OpenstarTs >
Ricerca >
Tesi di dottorato >
Scienze fisiche >

Please use this identifier to cite or link to this item: http://hdl.handle.net/10077/2616

Title: Crowding effects on biochemical reactions of surface-bound DNA.
Authors: Castronovo, Matteo
Supervisor/Tutor: Scoles, Giacinto
Casalis, Loredana
Co-supervisor: Morgante, Michele
Issue Date: 10-Apr-2008
Publisher: Università degli studi di Trieste
Abstract: Next-generation DNA detection arrays are expected to achieve unprecedented sensitivity, reducing the minimum amount of genetic material that can be directly (PCR-free and label-free) and quantitatively detected, up to the single cell limit. To realize these goals, we propose a new method for the miniaturization of DNA arrays to the nano-scale, which has the unique capability of controlling the packing quality of the deposited bio- molecules. We used NanoGrafting, a nano-lithography technique based on atomic force microscopy (AFM), to fabricate well ordered thiolated single stranded (ss)-DNA nano-patches within a self-assembled monolayer (SAM) of inert thiols on gold surfaces. By varying the “writing” parameters, in particular the number of scan lines, we were able to vary the density of the supported DNA molecules inside the nano-patches in a controlled manner. Our findings can be resumed in two parts: 1) Combining accurate height and compressibility measurements, before and after hybridization, we demonstrate that high-density ss-DNA nanografted patches hybridize with high efficiency, and that, contrary to current understanding, is not the density of probe molecules to be responsible for the lack of hybridization observed in high density ss-DNA SAMs, but the poor quality of their structure. 2) Dpn II enzymatic reactions were carried out over nanopatches with different molecular density and different geometries. Using nanopatch height measurements we are able to show that the capability of the Dpn II enzyme to reach and react at the recognition site significantly depends on the molecular density in the nanopatches. In particular the inhibition of the reaction follows a step-wise fashion at relatively low DNA densities. These findings suggest that, due to the enzyme size, it is possible to tune the efficiency of an enzymatic reaction within surface-bound DNA nanostructures by changing only the crowding of DNA on the surface and without introducing any further physical or chemical variable.
PhD cycle: XIX Ciclo
PhD programme: NANOTECNOLOGIE
Description: 2006/2007
Keywords: AFM
nanografting
nanopatterning
self-sssembly
monolayers
SAM
surfaces
DNA
DNA nanostructures
DNA chip
DNA nanoarrays
enzimatic reactions
restriction digestion
hybridization
steric hindrance
crowding
model membrane
mechanical resistance
bioresistance
compressibility
ordering
Main language of document: en
Type: Tesi di dottorato
Doctoral Thesis
Scientific-educational field: FIS/01
NBN: urn:nbn:it:units-7240
Appears in Collections:Scienze fisiche

Files in This Item:

File Description SizeFormat
PhD-thesis_Castronovo_final.pdfPhD thesis in english23.05 MBAdobe PDFView/Open
sommario in italiano.pdffrontespizio firmato + sommario della tesi165.45 kBAdobe PDFView/Open
abstract in english.pdfcover and signatures+ abstract of the thesis166.78 kBAdobe PDFView/Open
View Statistics

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.