Microtubule-Mediated Regulation of ß2AR Translation and Function in Failing Hearts.

BACKGROUND: ß1AR (beta-1 adrenergic receptor) and ß2AR (beta-2 adrenergic receptor)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac ß-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that ß-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. METHODS: The localization pattern of ß-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on ß-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible ß-AR translation sites in cardiomyocytes. The mechanism by which ß-AR mRNA is redistributed post-heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post-myocardial infarction and detubulated cardiomyocytes. RESULTS: ß1AR and ß2AR mRNAs show differential localization in cardiomyocytes, with ß1AR found in the perinuclear region and ß2AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of ß2AR transcripts toward the perinuclear region. The close proximity between ß2AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of ß2AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both ß1AR and ß2AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to ß-AR mRNA redistribution and impaired ß2AR function in failing hearts. CONCLUSIONS: Asymmetrical microtubule-dependent trafficking dictates differential ß1AR and ß2AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 ß-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from transverse tubule remodeling, leading to distorted ß2AR-mediated cyclic adenosine monophosphate signaling.

Selective Single-Molecule Nanopore Detection of mpox A29 Protein Directly in Biofluids.

Single-molecule antigen detection using nanopores offers a promising alternative for accurate virus testing to contain their transmission. However, the selective and efficient identification of small viral proteins directly in human biofluids remains a challenge. Here, we report a nanopore sensing strategy based on a customized DNA molecular probe that combines an aptamer and an antibody to enhance the single-molecule detection of mpox virus (MPXV) A29 protein, a small protein with an M.W. of ca. 14 kDa. The formation of the aptamer-target-antibody sandwich structures enables efficient identification of targets when translocating through the nanopore. This technique can accurately detect A29 protein with a limit of detection of ∼11 fM and can distinguish the MPXV A29 from vaccinia virus A27 protein (a difference of only four amino acids) and Varicella Zoster Virus (VZV) protein directly in biofluids. The simplicity, high selectivity, and sensitivity of this approach have the potential to contribute to the diagnosis of viruses in point-of-care settings.

Nanopore Detection Using Supercharged Polypeptide Molecular Carriers.

The analysis at the single-molecule level of proteins and their interactions can provide critical information for understanding biological processes and diseases, particularly for proteins present in biological samples with low copy numbers. Nanopore sensing is an analytical technique that allows label-free detection of single proteins in solution and is ideally suited to applications, such as studying protein-protein interactions, biomarker screening, drug discovery, and even protein sequencing. However, given the current spatiotemporal limitations in protein nanopore sensing, challenges remain in controlling protein translocation through a nanopore and relating protein structures and functions with nanopore readouts. Here, we demonstrate that supercharged unstructured polypeptides (SUPs) can be genetically fused with proteins of interest and used as molecular carriers to facilitate nanopore detection of proteins. We show that cationic SUPs can substantially slow down the translocation of target proteins due to their electrostatic interactions with the nanopore surface. This approach enables the differentiation of individual proteins with different sizes and shapes via characteristic subpeaks in the nanopore current, thus facilitating a viable route to use polypeptide molecular carriers to control molecular transport and as a potential system to study protein-protein interactions at the single-molecule level.

In situ solid-state nanopore fabrication.

Nanopores in solid-state membranes are promising for a wide range of applications including DNA sequencing, ultra-dilute analyte detection, protein analysis, and polymer data storage. Techniques to fabricate solid-state nanopores have typically been time consuming or lacked the resolution to create pores with diameters down to a few nanometres, as required for the above applications. In recent years, several methods to fabricate nanopores in electrolyte environments have been demonstrated. These in situ methods include controlled breakdown (CBD), electrochemical reactions (ECR), laser etching and laser-assisted controlled breakdown (la-CBD). These techniques are democratising solid-state nanopores by providing the ability to fabricate pores with diameters down to a few nanometres (i.e. comparable to the size of many analytes) in a matter of minutes using relatively simple equipment. Here we review these in situ solid-state nanopore fabrication techniques and highlight the challenges and advantages of each method. Furthermore we compare these techniques by their desired application and provide insights into future research directions for in situ nanopore fabrication methods.

Understanding Electrical Conduction and Nanopore Formation During Controlled Breakdown.

