AU2004224246B2 - Method of enhancing fluorescence - Google Patents

Description

WO 2004/085546 PCT/AU2004/000370 -1 METHOD OF ENHANCING FLUORESCENCE Technical Field This invention relates to a method of enhancing fluorescence, especially in organic molecules, such as proteins -and nucleic acids, stained or labelled with fluorescent dyes. 5 Background Art Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Compounds that fluoresce have many uses and are known to be particularly suitable 10 for biological applications where fluorescence is intrinsically more sensitive than absorption as the incidence and observed wavelengths are different. Fluorescence can be used for the detection of whole cells, cellular components, and cellular functions. For example, many diagnostic and analytical techniques require the samples to be fluorescently tagged so that they can be detected. This is achieved by using fluorescent dyes or probes 15 which interact with a wide variety of materkis such as cells, tissues, proteins, antibodies, nme drug, horm pid, nucleotides, nucleic acida, carbohydrates, or naturA or synthetic polymers to mahe fluorescent conijugates. With rynthedi fluorercent probes, ligands are frequenLy used to confer a pCifity for a biochemical reaction that is to be observed and the fluorescent dye provides the means 20 of detect or quantify the interaction, These applications include, among others, the detection ofproteins (fer ezxarnple in gels, on :urfaice or agneous solution), cell tracing, the assessment of enzymatic activity and the staining of nucleic acids or other biopolymers. Long wavelength absorbance usually increases the utility of a fluorescent probe since it reduces the interference from cellular autofluorescence and is less likely to cause photo 25 damage of labelled bioinolecules. Although lasers are particularly useful as a concentrated light source for the excitation of fluorescence, at present the output of powerful lasers is restricted to particular wavelengths of light. Compounds whose excitation spectrum coincide with laser output are therefore of high utility. The argon laser is the most common light source for excitation of fluorescence, and has principal output at 488 nm and a weaker 30 output at 514 nm. Fluorescent compounds that are excited by either of these wavelengths are therefore of particular utility. YAG lasers (532 nm or 473) and HeNe (543 nm, 633 nm) are also becoming common.

WO 2004/085546 PCT/AU2004/000370 -2 Red fluorescent compounds are used extensively in many fields of biological study Many of these, including Texas red, Tetramethyl rhodamine or red emitting BODIPY dyes require excitation at green wavelengths such as 542 nm. This limits their use in many applications, especially those where the argon-ion laser is used for excitation. 5 Compounds such as ethidium bromide, can be excited with light from the argon-ion laser, but are not generally suitable for tagging of organic molecules other than nucleic acids. Other compounds such as phycoerythrin, can be excited using the argon-ion laser (488 nm), and emits in the orange wavelengths (ca 580 nm). Phycoerythrin, however, has poor stability and a high molecular weight (ca 240,000Da) making it unsuitable for many 10 applications such as cell tracking, labelling of nucleic acids or staining proteins. For staining of proteins, there are a number of methods available. These methods can utilise non-fluorescent compounds, or fluorescent compounds, The most commonly used method utilises Coomassie blue (Bradford assay), which is non-fluorescent. Fluorescence based protein-detection methods utilise fluorescent dyes, which form a complex with the 15 protein and are intrinsically more sensitive than non-fluorescent methods. Fluorescent staining of proteins has a number of advanges ov er traditional Silver or Coomasile staining. These advantages inclu& greater ensitit lower background interference and greater dynamic range. For staining of nucleic acids such as DNA and RNA ethidium bromide as a 2 fluorescent stain has been most widely used due to its cost effectiveness and high sensitivity (2ng/ban of dsDNAK). It ues among researchers have been :omewhat limited because it is thought to be carcinogenic. Other fluorescent nucleic acid stains are currently available for quantification of nucleic acids as well as gel staining however in use such stains also have significant disadvantages. 25 WOO 1/81351, incorporated herein by reference, describes fluorescent dye compounds based on a furo[3,2-g][2]benzopyran-2,9(9aH)dione core. Fluorescent dyes are particularly useful in the field of electrophoresis. Electrophoresis allows the separation of charged biomolecules, such as DNA, RNA and/or proteins, by making use of the relative mobilities of the charged molecules in a gel matrix 30 after the application of an electrical field. The distance moved by each molecule in the electrical field depends on the charge, shape and weight of the molecule. The most commonly used gel matrix for the separation of proteins is polyacrylamide (PAGE electrophoresis). SDS-PAGE is a technique whereby proteins are treated with the -3 anionic detergent sodium dodecyl sulfate (SDS) before electrophoresis. SDS denatures the proteins and coats them with a uniform negative charge. This means that separation is based solely on molecular weight, and SDS-PAGE is typically used to determine the molecular weights of proteins'. 5 In contrast, nucleic acid bear a single negative charge for every nucleotide (MW approx 500 Daltons) so there is a reasonably constant mass / charge ratio. In the case of nucleic acids it is not necessary to normalise the charge with a detergent. Whilst a number of fluorescent dyes are known in the art, there is still a need to improve the signal intensity, the signal to background ratio and the sensitivity of 10 fluorescent dyes. There is an additional need to improve the stability of fluorescent complexes formed on an electrophoresis gel matrix. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. 15 Summary of the Invention According to a first aspect, the present invention provides a method of enhancing the fluorescence of a fluorescent dye, said method comprising combining or contacting the dye with a base and/or a detergent. 20 According to a second aspect, the present invention provides a method of increasing the emission wavelength of a fluorescent dye, said method comprising combining or contacting the dye with a base and/or a detergent. According to a third aspect, the present invention provides a method of improving the stability of fluorescent dye/organic molecule complex, said method comprising 25 contacting the fluorescent dye/organic molecule complex with an acid. According to a fourth aspect, the present invention provides a method of mobilising and detecting proteins comprising the steps of: (a) applying a solution of a protein to a matrix; (b) mobilising the protein on the matrix; 30 (c) forming a fluorescent complex between the mobilised protein and a fluorescent dye as described in any one of the first to third aspects; and (d) detecting the so-formed fluorescent complex, - 3a wherein the fluorescent complex is formed in the presence of a base and/or the fluorescent complex is treated with a base after its formation. According to a fifth aspect, the present invention provides a method of mobilising and detecting nucleic acids comprising the steps of: 5 (a) applying a solution of a nucleic acid to a matrix; (b) mobilising the nucleic acid on the matrix; (c) forming a fluorescent complex between the mobilised nucleic acid and a fluorescent dye as described in any one of the first to third aspects; and (d) detecting the so-formed fluorescent complex, 10 wherein the fluorescent complex is formed in the presence of a base and/or the fluorescent complex is treated with a base after its formation. The present invention provides methods for enhancing the fluorescence of fluorescent dyes, producing an increased Stokes' shift (ie. a further increase in the usual difference between the excitation and emission wavelengths) and improving stability of 15 fluorescent dye/organic molecule complexes thus increasing intensity and/or longevity of fluorescence of stored samples in solution or on gel matrices. Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the 20 sense of "including, but not limited to". As used herein, the term "enhancing the fluorescence" generally means increasing fluorescent emission from a fluorescent dye. The increase may be at the dye's usual emission wavelength or at a different wavelength. This may be evident by an increase in the fluorescent intensity, an increase in the signal to background ratio or an increase in 25 the detection sensitivity limit of the fluorescent dye (i.e. greater sensitivity). As used herein, the term "Stokes' shift" refers to the well-known phenomenon of a shift in the emission wavelength from the excitation wavelength of a fluorescent dye. The shift is towards a higher wavelength (i.e. a red shift). As used herein, the term "azaphilone fluorescent dye" means any fluorescent dye 30 which is obtained or is obtainable from a polyketide biosynthetic pathway. Examples o WO 2004/085546 PCT/AU2004/000370 -4 dyes obtainable from a polyketide biosynthetic pathway are described in WO01/81351, incorporated herein by reference. Moreover, unless the context clearly requires otherwise, the term "fluorescent dye" refers to fluorescent dye compounds, as well as fluorescent complexes or conjugates S formed when a fluorescent dye compound is associated with or conjugated with an organic molecule, such as a protein or a nucleic acid. Fluorescent complexes or conjugates may be formed with organic molecules by non-covalent and/or covalent interactions. The present inventors have found that the presence of a base, particularly a nitrogen containing base, causes a shift in the usual emission wavelength of certain fluorescent dyes, 10 particularly azaphilone fluorescent dyes, from green to red fluorescence. This Stokes' shift is advantageous because it shifts the emission wavelength of the fluorescent dye further from the excitation wavelength of a typical argon ion laser (488 nm). Long Stokes' shift fluorescent dyes are important biochemical reagents since their fluorescence emission may be detected with mininum interference from the excitation light source and are less prone 15 to self-quenching as there is less overlap between ercitation and emission profiles. Furthermore, long Stoe shift dyes are lessr prone to inerferene from wnofluorecnce which is due to the presence of shnrt S1oke slhift fluorophores present in many biological mnple,. Known flucrescent dye haing a long tok& shift are typically high molecular weight molecules, which severely limits their application as fluorescent labels due to poor 20 permeability and proteolysis. MAore significantly, in the present inCntion, thie Stoke Ehift, and/or the increase in Stokes shift, can also be accompanied by an increase in signal intensity, which in turn leads to a higher signal to background ratio (i.e. a reduction in non-specific background fluorescence) and/or an increase in the detection sensitivity limit of the fluorescent dye. 25 Therefore, the presence of the base significantly improves on known fluorescent techniques, and especially fluorescent staining techniques for the detection of organic molecules. Further in the present invention, it has been found that the choice of base can influence the magnitude of increase in the Stokes' shift. 30 Preferably, the fluorescent dye is of the formula (Ia), or isomer thereof: WO 2004/085546 PCT/AU2004/000370 -5 02 R R3 (Ia) Preferably, X is 0, NR 4 or C. Preferably, R' is a straight or branched chain Cao conjugated alkenyl group optionally substituted 1-6 groups independently selected from 5 hydroxy or oxo groups. Preferably, R2 is a straight or branched chain C 1

.

