Flourescence intramolecular energy transfer conjugate compositions and detection methods

• US 5,254,477 A
• Filed: 09/19/1991
• Issued: 10/19/1993
• Est. Priority Date: 06/25/1986
• Status: Expired due to Term

• Current Assignee: Trustees of Tufts College
• Original Assignee: Trustees of Tufts College
• Inventors: Walt, David R.
• Primary Examiner(s): Scheiner, Toni R.
• Application Number:
  US07/762,245
• Time in Patent Office: 761 Days
• Field of Search: 436/536, 436/537, 436/501, 436/56, 436/164, 436/165, 436/172, 436/518, 436/528, 436/546, 436/800, 436/805, 436/95, 436/71, 435/4, 435/11, 435/14, 435/15
• US Class Current: 436/172
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 2021/7786 Fluorescence
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• 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 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B
• CPC Class Codes: G01N 21/80 Indicating pH value
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

A variety of conjugated compositions and methods for the detection of an analyte of interest in a fluid sample is provided which relies upon an intramolecular energy transfer between a conjugated fluorophore and a chromophoric light-absorbing ligand for qualitative and quantitative results. The detection methods preferably employ fiber optic sensors in combination with analyte-insensitive fluorophores and analyte-sensitive absorber ligands in conjugated form. The methods and compositions rely upon the ability of the absorbing ligands to absorb energy which is transferred non-radiatively by the fluorophore when in an excited state.

Claims

1. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:

admixing the fluid sample containing the analyte of interest with a fluid containing a preformed, fluorophore-absorber linked conjugate composition whose spectral characteristics are affected by contact with the analyte of interest to form a fluid reaction mixture, said preformed, fluorophore-absorber linked conjugate composition comprising

(a) at least one fluorophore which is non-reactive with the analyte of interest, said fluorophore having characteristic and definable light absorption and light emission spectra, and which absorbs exciting light energy of a first wavelength, which transfers non-radiatively at least a portion of said exciting energy to an absorber, and which emit another portion of said exciting energy as emitted light of a second wavelength,

(b) at least one absorber which is reactive with the analyte of interest and whose spectral characteristics are modulated by reactive contact with the analyte of interest, said absorber having a light absorption spectrum which overlaps in some degree with the definable light emission spectrum characteristic of said analyte non-reactive fluorophore, and which absorbs energy transferred non-radiatively by said analyte non-reactive fluorophore, and which is held at a spatial distance of not more than 100 Angstroms from said analyte non-reactive fluorophore, and

(c) chemical linking agent means for holding said analyte reactive absorber at said spatial distance from said analyte non-reactive fluorophore within said conjugate composition;

introducing light energy of the first wavelength to said reaction mixture whereby said analyte non-reactive fluorophore of said conjugate composition absorbs said exciting light energy and emits an intensity of light of the second wavelength and wherein at least a portion of said exciting energy is non-radiatively transferred to and absorbed by said analyte reactive absorber of said conjugate composition; and

determining the intensity of said emitted light of the second wavelength from said reaction mixture, said intensity of emitted light being a measure of the analyte of interest in the sample.

3. The detection method as recited in claim 1 or 2 wherein said introduction of light energy is achieved using an optical fiber.

4. The detection method as recited in claim 1 or 2 wherein said determing of emitted light is made using a fiber optic sensor.

2. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:

admixing the fluid sample containing the analyte of interest with a fluid containing a preformed, fluorophore-absorber linked conjugate composition whose spectral characteristics are affected by contact with the analyte of interest to form a fluid reaction mixture, said preformed, fluorophore-absorber linked conjugate composition comprising

(a) at least one fluorophore which is non-reactive with the analyte of interest, said fluorophore having characteristic and definable light absorption and light emission spectra, and which absorbs exciting light energy of a first wavelength, and which transfers non-radiatively substantially all of said exciting energy to an absorber such that there then is an absence of emitted light energy of a second wavelength,

(b) at least one absorber which is reactive with the analyte of interest and whose spectral characteristics are modulated by reactive contact with the analyte of interest, said absorber having la light absorption spectrum which overlaps in some degree with the definable light emission spectrum characteristic of said analyte non-reactive fluorophore, and which absorbs energy transferred non-radiatively by said analyte non-reactive fluorophore, and which is held at a spatial distance of not more than 100 Angstroms from said analyte non-reactive fluorophore, and

(c) chemical linking agent means for holding said analyte reactive absorber at said spatial distance from said analyte non-reactive fluorophore within said conjugate composition;

introducing light energy of the first wavelength to said reaction mixture whereby said analyte non-reactive fluorophore of said conjugate composition absorbs said exciting light energy without subsequently emitting light energy of a second wavelength and wherein substantially all of said exciting energy is non-radiatively transferred to and absorbed by said analyte reactive absorber of said conjugate composition; and

determining the absence of emitted light energy of the second wavelength from said reaction mixture, said absence of emitted light energy being a measure of the analyte of interest in the sample.