WO2006061600A1 - Particles for use as a solid support and process for their preparation - Google Patents

Particles for use as a solid support and process for their preparation Download PDF

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Publication number
WO2006061600A1
WO2006061600A1 PCT/GB2005/004681 GB2005004681W WO2006061600A1 WO 2006061600 A1 WO2006061600 A1 WO 2006061600A1 GB 2005004681 W GB2005004681 W GB 2005004681W WO 2006061600 A1 WO2006061600 A1 WO 2006061600A1
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WO
WIPO (PCT)
Prior art keywords
particle
material
particles
diffraction pattern
process according
Prior art date
Application number
PCT/GB2005/004681
Other languages
French (fr)
Inventor
Hywel Morgan
David Cameron Neylon
Peter Roach
Nikolay Zheludev
Gabriel Cavalli-Petraglia
Gerasim Stoychev Galitonov
Original Assignee
University Of Southampton
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to GB0426809A priority Critical patent/GB0426809D0/en
Priority to GB0426809.0 priority
Priority to GB0507181.6 priority
Priority to GB0507181A priority patent/GB2421076A/en
Priority to GB0518478.3 priority
Priority to GB0518478A priority patent/GB2422686A/en
Application filed by University Of Southampton filed Critical University Of Southampton
Publication of WO2006061600A1 publication Critical patent/WO2006061600A1/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584Control arrangements for automatic analysers
    • G01N35/00722Communications; Identification
    • G01N35/00732Identification of carriers, materials or components in automatic analysers
    • GPHYSICS
    • G03PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03FPHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/26Processing photosensitive materials; Apparatus therefor
    • G03F7/40Treatment after imagewise removal, e.g. baking
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00427Means for dispensing and evacuation of reagents using masks
    • B01J2219/00432Photolithographic masks
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
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    • B01J2219/00497Features relating to the solid phase supports
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • BPERFORMING OPERATIONS; TRANSPORTING
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    • BPERFORMING OPERATIONS; TRANSPORTING
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    • BPERFORMING OPERATIONS; TRANSPORTING
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    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/0072Organic compounds
    • B01J2219/00722Nucleotides
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2219/00718Type of compounds synthesised
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    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES, IN SILICO LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/06Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES, IN SILICO LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/10Libraries containing peptides or polypeptides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES, IN SILICO LIBRARIES
    • C40B60/00Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N35/00584Control arrangements for automatic analysers
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    • G01N35/00732Identification of carriers, materials or components in automatic analysers
    • G01N2035/00742Type of codes
    • G01N2035/00772Type of codes mechanical or optical code other than bar code

Abstract

The invention relates to a process for the preparation of particles (73) suitable for use as supports for solid supported chemical reactions. The use of a metal-containing sacrificial layer to liberate the photolithographically-produced particles (73) results in their having superior utility in multi-step reaction sequences. The particles may comprise a predetermined diffraction pattern identifier. A method for reading such an encoding is also disclosed.

Description

PARTICLES FOR USE AS A SOLID SUPPORT AND PROCESS FOR THEIR PREPARATION

Field

The present invention relates to a process for the preparation of a particle for use in solid supported chemistry, to particles obtained by such a process, and to a plurality of uses of such particles.

Background

The use of solid supports in synthetic chemistry has been known for a number of years. The basic concept of attaching a starting material to a functionalised polymeric support, and performing transformations of the supported starting material confers a number of advantages over conventional solution-phase synthetic methods, including:

ease of separation of products from reaction mixture;

amenability to iterative synthetic sequences;

ease of purification of final product.

The technique was originally developed for the synthesis of peptides (R.B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963)). However, it has been substantially expanded and is now routinely used in the preparation of other chemical species such as oligonucleotides, oligosaccharides, heterocycles and others.

More recently, the methods of solid phase synthesis have found utility in the field of combinatorial chemistry. Combinatorial chemistry is a technique for generating large numbers of chemical compounds using relatively few chemical reaction steps. In tandem with high throughput screening, combinatorial chemistry has greatly accelerated the process of generating new compounds for leads as pharmaceuticals, agrochemicals and the like.

In various combinatorial chemistry and biological assay techniques it is known to use solid support particles (e.g. beads) to synthesise chemical entities (such as, for example, polypeptides, carbohydrates, nucleotides and other oligomeric and non- oligomeric compounds) for inclusion as predetermined chemical entities in a chemical library. Additionally, it is also known that such particles can be tagged to make them identifiable so that they may then be used to test various substances for the presence, or absence, of chemical entities in those substances.

Since identifiable particles are especially useful for high throughput screening, combinatorial chemistry, genomic and proteomic scientific applications, much effort has gone into the development of tagging techniques used to provide or encode an identifiable particle, or set of identifiable particles, with an identifier. Such identifiers can be read following chemical interactions at the identifiable particles in order that the identifiers can be matched to those indicating the chemical entities in the chemical library or other chemical entities with which the library chemical entities interact.

Numerous tagging techniques exist for tagging or coding identifiable particles, such as beads. For example, it is known that particles can be coded using transponders (US5736332, US5981166, US6051377, US6361950, US6376187), magnetic tags, biological tags and various optical techniques. One example uses unique short sequences of DNA that are attached to different tagged particles which are decoded after chemical processing by using a polymerase chain reaction (PCR). Conventional optical techniques that are currently used to identify particles include the following: fluorescent tagging; infrared (IR) tagging; optical image pattern recognition (GB2306484); Raman tagging (J. Am. Chem. Soc, 25, 10546-10560 (2003)); and quantum dot encoding (Anal. Chem., 76, 2406-2410, (2004)).

Prior art

GB2306484 discloses particles suitable for use in combinatorial chemistry techniques. The support particle preferably comprises a first phase comprising a solid support suitable for use in combinatorial chemistry techniques, and a second phase containing a machine readable code.

GB2334347 discloses a process for the preparation of coded particles comprising steps of

i. coating a face of a wafer of silicon or a similar crystalline material or inert metal or metal alloy with a photo-resist polymer ii. exposing the coated face of the wafer to ultra-violet radiation through a photolithographic mask, said mask defining the particle size and/or the position of code sites on the particles;

iii. dissolving or otherwise removing either the UV-Exposed or the UV- Unexposed areas of photo-resist polymer;

iv. etching the exposed areas of the wafer, from which the photoresist polymer has been removed, using an appropriate etching agent; and

v. liberating the particles

The processes of this disclosure involves the etching of the silicon wafer (or other suitable solid) itself to form the particles.

US2003/0153092 discloses a process for the preparation of coded microparticles comprising

i. providing a sheet of polymeric material on a substrate;

ii. delineating the sheet into a plurality of particles without destroying the integrity of the substrate;

iii. machine-readably encoding the particles;

iv. removing the particles from the substrate.

Amongst the techniques suitable for removing the particles from the substrate is the removal of a sacrificial layer. A preferred sacrificial layer is a 3 μm layer of photoresist. Release is effected using a diluted developer.

A problem that remains is to provide particles suitable as supports for multi-step synthetic sequences.

A further problem that remains is to provide improved particles suitable for use in solid supported chemical reaction sequences.

The present invention addresses the problems of the prior art. Summary of the invention

According to a first aspect, there is provided a process for the preparation of a particle for use as a solid support in chemistry comprising

i. coating a laminar support material comprising at least a sacrificial layer with a photopolymerisable material;

ii. exposing the coated face of the support to radiation through a photolithographic mask, said mask defining the particle size and shape;

iii. selectively removing the exposed or unexposed areas of photopolymerisable material;

iv. reducing the integrity of the sacrificial layer to liberate said particle;

characterised in that the sacrificial layer comprises a metal or metals.

According to a second aspect, there is provided a particle obtainable by a process of the invention.

According to a third aspect, there is provided the use of a particle of the invention as a support for solid supported chemistry.

According to a fourth aspect, there is provided the use of a particle of the invention in the preparation of an oligopeptide.

According to a fifth aspect, there is provided the use of a particle of the invention in the preparation of an oligonucleotide.

According to a sixth aspect, there is provided the use of a particle of the invention in the preparation of a chemical library.

According to a seventh aspect, there is provided a process for the preparation of a library of compounds comprising steps of

i. providing a plurality of encoded particles of the invention;

ii. dividing the particles into a plurality of portions; iii. subjecting each portion to a specific chemical reaction;

iv. reading the code of a particle;

v. recombining the portions; and

vi. repeating steps i to v n times, wherein n is an integer.

Brief description of the figures

Figure 1 shows particles according to embodiments of the present invention;

Figure 2 shows an identifiable particle according to an embodiment of the present invention;

Figure 3 shows scanning electron microscope (SEM) images of gratings that encode predetermined diffraction pattern identifiers according to embodiments of the present invention;

Figure 4 shows a plot of how encoding capacity varies with grating length for various gratings that encode predetermined diffraction pattern identifiers according to embodiments of the present invention;

Figure 5 shows schematically various one-dimensional and two-dimensional gratings that may encode predetermined diffraction pattern identifiers according to embodiments of the present invention;

Figure 6 illustrates a method of making a master template for producing particles according to an embodiment of the present invention;

Figure 7 illustrates a process for the preparation of particles according to an embodiment of the present invention;

Figure 8 illustrates an optional second part of a method of making tags for particles using a master template according to an embodiment of the present invention;

Figure 9 illustrates a method of making tags for particles according to an embodiment of the present invention; Figure 10 illustrates a method of making tags for particles according to an embodiment of the present invention;

Figure 11 illustrates a method of making tags for particles according to an embodiment of the present invention;

Figure 12 schematically shows a reader for reading an identifier from particles according to the present invention in perspective view;

Figure 13 schematically shows the reader of Figure 12 in plan view; and

Figure 14 schematically shows alternative detector positions in a reader for reading identifiers from particles according to the present invention.

Detailed description

Laminar support material

As used herein, the term "laminar support material" refers to a material comprising one or more layers and having a substantially uniplanar face.

Preferably, the laminar material comprises at least a substrate material. More preferably, the substrate material comprises silicon or a glass material. Most preferably, the substrate material consists of silicon or a glass material.

If the laminar support material comprises more than one layer, the layers may be held together using any suitable means. For example, an intermediate adhesive layer may be necessary or expedient.

In some embodiments, the laminar support material may consist of only a sacrificial layer.

