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Putting diagnostic tests on paper

BioPhotonics
Mar 2007
Michael J. Lander

Basic medical diagnosis may seem simple. Visit a hospital or clinic, leave a fluid sample, and you will know your malady within a day. In impoverished regions, however, detailed laboratory analysis becomes nearly impossible. When hundreds or thousands of individuals await screening, such tests also prove incredibly expensive and time-consuming.

These sobering facts motivated scientists led by George M. Whitesides at Harvard University in Cambridge, Mass., to develop an inexpensive and portable paper-based diagnostic test. Through an application of optical technology, the team created a bioassay that not only yields rapid and accurate results under field conditions, but also requires very small samples.

The researchers used cellulose chromatography paper as the bioassay’s foundation. After infusion of a paper disk in negative photoresist and subsequent baking, they used a mask aligner from AB-M Inc. of San Jose, Calif., to place a photomask over it and irradiate it with UV light at 405 nm. Another baking step caused photoresist that had been exposed to light to undergo polymerization and to bind firmly to the paper.

Treatment with developer and a washing solution removed the unbound compound and left the researchers with an absorbent millimeter-wide channel that diverged into three test areas, all bordered with hydrophobic photoresist. An additional treatment with oxygen plasma increased the hydrophilic character of the channel and test zones.

The researchers then trimmed away the excess paper and applied potassium iodide and a solution of glucose oxidase and horseradish peroxidase — glucose assay reagents — to the first test area. They spotted citrate buffer solution and tetrabromophenol blue in the opposite well to make a protein assay and added an incomplete test to the upper chamber as a control.

The group exposed prepared assays to 5-μL samples of artificial urine and allowed them to dry for 10 minutes. When exposed to urine with glucose and protein concentrations of 5 mM and 0.75 μM, respectively, the assays demonstrated noticeable changes. This sensitivity is comparable to that of commercial dipstick assays. Storage conditions affected only the glucose assay, which lost sensitivity after one day at room temperature, but not at freezing.

Contamination failed to alter the assay’s performance, a characteristic that is essential in the field. Samples of urine that contained the lower-threshold concentration of glucose and protein were mixed with pollen, soil and powdered graphite, yet the contaminated solution changed the assays’ color to the same degree as the unadulterated solution.

BNPaper_Paper-device.jpg
On a base of absorbent paper, researchers have created a durable and inexpensive bioassay that requires no laboratory equipment to interpret. In this prototype, reagents in the left compartment changed color from clear to brown upon exposure to glucose. A protein assay in the right compartment turned from yellow to blue. The upper chamber served as a control — it contained only one of two solutions needed for the glucose test. Image courtesy of Andres W. Martinez.

According to Andres W. Martinez, a graduate student working on the project, the bioassay has advantages beyond those outlined above. Different designs can be prototyped within a day. Furthermore, the device operates on capillary action alone and allows simultaneous multiplex assaying. On the other hand, the bioassay is limited by its simple design to tests that are readily visualized, such as those the researchers chose for the prototype.

The technology could have the greatest impact in the Third World, where it could help officials efficiently diagnose and monitor disease. In the developed world, the team envisions the technology playing a role in home health care and in first-response and backcountry applications.

Martinez explained that one of the group’s goals is to expand the platform to detect more analytes. “The surface [of the paper] is rich in functional groups, and you can use all kinds of chemistry to modify them,” he said. The researchers also seek to produce the channels more inexpensively, ideally through a printing process. Above all, however, they hope to see the bioassays in preliminary field testing in the near future.

Angewandte Chemie International Edition, Feb.12, 2007, pp. 1318-1320.


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