Publications
86 results found
Wang X, Ivanov AP, Edel JB, 2024, Biocompatible Biphasic Iontronics Enable Neuron-Like Ionic Signal Transmission., Research (Wash D C), Vol: 7
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.
Cai S, Ren R, He J, et al., 2023, Selective single-molecule nanopore detection of mpox A29 protein directly in biofluids, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 23, Pages: 11438-11446, ISSN: 1530-6984
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.
Koch C, Reilly-ODonnell B, Gutierrez R, et al., 2023, Nanopore sequencing of DNA-barcoded probes for highly multiplexed detection of microRNA, proteins and small biomarkers, Nature Nanotechnology, Vol: 18, Pages: 1483-1491, ISSN: 1748-3387
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.
Liu Y, Wang X, Campolo G, et al., 2023, Single-molecule detection of α-Synuclein oligomers in Parkinson's disease patients using nanopores, ACS Nano, Vol: 17, Pages: 22999-23009, ISSN: 1936-0851
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.
Ren R, Cai S, Fang X, et al., 2023, Multiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes, Nature Communications, Vol: 14, ISSN: 2041-1723
We report on single-molecule nanopore sensing combined with position-encoded DNA molecular probes, with chemistry tuned to simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity. We show that this sensing strategy can directly detect spike (S) and nucleocapsid (N) proteins in unprocessed human saliva. Moreover, our approach enables the identification of RNA fragments from patient samples using nasal/throat swabs, enabling the identification of critical mutations such as D614G, G446S, or Y144del among viral variants. In particular, it can detect and discriminate between SARS-CoV-2 lineages of wild-type B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.1.539 (Omicron) within a single measurement without the need for nucleic acid sequencing. The sensing strategy of the molecular probes is easily adaptable to other viral targets and diseases and can be expanded depending on the application required.
Kwan Z, Paulose Nadappuram B, Leung MM, et al., 2023, Microtubule-mediated regulation of β2AR translation and function in failing hearts, Circulation Research, Vol: 133, Pages: 944-958, ISSN: 0009-7330
BACKGROUND: β1AR (beta-1 adrenergic receptor) and β2AR (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 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: β1AR and β2AR mRNAs show differential localization in cardiomyocytes, with β1AR found in the perinuclear region and β2AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of β2AR transcripts toward the perinuclear region. The close proximity between β2AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of &be
Jiang T, Yi L, Liu X, et al., 2023, Fabrication of electron tunneling probes for measuring single-protein conductance, Nature Protocols, Vol: 18, Pages: 2579-2599, ISSN: 1750-2799
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.
Koch C, Reilley-O'Donnell B, Gutierrez R, et al., 2023, Novel screening platform for highly multiplexed biomarker detection, Publisher: SPRINGER, Pages: S190-S190, ISSN: 0175-7571
Sahota A, Monteza Cabrejos A, Kwan Z, et al., 2023, Recent advances in single-cell subcellular sampling., Chemical Communications, Vol: 59, Pages: 5312-5328, ISSN: 1359-7345
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.
Wang X, Thomas T-M, Ren R, et al., 2023, Nanopore detection using supercharged polypeptide molecular carriers, Journal of the American Chemical Society, Vol: 145, Pages: 6371-6382, ISSN: 0002-7863
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.
Fried JP, Swett JL, Nadappuram BP, et al., 2022, Localised solid-state nanopore fabrication via controlled breakdown using on-chip electrodes, Nano Research, Vol: 15, Pages: 9881-9889, ISSN: 1998-0124
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.
Tang L, Yi L, Jiang T, et al., 2022, Measuring conductance switching in single proteins using quantum tunneling, Science Advances, Vol: 8, Pages: 1-8, ISSN: 2375-2548
Interpreting the electrical signatures of single-proteins in electronic junctions has facilitated a better understanding of the intrinsic properties of proteins that are fundamental to chemical- and biological processes. Often such information is not accessible using ensemble and even single-molecule approaches. However, the fabrication of nanoscale single-protein junctions remains challenging as they often require sophisticated methods to ensure only a single protein is detected. We report on the fabrication of tunneling probes, direct measurement, and active control (switching) of single-protein conductance with an external field in solution. The probes allowed us to bridge a single streptavidin molecule to two independently addressable, biotin terminated electrodes and measure single protein tunneling response over long periods (up to 2 hours). We show that charge transport through the protein has multiple conductive pathways that depend on the magnitude of the applied bias. These findings open the door for the reliable fabrication of protein-based junctions, a better understanding of electronic properties of single protein molecules, and can enable their use in future protein-embedded bioelectronics applications.
