U.S. patent application number 11/213362 was filed with the patent office on 2006-01-05 for microfludic devices with new inner surfaces.
Invention is credited to Helene Derand, Frida Jernstrom.
Application Number | 20060002825 11/213362 |
Document ID | / |
Family ID | 29423542 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060002825 |
Kind Code |
A1 |
Derand; Helene ; et
al. |
January 5, 2006 |
Microfludic devices with new inner surfaces
Abstract
A microfluidic disc having one or more enclosed microchannel
structures, and the microchannel structures are intended to be used
for transport of transporting liquids. The device is characterized
in that at least a part of the inner walls of each of one or more
microchannel structures are treated with a gas plasma having one or
more organic precursor compounds.
Inventors: |
Derand; Helene; (Taby,
SE) ; Jernstrom; Frida; (Uppsala, SE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
29423542 |
Appl. No.: |
11/213362 |
Filed: |
August 26, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10409820 |
Apr 9, 2003 |
6955738 |
|
|
11213362 |
Aug 26, 2005 |
|
|
|
60371080 |
Apr 9, 2002 |
|
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|
Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01J 2219/00833
20130101; B01L 3/502707 20130101; B01J 19/0093 20130101; B01J
2219/00831 20130101; B01J 2219/0086 20130101; B01L 2300/165
20130101; B01L 2200/12 20130101; B01J 2219/00828 20130101; B01J
2219/00837 20130101 |
Class at
Publication: |
422/100 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Claims
1-17. (canceled)
18. A microfluidic device comprising one or more enclosed
microchannel structures, each of which comprises a section that is
defined between two essentially planar substrates wherein one
surface in at least one substrate comprises microstructures in the
form of grooves or projections that match each other so that they
together define said section one or more microchannel structures
when the two surfaces are apposed in the microfluidic device, said
microchannel structures being intended for transporting a liquid,
wherein a) a non-wettable and a wettable coat are present
edge-to-edge in at least one of the microchannel structures, b) at
least one of the coats has been introduced by the use of a gas
plasma comprising an organic precursor compound, and c) the
non-wettable coat defines a valve function or a non-wicking
function or a vent function to ambient atmosphere.
19. The microfluidic device of claim 18, wherein said section
comprises a complete microchannel structure.
20. The microfluidic device of claim 18, wherein wettable coat has
a water contact angle that is .ltoreq.40.degree..
21. The microfluidic device of claim 18, wherein the non-wettable
coat has a water contact angle that is .gtoreq.100.degree..
22. The microfluidic device of claim 18, wherein the coat is
anti-fouling with respect to bovine serum albumin with a decrease
ratio that is .ltoreq.0.50.
23. The microfluidic device of claim 13, wherein the wettable coat
has a water contact angle that is .ltoreq.60.degree..
24. The microfluidic device of claim 13, wherein the wettable coat
is anti-fouling with respect to bovine serum albumin.with a
decrease ratio .ltoreq.0.75.
25. The microfluidic device of claim 13, wherein the difference in
wettability between the coats .gtoreq.50.degree..
26. The microfluidic device of claim 13, wherein the non-wettable
coat has a water contact angle that is .gtoreq.90.degree..
Description
[0001] This Application is a divisional application of U.S.
application Ser. No. 10/409,820 filed Apr. 9, 2003, which claims
priority to U.S. Provisional Application No. 60/371,080 filed on
Apr. 9, 2002, which is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] I. Field of Invention
[0003] The present invention concerns a microfluidic device that
has inner surfaces with chemical surface characteristics that have
been introduced using gas plasmas having one or more organic
precursor compounds.
[0004] II. Related Art
[0005] A number of different techniques for modifying substrate
surfaces are well known. One common method is to subject a
substrate surface, for instance made in plastics, to various forms
of plasma treatment (Chan et al., Surface Science Reports 24 (1996)
1-54; and Garbassi et al., Polymer Surfaces--From Physics to
Technology, John Wiley (1998) 238-241). This is done in a plasma
reactor, which is a vacuum vessel containing a gas at low pressure
(typically 10 to 1000 mTorr). When a high frequency electric
excitation field is applied over the reactor, a plasma (also called
glow discharge) is formed, containing reactive species like ions,
free radicals and vacuum-UV photons. These species may react with
other species and/or with the surface and cause a chemical
modification of the substrate surface with properties depending on
the nature of the gas and on the plasma parameters. Gases like
oxygen and argon are typically used for hydrophilization and/or
adhesion improvement on plastics, while vapors of organic precursor
compounds can be used to apply thin coatings for a number of
different purposes (Yasuda, Plasma Polymerization, Academic Press
1985).
[0006] Previously, vapors of organic precursor compounds have been
used to produce surfaces that are wettable by aqueous liquids but
the hydrophilicity has been moderate and not utilized to facilitate
transport of aqueous liquids, in microchannels. In some cases, the
primary goal has been to introduce coats that have a low
non-specific adsorption, for instance of proteins and/or other
biopolymers and/or other bioorganic molecules. See for instance
discussions U.S. Pat. No. 5,153,072 (Ratner et al.), U.S. Pat. No.
5,002,794 (Ratner et al.), U.S. Pat. No. 6,329,024 (Timmons et
al.), U.S. Pat. No. 5,876,753 (Timmons et al.), EP 896035 (Timmons
et al.). Strictly hydrophobic surfaces have also been produced. See
for instance U.S. Pat. No. 5,171,267 (Ratner et al.).
[0007] WO 0056808 (Ocklind, Larsson and Derand, Gyros AB) describes
microfluidic devices comprising hydrophilic microchannel structures
defined between two essentially planar substrates that are apposed.
Before being apposed the surface of at least one of the substrates
has been hydrophilized in gas plasma, which comprises a
non-polymerizable gas. The surfaces obtained are hydrophilic and
can be coated subsequent to gas plasma treatment in order to
introduce further functionalities.
