Ανίχνευση C-αντιδρώσας πρωτεΐνης και D-διμερών σε μικροσυστοιχίες με φασματοσκοπία ανάκλασης λευκού φωτός

Abstract

Cardiovascular diseases are one of the main death causes in the modern world. Forthis 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 anischemic incident as well as for predicting a cardiac disease. The aim of this workwas to develop a method to simultaneously detect two markers that can help inpredicting a heart attack, namely C-reactive protein (CRP) and D-Dimers usingantibodies microarrays in combination with an optical immunosensor based on whitelight reflectance spectroscopy and allows the detection of biomolecular reactions inreal time without the use of labels. In particular, detection is based on monitoringchanges in interference spectrum created upon reflection of white light on a siliconsurface covered with a suitable film of transparent material on which the analytespecificantibodies are immobilized. At first, suitable antibodies were selected throughthe development of non-competitive enzyme immunoassays for the two analytes inmicrotitration wells. After that, the immunochemical assays were transferred to theoptical sensor, wherein the thickness and composition of the transparent filmdeposited on the silicon surface as well as the method for the chemical modificationof the surface and immobilization of antibodies were optimized. Subsequently, all theparameters of immunochemical determinations with the sensor were optimizedincluding antibody concentrations, immunoreaction time, flow rate, etc. By applying the optimal conditions, calibration curves for both markers were obtained and therepeatability and reliability of the measurements performed with the developedimmunosensor were evaluated. Finally, discrete zones of the two specific antibodiesagainst CRP and D-Dimers were created by spotting them onto the same surfaceand the surfaces were used for the simultaneous detection of the two analytes inserum samples. The values determined for both analytes were in good agreementwith those determined for the same samples with reference methods, demonstratingthe reliability of the determinations performed with the developed immunosensor andits potential for application in the simultaneous determination of two markers inclinical samples.