GB2617821A

GB2617821A - Monomer and light-emitting polymer

Info

Publication number: GB2617821A
Application number: GB2204604.9A
Authority: GB
Inventors: Kamtekar Kiran; Islam Nazrul
Original assignee: Sumitomo Chemical Co Ltd
Current assignee: Sumitomo Chemical Co Ltd
Priority date: 2022-03-30
Filing date: 2022-03-30
Publication date: 2023-10-25
Also published as: WO2023187020A1; GB202204604D0

Abstract

A compound of formula (I): wherein Ar1 is a C6-20 aromatic group which is unsubstituted or substituted with one or more substituents and X is selected from halogen, boronic acid, ester of boronic acid, trifluoroborate salt, sulfonic acid, ester of a sulfonic acid, Sn(R9)3 wherein R9 C1-12 hydrocarbyl group, or SnCl3. Also disclosed is a method for forming a polymer comprising polymerising formula (I) with a monomer of formula M1, where M1 is a monomer of Ar1 as seen in formula (I), disubstituted with Y, where Y is selected from the same list as X, but X and Y are not the same. Also disclosed is a conjugated light-emitting polymer of the formula where the thiophene ring and Ar1 repeat in an [A-B-A-B]n formula. The light-emitting polymer can form a composite particle comprising the light-emitting polymer and a matric material. Also disclosed is a light-emitting marker comprising the light-emitting polymer or composite particle and a binding group configured to bind to a target material. The light-emitting marker is used in a method of detecting a target analyte by contacting the light-emitting marker with a sample.

Description

Monomer and Light-Emitting Polymer Background

Embodiments of the present disclosure relate to conjugated light-emitting polymers, and the use thereof as a luminescent marker.

Light-emitting polymers have been disclosed as labelling or detection reagents.

J. Mater. Chem., 2013, vol. 1, pp 3297-3304, Behrendt et al. describes 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 with the silica monomer during formation of the nanoparticles.

Chem. Mater., 2014, vol. 26, pp 1874-1880, Geng et al. discloses poly(9,9-dihexylfluorene-alt-2,1,3-benzothiadiazole) (PFBT) loaded nanoparticles.

Light emitting marker particles are described in W02020/058668.

Summary

In some embodiments, the present disclosure provides a compound of formula (I): (I) wherein At.' is a C620 aromatic group which is unsubstituted or substituted with one or more substituents and Xis selected from the group consisting of halogen, boronic acid, an ester of boronic acid, trifluoroborate salt, sulfonic acid, an ester of a sulfonic acid, Sn(R9)3 wherein R9 independently in each occurrence is a C112hydrocarbyl group, and SnC13.

In some embodiments, the present disclosure provides a conjugated light-emitting polymer comprising a repeating structure of formula (P): (1) wherein Art is a C62, aromatic group which is substituted with one or more substituents including at least one polar group substituent comprising a group of formula -O(R30)-R4 wherein R3 in each occurrence is a Cis, alkylene group, R4 is H or Cis5 alkyl, and v is o or a positive integer, and q is a positive integer.

Description of the Drawings

The disclosed technology and accompanying figures describe some implementations of the disclosed technology.

Figure lisa graph of absorption and emission spectra for a light emitting polymer according to some embodiments.

Figure 2 is graph of absorption and emission spectra for a fluorescent nanoparticle according to some embodiments.

The drawings are not drawn to scale and have various viewpoints and perspectives. The drawings are some implementations and examples. Additionally, some components and/or operations may be separated into different blocks or combined into /5 a single block for the purposes of discussion of some of the embodiments of the disclosed technology. Moreover, while the technology is amenable to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are described in detail below. The intention, however, is not to limit the technology to the particular implementations described. On the contrary, the technology is intended to cover all modifications, equivalents, and alternatives falling within the scope of the technology as defined by the appended

Detailed Description

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to." As used herein, the terms "connected," "coupled," or any variant thereof means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, electromagnetic, or a combination thereof.

Additionally, the words "herein," "above," "below," and words of similar import, when -3 -used in this application, refer to this application as a whole and not to any particular portions of this application. Where the context permits, words in the Detailed Description using the singular or plural number may also include the plural or singular number respectively. The word "or," in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list. References to an atom include any isotope of that atom unless stated otherwise.

The teachings of the technology provided herein can be applied to other systems, not necessarily the system described below. The elements and acts of the various examples rn described below can be combined to provide further implementations of the technology. Some alternative implementations of the technology may include not only additional elements to those implementations noted below, but also may include fewer elements.

These and other changes can be made to the technology in light of the following /5 detailed description. While the description describes certain examples of the technology, and describes the best mode contemplated, no matter how detailed the description appears, the technology can be practiced in many ways. Details of the system may vary considerably in its specific implementation, while still being encompassed by the technology disclosed herein. As noted above, particular terminology used when describing certain features or aspects of the technology should not be taken to imply that the terminology is being redefined herein to be restricted to any specific characteristics, features, or aspects of the technology with which that terminology is associated. in general, the terms used in the following claims should not be construed to limit the technology to the specific examples disclosed in the specification, unless the Detailed Description section explicitly defines such terms.

