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<p>Protein nanopores have evolved into a powerful biophysical tool, from direct DNA sequencing to versatile molecular sensors that detect RNA, proteins, aptamers, and low-molecular-weight analytes. This review summarizes the current state of nanopore technologies for biopolymer sequencing and metabolite detection and discusses prospects for developing universal sensors based on engineered protein channels. We trace the evolution of nanopore applications from reading genomic DNA to direct detection of diverse molecules, including RNA, proteins with post-translational modifications, and metabolites. We analyze key physicochemical parameters of the pore that determine selectivity and sensitivity and outline engineering strategies to optimize constriction geometry, charge patterning, signal-to-noise ratio, and stability. Mechanisms for controlled molecular translocation are considered, including enzyme-driven pulling, electroosmotic capture, and other motor-free approaches. Different membrane systems, from lipid bilayers to polymer and hybrid matrices, are compared in terms of protein compatibility, noise, and lifetime. We also describe advances in electronic readout, including parallel recording, integrated analog-to-digital conversion, adaptive sampling, and embedded real-time processing. Finally, we highlight emerging applications, from protein sequencing with modification mapping to multiplexed biomarker assays and hybrid nanopore devices, and discuss how modelling, de novo design, and directed evolution can accelerate the development of universal nanopore biosensors for biomedical diagnostics.</p>

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Biophysics of nanopore technologies: a roadmap for engineering protein channels as universal molecular sensors

  • 期刊论文

  • 发表日期：

    2026年2月17日

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  • Aleksandr S. Petrov,
  • Vasiliy V. Belov,
  • Alexander V. Sergeev,
  • Maria I. Zvereva

摘要

Protein nanopores have evolved into a powerful biophysical tool, from direct DNA sequencing to versatile molecular sensors that detect RNA, proteins, aptamers, and low-molecular-weight analytes. This review summarizes the current state of nanopore technologies for biopolymer sequencing and metabolite detection and discusses prospects for developing universal sensors based on engineered protein channels. We trace the evolution of nanopore applications from reading genomic DNA to direct detection of diverse molecules, including RNA, proteins with post-translational modifications, and metabolites. We analyze key physicochemical parameters of the pore that determine selectivity and sensitivity and outline engineering strategies to optimize constriction geometry, charge patterning, signal-to-noise ratio, and stability. Mechanisms for controlled molecular translocation are considered, including enzyme-driven pulling, electroosmotic capture, and other motor-free approaches. Different membrane systems, from lipid bilayers to polymer and hybrid matrices, are compared in terms of protein compatibility, noise, and lifetime. We also describe advances in electronic readout, including parallel recording, integrated analog-to-digital conversion, adaptive sampling, and embedded real-time processing. Finally, we highlight emerging applications, from protein sequencing with modification mapping to multiplexed biomarker assays and hybrid nanopore devices, and discuss how modelling, de novo design, and directed evolution can accelerate the development of universal nanopore biosensors for biomedical diagnostics.