Langmuir 2007, 23, 1147-1151
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Thermal Stability of Lysozyme Langmuir-Schaefer Films by FTIR Spectroscopy E. Pechkova,†,‡ P. Innocenzi,§ L. Malfatti,† T. Kidchob,§ L. Gaspa,§ and C. Nicolini*,†,‡ Fondazione ELBA, Piazza Santissimi Apostoli 66, 00187 Roma, Laboratorio di Scienza dei Materiali e Nanotecnologie, UniVersita` di Sassari e Nanoworld Institute, Palazzo Pou Salit, Piazza Duomo 6, 07041 Alghero (SS), and Nanoworld Institute and Biophysics Eminent Chair, UniVersity of GenoVa, Corso Europa 30, 16132 GenoVa, Italy ReceiVed July 7, 2006. In Final Form: October 30, 2006 Fourier transform infrared spectroscopy has been applied to study the thermal stability of multilayer LangmuirSchaefer (LS) films of lysozyme deposited on silicon substrates. The study has confirmed previous structural findings that the LS protein films have a high thermal stability that is extended in a lysozyme multilayer up to 200 °C. 2D infrared analysis has been used here to identify the correlated molecular species during thermal denaturation. Asynchronous 2D spectra have shown that the two components of water, fully and not fully hydrogen bonded, in the high-wavenumber range (2800-3600 cm-1) are negatively correlated with the amine stretching band at 3300 cm-1. On the grounds of the 2D spectra the FTIR spectra have been deconvoluted using three main components, two for water and one for the amine. This analysis has shown that, at the first drying stage, up to 100 °C, only the water that is not fully hydrogen bonded is removed. Moreover, the amine intensity band does not change up to 200 °C, the temperature at which the structural stability of the multilayer lysozyme films ceases.
Introduction Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques represent a “classical” tool for biofilm engineering recently improved and extended to a wide variety of proteins.1 Indeed, new properties have been emerging in terms of the heatproof characteristic2 and long-range stability.3 Furthermore, on the basis of this technology, over the past few years new nanostructured biomaterials have been introduced from the use of the above technology in several fields of science and technology with far reaching implications.4-6 Protein layers obtained by the LB technique have shown good mechanical and thermal stability that can be used for functional biodevices and biomaterials.7 The deposition of protein films by the LB technique is an important process extendable to all classes of proteins for a wide range of applications1,7 alternative to the self-assembly technology yielding similar heat-proof materials but only in rhodopsin-based membranes.8 Using lysozyme as a model system, water uptake and release from protein thin films was originally studied by a combination of gravimetric and infrared techniques9 and later by a combined gravimetric and calorimetric technique,10 pointing both to a quite slower dynamics than in polymer films of * To whom correspondence should be addressed. Phone: +3901035338217. Fax: +3901035338215. E-mail:
[email protected]. † Fondazione ELBA. ‡ University of Genova. § Universita ` di Sassari e Nanoworld Institute. (1) Nicolini, C. Trends Biotechnol. 1997, 15, 395-401. (2) Nicolini, C.; Erokhin, V.; Antolini, F.; Catasti, P.; Facci, P. Biochim. Biophys. Acta 1993, 1158, 273-278. (3) Paddeu, S.; Erokhin, V.; Nicolini, C. Thin Solid Films 1996, 284-285, 854. (4) Nicolini, C. Ann. N. Y. Acad. Sci. 1996, 799, 297-311. (5) Nicolini C. Ann. N. Y. Acad. Sci. 1998, 864, 435-441. (6) Nicolini, C.; Erokhin, V.; Ram, M. K. In Nano-surface chemistry; Rosoff, M., Ed.; Marcel Dekker: New York, 2001; pp 141-212. (7) Nicolini, C.; Pechkova, E. J. Nanosci. Nanotechnol. 2006, 6, 2209-2236. (8) Shen, Y. C.; Safinya, R.; Liang, K. S.; Ruppert, A. F.; Rothschild, K. J. Nature 1993, 366, 48-50. (9) Smith A. L.; Shirazi H. M., Mulligan S. R. Biochim. Biophys. Acta 2002, 1594, 150-159. (10) Careri, G.; Giansanti, A.; Gratton, E. Biopolymers 1979, 18, 1187-1203.
