Abstract
Nanopore sensing is a promising tool well suited to capture and detect DNA and other single molecules. DNA is a negatively charged biomolecule that can be captured and translocated through a constricted nanopore aperture under an applied electric field. Precise assessment of DNA concentration is of crucial importance in many analytical processes and medical diagnostic applications. Recently, we found that hydrodynamic forces can lead to DNA motion against the electrophoretic force (EPF) at low ionic strength. This study utilized glass nanopores to investigate the DNA capture mechanism and detect DNA molecules due to volumetric flow at these low ionic strength conditions. We measured the DNA capture rate at five different pico-molar concentrations. Our findings indicated that the translocation rate is proportional to the concentration of DNA molecules and requires no calibration due to the volumetric flow rate and DNA counting directly correlates with concentration. Using finite element analysis, we calculated the volumetric flow and proposed a simple, straightforward approach for accurate DNA quantification. Furthermore, these experiments explore a unique transport mechanism where one of the most highly charged molecules enters a pore against electric field forces. This quantitative technique has the potential to provide distinct insight into nanopore-based biosensing and further enhance the nanopore’s capability as a biomolecule concentration sensor.
Highlights
Classical electrostatics play a significant role in molecular biology
The direction of fluid flow velocity was toward the pore in negative applied voltages, which resulted in electroosmotic flow (EOF)-driven DNA captures
Our findings indicate that the electrophoretic force (EPF) decayed faster in comparison to EOF, which results in the dominance of the EOF inside the nanopore
Summary
Classical electrostatics play a significant role in molecular biology. The strength and unique structure of molecules are due to the electrostatic forces. The electric field direction indicates a spherical shape surrounding the pore mouth in EPF dominant capture zone at high salt (Figure 1B) We investigated both contributed factors at low ionic strength (10 mM KCl) using finite element analysis. The direction of fluid flow velocity was toward the pore in negative applied voltages, which resulted in EOF-driven DNA captures. We performed the λ-DNA translocation experiment at low ionic strength (10 mM KCl solution) and calculated the mean capture rate under multiple applied voltages. There was a volume fluid flow; quantifying the DNA concentration were achieved by dividing the flow rate (nm3s−1) over the number of DNA events (s−1) captured by the nanopore sensor. The number of EOFdriven translocation events recorded per each analysis was 932, 1445, and 994 for 100, 500, and 1000 pM concentration of DNA, respectively
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