WO2024028470A1

WO2024028470A1 - Particulate probe

Info

Publication number: WO2024028470A1
Application number: PCT/EP2023/071624
Authority: WO
Inventors: Guy MAYNEORD
Original assignee: Cambridge Display Technology Ltd; Sumitomo Chemical Co Ltd
Current assignee: Cambridge Display Technology Ltd; Sumitomo Chemical Co Ltd
Priority date: 2022-08-05
Filing date: 2023-08-03
Publication date: 2024-02-08
Anticipated expiration: 2025-02-05
Also published as: GB202211481D0; GB2621185A

Abstract

A method of forming a particulate probe, the method comprising the steps of: providing a biomolecule; providing a particulate light-emitting marker comprising at least one azide group at a surface thereof; providing a linker, the linker being capable of reacting with the at least one azide group and the biomolecule; reacting the biomolecule and the linker to form a biomolecule linker conjugate; reacting the biomolecule linker conjugate with at least one of the at least one azide group to form a particulate probe; wherein the biomolecule linker conjugate comprises from one to five first attachment groups capable of reacting with the at least one azide group.

Description

PARTICULATE PROBE

BACKGROUND

In some embodiments, the present disclosure provides a method of forming a particulate probe. The particulate probes may be used as markers in bio-sensing applications. Pickens CJ et al. “Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide-Alkyne Cycloaddition” Bioconjug Chem. 2018 Mar 21, 29(3), 686-701 describes the emergence of click chemistry as it relates to bioconjugation.

WO 2020/058668A1 describes light-emitting marker particles comprising a lightemitting core, first surface groups bound to the light-emitting particle core and second surface groups bound to the light-emitting particle core.

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 a method of forming a particulate probe.

In some embodiments, the present disclosure provides a method of forming a particulate probe, the method comprising the steps of: providing a biomolecule; providing a particulate light-emitting marker comprising at least one azide group at a surface thereof; providing a linker, the linker being capable of reacting with the at least one azide group and the biomolecule; reacting the biomolecule and the linker to form a biomolecule linker conjugate; reacting the biomolecule linker conjugate with at least one of the at least one azide group to form a particulate probe; wherein the biomolecule linker conjugate comprises from one to five first attachment groups capable of reacting with the at least one azide group. Optionally, the biomolecule linker conjugate comprises from one to three first attachment groups capable of reacting with the at least one azide group, optionally wherein the biomolecule linker conjugate comprises from one to two first attachment groups capable of reacting with the at least one azide group.

Optionally, the linker comprises a first attachment group capable of reacting with the at least one azide group and a second attachment group capable of reacting with the biomolecule. Optionally, the biomolecule comprises a linker attachment group capable of reacting with the second attachment group of the linker.

Optionally, the first attachment group comprises an alkyne group and/or the second attachment group comprises an NHS ester.

Optionally, the particulate light-emitting marker is a light-emitting particle comprising a core and a light-emitting material.

Optionally, the core of the light-emitting particle comprises a matrix material, optionally an inorganic matrix material, optionally an inorganic oxide, optionally silica.

Optionally, the particulate light-emitting marker is a light-emitting nanoparticle.

In some embodiments, the present disclosure provides a method of forming a particulate probe, the method comprising the steps of: providing a particulate light-emitting marker comprising at least one azide group at a surface thereof; providing a linker, the linker being capable of reacting with at least one of the at least one azide group and a biomolecule; providing a biomolecule capable of reacting with the linker; reacting the linker and at least one of the at least one azide group to form a light-emitting conjugate; and reacting the light-emitting conj ugate with the biomolecule to form a particulate probe; wherein the method further comprises a quenching step after the light-emitting conjugate reacts with the biomolecule. Optionally, the linker comprises a first attachment group capable of reacting with the at least one azide group and a second attachment group capable of reacting with the biomolecule. Optionally, the light-emitting conjugate comprises a second attachment group capable of reacting with the biomolecule.

Optionally, the biomolecule comprises a linker attachment group capable of reacting with the second attachment group of the linker and/or light-emitting conjugate.

Optionally, the quenching step comprises reducing or eliminating the ability of the second attachment group of the light-emitting conjugate and the biomolecule to react together.

Optionally, the quenching step comprises providing a second attachment group quenching agent.

Optionally, the second attachment group comprises an NHS ester and the second attachment group quenching agent comprises a primary amine. Optionally, the first attachment group comprises an alkyne group and/or the second attachment group comprises an NHS ester.

In some embodiments, the present disclosure provides a particulate probe obtainable by a process as described herein. In some embodiments, the present disclosure provides a method of identifying the presence and/or concentration of a target in a sample comprising contacting the sample with a particulate probe as described herein and detecting emission from the particulate probe.

In some embodiments, the present disclosure provides a method of sequencing nucleic acids comprising: contacting a primed template nucleic acid molecule with a polymerase and a test nucleotide; incorporating the test nucleotide into a primed strand of the primed template only if it comprises a base complementary to the next base of the template strand; irradiating the primed strand; and determining from luminance of the primed strand if the test nucleotide has been incorporated into the primed strand, wherein the test nucleotide of the irradiated primed strand is bound to a particulate probe 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 graph showing the N-average particle size percentage increase of particulate probes formed according to some embodiments. Figure 2 is a graph showing the FITC adjusted signal for assays using particulate probes formed according to some embodiments and the comparative examples described herein.

Figure 3 is a graph showing the signal to noise ratios for assays using particulate probes formed according to some embodiments and the comparative examples described herein.

