GB2421076A

GB2421076A - Identifiable particles and uses thereof

Info

Publication number: GB2421076A
Application number: GB0507181A
Authority: GB
Inventors: Nikolay Zheludev; Hywel Morgan; Gerasim Galitonov
Original assignee: University of Southampton
Current assignee: University of Southampton
Priority date: 2004-12-07
Filing date: 2005-04-08
Publication date: 2006-06-14
Also published as: GB0507181D0; GB0426809D0

Abstract

The invention relates to an identifiable particle 10 for various uses such as in combinatorial chemistry cell tagging, various other combinatorial chemistry applications, security and product labelling. The identifiable particle 10 comprises a particle material 12. The particle material 12 comprises material variations (such as one or more gratings) that encode a predetermined diffraction pattern identifier. In various embodiments, the predetermined diffraction pattern identifier can be read optically by analysing a pattern that is generated when the identifiable particle 10 is exposed to optical radiation. Identifiable particles 10 may comprise a receptor material 14 for binding to a predetermined chemical entity. The identifiable particles 10 are particularly useful for combinatorial chemistry applications (for example, biological assays and DNA sequencing) as they can be made small and readily tracked or identified using far-field imaging techniques.

Description

Identifiable particles and uses thereof

Field

The invention relates to identifiable particles and uses thereof. In particular, the invention relates to identifiable particles that can be identified by reading an identifier that is encoded within the particles. Such particles find uses in many different applications, such as, for example, in biological assays for DNA sequencing and analysis, cell tagging and identification, non-viral gene transfection, various other combinatorial chemistry applications, or in security applications or for product labelling.

Background

In various combinatorial chemistry and biological assay techniques it is known to use particles to synthesise chemical entities (such as, for example, polypeptides, carbohydrates, nucleotides and other 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 [1-5], 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 (fIR) tagging; optical image pattern recognition [6-8]; Raman tagging [9]; and quantum dot encoding [10, 11].

Conventional techniques are, however, not without certain drawbacks.

For example, magnetic tags are difficult to read, particularly where they are read in a manner similar to that used in a computer hard-disc drive using a read-head placed proximal to the magnetic tags. Such magnetic tags require close proximity to the reading head in order to read any tag identifiers. Magnetic tags, or tags that require the use of transponders, can also be bulky, expensive and difficult to attach or incorporate with identifiable particles.

Where fluorescent tags encode identifiers using fluorescent objects (such as quantum dots) on a tag, it is difficult both to manufacture the tag, so as to provide the correct number of fluorescent objects, and to read the tag so as to distinguish between various fluorescent colours and intensities.

Tagging using Rarnan barcoded beads, comprising spectroscopic bar codes that incorporate infrared and Raman-active groups that are chemically inert and identifiable using standard infrared or Raman spectrometers, enables identifiable particles to be provided having predetermined identifiers. However, such identifiers can only be read slowly because of the low intensity of the Raman signal.

Optical image pattern recognition has also been used to provide identifiers in a manner similar to conventional product barcoding. For example, linear barcodes or two dimensional patterns can be provided having features on a scale larger than an interrogation wavelength. These features encode identifiers that can be read by modulating the amplitude, or intensity, of interrogation radiation incident on the particles, so as to provide a one or two-dimensional optical image. However, the nature of these features imposes a fundamental minimum limit on the size of tags that use optical image pattern recognition and limits the number of unique identifiers that can be provided to differentiate between tags.

The present invention has been devised bearing the above-mentioned drawbacks associated with conventional tagging techniques in mind.

