U.S. patent application number 11/651269 was filed with the patent office on 2007-05-17 for enzyme immobilization for electroosmotic flow.
Invention is credited to Jonathan S. Dordick, Bosung Ku, Moo-Yeal Lee, Aravind Srinivasan.
Application Number | 20070108057 11/651269 |
Document ID | / |
Family ID | 37588180 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070108057 |
Kind Code |
A1 |
Dordick; Jonathan S. ; et
al. |
May 17, 2007 |
Enzyme immobilization for electroosmotic flow
Abstract
Disclosed herein is a method and apparatus of immobilizing a
biocatalyst on a microfluidic biochip for conducting reactions in
the presence of electroosmotic flow. The biochip includes a polymer
on its microfluidic flow surfaces, wherein the polymer includes a
first substituent selected from ionic groups of the same polarity
or precursors thereof, a second substituent that is a hydrophobic
group, and a third substituent comprising an immobilized
biocatalyst-or precursor thereof. The biochip can be used to
conduct multiple sequential biocatalyzed reactions in the presence
of electroosmotic flow.
Inventors: |
Dordick; Jonathan S.;
(Schenectady, NY) ; Lee; Moo-Yeal; (Troy, NY)
; Srinivasan; Aravind; (Troy, NY) ; Ku;
Bosung; (Troy, NY) |
Correspondence
Address: |
ELMORE PATENT LAW GROUP, PC
209 MAIN STREET
N. CHELMSFORD
MA
01863
US
|
Family ID: |
37588180 |
Appl. No.: |
11/651269 |
Filed: |
January 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10351976 |
Jan 24, 2003 |
7172682 |
|
|
11651269 |
Jan 9, 2007 |
|
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Current U.S.
Class: |
204/601 |
Current CPC
Class: |
C07K 17/14 20130101 |
Class at
Publication: |
204/601 |
International
Class: |
G01N 27/00 20060101
G01N027/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by grants
from the Defense Advanced Research and Projects Agency, the
Biotechnology Research and Development Corporation and the National
Science Foundation under grant number BES-0118820. The Government
has certain rights in the invention.
Claims
1. A microfluidics biochip, comprising: a) a microfluidic component
comprising a flow surface; b) a plurality of electrodes, whereby an
electroosmotic flow can be generated at the flow surface; c) an
immobilizing polymer coated on the flow surface, wherein the
polymer is substantially adhered to the surface, wherein the
polymer comprises a first substituent selected from ionic groups of
the same polarity and covalent precursors of the ionic groups; a
second substituent that is a hydrophobic group; and an immobilized
biomolecule.
2. The biochip of claim 1, further comprising a microfluidic
channel, wherein a cross-sectional area of the channel is less than
about 0.1 mm.sup.2.
3. The biochip of claim 2, wherein the hydrophobic group is an
optionally substituted C8-C30 alkyl, polycyclic alkyl, polycyclic
aryl, or polycyclic heteroaryl, or a C8-C30 alkyl optionally
interrupted by a cyclic or polycyclic alkyl, aryl, or
heteroaryl.
4. The biochip of claim 3, wherein the polymer is substantially
adhered to the flow surface by hydrophobic interactions,
electrostatic interactions, or by a covalent bond.
5. The biochip of claim 4, wherein the polymer is substantially
adhered to the flow surface by covalent attachment.
6. The biochip of claim 5, wherein the biomolecule is a biocatalyst
comprising a catalytically functional portion of an antibody, an
enzyme, a peptide, or an oligonucleotide; and optionally, a
cofactor.
7. The biochip of claim 6, wherein the biocatalyst comprises a
catalytically functional portion of an oxidoreductase, a
transferase, a hydrolase, a lyase, an isomerase, or a ligase; and
optionally, a cofactor.
8. The biochip of claim 7, wherein the biocatalyst is immobilized
to the polymer through an amide bond.
9. The biochip of claim 8, wherein the flow surface comprises
surface --Si--O-- groups.
10. The biochip of claim 9, wherein the covalent attachment of the
polymer to the flow surface is represented by structural formula B:
##STR8## wherein R.sup.s represents one of the surface --Si--O--
groups; R.sup.a is a C1-C8 alkylene chain; each R.sup.b is
independently a C1-C4 alkyl group; and D represents an amide bond
to the polymer.
11. The biochip of claim 10, further comprising at least two
catalytically distinct immobilized biocatalysts.
12. The biochip of claim 11, further comprising at least two
spatially distinct flow surfaces, wherein the flow surfaces are in
microfluidic communication; each flow surface is coated with the
polymer; at least two said catalytically distinct biocatalysts are
respectively immobilized to the polymer at the spatially distinct
flow surfaces; and whereby the biocatalysts are catalytically and
spatially distinct.
13. The biochip of claim 12 wherein the polymer is represented by a
structure derived from a copolymer of ethylenically unsaturated
monomers, wherein the monomers are substituted with groups selected
from the ionic groups or precursors thereof, the hydrophobic
groups, the biomolecules, and the linker.
14. The biochip of claim 13, wherein the ionic groups are selected
from carboxylate, carbamate, sulfate, thiosulfate, sulfonate,
phosphate, phosphonate, and hydroxyl; the precursors are selected
from optionally substituted alkyl esters, alkyl anhydrides, cyclic
anhydrides, alkyl ethers, aryl esters, aryl anhydrides, and aryl
ethers of the ionic groups.
15. The biochip of claim 13, wherein the ionic groups are selected
from ammonium, phosphonium, and sulfonium groups substituted with
one or more groups selected from C1-C8 alkyl and aryl.
16. The biochip of claim 14, wherein the polymer is derived from a
maleic anhydride-.alpha.-olefin polymer, wherein the ionic groups
consist of anhydride or carboxyl derived from a maleic anydride
monomer; the hydrophobic substituent is a C12-C28 alkyl chain; the
biocatalysts are each immobilized through an amide bond to a
carboxyl derived from a maleic anhydride monomer; the amide bond
represented by D comprises a carboxyl derived from a maleic
anhydride monomer.
Description
RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 10/351,976, filed Jan. 24, 2003. The entire teachings of the
above application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Microfluidics is the field of microscale fluid flow and
control, typically through microscale fluid control features
constructed on a substrate such as a glass chip. These devices can
be used to manipulate liquid chemical and biological samples in
order to conduct analysis, perform synthetic reactions, and the
like.
[0004] A motive force often used in microfluidics is a phenomenon
known as electroosmotic flow (EOF). EOF depends strongly on various
aspects of charge mobility in these systems, and in particular,
depends on having a high density of ionic groups of the same
polarity on the walls or flow surfaces of a microfluidics
component. For example, a typical microfluidics feature is a
microchannel etched in a glass substrate. The interior surface of
the channel is treated to expose free silanol groups, which can
then be deprotonated to provide siloxide anions. A voltage is
applied across the flow direction of the channel causing solvated
counterions of the charged groups on the channel walls to move.
Because the dimensions of a microfluidic channel are small, the
layer of counterions at the flow surface contact a significant
portion of the fluid in the channel. Thus, when the counterion
layer moves, the entire volume of the channel moves nearly
simultaneously, a mechanism known as "plug flow". For many
applications, plug flow is advantageous because it means that the
components of a particular volume portion of the flow travel
together, and are not spread out as they would be in a conventional
pressure driven flow. Thus, on a microfluidics chip, precise
volumes can be delivered from one location to another with a high
degree of control.
[0005] Many features of a macroscale laboratory can thus be
miniaturized using microfluidics, allowing significant reductions
in the amount of costly, rare, or hazardous materials that are
used. For example, microfluidics has the potential to make
efficient use of biomolecules such as enzymes or catalytic
antibodies, which are typically expensive or difficult to prepare
in large quantities. It is particularly desirable that these
molecules be reusable or recoverable, for example, by immobilizing
them to a solid support, in order to further limit the quantities
that are required.
[0006] A significant problem that must be solved, however, is the
difficulty of immobilizing biomolecules with high biological
activity while simultaneously maintaining acceptable EOF
capability.
[0007] For example, enzymes have been attached to glass chips by
covalent attachment to free silanol groups on the glass surface.
