WO2017015693A1 - Luminescent biomolecular complex and use thereof - Google Patents

Abstract

The technology relates to biomolecular complex having an antibody-binding protein; a polypeptide linker capable of receiving a luminescence-activating unit; and a luminescence- activating unit. Also disclosed is the use of the biomolecular complex to detect biological target entities.

Description

LUMINESCENT BIOMOLECULAR COMPLEX AND USE THEREOF

Related Application

[001] This application claims the benefit of Australian Provisional Patent Application No 2015902978, filed on 27 July 2015, the disclosures of all of which are hereby expressly incorporated by reference in their entireties.

Technical Field

[002] The present invention relates generally to luminescent probes and their use for detecting the presence or identity of a biological target entity. More particularly, the invention relates to luminescent antibody conjugates and their use in detecting target antigens and cells.

Background

[003] Labelled antibodies have been used for many years as a means for detecting and visualizing target biologicals. There are many situations where directly-labelled antibodies do not bind well due to interference by the label or due to problems with chemical binding of the detectable labels to the antibodies.

[004] Lanthanide ions (e.g. Eu³⁺, Tb³⁺) are of growing interest as luminescent probes for temporally resolving labelled target entities from intrinsic fluorescence (autofluorescence) through the use of delayed imaging (time gated) techniques such as time-gated luminescence (TGL) bioimaging.

[005] The outstanding luminescent properties of lanthanide ions are characterised by their sharp emission profiles (< 10 nm width), large Stokes shifts (> 150 nm) and long (millisecond) excited-state lifetimes. For example, the long-lived excited state of trivalent europium ions results in a characteristic spiked luminescence profile at 618 nm (corresponding to the 5D0 to 7F1 transition) persisting for hundreds of microseconds. Such features, in conjunction with pulsed excitation and time-gated measurements, allow temporal discrimination against fast decaying (nanosecond) autofluorescence and scattered excitation light. Time gated

luminescence ( TGL) techniques rely upon a brief but intense excitation pulse followed by a short interval to allow short-lived fluorescence to decay. (J. R. Lakowicz, Principles of

Fluorescence Spectroscopy - Second Edition, 2nd ed. ed., Plenum Press, New York, N. Y., 1999). By exploiting the long luminescence lifetimes of lanthanide ions, TGL microscopy can be used to visualise biomolecules and cells in autofluorescent environments.

[006] Trivalent lanthanide ions (Ln³⁺) have intrinsically low absorption cross-sections, and as a consequence direct excitation yields only low levels of luminescence. As a result, lanthanide ions need to be excited indirectly through a method known as sensitization. One way of achieving this is to tether a lanthanide ion chelate to an organic chromophore that acts as an antenna to sensitize the adsorption of light and transfer of excitation energy to the chelated lanthanide ion, resulting in higher luminescence and extended emission lifetimes.

[007] Luminescent Eu³⁺ chelates have been used to label antibodies in TGL bioimaging. However, direct incorporation of lanthanide chelates onto antibodies encounters significant difficulties and drawbacks. For example, direct labelling of antibodies with lanthanide chelates can cause antibody inactivation and/or precipitation due to poor aqueous solubility, over modification and variations in antibody reactivity and sensitivity. Attempts to determine the optimal ratio of chelate to antibody that delivers sufficient luminescent signal with retention of antibody specificity and avidity can also prove difficult. This is largely due to the random nature of chelate insertion at various residues within the protein molecule and, in addition, it is likely that chelate residues may be inserted into the recognition cleft of the antibody itself, thus reducing or destroying the ability of the antibody to recognise the antigen. For these reasons direct antibody modification is often inefficient and not an ideal approach.

[008] Indirect methods of incorporating lanthanide chelates that deliver sufficient luminescent signal with retention of antibody function have also been investigated. In one approach, secondary antibodies were labelled with luminescent tetradentate Eu³⁺ chelates for indirect detection of Giardia using TGL microscopy. However, direct modification of a secondary antibody also makes it susceptible to inactivation. Another approach involved the use of lanthanide-labelled streptavidin as a universal detection reagent to label biotinylated secondary antibodies with detectable luminescence for TGL bioimaging. However, this method requires the specific modification of antibodies with biotin and relies on the biotin-streptavidin binding interaction, which can be difficult to control.

[009] One approach to maximize luminescence is to attach multiple lanthanide chelates onto a carrier molecule that tolerates a high degree of labelling and which can be chemically cross- linked to a detection reagent. For example, streptavidin (SA) can be conjugated to bovine serum albumin (BSA), and then the SA-BSA conjugate can be labelled with lanthanide chelates prior to use as an indirect detection reagent in TGL bioimaging. Another potential universal detection reagent is the recombinant fusion protein, Linker-protein G (LPG). LPG has been used as an anchorage point for the oriented immobilisation of antibodies onto silica-containing materials without the need for complex surface chemical modification.

[010] There is a need for alternative detection agents for detecting target biological entities. There is also a need for luminescent molecular complexes that enable the formation of antibody conjugates having improved aqueous solubility and stability whilst maintaining antibody specificity and avidity. The present inventors have developed new biomolecular complexes suitable to probe biological targets.

Summary

[011] In a first aspect, there is provided a biomolecular complex comprising:

an antibody-binding protein;

a polypeptide linker capable of receiving a luminescence-activating unit; and a luminescence-activating unit.

[012] The antibody-binding protein may be selected from protein A, protein A G, protein G, protein L, and any combination thereof.

[013] In one embodiment the antibody-binding protein is protein G.

[014] In one embodiment the polypeptide linker is fused to the antibody-binding protein at the amino or carboxyl terminal of the protein.

[015] In one embodiment the polypeptide linker which has a specific binding affinity towards silica-containing materials. In this form the complex can be used, for example, to attach labelled antibodies to a silica shell encapsulating a magnetic core.

[016] In one embodiment the polypeptide linker comprises one or more amino acids comprising a side group having a functional group selected from NH₂, SH, OH.

[017] In one embodiment the polypeptide linker comprises one or more lysine residues.

[018] In an embodiment the polypeptide linker comprises at least about 20 amino acids.

[019] In one embodiment the polypeptide linker comprises the sequence of SEQ 1 - (VKTQATSREEPPRLPSKHRPG) (SEQ ID NO 1 ).

[020] In one embodiment the polypeptide linker comprises the sequence of SEQ 2 - (VKTQATSREEPPRLPSKHRPG)₄VKTQATS (SEQ ID NO 2).

[021] In one embodiment the polypeptide linker comprises the sequence of NL1 - (GKSSGSSKGSPPKGPSKHKGP)₄ (SEQ ID NO 3). [022] In one embodiment the polypeptide linker comprises the sequence of NL2 - (GKSQGQSKGGPPKGPSKHKGP)₄ (SEQ ID NO 4).

[023] In one embodiment the polypeptide linker comprises the sequence of CON - (QTVTHRGRHEGKAPKGPELHRP)₄ (SEQ ID NO 5).

[024] In one embodiment the polypeptide linker comprises combinations of the linker sequences such as:

CON - (NL1 )₃ - QTVTHRGRHEGKAPKGPELHRP (GKSSGSSKGSPPKGPSKHKGP)₃ (SEQ ID NO 6);

(CON)2 - (NL1 )₂ - (QTVTHRGRHEGKAPKGPELHRP)₂ (GKSSGSSKGSPPKGPSKHKGP)₂ (SEQ ID NO 7);

(CON)3 - NL1 - (QTVTHRGRHEGKAPKGPELHRP)s GKSSGSSKGSPPKGPSKHKGP (SEQ ID NO 8);

CON - (NL2)₃ - QTVTHRGRHEGKAPKGPELHRP (GKSQGQSKGGPPKGPSKHKGP)₃ (SEQ ID NO 9);

(CON)₂ - (NL2)₂ - (QTVTHRGRHEGKAPKGPELHRP);,

(GKSQGQSKGGPPKGPSKHKGP)₂ (SEQ ID NO 10);

(CON)₃ - NL₂ - (QTVTHRGRHEGKAPKGPELHRP)3 GKSQGQSKGGPPKGPSKHKGP (SEQ ID NO 1 1 );

SEQ 1 - (NL1)₃ - QTQATSREEPPRLPSKHRPG (GKSSGSSKGSPPKGPSKHKGP)3;

(SEQ ID NO 12)

(SEQ 1)₂ - (NL1 )₂ - (QTQATSREEPPRLPSKHRPG)₂ (GKSSGSSKGSPPKGPSKHKGP)₂ (SEQ ID NO 13);

(SEQ 1)₃ - NL1 - (QTQATSREEPPRLPSKHRPG)₃ GKSSGSSKGSPPKGPSKHKGP (SEQ ID NO 14);

SEQ 1 - (NL2)₃ - QTQATSREEPPRLPSKHRPG (GKSQGQSKGGPPKGPSKHKGP)₃ (SEQ ID NO 15);

(SEQ 1)₂ - (NL2)₂ - (QTQATSREEPPRLPSKHRPG)₂ (GKSQGQSKGGPPKGPSKHKGP)₂ (SEQ ID NO 16);

(SEQ 1)₃ - NL2 - (QTQATSREEPPRLPSKHRPG)₃ GKSQGQSKGGPPKGPSKHKGP (SEQ ID NO 17).

[025] It will be appreciated that other combinations are contemplated and suitable for the present invention.

[026] The polypeptide linker may comprise more than one occurrence of the polypeptide sequence, for example (Linker Sequence^ where n can be any number. Typically n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15. [027] In an embodiment the biomolecular complex is linker-protein G (LPG).

[028] In an embodiment the LPG is a genetically engineered biomolecule comprising two functional regions: (a) a polypeptide linker sequence having specific binding affinity towards silica-containing materials; and (b) Streptococcus protein G, which has specific binding affinity towards an antibody.

[029] In one embodiment the luminescence-activating unit is attached to the polypeptide linker.

[030] In one embodiment the luminescence-activating unit is attached to the antibody-binding protein.

[031] In one embodiment a plurality of luminescence-activating units are attached to the biomolecular complex.

[032] In one embodiment the luminescence-activating unit is covalently attached to the polypeptide linker moiety by an amide bond.

[033] In one embodiment at least one luminescence-activating unit is attached to the antibody- binding protein.

[034] The luminescence-activating unit can include one or more of conventional or lanthanide- based fluorophores. Examples include but not limited to BHHST, BHHCT, BPPCT, BTBCT, BHHBCB, BPPBCB, or BTBBCB as their lanthanide complexes. Further, conventional fluorophores can be used and examples include: all members of the Alexa Fluor family, all members of the BODIPY family, all members of the Cyanine Dye "Cy dyes" family (eg Cy3, Cy5, Cy7), all members of the Quasar dye family. Full description of BHHBCB, BPPBCB, BTBBCB can be found in L. Zhang, Y. Wang, Z. Ye, J. Dayong, J. Yuan, Bioconj. Chem. 2012, 23, 1244-1251 .

[035] In one embodiment the luminescence-activating unit is a lanthanide-activating unit.

[036] In one embodiment the luminescence-activating unit comprises a tetradentate ligand moiety capable of chelating a lanthanide ion (eg, Eu³⁺ or Tb³⁺), and an antenna moiety capable of sensitising lanthanide emission when a lanthanide ion is chelated to the tetradentate ligand moiety.

[037] When a lanthanide ion is chelated to the tetradentate ligand moiety the luminescence- activating unit may be referred to herein as a 'luminophore'.

[038] In an embodiment the luminescence-activating unit has a reactive functional group 'A' selected from tosylate, mesylate, -halo, -N₃, -NH₂, -CN, N-succinimidyl carbonate, isocyanate, isothiocyanate, sulfonyl

chloride, -C(0)N₃, -C(0)halo, -C0₂H,

alkyl)-R^a, -C(0)NH(d_₆ alkyl)-R^a, -NHC(0)(d _₆ alkyl)-R^a, -C(NH)0(C₁_₆ alkyl)-R^a, aziridine, maleimide, C₂-₄ alkyne; wherein each R^a is independently selected from H, OH, halo, C0₂H, C0₂(Ci_-6 alkyl).

[039] Reactive functional group 'A' facilitates covalent attachment of the luminescent activating unit to the antibody-binding unit of the biomolecular complexes by reacting with functional groups on the polypeptide linker and antibody-binding protein.

