Nanopore ion sources deliver individual ions of amino acids and peptides directly into high vacuum.

Electrospray ionization is widely used to generate vapor phase ions for analysis by mass spectrometry in proteomics research. However, only a small fraction of the analyte enters the mass spectrometer due to losses that are fundamentally linked to the use of a background gas to stimulate the generation of ions from electrosprayed droplets. Here we report a nanopore ion source that delivers ions directly into high vacuum from aqueous solutions. The ion source comprises a pulled quartz pipette with a sub-100 nm opening. Ions escape an electrified meniscus by ion evaporation and travel along collisionless trajectories to the ion detector. We measure mass spectra of 16 different amino acid ions, post-translationally modified variants of glutathione, and the peptide angiotensin II, showing that these analytes can be emitted as desolvated ions. The emitted current is composed of ions rather than charged droplets, and more than 90% of the current can be recovered in a distant collector.

Combining iontronic, chromatography and nanopipette for Aß42 aggregates detection and separation.

In this work, we aim to capture, detect and analysis at single molecule level Aß42 aggregates. To this end, two strategies of track-etched nanopore membranes functionalization were investigated. The first one uses an aptamer and requires only three steps, whereas the second strategy uses Lecanemab antibodies and requires six steps. Out of the two presented strategies, the second one was found to be the most suitable to detect Aß42 aggregates using a quick current-voltage readout. The resulting single nanopore was then upscale to multipore membranes to capture the Aß42 aggregates before analysis through them through a single-molecule approach. By comparing the species present in the retentate and filtrate, we confirmed the membrane's affinity for the larger Aß42 aggregates present in the sample. We found that chromatographic membranes combined with an ionic diode for binary on/off readout are powerful tools for detecting rare biomarkers before single molecule analysis.

Methylene Blue-Loaded NanoMOFs: Accumulation in Chlamydia trachomatis Inclusions and Light/Dark Antibacterial Effects.

Metal-organic framework nanoparticles (nanoMOFs) are promising nanomaterials for biomedical applications. Some of them, including biodegradable porous iron carboxylates are proposed for encapsulation and delivery of antibiotics. Due to the high drug loading capacity and fast internalization kinetics, nanoMOFs are more beneficial for the treatment of intracellular bacterial infections compared to free antibacterial drugs, which poorly accumulate inside the cells because of the inability to cross membrane barriers or have low intracellular retention. However, nanoparticle internalization does not ensure their accumulation in the cell compartment that shelters a pathogen. This study shows the availability of MIL-100(Fe)-based MOF nanoparticles to co-localize with Chlamydia trachomatis, an obligate intracellular bacterium, in the infected RAW264.7 macrophages. Furthermore, nanoMOFs loaded with photosensitizer methylene blue (MB) exhibit complete photodynamic inactivation of C. trachomatis growth. Simultaneous infection and treatment of RAW264.7 cells with empty nanoMOFs resulted in a bacterial load reduction from 100 to 36% that indicates an intrinsic anti-chlamydial effect of this iron-containing nanomaterial. Thus, our findings suggest the use of iron-based nanoMOFs as a promising drug delivery platform, which contributes to antibacterial effect, for the treatment of chlamydial infections.

Metal-Organic Framework-Based Nanomedicines for the Treatment of Intracellular Bacterial Infections.

Metal-organic frameworks (MOFs) are a highly versatile class of ordered porous materials, which hold great promise for different biomedical applications, including antibacterial therapy. In light of the antibacterial effects, these nanomaterials can be attractive for several reasons. First, MOFs exhibit a high loading capacity for numerous antibacterial drugs, including antibiotics, photosensitizers, and/or photothermal molecules. The inherent micro- or meso-porosity of MOF structures enables their use as nanocarriers for simultaneous encapsulation of multiple drugs resulting in a combined therapeutic effect. In addition to being encapsulated into an MOF's pores, antibacterial agents can sometimes be directly incorporated into an MOF skeleton as organic linkers. Next, MOFs contain coordinated metal ions in their structure. Incorporation of Fe2/3+, Cu2+, Zn2+, Co2+, and Ag+ can significantly increase the innate cytotoxicity of these materials for bacteria and cause a synergistic effect. Finally, abundance of functional groups enables modifying the external surface of MOF particles with stealth coating and ligand moieties for improved drug delivery. To date, there are a number of MOF-based nanomedicines available for the treatment of bacterial infections. This review is focused on biomedical consideration of MOF nano-formulations designed for the therapy of intracellular infections such as Staphylococcus aureus, Mycobacterium tuberculosis, and Chlamydia trachomatis. Increasing knowledge about the ability of MOF nanoparticles to accumulate in a pathogen intracellular niche in the host cells provides an excellent opportunity to use MOF-based nanomedicines for the eradication of persistent infections. Here, we discuss advantages and current limitations of MOFs, their clinical significance, and their prospects for the treatment of the mentioned infections.