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 method of forming a particulate probe. It is desirable for particulate probes to form a colloid which is stable, and in particular which does not aggregate, when in use and/or when in storage. The present inventors have found that maintaining stability of such colloids is particularly problematic in aqueous solutions commonly used in biological assays such as aqueous buffer solutions.

The present inventors have surprisingly found that stable particulate probes may be formed using the methods described herein. Accordingly, in some embodiments there is provided a method of forming a particulate probe. In some embodiments, the present disclosure provides a method of forming a particulate probe, the method comprising the steps of: providing a particulate light-emitting marker comprising at least one azide group at a surface thereof; providing a linker, the linker being capable of reacting with at least one of the at least one azide group and a biomolecule; providing a biomolecule capable of reacting with the linker; reacting the linker and at least one of the at least one azide group to form a light-emitting conjugate; and reacting the light-emitting conjugate with the biomolecule to form a particulate probe; wherein the method further comprises a quenching step after the light-emitting conjugate reacts with the biomolecule. In some embodiments, the present disclosure provides a method of forming a particulate probe, the method comprising the steps of: providing a biomolecule; providing a linker, the linker being capable of reacting with an azide group and the biomolecule; reacting the biomolecule and the linker to form a biomolecule linker conjugate; providing a particulate light-emitting marker comprising at least one azide group at a surface thereof; reacting the biomolecule-linker conjugate with at least one of the at least one azide group to form a particulate probe; wherein the biomolecule-linker conjugate comprises from one to five first attachment groups.

Particulate light-emitting marker

A particulate light-emitting marker as described herein may be, without limitation, a micro- or nano-particulate light-emitting marker.

In some embodiments, the particulate light-emitting marker comprises or consists of a quantum dot. Exemplary light-emitting quantum dot materials include, without limitation, metal chalcogenides. Quantum dots include, without limitation, core, coreshell and alloyed quantum dots. In some embodiments, the particulate light-emitting marker is a collapsed light-emitting polymer.

In some embodiments, the particulate light-emitting marker comprises or consists of a light-emitting particle. In some embodiments, light-emitting particle of the particulate light-emitting marker may comprise or consist of a light-emitting material and a matrix. The light-emitting material may be a fluorescent or phosphorescent light-emitting material. The light-emitting material be polymeric or non-polymeric.

Exemplary non-polymeric fluorescent materials include, without limitation: fluorescein, fluorescein isothiocyanate (FITC); fluorescein NHS; Alexa Fluor 488; Dylight 488; Oregon green; DAF-FM; 6-FAM; 2, 7-di chlorofluorescein; 3’-(p- aminophenyl)fluorescein; 3’-(hydroxyphenyl)fluorescein; rhodamines, for example Rhodamine 6G and Rhodamine 110 chloride; coumarins; boron-dipyrromethenes (BODIPYs); naphthalimides; perylenes; benzanthrones; benzoxanthrones; benzothiooxanthrones; 2-(4-pyridyl)-5-phenyl-oxazole; 2-quinolinyl-5-phenyl-oxazole; 2-(4-pyridyl)-5-naphthyl-oxazole; 2-(4-pyridyl)-5-phenyl-thiazole; 2-quinolinyl-5- phenyl-thiazole; 2-(4-pyridyl)-5-naphthyl-thiazole; 2-(4-pyridyl)-5-phenyl-thiophene; 2- quinolinyl-5-phenyl- thiophene; 2-(4-pyridyl)- 5-naphthyl- thiophene and salts thereof, each of which may be unsubstituted or substituted with one or more substituents. Exemplary substituents are ionic or non-ionic substituents as described herein, optionally chlorine, alkyl amino; phenylamino; and hydroxyphenyl.

In the case where a light-emitting marker as described herein comprises a light-emitting polymer, the light-emitting polymer is optionally selected from light-emitting polymers described below.

Preferably, the light-emitting particles as described herein have a number average diameter of no more than 500nm or 400 nm in methanol as measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Details of measurement in the Examples). Preferably, the particles have a number average diameter of between 5-500 nm, optionally 10-200 nm, optionally 5-200 nm, preferably between 5-150 nm, as measured by a Malvern Zetasizer Nano ZS. For example, the particles have a number average diameter of between 5-100 nm, optionally between 5-50 nm.

In some embodiments, the light-emitting particles as described herein are light-emitting nanoparticles. A light-emitting particle as described herein may have a core comprising a light-emitting material. The core may comprise a matrix material, optionally an inorganic matrix material, optionally an inorganic oxide, optionally silica.

If a matrix material is present then in some embodiments the light-emitting material may be covalently bound, directly or indirectly, to the matrix material. In some embodiments, the light-emitting material may be mixed with (i.e. not covalently bound to) a matrix material. The core may comprise a silica shell partially or completely covering an inner core comprising or consisting of a light-emitting material, preferably a light-emitting polymer. The core may contain one or more light-emitting polymer chains mixed with and extending through the matrix material. One or more light-emitting polymer chains may protrude beyond a surface of the core defined by the matrix material.

A nanoparticle may comprise a nanoparticle core which 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.

Surface groups

The particulate light-emitting marker has an azide group at a surface thereof. In some embodiments, the light-emitting marker comprises a light-emitting particle and the lightemitting particle is substituted with a surface group which comprises an azide group. Light-emitting particles as described herein are substituted with at least one surface group. At least one of the at least one surface groups comprises an azide group.

Following formation of the particle cores, surface groups may be bound to the surface of the particles. Surface groups include, without limitation, surface groups for preventing aggregation of the particles, 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 surface of the particle core. To form a surface group, the particles may be brought into contact with a reactive compound for forming the surface group having a reactive group capable of reacting with a Si-0 groups at the surface of the particle core. The reactive group may be a group of formula -Si(OR⁷)s 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 particle 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 Ci-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 Ci-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, at least one end group EG is an azide group.

