Luminescence

Field of the Invention

The present invention relates to luminescent molecules and/or complexes, particularly chemiluminescent molecules and/or complexes and to methods involving such molecules and/or complexes .

Background to the Invention Assays that utilize luminescence emissions are highly sensitive, quantitative, reproducible, less hazardous and less costly than radio-isotope-based methodology. They also have extremely broad applications in fields such as food testing, forensic science, environmental contaminant screening, microbiology and biomedical analysis.

Luminescence is the emission of light from excited electronic states of atoms or molecules. Luminescence generally refers to all forms of light emission, except incandescence, and may include photoluminescence, chemiluminescence and electroluminescence. In photoluminescence, (such as fluorescence and phosphorescence) the excited electronic state is created by absorption of electromagnetic energy (e.g. light) . However in chemiluminescence, the excited electronic state is created by the transfer of chemical energy.

Chemiluminometric-based assays have a number of advantages over photoluminometric-based assays including simplicity of detection and sensitivity. Unlike fluorescence which requires an incident light source for its induction, chemiluminescence is by definition its own light source. Thus, luminoiuetric assays only require instrumentation that detects light. Such simple requirements make chemiluminescent assays robust and easy to use. In addition, the absence of an incident or background light signal greatly enhances the sensitivity of

chemiluminometric assays. Detection limits are often in the pico or feitito molar range.

The incorporation of a chemiluminescent signal into an assay can take many forms. In bioassays this is commonly achieved using compounds that produce chemiluminescence when acted upon by an enzyme label coupled to a reporter molecule. Thus, for example, an antibody which binds a specific protein of interest will be labelled with a signal generating enzyme such as horse radish peroxidase (HRP) . In the presence of an appropriate luminescent substrate, the HRP-antibody conjugate will generate chemiluminescence which is directly proportional to the target protein present. This method of generating chemiluminescence is indirect.

It is, however, often advantageous to label the reporter molecule with a compound that is directly chemiluminescent. Labelling a reporter molecule with a compound that undergoes direct chemiluminescence upon induction offers the advantages of increased simplicity, increased rate of light generation, the potential for the attachment of multiple labels to the reporter, and the avoidance of having to attach large enzyme labels to the reporter molecule which can significantly affect its affinity for the target.

Common labelling compounds include:

A) Acridinium derivatives (esters and sulphonamides) . These are used extensively in chemiluminescent assays but are unstable, particularly in alkaline solutions.

B) Acridan phosphates. These are more stable than acridinium derivatives but require harsh methods for chemiluminescent induction.

C) Luminol (3-aminophthalhydrazide) and isoluminol (5- aminophthalhydrazide) . These are widely used in indirect

luminescent assays, however the use of luminol is limited as a direct label because once covalently linked to a reporter molecule, the efficiency and rapidity of light emission of this compound is significantly reduced.

Direct chemiluminescent labelling has been limited by problems associated with the chemiluminescent compounds that are in current use, which primarily include label instability and inefficient light production. There is, therefore, a need for new chemiluminescent labels that overcome these limitations.

Summary of the Invention

It has now been found that 3-aminophthalimides (API) may be used as effective chemiluminescent labels. 3- aminophthalimides are stable fluorescent compounds that are readily synthesized by the reduction of nitrophthalimides . Although not directly chemiluminescent, 3-aminophthalimides can be considered as a pro-chemiluminescent molecules because they react with a developing agent to form 'free', i.e. non- covalently linked, chemiluminescent substrates. 3- aminophthalimides yield unconjugated luminol, which is a well- known chemiluminescent substrate, when treated with hydrazine (see Figure 1) .

3-aminophthalimides will have broad application in the biomedical sciences. It is shown here that a number of compounds have successfully been labelled with 3- aminophthalimides including protein (insulin) , polysaccharide (heparin) , lipids (palmitic acid) , and nucleic acid. Also shown here is the use of 3-aminophthalimides to quantitatively observe protein binding, protein uptake into cells, polysaccharide uptake, lipid metabolism and uptake in mitochondrial organelles. 3-aminophthalimides have also been used to qualitatively localise molecules in cells.

Advantages of using this system in direct chemiluminescence bioassays include:

A) Ease of synthesis: 3-aminophthalimide derivatives containing a variety of functional groups can be readily synthesized allowing for ease of coupling to a host of molecules such as proteins, sugars, lipids, nucleotides, peptides, nanoparticles and cellular probes.

B) Stability: 3-aminophthalimides are stable in bioassay solutions and over a wide pH range. C) Chemiluminescent efficiency: in the presence of hydrazine, luminol formed from 3-aminophthalimides rapidly releases light upon induction in a far more efficient manner than bound luminol resulting in increased sensitivity. D) Fluorescence properties: 3-aminophthalimides are highly fluorescent molecules (395 _ex /475 _em ) allowing for studies which require both fluorescence and chemiluminescence approaches.

In addition, 3-aminophthalimides are less expensive than radio-isotope based methodology, and they have neutral charge allowing for endocytosis and lipid studies.

It is also clear that analogues of 3-aminophthalimides will provide effective pro-chemiluminescent labels, in particular, analogues that upon reaction with a developing agent, e.g. hydrazine, produce free luminol or luminol analogues that are chemiluminescent substrates. Thus, in addition to 3- aminophthalimides, other phthalimides may be used as pro- chemiluminescent labels; in particular phthalimides that produce luminol or an analogue of luminol, e.g. a chemiluminescent phthalhydrazide, that is chemiluminescent upon reaction with hydrazine.

Phthalimides are cheap, easy to synthesise, stable and work effectively. Thus, phthalimides provide an important new tool which has broad application in the biochemical sciences. They may also have application in fields such as food testing,

forensic science, environmental contaminant screening, microbiology and biomedical analysis.

In some aspects the invention provides molecules that are labelled with a phthalimide, use of phthalimides as cherαiluminescent labels and methods of detecting molecules that are labelled with phthalimides.

In one aspect of the invention, there is provided a complex comprising a molecule and a phthalimide.

In all aspects of the invention, the term "phthalimide" preferably refers to aminophthalimides such as 3- aminophthalimides and 5-aminophthalimides, e.g. it refers to 3-aminophthalimides, and particularly 3-aminophthalimide and 5-aminophthalimide . The phthalimide may not be a 5- (N, N- dimethy1amino) -phthalimide, in particular, the complex may not be a peptide linked to 5- (N, N-dimethylamino) -phthalimide.

In all aspects of the invention the phthalimide is preferably a pro-chemiluminescent phthalimide, e.g. a chemiluminescent substrate is produced in a reaction with a developing agent such as hydrazine or a hydrazine derivative. Preferably, reaction of the phthalimide with hydrazine produces a phthalhydrazide . Preferably the phthalhydrazide is a chemiluminescent substrate such as luminol, or an analogue of luminol which is a chemiluminescent substrate.

A chemiluminescent substrate is a molecule that is a chemiluminescence "fuel". A chemiluminescent substrate is a molecule that undergoes a reaction, e.g. with an oxidising agent, to produce a molecule in an excited state. The molecule in an excited state may be a chemiluminescent molecule, and as such it may emit a photon as it loses its excitation thereby producing chemiluminescence. Thus, the chemiluminescent

"emitter" may be a "direct descendant" of the oxidation of the

chemiluminescent substrate. If the fuel is luminol, the emitting species is 3-aminophthalate. Reaction of a chemiluminescent substrate with an oxidising agent to produce chemiluminescence is commonly referred to as induction of the chemiluminescent substrate.

The oxidising agent may be hydrogen peroxide or hydrogen peroxide and a hydroxide salt, e.g. hydrogen peroxide under alkaline conditions. However, the oxidising agent may simply be oxygen dissolved in solution. Where dissolved oxygen is used as the oxidising agent no oxidising agent need be added to the reaction. Other oxidising agents that have been used include perborate, permanganate, hypochlorite and iodine.

A catalyst may be employed to catalyse the oxidation reaction, e.g. oxidation of the chemiluminescent substrate to a chemiluminescent molecule. A metal cation may be used, for example a transition metal cation, e.g. Fe(III) salts such as potassium ferricyanide.

An enhancer may also be included in the reaction to increase the chemiluminescent signal. For example, iodophenol may be included which may prolong the duration of chemiluminescence, e.g. it may produce a chemiluminescent molecule with a longer half-life.

The phthalimide may be a phthalimide that produces a chemiluminescent molecule in a reaction with a peroxyoxalate, such as those described below, e.g. a phthalimide in which contact with a peroxyoxalate such as bis (2,4,6- trichlorophenyl) oxlate (TCPO) causes the phthalimide to emit chemiluminescence .

The phthalimide may also be fluorescent, e.g. it may have fluorescent properties, whilst part of the complex and/or when released from the complex as a chemiluminescent substrate.

Phthalimide molecules that have fluorescent properties, in addition to pro-chemiluminescent properties, allow greater utility, e.g. they allow the phthalimide to be detected prior to developing with the developing agent. This may be useful if it is desired to detect the location of the phthalimide, as well as to quantify the amount of phthalimide. Fluorescence may be detected qualitatively and/or quantitatively, e.g. prior to developing with a developing agent, and chemiluminescence may be detected qualitatively and/or quantitatively after developing with the developing agent. 3-aminophthalimides are both fluorescent and pro- chemiluminescent .

In a complex of a molecule and a phthalimide the molecule may be associated and/or labelled with the phthalimide, e.g. the molecule may be linked, coupled, bound, and/or attached to the phthalimide. The molecule may be associated with the phthalimide by covalent bonding and/or non-covalent bonding, e.g. ionic bonding, and/or dipole-dipole attraction e.g. hydrogen bonding. Preferably, the complex is a covalent complex, e.g. the phthalimide is covalently bound to the molecule. The molecule may be linked to the phthalimide via a linking moiety.

A molecule labelled with the phthalimide means that the molecule is associated with the phthalimide such that the presence of the phthalimide, e.g. in an assay, indicates the presence of the molecule.

The molecule complexed with the phthalimide may be any molecule of interest. In particular, the molecule may be a reporter molecule and/or a probe, e.g. a cellular probe. A reporter molecule is any molecule that has binding affinity for a target. A cellular probe is a molecule is that may used to observe a cellular function and/or process, e.g. the molecule may be processed by a cell. Processing of a cellular

probe by a cell may be by active or passive transport, but is preferably active transport. A cellular probe is preferably recognised by the cell, e.g. the probe activates cellular machinery that interacts, e.g. specifically interacts, with the probe. In other words, transport of the cellular probe is preferably mediated by the cell.

A complex comprising a phthalimide and a molecule may be used to detect a target, e.g. such that observing chemiluminescence indicates the presence of the phthalimide and therefore that the molecule has bound to the target and that the target is present, e.g. in an assay. Such complexes will have utility in assays such as ELISA-type assays, sandwich assays, chemiluminescent bioassays, kinase assays and enzyme assays. Assays are discussed in more detail below.

The molecule that is complexed with the phthalimide may have specific binding affinity for a target. For example, the molecule may be a target binding agent. Specific binding affinity for a target means that the molecule selectively binds to the target, e.g. it preferentially binds to the target, and/or it binds to the target with a binding affinity that is higher than that due to non-specific binding.

Molecules that have binding affinity for a target include, for example, specific binding proteins such as antibodies, antibody binding domains, receptor binding domains, and receptor ligands, as well as aptamers and nucleic acids that bind to nucleic acids via hybridisation. Where the molecule has binding affinity for a target, the molecule preferably binds the target with a Kd of less than, or more than, ImM, lOOμM, lOμM, 5μM, 4μM, 3μM, 2μM, lμM, 90OnM, 80OnM, 70OnM, 60OnM, 50OnM, 40OnM, 30OnM, 20OnM, 10OnM, 9OnM, 8OnM, 7OnM, 6OnM, 5OnM, 4OnM, 3OnM, 2OnM, or less than 1OnM. For example, the molecule may bind the target with a Kd in the range ImM to

InM, lOOμM to InM, lOμM to InM, lμM to InM, 10OnM to InM, lμM to 1OnM or 10OnM to 1OnM.

Molecules that are cellular probes and which are complexed with a phthalimide may be probes for a particular organelle and as such may be used to assess the function of that organelle, e.g. mitochondria. For example, the molecule may be a molecule that is taken-up, processed and/or metabolised by a cell organelle. Assays may involve contacting the molecule with a cell organelle and detecting whether the molecule has been taken up, e.g. internalised by the cell organelle.

