Overcoming evanescent field decay using 3d-tapered nanocavities for on-chip targeted molecular analysis

ABSTRACT Enhancement of optical emission on plasmonic nanostructures is intrinsically limited by the distance between the emitter and nanostructure surface, owing to a tightly-confined and

exponentially-decaying electromagnetic field. This fundamental limitation prevents efficient application of plasmonic fluorescence enhancement for diversely-sized molecular assemblies. We

demonstrate a three-dimensionally-tapered gap plasmon nanocavity that overcomes this fundamental limitation through near-homogeneous yet powerful volumetric confinement of electromagnetic

field inside an open-access nanotip. The 3D-tapered device provides fluorescence enhancement factors close to 2200 uniformly for various molecular assemblies ranging from few angstroms to 20

nanometers in size. Furthermore, our nanostructure allows detection of low concentration (10 pM) biomarkers as well as specific capture of single antibody molecules at the nanocavity tip

for high resolution molecular binding analysis. Overcoming molecule position-derived large variations in plasmonic enhancement can propel widespread application of this technique for

sensitive detection and analysis of complex molecular assemblies at or near single molecule resolution. SIMILAR CONTENT BEING VIEWED BY OTHERS PHOTONIC CRYSTAL ENHANCED FLUORESCENCE EMISSION

AND BLINKING SUPPRESSION FOR SINGLE QUANTUM DOT DIGITAL RESOLUTION BIOSENSING Article Open access 08 August 2022 DUAL-WAVELENGTH METALENS ENABLES EPI-FLUORESCENCE DETECTION FROM SINGLE

MOLECULES Article Open access 02 January 2024 SINGLE-MOLECULE FLUORESCENCE MULTIPLEXING BY MULTI-PARAMETER SPECTROSCOPIC DETECTION OF NANOSTRUCTURED FRET LABELS Article Open access 15 May

2024 INTRODUCTION While enhancement of optical signals such as fluorescence using plasmonic nanostructures promised breakthroughs in areas such as single molecule fluorescence-driven DNA

sequencing1,2, rapid disease detection3,4,5, as well as observation of biological reactions6,7, their widespread application towards bioassays has remained lacking. One major reason for the

lack of applicability of plasmonic fluorescence enhancement remains the wide variability in enhancement efficiency for molecular assays. Fluorescence enhancement obtained from plasmonic

nanostructures is intrinsically dependent on both the efficiency of electromagnetic (EM) field confinement at the plasmonic hotspot as well as the distance between the optical emitter

(fluorophore) and the plasmonic hotspot8,9,10,11,12. These fluorophores are typically attached to biorecognition elements such as antibodies or nucleic acid aptamers that recognize and

specifically bind to other target molecules. Therefore the position or distance of light-emitting fluorophores with respect to a plasmonic hotspot is dependent on size as well as number of

the molecules within the biomolecular complex, and can drastically alter the plasmonic enhancement of the emitted signal due to changes in the radiative and non-radiative field

components11,12,13,14. Inconsistent or weak enhancement of signal due to these variations limits both the accuracy and efficiency of molecular binding analysis on chip. While several reports

have discussed novel nanoscale geometries that improve the confinement of EM fields leading to strong fluorescence enhancements15,16,17,18, engineering a hotspot that resolves the distance

challenge between the emitter and the nanostructure surface has remained elusive. Powerful fluorescence enhancement within a nanostructure independent of variation in molecule size and

position can be expected to rely on several important factors: (a) strong confinement of electromagnetic field (b) powerful coupling of the emitter to the field for enhancement of emission

and (c) a hotspot geometry, which generates uniform electromagnetic field distribution. At the same time, the hotspot geometry needs to be large enough for commonly used protein–protein

binding assays (i.e. larger than antibodies, ~15 nm). Metal–insulator–metal (MIM) structures utilizing surface-plasmon-polariton (SPP) propagation have been known to enable efficient

confinement of EM energy19,20,21,22. Specifically, waveguides with a 3D taper that rely on adiabatic compression of the SPP mode inside the MIM gap have been shown to provide extreme

volumetric nanoscale confinement of EM fields23,24,25,26,27. While extremely promising for energy confinement and transfer, the potential of 3D-tapered designs for coupling with fluorescent

emitters and bioassays had remained unrealized primarily due to closed monolithic structures, which prevented molecular integration with these devices. Furthermore, as electromagnetic field

intensity on a plasmonic surface is maximum at the metal–dielectric interface, close packing of multiple metal–dielectric interfaces such as in very thin MIM gaps, can result in integration

of these multiple field profiles within the gap creating a more homogeneous field distribution. Therefore, a 3D-tapered structure provides unutilized potential toward these goals, for

enabling confinement of a large amount of incident electromagnetic energy into a tiny MIM gap. In order to take advantage of the previously known as well as unexplored abilities of