[0008] WO 9958245 (Larsson et aL.) and WO 97 21090 (Mian et al.)
are examples of publications that in general terms suggest
microfluidic devices in which the inner surfaces of the
microchannel structures have been made hydrophilic by gas plasma
treatment, coating of hydrophobic surfaces with hydrophilic
polymer, etc.
BRIEF SUMMARY OF THE INVENTION
[0009] A first object of the invention is to present a surface
modification method. Accordingly, the first aspect of the invention
is a method for the manufacture of a microfluidic device to
introduce a predetermined degree of wettability (hydrophilicity
and/or hydrophobicity) on an inner surface of said microchannel
structures. The method is characterized in comprising the steps of:
(i) providing two essentially planar substrates (I and II); (ii)
placing either one or both of the substrates in a gas plasma
reactor, and creating within said plasma reactor a gas plasma
containing an organic precursor compound, said organic precursor
compound and the conditions in the reactor being selected such that
a coat of the predetermined degree of wettability is formed on a
selected part of the surface of the substrate/substrates; (iii)
removing the substrate/substrates from the plasma reactor; (iv)
adhering the surface of substrate I to the surface of substrate II
so that at least an enclosed section of each of microchannel
structures are formed between the two surfaces; and (v) optionally
joining further planar substrates to complete the microchannel
structures. In the simplest variant complete enclosed microchannel
structures are defined between substrate I and II.
[0010] A second object of the invention is to provide new surface
modifications that have a sufficient wettability combined with a
sufficiently low non-specific adsorption for a reliable and
reproducible mass transport and processing of reagents by a liquid
flow through a microchannel structure. This object, thus, aims at
optimizing wettability and anti-fouling in relation to each
other.
[0011] A third object is directed to a microchannel structure that
is present in a microfluidic device and comprises two or more
different functional parts, at least one of which comprises inner
surfaces of a sufficient hydrophilicity for a liquid aliquot to
penetrate completely the functional part by capillary force once
having wetted the entrance of the part. The demand for a
sufficiently low non-specific adsorption remains.
[0012] A fourth object is to accomplish a microfluidic device
comprising coats that can be stored for .gtoreq.7 days, such as
.gtoreq.30 days, while retaining the intended functionality of the
surface, i.e., the surface may still be used for the intended
purpose (= is essentially unchanged).
[0013] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawings.
[0015] FIG. 1 shows Total Internal Reflection Fluorescence (TIRF)
with Fluorescence-5-isothiocyanate-bovine serum albumin (FITC-BSA)
on untreated Polycarbonate (PC) (squares), and on PC treated with
diglyme (24 W) in the plasma reactor (circles). Protein solution
(400 ppm) enters the flow cell (filled arrow) and is replaced by
PBS buffer (dashed arrow).
[0016] FIG. 2 shows TIRF with FITC-BSA on PC treated with diglyme
(24 W), and allylic alcohol (12 W) in the plasma reactor. Protein
solution (400 ppm) enters the flow cell (filled arrow) and is
replaced by PBS buffer (dashed arrow).
[0017] FIG. 3 shows TIRF with FITC-BSA on PC treated with ethylene
glycol vinyl ether-plasma (24 W) in the Gyros reactor. Protein
solution (400 ppm) enters the flow cell (filled arrow) and is
replaced by PBS buffer (dashed arrow).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0018] As used herein, the use of the word "a" or "an" when used in
conjunction with the term "comprisng" in the sentences and/or the
specification may mean "one," but it is also consistent with the
meaning of "one or more," "at least one," and "one or more than
one."
[0019] As used herein, the term a "microfluidic device" typically
comprises one, two or more microchannel structures, which are
defined between two essentially planar and parallel substrates that
are apposed to each other. Thus, either one or both of the two
substrate surfaces that define the microchannel structures comprise
microstructures in the form of grooves and/or projections such that
the microchannel structures can be formed when the two surfaces are
apposed. The device is microfluidic in the sense that one or more
liquid aliquots can be transported between different functional
parts of the individual microchannel structures in order to process
the aliquots. The liquid aliquots are in the .mu.-range with
preference for the nl-range. The purpose of the transport is to
carry out predetermined process protocols, for instance for
assaying one or more constituents of a sample aliquot or to
synthesize an organic or an inorganic compound. The liquid aliquots
are typically aqueous, i.e., based on water and mixtures between
water and water-miscible organic solvents.
[0020] As used herein, the term "microchannel structure" relates to
the structure that is defined between the surfaces of two or more
planar substrates that are layered on top of each other. If
different sections of a microchannel structure are defined between
different pairs of planar substrates, there typically are holes in
the substrates so that the sections are in communication with each
other. Either one or both of the surfaces that are to define a
section of a microchannel structure comprises microstructures such
that the desired section of a microchannel structure will be formed
when the surfaces are joined together. Separate microchannel
structures may be defined between additional essentially planar
substrates.
[0021] As used herein, the term "plurality" means two, three, four,
five or more microchannel structures. Preferably "plurality" means
that the number of microchannel structures on the microfluidic
device is .gtoreq.10, such as .gtoreq.25 or .gtoreq.90 or
.gtoreq.180 or .gtoreq.270 or .gtoreq.360.
[0022] As used herein, the terms "microchannel", "microconduit",
etc., contemplate that a channel structure comprises one or more
cavities and/or channels/conduits that have a cross-sectional
dimension that is .ltoreq.10.sup.3 .mu.m, preferably
.ltoreq.0.5.times.10.sup.3 .mu.m or .ltoreq.10.sup.2 .mu.m. The
lower limit for cross sectional dimensions is typically
significantly larger than the size of the largest constituent of a
liquid that is to pass through a microchannel of the innovative
device. The volumes of microcavities/microchambers are typically in
the nl-range, i.e., .ltoreq.5000nl, such as .ltoreq.1000 nl or
.ltoreq.500 nl or .ltoreq.100 nl or .ltoreq.50 nl or .ltoreq.25 nl.