20 alkyl group. Preferably, R 3 is a straight or branched chain Cp-o alkyl group, optionally substituted with a hydroxyl group. Preferably, R 4 is N, 0, straight or branched chain Ciao alkyl and/or aryl group, optionally substituted with a hydroxyl, halide, amine, carboxyl, carboxyl related or heteroaryl group or groups. 10 Preferably, the dye is of formula (Tb), including isomers thereof: 0 OO H OH (Ib) The compound of formula (Jb) is 5,6-dihydro-3- [(Z, 4E, 6

E

, SE)-t-hydroxy-3- Qxo 1, -, 6, -ecattsny1]-6-hdromthyl-a-methyl-2H-furo [S, 2g r2] benprn-2-9' .1 . ~~ - ()b-MOym4 i1 (9aH)-dione. However, this compound vill be hereinafier referred to by its trivial name., which is "epicocconone". In the context of the present invention isomers of compounds of formulae Ia and Ib include tautomerrs and stereoisomers among other isomers. Epicocconone and epicocconone-containig dye mixtures and extracts are preferred. 20 The base used in the present invention is preferably selected from ammonia, and a variety of amines. Thus, the preferred base is a nitrogen-containing base. As used herein, the term "amine" refers to any compound containing one or more amino groups. Hence, the term includes monoamines, diamines, triamines etc. The amine may be primary, secondary, tertiary or quaternary. Further, salts of amines (e.g. HCl salts) are included within the 25 meaning of the term "amine". Metal carbonates and metal hydrogen carbonates, or combinations thereof may also be used.

WO 2004/085546 PCT/AU2004/000370 -6 Preferably, the base is ammonia, a primary amine, a secondary amine, a tertiary amine; a quaternary amine salt, or a combination thereof. The base used in the present invention is preferably ammonia or C-20 amines and dianmines, such as methylamine, ethylamine, propylamine, butylamine, ethylamine, propylamine, butylamine, pentylamine, 5 hexylamine, heptylamine, octylamine, nonylamine, deoylamine, undecylamine, dodecylamine and their isomers and allyl amine, aniline, benzylamine, 2 phenylethylamine, 4-phenylbutylamine, hydrazine and 1,2-diaminoethane, 1,3 diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7 diaminoheptane, 1,8-diaminooctane, 1,9-diaminononane, 1,10-diaminodecane, 1,12 10 diaminododecane and their isomers and dimethylamine, diethylamine, dipropylamin&, dibutylamine, dipentylamine, dihexylamine, dioetylamine, didecylamine; N-methylaniline, N-ethylaniline, N-propylaniline, N-butylaniline and their isomers and trimethylamine, triethylamine, tripropylamine, tributylamine, tripentylamine, trihexylamine, trioctylamine, tridecylamine, tridodecylamine and their isomers and tetramethylemmonium acetate, 15 tetramethylammoniumn bromide, tetramethylammonium carbonae.ttramethylammonium chloride, tetmmtylamnoniun fluoride, tetrmetylmmonim forma.te, tetramthylamnoniuim hydrogensulphate, tetramethylaxnmoniuam iodide, tetramedhylamnonium iodide, tetrameLylamroniumnuitate, erianethylarmnonium sulfate, tetraethylammonium acetate, tetraethylammonium bromide, tetraethylammonium 20 chloride, tetraethylamonium cyanide, tetraethylammonium fluoride, tetraethylammonium hydrozide. tetrnethylmmonium ic'dide, terathylanmmonium nitrate, tetrapropylmmonium bromide, tetrapropylammonium hydroxide, tetrabutylammonium hydrodde, tetrabutylammonium iodide, pyrrolidine, piperidine, pyridine, imidazole, indole, purine, quinoline, pyrimidine, pyrazole, tris(hydroxyTmethyl)aminomethane ("Tris") 25 or aminododecylamine. Preferably, the metal carbonates and metal hydrogen carbonates are salts of alkali and alkaline earth metals, such as sodium carbonate, potassium carbonate , sodium hydrogen carbonate etc. More preferably, the metal carbonate is sodium carbonate. Preferably, the method of the present invention is part of any chemical or 30 biochemical technique using a fluorescent dye. Typical examples of chemical or biochemical technique where the present invention may be used are electrophoresis, flow cytometry, pH sensing, analysing protein-protein interactions, fluorescent protein quantitation, assaying protein arrays or protein chips, assaying gene arrays or gene chips, WO 2004/085546 PCT/AU2004/000370 -7 assaying/detecting/quantifying nucleic acids, fluorescence microscopy and fluorescent antibody staining, and the like 2- 1 0 ,14-. The relevant publications are incorporated herein by reference. In one embodiment, the method described above is part of a method of staining 5 and/or labelling an organic molecule, the method comprising forming a fluorescent complex between the organic molecule and the fluorescent dye, wherein the fluorescent complex is formed in the presence of a base and/or the fluorescent complex is treated with a base after its formation. In another embodiment, the method described above comprises forming a fluorescent 10 complex between the organic molecule and the fluorescent dye in the presence of a base and/or a detergent, and/or the fluorescent complex is treated with a base and/or detergent after its formation. The organic molecule may be any molecule requiring staining or labelling by a fluorescent dye. However, the organic molecule is typically a protein, a peptide, a 15 nucleotide or a nucleic acid (DNA or RA and the like), which may or may not be included in another complex molecule auch as an en sm , cellular receptor, a growth factor, an antibody, or be part of a tissue, an organ or a cell, The base may be added to a ,olution containing the organic molecule before forming the fluorescent complex. The final concentration of base in this solution is preferably 20 between 0.001 and 10%, more preferably between 0.02% and 5%. The % values are given either w/ or v/v ra.rsomts, depending on the base used Alternatvely, th final concentration of base is preferably between 100 pMA and 2 M. more preferably between 1 mM and 100 mM. In another embodiment, the base is added to the fluorescent complex after it has been 25 formed. The concentrations of base used in this embodiment will be similar to those described above. In one aspect, the method of the present invention is used as part of a method of mobilising and detecting an organic molecule on a matrix, such as electrophoresis separation on a gel matrix. As described above, electrophoresis is typically carried out on a 30 polyacrylamide gel matrix and is used for separation and/or molecular weight determination of proteins. A protein solution is generally loaded onto the gel and an electrical field applied, which causes the negatively charged molecules to migrate towards the anode. In SDS-PAGE electrophoresis, the protein is complexed with an anionic WO 2004/085546 PCT/AU2004/000370

-

detergent, such as SDS, to give a uniform negative charge over the protein. Both conventional PAGE electrophoresis and SDS-PAGE electrophoresis methods are included within the scope of the present invention. It is also contemplated within the present invention that the organic molecule and/or 5 gel matrix may be treated with the base after electrophoresis. For example, the post electrophoresis gel may be washed with the base either before or after being treated with the fluorescent dye. Post-electrophoresis washing is preferably performed using fatty amines (C4 - C20 primary amines) or ammonia, since these bases will tend to partition to SDS micelles surrounding separated proteins in the gel. Typically, the gel is given 2 x 10 10 minute washes with a basic solution of appropriate concentration, although the number of washes and wash time will vary depending on the base used, the size of the gel plate, the concentration of base etc. In another embodiment, the detergent, whether used separately or in combination with the base, may be added to a solution containing the organic molecule before forming 15 the fuorescent complex. Typically this approach may be vsed where the organic molecule is a protein or a peptidek The f&Al concentration of detergent in tis solution i preferably between 0.001% and 10% more prnrably between 0.01 nd 1%. The % values are gven either as v or 7A amount, depending on the detergei ued. Altrnatively, the final concentration of detergent is preferably between 20 jM and 200 mM, m6re preferably 20 between 200 pM and 20 mM. In et another embodimeat, the detergenis h added ic the fluorescent complex (dhe complex may have been formed in the presence of a base or be exposed to a base after its formation) after it has been formed. This approach would typically be used where the organic molecule is a nucleic acid, but it may also be used with proteins. The 25 concentrations of detergent used in this embodiment will be similar to those described above. Similar to the methods described above for proteins, the methods of the present invention can be used as part of methods of mobilising and detecting nucleic acids (eg. DNA or RNA and the like) either in solution or following electrophoretic separation on a 30 gel matrix..Electrophoresis of nucleic acids is typically cared out on an agarose or polyacrylamide gel matrix and is used for separation, purification and/or molecular weight determination of nucleic acids. A nucleic acid solution is generally loaded onto the gel and WO 2004/085546 PCT/AU2004/000370 -9 an electrical field applied, which causes the migration of the nucleic acids towards the anode, similar to the protein electrophoretic technique described above. As for the base described above, the detergent used in the present invention may be incorporated at any suitable stage of electrophoresis prior to detection of the fluorescent 5 complex. For example, in case of methods used with proteins, the detergent may be admixed with the protein (preferably the detergent is SDS but other examples are provided herein) in solution before being loaded onto the gel. Alternatively, the gel matrix may be washed with the detergent, in the absence or presence of a base, after electrophoresis, This latter technique is typically used with nucleic acid separation and analysis. 10 In a further aspect of the present invention, the methods described above further comprises treating the fluorescent complex with an acid. This further treatment step is particularly suitable for fluorescent complexes formed on electrophoresis gels but may also be used for in-solution complexes. Treatment with an acid, surprisingly, stabilises the fluorescent complex, further 15 increases the fluorescent intenity, prevents or minimiose ihe loss of fluorescence and/or father increase the signal to bkzground ratio of the fluorescent complex. The acid may be elected from s rinerl cid, an organic acid, or combination thereof. Suitable mineral acids are sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, orthophosphoric acid. Suitable organic acids are alkanoic acids (e.g. a Ci 20 alkanoic acid), halogenoalkanoic 20 acids (e g a Cio alkanoio haig 1, 2 3, 4. 5 or 6 groups selected from F, Cl, Br or I, awc*rbie acid or triflir accid, propionic acid. ascorbic acid, hydrochloric acid, orthophosphoric acid, trifluoroacetic acid, trichloroacetic acid or chloroacetic acid. The concentration of the acid used is typically between about 0,01% and 20% (v/v), 25 preferably between about 0.02% and 10% (v/v). Alternatively, the concentration of acid is typically between about 0.1mM and 2 M depending on the acid used, although acid concentrations of about 10 mM are generally preferred. In a typical acid treatment step, an electrophoresis gel is incubated for about 10 minutes in acid (e.g. 10 mM sulfuric acid) prior to imaging. 30 Preferably, the fluorescent complex formed in the present invention is detected using any standard technique known in the art, Typically, the fluorescence of the fluorescent complex is detected by transillumination, spectroscopy, microscopy, scanning, photography or cytometry.

