Ultrasensitive methods for the detection of biomolecular systems at the micro/nano scale

Abstract

A great effort has been devoted, during the past decade, to the development of new devices for specific DNA sequences detection, due to the relevance of nucleic acid analysis in many areas, including clinical diagnostics, environmental monitoring and food-quality control. However, diagnostic tools used for the detection of nucleic acids need to fulfil specific requirements in term of sensitivity, selectivity and high-throughput in order to expand their applicability and to minimize the cost of the assay. In fact, very small amount of DNA is typically available for the analysis and a detection scheme capable of directly transducing the hybridization events with a proper sensitivity is required. In addition, the sequence to be targeted is often present in a complex environment such as the whole genomic DNA and the use of very specific and highly efficient probes is required in order to build highly selective detection scheme. The purpose of my research activity was the identification of new methodologies useful for DNA detection with the principal aim to perform the analysis with a very small amount of samples. In this perspective, interesting results were obtained by using microfluidic systems, characterized by the presence of one or more microchannels where fluid reagents are easily and carefully manipulated. In particular, the combined use of a droplet-based microfluidics, PNA-MBs and fluorescence microscopy enabled the selective and sensitive detection of DNA in an innovative and efficient platform. The experiments were carried out by using both oligonucleotides and PCR amplification products and were aimed at detecting DNA sequences of different olive cultivar. The ultrasensitive detection of DNA was also investigated by coupling microfluidic devices with the SPRi apparatus. A similar approach allowed a real time label-free detection of specific genomic DNA sequences carrying the â°39 thalassemia mutation. The capacity in term of selectivity and sensitivity was investigated by employing surface-immobilized PNA probes in combination with gold nanoparticle-enhanced SPRi detection. The DNA concentrations measured by this method are in a range suitable for detecting DNA samples without amplification.