This does not exclude larger chambers/cavities, for instance in the
intervals 1-1000 .mu.l, such as 1-100 .mu.l or 1-10 .mu.l which
typically are directly connected to inlet ports and intended for
application of sample and/or washing liquids.
[0023] As used herein, the term "microformat" means that one, two,
three or more liquid aliquots that are transported within the
device are within the intervals specified for the micro
chambers/micro cavities.
[0024] As used herein, the terms "non-specific adsorption" and
"fouling", which are interchangeable, mean undesired adsorption of
compounds to inner walls of the microchannel structures. The terms
may also include inactivation of bioactive compounds by the walls,
for instance denaturation of proteins. The compounds are present in
the liquid used and are primarily reagents. For aqueous liquids the
reagents may be proteins and/or other biopolymers and/or other
bioorganic and synthetic organic compounds.
[0025] As used herein the term "anti-fouling" refers to reduction
in non-specific adsorption (undesired adsorption) of reagents
compared to a reference surface that in the context of the
invention is the surface before being treated in a gas plasma
comprising an organic precursor.
[0026] As used herein, the term "analytes" are also included in the
term "reagent".
[0027] As used herein, the term "wettable" refers to a surface
having a water contact angle that is .ltoreq.90.degree.
(hydrophilic surface).
[0028] As used herein, the term "non-wettable" refers to a surface
that has a water contact angle .gtoreq.90.degree. (hydrophobic
surface).
[0029] As used herein, the term "wettability" refers to the "degree
of wettability" and may include a highly wettable surface, a highly
non-wettable surface or any variation therebetween.
[0030] As used herein, the term "predetermined degree of
wettability" refers to the wettability of a coat that is important
for the function of a microchannel structure. The predetermined
degree of wettability may imply a wettable surface that will allow
for capillary flow, a non-wettable surface that will act as a
valve, a vent, an anti-wicking means, etc. Typically, the
expression means that the wettability of the coat is different from
the wettability of the surface without the coat.
[0031] As used herein, the term "organic precursor" refers to an
organic compound that forms reactive species in a gas plasma.
[0032] As used herein, the term "masking" refers to placing a
removable protective coat/mask on surface parts that are not to be
coated by the subsequently applied coating method.
II. Method of Manufacture
[0033] During the last decade sophisticated microfluidic devices
have appeared with the goal to fully integrate complete process
protocols in miniaturized form. This means integration of all steps
of a protocol from sample preparation to recording of the results
in one and the same microchannel structure. Thus, is advantageous
if the same kind of equipment is used to produce surfaces
corresponding to a spectra of chemical surface characteristics, for
instance from extremely hydrophobic to extremely hydrophilic
surfaces, and preferably with anti-fouling properties.
[0034] The present inventors have recognized that the
above-mentioned objects can be achieved by treating the channel
surfaces with gas plasma, which comprises one or more organic
precursor compounds in gas form. The obtained surface
characteristics (for instance hydrophilicity or hydrophobicity) is
determined by the selection of the organic precursor compound
and/or the process parameters applied to create the gas plasma as
outlined below.
[0035] Accordingly the first aspect of the invention is a method
for the manufacture of a microfluidic device of the kind described
above in order to introduce a predetermined degree of wettability
(hydrophilicity and/or hydrophobicity) on an inner surface of said
microchannel structures. The method is characterized in comprising
the steps of: (i) providing two essentially planar substrates (I
and II); (ii) placing either one or both of the substrates in a gas
plasma reactor, and creating within said plasma reactor a gas
plasma containing an organic precursor compound, said organic
precursor compound and the conditions in the reactor being selected
such that a coat of the predetermined degree of wettability is
formed on a selected part of the surface of the
substrate/substrates; (iii) removing the substrate/substrates from
the plasma reactor; (iv) adhering the surface of substrate I to the
surface of substrate II so that at least an enclosed section of
each of microchannel structures are formed between the two
surfaces; (v) optionally joining further planar substrates to
complete the microchannel structures. In the simplest variant,
complete enclosed microchannel structures are defined between
substrate I and II.
[0036] Microchannels are typically defined by a limited number of
well-defined walls, for instance a bottom wall, a top wall and two
sidewalls. These walls may derive from different substrates.
Locally at least the walls derived from the same substrate are
wettable/non-wettable to the same extent. In the case the surface
characteristics of a channel is intended to facilitate liquid
transport, and the walls derived from one of the substrates is
non-wettable this can be compensated if the wall(s) derived from
the other substrate is(are) sufficiently wettable (i.e., has/have a
sufficiently low water contact angle).
[0037] In order to facilitate good transport of a liquid between
different functional parts of the inventive microfluidic devices,
the liquid contact angle in the individual parts should primarily
be wettable, preferably with a water contact angle
.ltoreq.60.degree. such as .ltoreq.50.degree. or .ltoreq.40.degree.
or .ltoreq.30.degree. or .ltoreq.20.degree.. Local surface breaks
that are to be used for valving and/or anti-wicking, for instance,
are important exceptions from this general rule. Local surface
breaks are typically non-wettable with water contact angles
.gtoreq.90.degree., such as .gtoreq.100.degree. or
.gtoreq.110.degree. or .gtoreq.120.degree.. Typically the
difference in wettability (in water contact angles) between a local
surface break and a bordering surface are .gtoreq.50.degree., such
as .gtoreq.60.degree. or .gtoreq.70.degree.. All figures refer to
values obtained at the temperature of use, typically 25.degree. C,
and with water as the liquid.
