biosensors Review Plasmonic Biosensors for Single-Molecule Biomedical Analysis Elba Mauriz 1,2, * and Laura M. Lechuga 3 1 Department of Nursing and Physiotherapy, Universidad de León, Campus de Vegazana, s/n, 24071 León, Spain 2 Institute of Food Science and Technology (ICTAL), La Serna 58, 24007 León, Spain 3 Nanobiosensors and Bioanalytical Applications Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, BIST, and CIBER-BBN, Campus UAB, 08193 Barcelona, Spain; laura.lechuga@icn2.cat * Correspondence: elba.mauriz@unileon.es; Tel.: +34-987-293617 Abstract: The rapid spread of epidemic diseases (i.e., coronavirus disease 2019 (COVID-19)) has con- tributed 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 ad- vances 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 devel- opments 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: single-molecule analysis; biosensors; plasmonics; nanoparticle; nanostructure; nucleic   acids; virus; living-cells Citation: Mauriz, E.; Lechuga, L.M. Plasmonic Biosensors for Single- Molecule Biomedical Analysis. 1. Introduction Biosensors 2021, 11, 123. https:// doi.org/10.3390/bios11040123 The detection of clinical biomarkers with relevant sensitivity is becoming a great concern for biomedical analysis [1–3]. The objective quantification of biological targets Received: 27 February 2021 (i.e., nucleic acids, proteins, cells and microorganisms) enables both the identification of Accepted: 13 April 2021 normal or pathological diagnostic outcomes and the monitoring of the biological response Published: 15 April 2021 to therapeutic drugs or environmental agents. Typically, chemical and biological species are present in clinical samples at trace concentrations as low as attomolar levels [1,2,4]. Publisher’s Note: MDPI stays neutral Measuring such small amounts of biomolecules is particularly relevant not only for the with regard to jurisdictional claims in early identification of cancer biomarkers but also for the rapid recognition of pathogenic published maps and institutional affil- agents and the prevention of potential outbreaks (Figure 1) [2,5]. For instance, the precise iations. diagnosis of bacterial and virus infections such as tuberculosis, hepatitis, Acquired Immune Deficiency Syndrome (AIDS) and more recently coronavirus disease 2019 (COVID-19) may require the transduction of independent events occurring at single-molecule or cellular lev- els [2,4]. Single-molecule analysis is also needed when only a minimum volume of sample Copyright: © 2021 by the authors. is available or multiplexed analysis is demanded to target various analytes simultaneously Licensee MDPI, Basel, Switzerland. in a short time [3,6]. This article is an open access article Meeting these challenges involves the monitoring of single-molecule interactions distributed under the terms and between reacting species via sensing devices that can distinguish binding kinetics in conditions of the Creative Commons heterogeneous samples with sufficient specificity [1,4]. The observation of a signal from an Attribution (CC BY) license (https:// individual molecule within the ensemble also relies on the transport of the analytes to the creativecommons.org/licenses/by/ sensing region. Therefore, strategies to improve single molecule detection should take into 4.0/). Biosensors 2021, 11, 123. https://doi.org/10.3390/bios11040123 https://www.mdpi.com/journal/biosensors Biosensors 2021,11, Biosensors2021, 11,123 x 22 of of 23 24 consideration bothboth into consideration the sensing mechanism the sensing and and mechanism the diffusion rate,rate, the diffusion analyte concentration analyte concentra- and tion sample volume and sample [6]. [6]. volume Figure 1. Figure 1. Concentrations of common common analytes analytesin inbiological biologicalsamples samplesexpressed expressedasascells cellsororparticles particles per milliliter per milliliter of of sample sample (bottom (bottom axis), and molarity (top axis). Bacterial concentrations are outlined in green, in green, viral viral targets targets are are outlined outlined in in purple, purple, circulating circulating tumor tumor cells cells (CTCs) (CTCs) are are shown shown in in blue blueand and circulating biomarkers circulating biomarkers including including proteins proteinsand andnucleic nucleicacids acidsin inred. red.Reprinted Reprintedwith withpermission permissionfrom from Kelley et al. [2] Copyright © 2017 American Chemical Society. Kelley et al. [2] Copyright © 2017 American Chemical Society. Current research Current research in in single-molecule single-molecule bioanalytical bioanalytical technologies technologies has has been been primarily primarily fo- fo- cused on cused onconfigurations configurationsbased basedon ontotal totalinternal internal reflection reflection fluorescence fluorescence (TIRF) (TIRF) andand fluores- fluorescent- cent-related methods [1,7,8]. Other optical detection techniques related methods [1,7,8]. Other optical detection techniques such as surface-enhanced Ra- such as surface-enhanced Raman man scattering scattering (SERS), (SERS), total total internalreflection internal reflectionscattering scattering(TIRS), (TIRS),dark-field dark-fieldmicroscopy microscopy (DFM), or near-field scanning optical microscopy (NSOM) (DFM), or near-field scanning optical microscopy (NSOM) have also demonstrated theirhave also demonstrated their value for value for the the determination determination of of single-molecule single-molecule interactions interactions [9,10]. [9,10]. From this From this perspective, perspective,plasmonic-based plasmonic-basedtechniquestechniquestake takeadvantage advantageofofthe theunique uniqueopti- op- ticaland cal and electronic electronic properties properties of plasmonic of plasmonic nanostructures nanostructures to assess to assess the signals the signals and and kineticki- netic distributions distributions of single-molecule of single-molecule bindingbinding events events occurring occurring in real-time in real-time [11,12]. [11,12]. Nano- Nanoplas- plasmonic monic sensing sensing principles principles relyrely on onthethe collective collective oscillationofoffree oscillation freeelectrons electronsknownknown as as localized surface plasmons at metallic metallic interfaces interfaces or or within within nanostructures nanostructures under under light light stimulation [13–15]. [13–15]. The Thepossibility possibilityofof providing providing sensitive sensitive andandselective selectiveanalysis analysisusing sin- using gle-molecule sensing single-molecule mechanisms sensing mechanisms depend depend on onthethesignal amplification signal amplification resulting resulting from the from the enhancement enhancement of the of the intensity intensity of the of the optical optical fieldfield [8]. [8]. Therefore, Therefore, the the manipulation manipulation of of the the physicochemical physicochemical environment environment andand composition composition of plasmonic of plasmonic nanostructures nanostructures allowallowthe the precise precise confinement confinement of theofelectromagnetic the electromagnetic fieldnanoscale field into into nanoscalevolumes volumes of onlyof onlyna- a few a few nanometers, which are named “hot spots”. The generation nometers, which are named “hot spots”. The generation of extreme electric field gradients of extreme electric field gradients facilitates facilitates optical trapping optical trapping of singleofmolecules single molecules while providing while providing longerlonger dwelling dwelling times times [16,17].[16,17]. Particularly, Particularly, the interaction the interaction between between a biological a biological receptorreceptor and its andtarget itsanalyte target analyte near thenear the optical-field optical-field can be detected can be detected through the through shift ofthetheshift of thepolariton plasmon plasmon resonance polariton resonance wavelength induced by a local refractive wavelength induced by a local refractive index change [13]. index change [13]. The The design designof ofhigh-spatial-resolution high-spatial-resolutionsurface surface structures structures is very valuable is very valuable for the for surface- the sur- enhanced face-enhanced sensing of individual events (Figure 2) [18]. In this sense, plasmonic single- sensing of individual events (Figure 2) [18]. In this sense, plasmonic single- molecule moleculeapproaches approachesmainlymainly rely upon rely uponthe the extinction extinctionspectra of metallic spectra nanoparticles of metallic (NPs) nanoparticles and (NPs) theand capacity of optical the capacity nanoapertures of optical nanoapertures(i.e., arrays of nanoholes, (i.e., arrays nanopores, of nanoholes, nanowells) nanopores, nan- to confine the electromagnetic field into ultrasmall subwavelength owells) to confine the electromagnetic field into ultrasmall subwavelength dimensions dimensions [1,8,9,19–23]. Both types of Both [1,8,9,19–23]. plasmonic types nanostructures have been successfully of plasmonic nanostructures have beenapplied to the applied successfully label-free to quantification of single-proteins in the diagnosis of acute and chronic diseases in early Biosensors 2021, 11, x 3 of 24 Biosensors 2021, 11, 123 3 of 23 the label-free quantification of single-proteins in the diagnosis of acute and chronic dis- eases in early stages, the recognition of single-nucleotide specificity and the tracking of stages, the recognition independent biological of single-nucleotide processes specificity in living cells insertedand the tracking in nanopores of independent [24–26]. biological processes in living cells inserted in nanopores [24–26]. (a) (b) Figure2.2.Schematic Figure Schematicrepresentation representationof ofplasmon-enhanced plasmon-enhancedsingle-molecule single-moleculesensing sensingusing usingnanoapertures nanoaperturesand andnanoparticles: nanoparticles: (a) Nanoapertures (rectangle in an etched silicon film) in metal films (gold) for enhanced interaction with (a) Nanoapertures (rectangle in an etched silicon film) in metal films (gold) for enhanced interaction with single emitters single emitters (shown as red glowing circles). The anisotropic etch produces a horn antenna for directive coupling. The (shown as red glowing circles). The anisotropic etch produces a horn antenna for directive coupling. The aperture provides aperture provides plasmonic enhancement for optical trapping. The aperture also serves as a nanopore for nanofluidic functionality, like 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 © 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 2020 Wiley; (b) particles. TheNanoparticles: field shown here Theisplasmon for a goldresonance nanorodinduces that is aexcited strongly onenhanced resonance.and Thetightly local confined local field field mediates plas- around the particles. The field shown here is for a gold nanorod that is excited on resonance. mon−molecule interactions, enabling enhanced single-molecule detection by monitoring plasmon-induced changes The local field mediates of the plasmon molecule −molecule (resultinginteractions, enabling enhanced in, e.g., fluorescence enhancement)single-molecule detection or by monitoring by monitoring plasmon-induced molecule-induced changes(re- changes of the plasmon of sulting the in frequency-shifts molecule of the (resulting in, e.g., plasmon). Adapted fluorescence with permission enhancement) from Taylor or by monitoring et al. [4] Copyright molecule-induced changes©of2017 the American plasmon Chemicalin (resulting Society. frequency-shifts of the plasmon). Adapted with permission from Taylor et al. [4] Copyright © 2017 American Chemical Society. Although the fundamentals and functionalities of single-molecule biosensing have Although been extensively theaddressed fundamentals and functionalities in previous of single-molecule reviews [1,8,22], the specific rolebiosensing of plasmonichave bi- been extensively osensors addressed in the detection in previous reviews of single-molecule [1,8,22], biological the has targets specific rolebeen still not of plasmonic satisfacto- biosensors in the rily resolved. detection Therefore, ofaim the single-molecule of this work is biological targetsrecent to summarize has still not been progress satisfacto- in plasmonic rily resolved. Therefore, single-molecule the aim biosensing of this schemes work for is to summarize biomedical recent applications. progress This reviewinparticularly plasmonic single-molecule concentrates onbiosensing schemes for the quantification of biomedical applications. clinically relevant This review biomarkers usingparticularly plasmonic concentrates on the quantification of clinically relevant biomarkers using plasmonic nanostructures such as metallic nanoparticles and optical nanoapertures. Recent research nanos- tructures in the design of the new generation of plasmonic platforms for practical sensing ofininde- such as metallic nanoparticles and optical nanoapertures. Recent research the design of the new generation of plasmonic platforms for practical sensing pendent molecular interactions from the perspective of point of care testing is also re-of independent molecular viewed. interactions from the perspective of point of care testing is also reviewed. 