The instructions for building all of the bodyâs proteins are contained in a personâs DNA, a string of chemicals that, if unwound and strung end to end, would form a sentence 3 billion letters long. Each personâs sentence is unique, so learning how to read gene sequences as quickly and inexpensively as possible could pave the way to countless personalized medical applications.
Researchers at the University of Pennsylvania have now made an advance towards realizing a new sequencing technique based on threading that string through a tiny hole and using a nearby sensor to read each letter as it passes through.
Their DNA sensor is based on graphene, an atomically thin lattice of carbon. Earlier versions of the technique only made use of grapheneâs unbeatable thinness, but the Penn teamâs research shows how the Nobel Prize-winning materialâs unique electrical properties may be employed to make faster and more sensitive sequencing devices.
Critically, the teamâs latest study shows how to drill these nanopores without ruining grapheneâs electrical sensitivity, a risk posed by simply looking at the material through an electron microscope.
The team includes Marija DrndiÄ, professor of physics in the School of Arts and Sciences, and members in her laboratory, including graduate student Matthew Puster and postdoctoral researchers Julio RodriÌguez-Manzo and Adrian Balan.
Their research was published in the journal ACS Nano.
DrndiÄâs group has previously demonstrated a series of advancements towards reading genes by passing them through a tiny hole, or nanopore. Their 2010 study involved drilling a hole in a sheet of graphene, then putting it in an ionic bath along with the strands of DNA to be detected. Because each of the four bases, the letters in DNAâs alphabet, have a different size, a different number of ions would be expected to squeeze through along with each base as the strand passes through the pore. Researchers could then interpret the sequence of the DNAâs bases by measuring the electrical signal of the ions. However, those current signals are weak, limiting the speed at which DNA could be sequenced.
Many research groups are now exploring multiple ways to improve the sensitivity and speed of the technique, including new materials and new ways of fashioning nanopores in them. DrndiÄâs group has experimented with different membranes, as well as adding improved electronics to measure at faster speeds, but its latest study represents an entirely new way of generating an electrical signal unique to each base.
âOur latest attempt at improving the technique is a departure from our previous work, however,â DrndiÄ said. âWeâre now trying to measure current directly from the graphene, whereas before we measured ionic current in the solution as it goes through the pore.â
The Penn team wanted to see if nanopores in graphene, the most conductive material known, would be capable of sensing the difference between bases directly. Instead of their different sizes, this method would rely on the bases altering the electric charge in the nearby material. In this case, the material would be a thin, wire-like ribbon of graphene. As each base passes through the pore, it would modulate the electrical current flowing through the ribbon. The changes in current would then be matched to their corresponding bases, allowing the researchers to decipher the sequence.
âThe advantage,â Balan said, âover the ionic method is that the current in the graphene ribbon is a thousand times higher. That means we can measure a thousand times faster. We wouldnât need to slow down the DNA to make an accurate measurement of each base.â
After fabricating the graphene ribbons on a silicon nitride membrane and attaching metal contacts, the researchers wired them to measure their resistance and then put them in a transmission electron microscope, or TEM. This type of microscope uses a broad beam of electrons to produce images with nanoscale resolution by measuring the electrons as they pass through the sample, but it can also be used like a drill by focusing the beam.
The researchers had used a TEM to drill nanopores in sheets of graphene for their earlier sequencing experiments but encountered an unexpected challenge this time. When they put their ribbons in the TEM, they found resistances significantly increased, limiting sensitivity.
âJust looking at the graphene ribbons with the TEM caused them to degrade,â DrndiÄ said. âThe wide beam we use for imaging was damaging them by introducing defects in the pattern of carbon atoms. It was almost not graphene any more.â
âIt didnât matter in our earlier experiments,â Puster said, âsince we were just using the graphene for its thinness and mechanical properties. We were creating these defects and raising the resistance, but we didnât realize it because we werenât measuring the grapheneâs electrical properties.â
But with grapheneâs ultra-low resistance key to their proposed sequencing device, the team was presented with a quandary; they needed to poke a hole in a precise spot on a ribbon 10,000 times thinner than a human hair while effectively blindfolded.
âThis was a real roadblock,â DrndiÄ said. âHow were we going to drill these pores when just looking at the ribbon kills the device?â
The teamâs solution was to use a different imaging mode in the TEM, which produced a rough scan rather than high-definition picture.
âInstead of opening up the beam valve and flooding the ribbon with electrons,â RodriÌguez-Manzo said, âwe use a scanning mode that just takes one snapshot. By taking the fuzziest picture that still tells us where the edge of the ribbon is, we limit the amount of electrons that hit it.â
âThe image we get back is very pixelated,â Puster said. âBut then we just need to pick the pixel where we want to put the pore or notch.â
The team simultaneously measured the ribbonsâ resistances as they took these snapshots, clearly showing that they remained undamaged throughout the process. They also simulated the presence of a strand of DNA by using an electric field to test that the device would be sensitive enough to conduct DNA experiments with.
âI think this may solve problems for a lot of different nanosensors,â DrndiÄ said. âWhether theyâre made of graphene, nanowires, carbon nanotubes or other nanostructures, this will help keep them in working order while in a TEM. The main trick here is to drill the nanopore with as little imaging as possible, just taking a quick peek out from under the blindfold.â