Accordingly, the actual scope of the technology encompasses not only the disclosed examples, but also all equivalent ways of practicing or implementing the technology under the claims.

To reduce the number of claims, certain aspects of the technology are presented below in certain claim forms, but the applicant contemplates the various aspects of the technology in any number of claim forms. For example, while some aspect of the technology may be recited as a computer-readable medium claim, other aspects may likewise be embodied as a computer-readable medium claim, or in other forms, such as being embodied in a means-plus-function claim. -4 -

In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of implementations of the disclosed technology. It will be apparent, however, to one skilled in the art that embodiments of the disclosed technology may be practiced without some of these specific details.

Monomer In some embodiments there is provided a compound of formula (I): (I) io wherein An is a C620 aromatic group which is unsubstituted or substituted with one or more substituents and Xis selected from the group consisting of halogen, boronic acid, an ester of boronic acid, trifluoroborate salt, sulfonic acid, an ester of a sulfonic acid, Sn(R9)3 wherein R9 independently in each occurrence is a C112 hydrocarbyl group, and SnC13.

In some embodiments, the compound of formula (I) is a solid room temperature (2022 °C) and pressure.

In some embodiments, the ester of a sulfonic acid is a -0S021{6 group, wherein R6 in each occurrence is independently a C112 alkyl group which is unsubstituted or substituted with one or more F atoms; or phenyl which is unsubstituted or substituted with one or more F atoms. -0S02126 is preferably tosylate or triflate.

In some embodiments 129 is C1-5 alkyl.

An exemplary trifluoroborate salt is -BF,K. Exemplary boronic esters have formula (VIII): pR7 *-B (VIII) -5 -wherein It in each occurrence is independently a C20 alkyl group, represents the point of attachment of the boronic ester to an aromatic ring of the monomer, and the two groups R7 may be linked to form a ring which is unsubstituted or substituted with one or more substituents, e.g. one or more C16 alkyl groups.

Optionally, 127 independently in each occurrence is selected from the group consisting of C2 alkyl; unsubstituted phenyl; and phenyl substituted with one or more C16 alkyl groups.

In a preferred embodiment, the two groups 127 are linked, e.g. to form: / *-B N-O 0 0 \ / * -Br 1 \ OF 0 OF 0 or A halogen leaving group is preferably Br or I. Exemplary compounds of formula (I) are:

OH Ar' -6 -

Optionally Art may be a C6-C14 arylene, for example selected from phenylene, fluorene, benzofluorene, phenanthrene, dihydrophenanthrene, naphthalene or anthracene.

The Arl may be unsubstituted or substituted. Substituents may be selected from polar and non-polar substituents. In some preferred embodiments, Art is substituted with one or more polar substituents, optionally one or more ionic substituents.

Optionally, Art is a group of formula (II): wherein Ar2 is an arylene group, e.g. a C6 14 arylene group; Sp is a spacer group; m is 0 /0 or 1; R., independently in each occurrence is a polar group; n is 1 if m is o and n is at least 1, optionally 1, 2, 3 or 4, if m is i; R2 independently in each occurrence is a non-polar group; p is o or a positive integer; q is at least 1, optionally 1, 2, 3 or 4; and wherein Sp, RI and R2 may independently in each occurrence be the same or different.

In some embodiments, q is 1 or 2. Preferably, m is iand n is 1-4.

/5 Preferably p is 0.

Preferably, Sp is selected from: - C1_20 alkylene or phenylene-C,,, alkylene wherein one or more non-adjacent C atoms may be replace with 0, S, N or C=0; - a C620 arylene or 5-20 membered heteroarylene, more preferably phenylene, which, other than the one or more polar groups Ri, may be unsubstituted or substituted with one or more non-polar substituents, optionally one or more CI-20 alkyl groups.

More preferably, Sp is selected from: -alkylene wherein one or more non-adjacent C atoms may be replaced with 0, S or CO; and - a C6_20 arylene or a 5-20 membered heteroarylene, even more preferably phenylene, which may be unsubstituted or substituted with one or more may be an ionic group or a non-ionic polar group.

An exemplary non-ionic polar group has formula -0(R30),-124 wherein R3 in each occurrence is a C,,0 alkylene group, optionally a 0_5 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 05 alkyl, and v is 0 or a positive integer, optionally 1-10. Preferably, v is at least 2. More preferably, v is 2 to 5. The value of v may be the same in all the polar groups of formula -0(R30)-R4. The value of v may differ between polar groups of the same polymer.

Optionally, the non-ionic polar group has formula O(CH2CH20),R4 wherein v is at least 1, optionally 1-10 and R4 is a C5 alkyl group, preferably methyl. Preferably, v is at least 2. More preferably, v is 2 to 5, most preferably v is 3.

By "C,1, alkylene group" as used herein with respect to R3 is meant a group of formula -(C112)1-wherein f is from 1-10.

By "non-terminal C atom" of an alkyl group as used herein means a C atom other than 20 the methyl group at the end of an n-alkyl group or the methyl groups at the ends of a branched alkyl chain.

