U.S. patent application number 11/501900 was filed with the patent office on 2006-11-30 for dendrimer-based dna extraction methods and biochips.
This patent application is currently assigned to YOKOGAWA ELECTRIC CORPORATION. Invention is credited to Kazuhisa Fukushima, Tadashi Matsunaga, Saya Satou, Haruko Takeyama.
Application Number | 20060269961 11/501900 |
Document ID | / |
Family ID | 34650678 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060269961 |
Kind Code |
A1 |
Fukushima; Kazuhisa ; et
al. |
November 30, 2006 |
Dendrimer-based DNA extraction methods and biochips
Abstract
The present invention provides a dendrimer-based biochip,
wherein a flow channel through which a solution containing
biopolymer molecules is flowed is formed in the substrate of the
biochip, a plurality of dendrimer molecules one end of each of
which is bound to the walls of the flow channel are formed thereon,
and probe biopolymer or antibody molecules are bound to the tips of
the dendrimer molecules so that, if the probe biopolymer molecules
are bound, then target biopolymer molecules can be captured by
means of a complementary combination and, if the antibody molecules
are bound, then protein can be extracted by means of
antigen-antibody reaction, whereby biopolymers can be retrieved in
a highly efficient manner.
Inventors: |
Fukushima; Kazuhisa; (Tokyo,
JP) ; Satou; Saya; (Tokyo, JP) ; Matsunaga;
Tadashi; (Tokyo, JP) ; Takeyama; Haruko;
(Tokyo, JP) |
Correspondence
Address: |
WESTERMAN, HATTORI, DANIELS & ADRIAN, LLP
1250 CONNECTICUT AVENUE, NW
SUITE 700
WASHINGTON
DC
20036
US
|
Assignee: |
YOKOGAWA ELECTRIC
CORPORATION
Tokyo
JP
Tadashi MATSUNAGA
Tokyo
JP
|
Family ID: |
34650678 |
Appl. No.: |
11/501900 |
Filed: |
August 10, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10928183 |
Aug 30, 2004 |
|
|
|
11501900 |
Aug 10, 2006 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/287.2; 977/924 |
Current CPC
Class: |
G01N 33/54353 20130101;
C12Q 1/682 20130101; Y10T 436/143333 20150115; C12Q 1/682 20130101;
C12Q 2565/501 20130101; C12Q 2565/629 20130101; C12Q 2565/629
20130101; C12Q 1/6837 20130101; C12Q 1/6837 20130101; C07H 21/04
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 977/924 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 2003 |
JP |
2003-417848 |
Claims
1. A dendrimer-based biochip, wherein a flow channel through which
a solution containing biopolymer molecules is flowed, is formed in
the substrate of said biochip, a plurality of dendrimer molecules,
one end of each of which is bound to the walls of said flow
channel, are formed thereon, probe biopolymer or antibody molecules
are bound to the tips of said dendrimer molecules and, if said
probe biopolymer molecules are bound, then target biopolymer
molecules are captured by means of a complementary combination and,
if said antibody molecules are bound, then protein is extracted by
means of antigen-antibody reaction.
2. The dendrimer-based biochip of claim 1, wherein said dendrimer
is a multibranched polyamide amine dendrimer produced by providing
aminosilane treatment on the walls of said flow channel and
overlaying a film of amidoamine, which is produced by the reaction
of methyl acrylate with ethylenediamine, upon the
aminosilane-treated area as a dendron unit.
3. The dendrimer-based biochip of claim 1, wherein the generation
of said dendrimer molecules is second or higher.
4. The dendrimer-based biochip of claim 2, wherein the generation
of said dendrimer molecules is second or higher.
5. The dendrimer-based biochip of claim 1, wherein concavities and
convexities are formed on the walls of said flow channel.
6. The dendrimer-based biochip of claim 2, wherein concavities and
convexities are formed on the walls of said flow channel.
7. The dendrimer-based biochip of claim 3, wherein concavities and
convexities are formed on the walls of said flow channel.
