Hemoglobin adsorption isotherm at the silica-water interface with evanescent wave cavity ring-down spectroscopy

Abstract

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is used to observe the adsorption isotherm for hemoglobin (Hb) from controlled urine samples to assess the potential for rapid diagnosis in hemoglobinuria. The absorbance of Hb at 425 nm is monitored using an alexandrite laser-pumped, room temperature, LiF:F2+** color-center pulsed laser. A minimum absorbance detection level of 2.57 x 10(-4) is achieved, corresponding to a minimum detectable concentration of Hb in urea of 5.8 nM. A multilayered Hb biofilm is formed, and a minimum of eight layers are required to model the adsorption isotherm, allowing for cooperative binding within the layers and extending 56 nm into the interface. A binding constant for Hb to silica 18.23+/-7.58 x 10(6) M is derived, and a binding constant for Hb to Hb in subsequent layers is determined to be 5.631+/-0.432 x 10(5) M. Stoichiometric binding coefficients of 1.530+/-0.981 for layer one and 1.792+/-0.162 for subsequent layers suggest that cooperative binding both to the silica surface and between the layers of the biofilm is important.

Evanescent-Wave Cavity Ring-Down Spectroscopy for Enhanced Detection of Surface Binding under Flow Injection Analysis Conditions

“…Furthermore, the adsorption isotherm of hemoglobin from urine samples was determined in order to assess the feasibility of using EW-CRDS diagnosis in hemoglobinuria. 16 Adsorption of a charged and an uncharged dye at the silica/CH 3 . Everest et al used a custom-made Dove prism with entrance and exit faces at normal incidence.…”

Liquid-Phase and Evanescent-Wave Cavity Ring-Down Spectroscopy in Analytical Chemistry

“…As discussed above, aspecific EW-CRDS biosensing has already been demonstrated (13). This study exploits the fact that the positively charged hemoglobin molecules in a urine sample attach to the bare silica surface.…”

“…A potential medical application of EW-CRDS is the rapid diagnosis of hemoglobinurea by measuring the adsorption of hemoglobin, which is present in the urine of hemoglobinurea patients, to the silica interface. Using this technique, one can detect concentrations of hemoglobin as small as 6 nM (13). An elegant way to probe the interfacial pH (which may be different from its bulk value) is through the covalent attachment of a pH-sensitive dye containing a triethoxysilane group to the silica surface.…”

“…As with liquid-phase CRDS, the development of EW-CRDS has only recently begun. EW-CRDS has shown its potential in various studies of interactions of analytes with bare silica surfaces (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Modification of the TIR surface (19) may become a useful tool for studying (bio)molecular interactions and in label-free biosensing, analogous to, for example, surface plasmon resonance spectroscopy.…”

Evanescent-Wave Cavity Ring-Down Spectroscopy for Enhanced Detection of Surface Binding under Flow Injection Analysis Conditions

“…Until now, liquid-phase EW-CRDS studies have included steady-state measurements 16,21 or measurements with an extremely low continuous flow (on the order of mL/h) of sample in a 190 lL sized flow cell. 14,15 The experimental conditions were such that adsorption equilibrium could be reached and thermodynamic properties could be measured; reversibility was not a critical factor.…”