Characterizing Chemical, Environmental, and Stimulated Subcellular Physical Characteristics of Size-Fractionated PMs Down to PM0.1.

Air pollution, especially particulate matter (PM), is a significant environmental pollution worldwide. Studying the chemical, environmental, and life-related cellular physical characteristics of size-fractionated PMs is important because of their different degrees of harmful effects on human respiratory tracts and organ systems, causing severe diseases. This study evaluates the chemical components of size-fractionated PMs down to PM0.1 collected during a biomass-burning episode, including elemental/organic carbon and trace elements. Single particle sizes and distributions of PM0.1, PM0.5-0.1, PM1.0-0.5, and PM2.5-1.0 were analyzed by scanning electron microscopy and Zeta sizer. Two commonly used cell lines, e.g., HeLa and Cos7 cells, and two respiratory-related cell lines including lung cancer/normal cells were utilized for cell cytotoxicity experiments, revealing the key effects of particle sizes and concentrations. A high-speed scanning ion conductance microscope explored particle-stimulated subcellular physical characteristics for all cell lines in dynamics, including surface roughness (SR) and elastic modulus (E). The statistical results of SR showed distinct features among different particle sizes and cell types while a E reduction was universally found. This work provides a comprehensive understanding of the chemical, environmental, and cellular physical characteristics of size-fractionated PMs and sheds light on the necessity of controlling small-sized PM exposures.

Nanopipette dynamic microscopy unveils nano coffee ring.

Liquid-phase electron microscopy (LP-EM) imaging has revolutionized our understanding of nanosynthesis and assembly. However, the current closed geometry limits its application for open systems. The ubiquitous physical process of the coffee-ring phenomenon that underpins materials and engineering science remains elusive at the nanoscale due to the lack of experimental tools. We introduce a quartz nanopipette liquid cell with a tunable dimension that requires only standard microscopes. Depending on the imaging condition, the open geometry of the nanopipette allows the imaging of evaporation-induced pattern formation, but it can also function as an ordinary closed-geometry liquid cell where evaporation is negligible despite the nano opening. The nano coffee-ring phenomenon was observed by tracking individual nanoparticles in an evaporating nanodroplet created from a thin liquid film by interfacial instability. Nanoflows drive the assembly and disruption of a ring pattern with the absence of particle-particle correlations. With surface effects, nanoflows override thermal fluctuations at tens of nanometers, in which nanoparticles displayed a "drunken man trajectory" and performed work at a value much smaller than kBT.

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.

In situ self-referenced intracellular two-electrode system for enhanced accuracy in single-cell analysis.

Since the emergence of single-cell electroanalysis, the two-electrode system has become the predominant electrochemical system for real-time behavioral analysis of single-cell and multicellular populations. However, due to the transmembrane placement of the two electrodes, cellular activities can be interrupted by the transmembrane potentials, and the test results are susceptible to influences from factors such as intracellular solution, membrane, and bulk solution. These limitations impede the advancement of single-cell analysis. Here, we propose a highly miniaturized and integrated in situ self-referenced intracellular two-electrode system (IS-SRITES), wherein both the working and reference electrodes are positioned inside the cell. Additionally, we demonstrated the stability (0.28 mV/h) of the solid-contact in situ Ag/AgCl reference electrode and the ability of the system to conduct standard electrochemical testing in a wide pH range (pH 6.0-8.0). Cell experiments confirmed the non-destructive performance of the electrode system towards cells and its capacity for real-time monitoring of intra- and extracellular pH values. Moreover, through equivalent circuits, finite element simulations, and drug delivery experiments, we illustrated that the IS-SRITES can yield more accurate test results and exhibit enhanced resistance to interference from the extracellular environment. Our proposed system holds the potential to enable the precise detection of intracellular substances and optimize the existing model of the electrode system for intracellular signal detection, thereby spearheading advancements in single-cell analysis.

Dual-signal readout sensing of ATP content in single dental pulp stem cells during differentiation via functionalized glass nanopipettes.

