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 Ca2+-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.

Through a Window, Brightly: A Review of Selected Nanofabricated Thin-Film Platforms for Spectroscopy, Imaging, and Detection

“…Fluorescence is the most likely optical technique to be used in conjunction with a nanopore, but one could imagine that a suitably structured gold film, separated by a well-defined nanopore gap containing an analyte, might also be used for surface-enhanced Raman spectroscopy. 20,27,46,61,90,[267][268][269][305][306][307][308][309][310][311][312][313][314][315] Extensions to Nanopore Arrays. We conclude our discussion of the merging of physical-and optical-based nanopore sensing and manipulation approaches, with the consideration of nanopore arrays.…”

Optical sensing and analyte manipulation in solid-state nanopores

“…53 This provided for higher photon count rates while utilizing pure digital photon counting. 53 This provided for higher photon count rates while utilizing pure digital photon counting.…”

Optofluidic devices with integrated solid-state nanopores

“…[42, 43] Ca 2+ sensitive dyes are also used for optical detection of DNA. [44, 45] The Ca 2+ ion gradient across the nanopore ensures a strong fluorescence signal only within the region close to the nanopore. These successful demonstrations of electro-optical detection have proven the power of combining nanopores and optical detection.…”