WO2023144394A1

WO2023144394A1 - Light-emitting nanoparticles

Info

Publication number: WO2023144394A1
Application number: PCT/EP2023/052203
Authority: WO
Inventors: Narul ISLAM; Jonathan BEHRENDT; Ruth Pegington
Original assignee: Sumitomo Chemical Co., Ltd; Cambridge Display Technology Limited
Priority date: 2022-01-31
Filing date: 2023-01-30
Publication date: 2023-08-03
Also published as: GB2615133A; GB202201234D0

Abstract

Light-emitting nanoparticles comprising a core comprising silica and a light-emitting material wherein a weight ratio of the light-emitting material : silicon in a silica-forming material used in formation of the nanoparticles is at least 2 : 15; and a number average diameter of the nanoparticle cores as determined by dynamic light scattering is no more than 40 nm. The light-emitting nanoparticles may be formed by reaction of the silica-forming material in a solution of the light-emitting material in which the solution contains a first solvent is a protic material in which the light-emitting material is soluble and a second solvent which is miscible with the first solvent, wherein the light-emitting material is at least 10 times less soluble in the second solvent as compared to the first solvent.

Description

LIGHT-EMITTING NANOPARTICLES

BACKGROUND

In some embodiments, the present disclosure provides light-emitting nanoparticles. The nanoparticles may be used as markers in biosensor applications. Nanoparticles of silica and a light-emitting material have been disclosed as labelling or detection reagents.

WO 2018/060722 discloses composite particles comprising a mixture of silica and a light-emitting polymer having polar groups.

Ow et al, “Bright and Stable Core-Shell Fluorescent Silica Nanoparticles”, Nano Letters, 2005, Vol. 5, No. 1, p. 113-117.discloses nanoparticles formed from dye molecules covalently attached to a silica precursor.

US 2013/039858 discloses a fluorescent dye comprising metal oxide nanoparticles are prepared where the nanoparticles are as small as 3 nm or up to 7000 nm in diameter and where the dye is bound within the metal oxide matrix. WO 2021/176210 discloses formation of silica nanoparticles containing a light-emitting polymer.

WO 2021/157475 discloses fluorescent silica nanoparticles comprising silica nanoparticles and a fluorescent dye encapsulated by the silica nanoparticles, wherein the total volume of the fluorescent dye is at least 5% of the total volume of the fluorescent silica nanoparticles and the luminescent quantum yield of the fluorescent silica nanoparticles is at least 10%.

SUMMARY

A summary of aspects of certain embodiments disclosed herein is set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of these certain embodiments and that these aspects are not intended to limit the scope of this disclosure. Indeed, this disclosure may encompass a variety of aspects and/or a combination of aspects that may not be set forth. The present disclosure provides light-emitting nanoparticles comprising a core comprising silica and a light-emitting material. A weight ratio of the light-emitting material : silicon in a silica-forming material used in formation of the nanoparticles is at least 2 : 15. A number average diameter of the nanoparticle cores as determined by dynamic light scattering is no more than 40 nm.

Optionally, the weight ratio of the light-emitting material : mass of silicon in a silica- forming material used in formation of the nanoparticles is at least 3 : 15.

Optionally, the weight ratio of the light-emitting material : mass of silicon in a silica- forming material used in formation of the nanoparticles is 2 : 15 - 10 : 15. Optionally, the number average diameter of the light-emitting nanoparticle cores as determined by dynamic light scattering is no more than 35 nm.

Optionally, the polydispersity index (PDI) of the light-emitting nanoparticles as determined by dynamic light scattering is less than 0.2.

Optionally, the polydispersity index (PDI) of the light-emitting nanoparticles as determined by dynamic light scattering is less than 0.1.

In some embodiments, the light-emitting material is bound to the silica.

In some embodiments, the light-emitting material is not bound to the silica.

Optionally, the light-emitting material is a light-emitting polymer, optionally a conjugated light-emitting polymer. Optionally, the nanoparticle core comprises at least one shell surrounding the silica and light-emitting material.

Optionally, the silica-forming material is a tetraalkylorthosilicate.

Optionally, a first surface group is bound to a surface of the nanoparticle core.

Optionally, the first surface group is capable of attaching to a probe group for detection of a target. Optionally, the light-emitting nanoparticle cores comprise a probe group for detection of a target attached to a surface thereof.

The present disclosure provides a method of forming a particulate probe comprising attachment of the probe group to the first surface group of the light-emitting nanoparticles.

The present disclosure provides a suspension comprising the light-emitting nanoparticles as described herein in a protic solvent. The protic solvent may be water, an alcohol, or a mixture thereof.