Description
[0001] This application is a divisional of application Ser. No.
10/928,183, filed Aug. 30, 2004.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method and a biochip for
efficiently retrieving (or alternatively referred to as
"extracting") biopolymers such as DNA, RNA and protein from
affected cells, for example, by producing dendrimer molecules in
the flow channel of the biochip's preprocessing area.
[0004] 2. Description of the Prior Art
[0005] The prior art is explained hereinafter by taking DNA as an
example of a biopolymer. The target DNA used for DNA chips is
retrieved by means of preprocessing.
[0006] Methods of retrieving DNA from affected cells or the like
dissolved in blood serum, lymphocyte-clastic solution or lysis
buffer solution, for example, are roughly classified into two
types: the centrifugal separation method and the magnetic bead
method.
[0007] Since the centrifugal separation method involves the use of
large-scale apparatus, the magnetic bead method is expected to
become mainstream in the future where downsizing is required.
[0008] Accordingly, the magnetic bead method is discussed
hereinafter. (See non-patent document 1, for example, for details
on examples of applications of magnetic beads. It should be noted
that the material on which this method is based, is referred to not
only as magnetic bead, but also as magnetic particle or magnetic
body. See patent document 1, for example, for details on examples
of applications of the method in which the material is referred to
as magnetic particles. Likewise, see patent document 2, for
example, for details on examples of applications of the method in
which the material is referred to as magnetic body.)
[0009] The magnetic bead method is a DNA retrieval method in which
molecules of probe DNA or probe antibody are bound to the surfaces
of magnetic beads at a specific density, and DNA in a solution is
retrieved by means of a complementary combination of target DNA
molecules in the solution and probe molecules. Then the magnetic
beads are collected and cleaned using a magnet and the DNA is
dissociated from the surfaces of the magnetic beads using a
solution and is thus retrieved.
[0010] Non-Patent Document 1
Haruko Takeyama and Hideki Nakayaka, Chapter 7 "DNA Chips Using
Magnetic Beads" in "DNA Chips and It's Application" published by
CMC Publishing Co., Ltd. in July 2000 under the editorship of
Tadashi Matsunaga
[0011] Patent Document 1
Japanese Laid-open Patent Application 1996-176212
[0012] Patent Document 2
Japanese Laid-open Patent Application 1999-313670
[0013] However, while apparatus using the magnetic bead method is
smaller and more convenient than those using the centrifugal
separation method, the magnetic bead method has difficulty in
binding probes to the surfaces of magnetic beads at an appropriate
density. Thus, the efficiency of DNA retrieval is currently at
issue. In addition, it is cumbersome to collect magnetic beads with
a magnet and then retrieve DNA from the surfaces of the beads.
Therefore, an even simpler method of DNA retrieval is required in
order to transform the preprocessing area into a chip in the
future.
SUMMARY OF THE INVENTION
[0014] The present invention is intended to solve the
aforementioned problems. One object of the present invention
therefore, is to provide a method for retrieving biopolymers in a
highly efficient manner by taking advantage of the dendrimers'
ability to achieve highly efficient densities because of their
freely controllable structural density, as well as a biochip using
dendrimers.
[0015] Another object of the present invention is to provide a
dendrimer-based biochip that has no mechanical moving parts and
whose preprocessing area can be easily miniaturized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic view illustrating one embodiment of a
flow channel formed in the preprocessing area of a dendrimer-based
biochip in accordance with the present invention.
[0017] FIG. 2 is a schematic view illustrating another embodiment
of the flow channel.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] In the present invention, a dendrimer structure is formed in
a flow channel through which a DNA solution flows, by taking
advantage of the ability of dendrimers to achieve highly efficient
densities because of their freely controllable structural density.
