Dual Friction Force/Fluorescence Microscopy.

Friction force microscopy (FFM) is a mode of atomic force microscopy (AFM) that quantifies both normal and horizontal forces against substrates. Recent improvement in its accuracy at nanonewton ranges and the possibility of combining AFM with fluorescence microscopy enabled the simultaneous characterization by FFM and fluorescence microscopy. This Tutorial describes the operation principle of the dual friction force/fluorescence microscopy setup and highlights its emerging applications in mechanochromic materials.

Dual Nanofriction Force Microscopy/Fluorescence Microscopy Imaging Reveals the Enhanced Force Sensitivity of Polydiacetylene by pH and NaCl.

Polydiacetylene (PDA) is a popular mechanochromic material often used in biosensing. The effect of its headgroup-headgroup interactions on thermochromism such as pH or salt concentration dependency has been extensively studied before; however, their effect on mechanochromism at the nanoscale is left unstudied. In this work, nanofriction force microscopy and fluorescence microscopy were combined to study the effect of pH and ionic strength on the polydiacetylene (PDA) force sensitivity at the nanoscale. We found that the increase in pH from 5.7 to 8.2 caused an 8-fold enhancement in force sensitivity. The elevation of NaCl concentration from 10 to 200 mM also made the PDA 5 times more force-sensitive. These results suggest that the PDA force sensitivity at the nanoscale can be conveniently enhanced by "pre-stimulation" with pH or ionic strength.

Role of Lipid Composition in the Antimicrobial Peptide Double Cooperative Effect.

The antimicrobial peptide double cooperative effect, where the mixture of two major antimicrobial peptides LL-37 and HNP1 kills bacteria more efficiently while minimizing the host damage by suppressing mammalian cell membrane lysis, has garnered attention due to its potential applications toward efficient and safe antibiotics. However, its mechanism is completely unknown. In this work, we report that the double cooperative effect can be partially recapitulated in synthetic lipid systems just by varying the lipid composition between eukaryotic and Escherichia coli membranes. Although real cell membranes are so much more complex than just lipids, including, e.g., membrane proteins and polysaccharides, our data implicates that one of the main driving forces of the double cooperative effect is a simple lipid-peptide interaction.

Quantitative and Anisotropic Mechanochromism of Polydiacetylene at Nanoscale.

Quantitative and anisotropic mechanochromism of polydiacetylene over nanoscale distances remains unaddressed even after 50 years of extensive research. This is because its anisotropic structure on substrates necessitates the application of both vertical and lateral forces (shear forces) to characterize it, whereas atomic force microscopy, which is the usual technique used to investigate nanoscale forces, is only capable of quantifying vertical forces. In this study, we address this lacuna by utilizing quantitative friction force microscopy that measures lateral forces. Our data confirm that polydiacetylene reacts only to lateral forces, F//, and disprove the previously claimed hypothesis that the edges of the polymer crystals exhibit higher force sensitivity than the rest of the crystal. In addition, we report a correlation between mechanochromism and thermochromism, which can be attributed to the fact that both work and heat are different means of providing the same transition energy.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Cooperative Function of LL-37 and HNP1 Protects Mammalian Cell Membranes from Lysis.

LL-37, cleaved from human cathelicidin, and human neutrophil peptide-1 (HNP1) from the defensin family are antimicrobial peptides that are occasionally co-released from neutrophils, which synergistically kill bacteria. We report that this couple presents another type of cooperativity against host eukaryotic cells, in which they antagonistically minimize cytotoxicity by protecting membranes from lysis. Our results describe the potential of the LL-37/HNP1 cooperativity that switches from membrane-destructive to membrane-protective functions, depending on whether the target is an enemy or a host.

Anion Transport with Pnictogen Bonds in Direct Comparison with Chalcogen and Halogen Bonds.

In this Communication, we introduce transmembrane anion transport with pnictogen-bonding compounds and compare their characteristics with chalcogen- and halogen-bonding analogues. Tellurium-centered chalcogen bonds are at least as active as antimony-centered pnictogen bonds, whereas iodine-centered halogen bonds are 3 orders of magnitude less active. Irregular voltage-dependent single-channel currents, high gating charges, and efficient dye leakage support for the formation of bulky, membrane-disruptive supramolecular amphiphiles due to "too strong" binding of anions to tris(perfluorophenyl)stibanes. In contrast, the chalcogen-bonding bis(perfluorophenyl)tellanes do not cause leakage and excel as carriers with nanomolar activity, with P(Cl/Na) = 10.4 for anion/cation selectivity and P(Cl/NO3) = 4.5 for anion selectivity. The selectivities are lower with pnictogen-bonding carriers because their membrane-disturbing 3D structure also affects weaker binders ( P(Cl/Na) = 2.1, P(Cl/NO3) = 2.5). Their 2D structure, directionality, hydrophobicity, and support from proximal anion-π interactions are suggested to contribute to the unique power of chalcogen bonds to transport anions across lipid bilayer membranes.

