U.S. patent application number 10/535894 was filed with the patent office on 2006-03-09 for dna hybrids and environment cleaning system employing dna hybrids.
This patent application is currently assigned to CANON KABUSHIKI KAISHA. Invention is credited to Yoshinori Kotani, Norio Nishi, Teigo Sakakibara, Zuyi Zhang.
Application Number | 20060051765 10/535894 |
Document ID | / |
Family ID | 33487264 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060051765 |
Kind Code |
A1 |
Zhang; Zuyi ; et
al. |
March 9, 2006 |
Dna hybrids and environment cleaning system employing dna
hybrids
Abstract
A DNA hybrid which comprises a porous oxide matrix and DNA
immobilized thereon, and is useful for environmental clean-up,
where the hybrid is prepared by removing a dispersion medium from a
dispersion of colloidal oxide and DNA.
Inventors: |
Zhang; Zuyi; (Yokohama-shi,
JP) ; Sakakibara; Teigo; (Yokohama-shi, JP) ;
Kotani; Yoshinori; (Yokohama-shi, JP) ; Nishi;
Norio; (Sapporo-shi, JP) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Assignee: |
CANON KABUSHIKI KAISHA
Tokyo
JP
|
Family ID: |
33487264 |
Appl. No.: |
10/535894 |
Filed: |
May 25, 2004 |
PCT Filed: |
May 25, 2004 |
PCT NO: |
PCT/JP04/07472 |
371 Date: |
May 23, 2005 |
Current U.S.
Class: |
435/6.11 ;
435/287.2; 536/24.3 |
Current CPC
Class: |
B01J 20/3297 20130101;
C07H 21/00 20130101; B01J 20/08 20130101; B01J 20/3274 20130101;
B01J 20/3293 20130101; B01J 2220/58 20130101; B01J 20/28061
20130101; B01J 20/3078 20130101; B01J 20/3204 20130101; B01J 20/06
20130101; B01J 20/103 20130101; C12Q 1/6834 20130101; C02F 1/286
20130101; C02F 1/288 20130101; B01J 2220/42 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 536/024.3 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12M 1/34 20060101
C12M001/34; C12M 3/00 20060101 C12M003/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 29, 2003 |
JP |
2003-152619 |
Claims
1. A DNA hybrid comprising a porous oxide matrix and a DNA
immobilized by the matrix, the DNA hybrid prepared by removing a
dispersion medium from a dispersion of a colloidal oxide and
DNA.
2. The DNA hybrid according to claim 1, wherein the dispersion
medium is removed by heating the dispersion at 25.degree. C. to
200.degree. C.
3. The DNA hybrid according to claim 1, wherein the DNA content in
the hybrid is 0.01 to 15% by mass.
4. The DNA hybrid according to claim 1 or 3, wherein the colloidal
oxide is a mixture of a colloidal silica and at least one colloidal
oxide of a metal whose valence is 3 or 4.
5. The DNA hybrid according to claim 1, wherein the oxide comprises
at least one oxide selected from the group consisting of aluminum
oxide, iron oxide, titanium oxide and zirconium oxide.
6. A method for producing a DNA hybrid comprising the steps of:
preparing a dispersion comprising a colloidal oxide, a DNA and a
dispersion medium; and, removing the dispersion medium from the
dispersion to immobilize the DNA in a porous matrix made of the
oxide.
7. The method according to claim 6, wherein the dispersion medium
is removed by heating the dispersion at 25.degree. C. to
200.degree. C.
8. The method according to claim 6, wherein the DNA content of the
DNA hybrid is 0.01 to 15% by mass.
9. The method for producing a DNA hybrid according to claim 6 or 8,
wherein the colloidal oxide is a mixture of colloidal silica and at
least one colloidal oxide of a metal whose valence is 3 or 4.
10. The method according to claim 9, wherein the oxide comprises at
least one oxide selected from the group consisting of aluminum
oxide, iron oxide, titanium oxide and zirconium oxide.
