Nanostructured Surfaces for the Detection of Single-Particles and Single-Molecules

Biomarker detection and diagnosis are important steps in the prevention and the treatment of a disease. There is an increasing need for utmost sensitive and robust sensing devices; in this framework, subwavelength (metal) pores could contribute to a conceptually new analytical tool. Such nanometer-sized holes milled in solid-state membranes offer unique possibilities for label-free detection of single-particles and single-molecules, such as proteins and nucleic acids, providing new biophysical insights and allowing the so-called ‘third-generation DNA sequencing’. These nanostructures are issued from the semiconductor industry and benefit from highly portable and rather low-cost manufacturing attributes which are required for point-of-care diagnostics. Owing to their simple format, single or arrays of such holes are easily combined with well-established biophysical techniques such as fluorescence microscopy, electrical recordings and force spectroscopy, expanding the capacities of the existing methods. In particular, nano-sized wells in a metallic support enable to circumvent the diffraction limit of light in confining the observation volume inside their nanometric core. Moreover, the coupling of light with the electronic shell of the metal confers plasmonic characteristics to the system which are currently driving an entire new generation of sensors. The first part of this thesis presents the combination of a particular class of fluorescence microscopy, known as fluorescence fluctuation spectroscopy, with subwavelength apertures in a gold layer. Thanks to the unique properties of metal wells, nano-sized objects such as polystyrene beads and proteins were detected in a label-free format and the device was able to address mobility, concentration and volumes of the analytes. In addition, the multiplexing of the approach was realized through the arrays of holes and a home-built wide-field setup which allowed the reduction of the time of acquisition by a factor equal to the number of analyzed holes, usually 25. In the second part, the manuscript describes an interesting application for sensing important biomarkers known as microvesicles. Here the resistive-pulse technique was used to electrically measure single translocation events through a single nanometric hole of nano-sized objects such as polystyrene beads and artificial lipid vesicles. In interpreting the magnitude, the frequency and the duration of such events, the size and the concentration of a particle as well as its time of translocation were retrieved. In addition of the pore functionalization, the capture of artificial and cell-derived vesicles on substrates modified with a functional supported lipid bilayer is presented.