WO2002091081A2 - Functionalising polymeric materials - Google Patents

Abstract

The invention provides a method for functionalising all or part of a surface of a structure fabricated from a crosslinkable polymeric material comprising the steps of; defining a structure in a material that is crosslinkable by undergoing a curing process; the curing process involving cross-linking of reactive functional groups in the polymeric material; performing the curing process under conditions where at least part of the surface of the defined structure is in contact with a polymeric material that is non-crosslinkable under the conditions of the curing process; removing the non-crosslinkable polymeric material from the surface of the defined structure after curing, whereby said surface comprises unreacted functional groups. The structure may be a shaped, coded, microparticle and may have attached thereto chemical moieties and/or biomolecules.

Description

Functionalising Polymeric Materials

Introduction

The present invention relates to functionalising polymeric materials, such as photoresists, in order to modify the surface properties of structures, such as particles, made from the materials. Such particles may be of a size which is visible or invisible to the naked eye (eg having a dimension larger than about 50μm-100μm or smaller than about 50μm respectively). The particles may carry a code for identifying the particles, such as a machine readable code. The machine readable code may, for example, be in the form of an applied binary code (such as a bar code), or formed by holes, pits, notches etc in the particle. The invention particularly relates to a machine readably encoded particle, on which the code is created by at least one shape (such as a castellated shape) applied to at least one edge surface of the particle, which particle has functional groups which are attached or can be attached to chemical moieties and/or biomolecules.

Background: Polymeric Materials

Many polymeric materials are known in the art, having potentially reactive functional groups which can undergo cross-linking under appropriate conditions; thereby creating solid structures for various purposes. Examples of such polymers are polystyrene, polyimide, polycarbonate etc. The step or steps which lead to cross-linking of the reactive functional groups may be referred to by the general term of "curing".

Uncured polymeric materials are generally fluid, having many possibilities for characteristics such as density, viscosity, temperature stability, means of curing (eg by chemicals, heat, light) and speed/ease of curing. For a chosen application the starting material may be chosen to be more or less "gooey" and the final product may be more or less "hard" or "rigid".

Generally, cured polymeric materials are expected to be relatively un-reactive, even inert, by virtue of the fact that most or all reactive functional groups ("cross-linkable groups") are expected to have taken part in the curing/cross-linking process.

It is known that cured (un-reactive) polymeric materials may be rendered at least partially reactive by treating them with aggressive chemicals and/or high energy (eg plasma treatment) so as to "break" some cross-linkages and hence re-create new reactive functional groups. These may then allow functionalisation of the material by attachment of chemical moieties or biomolecules to the newly formed functional groups. Background: Photoresists

Photoresists are used in the manufacture of silicon chips. In brief, a layer of photoresist is spun onto a silicon wafer and partially exposed to radiation that initiates a chemical transformation in the photoresist. Negative photoresists are made less soluble by exposure to radiation allowing the exposed portion of the resist to remain on the silicon when unexposed areas are dissolved in a suitable solvent (see Review article: Shaw et al., "Negative Photoresist for Optical Lithography", Optical Lithography Vol. 41 , (1997)). Positive photoresists are made more soluble by exposure to radiation, so the unexposed portion of the resist remains after washing with solvent.

In addition to their use in silicon processing, photoresists can also be used in their own right to make miniature devices, such as gears and channels for microfluidic flow (e.g. Zhang et al., J. Micromechanics and Microengineering, 11, pp20-26, 2001) or polymer beads with a characteristic barcode shape (WO 01/78889: A Method of Fabricating Coded Particles). Where legally permissible, the content of WO 01/78889 is incorporated herein by reference.

There is a desire to be able to introduce various kinds of surface functionality to such photoresist structures. This would enable new applications of these structures by the attachment of chemical moieties thereto, including, but not limited to, attaching DNA and proteins and modifying their physico-chemical surface properties.

However, most structures made from photoresists are essentially inert, and special steps would be expected to be needed to introduce or re-create new functional groups.

Background: Functionalisation using silanes

Silane modification is commonly used to modify inorganic supports or substrates, such as glass slides, to enable the attachment of organic compounds such as proteins and DNA. The silane compounds react with hydroxyl groups on the surface of the inorganic substrate. Silanes are available with a range of different functional moieties, for example amines, to which biological molecules can be attached.

Successful silanation of glass substrates requires the surface to be cleaned using strong acids to remove oils and detergents in order to activate the hydroxyl groups; insufficient cleaning results in the deposition of a non-uniform silane layer.

