Development of rolling circle amplification based surface-enhanced Raman spectroscopy method for 35S promoter gene detection

• RCA based SERS sandwich assay systems were formed.
• Traditional vs. RCA based SERS sandwich assays were compared.
• The detection limit of RCA–SERS sandwich method was found to be 6.3 fM.
• The selectivity and specificity tests of RCA–SERS sandwich assay were evaluated.

In this study, we developed the genetically modified organism detection method by using the combination of rolling circle amplification (RCA) and surface-enhanced Raman spectroscopy (SERS). An oligonucleotide probe which is specific for 35S DNA promoter target was immobilised onto the gold slide and a RCA reaction was performed. A self-assembled monolayer was formed on gold nanorods using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and the second probe of the 35S DNA promoter target was immobilised on the activated gold coated slide surfaces. Probes on the nanoparticles were hybridised with the target oligonucleotide. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. SERS spectra of target molecules were enhanced through the RCA reaction and the detection limit was found to be 6.3 fM. The sensitivity of the developed RCA–SERS method was compared with another method which had been performed without using RCA reaction, and the detection limit was found to be 0.1 pM. The correlation between the target concentration and the SERS signal was found to be linear, within the range of 1 pM to 10 nM for the traditional assay and 100 fM to 100 nM for the RCA assay. For the developed RCA–SERS assay, the specificity tests were performed using the 35S promoter of Bt-176 maize gene. It was found out that the developed RCA–SERS sandwich assay method is quite sensitive, selective and specific for target sequences in model and real systems.

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