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.

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.

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.

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.

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.

Label-free in-flow detection of single DNA molecules using glass nanopipettes.

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

Single molecule sensing with solid-state nanopores: novel materials, methods, and applications.

This tutorial review will introduce and explore the fundamental aspects of nanopore (bio)sensing, fabrication, modification, and the emerging technologies and applications that both intrigue and inspire those working in and around the field. Although nanopores can be classified into two categories, solid-state and biological, they are essentially two sides of the same coin. For instance, both garner popularity due to their ability to confine analytes of interest to a nanoscale volume. Due to the vast diversity of nanopore platforms and applications, no single review can cover the entire landscape of published work in the field. Therefore, in this article focus will be placed on recent advancements and developments taking place in the field of solid-state nanopores. It should be stated that the intention of this tutorial review is not to cite all articles relating to solid-state nanopores, but rather to highlight recent, select developments that will hopefully benefit the new and seasoned scientist alike. Initially we begin with the fundamentals of solid-state nanopore sensing. Then the spotlight is shone on the sophisticated fabrication methods that have their origins in the semiconductor industry. One inherent advantage of solid-state nanopores is in the ease of functionalizing the surface with a range of molecules carrying functional groups. Therefore, an entire section is devoted to highlighting various chemical and bio-molecular modifications and explores how these permit the development of novel sensors with specific targets and functions. The review is completed with a discussion on novel detection strategies using nanopores. Although the most popular mode of nanopore sensing is based upon what has come to be known as ionic-current blockade sensing, there is a vast, growing literature based around exploring alternative detection techniques to further expand on the versatility of the sensors. Such techniques include optical, electronic, and force based methods. It is perhaps fair to say that these new frontiers have caused further excitement within the sensing community.

Rapid ultrasensitive single particle surface-enhanced Raman spectroscopy using metallic nanopores.

Nanopore sensors embedded within thin dielectric membranes have been gaining significant interest due to their single molecule sensitivity and compatibility of detecting a large range of analytes, from DNA and proteins, to small molecules and particles. Building on this concept we utilize a metallic Au solid-state membrane to translocate and rapidly detect single Au nanoparticles (NPs) functionalized with 589 dye molecules using surface-enhanced resonance Raman spectroscopy (SERRS). We show that, due to the plasmonic coupling between the Au metallic nanopore surface and the NP, signal intensities are enhanced when probing analyte molecules bound to the NP surface. Although not single molecule, this nanopore sensing scheme benefits from the ability of SERRS to provide rich vibrational information on the analyte, improving on current nanopore-based electrical and optical detection techniques. We show that the full vibrational spectrum of the analyte can be detected with ultrahigh spectral sensitivity and a rapid temporal resolution of 880 µs.

Biocompatible Biphasic Iontronics Enable Neuron-Like Ionic Signal Transmission.

Biocompatible connections between external artificial devices and living organisms show promise for future neuroprosthetics and therapeutics. The study in Science by Zhao and colleagues introduces a cascade-heterogated biphasic gel (HBG) iontronic device, which facilitates electronic-to-multi-ionic signal transduction for abiotic-biotic interfaces. Inspired by neuron signaling, the HBG device demonstrated its biocompatibility by regulating neural activity in biological tissue, paving the way for wearable and implantable devices, including brain-computer interfaces.

Single-Molecule Detection of α-Synuclein Oligomers in Parkinson's Disease Patients Using Nanopores.

α-Synuclein (α-Syn) is an intrinsically disordered protein whose aggregation in the brain has been significantly implicated in Parkinson's disease (PD). Beyond the brain, oligomers of α-Synuclein are also found in cerebrospinal fluid (CSF) and blood, where the analysis of these aggregates may provide diagnostic routes and enable a better understanding of disease mechanisms. However, detecting α-Syn in CSF and blood is challenging due to its heterogeneous protein size and shape, and low abundance in clinical samples. Nanopore technology offers a promising route for the detection of single proteins in solution; however, the method often lacks the necessary selectivity in complex biofluids, where multiple background biomolecules are present. We address these limitations by developing a strategy that combines nanopore-based sensing with molecular carriers that can specifically capture α-Syn oligomers with sizes of less than 20 nm. We demonstrate that α-Synuclein oligomers can be detected directly in clinical samples, with minimal sample processing, by their ion current characteristics and successfully utilize this technology to differentiate cohorts of PD patients from healthy controls. The measurements indicate that detecting α-Syn oligomers present in CSF may potentially provide valuable insights into the progression and monitoring of Parkinson's disease.

Fabrication of electron tunneling probes for measuring single-protein conductance.

Studying the electrical properties of individual proteins is a prominent research area in the field of bioelectronics. Electron tunnelling or quantum mechanical tunnelling (QMT) probes can act as powerful tools for investigating the electrical properties of proteins. However, current fabrication methods for these probes often have limited reproducibility, unreliable contact or inadequate binding of proteins onto the electrodes, so better solutions are required. Here, we detail a generalizable and straightforward set of instructions for fabricating simple, nanopipette-based, tunnelling probes, suitable for measuring conductance in single proteins. Our QMT probe is based on a high-aspect-ratio dual-channel nanopipette that integrates a pair of gold tunneling electrodes with a gap of less than 5 nm, fabricated via the pyrolytic deposition of carbon followed by the electrochemical deposition of gold. The gold tunneling electrodes can be functionalized using an extensive library of available surface modifications to achieve single-protein-electrode contact. We use a biotinylated thiol modification, in which a biotin-streptavidin-biotin bridge is used to form the single-protein junction. The resulting protein-coupled QMT probes enable the stable electrical measurement of the same single protein in solution for up to several hours. We also describe the analysis method used to interpret time-dependent single-protein conductance measurements, which can provide essential information for understanding electron transport and exploring protein dynamics. The total time required to complete the protocol is ~33 h and it can be carried out by users trained in less than 24 h.

Recent advances in single-cell subcellular sampling.

Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells enable cells to remain in their standard microenvironment with little impact on their viability. This negates the requirement of lysing cells to access their contents, an advancement from previous single-cell manipulation methods. These novel methods have the potential to be used for dynamic studies on single cells, with many already providing high intracellular spatial resolution. In this article, we highlight key technological advances that aim to remove the contents of living cells for downstream analysis. Recent applications of these techniques are reviewed, along with their current limitations. We also propose recommendations for expanding the scope of these technologies to achieve comprehensive single-cell tracking in the future, anticipating the discovery of subcellular mechanisms and novel therapeutic targets and treatments, ultimately transforming the fields of spatial transcriptomics and personalised medicine.

Nanopore sequencing of DNA-barcoded probes for highly multiplexed detection of microRNA, proteins and small biomarkers.

There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance clinical diagnostics beyond the single biomarker model. Here we perform nanopore sequencing of DNA-barcoded molecular probes engineered to recognize a panel of analytes. This allows for highly multiplexed and simultaneous quantitative detection of at least 40 targets, such as microRNAs, proteins and neurotransmitters, on the basis of the translocation dynamics of each probe as it passes through a nanopore. Our workflow is built around a commercially available MinION sequencing device, offering a one-hour turnaround time from sample preparation to results. We also demonstrate that the strategy can directly detect cardiovascular disease-associated microRNA from human serum without extraction or amplification. Due to the modularity of barcoded probes, the number and type of targets detected can be significantly expanded.