An ancient Roman coin, depicting the two-faced god Janus. An approach that uses so-called Janus particles is one of two recently reported concepts for speeding up lab-based COVID-19 tests. [Image: claudiodivizia / Getty Images]
In the era of COVID-19, it’s become a familiar ritual. On the onset of symptoms, you tear open an antigen self-test kit. Then you swab each nostril for the mandated (and excruciating) 15 seconds; prepare and dribble out the test fluid; and await the appearance of the dreaded Second Pink Line.
But while rough-and-ready home testing of this sort is now a common annoyance, researchers continue the quest to improve on the more rigorous, complex and slow testing methods used in the lab. In that search, two recent studies from research groups in Japan have unveiled novel approaches that leverage light, nanoparticles and microfluidics in interesting ways.
Wanted: Better mousetraps
The target for both of the new studies is not the imprecise antigen testing of home test kits, but the “gold standard” techniques used for more quantitative evaluations. One such technique is reverse-transcriptase polymerase chain reaction (RT-PCR), in which fragments of viral RNA are transcribed to DNA and then massively amplified to reach detectable levels. Another is the enzyme-linked immunosorbent assay (ELISA), which measures the presence of antibodies for the virus in a blood sample and reads out the results colorimetrically (see “Optics and the COVID-19 Pandemic,” OPN, May 2020).
But while PCR and ELISA are generally more reliable than antigen testing, they have their disadvantages. Both require specialized equipment; are complex, multi-step processes; can take a long time to provide a detectable signal; tend to be expensive; and are vulnerable to potential contamination or other problems. Those defects have spurred an ongoing search for improvements to these techniques or for entirely new ones.
Two-faced nanoparticles
In one of the new studies, a team led by Hiroshi Yabu of Tohoku University, Japan, investigated the possibility of improving ELISA testing using a specific, clever flavor of nanoparticles—so-called Janus particles (Langmuir, doi: 10.1021/acs.langmuir.4c01911). Named for the two-faced god of Roman myth, Janus particles are essentially micro- or nanoparticles (typically spherical) that have different surface properties on their two sides.
In previous work, the Tohoku team had developed a technique for creating polymer Janus nanospheres that are magnetic on one side and coated with fluorescent dye on the other. The team found that, by applying a magnetic field, the fluorescent side of the nanoparticles could be uniformly oriented, considerably boosting the fluorescence signal.
The Tohoku University team's approach uses combined fluorescent and magnetic Janus nanoparticles (upper left), functionalized with COVID antibodies on the magnetic side, to detect a COVID-19-related antigen in a microfluidic system (upper right). The researchers have created a commercial system, the “Express Biochecker,” that uses the approach (lower left), which experiments showed can detect the viral antigens quickly and at low concentrations (lower right). [Image: H. Yabu]
The researchers wanted to see if the two-faced nanoparticles could be modded to build a system for detecting the COVID-19 virus, SARS-CoV-2. To do so, they started with their previously developed fluorescent/magnetic Janus particles, and cooked up a wet-chemistry technique to functionalize the magnetic side with antibodies that would bind to the SARS-CoV-2 nucleocapsid (N) protein. The result was particles with a magnetic-plus-antibody side, for detecting the virus, and a fluorescent side, for reading out the results.
Next, the team immobilized additional N-protein antibodies onto a fixed substrate within a microfluidic chip, and added samples of the SARS-CoV-2 antigen to the chamber, where they were caught and held in place by the immobilized antibodies. Finally, a solution containing the antibody-decorated fluorescent/magnetic Janus particles was run through the microfluidic system. As expected, the antibodies on the magnetic side of the Janus particles grabbed onto the captured SARS-CoV-2 antigens, tagging them for fluorescence measurement. Using this system, the team found that the Janus nanoparticles enabled sensing of the SARS-CoV-2 antigen at a detection limit of 3.1 nanograms per milliliter.
What’s more, applying a magnetic field during the immunoassay—basically, placing a magnet beneath the microfluidic chip—considerably speeds things up, by nudging the antibody-containing half of the particles closer to the fixed substrate and increasing the rate at which they grab onto the N-protein antigens captured there. At an antigen concentration of 400 ng/mL, a readable signal was available in as little as eight minutes. The magnetic field is “a key … to speed up the immunoassay process, by concentrating Janus particles on the surface of the microfluidic chip in a channel,” Yabu, the team leader, told OPN in an email. “Without the magnetic field, it takes a longer time to get accurate results.”
Yabu’s team has already designed a quantitative testing system, the “Express Biochecker,” that uses the approach described in the group’s recent paper. The authors believe the approach should “be applicable not only to SARS-CoV-2 but also to the quantitative measurement of various other disease marker proteins and biomolecules”—a prospect on which the researchers are working now.
COVID-catching bubbles
A second strand of work from a different Japanese team, led by Takuya Iida and Shiho Tokonami of Osaka Metropolitan University, uses light in a different way—as a mechanism to accelerate detection through faster, more efficient herding of SARS-CoV-2 viruses to test sites on a substrate (npj Biosensing, doi: 10.1038/s44328-024-00004-z). Specifically, the technique relies on localized surface plasmon resonance and a process the authors call optical condensation.
In the approach prototyped by the Osaka Metropolitan University team, low-power laser light, shone on gold surface dotted with round nanoimpressions, is plasmonically concentrated and converted to heat. This creates a nanobubble over the impression, and also leads to convective flow that concentrates nanoparticles (such as the SARS-CoV-2 virus) near the base of the bubble. [Image: Osaka Metropolitan University, CC-BY 4.0]
The system begins with a thin gold layer on a glass substrate, into which the researchers inscribed bowl-shaped depressions with a diameter on the order of 500 nm to serve as plasmonic amplifiers. When this gold-coated nanoparticle-imprinted plasmonic substrate (NPI-PS) is immersed in a liquid and illuminated with laser light, the nanobowls plasmonically concentrate the light energy and convert it to heat. The localized heat causes a bubble to grow over the nanobowl. It also leads to convective flow within the liquid, which in principle can drive nanoparticles to gather around the base of the bubble.
To put the approach to work in detecting the COVID-19 virus, the team used a two-step process. In the first step, they dropped 5 µL of a solution containing nanoparticles decorated with antibodies of the SARS-CoV-2 spike protein onto the NPI-PS, and irradiated it for one minute at a laser power of 3 mW. This caused enough convective flow to coat the NPI-PS surface with a layer of antibody-containing nanoparticles.
Then, the team dribbled in another 5 µL of a solution containing “virus-mimicking nanoparticles” (VMNPs)—fluorescent nanobeads with the virus’s spike protein tacked onto the surface. A second, much longer dose (four minutes) of 3-mW laser light built convective flow that crowded the nanoparticles around the base of the plasmonically induced nanobubble, where they were caught by the antibodies and could be detected by fluorescence readout. The entire two-step process thus, according to the team, takes five minutes—a dramatically shorter interval than most other assays.
“This study shows that we can shorten the cumbersome antibody coating process and perform rapid and highly sensitive protein detection,” Iida said in a press release reporting the work. And, while much clearly remains to be done before a commercial system could be devised, the team believes its findings “provide an important basis for the development of compact biochips for future portable light-induced acceleration systems”—and that such chips could prove applicable not only to the COVID virus but to biomarkers for a range of other diseases.