U.S. patent application number 13/026620 was filed with the patent office on 2012-01-12 for apparatus and methods for detecting nucleic acid in biological samples.
This patent application is currently assigned to HAI KANG LIFE CORPORATION LIMITED. Invention is credited to Lok Ting Lau, Ka Wai Wong, Cheung Hoi Yu.
Application Number | 20120010093 13/026620 |
Document ID | / |
Family ID | 34465492 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120010093 |
Kind Code |
A1 |
Yu; Cheung Hoi ; et
al. |
January 12, 2012 |
APPARATUS AND METHODS FOR DETECTING NUCLEIC ACID IN BIOLOGICAL
SAMPLES
Abstract
There are disclosed apparatus and methods for the field-assisted
acceleration of biological processes involving charged entities,
including in particular the detection of target DNA in a biological
sample. A reaction cell is provided with a dielectric surface, and
a field is generated by inducing charge-separation in the
dielectric material by applying a potential to an electrode in
contact with the dielectric material.
Inventors: |
Yu; Cheung Hoi; (Hong Kong,
HK) ; Lau; Lok Ting; (Kowloon, HK) ; Wong; Ka
Wai; (New Territories, HK) |
Assignee: |
HAI KANG LIFE CORPORATION
LIMITED
Kowloon
HK
|
Family ID: |
34465492 |
Appl. No.: |
13/026620 |
Filed: |
February 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12113392 |
May 1, 2008 |
7888109 |
|
|
13026620 |
|
|
|
|
10686252 |
Oct 16, 2003 |
7390622 |
|
|
12113392 |
|
|
|
|
Current U.S.
Class: |
506/9 ;
506/39 |
Current CPC
Class: |
C12Q 1/6834 20130101;
C12Q 2523/307 20130101; C12Q 1/6834 20130101; C07H 21/00
20130101 |
Class at
Publication: |
506/9 ;
506/39 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 60/12 20060101 C40B060/12 |
Claims
1. Apparatus for detecting target nucleic acid in a sample,
comprising a substrate formed with at least one reaction cell,
wherein said reaction cell includes an attachment surface formed of
a dielectric material for the attachment of nucleic acid capture
probes, and wherein a metal electrode is provided in direct contact
with said dielectric material.
2. Apparatus as claimed in claim 1 wherein said electrode is in
contact with a side of said dielectric material opposite from said
attachment surface.
3. Apparatus as claimed in claim 1 wherein said dielectric material
is an oxide.
4. Apparatus as claimed in claim 3 wherein said dielectric material
is selected from the group consisting of Al.sub.2O.sub.3, SiO.sub.2
and Ta.sub.2O.sub.5.
5. Apparatus as claimed in claim 1 wherein said electrode is formed
of aluminum.
6. Apparatus as claimed in claim 1 wherein said dielectric material
comprises Al.sub.2O.sub.3 and said electrode is formed of
aluminum.
7. Apparatus as claimed in claim 1 comprising a multilayer
structure comprising a first base layer, a second insulating layer
formed on said base layer, a third layer formed on said insulating
layer and comprising patterned conductive regions defining at least
one metal electrode, and a fourth layer comprising at least one
region of dielectric material, wherein each said metal electrode in
said third layer is covered by a region of dielectric material in
said fourth layer.
8. Apparatus as claimed in claim 6 wherein the patterned conductive
regions of said third layer are separated by regions formed of
dielectric material.
9. Apparatus as claimed in claim 6 wherein said regions of
dielectric material in said fourth layer are separated by regions
of a passivation material.
10. Apparatus as claimed in claim 8 wherein said regions of
passivation material extend over the edges of said regions of
dielectric material to define said reaction cells.
11. (canceled)
12. (canceled)
13. A method of performing field-assisted hybridization in the
detection of nucleic acid targets from a sample, comprising the
steps of providing a reaction cell having an attachment surface
formed of a dielectric material, providing a metal electrode in
direct contact with said dielectric material, attaching nucleic
acid capture probes to said attachment surface, adding the sample
to said reaction cell, and providing an electrical potential to
said electrode.
