Coated Microspheres

Field of the invention

The present invention relates to microspheres that include a functional layer, more particularly, but not exclusively, to microspheres that include a coloured, a fluorescent, a radioactive or a magnetizable layer. Such microspheres are useful for incorporating into paints, for constructing microspheres that may be used in separation procedures, or for use in methods of treating disorders such as cancer. The invention also relates to methods for making such microspheres.

Background to the invention

Microspheres that include a layer that covers the core of the microsphere in order to provide the microsphere with a specific function are used in many fields of technology. The term 'microsphere' is used herein as a synonym for microcapsule, microparticle, microballoon, and microsphere particles.

For example, microspheres that include a functional layer comprising a magnetizable agent can be used as the solid phase in separation methods. The layer of magnetizable agents enables the microspheres to be selectively recovered (and so selectively recover any analyte bound to the microspheres) from the sample in which an analyte is present by manipulation in a magnetic field. Thus, magnets may be used to selectively recover the microspheres from a liquid sample comprising the analyte. Under the appropriate optimal conditions the recovery is quantitative.

Microspheres that include a functional layer comprising a radioactive substance can be used in methods for treating disorders such as proliferative disorders. By targeting the binding of microspheres comprising such a radioactive layer to, for example, cancer cells, the radioactive microsphere may be used to selectively kill cancer cells in the body of a patient.

Microspheres that include a functional layer comprising a coloured or fluorescent agent

can be used as the basis for the preparation of paints, as components of cosmetic formulations, as tracers, for example in flow-visualization, and in diagnostics.

Unfortunately, however, due to the nature of the substances that are normally used to provide a functional layer (such as magnetizable agents, coloured agents, fluorescent agents and radioactive agents), these layers are poorly retained by the core of the microspheres. It has been found that such functional layers are easily stripped from microsphere cores by the physical forces that are exerted on the microspheres when in use.

For example, the forces exerted on microspheres as they are propelled from the nozzle of a paint spray gun are sufficiently high so as to strip any layer of coloured or fluorescent agent that may cover the core of the microspheres. The loss of such layers results in a deterioration, or eradication, of the colour delivered by the paint to the surface being painted.

It is also important for the structure of a microsphere that includes a functional layer comprising a radioactive agent or a magnetizable agent to be particularly robust. Any weakness in the attachment of a magnetizable layer or radioactive layer to the core of a microsphere seriously adversely affects the ability for such a microsphere to act as an effective solid phase in a separation method or as a therapeutic.

As well as attaching a functional layer to the core of microspheres for use in a separation procedure or a method of treatment, such microspheres may be modified so as to selectively bind to a selected analyte (e.g. by binding, to the surface of the microspheres, an agent with a high level of affinity for the selected analyte). It has, however, been found that after covering a microsphere with a layer of a functional agent it is difficult to modify the microsphere so that it selectively binds to a selected analyte, e.g. by binding an agent with a high level of affinity for the selected analyte to the surface of the coated microsphere. Previous attempts to bind agents with a high level of affinity for the analyte

to a coated microsphere have resulted in a low density of such agents deposited on the surface of the microsphere. Additionally, only a poor level of attachment of the functional agent to the core is achieved so that the functional layer may be easily ripped from the microsphere. In the case of microspheres with a functional layer comprising a radioactive agent, detachment of the radioactive agent from the microsphere can result in serious side-effects for the patient being treated. When detached from the microsphere, the radioactive agent will no longer selectively attack, for example, cancer cells. In the case of microspheres with a functional layer comprising a magnetizable agent, the detachment of the magnetizable layer would render it difficult to manipulate the microspheres using magnetic fields or to retain any agents that are attached to the microsphere via the magnetizable agents.

The importance of the robustness of construction of microspheres used in separation methods will be appreciated from a consideration of the physical conditions to which the microspheres may be subjected to in, for example, routine diagnostic applications. This point can be illustrated by reference to the following example of Cryptosporidium parvum oocyst recovery.

Microspheres made of borosilicate glass and with a buoyant density lower than that of water, when covered with an antibody that recognizes a Cryptosporidium parvum oocyst surface antigen, may be used for selectively extracting Cryptosporidium parvum oocysts from a sample. Cryptosporidium parvum is a recognized parasite of humans. The oocyst, radius 5 μm, is a massive ligand and the implication of this from simple physical considerations, is that (antibody mediated) binding of an oocyst to the surface of a microsphere places a very high stress loading on the surface. Indeed, if the magnetizable agent/ antibody layer is not sufficiently robustly assembled then the applied torque is sufficient to tear the magnetizable agent/antibody layer from the surface of the underlying glass microsphere. Indeed, it has been found that use of such ill-formed microspheres under the conditions of a typical Cryptosporidium oocyst detection assay, results in considerable loss of the magnetizable agent /antibody material (plus associated oocysts) from the microsphere, where it will be seen as a pellet of debris at the bottom of the

sample tube, separated from the buoyant microspheres cores. It is then impossible to either separate the oocysts from the pellet, or attempt to detect them from among the pellet.

Accordingly, it is an object of the present invention to provide a more robust microsphere that is able to be used without any or limited damage to the functional layer of the microsphere. It is an additional object of the present invention to provide a method for making such microspheres.

Summary of the invention

The present invention describes novel microspheres that comprise a functional layer, and novel methods for making such coated microspheres, which are sufficiently robust to, for example, enable them to be manipulated in a magnetic field (when a functional layer of a magnetizable agent is incorporated into the microsphere), be injected into a body (when a functional layer of a radioactive agent is incorporated in to the microsphere), be used in a separation procedures (when a layer of an effector molecule, such as an antibody, is incorporated into the microsphere), or be sprayed from a paint gun (when a functional layer of a coloured or fluorescent agent is incorporated into the microsphere), without damage to the integrity of the microsphere structure.

The key to the robust nature of the microspheres of the present invention is that they include a solid oxide coating. This solid oxide coating is capable of retaining materials that are provided as intermediate layers between the solid oxide coating and the core, and of acting as a structure through which subsequent "layers" (such as a layer comprising an effector agent such as an antibody) may be bound firmly to the microsphere. Thus, the forces exerted on the functional layers by, for example, a magnetic field, or the forces exerted on any other agents bound to the core via the solid oxide coating(i.e. effector agents such as antibodies), are not able to strip the functional layer from the core.

Thus, according to the first aspect of the present invention, there is provided a microsphere comprising a core, a functional layer and a solid oxide coating, wherein the functional layer forms an intermediate layer between the core and the solid oxide coating.

It is a preferred embodiment of the present invention that the solid oxide coating is substantially continuous over the surface of the microsphere and thus forming a structure whose integrity is not-dependent upon an anchorage to the underlying layer. Thus the oxide coating is conceived as an enclosing procedure to seal in one or more underlying components, comprising the functional layer that may merely cover the surface of the microsphere core and may not be chemically bonded to it. This invention therefore finds particular application for enclosing functional agents where chemical bonding would substantially change their properties in an undesirable manner. For example, chemical attachment of pigment molecules to a surface may change their UV-visible absorption spectra, and hence unfavourably change, or eliminate the colour properties of said pigment. The solid oxide coating may, therefore, act as a retaining layer.

The solid oxide coating is preferably less than 25, 20, 15, or 5 μm thick.

However, in a preferred embodiment of the current invention, ultrathin coatings are applied, that is, coatings that are no more that 100 nm thick, and may be as little as 0.2 nm thick. Such coatings may be applied either by slow continuous addition, or by batchwise addition in a single cycle, as described later.

It has been found that these relatively thin layers of solid oxide coating are still able to provide a substantially continuous coating of the microsphere such that the functional layer is retained below the solid oxide coating and is shielded from adverse reactions with substances in the environment in which the microsphere is placed. Surprisingly, such thin layers have the advantage that they present an increased surface area than microspheres constructed with a relatively thick solid oxide coating. Without wishing to be bound by theory, but in the interests of clarity, it would appear that functional layers, e.g. of superparamagnetic oxide nanoparticles (diameter < 100 nm) tend to produce a

rough finish to the microspheres, and when these layers are coated with a relatively thick layer of solid oxide (> 100 nm) the valleys and troughs that define this rough outer surface become buried within the smooth oxide coating. However, a thin layer of solid oxide coating retains the valleys and troughs established by the functional layer on the outer surface of the coated microsphere, thereby providing a large external surface area. Such a large surface area is beneficial. For example, a large surface area means that one can bind more ligands to the outer surface of the microspheres. A further advantage is that a thin, rather than thick, solid oxide layer will have less of an effect on the perceived intensity or colour of any functional layer.

