Plasmonic Biosensors for Single-Molecule Biomedical Analysis

Abstract

The rapid spread of epidemic diseases (i.e., coronavirus disease 2019 (COVID-19)) has contributed to focus global attention on the diagnosis of medical conditions by ultrasensitive detection methods. To overcome this challenge, increasing efforts have been driven towards the development of single-molecule analytical platforms. In this context, recent progress in plasmonic biosensing has enabled the design of novel detection strategies capable of targeting individual molecules while evaluating their binding affinity and biological interactions. This review compiles the latest advances in plasmonic technologies for monitoring clinically relevant biomarkers at the single-molecule level. Functional applications are discussed according to plasmonic sensing modes based on either nanoapertures or nanoparticle approaches. A special focus was devoted to new analytical developments involving a wide variety of analytes (e.g., proteins, living cells, nucleic acids and viruses). The utility of plasmonic-based single-molecule analysis for personalized medicine, considering technological limitations and future prospects, is also overviewed.

Keywords: biosensors; living-cells; nanoparticle; nanostructure; nucleic acids; plasmonics; single-molecule analysis; virus.

Figure 1

Concentrations of common analytes in biological samples expressed as cells or particles per milliliter of sample (bottom axis), and molarity (top axis). Bacterial concentrations are outlined in green, viral targets are outlined in purple, circulating tumor cells (CTCs) are shown in blue and circulating biomarkers including proteins and nucleic acids in red. Reprinted with permission from Kelley et al. [2] Copyright © 2017 American Chemical Society.

Figure 2

Schematic representation of plasmon-enhanced single-molecule sensing using nanoapertures and nanoparticles: (a) Nanoapertures (rectangle in an etched silicon film) in metal films (gold) for enhanced interaction with single emitters (shown as red glowing circles). The anisotropic etch produces a horn antenna for directive coupling. The aperture provides plasmonic enhancement for optical trapping. The aperture also serves as a nanopore for nanofluidic functionality, like monitoring ionic current from flow through the aperture Adapted with permission from Gordon et al. [27] Copyright © 2020 Wiley; (b) Nanoparticles: The plasmon resonance induces a strongly enhanced and tightly confined local field around the particles. The field shown here is for a gold nanorod that is excited on resonance. The local field mediates plasmon−molecule interactions, enabling enhanced single-molecule detection by monitoring plasmon-induced changes of the molecule (resulting in, e.g., fluorescence enhancement) or by monitoring molecule-induced changes of the plasmon (resulting in frequency-shifts of the plasmon). Adapted with permission from Taylor et al. [4] Copyright © 2017 American Chemical Society.

Figure 3

(A) Scanning electron micrograph (SEM) of an array of nanoholes in a gold film; (B) Extraordinary Optical Transmission (EOT) spectra for three arrays with different periodicities; (C) experimental setup to measure the EOT effect. The metal film is deposited on a glass slide, and the gold side of the array is exposed to solvents and aqueous solutions delivered by microfluidics. Adapted with permission from Gordon et al. [19] Copyright © 2008 American Chemical Society.

Figure 4

Graphene Quantum Dots (GQD) pores with nucleobases of (a) adenine, (b) cytosine, (c) guanine, and (d) thymine. For all the cases from (a–d), the length of the hexagonal GQD side was A = 1.8 nm and the side length of the pore was D = 1.0 nm. All the structures are relaxed and also binding atoms (hydrogen) to the pore and GQD edges are included. (e) Classification rates of unknown DNA nucleobases inserted into GQD (1.8,1.0) to nucleobases as a function of the signal-to-noise ratio (SNR). The classification rate is the percentage of the noisy samples, which are classified correctly to a type of DNA nucleobase. Adapted with permission from Abasifard et al. [35] Copyright © 2019 American Chemical Society.

