Modified ClyA Nanopore Biosensor Voltage-Dependent Gating
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Solution Overview
Problem
Current nanopore biosensors face challenges in achieving high sensitivity, water solubility, and stability across a range of potentials, particularly when analyzing large molecules like folded proteins, due to issues with mechanical stability, size limitations, and electrical noise.
Innovation Solution
A modified ClyA nanopore with specific amino acid substitutions, such as Cys to Ser or Ala, and additional residues like L99Q, E103G, and K294R, which forms oligomers with varying conductance properties, allowing for the analysis of proteins up to 70 kDa and maintaining stability from -60 to -150 mV, and reduced electrical noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the nanopore internal diameter is reduced to analyze smaller molecules, then measurement precision improves, but adaptability deteriorates because folded proteins and large nucleic acids cannot pass through
Solution Approach 1:
The patent introduces voltage-dependent gating behavior that allows the nanopore to dynamically adjust its effective aperture. At low voltages, the pore remains constricted for precise small molecule detection. At high voltages, the pore opens to accommodate large folded proteins and nucleic acids, enabling the system to adapt its detection capability based on operating conditions.
Solution Approach 2:
The patent modifies the nanopore's physical and chemical parameters through specific amino acid substitutions (Cys to Ser/Ala at positions 87 and 285, plus L99Q, E103G, K294R) to achieve augmented activity, solubility, and electrical properties. These parameter changes enable the pore to maintain stability while accommodating a broader range of molecule sizes.
2Reliability
If the nanopore is made more stable mechanically, then reliability improves, but ease of manufacture deteriorates due to complex engineering requirements
Solution Approach 1:
The patent achieves enhanced mechanical stability and solubility through specific amino acid parameter changes: substituting Cys with Ser or Ala at positions 87 and 285, and introducing L99Q, E103G, and K294R substitutions. These targeted parameter modifications improve reliability without requiring complex engineering processes.
3Productivity
If the nanopore remains open at high applied potentials, then productivity improves, but electrical noise increases
Solution Approach 1:
The patent modifies the electrical properties of the nanopore through amino acid substitutions that alter the vestibule environment and charge distribution. The K294R substitution particularly affects electrical characteristics, enabling the pore to maintain stability and reduce noise while operating at high potentials, thus improving productivity without excessive noise generation.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified ClyA nanopore demonstrates enhanced solubility, stability, and sensitivity, enabling the detection and analysis of protein analytes and nucleic acids, including dsDNA, with improved voltage-dependent gating and reduced noise, facilitating prolonged current blockades for precise molecular analysis.
Implementation Method 1
proteins such as thrombin (37 kDa) or malate dehydrogenase (dimer, 35 kDa monomer) can be electrophoretically trapped between the wide cis entrance (5.5 nm, table 1) and the narrower trans exit (3.3 nm, table 1), and can therefore be sampled for several minutes
Implementation Method 2
proteins such as thrombin (37 kDa) or malate dehydrogenase (dimer, 35 kDa monomer) can be electrophoretically trapped between the wide cis entrance (5.5 nm, table 1) and the narrower trans exit (3.3 nm, table 1), and can therefore be sampled for several minutes
Data Source
AI summary
Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromolecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.