Abstract
Nanoporous anodic alumina (NAA) attracts interest in nanotechnology[1]. Its physical and chemical properties combined with a cost-effective and scalable production make it a good for nanotechnology-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. 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 [3,4].
Biosensors engineered with aptamers as a bioreceptor are called aptabiosensors. Thrombin Binding Aptamer (TBA) has a well-known binding process and high affinity and is one of the most studied aptamers. Thrombin is the key factor of blood coagulation, whose activity is important in wounds and in blood circulation. The free thrombin-binding aptamer remains as a random-coil state in the absence of thrombin, while in the presence of thrombin the protein attaches to the aptamer changing its conformation to quadruplex. In this study we assess the capabilities of NAA with the inner pore surfaces modified with TBA to detect specifically the thrombin protein by means of the RIfS method.
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. Figure 1 shows SEM pictures of the NAA platforms prepared.
The NAA pore surfaces were initially functionalized with (3-aminopropyl)triethoxysilane (APTES) and then the surface mas modified in a three-step procedure monitored in real time thanks to the RIfS method. Figure 2 shows the change in EOT as a function of time during the surface functionalization of NAA: the covalent attachment of Sulfo-NHS-Biotin and subsequent streptavidin attachment (stages 1 and 2 in the figure) and the final immobilization of TBA in the pore walls of NAA (step 3 in the plot).. First, the flow of the Sulfo-NHS-Biotin solution produced a considerable increase in EOT. Then, a further increase in EOT is observed with the infiltration of streptavidin. Finally, EOT also shows clearly the immobilization of biotinylated TBA.
The aptamer-functionalized NAA substrates were employed to detect human thrombin protein. For this purpose, different substrates were used in RIfS experiments with human thrombin protein at different concentrations. Figure 3 shows one example of the evolution of the EOT signal upon infiltration of a 1.35 µM thrombin solution. Results show that after obtaining a baseline with constant EOT corresponding to the flow of binding buffer, the thrombin solution is injected causing a rapid increase in EOT, at a rate of 0,05 nm/s. After this increase a stable value is reached after 3600 s, with an absolute change in EOT of ∆EOT = 28 nm. The experiment corresponding to Figure 3 has been repeated for a set of concentrations to obtain a sensitivity curve of the aptamer-functionalized NAA and to determine the lower limit of detection. With this, the dissociation constant of the aptamer-thrombin reaction is determined as Kd = 0,9 µM, the sensitivity to be m = 45.5 nm/µM and the LOD = 72 nM.
Finally, in order to demonstrate the specificity of the NAA platform, experiments replacing the flow of thrombin by the flow of a different but similar protein, Bovine Serum Albumin (BSA) were conducted showing a much lower response. Furthermore, thrombin flow experiments with NAA lacking the aptamer functionalization step were also performed to demonstrate the protein does not bind non-specifically to the NAA inner pore walls.
[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] Laura Pol et al., Nanomaterials, vol. 9, p.478 (2019).
[4] Laura Pol et al, Sensors, vol. 19, p. 4543 (2019).
Figure 1