GB2474456A - Dendrimer functionalised nanoparticle label - Google Patents

Abstract

A label is disclosed comprising a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a target analyte to the functionalised nanoparticle, wherein the dendrimer has a stabilising effect on the zeta potential at the nanoparticle surface. The dendrimer may be a PAMAM or Newkome dendrimer, and the nanoparticle may be a silica or metal oxide nanoparticle. Nanoparticle labels are also disclosed as above with at least one activatable functional group for conjugating a detection molecule selective for a corresponding target analyte. The detection molecule may be an antibody. Methods of detecting target analytes using the nanoparticle dendrimer labels, and uses of the labels in biological discovery, biomedical detection, therapeutic applications, and in dermatological or in vivo diagnostic devices are also claimed. Methods of preparing the labels are also claimed.

Description

Title: Biomolecular Labels

Field of the Invention

The invention relates to biomolecule labels and labelling methods that may be used in bioanalytical applications. In particular, the invention relates to biomolecule labels based on nanoparticles, and more particularly, to the use of dendrimers, to bioconjugate biomolecule analytes to the detectable nanoparticle.

Description of Related Art

In the bioanalysis area, fluorescent labels are used for a range of applications including immunosorbent assays'2, immunocytochemistry34, flow cytometry56 and DNA/protein microarray analysis78. Fluorescence is preferred because it combines high sensitivity with a low limit of detection (LOD)9". Current research in biomedical diagnostics is moving to inexpensive devices, using biochips that require small samples volumes. To meet this new demand fluorescent labels with improved physical and chemical properties are required. For example, better photostability and high fluorescence intensity, with a reproducible signal under a variety of chemical and biological conditions.

Nanoparticles are useful as labels for antibodies in immunoassay procedures, where an antigen is captured onto a surface then visualised through its reaction with a labelled antibody, because they result in an intense signal. They can however suffer from some disadvantages in comparison with simple molecular labels, most notably effects of particle aggregation and, a related effect, of non-specific binding to the capture surface. Another issue in the use of nanoparticles as labels, is the fraction of the antibody that is coupled to the particle, that are in fact active for the reaction with antigen. This fraction can be rather small, which in turn can lead to diminished sensitivity and worsened non-specific binding.

In particularly, dye-doped silica nanoparticles'2'4 (NP) stand out as excellent candidates for improved labels, as it is possible to dope silica NP5 with a large number of fluorophores, increasing the total fluorescence of the label significantly'5'6. Moreover, the fluorophore is protected inside a silica matrix, increasing photostability'8'9 and quantum efficiency2021. Silica NP5 are also non-toxic, chemically inert, and can be prepared in a range of sizes22. It also relatively easy to functionalize silica NP with bioreactive groups such as aldehyde, cyano, isothiocyanate, carboxyl, amine, iodoacetamide, malemide, epoxide, thiol, carbodiimide, and many more. Fuctionalisation may be achieved by surface modification via post synthesis grafting onto the silica surface or during the growth phase of the silica nanoparticles but also after by enabling facile bioconjugation'5'2223.

It has been noted that problematically, bioassay data obtained using the same silica NP label with the same capture antibody, but prepared using different bioconjugation protocols can result in inconsistent results. For example, previous work involving glutaraldehyde gave inconsistent results. Assays lack precision with large standard deviations between the results.

Moreover, each step in the conjugation process can decrease colloidal stability. Loss of colloidal stability can often occur as a result of bioconjugation to a nanoparticle and is a significant stumbling block in the development of improved silica NP labels.

In 2006, Tan and colleagues'5 proposed a way of improving colloidal stability of silica NP5 through the addition of a negatively charged non-reactive organosilane alongside the bioreactive organosilane. It is also important to maintain a stable colloid after addition of a bi-linker which may be used to conjugate an antibody to the functionalised NP.

The functionality and number of functional groups on the bi-linker also affects the overall assay performance.

There is a great choice of commercial hetero-or homo-functional linkers available; however, issues may arise from their effect on NP stability, aggregation, and additionally, information relating to the solubility and the efficiency of bioconjugation can be lacking. There are many commercially available bi-linkers, for example glutaraldehyde24, adipic acid25, and succinic anhydride22'26. Using glutaraldehyde, the biomolecule of interest is immobilized directly, without use of activating reagents via Schiff base formation. In many other cases another issue that arises stems from that fact that additional chemicals are required for activation and that several steps may be required to conjugate a target to the nanoparticle. Stability may be adversely affected since the risk of decrease in the zeta potential is greater.

Furthermore, in a sensitive and functional bioassay, a NP labelled biomolecule must retain its specific binding to analyte, while keeping the non-specific adsorption (NSA) on the substrate to minimum. Therefore the linker must facilitate this requirement by being a linker specific for a particular functional group, for example, an amino acid functional group on a biomolecule.

It is desirable to provide improved bioanalytical systems, in which bioassays use superior labels comprising linkers capable of linking the biomolecular detection species to the nanoparticle label in a way that will not result in a reduction in activity, and which comprise a dispersion of the nanoparticles. Desirably the improved label will lead to high assay sensitivity and ultralow LODs for a desired analyte.

Summary of the Invention

According to the present invention, as set out in the appended claims, there is provided a label comprising: a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a target analyte to the functionalised nanoparticle, wherein the dendrimer has a stabilizing effect on the absolute value of the zeta potential at the nanoparticle surface.

The stabilizing effect is relative to the absolute value of the zeta potential, as the zeta potential can be positive or negative. The dendrimer functionalised nanoparticles of the invention of the invention can directly conjugate with the target analyte. Alternatively it may conjugate with one or more intermediate molecules which in turn may conjugate with the target analyte.

The invention provides an improved ultra sensitive label having an optimised linkage between the nanoparticle label and the biologically relevant analyte molecules of interest. The label of the invention advantageously allows for assays with improved binding, assay sensitivity, and LOD of the assay compared results achieved using alternative linkers. The nanoparticle label can easily be prepared while maintaining the integrity of a colloid dispersion of the nanoparticles.

In one embodiment, the stabilizing effect results from an increase in the absolute value of the zeta potential at the nanoparticle surface. In prior art labels, addition of an uncharged linker, for example, an succinimidyl ester linker, to the nanoparticle surface leads to a drop in the absolute zeta potential, as the uncharged linker becomes attached to the surface. The electrical stability of the nanoparticle is adversely affected. The dendrimer linkers overcome this problem since they typically have ionisable chemistries, for example, carboxylate or amine chemistries, which can be manipulated to stabilize the charge on the nanoparticle. High zeta potential will confer particle stability and accordingly, the colloidal dispersion of the nanoparticles will resist aggregation. When the zeta potential is low, attraction exceeds repulsion and the dispersion will break and the particles aggregate. Because a zeta potential can be positive or negative, an increase in zeta potential within the present invention is an increase in the absolute value thereof.

Dendrimers of the present invention will at least not destabilize a zeta potential (lower the absolute valve). A zeta potential having an absolute value in my of from about 20 and greater is desirable (a value that is greater than +20 or less than -20 is desirable). Suitably the zeta potential has an absolute value of about 25 or greater such as about 30 or greater. It will be appreciated that the positive effect observed on the absolute zeta potential by conjugation of the dendrimers of the invention to a nanoparticle surface may also be considered as a non-destabilizing effect.

Desirably when the dendrimer is linked to the nanoparticle there is an increase in the zeta potential that offsets any greater tendency of a dispersion of the dendrimer-nanoparticles to come out of their dispersed state. For example it may be desirable that an increase of greater than about 30%, desirably greater than about 35%, such as about 40%m in the zeta potential is achieved as compared to that of the non-functionalized NP Conjugation of the analyte to the nanoparticle by a dendrimer linker of the invention has the desirable effect of increasing the zeta potential at the nanoparticle surface. This may be zeta potential at the nanoparticle surface. These may be groups that are binding groups for binding other moieties to the nanoparticle. For example, these may be non-active groups that have been treated to make them active for bonding. For example, the dendrimer may already have, or may already be, functionalised with groups suitable to bind the dendrimer to a nanoparticle and/or target analyte.

Groups that allow such binding will be referred to herein as an "activated" or "activatable" group.

Typically, such activated or activatable groups include carboxylate or amine groups, which may be activated by methods known in the art. The activation generally renders the groups more reactive towards other corresponding groups, for example, amine or carboxylate groups on other moieties.

The dendrimer may need to be activated in order to conjugate it to the nanoparticle surface and/or to conjugate the dendrimer to an amino acid on a protein, a cell, antibody, antigen or other suitable analyte. Desirably, the activated groups on the dendrimer are susceptible to hydrolysis. Hydrolysis can be employed to form charged groups at physiological pH. Therefore, the electrostatic charge on the nanoparticle is increased and colloidal stability is maintained, despite the modification of the surface.

The invention is an improvement over prior art nanoparticle labels which require use of linkers, which when bound to the nanoparticle surface, can detrimentally affect the zeta potential of the nanoparticle. For this reason, prior art nanoparticle labels suffer from problems such as nanoparticle aggregation.

Accordingly, the invention provides a label comprising a nanoparticle wherein the nanoparticle surface is modified to comprise at least one dendrimer linker for conjugating a target analyte to the nanoparticle. A dispersion of dendrimer functionalised nanoparticles according to the invention will desirably have a zeta potential which is increased as a result of the conjugation of dendrimer to the nanoparticle surface. An increased zeta potential can offset a greater tendency of nanoparticles that are larger (by virtue of surface modification) to agglomerate. Such a stabilizing effect on the colloidal dispersion of the nanoparticles is desirable since undesirable aggregation or flocculation of the nanoparticles is hindered. This avoids problems that may occur during an assay, for example, particles "dropping out" of solution and too slow a flow rate where lateral flow microfluidic devices are used for analysis.

Furthermore, use of dendrimer linkers, having a plurality of reactive groups, improves the probability of binding of the analyte of interest to the nanoparticle. This increases the reaction yield and surface coverage to provide the basis for a surprisingly sensitive analytic technique which can be used to detect unexpectedly low levels of analyte.

The label of the invention comprises a silica nanoparticle functionalized with at least one dendrimer for conjugating a target analyte to the nanoparticle, so as to allow labelling of the analyte for detection thereof, wherein the dendrimer linker is selected so as to increase the zeta potential of the nanoparticles in a dispersion of the nanoparticles.

In one embodiment, the dendrimer functionalised nanoparticle may be conjugated to a detection molecule, which in turn detects a target analyte. The detection molecule may be highly selective for a corresponding target analyte. By highly selective it is meant that the selectivity for a moiety approaches near specific.

Accordingly, in one aspect of the invention, the label of the invention comprises a functionalised nanoparticle wherein the nanoparticle surface is modified to comprise at least one dendrimer linker for conjugating a detection molecule which is highly selective for a further target analyte thereto, wherein the dendrimer is selected to increase the zeta potential of the nanoparticle. The label comprising the detection molecule can then be used to bind a further analyte of interest in a highly selective manner. The detection molecule in this regard may be highly selective for the further analyte of interest. An example would be use of an antibody as a detection molecule (detection antibody) which is (highly) selective for an antigen (capture molecule) which is the target analyte of interest. It will be appreciated that the detection molecule may be an antigen, for which a particular antibody (capture molecule) is the target analyte.

Similarly, a particular antibody used as a detection molecule, may be used to detect a particular cell expressing a particular antigenic part of a protein.

