Nanoporous Anodic Alumina Pores Research Articles

Optical Detection of Streptavidin-Biotin Binding on Nanoporous Anodic Alumina Pore Surface

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

Nanoporous Anodic Alumina As a Three-dimensional Nanostructured material for the Remote Optical Sensing of Urea

Millions of people in the world suffer from some form of kidney disease [1]. Due to reduce the economic losses caused by this disease and also to improve the public health, the fabrication of a device to diagnose kidney disease is important for every government. Up to now, several biosensors have been reported for the determination of urea [2,3]. However, no of them is remote biosensor. The most advantage of the remote sensor is, analyzing the real samples without getting any fungal/virus/bacterial infection that can be in it. Infection. The interferometric reflectance spectroscopy (IRS) is one of the remote sensing devices. IRS is a physical method based on the interference of white light at porous thin substrates, which is used to investigate molecular interaction. This investigation is detected by the charge-coupled device (CCD) detector. Macroporous silicon (MPS) and nanoporous anodic alumina (NAA) are two of the interesting porous thin substrates for the fabrication of biosensor based on IRS [4-6]. However, NAA has become gradually influential in the IRS biosensors due to its high surface area, biocompatibility, easy functional ability, versatile, durable and low-cost substrate.Herein, we fabricated a remote optical biosensor for the determination of urea. For this purpose first, the self-orderednanoporous anodic alumina (NAA) was fabricated by electrochemical anodization of aluminum substrates in a two-step anodization process [7, 8]. The morphological characterization of the NAA was examined by SEM. The surface of NAA has a hexagonal multi-pore structure. The average diameter pore size of NAA was 41 nm. Then, the NAA pore walls were functionalized with 3-aminopropyl tri-methoxy silane (NAA-NH2). After that, the urease enzyme was immobilized to the pore walls of NAA-NH2 by the use of glutaraldehyde as cross-linker. After that, fluorescein 5(6)-isothiocyanate (FLITC) as the pH-sensitive optical probe was attached to urease enzyme to fabricate a novel urea and trypsin biosensor. The operation principle of the proposed biosensor is based on a change in the pH of the solution during the reaction of urease and urea and then change in the absorption properties of the FLITC. The reaction of urease with urea lets to increase the pH of its environment because of producing ammonia, which is basic. So, the absorption property of FLITC that is a pH-sensitive opt-probe will increase by increasing the urea in solution due to the increase of pH solution during urease/urea reaction. Consequently, the residence of light intensity that will be detected by the CCD detector in the presence of urea is less than in the absence of it. The proposed biosensor exhibited a good response to the concentration of urea in the range of 0.12 to 3.0 mM. The limit of detection (LOD) for urea was 0.06 mM (Fig.1).To examine its applicability in real sample analysis, the urease biosensor was used for the determination of urea in normal human urine with the standard addition method. Briefly, 1.0 mL of the filtered urine solution added to 9.0 mL of NaAC solution (0.1 M, pH 6.4) and then the solution was pumped into the analytical cell to be analyzed by NAA-NH-GLA-urease-FLITC. The concentration of urea was determined to be 192 mM. The recovery of the analysis was 92 %, considering the value determined by the fluorescent-based sensor [9].The NAA-NH-GLA-urease-FLITC exhibited good reproducibility in the detection of urea. The effect interfering compounds were also studied on the response of biosensor. No interference was observed with 0.1 mM of L-cysteine, dopamine, glucose, and nicotinamide adenine dinucleotide and common cations and anions (0.1 mM of Na+, Al+3, K+, Cl-, NO3 -, SO4 -2).The proposed biosensor exhibited good selectivity, linear range responsibility, and stability.References(1) https://www.worldkidneyday.org/2019-campaign/2019-wkd-theme/.(2) Mishra, G.K., Mishra, R.K., Bhand, S., 2010. Biosens Bioelectron.26, 1560-1564.(3) Parashar, U.K., Nirala, N.R., Upadhyay, C., Saxena, P.S., Srivastava, A., 2015. Appl Biochem Biotechnol. 176, 480-492.(4) Amouzadeh Tabrizi, M., Ferré-Borrull, J, Marsal, L. F., 2019. Biosens Bioelectron 137, 279-286.(5) Amouzadeh Tabrizi, M., Ferré-Borrull, J, Marsal, L. F., 2020. Biosens Bioelectron 137, 111828.(6) Ferré-Borrull, J, Pallarès, J, Macías, G, Marsal, L. F., 2014 Materials 7, 5225-5253.(7) Santos, A., Vojkuvka, L., Alba, M., Balderrama, V. S., Ferré‐Borrull, J, Pallarès, J., Marsal, L.F., Physica Status Solidi (a) 209, 2045-2048.(8) Santos, A., Macías, G., Ferré-Borrull, J., Pallarès, J., Marsal, L.F., 2012. Appl. Mater. Interfaces 4, 3584-3588.(9) Deng, H.-H., Li, K.-L., Zhuang, Q.-Q., Peng, H.-P., Zhuang, Q.-Q., Liu, A.-L., Xia, X.-H., Chen, W., 2018. Nanoscale 10, 6467-6473. Figure 1

