Much more than ever, nucleic acids are recognized as key building blocks in many of lifeʼs processes, and the science of studying these molecular wonders at the single-molecule level is thriving.
A new method of doing so has been introduced in the mid 1990ʼs. This method is exceedingly simple: a nanoscale pore that spans across an impermeable thin membrane is placed between two chambers that contain an electrolyte, and voltage is applied across the membrane using two electrodes.
This principle is based the Coulter Counter, also known as the resistive pulse sensor, was invented by Dr. Wallace H. Coulter in 1953.
The experimental conditions lead to a steady stream of ion flow across the pore. Nucleic acid molecules in solution can be driven through the pore, and structural features of the biomolecules are observed as measurable changes in the trans-membrane ion current.
In essence, a nanopore is a high-throughput ion microscope and a single-molecule force apparatus. Nanopores are taking center stage as a tool that promises to read a DNA sequence, and this promise has resulted in overwhelming academic, industrial, and national interest.
Regardless of the fate of future nanopore applications, in the process of this 16-year-long exploration, many studies have validated the indispensability of nanopores in the toolkit of single-molecule biophysics.
The nanopore sensor is perhaps the youngest single-molecule technique to be developed to date. In less than two decades, nanopore research has allowed a multitude of studies on small biomolecules, nucleic acids, and proteins.
Further, nanopores are on the verge of delivering new technologies that will undoubtedly improve health, the most important of them being DNA sequencing. This technology has the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. Many groups all around the world are trying to achieve the goal for nanopore-based DNA sequencing.
Graphene based Nanopores
In 2016, Researchers at the National Institute of Standards and Technology (NIST) have simulated a new concept for rapid, accurate gene sequencing by pulling a DNA molecule through a tiny, chemically activated hole in graphene — an ultra-thin sheet of carbon atoms — and detecting changes in electrical current.
NIST’s new proposal is to create temporary chemical bonds and rely on graphene’s capability to convert the mechanical strains from breaking those bonds into measurable blips in electrical current.
“This is essentially a tiny strain sensor,” says NIST theorist Alex Smolyanitsky, who came up with the idea and led the project. “We did not invent a complete technology. We outlined a new physical principle that can potentially be far superior to anything else out there.”
Graphene is popular in nanopore-sequencing proposals due to its electrical properties and miniaturized thin-film structure. In the new NIST method, a graphene nanoribbon (4.5 by 15.5 nanometers) has several copies of a base attached to the nanopore (2.5 nm wide). DNA’s genetic code is built from four kinds of bases, which bond in pairs as cytosine–guanine and thymine–adenine.
In simulations (see accompanying video) of how the sensor would perform at room temperature in water, cytosine is attached to the nanopore to detect guanine. A single-strand (unzipped) DNA molecule is pulled through the pore. When guanine passes by, hydrogen bonds form with the cytosine.
As the DNA continues moving, the graphene is yanked and then slips back into position as the bonds break.
Video: DNA sequencing through Graphene Nanopore
The NIST study focused on how this strain affects graphene’s electronic properties and found that temporary changes in electrical current indeed indicate that a target base has just passed by. To detect all four bases, four graphene ribbons, each with a different base inserted in the pore, could be stacked vertically to create an integrated DNA sensor.
The researchers combined simulated data with theory to estimate levels of measurable signal variations. Signal strength was in the milliampere range, stronger than in the earlier ion-current nanopore methods.
Based on the performance of 90 percent accuracy without any false positives (i.e., errors were due to missed bases rather than wrong ones), the researchers suggest that four independent measurements of the same DNA strand would produce 99.99 percent accuracy, as required for sequencing the human genome.
The NIST study suggests the method could identify about 66 million bases—the smallest units of genetic information—per second with 90 percent accuracy and no false positives. If demonstrated experimentally, the NIST method might ultimately be faster and cheaper than conventional DNA sequencing, meeting a critical need for applications such as forensics.