A molecule complexed with a phthalimide may be capable of being taken up by a cell and may be used to determine cellular uptake e.g. endocytosis, cell processing and/or cell metabolism of the molecule. This may involve contacting the molecule with a cell and detecting chemiluminescence to indicate whether the molecule has been taken up, internalised, processed, and/or metabolised by the cell.

Molecules complexed with a phthalimide may bind to the surface of a cell and may be used in cell surface binding studies. This may involve contacting the molecule with a cell and detecting chemiluminescence to indicate whether the molecule has bound to the cell, e.g. the cell surface and/or cell membrane .

The molecule complexed with a phthalimide may have a molecular weight of at least, or less than, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000Da. The molecule may have a molecular weight of at least, or less than, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, or even 50 kDa.

The molecule complexed with a phthalimide may be a molecule selected from the group consisting of: peptides,

oligopeptides, polypeptides, proteins, antibodies, antibody binding domains, nucleic acids (including RNA or DNA) , oligonucleotides, nucleotides, aptarαers, lipids e.g. phospholipids, glycolipids, sterols, carbohydrates e.g. sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, nanoparticles, and other molecules such as vitamins, hormones, neurotransmitters, prions, organic chemicals, viruses, and microbes such as bacteria. An organic chemical is, for example, a molecule that is more soluble in a non-polar solvent than in a polar solvent. The molecule may be a biomolecule, e.g. it may belong to a class of molecules or may be a specific molecule that occurs in nature, in particular in living organisms.

In some embodiments, the molecule complexed with a phthalimide may not be a molecule selected from the group consisting of: peptides, oligopeptides, polypeptides, proteins, antibodies, antibody binding domains, nucleic acids (including RNA or DNA), oligonucleotides, nucleotides, aptamers, lipids e.g. phospholipids, glycolipids, sterols, carbohydrates e.g. sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, nanoparticles, and other molecules such as vitamins, hormones, neurotransmitters, prions, organic chemicals, viruses, and microbes such as bacteria. The molecule may not be a biomolecule and/or may not be a molecule that occurs naturally in nature.

Preferably the phthalimide is linked to the molecule via the phthalimide imide nitrogen atom. This allows a chemiluminescent molecule to be released as a non-covalently bound molecule upon reaction with a developing agent such as hydrazine or hydrazine derivative. Reaction of the phthalimide with hydrazine or hydrazine derivative displaces the imide nitrogen atom from the phthalimide. The phthalimide imide nitrogen may be linked to the molecule via any suitable chemical linkage, e.g. the phthalimide may be coupled,

covalently linked, bound, covalently bound, and/or attached to the molecule. The linkage may be via any suitable functional group, e.g. an ester, amide or amine.

There may be a linking moiety between the molecule and the phthalimide, which may be, for example, any of a peptide, oligopeptide, polypeptide, protein, antibody, nucleic acid (including RNA or DNA) , oligonucleotide, nucleotide, lipid e.g. phospholipid, glycolipid, sterol, carbohydrate e.g. sugar, monosaccharide, disaccharide, oligosaccharide, polysaccharide, nanoparticle, or an organic chemical. The linking moiety may be linked to the molecule and/or phthalimide by covalent bonding, non-covalent bonding, ionic bonding, and/or dipole-dipole attraction e.g. hydrogen bonding. Preferably, the linking moiety is linked by covalent bonding.

Molecules that are cellular probes may be:

Probes for protein structure, e.g. probes for cytoskeletal proteins such as phalloidin. Probes for organelles, e.g.

- Mitochondria e.g. TPP (triphenyl phosphine) ;

- Lysosomal probes (e.g. probes that accumulate in low pH organelles, these would be weakly basic molecules, partially protonated at neutral pH e.g. DAMP);

- Peroxisomal organelles (e.g. probes that accumulate in these basic organelles, weakly acidic molecule) ;

- Stains for cytotoxicity measurements, e.g. cell impermeant molecules which only enter dead and dying with damaged membranes;

- Labelled lipid derivatives e.g. ceramides, for endoplasmic reticula;

- Labelled lectins e.g. for glyproteins in cell organelles, on the cell surface.

Probes for fatty acid uptake and metabolism, e.g. labelled sphingolipids, fatty acids.

Tracers for cell morphology and fluid flow.

Thiol reactive tracers for the below applications:

Cell adhesion, including adhesion on prosthetic surfaces ^'

Cell communication through gap junctions

Cell migration (chemotaxis) Cell tracing in mixed cultures

Platelet labeling without activation and their in vivo tracing for days

Long-term viability and cytotoxicity assays

Transplantation Multidrug resistance and transport of the glutathione adduct by the multispecific organic anion transporter

Tumor cell death in culture

Keratocytes in intact cornea

Glutathione content in cells Fungal growth and differentiation

Ingestion of live, fluorescently labeled protists by mixotrophic dinoflagellates

Cell-cell and cell-liposome fusion

Vacuolar morphology in yeast

Probes for Endocytosis, Receptors and Ion Channels.

In a further aspect of the invention, there is provided use of a phthalimide in an assay method that comprises detecting luminescence, e.g. chemiluminescence. Contact of the phthalimide with a developing agent, such as hydrazine or a hydrazine derivative or a hydrazine salt, preferably produces a chemiluminescent substrate. Contact of the phthalimide with a developing agent, such as a peroxyoxalate, may produce a chemiluminescent molecule.

The phthalimide may be a component of a complex comprising a molecule and a phthalimide as described above.

The use of the phthalimide may be to detect a target, e.g. in an assay. The phthalimide may form a complex comprising the phthalimide and target, which may optionally be linked to the target via a linking agent or target binding agent. Detection of the complex thereby indicates the presence of the target. The use of the phthalimide may additionally or alternatively be to detect migration of the molecule, e.g. across a lipid bilayer such as across a cell or cell organelle membrane. The use may comprise measuring the amount of chemiluminescence. The use may comprise detecting chemiluminescence and fluorescence. These uses are explained in more detail below.

In a further aspect of the invention, there is provided a method, e.g. an assay method, which method comprises the step of contacting a phthalimide with a developing agent to produce a chemiluminescent molecule or a chemiluminescent substrate. The method may also comprise producing the chemiluminescent substrate or chemiluminescent molecule.

A method of producing a chemiluminescent substrate may comprise contacting the phthalimide with a developing agent such as hydrazine, a hydrazine derivative, or a hydrazine salt. A method of producing a chemiluminescent molecule may comprise contacting a phthalimide with a developing agent such as a peroxyoxalate .

Contacting the phthalimide with the developing agent may comprise incubating, mixing, and/or reacting the phthalimide with the developing agent. Contacting the phthalimide may be performed in a mixture, reaction medium, aqueous solution, sample, composition, and/or assay, e.g. a bioassay.

Contacting the phthalimide with the developing agent preferably involves contacting the phthalimide with a developing agent under conditions that allow the developing agent to produce, e.g. generate and/or release, a chertiiluminescent substrate or a chemiluminescent molecule. A developing agent, such as hydrazine, may react with the phthalimide to release a non-covalently bound chemiluminescent substrate. For example, where the phthalimide is bound to another molecule, reaction with a developing agent preferably releases a free chemiluminescent molecule that is not covalently linked to the molecule. Suitable conditions may be those, for example, that allow hydrazine to react with 3- aminophthalimides to produce luminol or other phthalhydrazides, e.g. chemiluminescent phthalhydrazides . The skilled person is able to choose the appropriate conditions, which may vary depending upon the nature of the phthalimide and the presence of any other molecules.

The method may comprise the step of detecting luminescence, e.g. chemiluminescence and/or fluorescence, preferably chemiluminescence. Detecting luminescence may be qualitative and/or quantitative. Detecting luminescence may include observing, determining, measuring and/or quantitating any luminescence. Luminescence may be quantitated to determine the amount of the luminescence which may be indicative of the amount of phthalimide bound to the target or the amount of phthalimide in a complex with the target. As such, the amount of luminescence may be indicative of the amount of target. The method may therefore provide for quantitative detection of the target in a sample. Luminescence, e.g. chemiluminescence, may be detected using a luminometer or other suitable apparatus known to the person skilled in the art. The method may also comprise detecting, e.g. observing, determining, measuring and/or quantitating any fluorescence. Fluorescence may be detected prior to or after contacting the phthalimide with the

developing agent. Detection of fluorescence is preferably qualitative, but may be quantitative.

The method may include inducing, e.g. activating, the chemiluminescent substrate to produce a chemiluminescent molecule. Inducing the chemiluminescent substrate may convert it to a chemiluminescent molecule, thereby allowing production of a signal, e.g. emission of light. In particular, the method may include contacting the chemiluminescent substrate with an oxidising agent, e.g. to oxidise the chemiluminescent substrate to produce a chemiluminescent molecule. The method may also employ a catalyst, e.g. an oxidation catalyst. Contacting the chemiluminescent substrate with a catalyst and oxidising agent may catalyse oxidation of the chemiluminescent substrate, e.g. to produce a chemiluminescent molecule.

The method may be a method, e.g. an assay method, of detecting a target, which target may be any molecule of interest. Such a method may comprise the step of contacting the phthalimide with the target, e.g. to link the phthalimide to the target and/or form a complex of the phthalimide and target. This may be prior to, after, or at the same time as contacting the phthalimide with the developing agent. The presence of a chemiluminescent molecule after contacting with the developing agent may be indicative that target is present, e.g. in an assay.

For example, the target may be selected from the group consisting of peptides, oligopeptides, polypeptides, proteins, antibodies, antibody binding domains, nucleic acids (including RNA or DNA) , oligonucleotides, nucleotides, aptamers, lipids e.g. phospholipids, glycolipids, sterols, carbohydrates e.g. sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, nanoparticles, and other molecules such as vitamins, hormones, neurotransmitters, prions, organic chemicals, viruses, and microbes such as bacteria

Detecting a target may include observing, determining the presence of, quantitating, e.g. measuring the amount of, chemiluminescence. Detecting chemiluminescence may be indicative that target is present, e.g. in an assay. Detection may be qualitative and/or quantitative. The target may be detected in a mixture, reaction medium, aqueous solution, sample, composition, and/or assay, e.g. a bioassay.

A method of detecting a target may also comprise the step of partitioning, e.g. separating and/or removing, any phthalimide that is linked to target (e.g. bound to or complexed with the target) from any phthalimide that is not linked to target (e.g. not bound to or complexed with the target) . In particular, partitioning may include separating phthalimide that is bound to target from phthalimide that is not bound to target. Partitioning allows any phthalimide that is not linked to target to be removed so that chemiluminescence associated with target may be observed.

Phthalimide not linked to target may be partitioned from phthalimide that is linked to target in a variety of ways known to the person skilled in the art, e.g. by a washing step. Partitioning may, for example, be by chromatography, electrophoresis, and/or by immobilising the target on a solid support. Chromatography may, for example, be ion exchange, gel filtration, hydrophobic interaction, reverse phase, or affinity chromatography. Immobilising target on a support allows any phthalimide that is not linked to target to be removed, e.g. by washing the support. Target may be immobilised on a support by attaching antibodies, or any other substance that has specific binding affinity for the target to the support. The support may be any suitable solid support known to the person skilled in the art, e.g. a plate, column, paper, microtitre well, chip, etc..

Detecting a target may include detecting any fluorescence. Detecting fluorescence, e.g. after partitioning any phthalimide that is not linked to target, may be indicative that target is present. Quantitatively detecting any chemiluminescence after contacting the phthalimide with developing agent may allow determination of the amount of target, e.g. in the assay.

Generally, the phthalimide may be linked to the target via covalent, non-covalent, and/or ionic bonding, and/or via dipole-dipole attractions, e.g. hydrogen bonding.

The method may comprise linking the phthalimide to the target directly or indirectly. For example, the phthalimide is directly linked to the target when the phthalimide is part of a molecule that binds to the target. Binding may be via dipole-dipole attractions e.g. hydrogen bonds, ionic bonding, covalent bonding, and/or non-covalent bonding. The phthalimide is indirectly linked to the target when the phthalimide is linked to the target but is not part of a molecule that binds to the target. The phthalimide may be linked to the target in a sandwich arrangement. For example, the phthalimide may be part of a molecule that binds to a second molecule that is not the target, and the second molecule is linked to the target. The second molecule may bind to the target, e.g. it may be a target binding agent, or there may be one or more other molecules separating the second molecule from the target in a chain-like arrangement, with each subsequent molecule in the chain having affinity for the next.

Methods of the invention include detecting a target by detecting a complex comprising the phthalimide and the target. This may comprise forming complexes comprising the phthalimide and the target. In such a complex the target may be linked directly or indirectly to the target, for example, the phthalimide may optionally be linked to the target via one of

more linking agents, such as a target binding agent. Binding between components of the complex may be covalent or non- covalent. Such complexes may be detected by contacting the complex with developing agent to produce chemiluminescence. Observing chemiluminescence thereby indicates the presence of the complex, and therefore the presence of the target.

