U.S. patent application number 12/783477 was filed with the patent office on 2010-11-25 for flow-through method of functionalizing inner surfaces of a microfluidic device.
This patent application is currently assigned to NANYANG TECHNOLOGICAL UNIVERSITY. Invention is credited to Changming Li, Ling Yu.
Application Number | 20100297745 12/783477 |
Document ID | / |
Family ID | 43124821 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100297745 |
Kind Code |
A1 |
Li; Changming ; et
al. |
November 25, 2010 |
FLOW-THROUGH METHOD OF FUNCTIONALIZING INNER SURFACES OF A
MICROFLUIDIC DEVICE
Abstract
The present invention refers to a flow-through method of
functionalizing an inner surface of a microfluidic device. The
method can comprise transporting a silanizing solution through the
microfluidic device to silanize the inner surface of the
microfluidic device. The method can further comprise reacting the
silanized surface with an oxidized polysaccharide to bind the
oxidized polysaccharide to the silanized surface by transporting
the oxidized polysaccharide through the microfluidic device. The
present invention also refers to a microfluidic device which has
been subjected to a flow-through method of the present invention so
that at least a part of the inner surface or the entire inner
surface of the microfluidic device is functionalized.
Inventors: |
Li; Changming; (Singapore,
SG) ; Yu; Ling; (Singapore, SG) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
NANYANG TECHNOLOGICAL
UNIVERSITY
Singapore
SG
|
Family ID: |
43124821 |
Appl. No.: |
12/783477 |
Filed: |
May 19, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61296592 |
Jan 20, 2010 |
|
|
|
61179542 |
May 19, 2009 |
|
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Current U.S.
Class: |
435/287.2 ;
204/403.01; 422/52; 422/68.1; 422/82.08; 427/230 |
Current CPC
Class: |
C09D 105/02 20130101;
C09D 105/00 20130101; C09D 103/10 20130101; G01N 33/54393 20130101;
C09D 101/08 20130101; B01L 3/502707 20130101; C08B 37/0021
20130101; Y10T 428/24479 20150115 |
Class at
Publication: |
435/287.2 ;
204/403.01; 427/230; 422/68.1; 422/82.08; 422/52 |
International
Class: |
B05D 7/22 20060101
B05D007/22; G01N 27/26 20060101 G01N027/26; G01N 33/48 20060101
G01N033/48; C12M 1/34 20060101 C12M001/34; G01N 21/64 20060101
G01N021/64; G01N 21/76 20060101 G01N021/76 |
Claims
1. A flow-through method of functionalizing an inner surface of a
microfluidic device, wherein the method comprises: transporting a
silanizing solution through the microfluidic device to silanize the
inner surface of the microfluidic device; and reacting the
silanized surface with an oxidized polysaccharide to bind the
oxidized polysaccharide to the silanized surface by transporting
the oxidized polysaccharide through the microfluidic device.
2. The flow-through method of claim 1, wherein the oxidized
polysaccharide bound to the silanized surface is further oxidized
by transporting a glycol cleaving agent through the microfluidic
device.
3. The flow-through method of claim 1, wherein the inner surface of
the microfluidic device is oxidized before silanization.
4. The flow-through method of claim 1, wherein a part or the entire
inner surface of the microfluidic device is functionalized with the
flow-through method.
5. The flow-through method of claim 1, wherein the microfluidic
device is made of a material selected from the group consisting of
a silica based material, a ceramic material, a polymeric material
and combinations of the aforementioned materials.
6. The flow-through method of claim 5, wherein the silica based
material is a glass.
7. The flow-through method of claim 6, wherein the glass is
selected from the group consisting of borosilicate glass, fused
silica glass, and quartz.
8. The flow-through method of claim 5, wherein the polymeric
material is selected from the group consisting of poly(methyl
methacrylate) (PMMA), acetonitrile-butadiene-styrene (ABS),
polyethylene terephthalate (PET), poly-(dimethylsiloxane) (PDMS),
polycarbonate, polyethylene, polystyrene, polyolefin,
polypropylene, polyimide, and a polymeric organosilicon.
9. The flow-through method of claim 8, wherein the polymeric
organosilicon is poly(dimethyl siloxane) (PDMS) or poly(methyl
hydrosiloxane) (PMHS).
10. The flow-through method of claim 1, wherein the polysaccharide
is a glucan.
11. The flow-through method of claim 10, wherein the glucan is a
.alpha.-glucan or a .beta.-glucan or a mixture of both.
12. The flow-through method of claim 11, wherein the .alpha.-glucan
is at least one .alpha.-glucan selected from the group consisting
of dextran, glycogen, pullulan and starch.
13. The flow-through method of claim 11, wherein the .beta.-glucan
is at least one .beta.-glucan selected from the group consisting of
cellulose, curdlan, laminarin, chrysolaminarin, lentinan, lichenin,
pleuran and zymosan.
14. The flow-through method of claim 1, wherein at least one target
molecule is bound to the polysaccharide, wherein the at least one
target molecule is selected from the group consisting of a peptide,
a polypeptide, a peptoid, an inorganic molecule, a small organic
molecule, nucleotides, carbohydrates and a protein.
15. The flow-through method of claim 14, wherein the polypeptide is
selected from the group consisting of an antibody, a fragment of an
antibody and a proteinaceous binding molecule with antibody-like
functions.
16. The flow-through method of claim 14, wherein the antibody
fragment is selected from the group consisting of an Fab fragment,
a Fv fragment, a single-chain Fv fragment (scFv), a diabody and a
domain antibody.
17. The flow-through method of claim 14, wherein the proteinaceous
binding molecule is selected from the group consisting of a
lipocalin, an AdNectins, a tetranectin, an avimer and a
glubody.
18. The flow-through method of claim 14, wherein the protein is an
enzyme or an antibody or an antigen or a cytokine.
19. The flow-through method of claim 1, wherein the silanizing
solution comprises a silane coupling agent selected from the group
consisting of a vinylsilane, an acryloxysilane, a
methacrylatesilane, an isocyanatesilane, an epoxysilane, an
aminosilane and a mercaptosilane.
20. The flow-through method of claim 1, wherein the silanizing
solution comprises a silane coupling agent selected from the group
consisting of mercaptopropyltrimethoxysilane,
aminopropyltriethoxysilane, and
glycidoxypropyltrimethoxysilane.
21. The flow-through method of claim 19, wherein the silanizing
solution comprises the silane coupling agent in a concentration of
between about 10% to 100%.
22. The flow-through method of claim 3, wherein the surface
oxidizing agent is selected from the group consisting of a mixture
of hydrochloric acid and hydrogen peroxide in an aqueous solution,
piranha solution, UV/ozone oxidation, ultraviolet treatment, and
ultraviolet/ozone treatment.
23. The flow-through method of claim 2, wherein the glycol cleaving
agent is selected from the group consisting of a sugar oxidase,
sodium hypochlorite, tetra-acetate, hydrogen peroxide, periodic
acid, peracetic acid, alkali metal salts of periodate, or alkaline
earth metal salts of periodate, transition metal salts of
periodate, hypochlorite, perbromate, chlorite, chlorate, soluble
peroxide salts, persulfate salts, percarboxylic acids, oxyhalo
acids, and any combination of the aforementioned polysaccharide
glycol cleaving agents in any proportion thereof.