Black Lipid Membranes: Challenges in Simultaneous Quantitative Characterization by Electrophysiology and Fluorescence Microscopy.

Horizontal black lipid membranes (BLMs) enable optical microscopy to be combined with the electrophysiological measurements for studying ion channels, peptide pores, and ionophores. However, a careful literature review reveals that simultaneous fluorescence and electrical recordings in horizontal BLMs have been rarely reported for an unclear reason, whereas many works employ bright-field microscopy instead of fluorescence microscopy or perform fluorescence imaging and electrical measurements one after another separately without truly exploiting the advantage of the combined setup. In this work, the major causes related to the simultaneous electrical and fluorescence recordings in horizontal BLMs are identified, and several solutions to counteract the issue are also proposed.

Mechanosensitivity of polydiacetylene with a phosphocholine headgroup.

We have demonstrated colorimetric and fluorescence detection of a peptide, melittin, based on polydiacetylene (PDA) made of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC). The PDA used in this work has a phosphocholine headgroup, which mimics peptide-cell membrane interactions better than the conventional PDAs with carboxyl headgroups. The dose curve (colorimetric response vs. melittin concentration) showed a half maximum response at the melittin concentration of 0.1 mg ml-1, which is similar to that reported in traditional PDA assays. It suggests that the replacement of the headgroup was achieved without sacrificing the sensitivity. From the dose curve, the Hill coefficient was extracted as αHill = 2.1. The value is in agreement with the results from previous melittin studies with phospholipids, which reflects the benefit of having a biologically relevant headgroup. In addition, we found an unexpectedly slow spectral change when DC(8,9)PC-PDA was incubated with melittin. The origin of the time-dependent signal was studied by combining UV/VIS spectroscopy, fluorescence spectroscopy and dynamic light scattering.

Gold Nanowire Fabrication with Surface-Attached Lipid Nanotube Templates.

A high-throughput approach to fabricate gold nanowires on surfaces with a lipid nanotube template is demonstrated. Streptavidin-coated gold nanoparticles are attached to the biotin-tagged lipid nanotubes. After the chemical fixation, the samples are dried and treated with oxygen plasma to remove the organic template and connect the particles. The created nanowires are characterized by cryo-transmission electron microscopy, atomic force microscopy, and electrical measurements.

Self-assembled Lipid Structures as Model Systems for Studying Electrical and Mechanical Properties of Cell Membranes.

This mini review summarizes our recent works in the development of electrical and mechanical characterization tools for cell membranes. These research topics require the application of physical and chemical characterization tools to biological systems, thus are very interdisciplinary.

Freely drawn single lipid nanotube patterns.

LNTs are unique 3D structures made only of safe and abundant biomaterials by self-assembly. The current bottleneck for developing applications using LNTs is the lack of an easy technique to pattern them on substrates. We report a method to free-draw single lipid nanotube (LNT) patterns in any shape on surfaces with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) that takes an inverted hexagonal (HII) phase. We used pre-self-assembled LNTs or HII lipid blocks as a lipid reservoir from which new LNTs were pulled by applying a point load with a micromanipulator. The extreme simplicity of our technique originates from the fundamental nature of DOPE lipids that prefer a HII phase, while all the conventional approaches use PC lipids that form a lamellar phase. By adjusting the surface properties with polyelectrolyte multilayers, the created single LNT objects are able to remain adhered to the surface for over a week. Importantly, it could be shown that two vesicles loaded with caged fluorescent molecules were able to fuse well with a LNT, enabling diffusive transport of uncaged fluorescent molecules from one vesicle to the other.

Mechanical Properties of Polydiacetylene Multilayers Studied by AFM Force Spectroscopy.

The blue-to-red chromatic phase transition of polydiacetylene (PDA) is accompanied by the twist and rearrangement of its side chains, which results in shortening of the conjugation length in the backbone. However, how these morphological changes affect its mechanical properties remains elusive. In this work, force spectroscopy mapping by atomic force microscopy was employed to quantify mechanical parameters of PDA thin films such as breakthrough force and Young's modulus at the monomer, blue, and red phases during the chromatic transition. We found that the breakthrough force increased by 113% and Young's modulus decreased by 21% during the blue-to-red transition, highlighting that the subtle change in the side-chain configuration has a dramatic impact on its mechanical properties.