We have successfully demonstrated the modification of the surface of negative photoresist using silanes by attaching oligonucleotide DNA to the substrate (beads) following the treatment. In this experiment the polymer beads were floated off the silicon wafer using KOH to dissolve the sacrificial lift-off-release (LOR) layer and washed in distilled water to remove traces of the base. The silanation was carried out using a 2% amino-silane mix dissolved in dry acetone. Following a two-minute incubation, the beads were washed in acetone and then dried at 50°C for 30 minutes. The beads were then suspended in 48mg of cyanuric chloride and incubated for 5 minutes. Following surface activation, the beads were then re-suspended in carbonate buffer with an amino modified FAM labelled oligoucleotide and agitated overnight. On completion of the experiment the beads were visualised using a fluorescent microscope. (FAM = 6-carboxyfluorescein).

The degree of binding observed using the cyanuric chloride method was less than ideal. An alternative method used biotin and streptavidin following the modification of the surface using silane.

During this experimental programme N-Hydroxysuccinimidobiotin (NHS-Biotin) was attached to the amino modified surface, the beads were incubated for 30 minutes in the presence of NHS-Biotin, followed by 30 minutes with streptavidin-labelled Fluorescein. Following several wash steps the beads were visualised under a fluorescent microscope. A greater degree of fluorescence was observed using the biotin method compared to the cyanuric chloride coupling.

In a further experiment, the bead surface was pre-treated with HMDS (Hexamethyldisilazane), a chemical compound used as a pre-resist wafer treatment to improve adhesion of photoresist to wafers. Following this process, silanation of the photoresists occurs with higher yield. This method has the disadvantage that it does introduce an extra step into the procedure, however this is considered to be outweighed by the advantage of the increase in the level of fluorescence detected.

Ideally, the functionalisation of the surface, for example silanation, would homogeneously attach functional compounds to the entire surface of the photoresist structure.

Invention: Utilising un-reacted functions of the structure

We have observed that when we silanate structures made from polymeric materials, such as photoresists like the SU-8 negative resist made by Microchem Corporation, reproducible variations in the spatial distribution of the attached silane groups occur.

Contrary to expectations, silane groups are very effectively attached to the sides of the structures (i.e. surfaces made at the boundary between exposed and unexposed photoresist). Many fewer silane groups are attached to the top of the structure (formed at the boundary between exposed photoresist and air) or to the base of the structure (formed at the boundary between exposed photoresist and the support wafer, or a layer of lift-off resist, LOR).

There are many possible explanations for the above observation. There may be significant concentrations of unreacted photoresist functional groups at the edges of the structures, which then undergo reaction. Alternatively, the phenomenon could be due to the greater accessibility of photoresist functional groups at the edges of the structures, where polymer chains terminate, causing a ragged edge. Regardless of the mechanism, we believe that this observation identifies a novel route for preserving functional groups in a cured polymeric material and, where required, doing so in a spatially homogenous manner.

This invention provides a means of introducing chemical functionality to polymeric materials such as photoresists, by curing the material under conditions in which at least some unreacted functional groups are preserved during and after curing. This may be achieved by curing at least a portion of the material in contact with uncured material, thereby preserving functional groups at that portion, at the interface between cured and uncured material, which functional groups are capable of subsequent functionalisation by attachment to chemical moieties and/or biomolecules.

Statements of Invention

The present invention provides a method for functionalising all or part of a surface of a structure fabricated from a crosslinkable polymeric material comprising the steps of; defining a structure in a material that is crosslinkable by undergoing a curing process; the curing process involving cross-linking of reactive functional groups in the polymeric material; performing the curing process under conditions where at least part of the surface of the defined structure is in contact with a polymeric material that is non-crosslinkable under the conditions of the curing process; removing the non-crosslinkable polymeric material from the surface of the defined structure after curing, whereby said surface comprises unreacted functional groups.

The structure may be defined by a machining process.

The structure may be defined by a moulding process.

The structure may be defined by a forming process.

The crosslinkable polymeric material may be a photoresist material.

The non-crosslinkable polymeric material may be a photoresist material.

The structure may be defined in a photoresist material by exposure of the photoresist to photoradiation, where unexposed regions of the photoresist are non-crosslinkable in the curing step and the exposed regions of the photoresist are crosslinkable in the curing step.

The structure may be defined in a photoresist material by exposure of the photoresist to photoradiation, where unexposed regions of the photoresist are non-crosslinkable by undergoing the curing step and the exposed regions of the photoresist are crosslinkable by undergoing the curing step.

The exposure of the photoresist material may be through a mask, the shape of the structure being defined by the pattern of the photo-radiation through the mask.

The photoresist material may be in the form of a sheet. The photoresist material may have at least one surface contact with a non-photoresist polymeric material prior to the step of defining the structure.

The photoresist material may be sandwiched between layers of non-photoresist polymeric material prior to the step of defining the structure.

