WO2007047644A2 - Method for microchannel surface modification - Google Patents

Abstract

A method (10) for functionalizing microfluidic channels with specific characteristics by the introduction of hydroxyl groups onto the surface area of the channel using a solution of an acid and a peroxide (30) followed by a reaction of an essentially pure siloxane-based reagent (60) and a funtional group (70). Chemically modified channel surfaces can be designed to prevent non-specific binding or interaction of biological samples with the channel surfaces; surface wetting or dewetting with the control of the hydrophobicity and hydrophilicity of the channel surfaces; confer solvent resistance by attaching inert molecules to the interior channel; and tethering molecules that have available amine, thiol, isocyanato and isothiocyanato or similar groups to permit the attachment of biomolecules to channel surfaces.

Description

METHOD FOR MICROCHANNEL SURFACE MODIFICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application serial number 60/727,218, filed on October 14, 2005, incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

OR DEVELOPMENT [0002] This invention was made with Government support under Grant No.

NIH-NCI UCLA SPORE in Prostate Cancer, Grant No: P50CA92131 and NIH-NCI Nanosystems Biology Cancer Center, Grant No: U54CA119347-01. The Government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL

SUBMITTED ON A COMPACT DISC [0003] Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION [0004] A portion of the material in this patent document is subject to copyright protection under the copyright laws of the United States and of other countries. The owner of the copyright rights has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the United States Patent and Trademark Office publicly available file or records, but otherwise reserves all copyright rights whatsoever. The copyright owner does not hereby waive any of its rights to have this patent document maintained in secrecy, including without limitation its rights pursuant to 37 C.F.R. § 1.14. BACKGROUND OF THE INVENTION

1. Field of the Invention

[0005] This invention pertains generally to microchannel and nanochannel functionalization, and more particularly to a method for a solution phase surface modification of polymer based microfluidic devices.

2. Description of Related Art

[0006] Modern microfluidic devices have found application in biological and chemical analysis, medical diagnostics, clinical and forensic analysis, environmental monitoring and molecular diagnostics. Many of the functions of a typical laboratory have been successfully incorporated in an integrated microfluidic chip based on two dimensional and three dimensional channels having lateral dimensions ranging from 10 μm to 1000 μm. More complex devices incorporate multiple fluidic, electronic and mechanical components including microscale pumps, valves, detectors, sensors, heaters and voltage sources.

[0007] The reduction of scale to a chip sized substrate provides analytical performance improvements as well as reducing the consumption of reagents, reduced manufacturing costs and an increased ability to automate the processes as well as allow for the easy transportation of the device. The development of improved manufacturing techniques has enabled the production of devices with increased complexity, capability and versatility at greatly reduced costs.

[0008] Early microfluidic devices were manufactured on silicon or glass.

However these microfabrication techniques for microchannels in silicon or glass are very specialized requiring specialized machinery and facilities and the accompanying expense. Later devices were manufactured from a variety of polymers such as polycarbonate, polystyrene, poly(methyl methylacrylate) (PMMA) and poly (dimethylsiloxane) (PDMS). Among various microfluidic systems, poly(dimethylsiloxane) (PDMS)-based microfluidic devices have been gaining popularity due to advantages such as easy fabrication, low cost, practical scalability, optical transparency, and gas permeability. Additionally, the elasticity of PDMS matrixes enables the integration of pressure-driven valves and pumps with microfluidic channels, permitting execution and automation of complex chemical or biological processes within a single microfluidic chip. [0009] Despite the many advantages of PDMS-based microfluidics, certain issues with these devices remain. (1) Due to the inherent hydrophobicity of

PDMS materials, PDMS-based microfluidic channels are repulsive to aqueous solutions. (2) PDMS microchannel surfaces have a tendency to adsorb molecules from the fluid flow onto the surface and channels can be easily contaminated by biological samples such as, peptides, proteins, serum, blood, and cells (the so-called "biofouling" issue). Some molecules actually migrate into the polymer matrix. (3) inconsistent and poorly controlled electroosmotic flow and (4) solvent sensitivity.

[0010] Therefore, several approaches for surface modification of PDMS materials have been developed in an attempt to confer hydrophilicity and biomolecule-repelling properties to PDMS surfaces. However, the surface modifications that have been attempted in the past have been shown to be unstable and have limited effective lifetimes. The prior attempts have also provided heterogeneous surface properties with inconsistent characteristics. For example, PDMS surfaces are typically treated with oxygen plasma, UV radiation, and UV/ozone to introduce hydrophilicity by replacing silane (Si-Me) groups with silanol (Si-OH) groups. However, the properties of these silanol- covered surfaces are dynamic. As a result, progressive restoration of hydrophobicity occurs within a few minutes of the application. Furthermore, the exposure to UV and Ozone often requires the opening the device and exposure of the entire chip to UV light that could influence the activity of other microchannel surface coatings and potential down time for the apparatus. Consequently, most of the existing surface modification methods are not feasible for modifying the surfaces of intact (fully assembled) microfluidic channels that are deeply embedded in PDMS matrixes. In addition, from the standpoint of device fabrication, surface-modified PDMS components often face the challenges of device assembly and microchannel sealing that can interfere with the integrity and longevity of the modified surfaces. [0011] In addition, the UV/Ozone activation does not provide a homogenous covering of surfaces leaving patches of microchannel surface areas that are partially or completely untouched and not activated. Consequently, the surface characteristics of the microfluidic system are heterogeneous and unpredictable.

