GB2577405A

GB2577405A - Particle

Info

Publication number: GB2577405A
Application number: GB1915370.9A
Authority: GB
Inventors: Behrendt Jonathan; Bourcet Florence
Original assignee: Sumitomo Chemical Co Ltd
Current assignee: Sumitomo Chemical Co Ltd
Priority date: 2016-09-30
Filing date: 2016-09-30
Publication date: 2020-03-25
Anticipated expiration: 2036-09-30
Also published as: GB2577405B; GB201915370D0

Abstract

A particle comprising silica has polyether groups on a surface thereof. The particle may also include a polymeric light emitting material covalently bound to the particle. The particle may be included in a protic liquid.

Description

Particle

Field of the invention

The present invention relates to composite light-emitting particles and the use thereof as a luminescent marker. The present invention further relates to a method of preparing said composite particles.

Background of the Invention

Silica nanoparticles form highly stable suspensions in aqueous solvents, for example aqueous biological buffers, even at very high solid contents, due to their hydrophilic nature. Nanoparticles of silica and a light-emitting material have been disclosed as labelling or detection reagents.

Nanoscale Res. Lett., 2011, vol. 6, t7 328 disc r"atrment of a srrxafl molecule In a silica matrix.

Langmuir, 1992, vol. 8, pp 2921-2931 discloses coupling of a dye to a silane coupling t which is then incorporated into a silica sphere.

J. Mater. Chem., 2013, vol. 1, pp 3297-3304, Behrendt et al. describess silica-LEP nanoparticles where the LEP is covalently bound to the silica. The light emitting polymer has alkoxysilane groups pendant from the polymer backbone which react the silica monomer during formation of the nanoparticles.

Nanoscale, 2013, vol. 5, pp 8593-8601, Geng et al. describes silica-conjugated polymer (C. P) nanoparticles wherein the LEP has pendant non--polar alkyl side chains and where the nanoparticles have a "SiO4D.CK-41Sii0:," structure_ Chem. Mater., 2014, vol. 26, pp 1874-1880, Geng et al. discloses poly(9,0-di hexylfl uorene-all-2,1,3 zothi adi azole) (PFBT) loaded nanoparticles.

It is an object of the invention to provide structurally stable light--emitting particles.

It is a yet further object of t it invention to provide light-emitting particles having high colloidal stability.

It is a yet further object of the invention to provide a simple synthesis of said light-emitting particles

Summary invention

The present inventors have found that the combination of a silica polymer and a emitting polymer substituted with polar groups can provide stable light-emitting particles with good colloid forming properties.

Accordingly, in a first aspect of the invention provides a composite particle comprising a silica polymer and a light-emitting polymer comprising a backbone and polar groups pendant from the backbone.

In a second aspect the invention provides a colloidal suspension comprising composite particles according to the first aspect of the invention suspended in a liquid.

In a thira aspect the invention provides a process for preparing composite according to the first aspect of the invention, comprising formation of the silica polymer by polymerisation of a silica monomer in the presence of the light emitting polymer The present inventors have found that the colloidal stability of particles comprising silica, particularly colloidal stability in aqueous salt solutions, may be enhanced by providing polyether groups on the surface of the particles.

Accordingly, in a fourth aspect the invention provides particles comprising of silica having polyether groups on a surface thereof In a fifth aspect n provides a colloid comprising a liquid and particles of the fourth aspect. The liquid is preferably a protic liquid, optionally water or an alcohol.

The liquid may comprise one or more salts dissolved therein. The liquid may be a buffer solution.

In a sixth aspect the invention provides a method of forming particles according to the fourth aspect, the method comprising the step of reacting a reactive group of a compound comprising the reactive group and a polyether group with the particles to covalently bind the polyether group to the surface of the particles.

The reaction at the surface of the particles may be a reaction between the reactive group and silica at the surface or may be a reaction between another reactive group, optionally an amine, at the silica surface and the reactive group of the compound.

The particle of the fourth aspect stay comprise or consist of silica.

The particle the fourth aspect may be comprise silica and at least one light-err_itiing material. The light-emitting material may be polymeric or non-polymeric. The light-emitting material may or may not he covalently hound to the particle The particle may co be a composite particle according to the first aspect.

Description of the Drawings

The invention will now be described in more detail with reference to the drawings wherein: Figure 1 is a graph of mean number % vs. diameter (rim) for silica-LEP nanoparticles according to embodiments of the invention; Figure 2 is an absorption spectrum for silica-LEP nanoparticles according to embodiments of the invention; Figure 3 is a photoluminescence spectrum for silica-LEP nanoparticies according to embodiments of the invention; Figure 4 is a graph of size distributions of colloidal suspensions methanol of composite particles that have not been surface-treated and a composite particles that have been treated to form an amino group at the surface thereof; Figure 5 is a graph of size distributions of colloidal ensions in water of composite particles that have not been surface-treated and a composite particles that have been treated to form an amino group at the surface thereof; and -4 -Figure 6 is a graph of size distributions of colloidal suspensions in phosphate buffered saline. (pH 7.4) of composite particles that have not been surface-treated and a composite particles that have been treated to form a polvethyleneglycol chain at a surface thereof

Detailed description of the invention

A first aspect of the invention provides a composite particle comprising a mixture of a silica polymer and a light-emitting polymer comprising a backbone and polar groups pendant from the backbone.