3D-tapered MIM devices, we design and fabricate a fluidic channel-like 3D-tapered gap plasmon nanocavity, allowing ready access of the nanostructures to molecules in solution (Fig. 1a). We

demonstrate that a 3D-tapered gap plasmon nanocavity can overcome a long-standing limitation for plasmon-enhanced fluorescence demonstrating powerful emission enhancement independent of the

size or position of the molecules within the nanocavity. The 3D-taper results in confinement of the electromagnetic field collected throughout the body of the device into a tiny cavity

(~3300× smaller in volume) with a |E|2 enhancement close to 500. Furthermore, our analysis reveals that the 3D-taper geometry improves the coupling of molecular emitters to the

electromagnetic field, delivering up to 28% improvement in the radiative decay rates and thus leading to even stronger enhancement of fluorescence. We specifically trap and observe single

antibody molecules or arrays of molecular assemblies within the device, as well as detect low concentration (10 pM) protein molecules diffusing in solution. Significantly, optimizing the

taper angle and tip geometry of the device results in 40× improvement in uniformity of the electromagnetic field volume compared with a bowtie nanoantenna. Combination of the strong

electromagnetic confinement, powerful coupling of the emitter to the confined field and a homogenous electromagnetic field volume result in experimental enhancements of ~2200 compared with

glass chips for molecular heights ranging from few angstroms to 20 nm, which can be further extended to 50 nm using the current design approach. Overcoming the molecule placement limitation

for plasmonic enhancement of fluorescence such as presented in this manuscript can allow this technique to be reliably and widely applicable for a broad range of biological assays including

complex molecular assemblies. RESULTS DEVICE FABRICATION AND DESIGN OPTIMIZATION The 3D-tapered gap plasmon nanocavities were fabricated in gold- and silica-coated silicon substrates as

shown in Fig. 1a, b. Detailed description of the fabrication process can be found in the methods section. The sidewalls that are ion-milled in the gold layer and the exposed top surface of

the SiO2 base, together form a fluidic MIM nanocavity that tapers vertically and laterally into a nanoscale tip (Fig. 1c). The hydrophilic SiO2 base attracts fluid into the 3D-tapered

nanocavity channel, promotes efficient molecular delivery, and provides surface site-specific molecular binding. Repeating the fabrication process, we also produced 3D-tapered gap plasmon

nanocavity arrays with 20-nm wide and 500-nm long tips integrated onto a larger fluidic channel as shown in the scanning electron microscope images of Fig. 1e–g. The design of the device was

optimized for (a) efficient coupling of excitation light into the device body, (b) optimal transversal confinement of EM field- high and uniform |E|2 of the guided mode- through the taper,

and (c) efficient longitudinal confinement of EM field at the tip. We chose 750-nm as the target wavelength for fluorophore excitation. Finite-difference time-domain (FDTD) simulations were

utilized to accomplish efficient confinement of the fundamental anti-symmetric (AS) SPP mode at the tip of the 3D-tapered gap plasmon nanocavity. In the new design that allows hotspot access

to fluids, a pair of Au walls separated horizontally on the SiO2 substrate support the AS mode whose electric field is aligned parallel to the substrate (Supplementary Fig. 1a, b).

Parameters were set to _w_body = 150 nm, _h_body = 150 nm, and _l_body = 3 μm in order to accomplish efficient coupling of excitation light and low-loss guidance of the AS mode inside the

body with high efficiency (Supplementary Note 1). The taper angle (_α_) was set to 20° in order to achieve efficient transversal confinement of EM field, which results in large and a uniform

|E|2 profile at the tip (Supplementary Note 2, Supplementary Fig. 2). Figure 2a shows the profiles of EM energy density _u_ at the cross-sections of the body (top) and tip (bottom) at _α_ =

