(134b) Toehold-Mediated DNA Strand Displacement Reactions for Quantitative Paper-Based Diagnostics

Molecular techniques that specifically and quantitatively detect low copy numbers of nucleic acids are essential for identifying pathogens in patient and environmental samples and informing their treatment. Isothermal loop-mediated amplification (LAMP) is a promising nucleic acid detection method because it rapidly and exponentially amplifies dilute template DNA, performs robustly with complex samples, and requires minimal equipment. Primers are commonly tagged for post-amplification detection on a simple, lateral flow sandwich-based immunoassay (LFIA) that yields colored bands [1]. However, without an electronic reader, LFIAs provide only a qualitative readout of nucleic acid concentration. Moreover, we have found that the color intensity of the test bands significantly affects users’ accurate interpretation of even qualitative LFIA results [2]. Therefore, here we explore molecular mechanisms (toehold-mediated strand displacement) that could 1) permit more accurate qualitative assessment and 2) provide a quantitative readout of nucleic acids on paper-based test strips.

We recognized that tagging LAMP primers for subsequent LFIA detection frequently yields false positive results. Primer-dimers can cause spurious amplification products that are captured on the LFIA, causing widely varying test band intensities that are difficult for users to accurately interpret. To improve users’ instrument-free interpretation of paper-based diagnostics, we first incorporated toehold-mediated, strand displacement probes into LAMP [3]. We demonstrate that strand displacement probes prevent off-target LFIA capture caused by primer dimerization, thereby eliminating false positive results and permitting definitive interpretation of traditional, LFIA results. We find this simple modification to LAMP provides robust LFIA readout of multiple bacterial targets (Vibrio cholerae and Escherichia coli), even in the presence of complex sample matrices (pond water and human serum).

Second, we explored the utility of toehold-mediated strand displacement reactions to supplant the post-amplification LFIA for quantitative detection of pathogens’ DNA on paper strips. We printed low-cost DNA-hybrid hydrogels, functionalized by the same strand displacement probes described above, into paper strips. In the absence of a target pathogen’s DNA, we found that the hydrogel prevents flow through the paper strip; in the presence of the pathogen’s DNA, the hydrogel releases fluid. This unequivocally observed flow/no-flow response to pathogenic DNA could simplify diagnosis of infectious diseases on paper test strips. Furthermore, we demonstrate the hydrogel functionalized paper strips’ potential to yield a quantitative read-out by correlating DNA concentration with flow distance. In contrast to LFIA’s qualitative results, hydrogel functionalized paper strips offer an instrument-free, quantitative detection of pathogenic DNA. The integration of toehold-mediated DNA strand displacement reactions with paper strips constitutes a significant step towards clinically impactful pathogen detection outside of laboratories.

[1] Rodriguez, et al. Anal. Chem., 2015, 87 (15), pp 7872–7879.

[2] Phillips, et al. Anal. Chem. (in review).

[3] Yu, et al. Anal. Chem., 2015 (87), pp 3314-3320.