U.S. patent application number 12/131627 was filed with the patent office on 2009-01-01 for microfluidic device with multiple cognitive agents immobilized thereon and methods for its fabrication and method of use.
This patent application is currently assigned to National Science and Technology Development Agency (NSTDA). Invention is credited to Naoki ICHIKAWA, Vichuta LAURUENGTANA, Ryutaro MAEDA, Sohei MATSUMOTO, Sakon RAHONG, Apinan SOOTTITANTAWAT, Mana SRIYUDTHSAK, Yongyuth WANNA.
Application Number | 20090004746 12/131627 |
Document ID | / |
Family ID | 40161047 |
Filed Date | 2009-01-01 |
United States Patent
Application |
20090004746 |
Kind Code |
A1 |
SRIYUDTHSAK; Mana ; et
al. |
January 1, 2009 |
MICROFLUIDIC DEVICE WITH MULTIPLE COGNITIVE AGENTS IMMOBILIZED
THEREON AND METHODS FOR ITS FABRICATION AND METHOD OF USE
Abstract
A microfluidic device allowing for multiple discrete reactions
sites and allowing for sequential reactions and sample analysis
along with methods for device fabrication and use is provided. The
microfluidic device provides a micro-total analysis system on a
single substrate and has multiple reaction sites allowing for
cascade reactions and analysis on a single chip using
micro-quantities of sample and reagents. The microfluidic device
provides discrete sites for immobilization of cognitive agents
including enzymes, binding proteins, nucleic acids and the like as
well as methods for quantitative analysis. The invention also
provides methods for the fabrication of the device.
Inventors: |
SRIYUDTHSAK; Mana; (Pakred,
TH) ; SOOTTITANTAWAT; Apinan; (Bangkok, TH) ;
WANNA; Yongyuth; (Prachatipad, TH) ; RAHONG;
Sakon; (Changmai, TH) ; LAURUENGTANA; Vichuta;
(Klong Luang, TH) ; ICHIKAWA; Naoki; (Ibaraki,
JP) ; MATSUMOTO; Sohei; (Ibaraki, JP) ; MAEDA;
Ryutaro; (Tsuchiura, JP) |
Correspondence
Address: |
DORSEY & WHITNEY LLP;INTELLECTUAL PROPERTY DEPARTMENT
SUITE 1500, 50 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402-1498
US
|
Assignee: |
National Science and Technology
Development Agency (NSTDA)
|
Family ID: |
40161047 |
Appl. No.: |
12/131627 |
Filed: |
June 2, 2008 |
Current U.S.
Class: |
436/2 ; 156/247;
422/600; 422/68.1; 422/82.05; 422/82.12; 435/304.1 |
Current CPC
Class: |
B01L 2300/0887 20130101;
B01L 2200/10 20130101; B01L 2300/0663 20130101; B01L 2300/0636
20130101; B01L 2300/0816 20130101; B01L 2300/0645 20130101; B01L
3/502707 20130101; B01L 3/5027 20130101; C12Q 1/25 20130101 |
Class at
Publication: |
436/2 ; 422/193;
435/304.1; 422/68.1; 422/82.05; 422/82.12; 156/247 |
International
Class: |
G01N 33/00 20060101
G01N033/00; B01J 19/00 20060101 B01J019/00; C12M 1/40 20060101
C12M001/40; G01N 21/01 20060101 G01N021/01; G01N 25/00 20060101
G01N025/00; B32B 38/10 20060101 B32B038/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2007 |
JP |
2007-145346 |
Claims
1. A microfluidic device comprising a reaction channel fabricated
from a reaction layer on a substrate, including at least two
reaction channel networks, wherein the at least two reaction
channel networks are immobilized with at least one cognitive agent
allowing for a sequential reaction along the reaction channel.
2. The microfluidic device of claim 1, wherein said cognitive agent
is selected from a group of enzymes, antigens, antibodies,
proteins, receptors, nucleic acids or combinations thereof.
3. The microfluidic device of claim 1, wherein the at least two
reaction channel networks have different cognitive agents
immobilized therein.
4. The microfluidic device of claim 3, wherein the enzymes
immobilized in the reaction channel networks are invertase,
mutarotase, and glucose oxidase.
5. The microfluidic device of claim 1, wherein the reaction layer
is made from a polymer
6. The microfluidic device of claim 5, wherein the reaction layer
is made from plastic, polydimethyl siloxane, polycarbonate, acrylic
resin, polyvinyl chloride, polyacrylic amide or
polyacrylonitrile.
7. The microfluidic device of claim 1, wherein the substrate is
made from glass, silicon, polyvinyl chloride, polyester, polyimide,
acrylic resin, polycarbonate, cellulose acetate, polyacrylic amide,
polyacrylonitrile, polydimethyl siloxane or plastic.
8. The microfluidic device of claim 1, wherein the substrate has a
sensor for measurement.
9. The microfluidic device of claim 8, wherein said sensor is a
thermal sensor, electromagnetic sensor, mechanical sensor, chemical
sensor, optical sensor, ionizing radiation sensor or acoustic
sensor.
10. A method of fabrication of a microfluidic device comprising the
steps of: providing a substrate; attaching an immobilization layer
having one or more channel networks formed therein to the
substrate; immobilizing one or more cognitive agents onto the
substrate surface at the one or more channel networks; flowing one
or more solution of cognitive agents into the channel networks;
detaching the immobilization layer from the substrate; and
positioning a reaction layer including a single reaction channel to
the substrate; wherein the single reaction channel encompasses the
one or more channel networks.
11. The method of fabricating the microfluidic device of claims 10,
wherein the solution of cognitive agents is an enzyme solution, an
antigen solution, an antibody solution, a protein solution, a
receptor solution, a nucleic acid solution or combinations
thereof.
12. The method of fabricating the microfluidic device of claim 10,
further comprising a step of substrate surface pre-treatment, prior
to the step of attaching the immobilization layer.
13. The method of forming the microfluidic device of claim 10,
wherein the pre-treatment step is preformed prior to flowing the
cognitive through the channel network.
14. The method of claim 11 wherein the pre-treatment step comprises
deposition of a gold layer.