comparable thickness10 and to the relevance of these findings to protein dynamics.9,10 This paper concerns an in-depth study and characterization of a multilayer of chicken egg white lysozyme protein by temperature-dependent in situ Fourier transform infrared (FTIR) spectroscopy with new evidence on the physical properties of the correlated molecular species, extending earlier studies and findings with FTIR.11,12 By multilayer deposition it is possible to obtain thin protein films of several layers; up to 200 layers can be deposited with a good quality. The changes in the protein structure13 induced by LB deposition have been the subject of specific studies, and infrared spectroscopy has revealed a versatile and simple tool to investigate the modifications of the protein layers. Infrared spectroscopy can be used, in fact, to study the protein conformation14 and for probing the temperature-induced changes in proteins15,16 and protein LB layers. Materials and Methods For all the experiments we use chicken egg white lysozyme (enzyme code 3.2.1.17), molecular weight 14700, commercially available from Sigma (cod. L6876). LS Biofilm Engineering. The protein monolayer was prepared by using an in-house-built LB Teflon trough with a bath surface area of 0.44 × 0.11 m supplied with the appropriate software.1,2 The protein solution of about 40 mg/mL was prepared using the lysozyme protein and distilled water. The protein solution was filtered with a 0.22 µm filter. The Teflon trough was filled with pure distilled filtered water, and the paper Wilgelmi plate was stabilized for the surface pressure measurements. (11) Bramanti, E.; Benedetti, E.; Nicolini, C.; Berzina, T. S.; Erokhin, V.; D’Alessio, A.; Benedetti, E. Biopolymers 1997, 42, 227-237. (12) Pepe, I.; Ram, M. K.; Paddeu, S.; Nicolini, C. Thin Solid Films 1998, 327-329., 118-122. (13) Voet, D.; Voet, J.; Pratt, C. Fundamentals of Biochemistry; John Wiley & Sons Inc.: New York, 1999. (14) Dermirdoven, N.; Cheatum, C. M.; Chung, H. S.; Khalil, M.; Knoester, J.; Tokmakoff, A. J. Am. Chem. Soc. 2004, 126, 7981-7990. (15) Meersman, F.; Haremans K. Biochemistry 2003, 42, 14234-14241. (16) Van Stokkum, I.H.M.; Lindsell, H.; Hadden, J.M.; Haris, P. I.; Chapman, D.; Bloemendal, M. Biochemistry 1995, 34, 10508-10518.
10.1021/la061970o CCC: $37.00 © 2007 American Chemical Society Published on Web 12/09/2006
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Figure 1. Dispersion of the refractive index of multilayer LangmuirBlodgett films deposited on silicon. The film thickness is 230 nm. Protein molecules were placed at the air/water interface utilizing the fine Hamilton 100 µL syringe by small droplet deposition all over the bath surface. The two-dimensional system of protein molecules at the air/water interface was compressed by two Teflon barriers with a speed of 100 mm/min up to a surface pressure of 25 mN/m.17 The dependence of the surface pressure on the barrier position (a π-A isotherm) was obtained at a constant room temperature until a dense packing of molecules in the monolayer was reached. The floating monolayer was transferred onto the surface of the solid supports by the Langmuir-Schaefer (or horizontal lift) method,18 where the prepared substrate horizontally touches the monolayer, and the layer transfers itself onto the substrate surface. Langmuir-Blodgett multilayer films of lysozyme were deposited onto silicon wafers (dimensions about 5 × 5 mm), p-type borondoped (100) Jocam, one side polished, of 440 µm thickness. To obtain a sufficient FTIR signal, 200 layers were deposited onto the silicon substrate. The regularity and uniformity of deposition were controlled simultaneously by nanogravimetric measurements,19 including the area per molecule calculation. The resulting thickness of the lysozyme multilayer was about 200 nm. FTIR Measurements. Infrared absorption spectra were measured by an FTIR spectrophotometer (Nicolet Nexus) mounted with a KBr-DTGS detector, in the 5000-400 cm-1 range; 256 scans were obtained with a (4 cm-1 resolution. The spectra were recorded in absorption mode on Langmuir-Blodgett multilayer films of lysozyme. The background was recorded using a reference silicon wafer. In situ FTIR analysis as a function of the temperature was done employing a Hellma heating jacket. The measurements were performed from 25 up to 250 °C, with steps of 50 °C and a heating rate of 5 °C‚min-1. FTIR Data Fitting. A Gaussian peak fitting procedure has been applied to the FTIR spectra (Microcal Origin software). The quality of the fitting was evaluated on the basis of the χ2 values (on the order of 10-6) and correlation coefficient values g0.998. Baseline correction was performed by a concave rubberband correction method (OPUS 5.5 software) using 64 baseline points and 20 iterations. Two-Dimensional FTIR Correlation Spectroscopy. 