DNAzyme-Assisted the Detection of rps27l mRNA in Protein Nanopores

Analytica Chimica Acta, Journal Year: 2025, Volume and Issue: unknown, P. 343711 - 343711

Published: Jan. 1, 2025

Language: Английский

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Aerolysin Nanopore Electrochemistry

Accounts of Chemical Research, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 28, 2025

ConspectusIons are the crucial signaling components for living organisms. In cells, their transportation across pore-forming membrane proteins is vital regulating physiological functions, such as generating ionic current signals in response to target molecule recognition. This ion transport affected by confined interactions and local environments within protein pore. Therefore, can efficiently transduce characteristics of each into ion-transport-mediated with high sensitivity. Inspired nature, various pores have been developed high-throughput label-free nanopore sensors single-molecule detection, enabling rapid accurate readouts. particular, aerolysin, a key virulence factor Aeromonas hydrophila, exhibits sensitivity fingerprints detecting subtle differences sequence, conformation, structure DNA, proteins, polypeptides, oligosaccharides, other molecules. Aerolysin features cap that approximately 14 nm wide on cis side central pore about 10 long minimum diameter around 1 nm. Its lumen, 11 charged rings at two entrances neutral amino acids between, facilitates dwelling single analyte characteristic enables rich between well-defined residues analyte. As result, signal offers unique molecular fingerprint, extending beyond traditional volume exclusion model sensing. 2006, aerolysin was first reported discriminate conformational peptides, opening door rapidly growing field electrochemistry. Over years, mutant nanopores emerged, associated advanced instrumentation data analysis algorithms, simultaneous identification over 30 targets number still increasing. electrochemistry particular allows time-resolved qualitative quantitative ranging from DNA sequencing, proteomics, enzyme kinetics, reactions potential clinical diagnostics. Especially, feasibility dynamic would revolutionize omics studies level, paving way promising temporal omics. Despite success this approach so far, it remains challenging understand how correlate distinguishable signatures. Recent attempts added correction terms account variations mobility caused Account, we revisit origin blockade induced molecules inside nanopore. We highlight contributions noncovalent sensing ability through corrected conductance model. Account then describes design interaction networks nanopore, including electrostatic, hydrophobic, hydrogen-bonding, cation−π, ion–charged acid interactions, ultrasensitive biomolecular quantification. Finally, provide an outlook further understanding network improving manipulating fine-tuning toward broad range practical applications.

Language: Английский

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Single-Molecule Identification and Quantification of Steviol Glycosides with a Deep Learning-Powered Nanopore Sensor

ACS Nano, Journal Year: 2024, Volume and Issue: 18(36), P. 25155 - 25169

Published: Aug. 27, 2024

Steviol glycosides (SGs) are a class of high-potency noncalorie natural sweeteners made up common diterpenoid core and varying glycans. Thus, the diversity glycans in composition, linkage, isomerism results tremendous structural complexity SG family, which poses challenges for precise identification leads to fact that SGs frequently used mixtures their variances biological activity remain largely unexplored. Here we show wild-type aerolysin nanopore can detect discriminate diverse species through modulable electro-osmotic flow effect at varied applied voltages. At low voltages, neutral molecule was drawn stuck pore entrance due an energy barrier around R220 sites. The ensuing binding events enable majority species. Increasing voltage break cause translocation events, allowing unambiguous several pairs differing by only one hydroxyl group recognition accumulation from multiple sensing regions Based on data 15 SGs, deep learning-based artificial intelligence (AI) model created process individual blockage achieving rapid, automated, single-molecule quantification real samples. This work highlights value analysis complex glycans-containing glycosides, as well potential sensitive rapid quality assurance glycoside products with use AI.

Language: Английский

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Obtaining Narrow Distributions of Single-Molecule Peptide Signals Enables Sensitive Peptide Discrimination with α-Hemolysin Nanopores

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: March 10, 2025

Biological nanopore technology has emerged as a promising tool for analyzing peptides and post-translational modifications at the single-molecule level. However, broader application is currently limited by partial separation of low-throughput, mainly due to nonuniform peptide signals detected nanopores. Narrowing signal distribution crucial improving nanopore's sensing ability but remains bottleneck. Here, we demonstrate that capturing with electrophoretic force against electroosmotic flow can provoke more uniform blockades in α-hemolysin By using buffers 2 M KCl pH 3.8, obtain most signals, which may be correlated shape, linearization, actual dwelling position peptides. Five acetylation phosphorylation, including isomeric peptides, readily separated from each other. The citrullination replacement arginine β-hydroxybutyrylation modification another sequence are also discriminated mixture. A series different compositions induced when they were analyzed our method. Our work presents an efficient approach optimize analysis

Language: Английский

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Single-Molecule Resolution of Oligopeptides in Anti-Aging Cosmetics Combined with Nanopore Readouts and Deep Learning Model

Research Square (Research Square), Journal Year: 2025, Volume and Issue: unknown

Published: March 19, 2025

Oligopeptides in anti-aging cosmetics stand out as active ingredients to interact with skin cell and accelerate the collagen synthesis fibroblast proliferation. Some of them act neurotransmitter- or enzyme inhibitor, while others are signal carrier peptides. The ensemble techniques for extraction analysis bioactive peptides cosmetic production involve ultrafiltration, enzymatic hydrolysis, fermentation, high-performance liquid chromatography. This work provides a single-molecule approach resolution various typical oligopeptides cosmetics. A nanopore an aperture diameter ~ 2 nm is efficient shortest tripeptide achieve good noise ratio translocation frequency. Oligoeptides three eight amino acids could be discriminated single SiN_x nanopore, this also proved by AI modeling accuracy 90%, except argireline hexa-peptide-9 that hold similar behavior KCl. assay commercial reveals product from Viribati purer than other two harbors majority according comparison setups device. Our significant insight qualification quality control market.

