Molecular-level understanding of the rovibrational spectra of N2O in gaseous, supercritical, and liquid SF6 and Xe.

The transition between the gas-, supercritical-, and liquid-phase behavior is a fascinating topic, which still lacks molecular-level understanding. Recent ultrafast two-dimensional infrared spectroscopy experiments suggested that the vibrational spectroscopy of N2O embedded in xenon and SF6 as solvents provides an avenue to characterize the transitions between different phases as the concentration (or density) of the solvent increases. The present work demonstrates that classical molecular dynamics (MD) simulations together with accurate interaction potentials allows us to (semi-)quantitatively describe the transition in rotational vibrational infrared spectra from the P-/R-branch line shape for the stretch vibrations of N2O at low solvent densities to the Q-branch-like line shapes at high densities. The results are interpreted within the classical theory of rigid-body rotation in more/less constraining environments at high/low solvent densities or based on phenomenological models for the orientational relaxation of rotational motion. It is concluded that classical MD simulations provide a powerful approach to characterize and interpret the ultrafast motion of solutes in low to high density solvents at a molecular level.

Use of Nanoparticle Decorated Surface-Enhanced Raman Scattering Active Sol-Gel Substrates for SALDI-MS Analysis.

Spectroscopy and mass spectrometry techniques are sometimes combined into the same analytical workflow to leverage each technique's analytical benefits. This combined workflow is especially useful in forensic and medical contexts where samples are often precious in nature. Here, we adopt metal nanoparticle (NP) doped sol-gel substrates, initially developed for surface-enhanced Raman scattering (SERS) analysis, as surface-assisted laser desorption/ionization-mass spectrometry (SALDI-MS) substrates. Using dried blood and sample protocols previously developed for SERS analysis, we observe heme-related spectral features on both silver and gold NP substrates by SALDI-MS, demonstrating dual functionality for these orthogonal techniques. Modifying the dried blood extraction procedures also allows for the observation of blood triacylglycerols by SALDI-MS. This is the first demonstration of a SERS/SALDI-MS substrate based on a sol-gel scaffold and the first demonstration of a gold NP sol-gel substrate for SALDI-MS which features lower substrate-related SALDI-MS background compared to the silver substrate.

Ultrafast 2DIR comparison of rotational energy transfer, isolated binary collision breakdown, and near critical fluctuations in Xe and SF6 solutions.

The density dependence of rotational and vibrational energy relaxation (RER and VER) of the N2O ν3 asymmetric stretch in dense gas and supercritical Xe and SF6 solutions for near critical isotherms is measured by ultrafast 2DIR and infrared pump-probe spectroscopy. 2DIR analysis provides precise measurements of RER at all gas and supercritical solvent densities. An isolated binary collision (IBC) model is sufficient to describe RER for solvent densities ≤ ∼4M where rotational equilibrium is re-established in ∼1.5-2.5 collisions. N2O RER is ∼30% more efficient in SF6 than in Xe due to additional relaxation pathways in SF6 and electronic factor differences. 2DIR analysis revealed that N2O RER exhibits a critical slowing effect in SF6 at near critical density (ρ* ∼ 0.8) where the IBC model breaks down. This is attributable to the coupling of critical long-range density fluctuations to the local N2O free rotor environment. No such RER critical slowing is observed in Xe because IBC break down occurs much further from the Xe critical point. Many body interactions effectively shield N2O from these near critical Xe density fluctuations. The N2O ν3 VER density dependence in SF6 is different than that seen for RER, indicating a different coupling to the near critical environment than RER. N2O ν3 VER is only about ∼7 times slower than RER in SF6. In contrast, almost no VER decay is observed in Xe over 200 ps. This VER solvent difference is due to a vibrationally resonant energy transfer pathway in SF6 that is not possible for Xe.

Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity.

