Single-Molecule Spectroscopy Using Nanoporous Membranes

Abstract

We describe a novel approach for optically detecting DNA translocation events through an array of solid-state nanopores that potentially allows for ultra high-throughput, parallel detection at the single-molecule level. The approach functions by electrokinetically driving DNA strands through sub micrometer-sized holes on an aluminum/silicon nitride membrane. During the translocation process, the molecules are confined to the walls of the nanofluidic channels, allowing 100% detection efficiency. Importantly, the opaque aluminum layer acts as an optical barrier between the illuminated region and the analyte reservoir. In these conditions, high-contrast imaging of single-molecule events can be performed. To demonstrate the efficiency of the approach, a 10 pM fluorescently labeled lambda-DNA solution was used as a model system to detect simultaneous translocation events using electron multiplying CCD imaging. Single-pore translocation events are also successfully detected using single-point confocal spectroscopy.

Synchronous optical and electrical detection of biomolecules traversing through solid-state nanopores

“…These features make TIR illumination ideal for single-molecule spectroscopy. Although confocal imaging has been used to image stained -phage DNA molecules in ϳ250 nm channels, 26 using TIR illumination removes material restrictions for opaqueness and permits a higher signal/ background detection. Moreover, since large surfaces can be excited using TIR, a wide-field imaging device can be used for parallel imaging of many molecules simultaneously.…”

Synchronized Optical and Electronic Detection of Biomolecules Using a Low Noise Nanopore Platform

“…10 In addition, the nanopore platform provides the potential to enhance optical detection via control of throughput, incorporation of additional photonic structure or use as a zero-mode waveguide. 7,11 A number of optical configurations have so far been reported, these include the use of wide-field imaging 7 , liquid core anti-resonant reflecting optical waveguides 12 , total internal reflection fluorescence microscopy 13,14 and confocal fluorescence microscopy. 7 For structural information to be probed via resistive pulse sensing, high temporal resolution measurements are crucial due to the high translocation velocity of molecules.…”

Rapid Ultrasensitive Single Particle Surface-Enhanced Raman Spectroscopy Using Metallic Nanopores

“…Aside from electrical detection, optical fluorescence detection has also been used to successfully monitor translocation events using membranes made from SiN x or a combination of SiN x and aluminum 9,16,17 .…”

“…In all cases, the bias also reversed to confirm that events are indeed due to translocation events (ESI Figure 14). A more efficient means of increasing the number of translocation events is to use arrays of nanopores along with simultaneous detection 9 . Although outside the scope of this manuscript, this is something we intend to pursue in the future.…”