15. A method of component analysis comprising a step of introducing
a solution into the reaction channel of the device of claim 1 and
allowing a component in the solution to flow through the at least
two reaction channel networks with the one or more cognitive agents
in sequence, and measuring an amount of a reaction product.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to Japanese patent
application number 2007-145346 filed May 31, 2007 which is herein
incorporated in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to a microfluidic device for use with
a micro-total analysis system (.mu.-TAS) having discrete areas with
multiple cognitive agents immobilized thereon. More specifically,
the invention provides a lab-on-a-chip device that allows for
cascade or sequential reactions and product analysis on a
micro-scale.
BACKGROUND OF THE INVENTION
[0003] Chemical component analysis is extensively used in many and
varied fields. However, the uses for such chemical component
analysis have been limited due the requirement for special
equipment, physical space for the equipment, and the amount of time
necessary for preparing for and conducting the analysis. To address
these problems, there is much interest in developing "micro-total
analysis systems" (.mu.-TAS) having dimensions similar to a credit
card. Such systems combine a separating device, mixing device,
measuring device, and analyzing device on the same substrate.
Ideally, in use, the .mu.-TAS can deliver the sample solution to
the analysis equipment and then analyze the sample while requiring
microliters of sample at most. Moreover, the .mu.-TAS can be
operated using similarly small amounts of reagents. Such systems
have uniform reaction temperatures, superior controllability, and
are disposable improving safety and hygiene.
[0004] Analysis by .mu.-TAS is useful in a number of applications
including, but not limited to, medical, industrial, agriculture,
molecular, and forensic investigations. Examples of medical
applications include but are not limited to, measurement and
inspection of blood components and biochemical analysis, such as
measurement and inspection of various types of proteins, hormones,
and antibodies. Examples industrial applications include but are
not limited to, component analysis in manufactured products,
component analysis of waste-water and component analysis of raw
materials. Examples of use in agriculture include but are not
limited to, measurement of sugar content in vegetables and fruits
and measurement of chemical/pesticide residues both in the
environment and on produce. Examples for use with genetic analysis
include but are not limited to decoding of genetic information for
diagnosis and prevention of genetic diseases.
[0005] For chemical component analysis in solution, .mu.-TAS
employs microfluidic devices in which an internal channel is
immobilized with a cognitive agent, such as, for example, an enzyme
for a reaction catalyst or antibodies, binding proteins and
receptors for capturing desired substrates or analytes. Such
cognitive agents can be used to react specifically with a desired
analyte (detected substance). Currently, microfluidic devices using
the above mentioned cognitive agents such as enzymes, antibodies,
binding proteins, etc. are limited because only a single cognitive
agent is immobilized in the channels of the microfluidic device.
When used in chemical component analysis in solution and for which
multiple cognitive agents, e.g. enzymes or antibodies, receptors,
ligands or the like are used, the solution sample has to be
multiply contacted and reacted with different and multiple
immobilized cognitive agents. This requires several microfluidic
devices immobilized with single cognitive agent, as mentioned
above, that are linked by a connecting channel of each device to
form a composite device; or by connecting the microfluidic devices
and connecting the channels. The deficiencies of these approaches
are the difficulty of use and maintenance, solution leak at the
joints which requires substantially reinforced joint areas,
increases space needs and larger amounts of sample and reagent.
These deficiencies result in a large amount of time needed in
constructing and connoting the various devices, large amount of
time needed to perform the analysis as well as more costly devices
cost of analysis time and less consistent results.
[0006] Several methods of fabricating micro channel devices. These
include, methods of forming a resin layer on a substrate (JP nos.
2002-283293 and 2004-167607) by attaching a resin layer formed by
laser on a silicon, glass, or ceramic substrate; methods of
fabricating a channel using a photoresist and exposing it to
ultraviolet light through a mask, then removing non-exposed parts
(JP no. 2004-194652); methods of fabricating a channel by
micro-discharge; and methods of mechanical fabrication (etching)
using a hard material, e.g. diamond, as a tool for the
micro-fabrication. Additionally, methods of micro-channel
fabrication using a mould begins with coating the photoresist on
silicon substrate, exposing it through a mask, and removing the
photoresist to generate an embossed portion to form a mould for the
micro-channel on silicone substrate, adding a mixture of
polydimethyl siloxane (PDMS) and hardening material onto the mould,
obtaining a groove of the channel as the mixture hardens, detaching
the hardened layer off the mould, and attaching the hardened layer
on a substrate, e.g. silicone or glass (JP no. 2004-296099).
[0007] As with methods of fabricating microfluidic devices, methods
of immobilizing enzymes can also be performed in various ways.
However, a method of immobilizing different cognitive agents, such
as multiple enzymes or antibodies, onto a single channel in
discrete positions for use in continuous or cascade reactions is
not known. Currently, a screen printing technique can be used for
immobilizing different enzymes onto the same substrate, but the
accuracy of placement is limited to a range of 500 .mu.m to 1 mm.
This limitation is far in excess of what is required for .mu.-TAS
analysis.
[0008] In instances of reactions such as catalytic reactions,
binding reactions, or antigen-antibody reactions, that include
multiple steps that need to be performed continuously or
sequentially using multiple cognitive agents, e.g. enzymes,
antibodies; the use of a microfluidic device in these instances
should be capable of controlling the reaction, using only minimal
amounts of reactants/reagents as well as providing high efficiency.
Furthermore, in cases of cascade reactions occurring in a single
micro-channel it is a necessity to provide controlled and
reproducible environment. For example, in a small system, it is
difficult to separate the 1.sup.st reaction zone, from the 2.sup.nd
reaction zone. But, if one were capable of immobilizing different
cognitive agents at different positions along the micro-channel,
the product from the 1.sup.st reaction could be used in the
2.sup.nd reaction and so on. To achieve such cascade reactions, it
is necessary to immobilize the cognitive agents in different
positions along the channel path in a discrete and well-defined
manner. At present, it is difficult to immobilize cognitive agents
in different positions along a micro-channel path having a uniform
channel width with accuracy better than 500 .mu.m. However, the use
of narrower channels would result less reagents used and also
provide more consistent results, reproducible reaction in much less
time.
[0009] Therefore, it is desirable to provide a microfluidic device
having different cognitive agents immobilized in discrete positions
along a micro-scale channel that would allow sequentially reaction
to be performed thereon. In some instances, it would be further
desirable to utilize a method of quantitative component analysis to
analyze the results.