However, the high pH necessary to provide the charged siloxide
anions significantly decreases the stability and catalytic activity
of enzymes. Another attempt functionalized the silanol groups with
a linker group ending in an amine, which can then be covalently
attached to the enzyme. However, this leads to a reduction in the
number of available siloxide groups at the surface, and further,
shields the groups that are present from the flow, resulting in
poor EOF characteristics.
[0008] Other attempts have been made to provide polymers that
specifically enhance EOF, for example, by using a charged polymer
such as dextran sulfate. These polymers, however, are dynamically
unstable coatings, i.e., are not substantially adhered, and so are
eventually washed away by the flow. Furthermore, they are not
easily functionalized with biocatalysts such as enzymes, and they
do not maintain the catalytic activity of enzymes.
[0009] High enzyme activity can be maintained by encapsulating
and/or covalently attaching enzymes to matrices, such as solgels,
but such matrices typically fill the entire microfluidic channel,
providing a severe impediment to fluid flow. Furthermore, many
other examples of polymers exist to immobilize enzymes with high
activity, but they are not designed to support EOF.
[0010] Therefore, there is a need to immobilize biomolecules on a
microfluidics apparatus, while simultaneously maintaining high
biological activity and high EOF capability.
SUMMARY OF THE INVENTION
[0011] It has now been found that certain polymers containing both
ionic groups and hydrophobic groups can be substantially adhered to
microfluidic channels and can be used to simultaneously immobilize
biocatalysts with good catalytic activity while supporting
electroosmotic flow.
[0012] One embodiment of the invention is method of immobilizing a
biomolecule in the presence of electroosmotic flow. One step is
providing a microfluidic biochip. The biochip includes a
microfluidic component comprising a flow surface; at least two
electrodes whereby an electroosmotic flow can be generated at the
flow surface; and an immobilizing polymer that is substantially
adhered to the flow surface. The polymer includes a first
substituent selected from ionic groups of the same polarity and
covalent precursors of the ionic groups, wherein the first
substituent is optionally a biomolecule immobilizing group. The
polymer includes a second substituent that is a hydrophobic group.
The polymer optionally includes a third substituent that is a
biomolecule-immobilizing group. Between the first substituent and
the optional third substituent, the polymer includes at least one
substituent that is a biomolecule-immobilizing group. Another step
of the method is applying a motive force selected from pressure,
electroosmotic force, capillary action, and centrifugal force,
thereby generating flow. Yet another step is directing a
biomolecule from a source to the polymer by employing the flow. An
additional step is reacting the biomolecule with the
biomolecule-immobilizing group under suitable reaction conditions,
whereby the biomolecule is immobilized.
[0013] Another embodiment of the invention is the biochip, wherein
the substituents of the immobilizing polymer include a first
substituent selected from ionic groups of the same polarity and
covalent precursors of the ionic groups; a second substituent that
is a hydrophobic group; and a third substituent comprising an
immobilized biomolecule.
[0014] Another embodiment of the invention is a method for
conducting one or more reactions by using the biochip. The biochip
additionally includes at least one reservoir, wherein the reservoir
contains a starting reactant. All the microfluidic components are
in microfluidic communication. The substituents of the immobilizing
polymer include ionic groups of the same polarity; a hydrophobic
group; a first immobilized biomolecule; and optionally a second,
chemically distinct immobilized biomolecule;. A step of the method
is applying a voltage to the electrodes, thereby generating
electroosmotic flow. Another step is directing the reactant from
the reservoir to the first biomolecule by employing the
electroosmotic flow, then reacting the first reactant with the
first biomolecule under suitable reaction conditions, thereby
producing a first reaction product. Another step is optionally
contacting the first product with the second biomolecule, if
present, and reacting the first product with the second biomolecule
under suitable reaction conditions, thereby producing a second
reaction product.
[0015] The invention can be used to immobilize biomolecules on a
microfluidics biochip to conduct reactions. The invention retains
the activity of the biomolecules while simultaneously supporting
EOF. Also, the invention allows multiple reactions using
catalytically distinct biomolecules. Furthermore, the invention
allows sequential reactions using catalytically and spatially
distinct immobilized biomolecules. Thus, using the invention, a
wide array of sequential, stepwise reactions can be conducted with
high activity while minimizing the use of costly or dangerous
reactants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
[0017] FIG. 1 graphs enhanced electroosmotic flow versus voltage
for PMA-OL (poly (maleic anhydride)-alt-.alpha.-olefin) coated
channels of the invention (open circles) compared
glutaraldehyde-coated control channels (solid triangles), with
reference to glass EOF control channels (solid circles).
[0018] FIG. 2A graphs high retained biological activity of PMA-OL
immobilized soybean peroxidase (SBP) as shown by H.sub.2O.sub.2
consumption by on the biochip in 2% loading (filled circles) and
10% loading (open circles).
[0019] FIG. 2B graphs the kinetics of PMA-OL-immobilized soybean
peroxidase.
[0020] FIG. 3 graphs the high catalytic activity of two PMA-OL
immobilized enzymes for poly(p-cresol) production on the biochip
(filled circles) compared to a solution control (open circles).
[0021] FIG. 4 graphs the high catalytic activity of three PMA-OL
immobilized enzymes for poly(p-cresol) production on the biochip
(filled circles) compared to a solution control (open circles).
[0022] FIG. 5A depicts the 3-step multi-enzyme pathway incorporated
into a PDMS (polydimethyl siloxane) biochip, wherein the three
PMA-OD (poly (maleic anhydride)-co-1-octadecene) immobilized
enzymes are spatially separated.
[0023] FIG. 5B graphs poly(p-cresol) production in a three enzyme
system immobilized in distinct locations on a PDMS biochip (filled
circles) compared to a solution control (open circles).
[0024] FIG. 6 depicts the steps in fabrication of microfluidic
components on a glass slide for a biochip.
[0025] FIG. 7A depicts the chemistry of immobilizing an enzyme on a
glass microfluidic surface using PMA-OL and APTES (aminopropyl
triethoxysilane).
[0026] FIG. 7B depicts the chemistry of immobilizing an enzyme on a
glass microfluidic surface using glutaraldehyde and APTES.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The invention is generally directed to methods of
immobilizing biomolecules on a microscale device using an
immobilizing polymer. In particular, the invention is directed to
immobilizing biomolecules in microfluidic channels, using an
immobilizing polymer that simultaneously maintains good biological
activity of the biomolecule and good EOF characteristics.
[0028] A microfluidic device is a system of microscale fluid
control components, such as channels, reservoirs, junctions such as
T-junctions, and the like. Typically, these components are
incorporated into a single solid substrate, for example, by
chemically etching channels and reservoirs into the surface of a
chip, e.g., a glass microscope slide, a polymer slab, a silicon
wafer, and the like. These features can be made in such solid
substrates by other techniques known to the art, for example,
precision mechanical machining, laser machining, polymer molding
from a machined or etched master, and the like.
[0029] The cross-sectional area of a microfluidic channel is less
than 0.1 mm.sup.2, generally less than 0.05 mm.sup.2, more
preferably less than 0.025 mm.sup.2, and most preferably about
0.003 mm.sup.2. The length of a microfluidic channel can be at a
much larger scale, for example, a single channel could run up to
the length of the substrate, e.g. almost 75 mm on a standard
microscope slide, or a channel could loop back and forth on a chip,
becoming many times longer than the chip itself. One or more
reservoirs are typically connected to microchannel, e.g., to supply
buffer solution, reactant solution, to collect products or waste,
and the like. Fluid flow in a microfluidic chip can be generated by
applying a motive force, for example, pressure, electroosmotic
force, capillary action, centrifugal force, and the like.
Preferably, the force used is electroosmotic. The biochip typically
includes, or is used in combination with, a plurality of
electrodes, i.e., at least two, which can be used to apply a
voltage for causing EOF.
[0030] The ease of generating EOF in a microchannel depends on the
number of ionic groups attached to the interior flow surface of the
microchannel. When a voltage is applied to electrodes at opposite
ends of a channel, negatively charged ions tend to flow to the
anode and positively charged ions tend to flow to the cathode. In a
channel designed for EOF, the ionic groups attached to the flow
surface cannot move. However, the dissolved counterions of the
ionic groups can move, and cause the surrounding solvent to move,
resulting in EOF.
