Paper BI+NC-WeM11
Single-Molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets

Wednesday, October 22, 2008, 11:20 am, Room 202

Ultrasensitive biological sensors for low-abundant DNA and protein detection have emerged as an important tool for improving biomedical diagnostics, drug discovery, forensic analysis, but also advanced bioanalytical assays in fundamental research. We report on a single-molecule readout scheme based 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 DNA targets is accomplished by mediating the binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (~100 nm in diameter) to a DNA-modified substrate by unlabeled complementary single-stranded DNA. On top of rapid and sensitive detection, the technique is also shown capable of extracting kinetic data from statistics of the residence time of the binding reaction in equilibrium, i.e. 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 30-mer DNA target.¹ The proposed detection scheme is particularly appealing due to the simplicity of the sensor, which relies on self-assembly principles and conventional TIRFM. In contrast to most other single-molecule detection schemes the imaging mode also offers possibilities for multiple spots to be measured simultaneously in an array-based design. The proposed sensor holds particular promise in cases when information about binding kinetics is valuable, such as in single nucleotide polymorphism (SNP) diagnostics.

¹ Gunnarsson, A., et al., Single-molecule detection and mismatch discrimination of unlabeled DNA targets. Nano Letters. 8(1): p. 183-188, 2008.