NEW YORK ─ A pocket-sized point-of-care platform could serve as an affordable and sensitive test for a range of medical conditions and speed up receipt of results compared to lab-based systems, according to its developers.
Though the platform is in its early stages of development, it may one day be used to detect pathogen-based DNA sequences and genetic mutations while enabling affordable diagnostic testing at home or in low-resource communities, said Dan Luo, one of its developers who is a professor at Cornell University.
Luo and colleagues at medical research centers and universities in China described the development of the pocket-size detector in a research article published on Wednesday in Science Advances.
The investigators performed clinical sample testing at Southwest Hospital in Chongqing and further platform development at the Chinese Academy of Sciences in Suzhou. They used the test to detect DNA in blood, urine, plant matter, river water, and food, according to its developers.
Luo said that the group tested more than 170 clinical samples, and in addition to identifying pathogen-specific DNA sequences, it used the tool to analyze DNA mutations, including long-fragment and short-fragment deletions, and to detect single-base substitution, single-base insertion, and allele genes.
The platform has demonstrated high sensitivity reflected by a limit of detection of less than 1,000 copies per milliliter of pre-amplified DNA and a high specificity to the level of single-base differentiation. It provides results in less than two hours, he added.
Though the device is an early-stage prototype and will require further development as well as validation to establish its clinical utility, its developers believe it could eventually be made available as a commercial diagnostic test. Due to its portability and the internet connectivity of smartphones, the new platform could be used to locate and track the distribution of disease in real time, the researchers said. Such a device may eventually be used to detect DNA sequences associated with cancers, metabolic diseases, and infectious diseases, including COVID-19, Luo said.
In the Science Advances study, the investigators conducted preliminary validation of the device they call Pocket by testing samples for b-thalassemia, a hereditary anemia associated with different types of gene mutations. They also validated the platform's sensitivity for detecting allele genes that are strongly correlated with susceptibility to alcohol intoxication. In each case, the group said it obtained strong signals in the mutant samples and little-to-no signals in control samples. Results were validated with DNA sequencing.
To make the device affordable while retaining the high sensitivity of molecular diagnostic testing, Pocket – short for point-of-care for the entire test ─ leverages different parts of a smartphone and standard manufacturing techniques, including 3D printing and semiconductor fabrication, Luo said.
The platform consists of an integrated chip, called the i-chip, and a smartphone with an attached optical unit that has a lens, light emitting diode, and battery, all of which fit within a small, foldable box.
"What's different about this DNA detector is its use of a smartphone to generate heat that enables DNA amplification," Luo said. "You normally need an incubator in a lab to generate the heat you need for amplification, but because we are creating this platform for portable field and at-home use, we can't use a conventional incubator."
Generating a DNA signal strong enough that it can be clearly detected was a challenge due to the microscopic design features of the platform, Luo said, adding, "We needed more amplification than is accomplished in traditional molecular testing."
A point-of-care or laboratory-based molecular system might use a fluorometer within an instrument, for example, to detect fluorescence, but in developing the point-of-care test, the group wanted to eliminate restrictions imposed by traditional instrumentation, including high cost, space to run tests, and use of bulky, heavy equipment, Luo said.
The group developed reagents that have demonstrated a shelf life of greater than 10 weeks. The system's i-chip combines sample preparation with triple-signal amplification, Luo said, adding that three amplification phases are needed to boost the device's sensitivity and ensure strong DNA signals.
Pocket's triple amplification method combines isothermal recombinase polymerase amplification with gold and silver nanoparticle-based amplification to produce colorimetric amplified signals.
The overall testing process requires a drop of reagent for each test. In the context of commercial testing, reducing reagent volume contributes to reducing costs, Luo noted, but use of the smartphone is also an integral part of reducing expenses and potentially enabling more affordable point-of-care molecular testing.
In addition to functioning as a reagent heater, the smartphone serves as a DNA signal detector and a reader to show test results. After a specially designed smartphone application has been triggered, the phone takes about five minutes to heat up to 37 degrees C and that stimulates DNA amplification by warming the sample and reagents on top of the phone. When DNA of interest are present, the system's reagents create visual signals, and a dark image signals the presence of a pathogen, Luo said. The processing power and increased durability of modern-day smartphones make them more suitable than ever for use in testing, he said.
The overall platform uses small, lightweight parts that are easy to fabricate, Luo said, by combining 3D printing for plastic components, such as its amplification unit, with standard semiconductor fabrication for the device's integrated chip.
Luo said the researchers envision additional uses for the tool including DNA detection in environmental protection, food safety, and agricultural applications.
The group is in discussions with academic collaborators that have biosafety level 3 and level 4 labs, which take biocontainment precautions in isolating dangerous biological agents. It is looking to partner with labs that could help develop and implement a reverse transcription process for SARS-CoV-2 RNA suitable for use with the pocket-size device.
"We developed the prototype long before the outbreak of the coronavirus," Luo said. "But we see that all of the [SARS-CoV-2] molecular tests convert RNA to DNA, and though we haven't validated it yet, we should be able to develop our device for that application."
Other developers are looking to reduce or eliminate instrumentation for point-of-care molecular testing. They include Abington, UK-based Sense Biodetection, which is accelerating the development of an instrument-free platform for SARS-CoV-2, and Austin, Texas-based Nuclein, which is developing a handheld real-time PCR system and a prototype assay for SARS-CoV-2, both of which are disposable after a test has been completed.
Adding a smartphone as part of a testing system may contribute further to the platform's utility for coronavirus testing, Luo said. All smartphones, he noted, have built-in global positioning capabilities. "Of course, one needs to take user privacy issues into consideration, but from a pure research viewpoint … our smartphone-based platform can report test results in real time along with associated geographic information," he said.
Such a capability could be useful in both epidemiological investigations and field-based surveys, such as in environmental and food safety monitoring, he said. "It is quite easy to couple detection with a geographic location," Luo added.
With reference to the pocket-size platform, Gerald Kost, director of the point-of-care testing center at the University of California, said it is worth noting that heat consumes energy and very few smartphones have an excess of battery power to spare, so that is an important consideration in further development of the platform.
That aside, "there are two key points enabling use of this kind of technology," Kost said. "Smartphones come with connectivity, so what could be better for tracking asymptomatic positives in the pandemic while reporting negatives? Given that the platform produces colorimetric amplified signals, most current smartphones will be capable of reading out the detection signal."