Article pubs.acs.org/Langmuir
Directed Organization of DNA Filaments in a Soft Matter Template Luciano De Sio,*,† Patrizia D’Aquila,‡ Elvira Brunelli,§ Giuseppe Strangi,†,∥ Dina Bellizzi,‡ Giuseppe Passarino,‡ Cesare Umeton,† and Roberto Bartolino† †
Department of Physics, Centre of Excellence for the Study of Innovative Functional Materials, University of Calabria and CNR-IPCF UOS, Cosenza, 87036 Arcavacata di Rende, Italy ‡ Department of Cell Biology and §Department of Ecology, University of Calabria, 87036 Arcavacata di Rende (CS), Italy ∥ Department of Physics, Case Western Reserve University, Cleveland, Ohio 44106, United States S Supporting Information *
ABSTRACT: We have developed a noninvasive, all-optical, holographic technique for permanently aligning liquid crystalline DNA filaments in a microperiodic template realized in soft-composite (polymeric) materials. By combining optical intensity holography with a selective microfluidic etching process, a channelled microstructure has been realized which enables self-assembly of DNA. The striking chemicophysical properties of the structure immobilize the DNA filaments within the microchannels without the need of any kind of surface chemistry or functionalization. Polarized optical, confocal, and electronic microscopies have been used for characterizing the DNA geometry inside the microchannels in terms of birefringence, fluorescence, and nanoscale organization properties. In particular, observation of a far-field diffraction pattern confirms a periodic organization of the DNA filaments inside the polymeric template.
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INTRODUCTION The DNA is the fundamental genetic component of living matter and possesses the hereditary information on living organisms;1−3 investigation of this biomolecule is therefore carried out in many different scientific and technological fields.4−6 Where application oriented studies and DNA-based technologies are concerned, two important aspects concern its “immobilization” and “stretching”; in particular, the amount of immobilized DNA portion directly determines the accuracy, sensitivity, selectivity, and life of a DNA-based biosensor. For this reason, many solutions have been attempted, aiming to increase the amount of DNA that can be immobilized on the transducer surface; in this framework, also nanotechnologies have shown an emerging potential for realizing “on-chip” detection systems. Furthermore, also biosensors exploiting hybridization of complementary DNA oligonucleotides have been widely explored,7,8 and it has been found that one of the most accessible methods for producing miniaturized, high sensitivity biosensors is to synthesize individual oligonucleotides (thiol-modified oligos, amine-modified oligos, etc.) and attach them on a nanostructured surface (Au, CdS, Si, etc.).9 It is also worth noting that, the DNA molecule being a semiflexible polymer, it can be used for mapping or monitoring enzymatic activity. In this case, the resolution can be greatly © 2013 American Chemical Society
enhanced by stretching the DNA, usually by anchoring one of its ends and applying an extension force. In fact, extension forces have been used for monitoring the interaction with proteins that are not fluorescently labeled, and several techniques have been developed, which include the use of forces derived from light,10 magnetic and electric fields,11,12 and hydrodynamic flow.13 Recently, different approaches have been implemented, which can enable to both immobilize, unfold, and stretch molecular DNA by exploiting PDMS microfluidic channels,14 capillary tubes,15 and photoresist templates;16 however, most of the above techniques are quite cumbersome and require a preliminary chemical treatment of surfaces. From a different point of view, an interesting observation is represented by the tendency of flexible polymers in concentrated aqueous solutions to form liquid crystalline phases.17,18 Indeed, the ability of both long and ultrashort, hydrated, double-stranded DNA molecules to form liquid crystal phases has been known for more than 50 years and played a key role in the initial deciphering of the molecular Received: August 25, 2012 Revised: February 8, 2013 Published: February 19, 2013 3398
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structure19,20 (while, in the absence of water, neither long molecules nor short DNA segments exhibit a long-range order). In this paper, we show it is possible to stretch and immobilize DNA filaments inside a holographic grating, using the liquid crystalline properties of DNA filaments. The main idea has been conceived by considering the capabilities of a photopatterned platform, realized in a soft composite polymeric material, to induce a long-range organization in different liquid crystal phases.21 An important achievement of this approach is represented by the possibility of analyzing the diffraction pattern either in “real time” and “post-processing”; indeed, our experiments can be considered as a first step toward a total holographic DNA analysis, where information concerning the polymerization process or nucleation can be obtained by using noninvasive, all-optical, methods. In addition, from an application oriented perspective, it will be possible to use the obtained long-range liquid crystalline phase of DNA to induce order in nanomaterials, thus creating new materials, which possess nanoscale organization for technological applications, such as biologically inspired smart metamaterials. In these devices, the order degree of nanomaterials (e.g., gold nanoparticles, quantum dots, etc.) could be investigated by means of a simple spectral analysis, without using any kind of expensive, and sometime invasive, nanotechnology facility (e.g., scanning/transmission electron microscopy).