Accordingly, the labels of the invention are useful tools for biological discovery and biomedical detection, medical imaging and therapeutic applications such as cell labelling, targeted drug delivery, targeted gene delivery, biosensing, cell separation, cell purification and imaging.

Dendrimers are monodispersed compounds with a well-defined structure, in which the number and functionality of the dendrimer functional groups can be tailored to the highly selective bioconjugation required. Use of multivalent or multifunctional dendrimers as linkers in bioconjugated nanoparticle labels leads to a higher efficiency in bioconjugation of, for example, target analytes (e.g., antibodies or antigens), and overall facilitate an improved assay performance with ultra low LOD capability and in a highly sensitive and precise manner. Binding occurs more rapidly and so assays are completed in less time. These are attractive advantages, since such improvements are necessary for reducing costs in medical diagnostics, particularly where large numbers of samples are tested. The results obtained using the labels of the invention are also consistent. They do not vary depending on the batch of labels used.

Greater surface coverage and number of available binding sites for a target analyte leads to more efficient bioconjugation. Generally, the rate of the bioconjugation reaction will be proportional to the concentration of the reactants. In the case of the present invention, dendrimers provide more reactive groups and therefore a higher surface concentration, than would likely be achievable with other monovalent linkers that don't have multiplicity. Rates of bioconjugation of the target analyte to the nanoparticle label are improved. Furthermore, the multivalency of the dendrimer increases the probability that analyte will bind to the nanoparticle surface. These factors lead to superior nanoparticle label performance in assays.

The invention also relates to a dispersion of labels of the present invention in a suitable medium, desirably an aqueous medium.

In a preferred embodiment, the dendrimer is at least one dendrimer selected from the group consisting of Generation 0 to Generation 10 dendrimers, including half generations of dendrimers.

Desirably, the dendrimer is a Generation 0 dendrimer, a Generation 0.5 dendrimer, a Generation 1 dendrimer, a Generation 1.5 dendrimer, a Generation 2 dendrimer, a Generation 2.5 dendrimer, a Generation 3 dendrimer, a Generation 3.5 dendrimer, a Generation 4 dendrimer, a Generation 4.5 dendrimer, a Generation 5 dendrimer, a Generation 5.5 dendrimer, a Generation 6 dendrimer or a Generation 6.5 dendrimer. More preferably, the generation size of the dendrimer may be from Generation 0 to Generation 4.5. The most preferred size is Generation 4.5. Dendrimers of Generation 4.5 or smaller are desirable since, the cost of the higher generations is significant and accordingly they are not ideally suited for use in an inexpensive disposable diagnostic device.

Generation G4.5 dendrimers typically provide an optimum balance between cost and performance.

In a particularly preferred embodiment, the dendrimer is a Generation 4 dendrimer. In another preferred embodiment, the dendrimer is a Generation 0 dendrimer. In yet another preferred embodiment, the dendrimer is a Generation 2 dendrimer. In another preferred embodiment still, the dendrimer is a Generation 3 dendrimer. In an embodiment where Newkome type dendrimers are used, Generation 1 dendrimer are preferred.

In one embodiment, the dendrimer is a hydrophilic or a hydrophobic dendrimer. Desirably, the dendrimer is a hydrophilic dendrimer, since a number of hydrophobic dendrimers (including some starburst dendrimers) are comprised of conjugated aromatic rings. A number of these dendrimers may present water solubility issues. In a preferred embodiment, the dendrimer is a PAMAM dendrimer or a Newkome dendrimer. Suitably, the hydrophilic dendrimer is a PAMAM dendrimer. PAMAM dendrimers are quite water-soluble. Newkome dendrimers may require sonication to assist in dissolving them in water.

It will be appreciated that depending on the surface fuctionalisation of the nanoparticle, the dendrimer and the nanoparticle must have compatible surface chemistries. For example, nanoparticles may comprise non functionalised surfaces such as silica surfaces. However, the surfaces of the nanoparticles of the invention are typically functionalised with groups necessary to facilitate conjugation to the dendrimer. For example, the nanoparticles may be functionalised with amine groups. Accordingly, the dendrimer linker should comprise linking functionalities, for example, carboxylate surface chemistries (or other functionalities capable of linking to amine) so that the groups may react together to link the dendrimer to the nanoparticle surface. Examples of dendrimers having carboxylate surfaces include PAMAM whole generation, such as Generation 0, Generation 1, Generation 2, etc., or Newkome type dendrimers of half generation, e.g., Generation 0.5, Generation 1.5, Generation 2.5 etc. It will also be appreciated that if the nanoparticle surfaces comprise surface carboxylate groups, then dendrimers using amine surface chemistries may be used.

In a preferred embodiment, the nanoparticle may be a silica nanoparticle or a metal oxide nanoparticle. Suitably, the nanoparticle may be a silica particle. Silica is preferred in many applications since it is derivatisable with bioreactive functional groups, is biocompatible, while being a relatively inert substance that prevents agglomeration with other nanoparticles of in a dispersion.

In a preferred embodiment, nanoparticle may be at least one of dye-doped, magnetic, silica or metal doped. Suitably, the nanoparticle may be a dye-doped nanoparticle.

In a preferred embodiment, the nanoparticle is may be a hybrid nanoparticle comprised of a hybrid material and include, noble metal, quantum dot or magnetite doped silica nanoparticles.

Desirably, where the nanoparticle is a dye-doped nanoparticle, the dye-doped nanoparticle may be fluorescence optimized, for example, for near infrared (NIR) electromagnetic radiation.

This makes the label detectable by spectrophotometric techniques.

In a particularly preferred embodiment, the nanoparticle is a dye-doped silica nanoparticle.

Near-infrared dye doped silica NPs, in which the fluorescence has been optimized are preferred since at near infrared wavelengths there is low background interference from the fluorescence of biological molecules, solvent, and substrates. Furthermore, whole blood has a weak absorption in the NIR region, thus reducing the need for whole blood filtering for assays using whole blood. NIR light can also penetrate skin and tissue to several millimetres enabling fluorescence detection in vivo, for example, in dermatological or in-vivo diagnostic devices.

In a preferred embodiment, the nanoparticle may have a diameter in the range of about 10 nm to about 1000 nanometres. The size of the nanoparticle used will depend on the intended application and the size of the target analyte in question. Smaller nanoparticles may used for assay involving small proteins or nucleic acids, whereas larger nanoparticle labels may be used for application involving cells, for example.

The silica surface of the nanoparticle may be functionalised with molecules comprising reactive groups for example at least one one of silane, amines, thiols, phosphates, epoxy, carboxyl, cyano, isothiolcyanate, iodoacetamide, azido, NHS-ester groups. Suitably the molecule comprises at least one organosilane and desirably comprises an organo group comprising at least one of thiol, amine or carboxylate, phosphate, epoxide or isothiocynate functional groups. Desirably, the surface layer further comprises at least one of a Generation 0 to a Generation 10 dendrimer and half generations thereof.

It will be appreciated that the nanoparticle surface is modified to comprise desired functional groups. For example, a suitable organosilane may provide reactive groups, for example, amine functionalities on the surface of the nanoparticle which are suitable for binding to dendrimer having activated (or activatable) carboxylate chemistries. In addition to such silanes, it is desirable to provide phosphonate groups having negative charges on the nanoparticle surface, such phosphonate groups are necessary to stabilize the nanoparticle. An example of a phosphonate providing organosilane is trihydroxypropylsilanemethylphosphonate. The labels of the invention may comprise nanoparticles having such functionalised silica surfaces, wherein the reactive groups may be selected from the group of activatable biochemical groups consisting of: thiol, amine or carboxylate, phosphate, epoxide or isothiocynate.

The functionalizing group or groups will depend on the dendrimer linker required and/or other molecules of interest that can be added to the nanoparticles for various reasons. Suitably, the nanoparticle of the invention may be further functionalised with at least one of a protein, a nucleic acid, or a molecular label, a fluorophore, a nanoparticle or other moiety having a suitable group for binding to a functional group on a surface modified nanoparticle, for example, amine or carboxylic acid. In one embodiment, the label of the invention may comprise a nanoparticle having a modified silica surface comprising amine or carboxylic groups and a plurality of dendrimers. Such dendrimers may be all of the same generation, or may be of different generations, depending on the application. The surface layer may be further modified to comprise at least one of a protein, a nucleic acid, a molecular label, a molecular tag, a fluorophore or a nanoparticle. Suitably, the modified silica surface layer may have a thickness in the range of about 1 nm to about 100 nanometres.

In a preferred embodiment, the target analyte may be a cell, a pathogen, a protein, a molecular label, a molecular tag, a nucleic acid, a detection molecule or a secondary analyte highly selective for a further analyte species.

As mentioned above, a detection molecule highly selective for target analytes can be employed. An example of a detection molecule is a protein, such as an antibody, an antigen, or other moiety having a group suitable for attachment to a dendrimer of the invention, which may be used to selectively bind to a target analyte (for example, antibody, antigen) in an immunoassay.

Desirably, the detection molecule is highly selective for a target analyte selected from the group consisting of: a cell, a pathogen, a protein, a secondary analyte, a molecular label, a molecular tag, and a nucleic acid.

By secondary analyte it is meant a moiety that may selectivelty bind to the target analyte.

The secondary analyte may then be probed for, or detected by a detection molecule that is highly selective for the secondary analyte. This is useful where a detection molecule highly selective for the target analyte is not available.

Suitably, the target analyte is a protein, for example, green fluorescent protein. In a particularly preferred embodiment, the protein may be an antibody, such as a monoclonal antibody, a polyclonal, recombinant antibody, antibody fragment or an antigen. Desirably, the detection molecule may be a monoclonal antibody, a polyclonal antibody or an antigen. Suitably, the antibody is a detection antibody which is highly selective for a target analyte.

By molecular tag is it meant a peptide sequence which is removeably grafted onto a recombinant protein for reasons including facilitating protein detection in assays, protein purification, etc. By molecular label it is meant a chemical moiety with a detectable property, for example, a fluorescent or radioactive molecules which can be attached to a nucleic acid as a detectable group.

In a preferred embodiment, the molecular tag may be biton or streptavdin.

In a preferred embodiment, the molecular label may be a chemosensor, a fluorescent or a radioactive probe. Suitably the fluorescent probe is green fluorescent protein.

In a particularly preferred embodiment, the nucleic acid is a DNA or an RNA.

In a preferred embodiment, the target analyte, the detection molecule or the secondary analyte may be a protein, including a monoclonal antibody, a polyclonal, recombinant antibody, antibody fragment or an antigen.

Accordingly, in a related aspect the label of the invention comprises a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a detection molecule which is highly selective for a corresponding target analyte, wherein the dendrimer increases the zeta potential of the nan o particle.

In a preferred embodiment the detection molecule is a monoclonal antibody, a polyclonal antibody or an antigen.

Desirably, the antibody may be goat antihuman lgG. In a preferred embodiment, the antigen may be polyclonal human lgG.

In another aspect of the invention, there is provided a method of preparing a nanoparticle label for detecting a target analyte comprising the steps of: (i) activating at least one functional group on a provided nanoparticle to provide at least one activated functional group having enhanced reactivity; and (i) condensing the at least one activated functional group with a functional group of a provided dendrimer to link the dendrimer to the nanoparticle; and optionally, (ii) quenching the at least one activated function group.

