Colorimetric biosensing of nopaline synthase terminator using Fe3O4@Au and hemin-functionalized reduced graphene oxide

摘要

In this paper, we present a simple and label-free colorimetric biosensor for detection of the nopaline synthase (NOS) terminator in genetically modified (GM) plants. The "signal on" colorimetric biosensor was developed using a nanocomposite consisted of gold nanoparticles doped magnetic Fe3O4 nanoparticles (Fe3O4@Au NP), capture probe DNA (cDNA), and hemin-functionalized reduced graphene oxide nanosheets (H-GN). The nano-composite was successfully prepared by means of Au-S bonds and the strong pi interactions between cDNA and H-GN. The sensing approach is based on the excellent peroxidase-mimicking activity of H-GN and its different electrostatic interactions with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In presence of the target NOS, the cDNA in the nanocomposite will hybridize with its complementary sequence, and form dsDNA structure. Due to the weak pi interactions between dsDNA and H-GN, a portion of H-GN will be released from the surface of Fe3O4@Au NPs and transferred into solution. After magnetic separation was performed, the supernatant was incubated with 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The released HGN can catalyze the oxidation reaction of TMB and turn the colorless solution blue. This "signal-on" colorimetric biosensor shows a broad linear range of 0.5-100 nM for the target NOS, with a 0.19 nM detection limit. The application of the biosensor for determination of NOS segments in samples of GM and non-GM tomatoes shows that it can discriminate between GM and non-GM plants. The reliability of the method for samples of NOS-spiked GM tomato suggests satisfactory recoveries in the range of 93.6%-94.2%.