Assay procedure optimization of a rapid, reusable protein C immunosensor for physiological samples

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Immunosensing Technology

Background:

• Accurate quantification of protein C (PC) is crucial for diagnosing heterozygous PC deficiency.
• Existing diagnostic methods can be time-consuming and costly.
• Development of a rapid, cost-effective immunosensor is needed.

Purpose of the Study:

• To optimize key assay steps for a fiber-optic immunosensor designed for protein C (PC) quantification.
• To enhance the accuracy, speed, and cost-effectiveness of PC deficiency diagnosis.
• To report progress on the development of a novel PC detection tool.

Main Methods:

• Optimization of primary antibody concentration and assessment of its leaching.
• Determination of optimal fluorophore to secondary antibody ratio.
• Reduction of sample and secondary antibody incubation times.
• Evaluation of sensor performance with human plasma and under convective flow conditions.

Main Results:

• Optimal primary antibody concentration determined as 65 microg ml(-1) with minor leaching and stability over 30 days.
• Incubation times reduced to 5 min (sample) and 3 min (secondary antibody) while maintaining signal-to-noise ratio.
• Human plasma viscosity decreased signal intensity, but discrimination in the target range was achieved.
• Convective flow enhanced signal intensity by increasing PC mass transport.

Conclusions:

• The optimized fiber-optic immunosensor is a smaller, faster tool for PC detection in human plasma.
• The sensor provides a promising approach for accurate and cost-effective diagnosis of PC deficiency.
• Further development could lead to improved clinical diagnostic capabilities for thrombophilia.