Suitably, the label may be reactivated before use. Desirably, the method may further comprise the step of attaching at least one detection molecule to the activated label to form a label. Preferably, the detection molecule is highly selective for a target analyte. Desirably, the nanoparticle may be a dye-doped nanoparticle or a magnetic doped nanoparticle. It is preferred that dye-doped nanoparticles are used.

Desirably, the functional group provided on the dendrimer may be a carboxylate or an amine functional group (the nanoparticle needs to have a corresponding group on its surface, for example, amine or carboxylate, respectively). Preferably, the functional group on the dendrimer is a carboxylate group.

The functional group provided on the nanoparticle may be an amine or a carboxylate functional group. The nature of the groups are such that they complement each other, for example, where the dendrimer comprises an activatable carboxylate group, the nanoparticle will be provided with amine functional groups. On the other hand, where activatable carboxylate functional groups are provided on the nanoparticle, the dendrimer may suitably comprise amine groups.

The nanoparticle label formed will comprise an activated (or activatable, if quenched after initial preparation) dendrimer linked to a nanoparticle. In other words, an activated label is formed. The thus formed activated label may be used immediately in an assay. Alternatively, the activated label may be quenched and stored until required for use. The functional groups on the dendrimer may be activated at a later time, when required for use in an assay or other application.

In one aspect of the invention the label of the invention may be used in an immunoassay.

Preferably, the immunoassay is an immunosorbent assay, such as an [LISA, a FLISA, a fluorescent enzyme-linked immunosorbent assay, a sandwich enzyme-linked immunosorbent assay, a sandwich fluorescent enzyme-linked immunosorbent assay, a competitive enzyme-linked immunosorbent assay or a direct binding assay.

Desirably, the label of the invention may be used as a tool for biological discovery and/or biomedical detection, in medical imaging and/or therapeutic applications such as cell labelling, targeted drug delivery, targeted gene delivery, biosensing, cell separation, cell purification and imaging.

Suitably, the label of the invention may be used in a dermatological or in vivo diagnostic device.

In one embodiment, there is provided a method of detecting a target analyte in a test substance using the label of the invention comprising the steps of: (i) activating at least one of the activatable functional groups on the dendrimer to form an activated label; and (ii) exposing the activated label to the test substance comprising the target analyte; and (iii) analysing the test substance for bound label.

Preferably, the test substance is exposed to the activated label under conditions that allow the activated functional group to bind to the target analyte.

In a preferred embodiment, the label of the invention may be used in an immunoassay. By immunoassay, it is meant an assay which measures the concentration of a biochemical substance in a biological liquid, such as serum or urine. Such assays are generally based on the reaction of an antibody to an antigen.

Accordingly, in a related aspect of the invention there is provided a method of directly detecting a target analyte in an immunosorbent assay using the label of the invention comprising the steps of: (i) immobilising a target analyte onto a support; (ii) washing the support to remove unbound target analyte; (iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte; (iv) washing the support to remove unbound activated label; and (v) analyzing for bound target analyte.

Suitably, the target analyte may be may be a cell, a pathogen, a protein, a molecular label, a molecular tag, a nucleic acid, a detection molecule or a secondary analyte which is highly selective for a further analyte species.

Suitably, the immunoassay may be an immunosorbent assay, an [LISA, a FLISA, a fluorescent enzyme-linked immunosorbent assay, a sandwich enzyme-linked immunosorbent assay, a sandwich fluorescent enzyme-linked immunosorbent assay, a competitive enzyme-linked immunosorbent assay or a direct binding assay.

It is preferably that the immunosorbent assay is a plate-based assays for detecting and quantifying biochemical substances, for example, peptides, proteins, antibodies and hormones, etc. Suitably, the target analyte may then be directly detected by exposing it to a suitable label of the invention, for example a nanoparticle label having dendrimer linked to a primary antibody, which is highly selective for the target analyte. Alternatively, the target analyte may be indirectly detected, by exposing the target analyte to a secondary analyte molecule (for example, a primary antibody which is highly selective for a target antigen) before using a label of the invention comprising a primary antibody as a detection molecule that is highly selective to the secondary analyte molecule (nanoparticle label having dendrimer linked to a detection molecule (for example, primary antibody) which is highly selective for the secondary analyte molecule.

Immunosorbent assays do not usually use direct detection methods. Desirably, instead, a detection molecule (for example, a secondary antibody) that has been linked to a nanoparticle label is used to probe for a secondary analyte molecule (for example, a primary antibody which is highly selective for the target analyte), which is bound to the target analyte. Thus, the target analyte is detected indirectly through by probing with the detection molecule for a secondary analyte.

Alternatively, another form of indirect detection may suitably be used involves using a primary or secondary antibody that is labelled with an affinity tag such as biotin. Then a secondary (or tertiary) probe, such as streptavidin that is labelled with the nanoparticle label of the invention, can be used to probe for the biotin tag to yield a detectable signal.

Accordingly, in a related embodiment, there is provided a method of detecting a target analyte in an immunosorbent assay using the label of the invention comprising the steps of: (i) immobilising a target analyte onto a support; (ii) washing the support to remove unbound target analyte; (iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte; (iv) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule; (vi) exposing the activated label to the support so that the detection molecule binds to the secondary analyte molecule; (vii) washing the support to remove unbound activated label; and (viii) analyzing the support for bound label.

Suitably, in the immunosorbent assay, the target analyte (for example, an antigen) may be immobilized onto a support. Desirably, the support is a microplate or a plurality of polystyrene beads or a gel film or the like. Suitably, the immunosorbent assay may be performed in 96-well (or 384-well) polystyrene plates, which immobilize antibodies and proteins, including antigen. Using a plate assists in the separation of the bound material from the unbound material during the assay. Advantageously, unbound material may be washed away to facilitate the assay.

Suitably, the target analyte may be a protein, a detection molecule or a secondary analyte which is selective for a further analyte species. Preferably, the detection molecule and/or the secondary analyte is an antibody. It will be appreciated that an antibody that recognizes the target analyte is called the "primary antibody". It will be further appreciated that if the primary antibody is linked to a nanoparticle label of the invention, then direct detection of the analyte is possible. This technique is useful for immunohistochemical staining of tissues and cells.

Advantageously, the target analyte may then be complexed with the detection molecule (for example, an antibody), which then forms part of the label of the invention.

Suitably, the immunosorbent assay is a sandwich assay. This is desirable since it is one of the most powerful immunosorbent assay formats. This is a type of capture assay which is called a "sandwich" assay because the target analyte to be measured is bound between two primary antibodies, i.e., the capture antibody which is used to immobilize the target analyte onto a support and the label of the invention which comprises a detection antibody which is selective for the target analyte. The sandwich format is preferred since it is a sensitive and robust technique.

In a related embodiment, there is provided a method of detecting a target analyte in a sandwich immunosorbent assay using the label of the invention comprising the steps of: (i) immobilising a target analyte onto a support; (ii) washing the support to remove unbound target analyte; (iii) exposing the support to a secondary analyte molecule which binds to the target analyte; (iv) washing the support to remove any unbound secondary analyte molecule (iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte; (v) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule; (vi) washing the support to remove unbound activated label; (vii) analyzing for bound label.

This type of method involves an indirect immunosorbent assay procedure in which the analyte is detected, by actual detection of a secondary analyte, which is probed for, by the detection molecule of the label of the invention.

The key step of the assay is the immobilization of the target analyte to the support phase (for example, an antigen). Desirably, immobilization of the target analyte can be accomplished by direct adsorption to the assay plate. Alternatively, the target analyte may be immobilized onto the support phase indirectly through use of a capture molecule (for example, an antibody) that has been attached to the plate before it is exposed to the target analyte.

Accordingly, if desired, in the methods of the invention described herein, the first step of immobilising the target analyte onto the support may comprise: (i) immobilising a capture molecule selective for the target analyte onto the support; (ii) washing the support to remove unbound capture molecule; and (iii) exposing the support to the target analyte to immobilise the target analyte onto the immobilised capture molecule.

The method involves directly detects the target analyte through an immunosorbent assay, where the target analyte is bound to a capture molecule which is itself immobilised on the support. Suitably, the capture molecule may be an antibody or an antigen.

In all of the assay methods described herein involve a step of providing a nanoparticle label comprising at least one activatable dendrimer for linking the label to a target analyte, the dendrimer comprising at least one activatable functional group; and activating at least one of the activatable functional groups on the dendrimer to form an activated label. The activated label may then be immediately used in an assay.

Desirably, the methods may be carried out under conditions that allow the activated functional group of the dendrimer on the activated label to bind to the target analyte or to the secondary detection molecule. The target analyte therefore links to the label through the detection molecule or through the secondary analyte as the case may be. The test substance may then be analysed for target analyte bound nanoparticle.

In a particularly preferred embodiment, the nanoparticle label, such as a dye-doped nanoparticle. In a preferred embodiment, the target analyte may be protein, a nucleic acid, an antibody or an antigen. Suitably the target analyte is a protein, such as GFP. Preferably, the target analyte is an antigen, such as human lgG. Desirably, the capture molecule is an antibody, such as goat antihuman lgG. It is preferred that the detection molecule is an antibody, such as goat antihuman lgG. Suitably, the test substance may be tissue, blood or a tissue or blood derivate.

Preferable the support is a micro plate or a plurality of polystyrene beads. Desirably, the support is washed with a buffer, such as N-morpholinoethylsulfonic acid (MES) or Phosphate buffer saline (PBS), polysorbate materials (including polysorbate 20 and 80 such as those sold under the brand name Tween®), sodium dodecyl sulphate. Advantageously, the test substance may be tissue, blood, for example, whole blood or a blood or tissue derivate. Suitably, the test substance sample may be worked up (cleaned up) before assay.

In a particularly preferred embodiment, the activating step necessary to form an activated label involves treating the nanoparticle label (comprising activatable dendrimer, or activatable carboxylate groups on the nanoparticle, if provide in this order) with N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide, and a dehydrating agent. Sulfo N-hydroxysuccinimide and a dehydrating agent are the preferred activating reagents, since use of sulfo N-hydroxysuccinimide provides a more stable solution of the nanoparticle conjugates. Suitably, the dehydrating agent may be 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ([DC). This activating system is capable of activating carboxylate groups on the dendrimer to enhance their reactivity towards amine groups on the nanoparticle surface, (or vice versa, where the nanoparticle comprises activatable carboxylic acid groups and the dendrimer comprises amine surface functionalities). It will be appreciated that other activating system may be suitably used to activate other types of functional groups.

Desirably, the nanoparticle label may be activated by treating the nanoparticle label with N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide and a dehydrating agent 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ([DC). In this embodiment, the dendrimer carboxylate functional groups are made more reactive towards amines groups. Amine groups can be found on the nanoparticle surface. Amine groups can also be found on suitable target analyte or detection molecules. The reaction may take place in a MES buffer. Desirably, the buffer may have a concentration in the range about 1mM to about 1M. Suitably, the pH of the buffer may be in the range of about 3 to about 9. Desirably, the reaction may proceed for an activation period in the range about 10 seconds to about 30 minutes.

In embodiments where the dendrimer has a carboxylate surface chemistry, particular advantages arises from activating the carboxylate groups with N-hydroxysuccinimide (NHS) and a dehydrating agent 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ([DC) in 0.1 M MES buffer, at pH = 6.0, for 30 minutes. Desirably, the ratio of EDC:NHS may be 4:1. Preferably, the N-hydroxysuccinimide (NHS) may be present in a two fold molar excess of NHS reagent per carboxylate group (2:1). These conditions result in conversion of dendrimer carboxylate groups to semi-stable reactive NHS-esters. Typically those reactive NHS-esters have a maximum lifetime of about 60 minutes, an often of about 30 minutes. The availability and the lifetime of the NHS esters on the dendritic linker will determine the reaction efficiency and yields in the conjugation with the biomolecule that needs to be labelled. In a preferred embodiment of the invention, the label comprises activated dendrimer surface modified nanoparticle comprises semi-stable amine reactive NHS-esters.

