Single-Molecule Exchange inside a Nanocage Provides Insights into the Origin of π-π Interactions.

The attractive interactions between aromatic rings, also known as π-π interactions, have been widely used for decades. However, the origin of π-π interactions remains controversial due to the difficulties in experimentally measuring the weak interactions between π-systems. Here, we construct an elaborate system to accurately compare the strength of the π-π interactions between phenylalanine derivatives via molecular exchange processes inside a protein nanopore. Based on quantitative comparison of binding strength, we find that in most cases, the π-π interaction is primarily driven by dispersive attraction, with the electrostatic interaction playing a secondary role and tending to be repulsive. However, in cases where electronic effects are particularly strong, electrostatic induction may exceed dispersion forces to become the primary driving force for interactions between π-systems. The results of this study not only deepen our understanding of π-stacking but also have potential implications in areas where π-π interactions play a crucial role.

Correction: A bifunctional DNA probe for sensing pH and microRNA using a nanopore.

Correction for 'A bifunctional DNA probe for sensing pH and microRNA using a nanopore' by Yun Zhang et al., Analyst, 2020, 145, 7025-7029, DOI: 10.1039/D0AN01208D.

Simultaneous Sensing of Multiple Cancer Biomarkers by a Single DNA Nanoprobe in a Nanopore.

Both vascular endothelial growth factor (VEGF) and matrix metallopeptidase-9 (MMP-9) are key biomarkers in tumor angiogenesis. Determination of the overexpression of the two biomarkers would provide valuable information on the progression of tumor growth and metastasis, but their simultaneous quantification by a single probe is unprecedented. Here, we develop a triplex DNA-based nanoprobe for simultaneously quantifying VEGF and MMP-9 using an α-hemolysin nanopore. A DNA aptamer is used as the triplex molecular beacon (tMB) loop to bind VEGF, and a stem-forming oligonucleotide modified with a short peptide is used to recognize MMP-9. The sequential presence of VEGF and MMP-9 could also be identified by different patterns of current events. Besides, the characteristic current events generated by the DNA probe possess pH-dependent patterns that can be used to reflect the environmental pH. Success in the construction of such DNA nanoprobes will greatly facilitate the investigation of the mechanisms of different tumor angiogenesis processes and provide a useful approach for cancer diagnosis.

A bifunctional DNA probe for sensing pH and microRNA using a nanopore.

We have developed a bifunctional probe based on triplex molecular beacons for the measurement of environmental pH and quantification of microRNA-10b using a nanopore. The probe responds sharply to solution pH changes in the range of 6.0-7.5. The limit of detection for microRNA-10b is as low as 5.0 pM.

A Dual-Response DNA Probe for Simultaneously Monitoring Enzymatic Activity and Environmental pH Using a Nanopore.

Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of pH in a tumor environment remain challenging. Here, we develop a dual-response DNA probe that can simultaneously monitor protease activities and measure the local pH by translocation through α-hemolysin (αHL). The DNA probe bears a short peptide containing phenylalanine at a pre-designed position. Enzymatic cleavage of the peptide either exposes or removes the N-terminal phenylalanine that can form a complex with cucurbit[7]uril. Translocation of the DNA hybrid through αHL generates current signatures that can be used to quantify protease activities. Furthermore, the current signatures possess a pH-dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNA probe may be further explored for simultaneously measuring multiple parameters of a complex system such as single cells in the future.

Analyte-Triggered DNA-Probe Release from a Triplex Molecular Beacon for Nanopore Sensing.

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex-forming molecular beacon and a stem-forming DNA component that is modified with a host-guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α-hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point-of-care diagnostics with a portable nanopore kit in the future.

Simultaneous Quantification of Multiple Cancer Biomarkers in Blood Samples through DNA-Assisted Nanopore Sensing.

Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Herein, we show that the combination of a sandwich assay and DNA-assisted nanopore sensing could unambiguously identify and quantify several antigens in a mixture. We use five barcode DNAs to label different gold nanoparticles that can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer-associated biomarkers within a very small sample volume, which is critical for non-invasive early diagnosis of cancer.

