DEPOSITION AND CHARACTERIZATION OF LACCASE THIN FILMS OBTAINED BY MATRIX ASSISTED PULSED LASER EVAPORATION

We focused our attention on Laccase since it is an enzyme widely used as biological recognition component in biosensors. Laccase was deposited by Matrix Assisted Pulsed Laser Evaporation (MAPLE) working at conditions reported in Table 1, and a characterization study was carried out. We have considered the MAPLE, a laser deposition technique, exploitable within strategies for enzyme immobilization because it is able to produce thin films of organic material [1]. In MAPLE, a pulsed laser beam is focused inside a vacuum chamber and impinges on the surface of a rotating target. The target consists of a frozen solution of the material of interest that is diluted in an appropriate solvent. Thus, when the laser beam impacts the target, the laser pulsed energy is mainly absorbed by the solvent and converted to thermal energy, allowing the solvent to vaporize while the material of interest is deposited as a thin film [1]. In order to carry out a characterization study on Laccase thin films, Laccase was deposited on suitable substrates. Laccase layers deposited on silicon were investigated by Fourier Transform InfraRed (FTIR) spectroscopy and Atomic Force Microscopy (AFM) to evaluate the characteristic chemical bonds and morphology respectively. In order to estimate the amount of deposited enzyme, Quartz Cristal Microbalance (QCM) electrodes were used. Moreover, activity of the Laccase thin film was determined by colorimetric assay and amperometric detection by using glass and screen printed carbon electrode as deposition substrates. Figure 1 shows the images from AFM analysis, where films obtained by MAPLE are displayed at different magnifications. The morphology of films is characterized by a basal layer with grains of hundreds of nanometres (1A) that cover the whole silicon substrate, giving a Laccase thin film. Scanning on a smaller area (1B), randomly round shape grains with sizes of 50-100 nm, inhomogeneously chained together, can be observed. Figure 2 shows the comparison between the FTIR spectrum of Laccase obtained by drop-cast and those of Laccase deposited by MAPLE. The spectra are highly overlapped showing the same absorption peaks. FTIR analysis demonstrated that Laccase underwent no substantial modification in molecular structure during the deposition process. However, we have to underline that modifications of enzyme cannot be excluded even in the presence of identical FTIR spectra, indeed IR spectroscopy can give information only on primary and secondary structures of the enzyme [1, 2]. Therefore, the determination of the enzymatic activity is fundamental to claim structural integrity of the deposited enzyme. As far as the Laccase activity is concerned, the spectrophotometric assay, using syringaldazine as the enzyme substrate [3], showed that about 10% of deposited enzyme was active. Figure 3 shows the colour development due to the oxidation of syringaldazine. This result was also corroborated by electrochemical analysis. The chronoamperometric response with respect to the blank signal (Figure 4) showed a distinct signal due to the electrodic reduction of the ortho-quinone produced from catechol by the enzymatic conversion. These evidences demonstrate that MAPLE enables the Laccase thin film deposition, although partially active. Further investigations will be carried out to obtain improved performances of the enzyme deposited with MAPLE by optimizing the deposition conditions. References [1] A. P. Caricato, A. Luches, Appl. Phys. A 105, 2011, pp 565-582. [2] Y. Mei, L. Miller, W. Gao, R. A. Gross, Biomacromolecules 4, 2003, pp 70-74. [3] J. P. Ride, Physiol. Plant Pathol. 16, 1980, 187-196.