Detection of single-stranded nucleic acids by hybridization of probe oligonucleotides on polystyrene nanospheres and subsequent release and recovery of fluorescence

Abstract

In this contribution, we demonstrate that polystyrene (PS) nanospheres can serve as an effective sensing platform for fluorescence-enhanced DNA detection. This kind of assay can be completed by the following two steps: (1) PS quenches the fluorescence of dye-labeled single-stranded DNA (ssDNA) probes very effectively when they are brought into close proximity as a result of the adsorption of ssDNA on PS. The adsorption is ascribed to the strong π–π stacking between unpaired DNA bases and PS. (2) Upon presence of target ssDNA, the specific hybridization of the probe with its target produces a double-stranded DNA (dsDNA). The duplex detaches from PS due to its rigid conformation, the absence of unpaired DNA bases, and electrostatic repulsion between negatively charged dsDNA backbone and PS, leading to recovery of dye fluorescence. This assay system exhibits high selectivity and sensitivity with a detection limit as low as 5 nM. It suggests that this sensing platform can differentiate between perfectly complementary and mismatched targets. The fluorescence enhancement in response to single-base mismatched target T₂, two-base mismatched target T₃, and three-base mismatched target T₄ is about 71%, 63%, and 58% of that from complementary target T₁, respectively. The suggested method can also discriminate complementary and single-base mismatched sequences embedded in rather larger strands with short oligonucleotide probes. The fluorescence enhancement in response to single-base mismatched sequence embedded in large strand is 83% of that from the one embedded with complementary sequence. In further experiments, it is demonstrated that the present system can be used for multiple DNA detection. Finally, efforts are made toward its application in human blood serum system to evaluate the ability to withstand the interference arising from real sample.