Abstract: | Detection techniques for biosensors often require bulky instruments or cells that are not feasible for in‐field analysis. Our single‐drop cell design, optimized in this work, comprised a screen‐printed three‐electrode (SPE), strip in horizontal position onto which a volume of 100 μL of sample or substrate solution was placed to ensure electrical contact (complete circuit). Together with optimized linear sweep voltammetry (LSV), parameters for the detection of the enzyme alkaline phosphatase (AP), the system was applied to a biosensor for the analysis of polycyclic aromatic hydrocarbons (PAHs), in environmental samples. A limit of detection (LOD), of 0.15 ppb was achieved for a model system with an IC50 value of 0.885 ppb and a linear range (LR), of 0.2–10 ppb. Application of the single drop analysis (SDA), format to a PAH biosensor gave a LOD of 1.4 ppb for detection of phenanthrene with an IC50 value of 29.3 ppb and linear range of 2–100 ppb. Proof of concept is shown with spiked sample analysis of phenanthrene in matrices such as sea, river and tap water. |