Optical Detection of Thrombin on Nanoporous Anodic Alumina with Aptamer-Modified Surface

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

An optical biosensor for the determination of cathepsin B as a cancer-associated enzyme using nanoporous anodic alumina modified with human serum albumin-thionine.

An interferometric reflectance spectroscopy-based biosensor for the determination of cathepsin B (Cat B) as a cancer-related enzyme has been fabricated. For this purpose, the nanoporous anodic alumina (NAA) was fabricated electrochemically. The NAA was then modified with the amino-silane coupling agent. After that, human serum albumin (HSA) was immobilized into the NAA pores by using glutaraldehyde as a cross-linking agent. Subsequently, the carboxylic group of HSA was activated with N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to attach to thionine (TH) as a photoprobe to fabricate the labeled HSA (HSA-TH). HSA-TH plays a significant role in this sensor to determine cathepsin B as a model analyte for the development of theinterferometric reflectance spectroscopy-based biosensor for the measurement of protease. The attached TH adsorbed the illuminated white light on NAA modified with HSA-TH. Therefore, the intensity of the reflected light to the charge-coupled device (CCD) detector decreased in the wavelength range 450-1050nm. In the presence of Cat B, HAS-TH cleaved into short peptide fragments and washed away by flow cell system. Since TH was removed from NAA, the intensity of the reflected light increased. The peak area has a logarithmic relationship with the concentration of Cat B in the range 0.5 to 64.0nM. The limit of detection of the biosensor sensor was 0.08nM. The optical sensor was used for the determination of Cat B in a human serum sample. Graphical abstract Schematic presentation of biosensor for the determination of the cathepsin B which is based on nanoporous anodic alumina modified with HSA-thionine. The principle response of the optical biosensor is based on detecting changes in the intensity of the reflected light after cleaving the immobilized HSA-thionine by cathepsin B into short peptide fragments.

Experimental study and modeling of in vitro agrochemicals release from nanoporous anodic alumina

In this study, the ability of nanoporous anodic alumina (NAA) for the controlled release of agrochemicals including indole-3-acetic acid (IAA) and bentazon was investigated. This study was divided into two works. In the first work, the release of IAA loaded into the NAA pores was studied, and a new two-stage mechanism was proposed for the description of this release process. The first stage was modeled using the diffusion–dissolution equation with a moving boundary condition, while the second stage was correlated by the diffusion–dissolution equation with fixed boundary conditions. The proposed model predicts that the release rate in the first stage is first order when the pore diameter is small and is proportional to the reciprocal of the square root of time when the pore diameter is large. The results of the model also demonstrate that in the second stage, the increase in the pore diameter, at first, leads to an enhancement of the release rate; while a further increase in the pore diameter results in a decrease of release rate. In the second work of this study, NAA was used as a barrier membrane to form a reservoir system for the release of bentazon. The experimental data of this study were well fitted to the first-order release model. The results of this model confirm that an increase in the pore diameter leads to an enhancement of the release rate, while an opposite trend was found with increasing the pore length.

