Fluid imbibition-coupled laser interferometry (FICLI)[7] is an optofluidic technique that permits the investigation of the surface and geometrical properties of nanoporous structures by non-invasive optical methods. It consists of the measurement of interferometric light intensity fluctuations in a beam reflected in a nanoporous thin film membrane as it is being filled by a liquid. In FICLI, time-resolved interferograms are measured and then analyzed on the basis of mathematical models of the filling kinetics. With this the surface properties and the pore internal geometries can be estimated.
Recently, we established for FICLI an accuracy better than 2 nm in determining changes in NAA pore radius after different modifications involving radius increase (by chemical wet etching) or decrease (by the deposition of layer-by-layer coatings). Such accuracy leads to consider NAA and FICLI as a platform to develop biosensing systems. Such application depends critically on the functionalization of the pore surface, as it is necessary to enable the selective immobilization of the desired biomolecules.
In this work, we investigate the influence of three different NAA functionalization methods on the FICLI pore radius estimate: i) electrostatic binding of bovine serum albumin (BSA), ii) covalent binding for the attachment of streptavidin and biotin and iii) specific immune complexation of antibody-antigen pairs. The study was carried on NAA membranes produced in both oxalic and sulfuric acid electrolytes, and together a chemical wet etching step for a given amount of time (between 0 min. and 24 min.) permits to use a wide range of pore dimensions (between 15 nm and 44 nm). NAA inner pore surface was modified in different steps and the FICLI technique was applied to estimate the changes in filling dynamics and the consequent pore radius change after each step. For the electrostatic attachment of BSA, NAA membranes with different pore radius were used as-produced and measured before and after incubation in a BSA solution. For the covalent attachment of streptavidin, NAA samples with 40 nm initial pore radius were investigated after two steps: i) silanization with (3-aminopropyl)-triethoxysilane (APTES) with cross-linking activation with glutaraldehyde (GTA) and ii) incubation with streptavidin. Finally, immune complexation within NAA pores was tested on NAA samples with 40 nm initial pore radius attaching different kinds of immunoglobulin (IgG) to the inner pore surface. Several experiments were conducted, in all of them the first step was to attach electrostatically protein A, which has the ability to link with antibody or antigen portions. Then, samples were incubated following one of three possibilities: i) human IgG, ii) anti-human IgG and iii) anti-goat IgG from rabbit followed by incubation with goat IgG.
Results show that for the case of the electrostatic attachment, FICLI is able to detect the formation of a BSA layer on the NAA pores, independently of the initial pore radius with slight differences observed for different electrolyte used in the preparation. For covalent immobilization of streptavidin, FICLI estimates a much bigger radius reduction than expected from the molecule size, what can be explained by a multi-layer deposition of streptavidin. Finally, FICLI is able to demonstrate that different IgG attach differently to the NAA pores with a determinant influence of the pore size.
ACKNOWLEDGMENT
This work was supported by the Spanish MINECO (TEC2015-71324-R), the Catalan authority AGAUR (2014SGR1344), and ICREA under the ICREA Academia Award.
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