Metal ions play critical roles in wide range of biochemical and physiological processes, but they can cause toxicity if excessive ingestion or misregulation. Chelating agents offer an efficient mean for metal ions intoxication and therapeutics of diseases. Studies on metal ion–chelator interactions are important for understanding the reaction mechanism and developing new specific metal chelator drugs. However, it remains a significant challenge to detect the metal ion–chelator interactions at the molecular level. Here, we report a label-free nanopore sensing approach that enables single-molecule investigation of the complexation process. We demonstrate that the chemical reaction between Cu2+ and carboxymethyl-β-cyclodextrin (CMβCD) in a nanoreactor is completely different from in the bulk solution. The formation constant (Kf = 4.70 × 104 M–1) increases 14 417-fold in the nanopore than that in the bulk solution (Kf = 3.26 M–1). The bioavailable CMβCD as a natural derivative with higher affinity for Cu2+ could be used in the safe medicinal removal of toxic metal. On the basis of the different ionic current signatures across an α-hemolysin (α-HL) mutant (M113N)7 nanopore lodged with a CMβCD adaptor in the presence and absence of Cu2+, the reversible molecular binding events to CMβCD can be in situ recorded and the single-molecule thermodynamic and kinetic information can be obtained. Interestly, we found that the Cu2+ binding leads to the increase of the channel current, rather than the blocking as usual nanopore experiment. The uncommon (on/off) characteristic could be very useful for fabricating the nanodevice. Furthermore, the unique nanopore sensor can provide a highly sensitive approach for detecting metal ions.

A novel and simple nanopore sensing method has been developed for the detection of CuII ions using polyamine decorated cyclodextrin as the recognition element. The strong binding affinity between CuII and the amino groups of cyclodextrin inside an α-hemolysin pore causes the new current blockade events. The event frequency is linear for concentrations of CuII in the range 0.08–20 μM. The detection limit is as low as 12 nM. More significantly, the sensing system is highly specific for CuII and does not respond to other metal ions with concentrations up to 10 fold that of CuII. The applicability of this sensor has also been verified by the analysis of CuII ions in running water, suggesting the potential application of this sensing system.

Chiral recognition at single-molecule level for small active molecules is important, as exhibited by many nanostructures and molecular assemblies in biological systems, but it presents a significant challenge. We report a simple and rapid sensing strategy to discriminate all enantiomers of natural aromatic amino acids (AAA) using a metal–organic complex-functionalized protein nanopore, in which a chiral recognition element and a chiral recognition valve were equipped. A trifunctional molecule, heptakis-(6-deoxy-6-amino)-β-cyclodextrin (am7βCD), was non-covalently lodged within the nanopore of an α-hemolysin (αHL) mutant, (M113R)7-αHL. Copper(II) ion reversibly bonds to the amino group of am7βCD to form an am7βCD-CuII complex, which allowed chiral recognition for each enantiomer in the mixture of AAA by distinct current signals. The CuII plugging valve plays a crucial rule that holds chiral molecules in the nanocavity for a sufficient registering time. Importantly, six enantiomers of all nature AAA could be simultaneously recognized at one time. Enantiomeric excess (ee) could also be accurately detected by this approach. It should be possible to generalize this approach for sensing of other chiral molecules.