14. A method as claimed in claim 13 wherein said electrode is
provided in contact with a surface of said dielectric opposite from
said attachment surface.
15. A method as claimed in claim 13 wherein said electrical
potential is a continuously applied potential.
16. A method as claimed in claim 13 wherein said electrical
potential is applied as a series of pulses.
17. A method as claimed in claim 13 wherein said sample comprises
biological substances.
18. A method of attracting or repelling electrically-charged
entities to or from a surface of a reaction cell when performing a
biological reaction, comprising the steps of providing a dielectric
material as said surface, and generating an electrical field by
inducing charge-separation in said dielectric material.
19. A method as claimed in claim 18 wherein charge-separation in
said dielectric material is induced by placing an electrode in
direct contact with said dielectric material and applying a
potential to said electrode.
20. A method as claimed in claim 19 wherein a continuous potential
is applied to said electrode.
21. A method as claimed in claim 19 wherein a pulsed potential is
applied to said electrode.
22. A method as claimed in claim 19 wherein said electrode is
placed in contact with a surface of the dielectric material
opposite from the surface to which the charged entities are to be
attracted to or repelled from.
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation Application of U.S.
patent application Ser. No. 12/113,392, filed May 1, 2008, now U.S.
Pat. No. 7,888,109, issued Feb. 15, 2011, which is a Divisional
Application of U.S. application Ser. No. 10/686,252, filed Oct. 16,
2003, now U.S. Pat. No. 7,390,622, issued Jun. 24, 2008, both of
which are incorporated by reference herein in their entirety for
all purposes.
FIELD OF THE INVENTION
[0002] This invention relates to apparatus and methods for
detecting nucleic acid in biological samples. In particular the
present invention relates to a novel apparatus and method for
detecting DNA sequences using field-assisted nucleic acid
hybridization and to methods for optimizing the performance of such
apparatus, and further the present invention extends to the use of
field-assisted hybridization in any biological process that
includes charged entities.
BACKGROUND OF THE INVENTION
[0003] The emergence of high-density polynucleotide (eg DNA or RNA)
array technology has transformed the basic concepts of genomics and
protemics analysis. The transition from "dot blots" to "arrays on
glass slides" and then to DNA microarrays (also known as DNA chips)
has revolutionised the industry by making large-scale clinical
diagnostic testing and screening processes realistic for practical
applications. As is well-known, a typical microarray, with reactive
sites in a predetermined configuration on a substrate, will exhibit
a binding pattern when exposed to a sample with target nucleic acid
fragments having a base sequence complementary to that of the
capture fragments attached on the reactive sites. The binding
pattern and the binding efficiency can be detected by optical or
electronic methods when an appropriate detection mechanism is used,
which may include for example fluorescent labeling, current
detection or impedance measurement.
[0004] The use of electrically-assisted nucleic acid hybridization
is a known technique in the analysis of biological samples
containing DNA, e.g. blood, plasma, urine etc. Conventionally, a
chip for DNA detection is formed from one of a variety of materials
including glass, silica and metal. On the surface of the chip a
number of electrical contacts are formed using known techniques. To
detect a particular DNA sequence in a biological sample, capture
probes consisting of complementary DNA fragments are attached to
the chip surface by means of an attachment layer which is
conventionally an agarose gel. If a biological sample contains the
target DNA, the target DNA will bind to the complementary DNA
fragments by hybridization, and various imaging techniques may be
used to detect such hybridization and thus the presence in the
sample of the target DNA.
PRIOR ART
[0005] Nucleic acid fragments are electrically charged and thus can
be attracted towards a particular site by electrostatic attraction
by the use of electrodes and thus by the application of an
appropriate electrical current the hybridization process may be
accelerated and thus the detection process is also accelerated.
However, it is not possible to use the electrode in direct contact
with the nucleic acid fragments because of the danger of
electrochemical degradation or electrolysis of the sample.
