WO2008061363A1 - Biomolecule compatible silica particles - Google Patents

Abstract

The present invention relates to monodisperse biomolecule compatible silica particles prepared from organic polyol silanes in the presence of one or more stabilizing entities. The particles may optionally contain detection molecules and/or surface modifications. The particles are useful for the detection and/or killing of biological entities such as bacterial pathogens.

Description

B&P File No. 3244-166/PF

Title: BIOMOLECULE COMPATIBLE SILICA PARTICLES FIELD OF THE INVENTION

The present invention relates biomolecule compatible silica particles, methods of preparing them and to uses of the particles, in particular to capture, detect and, optionally, destroy biological entities. BACKGROUND OF THE INVENTION (a) Silica particles

The use of sol-gel techniques provides an exceptional degree of morphological control in the preparation of silica. t hus, total porosity, pore size and shape, regularity of pore distribution, etc. can be manipulated using a variety of starting materials, reaction conditions and dopants (Brinker, C. J.; Scherer, G. W. Sol-Gel Science, Academic Press: San Diego, 1990.). Recently, highly biocompatible monolithic silica materials have been prepared that are compatible with the incorporation of proteins and related biologically derived molecules.

Silica particles are also well known. Small, monolithic silica particles can be prepared by the hydrolysis of alkoxysilanes in the presence of alec hoi and water. Spherical silica particles are usually prepared in a sol-gel process first reported by Stober et al. using alkoxysilanes based on methanol or ethanol as precursors. These "Stober" particles are synthesized via base catalyzed hydrolysis and condensation in a water/alcohol solution and can be easily tuned in size by changing the initial concentrations of the silanes. Larger, less regular silica is available by the acidification of sodium silicates and related materials, which leads to precipitates. Thus, proper selection of starting materials and conditions allows the preparation of silica-based materials of a variety of sizes, morphologies and with surface chemistries. In the latter case, modification of the hydrophilic surface is well known to those skilled in the art. It is possible to introduce a variety of non-functional hydrophilic or hydrophobic groups, chemofunctional molecules, or biofunctional molecules, for example, peptides, proteins, DNA oligomers, etc. (b) Silica on Paper

Paper, a web formed from cellulose fibers, optionally containing lignin, is an inexpensive high surface area support, the structure of which is highly controllable by those skilled in the art. The surface nature of the material is readily modified to be more hydrophobic (e.g., for printing, by use of sizing agents), stronger (by addition of wet and dry strength polymers), brighter or colored by the addition of pigments, and made more opaque and stronger by the addition of mineral fillers (clay, silica). Paper is widely used as a filtration aid to separate materials, and as a medium to carry information (e.g., by printing of ink on paper), (c) Pathogen Detection

Although biological entities such as bacteria can be beneficial to human and animal health, such as part of the normal gastric flora, transmission of diseases through the air or contaminated water, or through contact with surfaces in hospitals, in food, and by other means is recogni/ed by public health organizations as an important challenge. It is thus necessary to devise methods not only to destroy pathogens, but to establish whether the destruction has been effective (i.e., is the water safe to drink, is the hospital bed safe to touch?). Although broad spectrum disinfection strategies are well known, it can be as important to detect which pathogens are or have been present in/on a given medium as to destroy them. Furthermore, there would be a benefit in destroying only the detrimental organisms, leaving beneficial organisrrs alone.

Several strategies exist for the specific detection of pathogens. Most of these rely on selective or explicit binding of molecules to receptors on cell surfaces. Thus, immunoassays can be based on the specific binding of antibodies to cell surfaces. Alternate strategies rely on the presence of molecules generated by a specific organism that can be detected directly, or more generally after amplification, by chromogenic, fluorescent, aggregation (turbidity or change in surface plasmon effects of gold nanoparticles), etc. Thus, enzyme based assays are also efficacious. SUMMARY OF TI IK INVENTION

Nearly monodisperse spherical silica particles have been prepared using an organic polyol silane as a precursor under methanol- and ethanol-free conditions. The colloidal stabilization of the particles was accomplished with the use cf poly(ethylene glycol) (PEG) or one of its derivatives, which provide steric stabiliza ion and further increases the biomolecule compatibility of the silica. The silica particles made under mild conditions optionally contain, within (i.e. inside) their matrix or attached to their surface, passive (colored) detecting groups, active detecting groups that respond to a chemical stimulus such as pH, active detecting groups that respond to a biochemical small molecule such as adenosine triphosphate (ATP) and enzymatic detecting groups. In addition to the entrained detection system, particles have been prepared in which capture molecules are tethered to the external surfaces of the silica material. Such capture molecules, such as antibodies, can selectively bind to specific molecules including molecules found on cell surfaces. Such silica materials can also optionally possess surface modification that leads to pathogen destruction. As an example, enzymes that cause cell lysis may be bound to the silica surface. The silica material thus has the potential to bind, kill and detect pathogens in a specific or more generic manner.

Accordingly the present invention includes a method of preparing monodisperse biomolecule-compatible silica particles comprising combining organic polyol silane precursors in the presence of one or more particle stabilizing entities, and optionally in the presence of one or more biomolecules and/cr one or more entities that stabilize the optional presence of biomolecules, under conditions sufficient for the formation of the particles. In another embodiment of the invention, the one or more particle stabilizing entities are water soluble polymers, for example pol> (ethylene glycol) (PEG) or modified derivatives of PEG. The particle stabilizing entities become incorporated within the particle itself, accordingly the biomolecule compatible silica particles comprise a silica matrix and particle stabilizing entities, such as water soluble polymers. In certain cases, for example, PEG modified with -L- Si(OR)₃ end groups, where R is any group that is hydrolyzable under the reaction conditions and L is a suitable linking group or a direct bond, the stabilizing entity may be chemically bound to a surface of the particle. In a further embodiment of the invention the method further comprises combining the polyol silane precursors and one or more particle stabilizing entities in the presence of one or more detector molecules so that the detector molecules become entrapped within (i.e. inside) or on the surface of the particle. In another embodiment of the invention, ihe biomolecule compatible silica particles are optionally surface modified. Such surface modifications include tethers that covalently or non-covalently link biomolecules to the silica surface, for example, through chelation of the biomolecule. I n embodiments of the invention the surface modification comprises the incorporation of non- functional hydrophilic or hydrophobic groups, chemofunctional molecules, or biofunctional molecules, for example, peptides, proteins, including antibodies, bacterial phage, DNA oligomers, DNA, etc. or on the surface of the particle.

The present invention also includes monodisperse, biomolecule compatible silica particles prepared using the method of the invention. In a further embodiment, the silica particles further comprise detector molecules or biomolecules entrapped within (i.e. inside) or on the surface of the particles. In another embodiment of the invention, the biomolecule compatible particles have a diameter of from about 100 nm to about 1000 nm, suitably from about 200 nm to about 500 nm. The present invention also includes methods of detecting one or more biological entities comprising contacting a sample suspected of containing the biological entity with a biomolecule compatible particle of the invention and detecting the presence of the entities. In one particular embodiment, the silica particles are associated with a paper surface, and the paper surface is used to detect the presence or absence of a biological entity, such as a pathogen.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only , since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRA WINGS

The invention will now be described in relation to the drawings in which: Figure IA shows a schematic for preparing silica particles under biomolecule compatible conditions using one embodiment of the present inventiDn. Figure IB shows the incorporation of detecting molecules in the sol in one embodiment of the present invention permits their entrapment in the final silica particles. Figure 2 show a conceptual model of detecting silica materials. Figure 3 shows SEM pictures of the three types of materials that were formed: (a) particles; (b) fused particles; and (c) monolithic pieces.

Figure 4 shows SEM images for 0.6K S-PEG-S, IK S-PEG-S and S-PEG-OH. Figure 5 shows SEM images of silica particles made from different end group modified PEGS. From top to bottom, 2K HO-PEG-OH, 2K A-PECJ-A and 2K S- PEG-S.

Figure 6 shows SEM images of silica particles made from different end group modified 600 MW PEGS. A. HO-PEG-OH; B. A-PEG-A; and C. S-PEG-S.

Figure 7 shows SEM pictures of: 10 K HO-PEG-OH prepared with A: stirring, B: ultrasound (right). C: 2K HO-PEG-OH prepared with ultrasound. IK A-PEG-A prepared with D: stirring, E: ultrasound (right).

Figure 8 shows SEM images of 10 K HO-PEG-OH with increasing armunts of water. Top to bottom: 250 μl, 500 μl, 1 ml, 1.25 ml.

Figure 9 shows the plaques formed by T4 bacterial phage attached to silica particles. A: from suspension, B: on paper.

Figure 10 shows osteoblasts bound to his-tagged antibodies tethered to a silica surface through ionic bonds to nickel-citrate chelates. DETAILED DESCRIPTION OF THE INVENTION (1) DEFINITIONS

The term "gel'^" as used herein refers to solutions (sols) that havj lost flow. The term "gel time" as used herein is the time required for flo vv of the sol-gel to cease after addition of the buffer solution, as judged by repeatedly tilting a test-tube containing the sol until gelation occurs.

The term '"cure'^" as used herein refers to the crosslinking process, the continued evolution of the silica matrix upon aging of the silica following gelation, until the time when the gel is treated (e.g., by washing, freeze drying, etc.).

The term "PEG" as used herein means poly(ethylene glycol^ which has the formula HO-(CH₂CH₂O)_n-H, wherein n can vary from one to several hundred thousand. It should be noted that the terms "oxide" (as in poly(ethyl _-ne oxide)) and

"glycol" (as in poly(ethylene glycol)) may be used interchangeably and the use of one term over the other is not meant to be limiting in any way.

The term "organic polyol silane precursor" refers to a non-cεtenated organic silane compound containing no significant quantities of siloxane (O-Si-0-Si) linkages. The term "monodisperse" as used herein refers silica particles of generally uniform size in a dispersed phase. The term "particle" as used herein refers to generally individual spherically shaped silica material. In an embodiment the particles are from about 100 nm to about 1000 nm, suitably from about 200 nm to about 500 nm, in diameter.