[0038] One important problem with respect to microfluidic devices
is to obtain surfaces with a sufficient hydrophilicity to support
liquid transport through a microchannel structure combined with a
sufficiently low non-specific adsorption (anti-fouling) of reagents
in order to accomplish reliable and reproducible results. The
severity of the fouling problem (non-specific adsorption) increases
with the surface to volume ratio, i.e., it increases when a cross
sectional dimension decreases, for instance from .ltoreq.1000 .mu.m
to .ltoreq.100 .mu.m to .ltoreq.10 .mu.m and/or from .ltoreq.1000
.mu.l to .ltoreq.100 .mu.l to .ltoreq.10 .mu.l to .ltoreq.1 .mu.l
to .ltoreq.100 nl to .ltoreq.50 nl. Even if it is often said that
hydrophobic surfaces have prominent non-specific adsorption there
are numerous systems for which also hydrophilic surfaces have a
disturbing non-specific adsorption.
[0039] A. Additional Steps and Variations
[0040] Between steps (i) and (ii), (ii) and (iii) and/or (iii) and
(iv) there may be one or more additional steps for introducing one
or more surface modifications with characteristics that are
different from the coat introduced in step (ii). These additional
steps may involve (a) a gas plasma treatment utilizing the same or
another precursor compound and/or the same or other conditions,
and/or (b) some other coating procedure. Depending on the kind of
surface modification, alternative (a) may be carried out without
removing and re-inserting the substrate/substrates from/into the
gas plasma reactor.
[0041] If only a part of a substrate surface is to be coated in
step (ii) or in any of the additional steps, appropriate masking
and/or unmasking may be done before or after such a coating step
(including sequence of steps). Parts that are masked/unmasked may
be present in either one or both of the substrate surfaces, for
instance on a part comprising microstructures. Washing steps may be
included between steps if appropriate.
[0042] One variant of step (ii) is to introduce a coat that is
wettable (hydrophilic) and/or has a pronounced resistance to
non-specific adsorption (= anti-fouling) on a major part of the
microstructured part of the surface. Microstructured areas that are
not going to be coated in this step are typically masked. The
precursor compound and the plasma conditions for the gas plasma are
selected as outlined below. After step (ii) and unmasking, the
uncoated areas thus exposed may be further processed, for instance
to render them non-wettable (hydrophobic) in order to create
passive (non-closing) valves and/or anti-wicking means and/or inlet
or outlet vents to ambient atmosphere. These kinds of
functionalities are illustrated in WO 9958245 (Larsson et al.,
Gyros AB), WO 0185602 (Larsson et al., Gyros AB & .ANG.mic AB),
WO 0146465 (Andersson et aL, Gyros AB), and WO 02074438 (Andersson
et al., Gyros AB), which are incorporated herein by reference. In
the case an uncoated area as such provides a sufficiently low
wettability (i.e., are non-wettable), the surface at these
positions may be used directly as a valve and/or as an anti-wicking
means and/or as a vent after step (iv) without any extra
processing. Many times, however, it is more appropriate to make
these non-treated areas more non-wettable (increase the
hydrophobicity), for instance by inserting steps according to
alternatives (a) or (b) between steps (ii) and (iv). In the case
alternative (a) is selected, the precursor and gas plasma
conditions are selected to give a non-wettable surface as known in
the field and also discussed below. Spraying or printing may also
be utilized as alternative (b). See for instance WO 0185602
(Larsson et al., Gyros AB & .ANG.mic AB), and WO 0146465
(Andersson et al., Gyros AB), which are incorporated by reference
herein. In order to secure that the valve and/or anti-wicking means
will be located to a desired position and/or have a desired
geometry, appropriate masking is advantageous for an additional
step.
[0043] Another variant of step (ii) is to introduce a coat that is
non-wettable (hydrophobic coat) on selected parts of the
microstructures. Areas on which other surface characteristics are
desired are then typically masked. The non-wettable coat may be
introduced for creating local surface breaks of the same type as
indicated in the preceding paragraph. The remaining parts may be
intended for liquid transport and therefore typically need to be
processed to surfaces that are wettable by inserting steps
according to either alternative (a) or alternative (b) above after
step (ii). Remasking for these additional steps is often
advantageous for similar reasons as for the first variant. In the
case the uncoated area after unmasking inherently comprises a
desired wettability (either by being wettable or non-wettable),
there is no need to introduce any additional surface treatment
steps before step (iv).
[0044] A third variant of step (ii) is to introduce a coat that is
sufficiently wettable or sufficiently non-wettable, but not with
sufficiently low non-specific adsorption (anti-fouling), or vice
versa. In this case, an additional step according to alternative
(a) or (b) may be used to modify the coat to exhibit the missing
characteristics while at the same time retaining an essential part
of the surface characteristics created in step (ii). In this case
the same masking can be utilized for the two coating steps.
Demasking and remasking between step (ii) and an additional step
may then not be required.
[0045] B. The Substrates
[0046] Each of the two planar substrates may comprise
microstructures in the form of projections and/grooves as discussed
above. In the preferred variants, however, only one of the two
substrates comprises microstructures that then are in the form of
open microchannel structures or open sections of the microchannel
structures. The other substrate is used to cover these open
structures. Either one or both of the substrates may have
through-going holes that are associated with individual
microchannel structures. These holes may be used as inlets or
outlets for liquids and/or as inlet or outlet vents for air. In the
case different sections of a microchannel structure are defined
between different pairs of substrates this kind of holes may
provide communication between the different sections.
[0047] The substrates may be made from inorganic or organic
material. Typical inorganic materials are silicon, quartz, glass,
etc. Typical organic materials are polymer materials, for instance
plastics including elastomers, such as silicone rubber (for
instance poly dimethyl siloxane) etc. Polymer material as well as
plastics comprises polymers obtained by condensation
polymerization, polymerization of unsaturated organic compounds
and/or other polymerization reactions. The microstructures may be
created by various techniques such as etching, laser ablation,
lithography, replication by embossing, moulding, casting, etc. Each
substrate material typically has its preferred techniques.
