Open AccessLitmus-Body: A Molecularly Targeted Sensor for Cell-Surface pH Measurements
Author(s) -
Joe Chin-Hun Kuo,
Marc C. Goudge,
Ann E. Metzloff,
Lingting Huang,
Marshall J. Colville,
Sangwoo Park,
Warren Zipfel,
Matthew J. Paszek
Publication year - 2020
Publication title -
acs sensors
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.055
H-Index - 57
ISSN - 2379-3694
DOI - 10.1021/acssensors.9b02080
Subject(s) - receptor , endocytic cycle , biophysics , endocytosis , microbiology and biotechnology , chemistry , fluorophore , fusion protein , epidermal growth factor receptor , litmus , antibody , fluorescence , biochemistry , biology , recombinant dna , immunology , physics , quantum mechanics , gene
Precise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the ability to localize and target specific receptor molecules required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-IgG nanobody is capable of piggybacking on IgG antibodies for molecular targeting to specific proteins on the cell surface. By normalizing a pH-dependent green fluorescent protein to a long Stokes shift red fluorophore or fluorescent protein, we readily report pH independent of sensor concentration using a single 488 nm excitation. Our Litmus-body showed excellent responsiveness in solution, with a greater than 50-fold change across the regime of physiological pH. The sensor was further validated for use on live cells and shown to be specific to the protein of interest. In complex with our Litmus-body, cetuximab therapeutic antibody retained its functionality in binding and inhibiting ligand interaction of its target epidermal growth factor receptor (EGFR), triggering receptor-mediated endocytosis that allowed tracking of local pH from the cell surface through the endocytic pathway.