The crosslinkable polymeric material may comprise epoxy groups.

The non-crosslinkable polymeric material may comprise epoxy groups.

The unreacted functional groups on the surface of the cured structure may be epoxy groups.

The non-crosslinkable polymeric material may be added to the surface of the crosslinkable polymeric material after the structure has been defined.

The structure defined may be a particle.

The structure defined may be a morphologically encoded particle.

The present invention also provides a functionalised structure fabricated from a crosslinkable polymeric material by cross-linking of reactive functional groups in the polymeric material, said structure having chemical moieties and/or biomolecules covalently attached to non-crosslinked functional groups on all or part of the surface thereof.

The one major surface (other than a side or edge surface) may be functionalised by attachment of chemical moieties and/or biomolecules.

The crosslinkable polymeric material may comprise epoxy groups.

The crosslinkable polymeric material may be a photoresist.

The structure may be a particle.

The particle may be morphologically encoded.

The present invention further provides a method of fabricating cured photoresist particles comprising: providing a sheet of uncured polymeric photoresist material on a substrate said sheet having first and second major surfaces in the plane of the substrate; delineating the sheet into a plurality of cured particles without destroying the integrity of the substrate using a mask by causing exposure of said material to photo radiation through said mask to cure said exposed material; and removing the particles from the substrate; the shape of the particles being defined by the pattern of the exposure photo-radiation through the mask; the curing reaction involving cross-linking of reactive photoresist functional groups; characterised in that at least one of said major surfaces in cured in contact with added uncured polymeric photoresist material; whereby curing of said surface(s) preserves at least some unreacted photoresist functional groups at the interface between said cured surface(s) and said uncured material.

There may be a step of machine-readably encoding the particles.

The particles may be morphologically encoded during the delineation.

The particles may be encoded after delineation but before removal form the substrate.

The adhesion between said sheet and the substrate may be produced by a radiation- sensitive sacrificial layer whose properties are modified to reduce the strength of the adhesion to release the particles.

The particle may be formed with at least one reading sense identifier.

The machine readable code may be formed by bleaching the fluorescence of a constituent of each particle.

The chemical or biological moieties may be covalently attached to photoresist functional groups on respective particles prior to removal from the substrate.

The chemical or biological moieties may be applied to or synthesised on said particles in a well of a micro titre plate.

The chemical or biological moiety may be applied to or synthesised on each particle after release of said particle from said substrate.

The two chemical moieties may be provided on each particle, each said moiety occupying its own respective space on said particle separate from the space occupied by the or each other moiety thereon.

The first and second major surfaces may be cured in contact with uncured photoresist material.

The major surface(s) and side surfaces of said particles (said side surfaces being in a plane orthogonal to the plane of the substrate and being parallel to the direction of exposure of photo-radiation through the mask) may have essentially homogeneously preserved photoresist functional groups.

The present invention yet further provides a particle obtained by the method above, said particle having at least 3 surfaces not sharing a common axis, said surfaces having preserved photoresist functional groups are curing; or having a continuous curved side edge surface and at least one major surface, said surfaces having preserved photoresist function groups after curing; or having a discontinuous curved side edge surface and at least one major surface, said surfaces having preserved photoresist functional groups after curing; having preserved photoresist functional groups on all surfaces after curing; having a surface not parallel to the direction of exposure to photo-radiation during curing, which surface has preserved photoresist functional groups. Figures:

In the accompanying drawings;

Figure 1 shows the pattern of attachment of silane groups to a cured photoresist u uυiuic, αi IVJ

Figure 2 shows sandwiched photoresist layers in an embodiment of the invention.

Non-Homogeneous Functionalisation

In the structure shown in Figure 1 significant numbers of silane groups would attach to the sides (1) which have been cured in contact with uncured photoresist, with little attachment of silane groups to the top and bottom surfaces (2) which conversely have not been cured in contact with uncured photoresist. The direction of mask illumination is shown by the large arrow (3) in Figure 1.

Most negative photoresists consist of large molecules - molecules like SU-8 with a M.W. of around 1400, oligomers, or polymer chains. These large molecules will be referred to as chains. Exposure to radiation forms reactive species such as free radicals, or strong acids. Radicals catalyse reactions such as the formation of crosslinks between polymer chains; acids catalyse ester hydrolysis and the formation of ether cross links from epoxy groups. The effect of the reaction is to effect a chemical transformation so that the exposed photoresist becomes insoluble in a suitable solvent, while the unexposed photoresist is soluble.

