WO2002083294A2 - Chemical libraries based on coded particles - Google Patents

Abstract

The chemical library, and a method of making said library, comprising particles each having thereon a respective chemical member of said library, each particle having markings serving to identify the particle, and thereby to identify the chemical member of the library on any selected particle, wherein said particles each comprise a fluorescent material which has been selectively bleached at selected locations to define said markings.

Description

Chemical Libraries based on Coded Particles

The present invention relates to a method of fabricating chemical libraries on coded particles. In a wide range of biochemical and chemical procedures there is a requirement for working with oligomers formed from monomer units of a similar type to one another but differing in detailed structure. The aim may be to discover with which sequence of monomer units forming an oligomer a particular chemical entity will react. In some cases this may be for identifying the sequence of an unknown oligomer of known (relatively short) length. The oligomers may be formed from nucleotides (RNA or DNA) , amino acids (peptides and proteins) , sugars or any other oligomerisable chemical compound.

With regard to oligonucleotides, one approach is to place an unknown (analyte) strand in the presence of all possible (target) strands of some shorter length. A small number of complementary target strands will bind to the analyte strand and this binding event may be identified by any suitable means, for example fluorescence, electro- chemiluminescence, chemiluminescence, biochemiluminescence or phosphorescence. In an existing technology, the target strands are spatially distributed on a surface. The sequence of each target strand is encoded in its location on the surface. The identity of the analyte strand can, therefore, be deduced from the physical location of the binding event in relation to the surface.

An alternative technology is based on small, physically differentiable beads. At least one target strand of known sequence is attached to a bead in such a way that all of the attached strands have the identical sequence. In this way, the sequence of a particular strand may be identifiable from the particular bead to which the strand is attached. The beads are then exposed to the analyte strand and binding between the analyte strand and any target strands may be detected using any suitable detection means, for example fluorescence. The beads upon which a binding event occurred may then be separated from the bulk of the beads and the sequence of the analyte strand deduced from the sequence of the bound target strand that is identifiable by a number of means .

In another procedure, for providing a chemical combinatorial library, peptide sequences are built-up on particles (one sequence per particle) and interaction between each peptide sequence and an active molecule such as a peptide cleaving enzyme is looked for. The substrate sequence for the enzyme may be discovered and inhibitors for it may be developed. A method of fabricating machine-readable beads by etching a silicon wafer is described in GB-A-2334347.

Each particle has a unique code, i.e. in the library there is only one particle present that has any particular code. GB 0009723 describes particles formed in plastics. Each generally bar shaped particle has a series of notches along each long side which form a machine readable bar code. One end of each particle is marked with a notch serving to differentiate that end from the other end of the particle. In a library of particles according to GB 0009723, each particle may bear a unique code or there may be a limited number of identically coded particles for each code. It is intended that each particle bearing a particular code should bear a known one of a library of chemical compounds.

It is proposed in GB 0009723 that the particles be coded by their shape and the shapes are to be defined by cutting using a laser. A layer of plastics for forming the particles is to be adhered to a supporting substrate via a photo- activated release layer. The shapes are cut into the particle forming layer, the chemical library is applied, and by photo-activation, the release layer is caused to release the particles. To identify the particles thereafter one would need to be able to detect the notches constituting the code .

It would be desirable to have an alternative method of encoding particles, especially one which may make it easier to read the code.

The present invention now provides a chemical library comprising particles each having thereon a respective chemical member of said library, each particle having markings serving to identify the particle, and thereby to identify the chemical member of the library on any selected particle, wherein said particles each comprise a fluorescent material which has been selectively bleached at selected locations to define said markings.

The fluorescent material may be a polymer constituting the particles or it may be a layer of material applied to such a polymer. It may be bleached locally by the application of a sufficient intensity of light energy, e.g. from a laser. A pattern of bleached spots or bars may be formed constituting a binary code. Alternatively, the entire area of the particle may be bleached except for a patern of spots or bars constituting a binary code. The laser bleaching operation may be conducted in place of the laser cutting operation described in GB 0009723, whilst the particles are retained on a substrate by a photo- activated release polymer layer. The divisions between the particles may be made by laser cutting as in GB 0009723 or by photo-lithography. For instance, a mask defining a multitude of micro-particles may be used for exposing a photolithographic photo-resist polymer layer supported on a substrate by a photo-actuable polymer release material. The photo-resist may then be developed with a solvent to produce channels therethrough demarcating releasable particles.

