326 MRS BULLETIN • VOLUME 38 • APRIL 2013 • www.mrs.org/bulletin © 2013 Materials Research Society Introduction In recent years, the urgent need for rapid and low-cost medical diagnostics—both in developed and developing countries—has become clear. One platform that addresses this need is paper- based microfluidics, which eliminates the need for external equip- ment to move fluids. Porous materials such as paper are ideal substrates for low-cost assays because they are inexpensive and disposable. One of these materials—porous nitrocellulose—is now one of the most commonly used materials in point-of-care (POC) devices, such as the OraQuick Advance HIV-1/2 test, the BinaxNow tests for a variety of infectious pathogens, and, most commonly, the home pregnancy test. This review focuses on the history and evolution of nitrocellulose-based assays, as well as critical aspects of assay performance: flow in porous media, protein adsorption, dry reagent storage, and analyte detection. Background History of nitrocellulose Nitrocellulose is a versatile polymer that has been broadly utilized since the 1800s. 1–3 Also known as cellulose nitrate, nitrocellulose is created commercially by the reaction of cellulose (purified from plants, commonly wood pulp and cotton) with nitric acid, replacing the cellulose hydroxyl groups with nitrate groups. 4 Today, nitro- cellulose membranes are created by phase inversion, 5–7 in which nitrocellulose is dissolved in an organic solvent that is evaporated in the presence of a nonsolvent, leaving a precipitated nitrocellu- lose membrane with high porosity 4,8 ( Figure 1a). The porosity and pore size of the membrane can be controlled by the solvents used, evaporation speed, temperature, and humidity. 8 The result is a material with the unique combination of tunable pore size, high surface-to-volume ratio, and very low cost. Porous nitrocellulose membranes were first used to immobilize biomolecules in the 1960s. 4 Nygaard and Hall demonstrated in 1963 that RNA-DNA complexes adsorb onto nitrocellulose membranes, while free nucleic acid strands pass through. 9 Oth- ers then began immobilizing nucleic acids on nitrocellulose membranes to probe for interactions between a nucleic acid of interest and other biomolecules. 10,11 In 1975, Southern demonstrated the transfer of DNA from polyacrylamide gels to nitrocellulose. 12 This groundbreaking technique, known as the “Southern blot,” allowed specific nucleic acid fragments to be captured for subsequent analysis. The Southern blot inspired the “Northern blot” for RNA transfer 13 and the “Western blot” for protein transfer to nitrocellulose. 14,15 These blotting techniques have been widely employed in biological research and highlight the unique ability of nitrocellulose to interact with three of the most important classes of biomolecules (proteins, DNA, and RNA). 4 The evolution of nitrocellulose as a material for bioassays Gina E. Fridley,* Carly A. Holstein,* Shefali B. Oza,* and Paul Yager The need to improve health outcomes in the developing world and to moderate healthcare costs in developed countries has resulted in an increased interest in sophisticated, inexpensive, and instrument-free point-of-care diagnostics using porous materials. One major segment of the paper-based diagnostics effort is focused on developing high-performance point-of-care tests using porous nitrocellulose membranes. This review provides a perspective on the nature, history, and future of nitrocellulose-based assays. Beginning as a protein blotting substrate, porous nitrocellulose membranes have grown to be the most commonly used lateral fow substrate and are the primary membranes used in two-dimensional paper networks for user-friendly multistep assays. In addition to the historical context, we examine assay development considerations, such as the physics of fow in porous media, reagent deposition and storage, and detection methods. Gina E. Fridley, Bioengineering Department, University of Washington; gfridley@uw.edu Carly A. Holstein, Bioengineering Department, University of Washington; cholst@uw.edu Shefali B. Oza, Bioengineering Department, University of Washington; shefali@uw.edu Paul Yager, Bioengineering Department, University of Washington; yagerp@uw.edu DOI: 10.1557/mrs.2013.60 * These authors all contributed equally to this article.