[0012] Accordingly, there is a need for a process that can functionalize microfluidic channel surfaces within an assembled microfluidic system. There is a further need for a process that will provide a microfluidic system that has predictable channel surface characteristics that have a long functional life. There is also a need for a method of functionalizing fluidic channels that is simple to apply and easy to accomplish.

[0013] The present invention meets these needs as well as others and generally overcomes the limitations of the prior art.

BRIEF SUMMARY OF THE INVENTION [0014] The present invention is directed to a method for functionalizing fluidic channels, preferably microchannels and nanochannels, with a solution phase activation of channel surfaces followed by the of a tether molecules that can couple with a second functional element or molecule or directly with a functional element. Thus, the physical and chemical characteristics of the microfluidic channel can be modified to provide specific conditions.

[0015] Microfluidic devices have been widely used to conduct microscale biomedical analyses and chemical reactions, often with a substantial improvement in performance over conventional bench top systems. Among the various microfluidic systems, poly(dimethylsiloxane) (PDMS)-based microfluidic devices are particularly popular. Accordingly, a PDMS microfluidic system is used to illustrate the procedure. However, it will be understood that the described process may be applied to polymer and other materials available in the art and suitable for use in microchannel or nanochannel systems that are susceptible to the methods of the invention. [0016] Generally, the invention comprises a method utilizing a solution-phase oxidation reaction with an acidic peroxide solution and a sequential silanization reaction preferably using neat silane reagents for surface modification of intact microfluidic channels that are deeply embedded in polymer based matrixes. The activated surface can receive a tether molecule that has an available amine or thiol groups that then couple with a functional element or the functional element can attach directly to the activated surface. Therefore, the physical and chemical characteristics of the fluidic channel can be determined by the selection of functional groups and tethers. [0017] In contrast to the conventional approaches, (i.e., reactive oxidation and then silanization by diluted silane reagents), the present approach includes the advantages of (i) simple and convenient handling suitable for routine practices in both chemistry and biology laboratories, with no requirement of specialized instruments (i.e., oxygen plasma cleaner, UV light source, and ozone generator), (ii) great stability and fidelity of the resulting surface modifications with no observed decay of surface performance, and (iii) the method is appropriate for use with intact polymer based microfluidic devices, with no device post-assembly required.

[0018] One aspect of the invention is that it is universally applicable to microfluidic devices based on different materials including polymers such as poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA)₁ polycarbonate, polystyrene as well as solid-state silicon. [0019] According to another aspect of the invention, a method is provided that will permit the introduction of functional groups to the interior surfaces of microchannel substrates by the use of silanization reactions on the freshly prepared silanol-covered channel surfaces and thereby provide desired surface properties to the channel. For example, poly(ethylene glycol) (PEG) can be attached onto PDMS substrates by carrying out silanization reactions on the acid peroxide solution treated substrates. The resulting PEG-grafted PDMS surfaces exhibit protein-repelling characteristics with comparatively long lifetimes (a few weeks) against the recovery of hydrophobicity. [0020] According to another aspect of the invention, functional groups may also be coupled to a tethering group such as molecules with amino (NH2) groups and thiol (S-H) groups to be grafted onto channel surface substrates for subsequent attachment of a variety of biomolecules. Consequently, the surface modification can be utilized for the chemical immobilization of probe molecules in polymer-based microfluidic channels for applications including miniaturized biological diagnostic arrays and assays.

[0021] Another aspect of the invention is a method that can functionalize channel surfaces for many different purposes, including (a) surface passivation for inhibiting non-specific binding of biomolecules, (b) surface modification for controlling the hydrophobicity to hydrophilicity of microfluidic channels; (c) surface functionalization for introducing amine, carboxylic, isocyanate and isothiocyanate and other reactive groups to the microfluidic surface for further functionalization, as well anchor specific biomolecules (e.g., cells, peptides, proteins, enzymes, antibodies, DNA and RNA) for the application in chip-based bioassays and (d) channel solvent resistance in the field of microfluidics. [0022] Another aspect of the invention is that it is simple, convenient and fast for large scale fabrications in any biological laboratory. The whole process can be finished in 10 to 15 minutes, and no special equipment such as plasma reaction chambers, UV lamps or ozone generators is required. [0023] One embodiment of the invention is provided for modification or functionalization of a fluidic channel by activating the surface of a fluidic channel with a solution of an acid and a peroxide; exposing the activated surface to a siloxane solution thereby coupling the siloxane molecules the surface of the fluidic channel. [0024] In a second embodiment of the invention, a method for surface functionalization of a fluidic channel is provided by activating the surface of a fluidic channel with a solution of an acid and a peroxide; exposing said activated surface to a siloxane solution coupling said siloxane molecules to said surface of said fluidic channel; and then binding a functional element to said siloxane molecules. [0025] According to another embodiment of the invention, a method for surface functionalization of a fluidic microstructure surfaces is provided with the steps of pretreating the surface of a fluidic microstructure with a solution of a hydroxide and a peroxide; activating the pretreated surface of the fluidic structure with a solution of an acid and a peroxide; binding a plurality of siloxane tethers to hydroxyl groups of said activated surface of the fluidic structure; and then coupling a functional element to the siloxane tether. [0026] Further aspects of the invention will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the invention without placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL

VIEWS OF THE DRAWINGS [0027] The invention will be more fully understood by reference to the following drawings which are for illustrative purposes only:

[0028] FIG. 1 is a flow chart of one embodiment of the method for a solution phase microchannel functionalization in accordance with the present invention. [0029] FIG. 2 is a view of an exemplary scheme for PEG-grafted and amine- grafted microchannels in accordance with the present invention. [0030] FIG. 3 shows an exemplary scheme for DNA hybridization of the example shown in FIG. 2.