Stimulated Raman scattering (SRS) microscopy allows for high-speed label-free chemical imaging of biomedical systems. The imaging sensitivity of SRS microscopy is limited to ~10 mM for endogenous biomolecules. Electronic pre-resonant SRS allows detection of sub-micromolar chromophores. However, label-free SRS detection of single biomolecules having extremely small Raman cross-sections (~10-30 cm2 sr-1) remains unreachable. Here, we demonstrate plasmon-enhanced stimulated Raman scattering (PESRS) microscopy with single-molecule detection sensitivity. Incorporating pico-Joule laser excitation, background subtraction, and a denoising algorithm, we obtain robust single-pixel SRS spectra exhibiting single-molecule events, verified by using two isotopologues of adenine and further confirmed by digital blinking and bleaching in the temporal domain. To demonstrate the capability of PESRS for biological applications, we utilize PESRS to map adenine released from bacteria due to starvation stress. PESRS microscopy holds the promise for ultrasensitive detection and rapid mapping of molecular events in chemical and biomedical systems.

Spectroscopic evidence for the origin of odd-even effects in self-assembled monolayers and effects of substrate roughness.

This paper reports the effects of substrate roughness on the odd-even effect in n-alkanethiolate self-assembled monolayers (SAMs) probed by vibrational sum frequency generation (SFG) spectroscopy. By fabricating SAMs on surfaces across the so-called odd-even limit, we demonstrate that differentiation of the vibrational frequencies of CH3 from SAMs derived from alkyl thiols with either odd (SAMO) or even (SAME) numbers of carbons depends on the roughness of the substrate on which they are formed. Odd-even oscillation in SFG susceptibility amplitudes was observed for spectra derived from SAME and SAMO fabricated on flat surfaces (RMS roughness = 0.4 nm) but not on rougher surfaces (RMS roughness = 2.38 nm). In addition, we discovered that local chemical environments for the terminal CH3 group have a chain-length dependence. There seems to be a transition at around C13, beyond which SAMs become "solid-like".

Rapid Detection of Bacteria from Blood with Surface-Enhanced Raman Spectroscopy.

Traditional methods for identifying pathogens in bacteremic patients are slow (24-48+ h). This can lead to physicians making treatment decisions based on an incomplete diagnosis and potentially increasing the patient's mortality risk. To decrease time to diagnosis, we have developed a novel technology that can recover viable bacteria directly from whole blood and identify them in less than 7 h. Our technology combines a sample preparation process with surface-enhanced Raman spectroscopy (SERS). The sample preparation process enriches viable microorganisms from 10 mL of whole blood into a 200 µL aliquot. After a short incubation period, SERS is used to identify the microorganisms. We further demonstrated that SERS can be used as a broad detection method, as it identified a model set of 17 clinical blood culture isolates and microbial reference strains with 100% identification agreement. By applying the integrated technology of sample preparation and SERS to spiked whole blood samples, we were able to correctly identify both Staphylococcus aureus and Escherichia coli 97% of the time with 97% specificity and 88% sensitivity.

The biochemical origins of the surface-enhanced Raman spectra of bacteria: a metabolomics profiling by SERS.

The dominant molecular species contributing to the surface-enhanced Raman spectroscopy (SERS) spectra of bacteria excited at 785 nm are the metabolites of purine degradation: adenine, hypoxanthine, xanthine, guanine, uric acid, and adenosine monophosphate. These molecules result from the starvation response of the bacterial cells in pure water washes following enrichment from nutrient-rich environments. Vibrational shifts due to isotopic labeling, bacterial SERS spectral fitting, SERS and mass spectrometry analysis of bacterial supernatant, SERS spectra of defined bacterial mutants, and the enzymatic substrate dependence of SERS spectra are used to identify these molecular components. The absence or presence of different degradation/salvage enzymes in the known purine metabolism pathways of these organisms plays a central role in determining the bacterial specificity of these purine-base SERS signatures. These results provide the biochemical basis for the development of SERS as a rapid bacterial diagnostic and illustrate how SERS can be applied more generally for metabolic profiling as a probe of cellular activity. Graphical Abstract Bacterial typing by metabolites released under stress.