[040] In one embodiment 'A' is selected from tosylate, mesylate, halo, N₃, NH₂,

isocyanate, -NHC(0)(C₁_₄ alkyl)-R^a, -C(NH)0(d_₄ alkyl)-R^a, maleimide, and

N-hydroxysuccinimidyl carbonate. In other embodiments 'A' is selected from N₃, halo, isocyanate, haloacetamide and N-hydroxysuccinimidyl carbonate. In one embodiment 'A' is N-hydroxysuccinimidyl carbonate. In another embodiment 'A' is maleimide. In a further embodiment 'A' is iodoacetamide. In another embodiment 'A' is iodo or bromo.

[041] In other embodiments R^a is selected from chloro, bromo, iodo, C0₂H, 0(0)0(0^ alkyl). In particular in some embodiments R^a is selected from bromo, iodo, C0₂H, 0(0)0(0^ alkyl).

[042] The compound of formula (I) comprises a functionalised terphenyl antenna unit and a hydrophilic linker moiety. The terphenyl antenna unit is capable of chelating a lanthanide ion (via the functionalised keto-enol groups) and sensitising lanthanide emission.

[043] The hydrophilic linker moiety of the luminescence-activating unit comprises a functionalised (poly)ethylene glycol in which the functional group 'A' facilitates attachment of the (poly)ethylene glycol linker to the antibody-binding unit to form the biomolecular complex. The linker may be functionalised with a functional group 'A' specifically chosen to react with one or more (preferably a plurality of) functional group(s) present on the polypeptide linker and/or antibody-binding protein of the antibody-binding unit. Thus, in various embodiments the functional group 'A' may comprise or consist of a leaving group capable of being displaced by a nucleophilic residue on the antibody-binding unit to form a covalent bond. In other

embodiments, 'A' may comprise a functional group capable of reacting with a reactive moiety on the antibody-binding unit to covalently attach the luminescence-activating unit to the antibody- binding unit. In some embodiments, at least one luminescence-activating unit is covalently attached to the polypeptide linker of the antibody-binding unit.

[044] In one embodiment the luminescence-activating unit is lanthanide-activating unit having the following structure:

or a salt, hydrate or tautomer thereof. The above compound may be referred to herein as BHHTEGST or Compound (1 ).

[045] Compound 1 is (4,4'-bis(1 ",1 ",1 ",2",2",3", 3"-heptafluoro-4",6"-hexanedion-6"- yl)sulfonylamino-tetraethyleneglycol-succinimidyl carbonate-o-terphenyl) BHHTEGST (based on lUPAC (International Union of Pure and Applied Chemistry) convention).

[046] In one embodiment the luminescence-activating unit includes a fluorescent nanoparticle. Examples of suitable fluorescent nanoparticles include nanodiamonds (NDs), nanorubies (NRs), silica-coated NRs, plasmonic nanoparticles (including gold, silver, and platinum), semiconductor nanocrystals (e.g., CdSe, CdS, ZnS,Ti0₂ and PbS), magnetic compounds (e.g., Fe₃0₄, CoFe₂0₄, CoPt), carbon nanoparticles, quantum dots (QDs), rare earth doped nanoparticles, and their combinations. These particles can be negatively charged by adding anionic groups to their surface or by incorporating an outer silica shell.

[047] In an embodiment the fluorescent nanoparticles are nanodiamonds, nanorubies or silica- coated nanorubies.

[048] In one embodiment the biomolecular complex further includes an antibody or binding fragment thereof bound to the antibody-binding protein.

[049] The antibody or binding fragment thereof can be selected to bind to a given target.

[050] Examples of suitable antibodies or binding fragments thereof include at least one of an IgG, IgM, IgD and IgA.

[051] Examples of suitable targets include cells, cell fragments, proteins.

[052] In a second aspect there is provided a detectable biomolecular complex for binding to a target, the complex comprising:

an antibody-binding protein;

an antibody or antibody fragment bound to the antibody-binding protein; a polypeptide linker attached to the antibody-binding protein; and

one or more luminescence-activating units associated with the polypeptide linker. [053] In one embodiment the antibody-binding protein is protein G.

[054] In an embodiment the polypeptide linker is selected from SEQ 1 , SEQ 2, NL1 , NL2, CON, CON - (NL1)_3l (CON)₂ - (NL1 )₂, (CON)₃ - NL1 , CON - (NL2)₃, (CON)₂ - (NL2)₂, (CON)₃ - NL2, SEQ 1 - (NL1)₃, (SEQ 1 )₂ - (NL1)₂, (SEQ 1)₃ - NL1 , SEQ 1 - (NL2)₃,

(SEQ 1)₂ - (NL2)₂, or (SEQ 1)₃ - NL2 (SEQ ID NOS 1 to 17) as defined herein.

[055] The polypeptide linker may comprise more than one occurrence of the polypeptide sequences defined, for example (Polypeptide Linker Sequence^ where n can be any number. Typically n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15.

[056] In one embodiment the luminescence-activating units are lanthanide-activating units. In one embodiment the lanthanide-activating units are BHHTEGST.

[057] In other embodiments the luminescence-activating units are selected from BHHST, BHHCT, BPPCT, BTBCT, BHHBCB, BPPBCB, or BTBBCB as their lanthanide complexes.

[058] In a third aspect there is provided a process for preparing a detectable biomolecular complex for binding to a target, the process comprising binding the biomolecular complex according to the first or second aspects to an antibody or antibody fragment.

[059] In a fourth aspect there is provided a method for determining the presence or identity of a target entity, the method comprising:

contacting a sample with a biomolecular complex having a lanthanide-activating unit, wherein the antibody-binding unit of the complex is capable of binding to the target entity, and wherein the contacting is performed under conditions such that if the target entity is present the antibody-binding unit binds to the target entity;

adding a lanthanide ion to the sample to such that the lanthanide ion chelates to the lanthanide-activating unit to form a luminescent bioconjugate;

exciting the luminescent bioconjugate at a selected wavelength:

detecting luminescence from the luminescent bioconjugate thereby determining the presence or identity of the target entity.

[060] In a fifth aspect there is provided use of a biomolecular complex having one or more luminescence-activating units in the detection of a target cell or tissue.

[061] The biomolecular complex is particularly suitable for direct labelling of antibodies. Other applications beside direct labelling of antibodies include bioimaging, histochemistry, flow cytometry, biosensing, rapid pathogen detection, nanomaterials functionalisation, cancer detection in body fluids, and pathogen detection in environmental samples [062] The current technology has widespread applications across various fields of research. It is compatible with numerous technologies that have been developed or are currently under development such as time-gated (TG) Orthogonal Scanning Automated Microscopy (OSAM), TG microscopy, TG flow cytometry, and upconversion nanoparticle functionalisation. This platform technology has the potential to expedite the development and application of these technologies for diagnostics, bioimaging and theranostics.

Definitions

[063] Throughout this specification, unless the context clearly requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[064] Throughout this specification, the term 'consisting of means consisting only of.

[065] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification.

[066] Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

[067] In the context of the present specification the terms 'a' and 'an' are used to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, reference to 'an element' means one element, or more than one element.

[068] In the context of the present specification the term 'about' means that reference to a figure or value is not to be taken as an absolute figure or value, but includes margins of variation above or below the figure or value in line with what a skilled person would understand according to the art, including within typical margins of error or instrument limitation. In other words, use of the term 'about' is understood to refer to a range or approximation that a person or skilled in the art would consider to be equivalent to a recited value in the context of achieving the same function or result.

[069] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. For the avoidance of doubt, the invention also includes all of the steps, features, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features and compounds.

[070] The term 'alkyl' as used herein means a group or part of a group and refers to a straight or branched aliphatic hydrocarbon group having 1 -6 carbon atoms, or 1 -4 carbon atoms, or 1 -3 carbon atoms, or 1-2 carbon atoms. Thus, the term alkyl includes, but is not limited to, methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1 ,2-dimethylpropyl,

1 ,1 -dimethylpropyl, pentyl, isopentyl, hexyl, and the like. The group may be a terminal group or a bridging (alkylene) group.

[071] The term 'alkyne' as used herein means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched and may have from 2-6 carbon atoms or 2-4 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl. In some embodiments the alkyne is a terminal group, eg, -C≡CH.

[072] The term 'acyl' as used herein refers to an alkyl-C(O)- group in which the term alkyl is as defined herein. The alkyl group may be a Ci-C₆ alkyl, C₁-C4 alkyl, or C C₃ alkyl group. Thus, acyl includes, but is not limited to, acetyl, propanoyl, and the like.

[073] The terms 'halogen' and 'halo' as used herein are synonymous and refer to fluorine, chlorine, bromine or iodine.

[074] In order that the present technology may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief Description of the Figures

[075] Figure 1. Illustration showing the luminescent labelling of cells using LA-PG- and LA- LPG coupled antibodies. Coupling of PG (A) and LPG (C) to an antibody results in a non- luminescent "PG or LPG + Antibody" complex that cannot impart luminescence to a target cell. Coupling of LA-PG (B) or LA-LPG (D) to an antibody results in a luminescent "LA-PG or LA- LPG + Antibody" complex that can be used to label a target cell with luminescence. The stars represent an unspecific number of BHHTEGST chelates (or ligands) incorporated onto the LA- PG (B) or LA-LPG (D) biomolecule. [076] Figure 2. A sample of Cryptosporidium oocysts was spiked with autofluorescent Synechococcus cells and then labelled with a "LA-LPG_MID + CRY104-FITC" complex (a) FITC channel (b) TGL microscopy.

[077] Figure 3. Scattering flow cytometry counting results of luminescence signal intensities of gated hAdMSC populations. Density maps showing shift in cell populations (A) Unlabelled control (no pre-incubation with LA-LPG_MID + anti-CD271 ) hAdMSC cells. (B) Labelled (pre- incubated with LA-LPG_MID +anti-CD271 ) hAdMSC cells.

[078] Figure 4. SDS-PAGE analysis of the formation of "LPG + FND" complexes. S - starting LPG sample ; U - unlabelled LPG; CF - "LPG + FND" complex.

[079] Figure 5. Fluorescent emission from FITC-conjugated antibodies (Ab-FITC) coupled to Nanorubies (NR) without (no peak) and with (high peak) LPG under 488 nm excitation.

Detailed Description

[080] The present technology broadly relates to the biomolecules suitable for use in detection of biological target entities.

[081] In a form, LPG is a genetically engineered biomolecule that contains two distinct functional regions; (a) a polypeptide linker and (b) and Streptococcus protein G, which has specific binding affinity towards antibodies. The polypeptide linker of LPG presents itself as a prospective lanthanide carrier molecule. It contains a number of accessible lysine residues that provide binding sites for succinimide-activated chelates (such as BHHTEGST), allowing the addition of multiple luminophores without affecting the antibody-binding function of the protein G region.

[082] This lysine-rich region of the polypeptide linker was exploited to produce a

Luminescence-Activating Linker Protein G (LA-LPG). LA-LPG is a purpose-functionalised biomolecule that has the unique property of mediating the attachment of luminescent chelates to antibodies. LA-LPG mediated luminescent lanthanide attachment does not affect the antibody's biological function (e.g., reduce antibody specificity or avidity). As such, LA-LPG represents the simplest and fastest tool to impart luminescence to antibodies in seconds and without the need for conventional chemical conjugation.

[083] The succinimide-activated chelate BHHTEGST referred to herein is a newly developed tetradentate β-diketone-europium chelate derived from BHHST and BHHCT. It has an improved aqueous solubility, excellent luminescent output and a mild succinimide reactive group that facilitates in a controllable manner covalent attachment to biomolecules that contain lysine residues. BHHTEGST also possesses an extended tetraethylene glycol tether that projects its hydrophobic luminescent moiety (BHHCT) away from the modified protein to prevent adverse interactions and enhance conjugate solubility.

Advantages of Technology

[084] The present invention may have one or more of the following advantages:

[085] High Specificity. No effect on the antibody's biological function; e.g., no reduction in specificity or avidity.

[086] Fast labelling. Less than 1 min, without the complications of direct or indirect streptavidin/biotin antibody labelling.

[087] Ultrasensitive Gated Auto-synchronous Luminescence Detector (GALD) detection. Sensitivity of 10^~12 - 10^~15 M can be achieved with lanthanide chelates in combination with , exceeding sensitivity achievable with conventional fluorophores.

[088] Reproducibility. In contrast to conventional methods, e.g. the biotin / streptavidin pair; LA-LPG is reproducible.

[089] Long life. LA-LPG can be freeze-dried (lyophilised) for potentially extended shelf-life.

[090] Wide range of applications. Any antibody able to afford an attachment to PG for application in immunodetection, biomarker/biotracking, cells/organisms labelling and detection, bioimaging and flow cytometry.

