13 research outputs found
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
Fabrication and characterization of dual function nanoscale pH-scanning ion conductance microscopy (SICM) probes for high resolution pH mapping
The easy fabrication and use of nanoscale dual function pH-scanning ion conductance microscopy (SICM) probes is reported. These probes incorporate an iridium oxide coated carbon electrode for pH measurement and an SICM barrel for distance control, enabling simultaneous pH and topography mapping. These pH-SICM probes were fabricated rapidly from laser pulled theta quartz pipets, with the pH electrode prepared by in situ carbon filling of one of the barrels by the pyrolytic decomposition of butane, followed by electrodeposition of a thin layer of hydrous iridium oxide. The other barrel was filled with an electrolyte solution and Ag/AgCl electrode as part of a conductance cell for SICM. The fabricated probes, with pH and SICM sensing elements typically on the 100 nm scale, were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and various electrochemical measurements. They showed a linear super-Nernstian pH response over a range of pH (pH 2–10). The capability of the pH-SICM probe was demonstrated by detecting both pH and topographical changes during the dissolution of a calcite microcrystal in aqueous solution. This system illustrates the quantitative nature of pH-SICM imaging, because the dissolution process changes the crystal height and interfacial pH (compared to bulk), and each is sensitive to the rate. Both measurements reveal similar dissolution rates, which are in agreement with previously reported literature values measured by classical bulk methods
Microtubule-mediated regulation of  β2AR translation and unction in failing hearts
Background: Beta-1 adrenergic receptor (β 1 AR)- and Beta-2 adrenergic receptor (β 2 AR)-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 β-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 β-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. Methods: The localization pattern of β-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on β-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 β-AR translation sites in cardiomyocytes. The mechanism by which β-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: β 1 AR and β 2 AR mRNAs show differential localization in cardiomyocytes, with β 1 AR found in the perinuclear region and β 2 AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of β 2 AR transcripts toward the perinuclear region. The close proximity between β 2 AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of β 2 AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both β 1 AR and β 2 AR 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 β-AR mRNA redistribution and impaired β 2 AR function in failing hearts. Conclusions: Asymmetrical microtubule-dependent trafficking dictates differential β 1 AR and β 2 AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 β-ARs on the plasma membrane. The localization pattern is altered post–myocardial infarction, resulting from t-tubule remodeling, leading to distorted β 2 AR-mediated cyclic adenosine monophosphate signaling
Quad-Barrel Multifunctional Electrochemical and Ion Conductance Probe for Voltammetric Analysis and Imaging
In situ solid-state nanopore fabrication
This review summarises the development of in situ solid-state nanopore fabrication techniques. These techniques are democratising solid-state nanopore research by providing rapid and accessible methods to fabricate nanopores
Gated single-molecule transport in double-barreled nanopores
Single-molecule methods have been rapidly developing with the appealing prospect of transforming conventional ensemble-averaged analytical techniques. However, challenges remain especially in improving detection sensitivity and controlling molecular transport. In this article, we present a direct method for the fabrication of analytical sensors that combine the advantages of nanopores and field-effect transistors for simultaneous label-free single-molecule detection and manipulation. We show that these hybrid sensors have perfectly aligned nanopores and field-effect transistor components making it possible to detect molecular events with up to near 100% synchronization. Furthermore, we show that the transport across the nanopore can be voltage-gated to switch on/off translocations in real time. Finally, surface functionalization of the gate electrode can also be used to fine tune transport properties enabling more active control over the translocation velocity and capture rates
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 Si3N4 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
Localised solid-state nanopore fabrication via controlled breakdown using on-chip electrodes
Controlled breakdown has recently emerged as a highly accessible technique to fabricate solid-state nanopores. However, in its most common form, controlled breakdown creates a single nanopore at an arbitrary location in the membrane. Here, we introduce a new strategy whereby breakdown is performed by applying the electric field between an on-chip electrode and an electrolyte solution in contact with the opposite side of the membrane. We demonstrate two advantages of this method. First, we can independently fabricate multiple nanopores at given positions in the membrane by localising the applied field to the electrode. Second, we can create nanopores that are self-aligned with complementary nanoelectrodes by applying voltages to the on-chip electrodes to locally heat the membrane during controlled breakdown. This new controlled breakdown method provides a path towards the affordable, rapid, and automatable fabrication of arrays of nanopores self-aligned with complementary on-chip nanostructures
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