Tip-Functionalized Au@Ag Nanorods as Ultrabright Surface

Article Cite This: J. Phys. Chem. C 2018, 122, 17983−17993

pubs.acs.org/JPCC

Tip-Functionalized Au@Ag Nanorods as Ultrabright SurfaceEnhanced Raman Scattering Probes for Bioimaging in OffResonance Mode Boris N. Khlebtsov,*,†,‡ Daniil N. Bratashov,‡ and Nikolai G. Khlebtsov*,†,‡

J. Phys. Chem. C 2018.122:17983-17993. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 09/24/18. For personal use only.

†

Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russia ‡ Saratov National Research State University, 83 Ulitsa Astrakhanskaya, Saratov 410012, Russia S Supporting Information *

ABSTRACT: Surface-enhanced Raman scattering (SERS) performance of Au nanorods (AuNRs) can be enhanced by the tip adsorption of Raman molecules (RMs) through anisotropic polymer stabilization or through the embedding of RMs between AuNR cores and Ag shells of AuNR@RM@Ag composite particles. We propose a new strategy to design ultrabright SERS probes composed of high-aspect-ratio AuNRs with anisotropic Ag coatings, with preferred adsorption of RMs to open AuNR tips. Specifically, for 4nitrobenzenethiol (NBT) concentrations above a threshold value c > ctr, the fabricated Au@NBT@Ag particles had NBT-functionalized open Au tips, as well as anisotropic Ag shells grown on the AuNR sides. The SERS response of these probes with an optimal Ag shell was highest in the off-resonance mode, when the excitation wavelength was far from the plasmon resonance of the Au@NBT@Ag composites. Growing the Ag shell further to completely cover the AuNRs decreased the SERS enhancement. For biocompatibility and stability, the probes were additionally covered with a thin silica layer. Under optimal conditions, the probes demonstrated superstrong and superstable SERS spectra, as compared to those from common SERS tags (AuNRs, nanostars, and Au@Ag NRs) with surface-adsorbed NBT. The excellent SERS performance of the developed ultrabright probes is illustrated by single-particle detection of SERS spectra, Raman imaging of living cells, and deep tissue imaging.

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INTRODUCTION Surface-enhanced Raman scattering (SERS) imaging labels hold great potential to provide highly sensitive, multiplex, and noninvasive diagnostic tools.1 In usual SERS labels, also called SERS tags, Raman molecules (RMs) are adsorbed on the surface of plasmonic metal nanoparticles (NPs), which can be covered with an additional protecting layer.1,2 The intensity of a SERS signal depends on the Raman cross section of the reporter molecules3,4 and on the enhancing properties of NPs,5 determined by the plasmonic enhancement of the local electromagnetic (EM) field.6 Typically, NPs with star,7,8 rod,9 prism,10 compass,11 and cube12 morphologies show stronger overall enhancement than do spherical NPs because of the strong EM field near sharp13,14 or junction points,15 acting as EM hot spots.16 Localization of RMs in EM hot spots is key to the development of SERS nanoplatforms with highest enhancement factors (EFs).17 In Au nanorods (AuNRs), the hot spots are localized only near the NPs’ ends. In particular, Chen et al.18 reported that about 65% of the overall SERS enhancement comes from the AuNR ends, occupying only a small part of the total surface area. Synthesis of AuNRs in single19 or binary20 surfactant mixtures gives rise to a stabilizing bilayer of cetyltrimethy© 2018 American Chemical Society

lammonium bromide (CTAB), with densely packed molecules on the facets and loosely packed ones on the ends.21 This provides AuNRs with site-specific chemical properties. For example, the AuNR ends can be selectively etched with chlorides22,23 or functionalized with analytes.18 Monothiol and dithiol derivatives of benzene adsorb on the AuNR ends but not on the facets;24 thus, adsorption enables the formation of NR dimers, chains, and other assemblies, generating EM hot spots.25,26 Effective generation of hot spots on AuNR tips and strong SERS responses are usually associated with on-resonance laser excitation, when the excitation wavelength overlaps the localized plasmon resonance (LPR).27,28 On the other hand, the resonance excitation of SERS tags during bioimaging can produce undesirable effects such as enhanced background metal fluorescence29 and photothermal degradation of labels and cells or tissues being imaged.30 Several SERS tags are available to decouple the LPR spectrum from SERS performance, thus ensuring minimal side effects and maximal SERS Received: May 19, 2018 Revised: July 14, 2018 Published: July 19, 2018 17983