It is noteworthy to mention that at these conditions, the optimum balance between the rate of aminolysis and the hydrolysis of the dendritic N-succinimidyl esters was observed. This is critical for maintaining high activity and specificity of a conjugated target capture antibody towards the antigen in an immunoassay, for example.

The dendrimers of the nanoparticle label of the invention may be activated more than one.

This is desirable, since the dendrimer label may be pre-prepared and sold in preformed form according to application. When required for use, the dendrimer can be activated in the laboratory.

There is also provided a kit for testing for target analyte, the kit comprising: (i) functionalised nanoparticles; (ii) activatable dendrimer of at least one predetermined Generation; (iii) optionally, capture molecule; (iv) optionally, detection molecule; (v) optionally, support; (vi) instructions for use.

The kit of the invention may further comprise activating reagents. Desirably, the kit may further comprise buffer and/or wash solutions.

Brief Description of the Drawings

The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:-Figure 1 shows examples of the monovalent and multivalent cross-linkers used in this work; Figure 2 shows a micrograph of dye-doped silica nanoparticles; Figure 3 shows dynamic light scattering (DLS) data showing the amount of nanoparticle aggregation (A) as effect of the corresponding linker attached to its surface and (B) change in zeta potential as a function of the change on the surface of the NIRNP; Figure 4 shows normalized fluorescence intensity measured after immobilization of target analyte Green Fluorescent Protein (GFP) onto the G2 modified NP surface under various conditions; Figure 5 shows normalized fluorescence intensity measured after immobilization of target Green Fluorescent Protein on the NP surface modified with sulfo-SMCC or the G2 dendrimer; Figure 6 shows fluorescence linked immunosorbent assays for the detection of hlgG using monovalent and bi-linkers and dendrimer multivalent linkers; Figure 7 shows the total fluorescence signal over background fluorescence signal, F/Fo combined with coefficient of variance, and LOD results for each linker in the fluorescence linked immunosorbent assay for detection of human lgG; Figure 8 shows the rate of change in signal as target concentration changes; Figure 9 shows comparison in the surface coverage of NIR-NP5 when using two different types of dendrimers; Figure 10 shows binding rate of NIR-NP conjugated through various generations of PAMAM dendrimers (left) and plot showing an optimal concentration of the detection antibodies-PAMAM-NP conjugates in a direct binding assay (right); Figure 11 shows the reaction parameters for the dendrimer-activated nanoparticles relative to those observed for an assay performed using antibody coupled directly to the fluorescent dye.

Detailed Description of the Invention

Experimental Results Referring now to the drawings and specifically Figures 1 to 11 inclusive and initially Figure 1.

Figure 1 schematically illustrates a number of the monovalent and multivalent cross-linkers used in this work. There is provided a view of activated dendrimer depicting the activation of the carboxylic acids and the two competing reactions occurring on the activated carbonyl group. The Generation 0 (GO) dendrimer is a tailor made Newkome type dendrimer having seven surface available carboxylate groups. Generation 1 and Generation 2 dendrimer are commercially available Newkome type dendrimers obtained from Frontier Scientific.

Referring now to Figure 2, there is presented a micrograph of dye-doped silica nanoparticles which are approximately 71.16 +1-3.83 nm in total diameter. The shell thickness is approximately 9.00 +/-1.80 nm. This layer comprises an area of silica on the surface of the nanoparticle comprising trihydroxypropylsilanemethylphosphonate (for stabilising the nanoparticle) and amino groups for attachment of the dendrimer linker to nanoparticle surface.

Referring now to Figure 3A and B, and initially Figure A which shows dynamic Light Scattering data showing the amount of nanoparticle aggregation as effect of the corresponding linker attached to its surface. It can be seen that bivalent linkers result in increased particle aggregation in comparison to dendrimer modified nanoparticles. Figure 3B shows the change in zeta potential as a function of the change on the surface of the NIRNP. The addition of the bilinker glutaraldehyde results in lowering of zeta potential relative to a nanoparticle without surface modification, and a modest increase in zeta potential where surface is modified with s-SMCC, whereas dendrimer surface modified nanoparticles show more pronounced increases in zeta potential.

Referring now to Figures 4A and 4B and initially Figure 4A in which there is presented normalized fluorescence intensity measurements taken after immobilization of target analyte Green Fluorescent Protein (GFP) onto the G2 modified NP surface under various conditions. In Figure 4A, it can be seen that by varying the stoichiometric ratio between EDC/NHS for the activation of G2 dendrimer carboxylate groups, the most target protein is bound when the ratio of EDC/NHS is 4:1. Figure 4B shows the effect of varying the activation time of the G2 dendrimer carboxylate groups before reacting with the amino functionalized surface of the NP5 in the first conjugation step. It can be seen that most target protein is bound when the -COOH groups on dendrimer modified nanoparticle are allowed to activate for 30 minutes before conjugation to the nanoparticle surface takes place. Referring now to Figure 5, which shows normalized fluorescence intensity measured after immobilization of target Green Fluorescent Protein on the NP surface modified with sulfo-SMCC or the G2 dendrimer. In these reactions, both the amount of the NIR-NP and the linker (sulfo-SMCC or the G2 dendrimer) were kept constant, and the concentration of the GFP was 1.5 or 3.0 equivalents respectively (1 equivalent represents an amount of protein (in ig) that is necessary for a surface saturation of 1mg of the nanoparticles). This value was calculated according to the formula in Reference (36); This means at the same concentration of target protein, the intensity of the fluorescent signal from the linker modified dyedoped nanoparticle is significantly higher when dendrimer is used as the linker. This results from the fact that more of the target is able to bind using the dendrimer linker that the sulfo-SMCC bilinker and since the target is GFP, more fluorescence is observed. This indicates that the G2 Newkome dendrimer binds more GFP per nanoparticle. Test results for Gi, G2 Newkome and G1.5 to G4.5 of PAMAM test result are presented in Figure 9.

Referring now to Figure 6, which shows results of fluorescence linked immunosorbent assays for the detection of hlgG using monovalent and bi-linkers and dendrimer multivalent linkers. Higher fluorescent signal are observed when label comprising a dendrimer surface modified dye-doped NP is used at the same concentration of target analyte, even at lower concentrations of target human lgG. This clearly indicates that dendrimer linkers allow a more sensitive assay.

Referring now to Figure 7, which shows the total fluorescence signal over background fluorescence signal, F/Fo combined with coefficient of variance, and LOD results for each linker in the fluorescence linked immunosorbent assay for detection of human lgG. It can be seen that the limit of detection of human lgG when label comprising a dendrimer surface modified dyedoped NP is significantly lower than the case when label comprising a monovalent surface modified dyedoped NP is used, or in the case where fluorescent dye directly attaches to the antibody.

Referring now to Figure 8, which shows that rate of change in signal as target concentration changes. The drawing shows a change in the sensitivity of fluorescence for immunosorbent assay with change in concentration of human lgG at low concentration. No sensitivity was observed for monovalent linkers and the organic fluorophore label, Cy5 at low concentrations; Signal was detected at low concentration of target when dendrimer surface modified dyedoped NP labels were used.

Referring now to Figure 9, which shows a comparison in the surface coverage of NIR-NPs when using two different types of dendrimers; For PAMAM dendrimer coverage from 2.5% of nanoparticle surface for GO to 25% coverage for G4 dendrimer, indicating that coverage is better for higher generations.

Referring now to Figure 10, which shows the binding rate of NIR-NP conjugated through various generations of PAMAM dendrimers (left) and plot showing an optimal concentration of the detection antibodies-PAMAM-NP conjugates in a direct binding assay (right). The Figure shows the reaction kinetics in a direct binding assay when the nanoparticles are sensitized through PAMAM G1.5 to G4.5. A proper fit was applied to the plot, based on which Kon and Koff rates were determined.

Figure 11, shows the comparison in binding rate constants (k = k0c+k0ff, offset corrected) obtained directly from the fit as well as the limiting value of relative fluorescence intensity (Iiim,rer offset) at long time (over 600 mm for CRP binding assays described in the present application). The on' rate, k0c, increased with increasing generation number of the dendrimer, consistent with the expected increase in the active surface area. The effect significantly outweighed the effect of decreased diffusion coefficient of the nanoparticle relative to that of the dye-couple antibody While there is a lot of work on the fuctionalisation of silica NP with a reactive organosilane'5'27, optimization of the linker chemistry has not been previously investigated.

Accordingly, the effect of the linker on NP stability and the performance of NP labelled molecules in a sandwich immunoassay was investigated. In particular, the LOD and sensitivity was determined for the detection of human lgG. The NP label results were compared against results from a duplicate assay performed using the same capture antibody labelled with organic fluorophore, Cy5.

For this study, three monovalent bilinkers (glutaraldehyde, sulfo-SMCC and SlAB), and three multivalent linker molecules (dendrimers, Generations zero, one and two) were selected for the bioconjugation of NP with an antibody. Multivalent dendrimers are attracting a great deal of interest in biomedical diagnostics2831, pharmacology/drug delivery32, cancer research33, and nanomedicine34, and their use in research is still unabated now, 30 years after their first reported synthesis by Vogtle35.

It has been found that multivalency of dendrimers leads to a higher efficiency in bioconjugation of detection antibodies and an improved assay performance. Such improvements are necessary for reducing costs in medical diagnostics, particularly where large numbers of samples are tested.

Near-infrared dye doped silica NP5, in which the fluorescence has been optimized were chosen for experimentation (Experimental and theoretical studies of the optimisation of fluorescence from near-infrared dye-doped silica nanoparticles, Nooney RI, McCahey CMN, Stranik 0, Guevel XL, McDonagh C, MacCraith BD, ANALYTICAL AND BIQANALYTICAL CHEMISTRY, Volume: 393, Issue: 4, Pages: 1143-1149, Published: FEB 2009). At near infrared wavelengths, there is low background interference from the fluorescence of biological molecules, solvent, and substrates.

Furthermore, whole blood has a weak absorption in the NIR region, thus reducing the need for whole-blood filtering for assays using whole blood. NIR light can also penetrate skin and tissue to several millimetres enabling fluorescence detection in dermatological or in-vivo diagnostic devices.

The three bivalent linkers used are shown in the top half of Figure 1. The structure and mechanism for attachment of the dendrimers is shown in the bottom half of Figure 1.

Nanoparticles were characterized using transmission electron microscopy and dynamic Light Scattering.

It should be noted that the number of dendrimers that may be accommodated on the nanoparticle surface will depend on the number of free amine groups provided on the nanoparticle surface. Typically, a nanoparticle of the invention may comprise surface amine groups on in the range of approximately 7 to 13% of the surface, with the remainder of the surface comprising stabilizing phosphate groups.

