NEW YORK (GenomeWeb) – Researchers from the University of California at San Diego are developing an electronic SNP detection method that they eventually hope to miniaturize for point-of-care diagnoses and/or in implantable biosensors to track disease development.
As the team reported recently in the Proceedings of the National Academy of Sciences, the approach brings together graphene field effect transistor (FET) technology with DNA strand displacement to identify SNPs of interest without labeling DNA in a relatively simple electrical system.
Using 47-base DNA probes — a target sequence made up of seven bases of single-stranded DNA and 40 bases bound to complementary DNA with intentionally weakened affinity to the target sequence — the group showed that it could pick up electrical current differences when perfectly matched test sequences move in and replace the weak strand, producing double-stranded sequence across all 47 bases, compared with challenge by a sequence containing one mismatch.
"Sensing of DNA with graphene is not new and sensing DNA with electricity is not new," co-senior author Gennadi Glinsky, a researcher with UCSD's Institute of Engineering in Medicine, told GenomeWeb. "The idea was to make this double-stranded design that is sensitive to the invading, duplicated strand and couple it with graphene."
But by applying their so-called dynamic DNA nanotechnology approaches and double helix design to this system, he explained, the researchers have made strand displacement reactions detectable within the graphene-based electrical system.
"This technology opens opportunities for the development of more reliable and efficient diagnostic tools," Glinsky and co-authors wrote, "including design and development of miniaturized, point-of-care, and implantable biosensors, for early detection of potentially life-threatening human diseases."
The team is interested in commercializing their methods, potentially with the help of investors and/or through partnerships with existing companies. The group is currently planning animal and other studies aimed at developing an implantable device targeting specific, disease-related mutations in relevant bodily fluids.
"We are very excited about that," Glinksy said. "In principle what you can do is put the chip somewhere where it has contact with bodily fluids — whatever bodily fluids happen to be informative in previous studies for a particular disease indication — and then, basically, even if the molecule exists in very small amounts … if you let the chip stay there for long enough it will capture and detect events."
He and his colleagues reasoned that SNP detection by conventional methods such as microarrays and sequencing is out of reach in many settings and situations, since it requires specialized equipment and can be time consuming.
They argued that sequencing-based genotyping approaches, while accurate, are "expensive and time-consuming," while the hybridization step in SNP detection with arrays may be complicated by cross-hybridization, particularly when long probes are used.
In an effort to minimize such cross-hybridization and boost the specificity possible by electrical detection, they pursued an approach centered on strand displacement, in which complementary DNA strand that perfectly matches a targeted DNA probe replaces the original complementary DNA.
To achieve this, a single-stranded seven-nucleotide sequence overhang, known as the toehold sequence, is attached to the end of each double-stranded DNA probe. The toehold latches each 40-base pair double helix probe onto the surface of a graphene FET sensor on a chip.
And when the sequence being tested matches the full 47 bases of the probe, it can replace the original, 40-base pair complementary strand (known as the weak side strand), since it matches both the probe and the single-stranded toehold sequence. In contrast, the weak side of the original helix is designed to contain inosine bases that decrease affinity to the target strand slightly.
"Inside the double helix in the probe, we do base substitution to make the bonding weaker on one strand," Glinsky said.
That strand displacement creates resistance in gate voltage being applied to the system, leading to a current change that does not occur when the sequence being tested contains a single sequence mismatch, he explained. "What happens when the strand gets displaced is there's a change in charge that we detect."
For the PNAS paper, the researchers used fluorescently labeled nucleotides to optimize their double-stranded DNA probe design and to the kinetics of DNA displacement when challenged with strands of DNA that were perfectly matched or differed by one base.
Next, the team tested the ability to measure such displacement in a system using two electrodes, a liquid gate chamber, DNA probes, and graphene. The two-dimensional graphene — an atom-thick layer on top of a silicon oxide-coated chip — was selected owing to its uniformity, batch-to-batch consistency, and ease of fabrication.
After recreating the strand displacement effects in a graphene FET setting, the team went on to document shifts in the current-voltage curve in the graphene FET system when challenged with different concentrations of matching DNA and DNA containing a single mismatch.
"To our knowledge, this is the first report of the successful electrical detection of strand displacement in long DNA strands by sensing the charge difference before and after strand displacement without any labeling or additional processes," the researchers wrote.
They have not done a formal analysis of probe design and productions costs, but suspect such systems would be cheaper than optical genotyping approaches.
Based on their results so far, the researchers believe it will be possible to extend the length of the double-stranded DNA probes used or parallelize probes to interrogate larger stretches of genomic sequence.
"That will give us the opportunity to cover basically the whole genome," Glinsky said. "And then we could do a separate set of probes for repeat sequences like retroviral sequences."
The latter sequences may be of interest for studies of retroviruses that are temporarily activated in embryonic stem cells and again in some cancers, which he and his team have studied in the past. The ability to detect both DNA and RNA might also help in detecting everything from gene regulatory sequences to specific microRNAs.
Glinsky noted that the double-stranded DNA probe detection approach might one day prove useful for tracking activity of treatments being targeted to tumors or other sites in the body by nanoball methods.
The group has gone on to tweak the system so that the strand displacement step can be done to test RNA rather than DNA, which Glinsky said is "much more broadly applicable in the diagnostics space."
In other work, which has not yet been published, the group has been adapting the system to work wirelessly — a next step toward a chip that could be implanted in the human body. Ultimately, the idea is to have a chip that can transmit SNP detection data from within the body to a cell phone or wearable device.
The researchers are more focused on the development of such implantable devices than in coming up with a system for scanning SNPs across the complete genome, though Glinsky said they would considering partnering with firms to achieve a range of potential applications.
"We are not working specifically on genome-wide coverage technology, simply because that would require significant investment of time and personnel," he said. "We are working right now on targeted [approaches] … in liquid biopsy applications."