A phthalimide that is part of a molecule that binds to the target or that binds to any other molecule may be simply be a phthalimide with a moiety, e.g. a reactive functional group, that allows the phthalimide to bind to the target or other molecule. Otherwise, a phthalimide that is part of a molecule that binds to the target or that binds to any other molecule may be a complex of a phthalimide and a molecule, as described above. For example, the phthalimide may be covalently bound to an antibody binding domain, antibody or aptamer.

A method of detecting a target may comprise the steps of contacting the phthalimide with a test substance and forming a complex comprising phthalimide and target.

A method of detecting target may comprise the steps of contacting a first complex comprising a phthalimide and a target binding agent with a test substance and forming a second complex comprising phthalimide, target binding agent and target.

The test substance may, for example, be a mixture suspected of containing the target, e.g. an aqueous solution.

The target binding agent may be a molecule that has binding affinity for a target, as described above. The target binding agent may be an antibody binding domain or an antibody, e.g. a human, rabbit, mouse, rat, or goat antibody binding domain or antibody. The antibody binding domain or antibody may be, or

may be derived from, an IgA, IgD, IgE, IgG, or IgM, but is preferably an IgG.

A method of detecting a target may also include determining the amount of target as an assay result. The amount of chemiluminescence may be indicative of the amount of target.

A method that comprises the step of contacting a phthalimide with a developing agent may be a method of detecting migration of a phthalimide, e.g. in an assay such as in an aqueous solution. Detecting migration may comprise detecting migration of the phthalimide from a first location to a second location and detecting phthalimide at the second location. The location of a phthalimide may be detected by contacting a phthalimide with a developing agent.

The method may be a method of detecting migration of a phthalimide, e.g. a complex comprising a phthalimide and a molecule. Migration may be from a first location to a second location. Phthalimide may be detected at the second location, e.g. detection of chemiluminescence at the second location may be indicative that the phthalimide has migrated to the second location. Migration of the phthalimide may be detected by contacting the phthalimide with a developing agent. The method may include placing the phthalimide at the first location.

The method may be a method of detecting migration of a molecule. The location of a molecule may be detected by linking a phthalimide to the molecule, as described above for the detection of targets. In this case the molecule is the target. Alternatively the molecule may migrate as a complex comprising the molecule and phthalimide. Complexes comprising a molecule and phthalimide are described above.

The method may be a method of detecting, observing, determining and/or quantitating migration of a phthalimide and/or molecule complexed with a phthalimide.

Migration of a phthalimide and/or molecule complexed with phthalimide refers to movement of the phthalimide from one location to a different location. For example, migration may be migration in chromatography, electrophoresis, in a gel, in solution, in a well. Migration of the phthalimide may be onto and/or across a lipid bilayer. Lipid bilayers of interest may include cell membranes, e.g. cell surface membranes, cell nuclear membranes and mitochondrial membranes. In particular, migration of the labelled molecule may be migration from outside a cell to inside a cell, e.g. cell-uptake such as endocytosis. Alternatively or additionally, migration may be from a particular location in a cell to a different location in a cell, e.g. from the nucleus to the cytoplasm.

A method of detecting migration of a phthalimide target may also include determining the degree of migration as an assay result. The location of chemiluminescence may be indicative of the location of the phthalimide.

The following method aspects may be understood in light of the above discussion:

In a further aspect of the invention there is provided a method of detecting a target, comprising the steps of:

(a) contacting a complex comprising a target binding agent and a phthalimide with a test substance suspected of containing target under conditions that allow the complex to bind to the target;

(b) partitioning complex that is bound to the target from complex that is not bound to the target; (c) contacting complex that bound to the target in (b) with a developing agent under conditions that allow the developing

agent to convert the phthalimide to a chemiluminescent molecule; and (d) detecting chemiluminescence .

An observation of chemiluminescence in step (d) is indicative that the assay contains target.

The complex comprising a target binding agent and a phthalimide may be a complex as described above. The phthalimide may be part of a molecule that binds to the target binding agent or may bind to the target binding agent directly.

The target binding agent is preferably a molecule that has binding affinity for the target, e.g. as described above.

The target binding agent may be an antibody binding domain or an antibody, e.g. a human, rabbit, mouse, rat, or goat antibody binding domain or antibody. The antibody binding domain or antibody may be, or may be derived from, an IgA, IgD, IgE, IgG, or IgM, but is preferably an IgG.

A phthalimide that is part of a molecule that binds to the target binding agent may be part of a molecule such as an antibody binding domain or antibody, e.g. a antibody binding domain or antibody that has affinity for a human, rabbit, mouse, rat, or goat antibody binding domain or antibody, e.g. an IgA, IgD, IgE, IgG, or IgM.

Step (a) may include forming a complex comprising phthalimide, target binding agent and target. Step (b) may include discarding target binding agent that is not bound to target, e.g. by washing. Step (b) may be performed whilst the target is immobilised, e.g. on a solid support. Step (d) may include contacting the phthalimide with an inducing agent and/or and enhancer.

In a further aspect of the invention there is provided a method of detecting a target, comprising the steps of:

(a) contacting a target binding agent with a test substance suspected of containing the target under conditions that allow the target binding agent to bind the target;

(b) partitioning target binding agent that is bound to the target from target binding agent that is not bound to the target;

(c) contacting target binding agent that bound to the target in (b) with a complex comprising a phthalimide and a molecule that has binding affinity for the target binding agent, under conditions that allow the complex to bind to the target binding agent;

(d) partitioning complex that is bound to the target binding agent from complex that is not bound to the target binding agent;

(e) contacting complex that bound to the target binding agent in (d) with developing agent under conditions that allow the developing agent to convert the phthalimide to a chemiluminescent molecule; and

(f) detecting chemiluminescence .

An observation of chemiluminescence in step (f) is indicative that the assay contains target.

The complex comprising a phthalimide may be a complex of a phthalimide and a molecule as described above. The complex may- comprise an antibody binding domain or antibody, e.g. a antibody binding domain or antibody that has affinity for a human, rabbit, mouse, rat, or goat antibody binding domain or antibody, e.g. an IgA, IgD, IgE, IgG, or IgM.

The target binding agent is preferably a molecule that has binding affinity for the target, e.g. as described above. The target binding agent may be an antibody binding domain or an antibody, e.g. a human, rabbit, mouse, rat, or goat

antibody binding domain or antibody. The antibody binding domain or antibody may be, or may be derived from, an IgA, IgD, IgE, IgG, or IgM, but is preferably an IgG.

Step (a) may include contacting the target binding agent with target to form a complex comprising target binding agent and target. Step (b) may include discarding target binding agent that is not bound to target, e.g. by washing. Step (b) may be performed whilst the target is immobilised, e.g. on a solid support. Step (c) may include contacting the complex comprising phthalimide with target binding agent to form a complex comprising phthalimide, target binding agent, and target. Step (d) may include discarding complex comprising phthalimide that is not bound to target binding agent, e.g. by washing. Step (f) may include contacting the phthalimide with an activating agent and enhancer.

In a further aspect of the invention there is provided a method of preparing a complex comprising a target binding agent and a phthalimide, which method comprises contacting a molecule comprising a phthalimide with a target binding agent, under conditions that allow the phthalimide to bind the target binding agent. Contacting the phthalimide with a target binding agent preferably forms a complex comprising phthalimide and the target binding agent. The target binding agent may be any target binding agent as discussed above.

In a further aspect of the invention there is provided a method of preparing a complex comprising a phthalimide, which method comprises contacting a molecule with a phthalimide under conditions that allow the phthalimide to bind to the molecule.

In the complex the molecule may be linked, coupled, covalently linked, and/or attached to the phthalimide. The molecule may be linked with the phthalimide by a covalent bond, ionic bond,

and/or dipole-dipole attraction e.g. a hydrogen bond. The complex may be a covalent complex.

Preferably the phthalimide is provided in a form that allows it to bind to a molecule. Preferably, the phthalimide binds to the molecule via its imide nitrogen atom. For example, the phthalimide may be a phthalimide derivative that comprises an activated functional group. The phthalimide imide nitrogen may be linked to the molecule via any suitable chemical linkage, e.g. it may be coupled to the molecule via an ester, amide or amine functional group. A convenient route for attaching the molecule to the phthalimide is to provide the phthalimide as an N-hydroxysuccinimde (NHS) ester. The NHS-phthalimide will then be amine reactive, e.g. it will react with amine groups on the molecule such that the molecule becomes labelled with the phthalimide.

In a further aspect of the invention, there is provided a method of detecting migration of a molecule comprising a phthalimide, comprising the steps of:

(a) adding a complex comprising a phthalimide to an assay at a first location;

(b) contacting the complex with a developing agent;

(c) detecting chemiluminescence in the assay at a second location.

Observing chemiluminescence at the second location is indicative that the molecule has migrated from the first location to the second location.

The complex comprising a phthalimide may be a complex comprising a phthalimide and a molecule as described above.

Step (a) may include allowing the phthalimide to migrate before contacting the phthalimide with developing agent. The first location and second location are preferably spatially

different locations. For example, the first location may be one side of a lipid bilayer and the second location may be on the other side of the lipid bilayer. Examples include migration across cell surface membranes, cell organelle membranes, or cell nuclear membranes, or migration by chromatography, electrophoresis, or in a gel. For example, the first location may be outside a cell and the second location may be inside a cell and vice versa.

In a further aspect of the invention, there is provided an assay method, e.g. an in vitro assay method, of detecting cell up-take, e.g. endocytosis, of a complex comprising a phthalimide, comprising the steps of:

(a) contacting a complex comprising a phthalimide with a cell;

(b) contacting the complex with a developing agent; and

(c) detecting chemiluminescence.

Observing chemiluminescence inside the cell is indicative that the cell takes up the molecule. Contacting the complex with a developing agent may comprise exposing the cell contents to the developing agent.

The complex comprising a phthalimide may be a complex comprising a phthalimide and a molecule as described above.

In a further aspect of the invention, there is provided an assay method, e.g. an in vitro assay method, of determining whether a complex comprising a phthalimide binds to a cell surface, comprising the steps of:

(a) contacting a complex comprising a phthalimide with a cell;

(b) contacting the complex with a developing agent; and

(c) detecting chemiluminescence.

Observing chemiluminescence on the cell surface is indicative that the cell surface binds the molecule. Contacting the complex with a developing agent may comprise exposing the cell contents to the developing agent.

The complex comprising a phthalimide may be a complex comprising a phthalimide and a molecule as described above.

Any method of the invention is preferably an in vitro method. The method may be a diagnostic method.

The invention also provides kits for carrying out the uses and methods of the invention.

In a further aspect of the invention, there is provided a kit comprising a complex of the invention, which complex comprises a molecule and a phthalimide. Such complexes are described above .

Preferably the kit comprises a developing agent. The developing agent may be hydrazine, a hydrazine derivative, a hydrazine salt, e.g. such that contact of the developing agent produces a chemiluminescent substrate. The developing agent may be a peroxyoxalate, e.g. such that contact of the developing agent produces a chemiluminescent molecule.

The phthalimide may be a component of a complex comprising a molecule and a phthalimide as described above. Alternatively or additionally, the phthalimide may be provided as an activated phthalimide to allow the imide nitrogen atom of the phthalimide to be linked to a molecule of interest. For example, the phthalimide may be provided as an N- hydroxysuccimide phthalimide.

The kit may be for detecting a target. The kit may include a target binding agent. A target binding agent may be a

component of a complex comprising the phthalimide, or a target binding agent may be provided separately, e.g. the phthalimide and target binding agent may be provided in separate containers .

The kit may be for detecting migration of a molecule. The kit may include a target binding agent which may be complexed to the phthalimide. The target binding agent may bind to a cellular probe. The phthalimide and cellular probe may be provided in separate containers. Alternatively or additionally the kit may include a complex comprising a phthalimide and a cellular probe, e.g. the phthalimide may be linked to the cellular probe.

Kits of the invention may include one or more of: a buffer solution, a wash solution, a stop solution, a developing agent such as hydrazine or a hydrazine derivative, an oxidising agent to oxidise the chemiluminescent substrate, an oxidation catalyst, e.g. an iron compound such as potassium ferricyanide may also be provided. The kit may also include an enhancer, e.g. iodophenol. Each of these may be provided in separate containers. Kits of the invention may also include instructions for contacting the phthalimide with a developing agent to produce a chemiluminescent molecule and/or for inducing the chemiluminescent molecule to emit light.

