IE20070722A1 - A hydrogel deposition method - Google Patents

Abstract

A method for forming a polymer coating such a hydrogel on a substrate comprises the steps of: applying immobilised photoinitiator onto the substrate surface; depositing a pre-polymer solution on the substrate and over the immobilised photoinitiator; and irradiating the immobilised photoinitiator to cause electron excitation nucleation points for polymer formation according to free radical polymerisation. In one example, a solid glass substrate (3) transparent to light used in polymerisation and sensing is coated with metal thin films (1, 2), one of a high refractive index (n>2.0) and the other of a low refractive index (n íÖ 1.5). Light is used for hydrogel polymerisation, being coupled into the waveguide using either a prism (4) on the back side of the waveguide or a period diffraction grating (6) embossed or etched in the solid support. For sensing applications monitoring changes in refractive index, the light can be coupled out of the waveguide using either a prism or diffraction grating 6 at the other end or with butt coupled optical fibres.

Description

The invention relates to deposition of polymers on surfaces. 1ST CL CO 8 F / /4 b A b I L 2>l( Q US 6841193 describes coating of micro fluidic wells with polymers that resist nonspecific adsorption of analytes. These anti-fouling agents allow for improved accuracy and repeatability of biological assays. The anti-fouling polymers are held in place via electrostatic interactions. It appears that good control of thickness of the anti-fouling agents is not achieved.

US 6632446 describes a method for coating substrates with an anti-fouling hydrogel using photochemical means. The hydrogel is formed from soluble macromers when illuminated by either blue or green light, depending on the photoinitiator used. Control of thickness is based on diffusion of photoinitiator adsorbed to contact surface into surrounding media, and is apparently only controllable at the millimetre regime.

US 6395558 describes the use of a waveguide optical sensing platform. The 20 waveguide layer is decorated with biomolecules. Upon binding of a specific ligand, the refractive index of the recognition layer is changed. This change serves as a method for detecting analytes in a sample. Gratings are used to couple in excitation radiation, and also to outcouple the signal.

EP 0832655 relates to coating stents and other biomaterials to a) confer biocompatible properties to the biomaterial and b) allow for controlled release of therapeutics to the surrounding biological matrix. Coating is performed by spraying or dipping using highly volatile solvents; drying leaves behind polymer material which can be cured if necessary.

The invention is directed towards providing an improved method for deposition of polymers. -2Jlo 70 722 Statements of Invention According to the invention, there is provided a method for forming a polymer coating on a substrate, the method comprising the steps of: applying immobilised photoinitiator onto the substrate surface; depositing a pre-polymer solution on the substrate and over the immobilised photoinitiator; and irradiating the immobilised photoinitiator to cause electron excitation nucleation points for polymer formation according to free radical polymerisation.

In one embodiment, the nucleation causes hydrogel formation.

In one embodiment, the substrate is transparent to the radiation, and the radiation is directed through the substrate so that an evanescent field causes nucleation.

In one embodiment, the substrate is configured to act as a sensor which operates with radiation propagating through it.

In one embodiment, the radiation is coupled by a prism into the substrate.

In one embodiment, the radiation is coupled by a grating into the substrate.

In one embodiment, the radiation is directed directly on the pre-polymer.

In another embodiment, the radiation is directed to a plurality of localised sites for selective polymer formation at those sites.

In one embodiment, the polymer formation is to selectively form structures within a micro fluidic channel. -3In one embodiment, the polymer is formed using a light source that is used as an excitation source to immobilise ligands or nanoparticles within the polymer.

In one embodiment, the ligands are biocomponents such as DNA - DNA, RNA, Antigen - Antibody, protein, or cells. The nanoparticles may be magnetic, optically active, or electrically active.

In one embodiment, the polymer formation in real time forms valves in a complex fluidic network selectively directing fluid flow based on feedback from previous physical, biological or chemical experiment results.

In another embodiment, the polymer is formed as part of an analysis system as an integral component.

In one embodiment, the step of irradiating the immobilised photoinitiator includes controlling the radiation depth of field to optimise electron excitation.

In another aspect, the invention provides an article such as a sensor whenever produced by any method defined above.