At a molecular level, the photoresists will consist of interlocking polymer chains, oligomers or large molecules. At the boundary between exposed and unexposed photoresist there will be chains of molecules which span this boundary. If the chains that are mostly in the unexposed region dissolve, leaving those chains that are mostly in the exposed region, the edges of the photoresist structures will be ragged, consisting of loose chains, extending into the bulk of the material. It is these chains, which are highly accessible and contain some unreacted photoresist functional groups (e.g. epoxy groups in SU-8, double bonds in KTFR resist etc.), that enable preferential functionalisation of the photoresist structures at the edges.

The present invention aims to utilise those unreacted functional groups (which would normally participate in the chemical amplification reaction if activated) at the interface between activated and inactivated material to chemically attach additional surface functionality. The attachment of a desired target functional group may occur by a single reaction between the edges of the structure and a chosen reactant molecule. Alternatively, a multi-step chemical synthesis may be required to convert the unreacted functional groups of the structure to the target functional group.

The above mechanism shown in Figure 1 does not operate at the upper and lower surfaces of a layer of polymeric material. The top of the layer is in contact with air, and the material at this surface will be almost or fully cured. Unreacted functional groups cannot be used to functionalise the surface because any photoresist functional groups will have either reacted following the curing, or will be sterically hindered and thus unable to react. A similar argument holds at the base of the layer, which is in contact with a supporting substrate such as a silicon wafer, or a cured layer of a sacrificial photoresist such as a LOR (e.g. polydimethylglutarimide).

High-Resolution Negative Resists

Materials with high levels of epoxy functionalities can provide high cross-linking density. A multifunctional glycidyl ether derivative of bisphenol-A novolac, available from Shell Chemical and known as EPON® resin SU-8, provides the highest epoxy functionality currently commercially available. Upon exposure to either UV, e-beam, or X-ray radiation, it forms a ladderlike structure with a high cross-linking density and a Tg of more than 200°C. Its versatile imaging capability has been used to fabricate advanced 0.25-μm and 0.1-μm devices using e-beam lithography. The low molecular weight of the resin provides high contrast and excellent solubility and planarizing capability, and the high epoxy functionality provides sensitivity. Although the pattern is developed using a solvent, it is one of the highest-resolution systems yet developed, and it demonstrates the capability of cross-linked negative resists.

One embodiment of the invention provides the introduction of surface functionalisation into negative photoresists suitable for the fabrication of high aspect ratio structures. An example of such a photoresist is the SU-8 photoresist. In this example surface functionalisation is introduced by reacting a suitable compound containing the surface functional group of interest with unreacted photoresist functional groups.

A fabrication method is now described for producing polymer based microparticles.

Preferably, in this method a silicon wafer is used as a substrate for the ensuing steps, although it will be understood by persons skilled in the art that any flat substrate could be used including porous substrates as described above. The silicon wafer is cleaned and baked and then a photoresist of thickness approximately 3 μm that acts as a sacrificial layer is spun on to the surface of the substrate. A typical example of a suitable photoresist for this step is SPOLR30B, available from MicroChem Corporation of Massachusetts.

This sub-assembly is then baked prior to the application of a further photoresist layer by spinning. The second photoresist layer is approximately 20 μm in thickness and is a chemically amplified negative photoresist such as SU8-25, also available from MicroChem Corporation as above. This is then subjected to a pre-exposure bake in accordance with the manufacturer's instructions. Then an etched chrome-on-glass mask is positioned over the SU8 layer which is then exposed using a UV exposure tool or similar excitation apparatus. A post-exposure bake is conducted, again in accordance with the manufacturer's instructions and the exposed material is then developed using an appropriate developer to leave an array of patterned or profiled microparticles on the substrate wafer.

The microparticles may be functionalised by attachment of chemical moieties and/or bimolecules prior to or subsequent to release from the wafer. Release is effected using diluted developer which dissolves the SPLOR30B layer. Using the techniques outlined above, microparticles having a length in the order of 50 to 200 μm can be obtained. Typical microparticle dimensions are as follows: length <100 μm

, V.V,;IU,-nL.I I

, (JιnI M-, thickness 20 μm

For SU-8, the photoresist functional groups are epoxy groups. These will react with amine groups to produce a β-hydroxyamine. Therefore if a compound containing two amine groups such as 1,6 diaminohexane is reacted with the epoxy group then one unreacted amine group will remain available for further reaction thereby altering the surface functionalisation of the structure. If the SU-8 structures are released from the wafer prior to functionalisation, it is necessary to use a suitable solvent to release the structures without causing unwanted reactions with the photoresist functional groups.

The hydrolysis of the epoxy group can occur as part of the existing manufacturing process when the cured photoresist structure is released from the LOR layer that attaches the photoresist to the supporting wafer. The development step to dissolve the LOR layer usually uses KOH, a strong base, which can hydrolyse the epoxy to a diol. If, however, it is preferable to preserve the epoxy functionality then non-KOH based developers are available which would preserve the integrity of the epoxy groups.