Summary of the invention

According to a first aspect of the invention, there is provided an identifiable particle for use in combinatorial chemistry. Such an identifiable particle may also be used in other applications such as cell tagging, security tagging or product labelling, for example. The identifiable particle comprises a particle material having material variations provided therein. The localised distribution of the material variations is used to encode a predetermined diffraction pattern identifier that may be used for identifying the identifiable particle. Such a diffraction pattern identifier is chosen from a number of possible diffraction pattern identifiers that could be encoded into the identifiable particle during its manufacture using a comnion 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 identifiable particle to various types of radiation such as, for example, optical radiation. Since the identifiable 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 identifiable particles may be sited remotely from the identifiable particles themselves. This has practical benefits for readers that are designed to read identifiers from the identifiable particle. Moreover, since the material variations that give rise to diffraction can be provided within the identifiable particle itself, no separate identifier-bearing tags need be attached to make particles identifiable.

The predetermined diffraction pattern identifier is 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.

According to a second aspect of the invention, there is provided a method of making identifiable 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 an identifiable particle. Many identifiable particles having the same or different identifiers may be made, as desired.

According to a third aspect of the invention, there is provided a kit for detecting the presence of a predetermined chemical entity comprising a plurality of identifiable 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 identifiable 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 fourth aspect of the invention, there is provided a reader for reading a predetermined diffraction pattern identifier from an identifiable particle. The reader comprises a source for exposing identifiable particles to radiation, a detector for detecting the diffraction pattern of radiation emitted from the identifiable particles, and an analyser for analysing the diffraction pattern of the radiation emitted from the identifiable 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 fifth aspect of the invention, there is provided a method for reading a predetermined diffraction pattern identifier from an identifiable particle. The method comprises exposing an identifiable particle to radiation, detecting the diffraction pattern of radiation emitted from the identifiable 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 sixth aspect of the invention, there is provided a method for identifying a chemical entity in a test substance. The method comprises introducing a plurality of identifiable particles comprising a plurality of predetermined receptor materials into the test substance, wherein respective different predetennined receptor materials bind to respective different predetermined chemical entities. The method also comprises reading the respective predetermined diffraction pattern identifiers of the identifiable 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 identifiable 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 identifiable particles.

Brief description of the drawings

Figure 1 shows identifiable 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 identifiable particles according to an embodiment of the present invention; Figures 7 illustrates a first part of a method of making tags for identifiable particles using a master template according to an embodiment of the present invention; Figure 8 illustrates a second part of a method of making tags for identifiable particles using a master template according to an embodiment of the present invention; Figure 9 illustrates a method of making tags for identifiable particles according to an embodiment of the present invention; Figure 10 illustrates a method of making tags for identifiable particles according to an embodiment of the present invention; Figure 11 illustrates a method of making tags for identifiable particles according to an embodiment of the present invention; Figure 12 schematically shows a reader for reading an identifier from identifiable 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 identifiable particles according to the present invention.

Detailed description

Figures la-d show identifiable particles lOa-d. The identifiable particles lOa-d are made of a particle material 12. The particle material 12 may include silicon dioxide, latex or various other polymeric materials such as, for example, the epoxy-based photoresist material SU8, or polymethylmethacrylate (PMMA), or combinations including metals or any other suitable material. Various bio-compatible materials can be used which may be taken up by cells without giving rise to toxic side-effects. In addition, for various applications, such as tracking of food products during or post- manufacture, the particle material 12 may be bio-degradable.

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 Ia illustrates a single one dimensional grating 1 6a provided in identifiable particle 1 Oa; Figure lb two overlapping one dimensional gratings 16b provided in identifiable particle lob; Figure lc a double overlapping two dimensional grating 1 6c provided in identifiable particle 1 Oc; and Figure id a complex pattern diffraction grating 16d provided in identifiable particle 1 Od.

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

The identifiable particles 1 Oa-d are rectangular in shape having a width of D, although in practise they can be made to have any practical shape that is desired. D is typically from about 100 nm up to less than about 1 pm, or up to about 10 tm or 100 pm, for identifiable particles that are to be used for biological or chemical applications. Where D is greater than about 100 nm, concerns regarding the toxicity of nano-particles in biological systems, which are not currently well understood, can be obviated.

