HFM-Tracker: a cell tracking algorithm based on hybrid feature matching.

Cell migration is known to be a fundamental biological process, playing an essential role in development, homeostasis, and diseases. This paper introduces a cell tracking algorithm named HFM-Tracker (Hybrid Feature Matching Tracker) that automatically identifies cell migration behaviours in consecutive images. It combines Contour Attention (CA) and Adaptive Confusion Matrix (ACM) modules to accurately capture cell contours in each image and track the dynamic behaviors of migrating cells in the field of view. Cells are firstly located and identified via the CA module-based cell detection network, and then associated and tracked via a cell tracking algorithm employing a hybrid feature-matching strategy. This proposed HFM-Tracker exhibits superiorities in cell detection and tracking, achieving 75% in MOTA (Multiple Object Tracking Accuracy) and 65% in IDF1 (ID F1 score). It provides quantitative analysis of the cell morphology and migration features, which could further help in understanding the complicated and diverse cell migration processes.

Emerging Data Processing Methods for Single-Entity Electrochemistry.

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.

Electrochemical Visualization of Single-Molecule Thiol Substitution with Nanopore Measurement.

Reactions involving sulfhydryl groups play a critical role in maintaining the structure and function of proteins. However, traditional mechanistic studies have mainly focused on reaction rates and the efficiency in bulk solutions. Herein, we have designed a cysteine-mutated nanopore as a biological protein nanoreactor for electrochemical visualization of the thiol substitute reaction. Statistical analysis of characteristic current signals shows that the apparent reaction rate at the single-molecule level in this confined nanoreactor reached 1400 times higher than that observed in bulk solution. This substantial acceleration of thiol substitution reactions within the nanopore offers promising opportunities for advancing the design and optimization of micro/nanoreactors. Moreover, our results could shed light on the understanding of sulfhydryl reactions and the thiol-involved signal transduction mechanisms in biological systems.

Wireless electrical-molecular quantum signalling for cancer cell apoptosis.

Quantum biological tunnelling for electron transfer is involved in controlling essential functions for life such as cellular respiration and homoeostasis. Understanding and controlling the quantum effects in biology has the potential to modulate biological functions. Here we merge wireless nano-electrochemical tools with cancer cells for control over electron transfer to trigger cancer cell death. Gold bipolar nanoelectrodes functionalized with redox-active cytochrome c and a redox mediator zinc porphyrin are developed as electric-field-stimulating bio-actuators, termed bio-nanoantennae. We show that a remote electrical input regulates electron transport between these redox molecules, which results in quantum biological tunnelling for electron transfer to trigger apoptosis in patient-derived cancer cells in a selective manner. Transcriptomics data show that the electric-field-induced bio-nanoantenna targets the cancer cells in a unique manner, representing electrically induced control of molecular signalling. The work shows the potential of quantum-based medical diagnostics and treatments.

Confined-Enhanced Raman Spectroscopy.

In 1997, the discovery of single molecule-surface enhanced Raman spectroscopy (SM-SERS) rekindled broad interests owing to its ultrahigh enhancement factor up to the 1014-1015 level. However, regretfully, the advantage of SM-SERS with an ultralow detection limit has not yet been fully utilized in commercialized applications. Here, we report a strategy, which we name confined-enhanced Raman spectroscopy, in which the overall Raman properties can be remarkably improved with in situ-formed active nanoshell on the surface of silver or gold nanoparticles. The nanoshell can confine and anchor molecules onto the surface of plasmonic nanoparticles and avoid desorption from hot spots so that the "on and off" blinking effect can be eliminated. It is the first time the single-molecule detection of analytes with super sensitivity, high stability, and reproducibility based on gold nanoparticles has been realized. In addition, this strategy is suitable for SERS detection in diverse molecule systems, including biomedical diagnosis, catalytic reaction, etc.

Paper-based substrates for surface-enhanced Raman spectroscopy sensing.

