Single-Molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets

We report on a single-molecule readout scheme on total internal reflection fluorescence microscopy (TIRFM) demonstrating a detection limit
in the low fM regime for short (30-mer) unlabeled DNA strands. Detection of unlabeled DNA targets is accomplished by letting them mediate
the binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (Ø ∼ 100 nm) to a DNA-modified substrate. On top
of rapid and sensitive detection, the technique is also shown capable of extracting kinetics data from statistics of the residence time of the
binding reaction in equilibrium, that is, without following neither the rate of binding upon injection nor release upon rinsing. The potential of
this feature is demonstrated by discriminating a single mismatch from a fully complementary sequence. The success of the method is critically
dependent on a surface modification that provides sufficiently low background. This was achieved through self-assembly of a biotinylated
copolymer, Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) on a silicon dioxide surface, followed by subsequent addition of streptavidin
and biotinylated DNA. The proposed detection scheme is particularly appealing due to the simplicity of the sensor, which relies on self-assembly principles and conventional TIRFM. Therefore, we foresee a great potential of the concept to serve as an important component in
future multiplexed sensing schemes. This holds in particular true in cases when information about binding kinetics is valuable, such as in
single nucleotide polymorphism diagnostics.