Language: Английский

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A novel MoS2/GA electrode based on hybrid capacitive deionization technology for ion removal in polypeptide solution

Separation and Purification Technology, Journal Year: 2025, Volume and Issue: unknown, P. 132658 - 132658

Published: March 1, 2025

Language: Английский

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Nanopore sensing of protein and peptide conformation for point-of-care applications

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: April 4, 2025

Abstract The global population’s aging and growth will likely result in an increase chronic aging-related diseases. Early diagnosis could improve the medical care quality of life. Many diseases are linked to misfolding or conformational changes biomarker peptides proteins, which affect their function binding properties. Current clinical methods struggle detect quantify these changes. Therefore, there is a need for sensitive sensors that can low-concentration analytes biofluids. Nanopore electrical detection has shown potential sensing subtle protein peptide conformation This technique single molecules label-free while distinguishing shape physicochemical property Its proven sensitivity makes nanopore technology promising ultra-sensitive, personalized point-of-care devices. We focus on capability detecting quantifying modifications enantiomers proteins discuss this as solution future societal health challenges.

Language: Английский

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Single-Molecule Nanopore Sensing of Proline cis/trans Amide Isomers

Chemical Science, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Molecules known as stereoisomers possess identical numbers and types of atoms, which are oriented differently in space. Cis-trans isomerization proline, a distinctive case stereoisomerism peptides proteins, includes the rearrangement chemical groups around an acyl-proline amide bond that bears partial double character. Many cellular processes affected by cis-trans proline associated conformational protein interconversions. This work explored conformer ratio natural chemically modified prolines using aerolysin pore nanosensor. Despite well-known involvement folding, stability, aggregation, highly demanding discrimination cis trans isomers Xaa-Pro peptide has not so far been reported at single-molecule level electrical detection with nanopore. For proline-rich 19 amino acid residue fragment Dynamin 2 protein, one subfamilies GTP-binding third sequence was substituted two stereoisomeric 4-fluoroprolines. The nanopore experiments were able to sense influence fluorination shifting cis/trans conformers' equilibrium compared proline: for 4-(R)-fluoroproline, isomer is more favored, while opposite shift observed 4-(S)-fluoroproline. NMR spectroscopy used validate results. Overall, our findings demonstrate high sensitivity sensing analytical tool stereoisomer identification within peptides.

Language: Английский

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Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision

ACS Nano, Journal Year: 2024, Volume and Issue: 18(31), P. 20468 - 20481

Published: July 25, 2024

Transcription factors often interact with other protein cofactors, regulating gene expression. Direct detection of these brief events using existing technologies remains challenging due to their transient nature. In addition, intrinsically disordered domains, intranuclear location, and lack cofactor-dependent active sites transcription further complicate the quantitative analysis critical processes. Here, we create a genetically encoded label-free sensor identify interaction between motif MYC factor, primary cancer driver, WDR5, chromatin-associated hub. Using an engineered nanopore equipped this motif, WDR5 is probed through reversible captures releases in one-by-one time-resolved fashion. Our single-molecule kinetic measurements indicate weak-affinity arising from relatively slow complex association fast dissociation tethered motif. Further, validate subtle by determinations ensemble single nanodisc-wrapped nanopores immobilized on biolayer interferometry sensor. This study also provides proof-of-concept for that reveals unique recognition signatures different binding sites. foundational work may be developed produce sensing elements analytical proteomics nanomedicine.

Language: Английский

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Single-Molecule Observation of Competitive Protein–Protein Interactions Utilizing a Nanopore

ACS Nano, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 24, 2024

Two or more protein ligands may compete against each other to interact transiently with a receptor. While this is ubiquitous phenomenon in cell signaling, existing technologies cannot identify its kinetic complexity because specific subpopulations of binding events different are hidden the averaging process an ensemble. In addition, limited time resolution prevailing methods makes detecting and discriminating among diverse interacting partners challenging. Here, we utilize genetically encoded nanopore sensor disentangle competitive protein–protein interactions (PPIs) one-on-one label-free fashion. Our measurements involve binary mixtures varying affinity same receptor, which was externally immobilized on tip. We use resistive-pulse technique monitor kinetics dynamics reversible PPIs without confinement, high-time bandwidth, at titratable ligand concentrations. way, systematically evaluate how individual take their turn reside receptor's site. Further, our single-molecule determinations these quantitatively compared data generated by two-ligand, one-receptor queuing model. The outcomes work provide fundamental basis for future developments aimed better mechanistic understanding PPIs. Moreover, they also form platform drug development pipelines targeting high-complexity mediated hubs.

Language: Английский

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