In further aspect of the invention, there is provided a kit comprising:

(a) a phthalimide; (b) a developing agent; and optionally

(c) instructions for contacting the phthalimide with the developing agent to produce a chemiluminescent molecule.

In further aspect of the invention, there is provided a kit for detecting a target comprising: (a) a phthalimide; and

(b) a target binding agent.

In further aspect of the invention, there is provided a kit for detecting a target comprising a complex comprising a target binding agent and a phthalimide.

In further aspect of the invention, there is provided a kit for detecting migration of a molecule comprising: (a) a phthalimide; and (b) a cellular probe.

In further aspect of the invention, there is provided a kit for detecting migration of a molecule comprising: a complex comprising a phthalimide and a cellular probe.

The peroxyoxalate system

The peroxyoxalate assay system is described, for example, in Kuroda et al . (9) . See also Milofsky et al . and Chokshi et al . (10 and 11) . Essentially, the method involves reacting a peroxyoxalate with an oxidising agent such as hydrogen peroxide to produce a high energy intermediate. The high energy intermediate may then interact with a fluorophore to excite the fluorophore such that the fluorophore may decay back to ground state by emission of chemiluminescence . The high energy intermediate may be a 1, 2-dioxetane, although the reaction may produce many intermediates and others have been proposed (10,11) . The peroxyoxalate may, for example, be bis (2, 4, 6-trichlorophenyl) oxlate (TCPO) . This molecule is shown in Figure 10 as molecule 18. Other peroxyoxalates that may be used are described in Kuroda et al. (9), and include bis (2, 6-difluorophenyl) oxalate (DFPO), bis (2, 4-dinitrophenyl) oxalate (DNPO), bis [2- (3, 6, 9-trioxadecyloxycarbonyl) -4- nitrophenyl] oxalate (TDPO), bis (pentafluorophenyl) oxalate (PFPO) , bis [2, 4, 5-trichloro-6 (pentyloxycarbonylphenyl) ] oxalate (TPPO), 4, 4'-OXaIyI- bis [ (trifluoromethylsuphonyl) imino] trimethylene-bis (4-

methylmorpholinium) trifluoromethane sulphonate (MPTQ) . The peroxyoxalate may not be TPPO.

Peroxyoxalates may be used to excite the fluorophore of the phthalimides of the invention, thereby producing chemiluminescence. Preferably peroxyoxalates are contacted with phthalimides in an organic solvent.

It is known that the 4-aminophthalimides exhibits solvatoflurochromism, i.e. the fluorescence wavelength changes depending upon the environment (17) . 4-aminophthalimides exhibit a blue shift in non-polar solvents such as benzene and lipids and a red shift (fluorescence wavelength increases and becomes greener) in polar solvents such as water or methanol. This property could be exploited using the peroxyoxalate system, for example, in order to tune the chemiluminescence of phthalimides, or to assess the environment of the phthalimide such as the polarity of environment. Observing a red shift or blue shift will indicate a more polar or non-polar environment respectively.

Peroxyoxalates may also be used to amplify the chemiluminescence of phthalimides that have been developed to produce a chemiluminescent substrate, e.g. with hydrazine.

Phthalimide analogues

A phthalimide of the invention may be any phthalimide that produces a chemiluminescent molecule in a reaction with a developing agent such as TCPO, or that produces a chemiluminescent substrate in a reaction with hydrazine.

The phthalimides of the invention include derivatives of phthalimide. For example, the benzene ring may be substituted with any substituent, and/or may be annelated.

A phthalimide of the invention is preferably a 3- aminophthalimide or a 5-aminophthalimide, which includes derivatives of 3-aminophthalimide and 5-aminophthalimide. Derivatives may be 3-aminophthalimides or 5-aminophthalimides that include other substituents on the benzene ring in addition to the amino group. For example, where the phthalimide is a 3-aminophthalimide there may be substituents other than hydrogen on the benzene ring at the 4, 5, or 6 position, or on the amino group at position 3. Where the phthalimide is a 5-aminophthalimide there may be substituents other than hydrogen on the benzene ring at the 3, 4, or 6 position, or on the amino group at position 5. In addition or alternatively, there may be substituents other than hydrogen on the benzene amino group in either 3- or 5-aminophthalimide.

Such substituents may be electron withdrawing groups such as a halide (F, Cl, Br, I) , nitro groups, sulphonate, and sulphonyl groups. In particular, an electron withdrawing group on the 4 position of the benzene ring relative to the amino group may achieve a higher degree of luminescence. Substituents may be electron donating groups such as alkyl groups, e.g. methyl, ethyl, propyl, butyl, ^fc butyl etc.. Alkyl substituents may be useful to link the phthalimide to another molecule.

In addition, or alternatively, additional 6 membered aromatic rings may be appended to the phthalimide benzene ring to create a fused ring, e.g. to create a naphthalene or anthracene system.

The presence of substituents on the benzene ring may change the wavelength of the emitted photons.

Preferably, the phthalimide is 3-aminophthalimide or 5- aminophthalimide . The phthalimide is preferably attached to the molecule of interest via the imide nitrogen atom or the nitrogen atom of the benzene amino group. Preferably the

molecule of interest is attached to the phthalimide via the imide nitrogen atom.

The phthalimide may be any of molecules (9), (11), (13), (15), or (17) which are shown in Figure 10. The R group is any molecule of interest. The R group indicates the point of attachment of a molecule to the phthalimide.

Preferably, the phthalimide is not 5- (N,N-dimethylamino) - phthalimide (see Vazquez et al . , reference 12, there this molecule is called 4- (N, N-dimethylamino) -phthalimide) . This is shown in Figure 10 as molecule 19. The phthalimide may not be a dimethylamino phthalimide, e.g. a 3- or 5-dimthyl amino phthalimide .

The phthalimides of the invention may form a chemiluminescent substrate, particularly a chemiluminescent phthalhydrazide, e.g. after reaction with hydrazine, that has a quantum yield of at least 1, or at least the same quantum yield as luminol (e.g. 1.25) . Preferably, the quantum yield is at least, or less than, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19,

1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29 or 1.30. In particular, the quantum yield is preferably at least 0.1. Preferably, the quantum yield is in the range 0.01-1.5, 0.1-1.3, 0.5-1.3, 0.8-1.3, or even 0.9-1.3.

Quantum yield is the probability of photon emission via the reaction of a single substrate molecule. Experimental determination of the quantum yield is achieved by dividing the absolute total number of luminescence photons, or the integrated quanta of luminescence, by the number of

consumed substrate molecules. The skilled person is able to determine quantum yield of a chemiluminescent substrate, see for example Ando et al. (18, 19) .

Chemiluminescent phthalhydrazides other than luminol are described in US 4,334,069, which are incorporated herein by reference. Chemiluminescent phthalhydrazides are also disclosed in Bruno et al. (14), Han et al. (15), and Whitehead et al. (16) . These are incorporated herein by reference. In particular, the phthalimides of the present invention may be the phthalimide analogue of any one of these phthalhydrazides. US 4,334,069 discloses 3 and 4 aminophthalhydrazides in which the benyl amino group is: NR ₁ R ₂ , in which R ₁ is hydrogen or straight chain alkyl containing 1,2,3 or 4 carbon atoms and R ₂ is L(CO) -HN- (CH ₂ ) _n -, wherein n is 2-8, and L (CO) -is any molecule (L), e.g. a hapten, bound through an amide bond.

Some phthalimides, such as phthalimide itself, may be chemiluminescent in the ultra-violet (UV) range rather than the visible range. These phthalimides may still be used in methods of the invention because luminometers are capable of detecting chemiluminescence in the UV spectrum. In particular, a phthalimide may be derivatised to tune the wavelength of the chemiluminescence. For example substituents on the benzene ring may increase or decrease the wavelength of emitted photon. Luminol produces blue light, having a wavelength in the region of 475nm. Changing the wavelength may be desirable in certain situations. For example, the phthalimide may be a prochemiluminescent molecule for a chemiluminescent molecule that emits red light, green light, or blue light. Red light has a wavelength in the region of 700nm.

Developing agents

The developing agent reacts with the phthalimide to produce either a chemiluminescent molecule or a chemiluminescent substrate. Developing agents that react with phthalimide to

produce a cheitiiluminescent molecule may be a peroxyoxalates, including TCPO, DFPO, PFPO, DNPO, TPPO, TDPO, or MPTQ. Developing agents that react with phthalimides to produce a chemiluminescent substrate include hydrazine, hydrazine derivatives and/or hydrazine salts. A hydrazine salt may be a monohydrazine salt or a dihydrazine salt. Dihydrazine salts are preferably used under alkaline conditions.

Assays The present invention may be used in a wide range of assays, including diagnostic assays. Examples of assays include but are not limited to target binding assays, sandwich assays, competition assays, kinase assay, enzyme assays, migration assays. These assays are well known to the person skilled in the art. See for example A Biologist's Guide to the Principles and Techniques of Practical Biochemistry (Cambridge Studies in Modern Biology) (Paperback) , Keith & Goulding, Kenneth H Wilson (Editor) .

In a target binding assay a target may be detected by binding to the target a molecule that has binding affinity for the target, "a target binding agent". The target binding agent may be labelled with a detectable signal. If the presence of the detectable label persists after immobilising the target and washing away any unbound target binding agent, this indicates the presence of target. The detectable signal may be a phthalimide which is bound to the target binding agent. Contacting the phthalimide with developing agent will cause emission of chemiluminescence, as described above. The target and target binding agent may be any molecules that have affinity for each other, e.g. peptide/antibody, nucleic acid/nucleic acid, receptor/ligand etc..

The target may be immobilised on a support, e.g. to facilitate washing phthalimide not bound to target from phthalimide bound to target. The support may be any suitable solid support known

to the person skilled in the art, e.g. a plate, column, paper, microtitre well, chip, resin, polymer, microsphere etc..

In a sandwich assay there may be one or more molecules separating the target and the agent that produces a detectable signal in a chain-like arrangement, each molecule having affinity for the next. For example, a molecule labelled with a detectable signal may bind to the target binding agent, which in turn is bound to the target. Commonly the target binding agent is an antibody that has been raised against a specific target, e.g. using a rabbit, mouse, rat, or goat etc.. The signal producing molecule may be attached to a molecule, e.g. an antibody or antibody binding domain that has binding affinity for the rabbit, mouse, rat, or goat antibody. Persistence of the label after immobilising the target and washing indicates the presence of the target.

Sandwich assays form the basis for ELISA assays, in which the signal producing molecule is an enzyme that acts on a substrate to make it visible. According to the present invention, the signal producing molecule may be a phthalimide. An advantage of the present invention over ELISA-type assays is that the amount of target is readily quantifiable because the amount of chemiluminescence is directly proportional to the amount of phthalimide present. In ELISA assays it is the rate of signal production that corresponds to the amount of target, which is difficult to measure with precision. In addition, enzymes may lose their activity over time, or if not stored correctly. As the phthalimide system does not require active enzymes, it does not suffer from this limitation.

Competition assays may involve contacting a target with a target binding agent and a target binding agent that is bound to a signal producing molecule in which both the labelled and unlabelled target binding agents compete for binding to the target. The assay reaches a dynamic equilibrium, at which

point a particular amount of the labelled target binding agent will be bound to the target and the remainder will be unbound in solution. One application of competition assays is in determining the amount of an unlabelled target binding agent present. For example, a known quantity of labelled target binding agent is added to a solution containing target and unlabelled target binding agent. The labelled target binding agent displaces the unlabelled target binding agent from the target and becomes bound to the target. The amount of unbound labelled target binding agent in solution will be inversely proportional to the amount of unlabelled target binding agent. Thus measuring the amount of free labelled target binding agent allows calculation of the amount of unlabelled target binding agent in the assay. The amount of labelled target binding agent may be measured by removing the target complexes from the solution, or by analysing a sample taken from the solution. As an example, the target and target binding agent may be a ligand and its cognate receptor.

Another application of competition assays is in determining the amount of a target binding agent in a sample. A sample of unlabelled target binding agent is added to a solution containing target and a known amount of labelled target binding agent. Unlabelled target binding agent will displace labelled target binding agent from the target thereby increasing the amount of free labelled target binding agent in solution. The increase in the amount of free labelled target binding agent will be proportional to the amount of unlabelled target binding agent in the sample. In these assays the label may be a phthalimide of the invention.