The present disclosure provides a method of forming light-emitting nanoparticles as described herein, the method comprising reacting the silica-forming material to form silica in the presence of the light-emitting material dissolved in a solvent mixture comprising a first solvent and a second solvent wherein the first solvent is a protic material in which the light-emitting material is soluble and a second solvent which is miscible with the first solvent, wherein the light-emitting material is at least 10 times less soluble in the second solvent as compared to the first solvent.

Optionally, at least one of the first and second solvents are alcohols.

Optionally, the first solvent is methanol.

Optionally, the second solvent is 1 -octanol.

Optionally, the silica-forming material is a tetraalkoxysilane. The present disclosure provides a method of identifying the presence and / or concentration of a target in a sample comprising contacting the sample with the lightemitting nanoparticles having a probe group as described herein, and detecting emission from the light-emitting nanoparticles.

The present disclosure provides a method of nucleotide sequencing in which nucleotides are substituted with a light-emitting nanoparticle having a probe group as described herein.

DESCRIPTION OF THE DRAWINGS The present disclosure is described in conjunction with the appended figures. It is emphasized that, in accordance with the standard practice in the industry, various features are not drawn to scale. In fact, the dimensions of the various features may be arbitrarily increased or reduced for clarity of discussion. In the appended figures, similar components and/or features may have the same reference label. Further, various components of the same type may be distinguished by following the reference label by a dash and a second label that distinguishes among the similar components. If only the first reference label is used in the specification, the description is applicable to any one of the similar components having the same first reference label irrespective of the second reference label.

The invention will now be described in more detail with reference to the drawings wherein:

Figure 1 is a plot of nanoparticle number average diameter vs. 1 -octanol volume % for light-emitting nanoparticles formed from a methanol / 1 -octanol solution containing tetraethylorthosilicate (TEOS) and a light-emitting polymer (LEP) at a fixed LEP loading; and

Figure 2 is a plot of LEP loading vs 1 -octanol volume % for light-emitting nanoparticles required to obtain nanoparticles with a number average diameter in the range of 30 - 35 nm and with a PDI <0.1 from a methanol / 1-octanol solution containing TEOS and a light-emitting polymer.

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. Additionally, the words "herein," "above," "below," and words of similar import, when 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. 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 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 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.

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.

The present disclosure relates to a nanoparticulate light-emitting material containing nanoparticles which contain a core including silica and a light-emitting material. One factor which can influence the average size of the nanoparticle cores is the amount of light-emitting material in the cores, with more light-emitting material typically resulting in a larger core. The presence of a large amount of light-emitting material is desirable in order to achieve a high brightness of the light-emitting nanoparticles, however an increase in average nanoparticle size to accommodate a larger amount of light-emitting material can be undesirable; for example, if the light-emitting nanoparticles are used in diagnosis or therapy in a living body then it may be harder for the body to excrete nanoparticles with a larger average size.

The present inventors have surprisingly found that the amount of light-emitting material incorporated into silica-containing light-emitting silica nanoparticles can be increased without a concomitant increase in the size of the nanoparticles.

Accordingly, the present disclosure provides light-emitting nanoparticles comprising a core comprising silica and a light-emitting material wherein a weight ratio of the lightemitting material : silicon in a silica-forming material used in formation of the nanoparticles is at least 2 : 15; and a number average diameter of the nanoparticle cores is no more than 40 nm.

It will be understood that a nanoparticle core as described herein is a nanoparticle comprising the silica and light-emitting material without any surface groups thereon. The core may comprise one or more shell layers, e.g. one or more silica layers, surrounding the silica and light-emitting material. Optionally, at least 7 wt% of total weight of the particle core consists of one or more light-emitting materials. Preferably 7-35 wt % of the particle core consists of the one or more light-emitting materials.

In some embodiments of the present disclosure, at least 70 wt% of the total weight of the particle core consists of the light-emitting material or materials and silica. Preferably at least 80, 90, 95, 98, 99, 99.5, 99.9 wt% of the total weight of the particle core consists of the light-emitting material or materials and silica. More preferably the particle core consists essentially of the one or more light-emitting materials and silica.

The, or each, light-emitting material may be polymeric or non-polymeric. Preferably, the nanoparticles comprise a light-emitting polymer. A light-emitting polymer may, due to its larger size, be less likely to leach out of the nanoparticle when dispersed in a liquid than a non-polymeric light-emitting material.

The light emitting material is preferably uniformly distributed within the core. In the case where the light-emitting material is a polymer, the polymer chains may all be contained within a surface of the core defined by the silica. One or more light-emitting polymer chains may protrude beyond a surface of the core defined by the silica.

Nanoparticle formation

To form the light-emitting nanoparticles, a silane substituted with C1-12 alkoxy groups, for example tetraethylorthosilicate (TEOS), may be reacted in in the presence of a light- emitting material a solvent or solvent mixture including at least one protic solvent.