[0031] The rate of fluid flow is (V.sub.EOF) is affected by several
factors (Eq. 1), where E is the electric field (equal to the
voltage divided by the distance between the electrodes for
microchannels of uniform resistance per unit length), .epsilon. is
the relative permittivity of liquid, .epsilon..sub.0 is the
permittivity of free space, .zeta. is the zeta potential of the
microchannel/liquid interface and .eta. is the liquid viscosity
(Fletcher, P D I; Haswell, S J; Paunov, V N. Analyst, 1999, 124,
1273-1282). V EOF = - E .times. .times. .times. .times. 0 .times.
.zeta. .eta. ( 1 ) ##EQU1## The sign of Eq 1 indicates the charge
of the ionic groups attached to the channel flow surface, i.e. when
the channel wall is negatively charged, the .zeta.-potential is
negative, the diffuse charge in the liquid is positive (i.e., due
to the positive counterions of the channel wall negative ions) and
the liquid flows towards the cathode.
[0032] The microfluidic biochip can be made of any material, for
example, glass, ceramics, polymers, silicon wafers, metals, and the
like, in which microfluidic features can be fabricated. The
microfluidic biochip can be a solid slab of a relatively
hydrophobic polymer (distinct from the immobilizing polymer) that
is readily coated by hydrophobic groups in the immobilizing
polymer. For example the biochip can be made of polydialkyl
siloxanes such as polydimethyl siloxane (PDMS), polyalkylenes such
as polyethylene and polypropylene, polystyrenes, polyvinyl alcohol
alkyl ethers, polyacrylate alkyl esters, and the like.
[0033] Alternatively, the microfluidic biochip is made of a
material that has groups on its surface that are reactive, or can
be made reactive, whereby the immobilizing polymer can be
covalently attached to the flow surface. For example, a biochip
made out of glass possesses --Si--O-- groups, which can be treated
to expose reactive siloxide anion or silanol surface groups that
can react with the immobilizing polymer or with a linker that
reacts with the immobilizing polymer. For example, siloxide anion
groups on a glass surface can be reacted with a linker, e.g.,
aminopropyl triethoxysilane (APTES), thus covering the surface with
amino groups: ##STR1## which can be reacted with the immobilizing
polymer. For example, in FIG. 7A (step 3), a maleic anhydride
moiety of the immobilizing polymer reacts with the amino group of
the APTES that is bound to the glass surface. Subsequently (step
4), the carboxyl group thus released, or another maleic anhydride
group (as shown) reacts with an amine on an enzyme (filled circle),
thus immobilizing the enzyme. The surfaces of other substrates can
be functionalized similarly, for example, surface
--CO.sub.2CH.sub.3 groups on a polymethyl methacrylate substrate
could be treated to expose carboxylates, --OH groups in a
phenol-formaldehyde substrate can be converted to phenolate anions,
and the like.
[0034] The "immobilizing polymer" is a multifunctional, substituted
polymer that is coated on, or exposed at, a flow surface of the
biochip. Through its substituents, the immobilizing polymer can
simultaneously provide four functions: biomolecule immobilization,
maintenance of biomolecule activity, support of EOF
characteristics, and substantial adherence of the polymer to the
flow surface of the biochip. The immobilizing polymer contains at
least two substituents that can perform these four functions.
Preferred polymers possess simple polymer structural chemistry,
such as a copolymer with the minimum set of substituents necessary
to achieve multiple functions, e.g., a minimal set can be an ionic
group (or precursor thereof) and a hydrophobic group. For example,
an immobilizing polymer can be substituted with carboxylate and a
C24 alkyl chain. The carboxylate can immobilize an enzyme by
forming an amide bond and can support EOF through its negative
charge, while the alkyl chain can help to maintain the enzyme's
activity and can help to adhere the polymer to a hydrophobic
microchannel wall through hydrophobic interactions. Alternatively,
adherence to the microchannel wall can be provided through the
carboxylate, either through ionic association with ionic groups on
the microchannel wall or through creation of a covalent bond to the
wall. An immobilizing polymer can also contain more than two
substituents to perform the four functions, for example, a polymer
could have a sulfonate for EOF, a carboxylate or thiol for enzyme
immobilization, and a hydrophobic group for enzyme stability and
adherence to the surface.
[0035] Substituents that support EOF are ionic substituents (or
precursors thereof) that are the same polarity. For example, ionic
groups can include negatively charged groups such as carboxylate,
carbamate, sulfate, thiosulfate, sulfonate, phosphate, phosphonate,
and hydroxyl. Alternatively, ionic groups include carboxylate,
carbamate, sulfate, and hydroxyl. Preferably, negatively charged
ionic groups are carboxylates.
[0036] In an alternative embodiment, ionic groups that are
positively charged are used. For example, positive ionic groups can
be ammonium, phosphonium, and sulfonium groups, optionally
substituted with one or more groups selected from C1-C8 alkyl and
aryl. Alternatively, positive ionic groups are selected from
ammonium groups optionally substituted with C1-C4 alkyl groups.
Preferably, positively charged ionic groups are ammonium
groups.
[0037] Precursors of ionic groups are uncharged groups that can be
reacted under appropriate reaction conditions to produce an ionic
group, for example, an ester can be hydrolyzed to produce a
carboxylate anion. Precursors of ionic groups can include
optionally substituted alkyl esters, acids, amides, alkyl
anhydrides, cyclic anhydrides, and aryl esters of ionic groups.
Alternatively, precursors of ionic groups include optionally
substituted alkyl esters, alkyl anhydrides, amides, and cyclic
anhydrides. More preferably, precursors are alkyl esters, alkyl
anhydrides, or cyclic anhydrides. Most preferably, a precursor is
derived from a maleic anhydride group.
[0038] Substituents that are biomolecule-immobilizing groups are
those that react to form a covalent bond with the biomolecule while
maintaining some biological activity. Biomolecule-immobilizing
groups can be ionic groups or precursors thereof, such as carboxyl
or an anhydride, or can be other groups separate from the ionic
groups used to support EOF. Biomolecule immobilizing groups include
aldehydes, amines, alcohols, acid halides, alkyl halides,
photoprecursors to radicals such as diazo and substituted diazenes,
carboxyls, esters, alkyl anhydrides, cyclic anhydrides, thiols,
disulfides, and the like. Alternatively, a biomolecule immobilizing
group is selected from aldehyde, amine, carboxyls, esters, alkyl
anhydrides, cyclic anhydrides, thiols, and disulfides. More
preferably, a biomolecule immobilizing group is selected from
amines, carboxyls, esters, alkyl anhydrides, cyclic anhydrides, and
thiols. Most preferably, a biomolecule immobilizing group is a
carboxyl or a cyclic anhydride, for example, maleic anhydride that
is part of the polymer backbone.
[0039] Substituents that maintain the biological activity of an
immobilized biomolecule include hydrophobic groups. A hydrophobic
group, as the term is used herein, means a group which, as a
separate entity, is more soluble in octanol than water. For
example, the octyl group (C8H17) is "hydrophobic" because its
parent alkane, octane, has greater solubility in octanol than in
water. Specific examples of suitable hydrophobic groups include
n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl,
n-tetradecyl, n-octadecyl, 2-ethylhexyl, 3-propyl-6-methyl decyl,
phenyl, (para n-dodecyl)phenyl, naphthyl, anthracyl, 3-cholesteryl,
and the like. The hydrophobic substituent can be an alkyl,
cycloalkyl, aliphatic, cycloaliphatic, non-aromatic heterocyclic,
aryl, or heteroaryl group, or combinations thereof. The hydrophobic
substituents can be the same, for example, all n-C18, or different,
such as a range, for example, C24 through C28, or a mixture of
structural isomers, for example, branched and linear C16, or
mixtures of different groups, such as alkyi and aromatic, and the
like. For example, a hydrophobic group can be C8-C30 aliphatic,
polycyclic aliphatic, polycyclic aryl, or polycyclic heteroaryl, or
a C8-C30 alkyl optionally interrupted or substituted by, for
example, a heteroatom, a cycloaliphatic, aryl, or heteroaryl group,
and the like. Alternatively, a hydrophobic substituent is an
optionally substituted C8-C30 alkyl, polycyclic alkyl, polycyclic
aryl, or polycyclic heteroaryl. Preferably, a hydrophobic
substituent is a C8-C30 alkyl, and most preferably, a C12-C28
alkyl.
