FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
“METHOD FOR FABRICATING MICROCHIP FOR
NUCLEIC ACID AMPLIFICATION REACTION”
SONY CORPORATION, of 1-7-1, Konan, Minato-ku, Tokyo
108-0075, Japan
The following specification particularly describes the invention and the manner in
which it is to be performed.
2
Description
Title of Invention
METHOD FOR FABRICATING MICROCHIP FOR NUCLEIC ACID
AMPLIFICATION 5 REACTION
Technical Field
[0001]
The present technology relates to a method for fabricating a microchip for
10 nucleic acid amplification reaction. More specifically, the present technology
relates to a microchip for nucleic acid amplification reaction in which a solidified
reagent that includes at least one or more kinds of the substances required for a
reaction is contained in a well that serves as a reaction site for a nucleic acid
amplification reaction.
15
Background Art
[0002]
In recent years, microchips have been developed in which wells and
channels for performing chemical and biological analyses are provided on a silicon
20 substrate or a glass substrate by applying micro-machining techniques used in the
semiconductor industry. These microchips have begun to be utilized for
electrochemical detectors in, for example, liquid chromatography, compact
electrochemical sensors in medical service locations and the like.
[0003]
25 Analytical systems using such microchips are called μ-TAS (micro-Total-
Analysis System), lab-on-a-chip, bio chip or the like. Attention is being paid to
such microchips as a technology that enables chemical and biological analyses to be
performed faster, with greater efficiency, and a higher level of integration, or that
enables the analyzing apparatuses to be reduced in size. μ-TAS, which enables
30 analysis with a small amount of sample and enables the disposable use of microchips,
is expected to be applied particularly in biological analyses where precious trace
3
amounts of samples or many specimens are handled.
[0004]
An applied example of μ-TAS is an optical detection apparatus in which a
substance is introduced into a plurality of areas arranged on the microchip, and the
substance is optically detected. Such an optical detection apparatus 5 may include a
reaction apparatus (for example, a real-time PCR apparatus) that causes a reaction,
such as a nucleic acid amplification reaction, between a plurality of substances to
proceed in a well on the microchip, and optically detects the produced substances.
[0005]
10 Microchip-type nucleic acid amplification apparatuses have conventionally
employed a method in which the reaction is performed by mixing in advance all of
the reagents and template DNA required for the nucleic acid amplification reaction,
and introducing the mixed solution into a plurality of wells arranged on the
microchip. However, with this method, since it takes a certain amount of time until
15 the mixed solution is introduced into the wells, there is the problem that during that
period the reaction proceeds in the mixed liquid, so that non-specific nucleic acid
amplification tends to occur, thereby reducing quantitative performance.
[0006]
In response to the above problem, for example, JP-A-2011-160728 discloses
20 a microchip in which a plurality of reagents required for a nucleic acid amplification
reaction are laminated and fixed in order in the wells.
Citation List
Patent Literature
25 [0007]
Patent Literature 1: JP 2011-160728A
Summary of Invention
Technical Problem
30 [0008]
It is a major object of the present technology to a method for fabricating a
4
microchip for nucleic acid amplification reaction that is capable of simple and highly
accurate analysis.
Solution to Problem
[0009]
According to the first aspect of the present invention in 5 order to achieve the
above-mentioned object, there is provided a method for fabricating a microchip for
nucleic acid amplification reaction, the method including a solidification step of
drying a reagent solution including at least a part of substances required for a nucleic
acid amplification reaction, and a containment step of arranging the solidified
10 reagent solution in wells that serve as a reaction site for a nucleic acid amplification
reaction.
It is preferred that the solidification step includes a step of freeze-drying the
reagent solution.
The method according to the present technology may also include a
15 preparation step of readying, prior to the solidification step, a plurality of the reagent
solutions that have different compositions, and the reagent solutions may include a
first reagent solution that includes an oligonucleotide primer but not an enzyme, and
a second reagent solution that includes an enzyme but not an oligonucleotide primer.
Further, the solidification step may also include a step of individually
20 freeze-drying the first reagent solution and the second reagent solution.
In addition, the containment step may include a step of containing the first
reagent solution that has been solidified and that includes two or more kinds of
oligonucleotide primer in each of a plurality of the wells.
According to another aspect of the present invention in order to achieve the
25 above-mentioned object, there is provided a method for fabricating a microchip for
nucleic acid amplification reaction, the method including, solidifying either one of
the first reagent solution and the second reagent solution in the solidification step,
and, prior to the containment step, a fixing step of adding the reagent solution not
used in the solidification step dropwise into the wells and drying in the wells.
30 It is preferred that the fixing step includes a step of vacuum-drying the
reagent solution.
5
Advantageous Effects of Invention
[0010]
According to the present technology, there is provided a microchip for
nucleic acid amplification that is capable of simple and highly accurate analysis.
5
Brief Description of Drawings
[0011]
[FIG. 1] FIG. 1 is a schematic view illustrating a configuration of a microchip 1a
according to a first embodiment of the present technology.
10 [FIG. 2] FIG. 2 is a schematic view illustrating a configuration in a well 43 of the
microchip 1a.
[FIG. 3] FIG. 3 is a flowchart illustrating a method for fabricating the microchip 1a.
[FIG. 4] FIG. 4 is a schematic view illustrating a configuration of a modified
embodiment of the microchip 1a.
15 [FIG. 5] FIG. 5 is a schematic view illustrating a configuration in a well 43 of a
microchip 1b according to a second embodiment of the present technology.
[FIG. 6] FIG. 6 is a flowchart illustrating a method for fabricating the microchip 1b.
[FIG. 7] FIG. 7 is a schematic view illustrating a configuration in a well 43 of a
microchip 1c according to a third embodiment of the present technology.
20 [FIG. 8] FIG. 8 is a graph illustrating a start time of nucleic acid amplification in a
microchip according to the present technology.
Description of Embodiments
[0012]
25 Hereinafter, preferred embodiments of the present invention will be
described in detail with reference to the appended drawings. Note that, in this
specification and the drawings, elements that have substantially the same function
and structure are denoted with the same reference signs, and repeated explanation is
omitted. The description will be made in the following order.