[0048] From the manufacturing point of view, substrates exposing
surfaces and microstructures in plastics are many times preferred
because the costs for plastics are normally low and mass production
can easily be done, for instance by replication. Typical
manufacturing processes involving replication are embossing,
moulding, casting, etc. See for instance WO 9116966 (Pharmacia
Biotech AB, Ohman & Ekstrom), which is incorporated herein by
reference. At the priority date of this invention, the preferred
plastics were polycarbonates and polyolefins based on polymerizable
monomeric olefins that comprise straight, branched and/or cyclic
non-aromatic structures. Typical examples are Zeonex.TM. and
Zeonor.TM. from Nippon Zeon, Japan. This does not outrule the use
of other plastics, for instance based on styrenes, methacrylates
and/or the like. Suitable polymers may be copolymers comprising
different monomers, for instance with at least one of the monomers
discussed above.
[0049] C. Plasma Variables and the Gas Plasma Reactor
[0050] The electric excitation field applied typically has a
frequency in the radiowave or microwave region, i.e., kHz-MHz or
GHz respectively. The modification on the polymer surface caused by
the plasma will depend mainly on a number of internal plasma
parameters such as: type of species present in the plasma, spatial
distributions, energy distributions and directional distributions.
The species typically derives from one or more organic precursor
compounds. In turn these parameters depend in a complex way on the
external plasma parameters: reactor geometry, type of excitation,
applied power, type of process gas, gas pressure and gas flow
rate.
[0051] The results of a treatment may depend on the design of the
reactor vessel used meaning that the optimal interval to a certain
degree may vary from one reactor design to another. The results may
also depend on where in the reactor the surface is placed during
the treatment.
[0052] A suitable reactor vessel should enable electric excitation
power input for instance in the microwave or radio wave ranges. The
required intensity of the plasma may depend on the variables
discussed above. Satisfactory gas plasmas may be found in the case
the electric excitation power applied is .ltoreq.300 W, with
preference for .ltoreq.100 W. The pressures are typically
.ltoreq.200 mTorr, with preference for .ltoreq.100 mTorr. The
design of the reactor vessel enables introduction of the vapor
phase of the organic precursor into the reactor chamber. This
includes the option of heating of the reactor chamber and/or flask
containing the organic precursor. The reactor vessel is designed to
facilitate homogenous plasma distribution in the reactor chamber.
More details on parameters influencing plasma polymerization can be
found in Inagaki, N., "Plasma surface modification and plasma
polymerization." Technomic Publishing company, Inc., USA, 1996.
[0053] The proper combination of different plasma and apparatus
parameters is typically found by varying the values for one or more
of these parameters and study how this affect the properties of the
modified substrate surface, i.e., the resulting hydrophilicity,
hydrophobicity, anti-fouling, stability, etc.
[0054] D. The Chemical Structure of the Coat
[0055] The chemical structure of the coat such as degree and type
of cross-linking, swelling, kinds of functional groups exposed to a
surrounding liquid, etc. determines the chemical surface
characteristics, primarily wetting/non-wetting ability including
hydrophilicity and hydrophobicity, and non-specific adsorption of
various compounds such as proteins and/or other biopolymers and
bioorganic compounds.
[0056] Surface characterisation of the coat can be carried out by a
number of methods, such as X-ray photoelectron microscopy (XPS),
static secondary ion mass spectrometry (static SIMS), liquid
contact angle methods, atomic force microscopy (AFM), near edge
X-ray adsorption fine structure (NEXAFS), FTIR and chemical
derivatization. For a review see Johnston et al. (J. Electron
Spectroscopy and Related Phenomena 81 (1996) 303-317).
[0057] Preferably, a sufficiently hydrophilic coat exposes neutral
hydrophilic groups to a liquid in contact with the coat, in
particular lower alkyl ether, such as ethylene oxy, hydroxy groups,
etc., and is essentially free of aromatic structures. The coat is
essentially free of charged or chargeable groups, in particular if
a low non-specific adsorption is required. Chargeable groups are
karboxy (--COOH), amino (--NH.sub.2), etc.). Non-chargeable groups
are hydroxy bound to sp.sup.3-hybridized carbon, ether, amido,
etc.
[0058] There is a relatively large number of publications related
to chemical structure of polymeric films deposited from gas plasmas
that are based on organic precursor compounds (e.g., U.S. Pat. No.
5,153,072 (Ratner et al.) and U.S. 5,002,794 (Ratner et al.). A
general idea has been that the incorporation of groups and/or
properties that derive from a precursor compound can be related to
the rate of fragmentation in the plasma and the rate of deposition
of the coat on a substrate surface. It has been discussed that a
lower power may decrease fragmentation and increase the
incorporation of groups and properties that derive from the
precursor compound. It has also been discussed that fragmentation
of the precursor compound depends on W/FM where W is the RF power
applied, and F and M are the flow rate and the molecular weight,
respectively, of the organic precursor compound. Other variables
that have been studied are: (a) the effect of pulsed radiofrequency
(RF) discharges on fragmentation of the precursor compound in
relation to an increase of the presence of precursor structures in
the deposited coat, (b) the location of the substrate in the gas
plasma reactor with the idea that a location adjacent but not
submersed in the plasma will increase the degree of precursor
structures in the coat, etc. An increase in precursor structures in
a deposited coat has also been suggested if there is a negative
temperature gradient between the plasma and the substrate to be
surface modified. See Ohkubo et al. (J. Appl. Polym. Sci 41 (1990)
349-), Lopez et al. (Langmuir 7 (1991) 766-, D'Agostino et al. (J.
Polym. Sci. Part A: Polym. Chem. Edn. 28 (1990) 3378-, Cho et al.
(J. Appl. Polym. Sci. 41 (1990) 1373-, Ward et al. (Short, Surfasce
Interface Anal. 22 (1994) 477-, Kiaei et al. (J. Biomater. Sci.:
Polym. Edn. 4 (1992) 35-, and Panchalingam et al. (ASAIO J. (1993)
M305).
[0059] The organic precursor compound typically is polymerizable by
which is meant that the compound is capable of forming a high
molecular weight insoluble aggregate on the surface of the
substrate. This may involve traditional polymerization reactions or
take place by degradation, rearrangement and extensive reactions of
the precursor compound and/or of the intermediary species formed in
the gas plasma.