In some embodiments, An may be substituted with a substituent consisting of an ionic group or comprising one or more ionic groups. Ionic groups may be anionic, cationic or zwitterionic. Preferably the ionic group is an anionic group.

Exemplary anionic group are -COO-, a sulfonate group; hydroxide; sulfate; phosphate; phosphinate; or phosphonate.

An exemplary cationic group is -N(R5)3+ wherein R5 in each occurrence is H or C1-12 hydrocarbyl. Preferably, each R5 is a 012 hydrocarbyl. -8 -

Cationic substituents may interact electrostatically with a target comprising one or more anionic groups, e.g. polysaccharides, polynucleotides, peptides and proteins carrying one or more anionic groups.

An Art 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 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.

An may 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 co u nterions.

In the case of an anionic group, the cation counterion is optionally a metal cation, /5 optionally Lit, Na+, K+, Cs, preferably Cs, or an organic cation, optionally ammonium, such as tetraalkylammonium, ethylmethyl imidazolium or pyridinium.

In the case of a cationic group, the anion counterion is optionally a halide; a sulfonate group, optionally mesylate or tosylate; hydroxide; carboxylate; sulfate; phosphate; phosphinate; phosphonate; or borate.

In some embodiments, the An comprises polar groups selected from groups of formula -0(R30),--R4 and / or ionic groups. Preferably, Ar' comprises polar groups selected from groups of formula -0(CH2C1-120),R4 and/or anionic groups of formula -COO-.

Preferably, at least one 121-is -COO-.

In the case where n is at least 2, each R' may independently in each occurrence be the or same or different. In some embodiments where n is at least 2 each Ri is different.

In the case where p is a positive integer, optionally 1, 2, 3 or 4, the group R2 may be selected from: -alkyl, optionally C1-20 alkyl; and

-

aryl and heteroaryl groups that may be unsubstituted or substituted with one or more substituents, preferably phenyl substituted with one or more C,_ -20 alkyl groups; - a linear or branched chain of aryl or heteroaryl groups, each of which groups may independently be substituted, for example a group of formula -(Ar3)s wherein each A 1.3 is independenlly an aryl or heLeroaryl 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 C1-20 alkyl groups; and - a crosslinkable-group, for example a group comprising a double bond such io and a vinyl or acrylate group, or a benzocyclobutane group.

Two R2 groups may be linked to form a ring, e.g. a 6-membered ring or 7-membered ring. Optionally, two R2 groups are linked to form a ring in which the linked R2 groups form a C4-or C5-alkylene chain wherein one or more non-adjacent C atoms of the alkylene chain may be replaced with 0, S, NIU° or Si(1210)2 wherein Rm in each /5 occurrence is independently a C1_20 hydrocarbyl group.

Preferably, each R2, where present, is independently selected from C140 hydrocarbyl, and is more preferably selected from C120 alkyl; unsubstituted phenyl; phenyl substituted with one or more C120 alkyl groups; and a linear or branched chain of phenyl groups, wherein each phenyl may be unsubstituted or substituted with one or more substituents; or two R2 groups are linked to form a ring as described herein Optionally, AP groups of formula (11) are selected from formulae (lla)-(11d): (11b) -10 - (R13)d (R13)d R13 R13 R13 R13 (Tic) (TId) wherein 12*3 in each occurrence is independently -(Sp)1-(R1)11 or R2 and two R13 groups may be linked to form a ring, with the proviso that at least one R13 is -(Sp)1-(R1)11; c is 0, 1, 2, 3 or 4, preferably 1 or 2; each d is independently o, 1,2 or 3, preferably o or 1; and e is 0,1 or 2, preferably 2.

In some preferred embodiments, the Ari group of formula (IIb) is a group of formula (IIb-1): (R2)p (R2)p Sp S\p (R1)/ (R1), wherein R2, p, Sp, Ri and n are independently in each occurrence as described above. Tn some preferred embodiments, n in each occurrence is 2. In some preferred embodiments, p in each occurrence is o.

Polymer synthesis and Monomers /5 Two examples of monomers that may be polymerised to give rise to polymers described herein are shown below. It may be beneficial to use the lower route as thiophenes, such 2,5-dibromothiophene, may be a liquid which can be difficult to purify and weigh out accurately compared to a solid monomer.

X and Y are reactive groups as described herein. X and Y are capable of selectively reacting with each other to create a direct carbon-carbon bond such as boronic acids/ester, boron trifluorides, trialkyl tin and chlorides, bromides, iodides and triflates.

A polymer as described herein may be formed by polymerising a compound of formula (I) and a monomer of formula (M1): io wherein AR, in Mi and AR, in the compound of formula (I) are the same; and Y is selected from the group consisting of halogen, boronic acid, an ester of boronic acid, trifluoroborate salt, sulfonic acid, an ester of sulfonic acid, Sn(R9)3 wherein R9 independently in each occurrence is a C112 hydrocarbyl group, and SnC13; provided that X and Y are not the same.

The polymerisation method includes, without limitation, methods for forming a carbon-carbon bond between an aromatic carbon atom of the compound of formula (I) and an aromatic carbon atom of Mi.