Surface-enhanced Raman scattering (SERS) has been recognized as one of the most sensitive analytical methods by adsorbing the target of interest onto a plasmonic surface. Growing attention has been directed towards the fabrication of various substrates to broaden SERS applications. Among these, flexible SERS substrates, particularly paper-based ones, have gained popularity due to their easy-to-use features by full contact with the sample surface. Herein, we reviewed the latest advancements in flexible SERS substrates, with a focus on paper-based substrates. Firstly, it begins by introducing various methods for preparing paper-based substrates and highlights their advantages through several illustrative examples. Subsequently, we demonstrated the booming applications of these paper-based SERS substrates in abiotic and biological matrix detection, with particular emphasis on their potential application in clinical diagnosis. Finally, the prospects and challenges of paper-based SERS substrates in broader applications are discussed.

Multiphase Chemistry under Nanoconfinement: An Electrochemical Perspective.

Most relevant systems of interest to modern chemists rarely consist of a single phase. Real-world problems that require a rigorous understanding of chemical reactivity in multiple phases include the development of wearable and implantable biosensors, efficient fuel cells, single cell metabolic characterization techniques, and solar energy conversion devices. Within all of these systems, confinement effects at the nanoscale influence the chemical reaction coordinate. Thus, a fundamental understanding of the nanoconfinement effects of chemistry in multiphase environments is paramount. Electrochemistry is inherently a multiphase measurement tool reporting on a charged species traversing a phase boundary. Over the past 50 years, electrochemistry has witnessed astounding growth. Subpicoampere current measurements are routine, as is the study of single molecules and nanoparticles. This Perspective focuses on three nanoelectrochemical techniques to study multiphase chemistry under nanoconfinement: stochastic collision electrochemistry, single nanodroplet electrochemistry, and nanopore electrochemistry.

Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore.

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site-specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine-based aminoacyl-tRNA synthetase-tRNA pair for a high yield of pore-forming protein. Both molecular dynamics (MD) simulations and single-molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches.

Observing Confined Local Oxygen-induced Reversible Thiol/Disulfide Cycle with a Protein Nanopore.

Disulfide bonds play an important role in thiol-based redox regulation. However, owing to the lack of analytical tools, little is known about how local O2 mediates the reversible thiol/disulfide cycle under protein confinement. In this study, a protein-nanopore inside a glove box is used to control local O2 for single-molecule reaction, as well as a single-molecule sensor for real-time monitoring of the reversible thiol/disulfide cycle. The results demonstrate that the local O2 molecules in protein nanopores could facilitate the redox cycle of disulfide formation and cleavage by promoting a higher fraction of effective reactant collisions owing to nanoconfinement. Further kinetic calculations indicate that the negatively charged residues near reactive sites facilitate proton-involved oxygen-induced disulfide cleavage under protein confinement. The unexpectedly strong oxidation ability of confined local O2 may play an essential role in cellular redox signaling and enzyme reactions.

Profiling the chemistry- and confinement-controlled sensing capability of an octameric aerolysin-like protein.

Octameric Aep1 was employed, for the first time to the best of our knowledge, as a nanopore to expand applications. After investigating the optimized conditions of Aep1 for single-channel recording, the sensing features were characterized. Cyclic and linear molecules of varying sizes and charges were employed to probe the radius and chemical environment of the pore, providing deep insights for expected future endeavors at predicting the structure of octameric Aep1. γ-CD showed unique suitability as an 8-subunit adapter in octameric Aep1, enabling the discrimination of ß-nicotinamide mononucleotide.

Simultaneous observation of the spatial and temporal dynamics of single enzymatic catalysis using a solid-state nanopore.

We developed a bipolar SiNx nanopore for the observation of single-molecule heterogeneous enzymatic dynamics. Single glucose oxidase was immobilized inside the nanopore and its electrocatalytic behaviour was real-time monitored via continuous recording of ionic flux amplification. The temporal heterogeneity in enzymatic properties and its spatial dynamic orientations were observed simultaneously, and these two properties were found to be closely correlated. We anticipate that this method offers new perspectives on the correlation of protein structure and function at the single-molecule level.