In Figure 1 f an identifiable 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. The applicant has 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 identifiable particle 12 can be uniquely identified, the reaction can be observed in situ whilst also being distinguished from any other reactions between different identifiable 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 identifiable 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, i03, i04, i05, 106, i07, etc. of uniquely identifiable particles may be provided). This enables, for example, sequencing of nucleic acids, such as DNA!RNAlprotein nucleic acids, to be performed rapidly and with minimal cost and effort.

In a variation upon the identifiable 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 identifiable 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 identifiable particles can be attached. In further embodiments, it envisaged to place identifiable particle material into living host cells, whereupon the cells themselves act as identifiable particles that can be individually identified.

The identifiable 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 an identifiable particle 20. The identifiable 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 mn to about 10 tm. 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 electronbeam lithography to provide a pattern on a PMMA film, provided on a metalcoated 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.im to a grating 49 comprising five superimposed single period gratings having an overall length of about 100 pm.

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 identifiable particles or tags for attaching thereto.

The first grating 52 has a length d1 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 d1 and is formed by two linearly superimposed gratings having respective pitches of p and P2.

The third grating 56 is based upon a two dimensional pattern of size d1 x d2, and is formed by two orthogonally superimposed gratings having respective pitches of p and P2. The fourth grating 58 is also based upon a two dimensional pattern of size d1 x 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 identifiable particles.

Figure 6 illustrates a method of making a master template 60 for producing identifiable particles. The master template can be used for embossing a pattern that provides an identifiable 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 need to be used to create a pattern for each identifiable particle (this would take many hours and be expensive).

The master template 60 is created from a pre-fonn 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 nrn 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, Aut0CADTM 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.

Following exposure of the resist 66, the resist 66 is developed to remove the non- exposed regions 68, as shown at step 2. Depending on the resist 66 that is used (positive or negative) this process may be reversed. After development at step 3, exposed underlying chromium in the exposed regions 68 is removed by wet or dry etching. In addition, the underlying substrate material 62 is also etched to a depth of typically 200 to 500 nm in order to create the master template 60. Optionally, the master template 60 can also be electroplated, or it may be used "as is" to stamp or emboss patterns into different materials, such as, for example, various polymers or plastics.

Figures 7 and 8 illustrate a method of making tags 73' for use with identifiable 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).

The extent of the tags 73' are defined using the following process, as shown in Figure 7. A glass or silicon wafer 70 is processed as per conventional lithography, i.e. cleaned etc. The surface is then coated with a release agent to provide a layer of sacrificial matenal 76. The release agent may comprise one or more layers of metal (e.g. aluminium or chromium), layers of photoresist (e.g. Si 818), or a thin coating of TeflonTM or any combination thereof A thick layer 75 of SU8 (for example of the order of ito 25tm thickness) is disposed over the thin layer of sacrificial material 76 onto the substrate 70, which has been pre- baked at 200 C 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 90 C for 20 minutes until the SU8 film is no longer sticky following a slow cooling.

The purpose of the release agent is to enable the encoded particle material to be removed from the substrate 70 after manufacture. The release agent may be a material that can be addressed in some manner, e.g. optically or electrically. Encoded particle material forming identifiable particles can then be selectively released from the substrate 70. This enables chemical moieties to be grown or deposited onto the encoded particle material whilst still on the substrate, with only selected ones being subsequently released during a test or experiment.

At stage 2, the SU8 film 71 is exposed to optical radiation (e.g. at 436 nm) for 40 seconds through a patterned chromium mask, mask 72. The mask 72 defines the size of the individual features (such as 10 tm x 50.tm bars, for example) that are used to define the extent of individual bare tags 73. At stage 3, the mask is released and the substrate 70 subjected to a post-exposure bake at 90 C for 15 minutes to complete cross-linking. At stage 4, development is performed using SU8 developer for several minutes until a clear pattern is observed, before rinsing the substrate 70 comprising the bare tags 73 in iso-propyl alcohol (IPA) for 10 seconds. The substrate 70 is then blow- dried in nitrogen.