24. The flow-through method of claims 1, wherein to obtain the
oxidized polysaccharide a polysaccharide is oxidized using a
polysaccharide oxidizing agent which is selected from the group
consisting of sodium hypochlorite, tetra-acetate, hydrogen
peroxide, periodic acid, peracetic acid, alkali metal salts of
periodate, or alkaline earth metal salts of periodate, transition
metal salts of periodate, hypochlorite, perbromate, chlorite,
chlorate, soluble peroxide salts, persulfate salts, percarboxylic
acids, oxyhalo acids, and any combination of the aforementioned
polysaccharide glycol cleaving agents in any proportion
thereof.
25. The flow-through method of claims 1, wherein the silanisation
is carried out at ambient temperatures.
26. A microfluidic device which has been subjected to a
flow-through method of claim 1 so that at least a part of the inner
surface or the entire inner surface of the microfluidic device is
functionalized.
27. The microfluidic device of claim 26, wherein the microfluidic
device is a portable microfluidic device.
28. The microfluidic device of claim 26 for carrying out an
enzyme-linked immunosorbent assay (ELISA), fluorescent,
chemiluminescent, electrochemical immunoassay.
29. The microfluidic device of claim 26 for detecting multiple
analytes simultaneously.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
provisional application No. 61/179,542, filed May 19, 2009 and U.S.
provisional application No. 61/296,592, filed Jan. 20, 2010, the
contents of which are hereby incorporated by reference in their
entireties for all purposes.
FIELD OF THE INVENTION
[0002] The present invention refers to the field of surface
chemistry, in particular the surface chemistry of materials used
for microfluidic devices in biotechnological applications.
BACKGROUND OF THE INVENTION
[0003] Advanced analytical techniques are towards micro-analyzers
with advantages of small sample requirement, reduced assay time,
low manufacturing cost, and portable flexibility. Up to date,
immunoassay is still the main stream in the clinical diagnostic
methods and is widely used in various selective and sensitive
detections of small and large molecules through specific bindings
of antibody and antigen. The research and development of
immunoassay continues to fuel up by the high demand of the society
for health care quality, food quality control, monitoring and
biological security. With current progress in versatile,
inexpensive, and reliable techniques for microfabrication and
bio-interaction, antibody microarrays and microfluidic
immunosensors, or their combinations bring about new generation of
powerful analytical tools for important applications in proteomics,
drug discovery and diagnostics.
[0004] Most of the early devices were made using silicon and glass
as the substrates, since their microfabrication including
patterning, etching and bonding can directly borrow the matured
technology from the semiconductor industry. As an economic
alternative, polymer substrate was employed to fabricate the
microfluidic device by simple molding instead of the expensive
patterning process used in semiconductor industry. However, the
surface properties of the polymer microdevices are varied due to
diversity of the physical and chemical properties of the polymeric
materials in use.
[0005] Thus, it is an object of the present invention to construct
further surfaces with adequate homogeneity and specificity which
allow effective biomolecule immobilization along with a low degree
of nonspecific binding.
SUMMARY OF THE INVENTION
[0006] In a first aspect, the present invention refers to a
flow-through method of functionalizing an inner surface of a
microfluidic device. The method can comprise transporting a
silanizing solution through the microfluidic device to silanize the
inner surface of the microfluidic device at ambient temperature.
The method can further comprise reacting the silanized surface with
an oxidized polysaccharide to bind the oxidized polysaccharide to
the silanized surface by transporting the oxidized polysaccharide
through the microfluidic device.
[0007] In another aspect, the present invention refers to a
microfluidic device which has been subjected to a flow-through
method of the present invention so that at least a part of the
inner surface or the entire inner surface of the microfluidic
device is functionalized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The invention will be better understood with reference to
the detailed description when considered in conjunction with the
non-limiting examples and the accompanying drawings, in which:
[0009] FIG. 1 discloses a specific embodiment of the flow-through
modification method of the present invention. In this embodiment
the surface of the microfluidic device is oxidized and silanized.
In parallel, a polysaccharide is activated for binding to the
silanized surface of the microfluidic device. After contacting the
oxidized polysaccharide with the silanized surface the
polysaccharide binds to the functional group at the silanized
surface of the microfluidic device. In this embodiment, the
functional group is an amino group. However, other functional
groups can also be used, such as thiol, chloro, amino, vinyl,
methacryl or epoxy groups. In a further oxidation step the
polysaccharide bound to the surface of the microfluidic device is
further oxidized. Aldehydes generated by the oxidation reaction
provide a reactive functional group for the attachment of a target
molecule, such as an antibody.
[0010] FIG. 2 shows a schematic of dextran flow-through
modification method for modifying of PDMS surface in a microchannel
of a microfluidic device. This embodiment is similar to the
embodiment shown in FIG. 1. In this embodiment, the polysaccharide
dextran has been used which is oxidized by mixture with sodium
periodate (NaIO.sub.4). The surface of the microfluidic device in
this embodiment is poly(dimethyl siloxane) (PDMS) which is oxidized
with hydrochloric acid and hydrogen peroxide before silanisation
with aminopropyltriethoxysilane (APS also named APTES or APTMS).
After binding of the oxidized polysaccharide to the silanized
surface via the aldehyde groups of the oxidized polysaccharide, the
polysaccharide is further oxidated using sodium periodate
(NaIO.sub.4). Aldehyde groups generated by the oxidation reaction
with NaIO.sub.4 provide a reactive functional aldehyde group for
the attachment of an antibody via an amino group of an amino acid
of the antibody.
[0011] FIG. 3 shows a top (a) and section (b) views of a
microfluidic ELISA device. A microfluidic device or microchip as
shown in FIG. 3 can comprise flow-through inlet and outlet
channels. The test channels can be in fluid communication with the
inlet and outlet channels, through inlets and outlets,
respectively. Each test channel can comprise one test site therein
for detection of specific molecules or molecular interactions. The
inlet in a test channel can be elevated from the outlet of the test
channel and the outlet can be elevated from a fluid level in the
outlet channel.
[0012] FIG. 4 shows spectra of PDMS: (A). FTIR, (B). Raman, (a)
Pristine PDMS, (b) APTES modified PDMS, and (c) aldehyde-dextran
functionalized PDMS.
[0013] FIG. 5 shows calibration curves of IL-5 detection with
dextran-functionalized PDMS microfluidic devices. (a) Absorbance
curve for the flow-through ELISA; (b) Enzymatic converted substrate
in different channel (insert), from left to right, the reaction
analytes concentration are: 0, 1 pg mL.sup.-1, 10 pg mL.sup.-1, 100
pg mL.sup.-1, 1 ng mL.sup.-1, 10 ng mL.sup.-1, 100 ng mL.sup.-1, 1
.mu.g mL.sup.-1, and 10 .mu.g mL.sup.-1; S.sub.RGB versus IL-5
solution concentrations.
