A fluorescent biosensor for protein detection based on poly(thymine)-templated copper nanoparticles and terminal protection of small molecule-linked DNA

In this paper, a fluorescent biosensor has been developed for protein detection based on poly(thymine) (poly T)-templated copper nanoparticles (Cu NPs) and terminal protection of small molecule linked-DNA. This strategy was demonstrated by using small molecule biotin and its binding protein streptavidin (SA) as a model case. In this assay, biotin-linked poly T (biotin-T30) probe was specifically bound to the target protein SA with strong affinity in the presence of SA. The selective binding events confirmed that biotin-T30 probe was protected against the hydrolysis by exonuclease I (Exo I), which could effectively template the formation of fluorescent Cu NPs. The results revealed that the developed strategy was highly sensitive for detecting SA in the concentration range from 0.5 to 1000 nM with a detection limit of 0.1 nM. In addition, the relative standard deviation was 3.6% in 5 repetitive assays of 50 nM SA, which indicated that the reproducibility of the method was acceptable. Besides desirable sensitivity, the developed biosensor also showed high selectivity, low cost, and simplified operations. Thus, it could hold considerable potential to construct a simple, selective and sensitive fluorescent platform for detection of small molecule-protein interactions in molecular diagnostics and genomic research.