Trends in Analytical Chemistry, Vol. 44, 2013 Trends Nanoporous anodic aluminum oxide for chemical sensing and biosensors Abel Santos, Tushar Kumeria, Dusan Losic Nanoporous anodic aluminum oxide (AAO) has become one of the most popular materials with potential applications in numerous areas, including molecular separation, catalysis, energy generation and storage, electronics, photonics, sensing, drug delivery, and template synthesis. The fabrication of AAO is based on simple, cost-effective, self-ordering anodization of aluminum, which yields verticallyaligned, highly-ordered nanoporous structures. Due to its unique optical and electrochemical properties, nanoporous AAO has been extensively explored as a platform for developing inexpensive, portable sensing and biosensing devices. This article reviews AAO-based sensing and biosensing technologies, highlighting key examples of different detection concepts and device performance. We conclude with a perspective on the exciting opportunities for further developments in this research field. Crown Copyright ª 2012 Published by Elsevier Ltd. All rights reserved. Keywords: Anodic aluminum oxide (AAO); Biosensor; Chemical sensor; Detection; Device performance; Electrochemical sensor; Nanopore; Nanoporous alumina; Optical biosensor; Self-ordering anodization 1. Introduction Abel Santos, Tushar Kumeria, Dusan Losic* School of Chemical Engineering, University of Adelaide, Adelaide SA 5005, Australia * Corresponding author. Tel.: +61 8 8313 4648; E-mail: [email protected] au The development of ultra-sensitive sensing and biosensing devices using nanomaterials has received a great deal of attention in recent years, due to their unique physical and chemical properties. In particular, nanoporous materials prepared by selfordering synthesis based on electrochemical anodization have enormous potential for the development of such devices . This process has successfully generated highly-ordered, vertically-aligned nanoporous and nanotubular structures with well-defined, controllable geometry, anodic aluminum (Al) oxide (AAO) and titania nanotubes (TNTs) being two outstanding examples [2,3]. Compared to conventional time consuming and expensive lithographic techniques, self-ordering anodization offers numerous advantages, including simple, cost-competitive fabrication, controllable pore structure with nanometric precision, structure with high aspect ratio and fast, industrially-scalable production [4,5]. In addition, AAO has a unique set of chemical, optical, mechanical, transport and electrical properties, which include chemical resistance, thermal stability, hardness, biocompatibility and large surface area. Nanopore structures demonstrate a dramatic increase in surface-to-volume ratio that enhances the signals corresponding to interaction between analyte and surfaces, including biomolecular reaction. The potential of nanopores to mimic protein nanochannels in cell membranes, which have single molecule sensitivity and selectivity is critical toward the development of novel bioinspired biosensors . The size and the surface chemistry of nanopores can provide selective molecular transport and attachment, which enables the integration of separation and sensing functions into one device. All these properties make AAO an excellent platform with exciting opportunities for development of advanced, smart, simple, cost-effective sensing devices for numerous analytical applications. Over the past decade, significant research efforts have been made to explore the properties and emerging applications of AAO, resulting in more than 2000 publications 0165-9936/$ - see front matter Crown Copyright ª 2012 Published by Elsevier Ltd. All rights reserved. doi:http://dx.doi.org/10.1016/j.trac.2012.11.007 25 Trends Trends in Analytical Chemistry, Vol. 44, 2013 in the past five years. However, this is the first literature review to highlight their sensing and biosensing applications. All this has led to the use of AAO for more sophisticated and relevant applications as selective molecular separators, chemical/biological sensing devices, catalysis, cell adhesion and culture, data storage, energy generation and storage, drug delivery and template synthesis [3,5–7]. These applications require AAO to have well-defined structural and chemical properties, and levels of complexity that can be achieved through structural engineering during fabrication and suitable functionalization in subsequent post-treatments. The use of AAO for sensing and biosensing is a very fast developing research area focused on different detection principles and broad applications, with potential in the near future to translate this research into commercial devices. Herein, we review the major advances and developments of chemical sensing and biosensing systems based on nanoporous AAO. First, we briefly describe the nanoporous structures of AAO prepared by self-ordered anodization, showing their key optical and electrochemical properties, which make AAO ideal for specific sensing applications. Second, we present the most outstanding examples of recent advances in optical and electrochemical sensors and biosensors, reporting their detection principles, performance and practical applications. Finally, we conclude with a prospective outlook over the future trends in this research field and future developments of AAO-based sensing and biosensing devices. 