Gill, Megha; (2025) Investigating an E.coli-based cell-free protein synthesis system as a biological amplification tool for lateral flow immunoassays. Doctoral thesis (Ph.D), UCL (University College London).

Text Gill_10213419_Thesis.pdf Access restricted to UCL open access staff until 1 October 2026. Download (9MB)

Abstract

Large-scale adoption of rapid diagnostic tests (RDTs), like paper-based lateral flow immunoassays (LFAs) are an important countermeasure for decentralized testing of infectious diseases. However, the sensitivity of current LFAs (μM-pM) lags significantly behind laboratory testing (ELISAs (pM-fM) & PCR (aM)). In recent work, novel bioengineering approaches have been employed to overcome the limitations of LFA sensitivity. In our research, we have engineered an ‘expression immunoassay’ with an E.coli-based cell-free protein synthesis system (CFPS) for signal enhanced SARs-CoV-2 nucleocapsid protein (NP) detection. Cell-free (CF) reactions from DNA-antibody-antigen complexes permitted the synthesis of a fluorescent reporter protein, SuperFolder GFP (sfGFP). Through cumulative production of CF-expressed sfGFP, the protein-substrate signal was enhanced. Fluorometric signals and LFA detection strategies were characterised and evaluated against traditional immunoassays. This thesis outlines the research framework, methodologies, and findings related to our exploration of an E.coli-based CFPS system as a suitable biological amplification platform for signal enhanced LFA antigen detection. The development pipeline for CF immunoassay was as follows, beginning with the characterization of sfGFP protein expression in the in-house E.coli-based CFPS system. Robust expression of sfGFP protein from linear dsDNA templates demonstrated sufficient amplification gains. Next, a paper-based LFA device was fabricated for CF sfGFP detection. Importantly, a strategy to handle CFPS sample matrices was explored. Lastly, both CF and immunoassay technologies were combined, and applied for SARs-CoV-2 NP detection. Overall, a 6x enhancement in antigen detection was achieved from the fluorometric readings (LOD ~8.45 ng/ml) compared to a traditional sandwich ELISA. Then, when coupled with LFA, an analytical sensitivity of ~ 1.64 ng/ml was achieved, demonstrating a 10x increase in sensitivity compared to a standard LFA test. In summary, the work provides a comprehensive study outlining the utility and shortcomings of integrating an E.coli CFPS system with ELISA and LFA reagents for infectious disease detection.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as JSONCSVXML

Archive Staff Only

View Item