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

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.