Self-assembled monolayers (SAMs) of single-stranded peptide nucleic acids (PNAs) containing 3 to 7 thymine (T) nucleotides, a C-terminus cysteine, and an N-terminus ferrocene group were formed on gold electrodes. The existence of two redox environments for the ferrocene was detected by cyclic voltammetry and was attributed to the presence of "lying-down" and "standing-up" PNA molecules. By exploiting the chemical instability of the ferrocenium ion, electrochemical cycling was used to destroy the ferrocene of "lying-down" molecules while keeping the ferrocene in the "standing-up" molecules intact. Electrochemical measurements were used to determine the electron-transfer rate through the "standing-up" PNA molecules. The tunneling decay constant for these SAMs was determined to be about 0.9 Å -1 . IntroductionThe interest in self-assembled monolayers (SAMs) 1,2 of nucleic acids has increased recently, largely because of their potential applications in molecular electronics, 3 materials science, 4 molecular recognition, 5 biotechnology, and biosensor development. [6][7][8] An understanding of the charge transport (CT) through such SAMs is needed to realize their potential in molecular electronics and biosensing. The past decade has seen progress in understanding charge transfer through deoxyribonucleic acid (DNA), which is believed to occur through either a superexchange mechanism, 9-19 which dominates at short distances, or a hopping mechanism, 20-25 which dominates at large distances.The weak distance dependence of the charge hopping mechanism and its prevalence in duplex DNA systems have motivated the exploration of charge transfer through DNA and its promise for molecular electronics by a large number of different research groups. Nevertheless, only a few research groups have studied CT in DNA monolayers, probably because of the difficulties in creating well-defined DNA assemblies on a metal surface. [26][27][28] For example, Hartwich et al. 29 used cyclic voltammetry to characterize charge transfer in mixed monolayers of DNA having a pyrroloquinoline-quinone redox probe attached to DNA through a spacer and linked to an Au(111) surface through an ethane-thiol linker. These studies determined that the CT rate constant for a 12-base-pair (bp) DNA duplex was 1.5 s -1 , while for the same duplex containing two mismatches it was 0.6 s -1 , and that charge transfer could not be detected for single-stranded (ss) DNA at a scan rate of >10 mV s -1 . Liu et al. 30 argued that CT through a monolayer of a 30-bp double-stranded (ds) DNA takes place through the nucleobase stack and does not involve the DNA backbone. They based their argument on the fact that the rate constant for CT of 30 s -1 was not affected by breaks in the sugar-phosphate backbone and was too small to be measured when a mismatch was introduced in the ds DNA. Interestingly, a similar rate constant for CT was measured for a monolayer of a 15-bp ds DNA. 31 CT rate constants for ss oligonucleotides are also quite high. For example, Kraatz and collaborators r...

Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.