2D correlation has been performed on the basis of the comparison of the spectral intensity changes as a function of the temperature (the perturbation parameter). The synchronous and asynchronous correlation maps have been plotted taking the first absorption spectrum as a reference using OPUS 5.5 software. Spectroscopic Ellipsometry. The refractive index and thickness of the films were measured by spectroscopic ellipsometry (SE R-Wollam). The data fitting procedure was performed by Wollam software. Circular Dichroism Measurements. Circular dichroism data of lysozyme in solution and a thin protein LS film were taken with a (17) Pechkova, E.; Nicolini, C. Trends Biotechnol. 2004, 22, 117-122. (18) Langmuir, I.; Schaefer, V. J. Am. Chem. Soc. 1938, 60, 1351. (19) Pechkova, E.; Nicolini, C. From art to Science in Protein crystallography by means of NanotechnologysOne year later. In Trends in Nanotechnology Research; Dirote, E. V., Ed.; Nova Publishers, Inc.: New York, 2004; pp 31-50.
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Figure 2. In situ FTIR absorption spectra, in the range 3020-2830 cm-1, of the lysozyme films deposited on silicon and treated at different temperatures. Jasco J-710 (Jasco, Japan) spectropolarimeter, equipped with a Peltier thermostatic cell holder. All spectra were recorded in a 0.05 cm path length quartz cell under a nitrogen atmosphere, using the following parameters: time constant 4 s, scanning speed 20 nm min-1, bandwidth 2 nm, sensitivity 10 mdeg, step resolution 0.5 nm. The photomultiplier voltage did not exceed 600 V in the spectral region measured. The instrument was calibrated with a standard solution of (+)-10-camphosulfonic acid. Each spectrum was averaged five times in the wavelength range 250-180 nm. Protein solution samples were prepared in 15 mM phosphate buffer, pH 8.0, at a protein concentration of about 0.1 mM. All the acquired spectra were corrected for the baseline but not normalized to the amino acid concentration. Therefore, the dependence of the mean residual ellipticity (deg cm2) on the temperature has been measured for lysozyme in an LS film on a quartz substrate (100 layers) and in solution. The film samples were analyzed at various temperatures by heating them according to the following procedure: each sample was heated for 5 min at the desired temperature in an oven. After 2 min was allowed for the samples to come back to room temperature, they were analyzed. The percentage of R-helix, β-sheet, β-turn, and random coil has been determined through the program Jwsse.exe.6,20
Results and Discussion The multilayer Langmuir-Schaefer film of lysozyme was characterized by spectroscopic ellipsometry. The dispersion of the refractive index as a function of the wavelength is shown in Figure 1. The refractive index has been obtained by a Cauchy model (nonabsorbing film on silicon), introducing surface roughness and native silicon oxide as free fitting parameters. The fit gave a thickness of 232 nm and a surface roughness of 10 nm. The FTIR absorption spectra, in the 3020-2830 cm-1 region, of the multilayer LS films are shown in Figure 2. In this range four main bands are observed, at 2962 cm-1 (CH3, νas), 2928 cm-1 (CH2, νas), 2872 cm-1 (CH3, νs), and 2855 cm-1 (CH2, νs).21-23 The effect of the thermal treatment on the LS lysozyme films can be clearly observed from these spectra. The intensities of the bands do not change up to 150 °C. At 200 °C a small decrease in intensity reveals that the thermal stability of the LS films starts to be compromised, while at 250 °C the strong intensity decrease indicates that the proteins are significantly thermally degraded. (20) Carrara, E. A.; Gavotti, C.; Catasti, P.; Nozza, F.; Berutti, Bergotto, L. L; Nicolini C. Arch. Biochem. Biophys. 1992, 294, 107-114. (21) Colthup, N. B.; Daly, L. H.; Wiberley, S. E. Introduction to Infrared and Raman Spectroscopy; Academic Press: San Diego, 1999. (22) Noda, I.; Ozaki, Y. Two-dimensional correlation spectroscopy; Wiley: New York, 2004. (23) Noda, I. Appl. Spectrosc. 1993, 47, 1329.
