Precise DNA Concentration Measurements with Nanoporesby Controlled Counting
2019
Using a solid-state
nanopore to measure the concentration of clinically
relevant target analytes, such as proteins or specific DNA sequences,
is a major goal of nanopore research. This is usually achieved by
measuring the capture rate of the target analyte through the pore.
However, progress is hindered by sources of systematic error that
are beyond the level of control currently achievable with state-of-the-art
nanofabrication techniques. In this work, we show that the capture
rate process of solid-state nanopores is subject to significant sources
of variability, both within individual nanopores over time and between
different nanopores of nominally identical size, which are absent
from theoretical electrophoretic capture models. We experimentally
reveal that these fluctuations are inherent to the nanopore itself
and make nanopore-based molecular concentration determination insufficiently
precise to meet the standards of most applications. In this work,
we present a simple method by which to reduce this variability, increasing
the reliability, accuracy, and precision of single-molecule nanopore-based
concentration measurements. We demonstrate controlled counting, a
concentration measurement technique, which involves measuring the
simultaneous capture rates of a mixture of both the target molecule
and an internal calibrator of precisely known concentration. Using
this method on linear DNA fragments, we show empirically that the
requirements for precisely controlling the nanopore properties, including
its size, height, geometry, and surface charge density or distribution,
are removed while allowing for higher-precision measurements. The
quantitative tools presented herein will greatly improve the utility
of solid-state nanopores as sensors of target biomolecule concentration.
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