Optionally Xis selected from one of group (a) and group (b), and Y is selected from the other of group (a) and group (b): (a) halogen or -0S02R8 wherein 128 is an optionally substituted C1-12 alkyl group or optionally substituted aryl group; (b) boronic acid and esters thereof; and -SnR9 wherein R9 independently in each occurrence is a C112hydrocarbyl group.

-12 -A carbon-carbon bond is formed during polymerisation between aromatic carbon atoms to which X and Y are bound.

Suitable polymerisation methods include, without limitation, Suzuki polymerisation and Stille polymerisation. Suzuki polymerisation is described in, for example, WO 5 00/53656.

In some embodiments, each X may be one of: (i) a halogen or -0S0 2R6; or (ii), a boronic acid or ester, and each Y may be the other of (i) and (ii).

In some embodiments, each X may be one of: (i) a halogen or -0SO2R6; and (iii) -SnR93, and each Y may be the other of (i) and (iii).

Optionally, 126 in each occurrence is independently a C12 alkyl group which is unsubstituted or substituted with one or more F atoms; or phenyl which is unsubstituted or substituted with one or more F atoms.

-0S02R6 is preferably tosylate or triflate. An exemplary trifluoroborate salt is -BF3K.

Exemplary boronic esters have formula (VIII): pR7 * -B'OR7 (VIII) wherein R7 in each occurrence is independently a C120 alkyl group, * represents the point of attachment of the boronic ester to an aromatic ring of the monomer, and the two groups 127 may be linked to form a ring which is unsubstituted or substituted with one or more substituents, e.g. one or more Cfi alkyl groups.

Optionally, 127 independently in each occurrence is selected from the group consisting of C112 alkyl; unsubstituted phenyl; and phenyl substituted with one or more C1-6 alkyl groups.

In a preferred embodiment, the two groups R7 are linked, e.g. to form: -13 -0 * -B 0 0 *-B * -B

D * -B

0 or 0 or 0 or A halogen leaving group is preferably Br or T. Polymer In some embodiments there is provided a conjugated light-emitting polymer comprising a repeating structure of formula (P):

S

wherein An is a C620 aromatic group which is substituted with one or more substituents including at least one polar group substituent Rl comprising a group of formula - 0(R30)"-R4 wherein 123 in each occurrence is a C1_5 alkylene group, R4 is H or C,5 alkyl, and v is 0 or a positive integer, and q is a positive integer.

By "conjugated polymer", e.g. a conjugated light-emitting polymer is meant a polymer having a backbone containing repeat units that are directly conjugated to adjacent repeat units in the polymer backbone. It will be appreciated that the polymer backbone /5 is not conjugated along its entire length, due to interruptions in conjugation arising from at least the conjugation-breaking repeat unit.

The polymer may have a solubility in water or a G_Fs alcohol at 20°C of at least 0.1 mg / ml, optionally at least 0.5 mg / ml or at least 1 mg/ml.

The polymer may have a solubility in a Cis, alcohol, preferably methanol, at 20°C of at least 0.1 mg / ml, optionally at least 0.5 mg / ml or at least mg/ml.

Solubility may be measured by the following method: The solid polymer is weighed out into a glass vial. The required amount of polar solvent (for example methanol) is added followed by a small magnetic stirrer. Then the vial is -14 -tightly capped and put on a preheated hot plate at 60 0C with stirring for 30 min. The polymer solution is allowed to cool to room temperature before use. The polymer solution can also be prepared by sonicating the polymer containing vial for 30 min at room temperature. The solubility of polymer was tested by visual observation and under white and 365 nm UV light.

Optionally, the light-emitting polymer may have an absorption spectrum having a maximum in the range of 400-500 nm, optionally in the range of 425-475 nm, optionally in the range of 440-460 nm, optionally about 450 nm.

Optionally, the light-emitting polymer may emit light having a peak wavelength in the io range of 460-520 nm, optionally in the range of 480-500 nm.

Optionally, the light-emitting polymer can be used as a water-soluble fluorescent tag for use in systems where an excitation source in the 440-460 region is used, optionally where an excitation source in about 450 nm is used.

Polymer-based dyes are desired due to their high extinction coefficients compared to small molecule dyes leading to high fluorescent tag brightnesses.

One or more repeat units of the polymer may be substituted with one or more water or C1-8 alcohol -solubilising substituents. A water or C18 alcohol solubilising substituent as described herein may enhance solubility of the polymer as compared to a polymer in which the water or C1.8 alcohol solubilising substituent is not present, e.g. in which the water or Cs alcohol solubilising substituent is replaced with H or a non-polar substituent such as an alkyl substituent.

The water or C18 alcohol solubilising substituent may consist of a polar group or may comprise one or more polar groups. Polar groups are preferably non-ionic groups capable of forming hydrogen bonds or ionic groups.

