doi:10.1016/j.bios.2007.07.011
Copyright © 2007 Elsevier B.V. All rights reserved.
Anodic porous alumina as mechanical stability enhancer for LDL-cholesterol sensitive electrodes
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Enrico Sturaa, Debora Bruzzeseb, Federica Valerioc, Valentina Grassoc, Pietro Perloc and Claudio Nicolinia, b, 
, 
aFondazione Elba, Piazza SS. Apostoli 66, 00100 Rome, Italy
bNanoworld Institute-CIRSDNNOB and Biophysics Division, University of Genova, Corso Europa 30, 16132 Genova, Italy
cCentro Ricerche FIAT, Advanced Product Technologies, Strada Torino 50, 10043 Orbassano (TO), Italy
Received 27 April 2007;
revised 10 July 2007;
accepted 24 July 2007.
Available online 28 July 2007.
Abstract
In this work, to improve the mechanical stability of electrodes based on P450scc for LDL-cholesterol detection and measure, anodic porous alumina (APA) was used. This inorganic matrix, which pores can be tuned in diameter modifying the synthesis parameters, was realized with cavities 275 nm wide and 160 μm deep (as demonstrated with AFM and SEM measurement), to allow the immobilization of P450scc macromolecules preserving their electronic sensitivity to its native substrate, cholesterol. Even if the sensitivity of the APA + P450scc system was slightly reduced with respect to the pure P450scc system, the readout was stable for a much longer period of time, and the measures remained reproducible inside a proper confidentiality band, as demonstrated with several cyclic voltammetry measures. To optimize the adhesion of P450scc to APA, a layer of poly-l-lysine, a poly-cathion, was successfully implemented as intermediate organic structure.
Keywords: Anodic porous alumina; P450scc; Modified electrode
Fig. 1. APA functionalised rhodium–graphite s.p.e. working electrode. Schematic view illustrating the direct electron transfer between the cytochrome P450scc catalytic ‘core’ and the APA modified working electrode. In the box is shown the specific interaction between the cytochrome P450scc negative surface (blue) and the positive charges of poly-l-lysine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 2. Topographic AFM image of rhodium–graphite s.p.e. working electrode before, after the APA deposition and after functionalization. (A) Top view of rhodium–graphite s.p.e. working electrode before the increase of its surface by APA membrane; (B) top view of the same rhodium–graphite s.p.e. after the APA deposition on its working electrode surface. Scale; (C) top view of APA nanopores after the working electrode surface functionalization (physical adsorption) with PLL and cytochrome P450scc. Scale bars correspond to 1 μm.
Fig. 3. SEM image of APA membrane. A cross-section view of APA membrane resulting by the anodization at the end of the second microstructuring process and before P450scc immobilization. Scale bar 10 μm.
Fig. 4. Current response of screen-printed electrode with APA functionalized by PLL (curve a) and with APA functionalized by PLL and cytochrome P450scc (curve b). All measurements investigated in K phosphate buffer (10 m M, pH 7.4) at a scan rate of 20 mV s−1, equilibration time 2 min. Results are representative of one of three similar experiments.
Fig. 5. Cyclic voltammograms (CV), showing the I–V current–voltage curves of s.p.e. of APA–P450scc Electrode in presence of substrate LDL-cholesterol. The s.p.e. of APA–P450scc electrode was investigated, after a month, in a 10 mM K-phosphate buffer pH 7.4 in presence of LDL-cholesterol. (a) P450, (b) LDL 0.5 mg/ml, (c) LDL 1.1 mg/ml and (d) LDL 1.6 mg/ml. Results are representative of one of three similar experiments.
Fig. 6. Time-stability: cyclic voltammograms (CV), showing the I–V current–voltage curves of s.p.e. of APA–P450scc. Electrode in presence of substrate LDL-cholesterol. The s.p.e. of APA/P450scc electrode was tested, after a month, in a 10 mM K-phosphate buffer pH 7.4 in presence of LDL. (a) P450, (b) LDL 0.5 mg/ml, (c) LDL 1.1 mg/ml and (d) LDL 1.6 mg/ml. Results are representative of one of three similar experiments.
Fig. 7. Multiple cycles of voltammetry: cyclic voltammograms (CV), showing the I–V current–voltage curves of s.p.e. of APA–P450scc. Electrode in presence of substrate LDL-cholesterol. The s.p.e. of APA/P450scc was tested, after 3 months of shelf life, in a 10 mM K-phosphate buffer pH 7.4 in presence of LDL 1 mg/ml.
Table 1.
Comparison among the results of the works about cholesterol detection using P450scc published by our group in the last years
Corresponding author at: Nanoworld Institute-CIRSDNNOB and Biophysics Division, University of Genova, Corso Europa 30, 16132 Genova, Italy. Tel.: +39 01035338220; fax: +39 01035338215.
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