Approach in aptamer based biosensors for agro-industrial applications

In recent years, thanks to advances in electronic technology and biotechnology, biosensors have become an interesting field of research in different areas ranging from molecular biology research, the identification of diseases, the detection of GMO contamination in food, environmental monitoring of pollutants. A biosensor is a device containing a biological component capable of detecting an analyte. It comprises a sensing element having high affinity to an analyte of interest, a detector that transforms the signal from interactions between the sensing element and the analyte, and an instrument that amplifies, translates and presents the signal in a user friendly manner (Wang et al., 2000). What makes these devices attractive to potential practical applications are its high sensitivity, high selectivity, low cost and the possibility of regeneration and reuse. Recently, biosensors based on the use of monoclonal or polyclonal antibodies have seen a great development in the field of small molecules analytical determination and specifically in the mycotoxins analyses for food safety application and in biomarkers detection for human health. Affinity to specific molecular targets (such as nucleic acids, proteins, small compounds, or cells) of antibodies is overcome by aptamers. Aptamers are short oligonucleotide sequences in DNA or RNA, of variable length generally between 15 and 80 nucleotides, which bind with high affinity and specificity to a wide range of target molecules, such as proteins or other organic and inorganic molecules. Aptamers are generated by an in vitro selection process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This technique combines the principles of combinatorial chemistry to molecular biology techniques, and allows to identify the specific DNA or RNA molecules of interest from a large population of oligomers in a random sequence. Aptamers offer many advantages compared to the proteins or to small drug molecules, therefore, they have potential application in many fields of research and technology both as active elements, able to interfere with the biological machinery of cells and tissues, both as elements "labeling" for agents of various diagnostic and analytical interest because, if equipped with suitable linker, can be easily immobilized on the surfaces of biosensors or bio-chip with a sufficient density and a very high precision. The purpose of this PhD thesis is to provide a study of a range of technologies and innovations in the field of the bio-sensible component of a biosensor, comparing different experimental approaches. At the beginning, we used a particular type of transducer as biosensors scales microgravimetric Quartz Crystal Microbalance (QCM), constituted by a thin disk of quartz fitted with two gold electrodes, one of which is functionalized so as to be sensitive to the presence of an analyte. Since the oscillation frequency of the quartz is proportional to the mass deposited on the electrode functionalized, such devices are able to provide qualitative and quantitative measures related to the presence / absence of an analyte (Tombelli, S. 2002). We used also an acoustic sensor in a TSM format (Thickness Shear Mode methods) for studying protein/aptamer interaction. We discussed different immobilization techniques, detection systems and advantages and disadvantages of these methods. During the second year, we approached a different transducer: the electrochemical techniques. An electrochemical biosensor ensures the signal recording on the basis of redox indicators which undergo oxidation or reduction reactions upon bioreceptoranalyte binding, and relates this signal to variations in analyte concentration. Electrochemical transduction presents considerable advantages over optical or thermal detection; for instance, it offers high sensitivity and selectivity, compatibility with novel microfabrication technologies, inherent miniaturization, low cost, disposability, simplicity in operation and low or negligible disturbance of the sample (Radi et al. 2011). In the last year, in order to examine the binding and the interactions between aptamer/gold surface and aptamer/proteins we used the Quartz Crystal Microbalance (QCM). During the PhD we moved in this direction, studying and defining different approaches to develop new biosensors for the most popular toxic and carcinogenic food contaminant class: mycotoxins.