The term "tether" or "tethering" as used herein means to attach either directly or via a linker, via covalent or non-covalent interactions. Suitably the tethering is by means of covalent linkages. Non-covalent interactions include, for example, ionic, electrostatic and hydrophobic interactions.

By "biomolecule compatible" it is meant that conditions jr a substance stabilizes proteins and/or other biomolecules against denaturation or does not facilitate their denaturation.

The term "biomolecule" as used herein means any of a wide variety of proteins, peptides, enzymes and other sensitive biopolymers including DNA oligomers, DNA aptamers, DNA and RNA, and complex systems i icluding whole plant, animal and microbial cells that may be entrapped in silica. In embodiments of the invention, the biomolecule is a protein, or fragment therecf. In further embodiments of the invention, the biomolecule is in its active form. By active form, it is meant that the biomolecule is in a conformation, configuiation or other arrangement found in natural state when the biomolecule is functional.

As used herein, the term "functional group" refers to an atom or group of atoms, acting as a unit (i.e., a chemical moiety), that has replaced a h>drogen atom in a hydrogen carbon molecule and whose presence imparts characteristic qualities to the resultant molecule. For example, acidic silica may be functionalized by reaction with an organosilane-containing hydrophilic functional groups, such as, but not limited to alcohol, diol, carboxylate, or ammonium, and the like, to produce a hydrophilic silica. In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, ntegers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their deriv ativ es. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it mod fies. (11) METHOD OF FORMING BIOMOLECULE COMPATIBLE SILICA PARTICLES

The preparation of biocompatible protein doped silica particles is challenged by the difficulty of incorporating biomolecules within the silica mesostructure without accompanying denaturation of the biomolecules. The preparation of spherical silica particles (diameter 200nm - 500nm) under highly biocompatible conditions in a one step process has been accomplished herein. Diglycerylsilane (DGIi) was reacted under ethanol or methanol-free conditions in pure water or in buffer solutions with or without glycerol as additive. Steric stabilization of the particles was obtained by using poly(ethylene glycol) (PEG) of various molecular weights and end groups as a co-reagent. The particles can be prepared at or below ambient temperature and are reasonably monodisperse in size.

Accordingly, the present invention includes a method of preparing monodisperse biomolecule-compatible silica particles comprising combining organic polyol silane precursors in the presence of one or more particle stabilizing entities, and optionally in the presence of one or more biomolecules and/cr one or more entities that stabilize the optional presence of biomolecules, urder conditions sufficient for the formation of the particles

In embodiments of the invention, the conditions sufficient for the formation of the particles comprise a water-containing medium and solutions prepared from about 0.1 - 10 weight percent polyol silane, for example DGS, in water-containing solvents, more suitably about 0.5 to about 5 weight percent polyol silane in solutions with about 50% to about 100% water content, and most suitably about 1 to about 2 weight percent polyol silane in aqueous solutions. The conditions also include entities necessary for the stabilization of particulate species, such as polymeic species, and optionally , for the stabilization of biomolecules (e.g., enzymes or proteins) which can include, humectants (such as glycerol), salts and appropriate nutrients. Conditions further comprise other factors for sol-gel condensations processes known to those skilled in the art. Catalysts are not required for the sol-gel process to occur. The pH ol^' the particle forming reaction is suitably between about 5 and about 9, suitable about 7 to about 9. Suitably buffered solutions are utilized. Buffers that are known to be used with biomolecules are suitable, for example, HEPES buffer.

The term "organic polyol silane precursor" refers to a non-catenated organic silane compound containing no significant quantities of siloxane (O-Si-0-Si) linkages, r he organic portion of the silane precursor is an organic polyol. The Drganic polyols may be selected from a wide variety of such compounds. By "polyol", it is meant that the compound has more the one alcohol group. The organic portion of the polyol may have an> suitable structure ranging from straight and branched ciain alkyl and alkenyl groups, to cyclic and aromatic groups. For the preparation of biomolecule compatible particles, it is a suitable embodiment for the organic polyol to be biomolecule compatible.

It is suitable for the polyol to be derived from natural sources. Particular examples of suitable polyols include, but are not limited to glycerol, glycerol derivatives, glycols, sugar alcohols, sugar acids, saccharides, oligosaccharides and polysaccharides. Simple saccharides are also known as carbohydrates or sugars. Carbohydrates may be defined as polyhydroxy aldehydes or ketones or substances that hydroyl/e to yield such compounds. The polyol may be a monosaccharide, the simplest of the sugars or carbohydrate. The monosaccharide may be any aldo- or keto-triose, pentose, hexose or heptose, in either the open-chained or cyclic form. Examples of monosaccharides that may be used in the present invent on include, but are not limited to, allose, altrose. glucose, mannose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, threose, erythrose, glyceraldehydes, sorbose, fructose, dextrose, levulose and sorbitol. The polyol may also be a disaccharide, for example, but not limited to, trehalose, sucrose, maltose, cellobiose and lactose. Polyols also include polysaccharides, for example, but not limited to dextran, (500- 50,000 MW), amylose and pectin. Other organic polyols that may be used include, but are not limited to glycerol, propylene glycol and trimethylene glycol. Suitably the polyol is glycerol. Specific examples of organic polyols that may be used in the methods of the invention, include but are not limited to, glycerol, sorbitol, maltose, trehalose, glucose, sucrose, amylose, pectin, lactose, fructose, dextrose and dextran ard the like. In embodiments of the present invention, the organic polyol is selected from glycerol, sorbitol, maltose and dextran. Some representative examples of the resulting polyol modified silane precursors include diglycerylsilane (DGS), and tetiaglycerylsilane (TGS). One of skill in the art can readily appreciate that other molecules including simple saccharides, oligosaccharides, and related hydroxylated compounds can also lead to viable silane precursors. Higher molecular weight water soluble polyol polymers do not leach from the silica particles, once formed, and therefore are a specific embodiment of the invention.

The preparation of organic polyol modified silanes is described in co-pending U.S. Patent Publication No. 20040034203.

In the method of the invention, the particles are prepared in ihe presence of one or more particle stabilizing entities such as water soluble polymers, for example poly(ethylene glycol) (PEG) or modified derivatives of PEG. The particle stabilizing entities become incorporated within the particle itself. In certain cases, for example, PEG modified with -L-Si(OR)₃ end groups, where R is any group that is hydrolyzable under the reaction conditions, (and the R groups may be the same or different), for example an alkyl group such as ethyl or methyl, and L is a direct bond or a suitable linking group, such as alkylene, for example propylene, the stabilizing entity may be chemically bound to a surface of the particle. The water soluble polymer may be selected from any such compound that stabilizes the particle formation and includes, but is not limited to, one or more of: polyethers, for example, poly(ethylene glycol) (PEG), modified PEG, amino- terminated poly(ethylene glycol) (PEG-NH₂), poly(propylene glycol) (PPG), poly(propylene oxide) (PPO), poly(propylene glycol) bis(2-amino-propyl ether) (PPG-NH2); polyalcohols, for example, poly(vinyl alcohol); polysaccharides; poly(vin> l pyridine); polyacids, for example. poly(acrylic acid); polyacrylamides e.g. poly(Λ'-isopropylacrylamide); (polyNIPAM); and polyallylamine (PAM). By '"water soluble'^' it is meant that the polymer is capable of being formed into an aqueous solution having a suitable concentration. In an embodiment of the invention, the water soluble polymer is selected from one or more of PEG or modified PEG. In an embodiment of the invention, modified PEG refers to PEG that has been modified by incorporation of functional groups on one or both ends of the polymer. Examples of such functional grovps include, for example, allyl and -L-Si(OR)₃, where L is a direct bond or a suitable linking group such as alkylene or alkylene interrupted by one or more heteroatoms selected from O, S, NH and NMe and R is any group that is hydrolyzable under the particle-forming reaction conditions, for example alkyl or organic polyol groups, and the three R groups may be the same or different. In an embodiment of the invent on, -L-Si(OR)₃ is propylene triethoxysilane.

In embodiments of the invention the molecular weight (MM') of the water soluble polymer has an effect on the formation of the biomolecule compatible particles. A person skilled in the art may adjust the MW of the water soluble polymer, depending on the identity of the polyol silane precursor and the identity of the polymer (for example whether it is mono- or bifunctionalized on the ends) and select a MW that stabilizes particle formation. In an embodiment of the invention, the MW of the water soluble polymer is greater than about 200 g/mol, suitably between about 200 and about 10,000 g/mol. If the polymer is monofunctionalized (i.e. has a functional grouping incorporated on only one end), a lower MV/ compared to bifunctionalized polymers (i.e. having functional groupings on both ends) may be used. For example, with mono and di-functionalized PEG, minimum MWs are about 500 g/mol and about 2000 g/mol, respectively. A schematic showing the general strategy for formation of silica particles under biomolecule compatible conditions in one embodiment of the present invention is shown in Figure IA. A schematic showing the general strategy far formation of silica particles in the presence of detector molecules under biomolecule compatible conditions in another embodiment of the present invention is shown in Figure IB. It is significant to note that the typical strategies utilized to stabilize particle formation known in the art did not work to form the biomolecule compatible particles of the present invention. Typical salts used to stabilize proteins, such as NaCl, magnesium salts and salts of buffers, such as citrates, caused significant flocculation and led to no monodisperse particle formation. In an embodiment of the invention the biomolecule compatible silica particles comprise one or more detector molecules entrapped within the particle. Accordingly, the method of the invention further comprises combining the reagents in the presence of one or more detector molecules under conditions sufficient for the incorporation of the detector molecules within the particle matrix. The detector molecules may be any such molecules, including, for example, passive detecting molecules ( or e.g. colored molecules), active detecting molecules that respond to a chemical stimulus such as pH, active detecting molecules that respond to a biomolecule small molecule such as adenosine triphosphate (ATP) and enzymatic detecting molecules. An example of a passive detecting molecule is the organic dye, Nile blue. An example of an active detector molecule is the organic dye neutral red which changes color due to an interaction with a chemical, for example, acids. An example of an ε.ctive detecting molecule that responds to a biomolecule small molecule such as adenosine triphosphate (ATP) is pyrocatechol violet ytterbium complex. An example of an enzymatic detecting molecule is horseradish peroxidase (HRP) whose activity is tested with tetramethyl benzidine.