Sacrificial layer

As used herein, the term "sacrificial layer" refers to the layer of the laminar support material which can be reduced in integrity in order to liberate the particle of the invention. The sacrificial layer of the present invention comprises a metal or metals. The metal may exist as a pure metal (i.e. a layer of elemental metal), as an alloy, or as a dispersion of metal particles within a matrix material. Preferably, the sacrificial layer consists of a metal or an alloy of metals.

Preferably, the sacrificial layer comprises at least aluminium or chromium or both. More preferably, the sacrificial layer is an alloy comprising aluminium. More preferably, the sacrificial layer consists essentially of aluminium.

Surprisingly, the use of sacrificial layers comprising a metal or metals results in a particle having markedly superior properties as a support for use in solid-supported chemistry, especially in multi-step reaction sequences.

Preferably, the thickness of the sacrificial layer is less than 1000 nm. More preferably, the thickness of the sacrificial layer is less than 500 nm. More preferably, the thickness of the sacrificial layer is less than 200 nm. More preferably, the thickness of the sacrificial layer is less than 100 nm. Most preferably, the thickness of the sacrificial layer is less than 50 nm.

Preferably, the thickness of the sacrificial layer is more than 1 nm. More preferably, the thickness of the sacrificial layer is more than 2 nm. More preferably, the thickness of the sacrificial layer is more than 5 nm. Most preferably, the thickness of the sacrificial layer is at least 10 nm.

Preferably, the thickness of the sacrificial layer is between 1 and 1000 nm. More preferably, the thickness of the sacrificial layer is between 5 and 100 nm. Most preferably, the thickness of the sacrificial layer is between 10 and 50 nm.

Preferably, the sacrificial layer is applied to the adjoining layer of the laminar support material by a sputtering process. The term sputtering refers to the process of dislodging atoms from a target material to coat a thin film onto a substrate.

Photopolymerϊsable material

As used herein, the term "photopolymerisable material" refers to a composition comprising at least one monomer which is capable of undergoing polymerisation when exposed to radiation. Preferably, the photopolymerisable material is a u.v. polymerisable material. That is, the composition is capable of undergoing polymerisation when exposed to ultraviolet light.

Preferably, the photopolymerisable material is present as a solution for the purposes of coating on to the laminar support material. Suitable solvents include water and organic solvents.

A preferred photopolymerisable material is SU-8. SU-8 comprises three components; an EPON epoxy resin, an organic solvent, and a photoinitiator. The EPON resin of SU-8 is a multifunctional glycidyl ether derivative of bisphenol-A novolac used to provide high-resolution patterning for semiconductor devices. The second component is gamma-butyrolactone (GBL), an organic solvent. The quantity of the solvent determines the viscosity of the solution, which determines final thickness of the spin- coated film. Along with GBL, cyclopentanone is also used as a solvent for SU-8. The third component is a triarylium-sulfonium salt, a photoinitiator which is approximately 10 wt % of EPON SU-8. SU-8 resin can be cationically polymerised by utilizing a photoinitiator which generates strong acid upon exposure to ultraviolet light (365 to 436 nm) and the acid facilitates polymeric cross-linking during post-exposure bake.

To enable the use of the particles formed from polymerised photopolymerisable material as supports in combinatorial chemistry, it is necessary that the polymerised material bears reactive functional groups. The term "reactive functional group" as used herein refers to a chemical group present in the polymerised photopolymerisable material capable of reacting with a small molecule (i.e. one present in solution) to form a covalent bond.

Examples of reactive functional groups are halogen (especially fluoro, chloro, bromo and iodo), hydroxy, carboxy (CO2H), carbonyl, amino, epoxy and thiol.

The reactive functional group may be introduced subsequent to polymerisation of the photopolymerisable material. For example, if the polymerised material comprises unfunctionalised aromatic rings, it may be chloromethylated in a manner analagous to the preparation of Merrifield resins (R.B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963)). However, it is preferred that the polymerised photopolymerisable material possesses reactive functional groups without the necessity of introducing these subsequent to polymerisation.

For example, when the photopolymerisable material comprises an epoxy resin (as in the case of SU-8), the polymerised material will comprise at least an amount of epoxy groups.

The photopolymerisable material may be coated on the laminar support material using any appropriate technique. It may be applied as a sheet that is simply adhered to the laminar support material. If the polymerisable material is a liquid, it may be applied as a spray or aerosol. Alternatively, it may be painted on using a brush or roller. A preferred technique for coating the laminar support material with photopolymerisable material is spin coating.

Spin-coating consists of dispensing photpolymerisable material solution over the wafer surface and rapidly spinning the wafer until it becomes dry. Typical spin- coating processes are conducted at final spin speeds of 3000-7000 rpm for a duration of 20-30 seconds.

The thickness of the coat of photopolymerisable material is preferably between 0.1 and 100 μm, more preferably between 0.5 and 50 μm, still more preferably between 1 and 25 μm.

Optionally, it may be necessary to pre-treat the laminar support material with a layer of adhesive or primer in order to achieve satisfactory binding of the support material to the photopolymerisable material. Preferably, such a primer comprises hexamethyldisilizane (such as Microposit primer).

Optionally, it may be necessary to heat treat the laminar support material (for example at about 200 0C for about 30 minutes) in order to effect dehydration. This also assists in achieving good adhesion of the photopolymerisable material to the laminar support material.

After the laminar support material has been coated with photopolymerisable material, it may be necessary or convenient to subject the coated material to one or more subsequent treatment steps prior to irradiation. When the photopolymerisable material is applied as a solution, a soft baking step is preferably employed. A soft bake is done to: i) drive away the solvent from the applied photopolymerisable material; ii) improve the adhesion of the photopolymerisable material to the laminar support material; and iii) anneal the shear stresses introduced during the coating. Soft baking may be performed using one of several types of ovens (e.g., convection, infrared, hot plate). Soft-bake ovens must provide well-controlled and uniformly distributed temperatures and a bake environment that possesses a high degree of cleanliness. The recommended temperature range for soft baking is between 90-100 0C, while the exposure time heeds to be established based on the heating method used and the resulting properties of the soft-baked resist. These factors will be readily determined by one skilled in the art.

Exposure to radiation

After the laminar support material has been coated with photopolymerisable material, and optionally subjected to one or more intervening treatments (preferably soft baking), the coated face is exposed to radiation

The radiation is of a wavelength and intensity to cause regions of photopolymerisable material exposed to it to polymerise. These factors will be dependent on the nature of the photopolymerisable material, and the skilled person will be able to determine them.

For example, when the photopolymerisable material is SU-8, polymerisation is suitably achieved by exposure to ultraviolet (u.v.) radiation for between 1 and 30, preferably about 18 seconds, at an intensity of between 1 and 50 mw cm"2, preferably about 13.6 mw cm"2.

Post exposure treatment

Preferably, subsequent to exposure to radiation, the polymerised photopolymerisable material is subjected to one or more post exposure treatment steps in order to make the polymerised material more physically and chemically durable during later stages. Preferably, the post exposure treatment steps comprise at least a post exposure bake. Such a post exposure bake serves to further cross-link the polymerised material and increase its strength and durability.

Preferably, the post exposure bake is conducted at about 65 0C for about 1 minute, followed by about 95 0C for about 5 minutes.

Photolithographic mask

The coated face of the laminar support material is exposed to radiation through a photolithographic mask. The principles of photolithography are well known from the field of semiconductor device fabrication. The photolithographic mask consists in essence of portions of material transparent to radiation, and portions of material opaque to radiation arranged in the desired pattern, such that when radiation is applied to the coated face of the laminar support material through the mask, the pattern is transposed onto the coated face.

The photolithographic mask defines the size and shape of the particles by means of the above-mentioned transparent and opaque portions.

Selective removal

Subsequent to exposure to radiation and optional post-exposure treatment, either the polymerised regions (i.e. those exposed to radiation) are removed to leave the unpolymerised regions, or the unpolymerised (i.e. unexposed) regions are removed to leave the polymerised regions. This is referred to as "development". Preferably, the unpolymerised regions are removed.

The person skilled in the art will be able to determine suitable conditions for the selective removal of either the exposed or unexposed regions.

Preferably, the unexposed photopolymerisable material is selectively removed from the coated laminar substrate by treatment with organic solvent. Preferably, the organic solvent is polypropylene glycol methyl ether acetate (PGMEA). It is particularly convenient to immerse the laminar substrate in a bath of such a solvent.

Again, the skilled person will be able to determine the optimum duration of such a treatment step to ensure substantially complete removal of unexposed regions of photopolymerisable material while having little or no effect on the exposed regions. After such a development step, the coated laminar substrate may be washed to remove excess solvent.

Optionally, after such development and optional washing steps, the developed coated laminar substrate may be subjected to a post-development bake.

Very preferably, the development step discloses or reveals portions of sacrificial material between the residual polymerisable material (i.e. the adhering particles).

Figure 7 illustrates a process for the preparation of particles of the invention. At step 1 , a layer of photopolymerisable material 75 is coated on to a thin layer of sacrificial material 76 on a substrate layer 70. At stage 2, a mask is superimposed on the layer 75, and the whole is exposed to radiation. The layer of photopolymerisable material is selectively exposed through transparent and opaque 71 regions of mask 72. At step 3 the mask 72 is removed, leaving polymerised 73 and unpolymerised regions of photopolymerisable material. At step 4, unpolymerised material is removed in a development process to leave particles 73 (i.e. regions of polymerised material) adhering to sacrificial material 76. In a subsequent step (not shown), the integrity of the sacrificial material 76 is reduced to liberate the particles.

Liberation of particles

After the abovementioned development step, the developed coated laminar substrate comprises particles of the invention adhering to the laminar substrate. In order to liberate the particles, the integrity of the sacrificial layer is reduced.

Reduction of the integrity of the sacrificial layer may be achieved by any one of a number of means. In a broad sense, any degree of reduction of integrity is acceptable provided that it serves to liberate the particles from the laminar support material.

Preferably, the integrity of the sacrificial layer is reduced by at least partially dissolving the metal component.

The metal component may be at least partially dissolved by treating the developed coated laminar substrate with an etch. Suitable etches for a variety of metals are known, and the skilled person will be able to select an appropriate one. When the sacrificial layer is or comprises aluminium, it is preferred that the etch comprises hydroxide ions. Highly preferably, the etch comprises tetraalkylammonium hydroxide. Very highly preferably, the etch comprises tetramethylammonium hydroxide.

