A fluorometric histidine biosensor based on the use of a quencher-labeled Cu(II)-dependent DNAzyme

The authors describe a biosensor for histidine that is based on the use of a DNAzyme catalytic beacon. The Cu(II)-dependent DNA-cleaving DNAzyme (Cu-Enzyme) was modified with a quencher (BHQ1) at its 5' end, and the corresponding substrate strand (Cu-Sub) was modified with a quencher and the FAM fluorophore at its 5' and 3' ends, respectively. The green FAM emission of the system is completely quenched after the Cu-Enzyme is hybridized with Cu-Sub. The presence of Cu(II) triggers the cleavage of the Cu-Sub so that fluorescence recovers. Histidine forms a complex with Cu(II) ion. The complex is not capable of cleaving Cu-Sub effectively so that the fluorescence of the system is not restored. These findings were exploited to design a robust and sensitive assay for the determination of histidine. Fluorescence intensity is linearly related to the concentration of histidine in the range between 0.05 and 40 mu M, and the detection limit is 20 nM. The method has been successfully applied to the determination of histidine in (spiked) human urine and gave satisfying results.