30 1. Configuration of the microchip for nucleic acid amplification reaction according to
a first embodiment of the present technology
6
2. Method for fabricating the microchip for nucleic acid amplification reaction
according to a first embodiment of the present technology
(1) Molding of the substrate layer
(2) Preparation of the reagent solutions
(3) Solidification of 5 the reagent solutions
(4) Reagent containment
(5) Bonding of the substrate layer
3. Configuration of the microchip for nucleic acid amplification reaction according to
a modified embodiment of the first embodiment
10 4. Configuration of the microchip for nucleic acid amplification reaction according to
a second embodiment of the present technology
5. Method for fabricating the microchip for nucleic acid amplification reaction
according to a second embodiment of the present technology
(1) Fixing of the reagent solutions
15 (2) Reagent containment
6. Configuration of the microchip for nucleic acid amplification reaction according to
a third embodiment of the present technology
[0013]
1. Configuration of the microchip for nucleic acid amplification reaction according to
20 a first embodiment of the present technology
FIG. 1 is a schematic view illustrating a configuration of a microchip 1a
according to a first embodiment of the present technology. FIG. 1A is a top face
schematic view, and FIG. 1B is a cross-sectional schematic view across the P-P crosssection
of FIG. 1A.
25 [0014]
The microchip for nucleic acid amplification reaction (hereinafter referred
to as “microchip”) denoted by reference numeral 1a includes, as an area into which a
sample solution is introduced, an introduction part 2 into which a liquid such as a
sample is externally introduced, wells 41 to 45 that serve as a reaction site for a
30 nucleic acid amplification reaction, and channels 31 to 35 connecting the
introduction part 2 and the respective wells. Further, as described below, reagents
7
R1 and R2 that include at least a part of the substances required in the nucleic acid
amplification reaction are contained in the wells 41 to 45 (reagents R1 and R2 are not
illustrated in FIG. 1B). In FIG. 1 and the description thereof, the five wells supplied
with sample solution by channel 31 are all referred to as wells 41. Similarly, each
of the five wells supplied with sample solution by channels 32, 33, 5 34, and 35 will be
collectively described as wells 42, 43, 44, and 45. Further, the term sample solution
refers to a solution that includes a nucleic acid such as DNA or RNA, which is a
template nucleic acid that is the target of amplification in a nucleic acid amplification
reaction.
10 [0015]
Examples of the “nucleic acid amplification reaction” performed using the
microchip according to the present technology may include a conventional PCR
(polymerase chain reaction) that employs thermal cycling, as well as various
isothermal amplification methods that do not involve thermal cycling. Examples of
15 isothermal amplification methods include methods such as LAMP (loop-mediated
isothermal amplification), SMAP (SMart Amplification Process), NASBA (nucleic
acid sequence-based amplification), ICAN® (isothermal and chimeric primerinitiated
amplification of nucleic acids) , TRC (transcription-reverse transcription
concerted), SDA (strand displacement amplification), TMA (transcription-mediated
20 amplification), RCA (rolling circle amplification) and the like. In addition, the
“nucleic acid amplification reaction” widely includes nucleic acid amplification
reactions that are based on varying temperature or constant temperature, which are
directed to the amplification of nucleic acids. Further, such nucleic acid
amplification reaction also include reactions that involve quantification of an
25 amplified nucleic acid, such as a real-time PCR method.
[0016]
The microchip 1a is formed by bonding a substrate layer 11 on a substrate
layer 12 on which the introduction part 2, the channels 31 to 35, and the wells 41 to
45 are formed, and then bonding a substrate layer 13 on the substrate layer 11 (refer
30 to FIG. 1B). In the microchip 1a, if the bonding of the substrate layer 11 and the
substrate layer 12 is carried out under a pressure lower than atmospheric pressure,
8
the interior of the introduction part 2, the channels 31 to 35, and the wells 41 to 45
can be hermetically sealed at a pressure lower than atmospheric pressure (1/100
atmospheric pressure). In the microchip 1a, by making the area into which the
sample solution is introduced have a pressure lower than atmospheric pressure, the
sample solution is sucked up due to the negative pressure inside 5 the microchip when
the sample solution is introduced. Consequently, the introduction of the sample
solution into the microchip 1a in which micro channel structures are formed can be
carried out in a shorter period of time.
[0017]
10 As the material of the substrate layers 11, 12, and 13, glass and various
kinds of plastic may be used. Preferably, the substrate layers 12 and 13 are formed
from a gas-impermeable material. By using a gas-impermeable material, such as
PC, for the substrate layers 12 and 13 that form the outer face of the microchip 1a,
the sample solution introduced into the wells 41 to 45 can be prevented from being
15 turned into a gas by the heat of the nucleic acid amplification reaction, and escaping
(fluid loss) through the substrate layer 11. Further, when the area of the microchip
1a into which the sample solution is introduced is hermetically sealed due to having a
lower pressure than atmospheric pressure, it is also preferred that the substrate layers
12 and 13 is formed from a gas-impermeable material in order to prevent the entry of
20 air from outside of the microchip 1a to maintain the internal negative pressure.
[0018]
Examples of the material forming the gas-impermeable substrate layers
includes glass, plastics, metals, and ceramics. Examples of plastics include PMMA
(polymethyl methacrylate acrylic resin), PC (polycarbonate), PS (polystyrene), PP
25 (polypropylene), PE (polyethylene), PET (polyethylene terephthalate), diethylene
glycol bis-allyl carbonate, SAN resin (styrene-acrylonitrile copolymer), MS resin
(MMA-styrene copolymer), TPX (poly(4-methyl penten-1)), polyolefin, SiMA
(siloxanyl methacrylate monomer)-MMA copolymer, SiMA-fluorine containing
monomer copolymer, silicon macromer-(A)-HFBuMA (heptafluorobutyl
30 methacrylate)-MMA terpolymer, disubstituted polyacetylene-based polymer and the
like. Examples of metals include aluminum, copper, stainless steel (SUS), silicon,
9
titanium, tungsten and the like. Examples of ceramics include alumina (Al2O3),
nitrogen aluminum (AlN), silicon carbide (SiC), titanium oxide (TiO2), zirconia
oxide (ZrO2), quartz and the like.
