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).

Nanoporous Anodic Alumina: A Versatile Platform for Optical Biosensors

“…A similar study was carried out by Koutsioubas et al [64], who characterised the adsorption and desorption of BSA onto a SPR-NAA system at different values of pH. Dhathathreyan demonstrated that this system is able to quantify real-time biological events, such as enzymatic reactions [65]. In this study, enzyme invertase was immobilised onto the nanoporous network of NAA, and its activity at different pHs and substrate concentrations was established when digesting sucrose to produce glucose and fructose.…”

Label-Free Real-Time Quantification of Enzyme Levels by Interferometric Spectroscopy Combined with Gelatin-Modified Nanoporous Anodic Alumina Photonic Films

“…[17][18][19][20] In particular, the use of nanoporous materials such as porous silicon and nanoporous anodic alumina (NAA) has resulted in several sensing systems featuring unique cutting-edge capabilities for detecting enzymes by means of different strategies. [21][22][23][24] Optical sensing platforms based on nanoporous materials can provide multiple advantages such as large specific surface area to accommodate chemically selective probes and enhance optical signals from analyte molecules, homogeneous and versatile nanoporous structure that can be engineered to create a broad range of photonic structures with precisely designed optical signals, chemical and physical stability and so forth. 25,26 The combination of gelatin-modified photonic structures based on porous silicon and reflectivity spectroscopy was introduced by Killian et al, who made a good use of this sensing concept to detect levels of metalloprotease enzyme released from stimulated human macrophage cells.…”

Advanced Structural Engineering of Nanoporous Photonic Structures: Tailoring Nanopore Architecture to Enhance Sensing Properties

“…As far as the different materials used to develop optical sensing platforms is concerned, nanomaterials have enabled the development of highly sensitive and sophisticated systems such as surface enhanced Raman spectroscopy (SERS), surface plasmon resonance spectroscopy (SPR), localized surface plasmon resonance spectroscopy (LSPR) and others. [6][7][8][9][10][11][12][13][14][15] These optical techniques can achieve detection limits as low as single molecules through the amplification of 3 electromagnetic fields generated by the excitation of localized surface plasmons. 16 Another example of sensitive optical technique extensively used to develop optical sensors is reflectometric interference spectroscopy (RIfS).…”