11. An environmental cleaning system for removing a harmful
substance in water comprising a DNA hybrid according to claim 1 and
means for bringing the DNA hybrid into contact with a water
containing a harmful substance.
12. An environmental cleaning method by removing a harmful
substance in a water comprising a step for bringing a DNA hybrid
according to claim 1 into contact with a water containing a harmful
substance to adsorb the harmful substance by the DNA hybrid.
Description
TECHNICAL FIELD
[0001] The present invention relates to a DNA hybrid. More
particularly, the invention relates to a DNA hybrid where DNA is
immobilized firmly in an oxide matrix, having water resistance,
retaining selective recognition function of DNA and allowing
intercalation into the double helix of DNA. It also relates to an
environmental clean-up system using this DNA hybrid.
BACKGROUND ART
[0002] DNA (deoxyribonucleic acid) carrying genetic information in
living organisms is one of the most important materials for life
process. DNA has the capability of extremely precise
molecular-recognition, because DNA forms a double-stranded
structure between two complementary strands via base pairs between
them. Based on this principle, genetic diagnosis that uses the DNA
chip has been developed. Application to biosensors and molecular
devices is also expected. Also since the DNA double helix allows
selective intercalation of an aromatic compound having a planar
chemical structure, it is useful in removing a carcinogenic
compound and the like, and is expected to be useful as an
environmental cleaning material for removing a harmful substance in
air or water (see FUNCTION & MATERIALS, Vol. 19, 1999).
[0003] For example, DNA chips carrying a lot of DNA fragments (DNA
probes) on the surface of a substrate like a slide glass have been
developed. Since DNA is naturally water-soluble, DNA must be
immobilized onto the substrate for such applications. In order to
immobilize a biological substance on a substrate, organic
crosslinking reaction is conventionally utilized. For instance,
there is a method of supporting a biological substance such as
nucleic acid, protein and peptide on the roughened surface of a
slide glass having a surface layer of a carbon-based material (see
Japanese Patent Application Laid-open No. 2002-211954). In this
method, first the carbon surface is carboxylized and subjected to
dehydration-polycondensation with carbodiimide and
N-hydroxysuccinimide to form active ester groups such as
N-hydroxysuccinimide ester group on the end of the hydrocarbons
through an amide bond, and then the biological substance to be
supported is bonded to the activated surface. Also a method and a
kit for quantitatively detecting nucleic acid are disclosed in
Japanese Patent Application Laid-open No. H11-148935.
[0004] Furthermore, a method for immobilizing nucleic acid on a
substrate is disclosed where the substrate is activated by using
atomic-oxygen plasma to immobilize nucleic acid thereon (see
Japanese Patent Application Laid-open No. 2002-218976). Any one of
these technologies has such problems that the supporting region is
limited to the surface of the substrate and the process is
complicated.
[0005] In addition to the organic crosslinking reaction, a
crosslinking reaction with a metal ion was also proposed. Japanese
Patent Application Laid-open No. H7-41494 discloses a method for
immobilizing a deoxyribonucleic acid by coagulating an alkaline
metal salt of the deoxyribonucleic acid and an alkaline metal salt
of alginic acid using a divalent metal-containing compound.
[0006] Meanwhile, concerning the use of DNA as an environmental
cleaning material, Japanese Patent Application Laid-open No.
2001-81098 proposes a material where DNA is solidified and
immobilized on a support by irradiating UV light of 250 to 270 nm
to a liquid film or thin layer of water-soluble DNA on the support.
Further, Japanese Patent Application Laid-open No. H10-175994
discloses a DNA complex where DNA is immobilized on an inorganic
solid. In these methods DNA is crosslinked to develop water
resistance, but there are such problems that the exposed area of
DNA is small and functions of DNA are not developed
efficiently.
[0007] U.S. Pat. No. 6,303,290 discloses a method for immobilizing
a biological material on a matrix of silica colloid. In this
method, although the matrix has a large number of micropores, the
matrix strength is not enough.