[0031] FIG. 4 shows an exemplary scheme for an Immunoassay of the example shown in FIG. 2. in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Referring more specifically to the drawings, for illustrative purposes the present invention is embodied in the apparatus generally shown in FIG. 1 through FIG. 4. It will be appreciated that the apparatus may vary as to configuration and as to details of the parts, and that the method may vary as to the specific steps and sequence, without departing from the basic concepts as disclosed herein. [0033] It can be seen that the method steps can be used for the modification or functionalization of fluidic devices and is particularly suited for the functionalization of micro and nano scale fluidic channels of a variety of cross- sectional sizes, shapes and lengths. For example, suitable microchannels may be an integral part of a variety of microfluidic devices known in the art manufactured from many different materials such as polymer matrixes, including poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), polycarbonate, polystyrene and solid-state silicon. Although PDMS is used to illustrate the functionalization methods, it will be understood that any material that is capable of channels that are susceptible to the solution phase oxidation reaction may be used.

[0034] Furthermore, the methods allow the modification of the surfaces of intact (fully assembled) microfluidic channels that are deeply embedded in polymer matrixes. Therefore, the surface-modified polymer surfaces will not experience damage during device assembly and microchannel sealing. The problems that have limited the further development of PDMS-based microfluidic devices in the biomedicine-related fields can now be avoided. In addition, the methods allow for integrated microfluidic systems with different channels that have been specifically functionalized with biomolecules in specific areas of the chip by the cooperation of a group of microfluidic channels and valves.

[0035] Turning first to FIG. 1 , a flow chart depicting the method steps 10 of one embodiment in accordance with the present invention. In the embodiment shown, hydroxy groups are formed on the side walls of preferably polymer-based microfluidic devices with three sequential solution-phase processes. At block 20 of FIG. 1 , an aqueous solution preferably containing NH₄OH and H2O2 (in a preferred mixing ratio of VNH4OH : V H202: V H20 approximately equal to 1 :1 :5 is introduced into the microfluidic channels. The solution is preferably introduced to the selected channels with a flow rate ranging from approximately 5 μl/min to approximately 50 μl/min for preferably a period of around 2 minutes. This preparatory step is optional and may not be necessary for some microchannel surface material compositions. [0036] At block 30, the initial flow of the first solution is followed by a second solution preferably comprising an aqueous solution of an acid, preferably hydrochloric acid (HCI), and a peroxide, preferably hydrogen peroxide (H₂O₂ ) in a preferred mixing ratio of V_Hcι : V H2₀₂: V _H2o of approximately 1 :1 :5 is introduced to the microchannel with a flow rate ranging from approximately 5 μl/min to approximately 50 μl/min for a period of approximately 2 minutes. In another embodiment, the channel is purged with deionized water and then dried with an inert gas before the second solution is dispensed through the channel. [0037] At block 40 of FIG. 1 , the acid peroxide solution is preferably followed by pure water with same loading rate of approximately 5 μl/min to approximately 50 μl/min for approximately 2 minutes. The water can optionally be purged with the use of a compressed gas such as Ar or air to remove the water and dry the microchannel or nanochannel. [0038] Although NH₄OH, HCI and hydrogen peroxide are used to illustrate suitable reagents at blocks 30 and 40, it will be understood that the initial solution and the acid peroxide solution can also be composed of other peroxides, acids and bases (e.g., NaOH, KOH and H₂SO₄) having generally similar characteristics. [0039] In another embodiment, a solution of acid peroxide and water in a preferred ratio VH_CI : V _H202: V _H20 of approximately 1:1 :5 is introduced to the microchannel with a continuous flow rate ranging from approximately 5 μl/min to approximately 50 μl/min for a period of approximately 5 minutes. The channel is then purged with de-ionized water and dry air. [0040] Referring now to block 50 of FIG. 1 , the hydroxyl groups that have been introduced on the activated surface area of the microchannels or other shaped chambers or arrays can react with many pure siloxane based reagents in the embodiment shown. At block 50, the hydroxyl group "anchors" are preferably reacted with neat siloxane reagents. Pure siloxane-based reagents (without dilution of any solvent) are preferably directly injected into the activated microchannels to react with the hydroxy-functionalized channels and may be followed by a treatment of pure water. Illustrative siloxane based reagents include 3,3,3-trifluoropropyl siloxane, octadecyl siloxane, ispbutyl siloxane, 3-glycodoxypropyl siloxane, PEG-based siloxane, (3-amin'opropyl)- triethoxysilane (APTES) and neat 2-[methoxy(polyethylenxy]propyl] trimethoxysilane. [0041] The selection of the siloxane based reagent will be influenced by the nature of the desired properties that are to be applied to the channel surface. Functional groups at block 70 may be directly applied or may be applied indirectly through the use of a tether at block 60. For example, direct functionalization of the channel may occur by the selection of siloxane compounds with suitable side groups or structure that will provide the desired surface characteristic. Alternatively, the selected siloxane compound at block 50 may serve as a tether at block 60 for the subsequent coupling of a functional group indirectly to the anchor on the channel surface at block 70. [0042] Accordingly, it is shown that the different chemically modified surfaces can serve many different purposes, including (i) surface passivation: attaching PEG onto the surfaces prevents the non-specific binding/interaction of biological samples (bio-fouling); (ii) surface wetting/dewetting: different attachment groups can be used to control the hydrophobicity and hydrophilicity of the microfluidic surfaces; (iii) further surface attachment: introducing tethers with amine, carboxylic, thiol, isocyanato, isothiocyanato and other reactive groups to the microfluidic surfaces can facilitate further surface attachments of a wide range of biomolecules (e.g., DNA₁ peptides, proteins, antibodies and cells) for bioassay applications, and (iv) solvent resistance: introducing Teflon-based or similarly inert molecules onto the surfaces of PDMS-based microfluidic channels can prevent the channels from being damaged by organic solvents. [0043] The attachment of a tethering group at block 60 to the activated hydroxyl anchors on the surface of the channel allows the introduction of a chemically active "site" (e.g. amine, carboxylic, thiol, isocyanato and isothiocyanato groups etc.) that can be utilized to attach other significant molecules indirectly to the surface. These tethers with chemically active grafting sites can be utilized for the immobilization of probe molecules for cell immobilization and incubation, semi-quantative DNA hybridization and immunoassays, for example. Being able to specifically control cell immobilization and cell repulsion in microfluidic channels should advance many types of cell cultures, cellular assays and microscale tissue engineering studies in microfluidic systems.

