Biomimetic ion channels formation by emulsion based on chemically modified cyclodextrin nanotubes.

Biomimetic ion channels can be made to display the high sensitivity of natural protein nanopores and to develop new properties as a function of the material used. How to design the best future biomimetic channels? The main challenges are to control their sensitivity, as well as their syntheses, chemical modifications, insertion and lifetime in a lipid membrane. To address these challenges, we have recently designed short cyclodextrin nanotubes characterized by mass spectrometry and high-resolution transmission electron microscopy. They form non-permanent ion channels in lipid bilayers. Here we show how to improve the nanotube insertion in order to limit multiple insertions, how to stabilize biomimetic channels into the membrane, and how to understand the ion dynamics in confined medium scale.

From current trace to the understanding of confined media.

Nanopores constitute devices for the sensing of nano-objects such as ions, polymer chains, proteins or nanoparticles. We describe what information we can extract from the current trace. We consider the entrance of polydisperse chains into the nanopore, which leads to a conductance drop. We describe the detection of these current blockades according to their shape. Finally, we explain how data analysis can be used to enhance our understanding of physical processes in confined media.

Dynamics of a polyelectrolyte through aerolysin channel as a function of applied voltage and concentration⋆.

We describe the behaviour of a polyelectrolyte in confined geometry. The transport of a polyelectrolyte, dextran sulfate, through a recombinant protein channel, aerolysin, inserted into a planar lipid bilayer is studied as a function of applied voltage and polyelectrolyte concentration and chain length. The aerolysin pore has a weak geometry asymmetry, a high number of charged residues and the polyelectrolyte is strongly negatively charged. The resulting current blockades were characterized by short and long dwelling times. Their frequency varies exponentially as a function of applied voltage and linearly as a function of polyelectrolyte concentration. The long blockade duration decreases exponentially when the electrical force increases. The ratio of the population of short events to the one of long events decreases when the applied voltage increases and displays an exponential variation. The long residence time increases with the polyelectrolyte chain length. We measure a reduction of the effective charge of the polyelectrolyte at the pore entry and inside the channel. For a fixed applied voltage, + / - 100 mV, at both sides of the protein pore entrance, the events frequency is similar as a function of dextran sulfate concentration. The mean blockade durations are independent of polyelectrolyte concentration and are similar for both entrances of the pore and remain constant as a function of the electrical force.

Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications.

Biomimetic membrane channels offer a great potential for fundamental studies and applications. Here, we report the fabrication and characterization of short cyclodextrin nanotubes, their insertion into membranes, and cytotoxicity assay. Mass spectrometry and high-resolution transmission electron microscopy were used to confirm the synthesis pathway leading to the formation of short nanotubes and to describe their structural parameters in terms of length, diameter, and number of cyclodextrins. Our results show the control of the number of cyclodextrins threaded on the polyrotaxane leading to nanotube synthesis. Structural parameters obtained by electron microscopy are consistent with the distribution of the number of cyclodextrins evaluated by mass spectrometry from the initial polymer distribution. An electrophysiological study at single molecule level demonstrates the ion channel formation into lipid bilayers, and the energy penalty for the entry of ions into the confined nanotube. In the presence of nanotubes, the cell physiology is not altered.

Temperature Effect on Ionic Current and ssDNA Transport through Nanopores.

We have investigated the role of electrostatic interactions in the transport of nucleic acids and ions through nanopores. The passage of DNA through nanopores has so far been conjectured to involve a free-energy barrier for entry, followed by a downhill translocation where the driving voltage accelerates the polymer. We have tested the validity of this conjecture by using two toxins, α-hemolysin and aerolysin, which differ in their shape, size, and charge. The characteristic timescales in each toxin as a function of temperature show that the entry barrier is ∼15 kBT and the translocation barrier is ∼35 kBT, although the electrical force in the latter step is much stronger. Resolution of this fact, using a theoretical model, reveals that the attraction between DNA and the charges inside the barrel of the pore is the most dominant factor in determining the translocation speed and not merely the driving electrochemical potential gradient.

Identification of Conformational Variants for Bradykinin Biomarker Peptides from a Biofluid Using a Nanopore and Machine Learning.

