Insights on using plastic-based dual in-plane nanopore sensors for differentiation and shape determinations of single...

Techniques to analyze proteins often involves complex workflows and/or sophisticated equipment with modest limits-of-detection. While fluorescence spectroscopy can interrogate single molecules, it often requires fluorescence labeling with lasers and microscopes. We report herein a label-free approach for analyzing intact proteins using resistive pulse sensing (RPS). RPS data were secured using a unique RPS device, which we call a dual in-plane nanopore sensor, fabricated in a thermoplastic. The nanopore sensor was produced via nano-injection molding with critical structures of 30 nm, enabling the detection of individual protein molecules and providing an approach toward their identification. Following nano-injection molding, the pore size could be reduced to ∼ 10 nm using thermal fusion bonding of a cover plate to the molded substrate. The device architecture contained two in-plane nanopores flanking a nanochannel (50 × 50 nm width × depth and 5 µm length) that facilitated the measurement of the apparent electrophoretic mobilities of protein molecules in a label free manner via their molecular-dependent time-of-flight (ToF; time-difference between two consecutive RPS events-peak pair). We investigated four model proteins and collected multiple characteristics including RPS peak amplitude and dwell time, as well as an RPS-independent value, which was the ToF. Furthermore, we analyzed the temporal profiles of RPS events revealing distinct peak shapes for spherical and non-spherical proteins that were influenced by their rotational motion when resident within the nanopore.