[0044] The solution-phase surface modification illustrated in FIG. 1 has been shown to provide stable and homogeneous surface functionalization in both preassembled and post assembled devices. The longevity of the surface modifications is also significantly longer than seen in the art. The direct attachment of functional groups as well as tethered functional groups has shown significant stability and longevity allowing the formation of a wide variety of functional applications on a micro or nano scale. [0045] The invention may be better understood with reference to the accompanying examples, which are intended for purposes of illustration only and should not be construed as in any sense limiting the scope of the present invention as defined in the claims appended hereto. The following examples illustrate the variety of possible uses and the viability of the methods.

EXAMPLE 1 (Surface Modification)

[0046] A test setup was constructed using experimental methods similar to the steps in the flow diagram shown in FlG. 1. Experiments for essentially all of the examples were conducted using single-channel microfluidic devices with channel heights of 25 μm, channel widths ranging from 100 μm to 200 μm and channel shapes of both linear and circular shapes were fabricated by soft lithography.

[0047] Fabrication of polymer based devices with microchannels using a number of different methods known in the art may be conducted that are optimized for the particular material that is selected. The microfluidic devices utilized in these examples were typically fabricated using a 25-μm thick negative photoresist (SU8-2025) that is spin coated on to a silicon wafer (Silicon Quest, San Jose, USA). After UV exposure and development, a fluidic mold with square-profiled patterns was obtained for the fabrication of PDMS-based microchannels. Before fabricating the device, the mold was exposed to trimethylchlorosilane (TMSCI) vapor for 2-3 minutes. A well-mixed

PDMS (GE, RTV 615 A and B in 5 to 1 ratio) was poured onto the mold located in a Petri dish to give a 5 mm-thick fluidic layer containing the microchannels transferred from the mold. The fluidic layer was then cured in an 80 ⁰C oven for 50 minutes. After curing, the fluidic layer was peeled off the mold, and holes were introduced onto the fluidic layer for access of reaction solutions. This fluid layer is then placed on top of a glass slide that was coated (2000 rpm, 60 s, ramp 15 s) with PDMS (GE RTV 615 A and B in 20:1 ratio) and then incubated for 45 minutes in an oven. The devices are normally ready for use after being incubated overnight in the oven. [0048] Introduction of hydroxyl groups on the interior surface of the PDMS- based microchannel was accomplished with three sequential solution-phase processes: (i) An aqueous solution containing NH₄OH and H₂O2 (in a mixing ratio of VNH4OH : V H202: V H20 = 1 :1 :5) was introduced into the microfluidic channels with a flow rate ranging from 5 to 50 μl/min for two minutes. This solution was followed by (ii) an aqueous solution of HCI and H₂O₂ (in a mixing ratio of VHCI ■ V H₂0₂: V H₂0 = 1 :1 :5) with a flow rate ranging from 5 to 50 μl/min for 2 min and purged by (iii) pure water (same loading rate for two minutes).

[0049] Several different siloxane compounds were applied onto their respective hydroxy-functionalized PDMS microfluidic channels. A bare channel was used as a control. Channels were functionalized by 3,3,3- trifluoropropyl siloxane, octadecyl siloxane, isobutyl siloxane, 3- glycodoxypropyl siloxane, and by PEG-based siloxane.

