Development of biosensors for early detection of anastomotic leak and sepsis

Anastomotic leak is a catastrophic surgical complication leading to high morbidity, mortality and cancer recurrence. Currently detection is difficult, with a paucity of available diagnostic tests that have variable sensitivity and specificity. The work described in this thesis evaluated the use of local biomarkers within the anastomotic environment coupled with a biosensor application to assess proof-of-concept feasibility as a point-of-care diagnostic tool for anastomotic leak. Using a small animal model of caecal ligation and puncture to replicate abdominal sepsis, local abdominal biomarkers lactate, TNFα, and E. coli were all found to significantly increase compared to sham control models at 24 and 36 hours. Chronoamperometry and electrochemical impedance spectroscopy (EIS) interrogation of biosensors were then used to detect and quantitate levels of these respective biomarkers in real patient samples, and data compared to that obtained by existing commercial assays to evaluate accuracy. Characterisation of each biosensor utilised cyclic voltammetry, SEM, Midland blotting, SDS-PAGE and dot blotting techniques to optimise the fabrication methodology. The lactate biosensor consisted of a pre-impregnated Prussian Blue carbon electrode with lactate oxidase enzyme immobilised onto the surface via polyethyleneimine. Using chronoamperometry, the lactate biosensor gave significantly similar results to a commercial enzyme-based lactate colorimetric assay in ten abdominal fluid patient samples. E. coli immunosensors were constructed using a polytyramine matrix onto which half polyclonal antibody fragments raised against multiple strains of E. coli were immobilised. EIS was used to measure the charge transfer resistance of the biosensors when incubated with a varying concentration of E. coli, with a limit of detection found to be 104 cells ml-1. EIS of E. coli biosensors in the ten patient samples showed statistically significant equivalent results to those from flow cytometry. Immunosensors to TNFα were constructed using a similar methodology to E. coli, with whole antibody to TNFα immobilised onto a polytyramine electrode surface. Initial EIS results in buffered solution showed good biosensor response to varying concentrations of TNFα, but further studies are required for complete biosensor development.