About mL of freshly prepared matrix guest

About 2mL of freshly prepared matrix/guest solution was placed in the target holder inside the vacuum chamber and the target was frozen by placing it in thermal contact with liquid nitrogen contained in a reservoir connected to the MAPLE deposition system. The target temperature was lowered to about −130°C and kept at this value during the whole deposition procedure. The vacuum chamber pressure was lowered, after target freezing, to about 10−4Pa, but its values increased by about one order of magnitude during deposition due to the presence of matrix vapor generated by laser pulses. The substrate was placed in front of the target at a distance of about 0.9cm. Such unusually short target to substrate distance was chosen to maximize the deposition yield for sample characterization, since here we are focused on the deposition of undamaged lipase rather than on the optimization of the film morphology. The Nd:YAG pulsed laser was operated at its fundamental wavelength (1064nm). Even if the Q-switched Nd:YAG laser can be also operated at wavelengths corresponding to second, third or fourth harmonics (532nm, 355nm, 266nm respectively), the IR laser wavelength was chosen in order to minimize photochemical damage of lipase, since it has a large adenosine triphosphate band [33], centered at 280nm, in the UV region mainly used for MAPLE deposition. The laser beam reached the target at an angle of 45°, partially focused to an ellipsoidal area of about 1.0mm by 1.4mm. The target was moved back and forth by a computerized 2D translation system so that, in order to avoid drilling, the beam scanned (one or more times, depending on the total number of pulses) a target area of about 1.5cm2. The values of the parameters of the laser beam (pulse repetition rate: 4 pulses per second, energy pulse: see Table 1, and number of pulses: 29720) have been discussed elsewhere [5]. Target composition for each sample is shown in Table 1 together with the lowest pulse energy required to get a MAPLE deposition. In order to facilitate subsequent FTIR analysis of the samples, 13mm diameter KBr pellets were chosen as substrates for MAPLE deposition. FTIR spectra were recorded, in the 4000–400cm−1 range, using a spectrometer equipped with a DTGS KBr (deuterated triglycine sulphate with potassium bromide windows) detector. A spectral resolution of 2cm−1 was chosen and each spectrum represented an average of 32 scans, corrected for the spectrum of the blank KBr pellet. Atomic force microscopy (AFM) analysis was performed by means of a Nanoscope IIIa AFM (Veeco Instruments Inc., USA), operating in tapping mode (scan size and rate of 1μm and 1Hz, respectively), equipped with a silicon tip having nominal curvature radius of about 5nm. In order to assess the functionality of the deposited lipase, hydrolysis tests were performed by emulsifying 1mL of soybean oil in 2mL of distilled water by means of the surfactant tween 80® and magnetic stirring (500rpm). After the emulsion was formed, the magnetic stirring was set at 200rpm, the biocatalyst anchored to the solid support were immersed in the reaction mixture kept under stirring for 24h. The reaction products were qualitatively characterized by reverse phase thin layer chromatography (RP-TLC), using precoated glass plates RP-18 and acetonitrile-ethylacetate 2.5:1 as mobile phase. 60μL of each reacted mixture and of the untreated soybean oil were dissolved into 1mL of dichloromethane and droplets of 2μL of these solutions were deposited on the pencil spot on the bottom of the chromatographic plate. The plates were immersed for about 1cm into the mobile phase and left for about fifteen minutes, then the solvent front was marked. The chromatographic plates were then dried at 150°C on a hot plate. The chromatograms were detected by spraying H2SO4 4N on the dried chromatographic plates and heating them at 150°C.
Results and discussion Last column in Table 1 reports the laser pulse energy required to obtain a MAPLE film on the substrate. The addition of m-DOPA actually lowered the ablation threshold of the target thus allowing using a lower laser energy pulse. This behavior can be explained since around 1064nm (the laser beam wavelength) there is the third overtone of C–H stretching of aromatic groups (catechol side chain of m-DOPA), while water does not have absorption bands near that wavelength: the presence of an even small amount of m-DOPA increased the overall laser energy absorption of the target.