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template with different channel width is reported in the Supporting Information 2). These conditions ensure that the influence of the untreated glass surfaces is minimized, while a long-range order of the DNA filaments can be induced by the chemical composition of the polymeric slices (thiol-based system); furthermore, the channel width L is well above the typical optical resolution (∼0.5 μm). Figure 1a
Figure 1. POM view of a droplet of DNA water solution on a glass substrate (a); “empty POLICRYPS” template between parallel polarizers before being infiltrated with the DNA water solution (b) (between crossed polarizers it appears as a uniform dark squarenot shown); the same, between crossed polarizers, after being infiltrated with the DNA water solution (c); transmitted intensity (bright fringes) for different angles between the analyzer and the optical axis of the sample (d−f). Dependence of the diffracted intensity of the structure on the polarization of the impinging probe light (g).
EXPERIMENTAL SECTION
The basic structure is obtained by starting from a periodic liquid crystalline composite material, called POLICRYPS (acronym of polymer liquid crystal polymer slices), which is a nano/microcomposite holographic grating made of slices of almost pure polymer alternated to films of well-aligned nematic liquid crystals (NLC).22,23 In a further step, the NLC is removed by using a microfluidic etching process, and the microchannels of the empty polymeric template can be filled in with different self-organizing materials. Upon considering the DNA strong similarity with liquid crystals (characterized by remarkable anisotropy and long-range order), a new method to align and immobilize DNA molecules has been conceived. In fact, we have found that the microarrays, characterized by a sharp morphology, induce a quite interesting sequence of effects, like stretching and immobilization of dehydrated DNA filaments, without using any kind of surface treatment or external perturbation. The striking feature of the photopatterned channels of our structure is related, in particular, to the hollowing process that removes branches and scaffolding elements, thus preparing cavities that are both sterically and chemically hospitable for DNA strands. In this way, we have immobilized and permanently aligned both ultrashort (27 bp) and long (6459 bp) DNA molecules in a geometry that depends on their length. This circumstance enabled us to investigate some DNA helices by means of a polarized optical microscope (POM), confocal microscope (CM), and an environmental scanning electronic microscope (ESEM). It is worth noting that characterization of samples has been carried out by using only optical and electronic noninvasive techniques, but without the need of exploiting those (quite complicated) “last generation” methods, which make use, for instance, of the properties of local field enhancement given by nanostructured materials.27 In a first attempt, we have used a human whole-genome DNA, which contains ∼48 500 base pairs and has a contour length of about 17.5 μm; detailed information on the DNA sample preparation is reported in the Supporting Information 1, while details on the preparation of the structure can be found in refs 21−23. To identify the optimal values of the geometric parameters of the structure, attempts performed on several, different sample cells, with varying cross-sectional dimensions, have shown that the best performances are exhibited by a structure of d = 10 μm thickness, with a channel width L = 2.5 μm (characterization of the DNA alignment in a polymeric
represents a POM view of a droplet of water solution of DNA deposited on a glass substrate: seemingly, the DNA is randomly aligned, since no macroscopic birefringence is exhibited. By capillary flow, we have injected the same solution into the microchannels of the polymeric template (Figure 1b). During the whole filling process, the sample is kept at a fixed high temperature (80 °C); in this way, the DNA strands are unpaired; meanwhile, the aqueous solution induces a hydrodynamic flow due to the surface tension between the solution and the microchannel surfaces. As a consequence, an extension force is induced, and the single strands are elongated along the channel direction. In fact, it has been shown24 that the desired extension length (defined as the ratio between the initial length and the extended length) of a DNA molecule inside a microfluidic channel can be obtained by controlling the extension force through control of the flow rate (mL/min). In our case, the DNA solution enters into the microchannels of the polymeric template by capillary flow, without using any external control from our side; however, a rough estimation of the extension length can be derived by measuring the flow rate (μm/s) of the DNA water solution in the microchannels under the influence of the capillary force. For a channel width and height of about 2 and 10 μm, respectively, we