WO2006125978A2

WO2006125978A2 - Assay particles

Info

Publication number: WO2006125978A2
Application number: PCT/GB2006/001896
Authority: WO
Inventors: Christopher Oldfield; Tom Johnston; David Nichols
Original assignee: Nanosphere Limited
Priority date: 2005-05-23
Filing date: 2006-05-23
Publication date: 2006-11-30
Also published as: GB2426583A; WO2006125978A3; GB0510461D0; GB0610209D0

Abstract

The invention relates to microspheres, more particularly magnetizable microspheres that may be used in separation procedures, or that may be used as the basis for constructing microspheres that may be used in separation procedures. The invention also relates to methods for making such microspheres. The microspheres comprise a core, an anchoring agent and a magnetizable agent, wherein the magnetizable agent is anchored to the core by binding to the anchoring agent.

Description

Assay Particles

Field of the invention

The present invention relates to microspheres, more particularly magnetizable microspheres that may be used in separation procedures, or that may be used as the basis for constructing microspheres that may be used in separation procedures. The invention also relates to methods for making such microspheres.

Background to the invention Separation technology is a large and diverse field that encompasses, on the one hand, relatively simple analytical separations, such as the recovery of a single analyte of interest from a clinical sample, to large scale preparative chromatography for the high purity recovery of chemicals, including biomolecules, for therapeutic use, on the other.

Microspheres are commonly used as the solid phase in separation methods. When preparing a microsphere for use in a separation method, the microsphere is modified so as to selectively bind to the selected analyte (e.g. by binding, to the surface of the microspheres, an agent with a high level of affinity for the selected analyte). The microspheres are also modified in order to enable them to be selectively recovered (and so selectively recover any analyte bound to the microspheres) from the sample in which the analyte is present. For example, the microspheres can be modified so that they may be manipulated by a magnetic field. For example, a magnet may be used to recover the microspheres from a liquid sample comprising the analyte. Under the appropriate optimal conditions the recovery is quantitative.

There are essentially two ways in which microspheres have been modified so that they can be manipulated by magnetic fields.

The first way involves the incorporation of magnetizable materials into the core of the microsphere. Microspheres prepared in this way are typically denser than water, and comprise an iron oxide core.

The production of microspheres that possess the aforementioned modified cores can be complicated and costly to manufacture. It is not possible to construct such microspheres from relatively cheap ready produced non-magnetizable microsphere cores, as a specialised core must usually be constructed. It should be noted that a relatively large amount of magnetizable material must be incorporated into the core of a microsphere in order to render the microsphere magnetizable to the degree that it can be easily manipulated by a magnetic field. It is not, however, appropriate to incorporate such large amounts of magnetizable materials into the core of many microspheres. The integrity of the structure of a glass microsphere may be adversely affected by the incorporation of magnetizable materials. Also, magnetizable materials are normally heavy and so large amounts of such a material cannot be incorporated in the cores of microspheres that are intended to be buoyant.

The alternative way of rendering a microsphere magnetically susceptible involves the coating of the surface of the microsphere with a magnetizable material. For example, US Patent No. 5,342,608 awarded to Moriya et al, in the field of ultrasound imaging, describes a metal-coated microsphere as a contrast agent. The microspheres are prepared by stirring glass microspheres with an aqueous solution of an iron salt under oxidising conditions, so depositing a layer of magnetite (a magnetic oxide of iron) on the surface. By contrast, US Patent No. 4, 624,923, awarded to Margel, describes a method for the preparation of metal-coated microspheres by electro-less deposition of the metal onto the surface of a polyaldehyde microsphere, using a procedure in which the microspheres are incubated with an aqueous solution of an appropriate metal salt, in the presence of a reducing agent. US Patent No. 4, 177, 253 describes glass microspheres which are rendered magnetically susceptible by electro-less deposition of a layer of nickel or cobalt on to the surface by stirring the microspheres with a solution of a suitable salt of the metal, in the presence of a reducing agent. US Patent No. 6,656,587 awarded to Johnson et al similarly describes, inter alia a method for coating iron oxide onto glass microspheres, using a vapour deposition process.

It has, however, been found that after coating a microsphere with a magnetizable material 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 has resulted in a low density of such agents deposited on the surface of the microsphere. Additionally, coating microspheres in the abovementioned manner relies on simple physical coating of the magnetizable material onto the surface of the core. Such a poor level of attachment means that the magnetizable layer may be easily torn from the microsphere when subjected to torsional forces, as for example following the attachment of a massive ligand, such as a cyst, oocyst or spore, thereby rendering it difficult to manipulate the microspheres using magnetic fields or to retain any agents that are attached to the microsphere via the magnetizable agents.

It is important for the structure of a microsphere used in a separation method to be particularly robust. The aforementioned weakness in the attachment of the magnetizable layer to the core of the microsphere seriously adversely affects the ability for the above discussed coated microspheres to act as an effective solid phase in a separation method.

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 relating to the recovery of Cryptosporidium parvum oocysts from water.

Microspheres, when coated 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 the (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, as is the case with the coated microspheres of the prior art discussed above, then the applied torque is sufficient to tear the magnetizable agent/antibody layer from the surface of the underlying microsphere. Indeed, it has been found that using microspheres prepared according to the methods described in the prior art by the separation procedures described in Example 12 results in a magnetizable agent /antibody material (plus associated oocysts) appearing as a pellet at the bottom of the Eppendorf tube separated from the cores. It is impossible to either separate the oocysts from the brown pellet, or attempt to detect them from among the brown pellet.

Accordingly, it is an object of the present invention to provide a microsphere that is much more robust than those of the prior art, which is capable of being straightforwardly magnetically manipulated and used in separation procedures, or that may be used as the basis for constructing microspheres that may be used in separation procedures. 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 magnetizable microspheres, and novel methods for making magnetizable microspheres, which are sufficiently robust to enable them to be manipulated in a magnetic field or used in separation procedures (when functionalised by the attachment of an effector molecule such as an antibody). The term 'microsphere' is used herein as a synonym for microcapsule, microparticle, microballoon, and microsphere particles.

The key to the robust nature of the microspheres of the present invention is that the agents that render the microspheres magnetizable, and which also may act as a structure through which subsequent "layers" including functionalizing "layers" (e.g. an effector agent such as an antibody) may be bound to the core, are firmly secured to the surface of the microsphere. Thus, the forces exerted on the magnetizable agents by a magnetic field, or the forces exerted on any other agents bound to the core via the magnetizable agent (e.g. torsional forces arising from binding of a ligand to a receptor attached to the magnetizable agent) do not result in the stripping of the "layer" of magnetizable agents from the core.

Thus, according to one aspect of the present invention, there is provided a microsphere comprising a core, an anchoring agent and a magnetizable agent, wherein the magnetizable agent is anchored to the core by binding to the anchoring agent.

Because of the nature of substances that are magnetizable, magnetizable agents are not usually able to chemically bind to materials conventionally used for making microspheres, such as glass. However, it has been found that by providing an anchoring agent as an intermediate between the core and the magnetizable agent it is possible to couple the magnetizable agent to the core via this intermediate using chemical bonding, thereby providing a more secure attachment than could be achieved by simply physically coating the core with a magnetizable agent by, for example, chemical vapour deposition or electrolytic deposition.

