WO1996026440A1

WO1996026440A1 - Specific binding materials

Info

Publication number: WO1996026440A1
Application number: PCT/GB1996/000410
Authority: WO
Inventors: Cameron Alexander; Evgeny Vulfson
Original assignee: The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority date: 1995-02-21
Filing date: 1996-02-21
Publication date: 1996-08-29
Also published as: AU4728196A; CA2211174A1; JPH11500824A; EP0811161A1

Abstract

Specific binding materials are provided that are adapted to specifically bind with a target material characterised in that the specific binding material has areas upon its surface corresponding to the size and/or shape of the target material. Preferably the target material is a biological target material; e.g., a microorganism, antibody or antigen. Preferably the specific binding material comprises a polymeric body on which areas corresponding in size and/or shape to the target have been formed.

Description

SPECIFIC BINDING MATERIALS.

The present invention relates to specific binding materials, to methods for their preparation and methods for their use. Particularly there are provided novel specific binding materials and methods that have application in separation and/or concentration of biological targets such as macromolecules and microorganisms, and particularly those targets found in water supplies, food and food derived materials.

It is known to immobilise biological targets, such as DNA, RNA, viruses and viral components, bacteria, antigens and antibodies, using specific binding materials. These materials typically comprise immobilised complementary species such as oligonucleotides, antibodies or antigens which have the capability to specifically bind the target. Commonly the species are immobilised on materials such as microtitre plates or wells, on latex or polymer beads or strips, on column materials such as polysaccharides, or on dipstick structures.

While the binding between these materials and their agents is primarily through charge interaction and hydrogen bonding with binding species, its efficacy is necessarily limited by the concentration of the binding species immobilised on the material surface. Thus the material will commonly require a high concentration of the species in order to ensure efficient capture of target from a liquid phase sample.

The present inventors have now provided novel specific binding materials that utilise target shape and/or size, as well as optionally using specific binding species, to enable capture a biological target from a liquid medium in specific fashion.

In a first aspect of the present invention there is provided a specific binding material adapted to specifically bind with a target material characterised in that the specific binding material has areas upon its surface corresponding to the size and/or shape of the target .

Preferably the target is a biological target, eg. a microorganism such as a virus particle, bacteria, yeast, antibody or antigen, and the areas on the binding material surface are shaped and sized to accommodate a substantial part of the target material. It will be understood that by size and shape it is intended to refer to more than just a molecular level interaction such as that between two molecules; a physical size and shape of a substantial part of the target being what is being accommodated by the binding material.

Preferably the material comprises a polymeric body on which areas corresponding in size and/or shape to the target have been formed. These areas preferably have a high affinity for binding the target; for example the size and/or shape specific area of the material may have functionalised species such as charge bearing groups or have antibodies or antigens bound to it, eg. by covalent bonding.

In a particularly preferred aspect the present inventors have found that the specificity and affinity of the specific binding material for the target material may be increased by treating the areas of the specific binding material surface that are not sized or shaped to correspond to the target material such as to reduce their ability to bind the target or any other material from a sample from which it is being specifically selected. In this fashion the specificity of the binding is increased as only targets having the correct size or shape can be effectively bound.

Such treated or 'poisoned' specific binding materials may be used to bind any desired entity; whether a particulate material such as a virus, bacteria or other microorganism, or a specific macromolecule or smaller chemical entity. Generally such treatments are those which remove or mask the species on the binding materials surface that cause the target and other materials to become bound. Thus hydroxy groups may be esterified or converted to ethers, while carboxyl groups could be esterified or reduced. Still more effectively these groups can be masked by treatment with groups such as silyl or perfluoro groups using entities as will be illustrated in the Examples below.

In a second aspect of the present invention there is provided a method for preparation of the specific binding materials of the present invention comprising binding a target material to the surface of the specific binding material such that the surface of the specific binding material becomes adapted the size and/or shape of the target material, then removing the target material from that surface.

