Improving the performance of immobilized molecular beacons through cleavage

Surface-immobilized molecular beacons (MBs) usually have relatively low fluorescence enhancement after hybridization, which causes significant limitations to their applications. Here we designed a molecular beacon with a restriction endonuclease recognition site at its loop structure, to study the behavior of the MB on solid matrix and to improve its performance. The maximum fluorescence enhancement of the MB caused by restricting cleavage after hybridization was 5.2, close to its corresponding enhancement 5.9 in solution, higher than its corresponding enhancement 2.7 that just caused by hybridization. This result showed that some of the MB molecules were still closed even though hybridization with target cDNA, this maybe due in part to the surface effects or the MB's conformation on solid matrix. Moreover, the cleavage induce fluorescence enhancement have been used to decrease the limit of detection from 10 to 2 nM cDNA.