CA1279562C - Use of polymeric mordants to increase the intensity of rigid fluorescent dyes - Google Patents

Abstract

USE OF POLYMERIC MORDANTS TO INCREASE THE
INTENSITY OF RIGID FLUORESCENT DYES
Abstract of the Disclosure Polymeric mordants are used with certain rigid fluorescent dyes or precursors thereof to in-crease fluorescence intensity. Compositions and analytical elements containing the mordants and dyes or dye precursors are useful in biological studies, such as cell and tissue staining and cell cytometry, and in analytical determinations for various ana-lytes, e.g. living organisms.

Description

~ ;~795~

USE OF POLYMERIC MORDANTS TO INCREASE THE
I NTENS I TY OF R I G I D E'LUORESCENT DYES
Field of~the Invention This invention rel~tes to compositions and 5 elements comprising polymeric mord~nts and rigid fluore~cent dyes or fluore~cent dye precursors. It ~lso rel~tes to the use of such materisl for biologi-csl ~tudies or analytical determin~tions. In psr-ticul~r, it relstes to clinic~l chemistry.
10 Background of the Invention The stsining of biologic~l cells snd tissues with dyes, especislly fluore~cent dyes, in order to render them more observsble or to differentiste types of cells, is well known in the ~rt. There is a con-15 tinuing need to find more efficient mean~ of stsining.

' .~795 Further, chemical ~nalysis of liquids, such 89 wster, milk and biologic~l fluids i~ often desir-able or necessary for heslth m~intensnce And disgnos-tic c~re. Vsriou3 compositions and elements to 5 ~acilitste ~uch analyses are known. Such composi-tions and elements generally include a resgent com-position for determinin8 a 3ubstance under analysis, identified 8s sn "~n~lyte" herein. The snalyte csn be 8 living organism "3uch as 8 bacterium or a yeast 10 cell, or a nonliving chemical substance. The resgent composition, upon intersction with the analyte, provides ~ detectable change (e.g. dye formstion).
Recently, much work h~s been directed to developing compositions ~nd elements which are useful 15 for rspid snd highly quantitative diagnostic or clin-icsl snslysis of biological fluids such as whole blood, blood sera, plasms, urine snd the like.
For example, for the rapid and effective disgnosis ~nd trestment of infectious disesses, it is ~ desirsble to be ~ble to detect the bscteris causing the disease as rapidly as possible. Infections of the urinary tr~ct are 3mong the most common bacterisl disesses, second in frequency only to infections of the respiratory tract. Most urinsry tract infections 25 sre associated with bacterisl counts of 100,000 or more organisms per ml of urine, a condition referred to ~s significsnt bacteriuris.
In Csnsdian Serial No. 501,256 filed February 6, 1986 of Belly et ~1, novel reducible 30 compounds sre described and clsimed which sre useful - in the detection of bscteria ~s well as other analytes. The compounds ~nd sssays described in this spplication csn be used to determine an an~lyte, e.g.
a bacterium, which reduces a reducible compound 35 thereby releasing a detectsble species, e.g. a - colored or fluorescent dye, at 8 pH of 9 or less.

1;~795~jX

In Csnadisn Serisl No. 507,327 filed April 23, 1986 of B~bb et al, certain reducible compounds sre de~cribed which ~re useful for relea~ing phenslenone snd benzphenslenQne fluorescent 5 dyes. The~e dyes c~n be u~ed to ~tsin biologicsl specimens and to determine an snalyte, such a~ a bscterium.
While those assays repre~ent signiflcant advances in the srt, it would be desirsble to improve 10 the ~ensitivity of the assays even further in order to extend the1r usefulne3~ to determ~ne anslytes at much lower concentrstions.
Summary of the Invention The present invention provides a means for 15 significsntly incressing fluorescent intensity using ~ composition which comprises 8 rigid phenalenone, benzphenalenone or 4-oxo-4H-benz-[d,e]snthrscene fluorescent dye or precur~or thereof snd a cstionic or nonionic polymeric mordant. Thi~ composition csn 20 be used for distinguishing cells by cont~cting a biologicsl sample with the composition.
Further, this invention provides 8 dry sn~-lytical element for the determination of an snalyte compri~ing an ~bsorbent csrrier msterisl snd contain-25 ing, in associstion, the rigid fluorescent dye or precursor thereof described sbove and a cstionic or nonionic polymeric mordsnt.
Still further, ~ method for the determina-tion of an snalyte compri~es the steps of:
A. contscting 8 ~mple of Q liquid ~uspected of contsining an analyte with A fluore~cent dye precursor which rele8seg 8 rigid phenalenone, benzphen~lenone or 4-oxo-4H-benz-td,e~anthrscene fluorescent dye in the 35 presence of the snslyte, and a cstionic or nonionic polymeric mordsnt, ~nd . .

1~'79~ ~

B. determining the fluorescent dye released as a result of the presence of the analyte.
This invention also provides a method for stsining a biological specimen comprising contacting the specimen with the composition described above.
The sdvantages of this invention are ob-tained by using a cationic or nonionic polymeric mordant with a particular rigid fluorescent dye or dye precursor thereof. While it is known that fluo-rescence of a nonplanar or nonrigid compound can beenhanced by making it more rigid~ e.g. with a mor-dant, it is unexpected that a mordant can intensify the fluorescence of phenalenone, benzphenalenone and 4-oxo-4H-benz-[d,e]anthracene compounds which sre lS already rigid.
This invention can also be used to advantage to significantly increase the fluorescent intensity of these rigid fluorescent dyes in biological studies, ~uch as staining biological specimens, e~g.
cells and tissues and in cell cytometry.
Brief DescriPtion of the Drawin~
The Figure contains graphical plots of the change in fluorescence over time for a composition of this invention and a Control composition. This Figure is discussed in more detail in Example 4 below.
Detsiled DescriPtion of the Invention The fluorescent dyes useful in the practice of the presen~ invention include any rigid substi-tuted or unsubstituted phenalenone, substituted on unsubstituted benzphenalenone or substituted or un-substituted 4-oxo-4H-benz-[d,e]anthracene which, when excited at a particular wavelength, fluoresce or emit detectsble radiation at another wavelength. As used in this application, the term "rigid" refers to com-pounds having two or more fused rings and which are 1~79t~