According to some embodiments, EG is optionally selected from H; C1-12 alkyl; C1-12 alkoxy; and esters, e.g. Ci-2ohydrocarbyl esters of COOH.

The particle core may be substituted with different surface groups, e.g. a first surface group comprising an azide group 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 40 mol %, less than 30 mol %, or less than 25 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 %, optionally, at least 1 mol %, optionally at least 10 mol %, of the total number of moles of the first and second surface groups.

Surface groups may be poly disperse.

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.

Colloids

The light-emitting particles may be provided as a colloidal suspension comprising the light-emitting particles suspended in a liquid. Preferably, the liquid is selected from water, Ci- 10 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 - 500 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. Linker

A linker as described herein is a compound that is capable of reacting with an azide group and a biomolecule. That is, the linker may have a first attachment group capable of reacting with an azide group and a second attachment group capable of reacting with a biomolecule. Preferably, the first attachment group is capable of reacting with an azide group to form a covalent bond. Preferably, the second attachment group is capable of reacting with a biomolecule to form a covalent bond.

Optionally, the first attachment group comprises an alkyne group such as a strained or terminal alkyne group. Optionally, the first attachment group comprises a group selected from the list consisting of a dibenzocyclooctytne group, an azadibenzocyclooctyne group, a cyclooctyne group, a monofluorinated cyclooctyne group, a difluorinated cyclooctyne group, a dibrominated cyclooctyne group, a 6,7-dimethoxyazacyclooct-4-yne group, and a bicyclo[6.1.0]nonyne group. For example, the first attachment group may comprise a dibenzocyclooctytne group. Optionally, the second attachment group comprises an amine, a thiol, an azide, acetal, tetrazine, carboxylic acid or a derivative thereof such as an amide or ester, preferably an NHS (N-hydroxysuccinimide) ester, acid chloride or acid anhydride group. The second attachment group may be activated before attachment to a biomolecule, e.g. activation of a carboxylic acid group using a carbodiimide, for example EDC. Optionally, the second attachment group comprises an amide or ester group. Optionally, the second attachment group comprises a group selected from the list consisting of an amide group, an ester group, an imidoester group, a maleimide group, a carboxylic acid group, and a tetrafluorophenyl ester group. For example, the second attachment group may comprise an NHS ester group. Optionally, the linker comprises an NHS ester group and a dibenzocyclooctytne group. For example, the linker may be a DBCO-NHS ester (dibenzocyclooctyne-sulfo-N- hydroxysuccinimidyl ester).

Biomolecule In some embodiments, the biomolecule may be a probe for detection of a target. In some embodiments, the biomolecule comprises a probe group for detection of a target attached to a surface thereof. In some embodiments, the target is a biomolecule.

The biomolecule may be, without limitation, a protein; 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.

The biomolecule comprises a linker attachment group. The linker attachment group is capable of reacting with the second attachment group of the linker or the light-emitting conjugate to form a covalent bond. Optionally, the linker attachment group comprises a group selected from the list consisting of a thiol group, a carboxylic acid group, a sugar group, a phosphate group, and an amine group (for example a primary amine group). For example, the linker attachment group may comprise an amine group. For example, the linker attachment group may be a lysine residue or a cysteine residue. Preferably, the biomolecule may be a protein comprising a lysine residue.

Light-emitting conjugate

In some embodiments, the method comprises reacting the linker and the at least one azide group to form a light-emitting conjugate. In some embodiments, the light-emitting conjugate comprises at least one second attachment group as described in relation to the linker.

Forming the particulate probe from the light-emitting conjugate

In some embodiments, the method comprises reacting the light-emitting conjugate with the biomolecule to form a particulate probe; wherein the method further comprises a quenching step after the light-emitting conjugate reacts with the biomolecule. For example, the light-emitting conjugate may comprise a second attachment group capable of reacting with the biomolecule. The quenching step may comprise reducing or eliminating the ability of the second attachment group of the light-emitting conjugate and the biomolecule to react together. For example, the quenching step may comprise providing a second attachment group quenching agent and reacting the second attachment group quenching agent with the light-emitting linker conjugate. Optionally, the second attachment group quenching agent comprises tris(hydroxymethyl)aminomethane, ethanolamine, methylamine, and/or aminoethanoic acid.

In some embodiments, when the second attachment group is an NHS ester, the second attachment group quenching agent comprises or consists of a primary amine.

Biomolecule linker conjugate

In some embodiments, the method comprises reacting the biomolecule and the linker to form a biomolecule linker conjugate and reacting the biomolecule linker conjugate with at least one of the at least one azide groups to form a particulate probe; wherein the biomolecule-linker conjugate comprises from one to five first attachment groups. Optionally, the biomolecule-linker conjugate comprises from one to three first attachment groups; optionally from one to two first attachment groups; optionally one first attachment group.

In some embodiments, the biomolecule is a population of biomolecules, i.e. a group of greater than one biomolecules. In such populations, the number of linker attachment groups in the population may be approximately one per biomolecule. That is, the average number of linker attachment groups in the population of biomolecules is approximately one.

In some embodiments, the biomolecule linker conjugate is a population of biomolecule linker conjugates, i.e. a group of greater than one biomolecule linker conjugates. In such populations, the number of first attachment groups in the population may be approximately one per biomolecule linker conjugate. That is, the average number of first attachment groups in the population of biomolecule linker conjugates is approximately one.