If glutaraldehyde, which may be considered a bi-linker having two potential linking sites, binds with one of its aldehyde groups to all of the free amine groups present on the nanoparticle surface having 13% amine covered surface, only 13% of the surface is available to bind to a target protein. On the other hand, if the Generation 0 dendrimer binds through one of its seven linking sites to all of the free amine groups present on the nanoparticle surface having 13%, there are still another six linking sites available to bind protein. This means that for nanoparticles having the same number of free amino groups on the surface, the number of potential protein or target binding sites is substantially increased by use of a dendrimer linker. While it is assumed that the dendrimer binds to the nanoparticle by a single linking -COOH group, it is possible that the dendrimer could bind to the nanoparticle surface by more than one carboxylic acid group. The multivalency of the dendrimer means that it is possible that each dendrimer may link to multiple amines on the surface of the NP, assuming that the amines are in close to each other.

It is assumed that steric effects would limit the number of target antibodies which are capable of binding to the dendrimer to about four per nanoparticle. The number will also depend on the size of the protein and will increase with increased generation of the dendrimer. The type of target is also an important factor, for example, the number will vary depending on the protein, it will be different for the whole antibody as for some antibody fragments such as Fab and ScFv.

Experimental Section Materials Triton ® X-100 (trademark Union Carbide), n-hexanol (anhydrous, >99%), cyclohexane (anhydrous 99.5 %), ammonium hydroxide (28 % in H20 > 99.99 %), tetraethylorthosilica (TEOS, 99.99 %), aminopropyltrimethoxysilane (APTMS, 97 %), aminopropyltriethoxysilane (APTES, 99 %), 3-(trihydroxysilyl)propyl methyl phosphonate, monosodium salt solution (THPMP, 42 wt % in water), triethylamine (>99 %) absolute ethanol, monobasic sodium phosphate, dibasic sodium phosphate. phosphate buffered saline (PBS, pH 7.4, 0.01 M), Tween ® 20 (trademark Uniqema), glutaraldehyde (25 wt % in water), sodium azide (99.99 %) and albumin from bovine serum (BSA, 98 %) were all purchased from Sigma Aldrich Ireland, sulfo-SMCC and sulfo-SIAB were purchased from Pierce Chemical Company, dendrimers Generations 1 and 2 were purchased from Frontier Scientific UK and used without further purification. However, it will be appreciated that any home-made or commercially available dendrimers that are soluble in the same solvent as the nanoparticles. Dendrimer Generation 0 was synthesized in two simple steps starting from cyanuric chloride as starting material and purified on silica gel chromatography column prior the use37.

Polyclonal CyS conjugated goat antihuman lgG (2.5 mg/mL in PBS), polyclonal goat anti human lgG (5 mg I mL in PBS) and polyclonal human lgG (5 mg/mL in PBS) were purchased from Biomeda Corp, California, USA. Black 96 well plates used in the immunoassay were purchased from AGB Scientific Ireland. Deionised water (< 18 MQ) was obtained from a Milli-Q system from Millipore Ireland.

The dye used in this work is 4,5-Benzo-1-ethyl-3,3,3,3-tetramethyl-1-(4-sulfobutyl) indodicarbocyanin-5-acetic acid N-succinimidyl ester, or more commonly referred to as NIR-664-N-succinimidyl ester (purchased from Sigma Aldrich).This dye has a quantum efficiency of 23 %, a molar absorptivity of 187,000 L mo[' cm' and fluorescence excitation and emission wavelengths of 672 nm and 694 nm, respectively, in isopropanol30. This dye is usually not used in immunoassays because it is not soluble in water.

Synthesis of silica NIR-NP5 Firstly, 15.6 mg NIR-664-N-succinimidyl ester was dissolved in 5 mL anhydrous n-hexanol.

To this solution, 5.021 iL of pure APTES and 3 iL of triethylamine were added. The mixture was agitated for 24 hours to ensure conjugation of the NIR dye to the organosilane. Briefly, the microemulsion was formed by mixing cyclohexane oil phase (15 mL), n-hexanol co-solvent (3.256 mL) and Triton ® X-100 surfactant (3.788 g) in 30 mL plastic bottles. To form the microemulsion, 0.96 mL of deionised water was added and the solution stirred for five minutes. Following this, 0.2 mL of TEOS and 0.l6mL of NH4OH were added to start the growth of the silica NP5. After thirty minutes, 0.344 mL of the NIR-664-APTES conjugate was added. The reaction was stirred for 24 hrs, after which 0.1 ml TEOS was added with rapid stirring.

After 30 minutes, 0.08 mL of the organosilane, 3-(trihydroxysilyl)propyl methyl phosphonate, monosodium salt solution (THPMP), (42 wt % in water) was added with stirring to prevent aggregation of the nanoparticles'9. After a further 5 minutes, 0.02 mL of bioreactive organosilane, aminopropyltrimethoxysilane, (APTMS) was added to and the solution stirred for a further 24 hours. The APTMS has a free primary amine group for crosslinking to biomolecules. The NPs were separated from the solution with the addition of excess absolute ethanol and centrifuged twice with ethanol and once with deionised water. Sonication was used between the washing steps to resuspend the NP5. The NP5 were dispersed in deionised water, at 2.0 mg I mL and stored in the dark at 4°C.

Bioconjugation All bioconjugation reactions were performed with the NIR-NP at 2mg/mL, linker concentration of 4 mM and with 268 pg of goat anti-human lgG. The conjugation protocols were optimized for each linker with respect to the nature of its reactive groups.

Mono valently Linked NIR NP Bioconju gates Glutaraldehyde (4 pmol) was added into a 1 mL solution of NIR-NP (2mg/mL) and gently shaken for 1 hour. The excess of unreacted linker was removed by means of centrifugation (3x) and the precipitates were dissolved in 946 p1 of 0. 1 M PBS buffer, pH=7.4. To this solution, 54 p1 (268 pg) of goat anti-human lgG was added and the mixture was allowed to shake for 4 hours. The excess of the unreacted protein was removed by repeated centrifugation (4x) and the final bioconjugation product was dissolved in 1 mL of 0.1 M PBS buffer, pH=7.4. Sodium azide (NaN3) was further added into the mixture so that its final concentration reached 0.01%.

Multivalently linked NIR NP Bioconju gates The bioconjugation reaction using the heterofunctional linkers comprised of two steps.

First, sulfo-SIAB or sulfo-SMCC (4pmol) were allowed to react with 1 mL solution of NIR-NP (2mg/mL) in 0.1 M PBS buffer, pH=7.2, containing 5 mM of EDTA for 30 minutes, after which the excess of the linker was removed by centrifugation. Second, 2-lminothiolane*HCI (16.8 nmol, Traut's reagent) was reacted with goat anti-human lgG (268 pg, 1.68 nmol) in 250 pL of 0.1 M PBS buffer, pH=8.0, containing 5 mM of EDTA for 1 hour, after which the excess of the unreacted iminothiolane was removed using Zeba desalting column (Pierce Chemical comp.).

Both fractions were subsequently mixed together in a reaction vessel for 4 hours, while keeping the total volume of the solution to 1 mL. The reaction mixture was then purified by repeated centrifugation (4x) and re-dissolved in 0.1 M PBS buffer, pH=7.4, 0.01% NaN3.

Dendrimer NIR NP Bioconju gates The nanoparticles as described herein comprise surfaces modified with -NH2 groups. The - COOH groups of all three generations of the dendrimers were first activated with EDC/NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC); N-hydroxysuccinimide (NHS)) mixture in 0.1 M MES buffer, pH=6.3 before reacting with the protein, goat anti human lgG.

For example, GO (4pmol, 7x -COOH) was dissolved in 0.5 mL of MES buffer. To this solution, NHS (42 pmol, 1.5 equiv. per one -COOH group) and [DC (168 pmol, 6.0 equiv. per one -COOH group) was added, the final volume was adjusted to 1 mL with MES buffer and the reaction was allowed to proceed for 30 minutes. White precipitates that were formed were separated in centrifuge. The clear liquid phase containing the NHS activated dendrimer was then directly added into the NIR-NP (2mg/mL), while keeping the total volume to 1 mL. This mixture was let to react for 30 minutes; subsequently the excess of solution-free dendrimer was removed by centrifugation. The dendritic-modified NIR-NP were re-dissolved in 946 p1 of MES buffer, pH7.O and detection antibody, goat anti-human lgG (135 pg, 54 p1 from stock) was added. The reaction was allowed to shake for 4 hours, then 100 p1 of MES buffer pH=9.O was added to convert the remaining NHS esters into carboxyls and the mixture was purified by centrifugation (4x). The NIR-NP -lgG bioconjugate was re-dissolved in 0.1 M PBS buffer, pH=7.4, with 0.01 % NaN3.

All reactions in which NIR-NPs were used were performed under reduced light (reaction vessels wrapped up in aluminium foil) to prevent photo-bleaching and all final samples were stored in dark at 4°C overnight before they were used in the immunoassay.

Fluorescence linked immunosorbent assay (FLISA) A sandwich assay format was used to test the performance of the NP5 compared to that of a single dye label. In the first step 100 iL of capture antibody, polyclonal goat anti human lgG, at 5 ig I mL was added to each well of a standard 96 well microplate. The plate was then incubated overnight at 4°C. To remove any non-adsorbed antibody the plate was rinsed three times with PBS and three times with PBS/0.05 wt % Tween ®. To prevent non-specific adsorption, 200 iL of 1 wt % BSA in PBS was added to each well and the plate incubated at 37 °C for 1 hour. The rinse cycle was then repeated. Following this, 100 pL aliquots of target analyte, antigen human lgG in 0.1 wt % BSA were added in a series of dilutions from 10,000 ng I mL to 0.1 ng I mL to each well and the plate incubated at 37 °C for 1 hour. The rinse cycle was repeated to remove any non-specifically bound human lgG. Finally, 100 iL aliquots of polyclonal goat anti-human lgG conjugated NP5 at 0.2 mg I mL were added to each well and the plate incubated for a further hour at 37 °C in the dark. Prior to analysis the plate was rinsed one more time. To compare the assay with a standard organic dye, the NP label was replaced with Cy5-conjugated goat antihuman lgG label at a concentration of 0.025 mg I mL.

The fluorescence signal including standard deviation at each concentration of human lgG was determined from seven replicate experiments. The complete assay was fitted using a standard sigmoidal logistics fit (see below, left hand side) and the low concentrations of human lgG were fitted using an allometric power function (see below, right hand side). / \m

10'max,-(C +fmax F=Fo+k-where, F is the fluorescence at concentration, C, F0 is the background fluorescence, Fmax is the maximum fluorescence, P is the power, and C0 is the point of inflection. For the allometric function, additional random variables, k, I, and m are required.

The LOD of human lgG was defined as the concentration corresponding to a fluorescence signal 3 times the standard deviation of the background signal. The sensitivity of the assay (change in fluorescence signal with concentration of human lgG) was determined at a range of concentrations through differentiation of the allometric power function. Previous work had indicated that the sensitivity of an assay is given without relating it to the concentration at which the sensitivity was measured. This can be misleading, since sensitivity does not change linearly with changing concentration.

Instrumentation TEM micrographs were obtained using a Hitachi 7000 Transmission Electron Microscope operated at 100 kV. Images were captured digitally using a Megaview 2 CCD camera. Specimens were prepared by dropping aqueous solutions of the NP5 onto a formvar carbon coated copper grid. Fluorescence measurements were performed on a Safire (Tecan) microplate reader. For NIR- 664-N-succinimidyl ester-doped NP5, the excitation and emission wavelengths were set at 672 nm and 700 nm, respectively. Dynamic light scattering (DLS) measurements were performed on a Zetasizer from Malvern instruments to yield values of zeta potential () for the NP5.

