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.

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.

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 Monitoring of Real-Time Vesicle Dynamics Induced by Tau in a Confined Nanopipette.

The microtubule-associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau's engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse-mimicking nanopipette to monitor vesicle dynamics induced by tau. A model vesicle of ~30 nm is confined within a lipid-modified nanopipette orifice with a comparable diameter to mimic the synaptic lipid environment. Both tau and phosphorylated tau (p-tau) present two-state dynamic behavior in this biomimetic system, showing typical ionic current oscillation, induced by lipid-tau interaction. The results indicate that p-tau has a stronger affinity to the lipid vesicles in the confined environment, blocking the vesicle movement to a higher degree. Taken together, this method bridges a gap for sensing synaptic vesicle dynamics in a confined lipid environment, mimicking vesicle movement near the synaptic membrane. These findings contribute to understanding how different types of tau protein regulate synaptic vesicle motility and to underlying its functional and pathological behaviours in disease.

Nanopore Deciphering Single Digital Polymers Towards High-Density Data Storage.

Sequence-defined polymer is one of the most promising alternative media for high-density data storage. It could be used to alleviate the problem of insufficient storage capacity of conventional silicon-based devices for the explosively increasing data. To fulfil the goal of polymer data storage, suitable methods should be developed to accurately read and decode the information-containing polymers, especially for those composed by a combination of the natural and unnatural monomers. Nanopore-based approaches have become one of the most competitive analysis and sequencing techniques, which are expected to read both natural and synthetic polymers with single-molecule precision and monomeric resolution. Herein, this work emphasizes the advances being made in nanopore reading and decoding of information stored in the man-made polymers and DNA nanostructures, and discusses the challenges and opportunities towards the development and realization of high-density data storage.

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.

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.

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.

Confined Nanopipet as a Versatile Tool for Precise Single Cell Manipulation.

The precise manipulation of single cells plays a fundamental role for single cell measurement, which is crucial for understanding the diverse cellular mechanisms. Unusual single cell behavior could thus be identified by integrating with advanced analytical methods such as single cell omics, unraveling the intrinsic cellular heterogeneity hidden in ensemble measurements. Herein, this technical note reports a nanopipet-based versatile method for manipulation of an ultrasmall volume of liquid, which further enables the precise manipulation of single cells. Femtoliter volumes of cytoplasm were extracted from single living cells and analyzed by time-of-flight secondary ion mass spectrometry. Moreover, several kinds of exogenous components were injected simultaneously into a cell, offering a delicate tool for multi-imaging in single living cells.

In situ food-borne pathogen sensors in a nanoconfined space by surface enhanced Raman scattering.

The incidence of disease arising from food-borne pathogens is increasing continuously and has become a global public health problem. Rapid and accurate identification of food-borne pathogens is essential for adopting disease intervention strategies and controlling the spread of epidemics. Surface-enhanced Raman spectroscopy (SERS) has attracted increasing interest due to the attractive features including simplicity, rapid measurement, and high sensitivity. It can be used for rapid in situ sensing of single and multicomponent samples within the nanostructure-based confined space by providing molecular fingerprint information and has been demonstrated to be an effective detection strategy for pathogens. This article aims to review the application of SERS to the rapid sensing of food-borne pathogens in food matrices. The mechanisms and advantages of SERS, and detection strategies are briefly discussed. The latest progress on the use of SERS for rapid detection of food-borne bacteria and viruses is considered, including both the labeled and label-free detection strategies. In closing, according to the current situation regarding detection of food-borne pathogens, the review highlights the challenges faced by SERS and the prospects for new applications in food safety. Graphical abstract In this review, the advances on the SERS detection of pathogens over the past decades have been reviewed, focusing on the improvements in sensitivity, reproducibility, specificity, and the performance of the SERS-based assay in complex analytical scenarios.

Confined Nanopipette Sensing: From Single Molecules, Single Nanoparticles, to Single Cells.

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.

Asymmetric Nanopore Electrode-Based Amplification for Electron Transfer Imaging in Live Cells.

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.

Characterization of the Dynamic Growth of the Nanobubble within the Confined Glass Nanopore.

