Jin He

Multi-parameter single-cell analysis and imaging are crucial for understanding fundamental cellular behavior and properties. Scanning ion conductance microscopy (SICM) is a powerful scanning probe microscope technique for studying the physical characteristics of individual cells under physiological conditions using a glass nanopipette as the scanning probe. Previously, SICM has been successfully developed for topography imaging, surface charge/potential mapping, and stiffness mapping of adherent cells. In this report, we demonstrated the integration of these three imaging modes to acquire three types of images simultaneously after optimizing the imaging conditions and data processing. Therefore, the morphology, electrical, and mechanical properties of cell surfaces can be investigated with a submicrometer spatial resolution. We successfully validated the method using various fixed and live cells, including HEK, CAL 27, and MoVas cells.

Antimicrobial peptides (AMPs), key components of innate immunity, kill bacteria by forming structured pores on their lipid membranes. Understanding how AMPs form pores is of fundamental interest and holds significant promise for various biomedical and biotechnological applications. However, the underlying molecular mechanism of pore formation remains elusive, and directly observing the dynamic pore formation process poses significant measurement challenges. Here, we report a method to probe the dynamic pore formation process driven by AMPs. This approach involves forming a stable suspended artificial planar phospholipid bilayer at the orifice of a glass nanopipette and monitoring the leakage ionic current across the membrane. By using nanoscale-sized membranes, we enhanced their stability, enabling longer time-resolved ionic current measurements with a high signal-to-noise ratio. We also added and optimized the cholesterol content in the diphytanoylphosphatidyl choline (DPhPC) bilayer to 20 mol %, which improved both the yield and stability of the membrane. We first validated this method by investigating voltage-controlled electroporation and subsequently applied it to study the pore-forming process of a model AMP, alamethicin. The interaction between alamethicin and the membrane, as well as its pore formation process, could be effectively controlled by the applied voltage and repeatedly probed. Analysis of the rich single-channel conductance changes revealed the alamethicin pore formation process in detail. The pore formation dynamics and the resulting pore structures were found to be sensitive to the applied voltage, alamethicin concentration, lipid composition, ionic strength, and cation size.

Probing intermolecular interactions between biomolecules (such as proteins and nucleic acids) or bioactive molecules (such as drug molecules) are important for both fundamental studies of living systems and various applications. Challenges remain for current methods in probing label‐free small (bio)molecules involved interactions at the single‐molecule level under physiological conditions. Nanopipette based nanopore sensing offers a powerful and cost‐effective approach for label‐free single‐molecule detection and analysis in solution. In a recent study, we have greatly improved the sensitivity of nanopipettes for the detection of small molecules when the tip is self‐crowded with molecules. Here, we applied this approach to study strong interactions involving small molecules, using streptavidin‐biotin interaction as a model system. After mixing streptavidin and biotin, unique multipeak ionic current spikes, containing one large peak and up to four small peaks, appeared. The relative peak height of these small peaks increased when larger biotin conjugates were used. Therefore, these multipeak current spikes reveal the stoichiometry and surface contour of the formed complex, highlighting the extremely high spatial resolution of our method. We further investigated how external factors affect the stoichiometry of the complex. The average number of small peaks within each multipeak spike increases with both higher crowding levels at the nanopipette tip (induced by greater applied voltage) and higher biotin‐to‐streptavidin concentration ratios in the premixed solution. This study demonstrates the promising potential of this nanopipette approach for investigating interactions involving label‐free small (bio)molecules in crowded environments and for high‐resolution structural characterization of the resulting complexes.

Recent innovations in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs) have unlocked a viable path to creating in vitro cardiac models. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) remain immature, leading many in the field to explore approaches to enhance cell and tissue maturation. Here, we systematically analyzed 300 studies using hiPSC-CM models to determine common fabrication, maturation and assessment techniques used to evaluate cardiomyocyte functionality and maturity and compiled the data into an open-access database. Based on this analysis, we present the diversity of, and current trends in, in vitro models and highlight the most common and promising practices for functional assessments. We further analyzed outputs spanning structural maturity, contractile function, electrophysiology and gene expression and note field-wide improvements over time. Finally, we discuss opportunities to collectively pursue the shared goal of hiPSC-CM model development, maturation and assessment that we believe are critical for engineering mature cardiac tissue.

The lantibiotic pore-forming peptide nisin is a promising candidate in the fight against multidrug-resistant bacteria due to its unique structure, which allows it to disrupt bacteria in two distinct ways─Lipid II trafficking and transmembrane pore formation. However, exactly how nisin and Lipid II assemble into oligomeric pore structures in the bacterial membrane is not known. Spontaneous peptide assembly into pores is difficult to observe in even the very long-time scale molecular dynamics (MD) simulations. In this study, we adopted an MD-guided modeling approach to investigate the nisin-nisin and nisin-Lipid II associations in the membrane environment. Through extensive microsecond-time scale all-atom MD simulations, we established that nisin monomers dimerize by forming β-sheets in a POPE:POPG lipid bilayer and oligomerize further to form stable transmembrane channels. We determined that these nisin dimers use Lipid II as a dimer interface to incur enhanced stability. Our results provide a clearer understanding of the self-assembly of nisin monomers within the membrane and insights into the role of Lipid II in the structural integrity of oligomeric structures.