[0040] Substituents that substantially adhere the immobilizing
polymer to the biochip are those that can create strong noncovalent
or covalent interactions between the polymer and the biochip.
"Substantially adhere" means that the polymer is not washed away
during normal operation of the biochip. For example, hydrophobic
substituents can cause substantial adherence to a hydrophobic
microchannel wall through hydrophobic interactions, e.g., between
octadecyl groups and a polydimethyl siloxane wall. In another
example, ionic substituents can cause substantial adherence to a
ionic microchannel wall through electrostatic interactions, e.g.,
between tetraalkyl ammonium cations and siloxide wall anions. In
yet another example, a group can form a covalent bond with a
microchannel wall, e.g, an ionic group such as a carboxyl can form
a covalent bond with a channel surface functionalized with an amine
as in structure A. Preferably, the immobilizing polymer is adhered
to the wall through hydrophobic interactions or through covalent
interactions. More preferably, the immobilizing polymer is
substantially adhered through covalent interactions. Most
preferably, the immobilizing polymer is covalently attached to the
microchannel wall through a linker containing an amide bond, e.g.,
an amide bond formed between a carboxylate on the polymer and an
amine on the linker. In a particularly preferred embodiment, the
immobilizing polymer is covalently attached to a glass microchannel
wall through a linker represented by structure B:1 ##STR2## wherein
R.sup.s represents a surface --Si--O-- group on the glass
microchannel, R.sup.a is a C1-C8 alkylene chain; each R.sup.b is
independently a C1-C4 alkyl group; and D represents an amide bond
to the immobilizing polymer. For example, amide D can be formed by
amidation of a group on the polymer, for example, an acyl halide,
an ester, or an anhydride. For example, if the polymer is derived
from a maleic anhydride copolymer, amide D can be formed by
amidation of one of the maleic anhydride groups on the polymer.
[0041] In another alternative, the microfluidic biochip is made of
the immobilizing polymer itself, and is thus effectively
"substantially adhered" to itself by virtue of being a solid
slab.
[0042] The immobilizing polymer itself, i.e, prior to reaction with
a biocatalyst or adherence to a biochip, can be represented as a
copolymer of monomers, wherein the collective substituents of the
monomers perform the four functions discussed above. As used
herein, a copolymer involves a combination of monomers wherein at
least one of each monomer are present in the resulting polymerized
polymer in any order. "Can be represented" means that the monomers
are the hypothetical monomers that describe the repeat units of the
polymer, not necessarily the actual monomer used to make the
polymer (i.e. acetylene is the hypothetical monomer of
polyacetylene, which can also be made from cyclooctatetraene). The
polymer could be made from a single monomer that has appropriate
substituents, for example, 2-octadecyl acrylate.
[0043] More preferably, the immobilizing polymer can be represented
as copolymer of a monomer with an ionic group, e.g., carboxylate,
and a monomer with a hydrophobic group, e.g., C16 alkyl. Even more
preferably, the immobilizing polymer can be represented as a
copolymer of ethylenically unsaturated monomers, e.g., acrylic acid
and 1-octadecene. For example, an immobilizing polymer can be
represented as a copolymer including a first monomer such as
acrylic acid, itaconic anhydride, methyl methacrylate, allyl amine,
vinyl alcohol, maleic anhydride, maleimide, carboxylate, vinyl
carbamate, allyl sulfate, vinyl thiosulfate, vinyl sulfonate, allyl
phosphate, vinyl phosphonate, vinyl alcohol, vinyl
trimethylammonium chloride, other ethylenically unsaturated
monomers substituted with ionic groups or precursors thereof, and
the like. A second monomer includes a hydrophobic substituent, for
example, styrene, C8-C30 .alpha.-olefin, acrylic acid-n-dodecyl
ester, vinyl naphthalene, vinyl anthracene, (para
n-dodecyl)styrene, other ethylenically unsaturated monomers
substituted with hydrophobic groups, and the like.
[0044] A particularly preferred polymer is a maleic
anhydride-.alpha.-olefin copolymer, for example, poly(maleic
acid-alt-.alpha.-olefin, C24-C28) (PMA-OL), poly (maleic
anhydride-alt-1-tetradecene) (PMA-TD), poly (maleic
anhydride-alt-1-octadecene) (PMA-OD), and the like.
[0045] Other alternatives for the immobilizing polymer can be any
polymer that includes the functional substituents described above,
i.e., at least an ionic group, or precursor thereof, and a
hydrophobic group. Such polymers include appropriately substituted
polyvinylalcohol, polyethers, polyacrylic acids,
polyalkylacrylates, polymethacrylic acids, polyalkylmethacrylates,
polystyrene, polystyrene sulfonates, polyvinylnaphthalene,
polyethylvinylbenzene, polyaminostyrene, polyvinylbiphenyl,
polyvinylanisole, polyvinylimidazolyl, polyvinylpyridinyl,
polydimethylaminomethylstyrene, polyvinylamine,
poly-N-alkylvinylamine, polyallylamine, poly-N-alkylallylamine,
polydiallylamine, poly-N-alkyldiallylamine, polyalkylenimine, other
polyamines, polyamides, polyacrylamides, polymethacrylamides,
poly-N-alkylacrylamides, poly-N-alkylmethacrylamides,
polydiallylmethylammonium chloride, polytrimethylammonium ethyl
methacrylate, polytrimethylammonium ethyl acrylate, and copolymers
thereof.
[0046] The molecular weight of the immobilizing polymer itself,
i.e., prior to reaction with a biocatalyst or attachment to a
biochip, should be large enough that there are significant
interpolymer hydrophobic interactions between hydrophobic groups to
allow the immobilizing polymer to stably coat the flow surfaces,
yet small enough so that the polymer can be dissolved in a suitable
solvent for coating. This is highly polymer dependent, e.g.,
polyacrylate can be much higher molecular weight than polystyrene
sulfonate. The number average molecular weight of the polymer can
be, for example, between about 2,000 and about 50,000,
alternatively between about 2,500 and about 25,000, more preferably
between about 5,000 and about 20,000, and most preferably between
about 7,500 and about 15,000. A suitable solvent includes any
solvent that can dissolve the immobilizing polymer, for example,
protic solvents such as water, and alcohols; aprotic polar solvents
such as N,N-dimethyl formamide, dimethyl sulfoxide, nitromethane;
halogenated solvents such as chloroform, carbon tetrachloride, and
trichloroethylene; ethereal solvents such as tetrahydrofuran; aryl
solvents such as benzene, toluene, and xylenes; and the like.
Suitable solvents can be made acidic or basic, for example, a
polymer with carboxyl substituents can be more soluble in a basic
alcohol.
[0047] The immobilizing polymer itself can be further, or
alternatively, characterized by the numerical ratio of ionic
groups, or precursors thereof, to hydrophobic groups. In this
characterization, ionic groups, or precursors thereof, are counted
by the number of charges that can be generated, e.g., a carboxylate
and a carboxylic acid are each counted as one, and a maleic
anhydride and a phosphate are each counted as two. Each hydrophobic
group counts as one. In this fashion, the ratio of ionic groups to
hydrophobic groups in the immobilizing polymer itself can be
between about 1:20 to about 20:1, alternatively from about 1:8 to
about 8:1, more preferably between about 1:5 to about 5:1, even
more preferably between about 1:2 to about 2:1, or alternatively
about 1:1. In a particularly preferred embodiment, the ratio of
ionic groups to hydrophobic groups in the immobilizing polymer
itself is about 2:1.
[0048] Alternatively, the polymer can be characterized by the molar
ratio of monomers, separated into ionic monomers, i.e., those
contributing an ionic group (or precursor thereof) and hydrophobic
monomers, i.e., those contributing a hydrophobic group. The ratio
between ionic and hydrophobic monomers can be between about 1:20 to
about 20:1, alternatively from about 1:8 to about 8:1, more
preferably between about 1:5 to about 5:1, even more preferably
between about 1:2 to about 2:1 or most preferably about 1:1. In a
particularly preferred embodiment, the ratio of ionic monomers to
hydrophobic monomers is about 1:1.