SUMMARY OF THE INVENTION
[0010] A microfluidic device allowing for multiple discrete
reactions sites and allowing for sequential reactions and sample
analysis along with methods for device fabrication and use is
provided. The microfluidic device provides a micro-total analysis
system on a single substrate and has multiple reaction sites
allowing for cascade reactions and analysis on a single chip using
micro-quantities of sample and reagents. The microfluidic device
provides discrete sites for immobilization of cognitive agents
including enzymes, binding proteins, nucleic acids and the like as
well as methods for quantitative analysis. The invention also
provides methods for the fabrication of the device.
[0011] Accordingly, in one exemplary embodiment, the invention
provides a microfluidic device comprising a reaction channel
fabricated from a reaction layer on a substrate, including at least
two reaction channel networks, wherein the at least two reaction
channel networks are immobilized with at least one cognitive agent
allowing for a sequential reaction along the reaction channel. In
various exemplary embodiments, the cognitive agent is selected from
enzymes, antigens, antibodies, proteins, receptors, ligands,
nucleic acids or combinations thereof. In some exemplary
embodiments, the at least two reaction channel networks have
different cognitive agents immobilized thereon. In various other
embodiments according to the invention, the reaction layer is made
from glass, silicone, polydimethyl siloxane, other silicone resins,
polymethyl methacrylate (PMMA), synthetic acrylic, or
polycarbonate. In various exemplary embodiments, the substrate is
made from glass, silicone, polyvinyl chloride, polyester,
polyimide, acrylic resin, polycarbonate, cellulose acetate,
polyacrylic amide, or polyacrylonitrile. In various exemplary
embodiments, the substrate has a sensor measurement. In some
exemplary embodiments, the sensor is an electrode, an optical
sensor, a thermal sensor, or a chemosensor.
[0012] In still other exemplary embodiments, the invention includes
a method of fabrication of a microfluidic device having discrete
regions of cognitive agents immobilized thereon comprising the
steps of providing a substrate, attaching an immobilization layer
having one or more channel networks formed therein to the
substrate; flowing one or more solution of cognitive agents into
the channel networks; immobilizing one or more cognitive agents
onto the substrate surface at the one or more channel networks;
detaching the immobilization layer from the substrate; positioning
a reaction layer including a single reaction channel to the
substrate and wherein the single reaction channel encompasses the
one or more channel networks. In some exemplary embodiments, the
solution of cognitive agents is an enzyme solution, an antigen
solution, an antibody solution, a protein solution, a receptor
solution, a ligand solution, a nucleic acid solution or
combinations thereof. In still other exemplary embodiments, the
method includes a step of substrate surface pre-treatment, prior to
the step of attaching the immobilization layer. In some embodiments
according to the invention, the pre-treatment step is performed
prior to flowing the cognitive agent solution through the channel
network. In various exemplary embodiments the pre-treatment step
comprises deposition of a gold layer.
[0013] In yet another, exemplary embodiment, the invention
comprises a step of introducing a solution to be analyzed into the
reaction channel of the microfluidic device according to the
invention and allowing a component in the solution to flow through
the at least two reaction channel networks with the one or more
cognitive agents in sequence, and measuring an amount of reaction
product.
[0014] These and other features and advantages of the present
invention will be set forth or will become more fully apparent in
the description that follows and in the appended claims. The
features and advantages may be realized and obtained by means of
the instruments and combinations particularly pointed out in the
appended claims. Furthermore, the features and advantages of the
invention may be learned by the practice of the invention or will
be apparent from the description, as set forth hereinafter.
BRIEF DESCRIPTION OF THE FIGURES
[0015] Various exemplary embodiments of the compositions and
methods according to the invention will be described in detail,
with reference to the following figures wherein:
[0016] FIG. 1 illustrates a top view of the microfluidic device
according to this invention.
[0017] FIG. 2 illustrates a cross section along X-X' of the
microfluidic device in FIG. 1.
[0018] FIG. 3 illustrates a top view of the substrate coated with a
pre-treatment membrane for cognitive agent immobilization.
[0019] FIG. 4a illustrates a top view of the immobilization layer
for cognitive agent immobilization.
[0020] FIG. 4b illustrates a cross section along Y-Y' of the device
in FIG. 4(a).
[0021] FIG. 5 illustrates a top view of the substrate with the
immobilization layer including the network channels for the
cognitive agent immobilization attached thereto.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0022] A microfluidic device allowing for multiple discrete
reactions sites and allowing for sequential reactions and sample
analysis along with methods for device fabrication and use is
provided. The microfluidic device provides a micro-total analysis
system on a single substrate and has multiple reaction sites
allowing for cascade reactions and analysis on a single chip using
micro-quantities of sample and reagents. The microfluidic device
provides discrete sites for immobilization of cognitive agents
including enzymes, binding proteins, nucleic acids and the like as
well as methods for quantitative analysis. The invention also
provides methods for the fabrication of the device.
[0023] Accordingly, in one exemplary embodiment, the invention
provides a microfluidic device comprising a reaction channel
fabricated from a reaction layer on a substrate, including at least
two reaction channel networks, wherein the at least two reaction
channel networks are immobilized with at least one cognitive agent
allowing for a sequential reaction along the reaction channel. In
various exemplary embodiments, the cognitive agent is selected from
enzymes, antigens, antibodies, proteins, receptors, ligands,
nucleic acids or combinations thereof. In some exemplary
embodiments, the at least two reaction channel networks have
different cognitive agents immobilized thereon. In various other
embodiments according to the invention, the reaction layer is made
from glass, silicone, polydimethyl siloxane, other silicone resins,
polymethyl methacrylate (PMMA), synthetic acrylic, or polycarbonate
a polymer. In various exemplary embodiments, the polymer includes
plastics polydimethyl siloxane, polycarbonate, acrylic resin,
polyvinyl chloride, polyacrylic amide or polyacrylonitrile. In some
exemplary embodiments, the substrate is made from glass, silicone,
polyvinyl chloride, polyester, polyimide, acrylic resin,
polycarbonate, cellulose acetate, polyacrylic amide, or
polyacrylonitrile. In various exemplary embodiments, the substrate
has a sensor measurement. In some exemplary embodiments, the sensor
is an electrode, an optical sensor, a thermal sensor, or a
chemosensor.