One or more biomolecules may also be entrapped within the matrix of the particles. Accordingly, the method of the invention, further comprises, combining the organic polyol silane precursors and one or more particle stabilizing entities in the presence of one or more biomolecules under conditions sufficient for the incorporation of the biomolecules inside the particle matrix. In another embodiment of the invention, incorporation of appropriate salts and other conditions to stabilize the presence of biomolecules is included in the method of the invention. In an embodiment of the invention the entities to stabilize biomolecules include humectants such as, glycerol, salts and buffer.

In another embodiment of the invention, the biomolecule compatible silica particles are optionally surface modified. Accordingly, the method of the present invention further comprises treating the particles under conditions to modify their surface. In embodiments of the invention the surface modification comprises the incorporation of non-functional hydrophilic or hydrophobic groups, chemofunctional molecules, or biofunctional molecules, for example, peptides, proteins, DNA oligomers, etc. For example the surface modification may include the tethering of biomolecules that will lyse cells, suitably to selectively lyse certain cells or the tethering of molecules to specifically bind certain types of biomolecules. Methods for modifying the surface of silica particles are known in the art. In an embodiment, the certain types of biomolecules are biomolecules on or associated with a pathogen.

In an embodiment, the particles contain detector molecules as defined above entrapped inside or on their surface. In another embodiment of the present invention, molecules that are bound to the surface of the particles are selective or non-selective capture molecules. The term "capture molecules" herein includes any of a wide variety of proteins, peDtides, enzymes and other sensitive biopolymers including small molecules, DNA oligomers, DNA aptamers, DNA and RNA, and complex systems including whole plant, animal and microbial cells that may be tethered to the external surfaces of the particles. In embodiments of the invention, the capture molecule is a protein, or a fragment thereof. In further embodiments of the invention, the capture molecule is in its active form.

Molecules may be tethered to the surface of the particles via covalent or non- covalent (e.g. ionic, electrostatic or hydrostatic) interactions. Typically a linker group is covalently attached to the surface of the particles using any of a number methodologies known in the art. For example, surface hydroxyl, carboxylic acid or other functional groups, including amino, allyl, or silane groups that may be incorporated in the stabilizing entity either before or after formation of the particle, may be used to react either directly with the molecule(s) to be attached, or to a linker group that contains functional groupings that will allow the attachment of the molecule(s) to the particle.

In an embodiment of the invention, the surface modification results from attaching amino groups to the surface of the particle. One way to do this is to react the particle with an aminoalkyltrialkoxysilane, such as aminopropyltriethoxysilane. The alkoxysilanes condense with the hydroxy groups on the surface of the silica particles, thereby covalently linking the aminoalkyl group to the surface of the particle. The amino group is therefore free to react with any suitable molecule, such as, for example, carboxylic acids on a protein or peptide using standard coupling reactions. In one embodiment, the amino group reacts with a carboxyl-functionalized biotin to incorporate the well know biotin group onto the particle, allowing access to a vvide variety of recognition/capture methodologies that have been developed around the biotin/ streptavidin moieties (see for example: Schetters, H. 199⁽). Avidin and streptavidin in clinical diagnostics. Biomol. Eng. 16:73-78; Weber, P. C, D. H. Ohlendorf, J. J. Wendoloski, and F. R. Salemme. 1989. Structural origins of high- affinity biotin binding to streptavidin. Science. 243:85-88). Accordingly in an embodiment of the invention, the capture molecule is biotin or streptavidin or a derivative thereof. In another embodiment of the invention, the surface modification is a citrate based coupling agent as described in PCT Patent Application Publication No. WO 2007/014471. Compounds such as this, and other chelating materials, may be bound to the surfaces of the silica particles. As is known in the art, once chelated with appropriate metal ions, such as nickel, such complexes will ior ically bind to complementary molecules, particularly histidine-modified molecules, including histidine-tagged antibodies, bacterial phage and other biomolecules.

The present invention also includes monodisperse biomolecule compatible silica particles prepared using the method of the invention. In an embodiment of the invention the biomolecule compatible silica particles are preparec from organic polyol silane precursors in the presence of one or more particle stabilizing entities. In an embodiment, the particle stabilizing entities become entrapped or entrained in the particle matrix, accordingly the biomolecule compatible silica particles comprise a silica matrix and particle stabilizing entities, such as water soluble polymers.

In another embodiment of the invention, the biomolecule compatible silica particles are optionally surface modified. In embodiments of the invention the surface modification comprises the incorporation of non-functional iydrophilic or hydrophobic groups, chemofunctional molecules, or biofunctional molecules, for example, peptides, proteins. DNA oligomers, etc. as described above. (HI) USIiS fhe monodisperse biomolecule silica particles of the preseni invention find many applications. All uses of these particles are included within the scope of the present invention.

The incorporation of detector molecules within or on the particles and/or the surface modification of the particles to incorporate biomolecules that recognize other molecules and/or cells, and potentially kill those cells, allows the particles of the invention to find applications in the detection, and optionally killing, of biological entities, such as pathogens. The present invention accordingly includes methods of detecting one or more biological entities comprising contacting a sample suspected of containing the biological entity with the monodisperse biomolecule compatible silica particles of the invention and detecting the presence of the entities. In embodiments of the invention, the detecting of the biological entities is done by observing a change in one or more detector molecules entrapped within (i.e. inside) or on the particle. In embodiments of the invention, the detecting of the biological entities is done by selectively binding of the particles of the invention to the entities via interactions with molecules specific for the entity on the surface of the particle. T he presence of the biological entities may then be detected by observing the presence of the particles (which may contain a colored detecting molecule within its matrix to facilitate detection).

The present method also renders silica particle surfaces capable of binding specific cell types. Such cell types include, but are not limited to, those that one wishes to avoid (e.g. pathogens) and those that are beneficial in applications such as tissue engineering. For example, stem cells can be tethered on such surfaces in the desired organization and then be transformed into cells of specific utility, including osteoblasts.

The term "biological entities" includes any biological molecule (for example, DNA oligomers. DNA aptamers, DNA, RNA, proteins, peptides, eic), cell and/or organism that one wishes to detect.

The definition of "detector molecules" remains the same as that used in Section II.

The biomolecule silica particles of the present invention are sufficiently large to be seen with the naked eye. The particles are also advantageously printed or coated onto various substrates including paper (for example a filter, trap or support). The paper thus coated with the particles of the invention is used to detect the presence or absence of a biological entity, such as a pathogen.

In one of its embodiments the present invention includes the use of the monodisperse biomolecule silica particles of the invention for the detection and disinfection of pathogens, for example bacterial pathogens. Selective detection of a specific pathogen is provided by an antibody tethered to the external surface of the particle. Bacterial cell walls are lysed by proteins such as lysozyme (e.g., which is found in the eye). Lysis of tethered bacteria occurs as a consequence of lysozyme tethered at the silica particle surface. Finally, detection may occur because the particle contains various amplification protocols entrapped in the interior of the particle. When the cell lyses, its contents can diffuse into the particle core, where a reaction occurs (typically an enzyme-catalyzed reaction, but it can also result from chemical changes, e.g., pH) that leads to a change in the particle colo\ A schematic showing the basic strategy of pathogen detection is shown in Figure 2.

In an embodiment of the invention, the particles of the invention comprise colored dyes entrapped therein so that colors show up. This allows the particles to be printed on paper and to be detected.

In another embodiment of the invention, the particles have antibodies to specific pathogens tethered to their surface. The particles are placed in specific places where the presence of pathogens are suspected and the binding of the pathogens to the particles is detected. In a further embodiment of the invention the detecting molecules include pH indicators which show a color change upon changes in pH. In this embodiment, particles at a suitable pH will change color, for example when a cell is lyzed.

In yet another embodiment of the invention, the detection mclecules involve ΛTP detection. ATP is released when cells are lyzed by surface mclecules and the ΛTP is detected by internal detecting molecules that change color in the presence of ATP so that particle location and the presence of a specific cell is demonstrated.

In a further embodiment of the invention, the particles of the invention may be used in biochemical tests. For example, the particles permit incorporation of proteins (demonstrated herein with the incorporation of HRP). The proteins within the particle are stabilized and respond to the presence of small molecules (in thϊ case of HRP, peroxide). This permits amplification of a response when a cell is killed at the external surface and indicates that a cell is present in the sample.

In a still further embodiment of the invention, an enzyme such as lysozyme, is tethered to the surface of the particle. Lysozyme kills bacterial cells by lysing their cell walls. When the contents of the bacterial cells are released, the contents can be detected by the detecting molecules in the inside of the particle. In another embodiment of the invention, an antibody is tethered to the surface of the particle so that selective targeting of cells or proteins can be done.

In another embodiment of the invention, bacterial phage is tethered to the surface of the particle so that the particles can selectively target and bind cells or proteins.

The following non-limiting examples are illustrative of the present invention: EXAMPLES

MA TERIALS AND METHODS Compounds Tetramethyl orthosilicate (TMOS), glycerol, polyethylene glycol) (HO-PEG-OH, MW 200, 600, 1000, 2000, 8000 and 10000 g mol^"1), tetrahydrofuran (THF), allyl bromide, potassium hydroxide, diethyl ether, sodium hydride, dichloromethane, platinum divinyl-tetramethyldisiloxane complex (3-5% platinum concentration, Karstedt's catalyst), triethoxysilane, cilite and active carbon were purchased from Aldrich. 4-(2-Hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) was purchased from Bioshop. Mono allyl poly(ethylene glycol) (HO-PEG-A, MW 550 and 1 100 g mol^"1) were a gift from Clariant.