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

The magnetizable agent can be coupled to the magnetizable-layer binding agent by a coupling agent (such as a silane). In such embodiments, the construction relies on a covalent bond to bind the magnetizable agent through the coupling agent to the magnetizable-layer binding agent. It has been surprisingly found, however, that a much simpler construction, whereby the magnetizable agent may bind directly to the magnetizable-layer binding agent, can produce a similarly robust structure. This is surprising as this direct binding between the magnetizable-layer binding agent and the magnetizable agent must be achieved through non-covalent interactions. In such embodiments no "pre-functionalising" of the magnetite is required (for example, by binding one half of a receptor pair (e.g. avidin) to the magnetizable agent and the other half of the pair (i.e. biotin) to the core. Thus, in such embodiments, an unmodified magnetizable agent may be bound straightforwardly, and non-covalently, directly to the core via a magnetizable-layer binding agent.

Microspheres that do not comprise an effector agent, for example those that consist of a core, an anchoring agent and a magnetizable agent arranged in the aforementioned manner, are useful as a "blank" starting point from which a skilled person may construct (e.g. by the methods described below) a functionalised microsphere for use in a separation procedure. The term "functionalised microsphere" means a microsphere that has been adapted in order to capture, for example, a specific analyte. Functionalisation is achieved by binding an effector agent to the microsphere (e.g. an agent that has specific affinity for the target analyte). Providing such "blank" microspheres gives the skilled person the ability to functionalise the microspheres by binding the appropriate effector agent for capturing the target analyte in any specific separation protocol.

Alternatively, the microspheres of the present invention can be prepared as functionalised microspheres. Thus, in one embodiment of the present invention the microspheres comprise an effector agent, which may be coupled to the magnetizable agent by a coupling agent (e.g. a silane).

It has been found, however, that it is possible for the magnetizable agent and the effector agent to adversely interact when they are in close proximity. Indeed, the ability for the effector agent to specifically bind to a target analyte may be impaired or lost as a result of such adverse interactions. Accordingly, in a preferred embodiment of the present invention, the microspheres comprise an effector binding agent (e.g. a protein such as gelatine) which binds to the effector agent and is coupled to the magnetizable agent by a coupling agent. The positioning of the effector binding agent between the magnetizable agent and the effector agent has been found to eliminate or substantially reduce any adverse interactions occurring between the magnetizable agent and the effector agent.

"Blank" microspheres may be provided that comprise an effector binding agent. Thus, according to a further embodiment of the present invention the microspheres may comprise an effector binding agent coupled to the magnetizable agent by a coupling agent.

As mentioned above, due to the nature of substances that are magnetizable, magnetizable agents are not usually able to chemically bind to materials conventionally used for making microspheres. Accordingly, applying a "layer" of magnetizable agents to a core as a first step in the process of producing microspheres results in a simple physical coating of the microspheres that is only weakly attached to the core. It has, however, been found that chemically modifying the surface of the core allows the magnetizable agent to bind chemically to the core.

Thus, according to a further aspect of the present invention there is provided a method for preparing a microsphere comprising the step of chemically modifying the surface of a core prior to the step of anchoring a magnetizable agent to the core.

The chemical modification may comprise the step of anchoring a magnetizable-layer binding agent to the core. Depending on the material used to make the core and the choice of magnetizable-layer binding agent, the magnetizable-layer binding agent may be able to bind directly to the core. However, the magnetizable-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 magnetizable-layer binding agent comprises the step of binding a coupling agent to the core. The step of anchoring the magnetizable-layer binding agent may comprise the step of binding the magnetizable- layer binding agent to the coupling agent. The coupling agent may be bound to the core prior to being bound to the magnetizable-layer binding agent. Alternatively, the coupling agent may be bound to the magnetizable-layer binding agent prior to being bound to the core.

The magnetizable agent can be coupled to the magnetizable-layer binding agent by a coupling agent. Thus, in a further embodiment of the present application, the step of anchoring the magnetizable agent to the core further comprises the step of coupling the magnetizable agent to the magnetizable-layer binding agent by a coupling agent. The coupling agent may be bound to the magnetizable agent prior to being bound to the magnetizable-layer binding agent. Alternatively, the coupling agent may be bound to the magnetizable-layer binding agent prior to being bound to the magnetizable agent. The magnetizable agent may instead bind directly to the magnetizable-layer binding agent. Thus the step of anchoring the magnetizable agent to the core may further comprises the step of coupling the magnetizable agent to the magnetizable-layer binding agent directly and without introducing a coupling agent. This step is so simple that it can comprise or consist of mixing the magnetizable agent with the magnetizable-layer binding agent coated microsphere in an aqueous solvent (such as water). This step may be carried out at standard room temperature. It has been surprisingly found that such a simpler construction process, whereby the magnetizable agent may bind directly to the magnetizable-layer binding agent, produces a similarly robust structure and represents a cheaper and faster method of production.

The above methods may be used to make, for example, microspheres that do not comprise an effector agent, for example, those that consist of a core, an anchoring agent and a magnetizable agent arranged in the aforementioned manner. Such microspheres are useful as a "blank" starting point from which a skilled person may construct (e.g. by the methods described below) a functionalised microsphere for use in a separation procedure.

Alternatively, the methods of the present invention may be used to make functionalised microspheres. Thus, in one embodiment of the present invention the method comprises the step of anchoring an effector agent to the microsphere, which may further comprise the step of coupling the effector agent to the magnetizable agent by a coupling agent.

It has been found, however, that it is possible for the magnetizable agent and the effector agent to adversely interact when they are in close proximity. Indeed, the ability for the effector agent to specifically bind to a target analyte may be lost as a result of such adverse interactions. Accordingly, in a preferred embodiment of the present invention, the method comprises the step of binding an effector binding agent to the effector agent and coupling the effector binding agent to the magnetizable agent using a coupling agent. The positioning of the effector binding agent between the magnetizable agent and the effector agent has been found to eliminate or substantially reduce any adverse reactions occurring between the magnetizable agent and the effector agent.

"Blank" microspheres that comprise an effector binding agent may also be produced by a method according to the present invention. Thus, according to an embodiment of the present invention there is provided a method that further comprises a step of coupling an effector binding agent to the magnetizable agent by a coupling 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 microspheres for use in conventional separation procedures. The core may be made of an inorganic material. Preferably, the core comprises any of the following; a synthetic polymer, a natural polymer, a co-polymer, a block co-polymer, polymethylmethacrylic acid, a protein, glass, a metal oxide, titania, zirconia, alumina, magnesia, 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 a magnetizable agent.

In a further preferred embodiment the core has a diameter that is less than 1000, 500, 100, 50, or 10 μm. The core may have a diameter from 0.1 to 1000 μm. For a microsphere optimally coated with an effector agent such as an antibody (i.e. such that the majority of bound antibodies are active), efficient capture of an analyte from a sample is primarily a function of the total antibody-coated surface area presented to the sample, which itself is the product of the surface area per microsphere and the number of microspheres introduced into the sample. For practical purposes, so that the volume of added microspheres is not so large that their manipulation/handling becomes difficult, the optimum size range has been found to lie in the range from 5 to 100 μm diameter, and the preferred size lies in the range of from 10 to 30 μm diameter.

