Sterically controlled nuclease enhanced DNA assembly in rapid sepsis diagnostics

Irving, Oliver James ORCID: 0000-0001-6617-2436 (2022). Sterically controlled nuclease enhanced DNA assembly in rapid sepsis diagnostics. University of Birmingham. Ph.D.

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Abstract

Sepsis is a life-threatening condition afflicting 250,000 patients in the UK alone and defined as the overstimulation of the immune system in response to a pathogen. As the condition progresses, symptoms worsen, from fever and respiration difficulties to organ failure and cardiovascular issues. The primary test for sepsis is blood cultures which can take up to 2 days to complete and can often provide a false negative result. There are currently no point of care devices available for sepsis diagnosis. While many biomarkers have been identified for sepsis, few have been incorporated into the clinic. Rapid and accurate diagnostics is the key to reducing patient mortality and improve prognosis. Nanopore sensing is a technique based on the principle of resistive pulse sensing, first described by Wallace H. Coulter. Nanopore sensing is a very sensitive technique often applied to single molecule sensing. Recent developments in biosensing demonstrate the potential for DNA modification for biomolecule capture and nanopore sensing. These techniques are often limited in their adaptability to capture multiple analytes.

This thesis discusses a newly developed technique for rapidly producing a diagnostic toolbox and testing multiple analytes simultaneously with nanopore sensing integration. It is possible to produce a DNA nanostructure containing multiple analyte binding sites that can bind IL-6 and procalcitonin proteins, detected using modified ELISA and resistive pulse sensing techniques. For a trimer structure, it is possible to capture proteins with an efficiency of 72.5%. From multiple assembly experiments it has been shown that structures of different sizes containing a variety of probes can be assembled. Most notably, this thesis shows that the limitations of the Gibson assembly method can be overcome using the developed technique. The inclusion of a biotin probe allowed for both DNA structure isolation and post assembly functionalisation.

Optimisation of nanopipette fabrication has allowed for the development of a reproducible protocol to produce nanopipettes with pore sizes at <10 nm, 10 - 30 nm, and finally <30 nm. To increase the sensing capability of the nanopipette, taper length was also optimised to consistently produce pipettes with taper lengths of ~3000 µm. Further optimisation of extrinsic electronic noise was performed to improve the signal to noise ratio for translocation experiments. It was determined that the primary contributor to noise was using equipment supplied by mains power. Therefore, optimisation explored altering the equipment to work off battery power alone. From these results, the experimental procedure used would involve using shorter, anodised electrodes, using a silver shielded data transfer cable, working on a granite slab, using a passive filter of 100 kHz or less, and working in an “off grid” approach using battery power where possible.

Standard DNA fragment translocation was performed, and the results compared against literature values. The results highlight similar translocation frequencies of ~1 per nM per second, event duration, and magnitude also. Through comparison between tetramer SCoNE DNA and bare DNA fragments, it was possible to identify significant subevents relating to probe structures at positions near 0, 0.25, 0.5, and 0.75 along the DNA backbone. Whilst it was not possible to confirm this difference for a decamer structure, subevents were observed at locations specific to probe sites. The difference was not significant between the decamer SCoNE structure and the 10 kbp fragment. The decamer structure was translocated prior to the development of the biotin probe. It is therefore possible to suggest that the gel extraction method used for isolation could have cleaved the probe structures from the backbone, limiting the potential for subevent detection.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):