Surface Plasmon Resonance Imaging Biosensors for Cancer Diagnosis: Detection of Circulating Tumor DNA

This doctoral thesis focused on the realization of Surface Plasmon Resonance Imaging (SPRI) biosensor for the rapid, simple and label-free detection of single point mutations in the KRAS gene, standard actionable cancer biomarkers for colorectal cancer, in human plasma samples. Initially, the SPRI assay included the immobilization of specific peptide nucleic acid (PNA) probes onto the gold sensor to ensure the hybridization reaction of PNA-DNA complexes. The spatially controlled immobilization of PNA probes has been obtained by injecting PNA wild-type and PNA mutated solutions into a microfluidic system coupled to SPR sensor chip. The extremely low concentration of genomic DNA required an improvement of SPRI detection capabilities, by using functionalized gold nanoparticles to amplify the hybridization signal between target analytes and corresponding PNA probes. Three representative single-point mutations, gDNA G12D, G12V and G13D, have been successfully detected. After preliminary results of nanoparticle-enhanced SPRI assay, a mixed-charge polymer based on Poly-L-lysine (PLL) polypeptide backbone modified with an anionic peptide, connected via a nonionic OEG spacer, has been synthesized in order to achieve control over the charge distribution of PLL-coated surfaces, and thus the antifouling property. The PLL backbone has been functionalized with different percentages (y%) of maleimide-OEG-NHS ester chains (PLL-mal(y%), from 13% to 26%), and the anionic oligopeptide CEEEEE, composed of one cysteine (C) and five glutamic acids (E), with a short sequence to limit the thickness of the mixed-charge polymer antifouling coating, has been attached to the maleimide units through the thiol maleimide Michael-type addition. The grafting density has been varied to tune the balance of charged groups at polymer backbone. PLL-mal(y%)-CEEEEE surfaces have been characterized by water contact angle and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). Complementary acoustic (quartz crystal microbalance with dissipation, QCM-D) and plasmonic (surface plasmon resonance imaging, SPRI) techniques have been employed to monitor the adsorption of bovine serum albumin (BSA), used as standard protein solution, and diluted human plasma samples. Hence, a new nanoparticle-enhanced SPRI assay for circulating tumour DNA (ctDNA) detection in human plasma samples using PLL-mal(y%)-CEEEEE layer as the antifouling coating has been devised. The PNA probes and the anionic peptide have been attached to the maleimide units through the thiol maleimide reaction using a microfluidic system coupled to SPR sensor chip. The analysis of ctDNA G12D target in diluted human plasma samples (5 pg uL-1), collected from cancer patients and healthy donors, has been carried out using the conjugated AuNPs system, with a minimal sampling handling to avoid any contamination and disruption of the antifouling activity of PLL-mal(y%)-CEEEEE layer. The combined use of PLL-mal(y%)-CEEEEE as the antifouling layer with functionalized gold nanoparticles for the amplification of target detection overcomes the limiting factors related to the biosensor in the clinical field and offers an excellent ctDNA discrimination in the bloodstream at attomolar level.