In a preferred embodiment of both aspects of the invention described above the magnetizable agent may be any agent that is capable of being manipulated by a magnetic field. Preferably, the magnetizable agents exhibit magnetic behaviour only in the presence of an externally applied magnetic field, and are not permanent magnets. 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 magnetic agglomeration of microspheres. This is undesirable in a diagnostic environment, where a thoroughly dispersed solid phase offering a 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 the avoidance of doubt, a superparamagnetic agent is one that becomes strongly magnetized in the presence of a 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. Whether 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. In the case of magnetite (magnetic iron oxide), superparamagnetism is exhibited by 30 micrometers or less; in the case of a spinel ferrite such as CoFe₂O₄ suerparamagnetism is exhibited by particles that are 10 micrometers or less.

The magnetizable agents may comprise any of the following; a paramagnetic agent, a superparamagnetic agent, iron oxide, a spinel ferrite, an alloy, or any combination thereof. In one preferred embodiment the magnetizable agent is a superparamagnetic material. For the avoidance of doubt, a spinal ferrite can have the generic formula MFe₂O₄, where M is a metallic element including, but not limited to, any of Co, Mg, Mn and Zn, or any combination thereof.

It is preferred that the 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. The magnetizable agents may have a diameter from 10 to 100, from 15 to 70, or from 20 to 50 nm.

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 suspension to the side of a 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 agent, e.g. magnetizable agents formed by sonication. The applicant has found that mixing high shear magnetite with bovine serum albumin (BSA)-coated microspheres (prepared, for example, in accordance with Example 5) yields dark, highly magnetizable and superparamagnetic particles, indicative of a high retention of magnetite particles on the surface. On the other hand, however, 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-shear" magnetite, for example as prepared according to Example 5 below, and in a preferred embodiment of 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 includes the high-shear mixing of the magnetizable agent. An alternative manner of producing magnetizable agent that has also been found to be superior to sonication is by precipitation from an ammoniacal Fe (II)/Fe (III) solution, as described for example, In Example 7. Thus, the pre-treatment step may instead include the aforementioned precipitation step.

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

In a further preferred embodiment of both aspects of the invention described above the magnetizable-layer binding agent comprises 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. Preferably, the magnetizable-layer binding agent is an inert protein, for example a serum albumin (e.g. bovine serum albumin, human serum albumin, chick serum albumin) or gelatine.

When the coupling agent is an amino silane, and the magnetizable-layer binding agent is an inert protein, direct binding between these two components may be achieved by means of a condensing agent such as a carbodiimide. (for example, as described in

Example 3). Carbodiimide-mediated condensation of the carboxyl groups of the protein with the amino-groups of the amino silane microsphere results in a microsphere in which the protein is covalently bound to the microsphere. Thus the polymer is preferably covalently bound to the microsphere.

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 glass, iron oxide, or 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. When the coupling agent is a silane and it is to be coupled to an inorganic molecule (e.g. when secured to a unmodified glass core) the silane is coupled to the inorganic molecule by mixing the two in the presence of an alcohol (e.g. methanol) and water. When the coupling agent is an amino silane and it is to be coupled to an protein the direct binding between these two components may be achieved by means of a condensing agent such as a carbodiimide. (for example, as described in Example 3). Carbodiimide-mediated condensation of the carboxyl groups of the protein with the amino-groups of the amino silane microsphere results in a microsphere in which the protein is covalently bound to the microsphere.

In a further preferred embodiment of both aspects of the present invention 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. In a preferred embodiment of both aspects of the invention described above the effector binding agent may comprise 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. Preferably, the effector binding agent is an inert protein, for example a serum albumin (e.g. bovine serum albumin, human serum albumin, chick serum albumin) or gelatine.

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 magnetizable binding agent, effector binding agent 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 1, 2, 3, 4, 5, 6, or 7 mg of protein per gram of microsphere. Preferably, the microspheres comprise from 4 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 magnetizable-layer binding agent to the microsphere, but prior to binding the magnetizable agent, 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. Following the step of coupling an effector binding agent to the magnetizable agent, but before the step of binding the effector agent, the microsphere preferably comprises no less than 1, 2, 4, 8 mg protein per gram of microsphere, more preferably from 8 to 25, from 12 to 25, from 8 to 20, from 10 to 20, or from 12 to 20 mg of 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. However, for simplicity it has been found that non-covalent bonding is preferred between the magnetizable agent and the magnetizable-layer binding agent.

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 applicant has found that not only are buoyant microspheres most useful as the solid phase in separation technologies (as they float to the top of a liquid medium along with the bound analyte, thereby permitting easy extraction from the surface of the liquid using a magnet that is passed over the surface), the methods of constructing the microspheres according to the present invention are far simpler and cheaper to carry out when the microsphere is a buoyant microsphere. Buoyant microspheres can be very easily retrieved from each step of the step-wise methods described above and 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 being also buoyant.

For the avoidance of doubt, a buoyant microsphere is one that is less dense than the liquid medium in which it is suspended, thus buoyant microspheres float to the surface of the liquid medium in which they are suspended. A buoyant microsphere is not isodense with the liquid medium in which it is suspended. The skilled person would understand that, by virtue of the conditions of the manufacturing process, smaller microspheres tend to have a higher buoyant density than do larger. For example, the microspheres used in Example 1 have a median diameter of 27 μm and the final density of the superparamagnetic microsphere, prepared using any of the means described in the Examples below, is approximately 0.5 g/cm³. On the other hand, a smaller base microsphere (diameter 18 μm) is much denser, and the final coated microsphere has a density of 0.6 g/cm³. Smaller microspheres have the advantage, vide supra, that fewer need be added to a sample to provide any given total (effector-coated) surface area. The exact choice of size and density for a specific application is one which is made on a case-by-case basis.

Not wishing to be restricted further, but in the interests of clarity, it has been found that the preferred microspheres of the present invention have a buoyant density of less than 0.9 g/cm³ , preferably from 0.05 to 0.85, from 0.2 to 0.8, from 0.3 to 0.7 g/cm³. The buoyant density of the microsphere may be 0.6 g/cm³. The microsphere may be hollow. The 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 the most preferred embodiment of the first aspect of the present invention there is provided a buoyant microsphere that comprises a hollow glass core that is coated with an inert protein (such as BSA or gelatine) via a silane, the protein being non-covalently bound to a superparamagnetic agent which has been prepared by high shear mixing or reduction of an Fe(II)/Fe(III) salt mixture. The density of the microspheres is preferably from 0.4 to 0.7 g/cm³. When functionalised, a protein is bound to the superparamagnetic agent via a silane and an effector agent, such as an antibody, bound to the protein via a further silane.

In the most preferred embodiment of the second aspect of the present invention there is provided a method that includes a simple mixing together of an aqueous suspension of the super-paramagnetic agent preparation and the protein-coated microspheres (for example, as described in Example 5).

It should be appreciated that the microspheres are used in their hundreds, thousands or more in separation techniques. Accordingly, the microspheres described above may exist as a multitude of microspheres all or substantially all sharing the physical characteristics described above.

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.

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

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

An example of the microspheres and methods according the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:-

Figure 1 shows Recovery of Cryptosporidium parvum oocysts from water samples using either the buoyant microspheres of the present invention (right speckled bar) or a commercially available test based on non-buoyant beads (left, hatched bar).

Example 1

Amino-functionalization of hollow glass microspheres

This procedure yields amino-functionalized glass microspheres.