Highly sensitive IRS based biosensor for the determination of cytochrome c as a cancer marker by using nanoporous anodic alumina modified with trypsin

The determination of cytochrome c in the human serum sample is a regular medical investigation performed to assess cancer diseases. Herein, we used interferometric reflectance spectroscopy (IRS) based biosensor for the determination of cytochrome c. For this purpose first, the nanoporous anodic alumina (NAA) was fabricated. Then, the NAA pore walls were functionalized with 3-aminopropyl trimethoxy silane (NAA-NH2). Subsequently, the trypsin enzyme was immobilized on the NAA pore walls. The sensing principle of proposed IRS sensor to cytochrome c is based on a change in the intensity of the reflected light to a charge-coupled device (CCD) detector after digesting of cytochrome c by immobilized trypsin enzymes on NAA-NH2 into the heme-peptide fragment. The heme-peptide fragment then oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to green color ABTS·− anion radical in the presence of hydrogen peroxide. The generated green color ABTS·− anion radical solution adsorbed the white light and therefore the intensity of the reflected light from NAA to the CCD decreased. The decrease in the intensity of the white light had a logarithmic relationship with the concentration of the cytochrome c in the range of 1–100 nM. The limit of detections (LOD) for cytochrome c was 0.5 nM. The proposed biosensor exhibited high selectivity, sensitivity, and good stability.

(Invited) Optical Detection of Streptavidin-Biotin Binding on Nanoporous Anodic Alumina Pore Surface

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 tunable 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 60nm 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., Materials, 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, et al. ACS Appl Mater Interfaces, vol. 10, p.10571 (2018). [4] Gerard Macías, et al. Analyst, vol. 140, p. 4848 (2015). Figure 1

Selective and Sensitive Probe Based in Oligonucleotide-Capped Nanoporous Alumina for the Rapid Screening of Infection Produced by Candida albicans.

A robust, sensitive, and time-competitive system to detect Candida albicans in less than 30 min in clinical samples based in capped nanoporous anodic alumina (NAA) is developed. In the proposed design, NAA pores are loaded with rhodamine B and then blocked with an oligonucleotide that is able to recognize C. albicans DNA. The capped material shows negligible cargo release, whereas dye delivery is selectively accomplished when genomic DNA from C. albicans is present. This procedure has been successfully applied to detect C. albicans in clinical samples from patients infected with this yeast. When compared with classical C. albicans detection methods, the proposed probe has a short assay time, high sensitivity and selectivity, demonstrating the high potential of this simple design for the diagnosis of infection produced by C. albicans.

Real-Time Monitoring of Biotinylated Molecules Detection Dynamics in Nanoporous Anodic Alumina for Bio-Sensing.

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.

Fluid-Imbibition Coupled Interferometry Study of Surface Modifications in Nanoporous Anodic Alumina for Biosensing