--5--therefore fixed-in apace in a re~tricted number of configuration~. The uaeful subatituted or unsubsti-tuted snthracene~ sre described in copending ~nd commonly ss~igned Csnsdisn Serisl No. 506,803 filed 5 APri1 16, 1986 bY GO5WAmi et al snd entitled FLUORESCENT DYES AND BIOLOGICAL AND ANALYTICAL USES
THEREOF.
The substituted or unsubstituted phenale-none3 and benzphenalenone~ described in Canadisn 10 Serisl No. 507,327 of 8abb et al noted sbove are preferred in the prsctice of this invention. These compound~ have the following structures:
R R
11 1 or i t fi wherein R is hydroxy, mercapto or amino tHN(R')-]-20 R' i~ hydrogen, substituted or unsubstituted ~lkyl (preferably of 1 to 10 carbon atoms, e.g. methyl, ethyl, $sopropyl, decyl,benzyl, etc.), ~ubstituted or unsubstituted cycloalkyl ~prefersbly of 5 to 12 c~rbon stoms, e.g. cyclopentyl, cyclohexyl, etc.), 25 sUbstituted or unsubstituted phenyl or a ~ubstituted or unsubstituted heterocyclic group, e.g. pyridyl or thienyL. Preferably, R' is hydrogen or substituted or unsubstituted alkyl of 1 to 3 carbon atoms. The compound~ can have one or more other substituents on 30 any of the fused rings as long a~ the fluorescence is not sdversely affected. Such ~ubstituents include substituted or unsubstituted alkyl (preferably of 1 to 12 carbon stoms, e.g methyl, ethyl, benzyl, etc.), ~ubstituted or un~ubstituted hydroxyalkyl 35 (preferably of 1 to 12 csrbon atoms, e.g. hydroxy-methyl, 2-hydroxyethyl, etc.), substituted or unsub-stituted slkoxyc~rbonyl (prefersbly of 2 to 12 csrbon 7~3S~

atoms, e.g. methoxycarbonyl, ethoxycarbonyl, etc.), halo (e.g. fluoro, chloro, bromo), cyano, carboxy, acyl, substituted or unsubstituted arylsulfonyl (pre-ferably of 6 to lO carbon atoms, e.g. phenylsulfonyl, tolyl~ulfonyl, etc.), substituted or unsubstituted alkylsulfonyl (preferably of l to 6 carbon atoms, e.g. methylsulfonyl, ethylsulfonyl, etc.), and other substituents known to one skilled in the art.
Representative phenalenone and benzphensle-none fluore cent compounds include:
OH NH
I ' I. i~ \i~ \li/ ~i snd IV. i~ \i~ \il/ ~i with compounds I and II being particularly useful.
Methods for preparing these compounds are described in the noted Babb et al application.
The rigid fluorescent dyes can also be derived from ~ dye precursor which can be sub~ected to some treatment or condition which will release the rigid fluorescent dye during the assay. For example, the dye precursor can be hydrolyzed chemically or enzymatically by an analyte or other reagents used in the assay. In one embodiment, dye precursors which are substrates for hydrolases, such as esterases (e.g. diacetinase~ and amidases (e.g. aminoacylase) can be used in the practice of this invention.

1;~795 The substrate~ h~ve the following structure~:
X-BLOCK X-BLOCK
5 !~ i SUB ` i~ I i SUB

wherein X i~ oxy, thio or imino t-N(R")-, wherein R"
is defined the ssme a8 R' sbove, i.e. hydrogen, sub-10 stituted or un~ub~tituted alkyl, substituted or un-substituted cyclo~lkyl, substituted or unsubstituted phenyl or a ~ubstituted or unsub tituted heterocyclic group, such as pyridyl or thienyl], SUB represents one or more substituent~ a9 defined above for the 15 phenalenone and benzphenslenone compound~, and BLOCK
is 8 blocking group, such as acyl, phosphono, thioxo-phosphono or a moiety derived from sn amino scid, peptide or mono- or polysacchsride which can be clesved from the remainder o`f the molecule by hydrolysis.
Preferred substrstes include:
O O
O-C-CH3 N~-C-CH3 .~
I 1l I and 1 11 Such dye precur~ors and their use as enzyme sub-30 strate~ are described in more detail ~n Canadian Serisl No. 507,335 filed April 23, 1986 of Babb et al.
In another embod~ment, the dye precur30r ha~
the structure CAR ( Rl)n wherein CAR- is a ~ub-~tituted or unsub~tituted aromatic or quinone nucle-35 U8, R comprises a ~ubstituted or un~ubstitutedphenalenone or benzphenalenone moiety derived from ~ ~.

the compounds described sbove and n is 1 or 2. This precursor is cap~ble of bein~ reduced at a pH of 9 or less. When Rl is replsced with H, CAR-~H)n has ~n El/2 of either at least ~bout +100 mV when measured 5 in w~ter. This El/2 value fAcilitstes the reduc-tion snd subsequent rele~se sf R from CAR- at physiological pH (i.e. pH of 9 or les~) in the determinations of biological an~lytes. Such mea-surementq sre made according to standard electr~-10 chemical techniques using either differential pulsepolarogrAphy or cyclic voltsmetry (see e.g. SPwyer and Robert, Jr., ExPerimentsl ElectrochemistrY for Chemists, John Wiley L Sons, New York, 1972).
Prefersbly, the El/2 i8 from sbout ~100 mV to about 15 +400 mV ss measured in water.
Such fluorescent dye precursors ~re described in considersble detail in Canadian Serial Nos. 5Ql,256 and 507,327 of Belly et al snd Babb et al noted above, respectively. Such precursors are 20 "shifted", i.e. the spectrs of the released dyes sre different than the spectrs of the dye precursors.
In a preferred embodiment, the reducible fluorescent dye precursors useful in this invention are RIND compounds, i.e. reducible compounds capsble 25 of undergoing intrsmolecular nucleophilic displace-ment st physiological pH to release one or more rigid fluorescent dyes when 8 nucleophilic group is gener-ated by ~t lesst a one electron reduction of the compound. In other words, such displacement occurs 30 when the RIND compound is reduced by a ~uitable reductant.
The term "intramolecular nucleophilic dis-placement" refers to a reaction in which ~ nucleo-philic center on a molecule rescts st another site in 35 the molecule, which site is sn Plectrophilic center, to effect displscement of 8 group or atom attached to 1~ 7~

the electrophilic center. Generally, the RIND com-pounds useful in this invention have the nucleophilic and el~trophilic groups ~uxtapo~ed in the three-dimensional configuration of the molecule in close proximity whereby the intramolecular reaction can take place and a ring is formed having from 4 to 7 atoms, and preferably having 5 or 6 atoms.
Particulsrly useful RIND compounds are those which have thé structure CAR~Rl wherein CAR- is o ll R \./ \./

R3/ \-/ \RZ
O

Rl i8 t R tmN Q FRAG

wherein m is O or l, and preferably 1. R5 is substituted or unsubstituted alkylene, preferably of 1 or 2 carbon atoms in the backbone (e.g. methylene, ethylene, alkoxymethylene, etc.). Most preferably, R is methylene. Q is carbonyl or thiocarbonyl and preferably carbonyl.
R6 is methyl.
FRAG is a shiftable fluorescent dye as defined above. That is, it has first spectral absorption and emission bands when attached to the RIND compound, and second spectral absorption and emission bands when released 8S a rigid fluorescent dye. This dye is released in an amount which can be directly related to the amount of reductant (i.e.
analyte) present.