Conventionally therefore a permeation/attachment layer is normally
coated on the electrode as shown in U.S. Pat. Nos. 5,605,662 and
6,306,348. The permeation/attachment layer is normally made from
porous materials, eg sol-gel materials, porous hydrogel materials,
porous oxides and serves to allow the selective diffusion of small
ions and also as an attachment surface for the capture probes.
Direct contact of the nucleic acid fragments with the electrode is
reduced owing to the size of the pores of the porous materials
which are generally too small to allow the larger nucleic acid
fragments to pass through.
[0006] When a voltage is applied to the electrode underneath the
permeation/attachment layer, the devices of the prior art can
provide electrophoretic transport effects without electrochemical
degradation of the sample and can thus enhance hybridization.
However, such prior techniques for enabling electrically-induced
hybridization are not without their drawbacks. For example, porous
materials such as hydrogels and polymers are vulnerable to
deterioration under contact with aqueous solutions, various
chemicals and a number of ambient factors. The preparation of
sol-gel materials are costly and complicated, increasing the
manufacturing costs. Furthermore the porous materials are naturally
fragile and susceptible to adsorption and the trapping of undesired
foreign materials such as moisture hydrocarbons in air, resulting
in a shorter-shelf-life of the devices.
SUMMARY OF THE INVENTION
[0007] According to the present invention there is provided
apparatus for detecting target nucleic acid in a sample, comprising
a substrate formed with at least one reaction cell, wherein said
reaction cell includes an attachment surface formed of a dielectric
material for the attachment of nucleic acid capture probes, and
wherein a metal electrode is provided in direct contact with said
dielectric material. The sample may comprise biological substances
and the sample may be wastewater, solution or reagent. The sample
may also be a biological sample such as blood, plasma or urine.
[0008] Preferably the electrode is provided beneath the attachment
surface, that is to say in contact with a side of the dielectric
material opposite from the attachment surface. Conceivably,
however, it could be applied in contact with a side of the
dielectric material or even in contact with the attachment surface
itself.
[0009] In preferred embodiments of the invention the dielectric
material is preferably an oxide, for example it may be selected
from the group consisting of Al.sub.2O.sub.3, SiO.sub.2 and
Ta.sub.2O.sub.5. The metal electrode for example may be formed of
aluminum.
[0010] In preferred embodiments of the invention the apparatus may
comprise a multilayer structure comprising a first base layer, a
second insulating layer formed on said base layer, a third layer
formed on said insulating layer and comprising patterned conductive
regions defining at least one metal electrode, and a fourth layer
comprising at least one region of dielectric material, wherein each
said metal electrode in said third layer is covered by a region of
dielectric material in said fourth layer. Preferably the patterned
conductive regions of the third layer are separated by regions
formed of dielectric material. Still more preferably the regions of
dielectric material in said fourth layer are separated by regions
of a passivation material, and the regions of passivation material
may extend over the edges of said regions of dielectric material to
define said reaction cells.
[0011] Viewed from another broad aspect the present invention
provides a method of performing field-assisted hybridization in the
detection of nucleic acid targets from a biological sample,
comprising the steps of providing a reaction cell having an
attachment surface formed of a dielectric material, providing a
metal electrode beneath in direct contact with said dielectric
material, attaching nucleic acid capture probes to said attachment
surface, adding a sample to said reaction cell, and providing an
electrical potential to said electrode. The sample may comprise
biological substances and the sample may be wastewater, solution or
reagent. The sample may also be a biological sample such as blood
plasma or urine.
[0012] The electrical potential may be applied as a continuous
potential, or may be a smoothly varying, or pulsed potential.
[0013] Viewed from another broad aspect the present invention also
provides a method of attracting or repelling electrically-charged
entities to or from a surface of a reaction cell when performing a
biological reaction, comprising the steps of providing a dielectric
material as said surface, and generating an electrical field by
inducing charge-separation in said dielectric material. The
electrically charged entities may be nucleic acid molecules.
