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Two color DNA barcode detection in photoluminescence suppressed silicon nitride nanopores.

ABSTRACT: Optical sensing of solid-state nanopores is a relatively new approach that can enable high-throughput, multicolor readout from a collection of nanopores. It is therefore highly attractive for applications such as nanopore-based DNA sequencing and genotyping using DNA barcodes. However, to date optical readout has been plagued by the need to achieve sufficiently high signal-to-noise ratio (SNR) for single fluorophore sensing, while still maintaining millisecond resolution. One of the main factors degrading the optical SNR in solid-state nanopores is the high photoluminescence (PL) background emanating from the silicon nitride (SiNx) membrane in which pores are commonly fabricated. Focusing on the optical properties of SiNx nanopores we show that the local membrane PL intensity is substantially reduced, and its spectrum is shifted toward shorter wavelengths with increasing e-beam dose. This phenomenon, which is correlated with a marked photocurrent enhancement in these nanopores, is utilized to perform for the first time single molecule fluorescence detection using both green and red laser excitations. Specifically, the reduction in PL and the concurrent measurement of the nanopore photocurrent enhancement allow us to maximize the background suppression and to detect a dual color, five-unit DNA barcode with high SNR levels.

SUBMITTER: Assad ON

PROVIDER: S-EPMC4296929 | biostudies-literature |

REPOSITORIES: biostudies-literature

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K(+) , Na(+) , and Mg(2+) on DNA translocation in silicon nitride nanopores.

Project description:In this work, we report on how salt concentration and cation species affect DNA translocation in voltage-biased silicon nitride nanopores. The translocation of dsDNA in linear, circular, and supercoiled forms was measured in salt solutions containing KCl, NaCl, and MgCl(2) . As the KCl concentrations were decreased from 1 to 0.1 M, the time taken by a DNA molecule to pass through a nanopore was shorter and the frequency of the translocation in a folded configuration was reduced, suggesting an increase in DNA electrophoretic mobility and DNA persistence length. When the salt concentration was kept at 1 M, but replacing K(+) with Na(+) , longer DNA translocation times (t(d) ) were observed. The addition of low concentrations of MgCl(2) with 1.6 M KCl resulted in longer t(d) and an increased frequency of supercoiled DNA molecules in a branched form. These observations were consistent with the greater counterion charge screening ability of Na(+) and Mg(2+) as compared to K(+) . In addition, we demonstrated that dsDNA molecules indeed translocated through a ∼10 nm nanopore by PCR amplification and gel electrophoresis. We also compared the dependence of DNA mobility and conformation on KCl concentration and cation species measured at single molecule level by silicon nitride nanopores with existing bulk-based experimental results and theoretical predictions.

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Lifetime and Stability of Silicon Nitride Nanopores and Nanopore Arrays for Ionic Measurements.

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Covalent Modification of Silicon Nitride Nanopore by Amphoteric Polylysine for Short DNA Detection.

Project description:In this work, we demonstrate a chemical modification approach, by means of covalent-bonding amphoteric poly-l-lysine (PLL) on the interior nanopore surface, which could intensively protect the pore from etching when exposed in the electrolyte under various pH conditions (from pH 4 to 12). Nanopore was generated via simple current dielectric breakdown methodology, covalent modification was performed in three steps, and the functional nanopore was fully characterized in terms of chemical structure, hydrophilicity, and surface morphology. I-V curves were recorded under a broad range of pH stimuli to evaluate the stability of the chemical bonding layer; the plotted curves demonstrated that nanopore with a covalent bonding layer has good pH tolerance and showed apparent reversibility. In addition, we have also measured the conductance of modified nanopore with varied KCl concentration (from 0.1 mM to 1 M) at different pH conditions (pHs 5, 7, 9, and 11). The results suggested that the surface charge density does not fluctuate with variation in salt concentration, which inferred that the SiN x nanopore was fully covered by PLL. Moreover, the PLL functionalized nanopore has realized the detection of single-stranded DNA homopolymer translocation under bias voltage of 500 mV, and the 20 nt homopolymers could be evidently differentiated in terms of the current amplitude and dwell time at pHs 5, 8, and 11.

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| S-EPMC4811674 | biostudies-literature

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