Immunoagglutinated particle rheology sensing on a microfluidic paper-based analytical device for pathogen detection

Abstract. Particle immunoagglutination assays have been successfully used in biological sensing for food, water, and environmental applications and medical diagnostics. In this method, interactions between antibody-conjugated particles and biological targets are typically quantified by optical-based sensing, including Mie scattering detection. While these optical methods demonstrate favorable sensitivity and specificity, those that measure light intensity changes are vulnerable to environmental perturbations, such as variations in ambient lighting or humidity. In this work, we investigated a new sensing method based on the particle rheology of immunoagglutinated samples, as seen in droplet spreading on a microfluidic paper-based analytical device (µPAD). By monitoring the overall bulk movement of a particle suspension on paper, these assays are not as critically affected by the sensing environment. Capillary flow of the particle suspension on µPAD channels was tuned by adjusting various parameters, including paper thickness, channel width, channel morphology, particle concentration, and particle size. We then tested the most favorable lateral flow channel design for E. coli K12 sensing in water samples, and applied this overall technique to Zika virus (ZIKV) sensing in biological matrices. From these assays, we achieved similar limits of detection as compared with other demonstrated methods (2 log CFU/mL E. coli; 0.510⁴ transcription copies/mL). Based on this work, direct detection of immunoagglutinated particle rheology through droplet spreading shows promise as a unique and simple method with applications in automated biosensors for environmental and health samples. (Funding provided by the BIO5 Institute and the U.S. National Science Foundation Graduate Research Fellowship, DGE-1143953).