[0014] Viewed from a still further aspect the invention also
extends to a method of forming an array of reaction cells for
performing biological analysis, comprising the steps of patterning
metal electrodes on an insulating substrate, depositing regions of
dielectric material on said metal electrodes, and forming a rim
around the edges of upper surfaces of said regions of dielectric
material so as to define said reaction cells.
[0015] Preferably, for example, the method may comprise depositing
a layer of metal on an insulating surface, covering a desired
pattern of said metal layer with a photoresist and removing the
remainder of said metal layer by an etching process, depositing a
layer of said dielectric material over said patterned metal whereby
said dielectric material covers said patterned metal and occupies
the areas between said patterned electrodes, depositing a
passivation layer over said layer of dielectric material, pattering
said passivation layer with a photoresist and removing said
passivation layer to open said dielectric material where it covers
said metal electrodes to define reaction said cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Some embodiments of the invention will now be described by
way of example and with reference to the accompanying drawings, in
which:--
[0017] FIG. 1 is a sectional view through a chip in accordance with
an embodiment of the present invention,
[0018] FIG. 2 is a view similar to FIG. 1 but showing the chip in
use,
[0019] FIG. 3 is a schematic illustration showing the underlying
principle of preferred embodiments of the present invention,
[0020] FIG. 4 illustrates the steps in a possible fabrication
process,
[0021] FIGS. 5(a) and (b) show first test results,
[0022] FIGS. 6(a) and (b) show second test results, and
[0023] FIGS. 7(a) and (b) show third test results.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0024] FIG. 1 shows in section a part of an embodiment of the
present invention that includes three cells for receiving a
sample-containing buffer solution, but it will be understood that
any number of cells could be provided, and they would normally be
formed in a rectangular array.
[0025] The device according to an embodiment of the invention is
fabricated by sequential deposition onto a silicon substrate using
conventional deposition techniques. Firstly, (FIG. 4(a)) an
insulating layer formed of SiO.sub.2 of a thickness of between
about 200 nm to 500 nm is formed on the Si substrate by any
suitable technique including thermal oxidation or by any suitable
deposition technique such as for example sputtering, electron beam
evaporation and the like. On top of the insulating layer is formed
(FIG. 4(b)) a layer of aluminum of a thickness of between about 500
nm to 1000 nm again using any conventional deposition
techniques.
[0026] Once the layer of aluminum has been formed, it is patterned
(FIG. 4(c)) using a layer of photoresist and the unmasked areas are
removed by etching (FIG. 4(d)) and the photoresist is removed (FIG.
4(e)). The chip is then coated (FIG. 4(f)) with Al.sub.2O.sub.3 to
a thickness of between 50-500 nm with regions of Al.sub.2O.sub.3
being formed between the aluminum regions that are formed on the
silicon dioxide substrate. A passivation layer of (for example)
Si.sub.3N.sub.4 is then deposited (FIG. 4(g)) on the
Al.sub.2O.sub.3 by means of plasma enhanced chemical vapor
deposition or similar techniques. The passivation layer is then
patterned with a photoresist (FIG. 4(h)) and the passivation layer
is then etched (FIG. 4(i)) to open up the Al.sub.2O.sub.3 areas
that are to become the attachment surfaces of the reaction cell.
Finally the photoresist is removed (FIG. 4(j)).
[0027] The result of this fabrication process is the multi-layer
structure of FIG. 1. Regions of aluminum are formed on the
insulating layer of silicon dioxide and these aluminum regions are
separated by Al.sub.2O.sub.3. Formed on top of the layer of
aluminum and Al.sub.2O.sub.3 is a layer that comprises regions of
Al.sub.2O.sub.3 located above the regions of aluminum and separated
from each other by the passivation material SiO.sub.2 which covers
the Al.sub.2O.sub.3 regions separating the aluminum regions on the
lower later. The passivation material also extends to cover the
edges of the top Al.sub.2O.sub.3 so as to define a surface for a
biological sample to be placed for analysis.
[0028] It will thus be understood that in the example shown in FIG.