2. 2. Plasmonic Plasmonic Nanoapertures Nanoapertures forfor Single-Molecule Single-MoleculeAnalysis Analysis The exploitation of plasmonic nanoapertures as sensing probes for single-molecule The exploitation of plasmonic nanoapertures as sensing probes for single-molecule analysis has attracted great interest over the last decade. The formation of nanoapertures analysis has attracted great interest over the last decade. The formation of nanoapertures in an opaque metal film permits the confinement of light in the subwavelength regime, in an opaque metal film permits the confinement of light in the subwavelength regime, thereby limiting the interaction volume to the nanometer scale [28,29]. thereby limiting the interaction volume to the nanometer scale [28,29]. Among nanoscale apertures, solid-state nanopores provide the basis for the develop- Among nanoscale apertures, solid-state nanopores provide the basis for the develop- ment of stable and durable sensing platforms capable of achieving extremely low single- ment of stable and durable sensing platforms capable of achieving extremely low single- molecule detection limits with high specificity [1,30]. Nanopore sensors consist of an molecule detection limits with high specificity [1,30]. Nanopore sensors consist of an elec- electrically thin insulating membrane traversed by 1–100 nm pores through which ions can trically thin insulating membrane traversed by 1–100 nm pores through which ions can cross [22,31]. When charged molecules pass through the pore, the application of a voltage cross [22,31]. When charged molecules pass through the pore, the application of a voltage between two electrodes produces the interaction with the membrane and the displacement between two electrodes produces the interaction with the membrane and the displace- of ions, causing the change of the ionic current. This displacement can be determined via ment oforions, optical causing electrical the change sensing ofThe [1,22]. the ionic current. optical This sensing displacement mainly dependscanon be thedetermined change of via refractive the optical orindex electrical aftersensing [1,22]. the arrival Thetarget of the optical sensingto molecule mainly depends the hotspot on plasmonic of the the change nanopore, wherein the electromagnetic field is strongly localized (Figure 3) [19]. Specifi- Biosensors 2021, 11, x 4 of 24 Biosensors 2021, 11, 123 4 of 23 of the refractive index after the arrival of the target molecule to the hotspot of the plas- monic nanopore, wherein the electromagnetic field is strongly localized (Figure 3) [19]. cally, the optical Specifically, thetransmission through through optical transmission the nanoaperture conductsconducts the nanoaperture the plasmon the resonance plasmon res-to the far field in which the individual molecule can be detected. Monitoring of onance to the far field in which the individual molecule can be detected. Monitoring of the plasmon resonance the plasmon redshift can either resonance redshiftbecan performed either beby registering performed by the plasmonthe registering resonance plasmonpeakreso- or by tracking the scattered light intensity at a fixed excitation wavelength. nance peak or by tracking the scattered light intensity at a fixed excitation wavelength.In this way,In plasmonic nanoapertures this way, plasmonic enable the ultrasensitive nanoapertures quantification enable the ultrasensitive of molecular of quantification interactions molecular by combiningbythe interactions acquisition combining theofacquisition bimodal optical and electrical of bimodal dataelectrical optical and [32]. data [32]. Figure 3. Figure 3. (A) (A)Scanning Scanning electron micrograph electron (SEM) micrograph of anofarray (SEM) of nanoholes an array in a gold of nanoholes in film; (B) film; a gold Ex- traordinary Optical Transmission (EOT) spectra for three arrays with different periodicities; (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 (C) experimental setup to measure the EOT effect. The metal film is deposited on a glass slide, and gold side of the array is exposed to solvents and aqueous solutions delivered by microfluidics. 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. Adapted with permission from Gordon et al. [19] Copyright © 2008 American Chemical Society. Since nanopore sensors facilitate the active transport of charged molecules at very Since nanopore sensors facilitate the active transport of charged molecules at very low concentrations towards the nanopore aperture from long distances, the combination low concentrations towards the nanopore aperture from long distances, the combination with plasmonic sensing can contribute to improve the diffusion of single-molecules at a with plasmonic sensing can contribute to improve the diffusion of single-molecules at a plasmonic hotspot. In addition, the nanoscale diameter of the nanopore is comparable to plasmonic hotspot. In addition, the nanoscale diameter of the nanopore is comparable to the the cross-section of the molecule, thus reducing the molecule motion to only one dimen- cross-section of the molecule, thus reducing the molecule motion to only one dimension. sion.feature This This feature allows allows both both the increment the increment of the of the dwelling dwelling time time andandthetheenhancement enhancementof of the signal-to-background ratio [22]. Therefore, by controlling the the signal-to-background ratio [22]. Therefore, by controlling the nanopore diameter, nanopore diameter, an- alytes can analytes canbebeconfined confinedtotoaarestricted restrictedspace spaceandandthe the capture capture time time increases significantly. increases significantly. Accordingly, the reduction in the nanopore size contributes to enhancing Accordingly, the reduction in the nanopore size contributes to enhancing the signal-to-noise the signal-to- noise ratio results enabling highly selective single-molecule ratio results enabling highly selective single-molecule detection. detection. Theseinteresting These interestingproperties properties have have paved paved thethe wayway for for a variety a variety of biological of biological applica- applications tions aimed at manipulating nanoscale molecules such as single-proteins, aimed at manipulating nanoscale molecules such as single-proteins, virus like-particles virus like-par- ticles or or nucleic nucleic acids. A acids. A detailed detailed descriptiondescription of the fabrication of the fabrication of nanoporesof nanopores has been has been compre- comprehensively hensively reviewed reviewed in previous in previous works and works it isand it is beyond beyond the scope theofscope of this [22,33]. this review review [22,33]. Novel Novel developments developments in plasmonic in plasmonic nanoporenanopore sensingfor sensing schemes schemes for evaluating evaluating bio- biological pro- logical processes including protein-protein interactions, translocation cesses including protein-protein interactions, translocation of single proteins, assessment of single proteins, assessment of of kinetic kinetic reactions, reactions, analysis of RNA analysis of RNA structure, singlestructure, nucleotidesingle nucleotideand polymorphisms polymor- DNA phisms andapproaches sequencing DNA sequencingare shownapproaches are shown in this section in this1). (see Table section (see Table 1). Biosensors 2021, 11, 123 5 of 23 Table 1. Key analytical features of plasmonic applications based on nanopores and nanohole arrays sensors classified according to the characteristics of target analyte, instrument configuration (namely sensing scheme or biological receptor) and detection format. Target Analyte Instrument Configuration Detection Strategy Reference Nanopore-nanowells with Double-stranded single DNA [34] fluorescence enhancement molecules adsorption Graphene quantum dots with a DNA-graphene interactions and nucleobase Nucleic acids nanopore (density functional (adenine, thymine, cytosine, and [35] theory calculations.) guanine) rotation Nanopore, bowtie, and bowtie-nanopore structures and SPR DNA nucleotides shifts in the SPR spectra [36] materials (chromium, aluminum, through Bowtie-nanopore structures rhodium and graphene) Single-DNA molecules in the nanopore gap Two elongated nanodiscs in a gold monitoring (l intensity of light backscattered [37] bowtie nanoantenna from the antenna) DNA-protein interactions and DNA Inverted-bowtie gold translocation (optical signals under different [38] plasmonic nanopore illumination conditions) Single-DNA origami triangles were captured on Nanohole arrays [39] SiO2 bottom surface of gold nanoholes Plasmonic nanopore tri-color Individual protein translocations, human plasma fluorescence simulation and [40] Proteins proteome and cytokine recognition pattern-recognition algorithms Plasmonic nanopore acting as Individual beta-amylase proteins [41] nano tweezer optical trapping Optical nanoantenna with direct Single nucleotide variations in Zika artificial Infectious agents [42] physical fluorescence amplification DNA origami buffer and human serum Single dengue virus-like particles and virucidal Nanohole array drug candidates (spectral shifts of [43] transmission peaks) Silver nanohole arrays and Individual rhodamine 6G (R6G) Organic compounds [44] microscopy imaging three-dimensional orientation 2.1. Single-Nucleic Acid Nanopore Detection Nucleic acid analysis is vital for the diagnosis of diseases such as cancer and in- fectious conditions. Recent technological advancements have permitted the emergence of DNA sequencing as a viable approach to obtain specific information about the dis- ease onset and progress. Specifically, the recognition of single nucleotides, adenine (A), thymine (T), cytosine (C), and guanine (G), is the primary aim of the synthesis-sequencing strategies [26,34,35,45,46]. In this vein, nanopore biosensors have proved to be compatible with the determination of DNA nucleotides at the single-molecule level with sufficient sensitivity and accuracy. Particularly, the sequence of DNA bases can be determined by monitoring current changes when the different DNA bases pass through the nanopore. Although DNA sequencing has been extensively investigated through nanopore detection strategies, several important challenges still remain unresolved. For example, single molecule analysis of nucleic acids using nanopores present various limitations due to the low control of certain parameters such as high salt conditions, high temperature and pH [34–36]. Likewise, the limited residence time of DNA nucleotides inside the nanopore and the elevated ionic current noise are also potential drawbacks of nanopore sensors [45]. The integration of an optical readout into nanopore structures offers potential benefits for the specific detection of DNA bases. A simulation approach was used to improve the detection efficiency of