CA2068002A1 - Merocyanine protein error indicators - Google Patents
Merocyanine protein error indicatorsInfo
- Publication number
- CA2068002A1 CA2068002A1 CA002068002A CA2068002A CA2068002A1 CA 2068002 A1 CA2068002 A1 CA 2068002A1 CA 002068002 A CA002068002 A CA 002068002A CA 2068002 A CA2068002 A CA 2068002A CA 2068002 A1 CA2068002 A1 CA 2068002A1
- Authority
- CA
- Canada
- Prior art keywords
- protein
- integer
- merocyanine
- sample
- error indicator
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/14—Styryl dyes
- C09B23/145—Styryl dyes the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/14—Styryl dyes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/81—Tube, bottle, or dipstick
Abstract
Abstract The present invention provides a merocyanine protein error indicator compound.
Merocyanine compounds are a new class of protein error indicators, providing an analytical tool useful in the detection of protein in a sample.
Merocyanine compounds are a new class of protein error indicators, providing an analytical tool useful in the detection of protein in a sample.
Description
2~8~2 Bac~roun~ O~ The Invention A. Field Of The Invention The present invention is related generally to the detection o~ protein; and more particularly, to a novel class of protein error indicators.
B. Description Of The Backaround Art The detection of protein is important in the diagnosis of di~ease, in medical research and in industry. Several methods exist for the detection of protein in a sample. With the exception of the methodologies using protein error indicators, all of the protein detection technigues are multi- tep processes, o~ten requiring highly complex equipment and several hours to complete.
,: ~
: One technique commonly used to measure protein in a sample is the Biuret method.
~ According ~o this method, th~ ~ample is first acidi~ied to precipitate any protein in the sample. The precipitat0d protein is th n re-~ solubilized in a moderately alkaline medium and : treated with~a solution containing cupric ions.
Th~ peptide bond of the protein and tha cupric ion react to ~orm a colored chelate. The ; absorbance of the treated solution is then determined using a spectrophotometer. From this data, the amount o~ protein in the sample is estimated using calibrated æpectrophotometric ~S-1655 .
absorbance curv~s. Thi~ method g~nerally take~
form 1 to 3 hours to perfor~.
A variation of the Biuret method i8 the Lowry method. According to the Lowry method, after the precipitated protein is re-solubilized, a phosphotungstomoly~dic acid reagent i8 added to the solution under alkaline conditions to oxidize any phenolic compounds in the ~olution. Inasmuch as ~ubstantially all proteins contain ~ome phenolic compounds, e.g., tyrosine, this technique is capable of measuring protein in a sample. The absorbance of the treated solution is th~n mea~ured with a spectrophotomet~r. Using calibrated spectro2hotometer curves, th~ measured absorbance is ther~aft~r used to estimat~ the amount of protein i~ the ~ample. ~ number of bu~fers and other co~pound~, h~wever, containing : amine groups, e.g., TRIS, glycine and amide . bur~ers, interfere with the test.
Anoth~r method u~ed to d~termine the presence o~ pr~tein:in a sample is measuring ~urbidity following sa~ple acidification.
According to the me~hod, the turbidity o$ the sample i~ measured using a spectrophoto~e~er following the addition o~ a protein precipitating agent, generally ~n acidifying agent, to ~he sampl~. Thç calculated tuxbidity of the sample i8 compared t~ ~pectrophotomet~ric standard curve~ to determine the presence of protein in th~ ~ampl~. Co~on preclpitating agents used in this method include ~ul~o~alicylic acid, trichloracetic acid and tannic acid.
M~-1655 ~0~80~2 Methodologies using prstein error indicators are widely used to determine the presence of protein in a sample. Methods using protein error indicators are inexpensive, fast, simple and convenient. Phenolsulfonephthalein compounds, such as bromophenol blue, bromocresol green and coosmassie blue, are, perhaps, the mos~ widely used protsin error indicators. Methodologies using protein error indicators often involve reagent strips which are impregnated with the protein error indicator. According to these methods, the reagent test strip is contactsd with a small quantity of the sample. If protein is present in the sample, the test strip will indicate this by ~imply changing color. The color observed may vary depending on the concentration of protein in the sample. This variable color change is used to quantify the protein in the sample. Reagent strips of the above-type require a minimum of training to use correctly. These reagent test strips provide an accurate, convenient, and rapid vehicle for the on-the-spot determination of protein. Test papers such as these are widely used by technicians in industry, research and clinical laboratories.
In more detail, protein error indicators are pH indicators including an ionizable group which has a pKa value that is displaced by the presence s of protein. In the case of the phenolsulfonephthaleins, the ionizable group is a phenolic hydroxyl. The release of the proton from ~he phenolic hydroxyl causes the observable color change which is indicative sf protein in the sample being tested. Protein error ~S-1655 , ... . . . . .
.
.. .
.. . .
- - ' ~ .
.
.
2 0 ~ 2 indicators which are generally considered u~eful for the analytical determination of protein in a sample are described in United States Patent No.
4,013,416.
8ummar~ O~ Th~ Inventlon The present invention provides a merocyanine protein error indicator. Merocyanin~ protein error indicator~ are a new class of protein error indicators. Until the present invention, merocyanine dyes were unknown as protein error indicators. Merocyanine protein error indicators are useful for the detection o~ protein. The merocyanine protein errox indicators of the invention are:
OER~ ~ OH
~CH2)m- T
~; 15 Q is -Cl, -Br, or - I;
-~ m is an integer from 1 to 6;
R is 5, Se, O, or C(CnH2n 1~2~ wherein n is an int~ger from 1 ~o 6; and T is -So3e or -H. In accordance with one embodiment of the invention m is 3 or 4; ~ i C(CH3)2; and T is -SO3e.
Another and important aspect of th~ present invention provide an analytical reagent strip including a merocyanine protein error indicator for the detection of protein in liquid samples.
,~
, ~ :
~ - MS-1655 , , .
,:~
;' 2~8~2 Still another aspect of the present invention is directed to a method ~or the det~ction of protein in a liquid sample. The method comprising the step of wetting an analytical reagent strip with the liquid sample.
The test strip is composed of an absorbent carrier impregnated with at least one of the m2rocyanine protein error indicators described above. The test strip is then observed to detect any color change. A color change is indicative of protein in the liquid sample.
Bri~ De~criptioD Of The Dr~wi~
FIG. 1 is a schematic of processes for the synthesis of merocyanine protein error indicators; and FIG. 2 illustrates the dose respon~e curve to albumin o~ an analytical test strip impregnated with 0.3 mM SPDIB (1~ sul~opropyl~- -2-(4'-hydroxy-3'~-5'-diiodostyryl)-3,3-dimethylindoleninium betaine) at p~ 2.5.
De~ription of rh- Pr0~rrR~ Embodi~cntY
Th~ pr~sent invention is dixected to the discovery of a new cla s of ompounds which are use~ul as protein error indicators. In Z~ accordance with one aspect o~ the invention, it ; has bPen discovered that the merocyanine compound o~ the invention react with protein resulting in an observable color change in the merocyanine compound. In accordance with another aspect o~ the invention, it has been discov2red that analytical reagent strips ~or the determination of protein in fluids can be ~:
' '' ' ; ~ , ' ' , ~8~
obtained by impregnating an absorbent paper tes~
strip with the novel protein error indicators o~
the present invention. In accordance with a further aspect of the invention, it has been discovered that the novel protein error indicators of the present invention are useful in aqueous liquid assays. This is particularly advantageous since prior art protein error indicators, suah as bromophenol blue, require organic solvents, which can react adversely with the protein in the sample being tested. The protein error of the invention also have pXa values above 3.5. This is advantageous since a wide variety of buffers can be used in constructing a liquid or dip-stick reagent including the inventive indicators.
The below-described novel prot2in error indicators are orange, and on contact with a ; sample containing protein become strongly colored pink. The intensity of the color reflecting the concentration of protein in the sample. The pinX
color produced is clearly distinct from the orange of a negative test. The protein error indicators of the invention positively det~ct a range of from about 15 to about 500 mg/dl of protein in a sample.
With reference to the observable color change from orange to pink, tests strips prepared in accordance with the present invention are a 30 ~ diagnostic aid for the detection of protein in biological fluids by producing a different and distinct color in the presence of protein which is clearly distinguishable from the orange o~ a negative test. This i8 distinguishable from :
i~ NS-1655 ' ~'' . , , g ~ ~ ~
other test stxips which ch~nge slightly from one shade of a cslor to another in th~ pre ~nc~ o~
albumin, e.g., yellow to yellow-green. The characteristic of orange for a neg~tive test, and pink in a positiYe test is seen as a significant departure from previous methods and indicators used to detect protein in liquid samples. More specifically, the invention provides clinicians with a reliable method for detecting protein in a sample. The change from orang~ to pink simplifies the interpretation of the re~ults.
This will result in le s ~isinterpretation, and accordingly, lower costs for the user.
The merocyan~ne protein error indicators o~
the present invention are th~ compound:
,~
~ ~0~ ' ~ ~CH2)m -T
;~ wherein: Q is -Cl, -Br, or -I; m is an integer rom 1 to 6; R is S, O, Se or C~CnH~ t l)Z~ wherein n i an integer from 1 to 6; and where T is -SO3 4 or -H. More preferably, Q is -Br or -I;m is an integer ~rom 2 to 4; R is CtCH3~2; and T is -SO30. Mo~t preferably, Q ic -I; and m i8 3.
It should be underctood that the present invention describes the first use o~ the merocyanine cla~s of chromogens as pro~ein error indicators and, accordingly, encompa ~ a wide ~ariety of sub~titut~d derivatives. It will be evident that the aro~atic rings in the formula an bear a vari~y o~ ~ubstituent groups without departing from the scopc of the present !'~. invention. Such substituent groups are limited ~ MS-1655 Y .
;
:
: . ..
20~80~
only by the ability of one of ordina~y skill in the art to prepare stable compounds which have the protein error indicator properties of the present invention, and include such groups as unsubstituted and substituted alkyl, unsub~tituted and substituted aryl, alkoxy, aryloxy, halo (e.g., fluoro, chloro, bromo), nitro and substituted amino such as dialkylamino.
In the context o~ the present invention, 'lalkyl" is in`tended to include linear and branched forms of unsubstitutPd hydrocarbon residues of the general formula ~CnH2n+~, preferably of the "lower alkyl" aliphatic type wherein n is 6 or less, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso~butyl, tert-butyl, n~hexyl, and the like, as well as ~- substituted forms thereof.
Further, in the context of the present invention "aryl" is intended to includs organic residues derived from an aromatic hydrocarbon ring or ring sy~tem by removaI of a hydrogen atom, and include the unsubstituted hydrocarbon ring residues such as phenyl and naphthyl, and substituted ~orms thereo~. For purpose of the present invention, aryl residues include those bearing one or more same or different functional groups or ~ubstituents which can be selected by one skilled in the art to provide the m2rocyanine protein error indicator compounds o~ the present invention.
