Plasmon-molecule remote coupling via column-structured silica layer for enhancing biophotonic analysis

We demonstrated remote plasmonic enhancement (RPE) by a dense random array of Ag nanoislands (AgNIs) that were partially gold-alloyed and attached with column-structured silica (CSS) overlayer of more than 100 nm in thickness. The physical and chemical protection of the CSS layer could lead to reducing the mutual impact between analyte molecules and metal nanostructures. RPE plate was realized just by sputtering and chemical immersion processes, resulting in high productivity. We found a significant enhancement on the order of 10$^7$-fold for Raman scattering and 10$^2$-fold for fluorescence by RPE even without the proximity of metal nanostructures and analyte molecules. We confirmed the feasibility of RPE for biophotonic analysis. RPE worked for dye molecules in cells cultured on the CSS layer, enabling the enhanced fluorescence biosensing of intracellular signaling dynamics in HeLa cells. RPE also worked for biological tissues, enhancing Raman histological imaging of esophagus tissues with esophageal adventitia of a Wistar rat attached atop the CSS layer. We also investigated the wavelength dependency of RPE on the on- or off-resonant with the dye molecular transition dipoles with various molecular concentrations. The results suggested that the RPE occurred by remote resonant coupling between the localized surface plasmon of AgNIs and the molecular transition dipole of the analyte via the CSS structure. The RPE plate affords practical advantages for potential biophotonic analyses such as high productivity and biocompatibility. We thus anticipate that RPE will advance to versatile analytical tools in chemistry, biology, and medicine.


INTRODUCTION
Fluorescence and Raman spectroscopy are essential analytical tools in biological and biochemical studies. [1][2][3][4][5][6][7][8] In fluorescence biosensing of live cells, various fluorescence probes offer information about intracellular metabolism and signaling mechanisms. [9][10][11][12] As for Raman spectroscopic biosensing of biological tissue, there has been considerable interest in the potential clinical application. [13][14][15][16][17][18][19][20] Currently available biophotonic methods are subject to some technical limitations. For fluorescence biosensing, since the sensor molecule is an additive that may disturb the natural metabolism in live cells, its dosage must be kept low, which causes a tradeoff between external perturbation and analytical sensitivity. The sensitivity problem could be serious in the case of tissue Raman imaging because intense and prolonged laser irradiation necessary for signal acquisition might induce photodamaging to specimens.
Plasmon-enhanced fluorescence and Raman spectroscopy offer a promising solution for analytes with weak optical signals. Surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEFL) have many important applications in biophotonic analyses. [21][22][23][24][25][26][27][28][29][30] Nevertheless, the application of localized surface plasmon resonance (LSPR) phenomena to bioanalysis has been limited because the nanostructured metal surfaces need to be in proximity to the analyte molecules, possibly leading to the mutual degradation between analyte molecules and metal nanostructures.
In this paper, we report plasmon-mediated long-range enhancement of fluorescence and Raman scattering via dielectric nanostructures, namely, remote plasmonic enhancement (RPE). The key element for the present RPE function is the column-structured silica (CSS) layer that protects the mutual adverse impact between a dense random array of Ag nanoislands (AgNIs) and analyte molecules. The experimental evidence presented in this paper establishes the proposed concept of 5 RPE. We have also demonstrated RPE-enhanced fluorescence biosensing for live cells and RPEenhanced Raman imaging of biological tissues as practical validation of RPE.

Materials
Most of the chemicals of special reagent grade, including basic fuchsine (FUC) dye, were used as received from Wako Pure Chemical Industries. Polyvinyl alcohol (PVA) was of polymerization degree of 500 or less and a saponification degree of 86-90%. Rhodamine 6G dye (R6G) was obtained from Exciton as a chloride salt. Intracellular Ca 2+ indicator fluo3-AM was from Dojindo Laboratories. An extracellular matrix (Matrigel) was from Corning. The frozen section compound (FSC 22) was from Leica Biosystems.

