DFA Livertest: Postage stamp-sized paper tests developed by the non-profit Diagnostics for All can screen for drug toxicity by measuring enzymes produced by liver cells (Courtesy of Diagnostics for All, Inc.)

When someone with a high fever walks into a rural African clinic, diagnosis could be murky. The symptoms could be those of dengue, Ebola, West Nile disease, malaria or flu, and blood work results from distant labs, if available, often takes days. Now a handful of researchers are separately working on inexpensive, paper-based diagnostic tests that accurately pinpoint the cause of a disease in minutes and could speed up treatment and prevent its spread. The lack of funds and commercial partners however, means most might languish in labs.

Experimental paper sensors that detect chemical or biological molecules have proved to be easy to use without the need for pricy equipment or trained specialists. They can cost pennies and they promise to be more sensitive than the rapid diagnostic kits on the market today. What’s more, they could have broader applications, such as treating neglected tropical diseases, mostly because pharmaceutical companies are focusing on widespread maladies that have a larger market. In addition to saving hundreds of thousands of lives each year in the developing world, these paper-based tests could stem health care costs by allowing home-based disease testing in developed regions.

Despite their numerous benefits, however, there’s a risk that most of these devices might never fulfill their promise.

Mini-laboratories

Pharmaceutical companies already sell millions of rapid, paper-based tests priced between $1 and $2 for HIV, hepatitis C and tuberculosis, among other diseases. These are simple lateral-flow systems akin to home pregnancy tests: A strip of paper wicks urine or blood from one end to the other where chemicals or antibodies in the sample interact with an appropriate reagent, creating a color change. But their simplicity is also a limitation. “They work, but they are black or white and they test for one thing,” says Harvard University chemist George Whitesides.

Whitesides and others have in the last decade pioneered new tests that are intricate mini-laboratories on paper. Like microfluidic chips, the paper devices can separate, mix, filter and concentrate fluids as well as perform timed reactions and control their sequence—all by patterning networks of fluid-wicking channels on paper. Whitesides does this patterning using wax on postage stamp–size pieces of filter paper patterned with wax to create tiny channels and compartments. His team uses inkjet printers to lay down these features, so they cost only a few pennies to make. He hasss also patented 3-D devices in which fluids flow along and between the layers for more complex processes.

Unlike lateral-flow tests sold today “paper microfluidic devices can do more complex tests that require multiple processing steps,” says Ali Yetisen, a chemical engineer and biotechnologist at Harvard Medical School. For instance, they could repeat a test for accuracy or for multiple diseases at a time or measure precise levels of target molecules. And, they require no sample preparation.

Paul Yager, a biochemist at the University of Washington, meanwhile, has developed a handheld plastic device the size of two stacked card decks that contains strips of patterned paper and wells containing reagents and dyes, and into which a user would insert a fluid sample. The patterns of dots that appear after 20 minutes could be read by a clinician or sent via smartphone camera to a physician elsewhere. Yager says that the box could cost as little as $1 to manufacture in bulk.

Killer app

With grants from the Defense Advanced Research Projects Agency (DARPA), both Yager and Whitesides are working on the killer app for paper microfluidics: nucleic acid testing. This would enable a medical practitioner to diagnosis a number of infectious and chronic diseases by detecting gene sequences or pathogen DNA. “The aim is to come up with a standard footprint for a paper-based nucleic acid test where you simply change one or two molecules to test for a different disease,” Yager says. Currently, nucleic acid detection is performed using the lab-based polymerase chain reaction (PCR) test, which makes copies of DNA strands. “If someone were to develop completely paper-based PCR, that would be revolutionary,” Yetisen says.

But because PCR requires a series of temperature cycles, Yager and Whitesides use what’s called isothermal amplification, which is carried out at a constant temperature range of 60 to 65 degrees Celsius. The challenge is to find a cheap, disposable heating mechanism. Yager’s group has made a prototype device that can accurately spot the antibiotic-resistant MRSA bacteria and are working on Zika virus test. But the device uses batteries to power a heating circuit. The researchers are experimenting with using tea bag–size sachets filled with 100-micrometer-wide iron and magnesium pellets, similar to the what’s used in hand warmers, to create the necessary heat chemically. Meanwhile Whitesides and his colleagues have made a paper machine that can detect nucleic acids using a handheld UV source and camera phone. The test costs less than $2 but requires an incubator for heating. The team is developing a built-in electronic heater.

A handful of companies are trying to test and deploy paper microfluidics in the developing world. Diagnostics for All, a nonprofit Whitesides started in 2007, is at the last stage of regulatory approval for a liver-toxicity test for patients who take potent liver-damaging drugs. The device measures the level of an enzyme released by liver cells when they break down. The firm is also developing a nucleic acid test for hepatitis C and HIV, says company CEO Marcus Lovell-Smith.

Bellevue, Wash.–based Intellectual Ventures is testing two products: One is a $2 malaria test that is over 100 times more sensitive than today’s lateral-flow tests, says Bernhard Weigl, a flow-based diagnostics researcher at the company. Researchers there are also developing an easy urine test for tuberculosis. Today’s strips require coughing up phlegm, which is difficult for sick patients.

Yet to date, most paper microfluidics remain proofs-of-concept. Part of the problem is taking a lab wonder to something that is robust and reliable in often hot, humid climates. “It’s very easy to make relatively cool devices but hard to make them reproducible,” Weigl says.

The biggest challenge is a lack of funding for trials, regulatory approvals and manufacturing. “We’ve shown a path and done exciting early work,” Smith says. “But these are immensely expensive projects. It’s tens of millions of dollars to get these tests approved.”

Yetisen points out the general lack of funding in the area of tropical diseases because the return for pharmaceutical companies is low. Whitesides is now in talks with two big non-U.S. industrial partners who are interested in paper microfluidic devices for uses other than tropical diseases. “The hope,” he says, “is that we can have a partner develop a platform for an application they’re interested in and then leverage the capital to develop what could be useful in Mumbai or Kinshasa.”

Source: Scientific American