Method and device for carbonyl detection and quantitation
Fluorescence chromophores such as phenylene diamine derivatives can undergo oxidative coupling and polymerization to form optical, colorimetric and fluorogenic, multimers and polymers. The presence of carbonyl containing moieties such as aldehydes and ketones under favorable environmental conditions can initiate, catalyze, accelerate and modulate this reaction which in turn provides a mechanism for the detection and quantitation of such moieties. Selected phenylene diamine derivatives can be used for the detection and quantitation of aldehyde and ketones via measurement of the reaction and the associated spectroscopic transformation. In particular, the use of meta-phenylene diamine (mPDA) and related compounds for aldehyde detection and quantitation is described. The method provides a convenient means for monitoring aldehyde and ketone levels without use of separation steps. The method is applicable to kinetic and quasi-endpoint detection assay formats.
1. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a phenylene diamine derivative to an aqueous salt solution to thereby form a phenylene diamine solution; adding a carbonyl containing moiety from the biological sample to the phenylene diamine solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution, wherein the detected fluorescence corresponds to the concentration of carbonyl containing moieties in the biological sample, and wherein the biological sample is human breath, the carbonyl containing moiety is an aldehyde and the phenylene diamine derivative is m-phenylene diamine.
2. The method of claim 1 wherein the carbonyl containing moiety is selected from the group consisting of aldehydes, ketones, carboxylic acids and mixtures thereof.
3. The method of claim 2 , wherein the aldehydes are aliphatic.
4. The method of claim 1 , wherein the biological sample is human breath.
5. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a phenylene diamine derivative to an aqueous salt solution to thereby form a phenylene diamine solution; adding a carbonyl containing moiety from the biological sample to the phenylene diamine solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution, wherein the phenylene diamine derivative is selected from the group of meta-phenylene diamine derivatives and mixtures thereof.
6. The method of claim 1 , wherein the fluorescence has a measurable emission band at 505 nm.
7. The method of claim 1 , wherein the fluorescing solution is maintained at below about 35° C.
8. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a phenylene diamine derivative to an aqueous salt solution to thereby form a phenylene diamine solution; adding a carbonyl containing moiety from the biological sample to the phenylene diamine solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution, wherein the aqueous salt solution comprises an alcohol, a salt, a surfactant and a buffer.
9. The method of claim 8 where the buffer is citrate.
10. The method of claim 8 wherein the alcohol is chosen from the group consisting of R—OH, where R is a straight or branched chain aliphatic group, and mixtures thereof.
11. The method of claim 9 , wherein the alcohol is ethanol.
12. The method of claim 8 , wherein the salt is NaCl.
13. The method of claim 8 , wherein the surfactant is sodium dodecyl sulfate.
14. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a phenylene diamine derivative to an aqueous salt solution to thereby form a phenylene diamine solution; adding a carbonyl containing moiety from the biological sample to the phenylene diamine solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution, wherein pH of the aqueous solution ranges from 2 to 4.5.
15. The method of claim 8 , wherein the molar ratio of the phenylene diamine derivative to the salt ranges from 0.03 to 0.5.
16. The method of claim 1 , wherein the carbonyl containing moiety is eluted from silica.
17. The method of claim 1 , wherein the carbonyl containing moiety is a C1 to C12 carbonyl containing organic molecule.
18. A method of detecting and measuring the concentration of a carbonyl containing moiety in a biological sample, the method comprising
a) isolating the carbonyl containing moiety from the biological sample;
b) adding the carbonyl containing moiety to an aqueous solution containing a phenylene diamine derivative to form a fluorescing solution; and
c) measuring the fluorescence emitted from the fluorescing solution at a pre-determined wave length.
19. The method of claim 18 , wherein the biological sample is a breath sample.
20. The method of claim 18 , wherein the concentration of the carbonyl containing moiety in the biological sample is determined using the fluorescence measurement.
21. The method of claim 1 , wherein the phenylene diamine derivative comprises pyridinium,4-[2-[4-(diethylaminio)phenyl-ethenyl]-1-[1-(3,5-diminobenzamide)-pentylamino-5-oxyhextyl].
22. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a phenylene diamine derivative to an aqueous salt solution to thereby form a phenylene diamine solution; adding a carbonyl containing moiety from the biological sample to the phenylene diamine solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution, wherein the phenylene diamine derivative comprises any one or mixtures of the following:
where R1, R2, R3, R4 are each independently selected from the group consisting of H, alkyl, substituted alky, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamine, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxylester) amino, (carboxy ester) oxy, cyano, halo, hydroxy, SO3-, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thioal, alkylthio, substituted alkylthio, acyl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycles, and substituted heterocycles.
23. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a fluorescence chromophore to an aqueous salt solution to thereby form a fluorescence chromophore solution; adding a carbonyl containing moiety from the biological sample to the fluorescence chromophore solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution.
24. The method of claim 23 , wherein the fluorescence chromophore is a phenylene diamine derivative.
25. The method of claim 24 , wherein the fluorescence chromophore is m-phenylene diamine.
26. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a phenylene diamine derivative to an aqueous salt solution to thereby form a phenylene diamine solution; adding a carbonyl containing moiety from the biological sample to the phenylene diamine solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution, wherein the phenylene diamine derivative is polymerized by the carbonyl containing moiety in the solution to form a fluorogenic species.
27. A method of detecting carbonyl containing moieties in a biological sample, the method comprising adding a phenylene diamine derivative to an aqueous salt solution to thereby form a phenylene diamine solution; adding a carbonyl containing moiety from the biological sample to the phenylene diamine solution to thereby form a fluorescing solution; and detecting fluorescence from the fluorescing solution, wherein the phenylene diamine derivative is housed in an ampule.
28. The method of claim 18 , wherein the fluorescing solution generates a broad optical absorbance band at 405 nm and an associated emission band at 505 nm.
29. The method of claim 18 , wherein the aqueous solution contains a surfactant, and the level of fluorescence over time varies with the concentration of the surfactant.
30. A method for detecting carbonyl containing moieties in a biological sample, the method comprising the steps of providing a substrate having an active reactive capture agent incorporated therein, capturing on said substrate carbonyl containing moieties from the biological sample, and forming a solution comprising painted carbonyl containing moieties.
31. The method of claim 26 , wherein there is no precipitate in the fluorescing solution which at the time of measurement, substantially interferes with the measurement of the fluorogenic species.