A highly preferred etch is Microposit MF 319 developer, available from Rohm and Haas. This is an aqueous solution comprising 97-98 wt. % water, 0.1 - 1 wt. % surfactant and balance tetramethylammonium hydroxide.

Preferably, the particles are liberated by immersing the developed coated laminar substrate in the etch. The liberated particles are then removed by filtration and washed. Preferably, the developed coated laminar substrate is immersed in the etch for a period of less than 1 minute. More preferably, the developed coated laminar substrate is immersed in the etch for a period of less than 30 seconds. More preferably, the developed coated laminar substrate is immersed in the etch for a period of less than 20 seconds. More preferably, the developed coated laminar substrate is immersed in the etch for a period of less than 10 seconds. It has been found that a reduction in the time the particles are exposed to the etch results in improved performance of the particles as substrates for solid supported chemistry.

Size and shape of particles

As will be appreciated, the thickness of the particles will be chiefly determined by the thickness of the coat of photopolymerisable material originally applied to the laminar substrate. The photolithographic mask determines the ultimate shape of the particles in the remaining two dimensions once liberated from the laminar substrate. The particles may be of any desired size and shape that is convenient for the intended end use.

Preferably, the particles are of square or rectangular cross section. More preferably, the particles are rectangles of from 1 to 500 μm by 1 to 500 μm.

Particle encoding

Optionally, and highly preferably, the particles may comprise an encoded identifier, or tag, which enables the individual particles to be distinguished from one another. In one embodiment, the particles of the invention are encoded by means of their shape. Such a scheme is described in US2003/0153092, which is incorporated herein by reference in its entirety.

Alternatively, each particle may be encoded by pits, grooves, notches, bumps, ridges or by fluorescent, coloured or monochrome markings (for example, bar codes). Any conventional technique may be used to apply such encoding motifs, for example by printing. Alternatively, magnetic or radiofrequency identifiers may be incorporated or attached.

The particles may also be encoded using Raman barcoding, comprising spectroscopic bar codes that incorporate infrared and Raman-active groups that are identifiable using standard infrared or Raman spectrometers. Such a scheme is described in J. Am. Chem. Soα, 25, 10546, 2003.

Encoding may be performed at any stage of the preparation of the particles. In a preferred embodiment, the photolithographic mask comprises encoding means, so that encoding occurs during exposure of the photopolymerisable material to radiation.

In an alternative preferred embodiment, the particles are encoded by embossing features into the photopolymerisable layer by means of a rigid stamp. This technique is referred to as nanoimprinting.

Alternatively, the encoding of the particle can be achieved by embossing, imprinting, injection moulding, laser ablation or direct write (e-beam) techniques.

In an alternative preferred embodiment, encoding occurs in a secondary coating / exposure / development sequence subsequent to development of the primary coating (i.e. after step iii).

The exposed and developed layer of photopolymerisable material is coated with a secondary coat of photopolymerisable material. The coated face is exposed to radiation through a second photolithographic mask, and developed in a similar manner to that described above.

Preferred Encoding Method Preferably, the particles are encoded with a diffraction pattern identifier such that the particles give an identifiable diffraction pattern when exposed to a particular wavelength of radiation.

The diffraction pattern identifier may be encoded by material variations provided in the particle material. Smoothly changing or stepped transparent refractive index variations may be used to provide patterns of material variations. Non-transparent materials may also be used where patterned material variations are provided in one or both of the absorptive strength or the reflectivity of the material. Reflective features may also be used. Various techniques can be used to provide material variations, such as, for example, embossing using a master template, nano-imprinting, laser- ablation, injection moulding, etching with a high energy beam, writing an interference pattern using an optical interference technique, or applying a pattern using a lithography technique. Such techniques lend themselves well to relatively rapid, simple and cheap mass production techniques that may be used when manufacturing identifiable particles.

The localised distribution of the material variations may be used to encode a predetermined diffraction pattern identifier that may be used for identifying the particle. Such a diffraction pattern identifier is chosen from a number of possible diffraction pattern identifiers that could be encoded into the particle during its manufacture using a common encoding scheme.

Each unique pattern of material variations in a particle which are provided according to the encoding scheme gives rise to a unique diffraction pattern. Such diffraction patterns are particularly suited to various forms of digital processing, since a large number of possible permutations in a spatially distributed pattern can be obtained using the same type of pattern of material variations provided by a particular encoding scheme. One example of an encoding scheme uses increasing numbers of superimposed refractive index gratings provided in the same physical space in a particle material to provide increasingly complex diffraction patterns. By increasing the number of superimposed gratings, such an encoding scheme enables an increasingly large number of different diffraction pattern identifiers to be encoded.

Various identifiers may be read by exposing the particle to various types of radiation such as, for example, optical radiation. Since in this embodiment the particle relies on diffraction techniques to provide the identifier, it need not rely upon near-field imaging techniques for particle reading and the material variations used to encode the identifier may be smaller in size than the wavelength of any exposing radiation that is used to read the identifier. This also means that a large number of different identifiers can be encoded in a relatively small volume of particle material.

Additionally, since the diffraction pattern identifier may be read using far-field detection, a reader for identifying the particles may be sited remotely from the particles themselves. This has practical benefits for readers that are designed to read identifiers from the particle. Moreover, since the material variations that give rise to diffraction can be provided within the particle itself, no separate identifier-bearing tags need be attached to make particles identifiable.

According to a further embodiment of the invention, there is provided a method of making particles comprising selecting a diffraction pattern identifier from a plurality of diffraction pattern identifiers that can be encoded using the same identifier encoding scheme, determining the material variations needed to encode the diffraction pattern identifier, and providing the material variations in a particle material to form a particle. Many particles having the same or different identifiers may be made, as desired.

According to a further embodiment of the invention, there is provided a kit for detecting the presence of a predetermined chemical entity comprising a plurality of particles. Such a kit may be used to "sift" genes or proteins in search of new drugs or drug targets. When attached with DNA sequences (oligomers), antibodies, peptide sequences, or other proteins or small molecules, the particles can home in on target molecules, tagging them with one or more identifiers that can subsequently be read. Such molecules may additionally be labelled with some other secondary molecule, such as, for example, a fluorescent tag, a magnetic tag, or an alternative means of identification.

According to a further embodiment of the invention, there is provided a reader for reading a predetermined diffraction pattern identifier from a particle. The reader comprises a source for exposing particles to radiation, a detector for detecting the diffraction pattern of radiation emitted from the particles, and an analyser for analysing the diffraction pattern of the radiation emitted from the particles in order to determine whether the diffraction pattern corresponds to a diffraction pattern identifier encoded according to a predetermined encoding scheme. In various embodiments the analyser comprises a data processing apparatus that uses one or more of: hardware components, firmware components, and software components.

According to a further embodiment of the invention, there is provided a method for reading a predetermined diffraction pattern identifier from a particle. The method comprises exposing a particle to radiation, detecting the diffraction pattern of radiation emitted from the particle, and analysing the detected diffraction pattern in order to determine whether it matches a diffraction pattern identifier encoded using a predetermined encoding scheme.

According to a further embodiment of the invention, there is provided a method for identifying a chemical entity in a test substance. The method comprises introducing a plurality of particles comprising a plurality of predetermined receptor materials into the test substance, wherein respective different predetermined receptor materials bind to respective different predetermined chemical entities. The method also comprises reading the respective predetermined diffraction pattern identifiers of the particles that have bound to respective chemical entities, and matching the identifiers that are read to respective predetermined chemical entities so as to identify the chemical entities present in the test substance.

Such a method may be used in the search for new drugs or drug targets, or for rapid parallel sequencing of nucleic acids, analogues of nucleic acids or modified nucleic acids. Moreover, the use of these particles can remove the need to perform PCR and gel migration steps when sequencing methods are performed.

The method may also be used to screen for proteins, to detect the binding of proteins to various immobilised bio-molecules or to detect proteins through interactions with bound ligands. It may be used to screen libraries for the activity of different agents, and peptide libraries may be grown or immobilised upon the particles.

Figures 1a-d show particles 10a-d. The particles 10a-d are made of a particle material 12.

Within the particle material 12 a grating 16 is provided that encodes a predetermined diffraction pattern identifier by providing a predicable diffraction pattern when exposed with laser-generated radiation. Figure 1a illustrates a single one dimensional grating 16a provided in particle 10a; Figure 1 b two overlapping one dimensional gratings 16b provided in particle 10b; Figure 1c a double overlapping two dimensional grating 16c provided in particle 10c; and Figure 1d a complex pattern diffraction grating 16d provided in particle 1Od.

The gratings 16 can be provided by inclusion of non-particle material 17 (as shown in Figure 1e) or by providing a relief pattern 18 in the particle material 12 (as shown in Figure 1f). The grating 16 may be written directly into the particle material 12 using techniques such as direct interference writing or by embossing the particle during manufacture.

In Figure 1f a particle 10 is further shown with a coating of receptor material 14 for binding to a predetermined chemical entity. The receptor material 14 is placed over the grating 16. It has been found that the grating 16 provides a suitable site for the stable hosting of various receptor materials 14 for use in combinatorial chemistry. Many types of receptor material 14 can be provided for binding to various nucleic acids, polypeptides, carbohydrates and other oligomeric compounds, for example. During a reaction, chemical entities bind to the receptor material 14 and, since the particle 12 can be uniquely identified, the reaction can be observed in situ whilst also being distinguished from any other reactions between different particles and their target chemical entities.

Where the receptor material 14 is an oligonucleotide that binds to a nucleic acid, an analogue of a nucleic acid or a modified nucleic acid, a highly parallel approach to sequencing is made possible, whereby a large number of particles having different receptor materials can be placed into a test substance, selectively bind to components in the test substance and subsequently be identified (for example, a large plurality greater than 10, 102, 103, 104, 105, 106, 107, etc. of uniquely particles may be provided). This enables, for example, sequencing of nucleic acids, such as D N A/RN A/protein nucleic acids, to be performed rapidly and with minimal cost and effort.

In a variation upon the particles 10 that are illustrated, the particle material 12 may incorporate various formations that can be attached to a further particle (not shown).

Such a further particle may be coated in receptor material so that the particle 10 acts as a tag that is separated from the active receptor material. For example, further particles may comprise coated spherical latex beads or the like to which tags comprising particles can be attached. In further embodiments, it envisaged to place particle material into living host cells, whereupon the cells themselves act as particles that can be individually identified.