Green tea polyphenol EGCG induces lipid-raft clustering and apoptotic cell death by activating protein kinase Cδ and acid sphingomyelinase through a 67 kDa laminin receptor in multiple myeloma cells.

EGCG [(-)-epigallocatechin-3-O-gallate], the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In the present study, we show that EGCG-induced apoptotic activity is attributed to a lipid-raft clustering mediated through 67LR (67 kDa laminin receptor) that is significantly elevated in MM (multiple myeloma) cells relative to normal peripheral blood mononuclear cells, and that aSMase (acid sphingomyelinase) is critical for the lipid-raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and PKCδ (protein kinase Cδ) phosphorylation at Ser664, which was necessary for aSMase/ceramide signalling via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell-death pathway triggered by EGCG for the specific killing of MM cells.

Colorimetric response in polydiacetylene at the single domain level using hyperspectral microscopy.

The structural variance of polydiacetylene (PDA) at the nanoscale level, even under the same fabrication conditions, is one of the origins of its poor reproducibility in chemo/biosensing. In this work, we present a spatial map of such structural distributions within a single crystal by taking advantage of the recent development of hyperspectral microscopy at visible wavelengths. Hyperspectral microscopy provides the distribution of absorption spectra at the spatial resolution of standard optical microscopy. By tracking the blue-to-red transition via this technique, we found that heat or pH stimulation leaves a unique pattern in the transition pathways.

Development of a desktop mask charger.

Polypropylene mesh, integrated in N95 respirators and surgical masks that are widely used in the current crisis of COVID-19, filters aerosols via electrostatics in addition to the physical block. However, any contact to water such as storage under high humidity, exposure to exhaling breath, and washing in water removes its charges and thus compromises its filtering efficiency. We developed a desk top device based on a Cockcroft-Walton's voltage multiplier that can restore the electrostatic charge of surgical masks within 1 min and recover the filtering efficiency of the polypropylene mesh from 87% to 97%. The device is easy to operate and safe, thus may be applied for the reuse of surgical masks towards reducing the plastic wastes.

Electrochemical plasmonic sensors.

The enormous progress of nanotechnology during the last decade has made it possible to fabricate a great variety of nanostructures. On the nanoscale, metals exhibit special electrical and optical properties, which can be utilized for novel applications. In particular, plasmonic sensors including both the established technique of surface plasmon resonance and more recent nanoplasmonic sensors, have recently attracted much attention. However, some of the simplest and most successful sensors, such as the glucose biosensor, are based on electrical readout. In this review we describe the implementation of electrochemistry with plasmonic nanostructures for combined electrical and optical signal transduction. We highlight results from different types of metallic nanostructures such as nanoparticles, nanowires, nanoholes or simply films of nanoscale thickness. We briefly give an overview of their optical properties and discuss implementation of electrochemical methods. In particular, we review studies on how electrochemical potentials influence the plasmon resonances in different nanostructures, as this type of fundamental understanding is necessary for successful combination of the methods. Although several combined platforms exist, many are not yet in use as sensors partly because of the complicated effects from electrochemical potentials on plasmon resonances. Yet, there are clearly promising aspects of these sensor combinations and we conclude this review by discussing the advantages of synchronized electrical and optical readout, illustrating the versatility of these technologies.

Recent progress in polydiacetylene mechanochromism.

Polydiacetylenes (PDAs) are a family of mechanochromic polymers that change color from blue to red and emit fluorescence when exposed to external stimuli, making them extremely popular materials in biosensing. Although several informative reviews on PDA biosensing have been reported in the last few years, their mechanochromism, where external forces induce the color transition, has not been reviewed for a long time. This mini review summarizes recent progress in PDA mechanochromism, with a special focus on the quantitative and nanoscopic data that have emerged in recent years.

Techniques for recording reconstituted ion channels.

This review describes and discusses techniques useful for monitoring the activity of protein ion channels in vitro. In the first section the biological importance and the classification of ion channels are outlined in order to justify the strong motivation for dealing with this important class of membrane proteins. The expression, reconstitution and integration of recombinant proteins into lipid bilayers are crucial steps to obtain consistent data when working with ion channels. In the second section recording techniques used in research are presented. Since this review focuses on analytical systems bearing reconstituted ion channels the industrial most important patch-clamp techniques of cells are only briefly mentioned. In section three, artificial systems developed in the last decades are described while the emerging technologies using nanostructured supports or microfluidic systems are presented in section four. Finally, the remaining challenges of membrane protein analysis and its potential applications are briefly outlined.