Recent advances in plasmonic nanocavities for single-molecule spectroscopy

Plasmonic nanocavities are able to engineer and confine electromagnetic fields to subwavelength volumes. In the past decade, they have enabled a large set of applications, in particular for sensing, optical trapping, and the investigation of physical and chemical phenomena at a few or single-molecule levels. This extreme sensitivity is possible thanks to the highly confined local field intensity enhancement, which depends on the geometry of plasmonic nanocavities. Indeed, suitably designed structures providing engineered local optical fields lead to enhanced optical sensing based on different phenomena such as surface enhanced Raman scattering, fluorescence, and Förster resonance energy transfer. In this mini-review, we illustrate the most recent results on plasmonic nanocavities, with specific emphasis on the detection of single molecules.


Introduction
Single-molecule spectroscopy is a central topic in nanoscience, and tremendous applications have been developed so far, from sequencing and trapping 1 to sub-nm control of quantum effects. [2][3][4][5] In parallel, during the last decade, metallic plasmonic nanocavities were extensively investigated as transducers for enhanced sensing, 6,7 optical trapping, 8 single-molecule imaging 9 and extreme nanophotonics. 10 Plasmonic Nicolò Maccaferri is sub-group leader of the Ultrafast Condensed Matter Physics Group at the University of Luxembourg. He received his PhD in Physics in 2016 from the University of the Basque Country (Spain). Before joining the University of Luxembourg, he worked as a researcher in Spain, Sweden and Italy. The main objective of his research is to develop novel concepts in materials science by investigating the physical properties of multifunctional metamaterials of high technological interest, in particular for information processing, opto-electronics and biomedical applications, using optical frequency-and timeresolved spectroscopy techniques, multi-physics numerical methods and bottom-up/top-down fabrication techniques.
Grégory Barbillon completed his PhD in Physics (2007) with the greatest distinction at the University of Technology of Troyes (France). Then, he obtained his Habilitation (HDR) in Physics (2013) from the University of Paris Sud (Orsay, France). He has been Professor of Physics at the Faculty of Engineering "EPF-Ecole d'Ingénieurs" (Sceaux, France) since his appointment in September 2017. His research interests are focused on plasmonics, nanooptics, non-linear optics, biosensing, optical sensing, condensed matter physics, nanophotonics, nanotechnology, surface enhanced spectroscopies, sum frequency generation spectroscopy, materials chemistry, physical chemistry, and uorescence. nanocavities enable the connement of visible and nearinfrared light to subwavelength volumes (typically a few tens of nm 3 ) simultaneously, and the amplication of optical eld intensity by several orders of magnitude. 3,11 This local eld intensity enhancement is possible thanks to the resonant excitation of surface plasmon polaritons generated from the coupling between the external electromagnetic (EM) radiation and the conduction electrons inside the metallic material. Thus, plasmonic nanocavities provide a powerful solution for reducing effective mode volumes and achieve, at the subnanometer scale, spatial control of the coupling with a single molecule in close proximity. 12,13 Conning light to a cavity is then used to enhance the interaction between the optical eld and low dimensional materials, including small molecules, 14 2D materials, 15 quantum dots, 16 nanoparticles 17,18 or quantum emitters 5 passing through or diffusing within the cavity.
A typical plasmonic nanocavity can be realized by coupling two nanostructures in a dimer-like fashion with a nanometer (or even sub-nanometer) gap. Alternatively, a dimer-like nanocavity can be achieved by placing a nanostructure above a metallic layer and separating the two building blocks by using a very thin (few nm) dielectric spacer. In this arrangement, also known as a nanoparticle-on-mirror (NPoM) 10,19 cavity, the metallic nanostructure interacts with its image induced on the other side of the metallic layer. This image-charge conguration generates a plasmonic hotspot centered between the nanoparticle and the metallic substrate. NPoM nanocavities, which can support multiple resonances, exhibit deep sub-diffraction mode volumes below 10 À7 (l/n) 3 (where l is the incident radiation wavelength and n is the refractive index of the cavity). 20,21 Finally, nanocavities can also be fabricated by engraving nanoholes of different shapes such as bow-tie, rectangular or circular, in thin metallic lms. 22 These geometries have been proved to be powerful platforms for many applications such as trapping and manipulation of nano-objects, 8 Since 2020 Denis Garoli has been an Assistant Professor at the applied science department of the University of Bozen where he works in the Environmental Sensing and Flexible Electronics group. He is also an affiliated researcher at Italian Institute of Technology where he works on the fabrication of plasmonic nanopores for enhanced spectroscopies. Prof. Garoli obtained his PhD degree from the plasmonic nanocavity architectures, such as apertures in metallic lms and zero-mode waveguides, pico-cavities with an atomic resolution, and nanocavities realized by using DNAbased nanofabrication techniques. In particular, we focus our attention on these architectures' extreme sensitivity capabilities to achieve single-molecule resolution.
