Controlling serum protein depletion in aqueous biphasic systems: interphase precipitation versus bottom phase...

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.

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.

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.