The or each protic solvent may be water or an alcohol.

Preferably, reaction takes place in a solvent mixture comprising or consisting of a first solvent and a second solvent wherein the first solvent is a protic material in which the light-emitting material is soluble and a second solvent which is miscible with the first solvent, wherein the light-emitting material is at least 10 times less soluble in the second solvent as compared to the first solvent.

The solubility of the light-emitting material in the first solvent is preferably at least 0.1 mg / ml, preferably at least 0.5 mg/ml, more preferably at least 1 mg / ml or at least 5 mg/ml. Solubility may be measured by the following method:

The solid polymer is weighed out into a glass vial. The required amount of solvent is added followed by a small magnetic stirrer. Then the vial is tightly capped and put on a preheated hot plate at 60°C with stirring for 30 minutes. The polymer solution is allowed to cool to room temperature. The polymer solution can also be prepared by sonicating the polymer containing vial for 30 minutes at room temperature. The solubility of polymer was tested by visual observation and under white and 365 nm UV light.

A preferred first solvent is methanol.

The second solvent may be selected taking into account the solubility of the lightemitting material in the first solvent. Exemplary second solvents are Ce-io alcohols, for example 1 -octanol. A first solvent : second solvent volume ratio may be selected according to a difference in solubilities of the first and second solvents. Optionally, the ratio is in the range of about 95 : 5 - 67 : 33, optionally about 90 : 10 - 67 : 33.

The first solvent is capable of dissolving the light-emitting material. Without wishing to be bound by any theory, the second solvent may have an antisolvent effect, causing the light-emitting material to adopt a more compact conformation in the solution, e.g. a coiled or ball-like conformation in the case of a light-emitting polymer, as compared to its conformation in the first solvent only. This may reduce the space occupied by the light-emitting material when it is is incorporated into the nanoparticle and therefore the overall size of the nanoparticle. This may also reduce the polydispersity of the nanoparticles.

The reaction is carried out in the presence of a base, e.g. a metal hydroxide, preferably alkali metal hydroxide, ammonium hydroxide or tetraalkylammonium hydroxide.

Surface groups

Nanoparticle cores as described herein may be substituted with surface groups. Following formation of the particle cores, surface groups may be bound to the surface of the nanoparticles. Surface groups include, without limitation, surface groups for preventing aggregation of the nanoparticles, surface groups comprising a binding group for binding to a target, and combinations thereof. Surface groups as described herein may be covalently bound to the silica surface of the nanoparticle core. To form a surface group, the nanoparticles may be brought into contact with a reactive compound for forming the surface group having a reactive group capable of reacting with Si-0 groups at the surface of the nanoparticle core. The reactive group may be a group of formula -Si(OR⁷)3 wherein R⁷ in each occurrence is independently H or a substituent, preferably a Ci-io alkyl.

The surface groups may comprise a group of formula (I):

-PG-EG (I) wherein PG is a polar group bound directly to the surface of the silica nanoparticle core or bound through an attachment group such as a group of formula -O-Si(R⁷)2-O; and

EG is an end group.

PG may be a linear or branched polar group.

PG may comprise heteroatoms capable of forming hydrogen bonds with water, optionally a linear or branched alkylene chain wherein one or more C atoms of the alkylene chain are replaced with O or NR⁶ wherein R⁶ is a C1-12 hydrocarbyl group, optionally a C1-12 alkyl group or C1-4 alkyl group.

Preferably, PG has a molecular weight of less than 5,000, optionally in the range of 130- 3500 Da.

Preferably, PG is a polyether chain. By “polyether chain” as used herein is meant a divalent chain comprising a plurality of ether groups.

Preferably, PG comprises a group of formula (II):

-((CR¹⁴R¹⁵)bO)_c-

(II) wherein R¹⁴ and R¹⁵ are each independently H or C1-6 alkyl and b is at least 1, optionally 1-5, preferably 2, and c is at least 2, optionally 2-1,000, preferably 10-500, 10-200 or

10-100, most preferably 10-50. Most preferably, PG comprises or consists of a polyethylene glycol chain.

In some embodiments, the end group EG is an attachment group for attachment to a probe group capable of binding to a target.

The attachment group may be a biomolecule, e.g. biotin, for attachment through streptavidin, neutravidin, avidin or a recombinant variant or derivative thereof to a biotinylated probe group

In some embodiments the attachment group may be an amine, a thiol, an azide, dibenzocyclooctyne (DBCO), acetal, tetrazine, carboxylic acid or a derivative thereof such as an amide or ester, preferably an NHS ester, acid chloride or acid anhydride group. The attachment group may be activated before attachment to a probe, e.g. activation of a carboxylic acid group using a carbodiimide, for example EDC.