There is a current need to develop methods for the sensitive detection of peptide biomarkers in complex mixtures of molecules, such as biofluids, to enable early disease detection. Moreover, to our knowledge, there is currently no detection method capable of identifying the different conformations of a peptide biomarker differing by a single amino acid. Single-molecule nanopore sensing promises to provide this level of resolution. In order to be able to identify these differences in a biofluid such as serum, it is necessary to carefully characterize electrical parameters to obtain specific signatures of each biomarker population observed. We are interested here in a family of peptide biomarkers, kinins such as bradykinin and des-Arg9 bradykinin, that are involved in many disabling pathologies (allergy, asthma, angioedema, sepsis, or cancer). We show the proof of concept for direct identification of these biomarkers in serum at the single-molecule level using a protein nanopore. Each peptide exhibits two unique electrical signatures attributed to specific conformations in bulk. The same signatures are found in serum, allowing their discrimination and identification in a complex mixture such as biofluid. To extend the utility of our experimental results, we developed a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used semisupervised classification to assign each event type to a specific biomarker at physiological serum concentration. In the future, single-molecule scale analysis of peptide biomarkers using a powerful nanopore coupled with machine learning will facilitate the identification and quantification of other clinically relevant biomarkers from biofluids.

Dynamically Mapping the Topography and Stiffness of the Leading Edge of Migrating Cells Using AFM in Fast-QI Mode.

Cell migration profoundly influences cellular function, often resulting in adverse effects in various pathologies including cancer metastasis. Directly assessing and quantifying the nanoscale dynamics of living cell structure and mechanics has remained a challenge. At the forefront of cell movement, the flat actin modules─the lamellipodium and the lamellum─interact to propel cell migration. The lamellipodium extends from the lamellum and undergoes rapid changes within seconds, making measurement of its stiffness a persistent hurdle. In this study, we introduce the fast-quantitative imaging (fast-QI) mode, demonstrating its capability to simultaneously map both the lamellipodium and the lamellum with enhanced spatiotemporal resolution compared with the classic quantitative imaging (QI) mode. Specifically, our findings reveal nanoscale stiffness gradients in the lamellipodium at the leading edge, where it appears to be slightly thinner and significantly softer than the lamellum. Additionally, we illustrate the fast-QI mode's accuracy in generating maps of height and effective stiffness through a streamlined and efficient processing of force-distance curves. These results underscore the potential of the fast-QI mode for investigating the role of motile cell structures in mechanosensing.

Identification and Detection of a Peptide Biomarker and Its Enantiomer by Nanopore.

Until now, no fast, low-cost, and direct technique exists to identify and detect protein/peptide enantiomers, because their mass and charge are identical. They are essential since l- and d-protein enantiomers have different biological activities due to their unique conformations. Enantiomers have potential for diagnostic purposes for several diseases or normal bodily functions but have yet to be utilized. This work uses an aerolysin nanopore and electrical detection to identify vasopressin enantiomers, l-AVP and d-AVP, associated with different biological processes and pathologies. We show their identification according to their conformations, in either native or reducing conditions, using their specific electrical signature. To improve their identification, we used a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used the Monte Carlo prediction to assign each event type to a specific l- or d-AVP enantiomer.

Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling.

The enzymatic degradation of long polysaccharide chains is monitored by nanopore detection. It follows a Michaelis-Menten mechanism. We measure the corresponding kinetic constants at the single molecule level. The simulation results of the degradation process allowed one to account for the oligosaccharide size distribution detected by a nanopore.

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification.

One of the most important health challenges is the early and ongoing detection of disease for prevention, as well as personalized treatment management. Development of new sensitive analytical point-of-care tests are, therefore, necessary for direct biomarker detection from biofluids as critical tools to address the healthcare needs of an aging global population. Coagulation disorders associated with stroke, heart attack, or cancer are defined by an increased level of the fibrinopeptide A (FPA) biomarker, among others. This biomarker exists in more than one form: it can be post-translationally modified with a phosphate and also cleaved to form shorter peptides. Current assays are long and have difficulties in discriminating between these derivatives; hence, this is an underutilized biomarker for routine clinical practice. We use nanopore sensing to identify FPA, the phosphorylated FPA, and two derivatives. Each of these peptides is characterized by unique electrical signals for both dwell time and blockade level. We also show that the phosphorylated form of FPA can adopt two different conformations, each of which have different values for each electrical parameter. We were able to use these parameters to discriminate these peptides from a mix, thereby opening the way for the potential development of new point-of-care tests.

Using Adhesive Micropatterns and AFM to Assess Cancer Cell Morphology and Mechanics.