[0014] FIG. 6 illustrates a multi-detection test of the
flow-through ELISA in human serum solution: (1). CCD image of the
enzyme catalyzed substrate, (2) Bar plot of RGB signal at each
microchannel. Immobilized probe in each microchannel: a. negative
control, b. anti-IL-5 antibody, c. anti-HBsAg antibody, d.
anti-rabbit IgG antibody. Test A: injected testing sample contains
IL-5 (100 ng mL.sup.-1), HBsAg (100 ng mL.sup.-1) and rabbit IgG
(100 ng mL.sup.-1), test B: injected testing sample contains IL-5
(100 ng mL.sup.-1) and HBsAg (100 ng mL.sup.-1), test C: injected
testing sample contains IL-5 (100 ng mL.sup.-1) and rabbit IgG (100
ng mL.sup.-1).
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0015] In a first aspect the present invention refers to a
flow-through method for functionalizing an inner surface of a
microfluidic device. The method can comprise transporting a
silanizing solution through the microfluidic device to silanize the
inner surface of the microfluidic device. In one embodiment,
silanisation is carried out at ambient temperature. The method can
further comprise reacting the silanized surface with an oxidized
polysaccharide to bind the oxidized polysaccharide to the silanized
surface by transporting the oxidized polysaccharide through the
microfluidic device.
[0016] The present invention provides a reliable process carried
out under mild reaction conditions, which is used to functionalize
the surface of microfluidic devices for the subsequent binding of
target (bio)molecules without risking unspecific binding at the
surface of the microfluidic device. Unspecific binding can for
example result in bio-fouling. Surface functionalization according
to the method claimed herein can substantially limit or even
eliminate crossover interference and unspecific binding at the
surface of the microfluidic device. The polysaccharide
functionalization of the surface of the microfluidic device and the
subsequent target molecule immobilization enhances the
hydrophilicity and target molecule immobilization efficiency at the
surface.
[0017] As described in the experimental section, a microfluidic
device subjected to the claimed method was used in one example to
simultaneously detect multiple important biomarkers IL-5, HBsAg,
and rabbit IgG in a flow-through process. A sensitivity of 100 pg
mL.sup.-1 and a dynamic range of 5 orders of magnitude was
obtained, which significantly improved the performance compared to
previously known hydrophobic and plasma-treated hydrophilic
flow-through immunoassay devices. Moreover, the microfluidic
devices modified with the claimed method were used exemplarily in
colorimetric detection of proteins, of which the results are in
good agreement with the absorbance measurements and could be
observed clearly by naked human eye.
[0018] In the present invention, a `microfluidic device` refers to
a device that can be used to control and manipulate fluids with
volumes on the order of microliters, or nanoliters or picoliters.
In one embodiment, the volume is on the order of nanoliter and
picoliters. The devices themselves have dimensions ranging from a
few centimeter or millimeters (mm) down to micrometers (.mu.m) and
are also often referred to as microchip or Lab-on-a-chip.
[0019] Microfluidic devices are often made using silicon and glass
as the substrates, since their microfabrication including
patterning, etching and bonding can directly borrow the matured
technology from the semiconductor industry. As an economic
alternative, polymer substrate can be employed to fabricate the
microfluidic device by simple molding instead of the expensive
patterning process used in semiconductor industry. Thus, a
microfluidic device can also be defined as an instrument that uses
very small amounts of fluid on a microchip to do certain laboratory
tests.
[0020] The method of the present invention functionalizes an inner
surface of a microfluidic device. `Functionalizing` of an inner
surface means to modify the surface characteristics of the
microfluidic device to subsequently allow immobilization of a
target molecule at the inner surface of the microfluidic device.
With `inner surface` it is referred to any surface of the
microfluidic device that is exposed to the fluid flowing or passing
through the microfluidic device. In one embodiment the entire inner
surface of a microfluidic device or only a part of the inner
surface of the microfluidic device is functionalized as described
herein. In one example, the inner surface refers to any surface of
the microfluidic device that is used for carrying out chemical
reactions for an analytical method carried out with the
microfluidic device.
[0021] A microfluidic device can be made of a silica based
material, ceramic material or a polymeric material or their
combinations. A silica based material can be a glass. Examples of
suitable glass materials can include, but are not limited to
borosilicate glass, fused silica glass, and quartz. Examples of
ceramic materials include, but are not limited to conductive metal
oxides, such as ReO, TiO.sub.2, ZnO, CrO.sub.2, V.sub.2O.sub.3, and
various forms of indium tin oxide, and conductive transition metal
nitrides, such as titanium nitride, zirconium nitride, and chromium
nitride. The polymeric material used for the manufacture of the
microfluidic device can include, but is not limited to poly(methyl
methacrylate) (PMMA), polyethylene terephthalate (PET),
poly-(dimethylsiloxane) (PDMS), acetonitrile-butadiene-styrene
(ABS), polycarbonate, polyethylene, polystyrene, polyolefin,
polypropylene, polyimide or a polymeric organosilicon. An examples
for a polymeric organosilicon can include, but is not limited to
poly(dimethyl siloxane) (PDMS) or poly(methyl hydrosiloxane)
(PMHS). Different parts or sections of the microfluidic device can
be made of different materials. Sections of the microfluidic device
which are not used for the immobilization of a target molecule can
even be made of any material even materials for microfluidic
devices not referred to herein.
[0022] The process of silanization is known in the art.
`Silanization` refers to the covering of a surface which contains
hydroxyl groups (metal oxides, glass, polymers etc.) with a coating
that contains silane-like molecules. Thus making the surface
chemically inert. For silanization a silane coupling agent is used
to covalently attach any kind of functional group, such as a
primary amine functionality, to a surface of a microfluidic
device.
[0023] The general formula of a silane coupling agent shows two
classes of functionality. RnSiX(4-n). n can be 0 or 1 or 2 or 3.
The X functional group is involved in the reaction with the
inorganic substrate. The bond between X and the silicon atom in
coupling agents can be replaced by a bond between the inorganic
substrate and the silicon atom. X can be a hydrolyzable group,
typically, alkoxy, acyloxy, amine, or chlorine. The most common
alkoxy groups can include methoxy and ethoxy, which give methanol
and ethanol as byproducts during coupling reactions. R can be a
nonhydrolyzable organic radical that possesses a functionality
which enables the coupling agent to bond with organic resins and
polymers. Most of the widely used organosilanes have one organic
substituent.
[0024] One group of silane coupling agents can include, but is not
limited to mercaptopropyltrimethoxysilane (MPS or MPTS or MPTMS),
aminopropyltriethoxysilane (APS or APTES or APTMS), and
glycidoxypropyltrimethoxysilane (GPS or GOPS). In another example,
a silane coupling agent comprises a carboxylic-ester group. Such
silane coupling agents can include, but are not limited to
.gamma.-methacryl oxypropyltrimethoxy silane,
.gamma.-acetoxypropyltrimethoxy silane, and
.gamma.-acryloxypropyltrimethoxy silane.
