By decorating tiny nanopores with short DNA strands called aptamers, researchers have created a sensor that distinguishes between infectious and non-infectious viruses (Sci. Adv. 2021, DOI: 10.1126/sciadv.abh2848). The device can detect a model virus that mimics SARS-CoV-2, for example, with a sensitivity close to the best conventional diagnostics.
The gold-standard method for detecting viruses is based on reverse transcription polymerase chain reaction (RT-PCR). But this tends to be time-consuming and expensive, and its high sensitivity can also be an Achilles’ heel. That’s because RT-PCR can pick up genetic material in the long-dead viral debris that may linger in patients for weeks after an infection. In the current pandemic, that means some patients are flagged as COVID-positive when they are no longer infectious.
“We really need a rapid method to tell people not only if they have a virus, but whether that virus is infectious or not,” says Yi Lu of the University of Illinois Urbana-Champaign, part of the team behind the new sensor. “To me, that is a really critical missing link.”
The usual way to determine the infectiousness of a virus is a laboratory test in which researchers infect a plate of cells with viruses, and then count how many areas of infection grow on the plate. This is labor intensive, takes several days, and does not work particularly well for viruses that struggle to thrive in cell cultures.
The sensor developed by Lu’s team takes a completely different approach. It spots infectious viruses using aptamers—DNA molecules that latch on to specific proteins, acting rather like synthetic antibodies. The researchers searched for aptamers that could bind only to infectious viruses, while ignoring non-infectious viruses. They first did this for human adenovirus (HAdV), and then for a lentivirus that incorporates the spike protein of SARS-CoV-2; this virus is often used as a safer model for SARS-CoV-2 itself.
For each virus, the team started with a pool of a quadrillion (1015) different aptamers with random sequences, each 45 nucleotides long, and mixed them with whole viruses that had been confirmed as infectious. Any aptamers that did not bind were discarded, while those that did were amplified to create a fresh batch.
Next, the researchers screened this batch of aptamers against non-infectious viruses, and removed any aptamers that bound to these duds. The remaining aptamers went through several more rounds of selection and counter-selection, until a winner emerged from the fray. The champion aptamers for each virus had 100–1000 times as much affinity for infectious virus as that of non-infectious virus.
The researchers used amine-based linkers to mount these aptamers on the walls of bullet-shaped nanopores etched into a thin polymer film. As viruses bind to the aptamers inside the nanopores, they change an electrical current running through the polymer film.
Virus-spiked samples of human serum and saliva produced a reliable change in current in the device, indicating the presence of infectious virus particles. Non-infectious viruses produced no signal.
The device could detect as few as 10,000 of the modified lentiviruses per ml, not far off the typical detection limit for RT-PCR. The technique also worked with spiked samples of drinking water and wastewater.
“If this result proves to be really correct, with real samples, it would be a great advantage,” says Arnaud Buhot at Grenoble Alpes University, who works on aptamer-nanopore sensors. He says that although there are a handful of prior examples of aptamer-nanopore sensors, none have been commercialized (Sensors 2020, DOI: 10.3390/s20164495).
For now, the assay procedure used in the new virus sensor may be too complicated for a point-of-care device, Buhot says. He also notes that the researchers do not know how the aptamers distinguish infectious from non-infectious viruses. “This is really a huge question,” Buhot says.
“We know that the aptamer binds to a protein on the surface of the virus, but we don’t know exactly where,” acknowledges team member Ana S. Peinetti at the University of Buenos Aires. The researchers are now working to answer that question, and are also testing the device with SARS-CoV-2 itself, in collaboration with researchers at the University of Illinois Chicago.