Ag nanocavity substrate for simultaneous plasmon-enhanced SM Raman and fluorescence spectroscopy
Figure 1A shows the schematic diagram of plasmon-enhanced SM spectroscopy enabled by a nanocavity composed of a Ag film coated with a 2-nm silica layer using atomic layer deposition (ALD) as the substrate with Ag shell-isolated nanoparticles (SHINs) deposited on top. RITC fluorescent probe molecules were covalently functionalized onto the substrate via isothiocyanate-amino chemistry (see text S1 and fig. S1 for the detailed description of the isothiocyanate-amino reaction). The average number density of RITC on the nanocavity substrate is ~3 × 10−5 to 14 × 10−5 dyes/nm2, which was achieved by using extremely low densities of the surface amino anchoring group and dye concentrations (see text S1 for calculation of number density of dyes on surface). A 532-nm continuous-wave laser was used to excite the plasmon, and a confocal Raman microscope was used to simultaneously acquire the fluorescence emission and resonant Raman scattering signals (see fig. S2 for experimental setup).
Typically, the far-field emission from a fluorophore is susceptible to being quenched by large nonradiative energy losses to the metal surface (14). However, with our method, the dielectric shell of the Ag SHIN prevents surface quenching of fluorescence signal intensity while still generating the plasmonic “hotspot” for enhancing quantum yield (23, 24). As shown on the right of Fig. 1A, discrete intensity fluctuations over time (“blinking” behavior) in the fluorescence emission intensity of a single RITC molecule in the nanocavity and a single-step photobleaching event (see fig. S3 for the corresponding spectra) show direct evidence of SM spectroscopy (14, 20, 25). However, as shown by finite-element method (FEM) calculations in fig. S4, the electric field and Raman enhancement in the hotspot are around 30-fold and 4 × 105–fold for a nanocavity substrate constructed with 10-nm shell Ag SHINs, which is not suitable to observe the SM vibrational spectroscopy. Therefore, to further enhance the local field intensity for vibrational Raman features, the size of nanocavity can be tuned by changing the dielectric shell thickness of SHIN from 10 to 2 nm (see fig. S5 for electron microscopy characterizations of SHINs). As a result, the electric field enhancement in a smaller nanocavity using 2-nm shell Ag SHIN is up to ~80-fold (Fig. 1B), enabling simultaneous collection of SM fluorescence and Raman signals and detection of a photo-induced bond cleavage reaction in real time (shown schematically in Fig. 1C).
Figure 2A shows the plasmon-enhanced SM emission spectrum of RITC in the nanocavity, where the plasmonic hotspot offers >1.0 × 107 Raman signal enhancement for SM resonant Raman scattering measurement. Therefore, several vibrational features can be distinguished from the fluorescence spectrum, i.e., the resonant Raman modes of the xanthene group of RITC, which are in good agreement with the literature (26) and of ensemble RITC experimental results in fig. S6. During illumination, both resonant Raman features and fluorescence band fluctuate over time before simultaneously disappearing after 37.30 s due to photo-induced degradation of the xanthene group (Fig. 2B). Moreover, there is a strong proportional correlation between the intensity fluctuations of the Raman peaks (gray area in Fig. 2A) and the fluorescence background (light red area in Fig. 2A). Correlating the intensities of the two strongest Raman peaks for the aromatic stretching modes (26) of the RITC xanthene group at 1534 and 1656 cm−1 with the corresponding fluorescence background gives R2 values of ~0.87 and ~0.83 (Fig. 2, C and D). These well-correlated Raman and fluorescence intensities can be attributed to the local field enhancement mechanism within the plasmonic hotspot, which further confirms that the Raman and fluorescence bands arise from the same SM (27–29). For comparison, two more representative SM spectra with Raman vibrational features are shown in fig. S7. In addition, Fig. 2E shows a Gaussian fit of the emission intensity distribution of 34 SM events observed using the 2-nm shell Ag SHIN substrate, which indicates the reproducible observation of RITC SMs. Compared with Ag SHINs with 10-nm shell, the thinner dielectric shell results in a stronger plasmonic coupling effect between the Ag core and the film, offering advantages in revealing vibronic features of SMs at room temperature and in real time.
