Desenvolvimento de metodologias analíticas para detecção de marcadores forenses em fluidos biológicos associados a crimes sexuais

Abstract

The identification of bodily fluids is a crucial step in criminal investigations, especially in sexual assault cases, which have a high incidence rate. According to the World Health Organization (WHO), it is estimated that one in three women worldwide has experienced physical and/or sexual violence. To confirm rape and identify the perpetrator, biological samples such as semen are collected from the crime scene or the victim. In this context, various methodologies have been evaluated to overcome the limitations of conventional assays, such as false-positive results. Therefore, techniques for identifying markers present in semen have been investigated. This study aimed to employ different methodologies, including solution-based systems, microfluidic paper-based analytical devices (μPADs), and a Sequential Injection Analysis (SIA) system, by exploring the colorimetric response variation of reactive systems in the presence of fructose, zinc ions, and acid phosphatase through the generation of characteristic chromophores. Several parameters were optimized during the development of these methods to enhance selectivity and sensitivity. For fructose detection, limits of detection (LOD) of 0.64 mg L⁻¹ and 6.25 mg L⁻¹ were obtained in solution and on the μPAD, respectively. For zinc ion detection, different azo dyes [2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (zincon), 1-(2-pyridylazo)-2-naphthol (PAN), 2-(5-bromo-2-pyridylazo)-5-(diethylamino)phenol (Br-PADAP) and 4-(2-pyridylazo) resorcinol (PAR)] were evaluated both in solution and on the μPAD, showing variations in sensitivity, but all were suitable for application in real samples. Among them, PAN exhibited the best analytical performance in both methodologies, with LODs of 0.05 µM (solution) and 3.44 µM (μPAD), and demonstrated 100% sensitivity in qualitative validation. Due to its higher sensitivity in solution, PAN was selected for use in the SIA system. The SIA approach aimed at the dual detection of acid phosphatase (AP) and zinc ions using 4-aminoantipyrine (4-AAP) and PAN, respectively. The SIA system showed high sensitivity and precision, with an LOD of 9.4 U L⁻¹ for AP and 3.1 μM for Zn(II). All applied methodologies presented relative standard deviations (RSD) below 3.8% and recoveries ranging from 88% to 120%, confirming their adequacy for the proposed application. Evaluation with real samples (semen, blood, menstrual blood, vaginal fluid, saliva, and urine) was conducted for the different markers, showing good sensitivity and reliability, allowing both qualitative and quantitative analyses with minimal sample volumes directly at the crime scene. These methodologies aim to overcome the limitations of commercially available kits, particularly regarding response time and false positives, thus representing a scientific innovation that can contribute to the resolution of sexual assault cases.