~ ore partiaularly, where "aryl" and "alXyl"
are substituted, such substitution is intended to include such groups or substituents when mono- or ns-l6ss , ",,, , 2 ~ 0 ~
polysubstituted with functional groups which do not substantially detract from the useful features of the present compounds. Such functional groups include chemical groups which may ~e introduced synthetically and result in the stable and useful merocyanine protein error indicator compounds of the present invention.
Examples of such functional groups include, but are not intended to be limited to, halo (e.g, fluoro, chloro, bromo~, substituted amino such as ; dialkylamino, nitro, alkoxy, aryloxy, alkyl, and aryl.
Illustrative merocyanine protein error indicators of the inventions include~
sulfopropyl)-~-(4'hydroxy-3',5'-dibromostyryl)-3,3-dimethylindoleninium betaine; 1-(~w-sulfobutylj-2~(4'-hydroxy-3',5'-diiodostyryl)-benzothiazolium betaine; 1-(w-sul~oethyl)-2-(4'-hydroxy-3',5'-diiodostyryl~-3,3-dimethylindoleninium~betaine; l-(~-sulfopropylj-2-(4i-hydro~y-3'-5'-diiodostyryl3-3,3-dimethylindoleninium~betaine; l~ sulfobutyl)-2-(4'~-hydroxy-3l,5'-diiodostyryl)-3,3-dimethylindoleninium be~aine; and 1-(n-butyl)-2-25~ (4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium iodide. Detailed protocols for preparing;the merocyanine protein error indicators listed above are set fort~ in the examples.
;, Fig. 1 generally illustrates the synthesis of several merooyanine protein indicator compounds of the presen~ invention. The chemistry is traight forward and generally involves the coupling of an aromatic ~S-1655 .., , :
:
, :
" ~ , :, hydroxyaldehyde with a heterocyclic quartenary salt under basic reaction conditions. Th merocyanine protein error indicators of the invention are water soluble. This is advantageous since these indicators can be added directly to aqueous systems to detect protein, e.g., urine, blood, serum, aq~teous gels (electrophoretic gels), aqueous solutions. The general procedures used in the preparing the merocyanine protein error indicators are illustrated in Fig. 1, and are discussed in detail in the examples bslow.
- One aspect of the presenk invention is - directed to an analytical test strip for the detection of protein in a liquid sample or gel comprising an absorbent carrier impregnated with ` one of the m~rocyanine protein error indicator compounds described above. The absorbent carri~r of the test strip is preferably a filter paper.
Other materials useful as the absorbent carrier include felt,~porous ceramic strips, and woven or matted glass fibers described in United States patent No. 3,846,247. Also suygested are the use of wood, cloth, sponge material and argillaceous substances described in United states Patent No.
3,552,92~). Alternatively, the ab~orbent carrier can be non-porous, ~uch as various polymeric films, glass and the like. All such ab~orbent carrier materials are feasible for use in the present invention, as are others. It has been found, however, that filter paper is especially suitable.
, :
-~ The absorbent strip is pre~erably ~ impregnated with a buffer. Any buffer system , ~ :
~ ~S-1655 - 11 2Q~8~
which can be adjusted to a pH of from about 1.5 to about 4.5 is useful in the practice of the present invention. Preferably, the buffer system is adjusted to a pH of from about 2.0 to abou~
B. Description Of The Backaround Art The detection of protein is important in the diagnosis of di~ease, in medical research and in industry. Several methods exist for the detection of protein in a sample. With the exception of the methodologies using protein error indicators, all of the protein detection technigues are multi- tep processes, o~ten requiring highly complex equipment and several hours to complete.
,: ~
: One technique commonly used to measure protein in a sample is the Biuret method.
~ According ~o this method, th~ ~ample is first acidi~ied to precipitate any protein in the sample. The precipitat0d protein is th n re-~ solubilized in a moderately alkaline medium and : treated with~a solution containing cupric ions.
Th~ peptide bond of the protein and tha cupric ion react to ~orm a colored chelate. The ; absorbance of the treated solution is then determined using a spectrophotometer. From this data, the amount o~ protein in the sample is estimated using calibrated æpectrophotometric ~S-1655 .
absorbance curv~s. Thi~ method g~nerally take~
form 1 to 3 hours to perfor~.
A variation of the Biuret method i8 the Lowry method. According to the Lowry method, after the precipitated protein is re-solubilized, a phosphotungstomoly~dic acid reagent i8 added to the solution under alkaline conditions to oxidize any phenolic compounds in the ~olution. Inasmuch as ~ubstantially all proteins contain ~ome phenolic compounds, e.g., tyrosine, this technique is capable of measuring protein in a sample. The absorbance of the treated solution is th~n mea~ured with a spectrophotomet~r. Using calibrated spectro2hotometer curves, th~ measured absorbance is ther~aft~r used to estimat~ the amount of protein i~ the ~ample. ~ number of bu~fers and other co~pound~, h~wever, containing : amine groups, e.g., TRIS, glycine and amide . bur~ers, interfere with the test.
Anoth~r method u~ed to d~termine the presence o~ pr~tein:in a sample is measuring ~urbidity following sa~ple acidification.
According to the me~hod, the turbidity o$ the sample i~ measured using a spectrophoto~e~er following the addition o~ a protein precipitating agent, generally ~n acidifying agent, to ~he sampl~. Thç calculated tuxbidity of the sample i8 compared t~ ~pectrophotomet~ric standard curve~ to determine the presence of protein in th~ ~ampl~. Co~on preclpitating agents used in this method include ~ul~o~alicylic acid, trichloracetic acid and tannic acid.
M~-1655 ~0~80~2 Methodologies using prstein error indicators are widely used to determine the presence of protein in a sample. Methods using protein error indicators are inexpensive, fast, simple and convenient. Phenolsulfonephthalein compounds, such as bromophenol blue, bromocresol green and coosmassie blue, are, perhaps, the mos~ widely used protsin error indicators. Methodologies using protein error indicators often involve reagent strips which are impregnated with the protein error indicator. According to these methods, the reagent test strip is contactsd with a small quantity of the sample. If protein is present in the sample, the test strip will indicate this by ~imply changing color. The color observed may vary depending on the concentration of protein in the sample. This variable color change is used to quantify the protein in the sample. Reagent strips of the above-type require a minimum of training to use correctly. These reagent test strips provide an accurate, convenient, and rapid vehicle for the on-the-spot determination of protein. Test papers such as these are widely used by technicians in industry, research and clinical laboratories.
In more detail, protein error indicators are pH indicators including an ionizable group which has a pKa value that is displaced by the presence s of protein. In the case of the phenolsulfonephthaleins, the ionizable group is a phenolic hydroxyl. The release of the proton from ~he phenolic hydroxyl causes the observable color change which is indicative sf protein in the sample being tested. Protein error ~S-1655 , ... . . . . .
.
.. .
.. . .
- - ' ~ .
.
.
2 0 ~ 2 indicators which are generally considered u~eful for the analytical determination of protein in a sample are described in United States Patent No.
4,013,416.
8ummar~ O~ Th~ Inventlon The present invention provides a merocyanine protein error indicator. Merocyanin~ protein error indicator~ are a new class of protein error indicators. Until the present invention, merocyanine dyes were unknown as protein error indicators. Merocyanine protein error indicators are useful for the detection o~ protein. The merocyanine protein errox indicators of the invention are:
OER~ ~ OH
~CH2)m- T
~; 15 Q is -Cl, -Br, or - I;
-~ m is an integer from 1 to 6;
R is 5, Se, O, or C(CnH2n 1~2~ wherein n is an int~ger from 1 ~o 6; and T is -So3e or -H. In accordance with one embodiment of the invention m is 3 or 4; ~ i C(CH3)2; and T is -SO3e.
Another and important aspect of th~ present invention provide an analytical reagent strip including a merocyanine protein error indicator for the detection of protein in liquid samples.
,~
, ~ :
~ - MS-1655 , , .
,:~
;' 2~8~2 Still another aspect of the present invention is directed to a method ~or the det~ction of protein in a liquid sample. The method comprising the step of wetting an analytical reagent strip with the liquid sample.
The test strip is composed of an absorbent carrier impregnated with at least one of the m2rocyanine protein error indicators described above. The test strip is then observed to detect any color change. A color change is indicative of protein in the liquid sample.
Bri~ De~criptioD Of The Dr~wi~
FIG. 1 is a schematic of processes for the synthesis of merocyanine protein error indicators; and FIG. 2 illustrates the dose respon~e curve to albumin o~ an analytical test strip impregnated with 0.3 mM SPDIB (1~ sul~opropyl~- -2-(4'-hydroxy-3'~-5'-diiodostyryl)-3,3-dimethylindoleninium betaine) at p~ 2.5.
De~ription of rh- Pr0~rrR~ Embodi~cntY
Th~ pr~sent invention is dixected to the discovery of a new cla s of ompounds which are use~ul as protein error indicators. In Z~ accordance with one aspect o~ the invention, it ; has bPen discovered that the merocyanine compound o~ the invention react with protein resulting in an observable color change in the merocyanine compound. In accordance with another aspect o~ the invention, it has been discov2red that analytical reagent strips ~or the determination of protein in fluids can be ~:
' '' ' ; ~ , ' ' , ~8~
obtained by impregnating an absorbent paper tes~
strip with the novel protein error indicators o~
the present invention. In accordance with a further aspect of the invention, it has been discovered that the novel protein error indicators of the present invention are useful in aqueous liquid assays. This is particularly advantageous since prior art protein error indicators, suah as bromophenol blue, require organic solvents, which can react adversely with the protein in the sample being tested. The protein error of the invention also have pXa values above 3.5. This is advantageous since a wide variety of buffers can be used in constructing a liquid or dip-stick reagent including the inventive indicators.
The below-described novel prot2in error indicators are orange, and on contact with a ; sample containing protein become strongly colored pink. The intensity of the color reflecting the concentration of protein in the sample. The pinX
color produced is clearly distinct from the orange of a negative test. The protein error indicators of the invention positively det~ct a range of from about 15 to about 500 mg/dl of protein in a sample.
With reference to the observable color change from orange to pink, tests strips prepared in accordance with the present invention are a 30 ~ diagnostic aid for the detection of protein in biological fluids by producing a different and distinct color in the presence of protein which is clearly distinguishable from the orange o~ a negative test. This i8 distinguishable from :
i~ NS-1655 ' ~'' . , , g ~ ~ ~
other test stxips which ch~nge slightly from one shade of a cslor to another in th~ pre ~nc~ o~
albumin, e.g., yellow to yellow-green. The characteristic of orange for a neg~tive test, and pink in a positiYe test is seen as a significant departure from previous methods and indicators used to detect protein in liquid samples. More specifically, the invention provides clinicians with a reliable method for detecting protein in a sample. The change from orang~ to pink simplifies the interpretation of the re~ults.