Detailed Description of the Invention The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only with reference to the accompanying drawings in which:Fig. 1 shows two examples of waveguide, each having waveguide layers (thin films of varied refractive index realised with materials such as metal, oxide nitride combinations) with radiation coupling elements such as gratings or prisms. -4fcO JQ 1 22 Fig. 2 is a perspective view of a planar waveguide substrate ready for polymerisation; Figs. 3 and 4 illustrate covalent attachment of a hydrogel layer using a prism and a grating respectively; Fig. 5 illustrates an alternative method, and a resultant patterned coating; Fig. 6 illustrates formation of a hydrogel layer in a microfluidic channel; Fig. 7 illustrates the relationship between gel height and the radiation exposure time; Fig. 8 is a set of plots showing normalised intensity for various samples; Fig. 9 illustrates fluorescence detection response to complimentary probe target olignucleotide hybridisation within a microfluidic channel; and Fig. 10 illustrates a gel produced by photo-initiation, drawing a tweezers across the surface tests for covalent attachment.

A process for photochemically depositing a controlled (on the micron scale) thickness of a hydrogel on a planar substrate is described. The planar substrate acts as an optical waveguide, and so the thickness of the gel can be controlled by the coupling angle of the incoming light. Changes in the angle of the incoupled light correspond to changes in the penetration of the evanescent wave into the surrounding medium, and because polymerisation will occur chiefly in the evanescent wave, control over layer thickness is observed. Other parameters affecting the evanescent wave include the waveguide layer materials and waveguide layer refractive indexes. -5If the planar substrate does not act as an optical waveguide, the exposure time of the planar surface to an external light source capable of exciting the covalently attached photoinitiator acts as the control mechanism for hydrogel thickness. This is because the photoinitiator is anchored to the surface, and the hydrogel forms outward from the surface. This process can be applied to any substrate that presents amine groups at its surface, and these amines are easily introduced via silanisation. These gels are biocompatible - in this context, meaning that the gels resist fibrotic responses in an in vivo environment, and resist the type of fouling so often seen in marine and open air environments. Further, these gels can be functionalized to present virtually any chemical or biological ligand to the solid-liquid interface, allowing it to be used as a platform for a biological sensing apparatus.

In the context of bioassay development, the gel can be formed using the very same light source that is used as an excitation source for fluorescent ligands bound to the gel. Finally, the three-dimensional characteristic of the gel allows for much higher signal per unit area on the substrate, offering a significant advantage over the conventional two-dimensional array format.

The waveguide apparatus with a functionalised hydrogel acts as a three-dimensional biosensor, by using refractive index or fluorescence excitation or a combination of both.

We use an evanescent field from an optical waveguide or an external light source to covalently attach a hydrogel to the waveguide surface.

Using the same light source for reading the output of the assay and for the actual deposition of hydrogel has two advantages: First, it removes the need to optimise the hydrogel film thickness using conventional fabrication techniques. This fabrication step is often expensive and cumbersome in related applications. Second, the hydrogel thickness conforms to the penetration of the evanescent field into the surrounding medium. The evanescent wave is also used to excite fluorophores in the gel layer for sensing events. Because the gel thickness matches the evanescent field, the balance between maximising the available electromagnetic radiation from the waveguide for sensing while keeping the gel as thin as possible for better diffusion is automatically struck. -6The invention also provides a method for controlling the thickness of a biologically inert hydrogel grown on a surface. It uses simple and easily adaptable chemistry that uses inexpensive light sources. Other methods for forming inert or antifouling coatings on a surface use serial coating and drying processes, or chemical grafting, which are more time consuming and use less flexible chemistries. The hydrogel height is controlled by the energy delivered to the photoinitiation process. This can be done by: (i) in direct source illumination, the exposure time of radiation may be varied by using a pulsed source or a shutter mechanism. The photoinitiation wavelength may also be varied off maximum efficiency to deliver less efficient hydrogel formation, offering the possibility of slower growth rate. (ii) For waveguide illumination the energy delivered to the photoinitiation process may be varied by controlling the evanescent field penetration depth by: - varying the coupling angle, - varying the waveguide structure to control evanescent field penetration depth, - varying the waveguide coupled radiation to achieve evanescent field of varied depth.

