Lateral Flow Assays (LFAs), having initially been popularized in the 1980s for use in home pregnancy testing, are now used in a wide array of diagnostic applications. LFAs are also rapid and simple point of care diagnostics of infectious diseases such as HIV, dengue fever or malaria that have high occurrence in low resource areas. Since their introduction in 2007, paper microfluidics combined the advantageous features typical of lateral flow point of care devices such as low cost, portability and ease of usage and result interpretation, with the advantageous features of microfluidics such as low sample volume requirements and the capacity to integrate multiple processing steps allowing increasingly complex tasks to be performed essentially without user intervention. Here, the wax printing method is used to design fluidic channels in Whatman chromatography paper, allowing washing and the time-delayed delivery of a reporter enzyme substrate to a specified region to be achieved without user intervention. Thus we can conveniently integrate our ultra-sensitive enzymatic LFA reporters into LFAs [1] maintaining the one-step hands-free approach typical of standard LFAs. Three separate inlets initially are filled simultaneously with the sample, wash buffer and reporter substrate and the system is left at rest until dry. The channel design allows the hands-free time-controlled delivery of sample, followed by the wash buffer and substrate without the sample and the reporter substrate coming into contact. Functionalization of the paper surface was also explored using a Carbohydrate Binding Module (CBM) fused with two Z domains of staphylococcal protein A (CBM-ZZ) that mediates the oriented coupling of the antibody to the cellulose-based paper surface [2]. An anti-biotin antibody coupled to the CBM-ZZ domain is spotted on the detection region of a wax printed channel and can capture a model analyte (a biotinylated dengue-specific RNA sequence). Gold nanoparticles functionalized with complementary DNA probes are used as reporters allowing the detection of as low as 0.5 μM of RNA target. 1] Adhikari, M. et. al. “Functionalized Viral Nanoparticles as Ultrasensitive Reporters in Lateral-Flow Assays.” Analyst 138, no. 19 (August 28, 2013): 5584–87. [2] Rosa, A. M. M. et al.“Capture and Detection of DNA Hybrids on Paper via the Anchoring of Antibodies with Fusions of Carbohydrate Binding Modules and ZZ-Domains.” Analytical Chemistry 86, no. 9 (May 6, 2014): 4340–47.

Paper microfluidics for the detection of infectious diseases

251st National Meeting (2016)

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