"Silica polymer" as used herein polymer comprising siloxane groups. 'the co silica polymer may have a. linear, branched or crosslinked backbone comprising or consisting of alternating Si and 0 atoms The silica polymer may form a matrix in which the light-emitting polymer is dispersed. The light-emitting polymer and the silica polymer of the composite are not covalently bound to one another Accordingly, there is no need for the silica polymer and /1 or 'the light-emitting polymer to be substituted with reactive groups fbr forming such covalent bonds during formation of the particles.

The light-emitting polymer may emit fluorescent light, phosphorescent light or a combination thereof The light-emitting polymer may be a homopolymer or may be a copolymer comprising 20 two or more different repeat units.

The light-emitting polymer may comprise li ght-emitting groups in the polymer backbone, pendant from the polymer backbone or as end groups of the polymer backbone. In the case of a phosphorescent polymer, a phosphorescent metal complex, preferably a phosphorescent iridium complex, may be provided in the polymer backbone, pendant from the polymer backbone or as an end group of the polymer backbone.

The light-emitting polymer may have a non-conjugated backbone or may be a conjugated polymer. "conjugated polymer is meant a polymer comprising repeat -5 -units in the polymer backbone that are directly conjugated to adjacent repeat units. Conjugated light-emitting polymers include, without limitation, polymers comprising one or more of arylene, heteroarylene and vinyl ene groups conjugated to one another along the polymer backbone.

The light-emitting polymer ay have a iinea.r, branched or crosslinked backbone.

The light-emitting polymer may comprise one or more repeat units in the backbone of the polymer substituted with at least one polar group. The one or more polar groups may be the only substituents of said repeat units, or said repeat units may be further substituted with one or more non-polar groups, optionally one or more C1-40hydrocarbyl groups. 'The repeat unit or repeat units substituted with one or more polar groups may be the only repeat units of the polymer or the polymer may comprise one or more further co-repeat units wherein the or each co-repeat unit is unsubstituted or is substituted with non-polar groups, optionally one or more C1.40hydrocarbyl groups.

C1.40 hydrocarbyl groups as described herein include, without limitat unsubstituted phenyl and phenyl substituted with one or more C1.20 grow As used herein "polar groups" may refer to one more groups which render the light-emitting polymer with a solubility of at least 0.0005 mg/ml in an alcoholic solvent, preferably at least 0.001, 0.01, 0.1, 1, 5 or 10 mg/ml. The solubility is measured at 25°C. Preferably, the alcoholic solvent is a Cpio alcohol, more preferably methanol.

Polar groups are preferably groups capable of forming hydrogen bonds or ionic groups.

In one embodiment of the first aspect of the invention, the light-emitting polymer comprises polar groups of formula -0(R.40)q-R3 wherein R° in each occurrence is a C1.

o alkylene group" optionally a Ci.< alkylene group, wherein one or more non-adjacent, non-terminal C atoms of the alkylene group may be replaced with 0, R4 is H or C1.5 alkyl, and q is at least 1, optionally 1-10. Preferably, q is at least 2. More preferably, q is 2 to 5. The value of q may he the same in all the polar groups of formula -0030),I-R4. The value of q may differ between polar groups of the same polymer.

By "C1.5 alkylene group" as used herein with respect to R3 is meant a group of formula -(CH2)/-wherein f is from 1--S.

Preferably, the light-emitting polymer comprises polar groups of formula 0(\CH2CH20),A4 wherein q is at least 1, optionally 1-10 and IC is a Ci_5 alkyl group, preferably methyl. :Preferably, q is at least 2.. More preferably, q is 2 to 5, most preferably q is 3.

In one embodiment of the first aspect of the invention, the light-emitting polymer comprises polar groups of formula -N(R5)2, wherein it" is H or C1_,2 hydrocarbyl. Preferably, each R5 is a C1-"hydrocarbyl.

lo In one embodiment of the first aspect of the invention, the light-emitting polymer comprises polar groups which are ionic groups which may be anionic, cationic or zwitterionie. Preferably the ionic group is an anionic Grow, Exemplary anionic group are --000-, a sulfonate group hydroxide; sulfate; phosphate; phosphinate; or phosphonate.

An exemplary cationic group is -N(115)35 wherein R5 in each occurrence is I)1-hydrocarbyl. Preferably, each R5 is a C1-12 hydrocarbyl.

A light-emitting polymer comprising cationic or anionic groups comprises counterions to balance the charge of these ionic groups.

An anionic or cationic group and counterion may have the same valency, with a an counterion balancing the charge of each anionic or cationic group.

The anionic or cationic group may be monovalent or polyvalent Preferably" the anionic and cationic groups are monovalent.

The light-emitting polymer m comprise a plurality of anionic or cationic polar groups wherein the charge of two or more anionic or cationic groups is balanced by a single counterion. Optionally, the polar groups comprise anionic or cationic groups comprising di-or trivalent counterions.

The counterion is optionally a cation, optionally a metal cation, optionally Li +,Na. , K.+, Cs-, preferably Cs+, or an organic cation, optionally ammonium, such as tetraalkylatnrnonium,Cthy IMethyl imidazolium orpyridinium.

The counterion is optionally an anion, optionally a halide; a sulfonate group, optionally mesyl ate or tosylate; hydroxide; carboxylate; sulfate; phosphate; phosphinate; phosphonate; or borate.