DISCLOSURE OF THE INVENTION
[0008] The DNA carriers obtained by the above methods are not
adequate as a DNA composite material applicable to selective
absorption process or environmental clean-up with high efficiency,
in view of the DNA-supporting strength and function expression. The
present invention has been made to solve such problems in the prior
arts, and an object of the present invention is to provide a DNA
hybrid where DNA is effectively immobilized and exhibits sufficient
function, a preparing method therefor, and further an environmental
clean-up system using the DNA hybrid.
[0009] The invention includes the following aspects.
[0010] (1) A DNA hybrid which comprises a porous oxide matrix and a
DNA immobilized on the matrix, where the DNA hybrid is
characterized by that it is prepared by removing a dispersion
medium from a dispersion of a colloidal oxide and DNA.
[0011] (2) The DNA hybrid described in the above (1) characterized
in that the dispersion medium is removed by heating the dispersion
at 25.degree. C. to 200.degree. C.
[0012] (3) The DNA hybrid described in the above (1), characterized
in that the DNA content in the hybrid is 0.01 to 15% by mass.
[0013] (4) The DNA hybrid described in the above (1) or (3),
characterized in that the colloidal oxide is a mixture of a
colloidal silica and at least one colloidal oxide of a metal whose
valence is 3 or 4.
[0014] (5) The DNA hybrid described in the above (1), characterized
in that the oxide comprises at least one oxide selected from the
group consisting of aluminum oxide, iron oxide, titanium oxide and
zirconium oxide.
[0015] (6) A method for producing a DNA hybrid which comprises the
steps of preparing a dispersion comprising a colloidal oxide, a DNA
and a dispersion medium; and removing the dispersion medium from
the dispersion to immobilize the DNA in a porous matrix made of the
oxide.
[0016] (7) The method described in the above (6), characterized in
that the dispersion medium is removed by heating the dispersion at
25.degree. C. to 200.degree. C.
[0017] (8) The method described in the above (6), characterized in
that the DNA content of the DNA hybrid is 0.01 to 15% by mass.
[0018] (9) The method described in the above (6) or (8),
characterized in that the colloidal oxide is a mixture of colloidal
silica and at least one colloidal oxide of a metal whose valence is
3 or 4.
[0019] (10) The method described in the above (9), characterized in
that the oxide comprises at least one oxide selected from the group
consisting of aluminum oxide, iron oxide, titanium oxide and
zirconium oxide.
[0020] (11) An environmental cleaning system for removing a harmful
substance in water, which comprises a DNA hybrid described in the
above (1) and means for bringing the DNA hybrid into contact with a
water containing a harmful substance.
[0021] (12) An environmental cleaning method by removing a harmful
substance in a water, characterized in that a DNA hybrid described
in the above (1) is brought into contact with water containing a
harmful substance to adsorb the harmful substance by the DNA
hybrid.
BEST MODE FOR CARRYING OUT THE INVENTION
[0022] In the invention, colloid aggregation or gelling occurs in
the DNA containing oxide colloid dispersion as of the dispersion
medium is evaporated or because of the presence of a third
component, and finally a porous oxide gel is formed after the
dispersion medium is removed. DNA is taken in the oxide colloid
during stages from dispersion to gellation to be immobilized in the
porous matrix.
[0023] DNA employed in the invention is not limited particularly
for its size or type, provided that it achieves the purpose of use
when being immobilized in the matrix. Such DNA may be single-strand
or double-stranded DNA obtained from, for instance, soft roe or
animal thymus; preferably obtained from soft roe (testis) of
salmon, herring or cod; and also preferably obtained from the
thymus of mammals or birds such as cow, pig and chicken.
[0024] Other example of water-soluble DNA may be a synthetic DNA,
for instance, poly (dA)-poly (dT) having base pairs of (dA)-(dT).
The molecular weight of DNA is preferably 100,000 or higher, more
preferably 500,000 or higher.
