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

• US 5,698,397 A
• Filed: 06/07/1995
• Issued: 12/16/1997
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: SRI International, Inc.
• Original Assignee: SRI International, Inc.
• Inventors: Kane, James, Faris, Gregory W., Dyer, Mark J., Ng, Steve Y., Schneider, Luke V., Zarling, David A., Rossi, Michel J., Peppers, Norman A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/482,203
• Time in Patent Office: 923 Days
• Field of Search: 435/5, 435/6, 435/7.1, 216/25, 250/581, 250/484.3, 313/467, 536/24.3, 530/387.1, 530/388.1
• US Class Current: 435/6.13
• 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 2219/00648 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 2021/6419 Excitation at two or more wavelengths
• 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 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6454 using an integrated detector array
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

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 composition comprising an up-converting inorganic phosphor which converts excitation radiation to emission radiation of a shorter wavelength and comprises at least one rare earth element in a host material and a probe selected from the group consisting of:

antibodies, avidins, lectins, Staphylococcus aureus Protein A, antigens, polypeptides and polynucleotides.

2. A composition according to claim 1, wherein the up-converting inorganic phosphor comprises ytterbium and an emitter selected from erbium, holmium, thulium, and terbium.

3. A composition according to claim 1, wherein the probe is attached to an up-converting inorganic phosphor by noncovalent linkage.

4. A composition according to claim 1, wherein the probe is streptavidin and the up-converting inorganic phosphor is attached to the probe by noncovalent linkage.

5. A composition of claim 4 further comprising a biotinylated magnetic bead, a streptavidin-coated magnetic bead, an avidin-coated magnetic bead, or an immunoglobulin-coated magnetic bead.

6. A composition for diagnostic detection of an analyte, comprising a microcrystalline up-convening phosphor covalently bound to antibodies, avidins, lectins, Staphylococcus aureus Protein A. antigens, polypeptides and polynucleotides.

7. A composition of claim 6, wherein the microcrystalline up-converting phosphor has the formula:

space="preserve" listing-type="equation">Na(Y.sub.x Yb.sub.y Er.sub.z)F.sub.4 ;

wherein x is 0.7 to 0.9, y is 0.09 to 0.29, and z is 0.05 to 0.01;

space="preserve" listing-type="equation">Na(Y.sub.x Yb.sub.y Ho.sub.z)F.sub.4 ;

wherein x is 0.7 to 0.9, y is 0.0995 to 0.2995, and z is 0.0005 to 0.001;

space="preserve" listing-type="equation">Na(Y.sub.x Yb.sub.y Tm.sub.z)F.sub.4 ;

wherein x is 0.7 to 0.9, y is 0.0995 to 0.2995, and z is 0.0005 to 0.001;

or

space="preserve" listing-type="equation">(Y.sub.x Yb.sub.y Er.sub.z)O.sub.2 S;

wherein x is 0.7 to 0.9, y is 0.05 to 0.12;

z is 0.05 to 0.12.

8. A composition of claim 6, wherein the microcrystalline up-converting phosphor has a formula selected from the group consisting of:

(Y₀.80 Yb₀.18 Er₀.02 F₃ ;

(Y₀.87 Yb₀.13 Tm₀.001)F₃ ;

(Y₀.80 Yb₀.198 Ho₀.002)F₃ ;

(Gd₀.08 Yb₀.18 Er₀.02)F₃ ;

(Gd₀.87 Yb₀.13 Tm₀.001)F₃ ;

(Gd₀.80 Yb₀.198 Ho₀.002)F₃ ;

(Y₀.86 Yb₀.08 Er₀.06)₂ O₂ S;

(Y₀.87 Yb₀.13 Tm₀.001)₂ O₂ S;

(Y₀.08 Yb₀.198 Ho₀.0022)O₂ S;

(Gd₀.86 Yb₀.08 Er₀.06)₂ O₂ S;

(Gd₀.87 Yb₀.13 Tm₀.001)₂ O₂ S; and

(Gd₀.08 Yb₀.198 Ho₀.002)₂ O₂ S.

9. A composition for diagnostic detection of an analyte, comprising an up-converting phosphor non-covalently bound to antibodies, avidins, lectins, Staphylococcus aureus Protein A, antigens, polypeptides and polynucleotides.

10. A composition of claim 9 wherein the microcrystalline up-converting phosphor has the formula:

space="preserve" listing-type="equation">Na(Y.sub.x Yb.sub.y Er.sub.z)F.sub.4 ;

wherein x is 0.7 to 0.9, y is 0.09 to 0.29, and z is 0.05 to 0.01;

space="preserve" listing-type="equation">Na(Y.sub.x Yb.sub.y Ho.sub.z)F.sub.4 ;

wherein x is 0.7 to 0.9, y is 0.0995 to 0.2995, and z is 0.0005 to 0.001;

space="preserve" listing-type="equation">Na(Y.sub.x Yb.sub.y Tm.sub.z)F.sub.4 ;

wherein x is 0.7 to 0.9, y is 0.0995 to 0.2995, and z is 0.0005 to 0.001;

or

space="preserve" listing-type="equation">(Y.sub.x Yb.sub.y Er.sub.z)O.sub.2 S;

wherein x is 0.7 to 0.9, y is 0.05 to 0.12;

z is 0.05 to 0.12.

11. A composition of claim 9, wherein the microcrystalline up-converting phosphor has a formula selected from the group consisting of:

(Y₀.80 Yb₀.18 Er₀.02)F₃ ;

(Y₀.87 Yb₀.13 Tm₀.001)F₃ ;

(Y₀.80 Yb₀.198 Ho₀.002)F₃ ;

(Gd₀.80 Yb₀.18 Er₀.02)F₃ ;

(Gd₀.87 Yb₀.13 Tm₀.001)F₃ ;

(Gd₀.80 Yb₀.198 Ho₀.002)F₃ ;

(Y₀.86 Yb₀.08 Er₀.06)₂ O₂ S;

(Y₀.87 Yb₀.13 Tm₀.001)₂ O₂ S;

(Y₀.80 Yb₀.198 Ho₀.002)₂ O₂ S;

(Gd₀.86 Yb₀.08 Er₀.06)₂ O₂ S;

(Gd₀.87 Yb₀.13 Tm₀.001)₂ O₂ S; and

(Gd₀.08 Yb₀.198 Ho₀.002)₂ O₂ S.