In recent decades, the field of nanoscale electrochemistry has advanced rapidly in ultrasensitive electroanalysis at the single-particle level. The glass/quartz nanopipette has emerged as an economical, readily accessible, versatile, and powerful tool for single-entity electrochemistry. We have integrated nanoelectrode and nanopore based techniques using the so-called multifunctional nanopipette, which includes both nanopore and nanoelectrode at the orifice of the nanopipette tip. The multifunctional nanopipette has been successfully used with both amperometric and potentiometric methods for protein and small biomolecule analysis and detection at the single-molecule level. The nanopipette has also been used for intracellular delivery and counting, as well as nanoelectrophyiology of excitable cells.

Engineered cardiac tissues show potential for regenerative therapy in ischemic heart disease. Yet, selection of soft biomaterials for scaffold manufacturing is primarily influenced by empirical and compositional factors, raising concerns about arrhythmic risks due to poor electrophysiological integration. Addressing this, we developed multiscale hybrid myocardial patches mimicking native myocardium's structural and biomechanical attributes, utilizing 3D printing and electrospinning techniques. We compared three patch types: pure silicone and silicone-poly(lactic-co-glycolic acid) (PLGA) with random (S-PLGA-R) and aligned (S-PLGA-A) fibers. S-PLGA-A patches with fiber orientation angles of 95-115° are achieved by applying a secondary electrical field using two parallel aluminum enhancers. With bulk and localized moduli of 350-750 and 13-20 kPa resembling the native myocardium, S-PLGA-A patches demonstrate a sarcomere length of 2.1 ± 0.2 μm, ≥50% higher strain motions and diastolic phase, and a 50-70% slower rise of calcium handling compared to the other two patches. This enhanced maturation and improved synchronization phenomena are attributed to efficient force transmission and reduced stress concentration due to mechanical similarity and linear propagation of electrical signals. This study presents a promising strategy for advancing regenerative cardiac therapies by harnessing the capabilities of 3D printing and electrospinning, providing a proof-of-concept for their effectiveness.

Gold nanorod embedded extracellular matrix significantly enhanced the maturation and lifetime of human engineered cardiac tissue.

Plasmonic molecular junctions can harvest visible light and effectively catalyze chemical reactions. The strong light field concentrated in the plasmonic junction also enables the application of surface enhanced Raman spectroscopy (SERS) to probe the catalyzed chemical reactions in situ and in real time down to single-molecule resolution. The benzyl radical produced from the aromatic methyl group through the dehydrogenation reaction is an important precursor for a large variety of reactions. Here, we used time-resolved SERS to conduct a mechanistic study of the plasmon-driven dehydrogenation reaction of the aromatic methyl group under ambient conditions under the illumination of red light on the apex of a gold nanoelectrode. Transient spectral changes with intensity bursts are frequently observed. Based on density functional theory and picocavity based local electric field enhancement calculations, they result from the plasmon mediated dehydrogenation reaction of aromatic methyl groups. The dehydrogenation reaction produces a benzyl radical, which is consequently converted to a benzyl anion. The benzyl anion is stabilized through strong interactions with gold, leading to the formation of dynamic gold adatoms and picocavities. In addition to the benzyl anion, we found spectral evidence that the benzyl radical generates dimers through a self-reaction. Furthermore, we demonstrated that the dehydrogenation reaction could be facially modulated by changing the electrode potential, which is attributed to the modulated inductive effect.

Glass nanopipettes have gained widespread use as a versatile single-entity detector in chemical and biological sensing, analysis, and imaging. Its advantages include low cost, easy accessibility, simplicity of use, and high versatility. However, conventional nanopipettes based on the volume exclusion mechanism have limitations in detecting small biomolecules due to their small volume and high mobility in aqueous solution. To overcome this challenge, we have employed a novel approach by capitalizing on the strong nanoconfinement effect of nanopipettes. This is achieved by utilizing both the hard confinement provided by the long taper nanopipette tip at the cis side and the soft confinement offered by the hydrogel at the trans side. Through this approach, we have effectively slowed down the exit motion of small molecules, allowing us to enrich and jam them at the nanopipette tip. Consequently, we have achieved high throughput detection of small biomolecules with sizes as small as 1 nm, including nucleoside triphosphates, short peptides, and small proteins with excellent signal-to-noise ratios. Furthermore, molecular complex formation through specific intermolecular interactions, such as hydrogen bonding between closely spaced nucleotides in the jam-packed nanopipette tip, has been detected based on the unique ionic current changes.