[0025] Another group of silane coupling agents that can be used
herein includes alkoxy- or aralkoxysilanes of the R--SiX.sub.3
type, wherein R is an alkyl or aralkyl group substituted with polar
groups for hydrophilic modification. X can be alkoxy, aralkoxy or
alkyl, but at least one X per silane molecule is alkoxy or
aralkoxy.
[0026] R can contain 1 to 20 C-atoms in the optionally branched
alkyl chain. The alkyl chains can also include 8 or 18 C atoms.
Typically, the aryl portions of R and X contain 6 to 10 C-atoms.
The X alkyl portions can also contain 1 to 20 C-atoms.
[0027] For hydrophilic modification of the surface, if R is
substituted by polar groups, examples of possible substituents are
hydroxyl, amino, epoxy, cyano, halogen, ammonium, sulfonium and
carboxyl. Epoxy or amino groups are preferably employed as
substituents.
[0028] Another group of silane coupling agents can include, but is
not limited to a vinylsilane, such as vinyltrichlorosilane,
vinyltris(.beta.-methoxyethoxy)silane, vinyltriethoxysilane,
vinyltrimethoxysilane; an acryloxysilane, such as
3-metacryloxypropyl-trimethoxysilane; an epoxysilane, such as
.beta.-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane,
.gamma.-glycidoxypropyl-trimethoxysilane,
.gamma.-glycidoxypropyl-methylidiethoxysilane; an aminosilane, such
as N-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane,
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,
3-aminopropylmethyldiethoxysilane, 3-aminopropyltriethoxysilane,
3-aminopropyltrimethoxysilane, (N-trimethoxysilylpropyl)
polyethyleneimine, trimethoxysilylpropyldiethylenetriamine;
methacrylatesilane, such as 3-[tris(trimethylsiloxy)silyl]propyl
methacrylate; isocyanatesilane, such as 3-(triethoxysilyl)propyl
isocyanate; or .gamma.-mercaptopropyl-trimethoxysilane,
.gamma.-chloropropyl-trimethoxysilane. Depending on the specific
surface and functional group which is to be provided at the surface
of the microfluidic device a person skilled in the art can
determine the suitable silane coupling agent. Functional groups
that can be introduced at the inner surface of a microfluidic
device can include, but are not limited to thiol, chloro, amino,
vinyl, methacryl or epoxy groups.
[0029] A silanizing solution, such as an aqueous silanizing
solution can comprise the silane coupling agent in a concentration
range from about 10% to about 100% (v/v) with absolute alcohol,
such as ethanol, as solvent. The silanization reaction can be
carried out by pumping the silanizing solution through the
microfluidic device for a time of between about 20 min to about 2
hours or for between about 30 min to about 1 hour. After
silanisation the microfluidic device can be washed with an
alcoholic solution, such as an ethanol solution, and with water to
remove excess of silane coupling agent.
[0030] Silanization can be carried out at ambient temperature.
Ambient temperature is normally in a range of between about
15.degree. to 35.degree. C. or 20.degree. C. to 25.degree. C.
[0031] For the surface of the microfluidic device to be responsive
to a silanisation the surface preferably comprises hydroxyl groups.
Hydroxyl groups can be introduced by oxidizing the surface of the
microfluidic device. Thus, in one embodiment, the inner surface of
the microfluidic device is oxidized before silanisation by
transporting, i.e., flowing or guiding, a surface oxidizing agent
through the microfluidic device. Any oxidizing agent known in the
art and adapted to react with the surface of the microfluidic
device to form a hydroxyl group at the surface can be used.
Non-limiting examples include solutions of HCl and H.sub.2O.sub.2.
In addition, oxidization of PDMS can be done by physical approach
such as UV/ozone oxidation or ultraviolet and ultraviolet/ozone
treatment, such as described by Berdichevsky, Y.; Khandurina, J.;
Guttman, A.; Lo, Y. H., UV/ozone modification of
poly(dimethylsiloxane) microfluidic channels. Sens. Actuators B.
2004, vol. 97, no. 2-3, pp. 402 and Efimenko, K.; Wallace, W. E.;
Genzer, J., Surface modification of sylgard-184 poly(dimethyl
siloxane) networks by ultraviolet and ultraviolet/ozone treatment.
J. Colloid Interface Sci., 2002, vol. 254, no. 2, pp. 306. Other
treatments include, for example a chemical approach, such as with
piranha solution (conc. H.sub.2SO.sub.4 and 30% H.sub.2O.sub.2 3:1)
described by Taylor Pietro, Xu, Chun, Chem. Phys., 2003, vol. 5,
pp. 4918.
[0032] Oxidation of the inner surface of the microfluidic device
can be carried out for at least 3 min or at least 5 min or for
between about 3 min to about 30 min or for between about 5 min to
20 min. In one embodiment, oxidation of the inner surface was
carried out for 5 min. After oxidation of the inner surface of the
microfluidic device with the surface oxidizing agent, the inner
surface of the microfluidic device can be washed with an alcoholic
solution, such as an ethanol solution, and with water to remove
excess of surface oxidizing agent.
[0033] The polysaccharide used in the method of the present
invention for binding to the silanized inner surface of the
microfluidic device on the one side and to the target molecule on
the other side can be any polysaccharide known in the art.
Polysaccharides are polymers consisting of chains of monosaccharide
or disaccharide units. Saccharides (carbohydrates) comprise any of
a series of compounds of carbon, hydrogen, and oxygen in which the
atoms of the latter two elements are in the ratio of 2:1,
especially those containing the group C.sub.6H.sub.10O.sub.5. In
their open chain form saccharides contain a number of hydroxyl
groups and either one aldehyde or one ketone group. The aldehyde or
ketone group can react with a hydroxyl group in the same molecule
to convert the molecule into a ring; in the ring form the carbon of
the original aldehyde or ketone group can be recognized as the only
one that is bonded to two oxygens. Once the ring is formed, this
carbon can become further linked to one of the carbons bearing a
hydroxyl group on another sugar molecule, creating a disaccharide.
The addition of more monosaccharides in the same way results in
oligosaccharides of increasing length (trisaccharide,
tetrasaccharide, and so on) up to very large polysaccharide
molecules with hundreds or thousands of monosaccharide units.
[0034] Examples of polysaccharides that can be used herein include,
but are not limited to xylan, arabinoxylan, mannan, fucoidan,
galactomannan, galactan, glucan or chitin. In one embodiment, the
polysaccharide is a glucan. A glucan used herein can be an
(.alpha.)alpha-glucan or a (.beta.)beta-glucan or a mixture of
both. Examples of an alpha-glucan include, but are not limited to
dextran, glycogen, pullulan or starch. Examples of a beta-glucan
include, but are not limited to callose, laminarin, cellulose,
curdlan, laminarin, chrysolaminarin, lentinan, lichenin, pleuran or
zymosan.
[0035] To link the polysaccharide to the silanized inner surface of
a microfluidic device, the polysaccharide is oxidized. Oxidization
introduces a reactive aldehyde group in the polysaccharide which
converts glucose residues to hemiacetal structures. The oxidation
is determined by the concentration of the oxidant and the reaction
time. In one embodiment, between about 2 to about 12 hours is
suitable to oxidize the polysaccharide. In one embodiment, the
molar ratio of oxidant to monosaccharide unit is from 1:5 to
1:2.