The complexation of glutathione (GSH) with divalent cadmium ion has been used as a typical model for investigating the coordination chemistry of sulfydryl-containing peptides and heavy metal ions, which is essential to understand the mechanism of intracellular cadmium detoxification. In this study, the single-molecule reaction between GSH molecule and Cd2+ ion was monitored in real time by a nanoreactor that formed by a mutant (M113R)7 α-hemolysin (αHL) protein nanopore equipped with a novel per-6-quaternary ammonium-β-cyclodextrin (p-QABCD). The reaction pathways, intermediates, and products could be recognized by analyzing the current fluctuations. The reaction between Cd2+ and GSH was highly dependent on solution pH value. Cd(GSH)2 was the only final product at pH 7.4, while both Cd(GSH)2 and Cd2(GSH)2 were present at pH 9.0. The formation of Cd2(GSH)2 follows two possible pathways: (1) one Cd2+ ion first coordinates with the thiol group of two GSH molecules to form Cd(GSH)2, and then the second Cd2+ ion quickly incorporates with the deprotonated amino group of Cd(GSH)2 to produce Cd2(GSH)2; (2) two Cd2+ ions separately coordinate with the thiol and deprotonated amino group of one GSH molecule to yield Cd2(GSH)1, and the second GSH molecule binds Cd2+ ions quickly to form Cd2(GSH)2. The free-labeling and free-modifying method for monitoring single-molecule chemical reaction was simple and sensitive, which would be important to further understand intracellular mechanisms of detoxification of heavy metals. This work greatly expands the research field of single-molecule nanopore technique.We reported a novel single-molecule nanoreactor and detector by utilizing per-6-quaternary ammonium-β-cyclodextrin (p-QABCD) as an adaptor lodged in the α-hemolysin-(M113R)7 nanopore, which can accurately discriminate various intermediates and products, and monitor the reaction pathways of GSH molecules and Cd2+ ions chelating reaction in real time.Download high-res image (115KB)Download full-size image

Anticancer activity and toxicity of doxorubicin (Dox) are associated with its DNA intercalation. To understand the role in gene regulation and the drug mechanism, it is a challenge to detect the DNA–Dox interaction at the single-molecule level without the use of laborious, time-consuming labeling assays and an error-prone amplification method. Here, we utilized the simplest and cheapest, yet highly sensitive, single-molecule nanopore technology to investigate the DNA–Dox interaction and explore in situ the intercalative reaction kinetics. Distinctive electronic signal patterns between DNA and the DNA–Dox complex allow protein nanopore to readily detect the changes in structure and function of DNA. After Dox insertion, nanopore unzipping time of DNA was elevated 10-fold while the blocking current decreased, demonstrating the higher affinity of the DNA–Dox complex (formation constant Kf = 3.09 × 105 M–1). Continuous rapid nanopore detection in real time displayed that Dox intercalation in DNA is a two-state dynamic process: fast binding and slow conformational adaption. The nanopore platform provides a powerful tool for studying small molecule–biomacromolecule interactions and paves the way for novel applications aimed at drug screening and functional analysis.

Nanopore technology, as the simplest and most inexpensive single-molecule tool, is being intensively developed. In nanopore stochastic sensing, KCl and NaCl have traditionally been employed as pore-filled electrolytes for recording the change of ion conductance in nanopores triggered by analyte translocation through the pore. However, some challenges limit its further advance. Here we used tetramethylammonium (TMA) chloride, instead of KCl, as a novel analysis system for nanopores. Some unique nanopore characteristics were observed: (1) The stability of the planar lipid bilayer for embedding the protein pores was elevated at least 6 times. (2) The TMA-Cl system could effectively reduce the noise of single-channel recording. (3) It was easy to control the insertion of protein pores into the lipid bilayer, and the formed single nanopore could last for a sufficiently long time. (4) TMA-Cl could be used as a DNA speed bump in the nanopore to slow DNA translocation speed. (5) The capacity of the nanopore capture of DNA (capture rate) increased significantly and simultaneously increased the translocation time of DNA in the pore. (6) The TMA-filled nanopore could discriminate between various polynucleotides.

In this communication, we assembled an electroactive p-hydroxythiophenol (p-HTP) monolayer on a gold nanoparticle surface and produced an amplified single particle-collision electrochemical signal, thus allowing us to sensitively detect the size and functions of inert gold particles in aqueous solution.

In this research, protein nanotubes with high affinity for DNA molecules were prepared by alternate layer-by-layer (LbL) assembly of human serum albumin (HSA) and polyethylenimine (PEI). The well-defined hollow cylinder (PEI/HSA)5PEI nanotubes with outer diameter 455 ± 13 nm, inner diameter 275 ± 35 nm, wall thickness 151 ± 7 nm and length 5.6 ± 0.9 μm were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and energy dispersive spectroscopy (EDS). Under optimal conditions, the maximum adsorption capacity of protein nanotubes for polyA25 was 606.0 mg g−1, which was far higher than the reported nanoparticles. The rates of adsorption were found to conform to the pseudo-second-order kinetics and intra-particle diffusion models with good correlation. The equilibrium adsorption data fitted well with the Langmuir isotherm model. The DNA release from DNA/(PEI/HSA)5PEI nanotubes was associated with the ionic strength and pH in the solution.