Controlled breakdown has recently emerged as a highly appealing technique to fabricate solid-state nanopores for a wide range of biosensing applications. This technique relies on applying an electric field of approximately 0.4-1 V nm-1 across the membrane to induce a current, and eventually, breakdown of the dielectric. Although previous studies have performed controlled breakdown under a range of different conditions, the mechanism of conduction and breakdown has not been fully explored. Here, electrical conduction and nanopore formation in SiNx membranes during controlled breakdown is studied. It is demonstrated that for Si-rich SiNx , oxidation reactions that occur at the membrane-electrolyte interface limit conduction across the dielectric. However, for stoichiometric Si3 N4 the effect of oxidation reactions becomes relatively small and conduction is predominately limited by charge transport across the dielectric. Several important implications resulting from understanding this process are provided which will aid in further developing controlled breakdown in the coming years, particularly for extending this technique to integrate nanopores with on-chip nanostructures.

Individually Addressable Multi-nanopores for Single-Molecule Targeted Operations.

The fine-tuning of molecular transport is a ubiquitous problem of single-molecule methods. The latter is evident even in powerful single-molecule techniques such as nanopore sensing, where the quest for resolving more detailed biomolecular features is often limited by insufficient control of the dynamics of individual molecules within the detection volume of the nanopore. In this work, we introduce and characterize a reconfigurable multi-nanopore architecture that enables additional channels to manipulate the dynamics of DNA molecules in a nanopore. We show that the fabrication process of this device, consisting of four adjacent, individually addressable nanopores located at the tip of a quartz nanopipette, is fast and highly reproducible. By individually tuning the electric field across each nanopore, these devices can operate in several unique cooperative detection modes that allow moving, sensing, and trapping of DNA molecules with high efficiency and increased temporal resolution.

Author Correction: Electrotunable nanoplasmonic liquid mirror.

In the version of this Article originally published, the last sentence of the acknowledgements incorrectly read 'L.V. acknowledges the support of a Marie Skodowska-Curie fellowship (N-SHEAD)'; it should have read 'L.V. and D.S. acknowledge the support of Marie Sklodowska-Curie fellowships, N-SHEAD and S-OMMs, respectively'.

Rapid Fragmentation during Seeded Lysozyme Aggregation Revealed at the Single Molecule Level.

Protein aggregation is associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The poorly understood pathogenic mechanism of amyloid diseases makes early stage diagnostics or therapeutic intervention a challenge. Seeded polymerization that reduces the duration of the lag phase and accelerates fibril growth is a widespread model to study amyloid formation. Seeding effects are hypothesized to be important in the "infectivity" of amyloids and are linked to the development of systemic amyloidosis in vivo. The exact mechanism of seeding is unclear yet critical to illuminating the propagation of amyloids. Here we report on the lateral and axial fragmentation of seed fibrils in the presence of lysozyme monomers at short time scales, followed by the generation of oligomers and growth of fibrils.

Chemically Modified Hydrogel-Filled Nanopores: A Tunable Platform for Single-Molecule Sensing.

Label-free, single-molecule sensing is anideal candidate for biomedical applications that rely on the detection of low copy numbers in small volumes and potentially complex biofluids. Among them, solid-state nanopores can be engineered to detect single molecules of charged analytes when they are electrically driven through the nanometer-sized aperture. When successfully applied to nucleic acid sensing, fast transport in the range of 10-100 nucleotides per nanosecond often precludes the use of standard nanopores for the detection of the smallest fragments. Herein, hydrogel-filled nanopores (HFN) are reported that combine quartz nanopipettes with biocompatible chemical poly(vinyl) alcohol hydrogels engineered in-house. Hydrogels were modified physically or chemically to finely tune, in a predictable manner, the transport of specific molecules. Controlling the hydrogel mesh size and chemical composition allowed us to slow DNA transport by 4 orders of magnitude and to detect fragments as small as 100 base pairs (bp) with nanopores larger than 20 nm at an ionic strength comparable to physiological conditions. Considering the emergence of cell-free nucleic acids as blood biomarkers for cancer diagnostics or prenatal testing, the successful sensing and size profiling of DNA fragments ranging from 100 bp to >1 kbp long under physiological conditions demonstrates the potential of HFNs as a new generation of powerful and easily tunable molecular diagnostics tools.

Double Barrel Nanopores as a New Tool for Controlling Single-Molecule Transport.

