Whitesides Research

Whitesides Group Research

Paper Diagnostic Systems

Background on paper-based microfluidic devices

We fabricate paper-based microfluidic devices by patterning paper using an epoxy-based negative photoresist (SU-8). Paper is impregnated with photoresist, dried, and exposed to UV light through a transparency mask, which can be printed using an inkjet printer or a photocopying machine, or drawn by hand. After a second baking step, the unexposed photoresist can be washed out of the paper with acetone and isopropanol. The only equipment required to pattern paper is a UV lamp and a hotplate. Channels patterned into paper wick micro-liter volumes of fluids by capillary action and distribute the fluids into test zones where independent assays take place (Fig 1 & 2). It is possible to pattern microfluidic channels in paper with dimensions down to 200 µm in width and 30 µm in height (the height of the channel is defined by the thickness of the paper). It also is possible to pattern different types of paper, such that the properties of the paper can be selected for specific applications (e.g., filtering, wicking fluids, pumping fluids, and storing reagents). The photoresist used to pattern paper can be prepared from commercially available materials for ~$60/L. Approximately 20 mL of photoresist are required to pattern a letter-sized piece of paper. Once paper is impregnated with photoresist, it can be stored in the dark for several months and presumably shipped. Another advantage of working with paper is that we believe it can be patterned in a reel-to-reel process by slightly modifying existing machines and techniques, which would allow for high-throughput fabrication of paper-based microfluidic devices. This new method of patterning would allow microfluidic devices to be prototyped in resource-limited settings (where they are needed most) and would require only paper impregnated with photoresist, a transparency mask, acetone and isopropanol.

Background on Three dimensional paper-based microfluidic devices

We stack layers of patterned-paper and double-sided adhesive tape to generate three dimensional (3D) microfluidic devices out of paper (Fig 3). Three dimensional devices can distribute fluids within layers of paper (in the x, y plane) and between adjacent layers of paper (in the z direction). Holes patterned into the tape and filled with cellulose powder allow fluids to wick from one layer of paper into an adjacent layer. Using 3D devices, it is possible to distribute samples from a single entry point into thousands of test spots where assays can take place (Fig 4 & 5). Three dimensional paper-based devices can be assembled on large scale by stacking large sheets of patterned paper and tape and by cutting the stacked layers into individual devices. The tape presumably can be patterned with a mechanical punch or knife cutter, and the holes in the tape could be filled by spraying cellulose powder into them using a spray gun. It also should be possible to mix the reagents for each assay into the cellulose powder before spraying it into the 3D device.

Paper-based Microfluidic Electrochemical Sensing

Microfluidic paper-based analytical devices (µPADs) are a new class of point-of-care diagnostic devices that are inexpensive, easy to use, and designed specifically for use in developing countries. Although colorimetric assays are the best choice for some applications, electrochemical sensing provides a more versatile and quantitative methodology for others. Our mission is to develop paper-based electrochemical system have the characteristics required to be useful in a range of applications, including human, animal and plant diagnostics, food-quality control, and environmental monitoring.

We have developed microfluidic paper-based electrochemical devices (we call these devices µPEDs) that are capable of quantitatively detecting various analytes (e.g., heavy metal ions, and glucose) in aqueous solutions, including biological fluids such as urine, serum and blood (Figure 6). The ?PEDs comprise microfluidic channels, fabricated from patterned chromatography or polyester/cellulose blend paper, and screen-printed electrodes on chromatography paper or polyester film. We are moving towards the implementation of such devices for electrochemical immunoassay for the detection of multiple preventable diseases.

We has also developed a simple, electroanalytical system—based on the combination of a commercial hand-held glucometer with easily fabricated Micro-Paper-based Analytical Devices (µPADs)—that is useful in quantitative analysis of metabolites such as glucose, cholesterol, and lactate in human plasma or whole blood, and ethanol (or acetaldehyde) in aqueous solution (Figure 7). The use of glucometers as readers for EµPADs substantially increases the scope of options for detection using paper–based analytical devices in the developing world, and in other resource-limited environments. This system can be interfaced with a cell phone (either by human reporting of the data, by photography the LCD display, or, in principle by a coded interface). It can also be used in home patient care with telephone or internet communications.

Synthesis of DNA on Paper for Gene Detection

For many diseases, early recognition is the key to increased survival rates. Diagnostic tests are indispensable tools for detecting many diseases such as HIV, hepatitis and tuberculosis. Making diagnostic tests available for a greater number of people, especially in the resource-limited areas of the world, is one of the top priorities of the medical community. We have developed an in situ DNA synthesis strategy using paper as the solid support, including paper pre-activation and protection, direct DNA synthesis using paper as the solid support, and post-synthesis deprotection. This strategy does not involve any purification steps; yet it still demonstrated high sensitivity and specificity against target genes. Using an E.coli. pUC 19 gene fragment as a model target, the detection limit of this method is about 1.0 nM, comparable to the current DNA detection methods (see Figure 8 & 9).

References

1. Martinez, A. W., Phillips, S. T., Whitesides, G. M. and Carrilho, E., Diagnostics for the developing world: microfluidic paper-based analytical devices, Anal. Chem., 2010, 82, 3-10.
2. Carrilho, E., Phillips, S. T., Vella, S. J., Martinez, A. W. and Whitesides, G. M., Paper Microzone Plates, Anal. Chem., 2009, 81, 5990-5998.
3. Martinez, A. W., Phillips, S. T. and Whitesides, G. M., Three-dimensional microfluidic devices fabricated in layered paper and tape, Proc. Natl. Acad. Sci. USA, 2008, 105, 19606-19611.
4. Martinez, A. W., Phillips, S. T., Wiley, B. J., Gupta, M. and Whitesides, G. M., FLASH: a rapid method for prototyping paper-based microfluidic devices, Lab Chip, 2008, 8, 2146-2150.
5. Martinez, A. W., Phillips, S. T., Carrilho, E., Thomas, S. W. 3rd, Sindi, H. and Whitesides, G. M., Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis, Anal. Chem., 2008, 80, 3699-3707.
6. Martinez, A. W., Phillips, S. T., Butte, M. J. and Whitesides, G. M., Patterned paper as a platform for inexpensive, low-volume, portable bioassays, Angew. Chem. Int. Ed., 2007, 46, 1318-1320.
7. Carrilho, E., Martinez, A. W. and Whitesides, G. M., Understanding wax printing: a simple micropatterning process for paper-based microfluidics, Anal. Chem., 2009, 81, 7091-7095.
8. Ellerbee, A. K., Phillips, S. T., Siegel, A. C., Mirica, K. A., Martinez, A. W., Striehl, P., Jain, N., Prentiss, M. and Whitesides, G. M., Quantifying colorimetric assays in paper-based microfluidic devices by measuring the transmission of light through paper, Anal. Chem., 2009, 81, 8447-8452.
9. Siegel, A. C., Phillips, S. T., Wiley, B. J. and Whitesides, G. M., Thin, lightweight, foldable thermochromic displays on paper, Lab Chip, 2009, 9, 2775-2781.
10. Nie, Z., Nijhuis, C. A., Gong, J., Chen, X., Kumachev, A., Martinez, A. W., Narovlyansky, M. and Whitesides, G. M., Electrochemical sensing in paper-based microfluidic devices, Lab Chip, 2010, 10, 477-483.
11. Cheng, C.-M., Martinez, A. W., Gong, J., Mace, C. R. Phillips, S. T., Carrilho, E., Mirica, K. A. and Whitesides, G. M., Paper-Based ELISA, Angew. Chem. Int. Ed., 2010, 49, 4771-4774.

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