In one embodiment of the first aspect of the invention, the light--emitting polymer comprises polar groups selected from groups of formula ---0(R30)g-Rt groups of formula -.N(R5)2, groups of formula OR' and/or ionic groups. Preferably, the light- in emitting polymer comprises polar groups selected from groups of formula -- 0(CH2C+FI20)gR4, groups of formula --N(102, and/or anionic groups of formula --COOT Preferably, the polar groups are selected from the group consisting of groups of formula -0(R30)q-R4, groups of formula -N(R5)1, and/or ionic groups. Preferably, the polar groups are selected from the group consisting of polyethylene glycol (PEG) groups of formula 0(C1-120420),Te, groups of formula -N(R5)7, and/or anionic groups of formula -COO-. R3, Ri, R5, and q are as described in relation to other embodiments of the in ti Optionally, the backbone of the light-emitting polymer is a conjugated polymer. Optionally, the backbone of the conjugated light-emitting polymer comprises repeat an units of formula (I): ci (I) wherein Ar' is an arylene group or heteroarylene group; Sp is a spacer group; m is 0 or 1; R1 independently in each occurrence is a polar group; n is 1 if in is 0 and n is at least -8 - 1, optionally 1, 2, 3 or 4, if m is 1; R2 independently in each occurrence is a non-polar group; p is 0 or a positive integer; q is at least I, optionally 1, 2, 3 or 4; and wherein Sp, R' and R2 may independently in each occurrence be the same or different.

Prefer, n is 1 and n is 2-4, more preferably 4. Preferably p is 0.

Arl of formula (1) is optionally a C6:,(1arylene group or a 5-20 membered heteroarylene group. Ari is preferably a 06.10 arylene group, optionally phenylene, fluorene, benzofluorene, phenanthrene, naphthalene or anthracene, more preferably fluorene or phenylene, most preferably fluorene.

Sp-(On may be a branched group" optionally a dendritic group, substituted with polar :to groups, optionally -NIT2 or -OH groups, for example polyethyleneimine.

Preferably, Sp is selected from: - Ci.20 alkylene or phenylene-C1.20 alkylene wherein one or more non-adjacent C atoms may be replace with 0, S. Nor C=0; - a Co.yo arylene or 5-20 membered heteroarylene, more preferably phenyiene, which, in addition to the one or more substituents R', may be unsubstituted or substituted with one or more non-polar substituents, optionally one or more C1-20 alkyl groups.

"alkylene" as used herein means a branched or linear divalent alkyl chain.

"non-terminal C atom" of an alkyl group as used herein means a C atom other ban the methyl group at the end of an n-alkyl group or the methyl groups at the ends of a branched alkyl chain.

More preferably, Sp is selected from: - alkylene wherein one or more non-adjacent C atoms may be replaced with 0, S or CO, and -9 - - a C6-20 arylene or a 5-20 membered heteroarytene, even more preferably phenylene, which may he unsubstituted or substituted with one or more non-polar substituents.