have measured (by means of an optical microscope) a flow rate of about 80 μm/s, corresponding to about 0.4 mL/min. Thus, by using the model proposed in ref 24, we have estimated a mean extension length of about 0.5; following the worm-like chain (WLC) model,25 which describes the DNA polymer dynamics, we can roughly estimate that this extension corresponds to a tension of ∼0.4 pN. It is worth noting that experiments reported in ref 24 have been performed by using a bacteriophage λ-DNA (≈48 000 bp), whose length is comparable to the length of the human whole genome, utilized in our first experiment (≈48 500 bp); thus, the model proposed in ref 24 can be utilized to roughly estimate the extension force also in our case. Further on, by keeping the filled sample at high temperature for a long time interval (20 min), evaporation of most of the aqueous solvent takes place. It is reasonable to suppose that, in these conditions, due to the presence of water, capillary forces can stretch the DNA along the microchannels, while no covalent bonding can 3399
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operate, since both the presence of water and the high temperature do not allow this process to occur. Subsequently, by slowly (0.5 deg/min) cooling down the sample to room temperature, we may assume that, due to the negative charge density of the DNA helix, a covalent bonding between the thiol group (S−H) present in the photopolymer (NOA-61) and the DNA ends is obtained (as it occurs in the thiolmodified DNA case26). As a matter of fact, it has been already shown, indeed, that the alkyl tails of the thiol induce a homeotropic alignment in liquid crystalline materials due to the electrostatic interaction with hydrophilic cyano groups that are present in the heads of LC compounds.27 Since electrostatic forces play a major role in DNA packaging processes, in our opinion, in analogy to the LC case, it is reasonable to assume that a strong electrostatic interaction between the thiol group and the DNA filaments occurs. In fact, a selforganization process takes place, which gives rise to a uniform and permanent (at least one year of bookshelf life) alignment of DNA filaments, with a considerable long-range order, as suggested by the optical birefringence (Δn = 0.011, measured by using the “optical compensator” method28) exhibited by the sample (POM micrograph of Figure 1c). A useful technique to check the organization of the DNA filaments within the polymeric template is based on POM observations; it includes illumination with polarized light which can be stopped by means of a second polarizer (called analyzer) oriented at 90° with respect to the illumination polarization direction. A birefringent material, located between a polarizer and an analyzer, acts as a retardation plate, rotating the impinging polarization and, thus, yielding an optical contrast with the background. If the sample exhibits a long-range order (that yields a uniform birefringence), the optical contrast can be changed by simply rotating the sample. An incontrovertible experimental evidence of the existence of a filament organization is represented, therefore, by the fact that, once realized, our sample behaves as a uniaxial retardation plate, with its optical axis aligned parallel to the microchannel direction. For an impinging white (noncollimeted) light, the noticeable difference between bright (Figure 1d) and dark (Figure 1f) states is obtained by rotating the sample between the crossed polarizers of the POM; this observation is a clear proof that the DNA filaments are aligned along the channel direction, reflecting a high degree of alignment (see the Supporting Information movie). The low measured value of birefringence, in comparison with typical values (∼0.2) obtained for very well-ordered LC phases, can be attributed to the low initial concentration of DNA in the aqueous solvent (1 mg/mL); indeed, for the used concentration, it has been already shown that the hydrated DNA exhibits a precholesteric phase, whose typical birefringence is of the order of 10−2.29 In addition, to further check the capability of our polymeric template to induce a long-range order in the DNA, we have measured a different physical quantity, that is, the sample optical contrast. This one turned out to be better than 20:1, a value that is comparable with the one measured in typical samples where the LC director is very well aligned. Since a way to investigate well-aligned birefringent materials inside a periodic structures is to observe its diffractive pattern,23 we have also monitored the intensity diffracted by our sample while varying the polarization of a probe impinging light (see ref 23 for the utilized setup). The sinusoidal behavior reported in Figure 1g is a well-known characteristic of periodic structures that contain an aligned birefringent material and is of relevance to us, since we believe it can represent the first step of an approach for a totally holographic DNA analysis and examination.