In each of these aspects the surface of the specific binding material is adapted such as to be capable of conforming or interacting with the shape or size of the target such that non-target materials not having the desired shape or being too large to fit into or onto the conformed area do not become bound or bind with decreased affinity. The conformation may be with any part of the target as long as the target can access the binding area so provided. Thus conveniently the adaptation is such that an imprint of the target is made in or on the surface of the binding material which is located and configured such to allow the target to access the binding surface.

In one embodiment of the second aspect of the invention the specific binding material is adapted to the shape and size of a target by forming a body of the specific binding material in the presence of the target material. It is known to imprint polymers with templates (see Wulff (1986) 'Polymeric Reagents and Catalysis' (ACS SympSer 308) Ed W T Ford. pl86 American Chemical Society) but not with labile materials such as the preferred biological targets of the present invention.

The condition for formation of the specific binding material body, eg. polymeric bodies, in the presence of biological target materials must meet several criteria if the specificity of the interaction for living or otherwise high temperature and chemically labile materials is to be maintained. For living material the formation preferably takes place at target physiological pH and temperature, with low toxicity conditions, and preferably with short reaction time and appropriate functionality for the purposes of binding the target. Furthermore, the formation must result in a mirroring of target surface features, eg. for bacteria preferably being on a 0.1 to 5 μm scale and more preferably on a 0.5 to 2μm scale. The material produced should preferably be durable and have easy and safe handling characteristics.

Preferably the specific binding material is functionalised at its binding surface by functional groups, eg. such as amino and/or hydroxy and/or carboxyl and/or amide groups; these allowing for direct binding and/or functionalising of the surface once formed.

It has further been found by the present inventors that commercially available polymeric beads used for immobilising antibodies, such as Dynabeads, will bind bacteria or viruses in non-specific fashion to their surfaces to various extents. Dynabeads as such are too small to be of use in binding bacteria by shape and size interaction, but can be used to bind smaller entities such as viral targets. Other larger commercial beads will of course be usable with bacteria and larger entities such as yeasts and protozoans. These commercially available beads are ideal starting materials for the surface poisoning aspect of the present invention, but other custom made materials may of course be used.

Preparation of polymer bodies at physiological/biological pH can be carried out by interfacial reactions using dispersed organic phase. One preferred method uses radiation cross-linking of monomer components to provide polymerisation. Once formed about the target material, the latter may be removed by a variety of means, eg. by subjecting the materials to a high vortex and/or use of acids or alkalis. In a preferred method of the invention however there is provided a method for production of preferred materials of the invention wherein the specific binding materials of the invention are preformed in the absence of target material; the preformed material exposed to target material, particularly in the absence of non- arget materials, to allow surface interaction, ie. binding; the two materials in bound form exposed to a further treatment whereby the surface of the specific binding material which is not covered by target material becomes wholly or partially inactivated with respect to its ability to bind target and non-target materials.

The form of the specific binding material is not limited to any specific type; convenient forms will include beads, capsules, strips, films and membranes, ie. semi-permeable films through which samples may be passed while retaining particulates.

The materials, methods and uses of the present invention will now be described by way of illustration only by reference to the following non-limiting Examples and Figures. Further embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these.

FIGURES

Figure 1: Shows a diagrammatic representation of the formation of a specific binding material body at the interface between an aqueous layer and an organic layer at which a bacteria is located.

Figure 2: Shows a diagrammatic representation of the four stages of formation areas of size and shape specific to target bacteria on polymeric beads as they form at a liquid interface.

Figure 3: Shows a diagrammatic representation of a variation of the method shown in Figure 2 wherein the beads formed from the second step, or preformed solid Deads that have had bacteria bound to them, are treated such as to 'poison' unbound surfaces such as to reduce or destroy their ability to bind the target and other materials.

Figure 4:

Plates la, lb: Confocal Laser Scanning Micrographs (Zeiss LSM ID). of ethidium bromide-stained Listeria monocvtogenes . and Staphylococcus aureus. respectively, attached to polyamide microcapsules. Plate la depicts the upper surface of a mircocapsule with Listeria monocytogenes showing clearly in fluorescence, indicating the density of cell coverage. Plat lb is an optical slice through the middle of a polymer microcapsule: the fluorescent Staphylococcus aureus cells delineate the outer surface of the polymer membrane.

