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Developing an Europium-based optical biosensor for detection of protein biomarkers

Al-Enezi, Eiman Ali (2020) Developing an Europium-based optical biosensor for detection of protein biomarkers. PhD thesis, University of Leeds.

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Abstract

Protein biomarker measurement has been well established using ELISA which offers good sensitivity and specificity but remains slow and expensive. Biosensors offer the prospect of reagent-less, processing-free measurements at the patient's bedside while recent developments in synthetic binding proteins have facilitated biosensor fabrication. The trivalent Europium ion (Eu3+) is a lanthanide that is attractive for use in optical biosensors due to its bright fluorescence when chelated by organic ligands. The aim of this project was to develop a platform for biosensing based on chelated Eu3+ against a range of proteins biomarkers for muscle injury/ heart attack, ischemic stroke, inflammation and colorectal cancer. Affimers against the biomarkers (human myoglobin, glial fibrillary acidic protein (GFAP), C-reactive protein (CRP) and carcinoembryonic antigen (CEA)) were used as targeting bioreceptors. The Affimers were purified and checked for selective binding using ELISA and immunoprecipitation assays. The Eu+3 was then chelated by pyromellitic dianhydride (PMDA) modified Affimers to form Eu+3 -Affimer complexes. The fluorescence characteristics of the complexes against the protein biomarkers were measured in diluted human serum at λex = 395 nm and λem = 590 nm and 615 nm. The Eu3+- Affimer complexes allowed sensitive detection of myoglobin, GFAP, CRP and CEA proteins as low as 100 fM in 1 % (v/v) human serum in 2 to 3 minutes. The Eu3+ complex was far more sensitive against CRP and CEA proteins as compared to GFAP and human myoglobin, and sandwich assays, using an Affimer against the biomarker plus a polyclonal antibody, suggested that the mass of the biomarker may have a substantial effect on quenching of the Eu3+ complex luminescence. These data are the first to demonstrate that the Affimer based Eu3+ complexes can function as nano-biosensors with potential analytical and diagnostic applications.

Item Type: Thesis (PhD)
Keywords: Biosensor; Lanthanide; Europium chelates; Affimer; biomarkers.
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
Depositing User: Dr. Eiman Ali Al-Enezi
Date Deposited: 09 Jun 2020 16:30
Last Modified: 09 Jun 2020 16:30
URI: http://etheses.whiterose.ac.uk/id/eprint/27029

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