[0019]
The substrate layer 11 is preferably formed from an elastic 5 material. In the
microchip 1a, by forming the substrate layer 11 that seals the introduction part 2
from an elastic material, a portion of a penetrating member, such as a needle, can
penetrate the introduction part 2 from outside the microchip 1a. If a syringe
connected to the needle is pre-filled with the sample solution, and the substrate layer
10 11 is penetrated by that needle, the sealed introduction part 2 and the interior of the
syringe are connected, and the sample solution can be introduced into the microchip
1a without air bubbles being formed.
[0020]
Further, when the area into which the sample solution is introduced is
15 hermetically sealed by having a lower pressure than atmospheric pressure, at the
point when the tip of the needle reaches the introduction part 2, due to the pressure
difference between outside the microchip 1a and the introduction part 2, the sample
solution in the syringe is automatically sucked into the introduction part 2.
[0021]
20 By forming the substrate layer 11 from an elastic material, when the needle
is withdrawn from the introduction part 2 after the sample solution has been
introduced, the penetrated location can be naturally sealed due to the self-sealing
ability of the substrate layer 11. In an embodiment of the present technology,
natural sealing of the penetrated location of the needle due to elastic deformation of
25 the substrate layer is defined as “self-sealing ability”.
[0022]
Examples of the elastic material includes acrylic-based elastomer, urethanebased
elastomer, fluorine-based elastomer, styrene-based elastomer, epoxy-based
elastomer, and natural rubber, in addition to silicon-based elastomer such as
30 polydimethylsiloxane (PDMS).
[0023]
10
Note that, in the case of optically analyzing the substances held in each well
of the microchip 1a according to an embodiment of the present technology, it is
preferred to select as the material for each of the substrate layers a material that is
light transmissive and that has little optical error due to having little intrinsic
fluorescence and a small wavelength 5 dispersion.
[0024]
Next, the reagents contained in the wells of the microchip 1a will be
described. In FIG. 2A, a well 43 is schematically illustrated as a representative of
the wells of the microchip 1a. The well 43 contains solid-phase reagents R1 and R2.
10 The reagents R1 and R2 include at least a part of the substances required to obtain an
amplified nucleic acid strand in a nucleic acid amplification reaction. Specific
examples include a component included in an oligonucleotide primer (hereinafter
sometimes also referred to as “primer”), a nucleic acid monomer (dNTPs), an
enzyme, and a reaction buffer solution that is complementary to at least a portion of
15 the base sequence of the DNA, RNA and the like that is the amplification target. In
addition, although not directly necessary in a nucleic acid amplification reaction, a
probe including a label, such as a fluorescent label, for detecting the amplified
nucleic acid strand, a detection reagent that intercalates with double-stranded nucleic
acid and the like may also be included in the reagents R1 and R2 as a substance that
20 is used for detection of an amplified nucleic acid strand.
[0025]
The components required for a nucleic acid amplification reaction that are
included in reagent R1 and reagent R2 may be a different composition to each other.
For example, reagent R1 may be a reagent solution (a first reagent solution) that
25 includes a primer but does not include an enzyme, and reagent R2 may be a reagent
solution (a second reagent solution) that includes an enzyme but does not include a
primer. By thus configuring so that an enzyme is not included in reagent R1 that
includes a primer, and so that a primer is not included in reagent R2 that includes an
enzyme, the primer and the enzyme do not mix until the sample solution is
30 introduced into the wells, which suppresses the occurrence of primer dimers.
Alternatively, reagent R1 may be the reagent solution (the second reagent solution)
11
that includes an enzyme but does not include a primer, and reagent R2 may be the
reagent solution (the first reagent solution) that includes a primer but does not
include an enzyme. The composition of reagents R1 and R2 may be freely selected.
It is noted that reagents R1 and R2 are not limited to the shapes illustrated in FIG. 2.
They may be any shape as long as they have a volume that can 5 be contained in the
well 43. Further, reagents R1 and R2 having the same composition may be
contained in the plurality of wells provided in the microchip 1a, or reagents R1 and
R2 having different compositions may be contained in each of the wells.
[0026]
10 2. Method for fabricating the microchip for nucleic acid amplification reaction
according to a first embodiment of the present technology
The method for fabricating the microchip 1a will now be described with
reference to the flowchart illustrated in FIG. 3.
(1) Molding of the substrate layer
15 In FIG. 3, reference symbol S1 represents a step of molding the substrate
layer. In this step, the introduction part 2, channels 31 to 35, and wells 41 to 45 are
formed on the substrate layer 12. The molding of the introduction part 2 and the
like onto the substrate layer 12 can be carried out by a known technique. For
example, the molding can be carried out by wet etching or dry etching of a glass
20 substrate layer, or by nano-printing, injection molding, or cutting of a plastic
substrate layer. Further, the substrate layer 12 and the like can be molded on the
substrate layer 11, or some parts may be molded on the substrate layer 11, and the
remaining parts molded on the substrate layer 12.
[0027]
25 (2) Preparation of the reagent solutions
In FIG. 3, reference symbol S2 represents a step of preparing a reagent
solution. In this step, a liquid or a gel-like reagent solution is prepared based on the
composition of the reagents R1 and R2 to be contained in the microchip 1a. It is
sufficient if the reagent solution only includes at least a part of the substances that are
30 required in the nucleic acid amplification reaction, and the composition of the
reaction solution may be arbitrarily set. For example, a reagent R1 that includes
12
only a primer and a reagent R2 that only includes an enzyme may be readied.
Further, the number of types of reagent solution that are prepared is not limited to
two. A single reagent solution may include just one type, or a plurality of types, of
the substances required in the nucleic acid amplification reaction.
5 [0028]
If a primer is included in the reagent solution readied in the preparation step,
one type of primer or a plurality of types of primer may be included. In the method
for fabricating the microchip 1a according to the present technology, it is noted that
primers including a different base sequence to a primer formed from a given base
10 sequence are counted as a different type of primer. Namely, for a target nucleic acid
that is the target of amplification, a primer set pairing a primer designed for a base
sequence of one nucleic acid strand with a primer designed for the base sequence of
that complementary strand is considered as including two types of primer. The
definition of these primer types is the same in the below-described second and third
15 embodiments.
