High performance system for protein assays: synergistic effect of terminal protection strategy and graphene oxide platform

Abstract

In this work, a novel fluorescent method for protein detection has been developed based on terminal protection of small molecule-linked DNA by target protein and the difference in affinity of graphene oxide (GO) for single-stranded DNA (ssDNA) containing different numbers of bases in length. A probe ssDNA, which is labeled carboxyfluorescein (FAM) at the 5′ end and a small molecule at the 3′ end, is designed for the detection of target protein. In the absence of target protein, the probe ssDNA can be hydrolyzed into mononucleotides by Exo I. The introduction of GO into the sensing solution results in weak quenching of the fluorescence of FAM due to the weak affinity of the short FAM-labeled oligonucleotide fragment to GO. Conversely, and very importantly, in the presence of target protein, the specifically binding of target protein to the small molecule of probe ssDNA can protect probe ssDNA from the Exo I-catalyzed digestion. Then the adsorption of the probe ssDNA on GO makes FAM close proximity to GO surface resulting in high efficiency quenching of fluorescence of FAM, and the fluorescence intensity gradually decreases with increasing concentration of target protein. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 1 to 80 ng mL⁻¹ with a detection limit of 0.81 ng mL⁻¹. Besides satisfactory sensitivity, the developed strategy also shows high selectivity, excellent reproducibility, and low cost, implying that this technique may have great potential applications in the future.