Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy.

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).

Synthetic oligonucleotides: AFM characterisation and electroanalytical studies.

One of the most important steps in designing more sensitive and stable DNA based biosensors is the immobilisation procedure of the nucleic acid probes on the transducer surface, while maintaining their conformational flexibility. MAC Mode AFM images in air demonstrated that the oligonucleotide sequences adsorb spontaneously on the electrode surface, showing the existence of pores in the adsorbed layer that reveal big parts of the electrode surface, which enables non-specific adsorption of other molecules on the uncovered areas. The electrostatic immobilisation onto a glassy carbon electrode followed by hybridisation with a complementary sequence and control with a non-complementary sequence was studied using differential pulse voltammetry and electrochemical impedance spectroscopy. Changes in the oxidation currents of guanosine and adenosine were observed after hybridisation events as well as after control experiments. Modification of the double layer capacitance that took place after hybridisation or control experiments showed that non-specific adsorption of complementary or non-complementary sequences occur allowing the formation of a mixed multilayer.