Conjugated polymers as described herein may be formed by polymerising monomers comprising leaving groups that leave upon polymerisation of the monomers to form conjugated repeat units. Exemplary polymerization methods include, without limitation, Yamamoto polymerization as described in, for example, T. Yamamoto, "Electrically Conducting And Thermally Stable pi-Conjugated Poly(arylene)s Prepared by Organometallic Processes", Progress in Polymer Science 1993, 17, 1153-1205, the contents of which are incorporated herein by reference and Suzuki polymerization as -15 -described in, for example, WO 00/53656, WO 2003/035796, and US 5777070, the contents of which are incorporated herein by reference.

The monomers may be formed by polymerisation of monomers containing boronic acid leaving groups or esters thereof, and halide or pseudohalide (e.g. sulfonate) leaving groups. Leaving groups may be selected to control which monomers may or may not form adjacent repeat units in the polymer. No thiophene repeat units are adjacent to one another in the polymer.

The polystyrene-equivalent number-average molecular weight (Mn) measured by gel permeation chromatography of the polymers described herein, preferably the polymers io described herein may be in the range of about ixicP to ixios, and preferably Duo) to 5x10o. The polystyrene-equivalent weight-average molecular weight (Mw) of the polymers described herein may be nno3 to mos, and preferably nno4 to Darr.

The photoluminescence spectrum of light-emitting materials or compositions as described herein may be as measured using an Ocean Optics 2000+ spectrometer.

Mechanisms for energy transfer include, for example, resonant energy transfer; Forster (or fluorescence) resonance energy transfer (FRET), quantum charge exchange (Dexter energy transfer) and the like.

Luminescent marker A luminescent marker may comprise a light-emitting polymer, and a binding group, preferably a biomolecule binding group, configured to bind to a target analyte.

Methods of preparing light-emitting markers comprising light-emitting materials are described in W02020/058668.

In some embodiments, the binding group is bound, preferably covalently bound, to the polymer. The binding group may be provided as a side group of a repeat unit of the 25 polymer or as an end-group of the polymer.

In some embodiments, the luminescent marker is dissolved in a sample to be analysed. In some embodiments, the luminescent marker is a particulate luminescent marker.

Formation of a luminescent nanoparticle marker may comprise collapse of a light-emitting polymer. A light-emitting particle may comprise a light-emitting polymer or a -16 -mixture of a polymer and a matrix. The matrix may at least partially isolate the light-emitting polymer from the surrounding environment. This may limit any effect that the external environment may have on the lifetime of the light-emitting polymer.

In some embodiments, the particle comprises the light-emitting polymer, 5 homogenously distributed through the matrix.

In some embodiments, the particle may have a particulate core and, optionally, a shell wherein at least one of the core and shell contains the light-emitting polymer. Preferably, the light-emitting particle contains the light-emitting polymer and a matrix material.

ro Polymer chains of the polymer may extend across some or all of the thickness of the core and / or shell. Polymer chains may be contained within the core and / or shell or may protrude through the surface of the core and / or shell.

In some embodiments, the particle comprises a core comprising or consisting of the light-emitting polymer and a shell comprising or consisting of the matrix.

/5 The matrix may be inorganic. The inorganic matrix may be an oxide, optionally silica, alumina or titanium dioxide.

Preferably, the matrix is not covalently bound to the polymer. Accordingly, there is no need for the matrix material and / or the polymer to be substituted with reactive groups for forming such covalent bonds, e.g. during formation of the particles.

In some embodiments, a silica matrix as described herein may be formed by polymerisation of a silica monomer in the presence of the light-emitting polymer.

In some embodiments, the polymerisation comprises bringing a solution of silica monomer into contact with an acid or a base. The acid or base may be in solution. The light-emitting composition may be in solution with the acid or base and / or the silica monomer before the solutions are mixed. Optionally, the solvents of the solutions are selected from water, one or more Ci s alcohols or a combination thereof.

Polymerising a matrix monomer in the presence of a polymer may result in one or more chains of the polymer encapsulated within the particle and / or one or more chains of the polymer extending through a particle.

-17 -The particles may be formed in a one-step polymerisation process.

Optionally, the silica monomer is an alkoxysilane, preferably a trialkoxy or tetraalkoxysilane, optionally a Cis,2 trialkoxy or tetra-alkoxysilane, for example tetraethyl orthosilicate. The silica monomer may be substituted only with alkoxy groups or may 5 be substituted with one or more groups.

In some embodiments, a luminescent marker as described herein comprises a biomolecule binding group is bound to a surface of a light-emitting particle. The biomolecule binding group may be bound directly to the surface of the particle group or bound through a surface binding group. The surface binding group may comprise polar io groups. Optionally, the surface binding group comprises a polyether chain. By "polyether chain" as used herein is meant a chain having two or more ether oxygen atoms.

Silica at the surface of the particles may be reacted to form a group at the surface capable of binding to a biomolecule binding group. Optionally, silica at the surface is reacted with a siloxane.

The biomolecule binding group of a soluble or a particulate light-emitting marker as described herein may be selected from the group consisting of: DNA, RNA, peptides, carbohydrates, antibodies, antigens, enzymes, proteins and hormones. The biomolecule binding group may be selected according to a target biomolecule to be 20 detected.