In more detail, a method for covalently attaching a biocompatible hydrogel with a controllable thickness on an optical waveguide surface has been developed. The method involves the silanisation of the surface of the planar waveguide with 1-(3aminopropyl)-trimethoxysilane, and covalently immobilising a photoinitiator to the amine groups presented by the silanized surface. These substrates are then immersed in a small volume of an aqueous prepolymer solution containing a,co-poly(ethylene glycol) diacrylate, a sensitizer, and an accelerant. Upon illumination with a green light source, polymerisation occurs, and is covalently attached to the surface. Two 72 -Ί strategies are described for controlling the morphology of the hydrogel. The first strategy involves directing the light at the surface of the substrate from a remote source (a laser, or possibly even a white light source). Hydrogel will develop only at areas exposed to green light. The exposure time of the substrate to the light source determines the thickness of the hydrogel layer. A second strategy involves coupling the green light into the optical waveguiding layer. Coupling is accomplished using a prism or a diffraction grating. The evanescent field produced by the green light propagating along the waveguide excites the immobilised photoinitiator, resulting in the formation of the hydrogel within this field. The depth of field can be controlled by adjusting the angle at which the light is coupled into the system, modifying the waveguide effective refractive indices (material refractive index or material thickness) or modifying the light source operating wavelength.

The hydrogel described above can be tailored to include virtually any biomolecule for sensing applications. Modification of the gel is accomplished by including a small fraction of a heterobifunctional PEG-based polymer in the prepolymer solution. The two functionalities consist of an acrylate group on one end of the chain, and a reactive functional group such as an activated ester or an alkyne group. An appropriately tagged biomolecule can be immobilised within and throughout the gel by introducing the conjugate before or after polymerisation.

After fabrication, the gel/waveguide system with an immobilized biomolecule probe (described above) is used as an optical waveguide biosensor. During - or after treatment with a target biomolecule, binding events are interrogated by directing light of an appropriate wavelength into the optical waveguide, much as described above for the hydrogel formation process. A signal can be recorded using an optical detection device (CCD camera, avalanche diode, etc.) a) positioned parallel to and above the substrate, or b) in the path of light outcoupled from the waveguide using a prism or diffraction grating. For refractive index measurement the hydrogel forms part of the guide structure to which radiation is coupled, the coupling angle is monitored to determine physical or chemical events in the hydrogel. This change in hydrogel structure may be a change in morphology (swelling) and / or change in optical properties such as refractive index or colour. This coupling angle may be monitored Z 0 Ζ 2 2 -8by measuring outcoupled radiation over angle, over space or the intensity from the guide output. It may also form a key element of the holographic sensor which alters its optical properties with changing morphology resulting from a chemical, physical or biological event.

Fig. 1 provides a side view of waveguides in one embodiment. A solid glass substrate 3 transparent to light used in polymerisation and sensing is coated with metal thin films 1, 2, one of a high refractive index (n>2.0) and the other of a low refractive index (n «1.5). The exact composition will depend on the actual waveguide platform being used. The light used for sensing and/or hydrogel polymerisation can be coupled into the waveguide using either a prism 4 on the back side of the waveguide or a period diffraction grating 6 embossed or etched in the solid support. For sensing applications monitoring changes in refractive index, the light can be coupled out of the waveguide using either a prism or diffraction grating 6 at the other end or with butt coupled optical fibres.

Fig. 2 is a three dimensional diagram of a planar waveguide substrate 11 ready for polymerisation. After activation of the waveguide / liquid interface, a suitable photo initiator 14 has been immobilised to the surface.

Referring to Fig. 3 there is a glass waveguide 11, metal / oxide / nitride coatings 12 and 13, and immobilized photoinitiator 14. A focused beam of light produced by source 17 is coupled into the waveguide using a prism 17. As the light propagates through the waveguide 11, an evanescent field 15 is created which excites an electron in the immobilised photoinitiator 14. Because the substrate is covered with prepolymer solution 16, the excited photoinitiator serves as a nucleation point for the hydrogel formation process using free radical polymerisation.

Fig. 4 illustrates a scheme for covalently attaching a hydrogel layer of defined thickness on the waveguide using a prism to couple in the light. A focused beam of light 18 produced by the source 17 is coupled into the waveguide using a diffraction grating 21. As the light propagates through the waveguide 11, an evanescent field 15 is created which excites an electron in the immobilised photoinitiator 14. Because the substrate is covered with prepolymer solution 16, the excited photoinitiator serves as a nucleation point for the hydrogel formation process using free radical polymerisation.