This will result in le s ~isinterpretation, and accordingly, lower costs for the user.
The merocyan~ne protein error indicators o~
the present invention are th~ compound:
,~
~ ~0~ ' ~ ~CH2)m -T
;~ wherein: Q is -Cl, -Br, or -I; m is an integer rom 1 to 6; R is S, O, Se or C~CnH~ t l)Z~ wherein n i an integer from 1 to 6; and where T is -SO3 4 or -H. More preferably, Q is -Br or -I;m is an integer ~rom 2 to 4; R is CtCH3~2; and T is -SO30. Mo~t preferably, Q ic -I; and m i8 3.
It should be underctood that the present invention describes the first use o~ the merocyanine cla~s of chromogens as pro~ein error indicators and, accordingly, encompa ~ a wide ~ariety of sub~titut~d derivatives. It will be evident that the aro~atic rings in the formula an bear a vari~y o~ ~ubstituent groups without departing from the scopc of the present !'~. invention. Such substituent groups are limited ~ MS-1655 Y .
;
:
: . ..
20~80~
only by the ability of one of ordina~y skill in the art to prepare stable compounds which have the protein error indicator properties of the present invention, and include such groups as unsubstituted and substituted alkyl, unsub~tituted and substituted aryl, alkoxy, aryloxy, halo (e.g., fluoro, chloro, bromo), nitro and substituted amino such as dialkylamino.
In the context o~ the present invention, 'lalkyl" is in`tended to include linear and branched forms of unsubstitutPd hydrocarbon residues of the general formula ~CnH2n+~, preferably of the "lower alkyl" aliphatic type wherein n is 6 or less, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso~butyl, tert-butyl, n~hexyl, and the like, as well as ~- substituted forms thereof.
Further, in the context of the present invention "aryl" is intended to includs organic residues derived from an aromatic hydrocarbon ring or ring sy~tem by removaI of a hydrogen atom, and include the unsubstituted hydrocarbon ring residues such as phenyl and naphthyl, and substituted ~orms thereo~. For purpose of the present invention, aryl residues include those bearing one or more same or different functional groups or ~ubstituents which can be selected by one skilled in the art to provide the m2rocyanine protein error indicator compounds o~ the present invention.
~ ore partiaularly, where "aryl" and "alXyl"
are substituted, such substitution is intended to include such groups or substituents when mono- or ns-l6ss , ",,, , 2 ~ 0 ~
polysubstituted with functional groups which do not substantially detract from the useful features of the present compounds. Such functional groups include chemical groups which may ~e introduced synthetically and result in the stable and useful merocyanine protein error indicator compounds of the present invention.
Examples of such functional groups include, but are not intended to be limited to, halo (e.g, fluoro, chloro, bromo~, substituted amino such as ; dialkylamino, nitro, alkoxy, aryloxy, alkyl, and aryl.
Illustrative merocyanine protein error indicators of the inventions include~
sulfopropyl)-~-(4'hydroxy-3',5'-dibromostyryl)-3,3-dimethylindoleninium betaine; 1-(~w-sulfobutylj-2~(4'-hydroxy-3',5'-diiodostyryl)-benzothiazolium betaine; 1-(w-sul~oethyl)-2-(4'-hydroxy-3',5'-diiodostyryl~-3,3-dimethylindoleninium~betaine; l-(~-sulfopropylj-2-(4i-hydro~y-3'-5'-diiodostyryl3-3,3-dimethylindoleninium~betaine; l~ sulfobutyl)-2-(4'~-hydroxy-3l,5'-diiodostyryl)-3,3-dimethylindoleninium be~aine; and 1-(n-butyl)-2-25~ (4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium iodide. Detailed protocols for preparing;the merocyanine protein error indicators listed above are set fort~ in the examples.
;, Fig. 1 generally illustrates the synthesis of several merooyanine protein indicator compounds of the presen~ invention. The chemistry is traight forward and generally involves the coupling of an aromatic ~S-1655 .., , :
:
, :
" ~ , :, hydroxyaldehyde with a heterocyclic quartenary salt under basic reaction conditions. Th merocyanine protein error indicators of the invention are water soluble. This is advantageous since these indicators can be added directly to aqueous systems to detect protein, e.g., urine, blood, serum, aq~teous gels (electrophoretic gels), aqueous solutions. The general procedures used in the preparing the merocyanine protein error indicators are illustrated in Fig. 1, and are discussed in detail in the examples bslow.
- One aspect of the presenk invention is - directed to an analytical test strip for the detection of protein in a liquid sample or gel comprising an absorbent carrier impregnated with ` one of the m~rocyanine protein error indicator compounds described above. The absorbent carri~r of the test strip is preferably a filter paper.
Other materials useful as the absorbent carrier include felt,~porous ceramic strips, and woven or matted glass fibers described in United States patent No. 3,846,247. Also suygested are the use of wood, cloth, sponge material and argillaceous substances described in United states Patent No.
3,552,92~). Alternatively, the ab~orbent carrier can be non-porous, ~uch as various polymeric films, glass and the like. All such ab~orbent carrier materials are feasible for use in the present invention, as are others. It has been found, however, that filter paper is especially suitable.
, :
-~ The absorbent strip is pre~erably ~ impregnated with a buffer. Any buffer system , ~ :
~ ~S-1655 - 11 2Q~8~
which can be adjusted to a pH of from about 1.5 to about 4.5 is useful in the practice of the present invention. Preferably, the buffer system is adjusted to a pH of from about 2.0 to abou~
4.0, and most preferably from about 3.5.
According to the method, the analytical test strip is contacted by the liquid sample or gel sample. The strip is then observed for a color change. A color change being indicative of protein in the sample.
The following Examples are presented to describe preferred embodiments and utilities of the pres~nt invention and are not meant to limit the present invention unless otherwise stated in the claims appended hereto.
EXAMPLES
Example 1. 1~ sulfopropyl)-2-(4'-hydroxv-3',5'-dibromostyryl)-3,3-dimethylindoleninium betaine A solution of 3,5~dibromo-4-hydroxybenzaldehyde (Lancaster Synthesis, Ltd., Windham, NH USA) (2.0 g, 7.14 mmole), 1~
sulfopropyl)-2,3,3-trimethylindoleninium betaine (Belg. 726,639; CA 73: P82538a) (2.0 g, 7.11 mmole) and piperidine (0.4 ml) in EtOH (30 ml~
z5~ wa maintained under an inert gas atmosphere.
The solutiun was refluxed for 50 minutes and cooled in an ice bath. The reaction mixture was evaporated to dryness in vacuo, and taken up in a ~: ~ minimum of methanol (MeOH). The solution was thereaftex chromatographed on silica gel (600 gram~) using MeOH/CHCl3 (1:4 v/v3 development.
Fractions cont ining the major purple product band were pooled and acidified with excess ~Cl in , . . .~,.. ..... ... .
2-propanol (i-PrOH~ to produc~ the color change from purple to golden yellow. The solution was evaporat~d to dryness in vacuo. The residue was taken up in hot EtOH (ca. 25 ml) and crystallized upon cooling. The solids that separated were collected by fil~ration, washed with ice-cold EtOH/h~xane (3:1 v/v), and vacuum dried to give the analytically pure compound 1-(~-sulfopropyl)-2-(4'-hydroxy-3',5'-dibromostyryl)-3,3-dimethylindolenini~m betain~ (0.96 g, 25%) as ~: goldsn yellow crystals. The compound had no :~ distinct melting point, but darXened at temperatures abova 200C. The above-described method for preparing the compound is generally illustrated as reaction A o~ Fig. 1.
~:~ Sp~ctros~opic data identifying the compound are :~ set forth below in Table 1.
able 1 IR (KBr) cm-1 3438, 3055, 1606, 1577, 20 : ~ 151g, 1475r 1406, 1372, -~
: : ~ 1305, 1277, 1212, 1173, 124, 739 NMR (DMso-d6)~ 8.58 (s, 2H), 8.29 (d, J=16.0 ~; Hz, 1~), 7.97 (d, ~=7.7 Hz, :~ 25 lH), 7.84 (d of d, J~=2.0 Hz : ~nd J2=6.6 Hz, lH), 7.67 (d, J=16 ~z, ~H~, 7.54 - 7.64 (m, : 2H), 4.81 (t, J=7.6 Hz, 2H), 3.77 (v. br. s, 1~), 2.63 (t, :~:30 ~ J=6.6 Hz, 2H), 2.10-2.22 (m, 2H), 1.76 ~s, 6H~
7C NMR(D~SO-d6)ppm 181.6, 155.6, 151.3, 143.8, ,,~ :
140.9, 135.1, 129.2, 129.1, 128.7, 123.0, 115.2, 112.5, :, -~ : M~-1655 - ' , ~
., ~ ., ~
2~8~
111.6, 52.2, 47.3, 45.5, 25.6, 24.8 (3 coincident bands) Anal. Calcd for C21XzlBrzNO4S-~EtOH: -C, 46.65; H,4.27; N,: 2.47 Found: C, 46.48; H, 4.50; N, 2.33.
-Example 2. 1-5~-sulfobutyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-benzothiazolium betaine A mixture of 3,5-diiodo-4 hydroxy-benzaldehyde (Lancaster Syntheses, Ltd., Windham, NH, U~) (3.74 g, 10 mmole), 3-(~-sulfobutyl)-2-methylbenzothiazolium betaine (Brit. 742, 112;
CA 50: P11149c3 f3.71 g, 13 mmole3 and piperidin0 (0.8 ml) in EtOH (30 ml) was maintained under an inert gas atmosphere. The solution was~refluxed for one hour then cooled to ambient temperature. ~The reaction mixture was aci~dified wikh suf~icient 1.93 M hydrochloric acid in~i-PrOH to effect a~ color change from 20~ purple to yallow~whereupon solids separated from the~solution~ The~solids~were;col~lected by filtration, washed~with~E~OH and dried. The solids~were~then~dissolved~in~warm (55C) EtOH/Me~N/H~0~(3~ v/v/v) ~(300 ml)~ containing 2 Z5 ~ M~;aqueous~sodium~hydroxlde~(5.2~ml), ~ilt~red through~Celite~(Johns-Manvill~e Corp., Denver, CO
USA) and~pre ipitated by~the~addition of 3M
agu~ous~hydrochlor~ic~acid~(6~ml3. Af~er cooling in an ice~bath, the~solids~wer:e collected by 30~ iltra~ion~,~washed with~EtOH and dried in vacuo.
The~`solids~were then boiled in acetic acid (HOAc~
(600ml)~l f~lltered~and dried in vacuo at 115C to af~rd the~analytically pure compound 1~
~ sul~obutyl)-2-(4'-hydroxy-3l,5'-diiodostyryl)-- 35 ~ benzothiazolium betaine ~5.10 g, 79%) as a yelIow ~ , , . . . .