Target biomolecules include without limitation DNA, RNA, peptides, carbohydrates, antibodies, antigens, enzymes, proteins and hormones. It will be understood that the biomolecule binding group may be selected according to the target biomolecule or binding agent.

The binding group of the light-emitting marker for binding to a target analyte may be attached to a functional group of a precursor of the light-emitting marker comprising the light-emitting polymer. In some embodiments, the functional group is covalently bound to the polymer. In some embodiments, the functional group is covalently bound to a matrix material of a precursor comprising the matrix material and the light-emitting polymer.

Optionally the functional group is selected from: -18 -amine groups, optionally -N12.112 wherein Ruin each occurrence is independently H or a substituent, preferably H or a C5 alkyl, more preferably H; carboxylic acid or a derivative thereof, for example an anhydride, acid chloride or ester, acid chloride, acid anhydride or amide group; -OH; -SH; an alkene; an alkyne; and an azide; and biotin or a biotin-protein conjugate.

The functional group may be reacted with or conjugated to a biomolecule to form a linking group linking the biomolecule to the rest of the light-emitting marker, the linking group being selected from esters, amides, urea, thiourea, Schiff bases, a primary rn amine (C-N) bond, a maleimide-thiol adduct or a triazole formed by the cycloaddition of an azide and an alkyne.

In the case where the functional group is biotin, it may be conjugated to a protein, e.g. avidin, streptavidin, neutravidin and recombinant variants thereof, and a biotinylated biomolecule may be conjugated to the protein to form the light-emitting marker.

The biotinylated biomolecule may comprise an antigen binding fragment, e.g. an antibody, which may be selected according to a target antigen.

In the case of a light-emitting particle, the functional group may be bound to a surface of the particle core, e.g. bound to a matrix material of the light-emitting particle core. Each functional group may be directly bound to the surface of a light-emitting particle core or may be spaced apart therefrom by one or more surface binding groups. The surface binding group may comprise polar groups. Optionally, the surface binding group comprises a polyether chain.

The surface of a light-emitting particle core may be reacted to form a group at the surface capable of attaching to a functional group. Optionally, a silica-containing particle is reacted with a siloxane.

Preferably, particulate luminescent markers or particulate luminescent marker precursors as described herein have a number average diameter of no more than 5000 nm, more preferably no more than 25oonm, r000nm, 900nm, 800nm, 700nm, 600 nm, 500nm or 400 nm as measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS. Preferably the particles have a number average diameter of between 5-5000 nm, optionally ro-r000 urn, preferably between 10-500 urn, most preferably between lo-roonm as measured by a Malvern Zetasizer Nano ZS.

Preferably, at least 50 wt% of the total weight of the particulate luminescent marker precursor consists of matrix material. Preferably at least 60, 70, 80, go, 95, 98, 99, 99.5, 99.9 wt% of the total weight of the particle consists of matrix material.

The particulate luminescent markers or particulate luminescent marker precursors as described herein may be provided as a colloidal suspension comprising the particles suspended in a liquid. Preferably, the liquid is selected from water, C,,F, alcohols and mixtures thereof. Preferably, the particles form a uniform (non-aggregated) colloid in io the liquid.

The liquid may be a solution comprising salts dissolved therein, optionally a buffer solution.

Optionally the polymer may be covalently attached to a biomolecule (such as a protein or antibody). Optionally a biomolecule can be attached via the polymer side chains or end-capping group. Optionally a biomolecule can be attached and the biomolecule and the polymer are attached via a linking group Z which may be a direct bond, a alkylene, a -0(W0),-group wherein R3 in each occurrence is a Gm3alkylene group and v is o or a positive integer, an arylene group or a heteroarylene group.

H 0 yo0 biomolecule N 0 polymer* Z 4)polymer* H2N-biomolecule +polymer*

H

H2N -b om o lecu le Zy N biomolecule 0 0 -Epolymer+

Z OH

Applications -20 -Luminescent markers comprising light-emitting polymers as described herein may be used as luminescent probes in an immunoassay such as a lateral flow or solid state immunoassay. Optionally the luminescent markers are for use in fluorescence microscopy or flow cytometry. Optionally the luminescent markers are for use in fluorescence microscopy, flow cytometry, next generation sequencing, in-vivo imaging, or any other application where a light-emitting marker is brought into contact with a sample to be analysed. The analysis may be performed using time-resolved spectroscopy. The applications can medical, veterinary, agricultural or environmental applications whether involving patients (where applicable) or for research purposes.

/0 Optionally, in use the light-emitting polymer is irradiated by light of two or more different wavelengths, e.g. wavelengths including at least two of 355, 405, 450, 488, 530,562 and 640 nm ± 10 nm. By use of polymers having well-defined absorption bands, absorptions at different wavelengths are readily distinguishable from one another.

/5 In some embodiments, dissolved light-emitting composition is brought into contact with a sample to be analysed.