Nanoporous anodic alumina (NAA) is a material with great perspectives in nanotechnology due to its cost-effective fabrication, its chemical stability and its versatility in applications such as biosensing or drug delivery[1]. Optical sensing with NAA has gained much attention in recent years[2,3]. Nano-optofluidic methods[4] based on a solid carrier matrix, a liquid carrying analytes and on the physico-chemical characteristics at their interfaces have been proven sensitive for substances as small as nanoparticles, viruses[5] and even ions[6]. 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. REFERENCES [1] J. Ferré-Borrull, E. Xifré-Pérez, J. Pallarès and L.F. Marsal in “Nanoporous Alumina: Fabrication, Structure, Properties and Applications”; Santos, A., Losic, D., Eds.; Springer-Verlag Berlin, 2015, p. 185. [2] G. Macias, J. Ferré-Borrull, J. Pallarès and L.F. Marsal, Analyst 140, 2015, p. 4848. [3] T. Kumeria, A. Santos, M.M. Rahman, J. Ferré-Borrull, L.F. Marsal, D. Losic, ACS Photonics 1, 2014, p. 1298. [4] D. Psaltis, S.R. Quake, C.H. Yang, Nature 442, 2006, p. 381. [5] A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, L. Novotny, ACS Nano 23, 2010, p. 1. [6] T. Kumeria, M.M. Rahman, A. Santos, J. Ferré-Borrull, L.F. Marsal, D. Losic, ACS applied materials & interfaces 6, 2014, p. 12971. [7] C. Eckstein, E. Xifré-Pérez, M. Porta-i-Batalla, J. Ferré-Borrull, L.F. Marsal, Langmuir 32, 2016, p. 10467.

Photonic Properties of Structurally-Engineered Nanoporous Anodic Alumina and Application to Biosensing

Nanoporous anodic alumina (NAA) is a material with growing interest in nanotechnology because of its high versatility and ease of preparation[1]. By electrochemical etching of aluminum in acidic electrolytes and under the adequate conditions of composition, anodization voltage or current and temperature, the NAA pores grow perpendicular to the aluminum surface in a self-arranged matrix. Pore radius and interpore distances can be modulated in the range of a few tens of nanometers and up to a few hundreds. A wide range of applications has been demonstrated in sensing[2], drug delivery[3], cell culture[4] or photovoltaics[5]. Technologies for NAA have evolved to tailor material properties to the desired applications, specifically photonic properties with a view to sensing[6]. These structures are applied to analytes such as glucose, proteins or antibodies, usually accomplished by reflection interference spectroscopy. This technique permits to monitor real-time changes in the optical properties of the NAA as they are infiltrated by solutions in a flow cell. Photonic properties are obtained by engineering the pore shape. For instance, by combining sequentially anodization steps with wet etching steps, two-layer systems can be obtained that provide self-calibration in sensing[7]. On the other hand, periodic modulation of the applied anodization voltage between two different values leads to periodic in-depth variations of pore morphology, and consequently to the formation of successive layers with different effective refractive index giving rise to distributed-Bragg reflectors (DBRs)[8]. Precise control of the anodization times for each voltage level and temperature permits to obtain high refractive index contrasts between the different layers in the DBR and consequently, wide photonic stop bands at the desired wavelengths in the spectrum. In this communication we report the more recent results in biosensing with NAA-engineered structures, focusing on NAA-based rugate filters (NAA-RF). These are obtained by the application of a periodic sinusoidal anodization current, composed of a constant offset current together with a sinusoidal component, which produce an in-depth continuous periodic variation in pore diameter. The consequent continuous variation in refractive index results in a 1-D photonic crystal structure with a stop band tunable within the UV-VIS-IR range. Figure 1 shows a SEM cross-section of a NAA-RF, where the pore modulation can be recognized. It has been shown that different sinusoidal components can be overlapped in the anodization current profile[9]. With this, photonic structures with several stop bands can be produced. Furthermore, such stop bands can be tailored at any position in the spectrum what permits to propose new applications such as information encoding. Nevertheless, this overlap of sinusoidal components has the drawback that the refractive index contrasts that can be achieved have to be shared among the different sinusoidal components, what results in stop bands with low reflectance. In this work, we study the preparation of NAA-RF based on the successive application of the different sinusoidal components. We call these structures stacked NAA-RF, since the different photonic crystals grow separately in the NAA. Figure 2 shows three selected sections of the applied sinusoidal current and of the measured electrochemical cell voltage. Figure 3 shows the spectrum resulting from such applied anodization current. Three clear stop bands with reflectances as high as 40% can be observed. In the communication we will report our results on the tunability of the stop bands and its sensitivity in biosensing applications. ACKNOWLEDGMENT This work was supported by the Spanish MINECO (TEC2015-71324-R), the Catalan authority AGAUR (2014SGR1344), and ICREA under the ICREA Academia Award. REFRENCES [1] J. Ferré-Borrull, J. Pallarès, G. Macias and L.F. Marsal, Materials 7, 2014, p. 5225. [2] G. Macias, J. Ferré-Borrull, J. Pallarès and L.F. Marsal, Analyst 140, 2015, p. 4848. [3] M. Alba, B. Delalat, P. Formentin, M.L. Rogers, L.F. Marsal and N. Voelcker, Small 11, 2015, p. 4626. [4] P. Formentin, U. Catalan, M. Alba, S. Fernandez-Castillejo, R. Sola, J. Pallares and L.F. Marsal, New Biotechnology 8, 2017, p. 675. [5] V.S. Balderrama, J. Albero, P. Granero, J. Ferre-Borrull, J. Pallares, E. Palomares and L.F. Marsal, Nanoscale 7, 2015, p. 13848. [6] J. Ferré-Borrull, E. Xifré-Pérez, J. Pallarès and L.F. Marsal in Nanoporous Alumina: Fabrication, Structure, Properties and Applications; Santos, A., Losic, D., Eds.; Springer-Verlag Berlin, 2015, p. 185. [7] G. Macias, L.P. Hernandez-Eguia, J. Ferré-Borrull, J. Pallarès and L.F. Marsal, ACS Applied Materials and Interfaces 5, 2013, p. 8093. [8] M. M. Rahman, L.F. Marsal, J. Pallares and J. Ferré-Borrull, ACS Applied Materials and Interfaces 5, 2013, p. 13375. [9] A. Santos, C.S. Law, T. Pereira and D. Losic, Nanoscale 8, 2016, p. 8091. Figure 1