DOI: 10.1021/acs.jpcc.8b04772 J. Phys. Chem. C 2018, 122, 17983−17993

The Journal of Physical Chemistry C

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performance under off-resonance excitation.31 Such decoupling can be achieved by embedding Raman-active molecules inside multilayered plasmonic NPs with spherical,32−42 polygonal,43 or anisotropic44−47 cores. In those structures, the maximal SERS enhancement is due to the plasmonic interaction between metal core and shell and is no longer associated with the LPR.38,48 Furthermore, SERS tags with embedded RMs have additional advantages: the RMs are protected from desorption and subjected to strong hot spots and their SERS response is stable, as compared to that from common “external” tags with surface-adsorbed RMs.49 Despite the advantages of anisotropic SERS tags with embedded RMs, several points remain unclear. First, it was pointed out previously45 that the Au-layered “nanomatryoshkas (NMs)” [Au(core)@RM@Au(shell)] have distinct hollow32 or bridged43 gaps (∼1 nm, depending on the structure of the dithiol insulator), whereas the Ag-coated Au(core)@RM@Ag(shell) structures have no gaps between Au and Ag layers, as shown by transmission electron microscopy (TEM) and high-resolution TEM (HRTEM).45 Second, the role of capping agents such as CTAB and thiolated reporter molecules in the formation of Ag shells on AuNR cores is complicated. For example, Wang et al.47 recently reported that when CTAB-stabilized AuNRs were washed in water, the thiolated RMs were found adsorbed to both tips and sides; by contrast, washing in 0.05 M CTAB resulted in preferential adsorption of the RMs to the tips of the AuNRs with anisotropic Ag shells. However, no study was made of other important factors, including the concentration of the thiolated RMs and the presence of open or Ag-coated tips in the AuNR@RM@Ag composites. We propose a new strategy to design ultrabright SERS probes with maximal SERS performance under off-resonance excitation. We have demonstrated previously45 that for RM concentrations below a threshold value c < ctr, the Au@RM@ Ag particles have a cuboid morphology with completely embedded RMs. Here, we show that for RM concentrations above a threshold value c > ctr, the fabricated Au@RM@Ag particles can have open Au- or Ag-coated tips, depending on the amount of Ag ions added. Remarkably, the highest SERS performance was found in the off-resonance mode for tags with open-functionalized Au tips and with an intermediate anisotropic Ag shell [hereinafter, these are referred to as tipfunctionalized Au@Ag nanorods (TFNRs)]. Growing the Ag shell further to completely cover the tips decreased the SERS enhancement. The designed probes demonstrated fundamental SERS EFs5 of about 107, much higher than the EFs of other resonant NPs, such as AuNRs, nanostars (NSTs), and Au@Ag NRs. For biocompatibility and stability, the probes were additionally covered with a thin silica layer. We show that our probes are suitable as single-particle SERS labels for the imaging of living cells and deep tissue structures. Compared to the previous report,31 this article presents the following main novel findings: (1) we provide experimental evidence for tip localization of Raman reporters that contribute the most to the SERS response. (2) We show superior SERS performance of the TFNRs in a colloidal format through comparison with other particle types and in a single-particle format by using colocalization of Raman and SEM images; and (3) we finally show the possibility of deep-tissue imaging with the TFNRs proposed.

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EXPERIMENTAL AND THEORETICAL METHODS

Materials. All chemicals were obtained commercially and were used without further purification. CTAB (>98.0%), cetyltrimethylammonium chloride (CTAC; 25% water solution), sodium oleate (NaOL; technical grade, >82% fatty acid), L-ascorbic acid (AA; >99.9%), 3-aminopropyltrimethoxysilane (APTMS; 98%), 4-nitrobenzenethiol (NBT), hydrochloric acid (HCl; 37 wt % in water), tetraethylortosilicate (TEOS; 98%), polyvinylpyridine (PVP; MW = 140 000), hyaluronic acid (HA; MW = 10−15 kDa), 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS), and sodium borohydride (NaBH4; 99%) were all purchased from Sigma-Aldrich. Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O) and silver nitrate (AgNO3; >99%) were purchased from Alfa Aesar. Ultrapure water, obtained from a Milli-Q Integral 5 system, was used in all experiments. Synthesis of TFNRs. AuNRs were first prepared by seedmediated growth in a binary surfactant mixture as described elsewhere20,52 and were redispersed in 20 mM CTAC solution with an Au concentration of 1 mM. Next, 300 μL of 2 mM NBT solution in ethanol was added to 10 mL of AuNRs, and the mixture was incubated at room temperature for 24 h. Unbound reporter molecules were washed off by centrifuging the NPs three times at 9000 g for 10 min and were redispersed in 0.08 M CTAC solution. Then, 4 mL of NBT-functionalized nanorods was mixed with 12 mL of H2O and with various amounts (10−80 μL) of 100 mM AgNO3 solution. This was followed by the addition of a fourfold molar excess of AA as a reductant. The mixture was incubated unstirred at 70 °C for 3 h. Synthesis of Au@Ag Cuboids. The cuboids were synthesized by using the same AuNRs as the templates. Then, 4 mL of as-prepared nanorods was mixed with 12 mL of H2O and with various amounts (10−80 μL) of 100 mM AgNO3 solution. This was followed by the addition of a fourfold molar excess of AA as a reductant. The mixture was incubated unstirred at 70 °C for 3 h. Next, 300 μL of 2 mM NBT solution in ethanol was added to 16 mL of cuboids, and the mixture was incubated at room temperature for 24 h. Synthesis of AuNRs with Plasmon Peaks at 785 and 633 nm. The samples were synthesized by controlled etching by using the same AuNRs as the templates.23 The initial AuNRs were centrifuged at 9000 g for 10 min and were resuspended in 0.1 M CTAB. A portion of 10 mM HAuCl4 (15 or 30 μL) was added to 10 mL of AuNRs. The etching was continued for 3 h until the LPR wavelength reached the designated values (785 and 633 nm). The resulting AuNRs were centrifuged at 9000 g for 10 min and were resuspended in 20 mM CTAC. Next, 300 μL of 2 mM NBT solution in ethanol was added to 10 mL of nanorods, and the mixture was incubated at room temperature for 24 h. Synthesis of Au NSTs with Plasmon Peaks at 785 and 650 nm. Au NSTs were prepared by a seed-mediated protocol.17 For synthesis of NSTs with an LPR peak at 785 nm, 200 μL of solutions of 15 nm citrate-stabilized seeds was added to 10 mL of 1 mM auric chloride (HAuCl4) with 40 μL of 1 M HCl in a 20 mL glass vial at room temperature under moderate stirring (700 rpm). At the same time, 300 μL of 2 mM AgNO3 and 200 μL of 100 mM AA were added quickly. The solution was stirred for 30 s as its color rapidly turned from light red to blue. For NSTs with an LPR peak at 650 nm, 17984