WO 2004/085546 PCT/AU2004/000370 -11 The present invention also provides a composition comprising of a fluorescent dye as described above, a base and/or a detergent as described above. The composition may further include an organic molecule, such as a protein and/or a nucleic acid. The present invention also provides a kit comprising of a fluorescent dye as described 5 <above, a base and/or a detergent as described above. The kit may further include an organic molecule, such as a protein and/or a nucleic acid. Brief Description of Figures Figure 1 shows the emission profile of epicocconone when excited at 390 nm with a) 100 mM aqueous acetic acid, b) 1 mM acetic acid, c) 10 pM acetic acid, d) 1 pM acetic 10 acid and e) no acetic acid. Figure 2: a) Deep PurpleTm with 1 mM acetic acid: excitation 300-500 nm/emission 524 nm and b) excitation 390 nm/emission 420-700 nm compared with c) Deep Purplei at the same concentration without acid: excitation 300-500 nm/emission 523 m and d) excitation 390 nm/emission 420-700 nm. 15 Figure 3: a) Epicocccon with 1 mM HzSO4: citation 300-490 nm/emnision 516 rim and b) excitation 390 nm/mission 420-700 n1m compared with c.) Epicocc.onone without avid: exitation 390 nm/emi:sion 420-700 nm. Figure 4: The emission profile of epicocconone when excited at 390 nm with a) 1.8% aqueous SDS (w/v), b) 0.8% SDS, c) 0.4% SDS, d) 0.1% SDS and e) 0.1% SDS. 20 Figure 5: a) Deep Purple" with 03% aquEous SDS (w/v): ex:citation 300-500 nm/emission 525 nm and b) excition 390 nm/,mision 420-700 nm compared with c) Deep Purple at the tame concentration without SDS: excitation 300-500 nm/emission 523 nm and d) excitation 390 nm/emission 420-700 nm. Figure 6: a) Epicocconone with CTAB: excitation 390 nm/emission 420-700 nm 25 compared with b) the same concentration of epicocconone without CTAB: excitation 300 500 nm / emission 516 inm and c) excitation 390 nm/emission 420-700 nm. Figure 7: a) Epicocconone with CPC: excitation 390 nm/emission 420-700 nm compared with b) Epicocconone at the same concentration without CPC: excitation 300 500 nm/emission 516 nm and c) excitation 390 nm/emission 420-700 Mn 30 Figure 8: a) Epicocconone with CHAPS: excitation 390 nm/emission 420-700 nm compared with b) Epicocconone at the same concentration without CHAPS: excitation 300-500 nm/emission 516 nm and c) excitation 390 nm/emission 420-700 nm.

WO 2004/085546 PCT/AU2004/000370 J0 The present invention also provides a method of mobilising and detecting proteins comprising the steps of: (a) applying a solution of a protein to a matrix; (b) mobilising the protein on the matrix; 5 (c) forming a fluorescent complex between the mobilised protein and a fluorescent dye as described above; and (d) detecting the so-formed fluorescent complex; wherein the fluorescent complex is formed in the presence of a base and/or a detergent. The preferred detergent to be used with proteins is an anionic detergent. 10 Another embodiment of the invention contemplates the above method in which the treatment of the fluorescent complex with a base and/or detergent is conducted after its formation. Preferably, the matrix used for proteins and peptides is a polyacrylamide gel matrix and the method is PAGE electrophoresis or SDS-PAGE electrophoresis. 15 The present invention further provides a mAhod of mobilising and detectin nucleic acids comprising the stepz of: (a)a sOlton of a nucleic acid to a matri;6 (b) mobilising the nucleic acid on the matri; (6) forming a fluorescent complex between the mobilised nucleic acid and a 20 fluorescent dye as described above; and (d) dtecting the so-formed fluorescent complex; wherein the fluorescent complex is formed in the presence of a base and/or a detergent, The preferred detergent for use with nucleic acids is a eationic detergent. Another embodiment of the invention contemplates the above method in which the 25 treatment of the fluorescent complex with a base and/or detergent is conducted after its formation. Preferably, the matrix used with nucleic acids is an agarose gel matrix. The above methods also contemplate a step in which the matrix is treated with an acid as the final step. The acid-treated matrices may be stored for periods of time without 30 significant loss of fluorescence intensity. The present invention also provides a fluorescent compound or complex obtainable by combining a fluorescent dye as described above with a base and/or detergent as described above.

WO 2004/085546 PCT/AU2004/000370 Figure 9: a) Epicocconone with Tween 80: excitation 300-500 nm/emission 526 nm, b) excitation 440 nm/emission 470-700 nm and c) excitation 390 an/emission 420-700 nm compared with d) Epicocconone at the same concentration without Tween SO: excitation 300-500 nm/emission 516 nm and E) excitation 390 rum/emission 420-700 na. 5 Figure 10: a) Epicocconone with Tween 20: excitation 3 00-500 nm/emission 524 nm and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone at the same concentration without Tween 20: excitation 300-500 nm/emission 516 nm and d) excitation 390 nm/emission 420-700 nm. Figure 11: a) Epicocconone with octyl D-glucoside: excitation 300-500 10 nm/emission 524 mn and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone at the same concentration without octyl D-glucoside: excitation 300-500 nn-/emission 516 nm and d) excitation 390 nm/emission 420-700 nm. Figure 12: a) Epicocconone with Triton X- 100: excitation 300-500 nni/emission 526 nm and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone at 1 the same concentration without Triton X-100: ex citation 300-500 nm/eMission 516 nm and d) excitaion 390 n/emission 420-700 nm. Figure 13: a) Epicoccnone with S nd acticM acid: excitation 00-500 n-memision 25 nrim and b) excitation 390 n/emision 420-700 nn compared with c) Epicocconone with SDS, but no acid: excitation 300-500 nm/emission 515 nm and d) 20 excitation 390 =x/emission 420-700 nm. Figure 14 : L) Epicocconaone wiAth CTAE an2ct2-cd xctto 00-300 nmnnission 516 nm compared with b) Epicocconooe with CTAB, but without acid: excitation 390 nm/emission 420-700 nm and c) Epicocconone at the same concentration without CTAB and without acid: excitation 300-500 nm/emission 516 nm and d) excitation 25 390 nm/emission 420-700 nm. Figure 15: Excitation 390 nm/emission 420-700 nm of a) Epicocconone with 1.625 mM DTAB and 1 mM acetic acid, b) Epicocconone with DTAB and without acid and c) Epicocconone without DTAB and without acid. Figure 16: a) Epicocconone with CHAPS and acetic acid: excitation 300-500 30 m/emission 525 nm and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone with CHAPS and without acid: excitation 300-500 nm/emission 528 nrn and d) Epicocconone without CHAPS and without acid: excitation 390 nm/emission 420-700 nm.