In competition assays the label is commonly a radioisotope, for example radioimmunoassasys (RIA) . These typically involve mixing known quantities of radioactive antigen (frequently labeled with gamma-radioactive isotopes of iodine attached to tyrosine) with antibody to that antigen, then adding unlabeled

or "cold" antigen and measuring the amount of labeled antigen displaced ( 13 ) . Use of the phthalimide system in competition assays has the advantage that it does not involve the hazards associated with radioactivity.

Kinase assays may be used to determine whether a particular substrate, e.g. a peptide, is phosphorylated by a kinase enzyme, to determine phosphorylation efficiency, or to determine location of phosphorylation. For example, a test substrate is contacted with a kinase enzyme in the presence of a phosphate donor e.g. ATP. Commonly the phosphate donor includes a detectable label which is usually the radioactive ³² P isotope. The substrate is then separated from the reaction mixture and the presence and/or quantity of label associated with the test substrate is observed. Detection of the label indicates that the substrate has been phosphorylated.

Kinase assays may employ phthalimides of the present invention instead of ³² P. For example, the test substrate may be labelled with a phthalimide of the invention. The test substrate is removed from the assay using a method that selects for the phosphate group, e.g. using a gallium chelate resin. In this way test substrate that is bound to a phosphate group may be selectively removed from the assay, and the presence and/or quantity of test substrate can be measured by virtue of the phthalimide label, which is bound to the test substrate.

Selection and enrichment of phosphopeptides is described in Collins et al. "Robust enrichment of phosphoylated species in complex mixtures by sequential protein and peptide metal- affinity chromatography and analysis by tandem mass spectrometry" (7) . This paper describes improvements to immobilised metal-affinity chromatography techniques for capturing phosphopeptides. Selection of phosphopeptides is also described in Bieber et al. "Metal ligand affinity

pipettes and bioreactive alkaline phophatase probes: tools for characterization of phosphoylated proteins and peptides" (8) .

Enzyme assays may be used to determine whether a test substrate includes a cleavage site for a particular enzyme. The assay may involve coupling one end of the test substrate to a detectable label, e.g. a phthalimide of the invention, and coupling the other end of the test substrate to a solid support. If the test substrate is cleaved by the enzyme the part of the test substrate coupled to the detectable label will become detached from the solid support and will be free in solution. If the detectable label is a microsphere or polymer conjugated with phthalimide, the detectable label may be removed from the assay by centrifugation thereby facilitating detection of the detectable label.

In an alternative arrangement one end of the test substrate may be bound to a solid support which is a microsphere or polymer and the other end is labelled with the detectable label. Any cleavage will release free detectable label, and any uncleaved test substrate may be removed from the solution by centrifugation by virtue of the presence of the microsphere or polymer. Suitable microspheres and polymers are commercially available and are well known to the person skilled in the art.

These assays may be used, for example, to determine whether a lipid has a lipase cleavage site, or whether a particular protein is a lipase. Alternatively, the test substrate and enzyme may be a peptide and protein respectively and the assay may involve determining whether the peptide is cleaved by the protein.

Other applications of the present invention include assays involving nanoparticles . For example, some metals have affinity for thiols, such as copper, silver, gold,

particularly gold. Phthalimid.es derivatised with a thiol group may thus be attached to such particles simply by mixing the metal with the derivatised phthalimide. As an example, a target binding agent may include one or more gold nanoparticles . Mixing the derivatised phthalimide with the target binding agent will lead to attachment of the phthalimide to the target binding agent, thereby allowing detection of the target. Colloid gold labelling offers a convenient way of labelling proteins, e.g. antibodies, with phthalimides . For example, proteins become labelled with gold in a reaction of gold chloride and antibody in the presence of a reducing agent such as cyanoborohydride or borohydride.

In another arrangement, a target may bind to a nanoparticle having affinity for thiol groups. Subsequent mixing of the nanoparticle with a phthalimide derivatised with a thiol group will allow the phthalimide to bind to the remaining binding sites on the gold particle. The amount of phthalimide on the nanoparticle will then be an indication of the amount of target in the assay.

The present invention also has utility in assays for detecting migration of a molecule. Examples of assays involving migration in which the present invention will be useful include migration by chromatography, by electrophoresis, and by gels. The phthalimide will allow a molecule of interest to be easily identified when a sample containing the molecule of interest is analysed by chromatography or electrophoresis. Furthermore, where a sample is run on a gel, the amount of the molecule of interest may be quantified by cutting out the region containing the molecule of interest.

Other migration assays include observing whether a molecule is taken up a cell, whether a molecule is processed by a cell, whether a molecule enters a particular cell organelle, whether a molecule moves between the nuclear and cytoplasmic regions,

whether a molecule localises to a particular region of the cell, and whether the molecule associates with the cell surface. The phthalimide label lends itself to these sorts of studies because it is very small, stable and neutral. Therefore it does not substantially interfere with the normal behaviour of the unlabelled molecule inside a cell and it does not break down when exposed to the cellular conditions. Examples the use of phthalimides of the invention in these sorts of applications are shown below.

Phthalimides of the invention may also be used in phagocytosis studies. For example, a molecule labelled with phthalimide may be presented to a phagocyte to determine the efficiency at which the molecule is internalised by the phagocyte.

There are a very diverse range of applications for assays involving phthalimides, in addition to applications in the biochemical and medical science. For example, phthalimides may be used in food testing, e.g. to analyse whether a particular component is present. Assays of this nature may be competition assays. In forensic science, phthalimides may, for example, be used to detect bodily fluids. Phthalimides may be used to detect the presence of contaminants in the environment, e.g. in drinking water.

The phthalimides of the present invention are preferably used in in vitro methods. In particular, if the methods of the present invention are performed on live cells, the method may result in death of the cell. The preferred pH range under which phthalimides are used, prior to addition of developing agent, is pH 4-9, although pHs outside this range, e.g. 3-10 may be used. Preferably, pHs below 0,1,2, or 3 are not used. Likewise, pHs above 10, 11, 13 or 14 are preferably not used. The use of lower pHs e.g. 2-4 may cause a change in the fluorescence of phthalimide which may be exploited, e.g. when observing passage of phthalimide into an acidic organelle. The

pH used when contacting the phthalimide with developing agent may be a pH outside these ranges.

Methods of the present invention may not involve using phthalimides in the presence of strong nucleophiles or powerful alkylating agents such as MeS.

Attachment of phthalimides to molecules of interest Phthalimides of the present invention may be attached to a molecule of interest via the imide nitrogen atom. Generally reaction of the phthalimide with hydrazine then displaces the imide nitrogen from the phthalimide leaving an amine group on the molecule of interest. However, phthalimides may also be attached to a molecule of interest via the benzene ring. This is particularly the case when, for example, the benzene ring is derivatised with an amino group, e.g. 3- or 5- aminophthalimide, and the molecule of interest is attached to the phthalimide via this amino group. The phthalimide may be cleavable from the molecule of interest to allow the phthalimide to become free in solution. This cleavage may be chemical cleavage if the molecule of interest is attached via an electron withdrawing group, or it may be enzymatic cleavage. Enzymatic cleavage may be feasible when, for example, there is a phospho- or sulfo- bridge between the amino group on the phthalimide benzene ring and the molecule of interest. A phosphatase enzyme or sulfatase enzyme will thus cleave the bond linking the phospho- or sulfo- group to the nitrogen atom, respectively. After release of the phthalimide molecule from the molecule of interest, developing agent, e.g. hydrazine, may then be added.

Phthalimides may be derivatised in order to attach the phthalimide to a molecule of interest. This may involve coupling phthalimide to a chemical group that is reactive with a chemical group that is present in the molecule of interest. Thus, if it is desired to couple a phthalimide to a protein,

this may be achieved by derivatising phthalimide with a chemical group that reacts with amines, e.g. the side chain of lysine amino acids, or alternatively with thiols, e.g. the side chain of cysteine amino acids. Derivatised phthalimides that may react with amines include isothiocyanates, succinimidyl esters, tetrafluorophenyl esters, and sulfonyl chloride derivatives. Derivatised phthalimides that may react with thiols include alkylhalides, and maleimides.

Derivatives for coupling to amines and thiols, other functional groups, and methods of preparing such derivatives are well known to the person skilled in the art.

Phthalimides may be coupled to a molecule of interest via photolabelling. For example the phthalimide is coupled to a arylazide. Upon irradiation with light the arylazide will react with amines and thiols, e.g. on proteins.

Phthalimide may be coupled to nanoparticles such as gold. A molecule of interest having exposed thiol groups may then be labelled with the nanoparticles bearing the phthalimide.

Phthalimide may also be coupled to nucleic acids, e.g. DNA or RNA, in a number of ways. For example, the nucleic acid may be synthesised, e.g. by polymerase chain reaction (PCR) or reverse transcription, with modified nucleotides that allow attachment of derivatised phthalimide. Examples of this approach include use of allylamine dUTP to which phthalimide may be attached. Digoxygenin may be attached to modified uridine bases, phthalimide may then be coupled to nucleic acid via anti-digoxygenen antibody. Another way of attaching phthalimide to nucleic acid is via 5' labelling. The 5' free phosphate group may be coupled to amines, e.g. an amine group linked to a phthalimide, using carbodiimides . Phthalimide may also be incorporated into DNA by incorporating modified bases

into the nucleic acid using "nick labelling" this involves using an enzyme with endonuclease and repair activity.

Coupling to RNA may exploit the viceryl hydroxyl groups e.g. by converting these to aldehyde by periodate oxidation.

The skilled person is well able to couple phthalimides to any molecule of interest. See, for example, The Handbook — A Guide to Fluorescent Probes and Labelling Technologies, Tenth Edition, Invitrogen Corporation.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1

Figure 1 shows the reaction of 3-aminophthalimide (1) with hydrazine (2) to give luminol (3) and R-NH ₂ . The R group is the molecule of interest that is labelled with the 3- aminophthalimide. Figure 1 also shows the chemical structure of 5-aminophthalirtιide (5) , isoluminol (6) and the basic

phthalimide structure (7) .

Figure 2

Figure 2A shows a scatchard plot of cell bound, API-labelled insulin ^* in cell lysates, normalized for total cellular protein (mg/ml) , over unbound labelled insulin remaining in the binding buffer versus cell bound, labelled insulin. HepG2 cells maintained in 12 well culture dishes until 80% confluence were used for insulin binding assays according to the method of Calzi et al. ⁵ After washing with HBSS, cells were exposed to ImI binding buffer ⁵ containing 5, 2.5, 1.25 and 0.625μM API and incubated at 4 ⁰ C in the dark for 19 hours. Thereafter, labelled binding buffer was removed from the cells, the cells were washed with 3xlml PBS, removed from the wells by scraping, pelleted, resuspended in 200μl water, sonicated and lOμl of the lysate used for protein quantification. For API measurement, cell lysates or labelled binding buffer (lOOμl) were mixed with an equal volume of 98% hydrazine hydrate, heated at 6O ⁰ C for 30minutes and 50μl of the hydrazine treated sample mixed with 15μl of a 0.2mg/ml iodophenol (in IN NaOH) . Luminescence was then quantified for 10 seconds using a Tropix TR717 multiplate luminometer, after luminescence induction by the addition of lOOμl 0.045% ferricyanide solution to each sample.

Figure 2B shows a comparison API and fluorescein ^* labelled insulin uptake by HepG2 cells. HepG2 cells maintained in 6 well culture plates until 80% confluence were exposed to 2ml binding buffer containing lOμM API or fluorescein-labelled insulin as above. After 19 hours, cells were washed with 2x2ml unlabelled binding buffer and incubated for a further hour at 37 ⁰ C to induce insulin uptake. Thereafter, cells were washed with 2x2ml PBS (pH 7.4), incubated with 2ml PBS (pH 3.8) for 10 minutes, washed twice more with the latter buffer to remove remaining externally bound insulin, pelleted and sonicated in 400μl water. Fluorescein fluorescence (grey bar) and API-

luminescence (black bar) in lOOμl cell lysate were determined and normalized for protein as above, n=2, ± SE.

*API and Fluorescein-labelled insulin were prepared as follows: Lyophilized human recombinant insulin (3.5mg) (Novo Nordisk) was resolubilized in ImI of 5OmM borate buffer (pH

9.0) to which was added 0.3μmoles of NHS coupled-API ^§ or

0.3μmoles fluorescein -5 (6) -carboxamidocaproic acid N- succinimidyl ester (NHS-Fluorescein) (Sigma UK) in 200μl DMSO. After 1 hour at room temperature the labelled insulin was purified on PD-IO columns and the concentration of eluted, labelled insulin determined by the BCA assay. The concentration of insulin-bound API or fluorescein was determined by measuring the absorbance of the labelled insulin fractions at 395 and 495nm respectively. Molar values were determined from API and fluorescein-albumin standard curves. The specific activity for API-labelled insulin (black bar) or fluorescein-labelled insulin (red bar) is expressed as the ratio of label concentration (inM) to insulin concentration (mg/ml), n=2.