1;~7'35~i~

FRAG is llnked to Q by means of a qingle bond through a bivalent monoatom linkage whlch is a part of FRAG. l-eferably, the monoatom linkage is oxy or thio, and most preferably it is oxy.
R2, R3 and R~ in the above quinone structure are independently hydrogen, sub-stituted or unsubstituted alkyl of 1 to 40 carbon atoms (e.g. methyl, ethyl, hydroxymethyl, methoxy-methyl, benzyl, etc.) substituted or unsubstituted aryl (e.g. phenyl, naphthyl, methylnaphthyl, ~-nitro-phenyl, m-methoxyphenyl, phenylsulfonamido, etc.) or an electron withdrawing group which generally has a positive Hammett sigma vslue, snd preferably has a sigma value greater than about 0.06. Hammett sigma values are calculated in accordance with standard procedures, e.g. described in Steric Effects in OrRanic ChemistrY, John Wiley & Sons, Inc., 1956, pp.
570-574 and Pro~ress in PhYsical Or~anic ChemistrY, Vol. 2, Interscience Publishers, 1964, pp. 333-339.
Representative electron withdrawing groups having positive Hammett sigma values include cyano, carboxy, nitro, halo (e.g. fluoro, bromo, chloro, iodo), tri-halomethyl (e.g. trifluoromethyl, trichloromethyl, etc.), trialkylammonium, carbonyl, carbamoyl, 8ul-fonyl, sulfamoyl, esters and others known in the art,or alkyl or aryl groups (defined above) substituted with one or more of these electron withdrawing groups. Preferred electron withdrawing groups in-clude P-nitrophenyl, m-nitrophenyl, ~-cyanophenyl and 2,5-dichlorophenyl. Aryl groups with methoxy or acetamido groups in the meta position are also useful.
R can also be Rl thereby potential-ly providing a 2:1 molar ratio of fluorescent dye molecules to original RIND compound molecules.

1~'79~

Alternatively, R3 ~nd R4, t~ken to-gether, c~n repre~ent the c~rbon atom~ neceQ~ary to complete a cub~tituted or unsub~tituted fu~ed carbo-cyclic r1ng stt~ched to the quinone nucleu~. For 5 ex~mple, ~uch 8 ring (mono- or bicyclic) c~n h~ve from 4 to 8, snd prefer~bly from 5 to 7, csrbon atom~
in the backbone.
Representative preferred RIND dye precur~ors ~re li~ted in Tsble I below ln reference to the fol-lowing ~tructure:

4 0 ~6 0 R ~ ,1! 1 11 f, ~,--CH2N--C--FRAG
R3/ ~-/ ~R2 o The El/2 vslue of Table I were mehsured in sn aqueous emul~ion of ths quinone di~solved in 2Q N,N-dimethylformamide, a nonionic surfsctsnt (TRITON
X-100~) ~nd sodium pho phste buffer (pH 7). A
normal hydrogen electrode wa~ u ed 8~ 8 ~tandard.

1~795~;~

^l _, O N `O
N t~l ~ ~) _ ~
S ~11 ~ + +

/i\ /~
¢ ~ ,~
~, i il ~, \,i ,,...

~ E E
~: o o 2 0 ~ s s ~ \~/- ~ \ I
C~
o~ ~o t~

~ I l! i l! I l!
~/ ~/\, I i i 3 5 ., ~o X l l l Z o P~
~,) H ~ H

1~7~5 ~ ~o ~
N ~ t~ _I
_ _~ N N
~ + + +

~ : : :

Ei C o ~ ~D ' 20 ~3 ~\
~ 11S\../

-r O O

l l l ~ :r:' "
Z o ~: E

, 1;~7956;~

The RIND dye precursors useful in this in-vention are prepared using a sequence of individually known reactions. Generally, the preparation sequen~e includes the following general steps: (1) prepara-tion of the substituted hydroquinone, (2) oxazinering formation, (3) oxazine ring opening, (4) pre-paration of the carbsmoyl chloride, and (5) re~ction of the FRAG moiety with the carbamoyl chloride~
Representative preparations are provided in the Belly et al and Bsbb et al applications noted above.
Other useful RIND compounds include those having the appropriate El/2 values and the structure CAR-tRl)n wherein:
(1) CAR- is a substituted or unsubstituted nucleus of a 1,2-naphthoquinone, 1,2-, 1,4- or - 9,10-anthraquinone, 4,4'-diphenoquinone, azuloquinone or l,6-[10~-anulenoquinone wherein R~ is attached to the nucleus one carbon atom distant or in the ~
position from one of the oxo groups of the nucleus.
The nucleus can be substituted with one or more elec-tron withdrawing groups as described above for R2 or have one or more fused rings as described above for R3 and R .

R~ is -tR5t-mN-Q-FRAG as defined ebove, and n is an integer of 1 or 2.

(2) CAR- is I i, il ./ \./
o I! Y
~j/ \~/
o o o .~^\./ \.~o ! I or ~./ \.~-\
o .~!\./-\.
l! 1 sny of which can be substituted with one or more electron withdrawing groups as described above for R2, R9 and R4 R~ is -tR5t-mN-Q-FRAG as defined above, and n is ]L or 2.
(3~ CAR- is a substituted or unsubstituted nitrobenzenoid nucleus of the structure R so2\.~ \.

l 02R~
wherein R' is substituted or unsubstituted slkyl of 1 to 20 csrbon atom~ (e.g. methyl, ethyl, methoxymethyl, 1 2~9'~

isopropyl, dodecyl, hexadecyl, octadecyl~ etc.), ~nd is -~R5 tmN-Q-FRAG as defined above and n is 1.
All of these reducible compounds csn ~e prepared using techniques and starting materials known in the art or readily apparent to a skilled synthetic chemist.
One or more of the fluorescent dyes or dye precursors described herein are used in combination with one or more cationic or nonionic polymeric mordants. Such materials are well known in the art, e.g. U.S. Patents 3,958,995 (issued May 25, 1976 to Campbell et al), 4,069,017 (issued January 17, 1978 to Wu et al), 4,124,386 (issued November 7, 1978 to Yo~hida et al), and 4,247,615 (issued January 27, 1981 to McGuckin et al) and Research Disclosure Publication 12045 (April, 1974), available from Kenneth Mason Publications, Ltd., The Old Harbourmsster's, 8 North Street, Emsworth, Hampshire, P010 7DD, England. The cationic mordants contain one or more charge-bearing cationic groups (e.g. cationic quaternary ammonium or phosphonium groups), while the nonionic mordants contain no charged groups.
Representative mordants useful in the practice of this invention include:
poly(N,N,N-trimethyl-N-vinylbenzylammonium chloride) poly[styrene-co-N-benzyl-N,N-dimethyl-N-(E-VinYlbenzyl)~mmonium chloride], poly(N,N,N-trioctyl-N-vinylbenzylphosphonium chloride) poly[styrene-co-N-vinylbenzyl-N,N,N-trihexyl-~mmonium chloride]
poly(styrene-co-N,N,N-trimethyl-N-vinyl-benzylammonium chloride), ~ ~ 7 ~