WO 2004/085546 PCT/AU2004/000370 -. 3 Figure 17: a) Epicocconone with cholic acid sodium salt and acetic acid: excitation 300-500 nm/emission 525 nm and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone with cholic acid sodium salt and without acid: excitation 390 nm/emission 420-700 mn and d) Epicocconone at the same concentration without cholic 5 acid sodium salt and without acid: excitation 390 nm/emission 420-700 nm. Figure 18: Excitation 390 un/emission 420-700 nm of a) Deep Purple with 0.3% SDS (w/v) and 1 mM acetic acid, b) Deep Purple tm with 0.3% SDS (w/v),-c) Deep PurpleJm with 1mM AcOH and d) Deep PurpleTM. Figure 19: a) Epicocconone with SDS and ammonia: excitation 310-500 10 nm/emission 605 mu and b) excitation 390 nm/emission 420-700 nm, compared with c) Epicocconone and SDS at the same concentration and without ammonia: excitation 300 500 nm/emission 520 nm and d) excitation 390 nm/emission 420-700 mn. Figure 20: a) Deep PurpleT with 0.3% SDS and 1 mM ammonia: excitation 300 500 nm/emission 605 unm and b) excitation 390 nm/emission 420-700 rn compared with c) 15 Deep Purple 1 M with 0,3% SDS and vithout ammonia at the same concentration: excitntion 300-500 nm/eriion 525 Rn and d) excitation 390 nm/emission 420-700 nm. Figure 21: a) Epicocconone with 3DS and ;thylamine: excitation 320-550 mn/emission 610 nr and b) ex;citation 390 nal/miss-ion 420-700 nm compared wit c) Epicocconone at the same concentration without SDS and without ethylamine: excitation 20 390 nm/emission 420-700 nm. Figure 22: a) Epicocconone with uD nd butlmine: exciation 390 nnemission 420-700 nm compared with b) Epicocconone at the same concentration without SDS and without butylamine: excitation 390 nm/emission 420-700 nm. Figure 23: a) Epicocconone with SDS and octylamine: excitation 320-550 25 nm/emission 600 n and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone at the same concentration without SDS and without octylamine: excitation 390 nm/emission 420-700 nm. Figure 24: a) Epicocconone with SDS and TRIS: excitation 320-550 nm/emission 600 un and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone at 30 the same concentration without SDS and without TRIS: excitation 390 nm/emission 420 700 nm. Figure 25: a) Epicocconone with SDS and benzylamine: excitation 320-550 nm/emission 600 nm and b) exdhation 390 nm/emission 420-700 nm compared with c) WO 2004/085546 PCT/AU2004/000370 -14 Epicocconone at the same concentration without SDS and without benzylamine; excitation 390 nm/emission 420-700 nm. Figure 26: a) Epicocconone with SDS and aniline: excitation 320-550 nm/emission 620 unm and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone at 5 the same concentration without SDS and without aniline: excitation 390 nm/emission 420 700 nm Figure 27: Excitation 390 nm/emission 420-700 nm of a) Epicocconone with SDS, DNA and 1,3-diaminopropane after 1 day and b) Epicocconone with SDS, DNA and 1,3 diaminopropane. 10 Figure 28: Excitation 390nm/emission 420-700 nm of a) Epicocconone with CHAPS and amnmonia, b) Epicocconone with CHAPS and without ammonia and c) Epicocconone without CHAPS and without ammonia, Figure 29: a) Epicocconone with SDS, acetic acid and aniline: excitation 320-600 nm/emission 621 nm and b) excitation 390 nm/emission 420-700 nm compared with c) 15 Epiocconone with SDS and aniline, but no acid: excitation 390 nra/emission 420-700 nm. Figure $0: a) Epicocconone Ath DS, acetic acid and TRIS: excitation 320-550 nm/mission 605 unm and b) esxcitation 390 nm/ermiszon 420-700 nm compared with c) Epicocconone with SDS and TRIS and without acid: excitation 390 nm/emission 420-700 nm. 20 Figre 31: a) Epicocconone with SDS. acetic acid and ethylaine: excitation 320 550 nm/emiesion 610 n and b) ecitation 390 nm/emizsion 420-700 nm compared with c) Epicocconone with SDS and ethylamine and without acid: excitation 390 nm/emission 420-700 nm. Figure 32: a) Epicocconone with DTAB, acetic acid and DNA: excitation 300-500 25 nm/emission 533 nm and b) excitation 390 nm/emission 420-700 unm compared with c)' Epicocconone with DTAB and acetic acid and without DNA: excitation 390 nm/emission 420-700 nm and d) Epicocconone alone: excitation 390 nm/emission 420-700 nmn. Figure 33: a) Epicocconone with CHAPS, ammonia and glucosamine hydrochloride: excitation 320-550 nm/emission 605 nm and b) excitation 390 nm/emission 420-700 rn 30 compared with c) Epicocconone with CHAPS and ammonia and without glucosamine hydrochloride: excitation 390 nm/emission 420-700 nm and d) Epicocconone alone: excitation 390 nm/emission 420-700 nrn.

WO 2004/085546 PCT/AU2004/000370 Figure 34: a) Epicocconone with DTAB and BSA: excitation 320-550 nm/emission 615 nm and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone alone: excitation 390 nm/emission 420-700 nm. Figure 35: a) Epicocconone in acetonitrile: excitation 300-500 nm/emission 532 5 nm and b) excitation 390 nm/emission 420-700 nm compared with c) Epicocconone at the same concentration in water: excitation 390 nm/emission 420-700 nm. Figure 36 shows an image of the microtitre plate captured by the Typhoon laser based imager (532 nm laser, 610 BP 30 emission filter). Figure 37: A) Plot of the raw fluorescence data against BSA concentration per ml 10 in the range of 12 ng - 100 g/mL. The ? value over the range is 0.985. B) Plot of the logo of fluorescence data against logio of BSA concentration per mL in the range of 781 ng - 800 pag/mL. The r2 value over the range is 0.997. Figure 38: A-G: Show stained gels and their corresponding intensity traces, following treatment of stained gels with water and different bases. 15 Figure 39: A and B: Show gel eent!s following staining with Deep Purple0 and treatnait with rnM amronia. Segment 1(A) wa dtored in C nM i &.mmonia under dark conditions for 46 h. replicate eegnqnt number 2 (B) a washed 3 10 min. in 3 mli ammonia and transferrd to 100 mI orthopiosphoric acid and stored under dark conditions also fbr 46 h. 20 Figure 40: A-D: gels imaged immediately after treatmt with ammonia () and fol]JqfLo g storage in 1 O Si 2ulphurie acid for periods of 2h (U), 2 1h (C) and 92h(D). E-H: show signal intensity trace of Lane 3 of the gel prior to placing in 10 n/IP sulfuric acid (E, Time 0) and at 2 h (F), 21 h (G) and 93 h (H) after addition of 10 mM sulfuric acid. Figure 41: A-C: gels imaged immediately after treatment with ammonia (A) and 25 following storage in 10mM acetic acid for periods of 46h(B) and 167h (C). Figure 42: Shows Typhoon-scanned images of the DNA gel strips (1: No detergent treatment, Epicocconone staining only; 2: SDS-incubation and Epicocconone staining; 3: DDTAB-incubation and Epicocconone staining; 4: SDS and DDTAB-incubation, and Epicocconone staining). 30 Figure 43: Typhoon image of red emission light. 532 nm laser, 560 LP filter. After addition of stain, 10 mM ammonia solution added to wells after addition of Deep PurpleTM formulated as described in Protocol.

WO 2004/085546 PCT/AU2004/000370 -16 Figure 44: A) Signal from Figure 43 plotted as raw data. B) plot of the logio transformed data. Figure 45: Typhoon image of green emission light. 532 nma laser, 526 SP filter. After addition of stain, 10 mM acetic acid solution added to wells after addition of Deep 5 Purplem formulated as described in Protocol. Figure 46: A) Signal from Figure 45 plotted as raw data. B) plot of the same data transformed logo. Figure 47: shows Typhoon-scanned images of the DNA gel (A and C, DNA MWM XVII; B, SPP- 1 DNA/Eco RI) strips. Figure 47-A, the DNA gel strips that were pre 10 incubated-in TDTAB (1, 0.001 mM; 2, 0.01 mM, 0.1 mM and 1 mM) before epicocconone staining; Figure 47-B, the DNA gel strips that were pre-incubated in CTAB (1, 0.001 mM; 2, 0.01 mM, 0.1 mM and 1 mM) before epicocconone staining; Figure 47-C, the DNA gel strips that were pre-incubated in CPC (1, 0.001 mM; 2, 0,01 mM, 0.1 mM and 1 mM) before epicocconone staining. 15 The invention will now be described with reference to non-limiting Eamples. To illustrate the enhancement and an increase in the Stokes' shift when epicocconone is in the presence of an amine, fluorescence measurements were recorded in 20 solution of p cconne with a variety of amines acid and detergen,. I was found that a combination ofi an amine and a detergent enhance the iluorescencet and causes an increase in the Stokes' shift. Inorganic base such as NaIHCOs caused a loss of fluorescence and the addition of a detergent did not recover the fluorescence. Fluorescence spectroscopy was carried out using a Perkin Elmer LS 50B 25 Luminescence Spectrometer (Perkin Elmer, Melbourne Australia). Freshly prepared solutions were placed into a Hellma Quartz SUPRASIL precision cell and the cell was then placed into the spectrometer. Typically a solution was 3 mL in volume and the components were added using a Gilson M- 1000 Micromian positive displacement pipette. The components and their final concentrations are given for each sample, All solutions are in 30 water (Millipore RiOS 5) unless otherwise stated.

WO 2004/085546 PCT/AU2004/000370 -17 Definitions and sources Deep PurplJ" (Amersham Biosciences, Australia, Cat.Nos. RPN6305 or RPN6306) is a partially purified form of epicocconone. It has an absorbance at 550 nm of 0.8. An aqueous stock solution was prepared from the methanol stock solution by diluting 5 it by a factor of 12.5 with water. Final solutions in the quartz cell are diluted further by a factor of 4. The total dilution of the Deep Purplem stock solution in methanol is 1 in 50. (Deep Purple front Amersham in methanol) Epicocconone was isolated by the method described in Bell JL and Karuso P 3 , incorporated herein by reference. A stock solution of epicocconone was prepared in 10 DMSO (28pg./mL or 42pg/mL in DMSO) and this was used to prepare stock solutions in water by diluting the DMSO solution by a factor of 5 or 10 respectively. Final solutions in the quartz cell are diluted further by a factor of 4. Final concentrations of epicocconone are reported with each example. Acetic acid: (APS, Asia Pacific Specialty Chemicals Ltd, formerly Ajax: 1-2.5L GL) 13 Acetcnitrile: (Aax Finechem - 231525 CL) Ammonia soiution- (Ajax Finechem - 43-2.5L GL) Aniiline: (Aldrich - 13.23-4) Benzylamine: (BDH - 27355) BSA - Bovine serum albumin: (Sigma - A-2153) 20 Butylamine: (Adrich- 47,130-5) rHPs - 3-[(3-Cholamidopropy)dimethylimmonio]-1-propancsulfate: (EDH - 30632) Cholic acid sodium salt: (Sigma - C-1254) CPC - Cetylpyridimium chloride: (Ajax - UL 0000145) CTAB - Cetyltrimethylammonium bromide: (Sigma - H-5882) 25 1,3-Diaminopropane: (Koch-Light Laboratories - 4774) DMSO -Dimethyl sulfoxide: (Aldrich - 27,043-1) DNA - Deoxyribonucleic acid sodium salt is from Salmon testes: (Sigma - D-1626) DTAB - Dodecyltrimethylammonium bromide: (Sigma - D-8638) TDTAB - Tetradecyltrimethylammonium bromide (Sigma - T-4762) 30 Ethylamine: (Lancaster - 10838) D-(+)-Glucosamine hydrochloride: (Sigma - G-4875) H2SO4 .Sulfuric acid : (Ajax Finechem -534) WO 2004/085546 PCT/AU2004/000370 Octylamine: (Lancaster - 8.06917.0250) Octyl-D-glucoside: (ICN Biomedicals - 153941) SDS - Sodium dodecylsulfate: (BDH - 301754) TRIS (HCi) - Tris(hydroxymethyl)aminomethane hydrochloride salt: (Sigma - T-3253) 5 Triton* X-100: (BDH -30632) Tween 20- Polyoxyethylenesorbitan monolaurate: (Sigma - P-1379) Tween 80 -Polyoxyethylenesorbitan monooleate: (BDH - 56023) The concentrations of the detergents other than SDS were such that they were 10 above their respective CMC values Stock concentrations of detergents used: CHAPS: 0.27% w/v Cholic acid sodium salt: 1.7% w/v (40 mM) CPC: 0.148% w/v (4.1 mM) 15 CTAB: 0.172% w/v (4.7 mM) DTAB: 0.2% w/v (6.5 nM) Octyl-D-glucoside: 3,1% w/v (106 mMA) SD: 1.2% uw (41.7 m) Triton* X-1 00: 0.27% w/v (4.35 mM) 20 Tween 20: 0.56 w/v Tween 80: 0.64% The detergents are diluted 1 in 4 in the final solution. (i) Enhancing the fluorescence of epicocconone and Deep Purple"" by acids The fluorescence of epicocconone in water can be enhanced by the addition of 25 acetic acid (see Figure 1). Using a fixed concentration of epicocconone (1.05 pg I mL; 2.56 x 10 M) in water fluorescence spectra were recorded with fmal acetic acid concentrations ranging from 100 nM to 100 mM. Figure 2 shows that the fluorescence of Deep Purple in water can also be enhanced by the addition of acetic acid. 30 The fluorescence of epicocconone in water can also be enhanced by the addition of other acids such as for example sulfuric acid. The fluorescence spectra shown in Figure 3 is with a fixed concentration of epicocconone (1.05 jg / mL; 2.56 x 106 M) in water and a final concentration of 1 mM H 2 S0 4