Nonlinear Midinfrared Photothermal Spectroscopy Using Zharov Splitting and Quantum Cascade Lasers.

We report on the mid-infrared nonlinear photothermal spectrum of the neat liquid crystal 4-octyl-4'-cyanobiphenyl (8CB) using a tunable Quantum Cascade Laser (QCL). The nonequilibrium steady state characterized by the nonlinear photothermal infrared response undergoes a supercritical bifurcation. The bifurcation, observed in heterodyne two-color pump-probe detection, leads to ultrasharp nonlinear infrared spectra similar to those reported in the visible region. A systematic study of the peak splitting as a function of absorbed infrared power shows the bifurcation has a critical exponent of 0.5. The observation of an apparently universal critical exponent in a nonequilibrium state is explained using an analytical model analogous of mean field theory. Apart from the intrinsic interest for nonequilibrium studies, nonlinear photothermal methods lead to a dramatic narrowing of spectral lines, giving rise to a potential new contrast mechanism for the rapidly emerging new field of mid-infrared microspectroscopy using QCLs.

Intensity-dependent exciton dynamics of (6,5) single-walled carbon nanotubes: momentum selection rules, diffusion, and nonlinear interactions.

The exciton dynamics for an ensemble of individual, suspended (6,5), single-walled carbon nanotubes revealed by single color E(22) resonant pump-probe spectroscopy for a wide range of pump fluences are reported. The optically excited initial exciton population ranges from approximately 5 to 120 excitons per ∼725 nm nanotube. At the higher fluences of this range, the pump-probe signals are no longer linearly dependent on the pump intensity. A single, predictive model is described that fits all data for two decades of pump fluences and three decades of delay times. The model introduces population loss from the optically active zero momentum E(22) state to the rest of the E(22) subband, which is dark due to momentum selection rules. In the single exciton limit, the E(11) dynamics are well described by a stretched exponential, which is a direct consequence of diffusion quenching from an ensemble of nanotubes of different lengths. The observed change in population relaxation dynamics as a function of increasing pump intensity is attributed to exciton-exciton Auger de-excitation in the E(11) subband and, to a lesser extent, in the E(22) subband. From the fit to the model, an average defect density 1/ρ = 150 nm and diffusion constants D(11) = 4 cm(2)/s and D(22) = 0.2 cm(2)/s are determined.

On the difference between surface-enhanced raman scattering (SERS) spectra of cell growth media and whole bacterial cells.

It has been recently suggested [N. E. Marotta and L. A. Bottomley, Appl. Spectrosc. 64, 601-606 (2010)] that previously reported surface-enhanced Raman scattering (SERS) spectra of vegetative bacterial cells are due to residual cell growth media that were not properly removed from samples of the lab-cultured microorganism suspensions. SERS spectra of several commonly used cell growth media are similar to those of bacterial cells, as shown here and reported elsewhere. However, a multivariate data analysis approach shows that SERS spectra of different bacterial species grown in the same growth media exhibit different characteristic vibrational spectra, SERS spectra of the same organism grown in different media display the same SERS spectrum, and SERS spectra of growth media do not cluster near the SERS spectra of washed bacteria. Furthermore, a bacterial SERS spectrum grown in a minimal medium, which uses inorganics for a nitrogen source and displays virtually no SERS features, exhibits a characteristic bacterial SERS spectrum. We use multivariate analysis to show how successive water washing and centrifugation cycles remove cell growth media and result in a robust bacterial SERS spectrum in contrast to the previous study attributing bacterial SERS signals to growth media.

Rapid point-of-care concentration of bacteria in a disposable microfluidic device using meniscus dragging effect.