Particulate probe The present disclosure relates to a method of forming a particulate probe. The particulate probe is a conjugate of a light-emitting marker, a linker and a biomolecule as described herein. Particulate probes formed by any method according to the present disclosure have a lesser propensity to aggregation and are more stable than those formed by known methods.

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-di chlorofluorescein, 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 Ci-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 Ci-40 hydrocarbyl substituents.

C 1-40 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 Ci- 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)s⁺ wherein R⁵ in each occurrence is H or C1-12 hydrocarbyl. Preferably, each R⁵ is a Ci- 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 trivalent counterions.

The counterion is optionally a cation, optionally a metal cation, optionally Li⁺, Na⁺, K⁺, Cs . preferably Cs⁺, or an organic cation, optionally ammonium, such as 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(CH2CH2O)tR⁴, 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

Z^J-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. The light-emitting material may be a monodisperse compound selected from are selected from formulae (XI)-(XV):

(A)u (XI)

(A-B)v (XII)

C-[(B-A)xl]yl (Xllla)

C-[(A-B)xl]yl (Xlllb)

C-[(B-A)xl]y2 (XlVa) C-[(A-B)xl]y2

C-[(A)zl)]y3 (XV) wherein:

A, B and C each independently represent a conjugated unit which is unsubstituted or substituted with one or more substituents; u is at least 2, preferably 2-10, more preferably 2-6; v is at least 1, preferably at least 2, more preferably 2-5; xl is at least 1, optionally 1, 2 or 3, preferably 1 or 2, more preferably 1; yl is at least 1, optionally 1, 2 or 3, preferably 1 or 2; y2 is at least 2, optionally 2 or 3, preferably 2. y3 is at least 2, optionally 2 or 3, preferably 2; and zl is at least 2, optionally 2 or 3, preferably 2.

Exemplary compounds of formula (XI) include dimers, trimers, tetramers and pentamers of A. Exemplary compounds of formula (XII) include A-B-A-B and A-B-A-B-A-B.

Exemplary compounds of formula (XIII) include A-B-A and A-B-A-B-A.

Exemplary compounds of formula (XIV) include A-B-C-B-A.

Exemplary compounds of formula (XV) include A- A-B-A- A.

It will be understood that a repeating unit A, B or C as described herein may be one or both of a monovalent repeating unit at a chain end of the compound and a divalent or higher valency repeating unit within a chain of the compound. For example, a compound of formula A-B-A-B-A contains two different repeating units: three units of formula A, of which one is divalent and two are monovalent; and two divalent repeating units of formula B.

In the case where a compound contains a repeating unit such as A, B or C that is present as both a monovalent and a divalent unit, the monovalent and divalent units differ only in their valencies. The terminal carbon atom of the monovalent unit corresponding to the binding carbon atom of the divalent unit may be unsubstituted or substituted with one or more substituents.

A, B and C each preferably and independently comprise or consist of an unsubstituted or substituted aromatic or heteroaromatic group. Aromatic groups as described herein are preferably fused or monocyclic C6-20 aromatic groups. Heteroaromatic groups as described herein are preferably 5-20-membered fused or monocyclic heteroaromatic groups.

Divalent groups A are preferably selected from formulae (XVIa) and (XVIb):

— -Ar²— -

(XVIa)

wherein Ar² is a monocyclic or fused C6-20 arylene group or a monocyclic or fused 5-20 membered heteroaromatic group. Ar² may be unsubstituted or substituted with one or more substituents.

Monovalent groups A are preferably selected from formulae (XVIc) and (XVId):

(XVIc) (XVId)

Divalent groups B are preferably selected from formulae (Vila) and (Vllb):

--_Ar3--

(Vila)

Monovalent groups B are preferably selected from formulae (Vile) and (VII d):

Ar³— -

(Vile)

wherein Ar³ is a monocyclic or fused C6-20 arylene group or a monocyclic or fused 5-20 membered heteroaromatic group. Ar³ may be unsubstituted or substituted with one or more substituents.

Groups C are preferably selected from formulae (Vllla)-(VIIIc):

Ar⁴— -

(Villa) (Vlllb) (VIIIc) wherein Ar⁴ is a monocyclic or fused C6-20 arylene group or a monocyclic or fused 5-20 membered heteroaromatic group, preferably a C6-20 arylene group. Ar⁴ may be unsubstituted or substituted with one or more substituents..

Preferably, a compound selected from formulae (XI)-(XV) comprises aromatic or heteroaromatic units directly linked to one another.

Preferred aromatic divalent groups Ar², Ar³ and Ar⁴ include:

wherein R⁹ and R¹⁰ are as described above. Preferably, compounds of formulae (XI)-(XV) are substituted with one or more ionic or non-ionic polar groups, preferably a polar group R¹ as described above. An ionic polar substituent of compounds of formulae (XI)-(XV) is preferably cationic.

Preferred divalent heteroaromatic groups Ar², Ar³ and Ar⁴ include:

wherein X is O or S.

Preferred monovalent groups Ar², Ar³ and Ar⁴ are monovalent analogues of divalent aromatic or heteroaromatic groups described above in which one of the binding positions is replaced with R⁹. Sp- R^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: - C1-20 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 C 1-20 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 heteroaryl 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 Ci-4ohydrocarbyl, 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, indenofluorene, 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 5x10⁶. 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.

Applications The particulate probes 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 particulate probes 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 particulate probes are for use in fluorescence microscopy, flow cytometry, next generation sequencing, in-vivo imaging, or any other application where a lightemitting 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 particulate probes, for example the particulate probes in a colloidal suspension.

