A spectroscopic assay based on surface enhanced Raman scattering (SERS) using silver nanorod array substrates has been developed that allows for rapid detection of trace levels of viruses with a high degree of sensitivity and specificity. This novel SERS assay can detect spectral differences between viruses, viral strains, and viruses with gene deletions in biological media. The method provides rapid diagnostics for detection and characterization of viruses generating reproducible spectra without viral manipulation.

Substrates consisting of silver nanorod arrays with an irregular surface lattice ͑i.e., random nucleation sites͒ and with varying rod lengths were fabricated by an oblique angle vapor deposition method. These arrays were evaluated as potential surface-enhanced Raman spectroscopy ͑SERS͒ substrates using trans-1,2-bis͑4-pyridyl͒ethene as a reported molecule. SERS activity was shown to depend upon the length of the nanorods. The Ag nanorods with average lengths of 508.29± 44.86 nm, and having aspect ratios of 5.69± 1.49 exhibited the maximum SERS enhancement factors of greater than 10 8. Theoretical calculations indicate that this large SERS enhancement may be partially explained by the shape, density, and lateral arrangement of the Ag nanorod arrays.

Aligned Ag nanorod arrays have been fabricated on glass substrates using an oblique angle vapor deposition (OAD) method. The surface morphology and structure of the Ag rods were studied by scanning electron microscopy, transmission electron microscopy, and X-ray diffraction. Individual nanorods were found to be primarily cylindrical, albeit with irregularly shaped surface protrusions of the rods. The surface enhanced Raman scattering (SERS) response as a function of nanorod length was investigated using trans-1,2-bis(4pyridyl)ethene as a probe molecule at an excitation wavelength of 785 nm. The enhancement factors reached a maximum of ∼5 × 10 8 as the nanorod length approached 868 nm and then decreased to ∼3 × 10 6 as the nanorod length increased further to 1900 nm. For the 868 nm nanorod array, signals collected over multiple spots within the same substrate resulted in a relative standard deviation (RSD) of 10%, while an RSD of 15% was measured in signals collected from different substrates. When stored in a food vacuum bag, the substrates are stable with respect to SERS intensity for greater than 2 months. This study demonstrates that the OAD Ag nanorod arrays are highly sensitive, uniform, and stable SERS substrates that are suitable for a variety of surface chemical analysis applications.

Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

Polarized surface-enhanced Raman scattering (SERS) and UV-vis absorbance spectra were measured for a nonplanar Ag nanorod array substrate prepared by oblique angle vapor deposition. The anisotropy of the SERS polarization was shown to differ from that of the polarized UV-vis absorbance. The maximum SERS intensity was observed in the polarization direction perpendicular to the long axis of the Ag nanorods, while the UV-vis absorbance was strongly polarized along the direction of the long axis of the nanorod array. Analysis of the polarization data showed that molecular orientation was not the cause of the anisotropic SERS scattering. Rather, the SERS anisotropy was primarily attributed to the lateral arrangement of the three-dimensional tilted nanorod lattice in which highly localized plasmon modes are created by strong electromagnetic coupling between adjacent metallic nanorods.

There is a critical need for a rapid and sensitive means of detecting viruses. Recent reports from our laboratory have shown that surface-enhanced Raman spectroscopy (SERS) can meet these needs. In this study, SERS was used to obtain the Raman spectra of respiratory syncytial virus (RSV) strains A/Long, B1, and A2. SERS-active substrates composed of silver nanorods were fabricated using an oblique angle vapor deposition method. The SERS spectra obtained for each virus were shown to possess a high degree of reproducibility. Based on their intrinsic SERS spectra, the four virus strains were readily detected and classified using the multivariate statistical methods principal component analysis (PCA) and hierarchical cluster analysis (HCA). The chemometric results show that PCA is able to separate the three virus strains unambiguously, whereas the HCA method was able to readily distinguish an A2 strain-related G gene mutant virus (DeltaG) from the A2 strain. The results described here demonstrate that SERS, in combination with multivariate statistical methods, can be utilized as a highly sensitive and rapid viral identification and classification method.

The angular dependence of surface enhanced Raman scattering (SERS) has been investigated for molecules adsorbed onto Ag nanorod array substrates fabricated using an oblique angle deposition technique. The strong SERS signal of trans-1,2-bis(4-pyridyl) ethene (BPE) was found to be strongly dependent on the incident angle of the excitation laser, with the maximum SERS intensity appearing at approximately 45° relative to the surface normal. A scattering model based on classical electrodynamic dipole radiation has been developed for BPE adsorbed onto these Ag nanorod substrates, and the theoretical SERS scattering intensity was found to be in good agreement with the experimental results.

Bovine pulmonary surfactant protein C (SP-C) is a hydrophobic, alpha-helical membrane-associated lipoprotein in which cysteines C4 and C5 are acylated with palmitoyl chains. Recently, it has been found that the alpha-helix form of SP-C is metastable, and under certain circumstances may transform from an alpha-helix to a beta-strand conformation that resembles amyloid fibrils. This transformation is accelerated when the protein is in its deacylated form (dSP-C). We have used infrared spectroscopy to study the structure of dSP-C in solution and at membrane interfaces. Our results show that dSP-C transforms from an alpha-helical to a beta-type amyloid fibril structure via a pH-dependent mechanism. In solution at low pH, dSP-C is alpha-helical in nature, but converts to an amyloid fibril structure composed of short beta-strands or beta-hairpins at neutral pH. The alpha-helix structure of dSP-C is fully recoverable from the amyloid beta-structure when the pH is once again lowered. Attenuated total reflectance infrared spectroscopy of lipid-protein monomolecular films showed that the fibril beta-form of dSP-C is not surface-associated at the air-water interface. In addition, the lipid-associated alpha-helix form of dSP-C is only retained at the surface at low surface pressures and dissociates from the membrane at higher surface pressures. In situ polarization modulation infrared spectroscopy of protein and lipid-protein monolayers at the air-water interface confirmed that the residual dSP-C helix conformation observed in the attenuated total reflectance infrared spectra of transferred films is randomly or isotropically oriented before exclusion from the membrane interface. This work identifies pH as one of the mechanistic causes of amyloid fibril formation for dSP-C, and a possible contributor to the pathogenesis of pulmonary alveolar proteinosis.