Abstract— The development of novel paper-based diagnostic tests has surged in recent years, due to the suitability of these tests for use at the point of care. These emerging paper-based tests retain the low cost and ease of use of traditional lateral flow tests, while offering increased sophistication and capabilities that approach those of traditional microfluidic devices. Here, we report on the development of a novel paper- based test for the diagnosis of influenza, commonly known as the flu. Influenza is a ubiquitously occurring infection, affecting 5-20% of Americans and resulting in an average of 23,000 deaths in the U.S., and up to 500,000 deaths globally, each year. Despite its prevalence, the diagnosis of influenza remains unsatisfactory, especially at the point of care. In particular, lateral flow tests for influenza suffer from poor sensitivity and provide only limited information about the infecting flu virus. Point-of-care testing of influenza therefore stands to benefit substantially from improved technology. To this end, we have developed two different versions of a paper-based flu assay, both using computationally designed affinity proteins, or “binders,” that bind to the influenza hemagglutinin (HA) protein. One version of the assay utilizes an HA stem-region binder and the other an HA head-region binder. With these assays, we demonstrate the detection of clinically relevant concentrations of recombinant HA and intact influenza virus, as well as the translation of this paper-based system to a two- dimensional paper network (2DPN) folding card device. I. INTRODUCTION Every year, influenza (flu) virus infects 5-20% of Americans, accounting for 15 million to 60 million infections [1]. These influenza infections lead to over 200,000 hospitalizations [1] and an average of 23,000 deaths [2] in the U.S. each year. This makes the flu more deadly in the U.S. than HIV and cervical cancer combined [3]. While the flu is especially problematic for young children, pregnant women, the elderly, and people with other health conditions, the flu can also be a severe respiratory disease for otherwise This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE – 0718124, by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI096184, and by the University of Washington Department of Bioengineering. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. C. A. Holstein, S. Bennett, A. Chevalier, and P. Yager are with the University of Washington Department of Bioengineering, Seattle, WA 98195, USA (corresponding author, C. A. Holstein: phone: 206-616-3129; e-mail: cholst@uw.edu). E. M. Strauch and D. Baker are with the University of Washington Department of Biochemistry, Seattle, WA 98195, USA. E. Fu was with the University of Washington Department of Bioengineering, Seattle, WA 98195, USA. She is now with Oregon State University School of Chemical, Biological, and Environmental Engineering, Corvallis, OR 97331, USA. healthy adults. In fact, during the most recent (2013-2014) flu season, adults (18-64 years old) have accounted for the majority of flu-related hospitalizations and deaths [4]. In addition to being a significant health problem, the flu is also a huge financial burden. A detailed study on the economic burden of flu by Molinari et al. found that influenza costs the U.S. economy $87B annually, with $10B in direct medical costs, $16B in lost productivity, and $61B in lost economic value due to early death [5]. Despite these grim numbers, the flu is treatable. Antiviral medications such as Tamiflu® are available for flu treatment and reduce the severity of symptoms, shorten the duration of illness, and decrease the risk complications—but only when prescribed early in the course of infection, specifically within the first 48 hours of symptoms [1], [6]. Timely diagnosis of influenza is therefore key for successful disease management. Given the importance of influenza diagnosis, several lateral flow-based rapid diagnostic tests (RDTs) have been developed for point-of-care (POC) detection of influenza. It has been shown that the correct use of these flu RDTs significantly reduces cost of treatment, time of hospitalization, and erroneous use of antibiotics versus antivirals [7]. Despite these benefits, current flu RDTs still suffer from low sensitivity, detecting flu infections only 10- 71% of the time [8], resulting in mistreatment, expensive follow-up testing, and often the inability to treat with antiviral medication within the critical 48-hour treatment window. Additionally, all currently available flu RDTs are based upon detection of the internal nucleoprotein and cannot subtype influenza, limiting the resulting diagnostic and epidemiological information. Current flu diagnostic tests are therefore unable to achieve effective early diagnosis for most flu-infected individuals. The key barrier to effective flu diagnosis and disease management is access to accurate, rapid flu testing. We therefore seek to develop a highly sensitive paper- based assay for POC diagnosis and subtyping of influenza based on the envelope protein hemagglutinin (HA). To do so, we have utilized a combination of our novel two- dimensional paper network (2DPN) platform and novel computationally designed affinity proteins that bind specifically to influenza HA. We have chosen the paper-based 2DPN platform for this influenza assay due to its ability to perform complex diagnostic testing in a simple, low-cost device. The 2DPN, as reported by others in our laboratory [9]–[13], combines the simplicity of traditional lateral flow tests with the sophistication of microfluidics-based tests to achieve an intermediate format that is highly suitable for POC use. In Development of a Paper-Based Diagnostic for Influenza Detection Carly A. Holstein, Steven Bennett, Eva-Maria Strauch, Aaron Chevalier, Elain Fu, David Baker, and Paul Yager 296 2014 Health Innovations and Point-of-Care Technologies Conference Seattle, Washington USA, October 8-10, 2014 U.S. Government work not protected by U.S. copyright