A novel method for the selective sensing of ATP was developed using ferrocenecarboxylic acid (FcA) as an electrochemical probe, based on the competitive reaction between ATP (or ADP, AMP) and FcA with per-6-ammonium-β-cyclodextrin (pABCD). pABCD was synthesized in four steps from β-cyclodextrin by an improved literature method. In the pABCD molecule, the primary hydroxyl groups at position 6 of β-cyclodextrin have been substituted by amino groups. The presence of amino groups increased the binding ability. pABCD showed the strongest binding ability towards adenosine triphosphate (ATP), not only due to the host–guest inclusion of the cavity of pABCD with the adenosine base, but also the interaction of the positively charged ammonium groups of pABCD with the phosphate anion moieties. FcA, as an excellent electroactive probe, can be included in the pABCD cavity and produce a decreased oxidation signal. However, when ATP was added to the pABCD–FcA system, the oxidation peak current increased with increasing concentrations of ATP. In this way, the pABCD–FcA system can recognize and sense ATP through the competitive interaction of ATP and FcA with pABCD. The increased signal of FcA upon the addition of ATP into the FcA–ABCD system was studied using differential pulsed voltammetry (DPV). The inclusion constants of pABCD with FcA, ATP, ADP and AMP were evaluated using DPV by application of the Langmuir equation 1/ΔI = 1/ΔImax + 1/(kcΔImax). Based on these results, under the optimized experimental conditions, the oxidation current of FcA in the FcA–pABCD system responding to the concentration of ATP was linear in the range of 3.12 × 10−7 to 1.68 × 10−6 mol L−1, with a correlation coefficient of 0.9978 and a detection limit (S/N = 3) of 1.43 × 10−7 mol L−1. These results demonstrate that this method is highly selective and sensitive for the determination of ATP.

Nanopore analysis has emerged as the simplest single-molecule technique. We combined DNA probes with a nanopore electrochemical sensor for the rapid and sensitive detection of pathogenic DNA. The novel nanopore biosensor allows the single-base discrimination and detection of picomolar DNA in serum samples.

Nanopore analysis has emerged as the simplest single-molecule technique. We combined DNA probes with a nanopore electrochemical sensor for the rapid and sensitive detection of pathogenic DNA. The novel nanopore biosensor allows the single-base discrimination and detection of picomolar DNA in serum samples.

A novel method for the selective sensing of ATP was developed using ferrocenecarboxylic acid (FcA) as an electrochemical probe, based on the competitive reaction between ATP (or ADP, AMP) and FcA with per-6-ammonium-β-cyclodextrin (pABCD). pABCD was synthesized in four steps from β-cyclodextrin by an improved literature method. In the pABCD molecule, the primary hydroxyl groups at position 6 of β-cyclodextrin have been substituted by amino groups. The presence of amino groups increased the binding ability. pABCD showed the strongest binding ability towards adenosine triphosphate (ATP), not only due to the host–guest inclusion of the cavity of pABCD with the adenosine base, but also the interaction of the positively charged ammonium groups of pABCD with the phosphate anion moieties. FcA, as an excellent electroactive probe, can be included in the pABCD cavity and produce a decreased oxidation signal. However, when ATP was added to the pABCD–FcA system, the oxidation peak current increased with increasing concentrations of ATP. In this way, the pABCD–FcA system can recognize and sense ATP through the competitive interaction of ATP and FcA with pABCD. The increased signal of FcA upon the addition of ATP into the FcA–ABCD system was studied using differential pulsed voltammetry (DPV). The inclusion constants of pABCD with FcA, ATP, ADP and AMP were evaluated using DPV by application of the Langmuir equation 1/ΔI = 1/ΔImax + 1/(kcΔImax). Based on these results, under the optimized experimental conditions, the oxidation current of FcA in the FcA–pABCD system responding to the concentration of ATP was linear in the range of 3.12 × 10−7 to 1.68 × 10−6 mol L−1, with a correlation coefficient of 0.9978 and a detection limit (S/N = 3) of 1.43 × 10−7 mol L−1. These results demonstrate that this method is highly selective and sensitive for the determination of ATP.