Colorimetric kit for presumptive detection of semen using ascorbic acid and acid phosphatase as (bio)chemical markers

The identification of biological fluids is a crucial step in forensic investigations. Semen is the most relevant bodily fluid in cases of sexual assault. Semen composition includes (bio)chemical components, such as the enzyme acid phosphatase (AP) and reduced ascorbic acid (AAᵣ), both of which are considered important markers of seminal fluid. In this work, a colorimetric kit was developed for the dual detection of AP and AAᵣ. For AP detection, ascorbic acid 2-phosphate (AAP) was employed as a substrate; in the presence of the enzyme (pH 5), it is hydrolyzed to inorganic phosphate and AAᵣ, which were subsequently used to monitor enzymatic activity. The AAᵣ, in the presence of Fe(III) and K₃Fe(CN)₆ under acidic conditions, leads to the formation of Prussian Blue (PB, Fe₄[Fe(CN)₆]₃), a dark blue colloid (λ = 710 nm). Linear range of 0.25-3.0 U L^-1 was obtained for AP detection, with a limit of detection (LOD) of 0.04 U L^-1. Similarly, AAᵣ could be quantified, yielding a linear range of 5-40 μM and LOD of 0.29 μM. Recovery assays (AAᵣ and AP) demonstrated quantitative performance, with recoveries ranging from 81 to 101% (RSD < 4%). The kit was qualitatively evaluated against potential interferents (biological fluids and food samples), with sensitivity, specificity, and efficiency exceeding 88%. The proposed kit demonstrated applicability both at crime scenes (in loco) and in laboratory environments, enabling rapid screening and supporting forensic decision- making.