2. Fabrication, structure and properties of nanoporous AAO 2.1. Fabrication and structure Self-ordered AAO can be described as a nanoporous alumina (Al2O3) matrix with close-packed arrays of hexagonally-arranged cells containing a cylindrical central pore, which grows perpendicularly to the surface of the underlying aluminum substrate . During the anodization process, an electrochemical equilibrium between the formation of Al2O3 and its dissolution takes place through a thin oxide-barrier layer located at the pore bottom tip (i.e. closed pores). After anodization, this oxide barrier layer therefore has to be removed, together with the remaining Al substrate to obtain free-standing AAO membranes (i.e. open pores). The pore structure of AAO can be defined by structural parameters [e.g., pore diameter (dp), interpore distance (dint), pore length (Lp) and oxide-barrier layer thickness (sobl)]. Fig. 1 shows a typical AAO structure (top and crosssection). These geometric pore features can be accurately tuned by the anodization conditions in the range 10–400 nm for dp, 50–600 nm for dint, from several nm to hundreds of lm for Lp and 30–250 nm for sobl. Other 26 http://www.elsevier.com/locate/trac characteristic structural parameters of AAO are the pore density (qp) and its porosity (P), which can be modified between 109–1011/cm2 and 5–50%, respectively . Over several decades, the influence of the anodization conditions on the AAO-pore structures was studied extensively in order to achieve highly-ordered pores with controllable dimensions [2,4,5,8,9]. The anodization voltage, electrolyte type, concentration and temperature have been recognized as the most critical parameters to control the self-ordering process and the geometry of the resulting porous structures. Generally, aqueous solutions of sulfuric acid (H2SO4) at 25 V, oxalic acid (H2C2O4) at 40 V and phosphoric acid (H3PO4) at 195 V are the most commonly used electrolytes for preparation of AAO by conventional anodization process, called ‘‘mild’’ anodization (MA). [4,5]. Several studies have reported on the self-ordering mechanism of AAO in other electrolytes (e.g., aqueous solutions of citric, maleic, malonic, tartaric and sulfamic acids), but they achieved poor pore ordering . The ground-breaking milestone in the fabrication of AAO with highly-ordered pore structures was first reported by Masuda and Fukuda, who introduced a twostep approach to anodization . In this method, after the first anodization step, the resulting porous oxide layer is removed to pre-structure the Al surface and make the propagation of well-defined pores from the top to bottom possible during the second anodization step. The main disadvantage of this conventional MA process is the slow pore-growth rate (i.e. 2–7 lm/h), which requires around two days to prepare well-ordered AAO structures. To address this problem, a new approach, so-called ‘‘hard’’ anodization (HA), was introduced by the Göseles group. Under these conditions, the pore-growth rate is considerably higher (50–100 lm/h) . Furthermore, this method spreads the fabrication of AAO with interpore distances not attainable by MA. These two anodization strategies, using different electrolytes and anodization conditions combined with chemical etching, provide broad scope to engineer AAO with desired pore dimensions and shapes. To fabricate AAO with complex pore geometries, several electrochemical approaches have been successfully implemented, including periodic changing of anodization conditions (voltage or current) with or without replacement of the acid electrolyte [5,13–15]. This has enabled the generation of AAO with many different morphologies, including funnel-type, branched pores, periodically-shaped pore structures, and hierarchical and multi-structured pores [14–18]. The structural engineering of AAO pores, combined with other modification methods, provides enormous scope to improve the properties of AAO for specific sensing applications. Trends Figure 1. Anodic aluminum oxide (AAO) structure. (a) Pore structure in AAO and definition of the main structural parameters: pore diameter (dp), interpore distance (dint), pore length (Lp), pore wall thickness (sW) and oxide barrier thickness (sobl). (b) SEM images showing the top and cross-section views of AAO structure. Trends in Analytical Chemistry, Vol. 44, 2013 2.2. Properties for sensing and biosensing applications The unique set of physical and chemical properties of porous structures makes AAO an excellent platform to develop sensing devices. The most characteristic optical and electrochemical properties of AAO are photoluminescence (PL), transmittance, reflectivity, absorbance, electron transfer, impedance, electric resistance, and conductance, which can be used as detection principles to fabricate highly sensitive, selective chemical and biological sensors. The miniaturization and easy integration of AAO films into microchips with current microfabrication methods used in silicon technology is another important feature of AAO. Nanoporous AAO can act not only as an active sensing layer, but also as a container to accommodate biological events inside its nanopores. It is worth noting that these properties of AAO, relevant to sensing applications, can be optimized by designing the pore geometry and its surface chemistry. Numerous soft and hard surface-modification techniques of nanoporous AAO (e.g., molecular self-assembly, layer-by-layer deposition, plasma polymerization, atomic layer deposition, dip coating, chemical-vapor deposition, sol-gel, physical and electrochemical metal deposition) have been demonstrated [19–22]. The capability of these modifications is not only to improve or to introduce new properties, but also to endow AAO with multifunctional properties (e.g., optical and electrochemical activity) provides flexibility to develop advanced sensing and biosensing devices for multiple-analyte detection. In terms of chemical composition, two main regions can be distinguished in the pore structure of AAO . The first region is an inner layer close to the aluminumalumina interface, which is mainly