The ability to control the motion of single biomolecules is key to improving a wide range of biophysical and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, molecular control and the possibility of slow readouts of long polymer molecules are still limited due to fast analyte transport and low signal-to-noise ratios. Here, we report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are separated by only a ∼ 20 nm gap. The nanopores can be addressed individually, allowing for two unique modes of operation: (i) pore-to-pore transfer, which can be controlled at near 100% efficiency, and (ii) DNA molecules bridging between the two nanopores, which enables detection with an enhanced temporal resolution (e.g., an increase of more than 2 orders of magnitude in the dwell time) without compromising the signal quality. The simplicity of fabrication and operation of the double-barrel architecture opens a wide range of applications for high-resolution readout of biological molecules.

Electrotunable nanoplasmonic liquid mirror.

Recently, there has been a drive to design and develop fully tunable metamaterials for applications ranging from new classes of sensors to superlenses among others. Although advances have been made, tuning and modulating the optical properties in real time remains a challenge. We report on the first realization of a reversible electrotunable liquid mirror based on voltage-controlled self-assembly/disassembly of 16 nm plasmonic nanoparticles at the interface between two immiscible electrolyte solutions. We show that optical properties such as reflectivity and spectral position of the absorption band can be varied in situ within ±0.5 V. This observed effect is in excellent agreement with theoretical calculations corresponding to the change in average interparticle spacing. This electrochemical fully tunable nanoplasmonic platform can be switched from a highly reflective 'mirror' to a transmissive 'window' and back again. This study opens a route towards realization of such platforms in future micro/nanoscale electrochemical cells, enabling the creation of tunable plasmonic metamaterials.

Single Molecule Trapping and Sensing Using Dual Nanopores Separated by a Zeptoliter Nanobridge.

There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.

Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.

Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein-peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.

Fundamentals and applications of self-assembled plasmonic nanoparticles at interfaces.

This tutorial review will introduce and explore fundamental and applied aspects of electrolytic interfaces incorporating nanoscale building blocks for use in novel applications such as sensors and tunable optics. In order to do this, it is important to understand the principles behind even the simplest of immiscible interfaces such as those of the liquid|liquid and solid|liquid. Qualitatively, the picture is simple however the complexity is easily compounded by the addition of electrolyte, and further compounded by the addition of more complex entities such as nanoparticles. Nevertheless combining all these components surprisingly results in an elegant solution, where the nanoparticles have the ability to self-assemble at the interface with a high level of control. Importantly, this opens up the door to the development of new types of materials with a range of applications which have only recently been exploited. Initially we begin with a description of the fundamentals related to liquid|liquid and solid|liquid interfaces both with and without electrolyte. The discussion then shifts to a description of biasing the interface by the application of an electric field. This is followed by an exploration of nanoparticle assembly and disassembly at the interface by controlling parameters such as ligand composition, charge, pH, and electric field. Finally a description of the state-of-the-art is given in terms of current applications and possible future directions. It is perhaps fair to say that these new frontiers have caused great excitement within the sensing community not only due to the simplicity of the technique but also due to the unprecedented levels of sensitivity and control.

Unravelling the optical responses of nanoplasmonic mirror-on-mirror metamaterials.

Mirror-on-mirror platforms based on arrays of metallic nanoparticles, arranged top-down or self-assembled on a thin metallic film, have interesting optical properties. Interaction of localized surface-plasmons in nanoparticles with propagating surface-plasmons in the film underpins the exotic features of such platforms. Here, we present a comprehensive theoretical framework which emulates such a system using a five-layer-stack model and calculate its reflectance, transmittance, and absorbance spectra. The theory rests on dipolar quasi-static approximations incorporating image-forces and effective medium theory. Systematically tested against full-wave simulations, this simple approach proves to be adequate within its obvious applicability limits. It is used to study optical signals as a function of nanoparticle dimensions, interparticle separation, metal film thickness, the gap between the film and nanoparticles, and incident light characteristics. Several peculiar features are found, e.g., quenching of reflectivity in certain frequency domains or shift of the reflectivity spectra. Schemes are proposed to tailor those as functions of the mentioned parameters. Calculating the system's optical responses in seconds, as compared to much longer running simulations, this theory helps to momentarily unravel the role of each system parameter in light reflection, transmission, and absorption, facilitating thereby the design and optimisation of novel mirror-on-mirror systems.

Precise attoliter temperature control of nanopore sensors using a nanoplasmonic bullseye.