Especially, an SM platform with a large spontaneous emission rate is a suitable candidate as a single-photon source for nanophotonics, and a fast emission rate can be achieved in a nanocavity due to the Purcell effect (5, 30). However, because of the remarkable nonradiative decay on the metal surface, it is desirable to have both a high quantum efficiency and a fast emission rate (30). To further understand the interaction between SM and optic field, FEM simulations of the Purcell factor and quantum yield in the nanocavity were carried out. As shown in Fig. 2F, the Purcell factor of a single emitter on the proposed substrate is around 1100, while the quantum yield remains higher than 0.65, which suggests an overwhelming dominance of fast radiative decay processes in the competition with nonradiative decay. Therefore, the largely enhanced emission decay rate indicates a possibility of applying the nanocavity substrate as a single-photon source at room temperature.
Application in real-time detection of SM reaction
Probing the intricate reaction pathways of an individual molecule is of profound importance for understanding intrinsic mechanisms and local environmental effects at the SM level (10, 11). In particular, photo-induced carbon─carbon bond cleavage reactions play a vital role in organic and catalysis chemistries (31). However, real-time structural characterization of SM photochemical reactions is challenging at room temperature and under ambient conditions. To apply the proposed method for SM reaction analysis, we further probe an SM photo-induced cleavage reaction by simultaneously correlating fluorescence spectrum and Raman features. Figure 3A shows the SM spectrum of an RITC molecule experiencing a photochemical reaction before the final photobleaching, where the vibrational modes are distinguished in the inset. As shown by the emission spectra contour and intensity trajectories in Fig. 3 (B and C), both the intensities of the fluorescence band and the corresponding Raman peaks fluctuate slightly from illumination onset (highlighted by the dashed pink rectangles) followed by a sudden signal drop to the background (“off” state) at 12.65 s and signal recovery at 15.05 s, until the final photobleaching event (highlighted by the pink solid rectangle). Similarly, strong intensity correlations (R2 = 0.95 and 0.94, as shown in fig. S8) between Raman peaks (~1654 and ~1519 cm−1, respectively) and the fluorescence band beneath along with the single-step photobleaching event indicate that the spectral bands are simultaneously coming from the same SM.
As is known, fluorescence is sensitive to molecular electronic structures. In Fig. 3D, discrete changes in the fluorescence band at 12.65, 15.05, and 15.70 s indicate four different molecular states due to SM structural evolution, denoted by I, II, III, and IV. Therefore, simultaneous spectral shifts in the Raman peaks and the fluorescence band were analyzed to determine these chemical structures. For structure I, the fluorescence band slightly varies around 576 nm, while frequency changes of Raman peaks around 1519 and 1654 cm−1 are also negligible, characteristic of an SM (20). For structure II, we notice that the off period lasts ~2.4 s, and both the fluorescence and resonant Raman signals disappear. Unlike triplet-state blinking of SMs, which has a typical lifetime on the millisecond time scale, this long off period can be probably ascribed to the structural transformation of the cationic form of RITC into the nonfluorescent/resonant lactonic form due to the basic local environment via slight hydrolysis of SiO2 shell of the Ag SHIN (32). The emission intensity then recovered at 15.05 s with a slightly shifted fluorescence band at around 580 nm, while the Raman peaks did not change significantly (structure III).