[0029]
Regarding the composition of the reagent solutions, for example, it is
preferred to prepare a reagent solution that includes a primer but does not include an
enzyme and a reagent solution that includes an enzyme but does not include a primer,
20 because this means the primer and the enzyme do not mix until the sample solution
introduced when the nucleic acid amplification reaction is started reaches the well,
which suppresses non-specific amplification of the nucleic acid by primer dimers.
Further, it is preferred that the primer-containing reagent solution includes two or
more types of primer.
25 [0030]
In the reagent solution preparation step S2, it is preferred to hold the reagent
solutions, and the primer solution and enzyme solution added to the reaction
solutions, at a cool temperature. This holding of the reagent solutions and the like
at a cool temperature can be carried out by placing the container containing the
30 reagent solutions and the like on ice, or by placing the equipment holding the tubes
of an aluminum block and the like in advance in a freezer, and using in a cooled state.
13
[0031]
(3) Solidification of the reagent solutions
In FIG. 3, reference symbol S3a represents a step of solidifying the reagent
solutions. In this step, the plurality of reagent solutions readied in preparation step
S2 are solidified. Namely, in this step the reagent solutions are 5 dried to produce
solid-phase reagents R1 and R2. Fixing step S3a will be described by dividing it
into two stages, which are, as illustrated in FIG. 3, in order, a “reagent solution
dropwise addition” step S3a-1, and a “freeze-drying” step S3a-2. Note that FIG. 3
is a flowchart illustrating a case in which two types of reagent solution were readied
10 in preparation step S2.
[0032]
Reagent solution dropwise addition step S3a-1
In this step, a reagent solution prepared in the above-described reagent
solution preparation step S2 is added dropwise to a solidification container to be used
15 in solidification step S3a. If a plurality of types of reagent solution were readied in
preparation step S2, each of these reagent solutions is individually added dropwise to
the solidification container, and individually solidified. Further, even for a case in
which reagents R1 with the same composition are to be contained in the plurality of
wells 41 to 45 of the microchip 1a, a number of solidification containers that matches
20 the number of wells are readied, and the reagent solutions are added dropwise to the
respective solidification container. Although the solidification container may be
any material, it is preferred that the solidification container is capable of
withstanding the temperature and air pressure set in the subsequent freeze-drying
step S3a-2.
25 [0033]
Freeze-Drying Step S3a-2
In this step, the above-described reagent solution added dropwise to the
container us solidified by drying. As the drying method, for example, freeze-drying
is preferred. Further, it is preferred that the freeze-drying includes steps such as
30 pre-freezing, primary drying (sublimation freezing), and secondary drying (removal
of bound water). The pre-freezing can be carried out if the freezing temperature is
14
at the eutectic point (temperature at which the reagent solution freezes) or lower.
However, in order to prevent enzyme deactivation and completely freeze the reagent
solution, it is preferred to freeze at about -40°C. In the primary drying, the reagent
solution frozen in the pre-freezing step is dried. At this point, dissolution during the
drying process can be prevented and the moisture included 5 in the reagent solution
can be sublimed by drying the reagent solution at the eutectic point or lower. The
degree of vacuum in the primary drying is desirably 100 Pa or less, for example.
The boiling point of water at 100 Pa is about -20°C, which is close to the abovedescribed
eutectic point of the reagent solution. Accordingly, dissolution during the
10 drying process is prevented. The degree of vacuum in the primary drying can be
appropriately selected based on the eutectic point of the prepared reagent solution.
In the secondary drying, water in a molecular state that is adhered to the components
included in the reagent solution after the primary drying is removed. The reagent
solution can be heated to a temperature at which the components in the reagent
15 solution are not deactivated, denatured or the like, to increase the degree of dryness
of the reagent solution. Note that in the method for fabricating a microchip for
nucleic acid amplification reaction according to the present technology, the drying
method of the solidification step S3a is not limited to freeze-drying.
[0034]
20 (4) Reagent containment
In FIG. 3, reference symbol S4 represents a step of containing the reagents
R1 and R2. In this step, the solid-phase reagents R1 and R2 produced in
solidification containers in the above-described reagent solution solidification step
S3a are removed from the solidification containers, and contained in any one of the
25 wells formed in the substrate layer in the substrate layer molding step S1. The
reagents R1 and R2 may be contained in any of the plurality of wells provided on the
substrate layer 12, either in one well or in a plurality of wells. Further, the number
and type of reagents R1 and R2 contained in one well can be freely set. Reagents
R1 and R2 with the same composition or reagents R1 and R2 with different
30 compositions may be contained in a plurality of wells. If a primer is included in
reagent R1 or in reagent R2, it is preferred that there is two or more types of primer
15
included in one reagent. For example, a reagent R1 and a reagent R2 each
containing different primers may be readied, and the reagents R1 and R2 contained
so that they are arranged in separate wells among the plurality of wells provided in
the microchip 1a. In such a case, the amplification of a plurality of nucleic acid
strands having different base sequences can be analyzed in a 5 single nucleic acid
amplification reaction, so that analysis using the microchip 1a is simpler.
[0035]
(5) Bonding of the substrate layer
In FIG. 3, reference symbol S5 represents a substrate layer bonding step.
10 In this step, another substrate layer is bonded on either of the substrate layers in
which the reagents R1 and R2 were contained. The bonding of the substrate layers
11, 12, and 13 can be performed by a known method, such as thermal fusion bonding,
with an adhesive, anodic bonding, bonding using a pressure-sensitive adhesive sheet,
plasma activation bonding, ultrasonic bonding and the like. Further, by carrying out
15 the bonding of the substrate layers 11, 12 and 13 under a pressure lower than
atmospheric pressure, the respective areas of the introduction part 2, the channels 31
to 35, and the wells 41 to 45 into which the sample solution is introduced can be
made to have a pressure lower than atmospheric pressure (e.g., 1/100 atmospheric
pressure). When a material, such as PDMS, that in addition to being elastic is also
20 impermeable to gases, is used for the substrate layer 11 that seals the wells 41 to 45,
if these layers are left under a negative pressure (vacuum) after the substrate layers
11 and 12 have been bonded, the air that is present in the respective areas, such as the
introduction part 2, passes through the substrate layer 11. Consequently, the interior
of the microchip 1a can be made to have a pressure lower than atmospheric pressure
25 (a vacuum). It is noted that the step of making the interior of the microchip 1a have
a pressure lower than atmospheric pressure is not a necessary step in the method for
fabricating the microchip according to an embodiment of the present technology.
