Fluorescent Determination of Calcium Ion by DNA Nanospheres Based Upon the EtNa DNAzyme

A Ca2+ sensor based on DNA nanospheres was developed. The DNA nanospheres were obtained by complementary pairing of Y-shaped DNA and DNAzyme, in which Y-shaped DNA is a triple-branched DNA with three sticky ends formed by three partially complementary single-stranded DNAs. DNAzyme is a recently reported RNA-cleaving DNAzyme, EtNa, that has high selectivity for Ca2+ and was used as linker in DNA nanosphere preparation. When EtNa DNAzyme was formed by enzyme chain (E) and substrate chain (S), the fluorescence signal was quenched significantly due to the proximity of fluorophore and quencher. In the presence of Ca2+, it bound to the E strand of EtNa DNAzyme, catalyzing the cleavage of S strand, which led to the separation of S from E and then the DNA nanosphere was disintegrated. At the same time, the fluorescence signal was restored and Ca2+ assay was realized. This detection system based on DNA nanospheres determined Ca2+ across a linear range from 20 to 400 μM with a detection limit of 1.6 μM and excellent selectivity. In addition, this proposed Ca2+ assay has been successfully applied to the assay of Ca2+ in human serum, thus demonstrating clinical application.