In some embodiments, the end group EG is not a reactive group for attachment (directly or indirectly) to a probe group. According to these embodiments, EG is optionally selected from H; C1-12 alkyl; C1-12 alkoxy; and esters, e.g. C1-20 hydrocarbyl esters of COOH.

The nanoparticle core may be substituted with different surface groups, e.g. a first surface group for attachment to a probe and a second, inert surface group.

Preferably, the number of second surface groups is greater than the number of first surface groups. Optionally, the number of moles of the second surface groups is at least 2 times, preferably 3 times, more preferably at least 5 times, the number of moles of the first surface groups. Most preferably, the number of first surface groups is less than 10 mol %, optionally up to 5 mol %, of the total number of moles of the first and second surface groups.

Preferably, the number of first surface groups is more than 0.1 mol%, optionally at least 0.5 mol %, of the total number of moles of the first and second surface groups.

The probe may be, without limitation, an antibody; an antigen-binding fragment (Fab); a mimetic, e.g. a minibody, nanobody, monobody, diabody or triabody or affibody; a DARPin; or a fusion protein, e.g. a single-chain variable fragment (scFv); a linear or cyclic peptide; annexin V; RNA or DNA; or an aptamer.

An antibody biomolecule may be selected according to the antigen to be detected. In the case of a biotinylated nanoparticle, wide range of biotinylated antibodies are known and commercially available, or may be prepared using techniques known the skilled person as disclosed in, for example, https://www.abcam.com/ps/pdf/protocols/biotin_conjugation.pdf, the contents of which are incorporated herein by reference.

Surface groups may be polydisperse. The surface groups may have a multimodal weight distribution, optionally a bimodal weight distribution. A multimodal weight distribution may be achieved by mixing polydisperse materials having different average molecular weights.

Nanoparticle shell

The nanoparticle cores may comprise one or more shell layers surrounding the silica and light-emitting material. If one or more shell layers are present then it will be understood that any surface groups as described herein are bound to the shell layer or, in the case that more than one shell layer is present, to the outermost shell layer.

A shell layer may comprise or consist of silica. A silica shell may be formed as described in, for example, WO 2021/176210, the contents of which are incorporated herein by reference.

Light-emitting materials

Light-emitting materials as described herein may emit fluorescent light, phosphorescent light or a combination thereof. Preferably, the light-emitting material is fluorescent. Preferably, the light-emitting material is a conjugated material. The light-emitting material may emit light having a peak wavelength in the range of 350-1000 nm. A blue light-emitting material as described herein may have a photoluminescence spectrum with a peak of no more than 500 nm, preferably in the range of 400-500 nm, optionally 400-490 nm.

A green light-emitting material as described herein may have a photoluminescence spectrum with a peak of more than 500 nm up to 580 nm, optionally more than 500 nm up to 540 nm.

A red light-emitting material as described herein may have a photoluminescence spectrum with a peak of no more than more than 580 nm up to 950 nm, optionally up to 630 nm, optionally 585 nm up to 625 nm. The light-emitting material may have a Stokes shift in the range of 10-850 nm.

UV/vis absorption spectra of light-emitting markers as described herein may be as measured in methanol solution or suspension using a Cary 5000 UV-vis-IR spectrometer.

Photoluminescence spectra of light-emitting particles as described herein may be measured in methanol solution or suspension using a Jobin Yvon Horiba Fluoromax-3.

The light-emitting material may be an inorganic light-emitting material; a non- polymeric organic light-emitting material; or a light-emitting polymer.

Exemplary non-polymeric fluorescent materials include, without limitation: fluorescein and salts thereof, for example, fluorescein isothiocyanate (FITC), fluorescein NHS, Alexa Fluor 488, Dylight 488, Oregon green, DAF-FM, 6-FAM2,7-dichlorofluorescein, 3’-(p-aminophenyl)fluorescein and 3’-(hydroxyphenyl)fluorescein; rhodamines, for example Rhodamine 6G and Rhodamine 110 chloride; coumarins; boron- dipyrromethenes (BODIPYs); naphthalimides; perylenes; benzanthrones; benzoxanthrones; and benzothiooxanthrones, each of which may be unsubstituted or substituted with one or more substituents. Exemplary substituents are chlorine, alkyl amino; phenylamino; and hydroxyphenyl.

A polymer as described herein is a material containing repeat units linked to one another in a linear or branched chain. A repeat unit is a unit that is present at a plurality of positions in the polymer chain. A light-emitting polymer as described herein may be a homopolymer, i.e. a polymer in which all repeat units are the same, or may be a copolymer comprising two or more different repeat units.

The light-emitting polymer may comprise light-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 polymers are preferred.

By “conjugated polymer” is meant a polymer comprising repeat 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 vinylene groups conjugated to one another along the polymer backbone.