dUTP; more information concerning the labeling process is reported in the Supporting Information 1. These labels give a fluorescent signal that will enable also CM analysis. First, a water solution of short DNA molecules (27 bp) was injected in the empty polymeric template by using the already described procedure. Figure 2a is an optical micrograph of the DNA
Figure 2. Optical micrograph (a), POM view (b), and ESEM picture of DNA bridge arrays (c) along with the confocal analysis image (d). Far-field 2-D diffraction pattern obtained by colaunching two laser beams of different color (e). High magnification of the “star-like” diffraction pattern (f). Intensity profile along one arm of the diffraction pattern (g).
microbridges stretched along the polymeric slices. In our opinion, the geometry can be explained by taking into account the ratio between the length of the ultrashort DNA filament and the width of the microchannels. When the sample is kept at high temperature, the denatured DNA short molecules are able to stick together “end-to-end”, thus forming rod-shaped aggregates that can behave like much longer segments of DNA. This hypothesized behavior is very similar to the one reported by Nakata et al. in ref 20, where short DNA oligomers are found to exhibit a LC phase due to the end-to-end adhesion and consequent stacking of the oligomers in anisotropic, roadshaped, aggregates. In our case, this process should be driven by the presence of the microconfinement, being very similar to the one that occurs in the case of DNA molecules digested with restriction endonucleases, which produces compatible cohesive ends. Indeed, a cohesive single-stranded end of linear duplex DNA molecules can promote hydrogen bond with a complementary single-strand base sequence from the end of another DNA molecule.30 In a further step, during the cooling down to room temperature, a local self-organization takes place, which follows the “least energy” path, facilitating molecular zippering.31 In order to confirm the capability of our polymeric template to induce DNA alignment, we have also analyzed the same area by means of POM technique; the result is reported in Figure 2b. The quasi-periodic birefringence confirms the organization of the DNA filaments along the microbridges. To investigate the envisaged geometry, after removing its top cover glass, we have analyzed the sample with an ESEM; a detailed description of the utilized equipment is reported in the
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RESULTS AND DISCUSSION In order to characterize the DNA geometry inside the single polymeric microchannel, we carried out new experiments by using both ultrashort and long DNA segments. In particular, we used a linear double-stranded 27 bp long (about 9 nm) oligonucleotide and a 6459 bp long (about 2.2 μm) plasmid DNA. Both samples were labeled at the 3′-terminal by using terminal deoxynucleotidyl transferase and fluorescein-123400
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this case, the DNA microbundles appear to be oriented along the channel direction. It seems that, due to the comparable channel width (L = 2.5 μm) with the length of the DNA filaments (about 2.2 μm), these ones prefer to orient along the microchannel direction, where a “least energy” path can be followed. This hypothesis is confirmed by the CM analysis, which highlights (Figure 3b) the presence of an array of bright spots, with a periodicity along the channel that is very close to the length of DNA filaments (the intensity distribution of Figure 3b is reported in the Supporting Information 4). An ESEM picture taken on the same sample (Figure 3c) shows, in different high magnifications (Figures 3d,e), the presence of stacks of coiled DNA helices, stretched along the channels. In particular, Figure 3e shows the presence of quite small crystals, which do dot appear in Figure 3d. We have performed a microanalysis of the area in Figure 3e, finding that the presence of these small crystals, surrounding the DNA helices, is due to an accumulation of salts (a very small amount of salts was present, indeed, in the DNA water solution). This accumulation is not evident in a different area of the sample, reported in Figure 3d. The ESEM characterization performed at high magnification (Figure 3d,e), indicator of high energy of the electronic beam, is a proof of the capability of our polymeric template to immobilize the DNA filaments. Indeed, when acted on by a “high energy” electron beam, the DNA is, in general, removed or, simply, it