composed of pure alumina. The second one is an outer layer located between the inner layer and the alumina-electrolyte interface. This layer is contaminated during the anodization process by anionic species incorporated into the alumina structure from the acid electrolyte (e.g., sulfate, oxalate, and phosphate) . The important function of these layers is that they provide AAO with specific optical properties (e.g., PL), which depend fundamentally on the acid electrolyte used during the anodization process (i.e. on the anionic species incorporated into the AAO structure). As stated previously, another outstanding characteristic of AAO is its capability to adjust the surface chemistry inside the pores in a controlled manner by surface functionalization with specific molecules. This process provides AAO with high selectivity toward target biomolecules (e.g., lipids, antibodies, DNA, proteins, and enzymes). In this way, the large specific surface area of AAO can be activated for interacting or capturing target molecules, which can be subsequently analyzed by optical or electrochemical techniques. These modifications can also improve the properties of AAO (e.g., http://www.elsevier.com/locate/trac 27 Trends Trends in Analytical Chemistry, Vol. 44, 2013 Table 1. Optical sensors and biosensors based on nanoporous anodic alumina oxide (AAO): detection principle, application and performance Optical technique Detection limit/concentration Ref. 6 DNA Oxazine 170 Glucose 5Æ10 M 40 lg/mL 0.1 mg/mL 100 mM 6.5Æ103 M 0.1 M       SPR-LSPR Avidin Anti-5-Fluorouracil Ru[BPhen3]2+ Fe[Phen3]2+ BSA Invertase Melittin 10 lg/mL 100 mg/mL 2 lM 1 lM 60 nM 10 nM 100 ng/mL        SERS p-aminothiophenol 4-mercaptopyridine 3-mercaptobenzoic 0.5 M 1Æ106 M 3 mM    RIfS H2S DNA Circulating tumor cells Immunoglobulin 0.5% v 2 nmol cm2 1000 cells/mL 0.1 mg/mL     PL Analyte Morin Trypsin reflectivity, hydrophobicity or hydrophilicity, antifouling, and accommodation and maintenance of biomolecules for specific and high-throughput assays) [23,24]. This review presents the classification and a brief description of nanoporous AAO sensors and biosensors on the basis of their optical and electrochemical-detection principles. 3. Optical biosensors Due to their dimensions, geometry and chemical composition, AAO structures show characteristic responses when interacting with light. This makes them an attractive material to develop optically-active devices. Many studies have demonstrated the applications of AAO for optical filters, waveguides, anti-reflective surfaces, resonators or microcavities [25,26]. AAO has been shown to be a particularly outstanding platform for the development of sensing devices with an exclusive set of optical properties including reflectance, transmittance, absorbance, PL, chemiluminescence, and wave-guiding. A new generation of optical sensing and biosensing devices based on the AAO recently emerged, and their performance was successfully explored for many analytical applications, including environmental and clinical analysis, industrial and food control, defense and homeland security. The most representative advances in development and applications of optical AAO sensors and biosensors are described in the following sections and summarized in Table 1 with their analytical performances. 28 http://www.elsevier.com/locate/trac 3.1. Photoluminescence (PL) spectroscopy The PL properties of AAO were reported several decades ago, followed by extensive studies to understand and to control PL behavior of AAO, but its origin is still in doubt [10,27,28]. It is generally accepted that this phenomena is related to two types of PL field centers: (1) F centers, which rely on the amount of carboxylate impurities incorporated into the AAO structure from the acid electrolyte in the course of the anodization process ; and, (2) F+ centers, which are related to ionized oxygen vacancies in the AAO structure [27,28]. Previous studies have demonstrated that PL of AAO depends on the acid electrolyte, anodization voltage, pore diameters, thermal treatment and anodization regime [10,29,30]. As an example, it has been observed that AAO fabricated in oxalic acid has a higher PL intensity than AAO produced in sulfuric or phosphoric acids . Pore widening or thermal treatment also increases the PL intensity in AAO . Another advantage of using AAO for developing PL-sensing devices is that, unlike other materials (e.g., porous silicon), AAO has a stable PL spectrum and it is not necessary to passivate AAO to prevent changes over the PL spectrum over the course of time. The unique fingerprint of PL in AAO is clearly useful for developing optical sensors, in which a high degree of resolution, sensibility and biocompatibility are required . In a basic configuration, AAO nanopores can be used as a nanocontainer to accommodate targeted molecules and record their PL response compared with the spectrum of unmodified AAO. It has been observed Trends Figure 2. Photoluminescent (PL) optical biosensors based on anodic aluminum oxide (AAO). (a) Experimental set-up used to perform PL measurements. (b) PL spectrum. (c) Resulting barcode generated from the PL spectrum. Trends in Analytical Chemistry, Vol. 44, 2013 that the presence of adsorbed molecules in the AAO can be detected by a shift in its PL spectrum. The adsorption of large biomolecules (e.g., morin, trypsin and human serum albumin) on AAO was studied by Jia et al. showing significant enhancement of PL signal . Feng et al. studied a more complex process of DNA hybridization by this method using quantum dots as biological markers . In this study, AAO nanopores were functionalized with mercapto-undecanoic acid followed by layer-by-layer deposition of positively or negatively charged dendrimers