Desenvolvimento de método colorimétrico para determinação da fosfatase ácida visando aplicações forenses

Abstract

Sexual crimes are those that threaten sexual dignity and freedom, characterized as a form of violence with a higher incidence of female gender. In this way, the identification biological fluids such as blood, saliva and semen are one of the crucial steps in the analysis of samples or crime scenes for forensics. Among these fluids, the presence of seminal fluid (semen) may constitute criminal evidence when found on the victim or on clothing, for example. Semen has a complex composition, however, the enzyme acid phosphatase (AP) can be used as a marker, since it has concentrations in seminal fluid upto 400 times higher compared to other body fluids. In this sense, the objective of this workwas to develop a methodology for colorimetric detection of AP using a small amount of sample, being able to be used in the field, with a quick and selective response, thus helpingto confirm evidence of sexual crimes. The proposed method is based on the detection of AP using ascorbic acid 2-phosphate (AAP) as substrate, which, in the presence of the enzyme, releases inorganic phosphate and reduced ascorbic acid (AAr) at pH 5.0, which is used to detect the enzyme in the medium. In this case, AAr reacts with Fe(III) ions leading to the formation of Fe(II) and oxidized ascorbic acid (AAo). Therefore, the reaction of Fe(II) ions generated in situ with K3[Fe(CN)6] in acid medium was explored,leading to the formation of Prussian blue (PB) (Fe4[Fe(CN)6]3), a dark blue colloid (λ = 710 nm). The use of PB as a colorimetric probe with λmax > 500 nm avoids possible spectral interference due to the composition of complex samples. Initially, the main reaction parameters were optimized using AAr, under the best analytical performance conditions Fe(III) at 1.5 µM and K3Fe(CN)6 at 1.5 mM were used, both in 0.45 M HNO3.To improve the stability of the PB generated, polyvinylpyrrolidone (PVP) 0.20% (w/v) was used. Then, the optimized conditions in the detection of AP were applied, using AAP (0.75 mM) as substrate at pH 5.0 (HAc/NaAc, 100 mM). Under these conditions, an analytical curve for AP was obtained, with a linear range from 0.25 to 3.0 U L-1 , corresponding to the equation A710 nm = 0.32 (±0.01) × CAP + 0.002 (±0.017) (n = 9, r = 0.9960) and LOD = 0.04 U L-1 , waiting for a time of 45 minutes. As a proof of concept, the method was successfully applied on a real semen sample for a qualitative evaluation after semen dilution in solution, applied on the surface of a tissue (cotton), swab and on the analytical device paper-based (PAD). The PAD presented a better condition since it was possible to monitor the color intensity using a volume of 0.5 µL of the sample. Furthermore, it was possible to determine figures of merit for ascorbic acid, a second marker species, in solution (LOD = 0.29 µM) and in PAD (LOD = 48.53 µM). In this line, AP enzyme was also evaluated in PAD with LOD = 18 .55 UL-1 . In addition, the method was evaluated against possible interferences such as saliva, blood, urine, lubricant, egg white, tomato sauce, pineapple juice and milk, for this test tomato sauce and pineapple juice showed values similar to the sample seminal, considering the ascorbicacid content, while for ascorbic acid it was egg white and tomato sauce. In the recovery assay for the ascorbic acid, values from 81 to 100%, were obtained, while AP ranged from 82 to 101%, both with RSD less than 4%. Finally, the proposed method proved to be capable of being applied in different situations, including a double response in relationto AP as well as reduced ascorbic acid, being a viable alternative for field applications, and contributing to a quick decision making when necessary.