Yüzey Plazmon Rezonans Temelli Trombin Biyosensörünün Geliştirilmesi

Abstract

Enhancing the performance of biosensors has become an important task. Some of the requirements for improving the sensitivity and selectivity of these systems includes overcoming the steric hinderance issue, providing resistance to nonspecific binding, stable biological recognition layer formation and providing upright position while retaining the biological activity of the biological recognition element during the surface immobilization process. Formation of mixed self-assembled monolayers (mSAMs) is a commonly used procedure for diluting thiol modified biological recognition elements, so that target recognition is enhanced due to reduction in steric hinderance. In this study, a surface plasmon resonance (SPR) based sensor was developed for thrombin detection via forming 3,3’ Dithiodipropionic acid di (N-hydroxysuccinimide ester) (DSP) :6-mercapto-1-hexanol (MCH) mSAMs on gold surface. During the development of the sensor surface, DSP was utilized together with MCH 1) To decrease the biological recognition element density and hence the steric hinderance effect for improving the target recognition sensitivity. 2) To minimize the risk of a loss in the biological activity of the biological recognition element by decreasing the chance of its random and nonspesific immobilization on the surface. For the chemical activation of the gold surface, different molar ratios of DSP:MCH (2.0:2.5, 2.0:5.0, 2.0:10.0) has been used for the formation of mSAMS and thrombin antibody or thrombin aptamer was immobilized on gold surface via DSP. The biosensor capability of the modified surface was tested by using the target molecule thrombin. The chemical and biological activation on the gold surface and the performance of the biosensor was tested using a flow-cell coupled SPR system. Both thrombin aptamer and thrombin antibody sensors’ performance were tested with increasing trombin concentrations and calibration curves were obtained. For the thrombin aptamer sensor, the linearity was observed in two different concentration regions, namely 0.0-20.0 nM and 20.0-100.0 nM range. R2 values for the first concentration region and the second concentration region were calculated as 0,998 and 0,961 respectively. The linear range for the thrombin antibody sensor was 22.0-100.0 nM. R2 value for the antibody sensor was calculated as 0,992. These ranges are within the physiological thrombin concentration range (1-500 nM) in serum during the coagulation process. Limit of Detection (LOD) for thrombin aptamer and thrombin antibody sensors were found to be 9.5 nM and 6.0 nM respectively. Limit of Quantification (LOQ) for thrombin aptamer and thrombin antibody sensors were found to be 30.0 nM and 22.0 nM respectively. Both sensors were also tested with thrombin spiked serum samples and compared with thrombin added PBS samples. For thrombin aptamer sensor, the sensor response (∆RU) obtained for the thrombin spiked serum samples was twice as that obtained from thrombin spiked PBS samples. As for the thrombin antibody sensor, sensor response (∆RU) obtained from thrombin spiked serum samples and thrombin spiked PBS samples were similar. In the light of these results, for the biosensor system prepared using DSP:MCH (2.0:2.5) as an intermediate layer, antibody usage as a biological recognition element is thought to be a better choice for the detection of thrombin in the complex serum matrixs. The regeneration capability of both sensors were also tested. For the aptamer based sensor, thrombin aptamer could be regenerated for five cycles. The change in the sensor response (∆RU) remained constant for five cycles and there was a decline afterwards. The antibody based sensor could not be regenerated. X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectroscopy were used for surface characterization of 2.0 mM DSP and 20.0 mM MCH self-assembled monolayers (SAMs), DSP:MCH (2.0:2.5) and DSP:MCH (2.0:10.0) mSAMs formed on gold surfaces. Tapping mode atomic force microscope (AFM) was utilized for the examination of surface morphology of DSP:MCH (2.0:2.5), BSA immobilized DSP:MCH (2.0:2.5) and blank surfaces. The roughness values of surfaces were determined and compared. The average surface roughness value for the gold surface were calculated as 1.20 nm. Following the formation of DSP:MCH (2.0:2.5) mSAMs on the surface, average surface roughness value increased from 1.20 nm to 2.20 nm and decreased to 1.81 nm following BSA binding. Based on the results of this thesis, it was shown that DSP:MCH interface could be used as a new immobilization platform for binding biological recognition elements for the development of biosensors.