Single Molecule Surface-Enhanced Raman Spectroscopy: A Critical Analysis of the Bianalyte versus Isotopologue Proof

Verification of single-molecule (SM) detection for surface-enhanced Raman spectroscopy (SERS) requires the use of two analytes via either the bianalyte or isotopologue approach. For both approaches, the preferential observation of the individual analytes over a combination of both analytes is used to conclude that SM detection has been achieved. Isotopologues are preferred because they have identical surface binding affinities and Raman cross sections, whereas bianalyte pairs typically do not. We conducted multianalyte SERS studies to investigate the limitations of the bianalyte approach. The bianalyte partners, Rhodamine 6G (R6G-<i>d</i>₀) and crystal violet (CV-<i>d</i>₀), were directly compared, while SM detection was verified (or disproved) using their corresponding isotopologues (R6G-<i>d</i>₄, CV-<i>d</i>₁₂). We found that the significant difference in counts between R6G and CV can provide misleading evidence for SMSERS. We then rationalized these results using a joint Poisson-binomial model with unequal detection probabilities and adjusted the relative concentrations of R6G and CV to achieve a comparable distribution of SMSERS counts. Using this information, we outlined the necessary considerations, such as accounting for the differences in molecular properties, for reliable SMSERS proofs. Moreover, we showed that multianalyte experiments at the SM level are achievable, opening the opportunity for new types of SM studies

Single Molecule Surface-Enhanced Raman Spectroscopy without Nanogaps

We provide conclusive proof of single molecule (SM) detection by surface-enhanced Raman spectroscopy (SERS) for discrete Ag triangular nanopyramids prepared via nanosphere lithography (NSL). While the observation of SMSERS has been well-demonstrated using various chemically synthesized nanoparticle substrates, they have a high degree of polydispersity in shape, size, and aggregation state resulting in an interest to develop more reproducible and uniform nanoparticles. Here triangular-based nanopyramids were characterized by scanning electron microscopy to confirm their geometry and interparticle spacing. Then the isotopologue approach with Rhodamine 6G was used to conclusively prove SM sensitivity for the individual nanoparticles. NSL’s straightforward, simple fabrication procedure produces large active arrays. Furthermore, the tunable localized surface plasmon resonance makes NSL prepared substrates effective for the detection of resonant molecules by SMSERS

Hydrophobic Collapse Initiates the Poly(<i>N</i>‑isopropylacrylamide) Volume Phase Transition Reaction Coordinate

The best-known examples of smart, responsive hydrogels derive from poly­(<i>N</i>-isopropylacrylamide) (PNIPAM) cross-linked polymer networks. These hydrogels undergo volume phase transitions (VPTs) triggered by temperature, chemical, and/or environmental changes. PNIPAM hydrogels can undergo more than 50-fold volume changes within ∼1 μs intervals. Studies have tried to elucidate the molecular mechanism of these extraordinarily large responses. Nevertheless, the molecular reaction coordinates that drive the VPT remain unclear. Using visible nonresonance Raman temperature-jump spectroscopy, we determined the molecular ordering of this VPT. The PNIPAM hydrophobic isopropyl and methylene groups dehydrate with time constants of 109 ± 64 and 104 ± 44 ns, initiating the volume collapse of PNIPAM. The subsequent dehydration of the PNIPAM amide groups is significantly slower, as our group previously discovered (360 ± 85 ns). This determination of the ordering of the molecular reaction coordinate of the PNIPAM VPT enables the development of the next generation of super-responsive materials

Aluminum Film-Over-Nanosphere Substrates for Deep-UV Surface-Enhanced Resonance Raman Spectroscopy

We report here the first fabrication of aluminum film-over nanosphere (AlFON) substrates for UV surface-enhanced resonance Raman scattering (UVSERRS) at the deepest UV wavelength used to date (λ_ex = 229 nm). We characterize the AlFONs fabricated with two different support microsphere sizes using localized surface plasmon resonance spectroscopy, electron microscopy, SERRS of adenine, tris­(bipyridine)­ruthenium­(II), and trans-1,2-bis­(4-pyridyl)-ethylene, SERS of 6-mercapto-1-hexanol (as a nonresonant molecule), and dielectric function analysis. We find that AlFONs fabricated with the 210 nm microspheres generate an enhancement factor of approximately 10^4–5, which combined with resonance enhancement of the adsorbates provides enhancement factors greater than 10⁶. These experimental results are supported by theoretical analysis of the dielectric function. Hence our results demonstrate the advantages of using AlFON substrates for deep UVSERRS enhancement and contribute to broadening the SERS application range with tunable and affordable substrates