poly(styrene-co-N~N-dimethyl-N-(P-vinyl-benzyl)ammonium chloride-co-divinylbenzene), poly[l-vinylimidazole-co-3-(2-hydroxyethyl)-1-vinylimidazolium chloride], poly[N-benzyl-N,N-dimethyl-N-(P-vinyl-benzyl)ammonium chloride], poly(vinylpyrrolidone), poly[acrylonitrile-co-l-vinylimidazole-co-3-(2-hydroxyethyl)-1-vinylimidazolium chloride], and poly(N-cyclohexyl--N,N-dimethyl-N-vinyl-benzylammonium chloride).
Each mordant may afect different rigid fluorescent dyes to a different degree. For example, some dyes may not be enhanced by every mordant. Some mordants may enhance the intensity of some dyes, but not others. It has been found, however, that each mordant unexpectedly increased the intensity of these particular rigid fluorescent dyes.
The mordants can be prepared using tech-niques and starting materials known to one skilled inthe art. Some of them are commercially available.
The rigid fluorescent dyes and mordants described herein are generally more soluble in water thsn the clye precursors. Therefore, the compositions of dyes and mordants can generally be prepared using an aqueous or buffer solution. In some instances, a water-misc:ible solvent or the combination of such a solvent and a surfactant may be needed to dissolve a given dye. The dye precursors have generally limited water solubility and must be dissolved in a solution or dispersion including either a water-miscible sol-vent or a surfactant or both.
Surfactants which are useful in the practice of this invention include any surfactants which do not inhibit analyte determination. Generally, for 1~795 determin~tion of living cells, the useful surf~ctants sre nonionic ~urfsctsnts including, for example, slkylarylpolyethoxy alcohol~ (e.g. TRITON X-lOQ~
~nd X-305~ avsil~ble from Rohm & Hsss, 5 Philadelphia, Pennsylvanis, U.S.A~), E-alkylaryl-oxypolyglycidols (e.g. SURFACTANT lOG ~v~ilable from Olin Corp., Stsmford, Connecticut, U.S.A.), TWEEN 80~ (av~ilable from ICI Americ~s, Inc., Wilmington, Delaware, U.S.A.), snd others known to 10 one ~killed in the ~rt.
Useful wster-miscible orgsnic solvents in-clude slcohols (e.g. methanol, ethanol, propsnol, etc.), N,N-dimethylformamide, dimethyl ~ulfoxide, acetonitrile, hexsmethylenephosphorsmide and the 15 like. The particulsr solvent to be used for a p~rticulsr composition can be readily determined by routine experimentation.
A dispersion can be prepared in the follow-ing genersl manner. The dye or dye precursor is dis-20 ~olved in the water-miscible solvent at a concentrs-tion which depends upvn it~ moleculsr weight, but generslly st from about 1 to ~bout 100, and prefer-ably from sbout 5 to about 80, mg per ml of solvent.
; The resulting solution is then mixed with a suitsble 25 surfactsnt in an amount generally of from about 0.1 to about 24, snd preferably from about 0.5 to ~bout 10~ mg surfactant per ml of dispersion. The result-ing solution is then mixed with buffer. A suitable mord~nt, dissolved ln buffer at gener~lly from sbout 30 0.1 to 20 mg/ml of buffer, is then mixed with the dye or dye precursor dispersion to obtain 8 composition of this invention. This prepsration is generslly csrried out at room temperature.
In many instances, it i~ desirable to use 35 the ~nalytical composition st physiologicsl pH (9 or less). The concentration of one or more buffers used ~X79S~

to maintain the pH in the dispersion can vary widely, but is generally from about 0.01 to about 1 molar.
Representative buffers include phosphates, borates and others reported by Good et al in BiochemistrY~ 5, 467 (1966), and Anal. Biochem., 104, 300 (1980).
The compositions de~cribed herein are useful in compositions for assay (i.e. qualitative or quan-titative detection) of aqueous and nonaqueous liquids, e.g. biological fluids, manufacturing pro-cesses, wastewater, food stuffs, etc. Determinationscan be made of various analytes via a single reaction or a sequence of reactions which bring about reduc-tion of the compound and release of the fluorescent moiety. The various analytes include living cells (e.g. bacteria, white blood cells, yeast, fungi, etc.), enzymes (e.g. hydrolases, glucose oxidase, lactate oxidase, creatine kinase, a-glycerophos-phate oxidase, lactate dehydrogenaQe, pyruvate dehydrogen~se, glucose-6-phosphate dehydrogenase, alanine aminotransferase, aspartate aminotran ferase and other NADH-based, FADH-bssed or oxidase-based assays which include dehydrogenase or reductase enzymes), biological or chemical reductants other than living cells which will reduce the preferred dye precursor (e.g. a9corbates, cysteine, glutathione, thioredoxim, etc.), metabolizable substances (e.g.
glucose, lsctic acid, triglycerides, cholesterol, etc.), imm~noreactants (e.g. antigens, antibodies, haptens, etc.).
The compositions of this invention are particularly u~eful in determining living cells in biological samples. Although any biological sample suspected of having living cells therein (e.g. food, tissue, ground water, cooling water, pharmaceutical products, sewsge, etc.) can be analyzed for bacteria, yeast, fungi, etc. by this invention, the invention is particularly useful for bacterial detection in aqueous liqu~ds, such as human and animal fluids ~e.g. urine, cerebral spinal fluid, whole blood, blood serum and plasma and the like as well as stool secretions) snd suspensions of humsn or animal tis-sue. The practice of this invention is particulsrly important for detection of urinary tract infections in urine (diluted or undiluted).
When determining living cells using this invention, it is preferable for rapid dye release that the living cells interact with an electron transfer agent (identified herein as an ETA). The presence of sn ETA may also provide more efficient dye formation for analytical determlnations of nonliving analytes. The ETA is a mobile compound which acts as an intermediary between the substance being deter~lned (e.g. living cell) and a reducible dye precursor.
In general, the ETA compounds useful in the practice of this invention have an El/2 in the range of from about -320 to about +400 mV as measured in aqueous buffer (pH 7) versus the normal hydrogen electrode using a differential pulse polarographic technique with a PAR Potentiostat (Princeton Applied Research, Princeton, New Jersey). In general, the potential of the ETA should be more positive than the potential of the analyte to be determlned and less positiv~ than the potential of the reducible dye pre-cursor (e.g. a RIND compound). The ETA is generallypresent at a concentration that is dependant upon the concentration of the analyte, and preferably at a concentration of from about 1 x 10 molar to about 1 x 10 molar.

