Biological and analytical uses of phenalenone and benzphenalenone compounds
AssigneeEastman Kodak Company
InventorsBruce E. Babb, Robert T. Belly, Albert J. Mura
Life of this patent
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Abstract
Certain phenalenone and benzphenalenone fluorescent compounds are useful in biomedical studies and analytical determinations. These compounds are particularly useful in assays for living organisms, e.g. microorganisms, carried out at a pH of 9 or less. For these determinations, the compounds can be attached to reducible compounds which will subsequently release the fluorescent moiety upon reduction. In the presence of an electron transfer agent, the reducible compounds are easily reduced by a microorganism.
Claims22
An analytical test composition buffered to a pH of 9 or less, said composition comprising a fluorescent dye selected from the group consisting of ##STR30## wherein R is hydroxy, mercapto or amino or salts thereof.
An analytical composition comprising a reducible compound of the structure CAR--R.sup.1).sub.n wherein CAR-- is an aromatic or quinone nucleus having a hydrogen atom removed to provide a valence through which R.sup.1 is attached to CAR--, R.sup.1 comprises a fluorescent moiety derived from a compound selected from the group consisting of ##STR32## wherein R is hydroxy or mercapto or salts thereof, and n is 1 or 2, said fluorescent moiety having an oxy or thio linkage, derived from said R group by removal of a hydrogen atom, through which said moiety is attached to said CAR-- nucleus,
- provided CAR--R.sup.1).sub.n is constructed so that it is capable of being reduced at a pH of 9 or less to release said derived fluorescent moiety, and
- further provided that when R.sup.1 is replaced with H, CAR--H).sub.n has an E.sub.1/2 of at least about +100 mV when measured in water.
A dry analytical element for the determination of an analyte comprising an absorbent carrier material, and containing a reducible compound of the structure CAR--R.sup.1).sub.n wherein CAR-- is an aromatic or quinone nucleus having a hydrogen atom removed to provide a valence through which R.sup.1 is attached to CAR--, R.sup.1 comprises a fluorescent moiety derived from a compound selected from the group consisting of ##STR34## wherein R is hydroxyl or mercapto or salts thereof, and n is 1 or 2, said fluorescent moiety having an oxy or thio linkage, derived from said R group by removal of a hydrogen atom, through which said moiety is attached to said CAR-- nucleus,
- provided CAR--R.sup.1).sub.n is constructed so that it is capable of being reduced at a pH of 9 or less to release said derived fluorescent moiety, and
- further provided that when R.sup.1 is replaced with H, CAR--H).sub.n has an E.sub.1/2 of at least about +100 mV when measured in water.
A dry analytical element for making a determination of living organisms comprising a support having thereon a porous spreading zone, and containing an electron transfer agent and a reducible compound of the structure ##STR36## wherein R.sup.2 and R.sup.4 are independently hydrogen, alkyl, aryl or an electron withdrawing group having a positive Hammett sigma value,
- R.sup.3 is R.sup.1, hydrogen, alkyl, aryl or an electron withdrawing group having a positive Hammett sigma value, or R.sup.3 and R.sup.4, taken together, represent the atoms necessary to complete a fused carbocyclic ring,
- R.sup.5 is alkylene of 1 or 2 carbon atoms,
- R.sup.6 is methyl,
- Q is carbonyl or thiocarbonyl,
- FRAG is a fluorescent moiety derived from a compound selected from the group consisting of ##STR37## wherein R is hydroxy or mercapto or salts thereof, and m is 0 or 1, said fluorescent moiety having an oxy or thio linkage, derived from said R group by removal of a hydrogen atom, through which said moiety is attached to said CAR-- nucleus,
- provided that when R.sup.1 is replaced with H, CAR--H has an E.sub.1/2 of at least about +100 mV when measured in water.
A method for the determination of an analyte comprising the steps of:
- A. at a pH of 9 or less, contacting a sample of a liquid suspected of containing analyte with a reducible compound of the structure CAR--R.sup.1).sub.n wherein CAR-- is an aromatic or quinone nucleus having a hydrogen atom removed to provide a valence through which R.sup.1 is attached to CAR--, R.sup.1 comprises a fluorescent moiety derived from a compound selected from the group consisting of ##STR38## wherein R is hydroxyl or mercapto or salts thereof, and n is 1 or 2, said fluorescent moiety having an oxy or thio linkage, derived from said R group by removal of a hydrogen atom, through which said moiety is attached to said CAR-- nucleus,
- provided CAR--R.sup.1).sub.n is constructed so that it is capable of being reduced at said pH to release said derived fluorescent moiety, and
- further provided that when R.sup.1 is replaced with H, CAR--H).sub.n has an E.sub.1/2 of at least about +100 mV when measured in water, and
- B. detecting said derived fluorescent moiety released from said compound upon its reduction as a result of the presence of said analyte.
A method for staining a biological specimen comprising contacting a biological specimen with a composition buffered to a pH of 9 or less, which composition comprises a fluorescent compound selected from the group consisting of ##STR41## wherein R is hydroxy, mercapto or amino or salts thereof.
A compound of the structure ##STR42## wherein R is hydroxy, mercapto or amino or salts thereof.
Dependencies are inferred from the claim text.