evaporates, and partial coating with metals is necessary for allowing an observation;32,33 in fact, to take the picture, it is necessary to have the DNA molecule firmly “imprisoned” in some structure. However, mechanisms (mainly photochemical) used to realize this immobilization can often damage or denature the sample;34,35 on the contrary, our technique realizes the goal after inducing self-organization at a quasi-molecular level, without the need of any kind of surface chemistry and apparently without damaging the sample (a DNA damage analysis is reported in the Supporting Information 5). It is worth pointing out that the ESEM analysis has been realized in environmental conditions, unlike other techniques which obtain the same, or even better, resolution but need to make use of cryo-electron microscopy.36
Supporting Information 3. The picture of Figure 2c supports an elongated shape of DNA bridges between the polymeric slices and also an average tilt angle, of about 22°, between the bridges and the normal to the polymeric slices. Furthermore, by using the fluorescence signal of the labeled filament heads, we have analyzed the sample under a CM (see Supporting Information 3); the result is reported in Figure 2d. Each microchannel appears quasi-periodically illuminated due to a strong fluorescence signal of DNA bridges, which possess several light responsive markers; indeed, the intensity distribution of Figure 2c (see Supporting Information 3) shows a quasiperiodic modulation of the light intensity along the channel with an average periodicity of about 2 μm. Finally, we have probed the sample with two, colaunched, laser beams of different color. The far-field view of the obtained twodimensional diffraction pattern, reported in Figure 2e, indicates the existence of a 2-D quasi crystal structure. Indeed, the pattern can be interpreted only as a result of the polymeric grating periodicity in one direction (Figure 1b) and a quasi periodic “zig-zag” distribution of the DNA bridges (which generates the “star-like” diffraction figure reported in Figure 2f). We can conclude that the presence of the two-dimensional diffraction pattern confirms the quasi-periodic distribution of the DNA bridges, since diffraction of light takes place only in presence of a periodic modulation of the refractive index (phase gratings). The important aspect of above observation is in the possibility of analyzing the diffraction pattern either “real time” and “post-processing”, with an approach that, in the future, can enable to obtain information concerning the polymerization process, or nucleation, by using a noninvasive all-optical method. A deeper insight into the mechanisms, driven by our polymeric template, which are responsible for the alignment and immobilization of DNA molecules, is given by the experiments repeated with a much longer DNA filament (6459 bp); results are reported in Figure 3. Figure 3a is a POM view of some channels filled with the long DNA filaments; in
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CONCLUSION In conclusion, we have reported on the realization and characterization of a holographic method for permanently aligning DNA filaments. This is obtained by exploiting the confining capabilities of a soft microarray structure, where DNA strands can be unfold and aligned, giving enough contrast to create an holographic DNA detector; this provides a tool to perform a real time analysis of the organization of short and long DNA strands. We have analyzed the DNA organization by means of optical and electronic microscopes, and results confirm the excellent capability of the polymeric template to induce long-range organization without the need of any kind of surface treatment (surfactant-free method). Our achievements represent a promising perspective for basic research, both on the DNA molecule and on a wide variety of cross disciplinary fields where complex molecule analysis is needed, as well as for application oriented studies. These are related, in particular, to the possibility of exploiting the immobilized DNA filaments to induce order in nanomaterials, thus creating new materials which possess nanoscale organization for technological applications, such as biologically inspired smart metamaterials.
Figure 3. POM view (a), ESEM picture of the sample (b), and confocal analysis (c) of the structure. (d) and (e) are an ESEM high magnification of a single bright spot reported in (c). 3401
