Cancer biomarkers detection in cell lysates by means of anisotropic fluorescence at the surface of 1D photonic crystal biochips

Novel disposable optical biochips based on one-dimensional photonic crystals (1DPC) sustaining Bloch surface waves (BSW) are an attractive tool for the detection of several disease-related biomarkers. In response to growing global burden of cancer, one of the most significant public health challenges of the 21st century is the prevention and early diagnosis of this disease. By diagnosing in advance such disease permits to increase the survival probability of patients and to improve their life perspectives. Consequently, cancer biomarkers have gained considerable attention. Within this framework, the herein proposed optical biochips can quantify low concentrations (sub ng/mL) of the ERBB2 breast cancer biomarker in biological complex matrices, such as cell lysate samples. The choice of focusing on this specific breast-cancer-related biomarker lies in the global issue represented by this type of cancer. According to the data provided by the World Cancer Research Fund, its incidence rate increases year per year establishing itself as the most commonly occurring cancer in women worldwide contributing 25% of the total number of new cases diagnosed in 2018 and as the second most common cancer overall after lung cancer. However, the versatility of the system opens up new possibilities for designing different assays, depending on the specific biomarker sought. To discriminate ERBB2 levels in several different cell lysate samples, we made use of 1DPC biochips and on a reading instrument that can work in both a label-free and a fluorescence detection mode. Such combined configuration provides the advantage of complementary information and lower limit of detection (LoD) in the fluorescence mode. In the label-free mode, the BSW excitation is achieved by a prism coupling system (Kretschmann-Raether configuration), like in the surface plasmon resonance (SPR) technique, resulting in a dip in the angular reflectance spectrum. According to the interactions that take place at the surface, the angular position of such a dip shifts as a function of refractive index change at the interface. Moreover, the fluorescence operation, in which fluorescence angular spectra are acquired, is obtained by making use of fluorophores or dye labelled antibodies bound at the 1DPC surface. Furthermore, coupling between the dye labels and the BSW results in strongly directional and enhanced fluorescence emission. The advantages brought by the 1DPC, when compared to metal structures, are the smaller energy losses and the narrower resonances. Despite the great sensitivity offered by the fluorescence detection mode, the measurements are affected by a phenomenon that cannot be neglected when quantitative and accurate information is needed, as occurs in biosensing assays, i.e. photobleaching. Presently, there is no study about photobleaching in experiments with BSW sustained by 1DPC, despite its evident effects. Photobleaching denotes the irreversible loss of fluorescence emitted energy of a dye that dramatically changes its absorption and emission properties. The rate of such a fluorescence bleaching is affected by several factors such as the power of the illumination beam, the exposure time, and the photonic crystal structure itself. In addition, it is also influenced by the molecule’s transition dipole moment. In particular, fluorophores having a transition dipole moment oriented parallel to excitation polarized light will be excited preferentially, and in turn will be strongly photobleached. As a consequence, the fluorescence emission will be polarized and no more isotropic. This effect is more or less significant depending on the binding strength of the fluorophores to the surface. In this dissertation, we report for the first time on cancer detection assays, carried out with our setup, in which the trustworthiness is guaranteed by a correct approach to data analysis, which accounts in a correct way for photobleaching, which could not only affect the overall emission intensity but also its polarization distribution via the TE and TM BSW modes provided by the 1DPC. To get to such a result, the experimental data is interpreted by means of a theoretical model for the orientational distribution of dye labels over time, taking into account the density of the optical states of the 1DPC, photobleaching and rotational diffusion of surface bound emitters. The approach permits to model anisotropic fluorescence emission and to manage photobleaching effects in biosensing assays, leading to a correct data interpretation. The theoretical description permits not only to manage photobleaching but also to exploit it as a new tool for probing rotational diffusion of any protein labelled with fluorescent emitters at the surface of 1DPC endowed with different chemistries. Such a possibility is related to the polarization dependent spectroscopic role played by the 1DPC, which permits to analyse simultaneously two polarizations, TE and TM, within a relatively simple optical layout and thus accessing either the orientation or the depolarization kinetics under non-stationary conditions of the resonant fluorescence signal. Such a type of measurement is not possible with conventional fluorescence anisotropy techniques or using other types of surface electromagnetic waves, such as surface plasmon polaritons, for example, that are only TM polarized