Theoretical and experimental investigation on sensing performance of protein C immuno-optical sensor for physiological samples.

摘要

Protein C (PC) is an anticoagulant and antithrombotic in blood plasma. PC deficiency can result in severe clotting complications and, therefore, early diagnosis is important for immediate treatment. For that reason, there is a clinical need for a real time assay to diagnose PC deficiency. A fiber optic immunosensor has been under development to quantify it in the range of PC deficiency (0.5–2.5 μg/ml). The sensor is composed of an optical fiber on whose surface a monoclonal antibody (10 mAb) against PC is immobilized. When the sample is applied to the sensor, PC molecules in the sample bind to the 10 mAb. The bound PC is probed with fluorophore tagged, another type of monoclonal antibody (20 mAb). Feasibility studies are completed by previous researchers. In this dissertation, following results are presented: (1) The assay protocol was optimized. The PC incubation times were reduced from 10 to 5 minutes. The sensor size was shortened from 12.5 to 6 cm. The sensor reusability was improved using TEA elution buffer. The effect of 1° mAb leaching on the sensor performance was investigated; (2) Sensor performance in physiological samples was studied. Plasma decreases the fiber sensitivity by 70%. The high plasma viscosity (1.9 cP) was found to be a cause in the signal decrease. The sensor was proven to be capable of quantifying PC level in the animal cell culture broth and the transgenic swine milk; (3) Convective flow was used for the sample and reagent application to the chamber. The signal intensity increased with the increase in flow velocity, while the effect of sample amount was insignificant. However, the binding kinetics changed from transport limited to reaction rate limited, at the flow velocity higher than 0.45 cm/sec; (4) The instrumentation was improved to develop an automated and user-friendly PC biosensor. An instrument was constructed for fiber tapering. A computer code was developed for user-friendly data analysis; (5) The PC sensor system was theoretically analyzed. The PC and 2° mAb binding kinetics were found to be diffusion limited. A mathematical model was established and sensing responses under various static assay conditions (viscosity of sample, reaction rate constant) were simulated using the model. The convective flow effect on the mass transfer increase was analyzed based on the film theory.; Through the systematic optimization and characterization, the fiber optic sensor provides a smaller (100 μl sample chamber), faster (10–15 minutes), and sensitive (0.5–2.5 μg/ml) tool for PC quantification in physiological samples.