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(14) Vasdekis, A. E.; O’Neil, C. P.; Hubbell, J. A.; Psaltis, D. Microfluidic Assays for DNA Manipulation Based on a Block Copolymer Immobilization Strategy. Biomacromolecules 2010, 11, 827−831. (15) Yamashita, K.; Yamaguchi, Y.; Miyazaki, M.; Shimizu, H.; Maeda, H. Direct Observation of Long-Strand DNA Conformational Changing in Microchannel Flow and Microfluidic Hybridization Assay. Anal. Biochem. 2004, 332, 274−279. (16) Marie, R.; Schmid, S.; Johansson, A.; Ejsing, L.; Nordström, M.; Häfliger, D.; et al. Immobilisation of DNA to Polymerised SU-8 Photoresist. Biosens. Bioelectron. 2006, 21, 1327−1332. (17) Onsager, L. The Effects of Shape on the Interaction of Colloidal Particles. Ann. N.Y. Acad. Sci. 1949, 51, 627−659. (18) Hagerman, P. J. Flexibility of DNA. Annu. Rev. Biophys. Biophys. Chem. 1988, 17, 265−286. (19) Franklin, R. E.; Gosling, R. G. Molecular Configuration in Sodium Thymonucleate. Nature 1953, 171, 740−741. (20) Nakata, M.; et al. End-to-End Stacking and Liquid Crystal Condensation of 6 to 20 Base Pair DNA Duplexes. Science 2007, 318, 1276−1279. (21) De Sio, L.; Ferjani, S.; Strangi, G.; Umeton, C.; Bartolino, R. Universal, Soft Matter Template for Photonic Applications. Soft Matter 2011, 7, 3739−3743. (22) Caputo, R.; De Sio, L.; Sukhov, A. V.; Veltri, A.; Umeton, C. Development of a New Kind of Holographic Grating Made of Liquid Crystal Films Separated by Slices of Polymeric Material. Opt. Lett. 2004, 29, 1261−1263. (23) Caputo, R.; Veltri, A.; Umeton, C.; Sukhov, A. V. Characterization of the Diffraction Efficiency of New Holographic Gratings with a Nematic Film−Polymer-Slice Sequence Structure. J. Opt. Soc. Am. B 2004, 21, 1939−1947. (24) Grané, li A.; Yeykal, C. C.; Prasad, T. K.; Greene, E. C. Organized Arrays of Individual DNA Molecules Tethered to Supported Lipid Bilayers. Langmuir 2006, 22, 292−299. (25) Marko, J. F.; Siggia, E. D. Stretching DNA. Macromolecules 1995, 28, 8759−8770. (26) Jin, B. K.; Ji, X. P.; Nakamura, T. Voltammetric Study of Interaction of Co(phen)33+ with DNA. Electrochim. Acta 2004, 50, 1049−1055. (27) Choi, M. C.; et al. Ordered Patterns of Liquid Crystal Toroidal Defects by Microchannel Confinement. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 17340−17344. (28) Leica Microsystems, http://www.leica-microsystems.com/, last accessed Dec 2011. (29) Rill, R. L. Liquid Crystalline Phases in Concentrated Aqueous Solutions of Na+ DNA. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 342− 346. (30) van Loenhout, M. T.; van der Heijden, T.; Kanaar, R.; Wyman, C.; Dekker, C. Dynamics of RecA Filaments on Single-Stranded DNA. Nucleic Acids Res. 2009, 37, 4089−4099. (31) Sambriski, E. J.; Ortiz, V.; de Pablo, J. J. Sequence Effects in the Melting and Renaturation of Short DNA Oligonucleotides: Structure and Mechanistic Pathways. J. Phys.: Condens. Matter 2009, 21, 034105−034109. (32) Dunlap, D. D.; Garcia, R.; Schabtach, E.; Bustamante, C. Masking Generates Contiguous Segments of Metal-Coated and Bare DNA for Scanning Tunneling Microscope Imaging. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 7652−7655. (33) Chen, N.; Zinchenko, A. A.; Yoshikawa, K. Probing Biopolymer Conformation by Metallization with Noble Metals. Nanotechnology 2006, 17, 5224−5232. (34) Elsner, H.; Mouristen, S. Use of Psorlens for Covalent Immobilization of Biomolecules in Solid Phase Assays. Bioconjugate Chem. 1994, 5, 463−467. (35) Millan, K. M.; Spurmanis, A. J.; Mikkelsen, S. R. Covalent Immobilization of DNA onto Glassy Carbon Electrodes. Electroanalysis 1992, 4, 929−932.
ASSOCIATED CONTENT
S Supporting Information *
(1) Detailed description of the DNA samples and labeling; (2) characterization of the DNA alignment by varying the polymeric channel width; (3) environmental scanning microscope technique; (4) confocal microscope analysis and working principle; (5) DNA damage analysis inside the polymeric template. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: luciano.desio@fis.unical.it. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Authors are grateful to Dr. Giovanni Desiderio for his help in the ESEM analysis as well as Prof.ssa Alessandra Crispini and Dott.ssa Federica Ciuchi for preliminary X-ray investigation. The ESEM utilized in this research belongs to the equipment acquired for the realization of the Project No. 1987/37, Umeton: “COSTITUZIONE DEL LABORATORIO ITALIANO CRISTALLI LIQUIDI”- PON: Misura II.1 Azione a - Interventi infrastrutturali (avviso 68) funded by Ministry of Education, Universities and Research (MIUR).
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