Antibody-Binding Protein

[091] The antibody-binding protein may be selected from the group comprising protein A, protein A/G, protein G, protein L, and any combination thereof.

[092] In one embodiment the antibody-binding protein is Streptococcus protein G.

Polypeptide Linker

[093] The polypeptide linker is capable of receiving a luminescence-activating unit so that the biomolecular complex can be detectable when bound to a target.

[094] In an embodiment the polypeptide linker comprises at least 20 amino acids. [095] In an embodiment the polypeptide linker comprises one or more amino acids comprising a side group having a functional group selected from NH₂, SH, OH.

[096] In an embodiment the polypeptide linker comprises one or more lysine residues.

[097] In one embodiment the polypeptide linker comprises the sequence of SEQ 1 - (VKTQATSREEPPRLPSKHRPG) (SEQ ID NO 1 ).

[098] In one embodiment the polypeptide linker comprises the sequence of SEQ 2 - (VKTQATSREEPPRLPSKHRPG)4VKTQATS (SEQ ID NO 2).

[099] In one embodiment the polypeptide linker comprises the sequence of NL1 - (GKSSGSSKGSPPKGPSKHKGP)4 (SEQ ID NO 3).

[0100] In one embodiment the polypeptide linker comprises the sequence of NL2 - (GKSQGQSKGGPPKGPSKHKGP)4 (SEQ ID NO 4).

[0101] In one embodiment the polypeptide linker comprises the sequence of CON - (QTVTHRGRHEGKAPKGPELHRP)4 (SEQ ID NO 5).

[0102] In one embodiment the polypeptide linker comprises combinations of the linker sequences such as:

CON - (NL1 )₃ - QTVTHRGRHEGKAPKGPELHRP (GKSSGSSKGSPPKGPSKHKGP)₃ (SEQ ID NO 6);

(CON)2 - (NL1 )₂ - (QTVTHRGRHEGKAPKGPELHRP)₂ (GKSSGSSKGSPPKGPSKHKGP)₂ (SEQ ID NO 7);

(CON)3 - NL1 - (QTVTHRGRHEGKAPKGPELHRP)s GKSSGSSKGSPPKGPSKHKGP (SEQ ID NO 8);

CON - (NL2)₃ - QTVTHRGRHEGKAPKGPELHRP (GKSQGQSKGGPPKGPSKHKGP)₃ (SEQ ID NO 9);

(CON)₂ - (NL2)₂ - (QTVTHRGRHEGKAPKGPELHRP)₂

(GKSQGQSKGGPPKGPSKHKGP)₂ (SEQ ID NO 10);

(CON)₃ - Nl_2 - (QTVTHRGRHEGKAPKGPELHRP)3 GKSQGQSKGGPPKGPSKHKGP (SEQ ID NO 1 1 );

SEQ 1 - (NL1 )₃ - QTQATSREEPPRLPSKHRPG (GKSSGSSKGSPPKGPSKHKGP)3; (SEQ ID NO 12)

(SEQ 1 )₂ - (NL1 )₂ - (QTQATSREEPPRLPSKHRPG)₂ (GKSSGSSKGSPPKGPSKHKGP)₂ (SEQ ID NO 13);

(SEQ 1 )₃ - NL1 - (QTQATSREEPPRLPSKHRPG)s GKSSGSSKGSPPKGPSKHKGP (SEQ ID NO 14); SEQ 1 - (NL2)₃ - QTQATSREEPPRLPSKHRPG (GKSQGQSKGGPPKGPSKHKGP)₃ (SEQ ID NO 15);

(SEQ 1)₂ - (NL2)₂ - (QTQATSREEPPRLPSKHRPG)₂ (GKSQGQSKGGPPKGPSKHKGP)₂ (SEQ ID NO 16);

(SEQ 1)₃ - NL2 - (QTQATSREEPPRLPSKHRPG)₃ GKSQGQSKGGPPKGPSKHKGP (SEQ ID NO 17).

[0103] It will be appreciated that other combinations are contemplated.

[0104] The polypeptide linker may comprise more than one occurrence of the polypeptide sequence, for example (Polypeptide Linker Sequence),, where n can be any number. Typically n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15.

Luminescence-Activating Unit

[0105] The luminescence-activating unit can include one or more of conventional or lanthanide- based fluorophores. Examples include but not limited to BHHST, BHHCT, BPPCT, BTBCT, BHHBCB, BPPBCB, BTBBCB as their lanthanide complexes. Further, conventional

fluorophores can be used and examples include: all members of the Alexa Fluor family, all members of the BODIPY family, all members of the Cyanine Dye "Cy dyes" family (eg Cy3, Cy5, Cy7), all members of the Quasar dye family. Full description of BHHBCB, BPPBCB, BTBBCB can be found in L. Zhang, Y. Wang, Z. Ye, J. Dayong, J. Yuan, Bioconj. Chem. 2012, 23, 1244-1251 .

[0106] Other examples include 1 ,2-Bis[4'-(1 ",1 ",1 ",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"- yl)-benzyl]-4-chlorosulfobenzene (BHHBCB);and 4,4"-Bis(4,4,5,5,6,6,6-heptafluoro-1 ,3- dioxohexyl)-o-terphenyl-4'-sulfonyl chloride (BHHCT).

[0107] In one embodiment the luminescence-activating unit is a lanthanide-activating unit.

[0108] In one embodiment the luminescence-activating unit comprises a tetradentate ligand moiety capable of chelating a lanthanide ion (eg, Eu³⁺ or Tb³⁺), and an antenna moiety capable of sensitising lanthanide emission when a lanthanide ion is chelated to the tetradentate ligand moiety. When a lanthanide ion is chelated to the tetradentate ligand moiety the luminescence- activating unit may be referred to herein as a 'luminophore'.

[0109] Advantageously, when a lanthanide ion (eg, Eu³⁺ or Tb³⁺) is chelated to the tetradentate ligand moiety of the luminescence-activating unit, the antenna unit can sensitise lanthanide emission which renders target entities highly visible under TGL conditions.

[0110] In one embodiment the luminescence-activating unit is a compound of general formula (I), or a salt, hydrate or tautomer thereof:

(I)

wherein

A is a reactive functional group selected from tosylate,

mesylate, -halo, -N₃, -NH₂, -CN, N-succinimidyl carbonate, isocyanate,

isothiocyanate, sulfonyl

chloride, -C(0)N₃, -C(0)halo, -C0₂H, -C(0)0(d_₆ alkyl)-R^a, -C(0)NH(C₁.₆ alkyl)-R^a, - NHC(0)(d_₆ alkyl)-R^a, -C(NH)0(d_₆ alkyl)-R^a, aziridine, maleimide, C₂-₄ alkyne; wherein each R^a is independently selected from H, OH, halo, C0₂H,

C0₂(d_₆ alkyl);

n is an integer from 1 -20 inclusive; and

each R¹ is independently an electron-withdrawing group.

[0111] Reactive functional group 'A' facilitates covalent attachment of the luminescent activating unit to the antibody-binding unit of the biomolecular complexes by reacting with functional groups on the polypeptide linker and antibody-binding protein.

[0112] In an embodiment A is selected from tosylate, mesylate, halo, N₃, NH₂,

isocyanate, -NHC(0)(C₁_₄ alkyl)-R^a, -C(NH)0(d_₄ alkyl)-R^a, maleimide, and

N-hydroxysuccinimidyl carbonate. In other embodiments A is selected from N₃, halo, isocyanate, haloacetamide and N-hydroxysuccinimidyl carbonate. In an embodiment A is N-hydroxysuccinimidyl carbonate. In another embodiment A is maleimide. In a further embodiment A is iodoacetamide. In another embodiment A is iodo or bromo.

[0113] In some embodiments R^a is selected from chloro, bromo, iodo, C0₂H, C(0)0(d_₄ a In some embodiments R^a is selected from bromo, iodo, C0₂H, C(0)0(Ci_₄ alkyl).

[0114] In some embodiments each R¹ is identical. In alternative embodiments each R¹ is different. In some embodiments each R¹ is independently selected from d_₆ haloalkyl, for example, d-6 fluoroalkyl. In one or more embodiments each R¹ is selected from

Ci-C₃ fluoroalkyl. In other [Bell ]embodiments each R¹ is selected from CF₃ and C₃F₇. In particularly some embodiments each R¹ is C₃F₇. [0115] In some embodiments n is an integer from 1 to 19, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 2 to 12, or 2 to 10, or 2 to 8, or 2 to 6 or 2 to 4, or 1 1 to 20, inclusive. In various some embodiments n is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and 20. In some embodiments n is 2, 3, 4, 5, 6 or 7. In other some embodiments n is 3, 4 or 5. In an embodiment n is 3.

[0116] The compound of formula (I) comprises a functionalised terphenyl antenna unit and a hydrophilic linker moiety. The terphenyl antenna unit is capable of chelating a lanthanide ion (via the functionalised keto-enol groups) and sensitising lanthanide emission.

[0117] The hydrophilic linker moiety of the luminescence-activating unit comprises a functionalised (poly)ethylene glycol in which the functional group 'A' facilitates attachment of the (poly)ethylene glycol linker to the antibody-binding unit to form the biomolecular complex. The linker may be functionalised with a functional group 'A' specifically chosen to react with one or more (preferably a plurality of) functional group(s) present on the polypeptide linker and/or antibody-binding protein of the antibody-binding unit. Thus, in various embodiments the functional group 'A' may comprise or consist of a leaving group capable of being displaced by a nucleophilic residue on the antibody-binding unit to form a covalent bond. In other

embodiments, 'A' may comprise a functional group capable of reacting with a reactive moiety on the antibody-binding unit to covalently attach the luminescence-activating unit to the antibody- binding unit. In some embodiments, at least one luminescence-activating unit is covalently attached to the polypeptide linker of the antibody-binding unit.

[0118] Numerous examples of reactive functional groups 'A' may be introduced onto the (poly)ethylene linker using techniques known to those skilled in the art. For example, in some embodiments 'A' comprises or consists of a leaving group. In other embodiments 'A' comprises or consists of a reactive moiety capable of being chemically transformed into a different reactive moiety. By way of illustration, a hydroxyl group may be replaced with a halide (eg, chloro, bromo, iodo) upon treatment with an appropriate haloacid, eg, hydrochloric acid, hydrobromic acid, hydroiodic acid, or with thionyl chloride or phosphorous tribromide. Alternatively, a hydroxyl group may be converted into a leaving group (eg, a tosylate or mesylate) which may then be displaced by a nucleophile such as an azide, cyanide or halide, etc, in a nucleophilic substitution reaction. Numerous other transformations can be envisaged and those skilled in the art will be familiar with such transformations. For example, a halide residue may be displaced by a maleimide residue or azide residue, among others. An azide residue advantageously offers access to 'click chemistry' protocols. Alternatively, an azide residue may be reduced to give an amino residue, which may be subsequently converted into numerous other functional groups such as amides, isocyanates, among others. In one embodiment, an amine residue may be reacted with a dicarboxylic acid or anhydride (eg, succinic anhydride) to give the corresponding carboxylic acid functionalised alkylamide. In other embodiments, A is a carboxylic acid group which may be converted into an acyl azide using methods known in the field (eg, see Kim et al. Synlett, 2008, 2072-2074). Routine reactions and conditions for achieving such transformations are within the general knowledge and capabilities of those skilled in the art and are described, for example, in March, J. Advanced Organic Chemistry, 4^th Ed (John Wiley & Sons, New York, 1992) and Vogel's Textbook of Practical Organic Chemistry, 5^th Ed (John Wiley & Sons, New York, 1989).

[0119] Non-limiting examples of some luminescence-activating units of formula (I) include:

[0120] In one embodiment the luminescence-activating unit is a compound of formula (I) having the following structure:

or a salt, hydrate or tautomer thereof. The above compound may be referred to herein as BHHTEGST or Compound (1 ).

[0121] In another embodiment the luminescence-activating unit is a compound of general formula (II)

(II)

wherein n and R¹ are as defined for formula (I), including some of the embodiments.

[0122] The compound of formula (II) may be used as a versatile starting precursor to compounds of formula (I), including compounds of formulae (la) to (li).