[0024] In still other exemplary embodiments, the invention includes
a method of fabrication of a microfluidic device having discrete
regions of cognitive agents immobilized thereon comprising the
steps of providing a substrate, attaching an immobilization layer
having one or more channel networks formed therein to the
substrate; flowing one or more solution of cognitive agents into
the channel networks; immobilizing one or more cognitive agents
onto the substrate surface at the one or more channel networks;
detaching the immobilization layer from the substrate; positioning
a reaction layer including a single reaction channel to the
substrate and wherein the single reaction channel encompasses the
one or more channel networks. In some exemplary embodiments, the
solution of cognitive agents is an enzyme solution, an antigen
solution, an antibody solution, a protein solution, a receptor
solution, a ligand solution, a nucleic acid solution or
combinations thereof. In still other exemplary embodiments, the
method includes a step of substrate surface pre-treatment, prior to
the step of attaching the immobilization layer. In some embodiments
according to the invention, the pre-treatment step is performed
prior to flowing the cognitive agent solution through the channel
network. In various exemplary embodiments the pre-treatment step
comprises deposition of a gold layer.
[0025] In yet another, exemplary embodiment, the invention
comprises a step of introducing a solution to be analyzed into the
reaction channel of the microfluidic device according to the
invention and allowing a component in the solution to flow through
the at least two reaction channel networks with the one or more
cognitive agents in sequence, and measuring an amount of reaction
product.
[0026] Before the present methods are described, it is understood
that this invention is not limited to the particular methodology,
protocols, cell lines, and reagents described, as these may vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention which will be
limited only by the appended claims.
[0027] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and equivalents thereof known to those skilled in the art, and so
forth. As well, the terms "a" (or "an"), "one or more" and "at
least one" can be used interchangeably herein. It is also to be
noted that the terms "comprising", "including", and "having" can be
used interchangeably.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the
chemicals, cell lines, vectors, animals, instruments, statistical
analysis and methodologies which are reported in the publications
which might be used in connection with the invention. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0029] As used herein, the term "sensor" refers to any a device
which measures a physical quantity and converts it into a signal
which can be read by an observer or by an instrument. For example,
an "optical sensor" as used herein is a sensing device that
measures changes in ultraviolet light (UV), infrared light (IR),
visible light, fluorescence, luminescence and the like. Further,
those of skill in the art will recognize that such sensors are well
known and may measure both the amount of light absorbed by a
compound or analyte, the amount of light reflected or the amount of
light emitted. A temperature sensor can be as simple as a
thermometer which simply allows visualization of the contraction
and expansion of mercury in a closed tube or a thermocouple which
converts temperature to an output voltage that is read by a
voltmeter. Briefly, sensors include thermal sensors,
electromagnetic sensors, mechanical sensors, chemical sensors,
optical sensors, ionizing radiation sensors and acoustic sensors.
Examples of chemical sensors include Chemical proportion sensors:
oxygen sensors, ion-selective electrodes, redox electrodes, and
carbon monoxide detectors. Odor sensors: Tin-oxide gas sensors, and
Quartz Microbalance sensors. An ion selective electrode is a
transducer which converts the activity of a specific ion dissolved
in a solution into an electrical potential which can be measured by
a voltmeter or pH meter. Such ion selective electrodes may be as
simple as a glass pH electrode that responds to single charged ions
such as H.sup.+, Na.sup.+, and Ag.sup.+. However there are more
complex electrodes available such as those that measure divalent
metal ions, such as Pb2.sup.+ and those that measure redox
potential etc.
[0030] As used herein the term "cognitive agent" means any molecule
capable of specific attraction another molecule. Cognitive agents
can be antibodies or fragments of antibodies that have binding
domains such as Fab's or partial Fab's. Capture molecules can be
"ligands" or "receptors" either partial or complete and whether or
not they are associated with other molecules or compounds. As used
herein the term "ligand" refers a molecule that binds to another
molecule. Generally, a ligand may be soluble and its binding
partner is referred to as a "receptor". Unless otherwise defined,
receptors are generally regarded as being associated with
particular cells. Classically, receptors transduce a signal
conferred by a ligand with the binding of the ligand to the
receptor resulting in a signal to the cell and a physiologic
response. Receptors may be on the cell surface on within the cells.
As a rule, peptide or protein hormones are ligands that bind to
receptors at the cell surface while steroid hormones are ligands
that pass through the cell membrane and bind to receptors in the
cell nucleus. Receptors can become soluble and act as a ligand.
Thus, the terminology of "receptor" and "ligand" may become
interchangeable. Generally, a receptor is thought to be larger than
its cognate ligand.
[0031] As used herein, the term "substrate" is used to refer to the
support layer of the microfluidic device. As such, the substrate
may be any material as long as it is strong enough to support the
overlying reaction layer and fabrication steps and also does not
interfere with or react with the components of the analysis. Such
substrates can include, but are not limited to, glass, silicone,
and polymers including generally plastics such as, for example
polyvinyl chloride, polyester, polyimide, acrylic resin,
polycarbonate, cellulose acetate, polyacrylic amide, and
polyacrylonitrile.
[0032] A detailed description of the embodiment of invention is
shown as follows:
[0033] Referring now to FIGS. 1 and 2, one exemplary embodiment of
a microfluidic device according to the invention is shown. FIG. 1
is a top-plan view of a microfluidic device according to the
invention. FIG. 2 is a cross-section of the exemplary embodiment of
the invention shown in FIG. 1 taken along X-X'. As illustrated, the
device 1 comprises a substrate 2 which includes a reaction layer 3,
a reaction channel 4 formed thereon, an inlet 5, an outlet 6 and a
sensor 7 for measurement such as for example, an electrode, a
thermal sensor, an optical sensor or a chemosensor such as are well
known to those of skill in the art. FIG. 2, further illustrates a
pre-treatment membrane 8 for cognitive agent immobilization, and a
cognitive agent 9 such as, for example, an enzyme, an antibody, a
protein or a receptor, immobilized thereon. FIG. 2 further shows
that the reaction layer 3 has a reaction channel or groove 4 formed
therein, the reaction layer 3 overlaying the substrate 2. Within
the channel 4, is a pre-treatment membrane 8 which assists in the
immobilization of cognitive agents 9.
