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.

Fluorinated Zinc and Copper Phthalocyanines as Efficient Third Components in Ternary Bulk Heterojunction Solar Cells.

Fluorinated zinc and copper metallophthalocyanines MPcF48 are synthesized and incorporated as third component small molecules in ternary organic solar cells (TOSCs). To enable the high performance of TOSCs, maximizing short-circuit current density (J SC) is crucial. Ternary bulk heterojunction blends, consisting of a polymer donor PTB7-Th, fullerene acceptors PC70BM, and a third component MPcF48, are formulated to fabricate TOSCs with a device architecture of ITO/PFN/active layer/V2O5/Ag. Employing copper as metal atom substitution in the third component of TOSCs enhances J SC as a result of complementary absorption spectra in the near-infrared region. In combination with J SC enhancement, suppressed charge recombination, improved exciton dissociation and charge carrier collection efficiency, and better morphology lead to a slightly improved fill factor (FF), resulting in a 7% enhancement of PCE than those of binary OSCs. In addition to the increased PCE, the photostability of TOSCs has also been improved by the appropriate addition of CuPcF48. Detailed studies imply that metal atom substitution in phthalocyanines is an effective way to improve J SC, FF, and thus the performance and photostability of TOSCs.

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.

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.

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.

Advances in Nanoporous Anodic Alumina-Based Biosensors to Detect Biomarkers of Clinical Significance: A Review.

There is a strong and growing demand for compact, portable, rapid, and low-cost devices to detect biomarkers of interest in clinical and point-of-care diagnostics. Such devices aid in early diagnosis of diseases without the need to rely on expensive and time-consuming large instruments in dedicated laboratories. Over the last decade, numerous biosensors have been developed for detection of a wide range of clinical biomarkers including proteins, nucleic acids, growth factors, and bacterial enzymes. Various transduction techniques have been reported based on biosensor technology that deliver substantial advances in analytical performance, including sensitivity, reproducibility, selectivity, and speed for monitoring a wide range of human health conditions. Nanoporous anodic alumina (NAA) has been used extensively for biosensing applications due to its inherent optical and electrochemical properties, ease of fabrication, large surface area, tunable properties, and high stability in aqueous environment. This review focuses on NAA-based biosensing systems for detection of clinically significant biomarkers using various detection techniques with the main focus being on electrochemical and optical transduction methods. The review covers an overview of the importance of biosensors for biomarkers detection, general (surface and structural) properties and fabrication of NAA, and NAA-based biomarker sensing systems.

Molecular gated nanoporous anodic alumina for the detection of cocaine.

We present herein the use of nanoporous anodic alumina (NAA) as a suitable support to implement "molecular gates" for sensing applications. In our design, a NAA support is loaded with a fluorescent reporter (rhodamine B) and functionalized with a short single-stranded DNA. Then pores are blocked by the subsequent hybridisation of a specific cocaine aptamer. The response of the gated material was studied in aqueous solution. In a typical experiment, the support was immersed in hybridisation buffer solution in the absence or presence of cocaine. At certain times, the release of rhodamine B from pore voids was measured by fluorescence spectroscopy. The capped NAA support showed poor cargo delivery, but presence of cocaine in the solution selectively induced rhodamine B release. By this simple procedure a limit of detection as low as 5 × 10-7 M was calculated for cocaine. The gated NAA was successfully applied to detect cocaine in saliva samples and the possible re-use of the nanostructures was assessed. Based on these results, we believe that NAA could be a suitable support to prepare optical gated probes with a synergic combination of the favourable features of selected gated sensing systems and NAA.

In Vitro Biocompatibility of Surface-Modified Porous Alumina Particles for HepG2 Tumor Cells: Toward Early Diagnosis and Targeted Treatment.

Porous alumina photoluminescence-inherent particles are produced and proposed for the development of biomarkers detectors and localized treatment of HepG2 cells. Nanoporous alumina particles (NPAPs) are amorphous, consist of hexagonally ordered nanometric pores in an alumina matrix, have high chemical stability in physiological pH, and exhibit a high inherent photoluminescence in the visible spectrum independently of their size, selectable from nanometers to tens of micrometers. The surface of NPAPs is chemically modified using two different functionalization methods, a multistep method with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GLTA) and a novel simplified-step method with silane-PEG-NHS. Fourier Transform infrared spectroscopy analysis confirmed the proper surface modification of the particles for both functionalization methods. HepG2 cells were cultured during different times with growing concentrations of particles. The analysis of cytotoxicity and cell viability of HepG2 cells confirmed the good biocompatibility of NPAPs in all culture conditions. The results prove the suitability of NPAPs for developing new label-free biomarker detectors and advantageous carriers for localized drug delivery.

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.

Optofluidic characterization of nanoporous membranes.

An optofluidic method that accurately identifies the internal geometry of nanochannel arrays is presented. It is based on the dynamics of capillary-driven fluid imbibition, which is followed by laser interferometry. Conical nanochannel arrays in anodized alumina are investigated, which present an asymmetry of the filling times measured from different sides of the membrane. It is demonstrated by theory and experiments that the capillary filling asymmetry only depends on the ratio H of the inlet to outlet pore radii and that the ratio of filling times vary closely as H(7/3). Besides, the capillary filling of conical channels exhibits striking results in comparison to the corresponding cylindrical channels. Apart from these novel results in nanoscale fluid dynamics, the whole method discussed here serves as a characterization technique for nanoporous membranes.

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).