Methods, means and kits for detecting scarce analytes

The invention relates methods, means and kits for detecting and quantitation of chemical and biological analytes using catalytic signal amplification. Provided are methods, kits, and reagents for detecting a target molecule or interacting target molecules in a sample based on catalytic signal amplification, comprising the steps of (i) providing at least a first non-fluorescent and a second non-fluorescent probe capable of binding to distinct binding sites on the target molecule(s), the first probe being provided with a first ligand and the second probe being provided with a second ligand, wherein the first and second ligand can form a catalytic complex in the presence of a catalytic compound and wherein the catalytic complex can catalyze a chromogenic reaction; (ii) contacting a sample known or suspected to contain the target molecule(s) with said at least a first and second non-fluorescent probe under conditions that allow for binding of the probes to the target molecule(s) such that, upon binding of the probes to the target molecule(s), the first and second ligand are brought in close proximity of each other and together with the catalytic compound form a catalytic complex immobilized on the target molecule(s); and (iii) detecting the formation of the catalytic complex based on a non-enzymatic conversion of a water- soluble precursor dye into a chromophoric reporter dye