[0034] Also shown in FIG. 1, the reaction channel 4 includes an
inlet 5 and an outlet 6. Thus, generally a solution to be analyzed
is entered into the device at the inlet and exits at the outlet.
The channel can be divided in three reaction channel
network--sections 4A, 4B, and 4C which, in some exemplary
embodiments comprise multiple folded reaction channels. In the
exemplary embodiment illustrated, each reaction network section can
be immobilized with the different cognitive agents. An electrode 7,
according to one exemplary embodiment of a sensor is used for
measuring components or analytes in a sample and, in some
embodiments provided on the substrate.
[0035] As disclosed herein, the substrate 2 is made from any
material, which does not react with components in the sample
solution and is not detrimental to the cognitive agent/s
immobilized thereon. For example, the substrate can be made from
inorganic materials, e.g. glass and silicone; or organic materials
such as synthetic resins, e.g. polyvinyl chloride, polyester,
polyimide, acrylic resin, polycarbonate, cellulose acetate,
polyacrylic amide, polyacrylonitrile, etc. Because most sample
solutions use water as a solvent, it is desirable to use a
synthetic resin, which has hydrophilic property. However, in
embodiments in which a hydrophobic material is used as a substrate,
surface modification or surface coating with a hydrophilic material
may be required to improve the hydrophilicity. Generally, there is
no particular requirement for the substrate thickness. However, it
will be appreciated the substrate provides adequate strength and
flexibility so as not to allow the solution to leak from the
surrounding channel. Obviously, in those instances where the
solvent is not water, the substrate may be chosen accordingly and
surface modification may not be necessary
[0036] Materials useful for forming the reaction layer 3 will be
recognized by those of skill in the art. Generally, such materials
should have similar properties to the substrate, which are
hydrophilic materials, and also should not react with sample
constituents and/or products from the sample reaction, nor be
harmful to the cognitive agent. Further, according to one exemplary
embodiment, because the reaction layer is attached to the substrate
which has a cognitive agent immobilized thereon, the material of
the reaction layer 3 should adhere well to the substrate, at least
transiently during the time of the analysis. Further, the reaction
layer should be amenable to forming a small groove therein thus
providing the channel for the solution to flow through. However,
those of skill in the art will recognize that the channel can also
be formed in the substrate 2, layer. Examples of materials useful
for forming the reaction layer include polydimethyl siloxane
(PDMS), which is a silicone resin material. However, in this
invention, the materials for forming the channel layer can include
any materials with stated properties, such as PDMS and other
silicone resins. Alternatively, other materials, can be used so
long as they are capable of bonding to the substrate surface by any
acceptable bonding or adhesive material so long as there is no
leakage of the sample entered therein. Examples of material used as
the channel layer are inorganic materials, e.g. glass, silicone; or
well-known synthetic resins, e.g. poly methyl methacrylate (PMMA),
synthetic acrylic, polycarbonate, etc.
[0037] As shown in FIG. 1, the channel 4 has one or more network
portions (4A, 4B, 4C) each have a width 4D. The network sections
provide increased length and therefore increased surface area and
reaction time for the sample in solution to react with the
cognitive agent. FIG. 1, illustrates one exemplary embodiment
wherein the network sections 4A, 4B, or 4C of the reaction channel
4 increases the surface area on which the cognitive agent is
immobilized and contacts the sample solution. However, it should be
appreciated that if less reaction time is needed the network
portion can be shortened or omitted altogether, i.e. the network
portion can have a short, straight line profile. Further, while as
illustrated in the exemplary embodiment shown in FIG. 1, the three
network sections 4A, 4B and 4C all have the same length for the
reaction channel network on which cognitive agents are immobilized,
those of skill in the art will appreciate that the length of
channel for each portion may be different according to the
component to be analyzed and measured or the cognitive agent to be
immobilized therein. Thus, the length of the channel for each
cognitive agent or reaction mixture used in each section can differ
and may be optimized as needed.
[0038] In various exemplary embodiments according to the invention,
several cognitive agents can be immobilized on the substrate in
different positions along the channel path as needed. In FIG. 1,
the channel provides reaction network sections 4A, 4B and 4C
allowing for the immobilization of three different cognitive agents
along the reaction network section of the reaction channel. In
practice, the number of cognitive agents may only be one or greater
than three. The number and type of cognitive agents can be changed
depending on the solution/compounds to be analyzed. The cognitive
agent may be an enzyme which functions in a catalytic reaction, but
cognitive agents are not limited to enzymes. For example, other
materials which specifically recognize the measured chemical
substance can be antigens, antibodies, proteins, receptors, or
nucleic acids including DNA, RNA and PNA. Further, examples of
enzymes used as cognitive agents include but are not limited to,
invertase, mutarotase, glucose oxidase, alcohol oxidase, lacto
oxidase, amino acid oxidase, catalase, uricase, cholesterol
oxidase, hexokinase, urease, trypsin, etc. Of course other enzymes
can also be used with the microfluidic device in accordance with
the invention. Since various cognitive agents are immobilized on
this device, the types and sequences of cognitive agents can also
be altered or optimized for analysis of each analyzed component.
For example, when the invention is used to analyze the sweetness of
fruits or sugar cane, one suitable fixing sequence useful is
invertase in section A, mutarotase in section B, and glucose
oxidase in section C.
[0039] As mentioned above, in some instances it may be difficult to
immobilize the cognitive agent on the substrate directly. Hence,
prior to the cognitive agent immobilization, surface modification
of the substrate may be made by providing a pre-treatment or a
fixation of an immobilization reagent. In these instances a
pre-treatment layer 8 may be applied. For example, any conventional
pre-treatment method generally used for cognitive agent
immobilization on a substrate of may be employed. Examples of
pre-treatment methods for immobilization of the enzyme include
vacuum evaporation of silver, gold, or platinum or flowing a
solution of an osmium polymer. However, it should be appreciated
that the method of pre-treatment in this invention is not limited
to the examples provided herein. Further, in the case of gold
coating by evaporation, it may be necessary to apply a chromium or
titanium layer before applying the gold layer to improve adhesion
on the glass substrate. In case of the osmium polymer, an osmium
polymer solution can be used. Of course, other materials can be
used as a pre-treatment layer too. Further, the chemical substance
for the immobilization may be flowed concurrently with the solution
of cognitive agent during the immobilization. One example of a
chemical substance useful for enzyme immobilization is
glutaraldehyde. In such cases, the coating of pre-treatment layer 8
is not required for the cognitive agent to be immobilized directly
on the substrate. Thus, in instances, the cognitive agent can
immobilized directly on the substrate, and the pre-treatment layer
is not necessary.