Buoyant glass microspheres are commercially available from a number of manufacturers. This Example utilizes Eccosphere® gas-filled borosilicate hollow glass microspheres

(Emerson and Cuming, Billerica, MA) Eccospheres are available in different size grades and the procedures below refer specifically to the grade SDT-40, which are smooth, perfectly spherical microspheres with a mean diameter of 27 micrometers.

The material is first cleaned thoroughly using the following procedure: 6 g of microspheres were weighed into a 250 ml Erlenmeyer flask, followed by 50 ml of hydrochloric acid solution (10% v/v), and placed on an orbital mixing table for 3 hours. The contents were then poured into a glass-sinter funnel attached to a Buchner flask and the acid solution was removed under vacuum. The vacuum was turned off and 250 ml de- ionized water was added to the funnel. The microspheres were re-suspended by stirring with a glass rod, and the water was removed under vacuum. This procedure was repeated until the filtrate tested pH-neutral using universal indicator paper. The microspheres were then left under vacuum for at least 3 hours to dry.

25 ml concentrated (> 99%) sulphuric acid was slowly added to 25 ml de-ionized water in a 250 ml Erlenmeyer flask, with stirring, followed by 50 ml ice-cold hydrogen peroxide solution (35% vol.) The acid-washed microspheres were added and the flask was placed in an ultrasound bath for 30 minutes, with swirling every 5 minutes. The suspension was poured into a glass-sinter funnel attached to a Buchner flask. The liquid was removed under vacuum. The microspheres were then washed by re-suspension in, successively, 2 x 250 ml de-ionized water, 250 ml methanol and 250 ml diethylether.

Following the last rinse, the material was left under vacuum for at least 3 hours to dry.

The product is a brilliant-white free-flowing powder with a buoyant density of about 0.3 g/cm³. 6 g of microspheres were weighed into a 250 ml separating funnel. 200 ml methanol was added and the separating funnel was stoppered. The separating funnel was shaken vigorously for 30 seconds and then rested. Settled solids (principally glass dust) were run off in the first 100 ml of methanol. The funnel was swirled gently to re-suspend the floating fraction of microspheres and then run off into a 500 ml screw-top conical flask. Additional methanol was added to the flask to bring the volume to 200 ml. 10 ml of de-ionized water was added, followed by 10 ml of 3-aminopropyltrimethoxysilane. The lid was screwed on and the flask swirled to re-suspend the microspheres and then placed on an orbital mixing platform for 3 hours at room temperature, at a speed sufficient to keep the microspheres in suspension. The suspension was then poured into a glass-sinter funnel attached to a Buchner flask and the liquid removed under vacuum. The vacuum was turned off and 250 ml methanol was added. The microspheres were dispersed by gentle stirring using a glass rod and the vacuum was turned to remove the methanol. This procedure was repeated once more, and then the vacuum left on for at least 3 hours to dry. The product is a brilliant-white free flowing powder with an amine content in the range 23-35 μmol NH₂ /g microspheres dry mass (Example 2) and is stable indefinitely when stored dry at room temperature.

Example 2

Determination of the amino 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 άl. (1981) 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 5 and 6 mg of amino- functionalized microspheres, as prepared in Example 1, were weighed into a 1.5 ml Eppendorf tube. A single no-microspheres control was included as a blank.

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 centrifuged at 12000 x g for 30 seconds in a microcentrifuge. The solution was then carefully pipetted out of the tube from underneath the plug of microspheres, to that the transferred liquid was free of microspheres. 100 μl of each solution was transferred to a clean Eppendorf tube and 900 μl of 60% v/v ethanol in water was added. The tubes were 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 NH₂/g microspheres) was calculated from (A₅₇o x 147)/ (exact mass of microspheres, in nig), and the mean of the triplicate estimates is stated.

Example 3 Coating of microspheres prepared in Example 1 with bovine serum albumin.

This procedure covalently couples bovine serum albumin (BSA) onto the microspheres.

375 mg of bovine serum albumin (BSA) was weighted out and transferred into a 250 ml conical flask. 75 ml deionized water was added and the flask swirled gently to dissolve the BSA. 185 mg of l-(3-dimethylaminopropyl)-3-ethylcarbodiirnide hydrochloride (EDAC) was weighed out and added to the flask. The flask was swirled gently on the orbital platform for exactly 15 min.

5 grams of the amine-functionalized microspheres prepared in Example 1 were weighed into a 500 ml screw-top conical flask, followed by 75 ml of 0.2 M borate buffer, pH 8.5. The flask was swirled gently to suspend the microspheres. The BSA solution was poured into the flask containing the microspheres and the flask was swirled gently on the orbital mixing platform at room temperature overnight.

The suspension was poured into a glass-sinter funnel attached to a Buchner funnel and the liquid removed under vacuum. In order to exhaustively remove non-covalently bound BSA, the microspheres were thoroughly washed in the sinter funnel using a cycle of re- suspension, by gentle stirring in the rinse buffer, followed by application of vacuum to remove the buffer, as follows: 2 x 250 ml de-ionized water followed by 2 x 250 ml 0.1 M acetate buffer pH 4.0 containing 0.5 M NaCl, followed by 2 x 250 ml de-ionized water, followed by 2 x 250 ml 0.1 M TRIS buffer, pH 9.5 containing 0.5 M NaCl, followed by 2 x 250 ml de-ionized water, followed by 3 x 250 ml 0.1% TWEEN® 20 solution in de- ionized water, followed by 3 x 250 ml de-ionized water, followed by 3 x 250 ml methanol. After the last methanol rinse, the vacuum was left on to for least 3 h to dry the microspheres. The product is a brilliant white free-flowing powder containing 4 - 7 mg BSA / gram microspheres, when assay by the bicinchoninic acid method (Example 4)

Example 4

Assay of BSA content of microspheres prepared in Example 3

This procedure enables the protein content of microspheres to be determined using the bicinchoninic acid (BCA) method (Pierce Chemical Co. Rockford, II).

The procedure was carried out as follows: In triplicate, between 5 and 6 mg of BSA- coated glass microspheres were weighed into 1.5 ml Eppendorf tubes. Standard protein (BSA) solutions at concentrations, 0, 25, 125, 250, 500, 750, 1000, 1500 & 2000 μg/ml were prepared by diluting a stock 2 mg/ml solution with de-ionized water. 50 μl of each standard was transferred to 1.5 ml Eppendorf tubes.

The BCA assay working reagent was prepared by mixing reagents 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 37⁰C 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 microsphere-sample tubes are centrifuged at 12000 x g for 30 seconds and the sample solution was transferred from below the surface plug of microspheres using a 2 ml syringe and needle. The needle was removed, a 0.2 μm filter fitted to the syringe body, and the needle replaced. The sample was then filtered directly into a 1 ml disposable cuvette. A new syringe and filter was used for each sample. The standard solutions were transferred directly to cuvettes. The Absorbance of each cuvette is read at 562 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 BSA content in mg per gram microspheres. The mean of the triplicate readings is stated.

Example 5

Procedure for the attachment of magnetite to BSA-coated microspheres In this procedure the microspheres prepared in Example 3 are coated with super- paramagentic iron oxide preparation (referred to herein as 'magnetite'), to yield a superparamagnetic microsphere.

Commercially available magnetite from a range of manufacturers is suitable for this method of preparation. The iron oxide (Fe₃O₄) nanoparticle preparation, (Nanostructured and Amorphous Materials Inc., Houston, Texas; stock no. 2650MY) is an example of a commercial material that is eminently suitable as a starting material for this procedure.

