Sensitive label-free and fluorescence cancer biomarker detection using one dimensional photonic crystal biochips

摘要

Biological and biochemical processes play a very important role in living organisms and their understanding is particularly important in medicine, biology and biotechnology. Optical biosensors hold great promise for solving challenging molecular recognition issues, such as the detection of biomolecules at very low concentration. In this framework, a direct measurement of the binding of analytes to a target molecule in biological samples is an essential step in diagnosis and in understanding how biomolecules interact under physiological conditions.udIn this thesis, I contributed to the development of an optical platform that combines label-free and fluorescence detection modes. Such a platform makes use of one-dimensional photonic crystals (1DPC) sustaining Bloch surface waves (BSW) to detect relevant cancer biomarkers in body fluids.udBSWs are surface electromagnetic waves that propagate along the truncation interface between a 1DPC and an external medium (the analyte) and can be strongly confined with a significantly enhanced field at the surface. By exploiting such features, 1DPC sustaining BSW (BSW biochips) are used as optical transducers that convert refractive index changes and fluorescence emission at their surface into a measurable optical signal.udAfter discussing the results of the platform development, I report on the use I made of BSW biochips to detect clinically relevant concentrations of Angiopoietin 2 and ERBB2 in different biological matrices. The aim of such a research endeavour is clear: to reveal cancer by means of integrated optofluidic structures before cancer reveals itself. In the case of breast cancer, for example, it is a fact that ERBB2 is a pivotal biomarker and targetable oncogenic driver associatedudwith several different aggressive subtypes. To quantitate Angiopoietin 2 and soluble ERBB2, I developed and implemented specific sandwich detection assays in which the BSW biochips’ sensitive surface is tailored with monoclonal antibodies for highly specific biological recognition.udIn a second step, a second antibody quantitatively detects the bound analytes. The strategy of the present approach takes advantage of the combination of both label-free and fluorescence techniques, making bio-recognition more robust and sensitive. In the fluorescence operation mode, the platform can attain the limit of detection 0.3 ng/mL (1.5 pM) for ERBB2 in cell lysates, which is the most complex biological matrix studied in the present work. Such a resolution meets the international guidelines and recommendations (15 ng/mL) for diagnostic ERBB2 assays that in the future may help to assign more precisely therapies counteracting cancer cell proliferation and metastatic spread.