[0025] The content (by mass) of DNA in the DNA hybrid according to
the present invention is in a range of 0.01 to 15% and preferably
of 0.1 to 10%. DNA content of 0.01% or more enables sufficient
expression efficacy of DNA-originated properties, and DNA content
of 15% or less can obviate the cost efficiency problem and allow
formation of pores in the DNA hybrid. The pores allow rapid
movement of gas and liquid into the DNA hybrid, so that DNA can
achieve its function not only on the surface but also in the
pores.
[0026] The colloidal oxide employed in the invention may be, for
example, colloidal silica, colloidal aluminum oxide, colloidal iron
oxide, colloidal gallium oxide, colloidal lanthanum oxide,
colloidal titanium oxide, colloidal cerium oxide, colloidal
zirconium oxide, colloidal tin oxide, colloidal hafnium oxide and
the like. Any of these can be used alone or in combination of two
or more. In view of the matrix stability and the cost, it is
preferred to use readily available silica colloid as the main
matrix component. More preferably, colloidal silica is used in
combination with at least one colloidal oxide of trivalent or
tetravalent metal. Addition of colloidal oxide of tri- or
tetravalent metal causes binding between the phosphate group of DNA
and the metal ion, which enhances the DNA-holding ability of the
matrix, preventing elution of DNA in the ordinary aqueous
environment. As a solid in the colloid, the 3- or 4-valent metal
oxide content is preferably 0.1 to 50% by mass. Among those listed
above, iron oxide, titanium oxide and zirconium oxide are
especially preferred. Any of these colloids can be synthesized by
the hydrothermal reaction. Alternatively, an aqueous colloidal
dispersion of oxide is commercially available.
[0027] Examples of colloidal silica include aqueous sols such as
SNOWTEX 20, SNOWTEX 30, SNOWTEX N, SNOWTEX O, SNOWTEX C (trade
names, provided by NISSAN CHEMICAL INDUSTRIES, LTD.) and the like,
methanolic sols, and solvent-based sols such as IPA-ST, EG-ST,
MEK-ST (trade name, provided by NISSAN CHEMICAL INDUSTRIES, LTD.),
OSCAL-1132, OSCAL-1432, OSCAL-1232 (trade name, provided by
SHOKUBAI KASEI KOGYO). Examples of colloidal aluminum oxide are
those marketed by NISSAN CHEMICAL INDUSTRIES, LTD. under the trade
name of ALUMINA SOL 100, ALUMINA SOL 520 and the like.
[0028] To immobilize DNA in the oxide matrix, first a mixture of a
DNA solution and a colloidal oxide dispersion is prepared and the
dispersion medium is removed from the mixture. The surface of
colloidal oxide particles may be modified in advance to prevent
agglomeration of colloid particles in the dispersion stage. The
modification can be accomplished by adding a chelating agent such
as a surfactant, titanium compound, silane coupling agent,
.beta.-diketone and the like and an organic acid such as acetic
acid as appropriate to a colloidal dispersion to partially modify
the surface of the colloid particles. The colloidal dispersion thus
obtained was combined with an aqueous solution of DNA to obtain a
DNA hybrid. In the step of immobilization, the dispersion medium is
removed by means of heating, spray drying, vacuum drying and the
like to form an oxide matrix. A drying process with heating is
especially preferred because it enhances the matrix strength. The
temperature of heat treatment for DNA hybrid is preferably
25.degree. C. or higher and 200.degree. C. or lower, more
preferably not lower than 30.degree. C. and not higher than
150.degree. C. If necessary, a third component may be added to
strengthen the bond between the oxide colloid particles. The third
component is not limited particularly, including acid or base,
water-soluble metal compound and metal alkoxide that can promote
the aggregation of colloid particles.
[0029] The DNA hybrid may be used in a form of powder or bulk. In
addition, it may be used as a coating film on a substrate such as
plate, tube, fiber, woven fabric and non-woven fabric, as needed.