[0050] The functionalized channels were then exposed to solutions of fluorophore-labeled Avidin (1.0 mg/mL). It was seen that the PEG- functionalized PDMS microfluidic channel shows the best capability for surface passivation from non-specific interaction with Avidin. EXAMPLE 2

(Protein Passivation)

[0051] Another approach composed of an oxidation reaction in acidic H2O2 solution and a sequential silanization reaction using neat silane reagents for surface modification of poly(dimethylsiloxane) (PDMS) substrates was developed. Using this approach, two different functional groups, poly(ethylene glycol) (PEG) and a molecule with an available amine (NH₂), were introduced onto PDMS surfaces for passivation of nonspecific protein absorption and attachment of biomolecules, respectively. X-ray electron spectroscopy and temporal contact angle experiments were employed to monitor functional group transformation and dynamic characteristics of the PEG-grafted PDMS substrates. Fluorescent protein solutions were then introduced into the PEG- grafted PDMS microchannels to test their protein repelling characteristics.

These analytical data indicate that the PEG-grafted PDMS surfaces exhibit improved short-term surface dynamics and robust long-term stability over the art. The amino-grafted PDMS microchannels are also found to be relatively stable and can be further activated for modifications with the attachment of a peptide, DNA, or protein on the surfaces of microfluidic channels. The resulting biomolecule-grafted PDMS microchannels can be utilized for cell immobilization and incubation, semi-quantitative DNA hybridization, and immunoassays. [0052] Turning now to FIG. 2, modification of the PDMS based microfluidic channels started with the solution-phase oxidation reaction of PDMS channel surfaces 100. This was carried out by continuously passing a mixture of H2O/H2O2 / HCI (in a volume ratio of 5:1 :1) through the microchannels for 5 minutes. After purging the microchannels with deionized water and dry Ar, hydrophilic silanol-covered PDMS surfaces 102 were obtained. Sequentially, neat 2-methoxy(polyethylenxy)propyl]trimethoxysilane was injected 104 into the hydrophilic microchannels 102 to perform silanization reactions at room temperature for approximately 30 min. The unreacted silane was flushed from the microchannels by deionized water to give the PEG-grafted microfluidic channels 106, which were then dried by Ar flow and preserved in Petri dishes under ambient environmental conditions for various periods of time (from 10 min to 8 weeks) prior to the protein repelling studies. It should be emphasized that the use of the neat (solvent free) silane reagents are very effective for providing long-lasting protein repelling properties of the PEG-grafted microchannels. [0053] Protein repelling studies suggest that the PEG-grafted PDMS surfaces exhibited improved short-term surface dynamics and robust long-term stability. A variety of fluorescent protein solutions were introduced into the PEG-grafted PDMS microchannels to test their protein repelling properties. The results indicate that the PEG-grafted PDMS microchannels exhibited novel protein repelling characteristics, which enhanced progressively over the initial period of 24 hours and then persisted for a significant time (>2 months). To study the surface dynamic property of these PEG-grafted PDMS surfaces 106, a time-dependent investigation on the protein repelling characteristics of the PEG-grafted microchannels 106 was performed. A number of freshly prepared PEG-grafted microchannels 106 were preserved at ambient environmental conditions for 10 min, 30 min, 2 hour, 1 day, 2 days, and 2-8 weeks, respectively. In this case, the intact PDMS microchannels 100 were employed as the control. The protein repelling study of each microchannel was carried out by first filling the channel with a concentrated solution (10 μL) of fluorophore-labeled proteins and incubating the microfluidic chip at 37 °C for 1 hour. Three fluorophore-labeled proteins, i.e., fluorescein- labeled Avidin (1.0 mg/mL solution in PBS), Alexa594- labeled fibronectin (0.5 mg/mL solution in PBS), and Alexa555-labeled bovine serum were utilized. The resulting protein contaminated microchannel was then cleaned by flushing a PBS solution (100 μL, for 30 s) through the microchannel. Finally, the nonspecific absorption of the fluorophore-labeled protein was quantified by fluorescent microscopy. For each microchannel, more than 30 fluorescent measurements were carried out at different locations where the channel widths and shapes are different. These measurement results showed a very small difference (<5%) through the entire microfluidic channel, suggesting that the PEG surface modification was quite homogeneous, without much influence by the channel widths and shapes. The time-dependent profiles for the protein repelling characteristics of the microchannels 4 showed consistent results for all three protein solutions. During the initial 24 h, the protein repelling property of the microchannels 4 improved progressively; thereafter, the protein repelling property lasted for more than two months. It was noted that the dynamic characteristics of protein repellency showed a good agreement in time scale with that observed for the temporal contact angle measurements. The consistency of these observations suggests that the robust cross-linked silane layers bear well oriented PEG chains exhibiting excellent protein-repelling property. [0055] This PEG modification approach was also performed in microfluidic devices incorporating a number of pressure-driven valves. These valves operated normally after the PEG modification, verifying that this approach is applicable for integrated microfluidic systems.

