Linda B. McGown

Molecular nanotube aggregates of β- and γ-cyclodextrins linked by diphenylhexatrienes

Linked by strings of diphenylhexatriene (DPH) molecules, β- and γ-cyclodextrins (CDs) can form nanotube aggregates that contain as many as ∼20 βCDs (20 nanometers long) or ∼20 to 35 γCDs (20 to 35 nanometers long). Nanotube formation was indicated in solution, by fluorescence anisotropy and light scattering results, and on graphite surfaces, by scanning tunneling microscopy. Tubes were not observed for the smaller αCDs. Molecular modeling shows that CD cavity size and the rodlike DPH structure are key factors in nanotube formation. Spectra generated by proton nuclear magnetic resonance indicate the inclusion of DPH in the interior of the CDs and formation of nanotubes in βCDs and γCDs only. The photophysical properties of DPH are affected by its arrangement into a one-dimensional array within the CD nanotube, possibly because of exciton formation.

Open‐tubular capillary electrochromatography of bovine β‐lactoglobulin variants A and B using an aptamer stationary phase

DNA aptamers that form a G‐quartet conformation were covalently attached to a capillary surface for open‐tubular capillary electrochromatographic separation of bovine β‐lactoglobulin variants A and B, which vary by 2 of their 162 amino acid residues. Separation was achieved using a 4‐plane, G‐quartet aptamer stationary phase with tris(hydroxymethyl)aminomethane (Tris) or phosphate buffer as the mobile phase. In control experiments, separation did not occur using either an oligonucleotide of similar base composition but which does not form a G‐quartet structure, or using capillary zone electrophoresis on a bare capillary under similar experimental conditions. Separation was achieved using a capillary coated only with the covalent linker molecule. In phosphate buffer, the separations were similar for aptamer‐coated and linker‐only stationary phases, while in Tris buffer, retention times were almost doubled for the linker‐only capillary. When Tris buffer is the mobile phase, there appears to be weaker interactions between the proteins and the stationary phase that may result in a gentler, less denaturing separation than is commonly achieved using hydrocarbon‐based stationary phases.

Capillary electrochromatographic separation of bovine milk proteins using a G-quartet DNA stationary phase

DNA oligonucleotides that form G-quartet structures were used as stationary phase reagents for separation of bovine milk proteins, including alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin and beta-lactoglobulin. Both artificial protein mixtures and a skim milk sample were analyzed. The separations were performed using open-tubular capillary electrochromatography, in which the oligonucleotides were covalently attached to the inner surface of a fused-silica capillary. Better resolution was achieved using the G-quartet-coated capillaries than was achieved using either a bare capillary or a capillary coated with an oligonucleotide that does not form a G-quartet structure. A 4-plane G-quartet-forming stationary phase was able to resolve three peaks for alpha-casein and to detect thermal denaturation of the proteins in the milk sample. The results suggest that G-quartet stationary phases could be used to separate very similar protein structures, such as those arising from genetic variations or post-translational modifications.

Phase-Resolved Fluorescence Spectral and Lifetime Characterization of Commercial Humic Substances

Phase-resolved excitation-emission matrices (PREEMs) are shown to provide a unique visual representation of the intrinsic fluorescence properties of humic acids under a variety of solution conditions. The calculation of spectral peak ratios in PREEMs as well as steady-state excitation-emission matrices provides a convenient means for quantitating differences between the spectra with good precision. Absorbance correction is shown to be essential for accurate comparison among spectral features. Increased detail is available from PREEMs at various modulation frequencies that reveal the distribution of fluorescence lifetime contributions across the spectral surface. Direct measurement of fluorescence lifetime recovered three ranges of lifetime components in the humic substances, <1 ns, 2–5 ns, and 8–14 ns, that are consistent with previously reported lifetimes. PREEMs, which provide a concise “survey” of how the lifetimes change across the spectrum, may aid in pinpointing spectral regions that provide the best lifetime discrimination among samples.

MOLECULAR FLUORESCENCE AND PHOSPHORESCENCE

Molecular luminescence spectroscopy can be used for fundamental studies of molecular excited states as well as for selective and sensitive analysis of luminescent samples. Luminescence processes su...

Separation of Trp-Arg and Arg-Trp using G-quartet-forming DNA oligonucleotides in open-tubular capillary electrochromatography

DNA oligonucleotides that form intramolecular G‐quartet structures were investigated as stationary phase reagents for separation of mixtures of the isomeric dipeptides Trp‐Arg and Arg‐Trp in open‐tubular capillary electrochromatography (OTCEC). The oligonucleotides included a thrombin‐binding aptamer that forms a biplanar G‐quartet structure and an oligonucleotide that forms a 4‐plane G‐quartet structure. Fluorescence, circular dichroism and UV‐visible absorbance spectroscopies were used in batch solution studies to indicate interactions between the dipeptides and the biplanar G‐quartet structure. Results for OTCEC separations were compared with results obtained for capillary zone electrophoresis separations on a bare capillary. Temperature studies suggest that resolution is improved when the G‐quartet structure is partially destabilized, but control experiments in which potassium chloride was not included in the mobile phase indicate that the G‐quartet structure nevertheless plays a role in the separations.