Polymer-metal-organic framework self-assembly (PMOFSA) as a robust one-step method to generate well-dispersed hybrid nanoparticles in water.

A new process, PMOFSA, is described here, that opens the way for the one-pot straightforward and versatile manufacture of polymer-MOF nanoparticles in water. It can be expected that this study will not only expand the scope of in situ preparation of polymer-MOF nano-objects but also inspire researchers in the field to prepare a new generation of polymer-MOF hybrid materials.

Solid-state and polymer nanopores for protein sensing: A review.

In two decades, the solid state and polymer nanopores became attractive method for the protein sensing with high specificity and sensitivity. They also allow the characterization of conformational changes, unfolding, assembly and aggregation as well the following of enzymatic reaction. This review aims to provide an overview of the protein sensing regarding the technique of detection: the resistive pulse and ionic diodes. For each strategy, we report the most significant achievement regarding the detection of peptides and protein as well as the conformational change, protein-protein assembly and aggregation process. We discuss the limitations and the recent strategies to improve the nanopore resolution and accuracy. A focus is done about concomitant problematic such as protein adsorption and nanopore lifetime.

Detection of Amyloid-ß Fibrils Using Track-Etched Nanopores: Effect of Geometry and Crowding.

Several neurodegenerative diseases have been linked to proteins or peptides that are prone to aggregate in different brain regions. Aggregation of amyloid-ß (Aß) peptides is recognized as the main cause of Alzheimer's disease (AD) progression, leading to the formation of toxic Aß oligomers and amyloid fibrils. The molecular mechanism of Aß aggregation is complex and still not fully understood. Nanopore technology provides a new way to obtain kinetic and morphological aspects of Aß aggregation at a single-molecule scale without labeling by detecting the electrochemical signal of the peptides when they pass through the hole. Here, we investigate the influence of nanoscale geometry (conical and bullet-like shape) of a track-etched nanopore pore and the effect of molecular crowding (polyethylene glycol-functionalized pores) on Aß fibril sensing and analysis. Various Aß fibril samples that differed by their length were produced by sonication of fibrils obtained in the presence of epigallocatechin gallate. The conical nanopore functionalized with polyethylene glycol (PEG) 5 kDa is suitable for discrimination of the fibril size from relative current blockade. The bullet-like-shaped nanopore enhances the amplitude of the current and increases the dwell time, allowing us to well discern the fibrils. Finally, the nanopore crowded with PEG 20 kDa enhances the relative current blockade and increases the dwell time; however, the discrimination is not improved compared to the "bullet-shaped" nanopore.

Machine Learning to Improve the Sensing of Biomolecules by Conical Track-Etched Nanopore.

Single nanopore is a powerful platform to detect, discriminate and identify biomacromolecules. Among the different devices, the conical nanopores obtained by the track-etched technique on a polymer film are stable and easy to functionalize. However, these advantages are hampered by their high aspect ratio that avoids the discrimination of similar samples. Using machine learning, we demonstrate an improved resolution so that it can identify short single- and double-stranded DNA (10- and 40-mers). We have characterized each current blockade event by the relative intensity, dwell time, surface area and both the right and left slope. We show an overlap of the relative current blockade amplitudes and dwell time distributions that prevents their identification. We define the different parameters that characterize the events as features and the type of DNA sample as the target. By applying support-vector machines to discriminate each sample, we show accuracy between 50% and 72% by using two features that distinctly classify the data points. Finally, we achieved an increased accuracy (up to 82%) when five features were implemented.

Unexpected ionic transport behavior in hydrophobic and uncharged conical nanopores.