and ZnCdSe quantum dots. A different approach was taken by Santos et al., who developed an optical barcode system for sensing based on the PL of AAO in the UV-visible region . The origin of these PL oscillations is the Fabry-Pérot effect, which amplifies the PL oscillations by enhancing the PL at wavelengths corresponding to the optical modes of the cavity formed by the system air–AAO–Al. In this way, the PL spectrum of AAO shows an abundance of narrow, well-resolved oscillations, which are very useful for biosensing. It is worth mentioning that the number, the intensity and the position of these oscillations can be tuned by modifying the pore length and diameters (i.e. by changing the effective medium). Fig. 2 shows this type of PL sensor, which has been tested with success for detecting biological substances (e.g., organic dyes, enzymes and glucose) [31,33]. This sensing method enables the generation of a wide range of PL barcodes, which are suitable for developing smart optical biosensors for a broad range of analytes. 3.2. Surface-plasmon resonance (SPR), waveguiding spectroscopy (WS) and localized surface-plasmon resonance (LSPR) SPR is recognized as the most popular analytical method to probe biological interactions. SPR is capable of realtime and in-situ measurements of a wide range of surface interactions, including ligand-binding affinity, association/dissociation kinetics, affinity constant, and highly sensitive surface-concentration measurements . SPR and WS systems are based on a Kretschmann configuration, where the excitation of surface plasmons is generated by an evanescent electromagnetic wave produced by the incidence of light on the surface of a prism coated with metal. SPR-AAO biosensors are integrated into this configuration, based on a prism on which a thin metallic film with AAO layer is grown. The plasmonic properties of this surface rely strongly on the refractive index of the adjacent medium within distances of 200 nm beyond the metal film surface. This makes it possible to use SPR with a thin layer of AAO for detecting biological binding events . Fig. 3(a) shows a typical SPR configuration used in SPR-AAO biosensing systems. These devices have been successfully used to study the adsorption and the desorption of bovine serum albumin (BSA) at different values of pH and bioaffinity interactions between http://www.elsevier.com/locate/trac 29 Trends Trends in Analytical Chemistry, Vol. 44, 2013 Figure 3. Surface-plasmon resonance (SPR) and waveguiding anodic aluminum oxide (AAO) sensors. (a) SPR set-up in a Kretschmann configuration for SPR-AAO sensors (left). Reflectance spectrum showing the different modes of the reflected light (right). (b) Waveguiding spectroscopy (WS) based on AAO (left). SEM image of AAO/Al layer and reflection spectra measured from this layer in contact with different concentration of bovine serum albumin (BSA) (right) (Reprinted from  with permission). biotin and avidin [36,37]. More recently, a SPR-AAO biosensor was used to measure the activity of immobilized enzyme invertase along the pores of AAO, which demonstrates that these devices are useful not only to perform qualitative analysis but also to quantify biological molecules or even to study biological events as enzymatic kinetics . Nanoporous AAO with a well-defined cylindrical pore geometry and pore diameters 0.1 times smaller than the wavelength of the incident light can confine light optical modes. This makes AAO an attractive sensing material for WS. Furthermore, AAO porous structures minimize scattering losses at visible and longer wavelengths, which is important to achieve sensitive probing of the binding of biomolecules inside nanopores . Fig. 3(b) shows a typical configuration of a WS-AAO biosensor and its signal response . Hotta et al. demonstrated that surface plasmons of WS-AAO biosensors can be exquisitely tuned and optimized by changing the AAO geometry via the anodiza30 http://www.elsevier.com/locate/trac tion process or by subsequent post-treatments (e.g., chemical etching) . Several new approaches to designing and optimizing SW-AAO sensors for detection of biomolecules using surface modification of AAO pores and their replication into polymer-nanorod structures were reported by the Knolls group [41,42]. Another configuration for developing optical biosensors is the so-called localized SPR (LSPR) or localized plasmon resonance (LPR), where surface plasmons are generated from metallic nanoparticles (NPs) when they are irradiated with light . The advantages of LSPR systems compared with SPR are that this is a simpler, inexpensive, portable system, which does not require a Kretschmann configuration (i.e. the prism element is not necessary). Highly ordered arrays of metallic NPs of gold or silver (i.e. nanocaps) can be easily fabricated by thermal deposition or other deposition and assembly methods on the top surface of the AAO. This makes AAO a very popular substrate for designing LSPR devices. Trends in Analytical Chemistry, Vol. 44, 2013 Trends Figure 4. Local surface-plasmon resonance (SPR) anodic aluminum oxide (LSPR-AAO) biosensor with metal-film (Au)-capped AAO substrate modified sensing biomolecules (antibody) (Reprinted from  with permission). Fig. 4 shows a typical LSPR-AAO label-free biosensor fabricated on a gold-capped AAO substrate for detection of antigen-antibody binding . The observed shift in the LSPR signal linearly depends on the adsorbed amount of biomolecules on the outer and inner surfaces of the AAO porous layer. Development of simple, costcompetitive and highly-sensitive LSPR-AAO biosensors for specific detections of proteins (e.g., BSA, avidin, DNA and thrombin) or their binding events with bioaffinity couples (e.g., biotin-avidin and 5-fluorouracil-anti-5fluorouracil) have been reported [36,44,45]. The combination of LSPR-AAO with an electrochemical system was also reported for sensitive detection of toxic peptide (i.e. melittin, the venom from the honey bees) with a limit of detection (LOD) of 10 ng/mL. This validates the flexibility of AAO to develop multi-detection sensing devices . 