New label-free detection technique digitally counts intact SARS-CoV-2 virus particles in saliva or exhaled breath

As health and research institutions continue to rapidly develop new methodologies for detecting SARS-CoV-2, researchers from the Holonyak Micro & Nanotechnology Laboratory have found themselves at both forefronts of discovery and featured on the cover of the Journal of the American Chemical Society with their paper: Label-free Digital Detection of Intact Virions by Enhanced Scattering Microscopy.

Label-free detection, an approach that utilizes a biosensor and detection instrument for viral load monitoring, is a solution that can capture and digitally count intact virus particles in saliva or exhaled breath to provide lower cost and reduced time to diagnose an infection.

Currently, the most widely used SARS-CoV-2 PCR testing method is the PCR assay, which uses enzymatic amplification to make many copies of a specific section of the virus’s RNA, which requires extraction of the viral genome and a complex laboratory procedure.  Instead, the new approach uses a specially designed nucleic acid molecule, called an “aptamer” attached to a biosensor that selectively recognizes one of the proteins on the virus outer surface, and captures it in a single step at room temperature, with no other reagents required.  Once captured, the viruses are counted, using a newly invented form of microscopy, that generates images from laser light that scatters from each captured virus.

“Our technique requires only the saliva sample, and avoids the need for any additional reagents, thus we expect the cost for a test to be significantly reduced and the overall process to be greatly simplified (less labor-intensive),” said co-author Nantao Li, a graduate student in electrical and computer engineering. “In addition, the aptamers we used can selectively differentiate between active viruses from inactive ones, thus providing more robustness for diagnosis results. Conventional techniques, such as PCR, detect the viral RNA sequence which can remain in bodily fluids even after infectious viruses are no longer present.”

To detect and count the captured viruses, the team recently invented a new imaging approach called Photonic Resonator Interferometric Microscopy (PRISM). PRISM uses a photonic crystal biosensor surface to enhance light scattering from virus particles. The photonic crystal is a nanostructured surface that provides two effects. First, it enables each virus to scatter more light from an illuminating laser, which increases their signal contrast.  Secondly, the photonic crystal directs the scattered light toward the microscope objective – allowing a larger fraction of the scattered light to be collected.

While label-free digital detection can be a promising alternative to traditional SARS-CoV-2 detection, principal investigator Brian Cunningham believes this approach can be widely applicable to other areas.

“We are already making plans soon to perform viral load monitoring of HIV in plasma, and we are building a more portable version of the detection system that would be small and inexpensive enough to perform well in biology labs or diagnostic lab facilities,” said Cunningham, Intel Alumni Endowed Chair in Electrical and Computer Engineering.

Moving forward, the research team – comprised of principal investigators Brian Cunningham, Yi Lu, Xing Wang, and co-authors Xioajing Wang, Nantao Li, and Joseph Tibbs, are designing and implementing a new type of capture molecule that will reduce the detection time to a few minutes. They envision the future capability for a person to exhale into a device, and for exhaled virus particles to be captured on the sensor.

To read more about Label-free Digital Detection of Intact Virions by Enhanced Scattering Microscopy, you can find it published in the Journal of American Chemical Society here. 

CUNNINGHAM GROUP DEVELOPS MICROSCOPE TECHNIQUE TO DETECT INDIVIDUAL VIRUSES FOR POWER RAPID DIAGNOSTICS

CHAMPAIGN, Ill. — A fast, low-cost technique to see and count viruses or proteins from a sample in real time, without any chemicals or dyes, could underpin a new class of devices for rapid diagnostics and viral load monitoring, including HIV and the virus that causes COVID-19.

Researchers at the University of Illinois Urbana-Champaign described the technique, called Photonic Resonator Interferometric Scattering Microscopy, or PRISM, in the journal Nature Communications.

“We have developed a new form of microscopy that amplifies the interaction between light and biological materials. We can use it for very rapid and sensitive forms of diagnostic testing, and also as a very powerful tool for understanding biological processes at the scale of individual items, like counting individual proteins or recording individual protein interactions,” said Illinois ECE Professor and study leader Brian T Cunningham, the Intel Alumni Endowed Chair of electrical and computer engineering and a member of the Holonyak Micro and Nanotechnology Lab and the Carl R. Woese Institute for Genomic Biology at Illinois.

In optical microscopes, light bounces off any molecules or viruses it encounters on a slide, creating a signal. Instead of a regular glass slide, the PRISM technique uses photonic crystal: a nanostructured glass surface that brilliantly reflects only one wavelength of light. Cunningham’s group designed and fabricated a photonic crystal that reflects red light, so that the light from a red laser would be amplified.

“The molecules we are looking at – in this study, viruses and small proteins – are extremely small. They cannot scatter enough light to create a signal that can be detected by a conventional optical microscope,” said graduate student Nantao Li, the first author of the paper. “The benefit of using the photonic crystal is that it amplifies the light’s intensity so it’s easier to detect those signals and enables us to study these proteins and viruses without any chemical labels or dyes that might modify their natural state or hinder their activity – we can just use the intrinsic scattering signal as the gauge for determining if those molecules are present.”

The researchers verified their technique by detecting the virus that causes COVID-19. PRISM detected individual coronaviruses as they traveled across the slide’s surface. The researchers also used PRISM to detect individual proteins such as ferritin and fibrinogen. The technique could allow researchers to study such biological targets in their natural states – watching as proteins interact, for example – or researchers could seed the surface of the photonic crystal slide with antibodies or other molecules to capture the targeted items and hold them in place.

“It takes 10 seconds to get a measurement, and in that time we can count the number of viruses captured on the sensor,” Cunningham said. “It’s a single-step detection method that works at room temperature. It is also fast, very sensitive and low cost. It’s very different from the standard way we do viral testing now, which involves breaking open the viruses, extracting their genetic material and putting it through a chemical amplification process so we can detect it. That method, called PCR, is accurate and sensitive, but it requires time, specialized equipment and trained technicians.”

Cunningham’s group is working to incorporate PRISM technology into portable, rapid diagnostic devices for COVID-19 and HIV viral load monitoring. The group is exploring prototype devices that incorporate filters for blood samples and even condensation chambers for breath tests.

“We are also going to use this as a research tool for biology and cancer,” Cunningham said. “We can use it to understand protein interactions that are parts of disease processes. We are interested in using it to detect these tiny vesicles that cancer cells shed, and to see what tissues they come from, for diagnosis, and also to study what cargo they are transporting from the cancer cells.”

The National Science Foundation and the National Institutes of Health supported this work. Cunningham is affiliated with the Beckman Institute and HMNTL.