Results and Discussion As seen in Figure 3, the monovalent linkers, glutaraldehyde and sulfo-SMCC have destabilizing effect on the NP with the tendency to form aggregates. Sulfo-SMCC shows two different populations, the first transition corresponds to the formation of nanoparticle trimers or tetramers, with an average diameter of 275 nm, and the second corresponds to larger aggregates with polydispersed characteristics.

On the contrary, NP modified with the multivalent dendrimers Gi and G2 show only a small increase in particle diameter, increasing the zeta potential from -29.8 to -47.9 and -46.0 for Gi and G2 respectively. The increased colloidal stability of dendrimer coated NP5 is due to the significant increase in the zeta potential on addition of the linker. This is because the NHS ester activated groups on the dendrimer are susceptible to hydrolysis, which leads to the formation of carboxylic groups that are charged at physiological pH. Therefore, the electrostatic charge on the NP is increased and colloidal stability is maintained.

Immobilization of a model protein, Green Fluorescent Protein (GFP), on the modified NP surface seemed to have little effect on the zeta potential of the NP modified with the Gi and G2, while the changes in case of Glu and sulfo-SMCC were more dramatic. The GFP attaches to the dendrimers. It was a surprising result to observe that the addition of the GFP did not detrimentally affect the zeta potential. It is therefore possible that there remains a large number of carboxyl groups on the surface to keep the electrostatic repulsion at a high level. This experiment was carried out to determine how much antibody would bind to the NP5. The GFP is used as a substitute for a real antibody. From the fluorescence of the GFP the number of GFP5 that are bound to the surface of our NP can be determined. The bioconjugation of the GFP on the dendrimer-modified surface was performed via a well-known technique for the conjugation of carboxyl (on the dendrimer) to amine groups (on the GFP) in peptides and proteins. The reaction between carboxylic acids and amines to form stable amides is commonly facilitated by an addition of a carbodiimide, such as 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ([DC).

However, [DC itself is not particularly efficient in linking because in the first step it forms an unstable 0-acylisourea ester that is prone to fast hydrolysis and regeneration of the carboxyl. The carboxylic acids of the dendrimers were therefore activated in the presence of a mixture of N-hydroxysuccinimide (NHS) and a dehydrating agent, [DC. NHS/EDC mixture is commonly used to convert -COOH groups to semi-stable amine reactive NHS-esters.

The availability and the lifetime of the NHS esters on the dendritic linker will determine the reaction efficiency and yields in the conjugation with the biomolecule that needs to be labelled.

Therefore, we decided to explore on the effect of some of the variables present in the EDC/NHS activation step such as the pH of the solution, the reaction time needed for the COOH activation, the molar excess of NHS per COOH group and the ratio of the EDC/NHS co-reactants.

It is noteworthy to mention that at these conditions, the optimum balance between the rate of aminolysis and the hydrolysis of the dendritic N-succinimidyl esters was observed. This turned out to be very important for maintaining high activity and specificity of the labelled antibody towards the antigen in the immunoassay.

After much experimenting with various parameters, the optimal conditions have been found and are as follows: * reaction medium -0.1 M MES buffer, pH = 6.0; * activation time -30 minutes; * two fold molar excess of NHS reagent per COOH group; and * 4:1 ratio of EDC:NHS (See Figure 4, in particular).

Another very important parameter in the bioconjugation protocol is the concentration of the biomolecule that will comprise a monolayer on the NP surface. This amount is usually estimated and depends upon factors such as the molecular weight of the protein, its relative affinity for the particle and the particle size36. While the amount of protein required to achieve surface saturation can be calculated, the actual protein concentration in the reaction may be substantially higher than the "monolayer = 1 equivalent of protein" value. The optimal value is usually given by the coupling efficiency, concentration of the target molecules and the amount of the non-specific binding and is usually determined experimentally. Therefore, the effect of the concentration of the protein on the reaction yield was investigated. The coupling efficiency of two linkers, sulfo-SMCC and G2 in the bioconjugation reaction using a model protein (GFP) was compared. The reactions were carried out at two different concentrations of the GFP. First reaction was run with the amount of GFP representing 1.5 equivalent of the theoretical value necessary for surface saturation, and second reaction was performed using 3.0 equivalents. Figure 5 shows the effect of the increased concentration of GFP from 1.5 to 3.0 equivalents on the conjugation efficiency. The normalized fluorescence signal for sulfo-SMCC has nearly doubled at the higher GFP concentration (3.0) when compared to the lower GFP concentration (1.5). Nevertheless, it was still significantly lower than the signal observed for the multivalent G2 linker, and that applies even for the case when the G2 sample contained only 1.5 equivalents of GFP. It is also noteworthy that the differences between the conjugation efficiency (number of protein units (GFP) bound per nanoparticle) of the two G2 samples were very small. It suggests that the multivalency of the dendrimer is a significant factor responsible for the improvement in the reaction yields of conjugate, even at lower protein concentration. This is very important, particularly when considering the cost of the bioorganic material that is used in bioconjugation reactions.

Fluorescence linked Immunosorbent assay To test the performance of the dye-doped silica NPs, a standard fluorescence linked immunosorbent assay for the detection of human lgG was carried out. A sandwich assay format was used, in which the capture and detection antibodies were polyclonal goat anti human lgG. Six different NP assays where performed using each of the mono-or multi-valent bilinkers. A further assay was performed using the same antibody labelled with the organic fluorophore Cy5. As seen in Figure 6, all assays showed standard sigmoidal behaviour, excluding NP5 prepared using G2 dendrimer, where it can be seen that the signal did not saturate at the highest concentrations.

Saturation was not achieved since, at the time of the assay, sufficient dendrimer material was not available to prepare the dendrimer standards at the highest concentrations. It is expected that when saturated standard solutions are used, the signal behaviour will be sigmoidal. The normalized fluorescence ratio, at the highest concentration of human lgG, (FmajFo) for multivalent NP labels and the organic Cy5 label was significantly greater than the fluorescence achieved from labels prepared using monovalent linkers (Figure 6 and Figure 7). Therefore, the number of NP labels bound to human lgG is much greater than the number bound non-specifically. Fmax is the total nanoparticle signal (from specifically bound NP5 and non specifically bound NP5), Fo is the signal from non specifically bound NP5. The normalized ratio is Fmax divided by Fo. If this ratio is high then this indicates that there is a lot more specifically bound sample than non specifically.

This is indicative of a good assay.

As seen in Table 1, all the multivalent labels and the Cy5 label showed lower LOD than the monovalent linkers. Moreover, the LOD for Gi dendrimer was greater than an order of magnitude lower than that for the Cy5 label. Therefore, multivalent NP are sensing lower concentrations of antigen human lgG and must be bound more strongly to the antigen coated surface on the plate. If lower concentrations of target are sensed, then the forces of interaction between the detection antibody coated NP and the antigen must be stronger. If binding is observed at lower concentrations, then the free energy is greater, indicating a stronger reaction between the nanoparticle and the target antigen.

From the work presented herein on the bioconjugation of GFP and other studies, it is likely the multivalent NP5 are coated with several detection antibodies. This would increase the number of binding sites per label, increasing the reactivity of the label. Furthermore, because the footprint of a single NP label is significantly larger than the diameter of antigen, it may be possible for two antigens to bind to a single NP at the same time. The LOD for the dendrimeric NP5 compares favorably with published data on the detection of biotinylated hlgG using avidin labelled silica NPs, where an LOD of 1.9 mg I mL was observed26.

For all three labels prepared using dendrimeric bi-linkers the assay showed good sensitivity at low concentrations of human lgG, with G1 showing the highest response (see Figure 8). For NP labels prepared using the monovalent bilinker and the Cy5 label no sensitivity was observed at concentrations of human lgG below 1 ng I mL. At higher concentrations all samples showed reasonable sensitivity.

Successful detection devices for biomedical diagnostics require high sensitivity and low LOD. Moreover, a device for point-of-care diagnostics, must be both inexpensive and reliable under a variety of experimental conditions. The data presented in this paper focused on fine-tuning the bioconjugation protocol in the labelling of biologically relevant material with NP5. We employed two types of linkers: monovalent linkers, glutaraldehyde; sulfo-SMCC; and sulfo-SIAB, dendrimers linker, in particular three generations of dendrimers with multivalent carboxylic functionality. The NP labels were compared against a commercial fluorophore label for the detection of human lgG. The NPs prepared using dendrimer Generation 1 showed a significantly lower LOD and higher sensitivity than the commercial label. We believe the multivalency of the dendrimers is one of the most significant factors behind the increase in the detection sensitivity.

As revealed in this work, dendrimers have a positive effect on NP stability and aggregation.

Moreover, they are more reactive with biological materials, whilst maintaining activity and specificity to the analyte of interest. These properties are required for managing cost, in the design and implementation of biomedical devices, where expensive biological materials are necessary.

Dendrimers are structurally appealing molecules that have already shown their high potential in the surface science of bioassay devices and this study confirmed that they are emerging as a powerful tool for the labelling of biomolecules in biomedical diagnostics as well.

It is shown how performance of an assay can be significantly improved when using multifunctional linkers such as dendrimers in the bioconjugation protocols. We demonstrated that with Newkome type dendrimers, Gen 1, the LOD was as low as 0.31 ng/mL. Nonetheless, the percentage of the NP surface area covered with the conjugated protein is still relatively low.

The conjugation protocol was further optimized and the surface coverage improved by using different type of dendrimers, polyamidoamine (PAMAM) dendrimers. PAMAM are the most common class of dendrimers suitable for many materials science and biotechnology applications.

They are inexpensive, well soluble in aqueous media and come readily available in a range of generations.

As seen on Figure 9, PAMAM dendrimers can increase the reaction yield and surface coverage by more than 20 times when compared to Newkome type dendrimers. It is believed that the protein coverage of the dye-doped nanoparticles is crucial element when designing point-of-care device as the key step in such a device is the binding rate of the detection antibodies. We hypothesize that the higher is the density of the detection antibody/protein on the NP surface, the faster is the kinetic response. This is very difficult to achieve with antibodies labelled with a single dye.

Preliminary data from a direct binding CRP assay demonstrate the clear advantage of using higher generation of PAMAM dendrimer. Generation 4 PAMAM has a binding rate nearly two times higher than its generation 1 PAMAM counterpart (Figure 10).

A point-of-care device requires low limit of detection, high sensitivity and ease of fabrication. But it also requires fast (usually within minutes) and precise detection, with a minimal error caused by a personnel manipulation. The use of dendrimers in the conjugation protocols seems to be a robust and reasonable way to prepare labelled antibodies necessary for [LISA type fluorescence assays with short response times and high sensitivity.

Conclusions

The labels of the invention present an opportunity for improvements of sensitivity in immunoassays through the use of dendrimers as antibody coupling agents for sensitisation of fluorescent nanoparticles.

Nanoparticles are useful as labels for antibodies in immunoassay procedures. As stated earlier, they can however suffer from some disadvantages including particle aggregation and non-specific binding to the capture surface. Another issue in the use of nanoparticles as labels is the fraction of the antibody that is coupled to the particle that is in fact active for the reaction with antigen. This fraction can be rather small, which in turn can lead to diminished sensitivity and worsened non-specific binding.

One important element that affects the activity of the bound antibody and the non-specific aggregation and surface binding of particles is the means used for attachment of the antibodies.