The light-emitting polymer may have a linear, branched or crosslinked backbone.

The light-emitting polymer may comprise one or more repeat units in the backbone of the polymer substituted with one or more substituents selected from non-polar and polar substituents.

Preferably, the light-emitting polymer comprises at least one polar substituent. The one or more polar substituents may be the only substituents of said repeat units, or said repeat units may be further substituted with one or more non-polar substituents, optionally one or more C1-40 hydrocarbyl groups. The repeat unit or repeat units substituted with one or more polar substituents 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 substituents, optionally one or more C1-40 hydrocarbyl substituents. Ci-40 hydrocarbyl substituents as described herein include, without limitation, C1-20 alkyl, unsubstituted phenyl and phenyl substituted with one or more C1-20 alkyl groups.

As used herein a “polar substituent” may refer to a substituent, alone or in combination with one or more further polar substituents, which renders the light-emitting polymer with a solubility of at least 0.01 mg/ml in an alcoholic solvent, optionally in the range of 0.01-10 mg / ml. Optionally, solubility is at least 0.1 or 1 mg/ml. The solubility is measured at 25°C. Preferably, the alcoholic solvent is a C1-10 alcohol, more preferably methanol.

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

In some embodiments, the light-emitting polymer comprises polar substituents of formula -O(R³O)t-R⁴ wherein R³ in each occurrence is a C1-10 alkylene group, optionally a C1-5 alkylene group, wherein one or more non-adjacent, non-terminal C atoms of the alkylene group may be replaced with O, R⁴ is H or C1-5 alkyl, and t is at least 1, optionally 1-10. Preferably, t is at least 2. More preferably, t is 2 to 5. The value of t may be the same in all the polar groups of formula -O(R³O)t-R⁴. The value of t 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)f- wherein f is from 1-5. Preferably, the light-emitting polymer comprises polar substituents of formula - O(CH2CH2O)t-R⁴ wherein t is at least 1, optionally 1-10 and R⁴ is a C1-5 alkyl group, preferably methyl. Preferably, t is at least 2. More preferably, t is 2 to 5, most preferably q is 3.

In some embodiments, the light-emitting polymer comprises polar substituents of formula -N(R⁵)2, wherein R⁵ is H or C1-12 hydrocarbyl. Preferably, each R⁵ is a C1-12 hydrocarbyl. In some embodiments, the light-emitting polymer comprises polar substituents which are ionic groups which may be anionic, cationic or zwitterionic. Preferably the ionic group is an anionic group.

Exemplary anionic groups 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 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 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 may comprise a plurality of anionic or cationic polar substituents wherein the charge of two or more anionic or cationic groups is balanced by a single counterion. Optionally, the polar substituents comprise anionic or cationic groups comprising di- or bivalent 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 tetraalkylammonium, ethylmethyl imidazolium or pyridinium.

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

In some embodiments, the light-emitting polymer comprises polar substituents selected from groups of formula -O(R³O)t-R⁴, groups of formula -N(R⁵)2, groups of formula

OR⁴ and/or ionic groups. Preferably, the light-emitting polymer comprises polar substituents selected from groups of formula -O CthCthOXR⁴, groups of formula - N(R⁵)₂, and/or anionic groups of formula -COO". Preferably, the polar substituents are selected from the group consisting of groups of formula -O(R³O)t-R⁴, groups of formula -N(R⁵)₂, and/or ionic groups. Preferably, the polar substituents are selected from the group consisting of polyethylene glycol (PEG) groups of formula -O(CH2CH2O)tR⁴, groups of formula -N(R⁵)2, and/or anionic groups of formula -COO". R³, R⁴, R⁵, and t are as described above.

Optionally, the backbone of the light-emitting polymer is a conjugated polymer. Optionally, the backbone of the conjugated light-emitting polymer comprises repeat units of formula (III):

wherein Ar¹ 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 substituent; n is 1 if m is 0 and n is at least 1, optionally 1, 2, 3 or 4, if m is 1; R² independently in each occurrence is a non- polar substituent; p is 0 or a positive integer, optionally 1, 2, 3 or 4; q is 0 or a positive integer, optionally 1, 2, 3 or 4; and wherein Sp, R¹ and R² may independently in each occurrence be the same or different. Two substituents of Ar¹ may be linked to form a ring.

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

Preferably q is at least 1.

Ar¹ of formula (III) is optionally a C6-20 arylene group or a 5-20 membered heteroarylene group. Ar¹ is preferably a dibenzosilole group or a C6-20 arylene group, optionally phenylene, fluorene, benzofluorene, phenanthrene, naphthalene or anthracene, more preferably fluorene or phenylene, most preferably fluorene.