5~i~

ETA compounds useful in the practice of thi~
invention include phenazine metho~ulfate, phenazine ethocul~ate and ~imil~r compound~ known to one ~killed in the ~rt. Combinstion~ of different ETA
5 compound~ can be u~ed if desired.
Preferred ETA compounds useful in the prac-tice of this invention which provide further sdvan-t~ge~ of low background sre those which are the sub-~ect of U.S. Patent No. 4,746,607 issued M~y 24, 1988 10 by Mura et al. In genersl, thoae compounds are aubstituted benzo- snd naphthoquinone~. Examples of this class of quinones include 2,3-dimethyl-5-hydroxymethyl-1,4-benzoquinone, 2,5-dimethoxy-1,4-benzoquinone, 2,3,5-trimethyl-1,4-benzoquinone, 15 2,6-dimethoxy-1,4-benzoquinone, 2-hydroxymethyl-1,4-naphthoquinone and 2-(2-hydroxyethyl~-1,4-naphtho-quinone.
The determination of living cells, and par-ticulsrly of bacterisl cells, is often carried out in 20 the presence of a nutrient for tho~e cella although its presence is not essenti~l. Any nutrient medium can be used which contsin~ useful carbon, and option-ally nitrogen, ~ources. Suitable nutrient media hav-ing proper component~ and pH are well known in the 25 art.
The present invention is adaptable to either solution or dry assays. In a solution 85say, a solution (or aqueous diapersion) containing a rigid fluorescent dye or precursor thereof, a mordant, and 30 preferably sn ETA, can be prepared and contacted, by mixing, with a liquid test sample containing the analyte to be determined. Generally the composition i~ mixed with the test ~ample in 8 suit~ble container (e.g. test tube, petri dish beaker, cuvette, test J~:

1 ~ ~9 device, etc.). The resulting solution (or di~per-sion) is gently mixed snd incubated for a relatively short time (i.e up to about 30 minutes) ~t a temper-~ture up to about 40C, and generally from about 20 to about 40~C. The test sample i5 then evaluated by measuring the resulting fluorescent dye with suitable detection equipment.
The solution assay can also be carried out by contacting a porous absorbent material, e.g. paper strip, containing the test ssmple with the composi-tion of this invention. The analyte in the test sample can migrate from the porous material into the composition and initiate the analytical reactions needed for determination. In solution assays, the amount of dye or dye precursor present is at least about 0.001, and preferably from about 0.01 to about 1.0, millimolar. The amount of mordant i-~ at least 0.1, and pre~erably from about 0.5 to ~bout 10%
(based on final weight). Other reagents can be pre-sent in amounts readily determined by one skilled inthe art.
Alternatively, the method of this invention can be practiced with a dry analytical element. Such an element can be an absorbent carrier material, i.e.
a thin sheet or strip of self-supporting absorbent or bibulous material, such as filter paper or strips, which contains the composition of this invention or a dried residue of a dispersion comprising same. Such elements are known in the art as test strips, diag-nostic elements, dip sticks, diagnostic agents andthe like. The rigid dye or dye precursor and the polymeric mordant must be in the element in ~ manner that they can interact with each other. This is termed "in association". If they are not in the same area of the element, the dye or dye precursor is capable of migrating to the mordant during the assay.

When employed in dry analytical elements, the compo~ition of this invention can be incorporated into a suitable absorben~ c~rrier material by imbi-bition or impregnation, or csn be coated on a suit-able material. Alternatively, it can be added to theelement during an assay. Useful carrier materials sre insoluble ~nd maintain their structural integrity when exposed to water or physiological fluids such as urine or serum. Useful carrier material can be prepared from paper, porous particulate structures, cellulose, porous polymeric films, wood, glass fiber, woven and nonwoven fabrics (synthetic and nonsynthe-tic) and the like. Useful materials and procedures for making such elements are well known in the art as exemplified by U.S. Patents 3,092,465 (issued June 4, 1963 to Adams et al), 3,802,842 (issued April 9, 1974 to Lange et al), 3,915,647 (issued October 28, 1975 to Wright), 3,917,453 (issued November 4, 1975 to Milligan et al), 3,936,357 (issued ~ebruary 3, 1976 to Milligsn et al), 4,248,829 (issued February 3, 1981 to Kita~lma et al), 4,255,384 (issued March 10, 1981 KitaJima et al), and 4,270,920 (issued June 2, 1981 to Kondo et al), and U.K. Pstent 2,052,057 (published January 21, 1981).
A dry assay can be practiced to particular advantage with fln analytical element comprising a support having thereon at least one porous spreading zone as the ab~orbent carrier material. The spread-ing zone can be prepared from any suitable fibrous or non-fibrous material or mixtures of either or both as described in U. S. Patent 4,292,272 (issued September 29, 1981 to Kit~ima et al), or from poly-meric compositions (e.g. blush polymers) or particu-late materials, with or without binding adhesives, as 3s described in U. S. Patents 3,992,158 (issued 12 79~

November 16, 1976 to Przybylowicz et al~, 4,258,001 (issued March 24, 1981 to Pierce et al) and 4,430,436 (issued February 7, 1984 to KoyaLa et al) snd Japanese Patent Publication 57(1982)-101760 (published June 24, 1982). It is desired that the spreading zones be isotropically porous, me~ning that the porosity is the same in each direction in the zone as created by interconnected spaces or pores between particles, fibers, polymPric strands, etc.
The dry analytical element of thi~ invention can be a single se-f-supporting porous spreading zone containing the composition of this invention, but preferably such zone is carried on a suitable non-porous support. Such a support can be any suitable dimensionally -~table, and preferably, transparent (i.e. radiation transmissive) film or sheet material which transmits electromagnetic radiation of a wave-length between about 200 snd about 900 nm. A support of choice for a particular element should be compst-ible with fluorescence spectroscopy and inert tochemical reagents and liquid samples used in the assay. Useful support materials include polystyrene, polyesters [e.g. poly(ethylene terephthalate)], poly-carbonates, cellulose esters (e.g. cellulose acetate), etc.
The elements can have more than one zone, e.g. a reagent zone, a registration zone, subbing zone, etc. The zones are generally in fluid contact with each other~ meaning that fluids, reagents and reaction products can pass between superposed regions of ad~acent zones. In particul~r, if the dye pre-cursor is not in the same zone as the mordant, the element can be designed to allow migration of the released dye to the mordant. Preferably, the zones are separately coated superposed layers, although two or more zones can be in a single layer. Besides the Przybylowicz et al and Pierce et al patents noted ..