Plates 2a, 2b: Scanning Electron Micrographs (Hitachi S570) showing polymer beads after cross-linking of the diacrylate-containing organic core. The microorganisms can be seen partially embedded in the surface. The retention of the rod and coccoid shapes indicates that the gross physical morphology of the cells was unaffected by the polymerization process.

Plates 3a. 3b: Scanning Electron Micrographs depicting the "lithographic prints" of the respective bacteria. The size and shape of the "prints" can be seen to correspond exactly to those of the microorganisms. In addition to the shape anisotropy, these sites were rendered distinct chemically by reaction of the beads with a diisocyanato-tipped perfluoropolyether, thus blocking the areas of the polymer surface not covered by the microorganisms. Following the hydrolysis step to remove the cells, the original functionality at the sites was exposed, allowing for further derivitization ("development" of the lithographic prints).

Plates 4a, 4b show the "chemically-amplified" prints of the bacteria. After removal of the bacteria, the beads were reached with a fluorescent-labelled lectin, FITC-Concanavalin A. Confocal Laser Scanning Microscopy has been used to define an optical section across the upper surface of the polymer beads, with the bright regions corresponding to areas reactive towards FITC-Concanavalin A. The anisotropic functionality of the surfaces can be seen to match exactly the dimensions of the bacteria in Plates 1 and 2, and the sites in Plate 3. thus establishing the lithographic prints of the bacteria both topologically and chemically.

EXAMPLES Purification of Reagents. The following reagents were purchased from Aldrich or Sigma and used as received: Polyallylamine (PAA) of V^ 100,000 and 1% 1 ,000; (3~[N] -morpholino)propylsulphonic acid (MOPS), dibutyl ether (DBE), lysozyme (chicken egg white), l-ethyl-3-(3^_diπιethylaminopropyl)carbodiimide (EDC) ; l.l'-azobis(cyclohexanecarbonitrile) (ACCN) F0MBLIN DIS0C (perfluoropolyether, diisocyanato terminated) , fluoroscein isothiocyanate (FITC), acridine orange, rhodamine 123 and sulforhodamine 101 acid chloride. 1,6-hexanedioldiacrylate (Aldrich) and divinylbenzene (80% tech. grade, Aldrich) were washed with dilute aqueous NaHCO , passed through activated neutral alumina and stored over dried 4A molecular sieves. Adipoyl chloride (Aldrich) was double -distilled in vacuo and stored under nitrogen in a Schlenk flask. Azobis(isobutyronitrile) (AIBN) was purchased from Fluka and recrystallised from methanol before use. 6-aminohexylmethacrylamide was prepared by reaction of methacrylic anhydride (1.0 equivalent) with 1,6-diaminohexane under Schotten-Baumann conditions followed by repeated extraction from chloroform with dilute aqueous acid.

Growth of microorganisms. Broth cultures obtained by overnight growth at 37°C in tubes containing 9ml of yeast-dextrose broth (YDB) ; containing 10 g/1 peptone; 8 g/1 beef extract; g/1 NaCl; glucose 5 g/1; yeast extract 3 g/1; pH6.8 for cultures of Staphylococcus aureus NCD0 9^9 and Salmonella enteritidis 010 37782, Nutrient broth (NB, Unipath) for Escherichia coli 4824/79 and coryneform broth (CB; containing 10 g/1 tryptone; yeast extract 5 g/1. NaCl 5 g/1; glucose 5 g/1; pH7.2) for Listeria monocvtogenes C200 type 2 and Listeria ivanovi C659-

Example 1: Preparation of polvamide-surf ce beads (Polvamide 1). A solution of MOPS buffer (0.6N, pH7.8, 250ml) was placed in a reaction vessel equipped with a magnetic bar and stirred at setting 5 over a IKA-MINI-MR stirrer plate whilst nitrogen was bubbled through for 10 minutes. A solution of adipoyl chloride (1.2 ml) in a mixed organic phase containing dibutyl ether (14.4 ml), 1.6-hexanedioldiacrylate (14.4 ml) and AIBN (300mg) was added and the stirrer speed was increased to setting 6 for 5 minutes before dropwise addition of polyallylamine solution (\ 100,000, 0.2N in 0.6N MOPS, 45ml) with MOPS (30ml of 0,6N). The capsules formed were irradiated (Blak-Ray B-100A lamp) with stirring for 12hrs and the resultant polymer beads filtered, washed with water (3 x 100ml) and methanol (3 x 100ml) and dried in air.

