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Chapter 5 times. The substrates were, then, treated using the same procedure shown before and imaged using a fluorescence microscope. The array was done printing water solution having different concentrations of PNA 1 (10, 20, 50, 100 µM) and PNA 3-4-5 (1, 5, 10, 20, 50, 100 µM) to test the selectivity performance of the fabricated platform. PNA 1 was visualized using a fluorescence microscope; since the array was too big to fit in one picture, the overall image was obtained pasting together many pictures (Figure 5-10). The results show that µCP technique can improve standard microarray fabrication, since a good homogeneity between the spots and within each spot was obtained. The fluorescence intensity of the first three replicates was almost the same, while after the fourth a decrease was observed, demonstrating the good reproducibility of the process. The possibility to apply the slides fabricated for DNA recognition assays was tested using two of them in hybridization experiments with fullmatch and mismatch oligonucleotides. TAMRA labeled DNA oligomer c3 and c5 100nM in SSC2x SDS 0.1% were used to incubate the slides at 55°C for 3 hours. The slides were then imaged using a laser scanner (Figure 5-11); the results showed the good performance of the platform in terms of selective recognition, since signals were seen only where expected. Figure 5-11: Hybridization of the devices fabricated as previously described with complementary and mismatched oligonucleotides. Image is taken using a laser scanner fluorescence system 104
PNA Microcontact Printing 5.3 Conclusions In this work the possibility to use microcontact printing techniques to derivatize different kind of surfaces with PNAs is demonstrated. In particular, chiral Arg-PNAs have been tested in patterning of surfaces and imaged by a fluorescein dye inserted in the molecule. The molecules linked on surfaces have been studied in order to evaluate their recognition properties, measuring for the first time a melting temperature of a PNA-DNA duplex directly on surface. All the techniques developed in the first part of the work have been exploited for preparing PNA microarrays by microcontact printing, demonstrating several improvements in comparison with standard microarrays. Further optimization of the method may allow to develop a commercial method for preparing PNA microarrays based on microcontact printing, reducing fabrication cost, time, and improving the quality. 5.4 Experimental Section 5.4.1 Materials and reagents PNA N-Boc protected standard monomers, N-[1H-1,2,3-(benzotriazol-1-yl)- (dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-Nmethylmethanaminium hexafluorophosphate N-oxide (HATU), dichloromethane, N– methylpyrrolidone (NMP) and N,N-dimethylformamide (DMF) were purchased from Applied Biosystem (Foster City, CA, USA). Carboxymethyl-O-benzyl-guanine was purchased by ASM (Hannover, Germany). Boc-Arg(Tos)-OH, Boc-D-Arg(Tos)-OH and m-cresol were from Fluka (Buchs, Switzerland). (4-Methylbenhydryl)amine (MBHA) PS resin was from Novabiochem (Laufelfingen, Switzerland). Diisopropylethylamine (DIPEA), N,O- Bistrimethylsilyl acetamide (BSA), trifluoromethane sulfonic acid (TFMSA), trifluoroacetic acid (TFA), 3-hydroxy-1, 2,3-benzotriazin-4-(3h)-one (DhBtOH) and diisopropylcarbodiimide (DIC) were from Sigma-Aldrich (St. Louis, MO, USA). Oligonucleotides were purchased from Sigma or MWG eurofins. All nucleotides were purified by HPLC and modified by the manufacturer. All buffers and immobilization solutions were prepared with 18 MΩ·cm distilled water (MilliQ). The following materials and 105
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Università degli studi di Parma Do
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2.4.4 Fluorescence measurements ...
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Chapter 1 PNAs: From birth to adult
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PNAs: from birth to adulthood amino
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Chapter 2 Label-free detection of S
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PNA Molecular Beacons In particular
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