.

WO 2004/085546 PCT/AU2004/000370 19 (ii) Enhancing the fluorescence of enicocconone and Deeg Purp1eT by detergents The fluorescence of epicocconone in water can be enhanced by the addition of SDS (anionic detergent). Increasing the SDS concentration increases the fluorescence observed (exciting at 390 nm, emission maximum at 525 nm). S Using a fixed concentration of epicocconone (1.05 pig / mL; 2.56 x 10 M) in water fluorescence spectra were recorded with final concentrations of SDS ranging from 0.1% to 1.8% (w/v). The fluorescence spectra in Figure 4 show that fluorescence increases with increasing SDS concentration. The fluorescence of Deep Purple in water can also be enhanced by the addition of 10 SDS (see Figure 5). The fluorescence of epicocconone in water can be enhanced by the addition of other detergents such as for example CTAB (cationic detergent) with an accompanying increase in Stokes' shift. The addition of CTAB to an epicocconone solution shows an increase in fluorescence at 625 nm with respect to epicocconone alone at 625 nm. There is 15 a decrease in fluorescence at 525 nm when CTAB is added. The fluoresceLnce spectra shown in Figure 6 were generated with concentrations of epiec cconone of 1.03 pg/mL, (2.55:6 x 1 M) in water and s final concentation o 'CTAB of 1.175 mMnj. The effect observed with CTAB is also seen with other detergents, eg CPC. The fluorescence of epicocconone in water can be enhanced by the addition of CPC (cationic 20 detegnt) w'it an accompanying Stokes' shift. The addition of CPC to a solution of epicoccconone increases its fluorescence~ at 630 n with repect to epicocconone alone at 630 nm. There is a decrease in fluorecence at 520 mu when CPC is added. The fluorescence spectra shown in Figure 7 were generated with concentrations of epicocconone of 1.05 pg / mL (2.56 x 104 M) and a final concentration of CPC of 1.025 25 mM. The fluorescence of epicocconone in water can also be enhanced by the addition of CHAPS (zwitterionic detergent). The fluorescence spectra shown in Figure 8 were generated with concentrations of epicocconone of 1.05 pg /mL (2.56 x 10 M) in water and a final concentration of CHAPS of 0.0675% (w/v). 30 The fluorescence of epicocconone in water can also be enhanced by the addition of a number of different non-ionic detergents (see list of detergents set out below). The fluorescence spectra shown in Figure 12 - 15 were generated with concentrations of epicocconone of 1.05 jig /mL (2.56 x 106 M) in water.

WO 2004/085546 PCT/AU2004/000370 -20 Tween 80 (final concentration of 0.16% w/v) - Figure 9 Tween 20 (final concentration of 0.14% w/v)-Figure 10 Octyl-D-glucoside (final concentration of 0.775% w/v) - Figure 11 Triton X-100 (final concentration of 0.0675% w/v) - Figure 12 S (iii) Enhancing the fluorescence of epicocconone and Deep PurpleM by detergents and acids The fluorescence of epicocconone in water can also be enhanced by the addition of acid and a number of different detergents (see list of detergents set out below). The fluorescence spectra shown in Figure 14 - 17 were generated with concentrations of 10 epicocconone of 1.1.4 pg / mL (3.4 x 106 M) in water and a final concentration of acetic acid of ImM. SDS (anionic detergent - final concentration of 0.03% w/v) - Figure 13 CTAB (cationic detergent - final concentration of 1.175mM) - Figure 14 DTAB (cationic detergent - fmal concentration of 1.625mM) - Figure 15 15 CiASt (zwritterionlc detergent - final concentration of 0.0675% wvi) - Figure 16 COllic acid, sodium salt (ninc deitrgent - final concentration of 10mM - Figureg 17 The fluorcen~ c'fLDeep Purle in water oi ako be enhanced by the combination of aceic acid and SDS (@ee Figure 1 G). (iv) Changes in Stokes'shift of epicocconone and Deep PurpleW in the presence of 20 bases and detergents Anmoni reduce th fiuorecnce of epicoccnn. The intenity of fluorecnce of tbis mixture can be enhanced by the addition of SDS. Geherally the fluorescence is of about the same intensity as for epicocconone alone, but the emission is shifted to a longer wavelength (increased Stokes' shift). The emission at a longer wavelength can also be 25 regarded as an enhancement as epicocconone alone generally emits weakly at the longer wavelength. The fluorescence of epicocconone in water with SDS emits with a longer Stokes' shift in the presence on ammonia. When the mixture is excited at 390 nm it has an emission maximum at 605 nm. The fluorescence spectra in Figure 19 is generated with a 30 concentration of epicocconone of 1.4 pg / mL (3.4 x 10- 6 M) in water and a final concentration of 0.3% SDS (w/v) and 1 mM aqueous ammonia.