3.3. Surface-enhanced Raman scattering (SERS) spectroscopy SERS spectroscopy is based on an increase in the local optical field, which excites the target molecules, multiplying and amplifying the radiated Raman scattered light . This effect generates a huge enhancement (i.e. of the order of 106) of the Raman signal from molecules adsorbed onto a metal surface with nanometric surface roughness (i.e. so-called ‘‘hot spots’’ or ‘‘hot junctions’’). For that reason, SERS is an extremely sensitive, espe- cially attractive system for detecting, identifying and quantifying trace amounts of molecules . The use of AAO for developing SERS-AAO active substrates has received significant attention in recent years because of its cost-competitive fabrication, reliable reproducibility, well-defined nanostructure and applicability over large surface areas. SERS-AAO devices are commonly fabricated by evaporating or sputtering a metallic layer of silver or gold on the top or bottom side of an AAO substrate. In this way, periodic metallic nm structures with well-controlled geometry can be produced. The size and the inter-distance of these structures can be tuned at will by changing the geometry of AAO pores and the conditions of the metal deposition, which enable optimization of these SERS-AAO sensors for specific sensing applications . Strong Raman signal enhancements (105–106) have been reported in AAO substrates with gold and silver NPs grafted onto or synthesized inside AAO nanopores and used for biosensing applications [49,50]. Ko et al. decorated AAO with gold NPs to detect trace amounts of 2,4dinitrotoluene, which were not detectable by conventional Raman spectroscopy . In another study, Lu et al. used silver NPs to decorate AAO and the performance of the resulting SERS-AAO sensor was studied by detecting p-aminothiophenol . A similar strategy was used by Ji et al. to detect other mercapto-based molecules (i.e. 4-mercaptopyridine). The metal-decoration process http://www.elsevier.com/locate/trac 31 Trends Trends in Analytical Chemistry, Vol. 44, 2013 was performed by Lu et al. using electrodeposition of silver NPs along the AAO pores . Other studies have shown that other metal nanostructures combined with AAO as nanowires and nanotubes can be used to develop SERS-AAO sensors [52,53]. Lee et al. prepared silver nanowires inside AAO templates by electrodeposition and showed their application to SERS detection to study the adsorption of 4-aminobenzenethiol . Valleman et al. applied electroless deposition of gold inside an AAO template in order to fabricate a metallic AAO/gold composite membrane, which was subsequently used as a SERS substrate . Furthermore, Kondo et al. used AAO templates as a deposition mask to grow 3D multi-layered gold-AAO cone-like arrays by vacuum evaporation deposition. The performance of this SERS biosensor was tested in analyzing pyridine, which is a precursor used in pharmaceutical and agrochemical applications and an important solvent and reagent . 3.4. Reflectometric interference spectroscopy (RIfS) RIfS is another highly sensitive optical sensing technique, which is based on the interaction of white light with thin films . In the past decade, nanoporous thin films based on porous silicon have emerged as more efficient, attractive substitutes for planar thin films used in earlier studies to develop RIfS biosensors [56,57]. Following this success, AAO was recently considered as an outstanding substrate, offering several advantages (e.g., better chemical stability and more controllable and defined pore structures) . This makes RIfS devices based on AAO more stable and reproducible than porous silicon RIfS systems . Likewise in PL, the RIfS spectrum of AAO presents fringes with well-resolved peaks generated by the Fabry–Pérot effect [58,59]. Fig. 5 shows a RIfS set-up used to detect binding molecules in AAO nanopores . The wavelength of each peak maxima in the RIfS spectrum follows the Fabry–Pérot relationship (i.e. 2neffLp = mk), where neff is the effective refractive index of AAO, Lp is the pore length and m is the order of the RIfS fringe, the maximum of which is located at wavelength k. These peaks in the RIfS spectrum are useful for sensing, as the binding molecules on the surfaces inside pores can be detected through shifts in the peak positions [56,58]. The RIfS spectrum of AAO (i.e. number, intensity and position of the fringes) can be tuned by modifying the AAO structures (i.e. pore length and its diameter). This enables the generation of multiple RIfS spectra, which are envisaged to develop accurate sensing devices with capability for qualitative, highly sensitive quantitative measurements within the UVvisible region. In the past few years, RIfS-AAO sensing and biosensing technology was rapidly progressed and successfully used for numerous sensing applications, including gases, organic molecules, label-free biological molecules (e.g., DNA, proteins, antibodies, and aptamers), and study of the binding of biomolecules (e.g., DNA and antibodyantigen) [58,60]. The pioneering work by Pan et al. showed the capability of the RIfS-AAO for label-free detection of complementary DNA with sensitivity to 1 nmol with a 0.5-cm2 probe area . Alvarez et al. developed an RIfS-AAO immunosensor to detect binding events Figure 5. Schematic diagram of a reflectometric interference spectroscopy anodic aluminum oxide (RIfS-AAO) set-up for biosensing applications showing reflection of light from the AAO structure, RIfS set-up, typical raw signal and processed interference signal. 