Small-scale nanopore sensor technology has enjoyed use in high-throughput and low-cost DNA sequencing. Now, University of Groningen scientists have demonstrated that a patented biological nanopore can read fingerprints of proteins and peptides, even at the single amino acid level.
In developing their nanopore methodology, the team addressed two crucial challenges: getting polypeptides into the pore and identifying differences in proteins by measuring changes in current.
"If the direction of the ion current can be controlled, a fluid flow strong enough to transport polypeptides can be generated,” said associate professor Giovanni Maglia. “We did this by tuning the charges inside the pore wall. By changing the pH of the medium, it was possible to fine-tune the balance between the electro-osmotic flow and the force of the electric field that was applied across the pore.”
Using this electro-osmotic flow to drive the polypeptides into the pores, the team was able to test five different different polypeptides ranging from 1 to 25 kilodaltons. These include biomarkers for a number of diseases, such as chymotrypsin (pancreatic cysts), endothelin-1 (bronchiolitis obliterans), and human EGF (chronic kidney disease).
As the tested polypeptides enter the pore, a current generated produces a unique fingerprint. Remarkably, the team has even managed to distinguish two versions for endothelin that differ by just one amino acid.
The key behind this technology is the nanopore, Fragaceatoxin C (FraC), which has been engineered to to allow the entry of polypeptides at a fixed potential regardless of the charge composition of the polypeptide. Moreover, while past pores have adopted a barrel-shaped structure, FraC’s alpha helical design features a narrow, funnel end from which measurements are made. This allows easier tuning for single amino acids.
“It is a major advantage that nanopore technology has already been developed for DNA sequencing. This technology is fast, cheap and robust: nanopore sequencing devices are used in the field and one has even been sent up to the International Space Station,” says Maglia. “Molecular diagnostics and biomarker discovery should benefit particularly from the single-molecule characterization of proteomes.”
These findings were published earlier today in Nature Communications.