1 the chip is formed with three cells 1-3 each formed of
Al.sub.2O.sub.3 with an underlying pad of aluminum. Although not
shown in FIG. 1 when the aluminum regions are formed by etching,
electrical connections may also be formed that allow an electrical
potential to be applied to the aluminum regions.
[0029] Once the chip of FIG. 1 has been fabricated it can be used
as the basis for a number of different biological tests and assays.
In particular each cell 1-3 in FIG. 1 may be provided with suitable
capture probes as shown in FIG. 2. Depending on the tests to be
performed, each cell may be provided with the same capture probes
or with different capture probes, the capture probes having nuclei
acid fragments that are complementary to fragments in the sample
that the test or assay is looking for. In the example of FIG. 2,
the cells 1-3 are all identical and a drop of sample containing
buffer solution is added to the cell so that it covers all three
cells.
[0030] As will be understood by those skilled in the art, if the
sample contains fragments of nucleic acid that are complementary to
the capture probes, they will bind to the capture probes by the
process of hybridization and this may be detected by known
techniques. Since the nucleic acid fragments are electrically
charged, this hybridization can be enhanced by providing an
electrical field that will attract desired nucleic acid fragments
towards the attachment surface and the capture probes. The
mechanism by which this may be done is shown in FIG. 3.
[0031] In particular, as shown in FIG. 3, if a potential is applied
to the aluminum electrode underlying a cell, then because
Al.sub.2O.sub.3 is a dielectric material charge separation will
occur within the Al.sub.2O.sub.3 the polarity of which will depend
on the polarity of the voltage applied to the aluminum electrode
beneath the Al.sub.2O.sub.3. As shown on the left of FIG. 3, if a
positive potential is applied to the aluminum electrode, then the
upper surface of the Al.sub.2O.sub.3 will also have a positive
potential which would attract negatively charged fragments, and
repel positively charged fragments. Conversely, if a negative
potential is applied to the aluminum electrode, then the upper
surface of the Al.sub.2O.sub.3 will also have a negative potential
which would attract positively charged fragments, and repel
negatively charged fragments as shown in the right-hand side of
FIG. 3. Thus selectively applying an electrical potential to the
aluminum electrodes that are directly underneath and in direct
contact with the Al.sub.2O.sub.3 attachment surface enables the
selective attraction/repelling of nucleic acid fragments and thus
enables electrically-induced hybridization. It should be understood
that the potential can be applied in many different ways. The
potential could for example be a constant continuous potential, may
be smoothly varying, or may be pulsed either with regular pulses or
in any desired pattern.
[0032] A particular advantage of the present invention, at least in
its preferred forms, in contrast with the prior art is that
undesired electrochemical reactions and/or electrolysis can be
completely avoided since there is no electron transfer between the
sample solution and the surface of the dielectric layer. The
nucleic acid fragments can thus be electrically drawn to the
attachment surface without electrochemical degradation. A further
important advantage of the field-assisted hybridization method and
apparatus of the present invention, at least in preferred forms, is
that the salt concentration and pH value of the sample will not be
changed. These parameters are crucial factors influencing the
hybridization efficiency and the stability of the hybridized
nucleic acid fragments. Prior art electrically-assisted
hybridization techniques lead to significant changes in the salt
concentration and the pH due to electrochemical reactions and other
techniques, such as special buffer solutions, are required in order
to compensate for these effects. A further advantage of the present
invention is that no electrochemical reactions will occur and in
turn this will mean that the solutions/reagents involved in the
detection process will not be disturbed. There will be no bubble
formation and/or precipitation during the detection process, which
is important in improving the quality of the detected signal.
[0033] It should also be understood that while preferred
embodiments of the present invention are described in the context
of accelerated hybridization in the detection of nucleic acid
fragments, the invention is more generally applicable to any
biological process that involves electrically-charged entities and
where it is desired to be able to control the movement of such
electrically-charged entities by attracting or repelling such
entities to or from a surface.