[0036] For oxidizing the polysaccharide to obtain an oxidized
polysaccharide a polysaccharide oxidizing agent can be used. A
polysaccharide oxidizing agent can include, but is not limited to a
sugar oxidase, such as galactose oxidase for galactose, sodium
hypochlorite, tetra-acetate, hydrogen peroxide, periodic acid,
peracetic acid, alkali metal salts of periodate, or alkaline earth
metal salts of periodate, transition metal salts of periodate,
hypochlorite, perbromate, chlorite, chlorate, soluble peroxide
salts, persulfate salts, percarboxylic acids, oxyhalo acids, or any
combination of the aforementioned polysaccharide oxidizing agents
in any proportion thereof. In one embodiment, sodium periodate is
used. Oxidization of the polysaccharide can be carried out by
soaking the polysaccharide in water before subjecting it to the
polysaccharide oxidizing agent for a time that allows oxidation of
the polysaccharide. This can take several hours. In one example,
oxidation of the polysaccharide by incubation of a mixture of
polysaccharide and aqueous solution of polysaccharide oxidizing
agent took place overnight, i.e., about 12 hours. In one
embodiment, the suitable time range is from about 2 to about 12
hours.
[0037] After the oxidized polysaccharide has been immobilized at
the inner surface of the microfluidic device, a further oxidation
step can be carried out to the oxidized polysaccharide to allow a
target molecule to bind to the oxidized polysaccharide. For
oxidization of the oxidized polysaccharide bound to the surface of
the microfluidic agent a glycol cleaving agent can be used. The
glycol cleaving agent oxidizes the previously already oxidized
polysaccharide to introduce further aldehyde groups which allow
coupling of a target molecule. Examples of glycol cleaving agents
can include, but are not limited to sodium hypochlorite,
tetra-acetate, hydrogen peroxide, periodic acid, peracetic acid,
alkali metal salts of periodate, or alkaline earth metal salts of
periodate, transition metal salts of periodate, hypochlorite,
perbromate, chlorite, chlorate, soluble peroxide salts, persulfate
salts, percarboxylic acids, oxyhalo acids, or any combination of
the aforementioned glycol cleaving agents in any proportion
thereof. In one embodiment, sodium periodate is used as glycol
cleaving agent.
[0038] The oxidation of already oxidized polysaccharide can be
controlled by varying the concentration and reaction time. In one
embodiment, the incubation time is between about 2 to 6 hours.
[0039] In one embodiment, the glycol cleaving agent, which is used
for oxidation of the oxidized polysaccharide, does not introduce an
additional coupling agent or linker molecule which forms a bridge
between the oxidized polysaccharide bound at the surface of the
microfluidic agent and a target molecule to be bound to the
oxidized polysaccharide. Such a linkage group would for example be
introduced when using carbonyldiimidazole. Thus, the target
molecule binds directly to the oxidized polysaccharide via an
aldehyde group formed during oxidation of the oxidized
polysaccharide bound at the surface of the microfluidic device. The
glycol cleaving agent forms aldehyde groups in the polysaccharide
which can bind a target molecule.
[0040] For example, carbonyldiimidazole derivatized dextran can be
prepared in a reaction between hydroxyl group of dextran and
carbonyldiimidazole. Thus, the derivatized dextran could then be
coupled to aminosilane functionalized surface. In contrast, in the
method of the present invention an aldehyde group is introduced
which can react with an aminosilane modified surface.
[0041] The surface of the microfluidic device thus modified can not
be reacted with a target molecule which is to be immobilized at the
surface of the microfluidic device by binding to it via the
aldehyde groups of the oxidized polysaccharide which is already
immobilized at the surface of the microfluidic device via the
method of the present invention.
[0042] A target molecule can be any molecule that is needed for any
kind of chemical or biochemical assay that one wishes to carry out
with a microfluidic device, such as an organic or inorganic
molecule. Those target molecules are selected to be capable of
binding to an aldehyde group found on the oxidized polysaccharide
immobilized at the surface of the microfluidic device.
[0043] A target molecule can for example include, but is not
limited to peptide, a polypeptide, a peptoid, an inorganic
molecule, a small organic molecule, nucleotides, carbohydrate, or a
protein. The concentration of the target molecule used dependends
only on the application and availability of target molecule.
[0044] Peptoids can have a much higher cell permeability than
peptides (see e.g., Kwon, Y.-U., and Kodadek, (2007) T., J. Am.
Chem. Soc. 129, 1508-1509). A peptide may be of synthetic origin or
isolated from a natural source by methods well-known in the art.
The natural source may be mammalian, such as human, blood, semen,
or tissue. A peptide or polypeptide may for instance be synthesized
using an automated polypeptide synthesizer. Illustrative examples
of polypeptides are an antibody, a fragment thereof and a
proteinaceous binding molecule with antibody-like functions. In one
embodiment, an antiobody can be directed against an interleukin,
such as IL-5, or a surface antigen of hepatitis B-virus, such as
HBsAg. Examples of (recombinant) antibody fragments are Fab
fragments, Fv fragments, single-chain Fv fragments (scFv),
diabodies or domain antibodies (Holt, L. J., et al., (2003) Trends
Biotechnol., 21, 11, 484-490). An example of a proteinaceous
binding molecule with antibody-like functions is a mutein based on
a polypeptide of the lipocalin family (WO 03/029462, Beste et al.,
(1999) Proc. Natl. Acad. Sci. U.S.A., 96, 1898-1903). Lipocalins,
such as the bilin binding protein, the human neutrophil
gelatinase-associated lipocalin, human Apolipoprotein D or
glycodelin, posses natural ligand-binding sites that can be
modified so that they bind to selected small protein regions known
as haptens. Examples of other proteinaceous binding molecules are
the so-called glubodies (see WO 96/23879), proteins based on the
ankyrin scaffold (Mosavi, L. K., et al., (2004) Protein Science 13,
6, 1435-1448) or crystalline scaffold (WO 01/04144) the proteins
described in Skerra, (2000) J. Mol. Recognit. 13, 167-187,
AdNectins, tetranectins, and avimers. Avimers contain so called
A-domains that occur as strings of multiple domains in several cell
surface receptors (Silverman, J., et al., (2005) Nature
Biotechnology 23, 1556-1561). Adnectins, derived from a domain of
human fibronectin, contain three loops that can be engineered for
immunoglobulin-like binding to targets (Gill, D. S. & Damle, N.
K., (2006) Current Opinion in Biotechnology 17, 653-658).
Tetranectins, derived from the respective human homotrimeric
protein, likewise contain loop regions in a C-type lectin domain
that can be engineered for desired binding (ibid.). Peptoids, which
can act as protein ligands, are oligo(N-alkyl) glycines that differ
from peptides in that the side chain is connected to the amide
nitrogen rather than the .alpha.-carbon atom. Peptoids are
typically resistant to proteases and other modifying enzymes and
can have a much higher cell permeability than peptides (see e.g.,
Kwon, Y.-U., and Kodadek, T., (2007) J. Am. Chem. Soc. 129,
1508-1509). Where desired, a modifying agent may be used that
further increases the affinity of the respective moiety for any or
a certain form, class etc. of target matter.
