WO2008035125A2 - Methods - Google Patents

Abstract

A method which involves or potentially involves organically-substituted silica particles in desorption of an adsorbate in a liquid medium, other than a method which is at least part of a process for making said silica particles. In some processes, which an analyte displaces a detectable substance, e.g. a dye, from organically modified/hydrophobic silica particles for which the analyte has affinity. Direct or indirect determination of the occurrence or amount of desorption may be used to provide a measure of the presence or amount (e.g. concentration) of analyte. Other processes comprise desorption of an analyte from organically modified/hydrophobic silica particles by elution, without displacement of a detectable substance from the particles. The methods may comprise one or more further activities, for example for determining the presence and/or amount of the analyte.

Description

METHODS

BACKGROUND

The present invention relates to silica particles and, in embodiments, to organically- substituted silica particles. The particles are useful for the adsorption of compounds, particularly the adsorption and desorption of compounds. The invention primarily relates to a method of using such particles. The invention further relates to the preparation of the particles and other subject matter.

Two reports describe the use of combinations of tetraethoxysilane (TEOS) and phenyltriethoxysilane (PTEOS) to produce relatively hydrophobic silica aerogels [E. R. Menzel, S. M Savoy, S. J. Ulvick, K. H. Cheng, R. H. Murdock and M. R. Sudduth, Photoluminescent Semiconductor Nanocrystals for Fingerprint Detection, Journal of Forensic Sciences (1999) 545-551] and the corresponding nanoparticles [E. R. Menzel, M. Takatsu, R. H. Murdock, K. Bouldin and K. H. Cheng, Photoluminescent CdS/Dendrimer Nanocomposites for Fingerprint Detection, Journal of Forensic Sciences (2000) 770-773] for use in bioanalysis and biosensor applications. The former report demonstrated that as the proportion of PTEOS increased, the hydrophobicity of the resulting sol gel also increased, whilst the latter report used the particles' hydrophobicity to incorporate the hydrophobic dye, rhodamine 6G into the resulting particles. The nanoparticles were highly fluorescent with the dye being strongly retained within the particles under aqueous conditions.

International patent application PCT/GB2006/050233 (WO2007/017700) describes a method for preparing hydrophobic silica particles, the method comprising reacting together in a single step a mixture of silane ether monomers and organically modified silane ether monomers with a hydrolysing agent, e.g. reacting together in a single step a mixture of TEOS (tetraethoxysilane) and PTEOS (phenyltriethoxysilane) monomers with the hydrolysing agent. The hydrolysing agent, typically an alkali, acts as a catalyst within the reaction. Preferably this catalyst is a hydroxide, for example ammonium hydroxide.

The mixture typically further comprises a water miscible solvent, for example ethanol, and water. The method is typically carried out at ambient temperature. The reaction may be performed overnight or for an equivalent time period, that is to say for between about 12 and about 18 hours. The length of the reaction has an effect on the size of silica particles produced. It is believed that the earlier a reaction is stopped, the smaller are the particles which are formed.

The silane ether monomer, for example TEOS, and the organically substituted silane ether monomer, e.g. PTEOS may be used in ratios (PTEOS:TEOS) of, in particular, 1.2:1 to 1 :1.2, preferably 1 :1 v/v.

Particles produced by the above method tend to be predominantly nanoparticles, that is to say, of an average diameter of approximately 100nm to about 900nm, typically about 200nm to 900nm, further typically about 300nm to 800nm and particularly 400nm to 500nm.

These nanoparticles can be subsequently processed to form microparticles, which can be considered coalesced nanoparticles. The microparticles may be produced using a method comprising the steps of: i) centrifuging a suspension of particles ; ii) transferring the suspension of hydrophobic silica particles into an aqueous phase; iii) extracting the suspension from the aqueous phase into an organic phase ; iv) evaporating the organic phase; and v) crushing and sieving the product obtained in (iv).

The organic phase may be dichloromethane.

In an alternative method of PCT/GB2006/050233 (WO 2007/017700), hydrophobic silica nanoparticles are isolated from a reaction product produced from carrying out the previously described method for their manufacture. The hydrophobic silica nanoparticles are isolated using a method which comprises centrifuging the reaction product and suspending it in an aqueous:solvent mixture, preferably a 50:50 mixture. The reaction product is removed from the aqueous: solvent mixture, centrifuged and suspended in a second aqueous:solvent mixture. Preferably, the second aqueous: solvent mixture has a similar proportion of solvent and aqueous component as the first mixture. The aqueous:solvent mixture is typically a mixture of water and a water-miscible solvent, e.g. ethanol.

The step of suspending the reaction product in an aqueous:solvent mixture and centrifuging it is repeated a plurality of times. Preferably, the composition of the aqueous:solvent mixture is altered to increase the proportion of solvent in the aqueous:solvent mixture over the course of repeated suspensions. The final step may comprise suspending the reaction product in an aqueous: solvent "mixture" which is 0% aqueous:100% solvent. The total number of suspensions is typically from 3 to 10, e.g. 4, 5, 6, 7, 8 or 9. Typically after each suspension except the final suspension the suspensions are centrifuged. The nanoparticles can be stored in the final ethanolic (or other) suspension.

However in order to be able to visualise the above-described particles it is advantageous to incorporate a variety of dyes within them.

As described earlier, the ratio of silane ether monomers, for example, TEOS and organically substituted silane ether monomers, for example PTEOS is preferably about 1 :1 v/v. It is at this ratio that the optimum incorporation and therefore retention of a dye molecule within the silica particle is demonstrated.

The particles may be magnetic or paramagnetic. For example, magnetisable microparticles can easily be dusted over fingerprints, using a magnetic wand or other appropriate tool. Thus, the methods for preparing hydrophobic silica particles may further comprise including magnetic or paramagnetic particles in a reaction mixture of silane ether monomers, for example TEOS monomers, and organically modified silane ether monomers, for example PTEOS monomers. The magnetic and/or paramagnetic particles may be any magnetic or paramagnetic component, for example metals, metal nitrides, metal oxides and carbon. Examples of magnetic metals include iron, whilst examples of a metal oxide include magnetite. Carbon may be in the form of, for example, carbon black, fullerene or carbon nanotubes (derivatized or non-derivatized carbon nanotubes).

The inclusion of carbon black results in the particles having a grey colour. The precise colour of the grey particles is dependent on the amount of carbon black included in the TEOS/PTEOS mixture during synthesis. A higher level of carbon black results in a darker particle.

A second international patent application, namely PCT/GB2006/050234 (WO 2007/017701 ), describes the use in fingerprint analysis of hydrophobic silica particles, for example particles as described in International patent application

PCT/GB2006/050233. The particles are applied to the fingerprint, usually after the fingerprint is lifted. The hydrophobicity of the silica particles enhances the binding of the particles to the fingerprint. The fingerprint and its associated silica particles are then subjected to matrix assisted mass spectroscopy, e.g. matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF-MS). The silica particles act as the matrix.

Many molecules have dipole moments due to non-uniform distributions of positive and negative charges on the various atoms. Such molecules include non-aromatic and aromatic organic compounds when, in either case, substituted by or containing heteroatoms, some examples of compounds containing both an aromatic ring structure and one or more heteroatoms considered in the following paragraphs.

Polyaromatic compounds such as dioxins (halogenated organic compounds comprising two benzene rings joined by a double oxygen bridge) and related furans (a single oxygen bridge joining two benzene rings) and estrogenic steroids are examples of potentially hazardous chemicals which require monitoring within industrial sites and within the wider environment. There are standard methods which have been developed for the former in a variety of sample types. These include airborne monitoring, in flue ash and soil and in food stuffs. These require complex extraction, clean up and concentration steps prior to gas chromatography (GC)-mass spectrometry (MS), which is generally based on high resolution GC-MS.

Estrogenic and other steroids are also found in environmental samples with the major estrogenic contribution probably deriving from excretion of the synthetic compound, ethynylestradiol (ETED) from users of the female contraceptive pill, and estrone (ES) and 17-β-estradiol which are excreted from females undergoing hormone replacement therapy. Again their analysis from environmental samples is complex following a similar approach to that described for dioxins and related compounds. Both types of analyte (polyaromatics and steroids) share structural similarities in that they are hydrophobic and have aromatic moieties. They also possess dipoles: dioxins and related furans carry poly chloro- or other halogens whereas the estrogenic steroids posses a phenolic substructure.

It would be desirable to provide additional products and methods for use in the processing of organic compounds, for example for the purpose of analysis or research, e.g. to provide methods which are more sensitive than, or are cheaper and/or easier than, current methods.

BRIEF SUMMARY OF THE DISCLOSURE

The present invention provides the use of organically modified (substituted) silica particles in a method in which the particles are involved or potentially involved in at least one activity selected from adsorption and desorption in a liquid setting. Particles are potentially involved in such an activity in methods in which the particles are contacted with a liquid which could contain species capable of participating in adsorption and/or desorption activities involving the particles but does not necessarily contain such species; this applies in particular to assays to detect or measure analytes, for example pollutants, whose very presence it is the function of the method to detect and, in some cases, to measure. The silica particles may normally be described as hydrophobic by virtue of having hydrophobic organic moieties.