[0060] In order for an organic precursor, compound to function in
the present invention it must have a sufficiently high vapor
pressure at the selected temperature within the plasma reactor.
This also means that precursor compounds that have a low tendency
for hydrogen bonding may have advantages compared to precursor
compounds of the same size that have a strong tendency for hydrogen
bonding.
[0061] Small precursor compounds may also have advantages, e.g.,
with molecular weights .ltoreq.2000 dalton, such as .ltoreq.1000
dalton or .ltoreq.500 dalton. The advantage of small compounds and
compounds with weak or no tendency for hydrogen bonding is based on
the fact that hydrogen-bonding and increased molecular weight tends
to increase the boiling point and the vapor pressure.
[0062] For hydrophilic coats, suitable precursor compound can be
found amongst organic compounds that have a high content of
heteroatoms selected amongst oxygen, nitrogen and sulphur, provided
that the other plasma parameters are properly set. By the term
"high content" in this context is meant that the ratio between the
total number of the heteroatoms, e.g. oxygen, and the number of
carbon atoms should be .gtoreq.0.1, such as .gtoreq.0.25 or
.gtoreq.0.5 or .gtoreq.0.75, in the precursor compound. From
theoretical considerations, this ratio is never larger than 2. In
the case that the organic precursor compound has certain properties
that one would like to incorporate into a coat, but a low content
of heteroatoms, this may be compensated for by including oxygen in
the gas plasma. Alternatively, one may include one or more other
organic compounds for which the content of heteroatoms is higher
than in the desired precursor compound. Typically, compounds for
creating hydrophobic coats are hydrocarbons and fluorinated
hydrocarbons (e.g., perfluoinated hydrocarbons (PFH))
[0063] For hydrophobic coats, suitable precursor compounds can be
found amongst organic compounds having a low content of heteroatoms
selected amongst oxygen, nitrogen and sulphur, provided that the
other plasma variables are properly set. A "low content" in this
context means that the ratio between the number of heteroatoms,
e.g., oxygen, and the number of carbon atom should be .ltoreq.0.75,
such as .ltoreq.0.50 or .ltoreq.0.25 or .ltoreq.0.10. In the case
organic precursor compound has certain properties that one would
like to incorporate into a coat, but a high content of heteroatoms,
this might be compensated for by including one or more organic
compounds for which the content of heteroatoms is lower than in the
desired precursor compound.
[0064] Suitable precursor compounds may also be found amongst
organic compounds that contain one, two or more structural units
that are present in polymers that are known to give coats that are
resistant to non-specific adsorption. These kinds of precursor
compounds are in the innovative method combined with gas plasma
conditions enabling this property to be retained in the coat
deposited on the substrate.
[0065] There are a large number of polymers that are known to
reduce non-specific adsorption. Typically, they are non-ionic and
hydrophilic, i.e., contains a plurality of neutral hydrophilic
groups, such as hydroxy, amido, and lower alkoxy including
alkyleneoxy (C.sub.1-3 in particular C.sub.2) and alkyl ether
groups. See for instance U.S. Pat. No. 6,337,212 (Caliper), WO
0147637 (Gyros AB), U.S. Pat. No. 4,680,201 (Hjerten), U.S. Pat.
No. 5,840,388 (Karger et al.), US 5,240,994 (Brink et al.), and
U.S. 5,250,613 (Bergstrom et al.), which are incorporated herein by
reference. Precursor compounds to be used in this variant of the
invention can, thus, be found amongst low molecular weight
compounds that comprise one or more of these structural units that
are present in polymers that reduce non-specific adsorption. At the
priority date, one of the most promising precursor compounds
comprise the structural unit --(CH.sub.2).sub.n O--, where (a) n is
an integer 1-3, with preference for 2, (b) the free valence at the
carbon binds to hydrogen or an oxygen, and (c) the free valence at
the oxygen binds to a hydrogen or a carbon. The carbon may be
sp.sup.3--, Sp.sup.2--or sp-hydridized and may thus be part of a
saturated or unstarurated hydrocarbon group such as alkyl (for
instance C.sub.1, C.sub.2, C.sub.3 to C.sub.5) and alkenyl, such as
vinyl). This is in-line with the findings of Ratner et al. (U.S.
Pat. No. 5,002,794 and U.S. Pat. No. 5,153,072) and Timmons et al.
(U.S. Pat. No. 6,329,024, U.S. Pat. No. 5,876,753, and EP 896053),
which are incorporated by reference for precursor compounds
comprising 1-4 repetitive ethylene oxide units either in straight
form or in cyclic form (crown ethers). According to the same
principles, one can envisage that other suitable candidate
precursor compounds can be found amongst low molecular weight
compounds which comprise structural units selected amongst
--CH.sub.2OH, --CH.sub.2 (OCH.sub.3), and [--CH.sub.2--CH
(OH)].sub.n'--, and [--CH.sub.2--CH(OR)].sub.n'-- and corresponding
monomers wherever applicable, where (a) n' is an integer 1-10 with
preference for 1-5, (b) R is lower alkyl (C.sub.1-5), such as
methyl, or lower acyl (C.sub.1-5, such as formyl or acetyl), and
(c) the free valences binds to atoms selected amongst hydrogen,
carbon, sulphur, nitrogen and oxygen. None of sulphur, nitrogen and
oxygen binds a hydrogen when two or more of them binds to the same
carbon. Other candidate precursor compounds are monomers or
oligomers (2-10, such as 2-5, repeating monomeric units)
corresponding to polymers that give coats that have low
non-specific adsorption.
[0066] In preferred variants, a coat providing low non-specific
adsorption can also have a sufficient hydrophilicity in order to
secure a reliable and reproducible transport of reagents by an
aqueous liquid flow. One can, thus, envisage that candidates of
precursor compounds can also be found among the precursor compounds
that are candidates for the creation of hydrophilic coats. See
above.
