Fluorescence detection of cysteine and homocysteine
Embodiments of probes for selectively detecting compounds having a thiol group and an amino group, e.g., cysteine and/or homocysteine, are disclosed, along with methods and kits for detecting the compounds in neutral media with the probes. The probes have a structure according to the general formula where R 1 -R 4 independently are hydrogen hydroxyl, halogen, thiol, thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to 4, and each R 5 independently is halogen, hydroxyl, thiol, thioether, lower aliphatic, or lower alkoxy. Embodiments of the disclosed probes are capable of undergoing condensation/cyclization reactions with cysteine and/or homocysteine. Cysteine and/or homocysteine can be selectively detected and identified by determining fluorescence emission of the probes at characteristic wavelengths.
1. A method for detecting at least one compound comprising a thiol group and an amino group, comprising:
combining a sample potentially comprising at least one compound comprising a thiol group and an amino group with a solution comprising a probe having a structure according to general formula II
where R 1 -R 4 independently are hydrogen, hydroxyl, halogen, thiol, thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to 4, and each R 5 independently is halogen, hydroxyl, thiol, thioether, lower aliphatic, or lower alkoxy;
allowing a reaction between the compound and the probe to proceed for an effective period of time; and
detecting the at least one compound by detecting fluorescence of the solution.
2. The method of claim 1 where the at least one compound is cysteine, homocysteine, or a combination thereof.
3. The method of claim 1 where detecting fluorescence of the solution comprises:
obtaining a fluorescence spectrum after the effective period of time; or
monitoring a fluorescence spectrum of the solution over a period of time ranging from zero minutes to a time greater than or equal to the effective period of time.
4. The method of claim 1 where R 1 is methoxy, R 2 -R 4 are hydrogen, and x is 0.
5. The method of claim 4 where detecting fluorescence of the solution comprises detecting the fluorescence at 377 nm, at 487 nm, or at 377 nm and 487 nm after the effective period of time or over a period of time ranging from zero minutes to the effective period of time.
6. The method of claim 4 where the at least one compound is cysteine and detecting the at least one compound comprises detecting fluorescence of the solution at 487 nm after the effective period of time.
7. The method of claim 4 where the at least one compound is homocysteine and detecting the at least one compound comprises detecting fluorescence of the solution at 377 nm after the effective period of time.
8. The method of claim 1 where the solution further comprises a surfactant.
9. The method of claim 8 where R 1 is methoxy, R 2 is hydrogen, and x is 0.
10. The method of claim 9 where the at least one compound is cysteine, the effective period of time is 8-10 minutes, and detecting the cysteine comprises detecting fluorescence of the solution at 487 nm after the effective period of time.
11. The method of claim 9 where the at least one compound is homocysteine, the effective period of time is 8-10 minutes, and detecting the homocysteine comprises detecting fluorescence of the solution at 377 nm 8-10 minutes after combining the sample and the solution comprising the probe.
12. The method of claim 9 where the at least one compound is homocysteine, the effective period of time is 8-10 minutes, and detecting the homocysteine comprises:
measuring fluorescence of the solution at 377 nm and 487 nm at a first time 8-10 minutes after combining the sample and the solution comprising the probe;
measuring fluorescence of the solution at 377 nm and 487 nm at a second time after combining the sample and the solution comprising the probe, wherein the second time is greater than 8-10 minutes; and
determining a difference in fluorescence at each of 377 nm and 487 nm at the first time and the second time, wherein a decrease in fluorescence at 377 nm and a proportional increase in fluorescence at 487 nm indicates presence of homocysteine.
13. The method of claim 9 where the at least one compound comprises cysteine, homocysteine, glutathione, one or more non-amino thiols, or a combination thereof, the effective period of time is greater than or equal to 9 minutes, and detecting the at least one compound comprises detecting fluorescence of the solution at 377 nm and 487 nm after the effective period of time.
14. The method of claim 13 further comprising identifying the at least one compound, wherein cysteine is identified based upon stable fluorescence intensity of the solution at 487 nm after 9 minutes and for a subsequent period of time of at least 5 additional minutes, glutathione and/or non-amino thiols are identified based upon stable fluorescence intensity of the solution at 377 nm after 9 minutes and for a subsequent period of time of at least 5 additional minutes, and/or homocysteine is identified based upon proportionally decreasing fluorescence intensity of the solution at 377 nm and increasing fluorescence intensity of the solution at 487 nm after 9 minutes and during a subsequent period of time of at least 5 additional minutes.