Amplification-free detection of genetically modified crops via spatial confinement CHA/HCR-aided surface-enhanced...

To distinguish genetically modified (GM) crops, we developed a target RNA amplification-free surface-enhanced resonance Raman scattering (SERRS) biosensor for the CaMV35S promoter RNA in GM crops by integrating enzyme-free, spatial confinement catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). A magnetic-plasmonic composite probe modified with the first sequence of CHA was first fabricated. A small amount of target RNA can activate it to initiate the CHA, and the CHA efficacy benefits from the spatial confinement effect provided by the composite probe. The resulting CHA products bridge the subsequent HCR, producing DNA nanowires above the probe. These nanowires exhibit a high loading capacity to methylene blue (MB), enabling the SERRS assay that benefits from both the plasmonic enhancement provided by the magnetic-plasmonic probe and the resonance effect under 633 nm wavelength excitation. The CHA/HCR-SERRS platform is highly specific and sensitive to the CaMV35S promoter RNA segments in genetically modified crops, exhibiting excellent linearity within the 0.01-100 pM range. The limit of detection is achieved as low as 10 fM (6 × 10³ copies/μL) with a minimum sensing duration of 60 min. Compared to other non-amplified nucleic acid detection, the sensitivity increased by 2-3 orders of magnitude. This study achieves highly sensitive, amplification-free RNA detection, which could serve as a universal nucleic acid analytical method in various other scenarios.