The chemical modification, or functionalization, of the surfaces of nanomaterials is a key step to achieve biosensors with the best sensitivity and selectivity. The surface modification of biosensors usually comprises several modification steps that have to be optimized. Real-time monitoring of all the reactions taking place during such modification steps can be a highly helpful tool for optimization. In this work, we propose nanoporous anodic alumina (NAA) functionalized with the streptavidin-biotin complex as a platform towards label-free biosensors. Using reflective interferometric spectroscopy (RIfS), the streptavidin-biotin complex formation, using biotinylated thrombin as a molecule model, was monitored in real-time. The study compared the performance of different NAA pore sizes in order to achieve the highest response. Furthermore, the optimal streptavidin concentration that enabled the efficient detection of the biotinylated thrombin attachment was estimated. Finally, the ability of the NAA-RIfS system to quantify the concentration of biotinylated thrombin was evaluated. This study provides an optimized characterization method to monitor the chemical reactions that take place during the biotinylated molecules attachment within the NAA pores.

BackgroundThe ability to direct the cellular response by means of biomaterial surface topography is important for biomedical applications. Substrate surface topography has been shown to be an effective cue for the regulation of cellular response. Here, the response of human aortic endothelial cells to nanoporous anodic alumina and macroporous silicon with collagen and fibronectin functionalization has been studied.MethodsConfocal microscopy and scanning electron microscopy were employed to analyse the effects of the material and the porosity on the adhesion, morphology, and proliferation of the cells. Cell spreading and filopodia formation on macro- and nanoporous material was characterized by atomic force microscopy. We have also studied the influence of the protein on the adhesion.ResultsIt was obtained the best results when the material is functionalized with fibronectin, regarding cells adhesion, morphology, and proliferation.ConclusionThese results permit to obtain chemical modified 3D structures for several biotechnology applications such as tissue engineering, organ-on-chip or regenerative medicine.

The fluid imbibition-coupled laser interferometry (FICLI) technique has been applied to detect and quantify surface changes and pore dimension variations in nanoporous anodic alumina (NAA) structures. FICLI is a noninvasive optical technique that permits the determination of the NAA average pore radius with high accuracy. In this work, the technique is applied after each step of different surface modification paths of the NAA pores: (i) electrostatic immobilization of bovine serum albumin (BSA), (ii) covalent attachment of streptavidin via (3-aminipropyl)-triethoxysilane and glutaraldehyde grafting, and (iii) immune complexation. Results show that BSA attachment can be detected as a reduction in estimated radius from FICLI with high accuracy and reproducibility. In the case of the covalent attachment of streptavidin, FICLI is able to recognize a multilayer formation of the silane and the protein. For immune complexation, the technique is able to detect different antibody-antigen bindings and distinguish different dynamics among different immune species.

Aptamer biosensors are one of the most powerful techniques in biosensing. Achieving the best platform to use in aptamer biosensors typically includes crucial chemical modifications that enable aptamer immobilization on the surface in the most efficient manner. These chemical modifications must be well defined. In this work we propose nanoporous anodic alumina (NAA) chemically modified with streptavidin as a platform for aptamer immobilization. The immobilization of biotinylated thrombin binding aptamer (TBA) was monitored in real time by means of reflective interferometric spectroscopy (RIfS). The study has permitted to characterize in real time the path to immobilize TBA on the inner pore walls of NAA. Furthermore, this study provides an accurate label-free method to detect thrombin in real-time with high affinity and specificity.

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