In embodiments, the methods of the invention do not include methods comprising at least part of a process for making the silica particles themselves.

Amongst other things, therefore, the invention provides a method which involves or potentially involves organically-substituted silica particles in desorption of an adsorbate in a liquid medium, other than a method which is at least part of a process for making said silica particles.

In one embodiment of the present invention, there is provided a method which involves or potentially involves organically-substituted silica particles in desorption of an adsorbate in a liquid medium, wherein the liquid medium is being tested, in a system having a detector for detecting a parameter indicative of occurrence of the desorption of the adsorbate, for the presence and optionally amount of a substance capable of causing the desorption.

The adsorbate may comprise a detectable adsorbate capable of being displaced by said substance. The displacement of the desorbate may be detected and/or measured by the detector as indirect detection and/or measurement of said substance. However, the invention does not require that the silica particles have a detectable adsorbate; in some embodiments a separate detection means can be used to detect and/or measure the amount of substance present in a sample. The detection means may include a second set of particles as described later, e.g. in combination with a detector or a different detection means.

Thus, the present invention provides the use of organically substituted silica particles as release agents, in a liquid phase process for releasing from the particles an organic substance. Also provided is the use of organically substituted silica particles as capture agents, in a process for capturing an organic substance in the liquid phase and, optionally but not necessarily, later determining the presence or amount of the organic substance and/or releasing it in the liquid phase.

The invention also provides a method which comprises performing a method which involves or potentially involves organically- substituted silica particles in desorption of an adsorbate in a liquid, namely passing an eluant over organically-substituted silica particles to elute an adsorbate from the particles, if the adsorbate is present; and then detecting the presence and/or amount of adsorbate in the eluate.

In one aspect of the invention, there is provided a method which comprises performing a method which involves or potentially involves organically- substituted silica particles in desorption of an adsorbate in a liquid, namely passing an eluant over organically-substituted silica particles to elute an adsorbate from the particles, if the adsorbate is present; then contacting the eluant with a solid phase having affinity for the adsorbate, whereby the adsorbate, if present in the eluant, associates with the solid phase; the method optionally comprising detecting the presence and/or amount of adsorbate associated with the solid phase. The solid phase used in the method described in the preceding paragraph may comprise a set of silica particles by which said adsorbate entrained in the eluant is sorbed, or it may, for example, comprise a resin. In some embodiments, the solid phase (whether or not silica particles) is responsive to sorption of said adsorbate to produce a detectable response, for example the adsorbate may quench a fluorescent dye of the solid phase or displace adsorbed dye molecules. The eluate may be contacted with the solid phase by being passed over it.

"Sorb" refers to the action of absorption or adsorption, or a combination thereof.

Thus, in embodiments, the methods of the present invention may additionally comprise the use of a second set of particles in addition to the organically-substituted silica particles from which an adsorbate is desorbed, if present; the second set of particles may be as described in more detail later.

Also included in the present invention is the use of organically substituted hydrophobic silica particles to concentrate a substance in the liquid phase by adsorption of the substance onto the particles. The use may further comprise detecting and/or measuring the concentrated substance using suitable detection means. For example, the adsorbed substance may eluted from the particles and the presence and/or amount of the substance in the eluate may then be determined.

Also included is the use of organically-substituted hydrophobic silica particles as an affinity substrate for substances in the liquid phase. Substances may become bound to and/or released from association with the particles.

In another aspect, there is provided a method comprising exposing organically- substituted silica particles to a liquid containing or suspected of containing an organic compound having affinity for the particles. Further provided is a method comprising exposing organically-substituted silica particles having an organic adsorbate to an organic solvent to cause desorption of the adsorbate. The solvent may be a water- miscible solvent in admixture with water.

In embodiments, the organically-substituted silica particles mentioned in this specification are hydrophobic and typically comprise an aromatic moiety and provide a substrate to which one or more aromatic compounds may be adsorbed and/or from which they may be desorbed. In some instances, both the aromatic moiety and the one of more of the compounds are polar, e.g. comprise a functional group which typically but not necessarily contains oxygen or nitrogen. In embodiments, therefore, the organically-substituted silica particles are substituted by an aromatic moiety and their use involves the adsorption and/or desorption of one or more aromatic compounds. In certain embodiments, the organically-substituted silica particles are substituted by an aromatic moiety having a substituent moiety and their use involves the adsorption and/or desorption of one or more aromatic compounds having a substituent capable of having affinity with said substituent moiety, for example both the particles and the aromatic compound(s) may have polar substituents capable of dipole interactions with each other.

The methods mentioned in the previous paragraphs may comprise separation and/or detection or measuring of an adsorbate. The methods mentioned in the previous paragraphs may be selected from liquid chromatography and fluid injection analysis. In some methods, the silica particles are pre-loaded with a detectable adsorbate and the method involves monitoring for and/or measuring displacement of the detectable adsorbate by an analyte in liquid by which the particles are contacted.

In some methods, a solid phase (e.g. a resin or a second set of silica particles) which includes a detectable molecule is used downstream from a first set of particles from which an adsorbate is desorbed, as described in more detail below under the heading "Use as a Capture Agent". The solid phase, therefore, may comprise a second set of particles, and these may comprise organically modified silica particles or silica hydrogel particles, particularly organically modified silica hydrogel particles. The solid phase may have selective affinity towards a specified substance or class of substances, whereby such substance(s) when present in liquid with which the solid phase is contacted associate with the solid phase. The solid phase may be adapted to produce a detectable response, responsive to a substance becoming associated with the solid phase. The detectable response may comprise a change in fluorescence, e.g. quenching. A particular solid phase comprises a second set of particles which comprise a detectable molecule e.g. integrated into the particle or coated on the surface of the particle. The second set of particles may be microparticles or nanoparticles.

The particles described herein may be used in a variety of techniques. One technique is Flow Injection Analysis. (FIA). FIA is a continuous flow technique which is ideally suited to rapid automated analysis of liquid samples. The technique was first reported by Ruzicka and Hansen in 1975, and research on this technique has subsequently been rapid and widespread, resulting in the publication of more than 10000 papers during the period 1975-2000.

A simple flow analyzer may comprise a pump or other injection apparatus which is used to propel a carrier stream through a conduit (in practice a tube, typically a narrow tube), an injection valve, a reaction zone (often called a reactor or microreactor) in which a sample zone disperses and, in conventional processes, reacts with components of the carrier stream, forming a species that is sensed by a flow-through detector and recorded. The height or area of the peak-shaped signal thus obtained can be used to quantify the analyte after comparison with the peaks obtained for solutions containing known concentrations of the analyte.

Aspects and embodiments of the invention are set forth in the following description and claims.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : MALDI-TOF-MS profile of 17-alpha-ethynylestradiol (ETED) in solution or adsorbed on different nanoparticulate matrix materials, one being a matrix material comprising polar (functionalised) hydrophobic silica particles, in each case in combination with conventional matrix material DHB.

Figure 2: MALDI-TOF-MS profiles of blank nanoparticles.

Figure 3: MALDI-TOF-MS profile of 17-alpha-ethynylestradiol (ETED) in solution or adsorbed on different DHB-free nanoparticulate matrix materials, one being a matrix material comprising polar (functionalised) hydrophobic silica particles.

Figure 4: A response curve of a flow injection analysis system, showing displacement of a fluorescent steroid by 17-α-estradiol.

Figure 5: A standard curve constructed from a set of 17- α-estradiol samples injected through the flow injection analysis system to which figure 4 relates.

Figure 6: Emission Spectrum of Rhodamine 6G PTEOS SG particles (Excitation wavelength = 530nm)

Figure 7: Response of Fiber Optic in Rhodamine immobilised PTEOS SG particle slurry to various concentrations of ETED

Figure 8: Emission spectrum for coumestrol adsorbed on different types of particles

DETAILED DESCRIPTION

Glossary

DHB: dihydroxybenzoic acid, a MALDI-TOF-MS matrix material.

MALDI-TOF-MS: Matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry, including SALDI-TOF-MS.

MALDI-TOF-MS/MS: Matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry-mass spectrometry, including SALDI-TOF-MS/MS. SALDI-TOF-MS: Surface assisted laser desorption/ionisation-time-of-flight mass spectrometry.

PTEOS NP: hydrophobic silica nanoparticles prepared by reacting PTEOS and TEOS.

PTEOS: phenyltriethoxysilane

TEOS: tetraethoxysilane.

Particles

The present invention relates in particular to uses and methods involving organically substituted silica particles. Such organically modified silica particles may be prepared from silane ether monomers and organically modified silane ether monomers. Exemplary silane ether monomers, as known in the art, include TEOS (tetraethoxysilane) and TMOS (tetramethoxysilane). The organically modified silane ether contains at least one organic moiety linked to silicon through a carbon atom of the organic moiety. An exemplary organically modified silane ether monomer is PTEOS (phenyltriethoxysilane).

Organically modified silica particles of the prior art have organic moieties which are dipole-free hydrocarbyl groups; such silica particles may be used in the present invention. Included in this disclosure are organically modified silica particles characterised by organic moieties having dipoles, i.e. polar organic moieties; such silica particles may also be used in the present invention.