Thus, in a preferred example of the present invention, the sold oxide coating may be less than lOOnm thick, preferably from 0.2 to 100, more preferably from 0.2 to 50 nm thick. Such thicknesses correspond roughly to between 1 complete and 100 complete molecular layers and correspond to between roughly 0.05% and 5% the mass of the final coated microsphere.

The functional layer may be directly associated with the core and/or the solid oxide coating. Alternatively, intermediate layers may exist between the core and the functional layer and/or between the functional layer and the solid oxide coating.

Where the precursor has an affinity for the functional layer (e.g. in the case of TEOS and magnetite) a priming layer may not be required. However, particularly where there is no or little affinity between the precursor, and the functional layer (e.g. in the case of TEOS and smooth silver-coated microspheres) it has been found that optimisation of the quality of the solid oxide coating (defined in terms of percentage surface area covering and thickness of deposited coating) may be achieved if the microsphere surface is first primed to accept the solid oxide coating. Accordingly, it is an embodiment of the present invention that the microsphere further comprises a priming layer, wherein the priming layer forms an intermediate layer between the solid oxide coating and the functional layer. The priming layer and the solid oxide coating may be directly associated with each other. The priming layer may be chemically attached to the functional layer via a

coupling agent, or it may simply be a coating, held in place by weak electrostatic and/or hydrophobic interactions.

The inventors have unexpectedly found that certain priming agents, such as gelatine, facilitate the dispersion of particles in the solvent for the subsequent solid oxide coating step. This is important because it prevents aggregation, and hence the formation of polymeric solid oxide coated particles, especially where that particle to be coated is substantially hydrophobic.

The microsphere may further comprise an anchoring agent, wherein the functional layer is anchored to the core by binding to the anchoring agent.

Because of the nature of the substances that are useful for constructing functional layers (i.e. magnetizable agents such as elemental nickel, or a pigment compound), functional layers are not usually able to chemically bind to materials conventionally used for making the core of microspheres, for example an inorganic material such as borosilicate glass or an inert polymer such as PVC. However, it has been found that by providing an anchoring agent as an intermediate between the core and the functional layer it is possible to chemically bind the functional layer to the core via this intermediate, thereby providing a more secure attachment than could be achieved by simply physically coating the core with a functional layer.

The anchoring agent may comprise a functional-layer binding agent (e.g. a protein such as gelatine) which binds to the functional layer. Depending on the material used to make the core and the choice of functional-layer binding agent, the functional-layer binding agent may be able to bind directly to the core. However, the functional-layer binding agent may be bound to the core by a coupling agent (e.g. a silane). Therefore, in one embodiment of the present invention, the anchoring agent further comprises a coupling agent which couples the functional -layer binding agent to the core.

The functional layer may bind directly to the functional-layer binding agent.

Alternatively, the functional layer can be coupled to the functional-layer binding agent by a coupling agent (such as a silane).

The microspheres of the present invention may comprise further layers that are external to the solid oxide coating. For example, microspheres that are prepared for use in a separation procedure can be adapted in order to capture a specific analyte, or microspheres that are prepared for treating a disorder can be adapted in order to bind to a specific target (i.e. a cancer cell), by binding an effector agent to the outer surface of the microsphere (e.g. an agent that has specific affinity for the analyte or target). Additionally, microspheres that are prepared for paint may include a coloured agent attached to the external surface. Such microspheres may be used as the basis for colour change paints; the externally attached colour agent may, over time, be removed from the microsphere by environmental factors leaving the microspheres to reflect only, for example, the colour of a colour agent (i.e. functional layer) trapped beneath the solid oxide coating.

Thus, in one embodiment of the present invention the microspheres comprise an effector agent, wherein the solid oxide coating forms an intermediate layer between the effector agent and the core. The effector agent may be directly associated with the solid oxide coating, or may be coupled to the solid oxide coating by a coupling agent (e.g. a silane). Alternatively, one or more layers may be interposed between the effector agent and the solid oxide coating.

Microspheres that have as their outer layer a coating of a solid oxide are also of use in a number of applications. For example, when the functional layer is a coloured or fluorescent layer trapped beneath the solid oxide coating, the microspheres may be useful for incorporating into paints and may not require any layer that is external to the solid oxide coating. It is also useful to produce microspheres for use in a separation protocol that have not been adapted in order to capture a specific analyte. Providing such "blank" microspheres gives the skilled person the ability to bind the appropriate effector agent (e.g. by the methods described below) for capturing the target analyte in any specific

separation protocol. With such applications in mind, the continuous or near continuous oxide coating confers the additional benefit of acting as a robust substratum for the incorporation of an appropriate effector layer. Methods for binding effector molecules to an oxide surfaces are well-described in the prior-art and can be straightforwardly applied to the present structure by those skilled in the art.

As mentioned above, functional layers applied to the core of a microsphere can easily be stripped from the core by various physical forces that are exerted on the microspheres when in use. However, it has been found that it is possible to construct microspheres that do not suffer from this problem by applying a coating of a solid oxide to the microspheres after the functional layer has been deposited on the core.

Thus, according to a second aspect of the present invention there is provided a method for preparing a microsphere comprising the steps of depositing a functional layer on a microsphere core prior to coating the microsphere with a solid oxide.

Any method of depositing a functional layer on a core would be appropriate for use in the method of the present invention. For example, the functional layer may be deposited by electrolytic or electroless deposition, by chemical precipitation, by chemical bonding, by spray-drying or by vapour phase coating.

Any method of applying the solid oxide coating would be appropriate for use in the method of the present invention. For example, the solid oxide coating may be applied by chemical precipitation. Preferably, the solid oxide coating is laid down by a sol-gel method. In such methods a chemical precursor or mixture of chemical precursors of the solid oxide are hydrolysed and so pass sequentially through a solution state and gel state before being dehydrated to a glass or ceramic. The solid oxide may be precipitated from an appropriate precursor of the solid oxide using the sol-gel approach. Methods for laying down of solid oxide coatings are known in the prior art. For example International Publication No. WO 98/12717 (to Merck) discloses one method for coating particles with

a solid silica layer, and US 61 10 528 to Kimura similarly discloses one method for coating particles with titanium dioxide (titania) layer.

For example, layers of silica can be grown by mixing a silica precursor such as tetraethoxyorthosilane (TEOS) in an appropriate solvent. Under optimal conditions the precursor migrates to the particle surface and polymerises to form a hydrated silica coat. The recovered particles are then heat-cured to form the final anhydrous silica shell. The exact conditions of the initial coating reaction are selected to ensure that the precursor migrates to the surface of the microsphere, and to minimise self-aggregation in the solvent, which gives rise to unwanted amorphous silica particles. Variables in the optimization procedure include the exact solvent composition and the temperature and the rate of addition of the precursor (from an external reservoir, in an appropriate solvent). Both the reservoir concentration of the precursor and the rate of addition can be manipulated to control the growth process. In general it is found that lower rates of addition, and lower concentrations favour growth of stable, smooth silica layers, whilst high concentrations and rates favour "chaotic" growth, leading to rough, uneven surfaces.

As an alternative to continuous addition, the precursor may be added batchwise in order to build up a shell of any desired final thickness.

The step of applying the solid oxide coating is preferably controlled in order to deposit a coating of pre-determined thickness (e.g. the preferred thicknesses described above). For example, control of the amount of solid oxide precursor that is added during the step of coating the microsphere in a solid oxide coating will control the thickness of that coating.

The precursor is preferably a low molecular-weight precursor of the solid oxide. For the avoidance of doubt a low molecular-weight molecule is one that has a molecular weight of 1000 amu or less. The skilled person would be aware of the appropriate precursors that could be used to lay down any chosen solid oxide coating. For example, if one is to lay down a silica coating then an alkoxysilane such as tetraethoxyorthosilane would be an

appropriate precursor. When laying down a titania coating, an alkoxytitanate such as tetraethoxyorthotitanate would be an appropriate precursor.

