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Hybrid Nanopore Combines Precision of Protein Pores with Robustness of Solid-State Membranes


By Monica Heger

This story was originally published Dec. 16.

Looking to combine the best of the protein nanopore and solid-state nanopore worlds, researchers from the Kavli Institute of Nanoscience in the Netherlands have created a hybrid nanopore by inserting an alpha-hemolysin protein pore into a silicon nitride membrane.

Integrating a biological pore into a solid-state device "opens up avenues towards the creation of wafer-scale parallel device arrays that may be useful for genomic sequencing," according to the researchers, who published the work last month in Nature Nanotechnology.

Protein pores are precise down to the atomic level, but the lipid bilayers in which they reside tend to be fragile. Solid-state nanopores, on the other hand, are much sturdier and can more easily be coupled to electronics; however the actual pores are difficult to reproduce, and tend to have different characteristics from each other, including the amount of noise, size of the pore, and charge.

In response, the Kavli team created a hybrid nanopore that "combines the precise structure and protein engineering possibilities associated with a biological pore with the robustness and potential required for the fabrication of an integrated device," the authors wrote.

"We have been trying to do the same thing, but they beat us to it," said David Deamer, a professor of chemistry at the University of California, Santa Cruz, and a leader in the protein nanopore field. The hybrid pore is a "great advantage," he said, and "it looks like they've got it working."

The key to the work was connecting a piece of double-stranded DNA to the alpha-hemolysin nanopore, which helped guide the pore into the silicon membrane when voltage was applied.

"It's not obvious at all that if you take a membrane protein like alpha-hemolysin out of its lipid bilayer surroundings, that it can survive and be functional," said Cees Dekker, a researcher at the Kavli Institute who led the study. So it was "an important surprise" that the pore was "stable enough to insert [in the silicon membrane] and not de-fold or deform."

"The challenge was to be able to reproducibly insert a protein into a hole. Your target is a couple of nanometers [in diameter] and you need to fit this protein channel into that hole and get it to seal well," said Mark Akeson, a professor of biomolecular engineering at UC Santa Cruz, who also works on protein nanopores. "It's not a trivial thing to do at all."

Akeson's team recently figured out a way to control the rate of translocation through a protein nanopore (IS 9/28/2010), and subsequently improved on that technique in a recent paper in the Journal of the American Chemical Society.

Creating a protein pore in a lipid membrane that is stable is still a major challenge, which is why researchers are exploring hybrid pores. To create the hybrid pore, Dekker's team first mutated the alpha-hemolysin pore so that it included an 11-amino acid loop. The loop also contained a 12-base DNA oligomer, which acted as an attachment point for a piece of double-stranded DNA with a complementary single-stranded end.

The team then made a hole in a silicon nitride membrane large enough to allow passage of double-stranded DNA and the stem of the alpha-hemolysin protein, but small enough so that the base of it could not fit. Applying an electric field to the membrane pulled the DNA, which was attached to the alpha-hemolysin pore, through the silicon nitride hole until the protein was lodged into the hole.

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"It's a neat idea because it's just a piece of DNA, not any specific sequence, and it is able to pull the alpha-hemolysin right into the hole and hold it there," Deamer said.

After inserting the pore into the solid-state membrane, the team next demonstrated that it was functional by translocating single-stranded DNA oligomers through the pore. The blockage current was similar to other demonstrations of DNA translocation through an alpha-hemolysin protein pore, suggesting that the pore was functional.

"Transient conductance blockades are observed, indicating the passage of individual nucleic acid molecules, and importantly, demonstrating the presence of a functional, non-denatured alpha-hemolysin protein within the structure," the authors wrote.

The next step is to demonstrate that the technique is highly reproducible so that researchers can build chips containing hundreds or thousands of these nanopores. In the current study, the team successfully inserted the alpha-hemolysin protein into the solid-state membrane in 21 out of 60 attempts, or approximately 35 percent of the time. This success rate will have to be improved upon in order to scale up the approach.

Nevertheless, Akeson added that the work opens up the possibility of integrating electronics with protein nanopores. "This combines the stability of the solid-state pore with the potential of bringing electronics to the protein nanopore," he said. "So it's conceivable now that someone could engineer electronics to the protein."

Hagan Bayley from the University of Oxford, whose work on nanopores has been licensed by Oxford Nanopore, is a co-author on the Nature Nanotechnology paper, but Dekker said he has no plans to license this technique.

Have topics you'd like to see covered in In Sequence? Contact the editor at mheger [at] genomeweb [.] com.