The identity of the polar or non-polar organic moiety is not critical to the invention but it is directly bonded to the silicon of the modified silane monomers through a carbon- silicon bond and, whilst not being bound by theory, it is believed that the organic moiety is similarly directly bound to silicon via a carbon-silicon bond in the silica particles.

The silica particles may be microparticles or nanoparticles.

In many embodiments, the organic moiety is a hydrocarbyl group substituted or not substituted by one or more functional groups. The size of the hydrocarbyl group is not critical to the invention but, for example, it may contain from one to eighteen carbon atoms. Typical organic moieties include a monocyclic, bicyclic or tricyclic ring, the rings for example being 5- or 6- membered. The hydrocarbyl moieties may be saturated or unsaturated and, in the latter case, commonly include an aromatic ring structure, for example phenyl or naphthyl.

The hydrocarbyl moieties may be aliphatic, for example alkyl or alkenyl, or alicyclic, for example cyclohexyl or cyclohexenyl.

The organic moiety may include one or more heteroatoms in order to impart polarity; the heteroatoms are typically selected from nitrogen, oxygen and the halogens, although other heteroatoms such as, for example, boron, phosphorus and sulphur are within the scope of the disclosure. The disclosure therefore includes silica particles containing organic moieties having at least one functional group.

The invention can be described, therefore, as having embodiments relating to silica particles having organic moieties capable of both hydrophobic-hydrophobic interactions and dipole-dipole interactions. In embodiments, and as mentioned further below, there are disclosed silica particles having organic moieties capable of both hydrophobic- hydrophobic interactions and hydrogen bonding interactions. In other embodiments there are disclosed silica particles having organic moieties capable of both hydrophobic- hydrophobic interactions and charge transfer (electron donor-acceptor) interactions. Exemplary particles therefore comprise an organic moiety having a hydrophobic hydrocarbyl domain or group, substituted by one or more substituents capable of participating in at least one of the interactions mentioned in this paragraph.

In particular embodiments, the particles comprise a polar organic moiety which is a substituted phenyl group.

It is therefore a feature of embodiments of the invention that the silica particles contain a hydrocarbyl group substituted by a functional group to impart polarity, for example functional groups containing nitrogen, oxygen or halogen, particularly fluorine or chlorine. Other embodiments have unsubstituted hydrocarbyl groups, for example those comprising aromatic and/or other rings as in the case of substituted hydrocarbyl groups. The functional group may by way of example be nitro, amino, hydroxy, chloro or fluoro. Amino groups are often unsubstituted but mono- or di- substituted amino is to be mentioned; substituents may be hydrocarbyl groups containing from one to six carbon atoms, e.g. alkyl groups. Other functional groups to mention are carbonyl, imine, oxime, N-oxide, carboxy, nitrile, azide, diazonium, isonitrile, cyanate, isocyanate, and the sulphur analogues of the aforementioned O-containing groups. As a further non-exhaustive list of examples may be mentioned phosphate, sulphate and boronyl.

In embodiments, the functional groups are H-bond donors; in other embodiments they are H-bond acceptors. H-bond donors and H-bond acceptors may associate respectively with H-bond acceptor and H-bond donor groups of adsorbates for the silica particles. Hydrogen bond acceptors include nitro, carbonyl (e.g. as -CHO), nitrile, boronyl and hydroxy. Hydrogen bond donors include primary and secondary amino groups; other hydrogen bond donors include hydroxy and amido groups.

Also contemplated as functional groups are those capable of electron donor-acceptor interactions, i.e. interactions in which an electronegative atom with a free pair of electrons acts as a donor and binds to an electron-deficient atom that acts as an acceptor for the electron pair of the donor. (See e.g. Karger et al., An Introduction into

Separation Science, John Wiley & Sons (1973), page 42). Typical acceptor atoms/groups are electron deficient atoms or groups, such as cyano, nitrogen in nitro etc, and include a hydrogen bound to an electronegative atom such as HO- in hydroxy and carboxy, -NH- in amides and amines, HS- in thiol etc. As donors may be mentioned nitrogen atoms of primary and secondary amines.

Although the invention does not prescribe a list of acceptable functional groups, it is practical for good sense that the hydrocarbyl groups of the silica particles should be substituted with functional groups which do not suffer from significant instability or unwanted chemical reactivity during the preparation, storage or use of the particles. It is also practical good sense that the substituent functional groups should normally be selected to provide the desired properties to the silica particles, for example improved matrix performance in matrix assisted laser mass spectroscopy or improved (or, if desired, worsened) adsorption properties for selected compounds. Stability and chemical reactivity of functional groups may be judged on the basis of common general knowledge of their chemistry, and functional performance in terms of properties imparted to the silica particles may be determined empirically.

If desired, the functional groups may in turn be derivatised. A hydrocarbyl or other organic moiety may be substituted by one or more functional groups, e.g. 2, 3 or 4 functional groups. In the case of substitution by multiple functional groups, the functional groups may be the same or different.

Also included are embodiments in which the organic moiety comprises a heterocycle. The heterocycle may be substituted by one or more functional groups as described above in relation to substituted hydrocarbyl groups. Alternatively, the heterocycle may be an unsubstituted polar heterocycle, e.g. nitrogen heterocycles, for example piperazine.

It will be appreciated from the aforegoing that the disclosure includes silica particles containing polar or non-polar moieties selected from aliphatic, alicyclic and aromatic moieties. Silica particles may include a combination of the aforesaid moieties.

The silica particles mentioned herein may be made by known methods for manufacture of organically modified or hydrophobic silica particles, for example as taught by Menzel et al (see above) or in PCT/GB2006/050233 (WO 2007/017700). These methods may be suitably modified to introduce functionality/heteroatoms, as desired.

Thus, a preparative method described in PCT/GB2006/050233 (WO 2007/017700) comprises reacting together in a single step a mixture of (1 ) silane ether monomers, for example, an alkoxysilane and (2) organically substituted silane ether monomers, for example a phenyl modified silicate, with a hydrolysing agent e.g. ammonium hydroxide or another alkali. The silane ether monomers may be tetraalkoxysilanes (abbreviated herein to TAOS). The TAOS's are particularly selected from TEOS (tetraethoxysilane) or TMOS (tetramethoxysilane). The organically substituted silane ether monomer has organic moieties appropriate to the desired end-product particles and may be phenyltriethoxysilane (PTEOS) or PTEOS derivatised with one or more functional groups, e.g. PTEOS substituted with nitro groups.

The reaction may be performed at ambient temperature and for a period of 12 to 18 hours in a medium which comprises water miscible solvent, for example ethanol, and water. It is believed that the earlier a reaction is stopped, the smaller the particles which are formed. The silane ether monomer and the organically substituted silane ether monomer may be used in ratios (e.g. PTEOS:TAOS) of 1 :1 v/v. The hydrophobic silica particles produced by the above method tend to be predominantly nanoparticles, that is to say, of an average diameter of approximately 100nm to about 900nm, typically approximately 200nm to about 900nm, further typically about 300nm to 800nm and particularly 400nm to 500nm. These nanoparticles can be subsequently processed to form microparticles, which can be considered coalesced nanoparticles. The microparticles may be produced using a method which for example comprises the following steps: i) centrifuging a suspension of particles ; ii) transferring the suspension of hydrophobic silica particles into an aqueous phase; iii) extracting the suspension from the aqueous phase into an organic phase; iv) evaporating the organic phase; and v) crushing and sieving the product obtained in (iv).

Thus, exemplary silica particles are microparticles obtained by reacting monomers as described above and forming a suspension of particles which are centrifuged, extracted into dichloromethane from water, and then dried by evaporation of the organic phase to yield a glass-like sheet of coalesced particles. This is crushed and sieved to obtain particles of the desired size, e.g. of from 1 to 100 μm, more often 10 to 100 μm, for example from 30 μm to 90 μm.

The organic phase preferably comprises an organic solvent which is non-polar or has low polarity. The organic phase may be dichloromethane or another organic solvent for example alkanes, e.g. hexane, toluene, ethyl acetate, chloroform and diethyl ether.

Alternatively, hydrophobic silica microparticles can be obtained from a reaction product containing hydrophobic silica nanoparticles using a method comprising: (a) centrifuging the reaction product; and (b) washing the reaction product in a fluid.

The method may comprise repeating steps (a) and (b) a plurality of times. Preferably, the fluid is an aqueous:solvent mixture and is typically a wateπorganic solvent mixture. Typically, the organic solvent is ethanol. Preferably the initial fluid comprises a mixture of water and organic solvent at a ratio of from about 60 (water):40 (solvent) to about a 40:60 v/v mixture. In other embodiments, the solvent can be, for example, dimethylformamide, n-propanol or iso-propanol.

Typically, the proportion of solvent in the mixture is increased between the initial washing (i.e. suspension) (b) and the final washing (suspension). To obtain microparticles which are coalesced nanoparticles, the final suspension is dried. The microparticles may then be sieved. Once sieved, the microparticles are ready for use.

