DNA Assay-on-a-String: Rapid Detection of Marker Panels Against Sepsis
Authors: Irving Oliver James , Albrecht Tim 
Published: May 2020

Sepsis is a life-threatening condition afflicting 250,000 patients in the UK alone. The condition is defined as the overstimulation of the immune system in response to an invasive microorganism, primarily bacterial infections1. As the condition progresses, symptoms worsen from fever and respiration difficulties to organ failure and cardiovascular issues. The primary test for sepsis is the use of blood cultures. These can take up to two days to complete and do not always provide a positive result. Whilst there have been many biomarkers identified for sepsis, few have been incorporated into the clinic. High sensitivity biomarkers include both proteomic and genetic biomarkers. In proteomic studies, interleukin 6, 8, and 10 proteins, c-reactive protein, and procalcitonin have proved to be statistically significant diagnostic markers for the disease. Two micro-RNAs have also been shown to be significant, these are miRNA-146a and miRNA-1502.
This research is focused on the development of a new, rapid diagnostic test for sepsis utilising many different biomarkers in combination. To achieve this the DNA-assay-on-a-string technique will be utilised to bind biomarkers of interest. The resolution required to monitor this modified DNA, requires nanopipettes with a pore size of 10 nm to be used3,4. This was achieved by use of a p2000 pipette puller along with quartz capillary tubes. All electrodes used were Ag/AgCl and pore diameter was determined by cyclic voltammetry5–7. The conductance was extracted from an I-V trace and applied to a pore size calculation equation by Steinbock et.al8.
To create a carrier for multiple biomarkers, double stranded DNA will be used as a backbone and single stranded DNA, antibodies and/or aptamer probes will be conjugated to the structure. The difference between unbound and bound probes will be determined through current changes during translocation.
It has been possible to produce nanopipettes reliably and reproducibly with an average pore diameter of 11 nm. Noise optimisation has focused in particular on the preparation protocols for the Ag/AgCl electrodes, which caused up to an order of magnitude difference in RMS current noise, depending on the method used. In particular, best performance was obtained by anodisation of the silver wire in LiCl electrolyte, which provided a less rough surface as illustrated by the reduction in noise amplitude in Figure 1. Translocation experiments with double-stranded DNA have been performed successfully (cf. Figure 1 A) and focus is now shifting towards target detection.
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8. L. J. Steinbock, A. Lucas, O. Otto, and U. F. Keyser, Electrophoresis, 33, 3480–3487 (2012).

Figure 1