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Scalable Production of DNA-Probe-Functionalized Heteromeric MspA Nanopores for Biosensing
Xialin Zhang1,2, Parker Hitt3, Meni Wanunu2,3,4
1Interdisciplinary Nanoscience Center, Aarhus University, Aarhus C 8000, Denmark.
ACS Applied Materials & Interfaces
|March 4, 2026
Summary
Researchers developed a new method to create specific nanopores for single-molecule sensing. This technique enables precise modification of Mycobacterium smegmatis porin A (MspA) for improved detection of analytes.
Area of Science:
- Biotechnology
- Nanotechnology
- Molecular Biology
Background:
- Biological nanopores offer potential for single-molecule sensing but often lack specificity due to similar analyte current signatures.
- Site-specific functionalization of multimeric nanopores, such as Mycobacterium smegmatis porin A (MspA), is challenging for improving selectivity.
- Current methods for isolating modified nanopores are labor-intensive, low-throughput, and can impair protein function.
Purpose of the Study:
- To develop a modular strategy for producing hetero-octameric MspA nanopores with single site-specific modifications.
- To establish a rapid, nondenaturing purification method for isolating functionalized MspA nanopores.
- To demonstrate the capability of engineered MspA nanopores for label-free and selective molecular detection.
Main Methods:
- Coexpression of MspA and a D56C MspA mutant in Escherichia coli (E. coli) to direct asymmetric pore assembly.
- Integration of magnetic bead capture with toehold-mediated DNA strand displacement for rapid, nondenaturing pore purification.
- Functionalization of purified MspA nanopores with DNA probes for targeting specific analytes (dopamine, microRNA, thrombin).
Main Results:
- Successful production of asymmetric MspA nanopores with defined subunit composition.
- Demonstration of selective enrichment of functionalized pores in under 3.5 hours, preserving structural integrity.
- Distinct current signatures observed upon target recognition, enabling label-free and selective detection of dopamine, microRNA, and thrombin.
Conclusions:
- The developed modular strategy enables robust and scalable production of site-specifically functionalized MspA nanopores.
- The rapid purification method effectively isolates functional nanopores while maintaining their structural integrity.
- Engineered MspA nanopores provide a versatile platform for sensitive and selective single-molecule detection across various applications.