R may he a polar group as described anywhere herein. Preferab R * is: - a polyethylene glycol (PEG) group of formula -0(CILCII20),,R4 wherein q is at least t, optionally 1-1.0 and R' is a -5 alkyl group, preferably methyl; - a group of formula1\KRD12, wherein R' is IT or Ci.0 hydrocarbyl; - an anionic grout offormula --000-. ;In the case where n is at least two, each R" may independently rn each occurrence be the ro same or different. Preferably, each R' attached to a given Sp group is different. ;In the case where p is a positive integer,optionally I, 2, 3 or 4, the group R.' may be selected from: - alkyl, optionally 01.70 alkyl; and - aryl and heteroaryl groups that may be unsubstituted or substituted with one rg or more substituents, preferably phenyl substituted with one or more C1-20 alkyl groups; a linear or branched chain of aryl or heteroaryl groups, "c. of which groups may independently be substituted, for example a group of formula -(Ar'), wherein each 4r3 is independently an aryl or heteroaryl group and s is at least 2, preferably a branched or linear chain of phenyl groups each of which may be unsubstituted or substituted with one or more C_20 alkyl groups; and - a crossii kable-group, for example a group comprising a double bond such and a vinyl or acrylate group, or a benzocyclobutane group -10 -Preferably, R-L, where present, is independently selected from C1-4011).,vdroc' b and is more preferably selected from C1_70alkyl; unusubstituted phenyl; phenyl substituted with one or more Ci_20 alkyl groups; and a linear or branched chain of phenyl groups, wherein each phenyl may be unsubstituted or substituted with one or more substituents. ;A polymer as described herein may comprise or consist of only one form of the repeating unit of formula (I) or may comprise or consist of two or more different repeat units of formula (I). ;Optionally, the polymer comprising one or more repeat units of t a (I) is a copolymer comprising one or more co-repeat units. ;If co-repeat units are present then the repeat units of formula (I) may form between 0 199 mol % of the repeat units of the polymer, optionally 50-99 mol % or 80-99 mol 11e,. Preferably, the repeat units of formula (I) form at least 50 mol% of the repeat units of the polymer, more preferably at least 60, 70, 80, 90, 95, 98 or 99 mol%. Most preferably the repeat units of the polymer consist of one or more repeat units of formula (I). ;The or each repeat unit of the polymer lay be selected to produce a desired colour of emission of the polymer. ;The backbone of a polymer comprising a unit formula (I) may be non-conjugated or conjugated. ;The polymer is preferably a conjugated polymercomprising repeat units of formula (I) conjugated to one another and / or conjugated to aromatic or heteroaromatic groups of co-repeat units adjacent to the repeat units of formula (I). Exemplary conjugated polymers include polymers comprising arylenevinylene repeat units; arylene repeat units; heteroarylene repeat units; and combinations thereof. ;If present, the or each co-repeat unit may be unsubstituted or substituted with one or more non-polar substituents, optionally one or more repeat units comprising or consisting of one or more groups selected from C16_70 arylene groups and 5-20 membered heteroarylene groups, wherein each of said arylene or heteroarylene groups independently in each occurencemay be unsubstituted or substituted with one or more non-polar substituents.. ;Arylene 'repeat units of the polymer include, without limitation, fluorene, preferably a 2,7-linked fluorene, phenylene, preferably a L4-linked phenytene, naphthalene, anthra.cene, indenottuorene, phenanthrene and dihydrophenanthrene repeat units. Arylene co-repeat units may be selected from repeat units of formulae (111)-(/11): (R13),,, (R13)d IV) (R13), (R I 3)d (R13)u (R13)e wherein R13 in each occurrence is independently a substituent; c is 0, or 4, preferably 1 or 2; each d is independently 0, 1,2 or 3, preferably 0 or I; and e is 0, or 2, preferably 2. ;Repeat units comprising or consisting of one or more unsubstituted or substituted 5-20 membered heteroaryiene groups in the polymer backbone include, without limitation, thiophene repeat units, bithiophene repeat units, benzothiadiazole repeat units, and combinations thereof Exemplary heteroarylene co-repeat units include reheat units oti formulae (VIE), (VEIL) and (IX): (IX) wherein R12 in each occurrence is independently a substituent d f is 0. 1 or 2. ;R12 in each occurrence may independently be a group comprising or consisting of a polar group, optionally a polar suhstituent -(Sp)",-(0n, or a non-polar substituent R2 wherein Sp, tn, Rt and R2 are as described with reference to Formula (I). ;Arylene repeat units or heteroarylene repeat units substituted with one or more polar groups, optionally repeat units of formulae (III)-(LX) substituted with one groups of lomtula -(Sp),,-(R5,3, are repeat units of formula (1). ;Arylene repeat units or heteroarylene repeat units, optionally repeat units of formulae (111)-(IX), which are unsubstituted or substituted only with one or more non-polar groups, are co-repeat units of the polymer. ;In the case of a phosphorescent conjugated polymer a phosphorescent group, preferably a metal complex, more preferably an iridium complex, may be provided in the main chain, in a side group and or as an end group of the polymer. An exemplary conjugating repeat unit comprising an iridium complex has formula: (I 13)f (1:213){ (R 3)t Preferably, he repeat unit of formula is e eat unit of form Oa). ;(R)p (R2)p (R1)n(R1)n Oa) wherein p, Sp, RI-and n are independently in each occurrence as described in relation to the repeat unit of formula fl). Preferably, n in each occurrence is 2. Preferably p in each occurrence is 0. ;An exemplary repeat unit of formula (Ia) is: ;O ;O ;1'O-Cs' Cs-0 H3C(OH2CH2C)30 0(CH2CH20)3CH3 OptionaHy, the silica polymer comprises repeat unit. [la and/or -14 -OR° OR" ORb (IIa) (Iib) wherein R° in each occurrence is independently selected from H or 01.12 hydrocarbyl, optionally 14 or Chp alkyl. Optionally, the silica polymer further comprises repeat units of formula (11c): (tic) It 11 be appreciated that the Si atom of the repeat unit of formula (11) is hound to an 0 lc) atom in the polymer backbone or a group of formula ORE. ;Preferably, at least 0.1 wt% of total weight of the composite particle consists of the light-emitting polymer. Preferably at least 1, 10, 25 or 50 wi% of the total weight of the composite particles consists of the light--emitting polymer. ;Preferably at least 50 wt% of the total weight of the composite particles consists of the silica polymer. Preferably at least 60, 70, 80, 90, 95, 98, 99, 99.5, 99.9 wt.% of the total weight of the composite particles consists of silica polymer. ;In one embodiment of th aspect of the invention, at least 70 of the total weight of the composite particles consists of the light-emitting polymer and silica polymer. Preferably at least 80, 90, 95, 98, 99, 99.5, 99.9 wt% of the total weight of the composite particles consists of the light-emitting polymer and silica. More preferably the composite particles essentially consists of the t -emitting polymer and silica polymer. ;In one embodiment of the first aspect of the invention, the composite particles are nanoparticulate. Preferably, the nanoparticies have a number average diameter of no more than 5000 nm, more preferably no more than 2500nm, 1000nm, 900nm, 800nm, 700nm, 600 mn, 500nm or 400 nm as measured by a Malvern Zetasizer Nano ZS. Preferably the nanoparticles comprises particles with a number average diameter of between 5-5000 nm, optionally 10-1000 am, preferably 25-600 nm, more preferably between 50-500 nm, most preferably between 75-400nm as measured by a.Malvern ro Zetasizer Nano ZS. ;The composite particles may be provided as a colloidal suspension comprising the composite particles suspended in a liquid. Preferably, the liquid is selected from water, Clem alcohols and mixtures thereof Preferably, the colloidal suspension does not comprise a surfactant. ;/5 The composite particles are fluorescent or phosphorescent. Preferably the composite particles are fluorescent. Preferably the composite particles are for use as a fluorescent probe, more preferably for use as a fluorescent probe in an immunoassay such as a lateral flow