Development of an Oligonucleotides-Based Sensor for Applications in Reconfigurable Robotics

The present work is a contribution in the framework of a NEST (New and Emerging Science and Technologies) European Project called ARES (Assembling Reconfigurable Endoluminal Surgical system), dedicated to the investigation and development of a reconfigurable diagnostic and surgical robot for the gastrointestinal tract. In particular, we focus on technological solutions allowing the realization of the “screening” module of the robot, consisting of a DNA-chip for the recognition of altered DNA sequences, related to the presence of cancerous cells in the gastrointestinal tract. In Chapter I, we analyze the characteristics of Colorectal Cancer, its ethiopathology and some traditional methods for diagnosis, screening and monitoring of this pathology. Then we report the correlations between the development of cancer and some specific alterations in the cellular genetic code, in particular concerning DNA methylation and k-ras mutation. Some DNA screening systems currently available in literature are described, showing that their limitations lead us to look for alternative technical solutions. In the second chapter we show some interesting solutions to the problem of cellular transport and positioning. In fact, a sensor analysis is effective only if we are able to lead the cell, and consequently its DNA, in the “sensitive” part of the biosensor. Weanalyze the way to transport colon cancer cells, possibly mixed with other cells and dispersed in blood, from the site of capture to the core of the biosensor’s module. Wepropose hydrodynamic focalization and plasma separation devices as possible solutions. We consider then how these cells can be exactly positioned on the biosensor’s surface and how we can extract genomic material from cells. We propose dielectrophoresis capture and applications of alternated electromagnetic fields for the breaking of cellular membrane. In Chapter III we explain the basic principles of quartz crystal microbalance, based on the Sauerbrey’s equation, and its possible fields of applications’, while in Chapter IV we describe in details the equipment used for our experiments: the quartz crystal analyzer and the quartz crystal microbalance with dissipative measurements (QCM-D). In this equipment a little variation in mass is directly related to a change in the resonating frequency of the crystal. This allows to measure phenomena related to a little changing in mass of the sample, such as DNA hybridization. In the last chapter we present the results of our experiments. We first show a test related to the detection of human serum albumin by using the quartz crystal analyzer, based on monoclonal antibodies immobilized onto the sensor’s surface. We describe the different phases of the activation of the surface, consisting of linking a thiol group to the surface, allowing the following linking of the monoclonal antibody, and finally we report the curves related to the addition of different concentrations of HSA (Human Serum Albumin) in the solution in contact with the sensor. We explain then the protocol used for the detection of DNA hybridization using the QCM-D device. We show the structure of the synthetic oligonucleotides used, and the protocol used for the activation of the sensor surface, consisting in the immobilization on the gold surface of the quartz crystal of simple DNA chains of 20down-SH oligonucleotides, by using the thiol group incorporated in one of the ends of the chains. These chains act as recognition elements for detecting different concentrations of 100up simple DNA chains, which present a section complementary to 20down chain. We describe the results obtained adding these complementary DNA sequences in the solution, evaluating the frequency response of QCM-D, corresponding to the hybridization of chains, and we finally evaluate the following recognition of the 80down DNA chain, complementary to the part of 100up that does not hybridize with the 20down chain. We show the important results obtained, demonstrating the possibility of detection of different concentrations of simple chains of 80 bases, and the meaning of this contribution in the ARES project whole conception, that paves the way to future detection of k-ras mutant sequences in a miniaturized system.