Exemplary Ar¹ groups of formula (III) include groups of formula (IV)-(X):

wherein

R⁹ in each occurrence is independently H, R¹ or R², preferably H;

R¹⁰ in each occurrence is independently R¹ or R²; R⁶ is a C1-12 hydrocarbyl group, optionally a C1-12 alkyl group or C1-4 alkyl group; c is 0, 1, 2, 3 or 4, preferably 1 or 2; d is 0, 1 or 2;

X independently in each occurrence is a substituent, preferably a substituent selected from the group consisting of branched, linear or cyclic C1-20 alkyl; phenyl which is unsubstituted or substituted with one or more substituents, e.g. one or more C1-12 alkyl groups; and F; and

Z¹ -Z²- Z³ is a C2 (ethylene) C3 alkylene (propylene) chain wherein one or two nonadj acent C atoms may be replaced with O, S or NR⁶.

Sp-(R¹)n may be a branched group, optionally a dendritic group, substituted with polar groups, optionally -NH2 or -OH groups, for example polyethyleneimine. Preferably, Sp is selected from:

C1-20 alkylene or phenylene-Ci-20 alkylene wherein one or more non-adjacent C atoms may be replace with O, S, N or C=O; a C6-20 arylene or 5-20 membered heteroarylene, more preferably phenylene, which, in addition to the one or more substituents R¹, 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 than 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:

Ci-20 alkylene wherein one or more non-adjacent C atoms may be replaced with O, 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 nonpolar substituents.

R¹ may be a polar substituent as described anywhere herein. Preferably, R¹ is: a polyethylene glycol (PEG) group of formula -O(CH2CH2O)tR⁴ wherein t is at least 1, optionally 1-10 and R⁴ is a C1-5 alkyl group, preferably methyl; - a group of formula -N(R⁵)2, wherein R⁵ is H or C1-12 hydrocarbyl; or an anionic group of formula -COO".

In the case where n is at least two, each R¹ may independently in each occurrence be the same or different. Preferably, each R¹ attached to a given Sp group 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 C1-20 alkyl groups; - a linear or branched chain of aryl or hetero aryl groups, each of which groups may independently be substituted, for example a group of formula -(Ar³)_s wherein each Ar³ 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 C1-20 alkyl groups; and a crosslinkable-group, for example a group comprising a double bond such and a vinyl or acrylate group, or a benzocyclobutane group.

Preferably, each R², where present, is independently selected from C1-40 hydrocarb yl, and is more preferably selected from C1-20 alkyl; unusubstituted phenyl; phenyl substituted with one or more C1-20 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 (III) or may comprise or consist of two or more different repeat units of formula (III).

Optionally, the polymer comprising one or more repeat units of formula (III) is a copolymer comprising one or more co-repeat units.

If co-repeat units are present then the repeat units of formula (III) may form between 0.1-99 mol % of the repeat units of the polymer, optionally 50-99 mol % or 80-99 mol %. 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 may be selected to produce a desired colour of emission of the polymer.

Arylene repeat units of the polymer include, without limitation, fluorene, preferably a 2,7-linked fluorene; phenylene, preferably a 1,4-linked phenylene; naphthalene, anthracene, indeno fluorene, phenanthrene and dihydrophenanthrene repeat units.

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 IxlO³ to IxlO⁸, and preferably IxlO³ to 5xl0⁶. The polystyrene-equivalent weight- average molecular weight (Mw) of the polymers described herein may be IxlO³ to IxlO⁸, and preferably IxlO³ or 5xl0³ to IxlO⁷.

Polymers as described herein are suitably amorphous polymers.

Colloids The particles may be provided as a colloidal suspension comprising the particles suspended in a liquid. Preferably, the liquid is selected from water, Ci-io alcohols and mixtures thereof.

The liquid may be a buffer solution. The salt concentration of a buffer solution may be in the range of about 1 mmol / L - 200 mmol / L. The concentration of the particles in the colloidal suspension is preferably in the range of 0.1-20 mg / mL, optionally 5-20 mg / mL.

Applications

Nanoparticles as described herein may be used as a luminescent probe, e.g. a fluorescent probe for detecting a biomolecule or for labelling a biomolecule or for use in DNA sequencing.

In some embodiments, the particles may be used as a luminescent probe, e.g. a fluorescent probe in an immunoassay such as a lateral flow or solid state immunoassay. Optionally the particles are for use in fluorescence microscopy, flow cytometry, next generation sequencing, in-vivo imaging, or any other application where a light-emitting marker configured to bind to a target analyte is brought into contact with a sample to be analysed. The applications can medical, veterinary, agricultural or environmental applications whether involving patients (where applicable) or for research purposes.

Preferably, the presence and / or concentration of a target analyte comprises measurement of any light-emitting markers dispersed or dissolved in the sample which are bound to the target analyte (as opposed to light-emitting markers bound to the target analyte and immobilised on a surface). Preferably, the presence and / or concentration of a target analyte comprises detection of light emitted directly from the light emitting marker.