In our experirhent the beads were released from the wafer in the presence of 1-methyl- 2-pyrrolidinone. Hexamethyl diamine was added to the solvent to enable introduction of a surface functionality to the bead. This process allowed a one step introduction of an amine functionality to the bead surface. The beads were then incubated with NHS- biotin followed by streptavidin and a biotinylated and fluorescently labelled oligonucleotide to attach a biological molecule.

It will be known to one skilled in the art that many other di or multi functional compounds can be reacted with epoxy groups to impart different surface functionalities to the photoresist structure. For example a bifunctional compound with one amine group and a latent carboxylic acid group (which could be protected if necessary or in the form of an anhydride) would provide an alternative surface functionality once the amine had reacted with the epoxy. The introduction of a carboxy functionality would enable the use of EDC coupling to be used, a standard technique used to covalently bond an amino modified molecule to a surface.

In addition, amino modified molecules have been directly linked to the surface of the beads utilising the available surface epoxy groups. In these experiments the beads were released from the wafer using 1-methyl-2-pyrrolidinone to preserve the epoxy groups followed by an overnight incubation using a high pH carbonate buffer. The resultant fluorescence was very strong and the best level of fluorescence observed of all the techniques developed. In addition to the reactions described, the reaction could equally well involve multiple steps. For example, epoxy groups can be hydrolysed to a diol containing two alcohol groups by either an acid or a base hydrolysis under suitable conditions. The alcohol groups can then be reacted with a range of compounds allowing flexibility in functionalising the surface.

Homogeneous functionalisation

For uniform introduction of functional groups to the structures we have developed a novel method to enable similar reaction conditions to be afforded to the top and/or bottom bead surfaces. This method gives rise to the same terminal functions seen on the side walls that enable covalent binding to occur. This method is applicable to crosslinkable polymeric materials in general, but will be exemplified in non-limiting fashion herein with reference to use of a polymeric photoresist material (such as SU- 8).

One method of producing unreacted photoresist functional groups on the top and bottom of the structure produced is to place a layer of the photoresist material that does not contain the crosslinking agent ("activator") both above and below the standard photoresist material containing the activator. For instance the SU-8 photoresist usually contains a photoacid generator (PAG). If a layer (4) of SU-8 including PAG is sandwiched between two layers (5) of SU-8 that do not contain the PAG, a structure shown in Figure 2 will be produced.

When this sandwich is exposed, acid will be generated in the middle layer of the sandwich initiating the crosslinking reaction to form a structure defined by the pattern of the exposure light. Curing of the layer above and below will only occur due to diffusion of the photoacid out of the middle layer during the post exposure bake and will therefore be very limited. This limited curing will leave significant numbers of unreacted photoresist functional groups on both the top and bottom of the structure produced and will be very similar to the surface produced on the sides of the structure.

Such a layered structure could be produced by repeating the process of spin coating and baking to remove residual solvent for each layer in turn. The baking temperature should be chosen to control the amount of diffusion between the two layers of photoresist. By keeping the baking temperature below the glass transition temperature for the photoresist, the layers will not interdiffuse, thus keeping the cross- linking agent (PAG in the SU-8 example) confined to one layer.

In other circumstances, a limited amount of diffusion and intermingling of polymer chains may be desirable. Once the layered structure has been created the mask exposure and subsequent processing steps can be carried out. The parameters for the baking processes must also be carefully controlled to ensure that the baked photoresist material does not stick to the illumination mask during the exposure step.

Such a process could be used to produce structures with unreacted functional groups on either or both the top and the bottom. Making Polymeric Materials "Non-Crosslinkable" Under the Conditions of Curing

As the post-exposure baking process is the step that allows the photoresist to cure other novel variations in the production process that result in the production of similar structures can be envisaged. For instance the addition of the top layer of photoresist material without activator could take place after the activated material has been exposed but prior to baking at a temperature above the glass transition temperature.

In another embodiment, if the aim was only to functionalise the top and side surfaces of the final photoresist structure then it would be possible to use standard activated photoresist material throughout the entire process. In this method the standard manufacturing process would be followed up to and including the mask exposure. At this point an additional layer of standard photoresist (containing the cross-linker, e.g. PAG) would be spin coated onto the top of the existing exposed layer. This would then be baked at a temperature below the glass transition temperature to drive off solvent from the new layer of material and finally the bake temperature increased above the glass transition temperature to allow the curing process to be completed.