32 http://www.elsevier.com/locate/trac Trends in Analytical Chemistry, Vol. 44, 2013 Trends Table 2. Electrochemical sensors and biosensors based on nanoporous anodic alumina oxide (AAO): detection principle, application and performance Electrochemical technique Analyte Voltammetry/amperometry Glucose Oxidase Detection limit/concentration Ref. DENV-2 WNV-DIII Glucose Cholesterol Na+/K+ DNA Immunoglobulin Thrombin Marker CA 15-3 Formaldehyde H2O2 100 ng/L 0.1 lM 1 pfU/mL 4 pg/mL 1 lM 0.5 mg/mL 5 mM 9.55Æ1012 M 50 ng/mL 1.8 ng/mL 52 U/mL 0.5 lM 0.1 lM              Impedance Escherichia coli DENV-2 1Æ102 CFU/mL 1 pfU/mL   Capacitance PCB77 H2O 8Æ108 M 15 pf/RH%   Resistance/conductance NO2 DNA 1 ppm 10 lM   between antibodies (i.e. rabbit anti-sheep IgG) and their corresponding antigens (i.e. sheep IgG) . These results confirmed that only selective antibody-antigen binding events were detected by a significant change in the optical thickness, demonstrating the capability of this system to design selective and sensitive, label-free immunosensors for broad applications. A similar approach using an RIfS-AAO microchip biosensor was demonstrated by Kumeria et al. fabricated by modification of gold-coated AAO with biotinylated anti EpCAM antibodies . This device was used to capture and to detect circulating tumour cells (CTCs) in one step. This group also demonstrated gas sensing of volatile sulfur compounds in oral malodor and hydrogen sulfide for biomedical applications, by using RIfS-AAO modified with a metal or other chemical-sensitive layer with affinity to gas adsorption . The combination of two techniques – RIfS and LSPR detection using AAO – was recently demonstrated using gold film on AAO . The results obtained showed exciting opportunities to develop smart, multi-functional optical devices with microchip design. 4. Electrochemical sensors and biosensors Electrochemical sensors based on AAO, depending on their detection principles, can be divided into several different types, including voltammetric, amperometric, impedometric, conductometric, capacitative, and resistive. The most relevant examples of AAO electrochemical sensors and their performances are presented in the following sub-sections and summarized in Table 2. 4.1. Voltammetric and amperometric Nanoporous AAO membranes are an excellent platform for the development of membrane-type nanosensors with amperometric or voltammetric detection, considering their large surface area for effective immobilization of sensing and biosensing elements inside pores, which allows optimal interaction with analyte molecules flowing through these pores. As was commented upon previously, AAO is an electrical insulating material and its pores have to be modified with a conventional electrode conductive layer to turn them into a transducer able to measure the changes in electrochemical response. To this end, several methods using metal or metal-oxide deposition (Au, Pt, SnO2), growth of carbon nanotubes (CNTs), conductive polymers (polypyrrole) or other electrochemically active materials (Prussian blue) were reported [3,22,64]. These AAO-based electrochemical devices were applied to a wide range of sensing applications, including gas detection (vinyl chloride, ammonia, and formaldehyde) , glucose , hydrogen peroxide , cholesterol , DNA, nucleotides, blood proteins, antibodies, cancer biomarkers [9,69–71], study of enzymatic activity , electron transfer  and detection of cells (viruses, bacteria and cancer cell) [74,75]. The electrochemical signals from AAO-modified electrodes were derived indirectly via redox mediator or redox reaction between the AAO substrate and the immobilized molecules or directly by electrical-conducting linkers http://www.elsevier.com/locate/trac 33 Trends 34 http://www.elsevier.com/locate/trac Trends in Analytical Chemistry, Vol. 44, 2013 Figure 6. Electrochemical anodic aluminum oxide (AAO) sensors. (a) AAO biosensor for electrochemical (voltammetric) sensing of DNA hybridization (Reprinted from  with permission). (b) AAO protein imunobiosensor showing the blood cells outside the nanopores, and the protein molecules entering inside to bind specific antibodies generating a blockage in the diffusion of electroactive species (left). The determination of cancer biomarker CA 16-3 in blood and buffer showing two functions of sensor, filtering (blood cells) and detection (right) (Reprinted from  with permission). Trends in Analytical Chemistry, Vol. 44, 2013 using amperometric and voltammetric methods (i.e. differential pulse voltammetry (DPV) and cyclic voltammetry (CV)). The first AAO-nanopore probe to detect DNA hybridization by voltammetric measurements of changing conductance through nanopores was demonstrated by Smirnovs group . The hybridization 21-mer singlestranded DNA (ssDNA) on internal pore structures modified by covalent immobilization of ssDNA was detected as result of the blocking effect in the diffusion of electroactive species [Fe(CN)6]4/[Fe(CN)6]3 measured by the decrease of the cyclic voltammetric peaks . The significant contribution in improving this voltammetric electrochemical biosensing concept based on AAO membranes came from Merkocis and Tohs groups, who developed AAO immunobiosensors [6,71,74,75,77,78]. The binding of antigen inside pores was successfully demonstrated and an limit of detection (LOD) of 100 ng/L was reached by measuring the change of the voltammetric signal caused by impeded diffusion of the redox probe (i.e. ferrocenemethanol) [74,75]. A similar biosensing device prepared by modifying AAO with 5 0 -aminated DNA probe showed an ultra-sensitive LOD of 3.1 · 1013 