These may be bleached to form code marks using a laser or other light source, the chemical library may be applied and the particles may then be released.

If desired, the particles may each have at least a first zone and a second zone, each said zone having thereon a respective chemical member of said library, each particle having markings service to identify the particle and serving to identify said zones of the particle, and thereby to identify the chemical member of the library on any selected zone.

By using each particle for more than one chemical entity within the library but marking the particle so that each chemical entity can be separately identified according to its readable position on the particle, the number of particles needed for a library containing a particular number of chemical entities can be at least halved.

Alternatively, for a given number of particles and a given number of chemical library members supported thereon, the number of physical locations within the particle library at which any particular chemical member can be encountered can be increased. For instance, if a library in accordance with GB-A-2334347 consists of n particles bearing n compounds (one compound per particle) , each compound can only be met with at one location within the mass of particles constituting the library. However, if in accordance with the present invention two compounds are present on each particle, without increasing the amount of particle material or the amount of each chemical compound in the library, it becomes possible to meet with each compound at two distinct locations. Thus, the time needed for reaction with the library may be reduced.

In principle, any shape of particle may be used. For instance the particle may be disc shaped with bleached marks being encoded at one or more positions around the periphery. Preferably however, each particle is of rod-like or barlike shape having bleached code marks formed along each long edge.

Each particle may have markings serving to identify the particle and an end marker or markers serving to identify a first end or the second end of the particle. The markings may be formed by shapes such as pits, grooves, notches or bumps. They may also be formed as fluorescent, or coloured or monochrome markings such as bars or spots which may be applied as surface markings, e.g. by printing. The particles may be morphologically encoded during the production process so that as the overall shape of the particles is defined, patterns of 3-dimensional features are defined that provide each particle with a machine readable code .

The particles preferably are relatively small, having a maximum dimension of not more than 500 μm, more preferably, not more than 250 μm. However, to provide room for markings which as a binary code are capable of differently encoding at least 32,000 different particles at a pitch of say 20 μm per mark, it is preferred that the particles have as their largest dimension a size of at least 50 μm, more preferably at least 100 μm. Particles within the size range of 100 μm to 250 μm are therefore preferred. Each library need not however contain as many as 32,000 different compounds and so beads capable of bearing fewer marker coding elements are still useful. For instance, a library of say 4000 beads (potentially bearing 8000 different compounds at two compounds per bead) could be coded by only 12 coding elements.

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

The deposition of the chemical members of the library may precede or follow the formation of the particle identifying marks and the division of a continuous sheet into separable particles.

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

The compounds of the library may be pre-synthesised and then placed on the particles or they may be synthesised on the particle surface. The compounds may be chemically bound to the surface of the particles or may be physically adsorbed thereon. The particles may be porous and the compounds of the library may be present within the pores of such a structure although it is preferred that the compounds be on the surface of the particle.

One option for forming the particles involves providing a sheet of polymeric material on a sacrificial substrate; delineating the sheet into a plurality of particles without destroying the integrity of the substrate; machine-readably encoding the particles by bleaching fluorescence at selected locations; and removing the substrate.

Fabrication of machine readable polymer beads is attractive for several reasons. Firstly, it provides a lower cost-manufacturing route than for silicon beads. Secondly, polymers (in particular: polystyrene, polyimide and polycarbonate) are preferred substrates for subsequent derivitisation with a wide variety of ligands.

The following is a description by way of example only and with reference to the accompanying drawings of presently preferred embodiments of the invention. In the drawings: Figure 1 is a cross-sectional view of a section of a sheet of polymeric material on a UV-release film;

Figure 2 is a plan view of an example of a coded particle; Figure 3 is a cross-sectional view of a section of a delineated sheet of polymeric material on a UV-release film;

Figure 4 is a cross-sectional view of a section of a delineated sheet of polymeric material on a UV-release film on the bottom layer of a conventional microtitre plate; Figure 5 is a plan view of a conventional microtitre plate incorporating the delineated sheet of polymeric material on a UV-release film;

Figure 6 is a side view of part of the microtitre plate depicted in Figure 5; and Figure 7 is a schematic representation of the side view of the microtitre plate depicted in Figures 5 and 6, together with means to remove the beads from the microtitre plate.

