Method for detecting analyte particles present in trace amounts in fluid sample

A method for detecting an analyte present in trace amounts in a fluid sample, the method comprising:

• causing a fluid sample containing an analyte to flow over a substrate having an array of microchambers formed on a surface thereof, with a first capture substance that specifically binds to the analyte immobilized in the microchambers;
• binding the analyte to the first capture substance in each microchamber of the array of microchambers;
• causing a second capture substance, which specifically binds to the analyte and binds to a signal-generating substance, to flow over the substrate to react the analyte with the second capture substance;
• causing the signal-generating substance to flow over the substrate to bind to the second capture substance;
• causing a substrate solution, which reacts with the signal-generating substance to generate a fluorescence signal, to flow over the substrate;
• causing a hydrophobic solvent to flow over the substrate to remove the substrate solution present outside the microchambers when the signal-generating substance and the substrate solution react in the microchambers;
• and
• counting and detecting the number of microchambers in which the fluorescence signal is generated.