Applications of acridinium compounds and derivatives in homogeneous assays

1. A homogenous assay method for detection or quantitation of an analyte in a sample, comprising the steps of:

(a) providing a conjugate comprising a chemiluminescent acridinium compound attached to an analyte or analyte analog, said acridinium compound comprising a carboxyl group at the C-9 position of the acridinium nucleus which is linked to a substituted aryl moiety to form a phenolic ester, acridinium compound having electron donating functional groups at the C2 and/or C7 position on the acridinium nucleus, the presence of which inhibit pseudo-base formation;

(b) adding a predetermined amount of said conjugate to a sample containing an unknown concentration of said analyte;

(c) adding an antibody specific to said analyte to form a binding complex with either said analyte or said conjugate;

(d) incubating the solution of said binding complex;

(e) triggering the chemiluminescence of said binding complex reaction mixture by adding peroxide in a pH range of about 6 to 10 to cause light emission without the addition of a strong acid or a strong base;

(f) measuring the amount of light emission; and

(g) calculating the concentration of said analyte by comparing the amount of light emitted from the reaction mixture with a standard dose response curve which relates the amount of light emitted to a known concentration of said analyte.

2. The assay of claim 1 , wherein said conjugate is formed by reacting said analyte with a chemiluminescent acridinium compound having the following structure:

wherein R 1 is an alky, alkenyl, alkynyl or aralkyl, each of which may contain up to 20 heteroatoms; or a sulfopropyl or sulfobutyl group;

where R 4 and R 5 are the same or different and are selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxyl (—OR), alkylthiol (—SR), or substituted amino groups (—NRA 2 ); where R is independently selected from alkyl, alkenyl, alkynyl, aryl, or aralkyl each containing up to 20 heteroatoms;

R 5 and R 7 are hydrogen or the same as R;

R 6 =—R 9 -R 10 ,

where R 9 is not required or is a branched or straight-chained alkyl, substituted or unsubstituted aryl or aralkyl, each containing up to 20 heteroatoms, and

R 10 is a leaving group or an electrophilic functional group attached with a leaving group selected from the group consisting of:

SO 2 Cl, —N 3 , —N 2 + Cl; a halide, or a carboxylic acid,

R 5 and R 6 , and R 6 and R 7 are interchangeable;

W1 and W2 are the same or different and are electron-donating functional groups selected from the group consisting of OR*, OH, SR*, SH, NH 2 , and NR′R″, wherein R*, R′ and R″ can be the same or different, and are selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl, each containing up to 20 heteroatoms;

A − is a counter ion which is introduced to pair with the quaternary nitrogen of said acridinium nucleus, and is selected from the group consisting of CH 3 SO 4 − , FSO 3 − , CF 3 SO 4 − , C 4 F 9 SO 4 − , CH 3 C 6 H 4 SO 3 − , halide, CF 3 COO − , CH 3 COO − , and NO 3 − .

3. The assay of claim 2 , wherein the acridinium compound has the structure:

wherein R 11 is selected from the group consisting of: —OH,

and —NH—R—NH 2 .

4. The assay of claim 2 , wherein the acridinium compound has the structure:

wherein R 11 is selected from the group consisting of: —OH,

and —NH—R—NH 2 ;

and R 12 is selected from the group consisting of hydrogen, amino, hydroxyl, halide, nitro, —CN, —SO 3 H, —SCN, —OR, NHCOR, —COR, —COOH, —COOR, or —CONHR.

5. The assay of claim 1 , wherein the analyte of interest is a small molecule selected from the group consisting of steroids, therapeutic drugs, vitamins, hormones and peptides.

6. The assay of claim 1 , wherein the peroxide is hydrogen peroxide, sodium peroxide, or bivalent peroxide salts.

7. The method of claim 1 , wherein the electron donating functional groups are selected from groups consisting of OR, OH, SR, SH, NH 2 , NR′R″; wherein R, R′ and R″ can be the same or different, and are selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl, each containing up to 20 heteroatoms.

8. The method of claim 7 , wherein said electron donating functional group is attached to the C2 position on the acridinium nucleus.

9. The method of claim 7 , wherein said electron donating functional groups are attached to the C2 and C7 positions on the acridinium nucleus.

10. The assay of claim 1 , wherein said acridinium compound exhibits a greater chemiluminescent efficiency when in said binding complex than when free in solution.