In any of the methods described above if it were found that the bake temperature required to remove solvent led to increased diffusion between layers then this temperature could be reduced by carrying out the bake at a pressure below atmospheric pressure. This reduced pressure will encourage the removal of residual solvent and if necessary could be carried out at room temperature.

A further embodiment of providing unreacted photoresist functional groups is to lower the percentage content of the activating compound (i.e. the photoacid generator for the SU-8 photoresist). This would leave more of the photoresist functional groups unreacted and available on the surface for reaction with suitable surface functionalisation compounds.

A further embodiment is to utilise standard photoresist containing the activating agent or compound in all of the layers spun onto the wafer, however in order to prevent the curing of particular layers (i.e. the top and bottom layers), to add an additional compound to the photoresist material for those layers. This compound would be chosen to react very rapidly with the reaction initiating agent; for example in SU-8 this compound would react with the anion produced by illumination of the PAG and thereby prevent or significantly reduce the initiation of the crosslinking reaction. Any strong base would serve to inhibit cross-linking by photoacids.

The advantages of this invention should be clear to one skilled in the art, and include, but are not limited to, great flexibility in the choice and spatial location of the functionalisation, flexibility in the choice of polymeric material, low cost, simplicity, and the reproducible attachment of the functional group by strong covalent bonds. Examples of functionalisation include, but are not limited to, alcoholysis of epoxides to form an ether link, amination to form an amine link, carboxylation, reaction with organometallics, and reaction with the hydrogensulphite ion to form sulphonic acids.

The above embodiment illustrates the introduction of a target functional group via a single-step reaction with the photoresist functional group. However, the reaction could equally well involve multiple steps. For example epoxy groups can be hydrolysed to a diol containing two alcohol groups by either an acid or a base hydrolysis under suitable conditions. These alcohol groups can then be reacted with a range of compounds allowing flexibility in functionalising the surface.

General: Carrying out the Invention and Utility

The present invention allows for the production of functionally reactive machine readably coded particles (or "beads") and for the application of such machine readable beads to the life sciences (combinatorial chemistry, proteomics, genomics, pharmacogenomics) by the further derivitisation of the beads with suitable ligands (antibodies, antigens, oligonucleotides, etc.), often along with the addition of label chemistries (e.g. fluorophores, ruthenium salts). Polymers provide a desirable substrate for many such applications.

The particles are preferably microparticles having a maximum dimension of 1 mm or less, preferably 500 μm or less, more preferably 250 μm or less. In a second and optionally in a third dimension such microparticles preferably are of 100 μm or less, more preferably 50 μm or less, e.g. 25 μm or less.

The particles may be of any shape, for example, plate or disc shaped, but bar, rod oblong or lozenge like particles are preferred.

To provide room for markings which as a binary code are capable of differently encoding at least 32,000 different particles at a pitch of say 20 μm per mark, it is preferred that the particles have as their largest dimension a size of at least 50 μm, more preferably at least 100 μm. Particles within the size range of 100 μm to 250 μm are therefore preferred. A library of particles may be formed, but it need not however contain as many as 32,000 different encoded particles and so particles capable of bearing fewer marker coding elements are still useful. For instance, a library of say 4,000 particles could be coded by only 12 coding elements.

Markings may be formed along at least two sides of each particle.

The particles may be morphologically encoded during the production process so that, as the overall shape of the particles is defined, so are patterns of 3-dimensional features that provide each particle with its machine readable code.

Alternatively, the particles may be encoded, whether morphologically or otherwise after the production of their overall shape but before separation from the substrate. Non-morphological methods of encoding include selective area bleaching, or the application of inks, paints or dyes which may be monochromatic or multi-coloured or, fluorescent or phosphorescent.

The substrate on which the particles are formed by the delineation process may be a disposable member such as a wafer that is discarded when the particles have been removed from it or it may be a member that forms a structural part of an apparatus in which the particles are subsequently used such as a base plate of a microtitre plate.

The substrate may comprise a base layer and a sacrificial layer on which the sheet of polymeric material for delineation into the particle is carried. The particles are then released by destruction of the adhesion of the particles to the sacrificial layer. This may be such that it is caused to release the particles by UV or other radiation or by other means such as solvent dissolution.

The particles may be formed in a photoresist polymer and are delineated therein by applying light via a mask defining the edge contours of the particles. Alternatively, they may be delineated by laser machining.

The coding applied to the microparticles may consist of a sequence of binary features, e.g. along edges of the particles. Each particle may be provided with one or more coding features serving as a reading sense identifier, i.e. indicating the direction in which the sequence of binary features is to be read.

The encoded particles may be used for various purposes, not limited to use in chemistry related applications.