Example 2: Preparation of polva ide surface beads with optimise.. physical properties (Pnlvfltmde 2). A solution of sodium carbonate buffer (0.5N, pH11.5. 400ml) was placed in a reaction vessel equipped with a magnetic bar and stirred at setting 5 over a IKA-MINI-MR stirrer plate whilst nitrogen was bubbled through for 10 minutes. A solution of adipoyl chloride (2.0ml) in a mixed organic phase containing chloroform (25ml). divinylbenzene (25ml), and ACCN (300mg) was added and the stirrer speed was increased to setting 6 for 5 minutes before dropwise addition of polyallylamine solution (M_y 100.000, 0.2N in 0.5N Na₂C0₃, 50ml). The capsules formed were irradiated (Blak-Ray B-100A lamp) with stirring for 12 hours and the resultant polymer beads were filtered, washed with water (3 x 100ml) and methanol (3 x 100ml) and dried in air.

Example 1 : Preparation of 'Imprinted' polymeric adsorbents (Imprinted Polvmer 1). A solution of MOPS buffer (0.6N, pH7.8. 250ml) was placed in a reaction vessel equipped with a magnetic stirrer bar and stirred at setting 5 over a IKA-MINI-MR stirrer plate whilst nitrogen was bubbled through for 10 minutes. A solution of adipoyl chloride (1.2ml) in a mixed organic phase containing dibutyl ether (l4.4ml), 1.6-hexandiol -diacrylate (l4.4ml) and AIBN (300mg) was added and the stirrer speed increased to setting 6 for 2 minutes before a suspension of Listeria monocvtogenes (ethidium dibromide stained, 200ml of 10¹⁰cfu. ml^'1), pre-stirred for ten minutes in MOPS buffer (50ml), was added and stirring continued for 3 minutes before dropwise addition of polyallylamine solution (M_w 100,000, 0.2N in 0.6N MOPS, 45ml) with MOPS (30ml of 0.6N). The capsules formed were assessed for bacterial binding by Confocal Laser Scanning Microscopy (CLSM) and irradiated (Blak-Ray B-100A lamp) with stirring for 12 hours. The resultant polymer beads were filtered, washed with water (3 x 100ml) and methanol (3 x 100ml) and dried in air.

Example 4: Preparation of 'Imprinted' polymeric adsorbents

(Imprinted Polvmer 2) . A suspension of Listeria monocvtogenes (5.0ml of 10¹⁰cfu/ml) was stirred in MOPS buffer (0.6N, 100ml) as nitrogen was bubbled through for 10 minutes. Stirring speed was increased to setting 6 (IKA-MINI-MR) as a solution of AIBN (lOOmg),

6-aminohexylmethacrylate (l.Og) in chloroform/1,6-hexanedioldiacrylate (50:50 v/v, 10ml) was added.

Stirring was continued under UV irradiation for 5 minutes at setting

4 (IKA-MINI-MR) and then at setting 1 until bead solidification occurred. The resultant beads were washed with methanol (5 x

100ml)and air dried.

Methods for removing bacteria from beads of Examples 1 to : Physical Shear method for detaching bacteria: this was carried out in flat-bottomed glass universals using a bench whirlimixer. DEFT counts were performed on bacteria released from samples of imprinted beads after vortexing. Beads were examined by DEFT and confocal microscopy to confirm removal.