EXAMPLE 3 (Tether Modification) [0056] Still referring to FIG. 2, similar to the preparation of the PEG-grafted microfluidic channels 106, the silanol-covered PDMS microchannels 102 were reacted with (3-aminopropyl)trimethoxy silane 108 for 30 minutes to generate the amino-grafted PDMS surfaces 109. The surface-grafted amino groups were converted to the isothiocyanate groups by introducing a 0.5% (v/ v) thiophosgen solution in MeCN into the amino-grafted microchannels 109 for 20 minutes at 40 ⁰C. Again, after purging with deionized water and dry Ar, the isothiocyanate-grafted PDMS microchannels 110 were then subjected to attachment reactions with a variety of amino-terminated biomolecules, including tripeptide RGD, single-stranded DNA (5¹- NH₂-(CH₂)6- TTTTTTGGTT-GGTGT-GGTTGG-3') and PSCA protein to produce RGD- grafted PDMS surfaces 114, DNA grafted PDMS surfaces 116, and PSCA- grafted PDMS surfaces 118, respectively. These biomolecule-grafted microfluidic channels 112 were washed with phosphate-buffered saline (PBS, pH 7.4) or Tris-buffer solutions (pH 7.4) and preserved at 4 °C for at least 24 hours prior to their respective studies. [0057] It was observed that the amino-grafted PDMS microchannels are fairly stable and can be further activated for surface modifications with three types of biomolecules, including tripeptide (arginine-glycine-aspartic acid, RGD), amino-terminated single-stranded DNA, and a soluble, recombinant form of prostate stem cell antigen, PSCA. Using these biomolecule-grafted PDMS microchannels, cell immobilization and incubation, semiquantitative DNA hybridization, and immunoassay were demonstrated in a miniaturized fashion, with the additional benefits of chemical/sample economy and operational efficiency.

[0058] By attaching different functional groups onto the surfaces of the microfluidic device, the surfaces can serve many different purposes. The attachment of a second stable molecular layer (tethers) can be used to provide an available reactive site (e.g. amine, carboxylic, thiol, isocyanato and isothiocyanato groups etc.) that is different from the channel wall and permits additional reactions and the attachment of a wide variety of biomolecules.

EXAMPLE 4 (Cell Adhesion) [0059] The ability to specifically control or immobilize cells and cell repulsion in microfluidic channels is important to many types of cell culture studies, cellular assays, and microscale tissue engineering studies in microfluidic systems. For example, the Tripeptide RGD, the smallest active fragment found in the extra cellular matrix, is known to be an important ligand for cell immobilization through the RGD-integrin (a transmembrane protein) interactions. RGD- grafted microchannels (such as 114 of FIG. 2) were produced to test the feasibility to immobilize cells in PDMS-based microfluidic channels and a PEG-grafted microchannel 106 was used for repelling cell adhesion. Intact PDMS microchannels 100 served as the control for both types of microchannels.

[0060] Here, A427 cells (colon cancer cell line, ATCC) suspended in

Dulbecco's modified Eagle medium (DMEM) cell culture media (Invitrogen) were utilized in all experiments. A427 cells in culture medium were introduced into the microchannels with surface configurations shown in 100, 106, 109 and 114 of FIG. 2, with a pressure of approximately 3 psi. The microfluidic chips were placed in the incubators at 37 °C for 4 hours. Culture medium was then slowly flushed through the microchannels to remove unattached A427 cells. It was observed that a large number of A427 cells were immobilized on the RGD-grafted microchannels 114. In contrast, no cells were immobilized in the PEG-grafted microchannels 106. A few cells were retained in the amino- grafted microchannels 109 and intact PDMS microchannels 100. The immobilized A427 cells survived for 4 days in the microchannels with the immobilized RGD 114, when these chips were kept at 37 °C and the cell culture medium was continuously and slowly introduced into the microchannels by gravity.

[0061] In another experiment, after the surface pretreatment, an isothiocyanato group was introduced onto the microfluidic surface by flushing the microfluidic channel with its trimethoxylsilane precursor. This group can readily react with amino groups for attachment of protein molecules. Fibronectin was attached onto this type of microfluidic channels and then employed for cell immobilization. A431 cells were introduced into PDMS- based microfluidic channels, which were functionalized by a) fibronectin, b) bare PDMS and c) PEG. The fibronectin functionalized microfluidic channel showed excellent properties for immobilization of the A431 cells. The result for bare PDMS microfluidic channel was ambiguous while the PEG-coated surface was totally inert to non-specific absorption of these cells. EXAMPLE 5

(DNA Microarray) [0062] Turning now to FIG. 3, one scheme for providing a microarray is generally shown. Although a DNA microarray is shown, it will be understood that arrays for nucleic acids and other biomolecules can be provided using the methods of the invention.

[0063] DNA microarray's are widely used tools in biomedical research and forensic diagnostics. This technology requires specific attachment of probe DNA fragments onto discrete locations within a two-dimensional surface. Using the improved PDMS surface modification, probe DNA fragment (5^J- NH₂-(CH₂)₆-TTTTTTGGTT-GGTGT-GGTTGG-3') was attached onto microfluidic channels for demonstration of a semiquantitative DNA hybridization using the technique shown in FIG. 2. A new type of PDMS- based microfluidic chip carrying parallel and individual accessible microfluidic channels (channel width, 100 μm) was fabricated and modified with the probe DNA fragment for this demonstration. These DNA-grafted microfluidic channels 116 were specifically exposed to different concentrations (i.e., 5 nM, 25 nM, and 50 nM, in Tris-buffer, pH 7.4) of the fluorophore-labeled target DNA (5'-5CyS-CCAACCACA-CCAACCA-S¹) solutions. An intact PDMS microchannel 1 was treated with 50 nM DNA solution was used as a control. [0064] To test specificity, another set of DNA-grafted microfluidic channels

116 as shown in FIG. 2 were exposed to the same concentrations of the fluorophore-labeled triple-mismatch DNA (5'-5Cy3-CGAACCACTCCAAGCA-

3') solutions 120. FIG. 3 is a schematic representation of the DNA hybridization inside the DNA grafted microchannels 116 as shown in FIG. 2. [0065] After carefully washing the channels with Tris-buffer solution, the resulting DNA hybridizations were measured and quantified under fluorescent microscopy. The observed integrations of fluorescence intensity across the microchannels suggest that the target DNA fragments can be detected semiquantitatively with reasonable specificity.