9.2 g of magnetite was weighed into a wide-necked bottle containing 460 ml de-ionized water and subjected to 15 minutes of high-shear mixing. Several commercially available high-shear mixing machines are suitable for this application. The Silverson SL2

(Silverson Machines Ltd., Chesham UK) was used in this Example. The high-shear suspension was then transferred to a 1 -litre Duran bottle. 4.6 g of microspheres prepared in Example 3 were suspended in 50 ml de-ionized water and added to the Duran bottle, with swirling to keep the magnetite in suspension. The bottle was placed on an orbital mixing table for 30 minutes at room temperature. The contents of the bottle were then poured into a 1 -litre glass beaker and left to stand for 30 minutes.

A magnetic device was used to transfer the magnetite coated microspheres, equally, between 2 x 200 ml de-ionized water in centrifuge bottles. The lids were screwed on and the bottles were shaken for 30 seconds. The bottles were centrifuged at 900 x g for 3 minutes. The bottles were then gently swirled in order to re-suspend the surface plug of microspheres, taking care not to disturb the pellet of magnetite, and centrifuged again. This procedure was repeated twice more.

Using a magnetic device, the buoyant microspheres were transferred into two fresh centrifuge bottles containing 200 ml of methanol, and the procedure just described was repeated. A glass-sinter funnel attached to a Buchner flask was filled with 250 ml methanol and the buoyant microspheres from both centrifuge bottles transferred into it using the magnetic device. The methanol was removed under vacuum. The microspheres were dried under vacuum for at least 3 hours. The product is a dark brown free-flowing powder that is stable indefinitely when stored dry at room temperature. The iron content, as determined by the assay with 1,10-phenanthroline (Example 6) is more than 20 mg Fe/g micro-spheres, and typically 200 — 300 mg Fe/g microspheres, dry mass.

The magnetite spontaneously binds irreversibly to the protein layer. Although no covalent bonds are formed between the protein layer and the oxide layer, the resulting structure is as robust as that obtained by a bonafide covalent attachment method, such as that disclosed in Example 9. Thus by reason of its inherent simplicity, the method disclosed in Example 5 is the preferred route to this structure.

The microspheres produced according to this example have been found to have a density of 0.5 g/cm³ +/-10%.

Example 6

Determination of the iron content of magnetite-coated microspheres

The iron content of magnetite-coated microspheres is determined 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 90⁰C 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 K₂HPO₄ 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 80⁰C 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_5I0 x 1136)/(mass of microspheres in grams). The stated value is the mean of triplicate readings.

Example 7

Alternative method for the preparation of magnetite. As an alternative to using commercially available magnetite preparations, colloidal magnetite was prepared de novo, by the reduction of an aqueous Fe(II)/Fe(III) mixture. A very convenient route to this synthesis is taught by Koneracka and co-workers in Journal of Molecular Catalysis B: Enzymatic, 2002, 18:13), which is incorporated by reference herein, in its entirety.

5.56 g of iron (II) sulfate heptahydrate was dissolved in 20 ml water and 10.8 g iron (III) chloride hexahydrate was dissolved in 20 ml water. A 150 ml beaker containing 15 ml of 7M ammonium hydroxide equipped with a mechanical stirrer and pH probe. The iron solutions were mixed together and then added dropwise to the beaker with parallel addition of concentrated ammonium hydroxide was added to maintain the pH at approx 9.5. The precipitated magnetite was washed thoroughly with de-ionized water by placing a strong magnet under the beaker to collect the solids and decanting off the supernatant. This product can be substituted for the commercial material in Example 5 to yield a microsphere product of an equivalent quality.

Example 8 Amino-functionalization of magnetite-coated microspheres

In this procedure the magnetite coating of the product disclosed in Example 5 is amine- functionalised.

6 g of magnetite-coated microspheres were placed in a 500 ml screw-top conical flask, followed successively by 200 ml methanol, 10 ml de-ionized water and 10 ml of 3- aminopropyltrimethoxysilane, with swirling to keep the microspheres in suspension. The flask was the placed on an orbital mixing platform for 3 hours at room temperature. The contents of the flask were then poured into a sinter funnel fitted to a Buchner flask, and the vacuum switched on to remove the reaction liquid. The vacuum was then turned off and 200 ml of methanol added, with gentle stirring using a glass rod, to re-suspend the microspheres. The vacuum was then turned on again to remove methanol. This procedure was repeated twice. The microspheres were then re-suspended in 100 ml of de-ionized water and poured into a centrifuge bottle. The sinter funnel was washed with a further 100 ml de-ionized water and the washings were added to the centrifuge bottle. Half of the contents of the centrifuge bottle (100 ml) were transferred to a second centrifuge bottle, with swirling, and 100 ml de-ionized water added to each bottle. The caps were placed on the bottles and they were then shaken vigorously for about 30 seconds and then at 900 x g for 3 minutes. The bottles were then swirled gently so as to re-suspend the surface plug of microspheres, but not the small pellet of dislodged magnetite, and centrifuged again. This procedure was repeated once more. By means of a magnetic device the microspheres in each bottle were transferred to fresh centrifuge bottles containing 200 ml methanol, shaken vigorously for about 30 seconds, and centrifuged at 900 x g three times, with swirling in between the re-suspend the microspheres. By means of the magnetic device, the microspheres were transferred into a glass-sinter funnel fitted to a Buchner flask containing 250 ml methanol. The microspheres were gently dispersed by stirring with a glass rod, and the vacuum turned on to suck away the methanol. The vacuum was left on for at least 3 hours to dry the microspheres. The product is a dark brown free-flowing powder that is stable indefinitely when stored dry at room temperature, and have an amino content, as determined using the ninhydrin assay (Example 2) in the range 15 - 25 μmol NH₂ /g microspheres dry mass. Example 9

Alternative procedure for the coating of microspheres with magnetite.

The magnetite is first amino-functionalized by treatment with 3- aminopropyltrimethoxysilane, and then used to coat microspheres prepared in Example 3, using a water-soluble carbodiimide to catalyse amide bond formation between the magnetite amino groups and the microsphere BSA-derived carboxylate groups. The product is a microsphere that is covalently coated with a layer of amino-functionalised superparamagnetic iron oxide that can be directly BSA-coated according to the method given in Example 10.

1.0 g of magnetite (Nanostructured and Amorphous Materials Inc., Houston, Texas; stock no. 2650MY) and 200 ml acetone were placed in a 250 ml Erlenmeyer flask, and then thoroughly mixed by sonication in an ultrasound bath for 1 hour. 5.0 ml of 3- aminopropyltrimethoxysilane was added, and the mixture sonicated for a further 2 hours. The flask was then placed on a magnetic plate strong enough to settle the magnetite. The supernatant was aspirated off and replaced with 150 ml acetone. The flask was removed from the plate, swirled vigorously to re-suspend the magnetite, and returned to the magnetic plate. The acetone was aspirated off and replaced with 35 ml borate buffer, with vigorous swirling to re-suspend the magnetite.