The coating of the microsphere with solid oxide is preferably carried out by adding an oxide precursor to a mixture of microspheres and a hardener in a solvent system. The hardener, otherwise known as an accelerator, can be, for example, ammonia, hydrochloric acid, acetic acid, ammonium carbonate, triethanolamine, calcium hydroxide, magnesium oxide, dicyclohexylamine, ammonium acetate, tributyltin, or any combination thereof. The solvent system preferably comprises a water-miscible organic solvent (e.g., isopropanol, methanol, ethanol, butanol, or any combination thereof). Optionally, the solvent system also includes water.

In a preferred embodiment, the solid oxide precursor is tetraethoxysilane, the organic solvent is propan-2-ol, the hardener is ammonia, and water is present.

Where the intention is to optimise the coating of a surface, conditions where deposition of the newly formed solid oxide coating onto the microsphere surface is preferred over the alternative, which is the formation of a separate fraction of amorphous solid oxide. It has been found that the quality of the coating, defined in terms of percentage surface area covering and thickness of deposited layer can be optimised by the choice of reaction conditions. For example:-

When the hardener is ammonia, the ammonia concentration may be in the range from 3.5 to 5.5 mol per litre of reactant mixture. Preferably, the ammonia concentration is not less than 3 mol per litre of reactant mixture

The water concentration may be in the range from 10 to 14 mol per litre of reactant mixture. Preferably, the water concentration is not less than 8 mol per litre of reactant mixture.

The concentration of microspheres may be in the range from 20 to 500 grams per litre of reactant mixture.

Preferably the precipitation reaction is carried out at a temperature in the range from 30 to 50 ⁰ C. It is also preferred that the reactant mixture is stirred at a rate that keeps the microspheres well dispersed. The precursor may be added continuously. Alternatively it can be added batchwise for example at 10 - 60 minute intervals). Typically, the precursor (e.g. TEOS) is added to the reaction mixture in from 0.84 - 450 mg per g of microsphere in order to achieve a coating of 0.2-100nm solid oxide, and from 450mg - 9Og per g of microsphere in order to achieve a solid oxide coating of from 0.1 to lOμm solid oxide coating.

The exact conditions of the reaction, as elaborated in the Examples, have the advantage that they permit the coating process to proceed rapidly, with almost complete incorporation (> 99%) of the solid oxide onto the surface layer, and no significant formation of unwanted amorphous silica.

It has been found that the ability to provide a continuous layer of solid oxide that, for example, can act as a barrier to leaching of the functional layer by an acid, can be optimised by a final curing step in the process of making microspheres. Thus, preferably the method according to the second aspect of the present invention includes a curing step. A curing step would involve ageing the solid oxide coated microspheres after the (at this stage, hydrous) solid oxide coating has been added for a period of time with the application of heat. The curing step may takes place over 2 to 24 hours, preferably more than 6, 8, 10 or 12 hours, and most preferably about 15 hours. The curing step may be carried out at a temperature of 40 - 800 ⁰ C, preferably at a temperature of over 60 ⁰ C, 80 ⁰ C, 100 ⁰ C and most preferably at from 12O ⁰ C to 150 ⁰ C. This curing step may be carried out under vacuum. Additionally, a wash step may follow the curing step.

As mentioned above, it has been found that the total surface area coated by the solid oxide and the ability for the solid oxide coating to retain layers of material that are

trapped between the solid oxide coating and the core may be optimised by the provision of a priming layer.

Accordingly, in one embodiment of the present invention the method further comprises the step of applying a priming layer to the microsphere as an intermediate step between the steps of depositing a functional layer and coating with a solid oxide. It is known how to coat particle surfaces with functional agents such as proteins. For example, Harrison et al (J. Coll. Interface Sci 217 203-207 [1999])disclose a method for coating iron particles with gelatine by refluxing the particles for 2h in an aqueous gelatine solution. However it has been surprisingly found that such extreme conditions are not in general necessary, and that good coating of, for example glass microspheres, can be obtained simply by stirring them in 1% aqueous gelatine at room temperature for 1 hour. Furthermore, by precisely controlling the ratio of gelatine (by mass) to particles (by total surface area), the amount of loosely bound (as opposed to very tightly bound) gelatin can be minimised, and thus, straightforwardly removed by a facile washing procedure. In the case of hollow microspheres, for example, with a median diameter of 27 μm, gelatin binding as a function of added gelatin concentration follows a standard rectangular-hyperbolic saturation curve, with optimum ratio of 1.2 mg gelatine per g microspheres.

As mentioned above, due to the nature of substances that are used to make a functional layer, functional layers are not usually able to chemically bind to materials conventionally used for making microsphere cores. It has, however, been found that chemically modifying the surface of the core allows the functional layer to chemically bind to the core produces a more robust attachment.

Thus, according to an embodiment of the present invention, the method further comprises the step of chemically modifying the surface of a core prior to the step of depositing the functional layer onto the core.

The chemical modification may comprise the step of anchoring a functional-layer binding agent to the core. Depending on the material used to make the core and the choice of

functional-layer binding agent, the functional-layer binding agent may be able to bind directly to the core. However, the functional-layer binding agent may need to be bound to the core by a coupling agent. Therefore, in a preferred embodiment of the present invention, the step of anchoring of the functional-layer binding agent comprises the step of binding a coupling agent to the core. The step of anchoring the functional-layer binding agent may comprise the step of binding the functional-layer binding agent to the coupling agent. The coupling agent may be bound to the core prior to being bound to the functional-layer binding agent. Alternatively, the coupling agent may be bound to the functional-layer binding agent prior to being bound to the core.

The functional agent may bind directly to the functional-layer binding agent. Alternatively, the functional agent can be coupled to the functional-layer binding agent by a coupling agent. Thus, in a further embodiment of the present application, the step of anchoring the functional agent to the core further comprises the step of coupling the functional agent to the functional-layer binding agent by a coupling agent. The coupling agent may be bound to the functional agent prior to being bound to the functional-layer binding agent. Alternatively, the coupling agent may be bound to the functional-layer binding agent prior to being bound to the functional agent.

The structure of the microsphere may be strengthened further by coupling the functional layer to the priming layer by a coupling agent. In this embodiment, the coupling agent may be bound first to the functional layer or priming layer.

The methods of the present invention may have as their final step the coating of the solid oxide. However, the methods of the present invention may also comprise further steps in order to add layers that are external to the solid oxide coating.

Thus, in one embodiment of the present invention the method comprises the step of anchoring an effector agent to the solid oxide coating. This step may be carried out after the step of coating the microsphere with the solid oxide. The step of anchoring the effector agent may comprise the step of coupling the effector agent to the oxide coating

by a coupling agent. The coupling agent may be bound first to the solid oxide coating or first to the effector agent.

The above described methods allow for total control at each step in the manufacturing process as it relies upon the stepwise and sequential construction upon the surface of the microsphere. The stepwise nature of the construction permits quantitative assessment of the quality of each layer by an appropriate chemical analysis (e.g. protein content; amine- content; iron content) and / or physical analysis (e.g. magnetic moment) following each completed step.

In a preferred embodiment of both aspects of the present invention described above the core may be made of any material or combination of materials used in the manufacture of conventional microspheres that are used in the aforementioned procedures (e.g. separation protocols). The core may be made of an inorganic material. Preferably, the core comprises or consists of any of the following: a synthetic polymer, a natural polymer, a co-polymer, a block co-polymer, polymethylmethacrylic acid, a protein, a glass, a ceramic, a ceramic oxide, or any combination thereof. When the core is made of a co-polymer, the co-polymer may comprise polymethylmethacrylic acid. In one embodiment, the core is a borosilicate glass microsphere. The core may be buoyant, and preferably hollow. Preferably, the core does not comprise or consist of a magnetizable agent. Porous cores are not preferred as these "honeycomb" like structures may permit a solvent to access the internal cavity of the core. What is preferred is a core that comprises an outer shell defining an internal void when the internal void is filled with a gas that is retained within the outer shell. The internal void may have a pressure that is less than one atmosphere.