or solid state immunoassay. Optionally the composite particles are for use in fluorescence microscopy or flow cytometry. ;According to the third aspect of the invention, the composite particles of any embodiment of the first aspect of the invention may be formed by polymerisation of a silica monomer in the presence of the light emitting polymer. ;In one embodiment, the process comprises treating a solution of silica monomer and light emitting polymer with a base, or by adding a solution of silica monomer to a solution of the light-emitting polymer and a base, wherein the solvents of the solutions are water, one or more C1.:0 alcohols or a combination thereof In another ernbodirnent,. the process comprises polymerising silica monomer in a solution of the monomer and light emitting polymer under acidic conditions. ;It will be appreciated that the mixture of the silica polymer and light-emitting polymer of the composite particles so formed may or may not be homogeneous and may include, without limitation, one or more chains of light-emitting polymer encapsulated within the particle and / or one or more chains extending through a particle. ;The polar groups of the light-emitting; polymer may enhance solubility of the polymer in polar solvents and may prevent the polymer from assuming a tightly coiled formation as compared to the case where a light-emitting polymer in which the polar groups are absent is placed in a polar solvent. ;The composite particle may be formed from the light-emitting polymer and the silica monomer in a one-step process of polymerisation of the silica monomer in the presence of the light-emitting polymer. ;Optionally, the silica monomer is an alkoxysilane, preferably a trialkox alkoxysilane, optionally a 01.12 trialkoxy or tetra-alkoxvsilane, for example tetraethyl orthosilicate. The silica monomer may be substituted only with alkoxy groups or may be substituted with one or more groups. In one embodiment, the silica monomer is substituted with a polyether group. In another embodiment, the silica monomer is substituted with a reactive binding group, as described in more detail below, which does not react during polymerisation of the silica monomer or which is protected during polymerisation of the silica monomer. ;Optionally, the solution comprises or consists of an ionic solvent or a protic solvent; preferably a solvent selected from water, alcohols and mixtures thereof. Exemplary alcohols include, without limitation, methanol, ethanol, 1-propanol, isopropanol. butanol, 2-butanol, t-butanol and mixtures thereof Preferably the solution comprises or consists of an alcoholic solvent selected from methanol, ethanol, iscpropanol or mixtures thereof, more preferably the solution comprises or consists of a solvent selected front methanol, ethanol or mixtures thereof. Preferably, the solvent system does not comprise a non-alcoholic solvent other than water. ;In one embodiment of the third aspect of the invention, the base is an aqueous base preferably, a solution of a hydroxide such as a metal hydroxide, preferably alkali metal hydroxide, ammonium hydroxide or tetraalkylammonium hydroxide in water, preferably 10-40% wlw NH3 in water, preferably 20-30% wlw NH3 in water. ;in one embodiment of the aspect of the invention, the light emitting polymer: silica monomer weight ratio is in the range 1 I to 1 500, preferably 1 3 to I to 1: 200, most preferably I 10 to 1: 100. The present inventors have found that the diameter of the particles can be tuned by selection of the light-emitting polymer: silica weight ratio. ;In one embodiment of the third aspect of the invention, the concentration of the light emitting polymer in the solution is at least 0.0005 mgiml, preferably at least 0.001, 0.01, 0.1, 1 or 10 mg/nil at 25°C. ;Optionally, the ss of forming the composite pal titles comprises the steps of: (a) dissolving the light-emitting polymer in a solvent system selected from one or more prone solvents, optionally water, alcohols and combinations thereof; (b) adding a base to the solution obtained in step (a); and (c) adding a solution of silica monomer to the solution of step (b). ;Optionally, the process is conducted in a homogeneous solution. ;The composite particles tnay be isolated following formation and resuspended in an aqueous solvent, an organic solvent or a mixture thereof The composite particles may be isolated from the reaction mixture by centrifuging. ;Silica at the surface of the composite compositeparticles may be reacted to coyalen ly bind a receptor to the surface of the silica. The receptor may be directly bound to the silica surface or spaced apart therefrom. ;A chain binding the receptor to the silica surface preferably comprises or consists of a vJ colloid stabilising group that enhances stability of a colloid comprising the composite particles in a. prone liquid such as water or an alcohol in which one or more solutes may be dissolved. The liquid may be a buffer solution. ;In one embodiment, the receptor is covalently bound to the composite nanopanicie in a process comprising e steps of 3:brining a first reactive group RCH at a surface of the silica; - reacting the reactive group with a compound comprising a second reactive group R02 capable of reacting with the first reactive group to form a covalent bond and a third reactive group RG3; - reacting the third reactive group RG3 v; h the eceptor to covalently bind the so receptor to the composite nanoparticic Silica at the surface of the composite particles may be reacted with an organosilane substituted with reactive binding group 1301, optionally an organosilane of formula (X): (-ft 0)3Si-Spa-R0 I wherein R' is H or a substituent, preferably a C1-111 alkyl group; Spi is a spacer group; and R01 is a first reactive group. ;Optionally,R01 is selected from the group consisting of: amines, preferably -N(RR)2 wherein R8 occurrence is H or a subs truer preferably H or a C1-5 alkyl, more preferably El; carboxylic acid or an ester thereof, optionally N-hydroxysuccinimide est alkene; alkvne; SE[;, or azide. ;An exemplary organosilane is 3-amiaiopropyl triethoxysilane. ;The reactive binding is reacted with a con pound of formula (XI RG2-Sp2-RG3 (XI) wherein RG2 is a group capable of reacting with RG1 to form a covalent bond; Sp2 is a spacer group; and RG3 is a reactive binding group capable of binding to a receptor. ;Optionally, RG1 is an amine and RG2 is a group capable of reacting with the amine, optionally a group capable of reacting with the amine to foulr an amide, optionally a carboxylic acid or acid chloride. ;Spl and Sp2 may each be selected according to their colloid stabilising properties. ;_to The present inventors have found that an polyether chain spacer group at the surface of a silica particle may stabilise collids comprising the particles, particularly in aqueous buffer solution liquids, such as &icons buffers having a salt concentration greater than 10 inNi. ;By "poiyether chain" as used herein is meant a divalent chain comprising at least two ether groups. ;Optionally, Sp' and Sp2 are each independently selected from a linear or branched divalent alkyl ene chain wherein one or more non-adjacent C atoms may be replaced with 0, S, C(=010, C(=0)NR12 or KR", wherein R12 in each occurenc,e is independently selected from H and C1-12 hydrocarbyl, optionally Cl -12 alkyl. ;Preferably, at least one of SO and Sp2 comprises or consists of a repeating unit of formula (Xl): C.R1410511 (Xl) wherein R'4 and 1215 are each independently fl or C14, alkyl and b is at least I, optionally 1-5, preferably 2, and c is at least 2, optionally 2-1,000, preferably 10-500, 10-200 or 10-100. The group of formula (XI) may be polydisperse. The group of formula (X0 may have a Mn of at least 500, optionally at least 2,000 Preferably, at least one of Sp,and Sp-comprises or consists of a polyethyleneglycol chain Optionally-, one of groups Spl-and Sp2 has chain length of 1-10 atoms, optionally a CI. ra alkylene chain, and the other of Sp' and Sp2 comprises a repeating, unit of formula (X1). ;The binding group 13(i3 may be reacted with a receptor which may be synthetic group or a receptor including, without limitation, biological material, optionally peptides, so carbohydrates, antibodies, antigens, enzymes; proteins, cell receptors, DNA, RNA, PNA" aptamers and natural products; biologically derived