NEW YORK ─ UK-based researchers are collaborating on the development of a low-cost, rapid screening platform to detect infectious disease pathogens with the aim of enabling broader access to diagnostic testing at the point of care.
In a recent study published in Nature Communications, researchers at Imperial College London and Moredun Research Institute in Edinburgh, Scotland, reported on the development of a highly sensitive proof-of-concept prototype that uses the properties of silicon to implement important functions such as target amplification and detection.
They produced a disposable, low-cost device using a fabrication process that didn't require expensive equipment normally needed to make silicon chips in a foundry, Firat Güder, the corresponding author for the study, said in an interview.
Their prototype could be the basis for a point-of-care testing platform able to detect many infectious diseases and that circumvents challenges associated with other electrochemical detection methods and traditional PCR-based testing, he said.
"We want to make it possible to run a molecular test at home, or take it with you if you are traveling," Güder said. "That's why we started this project four years ago, to develop a platform that is handheld, integrated, and does amplification as well as detection, and this prototype is the first step toward that goal."
The researchers believe that if they are successful, they could have a commercial test in a few years that offers an alternative to current molecular tests for infectious diseases used at the point of care.
The manufacturing techniques, materials, and form factor used in the test were specifically selected to enable a testing platform that would be battery-powered, handheld, and inexpensive enough to be disposable. In the study, their prototype ran 35 tests with a battery equivalent in power to that needed for a modern smartphone, the researchers said.
The ideal, miniaturized, low-cost portable detection system for nucleic acid testing, according to Güder, must be able to heat up the patient sample to specific temperature setpoints with high precision and measure the results of the amplification reactions quantitatively.
The group named the new test TriSilix because it uses silicon to enable three important functions ─ electrochemical detection, heating, and temperature control.
Because silicon is conductive, the test system uses electric current to heat the silicon chip and make it a temperature sensor, Güder said.
Heating initiates chemical reactions that amplify target DNA extracted from the sample. Güder noted that in the platform, PCR requires cycling the temperature to three setpoints, and to achieve this, the temperature of the silicon chip is precisely elevated and reduced during each amplification cycle.
"Temperature control and DNA amplification are critical, and the faster we can reach a temperature setpoint and maintain it, the quicker we can run a test," he said. "Our device is tiny with low thermal mass so it can be heated and cooled quickly."
Silver-plated copper electrodes near the silicon chip detect changes in current when target DNA are in the sample, enabling electrochemical detection and quantification. Coated dyes remain electrochemically active when they are not bound to target DNA but become electrochemically inactive and lead to a measurable drop in current intensity when they are bound to the target DNA.
In the Nature Communications study, the researchers reported that TriSilix quantitatively detected a 563 bp fragment of genomic DNA of Mycobacterium avium paratuberculosis through real-time PCR with a limit of detection equivalent to a single bacterium.
The group selected Mycobacterium avium paratuberculosis, a bacterial parasite and the causative agent of paratuberculosis, a disease predominately found in cattle and sheep, largely because they had access to research samples and wanted to show that the test platform would work with bacteria that are both difficult to detect and to culture.
Researchers developing electrochemical diagnostic tests generally don't leverage the intrinsic properties of silicon, and instead rely on the deposition of materials and new layers to add new functions to the device, all of which adds to cost and makes fabrication more complicated, Güder noted.
"[The proof-of-concept] work highlights a longstanding unmet need amplified by the current pandemic — to rapidly diagnose infections at the point of care and exploit the myriad attendant benefits," said Cesar Castro, director of the cancer program at the Massachusetts General Hospital Center for Systems Biology and an assistant professor of medicine at Harvard Medical School.
"The investigators cleverly integrate two modalities ─ electrical and chemical sensing ─ to amplify and detect pathogen-unique nucleic-acid sequences, by exploiting the properties of the semiconductor silicon within a purported point-of-care platform," said Castro, who is not involved in the development of the test.
