Split aptamer-based sandwich fluorescence resonance energy transfer assay for 19-nortestosterone

In order to develop an aptamer based fluorescence resonance energy transfer (FRET) assay for 19-nortestosterone, a 76-mer 17 beta-estradiol aptamer was split into two pieces (referred to as P1 and P2, respectively). P1 was labeled with a quencher (BHQ), and P2 with a fluorophore (6FAM). The two aptamer pieces were employed to detect NT via FRET quenching in a homogeneous solution. This method has a low detection limit (5 mu M) within a wide dynamic range (5 to 1000 mu M). The approach was used to analyze spiked urine samples, and the results showed that the average recovery of three samples containing different NT concentrations ranged from 58 to 118 % with a relative standard deviation (RSD) of less than 1 %. In our perception, the method has a wide scope for future applications to other analytes by using dually labeled split aptamers.