The applicant has found that not only are buoyant microspheres most useful as the solid phase and separation technologies (as they float to the top of a liquid medium along with a bound ligand, thereby permitting easy extraction from the surface of the liquid using a magnet that is passed over the surface). The methods of constructing the microsphere according to the present invention are far simpler and cheaper to carry out when the

microsphere is a buoyant microsphere. Buoyant microspheres can very easily be retrieved from each step of the step-wise methods described above and are introduced to the next step of the method when buoyant microspheres are used. It is surprising that, given the density of magnetizable agents, microspheres can be produced that are both capable of being removed from a liquid surface by a reasonably sized magnet and of also being buoyant.

For the avoidance of doubt, a buoyant microsphere is one that is less dense than, or in some cases isodense with, the liquid medium in which it is being suspended. Buoyant microspheres float to the surface of the liquid medium in which they are suspended. Isodense microspheres may be suspended in a liquid medium. The skilled person would understand that, by virtue of the conditions of the manufacturing process, smaller particles tend to have higher buoyant densities than do larger. For example, microspheres with a medium diameter of 27 micrometers and a final density of approximately 0.5 g/cm ³ . On the other hand, a smaller base microsphere (diameter 18 micrometers) is much denser, and the final coating microsphere has a density of 0.6 g/cm ^J . Smaller microspheres have the advantage, vide supere, that fewer need be added to a sample to provide any given total surface area. Exact choice of size and density of a specific application is one which is made on a case-by-case basis. Not wishing to restrict it further, but in the interest of clarity, it has been found that the preferred microsphere of the present invention of a buoyant density of less than 0.9 g/cm ³ , preferably from 0.05 to 0.85, from 0.2 to 0.8, from 0.3 to 0.7 g/cm ³ . The buoyant density of the microsphere may be 0.6 g/cm ³ . The microsphere may be hollow. Final coated microspheres may be hydrophilic and therefore mix well with water and other polar liquids and liquid mixtures. The microspheres may be stable in solvents over a wide range of temperatures and pH conditions.

A preferred core for the current invention is a hollow silica microsphere. Such microspheres, which are commercially available, will be more than lμm in diameter. As the core forms the greater part of the microspheres of the present invention, it will also have a diameter of greater than lμm. The applicant has found that it is important to use

microspheres and cores of such dimensions. Smaller cores, for example, are disproportionately small compared to ligands one may wish to bind the microsphere too, or present too small a surface area to be useful for paints. In a further preferred embodiment the core has a diameter that is less than 1000, 500, 100, 50, or 10 μm. The diameter of the core is preferably greater than 1, 5, 10 or 15, μm, and most preferably 5 - 20 μm, in diameter. The core is preferably roughly spherical.

In a preferred embodiment of both aspects of the invention described above the coupling agent is any agent that is capable of binding an inorganic molecule to an organic molecule. More specifically, the coupling agent may be any agent that is capable of binding a glass, iron oxide, spinel ferrite to an organic molecule. The coupling agent may be a silane, a germane, or a combination thereof. The silane may be any of the following; an amino silane, a carbonyl silane, a carboxy silane, a hydroxyphenyl silane, a sulfhydryl silane, 3-aminopropyltrimethoxysilane, or any combination thereof.

In a further preferred embodiment of both aspects of the invention described above the functional-layer binding agent comprises or consists of any of the following; a synthetic polymer, polymethylmethacrylic acid, a co-polymer, a block co-polymer, a natural polymer, a protein, a peptide, an amino acid, or any combination thereof. Essentially, the functional-layer binding agent is preferably an agent, that includes an active group such as -NH ₂ , -C=O, -COOH, -OH, or any combination thereof. Preferably, the functional- layer binding agent is a protein such as a serum albumin (e.g. bovine serum albumin, human serum albumin, chick serum albumin) or gelatine, or collagen. Alternatively a man-made polymer, such as polyvinylalcohol, may be used.

The skilled person would understand that any agent that could confer a useful function on a microsphere once that agent is coated on the microsphere would be useful for incorporating into the functional layer of the present invention. Although a number of specific examples have been provided (e.g. magnetizable agents, coloured agents, fluorescent agents, radioactive agents, fire-retardents) it should be understood by the above discussion that the invention could be realised using a wide array of functional

layers that would be known to the person skilled in the art and so should not be limited to any one of the specific examples provided.

In a preferred embodiment of both aspects of the invention described above the functional layer may comprise or consist of any agent that is capable of being manipulated by a magnetic field, and so is made of a magnetizable agent. Preferably, the magnetizable agents exhibit magnetic behaviour only whilst being exposed to a magnetic field, and are not permanent magnets. Thus, the microspheres do not aggregate together when a magnetic field is not present. Induced permanent magnetism following the application and then removal of an externally applied magnetic field to microspheres in suspension (e.g. in a liquid sample) may lead to a magnetic agglomeration of the microspheres. This is undesirable in a diagnostic environment where a thoroughly dispersed solid phase offering high surface area is essential for rapid and efficient (quantitative) binding of the analyte. Preferably, therefore, these agents are superparamagnetic, rather than simply paramagnetic for avoidance of doubt a superparamagnetic agent is one that becomes strongly magnetized in the presence of the magnetic field, but loses that induced magnetism entirely when the applied field is withdrawn. By contrast a paramagnetic agent is one that is likely to retain a degree of residual magnetism after the magnetic field is withdrawn.

When an agent is paramagnetic or superparamagnetic chiefly depends on the size of the individual particles, where smaller particles that is, those most closely constituting a single magnetic domain, will be superparamagnetic and larger particles (constituting an assembly of many magnetic domains) will be paramagnetic. Typically, a superparamagnetic particle, that is constituting a single magnetic domain, will have a diameter of less than 100 ran, and typically 10 - 30 nm. The magnetite (magnetic iron oxide particles used in Example 8 have a median diameter of 30 μm, for example.

The magnetizable agents may comprise any of the following; a paramagnetic agent, a superparamagnetic agent, iron, nickel, iron oxide, a spinel ferrite, an alloy, or any combination thereof. For the avoidance of doubt, a spinal ferrite can have the generic

formula MFe ₂ O ₄ , where M is a metallic element including, but not limited to, any of Co ₅ Mg, Mn and Zn, or any combination thereof.

It is preferred that magnetizable agents are in the form of nanoparticles. Accordingly, the magnetizable agents preferably have a diameter of less than 300nm, 200nm, lOOnm, 50nm or 30nm. Magnetizable agents may have a diameter from 10 to 100, from 15 to 70, or from 20 to 50nm.

Preferably the magnetizable agent coated microspheres have magnetic moments that are sufficiently high that the application of a magnet is sufficient to remove the microspheres from the surface of an aqueous sample, or to draw them quantitatively from suspensions to the side of sample tube under conditions of agitation.

It has surprisingly been found that preparing the magnetizable agents using a high-shear mixing process, preferably wherein the magnetizable agent is mixed in an aqueous medium in a high-shear mixing machine, is particularly preferred. Magnetizable agents prepared in this way exhibit a higher level of retention on the final constructed microsphere than other forms of magnetizable agents, e.g. magnetizable agents formed by sonication. The applicant has found that mixing high-shear magnetite with bovine serum albumen (BSA)-coated microspheres (prepared, for example, in accordance with the example 8) yields dark, highly magnetizable and superparamagnetic particles, indicative of a high retention of magnetite particles on the surface. On the other hand, magnetite pre-treated by sonication gives a very sparsely coated, and therefore weakly magnetizable microsphere, indicative of a low retention of magnetite in the final particle. Therefore, in a preferred embodiment of the first aspect of the present invention, the magnetizable agent is a high-sheared magnetite. In a preferred embodiment in the second aspect of the present invention the method includes a pre-treatment step for the magnetizable agent, prior to introducing the magnetizable agent to the microspheres, that it includes the high- shear mixing of the magnetizable agent. An alternative manner of producing a magnetizable agent that has also been found to be superior to sonication is by

precipitation from an ammoniacal Fe(II)/Fe(III)solution. Thus, the pre-treatment step may instead include the aforementioned precipitation step.