 20°. The total transversal EM energy stored inside the body (_U_A_body) is efficiently confined inside the tip with minimal loss (_U_A_body ~_U_A_tip), which significantly increases the

average transversal EM energy density \(\overline u _{\mathrm{A}}\) at the tip (Fig. 2b, Supplementary Note 2). The 3D-tapered gap plasmon nanocavity showed greater confinement of EM energy

(greater \(\overline u _{\mathrm{A}}\) at the tip) than the 2D-tapered nanocavity and the tip-only structure (MIM structure without a taper) of the same tip size due to a larger

cross-sectional area of the body being capable of storing greater _U_A_body (see “Methods”). This extreme confinement of EM energy through the 3D taper suppresses evanescent-field type decay

inside the 20 × 50 nm2 tip (MIM gap), which is generally observed in high-|E|2 plasmonic structures such as a bowtie nanoantenna15,28 or tip-only structures (MIM waveguides without a

taper)29 (Fig. 2c, d and Methods). The optimized 3D taper achieved four times greater \(\overline u _{\mathrm{A}}\) and 40× improvement in uniformity _σ_|E|2 than a bowtie nanoantenna of the

same gap size (Fig. 2e). All of the gap plasmon structures studied in the FDTD simulations -3D-, 2D-tapered nanocavities, and the tip-only structure showed a trend that a greater

\(\overline u _{\mathrm{A}}\) enabled a more uniform hotspot (a smaller _σ_|E|2) and greater average |E|2 (\(\overline {\left| {\mathbf{E}} \right|^2}\)). As a result, the optimized

3D-tapered nanocavity provided a large (\(\overline {\left| {\mathbf{E}} \right|^2}\) = 230), uniform (_σ_|E|2 = 0.05) |E|2 profile at the tip along with an 11% net coupling efficiency.

Experimental analysis of the taper angle and body width also matched the trend predicted by simulations, as maximal emission output was obtained from devices with taper angle _α_~20° and

_w_body = 150 nm (Supplementary Fig. 3). The volume and |E|2 profile of the hotspot can be further optimized for capture of targeted number of molecules by varying _l_tip. A shorter _l_tip

allows the coupled EM energy to be more densely packed longitudinally inside the tip, which results in greater |E|2 within a smaller hotspot volume. A 20-nm long tip provided a

\(\overline{\left| {\mathbf{E}} \right|^2}\) magnitude of 550 within a 20 × 50 × 5 nm3 hotspot (Fig. 2f, g, Supplementary Note 3, Supplementary Fig. 4). Based on the simulations,

experimental results and considerations, devices were fabricated with the aforementioned dimensions (as shown in Fig. 1) and fluorescence enhancement was studied. VOLUMETRIC OPTICAL

CONFINEMENT AND BIOSENSING We experimentally tested coupling of incident light into the nanocavity and gap plasmon-mediated volumetric confinement at the tips using surface-linked molecular

layers and fluorescent labels. A molecular monolayer of biotin was assembled along the exposed silica surface of the 3D-tapered nanocavity, using silane-polyethylene glycol-biotin (SPB) as

the reagent to form silane–silica covalent linkages (Supplementary Fig. 5a). Streptavidin linked with Alexa Fluor 750 (S-AF 750) was then used as a fluorescent label for detection of biotin

in the tips, taking advantage of the well-known strong and highly-specific molecular interaction between biotin and streptavidin30,31. This two-step binding reaction allows the formation of

a monolayer of fluorescently-tagged streptavidin on the silica surface along the length of the 3D-tapered nanocavity. We observed the capture of diffusing streptavidin molecules at high

concentrations (1 µM) and resultant enhancement in fluorescence once a molecule binds to the tip region (Supplementary Fig. 5b, c). Detection of molecules diffusing in solution at micromolar

concentrations has been an important target for biological tracking and has been demonstrated using nanostructures with zeptoliter detection volumes16. However, these detection methods

lacked molecular specificity commonly required for bioassays. The silica base of our device serves as a targeted functionalization region inside the gap plasmon nanocavity, allowing specific

capture of molecules diffusing in solution and differentiating them from the background through enhanced fluorescence signal. After molecular binding, chips were washed to remove unbound

molecules and interrogated using tail-end and full illumination modes (Fig. 3a, b). The tail-end illumination mode involves illumination of the back-end of the 3D-tapered nanocavity, whereas

full illumination mode allows the complete device to be placed under illumination (Supplementary Note 4). Excitation light from a near-infrared light-emitting diode light source (750 nm)