Preparation of RPE Plates
A dense array of AgNIs was grown by direct-current Ar + ion sputtering onto the smoother side of a float slide glass plate (Type S7213; Matsunami) in an apparatus similar to that used elsewhere. 31 A glow discharge at a negative voltage of 1.4 kV or less supplied to the cathode as an Ag target and a discharge current of 15 mA produced a dense random array of AgNIs in 5 min.
The deposition of a CSS layer on the AgNIs layer was conducted using radio-frequency sputtering (model RFS-200; Ulvac) at an Ar pressure of 1.0 Pa. The radio-frequency power was adjusted to 100 W, at which we gained a SiO 2 deposition rate of 10 nm/min. The high-energy discharge plasma caused the substrate temperature up to 160 °C.

Gold(I)/Halide Bath Treatment on AgNIs
The gold latensification has long been used in the silver halide photographic technology, which renders the Ag latent image a higher catalytic ability in photographic development. [32][33] This function stems from a nanoalloying of an (Ag) n latent image center into (Ag) n-x (Au) x by replacement or into (Ag) n (Au) m by plating. The standard protocol for the preparation of the gold(I)thiocyanate latensification solution is as follows; 0.5 g of KSCN is added to 40 mL of 0.1 wt% NaAuCl 4 ·2H 2 O solution, is heated to boiling for a few minutes, 0.6 g of NaBr is added, and dilutes with water to 1 L. In the present study, the optimum solution was prepared by diluting this standard solution with 0.2 M NaBr by 30 times. We thus obtained a gold latensification bath containing Au(SCN) 2− at a concentration of 3.3 μM. The CSS-protected AgNIs were processed for 1-2 min at 65 °C, quickly rinsed with water, immersed in 0.2 M NaCl solution for 5 min at 75 °C, rinsed again with water, and dried under air conditions. The follow-up processing in 0.2 M NaCl solution at 75 °C led to substantial uptake of NaCl along the intercolumn boundaries.

Spectroscopic Measurements
A large body of the fluorescence and Raman spectra were measured using a simple laboratorymade spectrometer. It comprised a low-power He-Ne laser (633 nm, 0.7 mW) or a green diode laser (532 nm, 3 mW), two lenses in series to collect the emission from the sample on the RPE 7 plate onto an aperture of a light receiver head, connected via fiber optics to the cooled multichannel analyzer (PMA-11 and C5966-31; Hamamatsu Photonics). The other one, a commercial portable spectrometer, was a coaxial probe Raman spectrometer (Raman-Probe-785; StellarNet) operated at the excitation wavelength of 785 nm at a high laser power of 600 mW.
High-resolution Raman spectra for the excitation laser wavelength of 633 nm were acquired with a Raman spectrometer (DXR3 SmartRaman Spectrometer; ThermoFisher Scientific). Highresolution Raman spectroscopy and imaging were conducted using a line illumination confocal Raman microscope (Raman-11; Nanophoton) with the excitation laser operating at 532 nm.
Fluorescence images of the cells were captured by an electron-multiplying charge-coupled device camera (ImagEM; Hamamatsu Photonics) operated with MetaFluor software (Molecular Devices). The excitation wavelength was 480 nm.

Cultivation of Cells on RPE Plates and Incorporation of Fluorescent Biosensors
HeLa cells were obtained from ATCC and were cultured routinely with Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 30 units/mL penicillin, and 30 μg/mL streptomycin under 95% air, 5% CO 2 atmosphere at 37 °C. For Ca 2+ measurement in HeLa cells, they were trypsinized and 5×10 4 cells were plated onto a Matrigel-coated RPE plate or glass. After

