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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.

Biomarkers are present in various metabolism processes, demanding precise and meticulous analysis at the single-molecule level for accurate clinical diagnosis. Given the need for high sensitivity, biological nanopore have been applied for single biomarker sensing. However, the detection of low-volume biomarkers poses challenges due to their low concentrations in dilute buffer solutions, as well as difficulty in parallel detection. Here, a droplet nanopore technique is developed for low-volume and high-throughput single biomarker detection at the sub-microliter scale, which shows a 2000-fold volume reduction compared to conventional setups. To prove the concept, this nanopore sensing platform not only enables multichannel recording but also significantly lowers the detection limit for various types of biomarkers such as angiotensin II, to 42 pg. This advancement enables direct biomarker detection at the picogram level. Such a leap forward in detection capability positions this nanopore sensing platform as a promising candidate for point-of-care testing of biomarker at single-molecule level, while substantially minimizing the need for sample dilution., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The study of a single-molecule reaction under nanoconfinement is beneficial for understanding the reactive intermediates and reaction pathways. However, the kinetics model of the single-molecule reaction under confinement remains elusive. Herein we engineered an aerolysin nanopore reactor to elaborate the single-molecule reaction kinetics under nanoconfinement. By identifying the bond-forming and non-bond-forming events directly, a four-state kinetics model is proposed for the first time. Our results demonstrated that the single-molecule reaction kinetics inside a nanopore depends on the frequency of individual reactants captured and the fraction of effective collision inside the nanopore confined space. This insight will guide the design of confined nanopore reactors for resolving the single-molecule chemistry, and shed light on the mechanistic understanding of dynamic covalent chemistry inside confined systems such as supramolecular cages, coordination cages, and micelles., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Reading the primary sequence directly using nanopores remains challenging due to the complex building blocks of 20 proteinogenic amino acids and the corresponding sophisticated structures. Compared to the uniformly negatively charged polynucleotides, biological nanopores hardly provide effective ionic current responses to all heterogeneously charged peptides under nearly physiological pH conditions. Herein, we precisely design a N226Q/S228K mutant aerolysin which creates a new electrostatic constriction named R3 in-between two natural sensing regions for controlling the capture and translocation of heterogeneously charged peptides. At nearly physiological pH, the decoration of positive charges at this constriction gives a large velocity of electroosmotic flow (EOF), leading to a maximum 8-fold increase in frequency for the heterogeneously charged peptides with the net charge from +1 to -3. Even the duration time of the negatively charged peptide Aβ35-25D4 in N226Q/S228K AeL also rises from 0.07 ± 0.01 ms to 0.63 ± 0.01 ms after introducing the third electrostatic constriction. Therefore, the N226Q/S228K aerolysin nanopore with three electrostatic constrictions realizes the dual goals of both capturing and decelerating heterogeneously charged peptides without labelling, even for the folded peptides., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Nanopore electrochemistry, as one of the promising tools for single molecule sensing, has proved its capability in DNA sequencing and protein analysis. To achieve a high resolution for obtaining molecular information, the nanopore electrochemical technique not only urgently requires an appropriate nanopore sensing interface with atomic resolution but also requires advanced instrumentation and its related data processing methods. In order to reveal the fundamental biological process and process the point-of-care diagnosis, it is necessary to use a nanopore sensing instrument with a high amperometric and temporal resolution as well as high throughput. The development of the instrumentation requires multi-disciplinary collaboration involving preparing a sensitive nanopore interface, low-noise circuit design, and intelligent data analysis. In this review, we have summarized the recent improvements in the nanopore sensing interface as well as discussed the higher throughput achieved by nanopore arrays and intelligent nanopore data analysis methods. The parallelized nanopore instrumentation could be popularized to all ranges of single-molecule applications.