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 im to 2 tm) 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 UV light for 24 seconds through the mask 72 (appropriately aligned) and then post-exposure baked at 90 C for minutes on a hotplate. A development step is then carried out in SU8 developer for 2 to 7 minutes with periodic rinsing in WA. 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 80 C). 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 thepattern is embossed. For example, for poly(benzyl methacrylate) hard baking could be performed at 54 C, whereas for poly(cyclohexyl acrylate) it could be performed at 100 C.

Figure 9 illustrates a method of making tags 83 for use with identifiable 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 may comprise one or more layers of metal (e.g. aluminium or chromium), layers of photoresist (e.g. Si 818), or a thin coating of TeflonTM or any combination thereof A layer of SU8 85 (for example of the order of 1 to 25.tm thickness) is disposed over a thin layer of sacrificial material 86 onto the substrate 80, which has been pre-baked at 200 C 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 90 C 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 80 C 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 identifiable 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 may comprise one or more layers of metal (e.g. aluminium or chromium), layers of photoresist (e.g. Si 818), or a thin coating of TeflonTM or any combination thereof A layer of SU8 95 (for example of the order of 1 to 25 jim thickness) is disposed over a thin layer of sacrificial material 96 onto the substrate 90, which has been pre-baked at 200 C 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 90 C 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, LIV 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 chemicals linked to thereto using conventional methods or by use of split and mix combinatorial chemistry techniques.

Figure 11 illustrates a method of making tags for identifiable 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 identifiable 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 identifiable particles 110 are transported.

The source 160 currently operates using optical radiation at 630 nm. However, use of a shorter wavelength such as, for example, 530 nrn, would allow the reader 100 to work with identifiable 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 JR 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 diffractionpattern 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 Brogue wavelength of the electrons, in order to obtain a characteristic electron diffraction pattern.

Laser light passing through the identifiable 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 twodimensional spatial distribution pattern that encodes the identifiers. The detector 120 can detect a diffraction pattern spatial distribution of radiation emitted from the identifiable 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 identifiable 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 identifiable particles 110.

In this reader 100, the identifiable 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 identifiable 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 Münster, Germany).

The electrokinetic orientation device 170 orientates the identifiable 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 identifiable 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 identifiable 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 identifiable 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 identifiable 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=0, 1,2. . . etc). The encoding capacity E (i.e. the number of different distinguishable identifiers) is of the order of ml {d1/A}, where mt {} is the integer operator, d1 is the grating length and 2 is the wavelength of the exposure radiation,. For example, if d1 = m, and 2= 1 Jim, then E= 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 E is of the order of mt {d1/2}", where N is number of superimposed gratings.

For example, ifdj = 50.tm, 2 = 1 m, and N= 5, then E= 3 x In a third encoding strategy, superimposed gratings are provided in two different directions with pitches p' and P2. 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 Xis of the order of mt {d,/2}.Int (d2/2}, where d1 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 E is of the order of mt {d/A.} !, where d is the smallest dimension of the grating pattern.

For example, , if d = 50 tm, and A. 1 pm, then E= 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 identifiable 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 identifiable particles 110 parallel to the plane containing the CCD detector 122 and is also insensitive to the actual position of the identifiable particles 110 relative to the CCDarray 122.

Figure 14 schematically shows alternative detector positions in a reader 200 for reading identifiers from identifiable 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', c' and d' 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 a', b', c' and d' 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 identifiable 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, identifiable 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. Identifiable 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 one example, patterns may be fabricated in a photoactive polymer. A thin film of the material gelatin can be deposited onto a glass or silicon substrate, and exposed in a holographic exposure system to record an interferometrically produced wave pattern of the desired fringe frequency and orientation to provide an identifier. Wet processing (development and or etching) may then be used to transform the exposed fringes into modulations in surface profile within the polymer. The substrate may be any glass material, such as, for example, optical glasses like BK7 and fused silica. Anti- reflection coatings may also be applied to substrates etc. to reduce reflection losses. It may also be desirable to use prisms and piano- convex or piano-concave lenses in the manufacture of various grating assemblies.

