Enhancing Paper-Based Diagnostics through Quantitative Experimentation, Modeling, and Design

Infectious diseases are a significant problem, accounting for 1 in every 4 deaths worldwide. The field of bioengineering is constantly innovating and advancing diagnostic technology; however, more often than not, these innovations are accessible only to communities with privileged resources. This has led to a growing focus on point-of-care (POC) diagnostics. Due to their ease of use, speed, and low cost, POC diagnostics can effectively test patients in resource-poor settings. One of the most well known POC technologies is the lateral-flow immunoassay (LFA). Most easily recognized for its use in pregnancy tests, LFA is a paper-based diagnostic that produces visually interpreted results using a colorimetric indicator decorated with antibodies specific to the target. Recently, we have seen rapid advancement in the fields of paper fluidics and paper diagnostics, which can have a tremendous impact on the future of LFA technology. In light of this, we focused our work in 3 distinct directions, which involves development of quantitative experimental methodologies, mathematical modeling, and improving the ease-of-use of the advanced LFA technology.Development of new paper-based devices would benefit significantly from being able to quantitatively assess the effects of engineering the device on important LFA parameters. For example, it would be useful to know the effects of manipulating the gold nanoprobes and test strips on the forward (kf,s) and reverse (kr,s) rate constants for the probe binding to and dissociating from the test line, respectively. We discuss our novel approach for determining these rate constants and the volumetric flow rate by using mathematical modeling and radioactive iodine-125 (125I). Moreover, we demonstrate how radioactivity and paper strips can also be used to determine the volume of fluid in and before the test line, the concentration of gold nanoprobes, and the number of antibodies per gold nanoprobe. As the field of paper-based diagnostics continues to rapidly expand, it becomes more important to incorporate modeling into their design. A model can be used to determine the effects of LFA parameters on desired outputs, such as the amount of probe bound to the test line. Such predictions become increasingly important as systems become more complicated, and the effects of changing different operating conditions become less intuitive due to the many physical, chemical, and biological processes that are simultaneously occurring. Moreover, a mathematical model allows the engineer to quantitatively predict the influence of well-defined changes in certain parameters. We have derived a simple model that could be used in combination with our novel estimation methods for LFA parameters to predict the amount of probe binding to the test line, an important performance indicator of LFA. We will discuss the derivation of the model, and demonstrate the model’s ability to predict empirical results.Our lab’s recent innovations have improved LFA sensitivity by utilizing aqueous two-phase systems (ATPSs) to thermodynamically concentrate the target molecule prior to detection. In the third direction, we describe a diagnostic design that dehydrates the ATPS components within the paper, creating a non-dilutive, one-step diagnostic process. We investigate the importance of ATPS component dehydration order and demonstrate an improvement in the limit of detection for Chlamydia trachomatis LFA using our dehydrated ATPS diagnostic design