In some embodiments, a sample to be analysed may brought into contact with the particles, for example the particles in a colloidal suspension. In some embodiments, the sample following contact with the particles is analysed by flow cytometry. In flow cytometry, the particles are irradiated by at least one wavelength of light, optionally two or more different wavelengths, e.g. one or more wavelengths including at least one of 355, 405, 488, 562 and 640 nm. Light emitted by the particles may be collected by one or more detectors. Detectors may be selected from, without limitation, photomultiplier tubes and photodiodes. To provide a background signal for calculation of a staining index, measurement may be made of particles mixed with cells which do not bind to the particles.

In some embodiments, e.g. a plate assay, any target antigen in the sample may be immobilised on a surface which is brought into contact with the particles. Examples

Measurements

Particle size as described herein is a number average diameter, or as an intensity average diameter (x), as measured by dynamic light scattering (DLS), for example using a Malvern Zetasizer Nano ZS, using a 4mW 633 nm He-Ne laser. Nanoparticle suspensions were measured either in methanol or water in single use UV transparent plastic cuvettes. The machine was operated in Backscatter mode at an angle of 173°. Samples are equilibrated to 25°C if the solvent is methanol or 20°C if the solvent is water for 60 seconds prior to measurement. The viscosity values of methanol (0.5476cP) and water (l.OOOOcP) and refractive index values of methanol (1.326) and water (1.330) were inputted into the software SOP before measurement. The sample is defined as Polystyrene 10 latex (RI: 1.590, Absorption: 0.0100). The automatic measurement duration setting is used, with automatic measurement positioning and automatic attenuation. The ‘general purpose’ analysis model is used, with the default size analysis parameters along with a refractive index of 1.59 for the sample parameter.

A single measurement is taken for each sample.

Cumulants analysis calculation is defined in ISO 13321 and ISO 22412 and provides a width parameter known as the Polydispersity Index (Pdl). Polydispersity Index (PDI) is calculated as the standard deviation divided by the intensity average squared (c/x²).

Nanoparticle Synthesis General Method

Silica nanoparticles containing LEP1 or LEP2, illustrated below, were formed.

Synthesis of LEP1 and LEP2 are described in, respectively, WO 2021/001663 and WO 2012/133229, the contents of which are incorporated herein by reference.

R¹³ =

LEP1

LEP2

1.0 mg/mL LEP solution was prepared by dissolving the LEP in methanol in a sealed vial. The solution was heated at 60°C followed by sonication for 5 min. The process was repeated until a clear solution was obtained. The solution was filtered using 0.45 pL syringe filter before use.

The required amount of LEP solution was taken in a screw cap amber vial containing a 4.5 x 12.5 mm stirrer bar. The required amounts of methanol and 1-octanol were added and mixed properly before addition of ammonium hydroxide (aq. 28-30%). The reaction vial was heated at 60°C in a preheated oil bath for 10 min then a methanol solution of tetraethyl orthosilicate (TEOS) was injected into the reaction mixture using a long needed and syringe. After 1 hour a second methanolic TEOS solution was injected to form a shell around the silica and light-emitting polymer, and heating was continued for additional 1 hour before cooling down to room temperature. Zeba desalting columns (7K MWCO) were equilibrated with ultrapure water according to the manufacturer’s recommendations. The crude reaction mixture was loaded on top of column and eluted to collect nanoparticles in water. The size of the nanoparticles was measured by DLS using a Malvern Zetasizer Nano S.

Nanoparticles containing LEP1 were formed by the general method using amounts of solvents and light-emitting polymer solutions as set out in Table 1.

In all cases, the number average diameter for nanoparticles shown in Table 1 is 30-35 nm and PDI is less than 0.1.

The amount of TEOS in the LEP : TEOS ratio in Table 1, and as described throughout these experiments, is based on the amount of TEOS used in the first addition of TEOS. Referring to the first entry, 1 mg of light-emitting polymer is used and 0.1 mL of TEOS is used. TEOS has a density of 0.93 g / mL, giving a LEP : TEOS weight ratio of 0.1 : 0.93 and, based on a molecular weight of 208 for TEOS and an atomic mass of 28 for silicon, a LEP : Si weight ratio of 1 : (93 x 28/208) = 1 : 12.5.

The percentage of 1 -octanol in Table 1, and as described throughout these experiments, is the volume percentage of 1 -octanol in the total volume of methanol + 1 -octanol present when silica formation starts with the first addition of TEOS. As shown in Table 1, a number average diameter of 30-35 nm and a PDI of less than 0.1 can surprisingly be maintained by selecting the amount of 1 -octanol used in the reaction, even at high LEP loadings.