Typically, the rhodamine dyes tend to decompose via N-dealkylation (e.g., the removal of N-ethyl groups of xanthene segment) under illumination, which results in a fluorescence shift toward shorter wavelength in ensemble experiments (33, 34). However, for structure IV in our SM experiment, a large shift in the fluorescence band from ~580 to ~591 nm and a concomitant shift in the symmetrical C─C stretching vibration modes from 1519 and 1654 cm−1 to 1500 and 1652 cm−1 occur, respectively. This suggests a possible bond cleavage reaction that influences the xanthene segment of the molecule producing the resulting shifts. For structures III and IV, observation of resonant Raman C─C stretching vibrations indicates that the xanthene segment of the molecule is not destroyed. From the literature, visible light irradiation can induce C─C bond cleavage in the pyronin analogs (35). Hence, we suggest that the phenyl carboxylic group attached to the anthracene of RITC undergoes cascading carboxylic group removal followed by a phenyl breaking reaction.
To prove the proposed reaction pathway, in Fig. 4A, representative SM spectra of the four structures, along with the ensemble spectra of RITC and the proposed product after removing the phenyl carboxylic group, i.e., pyronine B (Pyr B), are depicted. Because the molecule probed was in the plasmonic hotspot, the different states of this molecule can be distinguished by the changes in surface-enhanced spectral features. First, in the slightly basic local environment, RITC can readily form the nonfluorescent/resonant lactone, structure II, according to an inner nucleophilic addition reaction (32) (proposed pathway is shown in Fig. 4D). During laser illumination, the ensuing cascade ring-opening and decarboxylation processes result in structure III with a slightly red-shifted fluorescence band and unchanged xanthene resonant Raman frequencies, which are consistent with our density functional theory (DFT) calculations (fig. S9) and the literature (36). Last, as shown in Fig. 4 (A and D), structure III undergoes a subsequent phenyl group removal reaction, producing structure IV with a concomitant fluorescent red-shift and significant variation of Raman spectral bands that match well with the plasmon-enhanced emission spectra of Pyr B (structure IV is the cation of Pyr B, which represents the chromophore in Pyr B).
Comparing the aromatic stretching modes in structures I and IV in Fig. 4B, the peak at 1654 cm−1 slightly shifts to 1652 cm−1, while a noteworthy shift is also observed for the peak at 1519 cm−1. DFT calculations show two near-degenerate symmetrical stretching modes of the C─C bonds in side phenyls (
with the calculated frequencies of 1513 and 1521 cm−1; see inset illustrations in Fig. 4B), which contribute to the observed peak at around 1519 cm−1 in the RITC spectrum. Because of the C─C bond cleavage reaction removing the phenyl group,
that has considerable normal mode distribution in the carboxyphenyl shifts to 1500 cm−1, which is responsible for the appearance of a broad Raman band at ~1500 cm−1 in structure IV. Meanwhile,
remains around 1520 cm−1 because it is not directly affected by the carboxyphenyl group. What is more important is the disappearance of the stretching mode of the central xanthene (νscx) in structure IV. Because νscx is strongly associated with the stretching of C─C bond between central xanthene and carboxyphenyl (νscx is at 1564 cm−1 in structure I; see inset in Fig. 4B), providing direct evidence of removal reaction of the carboxyphenyl group. Meanwhile, the conformational transformation and removal of the carboxyphenyl group cause the fluorescence emission band to shift to ~590 nm (Fig. 4C). As revealed by the calculated molecular orbitals in fig. S10, the effect of C─C bond cleavage on the HOMO (highest occupied molecular orbital) is negligible, while the LUMO (lowest unoccupied molecular orbital) becomes more stable, which leads to a reduced energy gap in structure IV (2.75 eV) as compared with structures I (2.82 eV) and III (2.79 eV). This bond cleavage reaction is reproducible as shown by another data set in fig. S11. Hence, direct observation of an SM photochemical reaction is demonstrated, and the proposed detection method has potential as a routine laboratory SM reaction characterization platform. In addition, it has been reported that a strong electric field would affect the reaction, such as an electrostatic field at 108 V/m level (37). In this work, the strength of the optical electric field is estimated to only ~3.9 × 104 V/m. To shed the light on how to mediate the SM photochemical reactions by electric field, the laser power– and temperature-dependent experiments will be carried out in the near feature.