The steps of binding the magnetizable agent to the microsphere preferably do not involve the application of heat (i.e. by a furnacing step).

Alternatively, the functional layer may comprise or consist of one or more coloured or fluorescent agents. Such agents may be conventional coloured or fluorescent agents (for example, pigments, paints, inks, preferably with a low molecular-weight i.e. 1000 amu or less), polymeric organic pigments, including lakes, inorganic substances such as transition metal salts, such as chromium dioxide or ammonium polyphosphate, organometallic substances such as a zinc porphyrin, or any combination thereof.

Alternatively, the functional layer comprises or consists of one or more radioactive agent. The radioactive agents may be made from any radioactive substance that is suitable for killing cells. For example, the radioactive agent may comprise iodine-125, iodine-131, palladium, radium, iridium, or cesium.

It is preferred that the functional layer comprises functional agents that are in the form of nanoparticles. Accordingly, the functional agents used to make the functional layer preferably have a diameter of less than 300nm, 200nm, lOOnm, 50nm or 10nm. The functional agents may have a diameter from 10 to 100, from 15 to 70, or from 20 to 50 ran. It is also preferred that the formation of the layer using nanoparticles produces a rough textured surface on the microsphere. As has been discussed above, the rough textured surface may increase the surface area of the microsphere and so can optimise the deposition and retention of any further layers on the microsphere. The functional agents are preferably roughly spherical.

In a preferred embodiment of both aspects of the present invention the priming layer may comprise or consist of any of the following; a synthetic polymer, polymethylmethacrylic acid, a co-polymer, a block co-polymer, a natural polymer, a protein, a peptide, an amino

acid, or any combination thereof. Essentially, the priming layer should be an agent that includes an active group such as -NH ₂ , -C=O, -COOH, -OH, or any combination thereof. Preferably, the functional-layer binding agent is a serum albumin (e.g. bovine serum albumin, human serum albumin, chick serum albumin) or gelatine.

For the avoidance of doubt, the term "solid", as used in the term "solid oxide", should be understood to mean that the oxide is in a solid state at the temperature and pressure at which the microspheres are used. Preferably, the microspheres are used at Standard Ambient Temperature and Pressure, and so the oxide coating would be solid at 25°C and at a pressure of lOOkPa. In a preferred embodiment of both aspects of the present invention the solid oxide is an amorphous glass or ceramic coating. The skilled person would understand which oxides are capable of forming amorphous glass or ceramic coatings. However, in the interests of clarity, but not wishing to be restricted further, the solid oxide may be any of the following: silica, titania, zirconia, alumina, magnesia, or any combination thereof. In a preferred embodiment, the coating is polymeric silicon dioxide (silica) and silica particles and coatings are conveniently generated by the hydrolysis of an alkoxysilane such as tetraethoxysilane.

The solid oxide laid down in the above methods and coated on the above microspheres preferably forms a substantially continuous coating over the surface of the microsphere. ^• The solid oxide may coat from, 70-100, 80-100, 90-100, 95-100, or 100% of the surface area of the microspheres, preferably 100%. The percentage coating of a microsphere may be determined by, for example, microscopy..

It has been surprisingly found that it is possible to apply substantially continuous coating of the solid oxide over the surface of the microsphere according to the present invention such that external environmental forces cannot interact with the enclosed functional layer. For example, the silica coated protects the underlying functional layer from acid-attack, as described in example 9 below.

In a further preferred embodiment of both aspects of the present invention described above the effector agent is an affinity binding agent. Affinity binding agents have a binding affinity for a selected target, e.g. an analyte, for example a specific molecule or cell. Accordingly, the effector agent can be one partner of any binding partnership known to the skilled person, where the other partner is associated with or is the target. Not wishing to be limited further, but in the interests of clarity, the effector agent may comprise any of the following; a protein, an antibody, a lectin, an enzyme, a polypeptide, a nucleotide, a polynucleotide, a polysaccharide, a metal-ion sequestering agent, biotin, avidin, or any combination thereof.

It has been found that the microspheres according to the first aspect of the present invention, and the microspheres prepared according to the method of the second aspect of the present invention can include a surprisingly large amount of functional layer, and effector agent when compared to the amounts that would have been possible if the microspheres were constructed in accordance with prior art methods.

Thus, in a further preferred embodiment of both aspects of the invention described above the microspheres comprise no less than 0.1, 1, 2, 3, 4, 5, 6, or 7 mg of protein per gram of microsphere. Preferably, the microspheres comprise from 0.1 to 30, from 6 to 24, from 10 to 20, or from 12 to 24 mg protein per gram of microsphere. Following binding of the functional-layer binding agent to the microsphere, but prior to binding the functional layer, the microsphere preferably comprises at least 0.5, 1, 2, 3, 4 mg of protein per gram of microsphere, more preferably from 4 to 15, from 4 to 10, from 4 to 7 mg protein per gram of microsphere.

Although not wishing to be bound by theory, it is suggested that the ability for the microspheres to incorporate such large amounts of protein is as a result of the robust nature of the construction of the microspheres. Any "layer" of construction of the microspheres is preferably bound to the next by a chemical bond. The chemical bond is preferably a covalent bond, an electrostatic bond, hydrophobic interaction, or any combination thereof.

In a preferred embodiment of both aspects of the invention described above the microsphere may comprise at least 10, 20, 40, 80, 100 mg of magnetizable agent per gram of microsphere, more preferably from 100 to 500, from 150 to 400, or from 200 to 300 mg of magnetizable agent per gram of microsphere.

In a preferred embodiment of both aspects of the invention described above the microsphere is a buoyant microsphere. The microsphere may have a buoyant density from 0.05 to 0.99, from 0.2 to 0.8, from 0.3 to 0.7 g/cm ³ . The buoyant density of the microsphere may be 0.6 g/cm ³ . The buoyant density of the core may be 0.3 g/cm ³ . The final microspheres may be hydrophilic and therefore mix well with water and other polar liquids and liquid mixtures. They may be stable indefinitely in such solvents over a wide range of temperature and pH conditions.

In a further aspect of the present invention, there is provided a composition for use as a paint comprising any of the aforementioned microspheres and one or more binder. The binder may be any binder known in the art and preferably one that is capable of acting as a vehicle for the microspheres and eventually solidifies to form the dried paint film.

Examples of suitable binders are synthetic or natural resins such as acrylics, polyurethanes, polyesters, melamines, oils, latex, or any combination thereof. The composition may also comprise a diluent and/or an additive. The diluent is used in order to adjust the viscosity of the paint and may, for example, include water or an organic solvent (such as alcohols, ketones, esters, glycol ethers, or any combination thereof).

Additives may include pigments or colouring agents, catalysts, thickeners, stabilizers, emulsifiers, texturizers, adhesion promoters, flatteners, or any combination thereof.

In further aspect of the present invention, there is provided a microsphere prepared according to any of the above described methods.

In a further aspect of the present invention, there is provided a microsphere according to the first aspect of the present invention that is prepared according to the methods of the

second aspect of the present invention.

As discussed above, it can be expected that the microspheres according to the present invention that comprise a radioactive functional layer are able to be used in a method of treatment, e.g. treating cancer.

Thus, in a further aspect of the present invention, there is provided a pharmaceutical composition comprising the microspheres according to the first aspect of the present invention. Particularly, those microspheres that include a functional layer that comprises or consists of a radioactive agent and an effector agent. The effector agent preferably targets a specific cell type, most preferably a cancer cell and is an antibody. The pharmaceutical composition may further comprise any pharmaceutically acceptable excipients, binders, fillers, stabilizers, or any combination thereof.

In a further aspect of the present invention there is provided a method for treating a proliferative disorder (such as cancer), by administering a composition comprising an effective amount of a microsphere according the first aspect of the present invention to a subject suffering from a proliferative disorder. Preferably, microspheres are those that include a functional layer that comprises or consists of a radioactive agent and an effector agent. The effector agent preferably targets a specific cell type, most preferably a cancer cell and is an antibody. The composition may comprise any pharmaceutically acceptable excipients, binders, fillers, stabilizers, or any combination thereof.