The microparticles may be considered to be aggregates of smaller silica nanoparticles. It is desirable that the microparticles are of sufficient size to be efficiently captured using face masks and hence not inhaled. Thus, in one embodiment, the silica microparticles have an average diameter of at least 10 μm, typically at least 20 μm. Typically, the microparticles have an average diameter of from about 30-90μm. In some embodiments, the microparticles have an average diameter of between about 45-65μm or from about 65 to 90μm.

The silica particles may be nanoparticles, for example as described above under the heading "BACKGROUND".

Such hydrophobic silica nanoparticles may be stored in a suspension. The fluid may be an ethanolic aqueous suspension. Alternatively, other organic solvents may be used in place of ethanol in the suspension e.g. dimethylformamide, n-propanol or iso-propanol.

Alternatively, the hydrophobic silica particles can be obtained using other methods in the art, (see for example, Tapec et al NanoSci. Nanotech. 2002. Vol. 2. No. 3 / 4 pp405-409; E. R. Menzel, S. M Savoy, S. J. Ulvick, K. H. Cheng, R. H. Murdock and M.

R. Sudduth, Photoluminescent Semiconductor Nanocrystals for Fingerprint Detection,

Journal of Forensic Sciences (1999) 545-551 ; and E. R. Menzel, M. Takatsu, R. H.

Murdock, K. Bouldin and K. H. Cheng, Photoluminescent CdS/Dendrimer Nanocomposites for Fingerprint Detection, Journal of Forensic Sciences (2000) 770-

773).

The term "average diameter" can be taken to mean a "mean diameter" of particles typically formed from the methods of the invention. The term "mean" is a statistical term that is essentially the sum of all the diameters measured divided by the number of particles used in such measurements. The diameters of nanoparticles can be estimated from SEM pictures and the scale used in pictures, and for microparticles the diameter can be estimated from a combination of the sieve size, the results from particle size distribution measurements and from SEM pictures. One way a mean diameter can be determined is by using a Malvern Mastersizer (Malvern Instruments Ltd.)

Modification of synthesis to incorporate a polar organic moiety (where present)

In one class of embodiments, a polar organic moiety is incorporated by using in the synthesis an organically modified silane monomer comprising an organic moiety having the characteristics desired in the end product particles, for example the organic moiety may be a polar organic moiety such as, for example, a heterocycle or a carbocycle substituted by a functional group.

In another class of methods, a heteroatom is introduced into the organic moiety after preparation of the silica particles. For example, functional groups or other heteroatom- containing groups (e.g. heterocycles) may be introduced using conventional functional group chemistry and/or functional group transformations may be performed as known to the skilled chemist. By way of example, aromatic compounds may be substituted using, for example, aromatic substitution reactions familiar to the skilled chemist. For example, aromatic moieties contained in silica particles may be nitrated using a mixture of concentrated nitric and sulphuric acids. If desired, the nitro group may be reduced to an amino group. The amino group may in turn be diazotised and used to prepare azo- bridged derivatives with phenols and other compounds, for example phenol or tyrosine. Aromatic compounds may be halogenated by the action of halogen in the presence of a Lewis acid or, in the case of fluorine, using the techniques commonly known to organofluorine chemists. Aromatic compounds may be converted to phenyls by reaction with sulphuric acid to create the corresponding aromatic sulphuric acid, followed by fusion with alkali (e.g. KOH).

In the other methods, functional groups or other groups capable of participating in a desired non-hydrophobic interaction are added and/or transformed at an intermediate step in the preparation, e.g. after preparation of organically-substituted silicon nanoparticles but prior to their coalescence into microparticles.

After preparation of the derivatised particles, i.e. after addition of the desired functional group, the particles are conveniently separated from the reaction medium, e.g. by centrifugation, and then washed. After washing, the particles may be dried and crushed, and optionally sieved.

Use

1. General

The invention provides methods which comprise the use of silica particles as described herein and are characterised by contacting of the particles by a liquid. These methods by definition exclude synthesising or modifying the particles themselves, since such activities are not use of the particles.

More particularly, the particles find application as an adsorbent or a substrate for affinity binding of substances. Typically, therefore, the methods of the invention may involve adsorption of one or more substances onto silica particles as described herein. More particularly, the typical methods involve desorption of substances from particles carrying an adsorbate or an affinity-bound substance.

The silica particles may be used in an aqueous environment. That is, the described processes may take place in an aqueous liquid which contains, or may contain, at least one organic compound for adsorption on the particles or which have been desorbed from the particles. The aqueous liquid may for example be water or an aqueous buffer.

In embodiments, the silica particles are used in organic liquids, for example polar organic solvents, e.g. ethanol, methanol, acetic acid, acetone, acetonitrile, DMF, DMSO. The liquid may be a mixture of water and a water miscible solvent, e.g. ethanol, methanol, DMF, DMSO, acetone or acetonitrile.

Such adsorption, desorption and/or affinity binding processes are common in chemistry and biology, in settings relating to separation, purification, synthesis or assays, or detection or measurement of analytes. Many of the liquid phase methods involving use of organically-modified silica particles can fairly be described as liquid chromatography, and a particular liquid chromatographic method is discussed in the following paragraph.

Fluid injection analysis is a liquid phase technique which requires no introduction to the skilled reader but is, nonetheless, briefly explained above under the heading "BRIEF

SUMMARY OF THE DISCLOSURE". The present invention includes techniques in which the reactor of a fluid injection is loaded with silica particles as disclosed herein. The reactor may be a tube or column, the term "column" being convenient because of its use in chromatography to refer to the space in which the chromatographic process itself occurs. It will be understood, therefore, that the term "column" does not refer to a device having any particular shape or orientation, but merely to a zone in which contains or may be loaded with silica particles as described herein.

2. Displacement Processes

The invention provides inter alia processes in which an analyte displaces, or at any rate causes desorption or release of, a detectable substance from organically modified/hydrophobic silica particles for which the analyte has affinity. Direct or indirect determination of the occurrence or amount of desorption may be used to provide a measure of the presence or amount (e.g. concentration) of analyte. Suitable analytes include those organic compounds previously mentioned for adsorption to organically modified/hydrophobic silica particles. In embodiments the silica particles comprise an aromatic substituent, and both the analyte and the detectable substance comprise aromatic moieties, e.g. are aromatic compounds.

In particular embodiments, apparatus (for example the column or reactor of a fluid injection analysis system) is loaded with particles having a detectable adsorbate. A sample is then passed over the particles (injected into the system in the case of a fluid injection analysis system) and, if the sample contains a substance having sufficient affinity for the silica particles, at least a portion of the detectable adsorbate will be displaced, allowing the detectable adsorbate to be detected and optionally quantified. This technique, particularly the fluid injection analysis technique, may be used to measure one or more selected analytes by comparing the measured displacement of the detectable analyte against its standard. For this purpose, it is convenient to prepare standard curves using a set of analyte samples of known concentration.

As is demonstrated by example 5 of the specification, silica particles as described herein may be used in a liquid setting to distinguish between different compounds in a sample. For example, particles comprising aromatic moieties have relatively strong affinity for large hydrophilic molecules containing a fused substantially planar ring system, whereas smaller hydrophobic molecules such as those consisting of a single benzene ring and simple substituents have relatively weak affinity such that they cannot displace a large hydrophobic detectable molecule, for example coumesterol.

The invention therefore permits "fine tuning" of the particles to modify their affinity for different analytes by changing the affinity properties of the organic moiety contained in the particles, for example by introducing functional groups capable of dipole-and/or hydrogen bonding interactions, or by increasing or decreasing the size of a hydrophobic moiety. Suitable modifications for particular analytes or classes of analytes bay be determined empirically.

In particular embodiments, hydrophobic silica particles, especially those having aromatic hydrophobic moieties, are loaded with detectable polycyclic, particularly polyaromatic, molecules and are used to separate, detect or measure polycyclic compounds, particularly polyaromatic compounds in a sample by virtue of the ability of such polycyclic compounds in the sample to displace the detectable molecule. It will be understood that where the system is used solely for the separation of molecules, the particles may be pre-loaded with a hydrophobic particle which is not adapted for detection, although detectable molecules are useful also in this case for the purposes of monitoring the process. A detectable compound or adsorbate is one which can be detected using, for example, conventional detection techniques. They may be detectable by, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radiological or chemical means. Conveniently, detectable compounds are optically detectable, as in the case of fluorescent or luminescent moieties. Substances used as detectable adsorbates may be inherently detectable, as in the case of fluorescent or luminescent dyes, for example xanthenes. Alternatively, they may be coupled directly or indirectly to a compound having affinity for silica particles. A wide variety of labels is available, with the choice of labels depending on the sensitivity required, use of conjugation with the compound, stability, available instrumentation and disposal provisions. As useful labels may be mentioned dyes, particularly fluorescent or luminescent dyes, and radiolabels.

Detectable compounds may be selected empirically or otherwise to have a desired binding affinity for selected silica particles, as discussed above in relation to analytes. For example, detectable polyaromatic substances have strong affinity for phenyl- substituted particles whereas compounds containing a single benzene ring have lower affinity and, depending on whether higher or lower affinity is desired, the appropriate detectable molecule may be selected. The detectable molecules may contain functional groups to interact with functional groups on the hydrophobic particles, as previously discussed.