The technique to be described can create an easy-to- handle array of discrete beads within a polymer material . Monomers such as nucleotides can be printed on the top surface of the beads using an ink-jet printer type system. Oligomers may be built up by reaction of selected monomers sequentially at each location. Alternatively, preformed oligomers may be deposited on the surface. The beads may be of any suitable shape. Preferably, the beads are designed to be thin, typically 25 μm, rectangular shapes with typical lengths of 250 μm and widths of 40 μm.

Once the oligomers or other compounds have been applied to the beads, individual groups of beads can be released and processed e.g. using flow cytometry. With rectangular beads, the long aspect ratio lends itself easily to good mixing within the flow cell, thereby promoting effective binding of the bases of an analyte oligonucleotide onto a complementary target sequence. Each bead may have features defined around its periphery to give it a unique code. The structural embodiment discussed below is designed to be compatible with current micro-titre plates having 96-wells although the techniques mentioned are equally applicable to larger well sizes. At such dimensions, each 3.5 mm-square well could easily contain an array of 100 by 40 (or 4000) beads. The total number of beads defined within a commercially available 96-well structure would then be in the region of 384,000.

Example 1

In the embodiment of the invention depicted by Figure 1, a sheet of plastics material (10) , say 25 μm thick polyester or polycarbonate is placed upon another plastics sheet (12) having the specific property of being a UV-release material (e.g. 130 μm thick Furukawa UV tape-SP series) . The two sheets are placed one on top of the other so as to exclude all air gaps.

This sandwiched structure is laid down on a flat surface vacuum chuck positioned on the x-y stage of a laser micro- machining system. Preferably, the laser system is a carbon dioxide laser system with a galvanometer scan head.

The laser is operated in a first mode to cut around the rectangular periphery of each bead. It is operated at a lower power to bleach rectangular (as shown) or circular zones 20 (Fig. 2) , thus bleaching the natural fluorescence of the polymer.

Typical galvanometer scanning fields are of the order of 50mm x 50mm with typically 500 features fabricated per second. This concept permits the use of, for example, an 18 bit coding system through the creation of 18 "elements" that can be turned on or off as required. With an element width of 10 μm and an inter-element spacing of 10 μm if all the elements are defined on the same side, the total length of a bead would be just under 500 μm which may be too long. This length can be reduced if elements are defined on both sides. The reader would then need information relating to the reading sense of the bead to prevent inaccurate reading of the code when the bead flips over. However, a technique is provided here whereby the addition of two further features on the bead caters for all combinations of reading sense .

Considering a pitch of 20 microns and 9 elements on each side, the length of the bead is now about 250 microns which is an acceptable length. An example of an 12-bit bead is shown in Figure 2.

The illustrated bead has its upper surface demarcated into two zones, one bearing a first chemical library member ( "Biomolecule 1') and the other bearing a second chemical library member ( 'Biomolecule 2'). The left hand end of the particle as shown in the drawing is provided with a long marking 22 so that the ends are distinguishable. The presence of the Reference Marks 22 also serve to identify one long edge of the particle.

In the cutting mode the peak power of the laser is adjusted so that the plastic material (10) is cut at (16) all the way through but the UV sensitive tape (12) is just "nicked" by a few microns and is for all intents and purposes quite intact . Figure 3 shows that the integrity of the UV- release layer is substantially unaffected. This is preferred for all embodiments.

The machined sandwich is taken off the vacuum chuck and placed onto the bottom plate (18) of a conventional 96-well micro-titre plate (24) (Figure 4) . Generally, there are plates that are fabricated by injection molding or those whose base plate is modified. The latter are fabricated in two parts with baseplate being ultrasonically welded to an upper plate (20) which is formed with an array of holes which become the wells. In the present embodiment, nucleotides or oligonucleotides or other library chemicals (20) are applied to the surface of the machined sandwich at this stage and the machined sandwich serves as the base plate for a micro-titre plate.

The top plate of the micro-titre plate is now placed on top of the machined plastic layer. The resultant micro-titre plate is depicted in Figures 5 and 6.