"An added advantage lies in the ability to produce them outside of fabrication cleanrooms and directly in standard wet-labs," Castro added.
Giuseppe Spoto, a professor of chemical sciences at the University of Catania, who is developing a photonic detection platform, said TriSilix's use of PCR or isothermal amplification with the electrochemical detection of amplicons is a "critical advantage of the presented system. Electrochemical detection helps in implementing nucleic acid detection in miniaturized devices and facilitates the integration of the whole assay in portable and easy to use devices."
The TriSilix platform's silicon-based technology, unlike most silicon-based electronic products, doesn't require expensive lithography, deposition, and etching equipment. Also not needed is a cleanroom or a foundry for fabrication. The group used a standard laboratory with off-the-shelf equipment, including a computer-controlled laser and material bonding equipment that are broadly available, Güder said.
Using the less sophisticated manufacturing facility, the developers have been able to fabricate TriSilix chips for about $.35 each, an important contributor to future tests being affordable and disposable, he said.
The device is easily translatable to diseases that affect animals or humans, and would work equally well with human testing for tuberculosis, malaria, SARS-CoV-2, or other coronaviruses, Güder said.
Further work on the development of the test is ongoing. The researchers need to develop and integrate a sample handling system for the test, and they are considering manual or automated processes for that purpose, Güder said. The group is looking to first develop capabilities to handle saliva, followed by nasal swabs, blood, and urine.
"Ideally, we want to focus on integrating everything onto the chip," he said. "We can massively automate the device but that increases complexity and cost. Alternatively, we can develop sample preparation procedures that the user can implement manually."
As they look to integrate more challenging sample types into the system, they plan to reduce the level of automation to keep costs down.
The group is doing further development on a SARS-CoV-2 assay for the platform on which it has done preliminary research, and it is developing a multiplexed respiratory panel that includes SARS-CoV-2 and other common coronaviruses and can differentiate among them, Güder said.
"As a next step to advance this technology, we also plan to demonstrate its capability as a direct sample-to-answer platform, and we are developing its capabilities to do reverse transcription so that we can convert RNA to DNA for detection," he added.
"When RNA viruses are going to be detected, retrotranscription is needed," Spoto noted. "Such aspects need to be taken into account for the development of a device operating at the point-of-need."
Further, as the group continues to develop the technology, it will need to prioritize resources. It is seeking collaborations with funding organizations and diagnostic companies to move the test toward commercialization.
The current project was initiated as a result of a £100,000 ($134,240) grant from the Welcome Trust, a London-based charitable foundation that provides research support and funding.
If the group manages to obtain funding and collaborate with a suitable diagnostic industry partner, a commercial test could be available in Europe with CE marking within about two years, and because markets for the platform and its tests are international, the group would pursue US Food and Drug Administration clearance, Güder added.
Molecular point-of-care systems "have revolutionized nucleic acid testing," he said, but most are benchtop systems that use fluorescence-based optical detection, which adds expense and reduces portability.
Such systems on the market include Cepheid's GeneXpert, the Roche Cobas Liat, and Abbott's ID Now. San Diego-based Mesa Biotech's enables near-patient testing, including for SARS-CoV-2, with its Accula molecular platform, which fits in the palm of the hand and uses a disposable cartridge.
The challenge ahead for the developers of TriSilix lies in traversing the "translational valley of death …  where promising technologies tested within the confines of the highly regulated laboratory environment by skilled personnel fail to reach fruition in the clinical arena due to workflow, operator, [or] performance concerns under real-world conditions," Castro said. "As such, the work will benefit from continued hardware integration such as enhanced microfluidics and device miniaturization for on-chip sample processing to enable readouts with minimal manipulation."
He added that testing under arduous, real-world conditions across geographies will be needed, which generate not only temperature extremes but test the durability and performance of the platform.
"Ultimately, adoption by key stakeholders and end users will reflect a function of its ease of use and performance," Castro noted.