[0045] In one embodiment, the protein which can be immobilized at
the surface of the microfluidic device via the oxidized
polysaccharide can be an enzyme, an antibody, an antigen, a
cytokine. Such proteins can be proteins related to various diseases
or cell signaling pathways.
[0046] In another embodiment, it is also possible to add further
blocking agents to the surface of the microfluidic device before
binding of the target molecule to the oxidized polysaccharide. Such
blocking agents are known in the art and can include, but are not
limited to bovine serum albumin (BSA), skimmed milk, goat serum,
rabbit serum or casein. Such blocking agents are for example often
used in immunoassays.
[0047] In another aspect, the present invention is directed to a
microfluidic device which has been subjected to a flow-through
method described herein so that at least a part of the inner
surface or the entire inner surface of the microfluidic device is
functionalized to allow binding of a target molecule. The
microfluidic device can be a portable microfluidic device. Portable
microfluidic devices are suitable for point of care services.
[0048] The method described herein provides simple flow-through
surface functionalization of microfluidic devices for different
applications, such as highly sensitive immunoassays. In one
embodiment the microfluidic device obtained by a method described
herein can be used for different kinds of enzyme-linked
immunosorbent assays (ELISA), a fluorescent immunoassay, a
chemiluminescent immunoassay, or an electrochemical immunoassay.
Due to the method of their manufacture ELISAs carried out on
microfluidic devices described herein can detect multiple analytes
simultaneously without crossover interference from different
analytes.
[0049] Such microfluidic devices can be manufactured with lower
manufacturing costs due to the easy flow-through functionalization
of a pre-formed microfluidic device. No commercial desk or portable
optical or electronical equipments are needed for their
manufacture. The microfluidic devices described herein can be used
for the detection of infectious diseases, such as hepatitis.
[0050] An example of a microfluidic device which can be used in
combination with the method of the present invention is the type of
microchip referred to in WO 05/095262 A1. Such a microchip
comprises a substrate, an inlet channel in the substrate, an outlet
channel in the substrate for guiding fluid below a fluid level, a
plurality of test channels in the substrate for guiding fluid flow
from the inlet channel to the outlet channel, each one of the test
channels having an inlet for fluid communication with the inlet
channel and an outlet channel for fluid communication with the
outlet channel, the inlet elevated from the outlet for inhibiting
back flow through the inlet, the outlet elevated from the fluid
level for inhibiting back flow through the outlet; and one test
site in each one of the test channels for detection of at least one
of a specific molecule and a molecular interaction at the test
site.
[0051] Each one of the channels can have a substantially flat
bottom, the bottom of the inlet channel elevated from bottoms of
the test channels, the bottoms of the test channels elevated from
the bottom of the outlet channel. In one embodiment, the at least
one of the test channels comprises a well for holding fluid
therein.
[0052] In another embodiment, the inlet and outlet channels are
adapted to allow a liquid to flow at a greater rate in said outlet
channel than in said inlet channel. Also, each one of said test
sites comprises a surface suitable for immobilizing specific
molecules thereon.
[0053] The microchip can further comprise probe molecules
immobilized at each one of said test sites, said probe molecules
having specific affinity to selected target molecules. Different
probe molecules can be immobilized at different ones of said test
sites for detecting different target molecules. The microchip can
further comprise a plurality of electrodes, one of said electrodes
at each one of said test sites.
[0054] In one embodiment, at least one of said test channels
comprises a narrowed section proximate said test site in said at
least one test channel for guiding fluid towards said test site.
The plurality of electrodes can be first electrodes, said microchip
can further comprise a plurality of second electrodes, one of said
second electrodes in each one of said test channels.
[0055] In another embodiment, the first electrodes can be
interconnected and said second electrodes can be interconnected,
such that each pair of said electrodes in each one of said test
channels is uniquely addressable.
[0056] The microchip can comprise probe molecules immobilized
proximate said first electrodes. The test channels can extend
substantially in parallel.
[0057] The microchip can further comprise a dilution channel formed
in said substrate, said dilution channel in fluid communication
with said inlet channel and having a first inlet for reception of a
first fluid and at least one second inlet for reception of a second
fluid to dilute said first fluid. The at least one second inlet can
comprise a plurality of second inlets. The substrate can be a
polymeric substrate as described herein. The substrate can comprise
polydimethylsiloxane (PDMS).
[0058] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including", "containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0059] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0060] Other embodiments are within the following claims and
non-limiting examples. In addition, where features or aspects of
the invention are described in terms of Markush groups, those
skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group.
EXPERIMENTAL SECTION
Materials
[0061] IL-5 and monoclonal anti-IL-5 antibodies were kindly provide
as gifts by BD Biosciences, USA and used as received. Rabbit IgG,
anti-rabbit IgG antibody, anti-rabbit IgG antibody peroxidase (HRP)
conjugate, phosphate buffered saline (PBS, 0.01M pH7.4), Tris
buffered saline (TBS, 0.01 M pH 8.0) containing 0.05% detergent
Tween 20 and dextran were purchased from Sigma-Aldrich, USA. HBsAg,
anti-HBsAg antibody, anti-HBsAg antibody HRP conjugate were
purchased from USBioscience, USA. Blocker.TM. Casein in PBS (pH
7.4) was purchased from Pierce, Germany. Sodium metaperiodate and
3,3',5,5'-tetramethyl benzidine, TMB, (1.25 mM TMB, 2.21 mM
H.sub.2O.sub.2, pH 3.1) were purchased from Merck KGaA, Germany.
Sylgard.RTM. 184 PDMS pre-polymer and the curing agent were
purchased from Dow Corning, USA. The deionized water used in all
experiments was produced by a water purification system, QGrad.RTM.
1, from Millipore Corporation, USA. All other used chemicals were
of analytical grades and obtained from common commercial
supplies.
Microfluidic Device Fabrication
[0062] FIG. 3a and b show the top and section views of a fabricated
PDMS microfluidic ELISA device, respectively. The device comprises
a plurality of microchannels in the substrate for guiding a fluid
flow from the inlet channel into detection well, and then to the
outlet channel. FIG. 3b shows the detail structure of the detection
well and its related connections, in which a flow is introduced
through the inlet manifold microchannel into the detection well and
then drains out to the waste outlet manifold microchannel by
overflow. The overflow outlet of the detection well is designed in
such a way (FIG. 3b) that its flow level is lower than the inlet
but significantly higher than the waste outlet microchannel, thus
totally eliminating backflow of the reacted fluid and inhibiting
cross-contaminations of the reacted fluid from one detection well
to another one due to the extremely high fluidic resistances in
such a structure. This unique feature can also allow enough contact
time for interactions of target and capture proteins in the
detection well. The device in more details is previously described
in WO 05/095262. This simple but delicate design can automatically
and efficiently conduct sampling, incubation, washing and detection
within the plurality of test sites for multiple immunoassay
screening. To fabricate the three-dimensional architectured
microfluidic device, a lithography process involving multiple-step
deep reaction ion etch (DRIE) was carried out to make the master
mold, which could be used to manufacture the device in FIG. 3a and
b through soft lithography. In the fabrication, the silicon wafer
was thermally oxidized to form a 1 mm oxide layer on its surface as
the sacrificial layer in the DRIE process and photoresist AZ7220
was applied for the patterning.
