By Deborah Borfitz
September 15, 2021 | Many of the biological assays currently done in clinical labs could be performed at the point of care (POC), says Rashid Bashir, Ph.D., dean of the Grainger College of Engineering at the University of Illinois at Urbana-Champaign, speaking at the recent Next Generation Dx Summit. He was referencing his work on a microfluidic lab-on-chip device for the rapid detection of pathogens, which might also be useful for pathogen identification, antibody susceptibility testing, or to measure host response based on cell count, proteins, or other biological entities.
Steps in the diagnostic process that Bashir and his colleague are attempting to miniaturize and speed up include bacterial culture, blood cell count, and pathogen detection using nucleic acid technology or antigen-based testing, as well as the functionality of a flow cytometer, he says.
When it comes to testing for SARS-CoV-2, the relevant diagnostics are molecular, antigen, and antibody tests, says Bashir. Molecular tests, where the target gets amplified, are the gold standard and typically take 30 to 90 minutes depending on whether an isothermal or PCR process is being used.
The antigen tests are inherently less sensitive but more available at the POC, he continues. A lot of new technologies are now emerging that can increase the tests’ sensitivity.
What the market is looking for, says Bashir, is a low-cost, antigen test format (e.g., lateral flow) with the sensitivity of the molecular test.
Pivot To COVID
Pre-pandemic, Bashir’s group was working on viral load detection on a cartridge that was readable by a cell phone camera, he says. The team has already published on use of the device for HIV, hepatitis C, Zika, Dengue, and Chikungunya.
For this use case, the process involves taking a drop of blood from a fingerprick that gets injected into a device, he explains. The blood is lysed, and the constituents end up in a chamber where primers are spotted, and reagents get used. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) process is deployed for amplification.
“We can do multiplex detection to look for multiple pathogens… by splitting the sample into different channels where the primers of the target are already spotted,” Bashir says. “Using this, we demonstrated ranges of 100 to 250 particles per microliter of whole blood.”
When COVID-19 hit, and as soon as the sequence was available for SARS-CoV-2, the team got to work looking at a couple different primer sets for LAMP and optimizing them for the different genes, he says. They subsequently published on a primer set that targeted Gene N, first characterizing the assay off-chip and then on-chip.
Researchers also put the assay on a chip and cartridge while using clinical samples, per a published study last year, says Bashir, where the VTM (viral transport media) was collected from patients and half of the samples were run through the standard RT-PCR process and the other half through RT-LAMP.
The samples got injected into one of two ports in the additively manufactured cartridge (the other being for the reagent), he says. The samples move from a mixing region to an “amplification and diagnostic” region. Ultimately, the cartridge gets inserted into a reader with a heater, optics, and a cell phone camera able to detect the increase in fluorescence from the LAMP assay.
The reader would cost “a few hundred dollars, or less, if mass-produced,” says Bashir. The additive manufacturing step “allowed us to very quickly prototype and optimize and refine the design.”
In 20 to 30 minutes, the microfluidic lab-on-a-chip could “very clearly detect the presence of positive samples,” he adds. “We [demonstrated] very high predictive power and very high sensitivity and specificity.”
RT-LAMP is “slightly less sensitive” than PCR, Bashir continues, so the team optimized the two-step process earlier this year so that cDNA synthesis is done separately. The approach delivered some “promising data,” including the ability to go down to between one and four copies per microliter off the SARS-CoV-2 viral RNA from the VTM.
Similarly good results were seen using saliva, he adds. A signal could be detected from a few copies, both with the optimized process and conventional, one-step RT-LAMP process.
In work not yet published, the development team further modified the cartridges to spot the LAMP primers for the N gene as well as the S gene, and to detect both down to 10 copies per microliter, Bashir reports. Off-chip control and on-chip amplification time for the N and S genes “have pretty good correlation and we’re continuing to improve it.”
The goal, he says, is to be able to detect the SARS-CoV-2 variant B117 using LAMP and differentiate it from the wild type.
Curling Graphene
Another platform the team has been actively working on is designed for the ultrasensitive detection of biomolecules using “deformed” graphene channel field effect biosensors, Bashir says. These are electrical sensors which, unlike traditional flat graphene, are curved.
New properties are manifested by bending and crumpling the graphene, Bashir explains, such that “the Debye lengths [distance over which ions and electrons can be separated in a plasma] in the curved regions are extended and there is consequently less charge shielding, allowing for a higher signal [and] more sensitive detection of charged molecules.” There is also a possible bandgap opening of graphene when it is curled, he adds.
By making a transistor out of the deformed graphene, Bashir and his colleagues were able to detect microRNA down to the atomolar range. Further, they showed the platform could be used to detect proteins, including interleukin-6 and the COVID-9 N and S proteins, down to atomolar range concentrations from phosphate-buffered saline (akin to VTM).