[0034] FIGS. 5 to 7 show a number of experimental results using the
structure of FIGS. 1 to 3 with and without an electrical potential
applied to the aluminum electrodes beneath the cells. It will of
course be understood that in all of these examples the reaction
times, applied voltages and other parameters are purely exemplary
and may be varied as desired.
[0035] FIGS. 5(a) and (b) show a control in which in neither case
is an electrical potential applied to the aluminum electrodes and
therefore the hybridization proceeds without electrical assistance.
In this Example the target oligomers in the sample are synthetic
.beta.-actin (91 bases, pure) and the hybridization time is 90
minutes. The target oligomers are present in the sample of FIG.
5(a) and not present in the sample of FIG. 5(b). In neither FIG.
5(a) or 5(b) is an electrical potential applied to the aluminum
electrode, but the cells are clearly darker in FIG. 5(a) than 5(b)
owing to the presence of the target oligomers in the sample of FIG.
5(a).
[0036] In FIGS. 6(a) and (b) the conditions are the same as in FIG.
5(a) and (b) in that the same target oligomers are provided in the
sample of FIG. 6(a) and no target oligomers are provided in the
sample of FIG. 6(b). In this example, however, a potential of +10V
is applied to the aluminum electrodes and the hybridization time is
reduced to 10 minutes. A comparison of FIGS. 5(a) and 6(a) shows
that in FIG. 6(a) the cells are much darker clearly illustrating
even though the hybridization time has been substantially reduced,
demonstrating the effectiveness of the applied voltage in
accelerating the hybridization. The similarity between FIGS. 5(b)
and 6(b) where no target oligomers are present shows that the
applied voltage does not lead to any false positive results.
[0037] FIG. 7(a)-(c) illustrate a third example in which the target
oligomers in the sample are avarian influenza virus (AIV) H5
subtype (250 bases mixed with other non-specific oligomers). In all
three cases (a)-(c) the hybridization time is 10 minutes. The
differences between
[0038] FIGS. 7(a)-(c) are as follows: In FIG. 7(a) target oligomers
are present in the sample and an electrical potential of +10V is
applied to the aluminum electrodes beneath the cells; In FIG. 7(b)
no target oligomers are present in the sample and an electrical
potential of +10V is applied to the aluminum electrodes beneath the
cells; and in FIG. 7(c) target oligomers are present in the sample
but no electrical potential is applied to the aluminum electrodes
beneath the cells. Again this example shows that with a
hybridization time of only 10 minutes, the application of a +10V
potential to the electrode results in accelerated hybridization and
strong signal (very dark areas in the cells of FIG. 7(a)). In
comparison the similarity in appearance between FIG. 7(b) (without
target and with applied potential) and FIG. 7(c) (with target but
without applied potential) shows that it is not possible to obtain
effective hybridization in the same time period (10 minutes)
without electrically assisted hybridization.
[0039] The present invention at least in its applied forms provides
a simple low-cost device that allows nucleic acid field-assisted
hybridization and/or other biological processes to proceed at a
much faster rate with high performance that can be applied to a
large number of possible applications. The present invention
employs the principle of charge-separation in a dielectric material
that is in contact with an electrode to which a potential is
applied. In the embodiments described above the electrode is
aluminum and the dielectric material is Al.sub.2O.sub.3, but other
combinations of metal electrode and dielectric attachment surface
are also possible. For example, SiO.sub.2 and Ta.sub.2O.sub.5 may
be used as oxide based dielectric materials for the attachment
surface.
[0040] In contrast with the prior art devices that use a permeation
layer, the oxide based dielectric layer in direct contact with the
electrode provides a structure that is robust, compact, chemically
inert towards most of the acids, alkalis and other reagents
commonly used in biological reactions. The structure is also stable
with regards to ambient factors such as temperature and humidity
and is less vulnerable to physical damage. The production costs are
lower and the device can be manufactured very easily using standard
deposition techniques and other microelectronics fabrication
techniques. Indeed the use of such microelectronics deposition and
fabrication techniques in the manufacture of the devices of the
present invention also has the advantage that the devices can
readily be incorporated into other devices made using the same or
similar technology.
* * * * *