Adenosine triphosphate (ATP) is regarded as the "energy currency" in living cells, so real-time quantification of content variation of intracellular ATP is highly desired for understanding some important physiological processes. Due to its single-molecule readout ability, nanopipette sensing has emerged as a powerful technique for molecular sensing. In this study, based on the effect of targeting-aptamer binding on ionic current, and fluorescence resonance energy transfer (FRET), we reported a dual-signal readout nanopipette sensing system for monitoring ATP content variation at the subcellular level. In the presence of ATP, the complementary DNA-modified gold nanoparticles (cDNAs-AuNPs) were released from the inner wall of the nanopipette, which leads to sensitive response variations in ionic current rectification and fluorescence intensity. The developed nanopipette sensor was capable of detecting ATP in single cells, and the fluctuation of ATP content in the differentiation of dental pulp stem cells (DPSCs) was further quantified with this method. The study provides a more reliable nanopipette sensing platform due to the introduction of fluorescence readout signals. Significantly, the study of energy fluctuation during cell differentiation from the perspective of energy metabolism is helpful for differentiation regulation and cell therapy.

Influence of Seed structure on Volume distribution of α-Synuclein Oligomer at Early Stages of Aggregation using nanopipette.

Understanding α-synuclein aggregation is crucial in the context of Parkinson's disease. The objective of this study was to investigate the influence of aggregation induced by preformed seeding on the volume of oligomers during the early stages, using a label-free, single-molecule characterization approach. By utilizing nanopipettes of varying sizes, the volume of the oligomers can be calculated from the amplitude of the current blockade and pipette geometry. Further investigation of the aggregates formed over time in the presence of added seeds revealed an acceleration in the formation of large aggregates and the existence of multiple distinct populations of oligomers. Additionally, we observed that spontaneously formed seeds inhibited the formation of smaller oligomers, in contrast to the effect of HNE seeds. These results suggest that the seeds play a crucial role in the formation of oligomers and their sizes during the early stages of aggregation, whereas the classical thioflavin T assay remains negative.

Spatiotemporal Controlled Formation of Synthetic Nanoreactors in Single Living Cells.

Cellular compartments provide confined environments for spatiotemporal control of biological processes and enzymatic reactions. To mimic such compartmentalization of eukaryotic cells, we report an efficient and general platform to precisely control the formation of artificial nanoreactors in single living cells. We introduce an electroosmotic controlled strategy for the synthesis of ZIF-8 at the nanoscale liquid-liquid interface around the tip of a nanopipet, whereby the formed ZIF-8 nanoparticles are driven into a single living cell by the electroosmotic flow. The porous ZIF-8 nanoparticles, as synthetic nanoreactors, are not only able to harvest fluorescent molecules from peripheral cytoplasm but also perform the subsequent photocatalytic degradation, mimicking compartmentalized chemical reactions in eukaryotic cells. Our strategy provides a useful tool for spatiotemporal controlled synthesis of artificial nanoreactors with on-demand functions in single living cells with versatile applications in chemical biology.

Scanning Ion-Conductance Microscopy for Studying Mechanical Properties of Neuronal Cells during Local Delivery of Glutamate.

Mechanical properties of neuronal cells have a key role for growth, generation of traction forces, adhesion, migration, etc. Mechanical properties are regulated by chemical signaling, neurotransmitters, and neuronal ion exchange. Disturbance of chemical signaling is accompanied by several diseases such as ischemia, trauma, and neurodegenerative diseases. It is known that the disturbance of chemical signaling, like that caused by glutamate excitotoxicity, leads to the structural reorganization of the cytoskeleton of neuronal cells and the deviation of native mechanical properties. Thus, to investigate the mechanical properties of living neuronal cells in the presence of glutamate, it is crucial to use noncontact and low-stress methods, which are the advantages of scanning ion-conductance microscopy (SICM). Moreover, a nanopipette may be used for the local delivery of small molecules as well as for a probe. In this work, SICM was used as an advanced technique for the simultaneous local delivery of glutamate and investigation of living neuronal cell morphology and mechanical behavior caused by an excitotoxic effect of glutamate.

Aptamer Conformational Dynamics Modulate Neurotransmitter Sensing in Nanopores.