[0040] Fabrication of the Microfluidic Device
[0041] In another embodiment, the invention provides a method of
fabricating a microfluidic device according to the invention. FIG.
3 illustrates a top view of the substrate coated with a
pre-treatment layer prior to cognitive agent immobilization. FIG.
4(a) illustrates a top-plan view of the immobilization layer 11
used for the cognitive agent immobilization. FIG. 4(b) illustrates
a cross-section along Y-Y' shown in FIG. 4(a). FIG. 5 is a top-plan
view of the substrate with the immobilization layer 11 attached for
cognitive agent immobilization.
[0042] As shown in FIG. 3, pre-treatment membrane 8 includes
channel network sections 8A, 8B, and 8C. It should be noted that in
some exemplary embodiments, the pre-treatment channel width may be
smaller (8D not shown) than the width 4D of the reaction channel.
The pre-treatment network sections 8A, 8B and 8C are formed as a
line pattern at three discrete locations corresponding to the
reaction channel path 4 on the substrate 2. In FIG. 3, the
pre-treatment networks are formed at three discrete locations.
However, as mentioned above, if only two cognitive agents are
needed, then only two pre-treatment network sections at two
locations will be necessary etc. But if more than four cognitive
agents are needed, more than four pre-treatment networks will be
used. As discussed above, if the cognitive agent can be immobilized
directly on the substrate, it is not necessary to modify the
substrate surface using a pre-treatment layer. However, in those
instances when the cognitive agent is an enzyme, antibody, nucleic
acid, binding protein or receptor, etc., it is difficult to
immobilize the agent directly on the substrate. In addition, it
should be appreciated that each network section may have different
cognitive agents immobilized thereon and that therefore, each
pre-treatment network section may be optimized for the specific
cognitive agent to be immobilized thereon. In addition, in most
cases the substrate pre-treatment will be performed prior to
immobilization of the cognitive agent onto the substrate. Of
course, any known pre-treatment method useful for cognitive agent
immobilization can be used.
[0043] For example, a method for the deposition of a gold layer
using evaporation is one example of a pre-treatment method useful
for cognitive agent immobilization. One method of forming a gold
evaporated layer as a fine line pattern onto the substrate can be
conducted by coating a photoresist or attaching a photosensitive
dry film onto the substrate, exposing light through a mask with the
line pattern, and developing the film to form a photoresist layer
with a network channel line on the substrate, then evaporating the
gold layer, and removing the photoresist to form the desired
pattern for the reaction channel network. An alternative method is
evaporating the gold on an entire surface of the substrate, coating
the photoresist onto the gold layer, exposing light through a mask
with the network pattern, and developing the photoresist to form
with a network pattern on the substrate, then etching the gold that
does not have the photoresist. Another method is a direct
evaporation of the gold through a stencil mask having apertures
facilitating evaporation where the stencil is attached to the
substrate. In the case of forming a gold evaporation layer as the
pre-treatment layer, it may be formed, if necessary, simultaneously
with the forming of the sensor 7 which is used as a measuring
device. The sensor used as the measuring device may be formed in
advance on the substrate. Furthermore, the alignment marker 10 can
be prepared in the same step, and certainly the alignment marker 10
can be prepared in advance on the substrate.
[0044] Next, as shown in FIG. 4, to immobilize the cognitive agent,
such as, for example, an enzyme, an immobilization layer 11 is used
for cognitive agent immobilization. The immobilization layer 11 has
networks or grooves 12A, 12B, 12C corresponding to the reaction
channel network 4A, 4B and 4C and pre-treatment channel network 8A,
8B and 8C and is attached onto the substrate so as to align with
the pre-treatment networks or grooves formed by the pre-treatment
layer. The immobilization layer 11 contains a channel networks as
necessary, for cognitive agent immobilization and is attached on
the substrate 2 as shown in FIGS. 4 and 5. As with the
pre-treatment layer, the channel networks have a width 12D that, in
exemplary embodiments is about the same or less than the width 4D
of the reaction channel networks 4A, 4B and 4C. The immobilization
layer 11 is generally detachably fixed to the substrate to prevent
leakage of the cognitive agent solution from the immobilization
channel network during flow of the cognitive agent solution through
the channel. Hence, the immobilization layer 11 for the cognitive
agent immobilization should have excellent adhesion but also be
easy to detach. In most cases adhesives or coupling agents are not
easily detachable.
[0045] Therefore, some exemplary embodiments, the immobilization
layer 11 for the cognitive agent immobilization is not attached to
the substrate use adhesives or bonding agents. However, it should
be appreciated that in those cases where adhesives or bonding
agents used are detachable, and also do not allow leakage from the
channel, their use during cognitive agent immobilization is
contemplated by the invention. In these cases, it is possible to
use an adhesive that allows the immobilization layer to be detached
from the substrate. Hence, it is possible to use an inorganic
material, e.g. glass and silicon; general casting materials, e.g.
polycarbonate, acrylic resin as the substrate. Polydimethyl
siloxane (PDMS) is an example of material having excellent adhesion
and is easy to remove from the above mentioned substrates. One
method of forming a channel or groove in polydimethyl siloxane
useful for flowing a cognitive agent solution along the
pre-treatment network is to first, prepare the photoresist layer on
the substrate, e.g. glass, with the same thickness as the
pre-treatment network, exposing UV light through a patterned mask
having the network pattern of the pre-treatment layer, developing
the photoresist layer having the same or slightly greater width
than the pre-treatment layer. The result is a photoresist layer
that is used as a mould. Next, a mixture of polydimethyl siloxane
solution and hardener is poured into the mould and cured providing
the polydimethyl siloxane layer with the multiple network channels
comprising the network pattern of the pre-treatment layer. The
obtained immobilization layer is removed from the mould, and then
apertures comprising an inlet 13 and outlet 14 are created using a
suitable device such as a needle, scalpel etc. providing an
immobilization channel network 12A, 12B and 12C with width 12D in
the immobilization layer 11 suitable for cognitive agent 9
immobilization. In some exemplary embodiments, an alignment marker
15 is also formed in the immobilization layer 11 to help align the
position of the immobilization layer 11 on the substrate 2. Hence,
it is suitable to prepare the alignment marker on the mould, for
copying and forming a replica of the reaction layer and providing a
channel for cognitive agent immobilization.