Thermal Stability of Lysozyme LS Films
Figure 3. Asynchronous 2D infrared correlation spectra in the 30003700 cm-1 interval of LS films during thermal treatment from 25 to 250 °C. The analysis was done in transmission mode and in situ.
Figure 3 shows the contour map of the asynchronous 2D correlation spectrum24 obtained from the transmission spectra recorded during in situ FTIR analysis at different temperatures, from 25 to 250 °C. Blue graded areas indicate negative correlation cross-peaks and green-yellow areas positive correlation crosspeaks. In an asynchronous contour map the sign of the crosspeak gives information about the sequential order of the events along the temperature change. The sign of an asynchronous peak is positive if the intensity change at ν1 occurs before that at ν2. Two correlation squares can be built, between the positive crosspeak centered at 3330 cm-1 and the negative one at 3500 cm-1 and between the negative cross-peak centered around 3250 cm-1 and the positive one at 3330 cm-1. The band centered at 3300 cm-1 is attributed to the N-H stretching mode of the amide (amide A band),25 while the other two bands are attributed to different species of water, the one at lower wavenumbers (∼3250 cm-1) to O-H stretching in a fully H bonded environment and the one at higher wavenumbers (∼3500 cm-1) to O-H stretching of water molecules that are not fully H bonded.26 The asynchronous map shows that the water bands are asynchronously correlated with the amine band and that these three bands respond with a change in intensity with temperature variations. On the basis of correlation analysis, deconvolution of the spectra has been done using three components, one for amines and two for water. To optimize the fit, different numbers of components were used for spectral deconvolution, five Gaussian fitting curves for the spectrum taken at 25 °C and four for the others. The additional component at 25 °C has been introduced because of the change in the baseline induced by the high intensity and broadness of the 3400 cm-1 component (3750 cm-1, O-H stretching in crystallization water11) with respect to the spectra taken at higher temperatures. The 3080 cm-1 component (C-H stretching region) has also been introduced in the deconvolution because of its overlapping within the spectra at lower temperatures. Figure 4 shows the deconvolutions of the FTIR absorption spectra recorded in situ at different temperatures. The three main components, at ∼3400, 3330, and 3200 cm-1, exhibit a change in intensity and position as a function of the thermal treatment and are shifted to lower wavenumbers with respect to the peak value deduced from the 2D correlation spectra (3500, 3330, and 3250 cm-1, respectively). The main peak at 3400 cm-1 shifts to (24) Noda, I.; Dowrey, A. E.; Marcott, C.; Story, G. M.; Ozaki Y. Appl. Spectrosc. 2000, 236A. (25) Liu, Y.; Cho, R.; Sakurai, K.; Miura, T.; Ozaki, Y. Appl. Spectrosc. 1994, 48, 1249. (26) Bayly, J. G.; Kartha, V. B.; Stevens; W. H. Infrared Phys. 1963, 3, 211.