The mechanical properties of living cells reflect their physiological and pathological state. In particular, cancer cells undergo cytoskeletal modifications that typically make them softer than healthy cells, a property that could be used as a diagnostic tool. However, this is challenging because cells are complex structures displaying a broad range of morphologies when cultured in standard 2D culture dishes. Here, we use adhesive micropatterns to impose the cell geometry and thus standardize the mechanics and morphologies of cancer cells, which we measure by atomic force microscopy (AFM), mechanical nanomapping, and membrane nanotube pulling. We show that micropatterning cancer cells leads to distinct morphological and mechanical changes for different cell lines. Micropatterns did not systematically lower the variability in cell elastic modulus distribution. These effects emerge from a variable cell spreading rate associated with differences in the organization of the cytoskeleton, thus providing detailed insights into the structure-mechanics relationship of cancer cells cultured on micropatterns. Combining AFM with micropatterns reveals new mechanical and morphological observables applicable to cancer cells and possibly other cell types.

Circulating Tumor Cells in Cancer Diagnostics and Prognostics by Single-Molecule and Single-Cell Characterization.

Circulating tumor cells (CTCs) represent an interesting source of biomarkers for diagnosis, prognosis, and the prediction of cancer recurrence, yet while they are extensively studied in oncobiology research, their diagnostic utility has not yet been demonstrated and validated. Their scarcity in human biological fluids impedes the identification of dangerous CTC subpopulations that may promote metastatic dissemination. In this Perspective, we discuss promising techniques that could be used for the identification of these metastatic cells. We first describe methods for isolating patient-derived CTCs and then the use of 3D biomimetic matrixes in their amplification and analysis, followed by methods for further CTC analyses at the single-cell and single-molecule levels. Finally, we discuss how the elucidation of mechanical and morphological properties using techniques such as atomic force microscopy and molecular biomarker identification using nanopore-based detection could be combined in the future to provide patients and their healthcare providers with a more accurate diagnosis.

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to ß-cyclodextrin (MBP-ßCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-ßCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Thermal unfolding of proteins probed at the single molecule level using nanopores.

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.

Transport of long neutral polymers in the semidilute regime through a protein nanopore.

We investigate the entrance of single poly(ethylene glycol) chains into an α-hemolysin channel. We detect the frequency and duration of the current blockades induced by large neutral polymers, where chain radius is larger than pore diameter. In the semidilute regime, these chains pass only if the monomer concentration is larger than a well-defined threshold. Experiments are performed in a very large domain of concentration and molecular mass, up to 35% and 200 kDa, respectively, which was previously unexplored. The variation of the dwell time as a function of molecular mass shows that the chains are extracted from the semidilute solution in contact with the pore by a reptation mechanism.

Focus on using nanopore technology for societal health, environmental, and energy challenges.

With an increasing global population that is rapidly ageing, our society faces challenges that impact health, environment, and energy demand. With this ageing comes an accumulation of cellular changes that lead to the development of diseases and susceptibility to infections. This impacts not only the health system, but also the global economy. As the population increases, so does the demand for energy and the emission of pollutants, leading to a progressive degradation of our environment. This in turn impacts health through reduced access to arable land, clean water, and breathable air. New monitoring approaches to assist in environmental control and minimize the impact on health are urgently needed, leading to the development of new sensor technologies that are highly sensitive, rapid, and low-cost. Nanopore sensing is a new technology that helps to meet this purpose, with the potential to provide rapid point-of-care medical diagnosis, real-time on-site pollutant monitoring systems to manage environmental health, as well as integrated sensors to increase the efficiency and storage capacity of renewable energy sources. In this review we discuss how the powerful approach of nanopore based single-molecule, or particle, electrical promises to overcome existing and emerging societal challenges, providing new opportunities and tools for personalized medicine, localized environmental monitoring, and improved energy production and storage systems.

Dynamics of DNA Through Solid-state Nanopores Fabricated by Controlled Dielectric Breakdown.

Controlled dielectric breakdown (CDB) is gaining popularity for fabricating solid-state nanopores in situ with size control in a simple, low-cost, and scalable way. This technique could be used for a broad type of applications in the field of nucleic acid analysis and even for protein studies. In this work, we studied the entry and transport of double-stranded DNAs using a solid-state nanopore fabricated by CDB as a function of applied voltage for two different DNA lengths. We showed that the blockade rate increases exponentially with voltage up to 120 mV. The energy barrier depends on the chain length, and the dwell times decrease with applied voltage up to 120 mV. Moreover, no matter the chain length, it is possible to differentiate two families of blockade amplitudes, high and low ones, due to DNA folding.

Dynamics of unfolded protein transport through an aerolysin pore.

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.