Chemical methods for detaching bacteria from polvmer surfaces: Solutions of NaCl (1.0M), Urea (8.0M), citrate and borate buffers (1.0M, varying pH) were prepared in deionized water and sterilized by autoclaving. Phosphate buffer (1.0M. varying pH) was filter sterilised. Imprinted beads were resuspended in solutions of varying pH (2.0-11.0). concentration of phosphate (0-1.0M), NaCl (0-1.0M) and urea (0-8.0M) . Liberated bacteria were detected by DEFT. Samples of beads were boiled in 2.0M HC1 or 2.0M NH^OH for 2 hours prior to DEFT analysis.

Example 5: Preparation of 'Imprinted' polymeric adsorbents (Imprinted Polvmer Film ) . A suspension of Listeria monocvtogenes (5.0ml of 10⁸cfu/ml) was stirred in MOPS buffer (0.6N, 100ml) as nitrogen was bubbled through for 10 minutes. The suspension was then poured carefully onto a solution of AIBN (lOOmg), 6-aminohexylmethacrylate (l.Og) in chloroform/1,6-hexandioldiacrylate (50:50 v/v, 10ml) in a beaker. Irradiation of the two-phase system was carried out until film solidification occurred. The resultant film was washed with methanol prior to examination by Scanning Electron Microscopy. Samples were then washed with either 6M HCl/MeOH or 50% ammonia 880/MeOH solution to remove bacteria.

Example 6: Preparation of imprinted polvmer beads with 'poisoner-' surface. (Imprinted Pnlvmer 4). A suspension of imprinted polymer beads (l.Og) was stirred in 1,1,2 -trichlorotrifluoroethane (250ml) was stirred rapidly as a solution of F0MBLIN DIS0C (1.5g perfluoropolyether, diisocyanato terminated) in

1,1,2-trifluorotrichloroethane (20ml) was added dropwise via a funnel equipped with a drying tube. Stirring was continued for 3 hours before addition of the suspension to methanol (250ml) and the solvent was removed by decanting before washing the beads with further methanol (5 x 100ml).

Example 7: Removal of bacteria from 'Imprinted' polvmer beads. (Imprinted polvmer 5) . A suspension of imprinted polymer beads (250mg) was refluxed in 6M HCl/methanol (150ml) for _β hours with regular monitoring of the extent of cell removal by Scanning Electron Microscope. The beads were then repeatedly washed in methanol and dried in air.

Example 8: Preparation of 'Fnotprinted' polvmer beads having size and shape adaptation 'on' bead surface. Preformed polymer polyamide beads (l.Og) were added to 50ml of 1/4 strength Ringer's solution in the presence of bacteria serially diluted in 0.1/. peptone to give a final concentration of 10⁸ cfu/ml. The beads were incubated for 2 hours at 4°C with rolling.

Example Q: Preparation of 'Footprinted' beads with 'poisoned' surface (Footprinted Polvmer 1). Polymer beads with adsorbed bacteria (l.Og) were stirred in 1 ,1,2-trichlorofluoroethane (250ml) was stirred rapidly as a solution of F0MBLIN DIS0C (1.5g perfluorpolyether, diidocyanato terminated) in 1.1,2-trichloroethane (20ml) was added dropwise via a funnel equipped with a drying tube. Stirring was continued for 3 hours before addition of the suspension to methanol (250ml), the solvent was removed by decanting and the beads were washed with further methanol (5 x 100ml).

Example 10: Removal of bacteria from 'Footprinted' beads with 'poisoned' surface (Footprinted polvmer 2). A suspension of imprinted polymer beads (250mg) was refluxed in 1M HC1 /methanol (150ml) for 4 hours with regular monitoring of the extent of cell removal by Scanning Electron Microscopy. The beads were then repeatedly washed in methanol and dried in air.

Example 11: Use of Specific binding material beads of the invention. Specificity of beads of the invention was determined (see Table 1). Plate count: Serial dilutions of Escherichia coli. Staphylococcus aureus. Listeria monocvtogenes and Salmonella enteritidis in 0.1 peptone were plated using Yeast-Dextrose Agar (Unipath Ltd. Basingstoke UK) Colonies were counted after 24 to 48 hours incubation at 30°C.

Direct Epjfluorescent Technique (DEFT Bacterial Count: The DEFT was performed according to British Standard Methods BS 4285- The pre-filtration step using 5-0 micron nylon mesh to remove particulate matter from suspension was ommitted.