WO 2004/085546 PCT/AU2004/000370 -21 The fluorescence of Deep Purple in water with SDS can also emit with a longer Stokes' shift in the presence on ammonia. When the mixture is excited at 390 nm it has an emission maximum at 605 nm (see Figure 20). The Stokes' shift of epicocconone, when excited at 390 nm, can be increased by the 5 addition of bases such as ethylamine, butylamine, octylamine, TRIS, benzylamine and aniline. The fluorescence spectra in Figures 21 to 26 were generated with a concentration of epidocconone of 1.05 pg / mL (2.56 x 106 M) in water and a final concentration of 03% SDS (w/v). The following final concentrations of the bases were used: Ethylamine - 1mM aqueous (Figure 21) 10 Butylamine - 10mM aqueous (Figure 22) n-Octylamine - 10mM aqueous (Figure 23) TRIS - 100mM aqueous (Figure 24) Benzylamine - I0mM aqueous (Figure 25) Aniline - 10mM aqueous (Figure 26) 15 In this series of experiments, aniline yielded the Jar:gest Stokes" Juift (km=620 nm). Th tok Ahif of epicocconone, when cited at 390 nm. can be inornsed by ihe adclition ,3-danoprop:ne. The fincresence spectm in Figure 27 were recorded unga concentration of epicocconone of 1.05 pg / mL (2.56 x 10-* M), 0.3% SDS, DNA (1 mg/ mL) and 1 mM 1,3-diaminopropane. The emission was enhanced further when the sample 20 was left for a day 21 4C nd re-n (see Figura 27). The fluorescee of spicocconone in -water containing CKAPS (ziterionic .detergent) emits ata longer Stokes' shift when ammonia is added. The addition of ammonia to an epicocconone / CHAPS solution shows an increase in fluorescence at 630 nm with respect to epicocconone alone at 630 mm. The fluorescence spectra in Figure 28 25 was generated with a concentration of epicocconone of 1.05 pg / mL (2.56 x 106 M) in water and a final concentration of 0.0675% CHAPS (w/v) and 1 mM aqueous ammonia. The Stokes' shift of epicocconone/detergent mixtures, when excited at 390 nm, can be increased by the addition of bases and enhanced with acetic acid. The fluorescence spectra of Figures 32 to 34 were generated with a concentration of epicocconone of 1.05ptg 30 / mL (2.56 x 106 M), final concentration of SDS of 0.3% (w/v) and final concentration of acetic acid of 1mM. The following bases were used, at a final concentration stipulated. Aniline- I 0mM aqueous (Figure 29) TRIS - 100mM aqueous (Figure 30) WO 2004/085546 PCT/AU2004/000370 -22 Ethylamine - 1mM aqueous (Figure 31) In each case the addition of acetic acid further enhanced the fluorescence following an increase in Stokes'shift. (v) Other fluorescence enhancements and/or changes in the Stokes' shift 5 The emission of an aqueous solution containing epicocconone, acetic acid and DTAB can be enhanced with DNA when excited at 390 nrm with a concomitant Stokes' shift from 520 to 540nm. The fluorescence spectra of Figure 32 were recorded using a concentration of epicocconone of 1.05 pg / mL (2.56 x 10 M), 1.625 mM DTAB, 1 mM acetic acid and DNA (1 mg/mL) as a representative nucleoic acid. This indicates that 10 epicocconone, in combination with detergent and/or acid can be used in detection/analysis of DNA and other nucleic acids. The emission of an aqueous solution containing epicocconone, CHAPS and ammonia can be enhanced with glucosamnine hydrochloride when exoitated at 390 nm. As well as an enhancement there is an increase in the Stokes' shift. The fluorescence spectra in i Figure 33 were recorded using a concentration of epicocconone of 1.05 pg / mL (2.56 x 10 M), o.n7 CHAP, 1 mM amonriIA and glucosamine hydrochloride (13.25 nM). The emisicn of an aqueou-LnorLtion containing eic oc-on, DTAE anbe enhanced with protein when excited at 390 rim, As well as an enhancement there is an increase in the Stokes' shift. The fluorescence spectra in Figure 34 were recorded using a 20 ConcentrfAion of epioocone oq 1.05 g i/mL (2.5!6 10 M), 1.625 mI DTAB and BA (1 mg /nL). A solution of epicocconone (1.05 pg /mL; 2.56 x 10c M) in acetonitrile was prepared from the stock solution of epicocconone in DMSO. epicocconone in organic solvent fluoresces more than in -water (L.= 520 nm) (see Figure 35). 25 Summary of Emission Wavelengths for samples excited at 390 nm. Epicocconone and SDS (X, 525 nm) with: Amine X= (nm) a) Dodecylamine 584 b) Benzylamine 600 30 c) Butylamine 600 d) 1,6-Diaminohexane 600 /447 e) Octylamine 600 WO 2004/085546 PCT/AU2004/000370 -23 f) TRIS 600 g) Ammonia 605 h) Ethylamine 610 i) Aniline 620 5 Summary of preliminary results using other bases Diethylamino (20 amine) shift to red Triethylamine (3o amine) shift to red Hydrazine emissions in the green and red 1,6-diamninohexane shift to red 10 Aniline shift to red The extent of the enhancement of fluorescence of epicocconone in water can be affected by the order of addition of reagents. The optimal conditions and sequence of steps will be govemed by the type of analytical or quantitative technique used and can be easily determined by simple trial, to suit any such technique. 15 Example 2 - Mearurement oi protein eomntratirA uing flurecerne t enhancing Components Part A: Deep PurpleTM formulated in 80% (v/v) dimethyl sulfoxide and 20% (vlv) aconitrile atc'Arbanc550 ni = 0.30. 20 Part B: A 1Ox solution is prepared as shown in Table 1. Reagent Concentration SDS 3% w/v NaHCO3 200 mM Acetonitrile 25% v/v Water 75% v/v A 1x working solution of the kit is prepared by mixing together 8 parts water and 1 part of each of component Part A and Part B. Protocol 25 A two-fold dilution series of protein standard of bovine serum album was prepared in water over the range 10 ng/mL - 100 pg/nL. Aliquots (50 pL) of protein standard are WO 2004/085546 PCT/AU2004/000370 pipetted in duplicate into wells of microtitre plates. Fifty-pL aliquots of water are added as protein free controls. To each well is added an equal volume (50 pL) of lx working solution. Fluorescence is then allowed to develop for at least 5 min prior to measurement. 5 Protein standards and experimental samples are prepared and incubated for equal amount of time prior to recording fluorescence. For laser-based imaging system 532 nm laser light excitation with 610 BP 30 mn or -similar emission filtering is used. For plate-based fluorescent measuring systems, such as, the BMG Fluostar (BMG Labtech, Mornington,VIC, Australia), 540 um excitation filtering 10 together with 630-12 nm emission filtering or similar is recommended. Example Sigma bovine serum albumin (Castle Hill, NSW, Australia; Cat. A3059) was suspended at a concentration of 800 tg/mL in water and two-fold diluted to final concentration of 0.76 ng/mL. Fifty-pL aliquots were plated out, in triplicate, into a Greiner 15 (interpath Serices, West Heidelberg, VIC) 96-well plate (C at, 655096). Figure 36 hows an image of the mic-rotitre plate captured by the Typhoon Iaer-based imagery (532 nm laser, 6310 BP 30 iion filte) Figureas 37A 7 37B uhow plot: of fluorenee dt plotted against BSA concentration per mL in the range of 12 ng - 100 pg/mL Gel Staining 20 Ri >t4McWo Invitrogen Eis-Tris 10-well 12% polyaerylniide gels were prepared and run according to the manufacturer's instructions (NuPAGE Technical Guide, Version D, August 26 2002. IM-1001). Amersham Biosciences (Castle Hill, NSW, Australia) SDS Low Molecular Weight protein markers were prepared and two-fold diluted in Invitrogen 25 LDS sample buffer (Mt Waverly, VIC, Australia, Cat. NP0007) containing 50 mM dithiothreitol (Bio-Rad, Regents Park, NSW, Australia). Samples were heated at 70' C for 10 minutes. Gels were separated using 1 x MES (Sigma, Castle Hill, NSW, Australia; Cat M2933) buffer. Invitrogen Antioxidant (Cat. NP 0005; 500 pL) was added to the cathode chamber of the Invitrogen X-cell Sure-Lock Mini Cell and gels were run at constant 200 V 30 until tracking dye'reached the base of the gels. The gels were fixed in 100 mL of 7,5 % acetic acid (v/v) for 1 hour and gels were then washed in equal volumes of double distilled (dd) water 2 x 30 minutes.

WO 2004/085546 PCT/AU2004/000370 -25 Gels were then transferred to 50 mL volumes of fresh dd water and 250 pL of Deep Purplem (Amersham Biosciences) and stained for 1 h at room temperature. For the ammonia gel-developing step the gel stain was removed and replaced wita 100 mL volumes of 8 mM (0.05% v/v) concentrated ammonia. The gels were washed for 2 S x 10 minutes. An acid stabilizing step then involved replacing the ammonia solution with an equal volume of 0.05 % (9 mM) sulphuric acid and washing for a father 10 minutes. Gels were then ready for imaging and storage. During storage, gels were kept in the dark at room temperature. 10 Gels were imaged with an Typhoon imaging system (Amersham Biosciences, Castle Hill, NSW, Australia) using a 532 nm YAG laser with the photomultiplier tube set at 540 V and with 100 pm2 pixel size. Either a 560 nm Long Pass or 610 BandPass 30 nm emission filter was used for obtaining the image. Example 3 - Deep Purplemi Protein Cel Staining 1 5 L : Eo costing of intens Iiy o f Fel stin 0 Protocol Invitrogen Bi:-Tri 10% 12-well plcLry id gels wr ldd with reu (50 mM dithiothreitol). Amersham Biosciences Low Molecular Weight Markers prepared and heated (10 min., 70*C) in Invitrogen 1x LDS buffer. The amounts of Soybean Trypsin 20 Ithibilor (Cne of t i low ma rer) poded per 5 uL li quotaf Lane 2 3 4 15 16 17 8 9 10 1 ng 0.125 0.25 0.5 1 2 16 32 64 128 256 Gels were run at 200 V constant and transferred to 60 mL of 7.5% (v/v) acetic acid for 30 minutes followed by three sequential 30 minute washes in 60 mL RO water. Gels were placed in 50 mL of RO water together with 250 pL. Deep PurpleT" and stained in dark for 1 h. Gels were independently washed 3 x 10 minute in 16 mM solutions listed in Table 25 2 and imaged with Amersham Biosciences Typhoon using 532 nm laser, a 560 LP emission filter and 520 V at the photomultiplier tube. Using ImageQuant 5.2 (Amersham Biosciences, Castle Hill, NSW, Australia) a trace was made though Lane 9 of each gel so the absolute intensity of the signal could be compared across gels.

WO 2004/085546 PCT/AU2004/000370 .26 Table 2 Gel No. 16 mM reagent 1. Octylamine 2. Tris base 3. Butylamine 4. Aniline 5. 1,3 diaminopropane 6. Ammonia 7. Water Results are shown in Figures 38A to G. In each case a 3 x 10 minute wash with the listed 16 mM reagent caused an absolute increase in fluorescent signal relative to that of a gel washed 3 x 10 minute in water. 2. Boosting and stabilising fluorescence intensity in gels using orthophosphoric acid 5 Potol Initoen Bis-Tris 12% 10-wesll gels4c w'era loaded with re duced (50 mM-1 dithiothreitol) -merham Low iolecvhr Weight Marnrd prepare and heated (10 min., 70*C) in Invitrogen 1 x LDS buffer. Replicate gel loaded with 100 ng soybean trypsin inhibitor per lane. Gels were run in lx MES running buffer at 200 V. Gels were then 10 tramnfrred to 100 mL of 7.5% (v/v) a ceti acid for 1 hour followed by-Vtwo equenti-a 30 minute washes in 100 mL RO waer, Gel were then placed in 50 mL of RO vQwaer ioget'her with 250 ptL Deep Purplem and stained in dark for 1 h. On removing the gel stain, gel segment number 1 (see Figure 39A) was washed 3 x 10 minutes in 8 mM ammonia and stored in 8 mM ammonia under dark conditions for 46 15 h. Replicate gel segment number 2 (Figure 39B) was washed 3 x 10 min. in 8 mM ammonia and transferred to 100 mM orthophosphoric acid and stored under dark conditions also for 46 h. Gels were imaged on an Amersham Bioscience Typhoon with a 532 nm laser, 560 LP filter, and 540 Volts at the photomultiplier tube. Orthophosphoric acid increased fluorescence intensity of gels stained with 20 epicocconone after development with ammonia (Figure 39A) as compared to ammonia only (Figure 39B).