[0036]
In the microchip 1a for nucleic acid amplification reaction according to the
30 present technology, reagents R1 and R2 that include a part of the substances required
for the nucleic acid amplification reaction are contained in advance in the wells 41 to
16
45 that are analysis sites. Consequently, a nucleic acid amplification reaction can
be started just by supplying the remaining substances required for the nucleic acid
amplification reaction and the sample solution including the target nucleic acid
amplification strand to the wells 41 to 45. Further, by containing the plurality of
solid-phase reagents R1 and R2 in wells 41 to 45, the plurality of 5 substances required
for the nucleic acid amplification reaction can be held in the microchip 1a in a
separated state until the start of analysis. Consequently, in a nucleic acid
amplification reaction that uses the microchip 1a, the occurrence of primer dimers
and the like due to primers annealing to each other can be suppressed, and non10
specific amplification of the nucleic acid is reduced. In addition, by performing the
preparation of the reagents R1 and R2 in individual containers, it is simple to solidify
the substances that are used in the nucleic acid amplification reaction individually.
Consequently, the method for fabricating the microchip for nucleic acid amplification
reaction according to the present technology enables the fabrication of a microchip
15 for nucleic acid amplification reaction that is capable of simple yet highly accurate
analysis.
[0037]
3. Configuration of the microchip for nucleic acid amplification reaction according to
a modified embodiment of the first embodiment
20 In FIG. 4, a well 43 is schematically illustrated as a representative for a
reagent R that is contained in the wells of a microchip 1a-2 according to a modified
embodiment of the first embodiment. Except for the composition of the reagent R
contained in the respective wells, such as the wells 43, the microchip 1a-2 is the
same as in the first embodiment. The parts that are the same as in the first
25 embodiment are denoted with the same reference numerals, and a description thereof
is omitted here. Further, the material of the substrate layers 11, 12, and 13
configuring the microchip 1a-2 is the same as the substrate layers denoted with the
same reference numerals for the microchip 1a.
[0038]
30 One type of reagent R is contained in the wells 43 of the microchip 1a-2.
The fabrication steps of the microchip 1a-2 are the same as the flowchart illustrated
17
in FIG. 3, except for the type of reagent solution that is prepared. Accordingly, a
description of the fabrication steps will be omitted. As illustrated in well 43 of FIG.
4, the reagent R contained in the microchip 1a-2 may be a single type. For example,
a reagent R including an enzyme may be contained in well 43, and the other
components required for the nucleic acid amplification reaction, 5 such as a primer,
may be introduced into the microchip 1a-2 by mixing with the sample solution when
the nucleic acid amplification reaction starts.
[0039]
In the microchip 1a-2 according to the present technology, a part of the
10 components required for a nucleic acid amplification reaction are contained in
advance in the wells 41 to 45, so that the components included in the reagent R in the
wells can be kept separate from the other components until the sample solution is
introduced into the wells. Consequently, for example, an enzyme and a primer can
be separated until the nucleic acid amplification reaction starts, so that non-specific
15 nucleic acid amplification caused by primer dimers and the like is suppressed, which
enables highly accurate analysis using the microchip 1a-2.
[0040]
4. Configuration of the microchip for nucleic acid amplification reaction according to
a second embodiment of the present technology
20 In FIG. 5, a well 43 is schematically illustrated as a representative for
reagents R1 and R2 contained in the wells of a microchip 1b according to a second
embodiment of the present technology. Except for the composition of the reagents
R1 and R2 contained in the respective wells, such as the wells 43, the microchip 1b
is the same as in the first embodiment. The parts that are the same as in the first
25 embodiment are denoted with the same reference numerals, and a description thereof
is omitted here. Further, the material of the substrate layers 11, 12, and 13
configuring the microchip 1b is the same as the substrate layers denoted with the
same reference numerals for the microchip 1a.
[0041]
30 Similar to the reagents contained in the microchip 1a, the reagents R1 and
R2 illustrated in FIG. 5 are solid-phase reagents that include at least a part of the
18
substances required for a nucleic acid amplification reaction. Since the composition
of the reagents R1 and R2 is the same as that of the reagents R1 and R2 contained in
the microchip 1a, a description thereof is omitted. The difference between the
reagents R1 and R2 contained in the microchip 1b and the reagents R1 and R2
contained in the microchip 1a is that a part of the reagents contained 5 in the wells 43
is fixed in the well (refer to FIG. 5).
[0042]
5. Method for fabricating the microchip for nucleic acid amplification reaction
according to a first embodiment of the present technology
10 The method for fabricating the microchip 1b will now be described with
reference to the flowchart illustrated in FIG. 6. Since the substrate layer molding
step S1, the reagent solution preparation step S2, and the substrate layer bonding step
S5, respectively, are the same as in the first embodiment, a description thereof will be
omitted. The reagent solution fixing step S3b and the reagent containment step S4
15 will be described.
[0043]
(1) Fixing of the reagent solutions
In FIG. 6, reference symbol S3b represents a step of fixing the reagent
solutions. In this step, among the plurality of types of reagent solution readied in
20 the preparation step S2, one type of reagent solution is fixed in the wells 43.
Namely, a reagent solution is dried in the wells 43, and the dried reagent solution is
fixed in the well. The fixing step S3b will be described in order of a “reagent
solution dropwise addition” step S3b-1 and a “vacuum-drying” step S3b-2 as
illustrated in FIG. 6. Further, in the fabrication of the microchip 1b, the other
25 reagent solution not used in the reagent solution fixing step S3b is turned into a solid
state by the reagent solution solidification step S3a in the same manner as in the first
embodiment.