EXAMPLE 6 (Immunoassay) [0066] Referring also to FIG. 4, a schematic representation of an immunoassay for detection and quantification of anti-PSCA using the PSCA- grafted microchannels 118 illustrated in FIG. 2 is generally shown. Traditional immunoassays are used to detect protein molecules with high selectivity and specificity. An immunoassay is normally carried out in a 96-well plate, requiring the use of microliter-level samples. Performing a miniaturized immunoassay on a microfluidic chip offers the advantages of lower sample and reagent consumption, enhanced reaction efficiency, reduced operation time, and a portable operation platform. In the scheme of FIG. 4, parallel PSCA-grafted microchannels 118 of FIG. 2 were utilized to demonstrate an immunoassay for detection and quantification of a prostate cancer biomarker

122, anti-PSCA, with a sensitivity of 1.0 nM.

[0067] The PSCA-grafted microchannels 118 trapped the target molecule, anti-PSCA, 35 from the anti-PSCA solutions (PBS, pH 7.4) of three different concentrations (1.6 nM, 3.2 nM, and 12.5 nM), as detected by a one hour exposure to fluorophore-labeled secondary anti-PSCA (fluorescent Ab goat anti-human IgG (H+L), Molecular Probes, 6.7 nM) for detection under fluorescent microscopy. Each microchannel was washed with 100 μl_ of PBS buffer at room temperature to remove the unattached anti-PSCA. The integration of fluorescent intensity across the micrograph of three parallel microchannels indicated the feasibility for semiquantitative detection of anti- PSCA. [0068] Accordingly, the method of providing a two step approach of introducing hydroxy groups on the microchannel surfaces using an oxidation reaction in acidic peroxide solution and then a sequential silanization reaction using preferably neat silane reagents for surface modification of poly(dimethylsiloxane) (PDMS) substrates provides a stable and enduring channel functionalization. This solution-phase approach is simple and convenient for many routine analytical applications in chemistry and biology laboratories and is designed for intact PDMS-based microfluidic devices, with no device post assembly required. [0069] Illustrating the methods, two different functional groups, poly(ethylene glycol) (PEG) and amine (NH₂), were introduced onto PDMS surfaces for passivation of nonspecific protein absorption and attachment of biomolecules, respectively. X-ray electron spectroscopy and temporal contact angle experiments were employed to monitor functional group transformation and dynamic characteristics of the PEG-grafted PDMS substrates. Fluorescent protein solutions were also introduced into the PEG-grafted PDMS microchannels to test their protein repelling characteristics. These analytical data indicate that the PEG-grafted PDMS surfaces exhibit improved short- term surface dynamics and robust long-term stability. The amino-grafted PDMS microchannels are also relatively stable and can be further activated for modifications with peptide, DNA, and protein on the surfaces of microfluidic channels. The resulting biomolecule-grafted PDMS microchannels can be utilized for cell immobilization and incubation, semiquantitative DNA hybridization, and immunoassays. [0070] Although the description above contains many details, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention. Therefore, it will be appreciated that the scope of the present invention fully encompasses other embodiments which may become obvious to those skilled in the art, and that the scope of the present invention is accordingly to be limited by nothing other than the appended claims, in which reference to an element in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." All structural, chemical, and functional equivalents to the elements of the above-described preferred embodiment that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Moreover, it is not necessary for a device or method to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed under the provisions of 35 U. S. C. 112, sixth paragraph, unless the element is expressly recited using the phrase "means for."

Claims

CLAIMS What is claimed is:

1. A method for surface modification of a fluidic surface, comprising: activating the surface of a fluidic channel with a solution of an acid and a peroxide; and exposing said activated surface to a solution of siloxane molecules whereby said siloxane molecules are coupled to said surface of said fluidic channel.

2. A method as recited in claim 1, further comprising: binding a functional element to said siloxane coupled to said surface of a

■ fluidic channel.

3. A method as recited in claim 1 , wherein said acid peroxide solution is a solution comprising hydrochloric acid, hydrogen peroxide and water.

4. A method as recited in claim 3, wherein said acid peroxide solution is a solution comprising hydrochloric acid, hydrogen peroxide and water in a ratio of 1:1 :5.

5. A method as recited in claim 1 , wherein said siloxane has a chemically active group selected from the group of chemically active groups consisting essentially of: an amino group, a hydroxy group, a carboxylic group, a isocyanato group, a isothiocyanato group and a thiol group.

6. A method as recited in claim 1, wherein said siloxane molecules are selected from the group of molecules consisting essentially of: neat 2-[methoxy(polyethylenexy)propyl] trimethoxysilane, 3,3,3-trifluoropropyl siloxane, octadecyl siloxane, isobutyl siloxane, 3-glycodoxypropyl siloxane, and PEG-based siloxane.

