Label-Free Optical Analysis of Biomolecules in Solid-State Nanopores: Toward Single-Molecule Protein Sequencing

[Image: see text] Sequence identification of peptides and proteins is central to proteomics. Protein sequencing is mainly conducted by insensitive mass spectroscopy because proteins cannot be amplified, which hampers applications such as single-cell proteomics and precision medicine. The commercial success of portable nanopore sequencers for single DNA molecules has inspired extensive research and development of single-molecule techniques for protein sequencing. Among them, three challenges remain: (1) discrimination of the 20 amino acids as building blocks of proteins; (2) unfolding proteins; and (3) controlling the motion of proteins with nonuniformly charged sequences. In this context, the emergence of label-free optical analysis techniques for single amino acids and peptides by solid-state nanopores shows promise for addressing the first challenge. In this Perspective, we first discuss the current challenges of single-molecule fluorescence detection and nanopore resistive pulse sensing in a protein sequencing. Then, label-free optical methods are described to show how they address the single-amino-acid identification within single peptides. They include localized surface plasmon resonance detection and surface-enhanced Raman spectroscopy on plasmonic nanopores. Notably, we report new data to show the ability of plasmon-enhanced Raman scattering to record and discriminate the 20 amino acids at a single-molecule level. In addition, we discuss briefly the manipulation of molecule translocation and liquid flow in plasmonic nanopores for controlling molecule movement to allow high-resolution reading of protein sequences. We envision that a combination of Raman spectroscopy with plasmonic nanopores can succeed in single-molecule protein sequencing in a label-free way.