Surface Plasmon Resonance Imaging (SPRi) is a versatile biosensing platform for protein microarrays. It enables high-throughput, label-free analysis of various analytes in a multiplex format. However, the critical process of protein immobilization poses challenges, particularly in ensuring a reliable and reproducible receptor layer formation, which is essential for the validity of analyte determination. In the SPRi technique, protein immobilization on the chip occurs ex situ, making it challenging to monitor and resulting in an undetermined surface density of immobilized receptors. An optimal protein receptor layer requires reproducible fabrication with controllable density and homogeneous distribution of proteins while preserving their biological activity. A method of protein microarray quality control is yet to be developed, which emphasizes the need for a reliable assessment approach.

This study utilized commercially available protein dyes (Coomassie Brilliant Blue G, Amido Black, and Ponceau S) to map and characterize the quality of protein receptor layers by means of SPR and SPRi studies. Various factors influencing dye–protein binding were investigated using covalently immobilized model proteins (rabbit IgG and transferrin) on mixed -COOH monolayers or carboxylated PEG. Additionally, DNA-directed immobilization, allowing for protein–DNA conjugate immobilization through hybridization with complementary sequences, was explored. This study examined the appropriate pH and ionic strength for efficient protein staining and tested the effect of buffer composition on DNA double helix stability and dye intercalation. The association and dissociation constants of the dyes, along with the correlation between dye–protein and protein–antibody interactions, were determined. The results contribute valuable insights for developing a versatile quality control method for protein receptor layers immobilized using varied procedures. The presented approach seems to be very promising for evaluating the reliability of SPRi biosensors.