Richard A. Dluhy

Aligned silver nanorod arrays produce high sensitivity surface-enhanced Raman spectroscopy substrates

Substrates consisting of silver nanorod arrays with an irregular surface lattice (i.e., random nucleation sites) and with varying rod lengths were fabricated by an oblique angle vapor deposition method. These arrays were evaluated as potential surface-enhanced Raman spectroscopy (SERS) substrates using trans-1,2-bis(4-pyridyl)ethene 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±44.86nm, and having aspect ratios of 5.69±1.49 exhibited the maximum SERS enhancement factors of greater than 108. Theoretical calculations indicate that this large SERS enhancement may be partially explained by the shape, density, and lateral arrangement of the Ag nanorod arrays.

Rapid microRNA (miRNA) detection and classification via surface-enhanced Raman spectroscopy (SERS)

microRNAs (miRNA) are recognized as regulators of gene expression during development and cell differentiation as well as biomarkers of disease. Development of rapid and sensitive miRNA profiling methods is essential for evaluating the pattern of miRNA expression that varies across normal and diseased states. The ability to identify miRNA expression patterns is limited to cumbersome assays that often lack sensitivity and specificity to distinguish between different miRNA families and members. We evaluated a surface-enhanced Raman scattering (SERS) platform for detection and classification of miRNAs. The strength of the SERS-based sensor is its sensitivity to detect extremely low levels of analyte and specificity to provide the molecular fingerprint of the analyte. We show that the SERS spectra of related and unrelated miRNAs can be detected in near-real time, that detection is sequence dependent, and that SERS spectra can be used to classify miRNA patterns with high accuracy.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the α- and β-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both α- and β-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Cytomimetic Biomaterials. 1. In-Situ Polymerization of Phospholipids on an Alkylated Surface

A stabilized, phosphatidylcholine-containing polymeric surface was produced by in-situ polymerization of a self-assembled lipid monolayer on an alkylated substrate. The phospholipid monomer 1-palmitoyl-2-[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphorylcholine was synthesized, prepared as unilamellar vesicles, and fused onto alkylated glass. Free-radical polymerization was carried out in aqueous solution at 70 °C and characterized using either the water-soluble initiator 2,2′-azobis(2methylpropionamidine) dihydrochloride (AAPD) or an oil-soluble initiator 2,2′-azobis(isobutyronitrile) (AIBN). Under optimized conditions, the supported monolayer displayed advancing and receding water contact angles of 64 and 44°, respectively. Angle-dependent ESCA results confirmed the presence of phosphorus and nitrogen and were consistent with theoretical predictions for close-packed monolayer formation with near-normal alignment of lipid chains. In the absence of network formation, polymeric films demonstrated acceptable stability under static conditions in water and air, as well as in the presence of a high shear flow environment. Blood compatibility was assessed in a baboon arteriovenous shunt model, which revealed miminal platelet deposition over a 2 h observation period.

Identification and classification of respiratory syncytial virus (RSV) strains by surface-enhanced Raman spectroscopy and multivariate statistical techniques

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 posses 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 (ΔG) 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 interfacial structure of phospholipid monolayer films: an infrared reflectance study

Abstract Monolayer films of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the air-water (A/W) interface have been investigated in situ using external reflection-absorption IR spectroscopy. The IR spectra of the monolayer films were monitored as a function of the two-dimensional molecular area of the phospholipd molecules on the surface of a Langmuir trough. Examination of the conformation-sensitive CH stretching bands of the molecules hydrocarbon chains reveals the existence of two phase transitions during film compression. The first transition corresponds to the main liquid-expanded-to-liquid-condensed monolayer phase transition (at ∼58–78 A2 molecule−1), while the second transition occurs at ∼40–57 A2 molecule−1 and is indicative of a transition to a solid-condensed phase under increasing pressure. The headgroup bands from the phosphate ester group were also monitored during film compression. The asymmetric phosphate stretching band of the DPPC monolayer film is composed of two bands, one at ∼1220 cm−1 and the second at ∼1257 cm−1. These bands are also seen, and frequency shifts noted, for the interaction of the soluble cations Ca2+ and Pr3+ with the phospholipid phosphate group. The band splitting and frequency shifts suggest a mixture of hydration states for the lipids phosphate group. The equilibrium between these hydration states can be changed by film compression and/or cation interaction with the lipid headgroup.

Infrared spectroscopic investigations of pulmonary surfactant. Surface film transitions at the air-water interface and bulk phase thermotropism

The molecular structure of the phospholipid component of intact pulmonary surfactant isolated from bovine lung lavage has been examined by Fourier transform infrared spectroscopy. Two different physical states of the surfactant were examined by means of different infrared spectroscopic sampling techniques. Transmission infrared experiments were used to study the surfactant in the bulk phase. In these experiments, the thermotropic behavior of the bulk surfactant was monitored by temperature-induced variations in the phospholipid acyl chain CH2 stretching frequencies. A broad phase transition (confirmed by differential scanning calorimetry) was noted with an onset temperature near 15 degrees C and a completion temperature near 42 degrees C. In addition to the bulk transmission experiments, external reflection infrared spectroscopy was used to examine surfactant films in situ at the air-water interface. As surface pressure was increased from 0 to 43 dyn/cm, a gradual and continuous decrease in the CH2 stretching frequency was noted for the surfactant. Thus, under surface pressures which correspond to large lung volumes in vivo, the surfactant acyl chains exist mostly in the ordered (trans) configuration. The frequency shift in the CH2 stretching mode is consistent with a continuous ordering of the acyl chains upon compression over the pressure range 0-43 dyn/cm, and implies that a weakly cooperative phase transition occurs in the hydrocarbon region of the surface film. The surface film transition is especially noted in the pressure-area curve of the surfactant and approximates in two dimensions the broad thermotropic phase transition of the bulk phase surfactant. Substantial differences were observed between the response to surface pressure changes of intact surfactant compared with the main surfactant phospholipid, 1,2-dipalmitoyl-sn--glycero-3-phosphocholine. The changes in response are attributed to the presence of additional surfactant components. The current work demonstrates the ability of infrared spectroscopy to obtain structural information on the surfactant in physical states that directly relate to those in vivo.

Fabrication and characterization of a multiwell array SERS chip with biological applications.

Uniform, large surface area substrates for surface-enhanced Raman spectroscopy (SERS) are fabricated by oblique angle deposition. The SERS-active substrates are patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-di(4-pyridyl)ethylene (BPE) >or=98%, we show that this device provides a uniform Raman signal enhancement from well to well with a detection limit of at least 10(-8)M of the BPE solution or 10(-18)mol of BPE. The SERS intensity is also demonstrated to vary logarithmically with the log of BPE concentration and the apparent sensitivity of the patterned substrate is compared to previous reports from our group on non-patterned substrates. Avian influenza is analyzed to demonstrate the utility of SERS multiwell patterned substrates for biosensing. The spectra acquired from patterned substrates show better reproducibility and less variation compared to the unpatterned substrates according to multivariate analysis. Our results highlight potential advantages of the patterned substrate.

Effect of Hydrophobic Surfactant Peptides SP-B and SP-C on Binary Phospholipid Monolayers. I. Fluorescence and Dark-Field Microscopy

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.

Angle dependent surface enhanced Raman scattering obtained from a Ag nanorod array substrate

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.