End-selective functionalization of carbon nanotubes. Use of DOE for the optimization of a DNA probe attachment and hybridization using an enzymatic amplifying system

Self-assembling functionalized multi/single-walled carbon nanotubes (MWCNTs/SWCNTs) in a controlled manner may be an interesting route towards novel nanoscaled supramolecularly arranged MWCNT/SWCNT-based architectures as sensing constructs. Due to the presence of fullerene-like end-caps sensitive to oxidation, the oxidation of MWCNTs/SWCNTs may be readily performed to create oxidized MWCNTs/SWCNTs (MWCNTs_ox/SWCNTs_ox). MWCNTs _ox/SWCNTs_ox contain two different areas that are well-defined topologically, i.e. an outer hydrophobic sidewall containing oxygenated defects (COOH, CHO, and C-O-C functions), and an end-localized cluster of COOH groups. They have been readily derivatized following the simultaneous chemical activation of both end cluster and sidewall carboxylic groups by carbodiimides. This derivatization scheme and related variants were commonly used for the covalent attachment of numerous types of particles, polymers, and biomolecular probes, but in a non-selective way. In contrast, sidewall pegylation of MWCNTs_ox/SWCNTs_ox enabled the end-selective covalent attachment of an aminated 20-mer DNA probe and its hybridization with a fluoresceine (FITC)-labeled complementary 2nd DNA sequence. End-selective DNA hybridizations of resulting pegylated MWCNTs _ox/SWCNTs_ox have been assayed using a blue-colored (optical readout at 620 nm) amplifying horse radish peroxidase (HRP)-based enzymatic system. The optimization of the whole functionalization sequence, i.e. MWCNT/SWCNT oxidation, pegylation of oxididized MWCNTs_ox/SWCNTs _ox, DNA attachment and subsequent hybridization, has been performed using a DOE (design of experiments) approach. The most influential factors were found to be the oxidation temperature of MWCNTs/SWCNTs, the molecular weight of PEG polymers used for sidewall passivation, and, quite unexpectedly, the commercial source of the starting MWCNTs/SWCNTs.