The disclosed particles having polar organic moieties are useful for adsorbing polar materials. Suitably, such particles will have enhanced affinity and/or intimacy with one or more polar organic compounds, thereby providing a use for the nanoparticles in the processing of polar organic compounds, whether for example in analysis or synthesis.

The silica particles of the invention may be used in a liquid chromatographic setting, for example in affinity chromatography or fluid injection analysis (FIA). For example, silica particles of the invention may have adsorbed thereon a polar organic compound which is linked to or comprises a label, for example a fluorescent dye. These particles are placed in a reactor of a fluid analysis system and a sample containing a suspected analyte having affinity for the microparticles is injected into the system. If the analyte is present, it will displace labelled molecules, which will be detectable at the output of the

FIA system and potentially quantifiable using a standard curve. Of course, the particles having an adsorbed label may be used in other environments than FIA systems, e.g. in chromatography columns.lt is contemplated also, therefore, that the particles of the invention may find application in affinity chromatography.

Other formats may be mentioned for assays using the particles and a detectable analyte, e.g. a fluorescent label. These include a liquid flow system similar to FIA but using a microfluidic chip connecting two micro-reservoirs, or a plurality of such interconnected reservoir pairs. At a first of the reservoirs the particles are held (e.g. using hydrophobic magnetisable silica particles and a magnet under the chip's surface at this point) and the surface of the second of the reservoirs is sealed to generate an air lock across the channel. An aliquot of particle suspension pre-loaded with the detectable analyte (in this instance, fluorescent tracer) is added to the open reservoir followed by an aliquot of a sample or standard. If a rotating magnet is used then the particles will follow the magnet and also rotate, assisting mixing of reagents. Displacement of label into solution will take place. On opening the sealed second reservoir, fluid transfer will take place along the channel into the second reservoir with the particles being retained at the first by the magnet.

This concept has been demonstrated with magnetisable particles and determined the appearance of detectable analyte in the second wells by removing aliquots and detecting for the analyte, more particularly by determining their fluorescence using a multiwell fluorescence scanner. Magnetisable particles are described above under the heading "BACKGROUND".

Another format is envisaged in which the same interactions take place but with the components dispensed into separate reservoirs, e.g. micro-test tubes. We have set up a displacement assay using fluorescently-labelled and non-labelled analytes selectively adsorbed onto silica particles and measuring the non-bound/displaced fluorescence in the supernatant solution following incubation and centrifugation steps.

The organically substituted particles may be used to concentrate the captured substance, which concentrating may aid in detection and/or measurement of the substance downstream. In other words, the particles may be used firstly to capture a substance which has affinity for them and which is at a relatively low concentration in a liquid medium by which the particles are contacted; secondly, the captured substance is eluted at a relatively high concentration in an eluant.

3. Use as a Capture Agent.

A different class of methods comprises desorption of an analyte from organically modified/hydrophobic silica particles by elution, without displacement of a detectable substance from the particles. Included also are methods which comprise adsorption of an analyte to organically modified/hydrophobic silica particles for which the analyte has affinity without displacement of a detectable substance from the particles. The methods may comprise one or more further activities, for example for determining the presence and/or amount of the analyte. In particular, the invention relates to processes which involve desorption of analyte or other adsorbate from organically modified/hydrophobic silica particles. Suitable analytes include those organic compounds previously mentioned for adsorption to organically modified/hydrophobic silica particles. In embodiments the silica particles comprise an aromatic substituent and the analyte is an aromatic compound. The teachings above under the heading "Displacement Processes" in relation to hydrophobic substituents, analytes and detectable compounds, their selection and interactions are applicable mutatis mutandis to this section of the specification (as of course earlier teaching of the specification on such subjects are also applicable across the entire scope of the disclosure). As methods of this class may be mentioned multi-step (two or more step) sorption or affinity processes. As will already be apparent to the reader, the disclosed methods may comprise use of organically modified silica particles to capture a substance having affinity for the particles, if the substance is present, from a liquid medium prior to performance of one or more additional activities. Such additional activity may comprise detecting and/or measuring the amount of captured substance by a direct or indirect method, for example after desorbing the substance. In the methods described next, the at least one additional activity comprises desorbing the adsorbed substance into a liquid and then contacting the liquid with a solid phase with which the desorbed substance associates, to enable or facilitate detection or measurement of the desorbed substance. Such desorption itself is an aspect of the invention, without necessarily including the previous adsorption of the substance.

The organically substituted particles may be used to concentrate a substance from liquid medium, in a process in which the substance is captured by the particles, the captured substance is eluted and the eluate is exposed to a solid phase with which the substance in turn associates, for example as part of a process for determining the presence and optionally amount of the substance in the liquid medium. The solid phase may be a further or second set of particles e.g. silica particles. The solid phase is suitably hydrophobic, for example comprises aromatic groups with which an aromatic substance may associate; as previously described in relation to the organically- substituted particles, such aromatic groups may have a substituent capable of interacting with a substituent on the aromatic substance by dipole-dipole interactions, H- bonding or electron donor-acceptor interactions.

In any event, the method may comprise first contacting a liquid medium which may contain an organic analyte with organically-modified silica particles. As described above, if the substance is present, it may be adsorbed on to the organically modified silica particles, thus "capturing" the substance. This part of the method may be used to concentrate the substance to enhance detection and optionally measurement of the substance later on. The liquid medium and the silica particles may contact each other continuously (in a flow system). Alternatively, the liquid medium and the silica particles may contact each other as part of a batch process. The adsorbed substance is then desorbed or eluted from the organically modified silica particles. Some or all of the adsorbed substance may be desorbed or eluted but in other embodiments substantially all is desorbed or eluted. In one embodiment, desorption or elution is achieved by altering the hydrophobicity of the liquid medium or by contacting the silica particles with a liquid which is of different hydrophobicity than the liquid medium. Typically, the process is carried out by exposing the particles to a liquid of greater hydrophobicity than the original medium. In one embodiment, the initial medium from which the substance is adsorbed, if present, is aqueous or comprises a mixture of water and organic solvent, for example a mixture of water and one or more water-miscible solvents, suitable water-miscible solvents being ethanol or other alcohol in some methods; the eluant (desorbant) comprises a greater proportion of organic solvent, up to 100% organic solvent, as for example in the case of an increase in the alcohol content of water or a water-alcohol mixture, e.g. water- ethanol mixture.. In one embodiment, elution (desorption) is achieved by contacting the silica particles, on which the substance may be adsorbed, with a liquid comprising approximately 100% ethanol or other suitable solvent.

As mentioned, the liquid into which the substance is desorbed is in some methods subsequently contacted with a solid phase, the solid phase optionally being a second set of particles. The particles of the second set may be of the same or different constitution from the set of particles used to capture the substance. For example, the particles of the second set of particles may be silica particles, e.g. the organically substituted silica particles described herein. In one embodiment, the second set of particles includes silica particles which are not organically substituted. The particles of the second set are typically adapted for sorption e.g. absorption or adsorption of the substance. In one embodiment, the particles or the surfaces thereof are hydrophobic. In one embodiment, the second set of particles comprise silica gel particles, e.g. silica gel microparticles, for example those produced using organically substituted silane ether monomers, for example PTEOS. Exemplary are silica hydrogel particles, for example silica hydrogel microparticles. In one embodiment, the second class of particles are selected from microparticles and nanoparticles, e.g. silica nanoparticles or microparticles.

In embodiments, the solid phase, e.g. second set of silica particles, comprises a detectable molecule which is capable of detecting and/or measuring the amount of substance sorbed by the solid phase, e.g. by a particle of the second set of particles. Thus, in one embodiment, the detectable molecule is the same compound as a detectable adsorbate as described above, i.e. it may be detectable by, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, radiological or chemical means.

In particular embodiments, the detectable molecule has a detectable property which is altered as a result of sorption of the substance by the particle. Thus, the detectable molecule may be responsive to sorption of the substance by the particles to produce a detectable response. As an example may be mentioned fluorescent dyes, for example rhodamines, e.g. rhodamine 6-G. Rhodamines are an example of a class of suitable dyes which undergo a change in fluorescent properties as a result of association with organic substances. In one embodiment, the detectable molecule is adapted to decrease in fluorescence intensity, i.e. be quenched, when the substance associates with the molecule, as in the case of being sorbed by a silica particle. The decrease may be concentration-dependent, thus allowing for measurement of the amount of substance present in the liquid medium.

This "multi-step" process may be used in a liquid chromatographic setting, for example in affinity chromatography or fluid injection analysis (FIA). In some methods, organically substituted particles are placed in a reactor of a fluid analysis system and a sample containing a suspected analyte having affinity for the microparticles is injected into the system. If the analyte is present, it will be adsorbed onto the surface of the silica surface. The particles may then be contacted with an eluant, after which any desorbed analyte in the eluant is detected or measured, as for example when the eluate contacts a second class of particles which includes a detectable compound. In some methods applicable to FIA and other settings, analyte present in the eluate will displace detectable compound, which will be detectable (at the output of the FIA system, when present) and potentially quantifiable using a standard curve. Alternatively, and as discussed above, the label is not displaced by the analyte and instead undergoes a detectable change in a property which corresponds to the amount of analyte present in the sample. Of course, the particles having an adsorbed label may be used in other environments than FIA systems, e.g. in chromatography columns.

