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

IF 4.1 3区工程技术 Q2 CHEMISTRY, APPLIED

Dyes and Pigments Pub Date : 2025-04-06 DOI:10.1016/j.dyepig.2025.112806

Jéssica Raimundo da Rocha, Marcone Gomes dos Santos Alcântara, Verônica Diniz da Silva, Dimas José da Paz Lima, Josué Carinhanha Caldas Santos

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引用次数: 0

Abstract

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.

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基于偶氮化合物的比色探针检测酸性磷酸酶在精液鉴定中的法医应用

酸性磷酸酶（AP）是一种存在于人体各种组织中的酶，在精液中发现浓度很高。因此，它可以很容易地用作精液存在的法医标记，协助刑事调查和临床诊断测试。本工作开发了一种简单的比色法，使用偶氮染料衍生的探针检测精液中的AP。所提出的方法利用(E)-4-（苯基二氮基）磷酸苯二氢（AZO1- p）作为底物，经酶解产生黄色的(E)-4-（苯基二氮基）苯酚（AZO1）（λmax = 423 nm）。然而，对于涉及复杂矩阵的应用，由于比色对比度低，这种着色不理想。因此，AZO1与4-氨基安替比林（4-AAP）之间进行了偶联反应，以提高比色分辨率。所生成的产物具有较好的比色对比度（红色，λmax = 516 nm），并且易于可视化。在理想条件下，该方法对AP的检测范围为1 ~ 6 U L−1，检测限为0.42 U L−1，相对标准偏差（RSD）为3%。经典AP抑制剂，如钨酸钠和钼酸钠，其IC50值分别为11.7±0.5和2.9±0.7 mM。此外，该方法被应用于真实样本和可能在性犯罪现场发现的干扰物，证明对检测精液中的AP（从1000倍稀释）具有敏感性和选择性。最后，所提出的方法可用于实验室和现场的AP定量和定性检测，作为与性犯罪相关的法医学推定测试的替代方法。

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来源期刊

Dyes and Pigments 工程技术-材料科学：纺织

CiteScore

8.20

自引率

13.30%

发文量

933

审稿时长

33 days

期刊介绍： Dyes and Pigments covers the scientific and technical aspects of the chemistry and physics of dyes, pigments and their intermediates. Emphasis is placed on the properties of the colouring matters themselves rather than on their applications or the system in which they may be applied. Thus the journal accepts research and review papers on the synthesis of dyes, pigments and intermediates, their physical or chemical properties, e.g. spectroscopic, surface, solution or solid state characteristics, the physical aspects of their preparation, e.g. precipitation, nucleation and growth, crystal formation, liquid crystalline characteristics, their photochemical, ecological or biological properties and the relationship between colour and chemical constitution. However, papers are considered which deal with the more fundamental aspects of colourant application and of the interactions of colourants with substrates or media. The journal will interest a wide variety of workers in a range of disciplines whose work involves dyes, pigments and their intermediates, and provides a platform for investigators with common interests but diverse fields of activity such as cosmetics, reprographics, dye and pigment synthesis, medical research, polymers, etc.