Preparation of Biological Tissues
All animal experiments were conducted with the approval of and in accordance with guidelines from the Committee for Animal Research, Kyoto Prefectural University of Medicine (Permission No. M25-109). The esophagus with esophageal adventitia was excised from a Wistar rat after euthanasia. The esophagus with esophageal adventitia was immediately embedded in the frozen section compound, snapfrozen in dry ice-acetone, and stored at −80°C until cryostat sectioning.
The frozen samples were sliced into 5 μm in thickness using a cryostat microtome (CM1900; Leica) and mounted without any fixation on an RPE plate or a 0.17-mm thickness cover glass (No.1; Matsunami). Figure 1a shows a two-dimensional dense random array of AgNIs, which we used as the basal layer constituting an RPE plate. Each AgNI measured 50-150 nm in lateral dimension and less than 20 nm in height. Unless otherwise protected, such AgNIs are highly vulnerable that they do not even withstand aerobic corrosion and undergo immediate corrosive dissolution in saline, i.e., under a common aqueous environment for biological experiments. The CSS layer, ~100 nm or more in thickness, worked as a robust protection layer for the AgNIs. Figure 1b

Fundamental Confirmation of RPE
AgNIs protected with the as-grown CSS layer already acquired the capability to bring significant Raman enhancement for various molecules, such as R6G, 2-naphthalene thiol, 1-hexadecane thiol, 10 and cysteamine, adhered on top of the CSS layer, as shown in Figure 2. This pristine plate was not treated in the gold(I)/halide bath. The RPE-enhanced Raman spectrum of a spin-coated R6G in Figure 2a, compared to a spectrum of a 1.25 mM ethanolic solution of R6G on a slide glass (8×10 16 molecules/cm 2 in scattering volume, see Figure S1), suggested that the corresponding Raman enhancement factor (EF) typically reached the order of 10 6 , which was comparable to that achieved in the conventional SERS. In practice, the RPE functioned not just for molecules adhered to the CSS layer, as shown in Figure 2, but also for various analyte species embedded or dispersed in certain polymers (typically PVA) laid on the CSS protection layer.

11
As for molecular species that were weakly bound to the CSS surface, they were readily washed out from the surface, and thereby the enhanced Raman signals were extinguished concomitantly.
This proved that the observed enhancement occurred on the Raman-active species separated from the AgNIs by a more than 100 nm-thick CSS layer. For further proof of this long-range Raman enhancement, we also carried out an adhesive tape test ( Figure S2), where we lightly dispersed 2naphthalene thiol fine powders onto the adhesive side of a tape. We obtained enhanced Raman signals of 2-naphthalene thiol only when the tape was attached to the CSS surface of the RPE plate, and besides, the signals reversibly appeared and disappeared with the tap on and off the surface.
Furthermore, we found that the RPE function was upgraded by the gold(I)/halide bath treatment of the RPE plate. Figure

Enhancement characteristics of RPE
To characterize the enhancement effect of RPE, we first evaluated the effect of two opposite optical configurations, i.e., front-or rear-side excitation and on the same side collection of emission, as shown in Figure 5a. The RPE-enhanced spectra exhibited analogous signal intensities for the two opposite optical configurations for both Raman scattering and fluorescence, as shown in Figures 5b and 5c. The fact that the front/rear excitation and signal collection resulted in the same spectral intensity despite the 90% light attenuation by the dense AgNI array of the RPE plate suggested that LSPR of the AgNIs was intimately involved both in the excitation and emission processes. the fluorescence of FUC would have to show EF of ~4 likewise. However, it was ~20-fold which was much higher than that of R6G in the given excitation condition, as shown in Figure 5c. FUC has a small fluorescence quantum yield compared with R6G, even in the PVA matrix. These dependencies of EFs indicated that RPE was not just an electric field enhancement. 15 We also investigated the molecular concentration dependency for RPE. Figure 6a shows a FUC concentration series of RPE-enhanced emission spectra in a logarithmic intensity scale with overlapping Raman and fluorescence spectra taken at the excitation wavelength of 532 nm. In the lower concentrations (<10 μM), the Raman and fluorescence intensities increased almost proportional to the molecular concentration in both the front-and rear-side measurements. In contrast, in higher concentrations (> 100 μM), nonlinear dependence of the emission spectra on molecular concentration was observed. Namely, the Raman signals became invisible in both configurations in the logarithmic intensity scale, and the fluorescence signal was saturated in the rear-side measurement. This means that the equivalence between the two opposite optical configurations, characteristic of RPE, broke up in the high concentration regime (cf. Figure S3 for the R6G series of emission spectra given in linear intensity scale).  The Raman enhancement by RPE was also strongly affected by excitation wavelength. Since FUC had an absorption peak at around 550 nm, the results with the excitation wavelength of 532 nm shown in Figure 6 were in an almost rigorous electronic resonance condition of the excitation wavelength with the FUC molecular transition dipole. In a pre-resonance condition with the excitation wavelength of 633 nm, in which the excitation photon energy was near but lower than the absorption band of the FUC molecular transition dipole, the Raman EF by RPE was still large but decreased by an order of magnitude from the rigorous resonant case at the wavelength of 532 nm, as shown in Figure 7a. Conversely, the proportional Raman enhancement against concentration was observed up to 100 μΜ for the excitation wavelength of 633 nm, which was a much higher concentration than the corresponding upper limit to sustain such linearity for the excitation wavelength of 532 nm (~10 μM). In a further off-resonance condition of the excitation

