FRET based biosensor for detection of active NF-kB

The Nuclear Factor kB is a transcription factor, ubiquitously expressed, involved in the regulation of a large number of genes and in a variety of human disease including inflammation, asthma, atherosclerosis, AIDS, septic shock, arthritis and cancer. The critical need for a simple and direct method to evaluate the quantity of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. A single-stranded DNA (ssDNA) with auto-complementary sequence has been properly designed and synthesized. In order to evaluate FRET due to the DNA/protein binding interaction taking place between double-stranded DNA (dsDNA) immobilized in a capillary wall and NF-kB proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used. Preliminary results show that our system was capable of detecting the active form of NF-kB protein with a detection efficiency of about 90% and that the system has a good regenerability.