They may also of course be used as substrates in forming a chemical library in which a large number of chemically distinct materials are placed on respective particles or portions of particles. These may be oligomeric compounds differing one from another in sequences of monomer units, such as nucleic acids or their analogues (including DNA, RNA, PNA and other modified backbone nucleic acid analogues or hybrids thereof), proteins, polypeptides or peptides, and oligosaccharides.

In general, the compounds of the library may be either synthesised first and then bound to their particles or else may be synthesised stepwise on the particles. In either method, during the attachment of the compounds the particles may be still attached to the substrate on which they were delineated or may have been separated therefrom.

Generally, in a chemical library, it is intended that each particle bearing a particular code should bear a known one of a library of chemical compounds.

The term "chemical library" is to include "biological libraries" and may include DNA, RNA, PNA, antibodies, phages, and cells.

The chemical members of the library may be oligomeric compounds such as oligonucleotides or peptides in which monomer units selected from a limited range of chemically related compounds are arranged in a sequence characterising the oligomer. They may be non-oligomeric compounds, possibly being related to other members of the library by some common structure or actual or potential property. The compounds may be of complex structure, e.g. may be antibodies or other biomolecules.

Once the oligomers or other compounds have been applied to the beads, individual groups of beads can be released and processed e.g. using flow cytometry. With rectangular beads, the long aspect ratio lends itself easily to good mixing within the flow cell, thereby promoting effective binding of the bases of an analyte oligonucleotide onto a complementary target sequence.

The beads may be designed to be compatible with current micro-titre plates having 96- wells although the techniques mentioned are equally applicable to larger well sizes. At such dimensions, each 3.5 mm-square well could easily contain an array of 100 by 40 (or 4000) beads. The total number of beads defined within a commercially available 96-well structure would then be in the region of 384,000.

Particles carrying members of chemical libraries may be employed in any of the several fields in which chemical libraries have been used or proposed for use in the past including drug discovery assays, DNA sequencing, immunoassays, and combinatorial chemistry.

One method of reading codes on morphologically encoded (shaped) particles or beads is as follows:

Each bead has notches defined around its periphery to give it a castellated edge shape which imparts a code. The output of a laser beam of suitable wavelength is passed through the optical elements that transform the beam into a thin fan-shaped beam. The notches in the bead flowing through the flow cell obstruct the beam. To detect the forward-scattered energy, a sensor may be situated in line with the incident energy and beyond the bead. The output of this sensor varies according to the amount of energy falling on it. In the case of the forward-scattered energy, the notches prevent energy from reaching the detector and the gaps between the notches permit the energy to reach the detector. Either the forward or side scattered signals, or a combination of these signals may be used to deduce the code on the bead. This system allows for machine-reading of a signal from each coded particle so as to identify remotely said particle.

Claims

Claims

1. A method for functionalising all or part of a surface of a structure fabricated from a crosslinkable polymeric material comprising the steps of; defining a structure in a material that is crosslinkable by undergoing a curing process; the curing process involving cross-linking of reactive functional groups in the polymeric material; performing the curing process under conditions where at least part of the surface of the defined structure is in contact with a polymeric material that is non-crosslinkable under the conditions of the curing process; removing the non-crosslinkable polymeric material from the surface of the defined structure after curing, whereby said surface comprises unreacted functional groups.

2. A method according to claim 1 , wherein the structure is defined by a machining process.

3. A method according to claim 1 , wherein the structure is defined by a moulding process.

4. A method according to claim 1, wherein the structure is defined by a forming process.

5. A method according to claim 1 , wherein the crosslinkable polymeric material is a photoresist material.

6. A method according to claim 1 or 5, wherein the non-crosslinkable polymeric material is a photresist material.

7. A method according to claims 5 or 6, wherein the structure is defined in a photoresist material by exposure of the photoresist to photoradiation, where unexposed regions of the photoresist are non-crosslinkable in the curing step and the exposed regions of the photoresist are crosslinkable in the curing step.

8. A method according to claims 5 or 6, wherein the structure is defined in a photoresist material by exposure of the photoresist to photoradiation, where unexposed regions of the photoresist are non-crosslinkable by undergoing the curing step and the exposed regions of the photoresist are crosslinkable by undergoing the curing step.

9. A method according to claims 7 or 8, wherein the exposure of the photoresist material is through a mask, the shape of the structure being defined by the pattern of the photoradiation through the mask.

10. A method according to claims 5-9, wherein the photoresist material is in the form of a sheet.

11. A method according to any of claims 5-10, wherein the photoresist material has at least one surface in contact with a non-photoresist polymeric material prior to the step of defining the structure.

12. A method according to any of claims 5-11, wherein the photoresist material is sandwiched between layers of non-photoresist polymeric material prior to the step of defining the structure.

