Sputum is an increasingly promising noninvasive matrix for diagnosing and monitoring patients with lung conditions. Tuberculosis drug resistance is determined by culture or molecular analysis of bacteria in sputum;lung cancer cells can appear in sputum as an early indicator of disease;absence of bacteria in sputum is a rule-out criterion for broad-spectrum antibiotics in pneumonia. Yet the enormous challenge of extracting nucleic acids from sputum-with its exceptional viscoelasticity, heterogeneity of mucin amounts and cross-linking, and hydration levels-is a barrier to advances in lung disease clinical research and patient care. This Phase I SBIR encompasses early work on a novel tool for sputum processing: a cartridge that automatically carries out a sequence of chemical, mechanical, and biochemical processes on a sputum sample, culminating in the output of isolated DNA and RNA from the cells contained in the starting sample. The sputum sample processing method to be developed is itself wholly novel, while leveraging two innovative technologies pioneered by the Applicant Organization: slit capillary array fluidic actuators (SCAFAs) and solid- phase extraction. A key step in the sputum sample processing is the disruption of cell membranes by electrolytic effects induced through the passage of electrical current through a specially formulated buffer;in this newly prominent area of research, the Applicant Organization is one of a small number of groups leading the effort to characterize and understand the relevant effects. The starting point for the project is preliminary work which has led to the development of a baseline protocol for homogenizing sputum to degrade the elastic component of its mechanical response and reduce its viscosity to a level where it can be readily manipulated by SCAFA chips. Project work encompasses a parameter space study of geometry and other conditions for initial sputum homogenization;development of a filtration/elution process for concentrating cells; and detailed experimentation around electrical lysis chamber configuration, buffer conditions, and electrical input. The subsequent process steps, including glycogen-facilitated precipitation, washing, and elution, will be developed in Phase II, based on a framework developed by the Applicant Organization for diverse pathogen and host DNA/RNA.
The sputum produced in the respiratory tract of individuals with respiratory or lung conditions consist of not just mucous, but a variety of cells whose genetic material contains a wealth of information about the health of the patient's lungs, from bacterial infections to cancer risk. Because the genetic material is buried deep within the cells, and the cells enmeshed in the viscous, sticky mass of mucous, extracting the cellular genetic material requires specialized processing. This project develops an easy-to-use device that, upon introduction of a sputum sample by a clinician or researcher, executes a sequence of steps to automatically extract genetic material.
