Performance metrics and enabling technologies for nanoplasmonic biosensors

Nanoplasmonic structures can tightly confine light onto a material’s surface to probe biomolecular interactions not easily accessed by other sensing techniques. New and exciting developments in nanofabrication processes, nano-optical trapping, graphene devices, mid-infrared spectroscopy, and metasurfaces will greatly empower the performance and functionalities of nanoplasmonic sensors.

expense of reduced photon counts or higher system noise, the overall detection limit may be degraded 4 .

New generation of nanoplasmonic sensors
While conventional SPR sensors excel at measuring molecular binding interactions over large ensembles and rather macroscopic areas, nanoplasmonic structures can perform other unique tasks such as single-molecule detection, optical trapping, and rapid preconcentration, thus creating their own new set of performance metrics. By pushing the plasmonic field confinement in all three dimensions via a gold nanorod, researchers have accomplished label-free detection of a single protein molecule (Fig. 1a, b) 5 . Nanoplasmonic structures can also boost gradient optical forces 6,7 . Pushing the limit of this principle, a double-nanohole was used for low-power optical trapping of a single protein (Fig. 1c, d) 8 . With large-scale fabrication of single-molecule trap arrays based on coaxial nanoapertures (Fig. 1e) 9 , next-generation sensors could enable massively parallel trapping and conformation dynamics sensing of single molecules without tethers/fluorophores. One potential limitation of surface-based trapping is an immobilization of the molecule upon trapping. One could overcome this challenge by covering nano-optical traps with fluid biomembranes (Fig. 1f), which will open up possibilities to trap single membrane proteins and study their interactions with lipid rafts and nanovesicles.
Even with their remarkable sensitivity and strong near-field trapping capabilities, plasmonic sensors can suffer from the fundamental limitations of diffusion. For sensing low-concentration analytes, their diffusion into the nanometric sensing 'hotspot' can lead to long waiting times. Fortunately, many nanoplasmonic structures (sharp tips, holes, nanogaps) can be designed to generate long-range forces to speed up the delivery of analytes to hotspots. Researchers have added innovative schemes onto nanoplasmonic sensors to accelerate mass transport, including electrokinetic pre-concentration or flow-through sensing 1 . With on-chip integration of these concentration schemes, nanoplasmonic sensors may be able to surpass conventional SPR when key performance metrics-limit-of-detection, response time, and minimum sample volume-are considered simultaneously.

Surface chemistry and biological interfacing
While the sensitivity, resolution, and detection speed are important metrics to evaluate the nanoplasmonic "hardware", another critical element is the surface chemistry to interface the hardware with 'soft' biological matter. Regardless of how well the underlying sensor performs, the outcome of biosensing is determined via molecular recognitions between receptors and targets. Nanoplasmonic sensors often exhibit nonplanar and heterogeneous surfaces, presenting challenges for surface modification strategies. This issue can be addressed by adding a thin conformal silica shell to enable well-established silane chemistry and facilitate biomembrane formation 10,11 . Interfacing nanoplasmonic sensors with biomembranes can also allow the incorporation of membrane-bound receptors and passivate the surfaces against nonspecific binding 4 .
Microarray technologies for nucleic acids and proteins have been revolutionizing screening efforts in many ways, and we consider this application one of the more promising avenues for integration with nanoplasmonic sensors toward parallel and label-free measurements of molecular binding kinetics 1 .
Nanoplasmonics for infrared spectroscopy Infrared (IR) absorption spectroscopy probes vibrational modes associated with the molecular bonds of a sample and provides nearly unparalleled chemical information. Despite this unique advantage, poor sensitivity and difficulties in sampling in aqueous solutions limit the application of infrared spectroscopy for measuring biological analytes in real time. The limited sensitivity is due to inefficient coupling of long-wavelength mid-IR radiation to the small absorption cross-section of molecular vibrations. This poses a significant challenge to probe thin layers of biomembranes, protein monolayers, and low-concentration analytes. For measurement in aqueous solutions, the strong O-H absorption bands of water molecules can overwhelm the signal from biological samples.
While SPR can measure receptor-ligand binding kinetics, it cannot identify bound analyte molecules. As one of the promising options to perform such molecular "fingerprinting", we see surface-enhanced infrared absorption spectroscopy (SEIRA) as an exciting opportunity with many open challenges. Nanoplasmonic structures can provide well-defined resonances with strong nearfield enhancements and sub-wavelength light confinement in a reproducible manner. By overlapping these resonances with the absorption bands of targeted molecules, specific molecular bonds can be selectively excited and large signals from ultra-small sample volumes can be achieved. Researchers have been adapting SEIRA for ultrasensitive detection of various thin-film specimen [12][13][14][15] . In addition, the strong light confinement of plasmonics brings advantages for in-situ measurements. The field intensity decays exponentially from the plasmonic substrate surface with a penetration length of <100 nm. Thus, one can selectively probe surface-adsorbed molecules while minimizing the interference from the rest of the sample (i.e., water molecules in solution). Interestingly, the field enhancement for SEIRA extends sufficiently away from the surface (~100 nm) 16 in comparison to SERS whose enhancement is limited to only a few nanometers. This feature makes SEIRA ideal for studying proteins, biomembranes, as well as nanovesicle cargo while monitoring their dynamic interactions in real-time.