[0123] For example, compounds of formula (la) may be prepared from the compound of formula (II) by converting the hydroxyl group on the (poly)ethylene glycol linker to a leaving group (eg, a tosylate or mesylate, etc), followed by treatment with the desired haloacid (eg, hydrochloric acid, hydrobromic acid, hydroiodic acid). In other embodiments compounds of formula (lb) may be prepared from a compound of formula (la) by treatment of the compound of formula (la) with maleimide in a nucleophilic substitution reaction. In other embodiments compounds of formula (lc) may be prepared from the compound of formula (II) by converting the hydroxyl to a leaving group (eg, tosylate, mesylate, etc) followed by treatment with an azide salt (eg, sodium azide). In other embodiments compounds of formula (Id) may be prepared from compounds of formula (lc) by reduction of the azide residue. Suitable reducing agents are well known to those skilled in the art and include sodium borohydride, lithium aluminium hydride, hydrogenation in the presence of Pd/C catalyst, zinc/NH₄OH, among others. In other embodiments compounds of formula (le) may be prepared from a compound of formula (Id) by treatment of the amino compound with the relevant halocarboxylic acid (eg, chloroacetic acid, bromoacetic acid, iodoacetic acid). In other embodiments compounds of formula (If) may be prepared by reacting compounds of formula (Id) with phosgene (COCI₂). In other embodiments compounds of formula (Ig) may be prepared starting from a compound of formula (II) by conversion of the hydroxyl group on the (poly)ethylene glycol linker to a leaving group (eg, tosylate, mesylate, etc) followed by displacement of the leaving group by a cyano group, eg, by treatment with a cyanide salt (eg, NaCN, KCN, LiCN) in a nucleophilic substitution reaction, followed by reaction of the resultant nitrile with the desired Ci_₆alkyl alcohol. In other embodiments compounds of formula (Ih) may be prepared by treating a compound of formula (Id) with a dicarboxylic acid (eg, malonic acid, succinic acid, adipic acid, etc) or anhydride (eg, succinic anhydride). In other embodiments compounds of formula (li) may be prepared by reacting the compound of formula (II) with Ν,Ν-disuccinimidyl carbonate.

[0124] Suitable reaction conditions (including solvents, temperatures and reagents) for the foregoing transformations are well known to those skilled in the art. Typical solvents that may be utilised include acetonitrile, tetrahydrofuran, toluene, dichloromethane, acetone, water, alcohols (eg, methanol, ethanol, 1 -propanol, isopropanol, etc) and mixtures thereof, and can be selected to suit the particular reaction being performed.

[0125] A compound of formula (I) can be complexed with a lanthanide ion to form a lanthanide ion chelate. In some examples the lanthanide ion is Eu³⁺ or Tb³⁺.

[0126] A process for preparing luminescence-activating units of general formula (I) is illustrated in general schemes A-C below. In the embodiment shown, the initial step is the formation of a compound of general formula (II), which may then be converted into a compound of general formula (I).

Scheme A

[0127] The first step of the process illustrated in Scheme A comprises acylation of terphenyl (a) to give the diacylated compound (b). The acylation reaction may be performed using standard electrophilic substitution conditions, eg, between o-terphenyl and acetyl chloride in the presence of anhydrous aluminium chloride in dry solvent (eg, dichloromethane) at a

temperature in the range -5 to 10°C, typically 0°C, for about 1-2 hours. The next step involves a claisen condensation reaction of the acylated compound (b) with an alkyl ester functionalised with an electron-withdrawing group in the presence of a base (eg, lithium, sodium or potassium alkoxide, such as methoxide, ethoxide, tert-butoxide, etc) in a suitable solvent (eg,

tetrahydrofuran, acetonitrile) at a temperature of about 20-40°C (typically room temperature) to give the di(keto-enol) compound (c). The terphenyl moiety is then functionalised by treatment with chlorosulfonic acid (eg, at room temperature for about 1 -6 hours, typically about 2-4 hours) to give the chlorosulfonate compound (d).

[0128] The (poly)ethylene glycol linker compound may be prepared as illustrated in general Scheme B.

n = 1-19 R = NH₂

Scheme B. i. base, TsCI or MsCI ii. azide salt iii. reduction

[0129] The first step of the process illustrated in Scheme B is to select a (poly)ethylene glycol of suitable length (eg, wherein the number of 0-CH₂CH₂ units is between 1 -20, tetraethylene glycol, hexaethyleneglycol, nonaethyleneglycol, dodecaethyleneglycol, and the like) after which a terminal hydroxyl group is converted to a suitable leaving group, eg, a tosylate or mesylate by reacting with a tosylhalide or mesylhalide (eg, tosylchloride or mesylchloride), followed by conversion of the leaving group to an azide by treatment with an azide salt (eg, sodium azide), in a nucleophilic substitution reaction. The azide may then be reduced, for example by catalytic hydrogenation, or catalytic transfer hydrogenation using a Pd/Catalyst to give the corresponding amino-substituted linker compound.

[0130] The next step involves reacting the amino-substituted linker compound with the chlorosulfonate compound (d) in the presence of a base (eg, dimethylaminopyridine, triethylamine, DBU, etc) in a suitable solvent (eg, acetonitrile, THF) at a suitable temperature (eg, 20-50°C, typically room temperature) for about 10 minutes to about 2 hours to give a compound of formula (II) as illustrated in Scheme C.

Scheme C

[0131] The compound of formula (II) can be converted into compounds of formula (I) by reaction with suitable reagents to replace the terminal hydroxyl group on the (poly)ethylene glycol moiety with a functional group A.

[0132] Suitable reagents and reaction conditions for performing the above reactions are known to the skilled person and are described in the literature and text books, including for example March, J. Advanced Organic Chemistry, 4^th Ed (John Wiley & Sons, New York, 1992) and Vogel's Textbook of Practical Organic Chemistry, 5^th Ed (John Wiley & Sons, New York, 1989).

[0133] Compounds of formulae (I), (II) and (III), as well as their lanthanide chelates, may be purified using standard techniques known to those skilled in the art, including chromatography (eg, flash chromatography, HPLC), crystallization, recrystallization, lyophilisation, and the like. After purification the compounds of formulae (I), (II) and (III), as well as their lanthanide chelates may be isolated in substantially pure form. For example, the compounds and chelates may be isolated in a form which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% pure.

[0134] The following is a detailed description of a luminescence-activating unit that can be used for the present invention.

[0135] An example of formula (I) is the compound BHHTEGST having the structure shown below:

BHHTEGST or Compound (1 )

[0136] BHHTEGST is a tetradentate-bis(keto-enol) ligand which functions as a sensitising antenna molecule for the excitation of a lanthanide ion chelated to the ligand. The lanthanide ion can be a trivalent europium cation (Eu³⁺). Those skilled in the art will recognise that the keto-enol moiety is a tautomer of the corresponding diketone. Advantageously, the compound of formula (I) has electron-withdrawing groups that flank each diketo moiety and help to promote enol formation, which facilitates strong binding of the lanthanide ion. In some examples the electron-withdrawing groups are heptafluoropropyl groups. The tetraethyleneglycol linker advantageously enhances aqueous solubility. BHHTEGST comprises an N-hydroxysuccinimide carbonate as an activated leaving group which facilitates the substitution reaction with functional residues on the polypeptide linker and antibody-binding protein, to covalently attach the luminescence-activating unit to the linker and/or antibody-binding protein.

[0137] In one embodiment the luminescence-activating unit includes a fluorescent nanoparticle. Examples of suitable fluorescent nanoparticles include nanodiamonds (NDs), nanorubies (NRs), silica-coated NRs, plasmonic nanoparticles (including gold, silver, and platinum), semiconductor nanocrystals (e.g., CdSe, CdS, ZnS,Ti02 and PbS), magnetic compounds (e.g., Fe₃0₄, CoFe₂0₄, CoPt), carbon nanoparticles, quantum dots (QDs), rare earth doped nanoparticles, and their combinations. These particles can be negatively charged by adding anionic groups to their surface or by incorporating an outer silica shell. [0138] The technology can be used for imaging and sensing, in vitro and in vivo labelling in cells, tissues, and organisms. Application in biology and medicine includes proteomic and genomic studies, disease diagnostics, pharmaceutical screening, drug delivery, protein purification, medical imaging (sensing in cancer research and selective tumor targeting), several detection techniques such as fluorescence, fluorescence resonance energy transfer (FRET) and multiphoton microscopy.

Synthesis of BHHTEGST

[0139] The synthesis of BHHTEGST 1 initially commences with the preparation of the compound of formula 2 according to the method of J. L. Yuan, K. Matsumoto, H. Kimura, Anal. Chem. 1998, 70, 596-601 . The literature procedure can be modified to improve the yield and purity in each step of the synthesis. BHHTEGST 1 synthesis was initiated by preparation of 4,4'-diacetyl-o-terphenyl 4 using standard electrophilic substitution conditions between o- terphenyl and acetyl chloride in the presence of anhydrous aluminium chloride in dry dichloromethane (Scheme 1 ). This reaction can be optimized by addition of a solution of o- terphenyl 3 in dichloromethane in a drop-wise fashion to a mixture of anhydrous aluminium chloride and acetyl chloride in dry dichloromethane at 0 °C to give 4. Claisen condensation of 4 was performed by using sodium methoxide in dry tetrahydrofuran and the crude product was converted to compound 5 upon the addition of absolute ethanol. Compound 2 was synthesized from compound 5 by treatment with chlorosulfonic acid.

Scheme 1 : i. AICI₃, acetyl chloride, dry CH₂CI₂. ii. ethyl heptafluorobutyrate, NaOMe, THF. iii. CIS0₃H.

[0140] The amine-substituted tetraethylene glycol (TEG) linker was synthesized via mono- tosylation of tetraethylene glycol. The literature procedure of B. C. Sanders, et al., J. Am.

Chem. Soc. 2011 , 133, 949-957 can be improved by grinding of sodium hydroxide under anhydrous conditions, followed by rapid addition to the reaction mixture. The tosyl group was then converted to an amine via the azide to afford the amino terminated linker 6 using the method of M. J. Hynes, et al., Angew. Chem. Int. Ed. 2012, 51, 2151 -2154. (Scheme 2).

Scheme 2: i. NaOH, THF, p-TsCI. ii. NaN₃, EtOH, 70°C. iii. Pd/C, EtOH.

[0141] The next step is the formation of the precursor compound 7, which is a compound of general formula (II), followed by the conversion of compound 7 to compound 1 (BHHTEGST) as illustrated in general Scheme 3.

Scheme 3: i. DMAP, TEA, CH₃CN. ii. DSC, DMAP, TEA, MeCN.

[0142] Advantageously, the yield of the immediate precursor to BHHTEGST, compound 7, was improved by addition of compound 2 in a drop-wise fashion to a solution of 6,

dimethylaminopyridine (DMAP), triethylamine ( TEA) in dry acetonitrile (Scheme 3). The final stage of BHHTEGST 1 synthesis was performed by reacting compound 7 with

Λ/,/V-disuccinimidyl carbonate (DSC), DMAP, TEA in dry acetonitrile. The crude reaction mixture can be purified using standard techniques (eg, flash chromatography, optionally followed by preparative HPLC and optionally lyophilized) to give purified BHHTEGST 1. The above synthesis advantageously enables useful (eg, multi-milligram) quantities of compounds of formula (II) and compounds of formula (I), eg, BHHTEGST, to be prepared using readily available standard techniques.

[0143] Lanthanide chelates (eg, Eu³⁺, Tb³⁺) of compounds of formula (I), including BHHTEGST 1 , are intensely luminescent and easily synthesized using the methods disclosed herein. Those skilled in the art will recognise that the process illustrated above for the synthesis of

BHHTEGST may be readily adapted to prepare other compounds of formula (I) having alternative electron withdrawing groups (eg, ^-ΰ₆ fluoroalkyl), alternative linker groups (eg, - [(CH₂CH₂0)]_n where n is 1 -20) and alternative functional groups TV, using reactions, techniques, reagents and solvents known in the field. Compounds of general formula (II) are useful and versatile precursors for the synthesis of compounds of formula (I).

Synthesis of the Biomolecular Complex

[0144] The biomolecular complex may be prepared by attaching one or more, preferably two or more, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, luminescence-activating units to the complex. A plurality of luminescence-activating units can be attached to the polypeptide linker, the antibody-binding protein or both the polypeptide linker and the antibody-binding protein.

[0145] Polypeptide linkers found to be particularly suitable were SEQ 1 , SEQ 2, NL1 , NL2,

CON, CON - (NL1 )₃, (CON)₂ - (NL1 )_2l (CON)₃ - NL1 , CON - (NL2)₃, (CON)₂ - (NL2)₂, (CON)₃ - NL2, SEQ 1 - (NL1)₃, (SEQ 1 )₂ - (NL1)_2l (SEQ 1)₃ - NL1 , SEQ 1 - (NL2)₃,

(SEQ 1)₂ - (NL2)_2l or (SEQ 1)₃ - NL2 (SEQ ID NOS 1 to 17) as defined herein.