double- stranded single DNA molecules by means of subwavelength plasmonic nanopore-nanowell devices with fluorescence enhancement [34]. The most important benefits of the method rely upon the ability to synchronize the optical and electrical signals while providing the offers potential benefits for the specific detection of DNA bases. A simulation approach was used to improve the detection efficiency of double- stranded single DNA molecules by means of subwavelength plasmonic nanopore-nan- owell devices with fluorescence enhancement [34]. The most important benefits of the Biosensors 2021, 11, 123 method rely upon the ability to synchronize the optical and electrical signals while 6 of 23 provid- ing the suppression of the fluorescence background and the enhancement of the fluores- cence signal. This study demonstrated that the signal was stronger and the background smaller in several orders of magnitude due to the abolition of the stochastic activity asso- suppression of the fluorescence background and the enhancement of the fluorescence signal. Thisciated with the DNAthat study demonstrated motion nearwas the signal the stronger nanopore andand thethe enhancement background smallerin in the observed several fluorescence intensity. orders of magnitude due to the abolition of the stochastic activity associated with the DNA motion nearAnother singular and the nanopore strategy made use of experimental the enhancement in the observed calculations fluorescence to study the effects intensity. of the optical absorption spectrum of DNA bases on the interband Another singular strategy made use of experimental calculations to study the effects plasmons of graphene quantum of the dots [35]. The optical absorption work of spectrum also DNAinvestigated bases on the the interband sensitivityplasmons and selectivity of graphenefactors as quantum dots [35]. The work also investigated the sensitivity and selectivity factors as of well as the association between the insertion of DNA bases and the optical excitation wellgraphene quantumbetween as the association dots within nanopores the insertion of (Figure DNA bases 4). The andresults indicated the optical that among excitation of the four hexagonal graphene quantum dots’ structures examined, graphene quantum dots within nanopores (Figure 4). The results indicated that among the inserted DNA mole- four hexagonal graphene quantum dots’ structures examined, inserted DNA molecules a cules provided higher shift increments in the energy of the peaks for structures with pore side provided higherlength shiftofincrements 1.0 nm. Similarly, the effect in the energy of of theDNA-graphene peaks for structures interactions with aand porenucle- sideobase lengthrotation of 1.0 nm. wasSimilarly, studied in theorder effecttoofexplore the applicability DNA-graphene of the interactions andproposed nucleobase method for DNA rotation sequencing. was studied Specifically, in order to exploreall the the DNA nucleobases applicability were inserted of the proposed method intoforthe DNApore in a rotated mode, yielding almost no effect on the energy of the sequencing. Specifically, all the DNA nucleobases were inserted into the pore in a rotated peaks for G and T, in the case of 90° rotation. Regarding A and C, most of the peaks mode, yielding almost no effect on the energy of the peaks for G and T, in the case ofwere affected by the rotation 90◦ rotation. of the nucleobase Regarding andA could and C,bemost easilyof differentiated the peaks were from the peak affected by theshifts of theof rotation other the nu- cleobases nucleobase and incould both rotated be easilyand nonrotatedfrom differentiated modes. theFinally, peak shiftsthe of noise the level otherwas investigated nucleobases in order in both rotatedto and classify any unknown nonrotated modes.inserted Finally, nucleobase the noise level intowasoneinvestigated of the possible typestoA, C, in order G, and classify anyT.unknown A true classification rate greater inserted nucleobase intothan one95% was of the obtained possible when types theG, A, C, signal and T. to-noise A trueratio was higher classification than rate 12 dBthan greater (Figure 95%4). Therefore, was obtainedthe proposed when method the signal suggests to-noise ratiothat washigh- higher thanplasmonic speed 12 dB (Figure 4). and signals Therefore, forces thecan proposed overcomemethod currentsuggests limitations thatofhigh-speed solid-state na- plasmonic nopores signals and forces can overcome and two-dimensional materialscurrent suchlimitations as fast-DNA of solid-state nanopores translocation, slow and sensing two-dimensional mechanisms and materials suchnoise increased as fast-DNA levels. translocation, slow sensing mechanisms and increased noise levels. (e) Figure Figure 4. Graphene 4. Graphene Quantum Quantum Dots (GQD) Dots (GQD) poresnucleobases pores with with nucleobases of (a) adenine, of (a) adenine, (b) cytosine, (b) cytosine, (c) guanine, (c) guanine, and (d) thy- and (d) thymine. mine. For all the cases from (a–d), the length of the hexagonal GQD side was A = 1.8 nm and the side 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 length of was the pore 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. The application of plasmonics to the sensing of individual DNA molecules can be particularly promising for DNA sequencing. An interesting strategy investigated the effect of different materials like chromium (Cr), aluminum (Al), rhodium (Rh), and graphene (Gr) on the changes of the surface plasmon resonance (SPR) spectra in the UV region of light when single nucleotides pass through: nanopore, bowtie, and bowtie-nanopore compound structures (Figure 5) [36]. The work focused on the optical characterization of the proposed structures with regard to the plasmonic properties of distinct materials particularly promising for DNA sequencing. An interesting strategy investigated the ef- fect of different materials like chromium (Cr), aluminum (Al), rhodium (Rh), and gra- phene (Gr) on the changes of the surface plasmon resonance (SPR) spectra in the UV re- gion of light when single nucleotides pass through: nanopore, bowtie, and bowtie-na- Biosensors 2021, 11, 123 nopore compound structures (Figure 5) [36]. The work focused on the optical characteri- 7 of 23 zation of the proposed structures with regard to the plasmonic properties of distinct ma- terials using a discrete dipole approximation method. However, the sensitivity and selec- tivity factors were also calculated taking into account the dimension of the nanostructures. using a discrete dipole approximation method. However, the sensitivity and selectivity The nanopore structure, which showed higher sensitivity to nucleotides was the Gr-based factors were also calculated taking into account the dimension of the nanostructures. The bowtie, whereas Cr-based compound structures displayed better selectivity. Therefore, nanopore structure, which showed higher sensitivity to nucleotides was the Gr-based when increasing the pore diameter or gap distance, both sensitivity and selectivity factors bowtie, whereas Cr-based compound structures displayed better selectivity. Therefore, decrease. These results indicate that the presentation of DNA nucleotides through bowtie- when increasing the pore diameter or gap distance, both sensitivity and selectivity factors nanopore compound structures could generate more shifts in the plasmonic spectra than decrease. These results indicate that the presentation of DNA nucleotides through bowtie- the nanopore or bowtie structures. This is the crucial aspect for DNA sequencing, since nanopore compound structures could generate more shifts in the plasmonic spectra than the selectivity factor enables the differentiation between all the four possible nucleotides the nanopore or bowtie structures. This is the crucial aspect for DNA sequencing, since the from a single-stranded selectivity factor enablesDNA. In contrast, the the differentiation sensitivity between all thefactor is more important four possible nucleotidesfor DNA from a sensing or translocation detection. Consequently, the plasmonic properties of nanostruc- single-stranded DNA. In contrast, the sensitivity factor is more important for DNA sensing tures or contribute to translocation reduce DNA detection. translocation Consequently, thespeed while permitting plasmonic properties higher ionic or tun- of nanostructures neling signals in the pore in comparison to nanopore DNA sequencers, thus contribute to reduce DNA translocation speed while permitting higher ionic or tunneling laying the basis for signals inpractical the pore DNA sequencing. in comparison to nanopore DNA sequencers, thus laying the basis for practical DNA sequencing. (d) Figure5.5.(a) Figure (a)Schematic Schematicillustration illustrationof ofthe theproposed proposedstructures structuresand andthe theDNA DNAmolecule moleculepresent. present.(a) (a)Nanosheet Nanosheetwith withaapore pore (nanopore structure); (b) Bowtie structure. (c) Bowtie-nanopore compound structure. (d) Bowtie-nanopore (nanopore structure); (b) Bowtie structure. (c) Bowtie-nanopore compound structure. (d) Bowtie-nanopore combination, 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 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 average sensitivity. Graph (down left) shows total peak wavelength shifts relative to the condition where no nucleotide was was present. Graph (Upper right) shows SPR spectra with no nucleotide. Graph (down left) shows SPR spectra in the present. presenceGraph (Upper right) of nucleotide shows with A. Adapted SPR spectra withfrom permission no nucleotide. Fotouhi etGraph al. [36](down left) shows Copyright © 2016SPR spectra Optical in the Society of presence America. of nucleotide A. Adapted with permission from Fotouhi et al. [36] Copyright © 2016 Optical Society of America. Another plasmonic nanopore sensor demonstrated the optical detection of single DNA molecules at high acquisition rates by monitoring the backscattered light intensity from the plasmonic nanoaperture [37]. This approach took advantage of both the electrophoretic properties of the nanopore to attract molecules through it and the enhanced electromag- netic field hotspots of the nanoantenna (Figure 6). The translocation of individual DNA molecules in the nanopore gap was monitored due to the changes of the intensity of light backscattered from the nanoantenna. The principal benefit of the proposed method was the precision for delivering DNA molecules into plasmonic hotspots through a solid-state nanopore. The structure of the plasmonic nanopore, consisting of two elongated nanodiscs in a gold bowtie nanoantenna, also allows the efficient reuse of the sensor by driving biomolecules away from the hotspot after signal acquisition. Likewise, the ability to reduce the waiting time to sub-milliseconds while improving the time resolution and maximum DNA molecules in the nanopore gap was monitored due to the changes of the intensity of light backscattered from the nanoantenna. The principal benefit of the proposed method was the precision for delivering DNA molecules into plasmonic hotspots through a solid- state nanopore. The structure of the plasmonic nanopore, consisting of two elongated Biosensors 2021, 11, 123 nanodiscs in a gold bowtie nanoantenna, also allows the efficient reuse of the sensor by 8 of 23 driving biomolecules away from the hotspot after signal acquisition. Likewise, the ability to reduce the waiting time to sub-milliseconds while improving the time resolution and maximum number of events in several orders of magnitude are additional advantages number of eventsreported over previously in severalplasmon orders ofresonance-based magnitude are additional advantages single-molecule over sensing previously methods [47– reported plasmon resonance-based single-molecule sensing methods [47–50]. 