Spatially Confined Biochemical Reactions

We developed an electrochemical impedance biosensor based on capacitance readout for the detection of biomolecules in small sample volumes. We demonstrated that our three-electrode setup is stable, reproducible, and suitable for real-time measurements. With this device we were able to follow in situ and in real time the process of DNA hybridization.  L. Ianeselli et al., Biosens. Bioelectronics 55 1 (2014).
      

		We use Atomic Force Microscopy nanografting to generate monolayer patches of 44-bp dsDNA molecules with variable density (dsDNA matrices), within bioresistant alkylthiol self-assembled monolayers (SAMs), on an ultraflat gold surface. Nanografting is a technique in which the AFM tip induces, at selected locations, a substitutional exchange of the molecules of the original SAM matrix with new molecules present in solution (thiol-DNA in our case). By changing the number of times the tip is overwriting the same area during nanografting, we can fine-tune the molecular density in that area. The topographic height of such dsDNA matrices increases with the dsDNA packing and saturates to the length of dsDNA molecules (~15 nm) as they stand vertically in crowded monolayers. The dsDNA sequences are designed such that they are cut at half height by a restriction enzyme. We assess the reaction by measuring the dsDNA matrix height before and after enzyme incubation. We tested the action of different restriction enzymes, namely, DpnII, BfaI and BamHI over dsDNA matrices, as a function of their density.		Retrieve article Two-dimensional enzyme diffusion in laterally confined DNA monolayers Matteo Castronovo, Agnese Lucesoli, Pietro Parisse, Anastasia Kurnikova, Aseem Malhotra, Mario Grassi, Gabriele Grassi, Bruna Scaggiante, Loredana Casalis & Giacinto Scoles, Nature Communications 2, 297 (2011)