All chemicals were used without further purification. The THF was dried through an alumina column prior to use. Liquid PEG was dried under vacuum for two days before use. Equipment

¹ I I and ¹³C NMR were recorded at room temperature on a ESruker AC-200 spectrometer. FT-IR spectra were obtained on a Perkin-Elmer 283 spectrometer. Pore- size analysis of completely dried monoliths was performed on a Quantachrome Nova 2000 surface area/pore size analyzer. Before analysis, the particles were washed as described below and outgassed at 120 ⁰C for 7 h to remove air and bound water from the surface. The isotherms were measured with nitrogen at liquid nitrogen temperature. The mesopore surface area was calculated using the BET (Brunauer, Emmett and feller) method and from the desorption branch of the isotherm, the average pore size and distribution of pore sizes were determined using the BJH (Barrett. Joyner, and Halenda) calculation. The total pore volume was found at the last point of the adsorption branch. Example I : Precursor and additive Syntheses (a) Diglycerylsilane

Diglycerylsilane was prepared from TMOS and glycerol as described in U.S. Patent Application Publication No. 20040034203. (b) General method for Di-allyl-terminated PEG

PEG (1 mmol) with the desired molecular weight was dissolved in dry THF (150 ml) under a nitrogen atmosphere. To this solution potassium hydroxide (0.17 g, 3 mmol) and allyl bromide (0.36 g, 3 mmol) were added. For low molecular weight PEG the solution was stirred at room temperature, for high molecular weights at 60 ⁰C, under nitrogen overnight. The THF and unreacted allyl bromide were removed under vacuum and the residue dispersed in dichloromethane. The white solid was filtered off and the solvent was removed to give the crude product. The allyl-terminated PEG was purified either with silica column chromatography (low molecular weight) in dichloromethane/diethyl ether or through precipitation (higher molecular weights) with diethyl ether from dichloromethane to give 70 to 80% pure product.

(c) Di-allyl-terminated PEG (2) A-PEG-A;

PI-XJ ( 1 mmol) was dissolved in dry THF ( 150 ml) under nitrogen atmosphere. Sodium hydride (0.05 g, 2.2 mmol) was slowly added until no further gas formation could be observed. Allyl bromide (0.36g, 3 mmol) was added dropwise into the solution and the reaction was stirred at room temperature for over night. The THF and unreacted allyl bromide were removed under vacuum and the residue dispersed in dichloromethane. The white solid was filtered off and the solvent was removed to give the crude product. The allyl-terminated A-PEG-A was purified either with column chromatography in dichloromethane/diethyl ether or through precipitation (higher molecular weights) with diethyl ether from dichloromethane to give 70 to 80% pure product.

(d) Mono-allyl-terminated PEG

Mono-allyl terminated HO-PEG-A was prepared after one of the methods discribed above with lower amounts of based, e.g., potassium hydroxide (0.06g, lmmol) and allyl bromide (0.12g, 1 mmol). (e) Di-triethυxysilyl propyl-terminated S-PEG-S

Dried di-allyl-terminated PEG (lmmol) was dissolved in dry THF (20 ml) under nitrogen atmosphere. To this solution triethoxysilane (0.49 g, 3 mmol) and 2 drops of Karstedt's catalyst were added. The PEG solution was stirred jnder nitrogen overnight (at room temperature for low molecular weights and at 60 ⁰C for high molecular weights). The THF and unreacted triethoxysilane were removed under vacuum and the residue dispersed in dichloromethane. Active carbon was added to the solution and everything was stirred for at least 3 h to remove the catalyst. The carbon was filtered off over Celite and the solvent removed to give the final product with approximately 90% yield.

(/) Monυ-triethυxysilyl propyl-terminated PEG

Mono-triethoxysilyl propyl-terminated PEG was prepared in the same way as the di- triethoxysilyl propyl-terminated PEG with triethoxysilane (0.25g, 1.5 mmol). EXAMPLE 2: SILICA MA TERIAL PREPARATION In a typical synthesis the desired dried mono or di PEG (25 mg) was dissolved in deionized water (1.25 ml) or HEPES buffer (1.25 ml) in an ultrasound bath at room temperature. All polymer solutions were prepared just before use Glycerol was optionally added to the solution. Its presence can additionally lead to improved biocompatibility of the resulting materials. DGS (100 mg, 0.5 mmol) was dissolved in deionized water (1.25 ml) at neutral pH in an ultrasound bath at room temperature. The aqueous PEG and DGS/water solution were mixed under the desired conditions. The final solution was cured at room temperature overnight. The dispersions were centrifuged to precipitate the particles and washed repeatedly with deionized water or HEPES buffer (up to 5 times) and dried to give the final materials. In order to remove all the PEG some samples were calcined at 450 ⁰C for 4 hours in air. EXAMPLE 3: GENERAL PROCEDURE FOR DYESAND COMPLEXES

In a typical synthesis, the desired dried mono or di PEG (25 mg) was dissolved in deionized water (1.25 ml) in an ultrasound bath at room temperature and the desired detector was added (see Table 1 for detectors and concentrations). All polymer solutions were prepared just before use. DGS (100 mg, 0.5 mmol) was dissolved in deionized water (1.25 ml) in an ultrasound bath at room temperature. The aqueous PEG and DGS/water solution were mixed under the desired conditions. The final solution was cured at room temperature overnight. The dispersions were centrifuged to precipitate the particles which were washed repeatedly with deionized water and dried at room temperature to give the final materials with 95% yield. (a) Incorporation of methylene blue as an example of a passive dye

Methylene blue, a passive indicator, leads to a blue coloration of the particles. Such a dye is useful to detect the position of particles on white backgrounds like paper or in mixtures with other particles.

Particles containing methylene blue were prepared using E O-PEG-OH at molecular weights of 1 ,000 and 8,000 g/mol. Concentrations between 100 μmol and 5 μmol of dye were added to the particle preparation, which is between 20 and 1 mol% of silica formed in the reaction. All formed particles were blue with the intensity of the color decreasing with decreasing concentration of dye. The amount of dye incorporated during the process was 89% in the case of 100 μmol in the solution and 8,000 g/mol HO-PEG-OH. The dye was stable to washing with water. Leaching was tested by exposing the particles to aqueous solutions with pH of 3 and 10, to salt solutions containing 1 mol to 0.01 mol of NaCl and phosphate buffer at pH 7.4 and concentrations of 0.1 M and 0.0 IM for an overnight period. Absorbance measurements at 665 nm wavelength were taken and the results are shown in Table 2. It can be seen that the amount leached was highly dependent on the pE of the solution and the salt concentration. Eor low salt concentrations and pHs close to neutral the leaching was very low, when the salt concentration was increased or .he pH changed from neutral the leaching increased significantly. While not wishing to be limited by theory, in the case of NaCl, the leaching may have been due to the screening of the negative charge of the silica with Na-ions. In the case of a pH change, the leaching may have been due to the change of the methylene blue charge wilh pH. The low leaching at neutral pH and low salt concentrations makes these particles very useful for further reactions under biocompatible conditions.

SEM of the modified particles shows no large influence of the dye on the shape and size of the silica material, when compared with unmodified particles. In both cases spherical and fairly monodisperse particles with a mean diameter of 318±74 nm for modified particles and 415±56 nm for unmodified were found. The polymer used in both cases was 8,000 g/mol HO-PEG-OH. The smaller size and the slightly higher polydispersity of the dye containing particles shows that the positive charge of methylene blue influences the particle formation. (b) Incorporation of a pH indicator like neutral red Neutral red is a pH indicator that switches color from red to yellow at pH ~7.

It was added to the silica particle preparation at concentrations of 10 nmol or higher with an optimum concentration of ca. 500 μmol. At the optimum concentration, 94% of dye was incorporated into the silica. Leaching was low at 2.5 to 3.1% in NaCl solution and 2.2 to 2.5% in phosphate buffer. In this case the leaching increased with increasing concentration.

The incorporated dye was still able to react to pH changes by tinting the particles red-purple at low pH and yellow at high pH with an orange intermediate at pH 7. The dye in its acidic and base form was still bound to the panicles with only low amounts leaching out into solution. This leaching was lower at high pH than at low pi I, 0% and 1.5% respectively. This makes the indicator reversible with high accuracy.

The pi I change was not only done in solution, but also on paper. There are two different procedures possible: first the suspension is brought onto paper and the pH is changed without drying and second the particles are let to dry and then a solution of different pH is added. Both procedures led to a change in color of the particles and could be used in applications of pH papers. However the change with the wet particles was much faster than with the particles in the dry state. Also, the kind of paper played a role in the speed of changing. Whereas dry particles on Whatman filter paper nearly instantaneously changed their color, dried particles on normal office paper took a couple of minutes to change. In both cases, the change was reversible in the wet as well as in the dry state and the particles were strongly adsorbed onto the paper substrate and were not removed during the process. The use of neut'al red in silica instead of neutral red directly on paper has the advantage that the dye is not desorbed from the substrate during the analysis and the indicator can be reused. Silica is also a very good material for use in combination with paper, since it can be easily incorporated during the papermaking process or printed or coated onto it afterwards. SEM showed no change in particle morphology due to the incorporation of neutral red. (c) Incorporation of an A TP -sensing dye complex

Yin et al. (Caixia Yin, Fei Gao, Fangjun Huo and Pin Yang, Chem. Comm. 2004, 934) have described an active detector molecule which changes color on exposure to adenosine triphosphate (ATP). This detector is a small mclecule found in cells. Yin et al. report that, in aqueous buffer the complex formed ty pyrocatechol violet (PV) and ytterbium ions changes its color from blue to yellow in the presence of ATP. This is due to the stronger complexation of ATP with ytterbium leaving the PV free in solution. According to the mechanism 2 moles of ATP are needed per mole of PV-ytterbium complex. At least 625 μmol of the complex were needed to color the particles in the standard preparation. The final particles were first blue in color, but turn purple in a matter of 2 days, which was possibly due to the exchange of water molecules in the complex with silanol groups from the material. Both components must be present during particle synthesis in order to be entrained in the monolith subsequently formed. The colored particles were stable to washing with buffer and to changes in pH.