In some embodiments, the sample following contact with the particulate probes 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 particulate probes 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 particulate probes mixed with cells which do not bind to the particulate probes. 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 particulate probes.

Particulate probes as described herein may be particularly advantageous in nucleic acid sequencing due to a good balance of high brightness and small average nanoparticle size and / or low PDI. A method of sequencing nucleic acids may comprise: contacting a primed template nucleic acid molecule with a polymerase and a test nucleotide; incorporating the test nucleotide into a primed strand of the primed template only if it comprises a base complementary to the next base of the template strand; irradiating the primed strand; and determining from luminance of the primed strand if the test nucleotide has been incorporated into the primed strand, wherein the test nucleotide of the irradiated primed strand is bound to a particulate probe as described anywhere herein.

In some embodiments, the sequencing method comprises the detection of single nucleotides as they are incorporated at the 3’ end of the primer or 3’ end of the nascent growing strand.

In some embodiments, nucleotides are added to a nucleic acid primer thereby extending the primer in a template-dependent manner. Detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template.

At least some of the nucleotides are labelled with a particulate probe as described herein which may be used to detect and identify the nucleotide.

During each sequencing cycle, a nucleotide which is the next complementary nucleotide (i.e. comprises a base complementary to the next base of the template strand), is incorporated into the nucleic acid chain in a template-dependent manner due to complementary hydrogen bonding with the corresponding nucleotide in the template fragment.

A polymerase enzyme may subsequently catalyze the chemical addition of the next complementary nucleotide into the nucleic acid chain.

In each sequencing cycle, the next complementary nucleotide is identified by irradiation of the nucleotide and detection of any luminescence attributable to a marker that the nucleotide is labelled with.

A plurality of different species of test nucleotide may be included in the reaction mixture in a single sequencing cycle. Thus, labels associated with each different nucleotide species may be distinguishable and identifiable, for example on the basis of different luminescence emission spectra. In these embodiments, the luminescence emission that is detected is used to determine which of the different species of test nucleotides is incorporated into the primed strand and, thus represents the next complementary nucleotide. In these embodiments, at least one of the different nucleotide species is labelled with a particulate probe as described herein. In some embodiments, one or more of the nucleotides may be pre-labelled with the relevant marker prior to inclusion in the reaction mixture and/or prior to incorporation into the primer or nascent strand of the nucleic acid that is being sequenced, wherein at least one of the nucleotides is pre-labelled with a particulate probe as described herein. In other embodiments, one or more of the nucleotides may be labelled after correctly base pairing with the relevant nucleotide in the template strand, and/or after incorporation into the growing strand, wherein the labels include at least one particulate probe as described herein. For example, one of more of the nucleotides may comprise a moiety, such as a linker, to which a detectable label may be directly or indirectly attached to thereby detect and identify the nucleotide.

In some sequencing methods, each nucleotide is associated with a different luminescent label that may be used to detect and identify the nucleotide, wherein at least one of the different luminescent labels is a particulate probe as described herein. Thus, the number of possible different nucleotides to be detected is equal to the number of different labels used, and the number of detection channels required.

In some sequencing methods, the number of possible different nucleotides is greater than the number of different labels used and/or the number of detection channels required, wherein at least one of the different luminescent labels is a particulate probe as described herein. In some embodiments, one of the nucleotides is not labelled and all other nucleotides are labelled. The presence of the unlabeled nucleotide may be determined if no luminescence arising from a luminescent marker is detected.

In some sequencing methods, emissions from at least two different markers are detectable by the same detector, i.e. emissions from the at least two different markers are the a single nucleotide is labelled with a first and second marker, wherein the first and second markers can be excited by the different laser frequency to produce emissions having first and second peak wavelengths wherein the first and second peak wavelengths are detectable by the same detector.

In any event, at the point of detection, such as for example, when electromagnetic radiation at an excitation wavelength is applied to an emitter, the nucleotide that is being detected comprises the label, in the sense that the label is associated with the nucleotide in such a way that it may be used to specifically detect and identify the nucleotide.

The linker attaching a particulate probe to the nucleotide in the disclosed method may be cleavable or otherwise arranged to allow dissociation of the label from the nucleotide. Thus, after incorporation and identification of the test nucleotide, the emitter or other label may be removed to allow the next sequencing cycle to commence and the next complementary nucleotide to be identified. The result is base-by-base sequencing of the template fragment nucleic acids.

The terms “cleave”, “cleavage site”, and similar terms, refer to a moiety in a molecule, such as a linker, that can be modified or removed to physically separate two other moieties bound to the molecule.

EXAMPLES

Measurement of nanoparticle size

DLS measurements were performed on samples with a Malvern Zetaziser Nano ZS, using a 4mW 633 nm He-Ne laser according to the following procedure. Suspensions of nanoparticles/particulate probes in aqueous 50mM borate buffer or water were tested in single use UV -transparent plastic cuvettes. The machine was operated in Backscatter mode at an angle of 173°. Samples in methanol dispersant were equilibrated to 25 °C for 60 seconds prior to measurement and samples in water-based dispersants were equilibrated to 20 °C for 60 seconds prior to measurement. Values for the methanol dispersant in the software were 0.5476 cP for viscosity and 1.326 for the refractive index. Values for the water-based dispersants in the software were 1.0000 cP for viscosity and 1.330 for the refractive index. The sample was defined as Polystyrene latex (RI: 1.590, Absorption: 0.0100). The automatic measurement duration setting was used, with automatic measurement positioning and automatic attenuation. The ‘general purpose’ analysis model was used, with the default size analysis parameters along with a refractive index of 1.59 for the sample parameter. A single measurement was taken for each sample.