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in a water jacket incubator at 37° C with a 5% CO2-humidified atmosphere in 25 cm2 tissue culture flasks. Functionalization of TFNRs with HA. First, the mesoporous silica-coated TFNRs were functionalized with amino groups. To this end, 100 μL of APTMS was added to 10 mL of TFNR/mSiO2 in ethanol. The mixture was incubated overnight, after which the particles were washed with water. The amino-functionalized NPs were chemically conjugated with HA.72 Two milligrams of HA was added to 10 mL of the amino-functionalized TFNR/mSiO2, and the mixture was stirred for 2 h. This was followed by adding aqueous solutions of EDC (100 μL, 3 mg/mL) and NHS (100 μL, 1 mg/mL). The mixture was stirred for 12 h, after which it was centrifuged and redispersed in phosphate-buffered saline (PBS) or in serum-free DMEM. Cell Viability Assay. The toxicity of the silica-coated TFNRs was checked by the MTT assay. HeLa cells were grown in 96-well immunological plates. After the cells had formed a monolayer, a suspension of various concentrations of silica-coated TFNRs was added. After incubation with the TFNRs, the cells were washed with PBS and 0.2 mL of MTT (Sigma, USA) was added. The mixture was incubated at 37 °C for 3 h in the dark. Then, the cells were washed with PBS, and 0.2 mL of dimethylsulfoxide was added to dissolve formazan crystals. Absorbance spectra were collected with a Tecan spectrophotometer equipped with a microplate reader. All experiments were repeated six times. SERS Living Cell Imaging. HeLa cells were plated at a density of 4 × 104 cells on 0.5 cm × 0.5 cm silicon substrates in flat-bottomed 24-well plates. For imaging, 200 iL̀ of HAconjugated TFNR/mSiO2 was resuspended in 1 mL of the culture medium and was added to each well. Then, the cells were incubated with TFNRs at 37 °C for 4 h and were washed with PBS to remove unbound particles. SERS scanning was done with a Renishaw InVia confocal Raman microscope (laser wavelength, 785 nm; water immersion objective, 63×; laser power, 7 μW; and integration time per pixel, 10 ms). Ex Vivo Tissue Imaging. A slice of pork skin with hypodermic fat was used as the model tissue. The skin was about 2 mm thick, and the fat was 5 mm thick. A 10 μL portion of silica-coated TFNRs was applied on the skin at the selected point (i). A 100 μL portion of the same particles was subcutaneously injected into the fat layer at the selected point (ii). The area both points was mapped with a Renishaw inVia confocal Raman microscope (laser wavelength, 785 nm; laser power, 7 μW; and integration time per pixel, 100 ms). The optical part of the device was equipped with a 5× long-focus objective lens with a low numerical aperture, 0.12NA. FDTD Simulations. FDTD simulations of extinction spectra and EM field distributions were made with a commercially available software Lumerical FDTD Solutions 8. The simulation volume was a 200 × 100 × 100 nm parallelepiped with a uniform mesh of 0.5 nm; the input wavelengths ranged from 300 to 1100 nm.

we used the same protocol except that the amount of seeds was increased to 600 μL. The NSTs were further mixed with CTAC (final concentration, 0.1 M) and were functionalized with NBT by the same protocol as used for AuNRs. Silica Coating of TFNRs and NSTs. The NPs were coated with mesoporous silica by the method of Gorelikov and Matsuura.50 A 0.1 M portion of NaOH was added to 16 mL of NPs to adjust the pH to 11. Three 30 μL injections of 20% TEOS in methanol were made under gentle stirring at 30 min intervals, and the reaction was allowed to proceed for 12 h. After that, the obtained particles were washed three times with water and methanol and finally were redispersed in 4 mL of water. NP Characterization. Extinction spectra were measured with a Specord 250 spectrophotometer (Analytik, Jena, Germany). TEM images were recorded on a Libra-120 transmission electron microscope (Carl Zeiss, Jena, Germany) at the Simbioz Center for the Collective Use of Research Equipment in the Field of Physical−Chemical Biology and Nanobiotechnology, IBPPM RAS, Saratov. For TEM measurements, 10 μL of as-prepared core/shell nanorods was deposited onto a microscopic grid. HRTEM images and energy-dispersive X-ray spectra were recorded with a Tecnai G2 200 kV electron microscope (FEI, USA). Normal Raman and SERS spectra were acquired with a Peak Seeker Pro 785 Raman spectrometer (Ocean Optics) in 1 cm quartz cuvettes by using 785 nm irradiation (30 mW). The acquisition interval was 2 s, and all spectra were averaged over 10 independent runs. Catalytic Reduction of Nitrophenol. The catalytic reduction of nitrophenol near the NPs’ tips was investigated with TFNRs with the thickest (16 nm) Ag shells. A 1 mL portion of TNFRs was mixed with 100 μL of 100 mM NaBH4. SERS spectra were acquired from the colloids before and 5 min after the addition of sodium borohydride. SEM-Guided SERS from Individual TFNRs. Before use, the silicon substrates were submerged in a piranha solution for 30 min, thoroughly washed with deionized water and ethanol, and dried in a stream of nitrogen. The clean substrates were immersed into a 0.1% (v/v) solution of PVP in ethanol for 3 h,51 after which they were removed from the PVP solution, rinsed extensively with ethanol, and dried in a stream of nitrogen. Next, the substrates were marked by scrabbling a cross in the center. The marked substrates were placed into 3 mL of silica-coated TFNRs and were left to stand for 5 min to form a loosely packed NP layer. After that, the substrates were removed from the NP suspension, rinsed copiously with water, and dried at room temperature. The selected area (15 × 15 μm) near the cross was examined with a FEG SEM MIRA 3 microscope (Tescan, Czech Republic) at an acceleration voltage of 30 kV. The same area was examined by Raman mapping by using a Renishaw inVia confocal Raman microscope (Renishaw, UK). The optical part of the device was equipped with a Leica DM 2500 (Leica, Germany) optical microscope with a 50× objective lens. The measurement conditions were as follows: laser spot diameter, 1.5 μm; power on sample, 300 μW; and accumulation time in the point, 100 ms. Cell Culture. HeLa human cancer cells were purchased from Biolot (Russia) and were cultured in a Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum and with antibiotics (100 μg/mL penicillin and 100 μg/ mL streptomycin) (Sigma, St. Louis, MO). Cells were grown

