Protein C Tayinine Yönelik Yüzey Plazmon Rezonans Sensörlerin Hazırlanması
Abstract
Protein C is a vitamin K-dependent plasma protein. Activated protein C is a glycoprotein derived from its precursor, protein C and formed by the cleavage of an activation peptide by thrombin bound to thrombomodulin. Activated protein C is a potent anticoagulant and enhances blood clot lysis. Activated protein C, exerts its anticoagulant function by inhibiting the cofactors in the clotting cascade factors Va and VIIIa. PC is a trace protein at a concentration of 4 µg/mL in human blood. Serious problems can occur when the PC level in blood is lowered. Patients with PC deficiency are at risk of deep vein thrombosis (DVT), and other clotting complications, resulting in tissue oxygen deprivation, some of which can be life-threatening. When these blood clots break away from the surface of the vein and enter the blood stream, they will induce strokes, heart attacks, and pulmonary embolism. Surface plasmon resonance (SPR) is a highly sensitive method based on the real-time measurement of the refractive index on a metal surface. Surface plasmon resonance sensors are prepared by forming target-specific regions on the metal surface. Monoclonal antibodies and aptamers are two of the most prominent biorecognition elements for this purpose. SPR sensors prepared by this method do not require labeling. Nucleic acid aptamers (DNA or RNA) are ~15-100 nucleotides in length oligonucleotides that are partitioned from a large random pool. These isolated nucleotides exhibit high affinity and specificity for a wide range of targets. Because of their binding abilities, aptamers have great potential in many applications, such as biosensors, imaging probes, and pharmaceutical agents. The aim of the presented study is preparation of the surface plasmon resonance (SPR) sensor for the detection of Protein C from the artificial plasma and PC solution. Prepared poly(HEMA-APC Apt) polymer film was modified on the gold surface of the SPR sensor. In the first step, APC aptamer was complexed with MAC (N-methacryloyl-L-cysteine) monomer. Then, cyanamide and HEMA (2-hydroxyethyl methacrylate) solution was mixed with the APC Apt/MAC complex. Azobisisobutyronitrile was added to polymer mixture as the initiator. The mixture was dropped onto the gold surface of the SPR sensor and polymerized under an ultraviolet (UV) lamp. SPR sensors with poly(HEMA-Random DNA) and poly(HEMA-MAC) films were also prepared by same experimental procedure. Prepared SPR sensors were characterized with atomic force microscopy (AFM), the ellipsometer, and contact angle measurements. Selectivity studies and concentration studies were used performed with poly(HEMA-APC Apt) and poly(HEMA-Random DNA) SPR sensor, and selectivity studies were performed poly(HEMA-MAC) SPR sensor. Desorption studies were performed by using 0.025 M NaCl solution. The limit of detection (LOD), limit of quantitation (LOQ) values of prepared sensors were determined. SPR sensor showed high selectivity and sensitivity for PC in the artificial plasma.