RPE-Enhanced Fluorescence Biosensing for Live Cells
For the application of the RPE in fluorescence biosensing, we evaluated the RPE-enhanced fluorescence detection capacity of Ca 2+ oscillation of live cells. Precoating of the RPE plate with protein Matrigel led to a healthier growth of HeLa cells as like a general Matrigel-coated slide glass, as shown in Figure 8a. We employed fluo3-AM dyes as a cytosolic Ca 2+ sensor (Figure 8b), which has been extensively used as one of the most potent Ca 2+ indicators of the current biosensing technology. 35 We used a wide-field fluorescence microscope at the excitation wavelength of 480 nm.

Enhanced Tissue Raman Imaging
We show next how RPE works in histological Raman imaging. A tissue section of an esophagus with esophageal adventitia of a Wistar rat was attached onto an RPE plate, as shown in Figure 10a.
We used a line illumination confocal Raman microscope operating at the excitation wavelength of 532 nm.
Raman spectra of the respective tissue domains distinguished in the optical image are shown in Figure 10b. The distinct Raman spectra of vagus nerves, adipose tissues, blood vessels, and smooth muscles were clearly observed by using the RPE plate (red spectra). Whereas on slide glass (black spectra), we could obtain discernable Raman signals for only adipose tissues and blood vessels by  We also evaluated the tissue imaging capability of RPE, as shown in Figure 11. RPE-enhanced Raman images represented unique patterns that highlighted tissue domains. Table 1 summarizes this relationship along with the most probable vibrational assignments. In particular, the two 23 images at 1431 and 1450 cm −1 showed a sudden turn of contrast from that in favor of vagus nerves to that of adipose tissue, despite the minor difference in Raman shift. This suggested that the RPEenhanced Raman imaging sensitively reflected a subtle difference in the Raman shift associated with δ(CH) between the vagus nerve and adipose tissue domains.  CT, connective tissue; FSC, frozen section compound; VN, vagus nerve; AT, adipose tissue; BV, blood vessel; PEG, polyethylene glycol.