We have demonstrated significant improvements in the antigen-recognition activity of the sensitised particles, that correlates directly with the generation number of the dendrimer used for coupling.

We have also demonstrated that the use of dendrimers as the coupling agents suppresses non-specific binding effects to less than the level exhibited by a molecularly-labelled antibody.

The net effect is a significantly improved signal/background.

The capture reaction, of particles onto the capture surface, can be written P + S -*PS with forward rate constant k0 and reverse rate constant k0ff. Here, S denotes an active site on the capture surface. If the particle is bound to the surface by a single antibody-antigen interaction, then k0ff will simply be the dissociation rate constant for the antigen-antibody complex. The association rate, k0, will be determined by the probability of a reactive collision between a particle and an un-occupied reactive site on the surface. Particles are in a continuous state of collision with the surface, at a rate determined by the diffusion coefficient of the particles and by their concentration. A reactive collision, leading to coupling of the particle to the surface, would occur when a reactive part of the surface of the particle (an antibody, oriented with the binding site exposed) collides with a reactive part of the surface (an antigen, oriented with the epitope exposed).

In the absence of any forces favouring a particular orientation of the particle with respect to the surface during approach and collision, the probability that the collision will be with an active site on the particle surface will simply be the fraction of the particle surface area that is covered by active antibody. As the particle concentration on the surface builds up, then the particles not only occupy sites but also physically block the surface area, on account of their size. So, the probability that the collision will be with an active site on the capture surface will simply be the fraction of the total surface that is unblocked by bound particles, times the fraction of this available capture surface area that is covered by active antigen, times a coverage-dependent factor that expresses the requirement that a particle requires a space whose smallest dimension is at least as large as the particle diameter, in order that the particle can fit into the space. This statement assumes that the approach and collision of particles is unaffected by the presence of previously bound particles except insofar as these occupy surface area. II k0 is sufficiently low, then the requirement that particles can fit into the spaces available leads to a limiting coverage -the jamming limit' -which is approximately 53% of the total surface area for random sequential irreversible absorption of objects of circular cross-section.

If the coverage of the capture surface by bound particles is sufficiently low, then the effects of particles jamming the surface can be ignored, and the increase of particle coverage, and hence of surface-bound fluorescence, will follow a first-order rate equation: " {i -e-c+t) (1) L;_ Where I denotes the bound fluorescence intensity at time, t, with hypothetical value max if the surface were fully covered with fluorophore, and c denotes the particle concentration in the solution. The capture rate constant, k0 can be written = where kD is the diffusion-limit rate constant for particle consumption by the surface, dependent on the particle radius and the viscosity of the medium, e is the reaction efficiency for a reactive collision, q is the fraction of the capture surface that is active for the capture reaction, in this case dependent on the surface coverage of adsorbed antigen, and qp is the active fraction of the particle surface, in this case dependent on the surface coverage of active antibody attached to the particle.

The objective of surface fuctionalisation with dendrimer is to increase qp. The exponential increase of average cluster volume with time in the reaction-limited aggregation of antibody-sensitised particles in the presence of the multi-valent antigen can similarly be described in terms of the probability of capture of antigen onto the surface of a particle or onto a particle at the outside of a cluster times the probability that this site will collide with an active site on another particle or cluster. Thus the rate constant for average cluster volume increase should increase approximately as (qp)2 for a given initial concentration of particles and antigen.

Figure 10 shows the fit of the bound fluorescence intensity to equation 1, with the addition of a fixed offset that we take to represent a non-specific adsorption of the sensitised particles.

In Figure llwe present the reaction parameters for the dendrimer-activated nanoparticles relative to those observed for an assay performed using antibody coupled directly to the fluorescent dye rather than to the nanoparticles loaded with fluorescent dye. The limiting value of relative fluorescence intensity at long time (over 600 mm for CRP binding assays described in the present application) is -= , where F is a scaling factor.

The rate constant k = k0c+k0ff is obtained directly from the fit. Thus the values of k0c scaled relative to one another can simply be obtained from the relative values of the product k(Iiim,reil offset). The similarity of the values of (11im,rerloffset) scaled relative to the dye-coupled antibody with those of k0c similarly scaled implies that k0ff >> k0c. The variation of the rate constant, k, is thus that of the off rate, kQff: as expected, this was within the experimental uncertainty the same for the dye-labelled and particle-labelled antibodies, and not affected by the nature of the dendrimer coupling compound (Figure 11). The on' rate, k0c, increased with increasing generation number of the dendrimer, consistent with the expected increase in the active surface area. The effect significantly outweighed the effect of decreased diffusion coefficient of the nanoparticle relative to that of the dye-couple antibody.

The other important characteristic of the labels to consider for use in an assay is the ratio of signal to background: in this case, the ratio of the equilibrium fluorescence (offset corrected) to the non-specific offset. Again, a significant advantage is noted for the dendrimer-sensitised nanoparticle labels. The offset was hardly affected, indeed may have been reduced. The signal was significantly enhanced so the relative value of signal/non-specific background was increased by a factor of 3 as a consequence of the use of the dendrimer-sensitised nanoparticles as the label.

All in all, these effects lead to the provision of superior labels for use in assays capable of detecting target analyte to the ultra low LOD level.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

It will be readily apparent to one of ordinary skill in the art that the examples disclosed herein below represent generalised examples only, and that other arrangements and methods capable of reproducing the invention are possible and are embraced by the present invention.

References (1) Schaferling M.; NagI, S.; Anal Bioanal Chem 2006, 385, 500-517.

(2) Thaxton C. S.; Georganopoulou D. G.; Mirkin C. A.; Clinica Chimica Acta 2006, 363, 120-126.

(3) Lim M. L. R.; Lum M. G.; Hansen T. M.; et al.; J. Biom. Sc. 2002, 19, 488-506.

(4) Davidson, R. S.; Hilchenbach, M. M.; Photochemistry & Photobiology 1990, 2, 431-438.

(5) Yang, H-H.; Qu, H-Y.; Lin, P.; Li, S-H.; Ding; M-T, Xu, J-G.; Analyst 2003, 128, 462-466.

(6) Lowe, M.; Spiro, A.; Zhang, Y. Z.; Getts, R.; CytometryPartA 2004, 60A, 135-144.

(7) Binder, H.; Kirsten, T.; Hofacker, I. L.; Stadler, P. F.; Loeffler, M.; J. Phys. Chem. B 2004, 108, 18015-18025.

(8) Takahashi, M.; Nokihara, K.; Mihara, H.; Chemistry & Biology 2003, 10, 53-60.

(9) Zhang, 0.; Guo, L-H.; Bioconjugate Chem. 2007, 18, 1668-1672.

(10) Sukhanova, A.; Devy, J.; Venteo, L.; Kaplan, H., Artemyev, M.; Oleinikov, V.; Klinov, D.; Pluot M.; Cohen J. H. M.; Nabiev, I.; Analytical Biochemistry 2004, 324, 60-67.

(11) Hun, X.; Zhang, Z.; Biosensors and Bioelectronics 2007, 22, 2743-2748.

(12) Burns, A.; Ow, H.; Wiesner, U. Chemical Society Reviews 2006, 35, 1028-1042.

(13) Yan, J. L.; Estevez, M. C.; Smith, J. E.; Wang, K. M.; He, X. X.; Wang, L.; Tan, W. H. Nano Today 2007, 2, 44-50.

(14) Fowler, C. E.; Lebeau, B.; Mann, S. Chemical Communications 1998, 1825-1826.

(15) Bagwe, R. P.; Hilliard, L. R.; Tan, W. H. Langmuir 2006, 22, 4357-4362.

(16) Smith J. E.; Wang, L.; Tan, W. T.; Trac-Trends in analytical Chemistry 2006, 25, 848-855.

(17) He, X.; Chen, J.; Wang, K.; Qin, D.; Tan, W.; Talanta 2007, 72, 1519-1526.

(18) Santra, S.; Liesenfeld, B.; Bertolino, C.; Dutta, D.; Cao, Z.; Tan, W.; Moudgil, B. M.; Mericle, R. A.; J. Luminescence 2006, 117, 75-82.

(19) Zhou, X.; Zhou, J.; Anal. Chem. 2004, 76, 5302-5312.

(20) Gouanve, F.; Schuster, T.; Allard, E.; Meallet-Renault, R.; Larpent, C.; Adv. Funct. Mater.

2007, 17, 2746-2756.

(21) Stranik, 0.; Nooney, R.; McDonagh, C.; McCraith, B. D.; Plasmonics 2007, 2, 15-22.

(22) Qhobosheane, M.; Santra, S.; Zhang, P.; Tan, W.; Analyst 2001, 126, 1274-1278.

(23) Santra, S.; Dutta, D.; Walter, G. A.; Moudgil, B. M.; Technology in Cancer Research & Treatment 2005, 4, 593-602.

(24) Yang, H. H.; Qu, H. Y.; Lin, P.; Li, S. H.; Ding, M. T.; Xu, J. G. Analyst 2003, 128, 462-466.

(25) Corrie, S. R.; Lawrie, G. A.; Trau, M. Lan gmuir 2006, 22, 2731-2737.

(26) Lian, W.; Litherland, S. A.; Badrane, H.; Tan, W. H.; Wu, D. H.; Baker, H. V.; Gulig, P. A.; Lim, D. V.; un, s. G. Analytical Biochemistry 2004, 334, 135-144.

(27) Borchers, K.; Weber, A.; Brunner, H.; Tovar, G. E. M.; Anal. Bioanal. Chem. 2005, 383, 738-746.

(28) Rozkiewicz, D. I.; Brugman, W.; Kerkhoven, R. M.; Ravoo, B. J.; Reinhoudt, D. N. Journal of the American Chemical Society 2007, 129, 11593-11599.

(29) Bocking, T.; Wong, E. L. S.; James, M.; Watson, J. A.; Brown, C. L.; Chilcott, T. C.; Barrow, K. D.; Coster, H. G. L. Thin Solid Films 2006, 515, 1857-1863.

(30) Benters, R.; Niemeyer, C. M.; Wohrle, D. Chembiochem 2001, 2, 686-694.

(31) Ajikumar, P. K.; Kiat, J.; Tang, Y. C.; Lee, J. Y.; Stephanopoulos, G.; Too, H. P. Langmuir 2007, 23, 5670-5677.

(32) Beezer, A. E.; King, A. S. H.; Martin, I. K.; Mitchel, J. C.; Twyman, L. J.; Wain, C. F. Tetrahedron 2003, 59, 3873-3880.

(33) Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R. Biomacromolecules 2006, 7, 572-579.

(34) Majoros, I. J. B., J. R.; Dendrimer-based Nanomedicine; Pan Stanford Publishing, 2008.

(35) Buhleier, E.; Wehner, W.; Vogtle, F. Synthesis-Stuttgart 1978, 155-158.

(36) a) Bangs Laboratories, Inc.; TechNote 205, Rev. 003, www.bangslab.com; b) Cantarero, L. A.; Butler, J. E.; Osborne, J. W. Anal. Biochem. 1980, 105, 375-382 (37) Intermediate 1: To a pressure vessel containing 100 mL of CH2CI2:MeOH (1:1), 2-Amino-2- (hydroxymethyl)-1,3-propanediol (3.9 g, 33 mmol) and Hunig's base (8.4 g, 65 mmol) were added. The mixture was cooled in an ice bath before addition of cyanuric chloride (3 g, 16 mmol). The reaction was then allowed to warm up to room temperature and stirred for 24 hours before 4-piperidine carboxylic acid (8.4 g, 65.2 mmol) and NH4OH (5 mL) were added.

