A generalizable nanopore sensor for highly specific protein detection at single-molecule precision

Abstract

Protein detection has wide-ranging implications in molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific interfaces for detecting proteins without the steric hindrance of the pore interior. Here, we formulate a class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. The outcomes of this work could impact biomedical diagnostics by providing a fundamental basis for biomarker detection in biofluids.

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

“…Here, we emphasize that the orientation of the recognition element with respect to the pore opening and the geometry of its complex with the protein analyte play a pivotal role in the overall sensor design. For example, the adaptor is sometimes unnecessary for transducing the protein captures into an electrical readout. ,, In this study, we clarify that protein detection cannot be achieved without an adaptor peptide. Furthermore, using ND-BLI facilitated the outcome that weak nonspecific interactions between the adaptor and tFhuA do not impact the real-time kinetics.…”

“…All MLL4 Win tFhuA proteins were refolded in n -dodecyl-β- d -maltopyranoside (DDM; Anatrace, Maumee, OH) as previously described. , After 72 h of dialysis in 200 mM KCl, 20 mM Tris-HCl, pH 8 at 4 °C, the refolded proteins were centrifuged to remove unfolded precipitates.…”

“…A single nanopore is a versatile sensing element for numerous tasks in protein analytics. − Significant progress has been accomplished in basic research and biosensing technology using nanopores − fabricated in various scaffolds and materials. − The readout signal in these sensors is the transmembrane current through a nanopore . Key advantages that make this approach influential include the following: (i) this label-free method probes time-resolved molecular events at a single-molecule level; − (ii) the nanopore structure and composition can be altered with atomic precision; , (iii) nanopores are amenable to automated microelectrode recording technologies; − (iv) electrical recordings with single nanopores can be conducted in a broad dynamic range of interactions and analyte concentrations; (v) specific and sensitive detection can be performed in challenging heterogeneous solutions, such as biofluids, − or in complex mixtures of proteins . Therefore, this approach shows promise in the wide-time bandwidth evaluation of single-protein dynamics.…”

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection

“…Yet, we are confident that the presented method is extremely versatile and amenable for optimization and opens new horizons, such as (i) the existence of multiple branched moieties in the form of Alm-PNA subunits from a membrane-residing oligomer could enable multivalent capture and therefore further signal amplification during detection; 65 (ii) by employing linkers of various lengths between the voltage-gated alamethicin-based peptide and the probe PNA segment, it may be possible to improve the spatial mobility of the attached PNA moieties and augment the hybridization detection; (iii) the ability to programmatically engineer multiple affinities for targets binding, namely, to generate a mixture of Alm-PNA fragments, where PNAs are designed as replaceable moieties to target distinct ssDNA fragments, may facilitate the simultaneous detection of nucleic acid fragments of diverse lengths and primary sequence; (iv) the proposed biosensor design may extend its versatility toward detecting widespread metal pollutants, e.g., Hg 2+ , by relying on thymidine−Hg 2+ −thymidine coordination chemistry modulating ssDNA−PNA receptor chimera interactions. In other words, the deliberately designed T−T mismatches between aqueously added ssDNAs and PNA-appended receptor chimeras will effectively bind Hg 2+ ions in solution and generate hybridized duplexes near oligomer's entrance, leading to Hg 2+ -induced changes in the oligomer's gating dynamics and therefore detection; and (v) last but not least, as a clear advantage of our solution, we emphasize that the straightforward fabrication procedures of synthetic receptors resembling a barrel-stave-like dynamic nanopore render its diameter adjustable, which enables a wider sensing range, to encompass not only small ssDNAs but also protein targets, 65,66 without sacrificing the sensitivity or reproducibility.…”

Highly parallel, wash-free, and ultrasensitive centrifugal droplet digital protein detection in sub-microliter blood

“…Nanopore-based technology offers an alternative single molecule approach for the sensitive detection of proteins as well as for the identification of post-translational modifications of proteins (and peptides). 12–15 At present, this powerful technology is still evolving towards the development of diagnostic devices compatible with POCT.…”