Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens.

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/µL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics.

Effects of Relative Humidity and Paper Geometry on the Imbibition Dynamics and Reactions in Lateral Flow Assays.

Lateral flow assays and paper microfluidics have the potential to replace benchtop instrumented medical diagnostic systems with instrument-free systems that rely on passive transport of liquid through micro-porous paper substrates. Predicting the imbibition dynamics of liquid through dry paper substrates is mostly modeled through the Lucas-Washburn (LW) equations. However, the LW framework assumes that the fluid front exhibits a sharp boundary between the dry and wet phases across the liquid imbibition interface. Additionally, the relative humidity in the environment results in moisture trapped within the pores of the paper substrates as the paper attains an equilibrium with the ambient air. Here, we apply a two-phase transport framework based on Brooks and Corey's model to capture imbibition dynamics on partially saturated paper substrates. The model is experimentally validated and is then used to predict the liquid-paper imbibition dynamics in simulated environments with 1-70% relative humidity. The model was also used to determine the saturation gradient of liquid along the imbibition interface of the paper substrate. Insights from these studies enabled us to determine the mechanism of the liquid transport in partially saturated porous paper substrates. The model also enabled us to evaluate the optimal paper shapes and relative humidity of the environment that maximize imbibition rates and minimize imbibition front broadening. Finally, we evaluate the effect of moisture content of paper on the rate of paper-based biochemical reaction by amplifying a sequence of the SARS-CoV-2 RNA target via reverse transcriptase loop-mediated isothermal amplification. Taken together, this study provides some important guidelines to academic and applied researchers working in point-of-care diagnostics to develop paper-based testing platforms that are capable of functioning in a robust manner across multiple environmental conditions.