^§ 6- [N- (3-amino) phthalimide-5 (6) -carboxamido] hexanoic acid N- hydroxysuccinimide ester was synthesized by the esterification of 6- [N- (3-amino) phthalimide] hexanoic acid with Di (N- succinimidyl) carbonate.

Figure 3

Figure 3A and B shows fluorescence microscopy of HepG2 cells exposed to the API-labelled mitochondrial probe Triphenylphosphonium bromide (TPP) . HepG2 cells cultured on

Falcon tissue culture treated glass slides in 0.5ml DMEM were stained with 65μM API-labelled TPP ^¥ for 45 minutes in the absence (A) or presence (B) of a respiration inhibitor cocktail (13μg/ml Oligomycin, lOμg/ml Antimycin A and 5μM Rotenone, final concentration) , washed with PBS and viewed by

fluorescence microscopy using a Lucifer yellow filter at 4Ox magnification .

^¥ API-labelled TPP was synthesized by the coupling of N- [4- aminobutyl] - (3-amino) phthalimide with [2- (1,3 Dioxan-2-yl) ethyl triphenyl phosphonium bromide (Sigma-Aldrich) by reductive amination, following hydrolysis of the dioxan.

Figure 3C shows the effect of respiration inhibitors on the accumulation of API-labelled TPP by HepG2 mitochondria. HepG2 cells were cultured in 25cm ² tissue culture flasks until 100% confluence. Cells were then stained with 65μM API-labelled TPP for 45 minutes in the absence (black bar) or presence (gray bar) of a respiration inhibitor cocktail as above. Thereafter cells were washed with PBS (pH 7.4), removed from flasks by scraping and cell mitochondrial fractions obtained by homogenization and centrifugation as previously described. Mitochondrial pellets were sonicated in 200μl water and 50μl of the resultant lysate hydrazine treated and assayed for luminescence as in Fig. 2. Luminescence values are normalized for total mitochondrial protein (mg/ml) , n=2, +SE.

Figure 4

Figure 4A shows API-heparin run on a 4-20% TBE polyacrylamide gel and visualised using a transilluminator (arrow) . Labelled Heparin was prepared by periodate oxidation. Porcine heparin (2mg) was oxidised with 1OmM sodium periodate (final concentration) in 500μl, 10OmM Acetate buffer (pH 5.5) for 10 minutes. Thereafter, 0.5ml of 2% Borate buffer (pH 9.3) and lOμl 0.5M sodium hydroxide was added to the heparin to bring the pH to 9.3. To this ImI solution the following was then added: 107μl of a IM sodium cyanoborohydride solution in 1% borate buffer (pH 9.3) and 2mg N- [4-aminobutyl] 3- aminophthalimide dissolved in DMSO. Coupling of the API-amine to the oxidised heparin was carried out for 18 hours at RT. Labelled heparin fractions were then collected using a PDlO

column as for labelled insulin. Heparin concentrations in labelled fractions were determined by the carbazole assay (21) , while the concentration of API was determined by measuring the absorbance of the labelled heparin fractions at 395nm.

Figure 4B shows fluorescence microscopy of HepG2 cells on slides, exposed to 5μg of API-labelled heparin for 18 hours and viewed by fluorescence microscopy as above, indicating discrete cytosolic and nuclear staining.

Figure 4C shows the effect of unlabelled heparin on API- heparin nuclear transport. Cells were exposed to lOμg API- heparin for 18 hours in the absence (black bar) or presence (grey bar) of excess (500μg) unlabelled heparin. Thereafter, cells were washed with PBS, lysed with 1% NP40 and nuclear pellets obtained by centrifugation at 90Og. Pellets were sonicated in 200μl PBS, half the sample treated with hydrazine and assayed for chemiluminescence as above. Samples are normalised for total protein (BCA assay) mg/ml, n=3, +SD.

Figure 4D shows the results of API-heparin nuclear transport. Cells were exposed to lOμg API-heparin (grey bar) or unlabelled heparin (black bar) for 18 hours. Thereafter, cells were washed with PBS, lysed with 1% NP40 and nuclear pellets obtained by centrifugation at 90Og. Pellets were sonicated in 200μl PBS, and lOOμl samples treated with hydrazine and assayed for chemiluminescence as for insulin studies. Samples are normalised for total protein (mg/ml) , n=3, +SD.

Figure 5

Figure 5A shows fluorescence microscopy of HepG2 cells maintained on tissue culture-treated slides exposed to 24μM API-palmitic acid for 23 hours, indicating membrane staining and incorporation into discrete lipid bodies (4Ox magnification) . API-palmitic acid was synthesized from 16-

aminopalmitic acid and 3-nitrophthalic anhydride by heating 1.36mmols of the latter with 2.6mmols of the anhydride for 5 minutes at 180 ⁰ C. The resultant crude nitrophthalimide was then reduced with dithionite and after washing with brine and lyophalization, the crude API-palmitic acid was purified by silica chromatography using ethyl acetate as the mobile phase.

Figure 5B shows lipid profiles of HepG2 cells exposed to 240μM API-palmitic acid for 23 hours. After labelling, cells were washed with PBS and the incubation medium (10ml) collected and lyophalized. Cells along with dried medium were Folch extracted using 2Ox the volume of Folch reagent to cell pellet mass (22) . The lower extraction phases from cell pellets and medium was dried under nitrogen, lyophalized and resuspended in 20μl chloroforiti/methanol (2/1) . Total lipid extracts (6μl) from culture medium (CM) and cell lysates (CL) were separated on C18/silica TLC plates using 95% methanol as the mobile phase and visualised by transillumination. Mobility is inversely proportional to hydrophobicity . The uppermost arrow indicates an API-palmitic acid reference; the lowest arrow indicates poorly mobile, fluorescent API-lipid species in cell lysates and the arrow lying between the uppermost and lowest arrows indicates cellular export of the latter species into cell culture medium.

Figure 5C shows a comparison of lipid extracts untreated (L-) or treated with 28units of lipase for 60 minutes at 30 ⁰ C (L+) , separated on C18/silica TLC plates and visualised by transillumination. Arrows indicate fluorescent acyl-glyceride lipid species susceptible to lipase degradation.

Figure 5D shows the effect of fillipin on API-palmitate transport in HepG2 cells. Cells were exposed to 24μM API- palmitic acid for 30 minutes alone (black bar) or with 5μg/ml fillipin complex (grey bar) . Thereafter, cell pellets were sonicated, treated with development agent and assayed for

chemiluminescence as above. Values are normalized for total protein (mg/ml) , n=3, ±SD.

Figure 6 Figure 6 shows an assay scheme that utilises a 3- aminophthalimide labelled probe. Figure 6A shows the target molecule (T) bound to the surface of a microwell or tube. A reporter molecule (R) is labelled with 3-aminophthalimide (API) . In Figure 6B the labelled reporter molecule is added to the tube and it binds to or enters the target molecule. In Figure 6C hydrazine is added to the tube which converts 3- aminophthalimide to free (unconjugated) luminol (L) which releases light upon chemiluminescent induction. Light production is measured with a luminometer. The amount of light produced is directly proportional to the amount of target molecule present.

Figure 7

Figure 7 shows a 529bp AMPKα2 fragment that was amplified by PCR in the presence allylamine-dUTP with varying amounts of dTTP, or with dTTP alone (control) . The products were subsequently labelled with an amine reactive API- NHS ester. DNA (10-15μg) was labelled with 300μg NHS-API in lOOμl 0.15M bicarbonate buffer (pH 9.0) for 40 minutes. Labelled DNA was then purified on a Qiaquick DNA column (Qiagen) and DNA concentration determined using a nanodrop microspectrophotometer . Labelled DNA was run on a 1% agarose TAE gel and visualised with a transilluminator . Lane A shows the control (dTTP alone) did not label with API, whist lanes B and C exhibit API labelling, with degree of labelling reflective of the allylamine dUTP : dTTP ratio, lane B 1:1 and lane C 1:3, (allyl-dUTP:dTTP respectively) .

Figure 8 Figure 8 shows HepG2 cells that were transfected with 0.8μg API-labelled DNA using lipofectamine for 5 hours as per

manufacturer's instructions (Invitrogen) and viewed by fluorescence microscopy as above. Fluorescent DNA/lipofectamine complexes were incorporated into membranes (white arrows, Figure 8A and B) . In addition, fluorescent DNA/lipofectamine complexes were evident on the surfaces of both cells and slide (Figure 8B black arrows) .

Figure 9

Figure 9 shows API-DNA uptake quantified from cells transfected with 1.6μg of labelled DNA or 1.6μg of unlabelled control DNA, with or without lipofectamine for 16 hours. Cells grown in 12 well plates were exposed to DNA or DNA lipofectamine complex, washed with PBS, pelleted and sonicated in 300μl PBS. Lysates (lOOμl) were then exposed to hydrazine and assayed as above. Cells exposed to API-DNA exhibited significant luminescence whilst all controls displayed only background levels as per the medium only control. n=4, ±SD.

Figure 10 Figure 10 shows the chemical structure of various molecules.

Molecule (8) is an annelated derivative of luminol that is reported in Whitehead et al . (reference 16) to be chemiluminescent .

Molecule (9) is the phthalimide analogue of molecule (8) . Molecule (10) is the probable structure of chemiluminescent polymer diazoluminomelanin (DALM) as reported in Bruno et al.

(reference 14) .

Molecule (11) is the phthalimide analogue of molecule (10) .

Molecules (12) , (14) , and (16) are chemiluminescent molecules reported in Han et al . (reference 15) .

Molecules (13) , (15) and (17) are the phthalimide analogues of molecules (12) and (14) and (16) respectively.

Molecule 18 is bis (2, 4, 6-trichlorophenyl) oxlate (TCPO) .

Molecule 19 is 5- (N, N-dimethylamino) -phthalimide (referred to as 4- (N, N-dimethylamino) -phthalimide in Vazquez et al . ) .

Figure 11

Figure HA shows conversion (%) of 5βnmols of API-palmitate in lOOμl cell lysate (grey bar) or PBS (black bar) to luminol after treatment with an equal volume of hydrazine as described for insulin studies. Complete (100%) conversion is assumed to yield 56nmols (lOμg) of luminol. After hydrazine treatment, luminol was detected and quantified by GC-MS using a luminol standard curve (inset), n=3,±SD.

Figure HB shows a mass spectrum for silylated luminol. The major fragment ion for derivatized luminol has a m/ z of 404, corresponding to the de-protonated molecular ion for the silylated derivative.

Figure HC shows a Standard curve for API. NHS-API stocks were doubly diluted in 1 : 1 DMSO: Tris.HCl (pH 8.5) solution. Dilutions were treated with an equal volume of hydrazine and chemiluminescence determined as for insulin studies.

Figure 12

Figure 12a shows HepG2 cells exposed to the fluorescent mitochondrial probe, JC-I. Cells cultured on Falcon tissue culture treated glass slides in 0.5ml DMEM were stained with 6.5μM JC-I for 45 minutes, washed with PBS and viewed by fluorescence microscopy using a PE filter at 4Ox magnification. White arrow indicates fluorescent foci.

Figure 12b shows HepG2 cells exposed to the mitochondrial probe JC-I as in Fig. 12a but in the presence of a respiration inhibitor cocktail (13μg/ml Oligomycin, lOμg/ml Antimycin A and 5μM Rotenone final concentration) .

Figure 13

Figure 13 shows the chemical structure of various API derivatives and precursor molecules. These are referred to in the synthesis methods discussed herein.

Detailed Description of the Invention

Specific details of the best mode contemplated by the inventors for carrying out the invention are set forth below, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.

The following examples illustrate the use of the API system in fluorescence and chemiluminescence-based bioassays. In all examples "API" refers to 3-aminophthalimide and the developing agent is hydrazine.

Example 1 Insulin Binding and Uptake

An amine reactive, N-hydroxysuccinimide (NHS)-API ester was synthesized (see Example 7) and used to label human recombinant insulin. The resultant API-labelled insulin was used in insulin binding and uptake studies using HepG2 cells according to Calzi et al. (1) .

1) Insulin receptor-dependent insulin binding was assessed by quantifying cell bound label relative to unbound, or free label, still present in the binding buffer.