In certain embodiments of identifiable particles, no receptor material may be provided, or a coating applied that is not for binding to a predetermined chemical entity. Such identifiable particles may be used with a detection system that checks for the presence or absence of identifiable particles. One possible use of such identifiable 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 identifiable 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 identifiable particles and a detector, including those in which the detector is movable and the identifiable particles remain stationery.

Additionally, they will be aware that techniques other than electrokinetic/electrostatic orientation may be used to orientate identifiable 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 identifiable 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 identifiable particle.

Those skilled in the art will also realise that identifiable particles of the type described herein may be used in combination with various other techniques (e.g. with nano- particles, magnetic techniques, etc.) The identifiable 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 identifiable 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 identifiable 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 errorcorrection capabilities.

References 1. US-5,736,332 2. US-5,981,166 3. US-6,051,377 4. US-Bl-6,361,950 5. US-B1-6,376,187 6. Hoffman D. et a!, "Development of optically encoded micro-beads in microfluidic systems," Proc. uTAS 2004, Editor T. Laurel!, ISBN 0854048960 7. Evans M. et a!, "An encoded particle array tool for multiplex bioassay, " Assay and Drug Development Technologies, Vol. 1 (1-2), p.1, 2003 8. GB-A-2,306,484 9. Fenniri H. et al, "Preparation, physical properties, on-bead binding assay and spectroscopic reliability of 25 barcoded polystyrene- poly(ethylene glycol) graft copolymers," JACS, Vol. 25 (35), pp. 10546- 10560, 2003 10. Han M. et al, "Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules," Nature Biotechnology, Vol. 19, pp. 63 1-635, 2001 11. Xiaohu Gao and Shuming Nie, "Quantum dot-encoded high brightness mesoporous beads with high brightness and uniformity: rapid readout using flow cytometry," Anal. Chem., Vol. 76, pp. 2406-24 10, 2004 Where permitted, the content of the above-mentioned references are hereby also incorporated into this application by reference in their entirety.

Claims

1. 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.

2. The particle of claim 1, wherein the material variations include a pattern of refractive index variations that give rise to a predetermined diffraction pattern diffraction pattern.

3. The particle of claim 1 or claim 2, wherein the material variations include one or more reflective element disposed within the particle material.

4. The particle of any one of claims 1 to 3, wherein the identifier encoding scheme comprises providing a plurality of superimposed gratings in a particle material.

5. The particle of any one of claims I to 4, wherein the particle material is bio- compatible.

6. The particle of any one of claims 1 to 5, wherein the particle material is bio- degradable.

7. The particle of any one of claims 1 to 6, wherein the size of the particle is from about 100 nm to about 100 tm.

8. The particle of any one of claims 1 to 7, further comprising a receptor material for binding to a predetermined chemical entity.

9. The particle of claim 8, 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.

10. A method for making identifiable 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 an identifiable particle.

11. The method of claim 10, wherein the material variations are refractive index variations that form a pattern in the particle material.

12. The method of claim 10 or claim 11, wherein the material variations are provided by one or more of: i) embossing the particle material using a master template; ii) etching the particle material with a high energy beam; iii) writing an interference pattern into the particle material using an optical interference technique; iv) nano- imprinting; and v) applying a pattern to the particle material using a lithography technique.

13. The method of any one of claims 10 to 12, further comprising making a plurality of identifiable particles, each particle encoding a different permutation of the identifier encoding scheme.

14. The method of any one of claims 10 to 13, further comprising providing the identifiable particle with a receptor material for binding to a predetermined chemical entity.

