A graphene oxide-based paper chip integrated with the hybridization chain reaction for peanut and soybean allergen gene detection

Allergen genes of the peanut and soybean were selected as model targets. Four hairpin DNA probes, H1, H2, H3, H4 were designed. Cy3-labeled H1 and H2 were used to detect peanut DNA, while FAM-labeled H3 and H4 were used to detect soybean DNA. Graphene oxide (GO) was used as the adsorption material for capturing the hairpin probes, and as a selective fluorescence quencher to reduce the background signal. To develop an allergen gene detection system with a GO-based paper chip format, we integrated the hybridization chain reaction (HCR) with fluorescence resonance energy transfer (FRET) in our design. The results showed that in the absence of peanut DNA (T-P) and soybean DNA (T-S), the detection probes attached to the GO surface, which quenched their fluorescence. In the presence of T-P or T-S, however, complementary probe binding to the targets initiated HCR, producing long double-stranded DNA products that could not be absorbed onto the GO surface. Hence, a strong red or green fluorescent signal was generated. The detection limit for both peanut and soybean DNA was 1 nM using this method, indicating the high sensitivity of our approach. This method also exhibited good specificity and a single chip could be used to simultaneously detect two different targets.