Then, probe polymer molecules complementary with target biopolymer
molecules are bound to the tips of dendrimers so that these
biopolymer molecules are retrieved in a highly efficient manner. In
addition, the conventional method using magnetic beads requires
mechanical moving parts since magnetic beads are first collected
and then DNA is retrieved from their surfaces. The present
invention eliminates the need for such mechanical moving parts,
thereby enabling the preprocessing area to be easily
miniaturized.
[0019] The present invention will hereinafter be described in
detail with reference to the accompanying drawings. FIG. 1 is a
schematic view illustrating one embodiment of a flow channel formed
in the preprocessing area of a dendrimer-based biochip in
accordance with the present invention, wherein FIG. 1(a) is a
perspective projection (partially perspective view) of the surfaces
of a substrate, and FIG. 1(b) is an enlarged view of a dendrimer
molecule.
[0020] In FIG. 1, numeral 1 denotes the substrate of a biochip and
numeral 2 denotes a flow channel through which a DNA solution
flows, and numeral 3 denotes a dendrimer molecule bound to flow
channel 2.
[0021] Substrate 1 is made of such materials as glass or plastics
and U-shaped flow channel 2 is formed in substrate 1. A plurality
of molecules of dendrimer 3 is formed on the walls of flow channel
2.
[0022] Dendrimer 3 is, for example, a multibranched polyamide amine
dendrimer and is produced by providing aminosilane treatment on the
surfaces of flow channel 2 and overlaying a film of amidoamine,
which is produced by the reaction of methyl acrylate with
ethylenediamine, upon the aminosilane-treated area as a dendron
unit.
[0023] Probe DNA is bound to the tip (surface) of dendrimer 3 which
goes into a complementary combination with target DNA.
[0024] When a solution containing target DNA is poured into the
biochip structured as described above and is made to flow through
flow channel 2, the target DNA combines with the probe DNA in a
complementary manner. Consequently, the target DNA is captured in a
highly efficient manner.
[0025] It should be noted that the density of probe DNA molecules
bound to the tips of dendrimer molecules has the optimum value,
depending on the type of target (DNA, mRNA or protein). This
optimum value can be obtained by changing the number of dendrimer
layers (generations). Under normal conditions, second or higher
generation dendrimers are used.
[0026] It is to be understood that the present invention is not
restricted to the foregoing embodiments; rather, many other
alterations and modifications thereof may be made without departing
from the spirit and essential characteristics thereof. It is
therefore intended that such alterations and modifications be
covered by the appended claims.
[0027] For example, a plurality of concavities and convexities 4
may be formed on the walls of flow channel 2 so that turbulence
takes place when a solution containing target DNA flows through
flow channel 2, as illustrated in FIG. 2. This turbulence further
increases the efficiency of the complementary combination between
target DNA and probe DNA.
[0028] In the foregoing embodiments, an example has been shown
wherein DNA segments are bound to the surfaces of dendrimer
molecules and target and probe molecules are combined by means of
hybridization. Alternatively, molecules of a biopolymer probe other
than DNA may be bound so that target molecules combine with the
probe molecules. Alternatively still, antibody molecules may be
bound to the surfaces of dendrimer molecules as probe molecules so
that the protein of target molecules is extracted by means of
antigen-antibody reaction.
[0029] Alternatively still, it is possible to adopt the desired
number and shape of flow channels 2 formed in substrate 1, without
being restricted to those discussed in the foregoing
embodiments.
[0030] As is evident from the above description, the following
advantageous effects are achieved according to the present
invention. [0031] (1) By forming dendrimer molecules in a flow
channel through which a biopolymer solution flows and binding
molecules of a probe biopolymer or antibody to the tips of the
dendrimer molecules, it is possible to easily and efficiently
retrieve molecules of a target biopolymer or protein by means of a
complementary combination or antigen-antibody reaction. [0032] (2)
While the conventional method using magnetic beads requires
mechanical moving parts, the present invention eliminates the need
for such mechanical moving parts, making it possible to easily
miniaturize the preprocessing area where a target biopolymer or
protein is retrieved.
* * * * *