WO 2004/085546 PCT/AU2004/000370 -27 3. Boosting and stabilising fluorescence intensity in gels using sulfuric acid Protocol Storage of gels in sulfuric acid was investigated Invitrogen Bis-Tris 10% 15-well gels were equivalently loaded with reduced (50 mM dithiothreitol) Amersham Low 5 Molecular Weight Markers prepared and heated (10 min., 70DC) in Invitrogen 1x LDS buffer. Replicate gels were loaded with soybean trypsin inhibitor at 100 ng in 5 pL per lane. Gels were run at 200 V constant and transferred to 100 mL of 7.5% (v/v) acetic acid for 1 hour followed by two sequential 30 minute washes in 100 mL RO water. Gels were then placed in 50 mL of RO water together with 250 pL Deep Purple tm and stained in dark 10 for 1 h. Gels were then washed 3 x 10 minutes in 8 mM ammonia and imaged on the Amersham Biosciences Typhoon with a 532 nm laser, 560 LP emission filter and 540 V at the photomultiplier tube (Figure 40A). The gels was stored at room temperature in 10 mM sulfuric acid and re-imaged as described in Example 2 after 2 h, 21 h and 93 h (Figs. 40B - 40D). Intensity traces were plotted of the same lane of the gel at different time-points 15 (Figs. 40E-40H). Figurts 40 E-H ohov signal intensive irace of Lane 3 of the gel above prior to placing in 10 md ulfuric acid (E, Time 0) and at 2 h (F), 21 h (G) and 93 h (H) after Addition of 10 n slfuric acid, iote incre e in rtain ircity r 2 h. Result 10 mM sulfuric acid after increased in the intensity of fluorescence over and above 20 that rodced by armmoi washes post-:tkining. Store: of tha gJ in sufmic acid maintained the signal at leveL higher than if gel wesre in stored in 83 mMi anmonia or water. 4. Boosting and stahlising fluorescence intensity in gels using acetic acid Storage of gels in acetic acid was investigated. Invitrogen Bis-Tris 12% 10-well gels were equivalently loaded with reduced (50 mM dithiothreitol) Amersham Biosciences 25 Low Molecular Weight Markers where soybean trypsin inhibitor was at a concentration of 400 ng/5 pL, The protein samples were two-fold diluted to final concentration of 0.76 ng/5 pL and heated (10 min., 70*C) in Invitrogen lx LDS buffer and loaded into gels. Gels were run at 200 V constant and transferred to 100 mL of 7.5% (v/v) acetic acid for 1 hour followed by two sequential 30 minute washes in 100 mL RO water. Gels were then placed 30 in 50 mL of RO water together with 250 pL Deep Purplem and stained in dark for 1 h. On removing the gel stain, the gel Was washed 2 x 10 minutes in 8 mM ammonia and imaged on an Amersham Biosciences Typhoon with 532 nm laser, 560 LP emission filter and 560 WO 2004/085546 PCT/AU2004/000370 -28. V at the photomultiplier tube (Figure 41A). The gel was placed in 10 mM acetic acid and re-imaged after 46 h and 167 h (Figs. 41B -41C). Result 10 mM acetic acid after washes in 8 mM ammonia resulted in an increase in the 5 stain intensity over and above that produced by ammonia washes post-staining. Storage of the gel in acetic acid maintained the signal at levels higher than if gel were in stored in 8 mM ammonia or water. Example 4 Epicocconone-staining of DNA fragments in an agarose gel: the effect of detergent on epicocconone-stalning 10 Materials Epicocconone (0.042 mg/ml DMSO) DNA fragments:: SPP-1 Phage/Eco RI (cat DWM-S1, Geneworks), DNA Molecular Weight Marker XVII (cat # 1855646, Roche). DTAE (1.625 ml) 15 TDTAB (0.001, 0.01, 0.1 and 1rmMf) CTAB (0.001, 0.01, 0.1,and 2 mM) CPC (0.001, 0.01, 0.1 and 1mM) SDS (1.625 mM) Acetic acid (1! mM) 20 Reverse Oamosis (RO) watr Agarpse gel (1.5 %) (DNA grade agrose, Progen) TAE electrophoresis buffer (Tris-acetate/EDTA, pH 8.3) DNA sample loading buffer (cat number 200-0424, Progen) DTAB, TDTAB, CTAB and CPC were used in this nucleic acid experiment as 25 suitable examples of cationic detergents which, because of their cationic nature, are likely to react appropriately with DNA 1"1. The relevant publications are incorporated herein by reference. SDS was used as an example of an anionic detergent which, although useful in protein methodology, is unlikely to react appropriately with nucleic acid.

WO 2004/085546 PCT/AU2004/000370 Staining method 1. DNA molecular marker samples were prepared in Progen sample loading buffer. The concentration was adjusted to 1000 ng/sample. The samples were loaded into an agarose gel (1.0 % or 1.5 %) and run at 100 V for 1.5 hours. 5 2. After running the gel, the gel was rinsed off in 1-L of RO water. 3. Lanes of the DNA gel were out into strips. 4. Each gel strip containing DNA fragments was then placed into a 15-mL Falcon tube containing 10-mL of varying concentrations of different cationic detergents specified in the material section, The detergent incubation was done at room 10 temperature for 30 min. 5. After 30-min-incubation, the initial incubating solutions were decanted from each tube. Ten milliliter of epicocconone staining solution made in RO water (0.0042 mg/mL) was then replaced into the tubes. The gel strip tubes were stained at room temperature (dark) for 1 hour. 15 6. After 1-hour-epicocconone staining, Ihe staining solution was decanted, replaced with 10-mL of acetic acid (1 mM), and incubatcd at room temperature for 30 min. (thr ties of 10-min-inrubation) 7. After acetic acid-treatment, the gel strips were scanned by Typhoon scanner (Model 9200, Amersham Biosciences). The scanning conditions were: 550 V, normal 2eniitya 610 BP 30G:IGreen (532 nm). Results Figure 42 shows Typhoon-scanned images of the DNA gel (SPP-1 DNA /Eco RI) strips (1: No detergent treatment, epicocconone staining only; 2: SDS (1.625 mM) -incubation and epicocconone staining; 3: DTAB (1.625 mM)-incubation and epicocconone staining; 4: 25 SDS and DTAB-inoubation, and epicocconone staining). 1. The molecular DNA bands of gel strip 1 were not stained with epicocconone only. 2. The molecular DNA bands of gel strip 2 were not stained with epicocconone when the DNA had been initially incubated with SDS. 3. The molecular DNA bands of gel strip 3 were stained with epicocconone when the 30 DNA had been initially incubated with DTAB. 4. The molecular DNA bands of gel strip 4 were stained with epicocconone when the DNA had been initially incubated with SDS and DTAB.

WO 2004/085546 PCT/AU2004/000370 Figure 47 shows Typhoon-scanned images of-the DNA gel (A and C, DNA MWM XVII; B, SPP-l DNA/Eco RI) strips. Figure 47-A, the DNA gel strips that were pre-incubated in TDTAB (1, 0.001 mM; 2, 0.01 mlM, 0.1 mM and 1 mM) before epicocconone staining; 5 Figure 47-B, the DNA gel strips that were pre-incubated in CTAB (1, 0.001 mM- 2, 0.01 mM, 0.1 mM and 1 mM) before epicocconone staining; Figure 47-C, the DNA gel strips that were pre-incubated in CPC (1, 0.001 mM; 2, 0.01 mM, 0.1 mM and 1 mM) before epicocconone staining. 1. TDTAB pre-incubation stained DNA fragments with epicocconone, when the 10 detergent concentration was ranged from 0.1 to 1 mM. 2. CTAB pre-incubation stained DNA fragments with epicocconone, when the detergent concentration was ranged from 0.01 to 0.1 mM. 3. CPC pre-incubation stained DNA fragments with epicocconone, when the detergent concentration was ranged from 0.01 to 0,1 mM. 15 Ceiielasion 1. The anionic detergent used (0DS) did not aid staining of DIIAI w'ith picoocconone whers2 different cati ddtrgentc (DTAB, TDTA.B, 'TA2, and CFC) er effective in revealing DNA fragments stained with epicocconone. 2. The present experiment shows that epicocconone can be used as a DNA and other 20 nucic acid tin wen they aretre d with a wide range of diffrent aioniK detergent such as DTAB, TDTAB, CTA, ad CPC. Other suitable cationic detergents may be used and will be kmown to those skilled in the art. Some of these detergents are disclosed in, for example, Bhairi SM, incorporated herein by reference ' , but other sources of suitable detergents and surfactants will be 25 know to those skilled in the art. Although DNA was used as a convenient and stable example of a nucleic acid because of ease of handling, the above principles apply equally well to RNA and other nucleic acids and their derivatives, including single and double stranded nucleic acids. Further, these concepts and principles can be applied to a number of known nucleic acid analytical and 30 quantitative techniques, such as for example those described in Old RW 'and Primrose SB't' Innis MA et al 5 and Samubrook J et al 16 WO 2004/085546 PCT/AU2004/000370 31 Example 5. Measurement of DNA concentration Components Deep PurpleTm formulated in 80% (v/v) dimethyl sulfoxide and 20% (v/v) acetonitrile at Absorbance55O nm =0.30. 5 Protocol Salmon double stranded DNA (Sigma, D1626) was prepared at a concentration of 500 pg/mL in water and two-fold diluted in water to a final concentration of 488 ng/mL. Aliquots (25 ptL) of DNA were pipetted in duplicate into the wells of a 96-well microtitre plate (Greiner, Cat. 655096). 25 pL aliquots of water were also included as DNA-free 10 controls. To duplicate rows of the plate were then added 25 pL of the cationic surfactant dodecyl trimethyl ammonium bromide (Sigma, D5047) at concentration of 3 mM in water. To individual rows was then added, depending on the particular experiment, 25 pL aliquots of, 10 mM acetic acid, 10 mM ammonia solution, or 40 mM NaHCO 3 . To wells were then 15 added 25 pL aliquot of Part A diluted 1:10 in water. Plates were incubated in dark for approximately 30 minutes and viualised on a TVA and UVE transilluminator and imaged on an IAmerIIham Biocience TpThoon wAih a. 532 nra excitAion lar and itr 5 Z60 LP or 526 SP emission filter to measure red and green emission light respectively. By the above procedure it was possible to quantify DNA in solution. For results see Figures 20 43, 44A- 44FE,435 aind 46A -46E. Figure 4t &hows 2 Typhoon img of red emisson light. 532 nm laser, 560 LE filter. After addition of tin, 10 mM4 ammonia solution added to wells after addition of Deep Purple formulated as described in Protocol. Figures 44A shows signal from Figure 43 plotted as raw data. Figure 44B is a plot of the logio transformed data. Figure 45 is a Typhoon image of green emission light. 532 nm laser, 526 25 SP filter. After addition of stain, 10 mM acetic acid solution added to wells after addition of Deep Purple formulated as described in Protocol. On UVA and UV/B transilluminators the intensity of the wells treated with acetic acid was intensely bright green and substantially brighter than the red fluorescence from wells treated with 10 mM ammonia or 40 mM NaHCO 3 .Due to its hardware configuration the Typhoon instrument was poor at 30 exciting and capturing this information. Figure 46 A is a signal from Figure 45 plotted as raw data. Figure 46B is a plot of the same data transformed logio. The results clearly indicate the utility of the dye and the use of detergents and/or bases and/or acids in improving detection and quantitation of nucleic acids.

