Christine M. Ingersoll

Interactions between Methylene Blue and Sodium Dodecyl Sulfate in Aqueous Solution Studied by Molecular Spectroscopy

The interactions of methylene blue (MB, a cationic redox indicator and biological stain) and sodium dodecyl sulfate (SDS, a micelle-forming, anionic surfactant) in aqueous solution have been examined by using Rayleigh scattering, UV-visible absorption, and fluorescence spectroscopy. At SDS concentrations significantly below the critical micelle concentration (cmc), MB forms noncovalent dimers and aggregates with SDS that scatter light but do not fluoresce. For solutions containing 1 μM MB and < 3–5 mM SDS, shifts in the absorption spectrum characteristic of the formation of MB H-aggregates are noted. There appears to be little effect on the fluorescence emission spectrum, indicating that these MB aggregates do not fluoresce appreciably. At and above the known SDS cmc, MB is observed to interact with the micelles. The MB excited-state fluorescence lifetime (380 ps) remains constant until SDS micelles form, then increases to 615 ps. The MB rotational reorientation time similarly increases from 105 to 500 ps between 6 and 8 mM SDS. This finding suggests that the MB is encountering, on average, a microenvironment in the SDS micelles that is 5-fold more viscous than liquid water or the molar volume of the MB/SDS species that is reorienting is 5-fold larger than MB in water.

Toward Sol-Gel-Processed Chemical Sensing Platforms: Effects of Dopant Addition Time on Sensor Performance

The development of new chemical and biochemical sensing schemes has been a topic of growing interest. Simplicity of preparation and mild processing conditions have made sol-gel-derived composites attractive for many chemical sensing schemes. A portion of our research centers on using sol-gel-processed materials for the development of selective sensors. Over the years we have aimed to characterize the analytical performance of these types of sol-gel-based sensing platforms. In the course of this work we recently discovered that the time (prior to casting) when the sensing chemistry is actually doped into the sol-gel processing solution plays a critical role in a given sensors analytical performance. In this paper we report on the effects of doping time on the behavior of a model organic dopant (pyrene) sequestered within sol-gel-derived microfiber tips and films. We use O2 as the analyte and determine the sensor sensitivity and temporal response as a function of doping time. We also quantify the local dipolarity of the immediate environment surrounding the average pyrene molecule as a function of doping time.

Tracking Nanosecond and Subnanosecond Protein Dynamics On-the-Fly Using Frequency-Domain Fluorescence

Fluorescence anisotropy and intensity decay experiments on proteins can provide detailed information on biomolecule dynamics and function. However, experiments of this sort are normally performed while the biomolecule is at or near equilibrium. Although information on protein dynamics under equilibrium conditions is extremely important, details about the protein behavior while it is actually undergoing change can provide significantly more insight into the overall protein behavior. Multiharmonic Fourier frequency-domain fluorescence provides a means to acquire fluorescence anisotropy and intensity decay information on a reasonably rapid time scale. As a result, one can potentially track protein nanosecond and subnanosecond dynamical processes on-the-fly as they undergo change(s) during, for example, protein–ligand binding, enzymatic reactions, or antigen/hapten–antibody association. To illustrate the potential of the frequency-domain on-the-fly methodology, we report here on the behavior of a model protein, bovine serum albumin, that has been labeled site-selectively with the fluorescent probe acrylodan (BSA-Ac). Conformational changes in the BSA-Ac are effected by using trypsin or β-mercaptoethanol (BME). BME is a disulfide interchange reagent, and trypsin cleaves and excises from the entire BSA molecule a 21 amino acid peptide segment that contains the covalently attached Ac residue. This paper focuses on the time course of the fluorescence anisotropy and intensity decay kinetics of BSA-Ac as it reacts with trypsin or BME.