Green preparation of graphene oxide-incorporated monoliths for pH-controlled protein enrichment and clinical biomarker detection.

[BACKGROUND] The enrichment of specific proteins is essential for biomarker discovery, mechanistic studies, and the development of diagnostic and therapeutic strategies. In complex biological matrices such as plasma and cell lysates, target proteins are often present at trace levels relative to abundant background proteins, leading to low signal-to-noise ratios and compromised analytical sensitivity. Although affinity-based enrichment methods offer high specificity, they typically depend on prior knowledge of protein epitopes or ligands. Consequently, efficient enrichment of poorly characterized proteins remains a significant analytical challenge that this work aims to address.

[RESULTS] To overcome this limitation, a modified graphene oxide-incorporated monolith, PAS-GO-poly (BMA-co-EDMA) monolith, was fabricated using a green binary porogen system composed of deep eutectic solvents (DESs) and ionic liquids (ILs). The resulting monolith enabled selective protein enrichment through pH-controlled interactions. Its environmental performance, evaluated using a modified GAPI approach, demonstrated excellent sustainability with scores exceeding 92. The PAS-GO-poly (BMA-co-EDMA) monolith exhibited a high adsorption capacity for bovine serum albumin (26.13 mg/g), an enrichment factor of 61.32, and good reproducibility (RSD ≤1.08%). Langmuir isotherm analysis revealed a 55.7% increase in maximum adsorption capacity (R = 0.9629). Compared with the GO-free monolith, the GO-incorporated monolith increased the proportion of proteins with isoelectric point near the loading pH by more than 10% in HeLa cell lysate, and was successfully applied to selectively enrich neuron-specific enolase from small cell lung cancer patient plasma.

[SIGNIFICANCE] This study presents a green PAS-GO-poly (BMA-co-EDMA) monolith that enables selective enrichment of proteins based on isoelectric point through pH control, circumventing key limitations of conventional affinity-based methods. By integrating high adsorption capacity, robustness, and environmental sustainability, the developed platform enhances enrichment of low-abundance proteins in complex biological samples and demonstrates clinical relevance through effective isolation of neuron-specific enolase from patient plasma.