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Researchers Develop 'Nanotweezer' Microchip for Real-Time SNP Detection


NEW YORK (GenomeWeb) – A group led by University of California, San Diego researchers has developed an electronic DNA biosensor tool that it plans to apply for highly sensitive point-of-care SNP detection and infectious disease diagnostics.

Integrating DNA nanotweezer-based nucleic acid-sensing probes and a graphene field effect transistor (FET) chip, the real-time platform can be used to identify SNPs of interest, send data to personal electronic devices, and even potentially be integrated into implantable biosensors.

In addition, Ratnesh Lal, one of the inventors of the technology and a bioengineering professor at UCSD, has founded a company called Ampera Life to commercialize the biosensor as a diagnostic tool to monitor pathogen progression and detect bioterrorism threats.

In a study published this week in Advanced Materials, Lal and his team used a graphene FET as a DNA biosensor on human blood samples. In addition, the researchers demonstrated that they could wirelessly transfer an electric signal generated by electrical current differences to a smartphone.

First, Lal and his team tested the design of their DNA nanotweezer-based probes — which are at maximum about 50 nucleic acids long — for SNP detection with fluorescently labeled DNA nanotweezers. The researchers connect the probes with a loop that serves as the tweezer's hinge. The team then immobilized the DNA tweezers without a fluorescent label onto the graphene surface and verified their patterns and efficiency.

"When the zipper part of our nanotweezer, which acts like a clamp at one end, opens up," it causes the double strand to unwind and "flatten" onto the graphene probe, Lal explained. This allowed the researchers to introduce an artificial "test strand" that interacts with the double-stranded DNA.

If the test strand matches the normal strand, the normal strand releases the weaker strand and hybridizes with the new strand. The strand-displacement process pushes the negatively charged DNA strands closer to the graphene layer, and improves interference with the electrical current.

In order to enable electrical sensing of DNA using the nanotweezer-based probes, the team devised the graphene FET with two electrodes and a liquid gate chamber. The team found that high-quality graphene "grown in a defined condition" increases the target signal and improves the chip's sensitivity. The transistor's characteristics then changed depending on the number of mismatched nucleotides.

Lal and his team connected the graphene DNA chip to a wireless system with a microcontroller board. While a perfect DNA match generated a strong voltage signal, a single mismatched DNA produced a weaker voltage signal. Therefore, using the resisting wireless system, they showed that voltage emitted by strand-displacement could be sent to wireless devices for further analysis and reporting.

"All we're doing here is using the system to record and transmit the charge from the transistor," Lal explained. "Now we are integrating the wireless [portion] in the chip itself and sending the signal to a smartphone or laptop."

In a previous study, Lal and his team detected SNPs using double-stranded DNA displacement-based "zipper" probes on graphene FET, detecting relatively long nucleic acid sequences with improved specificity than standard methods. While the previous assay had nanomolar sensitivity, the team altered the process in order to increase the sensitivity to picomolar while relatively maintaining the same sensitivity. Lal explained that reducing the distance between graphene and the sensing part of the DNA tweezers therefore improved the tool's sensitivity.

Lal noted that while the graphene FET chip is very "adaptable," graphene has been a challenging substance to make, since graphene, "especially a single layer, is a complicated process and needs a controlled [setting]."

Lal and his team filed for patents on the nanotweezer and zipper technology in December 2016, followed by a later IP filing for the graphene-based FET.

In order to commercialize the real-time nanotweezer-based biosensor, Lal and his team founded Ampera Life earlier this year. While Ampera is currently using UCSD incubator space, Lal noted that the company eventually plans to move off campus. Ampera is also negotiating the patent-transfer process for the assay and securing small business innovation research funding for a prototype handheld version of its technology.

Emphasizing that the platform allows for continuous monitoring of samples, Lal envisions the SNP detection assay being used in the emergency room setting to quickly diagnose infectious disease in real time and detect bioterrorism threats in the field.

"The eventual microfluidic system will hopefully be able to detect toxins in the air, which could be converted into fluid samples," Lal said.

While Lal declined to mention which pathogens Ampera is targeting, he noted that a validation study on this application will be released later this year. The team aims to detect more than one molecular target per run, searching for specific bacterial and viral biomarkers.

"As long as you can record the changing charge, you can track the probe and sense the target," Lal said. "We plan to eventually have a chip that could have five to ten [different  targets] in a single sample."

In addition, the firm also hopes to "eventually have a wireless integrated electronic chip with a small microfluidic chamber, implanted under the skin, the size of a fingertip, and send real-time signals sent to a smartphone," Lal explained, noting that the chip will detect SNPs. "We want [the small amount] of test sample to flow continuously for sensing rare number of target molecules, including DNA, RNA, and viruses."

In the future, the researchers aim to develop and implement the technology to improve diagnosis of diseases including degenerative, genetic, cancer, and other SNP disorders. While currently only able to use a patient's blood samples, Lal hopes to develop the assay further so that it can use a patient's buccal sample and solid tissues as well.

"Patients will be freed from the constraint of wired facilities and would have comfortable testing experiences and minimized impact on their normal activities," the study's authors noted in the paper.

In addition, Lal's team believes that the FET-based SNP detection system will allow real-time SNPs detection in genetic material, such as somatic mutations in Alzheimer's disease and age-related macular degeneration, and SNPs linked to HIV drug-resistance mutations for quicker antiretroviral therapy.

"The technology is adaptable to any disease and any biomolecules, as we have a very broad platform," Lal said. "The assay [will] provide ... more precise, defined health results [for patients]."

Lal and his team aim to eventually reduce time of biomarker detection to 10 minutes. Lal also estimates the price of the chip for diagnostic use to eventually be between $15 and $60 for the end user.

According to Lal, the company "hopes to have the first generation system ready in a year or so, depending upon the funding for manpower." In addition, he said that the commercial timeline for the in vitro system will "likely be two to three years depending on funding" and FDA 510(k) approval. In order to develop and commercialize an implantable chip, however, Lal said that Ampera will need "much more quality control and biocompatibility testing."