title: Probing solid-state nanopores with light for the detection of unlabeled analytes.
abstract: Nanopore sensing has enabled label-free single-molecule measurements on a wide variety of analytes, including DNA, RNA, and protein complexes. Much progress has been made toward biotechnological applications; however, electrically probing the ion current introduces nonideal noise components. Here we further develop a method to couple an ionic current to a photon-by-photon counting of fluorescent signal from Ca(2+)-sensitive dyes and demonstrate label-free optical detection of biopolymer translocation through solid-state nanopores using TIRF and confocal microscopy. We show that by fine adjustment of the CaCl2 gradient, EGTA concentration, and voltage, the optical signals can be localized to the immediate vicinity of the pore. Consequently, the noise spectral density distribution in the optical signal exhibits a nearly flat distribution throughout the entire frequency range. With the use of high-speed photon counting devices in confocal microscopy and higher photon count rates using stronger light sources, we can improve the signal-to-noise ratio of signal acquisition, while the use of wide-field imaging in TIRF can allow for simultaneous quantitative imaging of large arrays of nanopores.
authors.affiliation: Department of Biomedical Engineering Boston University Boston, Massachusetts 02215, United States.
authors.forename: Brett N
authors.initials: BN
authors.lastname: Anderson
authors.forename: Ossama N
authors.initials: ON
authors.lastname: Assad
authors.forename: Tal
authors.initials: T
authors.lastname: Gilboa
authors.forename: Allison H
authors.initials: AH
authors.lastname: Squires
authors.forename: Daniel
authors.initials: D
authors.lastname: Bar
authors.forename: Amit
authors.initials: A
authors.lastname: Meller
mesh_terms: D001704:Biopolymers / Q000737:chemistry; D008027:Light; D018613:Microscopy, Confocal; D058608:Nanopores
chemical_list: D001704:Biopolymers
keywords: confocal microscopy; optical sensing; photon counting; solid-state nanopores; total internal reflection fluorescence