Abstract
Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 μm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls.
Highlights
Biosensors based on the specific binding of proteins on receptor-functionalized surfaces are among the most widespread analytical tools in biorecognition research.1À5 The goal is either to quantify the interaction with the surface in terms of rate constants or association/dissociation constants to determine the protein concentration in the bulk phase
We show that the dominant linear behavior of the time-resolved change in surface concentration can be explained in terms of boundary layer theory,[38,40] i.e., adsorption kinetics are predominately driven by the flux into the pores
The increasing interest in the applications of nanoporous media in biosensor research motivated our studies of the adsorptionÀdesorption kinetics of proteins in porous anodic aluminum oxide (AAO)
Summary
All FES were carried out using COMSOL within an experimentally accessible parameter space. 0.5 nm, averaged over several experiments (n = 20) and assuming εprotein = 2.1 This corresponds to about 50% surface coverage with avidin with dimensions of 4.0 Â 5.5 Â 6.0 nm3.45 We corroborated the OWS adsorption kinetics (Figure 4A,B) with time-resolved confocal laser scanning fluorescence microscopy. Desorption kinetics bears much richer physics and allows, in principle, one to obtain both kad and kdes from flushing the surface with protein-free buffer solution It requires, numeric solutions of the governing mass balance equation due to the absence of a stationary gradient. Sensing schemes that rely on the pore exit in response to external stimuli can be an effective method of utilizing the advantages of a nanoporous substrate for practical applications
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