The particles 10 may be manufactured from materials that enable the identifier to be determined only after exposure to some form of radiation, heat, chemical change (e.g. pH or salt concentration) or developing agent. In various embodiments, the identifier may be erased following exposure to radiation, heat, pH or electrolyte concentration.

Figure 2 shows a particle 20. The particle 20 is made of a particle material 22 embossed with a master made using a phase grating 26 that encodes a predetermined diffraction pattern identifier. The particle material 22 is of elongate shape having a longest dimension D. Typically D will lie in the range from about 100 nm to about 10 μm. The particle material 22 is additionally coated with a receptor material 24 for binding to a predetermined chemical entity.

Figure 3 shows scanning electron microscope (SEM) images of gratings 30, 32, 34 that encode respective predetermined diffraction pattern identifiers. The gratings are created by using direct writing electron-beam lithography to provide a pattern on a PMMA film, provided on a metal-coated silica substrate, followed by a standard Deep Reactive Ion Etching (DRIE) process. Information relating to the identifier is encoded by the grating period. Thus, in order to increase the encoding capacity of the gratings 30, 32, 34, the pattern that is written comprises a superposition of separate gratings each having a different periodicity.

Figure 4 shows a plot 40 of how encoding capacity of grating-based identifiers varies with grating length for various different gratings 41, 43, 45, 47, 49 that encode predetermined diffraction pattern identifiers. The encoding capacity is calculated as the number of bits of information that the particular grating 41 , 43, 45, 47, 49 can encode and is calculated assuming that the gratings are to be read using optical radiation having a wavelength of 532 nm.

The plot 40 shows how the encoding capacity increases by some five orders of magnitude when the grating changes from a single period grating 41 having a length of about 100 μm to a grating 49 comprising five superimposed single period gratings having an overall length of about 100 μm.

Figure 5 shows schematically various one-dimensional and two-dimensional gratings 52, 54, 56, 58 that may be used to encode predetermined diffraction pattern identifiers in particles or tags for attaching thereto.

The first grating 52 has a length di and comprises a regularly spaced series of either reflective and less reflective elements or a series of alternating refractive index materials of pitch p. The second grating 54 has a length d-i and is formed by two linearly superimposed gratings having respective pitches of P1 and p2.

The third grating 56 is based upon a two dimensional pattern of size d-i x d2, and is formed by two orthogonally superimposed gratings having respective pitches of pi and p2. The fourth grating 58 is also based upon a two dimensional pattern of size di x d2, but is not formed of regular gratings, comprising instead a predetermined arbitrary pattern that gives rise to a calculable (or measurable) far-field diffraction pattern identifier. Use of such two-dimensional patterning for gratings enables an even larger number of identifiers to be encoded with particles.

Figure 6 illustrates a method of making a master template 60 for producing particles. The master template can be used for embossing a pattern that provides a particle with an identifier. The pattern can have sub-micron feature sizes that cannot be made directly by simple or conventional photolithography. By creating a master template 60 that can be used for embossing, direct write electron beam lithography does not need to be used to create a pattern for each particle (this would take many hours and be expensive).

The master template 60 is created from a pre-form 61. The pre-form 61 comprises a substrate 62 made of silicon or glass. The substrate is coated in a thin layer of chromium 64 (~100 nm thick) overlaid with a layer of electron-beam sensitive resist 66.

The required pattern may be designed using a computer-aided design (CAD) package, such as, for example, AutoCAD™ available from Autodesk, Inc. of San Rafael, CA, USA. Such a pattern defines the variations needed to encode a particular predetermined diffraction pattern identifier. Once the pattern has been determined, it is then written at step 1 using an electron beam into the electron-beam sensitive resist 66. A direct write E-beam lithography system, model number SB350-DW, and which is available from Leica Microsystems Semiconductor GmbH of Wetzlar, Germany was used for this purpose.

Figure 7 illustrates a process for the preparation of a particle of the invention as described above.

Figure 8 illustrates a method of making tags 73" for use with particles using a master template 60. The tags 73' may be made from a polymer material such as SU8 (which is an Epoxy-based resist that can be used to produce high aspect ratio features), or polyimide or PMMA or some other resist material, for example. The tags 73' may be embossed into sheets or rolls of plastic material on a production line. A two step process is used, one to define the extent of the tags 73' (shown in Figure 7) and another to encode an identifier onto the tags 73' (shown in Figure 8).

Figure 8 shows how an identifier is encoded by nano-imprint lithography or nano- embossing onto the bare tags 73 produced on the substrate 70 using the method shown in Figure 7 and described above.

At stage 5, a thin second layer of SU8 (e.g. 0.5 μm to 2 μm) is deposited over the bare tags 73. As previously, the substrate 70 comprising the bare tags 73 is spun at 500 rpm for 5 seconds, accelerated to 1000 rpm within 5 seconds and kept at 1000 rpm for 30 seconds, followed by the soft-baking process already described.

Using the mask 72, the second layer of SU8 is exposed to remove the material between bare tags 73, at stage 6. The substrate 70 is exposed to L)V light for 24 seconds through the mask 72 (appropriately aligned) and then post-exposure baked at 9O0C for 15 minutes on a hotplate. A development step is then carried out in SU8 developer for 2 to 7 minutes with periodic rinsing in IPA. Finally, the substrate 70 is blow-dried with nitrogen leaving a thin layer of SU8 on the bare tags 73.

The bare tags 73 are then embossed to impart a pattern and convert them into tags 73" that encode a predetermined diffraction pattern identifier. To do this, a silicon or metal master template 60 is aligned and brought into contact with the substrate 70 before external pressure is applied (e.g. 50 kg/cm2 at 8O0C). Subsequently, the template is released and the substrate 70 is hard baked to affix the embossed pattern encoding the identifier into the tags 73'. The hard baking process is performed at an elevated temperature lower than the Tg of the material in which the pattern is embossed. For example, for poly(benzyl methacrylate) hard baking could be performed at 540C, whereas for poly(cyclohexyl acrylate) it could be performed at 1000C.

Figure 9 illustrates a method of making tags 83 for use with particles using a combined photolithography and nano-imprint lithography technique. A glass or silicon wafer 80 is processed as per conventional lithography, i.e. cleaned etc. The surface is then coated with a release agent. The release agent provides layer of a sacrificial layer 86 that comprises one or more layers of metal (e.g. aluminium or chromium), together with layers of photoresist (e.g. S1818), or a thin coating of Teflon™ or any combination thereof.

A layer of SU8 85 (for example of the order of 1 to 25 μm thickness) is disposed over a thin layer of sacrificial material 86 onto the substrate 80, which has been pre-baked at 2000C for 30 minutes for dehydration purposes. The substrate is spun at 500 rpm for 10 seconds, then accelerated to 2500 rpm within 10 seconds and keep at 2500 rpm for 10 seconds. Then it is soft-baked on a hotplate at 9O0C for 20 minutes until the SU8 film is no longer sticky following a slow cooling.

A mask 82 is pressed onto the layer of SU8 85. The mask 82 is similar to that described above, with the exception that it comprises embossing formations 87 for forming a pattern that encodes an identifier onto the tags 83. The layer of SU8 85 is imprinted, for example at 8O0C and a pressure of 50kg/cm2. Once the mask 82 has imprinted the pattern into the layer of SU8 85, the layer of SU8 85 is exposed to a suitable wavelength (e.g. 365 nm) through the mask 82 in order to cross link the exposed SU8. Following post-exposure baking on a hotplate for some minutes, the mask 82 and substrate 80 are separated. Thereafter, the layer of SU8 85 is developed as normal to remove any unexposed resist 88. This provides a substrate 80 supporting one or more tags 83 that can subsequently be released by etching the sacrificial material 86 or by being diced and then released as desired.

Figure 10 illustrates a method of making tags 93 for use with particles using a UV initiated imprinting technique. A glass or silicon wafer 90 is processed as per conventional lithography, i.e. cleaned etc. The surface is then coated with a release agent. The release agent provides layer of a sacrificial layer 96 that comprises one or more layers of metal (e.g. aluminium or chromium), together with layers of photoresist (e.g. S1818), or a thin coating of Teflon™ or any combination thereof.

A layer of SU8 95 (for example of the order of 1 to 25 μm thickness) is disposed over a thin layer of sacrificial material 96 onto the substrate 90, which has been pre-baked at 2000C for 30 minutes for dehydration purposes. The substrate is spun at 500 rpm for 10 seconds, then accelerated to 2500 rpm within 10 seconds and keep at 2500 rpm for 10 seconds. Then it is soft-baked on a hotplate at 9O0C for 20 minutes until the SU8 film is no longer sticky following a slow cooling.

UV initiated imprinting can also be used, by providing a low viscosity resist 99 having embossing formations 97 formed thereon for forming a pattern that encodes an identifier onto the tags 93. In this case, the resist 99 is provided between the mask 92 and the substrate 90 supporting the layer of SU8 95. The resist 99 may be placed on the mask 92, and the mask 92 brought into contact so that the resist 99 spreads under the influence of surface tension.

Once the mask 92 is in place, UV light is used to cross-link the resist 99 so as to define a pattern that encodes the desired identifier(s) when the mask is released from contact with the substrate 90.

Once the tags 93 have been manufactured and released, they can be processed to make them chemically active. This can be done whilst they are still in situ on the substrate 90, e.g. using robot "spotting", or by directed growth of compounds, e.g. using a robot or light mediated synthesis. Alternatively, the tags 93 may be released from the substrate 90 and processed as described herein.

Figure 11 illustrates a method of making tags for particles. Figure 11 shows a similar method to that shown in figure 10, with the difference that a low viscosity resist 91 is used.

Figure 12 schematically shows a reader 100 for reading identifiers 116 from particles

110. The reader 100 comprises a source 160 that generates a laser beam of infrared

(IR) or near-infrared (NIR) radiation (for example, using a frequency doubled Nd3+ laser) that is exposed to a region 164 through which the particles 110 are transported. The source 160 preferably operates using optical radiation at 630 nm. However, use of a shorter wavelength such as, for example, 530 nm, would allow the reader 100 to work with particles 110 that have a higher encoding capacity. Although ultra-violet light could be used in various applications, its effect on various biological molecules, by way of inducing chemical reactions and degradation, may make it unsuitable for use with certain receptor materials. Use of IR or NIR radiation, e.g. from about 650 nm to about 1000 nm, is particularly useful for biological applications since it corresponds to a wavelength window in which radiation can penetrate cells without causing cell damage.

