Abstract: An optical sensor is used to detect a target in a sample liquid. The optical sensor has an optical sensing layer including, in the same layer, a fluorescent reporter, a fluorescent reference, and an optical isolating reagent. The optical sensing layer has an outer surface that contacts the sample liquid and an opposing surface through which a light source irradiates the optical sensing layer with excitation light, and a detector detects fluorescence emitted from within the optical sensing layer. The optical isolating reagent reduces the amount of (i) excitation light that passes through the optical sensing layer and reaches the sample liquid and (ii) background fluorescence that is emitted within the sample liquid and passes back through the optical sensing layer to the detector. Accordingly, the optical reporter and optical reference fluorescence can be detected with higher signal to noise ratios than in the absence of the optical isolating reagent.