Controlling serum protein depletion in aqueous biphasic systems: interphase precipitation versus bottom phase retention for bisphenol A monitoring

BACKGROUND: Human serum is a clinically relevant yet analytically challenging matrix. Its high abundance of proteins interferes with the detection of low-concentration biomarkers and environmental contaminants, including bisphenol A (BPA), an endocrine disruptor monitored as a marker of human exposure. High-abundance proteins introduce matrix effects that can enhance, distort or suppress analytical signals, ultimately compromising assay performance. Aqueous biphasic systems (ABS) provide a promising pretreatment strategy to address these limitations by selectively depleting high-abundance proteins and generating a cleaner matrix that supports effective analyte recovery and improved detection sensitivity. RESULTS: In this work, a dual-strategy ABS platform was introduced and shown to deliberately direct protein depletion via two complementary mechanisms: (i) interphase precipitation, where serum proteins aggregate at a solid interphase, and (ii) bottom phase retention, where proteins accumulate in the salt-rich bottom phase. By combining polypropylene glycol (PPG) with either cholinium or sodium salts, the salt composition tunes phase behavior, selectively depletes high-abundance proteins, and enhances BPA recovery. Sodium salts generally outperformed cholinium salts due to stronger hydration and salting-out effects. Between the two implemented strategies, systems promoting interphase precipitation achieved up to 94% protein depletion, while simultaneously driving BPA into the largely protein-depleted PPG-rich top phase, minimizing matrix bias and enabling more efficient detection. SIGNIFICANCE: This work demonstrates, for the first time, that rational control of ABS composition can selectively switch between protein precipitation and liquid-liquid partition, establishing a versatile and tunable serum pretreatment platform. This approach enables efficient depletion of high-abundance proteins and sensitive recovery of BPA and potentially other low-concentration analytes. Supported by green and blue analytical chemistry metrics, it represents a significant advance in bioanalytical workflows and environmental exposure assessment, while offering a more sustainable alternative to conventional methods.