In some embodiments, particles containing the light-emitting polymer, for example the particles in a colloidal suspension, are brought into contact with a sample to be analysed. The particles may comprise a matrix and the light-emitting polymer as described herein. A target analyte may be immobilised on a surface carrying a group capable of binding to the target analyte, either before or after the target analyte binds to a component of the dissolved light-emitting polymer, or to particles containing the light-emitting polymer. The target analyte bound to the light-emitting polymer, or one of a polymer or a light-emitting group mixed with the polymer, may then be separated from any light-emitting composition which is not bound to the target analyte.

In some embodiments, the particles may be stored in a dry, optionally lyophilised, form.

Examples

Monomer Synthesis -21 -Br 0E1 0(CH2CH20)3Me (s SnEfus l'cl(PP113)4 Me(OH2CH2C)30 0E1

OR

Me(OH2CH2C)30 0(CH2CH20)3Me OEt Stage I OEt Monomert Stage 1 Dibromofluorene derivative 1 can be made as described in US9536633.

Dibromofluorene derivative 1 (25 g, 26.4 mmol) was dissolved in DMF (250 mL). 2-Tributylstannylthiophene (45.1 g, 121 mmol) was added and the reaction mixture degassed with a stream of nitrogen for 1 h. Tetrakis(triphenylphosphine) palladium(o) (i.o6 g 0.9 mmol) was added and the reaction was stirred at 90 °C for 16 h. After cooling the reaction mixture was poured into water and extracted with ethyl acetate. The ethyl acetate extract was passed through a celite plug and washed with water. The /o aqueous layer was extracted with a further two portions of ethyl acetate. The combined ethyl acetate extracts were washed with brine, dried with sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography on silica eluting with 0-25% methanol in ethyl acetate. The product-containing fractions were combined to yield 19 g (76% yield) stage 1 material with >97% HPLC purity.

Monomer 1 Stage 1 material (15 g, 15.7 mmol) was dissolved in THF (375 mL). NBS (5.57 g, 31.3 mmol) was added in three lots and the reaction mixture stirred for 6 h. The reaction mixture was diluted with water and the product was extracted with ethyl acetate twice. The combined organics were washed with sodium bisulfate and brine, dried over sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography on silica eluting with o-io% methanol in ethyl acetate. The product-

NBS

Me(OH2CH2C)30 OEt 0(CH2CH20)3Me -22 -containing fractions were then purified by reverse-phase column chromatography eluting with acetonitrile-water with the product eluting at 100% acetonitrile. The product-containing fractions were combined to yield 9.6 g (56% yield) Monomer 1 with HPLC of 99.6% Polymer Synthesis A polymer was made by the polymerisation of: and OEt Me(OH2CH2C)30 0(CH2CH20)3Me OEt with a phenylboronic acid end-capper and then hydrolysed with cesium hydroxide. The process used was analogous to those described in US9536633. The absorption and emission of the resultant polymer are shown in Figure 1.

The polymer was dissolved in water at 1 mg/mL and incorporated into silica nanoparticles by reacting with tetraethoxy orthosilicate (TEOS) and ammonium /5 hydroxide.

MiliQ water was taken in a screw cap amber vial with a septum. The required amount of base (aq. ammonium hydroxide) was added followed by an oval stirrer bar. The pH of the reaction mixture was found to be 10.1-10.3 at 21 °C. The reaction mixture was -23 -stirred to mix well and to make a clear solution. The vial was capped tightly and heated for 10 minutes on a preheated oil bath at 60 °C, with stirring at 550 rpm. Tetraethyl orthosilicate (TEOS) was transferred into a small vial. Using a long needle and syringe, TEOS was injected into the solution by submerging the needle in the solution. To ensure all TEOS was injected, some solution was withdrawn and reinjected several times. The needle/syringe was retracted with the needle left in the headspace of the reaction vessel. The reaction was left for 16 hours at 6o °C with stirring at 550 rpm. The stirring was stopped, the vial was removed from the oil bath and allowed to cool down to room temperature. The needle/syringe was removed. A Zeba desalting column On io mL, 7K MWCO) was equilibrated with MiliQ water according to the manufacturer's recommendation. The crude sample was loaded and spun down to collect the desalted sample.

The nanoparticles were treated with ammonium hydroxide then sonicated. The nanoparticle suspension was filtered using 0.45 pm syringe filter. The fluorescent nanoparticles obtained have the absorption and emission as shown in Figure 2.