was incident on the 3D-tapered nanocavities and a visible hotspot in the form of enhanced fluorescence was observed from the sub 20-nm tip region as a result of volumetric field confinement

at the tips (Fig. 3b). Due to improved coupling and light collection through the body of the device, about an order of magnitude improved intensity (~9×) at the tip was obtained using full

illumination mode as compared with tail-end illumination mode, which agrees with the calculation based on the FDTD simulations (~10×) (Supplementary Fig. 6). The role of the 3D-tapered

nanocavity body towards fluorescence enhancement was further verified by fabricating dimensionally-varying structures (Supplementary Note 5). Fluorescence intensity comparison between the

samples indicated that stand-alone taper and tip structures have a significantly weaker performance for optical molecular analysis as compared with the complete device (Supplementary Fig. 

7). This observation can again be attributed to improved coupling efficiency for the full device (Supplementary Fig. 8), in addition to light collection through the device body. For

subsequent molecular detection and analysis of plasmonic enhancement, full illumination mode was implemented. Limit of detection for biomolecules on the device was examined using two types

of sensing experiments. First, detection of low concentration protein molecules in solution (10 pM–1 nM) was performed, testing device suitability for on-chip diagnostics of rare

disease-specific biomarkers (Supplementary Fig. 9). The detection limit in this case is governed by diffusive transport of molecules to the plasmonic hotspot and can be accelerated by

improving molecule transport using convective flow, magnetic or dielectrophoretic trapping. Second, we examined the capture and detection of individual or small array of molecules at the

hotspot, while high concentration of molecules were present in solution. User-controlled analysis of single or small array of molecules remains an important target for high-resolution

analysis of protein function and behavior32,33,34. This is especially important in cases where ligands and biomolecules are physiologically present at higher (μM–mM) concentrations35. We

control the number of molecules captured within the tips by altering the tip length. After formation of the biotin monolayer within the 3D-tapered nanocavities, we utilized an anti-biotin

IgG primary antibody (tagged with DyLight 755 ~spectral response similar to AF-750) for performing fluorescence assays36. In order to compare effect of tip length on capture of molecules, we

performed these antibody binding experiments using devices with _l__tip_ = 500 nm as well as _l__tip_ = 20 nm (Fig. 3c, d). The dimensions of a 3D-tapered nanocavity with a short tip

(_l_tip = 20 nm, _w_tip = 20 nm) compared with those of an IgG antibody (length ~15–20 nm)36,37 indicate that a single antibody should be specifically trapped within the tip region. We

performed atomic force microscopy (AFM) which demonstrated uniform monolayer of PEG-biotin and antibodies on flat silica surfaces and indicated that only one antibody should specifically

occupy the 20 nm × 20 nm tip (Supplementary Fig. 10). We obtained 1.5% variation between expected and measured fluorescence intensity for a single molecule at the short tip, further

indicating presence of a single antibody at the short tip-end. The calculation was based on integrated fluorescence intensities from the tips and simulation-predicted |E|2 profiles (Fig. 2a)

along with an assumption of packed molecular arrangement within the tips. EM FIELD DECAY-RESISTANT PLASMON-ENHANCED FLUORESCENCE We analyzed the enhancement of fluorescence experienced by

an emitter within the tip, as compared with a non-enhancing substrate (silica or glass surface). As discussed previously, the tip region of the device exhibits a hotspot with uniformly

distributed, highly confined electromagnetic field (Fig. 4a). The emission enhancement was calculated using FDTD simulations (Fig. 4b, c), where the net fluorescence enhancement experienced

by a fluorophore is defined as a product of EM field intensity and quantum yield gain (Supplementary Note 6, Supplementary Figs. 11, 12). As discussed in detail in Fig. 2 and Supplementary

Fig. 2e, the 3D-tapered gap plasmon nanocavity provides superior |E|2 uniformity within the gap compared with conventional MIM structures. Our analysis also shows that the 3D-tapered gap

plasmon nanocavity allows greater radiative decay rate that results in up to 28.2% enhanced quantum yield gain along the width of the channel as compared with the tip-only structure, thereby

enabling ~500× greater net fluorescence enhancement (Supplementary Fig. 13, Fig. 4b, c). For a 500 nm long tip, the expected fluorescence enhancement is >1000 for about 70 % of the

channel width. The full-width half-maximum (FWHM) covers 95.5% of the _x_-axis with indication of fluorescence quenching very close to the metallic sidewalls (Fig. 4b). Fluorescence

