Development of 2-D and 3-D Paper-based Microfluidic devices for the detection of Cryptosporidium and Giardia
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In developing countries, morbidity due to infectious diseases such as diarrheal illness can cause major deterioration of physical and cognitive impairment in young children under the age of five and individuals with poor immune system. In such regions, proper diagnosis and treatment can help in changing the mortality and morbidity rates. Current tests used to detect diarrhea-causing pathogens are often expensive, time consuming, require a well-maintained centralized laboratory with continuous power supply, highly skilled laboratory personnel and good bio-safety practices, which are often limited in resource poor settings in both developed and developing countries. Real-time PCR, immunoassays (ELISA, or EIAs, lateral-flow test strips), microscopy, and flow cytometry are few examples of traditional tests available. The goal of this project was to develop a paper-based microfluidic device for detection of Cryptosporidium and Giardia, two protozoan pathogens that cause persistent to chronic diarrhea worldwide that is inexpensive and easy to use. Toward this goal, we designed and optimized a wax-printing technique to create microfluidic channels in paper that direct fluid flow via capillary action in defined patterns for colorimetric immunoassay detection of individual and multi-plexed pathogens. Initial results suggest that a minimum printed width of 300 µm was necessary to form an impermeable barrier in chromatography paper when heated at 95°C for 10 min, while a minimum channel width of 1500 µm was necessary to wick fluids through the microfluidic channels. Next, we performed a concentration series of immunolabeled Cryptosporidium oocysts and Giardia cysts to determine the lowest detectable number of oocysts in an enzyme-based colorimetric assay. Our results indicate that as few as 250 oocysts were visibly detectable for Cryptosporidium and 5000 cysts for Giardia. We further fabricated 3- dimensional (3-D) paper-based devices with a size- selective filter that excluded the use of cumbersome pre-labeling protocol to remove unbound-free antibodies by retaining enzyme-bound pathogens and measuring the amount of enzyme that reaches the bottom layer. Tests for size-selective membrane and 3-D retention assay using in-line filter holder showed that cellulose acetate membrane with 1.2 µm pore size was able to retain Cryptosporidium oocysts of size 4-6 µm. Follow-up assays for detection of Cryptosporidium in paper devices using enzyme retention assay showed potential for further improvement; however, this research provides initial proof-of-concept for paper-based microfluidic assays to detect infectious pathogens with high sensitivity using low cost materials and simple fabrication techniques.
CitationMadadi, S. (2015). Development of 2-D and 3-D paper-based microfluidic devices for the detection of cryptosporidium and giardia (Unpublished thesis). Texas State University, San Marcos, Texas.