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Georgia Tech Team Develops New Method for More Complex Lateral Flow Testing


NEW YORK – With the use of simple materials, a team from the Georgia Institute of Technology was able to create a method to increase the complexity of lateral flow tests, allowing for multistep assays that require few resources.

The team's work, laid out in two recent papers, draws on flow control technology to apply multiple reagents in a specific sequence, with specific time delays to allow reactions to occur. A paper published in Science Advances details the method itself, while a paper from ACS Sensors applies the technology for a multiplex point-of-care test for SARS-CoV-2 and influenza A/B.

Fatih Sarioglu, the lead author on both studies and a professor in Georgia Tech's school of electrical and computer engineering, said that his team works on developing point-of-care tests for diagnostics, with an emphasis on traditional microfluidics. His team hasn't used paper-based diagnostics much before, so this method is a "new direction for us," he said.

The impetus for developing the new method, he said, was a desire to see if the team could make lateral flow tests smarter by "taming capillary flow" and directing it to produce more complex tests.

Traditional lateral flow tests use spontaneous capillary-driven flow to induce a reaction and produce a result, but the lack of control means those tests aren't able to simultaneously screen for multiple analytes in a sample, Sarioglu said.

Using special ink to delimit parts of the paper in a lateral flow test, along with laminated tape to create sections where reagents could be kept, the team was able to coordinate the flow of reagents. The researchers tested the method with a variety of sample types, including saliva, urine, and whole blood, and it worked with each type, he said.

According to the Science Advances paper, the water-insoluble ink is applied to the paper strip of the test, which "imprint[s] roadblocks on the flow path" and gradually forms "a void between wetted paper and a sheath polymer tape to create timers." The void formed is the only pathway for the flow to move. Those timers are drawn at "strategic nodes" to hold the flow for a specific amount of time, enabling "multiple liquids to be introduced into multistep chemical reactions following a programmed sequence," they wrote.

The test can be programmed to "coordinate different capillary flows, sequentially introduce different reagents into a reaction leaving optimal incubation times in between, and autonomously perform complex assays that could otherwise not be possible with conventional lateral flow assays," the researchers wrote. They found that the immunoassays they created were "an order of magnitude more sensitive than commercial counterparts," they added.

The researchers also developed a separate device for DNA purification that used specialized filter paper pretreated with chemicals for lysing human cells and preserving extracted DNA. It moved the extracted DNA to different spots — one that had been treated with a solution for denaturing proteins; one with water to wash the proteins, cell debris, and potential inhibitors away; and one with a buffer to stabilize the DNA for downstream analysis.

For the SARS-CoV-2 and influenza multiplex test, which uses saliva, Sarioglu said the reagents and sample are loaded onto the lateral flow device at the same time, but the assay is programmed to delay the times certain reagents move through the device, allowing for a more complex reaction to occur. The device provides a colorimetric readout, like many traditional lateral flow tests, which can be read using a smartphone camera, he added. The COVID and flu test was able to detect the viruses at concentrations as low as 50 copies per microliter.

While many lateral flow tests are based on antibodies or antigens, Sarioglu's team built a test that required amplification. The amplification steps were able to be achieved by keeping the reagents in the specific delineated sections on the paper strip and applying amplifying reagents, which generated heat internally, he said. The method can be used to extract and stabilize RNA, which could then be put into a different device for testing, Sarioglu noted.

The COVID and flu test takes close to an hour to return a result, but if an amplification step isn't needed, a test's turnaround time could be much faster — Sarioglu said it depends on the test itself. "We think it can build a lot of different assays," including ones beyond infectious disease, he said.

He added that the team is interested in commercializing the COVID and flu test, especially since it can be used at home and doesn't require any external instruments. It can be produced easily and would cost "cents," since the only needs are for paper, paint, water, and the reagents, he said. The material cost is the "lowest of the low," he continued, and is simpler than other microfluidic or lab-on-a-chip methods.

Weijin Guo, a lecturer in the department of biomedical engineering at Shantou University in China who works on lateral flow technology, said via email that flow rate control is important for lateral flow tests because "it can affect almost all the steps" on the tests, "including dissolution of reagents, mixing of reagents, immuno/chemical reaction, and incubation."

However, Guo said that based on the published results, the variations of signal for the method are "slightly big, which may come from the substrate material itself, since microstructures of the paper are not uniform." He recommended the team look for another substrate material "with more uniform microstructures, to reduce the signal variations and improve the performance on sensitivity."

He also recommended the researchers try expanding the technique beyond colorimetric immunoassays, using it with fluorescent immunoassays to see if the performance of those types of assays can be improved.

Tests like the COVID and flu test could also be used in low income countries due to the low cost, and Sarioglu said the team is very interested in the "global health opportunities" that could be available beyond the US. It can also easily be multiplexed with other viruses, Sarioglu said.

Guo said that the method should be feasible to reproduce at a larger scale because the patterning of the tape ink is similar to the patterning of liquid immunoassays on lateral flow test substrates. As a result, "the spotting machines used to pattern liquid immunoassays on lateral flow test substrates in industry can also be used to pattern the tape ink," he said.

Sarioglu's team has applied for a patent in the US for the method and is currently talking with partners who could help commercialize it, Sarioglu said. Sarioglu is now looking for funding to continue the research and commercialize the test.

While the test isn't ready for Emergency Use Authorization submission to the US Food and Drug Administration, Sarioglu said that's the ultimate goal for the team.

They have also seen interest from the federal government in the test, he said. The method would also allow for cheaper versions of existing assays that currently require expensive instruments, he said.