[0046] As described herein a solution of different cognitive agents
can flow from the inlet 13 to the outlet 14 of each immobilization
network section 12A, 12B, 12C formed by the immobilization layer 11
and allowing for cognitive agent 9 immobilization for each network
section 12A, 12B or 12C or other region of the channel 4 as
desired. In some exemplary embodiments, to facilitate the solution
flow, the outlet pressure is reduced, such as, for example, by use
of a vacuum or suction. In addition, decreasing the outlet
pressure, can be useful in preventing the immobilization layer 11
from detaching from the substrate 2 during cognitive agent
immobilization. For example, a tube can be used as a connection to
prevent the solution containing the cognitive agent from leaking at
the inlet 13 and outlet 14, and to facilitate the flow of the
desired cognitive agent for each of the immobilization networks of
the channel. Using the tube at the outlet 14 also facilitates a
reduction of outlet pressure and ease of collection of used
solution. Further, the inlet and outlet positions can be exchanged
if it is helpful to apply the cognitive agent solution from either
or both ends of the immobilization network. For example, in the
case of using enzyme mutarotase as a cognitive agent, a commercial
mutarotase solution is passed into the inlet, then the pressure at
the outlet is reduced, a hazy solution can be observed in the
interior of channel within about a minute. Furthermore, in cases
where the interior of the channel is pre-treated to be hydrophilic
to assist the solution flow, the solution will flow easily
providing contact with the channel within about a minute. The
effluent solution is then collected and the interior of the channel
is allowed to dry. After detaching the immobilization layer 11 used
for cognitive agent immobilization, the substrate 2 immobilized
with the cognitive agent/s is obtained.
[0047] The reaction layer 3 is then attached onto the substrate 2
immobilized with the cognitive agent thereby providing the
microfluidic device as shown in FIG. 1. Polydimethyl siloxane
(PDMS) is one of suitable materials for forming reaction layer 3,
however; other materials can be applied too. In case of using
polydimethyl siloxane as the material for the reaction layer 3, it
can also be prepared in the same method as the one for forming the
immobilization layer 11. In some instances, the channel width 4D of
the reaction channel 4 should be about the same or slightly greater
than the channel width 12D of immobilization channel networks 12A,
12B, 12C. In these cases, the reaction channel 4 or groove width of
the reaction layer 3 should be larger than the channel width of the
immobilization layer channel 12A.
[0048] Additionally, the method of forming the channels or grooves
12A, 12B, 12C of the immobilization layer 11 or the channels or
grooves of the reaction network sections 4A, 4B and 4C of the
reaction layer 3 are not limited to aforementioned ones. Other
methods compatible with the material used can be exploited, for
example, such methods include but are not limited to forming the
channels by laser or photoresist.
[0049] The microfluidic device according to this invention can be
used for component analysis or inspection various solutions. An
example of component analysis is a measurement of sucrose
concentration in fruits or sugar cane. In this case, the invention
can be used by the immobilization of invertase, mutarotase, and
glucose oxidase enzymes at the reaction channel networks in
sections 4A, 4B and 4C respectively. A juice squeezed from sugar
cane is dropped at a volume of as low as about 2-5 .mu.l t into
inlet 5 of the microfluidic device 1, reducing the outlet 6
pressure and facilitating the solution flow into the reaction
channel 4. When the solution flows along network section 4A, the
sucrose is converted to .alpha.-D glucose and fructose by a
hydrolysis reaction catalyzed by invertase; in section 4B,
.alpha.-D glucose is converted to .beta.-D glucose catalyzed by
mutarotase; and then into section 4C, where .beta.-D glucose
converting to hydrogen peroxide and glucuronic acid catalyzed by
glucose oxidase. Then, at the sensor, an electrode 7 measures an
amount of hydrogen peroxide as to determine the sucrose
concentration in of the plant extract.
[0050] Of course, those of skill in the art will recognize that
other methods for the measurement of analyte concentration or other
compounds in solution, can be used besides measurement of
electrical conductivity. For example, such methods known in the art
include changes in optical parameters, thermal parameters and
frequency changes of piezoelectric devices, etc. Specific methods
of measurement include, but are not limited to the measurement of
optical luminescence including chemiluminescence of an
enzyme-immunoassay, the measurement of light absorption and emitted
fluorescence to name a few.
EXAMPLES
[0051] Various exemplary embodiments of the devices, compounds and
methods as generally described above according to this invention,
will be understood more readily by reference to the following
examples, which are provided by way of illustration and are not
intended to be limiting of the invention in any fashion.
Example 1
Pretreatment of the Substrate
[0052] As described above, a first step in the fabrication of the
device is the pretreatment of the substrate surface. Referring now
to FIG. 3, three sets of evaporated gold layers are shown at 8A, 8B
and 8C. In the embodiments shown, each of the gold layers has a
four-part network portion having 100 .mu.m width which were
fabricated on a glass substrate using the photolithography
technique described above.
[0053] Briefly, the substrate is prepared by cleaning with a
solvent such as acetone or methylene chloride and allowed to dry.
Substrate surface modification with a pre-treatment layer was made
by using a SU-8 thick film photoresist (MicroChem Corp., Newton,
Mass.) coated by spinning on the glass substrate. The dried layer
with 40 .mu.m thickness was pre-heated at 90.degree. C. for one
hour, then exposed to ultraviolet light through a mask having the
three sets of channel networks 8A, 8B and 8C (as illustrated in the
exemplary embodiment of FIG. 3) with 150 .mu.m width, followed by
hardening the exposed portion by curing at 90.degree. C. for one
hour. The photoresist was developed with PGMEA (propylene glycol
monomethyl ether acetate), the uncured portion is removed with
water. The cured portion was dried to form an embossed SU-8 layer
on the glass substrate (embossed glass substrate). This embossed
portion was used as a mould for forming the immobilization layer of
the enzyme immobilization channel.