50]. Further advancement of the method in terms of sensitivity and selectivity could Further be advancement of the method achieved by improving in terms of sensitivity the nanoantenna and design and theselectivity could biochemical be achieved modification of by the improving the nanoantenna design and the biochemical modification of the gold surface. gold surface. Figure6.6.Plasmonic Figure Plasmonicnanopores nanoporesfor forsingle-molecule single-moleculeoptical opticalsensing. sensing.(a)(a)Schematic Schematicside-view side-viewillustra- illus- tration of a DNA molecule that is electrophoretically driven through a plasmonic nanopore tion of a DNA molecule that is electrophoretically driven through a plasmonic nanopore and detected and detected by optical backscattering from the plasmonic antenna. (b) Illustration of the sensing prin- by optical backscattering from the plasmonic antenna. (b) Illustration of the sensing principle. The ciple. The temporary presence of the DNA in the hotspot region of the plasmonic antenna induces temporary presence a shift of the of the resonance DNA in theofhotspot wavelength regionhence the antenna, of thedecreasing plasmonic antenna induces the scattering a shift of intensity theis that resonance detected at wavelength of the the excitation antenna, laser hence decreasing wavelength. the scattering (c) Transmission electronintensity that is microscopy detected (TEM) imageat the of excitation laser nanopore the plasmonic wavelength. (c) Transmission devices electron microscopy used in the experiments. (TEM)nanopore The plasmonic image of consists the plasmonic of a nanopore gold dimer devices usedwith antenna in the experiments. a ~5 nm nanopore Theatplasmonic nanopore the gap center. The consists of a agold inset shows dimer false antenna colored TEM image with ofnm a ~5 a zoom of theatnanogap nanopore region,The the gap center. highlighting inset showsthea nanopore. false colored(d)TEM Simulated image electromagnetic of a zoom of the field distribution nanogap of the plasmonic region, highlighting nanopore(d) the nanopore. in Simulated longitudinal excitation withfield electromagnetic a wavelength distributionof of 785the nm. The simulation shows the extremely enhanced and confined electromagnetic plasmonic nanopore in longitudinal excitation with a wavelength of 785 nm. The simulation shows field within the gap of the dimer antenna, which is required for label-free optical sensing of single molecules. Re- the extremely enhanced and confined electromagnetic field within the gap of the dimer antenna, printed with permission from Shi et al. [37] Copyright © 2018 American Chemical Society. which is required for label-free optical sensing of single molecules. Reprinted with permission from Shi et al. [37] Copyright © 2018 American Chemical Society. The translocation of single DNA molecules is described by Dekker’s group [38] using an inverted-bowtie The translocationgoldof plasmonic single DNA nanopore of 100 molecules nm. In thisby is described work, the sensing Dekker’s groupprobe [38] examined DNA−protein interactions at physiological conditions. In particular, using an inverted-bowtie gold plasmonic nanopore of 100 nm. In this work, the sensing DNA translocation probe examined wasDNA evaluated −proteinthrough the characterization interactions of optical at physiological signals under conditions. different In particular, illumination conditions, showing no significant association with driving DNA translocation was evaluated through the characterization of optical signals under voltages and electrolyte different concentrations. illumination Although conditions, the proposed showing method no significant demonstrated association that the with driving optical voltages and electrolyte concentrations. Although the proposed method demonstrated that the optical transmission was induced by translocating DNA molecules, the improvement of the detection scheme was also suggested in order to enhance the plasmon resonance shift when the DNA molecule was present in the gap of the nanoantenna. 2.2. Single-Protein Nanopore Detection Solid-state nanopores sensors have been utilized in the monitoring of single-protein interactions by a significant number of developments [51–53]. Conversely, the integration of plasmonic nanoapertures and solid-state nanopores still has limited applications in the transcriptomics field for the determination of protein size, charge and structure. Recent research in combining plasmonic nanopore sensing is transforming the pro- teomics field and a few works have developed singular detection methods to discriminate proteins at the molecular level. In this sense, Ohayon et al. reported a plasmonic-nanopore biosensor that made use of a simulation approach based on tri-color fluorescence and pattern-recognition algorithms for the identification of nearly 108 individual protein translo- of plasmonic nanoapertures and solid-state nanopores still has limited applications in the transcriptomics field for the determination of protein size, charge and structure. Recent research in combining plasmonic nanopore sensing is transforming the pro- teomics field and a few works have developed singular detection methods to discriminate proteins at the molecular level. In this sense, Ohayon et al. reported a plasmonic-nanopore Biosensors 2021, 11, 123 9 of 23 biosensor that made use of a simulation approach based on tri-color fluorescence and pat- tern-recognition algorithms for the identification of nearly 108 individual protein translo- cations [40]. This method was applied to the recognition of human plasma proteome and commercial cations [40].cytokine panelswas This method withapplied an accuracy to theof ~98%. Theof recognition model humanfirst evaluated plasma the suit- proteome and commercial ability cytokine panels of the plasmonic with structure onantheaccuracy top of theof nanopore ~98%. Thebymodel using first evaluated Finite Difference the suitability Domain of the (FDTD) plasmonic structure computations. Secondly,onpartial the top of the nanopore fluorophore byofusing labelling each Finite Differ- of the three ence amino target Domain (FDTD) acids computations. (lysine, cysteine and Secondly, methionine) partial was fluorophore labelling achieved applying theofsimula- each of the three target amino acids (lysine, cysteine and methionine) was tion to each protein. Lastly, negatively charged SDS (sodium dodecyl sulfate polyacryla- achieved applying the simulation to mide)-denatured each protein. proteins Lastly, negatively were illuminated at threecharged wavelengthsSDS (sodium dodecyl when passing sulfate through polyacrylamide)-denatured the plasmonic nanopore (sub-5 proteins were illuminated nanometer). This passage at allowed three wavelengths whenofpassing the generation opti- calthrough the plasmonic fingerprints nanopore (three-color (sub-5 time fluorescence nanometer). Thisthe traces) and passage allowed subsequent the generation analysis with a of optical fingerprints deep-learning algorithm(three-color fluorescence (Figure 7). The proposedtime traces) method and the suggests subsequent potential analysis advantages with a deep-learning algorithm (Figure 7). The proposed method suggests over current detection techniques in terms of accuracy and robustness. Additionally, potential advan- the tages over current detection techniques in terms of accuracy and robustness. versatility of this strategy allows the identification of the whole proteome without de- Additionally, the versatility pending of this exclusively on strategy allows measurements the ion-current the identification of thethe through whole pore,proteome without thus leading to depending the exclusively parallel readout on theby provided ion-current multi-pixel measurements single-photonthrough sensors.the pore, thus leading to the parallel readout provided by multi-pixel single-photon sensors. (a) (b) Figure Figure7. 7.(a)(a) Schematics Schematicsof the nanopore of the chipchip nanopore andand optical system, optical which system, includes which a high includes numerical a high apertures numerical waterwater apertures im- mersion objective lens, three excitation laser lines and corresponding Silicon avalanche photodiodes (APDs). The 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 nanopore chip is made of four consecutive layers: silicon (grey), silicon nitride (green) in which the nanopore is drilled, oxide (grey blue) and gold (orange); (b) optical signals simulated using three distinct spatial resolutions: 10, 30 and 50 nm titanium oxide (grey blue) and gold (orange); (b) optical signals simulated using three distinct spatial resolutions: 10, 30 (from left to right). Adapted with permission from Ohayon et al. [40] Copyright © 2019 PLOS. and 50 nm (from left to right). Adapted with permission from Ohayon et al. [40] Copyright © 2019 PLOS. Another interesting approach described a plasmonic nanopore biosensor that took advantage of the combined effect of the electrophoretic and the optical trapping force from the nanopore and the nanoaperture, respectively. In particular, this work utilized an inverted-bowtie plasmonic nanopore to retain individual beta-amylase proteins [41]. The plasmonic nanoaperture shaped in a 100 nm/20 nm thick gold/silicon-nitride film allowed the electrical distribution of molecules to the nanopore while ensuring the nano-optical trapping of single-proteins and the collection of the enhanced optical transmission signal as a readout. The capacity of the plasmonic nanopore to act as nano tweezer was first demonstrated by trapping 20 nm polystyrene (PS) nanoparticles inside the nanopore. On the other hand, the optical trapping of beta-amylase protein, a globular 200 kDa protein of around 10 nm in size, in the plasmonic nanopore yielded larger residence times and significant surface interactions. The detection signal of the protein was also characterized by applying a transmembrane bias voltage that resulted in the increase in the event rate over an order of magnitude and the decrease in the residence times. Hence, the combination Biosensors 2021, 11, 123 10 of 23 of the optical trapping and the electrophoretic forces contributed to enlarge the control over single molecules and their motion toward the sensor. Finally, the investigation on the effect of protein-surface interactions demonstrated the absence of consistent trapping for larger proteins in comparison to beta amylase, thus indicating that the optical forces need to be strengthened in the absence of surface interactions. Therefore, although this method provided external control at the single-molecule level for proteins below 200 kDa, future work is needed to ascertain the contribution of nano tweezing for a wider variety of different biomolecules. 2.3. Single-Molecule Detection of Pathogenic Agents Infectious diseases are a major cause of global concern in the present day due to their persistence and rapid transmission among populations [5,54]. Their enormous impact on the global economy and public health result in a prevalence of over 3.1 billion cases and a mortality rate of ~15% worldwide (8.2 million) in 2016 [54] Therefore, the need for efficient diagnostic strategies has dramatically increased owing to the health risks posed by recent disease outbreaks, and the threat of global pandemics such as that caused by COVID-19. In this context, ultrasensitive quantification of pathogenic microorganisms via high throughput analytical methods at the single-molecule level is strongly needed. The development of plasmonic nanopore technologies has permitted the detection of various infectious agents including viruses and bacteria. An optical nanoantenna was applied to the detection of Zika nucleic acids using a direct physical fluorescence amplification mechanism (Figure 8) [42]. Specifically, a DNA origami scaffold that incorporated noble metal nanoparticles was designed to fabricate a plasmonic hotspot. The modular structure of the DNA origami provides the possibility of placing a fluorescence-quenching hairpin (FQH) in the plasmonic hotspot, near a noble metal nanoparticle or in the gap between two nanoparticles that act as the optical antenna. The detection of Zika artificial DNA and RNA sequences with fluorescence enhancement was performed in either buffer or human blood serum. The assay sensitivity as well as the multiplexing capacity was assessed by examining single nucleotide variations using a fluorescence barcode in the origami base. The comparison between fluorescence scans from two different optical nanoantennas could be differentiated by the signal arising from green excitation. The two types of optical antennas were immobilized on the same surface and the signal was recorded after an incubation with both target DNAs overnight. Although the detection of DNA-spiked human serum samples and multiplexing analysis of different targets are remarkable advancements, the method proposed the optimization of the optical antenna by the introduction of multiplication processes for further enhancement of the fluorescence signal. Biosensors 2021, 11, x 11 of 24 Biosensors 2021, 11, 123 11 of 23 (a) (b) Figure Figure8. 