The absorption and emission spectra were measured for Cy™5 and Alexa™ 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret these red shifts in terms of a change in the polarizability and polarity of the effective solvent. A formula is given that can be used to estimate expected shifts in absorption and emission maxima for surface-immobilized fluorophores. Spectroscopic ellipsometry measurements provide identification of the fluorophores confined on a glass surface. These results suggest that the design of microarray detection systems should be based on the optical properties of fluorophores attached to the surface and not on the optical properties of fluorophores in solution.
Fluorophores at surfaces are an integral part of many biological assays. For several decades, microspheres with immobilized fluorophores were used to calibrate flow cytometers (1). Images of cells obtained in epifluorescent microscopes contain fluorophores at the surface of various organelles (2) and provide information on cellular structure. More recently, there has been a large surge in microarray technology (3), which was first applied to the analysis of gene expression (4,5,6,7). The microarray paradigm has been extended to the study of protein activities (8,9) and whole tissues (10). In most cases, the microarray format involves biomolecules labeled with fluorophores and constrained on dried surfaces.
The desire to quantitate the fluorescence intensity from fluorophores at surfaces requires a thorough understanding of the surface effect on the photophysical properties of fluorophores. Spectral shifts, quantum yield changes, and absorbance changes are some of the properties that could be modified and affect quantitation of fluorescence intensity. In this work, we have measured the absorption and emission spectra of Alexa™ 488 and Cy™5 confined on a glass surface and discuss possible ramifications for fluorescence intensity quantitation. We use conventional fluorescence techniques as well as spectroscopic ellipsometry, which has been traditionally used for the study of thin film optical constants (11). With proper accounting of the glass surface, the ellipsometer data clearly identifies the surface-attached fluorophore electronic transitions and provides a method for estimating the effects of the surface on the fluorophore electronic states.Materials and Methods Preparation of Slides
UltraGAPS™ slides obtained from Corning (Corning, NY, USA) and SuperChip™ slides obtained from Erie Scientific (Portsmouth, NH, USA) were used for immobilization of two fluorophores, Alexa 488 (Molecular probes, Eugene, OR, USA) and Cy5 monofunctional Dye (Amersham Biosciences, Piscataway, NJ, USA). Both dyes had succinimidyl ester groups to facilitate covalent immobilization to the amine groups on the glass surface. The immobilization was performed for 1 h with approximately 1×10-3 mol/L dye dissolved in 0.1 mol/L sodium carbonate buffer, pH 8.9. After the reaction, the slides were washed intensively with the immobilization buffer and then with 0.1 mol/L phosphate-buffered saline (PBS), pH 7.2. Finally the slides were rinsed thoroughly with water and dried in desiccators overnight.Spectroscopic Ellipsometer Measurements
Ellipsometry is a technique that directly measures changes in light polarization parameters upon specular reflection from the surface (12). The reflecting surface is modeled as a stratified system, and Fresnel equations are used to calculate the thickness and optical constants of each layer. Optical constants of films of immobilized fluorophores were measured in the air, using a Model M2000 rotating compensator spectroscopic ellipsometer (J.A. Wollam, Lincoln, NE, USA). Before fluorophore deposition, bare glass slide index of refraction was determined at several spots. We then measured the thickness and real index of refraction of the silane coating. Finally, ellipsometric parameter (Δ and Ψ) readings in the range 700–800 nm were used to determine the film thickness of the layer containing the fluorophores. We assumed that the index of refraction, n, can be treated as a real quantity far from the fluorophore absorbance band and that the absorbance, k, is significant only in a small region of wavelengths around the fluorophore absorbance band. The average thickness for Alexa 488 was 4 nm and 5.3 nm for Cy5. Both components of the fluorophore complex refractive index, n+ik, were obtained in the wavelength range of 400–800 nm using a three-layer model (glass-silane film-fluorophore containing film).Fluorimeter Measurements
The fluorescence measurements were made with a Fluorolog®-3 spectrofluorometer from Jobin Yvon (Edison, NJ, USA) equipped with a sample turret for the surface measurements. The slides were placed so that the excitation beam reached the slides at a 60° angle relative to the surface normal. The background from unlabeled slides was subtracted from the signal from the labeled slides.
For the solution measurements, the deactivated Alexa 488 and Cy5 that were used for immobilization of fluorophores on surfaces was diluted in 0.1 M PBS, pH 7.2. The deactivation consisted of leaving the two fluorophores in an aqueous environment overnight. The concentration of both fluorophore solutions was approximately 0.6 µmol/L.