An assay method for the detection or quantitation of a biological molecule in a biological sample comprises binding a binding partner with a tracer to form a binding complex between the binding partner and the tracer, and wherein chemiluminescence is triggered from said tracer following said binding, wherein the improvement comprising detecting a light signal emitted at a wavelength maximum longer than 590 nm from said binding complex, and relating said detected light signal to the presence or an amount of said biological molecule in said biological sample,
- wherein said tracer is formed by covalently coupling, either directly or indirectly, a near-infrared acridinium compound to said biological molecule or analog, wherein said biological molecule is selected from the group consisting of small organic biomolecules, haptens and ligands,
- wherein said near-infrared acridinium compound comprises an acridinium nucleus having an electron-donating substituent directly attached to the acridinium nucleus, with the electron-donating substituent attached at the C2 position, the near-infrared acridinium compound having the structure:
- wherein
- R1 is alkyl, alkenyl, alkynyl or aralkyl, or alkyl, alkenyl, alkynyl or aralkyl having up to 20 heteroatoms;
- R2 and R3 are identical or different, and are selected from the group consisting of hydrogen, R, substituted aryl, unsubstituted aryl, halide, amino, hydroxyl, nitro, sulfonate, —CN, —COOH, —SCN, —OR, —SR, —SSR, —C(O)R, —C(O)OR, —C(O)NHR and —NHC(O)R;
- R is alkyl, alkenyl, alkynyl, aryl or aralkyl, or alkyl, alkenyl, alkynyl, aryl or aralkyl having up to 20 heteroatoms;
- alternatively, R2 and R3 can be bridged so as to form an additional ring fused to the acridinium nucleus;
- C1 and C3 through C8 of the acridinium nucleus can be substituted with substituents as defined by R2 and R3, and wherein the substituents defined by R2 and R3 are identical or different if more than one substituent is used;
- W is an electron-donating substituent that can donate an electron pair selected from the group consisting of OH, SH, NR′R″ and —CHp(EWG)m where p+m=3 and EWG is an electron withdrawing group selected from the group consisting of —NO2, —NO, —CN, —CHO, —C(O)R, +NR′R″R′″, —COOR, —COOH, —S(O)R, —SO2R, —SO2OR, —SO2NHR, —SO2NR′R″, —SO2OH and F;
- R′, R″ and R′″ are hydrogen or low alkyl and can all be the same or different;
- R3 can also be the same as W in which case the acridinium nucleus has two electron donating substituents instead of one;
- a counter-ion to pair with the quaternary nitrogen of the acridinium nucleus, wherein said counter-ion may be part of R1 or may be a group A−;
- X is nitrogen, oxygen or sulfur, such that when X is oxygen or sulfur, Z is omitted and Y is a polysubstituted aryl moiety of the formula:
- where R4 and R8 are alkyl, alkenyl, alkynyl, alkoxyl (—OR), alkylthiol (—SR) or substituted amino groups that serve to stabilize the —COX— linkage between the acridinium nucleus and the Y moiety through steric and/or electronic effect;
- R5 and R7 are any of R2 and R3 as defined above,
- R6=—R9-R10, where R9 is not required but optionally can be a branched or straight-chained alkyl, substituted or unsubstituted aryl or aralkyl containing optionally up to 20 heteroatoms;
- and
- R10 is selected from the group consisting of:
- a halide, —COOH, —Q—R—Nu, —Q—R—(I)nNu—, —Q—Nu, —R—Nu and —Nu, where n is a integer of at least 1, Nu is a nucleophilic group, Q is a functional linkage, and I is an ionic or ionizable group;
- and wherein
- R5 and R6 and/or R6 and R7 are interchangeable;
- and
- when X is nitrogen, then Z is —SO2—Y′, and Y and Y′ are each the polysubstituted aryl moiety defined above, or Y′ is the polysubstituted aryl moiety defined above and Y is a branched or straight-chained alkyl containing optionally up to 20 carbon atoms, halogenated or unhalogenated, a substituted aryl or heterocyclic ring system.