PET Foils Functionalized with Reactive Copolymers as Adaptable Microvolume ELISA Spot Array Platforms for Multiplex Serological Analysis of SARS-CoV-2 Infections

Microvolume ELISA platforms have become vital in diagnostics for their high-throughput capabilities and minimal sample requirements. High-quality substrates with advanced surface properties are essential for these applications. They enable both efficient biomolecule immobilization and antifouling properties, which are critical for assay sensitivity and specificity. This study presents PET-based microvolume ELISA spot arrays coated with amine- and DBCO-reactive copolymers MCP-2 and Copoly Azide. The platforms were designed for the sensitive and specific detection of specific antibodies such as COVID-19 biomarkers. Supporting robust attachment of the SARS-CoV-2 nucleoprotein (NP), these arrays outperform traditional approaches. It was demonstrated that covalent attachment methods proved more efficient than passive adsorption, together with the reduction of non-specific binding. Analytical performance was verified with classical ELISA and real-time Surface Plasmon Resonance (SPR) analysis. It enables sensitive detection of IgG and IgA antibodies, including IgG subclasses, in human serum. Clinically, the platform achieved 100.0% sensitivity and 92.9% specificity for anti-NP antibody detection in COVID-19-positive and negative samples. Additionally, DNA-directed immobilization extended the platform's utility to multiplex serological measurements. These findings underscore the potential of PET-based microvolume ELISA arrays as scalable, high-throughput diagnostic tools suitable for detecting multiple biomarkers in a single assay and easily integrated into microfluidic devices.