[0049] The polymer can be produced in situ, or can be produced in
whole or in part and then coated and adhered to the solid
substrate. For example, maleic anhydride and an .alpha.-olefin can
be polymerized using a radical initiator in the presence of the
amine-functionalized biochip of structure A. The biomolecules can
then be reacted with the immobilized polymer under suitable
reaction conditions. Alternatively, the polymer can be produced
separately (e.g., polymerizing maleic anhydride and an
.alpha.olefin) added to the amine-functionalized biochip, thereby
immobilizing the polymer and coating the flow surface.
Subsequently, the biomolecule can be added under suitable reaction
conditions, thereby immobilizing the biomolecule. Alternatively,
the polymer can be produced, the biomolecule can be immobilized
thereon, and the biomolecule-polymer product reacted with the
amine-functionalized biochip.
[0050] As used herein, a biomolecule is any biologically derived
molecule that has biological activity, for example, binding
activity, catalytic activity, and the like. A biomolecule is
derived from any appropriate source, such as an enzyme domain
extracted from a natural organism, a protein genetically engineered
to be expressed in a different organism, a naturally derived
peptide synthesized to contain an unnatural amino acid, and the
like. A biomolecule can be a protein, a peptide, an antibody, an
enzyme, an enzyme cofactor, a catalytic antibody, an
oligonucleotide such as ribonucleic acid or deoxyribonucleic acid,
a lipid, an oligosaccharide, a polysaccharide, a purified cell
component, and the like. A biocatalyst is a biomolecule that can
catalyze a chemical or biochemical reaction, and includes any
associated cofactors necessary to carry out a particular catalytic
transformation. A biocatalyst can include, for example, a
catalytically functional portion of a catalytic antibody, an
enzyme, an enzyme domain, a catalytic peptide, an oligonucleotide
enzyme such as a ribonucleic acid enzyme or a deoxyribonucleic acid
enzyme, and the like. Alternatively, the biocatalyst can be a
catalytic antibody, an enzyme, or an enzyme domain. More
preferably, the biocatalyst is an enzyme, most preferably, a
catalytically functional portion of an oxidoreductase, a
transferase, a hydrolase, a lyase, an isomerase, or a ligase, i.e.,
IUBMB(International Union of Biochemistry and Molecular Biology,
www.chem.qmul.ac.uk/iubmb/ systematic enzyme classification (EC)
numbers 1, 2, 3, 4, 5, and 6, respectively).
[0051] In one embodiment, a biomolecule is directed from a source
on the biochip to a biomolecule-immobilizing group under suitable
reaction conditions, thereby immobilizing the biomolecule to the
immobilizing polymer at a flow surface. For example, FIG. 5A shows
a schematic of a biochip where an enzyme, invertase, is directed
from reservoir 20 to a flow surface coated with immobilizing
polymer (PMA-OD), i.e., region 24, where the invertase is
immobilized.
[0052] In another embodiment, at least two chemically distinct
biomolecules are immobilized on a polymer at a flow surface. The
chemically distinct biomolecules can be immobilized in the same
location or in distinct locations. Alternatively, at least two
chemically distinct biomolecules are separately directed and
immobilized on polymers at spatially distinct flow surfaces,
whereby the biomolecules are catalytically and spatially distinct.
For example, FIG. 5A shows a schematic of a biochip where two
catalytically distinct enzymes, invertase and glucose oxidase, are
directed separately from reservoirs 20 and 30, respectively, and
immobilized separately at locations 24 and 34.
[0053] As used herein, "chemically distinct" means biomolecules
that differ in chemical structure, molecular formula, molecular
weight, isotopic composition, cofactors, sequence (of monomers,
amino acids, DNA base pairs, RNA base pairs, and the like),
function, i.e., catalysis or binding, oxidation state, secondary
structure, tertiary structure, and the like. "Catalytically
distinct" or "distinct biocatalysts" means that two catalysts are
capable of catalyzing different chemical or biochemical
reactions.
[0054] The biomolecule immobilized on the polymer at the flow
surface can be characterized relative to the amount of biomolecule
theoretically needed to form a monolayer on the flow surface. It is
believed that interpolymer hydrophobic interactions form a highly
porous, three dimensional matrix at the flow surface, allowing
immobilization of more biomolecules than needed for a monolayer.
Thus, in one embodiment, the amount of biomolecule immobilized on
the polymer at each flow surface is between about 100% and about
800% of a theoretical monolayer, alternatively between about 110%
and about 500% of a theoretical monolayer, more preferably between
about 125% and about 500% of a theoretical monolayer, and most
preferably between about 150% and about 300% of a theoretical
monolayer. Of course, in many embodiments, much less biomolecule
will be required.
[0055] The biochip with an immobilized biomolecule can be used to
conduct chemical or biochemical reactions, for example, catalysis,
binding, polymerization, molecular recognition, and the like. For
example, in one embodiment, a reactant is directed from a source to
an immobilized biocatalyst under suitable reaction conditions,
thereby producing a first reaction product. In another embodiment,
the first reaction product is reacted with a second catalytically
distinct immobilized enzyme, thereby producing a second reaction
product. Alternatively, the first reaction product is directed to a
second catalytically distinct biocatalyst that is immobilized at a
spatially distinct flow surface, thereby producing a second
reaction product in a sequential manner.
[0056] One particularly preferred embodiment of the invention is a
microfluidics biochip, comprising at least one microfluidic
reservoir and a plurality of microfluidic channels. The channels
are each less than about 0.1 mm.sup.2 in cross-sectional area, and
the channels each comprise a flow surface. Furthermore, the
channels are in microfluidic communication with each other and with
the reservoir, and each channel is in microfluidic communication
with at least two electrodes, whereby an electroosmotic flow can be
generated at the flow surfaces. A polymer is coated on at least one
flow surface, wherein the polymer is substantially adhered to the
surface. The polymer comprises one or more ionic substituents of
the same polarity selected from optionally substituted carboxylate,
carbamate, sulfate, thiosulfate, sulfonate, phosphate, phosphonate,
or hydroxyl. The polymer also includes one or more hydrophobic
substituents selected from optionally substituted C8-C30 alkyl, a
polycyclic alkyl, or a polycyclic aryl. The polymer also includes
at least two catalytically distinct biocatalysts that are each
immobilized to the polymer through an amide bond. The electrodes
can be used to generate an electroosmotic flow to direct a reactant
solution from the reservoir to a first catalytically distinct
biocatalyst, whereby a first reaction product can be produced; and
the first product from the first biocatalyst can be contacted with
a to a second catalytically distinct biocatalyst, whereby a second
reaction product can be produced.
[0057] As used herein, "suitable reaction conditions" include
appropriate values for temperature, pressure, reaction time, pH,
solvent, presence of biocatalyst cofactors, consumable reagents
required such as adenosine triphosphate (ATP) or nicotinamide
adenine dinucleotide phosphate (NADPH), and the like, that permit
biological or biocatalytic activity.
EXEMPLIFICATION
[0058] Several microfluidic biochips were constructed by etching
microfluidic channels into glass microscope slides or by molding
channels into polydimethyl siloxane slabs. Slides were prepared as
EOF controls (i.e., plain glass microchannels treated to activate
siloxide anions on the channel surfaces), as immobilization
controls (i.e., siloxide-activated channels functionalized with
aminopropyl triethoxysilane (APTES) and then with glutaraldehyde),
and as a test system to demonstrate the invention (i.e.,
siloxide-activated channels functionalized with APTES and PMA-OL,
and PDMS (polydimethyl siloxane) channels coated with PMA-OD). The
enzymes that were immobilized included soybean peroxidase (SBP),
lipase, invertase, and glucose oxidase.
[0059] In these experiments, the ionic groups necessary to support
the electroosmotic flow in the PMA-OL and PMA-OD coated biochips
are provided by carboxyl groups. These groups are freed from the
maleic anhydride groups in the PMA polymer when the maleic
anhydride group reacts with either the enzyme, or, in the case of
the APTES-functionalized glass biochips, when the anhydride reacts
with the surface amine groups. The resulting negative charges
ensured significant flow in the enzyme-bound microchannels.