8.(a) DNA (a) DNAorigami pillarpillar origami with awith totalaheight total of 125 nm height of is immobilized 125 via biotin modifications nm is immobilized on a BSA-biotin/neu- via biotin modifications on a BSA- travidin surface in a surface biotin/neutravidin low concentration to ensure single in a low concentration molecule to ensure singledetection. An ATTO An molecule detection. 542ATTO dye is542 incorporated into its dye is incorporated bottom part to localize the origami (green dot in sketch). On its upper part, a fluorescence-quenching hairpin (FQH) is into its bottom part to localize the origami (green dot in sketch). On its upper part, a fluorescence-quenching hairpin (FQH) bound to the pillar by extending its strand on the 5′ end. This allows incorporation of the FQH into the origami pillar is bound to the pillar by extending its strand on the 50 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 induring folding of the red fluorescence. Tonanostructures (insets). Upon enhance the fluorescence the addition arising from theofFQH specific targetnanoparticle a silver DNA/RNA,(NP, the FQH 80 nm is diameter) opened resulting is at- tached to the origami as shown in the top view. The thiol-labeled oligonucleotides hybridize with the protrudingisstrands in red fluorescence. To enhance the fluorescence arising from the FQH a silver nanoparticle (NP, 80 nm diameter) attached oftothe theDNA origami as (b) pillar; shown in theof Influence top view. The thiol-labeled single-nucleotide variationsoligonucleotides hybridize on hairpin opening, withthe showing theschematic protruding strands of the representation ofDNA pillar; (b) mismatched Influence positions of single-nucleotide of the variations on target DNA complementary to hairpin the FQHopening, sequence. showing the schematic Mis1 target representation has one mismatch locatedof atmismatched position 1, Mis2 targetof positions two themismatches target DNAatcomplementary position 2, and Mis3 to thetarget FQH three mismatches sequence. at position Mis1 target 3. mismatch has one Fraction oflocated openedat FQHs after1,18 position h oftarget Mis2 incubation with 1 nM of two mismatches at respective position 2,target DNA and Mis3 and with target threeand without the mismatches addition3.ofFraction at position NPs. Unpaired of openedt test results:* p > 0.05 and ** p < 0.05.Adapted with permission from Ochmann et al. [42] Copyright FQHs after 18 h of incubation with 1 nM of respective target DNA and with and without the addition of NPs. Unpaired © 2019 American Chem- ical Society. t test results: * p > 0.05 and ** p < 0.05. Adapted with permission from Ochmann et al. [42] Copyright © 2019 American Chemical Society. 3. Single-Molecule Biosensing Strategies Based on Nanohole Arrays Nanohole arraysBiosensing 3. Single-Molecule are nanostructures Strategies fabricated Based onon metallic Arrays Nanohole materials that exhibit a high aspect-ratio and large electric field intensities in the subwavelength Nanohole arrays are nanostructures fabricated on metallic materials regime thatbased exhibiton a extraordinary optical high aspect-ratio andtransmission large electric (EOT) [55]. The in field intensities utilization of metallicregime the subwavelength surfaces with based on EOT implies that surface plasmons could be excited with an incoherent extraordinary optical transmission (EOT) [55]. The utilization of metallic surfaces withsource of light. The EOTspectral impliesanalysis of theplasmons that surface transmission could peaks generated be excited withbyanchanges incoherentin the localofrefractive source light. The index can be monitored through a portable spectrometer using various plasmonic spectral analysis of the transmission peaks generated by changes in the local refractive modes index [56]. In comparison to other SPR arrangements, nanohole arrays enable can be monitored through a portable spectrometer using various plasmonic modes [56]. several scattering orders that rely on In comparison to the othershape, SPR size, periodicity,nanohole arrangements, dielectricarrays properties enableandseveral composition of scattering the materials orders [55]. on that rely These the unique shape, optical propertiesdielectric size, periodicity, have contributed propertiesto improving the sen-of and composition sitivity of conventional the materials [55]. These SPR unique biosensors [33,43]. optical properties have contributed to improving the For example, the adsorption of immobilized sensitivity of conventional SPR biosensors [33,43]. molecules is described using DNA ori- gami nanostructures For example, the as molecular adsorptionprobes and nanohole of immobilized arrays is molecules as described sensing mechanisms using DNA origami nanostructures as molecular probes and nanohole arrays as sensing mecha- Biosensors 2021, 11, 123 12 of 23 nisms [39]. In particular, DNA origami triangles were captured on the exposed SiO2 bottom surface of gold nanoholes. The characterization of the adsorption process involved the investigation of several parameters including nanohole diameter, DNA origami con- centrations and adsorption time. Experimental results showed that the buffer strength had a strong influence on DNA origami adsorption. Similarly, the utilization of low buffer strengths combined with low Mg2+ concentrations, low DNA origami concentrations, and long incubation times produced nanoholes occupied by only a single DNA origami triangle. However, the low surface mobility of the adsorbed DNA origami led to reduced localiza- tion accuracy inside the nanoholes, thus preventing their contact with the oxide surface. To overcome these limitations, the authors proposed the modification of the adsorption buffer and the employment of other substrate materials for the fabrication of the nanohole arrays. Jackman et al. designed a periodic nanohole array platform to capture single virus- like particles and virucidal drug candidates [43]. The selection of the suitable nanohole dimensions including diameter and height was calculated by nanoparticle tracking analysis of dengue virus-like particles before and after treatment with a virucidal drug candidate (Figure 9). The work specifically focused on the immobilization over gold nanoholes of lipid bilayer membranes consisting of cholesterol-enriched model virus particles. The low surface coverage of single virus particles was achieved due to the functionalization of nanoholes with thiol-terminated methoxy polyethylene(glycol) (mPEG-SH). The antifoul- ing properties conferred by the mPEG-SH functionalized nanoholes ensured that vesicle adsorption only occurred inside the nanohole preventing the adsorption on the sidewalls and the top surface of gold nanoholes. The efficiency of virus-like particle adsorption within the nanoholes was evaluated by tracking changes in the wavelength position of transmission peaks. The increment of refractive index resulted in the shift of all three trans- mission peaks toward longer wavelengths. Further experiments verified with Scanning electron microscopy (SEM) images confirmed that 82% of the virus particles were mainly inside the nanohole. This strategy also enabled the specific and ultrasensitive detection of the virucidal peptide-induced particle rupture in the two nanohole configurations. The virucidal activity of α-helical (AH) peptide is capable of the rupture of lipid vesicles while promoting lipid bilayer formation on the sidewalls of the nonfunctionalized gold nanoholes. Therefore, monitoring of the transmission peaks served to detect virus-like particle ad- sorption and peptide-induced rupture on the more sensitive position of the nanohole. The work contributed to the characterization of single lipid layers according to the size of the deposited molecules. The investigation also concentrated on single particle detection and receptor–ligand interactions, however the study of virus particle conformation on the activity of virucidal drugs will help to improve the contribution of nanoplasmonic biosensing to the analysis of single particles. A hybrid approach consisting of plasmonic silver nanohole arrays and second har- monic generation (SHG) microscopy imaging was used to visualize three-dimensional (3D) orientation of individual rhodamine 6G (R6G) molecules [44]. To demonstrate single- molecule detection, SHG and two-photon fluorescence microscopy experiments along with finite-difference time-domain (FDTD) simulations were conducted. The numerical simulation results indicated a ~106 -fold nonlinear enhancement of the electromagnetic field at the surface hot spots on the plasmonic nanohole substrate in comparison to R6G molecules immobilized on a glass. The comparison of experimental data with dipole emission images using the template matching algorithm revealed the position and 3D ori- entation of R6G molecules. Although this work mainly focused on the characterization of orientation imaging, it also provides the basis to improve the resolution of SHG signatures for single molecule detection of biological cells thanks to the integration of plasmonic silver nanohole structures. Biosensors 2021, 11, 123 13 of 23 Biosensors 2021, 11, x 13 of 24 Figure 9. Figure 9. This Thisisisa figure. Selective a figure. adsorption Selective of virus-like adsorption particles of virus-like into functionalized particles nanoholes. into functionalized Nanohole nanoholes. SPR experi- Nanohole SPR ments were performed on (a) nonfunctionalized and (b) mPEG-SH-functionalized nanohole arrays. Spectral shifts as a experiments were performed on (a) nonfunctionalized and (b) mPEG-SH-functionalized nanohole arrays. Spectral shifts function of time were recorded for all three transmission peaks. The baseline signal corresponds to aqueous buffer solu- as a function of time were recorded for all three transmission peaks. The baseline signal corresponds to aqueous buffer tion. Then, 0.3 mg mL−1 virus-like particles were added (denoted by arrow) and the particle rupture process was tracked. −1 virus-like particles were added (denoted by arrow) and the particle rupture process was solution. Then, 0.3 mg mL SEM images obtained at (c) 20,000× and (d) 40,000× magnification demonstrate that single virus-like particles adsorbed tracked. SEM images into individual obtained nanoholes (c) 20,000× and (d) 40,000nanohole on theatmPEG-SH-functionalized × magnification array. Indemonstrate part (d), arrowsthatindicate single virus-like the three particles cases: (i) adsorbed no particleinto individual in the nanohole;nanoholes (ii) one on the mPEG-SH-functionalized particle nanohole partially inside the nanohole; and array. In particle (iii) one part (d),predominately arrows indicateinside the three the cases: (i) no nanohole. particle Aside fromin the nanohole; (ii) one particle nanohole positions, partially nonspecific inside theofnanohole; adsorption virus-likeand (iii) one particles was particle predominately not observed on the mPEG-SH-functionalized inside the nanohole. Asidegold fromsurface. Adapted the nanohole with permission positions, from nonspecific Jackmanofetvirus-like adsorption al. [43] Copyright © 2016 particles was not Wiley. observed on the mPEG-SH-functionalized gold surface. Adapted with permission from Jackman et al. [43] Copyright © 2016 Wiley. A hybrid approach consisting of plasmonic silver nanohole arrays and second har- monic generation (SHG) 4. Single-Molecule microscopy Biosensing imaging Strategies Based wason used to visualize Plasmonic three-dimensional Nanoparticles (3D) orientation of individual rhodamine 6G (R6G) molecules [44]. To demonstrate single- Metallic nanoparticles (e.g., nanorods, Ag NPs) offer important benefits for single- molecule detection, SHG molecule biosensing due toand two-photon their optical andfluorescence electromagneticmicroscopy experiments properties along at the nanoscale with finite-difference time-domain (FDTD) simulations were conducted. level [9]. Nanoparticle-based plasmonic biosensors usually exploit the unique scattering The numerical simulation and absorptionresults indicated spectra a ~106-fold of localized nonlinear surface plasmonenhancement of the resonance (LSPR) electromagnetic [57]. In the case of field at the surface hot spots on the plasmonic nanohole substrate in comparison gold and silver nanoparticles, localized surface plasmon occurs at visible-NIR wavelengths to R6G molecules (400−100 nm),immobilized which ison a glass. The compatible comparison with of experimental optical microscopy data with dipole for subwavelength emis- imaging sion images using the template and single molecule visualization [4]. matching algorithm revealed the position and 3D orienta- tion of The R6G molecules. variety Althoughchemical of dimensions, this work mainly focused composition, on theconstants dielectric characterization of ori- and boundary entation imaging, it also provides the basis to improve the resolution of conditions can influence the collective oscillation modes of the conduction electrons inSHG signatures for the single molecule particle, therebydetection of biological determining cells thanks the frequency of theto the integration plasmon resonanceof plasmonic [4]. Owingsil- to ver the nanohole structures. electric field associated with the plasmon resonance, the interaction between individ- ual molecules and metallic particles leads to frequency shifts on the plasmon resonance. 4. Single-Molecule Nanoparticle Biosensing morphology Strategies can also affect Based on Plasmonic the efficiency Nanoparticles of plasmonic sensing for moni- toring biomolecular Metallic interactions nanoparticles (e.g., [8]. Therefore, nanorods, the composition Ag NPs) and shape offer important of plasmonic benefits for single- nanoparticles molecule are critical biosensing due aspects to their for the design optical of single-molecule and electromagnetic plasmonic properties at thesensors. In nanoscale general, level [9].plasmonic nanoparticles Nanoparticle-based based on plasmonic sphere and biosensors rod geometries usually areunique exploit the commonly used scattering in biomedical and absorptionanalysis spectradue to their relatively of localized narrow resonance surface plasmon size distribution (LSPR)(Figure [57]. In10) the[58]. case of electric field associated with the plasmon resonance, the interaction between individual molecules and metallic particles leads to frequency shifts on the plasmon resonance. Na- noparticle morphology can also affect the efficiency of plasmonic sensing for monitoring biomolecular interactions [8]. Therefore, the composition and shape of plasmonic nano- Biosensors 2021, 11, 123 particles are critical aspects for the design of single-molecule plasmonic sensors. In14gen- of 23 eral, plasmonic nanoparticles based on sphere and rod geometries are commonly used in biomedical analysis due to their relatively narrow size distribution (Figure 10) [58]. Figure Figure 10. 10. Synthesis-by-design Synthesis-by-design of of plasmonic plasmonic nanoparticles nanoparticles (NPs) (NPs) in in solution. solution. (a) (a) Illustrations Illustrations of of the the designed NP models (upper; dimensional unit, nm) and plasmon resonance electric designed NP models (upper; dimensional unit, nm) and plasmon resonance electric field patterns field patterns (below; unit, Vm− −1) generated by numerical simulations. (b) Linear fits to localized surface plas- (below; unit, Vm 1 ) generated by numerical simulations. (b) Linear fits to localized surface plasmon mon resonance (LSPR) wavelength shifts vs. changes in the refractive index (RI) of the NP sur- resonance (LSPR) wavelength shifts vs. changes in the refractive index (RI) of the NP surroundings. roundings. (c) Schematic diagram showing the early stage of direction-specific, contour-following, andSchematic (c) diagramcrystallization shape-controlled showing the early of Austage atoms ofby direction-specific, contour-following, reducing AuCl4−with NH3OH+.Theand shape- water controlled crystallization of Au atoms by reducing AuCl4 − with NH3OH + .The water interface of interface of DNA provides precise controllability under the synthetic conditions at pH 5 and 4, DNA provides generating NPsprecise controllability with nanobridges andunder the synthetic nanogaps conditions respectively. Adapted at pH 5 and with 4, generating permission NPs from Ma et al. [58] Copyright © 2019 Nature. with nanobridges and nanogaps respectively. Adapted with permission from Ma et al. [58] Copyright © 2019 Nature. Metallic nanoparticles require surface biofunctionalization to enable biomolecule de- Metallic tection. nanoparticles Because require of their physical surface biofunctionalization properties, to enable noble metal nanostructures biomolecule can de- be chemically tection. Because of their physical properties, noble metal nanostructures can be activated through distinct procedures involving strategies from covalent thiol-chemistrieschemically activated to throughglycol polyethylene distinct procedures (PEG). involving strategies Their versatility from covalent and biocompatibility thiol-chemistries also ensure that a to polyethylene broad glycol (PEG). range of biological Their receptors versatility can and biocompatibility be effectively also surface. immobilized on their ensure that a These broad range of biological receptors can be effectively immobilized on their surface. particular features along with the large surface area-to-volume ratio make them perfectly These particular suited features along for capturing withatthe particles thelarge surface area-to-volume single-molecule level. ratio make them perfectly suited for capturing particles at the single-molecule level. This section provides an overview of technological advancements and applications This section of metallic provides nanoparticles foranthe overview analysisofoftechnological advancements single molecules and applications using plasmonic biosensing of metallic nanoparticles schemes (see Table 2). for the analysis of single molecules using plasmonic biosensing schemes (see Table 2). Table 2. Key analytical features of plasmonic nanoparticle-based sensors classified according to the characteristics of target Table Key analytical 2. instrument analyte, features of plasmonic configuration nanoparticle-based (namely sensing sensors classified scheme or biological receptor) according to the and detection characteristics of target format. analyte, instrument configuration (namely sensing scheme or biological receptor) and detection format. Target Analyte Instrument Configuration Detection Format Reference Target Analyte Instrument Configuration Detection Format Reference Single base mismatched detec- LSPR LSPR platform platform with withnanostructures 3D plasmonic 3D plasmonic Single base mismatched detection of microRNAs Nucleic acids tion of microRNAs molecules at [59][59] nanostructures fabricated fabricated on gold strips on gold strips molecules at different melting temperatures LSPR platform with silicon oxide deposited on top of different meltingperfect Single nucleotide temperatures matching Nucleic acids [60] gold nanodisks and mismatching Gold nanoparticles attached to photonic crystals Single-base mismatches of5 different [61] (Photonic Resonator Absorption Microscopy) single-nucleotide variants of microRNA-375 Core−satellite plasmon rulers (gold nanoparticles) and microRNA-21 expression in living HeLa cells at [62] dark-field microscopy imaging single molecule level LSPR-induced fluorescence (of gold nanoparticles-SiO2 -quantum dots conjugated to a Single-base nucleotide DNA mismatch [63] molecular beacon) LSPR platform with gold nanorods (CMOS camera and Single-molecule equilibrium fluctuations [64] Proteins an imaging spectrometer) (PEG/anti-PEG interactions) Kinetics and binding position of single LSPR platform with gold nanorods [65] thyroglobulin and glycoprotein molecules LSPR with Nanotriangle arrays and Single-molecule fluorescence detection of Cells [66] fluorescence imaging photoactivable Vibrio cholerae living cells Binding of cell-secreted MMP3 proteins to aptamer Plasmon rulers linked by a single-aptamer [67] at the single-molecule level Biosensors 2021, 11, 123 15 of 23 4.1. Single-Nucleic Acid Detection Among nucleic acids, microRNAs (miRNAs) have become essential biomarkers in the diagnosis of pathological processes associated with cancer, diabetes or neurodegenerative diseases. Since miRNAs are noncoding RNAs short in length (19–22 nucleotides) and little abundant in biological fluids, detection of their expression levels could mainly be achieved by a real-time quantitative polymerase chain reaction (qRT-PCR) and other electrochemical or fluorescence-dependent methods. Recent label-free plasmonic sensing approaches have demonstrated their capacity for the quantitative determination of miRNA. Thus, Na et al. proposed a scalable and flexible LSPR platform capable of discriminating single-base mismatches in clinical samples [59]. The biosensing scheme involved the fabrication of 3D plasmonic nanostructures on gold strips wherein self-assembled monolayers (SAM) with a hairpin locked nucleic acid (LNA) probe were formed. The hybridization with miRNAs at different melting temperatures enable the determination of perfectly matched or single base mismatched miRNAs. The method also made use of a signal amplification procedure consisting of a treatment with a biotinylated signaling probe, followed by HRP-streptavidin binding and the enzymatic reaction that converted the soluble substrate to insoluble precipitate (Figure 11). The reaction resulted in a distinguishable LSPR peak shift allowing the detection of the target miRNA detection at attomole levels. Likewise, miRNA was also quantified in total RNA extracts from primary cancer cell lines showing high specificity with regard to structurally similar RNA molecules in biological samples. However, the main advantage of the proposed method over qRT-PCR is the possibility of discriminating single base mismatch without labeling, reverse transcription, or gene amplification. The Biosensors 2021, 11, x plasmonic biosensor demonstrated the ability to distinguish miRNA nonspecific sequences 16 of 24 containing only a single nucleotide substitution due to the denaturation of thermally unstable base pairing after incubation at elevated temperatures. Figure 11. Schematic of the signal-amplified LSPR miRNA sensing platform on a scalable, flexible, transparent three- Figure 11. Schematic of the signal-amplified LSPR miRNA sensing platform on a scalable, flexible, transparent three-di- dimensional (3D) plasmonic nanostructure. (a) Self-assembled monolayer (SAM) formation with a hairpin LNA probe, mensional (3D) plasmonic nanostructure. (a) Self-assembled monolayer (SAM) formation with a hairpin LNA probe, (b) (b) hybridization hybridization withwith miRNAsmiRNAs at elevated at elevated temperature temperature followed followed by treatment by treatment withwith a biotinylated a biotinylated signaling signaling probe, probe, andand (c) (c) binding with HRP-streptavidin followed by enzymatic reaction converting soluble substrate to insoluble binding with HRP-streptavidin followed