Included as an aspect of the invention are particles comprising a fluorescent compound. The particles may be silica particles, for example hydrophobic silica particles as described herein or silica gel particles, e.g. hydrophobically-modified silica gel particles. Such silica gel particles may incorporate the fluorescent dye. Also included as an aspect of the invention is a method for determining the presence and/or amount of an organic compound having affinity for a fluorescent dye, comprising contacting the organic compound with particles comprising the dye. The particles may be silica particles, for example hydrophobic silica particles as described herein or silica gel particles, e.g. hydrophobically-modified silica gel particles. Such silica gel particles may incorporate the fluorescent dye.

In an alternative embodiment, a different detection and/or measurement means can be used in place of the second set of particles. Thus, the desorbed substance may be detected and/or measured by a suitable sensor, for example an electrochemical, microbial or optical sensor. In other embodiments, a semipermeable membrane device may be used to accumulate a desorbed substance for subsequent analysis of the membrane device.

EXAMPLES

Example 4 does not relate directly to the invention but is included because it demonstrates that organically-substituted silica particles in which the organic moiety is polar (is substituted by a functional group) are effective adsorbents of hydrophobic compounds which are themselves polar (substituted by a functional group), indicating that such particles may be used in a liquid phase context.

Example 6 describes the preparation of silica gel particles having a detectable molecule, useful as a solid phase in a multi-stage sorption process.

EXAMPLE 1 - PREPARATION OF HYDROPHOBIC SILICA PARTICLES

Methods

Carbon black suspension was supplied by Cabot Corp, Cheshire UK. All other chemicals were purchased from Sigma-Aldrich, Dorset UK.,

This method was adapted from the preparation of blank silica based nanoparticles. [W.

Stobe, A. Fink and E. Bohn, J. Colloid Interface Science, vol 26, 62 (1968).]. The basic method is as follows; 30 ml ethanol, 5 ml dH₂O, 2.5 ml tetraethoxysilane and 2.5 ml phenyltriethoxysilane were mixed in a centrifuge tube. To this was added 2 ml ammonium hydroxide solution (28%) to initiate nanoparticle formation and the solution rotated overnight. The resulting particulate suspension was extracted repeatedly with methylene dichloride/water or ethanol/water (50:50 in both cases). The suspension was centrifuged (5 min at 3000 rpm). The supernatant was removed and 10 ml dH₂O and the same volume of dichloromethane were added. The suspension was rotated for a further 10 minutes, prior to the suspension being centrifuged again. The aqueous upper layer of the solution was removed and further aliquots of water and dichloromethane added. This process of rinsing and centrifugation was repeated 4 times until no further water: dichloromethane could be added. After such time, the particles were dried down from the dichloromethane in an incubator at 40 ⁰C.

Once dry, the particles were crushed in a mortar and pestle prior to being sieved to produce suitable particle sizes. The hydrophobic particles were sieved through brass test sieves with bronze mesh (Endecot Ltd., London UK) by hand. The particle size fractions used in this study were below 63 μm. A Malvern Mastersizer (Malvern Instruments Ltd., Malvern, UK) was used to verify the particle size distributions.

EXAMPLE 2 - PREPARATION OF CARBON BLACK-CONTAINING PARTICLES

For carbon black-containing particles, 5 ml of a 1 :100 fold dilution of the supplied carbon black suspension in water was added to the precursor solution placed in a centrifuge tube, prior to the addition of the silanisation reagents.

EXAMPLE 3 - DERIVATISATION OF ORGANICALLY MODIFIED SILICA PARTICLES

To a clean dry round bottomed flask weighed 300mg of PTEOS NP and added 2ml of cone. H₂SO₄. Placed on a ice bath with a magnetic stirrer and added 500 μl of cone. HNO₃ dropwise. After addition left the reaction to proceed for 1 hr at 4°C. After 1 hr the mixture was poured into 40ml of deionised cold water taken in a 50ml centrifuge tube. Centrifuged the particles for 3 minutes at 3000 rpm followed by removal of the supernatant. Added 40ml of deionised water, vortexed the nanoparticles for 30s, centrifuged for 3 minutes at 3000 rpm followed by the removal of the supernatant. The washing step was carried out 6 more times. After the final wash, transferred the nanoparticles to an evaporating dish and air dried for 2 days. Once the particles were dry, grounded in a mortar and pestle and stored in a air tight container. EXAMPLE 4 - ADSORPTION EXPERIMENTS

Particles were produced following modified synthetic routes. Four types of particles were formed 1. Hydrophilic particles based on TEOS only

2. Hydrophobic particles based on mixtures of PTEOS and TEOS as described above

3. Hydrophobic particles based on nitrated TEOS/PTEOS-derived particles ("PTEOS NP")

Method -4. Particles based on (2) above but embedded Carbon Black nanoparticles

a) Ethynylestradiol (ETED) onto silica nanoparticles

A stock solution of ETED (from Sigma Aldrich) (1 mg/ml) was prepared in absolute ethanol. A suspension of silica nanoparticles (5 mg/ml) was prepared in a 1/1 by volume mixture of ethanol and deionised water. An aliquot (200 μl) of this suspension was added to a polypropylene microcentrifuge tube (1.5ml polypropylene microcentrifuge tube from Sterilin), containing ethanohwater mixture (1/1 by volume) (700 μl). To this was added an aliquot of ETED (100 μl) containing 100 μg of ETED. The tube was closed using the integral stopper and inverted for 1 h using a Rotator Drive STR4, Stuart Scientific Supplies, UK inverter set at speed 1. This experiment was repeated with examples of each type of silica nanoparticle described above, and as a control the experiment was performed without any particles when the particle suspension was replaced with an aliquot of ethanol/water (200 μl). At the end of the incubation step the tubes were centrifuged for 3 min at 3000 rpm in a microcentrifuge (Jouan, BR4 i, Thermo Electron Corporation). The supernatant was aspirated off and d-H₂0 added (1 ml). The particles were resuspended by vortex mixing for 30 sec when the tubes were re-centrifuged as before. This wash cycle was repeated a further three times and the final supernatant of d- H₂O removed. At this point the washed particles were re- suspended in 100 μl of ethanol/water mixture as above.

MALDI-TOF-MS analysis

A Kratos Axima-CFR MALDI-TOF-MS (Shimadzu Biotech, Manchester UK.) system was used throughout with the following settings; laser power 90, reflectron positive mode, ion gate off, P. Ext 250, and mass range 1-500. Samples were pipetted onto stainless steel target plates also supplied by Shimadzu. For calibration, aliquots of the target analyte ETED 10 μl of stock solution containing 1 mg/ml of the analyte) were mixed with aliquots of 2,5- dihydroxybenzoic acid (DHB) (10 μl of a stock solution ethanol containing 1 mg/ml), and 1 μl of this mixture was pipetted onto the pre-cleaned surface of the target plate. The spots were air dried for 30 min.

An aliquot (1 μl) of the washed suspension from the absorption experiment with each type of silica particle was deposited onto the target plate together with the pre- dispensed standard. Finally an aliquot (1 μl) of the stock ETED solution was directly applied to the target plate and allowed to dry without any added DBH matrix. These were allowed to dry for 30 min prior to MS-analysis

As seen in the ETED solution profile of Fig 1 , ETED exhibits a molecular ion at m/z at 296 and the DHB matrix does not interfere with this peak. The spectra for the MALDI- TOF-MS of the nanoparticles with adsorbed ETED are also shown in Fig 1. No peak at 296 is seen with the hydrophilic particles derived from TEOS but peaks are seen for ETED in the hydrophobic particles derived from PTEOS and the nitrated PTEOS- derived particles, demonstrating that ETED binds to these particles. A clear peak for ETED at 296.23 is observed indicating that hydrophobic silica nanoparticles can be used to both adsorb ETED and as an agent for enhancing the MALDI-TOF-MS of the adsorbed chemical.

The spectra for the particles themselves plus DHB are shown in Fig 2. No peaks at 296 are seen indicating that the particles do not interfere with the spectra of the adsorbed ETED. Figure 2 shows the spectra for the three types of particles but in the absence of DHB matrix.

Figure 3 shows the spectra for the three types of particles but in the absence of DHB matrix. A clear peak for ETED at 296.28 is observed in the case of the nitrated particles indicating that this type of particle can be used to both adsorb ETED and as an agent for enhancing the MALDI-TOF-MS of the adsorbed chemical. It is to be noted in Figure 3 that the peak at 296.28 in the case of the nitrated particles does not have a corresponding peak for the non-nitrated particles.

The examples therefore demonstrate the surprisingly improved performance of functionalised particles as disclosed herein when used with a functionalised (polar) organic compound.