7. A method as recited in claim 2, wherein said functional element is an element selected from the group of elements consisting essentially of: a cell, a nucleic acid, a protein, a peptide, an enzyme, a ligand, an antibody, and an antigen.

8. A method as recited in claim 2, wherein said functional element is an element selected from the group of elements consisting essentially of: fragments of a cell surface, a nucleic acid, a protein, a ligand, an antibody and an antigen.

9. A method as recited in claim 1 , wherein said fluidic channel is surface is composed of a material selected from the group of materials consisting essentially of: poly(dimethylsiloxane) (PDMS) , poly(methyl methacrylate) (PMMA), polycarbonate, polystyrene and solid-state silicon.

10. A method as recited in claim 1 , further comprising:

pretreating a surface of a fluidic channel with a hydroxide and a peroxide

solution prior to activating said channel surface.

11. A method for surface modification of a fluidic surface, comprising: activating the surface of a fluidic channel with a solution of an acid and a peroxide; exposing said activated surface to a siloxane solution coupling said siloxane molecules to said surface of said fluidic channel; and binding a functional element to said siloxane molecules.

12. A method as recited in claim 11 , wherein said acid peroxide solution is

a solution comprising hydrochloric acid, hydrogen peroxide and water.

13. A method as recited in claim 12, wherein said acid peroxide solution is

a solution comprising hydrochloric acid, hydrogen peroxide and water in a ratio of

1 :1 :5.

14. A method as recited in claim 1 1 , wherein said acid peroxide solution is

a solution comprising sulfuric acid, hydrogen peroxide and water.

15. A method as recited in claim 11 , wherein said siloxane has a

chemically active group selected from the group of chemically active groups

consisting essentially of:

an amino group, a hydroxy group, a carboxylic group, a isocyanato group, a

isothiocyanato group and a thiol group. ιe>. A method as recited in claim 11, wherein said siloxane molecules are selected from the group of molecules consisting essentially of: neat 2-[methoxy(polyethylenexy)propyl] trimethoxysilane, 3,3,3-trifluoropropy! siloxane, octadecyl siloxane, isobutyl siloxane, 3-glycodoxypropyl siloxane, and PEG-based siloxane.

17. A method as recited in claim 11 , wherein said functional element is an element selected from the group of elements consisting essentially of: a cell, a nucleic acid, a protein, a peptide, an enzyme, a ligand, an antibody, and an antigen.

18. A method as recited in claim 11, wherein said functional element is an element selected from the group of elements consisting essentially of: fragments of a cell surface, a nucleic acid, a protein, a ligand, an antibody and an antigen.

19. A method as recited in claim 11 , wherein said fluidic channel is surface is composed of a material selected from the group of materials consisting essentially of. poly(dimethylsiloxane) (PDMS) , poly(methyl methacrylate) (PMMA),

polycarbonate, polystyrene and solid-state silicon. ZΌ. A method as recited in claim 11 , further comprising:

pretreating a surface of a fluidic channel with a hydroxide and a peroxide prior

to activating said surface.

21. A method as recited in claim 20, wherein said hydroxide is a hydroxide

selected from the group of hydroxides consisting essentially of:

ammonium hydroxide, sodium hydroxide and potassium hydroxide.

22. A method for surface functionalization of a fluidic microstructure surfaces, comprising: pretreating a surface of a fluidic microstructure with a solution of a hydroxide and a peroxide; activating the surface of said fluidic structure with a solution of an acid and a peroxide; binding a plurality of siloxane tethers to hydroxyl groups of said activated surface of said fluidic structure; and coupling a functional element to said siloxane tether.

23. A method as recited in claim 22, further comprising:

purging the pretreating solution and the activating solution with water; and

drying said fluidic micriostructure with a dry gas after purging.

24. A method as recited in claim 22, wherein said hydroxide is a hydroxide

selected from the group of hydroxides consisting essentially of:

amonium hydroxide, sodium hydroxide and potassium hydroxide. 2b. A method as recited in claim 22, wherein said hydroxide peroxide solution is a solution comprising water, ammonium hydroxide and hydrogen peroxide in a ratio of 5:1:1.

26. A method as recited in claim 22, wherein said acid peroxide solution is a solution comprising hydrochloric acid, hydrogen peroxide and water.

27. A method as recited in claim 22, wherein said acid peroxide solution is a solution comprising hydrochloric acid, hydrogen peroxide and water in a ratio of 1 :1 :5.

28. A method as recited in claim 22, wherein said acid peroxide solution is a solution comprising sulfuric acid, hydrogen peroxide and water.

29. A method as recited in claim 22, wherein said siloxane has a chemically active group selected from the group of chemically active groups consisting essentially of: an amino group, a hydroxy group, a carboxylic group, a isocyanato group, a isothiocyanato group and a thiol group.

30. A method as recited in claim 22, wherein said siloxane molecules are selected from the group of molecules consisting essentially of: neat 2-[methoxy(polyethylenexy)propyl] trimethoxysilane, 3,3,3-trifluoropropyl siloxane, octadecyl siloxane, isobutyl siloxane, 3-glycodoxypropyl siloxane, and PEG-based siloxane.

31. A method as recited in claim 11 , wherein said functional element is an element selected from the group of elements consisting essentially of: a cell, a nucleic acid, a protein, a peptide, an enzyme, a ligand, an antibody, and an antigen.