Mechanism Discussion on RPE
In this study, we have demonstrated RPE by AgNIs with CSS overlayer with more than 100 nm in thickness in fluorescence and Raman spectroscopy. The physical and chemical protection by the CSS layer could lead to reducing the mutual impact between analyte molecules and metal nanostructures. The CSS-protected AgNIs enabled a long-range plasmonic enhancement of fluorescence and Raman scattering, which we referred to as RPE. We found that the gold(I)/halide bath pre-treatment of the RPE plate intensifies the RPE activity. The enhancement effect by RPE has been confirmed with various molecules, including biological specimens.
The conventional plasmonic enhancement of fluorescence and Raman scattering has been realized by metal nanostructures with sufficient proximity of the metal surfaces to analyte molecules. Whereas in the present study, we found a possibility for enhancement without such a range limitation, namely, RPE. We obtained comprehensive experimental support for RPE, such as 1) the markedly upgraded RPE performance after the gold(I)/halide bath treatment, 2) the excitation wavelength dependency manifesting a huge difference in the Raman EF between the resonant and off-resonant excitation conditions, 3) the analyte molecular concentration dependency uncovering a strong preference for a low analyte concentration, and 4) the correlation between the front-and rear-side optical configurations suggesting the intimate role of LSPR both in the excitation and emission processes.
Further confirmation of RPE was performed with the biological samples, demonstrating enhanced fluorescence imaging of Ca 2+ signaling of living HeLa cells and enhanced Raman imaging of the histology of a rat esophageal tissue. We thereby found also that the RPE plate could provide the spatial resolution that was sufficient for identifying and distinguishing the cells and tissues. This novel enhancement modality of fluorescence and Raman spectroscopy will provide 25 an important insight in terms of the novel mechanism of plasmonic enhancement and for extending the applications of plasmon-enhanced fluorescence and Raman spectroscopy.
As for a possible mechanism of RPE, the resonant coupling between the metal nanoislands and the analyte molecules or the dipolar coupling between the giant plasmon dipole of LSPR and the molecular transition dipole of the analyte may play an important role, as indicated by the correlation between the front-and rear-side optical configurations. This possible mechanism of resonant coupling can also be inferred from the molecular concentration dependency and the wavelength dependency. At higher molecular concentration conditions, the intermolecular distance of analyte molecules becomes shorter, and the Förster-type transition dipole-dipole coupling between the analyte molecules may frequently occur by resonantly exciting the molecular dipole with light. 36 Under these conditions, the resonant coupling between the metal nanoislands and the analyte molecules, if any, would be disturbed. This effect may reflect in the molecular concentration dependency in the resonant case shown in Figure 6. At the concentration of 100 μM or higher of FUC, the average intermolecular distance was expected to be 25 nm or less, resulting in the random pairing of molecules within the Förster distance of ~6 nm would quite frequently occur. As a result, the concentration-dependent Raman enhancement might be no longer observed in the higher molecular concentration condition, possibly due to the disturbance of the resonant coupling between the metal nanoislands and the analyte molecules. In the off-resonant excitation condition, the molecular transition dipole becomes very small, and the intermolecular coupling may become insignificant compared to the plasmon-molecule coupling, resulting in the Raman enhancement according to the molecular concentration was observed even in the relatively higher molecular concentration conditions, as shown in Figure 7. However, the minor molecular transition 26 dipole involved in the plasmon-molecule coupling makes the Raman EF decrease accordingly, as demonstrated in Figures 6 and 7.
As for PVA, we used it as a polymer matrix to embed R6G and FUC dyes. We nevertheless observed no significant Raman signals associated with PVA under any excitation conditions. This is also consistent with the expected mechanism of RPE since PVA is transparent for wavelengths down to the far UV region affording minor transition dipoles by visible light excitation. PVA thus serves as an RPE-inert polymer matrix to disperse arbitrary analyte molecules of interest therein.
In the case of RPE-enhanced fluorescence biosensing of HeLa cells, fluorescent molecules incorporated in the host cells gain a large transition dipole in the given excitation condition and expect to undergo a strong enough coupling with metal nanoislands to bring the fluorescence enhancement. However, an excess amount of the fluorescence probes likely results in the failure of RPE due again to the interference by the intermolecular coupling.

Role of CSS
The aforementioned resonant coupling between metal nanoislands and analyte molecules is not mediated over long distances in common bulk materials. Thus, CSS is likely to act as a structure mediating long-range resonant coupling between the metal nanoislands and the analyte molecules.
The CSS divided by the directional grain boundaries offers a strongly anisotropic solid medium.
This anisotropy of the CSS expects to play an essential role in mediating the long-range resonant coupling. One evidence is the further enhancement of RPE by the gold(I)/halide bath treatment.
The gold(I)/halide bath treatment might decorate the intercolumn boundaries of CSS with the strongly polarizable halide ions (see Figure 3a). The resultant dielectric modification of the intercolumn boundaries of CSS would facilitate the long-range resonant coupling between metal nanoislands and analyte molecules.
If this hypothesis is correct, a similar promotion effect of the RPE could also be obtained with a gold-free halide bath treatment. Actually, we observed that plain halide bath treatment also brought about a remarkable increase in the RPE activity (data not shown). However, halide ions taken up into the CSS layer, especially at the considerably high processing temperatures, simultaneously caused a rapid corrosive halogenation of the AgNIs to deprive them of the RPE activity. The gold(I)/halide bath treatment allowed us to circumvent this problem, which we attributed to a partial nanoalloying of the AgNIs by the Au(SCN) 2− ions protecting against the corrosive halogenation by halide ions, as a similar manner of the process of gold latensification in the silver halide photography.