A fundamental question relating to protein folding/unfolding is the time evolution of the folding of a protein into its precisely defined native structure. The proper identification of transition conformations is essential for accurately describing the dynamic protein folding/unfolding pathways. Owing to the rapid transitions and sub-nm conformation differences involved, the acquisition of the transient conformations and dynamics of proteins is difficult due to limited instrumental resolution. Using the electrochemical confinement effect of a solid-state nanopore, we were able to snapshot the transient conformations and trace the multiple transition pathways of a single peptide inside a nanopore. By combining the results with a Markov chain model, this new single-molecule technique is applied to clarify the transition pathways of the β-hairpin peptide, which shows nonequilibrium fluctuations among several blockage current stages. This method enables the high-throughput investigation of transition pathways experimentally to access previously obscure peptide dynamics, which is significant for understanding the folding/unfolding mechanisms and misfolding of peptides or proteins., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In this study, we designed SiN X solid-state nanopores to detect the temperature effect on the hydrogen nanobubble formation. Here, we integrated a temperature controller with the highly sensitive nanopore. As the temperature decreases from 25 °C to 5 °C, the occurrence of the nanobubble nucleation inside a 12.3 nm SiN X nanopore confined space decreased from 102 s -1 to 23 s -1 , and the life-time of nanobubbles increased from 1.16 ms to 4.78 ms. The results further gave the activation energy for nanobubble nucleation which was 8.1 × 10 -20 J with a 12.3 nm SiN X nanopore. Our method provides an efficient analytical tool for revealing the temperature-dependent nanobubble nucleation, which further benefits the fundamental understanding of nanobubble nucleation.

Biological nanopore technology has the advantages of high selectivity and high reproducibility for characterizing single biomolecules. However, it is challenging to achieve protein sequencing owing to the heterogeneous charge distributions of the protein and the small structural difference from each amino acid. Here, we took the inherent electrochemically confined sensing interface of the aerolysin nanopore to enhance its interaction with single amino acids. The results showed that single cysteine molecules, a highly reactive amino acid in aging and neurodegenerative diseases, could be captured and monitored by an aerolysin nanopore as it produced distinctive current blockages with a prolonged statistical duration of 0.11 ± 0.02 ms at +120 mV. This is the first report of the detection of a single amino acid molecule by a biological nanopore directly without any modification and labelling. This study facilitates the direct detection of single amino acids by regulating the characteristic interaction between the single amino acids and the designed sensing interface of aerolysin nanopores.

We have developed a glass nanopore based single molecule tool to investigate the dynamic oligomerization and aggregation process of Aβ1-42 peptides. The intrinsic differences in the molecular size and surface charge of amyloid aggregated states could be distinguished through single molecule induced characteristic current fluctuation. More importantly, our results reveal that the neurotoxic Aβ1-42 oligomer tends to adsorb onto the solid surface of nanopores, which may explain its instability and highly neurotoxic features., (This journal is © The Royal Society of Chemistry 2019.)

Flavin adenine dinucleotide (FAD) as a cofactor is involved in numerous important metabolic pathways where the biological function is intrinsically related to its transient conformations. The confined space of enzymes requires FAD set in its specific intermediate conformation. However, conventional methods only detect stable conformations of FAD molecules, while transient intermediates are hidden in ensemble measurements. There still exists a challenge to uncover the transient conformation of each FAD molecule, which hinders the understanding of the structure-activity relationship of the FAD mechanism. Here, we employ the electrochemically confined space of an aerolysin nanopore to directly characterize a series of transient conformations of every individual FAD. Based on distinguishable current blockages, the "stack", "open", and four quasi-stacked FADs are clearly determined in solution, which is further confirmed by temperature-dependent experiments and mutant aerolysin assay. Combined with molecular dynamics simulations, we achieved a direct correlation between the residual current ratio ( I / I 0 ) and FAD backbone angle. These results would facilitate further understanding of the structure-activity relationship in the flavoprotein., (This journal is © The Royal Society of Chemistry 2019.)