The addition of ATP led to the loss in color in the particles and the formation of a yellow supernatant. Accordingly, like in buffer solution, ATP bound to the ytterbium and PV was released. Since PV has a negative charge the molecule is not able to bind to the silica surface and leached into solution. The discoloration of the particles was dependent on the concentration of the ATP added, but was only visible by eye if a molar ratio of 1 : 1 for complex:ATP was reached (corresponding to 625 μmol for 30 mg of particles) and the loss in color was complete at mol ratio of 1 :2. This example demonstrates that the particles of the present invention can be modified in such a way so that they are sensitive to biologically relevant molecules. EXAMPLE 4. GENERAL PROCEDURE FOR INCORPORATION OF PROTEINS SHOWN FOR HORSERADISH PEROXIDASE

In a typical synthesis, the desired dried mono or di PEC (25 mg) was dissolved in deionized water (pH 5-9, 1.25 ml) or HEPES buffer (pH 5.1-7.4, 1.25 ml) in an ultrasonic bath at room temperature. All polymer solutions we^*e prepared just before use. Glycerol (0.5-1 g) was optionally added to the solution. DGS (100 mg, 0.5 mmol) was dissolved in deionized water (1.25 ml) in an ultrasound bath at room temperature. Horseradish peroxidase (HRP, 2.5 μg) (or other proteins) was added to the PPXJ solution just before adding the DGS. The aqueous PEG and DGS/water solution were mixed under the desired conditions. The final solutioi was cured at room temperature or in the fridge overnight. The dispersions were centrifuged to precipitate the particles which were washed repeatedly with HEPES buffer (up to 5 times) and dried at room temperature to give the final materials. The structures formed can be grouped into the following three categories: (i) Particles (Figure 3a); (ii) Fused Particles (Figure 3b); and (iii) "monolithic pieces" (Figure 3c). A summary of the conditions and structures of particles with HRP incorporated is provided in Table

S) . EXAMPLE 5: A CTIVITY TEST OF HRP IN PARTICLES

After refrigeration overnight, particles were washed with HEPES buffer (25 mM, pH^^"7.2) at least three times to ensure all unbound protein was removed. 100 μL of wash water was taken and combined with 30 μL of tetramethyl benzidine (TMB) to determine if protein was still present. When no color change to blue was seen, the test tube was centrifuged and water was removed with a pipette. 4.00 mL of HEPES buffer (25 mM, pH=7.2) and 1.00 mL of TMB liquid substrate system were added to the washed particles. The test tube was shaken at 250 rpm for 30 minutes to disperse and incubate particles. 2.5 mL of 0.5 M H2SO4 was then added to draw the chromogen out of the particles and stop the reaction. The test tube was then centrifuged for ~3 minutes (as little time as possible) and three separate 187.5 μL aliquots were taken and dispensed into a 96 well plate. Absorbance readings were then taken at 450 nm on a TECAN plate reader. Comparison with a calibration curve shows that 0.005 μg or 0.2% of the initial HRP was incorporated, accessible and active during the synthesis. A summary of the activity of HRP in silica particles is provided in Table 3.

EXAMPLE 6: LIFETIME STUDY

For lifetime experiments, particles (30 mg) with HRP were suspended in FlEPES (25mM, pH 7.2, 4 ml) and left at room temperature or in the fridge and the activity assay was run after certain intervals. The HRP had 27% of the initial activity after 2 weeks at r.t. and 21% after 1 month; it does not show activity after 70 days. In the fridge, the activity was 70% after 2 weeks, 40% after 70 days anc 27% after 100 days: the protein was still active after 12 months. EXAMPLE 7: ATTACHMENT OF PROTEINS ON THE OUTSIDE OF PARTICLES

(a) Preparation of mono-allyl mono-succinate PEO (MW 8, 000 g/mol)

Monoallyl functionalized PEO (1 O g, MW 8,000 g/mol) was dissolved in dichloromethane (45 ml), and succinic anhydride (Ig) and diisopropylethylamine (2.7 ml) were added to the solution. The mixture was refluxed at 60 ⁰C for 24h. After this time, 2/3 of the dichloromethane was removed and an excess of cold diethyl ether added. The white precipitate was washed with ether and dried under vacuum.

(b) Preparation of particles

In a typical synthesis, mono-allyl mono-succinate PEG (25 ing, MW 8,000 g/mol) was dissolved in HEPES buffer (10 mM, pH 7.4, 1.25 ml) in an ultrasonic bath at room temperature. All polymer solutions were prepared just before use. DGS (100 mg, 0.5 mmol) was dissolved in deionized water (1.25 ml) in an ultrasound bath at room temperature. The aqueous PEG and DGS/water solution were mixed under the desired conditions. The final solution was cured at room temperature for overnight. The dispersions were centrifuged to precipitate the particles and washed repeatedly with water and dried at room temperature to give the final materials.

(c) Activation of particles with an EDC/NHS system l -Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (750 mg, EDC) and N- hydroxysuccinimide (750 mg, NHS) were dissolved in phosphate buffer (10OmM, pH 6.8, 10 ml). To this solution was added acid PEG containing silica particles (30 mg) in phosphate buffer (10OmM, pH 6.8, 5 ml) and the mixture was stirred for 30 minutes at room temperature. The dispersion was centrifuged and washed with water (2 times).

(d) Addition of lysozyme To freshly activated particles (15mg), a solution of lysozyme (0.685mg) in phosphate buffer (100 mM, pH 7.4, ImI) was added and the mixture was shaken at 34 ⁰C for 4 hours. The dispersion was centrifuged and washed with the reaction buffer (2 times). To remove physically adsorbed lysozyme, the particles were suspended in sodium dodecyl sulfate solution (2% in reaction buffer, 2ml) and shaken overnight. After this, the dispersion was centrifuged and washed with the reaction buffer (2 times) to give the final particles.

(e) Addition of radiolabeled lysozyme The protein was added to activated particles in the same way ε.s described for non-labeled lysozyme above, only that 5% of the protein was replaced by radiolabeled lysozyme. Counting of the radioactivity leads to the values shown in Table 4 for the attachment, when 340mg of lysozyme is added to 15mg of particles. (J) Lysozyme activity assay

Dried M. lysodeikticus cells (9.0 mg) were dissolved in potassium phosphate buffer (30.0 mL, 0.1 M, pH 7.4). Lysozyme modified silica particles (5mg) in phosphate buffer (50 μl, 0.1 M, pH 7.4) and cell suspension (50 μl) were mixed in a TECAN 96- well plate and the absorbance at 450 nm was measured every 23 seconds with a 5 second shaking of the plate between cycles. This reduces loss of turbidity do to settling of the silica particles.

EXAMPLE 8: MODIFICATION OF SILICA FOR BINDING OF BIOTlN CONTAINING BIOMOLECULES. (a) Amine surface modification of silica particles Silica particles (180 mg) were suspended in deionized water (15 ml) containing glacial acidic acid (0.6 ml). To this suspension was added aminopropyltriethoxysilane (0.6 ml, 2.6 mol) and the mixture was stirred at room temperature overnight. The particles were cleaned by centrifugation and washing with water (3 times) to give the final product in 97 % yield. (b) Biotin surface modification of silica particles

Amine modified silica particles ( 180 mg) were suspended in 4- morpholineethanesulfonic acid buffer (30.6 ml, 0.1M, plϊ 6.8) to which biotin (18 mg, 0.07 mmol), Λ^L(3-dimethylaminopropyl)-Λ^'-ethylcarbodiimide hydrochloride (180 mg, 0.9 mmol) and /V-hydroxysuccinimide (450 mg, 3.9 mmol) were added. The mixture was stirred overnight at room temperature and cleaned by centrifugation and washing with 4-morpholineethanesulfonic acid buffer to give the final product in 98% yield. (c) Streptavidin modification of silica particles

Biotin modified particles (30 mg) were suspended in phosphate buffer (1.5 ml, 0.1 M, pH 7.4) and streptavidin (15 μg) in water (15 μl) was added. The suspension was shaken at 150 rpm for 2 hours at room temperature. The particles were cleaned by centrifugation and washing with phosphate buffer (3 times) to give the final product in 98 % yield.

(d) T 4 phage modification of silica particles

Streptavidin modified particles (30 mg) were suspended in trishydroxymethylamino-methane buffer (1.5 ml, 0.1M, pH 7.4) and T4 phage in lambda buffer (50 μl, 10^b PFU/ml) were added. The mixture was shaken at 150 rpm for 1 hour and the particles were cleaned by centrifugation and washing with trishydroxymethylaminomethane buffer to give the final product in 9 S % yield. The final particles were stored in trishydroxymethyl-aminomethane buffer. Activity was tested by plating with E.coli (Figure 10).

(e) T 4 phage activity test

Agar plates were prepared using standard procedures with yec.st extract (5g), tryptone (1 Og), sodium chloride (1 Og), sodium hydroxide (200μl, 5M) and agar (15g) in aqueous solution (I L). Top agars were prepared using standard procedures from tryptone (1Og), sodium chloride (8g) and agarose (5g) in aqueous sol αtion (IL). The top agar was divided in aliquots (5ml) before autoclaving.