[0067] The thickness of the coat can be .ltoreq.50%, for instance
.ltoreq.20% or .ltoreq.10%, of the smallest distance between two
opposing sides of a microchannel part comprising the innovative
coat. An optimal thickness is typically be .ltoreq.1000 nm, for
instance .ltoreq.100 nm or .ltoreq.50 nm, with the provision that
the coat shall permit a desired flow to pass through. A lower limit
is typically 0.1 nm. The figures of present invention refer to
thickness after saturation with the liquid intended to pass through
a microchannel part comprising the coat. The coat may or may not
swell in contact with the liquid, which is passing through a
microchannel structure.
[0068] It is important to control the selected process parameters
so that they lead to predetermined surface characteristics, for
instance preselected wetting/or non-wetting properties and/or
ability to reduce non-specific adsorption (anti-fouling). This can
be accomplished as outlined in the experimental part that describes
the determination of a) liquid contact angles, and b) adsorption of
albumin, which is a measure of non-specific adsorption. Once the
proper values/ranges of the process parameters have been found for
the predetermined surface characteristics, the process can be run
without testing.
[0069] For aqueous solutions the term "a reduction in non-specific
adsorption" (anti-fouling effect) refers to bovine serum albumin as
a reference/model substance and means that the ratio between
adsorption of bovine serum albumin after and before a gas plasma
treatment of a surface according to the invention is .ltoreq.0.75,
such as .ltoreq.0.50 or .ltoreq.0.25 (decrease ratio). The ratio
can be even lower, for instance .ltoreq.0.10.
[0070] E. Adhering the Substrate Surfaces
[0071] There are a number of techniques suggested in the
literature. Thus conventional bonding without use of a particular
adhesive may be utilized, for example, in the case that the
substrates are made of inorganic material such as silicon, glass,
quartz and the like. In the case that the substrate surfaces
comprise plastics, the two surfaces can be fixed to each other by
pressing the surfaces together while heating selectively the
surface not containing microstructures above its transition
temperature, while the surface with the microstructures are
maintained below its transition temperature. In other alternatives,
various kinds of adhesives or glues may be used. See further WO
9424900 (Ove Ohman), WO 9845693 (Soane et al.), U.S. Pat. No.
6,176,962 (Soane et al.), WO 9956954 (Quine), and WO 0154810
(Derand et al., Gyros AB), which are incorporated herein by
reference. Thermolaminating is important because this technology
has been shown to be capable minimizing destruction of differences
in chemical surface characteristics that are to be retained in the
microfluidic device obtained after step (iv). See WO 0154810
(Derand et al., Gyros AB).
[0072] Problems with so-called bond voids can be minimized if the
open microchannel structures in a substrate surface is defined by
walls arising from the surface. See WO 9832535 (Lindberg et al.)
and WO 0197974 (Chazan et al., Caliper).
[0073] In order to avoid that an adhesive is pressed into a
microchannel during steps (iv) and (v) the microchannel structures
are preferably defined by relief patterns that are present in
either one or both of the substrate surfaces as outlined in
PCT/SE02/0243 1 (Derand et al.), which is incorporated by
reference.
[0074] In principle the adhesive may be selected as outlined in
U.S. Pat. No. 6,176,962 and WO 9845693 (Soane et al.), which are
incorporated by reference. Thus, suitable bonding materials include
elastomeric adhesive materials and curable bonding materials. These
kinds of bonding material as well as others may be in liquid form
when applied to a substrate surface. Bonding materials including
adhesives thus comprises liquid curable adhesive material and
liquid elastomeric material. After application, the adhesive
material is rendered more viscous or non-flowable for instance by
solvent removal or partial curing before the other substrate is
contacted with the adhesive. The term "liquid form" includes
material of low viscosity and material of high viscosity. Curable
adhesive includes polymerizable adhesives and activatable
adhesives. Thermo-curarable, moisture-curable, and bi-, three- and
multi-component adhesives are also examples of curable
adhesives.
[0075] III. The Microfluidic Device
[0076] This aspect of the invention is primarily characterized in
that a part of the inner surface of at least one of the
microchannel structures has been modified by the use of gas plasma
comprising an organic precursor compound selected according to the
principles outlined for the first aspect, i.e. has one or more
surface characteristics that is achievable by a plasma
polymerization coating method. Additional characteristic features
are defined below.
[0077] The microfluidic device preferably contains a plurality of
microchannel structures, each of which is defined between two or
more planar substrates. Each microchannel structure may comprise
one, two, three or more functional parts selected among: a)
application chamber/cavity/area, b) conduit for liquid transport,
c) reaction chamber/cavity; d) volume defining unit; e) mixing
chamber/cavity; f) chamber for separating components in the sample,
for instance by capillary electrophoresis, chromatography and the
like; g) detection chamber/cavity; h) waste conduit/chamber/
cavity; i) internal valve; j) valve to ambient atmosphere; etc.
Many of these parts may have one or more functionalities. There may
also be collecting chambers/cavities in which a compound, which has
been separated, formed or otherwise processed in a microchannel
structure are collected and transferred to some other instrument,
for instance an analytical instrument such as a mass spectrometer.
In addition, there are also one or more outlet vents for air.
Inlets and outlets for liquids may also function as vents (inlet
vent or outlet vent).
[0078] The preferred devices are typically disc-shaped with
sizes/surface areas and/or forms similar to the conventional
CD-format, e.g., their surface areas are in the interval from 10%
up to 300% of the surface area of a CD of the conventional
CD-radii. The upper and/or lower sides of the disc may or may not
be planar.