Confocal Examination of Samples: Samples of imprinted beads were dual stained in ethidium bromide and acridine orange, prior to examination by confocal microscopy. Samples were initially stained for 2 minutes by immersion in 0.1. ethidium bromide prepared in 0.05% benzalkonium chloride and were rinsed three times with deionised water before staining in acridine orange (0.025% in 0.1M citrate/NaOH buffer, pH6.6). After 2 minutes samples were washed twice in 0.1M citrate/NaOH at pH3-0. Stained preparations were mounted onto a microscope slide and covered with a coverslip. Microscopic examination was made using a Zeiss confocal laser scanning microscope (LSM ID) , operating at an excitation wavelength of 488nm using an Argon ion laser. Images of 512 x 512 pixels were photographed directly from a high resolution videophotometer using a Nikon F301 camera.

TABLE 1: Bacterial Adsorption by Beads of the Examples.

Polymer Bacterial species Cone. Bacteria %Bacteria added added cfu/assay extracted

Polyamide 1 Listeria 10" 52 Non-imprinted monocytogenes

Polyamide 1 Salmonella 10" 20 Imprinted enteritidis

Footprinted Listeria 10" 9 polymer 1 monocytogenes 'poisoned'

Footprinted Salmonella 10" 0 polymer 1 enteritidis 'poisoned'

Listeria Listeria 10" 48 Footprinted monocytogenes Polymer 2

Listeria Salmonella 10" 13 Footprinted enteritidis Polymer 2

Salmonella Listeria 10" 10 Footprinted monocytogenes Polymer 2

Salmonella Salmonella 10" 21 Footprinted enteritidis Polymer 2 Samples contained 5ml buffer (lOmM MOPS pH7.0), 50mg polymer beads, 100ml bacteria (lθ" cfu/ml). Rotation was carried out for 2 hours followed by settling of beads. Samples of supernatant (100ml) were drawn and plated for bacterial counting.

In order to demonstrate the increase in specific binding provided by these treatments over the non-specific binding provided using commercially available untreated beads, a comparative test was carried out using Dynabeads available from Dynal A/S P0 Box 158. Skoyen. N0212 Oslo, Norway that had Salmonella antibodies immobilised upon them.

TABLE 2: Bacterial Extraction using antibodies on Dynabeads.

Bacterial species Cone, added cfu/ml %Extracted

S. typhimurium 10⁵ 24 10" 27 10³ 24 10² 0

E. coli 10" 13 10³ 13 10² 8

S. enteritidis 10³ 20 10² 37

L. monocytogenes 10" 28 10³ 24

Assays were carried out in accordance with the manufacturers instructions. Example 12: Attachment of enzvmes to polvamines.

The method of forming a specific binding material shown diagrammatically in Figure 1 and Figure 2 wherein ligand L is lysozyme was carried out by dissolving lysozyme (l4.4mg, O.OOlmmol) and polyallylamine.HC1 (M_y 14,000) (lOO g-lmmol) in MOPS buffer (50mM, 5ml) and adjusting the pH of the solution to 4.5. A solution of EDC (19.2mg, O.lmmol) in water (1ml) was added and the reaction vessel was stirred gently at room temperature overnight with maintenance at pH4.5 throughout. The solution was then dialysed against MOPS buffer (30mM pH6.8. 3 x 100ml) before concentration (Amicon PM10 membrane) and purification by gel filtration (Sephacryl 200; elution with 30mM MOPS, lOOmM KC1, ImM EDTA, pH6.8). Fractions exhibiting UV absorption at 28θnm were combined, concentrated (Amicon PM3 membrane) and lyophilised to leave a white powdery solid (50mg).

This material can be used to form the beads of the invention by substitution for the polyallylamine reactants in each case.