[0063] For PDMS casting, the precursor prepared by mixing PDMS
elastomer with the curing agent from Sylgard.RTM. 184 kit at a
weight ratio of 10:1 was poured onto the DRIE fabricated silicon
mold and cured at 80.degree. C. for 2 h. After the PDMS replica was
peeled from the mold, holes were made by a drill at the inlet and
outlet channels for connecting a syringe pump. A flat PDMS slab,
which was used to cover the replicated PDMS microstructure, was
obtained by casting and curing the precursor PDMS on a clean glass
dish. The replicated PDMS microstructure and flat PDMS cover were
washed with ethanol and deionized (DI) water successively, followed
by baking at 70.degree. C. for 1 h. Then they were treated by
oxygen plasma for 17 s in a plasma generator (Oxford Instruments,
UK). After plasma treatment, the flat PDMS cover was put on top of
the replicated PDMS microstructure followed by baking at 70.degree.
C. for 30 min. Silicon tubes were employed to further connect the
main inlet with a syringe pump for sample delivery and washing.
Dextran-Flow-Through Functionalization of PDMS Microchannels in
Microfluidic Device
[0064] Functionalization of the PDMS microchannels in the sealed
microfluidic device for protein immobilization was conducted with a
flow-through polysaccharide solution, such as a dextran solution
and the detailed chemical reactions are elucidated in FIG. 2. The
solution was prepared by dissolving 0.475 g dextran in 10 mL
deionized water, to which 0.232 g sodium periodate (NaIO.sub.4)
were added as the oxidant. The NaIO.sub.4/dextran mixture was
stirred overnight for partial oxidization of dextran to produce
aldehyde groups. After the oxidation process it was immediately
used for the microchannel modification. It was worthy a note that
all treatment and washing steps described below for the
microchannel functionalization were totally conducted by a
flow-through process. Before the functionalization, an oxidative
solution prepared by mixing hydrochloric acid and hydrogen peroxide
with H.sub.2O/HCl/H.sub.2O.sub.2 in a volume ratio of 5:1:1 was
pumped into the channels for 5 min reaction to oxidize PDMS
surface, followed by rinsing with 15 mL deionized (DI) water
min.sup.-1 for 10 min and with ethanol for 5 min. Then,
3-aminopropyltriethoxylsilane (APTES)/ethanol (50%, v/v) solution
was pumped at a flow rate of 2 .mu.L min.sup.-1 into the
microchannels for 30 min-pretreatment. The excess APTES was removed
by ethanol washing for 3 min followed by DI water washing for 6 min
(flow rate of 15 .mu.L min.sup.-1 in both operations).
[0065] For the functionalization, the freshly prepared partially
oxidized dextran solution was pumped at a flow rate of 2 .mu.L
min.sup.-1 into the APTES-treated microchannels for 1 h, allowing
dextran conjugate to PDMS via the formation of Schiff's base
between aldehydes and amines. The unattached dextran was flushed
away by 15 mL DI water min.sup.-1 for 5 min. The grafted dextran
was further oxidized by pumping NaIO.sub.4 (0.1 M) into the
microchannels for a 2 h treatment. Then the excess NaIO.sub.4 was
washed away by DI water for 5 min, and the channels were ready for
the protein covalent immobilization.
[0066] FTIR spectra of the pristine and dextran functionalized PDMS
were acquired by using a spectrometer (PerkinElmer, USA) in a range
of 600 to 4000 cm.sup.-1 with a signal resolution of 0.5 cm.sup.-1.
The Raman spectra of the PDMS specimens were recorded by Raman
microscope CRM2000 (Witek, Germany) equipped with a 488 nm laser
and 20 s integrated time. The contact angle measurements were
conducted with a contact angle analyzer (Dataphysics OCA20,
DataPhysics Instruments GmbH, Germany).
Flow-Through ELISA with Dextran-Functionalized PDMS Device
[0067] To demonstrate the performance of the PDMS microfluidic
ELISA device, IL-5 as a model analyte was measured and the
calibration curve was plotted. A 10 .mu.g mL.sup.-1-anti-IL-5
solution was pumped into the device for a 15 min incubation to
attach the probes in different channels. A flow-through washing was
performed with 15 mL TBS min.sup.-1 for 3 min to remove the
unbounded proteins. Casein was then pumped at a flow rate of 2
.mu.L min.sup.-1 into the device for a 20 min incubation to block
possible non-specific bindings. The analyte, IL-5 diluted in human
serum ranging from 1 pg mL.sup.-1 to 100 .mu.g mL.sup.-1 was pumped
into the device at a flow rate of 2 .mu.L min.sup.-1 and incubated
for 15 min, followed by flow-through washing with 15 mL TBS
min.sup.-1 for 4 min. The HRP-labeled monoclonal anti-IL-5 antibody
(2 .mu.g mL.sup.-1) was then pumped at a flow rate of 2 .mu.L
min.sup.-1 into the channels for a 15 min reaction followed by 5
min flow-through washing. Finally, the enzyme substrate, 1 mL TMB
min.sup.-1 was pumped into the microfluidic channels and then
converted to product by the captured HRP for colorimetric detection
(read by the naked eye). To confirm the colorimetric results read
by the naked eye, the solution of HRP-converted product in
different channels were collected into different wells of a
384-well ELISA-plate for absorbance measurement with ELISA reader
GENios Plus made by Tecan (USA).
[0068] The diagnosis of multiple proteins with the microfluidic
ELISA device was demonstrated by detection of biomarkers IL-5,
HBsAg and rabbit IgG in different microchannels. The probe
antibodies (capture proteins), anti-IL-5 (10 .mu.g mL.sup.-1),
anti-HBsAg (10 .mu.g mL.sup.-1), and anti-rabbit IgG (10 .mu.g
mL.sup.-1) were injected into different microchannels respectively
for 15 min of immobilizations. The second antibodies for the
sandwich detection were HRP conjugated anti-IL-5 (2 .mu.g
mL.sup.-1), anti-HBsAg (2 .mu.g mL.sup.-1) and anti-rabbit IgG (2
.mu.g mL.sup.-1) antibodies, respectively. The washing, blocking
and signal detection processes in the multiple detection were
conducted with the same method as that used for preparing the IL-5
calibration curve.
Results--Characterization of Dextran Functionalized PDMS
[0069] The FTIR spectra recorded from pristine and modified PDMS
are shown in FIG. 4A, in which the peaks at 1259, 1015, and 851
cm.sup.-1 are assigned to --CH.sub.3, Si--O--Si and Si--C. The
spectrum of the APTES-treated PDMS (FIG. 4A.b) shows a new broad
band from 3400 to 3000 cm.sup.-1, which can be assigned to
--NH.sub.2 stretch vibration. The existence of nitrogen on the
surface of APTES-treated PDMS suggests a successful chemical
grafting of aminosilane. New peaks at 3550 to 3300 cm.sup.-1
observed after dextran immobilization are assigned to the
stretching of the primary functional groups of alcohols, indicating
that dextran is covalently attached on the APTES treated PDMS (FIG.
