Biophotonic Sensor Technologies for Systems Biology
Date: 29 August 2023
Thesis or dissertation
University of Exeter
Doctor of Philosophy in Biological Sciences
Profiling of complex systems responses requires multiplexed testing of analytes, ideally with rapid results from low volumes. The ability to analyse samples at the point of collection is especially important when testing frangible samples, where systems such as the complement cascade or acute phase response have been activated. The ...
Profiling of complex systems responses requires multiplexed testing of analytes, ideally with rapid results from low volumes. The ability to analyse samples at the point of collection is especially important when testing frangible samples, where systems such as the complement cascade or acute phase response have been activated. The combination of rapid sampling near the point of collection also allows for evolving systems to be evaluated in near real-time. The system used for this work was based on a gold nanoparticle sensor, with the specificity of response provided by functionalising the nanoparticles with proteins to allow an immuno-kinetic sandwich assay to be observed in real-time. The first study utilised a biosensor for C-reactive protein (CRP), a sensitive marker for the acute phase response, as it has proven use in diagnostics and an already working assay was assembled. The assay provided results in 8 minutes from a sample of 5 μL of whole blood. The analytical performance showed an accuracy of 0.42 mg L-1 (95% Confidence Interval – from here referred to as CI -14.7-13.8 mg L-1) (and precision of a CV of 10.6% (CI 0.9-20.2%) over the diagnostically useful range of 2-160 mg L-1. With the emergence of SARS-CoV-2, the flexible, multiplexed nature of the biophotonic LiScAR platform allowed for the CRP assay to be extended to include a profile of the antibody response to infection. During testing at St Thomas’ Hospital in March 2020, this semi-quantitative assay, conducted for IgM, IgA and IgG antibody concentrations against nucleocapsid (N), spike 1 (S1), spike 2 (S2), membrane (M) and envelope (E) proteins, was found to have a clinical sensitivity of 95% (CI 71- 97%) when compared to RT-PCR results. This assay was then developed into a fully quantitative assay for IgG against S, S (omicron), N and receptor binding domain (RBD) proteins that received a CE Mark in 2021, and a protective threshold of 3.4 mg L-1 calculated. As SARS-CoV-2 variants evolved, an assay was constructed to measure the quantitative IgG response to the S variants from Wuhan, Alpha, Beta, Gamma, Delta, BA.1, BA.2.12.1, BA.2.75, BA.4 and BA.5. The analysis of the concentration profile for each variant led to a classification of the serology response into a series of endotypes with the universal positive endotypes, U(+), having a prevalence of 22% (CI 12-37%) in the double-vaccinated cohort rising to 54% (CI 39-68%) in the triple-vaccinated cohort, compared to 11% (CI 4-25%) in the cohort infected with the Wuhan strain. Finally, a prospective cohort study was performed for a cohort of educational staff profiling both the concentration and pH-dependent affinity of the IgG response to the ten variant proteins. This found a U(+) prevalence of 78% (CI 60-88%), but fell to 25% (CI 13-43%) when only high-affinity antibodies were measured, potentially signifying a vulnerability masked by simple concentration measurements. Signs of antibody maturation against prevalent variants were also observed. Multiplexed profiling of the immune serological response to SARS-CoV-2 infection across different virus variants and patient cohorts has provided new information on the changing immune response to vaccination and disease which may be of help in managing the ‘immunity shield’ as SARS-CoV-2 becomes an endemic disease similar to influenza or respiratory syncytial virus.
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