Research Projects

This project focuses on the development of a two-dimensional (2D) paper-based diagnostic device that integrates CRISPR/Cas technology with Loop-Mediated Isothermal Amplification (LAMP) for the rapid and precise detection of S. pneumoniae, the leading bacterial cause of pneumonia. The device will enable point-of-care (POC) diagnosis using various clinical samples, such as sputum, and saliva, providing a cost-effective solution, particularly for resource-limited healthcare settings. The integration of CRISPR/Cas and LAMP is aimed at overcoming challenges associated with traditional diagnostic methods such as PCR, which require expensive equipment and are time-consuming. The 2D paper-based platform will be structured to support sample addition, LAMP amplification, and CRISPR/Cas detection at designated locations. Different types of cellulose filter paper substrates will be chosen based on their fluid dynamics, capillary flow properties, and compatibility with biochemical reactions. A CO2 laser cutter will be used to create defined flow pathways to fabricate the device, directing the sample and reagents to specific zones. This structural design enables the device to perform isothermal LAMP amplification and subsequent CRISPR-based detection on a single, self-contained platform. The LAMP amplification process will be optimized initially by testing in solution to establish baseline parameters, such as optimal buffer conditions, reaction temperature, and primer selection. Once optimized, LAMP will be adapted to the paper substrate, where its isothermal nature allows for robust amplification. Following amplification, the CRISPR/Cas system will be introduced to detect the presence of target DNA with high specificity. Customized guide RNAs will be designed to recognize sequences specific to the DNA of S. pneumoniae, triggering a collateral cleavage activity that provides a visual readout, such as a fluorescence, which is easily interpreted at any point of care. A significant focus of this project is the investigation of LAMP-CRISPR/Cas kinetics on paper. This entails studying the rate of target amplification, the efficiency of CRISPR-based recognition, and the timing required to produce the fluorescence. By optimizing these kinetics, the device can achieve rapid detection within a clinically relevant timeframe. This phase also includes experiments to evaluate the reaction under various conditions, such as different environmental temperatures, sample types, and pathogen loads, ensuring that the device performs consistently in diverse settings. To further enhance understanding and optimization, the project will develop an advection-diffusion-reaction (ADR) model which will simulate how reagents flow through the paper substrate, how they diffuse to reach the reaction zones, and how they interact in these zones to produce a detectable signal. Simulations from this model will be helpful in optimizing the device layout.