Label-free quantification of single-stranded DNA utilizing enzymatic digestion and an off-the-shelf glucose test strip

Abstract

A method was developed for quantifying single-stranded DNA (ssDNA) through enzymatic digestion and using commercially available glucose test strips. The process involves the initial digestion of ssDNA using a combination of exonuclease 1 and alkaline phosphatase enzymes, leading to the liberation of phosphates from the ssDNA backbone as free orthophosphate. Subsequently, the orthophosphates react with maltose and maltose phosphorylase, producing equivalent amounts of glucose to orthophosphate. The resulting glucose, which can be related to the ssDNA concentration, can be measured amperometrically with an off-the-shelf glucose test strip connected to a mini potentiostat. This method offers versatility, allowing the determination of ssDNA, regardless of nucleotide-count or sequence, with increased sensitivity as the number of nucleotides (NT) in the DNA increases. The method exhibits a limit of detection of 780 nM for 22-NT, 527 nM for 53-NT, 422 nM for 75-NT, and 329 nM for 87-NT ssDNA, and a linear range of 0–2 μM. To selectively quantify a specific ssDNA target, a magnetic microparticle-based isolation step was incorporated, demonstrating high selectivity for quantifying a particular ssDNA target from a mixture. The method holds potential for label-free quantification of ssDNA that can have an impact in myriad fields.