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.

Monitoring nanobubble nucleation at early-stage within a sub-9 nm solid-state nanopore.

Nanobubble nucleation study is important for understanding the dynamic behavior of nanobubble growth, which is instructive for the nanobubble applications. Benefiting from nanopore fabrication, herein, we fabricated a sub-9 nm SiNX nanopore with the comparable size to nanobubbles at early-stage. The confined nanopore interface serves as a generator for producing nanobubbles by the chemical reaction between NaBH4 and H2 O and as an ultra-sensitive sensor for monitoring the H2 nanobubble nucleation process. By carrying out the NaBH4 concentration-dependent experiments, we found the life-time of nanobubbles decreased 250 times and the frequency of nanobubble generation increased 38 times with the NaBH4 concentration increasing from 6 to 100 mM. The long-time equilibrium between gas molecules inward flux and outward flux could prolong the life-time of nanobubbles to hundreds of milliseconds at low NaBH4 concentration. The raw current trace depicted that the transient accumulation and dissolution of cavity occurred during all the life-time of nanobubbles. Therefore, the sub-9 nm SiNX nanopore shows a strong ability for real-time monitoring the nanobubble nucleation at early-stage with high temporal and spatial resolution. This work provides a guide to study the dynamic and stochastic characteristics of nanobubbles.

Measuring temperature effects on nanobubble nucleation via a solid-state nanopore.

In this study, we designed SiNX 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 SiNX 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 SiNX nanopore. Our method provides an efficient analytical tool for revealing the temperature-dependent nanobubble nucleation, which further benefits the fundamental understanding of nanobubble nucleation.

Detection of structured single-strand DNA via solid-state nanopore.

Nanopore is a single-molecule analysis method which also employed electrophoresis has achieved promising single-molecule detections. In this study, we designed two kinds of confined spaces by fabricating solid-state nanopores with desirable diameters to study the structured single-strand DNA of C-rich quadruplex. For the nanopore whose diameter is larger than the quadruplex size, the DNA molecule could directly translocate through the nanopore with extremely high speed. For the nanopore whose diameter is smaller than the quadruplex size, DNA molecule which is captured by nanopore could return to the solution without translocation or unzip the quadruplex structure into single-strand and then pass the nanopore. This study certifies that choosing a suitable sensing interface is the vital importance of observing detailed single-molecule information. The solid-state nanopores hold the great potential to study the structural dynamics of quadruplex DNA molecule.

Measuring a frequency spectrum for single-molecule interactions with a confined nanopore.

Nanopore analysis is a powerful technique for single molecule analysis by virtue of its electrochemically confined effects. As a single molecule translocates through the nanopore, the featured ionic current pattern on the time scale contains single molecule characteristics including volume, charge, and conformational properties. Although the characteristics of a single molecule in a nanopore have been written to the featured ionic current, extracting the dynamic information from a complex current trace is still a big challenge. Here, we present an applicable nanopore analysis method employing the Hilbert-Huang Transform (HHT) to study the vibrational features and interactions of a single molecule during the dynamic translocation process through the confined space of a nanopore. The HHT method is specially developed for analyzing nonlinear and non-stationary data that is highly compatible with nanopore data with a high frequency resolution. To provide proof-of-concept, we applied HHT to measure the frequency response for the wild-type (WT) aerolysin and mutant K238E aerolysin nanopores with and without the presence of poly(dA)4, respectively. The energy-frequency-time distribution spectra demonstrate that the biological nanopore contributes greatly to the characteristics of the high frequency component (>2 kHz) in the current recording. Our results suggest that poly(dA)4 undergoes relatively more consistent and confined interactions with K238E than WT, leading to a prolonging of the duration time. Therefore, the characteristics in frequency analysis could be regarded as an "single-molecule ionic spectrum" inside the nanopore, which encodes the detailed behaviours of single-molecule weak interactions.

In Situ Oxygen Generation via a Platinum-Based Wireless Nanopore Electrode for Single-Cell Manipulation.

Oxygen production within human cells plays a critical role in cellular metabolism and is implicated in various diseases, including cancer. Investigating cellular heterogeneity under oxygen stimulation is crucial for elucidating disease mechanisms and advancing early therapeutic design. In this study, the platinum-based wireless nanopore electrode (WNE) with a diameter of ≈200 nm is employed as a powerful tool to produce oxygen molecules near the cell nucleus. The oxygen production can be quantitatively controlled by adjusting the applied voltage. Through delivering oxygen near the cancer cell nucleus, this technique shows the capacity to alleviate the hypoxia microenvironment, a key factor in chemotherapy resistance. Furthermore, by modulating oxygen levels within individual living cells and delivering chemotherapeutic agents to the cancer cell nucleus, this approach offers significant potential for single-cell manipulation and the investigation of cellular heterogeneity under oxygen stimulation.

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.

Snapshotting the transient conformations and tracing the multiple pathways of single peptide folding using a solid-state nanopore.

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 ß-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.