Although the source 160 in this embodiment uses IR or NIR radiation, it is envisaged that other sources may be used. For example, a diffraction-pattern based tag that encodes an identifier may be read by an electron beam. Such a tag can comprise material variations that are provided on a scale that is even smaller than for an optically-based tag. In this case, the material variations can be provided on a scale that is of the same order as the de Broglie wavelength of the electrons, in order to obtain a characteristic electron diffraction pattern.

Laser light passing through the particles 110 is scattered, and forms a diffraction pattern at a detector 120. The detector 120 comprises a CCD array 122, which is ideally suited to detecting the two-dimensional spatial distribution pattern that encodes the identifiers. The detector 120 can detect a diffraction pattern spatial distribution of radiation emitted from the particles 110, and can therefore be sited remotely from the region 164. The detector 120 also comprises a high numerical aperture lens 124 that enhances the amount of light that can be collected, and which therefore enables the diffraction pattern radiation pattern to be detected using a relatively short signal integration time. This speeds detection and is thus useful when large numbers of particles 110 need to be rapidly identified.

The output from the detector 120 is analysed by analyser 150 to determine whether the spatial pattern matches that of any known predetermined identifier. The analyser 150 comprises a computer system that is programmed to match a diffraction pattern map from the detector 120 with corresponding records of known identifiers 116 stored in a database. The records in the database associate the known identifiers 116 with any chemical receptors, treatments etc. that have been imparted to the particles 110. In this reader 100, the particles 110 transported through the region 164 in a fluid, such as air or water, for example. A micro-fluidic transport channel 130 is used to guide the particles 110 suspended in the fluid from a storage tank (not shown), via an electrokinetic orientation device 170, through the region 164 and into a micro-sorter 140 (e.g. of the type available from Partec Gmbh of Mϋnster, Germany).

The electrokinetic orientation device 170 orientates the particles 110 toward a predetermined direction, so that they are all aligned in substantially the same direction with their largest dimension parallel to the bore of the micro-fluidic transport channel 130. The bore is configured to prevent the particles 110 from rotating by a large amount so that they remain oriented in substantially the same direction as they are transported through the region 164. This reduces the amount of processing that is required to determine the identifiers, since it helps ensure that the measured diffraction pattern patterns from different particles 110 are directly comparable with those used for matching at the analyser 150. The reader 100 may additionally, or alternatively, include a means for identifying the spatial orientation of the particles 110.

The identifiers 116 can be encoded using a variety of encoding strategies, such as those illustrated schematically in Figure 5, which are discussed further below. In various embodiments, the analyser 150 may be programmed to identify one or more type of particle having an identifier encoded according to one or more of these encoding strategies.

In a first encoding strategy, a single grating of varying optical density, reflectivity, refractive index or height is provided. Identifier information is encoded in the grating pitch p and can be read by detecting the diffraction angle of a single beam of Nth order (N=O, 1,2... etc). The encoding capacity Σ (i.e. the number of different distinguishable identifiers) is of the order of lnt {d-i/Λ}, where lnt {} is the integer operator, d-i is the grating length and λ is the wavelength of the exposure radiation,. For example, if O1 = 50 μm, and λ = 1 μm, then 2= 50.

In a second encoding strategy, superimposed parallel gratings with pitches p? and p2 are provided. Identifier information is encoded in the values of two pitches and can be read by detecting two diffracted beams at various angles in one plane. The encoding capacity Σ is of the order of lnt {di/λ}N, where N is number of superimposed gratings. For example, if Cl1 = 50 μm, λ = 1 μm, and N = 5, then Σ= 3 x 108.

In a third encoding strategy, superimposed gratings are provided in two different directions with pitches pi and p. Identifier information is encoded in the values of pitches, and is read by detecting two diffracted beams at various angles in different planes. The encoding capacity ∑\s of the order of lnt (d-j/λj.lnt {d2/λ}, where O1 and d2 are the respective grating lengths.

In a fourth encoding strategy, a complex pattern (for instance one that is computer generated to give a certain distinct diffraction pattern) is provided. Identifier information is read by detecting a complex diffraction pattern. The encoding capacity ∑\s of the order of lnt {d/λ}\, where d is the smallest dimension of the grating pattern. For example, if of = 50 μm, and λ = 1 μm, then Σ= 3 x 1065. Essentially this is equivalent to a number of distinctively different patterns that may be created on a matrix of d x d pixels.

Figure 13 schematically shows the reader 100 as a stream of particles 110 are passed through the region 164. A two-dimensional Fourier transform analysis of the diffraction pattern measured by the CCD array 122 is used by the analyser 150 to map the spatial diffraction pattern into k-space in order to determine the identifier. Moreover, the analysis of the diffraction pattern is not sensitive to the motion of particles 110 parallel to the plane containing the CCD detector 122 and is also insensitive to the actual position of the particles 110 relative to the CCD array 122.

Figure 14 schematically shows alternative detector positions in a reader 200 for reading identifiers from particles comprising a diffraction encoding structure 203. One or more reading optical beam 201 are shown, which may comprise beams at different wavelengths. The optical beam 201 is conditioned by an optical element 202 that comprises a lens, a spatial filter and a mask. The conditioned optical beam 201 is then incident upon the diffraction encoding structure 203.

Various diffracted beams are generated, including backwardly diffracted beams a', b", c1 and d1 and forwardly diffracted beams a, b, c and d. The various diffracted beams may provide a continuous distribution of light. A first optical element 204 is shown which conditions the forwardly diffracted beams a, b, c and d and couples them to a first detector 205. Also shown is a second optical element 206 which conditions the backwardly diffracted beams a1, b1, c1 and d1 and couples them to a second detector 207. One or more of these optical arrangements may be used in various readers provided as embodiments of the present invention.

Various readers described herein may be used as the basis of a device that identifies particles and acts upon the identity of the particles to sort them into various outlet channels. Such a device may be used for combinatorial chemical synthesis applications. For example, where combinatorial analysis of oligonucleotide sequences is performed, particles can be identified and sorted into one of four outlets corresponding to the bases of DNA (ACGT).

Those skilled in the art will be aware that many different embodiments of particles manufactured according to many different techniques are possible with many varied applications. Various ways of providing material variations may be used, including, for example, by the provision of reflective or non-reflective elements. Additionally, particles that are non-spherical may be provided, and any anisotropy in the shape exploited to align gratings in various reader devices.

Identifiable particles having feature sizes greater than about 100-200 nm for encoding identifiers may be manufactured in various materials, such as, for example, polymers, using direct write laser ablation methods. Particles may also be manufactured using holographic phase writing techniques. For example, two or more crossed laser beams may be used to produce an interference pattern with characteristic period less than the wavelength of the laser light. These methods may be used to write patterns of diffraction gratings into polymers, for example. They may also be used as a direct write method for providing patterns encoding identifiers in photo-active polymers, for example.

In certain embodiments of the particles, no receptor material may be provided, or a coating applied that is not for binding to a predetermined chemical entity. Such particles may be used with a detection system that checks for the presence or absence of particles. One possible use of such particles is in the field of checking for counterfeiting of manufactured articles. For example, genuine articles (such as, for example, vehicle parts) may be coated with a "dust" made up of one or more particles. The absence of such "dust" from manufactured articles may thus indicate that the articles are counterfeit, whilst the presence of such a "dust" could indicate that they are genuine.

Those skilled in the art will also realise various embodiments of readers may be made which use relative movement between particles and a detector, including those in which the detector is movable and the particles remain stationary. Additionally, they will be aware that techniques other than electrokinetic/electrostatic orientation may be used to orientate particles. In addition, they will be aware that reader devices may be operable to identify chemical entities as well as identifiers encoded by a diffraction pattern. Such chemical entities may be identified, for example, using one or more of fluorescence, FRET, Raman, magnetic, electrokinetic, etc. techniques.

Those skilled in the art would also understand that in various embodiments, the diffraction pattern identifier may be identified by analysing the diffraction pattern produced by the particle in momentum space, or k-space. Such a diffraction pattern identifier may be derived by calculating a Fourier transform of a spatial distribution of at least part of a diffraction pattern, which can then be compared to previously determined diffraction pattern identifiers in order to uniquely identify the particle.

Those skilled in the art will also realise that particles of the type described herein may be used in combination with various other techniques (e.g. with nano-particles, magnetic techniques, etc.) The particles may be wrapped around magnetic bar codes or incorporated into other elements such as, for example, chemicals for providing drug release at targeted sites. Such drugs could be released in response to exposure to radiation, for example. The particles may be made radioactive and used to target tumours, or contain DNA or drugs that are released at specific sites in a body or cell. They may, for example, contain other particles, such as QDS, for release at specific sites. Further, the particles may be used for non-viral gene transfection.

Moreover, it is understood that multi-colour/colour-selective CCD arrays may be used in various reader embodiments. These can be used simultaneously to distinguish and detect several different wavelength components, thereby increasing reader capacity and improving error-correction capabilities. Initial functionalisation of particles

In order to conduct subsequent solid supported chemical reactions, the particles may bear reactive functional groups or may have these introduced as described above, for example by chloromethylation.

Preferably, the particles are further functionalised by reaction with a functionalising agent.

As used herein, the term "functionalising agent" refers to a reagent or mixture of reagents which are capable of reacting with the reactive functional groups to render the particles more suitable for use as a solid support in subsequent reaction sequences.

Examples of functionalising agents are those comprising thiols, alcohols, and amines.

Preferably, the functionalising agent comprises one or more amines. Preferably, the amine is selected from ammonia, alkylamines and arylamines (anilines). Preferably, the amine comprises one, two, three or more than three amine groups.

Preferably, the amine is a polyoxyalkyleneamine or polyether amine. A more preferred amine is a Jeffamine. A still more preferred amine is Jeffamine8oo-

Preferably, the reactive functional group is an epoxide, and the functionalising agent is an amine. In this case, the functionalised particles will bear amine groups that are capable of undergoing further reaction.

Attachment of linker group

Subsequent to liberation of the particles and optional functionalisation, a linker group may optionally be attached.

