Category: Micro- and Nanotechnologies
There is a great need to develop a device for infectious disease detection that is rapid, sensitive, and accurate. Current gold standard screening often involves fluorescence-based DNA amplification readings or antibody-based assays to confirm pathogen presence in either patient or environmental samples. These assay methods are robust but are often not rapid (fluorescence DNA amplification) or sensitive enough (lateral flow antibody-based assays). We have developed an alternative method, particle diffusometry (PD), where the presence of pathogens are detected by measuring changes in the diffusivity of fluorescent particles, which results from pathogen DNA amplification. V. cholerae, S. aureus, and K. pneumoniae are combined with specialized oligonucleotide primers, allowing for DNA amplification with loop-mediated isothermal amplification (LAMP). This amplification increases the length of polynucleotides in the solution, which in turn makes the fluid sample more viscous. Furthermore, by using biotinylated primers and streptavidin coated particles, an increase in particle size will occur due to the polynucleotides hybridizing and clustering the particles together. We suspend 220nm streptavidin coated particles in a fluid sample and image their Brownian motion with fluorescence microscopy. In the presence of amplified pathogen DNA, these particles exhibit slower Brownian motion, due to size and viscosity changes, indicating a change in the particle diffusion coefficient. PD calculates the diffusion coefficient of the particles by correlating successive particle images.
We compare PD measurements to traditional measurements of fluorescent molecule intercalation into amplified DNA and discuss the development of a point-of-care platform. PD measurements of polymerized DNA solutions are compared to quantitative fluorescence measurements (SYBR/ROX) over a concentration range of pure DNA spanning 1-100,000 cells/mL; concentrations which are relevant for a variety of bacterial strains in patient and environmental samples. From this we achieve a lower limit of detection of 1,000 DNA copies/reaction (using a 25 microliter LAMP reaction volume with a 20-minute limited amplification time). Further, we determine the change in diffusivity due to DNA amplification in the presence of whole cells for V. cholerae, K. pneumoniae, and S. aureus with a lower limit of detection of 1 cell/reaction (25 microliter LAMP reaction volume, 20-minute amplification time). We also successfully measure the diffusivity of amplified DNA from V. cholerae cells in environmental water sources, such as pond water, a common habitat for this bacterium.
With this initial framework in place, we translate PD from the microscope onto a smartphone-based detection platform, enabling diffusivity measurements to be performed at the point of care. PD algorithms are integrated into a smartphone application that captures images of particle movement, correlates the images, and outputs pathogen detection results. This portable platform will enable detection of pathogens at the point of care in less than 30-minutes.
Katherine Clayton– Postdoctoral Scholar, Purdue University, West Lafayette, IN
West Lafayette, IN
Dr. Katherine N. Clayton is a Postdoctoral Researcher with Dr. Tamara L. Kinzer-Ursem, Dr. Steven T. Wereley, and Dr. Jacqueline C. Linnes at Purdue University. She has a PhD in Mechanical Engineering from Purdue University and a B.S. and M.S. in Biomedical Engineering from California Polytechnic State University. She is passionate about the development and translation of quantitative devices for point-of-care disease diagnostics. Additionally, Katherine has experience in applying algorithms she developed for biotherapeutic analysis, nanoparticle sizing, and protein-protein interactions and has additional experience in novel bioconjugation techniques, optical device design, and paper-based microfluidics.