13. A method according to any previous claim, wherein the crosslinkable polymeric material comprises epoxy groups.

14. A method according to any previous claim, wherein the non-crosslinkable polymeric material comprises epoxy groups.

15. A method according to any previous claim, wherein the unreacted functional groups on the surface of the cured structure are epoxy groups.

16. A method according to any previous claim, wherein non-crosslinkable polymeric material is added to the surface of the crosslinkable polymeric material after the structure has been defined.

17. A method according to any previous claim, wherein the structure defined is a particle.

18. A method according to any previous claim, wherein the structure defined is a morphologically encoded particle.

19. A functionalised structure fabricated from a crosslinkable polymeric material by cross-linking of reactive functional groups in the polymeric material, said structure having chemical moieties and/or biomolecules covalently attached to non-crosslinked functional groups on all or part of the surface thereof.

20. A structure according to claim 19 wherein at least one major surface (other than a side or edge surface) is functionalised by attachment of chemical moieties and/or biomolecules.

21. A structure according to claim 19 or 20, wherein the crosslinkable polymeric material comprises epoxy groups.

22. A structure according to claim 19, 20 or 21 wherein the crosslinkable polymeric material is a photoresist.

23. A structure according to any of claims 19 to 22, wherein the structure is a particle.

24. A structure according to claim 23, wherein the particle is morphologically encoded.

25. A method of fabricating cured photoresist particles comprising: providing a sheet of uncured polymeric photoresist material on a substrate said sheet having first and second major surfaces in the plane of the substrate; delineating the sheet into a plurality of cured particles without destroying the integrity of the substrate using a mask by causing exposure of said material to photo-radiation through said mask to r-i irα cαiH Qvn sorl manorial- anrl romnwinn tha nartiHoc frnm tha 01 ihcfrato" tho chap*³ of the particles being defined by the pattern of the exposure photo-radiation through the mask; the curing reaction involving cross-linking of reactive photoresist functional groups; characterised in that at least one of said major surfaces in cured in contact with added uncured polymeric photoresist material; whereby curing of said surface(s) preserves at least some unreacted photoresist functional groups at the interface between said cured surface(s) and said uncured material.

26. A method as claimed in claim 25, wherein there is a step of machine-readably encoding the particles.

27. A method as claimed in claim 26, wherein the particles are morphologically encoded during the delineation.

28. A method as claimed in claim 26, wherein the particles are encoded after delineation but before removal from the substrate.

29. A method as claimed in any one of the preceding Claims, wherein adhesion between said sheet and the substrate is produced by a radiation-sensitive sacrificial layer whose properties are modified to reduce the strength of the adhesion to release the particles.

30. A method as claimed in any one of the preceding claims, wherein each particle is formed with at least one reading sense identifier.

31. A method as claimed in claim 25 wherein the machine readable code is formed by bleaching the fluorescence of a constituent of each particle.

32. A method as claimed in any one of the preceding claims, wherein chemical or biological moieties are covalently attached to photoresist functional groups on respective particles prior to removal from the substrate.

33. A method as claimed in claim 32, wherein chemical or biological moieties are applied to or synthesised on said particles in a well of a micro titre plate.

34. A method as claimed in any one of claims 25 to 31 , wherein a chemical or biological moiety is applied to or synthesised on each particle after release of said particle from said substrate.

35. A method as claimed in any one of claims 32 to 34, wherein at least two chemical moieties are provided on each particle, each said moiety occupying its own respective space on said particle separate from the space occupied by the or each other moiety thereon.

36. A method as claimed in any preceding claim, wherein both first and second major surfaces are cured in contact with uncured photoresist material.

37. A method as claimed in any preceding claim wherein said major surface(s) and side surfaces of said particles (said side surfaces being in a piane orthogonal to the plane of the substrate and being parallel to the direction of exposure of photo-radiation through the mask) have essentially homogeneously preserved photoresist functional groups.

38. A particle, obtained by the method of any preceding claim, said particle having at least 3 surfaces not sharing a common axis, said surfaces having preserved photoresist functional groups after curing.

39. A particle, obtained by the method of any of claims 25-37, said particle having a continuous curved side edge surface and at least one major surface, said surfaces having preserved photoresist functional groups after curing.

40. A particle, obtained by the method of any of claims 25-37, said particle having a discontinuous curved side edge surface and at least one major surface, said surfaces having preserved photoresist functional groups after curing.

41. A particle, obtained by the method of any of claims 25-37, said particle having preserved photoresist functional groups on all surfaces after curing.

42. A particle, obtained by the method of any of claims 25-37, said particle having a surface not parallel to the direction of exposure to photo-radiation during curing, which surface has preserved photoresist functional groups.