In a further aspect of the present invention, there is provided a composition comprising a microsphere according to the first aspect of the present invention for use in a method of treatment. Preferably the method of treatment is for treating a proliferative disorder (such as cancer). The microspheres are preferably those that include a functional layer that comprises or consists of a radioactive agent and an effector agent. The effector agent preferably targets a specific cell type, most preferably a cancer cell and is an antibody. The composition may comprise any pharmaceutically acceptable excipients, binders, fillers, stabilizers, or any combination thereof.

In the most preferred embodiment pf the first aspect of the present invention, there is provided a buoyant microsphere that comprises a core, a functional layer and a solid oxide, wherein the functional layer forms an intermediate layer between the core and the solid oxide coating and the solid oxide coating forms a substantially continuous coating of form 0.2 to 100, preferably from 0.2 to 50 nm thick. The most preferred embodiment of the second aspect of the present invention is a method of making the aforementioned buoyant microsphere.

It should be understood that the microspheres described in the first aspect of the present invention, and those produced by the method according to the second aspect of the present invention are used in large numbers. Accordingly in a further aspect of the present invention there is provided a multitude of microspheres consisting of or comprising a population of the microspheres described above.

In a further aspect of the present invention, there is provided a method substantially as herein described.

In a further aspect of the present invention, there is provided a microsphere substantially as herein described and shown in the figures.

In yet a further aspect of the present invention there is provided a composition substantially as herein described.

An example of the microspheres and methods according to the present invention will now be described, by way of example only, and will make reference to the following drawings.

Figure 1 shows a microscope photograph of a nickel coated microsphere prior to applying a silica coating.

Figure 2 shows a microscope photograph of a nickel coated microspheres with an outer coating of silica.

Figure 3 shows a microscope photograph of a nickel coated microsphere with an outer coating of silica.

Figure 4 shows microscope photograph of a nickel coated microsphere with an outer coating of silica, after the nickel has been removed by treatment with hydrochloric acid. Magnification x 400. The core microsphere (diameter 50 μm is coated with a silica layer of uniform thickness 17 μm, approximately. A is the hollow microsphere core, B is a space left by the removal of the nickel layer, C is the deposited silica layer.

Example 1

Coating of a sample of Nickel-coated glass microspheres with a layer of gelatine

Glass microspheres are commercially available from a number of manufacturers. This Example utilizes a hollow gas-filled borosilicate glass microsphere (Eccosphere® brand; Emerson and Cuming, Billerica, MA) Eccospheres are available in different size grades and the procedures below refer specifically to the grade SI-200, which are smooth, perfectly spherical microspheres with a mean diameter of 55 micrometers. The procedures below refer to a batch of said microspheres that were nickel-coated by chemical vapour deposition process, by Powdermet Inc, Euclid OH.). The resultant material contained 52% wt nickel, a mean true particle density of 0.634g/cc and a mean diameter of 57 micrometers.

11.6 grams of a 45% w/v solution of cold-water fish gelatin solution (Sigma (RTM) Aldrich cat. no. G7765), was diluted with 520 ml deionized water (final gelatin concentration, 1% w/v). 5g of microspheres were added and the mixture warmed to 9O ⁰ C on a hot-plate, with occasional stirring, for 90 min. The suspension was poured into a No.

2 glass sinter funnel attached to a Buchner flask, to which had been added 50 ml de- ionized water, with stirring. The water was removed under vacuum and 150 ml de- ionized water added to the funnel. The microspheres were re-dispersed by gentle stirring and the water removed under vacuum. This procedure was repeated with de-ionized water a further four times. The microspheres were finally split into four equal batches and each was suspended in 50 ml de-ionized water. They were centrifuged at 2500 rpm for 5 min. The surface pellet was gently stirred to re-disperse, without disturbing the pellet, and centrifuged again. This procedure was repeated twice more.

The buoyant microsphere fraction was transferred, using a magnetic pick-up, to a 1 Litre beaker containing 450 ml de-ionized water. The beaker placed on a ring magnet, with gentle stirring to facilitate the settling of the highly magnetizable microsphere fraction.

The supernatant, including a fraction of weakly magnetizable material, was decanted off and the settled material was re-suspended in a further 450 ml de-ionized water and the above procedure was repeated a further three times. The recovered microspheres were transferred to a No. 2 sintered glass funnel attached to a Buchner flask, to which had been added 50 ml methanol, with stirring. The methanol was removed under vacuum and 20 ml methanol was added to the funnel. The microspheres were re-dispersed by gentle stirring and the methanol removed under vacuum. This procedure was repeated a further four times with 20 ml methanol and then five times with 20 ml diethyl ether. The recovered microspheres (2.75 g) were cured overnight at 12O ⁰ C under vacuum to yield a dark-grey free-flowing powder.

Figure 1 clearly shows the layer of nickel surrounding the core. As is demonstrated by this figure, the nickel layer provides the surface of the microsphere with a particularly rough textured surface.

Example 2

Gelatin assay of microspheres

This procedure enables the gelatin content of microspheres to be measured using a modification of the bicinchoninic acid (BCA) method (Pierce Chemical Co. Rockford, II).

The procedure was earned out as follows: Assay standards were first of all prepared using gelatin from cold water fish skin (45% w/v in water; Sigma (RTM) product # G7765). A working stock was prepared at 2000μg/ml and diluted in triplicate to 0, 25, 125, 250, 500, 750, 1000, 1500 & 2000μg/ml by dilution with deionized water. 50 μl of each standard was transferred to 1.5 ml Eppendorf tubes.

In triplicate, between 8 and 10 mg of gelatin-coated glass microspheres were weighed into 1.5 ml microfuge tubes.

The BCA assay working reagent was prepared by mixing reagents A and B (standard reagents presented in the bicinchoninic acid (BCA) tests of Pierce Chemical Co.

Rockford, Il as A and B) in the ratio 50:1, then vortexed to mix. 1.0 ml of working reagent was added to each of the standard tubes and to each of the microsphere sample tubes. The tubes were capped and vortexed, and then incubated at 37°C for 30 minutes.

At 10 minute intervals throughout the incubation period, the tubes were removed from the incubator and inverted several times to mix.

At the end of the incubation period, the tubes were inverted for a final time. The tubes containing microspheres were then placed against a magnet, so clearing the solution, and the solutions transferred to 1 ml disposable spectrophotometer cuvettes. The standard solutions were transferred directly to cuvettes.

The Absorbance of each cuvette was read at 526 nm, without delay, using the 0 μg/ml standard as a blank. The Absorbance reading of the sample solutions was converted to a mass of protein (in mg) from the derived standard curve, and then divided by the

recorded mass of microspheres (in grams) for that sample, to give the gelatin content in mg per gram microspheres.

The gelatin-content of the microspheres prepared in Example 2 was found by this method to be 7.8 mg / gram microspheres.

Example 3

Silica-coating of microspheres.

The following procedure may be used to coat microspheres that have or have not been prior-treated by coating with gelatin

0.8g gelatin-coated microspheres prepared in Example 1 were stirred at 300 rpm with a mixture of 30ml isopropanol and 12.6ml concentrated aqueous ammonia (density 0.9g/cc), in a 250 ml three-necked round bottomed flask at 4O ⁰ C. 8 x 0.171 ml (0.767 mmol) of tetraethoxysilane [78-10-4] were added at 45 min. intervals by means of a graduated pipette. Each cycle of tetraethoxysilane generates a deposited silica layer of approximately 1.7 μm, so that the total layer thickness after eight additions is approximately 14 μm. After the final addition the reaction mixture was stirred for an additional 45 min. and then poured with stirring into methanol (100 ml) which was in a N ⁰ 2 sintered glass funnel attached to a Buchner flask, and the methanol removed under vacuum. The vacuum was turned off, 30 ml methanol was added with brief stirring to disperse the product, and the methanol removed under vacuum. This procedure was repeated five times in total with methanol and five times with diethyl ether. The microspheres were cured in a vacuum oven at 12O ⁰ C overnight to yield a light grey free flowing powder (1.1 g). This material was suspended in 50 ml de-ionised water and centrifuged at 100 x g for 3 min. The surface plug was stirred gently to re-disperse, without disturbing the bottom pellet, and centrifuged again. The buoyant material was transferred with a magnetic pen to a new 50 ml centrifuge tube and 35 ml de-ionised water added and the tube was vortexed for 1 min. 15 ml of de-ionised water added then

