Cyanine dyes as labeling reagents for detection of biological and other materials by luminescence methods

• US 5,627,027 A
• Filed: 09/22/1992
• Issued: 05/06/1997
• Est. Priority Date: 04/18/1986
• Status: Expired due to Term

• Current Assignee: Carnegie Mellon University
• Original Assignee: Carnegie Mellon University
• Inventors: Waggoner, Alan S.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US07/831,759
• Time in Patent Office: 1,687 Days
• Field of Search: 435/6, 436/111, 436/131, 436/800, 436/86, 544/212, 548/465, 548/469, 430/581
• US Class Current: 435/6.11
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups

Abstract

Cyanine and related dyes, are strongly light absorbing and highly luminescent. These dyes are covalently attached to proteins and other biological and nonbiological materials to make these materials fluorescent so that they can be detected. The labeled materials can then be used in assays employing excitation light sources and luminescence detectors. Avidin labeled with cyanine type dyes can be used to quantify biotinylated materials and antibodies conjugated with cyanine-type dyes can be used to detect and measure antigens and haptens. In addition, cyanine-conjugated lectins can be used to detect specific carbohydrate groups. Also, cyanine-conjugated fragments of DNA or RNA can be used to identify the presence of complementary nucleotide sequences in DNA or RNA. The cyanine dyes have the advantage that by synthesizing structural modifications of the chromophore portion the molecule, different fluorescent labeling reagents can be made that will absorb and fluoresce light at many different wavelengths in the visible and near infrared region of the spectrum. Also, the cyanine and related dyes have an advantage in their structural versatility. That is, they can be synthesized in many structural forms and with a variety of functional groups attached. This versatility permits control over such factors as the solubility of the dye and labeled product and helps reduce nonspecific binding of the labeled material to irrelevant components in the assay mixture. This versatility also allows for selection of labeling reagents that minimally perturb the function of the labeled product.

Claims

1. A method for imparting luminescence for luminescence detection to a component which contains or is derivatized to contain an amine or hydroxy group in a liquid, said method comprising the steps of:

adding to a liquid containing said component, wherein said component is selected from the group consisting of proteins, cells, nucleic acids and DNA, a luminescent cyanine dye having the structure ##STR13## wherein;

the dotted lines each represent carbon atoms necessary for the formulation of said cyanine dye;

X and Y are selected from the group consisting of O, S and CH₃ --C--CH₃ ;

m is an integer selected from the group consisting of 1, 2, 3 and 4;

at least one of R₁, R₂, R₃, R₄ and R₇ contain a reactive group to make said dye covalently reactive with said amine or hydroxy group available for covalent bonding on said component, said reactive group selected from the group consisting of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido ester, glyoxal and aldehyde; and

reacting said cyanine dye with said component at said amine or hydroxy group under reaction conditions sufficient for forming a covalent bond between said reactive group and said amine or hydroxy group so that said dye labels said component, wherein said labeled component is luminescent and absorbs and emits light in the 400 to 900 nm spectral range for luminescence detection.

2. The method of claim 1 wherein said labeled component has a molar extinction coefficient of at least 50,000 liters per mole centimeter and a quantum yield of at least 5 percent.

3. The method of claim 1 wherein said labeled component has a molar extinction coefficient of at least 120,000 liters per mole centimeter and a quantum yield of at least 10 percent.

4. The method of claim 1 wherein said at least one R₁, R₂, R₃, R₄ and R₇ groups contains at least one reactive moiety selected from the group consisting of:

##STR14## where at least one of Q or W is selected from the group consisting of Cl;

Br;

I; and

n is zero or any integer.

5. The method of claim 1 wherein at least one of said R₁, R₂, R₃, R₄ and R₇ groups also contains at least polar moiety to increase solubility in water selected from the group consisting of hydroxy, sulfonate, sulfate, carboxylate and substituted amine or quaternary amine groups.

6. The method of claim 1 wherein the dye in said labeled component has a quantum yield of at least 5 percent.

7. The method of claim 1 wherein said amine or hydroxy group is added to said component before said dye reaction step.

8. A method for labeling an amine or hydroxy group of a component selected from the group consisting of proteins, cells, nucleic acids and DNA comprising:

adding (i) to a liquid containing said component, (ii) a cyanine dye having a structure consisting essentially of;

##STR15## wherein;

the dotted lines each represent carbon atoms necessary for the formation of said cyanine dye;

X and Y are selected from the group consisting of O, S and CH₃ --C--CH₃ ;

m is an integer selected from the group consisting of 1, 2 and 3;

R₁ and R₂ contain a substituent selected from the group consisting of --(CH₂)_n --SO^-₃, --CH₃, or a first reactive group selected from the group consisting of --(CH₂)_n --N═

C═

S, and ##STR16## where n equals 2, 3, 4 or 5; and

R₃ and R₄ are hydrogen or contain a second reactive group selected from the group consisting of ##STR17## wherein at least one of R₁, R₂, R₃, R₄ contains said first or second reactive groups; and

imparting luminescence to said component by reacting said cyanine dye with said component at said available amine or hydroxy group under reaction conditions so that at least one of said reactive groups of said cyanine dye covalently binds to said available amine or hydroxy group for providing a detectable luminescent labeled product for luminescence detection which absorbs and emits light in the 400 to 900 nm spectral range.

9. The method recited in claim 8 further comprising the step of detecting the luminescent labeled product.

10. The luminescent water soluble photostable reaction product of proteins, cells, nucleic acids or DNA which contain or are derivatized to contain an amine or hydroxy group available for covalent bonding and a luminescent cyanine dye which contains a substituent to make it covalently reactive with said amine or hydroxy group on said protein, cell, nucleic acid or DNA, said cyanine dye having the structure ##STR18## wherein:

the dotted lines each represent carbon atoms necessary for the formulation of said cyanine dye;

X and Y are selected from the group consisting of O, S and CH₃ --C--CH₃ ;

m is an integer selected from the group consisting of 1, 2, 3 and 4;

at least one of R₁, R₂, R₃, R₄ and R₇ contain a reactive substituent selected from the group consisting of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono-or di-halogen substituted diazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido ester, glyoxal and aldehyde, wherein said luminescent reaction product is prepared for luminescence detection of said protein, cell, nucleic acids or DNA and has a molar extinction coefficient of at least 50,000 liters per mole centimeter, a quantum yield of at least 5 percent and absorbs and emits light in the 400 to 900 nm spectral range.