4A.c) surface. Another new peak in the region of 1720 to 1760
cm.sup.-1 displayed after dextran immobilization can be assigned to
the aldehyde group, which is the key functional group for
efficiently and covalently immobilizing proteins for a highly
sensitive flow-through ELISA device.
[0070] The Raman results (FIG. 4B) are used to further confirm the
dextran immobilization on PDMS surface. The new peak observed after
dextran modification at 3345 cm.sup.-1 is assigned to OH stretching
(FIG. 4B.c), one of the characteristic peaks of dextran. The
imported hydroxyl groups can improve the hydrophilicity of the PDMS
surface. This result supports the conclusion that dextran
immobilization not only can enable the covalent binding through its
aldehyde group, but also enhances the hydrophilicity through its OH
groups.
[0071] The water contact angle was 109.28.degree. for the pristine
PDMS and 22.82.degree. for hydrochloric acid/H.sub.2O.sub.2 treated
PDMS. After APTES-modification, however, the contact angle was
raised to 66.97.degree. due to formation of the silane layer on
PDMS surface. After the subsequent dextran-modification, the
contact angle was reduced to 48.63.degree., since the attachment of
dextran improved the hydrophilicity by introducing hydrophilic
hydroxyl groups and polymer chains on the surface.
Results--Flow-Through ELISA with the Dextran-Functionalized PDMS
Device
[0072] FIG. 5a shows the calibration curve of IL-5 over a
concentration range of 1 pg mL.sup.-1 to 100 .mu.g mL.sup.-1. The
experiments were repeated 5 times to create the error bars and the
standard deviations were calculated by Origin 7.0. A sigmoid can be
seen over the whole detected concentration range and the linearity
lies between 1.times.10.sup.2 to 1.times.10.sup.6 pg mL.sup.-1,
illustrating a typical immunoassay characteristics. The limit of
detection (LOD) is determined as three standard deviations above
the background and is about 100 pg mL.sup.-1. The
dextran-functionalized PDMS microfluidic device demonstrated much
better performance than that of the hydrophobic PDMS based ELISA
device (LOD: 15 to 35 ng mL.sup.-1 and dynamic range: 1 to 3 orders
of magnitude). The high sensitivity of the device fabricated in
this embodiment is likely ascribed to the dextran-modified PDMS
surface with hydrophilic nature and efficient covalent probe
immobilization. The device performance is also much better than the
plasma-treated PDMS based microfluidic immunoassay device (LOD: 100
ng mL.sup.-1 and dynamic range: 3 orders of magnitude), which has
covalently immobilized proteins as probes, indicating its
significant improvement over the existing flow-through devices.
However, the LOD is still higher than the conventional ELISA
test-kit by one order of magnitude.
[0073] Interestingly, the conjugated-HRP oxidized product of TMB
had a blue color and the colorimetric dose responses could be
assessed by the human eye as shown in the inset of FIG. 5b,
demonstrating a great potential for fabrication of a potable
microfluidic ELISA device with multiple detections by naked human
eyes. In fact, quantitative analysis of the recorded colorimetric
images by means of RGB brightness based on color of red, green, and
blue (Nikon ACT-2U version 1.3, where
S.sub.RGB=0.229.times.red+0.587.times.green+0.114.times.blue) shows
a result (FIG. 5b) identical to that measured by absorbance (FIG.
5a), strongly supporting that the colorimetric detection is
accurate and could be used to simply assess the positive or
negative nature of the diagnostic biomarker by the naked eye.
[0074] A three 4-channel PDMS microfluidic ELISA moduled device
fabricated herein were used for simultaneously, colorimetrically
detecting IL-5, HBsAg and rabbit IgG in a flowthrough scheme. The
results are shown in FIGS. 6(1) and (2), where A, B and C
represents the three modules, while a, b, c, and d denotes the
different channels having different immobilized probes (a for
negative control where no capture protein was immobilized and b, c,
d for anti-IL-S, anti-HBsAg, and antirabbit IgG, respectively). In
order to demonstrate the simultaneously multiple detection, module
A was used to detect a sample containing mixed IL-5, HBsAg, and
rabbit IgG with an equal concentration of 100 ng mL.sup.-1, while B
and C were employed to detect samples containing mixed IL-5/HBsAg
and IL-5/rabbit IgG, respectively, but with an equal concentration
of 100 ng mL.sup.-1. Results shown in FIG. 6(1) illustrate that no
blue color is observed in the channel a where no capture proteins
were immobilized. There also is no observable blue color in channel
d of module B and channel c of module C since no specific analyte
in sample B (no rabbit IgG) and C (no HBsAg), demonstrating
expected negative responses. Other channels in module A, B, C show
a significantly blue color, indicating specific antigen-antibody
interaction. The significant response to the specific analytes in
comparison with nonspecific ones indicates good selectivity. FIG.
6(2) displays the bar plots of RGB brightness versus target
biomarker for all three modules, which clearly demonstrates that
all the measured background and negative responses are
significantly lower than the positive responses, which are expected
from the design. According to the colorimetric calibration curve in
FIG. 5b, the measured concentrations of the IL-5 in module A, B,
and C are 90.04, 98.67 and 94.34 ng mL.sup.-1, respectively, which
are in good agreement with the target concentration in the samples
(100 ng mL.sup.-1), showing a good assay accuracy and high
specificity in the multiple detection. Since the color changes for
multiple protein detection can be observed directly by naked eyes,
the microfluidic device demonstrates a great potential to fabricate
a portable, flow-though lab-on-chip system for high-throughput
screening of infectious diseases in health point-of-care
applications.
[0075] Thus, the device referred to in this embodiment is a
dextran-modified PDMS microfluidic ELISA device with a unique
architecture for eliminating crossover interference. The dextran
functionalization and the followed protein immobilization were
conducted with a simple, economical and fast flow-through process,
which greatly improved the hydrophilicity and protein
immobilization efficiency of the PDMS microfluidic ELISA device.
The device was used to simultaneously detect multiple important
biomarkers IL-5, HBsAg, and rabbit IgG by a flow-through process,
showing an excellent sensitivity of 100 pg mL.sup.-1 and a dynamic
range of 5 orders of magnitude, which significantly improved the
performance of the reported hydrophobic and plasma-treated
hydrophilic PDMS flow-through immunoassay devices. Moreover, the
fabricated PDMS devices were used in colorimetric detection of
proteins, of which the results are in good agreement with the
absorbance measurements and could be observed clearly by naked
human eye. Thus, the dextran-modified PDMS microfluidic device
referred to in this specific embodiment not only demonstrates a
great potential to fabricate an economical and sensitive
lab-on-chip system for applications in screening of various
infectious diseases, but also provides an opportunity to develop a
portable microfluidic ELISA device via human eye examination for
heath point-of-care services.
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