Peptide sequencing based on host-guest interaction-assisted nanopore sensing.

Direct protein sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose an alternative method for peptide sequencing based on enzymatic cleavage and host-guest interaction-assisted nanopore sensing. We serendipitously discovered that the identity of any proteinogenic amino acid in a particular position of a phenylalanine-containing peptide could be determined via current blockage during translocation of the peptide through α-hemolysin nanopores in the presence of cucurbit[7]uril. Building upon this, we further present a proof-of-concept demonstration of peptide sequencing by sequentially cleaving off amino acids from C terminus of a peptide with carboxypeptidases, and then determining their identities and sequence with a peptide probe in nanopore. With future optimization, our results point to a different way of nanopore-based protein sequencing.

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.

Construction of a pH-Mediated Single-Molecule Switch with a Nanopore-DNA Complex.

A molecular switch is one of the simplest examples of artificial molecular machines. Even so, the development of molecular switches is still at its very early stage. Currently, building single-molecule switches mostly rely on the molecular junction technique, but many of their performance characteristics are device-dependent. Here, a pH-mediated single-molecule switch based on the combination of an α-hemolysin (αHL) nanopore and a hexacyclen-modified DNA strand is developed. The single-stranded DNA is suspended inside an αHL through biotin-streptavidin linkage and the hexacyclen-modified nucleobase interacts with amino acid residues at positions 111, 113, and 147 to cause current oscillations. Distinct current transitions are observed when pH is tuned back and forth in the range of 3.0-7.4, with a typical "up" level when pH > 6.5 and a "down" level when pH < 4.5. This nanopore-DNA complex possesses membrane-bound advantages and may find applications in single-cell studies where pH could be readily tuned to control ON-OFF functions.

A Nanopore Sensing Assay Resolves Cascade Reactions in a Multienzyme System.

Enzymatic cascade reactions are widely used to synthesize complex molecules from simple precursors. The major underlying mechanism of cascade reactions is substrate channelling, where intermediates of different enzymatic steps are not in equilibrium with the bulk solution. Here, we report a nanopore sensing assay that allows accurate quantification of all the reaction intermediates and the product of an artificial three-enzyme system. A DNA-peptide complex is used as the initial substrate which undergoes sequential enzymatic cleavages in solution. All the temporal changes of the intermediates and product can be obtained through nanopore translocation recordings. Furthermore, we find that in a confined environment such as liposome, substrate channelling occurs between two sets of the three enzymes. Our results demonstrate a novel and powerful approach to determine and quantify substrate channelling effects, which is potentially useful for designing and evaluating multienzyme systems.

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.

Probing Conformational Polymorphism of DNA Assemblies with Nanopores.

Single-stranded DNA (ssDNA) can be designed to assemble into duplexes and other high-order structures through Watson-Crick hydrogen bonds. Incorporation of unnatural nucleobases or binding with small molecules can also introduce new interactions that give rise to novel DNA assemblies. However, the methods for determining the conformational properties of DNA assemblies are still very limited. Here we develop a new strategy for probing conformational polymorphism of different DNA assemblies. By installing poly(dC)30 tails to the ends of individual ssDNA that assemble into duplex, triplex, or other complex structures, we are able to observe different current blockade patterns corresponding to specific DNA nanostructures when the DNA assemblies are lodged inside α-hemolysin vestibule. We can also monitor the disassembly of the DNA nanostructures in solution. This method complements the existing traditional technologies such as circular dichroism spectroscopy, fluorescence labeling, and NMR spectroscopy, and shows distinct advantages of high accuracy and general applicability.

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.

Measuring Enzymatic Activities with Nanopores.

Nanopores have become powerful and versatile tools for measuring single molecules since their emergence in the mid-1990s. They can be used to sense a wide variety of analytes including metal ions, small organic molecules, DNA/RNA, proteins, etc. to monitor chemical reactions, and to sequence DNA. Recently, enzymes have been studied by using nanopore technologies. In this Minireview, we highlight recent efforts in developing nanopore enzymology and categorize the related work into three groups: 1) measuring enzymatic activities with nanopore-enzyme hybrids; 2) measuring enzymatic activities through sensing their catalytic products with nanopores; 3) the use of enzymes for DNA sequencing and DNA/protein translocation. At the end, we discuss the challenges and opportunities in nanopore enzymology.

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.

Enhanced Sensitivity in Nanopore Sensing of Cancer Biomarkers in Human Blood via Click Chemistry.

Cancer biomarkers are expected to be indicative of the occurrence of certain cancer diseases before the tumors form and metastasize. However, many biomarkers can only be acquired in extremely low concentrations, which are often beyond the limit of detection (LOD) of current instruments and technologies. A practical strategy for nanopore sensing of cancer biomarkers in raw human blood down to the femtomolar level is developed here. This strategy first converts the detection of cancer biomarkers to the quantification of copper ions by conducting a sandwich assay involving copper oxide nanoparticles. The released Cu2+ is then taken to catalyze the "click" reaction which ligates a host-guest modified DNA probe. Finally, this DNA probe is subjected to single-channel recordings to afford the translocation events that can be used to derive the concentrations of the original biomarkers. Due to the amplification effects of nanoparticle loadings and the "click" reaction, the LOD of this strategy can be as low as the subfemtomolar level. Further, the acid treatment step could effectively eliminate the interferences from plasma proteins in raw human blood and make the strategy highly suitable for the detection of cancer biomarkers in clinical samples.

Investigating the effect of mono- and multivalent counterions on the conformation of poly(styrenesulfonic acid) by nanopores.

