Οπτικοί βιοαισθητήρες για την ανίχνευση βιομορίων χωρίς τη χρήση ιχνηθετών

Abstract

Recent developments in the fields of bioanalytical chemistry and microelectronics have resulted in a growing trend of transferring the classical analytical methods from the laboratory bench to the field through the development of portable devices or microsystems based on biosensors. Biosensors are self-contained integrated devices capable to provide analytical information using biological recognition molecules in direct spatial contact with a transducer. Biosensors using antibodies or antigens as biological recognition elements are termed as immunosensors and they are based on the same principle as the classical solid-phase immunoassays.The aim of this thesis was to develop and evaluate an optical immunosensor based on Mach-Zehnder Interferometry and integrated on silicon substrate for the immunochemical determination of clinical analytes. The optical sensor developed is fabricated entirely by mainstream silicon technology by the Optical Biosensors group of the Institute of Nanoscience and Nanotechnology of NCSR “Demokritos” and combines arrays of ten sensors in a single silicon chip. Each sensor consists of an integrated on silicon light source that emits a broad spectrum in visible-near ultraviolet range and it is coupled to an integrated silicon nitride waveguide which has been patterned into Mach-Zehnder interferometer. The signal is recorded either through a photodetector monolithically integrated onto the same silicon chip (fully integrated configuration) or through an external spectrometer (semi-integrated configuration). In the fully integrated configuration, the signal recorded is the total photocurrent across the whole spectral range, while in semi-integrated configuration the whole transmission spectrum is continuously recorded and is mathematically transformed (Fourier Transform) to phase shift. As in the classical Mach-Zehnder interferometers, the waveguide in the proposed sensor is split into two arms, the sensing one which is appropriately modified with recognition biomolecule and the reference arm that is covered by a protective layer. The specific binding of the analyte with the immobilized onto the surface recognition biomolecule causes an effective refractive index change at the surface of the sensing arm thus affecting the phase of the waveguided light with respect to the reference arm. Thus, when the two arms converge again, an interference spectrum is generated that is altered during bioreaction providing the ability of monitoring in real-time and without using labels. The main difference of the sensor developed with respect to classical Mach-Zehnder interferometers is that the light source is monolithically integrated on the same silicon substrate with the waveguides and the waveguided light is not monochromatic, but broad spectrum.At first in this study, the method for chemical activation of biofunctionalization of chips was optimized. It was found that the highest signals were obtained when chips where activated by (3-aminopropyl)triethoxysilane and deposition of biomolecules solutions using a microarray spotter. Then, a comparison of the two sensor configurations, i.e. the fully and the semi-integrated configuration was performed using a model binding assay namely the streptavidin-biotin reaction. Semi-integrated configuration provided higher detection sensitivities mainly due to lower between-sensor signal variation in the same chip and between different chips. Thus, this configuration was selected for further evaluation with respect to the determination of analytes of clinical interest and especially of immunochemical determination of C-reactive protein in human serum samples. CRP is a marker of inflammation widely used in everyday clinical practice for diagnosis and therapy monitoring of inflammatory situations. Nevertheless, CRP has been also proposed as a prognostic marker of myocardial infraction and three risk levels have been established; low risk for serum CRP concentrations < 1 μg/mL; medium risk for concentrations in the range 1-3 μg/mL; and high risk for concentrations >3 μg/mL. In the frame of the present thesis, enzyme immunoassays for the determination of CRP in microtitration plates both competitive and non-competitive were developed in order to select the most appropriate reagents and define the immunoassay conditions. Then both assay format were transferred and evaluated on the sensor. It was found that the non-competitive format offered higher responses and ability for regeneration of immobilized onto the sensor antibody against CRP and was therefore selected for the final sensor evaluation. The assay developed following the competitive format was sensitive and accurate as was demonstrated through recovery and dilution linearity experiments, and provided for analysis of samples with a wide range of CRP concentrations since it was immune to the presence of serum. In addition, the CRP values determined with the immunosensor developed in serum samples from unknown donors were in good agreement with those determined for the same samples by commercially available kits and instruments showing the reliability of the determinations performed with the immunosensor developed and its potential for analysis of clinical samples.