[0054] The pattern of the evaporated gold layers was formed to
correspond to the reaction network channel path of the final
fabricated microfluidic device. Moreover, the three sets of the
network sections of evaporated gold layer 8A, 8B and 8C are
immobilized with various enzymes as described in the following
EXAMPLES. In this exemplary embodiment, an electrode made from
platinum 7 is formed on the substrate at the time of the
pre-treatment to analyze a desired component of a solution. Those
of skill in the art will appreciate that the sensor can be mounted
to the substrate at any convenient time.
Example 2
Fabrication of the Immobilization Layer
[0055] As described above, surface treatment of the substrate
results in an SU-embossed glass substrate. This embossed portion
was used as a mould for forming the immobilization layer of the
enzyme immobilization channel.
[0056] The PDMS (polydimethyl siloxane) layer with three sets of
network portions was obtained by pouring a solution having a
mixture of 10 parts of PDMS (polydimethyl siloxane) solution
(Sylguard.TM. 184, Dow Corning Corp.) and 1 part of hardening
compound onto the embossed glass substrate mould described in
Example 1 to a thickness 1-2 mm. The air was evacuated using a
vacuum system and the PDMS layer allowed to harden for one hour at
70.degree. C. The hardened film was then gradually removed from the
embossed glass substrate. The cured PDMS layer having a total
thickness of about 230 .mu.m then forms a image of the embossed
substrate. The channel networks 12A, 12B and 12C had a height or
diameter of approximately and 30-40 .mu.m channel and a width 12D
about the same or slightly larger than the width of the embossed
substrate used as the mould.
[0057] Apertures were opened at each inlet 13 and outlet 14 of the
three fabricated immobilization channel networks 12A, 12B and 12C.
The obtained immobilization layer 11 was attached on the glass
substrate such that the pre-treatment channel networks 8A, 8B and
8C having the evaporated gold layer aligns with the immobilization
channel networks 12A, 12B and 12C thereby forming closed channels.
To facilitate an accurate position alignment, the alignment markers
15 were prepared on the glass substrate 10 and the embossed glass
substrate 10. As will be recognized by those of skill in the art,
use of the alignment markers on the embossed glass substrate aids
in the ease of copying and forming the replica of the PDMS
layer.
Example 3
Immobilization of the Enzyme
[0058] After attaching the immobilization layer for the enzyme
immobilization, each of the different enzyme solutions was injected
at the aperture of the inlet of each channel network 12A, 12B, 12C
in the order desired. The pressure at the outlet was reduced by
applying a vacuum or using a syringe (for example) or aspirator to
introduce the enzyme solution to the channel providing enzyme
immobilization on the evaporated gold layer. In addition, by
reducing the pressure, the grooved PDMS (polydimethyl siloxane)
immobilization layer 11 has greater adherence to the glass
substrate, preventing a leak of enzyme solution from the channel,
and allowing better solution flow in the channel. Thus, in one
exemplary embodiment, invertase solution was allowed to flow into
channel the first channel 12A, mutarotase solution was flowed into
the second channel, 12B and glucose oxidase solution was flowed
into the third channel 12C allowing each enzyme to be immobilized
on the evaporated gold layer. In this embodiment, the concentration
of each enzyme is approximately 3 mol/l. However, it should be
appreciated that for different cognitive agents, different
substrates or different assays, etc. different concentrations of
cognitive agents may be desirable. Following cognitive agent
immobilization, the grooved PDMS (polydimethyl siloxane) layer is
removed from the glass substrate. The substrate is then washed with
distilled water to remove excess enzyme to provide the enzyme
immobilized (cognitive agent) substrate.
[0059] Fabrication of the Microfluidic Device
Example 4
Fabrication of the Reaction Channel Layer
[0060] A glass substrate with an embossed portion is prepared by
the same method described in Examples 1 and 2 to form a reaction
channel layer having corresponding network sections as the
pre-treatment and immobilization layers. In this exemplary
embodiment the reaction layer is formed of PDMS (polydimethyl
siloxane). This channel bearing layer becomes the reaction layer.
The reaction channel, is formed using the embossed mould as
described in Examples 1 and 2. In this particular Example, the
dimension of the reaction channel is 250 .mu.m width and 70 .mu.m
depth (height) although those of skill in the art will recognize
that the dimensions can be optimized for each desired use. As
illustrated in FIGS. 1 and 4, the difference between the reaction
layer and the immobilization layer used for enzyme immobilization
is the reaction layer provides a continuous channel connecting all
of the reaction networks sections 4A, 4B and 4C as shown in FIG.
1.
[0061] Following the opening of an aperture at the inlet and outlet
of the reaction layer channel 4, the reaction layer 3 was attached
onto the glass substrate by aligning each of the position markers
on the reaction layer and the substrate. This forms the
microfluidic device. As with the immobilization layer, alignment
markers were formed on the grooved layer, and then the alignment
markers were used to align the immobilized enzyme substrate with
the reaction layer.
Example 5
Sucrose Concentration Measurement
[0062] The microfluidic device of this invention was used to
measure the sucrose concentration in sugar cane. The procedure
starts from using squeezed sugar cane juice as a solution sample;
dropping the solution sample (about 2-5 .mu.l/drop) in the inlet of
the microfluidic device where the solution sample flows into the
channel by reducing the outlet pressure. An amount of hydrogen
peroxide as a final reaction product was measured using a hydrogen
peroxide sensitive electrode and thereby determining the sucrose
concentration in the sugar cane.
[0063] While this invention has been described in conjunction with
the various exemplary embodiments outlined above, various
alternatives, modifications, variations, improvements and/or
substantial equivalents, whether known or that are or may be
presently unforeseen, may become apparent to those having at least
ordinary skill in the art. accordingly, the exemplary embodiments
according to this invention, as set forth above, are intended to be
illustrative not limiting. various changes may be made without
departing from the spirit and scope of the invention. therefore,
the invention is intended to embrace all known or later-developed
alternatives, modifications, variations, improvements and/or
substantial equivalents of these exemplary embodiments.
* * * * *