title: Synthesis and chromatography-free purification of PNA-PEO conjugates for the functionalisation of gold sensors.
abstract: Peptide Nucleic Acids (PNAs) linked to high molecular weight (MW) poly(ethylene oxide) (PEO) derivatives could be useful conjugates for the direct functionalisation of gold surfaces dedicated to Surface Plasmon Resonance (SPR)-based DNA sensing. However their use is hampered by the difficulty to obtain them through a convenient and economical route. In this work we compared three synthetic strategies to obtain PNA-high MW PEO conjugates composed of (a) a 15-mer PNA sequence as the probe complementary to genomic DNA of ]Mycobacterium tuberculosis, (b) a PEO moiety (2 or 5 KDa MW) and (c) a terminal trityl-protected thiol necessary (after acidic deprotection) for grafting to gold surfaces. The 15-mer PNA was obtained by solid-phase synthesis. Its amino terminal group was later condensed to bi-functional PEO derivatives (2 and 5 KDa MW) carrying a Trt-cysteine at one end and a carboxyl group at the other end. The reaction was carried out either in solution, using HATU or PyOxim as coupling agents, or through the solid-phase approach, with 49.6%, 100% and 5.2% yield, respectively. A differential solvent extraction strategy for product purification without the need for chromatography is described. The ability of the 5 KDa PEO conjugate to function as a probe for complementary DNA detection was demonstrated using a Grating-Coupling Surface Plasmon Resonance (GC-SPR) system. The optimized PEO conjugation and purification protocols are economical and simple enough to be reproduced also within laboratories that are not highly equipped for chemical synthesis.
authors.affiliation: Department of Industrial Engineering, University of Padova, via Marzolo 9, 35131 Padova, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Dettin
authors.forename: Davide
authors.initials: D
authors.lastname: Silvestri
authors.forename: Roberta
authors.initials: R
authors.lastname: Danesin
authors.forename: Erica
authors.initials: E
authors.lastname: Cretaio
authors.forename: Gianluca
authors.initials: G
authors.lastname: Picariello
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Casarin
authors.forename: Agnese
authors.initials: A
authors.lastname: Sonato
authors.forename: Filippo
authors.initials: F
authors.lastname: Romanato
authors.forename: Margherita
authors.initials: M
authors.lastname: Morpurgo
mesh_terms: D015374:Biosensing Techniques; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D020135:Peptide Nucleic Acids / Q000138:chemical synthesis / Q000737:chemistry; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry; D020349:Surface Plasmon Resonance
chemical_list: D004269:DNA, Bacterial; D020135:Peptide Nucleic Acids; D011092:Polyethylene Glycols; D006046:Gold