New approach for the determination of the hybridation efficiency of ssDNA nanopatches
published: Feb. 12, 2008, recorded: October 2007, views: 135
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Films of single-stranded DNA (ssDNA) immobilized on surfaces form the basis of a number of important biotechnology applications, including DNA micro- and nano-arrays. However, the question about how the film structure affects the hybridization process is not properly addressed in literature. While it is commonly accepted that the hybridization efficiency in extensive self assembled monolayers of ssDNA is basically inversely proportional to the molecular density of the probes on the surface, the same agreement has not been reached when the monolayers are confined in nanometric structures. Using nanografting (a nanolithographic technique performed in liquid environment with an AFM tip) we are able to fabricate ssDNA micro- and nanostructures with well defined height, lateral size. The hybridization causes an increase of height that can be monitored by AFM microscopy. Surprisingly, we observed the highest efficiency of ssDNA hybridization at densities halfway to saturation; moreover, in this regime the mechanical properties of the ssDNA hybridized film and of a nanografted double-stranded DNA film are equivalent. To quantify the hybridization efficiency on our nanostructures, and, more in general, the efficiency of chemical processes which involve only few molecules, the analytical chemistry techniques are no longer effective. On the contrary, a physical approach offers many suitable solution. In particular micro- and nano-mechanical resonating sensors experienced a significant progress in the last 5 years, allowing the measurement/enumeration of single molecules and a mass sensitivity of 10-21 g. We fabricate several devices consisting of silicon cantilevers 5x2x15m3 and 5x2x25m3 in size with mass of 350 and 580 pg and resonance frequency at 10.3 and 3.7 MHz respectively. The top side was covered with an ultraflat Au layer (0.8nm RMS) on which an organic self assembled monolayer was formed. ssDNA nanostructures on the free end of the cantilevers are fabricated by nanografting. When the ssDNA hybridizes the mass of the cantilever increases and the resonance frequency shifts toward lower values. Operating in vacuum conditions we obtained a quality factor better than 104 that allowed us to attain a mass sensitivity of 5x10-16 g, corresponding to the hybridization of only 1% of a 3x3m2 ssDNA nanostructure.
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