Optical microchips based on high-affinity recombinant protein binders—Human serum albumin detection in urine

摘要: Abstract Recent developments in molecular evolution technologies have led to novel types of high-affinity recombinant protein binders (PB) able substitute antibodies many diagnostic and therapeutic applications. Despite almost a decade research, they so far only been sporadically used for biosensor construction. Here, we present proof-of-principle comparative study focused on the application three PB recognizing human serum albumin (HSA) fabrication optical microchips detecting clinically relevant HSA levels urine. The tested were: (i) biotinylated anti-HSA Affibody (AF) (IgG binding domain A, Staphylococcus aureus); (ii) construct based albumin-binding (ABD) G (Streptococcus G148) fused with long TolA spacer (6xHis-WT-ABD-TolA-AviTag) (iii) WT-ABD-Trp leader-streptavidin tetrameric fusion (SA-ABD-WT). Open glass 24 independent microwells (volume 8 μL) micropatterned detection zones were prepared oriented proteins through biotin/streptavidin chemistry. analytical performance was by performing direct specific fluorescently labelled various environments. Results show that length peptide between sensor surface is key factor influencing performance. SA-ABD-WT reached limit (LOD) urine (LOD = 0.65 μg/ml) sufficient identify chronic kidney disease caused high blood pressure or diabetes. Furthermore, it offers highest signal intensity, low noise significant simplification microchip preparation due simple one-step immobilization procedure. Our results may be further exploited development dedicated wide range targets recognized ABD binders.