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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- "Sucrose/invertase interaction is monitored by surface plasmon resonance (SPR) measurement. Reproduced with permission, (Dhathathreyan 2011). Copyright 2011 American Chemical Society device, for the detection of either biomolecules such as proteins and nucleic acids or heavy metal contaminants in water (Prakash et al. 2012a, b). "
ABSTRACT: Species transport in nanocapillary membrane systems has engaged considerable research interest, presenting technological challenges and opportunities, while exhibiting significant deviations from conventionally well understood bulk behavior in microfluidics. Nonlinear electrokinetic effects and surface charge of materials, along with geometric considerations, dominate the phenomena in structures with characteristic lengths below 100 nm. Consequently, these methods have enabled 3D micro- and nanofluidic hybrid systems with high-chemical selectivity for precise manipulation of mass-limited quantities of analytes. In this review, we present an overview of both fundamental developments and applications of these unique nanocapillary systems, identifying forces that govern ion and particle transport, and surveying applications in separation, sensing, mixing, and chemical reactions. All of these developments are oriented toward adding important functionality in micro-total analysis systems.Journal of Nanoparticle Research 08/2012; 14(8-8). DOI:10.1007/S11051-012-0951-0 · 2.28 Impact Factor
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ABSTRACT: 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.ACS Applied Materials & Interfaces 06/2012; 4(7). DOI:10.1021/am300648j · 6.72 Impact Factor
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ABSTRACT: Sol-gel copolymer-templated mesoporous silica films with a thickness of 70 nm and interpore spacing of 4.34 nm were fabricated on gold layer covered glass substrates for application as a wavelength-interrogated surface plasmon resonance (SPR) sensor. The resonance wavelength (λ(R)) of the sensor with a solution sample was determined by absorptiometry at a given incident angle. A comparison between the experimental data obtained with the coated and uncoated SPR chips demonstrated that the mesoporous silica film effectively enhanced sensor response to individual adsorption of cysteamine molecules and lead(ii) ions. An approximate proportional relationship between the resonance-wavelength shift of the sensor and the volume fraction of analyte molecules adsorbed in the mesoporous silica film was obtained by numerical simulation. Porosities of 0.865 and 0.785 for the two silica films used as well as the volume fractions of 0.048 and 0.116 for adsorbed lysozyme and cysteamine molecules were determined by fitting the simulation results to the experimental data. The adsorbed amount of cysteamine (∼0.5 nm) is equivalent to more than 16 full monolayers on the geometric surface of the mesoporous silica film used. In contrast, an equivalence of less than 2 full monolayers for adsorbed lysozyme molecules (3 nm × 3 nm × 4.5 nm) suggests that the mesoporous silica film has good size-selective adsorption capability due to its uniform pore size distribution. Cysteamine modification of the mesoporous silica film renders the SPR sensor able to detect lead(ii) ions at concentrations as low as 1 nM.The Analyst 08/2012; 137(20):4822-8. DOI:10.1039/c2an16057a · 4.11 Impact Factor