Dissertation committee:
Αθανασία Σιαφάκα-Καπάδαη Καθηγήτρια (Επιβλέπουσα), Σωτήριος Κακαμπάκος Ερευνητής Α΄, Παναγιώτα Πέτρου Ερευνήτρια Α΄
Summary:
Cardiovascular diseases are one of the main death causes in the modern world.
For this reason, the determination of the concentration in the serum of
particular proteins, called cardiac markers, is vital both for determining the
extent of the damage after an ischemic incident as well as for predicting a
cardiac disease. The aim of this work was to develop a method to simultaneously
detect two markers that can help in predicting a heart attack, namely
C-reactive protein (CRP) and D-Dimers using antibodies microarrays in
combination with an optical immunosensor based on white light reflectance
spectroscopy and allows the detection of biomolecular reactions in real time
without the use of labels. In particular, detection is based on monitoring
changes in interference spectrum created upon reflection of white light on a
silicon surface covered with a suitable film of transparent material on which
the analyte-specific antibodies are immobilized. At first, suitable antibodies
were selected through the development of non-competitive enzyme immunoassays
for the two analytes in microtitration wells. After that, the immunochemical
assays were transferred to the optical sensor, wherein the thickness and
composition of the transparent film deposited on the silicon surface as well as
the method for the chemical modification of the surface and immobilization of
antibodies were optimized. Subsequently, all the parameters of immunochemical
determinations with the sensor were optimized including antibody
concentrations, immunoreaction time, flow rate, etc. By applying the optimal
conditions, calibration curves for both markers were obtained and the
repeatability and reliability of the measurements performed with the developed
immunosensor were evaluated. Finally, discrete zones of the two specific
antibodies against CRP and D-Dimers were created by spotting them onto the same
surface and the surfaces were used for the simultaneous detection of the two
analytes in serum samples. The values determined for both analytes were in good
agreement with those determined for the same samples with reference methods,
demonstrating the reliability of the determinations performed with the
developed immunosensor and its potential for application in the simultaneous
determination of two markers in clinical samples.
Keywords:
C-reactive protein, D-Dimers, Microarrays, Biosensor