[0044]
Reagent solution (R2) dropwise addition step S3b-1
30 In this step, among the above-described reagent solutions prepared in the
reagent solution preparation step S2, one type of reagent solution is added dropwise
19
to each well formed in the substrate layer 12 and the like in molding step S1. At
this stage, it is preferred that the substrate layer 12 in which the wells are formed has
been cooled.
[0045]
Vacuum-5 drying step S3b-2
In this step, the reagent solution is dried by placing the substrate layer 12
onto which the above-described reagent solution was added dropwise under a
vacuum (600 to 1,000 Pa). Unlike the reagent solution solidification step S3a in the
first embodiment, in this method it is necessary to select a drying method that does
10 not change the shape of the substrate layer 12, so that vacuum-drying, for example, is
preferred. The drying method may also be carried out by air drying, for example,
according to the nature of the substances included in the reagent solution.
[0046]
(2) Reagent containment
15 In FIG. 6, reference symbol S4 represents a reagent containment step. As a
result of the above-described reagent solution fixing step, unlike the first
embodiment, the reagent R2 is present in the wells 43 in the microchip 1b. In this
step, the reagent R1 readied in the reagent solution solidification step S3a is
separately contained in the well in which this reagent R2 has been fixed. The
20 solidified reagent solution R1 to be contained in the microchip 1b is not limited to
being one type, it may be freely selected.
[0047]
In the microchip 1b according to the present technology, reagents R1 and R2
that include a part of the substances required for a nucleic acid amplification reaction
25 are held in advance in the wells 41 to 45 that are analysis sites. Consequently,
similar to the microchip 1a, when performing a nucleic acid amplification reaction
using the microchip 1b, the nucleic acid amplification reaction can be simply
performed by introducing just the remaining substances required for the nucleic acid
amplification reaction and the sample solution including the target nucleic acid
30 amplification strand into the wells 41 to 45. Further, the components included in
the plurality of solid-phase reagents R1 and R2 having different compositions that
20
are held in the wells 41 to 45 and that have difference compositions can be
maintained in separate states until the start of the nucleic acid amplification reaction.
Consequently, by including, for example, an enzyme and a primer as the components
included in the reagent R1 and reagent R2, respectively, non-specific amplification of
the nucleic acid due to the occurrence of primer dimers 5 can be suppressed.
[0048]
6. Configuration of the microchip for nucleic acid amplification reaction according to
a third embodiment of the present technology
In FIG. 7, a well 43 is schematically illustrated as a representative for a
10 reagent R that is contained in the wells of a microchip 1c according to a third
embodiment. Except for the composition of the reagent R contained in the
respective wells, such as the wells 43, the microchip 1c is the same as in the first
embodiment. The parts that are the same as in the first embodiment are denoted
with the same reference numerals, and a description thereof is omitted here. Further,
15 the material of the substrate layers 11, 12, and 13 configuring the microchip 1c is the
same as the substrate layers denoted with the same reference numerals for the
microchip 1a.
[0049]
The reagent R, which includes at least a part of the substances required to
20 obtain a nucleic acid amplification strand in a nucleic acid amplification reaction, is
fixed in the wells 43 of the microchip 1c (FIG. 7). The components required for the
nucleic acid amplification reaction that are included in the reagent R may be a single
type, or a plurality of types.
[0050]
25 In the fabrication steps of the microchip 1c, since the substrate layer
molding step S1, the reagent solution preparation step S2, and the substrate layer
bonding step S5 are the same as in the first embodiment, a description thereof will be
omitted. Similar to the reagent solution fixing step S3b in the second embodiment,
the step of fixing a reagent solution in the wells 43 is carried out by adding a reagent
30 solution prepared to a predetermined composition dropwise to each well provided on
the substrate layer 12, and fixing the reagent solution in the wells 43 by vacuum21
drying or the like.
[0051]
During the dropwise addition of the reagent solution, it is preferred that the
prepared reagent solution is stored at a cool temperature. Further, it is preferred that
the substrate layer 12 in which the respective wells are formed is also 5 stored at a cool
temperature. For example, the equipment holding the substrate layer 12, such as an
aluminum block, may be cooled in advance in a freezer, and the dropwise addition of
the reagent solution carried out by placing the substrate layer 12 on the cooled
equipment. In the microchip 1c, the reagent R fixed in the wells 43 and the like
10 may be a single type, or may be reagents R1 and R2 having different compositions.
If a plurality of reagents R1 and R2 are fixed in the wells 43, the dropwise addition
of the reagent solution may be carried out by adding either of the reagent solutions
dropwise into the wells 43 and fixing by vacuum-drying or the like, then adding
dropwise the next reagent solution onto the fixed reagent R1 and drying, and then
15 repeating these steps.
[0052]
By maintaining the reagent solutions at a low temperature from the reagent
solution preparation step until the reagent solution drying step, bonding of the
substances and enzyme activity are suppressed for the components that are required
20 for the nucleic acid amplification reaction which are included in the reagent solution.
Consequently, the occurrence of primer dimers is suppressed, and non-specific
amplification of the nucleic acid is reduced.
[0053]
Aspects of the present technology may include the following.
25 (1) A method for fabricating a microchip for nucleic acid amplification reaction,
including a solidification step of drying a reagent solution including at least a part of
substances required for a nucleic acid amplification reaction, and a containment step
of arranging the solidified reagent solution in wells that serve as a reaction site for a
nucleic acid amplification reaction.
30 (2) The method for fabricating a microchip for nucleic acid amplification reaction
according to (1), wherein the solidification step includes a step of freeze-drying the
22
reagent solution.
(3) The method for fabricating a microchip for nucleic acid amplification reaction
according to (1) or (2), further including a preparation step of readying a plurality of
the reagent solutions that have different compositions, wherein the reagent solutions
are a first reagent solution that includes an oligonucleotide primer 5 but not an enzyme,
and a second reagent solution that includes an enzyme but not an oligonucleotide
primer.
