A photo of the first PathTracker clip-on box. [Photo courtesy Brian Cunningham]

Researchers headed by a team at the University of Illinois, Urbana-Champaign, have developed what they claim is an inexpensive, sensitive smartphone-based device that can detect viral and bacterial pathogens in about 30 minutes, and could be adapted to test for SARS-CoV-2. The platform comprises a cartridge-housed microfluidic chip that carries out isothermal amplification of viral nucleic acids from nasal swab samples, which are then detected using the smartphone camera. The investigators report on their use of the system to detect equine viruses as a non-biohazard surrogate for SARS-CoV-2, but say that when adapted to test for coronavirus, the smartphone accessory, costing about $50, could be used to reduce the pressure on testing laboratories during pandemics such as COVID-19.

“This test can be performed rapidly on passengers before getting on a flight, on people going to a theme park, or before events like a conference or concert,” said University of Illinois, Urbana-Champaign electrical and computer engineering professor Brian Cunningham, PhD, who, together with bioengineering professor Rashid Bashir, PhD, led the development of the device. “Cloud computing via a smartphone application could allow a negative test result to be registered with event organizers or as part of a boarding pass for a flight. Or, a person in quarantine could give themselves daily tests, register the results with a doctor, and then know when it’s safe to come out and rejoin society.”

The multi-institutional researchers described their development and use of the device, in Lab on a Chip. The paper is titled, “Smartphone-Based Multiplex 30-minute Nucleic Acid Test of Live Virus from Nasal Swab Extract.”

As the COVID-19 pandemic has escalated, a “key failure” of health systems across every country has been the ability to rapidly and accurately diagnose disease, the authors stated. Contributing factors include “ … a limited number of available test kits, a limited number of certified testing facilities, combined with the length of time required to obtain a result and provide information to the patient.”

Most viral test kits rely on labor- and time-intensive laboratory preparation and analysis techniques, they continued. Testing for SARS-CoV-2 from nose swabs can take days. And “because available technologies remain expensive (in terms of capital equipment and reagents), technically challenging, and labor intensive, there is an urgent need for low-cost portable platforms that can provide fast, accurate, and multiplex diagnosis of infectious disease at the point of care,” the researchers pointed out. “The challenges associated with rapid pathogen testing contribute to a lot of uncertainty regarding which individuals are quarantined and a whole host of other health and economic issues,” Cunningham said.

Nucleic acid tests (NATs) represent an important class of point-of-care (POC) technologies for pathogen sensing that can achieve high specificity for the detection of pathogenic nucleic acid sequences, the authors noted. Such tests can also be designed to tag the amplified sequences using fluorometric or colorimetric markers. “Due to their success in laboratory settings, considerable efforts have been devoted to performing NATs in POC settings,” the investigators commented. While most NAT methods are based on polymerase chain reaction (PCR) amplification, which requires repeated heating and cooling cycles that are not ideal for POC applications, NATs that use isothermal nucleic acid amplification approaches, such as loop-mediated isothermal amplification (LAMP) are now being harnessed to develop simple, miniaturized POC devices. LAMP can rapidly amplify nucleic acids at a constant temperature, with just one type of enzyme and four to six primers.

The device developed by Cunningham and Bashir’s team started out as a project to detect a panel of equine viral and bacterial pathogens, including those that cause severe respiratory illnesses in horses, which are similar to those presented in COVID-19. “Utilizing the system in the context of equine respiratory diseases represents a model system for human pathogens such as SARS-CoV-2, which does not pose biosafety issues, but preserves the main features of a human COVID-19 testing protocol,” the researchers indicated. “Horse pathogens can lead to devastating diseases in animal populations, of course, but one reason we work with them has to do with safety,” Cunningham noted.

“The horse pathogens in our study are harmless to humans.” The researchers developed a device that could detect multiple horse viral pathogens quickly and cost-effectively, using LAMP technology. The device comprises a small cartridge containing the testing reagents and a port to insert a nasal extract or blood sample. The whole unit then clips to a smartphone. The test reagents break open the viral pathogens to gain access to the RNA. A primer molecule then amplifies the genetic material into many millions of copies in about 10 or 15 minutes. A fluorescent dye then stains the copies and glows green when illuminated by blue LED light, which is detected by the smartphone’s camera.

Electrical and computer engineering professor Brian Cunningham, PhD, co-led a multi-institutional team to demonstrate an inexpensive and rapid smartphone-based pathogen testing device designed to ease pressure on testing laboratories during pandemics such as COVID-19. [Photo by L. Brian Stauffer]
Recent research has consistently shown that image sensors integrated within today’s smartphones have enough sensitivity to detect fluorescence in the contexts of fluorescence microscopy of cells, viruses, and bacteria. Smartphone cameras can also sense fluorescence signals from a wide variety of biological assays, including LAMP, within microfluidic compartments, the authors noted. “The advantage of using a smartphone as the detection instrument for POC analysis is that it is possible to take advantage of the integrated optics, image sensor, computation power, user interface, and wireless communication capabilities of mobile devices, thus minimizing cost,” the team wrote.” With assistance from an inexpensive snap-in cradle or clip-on instrument, anyone that carries a smartphone would have the ability to perform testing.”

The investigators used their prototype device to detect nucleic acids from five different pathogens that cause equine respiratory infectious diseases. “Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ~30 minutes,” they wrote. And while the test system reported in the paper was used to detect pathogenic DNAs, the assay could be easily adapted for detecting RNA viruses, they suggested, by using a one-step RT-LAMP protocol that adds reverse transcriptase to the LAMP reaction mix without modifying the buffer or reaction conditions.

A schematic drawing of the new device system.[Graphic courtesy Brian Cunningham]
They suggest that using a smartphone in conjunction with a cradle that enables the phone’s camera to quickly gather a fluorescent endpoint image of the LAMP reaction, it will be possible to generate a positive/negative result, and incorporate integrated experimental controls and replicates to assure that the test has been carried out correctly. “ … we envision a detection instrument that clips onto a smartphone, with mechanical adapters that will align the rear-facing camera correctly with several popular phone models,” the scientists explained.

Using a mobile device as a detection instrument will also make it possible to report the data via integration with telemedicine platforms, both for epidemiology reporting and for sharing test results with doctors.

The researchers’ system does currently require a few preparatory steps to be performed outside of the device, but they are working on a cartridge that has all of the reagents needed to create a fully integrated system. “In future work, our plans include integrating the functions of viral lysis, LAMP buffer mixing, and LAMP reaction into a single cartridge with the reagents held within on-cartridge reservoirs,” they wrote.

Other researchers at the University of Illinois are using the novel coronavirus genome to create a mobile test for COVID-19, and making an easily manufactured cartridge that Cunningham said would improve testing efforts.

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