Quantitative Determination of Prostate-Specific Antigen in Forensic Samples Using Antibody Microarrays on 3D Plasma Nanotextured Polymeric Slides

Semen identification in forensic samples using prostate-specific antigen (PSA) overcomes limitations of conventional methods such as UV illumination and spermatozoa detection. In this study, we describe an antibody microarray for the quantitative immunochemical detection of PSA in forensic samples using 3D oxygen plasma-nanotextured polymer-coated glass slides. The analytical performance of these substrates was compared with that of silane-modified glass slides and demonstrated improved intra- and inter-spot homogeneity, as well as an extended linear dynamic range. Based on the 3D nanotextured slides, a two-step non-competitive immunoassay was developed using a biotinylated anti-PSA antibody and Alexa Fluor 546-labeled streptavidin for fluorescence detection. The assay was completed within 1 h, achieved a detection limit of 0.05 ng/mL PSA, and enabled semen detection at dilutions of up to 2 × 106. Incorporation of biotinylated bovine serum albumin spots as internal positive controls reduced slide-to-slide fluorescence variability, allowing cost-effective analysis of 24 samples per slide. The method was successfully applied to the detection of semen stains on spiked swabs and textile substrates of different compositions following a 1 h extraction step, demonstrating its suitability for rapid semen detection in forensic casework.