Nanoporous Anodic Alumina Surface Modification by Electrostatic, Covalent, and Immune Complexation Binding Investigated by Capillary Filling.

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.

Fabrication and Optimization of Bilayered Nanoporous Anodic Alumina Structures as Multi-Point Interferometric Sensing Platform.

Herein, we present an innovative strategy for optimizing hierarchical structures of nanoporous anodic alumina (NAA) to advance their optical sensing performance toward multi-analyte biosensing. This approach is based on the fabrication of multilayered NAA and the formation of differential effective medium of their structure by controlling three fabrication parameters (i.e., anodization steps, anodization time, and pore widening time). The rationale of the proposed concept is that interferometric bilayered NAA (BL-NAA), which features two layers of different pore diameters, can provide distinct reflectometric interference spectroscopy (RIfS) signatures for each layer within the NAA structure and can therefore potentially be used for multi-point biosensing. This paper presents the structural fabrication of layered NAA structures, and the optimization and evaluation of their RIfS optical sensing performance through changes in the effective optical thickness (EOT) using quercetin as a model molecule. The bilayered or funnel-like NAA structures were designed with the aim of characterizing the sensitivity of both layers of quercetin molecules using RIfS and exploring the potential of these photonic structures, featuring different pore diameters, for simultaneous size-exclusion and multi-analyte optical biosensing. The sensing performance of the prepared NAA platforms was examined by real-time screening of binding reactions between human serum albumin (HSA)-modified NAA (i.e., sensing element) and quercetin (i.e., analyte). BL-NAAs display a complex optical interference spectrum, which can be resolved by fast Fourier transform (FFT) to monitor the EOT changes, where three distinctive peaks were revealed corresponding to the top, bottom, and total layer within the BL-NAA structures. The spectral shifts of these three characteristic peaks were used as sensing signals to monitor the binding events in each NAA pore in real-time upon exposure to different concentrations of quercetin. The multi-point sensing performance of BL-NAAs was determined for each pore layer, with an average sensitivity and low limit of detection of 600 nm (mg mL−1)−1 and 0.14 mg mL−1, respectively. BL-NAAs photonic structures have the capability to be used as platforms for multi-point RIfS sensing of biomolecules that can be further extended for simultaneous size-exclusion separation and multi-analyte sensing using these bilayered nanostructures.

