Team:Washington/Paper Device

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• Methods
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Introduction

The Rise of Paper Diagnostics

<p>Paper diagnostics is a fast-growing area of bioengineering. It is especially appealing for detecting pathogens, such as markers for infectious diseases, in low-resource settings. However, paper test strips can be engineered for a wide range of uses, including to detect for heavy metals, environmental toxins, hormones, and ions. Paper-based technologies are generally affordable, fast-acting, easy to use, and easy to produce. Because of these benefits, they have recently begun to be explored as low-cost alternatives to more complex diagnostic technologies, such as PCR and ELISA.

Paper as a Platform for Cell Culture

While many have functionalized paper with proteins, fluorescent tags, and other molecules, few have tried to grow cells or use cellular detection pathways on paper. There are many benefits to using a paper platform in conjunction with synthetic biology; biological sensing systems are cheap when produced en masse and can be engineered for high specificity. They can often sense for long periods of time, and can form more complex signaling and receiving systems than can be achieved through chemistry. We took inspiration from the work of the Derda Research Group at the University of Alberta, which has performed some work in growing bacteria and mammalian cells on paper []. We especially drew from a 20XX article in which the group developed a low-cost test strip mainly using products that could be found in the home []. This makes our device very cheap and accessible to make.

S. Cerivisiae as a Biosensor

Saccharomyces Cerivisiae, commonly known as baker’s yeast, was engineered as the biosensing organism in this platform. This cell has an activity > 0 for pH from 2.1 to 7 (Arroyo et al. 2009). The activity is above 0 and increasing from 12°C to 36°C (Arroyo et al. 2009). The wild type Saccharomyces Cerivisiae is not known to be mutagenic. Another crucial characterization for test strip media is longevity. S. Cerivisiae can live for approximately 20 to 120 hours (Minois et al. 2004). In a dehydrated dormant state, however, the yeast can survive for years (Fabrizio & Longo 2003). The genome of this model organism has been sequenced, it is easy to obtain in the lab, and the genomic structure is easy to modify.

Methods

Making the Paper Device

The device has three basic layers - the filter paper platform, the yeast with media, and the PDMS (Polydimethyl Siloxane) window. Filter paper was chosen because of its predictable and even wicking properties, it’s durability when wet, and the fact that yeast cannot travel through the tight mesh of paper fibers. The PDMS provides a barrier from the outside that allows the yeast to be contained in a sterile environment. It also helps prevent evaporation of media from within a loaded device; while oxygen and other gases can diffuse through the PDMS, water and contaminates cannot. Squares of PDMS were secured to each side of the paper strip using heat-resistant cloth electrical tape, and the partially-built devices were autoclaved.

After they had been sterilized, media and yeast could be added. Media was added first by pipetting liquid or gel between the PDMS and the paper on one side. Using a pipette tip, yeast were then placed in the center of the window, and the device was sealed.

Performing Assays

Assays were performed using two different methods for the application of the target molecule; First, a small volume of target molecule was pipetted directly into the cell growth area behind the PDMS window. Secondly, the paper device was allowed to stand upright in a beaker containing 3mL of target solution, so that the target molecule could travel up the paper to the cells by wicking.

Our device could be easily modified to accommodate a wide range of biological systems. For different strains of cells, different media was added depending on the yeast marker. Additives, such as inducers, could also be pipetted into the media or wicked up the paper, making systems with inducible promoters viable. Cells could also be lysed on paper with the addition of 0.2% SDS and gentle massaging of the PDMS window.

Results

Conclusions

References