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference with a 5'-benzaldehyde poly(dA) 4 probe. Our strategy allows potential detection and characterization of various amino acids and their modifications, and provides a crucial step towards developing nanopore protein sequencing devices.

Wild-type aerolysin (AeL) nanopores allow direct single nucleotide discrimination of very short oligonucleotides (≤10 nt) without labelling, which shows great potential for DNA sensing. To achieve real applications, one major obstacle of AeL is its poor capture ability of long single-stranded DNA (ssDNA, >10 nt). Here, we have proposed a novel and robust strategy for the electrostatic focusing of long ssDNA into a lithium-chloride (LiCl)-active AeL. By using this method, for the first time we have demonstrated AeL detection of ssDNA longer than 100 nt. Due to screening more negative charges, LiCl improves AeL capture ability of long ssDNA ( i.e. 60 nt) by 2.63- to 10.23-fold compared to KCl. Further calculations and molecular dynamics simulations revealed that strong binding between Li + and the negatively charged residue neutralized the AeL, leading to a reduction in the energy barrier for ssDNA capture. These findings facilitate the future high-throughput applications of AeL in genetic and epigenetic diagnostics.

To enable electrochemistry-based sensors to be applied under size and cost sensitive conditions, we developed a thumb-size electrochemistry instrument system (MiniEC) with integrated components including an electrochemical sensing interface, signal transduction, amplification, filtering, processing and transmission with a cost of around only $15. We showed its potential application for electrochemical studies and on-site environmental monitoring in a wide range of environments such as laboratory, household, countryside, industry or wild settings. By using the MiniEC with screen printed electrodes, the LOD (limit of detection) for Cd2+ and Pb2+ is as low as 1 μg L-1 and 0.5 μg L-1, respectively.

Here, we show a designed solid-state nanopore sensor for the direct sensing and quantification of prostate-specific antigen (PSA) as cancer biomarker in serum without any pretreatment. This nanopore technique provides a convenient, fast, and low-cost quantification of cancer biomarkers in clinical samples.

To achieve accurate detection of cancer biomarkers with nanopore sensors, the precise recognition of multi-level current blockage events (signature) is a pivotal problem. However, it remains rather a challenge to identify the multi-level current blockages of target biomarkers in nanopore experiments, especially for the nanopore analysis of serum samples. In this work, we combined a modified DBSCAN (Density-Based Spatial Clustering of Applications with Noise) algorithm with the Viterbi training algorithm of the hidden Markov model (HMM) to achieve intelligent retrieval of multi-level current signatures from microRNA in serum samples. The results showed that the developed intelligent data analysis method is highly efficient for processing the large-scale nanopore data, which facilitates future application of nanopores to the clinical detection of cancer biomarkers.

Herein, the structural stability of single azobenzene modified DNA duplexes, including the trans form and cis form, has been examined separately based on their distinguishable unzipping kinetics from the mixture by an α-hemolysin nanopore. Furthermore, the accurate isomerization efficiency between the trans and cis form was obtained with single molecule resolution.

Understanding the single molecular protein-protein interaction has great significance in evaluating the affinity of a specific antibody. Herein, the interaction between single α-fetal protein (AFP) and its antibody was monitored via transient ionic current recording by using the antibody functionalized nanopore sensors. More importantly, the kinetic evaluation was performed at the single molecule level to determine the dissociation constant of this interaction. This method enables the monitoring of the kinetic antigen-antibody interaction in their heterogenetic state without any labelling. Our results provided new insights into the evaluation of the antibody's binding affinity and more into the development of immunoassays for diagnostics.

Carbon quantum dot wrapped gold nanorods were fabricated on an ITO electrode surface via electrostatic interactions. The electron transfer properties of carbon quantum dots on gold nanorod surfaces were systematically investigated by plasmonic resonance scattering spectroscopy.

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.