500 mg of BSA coated microspheres prepared in Example 3 were suspended in 20 ml MES-NaCl buffer (0.1M MES buffer, pH 4.7, containing 0.15 M NaCl) in a 50 ml centrifuge tube, followed by 250 mg l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride dissolved in 5 ml MES-NaCl buffer. The tube was then placed on a blood- tube rotator, with the wheel set vertically, and incubated for 10 min. at room temperature and 30 rpm. The tube was then centrifuged at 800 x g for 3 min and the subnatant was pipetted off and replaced with 25 ml of borate buffer (0.2 M borate titrated to pH 8.5 with NaOH). The tube was shaken to re-suspend the microspheres and then centrifuged at 800 x g for 3 min. The subnatant was removed with a pipette and the magnetite suspension in borate buffer added. The tube was incubated overnight on the blood tube rotator (30 rpm, room temperature) and then centrifuged at 800 x g for 5 minutes. The floating microspheres were transferred to a 50 ml screw-cap centrifuge tube containing 25 ml de-ionized water, and shaken for 5 seconds. The tube was then allowed to stand for 5 minutes. This procedure was repeated further 5 times. The microsphere suspension from the last wash was poured into a glass-sinter funnel attached to a Buchner flask and allowed to dry under vacuum for at least 1 hour. The product is a dark brown free- flowing powder that is stable indefinitely when stored dry at room temperature.

Example 10

BSA Coating of amino-functionalized magnetite-coated microspheres

In this Example, magnetite coated amino-functionalized microspheres as disclosed, for example in Examples 8 and 9 are covalently coated with BSA. The BSA coating serves as a substratum for the functionalization of the microsphere with the desired effector.

375 mg of BSA was dissolved in 75 ml de-ionized water in a 250 ml Erlenmeyer flask. The flask swirled gently to allow the BSA to dissolve completely and 185 mg of l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added. The flask was then incubated on an orbital platform for 15 minutes at room temperature. 5 g of amino- functionalized magnetite-coated microspheres prepared in Example 8 were placed in a 500 ml screw-top conical flask followed by 75 ml 0.2 M borate buffer, pH 8.5, with gentle swirling to suspend the microspheres. The BSA solution was poured into the flask containing the microspheres and incubated on an orbital platform overnight at room temperature.

The microspheres were separated from the reaction mixture by pouring them into a glass- sinter funnel under vacuum. In order to exhaustively remove non-covalently bound BSA, the microspheres were then thoroughly washed using the procedure exactly as described under Example 3. After the third and final wash with 250 ml methanol, 100 ml methanol was added and the microspheres were re-suspended by gentle swirling. 50 ml of the suspension was added to each of two 250 ml centrifuge bottles and a further 100 ml methanol was added to each bottle, with gentle swirling to mix. The bottles centrifuged at 900 x g for 3 minutes and then swirled gently to re-suspend the surface plug of microspheres, taking care not to disturbing the pellet of magnetite. This procedure was repeated twice more.

By means of a magnetic device the buoyant microspheres were transferred to two fresh 250 ml centrifuge bottles each containing 200 ml of 0.1% TWEEN® 20. The caps were replaced, and the bottles inverted gently several times, taking care to avoid excessive foaming. The bottles were centrifuged at 900 x g for 3 minutes, then swirled gently to re- suspend the surface plug of microspheres and centrifuged again. This procedure was repeated once more.

By means of a magnetic device, the buoyant microspheres were transferred to two fresh 250 ml centrifuge bottles each containing 200 ml of methanol. After replacement of the caps, the bottles were shaken gently for 30 seconds and then centrifuged at 900 x g for 3 minutes. The bottles were then swirled gently to re-suspend the surface plug of microspheres and centrifuged again. This procedure was repeated once more.

The buoyant microspheres layer from both centrifuge bottles was transferred, using the magnetic pen, to a glass-sinter funnel containing 250 ml ethanol. The vacuum was turned on to remove the methanol left for at least 3 hours to air-dry the microspheres. The product is a dark brown free-flowing powder with a total protein content, determined using the BCA assay (Example 4), of between 12 and 24 mg / gram microspheres (dry mass). The microspheres are stable indefinitely when stored dry at room temperature.

The microspheres of the preferred embodiments, as developed through the above Examples can be characterised by reference to the set of physical and chemical properties as listed in table 1.

Table 1: Physical and chemical properties of microspheres of preferred embodiments

Example 11

Method of coating microspheres with antibody In this procedure antibody is coupled directly to the microsphere surface by random glutaraldehyde cross-linking between surface- and protein-amino groups.

5.5 mg of microspheres prepared in Example 10 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 Na₂CO₃ plus 0.1M NaHCO₃, 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 or 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 ipm, 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 of 5OmM₅ and the tube returned to the wheel. The microspheres were finally washed by successive transfers into 5 x ImI of PBS-azide (1OmM Phosphate, 2.7 mM KCl and 137 mM NaCl; pH7.4 containing 0.02% sodium azide) with 5 min. on the wheel between transfers and stored at 4°C. Example 12

Use of buoyant beads coated with anti-Cryptosporidium parvum antibody to enumerate Cryptosporidium parvum oocysts in a sample of drinking water.

Water Sample Preparation

Cryptosporidium parvum oocyst stock suspensions were prepared by diluting a commercial preparation of freshly harvested oocysts (ca. 4 x 10⁶ oocysts/ml) obtained from Moredun Scientific Ltd, Edinburgh UK). Sequential serial dilutions of 1:100, 1:10 and then 1:2, with vortexing for 3 minutes at each step, gave a working stock solution containing ca. 100 oocysts per 50μl. The exact number was determined by transferring

50 μl of the stock to a microscope slide, and then fixing, fluorescent staining and counting as described below. 6 repeats were made to give a mean value and standard deviation.

In order to prepare the 'spiked' water sample 50μl of the working stock oocyst suspension was added to 35 ml of the water sample, which was then vortexed for 3 minutes.

Buoyant microspheres stock solution

The buoyant microspheres prepared according to Example 5 are stored at 4°C in 75% aqueous glycerol, in a dropper bottle. During storage they float to the surface and must be re-dispersed immediately prior to use. This is done by means of gentle end-over-end rotation of the bottle on a rotating tube rack. For this purpose the rack is set to rotate at a speed of 10 rpm for 5 minutes.

Recovery of oocysts using buoyant microspheres 35 ml of the test sample, contained in a polypropylene, disposable 50 ml screw-cap centrifuge tube was diluted with 3.5 ml of Buffer A (buffer A consists of 500 mM N₅N- Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, neutralised to pH 7.14 with sodium hydroxide, 1.5 M NaCl and 1% v/v Tween 80). 3 drops, that is approximately 60 μl, of the buoyant microspheres stock solution was then added, taking care to ensure that the microspheres were thoroughly dispersed.

The sample tube was tightly capped and attached to a rotating wheel (blood-tube wheel). The wheel was set at an angle of approximately 20° from the horizontal and the rotation rate adjusted to 34 rpm.

After 1 hour the tube was removed and placed into a rotating tube rack with a magnetic field disposed at one side and rotated end-over-end at a speed of 15 rpm.

The tube was rotated for 1 minute. During this time the buoyant microspheres migrate to the wall of the sample tube. The rotator was then stopped with the tube in the vertical position and the magnetic field was removed. The tube was allowed to stand until all the buoyant microspheres have 'streamed' up the side of the tube to the surface of the sample. This process can be observed directly, since the microspheres are intensely dark brown in colour.

The tube was then centrifuged at 500 x g for 3 minutes. The microspheres can then be seen as a brown ring on the surface, and the debris (fine sand or silt from the water- concentrate) can be seen as a loose pellet in the bottom of the tube.