, . . . . .
:~ ' ' ` ' ' .
:
- ~&~Q2 powder. The above-described method ~or preparing the compound is generally illustrated as reaction B of Fig. lo Spectroscopic data identifying the compound are set forth below in Table 2.
Table 2 IR (KBr) cm~l 3436, 1608, 1572, 1529, 1497, 1458, 1396, 1318, 1267, 1208, 1038 H NMR (DMSO d6)~ 8.55 (s, lH), 8.30-8.50 (m, 3H), 7.92-8.16 (m, 3H), 7.73- 7.88 (m, 2h), 4.95 (br.
t, J=7.5Hz, 2H), 2.53 (t, J=7.1 Hz, 2H), 1.98 (br. m, 2H), 1.81 Sq, J=7.0Hz, 2H) . ~
~ 13C NMR (DMSO-d6jppm 171.4, 159.1, 14801, 146.1, 141.0, 140.6, 131.5 129.2, 12~.0, 123.9,116.7, 11202, 86.4, 50.0, 48.8, 27.2, 2I.9 (2 coincident bands).
Analysis calculated for ClsH17I~NSa4:
C, 35.58; H, 2.67; N, 2.18 - Found:~ C, 35.52; ~, 2.75; N, 2.06.
~ple 3. 1- r~-sulfoethyl) -2-(4'-hydroxy-3',S'-25~ diiodostyryl)-3 3-d~ethylind~oleninium betaine A mixture of 3,5-diiodo-4-hydroxy-benzaIdehyde ~(3.~73 g, 10 mmole), 1~
ulfoethyl)-2,3,3-trimethyl-indoleninium bromids (US 2,503,776; CA 44: P5738i) (6.61 g; 19 mmole) 30 ~ ~ and piperidine (2.0mI) in EtOH/MeOH (2:1 v/v~
(60 ml) was maintained under an inert gas atmosphere. ~he ~olution was refluxed for 4 hours, cosled to an ambient temperature, and , :
''~
~, .
, .. . .. .. . .
: ' . . ' . -.: ' -, ' . . , , ' ' , . , .~ ~ , - .
2~sg~
evaporated to dryness in vacuo l~aving a brown residue. The brown residue was taken up in MeOH
(2-3 ml). This solution was treated with triethylamine (NEt3) (2 ml) and chromatographed on silica gel using MeOH/CHC13 (1:4 v/v) development.
Fractions containing the purple product band wer~
pooled and evaporated to dryness in vacuo. This crude product was taken up in EtOH (10 ml), acidified with sufficient 1.93 M HCl in i-PrOH to effect a color change from purple to yellow.
~ This solution was evaporated to dryness. The -' residue was then tak n up in EtOH/hexane ~3:1 v/~), and refrigerated until the solution - -crystallized. The crystalline solids that separated were collected by filtration. These solids wer~ washed with ice-cold EtOH and then ; EtOH/hexane. The remaining solids were vacuum dried to give the compound 1~ sulfoethyl)-2-(4'-hydroxy-3',5'-diiodostyrylj-3,3-dimethylindoleninium betaine (0.80 g; 12.8%).
Recrystallization from EtOH/HOAc afforded the analytically pure compound~as a dark reddish-ro~n powder.~ The~above-described method for preparing the~compound is generally illustrated 25 ~ ~ as reaction~C`of Fig. 1. Spectroscopic data identi~ying the aompound are set ~orth below in Table 3.
:: IR (KBr) cm-l ~ 3444, 2992, 1608, 1574, 1530, 30~ 1469, 1399, 1371, 1327, 129~, -1282, 1230, 1212, 1178, 1141, , : ~
86, 1033, 964 ', ': ~' ` :
H NMR (DMso-d6)~ 8.58 (s, 2H), 8.18 td, J=16.3 Hz, lH), 7.69-7.88 (m, 4H), 3~5 - ~ 7.51-7.62 (m, 2H), 4.82 (t, ,,, ~ . . , , , : , ~, , J=5.7 Hz, 2H), 3.04 (t, J=6.1 Hz, 2H), 1.73 (s, 6H);
3C ~MR ~DMSO-d~)ppm 182.4, 16~.0, 149.1, 143.6, 141.4, 140.7, 130.5, 128.8, 122.8, 115.1, 112.9, 87.3, 52.0, 47.7, 43.7, 2S.4 (4 coincident bands) Analysis calculat~d for C20~19I2NO~S~tOH:
C, 39~Q2; H, 3.43; N, 2.16 Found: C, 39.25; H, 3.47; N, 2.25.
Example 4. 1~ ulfopropy~ 2-(4'-hydroxy-3',-5'-diiodostYryl)-3,3-dimethYlindoleninium ~etaine ~spDIs~ -A mixture of 3,5-diiodo-4-hydroxy-: benzald~hyde :(3.73 g, 10 ~mole), l-S~-sulfopropyl)-2,3,3-trimethyli~dolenini~m betaine 3.Ç5 g, 13 mmolej and piperidine (0.8 ml~ in ~: .
EtOH~(ca. 50 ~1) was maintained under an inert ~: gas atmo~phereO The solution wa~ re~luxed for .
::~: : 2.75 .hour~ and then cooled in an ice bath. The 0 solution was acidified with 1.93 ~ HCl in i-PrOH
5.0 ml).: A dark:~ar separated and was collected by filtration and triturated with boiling HOAc.
The co~bin2d triturates were e~aporated to dryness in v~cu~ ak~n up in ~O~c (20 ml~ and ~5 allowed to crystallize. The soIids that separatsd were :collected by filtration, washed with ~OAc and vacuum dried:to give the compound ul~prspyl)-2-~4'-hydroxy-3'-5'-diiodostyryl)-3,3-dimethylindolsninium betaine (4.~7 g, 68%J as an:orange powder. The compound wa~ r~rrysta~ d from HOAr to af~ord the anal~tically pure compound. The above-descr~bed method for prBparing the co~pound i~ generally illustrated as reaction D o~ Fig. 1.
"
~' ' ' ' ' , .: .
~: ' o ~
Spectroscopic data identifying the compound are set forth below in Table 4.
Tabl0 4 IR (KBr) cm-1 1604, 1572, 1526, 1468, 1402, 1376, 1274, 1214, 1173, 766, H NMR ~DMSO-d6~ 8.71 (s, 2H), 8.21 (d, J=15.5 Hz, lH), 7.92 (d, ;~ J=7.2 Hz, lH), 7.81 (d, J-6.6 Hz, lH), 7.50-7.65 ; (m, 3H), 4.72-4.82 (v. br.
; m, 2H), 3.57 (v.br. s, lH), ;~ 2.61 (t, J=6.5 Hz, 2H), 2.07-2.20 (v. br. m, 2H) 15 ~ 1.76 (s, 6H) 3C NMR (DMSO-d6)pp~ 181.3, 160.6, 151.1, 143.7, 142.0, 140.9, 130.0, 12~.1, 2.g, 115.0, 110.7, 87.4, 52.0, 47.3,~45.4, 25.7, ~20 ~ 24.7 ~4 coincident bands) ,~,: , Analysis calculated for C21~21IzNOhS-~H2O:
C,39.02; H, 3.43; N, 2.17 ~-~ Found: ~,39.01; H, 3.46; N, 1.94.
E~a~ple 5. 1-(~-sulfobutyl~-2-(4'-hvdroxy-3'.5'-~ ~io~tyryl)-3r;3-dimethylindeleninium betaine A mixtu~e of 3,5-diiodo-4-hydroxy-benzaldehyde (1.87 g, 5 mmole), l-(~-sulfobutyl)-3,3-trimethylindoleninium betaine (R.B.
~ Mujumdar et al., tometry_~o, 11-9 (1~89)) "~ ;30~ (2.36 g, 8 mmole) and piperidine (0.4 ml) in EtOH
(35 ml) was maintained under an iner~ gas atmosphere. Thè solution was refluxed for 2.5 ~S-1655 .
,., ,, ,.
~, 18 ~0~80~
hours and then cooled to an ambient t~mperature.
The reaction mixture was acidified with an exc2ss of 1.93 M HCl in i-PrOH, and evaporated to dryness in vacuo, leaving a residue. The residue was taken up in EtOH (lOml). on standing in a refrigerator, solids separated from the mixture.
The solids were collected by filtration, washed with ice-cold EtOH/hexane (3:1 v/v), and vacuum dried to afford an or~nge solid (3.36 g). The crude product was taken up in boiling EtOH (ca.
; 30 ml) and i~mediately reprecipitated.
Additional boiling EtOH was added, (ca. 220 mL3 but the solid did not redissolve. After cooling in ice the solids were collected by filtration, washed with EtOH and vacuum dried to afford the analytically pure compound 1~ sulfobutyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium betaine (1.54 g, 47%) as an orang~ powder~ The abo~e-described method for 20~ preparing the compound is generally illustrated as reaction E of Fig. 1. Spectroscopy data identifying the compound is set forth below in Table 5.
Tabl~5 ~25 IR (KBr) cm~l ~2977, 1605, 1572, I525, -~ 1469, 1401, 1372, I308, 1271, 1214, 1182, 1120, 1034, 769, 714 H NMR (D~SO-d6)3 8.71 (s, 2H), 8.24 ~d, J=16.0 H2, lH), 7.90-7.97 (m, lH), 7.81-7.87 tm, lH), 7.~3-7.64 (m, 3H), 4.68 (t, ,:
J-7.2 Hz, 2H), 2.45-2.55 ~ ~m, 2H), 1.8g- 2.0G (m, - ~ ~5-1655 '-, . . . . .
'".;
~ , ' 2~8~
2~), 1.75 1.83 (m, 2H), 1.76 (s, 6H) 3C NMR (DMSO-d6)ppm 181.3, 160.5, 151.1, 143.7, 1~1.9, 140.8, 130.0, 129.0, 122.9, 115.2, 110.7, 87.3, 52.0, 50.3, 46.2, 27.2, 25.8, 22.3 (4 coincident band~) ~nalysis calculated for C22~I23I2NOjS
1~ C, 40.57; ~, 3.~6; N, 2.15 Found: C, 40.59; H, 3.50; N,1.99.
,:
- Example 6. l~ln-b~tYl)-2-L~ -hydroxv-3 ~ 5'-diiodostyryl~3 3-dimethvlindolenini~m iodide, A mix*ure of 3,5-diiodo-4-hydroxy-~; 15 benzaldehyde (3.73, 10 mmole), 1-(n-butyl)-2,3,3-trimethylindoleninium iodide ~D.P. Maisuradze e$
al., Soobschch. ~kad. Nauk Gru~ SSR 50, 77-82 : ~ :
~- (1968); CA 69: 106526r) (4.46 g, I3 mmole) and ?
piperidine (0.~8 ml)~ in EtOH ~40 ml) was 20~ maintained under~an inert-gas atmosphsre. The solution was re~luxed~for l hour, and cooled to n ambient temperature. The solu~ion w s evaporated t~dryness ~n~v~uo, leaving a residue. ~The~residue was taken up in EtOH (10 25 ~ ml) and treated with 1.93 M HCl in i-PrOH (3.9 ~1). The solution was therea*ter again eYaporated to dryness~1n vacuo, leaving a residue. The residue was taken up in ~tOH (4ml).