(4) The method for fabricating a microchip for nucleic acid amplification reaction
according to (3), wherein the solidification step includes a step of individually
10 freeze-drying the first reagent solution and the second reagent solution.
(5) The method for fabricating a microchip for nucleic acid amplification reaction
according to (3) or (4), wherein the containment step includes a step of containing
the first reagent solution that has been solidified and that includes two or more kinds
of oligonucleotide primer in each of a plurality of the wells.
15 (6) The method for fabricating a microchip for nucleic acid amplification reaction
according to (3), further including, before solidifying either one of the first reagent
solution and the second reagent solution in the solidification step and performing the
containment step, a fixing step of adding the reagent solution not used in the
solidification step dropwise into the wells and drying in the wells.
20 (7) The method for fabricating a microchip for nucleic acid amplification reaction
according to (6), wherein the fixing step includes a step of vacuum-drying the
reagent solution.
Examples
[0054]
25
1. Detection of non-specific amplification in a nucleic acid amplification reaction
The suppression of non-specific amplification of a nucleic acid strand in a
nucleic acid amplification reaction in which the microchip according to the present
technology is used was verified.
30 [0055]
Materials and methods
23
1. Microchip fabrication
Four types of microchip whose methods and the like of producing the
reagent to be contained were different were used as the microchips used in the
present example. For all four types of microchip, substrates made from PDMS and
glass were used as materials. Further, as the reagent required for 5 the nucleic acid
amplification reaction performed in the present example, four types of primer used
for amplification of influenza type A, Bst DNA polymerase, dNTPs, and a reaction
buffer solution were readied. The steps from the reagent solution preparation step
until the containment step will be described below for each microchip.
10 [0056]
<1> Microchip 1
As a comparative example of the microchip for nucleic acid amplification
reaction according to the present technology, microchip 1 (hereinafter referred to as
“M1”) was fabricated. In the fabrication of M1, a reagent solution including four
15 types of primer, Bst DNA polymerase, dNTPs, and a reaction buffer solution was
prepared. 1.2 μL of the reagent solution was added dropwise into the wells formed
in the substrate layer, and the reagent solution in the wells was fixed by a vacuumdrying
treatment (about 1,000 Pa) of about 2 hours.
[0057]
20 <2> Microchip 2
Microchip 2 (hereinafter referred to as “M2”) is a microchip in which a
solidified reagent is contained in the wells. In the fabrication of M2, the
preparation of a reagent solution including four types of primer, Bst DNA
polymerase, dNTPs, and a reaction buffer solution was carried out under cooling by
25 placing the solidification container over ice. The reagent solution was frozen by
leaving the solidification container containing 1.2 μL of reagent solution for 6 hours
at -40°C. After the reagent solution had frozen, the solidification container was set
in a freeze dryer (FDU-2200, EYELA). The reagent solution was dried for 12
hours or more in a vacuum (about 6 to 8 Pa) with the reagent solution kept in a
30 frozen state. Then, the temperature of the drying chamber was set to 30°C, and the
reagent solution was dried for a further 6 hours or more. The reagent solidified by
24
the freeze-drying was removed from the solidification container, and placed in the
wells formed in the substrate layer.
[0058]
<3> Microchip 3
Microchip 3 (hereinafter referred to as “M3”) is a microchip 5 in which a
plurality of solidified reagents containing different substances are contained in the
wells. In the fabrication of M3, a reagent solution including, among the
components required for the nucleic acid amplification reaction of four types of
primer, Bst DNA polymerase, dNTPs, and a reaction buffer solution, the primers
10 (hereinafter referred to as “FluA”) was prepared while cooling. Further, a reagent
solution including the Bst DNA polymerase, the dNTPs, and the reaction buffer
solution (hereinafter referred to as “RM”) was prepared while cooling. The
prepared reagent solutions were added dropwise (0.4 μl for FluA and 0.8 μl for RM)
into separate solidification containers. The respective reagent solutions in the
15 solidification containers were solidified by freeze-drying in the same manner as for
M2. The solidified FluA and RM were removed from the solidification containers,
and placed in each of the wells formed in the substrate layer so that both FluA and
RM were contained in each well.
[0059]
20 <4> Microchip 4
Microchip 4 (hereinafter referred to as “M4”) is a microchip in which
reagents containing different components were fixed in the wells over a plurality of
times. In the fabrication of M4, the reagent solution FluA and the reagent solution
RM were prepared in the same manner as M2. 0.4 μl of FluA was added dropwise
25 into the wells, and fixed in the wells by vacuum-drying in the same manner as for
M1. The substrate layer having the wells in which the FluA had been fixed was
cooled and maintained at a low temperature, and in that state 0.8 μl of RM was added
dropwise into the wells in which the FluA was fixed. Vacuum-drying was carried
out again in the same manner as for M1 to fix the RM in the wells.
30 [0060]
The substrate layer having the wells in which the reagent was contained or
25
had been fixed of the above four types of microchip was laminated with another
substrate layer to seal the wells. The surface of each substrate layer was treated by
oxygen plasma irradiation (O2: 10 cc, RF output: 100 W, RF irradiation time: 30
seconds) and bonded under a vacuum to complete the microchips M1 to M4.
5 [0061]
2. Nucleic acid amplification reaction
A nucleic acid amplification reaction was carried out using the microchips
M1 to M4 fabricated by the above-described steps. A LAMP method was employed
for the nucleic acid amplification. A sample solution was charged into M1 to M4,
10 and the nucleic acid amplification reaction was carried out at 63°C. For the sample
solutions, an influenza type A positive specimen (positive control, hereinafter
referred to as “PC”), an influenza type A negative specimen (negative control,
hereinafter referred to as “NC”), and water (non-template control, hereinafter
referred to as “NTC”) were used. Detection of the nucleic acid strands was carried
15 out by fluorescence detection, and SYBR Green was used for the detection reagent.
[0062]
Results
The results of the present example are shown in FIG. 8. FIG. 8 shows the
start of nucleic acid amplification in each of the microchips M1 to M4 for each
20 sample solution. The nucleic acid amplification start time is defined as the time at
which an amplification curve plotting the fluorescence intensity obtained by SYBR
Green rises up and reaches a predetermined threshold. It is noted that the M1’ in
FIG. 8 is a microchip fabricated by the same fabrication steps as for M1, and which
was used in a nucleic acid amplification reaction in the same manner as M1.