EXAMPLE 5 - FLOW INJECTION ANALYSIS A polypropylene column (1 ml capacity SPE tube), was fitted with a polyethylene frit (20 μm pore size from Supelco) was packed by gravity with a slurry of the nanoparticles from synthetic route 2 above (approximately 20 mg in 400 μl of a 1 :1 by volume of ethanol/water). The column was washed with about 5 ml of PBS buffer (phosphate buffered saline, pH 7.4) and connected to a flow injection analysis system consisting of a rheodyne injector with a 100 μl loop, upstream of the column, with a spectrofluorimeter (Perkin Elmer LS 50B), set at 380 nm excitation and 437 nm emission. A reservoir containing PBS buffer was connected at the front of the system and this was continuously pumped through the system at 0.7 ml/min using a peristaltic pump. A solution of coumesterol in PBS (500 ng/ml) was prepared and 750 ng was added via 15 injections of 100 μl of stock solution. No signal was seen until the 15th injection, indicating that the fluorescent steroid tracer had been adsorbed onto the column stationary phase and that saturation and breakthrough was then seen following the 15th injection.

A number of samples were prepared as 500 ng/ml solutions in PBS from ethanol stock solutions. These were 17-α-estradiol, 17-β-estradiol, 17-α-ethynylestradiol, estrone, dexamethasone, prednisolone, squalene, nitrobenzene, chlorobenzene, and toluene. Single injections of each (100 μl) sample were performed and the peak intensities noted. For 17-α-estradiol a set of standards over the range 0-500 ng/ml were prepared in PBs and these were used as duplicates and the results used to construct a standard curve

The results from injecting estradiol at varying concentrations through the pre-loaded bioreactor within the FIA system, are shown in Fig 4. A concentration dependent response with good reproducibility is seen and when a standard curve is constructed using this data then a linear plot is obtained (Fig 5). These results indicate that this steroid displaces the fluorescent coumesterol pre-loaded onto the hydrophobic column in consistent and reproducible manner. When injections containing 50 ng amounts of other chemicals were passed through the bioreactor within the FIA system, peaks of equivalent intensity were seen for 17-β-estradiol, 17-α-ethynylestradiol, estrone, dexamethasone, prednisolone and squalene whereas no peaks were seen for nitrobenzene, chlorobenzene, and toluene. This indicates that the FIA system can be used to detect large hydrophobic molecules and that small hydrophobic molecules do not appear to displace the adsorbed coumesterol and hence are unlikely to be detected in this system.

Discussion

The results clearly demonstrate that silica particles which include polar hydrophobic groups, in this case nanoparticles formed by nitration of particles synthesised using TEOS and PTEOS, can be used to adsorb large hydrophobic but dipole-containing molecules such as estrogenic steroids and related steroids and hydrophobic polychlorinated molecules such as dioxins from aqueous solutions.

In Example 5 the hydrophobic particles are packed into columns and then used with a flow injection analysis system for the analysis of steroids and related hydrophobic compounds. This employs detection of a fluorescent steroid such as coumesterol that is pre-adsorbed onto the particles and is then displaced when the target analytes are injected into the bioreactor.

EXAMPLE 6 - PREPARATION OF DYE-INCORPORATED PTEOS PARTICLES

PTEOS silica gel particles incorporating rhodamine 6G (R-6G) were prepared by following method:

Stage 1. Activation of Silica gel - About 6g of 40 -60 μm silica gel was weighed in a 100 ml glass beaker and added 50 ml of 27% hydrogen peroxide and stirred for 2 hrs at room temperature in a fume cupboard. After 2 hrs, washed the particles with deionised water for 4 times.

Stage 2. Preparation of R-6G PTEOS silica gel particles - About 1g of activated silica gel was taken in a 50 ml centrifuge tube and added 19 ml of ethanol, 1 ml of 25mg/ml of rhodamine 6G, 15 ml of deionised water, 2.5 ml of PTEOS and 2 ml of 28% NH3. Rotated overnight and washed with 50:50 ethanol:0.1 M potassium phosphate buffer mixture once followed by 30:70 ethanol:0.1 M potassium phosphate buffer mixture twice. Then washed twice with 100% ethanol and the slurry was air dried in a fume cupboard for 4 hrs and dried in an oven overnight. The R-6G PTEOS silica gel particles were stored in an air tight sterilin tubes and stored at 4⁰C in dark. EXAMPLE 7 - EMISSION SPECTRUM OF R-6G-CONTAINING SILICA GEL

The R-6G PTEOS silica gel particles slurry from Example 6 was packed in a microcuvette and its fluorescence spectrum is shown in Figure 6 in comparison with that of free rhodamine 6G.

Results: The emission wavelength of the R-6G shifts from 550 nm to 591 nm when it is incorporated in the PTEOS silica gel particles. Hence this wavelength combination was used for further studies which involve the use of R-6G PTEOS silica gel particles.

EXAMPLE 8 - MEASUREMENT OF BINDING OF ESTROGENIC STEROIDS.

A mini bioreactor (5 mm x 1 cm external dimensions) was packed with R-6G PTEOS silica gel slurry and a bifurcated fiber optic cable was introduced into the centre of the slurry. The bifurcated ends of the fiber optic were fitted to a fluorescence system (FIAIab Inc.), the output from which was connected to a computer. The solvent used was a 10% ethanohwater mixture and this was pumped using a peristaltic pump through the bioreactor at a flow rate of 0.4 ml/min. The inlet tubing to the bioreactor was also connected to a manual Rheodyne injection port with 100 μl sample loop. Different concentrations of 17 α-ethynylestradiol were injected into the bioreactor system and the change in the intrinsic fluorescence intensity of the R-6G PTEOS silica gel particles was measured (excitation 530 nm, emission 591 nm). The resulting changes in fluorescence intensities are depicted in Figure 7.

Table 1

Result: Adsorption of ethynylestradiol onto R-6G PTEOS particles decreases the fluorescence intensity and the decrease is concentration dependent. The sensitivity was in the ug/ml range.

EXAMPLE 9: ADSORPTION OF COUMESTROL

The adsorption of coumestrol on different types of nanoparticles was studied by taking 3 types of nanoparticles differing in organic functional moieties present on the silica backbone. 20 mg of: (A) TEOS nanoparticles, (B) PTEOS nanoparticles and (C) nitro- substituted PTEOS silica gel particles were taken in respective microcentrifuge tubes and suspended in 100 ul of ethanol followed by 20 ul of 50 ug/ml of coumestrol and 880 ul of deionised water. The tubes were rotated for 1 hr end to end on a rotator. After an hour, the tubes were centrifuged and the supernatant was removed and analysed for the coumestrol. Figure 8 shows the intensity of the supernatants.

Result: The adsorption of coumestrol on nitro PTEOS silica gel particles was 100 % and about 97% for the PTEOS nanoparticles. There was no adsorption of coumestrol on TEOS nanoparticles.

The invention includes the subject matter of the following paragraphs:

1. A method which involves or potentially involves organically-substituted silica particles in at least one activity in a liquid medium, the activity or activities being selected from adsorption and desorption, other than a method which is at least part of a process for making said silica particles.

2. A method of paragraph 1 wherein the activity comprises adsorption.

3. A method of paragraph 1 or paragraph 2 wherein the activity comprises desorption.

4. A method of any preceding paragraph wherein the liquid medium is being tested, in a system having a detector for detecting a parameter indicative of occurrence of said at least one activity, for the presence and optionally amount of a substance capable of participating in a said activity. The parameter may be the presence or amount of a detectable adsorbate desorbed from the particles, for example because of displacement by an analyte, when the analyte is present.

5. A method of paragraph 4 wherein the particles carry a detectable adsorbate capable of being displaced by said substance, whereby the displaced adsorbate may be detected and optionally measured by the detector as indirect detection and optionally measurement of said substance.

6. A method of paragraph 5 wherein the adsorbate is spectroscopically, optically or radiologically detectable.

7. A method of paragraph 6 wherein the adsorbate comprises a dye.

8. A method of any of paragraphs 4 to 7 wherein a said substance is present in an amount such that said at least one activity occurs to an extent detectable by the detector.

9. A method of any of paragraphs 4 to 7 wherein such substance is not present in an amount such that at least one activity occurs to an extent detectable by the detector.

10. A method of any preceding paragraph which is a liquid chromatographic method.

1 1. A method of any of paragraphs 1 to 9 which is a fluid injection analysis method.

12. A method of any preceding paragraph in which the particles are substituted by a moiety having at least one functional group.

13. A method of paragraph 12 in which the functional group is capable of at least one interaction selected form dipole-dipole interactions, hydrogen bonding interactions and charge transfer interactions.

14. A method of any preceding paragraph in which the particles are substituted by a hydrocarbyl group having from 5 to 30 carbon atoms. 15. A method of paragraph 14 in which the hydrocarbyl group comprises a ring structure having from 5 to 18 carbon atoms.

16. A method of paragraph 14 or paragraph 15 wherein the hydrocarbyl group is substituted as specified in paragraph 12 or paragraph 13.

17. A method of paragraph 15 in which the ring structure is aromatic.

18. A method of paragraph 15 or paragraph 16 in which the hydrocarbyl group is phenyl.

19. A method of paragraph 17 or paragraph 18 wherein said at least one activity comprises adsorption or desorption of an aromatic compound.

20. A method of paragraph 19 wherein the silica particles are pre-loaded with a detectable polyaromatic adsorbate and the liquid medium is being tested for the presence and optionally amount of at least one polyaromatic analyte, whereby the at least one polyaromatic analyte, when present, will displace the detectable polyaromatic adsorbate and the displaced adsorbate may be detected and optionally measured.