We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from liquid samples using enhanced evaporation targeted at downstream detection using surface enhanced Raman spectroscopy (SERS). The device is composed of a poly(dimethylsiloxane) (PDMS) liquid sample flow layer, a reusable metal airflow layer, and a porous PTFE (Teflon™) membrane sandwiched in between the liquid and air layers. The concentration capacity of the device was successfully demonstrated with fluorescently tagged Escherichia coli (E. coli). The recovery concentration was above 85% for all initial concentrations lower than 1 × 10(4) CFU mL(-1). In the lowest initial concentration cases, 100 µL initial volumes of bacteria solution at 100 CFU mL(-1) were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min. Subsequent tests with SERS on clinically relevant Methicillin-Sensitive Staphylococcus aureus (MSSA) after concentration in this device proved more than 100-fold enhancement in SERS signal intensity compared to the signal obtained from the unconcentrated sample. The concentration device is straightforward to design and use, and as such could be used in conjunction with a number of detection technologies.

Engineered SERS substrates with multiscale signal enhancement: nanoparticle cluster arrays.

Defined nanoparticle cluster arrays (NCAs) with total lateral dimensions of up to 25.4 microm x 25.4 microm have been fabricated on top of a 10 nm thin gold film using template-guided self-assembly. This approach provides precise control of the structural parameters in the arrays, allowing a systematic variation of the average number of nanoparticles in the clusters (n) and the edge-to-edge separation (Lambda) between 1 < n < 20 and 50 nm < or = Lambda < or = 1000 nm, respectively. Investigations of the Rayleigh scattering spectra and surface-enhanced Raman scattering (SERS) signal intensities as a function of n and Lambda reveal direct near-field coupling between the particles within individual clusters, whose strength increases with the cluster size (n) until it saturates at around n = 4. Our analysis shows that strong near-field interactions between individual clusters significantly affect the SERS signal enhancement for edge-to-edge separations Lambda < 200 nm. The observed dependencies of the Raman signals on n and Lambda indicate that NCAs support a multiscale signal enhancement which originates from simultaneous inter- and intracluster coupling and |E|-field enhancement. The NCAs provide strong and reproducible SERS signals not only from small molecules but also from whole bacterial cells, which enabled a rapid spectral discrimination between three tested bacteria species: Escherichia coli, Bacillus cereus, and Staphylococcus aureus.

Nitrous oxide vibrational energy relaxation is a probe of interfacial water in lipid bilayers.

Ultrafast infrared spectroscopy of N 2O is shown to be a sensitive probe of hydrophobic and aqueous sites in lipid bilayers. Distinct rates of VER of the nu 3 antisymmetric stretching mode of N 2O can be distinguished for N 2O solvated in the acyl tail, interfacial water, and bulk water regions of hydrated dioleoylphosphatidylcholine (DOPC) bilayers. The lifetime of the interfacial N 2O population is hydration-dependent. This effect is attributed to changes in the density of intermolecular states resonant with the nu 3 band ( approximately 2230 cm (-1)) resulting from oriented interfacial water molecules near the lipid phosphate. Thus, the N 2O VER rate becomes a novel and experimentally convenient tool for reporting on the structure and dynamics of interfacial water in lipids and, potentially, in other biological systems.

Optical heterodyne detected accumulated acoustic grating responses in near supercritical fluids.

A novel third-order polarization effect due to an accumulated optical heterodyne detected (OHD) transient acoustic grating response in near critical fluids was observed and experimentally characterized. Femtosecond pump-probe responses in near critical CO2 and CHF3 illustrate this phenomenon. This large optically generated acoustic response due to electrostrictive coupling appears only when pump and probe pulses are temporally overlapped and is pi out-of-phase with the normal optical Kerr effect (OKE) birefringent signal. The local oscillator, the laser intensity, and the modeled experimental repetition rate dependence identify the accumulated heterodyne origin of these responses. The observed OHD accumulated acoustic birefringent signal is inversely dependent on sound velocity to the fifth power. A corresponding sound velocity dependent dichroic (in-phase) response was also observed for these electronically nonresonant samples. The accumulated effect described here may have applications for the design of efficient modulators and as a simple and sensitive experimental technique for the measurement of near critical fluid thermodynamic and acoustic parameters.

Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction.

Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.