Targeted temperature control in nanopores is greatly important in further understanding biological molecules. Such control would extend the range of examinable molecules and facilitate advanced analysis, including the characterization of temperature-dependent molecule conformations. The work presented within details well-defined plasmonic gold bullseye and silicon nitride nanopore membranes. The bullseye nanoantennae are designed and optimized using simulations and theoretical calculations for interaction with 632.8 nm laser light. Laser heating was monitored experimentally through nanopore conductance measurements. The precise heating of nanopores is demonstrated while minimizing the accumulation of heat in the surrounding membrane material.

Electrodeposition and bipolar effects in metallized nanopores and their use in the detection of insulin.

Solid-state nanopore devices with integrated electrodes are an important class of single-molecule biosensors, with potential applications in DNA, RNA, and protein detection and sequence analysis. Here we investigate solid-state nanopore sensors with an embedded gold film, fabricated using semiconductor processing techniques and focused ion beam milling. We characterize their geometric structure in three dimensions on the basis of experimental conductance studies and modeling as well as transmission electron microscopy imaging and tomography. We used electrodeposition to further shrink the pores to effective diameters below 10 nm and demonstrate how bipolar electrochemical coupling across the membrane can lead to significant contributions to the overall pore current and discuss its implications for nanopore sensing. Finally, we use metallized nanopores modified with homocysteine for the detection of insulin. We show that adsorption of the protein to the chemically modified nanopores slows down the translocation process to tens of milliseconds, which is orders of magnitude slower than expected for conventional electrophoretic transport.

3D Droplet Microfluidic Systems for High-Throughput Biological Experimentation.

Herein, we describe the development of a multilayer droplet microfluidic system for creating concentration gradients and generating microdroplets of varying composition for high-throughput biochemical and cell-based screening applications. The 3D droplet-based microfluidic device consists of multiple PDMS layers, which are used to generate logarithmic concentration gradient reagent profiles. Parallel flow focusing structures are used to form picoliter-sized droplets of defined volumes but of varying composition. As proof of concept, we demonstrate rapid enzymatic activity assays and drug cytotoxicity assays on bacteria. The 3D droplet-based microfluidic platform has the potential to allow for high-efficiency and high-throughput analysis, overcoming the structural limitations of single layer microfluidic systems.

Fine tuning of nanopipettes using atomic layer deposition for single molecule sensing.

Nanopipettes are an attractive single-molecule tool for identification and characterisation of nucleic acids and proteins in solutions. They enable label-free analysis and reveal individual molecular properties, which are generally masked by ensemble averaging. Having control over the pore dimensions is vital to ensure that the dimensions of the molecules being probed match those of the pore for optimization of the signal to noise. Although nanopipettes are simple and easy to fabricate, challenges exist, especially when compared to more conventional solid-state analogues. For example, a sub-20 nm pore diameter can be difficult to fabricate and the batch-to-batch reproducibility is often poor. To improve on this limitation, atomic layer deposition (ALD) is used to deposit ultrathin layers of alumina (Al2O3) on the surface of the quartz nanopipettes enabling sub-nm tuning of the pore dimensions. Here, Al2O3 with a thickness of 8, 14 and 17 nm was deposited onto pipettes with a starting pore diameter of 75 ± 5 nm whilst a second batch had 5 and 8 nm Al2O3 deposited with a starting pore diameter of 25 ± 3 nm respectively. This highly conformal process coats both the inner and outer surfaces of pipettes and resulted in the fabrication of pore diameters as low as 7.5 nm. We show that Al2O3 modified pores do not interfere with the sensing ability of the nanopipettes and can be used for high signal-to-noise DNA detection. ALD provides a quick and efficient (batch processing) for fine-tuning nanopipettes for a broad range of applications including the detection of small biomolecules like RNA, aptamers and DNA-protein interactions at the single molecule level.

Single molecule ionic current sensing in segmented flow microfluidics.

Herein, we describe the integration of two glass nanopores into a segmented flow microfluidic device with a view on enhancing the functionality of label free, single molecule nanopore sensors. Within a robust and mechanically stable platform, individual droplet compositions are distinguished before single molecule translocations from the droplet are detected electrochemically via the Coulter principle. This result is highly significant, combining the sensitivity of single molecule methods and their ability to overcome the clouding of the ensemble average with the "isolated microreactor" benefits of droplet microfluidics. Furthermore, devices as presented here provide the platform for the development of systems where the injection and extraction of single molecules allow droplet composition to be controlled at the molecular level.