Nanopipettes provide a promising confined space that enables advances in electrochemical, optical, and mass spectrometric measurements at the nanoscale. They have been employed to reveal the hidden ...population properties and dynamics of single molecules and single particles. Moreover, new detection mechanisms based on nanopipettes have led to detailed information on single cells at high spatial and temporal resolution. In this Minireview, we focus on the fabrication and characterization of nanopipettes, summarize their wide applications for the analysis of single entities, and conclude with an outlook for advanced practical sensing.
The preparation and characterization of nanopipettes—promising devices for electrochemical, optical, and mass spectrometric measurements at the nanoscale—are the themes of this Minireview. Their application enables the dynamics and properties of single molecules and particles to be uncovered. Furthermore, new detection mechanisms based on nanopipettes have led to detailed information on single cells at high resolution.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Single-molecule measurements have greatly enhanced our understanding of living systems. Biological systems offer nanopores, a sub-class of membrane proteins, the well-defined confined space for ...accommodating a single molecule. The biological nanopore acts as a single-biomolecule interface for capturing and identifying a single molecule of interest, and thus it can be used as a single-molecule sensor. In this Perspective, we focus on biological nanopore-based single-biomolecule interfaces for single-biomolecule detection. First, we outline the design of the nanopore-based single-biomolecule interface, which provides rich stochastic information regarding each biomolecule. Next, we highlight future research directions beyond DNA sequencing, including detection of rare species, identification of hidden intermediates, spectral analysis of covalent/noncovalent interactions, and tracing of the dynamic pathways of single-biopolymer behaviors. The concept of a “single-molecule ionic spectrum” is discussed, which may allow mapping of noncovalent interactions at an atomic level in the future. We also discuss the challenges and goals for the future to make this measurement possible for addressing entirely new types of biological questions, which would be an exciting area of future research.
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IJS, KILJ, NUK, PNG, UL, UM
Proteins are responsible for the occurrence and treatment of many diseases, and therefore protein sequencing will revolutionize proteomics and clinical diagnostics. Biological nanopore approach has ...proved successful for single‐molecule DNA sequencing, which resolves the identities of 4 natural deoxyribonucleotides based on the current blockages and duration times of their translocations across the nanopore confinement. However, open challenges still remain for biological nanopores to sequentially identify each amino acid (AA) of single proteins due to the inherent complexity of 20 proteinogenic AAs in charges, volumes, hydrophobicity and structures. Herein, we focus on recent exciting advances in biological nanopores for single‐molecule protein sequencing (SMPS) from native protein unfolding, control of peptide translocation, AA identification to applications in disease detection.
Nanopore electrochemistry offers a bright prospect for single‐molecule protein sequencing by measuring specific interactions between amino acids based on their natural structure and chemistry continuity and diversity. This Minireview focusses on recent advances in biological nanopores from protein unfolding, peptide translocation, amino acid identification to diagnostic application.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Nanopore‐based techniques, which mimic the functions of natural ion channels, have attracted increasing attention as unique methods for single‐molecule detection. The technology allows the real‐time, ...selective, high‐throughput analysis of nucleic acids through both biological and solid‐state nanopores. In this Minireview, the background and latest progress in nanopore‐based sequencing and detection of nucleic acids are summarized, and light is shed on a novel platform for nanopore‐based detection.
Adopted from nature: The functions of natural ion channels can be mimicked with both biological and solid‐state nanopores, which have attracted increasing attention for their possible use in the detection and sequencing of DNA. In this Minireview, background and latest progress in this area are summarized and a novel platform for nanopore‐based detection is discussed.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Inspired by the biological processes of molecular recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive analytical tools for individual ...molecules. In particular, nanopore-based single-molecule DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in molecular sensing and sequencing, chemical catalysis and biophysical characterization. We highlight the prospects of applying nanopores for single-protein analysis and sequencing, single-molecule covalent chemistry, clinical sensing applications for single-molecule liquid biopsy, and the use of synthetic biomimetic nanopores as experimental models for natural systems. We suggest that nanopore technologies will continue to be explored to address a number of scientific challenges as control over pore design improves.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
A protein–nanopore machine that can unfold, thread and degrade a protein has now been developed. Fabricated in a bottom-up fashion, the nanopore machine is assembled from three proteins and provides ...an important step towards deciphering the sequence of single proteins via nanopores.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular ...communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.
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IJS, KILJ, NUK, PNG, UL, UM
Nanoelectrodes, with dimensions below 100 nm, have the advantages of high sensitivity and high spatial resolution. These electrodes have attracted increasing attention in various fields such as ...single cell analysis, single-molecule detection, single particle characterization and high-resolution imaging. The rapid growth of novel nanoelectrodes and nanoelectrochemical methods brings enormous new opportunities in the field. In this perspective, we discuss the challenges, advances, and opportunities for nanoelectrode fabrication, real-time characterizations and high-performance electrochemical instrumentation.
In this perspective, we discuss the challenges, advances and opportunities in electroanalytical chemistry at nanoelectrodes, including nanoelectrode fabrication, real-time characterizations, and high-performance electrochemical instrumentation.
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IJS, KILJ, NUK, UL, UM, UPUK
Single‐entity electrochemistry is a powerful tool that enables the study of electrochemical processes at interfaces and provides insights into the intrinsic chemical and structural heterogeneities of ...individual entities. Signal processing is a critical aspect of single‐entity electrochemical measurements and can be used for data recognition, classification, and interpretation. In this review, we summarize the recent five‐year advances in signal processing techniques for single‐entity electrochemistry and highlight their importance in obtaining high‐quality data and extracting effective features from electrochemical signals, which are generally applicable in single‐entity electrochemistry. Moreover, we shed light on electrochemical noise analysis to obtain single‐molecule frequency fingerprint spectra that can provide rich information about the ion networks at the interface. By incorporating advanced data analysis tools and artificial intelligence algorithms, single‐entity electrochemical measurements would revolutionize the field of single‐entity analysis, leading to new fundamental discoveries.
This Minireview summarizes the latest advances in data processing techniques for single‐entity electrochemistry towards achieving automation, real‐time monitoring, increased sensitivity, as well as improved temporal and current resolution.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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