Real-Time Monitoring of Doxorubicin Release from Hybrid Nanoporous Anodic Alumina Structures.

This work demonstrates an advanced approach to fabricate Hybrid nanoporous anodic alumina gradient-index filters (Hy-NAA-GIFs) through a heterogeneous anodization process combining sinusoidal current-density anodization and constant potential anodization. As a result, the hybrid structure obtained reveals a single photonic stopband (PSB), which falls within the absorption region of the drug molecule and the intensity of the spectrum that are far from such absorption range. The prepared structures were loaded with the doxorubicin (DOX) drug through the drop-casting method, which allows for evaluating the maximum reflectance of the relative height of the PSB with the average reflectance of the spectrum intensity. Thereafter, this property has been applied in a flow cell setup connected to a reflectance spectrophotometer where different drug-loaded samples were placed to study the behavior and kinetics of the drug release in real-time by varying two parameters, i.e., different pore length and flow rates. As such, obtained results were analyzed with a model that includes a sum of two inverted exponential decay functions with two different characteristic time releases. Overall, this study opens up several possibilities for the Hy-NAA-GIFs to study the drug kinetics from nanoporous structures.

Advances in Optical Biosensors and Sensors Using Nanoporous Anodic Alumina.

This review paper focuses on recent progress in optical biosensors using self-ordered nanoporous anodic alumina. We present the fabrication of self-ordered nanoporous anodic alumina, surface functionalization, and optical sensor applications. We show that self-ordered nanoporous anodic alumina has good potential for use in the fabrication of antibody-based (immunosensor), aptamer-based (aptasensor), gene-based (genosensor), peptide-based, and enzyme-based optical biosensors. The fabricated optical biosensors presented high sensitivity and selectivity. In addition, we also showed that the performance of the biosensors and the self-ordered nanoporous anodic alumina can be used for assessing biomolecules, heavy ions, and gas molecules.

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 the interferometric 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 450-1050 nm. 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 64.0 nM. The limit of detection of the biosensor sensor was 0.08 nM. 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.

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.

Aptamer-Based Nanoporous Anodic Alumina Interferometric Biosensor for Real-Time Thrombin Detection.

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.

Highly sensitive aptasensor based on interferometric reflectance spectroscopy for the determination of amyloid ß as an Alzheimer's disease biomarkers using nanoporous anodic alumina.

It is well known that Alzheimer's disease is one of the global challenges for the 21st century. Therefore, it is urgent to develop a reliable biosensor for the detection of this disease. Here in, we have developed for the first time, an aptasensor based on interferometric reflectance spectroscopy (IRS) for the determination of amyloid ß (Aß) oligomers that is an Alzheimer's disease biomarker. For this purpose, the nanoporous anodic alumina (NAA) was first fabricated. After that, the pore walls of the NAA were modified with (3-aminopropyl) trimethoxysilane (NAA-NH2). The amino-terminal aptamers probe were then attached to the pore walls of the NAA-NH2 by using glutaraldehyde (GA) as the cross-linking agent. Subsequently, methylene blue (MB) was immobilized into the aptamer as the photo-probe, generating the MB/G-quadruplex complex. Since MB has a high absorption coefficient, the intensity of the reflected white light to the charge-coupled device (CCD) detector decreased. In the presence of the Aß oligomers that have high affinity to the immobilized aptamer, the MB/quadruplex complex broke and MB washed away from the aptasensor. Therefore, the intensity of the reflected white light to the CCD detector increased. The increased signal intensity of the aptasensor has a logarithmic relationship with the concentration of Aß oligomers. The proposed aptasensor exhibited a good response to the concentration of Aß oligomers in the range of 0.5-50.0 µg × mL-1. The experimental detection limit was of 0.02 µg × mL-1 (at 3σ/S). The proposed optical aptasensor exhibited good selectivity, linear range, and stability.

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.

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.

Structural and optical nanoengineering of nanoporous anodic alumina rugate filters for real-time and label-free biosensing applications.

In this study, we report about the structural engineering and optical optimization of nanoporous anodic alumina rugate filters (NAA-RFs) for real-time and label-free biosensing applications. Structurally engineered NAA-RFs are combined with reflection spectroscopy (RfS) in order to develop a biosensing system based on the position shift of the characteristic peak in the reflection spectrum of NAA-RFs (Δλpeak). This system is optimized and assessed by measuring shifts in the characteristic peak position produced by small changes in the effective medium (i.e., refractive index). To this end, NAA-RFs are filled with different solutions of d-glucose, and the Δλpeak is measured in real time by RfS. These results are validated by a theoretical model (i.e., the Looyenga-Landau-Lifshitz model), demonstrating that the control over the nanoporous structure makes it possible to optimize optical signals in RfS for sensing purposes. The linear range of these optical sensors ranges from 0.01 to 1.00 M, with a low detection limit of 0.01 M of d-glucose (i.e., 1.80 ppm), a sensitivity of 4.93 nm M(-1) (i.e., 164 nm per refractive index units), and a linearity of 0.998. This proof-of-concept study demonstrates that the proposed system combining NAA-RFs with RfS has outstanding capabilities to develop ultrasensitive, portable, and cost-competitive optical sensors.

Gold-coated ordered nanoporous anodic alumina bilayers for future label-free interferometric biosensors.