Figure 1 illustrates the dependence of nanoparticle size on the volume percent of 1- octanol used in nanoparticle formation using a 4 : 93 LEP : TEOS ratio. Similarly, Figure 2 illustrates the amounts of 1 -octanol and light-emitting polymer required to obtain nanoparticles with a number average diameter in the range of 30 - 35 nm and with a PDI <0.1. Accordingly, particles of a desired size may be selected by selection of amounts of light-emitting polymer and 1-octanol.

Table 2 shows the effect of not adjusting the amount of 1-octanol in accordance with the amount of light-emitting polymer.

Table 2

As shown in Table 2, increasing the amount of light-emitting polymer increases the average particle size for a given methanol : 1 -octanol ratio. Surprisingly, however, appropriate selection of the methanol : 1 -octanol ratio results in a reduction in particle size.

The higher amount of light-emitting polymer that can be incorporated into the nanoparticles increases the extinction co-efficient of the nanoparticles, as shown in Tables 3-5 below.

Table 3 - nanoparticles containing LEP1

Table 4 - nanoparticles containing LEP2

Claims

1. Light-emitting nanoparticles comprising a core comprising silica and a lightemitting material wherein a weight ratio of the light-emitting material : mass of silicon in a silica-forming material used in formation of the nanoparticles is at least 2 : 15; and a number average diameter of the nanoparticle cores as determined by dynamic light scattering is no more than 40 nm.

2. The light-emitting nanoparticles according to claim 1 wherein the weight ratio of the light-emitting material : mass of silicon in a silica-forming material used in formation of the nanoparticles is at least 3 : 15.

3. The light-emitting nanoparticles according to claim 1 or 2 wherein the number average diameter of the light-emitting nanoparticle cores as determined by dynamic light scattering is no more than 35 nm.

4. The light-emitting nanoparticles according to any preceding claim wherein the polydispersity index (PDI) of the light-emitting nanoparticles as determined by dynamic light scattering is less than 0.2.

5. The light-emitting nanoparticles according to any preceding claim wherein the polydispersity index (PDI) of the light-emitting nanoparticles as determined by dynamic light scattering is less than 0.1.

6. The light-emitting nanoparticles according to any preceding claim wherein the light-emitting material is bound to the silica.

7. The light-emitting nanoparticles according to any one of claims 1-5 wherein the light-emitting material is not bound to the silica.

8. The light-emitting nanoparticles according to any one of the preceding claims wherein the light-emitting material is a light-emitting polymer.

9. The light-emitting nanoparticles according to claim 8 wherein the light-emitting polymer is a conjugated light-emitting polymer.

10. The light-emitting nanoparticles according to any one of the preceding claims wherein the nanoparticle core comprises at least one shell surrounding the silica and light-emitting material.

11. The light-emitting nanoparticles according to any one of the preceding claims wherein the silica-forming material is a tetraalkylorthosilicate.

12. The light-emitting nanoparticles according to any one of the preceding claims wherein a first surface group is bound to a surface of the nanoparticle core.

13. The light-emitting nanoparticles according to claim 12 wherein the first surface group is capable of attaching to a probe group for detection of a target.

14. The light-emitting nanoparticles according to any one of claims 1-12 wherein the light-emitting nanoparticle cores comprise a probe group for detection of a target attached to a surface thereof.

15. A method of forming a particulate probe comprising attachment of a probe group according to claim 14 to the first surface group of the light-emitting nanoparticles according to claim 13.

16. A suspension comprising the light-emitting nanoparticles according to any one of the claims 1-14 in a protic solvent.

17. The suspension according to claim 16 wherein the protic solvent is water, an alcohol, or a mixture thereof.

18. A method of forming light-emitting nanoparticles according to any one of claims

1-11, the method comprising reacting the silica-forming material to form silica in the presence of the light-emitting material dissolved in a solvent mixture comprising a first solvent and a second solvent wherein the first solvent is a protic material in which the light-emitting material is soluble and a second solvent which is miscible with the first solvent, wherein the light-emitting material is at least 10 times less soluble in the second solvent as compared to the first solvent.

19. The method according to claim 18 wherein at least one of the first and second solvents are alcohols.

20. The method according to claim 19 wherein the first solvent is methanol.

21. The method according to claim 19 or 20 wherein the second solvent is 1 -octanol.

22. The method according to any one of claims 18-21 wherein the silica- forming material is a tetraalkoxysilane.

23. A method of identifying the presence and / or concentration of a target in a sample comprising contacting the sample with the light-emitting nanoparticles according to claim 14, and detecting emission from the light-emitting nanoparticles.

24. A method of nucleotide sequencing in which nucleotides are substituted with a light-emitting nanoparticle according to claim 14.