by enzymatic reaction converting soluble substrate to insoluble precipitate.; (d) precipitate.; (d) LSPR LSPR spectra spectra of patterns of patterns afterafter incubation incubation inabsence in the the absence (gray) (gray) or presence or presence of target of target miR-NA miR-NA (black). (black). Adapted Adapted with with per- permission mission fromfrom Naal.et[59] Na et al. [59] Copyright Copyright © 2018 © 2018 Elsevier. Elsevier. Single nucleotide mismatch detection has also been performed by an LSPR biosensor using thiolated single strand oligonucleotides immobilized on nanostructured LSPR sen- sors chips [60]. Two distinct functionalization strategies involving the utilization of gold nanodisks were evaluated. First, glass slides decorated with gold nanodisks provided op- tical signals corresponding to a single particle due to distribution and separation of Biosensors 2021, 11, 123 16 of 23 Single nucleotide mismatch detection has also been performed by an LSPR biosen- sor using thiolated single strand oligonucleotides immobilized on nanostructured LSPR sensors chips [60]. Two distinct functionalization strategies involving the utilization of gold nanodisks were evaluated. First, glass slides decorated with gold nanodisks provided optical signals corresponding to a single particle due to distribution and separation of nanostructures on the substrate, avoiding both far and near field coupling between the particles. The second type of sensor chips involved silicon oxide films (thickness 10 nm) deposited on top of gold nanodisks. Both Au and SiO2 sensor chips yielded LSPR signals because of the refractive index changes following the DNA hybridization processes. The immobilization of 5’-thiol modified 35-mer ssDNA probes onto the Au nanodisks array exhibited high efficiency displaying a surface coverage of 2.8 ± 0.1 × 1012 /cm2 . However, the work primarily focused on the investigation of the hybridization rate constants between surface-bound DNA probes and DNA target strands in solution. Particularly, a limit of detection of 10 nM and 13 nM was obtained when detecting perfect matching (PM) and mismatching (MM) targets for 93-mer sequences, respectively. The kinetic analysis and time dependence of two-component systems (PM and MM) were studied in comparison to a single-component system. Results showed that the MM strands fractions could be quanti- fied in PM-MM binary solutions by monitoring the intensity of the relative LSPR signal at a fixed time. In addition, the hybridization kinetics displayed lower rates for MM with respect to PM target, whereas the competitive hybridization showed that MM sequences could kinetically hinder the hybridization rate of PM strands. These findings suggest that LSPR measurements could be used for detecting single nucleotide polymorphisms in a single step by means of the time response. The capability to discriminate single-base mismatches was also demonstrated by a hybrid plasmonic-photonic coupling approach consisting of DNA modified gold nanoparti- cles attached to photonic crystals (PC) [61]. The proposed biosensor provided the molecular recognition of individual miRNA targets (LSPR wavelength of ~625 nm) with digital res- olution via so-called Photonic Resonator Absorption Microscopy (PRAM). The coupling between the resonant wavelength of the conjugated-gold nanoparticle and the PC nanos- tructure enabled the local quenching of the reflected light intensity from the PC due to the presence of individual nanoparticles (Figure 12). Dynamic PRAM imaging allowed the quantification of particle count as a function of miR-375 concentration at 2 h (prostate cancer biomarker). The selectivity of the assay for single-mismatch discrimination was investigated using five different single-nucleotide variants of miRNA-375. A remarkable decrease in particle count was observed for all five single nucleotide variants over time, with a range of ~83–94% signal reduction at 2 h. Therefore, through the synergistic coupling between the two resonators, digital detection of AuNPs served to selectively discriminate single-base mismatches, building the basis for direct diagnostics in complex background. A singular approach involving core−satellite plasmon rulers and dark-field imaging was used to study the dynamics of DNA/RNA displacement at the surface of gold nanopar- ticles [62]. The synthesis of core-satellite plasmon rulers implied the assembly of “satellite” gold nanoparticles to a “core” nanoparticle by duplex DNA. The separation of the satellite from the core due to the strand displacement induced a shift of scattering wavelength along with a change of color that was monitored by dark-field microscopy. The kinetics of toehold-mediated strand displacement was studied at the single-molecule level using microRNA-21 (miRNA-21) as invader strands. The calculation of reaction constants in cell lysates also allowed deducing the expression of miRNA-21 in living HeLa cells. Results were confirmed with a conventional fluorescence method, enabling the kinetics study at lower concentrations, even at the single-molecule level, with the proposed method in comparison to the fluorescence assay. Therefore, these findings suggest that the utilization of plasmon rulers in combination with microscopy imaging offers significant advances for single-molecule over fluorescence and sequencing methods. tion was investigated using five different single-nucleotide variants of miRNA-375. A re- markable decrease in particle count was observed for all five single nucleotide variants over time, with a range of ~83–94% signal reduction at 2 h. Therefore, through the syner- gistic coupling between the two resonators, digital detection of AuNPs served to selec- Biosensors 2021, 11, 123 tively discriminate single-base mismatches, building the basis for direct diagnostics in23 17 of complex background. (G) Figure Figure 12. Schemes 12. Schemes follow follow another another format. format. If there If there areare multiplepanels, multiple panels,they theyshould shouldbe belisted listed as: as: (A) (A) Components Componentsofofthe the toehold DNA–AuNP and miR detection by PC biosensors. DNA hybridization probes are conjugated to 100-nm diameter 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 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 dis- binding to the PC placement sensor of the (purple/blue protector structure). and exposing (B–D) additional miR probe (red) binds sequence at the probe (C), which toeholdprobe (D) stabilizes (B), resulting binding tointhe strand PC displacement capture DNAof theonprotector and surface the biosensor exposing additional (D). probe of The free energy sequence (C), which the activation (D)can reaction stabilizes be tunedprobe by thebinding to the protector PC (blue) stoichiometry, capture DNA on the thus enhancing biosensor mismatch surface selectivity. (D). The Bound free energy of particles (E) canreaction the activation be measured can beby a shift tuned byinthe theprotector PC resonance (blue) wavelength stoichiometry, (F). enhancing thus All images mismatch are not to scale. The PRAM selectivity. Boundassay images(E) particles thecan number of surface-captured be measured by a shift inparticles the PC over time resonance (after miR addition); (G) AuNP count quantification of miR-375 and the SNV cases. Adapted with permission from wavelength (F). All images are not to scale. The PRAM assay images the number of surface-captured particles over time Canady et al. [61] Copyright © 2019 PNAS. (after miR addition); (G) AuNP count quantification of miR-375 and the SNV cases. Adapted with permission from Canady et al. [61] Copyright © 2019 A PNAS. singular approach involving core−satellite plasmon rulers and dark-field imaging was used to study the dynamics of DNA/RNA displacement at the surface of gold nano- Another single-nucleotide mismatch distinguishing strategy was developed using a particles [62]. The synthesis of core-satellite plasmon rulers implied the assembly of “sat- nanohybrid system based on LSPR-induced fluorescence [63]. The detection of nucleotide ellite” gold nanoparticles to a “core” nanoparticle by duplex DNA. The separation of the DNA sequences benefited from the formation of a sensing probe composed of gold nanopar- ticles functionalized with SiO2 -quantum dots and subsequently conjugated to a molecular beacon. The biosensing mechanism relied specifically on the triggering of fluorescence changes in the quantum dots owing to the LSPR signal induced from gold nanoparticles upon hybridization of the targeted DNA. Although the method concentrated mostly on the characterization of the colloidal stability of gold nanoparticles, the efficiency of the probe for targeting non-complementary sequences was also demonstrated. Therefore, the plasmon-enhanced fluorescence effect of AuNPs on SiO2-Quantum dots upon binding offered either the opportunity to detect perfect complementary nucleotide sequences at 10 fg/mL or single-base nucleotide mismatch and non-complementary sequence target. 4.2. Single-Protein Interaction Detection The quantification of proteins at ultra-low concentrations is of paramount importance for the diagnosis of diseases at an early stage. In this regard, plasmonic nanoparticles are perfectly suited not only for the detection of single protein at molecular level but also for the investigation of the interaction between proteins and their specific recognition element. Biosensors 2021, 11, 123 18 of 23 Monitoring single-molecule binding events is of particular interest for understand- ing the dynamics of antibody-antigen interactions in the immune-related response. In this sense, LSPR biosensing offers the possibility of tracking spectral shifts over time en- abling the study of antibody-antigen kinetics such as association constants, equilibrium fluctuations or denaturation dynamics of large proteins [64]. Particularly, a LSPR based- approach investigated the interaction dynamics between PEG-specific immunoglobulin antibody (Anti-PEG) and surface-thiolated polyethylene glycol (PEG) brushes coated on gold nanorods. The device made use of an opto-mechanical set-up comprising a standard inverted microscope with a CMOS camera and an imaging spectrometer for LSPR detec- tion. Analysis of PEG/Anti-PEG interaction kinetics revealed a strong association between Anti-PEG concentrations and surface coverage, showing a hierarchical-like evolution trend (Figure 13). The quantification of single-molecule equilibrium fluctuations required the collection of LSPR data at the single nanoparticle level during periods of time of more than 24 h, much longer than previous experiments based on short time scales. This special feature allowed the calculation of association rates as a function of the arrival and binding times of molecules to the sensor surface. The possibility of using different set-ups whenever the amplitude of white noise (obtained before calculating the spectra) was larger than the single-molecule signal confirmed the flexibility of the optofluidic system. Similarly, the capability for analyzing not only interaction kinetics but also other dynamic processes Biosensors 2021, 11, x involved in binding events such as surface diffusion or conformational changes 19 of 24are other potential advantages of this plasmonic nanoparticle biosensor. Figure Figure 13. 13. (a–e) (a–e) Nanoplasmonic LSPR Nanoplasmonic LSPR sensing sensingofofsingle-molecule single-molecule equilibrium fluctuations. equilibrium (f) Simulated fluctuations. responseresponse (f) Simulated of a of a nanosensor to binding of analyte molecules (blue) and the corresponding average over many sensors (orange). ① A na- nanosensor to binding of analyte molecules (blue) and the corresponding average over many sensors (orange). 1 A nanorod norod covered with a receptor layer is exposed to flow of analyte solution. ② Analyte molecules bind to the receptors and covered with a receptor layer is exposed to flow of analyte solution. 