As used herein, the term "linker group" refers to a chemical moiety capable of forming a chemical bond with both the particles (optionally functionalised) and partaking in further chemical reactions. Preferably, the linker group is cleavable at the end of the reaction sequence to furnish the desired product.

Preferably, the linker group is introduced by reaction of the optionally functionalised particles with a compound containing at least two functional groups. Suitable linker groups are those derived from amino acids (for example α amino acids such as the natural amino acids, β amino acids, γ amino acids, δ amino acids, 1 ,5 amino acids and 1 ,6 amino acids), dicarboxylic acids (such as oxalic, maleic, fumaric and succinic acids), and derivatives and protected versions thereof. The person skilled in the art will be able to determine which conditions to use to attach a particular linker group to the particles of the invention

In a preferred embodiment, the linker is an amino acid. A preferred linker group is 6- aminohexanoic acid.

In the embodiment wherein the particles bear an amine reactive functional group, a linker group bearing a carboxylic acid moiety is conveniently introduced using standard amide coupling conditions. Suitable techniques are described in "The Chemical Synthesis of Peptides" by J Jones, Oxford University Press, 1994.

It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional techniques, for example as described in "Protective Groups in Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by P.J.Kocienski, in "Protecting Groups", Georg Thieme Verlag (1994).

Preferably, the linker group is attached by reaction of the particles with N-protected 6- aminohexanoic acid, or a reactive derivative thereof, followed by subsequent deprotection. Preferably, the protecting group is fluorene-9-yl methoxycarbonyl (FMOC). Suitable reactive derivatives are those formed by the reaction of the N- protected 6-aminohexanoic acid with a diisocyanate (particularly hexamethyldiisocyanate (DIC)), or hydroxybenzotriazole (HOBt).

In an alternative preferred embodiment, the linker group is derived from a hydroxycarboxylic acid (a molecule possessing both a hydroxyl and carboxylic acid group). A highly preferred hydroxycarboxylic acid group is 4-hydroxymethyl-phenoxy acetic acid (HMPA). HMPA may be coupled to particles bearing reactive amine groups using standard amide bond formation conditions as described above. Solid supported synthesis

The particles of the invention may be used as supports in an identical manner to conventional solid supports. The particles of the invention are amenable to multi-step synthetic sequences, and provide products in good purity and yield.

Peptide synthesis

A typical solid phase peptide synthesis scheme involves attaching a first amino acid or peptide group to the particles of the invention via the carboxyl moiety of the peptide or amino acid. This leaves the amine group of the resin bound material available to couple with additional amino acids or peptide material. Thus, the carboxyl moiety of the additional amino acid or peptide desirably reacts with the free amine group of the particle bound material. To avoid side reactions involving the amine group of the additional amino acid or peptide, such amine group is masked with a protecting group during the coupling reaction. Two well-known amine protecting groups are the BOC group and the FMOC group. Many others also have been described in the literature. After coupling, the protecting group on the N-terminus of the resin bound peptide may be removed, allowing additional amino acids or peptide material to be added to the growing chain in a similar fashion. In the meantime, reactive side chain groups of the amino acid and peptide reactants, including the particle bound peptide material as well as the additional material to be added to the growing chain, typically remain masked with side chain protecting groups throughout the synthesis.

After the desired peptide has been assembled in this fashion, it may be liberated from the particles. Suitable conditions for this liberation step depend on the nature of the particle and in particular the linker group as defined above (where present). The skilled person will be able to determine conditions for the liberation of the peptide.

A preferred class of peptides of the present invention are those that incorporate from about 2 to about 100, preferably from about 4 to about 50, residues of one or more amino acids. Residues of one or more other monomeric, oligomeric, and/or polymeric constituents optionally may also be incorporated into a peptide. Non-peptide bonds also may be present. For instance, the peptides of the invention may be synthesized to incorporate one or more non-peptide bonds. These non-peptide bonds may be between amino acid residues, between an amino acid and a non-amino acid residue, or between two non-amino acid residues. These alternative non-peptide bonds may be formed by utilizing reactions well known to those in the art, and may include, but are not limited to imino, ester, hydrazide, semicarbazide, and azo bonds.

The principles of the present invention may be advantageously used to synthesise the following peptide material, fragment intermediates thereof, and/or analogs from a particle after solid phase synthesis: enkephalins, oxytocin, vasopressin, felypressin, pitressin, lypressin, desmopressin, terlipressin, atosiban, adrenocorticotropic hormone, insulin, secretin, calcitonins, luteinizing hormone-releasing hormone (LH- RH) and analogues, leuprolide, deslorelin, triptorelin, goserelin, buserelin, nafarelin, cetrorelix, ganirelix, parathyroid hormone, human coriticotropin-releasing factor, ovine coriticotropin-releasing factor, growth hormone releasing factor, somatostatin, lanreotide, octreotide, thyrotropin releasing hormone (TRH), thymosin-1, thymopentin (TP-5), cyclosporine and integrilin. A particularly preferred peptide is Leu-enkephalin.

The nature and use of protecting groups for peptide synthesis is well known in the art. Generally, a suitable protecting group is any sort of group that that can help prevent the atom or moiety to which it is attached, e. g. , oxygen or nitrogen, from participating in undesired reactions during processing and synthesis. Protecting groups include side chain protecting groups and amino-or N-terminal protecting groups. Protecting groups can also prevent reaction or bonding of carboxylic acids, thiols and the like.

A side chain protecting group refers to a chemical moiety coupled to the side chain (i.e., R group in the general amino acid formula H2N-C(R)(H)-COOH) of an amino acid that helps to prevent a portion of the side chain from reacting with chemicals used in steps of peptide synthesis, processing, etc. The choice of a side chain-protecting group can depend on various factors, for example, type of synthesis performed, processing to which the peptide will be subjected, and the desired intermediate product or final product. The nature of the side chain protecting group also depends on the nature of the amino acid itself. Generally, a side chain protecting group is chosen that is not removed during deprotection of the amino groups during the solid phase synthesis ("orthogonal protection"). Therefore the amino protecting group and the side chain protecting group are preferably not the same.

In some cases, and depending on the type of reagents used in solid phase synthesis and other peptide processing, an amino acid may not require the presence of a side- chain protecting group. Such amino acids typically do not include a reactive oxygen, nitrogen, or other reactive moiety in the side chain.

Examples of side chain protecting groups include acetyl (Ac), benzoyl (Bz), tert-butyl, triphenylmethyl (trityl), tetrahydropyranyl, benzyl ether (BzI) and 2,6-dichlorobenzyl (DCB), t-butoxycarbonyl(BOC), nitro, p-toluenesulfonyl (Tos), adamantyloxycarbonyl, xanthyl (Xan), benzyl, 2,6-dichlorobenzyl, methyl, ethyl and t-butyl ester, benzyloxycarbonyl (Z), 2-chlorobenzyloxycarbonyl (2-CI-Z), t- amyloxycarbonyl (Aoc), and aromatic or aliphatic urethane type protecting groups, and photolabile groups such as nitroveritryl oxycarbonyl (NVOC); and fluoride labile groups such as trimethylsilyl oxycarbonyl (TEOC).

Preferred side chain protecting groups include t-Bu group for Tyr (Y), Thr(T) ; Ser (S) and Asp (D) amino acid residues; the Trt group for His (H), GIn (Q) and Asn (N) amino acid residues; and the Boc group for Lys (K) and Trp (W) amino acid residues.

An amino-terminal protecting group includes a chemical moiety coupled to the alpha amino group of an amino acid. Typically, the amino-terminal protecting group is removed in a deprotection reaction prior to the addition of the next amino acid to be added to the growing peptide chain, but can be maintained when the peptide is cleaved from the support. The choice of an amino terminal protecting group can depend on various factors, for example, type of synthesis performed and the desired intermediate product or final product.

Examples of amino-terminal protecting groups include acyl-type protecting groups, such as formyl, acrylyl (Acr), benzoyl (Bz) and acetyl(Ac); aromatic urethane-type protecting groups, such as benzyloxycarbonyl (Z) and substituted Z, such as p- chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p- methoxybenzyloxycarbonyl; aliphatic urethane protecting groups, such as t- butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; cycloalkyl urethan-type protecting groups, such as 9-fluorenyl-methyloxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; and thiourethane-type protecting groups, such as phenylthiocarbonyl. Preferred protecting groups include 9-fluorenyl- methyloxycarbonyl (Fmoc), 2-(4-biphenylyl)-propyl (2) oxycarbonyl (Bpoc), 2- phenylpropyl (2)-oxycarbonyl (Poc) and t-butyloxycarbonyl (Boc). Optionally, the peptide may incorporate one or more labels such as a chromophore, fluorophore, biotin or a magnetic or electron-dense entity. Preferred fluorophores include fluorescein, eosin, Alexa Fluor, Oregon Green, Rhodamine Green, tetramethylrhodamine, Rhodamine Red, Texas Red, coumarin and NBD.

Oligonucleotide synthesis

Solid phase chemical synthesis of DNA fragments is conveniently performed using protected nucleoside phosphoramidites. In this approach, the 3'-hydroxyl group of an initial 5'-protected nucleoside is first covalently attached to the particle of the invention, optionally via a linker (preferably a succinic acid derivative). Synthesis of the oligonucleotide then proceeds by deprotection of the 5'-hydroxyl group of the attached nucleoside, followed by coupling of an incoming nucleoside-31- phosphoramidite to the deprotected hydroxyl group. Alternatively, sythesis may be conducted in the 5'-3' direction. Suitable methodologies are for example disclosed in "Oligonucleotide synthesis: A practical approach" M.J. Gait, IRL Press, 1992 which is incorporated herein in its entirety.

Residues of one or more other monomeric, oligomeric, and/or polymeric constituents optionally may also be incorporated into an oligonucleotide. Non-nucleotide bonds also may be present. For instance, the oligonucleotides of the invention may be synthesized to incorporate one or more non-nucleotide bonds. These non-nucleotide bonds may be between nucleotide residues, between a nucleotide and a non- nucleotide residue, or between two non-nucleotide residues. Non-nucleotide linkages include phosphorothioates, phosphonates, amides and others. Non-DNA base phosphoramidates may also be incorporated, and may function as linkers, labels, or functional groups (such as biotin).

Coupling is achieved with any one of a number of coupling reagents known to those skilled in the art. Preferred coupling reagents are uronium salts, particularly TBTU, and diisocyanates, particularly DIC, and ETT (5-ethylthio-1f/-tetrazole).

