A combination of scanning and imaging surface plasmon
resonance (SPR) experiments is used to characterize DNA
hybridization adsorption at gold surfaces and the subsequent immobilization of streptavidin. Single-stranded
oligonucleotides are immobilized at gold surfaces, and the
hybridization of biotinylated complements from solution
is monitored with SPR. The subsequent attachment of
streptavidin to the biotinylated complements provides a
method of enhancing the SPR imaging signal produced
as a result of the hybridization and leads to a 4-fold
improvement in the hybridization detection limit of the
SPR imaging apparatus. In situ scanning SPR experiments are used to measure a 60 ± 20% hybridization
efficiency between immobilized single-stranded DNA and
biotinylated complements. From the information provided by both the in situ imaging and scanning SPR
experiments, an absolute surface coverage of immobilized
single-stranded DNA is estimated to be ∼3 ×
1012
molecules/cm2. The SPR signal resulting from
hybridization onto immobilized probes is further amplified by the
formation of streptavidin/DNA multilayers which grow by
a combination of DNA hybridization and biotin−streptavidin binding. DNA/DNA multilayers without
streptavidin
are used as an additional method of amplifying the SPR
signal.
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