Example 1
PMA-OL Coating Enhances EOF Versus Glutaraldehyde Control Chips
[0060] In FIG. 1, the flow rate as a function of EOF voltage
applied to the biochip is shown for the EOF control (solid circle),
the glutaraldehyde-control (solid triangle), and PMA-OL-coated
channels (open circle). A linear relationship was obtained for all
three cases. For 5% PMA-OL coated glass at 1500 V, a flow rate of
about 100 nL/min (aqueous buffer containing 20% (v/v) DMF) was
obtained. Under the same conditions, the flow rate for the 5%
glutaraldehyde-control was only 10 nL/min. Such low flow rates are
most likely due to the loss of negative charges on the microchannel
walls, thereby slowing EOF-driven flow. By comparison, the EOF
control (solid circle) is about 200 nL/min under the same
conditions. Thus, the addition of negative charges by the free
carboxylates in the PMA-OL provide a substantial fraction of the
flow rate of the EOF control chip.
Example 2
PMA-OL Immobilized Eenzyme Retains High Biological Activity
[0061] A single step reaction consisting of SBP catalyzed oxidation
of p-cresol was examined: ##STR3##
[0062] The reactions were conducted by SBP catalyzed reaction of
cresol and H.sub.2O.sub.2 using a 2% PMA-OL -coated chip with 0.4
.mu.g immobilized SBP, 20 mM p-cresol and 0.125 mM H.sub.2O.sub.2
in phosphate buffer containing 20% (v/v) DMF. Initially, the
microchannel, with immobilized SBP, and product reservoir were
filled with the DMF-buffer solution, and substrates were added to
both substrate reservoirs. Substrates were supplied to the
microchannel by EOF allowing product accumulation to take
place.
[0063] As shown in FIG. 2A, SBP was active on the PMA-coated
microchannel using either 2% (filled circles) or 10% (open circles)
(w/v) PMA loading, corresponding to 0.4 and 0.6 .mu.g SBP loaded,
respectively. For the higher enzyme loading, complete consumption
of H.sub.2O.sub.2 was achieved at residence times as short as 0.2
min. Hence, SBP retained high activity in the biochip, and the rate
of reaction was limited by the rate of delivery of H.sub.2O.sub.2
to the microchannel. For the lower amount of SBP loaded into the
microchannel, the enzyme became rate limiting, with up to ca. 80%
conversion for a 2 min residence time. This enabled evaluation of
the reaction kinetics of the immobilized SBP, which followed a plug
flow Michaelis-Menten rate model (FIG. 2B), based on Equation 2: -
( ln .times. .times. C Af + C Af K m ) = ( V max K m ) .times.
.tau. - ( C Ao K m + ln .times. .times. C Ao ) ( 2 ) ##EQU2## In
this equation, C.sub.Af is the residual H.sub.2O.sub.2
concentration in the microchannel, .tau. is residence time of
substrate in the microchannel, and C.sub.A0 is initial
H.sub.2O.sub.2 concentration (0.125 mM). The term
C.sub.Af/V.sub.max was assumed to be small relative to the other
term on the left-hand side of Eq. 4 and was, therefore, ignored to
simplify the expression. This expression can be used when the
limiting substrate (e.g., H.sub.2O.sub.2) conversion is high at
long residence times, and the plot in FIG. 2B allows calculation of
the V.sub.max/K.sub.m (slope) and a Y-intercept that provides a
value for K.sub.m.
[0064] Calculation of SBP's kinetics of p-cresol oxidation reveals
that the kinetics are, surprisingly, largely unaffected by
immobilization to PMA in the 90 nL microchannels. Specifically, the
K.sub.m of SBP in the biochip was 0.98.+-.0.12 mM, compared with
0.95.+-.0.14 mM for p-cresol oxidation in soluble forms in both
5-mL volumes (in 20 mL scintillation vials) and 20 .mu.L volumes in
384-well plates. Similarly, the V.sub.max was 0.21.+-.0.03 .mu.mol
H.sub.2O.sub.2 converted/mg SBP-min, which was essentially
identical (0.20.+-.0.03 .mu.mol H.sub.2O.sub.2 converted/mg
SBP-min) for the soluble enzyme at larger scales.sup.12. These
results indicate that SBP displays intrinsically native activity
even in the immobilized form at the microscale, and further attests
to the mild immobilization conditions afforded by a polymer
containing ionic and hydrophobic groups such as PMA-OL. Moreover,
the PMA-OL matrix appeared to be highly porous, as the enzyme is
immobilized at a higher concentration than would be expected in a
single monolayer, yet no diffusional limitations are apparent, as
the immobilized SBP in the microchannel has a V.sub.max identical
to that in solution.
Example 3
PMA-OL Biochip Immobilizes Two Distinct Enzymes with High
Activity
[0065] A coupled lipase/peroxidase biochip was prepared where the
enzymes were co-immobilized using 5% (w/v) PMA solution. Lipase B
from C. antarctica was used for the first reaction, the hydrolysis
of p-tolyl acetate, which yields acetic acid and p-cresol, followed
by coupling of the cresol to poly(p-cresol) using SBP and
H.sub.2O.sub.2: ##STR4## The lipase was used at a 10-fold higher
concentration than SBP because of the expected low reactivity of
the lipase on the cresol ester, which was not expected to be a
highly reactive substrate of the enzyme. Reactions were initiated
upon flowing solutions of 15 mM p-tolyl acetate and 0.25 mM
H.sub.2O.sub.2 in phosphate buffer (pH 7.0) containing 20% (v/v)
DMF. The solubility of the tolyl acetate was greatly facilitated by
the presence of DMF. Reactions were performed in the microchannel
with 0.6 .mu.g total enzyme, 15 mM p-tolyl acetate, 0.25 mM
H.sub.2O.sub.2 and in the 384-well plate with 100 .mu.g/mL lipase,
10 .mu.g/mL SBP, 15 mM p-tolyl acetate, and 0.25 mM H.sub.2O.sub.2
in phosphate buffer containing 20% (v/v) DMF.
[0066] The two-step enzymatic transformation was sufficiently
reactive to give complete conversion of the H.sub.2O.sub.2 supplied
with a residence time of 3 min FIG. 3 (filled circles). The K.sub.m
and V.sub.max values for lipase in the microchannel were calculated
to be 0.59 mM and 0.13 .mu.mol/mg lipase-min. Also shown in FIG. 3
is the reactivity of the two-step enzyme transformation in solution
in a 384-well plate using 100 .mu.g/mL lipase and 10 .mu.g/mL SBP
(open circles). In this case, consumption of H.sub.2O.sub.2 was
followed as a function of reaction time. Clearly the high loading
of lipase in the microchannel and its high activity coupled with
the high activity of SBP resulted in an efficient bienzymic
system.
[0067] The higher reactivity of the bienzymic system in the
microfluidic biochip is likely due to the higher concentration of
the rate limiting enzyme, in this case the lipase, in the biochip
than in free solution. Assuming 0.6 .mu.g total protein loading
(the lipase and SBP are of similar size), then ca. 0.55 .mu.g of
lipase was immobilized to the microchannel, giving ca. 5.5 mg/mL
lipase concentration. This is far higher than the 100 .mu.g/mL
lipase, even if the aforementioned calculation was an overestimate
(we did not determine the loading density of lipase in the
microchannel) used in free solution in 384-well plates.
Example 4
PMA-OL Biochip Immobilizes Three Distinct Enzymes with High
Activity
[0068] In Example 4, a three-enzyme biochip was constructed to
demonstrate a 3 step reaction sequence that is purely synthetic,
i.e., is a non-natural pathway. Invertase cleaves sucrose into
glucose and fructose: ##STR5##
[0069] glucose oxidase oxidizes glucose using O.sub.2 to produce
H.sub.2O.sub.2; ##STR6##
[0070] and SBP uses the H.sub.2O.sub.2 to produce poly(p-cresol):
##STR7## The immobilized and solution phase systems used the three
enzymes, invertase, glucose oxidase, and SBP in a ratio of 10:10:2.
Reactions were performed in the microchannel with 0.6 .mu.g total
enzyme, 1 mM sucrose, and 20 mM p-cresol and in solution with 100
.mu.g/mL invertase, 100 .mu.g/mL glucose oxidase, 20 .mu.g/mL SBP,
1 mM sucrose, and 20 mM p-cresol in phosphate buffer containing 20%
(v/v) DMF.