centrifuged at 100 x g for 3 min. This process was repeated three times. The buoyant material was transferred with a magnetic pen to a new 50 ml centrifuge tube and 35 ml 0.1M hydrochloric acid added and the tube was vortexed for 1 min. 15 ml of de-ionised water added then centrifuged at 100 x g for 3 min. This process was repeated three times. The buoyant material was transferred with a magnetic pen to a new 50 ml centrifuge tube and 35 ml de-ionised water added and the tube was vortexed for 1 min. 15 ml of de- ionised water added then centrifuged at 100 x g for 3 min. This process was repeated three times. The pH of the supernatant liquid was 7. The buoyant material was transferred into methanol (50 ml) which was in a N ⁰ 2 sintered glass funnel attached to a Buchner flask, and the methanol removed under vacuum. The vacuum was turned off, 30 ml methanol was added with brief stirring to disperse the product, and the methanol removed under vacuum. This procedure was repeated three times in total with methanol and three times with diethyl ether. The microspheres were cured in a vacuum oven at 12O ⁰ C for 30 min. to yield a light grey free flowing powder (0.51 g). Under the microscope an apparently continuous covering of silica is clearly visible, as shown in Figure 3.

The apparent gelatin content of the silica-coated microspheres was measured using the BCA assay as described in example 2. Compared with non-treated microspheres (scoring 100% in the BCA assay), the silica-coated microspheres scored only 5%. This result is consistent with a silica surface-covering of at least 95%, on the assumption that silica- coated gelatin is not accessible to the BCA reagent.

The silica coated microspheres produced by this method are shown in Figure 2.

Example 4

Amino-functionalization of silica-coated microspheres

0.25g of microspheres prepared in Example 2 were suspended in 2.5 ml 95% aqueous MeOH in a screw-cap 50 ml polypropylene centrifuge tube and placed in an ultrasound bath for 30 seconds to disperse thoroughly. 0.048 g 3-aminopropyltrimethoxysilane was added and placed on a blood-tube wheel for 30min. The suspension was then poured into a No.2 sintered glass funnel attached to Buchner flask and the solvent removed under vacuum. The vacuum was turned off and 20 ml methanol was added to the funnel with gentle stirring to re-disperse the microspheres. The vacuum was turned on to remove the methanol. This procedure was repeated a further three times with methanol, then three times with diethylether. The funnel was then placed in a vacuum oven at 12O ⁰ C for lhour. 0.185g microspheres were recovered as a light-grey free-flowing powder.

Using the procedure described in Example 5, the amino-group content of the microspheres was found to be 15.7 μmol -NH ₂ Zg.

Example 5 Determination of the amino-group content of microspheres

This assay takes advantage of the colorigenic reaction of ninhydrin with primary amines and the present procedure is modified after Sarin et a {\9%\) Anal. Biochem. 117: 147.

Two reagents are required as follows: Reagent A: 6.5 mg potassium cyanide was dissolved in 100 ml de-ionized water and 1 ml of this was diluted with 49 ml pyridine. 8 g of phenol was dissolved in 2 ml of ethanol (with warming), and the two solutions were mixed together. Reagent B: 500 mg ninhydrin was dissolved in 10 ml of ethanol.

The procedure was carried out as follows: In triplicate, between 8 and 10 mg of amino- functionalized microspheres, as prepared in Example 3, were weighed into a 1.5 ml Eppendorf tube. Triplicate no-microspheres control tubes were included as blanks.

180 μl of reagent A was added to each tube, followed by 40 μl of reagent B. The tubes were vortexed for two seconds and then placed in a heating block pre-heated to 70°C. For 3 successive 5 minute intervals each tube was removed in turn from the heating block, vortexed for two seconds, and then placed back in the block. Five minutes after the last round of vortexing the tubes were removed from the block, vortexed for two seconds and then placed on ice for 5 minutes. The tubes were then placed against a magnet, so pulling the microspheres to the side, and 100 μl of the solution was withdrawn to a clean microcentrifuge tube containing 900 μl of 60% v/v ethanol in water. The tubes were capped and vortexed for 5 seconds to mix. The samples were finally transferred to disposable plastic micro-cuvettes. A spectrophotometer set at 570 nm was set to zero with the blank sample then the absorbance of each sample is recorded. The amine content of the microspheres (in μmol NH ₂ /g microspheres) was calculated from (A ₅₇ o x 147)/ (exact mass of microspheres, in mg), and the mean of the triplicate estimates is stated.

Example 6

Removal of nickel from silica-coated nickel microspheres

Removal of the nickel layer from silica coated microspheres permits the exact thickness of the new silica layer to be measured by light microscopy, since the interface between the inner (core) and deposited outer layer is clearly seen.

Nickel-coated microspheres without a silica coating (0.5 g) were placed in 37% hydrochloric acid (10 ml) with gentle stirring. Hydrogen bubbles formed rapidly on the surface of the microspheres and the supernatant liquid turned green due to formation of Ni ²⁺ . The microspheres turned white as the reaction proceed to completion, in less than 1 hour. By contrast, when nickel microspheres coated with silica, as prepared in Example 3, were treated with 37% hydrochloric acid, development of the green colour proceeded much more slowly, and was not discernable until ca. 30 min; hydrogen bubbles did not appear, and the nickel was not completely removed (microspheres completely white) until 5-6 hours had elapsed.

The relative resistance of the nickel layer to acid treatment is consistent with protection of the nickel layer by the silica layer.

Microspheres from which the nickel has been completely removed are transparent and the interface between the original microsphere and the deposited silica layer, which is approximately 15 μm in thickness, is clearly visible (Figure 4).

Example 7

Coating of silica-coated nickel-coated microspheres with anti-Cryptosporidium antibody

In this procedure antibody is coupled to the microsphere surface by random glutaraldehyde cross-linking between surface- and protein-amino groups.

(a) antibody derealization. A number of anύ-Ciγptosporidium oocyst-specific antibodies are commercially available. This example utilizes the mouse monoclonal antibody 2C9, available from Waterborne Inc. (New Orleans, LA), which was purified from ascites fluid, as per the manufacturer's instructions, before use. The protein concentration was determined by measuring the absorbance at 280 nm (antibody concentration in mg/ml = A ₂₈₀ /1.3).

(b) Functionαli∑αtion of microspheres with antibody. 25.5 mg of silica-coated microspheres prepared in Example 3 were weighed into an Eppendorf tube, followed by 1 ml of carbonate buffer. The microspheres were dispersed by gentle shaking and allowed to stand 5 minutes. The floating microspheres were transferred to a second Eppendorf tube containing 1 ml carbonate buffer (0.1 M Na ₂ CO ₃ plus 0. IM NaHCO ₃ , pH 9.5), using a magnetic pen, followed by 500 μl 10% aqueous glutaraldehyde, prepared by diluting the 50% stock with carbonate buffer. The tube was attached to a blood-tube wheel, clamped vertically, and allowed to rotate for two hours at 30 rpm, room temperature. The microspheres were then washed by transferring successively to 6 x 1 ml carbonate buffer, with 5 min. on the blood-tube wheel between transfers. 500 μl of purified 2C9 antibody

(100 μg/ml in carbonate buffer) was then added, and the tube placed on the blood-tube wheel and incubated overnight (30 rpm, room temperature). Freshly prepared aqueous sodium cyanoborohydride was then added, to final concentration of 5OmM, and the tube returned to the wheel for 30 min. Unreacted aldehyde sites were blocked by addition of aqueous ethanolamine hydrochloride solution, pH 9.5 to final concentration 5OmM ₅ and the tube returned to the wheel. The microspheres were finally washed by successive transfers into 5 x ImI of PBS-azide (1 OmM Phosphate, 2.7 niM KCl and 137 niM NaCl; pH7.4 containing 0.02% sodium azide) with 5 min. on the wheel between transfers and stored at 4 ⁰ C.