2) Insulin receptor mediated insulin endocytosis was assessed by quantifying internalized label by luininometry, after treatment of cell pellets with developing agent.

Scatchard plots (bound ligand/free ligand versus bound ligand) revealed that API-Insulin produced a typical curvilinear plot (Fig. 2A) , previously shown to be indicative of insulin binding using ¹²⁵ I-labelled insulin (2) . Endocytosis studies showed that insulin receptor mediated insulin uptake for cells exposed to lOμM API-insulin was 24-fold greater than that for cells exposed to the same concentration of fluorescein-

labelled insulin (prepared from commercially available NHS- fluorescein) (Fig. 2B) .

Example 2 Mitochondrial probe

To illustrate the use of the API system in dual fluorescence and chemiluminescence studies, API was coupled to triphenylphosphonium bromide (TPP) (see Example 8) , a well established mitochondrial probe, which accumulates in mitochondria in a membrane potential proportional manner (3) . Cells stained with TPP-API and viewed by fluorescence microscopy exhibited green fluorescence with the appearance of brightly fluorescent foci (white arrow) indicating the accumulation of this compound within mitochondria (Fig 3A) . In the presence of a mitochondrial respiration inhibitor cocktail, focal staining was abolished (Fig. 3B) .

In comparison, cells stained with the established membrane- potential dependent mitochondrial dye, JC-I (Fig.12A), exhibited identical focal staining to TPP-API. JC-I staining was also significantly diminished, although not completely abolished by incubation of cells with the same mitochondrial respiration inhibitor cocktail (Fig.12B) . Therefore it is clear that like JC-I, TPP-API accumulated within mitochondria in a membrane potential-dependent manner.

To quantify TPP-API mitochondrial accumulation, HepG2 cells were labelled with 65μM TPP-API with or without mitochondrial respiration inhibitor cocktail for 45 minutes. Cells were then homogenised, the mitochondrial fraction obtained by centrifugation and mitochondria assessed for internalized label by luminometry, after treatment with developing agent. Mitochondria from cells exposed to inhibitor cocktail exhibited a 25% reduction in TPP-API chemiluminescence in comparison cells exposed to TPP-API alone (Fig. 3C) .

Example 3

Heparin studies To illustrate the use of the API-system in polysaccharide labelling, An API molecule containing a terminal amine group (see Example 9) was coupled to heparin by periodate oxidation and reductive amination. API-labelled heparin (Fig. 4A) was then used in cellular uptake and nuclear transport studies according to Dudas et al. 2000 (5) . HepG2 cells incubated with 5μg API-heparin for 18 hours and viewed by fluorescence microscopy exhibited fluorescent cytosolic and nuclear staining, indicating cellular uptake of heparin in a manner similar to that observed using biotinylated heparin and fluorescein-labelled streptavidin detection (Fig. 4A) (5) . To chemiluminometrically quantify heparin nuclear transport, cells were incubated with lOμg API-heparin with or without excess unlabelled heparin for 18 hours. Nuclear pellets were assessed for internalized label by luminometry, following sonication and treatment with developing agent. HepG2 nuclear transport of API-labelled heparin was significantly reduced in the presence of excess unlabelled polysaccharide indicating that the accumulation of the API label within nuclei was heparin-dependent (Fig. 4C) .

Confirmation that API-labelled heparin nuclear transport was responsible for the observed chemiluminescence was obtained for API-heparin and unlabelled heparin (Fig. 4D) .

Example 4

Lipid transport and metabolism

To illustrate the use of the API system in lipid studies, an API-labelled long chain fatty acid, API-palmitic acid, was synthesized (see Example 10) and used to investigate cellular uptake and lipid metabolism. HepG2 cells incubated with 24μM API-palmitic acid for 23 hours and viewed by fluorescence

microscopy exhibited both membrane staining and discrete cytosolic accumulation (Fig. 5A) .

Importantly, total lipid extracts from cells incubated with API-palmitate, and separated using C18/silica thin layer chromatography, contained fluorescent API-labelled lipid species with increased hydrophobicity . This indicated the incorporation of API-palmitic acid into more complex lipid species, a proportion of which was exported into culture medium (Fig. 5B) . In addition, a significant proportion of these fluorescent API-labelled lipid species was removed by lipase treatment, further indicating the incorporation of API- palmitic acid into acyl-glycerides (Fig 5C) . To quantify lipid transport using the API chemiluminescence system, HepG2 cells were incubated with 24μM API-palmitate for 30 minutes with or without filipin, an inhibitor of caveolin-dependent fatty acid transport (6) . Cell pellets were assessed for internalized fatty acid by luminometry, following sonication and treatment with developing agent. HepG2 transport of API- palmitic acid was reduced (20%) in the presence of fillipin, (Fig. 5D) , indicating that API-palmitic acid transport was partly caveolin-dependent. This is in agreement with a previous study which utilized NBD-labelled stearic acid (6) .

Example 5

API as a nucleic acid label

To illustrate the applicability of the API-system as a nucleic acid label, a 529 base pair DNA fragment was amplified from a plasmid containing the AMP-activated kinase alpha subunit (AMPKα2) by PCR. Primers used were: Forward: TGG

CTGAGAAGCAGAAGCAC Reverse: GGCGATCCACAGCTAGTTCG. PCR was carried out in the presence of alIyIamine-dUTP, a modified base containing an allylamine functional group. This allowed for the incorporation of terminal amine groups within the PCR product. The resultant allylamine PCR product was then labelled with an amine reactive API- NHS (N-

hydroxysuccinimide-coupled) ester (see Example 7) to generate a fluorescent API-DNA conjugate (Fig. 7) .

The API-labelled DNA was then used for transfection studies using HepG2 cells. HepG2 cells were transfected with O.δμg labelled DNA using Lipofeetamine 2000 (Invitrogen, as per manufacturers' instruction) for 5 hours and viewed by fluorescence microscopy. Discrete regions of fluorescent DNA/lipofectamine complex were visibly incorporated into membranes (white arrows, Fig 8A) . In addition, fluorescent

DNA/lipofectamine complexes were also evident on the surfaces of cells and on the slide surface (Fig8B black arrows) .

To quantitatively assess DNA uptake using lipofectamine transfection, cells were transfected with 1.6μg of labelled DNA or 1.6μg of unlabelled control DNA with or without lipofectamine for 16 hours. Cells were then sonicated, and assayed for chemiluminescence after hydrazine treatment, as previously described. Cells exposed to API-DNA exhibited significant luminescence whilst all controls displayed only background levels (Fig. 9) .

Details of the labelling of DNA with API are as follows.

Initially, to introduce allyl-amine dUTP into a DNA probe, which would allow for labelling with API, a 529bp AMPKα2 fragment was amplified by PCR from pcDNA3 plasmid construct containing the full length cDNA sequence. The following primers and conditions were used:

Forward (F) 5' -tggctgagaagcagaagcac-3' and Reverse (R) 5'- ggcgatccacagctagttcg-3'

PCR mix: Buffer (Qiagen) , 0.5mM MgCl ₂ , 0. ImM dATP, 0. ImM dCTP,

O.litiM dGTP, 0.05mM dTTP, 0.05mM allyl-amine dUTP (Sigma), 5ng

DNA, 0.5μM primer F, 0.5μM primer R, lunit Taq DNA polymerase

(Qiagen) . Final volume 20μl. Cycling conditions: 94 ⁰ C 3 minutes; (94 °C 30 seconds, 57 ⁰ C 30 seconds, 72 ⁰ C 1 minute) 30 cycles; 72 °C 10 minutes.

PCR amplified product (llx20μl samples) either having been synthesized using allyl-amine dUTP: dTTP in a 1:1 ratio or dUTP alone was pooled and concentrated using Qiaquick PCR purification kit (Qiagen) . Samples were eluted with 50μl 4mM potassium phosphate buffer (pH 8.5) to which was added, 30μl 0.3M bicarbonate buffer (pH 9.1) . To this, 20μl of a NHS-API stock was added (17mg/ml in a 1:11 pyridine: DMSO solution), the mixture mixed for 3 minutes and then incubated for a further 40 minutes at room temperature. Labelled DNA was purified again as above, with an additional 80% ethanol wash step, eluted with a final volume of 150μl (50μl 1OmM Tris.HCl (pH 8.5) and 2x50μl H ₂ O) and quantified using a Nanodrop microspectrophotometer . Samples were lyophilised and stored at -80 ⁰ C until use.

Example 6

Determination of API to luminol conversion efficiency by GC-MS analysis

Cell lysates (2mg/ml total protein) or PBS samples (lOOμl) were spiked with 56nmols of API-palmitate in DMSO such that 100% conversion of API to luminol by hydrazine treatment would yield 56nmols (lOμg) luminol. Samples were then incubated with an equal volume of 98% hydrazine hydrate for 30 minutes at 6O ⁰ C. Luminol was extracted from hydrazine-treated samples using ENV ⁺ reverse-phase extraction columns (Isolute SPE products) . In brief, the samples were dissolved in 1 ml Tris- HCl buffer (20 mM, pH 8.0) and extracted on an ENV ⁺ column, which had already been pre-washed with 1 ml methanol and 4 ml of Tris-HCl buffer (pH 8.0) . The columns were washed with water, and eluted with 2 ml of methanol. Purified samples were dried under vacuum and derivatized to the tert- butyldimethylsilyl derivative by the addition of 20 μl of di- isopropylethylamine, 30 μl dimethylformamide and 30 μl of N- (t-butyldimethylsilyl) -ZV-methyltrifluoroacetamide for 1 h at

60 ⁰ C. Derivatized samples were dried under nitrogen and re- dissolved in 20 μl of n-undecane. Samples were applied to a GC equipped with a 15-m DB-1701 (J&W Scientific, Folsom, CA) capillary column (0.25-mm internal diameter, 0.25-mm film thickness) interfaced with a mass spectrometer (Trio 1000;

Fisons Instruments, Beverly, MA) . The ion source and interface temperatures were set at 200 and 320 °C, respectively. Samples were analyzed in negative-ion chemical ionization mode with ammonia as the reagent gas, using 1 μl of each sample for injection. The initial column temperature was maintained at 150 ⁰ C for 1 min increasing to 300 ⁰ C at 20 °C/min. The mass spectrum obtained for silylated luminol is shown in Figure HB . The major fragment ion for derivatized luminol has a m/z of 404, which corresponds to de-protonated molecular ion for the silylated derivative. For quantitative measurement of luminol, ions were monitored by single ion monitoring at 404 m/z and their concentrations determined against known amounts of standards .

Thus, GC-MS analysis indicates that hydrazine treatment of

API-spiked cell lysates results in an approximately 40% API to luminol conversion efficiency and a typical assay is linear from O.βnmol to lpmol with a detection limit of 0.3pmol . (See Figures HA and HC) . Upon induction, maximum chemiluminescence occurs within 0.25 seconds.

The following examples relate to the synthesis of API derivatives useful in the assay methods of the present invention. The structures referred to are shown in Figure 13.

Example 7

NHS-API

6- [N- (3-amino)phthalimide-5 (6) -carboxamido] hexanoic acid N- hydroxysuccinimide ester (NHS-API) (2) was synthesized by the esterification of 6- [N- (3-amino)phthalimide] hexanoic acid (1) with di (N-succinimidyl) carbonate. Initially, (1) was

synthesized by fusing equal quantities (2.6mmols) of 3- nitrophthalic anhydride and 6-amino-n-caproic acid at 153 ⁰ C for 10-15 minutes. The melt (crude 6- [N- (3-nitro)phthalimide] hexanoic acid) was washed with boiling water and resuspended in 10ml 10% sodium dithionite. Reduction was carried out for at least 20 minutes with maintenance of the pH at 7-7.5, until most of the suspension had dissolved. After, the removal of insoluble residue, the pH was adjusted to 3.0 with HCl and crude (1) precipitated with brine. The resultant oily yellow pellet was washed with water, lyophilised and reacted with 2.6mmols di (N-succinimidyl) carbonate in 2ml pyridine/3ml acetonitrile for 1 hour at 6O ⁰ C, then dried at 60 ⁰ C for a further hour under nitrogen. The resultant crude (2) was purified on a silica gel 60 column using dichloromethane/ acetone (40/80) as the mobile phase and/or recrystallized from ethyl acetate. Lyophilized aliquots were stored desiccated at -2O ⁰ C until use.