The evolution of the nanobubble from gas molecules in liquid phase is of fundamental interest. However, the lack of sensitive tools hinders the study of growth dynamic of the bubble at nanoscale. Here, we employed a confined glass nanopore to real-time monitor the dynamics behavior of a single nanobubble generated by the reaction between NaBH4 and H2O. By analyzing the characteristic ionic current signal, the formation time and growth time of a single nanobubble could be estimated as 200 and 21 ms, respectively. Further, the nanopore size has been altered to modulate the growth behavior of the nanobubble. The results demonstrate the capability of the nanopore for sensitively tracking the behavior of single nanobubbles in liquid phase, which provides a powerful method for further understanding nanobubble evolution.

A 30 nm Nanopore Electrode: Facile Fabrication and Direct Insights into the Intrinsic Feature of Single Nanoparticle Collisions.

Clarifying the hidden but intrinsic feature of single nanoparticles by nanoelectrochemistry could help understand its potential for diverse applications. The uncontrolled interface and bandwidth limitation in the electrochemical measurement put the obstacle in single particle collision. Here, we demonstrate a well-defined 30 nm nanopore electrode with a rapid chemical-electrochemical fabrication method which provides a high reproducibility in both size and performance. A capacitance-based detection mechanism is demonstrated to achieve a high current resolution of 0.6 pA ±0.1 pA (RMS) and a high the temporal resolution of 0.01 ms. By utilizing this electrode, the dynamic interactions of every single particle in the mixture could be directly read during the collision process. The collision frequency is two orders of magnitude higher than previous reports, which helps reveal the hidden features of nanoparticles during the complex and multidimensional interaction processes.

Label-Free Monitoring of Single Molecule Immunoreaction with a Nanopipette.

The nanopipette has been employed for the single molecule analysis due to its advantage of easy fabrication and controllable diameter. Herein, we present that the single molecule immunoreaction could be monitored by using the quartz nanopipette through the discrimination of characteristic blockade current, which reflect the intrinsic character of the individual unlabeled protein molecules due to its heterogeneous motion in solution. Our methods show the ability to monitor the immunoreaction between single α-fetal protein (AFP) and its specific antibody in aqueous solution without any labeling. Our studies may open a new door to comprehensively understand the single molecule immunoreaction, which gain more insight into the molecular dynamic of elementary steps.

Real-Time Sensing of O-Phenylenediamine Oxidation on Gold Nanoparticles.

Real-time monitoring of chemical reactions is still challenging as well as important to study reaction mechanisms and reaction kinetics. Herein, we demonstrated the real-time monitoring of o-phenylenediamine (OPD) oxidation on the surface of gold nanoparticles by surface-enhanced Raman spectroscopy (SERS). The oxidation mechanism and the reaction kinetics were investigated on the basis of the SERS spectrum variation and the related density functionalized theory calculation. It was shown that the oxidation of OPD in the presence of copper ions was a two-step process of the deprotonation of the amino group on the aromatic rings and the rearrangement of the electron cloud to a π-conjugated system, which may open a new door to comprehensively understand the reaction process.

[One Carrier of Robinsoniella peoriensis in China].

OBJECTIVES: To study morphological feature, biochemical characteristic and antibiotic resistance of Robinsoniella peoriensis (R.peoriensis) strain. METHODS: The clinical R.peoriensis strain was isolated from ananus swab samples being screened in ICU. Biochemical characteristic of the strain was completed by fully automatic microbial identification and drug susceptibility analysis system (BioMérieux, Marcy-l'éE toile, France) with the ANC Vitek2. Antibiotic susceptibility in vitro was performed using agar dilution. RESULTS: The organism was found to be positive to saccharose, beta-galactopyr anosidase indoxyl, alpha-arabinosidase and so on, while negative to the others. The susceptibility test in vitro showed that this strain was resistant to clindamycin, rifampicin, moxifloxacin, while sensitive to vancomycin, metronidazole and tetracycline. CONCLUSIONS: R.peoriensis is positive to many biochemical products such as saccharose. The clinical isolate of R.peoriensis strain is resistant to clindamycin, rifampicin and moxifloxacin.

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.

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.

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.