Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays

Abstract

In recent years, nanopores have emerged as exceptionally promising single-molecule sensors due to their ability to detect biomolecules at subfemtomole levels in a label-free manner. Development of a high-throughput nanopore-based biosensor requires multiplexing of nanopore measurements. Electrical detection, however, poses a challenge, as each nanopore circuit must be electrically independent, which requires complex nanofluidics and embedded electrodes. Here, we present an optical method for simultaneous measurements of the ionic current across an array of solid-state nanopores, requiring no additional fabrication steps. Proof-of-principle experiments are conducted that show simultaneous optical detection and characterization of ssDNA and dsDNA using an array of pores. Through a comparison with electrical measurements, we show that optical measurements are capable of accessing equivalent transmembrane current information.

Single-molecule sensing with nanopores

“…They include optical methods involving the detection of fluorescent dyes attached to analytes or, as illustrated in figure 5a, the use of ion-sensitive dye gradients across the pore. 13 Plasmonic nanostructures can be integrated into the membrane to focus light onto the nanopore, as shown in figure 5b, and may allow Raman spectroscopy of translocating analytes. 14 Researchers are also pursuing alternative electrical methods of detection, including measuring the tunneling current between nanoelectrodes positioned opposite one another across the pore's diameter Nanopore sensing End-to-end, air-to-vacuum solutions... always in stock always in stock always in stock Accu-Glass Products, Inc.…”

Electrical trapping mechanism of single-microparticles in a pore sensor

“…On the other hand, combining optical microscopy observations with nanopore measurements have proven to be an effective technique to address this issue: fast translocation of dyed particles or molecules through a fluidic channel are traced by fluorescence imaging while recording the cross-pore ionic current simultaneously so that the ionic current signals obtained can be assigned unambiguously to the actual events occurred. [22][23][24][25][26][27] Mitsui et al 22 studied electrophoretic capture of single-molecule DNA into a nanopore by fluorescent observations alone. They observed motion of DNA around a nanopore with different gate voltages and presented the possibility for controlling the trajectory of analyte at the vicinity of a nanopore.…”

“…They observed motion of DNA around a nanopore with different gate voltages and presented the possibility for controlling the trajectory of analyte at the vicinity of a nanopore. Furthermore, several groups [23][24][25][26][27] have succeeded in detections of analyte translocations by simultaneous measurements of the ionic current and the fluorescence imaging. As described above, such simultaneous measurement utilizing fluorescent observation facilitates to conform the relationships between the experimental condition and dynamics of analyte and develops reliable high-throughput nanoscale device based on rich information of dynamic behavior of the targets.…”

An integrated system for optical and electrical detection of single molecules/particles inside a solid-state nanopore

“…Therefore, as Ca 2+ ions flow from one side of the pore to the other side, the intensity of the ionic current can be converted into the intensity of fluorescence at a small region around the nanopore. Recently, this method has been successfully applied in solid-state nanopores and has been used for solid-state nanopore readouts 37,38 .…”

“…To simultaneously acquire optical and electronic signals at a high bandwidth and high laser power, a low noise nanopore platform based on a Pyrex substrate and a silicon nitride membrane has been developed to reduce the background signal 40 . Meanwhile, the "optical patch clamping" methods have also been applied to simultaneous optical and electronic detection on a solid-state nanopore without labeling of the analyte 37,38 .…”