Presumptive semen detection using fructose as a forensic marker: comparison of colorimetric solution assays and paper-based analytical devices

Abstract

Identification of biological fluids is a fundamental step in criminal investigations, especially in sexual assault cases, which have a high global incidence. To prove the crime and identify the perpetrator, biological samples such as semen are collected from the crime scene or from the victim. Thus, this study aimed to develop an analytical methodology for detecting fructose (a chemical marker) in solutions and in paper-based analytical devices (PADs). The colorimetric response is due to the presence of fructose, which results in the formation of a blue chromophore (λ = 625 nm). Various parameters were optimized to improve the method's detectability and sensitivity, enabling a linear response over 0.5–3 mg L−1 (solution) and 12.5–150 mg L−1 (PAD). The limits of detection (LOD) were 0.17 mg L−1 and 6.25 mg L−1, respectively, demonstrating that the method fully meets the intended purpose. In addition, the methodologies showed recoveries between 91 and 102% and RSD ≤ 3.8%, evidencing the method's accuracy and precision. The methodologies evaluated on real samples demonstrated high sensitivity and reliability, even in the presence of potential interferences. A qualitative assessment (n ≥ 60) confirmed the strong performance of the assays, with a minimum sensitivity of 98%. Consequently, both methodologies allow for rapid qualitative and quantitative analyses directly at crime scenes, addressing key limitations of commercially available tests, particularly regarding response time and the risk of false positives. Moreover, these approaches have the potential to enhance public security by significantly reducing the time required to resolve criminal cases.