Accordingly, the DNA hybrid according to the present invention can
be used in these forms. For instance, DNA hybrid powder can be
obtained by spray drying of a dispersion containing colloidal oxide
and DNA. Alternatively, first DNA hybrid is formed in the bulk and
then the bulk is pulverized.
[0030] Furthermore, it is possible to make a module using the
above-described DNA hybrid powder, or plate, tube, fiber, woven or
non-woven fabric to which DNA hybrid is immobilized. For instance,
a column filled with the DNA hybrid powder can be used for
extracting a specific substance from a gas or a liquid. Or a filter
module may be contemplated using a fiber or fabric supporting the
DNA hybrid to filter cow's milk or mother's milk.
[0031] Such a module can be applied to an environmental clean-up
system for removing hazardous materials from various effluents,
waste water, and river or lake water. For instance, as described
above, a column containing the DNA hybrid powder, or DNA hybrid
held on a plate, a tube, a fiber, a woven or non-woven fabric can
be used to purify water containing a harmful substance by passing
it through the column.
EXAMPLES
Example 1
[0032] Five parts by mass of double-stranded DNA obtained from
salmon testis (molecular weight: 6.times.10.sup.6) was dissolved in
1000 parts by mass of ion exchange water over a day to obtain an
aqueous solution of DNA.
[0033] To 70 parts by mass of 30% silica sol (NISSAN CHEMICAL
INDUSTRIES, LTD., SNOWTEX CM), 20 parts by mass of 20% by mass
alumina sol (NISSAN CHEMICAL INDUSTRIES, LTD., ALUMINA SOL 520) was
added with stirring. The resultant sol mixture was combined with
100 parts by mass of the DNA solution, and stirred slowly for 10
minutes. Subsequently, the resultant DNA dispersion was dried for
24 hours at 50.degree. C. to obtain DNA Hybrid 1 whose DNA content
was about 2% by mass.
[0034] This DNA hybrid was subjected to an elution test. To 20
parts by mass of ion-exchanged water, 0.05 parts by mass of the DNA
hybrid powder was added and settled in a sealed condition at room
temperature for 48 hours. The absorbance (260 nm) of DNA in the
supernatant liquid measured by a spectrophotometer (U-3310,
Hitachi) was 0.05 or less. The DNA hybrid was proved to be holding
95 mass % or more of the DNA. When 0.5 parts by mass of the DNA
hybrid was put into an aqueous solution of 60 ppm ethidium bromide,
the DNA hybrid turned red after three hours, while the red color
due to ethidium bromide in the supernatant decreased. When
irradiated with ultraviolet light of 366 nm wavelength, the DNA
hybrid emitted an orange fluorescence, which ensured that the
intercalating function for a hazardous compound having a planar
structure was maintained.
[0035] The DNA hybrid showed a specific surface area of 135
m.sup.2/g, when measured with a nitrogen adsorption method.
Example 2
[0036] To 70 parts by mass of 30% silica sol (NISSAN CHEMICAL
INDUSTRIES, LTD., SNOWTEX CM), 40 parts by mass of 20% by mass
alumina sol (NISSAN CHEMICAL INDUSTRIES, LTD., ALUMINA SOL 520) was
added with stirring. The resultant sol mixture was combined with
100 parts by mass of the DNA solution prepared in Example 1, and
stirred slowly for 10 minutes. Subsequently, the resultant DNA
dispersion was dried for 24 hours at 50.degree. C. to obtain DNA
Hybrid 2 whose DNA content was about 1.7% by mass.
[0037] This DNA hybrid was subjected to an elution test. To 20
parts by mass of ion-exchanged water, 0.05 parts by mass of the DNA
hybrid powder was added and settled in a sealed condition at room
temperature for 48 hours. The absorbance (260 nm) of DNA in the
supernatant liquid measured by a spectrophotometer (U-3310,
Hitachi) was about 0.02. The DNA hybrid was proved to be holding 98
mass % or more of the DNA.