[0071] FIG. 4 shows a comparison of the biochip reaction (filled
circles) with that of a solution-phase reaction in the 384-well
plate (open circles), also performed in the presence of 20% (v/v)
DMF. As with Example 3 above, the microchannel reaction was much
better than the solution-phase reaction, again likely because of
the higher concentrations of the invertase and glucose oxidase in
the biochip than in free solution. Interestingly, the microchannel
reactions did not show a lag in product formed in the trienzymic
pathway. This is likely due to the higher concentration of the rate
limiting enzyme (invertase or glucose oxidase) in the microchannel
as compared to free solution, thereby resulting in faster formation
of H.sub.2O.sub.2 to feed the SBP reaction leading to
poly(p-cresol).
Example 5
Biochip Conducts Multiple Sequential Catalyzed Reactions
[0072] In Example 5, the three-enzyme system of Example 4 was
established on a PDMS biochip, wherein the three enzymes were
immobilized at distinct locations. FIG. 5A shows a schematic of the
PDMS biochip. The substrate sucrose is provided from reservoir 10
and is directed down channel 12 towards the product reservoir 46.
At region 24, the sucrose is cleaved by immobilized invertase into
glucose and fructose. At region 34, the glucose reacts with
immobilized glucose oxidase to release H.sub.2O.sub.2. The p-cresol
substrate is directed from reservoir 42 to region 44, where
immobilized SBP reacts the p-cresol and H.sub.2O.sub.2 from the
previous step to produce poly(p-cresol). The poly(p-cresol) product
is then directed to reservoir 46.
[0073] FIG. 5B shows a comparison of the biochip reaction (filled
circles) with that of a solution-phase reaction in the 384-well
plate (open circles), also performed in the presence of 20% (v/v)
DMF. As with the preceding examples, the microchannel reaction was
much better than the solution-phase reaction, again likely because
of the higher enzyme concentrations in the biochip than in free
solution. Here, the overall microchannel reaction is slower than in
Example 4, principally because the reaction sequence has been
spatially, and therefore temporally separated. However, the
microchannel reaction is still considerably faster than the
solution reaction.
[0074] As in Example 4, the immobilized and solution phase systems
used the three enzymes, invertase, glucose oxidase, and SBP in a
ratio of 10:10:2. Reactions were performed in the microchannel with
0.6 .mu.g total enzyme, 1 mM sucrose, and 20 mM p-cresol and in
solution with 100 .mu.g/mL invertase, 100 .mu.g/mL glucose oxidase,
20 .mu.g/mL SBP, 1 mM sucrose, and 20 mM p-cresol in phosphate
buffer containing 20% (v/v) DMF.
Materials
[0075] SBP (54 purpurogallin units/mg solid; RZ=1.3), fluorescein
isothiocyanate (FITC)-labeled horseradish peroxidase (HRP),
invertase from Saccharomyces cerevisiae and glucose oxidase from
Aspergillus niger were purchased from Sigma (St. Louis, Mo.).
Lipase B from Candida antarctica (Chirazyme L-2) was purchased from
BioCatalytics (Pasadena, Calif.). 3-Aminopropyltriethoxysilane
(APTES), poly(maleic anhydride-alt-.alpha.-olefin) (PMA-OL),
glutaraldehyde, p-cresol, tolyl acetate, toluene, methanol (MeOH)
and dimethylformamide (DMF) were purchased from Aldrich (Milwaukee,
Wis.). Hydrogen peroxide (H.sub.2O.sub.2), NH.sub.4OH, NaOH, and
borosilicate glass microscope slides were obtained from Fisher
Scientific (Pittsburgh, Pa.). Buffered oxide etch was purchased
from Doe and Ingalls, Inc (Boston, Mass.). All other solvents and
reagents were obtained commercially at the highest purity available
and used without further purification.
[0076] Unless otherwise specified, substrates (20 mM p-cresol and
0.125 mM H.sub.2O.sub.2) were dissolved in 0.1 M sodium phosphate
buffer (pH 7.0) containing 20% (v/v) DMF (to aid inp-cresol
solubility). The substrates were loaded into opposite reservoirs on
the upstream side of the T-channel; each with 80 .mu.L of 40 mM
p-cresol and 0.25 mM H.sub.2O.sub.2 in phosphate buffer containing
20% DMF. The product reservoir was initially filled with 40 .mu.L
of phosphate buffer containing 20% DMF. After initiating different
flow rates by applying different voltages between the substrate
(anode) and product (cathode) reservoirs for 1 h, 20 .mu.L of
sample was withdrawn from the product reservoir. Minimal
electrolysis gases were observed to form at voltages less than 2500
V.
Glass Biochip Fabrication
[0077] Borosilicate microscope slides were cleaned with isopropyl
alcohol and acetone in a Class 100 clean room facility. The biochip
consisted of a simple T-shaped microchannel with two substrate
reservoirs and a product reservoir, and was fabricated using
standard photolithographic techniques, shown schematically in FIG.
6. Briefly, the pattern from a photomask was transferred onto a
glass slide, which was spin-coated with a layer of positive
photoresist (Shipley 1813, Microlithography Chemical Co.,
Watertown, Mass.). Wet etching was performed using 10:1 buffered
oxide etch solution for 45 min.
[0078] The dimensions of the channel, as measured using a
profilometer (Alpha-Step 2000, Tencor Instruments, Mountain View,
Calif.), were 15 .mu.m deep and 200 .mu.m wide at the center. The
microchannel was 30 mm long (V=90 nL) and each arm of the T channel
was 10 mm long. Holes were drilled into the end of the channels to
act as reservoirs for sample withdrawal and addition of substrate
solutions. The drilling dust was removed in an ultrasonic bath
before cover plate bonding.
[0079] The silanol groups on the surfaces of the etched glass slide
and the cover plate were activated by treating with a 1:1 solution
of NH.sub.4OH and H.sub.2O.sub.2 at 70.degree. C. for 25 min.
Channels activated in this manner could be used as the EOF control
without further surface modification. After the glass slide and
cover plate were rinsed with distilled water and dried using a
nitrogen gun, bonding was performed using UV-cured glue, lens bond
type SK-9 (Summers Optical, Fort Washington, Pa.). The glue was
spread between the cover plate and the etched glass slide by
capillary action and cured at room temperature by UV irradiation at
365 nm for 1 h. Subsequently, additional glass tubes of 100 .mu.L
volume as substrate and product reservoirs and platinum electrodes
were attached to the drilled holes using 2-ton epoxy glue (ITW
Devcon, Danvers, Mass.).
Surface Modification of Glass Biochip with PMA-OL and
Glutaraldehyde
[0080] FIGS. 7A and 7B show schematically the steps involved in
derivatizing the surface of the microchannels with PMA-OL and
glutaraldehyde, respectively. The dark circle with the pendant
--NH.sub.2 group represents the enzyme. First, the internal surface
of the microchannel was washed with MeOH:HCl (1:1) and then
concentrated H.sub.2SO.sub.4 each for 5 min and removing the
solutions by vacuum. MeOH:HCl and H.sub.2SO.sub.4 were loaded into
substrate reservoirs (with a micropipette) with one outlet (product
reservoir) connected to a vacuum pump (25 mm Hg) to enable flow
into the microchannel. The microchannel was then rinsed with
deionized distilled H.sub.2O for 30 min to give a solution pH of
7.0 and to provide silanol OH groups on the surface. Excess water
in the microchannel was removed by washing with acetone and the
biochip was dried at 110.degree. C. for 3 h. Amino group
functionalization was achieved by addition of 10% (w/v) of APTES in
anhydrous toluene containing 0.5% (v/v) CH.sub.2Cl.sub.2, and 10
.mu.L of this solution was supplied to the microchannel by vacuum.
Concentrated APTES, therefore, was not exposed to air and humidity,
and this prevented oxidation and hydrolysis. The biochip was dried
at 90.degree. C. for 2 h and then the microchannel was rinsed three
times with toluene.
[0081] A thin PMA-OL coating was prepared on the microchannel using
5% (w/v) of PMA-OL in toluene, and supplying 4 .mu.L of this
solution by vacuum. After removing excess PMA-OL in the
microchannel, the biochip was dried at 90.degree. C. for 1 h. To
wet the microchannel, a series of washings were performed with
acetonitrile (ACN), ACN:H.sub.2O (1:1), and deionized distilled
H.sub.2O; each kept in the microchannel for 15 min and then removed
by vacuum. Excess PMA-OL in the substrate and product reservoir was
then removed by washing with toluene.
