Language

English

Publication Date

6-13-2025

Journal

Biosensors

DOI

10.3390/bios15060382

PMID

40558464

PMCID

PMC12190510

PubMedCentral® Posted Date

6-13-2025

PubMedCentral® Full Text Version

Post-print

Abstract

Microfluidic modulation spectroscopy-infrared (MMS) offers a label-free, high-sensitivity approach for quantifying changes in protein secondary structures under native solution conditions. MMS subtracts the solvent backgrounds from sample signals by alternately flowing proteins and matched buffers through a microfluidic chamber, yielding clear amide I spectra from microliter volumes. In this study, we validated MMS on canonical globular proteins, bovine serum albumin, mCherry, and lysozyme, demonstrating accurate detection and resolution of α-helix, β-sheet, and mixed-fold structures. Applying MMS to the intrinsically disordered protein Tau, we detected environment-driven shifts in transient conformers: both the acidic (pH 2.5) and alkaline (pH 10) conditions increased the turn/unordered structures and decreased the α-helix content relative to the neutral pH, highlighting the charge-mediated destabilization of the labile motifs. Hyperphosphorylation of Tau yielded a modest decrease in the α-helical fraction and an increase in the turn/unordered structures. Comparison of monomeric and aggregated hyperphosphorylated Tau revealed a dramatic gain in β-sheet and a loss in turn/unordered structures upon amyloid fibril formation, confirming MMS's ability to distinguish disordered monomers from amyloids. These findings establish MMS as a robust platform for detecting protein secondary structures and monitoring aggregation pathways in both folded and disordered systems. The sensitive detection of structural transitions offers opportunities for probing misfolding mechanisms and advancing our understanding of aggregation-related diseases.

Keywords

Spectrophotometry, Infrared, Cattle, Animals, Muramidase, Serum Albumin, Bovine, Protein Structure, Secondary, Microfluidics, Proteins, tau Proteins, Biosensing Techniques

Published Open-Access

yes

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