enhancement is very uniform (~1000) along the _y_-axis and does not decline below half of the maximum enhancement throughout the channel height (Fig. 4c). This is especially important as

variation along the y-axis represents the increase in fluorophore height from the bottom silica surfaces. This can be a result of the size of molecule the fluorophore is attached to or based

on number of molecules in the assembly. The simulations indicate that this variation should have minimal impact on the enhancement experienced by the emitter, which has been a long-standing

challenge for plasmonics-enhanced fluorescence. In order to experimentally verify the optimized fluorescence enhancement response predicted in Fig. 4b, c, we performed tests using

diversely-sized molecules including dye molecules, aptamers, smaller proteins and antibodies. These molecules were specifically bound to the 3D-tapered nanocavity, and fluorescence

enhancement was analyzed compared with non-structured SiO2 control samples. While the dye molecules were covalently bound to the bottom silica surface, all other molecules were specifically

bound to their respective recognition agents or antigens preassembled on the surface (Methods and Supplementary Note 7). The expected height of the fluorophore from the silica base of the

nanocavity is indicated in Fig. 4d and varies from <1 nm to ~20 nm. The experimentally calculated enhancement performance of the device showed a uniform response for various molecular

shapes and heights, which agrees with the simulation results shown in Fig. 4c. We expect this device to maintain the same enhancement for molecular assemblies with height up to 50 nm, which

is defined by the height of the tip for this device. The level of enhancement was dependent on the tip length as expected. For tips with length 500 nm, the enhancement was close to 950

whereas shorter tips (_l_tip = 20 nm) provided higher enhancement (EF~2200) due to stronger |E|2 enhancement at the tip region for diverse molecular sizes (Fig. 4e, Supplementary Fig. 14).

These results matched the trend and values predicted earlier by simulation-based analysis (Supplementary Fig. 3c, Supplementary Note 8). As fluorescence enhancement is dependent on the

quantum yield of the fluorophore used, we also used another metric, enhancement figure of merit—which normalizes the enhancement with respect to the dye quantum yield and allows comparison

of device performance to other nanostructures (Supplementary Table 1)18. The device showed an enhancement figure of merit close to 260, which is one of the highest values obtained for

fluorescence enhancement obtained using plasmonic nanostructures, and uniquely provides this enhancement independent of molecule size within the nanocavity tip. DISCUSSION We have

demonstrated 3D-tapered gap plasmon nanocavities, which provide one of the highest enhancement of fluorescence obtained by plasmonic nanostructures (EF: ~2200 with figure of merit ~260),

independent of the size of the molecular assemblies used in the assay. Overcoming molecule size and placement-dependent extreme variation in plasmonic fluorescence enhancement has been a

major challenge restricting widespread application of this method in bioassays. The nanostructure geometry presented in this work demonstrates a way to overcome this limitation thereby

improving the consistency and range of plasmon-enhanced emission for diversely sized assembly of molecules. Simultaneously, we also demonstrate capture and visualization of single antibodies

at the tip as well as sensing of proteins at low concentrations (10 pM). These advantages can be readily transferred towards applications such as highly sensitive biosensing using molecular

labels of varying sizes and analysis of single molecules or tightly controlled arrays of molecules for protein orientation, protein function, and biological polymer formation

studies32,33,34. While the device geometry promises several benefits, weaknesses of the current fabrication process include low throughput using focused ion beam lithography and high

footprint of the device compared with smaller nanostructures. Future improvements can target the replacement of current fabrication process with wafer-scale methods including nanoimprinting,

e-beam lithography, and template stripping for reproducible manufacturing of nanocavities in combination with anisotropic etching methods for the tapered portions. We may also see further

improvement in device performance after replacing ion beam milling with alternative methods mentioned above, which are known to yield smoother device surfaces, and have shown such

improvements in the past18. The presented device design can also provide advantages in other areas of nanophotonics for optical confinement, data-transfer, quantum optical communication, and

molecular sensing in mid-infrared and terahertz domains. METHODS 3D-TAPERED NANOCAVITY FABRICATION Single-side polished silicon wafers with thermally grown SiO2 (thickness: 1 µm) were

purchased from University Wafers, Boston, USA. E-beam evaporation was used to deposit 50 nm gold (Au) on the wafers. 3D-tapered nanocavity patterns were milled through the gold and silica