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RESULTS AND DISCUSSION Extinction and SERS Spectra of TFNRs with Different Ag Shell Thicknesses. The initial AuNR suspension was prepared in a binary surfactant mixture as described elsewhere.20,52 According to the TEM data, the width and length of the as-prepared AuNRs were 14 ± 3 and 76 ± 12 nm, respectively (Figure 1a-i). 17985


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We assume that the adsorption of Raman reporters on the tips protects them from deposition of Ag, whereas little or no reporters on the NR sides promote the formation of Ag shells there. As a result, we observed the formation of TFNRs, which resembled nanospindles. Figure 1a-ii−v shows TEM images of NBT-functionalized TFNRs with different Ag shell thicknesses (see more TEM images of the initial AuNRs and TFNRs with different Ag shells in Figure S1 of the Supporting Information). The Ag shell thickness along the particles was controlled with AgNO3 added to the reaction mixture. Typically, the Ag shell was homogeneous over the NR long axis and had an average thickness of about 1.7 nm when a small (10 μL) amount of AgNO3 was added (Figure 1a-ii). Then, the Ag shell gradually grew to become 4, 7.8, and 16 nm thick on average when 20, 40, and 80 μL of AgNO3 were added, respectively (Figure 1aiii−v). For simplicity, we will further label the samples according to their Ag shell thicknesses; that is, the designations TFNR/Ag1.7−TFNR/Ag16 will stand for nanostructures with Ag shell thicknesses ranging from 1.7 to 16 nm. The initial AuNR cores inside the final particles can be seen as semitransparent layers. Importantly, we did not observe any gaps between the Au and Ag layers in any types of TFNRs. Figure 1b shows well-known changes in the extinction spectra of TFNRs (from NIR to VIS) with increasing Ag shell thickness. Specifically, the dipolar longitudinal mode continuously blue shifts from 922 to 545 nm when the Ag shell thickness is increased from 0 to 16 nm. In agreement with the previous observations for other types of AuNR@Ag shell NPs (cuboids, dumbbells, and rods),56−58 the longitudinal resonance shift depends nonlinearly on the Ag shell thickness (Figure 1d). After a thick Ag shell had formed (Figure 1b-iv,v), additional strong plasmon bands, between 400 and 450 nm, could be observed. These bands are usually attributed to the dipolar excitation of the Ag shell.59 We next measured the SERS spectra of the AuNRs and TFNRs under the same optical (laser power and accumulation time) and geometrical (cuvette and focusing) conditions. In close agreement with the previous study,60 the SERS spectra for all samples were dominated by nitrobenzene peaks associated with the vibrational modes δ (CS) at 390 cm−1, γ (CCC) at 560 cm−1, π (CH) + π (CS) + π (CC) at 723 cm−1, π (CH) at 854 cm−1, ν (CS) at 1081 cm−1, δ (CH) at 1110 cm−1, ν (NO2) at 1345 cm−1, and ν (CC) at 1569 cm−1. Using the most intense Raman band at 1345 cm−1, we calculated the fundamental SERS EFs for all samples (Supporting Information). For calculations, we used the normal Raman signal of 0.1 M NBT solution as a normalization base, assuming that in SERS, the Raman-active molecules were adsorbed only on the AuNR tips (see details of EF calculations and Table S1 in the Supporting Information). For NBT-functionalized AuNRs, the EF was 2.8 × 104. The Ag shell coating significantly boosted the Raman signal of NBT, and the EF values were 3.7 × 105, 6.8 × 105, 2.1 × 106, and 6.1 × 106 when the Ag shell thicknesses were 1.7, 4, 7.8, and 16 nm, respectively. The maximal EF of the optimized TFNRs was 6.1 × 106, 3.6 times higher than that reported previously31 after appropriate correction.a This means that the SERS response of the TFNRs increased with increasing Ag shell thickness (Figure 1d). However, for shells thicker than 16 nm, the SERS enhancement started to decrease (Figures 1d and S2c in the Supporting Information; see also Figure 2I in ref 47). More importantly, we did not observe any correlation between the plasmon resonance laser wavelength (785 nm in our case) and

Figure 1. (a) Schematics and TEM images of TFNRs with increased Ag shell thicknesses and their corresponding extinction (b) and SERS (c) spectra. All scale bars are 100 nm. (d) Blue shift of the longitudinal resonance mode and the SERS intensity dependence on the Ag shell thickness of the TFNRs.

The axial ratio of the AuNRs was 5.4, in good agreement with the calculated53 and measured wavelengths of the longitudinal (922 nm) and transversal (507 nm) resonances (Figure 1b-i). First, the AuNRs were functionalized with thiolated NBT molecules through Au−sulfur (S) bonding. The thiolated NBTs were chosen as Raman reporters because they have the highest Raman cross section among other thiolated aromatic molecules.4 On the other hand, thiol derivatives preferentially bind onto the {111} facets of AuNRs, causing the monothiol molecules to be preferentially localized at the AuNR tips.47,54,55 The absorption spectrum of the AuNRs remained barely affected after the attachment of the NBT molecules (data not shown). After excess Raman reporters had been removed, Ag shells of desired thickness were grown on the AuNR seeds by sequential addition of CTAC, AgNO3, and ascorbic acid. The shape of the resulting NPs depends strongly on the reaction conditions and on the concentration of the reagents. For example, after the reaction rate had been adjusted by varying the pH, monodisperse Au−Ag cuboids and dumbbells were synthesized by using the same AuNRs as the templates56 for both types of particles. The addition of glycine to the reaction mixture gives rise to shuttle-like Au@Ag NPs,57 whereas replacing CTAC by CTAB affords cuboid- or rodlike particles.58 Following the procedures described in the experimental section, we observed the growth of anisotropic Ag shells on the nanorods’ sides and the absence of such growth on their tips. 17986


