Related Experiment Video
Updated: Jun 14, 2026
11:55
Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution
Published on: August 16, 2016
12.1K
Engineering Linker-Enhanced OmpG Nanopores for Rapid Single-Molecule Protease Detection
1Food Science Department, University of Massachusetts Amherst, Amherst, MA 01003, USA.
Sensors (Basel, Switzerland)
|November 13, 2025
Summary
Engineered nanopore sensors detect protease activity with 95% efficiency. Linker design in OmpG nanopores is crucial for sensitive, real-time enzymatic cleavage detection in diagnostics.
Area of Science:
- Biophysics
- Biotechnology
- Analytical Chemistry
Background:
- Single-molecule nanopore sensors offer real-time detection of enzymatic activity.
- Sensitive and specific protease analysis is vital for diagnostics but remains challenging.
Purpose of the Study:
- To engineer an OmpG nanopore sensor for sensitive and specific protease activity detection.
- To optimize nanopore constructs using varied linkers for improved thrombin cleavage.
Main Methods:
- Engineered OmpG nanopore constructs with thrombin recognition peptides and flexible/charged linkers.
- Analyzed cleavage efficiency using SDS-PAGE.
- Performed single-channel recordings to assess pore conductance and real-time protease activity.
Main Results:
- Dual linkers and specific peptide placement achieved up to 95% cleavage efficiency.
- Linker integration modulated pore conductance, with extended loops showing intermediate currents.
- Observed rapid, irreversible current drops upon thrombin addition, confirming real-time detection.
Conclusions:
- Linker design (flexibility, charge) is critical for optimizing nanopore protease sensors.
- The engineered OmpG nanopore platform enables sensitive, real-time protease activity detection.
- This versatile platform holds promise for various biomedical applications.
