TWO-DIMENSIONAL BIO-ACTIVE NANOSTRUCTURED INTERFACES AIMED AT ACCURATE MONITORING OF CLINICALLY RELEVANT CANCER BIOMARKER PROTEINS

Abstract

Circulating HER2 extracellular domain (HER2 ECD) levels were proposed as a surrogate for HER2 tissue expression to monitor breast cancer patients. Currently, analytical tools capable of detecting HER2 ECD level rely on commercially available ELISA (or ELISA-derived) assays employing antibodies recognizing undisclosed or unknown epitopes, providing sensitivity that very often is not adequate to detect the very low level the of cancer biomarker protein (< 12 ng/ml) in real samples. This is particularly true during the treatment follow-up to identify acquired resistance to HER2-targeted antibody therapy. Moreover, the analyses can be run only in centralized laboratories by qualified personnel, thus increasing cost and time. Therefore, there is an urgent need to develop new user-friendly analytical tools enabling a rapid, reliable analysis of real sample, thus reducing the costs and time required for the analyses. However, the sensitivity of SPR is limited by the nature of the receptors tethered on its biosensing platform. Heavy-weight antibodies (150 kDa) provide a dramatic change in the refractive index and dielectric constant at the gold layer/sample interface, so that a high number of antigens are required to be captured in order to provide a significant response. Nanobodies have been recently proved to be a valid alternative to antibodies, as the feature a lower molecular weight and a simpler molecular structure that can in principle allow devising a nanostructured biosensing platform of properly oriented receptors. In fact, a further functionalization of the nanometric layer of gold employed in the SPR with nanostructured and optically transparent materials, such as graphene, may be advantageous both in terms of further functionalization, exploiting the carbon chemistry, and electronic properties, enhancing the sensitivity. Two dimensional materials have emerged as ideal candidates for the realization of miniaturized biosensor platforms. However, a comprehensive understanding of the bioanalytical mechanisms at such atomically thin 2D material is central to such developments. In this work a graphene-based bio-nanointerface has been realized by transferring chemical vapor deposited (CVD) graphene on top of a gold SPR-active substrate. The graphene layers have been transferred in different stacked patterns and the interfacial inhomogeneities and topological variations, induced by the transfer process have been investigated by using imaging Ellipsometry, Atomic Force Microscopy and Surface Plasmon Imaging techniques. A bioanalytical framework has been created to quantify the influence of the 2D layer inhomogeneities on the bio interactions and to tune the accuracy of the biosensing. A microfluidic system together with the Kinetic-SPR have been deployed to investigate the biomolecular interactions between the HER2-ECD and three newly developed nanobodies.