WO 2004/085546 PCT/AU2004/000370 32 The present invention has been described with reference to specific examples, The skilled person will appreciate that the inventions may be embodied in many other forns, in keeping with the spirit of the inventive concept described herein.

WO 2004/085546 PCT/AU2004/000370 33 References 1 Hames, B.D. (1990) One-dimensional polyacrylamide gel electrophoresis, in "Gel electrophoresis of proteins: a practical approach" Second Ed., (Eds, B.D. Hames and D. Rickwood). pp. 1-139. IRL Press, Oxford 5 2 Bartoszek A, Sielenko A and Wesiora M. (2003). Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems. Anal Biochem. 313(1):53-9 3. Jaroszeski MJ, Gilbert R and Heller R (1994). Detection and quantitation of cell cell electrofusion products by flow cytometty. Anal Biochem 216: 271-275 10 4. Kamp F, Guo W, Souto P, Pilch PF, Corkey BE and Hamilton JA (2003). Rapid flip-flop of oleic acid across the plnma membrane of adipocytes. JBiol Chem 278:7988-95 5. Paulmurugan R, Massoud TF, Huang J and Gambhir SS (2004). Molecular imaging of drug-modulated protein-protein interactions in living subjects. Cancer Res. 64:2113 2119. 15 6. 14ersien B, Feilner T, Kramer A, Wehrmyer 23, Possling A, Witt . an'or T StrekeFt L in A Krentberger 1, Lerhrch H) Uill DJ. (2003). Gmrtion of Arabidopsis protein chips for antibody and serum screening. PlaniMol Biol. 52:999-1010. 7 Breadmore MC, Wolfe KA, Arcibal TG, Leung WK, Dickson D, Giordano BC, Power ME,. Ferrance JPB, Feldman 2fftK Norris PI and Landers JIP (2003). Microcip-based 20 purifiction of DN A from biological namples. Anal C.hem. 75:18S0-1886. 8 Ferrari BC, Attfield PV, Veal DA and Bell PJ. (2003). Application of the novel fluorescent dye Beijian red to the differentiation of Giardia cysts, JMicrobiol Methods. 52:133-5. 9 Graczyk TK, Grimes BH, Knight R, Da Silva AJ, Pieniazek NJ, Veal DA.(2003). 25 Detection of Cryptosporidium parvum and Giardia lamblia carried by synanthropic flies by combined fluorescent in situ hybridization and a monoclonal antibody. Am J Trop Med Hyg. 68:228-232 10 Shapiro, H.M. 2003 Practical Flow Cytometry, Forth Edition, John Wiley, New Jersey. 30 11 Bathaie SZ, Moosavi-Movahedi AA, Saboury AA. (1999). Energetic and binding properties of DNA upon interaction with dodecyl trimethylammonium bromide. Nucleic Acids Res. 1999 Feb 15;27(4):1001-5.

WO 2004/085546 PCT/AU2004/000370 34 12 Harrington LA, and Andrews, BJ. (1996) Binding to the yeast Swi4,6-dependent cell cycle box, CACGAAA, is cell cycle regulated in vivo Nucleic Acids Res. 24:558-565. 13 Bell PJL and Karuso P, (2003), Epicocconone, A Novel Fluorescent Compound from the Fungus Bpicoccum nignm, J Am Cehm Soc, 125: 9304-9305. 5 14 Old RW and Primrose SB, Principles of Gene manipulation: An Introduction to genetic engineering, Blackwell Scientific Publications, 4t Edition, 1989. 15 Innis MA, Gelfand DH, Sninsky JJ and White TJ, PCR Protocols: A guide to methods and applications, Academic Press Inc, 1990 16 .Sambrook J, Fritsch EF and Maniatis T. Molecular cloning, a laboratory manual. 10 Cold Spring Harbor Laboratory Press, NY, 1989. 17 Bhairi SM. Detergents: A guide to the properties and uses of detergents in biological systems, Calbiochem-Novabiochem Corp., 2001

Claims (14)

1. 2. A method of increasing the emission wavelength of a fluorescent dye, said method comprising combining or contacting the dye with a base and/or a detergent. 3 A method of improving the stability of fluorescent dye/organic molecule complex, 10 said method comprising contacting the fluorescent dye/organic molecule complex with an acid.
2. 4. The method according to claim 3 wherein the complex further comprises a base and/or a detergent. 15 5 The method according to claim 3 or claim 4, wherein the complex is contained within a matrix or on a surface.
3. 6. The method according to any one of claims I to 5, wherein the dye is of formula 20 (Ia), including isomers thereof: R2 O X R -a R3 (la) wherein X is 0, NR 4 or C, R is a straight or branched chain C 1 - 20 conjugated alkenyl 25 group optionally substituted 1-6 groups independently selected from hydroxy or oxo groups, R 2 is a straight or branched chain Cj-20alkyl group, R 3 is a straight or branched chain Ci-20 alkyl group, optionally substituted with a hydroxyl group, R 4 is N, 0, straight or branched chain C. 2 0 alkyl and/or aryl group, optionally substituted with a hydroxyl, halide, amine, carboxyl, carboxyl related or heteroaryl group or groups. 30 - 36 7. The method according to any one of claims I to 6 wherein the fluorescent dye is epicocconone (Ib): O OH O OH (I b) 5 or an epicocconone-containing dye mixture or extract.
4. 8. The method according to any one of claims 1 to 7, wherein the base is a nitrogen containing base. 10 9. The method according to any one of claims 1 to 8, wherein the base is selected from the group consisting of ammonia, amines, metal hydroxides, metal carbonates, metal hydrogen carbonates or combinations thereof, wherein the base increases the fluorescent intensity, the signal to background ratio and/or the sensitivity detection limit of the fluorescent dye. 15
5. 10. The method according to any one of claims 1 to 9 which is part of a chemical or biochemical technique using a fluorescent dye.
6. 11. The method according to claim 10, wherein said chemical or biochemical 20 technique is selected from electrophoresis, flow cytometry, pH sensoring, analysing protein-protein interactions, fluorescent Bradford assaying, protein quantitation, antibody labelling, ligand labelling, assaying protein arrays or protein chips, assaying gene arrays or gene chips, assay or detection of DNA or RNA, fluorescence microscopy or biological sensors. 25
7. 12. The method according to any one of claims I to 1 1 which is part of a method of staining and/or labelling a protein, a peptide, a nucleic acid or a nucleotide said method comprising forming a fluorescent complex between a protein, a peptide, a nucleic acid or - 37 a nucleotide and the fluorescent dye, wherein the fluorescent complex is formed in the presence of a base and/or a detergent to form an organic molecule/detergent complex.
8. 13. The method according to claim 12 wherein the detergent is selected from the group 5 consisting of sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), Triton, Tween, CHAPS, CTAB, DTAB, TDTAB, CPC, cholic acid and Octyl-D-glucoside.
9. 14. The method according to claim 12 or claim 1 3, further comprising the step of treating the fluorescent complex with an acid. 10
10. 15. The method according to claim 14, wherein the acid treatment stabilises the fluorescent complex, further increases the fluorescent intensity and/or further increases the signal to background ratio of the fluorescent complex. 15 16. The method according to any one of claims I to 15, further comprising the step of detecting the fluorescence of the fluorescent dye.
11. 17. A method of mobilising and detecting proteins comprising the steps of: (a) applying a solution of a protein to a matrix; 20 (b) mobilising the protein on the matrix; (c) forming a fluorescent complex between the mobilised protein and a fluorescent dye as defined in claim 6 or claim 7; and (d) detecting the so-formed fluorescent complex; wherein the fluorescent complex is formed in the presence of a base and/or the 25 fluorescent complex is treated with a base after its formation.
12. 18. The method according to claim 17, wherein the matrix is a polyacrylamide gel matrix and the method is PAGE electrophoresis or SDS-PAGE electrophoresis. 30 19. A method of mobilising and detecting nucleic acids comprising the steps of: (a) applying a solution of a nucleic acid to a matrix; (b) mobilising the nucleic acid on the matrix; - 38 (c) forming a fluorescent complex between the mobilised nucleic acid and a fluorescent dye as defined in claim 6 or claim 7; and (d) detecting the so-formed fluorescent complex; wherein the fluorescent complex is formed in the presence of a base and/or the 5 fluorescent complex is treated with a base after its formation.
13. 20. The method according to claim 19, wherein the matrix is an agarose gel matrix.
14. 21. A method of enhancing the fluorescence of a fluorescent dye according to claim 1; 10 a method of increasing the emission wavelength of a fluorescent dye according to claim 2; a method of improving the stability of fluorescent dye/organic molecule complex according to claim 3; a method of mobilising and detecting proteins according to claim 17; or a method of mobilising and detecting nucleic acids according to claim 19, substantially as herein described with reference to any one or more of the examples or 15 figures.