Apertures in metallic lms: from zero-mode waveguides to plasmonic nanopores In this section, we target a particular type of plasmonic nanocavity, which is oen used to perform single-molecule detection by means of uorescence. This architecture, a dubbed zeromode waveguide (ZMW) since it operates at wavelengths longer than its cut-off wavelength, is realized by engraving an aperture, usually a square-or circular-like nanohole (typically with a lateral size of 50-100 nm), in a thin metallic lm. This conguration allows guiding visible EM radiation into a volume smaller than its wavelength and conning it at the bottom of the aperture (Fig. 1). This extreme EM eld connement can reduce the effective detection volume down to 10 À21 liter, allowing parallel and rapid sensing of molecules at concentrations in the micromolar range, 22,25,[29][30][31] since the excitation of molecules outside this detection volume is screened by the metallic lm. Furthermore, besides affecting the excitation rate of the molecules inside the zeptolitre detection volume, ZMWs can also modify the uorescence photokinetics decay rates, 32,33 improving the net detected photon count rate per molecule. 22,34 Although the most explored ZMW geometry is a circular hole prepared on a metallic lm, 35 recently several groups investigated alternative ZMW designs. In particular, rectangular ZMWs realized either on Al or Au-Si bilayers ( Fig. 1(a) and (b), respectively), have been proved to yield signicant enhancement both in terms of uorescence signals and volume reduction. 22,29,30 Alternatively, the use of apertures in metallic lms and whose depth is partially engraved in a transparent substrate have been proved as a potential approach to increase the uorescence of molecules. 34 Current efforts in ZMW optimization are also devoted towards extending their working spectral range down to the UV, where several bio-molecules have intrinsic uorescence, thus enabling label-free detection. 26,31 Thanks to ZMW technology, a wide range of applications have been enabled, from DNA sequencing 36 to enzymatic reactions. 25 Moreover, the development of sensing architectures based on the ZMW concept paved the way for the realization of a new class of sensing platforms, so called solid-state nanopores, 37 and consequently the subclass of plasmonic nanopores. 1 Nanopore technology recently got massive attention, in particular for single molecule detection and sequencing. [38][39][40] In the last few years, several groups investigated different congurations of plasmonic nanopores. A very smart as well as simple geometry has been proposed by Meller and co-workers, who realized a ZMW on a transparent thin Si 3 N 4 membrane in a ow-through conguration by drilling a sub-10 nm hole in the membrane using a high-resolution transmission electron microscope 40 (Fig. 1(c)). This platform enables enhanced singlemolecule uorescence detection and can be integrated with an electrical read-out of DNA translocation through the nanopore. Several optimizations or variations of this design have been reported. In terms of both local eld connement and enhancement, an outstanding example is represented by a bowtie antenna (dimer made of Au triangles) fabricated in close proximity to a solid-state nanopore. 38 Spectroscopic techniques used in plasmonic-based singlemolecule sensing Although uorescence is the most used spectroscopic method in ZMW and nanopore-based single-molecule experiments, other phenomena have been investigated with very interesting outcomes. For instance, Förster resonance energy transfer (FRET) enhancement has been demonstrated in a ZMW. 25,26,41 The exploitation of the FRET mechanism in plasmonic nanopores has been recently proposed as an efficient approach to multiplex maximum uorescence wavelength channels by means of life-time/intensity multiplexing. 42,43 The latter can nd application in nanopore protein sequencing, where the high number of distinct amino acids to be discriminated (20)   In the case of non-uorescent molecules, surface-enhanced Raman scattering (SERS) spectroscopy performed on plasmonic nanocavities has been demonstrated to enable singlemolecule sensitivity. [46][47][48][49][50][51] The ability of SERS to detect these ngerprints with single-molecule sensitivity can be of paramount importance in plasmonic-based sequencing applications. In fact, by probing the SERS signals of four nucleotides and DNA oligonucleotides, the vibrational modes of single molecules very close ($1 nm) to a metallic nanopore can be detected, owing to the enhanced electromagnetic elds. 52 It is well-known that every molecule has its own Raman ngerprint, which is related to the building blocks of bio-molecules such as DNA, RNA, and proteins. In attempts to reach high and reproducible SERS signals, several types of plasmonic pores have been tested in recent years showing that the approach is potentially a winning strategy. As a signicant example, recently van Dorpe and colleagues were able to detect DNA adsorbed inside a plasmonic nanoslit, reporting a spectroscopic library of nucleotides identied with single-molecule sensitivity. 