Herein, the unzipping and translocation of DNA duplexes through a sub-2 nm silicon nitride (SiN x ) solid-state nanopore have been demonstrated by well-resolved three-level blockades. In order to examine our observations, we applied a simple model which is applicable to the unzipping and translocation processes of DNA duplexes through solid-state nanopores. The generation of these highly recognizable signatures is an important step towards the real applications of solid-state nanopores in complex samples.

Single nanoparticle (NP) electrochemical measurements are widely described, both theoretically and experimentally, as they enable visualization of the electrochemical signal of a single NP that is masked in ensemble measurements. However, investigating the behavior of individual NPs using electrochemical signals remains a significant challenge. Here we report experiments and simulations demonstrating that multiple distinct motion trajectories could be discerned from time-resolved current traces by dynamic Monte Carlo simulations. We show that continuous monitoring and quantification of electrochemical oxidation of individual AgNPs using a low-noise electrochemical measurement platform produce significantly distinguished current traces due to the size-dependent motions of AgNPs. Our findings offer a view of the electrochemical signals of individual NPs that are largely different from that in the literature, and underscore the significance of motion behaviors in single NP electrochemistry.

We employed an α-hemolysin (α-HL) nanopore as a single-molecule tool to investigate the effects of initial structure on the amyloidosis process. The differences in the initial structure of two β-amyloid (Aβ) peptides (Aβ25-35 and Aβ35-25) could be distinguished in real-time due to their characteristic blockades. More importantly, the distinct aggregate dynamics for these two kinds of Aβ fragments can be readily analyzed by monitoring the blockade frequency over time.

This work proposes a gold nanoparticle (AuNP) based probe to study the single-nanoparticle translocation behavior through a silicon nitride (SiNx) solid-state nanopore. The AuNP probe is functionalized with a rhodamine derivative molecule, termed Rhod-DPA, whose fluorescence can be activated in the presence of Cu(2+) due to the binding between Rhod-DPA and Cu(2+). The Cu(2+) triggered configuration change of Rhod-DPA on the probe surface can induce the plasmon resonance energy transfer (PRET) from single AuNPs to the transformed fluorescent molecules, which can be detected by the color change of AuNP probes under dark-field microscopy (DFM) and their scattering spectra recorded on a spectrometer. By analyzing the peak shifts before and after the addition of Cu(2+), evidence of single nanoparticle translocation through the nanopore has been obtained, proving the successful establishment of the tracking strategy.

In this communication, we provide a new method for characterizing the kinetics of a catalytic process on multiple sites of a single nanowire by dark-field-assisted surface-enhanced Raman spectroscopy (DFSERS). The differences in the reaction rate and the extent of the photocatalysis between sites of a single nanowire were observed.

The self-assembly process from a 1 : 1 to a 1 : 2 complex, facilitated by para-sulfonatocalix[6]arenes (SC6) as host and methyl viologen (MV(2+)) as guest, was analyzed at the single-molecule level through an α-hemolysin nanopore. Especially, the assembled complex structures were discriminated in real time in the mixture of SC6 and MV(2+).

Nanopore sensors provide a highly innovative technique for a rapid and label-free single molecule analysis, which holds a great potential in routing applications. Biological nanopores have been used as ultra-sensitive sensors over a wide range of single molecule analysis including DNA sequencing, disease diagnosis, drug screening, environment monitoring and the construction of molecule machines. This mini review will focus on the current strategies for the identification and characterization of an individual analyte, especially based on our recent achievements in biological nanopore biosensors.

A novel method for real-time monitoring of the oxidative response of a membrane-channel biomimetic system (MCBS) to free radicals is developed and the deduction of the buffering effect of MCBS is discussed.

Two peptide-oligonucleotide conjugates are studied using an α-hemolysin nanopore to investigate their structural properties at the single-molecule level.

The translocation time of poly(dT)(45) through an α-hemolysin pore was reduced in the presence of a DNA-binding Fab fragment. The Fab acts as a rudder to steer the DNA into the pore., (© The Royal Society of Chemistry 2011)