The resulting phosphite triester is finally oxidized to a phosphorotriester to complete the internucleotide bond. The steps of deprotection, coupling and oxidation are repeated until an oligonucleotide of the desired length and sequence is obtained. The completed oligonucleotide may optionally be cleaved from the particle. Again, the skilled person will be able to determine suitable conditions for achieving this result. The Chemical group conventionally used for the protection of nucleoside 5'-hydroxyls is preferably dimethoxytrityl ("DMT"), which is removable with acid.

Optionally, the oligonucleotide may incorporate one or more labels such as a chromophore, fluorophore, biotin or a magnetic or electron-dense entity. Preferred fluorophores include fluorescein, eosin, Alexa Fluor, Oregon Green, Rhodamine Green, tetramethylrhodamine, Rhodamine Red, Texas Red, coumarin and NBD fluorophores.

Small molecule synthesis

In addition to peptides and oligonucleotides, the particles of the invention may be used as supports in the solid phase synthesis of many other classes of compound which have been demonstrated to be amenable to solid phase synthesis using conventional supports. Heterocycles are a preferred class of compounds.

Library synthesis

The particles of the invention have special utility in the preparation of libraries of chemical compounds.

In a typical procedure, a suspension of particles is separated into three portions, each of which is then exposed to a specific reagent, A1, A2, or A3, which becomes chemically bound to the particles. The three suspensions of particles are then recombined within a single vessel and thoroughly mixed before being divided once again into three separate parts, each part containing a proportion of all three compound types. The large number of particles involved in the process ensures that statistically the numbers of each type of compound in each vessel are approximately equal. The three suspensions are then further exposed to the same or different reagents B1 , B2, or B3, and this results, for example in compounds A1B1 , A2B1 , and A3B1 in the first vessel; A1 B2, A2B2, and A3B2 in the second vessel; and A1 B3, A2B3 and A3B3 in the third. The suspensions are then remixed in a single vessel and the process repeated. In this way, a substantial number of different compounds may be produced in a relatively small number of reaction steps. At the end of the processing the identity of the compound(s) showing the desired properties may subsequently be deduced. The use of the particles of the invention, when tagged or encoded as described above, in such a sequence confers the advantage that the process steps to which each individual particle has been subjected, and consequently the nature of the chemical species appended thereto, can be determined by reading the tag or code.

Such libraries of compounds are advantageously employed in binding, inhibition, or other chemical or biochemical activities.

In a preferred embodiment, a compound library affixed to a plurality of particles of the invention is brought into association with a target species. Particles bearing chemical compounds which bind to the target species are identified, and the nature of the chemical compound is established. Highly preferably, the nature of the chemical compound is identified by means of the tag or encoding element present on the particle.

The target species may be a protein, an enzyme, receptor, micro organism, or a DNA or RNA fragment. The target species may bear a means for detection such as a radiolabel, fluorescence label, or visual label.

Examples

The present invention will now be described only by way of example. However, it is to be understood that the examples also present preferred embodiments of the present invention, as well as preferred routes for making same and useful intermediates in the preparation of same.

Example 1. Fabrication of particles

Microfabrication techniques were developed to construct bars based on SU-8 photolithographic process and lift off of the patterned SU-8 bars from the substrate (Si).

Processing of Al as a sacrificial layer The silicon substrates were prepared by cleaning them in fuming nitric acid for 20 minutes. 50 nm Al was coated by evaporation of pure aluminium by Al E-Gun Evaporator. An Aluminium sacrificial layer is preferred to S1813 which was also tested as particles made with the Al sacrificial layer performed better in later chemistry.

SU-8 photolithography process

The Al coated silicon wafers were baked at 200° C for 1 hour. To increase the adhesion of SU-8 with Al, Microposit primer was spin coated. SU-8 was then spin coated onto the silicon substrate coated with Al. Following the spin coating, a soft bake step was performed. The polymer was cross-linked by exposure to UV light for 18 seconds (13.6 mw cm"2). A post exposure bake of 65° C for 1 minute and 95° C for 5 minutes was performed to further cross-link the SU-8 to be resistant to solvent during processing and also to be resistant to the solvents used for later chemistry. Finally, the wafers were developed in polypropylene-glycol-methyl-ether-acetate (PGMEA) for 2 minutes.

Releasing of SU-8 bars

Rom and Haas MF 319 developer was used to dissolve the Al in order to release the bars from the substrates. After immersing the wafers into the solution and leaving them for few seconds, the bars were filtered and washed off with water followed by acetone.

Chemistry on SU-8 particles

Example 2. Initial functionalisation of particles.

SU8 (100 mg) was treated with the Jeffamine80o (500 mg) and acetonitrile (500 μl) and heated to 65 0C in an oven overnight. The support was washed with acetonitrile (7 x 800 μl) followed by THF (7 x 800 μl) and dried under vacuum at room temperature for 4 h. Jeffamine8oo is preferred to simply alkylamines.

Example 3. Attachment of N-Fmoc-6-aminohexanoic acid.

N-Fmoc-6-aminohexanoic acid (5.0 mg, 14 μmol) was dissolved in DMF (100 μl) and DIC (2 μl, 13 μmol) was added. The mixture was shaken for 8 min at room temperature. HOBt (2 mg, 15 μmol) was added and the mixture was shaken for 5 min at r.t. The mixture was added to Jeffamine functionalised SU8 particles (1.50 μmol based on free -NH2 groups) suspended in DMF (300 μl) and the mixture was heated to 60 0C for 1h. The support was washed with DMF (7 x 800 μl) followed by THF (7 x 800 μl) and dried under vacuum at room temperature for 4 h.

Example 4. Attachment of HMPA linker.

HMPA (17 mg, 90 μmol) was dissolved in DMF (100 μl) and DIG (14 μl, 90 μmol) was added. The mixture was shaken for 8 min at room temperature. HOBt (12 mg, 90 μmol) was added and the mixture was shaken for 5 min at room temperature. The mixture was added to amine functionalised SU8 (30 μmol based on free -NH2 groups) suspended in DMF (300 μl) and the mixture was shaken for 1h at room temperature. The support was washed with DMF (7 x 800 μl) and the procedure was repeated. The support was washed with DMF (7 x 800 μl) followed by THF (7 x 800 μl) and dried under vacuum at room temperature for 4 h.

Example 5. Attachment of first amino acid.

N-Fmoc-Leucine (36 mg, 100 μmol) was dissolved in DMF (50 μl) and DIC (16 μl, 100 μmol) was added. The mixture was shaken for for 8 min at room temperature. DMAP (1.5 mg, 10 μmol) was added and the mixture was added to HMPA SU8 (30 μmol based on -OH groups) suspended in DMF (300 μl) and the mixture was shaken for 1h at room temperature. The support was washed with DMF (7 x 800 μl) and the procedure was repeated twice. The support was washed with DMF (7 x 800 μl) followed by THF (7 x 800 μl) and dried under vacuum at room temperature for 4 h.

Example 5. Attachment of subsequent amino acids.

N-Fmoc-amino acid (25 μmol) was dissolved in DMF (50 μl) and TBTU (8 mg, 25 μmol), HOBt (0.5 mg, 4 μmol) and DIPEA (4 μl, 25 μmol) were added. The mixture was shaken for 2 min and the mixture was added to the deprotected SU8 (8 μmol based on -NH2 groups) suspended in DMF (300 μl) and the mixture was shaken for 1h at r.t. The support was washed with DMF (3 x 800 μl) followed by THF (2 x 800 μl) and Et2θ (3 x 800 μl). The completeness of the reaction was monitored by ninhydrin test. After a negative ninhydrin test the N-terminus Fmoc group was removed.

Example 6. Cleavage of peptides.

The support was treated with TFA/phenol (98/2 % v/w, 25 ml/g resin) for 90 min at room temperature. The support was filtered and washed with TFA (3 x 1 ml). The combined filtrates were evaporated under vacuum and the remainder oil was titrated with Et2O. The solid that precipitated was washed with Et2O and dried under vacuum at room temperature for 4 h.

Example 6. Attachment of succinylated nucleoside

2'-0-succinyl-5'-0-DMT-cytosine was dissolved in DMF and TBTU (8 mg, 25 μrnol), HOBt (0.5 mg, 4 μmol) and DIPEA (4 μl, 25 μmol) were added. The mixture was shaken for 2 min and the mixture was added to the SU-8 particles functionalised with Jeffamine and aminohexanoic acid linkers suspended in DMF (300 μl) and the mixture was shaken for 1h at room temperature. The support was washed with DMF (3 x 800 μl) followed by THF (2 x 800 μl) and Et2O (3 x 800 μl). The completeness of the reaction was monitored by ninhydrin test. The succinylated nucleoside can also be coupled using DIG and HOBt conditions.

Example 7. Automated DNA synthesis

SU-8 particles derivatised with succinylated base were subjected to multiple cycles of standard automated DNA synthesis using 5'-O-DMT-3'-O-phosphoramidite nucleosides. Coupling was performed with ETT as activator. After synthesis the DNA was deprotected and cleaved from the solid support by standard methods. The resulting solution was freeze dried and purified by HPLC.

Example 8. Coupling amino-modified oligonucleotides to SU-8 microparticles.

Amine modified SU-8 particles were treated with succinic anhydride to generate a carboxy modified surface, δ'-amino modified oligonucleotides (with or without fluorescent labels) were coupled to the surface with EDC.

Example 9. Synthesis of Leu-Enkephalin Following the procedures above amine functionalised SU-8 particles were prepared with Jeffamine and aminohexanoic acid linker and an HMPA linker. The initial amino acid Leucine was coupled on to this support followed by phenylalanine, 2 x glycine, and tyrosine. The peptide was cleaved from the resin as described. The dry solid was analysed by HPLC-MS and found to be identical to a synthetic standard.

Example 10. Synthesis of 5'-Ti5C

SU-8 particles derivatised with Jeffamine and aminohexanoic acid linker, and with succinyl-DMT-cytosine, were subjected to 15 cycles of DNA synthesis in which 15 x thymidine phosphoramidites were coupled. Following deprotection, cleavage and HPLC purification the oligonucleotide product was analysed by capillary electrophoresis which showed the presence of an oligonucleotide that co-migrated with a synthetic standard.

