Banca de DEFESA: BEATRIZ DO NASCIMENTO OLIVEIRA

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : BEATRIZ DO NASCIMENTO OLIVEIRA
DATE: 27/07/2023
TIME: 09:00
LOCAL: Google Meet
TITLE:

Development of a colorimetric method for detection of acid phosphatase aiming forensic applications.

KEY WORDS:

Sexual crimes; Acid phosphatase; Colorimetric detection; Forensic chemistry

PAGES: 80
BIG AREA: Ciências Exatas e da Terra
AREA: Química
SUMMARY:

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 of biological fluids such as blood, saliva and semen is one of the crucial steps in the analysis of crime scenes or samples for forensics. The presence of seminal fluid (semen) on the victim or on clothing, for example, may constitute criminal evidence. Semen has a complex composition, however, the enzyme acid phosphatase (AP) can be used as a marker, since it has concentrations in seminal fluid up to 400 times higher compared to other body fluids. In this sense, the objective is to develop a methodology with 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 helping to 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 0.50 to 3.0 U L-1 , corresponding to the equation A710 nm = 0.32×CAP + 0.016 (n = 8, r = 0.9964) and LOD = 0.004 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 (CAAE: 58121322.7.0000.5013) 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), among the analyzes the PAD presented better condition since it was possible to monitor the intensity of the color using the volume of 0.5 µL of the sample. In addition, the method was evaluated with possible interferences such as saliva, blood, urine, lubricant, egg white, tomato sauce, pineapple juice and milk, for this test the tomato sauce presented the values closest to the seminal sample, due to tomatoes being rich in ascorbic acid. Finally, the proposed method proved to be applicable in different situations, including a double response of the system in relation to acid phosphatase enzyme as well as reduced ascorbic acid, being an alternative to use in field applications, and contributing to a quick decision making when necessary.

BANKING MEMBERS:
Externo(a) à Instituição - JOSÉ LUIZ DA COSTA
Interno(a) - 1869296 - CINTYA D'ANGELES DO ESPIRITO SANTO BARBOSA
Presidente - 1613338 - JOSUE CARINHANHA CALDAS SANTOS