A cost-effective label-free optical biosensor based on gold-coated self-ordered nanoporous anodic alumina bilayers is presented. The structure is formed by two uniform nanoporous layers of different porosity (i.e., a top layer with large pores and a bottom layer with smaller pores). Each layer presents uniform pore size, regular pore distribution, and regular diameter along its pore length. To increase and improve the output sensing signals, a thin gold layer on the top surface was deposited. The gold layer increases the refractive index contrast between the nanoporous alumina layer and the analytical aqueous solution, and it results in a greater contrast in the interferometric spectrum and a higher sensitivity of the structure. From this structurally engineered architecture, the resulting reflectivity spectrum shows a complex series of Fabry-Pérot interference fringes, which was analyzed by the reflective interferometric Fourier transform spectroscopy (RIFTS) method. To determine the performance of this structure for biosensing applications, we tested bovine serum albumin (BSA) as the target protein. The results show a significant enhancement of the RIFTS peak intensity and position when a gold layer is on the top surface.

Photoluminescent enzymatic sensor based on nanoporous anodic alumina.

Herein, we present a smart enzymatic sensor based on nanoporous anodic alumina (NAA) and its photoluminescence (PL) in the UV-visible range. The as-produced structure of NAA is functionalized and activated in order to perform the enzyme immobilization in a controlled manner. The whole process is monitored through the PL spectrum and each stage is characterized by an exclusive barcode, which is associated with the PL oscillations. This characteristic property allows us to calculate the change in the effective optical thickness that takes place after each stage. This makes it possible to accurately detect and quantify the immobilized enzyme within the NAA structure. Finally, the NAA geometry (i.e., the pore length and its diameter) is optimized to improve the enzyme immobilization and its detection inside the pores. This enzymatic sensor can give quick and accurate measurements of enzyme levels, what is crucial in clinical enzymology to prevent and detect diseases at their primary stage.

Nanoporous anodic alumina barcodes: toward smart optical biosensors.

Toward a smart optical biosensor based on nanoporous anodic alumina (NAA): by modifying the pore geometry in nanoporous anodic alumina we are able to change the effective medium at will and tune the photoluminescence of NAA. The oscillations in the PL spectrum are converted into exclusive barcodes, which are useful for developing optical biomedical sensors in the UV-Visible region.

Understanding pore rearrangement during mild to hard transition in bilayered porous anodic alumina membranes.

We present a systematic study about the influence of the main anodization parameters (i.e., anodization voltage ramp and hard anodization voltage) on the pore rearrangement in nanoporous anodic alumina during mild to hard anodization regime transition. To cover the ranges between mild and hard regimes, the anodization parameters were each set to three levels (i.e., 0.5, 1.0, and 2.0 V s(-1) for the anodization voltage ramp and 80, 110, and 140 V for the hard anodization voltage). To the best of our knowledge, this is the first rigorous study about this phenomenon, which is quantified indirectly by means of a nickel electrodeposition. It is found that pore rearrangement takes place in a relatively random manner. Large areas of pores remain blocked when the anodization regime changes from mild to hard and, under certain anodization conditions, a pore branching takes place based on the self-ordering mechanism at work during anodization. Furthermore, it is statistically demonstrated by means of a design of experiments strategy that the effect of the anodization voltage ramp on the pore rearrangement is practically negligible in contrast to the hard anodization voltage effect. It is expected that this study gives a better understanding of structural changes in nanoporous anodic alumina when anodization is switched from mild to hard regime. Furthermore, the resulting nanostructures could be used to develop a wide range of nanodevices (e.g., waveguides, 1D photonic crystals, Fabry-Pérot interferometers, hybrid mosaic arrays of nanowires).

Ultraviolet optical and microstructural properties of MgF2 and LaF3 coatings deposited by ion-beam sputtering and boat and electron-beam evaporation.

Single layers of MgF2 and LaF3 were deposited upon superpolished fused-silica and CaF2 substrates by ion-beam sputtering (IBS) as well as by boat and electron beam (e-beam) evaporation and were characterized by a variety of complementary analytical techniques. Besides undergoing photometric and ellipsometric inspection, the samples were investigated at 193 and 633 nm by an optical scatter measurement facility. The structural properties were assessed with atomic-force microscopy, x-ray diffraction, TEM techniques that involved conventional thinning methods for the layers. For measurement of mechanical stress in the coatings, special silicon substrates were coated and analyzed. The dispersion behavior of both deposition materials, which was determined on the basis of various independent photometric measurements and data reduction techniques, is in good agreement with that published in the literature and with the bulk properties of the materials. The refractive indices of the MgF2 coatings ranged from 1.415 to 1.440 for the wavelength of the ArF excimer laser (193 nm) and from 1.435 to 1.465 for the wavelength of the F2 excimer laser (157 nm). For single layers of LaF3 the refractive indices extended from 1.67 to 1.70 at 193 nm to approximately 1.80 at 157 nm. The IBS process achieves the best homogeneity and the lowest surface roughness values (close to 1 nm(rms)) of the processes compared in the joint experiment. In contrast to MgF2 boat and e-beam evaporated coatings, which exhibit tensile mechanical stress ranging from 300 to 400 MPa, IBS coatings exhibit high compressive stress of as much as 910 MPa. A similar tendency was found for coating stress in LaF3 single layers. Experimental results are discussed with respect to the microstructural and compositional properties as well as to the surface topography of the coatings.