E.coli bacteria were grown using standard LB media for an overnight period fresh before every use. In a standard test E.coli in media (100 μl) wee added to one aliquot (5ml) of dissolved and temperature adjusted (52°C) top agar, which was poured onto an agar plate after mixing. After solidification of the top agar (minutes) the material containing phage to be tested was brought onto of the plate and everything was incubated at 37⁰C for an overnight period. Clear areas (plaques) on the plates indicated active phage (Figure 9). EXAMPLE 9: SILICA PARTICLE MODIFICATION FOR NICKEL BINDING (a) 3-Hydroxy-3-methoxycarbonylpentanedioic acid dimethyl ester

To a stirred solution of citric acid (9.00 g, 46.8 mmol) in absolute methanol at 0 ⁰C under a nitrogen atmosphere was carefully added thionyl chlcride (20.5OmL, 0.28 mol). The reaction mixture was stirred at O⁰C for an additional hcur then at room temperature overnight. Volatiles were removed in vacuo. The residual solid was recrystallized from hexane/ethyl acetate to yield 10.80 g (98%) of the title compound as white crystals. (b) 3-Allyloxy-3-benzyloxycarbonylpentanedioic uc id dimethyl ester

Under a nitrogen atmosphere, S-hydroxy-S-methoxycarbonyl-pentanedioic acid dimethyl ester (Example 8(a), 10.32 g, 22.3 mmol) was introduced in a round- bottomed flask, followed by allyl-terώutyl-carbonate (5.29 g, 33.5 mmol) and dry toluene ( 1 10 mL). Palladium acetate (30 mg, 0.13 mmol) and triphenylphosphine (0.31 g, 1.18 mmol) were then added, and this mixture was refluxed for 4 hours. After being cooled to room temperature, the reaction medium was washed with dilute aqueous NaIlCC^, water, then brine. The organic phase was dried OVΞT Na₂SCU, and the volatiles removed in vacuo. The residue was purified by chromatography over a silica gel column, eluting with increasing amounts of EtOAc in hexares (9: 1 to 4: 1), to afford 8.97g of the title compound (80%) as a clear oil.

(c) 3-Methoxycarbonyl-3-(tήethoxysilylpropyloxy)pentanedioic acid dimethyl ester

In a round-bottom flask was introduced 3-allyloxy-3-benzyloxycarbonyl- pentanedioic acid dimethyl ester (Example 8(b), 2.74g, lOmmol) in 25 mL of dry toluene, followed by triethoxysilane (2.63g, lόmmol). 0.02 mL of hydrosilylation catalyst (platinum-divinyl-tetramethyl-disiloxane complex, solution in xylenes) was added, and the mixture was stirred at 80 ⁰C in a dry atmosphere for 16 hours. The volatiles were then removed in vacuo. The residue was dissolved in dry dichloromethane, a spatula of activated carbon was added, and the mixture was stirred for 3 hours under nitrogen. The mixture was then filtered over a 0.45μm Teflon filter, and the solution was then evaporated to dryness, to yield the title compound (3.75 g, 86%) as an oil who was used without further purification.

(d) 3-Methυxycarbonyl-3-(triethoxysilylpropyloxy)pentanedioic acid dimethyl ester modified silica particles Silica particles ( 1.2 g) were suspended in toluene (20 ml) containing 3- methoxycarbonyl-3-(triethoxysilylpropyloxy)pentanedioic acid dimethyl ester (Example 8(c), 20 μl) and the resulting mixture was stirred at room temperature overnight. The particles were cleaned by centrifugation and washed with toluene (3 times) and dried at 1 10 ⁰C to give the title product. (e) 3-Meihoxycarbonyl-3-(triethoxysilylpropyloxy)pentanedioic acid modified silica particles

3-Methoxycarbonyl-3-(triethoxysilylpropyloxy)pentanedioic acid dimethyl ester modified silica particles (Example 8(d), 1.2 g) were suspended in LiOH (10 ml, pH 10.3) containing methanol (ImI) and stirred overnight at room temperature. The particles were cleaned by centrifugation and washed with water (3 times) to give the title product.

(J) Silica particles with bound nickel ions

3-Methoxycarbonyl-3-(triethoxysilylpropyloxy)pentanedioic acid modified silica particles (Example 8(e)) were suspended in phosphate buffer (10ml, 0.1M, pH

7.4) containing nickel chloride (IM) overnight. The particles were cleaned with centrifugation and washed with water (3 times) to give the title product.

(g) Binding of nickel-modified silica to his-tagged antibodies

The nickle-bound silica particles from Example 8(f) were imnersed into his- tagged antibody solution (2ml at 3μg/ml antibody) for 60 minutes at 4 ⁰C. The particle were then washed once with phosphate buffer (3ml, 0.1 M, pH 7.4) to give the final product.

(h) Attachment of cells to his-tagged antibody-containing silica particles

The modification of the silica particles of the present disclosure with citric acid based coupling agents was straightforward. Following complexaiion with nickel ions, the surface had a high affinity for certain ligands, key among which was histidine. For example, it was possible to bind different oligo-bistidine tagged antibodies to osteoblasts and mesenchymal stem cells (MSC), respectively. Exposing the antibody-functionali/ed silica surface to cell suspensions isolated from human blood and removal of unbound cells by washing done by respiration led to the selective attachment oϊ osteoblasts. Such cells bound to the surface were robust in their binding.

RESULTS ASD DISCUSSION

Polymer formation and behavior PEG was chosen as a steric stabilizer for the silica particles because of its biocompatibility. PEGylated proteins have much longer lifetimes than their unmodified analogues. Its polarity and specific interaction with the silica surface can be changed by modifying the end-groups. Hydroxy-terminated PEG ^PEG-OH) was compared in this study with its ally 1 (PEG-A) or propyltriethoxysilane (PEG-S) end- group modified derivatives. Depending on the ratio of polymer end groups to the other reactants. one or both termini of the PEG can be modified (e.g., monoallylPEG is A-PhG-OH; diallylPEG is A-PEG-A). The use of monofunctiorial PEG leaves residual hydroxy 1 groups that can be modified after the formation cf the particles. The allyl compounds are commercially available in various molecular weights, or can be prepared by standard Williamson synthesis from any HO-PEG-OH → A-PEG-OH —> A-PEG-A. The silyl-modified materials are prepared from the allyl derivatives by platinum catalyzed hydrosilylation of the PEG-A compounds (A-PEG-OH → S-PEG- OH; A-PEG-A → S-PEG-S). The short hydrophobic allyl end groups are unable to react with silanol groups during the particle growth. By contrast, the propyltriethoxysilane groups can condense with silanols leading to chemical incorporation of the PEG polymer into the silica matrix. The ability of these PEG polymers to self assemble in watc was analyzed using pyrene anisotropy measurements. A value of 0.005 for the unmodified 2K HO- PEG-OH indicated a fast rotation of the polymer within the solution and the absence of significant association. For 2K A-PEG-A, the value of 0.06 is much higher, indicating a much slower rotation of the polymer in solution due to the aggregation of the hydrophobically modified polymer to larger aggregates. Somewhat surprisingly, the 2K S-PEG-S similarly showed high values of aggregation. As noted below, this is not due to hydrolysis and condensation of the EtOSi groups, but rather due to hydrophobic association. A possible structure of these aggregates s the so-called flower-like micelle and the polymer chains are wrapped around the middle like the petals of a flower. Such aggregations have been reported in hydrophobically modified PhGs with much higher molecular weight.

Prior to particle synthesis, the degree to which the silyl precu'sors, DGS and S-PEG-S, had complementary reactivity was determined. ⁹Si-NMR was used to analyze the hydrolysis and condensation times of the silane-modified PEG. In order to simplify the measurement, 200MW S-PEG-S was chosen since it provides a high quantity of silyl groups per gram of polymer. In aqueous solution at neutral pH the hydrolysis takes place over a period of 3 hours, during which time little condensation occurred, through one silanol group per end group. The spectra did not change much over the next 17 hours with, on average, average condensation of 1 : 1 (one silanol group per end group). By contrast, under the same conditions, the hydrolysis of DGS in water was much faster, taking less than 10 minutes. The condensation process was also much faster for DGS than for S-PEG-S. These very differen. reaction time frames for reaction of the two starting materials lead to the conclusion ihat most of the PEG condenses at the end of the silica matrix formation, such that PEG will be ultimately tethered to interfaces either on the inside or outside surface of the particles. This surface functionalization provides steric stabilization for the particles. For the particles derived from mono functional PEG, free carbinol groups are also present on their surface (one silane group per PEG) that can be used for further modification. Particle preparation

Silica particles were prepared from DGS by dissolving DCiS in water at neutral pi I and adding an aqueous solution of PEG at various pH values (this solution can optionally be buffered) and the resulting solution was stirred at room temperature overnight. In certain cases, the presence of glycerol was also included to obtain reasonably monodisperse particles. Its presence can additionally lead to improved biocompatibility of the resulting materials. After stirring, the solution was stored in a refrigerator for aging. Excess polymer and other additives were removed by centifugation and repeated washing with water or buffer, after which the silica particles were analyzed by SEM, IR, TGA and porosimetry. In order to remove all the PEG, some samples were calcined at 450 ⁰C for 4 hours in air. General features of particles

The typical silica particles derived from HO-PEG-OH were quite monodisperse with a mean diameter of 400 nm as found by SEM. IFL measurements after extensive washing were notable for the strong signal from the PEG's ether groups, demonstrating its incorporation and adsorption to the surface of the particles. The porosity of the as prepared sample was quite low with a BET surface area of 20.5 m² g^{" 1} and a total pore volume of 0.04 cm g^"1 in the mesopore range. Calcination of the sample at 450 ⁰C leads to an increase in the mesopore surface area and macropore surface areas. The pore diameter distribution in the mesopore range after calcination is bimodal with peaks at 2.6 nm and 48 nm: in the macropore range This suggests that most of the PEG is strongly incorporated into and cannot be removed by washing even though there is no possiblity of forming hydrolytyically stable links to the silica substrate. The alternative possibility, that PEG is adsorbed onto the silica, is countered by previous experiments that show PEG to be easily removed from silica surfaces by washing. The amount of retained PEG was quantified with TGA, which showed an 18 % weight loss on heating from room temperature to 600 ⁰C. SEM analysis on the calcined sample demonstrates that the particles retain their shape and size and do not aggregate. Samples prepared from all the PEG compounds exhibited similar features to those described above. Influence of molecular weight and the number of end groups on steric stabilization

PEGs with molecular weights between 200 and 10,000 g/mo were used for steric stabilization during the particle synthesis. These polymers were modified on either one or both ends leaving, in the former case, free hydroxyl groups for further reactions on the silica particles. It can be seen in Figure 4 that the molecular weight and the extent of end group functionalization can have a large influence on the formation of the particles. In the case of bi-functionalized S-PEG-S, the 600 MW chain was not optimal in providing steric stabilization of the silica par.icles, the 1 ,000 MW was much better, and the 2,000 MW PEG (Figure 5) gave reasonably monodisperse spherical particles. For monofunctionalized polymers, a molecular weight of 550 g/mol was already enough to give a stable particle dispersion. These results suggests that the bifunctionalized polymers have both ends on the silica surface, and thus are somewhat less than half as efficient steric stabilizers. Influence of the end group