52 From nano to picocavities for high-resolution single-molecule imaging and spectroscopy Visualizing single molecules with chemical recognition represents a fundamental target in nano-biotechnology. Vibrational spectroscopy based on tip-enhanced Raman scattering (TERS) allows accessing the spectral signals of molecular species very efficiently via the strong localized plasmonic elds produced at the tip apex. 53 In this context, recently Jaculbia and co-workers reported nanocavity-based TERS as a versatile tool for single molecule chemical analysis at the nanoscale. 47 Similarly, nanocavities can be exploited to reconstruct single molecules' spatial locations within a plasmonic hotspot with an accuracy of 1 nm, thus enabling nanoscopy of their vibrational signatures. 54 Recently, Hou and colleagues were able to image individual vibrational modes at theÅngström level for a single Mgporphine molecule, revealing distinct characteristics of the vibrational modes in real space (Fig. 2). 55 The same group also demonstrated spatially and spectrally resolved photoluminescence imaging of a single phthalocyanine molecule, as well as the local mapping of the molecular exciton energy and linewidth, coupled to nanocavity plasmons in a tunnelling junction with a spatial resolution down to $8Å. 56 Similarly, Lee et al. reported similar results using TERS at the precisely controllable junction of a cryogenic ultrahigh-vacuum scanning tunneling microscope, showing thatÅngström-scale resolution is attained at subatomic separation between the tip atom and a molecule in the quantum tunneling regime of plasmons. 57 They were able to record the vibrational spectra of a single molecule, obtain images of normal modes and analyse at the atomistic level the intramolecular charges and currents driven by vibrations.
In this context, in pioneering work Baumberg and colleagues were able to place a self-assembled monolayer of biphenyl-4thiol molecules sandwiched in a picocavity made of a gold nanoparticle on top of a gold lm and able to localize light to volumes well below 1 nm 3 . This architecture was then used to experimentally record time-dependent Raman spectra from individual molecules at cryogenic temperature. 58 They reported extreme optical connement yielding a 100-fold enhancement, thus enabling optomechanical coupling between the cavity eld and the vibrations of individual molecular bonds. In the same year they also showed that by scaling the cavity volume to less than 40 nm 3 , they could achieve room temperature strong coupling at the single-molecule level. 59 Recently, the same group reported large-scale room-temperature single-molecule detection by using nanocavities to retrieve either the enhanced Raman scattering of the molecules (Fig. 3) 60 or their uorescence emission. 4 Finally, it is worth mentioning here that narrow ngerprint Raman peaks are promising for biomedical analysis. Raman sensing based on a ow-through scheme is desirable for many practical applications, including lab-on-chip diagnostics. Recently, Huang et al. introduced a new scheme to achieve on demand control and delivery of single plasmonic nanoparticles, which can be functionalized with Raman tags. Using an analogous strategy and exploiting the enhanced optical eld in a picocavity formed by translocating a gold nanoparticle coupled to a plasmonic nanohole, they were able to discriminate the SERS signal of single DNA bases in single oligonucleotides by an electro-plasmonic trapping mechanism. 27 They also used this approach to detect single amino acid residues in polypeptides. 61 Nanocavities realized by using DNA-based nanotechnology Over the last decade, the DNA origami technique has been consolidated into the state-of-the-art approach for the selfassembly of nanophotonic structures. 62,63 DNA origami is fabricated in a bottom-up manner by folding a "long" singlestranded DNA (ss-DNA) sequence (termed "scaffold", $8000 bases long) into a predesigned shape with the help of approximately 200 "short" ss-DNA sequences (termed "staples", $40 bases long and complementary to the scaffold sequencesee Fig. 4(a)). These structures can be used as breadboards where different species, including single-photon emitters such as organic uorophore molecules and quantum dots, together with colloidal metallic nanoparticles (MNPs) of different shapes, materials and sizes, can be incorporated with nanometric accuracy and stoichiometric control 64 to form nanocavities.