The ~olution~was~re~rigerat-d and crystals 3;0 spontaneously ~ormedO The crystalline solids that separated were collected by ~iltration, washed with ice-cold EtOH and YacuUm dried to ; give crude l-(n-Butyl3-2-~4'-hydroxy-3',5~-dilodostyryl)-3,3-dimethylindoleninium iodide :
, ~ , , 20~8~G'~
(4.90 g, 80.7~). The crude compound was taken up ~ in hot EtO~ (60 ml), filtered thrsugh pap~r and : concentrated in vacuo to about 30 ml. The solution was allowed to crystallize. The crystalline solids that separated were collected, washed and dried as above to afford the analytically pure compound l-(n-butyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium iodide (3.90 g, 56%) as a bright orange powd~r. The above-described m~thod for preparing the compound is g~nerally illustrated as reaction F of Fig. 1.
Spectroscopic data identifying the compound is set forth below in Table 6.
:
~bl~ 6 IR (KBr) cm~l 3361, 2979, 1605, 1574, : 1530, 1463, 1402, 1372, 1320, ~250, 1213, 1198, 1H N~R (DMSO d6)~ 8.63 (s, 2H), 8.23 ~d, J=15.9 Hz, lH), 7.33-7.87 (m, 6H), 4.65 (t, ~=7.0, 2H), 1.73-1.85 (m, 2H), ~ 1.76 (s, 6H~, 1.34-1.48 : 25 (m, 2H), 0.93 (t, J=7.3 Hzj 3H~
3C NMR ~DMSO-d6~ppm 181.2, 160.7, 150.9, 143.7, 141.7, 140.7, 129.8, 129.0, 123.0, 115.0, 110.4, 87.7, : 30 52.~, 46.1, 30.4, 25.~, 19.2, 13.7 (~ coincident : bands) '';, ~
"
" ~ , ' .
' ~B~
Analysis calculated for C2~H24I3NO~tOH:
C, 38.62; H, 4.06; N, 1.89 : Found: C, 38.55;~,3.96;N,1.91.
Example 7. Performance In Assay For Human Serum Albumin The utility of the merocyanine protein error indicator compounds of the present invention in a liquid assay for the d~termination of protein levels in a liquid test sample is illustrated in Table 7 b~low. A solution of the compounds, in 200 mM - 250 mM Na~ or K~ citrate buffer at a pH
:~ at least 0.5 unit below the pK~ of the compound, was prepared and its absorbance measured at the listed wav~length below. The solution was then treated with:sufficient human serum albumin to make the albumin con~entration 100 mg/dl and the absorbance was measured again. The reported increase in ab~orbance (~ abs) is proportional to the amount of:albumln present, and is indicative ~:~ 20 of the rPlative sensitivity of the dye for measuring pro~ein.
B~ 7 : C9MPD. COMPD. COMPD. ASSAY AS5AY ~ABS
:~ No. pKa. CONC.(M) pH ~ A:~n~) 3.59 l.OXIO-5 3.0: 5X2 0.156 B 4.~5 2.5Xl0-5 3.5 506 0.027 C 3.65 1.5Xl~-5 3.~ 543 0.255 : D 3.65 2.0~10-5 3.0 541 0.40a 3.52 1.8X10-5 3.0 535 0.322 :
`~ M~-1655 ~ , , , 22 206~2 Compound A is 1~ sulfopropyl)-2-(4'hydroxy-3',5'-dibromostyryl)-3,3-dimethylindoleninium ~staine Compound B is ~ -sulfsbutyl)-2-(4l-hydroxy-3',5'-diiodostyryl)-benzothiazolium betaine Compound C is 1-(~-sulfoethyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium betaine Compound D 1-(~-sulfopropyl)-2-(4'-hydro~y-3'-5'-diiodostyryl)-3,3-dimethylindoleninium betaine Compound E is 1-(~-sulfobutyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium betaine.
Example 8. ~eaqent Strip PreParation one method for the preparation of the analytical protein reagent strips discussed herein is shown below. The method described is a continuous method for mass producing protein reagent test strips.
According to the method, a thin absorbent strip of paper is moved through the line at a : preferred speed of about four feet per minute.
One preferred paper being E & D 237 ~Ahlstrom Filtration, Inc., Mount Holley Springs, PA, U.SO~.). The paper is dipped into a buffered bath, pH 2.5, including the merocyanine protein error indicator SPDIB
dissolved in ethan~l. According to one preferred method, the bath contains from about O.03 to about 0.08~M SPDIB, a 0.5 M potassium :~ ~ citrate buf~er pH 2.5, and 20% ethanol. The test strip is then passed through a dryer having an air pressure of one inch of water and a te~perature of 60C at a speed of four feet : per minute. The test strips are then cut and packaged.
2 ~ 0 ~
Example 9. The Dose Response of Reagent Strips Includina SPDIB
A buffered liquid sample (50 mM potassium citrate, pH 2.5) which was shown by immunoassay to be devoid of albumin, was spiked to various clinically significant l~vels with Pen~ex~
human sçrum albumin (HSA~ (Miles, Inc., ~; Elkhart, Indiana). Using a Clinitek ~00 Instrument (Miles, Inc., Elkhart, Indiana~
protein measurements were made using analytical test s~rips including 0.3 mM SPDIB.
- Resolution was~quantitatively expressed in delta K/S between albumin levels, as shown Fig.
2. K/S are calculated from~the formula:
R)2 K/S =
2~
wherein R is the fraction~of~reflectance from the test devioe,~K is~a aonstant, and S is the light sca~tering~coef~icient of the particular reflecting medium. ~The above equation is a simplified form of~thé well-known~Kubelka-Mu~k equation tSee~Gustav Kortum, "Reflectance Spe~ctroscopy,'l pp.~106~ springer Verlas, New York (1969). K/S was determined at 25 seconds.
; ~ , ~: : ~ : :
The reagent strip de cribed above were analyzed for their response to the different levels of protein. The results of this study are summarized as Fig. 2. As shown in Fig. 2, ths strip of the invention was sensitive to a wide range of protein concentrations.
:", ~ ~
~ MS-1655 , , .~ ' : ' ' 24 20~8~
Furthermore, a significant resolution between protein levels was seen with the invention.
While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example and were herein described in detail. It should be understood, however, that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the` intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
i ~ :
:~:
, , :
, ~:
'', :' : ~ :
', ~
' - -, ,~. - `
:
, ,~ . - . .
,
According to the method, the analytical test strip is contacted by the liquid sample or gel sample. The strip is then observed for a color change. A color change being indicative of protein in the sample.
The following Examples are presented to describe preferred embodiments and utilities of the pres~nt invention and are not meant to limit the present invention unless otherwise stated in the claims appended hereto.
EXAMPLES
Example 1. 1~ sulfopropyl)-2-(4'-hydroxv-3',5'-dibromostyryl)-3,3-dimethylindoleninium betaine A solution of 3,5~dibromo-4-hydroxybenzaldehyde (Lancaster Synthesis, Ltd., Windham, NH USA) (2.0 g, 7.14 mmole), 1~
sulfopropyl)-2,3,3-trimethylindoleninium betaine (Belg. 726,639; CA 73: P82538a) (2.0 g, 7.11 mmole) and piperidine (0.4 ml) in EtOH (30 ml~
z5~ wa maintained under an inert gas atmosphere.
The solutiun was refluxed for 50 minutes and cooled in an ice bath. The reaction mixture was evaporated to dryness in vacuo, and taken up in a ~: ~ minimum of methanol (MeOH). The solution was thereaftex chromatographed on silica gel (600 gram~) using MeOH/CHCl3 (1:4 v/v3 development.
Fractions cont ining the major purple product band were pooled and acidified with excess ~Cl in , . . .~,.. ..... ... .
2-propanol (i-PrOH~ to produc~ the color change from purple to golden yellow. The solution was evaporat~d to dryness in vacuo. The residue was taken up in hot EtOH (ca. 25 ml) and crystallized upon cooling. The solids that separated were collected by fil~ration, washed with ice-cold EtOH/h~xane (3:1 v/v), and vacuum dried to give the analytically pure compound 1-(~-sulfopropyl)-2-(4'-hydroxy-3',5'-dibromostyryl)-3,3-dimethylindolenini~m betain~ (0.96 g, 25%) as ~: goldsn yellow crystals. The compound had no :~ distinct melting point, but darXened at temperatures abova 200C. The above-described method for preparing the compound is generally illustrated as reaction A o~ Fig. 1.
~:~ Sp~ctros~opic data identifying the compound are :~ set forth below in Table 1.
able 1 IR (KBr) cm-1 3438, 3055, 1606, 1577, 20 : ~ 151g, 1475r 1406, 1372, -~
: : ~ 1305, 1277, 1212, 1173, 124, 739 NMR (DMso-d6)~ 8.58 (s, 2H), 8.29 (d, J=16.0 ~; Hz, 1~), 7.97 (d, ~=7.7 Hz, :~ 25 lH), 7.84 (d of d, J~=2.0 Hz : ~nd J2=6.6 Hz, lH), 7.67 (d, J=16 ~z, ~H~, 7.54 - 7.64 (m, : 2H), 4.81 (t, J=7.6 Hz, 2H), 3.77 (v. br. s, 1~), 2.63 (t, :~:30 ~ J=6.6 Hz, 2H), 2.10-2.22 (m, 2H), 1.76 ~s, 6H~
7C NMR(D~SO-d6)ppm 181.6, 155.6, 151.3, 143.8, ,,~ :
140.9, 135.1, 129.2, 129.1, 128.7, 123.0, 115.2, 112.5, :, -~ : M~-1655 - ' , ~
., ~ ., ~
2~8~
111.6, 52.2, 47.3, 45.5, 25.6, 24.8 (3 coincident bands) Anal. Calcd for C21XzlBrzNO4S-~EtOH: -C, 46.65; H,4.27; N,: 2.47 Found: C, 46.48; H, 4.50; N, 2.33.
