Reliable microspotting methodology for peptide-nucleic acid layers with high hybridization efficiency on gold SPR imaging chips

One-step direct immobilization of peptide-nucleic acid (PNA) probes onto gold surfaces through Au-S chemistry is critical in terms of generating self-assembled monolayers with high hybridization efficiency. We found that this problem is more severe if the immobilization is performed by contact microspotting to generate PNA arrays. Therefore, here we propose a novel microspotting-based immobilization method to generate PNA arrays with high hybridization efficiency on bare gold surface plasmon resonance imaging (SPRi) chips. The essence of the approach is to spot thiol labelled PNA strands prehybridized with a short complementary DNA strand instead of conventionally used single stranded PNA (ssPNA) probes. After immobilization the complementary DNA strands could be easily removed to activate the surface confined PNA probes. The incubation time and the type of spotting needle also have a marked influence on the hybridization efficiency of the PNA layers. However, we show that if all other conditions remain the same, PNA layers from prehybridized PNA probes exhibit superior hybridization efficiency than those from ssPNA probes.