Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.

Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer-target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics.

Ion Concentration-Dependent Surface Charge Density Inside a Nanopore.

Surface charges shape the electrical double layer (EDL) structure at solid-liquid interfaces, critically influencing the performance of energy storage and micro/nanofluidic devices. However, accurately measuring surface charge density in nanoconfined spaces continues to be a challenge. Here, we introduce a methodology via solid-state nanopores that can investigate the dependence of surface charge density on salt concentrations and nanopore diameters. Measurements, complemented by a theoretical model, reveal that the surface charge density decreases as both the salt concentration in bulk solutions and the nanopore sizes are reduced. Notably, when the salt concentration in the bulk solution drops below 10-3 M, protons dominate ion conductance in a nanopore, resulting in a constant surface charge density. This study introduces an effective approach to surface charge characterization and may serve in the design of electrokinetically driven nanofluidic systems.

Fast Probing Amyloid Polymorphism via Nanopore Translocation.

Neurodegenerative diseases are characterized by the presence of cross-ß-sheet amyloid fibrils and a rich mesoscopic polymorphism, requiring noninvasive detection with high fidelity. Here, we introduce a methodology that can probe via a sensitive synthetic nanopore the complex polymorphism of amyloid fibrils by an automated and fast screening protocol. Statistically analyzing the translocation events on two model amyloid systems derived from ß-lactoglobulin and lysozyme allows extracting the cross-sectional configuration of hydrated amyloid fibrils from current block amplitude and correlating dwell time with fibril length. These findings are consistent with the amyloid polymorphs observed in solution by atomic force microscopy. Furthermore, the ionic current signal of a single translocation event can reveal abnormally aggregated conformations of amyloid fibrils without potential artifacts associated with microscopy methods. This study introduces an effective approach to physically discriminating and separating amyloid and may serve in the rapid diagnosis of early aggregating pathological amyloidosis.

Length-Dependent Collective Vibrational Dynamics in Alpha-Helices.

The front cover artwork is provided by Prof. Yunfei Chen's group at the Southeast University. The image shows collective vibrational motions of alpha-helices inside a protein. Studying the vibrations allows connecting protein structure and function, and therefore benefits de novo protein design. Read the full text of the Research Article at 10.1002/cphc.202200082.

Length-Dependent Collective Vibrational Dynamics in Alpha-Helices.

Functions of protein molecules in nature are closely associated with their well-defined three-dimensional structures and dynamics in body fluid. So far, many efforts have been made to reveal the relation of protein structure, dynamics, and function, but the structural origin of protein dynamics, especially for secondary structures as building blocks of conformation transition, is still ambiguous. Here we theoretically uncover the collective vibrations of elastic poly-alanine α-helices and find vibration patterns that are distinctively different over residue numbers ranging from 20 to 80. Contrary to the decreasing vibration magnitude from ends to the middle region for short helices, the vibration magnitude for long helices takes the minimum at approximately 1/5 of helix length from ends but reaches a peak at the center. Further analysis indicates that major vibrational modes of helical structures strongly depend on their lengths, where the twist mode dominates in the vibrations of short helices while the bend mode dominates the long ones analogous to an elastic Euler beam. The helix-coil transition pathway is also affected by the alternation of the first-order mode in helices with different lengths. The dynamic properties of the helical polypeptides are promising to be harnessed for de novo design of protein-based materials and artificial biomolecules in clinical treatments.

Fast Fabrication of Solid-State Nanopores for DNA Molecule Analysis.

Solid-state nanopores have been developed as a prominent tool for single molecule analysis in versatile applications. Although controlled dielectric breakdown (CDB) is the most accessible method for a single nanopore fabrication, it is still necessary to improve the fabrication efficiency and avoid the generation of multiple nanopores. In this work, we treated the SiNx membranes in the air-plasma before the CDB process, which shortened the time-to-pore-formation by orders of magnitude. λ-DNA translocation experiments validated the functionality of the pore and substantiated the presence of only a single pore on the membrane. Our fabricated pore could also be successfully used to detect short single-stranded DNA (ssDNA) fragments. Using to ionic current signals, ssDNA fragments with different lengths could be clearly distinguished. These results will provide a valuable reference for the nanopore fabrication and DNA analysis.

Detection and Separation of Single-Stranded DNA Fragments Using Solid-State Nanopores.

Many biological assays require effectively and sensitively sorting DNA fragments. Here, we demonstrate a solid-state nanopore platform for label-free detection and separation of short single-stranded DNA (ssDNA) fragments (<100 nt), based on their length-dependent translocation behaviors. Our experimental data show that each sized pore has a passable length threshold. The negative charged ssDNA fragments with length smaller than the threshold can be electrically facilitated driven through the correspondingly sized nanopore along the direction of electric field. In addition, the passable length threshold increases with the pore size enlarging. As a result, this phenomenon is able to be applicable for the controllable selectivity of ssDNA by tuning nanopore size, and the selectivity limitation is up to 30nt. Numerical simulation results indicate the translocation direction of ssDNA is governed by the competition of electroosmosis and electrophoresis effects on the ssDNA and offer the relationship between passable length threshold and pore size.