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higher wavenumbers at increased temperatures of treatment. The position of this band is influenced by the O-H stretching of the different water components bonded to the protein, and the shift is attributed to a decreased water content in the lysozyme film. The variation of the band area of the three components represents the calculated average from the deconvoluted spectra of three different samples. Figure 5 shows the change of the area, which was calculated by the deconvolution, as a function of the temperature. It can be observed that the component at 3400 cm-1 (O-H stretching of water molecules that are not fully H bonded) decreases quite quickly at low temperature, within 100 °C, much faster than the other water component at lower wavenumbers, 3200 cm-1, which is attributed to fully H bonded water molecules. Figure 6 shows the FTIR absorption spectra as a function of the heating temperature in the 1850-1400 cm-1 range. In this region the amide I and amide II bands of lysozyme are observed.27 The amide I band (1700-1600 cm-1 region) is mainly due to CdO stretching modes, while the amide II band (1600-1480 cm-1 region) is assigned to the coupling of the N-H in-plane bending and C-N stretching modes. The thermal treatment induces a shift to higher wavenumbers and an intensity decrease of the amide I band centered around 1653 cm-1, which is characteristic of the R-helical structure.28 The shift to higher temperatures of this component in the solid state is attributed to the stabilization of the helical structure upon removal of water molecules. The band shifts from 1653 cm-1 at 25 °C up to 1661 cm-1 at 200 °C. At 250 °C the FTIR spectrum shows, instead, a significant difference, a strong decrease in intensity of the R-helix band that is accompanied by the appearance of new absorption bands at 1680, 1609, and 1716 cm-1. The two bands at 1680 and 1609 cm-1 are typical of intermolecular antiparallel β-sheet14 aggregation and are an indication that at temperatures higher than 200 °C part of the lysozyme proteins tend to form aggregates. The amide II band, which peaks around 1540 cm-1, shows a trend similar to that of the amide I band, a decrease in intensity with an increase of the heating temperature and a shift of the band produced by the protein denaturation. The shift in this case is toward lower wavenumbers. A shoulder at 1555 cm-1 is observed as an overlapped band of the amide II mode and is assigned to β-turns. A new band in the films after treatment at 200 and 250 °C is detected around 1512 cm-1 and assigned to antiparallel β-sheet vibrations due to aggregation of proteins in the LB films. 2D infrared spectroscopy has also been applied to investigate the synchronous changes in the amide region induced by the thermal treatment. In the synchronous correlation map a positive cross-peak at (ν1, ν2) indicates that the temperature-induced changes in intensity, at these wave numbers, are coincidental. Figure 7 shows the schematic contour map of a synchronous 2D FTIR correlation spectrum in the 1450-1850 cm-1 interval. A color scale is used to indicate the intensity changes. Six distinct autopeaks of different intensities are observed in the diagonal from the left top to right bottom of the contour map, at the wavenumbers 1515, 1530, 1550, 1630, 1655, and 1730 cm-1. The autopeaks correspond to the region of the spectrum that shows intensity changes induced by the change of the external variable, which is in this case the temperature variations from 25 up to 250 °C. The autopeaks are all positive, while the crosspeaks can be either positive or negative. Several correlation squares, joining pairs of cross-peaks located at the opposite sides of the diagonal line, can be drawn, indicating the existence of (27) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1960, 83, 712-719. (28) Krimm, S.; Bandekaer, J. AdV. Protein Chem. 1986, 38, 181.
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Figure 4. Deconvolutions of the FTIR absorption spectra recorded in situ at different temperatures.
Figure 5. Evolution of the average area of the three components at 3400, 3330, and 3200 cm-1 as a function of the temperature.
coherent changes of spectral intensities with temperature. In particular, correlation squares between the autopeaks at 1720 and 1655, 1630 and 1550, 1515 and 1550, and 1515 and 1630 cm-1 can be built. The correlation squares indicate a possible related origin of the spectral intensity changes induced by the temperature on the lysozyme films. While the signs in the correlation square between the autopeaks at 1630 and 1550 cm-1 and the relative cross-peaks are all positive, in the other correlation squares the signs of the autopeaks are always positive and the signs of the cross-peaks always negative. In particular, the correlation between the 1720 and 1655 cm-1 modes suggests that the thermal treatment produces acid denaturization in the protein, as shown by the different signs in the R-helix amide I band and the 1720 cm-1 band. Similarly, the 1515 cm-1 band, attributed to protein aggregation, is correlated with the amide II
Figure 6. Average in situ FTIR absorption spectra, in the 18501450 cm-1 range, of the lysozyme films deposited on silicon and treated at different temperatures.
band at 1550 cm-1. The different signs also indicate that the decrease in intensity of the amide II band is accompanied by an increase of the aggregation band. The same phenomenon is the cause of the correlation between the 1515 and 1630 cm-1 bands. On the other hand, the amide I and amide II bands are also correlated and have the same signs, as expected. For a comparison to what occurs in the same temperature range in the lysozyme LB film versus the same lysozymes in solution, we carried out a systematic circular dichroism study in the 180-250 nm spectra to monitor the percentages of R helices, β pleats, and random coils of the protein (Figures 8 and 9). Figure 8 shows the temperature-dependent continuous changes in the mean residual ellipticity versus temperature at 193 nm up to 100 °C, confirming that the lysozyme LS film secondary
Thermal Stability of Lysozyme LS Films
Langmuir, Vol. 23, No. 3, 2007 1151 Table 1. Temperature Dependence of the Lysozyme Secondary Structure in the LS Filma temp (°C)
[R helix] (%)
[β sheet] (%)
[turn] (%)
[coil] (%)
25 50 100 150 200 250
31.1 36.0 39.0 36.7 38.3 29.8
30.1 15.8 11.5 18.0 18.5 0.0
11.5 18.3 20.0 16.0 14.2 26.0
27.2 29.8 29.5 29.4 29.1 44.2
a Percentages of R helices, β sheets, β turns, and random coils determined from CD measurement prove highly reproducible in a wide range of temperature and conditions, with a mean error of 2.9%, comprehensive of three independent measurements and of the leastsquares fit of the wavelength-dependent CD measurements by the software being used.20
Figure 7. Synchronous 2D infrared correlation spectra in the 18001400 cm-1 interval of LS films during thermal treatment from 25 to 250 °C. The analysis was done in transmission mode and in situ.