21. A method of paragraph 21 wherein the at least one polyaromatic analyte consists of polyaromatic compounds as a class.

22. A method of paragraph 23 which is performed in an analytical apparatus having a detector for the detectable adsorbate and wherein the liquid medium includes at last one monoaromatic compound containing exactly one aromatic ring, namely a benzene ring, and the monoaromatic compound does not displace the adsorbate in an amount detectable by the detector.

23. A method of fluid injection analysis performed in a system comprising a reactor, characterised in that the reactor contains organically-substituted silica particles.

24. A method of liquid chromatography, characterised in that the chromatography column contains organically-substituted silica particles. 25. A fluid injection or liquid chromatography system, or a component of either, comprising organically-substituted silica particles.

26. The subject matter of any of paragraphs 1 to 25 wherein the particles comprise a polar organic moiety.

27. The subject matter of any preceding paragraph wherein the particles are selected from the group consisting of nanoparticles having a size of less than about 1 μm and microparticles having a size of from about 1 μm to less than about 1000 μm.

28. The subject matter of any preceding paragraph wherein the particles have a size of from about 200 nm to about 900 nm.

29. The subject matter of paragraph 28 wherein the particles have a size of from about 400 nm to about 500 nm.

30. The subject matter of any of paragraphs 1 to 26 wherein the particles are microparticles having a size of from about 10 μm to about 100 μm.

31. The subject matter of paragraph 26 optionally in combination with at least one of paragraphs 27 to 30 wherein the polar organic moiety comprises a hydrocarbyl moiety substituted by one or more functional groups.

32. The subject matter of paragraph 31 wherein the functional groups are selected from nitro, substituted or unsubstituted amino, hydroxy, halo, carbonyl, imine, oxime, N- oxide, carboxy, nitrile, azide, diazonium, isonitrile, cyanate, isocyanate, and the sulphur analogues of the aforementioned O- containing groups.

33. The subject matter of paragraph 32 wherein the functional groups are selected from nitro, amino, hydroxyl and halo.

34. The subject matter of any of paragraphs 31 to 33 wherein the hydrocarbyl moiety comprises an aromatic ring.

35. The subject matter of paragraph 34 wherein the hydrocarbyl moiety is phenyl or naphthyl. 36. The subject matter of paragraph 34 wherein the hydrocarbyl moiety is phenyl.

37. The subject matter of any of paragraphs 31 to 33 wherein the hydrocarbyl moiety comprises an aliphatic or alicyclic moiety.

38. The subject matter of paragraph 34 or paragraph 37 wherein the hydrocarbyl moiety has up to 18 carbon atoms.

39. The subject matter of any preceding paragraph wherein the particle is obtainable by a process involving reaction of a silane ether monomer and an organically- substituted silane ether monomer.

40. The subject matter of paragraph 39 wherein the silane ether monomer is a tetraalkoxysilane.

41. The subject matter of paragraph 39 or paragraph 40 wherein the organically- substituted silane ether monomer is an aryltrialkoxysilane of which the aryl moiety is optionally substituted by at least one functional group.

42. The subject matter of any of paragraphs 1 to 30 wherein the particles are aryl- substituted silica microparticles or nanoparticles optionally having aryl groups substituted by one or more functional groups.

43. The subject matter of paragraph 42 wherein the aryl groups comprise phenyl groups.

44. The subject matter of paragraph 42 or paragraph 43 wherein the functional groups are selected from nitro, amino, hydroxy and chloro.

45. The use of particles as defined in any of paragraphs 1 to 44 to adsorb a polar organic compound in a liquid.

46. The use of paragraph 45 which further involves at least partial desorption of the adsorbed compound. 47. The use of paragraph 45 or paragraph 46 which comprises chromatography or fluid injection analysis.

48. The use of paragraph 47 wherein the chromatography or fluid injection analysis involves monitoring for displacement of the adsorbed polar organic compound upon contacting of the particles with a sample.

Claims

1. A method which involves or potentially involves organically-substituted silica particles in desorption of an adsorbate in a liquid medium, other than a method which is at least part of a process for making said silica particles.

2. A method of claim 1 , wherein the liquid medium is being tested, in a system having a detector for detecting a parameter indicative of occurrence of the desorption of the adsorbate, for the presence and optionally amount of a substance capable of causing the desorption.

3. A method of claim 2 wherein the particles carry a detectable adsorbate capable of being displaced by said substance.

4. A method of claim 3, wherein displacement of the adsorbate is detected and optionally measured by the detector as indirect detection and optionally measurement of said substance.

5. A method of claim 3 or claim 4, wherein the adsorbate is spectroscopically, optically or radiologically detectable.

6. A method of claim 5 wherein the adsorbate comprises a dye, wherein optionally the adsorbate is an aromatic compound.

7. A method of any of claims 4 to 6, wherein the silica particles are pre-loaded with a detectable polyaromatic adsorbate and the liquid medium is being tested for the presence and optionally amount of at least one polyaromatic analyte, whereby the at least one polyaromatic analyte, when present, will displace the detectable polyaromatic adsorbate and the displaced adsorbate may be detected and optionally measured.

8. A method of claim 7 which is performed in an analytical apparatus having a detector for the detectable adsorbate and wherein the liquid medium includes at last one monoaromatic compound containing exactly one aromatic ring, namely a benzene ring, and the monoaromatic compound does not displace the adsorbate in an amount detectable by the detector.

9. A method of any preceding claim wherein said substance is present in an amount such that said desorption occurs to an extent detectable by the detector.

10. A method of any of claims 1 to 8, wherein such substance is not present in an amount such that desorption occurs to an extent detectable by the detector.

1 1. A method of claim 1 wherein the adsorbate is desorbed by passing an eluant over the organically-substituted silica particles to elute the adsorbate from the particles, if the adsorbate is present; the method then comprising directly or indirectly detecting the presence and/or amount of adsorbate in the resulting eluate.

12. A method of claim 1 , wherein said organically substituted silica particles constitute a first set of particles and the method further uses a second set of hydrophobic silica particles, wherein the method comprises performing the method of claim 1 such that the substance, if present, in the liquid medium is adsorbed by the first set of particles, passing an eluant over the first set of particles to elute any of said substance adsorbed to the first set of particles and then passing the eluant through the second set of particles, whereby said substance, if present in the eluant, is sorbed by the second set of particles, the particles of the second set being responsive to sorption of said substance to produce a detectable response

13. A method of claim 12, wherein the detectable molecule is comprised in the particles or is on the surface of the particles.

14. A method of claim 12 or claim 13, wherein the second set of particles comprise hydrophobic silica gel particles.

15. A method of any of claims 1 1 to 14, wherein the liquid medium is being tested for the presence and optionally amount of at least one polyaromatic analyte.

16. A method of any of claims 1 1 to 15 wherein said substance is present in an amount such that said desorption occurs to an extent detectable by the detector.

17. A method of any of claims 1 1 to 15, wherein such substance is not present in an amount such that desorption occurs to an extent detectable by the detector.

18. A method of any preceding claim which is a liquid chromatographic method.

19. A method of any of claims 1 to 16 which is a fluid injection analysis method.

20. A method of any preceding claim in which the organically substituted silica particles are substituted by a moiety having at least one functional group.

21. A method of claim 20 in which the functional group is capable of at least one interaction selected from dipole-dipole interactions, hydrogen bonding interactions and charge transfer interactions.

22. A method of any preceding claim in which the organically substituted silica particles are substituted by a hydrocarbyl group having from 5 to 30 carbon atoms.

23. A method of claim 22 in which the hydrocarbyl group comprises a ring structure having from 5 to 18 carbon atoms.

24. A method of claim 21 or claim 22 wherein the hydrocarbyl group is substituted as specified in claim 22 or claim 23.

25. A method of claim 24 in which the ring structure is aromatic.

26. A method of claim 22 or claim 23 in which the hydrocarbyl group is phenyl.

27. A method of fluid injection analysis performed in a system comprising a reactor, characterised in that the reactor contains organically-substituted silica particles.

28. A method of claim 27, wherein the system further comprises a second set of organically modified silica particles, the particles of the second set comprising a detectable molecule.

29. A method of liquid chromatography, characterised in that the chromatography column contains organically-substituted silica particles and in that the method comprises determination of the presence or amount of a compound in an eluate.

30. A fluid injection system or liquid chromatography system, or a component of either, comprising organically-substituted silica particles and one of both of (a) a detector for detecting a detectable parameter downstream of the particles and (b) a solid phase having affinity for hydrophobic compounds and arranged downstream of the particles.

31. A system or component of claim 30, wherein the solid phase comprises hydrophobic silica particles comprising a fluorescent dye, the particles optionally being hydrophobic silica gel particles.

32. A method for preparing a system or component of claim 30 or claim 31 , comprising putting the organically-substituted silica particles into apparatus which forms part of the system or component.

33. Analytical apparatus adapted for analysis of a liquid, or a component thereof, in either case containing organically-modified silica particles.

34. The use of silica particles comprising a fluorescent dye to determine the presence and/or amount of an organic compound in a sample by contacting the particles with the sample and detecting for quenching of fluorescence.