Ren R, Sun M, Goel P, et al., 2021, Single-molecule binding assay using nanopores and dimeric NP conjugates, Advanced Materials, Vol: 33, ISSN: 0935-9648
The ability to measure biomarkers, both specifically and selectively at the single-molecule level in biological fluids, has the potential to transform the diagnosis, monitoring, and therapeutic intervention of diseases. The use of nanopores has been gaining prominence in this area, not only for sequencing but more recently in screening applications. The selectivity of nanopore sensing can be substantially improved with the use of tags, but substantial challenges remain, especially when trying to differentiate between bound from unbound targets. Here we design highly sensitive and selective molecular probes made from NPs designed specifically for nanopores that self-assemble and dimerise upon binding to a biological target. We show that both single and paired NPs can be successfully resolved while improving the time and sensitivity of the biomarker detection. Nanopore sensing with NP conjugates can be used for applications such as antigen/antibody detection for sepsis screening and miRNA sequence analysis relevant to prostate cancer. We believe that such technology opens the doors to developing a highly sensitive and selective strategy for diagnosis and screening of diseases without the need for sample processing or amplification while requiring minimal sample volume.
Fried JP, Swett JL, Nadappuram BP, et al., 2021, Understanding electrical conduction and nanopore formation during controlled breakdown, Small, Vol: 17, Pages: 1-9, ISSN: 1613-6810
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.
Oh S-H, Altug H, Jin X, et al., 2021, Nanophotonic biosensors harnessing van der Waals materials, NATURE COMMUNICATIONS, Vol: 12, ISSN: 2041-1723
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Al Sulaiman D, Gatehouse A, Ivanov AP, et al., 2021, Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA, ACS APPLIED MATERIALS & INTERFACES, Vol: 13, Pages: 26673-26681, ISSN: 1944-8244
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- Citations: 7
Cai S, Pataillot-Meakin T, Shibakawa A, et al., 2021, Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum, Nature Communications, Vol: 12, ISSN: 2041-1723
MicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 μl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer.
Ying Y-L, Ivanov AP, Tabard-Cossa V, 2021, No small matter, NATURE CHEMISTRY, Vol: 13, Pages: 216-217, ISSN: 1755-4330
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Fried JP, Swett JL, Nadappuram BP, et al., 2021, In situ solid-state nanopore fabrication., Chemical Society Reviews, ISSN: 0306-0012
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.
Tang L, Paulose Nadappuram B, Cadinu P, et al., 2021, Combined quantum tunnelling and dielectrophoretic trapping for molecular analysis at ultra-low analyte concentrations, Nature Communications, Vol: 12, Pages: 1-8, ISSN: 2041-1723
Quantum tunnelling offers a unique opportunity to study nanoscale objects with atomic resolution using electrical readout. However, practical implementation is impeded by the lack of simple, stable probes, that are required for successful operation. Existing platforms offer low throughput and operate in a limited range of analyte concentrations, as there is no active control to transport molecules to the sensor. We report on a standalone tunnelling probe based on double-barrelled capillary nanoelectrodes that do not require a conductive substrate to operate unlike other techniques, such as scanning tunnelling microscopy. These probes can be used to efficiently operate in solution environments and detect single molecules, including mononucleotides, oligonucleotides, and proteins. The probes are simple to fabricate, exhibit remarkable stability, and can be combined with dielectrophoretic trapping, enabling active analyte transport to the tunnelling sensor. The latter allows for up to 5-orders of magnitude increase in event detection rates and sub-femtomolar sensitivity.
Ren R, Wang X, Cai S, et al., 2020, Selective sensing of proteins using aptamer functionalised nanopore extended field-effect transistors, Small Methods, Vol: 4, Pages: 1-8, ISSN: 2366-9608
The ability to sense proteins and protein‐related interactions at the single‐molecule level is becoming of increasing importance to understand biological processes and diseases better. Single‐molecule sensors, such as nanopores have shown substantial promise for the label‐free detection of proteins; however, challenges remain due to the lack of selectivity and the need for relatively high analyte concentrations. An aptamer‐functionalized nanopore extended field‐effect transistor (nexFET) sensor is reported here, where protein transport can be controlled via the gate voltage that in turn improves single‐molecule sensitivity and analyte capture rates. Importantly, these sensors allow for selective detection, based on the choice of aptamer chemistry, and can provide a valuable addition to the existing methods for the analysis of proteins and biomarkers in biological fluids.