A multiple fluorescein-based turn-on fluorophore (FHCS) identified for simultaneous determination and living imaging of toxic Al3+ and Zn2+ by improved Stokes shift.

A multiple turn-on fluorophore (FHCS), combining fluorescein, hydrazone, cyanuric chloride and salicylaldehyde chromone into a molecule, was identified and developed based on density functional theoretical calculation. It was expected that FHCS could express exclusive fluorescent signals and improved Stokes shifts when chelating Al3+ or Zn2+. After it was synthesized and characterized in detail, it was noted that FHCS could turn-on fluorescently discriminate trace Al3+ and Zn2+ under the optimized conditions, i.e., from no-fluorescence to strong blue fluorescence for Al3+ and to green fluorescence for Zn2+ with low detection limits of 5.37 × 10-8 M and 7.90 × 10-8 M respectively. Owing to its low toxicity, FHCS was successfully applied for quantitative determination of Al3+ and Zn2+ in natural aqueous samples and toxicity evaluation of Al3+ and Zn2+ in living cells and bio-tissues with excellent linear relationships. The action mechanisms for FHCS with Al3+ and Zn2+ were confirmed to form stable 5-member-co-6-member condensed rings between Al3+/Zn2+ and N/O atoms in FHCS by both theoretic and experimental methods, which resulted in turn-on fluorescence with different dipolar moments and improved Stokes shifts.

Measuring Binding Constants of Cucurbituril-Based Host-Guest Interactions at the Single-Molecule Level with Nanopores.

Cucurbiturils are one type of widely used macrocyclic host compound in supramolecular chemistry. Their peculiar properties have led to applications in a wide variety of research areas such as fluorescence spectroscopy, drug delivery, catalysis, and nanotechnology. However, the solubilities of cucurbiturils are rather poor in water and many organic solvents, which may cause accuracy problems when measuring binding constants with traditional methods. In this report, we aim to develop an approach to measure the binding constants of cucurbituril-based host-guest interactions at the single-molecule level. First, we covalently attach different guest compounds to the side-chain of DNA molecules. Then, excess cucurbiturils are incubated with DNA probes to form the host-guest complexes. Next, the modified DNA hybrids are threaded through α-hemolysin nanopore to generate highly characteristic current events. Finally, statistical analyses of the obtained events afford the binding constants of cucurbiturils with various molecules. This new approach provides a simple and straightforward method to compare binding strength of different host-guest complexes and may find applications for quantifying other macrocycle-based host-guest interactions.

Multiplexed discrimination of microRNA single nucleotide variants through triplex molecular beacon sensors.

Herein, we develop a new nanopore sensing strategy for the selective detection of microRNAs and single nucleotide variants (SNVs) based on triplex molecular beacon sensors. This sensing system shows very high specificity in discriminating microRNA SNVs and can be applied for the simultaneous detection of several microRNAs of the same family in a mixture.

Revealing different aggregational states of a conjugated polymer in solution by a nanopore sensor.

The functionalities of conjugated polymers are determined not only by local molecular structure, but also by the mesoscale conformational and morphological states of the polymer chains. Simulation studies have successfully established the connections between molecular structure and conformational states of certain conjugated polymers. However, experimental tools that can accurately discriminate between different conformational and morphological states of conjugated polymers are still scarce. Here, we use a nanopore sensor to analyze different aggregational states of a polythiophene derivative by threading the polymer through the pore under applied potentials. When the fluorescence of the polythiophene is quenched by pH tuning or the presence of Dy3+, the UV-vis and fluorescence spectra of the two solutions appear indistinguishable. However, threading the polymer molecules of these two solutions through an α-hemolysin nanopore affords entirely different translocation profiles owing to their different aggregational states. We further substantiate the results by conducting aggregational interconversion experiments and TEM measurements. This work has clearly indicated that nanopores are promising tools for the analysis of aggregational changes of conjugated polymers and may open new avenues for the investigation of aggregational states of biomacromolecules in the context of early disease diagnosis and prognosis.