Bioanalytical Impact of RPE
The present results suggest that RPE serves as a useful potential platform for enhanced biophotonic analyses. The most significant advance of RPE is the sizable enhancement of the Raman scattering and fluorescence signals without the proximity of metal nanostructures and the biological specimens. The CSS layer thicker than 100 nm serves as a robust protection layer for the vulnerable AgNIs to survive highly corrosive solution environments that are employed in biological experiments. It also becomes possible to cultivate live cells atop the CSS protection layer of the RPE plate in the standard manner employing saline as a culture medium. It can also easily attach thin-sliced biological tissues onto the RPE plate. The analyte molecules placed at least ~100 nm apart from the metal surface no longer suffer from short-range electronic 28 interference by the metal. Therefore, RPE enables fast and sensitive molecular sensing in a more biocompatible environment.
In addition, not only these advantages but also the following effects can be obtained. In the case of tissue Raman imaging, RPE may work with a resonate coupling between metal nanostructures and analyte molecules, resulting in sensitive reflection of the electronic properties of the respective tissue materials in the Raman EFs by RPE. Consequently, unique Raman imaging reflecting both the vibrational and electronic states of the analyte molecule can be performed, as shown in Figure   10.
In the case of fluorescence biosensing for live cells, the unique feature of RPE in that it strongly favors a low analyte concentration affords an extra advantage of RPE because one always has to care about the interference effects by the external fluorescence probes. In fact, an adverse effect of fluo3-AM load might appear at the highest fluo3-AM loading level of 1.0 μM. The fluorescence intensities of fluo3-AM of a substantial portion of the HeLa cells yield high enough (>1000 counts) to analyze Ca 2+ oscillation in both the RPE plate and slide glass. However, the number of oscillations tended to be lower than that at the lower concentration conditions in both the RPE plate and slide glass. This may be understood in terms of the positive feedback mechanism of cells in the regulation of intracellular Ca 2+ . In this mechanism, the initial Ca 2+ release from the internal Ca 2+ stores, corresponding to the first spike, promotes the Ca 2+ influx corresponding to the subsequent oscillations. The sequestration of free Ca 2+ by complexing with excess fluo3-AM in the overdosed cells results in insufficient initial Ca 2+ rise, thus impairing the intracellular dynamics of the positive feedback loops. Whereas on the RPE plate at the low concentration conditions, the weakened fluorescence signals were sufficiently amplified with the less adverse effect of fluo3-AM load on Ca 2+ signaling, enabling the Ca 2+ oscillations to be counted more accurately.

CONCLUSION
We demonstrated RPE by a dense random array of AgNIs with a CSS overlayer of more than 100 nm thickness. The present RPE plate affords practical advantages for potential biophotonic analyses. RPE plate is realized just by sputtering and chemical immersion processes, enabling large-area RPE plates to be easily fabricated. Biocompatible structure with CSS provides a more biocompatible molecular detection environment to reduce the mutual impact between analyte molecules and metal nanostructures. It promises high analytical sensitivity and a short acquisition time for biophotonic measurements without any instrumental modification or certain sample manipulation. We thus anticipate that RPE will advance to versatile analytical tools in chemistry, biology, and medicine.

Supporting Information
The following files are available free of charge.
Comparison of 633-nm excited Raman spectra on a slide glass and an RPE plate ( Figure S1), adhesive tape test to verify RPE-enhanced Raman signals ( Figure S2