Optical Detection of Streptavidin-Biotin Binding on Nanoporous Anodic Alumina Pore Surface
Authors: Pol Laura , Ferre-Borrull Josep , Xifre-Perez Elisabet , Pallares Josep , Marsal Lluis F. 
Published: July 2021

Nanoporous anodic alumina (NAA) is a material with a high interest in nanotechnology[1]. Its physical and chemical properties combined with a cost-effective and scalable production process make it a good candidate in many nanotechnologically-related applications. Its high and tuneable surface-to-volume ratio as well as its interesting optical properties[2] have been used as the basis of different biosensing schemes[3]. Biosensing, and specifically optical biosensing is of great interest in health and environmental applications. The reflection interference spectroscopy (RIfS) method based on NAA has demonstrated its ability in detecting many kinds of molecules (proteins, antibodies[4]).
One of the molecular systems with interest in biotechnological applications is the streptavidin-biotin system. The high affinity between both molecules is exploited in many applications for the binding of different biological species. For this reason, we have studied the streptavidin-biotin binding event with NAA-based RIfS, as part of a biosensing process. In our study we assess the optimal conditions for real time monitoring of the different steps (covalent streptavidin bonding, surface passivation, biotin complexation). In order to establish such optimal conditions, we have chosen the protein thrombin as a model molecule to be detected.
We prepared NAA with the usual anodization conditions under oxalic acid electrolyte to obtain porous layers with uniform pore diameter and with a thickness that permits the measurement with RIfS in a flow-cell where the different species in solution can be introduced. The experiments consisted of monitoring the change in effective optical thickness (EOT, the quantitative characteristic parameter obtained from RIfS) in the different steps of the biosensing process. The NAA pore surfaces were initially functionalized with (3-aminopropyl)triethoxysilane (APTES) with the carboxyl groups activated in order to form later a peptide bond with the streptavidin.
One of the parameters to be optimized was the pore diameter. Furthermore, in order to study the ability of RIfS to detect the covalent streptavidin binding to the NAA pore surface, different concentrations of streptavidin were tested. Finally, the biosensing capabilities were evaluated by detecting biotinylated thrombin at different concentrations.
An example of a complete experiment with all the steps in the streptavidin-biotinylated molecule attachment detection can be seen in Figure 1. After establishing a baseline with a PBS flow, streptavidin at 50 µg/ml is introduced in the RIfS setup and its covalent attachment to APTES is detected as an increase in EOT. Following this first step, PBS is introduced to wash away the non-specifically bound molecules and a 3% Bovine Serum Albumin (BSA) solution is flowed to passivate the remaining activated carboxyl groups of APTES and the remaining hydroxyl groups on the NAA pore surface. Such steps are detected by the RIfS procedure. Then, after a subsequent PBS wash, biotinylated thrombin at a concentration of 20 µg/ml is introduced causing a steady increase in EOT reaching a value of 3.5 nm above the initial one. The experiment in Figure 1 was repeated for different NAA pore sizes revealing a higher change in EOT for bigger pore sizes.
An important parameter in the detection of the biotinylated molecules binding is the concentration at the streptavidin immobilization step. Thus, different streptavidin concentrations were tested in 60 nm pore diameter NAA. Results are summarized in Figure 2: for the highest concentrations (50 µg/ml) the attachment of streptavidin shows a two-phase curve with an initial phase with a faster increase in EOT and a second phase with a slower increase after an inflection point in the EOT versus time curve. Instead, for a small concentration (1 µg/ml) an initial fast increase is followed by a stabilization of the EOT signal. These two behaviors are associated with the attachment of streptavidin to the activated carboxyl of APTES for the initial fast EOT increase, while the subsequent slower increase can be explained by the aggregation of streptavidin through amine group binding.
Finally, in order to demonstrate the capability of quantifying the attachment of biotinylated molecules to the NAA surface, the process was carried out using thrombin as the model molecule and for increasing concentrations. Figure 3 shows the change in EOT as a function of the thrombin concentration. It can be seen that RIfS based on NAA sensing platforms is able to quantify such attachment.
[1] Josep Ferré-Borrull, et al., Nanomaterials, vol. 7, p. 5225 (2014).
[2] Josep Ferré-Borrull et al., in NANOPOROUS ALUMINA: FABRICATION, STRUCTURE, PROPERTIES AND APPLICATIONS, Springer Series in Materials Science, vol. 219, p. 185 (2015).
[3] Chris Eckstein, ACS Applied Materials and Interfaces, vol. 10, p.10571 (2018).
[4] Gerard Macías, Analyst, vol. 140, p. 4848 (2015).

Figure 1