above, suitable element formats and components are described also, for example, in U. S. Patents 4,042,335 (issued August 16, 1977 to Cléme;lt) ~nd 4,144,306 (noted above) and Reissue 30,267 (reissued May 6, 1980 to Bruschi).
In the elements of this invention, the dye precursor, mordant and any other reagents can be in the same or different zones. During the assay, if in different zones, the released dye migrates through the element until it comes into contact with the mordant. The amount of the fluorescent dye or dye precursor can be varied widely, but it is generally present in a coverage of at least about 0.01, and preferably from about 0.05 to about 0.2, g/m2.
Other necessary or optional reagents are generally present in the following coverages:
mordant: generally at least about 0.1, and preferably from about 0.5 to about 5, glm2~
20 ETA: generally at least about 0.001, and preferably from about 0.01 to about 1, g/m2, nutrient: generally at least about 0.005, and preferably from about 0.1 to about 2, g/m2 (used only in living cell detection), buffer (pH< 9): generally at least about 0.1, and preferably from about 0.5 to sbout 2, g/m2, and 30 surfactant: generally at least about 0.1, and prefersbly from about 0.2 to about 5, g/m2.
The element zones can contain a variety of other desirable, but optional, addenda, including activators, binders (generally hydrophilic), anti-oxidants, etc. as ls known in the art.

In a preferred embodiment of this lnvention, an element for determination of microorganisms or cells (e.g. yeast, white blood cells, fungi, bacter-i~, etc.) in an aqueous liquid contains an electron transfer agent, a nonionic mordant and a reducible dye precursor described above (e.g. a RIND com-pound). It is desirable that these elements also contain a nutrient for the llving cells and a buffer which maintains physiological pH during the assay (e.g. when contacted with a 1-200 ~1 sample of test liquid). Such sn element can be used to detect bac-teria, for example, in a urine sample by contacting the sample and element in a suitable manner, and detecting the rigid fluorescent dye released from the dye precursor at the appropriate wavelength. A urine sample may have to be pretreated before testing to eliminate reductive interferents.
In another embodiment of this invention, an element for the determination of a nonliving biolog-ical or chemical an~lyte in an aqueous liquid com-prises the dye precursor and a mordant as described above, and optionally an ETA, a nonionic surfactant and a buffer which maintains physiological pH during the assay, all of which are described above.
Examples of such analytes are described above. The elements may also contain suitable interactive com-position compriqing suitable reagents which react with the analyte and effect release of the fluo-rescent dye. The amount of dye detected can be correlated to the amount of analyte presen~ in the liquid sample.
The element of this invention is also useful for determining reductants such as ascorbate (ascor-bic acid and equivalent alkali metal salts), cys-teine, glutathione, thioredoxin and the like.

~ ~ ~9 A variety of different elements, depending on the method of ~say, c~n be prep~red in accord~nce with the present invention. Elements can be con-figured in R vsriety of forms, includ~ng elongated 5 tspe~ of sny desired width, sheets, slide3 or chips.
The a~ssy of this invention c~n be msnual or automsted. In genersl, in using the dry elements, sn an~lyte or living cell determination is msde by tak-ing the element from a supply roll, chip packet or lO other source and physicslly contacting it with a sample (e.g. 1-~00 ~1) of the liquid to be tested so thst the ssmple is mixed with the resgents in the element. Such cont~ct csn br sccomplished in sny suitable manner, e.g. dipp1ng or immersing the ele-15 ment into the sAmple or, preferably, by spotting the element by hand or mschine with one or more drops of the ssmple with a suitable dispensing mesns so that the liquld s~mple mixes with the reagents within the element.
After s~mple ~pplication, the element is exposed to any conditioning, such as incubation,heating or the like, that may be desirsble to quicken or otherwise fscilitste obt~ining any test result.
Determination of an snalyte or living cell 25 is achieved when a fluorescent dye is relessed which csn be detected in a suitsble manner using conven-tionsl fluorometric sppsrstus snd detection proce-dures. Determinations can be msde at the msx~mum wavelength or ~t other wavelengths.
In the examples provided below to illustrate the practice of this invention, the msterisls used were obtained from the following sources:
ZONYL FSN~ surfactant from DuPont Co.
(Wilmington, Delawsre, U.S.A.), DAXAD 30~ surfactant from W. R. Grace Co.
(Lexington, Msssschusetts, U.S.A.), ~27956 TRITON X-100~ surfactsnt from Rohm snd Hsss (Philsdelphi~, Penn~ylvsnia), snd the rem~inder from Ea~tmsn Kodsk Compsny (Rochester, New York) or prepered using known 5 ~tsrting materials snd technique~.
As u~ed in the context of this disclo~ure ~nd the claim~, I.U. represents the Internstional Unit for enzyme ~ctivity defined ~ one I.U. being the amount of enzyme ectivity required to cstslyze 10 the conversion of 1 ~mole of sub~trste per minute under standsrd pH and temper~ture conditions for the enzyme.
ExsmPles 1-3: Fluorescence Enhancement of Phenslenone snd BenzPhenalenone Dyes Usin~ Mordants Compositions were prepared contsining esch of the fluore~cent dyes I, II snd III identified sbove snd esch of seversl polymeric mordsnts. The fluorescence inten~ity of e~ch composition wss com-pared to the inten~ity of a Control composition con-20 tsining the dye alone. The fluorescent ~cans were made using sn excitation msxima of 540 nm and emis-sion m~xima of 600 nm for dyes I ~nd II, and an exci-tstion maxima of 540 nm snd an emi~sion maximfi of ~70 nm for dye III.
The compositions were prepared in the following manner:
Compositions Ia, Ib, Ic snd Id of Exsmple 1 contsined dye I ~5.83 ~molar) snd poly(styrene- co-N,N,N-trimethyl-N-vinylbenzyl~mmonium chloride)(1.8 30 mg/ml), poly~N-cyclohexyl-N,N-dimethyl~N-vinylbenzyl-smmonium chloride)(l.8 mg/ml), poly(l-vinyl imida-zole-co-3-(2-hydroxyethyl)-1-vinylimidazolium chlo-ride)(l.8 mg/ml) snd poly(N-vinylpyrrolidone)(1.8 mg/ml), respectively, in 50 mmolar sodium phosphste 35 buffer (pH 7.5). A Control compo~ition cont~ined dye I (5.83 ~molar) in SO mmolar sodium phosphate buf-fer (pH 7.5).