Example 3
[0038] To 70 parts by mass of 30% silica sol (NISSAN CHEMICAL
INDUSTRIES, LTD., SNOWTEX CM), 60 parts by mass of a titanium oxide
sol (TAKI CHEMICAL CO., LTD., M-6, 6% by mass) was added with
stirring. The resultant sol mixture was combined with 100 parts by
mass of the DNA solution prepared in Example 1, and stirred slowly
for 10 minutes. Subsequently, the resultant DNA dispersion was
dried for 24 hours at 50.degree. C. to obtain DNA Hybrid 3 whose
DNA content was about 1% by mass.
[0039] This DNA hybrid was subjected to an elution test. To 20
parts by mass of ion-exchanged water, 0.1 parts by mass of the DNA
hybrid powder was added and settled in a sealed condition at room
temperature for 48 hours. The absorbance (260 nm) of DNA in the
supernatant liquid measured by a spectrophotometer (U-3310,
Hitachi) was about 0.05. The DNA hybrid was proved to be holding 95
mass % of the DNA.
Example 4
[0040] To 70 parts by mass of 30% silica sol (NISSAN CHEMICAL
INDUSTRIES, LTD., SNOWTEX CM), 100 parts by mass of a titanium
oxide sol (TAKI CHEMICAL CO., LTD., M-6, 6% by mass) was added with
stirring. The resultant sol mixture was combined with 100 parts by
mass of the DNA solution prepared in Example 1, and stirred slowly
for 10 minutes. Subsequently, the resultant DNA dispersion was
dried for 24 hours at 50.degree. C. to obtain DNA Hybrid 4 whose
DNA content was about 1.8% by mass.
[0041] This DNA hybrid was subjected to an elution test. To 20
parts by mass of ion-exchanged water, 0.1 parts by mass of the DNA
hybrid powder was added and settled in a sealed condition at room
temperature for 48 hours. The absorbance (260 nm) of DNA in the
supernatant liquid measured by a spectrophotometer (U-3310,
Hitachi) was about 0.07. The DNA hybrid was proved to be holding 96
mass % of the DNA.
Example 5
[0042] To 100 parts by mass of 30% silica sol (NISSAN CHEMICAL
INDUSTRIES, LTD., SNOWTEX CM), 50 parts by mass of a titanium oxide
sol (TAKI CHEMICAL CO., LTD., M-6, 6% by mass) was added with
stirring. The resultant sol mixture was combined with 20 parts by
mass of the DNA solution prepared in Example 1, and stirred slowly
for 10 minutes. Subsequently, the resultant DNA dispersion was
dried for 24 hours at 50.degree. C. to obtain DNA Hybrid 5 whose
DNA content was about 0.3% by mass.
[0043] This DNA hybrid was subjected to an elution test. To 20
parts by mass of ion-exchanged water, 0.1 parts by mass of the DNA
hybrid powder was added and settled in a sealed condition at room
temperature for 48 hours. The absorbance (260 nm) of DNA in the
supernatant liquid measured by a spectrophotometer (U-3310,
Hitachi) was not detected. The DNA hybrid was proved to be holding
almost all DNA.
Comparative 1
[0044] 5 Parts by mass of a silica powder whose particle size was
0.063 to 0.2 mm was combined with 5.6 parts by mass of the DNA
solution of Example 1 and mixed uniformly. The resultant paste was
dried for 24 hours at 50.degree. C. to obtain a silica powder whose
DNA supporting level was 0.56% by mass. 0.040 Parts by mass of the
resultant silica powder was combined with 5 parts by mass of ion
exchange water to conduct the elution test. The absorbance of the
supernatant at 260 nm was 0.56. Thus, it was revealed that about
61% by mass of the DNA was eluted.
[0045] The present invention is not limited to the above
embodiments and various changes and modifications can be made
within the spirit and scope of the present invention. Therefore to
apprise the public of the scope of the present invention, the
following claims are made.
* * * * *