A Raman spectral marker for the iso-octyl chain structure of cholesterol.

Raman spectroscopy provides label-free, specific analysis of biomolecular structure and interactions. It could have a greater impact with improved characterization of complex fingerprint vibrations. Many Raman peaks have been assigned to cholesterol, for example, but the molecular vibrations associated with those peaks are not known. In this report, time-dependent density functional theory calculations of the Raman spectrum of cholesterol are compared to measurements on microcrystalline powder to identify 23 peaks in the Raman spectrum. Among them, a band of six peaks is found to be sensitive to the conformational structure of cholesterol's iso-octyl chain. Calculations on 10 conformers in this spectral band are fit to experimental spectra to probe the cholesterol chain structure in purified powder and in phospholipid vesicles. In vesicles, the chain is found to bend perpendicular to the steroid rings, supporting the case that the chain is a dynamic structure that contributes to lipid condensation and other effects of cholesterol in biomembranes. Statement of Significance: Here we use density functional theory to identify a band of six peaks in cholesterol's Raman spectrum that is sensitive to the conformational structure of cholesterol's chain. Raman spectra were analyzed to show that in fluid-phase lipid membranes, about half of the cholesterol chains point perpendicular to the steroid rings. This new method of label-free structural analysis could make significant contributions to our understanding of cholesterol's critical role in biomembrane structure and function. More broadly, the results show that computational quantum chemistry Raman spectroscopy can make significant new contributions to molecular structure when spectra are interpreted with computational quantum chemistry.

Quantum Dot-DNA FRET Conjugates for Direct Analysis of Methylphosphonic Acid in Complex Media.

Rapid detection of nerve agents from complex matrices with minimal sample preparation is essential due to their high toxicity and bioavailability. In this work, quantum dots (QDs) were functionalized with oligonucleotide aptamers that specifically targeted a nerve agent metabolite, methylphosphonic acid (MePA). These QD-DNA bioconjugates were covalently linked to quencher molecules to form Förster resonance energy transfer (FRET) donor-acceptor pairs that quantitatively measure the presence of MePA. Using the FRET biosensor, the MePA limit of detection was 743 nM in artificial urine. A decrease in the QD lifetime was measured upon DNA binding and was recovered with MePA. The biosensor's flexible design makes it a strong candidate for the rapid detection of chemical and biological agents for deployable, in-field detectors.

Effects of Conformational Variation on Structural Insights from Solution-Phase Surface-Enhanced Raman Spectroscopy.

Surface-enhanced Raman scattering (SERS) spectra contain information on the chemical structure on nanoparticle surfaces through the position and alignment of molecules with the electromagnetic near field. Time-dependent density functional theory (TDDFT) can provide the Raman tensors needed for a detailed interpretation of SERS spectra. Here, the impact of molecular conformations on SERS spectra is considered. TDDFT calculations of the surfactant cetyltrimethylammonium bromide with five conformers produced more accurate unenhanced Raman spectra than a simple all-trans structure. The calculations and measurements also demonstrated a loss of structural information in the CH2/CH3 scissor vibration band at 1450 cm-1 in the SERS spectra. To study lipid bilayers, TDDFT calculations on conformers of methyl phosphorylcholine and cis-5-decene served as models for the symmetric choline stretch in the lipid headgroup and the C═C stretch in the acyl chains of 1,2-oleoyl-glycero-3-phosphocholine. Conformer considerations enabled a measurement of the distribution of double-bond orientations with an order parameter of SC═C = 0.53.

The orientation of a membrane probe from structural analysis by enhanced Raman scattering.

Small fluorescent molecules are widely used as probes of biomembranes. Different probes optically indicate membrane properties such as the lipid phase, thickness, viscosity, and electrical potential. The detailed molecular mechanisms behind probe signals are not well understood, in part due to the lack of tools to determine probe position and orientation in the membrane. Optical measurements on aligned biomembranes and lipid bilayers provide some degree of orientational information based on anisotropy in absorption, fluorescence, or nonlinear optical properties. These methods typically find the polar tilt angle between the membrane normal and the long axis of the molecule. Here we show that solution-phase surface enhanced Raman scattering (SERS) spectra of lipid membranes on gold nanorods can be used to determine molecular orientation of molecules within the membrane. The voltage sensitive dye 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-hydroxide, known as di-4-ANEPPS, is studied. Through the analysis of several peaks in the SERS spectrum, the polar angle from the membrane normal is found to be 66°, and the roll angle around the long axis of the molecule to be 305° from the original orientation. This structural analysis method could help elucidate the meaning of fluorescent membrane probe signals, and how they are affected by different lipid compositions.

Structural Analysis by Enhanced Raman Scattering.

Gold nanostructures focus light to a molecular length scale at their surface, creating the possibility to visualize molecular structure. The high optical intensity leads to surface enhanced Raman scattering (SERS) from nearby molecules. SERS spectra contain information on molecular position and orientation relative to the surface but are difficult to interpret quantitatively. Here we describe a ratiometric analysis method that combines SERS and unenhanced Raman spectra with theoretical calculations of the optical field and molecular polarizability. When applied to the surfactant layer on gold nanorods, the alkane chain is found to be tilted 25° to the surface normal, which matches previous reports of the layer thickness. The analysis was also applied to fluid phase phospholipid bilayers that contain tryptophan on the surface of gold nanorods. The lipid double bond was found to be oriented normal to the bilayer and 13 Å from the nitrogen atom. Tryptophan was found to sit near the glycerol headgroup region with its indole ring 43° from the bilayer normal. This new method can determine specific interfacial structure under ambient conditions, with microscopic quantities of material, and without molecular labels.