Particle synthesis Nanoparticle 1 synthesis

A nanoparticle (Particle 1) was synthesised by polymerisation of tetraethylorthosilicate (TEOS) in a methanol solution with a polymer of formulation Monomer 1 50 mol%, Monomer 240 mol%, Monomer 3 10 mol % dissolved therein, as described in GB 2554666 (incorporated herein by reference). The reaction was initiated with a ratio of 1 mg of polymer to 100 pL of tetraethylorthosilicate, with a subsequent addition of a further 60 pL of tetraethylorthosilicate after 1 hour. After a further 1 hour the mixture was purified in 3.5 mL portions through lOmL Zeba desalting columns.

Comparative example 1: Nanoparticle surface passivation and functionalisation

The nanoparticles (particle 1) were surface functionalised using 39.2 mg mPEG-Silane, MW Ik and 0.8 mg Biotin-PEG-Silane, MW Ik to 2 mg of nanoparticles in 1 mL of water. The mixture was stirred at 60 °C for 16 hours. After cooling the nanoparticles were isolated by pelleting using a centrifuge at 20,000xg for 20 minutes, the supernatant was removed by pipette and the pellet was resuspended in water using sonication. Streptavidin functionalisation

A volume calculated to contain Img of nanoparticles was pelleted using the centrifuge (20,000xg, 20 min), and the supernatant carefully removed using a pipette.

The resulting solid particle was resuspended in 1% BSA (bovine serum albumin)/borate buffer (1 ml) using a combination of vortex mixing and ultrasonication. Once the pellet had fully resuspended, a 7 mg/ml solution of streptavidin (350 pg) in borate buffer (50 pl) was added, and the resulting mixture was stirred together for 1 h.

The resulting streptavidin-attached nanoparticle was then pelleted using a centrifuge (20,000xg, 20 min), the supernatant removed carefully using a pipette and the pellet was then resuspended in a mixture of 97pl l%BSA/borate buffer and 3 pl of 0.1 M sodium azide solution using a combination of vortex mixing and ultrasonication.

The resultant particle suspension of particles (10 mg/ml in l%BSA/borate buffer), was analysed by dynamic light scattering (DLS) (Z avg: 80.76, 1 avg: 98.34, N avg: 46.63, PDI:0.172 ) to confirm that the particle was not aggregated. Example 1: Formation of particulate probe 1

Azide functionalisation of nanoparticle 1

Nanoparticles were formed as described in Comparative Example 1, except that azide- PEG-Silane, illustrated below, (Mw 1,000 g / mol) was used in place of Biotin-PEG- Silane.

Azide-PEG-Silane

Linker functionalisation of nanoparticle

Bioconjugation using a metal-free click reaction between the strained alkyne dibenzocyclooctyne [DBCO] and an azide group was used as described in Beilstein J. Org. Chem. 2018, 14, 11-24 and Polym. Chem. 2019, 10, 705-717 (incorporated herein by reference).

A volume calculated to contain Img of nanoparticles was pelleted using the centrifuge (20,000xg, 20 min), and the supernatant carefully removed using a pipette. The pellet was resuspended in 50mM MES buffer pH 5.0 using a combination of vortex mixing and ultrasonication.

Following this, 50pL of 42mM NHS-DBCO linker was added from a freshly made stock solution to give a final concentration of 2mM in the reaction, and the mixture was stirred for 20 minutes. The nanoparticles were then pelleted again using the centrifuge (20,000xg, 20 min), and the supernatant carefully removed using a pipette. The nanoparticles were then resuspended in 50mM MES buffer pH 5.0 using a combination of vortex mixing and ultrasonication before being centrifuged again (20,000xg, 20 min). The supernatant was removed using a pipette and the pellet re-suspended in 1ml of 50mM HEPES buffer (pH 7.0) using a combination of vortex mixing and ultrasonication.

Biomolecule functionalisation of nanoparticle lOOpL of 7mg/ml Streptavidin in 50mM HEPES buffer (pH 7.0) was then added to the tube and mixed for 1 hour.

Following this the sample had lOOpL of IM Tris buffer (pH 7.4) added as a quenching agent for 30 minutes. Once this incubation was complete, the sample was pelleted in a centrifuge (20,000xg, 20 min), the supernatant removed, and the pellet resuspended in 1.3ml of lOmg/ml BSA in 50mM Borate buffer (pH 8.5). This pelleting and resuspension was repeated twice more with the final resuspension in 97pL of lOmg/ml BSA in 50mM Borate buffer (pH 8.5). 3pL of sodium azide (0. IM) was then added as a preservative. Example 2: Formation of particulate probe 2

Linker functionalisation of biomolecule Streptavidin was dissolved in 50mM HEPES buffer, pH 7.4 to a concentration of 135pM. DBCO-NHS linker was prepared fresh by dissolving powder into 50mM HEPES buffer to a concentration of 12mM. This solution was then dissolved 4.5-fold, to give a concentration of 2.6mM. This dissolved solution was then mixed with the Streptavidin solution, lOOpL for every 1ml of Streptavidin solution to give final concentrations of 120uM for Streptavidin and 240uM for DBCO-NHS (Hence giving a 1 :2 ratio). This was incubated for 90 minutes. Following this, lOOpL of IM Tris buffer was added for every 1ml of Streptavidin solution and a further 30 minute incubation took place. This sample was then applied to a Sephadex G-25 desalting column equilibrated in 50mM Borate buffer, pH 8.5 to remove excess linker and blocking buffer. The resulting protein fractions from the desalting column were pooled and concentrated in a lOkDa spin concentrator to 60uM, and the average number of DBCO groups per Streptavidin measured using UV- Vis spectroscopy. The sample was then aliquoted and frozen until required for functionalization of the nanoparticle. Azide functionalisation of nanoparticle

Azide functionalised nanoparticles were formed as described in Example 1.