[0079] The preferred microfluidic discs have an axis of symmetry
(Cn) that is perpendicular to the disc plane, where n is an integer
.gtoreq.2, 3, 4 or 5, preferably .infin. (C.infin.). In other words
the disc may be rectangular, such as square-shaped, or have other
polygonal forms, but is preferably circular. Once the proper disc
format has been selected centrifugal force may be used for driving
liquid flow. Spinning the device around a spin axis that typically
is perpendicular or parallel to the disc plane may create the
necessary centrifugal force. In the most obvious variants at the
priority date, the spin axis coincides with the above-mentioned
axis of symmetry.
[0080] Different principles may be utilized for transporting the
liquid aliquots within the microfluidic device/microchannel
structures between two or more of the functional parts described
above. Inertia force may be used, for instance by spinning the disc
as discussed in the preceding paragraphs. Other forces that may be
used are electrokinetic forces and non-electrokinetic forces, such
as capillary forces, hydrostatic pressure, etc. In preferred
variants utilizing centrifugal force for liquid transport, each
microchannel structure comprises an upstream section that is at a
shorter radial distance than a downstream section relative to a
spin axis.
[0081] The microfluidic device may also comprise common channels
connecting different microchannel structures, for instance common
distribution channels for introduction of liquids and common waste
channels including waste reservoirs. Common channels including
their various parts such as inlet ports, outlet ports, vents, etc.,
are considered to be part of each of the microchannel structures
they are connecting. Common microchannels may also fluidly connect
groups of microchannel structures that are in different planes or
in the same plane.
IV. Examples
[0082] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLE 1
Plasma Treatment with Diethylene Glycol Dimethyl Ether
[0083] A polycarbonate CD disc (Macrolon DP-1265, Bayer AG,
Germany), and pieces cut from a polycarbonate CD disc were placed
in a plasma reactor (CVD Piccolo, Plasma Electronic, Germany), and
subjected to argon plasma treatment at 24 W for 2 min.
Subsequently, the polycarbonate surfaces were treated with plasma
of diethylene glycol dimethyl ether (diglyme; Aldrich, USA) at 24 W
for 5 min. The water contact angle (sessile drop) of the resulting
surfaces was measured on a Rame-Hart manual goniometer bench. The
average of six equilibrium measurements (three droplets) was
48.degree..
EXAMPLE 2
Plasma Treatment with Diethylene Glycol Dimethyl Ether and Allylic
Alchohol
[0084] A polycarbonate CD disc (Macrolon DP-1265, Bayer AG,
Germany), and pieces cut from a polycarbonate CD disc were placed
in a plasma reactor (CVD Piccolo, Plasma Electronic, Germany), and
subjected to argon plasma treatment at 24 w for 2 min.
Subsequently, the polycarbonate surfaces were treated with plasma
of diethylene glycol dimethyl ether (diglyme; Aldrich, USA) at 24 W
for 5 min. Finally, they were subjected to plasma of allylic
alcohol (Merck, Germany) at 12 W for 5 min. The water contact angle
(sessile drop) of the resulting surfaces was measured on a
Rame-Hart manual goniometer bench. The average of six equilibrium
measurements (three droplets) was <10.degree..
EXAMPLE 3
Plasma Treatment with Ethylene Glycol Vinyl Ether
[0085] A polycarbonate CD disc (Macrolon DP-1265, Bayer AG,
Germany), and pieces cut from a polycarbonate CD disc were placed
in a plasma reactor (CVD Piccolo, Plasma Electronic, Germany), and
subjected to argon plasma treatment at 24 w for 2 min.
Subsequently, the polycarbonate surfaces were treated with plasma
of ethylene glycol vinyl ether (Aldrich, USA) at 12 W for 5
min.
[0086] The water contact angle (sessile drop) of the resulting
surfaces was measured on a Rame-Hart manual goniometer bench. The
average of six equilibrium measurements (three droplets) was
22.degree..
EXAMPLE 4
Microfluidic Test
[0087] A silicone rubber lid (polydimethylsiloxane) was placed on a
polycarbonate CD with recessed microchannel pattern, (50-200 .mu.m
wide, 50-100 .mu.m deep), that had been treated either with diglyme
plasma, or with diglyme plasma with subsequent allylic alcohol
plasma treatment, as described above. Alternatively, silicone
rubber with recessed microchannel pattern (1000 .mu.m wide, 100
.mu.m deep) was placed on flat polycarbonate surfaces that had been
treated either with diglyme plasma, or with diglyme plasma with
subsequent allylic alcohol plasma treatment, as described above.
Resulting flow channels were examined using a solution of Cibacron
Brilliant Red (CIBA limited) in MilliQ water (Millipore). A drop
was placed at the channel inlet and it was concluded that flow rate
into channels on surfaces that had been subjected to diglyme plasma
with subsequent allylic alcohol plasma treatment was significantly
higher than on surfaces that had only been treated with diglyme
plasma.
EXAMPLE 5
Protein Adsorption Studied with Total Internal Reflection
Fluorescence (TIRF) Spectroscopy
[0088] The theory of TIRF spectroscopy, as well as the experimental
set-up used in the present work is described in Example 1.
[0089] Bovine serum albumin (BSA; fraction V, Sigma, USA) was
chosen as model protein for adsorption studies, and labelled with
fluorescein-5-isothiocyanate (FITC; isomer I; Molecular Probes), as
described in [Lassen, B. and Malmsten, M., Competitive protein
adsorption studied with TIRF and ellipsometry. Journal of colloid
and interface science, 1996. 179:p. 470-477]. The molar ratio of
FITC to proteins was found to be approximately unity in all
cases.
[0090] A TIRF fluorescence intensity graph resulting from
adsorption of 400 ppm FITC-BSA on untreated polycarbonate (PC) is
shown in FIG. 1, together with a graph representing the same
experiment on a diglyme plasma-treated surface. TIRF fluorescence
intensity graphs using diglyme plasma + allylic alcohol plasma
(FIG. 2), and ethylene glycol vinyl ether plasma (FIG. 3) are also
presented here.
[0091] It is apparent from the figures that the ratio between
adsorption of protein on the treated surface and the untreated
surface always is <0.25.
[0092] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
* * * * *