¹ H NMR (CDCl ₃ , 500 MHz) 8.11-8.05 (2H, m, 2 x ArH), 7.92 (IH, t, j = 7.7 Hz, ArH), 5.21, (2H, br. s, NH ₂ ), 3.72 (2H, t, j = 7.2 Hz, CH ₂ ), 2.83 (4H, br. s, 2 x CH ₂ ), 2.60 (2H, t, j = 7.2 Hz, CH ₂ CO), 1.82-1.61 (6H, m, 3 x CH ₂ ) ppm. 1 ³ C NMR (CDCl ₃ , 125 MHz) 170.3 (s, C=O), 169.2 (s, 2 x C=O), 165.9 (s, C=O), 163.0 (s, C=O) , 145.2 (s, C(Ar)), 135.2 (d, CH(Ar)), 128.5 (d, CH(Ar)), 127.0 (d, CH(Ar)), 38.4 (t, CH ₂ ) , 30.9 (t, CH ₂ ) , 27.9 (t, CH ₂ ), 25.6 (t, 2 x CH ₂ ), 24.1 (t, CH ₂ )

FTIR (Thin Film, cm ^"1 ) 3476 w, 3374 w, 2943 m, 1780 m, 1735 s, 1715 s, 1695 s, 1633 m, 1341 m.

LRMS (CI +ve) 404 (55%), 374 ([MH] ⁺ , 37%), 259 ([M-ONSu] ⁺ , 100%) .

Example 8

TPP-API

N- [4- [3- (triphenylphosphonium) propyl] aminobutyl] (3- amino) phthalirαide (TPP-API) (4) was synthesized by the coupling of N- [4-aminobutyl] (3-nitro) phthalimide (3) with [2- (1, 3dioxan-2yl) ethyl] triphenylphosphonium bromide (DETP) using reductive amination. Initially, crude (3) was prepared as follows: 6.2mmols of putrazine dihydrochloride was combined with 0.012mols of sodium hydroxide in ImI methanol and vortexed until dissolution of the hydroxide pellets. The suspension was centrifuged at 350Og for 5 minutes and the supernatant (ImI) placed in a thick walled 50ml glass tube to which was added 2.6mmols 3-nitrophthalic anhydride. The mixture was rapidly heated at 18O ⁰ C until converted to a yellow melt. The warm melt (crude (3)) was dissolved in 10ml methanol and dried under nitrogen yielding a yellow solid which was coupled to DETP as follows: DETP (3.1mmols) was stirred with 0.03g p-toluene sulphonic acid (in 20ml methanol) to allow for dioxan hydrolysis. After 1 hour, crude (3) (dissolved in 10ml water) was added to the above and the solution adjusted to pH 7. Reductive amination was initiated by the addition of 3.1mmols sodium cyanoborohydride and the pH maintained at 6.8- 7.0 for 1 hour with HCl, yielding crude N- [4- [3- triphenylphosphonium) propyl] aminobutyl] (3-nitro) phthalimide, which was reduced by the addition of 3g (10%) sodium dithionite. The resultant yellow precipitate (crude (4)) was washed extensively with water and lyophilized and stored dessicated at -2O ⁰ C until use or as frozen DMSO stocks.

Example 9

N- [4-aminobutyl] (3-amino) phthalimide (5) for heparin studies was prepared from monoBoc-protected putrazine and 3- nitrophthalic anhydride (3NPA) . Initially, equivalent amounts (0.023mols) of putrazine and (Boc) ₂ 0 were reacted in methanol

in the presence of an iodine catalyst (0.0023mols) according to the method of Varala et al. 2006 (20) . The resultant precipitate (crude mono-protected diamine) was washed extensively with water, lyophilised and 0.5g fused with an equivalent amount of 3NPA at 153 ⁰ C for 10 minutes. The melt

(crude N- [4-Boc-aminobutyl] (3-nitro) phthalimide) was dissolved in 10ml THF and reduced to Boc protected (5) with 10% dithionite. Phase separation was achieved by the addition of excess sodium bicarbonate and the organic phase collected and dried under nitrogen. Crude N- [4-Boc-aminobutyl] (3- amino) phthalimide was purified by silica gel chromatography, deprotected with trifluoroacetic acid for 10 minutes, dried under nitrogen and re-dissolved in water. The resultant water- soluble, N- [4-aminobutyl] (3-amino) phthalimide, trifluoroacetate salt was lyophilized and stored desiccated at -2O ⁰ C until use.

Characterisation Data for NH ₂ -API

FTIR (Thin Film, cm ^"1 ) 2950 m, 1686 s , 1634 m, 1535 m .

LRMS (CI +ve) 264 ( [M+MeOH] ⁺ , 20% ) , 234 ( [MH] ⁺ , 15% ) , 217 ( [M- NH ₂ ] ⁺ , 15% ) , 148 ( 100% ) .

HRMS (CI +ve) [MH] ⁺ , C ₁₂ H ₁₅ N ₃ O ₂ Requires 234 . 12425 . Found 234 . 12428 .

Example 10

Omega API-palmitic acid

Omega API-palmitic acid (6) was synthesized as follows: 16- Bromopalmitic acid (1.5mmols) (Aldrich) in 10ml ethanol was added to excess (20ml) 30% ammonia solution and heated in a boiling water bath for 1 hour in a 50ml capped tube with

constant mixing. The resultant white precipitate (crude 16- aminopalmitic acid, ammonium salt) was converted to the potassium salt with potassium hydroxide, lyophilised and 0.4g fused with 0.5g 3NPA at 18O ⁰ C until the formation of a light brown melt was evident. The cooled melt was washed with boiling water and reduced with 10% sodium dithionite solution as above, yielding a yellow solid (crude 16-N-[(3- amino) phthalimide] hexadecanoic acid) (API-palmitic acid) which was acidified and washed with water. Crude API-palmitic acid was purified by silica gel chromatography using ethyl acetate and/or recrystallized from dichoromethane and stored desiccated at -2O ⁰ C until use.

Characterisation Data for ω-16-API

¹ H NMR (CDCl ₃ , 500 MHz) 7.58 (IH, dd, j = 8.5, 7.2 Hz, ArH), 7.35 (IH, d, j = 7.2 Hz, ArH), 7.04 (IH, d, j = 8.5 Hz, ArH), 5.41 (2H, br. s, NH ₂ ) , 3.92 (IH, t, j = 7.4 Hz, NCHH), 3.85 (IH, t, j = 7.2 Hz, NCHH), 2.56 (2H, t, j = 7.5 Hz, CH ₂ CO ₂ H), 1.94-1.79 (4H, m, 2 x CH ₂ ), 1.61-1.42 (22H, m, 11 x CH ₂ ) .

¹³ C NMR (CDCl ₃ , 125 MHz) 179.8 (s, C=O), 167.5 (s, C=O), 166.0 (s, C=O), 145.2 (s, C(Ar)), 135.1 (d, CH(Ar)), 128.5 (d, CH(Ar)), 126.9 (s, C(Ar)) , 121.0 (d, CH(Ar)), 112.7 (s, C(Ar)), 38.9 (t, CH ₂ ) , 37.7 (t, CH ₂ ), 34.1 (t, CH ₂ ), 29.7 (t, 2 x CH ₂ ), 29.5 (t, 2 x CH ₂ ), 29.3 (t, 2 x CH ₂ ), 29.1 (t, 2 x CH ₂ ), 28.8 (t, CH ₂ ), 28.4 (t, CH ₂ ), 26.9 (t, CH ₂ ), 24.8 (t, CH ₂ ) .

FTIR (Thin Film, cm ^'1 ) 3394 w, 2918 s, 2847 s, 1753 m, 1718 s, 1697 s, 1634 m, 1541 m, 1467 m.

LRMS (EI +ve) 416 (M ⁺ , 32%), 175 ( [M- (CH ₂ ) ₁₄ CO ₂ H] ⁺ , 100%) . HRMS (EI +ve) M ⁺ , C ₂₄ H ₃₆ N ₂ O ₄ Requires 416.26696. Found 416.26604.

References

1. Calzi SL, Choice CV, Najjar SM. Differential effect of ppl20 on insulin endocytosis by two variant insulin receptor isoforms . Am J Physiol Endocrinol Metab 273, 801-808 (1997) .

2. Donner DB. Regulation of insulin binding to isolated hepatocytes: Correction for bound hormone fragments linearizes Scatchard plots. Proc Natl Acad Sci USA 77, 3176-3180 (1980) .

3. Ross MF, Kelso GF, Blaikie FH, James AM, Cocheme HM, Filipovska A, Ros T, Hurd TR, Smith RAJ, Murphy MP. Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry (Moscow) 70, 222-230 (2005) .

4. Pallotti F, Lenaz G, Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. In: Methods in Cell Biology, Volume 65 Mitochondria (Eds. Pon LA, Schon BA) Academic Press, London UK, pp 2-31 (2001) .

5. Dudas J, Ramadori G, Knittel T, Neubauer K, Raddatz D, Egedy K, Kovalszky I. Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: altered potential of hepatocellular carcinoma heparan sulphate. Biochem J. ; 350, 245-51 (2000) .

6. Pohl J, Ring A, Stremmel W. Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway. J Lipid Res. 43, 1390-9 (2002) .

7. Collins MO, Yu L, Husi H, Blackstock WP, Choudhary JS, Grant SG. Robust enrichment of phosphorylated species in complex mixtures by sequential protein and peptide metal-affinity chromatography and analysis by tandem mass spectrometry. Sci

STKE. 298, plβ (2005) .

8. Bieber AL, Tubbs KA, Nelson RW. Metal ligand affinity pipettes and bioreactive alkaline phosphatase probes: tools for

characterization of phosphorylated proteins and peptides MoI Cell Proteomics. 3, 266-72(2004) .

9. Kuroda N, Hosoki S, Nakashima K, Akiyama S, Givens RS, Photographic detection of fluorescent-labelled oigodeoxynucleotide in the blotting format by peroxyoxalate chemiluminescence, J. Biolumin. Chemilumin. 13, 101-105 (1998).

10. Milofsky RE, Birks JW, J. Am. Chem. Soc. 113, 9715-9723 (1991).

11. Chokshi HP, Barbush M, Carlson RG, Givens RS, Kuwana T, Schowen RL, Biomed. Chrom. 4, 96-99 (1990) .

12. Vazquez ME, Rothman DM, Imperiali B, A new environment- sensitive fluorescent amino acid for Fmoc-based solid phase peptide synthesis, Org. Biomol. Chem. 2, 1965-1966 (2004) .

13. Werner SC, Acebedo G, Radichevich I, Rapid radioimmunoassay for both T4 and T3 in the same sample of human serum. J Clin Endocrinol Metab. 38, 493-5 (1974) .

14. Bruno JG, Parker JE, Howlwitt E, Alls JL, Kiel JL, Preliminary Electrochemiluminescence studies of metal ion-bacterial diazoluminomelanin (DALM) interactions, J.

Biolumin. Chemilumin, 13, 117-123 (1998)

15. Han J, Jose J, Mei E, Burgess K, Chemiluminescent energy- transfer cassettes based on fluorescein and nile red, Angew. Chem. Int. Ed., 46, 1684-1687 (2004)

16. Whitehead TP, Kricka LJ, Carter TJN, Thorpe GHG, Analytical luminescence: its potential in the clinical laboratory, Clin. Chem. 25/9, 1531-1546 (1979)

17. Okamoto H, Kohno M, Satake K, Kimura M, An azacrowned phthalimide as a metal-ion sensitive and solvatoflurochromic

fluorophore: fluorescence properties and a mimic integrated logic operation, Bull. Chem. Soc. Jpn., 78, 2180-2187 (2005).

18. Ando Y, Niwa K, Yamada N, Irie T, Enomoto T, Kubota H, Ohrαiya Y, Akiyama H, Determination and Spectroscopy of

Quantum Yields in Bio/Chemiluminescence via Novel Light- Collection-Efficiency Calibration: Reexamination of The Aqueous Luminol Chemiluminescence Standard, Front for the arXiv, q-bio.QM/0610029.

19. Ando Y, Niwa K, Yamada N, Irie T, Enomoto T, Kubota H, Ohmiya Y, Akiyama H, Development of a Quantitative Bio/Chemiluminescence Spectrometer Determining Quantum Yields: Re-examination of the Aqueous Luminol Chemiluminescence Standard, Photochemistry and

Photobiology, 83, 1205-1210 (2007) .

20. Varala, R., Nuvula, S., & Matile, S. R. Molecular iodine- catalyzed facile procedure for N-Boc protection of amines. J. Org. Chem. 71, 8282-8286 (2006) .

21. Cesaretti, M., Luppi, E., Maccari, F. & Volpi, N. A 96- well assay for uronic acid carbazole reaction. Carbohydrate Polymers. 54, 59-61 (2003) .

22. Folch, J., Lees, M. & Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497-509 (1957) .