[0146] The polypeptide linker may comprise more than one occurrence of the polypeptide sequence, for example (Polypeptide Linker Sequence),, where n can be any number. Typically n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15.

[0147] The biomolecular complex can include a plurality of luminescence-activating units covalently attached to the polypeptide linker and/or the antibody-binding protein. In

embodiments the polypeptide linker and/or antibody-binding protein comprise one or more reactive functional groups capable of forming a covalent bond by reacting with or displacing functional group 'A' on the luminescence-activating unit. Suitable reactive functional groups include, for example, amino, hydroxyl, thiol, and the like. In some embodiments, the polypeptide linker comprises one or more (preferably a plurality) of lysine residues having an amino side group capable of forming a covalent bond by reacting with or displacing functional group 'A' on the luminescence-activating unit. In other some embodiments the antibody-binding protein has one or more amino groups capable of forming a covalent bond by reacting with or displacing functional group 'A' on the luminescence-activating unit.

[0148] In some embodiments the luminescence-activating unit is BHHTEGST. BHHTEGST has suitable aqueous solubility, excellent luminescent output and a mild succinimide reactive group that facilitates covalent attachment to biomolecules (such as the antibody-binding unit) containing lysine residues in a controllable manner. BHHTEGST also advantageously possesses an extended tetraethylene glycol tether that projects the hydrophobic lanthnide ligand antenna unit away from the antibody-binding protein of the antibody-binding unit to prevent or minimise adverse interactions, and enhance solubility of the biomolecular complex. In some embodiments one or more (preferably two or more, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25) BHHTEGST molecules are attached to the polypeptide linker and/or antibody- binding protein by a reaction between the succinimidyl carbonate of BHHTEGST and amino residues on the polypeptide linker and/or antibody-binding protein to form amide bonds. Two or more BHHTEGST molecules are attached to the amino side group of lysine residues in the polypeptide linker by the formation of amide bonds.

[0149] The di(keto-enol)-substituted terphenyl antenna moiety functions as a tetradentate ligand capable of chelating a lanthanide ion. Accordingly, in another aspect the invention relates to a lanthanide ion chelate comprising a lanthanide ion complexed to the compound of formula (I) or the biomolecular complex. In one or more examples the lanthanide ion is Eu³⁺ or Tb³⁺. [0150] Labelled nanoparticles can be purchased commercially or synthesised in the laboratory. It will be appreciated by persons skilled in the art that several procedures and techniques are available in the literature. Examples include but not limited to: Edmonds AM, Sobhan MA, Sreenivasan VKA, Grebenik EA, Rabeau JR, Goldys EwaM, Zvyagin AV (2013) Nano-Ruby: A Promising Fluorescent Probe for Background-Free Cellular Imaging. Part. Part. Syst. Charact. 30: 1521 -41 17; Say JM, van Vreden C, Reilly DJ, Brown LJ, Rabeau JR, King NJC (201 1 ) Luminescent nanodiamonds for biomedical applications. Biophysical Reviews 3:171 -184;

Geiselmann M, Juan ML, Renger J, Say JM, Brown LJ, de Abajo FJG, Koppens F, Quidant R (2013) Three-dimensional optical manipulation of a single electron spin. Nature nanotechnology 8:175-179; Lu X, Rycenga M, Skrabalak SE, Wiley B, Xia Y (2009) Chemical synthesis of novel plasmonic nanoparticles. Annu. Rev. Phys. Chem. 60:167-192; and Reiss P, Carriere M Lincheneau C Vaure L Tamang S (2016) Synthesis of Semiconductor Nanocrystals, Focusing on Nontoxic and Earth-Abundant Materials. Chem Rev. 2016 (DOI:

10.1021/acs.chemrev.6b001 16).

[0151] The present invention is illustrated by the following examples, which do not limit the scope of the invention in any way. It is to be understood that the particular examples, materials, amounts and procedures are representative and are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Use of the Biomolecular Complex

[0152] For use, an antibody or binding fragment thereof directed to a desired biological target is attached to the biomolecular complex via the antibody-binding protein.

Detection of biological target entities

EXAMPLES

Materials and instruments

[0153] General chemicals for organic synthesis were the highest grade available supplied by Sigma Aldrich Australia and they were used without further purification. Modified short DNAs were purchased from GeneWorks & Sigma Aldrich Australia. Printed microscope slides (G352104-W Teflon® printed slides, 21 wells, 4mm, white) supplied by ProSciTech Pty Ltd. Reverse phase (RP) HPLC was performed using a Shimadzu apparatus consisting of a DGV- 12A degasser, SIL-10AD auto injector, SPD-M10A tuneable absorbance detector. Preparative RP-HPLC (BHHTEGST purification) was performed using Econosil C18 column (10 μηι, 22 mm ID, 250 mm) with a flow rate of 9.0 mL/min. General procedures

[0154] Unless specified otherwise, all reactions were performed under an inert atmosphere of nitrogen with dry solvents under anhydrous conditions and monitored by TLC using aluminium plates coated with Merck Silica Gel 60 F₂₅₄, visualised using either UV light (254 or 365 nm) or a europium staining reagent [eg, a solution of EuCI₃ 1 .0 mg in Milli Q (MQ) water (100 mL)]. Ratios of solvent systems for TLC and column chromatography are expressed in v/v as specified. NMR spectra were recorded on 400 MHz Bruker Avance spectrometer. The solvent ¹ H and ¹³C signals, δ_Η 7.26 for residual CHCI₃ and 5_C 77.0 for CDCI₃ were used as internal references. Flash chromatography was performed on silica gel (Merck silica gel 60, 40-63 μιη).

Synthesis of BHHTEGST

1 .1 Preparation of 4,4'-Diacetyl-o-terphenyl

[0155] A solution of o-terphenyl (23 g, 100 mmol) in dry dichloromethane (20 mL) was added in a drop wise fashion, via use of a syringe, to a mixture of anhydrous aluminium chloride (30 g, 225 mmol) and acetyl chloride (20 g, 254 mmol) in dry dichloromethane (200 mL) at 0°C over a period of 1 h. The reaction mixture was stirred for 1 h at 0 °C, then warmed up to room temperature and stirring was continued overnight, followed by 2 h reflux. After cooling, the reaction mixture was slowly added into a separating funnel containing ice-hydrochloric acid solution [ice (200 g) and hydrochloric acid (3 M, 100 mL)]. The aqueous layer was extracted with dichloromethane (2 x 100 mL) and the combined organic layers were washed with saturated brine (1 x 100 mL), dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo to give crude product (30.0 g, > 97%). TLC analysis of crude product (hexane/ethyl acetate, 4/1 ) indicated the presence of the product 4,4'-diacetyl-o-terphenyl (R_f = 0.3) as a major spot with minority of side product/s (R_f = 0.6) with no presence of starting material o- terphenyl (R_f = 0.85). The crude product was then dissolved in butanone by slightly heating (240 mL, about 8-10 mL per gram of crude product) then left at room temperature over night to give yellow crystals (23 g, 75%). Alternatively, silica gel column purification using

CH₂CI₂/hexane (1 : 1 to 4: 1 ) was used to purify the crude product and after optimization 600 mg of crude product afforded 500 mg pure 4,4'-diacetyl-o-terphenyl. ¹ H NMR (400 MHz, CDCI₃) δ 2.55 (s, 6H), 7.21 (d, J = 8.4 Hz, 4H), 7.44 (m, 4H), 7.80 (d, J = 8.4 Hz, 4H); ¹³C NMR (100 MHz, CDCI₃) δ 26.47, 128.06, 128.23, 129.90, 130.46, 135.29, 139.35, 145.94, 197.62.

1.2 Preparation of (4,4'-Bis-(1 ",1 ",1 ",2",2",2",3",3"-heptafluoro-4",6"-hexandion-6"-yl)-o- terphenyl) (5)

[0156] Sodium methoxide (520 mg, 9.6 mmol) was added to a stirred solution of 4,4'-diacetyl-o- terphenyl (1 .0 g, 3.2 mmol) in dry tetrahydrofuran (30 mL) at room temperature under an atmosphere of nitrogen. The reaction mixture quickly turned to a light cloudy orange colour. Ethyl heptafluorobutyrate (1.4 mL, 8 mmol) was added drop wise via syringe over 10 min while the reaction mixture was stirring. TLC analysis of the reaction mixture after 16 h [using ethyl acetate/hexane (4/1 ) with one drop of trifluoroacetic acid in a 5 mL mixture] indicated the presence of starting material which was consumed over the next 4 h. The organic solvent was removed under reduced pressure, and the residue was dissolved in ether (50 mL) and quenched with hydrochloric acid (3 M, 20 mL), the ether layer was then dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure to give a pale yellow oil that was crystallized in absolute ethanol (5 mL) and collected by filtration to give compound 5 (1 .9 g, 85%) as a yellow powder. ¹ H NMR (400 MHz, CDCI₃) δ 6.59 (s, 2H), 7.29 (d, J = 8.5 Hz, 4H), 7.47-7.54 (m, 4H), 7.85 (d, J = 8.5 Hz, 4H); ¹³C NMR (100 MHz, CDCI₃) δ 93.82, 127.63, 128.75, 130.48, 130.66, 131.16, 139.09, 147.16, 179.02, 184.95.

1.3 Preparation of 4,4"-Bis-(4,4, 5,5,6, 6,6-heptafluoro-3-oxo-hexanoylH1 ,1 ':2',1 "l-o- terphenyl-4'-sulfonyl chloride) (2)

[0157] A 25 ml round bottom flask was dried at 180°C for 20 minutes and then charged with chlorosulfonic acid (5 ml). The flask was equipped with a stirrer bar and small portions of compound 5 (600 mg; 0.9 mmol) were added slowly. The reaction was allowed to proceed at room temp (4 h) whereon the reaction mix was quenched by drop wise addition to crushed ice. The ice/water mix was then extracted with ethyl acetate (3 x 60 ml), dried over anhydrous sodium sulfate, filtered and concentrated to give crude compound 2 (700 mg, 95%) that was used in future steps without further purification (based on NMR results it was more than 98% pure). ¹ H NMR (400 MHz, CDCI₃) δ 6.58 (s, 2H), 7.31 (dd, J = 2.6, J = 8.6 Hz, 4H), 7.72 (d, J = 8.3 Hz, 1 H), 7.89 (d, J = 7.8 Hz, 4H), 8.13 (d, J = 2.1 Hz, 1 H), 8.17 (dd, J = 2.1 , J = 8.3 Hz, 1 H) - need to scan out to 16-18 ppm to see the enolic OH; ¹³C NMR (100 MHz, CDCI₃) δ 94.02, 126.73, 127.90, 127.95, 128.02, 128.77, 130.06, 130.18, 130.29, 131.99, 132.41 , 132.56, 140.91 , 144.08, 144.24, 145.92.

1.4 Preparation of 2-[2-[2-(2-Hvdroxyethoxy)ethoxy1ethoxy1ethyl 4- methylbenzenesulfonate

H0^^₀^^0^^_0/-^OH NaOH, THF, p-TsCI _¾ HO^^_Q,_/^0^_/\0/^OTs

[0158] Sodium hydroxide (0.69 g, 17.13 mmol) was ground to a coarse powder and quickly added to a solution of tetraethylene glycol (21.95 g, 1 13 mmol) in tetrahydrofuran (5 ml.) at 0 °C, followed by a slow addition of a solution of p-toluenesulfonylchloride (2.08 g, 10.93 mmol) in tetrahydrofuran (20 ml). The reaction mixture was then stirred for 2 h at 0 °C and poured into a mixture of ice and water. The organic layer was separated, and the aqueous layer extracted with dichloromethane (3 x 100 ml). The combined organic layers were washed with water (2 x 50 ml), dried over anhydrous magnesium sulfate, filtered and evaporated in vacuo to yield 2-[2- [2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (3.29 g, 86%) as a yellow oil. ¹H NMR (400 MHz, CDCI₃) δ 2.42 (s, 3H), 3.58-3.63 (m, 14H), 4.13 (m, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 21 .57, 61.69, 68.66, 69.19, 70.29, 70.42, 70.61 , 70.69, 72.43, 127.93, 129.77, 132.98, 144.76.

