Method and apparatus for improved analytical fluorescent spectroscopy

• US 4,058,732 A
• Filed: 06/30/1975
• Issued: 11/15/1977
• Est. Priority Date: 06/30/1975
• Status: Expired due to Term

• Current Assignee: Irwin Wieder
• Original Assignee: Analytical Radiation Corporation
• Inventors: Wieder, Irwin
• Primary Examiner(s): Willis, Davis L.
• Application Number:
  US05/591,305
• Time in Patent Office: 869 Days
• Field of Search: 250/461 B, 250/301, 250/269, 424/7
• US Class Current: 250/461.1
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

A method is disclosed whereby the molecules of a chemical substance to be measured, referred to as target molecules, are combined with fluorescent molecules having a relatively long fluorescent decay lifetime. These tagged target molecules are excited with an intense pulse of ultraviolet or visible radiation, and a detection system is gated on only after background fluorescence has substantially decayed, but while the fluorescent tag attached to the target molecules is still actively decaying. The amount of fluorescent tag emission which is measured is indicative of the target molecule content of the sample. An apparatus for improved fluorescent spectrofluorometry includes a pulsed excitation source and a gating means connected to a detection system for gating the detection system on after tagged target molecules have been excited and competing background fluorescence has substantially decayed, but before tagged target molecules have substantially decayed.

Claims

1. A method for fluorescent spectroscopy of a target substance comprising:

isolating a target substance,

tagging said target substance with a fluorescent tag having a long fluorescent decay lifetime compared to the longest of the decay lifetimes of competing untagged ambient substances,

removing excess fluorescent tag,

exciting the tagged target substance with at least one pulse of radiation, said pulse having a pulse duration which is short compared to the fluorescent decay lifetime of said fluorescent tag,

detecting the fluorescence of said excited tagged target substances after the fluorescence of said ambient substances has substantially decayed.

2. The method of claim 1 whereby isolating said target substance is by means of chromatography.

3. The method of claim 1 whereby isolating said target substance is by means of combination with antibodies fixed on a substrate.

4. The method of claim 1 wherein said fluorescent tag is a fluorescent antibody specific to the target substance.

5. The method of claim 4 wherein said fluorescent antibody is an antibody conjugated with pyrenebutyrate.

6. The method of claim 1 wherein said fluorescent tag is a fluorescent rare earth-organo complex.

7. The method of claim 6 wherein said fluorescent rare earth-organo complex is europium benzoyltrifluoroacetonate.

8. The method of claim 1 wherein said fluorescent tag is pyrenebutyrate.

9. The method of claim 1 wherein said fluorescent detection system is gated on after five lifetimes of background fluorescence from the target.

10. The method of claim 1 wherein said exciting pulse is derived from a laser source.

11. The method of claim 10 wherein said exciting pulse is derived from a nitrogen laser source.

12. The method of claim 1 wherein said exciting pulse is derived from a pulsed dye laser excited by a nitrogen laser.

13. In a method of fluorescent spectroscopy for a target substance wherein at least one pulse of excitation radiation of relatively short duration is directed toward the target substance the improvement comprising, combining with said target substance a fluorescent tag specific to the target substance with fluorescent decay lifetime long compared with the longest of the fluorescent decay lifetimes of competing untagged ambient substances and detecting said tagged target substance after fluorescence of competing ambient substances substantially decayed.

14. The method of claim 13 wherein said fluorescent tag is a fluorescent rare earth-organo complex.

15. The method of claim 13 wherein said fluorescent tag is a fluorescent antibody specific to said target substance.

16. The method of claim 15 wherein the fluorescent antibody is an antibody conjugated with pyrenebutyrate.

17. A method for fluorescent spectroscopy of a target substance amid ambient substances comprising;

tagging a target substance with a fluorescent tag specific to the target substance having a long fluorescent decay lifetime compared to the longest of the decay lifetimes of competing untagged ambient substances,

removing excess fluorescent tag,

exciting the tagged target substance for fluorescence with at least one pulse of radiation, said pulse having a pulse duration which is short compared to the fluorescent decay lifetime of said fluorescent tag,

detecting the fluorescence of said excited tagged target substances after the fluorescence of said ambient substances has substantially decayed.

18. The method of claim 17 wherein said fluorescent tag is a fluorescent antibody specific to the target substance.

19. A fluorometer system for fluorescent spectroscopy of target substances comprising,

an amount of target substance amid untagged ambient substances, said target substance tagged with a fluorescent tag having a long fluorescent decay lifetime compared to the longest of the decay lifetimes of competing ambient substances,

radiation means having an output directed at said target substance for exciting said tagged target substance into fluorescence with at least one pulse of radiation, said pulse having a pulse duration which is short compared to the long fluorescent decay lifetime of said fluorescent tag,

a fluorescence detector, disposed for receiving fluorescence from said tagged target substance, having means for detecting said fluorescence after the fluorescence of competing ambient substances has substantially decayed and having gating means connected to said detecting means for gating on said fluorescence detector after said fluorescence of competing ambient substances has substantially decayed, and

means for correlating the detected fluorescence of the tagged target substance with the amount of tagged target substance

20. The apparatus of claim 19 wherein said target is supported on a solid matrix.

21. The apparatus of claim 19 wherein said radiation means is a pulsed dye laser.

22. The apparatus of claim 19 wherein said radiation means is a pulsed nitrogen laser.

23. The apparatus of claim 19 wherein said radiation means is a pulsed dye laser excited by a nitrogen laser.

24. The apparatus of claim 19 wherein said radiation means is a pulsed gas discharge.

25. The apparatus of claim 19 wherein said gating means is connected to a delay circuit means for establishing a selected time for fluorescent radiation from said ambient substances to substantially decay.