A variety of paper-based devices and sensors have emerged within the last decade as a strategy to help physicians and researchers in resource-poor areas carry out quick, inexpensive biological assays. Though inkjet printing the needed reagents would be a logical way to produce such devices, biomolecules can degrade and quickly clog ink cartridges. Now researchers have developed a polysaccharide-stabilized ink that’s easily printable and improves the shelf life of paper-based diagnostic tests by 50-fold (Anal. Chem. 2015, DOI: 10.1021/acs.analchem.5b01923).
Last year, John D. Brennan and Carlos D. M. Filipe of McMaster University, in Hamilton, Ontario, and their colleagues used a commercially available polysaccharide, pullulan, to encapsulate and stabilize assay reagents, including enzymes, within tablets for use in areas with limited refrigeration (Angew. Chem. Int. Ed. 2014, DOI: 10.1002/anie.201403222). This work and previous studies suggested that pullulan protects the proteins from oxygen. They wondered if developing an ink that contained pullulan might also provide a convenient way to protect enzymes and other molecules from decomposition in paper sensors.
To test the idea, postdoctoral researcher Balamurali Kannan suggested that the team build a sensor for lactate dehydrogenase (LDH), a biomarker that is elevated in people with disorders including cancer and organ failure. The reagents needed for the test include lactic acid, the cofactor NAD+, and iodonitrotetrazonium chloride, which converts to a bright red dye when LDH converts lactic acid to pyruvate.
To build the paper test, the researchers printed rings of wax in a 96-well array onto filter paper and heated it so the wax soaked into the paper to create barriers to liquid flow. Then they mixed the reagents with processed pullulan to create a viscous ink that they printed in 4-µL droplets in each well and allowed to dry. At each location they added serum samples, some spiked with LDH, and allowed the spots to develop for five minutes. Finally, the researchers took digital photos and analyzed the color change with software to determine the LDH concentration. The pullulan-based paper LDH sensors gave accurate, consistent values for LDH concentrations over a range of levels that occur in the body. The ink doesn’t clog the nozzles, is stable, and doesn’t gel until after printing, Brennan says.
At room temperature, the paper-based pullulan tests remain stable for up to three weeks—seven weeks if refrigerated—while assays printed without pullulan became inaccurate within a single day. The limit of detection for the pullulan-stabilized assay is one-sixth that of assays printed without pullulan. Brennan would like to see even better performance, but he also notes that the compounds—especially the cofactor NAD+— in this assay are particularly unstable, which makes a paper-based assay for this particular reaction especially challenging.
The ability of pullulan to immobilize and stabilize enzymes is interesting, says Ali Kemal Yetisen of Harvard Medical School and Massachusetts General Hospital, particularly considering that pullulan is inexpensive and FDA approved. However, Yetisen is not convinced that this particular assay would be useful in a low-resource setting because it doesn’t provide a definitive diagnosis and requires additional equipment—at least a smartphone camera—for analysis.
The group is currently working on stabilizing other challenging compounds, Brennan says. And industry partners are interested in licensing their pullulan-based printing technology.