M for quantification of single-stranded DNA sequences . Fig. 6 shows this DNA biosensor sensor. This sensing concept was further extended to develop ultra-sensitive nanobiosensors for the detection of viruses and pathogen bacteria (e.g., dengue type 2 mosquito virus, West Nile viral particles and Legionella pneumophila), using specific antibodies immobilized inside AAO pores [74,75,78]. DPV and redox probe were used to improve sensitivity, achieving the ultra-low LOD of 1 pfU/mL (dengue type 2) and 4 pg/mL (West Nile viral) in blood with an insignificant cross-reaction with nonspecific viruses. This concept of AAO immunobiosensor was further improved by the Merkocis group, by introducing gold NPs inside nanopores in order to enhance the blockage of the pores [71,78,79]. The reproducible detection of target ssDNA was achieved with linear correlation in the range 50–250 ng/mL and LOD of 42 ng/mL . Furthermore, it was shown that the nanopore blockage could be further enhanced by silver deposition to decrease the diffusion of the redox probe through the nanopores, and increase signal amplification and biosensor sensitivity . This concept was successfully applied [Fig. 6(b)] to develop more advanced AAO nanobiosensors with a filtration function and used for analysis of complex samples (e.g., whole blood) [71,79]. Efficient immunoassay of thrombin, immunoglobulin and cancer-biomarker CA 15-3 in blood samples with LODs of 1.8 ng/mL, 50 ng/mL and 52 U/mL, respectively, was demonstrated [71,79]. This separation and detection integrated on the same platform is particularly interesting for analysis of composite samples for biomedical, environmental and food analysis. Trends The development of electrochemical-based AAO sensors by modifying pores with Prussian blue (PB), ironhexacyanoferrate polymer, have gained tremendous attention in recent years due to the unique ion-exchange and electrocatalytic properties of PB [64,80]. PB was electrodeposited inside AAO membranes coated with gold on the bottom to form arrays of PB nanoelectrodes for amperometric measurements of glucose concentration . The resulting glucose biosensor showed excellent performance with a broad linear concentration range over three orders of magnitude, and a low LOD of 1 lM glucose, comparable with commercial enzymebased glucose biosensors. Furthermore, PB-AAO sensors were also successfully utilized for ion-selective detection of Na+ and K+ ions . Wong et al. recently reported development of an advanced PB-AAO amperometric nanotube sensor for the detection of hydrogen peroxide and glucose with a unique self-powered function . 4.2. Impedance spectroscopy (IS) IS measures the dielectric properties of a material, as a result of the interaction between the applied electric field (frequency) and the electric dipole moment of this material. The electrical behavior of AAO can be expressed by an equivalent circuit, the components of which depend on the model used. If the oxide-barrier layer of AAO is removed from the pore bottom tips, the electrical resistance of the system is significantly reduced and the current can flow exclusively through the pores. In this way, AAO pores can be used not only as containers to accommodate biological events, but also as an impedometric sensor by monitoring changes of conductance or impedance produced by binding reactions inside AAO pores. The most important applications of impedance AAO biosensors include DNA biosensing , study of protein and lipid membrane interactions , and detection of cancer cells and bacteria . IS using modified AAO to study DNA hybridization was reported by Smirnov et al. . First, the pore diameter at the pore mouth of produced AAO was reduced by a hydrothermal treatment with boiling water, followed by modification with 3-aminopropyltrimethoxysilane (APTES) and covalent attachment of complementary ssDNA inside AAO pores. DNA hybridization was studied by IS showing an impedance increment of 50% when that hybridization process took place inside the pores. The importance of surface-charge effect in controlling the ionic conductance through AAO nanopores is highlighted by the same group and applied as a convenient detection method for unlabeled DNA . Wang et al. used a more advanced approach by combining voltammetry and IS to characterize DNA hybridization through AAO pores . AAO pores were functionalized in a similar way with 5 0 -aminated ss-DNA http://www.elsevier.com/locate/trac 35 Trends Trends in Analytical Chemistry, Vol. 44, 2013 and, subsequently, a complementary target bacterium (i.e. Escherichia coli O157:H7) was detected by CV and IS, confirming significant blockage of pores as result of this hybridization process. Miniaturization and integration of AAO into microchips for the development of microchip impedance sensors to detect pathogens (e.g., E. coli O157:H7 and Staphylococcus aureus) were reported by Tan et al. . The AAO membrane was functionalized with 3-glycidoxypropyl)trimethoxysilane (GPMS) and embedded in a polydimethylsiloxane matrix. Nguyen et al. developed an impedance-based AAO sensor for dengue-virus particles by anodizing aluminum sputtered on platinum electrodes . The impedance change was recorded in response to the binding of dengue-virus particles serotype 2 (DENV2) with its serotype 2 immunoglobulin G antibody. 