Acid phosphatase detection using a colorimetric probe based on azo compound toward forensic applications for seminal fluid identification

Acid phosphatase (AP) is an enzyme present in various tissues of the human body and is found in high concentrations in seminal fluid. Therefore, it can be easily used as a forensic marker for the presence of semen, assisting criminal investigations and clinical diagnostic tests. This work developed a simple colorimetric method for detecting AP in semen using a probe derived from an azo dye. The proposed method utilizes (E)-4-(phenyldiazenyl)phenyldihydrogen phosphate (AZO1-P) as a substrate, which, upon enzymatic hydrolysis, yields the yellow-colored (E)-4-(phenyldiazenyl)phenol (AZO1) (λ_max = 423 nm). However, for applications involving complex matrices, this coloration is not ideal due to low colorimetric contrast. Thus, a coupling reaction was performed between AZO1 and 4-aminoantipyrine (4-AAP) to improve colorimetric resolution. The product generated provided greater colorimetric contrast (red color, λ_max = 516 nm) and was easily visualized. Under ideal conditions, the method exhibited a linear range of 1–6 U L⁻¹ for AP, with a detection limit of 0.42 U L⁻¹ and a relative standard deviation (RSD) of 3 %. Classical AP inhibitors, such as sodium tungstate and molybdate, were evaluated and showed IC₅₀ values of 11.7 ± 0.5 and 2.9 ± 0.7 mM, respectively. In addition, the method was applied to real samples and against possible interferents that can be found at sex crime scenes, proving to be sensitive and selective for detecting AP in semen (from 1000-fold dilution). Finally, the proposed method can be used for the quantitative and qualitative detection of AP in the laboratory and the field as an alternative to presumptive tests in forensic sciences associated with sex crimes.