**Background:** Food allergy is a growing global health problem, with dietary lectins posing a significant threat to consumers. While some allergenic lectins are regulated for labeling, many are not, creating a need for reliable analytical methods to detect and quantify allergenic proteins in foods. Stir bar sorptive extraction (SBSE) has been successfully applied to small organic compounds but had not been reported for large biomolecules like proteins, due to fouling issues and lack of selective coatings. Aptamers—single-stranded DNA or RNA ligands—offer high selectivity, stability, reproducibility, and low cost compared to antibodies or molecularly imprinted polymers. This study aimed to develop the first aptamer-functionalized SBSE coating for selective isolation and enrichment of the model allergenic lectin concanavalin A (Con A) from food samples, with detection by MALDI-TOF-MS.
**Methods:** Commercial polytetrafluoroethylene (PTFE) stir bars were chemically etched with sodium naphthalene (Fluoroetch®) to introduce hydroxyl groups, then vinylized via reaction with glycidyl methacrylate (GMA). A thiol-modified aptamer against Con A was covalently attached via "thiol-ene" click chemistry. Coupling efficiency was optimized by measuring aptamer concentration before and after immobilization over 30 min to 7.5 h. The coating was characterized by FT-IR spectroscopy and phosphorus determination by ICP-MS. The SBSE protocol was optimized for loading solution pH, extraction time (30 min), stirring rate (600 rpm), temperature (25 °C), and elution conditions (100 mM NH₄OH, pH 11.2, 45 min, 25 °C). MALDI-TOF-MS analysis was performed in positive ion mode over 5000–30,000 m/z using a sinapinic acid matrix with a dried-droplet multilayer method.
**Key Results:** Aptamer coupling reached a plateau at 5 h with approximately 80% coupling efficiency. FT-IR confirmed successful vinylization and aptamer immobilization, and ICP-MS showed significantly higher phosphorus content in aptamer-functionalized coatings (0.0498 ± 0.0008%) versus controls (0.0055 ± 0.0003%). Three Con A proteoforms were identified: α-chain (Mr theo 25,598.19), β-chain (Mr theo 12,936.36), and γ-chain (Mr theo 12,679.85). The method was linear from 1.5 to 50 μg mL⁻¹ (R² > 0.996). Limits of detection were 0.16–0.18 μg L⁻¹ for individual proteoforms (overall LOD 0.5 μg L⁻¹), and limits of quantification were 0.48–0.55 μg L⁻¹ (overall LOQ 1.5 μg L⁻¹). Inter-day precision (single stir bar) ranged from 2.7 to 4.5% RSD, intra-batch precision was <5.5% RSD, and inter-batch precision was <7.6% RSD. In selectivity tests using a mixture of Con A (5 μg mL⁻¹) with peanut agglutinin, phytohemagglutinin-L, and Pisum sativum agglutinin (each at 10 μg mL⁻¹), only Con A proteoforms were detected after SBSE. In spiked food samples (chickpea flour, lentil flour, wheat flour, white beans) at 5 mg Con A per 100 g food, recoveries ranged from 81 to 97% across all matrices and proteoforms. The stir bars were reusable for at least 10 cycles with standard solutions and 5 cycles with food extracts (recoveries >80%). Storage stability showed >85% recovery after 2 months at 4 °C in water. Up to 10 stir bars could be modified simultaneously in ~12 h, with an estimated cost of 0.6 €/stir bar for modification and up to 16 €/stir bar after aptamer functionalization.
**Clinical Implications:** This method enables selective, sensitive, and accurate detection of allergenic lectins in food matrices at levels relevant to health-based reference doses for allergenic proteins. The aptamer-functionalized SBSE platform addresses critical gaps in protein extraction from complex food samples, offering resistance to fouling and clogging that plague other microextraction devices. By demonstrating successful application to Con A detection in white beans, chickpea, lentil, and wheat flours, the method provides a template that can be adapted to other allergenic proteins using corresponding aptamers. The combination of selective SBSE with MALDI-TOF-MS identification prevents false positives and erroneous quantifications that can occur with non-MS-based biosensors. This approach has broad applicability in agri-food safety, clinical diagnostics, and environmental monitoring for large biomolecules.