Figure 5

(a) Schematic illustration of the proposed structures and the DNA molecule present. (a) Nanosheet with a pore (nanopore structure); (b) Bowtie structure. (c) Bowtie-nanopore compound structure. (d) Bowtie-nanopore combination, a = 3 nm, D = 2 nm, g = 1.5 nm and the bowtie side length was 2 nm. Graph (upper left) shows minimum selectivity and average sensitivity. Graph (down left) shows total peak wavelength shifts relative to the condition where no nucleotide was present. Graph (Upper right) shows SPR spectra with no nucleotide. Graph (down left) shows SPR spectra in the presence of nucleotide A. Adapted with permission from Fotouhi et al. [36] Copyright © 2016 Optical Society of America.

Figure 6

Plasmonic nanopores for single-molecule optical sensing. (a) Schematic side-view illustration of a DNA molecule that is electrophoretically driven through a plasmonic nanopore and detected by optical backscattering from the plasmonic antenna. (b) Illustration of the sensing principle. The temporary presence of the DNA in the hotspot region of the plasmonic antenna induces a shift of the resonance wavelength of the antenna, hence decreasing the scattering intensity that is detected at the excitation laser wavelength. (c) Transmission electron microscopy (TEM) image of the plasmonic nanopore devices used in the experiments. The plasmonic nanopore consists of a gold dimer antenna with a ~5 nm nanopore at the gap center. The inset shows a false colored TEM image of a zoom of the nanogap region, highlighting the nanopore. (d) Simulated electromagnetic field distribution of the plasmonic nanopore in longitudinal excitation with a wavelength of 785 nm. The simulation shows the extremely enhanced and confined electromagnetic field within the gap of the dimer antenna, which is required for label-free optical sensing of single molecules. Reprinted with permission from Shi et al. [37] Copyright © 2018 American Chemical Society.

Figure 7

(a) Schematics of the nanopore chip and optical system, which includes a high numerical apertures water immersion objective lens, three excitation laser lines and corresponding Silicon avalanche photodiodes (APDs). The nanopore chip is made of four consecutive layers: silicon (grey), silicon nitride (green) in which the nanopore is drilled, titanium oxide (grey blue) and gold (orange); (b) optical signals simulated using three distinct spatial resolutions: 10, 30 and 50 nm (from left to right). Adapted with permission from Ohayon et al. [40] Copyright © 2019 PLOS.

Figure 8

(a) DNA origami pillar with a total height of 125 nm is immobilized via biotin modifications on a BSA-biotin/neutravidin surface in a low concentration to ensure single molecule detection. An ATTO 542 dye is incorporated into its bottom part to localize the origami (green dot in sketch). On its upper part, a fluorescence-quenching hairpin (FQH) is bound to the pillar by extending its strand on the 5′ end. This allows incorporation of the FQH into the origami pillar during folding of the nanostructures (insets). Upon the addition of specific target DNA/RNA, the FQH is opened resulting in red fluorescence. To enhance the fluorescence arising from the FQH a silver nanoparticle (NP, 80 nm diameter) is attached to the origami as shown in the top view. The thiol-labeled oligonucleotides hybridize with the protruding strands of the DNA pillar; (b) Influence of single-nucleotide variations on hairpin opening, showing the schematic representation of mismatched positions of the target DNA complementary to the FQH sequence. Mis1 target has one mismatch located at position 1, Mis2 target two mismatches at position 2, and Mis3 target three mismatches at position 3. Fraction of opened FQHs after 18 h of incubation with 1 nM of respective target DNA and with and without the addition of NPs. Unpaired t test results: * p > 0.05 and ** p < 0.05. Adapted with permission from Ochmann et al. [42] Copyright © 2019 American Chemical Society.

