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Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3' dye moiety and a 5' thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ∼90% at intermediate coverages of (1-2) × 10(12) probes/cm(2) and dropping to ≤20% at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (∼(3-4) × 10(11) probes/cm(2)), but not at higher probe coverages (1 × 10(12)/cm(2)). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3' or 5' end of the complementary target sequence. Additional nucleotides on the 3' end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5' end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.

Hybridization Efficiency of Molecular Beacons Bound to Gold Nanowires: Effect of Surface Coverage and Target Length