Changing the end groups on the 2000 g/mol molecular weight bifunctionalized PEG from hydroxyl (OH) to allyl (A) to propyltriethoxysilane (S) hac little influence on the shape of the silica spheres (Figure 5). Although in the case of Si(OEt)₃ end groups some of the polymer can be chemically bound to the surface, the change of end groups seems to also have no effect on the size of the particles as seen by SEM (Figure 5). The end groups had a much larger influence at lower molecular weights of bifunctionalized PEG. At 600 MW, only bulk aggregates of porous silica were formed for FIO-PEG-OH and A-PEG-A, while for S-PEG-S spherical particles were prepared (Figure 6). This is due to the large influence of the end group on the hydrophobicity and molecular weight of short chain PEGs. For 600 MW PEG, the polymer consisted of approximately 10 ethyleneoxide (EO) units. It is understandable that an allyl group on each end would change the properties considerably. This is even more significant for the propyltriethoxysilane. Influence of mixing conditions

Changing the way of mixing of the PEG and the DGS solution from stirring to ultrasound led to a difference in the shape and size of particles formed. In the case of ultrasound, the particles were generally smaller and less spherical (Figure 7A,B), completely the opposite of the experience of making Stober particles, where ultrasound was necessary to achieve reasonable particle dispersities. In the case of 1OK HO-PEG-OH, the size changes from about 350 nm to 150 nm diameter. In the case of the higher MW material (2OK HO-PEG-OH), the distribution was biomodel with particle sizes centred at 180 nm and 500 nm suggesting secondary nucleation was occurring (Figure 7C).

An even larger influence in the use of ultrasound can be seen for 1 K A-PEG-A particles. In the case of stirring, the particles were relatively smooth and fused together (aggregate si/e ~ 500-800nm), while in the case of ultrasonication, the particles were rough, individual, and smaller than usual (average size ~ 200nm, Figure 7D,E). While not wishing to be limited by theory, the cavitation occurring during the ultrasound exposure probably led to a faster hydrolysis and condensation rate and thus to a higher number of nuclei and thus smaller particles. The rougher surface might be due to the increased probability of aggregation eve its followed by cavitation-induced separation. Influence of the amount of water

The amount of water used during the synthesis had an influence on particle formation. The influence of the amount of water on a 1OK HO-PEG-OH sample series was analyzed in detail, and shown to be influential for silica particle morphology. For example, as the amount of water, per 100 mg of DGS, was increased from 250 μl to 1250 μl the silica formed changed fiom aggregated particulate, to a mix of particles and aggregates to highly polydispcse and, finally, nearly monodisperse particles (Figure 8). This indicates that the particles are formed through the formation of primary particles and their aggregation to larger ones. In low water content, these larger particles fuse to form continous structures with macroporous spaces between them. In the case of high water content, ihe particles are held further apart and fusion only takes place if the steric stabilization is insufficient. Influence of pH

Proteins normally require pH ranging from pH 5 and 9. Therefore, the optimized reactions at pH 7 described above were re-examined at these pHs. Using I K HO-PEG-S as stabilizer, both pH 5 and 9 led to the formation of spherical particles which were intermixed with foam like silica structures. The r diameter was 230 nm, about the same as for pH 7. Thus, as with monoliths prepared from DGS, and unlike silica formation from TEOS, the influence of pH in the chemistry of the reaction is small. The combined hydrolysis and condensation times are essentially identical between pH 5-9. Use of buffered solutions in the preparation The isoelectric points, and pHs of highest activity of proteins and enzymes range from about pH 5-9. To test the ability to make particles at these different pHs silica particles were prepared in HEPES buffer at pHs ranging between 5.1 and 7.2 with varying ionic strength. Unlike the case at pH 7, it was necessary to add glycerol to the reaction in order to obtain particles. The rate of hydrolysis and condensation of DGS increased substantially with increasing ionic strength, while glycerol was found to have the opposite effect. Thus, there was exquisite control in the kinetics of gelation by balancing pi I and ionic strength effects with the glycerol concentration. While not wishing to be limited by theory, it is postulated that DGS hydrolyzes too quickly for PEG to act as steric stabilizer in high ionic strength buffer solutions, thus yielding aggregates. The addition of glycerol counteracts this effect, by retarding the kinetics of the condensation step, allowing individual particles to grow even at significant ionic strengths. Monofunctional PEGs were used in these reactions. At a pH of 5.1, particles were formed an ionic strengths up to 100 mM. Ely contrast, at a pH of 7.2 individual particles could be prepared up to 20 mM HEPES only as long as glycerol concentrations of between 0.5 and 1 g (per 100 mg DCJS) were used. Increasing the ionic strength further leads to fused particles. The MW of the stabilizing PEG also played a determining role in the formation of particles or aggregates. For example, a reaction using IK MW HO- PEG-A led to fused aggregates at much lower ionic strengths than the same reaction using 8,000 g/mol HO-PEG-A as a stabilizer. While not wishing to be limited by theory, this behavior can be explained with the better steric stabilization capability of the larger polymer. The inability to form discrete particles at ionic strengths above 2OmM at neutral pH can be explained by the sensitivity of the precursor DGS towards salt concentrations. Condensation rates of DGS increase dramatically with ionic strength, faster than the PEG interact with the evolving surface, such that steric stabilizers are not effectively formed. The role of salt content is multifunctional, however. In addition to mediating the kinetics of condensation, salts vvill electrically screen out surface charge, leading to particle aggregation. At lower pH, however the condensation in general is slower than at neutral conditions and the influence of the salt is smaller. Addition of glycerol for higher biocompatibility

Most biomolecules not only need near neutral conditions to stay functional, but also need a certain ionic composition of the solvent or organic additives. As noted above, glycerol could mediate silica production. Since glycerol is also used for the storage of biological moieties, as it generally extends their lifetime in solution, it was also added to the reaction mixture to increase the biocompatibilit) of the process. Particles with up to 1.1 g of glycerol (per 100 mg DGS) in the reaction mixture showed no difference in their structure to the ones prepared in pure water. Comparing this to the preparation of monolithic materials from DGS, it was surprising that the glycerol had no larger influence. Monolithic structures were prepared using similar recipes to that for the particles, with the key difference being the much lower water concentration during synthesis in the former case since for monolithic materials the reaction time was very dependent on the amount of glycerol added. In the particle preparation however the water content was so high that the shielding effects had no strong influence. (i) Summary of various effects on the formation of particles

While not wishing to be limited by theory, the observations described above can be rationalized by consideration of both synthetic and colloidal chemistry (See Scheme 1 ). Glycerol acts to distort the normal equilibrium A =- B, favouring starting material. However, this effect is removed at higher salt concentrations possibly because the metal ions can complex with glycerol. Once primary particles nucleate, they continue to grow D→E→F and to chemo- or physisorb PEG on the particle surface. However, beneficial steric stabilization provided by 1he PEG groups D→E→F are overcome at early stages of the reaction (D→G, or E→F ): i) if the PEG chains are too short; if the PEG concentration is too low; or if the salt concentration is too high. However, a wide variety of biocompatible conditions have been found, as noted above which permit the preparation of well dispersed silica particles.

Scheme 1

Lysυzyme binding and activity External modification of the silica particles of the present invention is additionally possible. In this case, the external surface was first modified with a linker or tether to which capture or detecting molecules were attached. PEO is known to become entangled within silica particles, and presenting the polymer at the surface. Without wishing to be bound by theory, free COOH groups presenting at the surface can be activated with NHS/EDC, which permits the grafting of lysozyme via amide groups. Reaction of such a surface with lysozyme results in 0.8 μg of lysozyme chemically bound to 15 mg of silica particles and 3.3 μg adsorbed protein, which can be removed by washing, as found by radiolabeling of the protein (Table 4). This was 16 times more bound and 3 times more adsorbed protein than was found on unmodified particles. A standard assay for activity testing of lysozyme, namely the disruption of Micrococcus lysodeikticus cells and the loss of turbidity in the solution, shows that the bound protein is active. A quantification of the activity is challenging, since the dense silica particles will spontaneously settle leading to a loss of turbidity in the solution (which is also the method by which activity is measured). However, the results show that the direct binding of proteins to the stabilizing polymer on the exterior of the silica particles was possible and the protein stays active. K. coll antibody binding and interaction with E. coll bacteria

Following the same procedure as described above for lysozyme, E.coli antibodies were chemically attached to the PEO on the surface of unmodified silica particles. Their presence was shown through the addition of E.coli bacteria with or without GFP expression and subsequent analysis with scanning electron microscopy and confocal microscopy using dye-loaded silica particles. Both (Df which show bacteria and particles in close proximity, however, no clear layer of silica on the bacteria surface can be seen. Biotinylatlon of silica particles Antibodies were alternatively tethered to the external surface of silica particles using established biotin/streptavidin technology. Biotinylated silica particles with diameters between 200nm - 500nm were prepared under mild conditicns in a two step process. In the first step, a silane coupling agent containing amine groups was hydrolyzed in the presence of the silica surfaces under mildly acidic aqueous conditions. After removing the excess unreacted silane coupling agent by centrifugation and washing, the samples were cured at elevated .emperatures to complete the crosslinking of siloxane binding of the coupling agent to the silica surface.

Biotin with its free carboxylic acid group readily reacts with amine groups to form amide bonds. As an example, amine-functionalized silica particles were suspended in an aqueous solution containing EDC and NHS to which biotin was added. After an overnight reaction period the final product was purified by centrifugation and repeated washing. The presence of amine groups was confirmed by energy dispersive x-ray spectroscopy and IR spectroscopy. Biotin-com aining surfaces were analyzed using an indirect method like binding of fluorescently labeled streptavidin.