Claims

-24 -Claims A compound of formula CO: (1) wherein Arl is a C620 aromatic group which is unsubstituted or substituted with one or more substituents and Xis selected from the group consisting of halogen, boronic acid, an ester of boronic acid, trifluoroborate salt, sulfonic acid, an ester of a sulfonic acid, Sn(R9)3 wherein R9 independently in each occurrence is a CI 12 hydrocarbyl group, and 2) The compound according to claim 1 wherein Ay-is substituted with at least one substituent selected from the group consisting of: - C1-40 alkyl wherein one or more non-adjacent, non-terminal C atoms may be replaced with 0, S, CO or COO and one or more H atoms may be replaced with F; - a group of formula -(Ar2)" wherein Ar2 in each occurrence is independently an aryl or heteroaryl group which is unsubstituted or substituted with one or more nonionic substituents; and 3) The compound according to claim 1 or 2 wherein Ay-is substituted with at least 4) The compound according to any preceding claim wherein Art is substituted with at least one R1 group and 121 independently in each occurrence is a polar group.5) The compound according to claim 4, wherein RI comprises a group of formula - 0(1230),-R4 wherein 10 in each occurrence is a C1,5 alkylene group, 124 is H or 0,5 alkyl, and v is o or a positive integer.6) The compound according to any preceding claim wherein Ai, is a group of formula (11b): -25 -(11b) wherein 129 is -(Sp)1-(121), or R2 and two R9 groups may be linked to form a ring, with the proviso that at least one 129 is -(Sp)1-(122); Sp is a spacer group; m is (7) or 1; 122 is a polar group; n is 1 if m is 0 and n is at least 1 if m is 1; R2 is a non-polar group; each d is (7), 1, 2 or 3, wherein R9, Sp, m, n, 12, and R2 may independently in each occurrence be the same or different.7) The compound according to claim 6 wherein Art is a group of formula (Jib-1): wherein Sp is a spacer group; 121 is a polar group; n is at least 1; R2 is a non-polar group; each p is is c), 1, 2 or 3, wherein Sp, n, p, RI-and R2 may independently in each occurrence be the same or different.8) The compound according to any preceding claim wherein the ester of a sulfonic acid is a -0S02126 group, wherein R6 in each occurrence is independently a CI 1, alkyl group which is unsubstituted or substituted with one or more F atoms, or phenyl which is unsubstituted or substituted with one or more F atoms.9) A method of forming a polymer comprising polyrnerisation of a compound of formula (I) according to any one of claims 1-8 and a monomer of formula (M1): R2)p R2)p -26 -(M1) wherein AR, in Mi and AR, in the compound of formula (I) are the same; and Y is selected from the group consisting of halogen, boronic acid, an ester of boronic acid, trifluoroborate salt, sulfonic acid, an ester of a sulfonic acid, Sn(R9)3 wherein Rg independently in each occurrence is a C112 hydrocarbyl group, and SnC13; provided that X and Y are not the same.io) The method according to claim 9 wherein X is selected from one of group (a) and group (b), and Y is selected from the other of group (a) and group (b): (a) halogen or -0S02128 wherein R8 is an optionally substituted C,,2 alkyl group or optionally substituted aryl group; (b) boronic acid and esters thereof; and -SnR9 wherein R9 independently in each occurrence is a Cfl, hydrocarbyl group.A conjugated light-emitting polymer comprising a repeating structure of formula (P): -Ets Ari ' (P) wherein Ar9 is a C620 aromatic group which is substituted with one or more substituents including at least one polar group substituent RI-comprising a group of formula -0(R30),-R4 wherein 12-1 in each occurrence is a 0_5 alkylene group, R4 is H or 0_5 alkyl, and v is o or a positive integer, and q is a positive integer.12) The conjugated light-emitting polymer according to claim 11 wherein An is substituted with at least one substituent selected from the group consisting of: 0_40 alkyl wherein one or more non-adjacent, non-terminal C atoms may be replaced with 0, S, CO or COO and one or more 1-1 atoms may be replaced with F;-a group of formula -(Ar2)q wherein Ar2 in each occurrence is independently an aryl or heteroaryl group which is unsubstituted or substituted with one or more nonionic substituents; and 13) The conjugated light emitting polymer according to claim 11 or 12 wherein Art is substituted with at least one ionic substituent.14) The conjugated light-emitting polymer according to any one of claims 11-13 wherein Ari is a group of formula (11b): (Jib) wherein is -(Sp),"-(R.0" or R2 and two R.13 groups may be linked to form a ring, with the proviso that at least one R-3 is -(Sp)",-(ROn; Sp is a spacer group; m is 0 or 1; 12,-is a polar group; n is 1 if m is o and n is at least 1 if m is 1; R2 is a non-polar group; each d is 0, 1, 2 or 3, wherein R13, Sp, m, n, R1 and R2 may independently in each occurrence be the same or different.15) The conjugated light-emitting polymer according to any one of claims 11-14 wherein Ari is a group of formula (11b-1): Sip Sp ( ),/, (R15n (R\2)p (R2)p (Jib-i) -28 -wherein Sp is a spacer group; 12,-is a polar group; n is at least 1; R2 is a non-polar group; each p is is c), 1, 2 or 3, wherein Sp, n, p, 121 and R2 may independently in each occurrence be the same or different.16) A light-emitting composite particle comprising a light-emitting polymer according to any one of claims 11-15 and a matrix material.17) The light-emitting composite particle according to claim 16 wherein the matrix material is silica.18) A light-emitting marker comprising the conjugated light-emitting polymer or the light emitting composite particle according to any one of claims 11-17 and a binding group configured to bind to a target material.19) A method of detecting a target analyte in a sample, the method comprising contacting a light-emitting marker according to claim 18 with a sample.20) The method according to claim 19 wherein the sample comprising the light-emitting marker is irradiated with light at an absorption wavelength of the light-emitting polymer and detecting emission from the light-emitting marker.