1.5 Preparation of 2-[2-[2-(2-Azidoethoxy)ethoxy1ethoxy1ethanol

HO^ _{/\ /}\_/ /_{\ /}\_/OTs NaN₃, EtOH, 70°C HC /_{\ /}\_/C /_\ \^N₃

[0159] Sodium azide (3.0 g, 46.15 mmol) was added to a solution of 2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (3.2 g, 9.18 mmol) in ethanol (50 ml) at room temperature. The reaction mixture was stirred overnight at 70°C. The reaction was then quenched by the addition of water (50 ml) and concentrated under vacuum to a third of its volume. The aqueous layer was extracted with ethyl acetate (3 x 50 mL) and the combined organic layers were then dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum. The residue was then purified by flash column chromatography on silica gel using a mixture of acetone and hexane (2:3) to yield 2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy]ethanol (1.63 g, 81 %) as an oil. ¹H NMR (400 MHz, CDCI₃) δ 3.39 (t, J = 6.0 Hz, 1 H), 3.54-3.73 (m, 14H) - OH not observed; ³C NMR (100 MHz, CDCI₃) δ 50.64, 61 .71 , 70.00, 70.31 , 70.55, 70.64, 70.67, 72.47.

1.6 Preparation of 2-[2-[2-(2-aminoethoxy)ethoxy1ethoxy1 ethanol (6)

HCX^₀^^0^^₀^_/N₃ Pd/C H₂, BOH _¾ HO.^_o^_/0^^_0/^NH₂

(6)

[0160] Palladium on carbon (10%, 0.1 g) was added to a solution of 2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy] ethanol (600 mg, 2.7 mmol) in methanol (20 ml). The flask was repeatedly evacuated and then filled with hydrogen gas three times, then the mixture was stirred under a hydrogen atmosphere for 6 h. The mixture was then filtered through a pad of celite, and the celite washed with methanol (2 x 50 ml). The filtrates were combined and the solvent removed under reduced pressure to give 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy] ethanol (6) (495 mg, 95%) as a colourless oil which was used without further purification. ¹H NMR (400 MHz, CDCI₃) δ 2.77 (t, J = 5 Hz, 2H), 3.49-3.74 (m, 14H) - OH not observed; ¹³C NMR (100 MHz, CDCIs) δ 48.94, 61.41 , 70.01 , 70.17, 70.20, 70.41 , 70.51 , 72.91 ; ESI-MS (positive mode) m/z = 194 ([MH]⁺, 100 %); calculated for C₈H₁₉N0₄: 194.1. 1.7 Synthesis of 4,4'-bis(1 ",1 ",1 ",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"-yl) N-(2-(2- [2-(2-hvdroxyethoxy)ethoxy1ethoxy>ethyl)-o-terphenyl-sulfonamide (7)

[0161] A stirred solution of 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethanol (6) (215 mg,

1.1 mmol), A/,/V-4-dimethylaminopyridine (24 mg, 0.2 mmol) and triethylamine (145 μΙ, 1.0 mmol) in dry acetonitrile (10 ml) were combined under anhydrous conditions. Then a solution of compound 2 (800 mg, 1 .0 mmol) in dry acetonitrile (5 mL) was added in drop wise via a syringe over a 10 minute period. The progress of the reaction was monitored by TLC

(chloroform:methanol, 9:1 and a drop of trifluoroacetic acid) which indicated completion of reaction in less than 1 h (compound 2 R, = 0.5, BHHCT-TEG-OH 7 R_f = 0.45). Acetonitrile was removed under reduced pressure at 45 °C and the residue was partitioned between ethyl acetate (50 mL) and an aqueous solution of potassium hydrogen sulfate (1 M, 20 ml). The organic layer was washed with potassium hydrogen sulfate (20 ml) and saturated brine (20 ml) and dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (chloroform:methanol, 9:1 and 0.01 % trifluoroacetic acid) to give compound 7 (800 mg, >80%) as a yellow oil. ¹H NMR (400 MHz, CDCI₃) δ 3.20 (m, 2H), 3.33 (m, 2H), 3.65 (m, 8H), 3.71 (m, 4H), 6.52 (s, 2H), 7.23 (m, 4H), 7.66 (m, 1 H), 7.85 (m, 4H), 7.95 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 43.2, 61.4, 69.7, 69.8, 69.9, 70.1 , 70.7, 72.4, 94.0, 126.7, 127.9, 128.0, 128.8, 130.1 , 130.3, 131.9, 132.4, 132.5, 140.9, 144.0, 144.2, 144.3, 145.9; ESI-MS (negative mode) m/z = 960 ([M-H]-, 100%); HRMS (ESI): calculated for CssHssF^NO^S a: 984.1499, Found 984.1483.

1.8 Synthesis of BHHTEGST (1 ):

[0162] To a stirred solution of compound 7 (125 mg, 0.13 mmol), 4-dimethylaminopyridine (DMAP) (12 mg, 0.1 mmol) and triethylamine (50μΙ, 0.4 mmol) in dry acetonitrile (10 ml) under anhydrous conditions was added Λ/,/V-disuccinimidyl carbonate (100 mg, 0.4 mol). The mixture was then stirred for 3 h at room temperature, after which time TLC analysis of the reaction mixture indicated the completion of reaction (chloroform:methanol, 9:1 and 0.01 % trifluoroacetic acid) (compound 7; R_f = 0.45, BHHTEGST (1 ); R_f = 0.55). Thesolvent (acetonitrile) was then removed under reduced pressure at 45 °C and the residue was purified by flash column chromatography (chloroform:methanol, 9:1 and a drop of trifluoroacetic acid) to give

BHHTEGST (1 ) as a light yellow powder (100 mg, 70%). HPLC of this partially purified material afforded pure BHHTEGST (1 ) as a light yellow powder (92 mg, 65% yield based on (7)). ¹H NMR (400 MHz, CDCI₃) δ 2.80 (m, 4H), 3.22 (m, 2H), 3.37 (m, 2H), 3.70 (m, 8H), 3.75 (m, 4H), 6.66 (s, 2H), 7.30 (m, 4H), 7.70 (m, 1 H), 7.85 (m, 4H), 7.95 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 25.4, 43.1 , 68.3, 68.7, 69.3, 70.1 , 70.3, 70.5, 70.9, 94.1 , 126.8, 127.9, 128.5, 128.9, 130.2, 130.5, 131.7, 132.6, 132.7, 140.9, 144.1 , 144.4, 144.5, 145.8; ESI-MS (negative mode) m/z = 1 101 ([M-HV, 100 %); HRMS (ESI): calculated for C₄3H36F₁4 ₂0₁₄S a: 1 125.1561 , Found 1 125.1545.

[0163] Analytical HPLC was performed using a Gemini-NY C18 column (5 μιη, 4.6 mm ID, 250 mm) with a flow rate of 1.0 ml/min. HPLC solvent A (water with 0.05% TFA), solvent B (acetonitrile) using 5 to 100% B gradient in 15 minutes. BHHTEGST (1 ) retention time is 25.8 mins.

[0164] Preparative HPLC was carried out using an Econosil C18 column (10 μιη, 22 mm ID, 250 mm) with a flow rate of 9.0 ml/min. HPLC solvent A (water with 0.05% TFA), solvent B (acetonitrile) using 5 to 100% B gradient in 15 minutes. BHHTEGST (1 ) retention time is 22.3 mins.

Synthesis of Europium BHHTEGST

The europium chelate 4,4'-bis (1 ",1 ",1 ",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"-yl) sulfo-o-terphenyl-tetraethylene glycol-N-hydroxysuccinimide (BHHTEGST) was

synthesized.

EXAMPLE 1 - Synthesis of the Biomolecular Complexes

[0165] Recombinant LPG was produced in E. coli and purified by ion exchange

chromatography as described in Sunna A. et al. A linker polypeptide with high affinity towards silica-containing materials, New Biotechnology 30 (2013) 485-492. Recombinant PG was purchased from Sigma-Aldrich (Australia). BHHTEGST contains an A/-hydroxysuccinimide carboxylate residue that enables its covalent attachment to the free amino group of lysine residues in the polypeptide linker of LPG. Assuming that all of the lysine residues were available, each protein (PG without linker, 19 lysine residues and LPG, 27 lysine residues) was reacted with three different molar equivalents of the BHHTEGST chelate per lysine residue. This is referred herein as the BHHTEGST:Lysine ratio. All reactions were performed in triplicate.

[0166] In each reaction, 100 g LPG was exchanged into 100 mM NaHC0₃, pH 8.5 and then mixed with a different molar excess of the BHHTEGST chelate. After incubation for 1 h at 37°C the reaction mixtures were passed through a Sephadex G-25 column in 0.1 M phosphate- buffered saline (PBS) to remove excess BHHTEGST. The fractions corresponding to biomolecular complexes (LA-LPG and LA-PG) were collected by visual detection using a spectrophotometer (280 and 320 nm), then combined and concentrated to 100 ί using an Amicon Ultra centrifugal filter (10 kDa cut-off, Millipore).

Relative Quantification of Ligand

[0167] UV adsorption analysis of the BHHTEGST ligand indicated maximum absorption at 340 nm and also partial absorption at 280 nm, which overlaps with that from both proteins (PG and LPG). Thus, to account for the partial adsorption of BHHTEGST in the biomolecular complex, molar extinction coefficients of the chelate at 320 nm and 280 nm were obtained separately from the calibration curves at 320 nm and 280 nm, respectively. The concentration of BHHTEGST ligand was then obtained by reading the absorbance of the biomolecular complexes at 320 nm and the molar extinction coefficient of ligand at 320 nm. Next, partial absorption of BHHTEGST ligand at 280 nm was identified by its calibration curve (at 280 nm). The final protein concentration was obtained by subtracting the absorbance of BHHTEGST ligand from the absorbance of labeled protein at 280 nm. The average number of BHHTEGST ligands per protein was obtained by dividing the chelate concentration by the protein concentration.

Cells and Antibodies

[0168] Cryptosporidium oocysts and Giardia cysts were purchased from BTF Pty Ltd (Sydney, Australia). Cryptosporidium monoclonal antibodies CRY104 and CRY104-conjugated to fluorescein isothiocyanate (CRY104-FITC) specific to the walls of Cryptosporidium oocysts, and the Giardia monoclonal antibody G203 specific to the walls of Giardia cysts also were purchased from BTF Pty Ltd. Synechococcus cells (Cyanobacterium) were kindly provided by Ms. Deepa Varkey (Macquarie University). Human adipose-derived mesenchymal stem cells were prepared by Peter Succar (Macquarie University, Sydney). Human anti-CD271 monoclonal antibody specific to the cell surface protein CD271 of human stem cells was purchased from BD Biosciences (Sydney, Australia). Human astrocyte and U251 astroglioma cells were propagated and harvested at the Australian School of Advanced Medicine (ASAM, Macquarie University) by Ms. Vanessa Tan and Ms. Ariel Seaton. Mouse anti-GFAP monoclonal antibody specific for the astrocyte glial fibrillary acidic protein (GFAP) and Alexa Fluor 488-Goat anti-Mouse IgG were purchased form Invitrogen (Sydney, Australia). The monoclonal antibody anti-GLAST specific to U251 astroglioma cancer cell membrane was purchased from (Miltenyi Biotec, Bergisch Gladbach, Germany). The blue-fluorescent DNA stain 4',6-diamidino-2-phenylindole (DAPI, ProLong Gold, Invitrogen) was used to stain astrocyte cell nuclei fluorescently.

Microscopy

[0169] All bright-field, fluorescence and time-gated luminescence imaging was performed on an Olympus BX51 fluorescence microscope with a UPLSAPO 100 X oil immersion objective lens. Colour images were captured on an Olympus 12.8 megapixel DP72 camera with a sensor resolution of 4140 x 3096 and stored in the TIFF format. To view time-gated luminescence a Gated Auto-synchronous Luminescence Detector (GALD) as described by Connally, R, A Device for Gated Autosynchronous Luminescence Detection, Analytical Chemistry 83 (201 1 ) 4782-4787was inserted into the DIC slot of the microscope nosepiece. Cells labelled with lanthanide chelate were detected by the GALD with exposure times of 3 seconds. FITC fluorescence imaging was carried out using a 100 W mercury arc lamp and a FITC filter set with 200 ms exposure times. DAPI staining images also were captured with the same UV lamp source and DAPI filter with 10 ms exposure time.