Example s

Preparation of a magnetite coated, silica-coated microsphere for diagnostic applications

(i) APS coating of hollow glass microspheres

294.46g of 27 μm hollow glass microspheres were treated with 59g 3- trimethoxysilylpropylamine (APS) in 3000ml 95% aqueous methanol. These beads (282.92g) are cured under vacuum at 70 ⁰ C for 3 hours.

(H) Assay of amino groups

The microspheres prepared above were assayed using the method described in Example 5. A value of 6.3 μmoles NH _s /g microspheres was obtained.

(iii) Gelatin-coating of amino functionalized microspheres

50.0Og of microspheres were boiled in an aqueous solution of 45% cold water fish gelatin (26 mg/ml) for 1 hour. The microspheres were then washed thoroughly with deionised

water, deionised:water:methanol (1:1) and finally methanol. The recovered microspheres (49.63g) were cured under vacuum at 12O ⁰ C overnight.

(iv) Protein determination of gelatin-coated microspheres

The gelatin-con tant of the microspheres prepared above was assayed as described in Example 2. A value of 3.6 mg gelatin/g microspheres was obtained).

(v) Magnetite-coating of microspheres

Magnetite 2Og was suspended in 1 litre deionised water and subjected to high shear mixing (7500 rpm) for 15 minutes.

The microspheres from (iii) were mixed with high shear magnetite in the ratio 1 :0.75 w/w in water. The suspension was placed on an orbital shaker at room temperature for 1 hour. The microspheres were recovered by centrifuging the mixture at 1500 x g for 15 minutes. The floating fraction was transferred with a magnetic pen to fresh centrifuge tube containing 50 ml distilled water, shaken to disperse and then centrifuged again. This procedure was repeated a further four times. After the last wash the microsphere were dispersed in 30 ml methanol in a Buchner funnel (No2. sinter) attached to a Buchner flask and vacuum line. The vacuum was turned on to remove the methanol. The vacuum was turned off. 30 ml methanol was added and the product resuspended by gentle stirring. The vacuum was turned on to remove the methanol. This procedure was repeated three times with 30 ml methanol and then once with 30 ml methanol/diethylether (1 :1 v/v). The vacuum was left on to air-dry. The material was dried in a vacuum at 12O ⁰ C, overnight.

(vi) Assay of bound magnetite

The iron content of magnetite-coated microspheres is deteπnined by nitric acid treatment of a sample of the microspheres, which quantitatively releases the iron into solution as a

mixture of Iron (II) and Iron (III). The iron is quantitatively reduced to Iron (II) using hydroxylamine and titrated using 1,10-phenanthroline. The concentration of the Fe(II)- 1,10-phenanthroline complex is determined spectrophotometrically.

Between 3 and 4 mg of magnetite-coated microspheres were weighed out in triplicate. 1 ml of 6 M nitric acid was added to each tube and vortexed to mix. The tubes were placed in a heating block at 90 ⁰ C for 4 hours, with vortexing every 30 minutes. The tubes were removed from the block from and centrifuged for 30 seconds at 12 000 x g. 25 μl of the solution from the middle of the reaction tube mixture (avoiding material at top and bottom of the tube) was transferred into an Eppendorf tube to which had been 175 μl deionized water. 400 μl of 1 M K ₂ HPO ₄ was added to neutralize the solution, followed by 400 μl of 1% hydroxylamine hydrochloride solution in water. 400 μl of 0.1% 1,10-phenanthroline in 12.5 mM sodium acetate was added and the tubes vortexed briefly to mix. The tubes were placed in a heating block at 80°C for 20 min, then removed and allowed to cool. 250 μl of the solution was transferred into a plastic disposable spectrophotometer cuvette, followed by 750 μl of de-ionized water. The absorbance at 510 nm was measured using water as the blank. The iron content, in mg iron per g microspheres is given by (A ₅₁₀ x 1 136)/(mass of microspheres in grams). The stated value is the mean of triplicate readings.

The 1,10-phenanthroline iron assay was used to determine the level of iron incorporation and typically a value of 230 mg Fe/g microspheres was obtained.

(vii) Silica glass coating of microspheres

37 ml iso-propanol, 30 ml concentrated aqueous ammonia and 1.0Og of magnetite coated microspheres prepared in (v) were placed in a 100ml round bottom flask. The was fitted with a mchenical stirrer and placed in a heating mantle se and heated to constant temperature 6O ⁰ C, with stirring at 300 rpm. 307.5 μl (0.2868 g) tetra-ethoxyorthosilicate (TEOS) was added and the mixture stirred for 20 minutes. The product was recovered by vacuum filtration through a Buchner funnel (No2. sinter). The vacuum was turned off. 30

ml methanol was added and the product resuspended by gentle stirring. The vacuum was turned on to remove the methanol. This procedure was repeated three times with 30 ml methanol and then once with 30 ml methanol/diethylether (1:1 v/v). The vacuum was left on to air-dry. The material was dried in a vacuum at 12O ⁰ C, overnight.

The final product was a free-flowing powder, yield 1.07 g (theoretical yield 1.08g). The weight increase corresponds to a continuous silica layer that is roughly 60 nm in thickness.

Example 9

Demonstration of acid resistance of magnetite-coated microspheres

In certain immunodiagnostic procedures it may be necessary to perform a release step to separate immunocaptured analyte from the solid phase. A common way of achieving this is a very low- or very high pH wash, which has the effect of inactivating the antibody. One such example is the assay of water samples for oocysts of Ciγptosporidium parvum, where the acid elution step is necessary to separate the oocysts from the microspheres prior to a microscope slide count. A procedure for the assay of Cryptosporidium oocysts using buoyant superparamagnetic microspheres was disclosed in co-pending application PCT/GB/2006/001896. Acid elution of the bound analytes from a 'bare' magnetite- coated buoyant microsphere can lead to partial dissolution of the magnetite and hence generate artefacts, particularly during microscopic evaluations, which render the slide examination more difficult. In principal, the silica coating should prevent this, and in order to test the hypothesis, the magnetite coated, silica-coated microspheres prepared in Example 8 (v) were subjected to an acid test. In addition, microspheres coated with anticryptosporidium antibody (using the means disclosed in Example 7 above) were used in to assay for Cryptosporidium ococysts in water, as described in PCT/GB2006/001896.

The acid test was carried out as follows: lOmg of the microspheres prepared in Example 8 (v) were placed in small glass tube together with 1 ml cone. HCl. A stopwatch was

started immediately and the time taken for the microspheres to turn white was recorded (this corresponded to the appearance of the intense yellow colour of FeCl ₃ in the medium). Magnetite-coated microspheres with no silica coating treated in this way become white immediately on contact with the acid solution. By contrast, the microspheres prepared above were still intact (brown, with no yellow FeCl ₃ in medium) after lOmin. The experiment was repeated with microspheres coated with a silica layer l/10 ^th , and l/100 ^th , that of these (corresponding to a continuous coat thickness of roughly 6nm, and 0.6 nm. These failed after 5 min. and 1 min, respectively.

Therefore it was concluded that thicker layers of silica confer significant protection of the magnetite layer against acid attack. It was surprising, but reproducible, that these exceptionally fine layers (corresponding to roughly 2, 20 and 200 molecular layers, should confer good resistance to concentrated acid.

The oocysts recoveries from distilled water using the standard Cryptosporidium assay described in PCT/GB2006/001896 were as follows: untreated (i.e. no silica layer): 79.3%; microspheres with nominally 60nm, 6 nm and 0.6 nm thick silica: 63.6%, 65.2% and 72.3%, respectively. These results are interpreted was follows: Relative to the uncoated control, recovered yields decrease as the thickness of the silica layer increases. This result is interpreted in terms of a smoothing out of the surface of the microsphere as the thickness of the layer increases. The decreasing recoveries of oocysts is intrepted as being due to loss of surface are per microsphere, and hence of total surface area, under the conditions of the assay (constant mass of microspheres added). In the oocyst assay that the silica layer confers the advantage that it prevents the magnetite from being dissolved off the microsphere at the acid wash stage which is used to separate the oocyts from the microspheres prior to the oocyst count. Therefore it is clear from this kind of experiment that there is an optimum silica layer thickness: one that imparts maximum acid-protection of the underlying magnetite layer, without unduly compromising the performance of the microsphere due to loss of surface area.