# Engineering Linker-Enhanced OmpG Nanopores for Rapid Single-Molecule Protease Detection

## TL;DR

Researchers improved OmpG nanopores by adding linkers, enabling more sensitive and efficient detection of protease activity at the single-molecule level.

## Contribution

The use of dual flexible and charged linkers in OmpG nanopores significantly enhances substrate accessibility and cleavage efficiency for protease detection.

## Key findings

- OmpG constructs with dual linkers achieved up to 95% cleavage efficiency, compared to limited cleavage without linkers.
- Linker integration modulated pore conductance, with extended loops showing intermediate open-state currents near 18 pA.
- Thrombin addition caused rapid and irreversible current drops, confirming real-time protease activity detection.

## Abstract

What are the main findings?
OmpG nanopores enable sensitive single-molecule detection of protease activity.Loop length and charge modifications via linkers boost substrate accessibility.

OmpG nanopores enable sensitive single-molecule detection of protease activity.

Loop length and charge modifications via linkers boost substrate accessibility.

What are the implications of the main finding?
Dual linkers enhance substrate accessibility and cleavage efficiency in nanopores.Engineered OmpG provides a versatile platform for protease biosensing applications.

Dual linkers enhance substrate accessibility and cleavage efficiency in nanopores.

Engineered OmpG provides a versatile platform for protease biosensing applications.

Single-molecule nanopore sensors have enabled real-time detection of enzymatic cleavage events, yet achieving sensitive and specific analysis of protease activity remains an important challenge for diagnostic applications. We engineered OmpG nanopore constructs incorporating thrombin recognition peptides into loop 6 with varied flexible and negatively charged linkers to optimize accessibility and cleavage. SDS-PAGE gel analysis showed that constructs with the recognition peptide placed after residue D225 and incorporating dual linkers achieved cleavage efficiencies up to 95%, whereas constructs without linkers showed limited cleavage. Single-channel recordings revealed that linker integration modulates pore conductance, with extended loops exhibiting intermediate open-state currents near 18 pA compared to 25 pA in wild-type OmpG. Upon thrombin addition, rapid and irreversible current drops confirmed real-time protease activity detection. These results demonstrate the critical role of linker design, particularly flexibility and charge, in optimizing nanopore protease sensors, providing a versatile platform for biomedical applications.