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Download fileQuantitative Measurements of Protein Volume and Concentration using Hydrogel-Backed Nanopores
journal contribution
posted on 11.03.2021, 17:05 authored by Reyhaneh Nazarian, Eric Lee, Brian Siegel, Chance Kuo, Shiv Acharya, Jacob SchmidtAccurate
identification and quantification of proteins in solution
using nanopores is technically challenging in part because of the
large fraction of missed translocation events due to short event times
and limitations of conventional current amplifiers. Previously, we
have shown that a nanopore interfaced with a poly(ethylene glycol)-dimethacrylate
hydrogel with an average mesh size of 3.1 nm significantly enhances
the protein residence time within the pore, reducing the number of
missed events. We used hydrogel-backed nanopores to sense unlabeled
proteins as small as 5.5 kDa in size and 10 fM in concentration. We
show that the frequency of protein translocation events linearly scales
with bulk concentration over a wide range of concentrations and that
unknown protein concentrations can be determined from an interpolation
of the frequency-concentration curve with less than 10% error. Further,
we show an iterative method to determine a protein volume accurately
from measurement data for proteins with a diameter comparable to a
nanopore diameter. Our measurements and analysis also suggest several
competing mechanisms for the detection enhancement enabled by the
presence of the hydrogel.
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nanopore diameterdetection enhancementmesh sizeprotein volumetranslocation eventsfrequency-concentration curvebulk concentration5.5 kDaprotein residence time3.1 nmprotein concentrationsevent timeshydrogel-backed nanoporesProtein VolumeHydrogel-Backed Nanoporesprotein translocation eventsQuantitative Measurementsnanopore interfacediterative methodmeasurement data10 fM