Compared to conventional "top-down" nanofabrication techniques, i.e., electron or ion beam lithography, the DNA origami technique has three main advantages. First, it is a bottom-up self-assembly process in which billions of structures can be fabricated in a parallel fashion without the need of costly equipment. Second, it employs colloidal MNPs, which are less prone to surface defects and can be fabricated with higher uniformity than evaporated metallic structures leading to improved reproducibility and performance. 65 Finally, with this technique, a single-photon emitter can be routinely placed in the hotspots of MNPs with nanometer precision, a key factor in controlling the coupling between single molecules and nanocavities. 66,67 These advantages were initially exploited to revisit experiments on uorescence-enhanced spectroscopy 68,69 and SERS 70-72 using dimer nanocavities made of Au or Ag, achieving enhancement values outperforming in some cases those obtained by using nanocavities fabricated with more complex top-down lithographic techniques. 73 While most DNA origami based dimer nanocavities are based on spherical MNPs, anisotropic geometries such as gold nanorods were also demonstrated. 74,75 One step further was recently taken by Ding's group by positioning and orienting triangular gold nanoplates onto two rectangular DNA origami structures in order to fabricate bowtie antennas 76 (see Fig. 4(b) and (c)). The advancement introduced by the DNA origami fabrication technique is re-ected by smaller and more homogenous gaps between the triangular plates reaching 5 AE 1 nm, which represents an extremely challenging gap to fabricate with lithographic techniques. Moreover, smaller gaps translate into a 200-fold higher electric eld enhancement. However, the main advantage of this approach is that single Cy3 and Cy5 Raman active molecules could be placed at the hotspot of the bowtie antenna in order to demonstrate single-molecule SERS (Fig. 4(d)).
Another improvement recently enabled by the DNA origami technique is the possibility to tune the gap of dimer nanocavities. The groups led by Liedl and Lohmüller showed that the distance between two Au 40 nm MNPs self-assembled onto a DNA origami structure can be adjusted by increasing the cavity temperature, see Fig. 5(a). An increase of approximately 200 C leads to a shrinking of the DNA molecule and thus to a reduction of the gap size from 2.5 to 1.4 nm. 49 The gap thermal shrinking was monitored by studying the redshi in the scattering cross-section of the dimer nanocavity ( Fig. 5(b)) and by  SERS measurements of a single Cy3.5 molecule placed at the hotspot.
An alternative approach for the fabrication of nanocavities, using the NPoM geometry, was proposed by Chikkaraddy and co-workers. 77 A rectangular DNA origami structure was used as a spacer in order to self-assemble a single spherical Au MNP onto an Au layer forming an NPoM nanocavity (Fig. 5(c)). This approach enabled the subsequent incorporation of single molecules at a well-dened position within the NPoM cavity gap in contrast to initial studies where neither the position nor the stoichiometry of uorescent molecules could be controlled. 78 This level of position control was exploited to map the hotspot of the NPoM cavity by placing single Cy5 molecules at different positions within the 5 nm gap between an 80 nm gold MNP and a thick gold layer using a rectangular DNA origami (Fig. 5(d)). By performing uorescence measurements at each position, the spatial prole of the local density of optical states was estimated with a resolution of approximately 2 nm. 77

Conclusions
In summary, since their development, plasmonic nanocavities have gained growing interest due to their unique capabilities to conne and concentrate the EM eld in the nanometer and subnanometer range. This effect has been exploited to apply spectroscopic techniques (e.g., SERS and uorescence) to detect single molecules with a strong impact on biomedical applications such as real-time DNA sequencing. ZMWs and plasmonic nanopores still represent the most valuable platforms for single diffusing/translocating molecule detection at high concentrations in the micromolar range. They can be used not only in single-molecule spectroscopy, but also integrated with additional functionalities such as optical trapping and thermoelectro-phoretic effects. Moreover, advanced nanofabrication approaches enable the preparation of hybrid devices that integrate both solid-state and biological elements (such as organic coatings or functional proteins). 79,80 Future development in nanocavities engineering might include the exploration of highindex dielectric materials for the fabrication of dimers, 81,82 and their integration with MNPs. 83 Other directions might include the combination of dimers and NPoM structures, so called dimers-on-lm, in order to obtain narrow resonances through the superposition of bright and dark modes. 84,85 DNA nanotechnology might also play a fundamental role in optimizing nanocavity hotspots. This paves the way for the combination of nanocavities with, for example, bio-assays for DNA sensing and diagnostics. 86 Finally, we also envision a synergistic combination of the DNA origami technique with nanoapertures 87,88 in order to control and address their occupation.

Conflicts of interest
There are no conicts to declare.