material, optionally recombinant antibodies, engineered proteins; and biornimics, optionally synthetic receptors, bionninetic catalysts, combinatorial ligands and imprinted polymers. A preferred bioreceptor is streptavidin. ;rg It will be appreciated that other methods may be used to covalently bind a receptor and or a colloid stabilising group to the surface of a silica particle including, without limitation, polymerising a silica monomer that is substituted with a colloid stabilising group and / or an unprotected or protected reactive group RGI; and reacting the composite particle with a compound of formula (R70)3Si-Spl--RC33 wherein Sp 1 comprises a colloid stabilising group. ;In use the particle having receptor groups at the surface thereof may bind to target biomolecules in a sample. Bi om ol call es include without limitation DNA, RNA, peptides, carbohydrates, antibodies, antigens, enzymes, proteins and hormones. A preferred bi ()molecule is biotin. ;The sample may be immobilised on a surface which is brought o contact with the composite nanoparticles described herein, preferably treated with a colloidal suspension comprising the composite nanoparticies described herein. ;The polystyrene-equivalent number-average molecular weight (Mn) measured by gel permeation chromatography of the light-emitting polymers or the silica polymers described herein may be in the range of about 1x103 to 1x108, and preferably 1x10' to 5x10P. The polystyrene-equivalent weight-average molecular weight (Mw) of the polymers described herein may be 1x103 to ix 108, and preferably ix 1_04 to ix 10'. ;Polymers as described herein are suitably amorphous polymers. ;Composite particles as described herein may be used in, without limitation, biological noising fluorescence microscopy, flow cytometry and fluorescence-based noassays. ;so Example ;Method "Mr form g silica-LEP composite ttiZt opciriicles Oa the StOher process: Structure of LE Pi EEP1, disclosed in VC) 2012/133229, the contents of which are incorporated herein by reference, was dissolved in methanol (either 1 mg/mL or 10 trig/mL) by heating to 50 'V for 1 hour and the solution was then cooled to room temperature. To 2 mL of this solution was added 0.15 mi... of ammonium hydroxide (30% aq.), followed by rapid addition of a solution comprised of tetraethylorthosilicate ETEOS, 0.2. mL) and methanol (0.5 mL), with stirring at room temperature. Stirring was continued for 1 h at room temperature, after which time the solution was centrifuged at 14,000 rpm for 10 minutes to isolate the resultant silica-LEP nanoparticies from the supernatant containing excess unreacted TEOS and ammonium hydroxide. The supernatant was removed by decantation and gentle sonication was used to redisperse the isolated pellet of Ce nanoparticies in of fresh methanol. Wash cycles consisting of centrifugation, decantation and redispersion in methanol (2.5 rnL) were repeated a further two times, followed by three similar washes using 2.5 ML of deionised water. Finally, the nanoparticles were redispersed in 1.5 mL of deionised water for measurement of particle size Yea dynamic light scattering using a Malvern Zetasizer Nano ZS. ;The solid content of the as-prepared nanoparlicle suspension (mass of nanoparticles/volume) was determined by isolating the solid nanoparticles from I mL of the dispersion by centrifugation at 14,000 rpm for 10 minutes. After washing twice with methanol by centrifugation, decantation and redispersi on (as above) and leaving the io solid pellet to dry overnight, the mass of solid was determined using a microbalance. ;The optical density of the as--prepared nanoparticle dispersion was determined using a Cary 5000 UV--vis-lit. spectrometer. ;A Hamamatsu C9920-02 PL quantum yield spectrometer equipped with integrating sphere accessory was used to determine the photoluminescence quantum yield of the nanoparticies in aqueous dispersion. ;Table I PLQY of silica-LEP nanoparticles prepared with varying ratios of LEN and TEOS 1:100 3.7 1:10 LEP/TEOS ratio Number average diameter (nm) Solid content of as-prepared dispersion (mg/ fl ;PLQY (04) ;The size distribution, absorption spectra. and photoluminescence spectra of these composite particles are shown in Figures 1-3. ;Due to their very high fluorescence brightness, stability in aqueous buffers and ease of surface attachment to hi mnolecules, the silica-LEP nanoparticles prepared are particularly well suited for use as fluorescent tracers or tags for optical sensing assays. ;odificatit of composite nanopartictes To a 3 niL suspension of composite nanoparticles in methanol (number average diameter by dynamic light scattering = 165 rum, solid content 4 mg/mL) was added 120 uL of (3-aminopropyetriethoxysilane and the suspension was stirred at room co temperature for 1 hour. The suspension was centrifuged at 14,000 rpm for 2 minutes to isolate the resultant silica-LEP nanoparticles from the supernatant containing excess unreacted. (3-aminopropyptriethoxysilane. The supernatant was removed by decantation and gentle sonication was used to redisperse the isolated pellet of nanoparticles in 3 min of fresh methanol. Wash cycles consisting of centrifugation, decantation and redispersion in methanol (3 triT) were repeated a further two times, before finally redispersing in 3 mL methanol. To prepare samples for dynamic light scattering analysis, 100 uL of the suspension was centrifuged and the supernatant ecanted as above and the isolated nanoparticles were resuspended in 1 of either methanol or water. ;PI Gyklii0 if am ino-m ed composite n °particles 1 mL of the suspension of amino-modified composite nanoparticles in methanol formed in the example above was centrifuged at 14, 000 rpm for 2 minutes to isolate the nanopartictes through decantation of the supernatant. A I mL solution of a, w-Bis{ 2-[(3-carboxyl -oxopropyl)aminoiethyl}polyethylene glycol (Mr = 2000 glinol, 10 mg), N(3-aminopropy1)-N-ethylc,arbodiimide (2.1 mg) and N-hydroxysuccinimide (2.5 mg) in methanol was used to redisperse the nanoparticle pellet by gentle sonication and the resultant suspension was stirred at room temperature for 1 hour. The suspension was centrifuged at 14,000 rpm for 2 minutes to isolate the resultant silica-LEP nanoparticles from the supernatant containing excess unreacted PEGylation reagents. The supernatant was removed by decantation and gentle sonication was used to redisperse the isolated pellet of nanoparticles in I mL of fresh methanol. Wash cycles consisting of centrifugation, decantation and redispersion in methanol (i nth) were repeated a further two times. Before the final centrifugation and decantation, the suspension was aliquoted into four 250 tiL portions and the resultant pellets were stored at -20 °C prior to use. ;Conjugation o,streptraidin to PEGylated composite nanoparticies One of the isolated Paiyiated composite nanoparticle pellets in the example above was resuspended in I int_ of phosphate buffered saline (pH 7 4) by gentle sonication, followed by immediate addition of 50 uL of a solution of streptavidin in the same buffer mg/mL), The suspension was stirred at room temperature for i. hour before adding to the top of a 4.5 cm height, 3 cm diameter column packed with Sephacryl S-300 HR. separation media (prewashed with 150 mL of phosphate buffered saline). The column was eluted with the same buffer collecting 1.5 mL fractions. The column fraction containing the highest concentration of nanoparticles (based on fluorescence intensity) was selected for use in a subsequent bio-assay. ;Assessing the colloidal stabil.ty of composite nanoparticles in rear ions c3isper*sants The following test was used to determine the relative stability of bare and functionalised composite nanoparticle (produced from the same batch) in various dispersants. Following centrifugation and decantation, isolated composite nanoparticles (--0.4 mg) were redispersed in the dispersant (I mL) by sonication in a bath sonicator for 5 minutes. Immediately prior to DLS analysis the nanoparticle suspension was sonicated for a further minute and was then analysed using a.1Malvern Zetasizer Nano ZS. Table 2 shows the polydispersity index (Rd]) of bare and surface modified composite nanoparticles in various dispersants, as determined by DLS and figures 4-6 show the corresponding number average size distributions.