-Example 2. 1-5~-sulfobutyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-benzothiazolium betaine A mixture of 3,5-diiodo-4 hydroxy-benzaldehyde (Lancaster Syntheses, Ltd., Windham, NH, U~) (3.74 g, 10 mmole), 3-(~-sulfobutyl)-2-methylbenzothiazolium betaine (Brit. 742, 112;
CA 50: P11149c3 f3.71 g, 13 mmole3 and piperidin0 (0.8 ml) in EtOH (30 ml) was maintained under an inert gas atmosphere. The solution was~refluxed for one hour then cooled to ambient temperature. ~The reaction mixture was aci~dified wikh suf~icient 1.93 M hydrochloric acid in~i-PrOH to effect a~ color change from 20~ purple to yallow~whereupon solids separated from the~solution~ The~solids~were;col~lected by filtration, washed~with~E~OH and dried. The solids~were~then~dissolved~in~warm (55C) EtOH/Me~N/H~0~(3~ v/v/v) ~(300 ml)~ containing 2 Z5 ~ M~;aqueous~sodium~hydroxlde~(5.2~ml), ~ilt~red through~Celite~(Johns-Manvill~e Corp., Denver, CO
USA) and~pre ipitated by~the~addition of 3M
agu~ous~hydrochlor~ic~acid~(6~ml3. Af~er cooling in an ice~bath, the~solids~wer:e collected by 30~ iltra~ion~,~washed with~EtOH and dried in vacuo.
The~`solids~were then boiled in acetic acid (HOAc~
(600ml)~l f~lltered~and dried in vacuo at 115C to af~rd the~analytically pure compound 1~
~ sul~obutyl)-2-(4'-hydroxy-3l,5'-diiodostyryl)-- 35 ~ benzothiazolium betaine ~5.10 g, 79%) as a yelIow ~ , , . . . .
, . . . . .
:~ ' ' ` ' ' .
:
- ~&~Q2 powder. The above-described method ~or preparing the compound is generally illustrated as reaction B of Fig. lo Spectroscopic data identifying the compound are set forth below in Table 2.
Table 2 IR (KBr) cm~l 3436, 1608, 1572, 1529, 1497, 1458, 1396, 1318, 1267, 1208, 1038 H NMR (DMSO d6)~ 8.55 (s, lH), 8.30-8.50 (m, 3H), 7.92-8.16 (m, 3H), 7.73- 7.88 (m, 2h), 4.95 (br.
t, J=7.5Hz, 2H), 2.53 (t, J=7.1 Hz, 2H), 1.98 (br. m, 2H), 1.81 Sq, J=7.0Hz, 2H) . ~
~ 13C NMR (DMSO-d6jppm 171.4, 159.1, 14801, 146.1, 141.0, 140.6, 131.5 129.2, 12~.0, 123.9,116.7, 11202, 86.4, 50.0, 48.8, 27.2, 2I.9 (2 coincident bands).
Analysis calculated for ClsH17I~NSa4:
C, 35.58; H, 2.67; N, 2.18 - Found:~ C, 35.52; ~, 2.75; N, 2.06.
~ple 3. 1- r~-sulfoethyl) -2-(4'-hydroxy-3',S'-25~ diiodostyryl)-3 3-d~ethylind~oleninium betaine A mixture of 3,5-diiodo-4-hydroxy-benzaIdehyde ~(3.~73 g, 10 mmole), 1~
ulfoethyl)-2,3,3-trimethyl-indoleninium bromids (US 2,503,776; CA 44: P5738i) (6.61 g; 19 mmole) 30 ~ ~ and piperidine (2.0mI) in EtOH/MeOH (2:1 v/v~
(60 ml) was maintained under an inert gas atmosphere. ~he ~olution was refluxed for 4 hours, cosled to an ambient temperature, and , :
''~
~, .
, .. . .. .. . .
: ' . . ' . -.: ' -, ' . . , , ' ' , . , .~ ~ , - .
2~sg~
evaporated to dryness in vacuo l~aving a brown residue. The brown residue was taken up in MeOH
(2-3 ml). This solution was treated with triethylamine (NEt3) (2 ml) and chromatographed on silica gel using MeOH/CHC13 (1:4 v/v) development.
Fractions containing the purple product band wer~
pooled and evaporated to dryness in vacuo. This crude product was taken up in EtOH (10 ml), acidified with sufficient 1.93 M HCl in i-PrOH to effect a color change from purple to yellow.
~ This solution was evaporated to dryness. The -' residue was then tak n up in EtOH/hexane ~3:1 v/~), and refrigerated until the solution - -crystallized. The crystalline solids that separated were collected by filtration. These solids wer~ washed with ice-cold EtOH and then ; EtOH/hexane. The remaining solids were vacuum dried to give the compound 1~ sulfoethyl)-2-(4'-hydroxy-3',5'-diiodostyrylj-3,3-dimethylindoleninium betaine (0.80 g; 12.8%).
Recrystallization from EtOH/HOAc afforded the analytically pure compound~as a dark reddish-ro~n powder.~ The~above-described method for preparing the~compound is generally illustrated 25 ~ ~ as reaction~C`of Fig. 1. Spectroscopic data identi~ying the aompound are set ~orth below in Table 3.
:: IR (KBr) cm-l ~ 3444, 2992, 1608, 1574, 1530, 30~ 1469, 1399, 1371, 1327, 129~, -1282, 1230, 1212, 1178, 1141, , : ~
86, 1033, 964 ', ': ~' ` :
H NMR (DMso-d6)~ 8.58 (s, 2H), 8.18 td, J=16.3 Hz, lH), 7.69-7.88 (m, 4H), 3~5 - ~ 7.51-7.62 (m, 2H), 4.82 (t, ,,, ~ . . , , , : , ~, , J=5.7 Hz, 2H), 3.04 (t, J=6.1 Hz, 2H), 1.73 (s, 6H);
3C ~MR ~DMSO-d~)ppm 182.4, 16~.0, 149.1, 143.6, 141.4, 140.7, 130.5, 128.8, 122.8, 115.1, 112.9, 87.3, 52.0, 47.7, 43.7, 2S.4 (4 coincident bands) Analysis calculat~d for C20~19I2NO~S~tOH:
C, 39~Q2; H, 3.43; N, 2.16 Found: C, 39.25; H, 3.47; N, 2.25.
Example 4. 1~ ulfopropy~ 2-(4'-hydroxy-3',-5'-diiodostYryl)-3,3-dimethYlindoleninium ~etaine ~spDIs~ -A mixture of 3,5-diiodo-4-hydroxy-: benzald~hyde :(3.73 g, 10 ~mole), l-S~-sulfopropyl)-2,3,3-trimethyli~dolenini~m betaine 3.Ç5 g, 13 mmolej and piperidine (0.8 ml~ in ~: .
EtOH~(ca. 50 ~1) was maintained under an inert ~: gas atmo~phereO The solution wa~ re~luxed for .
::~: : 2.75 .hour~ and then cooled in an ice bath. The 0 solution was acidified with 1.93 ~ HCl in i-PrOH
5.0 ml).: A dark:~ar separated and was collected by filtration and triturated with boiling HOAc.
The co~bin2d triturates were e~aporated to dryness in v~cu~ ak~n up in ~O~c (20 ml~ and ~5 allowed to crystallize. The soIids that separatsd were :collected by filtration, washed with ~OAc and vacuum dried:to give the compound ul~prspyl)-2-~4'-hydroxy-3'-5'-diiodostyryl)-3,3-dimethylindolsninium betaine (4.~7 g, 68%J as an:orange powder. The compound wa~ r~rrysta~ d from HOAr to af~ord the anal~tically pure compound. The above-descr~bed method for prBparing the co~pound i~ generally illustrated as reaction D o~ Fig. 1.
"
~' ' ' ' ' , .: .
~: ' o ~
Spectroscopic data identifying the compound are set forth below in Table 4.
Tabl0 4 IR (KBr) cm-1 1604, 1572, 1526, 1468, 1402, 1376, 1274, 1214, 1173, 766, H NMR ~DMSO-d6~ 8.71 (s, 2H), 8.21 (d, J=15.5 Hz, lH), 7.92 (d, ;~ J=7.2 Hz, lH), 7.81 (d, J-6.6 Hz, lH), 7.50-7.65 ; (m, 3H), 4.72-4.82 (v. br.
; m, 2H), 3.57 (v.br. s, lH), ;~ 2.61 (t, J=6.5 Hz, 2H), 2.07-2.20 (v. br. m, 2H) 15 ~ 1.76 (s, 6H) 3C NMR (DMSO-d6)pp~ 181.3, 160.6, 151.1, 143.7, 142.0, 140.9, 130.0, 12~.1, 2.g, 115.0, 110.7, 87.4, 52.0, 47.3,~45.4, 25.7, ~20 ~ 24.7 ~4 coincident bands) ,~,: , Analysis calculated for C21~21IzNOhS-~H2O:
C,39.02; H, 3.43; N, 2.17 ~-~ Found: ~,39.01; H, 3.46; N, 1.94.
E~a~ple 5. 1-(~-sulfobutyl~-2-(4'-hvdroxy-3'.5'-~ ~io~tyryl)-3r;3-dimethylindeleninium betaine A mixtu~e of 3,5-diiodo-4-hydroxy-benzaldehyde (1.87 g, 5 mmole), l-(~-sulfobutyl)-3,3-trimethylindoleninium betaine (R.B.
~ Mujumdar et al., tometry_~o, 11-9 (1~89)) "~ ;30~ (2.36 g, 8 mmole) and piperidine (0.4 ml) in EtOH
(35 ml) was maintained under an iner~ gas atmosphere. Thè solution was refluxed for 2.5 ~S-1655 .
,., ,, ,.
~, 18 ~0~80~
hours and then cooled to an ambient t~mperature.
The reaction mixture was acidified with an exc2ss of 1.93 M HCl in i-PrOH, and evaporated to dryness in vacuo, leaving a residue. The residue was taken up in EtOH (lOml). on standing in a refrigerator, solids separated from the mixture.
The solids were collected by filtration, washed with ice-cold EtOH/hexane (3:1 v/v), and vacuum dried to afford an or~nge solid (3.36 g). The crude product was taken up in boiling EtOH (ca.
; 30 ml) and i~mediately reprecipitated.
Additional boiling EtOH was added, (ca. 220 mL3 but the solid did not redissolve. After cooling in ice the solids were collected by filtration, washed with EtOH and vacuum dried to afford the analytically pure compound 1~ sulfobutyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium betaine (1.54 g, 47%) as an orang~ powder~ The abo~e-described method for 20~ preparing the compound is generally illustrated as reaction E of Fig. 1. Spectroscopy data identifying the compound is set forth below in Table 5.
Tabl~5 ~25 IR (KBr) cm~l ~2977, 1605, 1572, I525, -~ 1469, 1401, 1372, I308, 1271, 1214, 1182, 1120, 1034, 769, 714 H NMR (D~SO-d6)3 8.71 (s, 2H), 8.24 ~d, J=16.0 H2, lH), 7.90-7.97 (m, lH), 7.81-7.87 tm, lH), 7.~3-7.64 (m, 3H), 4.68 (t, ,:
J-7.2 Hz, 2H), 2.45-2.55 ~ ~m, 2H), 1.8g- 2.0G (m, - ~ ~5-1655 '-, . . . . .