Binding of biotinylated bacteria phage to streptavidin particles

Biotinylated silica particles readily bind streptavidin during exposure in buffer solutions as noted above. The streptavidin so tethered to the surface still has available biotin binding sites, as was shown by its ability to bind fluorescently labeled biotin. Given the large size of the silica in comparison to the protein, this is not unanticipated. 1 he free binding sites on the streptavidin give the possibility to attach biomolecules to the surface of the particles. One type of biomolecule employed was biotinylated bacteria phage, which will interact, infect and destroy E.coli bacteria.

The phage was bound to the streptavidin-silica by mixing both for 30 minutes at room temperature. Excess and physically adsorbed phage was remo ved by repeated washing with surfactant solutions. The activity of phage is tested by plating them onto E.coli containing agar plates using the procedure described in the experimental section. The formation of plaques shows the activity of the phage. Preliminary lifetime experiments indicate that these particles in suspension have phage activity for several weeks and on paper surfaces the particles are active for a week or more. Nickel modified silica and its use for binding his-tagged proteins

1 he modification of silica with citric acid based coupling agents is straightforward, f ollowing complexation with nickel ions, the surface has a high affinity for certain ligands, key among which is histidine. For examp e, it is possible to bind oligo-histidine tagged antibodies to osteoblasts and mesenchymal stem cells (MSC) Exposing the antibody-functionalized silica surface to c;ll suspensions isolated from human blood and removal of unbound cells by washing done by respiration leads to the selective attachment of osteoblasts. Such cells bound to the surface are fairly robust in their binding.

CONCLUSION

The preparation of silica particles under biocompatible condit ons is possible when organic polyol silanes, for example, DGS is used as the precursor and PEG is added as a steric stabilizer. The formation of the particles is affected by the PEG molecular weight, the end group functionality and the amount of wat=r. Larger PEG molecular weights lead to a better stabilization and thus to less aggregated and more monodisperse particles. The influence of the end group is significant only for short- chain PEGs (<2000 MW), which is believed to be due to end-group-mediated aggregation of the polymer. The particles are both meso- and microporous and have diameters between 200 and 500 nm. The size can be coarsely tuned by changing the mixing conditions under which the nucleation and growth are taking place. Changes in pH and addition of glycerol have no substantial influence on the size and shape of the particles. Buffered solutions can be used as a reaction medium, thus enabling the incorporation of biomolecules, which need specific conditions for their activity.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term. T ABLE 1

PVYb2 pyrocatechol violet-ytterbium complex; MB' methylene blue; RJ> rhodamine; NR: neutral red; NB' Nile blue TABLE 2

TABLE 3

TABLE 4

Claims

CLAIMS:

1. Λ method of preparing monodisperse biomolecule-compatible silica particles comprising combining organic polyol silane precursors in the presence of one or more particle stabilizing entities, and optionally in the presence of one or more biomolecules and/or one or more entities that stabilize the optional presence of biomolecules, under conditions sufficient for the formation of the particles.

2. The method according to claim 1, wherein the conditions sufficient fcr the formation of the particles comprise a water-containing medium and solutions prepared fom about 0.1 to about 10 weight percent polyol silane.

3. The method according to claim 2, wherein the conditions sufficient for the formation of the particles comprise about 0.5 to about 5 weight percent polyol silane in solutions with about 50% to about 100% water.

4. The method according to any one of claims 1 to 3. wherein the conditions sufficient for the formation of the particles further comprise entities for stabilization of biomolecules.

5. The method according to claim 4, wherein the entities for stabilization of biomolecules are selected from humectants, salts and appropriate nutrients.

6. The method according to any one of claims 1 to 5, wherein the conditions sufficient for the formation of the particles further comprise a pH between about 5 and about 9.

7. The method according to any one of claims 1-6, wherein the organic portion of the silane precursor is an organic polyol.

8. l^'he method according to claim 7, wherein the organic polyol is derived from natural sources.

9. The method according to claim 7. wherein the organic polyol is selected from glycerol, glycerol derivatives, glycols, sugar alcohols, sugar acids, saccharides, oligosaccharides and polysaccharides.

10. The method according to claim 9, wherein the organic polyol is a monosaccharide selected from allose, altrose, glucose, mannose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, threose, erythrose, glyceraldehydes, sorbose, fructose, dextrose, levulose and sorbitol.

1 1. The method according to claim 9, wherein the organic polyol is a disaccharide, selected from trehalose, sucrose, maltose, cellobiose and lactose.

12. The method according to claim 9, wherein the organic polyol is a Dolysaccharide, selected from dextran, (500-50,000 MW), amylose and pectin.

13. The method according to claim 9, wherein the organic polyol is selected from glycerol, propylene glycol and trimethylene glycol.

14. The method according to any one of claims 1-6 wherein the organic polyol silane is selected from diglycerylsilane (DGS) and tetraglycerylsilane (TGS).

15. The method according to any one of claims 1 -14, wherein the one cr more particle stabilizing entities are water soluble polymers.

16. The method according to claim 15, wherein the one or more water soluble polymers are selected from polyethers, polyalcohols, polysaccharides, poly(vinyl pyridine), polyacids, polyacr> lamides (polyNIPAM), and polyallylamine (PAM).

17. The method according to claim 16, wherein the one or more water soluble polymers are polyethers selected from poly(ethylene glycol) (PEG), modified PEG, anino-terminated polyethylene glycol (PEG-NH₂), poly(propylene glycol) (PPG), poly(propylene oxide) (PPO), and poly(propylene glycol) bis(2-amino-propyl ether) (PPG-NH₂).

18. The method according to claim 17 wherein the one or more water soluble polymers are selected from PEG and modified PEG.

19. rhe method according to claim 18, wherein the modified PEG is PEa that has been modified by incorporation of functional groups on one or both ends of the polymer.

20. The method according to claim 19, wherein the functional groups are selected from ally 1 and -L-Si(OR)₃, where L is a direct bond or a suitable linking group R is any group that is hydrolyzablc under the particle-forming reaction conditions.

21. The method according to any one of claims 15-20, wherein the molecular weight of the water soluble polymer is greater than about 200 g/mol.

22. The method according to claim 21, wherein the molecular weight of the water soluble polymer is between about 200 and about 10,000 g/mol.

23. The method according to claim 19, wherein the PEG is monofunctionalized and has a molecular weight of at least about 500 g/mol.

24. The method according to claim 19, wherein the PEG is bifunctiona ized and has a molecular weight of at least about 2000 g/mol.

25. The method according to any one of claims 1-24, further comprising combining the organic polyol silane precursors and one or more particle stabilizing entities in the presence of one or more detector molecules under conditions sufficient for the incoiporation of the detector molecules within the particle matrix.

26. The method according to claim 25, wherein the detector molecules are selected from passive detecting molecules, active detecting molecules that respond to a chemical stimulus, active detecting molecules that respond to a biomolecule small molecule and enzymatic detecting molecules.

27. The method according to any one of claims 1-26, further comprising combining the organic polyol silane precursors and one or more particle stabilizing entities in the presence of one or more biomolecules under conditions sufficient for the incorporation of the biomolecules inside the particle matrix.

28. The method according to any one of claims 1-27, further comprising treating the particles under conditions to modify their surface.

29. The method according to claim 28, wherein the surface modification comprises the incorporation of non-functional hydrophilic or hydrophobic groups, chemofunctional molecules, or biofunctional molecules.

30. The method according to claim 29, wherein the surface modification is the tethering of biomolecules that will lyse specific cells or the tethering of molecules to specifically bind certain types of biomolecules.

31. The method according to claim 30, wherein the cells or biomolecules are associated with a pathogen.

32. The method according to claim 29, wherein the surface modification is tethering of selective or non-selective capture molecules.

33. The method according to claim 32, wherein the capture molecule is a protein, or fragment thereof.

34. The method according to claim 33, wherein the protein is in active form.

35. The method according to claim 32, wherein the capture molecule is biotin or streptavidin or a derivative thereof.

35. l^'he method according to claim 29, wherein the surface modification h a citrate based coupling agent.

36. Monodisperse biomolecule compatible silica particles prepared using the method according to any one of claims 1 -35.

37. Monodisperse biomolecule compatible silica particles prepared using the method according to any one of claims 29-35

38. The particles according to claim 36 or 37 having a diameter of about 100 nm to about 1000 nm.

39. A method of detecting one or more biological entities comprising contacting a sample suspected of containing the biological entity with the monodisperse biomolecule compatible silica particles according to any one of claims 36-38 and detecting the presence of the entities.

40. A method of detecting one or more biological entities comprising contacting a sample suspected of containing the biological entity with the monodisperse biomolecule compatible silica particles according to claim 37 or 38 and detecting the presence of the entities.

41. The method according to claim 40, wherein the detecting of the biological entities is done by observing a change in one or more detector molecules entrapped inside or on the particle.

42. The method according to claim 40, wherein the detecting of the biological entities is done by selectively binding of the particles to the entities via interactions with molecules specific for the entity on the surface of the particle and the presence of the biological entities is detected by observing the presence of the particles.

43. The method according to any one of claims 40-42, wherein the biological entities are pathogens.

44. The method according to claim 43, wherein the particles have antibodies to specific pathogens tethered to their surface, the particles are placed in specific places where the presence of pathogens are suspected and the binding of the pathogens to the particles is detected.

45. The method according to claim 40, where the detecting of the biological entities is done using indicators which show a color change upon changes in pH.

46. The method according to claim 45 wherein changes in pH occur when a cell is lyzed.

47. The method according to claim 40, where the detecting of the biological entities is done using ATP detection.

48. The method according to claim 47, wherein ATP is released when cells are lyzed and the ΛTP is detected by detecting molecules inside the particle that change colour in the presence of ΛTP.

49. The method according to claim 40. wherein lysozyme is tethered to the surface of the particle and the lysozyme kills cells by lysing their cell walls releasing the contents of the bacterial cells and the contents are detected by the detecting molecules in the inside of the particle.

50. The method according to claim 40, wherein antibodies or bacterial phage are tethered to the surface of the particle so that the particles can selectively target and bind cells or proteins.