Example 13: visualisation of process with SEM

A solution of 3-[N]-morpholino/propylsulphonic acid (MOPS) buffer (pH 7.8, 0.6N, 250ml) was purged with nitrogen for 10 minutes before addition of adipoyl chloride (1.2ml) in a mixed organic phase containing dibutyl ether (14.4ml), 1,6-hexanedioldiacrylate (14.4ml), and azobis (isobutyronitrile) (300mg). A suspension of bacteria (Listeria monocytogenes or Staphylococcus aureus. ethidium bromide stained, 4 x lOδcfu.ml^"1 ) . in MOPS buffer (50ml), was then added and stirring continued for 3 minutes before dropwise addition of^ poly(allylamine) solution (0.12N in 0.6N MOPS, pH 7-8, 75ml). The resultant microcapsules with attached bacteria were irradiated (36θnm) with stirring for 12 hours to generate solid beads which were filtered, washed and water (3 x 100ml), methanol (3 x 100ml) and air-dried. The beads (5-0g) were then stirred in 1,1.2-trichlorotrifluoroethane (250ml) as a solution of diisocyanato-terminated perfluoropolyether (F0MBLIN Z DIS0C 1.5g) in 1,1,2-trifluorotrichloroehane (20ml) was added dropwise. Stirring was continued for 3 hours before the reaction was terminated by addition of suspension to methanol (250 ml). After filtration and methanol washing (5 x lOOmL) , the beads were refluxed in 6M HCl/methanol (150ml) to remove the bacteria. "Development" of the exposed lithographic prints was effected by stirring the beads (lOOmg) in pH 4.75 sodium acetate buffer (5ml, 50mM, containing 5mM MnCl₂, 5mM CaCl₂, and ethanol (500ml)) with fluorescein isothiocyanate-labelled Concanavalin A (lmg, ~ O.OOOlmmol) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (5mg, 0.025mmol).

At each stage of the procedure the appearance and morphology of the polymers was monitored using a combination of Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM) (Figure 4). Plate la and lb show the initial polyamide microcapsules, containing a liquid organic core, with surface-bound Listeria monocytogenes and Staphylococcus aureus as representative rod-shaped and coccoidal bacteria respectively. The cells were labelled with the fluorescent dye ethidium bromide for this experiment. It is evident from the optical slices shown that the microorganisms were located on the outside of the polymer capsules, and the degree of surface coverage was easily controlled by variation of initial microorganism concentration and polymerization conditions. Photomicrographs of polymeric beads obtained after irradiation of the respective capsules are presented in Plates 2a and 2b. Examination of the surface by SEM showed a certain degree of variation with regard to position of the bacteria at the surface, with some cells almost completely buried in the outer layer of the polyamide and the majority only slightly embedded in the surface. After removal of the template microorganisms, the presence of deep indentations (100-200nm) was readily apparent in SEM micrographs (Plates 3a and 3b) . These "sites" exhibited exact size and shape complementarity to the bacteria. However, to demonstrate the success of our lithographic procedure it was necessary to "develop" the difference in chemical functionality between the now exposed sites and the perfluoropolymer modified surfaces. The beads were therefore reacted with a fluorescent labelled lectin (FITC-Concanavalin A) via l-ethyl-3-(3-dimethylamιnopropyl) carbodiimide (EDC) mediated coupling of free amino groups on the unmodified polymer surfaces with carboxyl residues on the lectin. This enabled us to achieve a chemical amplification of the site functionality, visualised by CLSM, but exactly the same procedure can be used for the introduction of a specific ligand (antibodies or lectins) for selective recognition of the template microorganism. The results of this experiment are shown in Plates 4a and 4b where the lithographic image of the cells on the polymeric surface is enhanced and developed through the use of the fluorescent dye.

This experiment clearly shows that it is possible to recreate the shape and size of whole cells employed as templates during multi-component polymer synthesis. The resultant polymeric beads exhibiting functionally anisotropic patches, of dimensions defined by the template, can be further modified to adjust the chemistry in the sites and/or to introduce any further recognition elements required for a particular use. The materials obtained are expected to find wide ranging applications in the biomedical field and environmental/food analysis where the rapid, efficient separation and recovery of cells, microorganisms and viruses is of paramount importance. Incorporation of, for example, ferrofluids in the organic core of these polymer particles will enable the exploitation of these materials in diagnostic applications where magnetic separations are already in widespread use.

