Sana Jahanshahi-Anbuhi started her study with a trip to the supermarket to buy Listerine breath strips, the sort that melt on your tongue in seconds. But the chemical engineering postdoc at McMaster University in Hamilton, Canada wasn’t worried about bad breath. She hoped to repurpose the gooey starch contained in the strips to create easy-to-use chemical tests—whether employed to detect E. coli in water, for example, or the presence of drugs such as methamphetamines. Jahanshahi-Anbuhi and colleagues recently reported their success in the journal Lab on a Chip.
Chemical tests often require users to perform multiple steps, such as adding solutions, explains Carlos Filipe, Jahanshahi-Anbuhi’s advisor and study co-senior author. “We thought we would break down these steps in terms of layers of reagents,” Filipe says.
The key ingredient in both breath strips and Jahanshahi-Anbuhi’s layers is pullulan, a polysaccharide that easily forms films when air-dried—“like Jell-O,” says Filipe. It dissolves again, just as easily, when wet. Jahanshahi-Anbuhi’s idea was to suspend test reagents in that pullulan film, and stack the films like a layer cake atop a piece of paper. By dribbling a bit of solution on the stack, or simply dunking the paper, the liquid would dissolve the pullulan layers one by one, releasing the chemicals as needed. It’s also possible to line the pullulan films up side-by-side on a paper strip, so each reagent is released as liquid climbs the paper.
“Stacked layers soaked with different reagents are a known concept…[but] the use of water-soluble, totally safe and green pullulan is a major step forwards,” says David Avnir, a materials chemist at the Hebrew University of Jerusalem, who was not involved in the work. “It really is an ingenious idea!”
To make a test for fecal E. coli bacteria in water samples, the authors adapted the traditional two-step process. Normally scientists must first break open the bacteria in a sample, then use a paper-based test compound that turns red or purple when such contaminants are present. Instead, the engineers built the cell-opening enzymes right into a layer of pullulan atop the indicator paper. They pipetted a bit of bacterial culture on top, which dissolved the pullulan to release the necessary enzyme. After a 40-minute incubation, the test could detect as few as 100,000 bacteria in a milliliter of solution, which is comparable to commercial E. coli water tests.
The authors also incorporated a two-step assay for amphetamines—potentially useful for testing powders from a suspect or crime scene in a lab—into a dual-layer pullulan cake. The first layer contains acetaldehyde, which reacts with the drugs to form a compound called an enamine. The second layer contains sodium nitroprusside and sodium carbonate, which causes enamines to form a blue precipitate, hence confirming the drug’s presence.
Filipe was particularly delighted to see that the film securely held in acetaldehyde; though the compound is quite volatile, it didn’t evaporate away. All the compounds the authors tried to suspend in pullulan worked, Filipe says, while noting that the chemical must be water-soluble; a strongly hydrophobic molecule might not work.
Filipe says it would be easy to mass-produce pullulan tests, and envisions many uses. For example, by designing the right set of pullulan layers, engineers might perhaps design a way to prepare DNA—taken from a fecal sample—for sequencing, even at a field site.
“Sequential delivery [of test chemicals] can be a hard process to reduce to simple parts,” says Paul Yager, a bioengineer at the University of Washington in Seattle who wasn’t involved in the research. “This way adds a new tool to the arsenal of those of us working to produce simple, inexpensive point-of-care devices for detection of infectious diseases.”
The researchers also found that pullulan protects proteins from degradation, even at temperatures as high as 90 degrees Celsius. They are hoping to use pullulan to store vaccines during transport, eliminating the need for the “cold chain” of refrigeration that’s normally required to provide lifesaving vaccines to far-flung locales.