Fig. 5 shows a method for forming hydrogel structures on a waveguide surface. Light emitted from sources 35 is directed at the waveguide covered in a layer of prepolymer solution 34. Post photoinitiation, the pre-polymer solution is washed away using a wash solution e.g. deionised water 40. The remaining hydrogel structures 41 are formed where illumination occurs. The depth of the hydrogel structures can be controlled by varying exposure time (Energy delivered).

Fig. 6 shows a method to form the hydrogel structure on a waveguide surface, enclosed in a microfluidic channel. The prepolymer solution is introduced into the microfluidic channel 42 and hydrogel structures 41 are formed where illumination occurs.

Fig. 7 illustrates the relationship between the gel height and the exposure time of the radiation. Hydrogel heights are formed in the range 1 pm to 8 pm over the exposure time, 8 seconds to 300 seconds.

Fig. 8 shows normalised intensity readings from a hybridisation with complementary and non-complementary target DNA on gel immobilised probe DNA.

Fig 9 illustrates the suitability of this hydrogel to DNA hybridisation for (A) Sample 1 Cross linked Hydrogel (No DNA), (B) sample 2 Hydrogel with unbound DNA (i.e. no NHS PEG - Acrylate) after wash , (C) sample 3 Hydrogel with bound DNA (NHS PEG-Acrylate) (D) Sample 2 with unbound DNA after further rinsing in PBS buffer.

Fig 10 illustrates a hydrogel 43 covalently attached to a glass substrate surface using the surface chemistry, pre-polymer solution and irradiation technique outlined above.

This invention may be applied to low-cost binary output microarrays. This sensing platform has a three-dimensional design, allowing increased signal output per unit area. The use of the waveguiding platform significantly reduces background signal -10prevalent in top- or bottom-excited fluorescence detection platforms. Because the signal density is increased and background reduced, inexpensive light sources and fluorescence detectors could be integrated into this detection system to produce portable and disposable on-site diagnostics.

This process facilitates micron/nanometer thick barriers to be patterned in a rapid fashion on a surface or interface of any geometry a characteristic not available with hydrogels chemically or thermally initiated. Its application includes a DNA hybridisation platform within a microfluidic system compatible with rapid diagnostic applications such as Single Nucleotide Polymorphisms or SNiP analysis. It is compatible with applications for range of bio-molecules including DNA, antigens, antibodies, proteins and cells, where the hydrogel structure provides a morphology and suitable environment in the vicinity of such bio-molecules.

An additional application is in the field of developing protective anti-fouling coats. This method is particularly suited to coating substrates with a large surface area, as the chemicals are inexpensive (and could be sprayed on the substrate in bulk), and polymerisation can be effected with simple white light. Because the hydrogel is PEGbased, it will resist the fouling commonly seen in complex biological environments (such as bodily fluids, marine environments, etc.). Hydrogel coating is used to minimise non specific binding from biofluids (e.g. blood, saliva) preventing large biomolecules such as fats interacting with binding sites. Modifying the hydrogel porosity, increases water content minimising non specific binding effects and resulting inconsistencies in results in assays (microarrays or microfluidics). This Hydrogel has can also be used a selective membrane in sensors to improve selectivity and sensitivity.

This approach to hydrogel formation facilitates uniform coating to be formed on contoured structures for biomedical applications (e.g. implants, stents) as an anticoagulation coating inclusive of drug delivery I elution or structures for wound treatment / dressings where drug elution may also be applicable. -11It will be appreciated that the invention allows a polymer to be deposited on an aminated surface using photochemistry.

The thickness of the polymer layer can be controlled using either an evanescent wave from an optical waveguide or by controlling the exposure time of the surface from an outside light source that initiates the polymerisation.

The functionality of this hydrogel technology allows structures to be readily formed within microfluidic devices in a controlled fashion in milliseconds or microseconds, its characteristics (morphology) change size with water content making it an ideal candidate for micron or nanometer liquid valves in microfluidics. This can be used to define real time valves in a microfluidic platform implementing a sequence of chemical reactions or assays based on real time results. The fluidic path and reaction sequence of a sample may be altered in real time based on feedback for previous results (physical, chemical, biological) in the fluidic path taken by the liquid.