A label-free optical biosensor based on nanoporous anodic alumina for tumour necrosis factor-alpha detection in chronic wounds

Tumour necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine important to wound healing. In non-healing wounds, it has been suggested that the expression of TNF-α is prolonged and elevated which contributes to impaired healing responses. Hence it is of great interest to develop biosensors towards the detection of TNF-α in non-healing wounds. In this study, we have developed a label-free optical TNF-α biosensor based on interferometric reflectance spectroscopy (IRS) technique using a functionalized nanoporous anodic alumina (NAA) thin film transducer. The biosensor is fabricated by functionalizing NAA pore walls with anti-TNF-α antibodies using silanization chemistry. Binding of TNF-α to the bioreceptors within the pores causes a change in effective optical thickness (EOT) of the NAA thin film. Thus, analyte detection is achieved by monitoring EOT evolution with time. Label-free detection of TNF-α was demonstrated in buffer solution and in complex media such as simulated wound fluid. A limit of detection of 0.13μg/mL was achieved. This study provides proof-of-concept evidence which sets foundations for further development of biosensors as point-of-care (POC) diagnostic tools for chronic wound care.

3D Nanoporous Anodic Alumina Structures for Sustained Drug Release.

The use of nanoporous anodic alumina (NAA) for the development of drug delivery systems has gained much attention in recent years. The release of drugs loaded inside NAA pores is complex and depends on the morphology of the pores. In this study, NAA, with different three-dimensional (3D) pore structures (cylindrical pores with several pore diameters, multilayered nanofunnels, and multilayered inverted funnels) were fabricated, and their respective drug delivery rates were studied and modeled using doxorubicin as a model drug. The obtained results reveal optimal modeling of all 3D pore structures, differentiating two drug release stages. Thus, an initial short-term and a sustained long-term release were successfully modeled by the Higuchi and the Korsmeyer–Peppas equations, respectively. This study demonstrates the influence of pore geometries on drug release rates, and further presents a sustained long-term drug release that exceeds 60 days without an undesired initial burst.

Use of multivariate analysis on Fabry-Pérot interference spectra of nanoporous anodic alumina (NAA) for optical sensors purposes

Nanoporous anodic alumina (NAA) presents interesting optical properties such as the Fabry-Pérot interference phenomenon, which can be used for sensing purposes. Optical sensing with NAA requires the determination of the material’s effective refractive index from the interference spectra and its variation when the analyte interacts with NAA pores in a non-specific or selective way. In this work, we propose an alternative approach to deal with Fabry-Pérot interference spectra as an analytical signal for optical sensors: multivariate analysis. As a proof of concept, NAA substrates were employed in the determination of glucose by photoluminescence spectroscopy. Principal component analysis (PCA) and partial least-squares (PLS) regression were used to qualitatively investigate spectra profile changes with increasing concentrations of glucose, and to establish a quantitative regression model to determine glucose concentrations from the photoluminescence spectra, respectively.Results showed that multivariate analysis can be used in Fabry-Pérot interferences to obtain optical sensors responses.

Engineering of Surface Chemistry for Enhanced Sensitivity in Nanoporous Interferometric Sensing Platforms.