Aptamers that undergo conformational changes upon small-molecule recognition have been shown to gate the ionic flux through nanopores by rearranging the charge density within the aptamer-occluded orifice. However, mechanistic insight into such systems where biomolecular interactions are confined in nanoscale spaces is limited. To understand the fundamental mechanisms that facilitate the detection of small-molecule analytes inside structure-switching aptamer-modified nanopores, we correlated experimental observations to theoretical models. We developed a dopamine aptamer-functionalized nanopore sensor with femtomolar detection limits and compared the sensing behavior with that of a serotonin sensor fabricated with the same methodology. When these two neurotransmitters with comparable mass and equal charge were detected, the sensors showed an opposite electronic behavior. This distinctive phenomenon was extensively studied using complementary experimental techniques such as quartz crystal microbalance with dissipation monitoring, in combination with theoretical assessment by the finite element method and molecular dynamic simulations. Taken together, our studies demonstrate that the sensing behavior of aptamer-modified nanopores in detecting specific small-molecule analytes correlates with the structure-switching mechanisms of individual aptamers. We believe that such investigations not only improve our understanding of the complex interactions occurring in confined nanoscale environments but will also drive further innovations in biomimetic nanopore technologies.

θ-Nanopipette for Single-Cell Resistive-Pulse Profiling of DNA Repair Proteins Accompanied by Drug Evaluation.

Single-cell analysis of the DNA repair protein is important but remains unachieved. Exploration of nanopipettte technologies in single-cell electroanalysis has recently seen rapid growth, while the θ-nanopipette represents an emerging technological frontier with its potential largely veiled. Here a θ-nanopipette is first applied for single-cell resistive-pulse sensing (RPS) of the important DNA repair protein O6-alkylguanine DNA alkyltransferase (hAGT). The removal of alkyl mutations by hAGT could restore the damaged aptamer linking with a structural DNA carrier, allowing the selective binding of the aptamer to thrombin with precisely matched size to produce distinct RPS signals when passing through the orifice. Kinetic analysis of hAGT repair was studied. Meanwhile, the device shows the simultaneous on-demand infusion of inhibitors to inactivate the hAGT activity, indicative of its potential in drug screening for enhanced chemotherapy. This work provides a new paradigm for θ-nanopipette-based single-cell RPS of a DNA repair protein accompanied by drug evaluation.

An Integrated Dual-Functional Nanotool Capable of Studying Single-Cell Epigenetics and Programmable Gene Regulation.

Single-cell epigenetics is envisioned to decipher manifold epigenetic phenomena and to contribute to our accurate knowledge about basic epigenetic mechanisms. Engineered nanopipette technology has gained momentum in single-cell studies; however, solutions to epigenetic questions remain unachieved. This study addresses the challenge by exploring N6-methyladenine (m6 A)-bearing deoxyribozyme (DNAzyme) confined within a nanopipette for profiling a representative m6 A-modifying enzyme, fat mass and obesity-associated protein (FTO). Electroosmotic intracellular extraction of FTO could remove the m6 A and cause DNAzyme cleavage, leading to the altered ionic current signal. Because the cleavage can release a DNA sequence, we simultaneously program it as an antisense strand against FTO-mRNA, intracellular injection of which has been shown to induce early stage apoptosis. This nanotool thus features the dual functions of studying single-cell epigenetics and programmable gene regulation.

Beginner's Guide to Micro- and Nanoscale Electrochemical Additive Manufacturing.

Electrochemical additive manufacturing is an advanced microfabrication technology capable of producing features of almost unlimited geometrical complexity. A unique combination of the capacity to process conductive materials, design freedom, and micro- to nanoscale resolution offered by these electrochemical techniques promises tremendous opportunities for a multitude of future applications spanning microelectronics, sensing, robotics, and energy storage. This review aims to equip readers with the basic principles of electrochemical 3D printing at the small length scale. By describing the basic principles of electrochemical additive manufacturing technology and using the recent advances in the field, this beginner's guide illustrates how controlling the fundamental phenomena that underpin the print process can be used to vary dimensions, morphology, and microstructure of printed structures.

G-quadruplex-hemin DNAzyme functionalized nanopipettes: Fabrication and sensing application.

