Synthesis and immobilization of thiolated pyrrolidinyl peptide nucleic acids on gold-coated piezoelectric quartz crystals for the detection of DNA hybridization

Two novel thiolated pyrrolidinyl peptide nucleic acids (HS-PNA) carrying d-prolyl-2-aminocyclopentane-carboxylic acid (ACPC) backbones were synthesized and directly immobilized on gold-coated quartz crystals by self-assembled monolayer (SAM) formation (via S atom linkage) for the detection of DNA hybridization. The amount of immobilized HS-PNA was determined by quartz crystal microbalance (QCM), whilst the surface functionality and wettability of the gold surfaces before and after immobilization of the HS-PNA were characterized by reflection absorption infrared spectroscopy (RAIRS) and water contact angle analysis, respectively. Three different procedures for immobilization of HS-PNA were investigated; (I) HS-PNA without blocking thiol, (II) HS-PNA with subsequent blocking thiol and (III) co-immobilization with mixed HS-PNA and blocking thiol. The amount of immobilized HS-PNA on the gold electrode increased asymptotically with the HS-PNA concentration. The sequential blocking step (procedure II) was critical to the success of subsequent DNA hybridization. For hybridization, 0.5 mM phosphate buffer at pH 7.0 with no added NaCl was found to be optimal. Under these conditions, discrimination between complementary and single or multiple base mismatched DNA targets was achieved using a combination of pyrrolidinyl HS-PNA with QCM.