We investigated ionic transport behavior in the case of uncharged conical nanopores. To do so, we designed conical nanopores using atomic layer deposition of Al2O3/ZnO nanolaminates and then coated these with trimethylsilane. The experimental results are supported by molecular dynamics simulations. The ionic transport reveals an unexpected behavior: (i) a current rectification and (ii) a constant conductance at low salt concentration which are usually reported for charged conical nanopore. To explain these results, we have considered different assumptions: (i) a default of functionalization, (ii) the adsorption anion and (iii) the slippage. The first one was refuted by the study of the poly-l-lysine transport through the nanopore. To verify the second assumption, we investigate the effect of pH on the current rectification and the molecular dynamics simulations. Finally our study demonstrates that the unexpected ionic transport is provided to a predominant effect of slippage due to the water organization at the solid/liquid interface.

Impact of Polyelectrolyte Multilayers on the Ionic Current Rectification of Conical Nanopores.

Single conical nanopores were functionalised layer by layer with weak polyelectrolytes. We studied their influence on the ionic diode properties We have considered different couples of polyelectrolytes: poly-l-lysine/poly(acrylic acid) and poly(ethyleneimine)/poly(acrylic acid) as well as the influence of cross-linking. The results show that the nanopores decorated with poly(ethyleneimine)/poly(acrylic acid) exhibit an interesting behavior. Indeed, at pH 3, the nanopore is open only at the low salt concentration, while at pH 7, it is already open. The nanopores functionalized with poly-l-lysine/poly(acrylic acid) do not show an inversion of ionic transport properties with the pH as expected. After cross-linked to prevent large conformational changes, the ionic diode properties are dependent on the pH.

The nanopore mass spectrometer.

We report the design of a mass spectrometer featuring an ion source that delivers ions directly into high vacuum from liquid inside a capillary with a sub-micrometer-diameter tip. The surface tension of water and formamide is sufficient to maintain a stable interface with high vacuum at the tip, and the gas load from the interface is negligible, even during electrospray. These conditions lifted the usual requirement of a differentially pumped system. The absence of a background gas also opened up the possibility of designing ion optics to collect and focus ions in order to achieve high overall transmission and detection efficiencies. We describe the operation and performance of the instrument and present mass spectra from solutions of salt ions and DNA bases in formamide and salt ions in water. The spectra show singly charged solute ions clustered with a small number of solvent molecules.

Functionalization of single solid state nanopores to mimic biological ion channels: A review.

In nature, ion channels are highly selective pores and act as gate to ensure selective ion transport, allowing ions to cross the membrane. By mimicking them, single solid state nanopore devices emerge as a new, powerful class of molecule sensors that allow for the label-free detection of biomolecules (DNA, RNA, and proteins), non-biological polymers, as well as small molecules. In this review, we exhaustively describe the fabrication and functionalization techniques to design highly robust and selective solid state nanopores. First we outline the different materials and methods to design nanopores, we explain the ionic conduction in nanopores, and finally we summarize some techniques to modify and functionalize the surface in order to obtain biomimetic nanopores, responding to different external stimuli.

Structure and antibacterial activity relationships of native and amyloid fibril lysozyme loaded on layered double hydroxide.

Lysozyme from hen egg white is composed by a unique linear chain of 129 amino acids. It is known to inhibit Gram positive bacteria and to form amyloid fibrils at low pH, under 75°C. This work investigates the effect of the fibrillation and/or adsorption onto a layered double hydroxide material on the antibacterial properties of lysozyme. The kinetics of adsorption follows a behavior of pseudo second order model. The X-ray diffraction and the Fourier transform infrared spectroscopy highlight that adsorption occurs only on the external surface of the material. Interestingly, the amyloid fibrils of lysozyme retain their antibacterial properties when they are adsorbed on the layered double hydroxide; even if their activity is lowered, the active site of the enzyme is not fully denatured and is still accessible. This is confirmed by the study of the tryptophan using time-resolved fluorescence spectroscopy.

Diffusion dynamics of latex nanoparticles coated with ssDNA across a single nanopore.

The fundamental understanding of the transport mechanisms of objects across a single nanopore is one key point to develop Coulter counters at the nanoscale for macromolecule or nanoparticle detection. In this area, nanoparticles have been less investigated than biomacromolecules such as DNA or proteins due to their self-aggregation in the presence of salts. In this work, the transport of modified latex nanoparticles across solid-state nanopores was investigated. To prevent their aggregation, their surface was modified with a low molecular weight single strand DNA coating. Then the coated nanoparticles were successfully detected across a single pore material in 200 mM NaCl buffer. The experimental capture rate was compared to that of the predictive model. It reveals that the nanoparticle entrance inside the nanopore is mainly governed by diffusion and required a weak energy. For relative current blockades, the predictive model should take into account both the nanopore shape and the additional charge due to ssDNA coating.

