Development of fluoropolymer based biosensor for the optical detection of biomolecular interactions

The last two decades have witnessed remarkable progress in the development of devices enabling the detection of biomolecules in solution and the measurement of their interactions. These devices, generally named “biosensors”, are based on various detection principles, all enabling to transduce a localized quantity of specific molecules into a detectable signal. Most standard laboratory assays used to characterize the presence and the interactions of biomolecules require the labeling of the analyte with fluorescent or radioactive groups. However, the development of a label-based method requires additional time and cost allocation, a component of which is not required for true label-free assays, which typically rely on the direct measurement of the quantity of analyte (molecules) interacting with a specific ligand. Novel methods to realize label-free biosensors are still emerging and they are the object of increasing research efforts, since they represent an attractive alternative to the standard labelled assays to report interactions between bio-molecules. The aim of this thesis is to develop and test a new label-free method for the optical detection of molecular interactions taking place on the surface of a polymeric material with peculiar optical and chemical properties. The method proposed in this thesis is based on a simple optical phenomenon, still unexplored in biosensors applications: the intensity of light reflected from an interface between two materials with similar refractive index increases significantly when a thin molecular layer with different refractive index is placed at the interface. In particular, the method is based on the variation of the low intensity signal reflected from a surface of perfluoropolymer material isorefractive to water, as ligand and receptors are absorbed or immobilized on the surface. This phenomenon can be monitored simply illuminating the interface with a LASER or a LED and detecting the reflection with a photodiode or a CCD camera. Since the reflection from a film depends on this thickness, we can follow the gradual adsorption of several molecular layers onto the surface. The sensor is based on a Hyflon®AD perfluoropolymer produced by Solvay-Solexis in Bollate (MI), featuring peculiar properties: (i) its refraction index matches that of water, (ii) it is hydrophobic and slightly charged negatively, a combination that makes on it certain kind of proteins to spontaneously adsorb; (iii) it is transparent and (iv) inert to most chemical substances. These characteristics make Hyflon®AD an attractive substrate for developing a biosensor. The protein Avidin with positive net charge under physiological conditions spontaneously adsorbs on Hyflon AD, thus allowing a general strategy for immobilization of biotin-labeled receptors. In this way, the method can be used for the detection of biologically relevant molecules interacting with other receptors previously adsorbed on the sensor surface. Several stages of design, realization and test of the prototypes have been made. The activity has been mainly devoted to a large variety of tests aimed to optimize the hardware and protocols of the method, to assess the quality of the measurements and to treat the surface with functional linkers. A great effort has been devoted to the design, realization and characterization of the measuring cell, in particular for what concerns the transport of biomolecules into the fluidic system. One of the instrumental realization includes an interferometric, real-time measurement of the concentration of analyte in contact to the active surface. This innovation is based on the small change of refractive index present in the cell. This set-up is required to perform accurate kinetic measurements for which fully characterized fluidics is necessary. The final sensors has performances similar to other label-free optical biosensors recently developed. The limit of detection (30 pg/mm2) is comparable to most commercial biosensors. The sensitivity of the method might be further improved by reduction of stray light and electronic noise. Numerous biological experiments have been carried out using the method here developed. Antigen-antibody interaction has been tested with BSA conjugated with biotin and anti-BSA in solution; a peptide-protein interaction has been characterized with an immobilized phosphorylated peptide and a fragment of SRC with the SH2 domain. Moreover, test experiments aimed at the measurement of the concentration in solution of an antigen of biomedical relevance (PSA) have been also using three different strategies of surface immobilization. Measurements of the kinetics of interaction have been carried out between BSA and its antibody and between protein A and human IgG. The simultaneous measurement of different interactions has also been tested. An instrumental set-up based on the detection of the reflection image of the active surface has been developed. In this way, the reflectivity increase of multiple spots is measured simultaneously, thus allowing the detection of multiple interactions with a single cell. This device represents a proof of concept for a protein array based on optical label-free detection.