'".;
~ , ' 2~8~
2~), 1.75 1.83 (m, 2H), 1.76 (s, 6H) 3C NMR (DMSO-d6)ppm 181.3, 160.5, 151.1, 143.7, 1~1.9, 140.8, 130.0, 129.0, 122.9, 115.2, 110.7, 87.3, 52.0, 50.3, 46.2, 27.2, 25.8, 22.3 (4 coincident band~) ~nalysis calculated for C22~I23I2NOjS
1~ C, 40.57; ~, 3.~6; N, 2.15 Found: C, 40.59; H, 3.50; N,1.99.
,:
- Example 6. l~ln-b~tYl)-2-L~ -hydroxv-3 ~ 5'-diiodostyryl~3 3-dimethvlindolenini~m iodide, A mix*ure of 3,5-diiodo-4-hydroxy-~; 15 benzaldehyde (3.73, 10 mmole), 1-(n-butyl)-2,3,3-trimethylindoleninium iodide ~D.P. Maisuradze e$
al., Soobschch. ~kad. Nauk Gru~ SSR 50, 77-82 : ~ :
~- (1968); CA 69: 106526r) (4.46 g, I3 mmole) and ?
piperidine (0.~8 ml)~ in EtOH ~40 ml) was 20~ maintained under~an inert-gas atmosphsre. The solution was re~luxed~for l hour, and cooled to n ambient temperature. The solu~ion w s evaporated t~dryness ~n~v~uo, leaving a residue. ~The~residue was taken up in EtOH (10 25 ~ ml) and treated with 1.93 M HCl in i-PrOH (3.9 ~1). The solution was therea*ter again eYaporated to dryness~1n vacuo, leaving a residue. The residue was taken up in ~tOH (4ml).
The ~olution~was~re~rigerat-d and crystals 3;0 spontaneously ~ormedO The crystalline solids that separated were collected by ~iltration, washed with ice-cold EtOH and YacuUm dried to ; give crude l-(n-Butyl3-2-~4'-hydroxy-3',5~-dilodostyryl)-3,3-dimethylindoleninium iodide :
, ~ , , 20~8~G'~
(4.90 g, 80.7~). The crude compound was taken up ~ in hot EtO~ (60 ml), filtered thrsugh pap~r and : concentrated in vacuo to about 30 ml. The solution was allowed to crystallize. The crystalline solids that separated were collected, washed and dried as above to afford the analytically pure compound l-(n-butyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium iodide (3.90 g, 56%) as a bright orange powd~r. The above-described m~thod for preparing the compound is g~nerally illustrated as reaction F of Fig. 1.
Spectroscopic data identifying the compound is set forth below in Table 6.
:
~bl~ 6 IR (KBr) cm~l 3361, 2979, 1605, 1574, : 1530, 1463, 1402, 1372, 1320, ~250, 1213, 1198, 1H N~R (DMSO d6)~ 8.63 (s, 2H), 8.23 ~d, J=15.9 Hz, lH), 7.33-7.87 (m, 6H), 4.65 (t, ~=7.0, 2H), 1.73-1.85 (m, 2H), ~ 1.76 (s, 6H~, 1.34-1.48 : 25 (m, 2H), 0.93 (t, J=7.3 Hzj 3H~
3C NMR ~DMSO-d6~ppm 181.2, 160.7, 150.9, 143.7, 141.7, 140.7, 129.8, 129.0, 123.0, 115.0, 110.4, 87.7, : 30 52.~, 46.1, 30.4, 25.~, 19.2, 13.7 (~ coincident : bands) '';, ~
"
" ~ , ' .
' ~B~
Analysis calculated for C2~H24I3NO~tOH:
C, 38.62; H, 4.06; N, 1.89 : Found: C, 38.55;~,3.96;N,1.91.
Example 7. Performance In Assay For Human Serum Albumin The utility of the merocyanine protein error indicator compounds of the present invention in a liquid assay for the d~termination of protein levels in a liquid test sample is illustrated in Table 7 b~low. A solution of the compounds, in 200 mM - 250 mM Na~ or K~ citrate buffer at a pH
:~ at least 0.5 unit below the pK~ of the compound, was prepared and its absorbance measured at the listed wav~length below. The solution was then treated with:sufficient human serum albumin to make the albumin con~entration 100 mg/dl and the absorbance was measured again. The reported increase in ab~orbance (~ abs) is proportional to the amount of:albumln present, and is indicative ~:~ 20 of the rPlative sensitivity of the dye for measuring pro~ein.
B~ 7 : C9MPD. COMPD. COMPD. ASSAY AS5AY ~ABS
:~ No. pKa. CONC.(M) pH ~ A:~n~) 3.59 l.OXIO-5 3.0: 5X2 0.156 B 4.~5 2.5Xl0-5 3.5 506 0.027 C 3.65 1.5Xl~-5 3.~ 543 0.255 : D 3.65 2.0~10-5 3.0 541 0.40a 3.52 1.8X10-5 3.0 535 0.322 :
`~ M~-1655 ~ , , , 22 206~2 Compound A is 1~ sulfopropyl)-2-(4'hydroxy-3',5'-dibromostyryl)-3,3-dimethylindoleninium ~staine Compound B is ~ -sulfsbutyl)-2-(4l-hydroxy-3',5'-diiodostyryl)-benzothiazolium betaine Compound C is 1-(~-sulfoethyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium betaine Compound D 1-(~-sulfopropyl)-2-(4'-hydro~y-3'-5'-diiodostyryl)-3,3-dimethylindoleninium betaine Compound E is 1-(~-sulfobutyl)-2-(4'-hydroxy-3',5'-diiodostyryl)-3,3-dimethylindoleninium betaine.
Example 8. ~eaqent Strip PreParation one method for the preparation of the analytical protein reagent strips discussed herein is shown below. The method described is a continuous method for mass producing protein reagent test strips.
According to the method, a thin absorbent strip of paper is moved through the line at a : preferred speed of about four feet per minute.
One preferred paper being E & D 237 ~Ahlstrom Filtration, Inc., Mount Holley Springs, PA, U.SO~.). The paper is dipped into a buffered bath, pH 2.5, including the merocyanine protein error indicator SPDIB
dissolved in ethan~l. According to one preferred method, the bath contains from about O.03 to about 0.08~M SPDIB, a 0.5 M potassium :~ ~ citrate buf~er pH 2.5, and 20% ethanol. The test strip is then passed through a dryer having an air pressure of one inch of water and a te~perature of 60C at a speed of four feet : per minute. The test strips are then cut and packaged.
2 ~ 0 ~
Example 9. The Dose Response of Reagent Strips Includina SPDIB
A buffered liquid sample (50 mM potassium citrate, pH 2.5) which was shown by immunoassay to be devoid of albumin, was spiked to various clinically significant l~vels with Pen~ex~
human sçrum albumin (HSA~ (Miles, Inc., ~; Elkhart, Indiana). Using a Clinitek ~00 Instrument (Miles, Inc., Elkhart, Indiana~
protein measurements were made using analytical test s~rips including 0.3 mM SPDIB.
- Resolution was~quantitatively expressed in delta K/S between albumin levels, as shown Fig.
2. K/S are calculated from~the formula:
R)2 K/S =
2~
wherein R is the fraction~of~reflectance from the test devioe,~K is~a aonstant, and S is the light sca~tering~coef~icient of the particular reflecting medium. ~The above equation is a simplified form of~thé well-known~Kubelka-Mu~k equation tSee~Gustav Kortum, "Reflectance Spe~ctroscopy,'l pp.~106~ springer Verlas, New York (1969). K/S was determined at 25 seconds.
; ~ , ~: : ~ : :
The reagent strip de cribed above were analyzed for their response to the different levels of protein. The results of this study are summarized as Fig. 2. As shown in Fig. 2, ths strip of the invention was sensitive to a wide range of protein concentrations.
:", ~ ~
~ MS-1655 , , .~ ' : ' ' 24 20~8~
Furthermore, a significant resolution between protein levels was seen with the invention.
While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example and were herein described in detail. It should be understood, however, that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the` intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
i ~ :
:~:
, , :
, ~:
'', :' : ~ :
', ~
' - -, ,~. - `
:
, ,~ . - . .
,
Claims (20)
1. A merocyanine protein error indicator compound:
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3e or -H.
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3e or -H.
2. The compound of claim 1 wherein m is ths integer 3 or 4; Q is -Br or -I; R is C(CnH2n + 1)2, wherein n i5 an integer from 1 to 3; and T
is -SO3e.
is -SO3e.
3. The compound of claim 1 wherein m is the integer 3; and R is C(CH3)2.
4. The merocyanine protein error indicator compound 1-(.omega.-sulfopropyl)-2-(4'-hydroxy-3'-5'-diiodostyryl)-3,3-dimethylindoleninium betaine.
5. An analytical test strip for the detection of protein in a liquid sample comprising an absorbent carrier impregnated with a merocyanine protein error indicator.
6. An analytical test strip for the detection of protein in a liquid sample comprising an absorbent carrier impregnated with the merocyanine protein error indicator compound:
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3.THETA. or -H.
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3.THETA. or -H.
7. The analytical test strip of claim 6 wherein m is the integer 3 or 4; Q is -Br or -I; R is C(CnH2n + 1)2, wherein n is an integer from 1 to 3; and T is -SO3.THETA..
8. The analytical test strip of claim 6 wherein m is the integer 3; and R is C(CH3)2.
9. A method for the detection of protein in a liquid sample, the method comprising the steps of:
a) wetting an analytical test strip with the liquid sample, the test strip including an absorbent carrier impregnated with the merocyanine protein error indicator compound:
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3.THETA. or -H; and b) observing and recording any color change of the test strip, wherein a color change is indicative of protein in the liquid sample.
a) wetting an analytical test strip with the liquid sample, the test strip including an absorbent carrier impregnated with the merocyanine protein error indicator compound:
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3.THETA. or -H; and b) observing and recording any color change of the test strip, wherein a color change is indicative of protein in the liquid sample.
10. The method of claim 9 wherein the merocyanine protein error indicator compound is further defined as m is the integer 3 or 4; Q
is -Br or -I, R is C(CnH2n + 1)2, wherein n is an integer from 1 to 3; and T is -SO3.THETA..
is -Br or -I, R is C(CnH2n + 1)2, wherein n is an integer from 1 to 3; and T is -SO3.THETA..
11. The method of claim 9 wherein the merocyanine protein error indicator compound is further defined as m is the integer 3; and R is C(CH3)2.
12. The method of claim 9 wherein the merocyanine protein error indicator compound is 1-(.omega.-sulfopropyl) 2-(4'-hydroxy-3'-5'-diiodo tyryl)-3,3-dimethylindoleninium betaine.
