Real-Time Monitoring of Invertase Activity Immobilized in Nanoporous Aluminum Oxide
ABSTRACT In this work, we demonstrate the activity of enzyme invertase immobilized in the pores of nanoporous anodized 3 μm thick aluminum oxide (AA). The porous anodic alumina has uniform nanosized pores with an interpore distance of p = 100 nm, with pore diameters on the order of 60-65 nm. The pores trap the enzyme and continuous monitoring of the activity is carried out in a flow cell where the substrate is made to flow and the product is detected. The activity of the immobilized enzyme has been determined for the different concentrations of sucrose and for pH ranging from 3 to 6.5. Maximum activity was found for pH 4.5. Adsorption of the enzyme followed by its interaction with the substrate have been analyzed using confocal laser scanning microscopy (CLSM) and surface plasmon spectroscopy (SPR) and the results obtained show excellent correlation. SPR results show a biphasic kinetics for the adsorption of the enzyme as well as its interaction with the substrate with rates of adsorption for the enzyme at k = 2.9 × 10(5) M(-1) s(-1) and 1.17 × 10(5) M(-1) s(-1). The rate of interaction of the substrate with the invertase is initially rapid with k = 4.49 × 10(5) M(-1) s(-1) followed by a slower rate 1.43 × 10(4) M(-1) s(-1).
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ABSTRACT: In this study, we demonstrate that an optimal design of the pore geometry and shape of sensing platforms based on nanoporous anodic alumina (NAA) photonic structures is critical to develop optical sensors with improved capabilities. To this end, two types of NAA photonic structures featuring different pore geometries (i.e. pore lengths and diameters) and shapes (i.e. straight and modulated pores) are produced and their optical characteristics assessed systematically by reflectometric interference spectroscopy (RIfS). The geometric features (i.e. pore lengths, diameters and shapes) are systematically modified in order to establish the optimization paths for the sensitivity, low limit of detection and linearity of these optical sensing platforms. The obtained results reveal that an optimal design of these nanoporous photonic structures can enhance their sensitivity, achieve lower limit of detection and improve their linearity for both nonspecific and specific detection of analytes. Therefore, as this study demonstrates, a rational design of optical nanoporous sensing platforms is critical in the development of reliable, sensitive, robust, inexpensive and portable optical systems for a broad range of sensing applications.11/2014; DOI:10.1021/ph500316u
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ABSTRACT: Electrochemical anodization of pure aluminum enables the growth of highly ordered nanoporous anodic alumina (NAA) structures. This has made NAA one of the most popular nanomaterials with applications including molecular separation, catalysis, photonics, optoelectronics, sensing, drug delivery, and template synthesis. Over the past decades, the ability to engineer the structure and surface chemistry of NAA and its optical properties has led to the establishment of distinctive photonic structures that can be explored for developing low-cost, portable, rapid-response and highly sensitive sensing devices in combination with surface plasmon resonance (SPR) and reflective interference spectroscopy (RIfS) techniques. This review article highlights the recent advances on fabrication, surface modification and structural engineering of NAA and its application and performance as a platform for SPR- and RIfS-based sensing and biosensing devices.Sensors 07/2014; 14(7):11878-11918. DOI:10.3390/s140711878 · 2.05 Impact Factor
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ABSTRACT: A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature's enzymes-complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nm-scale electrodes at the pore entrances/exits of Anodic Aluminum Oxide Membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions - a one-step 'reverse' sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6-20 min-1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min-1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogenous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme-complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone (4Me-Umb) was supplied at the exit face of the reactor affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.ACS Nano 07/2014; 8(8). DOI:10.1021/nn502181k · 12.03 Impact Factor