2 Analyte molecules bind to the receptors and surface surface coverage eventually reaches equilibrium ③, at which time averaged numbers of associating and dissociating mol- coverage eventually ecules are reachesmolecules equal. However, equilibrium 3 , at continue ④ dissociate towhich and ⑤ associate time averaged numbers of associating at random times. Thisand dissociating causes molecules short time- are scale equal.fluctuations However,ofmolecules the surfacecontinue occupancy toaround the mean. 4 dissociate and(g)5Once equilibrium associate is reached, at random times. molecules associate This causes short and time-scale dissociateof fluctuations stochastically the surfacewith rates determined occupancy around the kon, koff,(g) by mean. andOnce ρ, causing single-molecule equilibrium is reached,fluctuations molecules around the equilib- associate and dissociate rium signal. In this regime, the system ceases to be mass transport limited. (h) Analysis of power spectrum of equilibrium stochastically fluctuationswith rates yields determined accurate values ofby kon rate , koff , and constants. ρ, causing Adapted withsingle-molecule fluctuations permission from Aćimović et al.around the equilibrium [64] Copyright © 2018 signal. In this regime, American the system Chemical ceases to be mass transport limited. (h) Analysis of power spectrum of equilibrium fluctuations Society. yields accurate values of rate constants. Adapted with permission from Aćimović et al. [64] Copyright © 2018 American Chemical Society. Another plasmonic application utilized gold nanorods for investigating the kinetics and binding position of protein biomarkers [65]. In particular, this method proposed the calculation of ideal detection volumes for single molecules. Simulation results showed that single molecule analysis required sufficiently small volumes (~990 nm3) to allow the detection of protein molecules (~5–10 nm.) while ensuring the suppression of the back- ground noise. To improve the assay sensitivity, the effect of the environment including buffer, substrate and type of ligand was also studied using streptavidin-R-phycoerythrin molecules. Whereas the substrate and buffer could drastically affect the sensitivity, the Biosensors 2021, 11, 123 19 of 23 Another plasmonic application utilized gold nanorods for investigating the kinetics and binding position of protein biomarkers [65]. In particular, this method proposed the calculation of ideal detection volumes for single molecules. Simulation results showed that single molecule analysis required sufficiently small volumes (~990 nm3 ) to allow the detection of protein molecules (~5–10 nm.) while ensuring the suppression of the background noise. To improve the assay sensitivity, the effect of the environment including buffer, substrate and type of ligand was also studied using streptavidin-R-phycoerythrin molecules. Whereas the substrate and buffer could drastically affect the sensitivity, the ligand diameter did not influence the assay performance. Nevertheless, the sensitivity was dependent on the length of the ligand since it determines the distance of the biomolecule from the gold nanorod. Finally, the detection of single molecules was further enhanced using a dielectric cladding layer around the nanorod except the two tips. This strategy pursued that the biomolecules were only attached to the tips, where the plasmonic field enhancement is at its maximum (hot spots). By taking advantage of the intense electric field confinement around tips, the sensitivity increased four orders of magnitude when monitoring thyroglobulin and glycoprotein biomarkers. Thus, LSPR biosensors based on the attachment of molecules on the tips of gold nanorods demonstrated higher sensitivi- ties for label-free detection of single protein molecules in comparison with other optical sensing approaches. 4.3. Single-Cell Detection A biosensor application involving plasmon-enhanced fluorescence has been used for single-molecule fluorescence imaging of Vibrio cholerae living cells [66]. The work demonstrated the enhancement of single-molecule fluorescence intensity due to the cou- pling of living cells to gold nanotriangle arrays. Since this enhancement depends on the nanoparticles’ properties and dimensions, the plasmonic coupling was studied as a function of the surface coverage and LSPR frequencies of gold nanoparticles. As a result, nanotriangle arrays fabricated by nanosphere lithography on glass coverslips were char- acterized according to their morphology and diameter before being used as substrates for the imaging of living Vibrio cholerae cells. The pathogenicity of Vibrio cholerae relies on the expression of photoactivatable fluorescent proteins. Therefore, by monitoring V. cholerae cells on top of extracellular nanoparticles, single-molecule fluorescence is further improved due to the proteins (TcpP-PAmCherry) being positioned within the enhanced near field of the plasmonic nanotriangle arrays. In this localization, the fluorescence excita- tion and emission maxima wavelengths of TcpP-PAmCherry overlap well with the NTA LSPR frequencies. The optimization of the nanoparticle geometry and coverage led to a two-fold single-molecule fluorescence enhancement in live cells, improving the resolution of standard fluorescence single-molecule imaging methods. Single-molecule sensitivity has also been proved using plasmon rulers for live-cell detection of secreted single protein molecules [67]. Plasmon rulers comprised dimers of identical gold nanoparticles linked by a single-aptamer that enabled the detection of single- molecule activity. In contrast to fluorescence-based single-molecule methods, plasmon rulers are capable of binding individual molecules without photobleaching or blinking. Their sensing mechanism relies on the generation of a light scattering spectrum when the surface plasmon resonance of the two gold nanoparticles couple due to their proximity. Therefore, the binding of target molecules to the aptamer originates conformational changes that induce the variation of interparticle distance and an observable change in the light scattering spectrum. In this work, a matrix metalloproteinase (MMP3) was selected as the target molecule because of its role in the dynamics involved in biochemical pathways in cancer. The kinetics of the binding between MMP3 proteins and the single aptamer of plasmon rulers were measured at the single-molecule level. It was observed that the binding of the MMP3 molecule to the aptamer between the two Au nanoparticles caused a decrease in the interparticle distance resulting in a spectral redshift. The reversibility of the aptamer-protein binding reaction represents an additional advantage over plasmon Biosensors 2021, 11, 123 20 of 23 rulers that rely on the cleavage of their biomolecule substrate to generate a change in their interparticle distance. Therefore, the dynamic response of these plasmon rulers demonstrated their capability to measure single secreted molecules with high specificity and in a reversible manner. 5. Conclusions and Future Outlook This work compiles recent progress in plasmonic applications for single-molecule biomedical analysis. The utilization of hybrid coupling technologies (i.e., electrical, flu- orescence or microscopy) has opened the way toward the development of plasmonic ultrasensitive detection methods expected to be applied in clinical settings. Although label-free plasmonic sensing has been extensively exploited in medical research, the role of single molecule-based plasmonic platforms as diagnosis tools in the medical field is still at an early stage. To overcome this limitation, current emerging technologies have prompted the fab- rication of new plasmonic structures aimed at addressing the peculiarities of targeting individual molecules. Among them, the complementarity of plasmonic nanomaterials and nanopores has enabled the monitoring of single binding events with high precision. The development of plasmonic nanopores has benefited from the extraordinary optical proper- ties of plasmonic surfaces and the molecular-scale dimensions of nanopores. Plasmonic nanopores have contributed to improving the dwelling times of analytes while facilitating molecule trapping into the active detection sites due to their enhanced field localization and thermophoretic capacity. Several meaningful works have attained single-molecule sensing of nucleic acids, proteins and pathogenic agents using plasmonic nanopores. However, the number of applications is still primarily limited to proof-of-concept demonstrations and optical characterizations supported by theoretical simulations. To date, most approaches conducted to investigate DNA sequencing and protein binding interactions do not exceed the requirements of commercially available instruments. Nonetheless, the possibility of fabricating nanopores integrated into plasmonic-nanofluidic platforms can lead to the multiplexed analysis of biomolecules with superior spectral resolution. The other plasmonic strategy commonly applied to single-molecule biosensing in- volves the utilization of nanoparticles. Plasmonic nanoparticles offer the opportunity of providing higher sensitivities in comparison with two dimensional metallic films because of the enhancement of surface-to-volume ratios. The efficiency of plasmonic nanoparticle- based applications also relies on both the manipulation of these strongly-enhanced near fields and the engineering of nanoparticles assemblies. Although plasmonic biosensors have been mostly employed for targeting single-base DNA mismatches, single-cell biosens- ing also provides promising advantages thanks to the incorporation of far-field optical microscopy and fluorescent techniques. By integrating additional readers, single-molecule biosensing has been effectively applied to monitoring the dynamics of biological processes. In particular, the investigation of conformational changes and binding kinetics of individual molecules has been reported in various protein detection approaches. In spite of these significant innovations, the state-of-the-art of single-molecule plas- monic biosensors is far from the clinical implementation as point-of-care devices. First, the fabrication of operational devices ready for personalized medicine requires the restriction of mass transport effects. The integration of nanofluidics into scalable platforms is essential for controlling sample volumes down to the subpicoliter range. Therefore, detection of single molecules involves the downscaling of fluidics to the dimensions of nanoplasmonic structures. In this sense, the selection of materials with specific geometries such as well- shaped nanoapertures enables the maximization of plasmon confinement and resonance intensity while leading to a substantial gaining of signal-to-background ratios. Similarly, further enhancement of detection rates and nanoscale volumes could help to design plas- monic sensors by fabricating arrays of nanoholes. Overall, building scaling nanosensors for biomolecular analysis of clinical samples demands high sensitivity and specificity. Since the presence of analytes in complex media can be outnumbered by non-target species, Biosensors 2021, 11, 123 21 of 23 plasmonic sensors should exhibit high precision when quantifying biomolecular targets at single molecule levels. By addressing this gap, the number of biosensing applications in the clinical field could increase significantly. The development of plasmonic sensing systems with higher spatiotemporal resolutions will contribute to broadening the spec- trum of single-molecule applications to other analytes such as exosomes or living cells. Furthermore, the search for nanomaterials with extended physicochemical stability and biocompatibility will refine the robustness of plasmonic configurations and support the progress towards functional devices at the point of need. Author Contributions: E.M. contributed to the conceptualization, methodology, formal analysis, investigation, writing—review and editing and supervision of this work. 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