Table 2

Composite Pd! in PdI in PdI in particle surface methanol water phosphate modification buffered saline (pH 7.4) None ("bare" 0.083 0.155 0.409 particle) amino 0.060 0-474 -PEG-COOH - 0.131 (Mr 2000) Preparing No it al?"e for io-A glass microscope slide functionaiised with a self-assembled monolayer of (3-aminopropyOsil ane was submersed in a solution containing succinic anhydride (1 g) and trimethylamine (1.3 mL) in acetonitrile (50 mi..) for 16 hours, before washing three times with fresh acetonitrile (50 After drying, a Grace-Biolabs Secure Seal imaging spacer was affixed to the surface of the resultant carboxy-functionalised glass slide in order to isolate four circular areas (diameter = 9 mm) for use in the subsequent binding assay. Within each isolated area of the slide was added 80 uL of a I mt.

ro solution containing N -(3-am inopropy1)-N-ethyl carbodii i de (77.0 mg) and N-hydroxylsultbsuecinimi de (33.0 mg). After leaving at room temperature for 30 ruins, the solutions were removed and isolated areas washed three times with water (80 uL). After removing the last wash solution, to two of the areas was added 80 iff, of a solution of biotinylated bovine serum albumin (50 ug/mL) in phosphate buffered saline (pH 7.4)and to the two remaining areas was added SO Lae of a blocking buffer containing bovine serum albumin (3 wt. ?/-b) in phosphate buffered saline (pH 7.4) containing 0.01 wt. % Tween-20. After 1 hour at room temperature, solutions were removed from the two areas containing hiotinylated bovine semm albumin solutions and in their place was added 80 u1_, of the blocking buffer described above. After a further hour at room temperature, solutions were removed from all four areas and each was washed three times with phosphate buffered saline (pH 7.4) containing 0.01 wt. % Tween-20.

