Optimization of a fully automated oligonucleotide assay to minimise metabolite interference

A dual hybridisation assay was developed on a fully automated microfluidic immunoassay platform. The hybridisation reaction was performed in the mixing chamber of the Mixing CD. It was shown that similar performance was achieve to offline incubations which allowed the assay to be fully automated.   

 One limitation of dual hybridisation assays is the potential to detect metabolites. Although the nuclease cutting assay format was tested, the dual hybridisation assay format gave improved sensitivity  

 Metabolite interference was assessed using N-1, N-2 and N-3 metabolites at both the 5’ and 3’ end of the parent oligo. Using the automated assay format, the only significant interference was demonstrated for the N-1 metabolite at the 5’ end. It was however possible to reduce the interference by the addition of a nuclease wash across the affinity column.   

 This Automated Microfluidic Nuclease Enzyme Wash Twin Oligonucleotide Probe (AM NEW TOP) assay format was shown to improve the metabolite interference. The flexibility of the microfluidic approach would allow for method modification to improve metabolite dependent on the expected levels and the chemistry of the analyte.

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