using a FEI Nova 600 dual beam system as shown in Fig. 1. Au (50 nm) was deposited again using e-beam deposition. Second round of milling was performed using Nova 600 to remove gold from the

bottom of the substrates, exposing the silica and to mill the tip. SIMULATIONS We used a commercial software developed by Lumerical Inc. for the FDTD analyses. In all analyses, the mesh

size was 1 nm and uniform throughout the device area. For the |E|2 and uniformity analyses, a 750-nm dipole source was placed at the tail-end of the 3D-tapered nanocavity body and the

intensity of the guided AS mode was monitored across the cross-section of the tip. \(\overline {\left| {\mathbf{E}} \right|^2}\) at each _α_ was calculated by averaging the |E|2 profile over

the cross-sectional area (20 × 50 nm2) of the tip. _σ_|E|2 was calculated by normalizing the |E|2 enhancement profiles (setting 1 as the highest) and calculating 2D standard deviation of

the profiles over the cross-sectional area. 2D-tapered nanocavity was designed to provide only lateral confinement (body cross-sectional area: 150 × 50 nm2) along with the same taper length

of 500 nm and tip cross-sectional area (20 × 50 nm2) as the 3D-tapered nanocavity. The tip-only structure was a simple MIM waveguide without a taper that provided the same cross-sectional

area (20 × 50 nm2). The bowtie antenna was a set of two equilateral Au triangles (side length 140 nm, thickness 50 nm) separated by 20 nm on a 3-nm thick Cr layer on top of a 25-nm thick

ITO15,28, which provided the same cross-sectional area at the gap (20 × 50 nm2). For the coupling efficiency into body analyses (Supplementary Figs. 6, 8), a 1.4-NA Gaussian source centered

at 750-nm was focused onto either the tail-end or varied longitudinal locations (_z_) throughout the body. In both cases, transmitted power through the cross-section was calculated at each

location of incidence. For the fluorescence enhancement analysis, a 750-nm dipole source was placed inside the tip and transmitted power through a closed box enclosing the dipole and tip was

monitored in order to obtain radiative and non-radiative decay rates (see Supplementary Note 6 and Supplementary Figs. 11,12). DYE (AF-750) BINDING 3-Aminopropyl-triethoxysilane (APTES) was

obtained from Sigma-Aldrich, USA. The nanocavity chips were cleaned with acetone, methanol, and isopropanol prior to binding experiments. A 1% APTES solution was prepared in toluene

(anhydrous) and allowed to bind on the chips for 30 min. This step functionalizes the silica surface with amino groups available for further binding. The substrates were washed with toluene

to remove weakly bound APTES molecules. The chips were baked at 110 °C for 30 min and then cleaned again with DI water for 15 min. The samples were then dried with nitrogen. Alexa Fluor 750

NHS ester was purchased from Thermofisher Scientific USA and dissolved in DMSO (1 mg/ml). The solution was added to the substrates with stirring and dye molecules were allowed to bind to the

functionalized substrates for one hour. The chips were then cleaned well with DMSO and water. A drop of water was placed on the chips, covered with a coverslip, and then imaged. APTAMER

SENSING Insulin from bovine pancreas was purchased from Sigma Aldrich (USA). Insulin was mixed in PBS at a concentration of 10 µM. Solution was added to the chips and left undisturbed for an

hour to allow physisorption of the peptide hormone to the surfaces. BSA (0.1%) was then added to the chips and allowed to interact with the surface to account for any nonspecific binding.

An insulin-binding aptamer (sequence: 5′ GGT GGT GGG GGG GGT TGG TAG GGT GTC TTC 3′) with AF-750 conjugated to the 5′ end was obtained from IDT technologies (St. Louis, USA). Aptamer was

dissolved at a concentration of 1 µM in a folding buffer (Tris: 10 mM; MgCl2·6H2O: 1 mM; NaCl: 100 mM; pH: 7.4) and added to the substrate for an hour. The samples were washed with folding

buffer after an hour and then imaged. SURFACE BIOTINYLATION SPB (MW: 1000 and 5000) were purchased from Laysan Bio Inc. (USA). For surface functionalization, SPB powder was dissolved in 50%

ethanol: DI water. This solution was added to substrates with exposed silica surface and left undisturbed for an hour. The substrates were then washed with DI water. STREPTAVIDIN BINDING