We explore new approaches to engineering the surface chemistry of interferometric sensing platforms based on nanoporous anodic alumina (NAA) and reflectometric interference spectroscopy (RIfS). Two surface engineering strategies are presented, namely (i) selective chemical functionalization of the inner surface of NAA pores with amine-terminated thiol molecules and (ii) selective chemical functionalization of the top surface of NAA with dithiol molecules. The strong molecular interaction of Au3+ ions with thiol-containing functional molecules of alkane chain or peptide character provides a model sensing system with which to assess the sensitivity of these NAA platforms by both molecular feature and surface engineering. Changes in the effective optical thickness of the functionalized NAA photonic films (i.e., sensing principle), in response to gold ions, are monitored in real-time by RIfS. 6-Amino-1-hexanethiol (inner surface) and 1,6-hexanedithiol (top surface), the most sensitive functional molecules from approaches i and ii, respectively, were combined into a third sensing strategy whereby the NAA platforms are functionalized on both the top and inner surfaces concurrently. Engineering of the surface according to this approach resulted in an additive enhancement in sensitivity of up to 5-fold compared to previously reported systems. This study advances the rational engineering of surface chemistry for interferometric sensing on nanoporous platforms with potential applications for real-time monitoring of multiple analytes in dynamic environments.

Optical Monitoring of the Capillary Filling Dynamics Variation in Nanoporous Anodic Alumina toward Sensing Applications.

Fluid imbibition-coupled laser interferometry (FICLI) is a technique in which the kinetics of a fluid infiltrating a nanoporous anodic alumina (NAA) membrane is monitored by the interference of a laser beam at the membrane top and bottom surfaces. Further processing of the measured data results in an estimate of the pore radius. In this work, we study the accuracy of FICLI in the detection of small changes in pore radius, and we evaluate the possibility of using such detection as a sensing paradigm. The accuracy is estimated by measuring samples with increasing pore radius, obtained by successive wet etching steps, and repeatability is evaluated by using different liquids. For decreasing pore radius, samples obtained by the successive deposition of polyelectrolyte double layers are used. With the aim of evaluating the possibility of the FICLI method to sense biological binding events, BSA attachment detection is demonstrated by applying FICLI to samples before and after immobilization of the protein. Results show that the technique permits an accurate estimation of the pore radius, the pore-etching rate (with a radius variation of retch,DI = 1.05 nm/min ± 0.11 nm/min), and the polyelectrolyte double layer thickness (with a radius variation of rPAH/PSS = 3.2 nm ± 0.2 nm per polyelectrolyte double layer). Furthermore, the pore radius reduction measured after BSA immobilization (dBSA = 4.9 nm ± 1.1 nm) is in good agreement with the protein size, as reported in the literature. With these results, we provide a sound basis for the applicability of FICLI as a sensitive technique for the characterization of NAA pore radius modifications.

Protein attachment to nanoporous anodic alumina for biotechnological applications: Influence of pore size, protein size and functionalization path

Nanoporous anodic alumina (NAA) is a material with great interest in nanotechnology and with promising applications to biotechnology. Obtaining specific and regularly functionalized NAA surfaces is essential to obtain meaningful results and applications. Silane-PEG-NHS (triethoxysilane-polyethylene-glycol-N-hydroxysuccinimide) is a covalent linker commonly used for single-molecule studies. We investigate the functionalization of NAA with silane-PEG-NHS and compared with two common, but not single-molecule, grafting agents, APTMS (3-aminopropylotrimethoxysilane) as an electrostatic linker, and APTMS-GTA (3-aminopropylotrimethoxysilane-glutaraldehyde) as covalent. Another outcome of this study is to show how two proteins (collagen and bovine serum albumin, BSA) with different properties differentially arrange for different functionalizations and NAA pore sizes. FTIR is used to demonstrate the surface modification steps and fluorescence confocal microscopy reveals that silane-PEG-NHS results in a more homogeneous protein distribution in comparison to the other linkers. Reflection interference Fourier transform spectroscopy confirms the confocal fluorescence microscopy results and permits to estimate the amounts of linker and linked proteins within the pores. These results permit to obtain uniformly chemical modified NAA supports with a great value in biosensing, drug delivery and cell biology.