Gold nanoparticle labelled DNA hairpin grafting on transparent and conductive oxide (TCO) films: Characterization of grafting and hybridization

Biosensors and biochips can be hybrid nanobiosystems involving different kinds of components, i.e. solid surface, bio-molecules and nanoparticles. These components are confined in a very small area (nanometre range). It is expected that interactions are produced between both components due to their close proximity. So to optimize the performance of these biosensors, it is very important to get a deeper insight of their surface characteristics. In this context, nanoparticles linked to DNA strands (in a ratio of 1:1) immobilized on a solid surface give the opportunity to combine complementary techniques to characterize the hybrid system. A typical example will be illustrated in this study. We have grafted DNA hairpins at their 3'-end via a silanisation process using aminopropyltriethoxysilane (APTES) on different transparent and conductive (TCO) oxide film surfaces. DNA hairpins comprise a stem in which both strands are complementary and a loop. These molecules exhibit a particularly high sensitivity for the detection of mismatches compared to the corresponding linear strands. They have been monolabelled at their 5'-end by a 1.4 nm gold nanoparticle. Because of the hairpin conformation, the label is close to the surface. Upon hybridization with a complementary target, the formation of a linear duplex structure with relative rigidity forces the label away from the surface. Due to their conductive properties, TCO films are attractive materials for biochips. They can advantageously replace the classical gold electrodes as working electrodes for direct electrochemical detection of DNA hybridization. As for silica, their surface chemistry allows the covalent and strong binding of DNA. Here, we used different TCO films: ITO films, doped SnO2 films as well as insulating SiO2 films. Thanks to the presence of gold nanoparticles bounded to DNA probes, the effects of grafting and hybridization of DNA could be studied on both conductive oxide surfaces. Particularly, we studied the modifications of surface morphology and chemistry as well as fluorescence results. By coupling AFM with SEM-FEG analyses, dispersed and well-resolved groups of gold nanoparticles linked to DNA were emphasized on the SnO2 films. Their surface density is 2.1 ± 0.3 x 1011 groups.cm2. TEM images obtained after silver enhancement of gold nanoparticles on ITO films revealed round spheres corresponding to silver coated gold nanoparticles. Their density was in agreement with the data obtained by AFM on SnO2 films. The evolution of the chemical state of the modified oxide surfaces was monitored using XPS and ToF-SIMS. As expected, the XPS N 1s peak intensity increased after grafting and hybridization of DNA. The Au 4d peak was detected only on samples modified with Au labelled hairpin probes. Its intensity decreased with probe concentration. From the ratio Au/Si (Si belonging to APTES), the surface DNA density was estimated to be 9.6x1011 cm2 and 3.7x1011 cm2 for SnO2 and ITO films respectively. The P 2p peak was observed only after hybridization with a weak intensity. Its presence was essentially correlated to phosphate residues originating from the hybridization solution. Positive and negative fragments of sugar, bases and phosphates from DNA probes were identified by TOF-SIMS. Positive and negative ions from Au nanoparticles were detected only in the case of Au labelled hairpin probes before and after hybridization. After hybridization of Au labelled hairpin probes with complementary Cy3 targets, quenching of the Cy3 fluorescence by gold nanoparticles was evidenced using fluorescence microscopy. This phenomenon was obtained for both oxides and is in agreement with the Nanometal Surface Energy Transfer (NSET) theory.