Single-Molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets

Abstract

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 approximately 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.

Application of nucleic acid–lipid conjugates for the programmable organisation of liposomal modules

“…Surface immobilization of liposomes allows the use of sensitive surface analytical techniques to probe the binding and properties of liposomes for biosensor applications [109,[117][118][119]. These techniques can also be applied to develop new biophysical techniques for membrane biophysics, for example in investigating intermembrane interactions of unsupported bilayer membranes [113,120].…”

“…Surface analytical techniques that have been applied to DNA-mediated surface-anchored liposomes include fluorescence interference contrast microscopy [63], Quartz Crystal Microbalance with Dissipation (QCM-D) [110], Total Internal Reflection Fluorescence Microscopy (TIRF-M) for label free detection of single base mismatches in DNA strands [117,118], DNA detection by imaging mass spectrometry [119] and surface plasmon resonance (SPR) [109].…”

Accounting for adsorption and desorption in lattice Boltzmann simulations

“…For molecules, the rates can vary widely. As an example, the dissociation rate of DNA double strands on a surface grafted with single-strand DNA ranges from 10 −5 to 10 −3 s −1 for a few tens of base pairs [9]. Their adsorption rate can be adjusted by changing the grafting density.…”

Microfluidic Assays for DNA Manipulation Based on a Block Copolymer Immobilization Strategy

“…For its superior protection from nonspecific adsorption and capacity to form large-area uniform coatings, PLL-g-PEG has found several successful applications in molecular sensing and in cell cultures. [30][31][32] In this study, we report the manipulation and visualization of single DNA molecules by combining microfluidics with the block copolymer PLL-g-PEG. Bacteriophage λ-DNA was stretched by both molecular combing and hydrodynamic flow in the same microfluidic chip.…”