Xue L, Yamazaki H, Ren R, et al., 2020, Solid-state nanopore sensors (Sep, 10.1038/s41578-020-0229-6, 2020), NATURE REVIEWS MATERIALS, Vol: 5, Pages: 952-952, ISSN: 2058-8437
Xue L, Yamazaki H, Ren R, et al., 2020, Solid-state nanopore sensors, Nature Reviews Materials, Vol: 5, Pages: 931-951, ISSN: 2058-8437
Nanopore-based sensors have established themselves as a prominent tool for solution-based, single-molecule analysis of the key building blocks of life, including nucleic acids, proteins, glycans and a large pool of biomolecules that have an essential role in life and healthcare. The predominant molecular readout method is based on measuring the temporal fluctuations in the ionic current through the pore. Recent advances in materials science and surface chemistries have not only enabled more robust and sensitive devices but also facilitated alternative detection modalities based on field-effect transistors, quantum tunnelling and optical methods such as fluorescence and plasmonic sensing. In this Review, we discuss recent advances in nanopore fabrication and sensing strategies that endow nanopores not only with sensitivity but also with selectivity and high throughput, and highlight some of the challenges that still need to be addressed.
Wang X, Wilkinson MD, Lin X, et al., 2020, Correction: Single-molecule nanopore sensing of actin dynamics and drug binding, Chemical Science, Vol: 11, Pages: 8036-8038, ISSN: 2041-6520
Correction for ‘Single-molecule nanopore sensing of actin dynamics and drug binding’ by Xiaoyi Wang et al., Chem. Sci., 2020, 11, 970–979, DOI: 10.1039/C9SC05710B.
Clark R, Nawawi MA, Dobre A, et al., 2020, The effect of structural heterogeneity upon the microviscosity of ionic liquids, Chemical Science, Vol: 11, Pages: 6121-6133, ISSN: 2041-6520
The behaviour of two molecular rotors, one charged – 3,3′-diethylthiacarbocyanine iodide (Cy3) and one neutral – 8-[4-decyloxyphenyl]-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY-C10), have been studied in various ionic liquids. The fluorescent decay lifetime has been used to elucidate the structure of the immediate region around the rotor. The neutral BODIPY-C10 was found to prefer the non-polar alkyl chain environment, leading to two trends in the lifetime of the dye: one when it was fully partitioned into the non-polar domain, and one when it also sampled polar moieties. The positively charged Cy3 dye showed a complex relationship between the bulk viscosity of the ionic liquid and lifetime of the molecular rotor. This was attributed to a combination of polarity related spectral changes, changes in anion cages around the dye, and temperature dependent fluorescent lifetimes alongside the dependence of the rotor upon the viscosity.
Cadinu P, Kang M, Paulose Nadappuram B, et al., 2020, Individually addressable multi-nanopores for single-molecule targeted operations, Nano Letters, Vol: 20, Pages: 2012-2019, ISSN: 1530-6984
The fine-tuning of molecular transport is a ubiquitous problem of single-molecule methods. The latter is evident even in powerful single-molecule methods 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 DNA molecules with high efficiency and increased temporal resolution.
Wang X, Wilkinson MD, Lin X, et al., 2020, Single-molecule nanopore sensing of actin dynamics and drug binding, Chemical Science, Vol: 11, Pages: 970-979, ISSN: 2041-6520
Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores, we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, filament-growth, and drug-binding at the single-molecule level demonstrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.
Zhang Y, Takahashi Y, Hong SP, et al., 2019, High-resolution label-free 3D mapping of extracellular pH of single living cells, Nature Communications, Vol: 10, Pages: 1-9, ISSN: 2041-1723
Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity >0.01 units, 2 ms response time, and 50 nm spatial resolution. The technology was incorporated into a double-barrel nanoprobe integrating pH sensing with feedback-controlled distance sensing via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.
Wang X, Wilkinson MD, Lin X, et al., 2019, Single-molecule nanopore sensing of actin dynamics and drug binding
<jats:title>Abstract</jats:title><jats:p>Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.</jats:p>
Cai S, Sze JYY, Ivanov AP, et al., 2019, Small molecule electro-optical binding assay using nanopores., Nat Commun, Vol: 10
The identification of short nucleic acids and proteins at the single molecule level is a major driving force for the development of novel detection strategies. Nanopore sensing has been gaining in prominence due to its label-free operation and single molecule sensitivity. However, it remains challenging to detect small molecules selectively. Here we propose to combine the electrical sensing modality of a nanopore with fluorescence-based detection. Selectivity is achieved by grafting either molecular beacons, complementary DNA, or proteins to a DNA molecular carrier. We show that the fraction of synchronised events between the electrical and optical channels, can be used to perform single molecule binding assays without the need to directly label the analyte. Such a strategy can be used to detect targets in complex biological fluids such as human serum and urine. Future optimisation of this technology may enable novel assays for quantitative protein detection as well as gene mutation analysis with applications in next-generation clinical sample analysis.
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