The tube was returned to the rotating rack and 1 ml of a 1:10 dilution of Buffer A was dispensed into a 2 ml microcentrifuge tube (siliconised with 'Sigmacoat'; product cat. No. T3531₅ Sigma Chemical Co) and placed in a microcentrifuge tube rack.

Separation of oocysts from buoyant microspheres The microspheres were removed from the surface of the sample using a magnet, and transferred into the microcentrifuge tube. To facilitate this procedure, the magnetic field was left in place. The magnetic field was then removed and the rack was rotated end- over-end for 30 sec at a speed of 15rpm. The magnetic field was then re-positioned and the rack was rotated end-over-end for a further 30 sec at a speed of 15rpm, to re-capture the buoyant microspheres.

The entire liquid sample was then withdrawn from the microcentrifuge the tube (including trapped in the cap) using a 1 ml micro-pipette, leaving the buoyant microspheres and attached oocysts behind, retained by the magnet.

The magnetic field was removed from the rack and 150 μl of 0.1 M hydrochloric acid was added to the microcentrifuge tube. The tube was vortexed for 1-2 seconds, left to stand for 5 minutes and then vortexed once again, for 1-2 seconds. The tube was then replaced in the microcentrifuge tube rack with the magnetic field in place. The rack was shaken gently until all of the buoyant microspheres were captured.

15 μl of 1.0 M sodium hydroxide was placed on a microscope slide with 11 mm diameter wells (Hendley & Co, Essex, UK). The contents of the tube were transferred to the microscope slide by means of a micropipette and the slide was incubated on a slide heater at 45 ⁰C until dry.

Staining of oocysts

The oocysts were fixed by adding one drop of methanol to the slide well and then allowing it to dry. The slide was then placed in a humid chamber; 50 μl of Fluorescein isothiocyanate (FITC)-conjugated anti-Cryptosporidium antibody solution without Evans Blue counter-stain (Crypto Cel®, Cellabs Inc.) was added to the well and the slide was incubated at 37°C for 1 hour. The antibody solution was aspirated off and 100 μl of phosphate-buffered saline (PBS) was added. The slide was allowed to stand for 1 min and the PBS was aspirated off. 100 μl of 4',6-diamidino-2-phenylindole (DAPI) solution (0.4 μg DAPI/ml PBS) was added to the well. The slide was allowed to stand for 2 minutes and then the DAPI solution was removed by aspiration. 100 μl of deionised water was then added, and aspirated away after a few seconds. One drop of mounting fluid was added, and a cover-slip was placed over the well. The oocysts were counted using an epifluorescence microscope.

The percentage efficiency of recovery from each water sample is defined as (a/b) x 100, where a = number of oocysts recovered from the water sample, b = the number of oocysts in the 50 μl spike, this being taken as the mean of the 6-fold control count.

The results of a study using the above protocol are shown in Figure 1. Figure 1 is a comparison of the performance of the Cryptosporidium parvans assay based on the buoyant microspheres of the present invention, and a commercially available test that utilizes non-buoyant beads comprising a polystyrene shell wrapped around a magnetite core with a typical particle size is 5.8 micrometers and a density around 1.2g/cm3. (Dynal, Oslo). The water samples were harvested on the dates shown in the locations given. The pellet sizes refer to packed pellet volumes recovered from 35 ml of sample by centrifugation. Each water sample (10 ml for the assay using the Dynal test-kit and procedure, or 35 ml using the buoyant microspheres and procedures of the present invention) were spiked with ca 100 Cryptosporidium parvum oocysts as described in Example 12. The Dynal assay was carried out according to the manufacturer's instructions. The buoyant microspheres of the present invention were used as disclosed in Example 12. For each entry (water sample) the left-hand (striped) bar represents the result of the test using Dynal' s kit and the right-hand (speckled) bar represents the results obtaining using the present invention. The Dynal test score for the sample 'Suffolk Jan 04' was zero.

The data show that on the microspheres of the current invention perform as well, within the error of the measurements, or better than, the commercially available product based on dense polystyrene beads, even though the water sample volume is considerably larger in the assay using the present invention than in the assay using Dynal' s kit (35 ml vs 10 ml), so that the oocyst concentration is correspondingly lower (ca 2.8 / ml vs 10 / ml).

Example 13

Determination of Densities of Microspheres

The skilled person would be well aware of how to measure densities of microspheres according to the present invention, for example, by gas or liquid pycnometery. However, in the interests of clarity, the following method may be used to determine the density of a sample of microspheres :-

A) Using a calibrated 4 decimal place balance, weigh a 10ml graduated cylinder or volumetric flask.

B) Add 1 gram of dry microspheres to the vessel.

C) Using deionized water (density lg/cm³), approximately half fill cylinder/flask and gently agitate for 5 min to completely wet out microspheres.

D) Add deionized water until the level of the microsphere suspension reaches the 10ml graduation.

E) Record the mass of the filled vessel and calculate the net mass of the microsphere suspension by subtracting the mass of the empty vessel. F) Subtract 1 gram from the calculated mass. This is the contribution of the water to the mass of the suspension and, as the density of water is lg/cm³, this also equates to the volume of water added to produce 10ml of microsphere suspension.

G) Subtract this volume from 10ml to give the volume contribution of the 1 gram of microspheres. H) Using the determined volume and the mass (lgram), calculate the density of the microsphere sample using O=MIV (D is density and is measured in g/cm³, M is mass and is measured in g, and V is volume and measured in cm³).

Claims

1) A microsphere comprising a core, an anchoring agent and a magnetizable agent, wherein the magnetizable agent is anchored to the core by binding to the anchoring agent .

2) A microsphere as claimed in claim 1, wherein the anchoring agent comprises a magnetizable-layer binding agent which binds to the magnetizable agent.

3) A microsphere as claimed in claim 2, wherein the anchoring agent further comprises a coupling agent which couples the magnetizable- layer binding agent to the core .

4) A microsphere as claimed in claim 2 or 3 , wherein the magnetizable agent binds directly to the magnetizable- layer binding agent .

5) A microsphere as claimed in claim 2 or 3 , wherein the magnetizable agent is coupled to the magnetizable- layer binding agent by a coupling agent.

6) A microsphere as claimed in any of the preceding claims, further comprising an effector binding agent coupled to the magnetizable agent by a coupling agent.

7) A microsphere as claimed in any of the preceding claims, further comprising an effector agent.

8) A microsphere as claimed in claims 7, wherein the effector binding agent binds to the effector agent. 9) A microsphere as claimed in claim 7, wherein the effector agent is coupled to the magnetizable agent by a coupling agent.

10) A microsphere as claimed in any of the preceding claims, wherein the core comprises any of the following; a synthetic polymer, a natural polymer, a co-polymer, a block co-polymer, polymethylmethacrylic acid, a protein, a glass, a metal oxide, titania, zirconia, alumina, magnesia, a ceramic, a ceramic oxide, or any combination thereof .

11) A microsphere as claimed in claim 10, wherein the core is a borosilicate glass microsphere.

12) A microsphere as claimed in any of the preceding claims, wherein the core has a diameter from 0.1 to 1000 μm.

13) A microsphere as claimed in any of the preceding claims, wherein the magnetizable agent comprises a superparamagnetic agent .

14) A microsphere as claimed in any of the preceding claims, wherein the magnetizable agent is prepared by high shear mixing or by reduction of Fe (II) /Fe (III) salt mixture.