Tunable Fluorescent Output

[0170] For use as universal detection reagents in TGL bioimaging, PG (without linker) and LPG must be able to withstand extensive conjugation with the BHHTEGST ligand and retain their capacity to bind antibodies. Thus, each protein was reacted with BHHTEGST at different BHHTEGST:Lysine ratios, yielding a total of three Luminescence-Activating (LA-) biomolecular complexes (Table 1 ), which are referred to herein as LA-PG_HIGH_, MID _OR LOW and LA-LPGHIGH, MID or LOW The average number of BHHTEGST ligands attached to LA-PG or LA-LPG at each ratio was then semi-quantified as described above and the results are summarised in Table 1. Table 1. Semi-quantification of the number of BHHTEGST ligands attached to PG and LPG after reactions were performed in triplicate with BHHTEGST:Lysine ratios between 1 .6-0.4.

BHHTEGST:Lysine

Relative Number of Chelates

Molar Ratio Ratio³

LA-PG CE(%)^b LA-LPG CE (%) LA-PG LA-LPG

15.5 ±

High 81 .6 ± 7.3 18.0 ± 0.7 66.7 ± 0.7 1 .5 1 .6

1 .4

9.7 ± 51 .2 ±

Mid 16.8 ± 0.6 62.1 ± 0.6 0.7 0.8

2.8 14.8

4.3 ±

Low 22.8 ± 3.1 12.3 ± 0.2 45.7 ± 0.2 0.4 0.4

0.6

a BHHTEGST:Lysine Ratio is the number of moles of BHHTEGST added per lysine residue in each protein.

b CE = Conjugation Efficiency (% of total lysine residues modified with BHHTEGST).

[0171] The BHHTEGST ligand is amine reactive and attaches covalently to lysine residues in the target proteins. PG contains 19 lysine residues whereas LPG has 27 due to the presence of an additional 8 lysine residues located in the linker region. Therefore, it was expected that LPG would be modified with a higher number of ligands at each BHHTEGST:Lysine ratio tested. As shown in Table 1 , in reactions performed at the lowest BHHTEGST:Lysine ratio PG was modified with only an average of 4.3 chelates, whereas LPG had 12.3. This difference

(8 ligands) correlates with the additional eight lysine residues present on the linker region of LPG. A similar difference (7.1 ligands) in the number of attached ligands was also observed at the mid BHHTEGST:Lysine ratio, in which PG and LPG contained on average 9.7 and 16.8 ligands, respectively. However, the difference was considerably smaller (2.5 ligands) at the highest BHHTEGST:Lysine ratio tested, which resulted in PG and LPG biomolecular complexes that contained on average 15.5 and 18.0 ligands, respectively. These results show that better and more consistent reaction efficiencies were achieved with LPG (45.7-66.7%) compared to PG (22.8-81 .6%) across the range of BHHTEGST:Lysine ratios tested.

Standard Cell-Labelling Procedure

[0172] To couple luminescence-activating biomolecular complexes to cell-specific antibodies, each complex (2 ig) was mixed with antibody (2 g) in 100 mM PBS, pH 7.4 to a final volume of 10 μ I and incubated for 30 s at room temperature to produce complex-coupled antibodies (Figure 1 ). [0173] To label cells, 5 μΙ_ of the complex-coupled antibodies (LA-LPG + Antibody or LA-PG + Antibody) were loaded onto a microscope slide containing fixed target cells and incubated for 1 min at room temperature. Cell fixation was carried out by gently drying a 10 μΙ cell sample on a slide with a hot plate at 40°C for 20 s. The slide was gently rinsed with MilliQ water. Then 3 μΙ of 22 mM europium chloride (EuCI₃)was added to the slide and allowed to react with the chelates for 30 s. The labelled cells were examined using bright-field, fluorescence and time- gated luminescence microscopy.

[0174] The luminescent labelling of cells was quantified by analysing the raw digital images with ImageJ software. In each image, a line-plot was placed across the cells. The 8-bit value of the pixels in the red channel was used to attain the peak intensity and was defined as the 'signal'. The mean intensity from a selected section in the darkest region (cell free) was defined as the 'noise'. These values were then used to calculate signal-to-noise ratios (SNR). SNRs were normalised to 1 by dividing the 'signal' value by the 'noise' value.

EXAMPLE 2 - Direct Luminescence Labelling of Antibodies For Bioimaging and Detection

[0175] The ability of lanthanide-activated biomolecular complexes to act as universal detection reagents which impart detectable luminescence to primary antibodies without compromising their function was investigated. This study also investigated which BHHTEGST:Lysine ratio was the most effective.

[0176] Using the strategy illustrated in Figure 1 , each of the biomolecular complexes (LA-PG and LA-LPG) from Example 1 was coupled to cell-specific antibodies for the luminescent labelling of Giardia cysts (Figure 2) and Cryptosporidium oocysts (Figure 3). Labelled cells were visualised by TGL microscopy using the GALD and the SNRs from the raw digital images were calculated using ImageJ software (Table 2).

[0177] LA-LPGMID produced the highest quality images of the complexes studied and showed high definition and SNRs of 76 for Giardia and 71 for Cryptosporidium. In contrast, the cells labelled with the LA-PG_MID complex exhibited low luminescence and poor resolution.

Accordingly, the SNRs in these images were significantly lower for both cell-types (Giardia = 7; Cryptosporidium = 16). The LA-LPG_MID complex provided more than a 10-fold enhancement in the SNRs of labelled Giardia luminescence and more than a 4-fold enhancement for

Cryptosporidium when compared to LA-PG_MID-

[0178] Cells labelled with LA-LPG_HIGH displayed similar SNRs (Giardia = 72; Cryptosporidium = 76) to those shown by LA-LPG_MID- However, the cells appeared oversaturated with poorly- defined boundaries. The LA-PG_HIGH control generated low SNRs {Giardia =16; Cryptosporidium = 21 ) and produced images that showed weak luminescence and low resolution. In comparison to LA-PGHIGH, the LA-LPGHIGH complex improved the SNRs of the labelled Giardia and

Cryptosporidium by more than 4-fold and 3-fold, respectively.

[0179] Low SNRs were obtained from cells labelled with the LA-LPG_Low complex (Giardia = 20; Cryptosporidium = 25) and the cells showed very weak luminescent output and poor definition with the weakest S Rs produced by cells labelled with LA-PG_Low (Giardia = 6; Cryptosporidium = 10). In these images the cells were difficult to detect and identify. When compared to LA- PGLOW, the LA-LPGLOW complex amplified SNRs by more than 3-fold and 2-fold for labelled Giardia and Cryptosporidium, respectively.

[0180] These results indicated that all of the modified complexes can bind antibodies and render them luminescent. The cells labelled with LA-LPG were significantly brighter in label intensity than LA-PG (irrespective of the conjugation concentration) an observation that can be attributed to the higher chelate content of the LA-LPG complexes (see Table 1 ). However, LA- LPGHIGH possesses the highest chelate content and produced images in which cells were highly luminescent but poorly defined. This lack of definition possibly could be due to a reduction in cell targeting efficiency.

[0181] The LA-LPG_M|_D complex provided clear, high-contrast and high definition images, making it the best detection reagent under these conditions for the attachment of luminescent BHHTEGST chelates to the antibodies tested. Therefore, all subsequent cell-labelling experiments were performed using LA-LPG_MiD-coupled antibodies.

Table 2. Signal to noise ratios (SNRs) for Giardia cysts and Cryptosporidium oocysts labelled with each of the LA-PG and LA-LPG biomolecular complexes. SNR analysis of raw image data was performed using ImageJ software.

Signal-to-Noise Ratios

Conjugatio Giardia cysts Cryptosporidium oocysts n ratio

LA-PG LA-LPG Fold diff. ^a LA-PG LA-LPG Fold diff.

High 16 72 4.5 21 76 3.6

Mid 7 76 10.9 16 71 4.4

Low 6 20 3.3 10 25 2.5

³ Fold diff. = Fold difference [0182] Luminescent labelling, detection and bioimaging experiments of Cryptosporidium and Giardia (oo)cysts were performed using LA-PGMID and LA-LPGMID with and without the addition of cell-specific antibodies (CR104 antibody for Cryptosporidium oocysts and G203 antibody for Giardia cysts). In the absence of cell-specific antibodies LA-PGMID and LA- LPGMID displayed no luminescent labelling of Cryptosporidium oocysts and Giardia cysts under TGL condition. However, when LA-PGMID and LA-LPGMID were coupled with the corresponding cell-specific antibodies, strong luminescent labelling of Cryptosporidium oocysts and Giardia cysts was observed under TGL condition. The absence of luminescent labelling in experiments without cell-specific antibodies confirmed that LA-PGMID and LA-LPGMID did not display non-specific binding to Cryptosporidium and Giardia (oo)cysts.

EXAMPLE 3 - Dual Labelling Immunofluorescence

[0183] The LA-LPGMID biomolecular complex was coupled to a FITC-conjugated antibody (CRY104-FITC) (fluorescein isothiocyanate) specific for the cell walls of Cryptosporidium oocysts. Cells were then labelled and visualised by either fluorescence microscopy or time- gated luminescence microscopy. Cells observed in the fluorescent FITC channel displayed bright green fluorescence and good definition. Under time-gated conditions the cells emitted red luminescence and maintained their definition. These results indicated that LA-LPG_MID can be coupled easily to a different fluorophore-conjugated antibody and applied as a dual-immuno- labelling technique based on emission lifetimes.

EXAMPLE 4 - Elimination of Intrinsic Autofluorescence

[0184] Lanthanides have long luminescence lifetimes that enable temporal discrimination of shorter-lived autofluorescence, unlike conventional fluorophores. It was tested whether LA- LPG_MiD-coupled antibodies in conjunction with time-gated luminescence microscopy can be used for background-free detection of cells in autofluorescent environments. Consequently, we used this methodology to compare the efficiency of a conventional fluorophore (FITC) and our lanthanide-carrier molecule (LA-LPG_MID) to label cells in an autofluorescent environment.

Cryptosporidium oocysts were mixed with a sample of autofluorescent Synechococcus cells and then labelled with the "LA-LPG_MID + CRY104-FITC" complex. The Cryptosporidium oocysts were fluorescent green in the FITC channel as expected, but difficult to discriminate against background autofluorescence provided by the Synechococcus cells (Figure 2a). Switching to time-gated microscopy suppressed the background autofluorescence and generated high- contrast images in which cells exhibited red luminescence and were identified readily against a fluorescence-free background (Figure 2b). These results show that the LA-LPG_MID complex in combination with time-gated luminescence microscopy can be used successfully for the sensitive detection of cell targets in the presence of background autofluorescence.

EXAMPLE 5 - Extracellular Visualization

[0185] As shown above, "LA-LPGMID + Antibody" complexes can label cells with strong luminescence for the background-free detection of protozoan pathogens (i.e., Cryptosporidium and Giardia). The same approach was tested to the detection and visualization of human cells, specifically stem cells and cancer cells. Human-adipose derived mesenchymal stem cells (hAdMSC) and U251 astroglioma cancer cells were labelled with luminescence using "LA- LPGMID + Antibody" complexes as described in "Cells and antibodies" and "Standard cell- labelling procedure" sections. Under UV light excitation (hAdMSCs displayed red luminescence. However, the interior of the cells exhibited autofluorescence as a consequence of cellular metabolism. This autofluorescence subsequently was eliminated under time-gated conditions. In addition, labelled U251 astroglioma cancer cells were shown to be rapidly and easily visualized under time-gated conditions with high contrast and without any background fluorescence.

EXAMPLE 6 - Immunohistochemistry

[0186] Immunohistochemical staining of glial fibrillary acidic protein (GFAP) in human astrocyte cells using LA-LPG_MID was performed as described below.

Immunohistochemistry Staining Method

[0187] Cell fixation and permeabilization. Astrocytes derived from human fetal brain were plated in glass chamber slides and grown in culture for 1 week. Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min, and washed 3 times with 0.2% BSA in PBS to remove excess PFA. Cells were permeabilized and blocked by incubation in 0.2% Triton X-100, 10% normal goat serum, and 2% BSA in PBS for 30 min at room temperature and then washed in PBS.

[0188] Primary antibody labelling of GFAP. Cells were incubated with the primary antibody, mouse anti-GFAP (1 :250 dilution in 2% BSA and 10% donkey serum in PBS), overnight at 4°C. Primary antibody solution was removed from the cells by washing with PBS three times.

[0189] "LA-LPGMID + Secondary antibody" labelling of GFAP. 4 g LA-LPG_MID was mixed with 0.3 g secondary antibody, Goat anti-Mouse (Alexa Fluor 488), in 100 μΙ PBS and incubated for 30 s at room temperature. The resulting "LA-LPG_M|_D + Antibody" complexes then were diluted to 300 μΙ in PBS and incubated with the fixed cells for 30-45 min at room temperature. Excess "LA-LPGMID + Antibody" complex was removed from the cells by washing with PBS three times. DAPI (4',6-diamidino-2-phenylindole) and europium chloride (22 mM) were applied to the cells, which were examined using bright-field, fluorescence and TGL microscopy.