Revisiting the Origin of Nanopore Current Blockage for Volume Difference Sensing at the Atomic Level.

Changes in the nanopore ionic current during entry of a target molecule underlie the sensing capability and dominate the intensity and extent of applications of the nanopore approach. The volume exclusion model has been proposed and corrected to describe the nanopore current blockage. However, increasing evidence shows nonconformity with this model, suggesting that the ionic current within a nanopore should be entirely reconsidered. Here, we revisit the origin of nanopore current blockage from a theoretical perspective and propose that the noncovalent interactions between a nanopore and a target molecule affect the conductance of the solution inside the nanopore, leading to enhanced current blockage. Moreover, by considering the example of an aerolysin nanopore discriminating the cytosine DNA and methylcytosine DNA that differ by a single methyl group, we completely demonstrate, by nanopore experiments and molecular dynamics simulations, the essential nature of this noncovalent interaction for discrimination. Our conductance model suggests multiplicative effects of both volume exclusion and noncovalent interaction on the current blockage and provides a new strategy to achieve volume difference sensing at the atomic level with highly specific current events, which would promote the nanopore protein sequencing and its applications in real-life systems.

An advanced optical-electrochemical nanopore measurement system for single-molecule analysis.

Nanopore measurement has advanced in single-molecule analysis by providing a transient time and confined space window that only allows one interested molecule to exist. By optimization and integration of the electrical and optical analysis strategies in this transient window, the acquisition of comprehensive information could be achieved to resolve the intrinsic properties and heterogeneity of a single molecule. In this work, we present a roadmap to build a unified optical and electrochemical synchronous measurement platform for the research of a single molecule. We design a low-cost ultralow-current amplifier with low noise and high-bandwidth to measure the ionic current events as a single molecule translocates through a nanopore and combine a multi-functional optical system to implement the acquisition of the fluorescence, scattering spectrum, and photocurrent intensity of single molecule events in a nanopore confined space. Our system is a unified and unique platform for the protein nanopore, the solid-state nanopore, and the glass capillary nanopore, which has advantages in the comprehensive research of nanopore single-molecule techniques.

Detection of Single Proteins with a General Nanopore Sensor.

Single protein sensing based on solid-state nanopores is promising but challenging, because the fast translocation velocity of a protein is beyond the bandwidth of nanopore instruments. To decelerate the translocation speed, here, we employed a common protein cross-link interaction to achieve a general and robust nanopore sensing platform for single-molecule detection of protein. Benefiting from the EDC/NHS coupling interaction between nanopore and proteins, a 10-fold decrease in speed has been achieved. The clearly distinguishable current signatures further reveal that the anisotropic translocation of a protein, which are horizontal, vertical, and flipping transit inside nanopore confinement. This strategy provides a general platform for rapid detection of proteins as well as exploring fundamental protein dynamics at the single-molecule level.

A lithium-ion-active aerolysin nanopore for effectively trapping long single-stranded DNA.

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.

In situ and real-time ToF-SIMS analysis of light-induced chemical changes in perovskite CH3NH3PbI3.

A hybrid light/ToF-SIMS system was used to analyze the dynamic chemical changes of perovskite CH3NH3PbI3 films under light illumination, in order to reveal the mechanism of light instability for perovskite materials. Real-time material degradation and quasi-reversible iodine migration were successfully observed.

Dynamics of a Molecular Plug Docked onto a Solid-State Nanopore.

Docking of a protein-DNA complex onto a nanopore can provide ample observation time, and has enabled collection of analytic applications of biological nanopores, including DNA sequencing. However, the application of the same principle to solid-state nanopores is tempered by poor understanding of the docking process. Here, we elucidate the behavior of individual protein-DNA complexes docked onto a solid-state nanopore by monitoring the nanopore ionic current. Repeat docking of monovalent streptavidin-DNA complexes is found to produce ionic current blockades that fluctuate between discrete levels. We elucidate the roles of the protein plug and the DNA tether in the docking process, finding the docking configurations to determine the multitude of the current blockade levels, whereas the frequency of the current level switching is determined by the interactions between the molecules and the solid-state membrane. Finally, we prove the feasibility of using the nanopore docking principle for single-molecule sensing using solid-state nanopores by detecting conformational changes of a tethered DNA molecule from a random coil to an i-motif state.

Structural stability of the photo-responsive DNA duplexes containing one azobenzene via a confined pore.

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.

Direct sensing of cancer biomarkers in clinical samples with a designed nanopore.

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.

Characterization of DNA duplex unzipping through a sub-2 nm solid-state nanopore.

Herein, the unzipping and translocation of DNA duplexes through a sub-2 nm silicon nitride (SiNx) 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.

A precise pointing nanopipette for single-cell imaging via electroosmotic injection.

The precise transportation of fluorescent probes to the designated location in living cells is still a challenge. Here, we present a new addition to nanopipettes as a powerful tool to deliver fluorescent molecules to a given place in a single cell by electroosmotic flow, indicating favorable potential for further application in single-cell imaging.