Single-Molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets

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• 作者：Anders Gunnarsson ; Peter Jö ; nsson ; Rodolphe Marie ; Jonas O. Tegenfeldt ; Fredrik Hö ; ö ; k
• 刊名：Nano Letters
• 出版年：2008
• 出版时间：January 2008
• 年：2008
• 卷：8
• 期：1
• 页码：183 - 188
• 全文大小：254K
• 年卷期：v.8,no.1(January 2008)
• ISSN：1530-6992

文摘

We report on a single-molecule readout scheme on total internal reflection fluorescence microscopy (TIRFM) demonstrating a detection limitin the low fM regime for short (30-mer) unlabeled DNA strands. Detection of unlabeled DNA targets is accomplished by letting them mediatethe binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (Ø ~ 100 nm) to a DNA-modified substrate. On topof rapid and sensitive detection, the technique is also shown capable of extracting kinetics data from statistics of the residence time of thebinding reaction in equilibrium, that is, without following neither the rate of binding upon injection nor release upon rinsing. The potential ofthis feature is demonstrated by discriminating a single mismatch from a fully complementary sequence. The success of the method is criticallydependent on a surface modification that provides sufficiently low background. This was achieved through self-assembly of a biotinylatedcopolymer, Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) on a silicon dioxide surface, followed by subsequent addition of streptavidinand 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 infuture multiplexed sensing schemes. This holds in particular true in cases when information about binding kinetics is valuable, such as insingle nucleotide polymorphism diagnostics.