Mass Spectrometry Imaging-Based Single-Cell Lipidomics Profiles Metabolic Signatures of Heart Failure.

Heart failure (HF), leading as one of the main causes of mortality, has become a serious public health issue with high prevalence around the world. Single cardiomyocyte (CM) metabolomics promises to revolutionize the understanding of HF pathogenesis since the metabolic remodeling in the human hearts plays a vital role in the disease progression. Unfortunately, current metabolic analysis is often limited by the dynamic features of metabolites and the critical needs for high-quality isolated CMs. Here, high-quality CMs were directly isolated from transgenic HF mice biopsies and further employed in the cellular metabolic analysis. The lipids landscape in individual CMs was profiled with a delayed extraction mode in time-of-flight secondary ion mass spectrometry. Specific metabolic signatures were identified to distinguish HF CMs from the control subjects, presenting as possible single-cell biomarkers. The spatial distributions of these signatures were imaged in single cells, and those were further found to be strongly associated with lipoprotein metabolism, transmembrane transport, and signal transduction. Taken together, we systematically studied the lipid metabolism of single CMs with a mass spectrometry imaging method, which directly benefited the identification of HF-associated signatures and a deeper understanding of HF-related metabolic pathways.

Pore confined time-of-flight secondary ion electrochemical mass spectrometry.

Molecular structure conversion concomitant with mass transfer processes at the electrode-electrolyte interfaces plays a central role in energy electrochemistry. Mass spectrometry, as one of the most intuitive, sensitive techniques, provides the capability to collect transient intermediates and products and uncover reaction mechanisms and kinetics. In situ time-of-flight secondary ion electrochemical mass spectrometry with inherent high mass and spatiotemporal resolution has emerged as a promising strategy for investigating electrochemical processes at the electrode surface. This review illustrates the recent advancements in coupling time-of-flight secondary ion mass spectrometry and electrochemistry to visualize and quantify local dynamic electrochemical processes, identify solvated species distribution, and disclose hidden reaction pathways at the molecular level. Moreover, the key challenges in this field are further discussed to promote new applications and discoveries in operando studying the dynamic electrochemical interfaces of advanced energy systems.

Editorial: Nanopore Electrochemistry.

This Special Collection highlights the most recent developments in nanopore electrochemistry and applications. In this Editorial, guest Editors Yi-Tao Long, Meni Wanunu, and Mathias Winterhalter briefly introduce the research published in this special collection.

Protein nanopore reveals the renin-angiotensin system crosstalk with single-amino-acid resolution.

The discovery of crosstalk effects on the renin-angiotensin system (RAS) is limited by the lack of approaches to quantitatively monitor, in real time, multiple components with subtle differences and short half-lives. Here we report a nanopore framework to quantitatively determine the effect of the hidden crosstalk between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) on RAS. By developing an engineered aerolysin nanopore capable of single-amino-acid resolution, we show that the ACE can be selectively inhibited by ACE2 to prevent cleavage of angiotensin I, even when the concentration of ACE is more than 30-fold higher than that of ACE2. We also show that the activity of ACE2 for cleaving angiotensin peptides is clearly suppressed by the spike protein of SARS-CoV-2. This leads to the relaxation of ACE and the increased probability of accumulation of the principal effector angiotensin II. The spike protein of the SARS-CoV-2 Delta variant is demonstrated to have a much greater impact on the crosstalk than the wild type.

Monitoring Photoinduced Interparticle Chemical Communication In Situ.