Figure 8. Temperature dependence of the circular dichroism signal at 193 nm of the lysozyme LS film (gray line) and lysozyme solution (black line). The CD signal for the lysozyme solution displays a melting temperature of about 83 °C. Instead, the lysozyme LB film even after heating up to 200 °C does not display a melting transition and maintains a constant circular dichroism signal at 193 nm.
Figure 9. Typical CD spectra of the lysozyme thin LS film at various temperatures.
structure is preserved at higher temperature contrary to the solution lysozyme. Figure 9 and Table 1 show, respectively, the molar ellipticity and the percentages of R helices, β sheets, β turns, and random coils determined from CD measurement as a function of temperature up to 250 °C in the LS film. The emerging picture is in perfect agreement with the reported FTIR data, whereby the R helix content and the overall structure of the protein remain preserved only up to 200 °C with a dramatic drop in the CD signal at 225 and 250 °C, confirming what was previously reported for all protein systems immobilized in LB thin films.1,7
Conclusions Analysis by FTIR spectroscopy of OH and NH vibration intensities allows the study of the thermal stability of lysozyme, proving that lysozyme LS films made of 200 layers are stable up to 200 °C. In summary, synchronous correlation gives additional information about the structure of lysozymes, while the 2D asynchronous IR analysis of CdO amide vibrations indicates that helical forms increase with temperature, and that above 200 °C lysozyme forms aggregates. Asynchronous 2D spectra have shown indeed that the two components of water, fully and not fully hydrogen bonded, in the high-wavenumber range (2800-3600 cm-1) are negatively correlated with the amine stretching band at 3300 cm-1. On the grounds of the 2D spectra the FTIR spectra have been deconvoluted using three main components, two for water and one for the amine. This analysis has shown conclusively that, at the first drying stage, up to 100 °C, only the water that is not fully hydrogen bonded is removed and that the amine intensity band does not change up to 200 °C, which represents the temperature at which the structural stability of the multilayer lysozyme films ceases. The present findings and earlier FITR studies on the formation of LB films of bovine rhodopsin12 confirmed the above conclusion explaining the striking properties such as maintaining the secondary structure of proteins at a temperature up to 200 °C as shown in Figure 7 and in refs 1, 8, and 29. The present FTIR study, which employs advanced tools for infrared data analysis, appear strikingly confirmed by circular dichroism and thermal denaturation studies (Figures 8 and 9 and Table 1) and further clarifies the organization of the protein film recently independently monitored by other independent biophysical probes7,30 and by µGISAXS technology.31 The emerging nature of the thermal stability induced in the proteins by the LS technology adds further details on the role of water previously suggested.1,7,9,10,32 Acknowledgment. This project was supported by a FIRB grantonOrganicNanosciencesandNanotechnologies(RBNE01X3CE) from MIUR (Ministero dell’Istruzione, Universita` e Ricerca) to Fondazione ELBA and CIRNNOB (Interuniversity Research Center on Organic/Biological Nanotechnology and Nanoscience) of the University of Genova and Sassari. LA061970O (29) Maxia, L.; Radicchi, G.; Pepe, I. M.; Nicolini, C. Biophys. J. 1995, 69, 1440-1446. (30) Pechkova E., Vasile F., Spera R., Fiordoro S., Nicolini C. J. Synchrotron Radiat. 2005, 12, 772-778. (31) Pechkova E., Nicolini, C. J. Cell. Biochem. 2006, 97, 544-552. (32) Rupley, J. A.; Gratton, E.; Careri, G. Trends Biochem. Sci. 1983, 8, 18-22.