Claims

CLAIMS ,

1. A specific binding material adapted to specifically bind with a target material characterised in that the specific binding material has areas upon its surface corresponding to the size and/or shape of the target material.

2. A specific binding material as claimed in claim 1 wherein the target is a biological target material.

3. A specific binding material as claimed in claim 1 wherein the target is a microorganism, antibody or antigen.

4. A specific binding material as claimed in any one of the preceding claims wherein the material surface has areas sized and shaped to accommodate a substantial part of the target material.

5. A specific binding material as claimed in any one of the preceding claims comprising a polymeric body on which areas corresponding in size and/or shape to the target have been formed.

6. A material as claimed in claim 5 wherein the areas have a high affinity for binding the target.

7- A material as claimed in claim 5 or 6 wherein the size and/or shape specific area of the material has functionalised species or have antibodies or antigens bound to it.

8. A material as claimed in claim 7 wherein the functionalised species include charge bearing groups.

9. A material as claimed in any one of the preceding claims wherein areas of the specific binding material surface that are not sized or shaped to correspond to the target material have been treated to reduce their ability to bind the target and/or any other material from a liquid phase sample from which it is being specifically selected. 10. A material as claimed in claim 9 wherein the treatment removes or mask the species on the binding material surface that cause the target and other materials to become bound.

11. A material as claimed in claim 19 wherein the species are reacted with an agent such as to attach a silyl or perfluoro group to the material surface.

12. A method for the preparation of a specific binding material as claimed in any one of the preceding claims comprising binding a target material to the surface of a material such that the surface of the material becomes adapted the size and/or shape of the target material, then removing the target material from that surface.

13- A method as claimed in claim 12 wherein the material is adapted such as to be capable of conforming or interacting with the shape or size of the target such that non-target materials not having the desired shape or being too large to fit into or onto the conformed area do not become bound or bind with decreased affinity.

14. A method as claimed in claim 12 or 13 wherein the adaptation is such that an imprint of the target is made in or on the surface of the binding material which is located and configured such to allow the target to access the binding surface.

15- A method as claimed in any one of claims 12 to 14 wherein the specific binding material is adapted to the shape and size of a target by forming a body of the specific binding material in the presence of the target material.

16. A method as claimed in claim 14 wherein the body is formed by polymerising monomers in the presence of the target at target physiological pH and temperature.

17- A method as claimed in claims 15 or 16 whetein the formation results in a mirroring of target surface features. 18. A method as claimed in any one of claims 15 to 17 wherein the polymerization is carried out by interfacial reactions using dispersed organic phase in an aqueous matrix.

19. A method as claimed in any one of claims 15 to 18 wherein the monomers are radiation cross-linked to produce the polymer.

20. A method as claimed in any one of claims 12 to 19 wherein the target material is removed from the formed material by subjecting the materials to a high vortex and/or use of acids or alkalis.

21. A specific binding material or method as claimed in any one of the preceding claims wherein the specific binding material is functionalised at its binding surface by functional groups.

22. A specific binding material or method as claimed in claim 18 wherein the functional groups are amino and/or hydroxy and/or carboxyl and/or amide groups.

23. A method as claimed in any one of claims 14 to 20 wherein a material capable of binding a target material is preformed in the absence of target material; the preformed material exposed to target material resulting in binding; and the two materials in bound form exposed to a further treatment whereby the surface of the specific binding material which is not covered by target material becomes wholly or partially inactivated with respect to its ability to bind target and non-target materials.

24. A specific binding material as claimed in any one of claim 1 to 13. 21 or 22 characterised in that it is in the form of a bead, capsule, strip, film, membrane or dipstick.

5. Use of a specific binding material as claimed in any one of claims 1 to 13. 21, 22 or 22 for the purpose of extracting specific target materials from a liquid sample. 26. Use as claimed in claim 25 wherein the specific target material is a microorganism.

27- A test kit characterised in that it comprises a specific binding material as claimed in any one of claims 1 to 13. 21, 22 or 23-