Up-converting reporters for biological and other assays using laser excitation techniques

• US 5,891,656 A
• Filed: 07/02/1997
• Issued: 04/06/1999
• Est. Priority Date: 09/14/1992
• Status: Expired due to Term

• Current Assignee: SRI International, Inc.
• Original Assignee: SRI International, Inc.
• Inventors: Dyer, Mark J., Ng, Steve Y., Schneider, Luke V., Zarling, David A., Rossi, Michel J., Peppers, Norman A., Kane, James, Faris, Gregory W.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): PORTNER, VIRGINIA ALLEN
• Application Number:
  US08/887,428
• Time in Patent Office: 643 Days
• Field of Search: 435/7.92, 435/7.21, 422/56, 422/82.08, 427/7
• US Class Current: 435/7.92
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/00 Nucleic acid detection characterized by the use of physical, structural and functional properties
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C40B 60/14 for creating libraries
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots

Abstract

The invention provides methods, compositions, and apparatus for performing sensitive detection of analytes, such as biological macromolecules and other analytes, by labeling a probe molecule with an up-converting label. The up-converting label absorbs radiation from an illumination source and emits radiation at one or more higher frequencies, providing enhanced signal-to-noise ratio and the essential elimination of background sample autofluorescence. The methods, compositions, and apparatus are suitable for the sensitive detection of multiple analytes and for various clinical and environmental sampling techniques.

Claims

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

contacting a sample containing a target analyte with a labeled probe to specifically bind the target analyte and form a labeled probe-target complex, wherein the labeled probe comprises a probe attached to an up-converting chelate complex comprising a rare earth ion and a ligand and being capable of converting excitation radiation to emission radiation of a shorter wavelength;

separating any bound labeled probe from the labeled probe-target complex;

illuminating the labeled probe-target complex with excitation radiation; and

detecting emission radiation of at least one label emission wavelength, wherein the emission radiation has a shorter wavelength than the excitation radiation.

2. A method of claim 1, further comprising, before the contacting step, the step of attaching the up-converting chelate complex to the probe to form the labeled probe.

3. A method according to claim 1, wherein the rare earth ion is selected from the group of an erbium ion, neodymium ion, thulium ion, a holmium ion and a praseodymium ion.

4. A method according to claim 3, wherein the ligand is selected from the group ethylenediaminetetraacetic acid, dipicolinic acid, diethylenetriaminetetraacetic acid, diethylenetriaminepentaacetic acid, tetraazacyclotetradecanetetraacetic acid, an antibiotic, a protein, a phthalocyanine and a cryptate.

5. A method for detecting an analyte in a sample, comprising the steps of:

contacting a sample containing a target analyte with a probe to specifically bind the target analyte and form a probe-target complex;

contacting the probe-target complex with a label to form a labeled probe-target complex, wherein the label is an up-converting chelate complex comprising a rare earth ion and a ligand and being capable of converting excitation radiation to emission radiation of a shorter wavelength;

separating any unbound label from the labeled probe-target complex;

illuminating the labeled probe-target complex with excitation radiation; and

detecting emission radiation of at least one label emission wavelength, wherein the emission radiation has a shorter wavelength than the excitation radiation.

6. A method for detecting an analyte in a sample, comprising the steps of:

contacting a sample containing a target analyte with a labeled probe to specifically bind the target analyte and form a labeled probe-target complex, wherein the labeled probe comprises a probe attached to an up-converting chelate complex comprising a rare earth ion and a ligand and wherein the up-converting chelate complex is capable of converting excitation radiation to emission radiation of a shorter wavelength;

differentiating the labeled probe-target complex from any unbound labeled probe in the sample;

illuminating the labeled probe-target complex with excitation radiation; and

detecting emission radiation of at least one label emission wavelength from the labeled probe-target complex, wherein the emission radiation has a shorter wavelength than the excitation radiation.