Detection of very low amounts of illicit drugs such as cocaine in clinical fluids like serum continues to be important for many areas in the fight against drug trafficking. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of cocaine in human serum and saliva samples based on target-induced strand release strategy. In this bioassay, an aptamer for cocaine was prehybridized with a short complementary DNA. Owing to cocaine specific binding with aptamer, the short DNA strand was displaced from aptamer and translocation of this output DNA through α-hemolysin nanopore generated distinct spike-like current blockages. When plotted in double-logarithmic scale, a linear relationship between target cocaine concentration and output DNA event frequency was obtained in a wide concentration range from 50 nM to 100 μM of cocaine, with the limit of detection down to 50 nM. In addition, this aptamer-based sensor method was successfully applied for cocaine detection in complex biological fluids like human saliva and serum samples with great selectivity. Simple preparation, low cost, rapid, label-free, and real sample detection are the motivating factors for practical application of the proposed biosensor.

DNA methylation catalyzed by DNA methyltransferase plays an important role in many biological processes. However, conventional assays proposed for DNA methyltransferase activity are laborious and discontinuous. We have proposed a novel method for real-time monitoring of the activity and kinetics of Escherichia coli DNA adenine methyltransferase (Dam) using nanopore technique coupled with enzyme-linkage reactions. A double-stranded DNA probe AB having a recognition sequence 5'-GATC-3' for both Dam and MboI restriction endonuclease was prepared. Dam catalyzed the methylation of substrate probe AB, which blocked the cleavage reaction of MboI, while the absence of Dam resulted in cleavage of nonmethylated probe AB into four ssDNA fragments by MboI. When tested with nanopore, double-stranded methylated probe AB generated long-lived events, distinguished clearly from MboI-cleavage-mediated ssDNA fragments that generated only spikelike events. The proposed method has a detection limit of 0.03 U/mL for Dam in a short assay time of about 150 min. This sensing system is easy to perform, simple to design and circumvents the use of radioactive substances, resulting in efficient detection of the activity of Dam even in complex matrixes like human serum sample. Furthermore, it has the potential to screen Dam-targeted inhibitor drugs which may assist in the discovery of new anticancer medicines. This method is general and could be extended easily for monitoring activity of a wide variety of methyltransferases by coupling with their corresponding methylation-sensitive endonucleases.

Ligands that selectively promote the formation of G-quadruplexes in human telomeres have great potential for cancer treatment by inhibiting the telomere extension by telomerase. Thus, understanding the interactions of the G-quadruplex ligands with the telomere sequence at the single-molecule level is of significant importance. Here, human telomere sequence interactions with a small molecule ligand pyridostatin (PDS) were analyzed via α-hemolysin protein nanopore, and a nanopore thermodynamic analytical method was proposed. The prolonged unraveling time of the telomeric DNA G-quadruplex after PDS binding demonstrated the potent stabilization effect of ligand on G-quadruplex structure. The signature two-level electronic blocks generated by K(+)-PDS-G-quadruplex complexes suggested a two-state unraveling process, including the dissociation of the interquartet cation and the unraveling of the cation-free ligand-bound G-quadruplex. The translocation studies and the analysis of free-energy changes demonstrated a ligand-binding mode in which PDS molecule and K(+) were simultaneously bound to one G-quadruplex structure with the coordinated effect on G-quadruplex stabilization. The single-molecular nanopore platform permits the efficient and accurate determination of ligand affinity constants without the requirement for labeling, amplification, or ligand/receptor titration, which provides a general analytical tool for effectively monitoring and quantifying the G-quadruplex/ligand interactions, possessing important implications for the design and screen of anticancer drugs.

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