Polyelectrolytes are useful materials that have many technical, medical, physiological and biological applications. The properties of polyelectrolytes are determined not only by their chemical composition but also by their conformational states. However, the conformations of polyelectrolytes in solution are very difficult to characterize. Herein, we propose to use a protein nanopore to investigate the effect of mono- and multivalent counterions on the conformational changes of a simple polyelectrolyte, sodium poly(styrenesulfonic acid) (NaPSS). High concentration of KCl induced a conformational transition of NaPSS from "swollen random coil" form in low salt concentration to "random coil" form and was evidenced by the changes of the translocation event pattern. Addition of Mg2+ in buffer solution did not cause notable changes of NaPSS translocation events, but Dy3+ and Y3+ were shown to have remarkable effects on the translocation profile of NaPSS. Bridging events caused by Dy3+ or Y3+ between polyelectrolyte chains largely affected current blockage and dwell time of the translocation events. Our results provide experimental evidence for the classical theories of conformational transitions of polyelectrolytes and may find applications in many other polyelectrolyte-related researches.

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.

Rapid and selective DNA-based detection of melamine using α-hemolysin nanopores.

We have developed a rapid and selective approach for the detection of melamine based on simple DNA probes and α-hemolysin nanopores. The limit of detection can be as low as 10 pM.

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.

Rapid and visual detection of heparin based on the disassembly of polyelectrolyte-induced pyrene excimers.

We have developed a ratiometric sensor based on polyelectrolyte-induced pyrene excimers for the rapid and visual detection of heparin in aqueous media. This sensor has a simple preparation method, fast response, low cost, and high sensitivity and selectivity. As a complementary strategy for the detection of heparin based on the formation of pyrene excimers, this approach could identify heparin specifically over chondroitin sulphate, which is the major interferent in most sensing systems for the detection of heparin. The detection limit of this approach can be as low as 0.14 µM using fluorescence measurements. To the best of our knowledge, this is among the lowest LOD for the detection of heparin reported in the literature. It enables the rapid and visual detection of heparin both in buffered solution and serum samples.

Approach Based on Polyelectrolyte-Induced Nanoassemblies for Enhancing Sensitivity of Pyrenyl Probes.

We have developed a unique approach for enhancing the sensitivity of pyrenyl probes based on polyelectrolyte-induced nanoassemblies and explored its sensing application toward 2,4,6-trinitrophenol (TNP). The key issue of the method is the formation of the nanoassemblies which possess high-density charges, specific surface area, and inner hydrophobic regions. These properties would help increase the loading of analytes and promote probe-analyte interactions, thereby leading to the prominent enhancement of the sensitivity. In the course of TNP detection, pyrene nanoassemblies can bind TNP efficiently through cooperative noncovalent interactions including electrostatic, π-π stacking, and charge-transfer interactions, resulting in the distinct fluorescent responses of pyrene moieties. This system has excellent selectivity and sensitivity for TNP detection. The detection limit is as low as 5 nM. It may be used for monitoring the TNP concentrations in real-world samples.

Selective Detection of 8-Oxo-2'-deoxyguanosine in Single-Stranded DNA via Nanopore Sensing Approach.

We have developed a nanopore sensing approach for the selective detection of 8-oxo-2'-deoxyguanosine (8-oxoG) in single-stranded DNA. First, 1,12-dodecanediamine is coupled with 8-oxoG-containing DNA molecules in high yield which leaves a free amine group for subsequent attaching of an adamantane moiety. After incubation with cucurbit[7]uril, the host-guest complex-modified DNA hybrid is translocated through an α-hemolysin nanopore. Highly characteristic events can be recorded and used to quantify the 8-oxoG-DNA content in a DNA mixture. Compared with the existing methods, this study provides a reliable, quick, and low-cost approach for the detection of 8-oxoG site in single-stranded DNA at the single-molecule level, particularly suitable for high-throughput screening of a massive number of samples.

Photocontrolled Reversible Luminescent Lanthanide Molecular Switch Based on a Diarylethene-Europium Dyad.

A new europium complex coordinated between a Eu(III) ion and an unsymmetrical diarylperfluorocyclopentene yields a light-controlled diarylethene-europium dyad, DAE@TpyEu(tta)3, whose photophysical properties can be reversibly switched by optical stimuli. When DAE@TpyEu(tta)3 is exposed to 365 nm UV light, an efficient intramolecular photochromic fluorescence resonance energy transfer (pc-FRET) occurs between the emission of the Eu(3+) donor (D) and the absorption of the diarylethene acceptor (A) in closed-form DAE@TpyEu(tta)3 accompanied by luminescence quenching. However, the pc-FRET process could be effectively inhibited by visible light (λ > 600 nm) irradiation, and the lanthanide emission of DAE@TpyEu(tta)3 is rapidly recovered. Furthermore, this luminescent lanthanide molecular switch could serve as a highly reliable and sensitive "turn on" fluorescent marker in living cells irradiated by red light without any optical interference.

DNA-Based Nanopore Sensing.

Nanopore sensing is an attractive, label-free approach that can measure single molecules. Although initially proposed for rapid and low-cost DNA sequencing, nanopore sensors have been successfully employed in the detection of a wide variety of substrates. Early successes were mostly achieved based on two main strategies by 1) creating sensing elements inside the nanopore through protein mutation and chemical modification or 2) using molecular adapters to enhance analyte recognition. Over the past five years, DNA molecules started to be used as probes for sensing rather than substrates for sequencing. In this Minireview, we highlight the recent research efforts of nanopore sensing based on DNA-mediated characteristic current events. As nanopore sensing is becoming increasingly important in biochemical and biophysical studies, DNA-based sensing may find wider applications in investigating DNA-involving biological processes.