13. The method of claim 9 wherein the liquid sample is a body fluid.
14. The method of claim 9 wherein the liquid sample is urine.
15. A method for the detection of protein in a sample, the method comprising the steps of:
a) adding to the sample the merocyanine protein error indicator compound:
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3.THETA. or -H; and b) observing and recording any color change, wherein a color change is indicative of protein in the sample.
a) adding to the sample the merocyanine protein error indicator compound:
wherein:
m is an integer from 1 to 6;
Q is -Br, -I, or Cl;
R is S, Se, O, or C(CnH2n + 1)2, wherein:
n is an integer from 1 to 6; and T is -SO3.THETA. or -H; and b) observing and recording any color change, wherein a color change is indicative of protein in the sample.
16. The method of claim 15 wherein the merocyanine protein error indicator compound is further defined as m is the integer 3 or 4; Q
is -Br or -I; R is C(CnH2n + 1)2, wherein n is an integer from 1 to 3; and T is -SO3.THETA..
is -Br or -I; R is C(CnH2n + 1)2, wherein n is an integer from 1 to 3; and T is -SO3.THETA..
17. The method of claim 15 wherein the merocyanine protein error indicator compound is further defined as m is the integer 3; and R is C(CH3)2.
18. The method of claim 15 wherein the merocyanine protein error indicator compound is 1-(.omega.-sulfopropyl)-2-(4'-hydroxy-3'-5'-diiodostyryl)-3,3-dimethylindoleninium betaine.
19. The method of claim 15 wherein the sample is an electrophoretic gel.
20. the method of claim 15 wherein the sample is a liquid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/710,955 US5264589A (en) | 1991-06-06 | 1991-06-06 | Merocyanine protein error indicators |
| US710,955 | 1991-06-06 |
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| Publication Number | Publication Date |
|---|---|
| CA2068002A1 true CA2068002A1 (en) | 1992-12-07 |
Family
ID=24856197
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002068002A Abandoned CA2068002A1 (en) | 1991-06-06 | 1992-05-05 | Merocyanine protein error indicators |
Country Status (17)
| Country | Link |
|---|---|
| US (2) | US5264589A (en) |
| EP (1) | EP0517055B1 (en) |
| JP (1) | JP3153831B2 (en) |
| KR (1) | KR100231079B1 (en) |
| AT (1) | ATE143145T1 (en) |
| AU (1) | AU646571B2 (en) |
| CA (1) | CA2068002A1 (en) |
| DE (1) | DE69213827T2 (en) |
| ES (1) | ES2092596T3 (en) |
| FI (1) | FI922587A (en) |
| GR (1) | GR3021141T3 (en) |
| IE (1) | IE921839A1 (en) |
| IL (1) | IL101665A (en) |
| NO (1) | NO922002L (en) |
| NZ (1) | NZ242732A (en) |
| TW (1) | TW198023B (en) |
| ZA (1) | ZA923805B (en) |
Families Citing this family (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69213826T2 (en) * | 1991-06-06 | 1997-01-30 | Bayer Ag | Test strips with merocyanine and nitro or nitroso substituted polyhalogenated phenolsulfonphthaleins as protein indicators |
| GB9312853D0 (en) * | 1993-06-22 | 1993-08-04 | Euro Celtique Sa | Chemical compounds |
| US5591776A (en) * | 1994-06-24 | 1997-01-07 | Euro-Celtique, S.A. | Pheynl or benzyl-substituted rolipram-based compounds for and method of inhibiting phosphodiesterase IV |
| US5922751A (en) * | 1994-06-24 | 1999-07-13 | Euro-Celtique, S.A. | Aryl pyrazole compound for inhibiting phosphodiesterase IV and methods of using same |
| US5665737B1 (en) * | 1994-10-12 | 1999-02-16 | Euro Celtique Sa | Substituted benzoxazoles |
| US6372770B1 (en) | 1994-10-12 | 2002-04-16 | Euro-Celtique, S.A. | Benzoxazoles |
| EP0814809B1 (en) * | 1994-12-13 | 2003-08-13 | Euroceltique S.A. | Aryl thioxanthines |
| DE69531555T2 (en) * | 1994-12-13 | 2004-06-17 | Euroceltique S.A. | TRIPLE SUBSTITUTED THIOXANTHINE |
| US6025361A (en) * | 1994-12-13 | 2000-02-15 | Euro-Celtique, S.A. | Trisubstituted thioxanthines |
| US5616502A (en) * | 1995-05-19 | 1997-04-01 | Molecular Probes, Inc. | Non-specific protein staining using merocyanine dyes |
| US6166041A (en) * | 1995-10-11 | 2000-12-26 | Euro-Celtique, S.A. | 2-heteroaryl and 2-heterocyclic benzoxazoles as PDE IV inhibitors for the treatment of asthma |
| US6075016A (en) * | 1996-04-10 | 2000-06-13 | Euro-Celtique S.A. | 6,5-fused aromatic ring systems having enhanced phosphodiesterase IV inhibitory activity |
| US5864037A (en) * | 1996-06-06 | 1999-01-26 | Euro-Celtique, S.A. | Methods for the synthesis of chemical compounds having PDE-IV inhibitory activity |
| US20030070927A1 (en) * | 1996-07-24 | 2003-04-17 | Merchant Mark E. | Reagents and methods for fluorescent analysis of serum proteins |
| US5744473A (en) * | 1996-09-16 | 1998-04-28 | Euro-Celtique, S.A. | PDE IV inhibitors: "bis-compounds" |
| DE60032853T2 (en) | 1999-10-28 | 2007-11-15 | Matsushita Electric Industrial Co., Ltd., Kadoma | Method for measuring the concentration of a solution |
| JP2001174457A (en) * | 1999-12-21 | 2001-06-29 | Matsushita Electric Ind Co Ltd | Solution concentration measurement method and device therefor, and urine examination method |
| JP2001249134A (en) * | 1999-12-28 | 2001-09-14 | Matsushita Electric Ind Co Ltd | Reagent for measuring protein concentration, method for measuring protein concentration using it, and urinalysis |
| US7776529B2 (en) | 2003-12-05 | 2010-08-17 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
| WO2006124816A1 (en) | 2005-05-11 | 2006-11-23 | Molecular Probes, Inc. | Fluorescent chemical compounds having high selectivity for double stranded dna, and methods for their use |
| US7485466B2 (en) * | 2005-05-31 | 2009-02-03 | The Clorox Company | Protein detection system |
| CN106349146A (en) * | 2016-08-11 | 2017-01-25 | 潘忠宁 | Novel synthesis method of photochromic material |
| CN111004166A (en) * | 2019-12-27 | 2020-04-14 | 苏州络森生物科技有限公司 | Preparation method of SPDIB |
| CN112876405B (en) * | 2021-01-18 | 2022-08-02 | 南开大学 | Preparation method and application of endoplasmic reticulum targeted activation type photosensitizer |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2102175A1 (en) * | 1971-01-18 | 1972-07-27 | Agfa Gevaert Ag | Sensitized electrophotographic layers |
| DE2510633C3 (en) * | 1975-03-12 | 1978-07-13 | Boehringer Mannheim Gmbh, 6800 Mannheim | Diagnostic agent for the detection of protein in body fluids and indicator dyes suitable therefor |
| US4239495A (en) * | 1979-02-12 | 1980-12-16 | Sherwood Medical Industries Inc. | Determination of protein |
| US4366241A (en) * | 1980-08-07 | 1982-12-28 | Syva Company | Concentrating zone method in heterogeneous immunoassays |
| JPS57200862A (en) * | 1981-06-05 | 1982-12-09 | Fuji Photo Film Co Ltd | Mutilayer analysis element utilizing unique binding reaction |
| US4568647A (en) * | 1983-10-11 | 1986-02-04 | Eastman Kodak Company | Method and element for albumin assay |
| US4774192A (en) * | 1987-01-28 | 1988-09-27 | Technimed Corporation | A dry reagent delivery system with membrane having porosity gradient |
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1991
- 1991-06-06 US US07/710,955 patent/US5264589A/en not_active Expired - Lifetime
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1992
- 1992-04-21 IL IL10166592A patent/IL101665A/en not_active IP Right Cessation
- 1992-04-21 TW TW081103100A patent/TW198023B/zh active
- 1992-05-05 CA CA002068002A patent/CA2068002A1/en not_active Abandoned
- 1992-05-14 NZ NZ242732A patent/NZ242732A/en unknown
- 1992-05-21 NO NO92922002A patent/NO922002L/en unknown
- 1992-05-23 ES ES92108738T patent/ES2092596T3/en not_active Expired - Lifetime
- 1992-05-23 DE DE69213827T patent/DE69213827T2/en not_active Expired - Fee Related
- 1992-05-23 EP EP92108738A patent/EP0517055B1/en not_active Expired - Lifetime
- 1992-05-23 AT AT92108738T patent/ATE143145T1/en not_active IP Right Cessation
- 1992-05-25 ZA ZA923805A patent/ZA923805B/en unknown
- 1992-06-04 FI FI922587A patent/FI922587A/en unknown
- 1992-06-04 AU AU18049/92A patent/AU646571B2/en not_active Ceased
- 1992-06-04 KR KR1019920009664A patent/KR100231079B1/en not_active IP Right Cessation
- 1992-06-05 JP JP17004092A patent/JP3153831B2/en not_active Expired - Fee Related
- 1992-07-01 IE IE183992A patent/IE921839A1/en unknown
- 1992-12-14 US US07/989,716 patent/US5328850A/en not_active Expired - Fee Related
-
1996
- 1996-09-25 GR GR960402507T patent/GR3021141T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPH05223825A (en) | 1993-09-03 |
| DE69213827T2 (en) | 1997-01-30 |
| JP3153831B2 (en) | 2001-04-09 |
| IL101665A (en) | 1996-10-16 |
| KR930000954A (en) | 1993-01-16 |
| ZA923805B (en) | 1993-03-10 |
| AU1804992A (en) | 1993-03-11 |
| ATE143145T1 (en) | 1996-10-15 |
| IL101665A0 (en) | 1992-12-30 |
| AU646571B2 (en) | 1994-02-24 |
| EP0517055A1 (en) | 1992-12-09 |
| ES2092596T3 (en) | 1996-12-01 |
| KR100231079B1 (en) | 1999-11-15 |
| NZ242732A (en) | 1994-11-25 |
| FI922587A0 (en) | 1992-06-04 |
| IE921839A1 (en) | 1992-12-16 |
| NO922002D0 (en) | 1992-05-21 |
| TW198023B (en) | 1993-01-11 |
| EP0517055B1 (en) | 1996-09-18 |
| US5328850A (en) | 1994-07-12 |
| FI922587A (en) | 1992-12-07 |
| GR3021141T3 (en) | 1996-12-31 |
| NO922002L (en) | 1992-12-07 |
| DE69213827D1 (en) | 1996-10-24 |
| US5264589A (en) | 1993-11-23 |
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