Label-free evaluation of short oligonucleotide bound alginate hydrogels using circular dichroism spectroscopy

Oligonucleotide-based biosensors have attracted interest due to the increasing global demand for disease detection, environmental monitoring, and detection of degraded samples such as those found in forensic contexts. In this study, we explore the efficacy of circular dichroism (CD) spectroscopy to detect DNA and RNA hybridization on label-free short oligo-alginate hydrogels, without the need for amplification. We use an EDC/NHS coupling reaction to synthesize alginate-azide molecules, which are then crosslinked to DNA-alkyne or RNA-alkyne oligonucleotides using a copper-catalyzed azide-alkyne cycloaddition (CuAAC). A complementary strand to the bound oligonucleotide is added to the hydrogel and hybridization is assessed using CD spectroscopy. We report a limit of detection of 0.73 nmol and 0.17 nmol for DNA- and RNA-based biosensors, respectively. Biosensor specificity is evaluated by adding solution mixtures containing up to four different non-complementary strands to the alginate-oligo hydrogels. Chemometric models are then used to assess biosensor specificity. Principal component analysis (PCA) is performed on the spectra collected from 156 samples and successfully differentiated samples with and without a bound complement. DNA-based biosensors can also be distinguished from RNA-based biosensors. Additionally, random forest classification models are computed to classify unknown samples based on complement binding, achieving prediction accuracies greater than 92 %. Our findings demonstrate the feasibility of label-free, amplification-free detection of short oligonucleotides in aqueous solutions by measuring hybridization within our biosensor with CD spectroscopy, supporting potential applications to more complex environmental matrices.