Philip B. Oldham

A Comparison of Fluorescence Inner-Filter Effects for Different Cell Configurations

Abstract In conventional fluorescence spectroscopy, fluorescence intensity at high fluorophore concentration is often not proportional to fluorophore concentration, owing to primary and secondary absorption (inner-filter effects). In this paper, fluorescence calibration curves for anthracene solutions were obtained using a conventional right angle cell, a frontal reflection cell, a short pass cell, and a total internal reflection fluorescence (TIRF) cell for comparing the observed primary inner-filter effects. Measurements were also made of a two-component mixture using the nonfluorescent 9-nitrofluorene with anthracene for comparing primary and secondary inner-filter effects. A conventional right angle cell exhibited the widest linear dynamic range and lowest detectable anthracene concentration, whereas the TIRF cell provided the best linearity at high concentrations. The TIRF cell was determined to have significant potential for quantitative analysis of highly concentrated and/or turbid solutions.

Investigation of Chromatographic Surface Viscosity Using Total Internal Reflection Fluorescence

Total internal reflection fluorescence (TIRF) spectroscopy was employed to determine the viscosity of planar chromatographic surfaces. Various n-alkyldimethylchlorosilanes were used to modify the surface of a UV-quartz plate, so as to obtain a covalently attached monomeric C1, C3, C8, or C18 surface. TIRF anisotropy data of 1, 6-diphenylhexatriene (DPH) were collected on the unmodified and modified surfaces in the presence of overlaying solvents covering a range of viscosities. The results indicate that DPH adsorbs onto C1 and C3, but clearly exhibits partitioning with the C18 phase. The C8 surface data indicate intermediate behavior between adsorption and partitioning. On the basis of these preliminary data, the estimated microviscosity of a planar C18 surface is approximately 18 cP.

Intrinsic fluorescence as a potential rapid scoring tool for protein crystals

Genomics is on the verge of creating a large number of proteins for structure determination. Since X-ray crystallography is the main tool for determining protein structure, a corresponding large number of X-ray diffraction experiments will have to be performed. However, X-ray analysis is labor intensive so that the question of which protein crystal to choose for X-ray analysis from the vast pool of candidates becomes important from an economics standpoint. It is desirable to have a rapid and efficient method to score protein crystals regarding their likelihood for being of diffraction quality. We propose intrinsic protein fluorescence as a scoring tool and report preliminary data that demonstrate correlation between fluorescence spectra of single crystals and their internal order as determined by X-ray crystallography. Specifically, fine structure and maxima of fluorescence emission spectra of lysozyme, hyaluronate lyase, and germination protease crystals were found to correlate with resolution and mosaicity determined by X-ray analysis. If the correlation exhibited by these three proteins reflects a general relationship for the majority of protein crystals, a rapid fluorescence assay for scoring crystals can be developed and adapted for a variety of configurations of high throughput crystal growth apparatus.

A total internal reflection fluorescence biosensor for aluminum (III)

Abstract Total internal reflection fluorescence (TIRF) is widely used for investigating interfacial interactions. It has proven to be a well-suited technique for biosensing applications due to its surface sensitivity and minimal sample consumption. In this paper, the protein phosvitin has been investigated as a molecular sensor for the detection of aluminum (III). Phosvitin was labeled with fluorescein isothiocyanate (FITC) and immobilized on a quartz surface via EDC/NHS chemistry. Phosvitin–FITC and aluminum complexation, both in free solution and surface-bound form, was studied. A TIRF biosensor selective for Al 3+ was constructed using surface-immobilized phosvitin–FITC. An enhancement of phosvitin–FITC fluorescence intensity was observed upon addition of Al 3+ . The response was linear over the range 0.1–10 μM of aluminum concentration.

Control of antibody-antigen binding or dissociation by electric field

The development of a biosensor with adequate sensitivity generally requires a biospecific interaction with high binding affinity. The affinity constants for most antibody- antigen interactions are determined largely by the dissociation constants, kd, with little variation observed in rates of associated. additionally, surface immobilization typically results in a reduced kd. In this case, the sensor binds analyte kinetically irreversibly preventing response to changes in analytic concentration or reuse. Regeneration of the sensor surface is difficult, at best. On the other hand, a higher dissociation rate which would lend itself to a linear and reusable sensor, results in lower affinity and poor sensitivity. Consequently, most biosensors are disposable devices and quantitation is obtained using multiple single-use sensors. In this work, a new reusable biosensor platform which provides simultaneous fluorescence detection and electrochemical control of biospecific binding has been developed. Biotin was covalently attached to a transparent indium tin oxide electrode, which also served as an integral part of a total internal reflection fluorescence (TIRF) flow cell. TIRF was used to monitor biospecific interactions while electrochemical polarization was employed to control the interactions. Two possible mechanisms of the electric field effect involving interactions with induced and permanent dipoles of proteins will be discussed.

The structure and conformation of 4-hydroxyphenyl terephthalate: a model compound for a liquid crystalline polyester

Abstract A theoretical analysis using ab initio calculations of the possible conformations of 4-hydroxyphenyl terephthalate which represents the repeat unit for poly( p -phenylene terephthalate) is reported. The study includes complete geometry optimizations and calculation of vibrational frequencies at the SCF/STO-3G level of ten distinct stationary points on the molecular potential surface. The calculations show that both the terephthalic and the hydroquinone fragments of the system prefer planar conformations. For the entire molecule, the low energy form is a twisted conformation where the hydroquinone portion is twisted by about 65° relative to the terephthalic portion of the molecule. The barrier to internal rotation around the ester bond is about 2 kcal mol -1 .

Effect of surface electrostatics on adsorption behavior and biospecific interactions of DNA oligonucleotides

A total internal reflection fluorescence (TIRF) flow system was used to detect DNA adsorption and hybridization of a single stranded target DNA with a surface immobilized DNA probe. A transparent SnO2 or indium tin oxide (ITO) film served as a spectroscopic surface in the TIRF flow system and simultaneously as a working electrode for electrochemical (EC) control. The SnO2 and ITO electrodes were chemically modified to provide hydrophilic or hydrophobic surfaces with different functional groups. Charge of the sensor surface was modulated by external electrochemical polarization. Results indicate that DNA oligonucleotides exhibit higher adsorption affinity to positively charged aminated sensor surfaces, while negative polarization stimulates desorption of DNA molecules. To investigate DNA hybridization at the TIRF EC sensor surface, a thiolated probe oligonucleotide, with the complementary sequence to that of the target DNA, was covalently attached to the aminated sensor surface using a heterobifunctional crosslinker. A solution phase 27-base target DNA labeled by fluorescein was exposed to the sensor surface. The kinetics of heterogeneous hybridization between target DNA and surface immobilized probe was studied at different electrode potentials. The results show that cathodic polarization can accelerate hybridization and, at the same time, suppress nonspecific DNA adsorption.