The reaction mixture was then heated up to 60°C and stirred overnight. Upon cooling, the precipitates were filtered and the solvent was removed by evaporation. The residue was re-dissolved in water (20 mL) and acidified to pH = 4-5. Precipitates were formed, filtered off and washed extensively with water to give off-white powder (3.44 g, 47 % yield). This intermediate was used in the second step without further purification.

Dendrimer GO: Intermediate 1 (0.5 g, 1.1 mmol) and diglycolic anhydride (3 g, 16 mmol) were placed in a sealed vessel and irradiated in a CEM microwave at 300 W, 120°C for 5 minutes.

Upon cooling, precipitates were formed, the whole mixture was dissolved in water and the pH was adjusted to 4-5. The solution was concentrated and the residue was purified on silica gel chromatography column with MeOH/CH2CI2 (5:95) as an eluent to yield oily product (440 mg, 35% yield).

Claims (60)

1. Claims 1. A label comprising: a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a target analyte to the functionalised nanoparticle, wherein the dendrimer has a stabilizing effect on the zeta potential at the nanoparticle surface.
2. 2. A dendrimer according to claim 1 wherein the stabilizing effect results from an increase in the zeta potential at the nanoparticle surface.
3. 3. A dendrimer according to any preceding claim wherein the zeta potential has an absolute value greater than 20 (mV).
4. 4. A label according to any preceding claim wherein dendrimer functionalised nanoparticle may be conjugated to a detection molecule which detects a target analyte.
5. 5. A label according to claim 4 wherein the detection molecule is selective for a particular target analyte.
6. 6. A label according to any preceding claim wherein the dendrimer is at least one dendrimer selected the group consisting of: Generation 0 to Generation 10, including half generations thereof.
7. 7. A label according to claim 6 wherein the dendrimer is at least one of a Generation 0 dendrimer, a Generation 0.5 dendrimer, a Generation 1 dendrimer, a Generation 1.5 dendrimer, a Generation 2 dendrimer, a Generation 2.5 dendrimer, a Generation 3 dendrimer, a Generation 3.5 dendrimer, a Generation 4 dendrimer, a Generation 4.5 dendrimer, a Generation 5 dendrimer, a Generation 5.5 dendrimer, a Generation 6 dendrimer or a Generation 6.5 dendrimer.
8. 8. A label according to any preceding claim wherein the dendrimer is a Generation 4 dendrimer.
9. 9. A label according to any one of claims 1 to 7 wherein the dendrimer is a Generation 0 dendrimer.
10. 10. A label according to any one of claims 1 to 7 wherein the dendrimer is a Generation 2 dendrimer.
11. 11. A label according to any preceeding claim wherein the dendrimer is a hydrophilic dendrimer.
12. 12. A label according to any preceeding claim wherein the dendrimer is a PAMAM dendrimer or a Newkome dendrimer.
13. 13. A label according to any preceeding wherein the nanoparticle is a silica nanoparticle or a metal oxide nanoparticle.
14. 14. A label according to any preceeding claim wherein the nanoparticle is at least one of dye-doped, magnetic, silica or metal doped.
15. 15. A label according to any preceeding claim wherein the nanoparticle is a hybrid nanoparticle comprised of a hybrid material and include, noble metal, quantum dot or magnetite doped silica nanoparticles.
16. 16. A label according to claim 14 wherein the dye-doped nanoparticle is fluorescence optimized for near infrared electromagnetic radiation.
17. 17. A label according to any preceeding claim wherein the nanoparticle has a diameter in the range of about 10 nm to about 1000 nanometres.
18. 18. A label according to any preceeding claim wherein the nanoparticle has a surface layer comprising an organosilane with one or more functional groups, for example, amines, thiols, phosphates, epoxy, carboxyl, cyano, isothiolcyanate, iodoacetamide, azido, NHS-ester.
19. 19. A label according to claim 18 wherein the organosilane comprises an organo group comprising at least one of thiol, amine or carboxylate, phosphate, epoxide or isothiocynate functional groups.
20. 20. A label according to any one of claims 18 to 19, wherein the surface layer further comprises at least one of a Generation 0 to a Generation 10 dendrimer and half generations thereof.
21. 21. A label according to any preceeding claim wherein the surface layer has a thickness in the range of about 1 nm to about 100 nanometres.
22. 22. A label according to any preceeding claim wherein the nanoparticle surface is further functionalised with at least one of an activatable biochemical group including thiol, amine or carboxylate, phosphate, epoxide or isothiocynate.
23. 23. A label according to any preceeding claim wherein the nanoparticle is further functionalised with at least one of a protein, a nucleic acid, a molecular label, a molecular tag, a fluorophore or a nanoparticle.
24. 24. A label according to any preceeding claim wherein the target analyte is a cell, a pathogen, a protein, a molecular label, a molecular tag, a nucleic acid, a detection molecule or a secondary analyte selective for a further analyte species.
25. 25. A label according to any one of claims 23 or 24 wherein the molecular tag is bioton or streptavadin.
26. 26. A label according to any one of claims 23 to 25 wherein the molecular label is a chemosensor or a protein fluorescent probe.
27. 27. A label according to any one of claim 23 to 26 wherein the protein is green fluorescent protein.
28. 28. A label according claim 24 wherein the target analyte, the detection molecule or the secondary analyte is protein, including a monoclonal antibody, a polyclonal, recombinant antibody, antibody fragment or an antigen.
29. 29. A label comprising a nanoparticle wherein the nanoparticle surface is functionalised to comprise at least one dendrimer having at least one activatable functional group for conjugating a detection molecule selective for a corresponding target analyte, wherein the dendrimer increases the zeta potential of the nanoparticle.
30. 30. A label according to claim 29 wherein the detection molecule is a monoclonal antibody, a polyclonal antibody or an antigen.
31. 31. A label according to any one of claims 24 to 30 wherein the antibody is goat antihuman lgG.
32. 32. A label according to any one of claims 24 to 31 wherein the antigen is polyclonal human lgG
33. 33. Use of a label according to any preceeding claim in an immunoassay.
34. 34. Use of a label according to claim 32 wherein the immunoassay is an immunosorbent assay, such as an [LISA, a FLISA, a fluorescent enzyme-linked immunosorbent assay, a sandwich enzyme-linked immunosorbent assay, a sandwich fluorescent enzyme-linked immunosorbent assay, a competitive enzyme-linked immunosorbent assay or a direct binding assay.
35. 35. Use of a label according to any one of claims 1 to 34 as a tool for biological discovery and/or biomedical detection, in medical imaging and/or therapeutic applications such as cell labelling, targeted drug delivery, targeted gene delivery, biosensing, cell separation, cell purification and imaging.
36. 36. Use of a label according to any one of claims 1 to 34 in a dermatological or in vivo diagnostic device.
37. 37. A method of preparing a nanoparticle label according to any one of claims 1 to 32 comprising the steps of: (i) activating at least one functional group on a provided nanoparticle to provide at least one activated functional group having enhanced reactivity; and (ii) condensing the at least one activated functional group with a functional group of a provided dendrimer to link the dendrimer to the nanoparticle; and optionally, (iii) quenching the at least one activated function group.
38. 38. A method according to claim 37 wherein the label is reactivated before use.
39. 39. A method according to any one of claims 37 or 38 further comprising the step of attaching at least one detection molecule to the activated label to form a label.
40. 40. A method according to claim 39, wherein the detection molecule is selective for a target analyte.
41. 41. A method of detecting a target analyte in a test substance using the label according to any one of claims 1 to 32 comprising the steps of: (i) activating at least one of the activatable functional groups on the dendrimer to form an activated label; and (ii) exposing the activated label to the test substance comprising the target analyte; and (iii) analysing the test substance for bound label.
42. 42. A method of detecting a target analyte in an immunosorbent assay using the label according to any one of claims 1 to 32 comprising the steps of: (i) immobilising a target analyte onto a support; (ii) washing the support to remove unbound target analyte; (iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte; (iv) washing the support to remove unbound activated label; (v) analyzing for bound target analyte.
43. 43. A method of detecting a target analyte in an immunosorbent assay using the label according to any one of claims 1 to 32 comprising the steps of: (i) immobilising a target analyte onto a support; (ii) washing the support to remove unbound target analyte; (iii) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte; (iv) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule; (vi) exposing the activated label to the support so that detection molecule binds to the secondary analyte molecule; (vii) washing the support to remove unbound activated label; and (viii) analyzing for bound label.
44. 44. A method of detecting a target analyte in an immunosorbent assay using the label according to any one of claims 1 to 32 comprising the steps of: (i) immobilising a target analyte onto a support; (ii) washing the support to remove unbound target analyte; (iii) exposing the support to a secondary analyte molecule which binds to the target analyte; (iv) washing the support to remove any unbound secondary analyte molecule (v) activating at least one of the activatable functional groups on the dendrimer of the label to form an activated label and exposing the activated label to the support so that activated label binds to the immobilised target analyte; (vi) exposing the activated label to a detection molecule which is selective for the secondary analyte molecule; (vii) washing the support to remove unbound activated label; (viii) analyzing for bound label.
45. 45. A method according to any one of claims 42 to 44, wherein the first step of immobilising the target analyte onto the support comprises: (i) immobilising a capture molecule selective for the target analyte onto the support; (ii) washing the support to remove unbound capture molecule; and (iii) exposing the support to the target analyte to immobilise the target analyte onto the immobilised capture molecule.
46. 46. A method according to any one of claims 42 to 45 wherein target analyte is extracted from a test substance, including blood, tissue or a blood derivative or a tissue derivative.
47. 47. A method according to any one of claims 37 to 46 wherein the activating step involves treating the nanoparticle label with N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide and a dehydrating agent.
48. 48. A method of detecting an analyte according to claim 47 wherein the dehydrating agent is 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ([DC).
49. 49. A method of detecting an analyte according to claims 48 wherein the nanoparticle label is activated by treating the nanoparticle label with N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide and a dehydrating agent 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride ([DC) in a MES buffer having a concentration in the range about 1mM to about 1M.
50. 50. A method according to claim 49 wherein the concentration of the MES buffer is 0.1M.
51. 51. A method of detecting an analyte according to claims 49 wherein the buffer has a pH in the range about 3 to about 9.
52. 52. A method according to claim 51 wherein the pH of the MES buffer is pH = 6.
53. 53. A method of detecting an analyte according to any one of claims 49 to 52 wherein the activation period is in the range about 10 seconds to about 30 minutes.
54. 54. A method according to claim 53 wherein the activation period is about 2 hours.
55. 55. A method of detecting an analyte according to any one of claims 37 to 54 wherein the ratio of [DC:NHS is in the range about 1:2 to about 4:1.
56. 56. A method of detecting an analyte according to any one of claims 49 to 56 wherein the N-hydroxysuccinimide (NHS) or sulfo N-hydroxysuccinimide is present in a molar excess in the range of NHS reagent per activatable group.
57. 57. A methods according to claim 56 wherein the molar excess is 2:1.
58. 58. A label prepared according to the method as claimed in any one of claims 37 to 57 wherein the activated dendrimer surface modified nanoparticle comprises semi-stable amine reactive NHS-esters.
59. 59. A label substantially as hereinbefore described with references to the accompanying drawings.
60. 60. A label substantially as hereinbefore described with references to the accompanyingexamples.spec2576