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solution of TFNRs/Ag16 with 100 mM sodium borohydride and measured the SERS spectra before and after the reaction. Scheme 1 explains the main idea behind the catalytic reduction experiment. We suppose that the total catalytic

the SERS response. For example, the magnitude of the SERS response was highest for the TFNRs with a longitudinal extinction resonance at 545 nm, whereas the on-resonance particles with an LPR at 780 nm had a moderate EF. Usually, the absence of a correlation between SERS response and resonance excitation is characteristic of complex SERS substrates or of NPs with embedded reporter molecules, socalled NMs.31 Localization of Raman NBT Molecules on AuNR Surface. A typical protocol of NM synthesis is similar to that used here and consists of three basic steps: (1) synthesis of a plasmonic core, (2) functionalization with Raman reporters and spacers, and (3) overgrowth of a secondary metal shell. The most evident confirmation that RMs have been successfully included in the NMs is a distinct gap between the metal layers. This gap is usually seen in Au@Au NMs embedded with thiolated molecules or with DNA/ fluorescent dyes inside.32,35,38,48 However, such gaps were not seen in our TFNRs or in other Au@Ag nanostructures.45 To additionally confirm that the particles contained reporter molecules only at the tips but not between Au and Ag layers, we conducted two other experiments. One experiment was HRTEM imaging of the NPs (Figure 2).

Scheme 1. Possible Localization of Raman NBT Molecules on Both AuNR Tips and Sides (Route 1) and Predominant Localization on the AuNR Tips (Route 2)a

a

Shown are the initial functionalized AuNRs before (A,D) and after (B,E) growth of an Ag shell, as well as after catalytic reduction of composite particles (C,F). The bottom plots show the corresponding expected SERS spectra. The red and green SERS spectra correspond to the intact and reduced NBT molecules, respectively; the spectrum (c) represents a superposition of the red and green spectra.

reduction of the nitro groups to amino groups may have taken place within 5 min (Figure 2e). Although a detailed study of the catalytic activity of TFNRs is beyond the main goals of this study, we note that the reaction rate was much faster than that usually observed for Au@Ag catalysts.64 The main question is whether NBT molecules can be immobilized on the AuNR sides, as well as on the tips (Scheme 1, Route 1). Another scenario is the predominant immobilization of NBT molecules on the AuNR tips (Scheme 1, Route 2). In Route 1, the formation of an Ag shell would result in an enhanced SERS spectrum (b) with two possible contributions from tip- and side-localized NBT molecules. The treatment of Au@NBT@Ag particles with NaBH4 would reduce the tiplocalized NBT molecules only, thus replacing the NO2 groups with NH3 ones. In contrast, the side-localized molecules would be protected by the Ag shell and their chemical structure should be unchanged. As a result, one can expect a superposition of the SERS spectra that would combine the spectral features of the NBT and 4-aminothiophenol (4-ATP) spectra and exhibit at least two major peaks, at 1345 and 1085 cm−1. In Route 2, we expect the appearance of a new SERS spectrum with lines characteristic of 4-ATP molecules with the major peak at 1085 cm−1. As seen in Figure 2f, the experimental observations are in line with the second scenario (Route 2 in Scheme 1). Indeed, the intensity and spectral features of the Raman signal changed greatly. After the reaction, the Raman peak intensities of the reduced molecules decreased fivefold and their spectral positions completely overlapped the characteristic peaks of 4-

Figure 2. HRTEM images of TFNRs with 7.8 nm (a,c) and 16 nm (b,d) Ag shells. The red arrows point to bare Au tips. The bars are 10 (a), 20 (b), and 5 nm (c,d). (e) Scheme of catalytic reduction of nitro groups of TFNRs by NaBH4. (f) SERS spectra of TFNRs/Ag16 before (f) and after (g) catalytic reduction. After the reduction, the characteristic peak of NO2 at 1345 cm−1 disappeared. The magnified inset in (f) shows the shape of doublet lines at 1081 and 1108 cm−1; the second line is absent from the spectrum (g).

Under high magnification, we observed no distinct gap between Au and Ag layers (Figure 2a−d). More importantly, the AuNR tips were not covered even with a thin Ag layer, whereas the Ag coatings on the sides were both relatively thin (7.8 nm) and thick (16 nm). On the other hand, the absence of a distinct gap inside Au@Ag NPs cannot be conclusive evidence that there are no RMs on the particles’ sides. For example, no gap structures have previously been reported for variously shaped Au@Ag NMs, such as nanospheres and nanorods.33,45,61−63 Recently, we have shown that an Ag shell can protect the molecules embedded inside Au@Ag nanorods from oxidation/ reduction.45 In this work, we used the protective properties of the Ag shell to study possible catalytic reduction of nitrophenol by NaBH4 on the Au@Ag particles. To this end, we mixed a 17987


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The Journal of Physical Chemistry C ATP,65 but not those of NBT. In particular, the major characteristic NBT peak of (NO2) at 1345 cm−1 disappeared. Of note, the minor peak of the doublet at 1081 and 1108 cm−1 (Figure 2f) was absent from the spectrum of the reduced NBT molecules with NH3 groups (Figure 2g). The only peak at 1079 cm−1 is seen in the spectrum of Figure 2g, and its magnitude is decreased by 40%, compared to that of the major line of the doublet in Figure 2f. The NBT peak at 1571 cm−1 is shifted to 1590 cm−1 after the catalytic reduction. Other evidence for the tip localization of the immobilized NBT molecules comes from the experimental data shown in Figure 3. The addition of NaBH4 to TFNRs with relatively thin