.

Compositions IIa, IIb, IIc ~nd IId of Exsmple 2 ~nd Control compositlon II were prep~red similarly except dye II wsq used.
Composition III of Ex&mple 3 contained dye III (9.2 ~molAr) snd poly(styrene-co-N,N,N-tri-methyl-N-vinylbenzylsmmonium chloride)(l.8 mg/ml) in 50 mmolsr sodium bicarbonate buffer (pH 10).
Control compos$tion III W3 S prep~red with dye III
only in buffer.
Table II below lists the incresse in fluo-rescence intensity for the compositions of this invention ovPr the intensitles of the respective Control compositions.
T A B L E II
ComPosition Intensity Incresse IQ 40%
Ib 35%

Id 19 IIs IIb 52%
IIc 64%
IId 51S
III 76%
25 ExamPle 4: Flourescence Enhancement in a Determination of an Esterase EnzYme This example demonstrates the use of the pre-sent invention to determine sn snalyte, discetinsse, with the composition of the present invention. It 30 slso shows the improved fluorescence intensity ob-tsined wlth thst composition.
The fluorescent dye precursor (4 mg) hsving the following structure snd prepsred according to the procedure described in Can~dian Serial No. 507,346 35 filed April 23, 1986 of Babb et al entitled HYDROLYZABLE FLUORESCENT SUBSTRATES AND ANALYTICAL
DETERMINATIONS USING SAME, :. O
O-C-CH
\./ ~.
` t t was dissolved in N,N-dimethylformamlde (250 ~l) contsining 0.1~ sulfuric acid. Some of this ~olution (75 ~1) was mixed with TRITON X-100 surfactant (600 ~1) snd sodium phosphate buffer (30 ml, 50 mmolar, pH 7) to form Solution A, and kept in the dark.
A Control composition was prepared with 3 ml f Solution A and 10 ~1 of diacetinase (0.2 I.U./ml of water). A composition of this invention was pre-pared from 3 ml of Solution A, 10 ~1 of diacetinase and 40 ~1 of poly(N-cyclohexyl-N,N-dimethyl-N-vinylbenzylammonium chloride)(l5 mg/ml of buffer).
2~ Fluorescent scans were run at 25C for 40 minute~ with re~dings taken at 10 minute intervals using a commercially available Per~in-Elmer fluoro-meter. The excitation maxima was 540 nm and the emlssion maxima was 598 nm.
The Figure shows the change in relative fluorescence vs. time for both compositions. It is clear that the present invention provides signifi-cantly improved fluorescence compared to the Control composition where the mordant was absent.
ExamPle 5: Stainin~ of White Blood Cells Usin~ a Com~osition of this Invention This example demonstrates the usefulnes of the present invention to stain white blood cells.
Human blood (5 ml) was collected in sterile vacutainer tubes containing 1.5 ml acid citrate dextrose as an anticoagulant, and 1.5-2 ml ~f 6 g%
dextran ~molecular weight 60,000-90,000, available from Sigmu Chemical ~o. St. Louis, M~ssouri, U.S.A.) was added to each tube. The tubes were mixed by inversion and allowed to settle at 25C for 1.5-2 hours. The plasma containing the buffy C08t was removed and transferred to u 7 ml plastic tissue culture tube, centrifuged at 1000 RPM for 10 minute~
and decanted. The cells were washed with phosphate buffer saline solution, (PBS) (8.7 sodium chloride in 0.05 molar potassium phosphate buffer, pH 7.3) and 10 ml of lysing solution [155 mmolar ammonium chloride, 10 mmolar potassium bicarbonate and 0.008 mmolar (ethylenedinitrilo)tetraacetic acid, disodium ~alt, pH 7.2] was added and the resulting solution was allowed to set at 25C for 5 minutes. The resulting lysed cell mixture was centrifuged, decanted and the pellet was washed with PBS solution and resuspended in 0. 5 ml PBS solution.
The white blood cells were treated with Dye Compound I identified above (23 mg) in 700 ~1 N,N-dimethylformamide and 35 ml of 0.1 molar potas-sium pho~phate buffer (pH 7) (final dye concentration of 1~2 x 10 ~ molar) and 0.18 g% (final concen-tration) of the mordant poly(styrene-co-N,N,N-tri-methyl-N-vinylbenzylammonium chloride) for 1-3 hour~. The stained cells were then dried and fixed on a glass slide with methanol and washed three times with water to remove excess dye.
The stained cell preparations were examined wlth a Zeiss fluorescence microscope against a dark background (excitation at 510-560 nm and emission a~
590 nm). Bright pink fluorescence of the white blood cells was observed ~7956 Example 6: Determin~tion of E coli With RIND I
snd ~ Mordsnt in 8 DrY Element Thi~ ex8mple i8 tsken from CQnsdian Serial No. 507,327 of Babb et ~1 (Exsmple 11), noted 8bove.
5 It 8how8 the use of the present invention to deter-mine ~ microorganism, E. coli. A dry element h8ving the following formst w~s used in thi8 exsmple.

Poly(vinyltoluene-co- 100-150 g/m2 ~--t-- butyl3tyrene--co--meth8crylic 8cid) Be~ds Poly(n-butyl ~cryl~te-co- 2-6 g/m2 Spresding/ 8tyrene- co - 2 - acrylamido-Re88ent Lsyer 2-methylprop8ne sulfonlc Acid, 80dium 881t) TRITON X-100~ 8urf~ct~nt 2-5 gJm2 Glucose 0.1-0.5 gJm2 : RIND I (of T~ble I ~bove) 0.1-0.5 g/m2 2,3,5-Trimethyl-1,4-0.8-4 g/m benzoquinone Gel8tin (h8rdened)1-lO g/m Reflection Tit~nium dioxide0 5_5 0 g/m2 L~yer ZONYL FSN~ 8urf8ct8nt 0.1-0.5 g/m2 DAXAD 30m 8urf8ctsnt 0.02-0.04 g/m2 Mord8nt/ Gel8tin (hsrdened~1-10 g/m2 Registr8tion Poly (~tyrene-co-N-benzyl-N,N-dimethyl-N~
vinylbenzyl)8mmonium chloride-co-divinyl-benzene) mordsnt0.5-5.0 g/m ; L~yer ZONYL FSN~ ~urf8ct~nt 0.1-0.5 g/m ~ ~ ~ oly(ethylene terephth~lste) /

j. jl 1 ~ 7 9 To evaluate this element, solutions of vary--ing E. coli cell concentrations in potassium phos-phate -uffer (pH 7.5) and a Control containing only buffer were prepared. These solutions were then spotted onto the element using 10 ~1 drops, and the element was incubated at 37C for up to 60 minutes.
The fluorescence was measured in a modified, commer-cially availsble fluorometer (excitation, 540 nm, emission, 620 nm) after 3 minutes and at 60 minutes.
The results, listed in Table III below, are the dif-ferences (~) in relative fluorescence at 3 and 60 minute~. They indicate that approximately 10 cells/ml can be detected using this element.
T A B L E III
E. coli ~ Relative Fluorescence Standard (Cells/ml) (57 Min., 37C) Devi~tion CV (%)*
1.0 x 10' 0.272 0.007 2.6 4.1 x 106 0.249 0.007 2.8 0 0.221 0.010 4.5 *CV = Coef~icient of Variation The invention has been described in detail with particular reference to preferred embodiments thereof, but lt will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims (24)