Example 11. DNA hybridisation on oligonucleotide modified SU-8 particles

DNA-modified particles were suspended in an aqueous buffer containing fluorescently labelled oligonucleotide complementary to that on the particles. The increase in fluorescence on hybridisation was monitored using a Fluorescence Activated Cell Sorter. Incubation of the particles with a non-complementary fluorescently labelled oligonucleotide gave significantly lower fluorescence measurements by FACS.

Example 12. Synthesis and enzymatic cleavage of HIV protease substrate

A peptide substrate of the HIV protease with a terminal fluorophore was synthesised on SU-8 particles. Upon treatment with the protease the particle fluorescence due to the fluorophore was lost due to peptide cleavage. The loss of fluorescence was monitored using a Fluorescence Activated Cell Sorter.

Example 13. Hybridisation of DNA to in situ synthesised oligonucleotides

DNA oligonucleotides were synthesised on SU-8 particles using a non-cleavable linker and deprotected in situ. The DNA-modified particles were then suspended in an aqueous buffer containing fluorescently labelled oligonucleotide complementary to that on the particles. The increase in fluorescence on hybridisation was monitored using a Fluorescence Activated Cell Sorter. Incubation of the particles with a non- complementary fluorescently labelled oligonucleotide gave significantly lower fluorescence measurements by FACS.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.

Claims

1. A process for the preparation of a particle for use as a solid support in chemistry comprising
i. coating a laminar support material comprising at least a sacrificial layer with a photopolymerisable material;
ii. exposing the coated face of the support to radiation through a photolithographic mask, said mask defining the particle size and shape;
iii. selectively removing the exposed or unexposed areas of photopolymerisable material;
iv. reducing the integrity of the sacrificial layer to liberate said particle;
characterised in that the sacrificial layer comprises a metal or metals.
2. The process according to claim 1 wherein the sacrificial layer consists essentially of a metal or metals.
3. The process according to claim 1 or 2 wherein one of the metals is aluminium.
4. The process according to claim 3 wherein the sacrificial layer consists essentially of aluminium.
5. The process according to any preceding claim wherein the sacrificial layer is less than 100 nm thick.
6. The process according to any preceding claim wherein the laminar support material further comprises a substrate layer.
7. The process according to claim 6 wherein the substrate layer comprises silicon.
8. The process according to any preceding claim wherein the photopolymerisable material comprises at least a reactive functional group when polymerised.
9. The process according to claim 8 wherein the photopolymerisable material comprises at least an epoxide group when polymerised.
10. The process according to any preceding claim comprising a further step of reacting the particle with a functionalising agent.
11. The process according to claim 10 wherein the functionalising agent comprises a polyoxyalkyleneamine or polyether amine.
12. The process according to any preceding claim comprising the further step of reacting the particle with a linker group.
13. The process according to claim 12 wherein the linker group is an amino acid.
14. The process according to claim 13 wherein the linker group is 6- aminohexanoic acid.
15. The process according to claim 14 wherein the linker group is a hydroxycarboxylic acid.
16. The process according to claim 15 wherein the hydroxycarboxylic acid is 4- hydroxymethyl-phenoxy acetic acid
17. The process according to any preceding claim comprising an additional step of incorporating an identifier.
18. The process according to claim 17 wherein the identifier comprises a pattern of refractive index variations that give rise to a predetermined diffraction pattern.
19. A process according to claim 18 wherein the predetermined diffraction pattern identifier is chosen from a plurality of diffraction pattern identifiers that can be encoded using the same identifier encoding scheme.
20. The process according to claim 18 or 19, wherein the variations include one or more reflective elements disposed within the particle material.
21. The process according to any one of claims 18 to 20, wherein the identifier encoding scheme comprises providing a plurality of superimposed gratings in a particle material.
22. A process for the preparation of a polymer particle for use as a solid support in chemistry comprising
i. coating a laminar support material comprising at least a sacrificial layer with a composition comprising monomer;
ii. selectively polymerising said monomer to define the particle size and shape;
iii. selectively removing unpolymerised material;
iv. reducing the integrity of the sacrificial layer to liberate said particle;
characterised in that the sacrificial layer comprises a metal or metals.
23. A particle obtainable by a process as claimed in any one of claims 1 to 22.
24. A particle obtained by a process as claimed in any one of claims 1 to 22.
25. An identifiable particle for use in combinatorial chemistry, the identifiable particle comprising a particle material having material variations provided therein that encode a predetermined diffraction pattern identifier, wherein the predetermined diffraction pattern identifier is chosen from a plurality of diffraction pattern identifiers that can be encoded using the same identifier encoding scheme.
26. The particle according to claim 25, wherein the material variations include a pattern of refractive index variations that give rise to a predetermined diffraction pattern diffraction pattern.
27. The particle according to claim 25 or 26, wherein the material variations include one or more reflective element disposed within the particle material.
28. The particle according to any one of claims 25 to 27, wherein the identifier encoding scheme comprises providing a plurality of superimposed gratings in a particle material.
29. The particle according to any one of claims 25 to 28, wherein the particle material is bio-compatible.
30. The particle according to any one of claims 25 to 29, wherein the particle material is bio-degradable.
31. The particle according to any one of claims 25 to 30, wherein the size of the particle is from about 100 nm to about 100 μm.
32. The particle according to any one of claims 25 to 31 , further comprising a receptor material for binding to a predetermined chemical entity.
33. The particle according to claim 32, wherein the predetermined chemical entity is a nucleic acid, an enzyme, a protein, a pharmaceutical product, a peptide, a ligand, a small molecule, an antibody, a receptor, a host-guest complex, a carbohydrate, a lectin, an analogue of a nucleic acid or a modified nucleic acid.
34. Use of a particle as claimed in any one of claims 23 to 33 as a support for solid supported chemistry.
35. Use of a particle as claimed in any one of claims 23 to 33 in the preparation of an oligopeptide.
36. Use of a particle as claimed in any one of claims 23 to 33 in the preparation of an oligonucleotide.
37. Use of a particle as claimed in any one of claims 23 to 33 in the preparation of a chemical library.
38. A process for the preparation of a library of compounds comprising steps of
i. providing a plurality of encoded particles as claimed in any one of claims 23 to 33; ii. dividing the particles into a plurality of portions;
iii. subjecting each portion to a specific chemical reaction;
iv. reading the code of a particle;
v. recombining the portions; and
vi. repeating steps i to v n times, wherein n is an integer.
39. A method for reading a predetermined diffraction pattern identifier from the particle as claimed in any one of claims 23 to 33, the method comprising:
exposing a particle to radiation;
detecting the diffraction pattern of radiation emitted from the particle; and
analysing the detected diffraction pattern of the radiation emitted from the particle in order to determine whether it matches a diffraction pattern identifier encoded using a predetermined encoding scheme.
40. The method according to claim 39, wherein analysing the detected diffraction pattern comprises calculating the Fourier transform of at least part of the diffraction pattern and determining whether the Fourier transform provides a k- space match with any predetermined identifiers.
41. The method according to claim 39 or 40, wherein the radiation is infrared (IR) or near-infrared (NIR) radiation.
42. The method according to any one of claims 39 to 41, further comprising transporting the identifiable particle suspended in a fluid through a region in which the identifiable particle can be exposed to radiation.
43. The method of any one of claims 39 to 42, further comprising orientating one or more identifiable particles towards a predetermined direction prior to exposure to radiation.
44. The method of any one of claims 39 to 43, wherein the scale of the material variations is less than half the wavelength of the radiation.
45. A process substantially as hereinbefore described with reference to the examples or figures.
46. A particle substantially as hereinbefore described with reference to the examples or figures.
47. A use substantially as hereinbefore described with reference to the examples or figures.
PCT/GB2005/004681 2004-12-07 2005-12-07 Particles for use as a solid support and process for their preparation WO2006061600A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
GB0426809A GB0426809D0 (en) 2004-12-07 2004-12-07 Nano-optical diffraction bar-code tagging for biological and chemical applications
GB0426809.0 2004-12-07
GB0507181.6 2005-04-08
GB0507181A GB2421076A (en) 2004-12-07 2005-04-08 Identifiable particles and uses thereof
GB0518478A GB2422686A (en) 2004-12-07 2005-09-09 Preparation of solid support particles for chemical reactions
GB0518478.3 2005-09-09

Publications (1)

Publication Number Publication Date
WO2006061600A1 true WO2006061600A1 (en) 2006-06-15

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006468A2 (en) * 1995-07-28 1997-02-20 Ely Michael Rabani Pattern formation, replication, fabrication and devices thereby
GB2306484A (en) * 1995-10-26 1997-05-07 Univ Hertfordshire Solid support particle marked with a machine-readable code for use in Combinatorial Chemistry Techniques
WO2002059603A2 (en) * 2001-01-26 2002-08-01 Aviva Biosciences Corporation Microdevices containing photorecognizable coding patterns and methods of using and producing the same thereof
US20030129654A1 (en) * 1999-04-15 2003-07-10 Ilya Ravkin Coded particles for multiplexed analysis of biological samples
US20030153092A1 (en) * 2000-04-19 2003-08-14 Skinner Nigel Guy Method of fabricating coded particles
GB2393785A (en) * 2002-10-03 2004-04-07 Toshiba Res Europ Ltd Method of making a free standing structure
WO2004066210A1 (en) * 2003-01-22 2004-08-05 Cyvera Corporation Hybrid random bead/chip based microarray

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006468A2 (en) * 1995-07-28 1997-02-20 Ely Michael Rabani Pattern formation, replication, fabrication and devices thereby
GB2306484A (en) * 1995-10-26 1997-05-07 Univ Hertfordshire Solid support particle marked with a machine-readable code for use in Combinatorial Chemistry Techniques
US20030129654A1 (en) * 1999-04-15 2003-07-10 Ilya Ravkin Coded particles for multiplexed analysis of biological samples
US20030153092A1 (en) * 2000-04-19 2003-08-14 Skinner Nigel Guy Method of fabricating coded particles
WO2002059603A2 (en) * 2001-01-26 2002-08-01 Aviva Biosciences Corporation Microdevices containing photorecognizable coding patterns and methods of using and producing the same thereof
GB2393785A (en) * 2002-10-03 2004-04-07 Toshiba Res Europ Ltd Method of making a free standing structure
WO2004066210A1 (en) * 2003-01-22 2004-08-05 Cyvera Corporation Hybrid random bead/chip based microarray

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