Figure 9

This is a figure. Selective adsorption of virus-like particles into functionalized nanoholes. Nanohole SPR experiments were performed on (a) nonfunctionalized and (b) mPEG-SH-functionalized nanohole arrays. Spectral shifts as a function of time were recorded for all three transmission peaks. The baseline signal corresponds to aqueous buffer solution. Then, 0.3 mg mL⁻¹ virus-like particles were added (denoted by arrow) and the particle rupture process was tracked. SEM images obtained at (c) 20,000× and (d) 40,000× magnification demonstrate that single virus-like particles adsorbed into individual nanoholes on the mPEG-SH-functionalized nanohole array. In part (d), arrows indicate the three cases: (i) no particle in the nanohole; (ii) one particle partially inside the nanohole; and (iii) one particle predominately inside the nanohole. Aside from the nanohole positions, nonspecific adsorption of virus-like particles was not observed on the mPEG-SH-functionalized gold surface. Adapted with permission from Jackman et al. [43] Copyright © 2016 Wiley.

Figure 10

Synthesis-by-design of plasmonic nanoparticles (NPs) in solution. (a) Illustrations of the designed NP models (upper; dimensional unit, nm) and plasmon resonance electric field patterns (below; unit, Vm⁻¹) generated by numerical simulations. (b) Linear fits to localized surface plasmon resonance (LSPR) wavelength shifts vs. changes in the refractive index (RI) of the NP surroundings. (c) Schematic diagram showing the early stage of direction-specific, contour-following, and shape-controlled crystallization of Au atoms by reducing AuCl4−with NH3OH⁺.The water interface of DNA provides precise controllability under the synthetic conditions at pH 5 and 4, generating NPs with nanobridges and nanogaps respectively. Adapted with permission from Ma et al. [58] Copyright © 2019 Nature.

Figure 11

Schematic of the signal-amplified LSPR miRNA sensing platform on a scalable, flexible, transparent three-dimensional (3D) plasmonic nanostructure. (a) Self-assembled monolayer (SAM) formation with a hairpin LNA probe, (b) hybridization with miRNAs at elevated temperature followed by treatment with a biotinylated signaling probe, and (c) binding with HRP-streptavidin followed by enzymatic reaction converting soluble substrate to insoluble precipitate.; (d) LSPR spectra of patterns after incubation in the absence (gray) or presence of target miR-NA (black). Adapted with permission from Na et al. [59] Copyright © 2018 Elsevier.

Figure 12

Schemes follow another format. If there are multiple panels, they should be listed as: (A) Components of the toehold DNA–AuNP and miR detection by PC biosensors. DNA hybridization probes are conjugated to 100-nm diameter AuNPs. (A) The gold-conjugated DNA probe (green) is bound by a partially complementary protector (blue) preventing binding to the PC sensor (purple/blue structure). (B–D) miR (red) binds at the probe toehold (B), resulting in strand displacement of the protector and exposing additional probe sequence (C), which (D) stabilizes probe binding to the PC capture DNA on the biosensor surface (D). The free energy of the activation reaction can be tuned by the protector (blue) stoichiometry, thus enhancing mismatch selectivity. Bound particles (E) can be measured by a shift in the PC resonance wavelength (F). All images are not to scale. The PRAM assay images the number of surface-captured particles over time (after miR addition); (G) AuNP count quantification of miR-375 and the SNV cases. Adapted with permission from Canady et al. [61] Copyright © 2019 PNAS.

Figure 13

(a–e) Nanoplasmonic LSPR sensing of single-molecule equilibrium fluctuations. (f) Simulated response of a nanosensor to binding of analyte molecules (blue) and the corresponding average over many sensors (orange). ① A nanorod covered with a receptor layer is exposed to flow of analyte solution. ② Analyte molecules bind to the receptors and surface coverage eventually reaches equilibrium ③, at which time averaged numbers of associating and dissociating molecules are equal. However, molecules continue to ④ dissociate and ⑤ associate at random times. This causes short time-scale fluctuations of the surface occupancy around the mean. (g) Once equilibrium is reached, molecules associate and dissociate stochastically with rates determined by k_on, k_off, and ρ, causing single-molecule fluctuations around the equilibrium signal. In this regime, the system ceases to be mass transport limited. (h) Analysis of power spectrum of equilibrium fluctuations yields accurate values of rate constants. Adapted with permission from Aćimović et al. [64] Copyright © 2018 American Chemical Society.