(Invited) Pathogen Agnostic Biodetection at the Point of Need
Authors: Mukundan Harshini 
Published: February 2024

Emerging pathogens are a constant threat to global health and human life, as exemplified by the recent COVID-19 pandemic. The availability of rapid testing - including in home tests - was instrumental in facilitating return to our 'normal' way of life. Diagnostics - especially those that can quickly and effectively inform on the condition at the point of need, enhance situational awareness and guide decision making and mitigation strategies can greatly impact our ability to counter emerging threats, epidemics and pandemics. Yet, current diagnostics are largely targeted- meaning they are tailored to identify a limited repertoire of anticipated threats. For true preparedness, it is important that the strategies we develop are pathogen agnostic and can be readily used against any organism quickly. Our team has sought inspiration from the human innate immune system in order to develop truly agnostic diagnostics. Innate immunity is medicated by pattern recognition receptors that have evolved to recognize evolutionarily conserved signatures on pathogens - allowing for their early identification. These germline receptors bind with conserved pathogen associated molecular patterns – PAMPs – produced by all pathogens, and result in the development of an associated cytokine and chemokine response. Innate immunity is thus a universal biosensor. Drawing inspiration from this natural system, our team has developed diagnostic assays for all pathogens. This involved understanding of common mechanisms of host-pathogen interactions, which then lead to the design of curated novel assay modalities - membrane insertion and lipoprotein capture - for the detection of pathogen. Beyond assay development and characterization of the response, we have evaluated the feasibility of these methods to detect signatures in blinded clinical samples for a variety of diseases (tuberculosis, invasive Salmonella, Shiga toxin carrying E. coli, Staphylococcal bacteremia, and others) with excellent sensitivity as compared to benchmark assays. These outcomes and strategies for pathogen agnostic detection will be presented.

Transitioning this platform for field use, our team engineered a portable waveguide-based optical biosensor platform with integrated microfluidics for sample processes. The platform named Portable EnGineered Analytical Sensor with aUtomated Sampling (PEGASUS, R&D100 2021). This platform uses single mode planar optical waveguides as the sensing element, and builds on a bench top instrument that has been developed at the Los Alamos National Laboratory. PEGASUS miniaturizes this benchtop sensor, with integrated sample processing, moving us toward the goal of developing a truly fieldable biosensor. PEGASUS uses a different, smaller waveguide mounting apparatus, miniaturized robust components, sensing hardware and software.PEGASUS integrates Catch-all, the first ever sample processing system capable of lipid separation with ultra-sensitive optical detection of complex bioanalytes at the point of need. a microfluidic device capable of centrifugal separation of serum from blood at the point of need with a system that is compatible with biomarkers that are both hydrophilic and hydrophobic. The cross-flow filtration device separates serum from blood as efficiently as traditional methods and retains amphiphilic biomarkers in serum for detection.Whereas the scope of targets is extremely broad, the platform still relies on the use of reagents, which complicates field use in some instances. Therefore, we are beginning to explore multi-wavelength spectroscopy as a tool for the measurement of complex clinical signatures - within seconds, without reagents. Preliminary data from this development will also be presented.