Biomolecule functionalisation of nanoparticle

Nanoparticles were formed and were surface functionalized with Azide as described in Example 1. A volume calculated to contain Img of nanoparticles was pelleted using the centrifuge (20,000xg, 20 min), and the supernatant carefully removed using a pipette. The pellet was resuspended in 550pL of 50mM Borate buffer pH 8.5 using a combination of vortex mixing and ultrasonication. 550pL of DBCO-Streptavidin was then added to the solution to give a final concentration of 30uM DBCO-Streptavidin and 50nM of Nanoparticle. This was incubated for 2 hours, then the sample was pelleted using the centrifuge (20,000xg, 20 min), the supernatant removed using a pipette and the sample resuspended in 1.4ml of lOmg/ml BSA in Borate Buffer. The sample was then pelleted, the supernatant removed and resuspended again in 1.4ml of lOmg/ml BSA in borate buffer. This was repeated twice more to give three washes of the nanoparticles and remove excess DBCO-Streptavidin. The final resuspension was in 97pL of lOmg/ml BSA in borate buffer, and 3pL of Sodium azide solution. This sample was then stored at 4°C until use.

Particle Size Stability

Biotinylated nanoparticles and particulate probes synthesized according to Example 1 and to Comparative Example 1 were stored at 4°C for a period of three days. Periodic measurements of the particle size were obtained using dynamic light scattering technique described above. Figure 1 shows the N-average size percentage increase of the respective particles over the three days. Figure 1 clearly shows that the methodologies described herein results in nanoparticles that are less prone to aggregation and therefore have increased colloidal stability.

Plate assays

Assay plates were generated as follows:

Sample wells:

• Incubating a mix of 0.2pg/ml Biotinylated-BSA and 54.8pg/ml BSA with a Greiner high and medium binding 96-well polystyrene plate (Black wells) for 1 hour.

• Remove solutions and replace with lOmg/ml BSA in 200mM Citrate buffer (pH 4.5) for 1 hour.

• Remove solutions and wash plates three times with 120pL of 200mM Citrate buffer (pH 4.5).

• Incubate nanoparticle solutions at varying concentrations from 15-250pg/ml for 30 minutes in lOmg/ml BSA in borate buffer

• Remove solutions and wash plates three times with 120pL of 50mM Borate Buffer, pH 8.5. Negative wells:

Incubate lOmg/ml BSA in 200mM Citrate buffer (pH 4.5) for 2 hours. • Remove solutions and wash plates three times with 120pL of 200mM Citrate buffer (pH 4.5).

• Incubate nanoparticle solutions at varying concentrations from 15-250pg/ml for 30 minutes in lOmg/ml BSA in borate buffer • Remove solutions and wash plates three times with 120pL of 50mM Borate

Buffer, pH 8.5.

Blank wells:

• Incubate lOmg/ml BSA in 200mM Citrate buffer (pH 4.5) for 2 hours.

• Incubate lOmg/ml BSA in borate buffer for 30 minutes.

• Remove solutions and wash plates three times with 120pL of 50mM Borate Buffer, pH 8.5.

Assay plates were imaged on a BMG Labtech CLARIOstar Plus plate reader. Wells were excited via between 460-490nm, with a dichoic filter of 507.5nm and emission measured between 525-625nm. Wells were scanned using this filter set in a 2x2 matrix scan with a diameter of 5mm and measuring the average of 10 repeat flashes per pixel. The average of the four pixels in each well was then given as the fluorescent signal for that well. The blank-corrected signal intensity for sample and negative wells was then given by the average well intensity minus the average of all the blank well values.

Signal-to-noise was calculated by dividing the sample wells by the negative wells for a given nanoparticle concentration.

Figure 2 shows the FITC adjusted signal for assays using particulate probes formed according to some embodiments and the comparative examples described herein. Higher signal is observed from azide-functionalized nanoparticles compared to biotin- functionalized nanoparticles within the same assay.

Figure 3 shows the signal to noise ratios for assays using particulate probes formed according to some embodiments and the comparative examples described herein. Within the same assay, azide nanoparticles display higher signal to noise over the biotinylated nanoparticles.

Nanoparticle 2 synthesis

Nanoparticles (particle 2) were synthesised by polymerisation of tetraethylorthosilicate (TEOS) in a methanol: octanol: ammonium hydroxide (29% aqueous) solution

(8.9: 1.1 :0.5 mL) in the presence of 1 mg of Compound 2 illustrated below. The reaction was initiated at 60 °C with the addition of 100 pL of tetraethylorthosilicate, with a subsequent addition of 60 pL of tetraethylorthosilicate after 1 hour. After a further 1 hour at 60 °C, the mixture was concentrated and passed through 2 rounds of water- equilibrated Zeba deslating columns (7k MWCO). The aqueous eluent was basified with ammonium hydroxide (29% aqueous) and sonicated at 80% amplitude for 2 mins to yield particle 2 nanoparticles.