Detecting conformational changes
Notably, the amide-bond signatures of proteins resulting from backbone vibrations can provide comprehensive insights on the protein structure and conformation. Detection of protein conformations is crucial to understand the underlying role of protein misfolding in incurable neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Critically, these conformational changes do not involve mass transfer and thus are largely inaccessible to other methods. The potentials of plasmonics to access rich mid-IR spectral content for functional protein studies are yet to be exploited. Specifically, we think that a critical scientific challenge on this front is to show real-time dynamic analysis of secondary structure changes in native proteins with single-molecule resolution. Another challenge is to analyze proteins and their conformations in heterogeneous biological samples. Differentiating and monitoring selected biomolecules in complex mixtures containing diverse analytes such as lipids, nucleic acids, and small molecules is a central goal in biosensing. Infrared spectroscopy, by providing exquisite chemical selectivity, is uniquely positioned for such tasks. A multi-analyte biosensor could be achieved with substrates supporting multiple resonances that are designed to match the characteristic vibrations of different analytes of interest. In this regard metasurfaces and metamaterials can provide new ways to engineer unconventional propagating and resonant modes. Recently, a multi-resonant metasurface enhancing simultaneously amide and methylene bands has been used to resolve the interactions of lipid membranes with polypeptides and peptide-induced neurotransmitter cargo release from synaptic vesicle mimics (Fig. 2b) in real-time 17 .
Another avenue to extend the functionality of plasmonic sensors is through dynamic tunability. Noble metals, in particular gold, have been the material choice for SEIRA due to their strong plasmonic resonances and biocompatibility. The resonances of metallic structures, however, are fixed upon fabrication. In contrast, two-dimensional (2D) materials such as graphene are emerging as a new platform to provide dynamically tunable resonances and reconfigurable biosensors via electrostatic biasing (Fig. 2c) 18 . Furthermore, graphene can simultaneously function as electronic sensors and even atomically sharp tweezers to trap molecules on its edges 19 . On the other hand, these materials come with their own limitations. Graphene supports weak plasmonic resonances because of its low oscillator strengths. The resonances of hexagonal boron nitride (hBN) are spectrally limited in device applications due to the fixed phonon bands. Hybrid substrates containing multiple materials such as graphene, hBN, or metallic antennas can be a viable solution to better exploit tunability and optical conductivity. Likewise, complementary materials such as low-loss dielectrics supporting naturally high-Q resonances are promising for SEIRA. For example, a recent work employs arrays of high-Q dielectric metasurface pixels to convert near-fieldenhanced absorption signatures into 2D molecular barcode-like images (Fig. 2d) 20 .
Compact on-chip integration of mid-IR sensors has been challenging partly due to the limited choice of efficient light sources and sensitive detectors. Fortunately, the field is advancing rapidly with the introduction of widely tunable roomtemperature quantum cascade lasers, optical parametric oscillator sources, on-chip frequency combs, broadband fiber sources, large-area imaging detectors, and maturing mid-IR waveguide technology. These advancements will bring new opportunities by eliminating the need for expensive and bulky Fourier Transform IR spectrometers and moving mechanical parts. As innovations by many researchers helped overcome the challenges of manufacturing nanoplasmonic structures, we expect that the next round of exciting developments will move towards compact systems integration, ultrasensitive spectroscopies of complex samples, and label-free measurements of single-molecule dynamics, which could advance fields as diverse as point-of-care diagnostics, drug discovery, structural biology, and environmental monitoring.
Received: 11 June 2018 Accepted: 4 September 2018 Fig. 2 New directions in surface-enhanced infrared spectroscopy. a The mid-infrared spectral range contains characteristic absorption signatures associated with the vibrational modes of a wide range of molecules and therefore provides rich chemical information. Adapted from ref. 13 with permission. Copyright 2017 American Chemical Society. b Plasmonic nanoantennas can focus IR light into nanoscale volumes to enhance and detect the absorption fingerprints of small amounts of molecules on the surface. Multi-resonant antennas simultaneously monitor lipid membranes and protein molecules to unravel their interaction kinetics. Adapted from ref. 17 with permission. c In contrast to static metallic antennas, novel materials such as graphene enable dynamically tunable resonances via external electrostatic biasing. Reprinted from ref. 18 with permission. Copyright 2015, American Association for the Advancement of Science. d High-Q dielectric metapixels can overcome intrinsic metal losses to convert absorption signatures into barcode-like molecular images without the need for spectrometry. Adapted from ref. 20 with permission. Copyright 2018, American Association for the Advancement of Science