15. The method of claim 14, wherein the receptor material is chosen to bind to a nucleic acid, an analogue of a nucleic acid or a modified nucleic acid.

16. A kit for detecting the presence of a predetermined chemical entity, the kit comprising a plurality of identifiable particles made according the method of claim 14 or claim 15.

17. A reader for reading a diffraction pattern identifier from the identifiable particle according to any one of claims 1 to 9, the reader comprising: a source for exposing identifiable particles to radiation; a detector for detecting the diffraction pattern of radiation emitted from the identifiable particle; and an analyser for analysing the diffraction pattern of the radiation emitted from the identifiable particle in order to determine whether the diffraction pattern corresponds to a diffraction pattern identifier encoded according to a predetermined encoding scheme.

18. The reader of claim 17, wherein the source generates infrared (IR) or near- infrared (NIR) radiation.

19. The reader of claim 17 or claim 18, wherein the detector comprises a CCD array, a CCD matrix or a linear diode array.

20. The reader of any one of claims 17 to 19, further comprising a fluid transport channel for transporting identifiable particles suspended in a fluid through a region exposed to radiation from the source.

21. The reader of any one of claims 17 to 20, further comprising an electrokinetic, electrostatic or microfluidic device for orientating identifiable particles towards a predetermined direction.

22. The reader of any one of claims 17 to 21, further comprising a sorter for separating identifiable particles that have undergone different chemical binding events from one another.

23. The reader of any one of claims 17 to 22, further comprising an orientation detector for detecting the orientation of the identifiable particles.

24. A method for reading a predetermined diffraction pattern identifier from the identifiable particle according to any one of claims 1 to 9, the method comprising: exposing an identifiable particle to radiation; detecting the diffraction pattern of radiation emitted from the identifiable particle; and analysing the detected diffraction pattern of the radiation emitted from the identifiable particle in order to determine whether it matches a diffraction pattern identifier encoded using a predetermined encoding scheme.

25. The method of claim 24, 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.

26. The method of claim 24 or claim 25, wherein the radiation is infrared (IR) or near-infrared (NIR) radiation.

27. The method of any one of claims 24 to 26, further comprising transporting the identifiable particle suspended in a fluid through a region in which the identifiable particle can be exposed to radiation.

28. The method of any one of claims 24 to 27, further comprising orientating one or more identifiable particles towards a predetermined direction prior to exposure to radiation.

29. The method of any one of claims 24 to 28, wherein the scale of the material variations is less than half the wavelength of the radiation.

30. A method for identifying a chemical entity in a test substance, comprising: introducing a plurality of identifiable 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; reading the respective diffraction pattern identifiers of the identifiable particles that have bound to respective chemical entities; and matching the diffraction pattern identifiers that are read to respective predetermined chemical entities so as to identify the chemical entities present in the test substance.

31. The method of claim 30, further comprising separating identifiable particles that have undergone different chemical binding events from one another.

32. The method of claim 30 or claim 31, wherein the chemical entity to be identified comprises a nucleic acid, an analogue of a nucleic acid or a modified nucleic acid.

33. A chemical entity identified using the method of any one of claims 30 to 32 or a receptor material for binding thereto.

34. A product comprising the chemical entity of claim 33.

35. The product of claim 34, wherein the product is a pharmaceutical or medicinal product.

36. An identifiable particle substantially as hereinbefore described with reference to the accompanying drawings.

37. A method of making identifiable particles substantially as hereinbefore described with reference to the accompanying drawings.

38. A kit substantially as hereinbefore described with reference to the accompanying drawings.

39. A reader substantially as hereinbefore described with reference to the accompanying drawings.

40. A method of reading substantially as hereinbefore described with reference to the accompanying drawings.

41. A method of identifying the presence of a predetermined chemical entity in a test substance substantially as hereinbefore described with reference to the accompanying drawings.