Although the above HRTEM and catalytic reduction data support the tip-localized NBT hypothesis, several important points need to be discussed. The covalent functionalization of the initial AuNRs with aromatic molecules can affect the formation of the Ag shell. Specifically, the surface functionalization of AuNRs with 4-ATP molecules yields irregular Ag shells if the ATP concentration is above a threshold concentration ctr, corresponding to a dense coating of the AuNR surface. By contrast, the use of low subthreshold concentrations results in Au@Ag cuboids with a regular symmetrical shape. As a rough approximation, the threshold concentration ctr can be estimated through the ratio between the surface area of AuNRs and the specific topological area of ATP molecules45 (see eq S10 in the Supporting Information). Also, even small variations in synthetic conditions, such as an additional washing of reporter-functionalized nanorods, can affect the position of the reporter molecules inside/outside the Ag shells.47 From our previous31,45 and present experiments, we note a large difference in the kinetics and peculiarities of Ag shell growth between the AuNRs functionalized with BDT and NBT molecules. In particular, NBT functionalization of AuNRs promotes easy Ag shell growth, as compared to BDT functionalization. This means that the formation of distinct gaps in the AuNR@BDT@Au core/shell structures and the absence of such gaps in AuNR@NBT@Ag structures can be attributed, at least in part, to the chemical nature of the BDT and NBT molecules. Keeping in mind the existence of the threshold concentration ctr of RMs, we have to differentiate between two possible routes for growing an Ag shell (Scheme 2).

Figure 3. SERS spectra of TFNRs with a thin (∼8 nm, a) and thick (∼40 nm, b) Ag shell before (red) and after (blue) catalytic reduction with NaBH4. Note the decreased SERS intensity for the thick Ag shell, as shown by ordinate scale bars. The stars and crosses mark the identical spectral peaks at 385 (a) and 389 cm−1 (b) and at 1592 (a) and 1588 cm−1 (b, looks like a shoulder, see the magnified inset), respectively.

Scheme 2. Formation of Au@RM-SH@Ag Cuboids (Top Route) and a Strongly Anisotropic Ag Shell (Bottom Route) at Subthreshold and Overthreshold Concentrations of RMsa

Ag shells and open Au tips (Figure 3a) resulted in the appearance of spectral features similar to those in Figure 2g. However, the addition of the same amount of NaBH4 to a solution of AuNR@Ag particles did not change the SERS spectra when the NBT molecules were completely covered by a thick Ag shell (Figure 3b). In fact, the blue spectrum in Figure 3b looks like the SERS spectra of untreated TFNRs with thin Ag shells, except that its intensity is twofold lower owing to the EM shielding effect (see Figure S2c, last experimental point for 5 mM AgNO3). We also observed the appearance of two new and practically identical peaks for both blue spectra in Figure 3a,b, recorded after catalytic reduction. These peaks are located near 387 and 1592 cm−1, and they are marked with stars and crosses, respectively (for the spectrum in Figure 3b, the new peak looks like a weak shoulder). For TFNRs with thick Ag shells, these peaks can be attributed to a minor fraction of particles with open AuNR tips, which can be seen in the TEM images. If one assumes that both blue and red spectra of the TFNRs with thick Ag shells (Figure 3b) originate from tip- and sidelocalized reporters, then it would be reasonable to expect a notable contribution from intact (nonreduced) NBT molecules, which are localized on the AuNR sides and are protected by Ag layers. Moreover, as the thickness of the Ag layer is smaller for the TFNRs in Figure 3a, the EM shielding is weaker than that in Figure 3b. Thus, the contribution from the sidelocalized NBT molecules is expected to be stronger than that in Figure 3b. However, the blue spectrum in Figure 3a does not support the side-localization assumption.

a

In both cases, it is assumed that the RMs are localized on the sides as well as on the tips.

At subthreshold NBT concentrations, the NBT molecules were localized on both tips and sides of the AuNRs. This localization ensures slow growth of Ag shells and the formation of symmetrical cuboidlike structures.45,56 At high overthreshold concentrations of the RMs, we observed the formation of strongly anisotropic Ag shells, thick on the sides and thin on the tips. Returning to the main question under discussion, we assume, in contrast to Scheme 1, that the NBT molecules are localized on both tips and sides of the AuNRs. However, it follows from the above discussion of Scheme 1 and Figure 3 that the side-localized molecules give no appreciable contribution to the SERS spectra, at least for the TFNR structures discussed here. 17988