Solid-nanopores/nanopipettes have the exquisite ability to reveal the changes in molecular volume due to the advantages of adjustable size, good rigidity and low noise. Herein, a new platform for sensing application was established based on G-quadruplex-hemin DNAzyme (GQH) functionalized gold-coated nanopipettes. In this method, GQH was immobilized on gold-coated nanopipette, which could be used as a catalyst for the reaction of H2O2 with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) to promote the conversion of ABTS to ABTS+ ions inside gold-coated nanopipette, and the change of transmembrane ion current could be monitored in real time. At the optimal conditions, there was a correlation between the ion current and the concentration of H2O2 in a certain range, which could be used for the hydrogen peroxide sensing. The GQH immobilized nanopipette provides a useful platform to investigate enzymatic catalysis in confined environment, which can be used in electrocatalysis, sensing and fundamental electrochemistry.

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection.

A glass capillary-based nanopore (G-nanopore), due to its tapered tip, easy tunability in orifice size, and especially its flexible surface modifications that can be tailored to effectively capture and enhance the ionic current signal of single entities (single molecules, single cells, and single particles), offers a powerful and nanoconfined sensing platform for diverse biological measurements of single cells and single molecules. Compared with other artificial two-dimensional solid-state nanopores, its conical tip and high spatial and temporal resolution characteristics facilitate noninvasive single molecule and selected area (subcellular) single cell detections (e.g., DNA mutations, highly expressed proteins, and small molecule markers that reflect the change characteristics of the tumor), as a small G-nanopore (≤100 nm) does negligible damage to cell functions and cell membrane integrity when inserted through the cell membrane. In this brief review, we summarize the preparation of G-nanopores and discuss the advantages of them as solid-state sensing platforms for single molecule and single cell detection applications as well as for cancer diagnosis and treatment applications. We also describe the current bottlenecks that limit the widespread use of G-nanopores in clinical applications and provide an outlook on future developments. The brief review will provide the reader with a quick survey of this field and facilitate the rapid development of a G-nanopore sensing platform for future tumor diagnosis and personalized medicine based on single-molecule/single-cell bioassay.

An ultrasensitive aptasensor of SARS-CoV-2 N protein based on ion current rectification with nanopipettes.

Since the outbreak of COVID-19 in the world, it has spread rapidly all over the world. Rapid and effective detection methods have been a focus of research. The SARS-CoV-2 N protein (NP) detection methods currently in use focus on specific recognition of antibodies, but the reagents are expensive and difficult to be produced. Here, aptamer-functionalized nanopipettes utilize the unique ion current rectification (ICR) of nanopipette to achieve rapid and highly sensitive detection of trace NP, and can significantly reduce the cost of NP detection. In the presence of NP, the surface charge at the tip of the nanopipette changes, which affects ion transport and changes the degree of rectification. Quantitative detection of NP is achieved through quantitative analysis. Relying on the high sensitivity of nanopipettes to charge fluctuations, this sensor platform achieves excellent sensing performance. The sensor platform exhibited a dynamic working range from 102-106 pg/mL with a detection limit of 73.204 pg/mL, which showed great potential as a tool for rapidly detecting SARS-CoV-2. As parallel and serial testing are widely used in the clinic to avoid missed diagnosis or misdiagnosis, we hope this platform can play a role in controlling the spread and prevention of COVID-19.

Operando Nanoscale Imaging of Electrochemically Induced Strain in a Locally Polarized Pt Grain.

Developing new methods that reveal the structure of electrode materials under polarization is key to constructing robust structure-property relationships. However, many existing methods lack the spatial resolution in structural changes and fidelity to electrochemical operating conditions that are needed to probe catalytically relevant structures. Here, we combine a nanopipette electrochemical cell with three-dimensional X-ray Bragg coherent diffractive imaging to study how strain in a single Pt grain evolves in response to applied potential. During polarization, marked changes in surface strain arise from the Coulombic attraction between the surface charge on the electrode and the electrolyte ions in the electrochemical double layers, while the strain in the bulk of the crystal remains unchanged. The concurrent surface redox reactions have a strong influence on the magnitude and nature of the strain changes under polarization. Our studies provide a powerful blueprint to understand how structural evolution influences electrochemical performance at the nanoscale.