1. A composition comprising a rigid phen-alenone, benzphenalenone or 4-oxo-4H-benz-[d,e]an-thracene fluorescent dye or precursor thereof and a cationic or nonionic polymeric mordant.

2. The composition of claim 1 wherein said fluorescent dye is a phenalenone or benzphenalenone dye.

3. The composition of claim 1 comprising a fluorescent dye precursor of the structure wherein CAR- is a substituted or unsubstituted aroma-tic or quinone nucleus, R1 comprises a substi-tuted or unsubstituted phenalenone or benzphenalenone moiety, and n is 1 or 2, provided that when R1 is replaced with H, has an E? of either at least about +100 mV when measured in water.

4. The composition of claim 3 wherein said dye precursor has the structure CAR-R2, wherein CAR- is R1 is R2 and R4 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, R3 is R1, hydrogen, substituted or un-substituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, or R9 and R4, taken together, represent the atoms necessary to complete a substituted or unsubstituted fused carbocyclic ring, R5 is substituted or unsubstituted alkylene of 1 or 2 carbon atoms, R6 is methyl, Q is carbonyl or thiocarbonyl, FRAG is said phenalenone or benzphenalenone moiety, and m is 0 or 1.

5. The composition of claim 4 wherein FRAG

is

6. The composition of claim 1 comprising a cationic polymeric mordant.

7. The composition of claim 1 buffered at a pH of 9 or less.

8. A composition for the determination of a living organism, said composition comprising an electron transfer agent, a cationic or nonionic mordant, and a reducible fluorescent dye precursor of the structure wherein CAR- is a substituted or unsubstituted aromatic or quinone nucleus, R1 comprises a substituted or unsubstituted phenalenone or benzphenalenone moiety and n is 1 or 2, provided said precursor is capable of being reduced at physiological pH.

9. The composition of claim 8 wherein said dye precursor has the structure CAR-R1, wherein CAR- is R1 is R2 and R4 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, R3 is R1, hydrogen, substituted or un-substituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, or R3 and R4, taken together, represent the atoms necessary to complete a substituted or unsubstituted fused carbocyclic ring, R5 is substituted or unsubstituted alkylene of 1 or 2 carbon atoms, R6 is methyl, Q is carbonyl or thiocarbonyl, FRAG is said phenalenone or benzphenalenone moiety, and m is 0 or 1, provided that when R1 is replaced with H, CAR-H has an E1/2 of at least about +100 mV when measured in water.

10. The composition of claim 8 comprising a surfactant.

11. A dry analytical element for the deter-mination of an analyte comprising An absorbent car-rier material, and containing, in association, a rigid phenalenone, benzphenalenone or 4-oxo-4H-benz-[d,e]anthracene fluorescent dye or precursor thereof and a cationic or nonionic polymeric mordant.

12. The element of claim 11 wherein said fluorescent dye precursor is a reducible compound of the structure CAR?R1)n wherein CAR- is a sub-stituted or unsubstituted aromatic or quinone nucle-us, R1 comprises a substituted or unsubstituted phenalenone or benzphenalenone moiety, and n is 1 or 2.

13. The element of claim 12 wherein said dye precursor has the structure CAR-R1, wherein CAR- is R1 is R2 and R4 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, R3 is R1, hydrogen, substituted or unsub-stituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, or R3 and R4, taken together, represent the atoms necessary to com-plete a substituted or unsubstituted fused carbo-cyclic ring, R5 is substituted or unsubstituted alkylene of 1 or 2 carbon atoms, R6 is methyl, Q is carbonyl or thiocarbonyl, FRAG is said phenalenone or benzphenalenone moiety, and m is 0 or 1, provided that when R1 is replaced with H, CAR-H has an E1/2 of at least about +100 mV when measured in water.

14. The element of claim 13 wherein FRAG is a substituted or unsubstituted phenalenone moiety.

15. The element of claim 11 comprising an interactive composition for said analyte.

16. The element of claim 11 comprising an electron transfer agent.

17. The element of claim 11 comprising a support carrying said absorbent carrier material.

18. A method for the determination of an analyte, said method comprising the steps of:
A. contacting a sample of a liquid suspected of containing an analyte with a phenalenone, benzphenalenone or 4-oxo-4H-benz-[d,e]anthracene fluorescent dye precursor which releases a rigid fluorescent dye in the presence of said analyte, and a cationic or nonionic polymeric mordant, and B. determining said dye released as a result of the presence of said analyte.

19. The method of claim 18 wherein said fluorescent dye precursor is a reducible compound of the structure CAR?R1)n wherein CAR- is a substituted or unsubstituted aromatic or quinone nucleus, R1 comprises a substituted or unsubstituted phenalenone or benzphenalenone moiety, and n is 1 or 2.

20. The method of claim 19 wherein said dye precursor has the structure CAR-R1, wherein CAR- is R1 is R2 and R4 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, R3 is R1, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or an electron withdrawing group, or R3 and R4, taken together, represent the atoms necessary to complete a substituted or unsubstituted fused carbocyclic ring, R5 is substituted or unsubstituted alkylene of 1 or 2 carbon atoms, R6 is methyl, Q is carbonyl or thiocarbonyl, FRAG is said phenalenone or benzphenalenone moiety, and m is 0 or 1, provided that when R1 is replaced with H, CAR-H has an E1/2 of at least about +100 mV when measured in water.

21. The method of claim 18 for the deter-mination of a living organism.

22. The method of claim 18 wherein said contacting is carried out in the presence of an electron transfer agent.

23. The method of claim 18 for the deter-mination of a nonliving substance in the presence of an interactive composition for said analyte.

24. A method for distinguishing cells com-prising contacting a biological sample containing cells with a composition comprising a rigid phenale-none, benzphenalenone or 4-oxo-4H-benz-[d,e]anthra-cene fluorescent dye or precursor thereof and a cationic or nonionic polymeric mordant.