Maximum entropy method for frequency domain fluorescence lifetime analysis. 1. Effects of frequency range and random noise

The maximum entropy method (MEM) provides a self-modeling fit to data in which minimization of the χ(2) goodness-of-fit parameter is coupled with maximization of a statistical entropy function. We have found that MEM provides an excellent visual description of the uncertainties, errors, and limitations associated with the distributions which it recovers. To more accurately interpret fluorescence lifetime distributions recovered by the MEM from frequency domain lifetime data, a detailed examination of the effects of frequency range, noise, data set size, and sample heterogeneity was carried out for both simulated and real data. Results clearly demonstrate that the frequency range in which data are collected can affect the number and nature of the fluorescence lifetime components that are recovered by MEM, and the quality of the data at the frequencies that are optimal for a given lifetime is also crucial. Expansion of sufficient data sets to include more frequencies, or more replicates at the same frequencies, provides little improvement over the original data set when the lifetimes are well-windowed by the frequency range. Synergism among multiple components in a sample can affect the recovered distribution, by shifting and splitting poorly windowed components and broadening the recovered peaks for all components. These effects are related to the number of components for which evidence must be found.

Total Lifetime Distribution Analysis for Fluorescence Fingerprinting and Characterization

A new technique, total lifetime distribution analysis (TLDA), is described for rapid, sensitive, and accurate lifetime characterization of complex samples. Multiharmonic Fourier transform technology in a commercial, frequency-domain fluorescence lifetime instrument allows rapid acquisition of TLDA data. High sensitivity derives from the use of the entire fluorescence emission from the sample in the lifetime measurement. The maximum entropy method (MEM) provides a consistent basis for modeling of the lifetime data for accurate recovery of the total lifetime distribution of the sample. Because MEM is self-modeling, it is not subject to the same sources of bias that influence nonlinear least-squares fits of lifetime data to a priori models. These features make TLDA an effective tool for sample characterization and fingerprinting that is based on the responsiveness of fluorescence lifetime to the chemical composition and dynamic processes that contribute to the uniqueness of the sample. TLDA results are presented for coal liquids and a humic substance. The effect of signal intensity on lifetime recovery is investigated, and comparison is made between MEM and conventional nonlinear least-squares for data analysis.

Multifrequency Phase-Modulation Fluorescence Lifetime Determinations On-the-Fly in HPLC

The first use of a multifrequency, phase-modulation spectrofluorometer for fluorescence lifetime determinations on-the-fly in HPLC is described. Direct, simultaneous measurements of fluorescence intensity, phase-shift, and demodulation are made at one-second intervals for polycylic aromatic hydrocarbons as they are eluted from the chromatograph. Fluorescence lifetime is calculated from both the phase-shift and the demodulation; the two independent values can be used to indicate the absence or presence of more than one component at any point in the chromatogram. Special considerations regarding data correction and calibration for phase-modulation lifetime determinations under continuous-flow conditions are discussed, along with effects of flow rate and mobile phase composition.

Spectroscopic studies of YO and YOYO fluorescent dyes in a thrombin-binding DNA ligand

The fluorescent, oxazole yellow dye YO-Pro-1 iodide (YO) and its homodimer YOYO-1 iodide (YOYO) were studied in a thrombin-binding DNA ligand, or aptamer, (tb-ligand) and in an oligomer with the same base composition in a scrambled sequence (s-ligand), both single strands of 15 bases in length. Binding constants for the dye-ligand complexes, assuming 1:1 stoichiometry, were determined to be on the order of 107M−1 for YOYO and 105M−1 for YO, which are approximately 105 smaller than estimated constants for YOYO in double-stranded DNA. In both ligands, YOYO assumes a folded conformation that promotes stability of the dye-ligand complex and excitonic coupling between the two YO groups. The folded conformation provides greater overlap of the YO groups than has been reported for YOYO in double-stranded DNA; the overlap is slightly greater in tb-ligand than in s-ligand. Both dyes exhibit bi-exponential fluorescence decay in the ligands and the lifetimes of YOYO (3–4 ns and 7–8 ns) are longer and more discrete than those of YO (1–3 ns and 4–5 ns). Fluorescence anisotropy of YOYO is a low, constant value in both ligands due to intramolecular energy transfer between the overlapping YO groups. Higher anisotropies are observed for YO, and the value is slightly higher in s-ligand than in tb-ligand. The addition of thrombin to the s-ligand affects the fluorescence intensity and anisotropy of YO but not of YOYO. The absence of intermolecular G-quartet formation of the s-ligand was demonstrated. This suggests that thrombin interacts weakly with the s-ligand but is not sensed by the fluorescence of YOYO, which is dominated by the coupling between the YO groups in the folded conformation of the bound dye. The results of these studies have implications for the application of these dyes for detection of single-stranded DNA ligands and their binding interactions.