Streptavidin Alexafluor 750 conjugate (SAF-750) was purchased from Thermofisher Scientific (USA). Streptavidin in PBS (pH 7.4, concentration: 0.1 mg/ml) was added to chips with biotin

monolayer and left undisturbed for an hour. The chips were then washed with DI water and imaged. A layer of water was maintained at all times on the chip through molecular functionalization

and imaging. For Streptavidin bioassays, the concentration of streptavidin in PBS was varied while maintaining the duration of incubation (1 h). ANTIBODY BINDING Anti-biotin antibody (Mouse

monoclonal (Hyb-8), IgG1, MW: 244 kDa) conjugated with DyLight 755 fluorophore (response similar to Alexafluor 750) were obtained from Novus Biologicals (USA). The antibody was diluted in

PBS at a concentration of 0.05 mg/ml and added to the chips. The solution was left undisturbed for 2 h. The samples were then washed with DI water. ATOMIC FORCE MICROSCOPY (AFM) Topology and

phase images of dried antibody monolayer on silica substrates were obtained on an AFM system (Bruker Dimension Icon, Santa Barbara, CA, USA) using a 100 µm long monolithic silicon

cantilever (All-In-One-Al, NanoAndMore USA Corp (BudgetSensors), Watsonville, CA, USA). All the experiments were conducted under ambient laboratory conditions using tapping mode with a

resonance frequency of about 350 kHz. Images were analyzed afterward by commercial software Nanoscope Analysis. IMAGING Fluorescence imaging was performed using a Leica DMI 6000 widefield

fluorescence microscope. Scanning electron micrographs were taken using FEI Nova 600 and 200 dual beam systems. IMAGE ANALYSIS Images were analyzed using Fiji (ImageJ) software38. Graphs

were created in Matlab and Graphpad Prism 5.01. DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author upon reasonable request.

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biological-image analysis. _Nat. Methods_ 9, 676–682 (2012). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS Device fabrication was performed at the Kavli Nanofabrication

Center at California Institute of Technology. Fluorescence imaging was performed at the Beckman Imaging center at California Institute of Technology. Funding for this research was provided

by HMRI Investigator award and Samsung GlobalResearch Outreach (GRO) program. AUTHOR INFORMATION Author notes * These authors contributed equally: Shailabh Kumar, Haeri Park. AUTHORS AND

AFFILIATIONS * Department of Medical Engineering, California Institute of Technology, 1200 E. California Blvd., MC 136-93, Pasadena, CA, 91125, USA Shailabh Kumar, Haeri Park, Radwanul H.

Siddique, Vinayak Narasimhan, Daejong Yang & Hyuck Choo * Image Sensor Lab, Samsung Semiconductor, Inc., 2 N. Lake Ave. Ste. 240, Pasadena, CA, 91101, USA Haeri Park, Radwanul H.

Siddique & Hyuck Choo * Department of Electrical Engineering, California Institute of Technology, 1200 E. California Blvd., MC 136-93, Pasadena, CA, 91125, USA Hyunjun Cho & Hyuck

Choo * Imaging Device Lab, Device & System Research Center, Samsung Advanced Institute of Technology (SAIT), Suwon, 16678, Republic of Korea Hyuck Choo Authors * Shailabh Kumar View

author publications You can also search for this author inPubMed Google Scholar * Haeri Park View author publications You can also search for this author inPubMed Google Scholar * Hyunjun

Cho View author publications You can also search for this author inPubMed Google Scholar * Radwanul H. Siddique View author publications You can also search for this author inPubMed Google

Scholar * Vinayak Narasimhan View author publications You can also search for this author inPubMed Google Scholar * Daejong Yang View author publications You can also search for this author

inPubMed Google Scholar * Hyuck Choo View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.K., H.P., and H.C. conceived the study. S.K.

performed the device fabrication and the molecular fluorescence experiments. H.P. performed the theoretical device design optimization and numerical simulations. HJ.C. and D.Y. helped

establish device fabrication protocol. R.H.S. helped with numerical simulations and device optimization. V.N. assisted with data visualization and validation. S.K., H.P., and H.C. wrote the

paper. CORRESPONDING AUTHOR Correspondence to Hyuck Choo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION

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copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Kumar, S., Park, H., Cho,

H. _et al._ Overcoming evanescent field decay using 3D-tapered nanocavities for on-chip targeted molecular analysis. _Nat Commun_ 11, 2930 (2020). https://doi.org/10.1038/s41467-020-16813-5

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