[0190] The intracellular immunohistochemical staining of glial fibrillary acidic protein (GFAP) was evaluated in human astrocyte cells. Following primary labelling of GFAP with Mouse anti- GFAP, a "LA-LPGMID + Goat anti-Mouse IgG (Alexa Fluor 488)" complex was used as a secondary luminescent label (see method above). The labelled GFAP was visualised by either fluorescence microscopy or time-gated luminescence microscopy.

[0191] GFAP displayed strong green fluorescence (Alexa Fluor 488) and good definition in the FITC channel. Similarly, under time-gated conditions, GFAP exhibited bright red luminescence (LA-LPGMID) and was well defined. These results show that LA-LPGMID can be applied in immunohistochemistry to label intracellular targets.

EXAMPLE 7 - Flow Cytometry

[0192] Fluorescent-dye conjugated antibodies are used extensively in flow cytometry methods. We investigated whether "LA-LPG_MID ⁺ Antibody" complexes can label cell populations with a detectable luminescent signal for flow cytometric analysis. hAdMSCs were harvested and diluted in 100 mM Tris-HCI pH 7.5 and centrifuged at 2000 x g for 5 min. The cells were washed twice in the same buffer then resuspended in Tris-HCI buffer with 2% BSA. Cells were labelled with the "LA-LPGMID + anti-CD271 " complexes and incubated at 4°C for 45 min. Cells were washed with ice cold Tris-HCI, centrifuged at 300 x g for 5 min and resuspended in Tris-HCI supplemented with excess EuCI₃. Labelled and unlabelled control cells were then analyzed by a FACS Aria flow cytometer (Becton Dickinson, Figure 3). In flow cytometry experiments, LA- LPGMID luminescence was excited at 355 nm (UV excitation), and emission was captured by a 670/30 nm bandpass filter (red emission). Scattering flow cytometry counting results of luminescence signal intensities of gated hAdMSC populations showed a phase shift in cell luminescence between the unlabelled and labelled cell populations. The results indicated that the cells labelled with "LA-LPG_MID + anti-CD271 " complexes were more luminescent than nonlabelled control cells. However, it is expected that a substantial improvement in signal and population shifts may be achieved with the use of a flow cytometer equipped with time-gated luminescence detection. EXAMPLE 8 - Cell Capture and Visualization

[0193] A cell capture system has been developed using LPG as an anchoring point for the binding of cell surface antibodies to silica-coated magnetic particles. It was tested whether LPG retained its capacity to functionalise silica-coated magnetic particles after the attachment of multiple BHHTEGST chelates.

[0194] Silica-coated magnetic particles (1 mg) were mixed with 20 μg of a "LA-LPG MID ⁺ anti- CD271" complex in PBS and incubated at room temperature for 15 min. Particles were collected using a magnet and any unbound material was removed. The functionalised particles (particle-bound "LA-LPG_MID + anti-CD271 " complex) were washed three times in PBS, followed by incubation with a sample of hAdMSCs diluted in PBS. Unbound cells were removed by washing with PBS three times. Particle-bound cells were visualised using TGL microscopy.

[0195] Cells were observed to be partially or completely covered in particles (Figure 10), indicating that the "LA-LPG_MID + anti-CD271 "complex was able to mediate the selective binding and recovery of cells from dilute solutions. Furthermore, these results show that the

modification of LPG with BHHTEGST does not prevent its capacity to bind and functionalise silica-coated particles. Cells that were completely covered in particles fluoresced under UV light excitation), and any auto-fluorescence was eliminated under time-gated conditions.

[0196] Examples of other luminescence-activating units include 1 ,2-Bis[4'-(1 ",1 ",1 ",2",2",3",3"- heptafluoro-4",6"-hexanedion-6"-yl)-benzyl]-4-chlorosulfobenzene (BHHBCB) and 4,4"- Bis(4,4,5,5,6,6,6-heptafluoro-1 ,3-dioxohexyl)-o-terphenyl-4'-sulfonyl chloride (BHHCT).

EXAMPLE 9 - Use of LPG as a Carrier for Fluorescent Nanodiamonds

[0197] Fluorescent nanodiamonds (FNDs) are a new and promising class of nanomaterials that can be used as fluorescence probes for bioimaging. As an alternative to luminescent lanthanide chelates, we tested whether the LPG biomolecule can be used as a carrier molecule for FNDs. To label LPG with FNDs, 1 mg of carboxylated FNDs were mixed with 20 g of LPG in Tris- HCL, pH 7, and incubated at room temperature for 15 min. "LPG + FND" complexes were collected by centrifugation and any unlabelled LPG was removed. LPG was then released from the "LPG + FND" complexes by heating at 99°C in SDS-PAGE loading buffer. Fractions containing LPG were visualised by SDS-PAGE with Coomassie Brilliant Blue staining (Figure 4). After interaction with FNDs, no unlabelled LPG (U) was observed and all of the starting (S) LPG was present in the "LPG +FND" complex fraction (CF). It has been shown that LPG, in particular the positively charged linker region, can be effectively labelled with negatively charged FNDs via physical adsorption. EXAMPLE 10 - Use of LPG as a Carrier for Fluorescent Nanorubies

[0198] Nanorubies (NRs) are a new class of nanoparticles that show great promise as fluorescent probes for bioimaging. As an alternative to luminescent lanthanide chelates, we tested whether the LPG biomolecule can act as a carrier molecule for silica-coated NRs and used to couple these particles to antibodies. NRs (1 mg) were mixed with 20 g of LPG in Tris- HCL, pH 7, and incubated at room temperature for 15 min. "LPG + NR" complexes were collected by centrifugation and any unlabelled LPG was removed. "LPG + NR" complexes and "NR without LPG" were incubated with an antibody-conjugated to fluorescein isothiocyanate (Ab-FITC). The emission spectra of each sample dispersed in water was measured by fluorescence spectrometry under excitation at 488 nm (Figure 5). As expected, the "LPG + NR" complexes in combination with Ab-FITC exhibited significant emission intensity in the green region (500-550 nm), indicating the effective coupling of Ab-FITC to the NRs. However, in the control sample without LPG, only negligible green emission was detected, indicated no coupling between Ab-FITC and the NRs. It has been shown that LPG, in particular the positively charged linker region, can be effectively labelled with negatively charged silica-coated NRs via physical adsorption. Furthermore, NR-labelled LPG can bind effectively to antibodies.

EXAMPLE 11 - Use of Linker-Barstar (L-Bs) as a Carrier for Fluorophore

Nanoparticles

[0199] As an alternative to LPG, the linker sequence was fused to the N-terminus of Barstar, a protein that binds strongly to its interaction partner protein Barnase (Bn), resulting in the fusion protein Linker-Barstar (L-Bs). Like LPG, the ability of L-Bs to act as a carrier molecule for negatively charged fluorescent nanoparticles was examined (see Example 9 and Example 10 above). The capacity of L-B to couple such particles to fusion proteins that contain Bn was studied. One mg of Texas-red doped silica nanoparticles (SNPs) that carry a negative charge were mixed with 20 g of L-Bs in PBS, pH 7.4, and incubated at room temperature for 15 min. "L-Bs + SNP" complexes were collected by centrifugation and any unlabelled L-Bs was removed. DARPin-Bn is a fusion protein that comprises two functionally distinct regions (i) a HER2/neu-specific DARPin peptide; (b) Barnase (Bn). Cells that were positive (SKBR-3) or negative (CHO) for the cancer-specific HER2/neu epitope were sequentially incubated with DARPin-Bn and "L-Bs + SNP" complexes. The cell suspensions were then analysed by fluorescence microscopy. The membranes of the positive SKBR-3 cells displayed bright fluorescence, whereas the negative CHO cells exhibited no detectable fluorescence. [0200] It has been shown that L-Bs acts similarly to LPG, and can be effectively labelled with negatively charged nanoparticles via physical adsorption. Furthermore, SNP-labelled L-Bs is able to impart fluorescence to fusion proteins that contain Bn for bioimaging applications.

[0201] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of technology as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive

Claims

Claims:

1. A biomolecular complex comprising:

an antibody-binding protein;

a polypeptide linker capable of receiving a luminescence-activating unit; and a luminescence-activating unit.

2. The biomolecular complex according to claim 1 wherein the antibody-binding protein is selected from protein A, protein A/G, protein G, protein L, or any combination thereof.

3. The biomolecular complex according to claim 2 wherein the antibody-binding protein is protein G.

4. The biomolecular complex according to any one of claims 1 to 3 wherein the polypeptide linker is fused to the antibody-binding protein at the amino or carboxyl terminal of the protein.

5. The biomolecular complex according to any one of claims 1 to 4 wherein the polypeptide linker comprises one or more amino acids comprising a side group having a functional group selected from NH₂, SH, OH.

6. The biomolecular complex according to any one of claims 1 to 5 wherein the polypeptide linker comprises one or more lysine residues.

7. The biomolecular complex according to claim 6 wherein the polypeptide linker comprises are selected from SEQ ID NOS: 1 to 17.

8. The biomolecular complex according to claim 7 wherein the polypeptide linker comprises a combination of sequences defined by (Polypeptide Linker Sequence^ where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15.

9. The biomolecular complex according to any one of claims 1 to 8 wherein the luminescence-activating unit is attached to the polypeptide linker, or attached to the antibody-binding protein, or attached to the polypeptide linker and the antibody-binding protein.

10. The biomolecular complex according to claim 9 wherein the luminescence-activating unit is covalently attached to the polypeptide linker moiety by an amide bond.

1 1 . The biomolecular complex according to any one of claims 1 to 10 wherein the luminescence-activating unit is one or more of conventional or lanthanide-based fluorophores.

12. The biomolecular complex according to claim 1 1 wherein the luminescence- activating unit is a lanthanide-activating unit.

13. The biomolecular complex according to claim 12 wherein the lanthanide-activating unit comprises a tetradentate ligand moiety capable of chelating a lanthanide ion, and an antenna moiety capable of sensitising lanthanide emission when a lanthanide ion is chelated to the tetradentate ligand moiety.

14. The biomolecular complex according to claim 1 1 wherein the luminescence- activating unit is selected from the group consisting of BHHST, BHHCT, BPPCT, BTBCT, BHHBCB, BPPBCB, and BTBBCB.

15. The biomolecular complex according to claim 1 1 wherein the lanthanide-activating unit has a reactive functional group 'A' selected from tosylate,

mesylate, -halo, -N₃, -NH₂, -CN, N-succinimidyl carbonate, isocyanate, isothiocyanate, sulfonyl

chloride, -C(0)N₃, -C(0)halo, -C0₂H, -C(0)0(Ci_6 alkyl)-R^a, -C(0)NH(d_₆ alkyl)-R^a, -NHC(0 )(Ci_6 alkyl)-R^a, -C(NH)0(Ci_₆ alkyl)-R^a, aziridine, maleimide, C₂-₄ alkyne; wherein each R^a is independently selected from H, OH, halo, C0₂H, C0₂(Ci_₆ alkyl).

16. The biomolecular complex according to claim 1 1 wherein the luminescence- activating unit is BHHTEGST having the following structure:

or a salt, hydrate or tautomer thereof.

17. The biomolecular complex according to any one of claims 1 to 10 wherein the luminescence-activating unit includes a fluorescent nanoparticle.

18. The biomolecular complex according to claim 17 wherein the fluorescent nanoparticle is selected from nanodiamond, nanoruby, silica-coated nanoruby, plasmonic nanoparticles of gold, silver, or platinum, semiconductor nanocrystals of CdSe, CdS, ZnS,Ti0₂ or PbS, magnetic compounds of Fe₃0₄, CoFe₂0₄, or CoPt, carbon nanoparticles, quantum dots, rare earth doped nanoparticles, or combinations thereof.

19. The biomolecular complex according to claim 18 wherein the fluorescent nanoparticle is selected from nanodiamond, nanoruby or silica-coated nanoruby.

20. The biomolecular complex according to any one of claims 1 to 19 further including an antibody or binding fragment thereof bound to the antibody-binding protein.

21 . The biomolecular complex according to claim 20 wherein the antibody or binding fragments thereof include at least one of an IgG, IgM, IgD and IgA.

22. Use of a biomolecular complex according to any one of claims 1 to 21 in the detection of a target cell or tissue.