Although significant advancements have been made with biological nanopores toward next-generation sequencing, the first commercial nanopore approaches will most likely utilize biological channels. But solid-state counterparts also pose attractive possibilities.
The solid-state membrane made of silicon nitride or other material can replace the lipid bilayer, and a focused electron beam is used to drill a nanometer scale pore through the membrane. Since the first report in 2001 by Golovchenko, many groups have reported forming pores and translocation of DNA under applied electric fields.
The solid-state pores possess superior mechanical and thermal characteristics compared to their biological counterparts. These devices have been used to study the fundamental biophysical phenomenon of molecules moving through nanopores, the detection of nucleic acid molecules such as miRNA, epigenetics for diagnostic applications and, of course, the quest for direct label-free detection of DNA sequences.
This approach, however, requires the ability to form ultrathin membranes and narrow pores comparable to the size of single nucleotides. Atomic layer deposition of solid-state materials is an attractive option to make thin membranes, as it can allow for precise control of membrane thickness and material composition. It has been used to make nanopores in aluminum oxide.
However, the goal to make a membrane thin enough to allow the interrogation of a single or just a few bases remained a challenge until the last few years.
Recent developments in the exploration of graphene materials offer exciting possibilities. Along with its unique physical structure and high electronic mobility, this two-dimensional sheet of carbon atoms has a thickness that is comparable to the spacing between nucleotides in ssDNA (0.32–0.52 nm). As a result, the use of graphene as a membrane is particularly attractive for electronic DNA sequencing.
Drndic and co-workers first demonstrated the fabrication of nanopores in suspended graphene films. Subsequently, groups led by Golovchenko, Drndic, and Dekker reported the DNA translocation through nanopores made of graphene. This field is quite nascent with only a handful of studies having been reported to date.
As graphene is electrically conducting in the plane of the 2D lattice, these ultrathin electrodes could be used to provide new device and sensing topologies such as graphene nano ribbons and graphene-based tunnel junctions. The speed of the molecule is still too fast and methods to slow down the molecule are being pursued.
The use of nanopore-based sensors to perform long base reads on unlabeled, single-stranded DNA molecules quickly and cheaply can revolutionize the fields of genomics and personalized medicine. Many challenges in sequencing with biological nanopores, such as the high translocation velocity and the lack of nucleotide specificity, already have been addressed and commercially viable approaches are being pursued.
Solid-state counterparts also promise to evolve into robust methodologies although the challenges of speed and specificity still exist. If the speed of the molecules could be reduced to a single nucleotide per millisecond, thus allowing high fidelity electronic measurements of single bases, and if nucleotides could have a unique electronic signature, then it might be possible to sequence molecules containing one million bases in less than 20 minutes.
The outlook for developing such nanopore-based next-gen sequencing tools remains bright and there is little doubt that nanopore-based sensors will continue to develop as strong candidates to join other third-generation sequencing technologies in the race toward low-cost personalized DNA sequencing.