US20100297745A1

US20100297745A1 - Flow-through method of functionalizing inner surfaces of a microfluidic device

Info

Publication number: US20100297745A1
Application number: US12/783,477
Authority: US
Inventors: Changming Li; Ling Yu
Original assignee: Nanyang Technological University
Current assignee: Nanyang Technological University
Priority date: 2009-05-19
Filing date: 2010-05-19
Publication date: 2010-11-25
Also published as: US8383025B2; US20100323924A1

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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

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

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.
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.
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

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.
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

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:

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.

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 (NaIO₄). 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 (NaIO₄). Aldehyde groups generated by the oxidation reaction with NaIO₄provide a reactive functional aldehyde group for the attachment of an antibody via an amino group of an amino acid of the antibody.

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.

FIG. 4 shows spectra of PDMS: (A). FTIR, (B). Raman, (a) Pristine PDMS, (b) APTES modified PDMS, and (c) aldehyde-dextran functionalized PDMS.

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⁻¹, 10 pg mL⁻¹, 100 pg mL⁻¹, 1 ng mL⁻¹, 10 ng mL⁻¹, 100 ng mL⁻¹, 1 μg mL⁻¹, and 10 μg mL⁻¹; S_RGBversus IL-5 solution concentrations.

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⁻¹), HBsAg (100 ng mL⁻¹) and rabbit IgG (100 ng mL⁻¹), test B: injected testing sample contains IL-5 (100 ng mL⁻¹) and HBsAg (100 ng mL⁻¹), test C: injected testing sample contains IL-5 (100 ng mL⁻¹) and rabbit IgG (100 ng mL⁻¹).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

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.
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.
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⁻¹and 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.
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 (μm) and are also often referred to as microchip or Lab-on-a-chip.
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.
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.
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, TiO₂, ZnO, CrO₂, V₂O₃, 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.
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.
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.
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 γ-methacryl oxypropyltrimethoxy silane, γ-acetoxypropyltrimethoxy silane, and γ-acryloxypropyltrimethoxy silane.
Another group of silane coupling agents that can be used herein includes alkoxy- or aralkoxysilanes of the R—SiX₃type, 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.
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.
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.
Another group of silane coupling agents can include, but is not limited to a vinylsilane, such as vinyltrichlorosilane, vinyltris(β-methoxyethoxy)silane, vinyltriethoxysilane, vinyltrimethoxysilane; an acryloxysilane, such as 3-metacryloxypropyl-trimethoxysilane; an epoxysilane, such as β-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane, γ-glycidoxypropyl-trimethoxysilane, γ-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 γ-mercaptopropyl-trimethoxysilane, γ-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.
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.
Silanization can be carried out at ambient temperature. Ambient temperature is normally in a range of between about 15° to 35° C. or 20° C. to 25° C.
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 H₂O₂. 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. H₂SO₄and 30% H₂O₂3:1) described by Taylor Pietro, Xu, Chun, Chem. Phys., 2003, vol. 5, pp. 4918.
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.
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 C₆H₁₀O₅. 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.
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 (α)alpha-glucan or a (β)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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 α-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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The microchip can comprise probe molecules immobilized proximate said first electrodes. The test channels can extend substantially in parallel.
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).
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.
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.
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

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™ 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 H₂O₂, pH 3.1) were purchased from Merck KGaA, Germany. Sylgard® 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® 1, from Millipore Corporation, USA. All other used chemicals were of analytical grades and obtained from common commercial supplies.

Microfluidic Device Fabrication

FIG. 3 a 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. 3 b 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. 3 b) 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. 3 a 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.
For PDMS casting, the precursor prepared by mixing PDMS elastomer with the curing agent from Sylgard® 184 kit at a weight ratio of 10:1 was poured onto the DRIE fabricated silicon mold and cured at 80° 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° 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° 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

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 (NaIO₄) were added as the oxidant. The NaIO₄/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 H₂O/HCl/H₂O₂in 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⁻¹for 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 μL min⁻¹into 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 μL min⁻¹in both operations).
For the functionalization, the freshly prepared partially oxidized dextran solution was pumped at a flow rate of 2 μL min⁻¹into 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⁻¹for 5 min. The grafted dextran was further oxidized by pumping NaIO₄(0.1 M) into the microchannels for a 2 h treatment. Then the excess NaIO₄was washed away by DI water for 5 min, and the channels were ready for the protein covalent immobilization.
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⁻¹with a signal resolution of 0.5 cm⁻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
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 μg mL⁻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⁻¹for 3 min to remove the unbounded proteins. Casein was then pumped at a flow rate of 2 μL min⁻¹into 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⁻¹to 100 μg mL⁻¹was pumped into the device at a flow rate of 2 μL min⁻¹and incubated for 15 min, followed by flow-through washing with 15 mL TBS min⁻¹for 4 min. The HRP-labeled monoclonal anti-IL-5 antibody (2 μg mL⁻¹) was then pumped at a flow rate of 2 μL min⁻¹into the channels for a 15 min reaction followed by 5 min flow-through washing. Finally, the enzyme substrate, 1 mL TMB min⁻¹was 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).
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 μg mL⁻¹), anti-HBsAg (10 μg mL⁻¹), and anti-rabbit IgG (10 μg mL⁻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 μg mL⁻¹), anti-HBsAg (2 μg mL⁻¹) and anti-rabbit IgG (2 μg mL⁻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

The FTIR spectra recorded from pristine and modified PDMS are shown in FIG. 4A, in which the peaks at 1259, 1015, and 851 cm⁻¹are assigned to —CH₃, 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⁻¹, which can be assigned to —NH₂stretch 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⁻¹observed 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⁻¹displayed 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.
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⁻¹is 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.
The water contact angle was 109.28° for the pristine PDMS and 22.82° for hydrochloric acid/H₂O₂treated PDMS. After APTES-modification, however, the contact angle was raised to 66.97° due to formation of the silane layer on PDMS surface. After the subsequent dextran-modification, the contact angle was reduced to 48.63°, 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
FIG. 5 a shows the calibration curve of IL-5 over a concentration range of 1 pg mL⁻¹to 100 μg mL⁻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 1×10²to 1×10⁶pg mL⁻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⁻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⁻¹and 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⁻¹and 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.
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. 5 b, 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_RGB=0.229×red+0.587×green+0.114×blue) shows a result (FIG. 5 b) identical to that measured by absorbance (FIG. 5 a), 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.
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⁻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⁻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. 5 b, the measured concentrations of the IL-5 in module A, B, and C are 90.04, 98.67 and 94.34 ng mL⁻¹, respectively, which are in good agreement with the target concentration in the samples (100 ng mL⁻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.
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⁻¹and 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.

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 α-glucan or a β-glucan or a mixture of both.

12. The flow-through method of claim 11, wherein the α-glucan is at least one α-glucan selected from the group consisting of dextran, glycogen, pullulan and starch.

13. The flow-through method of claim 11, wherein the β-glucan is at least one β-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.