Bic -binding assay 11.5111 t MilIMAS le.?pt nicks To each of the bovine serum albumin modified areas produced in the example above (two biotinylated and two non-biotinylated) was added 60 uL of the column fraction containing streptavidin-modified composite nanoparticies described in the previous example. After leaving for 1 hour at room temperature, the solution was removed and washed three times with 80 ti, of phosphate buffered saline (pH 7.4) containing 0.01 wt. % Tween-20 and once with 80 0_, of deionised water. After allowing to dry in air, the fluorescence intensity of each of the four assay regions was measured using a microscope-based spectrometer, using a mercury lamp as the excitation source Rex ro 365 nm) and a fibre-optic spectrometer for detection. As shown in figure X, the average integrated fluorescence intensity of the two assays containing biotin is higher than that for the non-hiotinylated control r4ons" demonstrating, that Si-LET nanoparti des have been immobilised on the surface through specific streptavidin-biotin interactions.

Although the present invention has been described in terms of specific exemplary embodiments, it will be appreciated that various modifications, alterations and/or combinations of features disclosed herein will be apparent to those skilled in the art without departing from the scope of the invention as set forth in the following claims.

Clauses A composite particle comprising a mixture of a silica polymer and a light-emitting polymer comprising a backbone and polar groups pendant from the backbone.

The composite particle according to clause 1, wherein the light-emitting polymer has a solubility of at least 0.001 mg/ml in an alcoholic solvent.

The composite particle ccording to clause 1 or 2, wherein the polar groups comprise or consist of groups of formula-0(R30)-R' wherein R' in each occurrence is a C140 alkylene group wherein one or more non-adjacent C atoms so may be replaced with 0, R" is H or Ck5 alkyl and q is at least 1.

The composite particle according to any one of the preceding clause, wherein the polar groups comprise or consist of ionic groups.

The composite particle according to clause wherein he ionic groups ar C00. groups.

6. The composite particle according to any one of the preceding clauses wherein the light-emitting polymer is a conjugated polymer.

The composite particle according to clause 6 wherein the backbone of the lig' emitting polymer comprises repeat units of formula (I): (0 wherein Art is an arylene group; Sp is a spacer group; m is 0 or 1; RI is a polar group; n is 1 if m is 0 and n is at least I if m is 1; R2 is a non-polar substituent; p is 0 or a positive integer; q is at least 1; and wherein Sp, RI and R2 may independently in each occurrence be the same or different.

The composite particle according to clause 7 wherein the repeat unit of formula (I) is a repeat unit of formula (Ia): (R)n (R1)ri (Ia) wherein R2, p, Sp: and n are independently in each occurrence as defined in clause 7.

9. The composite particle according to any one of the preceding precedini clauses, wherein to the silica comprises repeat units of formula lla and/or Ilb: OR° sio Si )Ro OR' (11a) wherein Rn in each occurrence is independently selected from H or Ci.p hydrocarbyl, The composite particle according to clause 9, wherein the silica further comprises repeat units of fmmula (R2 (R2)E, The composite particle according to any one of the preceding clauses, wherein the composite particle is nanoparticulate.

12. The composite particle according to any one of the preceding clauses,wherein the composite particle is fluorescent.

13. The composite particle according to one of the preceding clauses, comprising a receptor group for binding to a biomolecule covalently bound to the surface of the silica polymer.

lit. The composite particle according to any one of the preceding clauses wherein a polyether chain s cwaiently bound to the surface of the silica polymer.

The composite particle according to clauses 13 and 14 wherein the polyether chain is provided between the surface of the silica polymer and the receptor.

16. A colloidal suspension comprising composite particles according to any preceding clause suspended in a liquid.

17. A colloidal suspension.c ording to clause:h liquid is a protic liquid.

18. A colloidal suspension according to clause 17 wherein the prc comprises one or more salts dissolved therein.

00 19 A process for preparing composite particles according to any one of clauses 115, comprising formation of the silica polymer by polymerisation of a silica monomer in the presence of the light emitting polymer. -3 -

20. A process according to clause 19 wherein the silica monomer,dissolved in a solvent, is polymerised in the presence of a base.

The process according to clause 20, wherein the silica monomer is a trialkoxy or tetraalkoxv slime.

The process according to any one of clauses 19-21 wherein,-herein the light-emitting polymer and the silica monomer are dissolved in a polar solvent.

The process according to any one of clauses 19-22 wherein the light-emitting polymer and the silica monomer are dissolved in a solvent selected from water, alcohols and combinations thereof.

The process according to any one of clauses 19-23., wherein the light emitting polymer: silica monomer weight ratio is in the range 1: 1 to 1: 500.

The process according to any one of clauses 19-24, wherein the concentration of the light emitting polymer in the alcoholic solution is at least 0.1 e. The process according, to one f clauses 1 5, wherein the process comprises the steps of (a) dissolving the light-emitting polymer in a solvent (b) adding a base o the solution obtained in step (a), and (c) adding a solution of silica monomer to the solution of step (b).

The process according to any of clauses 19-26, wherein the surface of the composite particles is functionalisecl with a group capable of binding to a biomolecule.

2S. A method of marking a biomolecule, the method comprising the step of binding the biomolecule to a composite particle according to clause 13.