Probing RNA Conformations Using a Polymer-Electrolyte Solid-State Nanopore.

Nanopore systems have emerged as a leading platform for the analysis of biomolecular complexes with single-molecule resolution. The conformation of biomolecules, such as RNA, is highly dependent on the electrolyte composition, but solid-state nanopore systems often require high salt concentration to operate, precluding analysis of macromolecular conformations under physiologically relevant conditions. Here, we report the implementation of a polymer-electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer-electrolyte bath governs the translocation dynamics of the analyte which correlates with the physical properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer-electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiologically relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the co-transcriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer-electrolyte solid-state nanopore system will further enable structural and conformational analyses of individual biomolecules under physiologically relevant conditions.

Translocation of Specific DNA Nanocarrier through an Ultrasmall Nanopipette: Toward Single-Protein-Molecule Detection with Superior Signal-to-Noise Ratio.

The use of functional DNA nanostructures as carriers to ship proteins through solid-state nanopores has recently seen substantial growth in single-protein-molecule detection (SPMD), driven by the potential of this methodology and implementations that it may enable. Ultrasmall nanopores have exhibited obvious advantages in spatiotemporal biological detection due to the appropriate nanoconfined spaces and unique properties. Herein, a 6.8 nm DNA tetrahedron (TDN) with a target-specific DNA aptamer (TDN-apt) was engineered to carry the representative target of acetylcholinesterase (AChE) through an ultrasmall nanopipet with a 30 nm orifice, underpinning the advanced SPMD of AChE with good performance in terms of high selectivity, low detection limit (0.1 fM), and especially superior signal-to-noise ratio (SNR). The kinetic interaction between TDN-apt and AChE was studied and the practical applicability of the as-developed SPMD toward real samples was validated using serum samples from patients with Alzheimer's disease. This work not only presented a feasible SPMD solution toward low-abundance proteins in complex samples and but also was envisioned to inspire more interest in the design and implementation of synergized DNA nanostructure-ultrasmall nanopore systems for future SPMD development.

Gold nanoparticle functionalized nanopipette sensors for electrochemical paraquat detection.

A sensitive nanopipette sensor is established through a unique design of host-guest recognition, which could be further enhanced by the introduction of gold nanoparticles (Au NPs). Generally, the nanopipette is conjugated with caboxylatopillar[5]arenes (CP[5]) or carboxylated leaning pillar[6]arene (CLP[6]) to generate recognition sites. After the addition of pesticide molecules, they would be captured by CP[5] (or CLP[6]), resulting in a significant electronegativity change on the nanopipette's inner surface, which could be determined by the ionic current change. The CP[5]-modified nanopipette exhibited reliable selectivity for paraquat, while the CLP[6]-modified nanopipette showed an ability of detection for both paraquat and diquat. The addition of Au NPs improved the selectivity and sensitivity of the CP[5]-Au NP-modified nanopipette for paraquat sensing. After optimization by lowering the size of the Au NPs, CP[5]-Au NPs (3 nm)-modified nanopipettes achieved lower detection limits of 0.034 nM for paraquat. Furthermore, in real sample analysis, this sensor demonstrates exceptional sensitivity and selectivity. This study provides a new strategy to develop nanopipette sensors for practical small molecule detection. The gold nanoparticles enhanced quartz nanopipette sensor based on host-guest interaction was firstly established, which could achieve an excellent limit of detection of 3.4 × 10-11 mol/L for paraquat.

Pressure-Regulated Single-Entity Electrochemistry Inside Carbon Nanopipettes.

Conductive nanopipettes have been widely used as a multifunctional platform for emerging sensing applications in small spaces, although the electrochemical processes involved are not well controlled and fully quantified. Herein, we use an external pressure to precisely control the solution volume and regulate the electrochemical signals in carbon nanopipettes. In addition to polarizing the redox concentration profile, the pressure is found to generate a convective flow to control the transport processes of redox molecules and nanoparticles as well, and their quantitative correlation is established by a numerical simulation. The elucidated pressure-regulated electrochemistry in conductive nanopipettes would reveal the fundamental charge transport processes at the nanoscale and promote better usage of conductive nanopipettes for delivery and sensing applications in single-cell analysis.