biomolecule, and reducing non-specific binding of a
target biomolecule or impurities such as proteins. Thus, after a
probe biomolecule is immobilized on the microarray substrate, no
further post-treatment of the microarray substrate is required
Further, a crude sample from a PCR reaction can be directly applied
to the microarray substrate, thereby ensuring process simplicity
and cost-effectiveness in chip production and biomolecule
assay.
[0100] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. The terms "a" and "an" do not denote a limitation of
quantity, but rather denote the presence of at least one of the
referenced item. The term "or" means "and/or". The terms
"comprising", "having", "including", and "containing" are to be
construed as open-ended terms (i.e., meaning "including, but not
limited to").
[0101] Recitation of ranges of values are merely intended to serve
as a shorthand method of referring individually to each separate
value falling within the range, unless otherwise indicated herein,
and each separate value is incorporated into the specification as
if it were individually recited herein. The endpoints of all ranges
are included within the range and independently combinable.
[0102] All methods described herein can be performed in a suitable
order unless otherwise indicated herein or otherwise clearly
contradicted by context. The use of any and all examples, or
exemplary language (e.g., "such as"), is intended merely to better
illustrate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention as used
herein. Unless defined otherwise, technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs.
[0103] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
While the present invention has been particularly shown and
described with reference to exemplary embodiments thereof, 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.
Sequence CWU 1
1
5 1 15 DNA Artificial Sequence probe DNA 1 tgttctcttg tcttg 15 2 15
DNA Artificial Sequence target DNA 2 caagacaaga gaaca 15 3 25 DNA
Artificial Sequence probe DNA 3 aacatatgtg taagtaactg tgcac 25 4 20
DNA Artificial Sequence primer 4 yccakactcc tacgggaggc 20 5 22 DNA
Artificial Sequence primer 5 gtgccagcag yygcggtaat ac 22
* * * * *