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Au@Ag cuboids with NBT and compared the SERS response with that from the TFNRs. The TEM images and SERS intensities of the peak at 1345 cm−1 are shown in Figure S3 in the Supporting Information. Obviously, the TFNRs had a higher SERS response than the cuboids, and the difference became greater for thicker Ag shells. FDTD Simulations of Near Field Around TFNRs. To better understand the off-resonance properties of the TFNRs, we used the FDTD method to examine the extinction spectra and EM field distribution around the particles under 785 nm excitation by using the FDTD method. The model was a nanorod with a length of 74 nm and a width of 14 nm that was coated with a 0−16 nm thick anisotropic Ag shell. The schematic images of the particles and the simulation details and results are shown in Figure S4 of the Supporting Information. In general, there was good agreement between the plasmonic blue shift and the thickness of the Ag coating of the AuNRs. More importantly, an enhanced EM field was generated near the TFNR ends even under off-resonance conditions, which correspond to thick Ag shells when the LPR is far from 785 nm. In Table S2 (Supporting Information), we provide the normalized near-field amplitudes averaged over field distributions in the equatorial plane, which contains the particle axis and the direction of the exciting incident field. The maximal average value (12.9) was obtained for the TFNR structure “(iii)”, whereas for the model “v”, the average value was smaller (9.8). However, the calculated SERS EF was greater for model “v” than it was for model “(iii)”. The exact reasons for this contradiction are not clear. Possibly, the simulation models did not match the experimental particles, or there were additional chemical or charge-transfer67 contributions to the obtained EFs. In any case, we note that simplified EM simulations typically cannot explain experimental data on the observed plasmonic EFs.68 Single-Particle SERS Spectra of TFNRs. It is now believed that an EF value of 107 may be sufficient for singleparticle detection.69 To examine whether it is possible to detect a SERS signal from a single TFNR particle with a common Raman microscope, we first coated TFNR/Ag7.8 particles with a mesoporous silica layer (TFNR/Ag7.8@ mSiO2) and then adsorbed the particles on a silicon substrate. The surface density of the particles was chosen so as to make the interparticle separation larger than the laser beam spot. The same area was examined by SEM and by Raman mapping to couple the signal to the particle. In the enlarged SEM images in Figure 5aii, one can clearly see the adsorbed single TFNR/Ag7.8@mSiO2 particles (iii) and their aggregates (iv). The overlaid SERS and bright-field images in Figure 5ai show red spots, associated with Raman scattering from these objects. For example, the scattering from an individual particle (iii) and an aggregate (iv) is shown in Figure 5b. In agreement with the colloidal measurements shown in Figure 1, the SERS spectra of individual particles are dominated by nitrobenzene peaks associated with π (CH) + π (CS) + π (CC) at 723 cm−1, π (CH) at 854 cm−1, ν (CS) at 1081 cm−1, δ (CH) at 1110 cm−1, ν (NO2) at 1345 cm−1, and ν (CC) at 1569 cm−1. Between 900 and 1000 cm−1, there is also a background signal from silicon. Figure 5c shows the distribution of the SERS intensities at 1345 cm−1, recorded for spots that are associated with the particles shown in the corresponding SEM images. The most probable SERS

In summary, the absence of a gap between the metal layers and the fast catalytic reduction of the nitro groups of the Raman reporters constitutes strong evidence for the successful synthesis of tip-functionalized AuNRs with Ag-coated sides. SERS Performance of TFNRs in Comparison with Other NPs. Because a high SERS response under offresonance excitation is a key feature of TFNRs, we compared the SERS intensities of NBT-functionalized NPs with their LPR overlapping 785 nm or located below this value (for AuNRs with an LPR above 785 nm, the SERS spectra are shown in Figure 1c, i). For comparison, we chose as-prepared AuNRs with LPRs at 700 and 785 nm (the aspect ratios e = 3 and 3.8, respectively), Au NSTs (AuNSTs) with LPRs at 650 and 785 nm, and two samples of TFNRs (TFNR/Ag4 and TFNR/Ag16). The list of particles for comparison included both on- and off-resonance examples. In general, it is impossible to provide equal numbers of RMs for all samples; therefore, we normalized all samples on the basis of the Au concentration (1 mM for all AuNRs and AuNSTs). TFNRs were used as prepared, without dilution. TEM images of the particles and their extinction and SERS spectra are shown in Figure 4. As expected, we observed an

Figure 4. (a) Schematics and TEM images of on- and off-resonance AuNRs with aspect ratios e = 3 and 3.8, AuNSTs of different sizes, TFNR/Ag4, and TFNR/Ag16, as well as their corresponding extinction (b) and SERS (c) spectra. All scale bars are 100 nm.

increase in the SERS intensity for AuNRs with an LPR close to the laser excitation wavelength (curves (i) and (ii) in Figure 4b,c). The AuNSTs had a higher SERS response that did AuNRs, and again, the on-resonance NPs had a higher scattering intensity. By contrast, the picture changed greatly with TFNRs, for which off-resonance excitation was much more effective. Additionally, the TFNR/Ag16 NPs had the highest SERS response among all particle types examined. Another important advantage of off-resonance excitation is the absence of a fluorescent background on SERS spectra. This background was clearly seen in the resonance excitation of AuNRs (curve ii in Figure S6) and TFNR/Ag4 (curve v in Figure S6). The physical origin of this background is usually associated with the plasmonic heating of NPs during SERS measurements.66 To further extend the range of NPs used to compare the SERS responses, we synthesized Au@Ag nanocuboids with the same protocol as used for the TFNRs but with unfunctionalized nanorods as the templates. In this case, it is possible to prepare cuboidal Au@Ag nanorods with different Ag shell thicknesses.56 The concentrations of Au and Ag were the same as used for the TFNRs. We functionalized the surface of the 17989


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biocompatible and biodegradable, and it is widely used to deliver nanomaterials into cancer cells.72,73 The biocompatibility of HA-functionalized silica-coated TFNRs was tested by the MTT assay with different NP concentrations and incubation times. As shown in Figure 6c, the number of living cells cocultured with up to 8.5 × 1010 mL−1 of TFNRs for 72 h was more than 80% of the total cell number. We further used these highly SERS-active off-resonance TFNRs to image living cells (Figure 6b). Note that TFNRs retain their off-resonance properties after being coated with silica because that coating usually causes only a small red shift of about 10 nm.60 The bright-field images of HeLa cells incubated with silica-coated TFNRs indicate that the SERS probes were biospecifically bound to the cells. The overlaid images show that the SERS images fit well with the corresponding bright field images. The single-cell SERS image was acquired within 50 s (2500 spectra acquired from an area of 27 μm × 28 μm) with a 10 ms exposure time per pixel. These conditions are believed to be safe in the use of offresonance SERS probes.48 SERS Bioimaging of Model Pig Skin with OffResonance TFNRs. To show that it is possible to use TFNRs as labels for deep tissue imaging ex vivo, we used a slice of pork skin as a model. Silica-coated TFNR/Ag7.8 was spotted on the skin and was also injected in the hypodermic fat layer at selected points (i) and (ii), respectively (Figure 7a). The bright-field images of the skin near these points show the absence of NPs. On the SERS map, however, the area of deposition is clearly seen (Figure 7b, right top). Indeed, the SERS spectra in empty point 1 do not have visible SERS peaks (Figure 7c), whereas in the areas in which the particles were spotted or injected (2 and 3), the characteristic peaks of nitrobenzene are clearly seen. What is more, the skin layer of about 2 mm does not shadow the Raman signal of the TFNRs injected below the skin. Such deep imaging became possible because of the high SERS response from the TFNRs, if one works in the NIR tissue transparency window and uses a longfocus objective lens.