One may then proceed to remove a group of beads from the base surface of the plate to make them free in the wells (22) (exact number not important) and to process them within the buffer solution of the flow system. The first step is to locally destroy the adhesive property at the interface between the UV tape and a specific group of beads. Typical values for adhesive strengths of UV tape (currently available) are 2.5 N/25 mm before UV and 0.05 N/25 mm after UV. Typical UV dosages required to do this are of the order of 1000 mJ/cm². In this embodiment, a UV source (30) e.g. a pulsed laser beam delivering this magnitude type of energy per pulse, is located on a precision x-y stage and delivers its energy from below the microtitre plate. Once the requisite amount of energy has been delivered to a specific group of beads, the second task is to remove that specific group of beads .

The process of removing the beads must not damage the top surface containing the DNA bases . One way of doing this is to use a flat precision ground hollow needle (32) with an inner diameter chosen to be between, e.g. 200 to 500 μm in diameter. The aperture could be blocked with a micro-porous membrane. The concept is to mount this needle on a precision x-y stage and point down within a well towards the cluster of 4000 beads. The removal procedure is shown in Figure 7. Each well may contain a separate library, with one or more beads within the well bearing each chemical library member. Each well may contain an identical library or different wells may contain different libraries.

After exposure to a test compound which may bind to or react with a compatible compound on a particular bead in the library, the beads may be screened to identify on which bead and which end of the bead the binding or other reaction has occurred. This may be done by removing the beads from the well and passing them through a suitable flow system to a detector at which they are inspected one at a time. When the appropriate reaction is detected the bead code is read to identify the reacting library compound. Example 2

In an alternative embodiment, a layer of polymeric photoresist material (e.g. SU-8 or other polymer resist) is formed on a solid support substrate (e.g. a silicon wafer) , and particles are delineated by exposure to a suitable lightsource (e.g. to cross-link exposed polymeric material), followed by removal of particles from the substrate. In this embodiment the bleaching of the selected locations of the particle may remove or reduce the inherent fluorescence of the polymeric material at those selected locations.

Claims

1. A chemical library comprising particles each having thereon a respective chemical member of said library, each particle having markings serving to identify the particle, and thereby to identify the chemical member of the library on any selected particle, wherein said particles each comprise a fluorescent material which has been selectively bleached at selected locations to define said markings.

2. A library as claimed in Claim 1, wherein each particle is of elongate shape having a first end and a second end separated by first and second long sides with said markings being present along each of said long sides .

3. A library as claimed in Claim 2, wherein each particle has markings serving to identify the particle and an end marker serving to identify the first end or the second end of the particle.

4. A library as claimed in any preceding claim, wherein the maximum dimension of each particle does not exceed 500 μm.

A library as claimed in any preceding claim, wherein the maximum dimension of each particle does not exceed 250 μm.

A library as claimed in any preceding claim, wherein at least some of the markings are formed by shapes such as pits, grooves, notches or bumps.

A library as claimed in any of claims 1 to 5, wherein at least some of the markings formed as fluorescent, or coloured or monochrome markings such as bars or spots which may be applied as surface markings, e.g. by printing.

8. A library as claimed in any preceding claim, wherein at least some of the particles may be morphologically encoded during the production process so that as the overall shape of the particles is defined, patterns of 3-dimensional features are defined that provide each particle with a machine readable code.

9. A method of fabricating a chemical library, comprising providing a sheet of substrate material incorporating a fluorescent material, in either order or simultaneously (a) forming deposits of selected chemical library members at known respective spatial zones on the surface of said substrate material, and (b) bleaching said fluorescent material at selected locations to provide coded markings associated with each said spatial zone, and then (c) dividing said sheet to form separate particles each of which includes a said spatial zone bearing a said chemical library member, each particle being marked with said coded markings identifying the chemical library member on the particle.

10. A method as claimed in Claim 9, wherein selected areas of said fluorescent material are bleached by use of a laser.

11. A substrate sheet for use in fabricating a chemical library, which sheet comprises a substrate material incorporating a fluorescent material which has been bleached at selected locations to form coded markings which identify spatial zones of the sheet for receiving respective chemical library compounds.