Influence of Adsorption on Proteins and Amyloid Detection by Silicon Nitride Nanopore.

For the past 2 decades, emerging single-nanopore technologies have opened the route to multiple sensing applications. Besides DNA sensing, the identification of proteins and amyloids is a promising field for early diagnosis. However, the influence of the interactions between the nanopore surface and proteins should be taken into account. In this work, we have selected three proteins (avidin, lysozyme, and IgG) that exhibit different affinities with the SiNx surface, and we have also examined lysozyme amyloid. Our results show that the piranha treatment of SiNx significantly decreases protein adsorption. Moreover, we have successfully detected all proteins (pore diameter 17 nm) and shown the possibility of discriminating between denatured lysozyme and its amyloid. For all proteins, the capture rates are lower than expected, and we evidence that they are correlated with the affinity of proteins to the surface. Our result confirms that proteins interacting only with the nanopore surface wall stay long enough to be detected. For lysozyme amyloid, we show that the use of the nanopore is suitable for determining the number of monomer units even if only the proteins interacting with the nanopore are detected.

Biomimetic solution against dewetting in a highly hydrophobic nanopore.

A water molecule is the foundation of life and is the primary compound in every living system. While many of its properties are understood in a bulk solvent, its behavior in a small hydrophobic nanopore still raises fundamental questions. For instance, a wetting/dewetting transition in a hydrophobic solid-state or a polymer nanopore occurs stochastically and can only be prevented by external physical stimuli. Controlling these transitions would be a primary requirement to improve many applications. Some biological channels, such as gramicidin A (gA) proteins, show a high rate of water and ion diffusion in their central subnanochannel while their external surface is highly hydrophobic. The diameter of this channel is significantly smaller than the inner size of the lowest artificial nanopore in which water drying occurs (i.e. 1.4 nm). In this paper, we propose an innovative idea to generate nanopore wetting as a result of which the application of an external field is no longer required. In a nanopore, the drying or wetting of the inner walls occurs randomly (in experiments and in simulations). However, we have shown how the confinement of gA, in a dried hydrophobic nanopore, rapidly generates a stable wetting of the latter. We believe that this simple idea, based on biomimetism, could represent a real breakthrough that could help to improve and develop new nanoscale applications.

Influence of nanopore surface charge and magnesium ion on polyadenosine translocation.

We investigate the influence of a nanopore surface state and the addition of Mg(2+) on poly-adenosine translocation. To do so, two kinds of nanopores with a low aspect ratio (diameter ∼3-5 nm, length 30 nm) were tailored: the first one with a negative charge surface and the second one uncharged. It was shown that the velocity and the energy barrier strongly depend on the nanopore surface. Typically if the nanopore and polyA exhibit a similar charge, the macromolecule velocity increases and its global energy barrier of entrance in the nanopore decreases, as opposed to the non-charged nanopore. Moreover, the addition of a divalent chelating cation induces an increase of energy barrier of entrance, as expected. However, for a negative nanopore, this effect is counterbalanced by the inversion of the surface charge induced by the adsorption of divalent cations.

Combining a sensor and a pH-gated nanopore based on an avidin-biotin system.

Here we propose a new approach to tailor nanopores, which combines both pH gating and sensing properties. This strategy is based on PEG like-avidin grafting in nanopores designed by atomic layer deposition (ALD). Below pH 5 the nanopore is blocked. We show that the PEG chains are at the origin of these properties.

Controlling potassium selectivity and proton blocking in a hybrid biological/solid-state polymer nanoporous membrane.

Specific separations of protons and cations are usually performed by electromembrane processes, which require external electric energy. An easier process would be using a membrane able to separate both entities by passive diffusion. Presently, such synthetic nanoporous membranes do not exist. Here, we report the production of a robust hybrid biological/artificial solid-state membrane, which allows selective permeation of alkali metal cations without competing or concurrent permeation of protons. This membrane is simple to prepare and is based on the hydrophobic nature of the polymeric pore walls, and the confined gramicidin A molecules within. This work opens a new route for separation in the domain of nanobiofiltration, especially for tunable nanodevices based on differential ion conduction, with a fundamental understanding of the confinement mechanism.