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

Abstract

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.

Figure 1

Label-free detection using a plasmon resonance shift. (a) Scheme of a DNA molecule electrophoretically driven through a plasmonic nanopore and detected by optical backscattering from the plasmonic antenna. (b) Typical TEM image of the plasmonic nanopore which consists of a gold dimer antenna with a nanopore at the gap center. The inset shows a TEM image of zoom of the nanogap region. (a, b) Reprinted with permission from ref (38). Copyright 2018 American Chemical Society. (c) Scheme of a DNA translocated through an inverted bowtie nanoantenna and detected by both the ionic current through the nanopore and the light transmitted through an inverted bowtie nanoantenna. Reprinted with permission from ref (39). Copyright 2019 American Chemical Society. (d) Scheme of double nanohole apertures. (e) Single-strand DNA trapping event in the double nanohole with no intermediate step. (f) A hairpin DNA trapping event in the double nanohole shows the unzipping with an intermediate step of ∼0.1 s. (g) Energy reaction diagram of trapping and unzipping of a DNA hairpin; k, Boltzmann constant; T, temperature; U, energy. (d–g) Reprinted with permission from ref (47). Copyright 2014 OSA.

Figure 2

Surface-enhanced Raman spectroscopic sensing in nanopore/nanoslit. (a) Schematic illustration of DNA threading through a nanopore with a bowtie antenna, the SERS signal of a DNA base will be enhanced when it passes through the nanopore located in the hot spot. Reprinted with permission from ref (55). Copyright 2015 American Chemical Society. (b) Schematic illustration of gold plasmonic nanopores synthesized at the tip of a glass nanopipette. When molecules driven by electrophoresis translocate through the gold nanopores, SERS signals will be generated. Reprinted with permission from ref (61). Copyright 2019 American Chemical Society. (c) Schematic representation of the setup for nanoslit SERS. The inset shows a top-view SEM image of the nanoslit structure, consisting of an inverted prism nanoslit cavity with Bragg-mirror gratings. The scale bar is 1 μm. (d) SERS spectra of four DNA nucleotides. Each spectrum was averaged from 100 spectra, with a nucleotide solution of 1 × 10^–3 M. (c, d) Reprinted with permission from ref (63). Copyright 2018 Springer Nature; http://creativecommons.org/licenses/by/4.0.

Figure 3

Electroplasmonic trapping for single-molecule SERS. (a) Schematic of the flow-through setup that allows a single gold nanourchin to flow through and be trapped under transmembrane bias at a plasmonic resonance upon 785 nm laser excitation. (b) Under laser illumination hot spot forms between AuNU and the nanopore sidewall, inside of which the SERS signal of analytes will be generated. (c) TEM image of the AuNU. The scale bar is 10 nm. (a–c) Reprinted with permission from ref (70). Copyright 2020 Wiley-VCH. (d) Schematic illustration of the electro-plasmonic forces exerted on an AuNU in the nanohole under bias, both of which have negative surface charges. The trapping is due to a balance between the electrophoretic (F_EP), electro-osmotic (F_EO), and optical (F_OF) forces. White arrows indicate the zeta potentials on the AuNU (ζ_np) and the nanohole wall (ζ_hole), respectively, and d is the distance between the particle tip and nanohole wall. (e) Simulated electromagnetic field intensity distributions of the AuNU coupled with the nanohole. The color bar represents the enhancement of the electromagnetic field intensity. (f) Magnified view of the electromagnetic field intensity at one tip of the AuNU. The scale bar is 2 nm. (d–f) Reprinted with permission from ref (69). Copyright 2019 Springer Nature; http://creativecommons.org/licenses/by/4.0.

Figure 4

(a) Schematic illustration of AuNU trapped inside of a gold nanopore under laser illumination. (b) Schematic illustration of a SERS hot spot generated between the nanopore side wall and AuNU with physically adsorbed vasopressin molecules. (c) Schematic illustration of a vasopressin molecule partially excited by a subnanometer hot spot. Physically adsorbed on the gold surface, the molecule will change orientation, position, and conformation inside the subnanometer hot spot under laser illumination. (d) SERS time series extracted from 1400 spectra produced by adsorbing vasopressin submonolayer on the gold nanourchins and trapping them in the nanohole. The color bar represents the signal-to-baseline intensity of the Raman modes. The blue dotted lines indicate (e) the parts of Arg, Pro, and Cys that are excited by the hot spot. (f) The Arg and Pro are excited by the hot spot and (g) only the Pro is excited in the hot spot. The left panels are the SERS spectrum with peaks showing corresponding vibration modes. The right panels illustrate the corresponding molecule position and conformation inside of the hot spot. (a, b, and d) Reprinted with permission from ref (70). Copyright 2020 Wiley-VCH.

Figure 5

Single-molecule SERS spectra of 20 types of individual amino acids were collected by adsorbing amino acid submonolayer individually on the gold nanourchins (AuNUs) and trapping the AuNUs in the plasmonic nanohole. The 10 Raman spectra in the upper part of the figure (Lys, Val, Trp, Thr, Ser, Met, His, Glu, Asp, and Ala) are presented in this Perspective for the first time, while the 10 Raman spectra in the lower part of the figure (Arg, Pro, Ile, Gly, Asn, Leu, Gln, Tyr, Cys, and Phe) have been published in our previous paper. Reprinted with permission from ref (70). Copyright 2020 Wiley-VCH. The Raman spectra intensity has been normalized to allow for plotting on a comparable scale. The asterisks indicate vibration modes belong to citric acids that were surfactant residues on the AuNU surface.

Figure 6

(a) A subnanometer nanopore was used to detect a denatured protein with a rod-like structure. Reprinted with permission from ref (20). Copyright 2016 Springer Nature. (b) Current traces of a solid-state nanopore at different pH values for positive and negative applied bias. Reprinted with permission from ref (99). Copyright 2010 American Chemical Society. (c) Simulation of the electro-osmotic flow velocity distribution in a truncated pyramidal nanopore under a positive (left) and negative bias (right). Reprinted with permission from ref (100). Copyright 2019 Springer Nature. (d) Scheme of a DNA origami sphere docked on a nanopore under an electric bias inducing an electro-osmotic flow that traps proteins. Reprinted with permission from ref (12). Copyright 2021 Springer Nature.