Compound 2 Azide functionalisation of Nanoparticle 2

15 mg of Nanoparticle 2 in the dispersion made above was combined with 117 mg of mPEG silane, Mw Ik and 33 mg of azide-PEG silane, Mw IK in 7.5 mL of water. The reaction was stirred at 60 °C for 16 hours. After cooling, the mixture was concentrated, filtered through a 0.22 urn syringe filter and purified by size-exclusion chromatography on a Cytiva Akta Start using S400-HR media, eluting with borate buffer (50 mM, pH 8.5). The desired fractions were combined and concentrated and measured on a Malvern Zetasizer Nano ZS which showed a Z average diameter of 18.19 nm, a Pdl of 0.256 and an N average diameter of 11.30 nm.

Linker and biomolecular functionalisation of Nanoparticle 2

The bioconjugation and linker functionalisation of Nanoparticle 2 was carried out as per particulate probe 1. Due to the smaller size of Nanoparticle 2, instead of forming pellets, nanoparticles were handled by concentrating the samples and diluting into the desired buffers. Final purification after biomolecule functionalisation was carried out on a Cytiva Akta Start using S200-HR media, eluting with borate buffer (50 mM, pH 8.5) to separate probe 2 from the excess streptavidin. Probe 2 was found to be size-stable by DLS over a period of 2 weeks.

Claims

1. A method of forming a particulate probe, the method comprising the steps of: providing a biomolecule; providing a particulate light-emitting marker comprising at least one azide group at a surface thereof; providing a linker, the linker being capable of reacting with the at least one azide group and the biomolecule; reacting the biomolecule and the linker to form a biomolecule linker conjugate; reacting the biomolecule linker conjugate with at least one of the at least one azide group to form a particulate probe; wherein the biomolecule linker conjugate comprises from one to five first attachment groups capable of reacting with the at least one azide group.

2. A method of forming a particulate probe according to claim 1, wherein the biomolecule linker conjugate comprises from one to three first attachment groups capable of reacting with the at least one azide group, optionally wherein the biomolecule linker conjugate comprises from one to two first attachment groups capable of reacting with the at least one azide group.

3. A method of forming a particulate probe according to claim 1 or 2, wherein the linker comprises a first attachment group capable of reacting with the at least one azide group and a second attachment group capable of reacting with the biomolecule.

4. A method of forming a particulate probe according to claim 3, wherein the biomolecule comprises a linker attachment group capable of reacting with the second attachment group of the linker.

5. A method of forming a particulate probe according to claim 3 or 4, wherein the first attachment group comprises an alkyne group and/or the second attachment group comprises an NHS ester.

6. A method of forming a particulate probe according to any one of claims 1 to

5, wherein the particulate light-emitting marker is a light-emitting particle comprising a core and a light-emitting material.

7. A method of forming a particulate probe according to claim 6, wherein the core of the light-emitting particle comprises a matrix material, optionally an inorganic matrix material, optionally an inorganic oxide, optionally silica.

8. A method of forming a particulate probe according to any one of claims 1 to 7, wherein the particulate light-emitting marker is a light-emitting nanoparticle.

9. A method of forming a particulate probe, the method comprising the steps of: providing a particulate light-emitting marker comprising at least one azide group at a surface thereof; providing a linker, the linker being capable of reacting with at least one of the at least one azide group and a biomolecule; providing a biomolecule capable of reacting with the linker; reacting the linker and at least one of the at least one azide group to form a light-emitting conjugate; and reacting the light-emitting conjugate with the biomolecule to form a particulate probe; wherein the method further comprises a quenching step after the light-emitting conjugate reacts with the biomolecule.

10. A method of forming a particulate probe according to claim 9, wherein the linker comprises a first attachment group capable of reacting with the at least one azide group and a second attachment group capable of reacting with the biomolecule.

11. A method of forming a particulate probe according to claim 9 or 10, wherein the light-emitting conjugate comprises a second attachment group capable of reacting with the biomolecule.

12. A method of forming a particulate probe according to claim 10 or 11, wherein the biomolecule comprises a linker attachment group capable of reacting with the second attachment group of the linker and/or light-emitting conjugate.

13. A method of forming a particulate probe according to claim 11 or 12, wherein the quenching step comprises reducing or eliminating the ability of the second attachment group of the light-emitting conjugate and the biomolecule to react together.

14. A method of forming a particulate probe according to any one of claims 10 to 13, wherein the quenching step comprises providing a second attachment group quenching agent.

15. A method of forming a particulate probe according to claim 14, wherein when the second attachment group comprises an NHS ester and the second attachment group quenching agent comprises a primary amine.

16. A method of forming a particulate probe according to any one of claims 10 to 15, wherein the first attachment group comprises an alkyne group and/or the second attachment group comprises an NHS ester.

17. A method of forming a particulate probe according to any one of claims 9 to 16, wherein the particulate light-emitting marker is a light-emitting particle comprising a core and a light-emitting material.

18. A method of forming a particulate probe according to claim 17, wherein the core of the light-emitting particle comprises a matrix material, optionally an inorganic matrix material, optionally an inorganic oxide, optionally silica. A method of forming a particulate probe according to any one of claims 9 to 18, wherein the particulate light-emitting marker is a light-emitting nanoparticle. A particulate probe obtainable by a process according to any one of claims 1 to 19. A method of identifying the presence and/or concentration of a target in a sample comprising contacting the sample with the particulate probe according to claim 20 and detecting emission from the particulate probe. A method of sequencing nucleic acids comprising: contacting a primed template nucleic acid molecule with a polymerase and a test nucleotide; incorporating the test nucleotide into a primed strand of the primed template only if it comprises a base complementary to the next base of the template strand; irradiating the primed strand; and determining from luminance of the primed strand if the test nucleotide has been incorporated into the primed strand, wherein the test nucleotide of the irradiated primed strand is bound to a particulate probe according to claim 20.