Example In the polymer being deposited is a bioinert hydrogel comprising PEG-diacrylate, or any other biocompatible hydrogel The thickness of the polymer layer can be controlled between thicknesses of 100 nanometres and 100 micrometres or alternatively between thicknesses of 0.5 microns and 10 microns, or alternatively between thicknesses of 1 micron and 5 microns. The hydrogel can be functionalised to serve as a biosensing layer, detecting DNA, proteins and any other analyte of interest.

The protocol for surface silanisation and hydrogel formation The silanisation of the substrate surface follows a standard APTMS protocol.

Preparation of the Pre polymer solution: Buffer: Dilute 1:9 of 10X PBS solution with deionised water, pH should be in range 7.0-7.4. «Ο 7 0 7 22 -12For a 50ml prepolymer solution, add 12.5ml of PEG diacrylate to 37.5ml of deionised water. To this solution add 1.49 ml of triethanolamine, finally add 198μ1 of 1-vinylpyrrolidone to the solution.

Immobilisation of Photo-initiator to the substrate surface Use a lOmM sodium phosphate solution 25mM in EDC(1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 0.5mM Eosin -Y. Agitate the substrates in this solution for 60 - 90 minutes. Finally rinse the slides with deionised water This invention could be used as the basis for a biosensing technology, and is appropriate for both portable biosensors and low-cost bioassay equipment. The hydrogel coating could also be used to protect sensitive surfaces in environmentally challenging conditions. The highest-impact applications in this field would be the coating of medical devices with a biocompatible hydrogel (and in fact, an inert gel that could actually help its environment with anti-thrombogenic agents), the coating of sensors in air and sea, and the coating of ship hulls to resist the attachment of marine organisms.

The invention is not limited to the embodiments described but may be varied in construction and detail. -13ΙΪΟ 7 0 7 22

Claims (10)

1. A method for forming a polymer coating on a substrate, the method comprising the steps of: applying immobilised photoinitiator onto the substrate surface; depositing a pre-polymer solution on the substrate and over the immobilised photoinitiator; and irradiating the immobilised photoinitiator to cause electron excitation nucleation points for polymer formation according to free radical polymerisation.

2. A method as claimed in claim 1, wherein the nucleation causes hydrogel formation.

3. A method as claimed in claim 1, wherein the substrate is transparent to the radiation, and the radiation is directed through the substrate so that an evanescent field causes nucleation.

4. A method as claimed in claim 3, wherein the substrate is configured to act as a sensor which operates with radiation propagating through it.

5. A method as claimed in any preceding claim, wherein the radiation is coupled by a prism into the substrate.

6. A method as claimed in any of claims 1 to 4, wherein the radiation is coupled by a grating into the substrate.

7. A method as claimed in claims 1 or 2, wherein the radiation is directed directly on the pre-polymer. 10 7 0 7 2 2 -148. A method as claimed in any preceding claim, wherein the radiation is directed to a plurality of localised sites for selective polymer formation at those sites. 9. A method as claimed in any preceding claim, wherein the polymer formation is to selectively form structures within a microfluidic channel. 10. A method is claimed in any preceding claim, wherein the polymer is formed using a light source that is used as an excitation source to immobilise ligands or nanoparticles within the polymer. 11. A method as claimed is claim 10, wherein the ligands are biocomponents such as DNA - DNA, RNA, Antigen - Antibody, protein, or cells. 12. A method as claimed in claim 10, wherein the nanoparticles may be magnetic, optically active, or electrically active. 13. A method as claimed in any preceding claim, wherein the polymer formation in real time forms valves in a complex fluidic network selectively directing fluid flow based on feedback from previous physical, biological or chemical experiment results. 14. A method is claimed in any preceding claim, wherein the polymer is formed as part of an analysis system as an integral component. 15. A method is claimed in any preceding claim, wherein the step of irradiating the immobilised photoinitiator includes controlling the radiation depth of field to optimise electron excitation. 16. An article having a coating formed by a method as claimed in any preceding claim. 17. A sensor comprising a transparent substrate for propagation of radiation and a coating formed by a method as claimed in any preceding claim. 1/10 σ> LL CO CN 2/10 !i0 7 0 7 22 Fig.2 3/10 4/10 5/10 co co in 0) Ll 6/10 ijg® Ο Of 7/10 (wBbu uinuiixeuijo uoipejj se) souejsip MBnojj-q-^zed 6Ό Exposure time (s) (uitI ‘ uinuiixeui ie) I^O i^ 0) 'll

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