15) A microsphere as claimed in any of claims 2 to 12, wherein the magnetizable-layer binding agent comprises 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.

16) A microsphere as claimed in 15, wherein the magnetizable-layer binding agent is a serum albumin or gelatine .

17) A microsphere as claimed in any of claims 3 to 16, wherein the coupling agent is any agent that is capable of binding an inorganic molecule to an organic molecule .

18) A microsphere as claimed in claim 17, wherein the coupling agent is any agent that is capable of binding a glass, iron oxide or spinel ferrite to an organic molecule .

19) A microsphere as claimed in claim 18, wherein said coupling agent is a silane, a germane, or a mixture thereof .

20) A microsphere as claimed in claim 19, wherein the silane is any of the following; an amino silane, a carbonyl silane, a carboxy silane, a hydroxyphenyl silane, a sulfhydryl silane, 3 -aminopropyltrimethoxy-silane, or any combination thereof.

21) A microsphere as claimed in any of claims 7 to 20, wherein the effector agent comprises 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.

22) A microsphere as claimed in any of claims 6-8, and 10- 21, wherein the effector binding agent comprises any of the following; a synthetic polymer, polymethyl- methacrylic acid, a co-polymer, a block co-polymer, a natural polymer, a protein, a peptide, an amino acid, or any combination thereof.

23) A microsphere as claimed in 22, wherein the effector binding agent is a serum albumin or gelatine.

24) A microsphere as claimed in any of the preceding claims, wherein the microsphere comprises no less than 1 mg protein per gram of microsphere, preferably from 4 to 24 mg protein per gram of microsphere .

25) A microsphere as claimed in any of the preceding claims, wherein the microsphere comprises at least 10 mg of magnetizable agent per gram of microsphere, preferably from 150 to 400 mg of magnetizable agent per gram of microsphere .

26) A microsphere as claimed in any of the preceding claims, wherein the microsphere is a buoyant microsphere.

27) A microsphere as claimed in claim 26, wherein the microsphere has a buoyant density from 0.25 to 0.85 g/cm³. 28) A microsphere as claimed in claim 26 or 27, wherein the microsphere is hollow.

29) A method for preparing a microsphere comprising the step of chemically modifying the surface of a core prior to the step of anchoring a magnetizable agent to the core.

30) A method as claimed in claim 29, wherein the chemical modification comprises the step of anchoring a magnetizable-layer binding agent to the core.

31) A method as claimed in claim 30, wherein the anchoring of the magnetizable-layer binding agent comprises the step of binding a coupling agent to the core.

32) A method as claimed in claim 31, wherein the anchoring of the magnetizable-layer binding agent further comprises the step of binding the magnetizable-layer binding agent to the coupling agent.

33) A method as claimed in any of claims 30-32, wherein following the binding of the magnetizable-layer binding agent, but prior to the binding of the magnetizable agent, the microsphere comprises at least 0.5 mg magnetizable-layer binding agent per gram of microsphere, preferably from 4 to 7 mg of magnetizable- layer binding agent per gram of microsphere .

34) A method as claimed in any of claims 30 to 33, wherein the magnetizable agent binds directly to the magnetizable-layer binding agent.

35) A method as claimed in any of claims 30 to 33, wherein the step of anchoring the magnetizable agent to the core further comprises the step of coupling the magnetizable agent to the magnetizable-layer binding agent by a coupling agent.

36) A method as claimed in any of claims 29 to 35, further comprising the step of coupling an effector binding agent to the magnetizable agent by a coupling agent.

37) A method as claimed in any of claims 29 to 30, further comprising the step of anchoring an effector agent to the microsphere .

38) A method as claimed in claims 37, wherein the step of anchoring an effector agent comprises the steps of binding the effector agent to the effector binding agent .

39) A method as claimed in claim 37, wherein the step of anchoring the effector agent comprises the step of coupling the effector agent to the magnetizable agent by a coupling agent.

40) A method as claimed in any of claims 29 to 39, wherein the core comprises any of the following; a synthetic polymer, a natural polymer, a co-polymer, a block copolymer, polymethylmethacrylc acid, a protein, a glass, a metal oxide, titania, zirconia, alumina, magnesia, a ceramic, a ceramic oxide, or any combination thereof.

41) A method as claimed in claim 40, wherein the core is a borosilicate glass microsphere.

42) A method as claimed in any of the claims 29 to 41, wherein the core has a diameter from 0.1 to 1000 μm.

43) A method as claimed in any of claims 27 to 39, wherein the magnetizable agent comprises a superparamagnetic agent .

44) A method as claimed in any of claims 29 to 43, wherein prior to introducing the magnetizable agent to the microsphere, the magnetisable agent is prepared by high shear mixing or by reduction of Fe (II) /Fe (III) salt mixture .

45) A method as claimed in any of claims 29 to 44, wherein the magnetizable-layer binding agent comprises any of the following; a synthetic polymer, polymethyl- methacrylic acid, a co-polymer, a block co-polymer, a natural polymer, a protein, a peptide, an amino acid, or any combination thereof .

46) A method as claimed in 45, wherein the magnetizable binding agent is a serum albumin or gelatine.

47) A method as claimed in any of claims 31 to 46, wherein the coupling agent is any agent that is capable of binding an inorganic molecule to an organic molecule.

48) A method as claimed in claim 47, wherein the coupling agent is any agent that is capable of binding a glass, iron oxide, or spinel ferrite to an organic molecule.

49) A method as claimed in claim 48, wherein said coupling agent is a silane, a germane, or a mixture thereof.

50) A method as claimed in claim 49, wherein the silane is 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.

51) A method as claimed in any of claims 38 to 50, wherein the effector agent comprises 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.

52) A method as claimed in any of claims 36 to 38 and 40 to 51, wherein the effector binding agent comprises any of the following; a synthetic polymer, polymethyl- methacrylic acid, a co-polymer, a block co-polymer, a natural polymer, a protein, a peptide, an amino acid, or any combination thereof.

53) A method as claimed in 52, wherein the effector binding agent is a serum albumin or gelatine.

54) A method as claimed in any of claims 36 to 54, wherein following the step of coupling an effector binding agent to the magnetizable agent, but before the step of binding the effector agent to the effector binding agent, the microsphere comprises no less than 1 mg protein per gram of microsphere, preferably from 10 to 20 mg protein per gram of microsphere.

55) A method as claimed in any of claims 29 to 54, wherein the microsphere comprises at least 10 mg of magnetizable agent per gram of microsphere, preferably from 150 to 400 mg of magnetizable agent per gram of microsphere.

56) A method as claimed in any of claims 29 to 55, wherein the microsphere is a buoyant microsphere.

57) A method as claimed in claim 56, wherein the microsphere has a buoyant density from 0.25 to 0.85 g/cm³.

58) A method as claimed in claims 56 or 57, wherein the microsphere is hollow.

59) A microsphere prepared according to any of the methods claimed in claims 29 to 58.

60) A method as claimed in any of claims 29 to 58, wherein the microsphere prepared by any such method is a microsphere as claimed in any one of claims 1-28.

61) A method for preparing a microsphere substantially as hereinbefore described. 62 A microsphere according to claim 1, and substantially as hereinbefore described. 62) A microsphere comprising a magnetizable agent anchored thereto by chemical bonding substantially as hereinbefore described.