Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET)...

Neovascular pathologies in the eye like age-related macular degeneration (AMD), the diabetic retinopathie (DR), retinopathie of prematurity (ROP) or the retinal vein occlusion (RVO) are caused through a hypoxia induced upregulation of the vascular endothelial growth factor (VEGF). So far a correlation of intraocular VEGF concentrations to the impact of the pathologies is limited because of invasive sampling. Therefore, a minimally invasive, repeatable quantification of VEGF levels in the eye is needed to correlate the stage of VEGF induced pathologies as well as the efficacy of anti-VEGF treatment. Here we describe the development of three variants of enhanced BRET2 (eBRET2) based, single molecule biosensors by fusing a Renilla luciferase mutant with enhanced light output (RLuc8) to the N-terminus and a suitable eBRET2 acceptor fluorophore (GFP2) to the C-terminus of a VEGF binding domain, directly fused or separated with two different peptide linkers for the quantification of VEGF in vitro. The VEGF binding domain consists of a single chain variable fragment (scFv) based on ranibizumab in which the light- and the heavy- F(ab) chains were connected with a peptide linker to generate one open reading frame (orf). All three variants generate measureable eBRET2 ratios by transferring energy from the luciferase donor to the GFP2 acceptor, whereas only the directly fused and the proline variant permit VEGF quantification. The directly fused biosensor variant allows the quantification of VEGF with higher sensitivity, compared to the widely used ELISA systems and a wide dynamic quantification range in vitro. Our system demonstrates not only an additional in vitro application on VEGF quantification but also a promising step towards an applicable biosensor in an implantable device able to quantify VEGF reliably after implantation in vivo.