25 [0063]
Based on the results of the nucleic acid amplification reaction, nucleic acid
amplification was detected in the wells that the PC was introduced into for the
microchips M1 to M4 (regarding M1, refer to M1’). Namely, it was shown that the
reagent contained in the wells was stored in a state that could be used in a nucleic
30 acid amplification reaction. On the other hand, nucleic acid amplification was
observed also for the microchips M1 to M4 into which the NC and the NTC had been
26
introduced. This indicates that non-specific amplification of the nucleic acid strand
occurred in the wells of the microchips M1 to M4. In the nucleic acid amplification
reaction performed in the present example, specific amplification for a template
nucleic acid strand of a nucleic acid was detected within 30 minutes of the start of
the nucleic acid amplification reaction (FIG. 8). Consequently, 5 the fact that nucleic
acid amplification occurred within 30 minutes of the reaction starting in the wells
into which NC and NTC had been introduced, in which nucleic acid amplification
should not occur, impairs analysis using the microchips.
[0064]
10 As illustrated in FIG. 8, the start of non-specific nucleic acid amplification
in M3 was more than 50 minutes after the start of the nucleic acid amplification
reaction. On the other hand, the start of non-specific nucleic acid amplification in
the comparative example M1 was detected about 20 minutes after the start of the
reaction. From this result, it was shown that non-specific nucleic acid amplification
15 is suppressed in the nucleic acid amplification reaction using M3.
[0065]
The start of non-specific nucleic acid amplification in M2 and M4 was, in
some of the wells, after about 30 minutes had passed. Compared with the M3
results, in the results for M2 and M4 the start time of non-specific nucleic acid
20 amplification was earlier. However, nucleic acid amplification was not deemed to
have occurred for NTC and NC within 30 minutes of the start of the nucleic acid
amplification reaction. From this result, it was shown that in M2 and M4, nonspecific
nucleic acid amplification was suppressed more than in M1 (comparative
example). Further, the suppression effect of non-specific nucleic acid amplification
25 was about the same in M2 and M4.
[0066]
From the results of the present example, the suppression of non-specific
nucleic acid amplification in a nucleic acid amplification reaction by using a
microchip that contained a reagent including the substances required for the nucleic
30 acid amplification reaction in the wells was confirmed. Especially, non-specific
nucleic acid amplification was greatly suppressed for the microchip (M3) in which a
27
reagent solution that included a primer but did not include an enzyme and reagent
solution that included an enzyme but did not include a primer were individually
solidified and sealed in wells. Namely, with a microchip fabricated based on the
microchip fabrication method according to the present technology, non-specific
nucleic acid amplification is reduced and analysis accuracy 5 is improved.
[0067]
Further, even in the microchip (M2) that contained a solid-phase reagent
including an enzyme and a primer, and the microchip (M4) fabricated by adding a
reagent solution including an enzyme dropwise into wells in which a reagent
10 including a primer had been fixed, the suppression of non-specific nucleic acid
amplification was observed. This indicates that non-specific nucleic acid
amplification is suppressed in a nucleic acid amplification reaction that uses a
reagent that was dried after the mixing of a cooled enzyme and a primer in the
microchip fabrication steps. Based on the above, it was confirmed that the
15 microchip nucleic acid amplification reaction according to the present technology not
only enables analysis to be carried out simply just by the introduction of a sample
solution and the like, but since non-specific nucleic acid amplification is suppressed,
also enables highly accurate analysis.
20 Industrial Applicability
[0068]
According to the microchip for nucleic acid amplification reaction
according to the present technology, analysis based on nucleic acid amplification can
be carried out simply and accurately. Consequently, the microchip for nucleic acid
25 amplification reaction according to the present technology can be used as a device
that performs nucleic acid amplification for clinical genotyping and contagion
determination.
Reference Signs List
30 [0069]
R, R1, R2 reagent
28
1a, 1a-2, 1b, 1c microchip
11, 12, 13 substrate layer
2 introduction part
31, 32, 33, 34, 35 channel
41, 42, 5 43, 44, 45 wells
29
CLAIMS
Claim 1
A method for fabricating a microchip for nucleic acid amplification reaction,
the method comprising:
a solidification step of drying a reagent solution including 5 at least a part of
substances required for a nucleic acid amplification reaction; and
a containment step of arranging the solidified reagent solution in wells that
serve as a reaction site for a nucleic acid amplification reaction.
10 Claim 2
The method for fabricating a microchip for nucleic acid amplification
reaction according to claim 1, wherein the solidification step includes a step of
freeze-drying the reagent solution.
15 Claim 3
The method for fabricating a microchip for nucleic acid amplification
reaction according to claim 2, the method comprising:
a preparation step of readying, prior to the solidification step, a plurality of
the reagent solutions that have different compositions,
20 wherein the reagent solutions include a first reagent solution that includes an
oligonucleotide primer but not an enzyme, and a second reagent solution that
includes an enzyme but not an oligonucleotide primer.
Claim 4
25 The method for fabricating a microchip for nucleic acid amplification
reaction according to claim 3, wherein the solidification step includes a step of
individually freeze-drying the first reagent solution and the second reagent solution.
Claim 5
30 The method for fabricating a microchip for nucleic acid amplification
reaction according to claim 4, wherein the containment step includes a step of
30
containing the first reagent solution that has been solidified and that includes two or
more kinds of oligonucleotide primer in each of a plurality of the wells.
Claim 6
The method for fabricating a microchip for nucleic 5 acid amplification
reaction according to claim 3, the method comprising:
solidifying either one of the first reagent solution and the second reagent
solution in the solidification step, and, prior to the containment step, a fixing step of
adding the reagent solution not used in the solidification step dropwise into the wells
10 and drying in the wells.
Claim 7
The method for fabricating a microchip for nucleic acid amplification
reaction according to claim 6, wherein the fixing step includes a step of vacuum15
drying the reagent solution.