Figure 5. (a) Bright-field and SEM images of the silicon substrate with adsorbed TFNR/Ag7.8@mSiO2 particles. The same areas (i,ii) were chosen for SEM and Raman mapping. The red circles in the magnified SEM image are associated with single particles, whereas the blue circles are associated with aggregates. The scale bars are 100 nm. (b) SERS spectra recorded from spot (iii) (single particle) and spot (iv) (five-NP aggregate). (c) Distribution of SERS intensities from TFNR/Ag7.8@mSiO2 and from aggregates of such NPs.

intensities, of about 300 counts, are associated with individual particles. SERS Bioimaging of Living Cells with Off-Resonance TFNRs. To stimulate the biospecific uptake of silica-coated TFNRs into cancer cells, we coupled the NPs to HA by chemical conjugation (Figure 6a). HA is a natural polysaccharide of the extracellular matrix that contributes to cell proliferation. It interacts with three types of cell surface receptors: CD44 (cluster determinant 44), receptor for HAmediated motility, and intercellular adhesion molecule-1.70 CD44 is overexpressed in several cancer types (including the HeLa cell line) and is typically associated with cancerous angiogenesis and tumor progression.71 HA is also well

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CONCLUSIONS We have developed a new type of ultrabright plasmonic SERS probe composed of tip-functionalized AuNRs with anisotropic

Figure 6. SERS bioimaging of living cells with off-resonance TFNRs. (a) Scheme for conjugation of silica-coated TFNRs with HA. The inset shows a silica-coated NP (see also the Supporting Information for more images). (b) Bright-field images and their overlays with SERS images of HeLa cells incubated with TFNR/Ag7.8@SiO2@HA (i,ii) and untreated cells (iii,iv) as the control. The scale bars are 10 μm. (c) Evaluation of cell viability after incubation with the off-resonance SERS probe of silica-coated TFNRs, with concentrations of 0 (1), 2.5 (2), 4.5 (3), 6.5 (4), and 8.5 × 1010 (5) particles per milliliter. 17990


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distribution around TFNRs under 785 nm excitation, simulated extinction spectra of TFNRs, and the normalized near field amplitudes averaged over 2D projection of field distributions; and additional TEM images of silica-coated TFNRs at different magnifications (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (B.N.K). *E-mail: [email protected] (N.G.K). ORCID

Nikolai G. Khlebtsov: 0000-0002-2055-7784 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Russian Scientific Foundation (project no. 18-14-00016). We thank D. N. Tychinin (IBPPM RAS) for his help in the preparation of the manuscript.

Figure 7. SERS bioimaging of model pig skin with off-resonance TFNRs. (a) Image of skin with selected points on TFNRs after deposition (i) and subcutaneous injection (ii) of TFNRs. (b) Brightfield (left) and overlaid bright-field and SERS images (right) of skin near the selected points (i,ii). The scale bars are 3 mm. (c) SERS spectra recorded from selected points 1 (pure skin), 2 (skin with deposited TFNRs), and 3 (injected TFNRs).

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ADDITIONAL NOTE In ref 31, the mass of AuNRs was occasionally overestimated by an order of magnitude; accordingly, the number concentration was underestimated by an order of magnitude. Thus, the EF values reported in Table S1 and in the main text should be divided by 10.

a

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Ag shells for bioimaging in the off-resonance mode. By controlling the Ag shell thickness of the TFNRs, the LPR peak position can be tuned from NIR to VIS range to ensure a high SERS response under off-resonance conditions. Therefore, the unique NP geometry allows the LPR spectrum to be decoupled from SERS performance. The anisotropic Ag shell allows an enhanced EM field to be kept on the nanorods’ ends (which is where RMs were located) even under off-resonance excitation. The TFNRs demonstrate a higher SERS response than do AuNRs, AuNSTs, and Au@Ag cuboids operating in both onand off-resonance modes. As the EFs of the optimized TFNRs are about 107, they can be used as supercontrast NIR SERS agents for the imaging of living cells with minimized photothermal damage and for deep-tissue SERS imaging. Finally, an important note is in order here. Although the HRTEM images and the reduction experiments have provided strong evidence for tip localization of the NBT molecules, the ultimate explanation of the reported difference between Au@ RM-SH@Au nanostructures with distinct nanometer-sized gaps and Au@RM-SH@Ag particles without any gaps is still an open question. Further work is needed to resolve this point.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.8b04772. Additional TEM images of AuNRs and tip-functionalized AuNR@NBT@Ag particles; extinction and SERS spectra of AuNR@NBT@Ag particles with openfunctionalized Au tips and complete Ag shell; calculations of the SERS EFs; TEM images and comparison of SERS intensities at 1345 cm−1 for surface-functionalized Au@Ag@NBT cuboids and tip-functionalized Au@NBT@Ag particles; FDTD simulations of EM field 17991


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