[0082] For glutaraldehyde modification, the microchannel was rinsed
with toluene, ACN:H.sub.2O (1:1), and deionized distilled H.sub.2O
three times following APTES treatment.sup.8. Glutaraldehyde (5%,
v/v) in 0.1 M sodium phosphate buffer (pH 7.0) was loaded into the
substrate reservoirs at room temperature and supplied to the
biochip for 10 min by vacuum, and then for 2 h by EOF at 500 V.
Excess glutaraldehyde was rinsed out of the microchannel by vacuum
with 0.1 M sodium phosphate (pH 7.5) containing 1 mM MgCl.sub.2 for
30 min. A high voltage power supply (Model 215, Bertan Associates,
Inc., Syosset, N.Y.) was used to maintain a constant voltage
difference for EOF between substrate reservoirs and product
reservoir.
Enzyme Immobilization
[0083] Soybean peroxidase (SBP) (2 mg/mL) in 0.1 M sodium phosphate
buffer (pH 7) was supplied to the PMA-coated or
glutaraldehyde-derivatized microchannel for 8 h by EOF at 500 V to
immobilize enzyme. All enzyme and substrate solutions were filtered
prior to use to prevent particulates from clogging the
microchannel. A hydrophilic syringe-driven filter unit with 0.45
.mu.m pore size was used for the filtration (Millex-LCR, Millipore
Co., Bedford, Mass.). The enzyme solution was replaced every 2 h
due to the pH change in the reservoirs as a result of the
electrolysis of water generating H.sup.+ and OH.sup.- ions. Small
bubbles that often formed in the microchannel were removed by
vacuum. To remove excess enzyme after immobilization and before
reaction, the microchannel was rinsed with phosphate buffer
solution for 4 h by EOF at 1000 V. Immobilized FITC-labeled HRP was
visualized in the microchannel using a Spot RT camera attached to a
Nikon Eclipse TE 200 inverted microscope with TE-FM Epifluorescence
attachment (Micro Video Instruments, Avon, Mass.).
PDMS Biochip Fabrication
[0084] A 1:1 mask was created by printing a high-resolution drawing
of the desired microfluidic features as a negative (i.e., with the
desired microfluidic features transparent) on a standard
transparency film. A silicon master was created in a class-100
clean room facility from a silicon wafer (Silicon Quest
International, Inc., Santa Clara, Calif.). The silicon wafers were
cleaned with isopropyl alcohol and acetone, a layer of SU-8
negative photoresist (Microlithography Chemicals Co., MA) was
spin-coated at 2000 rpm and 40 sec, and the coated wafer was
pre-baked at 90.degree. C. for 20 min. The coated wafer was aligned
and exposed to ultraviolet light at 8.4 mW/cm.sup.2 (365nm) for 50
sec using a mask aligner through direct contact with the
transparency film mask. After post baking the wafer for 5 min at
90.degree. C., it was developed by immersion into SU-8 thinner
solution (Microlithography Chemicals Co., MA) for 1 min followed by
immersion into SU-8 developing solution (Microlithography Chemicals
Co., MA) until unmodified photoresist was removed, about 15 min.
The dimensions of the pattern on the silicon master were determined
using a profilometer (Alpha-Step 2000, Tencor Insturments, Mountain
View, Calif.).
[0085] A 10:1 weight ratio of monomer and curing agent (Sylgard
184, Dow Coming Co., MI) was mixed and degassed under vacuum. This
mixture was poured over the silicon master and cured at 70.degree.
C. for 2 hours. The patterened PDMS substrate thus formed was
peeled off, cut to working dimensions, and sealed using a cover
slab of PDMS. This was achieved by oxidizing the common surfaces of
the substrate and the cover slab using oxygen plasma for 10 sec and
then bringing them into conformal contact.
PDMS Biochip Surface Polymer Coating and Enzyme Immobilization
[0086] A thin PMA-OD coating of the microchannels was obtained by
flowing a 0.5% (w/v) of PMA-OD in acetone (4 .mu.L) through the
whole microfluidic network (FIG. 5A) using a vacuum. The enzymes
(10 mg/mL Invertase (reservoir 20), 10 mg/mL Glucose oxidase
(reservoir 30), and 2 mg/mL SBP (reservoir 40)) were supplied by
electro-osmotic flow (250 V and 4 hrs) to spatially immobilize them
in the microchannel at locations 24, 34, and 44, respectively.
Excess enzyme was washed with phosphate buffer (pH 7.0) containing
20% DMF for 30 min. The three-step enzymatic reactions were carried
out by supplying substrates 1 mM sucrose (from reservoir 10) and 20
mM p-cresol (from reservoir 42) for 1 hour.
Measurement of Enzyme Loading Density
[0087] To estimate the immobilized enzyme content in the
microchannel, the amount of immobilized SBP on a glass surface at
different PMA concentration was measured using the micro-BCA method
(Pierce Biotechnology, Inc., Rockford, Ill.). Glass (1 cm.times.1
cm) was incubated for 16 h in 10 mg/mL SBP solution after coating
with different concentrations of PMA and washed with phosphate
buffer and distilled water for 4 h before BCA analysis. The glass
squares with SBP covalently attached were incubated at 60.degree.
C. for 1 h in the micro-BCA working reagent (see
www.piercenet.com). The chromogenic product of the micro-BCA assay
is soluble in the assay reagent and was removed from the
immobilized enzyme preparation for spectrophotometric measurements.
The protein content was then determined based on a standard curve
with SBP in solution.
Fluorescence and Flow Velocity Measurement
[0088] Spectrofluorophotometry was used to determine the activity
of SBP. The intrinsic fluorescence of oligo- and polyphenols can be
used to monitor the formation of product as the monomeric phenols
have relatively minimal fluorescence. Sample analysis was performed
in a 384-well plate (Simport Plastics, Quebec, Canada) and the
reactions were monitored by measuring the relative fluorescence
intensity (RFU) using a BioAssay Reader HTS 7000 Plus (Perkin
Elmer, Norwalk, Conn.) at an excitation wavelength of 325 nm and
emission wavelength of 405 nm.
[0089] To determine the flow rate of substrate in the microchannel
at a particular voltage, a known concentration of the fluorescent
p-cresol oligomeric product (original RFU of 8,500) was transported
through the microchannel by EOF. The product reservoir was filled
with 40 .mu.L of phosphate buffer with 20% DMF. After 1 h of EOF
operation, 20 .mu.L samples were withdrawn from the product
reservoir and the RFU measured. The flow rate was calculated by
correlating the measured RFU as a function of the dilution ratio to
known oligomeric concentration.
Calculation of Reaction Yield
[0090] SBP catalysis gives a diverse array of oligomers that appear
to have widely different fluorescence properties. Moreover,
fluorescence intensity does not scale linearly with concentration,
as shown in Eq. 2, where where .phi. is the quantum efficiency,
I.sub.0 is the incident radiation power, .epsilon. is the molar
absorptivity, b is the path length of the cell, and c is the molar
concentration.sup.16. F=.phi.I.sub.0(1-e.sup..epsilon.bc) (2)
[0091] Thus, the biochip operating at different flow rates, thereby
resulting in different degrees of product dilution in the product
reservoir, will give different fluorescence intensities even if the
conversion is identical. To overcome this problem, we generated
poly(p-cresol) in a 5 ml reaction containing 20 mM p-cresol, 0.125
mM H.sub.2O.sub.2, and 20 .mu.g/ml SBP in phosphate buffer (pH 7.0)
with 20% (v/v) DMF to obtain a product with a net relative
fluorescence (RFU) of 8,500. This product was then diluted up to
100 fold and the fluorescence intensity measured as a function of
dilution. As expected, the RFU was logarithmically dependent on
dilution (curve not shown), and could be represented by the
expression in Eq. 3, where X is the dilution ratio in the product
reservoir and A is the original RFU of the sample after correcting
for dilution. Log Y=-0.85Log X+Log A (3) Based on this expression
the dilution ratio (X) in the product reservoir at different
voltages was calculated from the original RFU (8,500) in the
substrate reservoir (A) and diluted RFU measured in the product
reservoir (Y).
[0092] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *
References