4.3. Capacitive, conductometric and resistive sensors Porous metal oxides, including AAO, are attractive materials to develop capacitance-based gas-sensing devices because of their stability at large temperature, high surface area and affinity to adsorb gases and vapors. Relative humidity (RH) capacitance sensors based on AAO have been explored by several groups, but showed limitations caused by non-linearity of response at higher RH levels. This problem was successfully addressed by Juhász et al. by fabricating an AAO sensor for RH using a CMOS-MEMS process with AAO films grown on silicon wafers . The porous structure was used as the sensing layer, while two types of electrodes were grown on the AAO surface (i.e. a vapor-permeable palladium layer and a gold grid). The sensitivity of this sensor (i.e. 15 pF/RH%) was found to be much higher than that of many commercial humidity sensors. Jin et al. developed an AAO-based capacitance gas sensor to detect and quantify polychlorinated biphenyls (PCBs), which are important environmental contaminants, with an LOD of 8 · 108 M at room temperature . The behavior of capacitance in the AAO sensor was modeled on a parallel-plate capacitor and indicated an exponential increase of capacitance with the PCB concentration. The integration of AAO with other materials (e.g., tungsten-trioxide layers) was introduced by Khatko et al. to improve the sensitivity of a resistance-based sensor for toxic gas detection (i.e. nitrogen dioxide, ammonia and ethanol) . The resistance increased with the concentration of these gases in air with an LOD of 1 ppm. Apart from gas sensing, Wang et al. developed an AAO-conductometric biosensor for the label-free detection of DNA molecules based on changes of ionic conductance of nanopores as result of DNA binding . In another study, Yang et al. used a similar approach to fabricate AAO-conductometric sensors for establishing the activity of enzyme immobilized inside AAO pores 36 http://www.elsevier.com/locate/trac . The urease enzyme was used as a model and immobilized onto AAO by four different strategies (i.e. physical absorption, absorption and reticulation, absorption and chitosan covering and reticulation and chitosan covering) . 5. Miscellaneous principles for chemical sensing and biosensing In addition to optical and electrochemical methods, AAO has been used as a substrate for sensing applications based on other detection principles, of which surface acoustic wave (SAW), matrix assisted laser desorption ionisation (MALDI) and quartz crystal microbalance (QCM) are the most noteworthy examples. Several developments of SAW-AAO and QCM-AAO sensing devices for humidity, gas, organic vapor detection and enzyme activity have been reported, demonstrating high sensitivity and outstanding performance [90,91]. 6. Conclusions In this review, we summarized recent advances in the application of nanoporous AAO as a platform to develop chemical-sensing devices and biosensors. We presented relevant examples of detection concepts and developed devices based on optical and electrochemical detection principles. The key features of AAO include simple, inexpensive self-ordering fabrication, large surface area, well-defined and controllable porous structures at the nanometric scale, biocompatibility, easy functionalization of inner pore surfaces, and stable optical, thermal and chemical properties. The combination of these characteristics makes nanoporous AAO a highly attractive material for the development of a broad range of sensing devices for many applications. Furthermore, AAO can be structurally engineered with complex pore structure and chemically modified with desired functionalities, which can considerably improve its performance in multi-functional and smart sensing devices. We expect that further progress on structural and chemical modifications of AAO will facilitate the development of AAO-based devices with superior performances. This research field is broad and multidisciplinary, involving several disciplines from materials science, nanotechnology, optics, electrochemistry, cell biology and medicine. AAO chemical sensors and biosensors have been used in a broad range of applications from gases, vapors, organic molecules, biomolecules (DNA, proteins, antibodies) and cells (viruses, bacteria, cancer cell) in air, water and biological environments. In general, devices based on optical detection (SERS, SPR, LSPR or RIfS) showed lower LODs than those based on electrochemical Trends in Analytical Chemistry, Vol. 44, 2013 analytical systems. In particular, the development of small optical spectrometers (i.e. mobile spectrometers) and their cost-competitive price make them an attractive tool to develop portable point-of-care biosensing systems. Additional advantages of optical systems are their easy fabrication and implementation into more complex systems (e.g., microfluidics). Worth noting is that the speed of optical measurements is faster than that of electrochemical systems, although the choice of the biosensing system will depend on many factors (e.g., speed of measurements, price of equipment, analytes, measurement conditions, stability and accuracy). Finally, we conclude that there is a bright opportunity for further advances and developments of sensing and biosensing devices based on AAO, especially through further miniaturization and integration into lab-on-chip systems. The design of implantable biosensors with the ability to monitor biological systems in vivo and real time is promising for the application of AAO immunosensors, even though it is yet to be explored. Biomedical applications for point-of-care biodiagnostics and environmental detection of toxic agents are two areas of focus for future applications. Acknowledgments The authors acknowledge the financial support of the Australian Research Council (FT 110100711) and the University of Adelaide for this work. References                    Z. Dai, H. Ju, Trends Anal. Chem. 39 (2012) 149. A. Ghicov, P. Schmuki, Chem. Commun. 20 (20) (2009) 2791. H. Chik, J.M. Xu, Mater. Sci. Eng. R43 (2004) 103. A.P. Li, F. Müller, A. Birner, K. Nielsch, U. Gösele, J. Appl. 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