Monitoring interparticle chemical communication plays a critical role in the nanomaterial synthesis as this communication controls the final structure and stability of global nanoparticles (NPs). Yet most ensemble analytical techniques, which could only reveal average macroscopic information, are unable to elucidate NP-to-NP interactions. Herein, we employ stochastic collision electrochemistry to track the morphology transformation of Ag NPs in photochemical process at the single NP level. By further statistical analysis of time-resolved current transients, we quantitatively determine the dynamic chemical potential difference and interparticle communication between populations of large and small Ag NPs. The high sensitivity of stochastic collision electrochemistry enables the in situ investigation of chemical communication-dependent transformation kinetics of NPs in photochemical process, shedding light on designing nanomaterials.

Mass Transport and Electron Transfer at the Electrochemical-Confined Interface.

Single entity measurements based on the stochastic collision electrochemistry provide a promising and versatile means to study single molecules, single particles, single droplets, etc. Conceptually, mass transport and electron transfer are the two main processes at the electrochemically confined interface that underpin the most transient electrochemical responses resulting from the stochastic and discrete behaviors of single entities at the microscopic scale. This perspective demonstrates how to achieve controllable stochastic collision electrochemistry by effectively altering the two processes. Future challenges and opportunities for stochastic collision electrochemistry are also highlighted.

SmartImage: A Machine Learning Method for Nanopore Identifying Chemical Modifications on RNA.

RNA modifications modulate essential cellular functions. However, it is challenging to quantitatively identify the differences in RNA modifications. To further improve the single-molecule sensing ability of nanopores, we propose a machine-learning algorithm called SmartImage for identifying and classifying nanopore electrochemical signals based on a combination of improved graph conversion methods and deep neural networks. SmartImage is effective for nearly all ranges of signal duration, which breaks the limitation of the current nanopore algorithm. The overall accuracy (OA) of our proposed recognition strategy exceeded 90% for identifying three types of RNAs. Prediction experiments show that the SmartImage owns the ability to recognize one modified RNA molecule from 1000 normal RNAs with OA >90%. Thus our proposed model and algorithm hold the potential application in clinical applications.

In Situ Characterization of Oxygen Evolution Electrocatalysis of Silver Salt Oxide on a Wireless Nanopore Electrode.

Silver salt oxide shows superior oxidation ability for the applications of superconductivity, sterilization, and catalysis. However, due to the easy decomposition, the catalytic properties of silver salt oxide are difficult to characterize by conventional methods. Herein, we used a closed-type wireless nanopore electrode (CWNE) to in situ and real-time monitor the electrocatalytic performance of Ag7NO11 in the oxygen evolution reaction. The real-time current recording revealed that the deposited Ag7NO11 on the CWNE tip greatly enhanced the oxidative capacity of the electrode, resulting in water splitting. The statistical event analysis reveals the periodic O2 bubble formation and dissolution at the Ag7NO11 interface, which ensures the characterization of the oxygen evolution electrocatalytic process at the nanoscale. The calculated kcat and Markov chain modeling suggest the anisotropy of Ag7NO11 at a low voltage may lead to multiple catalytic rates. Therefore, our results demonstrate the powerful capability of CWNE in direct and in situ characterization of gas-liquid-solid catalytic reactions for unstable catalysts.

3D Blockage Mapping for Identifying Familial Point Mutations in Single Amyloid-ß Peptides with a Nanopore.

Accurate discrimination of amyloid-ß (Aß) peptides containing familial point mutations would advance the knowledge of their roles in early-onset Alzheimer's disease. Herein, we simultaneously identified the mutant A21G, E22G, E22Q, and the wild-type (WT) Aß18-26 peptides with aerolysin nanopore using a 3D blockage mapping strategy. The standard deviation of current blockade fluctuations (σb ) was proposed as a new supplement to current blockage (Ib /I0 ) and duration time (tD ) to profile the blockage characteristics of single molecules. Although the WT and A21G Aß18-26 are indistinguishable in a traditional Ib /I0 -tD 2D description, ∼87 % of the blockade events can be accurately classified with half reduction of false identification using a combination of Ib /I0 , tD, and σb . This work offers an easy and reliable strategy to promote nanopore sensitivity of peptide mutants, leading to a more precise analysis of pathogenic mutations for developing effective diagnosis and treatment.